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iEditors:Yael Heifetz, Amos Tandler, Sharon Schlesinger, Dimitrios Rizos, Tiziana Brevini

March 3-6, 2020 The Hebrew University,Mt. Scopus, Jerusalem, Israel

Proceedings of the 3rd CellFit Workshop

3D3C: Towards a new vista of in-vitro 3-dimensional organization and cell to cell communication

We would like to thank the following organisations for the support and sponsorship:

European Cooperation in Science and Technology

COST is supported by theEU framework Programme

Horizon 2020

The University of Milan

Publisher: Jointly published by The Hebrew University, The University of Milan, The National Institute for Agriculture and Food Research and Technology and the COST Action CA16119.

Book Title: 3D3C: Toward new vista of in-vitro 3-dimensional organization and cell 2 cell communication

Year of Publication: 2020ISBN: 978-965-599-199-4

Legal Notice:Neither the COST Office nor any person acting on its behalf is responsible for the use which might be made of the information contained in this publication. The COST Office is not responsible for the external websites referred to in this publication.

No permission to reproduce or utilise the contents of this book by any means is neces-sary, other than in the case of images, diagrams or other material from other copyright holders. In such cases the permission of the copyright holders is required.

Version: V1

Copyright © COST Office: 2020

Table of Contents About the European Co-operation in Science and Technology…... 1

Welcome from the Chairman…………………………………………. 2

Welcome from the local organizers………………………………….. 3

Time table………………………………………………………………. 5

Speakers…………………………………..………………………….… 11

Special sessions: Old challenges and new opportunities in engineering and analyzing complex 3D functional tissues………………...…………

42

Emerging live cell imaging technologies: cell biology models, microscopy techniques and bioimage analysis…………………….

47

Microfluidics – from basic concepts to organ-on-a-chip………….. 55

Poster presentations………………………………..…………………. 68

Participants…..……………………………………………………….... 108

About the European Co-operation in Science and Technology

The European Cooperation in Science and Technology (COST) is the oldest and widest European intergovernmental network for cooperation in research. Established by the Ministerial Conference in November 1971, COST is presently used by more than 30 000 scientists of 35 European countries to cooperate in common research projects supported by national funds. The financial support for cooperation networks (COST Actions) provided by COST is about 1.5% (30 million EUR per year) of the total value of the projects (2000 million EUR per year). The main characteristics of COST are:

• Bottom up approach (the initiative of launching a COST Action comes from the European scientists themselves),

• Á la carte participation (only countries interested in the Action participate)

• Equality of access (participation is also open to the scientific communities of countries which do not belong to the European Union) and

• Flexible structure (easy implementation and management of the research initiatives).

As precursor of advanced multidisciplinary research COST has a very important role in shaping the European Research Area (ERA). It anticipates and complements the activities of the current Framework Programme for Research and Innovation (Horizon 2020). COST activities create a bridge between the scientific communities of countries and increases the mobility of researchers across Europe in many key scientific domains.

From the Chairman Dear CellFit Member, Welcome to our third Workshop that will take place in Jerusalem, the city of earth and heaven. Located in the mountains that separate the Mediterranean from the Dead sea. Where old meets new, holy meets secular, and where nature and beautiful architecture merge into an incredible mix of scents, sounds and sights. Jerusalem is a fascinating city. It will enchant us all, but will also give us the opportunity to experience top science and an exciting scientific program, with great speakers from many different countries. Continuing a trend that started a couple of years ago, we have greatly encouraged the participation and involvement of Early Career Investigators, hoping that this will be beneficial for their present activities as well as future careers.

I do hope this meeting will lead to many active collaborations, implementing networking activities and synergies within the European excellence and beyond.

I would like to take this opportunity to thank Yael, Dimitrios and Amos for the effort they have addressed to the organization of this workshop. Also, many thanks to Alireza, for precious help and advice and all CellFit Grant Holder team for support on any financial issue. Last but not least, I gratefully acknowledge all members of the Executive Committee for their inputs and cooperation.

Shalom!

Prof Tiziana A.L. Brevini Chair of CellFit March 2020

Welcome from the organizing committee It is our pleasure to have you here on Mount Scopus from which you can see the "Golden Dome" and "El-Aqsa" mosques, among the holiest points for Muslims, the Judean desert where Christianity was born, and the wall of the demolished Jewish temple. This is the city of Jerusalem – the intersection of the three monotheist religions that generates so much energy, sometimes ends up in tragic conflicts, but many other times it provides reasons for human existence and strengthens unity and collaboration. This is also an opportunity to tell you that the Hebrew University, where the meeting is taking place is the first university in Israel, established in 1925 here on Mount Scopus. The organising committee trusts that you will find this place exciting and insightful. The 3D3C: Towards a new vista of in-vitro 3-dimensional organization and cell to cell communication workshop brings together junior and established world leading scientists from Europe, North America and Israel; from academic, governmental, and clinical sectors. Using different models; and with wide-ranging experimental approaches it would be possible to discuss recent advances and to consider present challenges in investigating new paradigms in the area of 3D tissue culture and cell to cell communication, setting health status in the next generation. Presentations at the 3D3C workshop will share new insights into cell-environment interactions and present cutting edge research that uses new approaches to study chemical signals and gradients to which cells are exposed. We envision that the data presented, and discussions held will lead to collaborations generating better understanding of the in vitro complex chemical environments. The present workshop also includes special sessions that will expose the attendees to cutting-edge technologies and solutions and scientific innovations on emerging live cell imaging technologies and

microfluidics from basic concepts up the development the organ-on-a-chip design. The 3D3C workshop could not have happened without the support of COST, The Hebrew University, Katzir-Katchalsky Center Funding, Rhenium, Biological Industries and AVBA. Sincere thanks go to the organizing committee of the special sessions, Yuval Garini, Assaf Zaritsky, Javier, A. Sanchez-Lopez, Ofra Benny and Shay Tal, for their professional help and stimulating discussions about the content of the sessions. We are thankful to the members of the Heifetz laboratory for their understanding, support, and help during the journey. We would like to thank particularly to Javier Arturo Sanchez-Lopez for his essential help in setting and organising the digital parts of the meeting and the abstract booklet. Amalia Shalom-Gothilf, Shani Kornhauser, Rita Shuhmaher, Ido Apel and Shai Twena for their professional support in executing other essential administrative tasks, and Carmit Feliks, from the Schlesinger laboratory, for her help with the budget. Finally, many thanks to the speakers, presenters of oral communications or posters; we believe that the exchange of information and discussion will provide a unique opportunity to create collaborations in the newly development of the 3D3C field. We hope that you will enjoy and look forward to meeting all of you, Best wishes, Yael Heifetz, Amos Tandler, Sharon Schlesinger, Dimitrios Rizos and Tiziana Brevini

3D3C: Towards a new vista of in-vitro 3-dimensional organization and cell to cell

communication

Time table

March 3 2020 17:00 – 19:30 Meeting Registration 20:30 – 21:45 Welcome Reception

March 4, 2020 08:00 – 09:00 Meeting registration & Poster setup

09:00 – 09:10 Welcome and opening remarks Tiziana Brevini, Amos Tandler, Yael Heifetz (Mandel Auditorium)

Epigenetic control governed by external or environmental factors - challenges in tissue engineering Moderators: Tiziana Brevini, Sharon Schlesinger,

Nicolas Ortega

09:10 – 09:40

Moshe Szyf, McGill University, Canada Epigenetic processes mediating between environment, exposures and phenotypes; implications for 3D3C; could we mimic the “natural epigenome” in vitro

09:40-10:10 Shoseyov Oded, The Hebrew University, Israel Plant produced human recombinant collagen a scaffold for regenerative medicine

10:10-10:40

Priscila Ramos, INIA Madrid, Spain A single-cell transcriptional atlas of pig embryogenesis; toward a new system to extend the window of embryo development in vitro

10:40-10:50 Miranda Joana P, University of Lisbon, Portugal

Towards liver-on-a-chip: the role of hepatocyte-like cells in a physiologically relevant model

10:50-11:11

Pušić Maja, University of Zagreb, Croatia In vitro cultivation of cartilage grafts using nasal chondrocytes and 3D chitosan scaffolds

11:00–11:30 Coffee/Tea Break & Poster Presentation

Cell-cell communication and microenvironmental homeostasis of cells, organs, and intact systems Moderators: Yael Heifetz, Dimitrios Rizos,

Tamara Kukolj

11:30-12:00 Neta Regev-Rudzki, Weizmann Institute, Israel 20S proteasomes exported by malaria parasite promote its growth

12:00-12:30 Assaf Zaritsky, Ben-Gurion University, Israel Defining the building blocks of collective cell behaviour

12:30-13:00

Angel Raya, Bellvitge Biomedical Research Institute, Spain Eliciting tissue-like behaviour on human cardiac constructs

13:00 – 14:45

Lunch & Poster Presentation

Cell environment and Chemobiological cues Moderators: Bart Gadella, Anne Navarrete-

Santos, Amos Tandler, Yoav Soen, Milda Alksne

14:45 – 15:15

Sabeth Verpoorte, University of Groningen, Netherlands Leveraging microfluidics to enhance in vitro experimentation with cells and tissue

15:15 – 15:25

Rohrer Jack, Zurich University of Applied Sciences, Switzerland Reporter constructs in primary human cells to generate in vitro model systems

15:25-15:35 Ortega Nicolás M, Karolinska Institutet, Sweden Elucidating lineage segregation on bovine preimplantation embryos

15:35-15:45

Šimoliūnas Egidijus, Vilnius University, Lithuania The Role of MAP Kinases in Human GMSCs in Response to Different Degrees of Matrix Stiffness

15:45-15:55

Alexandrova Radostina, Bulgarian Academy of Sciences, Bulgaria

Evaluation of cytotoxic / antitumor properties of new metal complexes with different ligands in 2D and 3D cell cultures

15:55- 16:25

Coffee/Tea Break & Poster Presentation

16:25-16:55 Nicola Elvassore, University of Padova, Italy Confinement environment and extracellular matrix proteins enable endodermal organoid culture

16:55-17:05

Žegura Bojana, National Institute of Biology, Slovenia Hepatic in vitro 3D cell model developed from HepG2 cells for the assessment of genotoxic activity of environmental pollutants

17:05-17:15

Krysiak Zuzanna Joanna, AGH University of Science and Technology, Poland Electrospun polyvinyl butyral-co-vinyl alcohol-co-vinyl acetate (PVB) patches for atopic skin treatment - cytotoxicity tests

17:15-17:45

Discussion Moderators: Tiziana Brevini, Sharon

Schlesinger, Yael Heifetz, Dimitrios Rizos, Bart

Gadella, Anne Navarrete-Santos, Amos Tandler,

Yoav Soen

18:30 –

Gala dinner – Jerusalem Sound and Light Show at the Citadel - Tower of David

March 5, 2020

Old challenges and new opportunities in engineering and analyzing complex 3D functional tissues Moderator: Moshe Szyf, Angel Raya

09:05-09:40 Shulamit Levenberg, Technion, Israel

Engineering 3D vascularized tissue constructs

09:40-10:10

Assaf Bar, Ben-Gurion University, Israel Live imaging flow bioreactor for elucidating the mechanism behind regeneration of articular cartilage after treatment with bioactive hydrogel

Emerging live cell imaging technologies: cell biology models, microscopy techniques, and bioimage analysis Moderators: Yuval Garini, Javier, A. Sanchez-

Lopez

10:15-10:40 Natan T. Shaked, Tel-Aviv University, Israel Rapid, label-free, high-resolution three-dimensional imaging of cells

10:40-10:55 Ori Avinoam, Weizmann Institute, Israel

Membrane sequestration and crumpling maintain membrane homeostasis during exocrine secretion

10:55- 11:25

Coffee/Tea Break & Poster Presentation

11:25-11:50

Yoav Shechtman, Technion, Israel Volumetric and high-throughput localization microscopy via wavefront shaping and deep learning

11:50-12:15 Yuval Garini, Bar-Ilan University, Israel Studying the dynamics of molecules in live cells

12:15-12:25 Newman, Abigail, Technion, Israel 3D Bioprinted Pancreatic Endocrine Tissue

12:25 – 14:10

Lunch & Poster presentation

Microfluidics – from basic concepts to organ-on-a-chip Moderators: Ofra Benny, Shay Tal

14:15 -14:40 Yaakov Nahmias, The Hebrew University, Israel Illuminating human function: development of a smart micro-physiological bioanalyzer

14:40 – 15:05 Nissan Yissachar, Bar-Ilan University, Israel Thinking outside the mouse: ex-vivo dissections of host-microbiome cross talks

15:05 – 15:30 Yoav Soen, Weizmann Institute, Israel Promoting desired biological functionality in live bioreactors

15:30 - 15:55 Arbel Artzy-Schnirman, Technion, Israel Lung-on-chip platforms: narrowing the gap between in vitro models to in-vivo

15:55-16:25

Coffee/Tea Break & Poster Presentation

16:25-16:50 Ben M. Maoz, Tel-Aviv University, Israel Organs-on-a-chip: A new tool for the study of human physiology

16:50 – 17:15 Amir Bitan, National Center for Mariculture, Israel Fish-AI- developing an artificial intestine for the sustainable farming of healthy fish

17:15 – 17:45 Discussion Moderators: Moshe Szyf, Yuval Garini, Javier, A. Sanchez-Lopez, Ofra Benny, Shay Tal

18:30 – Dinner & Social event

Day 4 – March 6, 2020

06:30 – Excursion - Jerusalem

Speakers

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Szyf, Moshe (1)

(1)Department of Pharmacology McGill University Montreal Canada

Epigenetic processes mediating between environment, exposures and phenotypes; implications for 3D3C; could we mimic the “natural epigenome” in vitro

The relationship between genotype and phenotype is mediated in many instances by epigenetic programming. Epigenetic processes involve direct covalent modification of DNA by methylation catalyzed by DNA methyltransferases (DNMT) and further modifications of the methyl moiety by a series of oxidations catalyzed by TET enzymes. In addition to these direct modifications of the DNA molecule itself, creating a different molecular entity than unmethylated DNA, other epigenetic elements control the packaging of DNA in chromatin through diverse enzymatic modifications of histones by methylation, acetylation, phosphorylation ubiquitination etc. regulatory noncoding RNAs such as microRNA and long noncoding RNAs contribute as well to epigenetic regulation. All these processes act together to set

12

up different cellular identities on identical DNA sequences during cellular differentiation and enable one DNA to encode numerous different programs that express themselves in highly predictable manner in space and time. The evolution of epigenetic programs is encoded in DNA, defined by evolution and is highly predictable but its execution requires interaction between different components of the evolving embryo. This poses the first immense challenge to 3DC. Studies that span the last decade and a half reveal that in addition to these innate processes the epigenome is shaped by external exposures and experiences including behavioral exposures. These exposures drive epigenetic changes and stably alter the phenotype. We will provide evidence from rodents, nonhuman primates and humans for epigenetic programming of the phenotype by environmental exposures. These altered epigenetic profiles have been implicated in many diseases posing the question of how 3D3C could mimic these processes in vitro. We will show examples of how culture environments alter the epigenome. Possible approaches to tackle these challenges will be discussed. Our ability to map epigenomes in a genomic scale provides tools to asses the epigenomic programming in vitro and measure its proximity to naturally occurring epigenetic modulations by exposure.

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Shoseyov, Oded (1)

(1)The Faculty of Agriculture, The Hebrew University of Jerusalem, Israel

Plant produced human recombinant collagen, a scaffold for regenerative medicine

In the last 200 years of modern chemistry, synthetic materials dominate the medical industry including orthopedic surgery, and pharmaceuticals. In my talk I will demonstrate the superiority of biomaterials and particularly plants as a novel tool for the bio-manufacturing of human recombinant Type I collagen as a scaffold for tissue engineering and regenerative medicine.

Applications and clinical data will be presented for its application in diabetic foot ulcer and chronic wounds. rhCollagen/PRP injections for treatment of tendonitis will also be discussed as well as collagen bio-ink for tissue and organ 3D bioprinting.

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Ramos-Ibeas, Priscila (1)

Ramos-Ibeas, P (1); Sang, F (2); Zhu, Q (3); Klisch, D (3); Loose, M (2); Surani, A (4,5); Bermejo-Álvarez, P (1); Alberio, R (3) (1) National Institute for Agricultural and Food Research and Technology (INIA), Spain; (2) School of Life Sciences, University of Nottingham, United Kingdom; (3) School of Biosciences, University of Nottingham, United Kingdom; (4) University of Cambridge, United Kingdom; (5) Wellcome Trust/Cancer Research UK Gurdon Institute, University of Cambridge, United Kingdom

A single-cell transcriptional atlas of pig embryogenesis; toward a new system to extend the window of embryo development in vitro

Early embryo development is a critical period in which key genetic and epigenetic mechanisms orchestrate the first cell fate decisions and the development of the epiblast, whose cells will form the proper fetus. The mechanisms regulating these processes in large mammals are still poorly understood, since most studies have been carried out in rodents, a classical model for developmental biology. Furthermore, the study of human embryos is limited to

15

pre-implantation stages because early post-implantation embryos are not accessible. However, ungulates and primates share conserved developmental principles, such as the development of the epiblast as an embryonic disc, in contrast to rodents. Recently, single-cell analyses have opened the doors to delineate the cellular foundations of mammalian embryogenesis. Here, we present the transcriptional map of pig early embryo development using single-cell RNA sequencing. We show progressive segregation of inner cell mass and trophectoderm in early blastocysts, and of epiblast and hypoblast in late blastocysts. We reveal the transcriptional circuitry of pluripotency characterized by a short naïve pluripotent signature followed by a protracted primed stage in advanced embryonic stages. In females, dosage compensation with respect to the X chromosome is accomplished via X-inactivation in the late epiblast before lineage priming. Detailed comparisons between human and pig embryo development were performed as a basis for further studies of human pluripotent stem cell differentiation in pig interspecies chimeras. Besides, this detailed transcriptional profile provides a blueprint to understand the requirements of the embryo at each stage of development. Taking advantage of this information and exploring different non-adherent tridimensional substrates, we aim to develop a chemically-defined in vitro culture system to support embryo development after the blastocyst stage in ungulates.

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Miranda, Joana P (1)

Rodrigues, JS (1); Cipriano M (1); Correia JC (2); Camões SP (1), Ruas JL (2) (1) Research Institute for Medicines (iMed.ULisboa), Faculdade de Farmácia, Universidade de Lisboa, Portugal; (2) Karolinska Institutet, Sweden

Towards liver-on-a-chip: the role of hepatocyte-like cells in a physiologically relevant model

Long-term and physiologically relevant human hepatic models constitute a great opportunity for personalised biomedical research. As such, this work focused on the optimization of a microfluidic model able to maintain hepatic features of human neonatal MSCs (hnMSCs)-derived hepatocyte-like cells (HLCs) under physiological media concentrations. Prior to cells adaptation to a double channel PDMS-based microfluidic device (MDs), HLCs were differentiated from hnMSCs through a 3-step protocol lasting 21 days. From day 21 (D21) to D34, HLCs were cultured in 4 media formulations: differentiation medium (DM-high), with 25 mM of glucose, 1.72 μM of insulin and 1μM of dexamethasone; maintenance medium (MM-high) with 25 mM of glucose, 1 nM of insulin and 100 nM of dexamethasone; DM-low, equal to DM-high with 5 mM of

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glucose; and MM-low, the more physiologically relevant medium, equal to MM-high with 5 mM of glucose. Phase I and phase II activities (EROD and UGT activities), glycogen storage ability (PAS assay); mitochondrial activity (Seahorse XF Cell Mito Stress Test); expression of hepatic-specific genes through RT-qPCR were assessed at D27 and D34. For MD adaptation, cells were seeded at day 17 (D17). Both coating and cell inoculum were optimized. HLCs’ albumin and urea production were evaluated up to D34. Herein, HLCs cultured in the 4 media expressed Alb, Cyp3a4 and Hnf4a at D34, maintained EROD and UGT activities and mitochondrial activity and presented glycogen storage ability, at both D27 and D34. Overall, HLCs were successfully adapted to MDs by inoculating 7.5 x 104 cells/channel using 0.2 mg/mL of type I collagen as coating, presenting stable urea and albumin production. Importantly, HLCs kept in more physiologically relevant conditions maintained the hepatic features herein assessed. Along with the successful adaptation of HLCs to MD, these results represent a step forward to obtain a model representative of the complex hepatic microenvironment.

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Pušić, Maja (1)

Rogina, A (1); Štefan L (1); Ivkovic A (1); Marijanovic I (1); Ivanković M (1); Ivanković H (1) (1) University of Zagreb, Croatia

In vitro cultivation of cartilage grafts using nasal chondrocytes and 3D chitosan scaffolds

Articular cartilage defects are common in population and are frequently caused by trauma, aging or disease. Articular cartilage tissue lacks blood, lymph and nerve supply so it has limited ability to repair or regenerate. Tissue engineering approach using scaffolds and cells presents as the most adequate method for cartilage repair. In this study, three commercially available chitosans modified by adhesive protein were used as a support for in vitro engineering of cartilage grafts from sheep nasal chondrocytes (NC) for the repair of knee cartilage lesions. Chitosan scaffolds used in this study were prepared by lyophilization from the medium molecular weight (MC75) and high molecular weight (HC75) chitosan with deacetylation degree >75% and chitosan derived from shrimp shells (MC95) with deacetylation degree >95%. The microstructure of the scaffolds was determined using SEM. The pore size distribution and porosity of scaffolds were estimated using the immersion method in absolute ethanol under vacuum. Adsorption and desorption of fibronectin (FN) on scaffolds were determined using BCA assay. Scaffolds were

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incubated in FN solution for 24 h and seeded with sheep NC. Seeded constructs were cultivated in chondrogenic medium for 7 or 14 days. Manufactured grafts were fixed in 4% formaldehyde, embedded in paraffin and sectioned on microtome. Sections were stained with hematoxylin-eosin and safranin O and immunostained for collagen type II and aggrecan. Microstructure of all scaffolds observed with SEM was highly porous with good pore interconnectivity. Average pore size for all three scaffold ranged from 100 to 300 μm while porosity measured had values between 96 and 98% with no significant difference between the scaffolds. All prepared scaffolds showed similar adsorption capacity of FN. Desorption of FN was lower for chitosan MC95 compared to scaffolds with deacetylation degree >75% (MC75 and HC75). Histological analysis revealed that grafts cultivated for 14 days had higher amount of glycosaminoclycans and cartilage specific proteins compared to grafts cultivated for 7 days. Best support for NC differentiation was determined with chitosan scaffold of high molecular weight and deacetylation degree >75% (HC75) without FN. Presence of FN on chitosan scaffold did not affect cell adherence or improved differentiation of chondrocytes. Chitosan scaffolds of different molecular weights and deacetylation degree support differentiation of NC. In this study, we have successfully engineered cartilage grafts that can be used in a translational in vivo study for articular cartilage repair.

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Regev-Rudzki, Neta (1)

Dekel, E(1), Yaffe, D(1), Goldian, I (2), Ziv, T(3), Sisquella, X (4,5), Pimentel, MA (4,5), Nebl, T (4,5), Kapp, E (4,5), Ohana-Daniel Y (1), Abou-Karam P(1), Malihi, S(1), Mullick D (6), Rudik, A(1), Gov N(6), Azuri I(7), Porat Z(8), Carvalho T(9), Cohen S(2), Sharon M(*1) 1Faculty of Biochemistry, Department of Biomolecular Sciences, Weizmann Institute of Science, Rehovot 76100, Israel 2Department of Chemical Research Support, Weizmann Institute of Science, Rehovot 76100, Israel 3Smoler Proteomics Center, Department of Biology, Technion - Israel Institute of Technology, Haifa, Israel 4The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, VIC 3052, Australia. 5Department of Medical Biology, The University of Melbourne, Grattan Street, Parkville, VIC 3010, Australia 6Department of Chemical and Biological Physics, Weizmann Institute of Science, Rehovot 76100, Israel 7Department of Materials and Interfaces, Weizmann Institute of Science, Rehovot 76100, Israel 8Flow Cytometry Unit, Life Sciences Core Facilities, Weizmann Institute of Science, Rehovot 76100, Israel 9Molecular Parasitology Laboratory, Department of Physiology, Anatomy and Microbiology, La Trobe University, Kingsbury Drive, Bundoora, VIC 3086, Australia.

20S proteasomes exported by malaria parasite promote its growth

The malaria parasite, Plasmodium falciparum (Pf), is one of the most virulent species, responsible for the greatest number of fatalities worldwide. The parasites in the merozoite

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stage of their blood cycle must invade naive Red Blood Cells (RBCs) to sustain a blood-stage infection. It has already been established that intracellular pathogens remodel the host membrane to mediate their invasion and that communication mechanisms with the host cell exist. However, it is still unknown whether Pf parasites, while growing inside the RBC host, remodel naïve surrounding RBCs to ease succeeding invasions. One known communication mechanism that pathogens employ to persist within their host is the release of multipurpose extracellular vesicles (EVs). These secreted vesicles carry different cargo components and are heterogeneous in size (30 to 500 nm in diameter) and target destination. Here we show that the secreted EVs are enriched with parasitic and host kinases as well as 20S proteasome subunits. Strikingly, using biochemical methods we demonstrated that the 20S proteasomes carried by the EVs (EV-20S) are assembled and active within the vesicles and their function promotes parasite growth in the treated uRBCs. Systematic mechanical analysis of the recipient host RBCs revealed that the 20S proteasome activity increase cell softness and that this is achieved through the degradation of cytoskeleton proteins of the RBC. Finally, we identified four target protein substrates of the EV-20S that belong to the cytoskeleton network of the RBC membrane: *-adducin, ankyrin-1, dematin and Epb4.1. Overall, our results indicate that EV-20S secreted from Pf-infected RBCs are able to prime naïve host RBCs for impending parasite growth.

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Zaritsky Assaf (1)

(1) Ben Gurion University

Defining the building blocks of collective cell behavior

A fundamental question in the study of collective cell behavior is how multicellular patterns emerge from the behavior and interactions of individual cells. Examples include collective cell migration, planar cell polarity, collective calcium signaling, and collective cell death. In each case, individual cells use basic cellular machineries to influence and respond to neighboring cells through a complex interplay of chemical and physical cues. Our understanding on how these local interactions are integrated in space and time to induce global collective behavior is limited due to phenotypic heterogeneity. Traditionally, the vast majority of studies have relied on population averages, treating all cells in a collectively behaving group as identical. However, behavioral heterogeneity is prevalent in cellular biological systems, both in health and in disease. For example, some cells within a group may function as super-influential, promoting the spread of information through the group, while others behave as

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followers or act individually, independently of other cells in their vicinity. Thus, to elucidate mechanisms for emergence of multicellular behavior it is essential to quantitatively evaluate how information propagates between single cells in multicellular systems. I will present a conceptual unified computational framework to measure the cell-cell information-transfer between all pairs of cells in dynamic multicellular systems and demonstrate its applicability in a variety of biological systems.

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Raya, Angel (1) (1) Bellvitge Biomedical Research Institute, Barcelona, Spain Eliciting tissue-like behavior on human cardiac

constructs In vitro surrogate models of human cardiac tissue hold great promise in disease modeling, cardiotoxicity testing, and future applications in regenerative medicine. However, the generation of engineered human cardiac constructs with tissue-like functionality is currently thwarted by difficulties in achieving efficient maturation at the cellular and/or tissular level. Recent developments in the engineering of miniaturized cardiac constructs result in greatly improved maturation of cardiac muscle cells, but at the expense of exhibiting rudimentary tissue-like behaviors. Here, I will discuss the design and implementation of a platform for the production of engineered cardiac macrotissues from human pluripotent stem cells (PSC), which we term ‘CardioSlice’. PSC-derived cardiomyocytes, together with human fibroblasts, are seeded into large 3D porous scaffolds and cultured using a parallelized perfusion bioreactor with custom-made culture chambers endowed with

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electrostimulation capabilities. Constructs cultured in this way develop into macroscopically contractile structures in which cardiomyocytes undergo further maturation. More importantly, continuous electrical stimulation of cardiac macrotissues for 2 weeks promotes cardiomyocyte alignment and synchronization, and the emergence of cardiac tissue-like properties. These include spontaneous electrical activity that can be readily measured on the surface of CardioSlice constructs as electrocardiogram-like signals, and a response to proarrhythmic drugs that is predictive of their effect in human patients.

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Sabeth Verpoorte (1)

(1) University of Groningen, The Netherlands

Leveraging microfluidics to enhance in vitro experimentation with cells and tissue

Confining molecular species in micro-, nano-, pico- and even femtoliter volumes can significantly improve our manipulation and control of them. In the past three decades, we have learned how to perform multistep chemical processing in microchannels, exploiting the exquisitely well-defined flows at this scale. This presentation will take a look at how manual experimentation in the lab has been transposed to the microfluidic format. In addition, I will consider the different ways we can apply this microfluidic toolbox to enhancing cell and tissue culture.

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Rohrer, Jack (1)

Filippi, BGH (1); Pally, J (1) (1) Zurich University of Applied Sciences

Reporter constructs in primary human cells to generate in vitro model systems

Tissue engineering aims at the generation of biological material to heal or replace damaged tissues and organs. Tissue engineering also includes the use of tissues to study complex 3D biological models, and to test the pharmacology and the toxicity of novel drugs. Reporter genes enable the precise measurement of specific cellular conditions (e.g. vitality, cellular stress, and activity of intra-cellular pathways) and numerous biological models consisting of immortalized cell lines modified with such reporter constructs (i.e. “reporter cells”) currently play an essential role as test systems to study the effect of various drugs on these cells. 3D models are physiologically more accurate than traditional 2D models, but can often not be produced using immortalized cell lines and rather require primary cells. However, conventional regulatory elements developed for the expression of reporter genes in immortalized cell lines produce mixed results in primary cells. Therefore, the choice of the regulatory

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elements is critical for the proper function of reporter genes in primary cells and each primary cell type requires a specific architecture for efficient expression of constitutive or conditional reporter genes. In this work, we developed constitutively and conditionally expressed reporter systems in primary human keratinocytes. Such modified primary human keratinocytes can be used for the production a 3D Nrf2/KEAP1 reporter human epidermal model, therefore showing that this approach can help to establish novel functionalized 3D models.

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Ortega, Nicolás M. (1)

Ortega,NM (1); Plaza-Reyes, A (1); Van Leeuwen, T (1); Gassol i Carbonell, A (1); Ahola, V (2); Jouneau, A (3); Duranthon, V (3) & Lanner, F (1). (1) Karolinska Institutet, Stockholm, Sweden; (2) Karolinska Institutet, Hong Kong, China; (3) INRA, Jouy en Josas Cedex, France.

Elucidating lineage segregation on bovine preimplantation embryos

Bovine embryos share morphological and temporal resemblance with human early development, still molecular and cellular key developmental mechanisms need to be further explored. From the human totipotent zygote, cleavage stage cells multiply, and take fate decisions undergoing an intermediate state of co-expression of lineage-specific genes, followed by a concurrent establishment of the trophectoderm (TE), epiblast (EPI), and primitive endoderm (PE) lineages, which coincide with blastocyst formation. Our single cell transcriptomics data showed that the first lineage segregation in the preimplantation bovine embryo occurs after cavitation at E6 in the early blastocyst, suggesting a similarity with the early human blastocyst. In addition, E7 showed distinctive and more mature inner cell mass (ICM) and TE profiles.

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Interestingly, we also found that the second lineage segregation, which segregates the EPI from the PE within the ICM, starts in bovine at E8 expanded hatched late blastocysts. Taken our scRNAseq data, we evaluated the role of different signaling pathways very well studied in the mouse embryo during the bovine lineage segregation. We performed genome editing using CRISPR/cas9 and Knockdown of mRNA using antisense LNA GapmeRs by cytoplasmic direct injection into bovine presumptive zygotes. We tested different embryo culture conditions and media containing small molecules followed by immunofluorescence analysis. Our results suggest roles in lineage segregations and embryo development different from the mouse, thus confirming how the cellular and genetic mechanisms that control fundamental processes are surprisingly distinct between the species. We aim to translate the gained knowledge from our bovine experiments for infertility treatments, and better understanding of the human embryo.

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Šimoliūnas, Egidijus (1)

Sakalauskaitė I (1), Bagdzevičiūtė L (1), Baltriukienė D (1) (1) Vilnius University, Lithuania

The Role of MAP Kinases in Human GMSCs in Response to Different Degrees of Matrix Stiffness

The mechanical properties of the extracellular matrix (ECM) in which cells reside have emerged as an important regulator of cell behavior. Cell interaction with the ECM initiate signaling pathways which depends on ECM mechanical stimulation. ECM generated mechanical forces can lead to changes in cell and tissue development, cell differentiation, physiology and contribute to the pathogenesis of various diseases: atherosclerosis, fibrosis, heart failure and cancer, etc. For this reason, it is important to find out how tissue cells respond to changes in the mechanical environment. To study the mitogen activated protein kinases (MAPK) role in human derived gingival mesenchymal stem cell (GMSC) on different degrees of matrix stiffness a series of experiments were carried out. GMSC morphology, cell cycle regulation, adhesion, amount of focal adhesion, proliferation, response to oxidative stress, MAPK expression, activation and the role of induced cell oxidative stress on the matrixes of different

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stiffness were studied. MSC extracted from gingival tissue were characterized by flow cytometry. All experiments were performed by growing cells on tissue cell culture plate surface (10 MPa); 40, 8 and 1 kPa elasticity polyacrylamide gels coated with collagen type I. The substrate stiffness had an impact to cell morphology, cells width and length increased on stiffer substrates. Matrix stiffness also affected cells adhesion, the amount of focal adhesions formed, cell cycle, and proliferation. Cell resistance to oxidative stress increases with stiffness of environment. Differences in MAPK expression and activation in GMSC had also been identified on hydrogels of different hardness effected by oxidative stress, ERK 1/2 and JNK MAPK participates in cell survival in stiffer environment, while p38 – in softer.

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Alexandrova, Radostina (1)

Zhivkova, T (1); Spasov, R (1, 2); Petrova, Z (1); Andonova-Lilova, B (1); Dinev, D (1); Abudalleh, A (1); Dyakova, D (3); Marinescu, G (4), Culita, D. (4); Podlipnik, C (5) (1) Institute of Experimental Morphology, Pathology and Anthropology with Museum, Bulgarian Academy of Sciences, Sofia, Bulgaria; (2) Faculty of Medicine, Sofia University “St. Kliment Ohridski”, Sofia, Bulgaria; (3) Institute of Neurobiology, Bulgarian Academy of Sciences, Sofia, Bulgaria; (4) Institute of Physical Chemistry “Ilie Murgulescu”, Romanian Academy, Bucharest, Romania; (5) University of Lubljana, Lubljana, Slovenia

Evaluation of cytotoxic / antitumor properties of new metal complexes with different ligands in 2D and 3D cell cultures

The need for effective new anticancer agents is one of the main challenges in modern biomedicine. Successful application of cisplatin in clinical oncology has encouraged the search for other metals and metal compounds with antineoplastic properties. The aim of this study was to evaluate the influence of newly synthesized metal [Zn(II), Cu(II), Co(II), Ni(II), Zn(II)/Ag(I), Zn(II)/Au(I)] complexes with various ligands (Schiff bases, non-steroidal anti-inflammatory

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drugs, bile acids) on viability and 2D/3D growth of cultured tumor cells. Cell lines established from rat sarcoma and human cancers of the lungs, breast, liver, uterine cervix, skin, brain; osteosarcoma as well as multidrug resistant cells, were used as model systems. Short-term (3 – 96h, with monolayer cell cultures) and long-term (> 2 weeks, with 3D cell colonies) experiments were carried out using cytotoxicity assays, cytological / immunocytochemical, biochemical and molecular-biological methods to assess the influence of the compounds on cell viability and proliferation and their ability to induce apoptosis/necrosis (in monolayr cell cultures) as well as colony-forming method (with 3D cell colonies) to examine their capacity to prevent 3D cell growth Cytotoxic concentration 50 (CC50, μM) that reduces viability and 2D growth of the treated cells by 50% as compared to the control and inhibitory concentration (IC, μM) that completely suppresses the formation of 3D cell colonies in a semi-solid medium were determined. The examined metal complexes express cytotoxic activity in 2D and 3D cell cultures that is time- and concentration-dependent and are more active than the corresponding ligands tested alone. Zn(II)/Au(I) and Zn(II)/Ag(I) complexes with Schiff base Salen are found to be the most promising cytotoxic agents being effective also in multidrug resistant cancer cells. Their cytotoxic activity is higher than those of cisplatin, oxaliplatin and epirubicin.

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Elvassore, Nicola (1)

(1)University of Padova, Italy

Confinement environment and extracellular matrix proteins enable endodermal organoid culture

The specification of cell identity during organ development is strictly controlled by extrinsic signals that restrict and define distinct cell fates. However, it is still not clear how cells, when exposed to exogenous signals, activate secretory cascades involving morphogens, growth factors and cytokines, extracellular matrix (ECM) deposition and remodeling. Here, we investigated the proteins secreted by cells in response to developmental exogenous signals, during the progression from endoderm to the hepatic lineage. A microfluidics-based approach coupled with SILAC-MS-based quantitative proteomic analysis revealed, among the 244 endogenously secreted proteins detected, high abundancy of ECM-associated proteins. Hepatocyte-like cells derived in microfluidics displayed a more mature hepatic transcriptomic signature and 1.5- fold higher ammonia detoxification capacity compared to conventional culture conditions. Moreover, in the microfluidics-confined environment we

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observed organized deposition of ECM proteins, consistent with observations in human fetal liver at 8-15pcw. Then, we tested if the exogenous supplementation of soluble ECM proteins identified by microfluidics-based SILAC-MS proteomic analysis, increase the 3D self-organization capacity of hepatic progenitor cells. We found that ECM proteins significantly enhance the hepatic organoid formation capacity of hiPSCs differentiated in conventional culture systems. Furthermore, we envisage that the use of complex extracellular matrix (ECM) hydrogels derived from decellularized tissues (DT) can provide an environment capable of directing cell growth. Gels from decellularized porcine small intestine (SI) mucosa/submucosa enable formation and growth of endoderm-derived human organoids, such as gastric, hepatic, pancreatic, and SI. These gels possess the bio- chemical signature of tissue-specific ECM and open up the potential for human organoids to be used clinically.

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Žegura, Bojana (1)

Hercog, K (1,2); Štampar, M (1,2); Štern, A (1); Filipič, M (1) (1) National Institute of Biology, Slovenia; (2) Jozef Stefan International Postgraduate School, Slovenia

Hepatic in vitro 3D cell model developed from HepG2 cells for the assessment of genotoxic activity of environmental pollutants

In the present study, the advanced in vitro hepatic 3D cell model developed from human hepatocellular carcinoma (HepG2) cells was used for the evaluation of cyto-/genotoxic activity of the cyanotoxin, cylindrosperompsin (CYN). CYN is considered as an emerging environmental pollutant and is pro-genotoxic meaning that metabolic activation is required for its cytotoxic and/or genotoxic effects. The spheroids were formed by the forced floating method and were cultured for three days under static conditions prior to the exposure to CYN (0.125, 0.25 and 0.5 μg/mL) for 72h. CYN influence on spheroid growth was measured daily and cell survival was determined by the MTS assay and live/dead staining of the whole spheroid and evaluated by confocal microscopy. The influence on the cell proliferation (Ki67 marker), cell cycle alterations (Hoechst staining) and induction of DNA damage

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(■H2AX foci) was determined by flow cytometry. Further, the expression of selected genes (qPCR) involved in the metabolism of xenobiotics, proliferation and DNA damage response was studied. The results revealed that CYN dose-dependently reduced the size of the spheroids and affected cell division by arresting HepG2 cells in G1 phase of the cell cycle. No increased induction of DNA double strand breaks compared to the control was determined at the applied conditions. Gene expression analysis revealed that CYN significantly deregulated genes encoding phase I (CYP1A1, CYP1A2, CYP3A4, ALDH3A) and phase II enzymes (NAT1, NAT2, SULT1B1, SULT1C2, UGT1A1, UGT2B7) as well as genes involved in cell proliferation (PCNA, TOP2α), apoptosis (BBC3) and DNA damage response (GADD45a, CDKN1A, ERCC4). The in vitro 3D HepG2 cell model due to its more complex structure and improved cellular interactions provides physiologically more relevant information and more predictive data for human exposure, and can thus contribute to a more reliable genotoxicity assessment of chemicals including environmental pollutants.

Acknowledgements: This study was supported by the Slovenian research agency, Program P1-0245 and Grant to KH and MŠ, and by the COST Action 16119.

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Krysiak, Zuzanna Joanna (1)

Stachewicz, U (1) (1) AGH University of Science and Technology, Cracow, Poland

Electrospun polyvinyl butyral-co-vinyl alcohol-co-vinyl acetate (PVB) patches for atopic skin treatment – cytotoxicity tests

Methods and Materials PVB was used with two molecular weight low LMw=70,000-100,000 and high HMw=170,000-250,000 g·mol -1 from Sigma Aldrich, UK. To prepare the solution for electrospinning PVB was dissolved in methanol, N,N-dimethyloformamide (DMF) and dimethyl sulfoxide (DMSO) to the final concentration of 10%. Fibers were electrospun (IME Technologies, The Netherlands) at controlled climate. Characterization and biocompatibility tests Electrospun fibers and cell morphology were analysed with SEM (Merlin Gemini II, Zeiss, Germany) and the average fiber diameter was calculated using ImageJ. Fibroblasts NIH 3T3 cells were cultured on PVB fibers at concentration 2·104 cells per sample at 37 °C, atmosphere with a concentration of 5% CO2 and approximately H = 90%. Biocompatibility of electrospun fibers was analyzed with cell proliferation and

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cytotoxicity assays. Results Via electrospinning we produced electrospun PVB fibers from LMw and HMw with the average fiber diameter 335 ± 86 nm and 965 nm ± 92 nm, respectively. LDH assay proved that there was no cytotoxic effect on murine fibroblasts up to third day of cell culture. MTS test and SEM images after cell culture showed that both PVB fibers mats enhance cell proliferation. Discussion and Conclusions PVB fibers with different diameter size, what was controlled by molecular weight of polymer, were obtained by electrospinning process. LDH and MTS assays proved PVB biocompatibility and their future perspective to use as skin patches. Acknowledgments This research was part of "Nanofiber-based sponges for atopic skin treatment" project is carried out within the First TEAM programme of the Foundation for Polish Science co-financed by the European Union under the European Regional Development Fund, project no POIR.04.04.00-00- 4571/18-00.

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Old challenges and new opportunities in engineering and analyzing complex 3D functional tissues

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Levenberg, Shulamit (1)

(1) Department of Biomedical Engineering, Technion, Israel

Engineering 3D vascularized tissue constructs

Living tissues require a vascular network to supply nutrients and gases and remove cellular waste. Fabricating vascularized constructs represents a key challenge in tissue engineering. Several methods have been proposed to create in vitro pre-vascularized tissues, including co- culturing of endothelial cells, support cells and cells specific to the tissue of interest. This approach supports formation of endothelial vessels and promotes endothelial and tissue- specific cell interactions. In addition, we have shown that in vitro pre-vascularization of engineered tissue can promote its survival and perfusion upon implantation. Implanted vascular networks, can anastomose with host vasculature and form functional blood vessels in vivo. Sufficient vascularization in engineered tissues can be achieved through coordinated application of improved biomaterial systems with proper cell types. We have shown that vessel network maturity levels and morphology are highly regulated by matrix composition. We also explored the effect of mechanical forces on vessels organization and analyzed the vasculogenic dynamics within

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the constructs. We demonstrated that morphogenesis of 3D vascular networks is highly regulated by tensile forces. Creating complex vascular networks with varying vessel sizes is the next challenge in engineering vascularized tissue constructs. 3D bioprinting, the controlled and automatized deposition of biomaterials and cells, represents a very attractive approach to solve this issue. This technique allows for combining different bioinks (biocompatible printable materials) in an organized fashion to attain native-tissue mimicking structures.

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Bar, Assaf (1)

Ruvinov, E (1); Cohen, S (1) (1) Ben-Gurion University of the Negev, Israel

Live imaging flow bioreactor for elucidating the mechanism behind regeneration of articular cartilage after treatment with bioactive hydrogel

Osteochondral defects (OCDs) are conditions affecting both cartilage and the underlying bone. Since cartilage is not spontaneously regenerated, our group has recently developed a strategy of injecting bioactive alginate hydrogel into the defect for promoting endogenous regeneration of cartilage via presentation of affinity-bound transforming growth factor β1 (TGF-β1). It was postulated that endogenous stem cells, possibly from the bone marrow (BM), infiltrate the hydrogel and differentiate to chondrocytes. However, the question arises whether these cells were attracted into the hydrogel by TGF-β1 signaling or whether they randomly arrived to the hydrogel by convective flow. The main obstacle in most in vivo model systems researchers face, is its inaccessibility for desired analyses in situ (i.e. live-imaging, different biochemical assays), making it a “black

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box” in many cases. Moreover, in vivo models exhibit high variability, making it difficult to isolate the influence of specific factors, limiting our understanding of time-dependent processes occur in vivo. In this work, we describe a novel flow bioreactor for the in vitro modeling of the OCD microenvironment, designed to promote cell recruitment from the simulated BM compartment into the hydrogel, under physiological flow conditions. Computational Fluid Dynamics modeling confirmed that the bioreactor operates in a relevant slow-flowing regime. Accessible through live imaging, the bioreactor enabled monitoring and discrimination between erosion rates and profiles of different alginate hydrogel compositions. Mathematical modeling of the erosion front progress kinetics predicted the erosion rate in the bioreactor up to 7 days post operation. Using quantitative real-time PCR (qPCR) of early chondrogenic markers, the onset of chondrogenic differentiation in hMSCs was detected after 7 days in the bioreactor. The bioreactor system was capable to shed light on the mechanisms leading to articular cartilage regeneration in the presented hydrogel system, suggesting an "outside in" mechanism. In conclusion, the designed bioreactor presents multiple attributes, making it an optimal device for mechanistical studies, serving as an investigational tool for the screening of other biomaterial-based, tissue engineering strategies.

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Emerging live cell imaging technologies: cell biology models, microscopy techniques and bioimage analysis

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Shaked, Natan T. (1)

(1) Department of Biomedical Engineering, Tel Aviv University

Rapid, label-free, high-resolution three-dimensional imaging of cells

I will present our latest advances in ultra-rapid refractive-index tomography of live cells, creating high-resolution three-dimensional imaging of live cells without staining, and opening new horizons to many prospective applications, such as label-free three-dimensional imaging flow cytometry and sperm selection for in vitro fertilization.

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Avinoam, Ori (1)

(1) Weizmann Institute of science, Rehovot, Israel

Membrane sequestration and crumpling maintain membrane homeostasis during exocrine secretion

Exocrine secretion is an essential process which relies on giant vesicles, several microns in diameter. Secretion of such giant vesicles adds large amount of membrane to the apical surface of cells. However, it remains unclear how hemostasis of the cell surface in terms of size, shape and composition is maintained under these extreme circumstances. To answer this question, we used the salivary gland of the Drosophila larvae, which secrets a viscous mucin-like protein called ‘glue’ via giant vesicles. Since, it is well established that a contractile actomyosin network is recruited the vesicular surface after its fusion, we used it as a marker to follow the fate of the vesicular membrane. Using live-gland super resolution microscopy, we observe that the vesicular membrane did not simply collapse into the apical surface but instead presented a crumpled appearance. To determine the precise ultrastructure of the vesicular membrane at different phases of secretion, we used correlative fluorescence and 3D-electron microscopy. We observed that as secretion

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advances the vesicle membrane becomes increasingly crumpled and folded suggesting that the membrane is not fully added to the apical surface. To quantify this phenomena, we used a correlative block-face and FIB-SEM approach and found that the compacted membrane indeed accounts for most of the original vesicular membrane before secretion. These results indicate that membrane homeostasis is maintained by actomyosin mediated crumpling and sequestration of the vesicular membrane. Furthermore, we observed that such sequestration of the vesicular membrane occurred for a prolonged period of time allowing clathrin- mediated endocytosis to recover the membrane in a that time frame, hence, preventing extreme perturbations to the apical surface. This is in contrast to the prevailing view in the field that the vesicular membrane is added to the apical surface. Thus, we have found a novel mechanism for maintaining membrane homeostasis in exocrine tissues.

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Shechtman, Yoav (1)

(1) Technion, Israel Institute of Technology

Volumetric and high-throughput localization microscopy via wavefront shaping and deep learning In localization microscopy, the positions of individual nanoscale point emitters (e.g. fluorescent molecules) are determined at high precision from their point-spread functions (PSFs). This enables highly precise single/multiple-particle-tracking, as well as super- resolution microscopy, namely single molecule localization microscopy (SMLM). To obtain 3D localization, we employ PSF engineering – namely, we physically modify the standard PSF of the microscope, to encode the depth position of the emitter. In this talk I will describe how deep learning enables unprecedented capabilities in super-resolution localization microscopy; specific applications include dense emitter fitting, multicolor imaging from grayscale data, and volumetric multi-particle tracking/imaging. Notably, our use of neural nets is not limited to image processing; we use nets to design the optimal optical acquisition system in a task-specific manner. Finally, I will discuss our most recent advance, where we combine PSF engineering with imaging flow cytometry, to enable 3D localization inside live cells at unprecedented throughput.

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Garini, Yuval (1) (1) Bar Ilan University, Israel

Studying the dynamics of molecules in live cells Proteins play the most important role in structuring the biological structures, as well as the metabolism processes. In order to understand the mechanism by which a protein acts, it is necessary to explore both its structure and function. While the structure is normally analyzed ex-vivo, exploration of the proteins function necessitates live-cell measurements. Different methods were developed for studying the dynamics of proteins and other entities in live cells, each has its own limitations and advantages. We describe a new method that we have developed termed time-resolved intensity photobleaching (TRIP). It combines few of the existing methods, including fluorescence life time imaging (FLIM), fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS). Combining these onto a single method provides significant advantages, including the ability to distinguish bound and free fraction of the tested molecules, as well as quantitative measurement of different parameters. We will describe the method principles and its application for studying the organization of the genome in the nucleus.

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Newman, Abigail (1)

Edri, S (1); Levenberg, S (1) (1) Technion - Israel Institute of Technology, Israel

3D Bioprinted Pancreatic Endocrine Tissue

Multiple challenges are associated with the diagnosis and treatment of pancreatic diseases. The pancreas is a lobular organ that is situated deep in the abdomen and comprises endocrine and exocrine regions. Diabetes develops in the endocrine pancreas and causes impaired glucose homeostasis. Disease progression can cause multiple problems including dental and gum diseases, sight loss, ulcers, strokes and, in severe cases, mortality. No cure currently exists for the Diabetes. A strong motivation exists to develop disease models and pharmaceuticals for pancreatic diseases. However, limited access to in vivo pancreatic tissue has impeded progress in this field. Three dimensional (3D) bioprinting is a promising new technology for in vitro engineering of biological tissue. It allows for accurate deposition of cell-laden biomaterials in a spatially defined manner to mimic the native tissue structure. Utilization of this technology could pave the way towards creating functional tissue ex vivo, greatly advancing the fields of tissue

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engineering and regenerative medicine. This research aims to 3D bioprint pancreatic endocrine tissue for use in disease modelling and pharmaceutical testing. We printed beta-like cells, the endocrine cells responsible for lowering blood glucose level, in a biocompatible cell-ink. We ascertained that these cells maintain their glucose sensitivity whilst encapsulated in the cell-ink, as they secreted insulin in a concentration dependent manner upon exposure to varying glucose levels. We combined endothelial cells with support cells in the cell-ink and saw vessel formation. The results of this research show exciting potential to create a 3D printed vascularized pancreatic endocrine tissue construct.

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Microfluidics - from basic concepts to organ-on-a-chip

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Nahmias, Yaakov (1)

(1) Grass Center for Bioengineering, The Hebrew University of Jerusalem

Illuminating Human Function: Development of a Smart Micro-Physiological Bioanalyzer

Organ-on-chip technology aims to replace animal toxicity testing, but thus far demonstrated few advantages over traditional methods. Current methods to evaluate product safety still rely on end-point assays measuring tissue damage and cell death, resulting in little to no mechanistic information that could make the development process iterative in nature. Here we present a platform technology capable of tracking the dynamics of tissue function from very onset of the molecular initiative event gaining critical information about compounds mechanism of action. This data rich approach enables the use of deep learning algorithms to track and identify risk factors without a priori information. Our microfluidic platform is capable of maintaining vascularized three-dimensional liver, cardiac, kidney and neural micro-tissues for over a month in vitro. Micro-tissues acquire the native architecture, physiological

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activity and show complex metabolic zonation of in vivo organs. Tissue-embedded metabolic sensors for oxygen, glucose, lactate and glutamine permit the real-time quantification of intracellular fluxes and tissue level function. Change in metabolic function is the first indication of physiological stress, preceding any detectable damage. Using the Tissue Dynamics bioanalyzer, we already explained the idiopathic nephrotoxicity of acetaminophen (Tylenol), and the idiosyncratic toxicity of valproate (Depakine) and troglitazone (Rezulin) whose post-market withdrawal cost upwards of $1B in legal settlements. Importantly, we will present a new mechanism of drug-induced nephrotoxicity and the resulting therapeutic approach to increase the safety of cyclosporine (Neoral) and cisplatin (Platinol). Our work marks the importance of tracing human organ function in real-time, demonstrating its specific advantages in lead optimization and predicative toxicology.

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Yissachar, Nissan (1)

Del Mare-Roumani, S (1); Bootz, H (1); Pearl, A (1); Abuchatsera, S (1); Amidror, S (1); Yissachar, N (1) (1) The Mina and Everard Goodman Faculty of Life Sciences Bar-Ilan Institute of Nanotechnology and Advanced Materials Bar-Ilan University, Israel

Thinking outside the mouse: ex-vivo dissections of host-microbiome cross talks

Key points for presentation:

• A novel ex-vivo gut organ culture system, as a unique tool for studying host-microbiome interactions (Yissachar et al., Cell 2017).

• Enteric neuro-immune-microbiome cross-talks control regulatory and effector T-cells development (Yissachar et al., Cell 2017; Yissachar, Curr. Opin. Neurobiol., 2020).

• The gut organ culture system provides insights into the functional consequences of microbial dysbiosis in mice and humans.

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• Development of a novel ‘on-chip gut permeability assay’, for tracking real-time changes in gut permeability.

• Dysbiotic, chemotherapy-disrupted microbiome alters gut permeability in a drug-independent manner, suggesting that barrier altering microbes may promote chemotherapy-induced gastrointestinal mucositis.

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Soen, Yoav (1)

(1)Weizmann Institute of science, Rehovot, Israel

Promoting desired biological functionality in live bioreactors

In vitro models provide simplified mimics of the in vivo system that focus the investigation on particular aspects of interest. This approach can be very effective when the in vivo system is sufficiently defined, but may be misleading when the configuration and/or state of the system is poorly defined or highly dynamic. To investigate how these complex scenarios are dealt with, we developed an in vivo model of nutritional challenge which promotes extensive variations in the gut microbiota of the fly, D. melanogaster. We find that newly forming variations in the microbiome enable host larvae to overcome a severe challenge of growing on carbon sources that cannot be metabolized by the larvae on their own. The acquisition of new and/or enhanced capabilities by newly-forming changes in the microbiome indicates that the host does not necessarily rely on a “well-define” microbiome; in this particular case, for instance, the host actually benefits from microbial variations that it cannot control or predict. The emergence of adaptive solutions to this challenge demonstrates the ability to acquire desired functions without having to specify the trajectory of changes or the underlying mechanisms. It also provides a proof-of-concept for the use of

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living hosts as live bioreactors for production of bacteria with a pre-specified function. Reproducing the key elements of this process in vitro, will give rise to a new class of powerful bioreactors.

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Artzy-Schnirman, Arbel (1)

(1) Technion, Israel

Lung-on-chip platforms: narrowing the gap between in vitro models to in vivo

In recent years, accumulating evidence on the limitations of in vivo animal models has directed many efforts to seek alternative in vitro humanized models. One of the promising avenues lie in organ-on-chips (OOC) platforms. In this talk, I will discuss our endeavors in developing lung-on-chips to advance basic research and translational medicine pertinent to the respiratory organ. The lung poses numerous challenges that cannot be easily addressed using traditional in vitro assays; chiefly the complex anatomy, constant mechanical stretches and physiological flows. Microfluidics models hold the potential to overcome those challenges by recapitulating more closely the specific anatomical features of the lungs, the cell populations lining it and together with engineering, mechanical stretches can be integrated and applied. I will present two distinct biomimetic models, namely bronchiole-on-chips and acini-on-chips. In each we emulate

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the specific relevant epithelium of human airways with critical cellular barrier functions at an air-liquid interface. Each of the models comes to advance our understanding of clinically-relevant problems, namely infection and drug delivery. Our bronchiole-on-chip enables simultaneous aerosol exposure across multiple airway chips integrated within a reconstructed larger bronchial airway tree, under physiological respiratory airflow conditions. Using this system we are able to mimic in situ-like aerosol deposition outcomes where deposition patterns exhibit a clear dependence on geometry and gravitational orientation. Our acini-on-chips mimic the native acinar airways at true scale with an anatomically-inspired, multigeneration alveolated tree. The usability and versatility of the platform is demonstrated by a realistic inhalation exposure assay mimicking bacterial infection. Our chips highlight the need to replicate inhalation exposure processes more accurately. These efforts underscore the potential to deliver advanced in vitro biosystems that can provide new insights into drug screening, disease model as well as toxicity assays.

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Maoz, Ben M. (1)

(1) Tel Aviv University, Israel

Organs-On-a-Chip: A New Tool for the Study of Human Physiology

Micro-engineered cell culture models, termed Organs-on-Chips, have emerged as a new tool to recapitulate human physiology and drug responses. Multiple studies and research programs have shown that Organs-on-Chips can capture the multicellular architectures, vascular-parenchymal tissue interfaces, chemical gradients, mechanical cues, and vascular perfusion of the body. Accordingly, these models can reproduce tissue and organ functionality and mimic human disease states to an extent thus far unattainable with conventional 2D or 3D culture systems. Here we exploit the micro-engineering technology in a novel system-level approach to decompose the integrated functions of the neurovascular unit into individual cellular compartments, while retaining their paracellular metabolic coupling. Using individual, fluidically- connected chip units, we have created a system that models influx and efflux functions of the brain vasculature and the metabolic

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interaction with the brain parenchyma. Results of proteomic and metabolic assays indicate that this system mimics the effect of intravascular administration of the psychoactive drug methamphetamine observed in vivo. Moreover, this model reveals a previously unknown role of the brain endothelium in neural cell metabolism: In addition to its well-established functions in metabolic transport, the brain endothelium secretes metabolites that are directly utilized by neurons. This discovery would have been impossible to achieve using conventional in vitro or in vivo measurements.

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Bitan, Amir (1)

Tandler, Amos (1); Koch, Liat (1); Gandolfi, Fulvio (2) (1) Israel Oceanographic & Limnological Research, Israel (2)University of Milan, Italy

Fish-AI- developing an artificial intestine for the sustainable farming of healthy fish

A healthy, balanced diet has a fundamental role in preventing a large range of chronic diseases and contributes to prolong life quality with obvious benefits for the individual as well as for the society. Aquaculture production plays a substantial role in this perspective because fish is an important source of well-balanced proteins and important nutrients such as marine derived omega-3 fatty acids. However, its sustainability generates concerns as farmed fish diet is largely based on fishmeal and fish oil. Fish-AI’s objective is to develop a next generation 3D scaffold for organoid culture that accurately mimics the complex functions of the intestinal mucosa. Its purpose is to develop a technology that helps predicting the health and nutritional value of innovative components of fish feeds. Current methods for such feed ingredients evaluation are lengthy, expensive and require the

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use of large number of animals. Furthermore, they do not provide the knowledge of the cellular and molecular mechanisms that determine the effect of ingredients on fish performance and health. The Fish-AI project aims at improving the present limited capacity of feed ingredients evaluation, which severely limits the capacity to understand and predict the biological value of the single raw ingredients and of their different combinations, presently impacting the environment, fish welfare and the economics of the aquaculture feed industry. The Fish-AI project fosters cross-fertilization and synergy among nutrition, physiology, bioengineering, cell and stem cell biology with academia and industry partners from Italy, Norway, Portugal, Belgium and Israel, to develop innovative technologies for sustainable livestock production.

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Poster presentations

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Alksne, Milda (1)

Kalvaityte, M (1); Simoliunas, E (1), Rinkunaite, I (1), Barasa, P (1), Gendviliene, I (1), Vygandas, R (1), Bukelskiene, V (1). (1) Vilnius University, Lithuania.

In vitro comparison of 3D printed polylactic acid/hydroxyapatite and polylactic acid/bioactive glass composite scaffolds designed for bone regeneration

Loss of bone tissue due various reasons such as inflammation, genetic diseases, etc. affects millions of people each year. The treatment of these large bone defects is still a major challenge for doctors. Artificial bone grafts, created by implementing main tissue engineering principles, could significantly improve clinical outcomes related to bone regeneration. Researchers recognize the importance of proper material selection and their biological impact to bone regeneration. Newly produced bone graft should not only demonstrate osteoconductive, osteoinductive, and biodegradable properties, but also the inner and outer structures of these grafts should be accurately and easily formed to mimic patient-specific bone defect morphology. Additionally, the manufacture of this kind of grafts should also

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be low in price and high in production speed. These requirements can be fulfilled by 3D printing technology. Polylactic acid (PLA) and bioceramic materials are perfectly compatible with 3D printing methods. Hydroxyapatite (HA) and bioglass (BG) bioceramics are the most promising materials as composite constituents, since they enhances osteogenesis and new bone formation processes. However, they are different materials, with different composition, having different osteogenesis inducing mechanisms. Thus, it is important to compare bone regeneration process induced by 3D printed PLA+HA and PLA+BG composite scaffolds in order to evaluate the strongest osteoconductive and osteoinductive properties possessing bioceramics. In this study, we analysed porous PLA+HA (10 %) and PLA+BG (10 %) composites’ bioactivity on rat’s dental pulp stem cells fate in vitro. Obtained results indicated, that PLA+BG scaffolds lead to weaker cell adhesion and proliferation than PLA+HA. Nevertheless, osteogenic differentiation related genes expression levels, ALP activity and extracellular matrix mineralization were more pronounced in cells grown on PLA+BG scaffolds. Overall, the results showed bioceramic BG advantage against HA, thus, 3D printed BG composite scaffolds could be a promising approach for patient specific, cheaper and faster artificial bone tissue production.

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Akalabya, Bissoyi (1)

Sirotinskay, V(1); Chasnitsky, M(1); Braslavsky, I(1) (1) Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot , Israel

Bioactivation of the substrate by antifreeze proteins to enhance the cryopreservation of attached cells In cell therapy one of the major challenges in cell preservation. Currently, there is no proven method for successful cryopreservation of adherent cells. Cells are currently stored in liquid medium in suspended conditions and ready-to-use intact cells have huge potential for therapeutically and research applications. The research work aims to explore the combination of controlled-rate freezing and surface-modified substrate using antifreeze proteins (AFPs) for the preservation of adherent cells. The result shows that AFP modified substrates are more effective. We observed that up to 80% of the cells were shown to survive after post-thaw, which is significantly higher than non-AFP modified substrates. Additionally, we explored change in the focal point of adhesion after post-thaw. In brief, this work provides a plausible methodology for preserving cells attached to the substrate and the use of AFPs in cryopreservation. Furthermore, this will allow more efficient use of cells based on lab-on-chip devices in a more efficient way. Keywords: nanosurface modified; the focal point of adhesion; anti-freezing protein, post-thaw

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Bjelogrlic, Snezana (1,2)

Todorovic, T (3); Muller, C.D. (2); Filipovic, N (4) (1) National Cancer Research Center of Serbia; (2) Institut Pluridisciplinaire Hubert Curien, France; (3) Faculty of Chemistry University of Belgrade, Serbia; (4) Faculty of Agriculture University of Belgrade, Serbia.

Changes of growth rate and Oct-4 expression in MCF-7 and AsPC-1 spheroids subjected to newly synthesized indole ligand and its metal complexes

Octamer-binding transcription factor 4 (Oct-4) is a protein of critical role in reprogramming differentiated somatic cells to pluripotent state, as its expression level in cancer was shown to be in positive correlation with worse clinical outcome (1). Here, we investigated the impact of newly synthesized indole ligand (HL) and its palladium (1), platinum (2) and copper (3) metal complexes on growth and Oct-4 expression in MCF-7 breast cancer and AsPC-1 pancreatic cancer 3D models, with cisplatin (CDDP) as the reference drug. According to growth rates that are computed for the 8-day incubation time, AsPC-1 spheroids reveal as very sensitive to treatment with HL, 2, and particularly 3, while 1 does not achieve any noteworthy effect. On the other hand, 3 remains the only one

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reducing the volume of MCF-7 spheres below their initial size, being even more efficient than CDDP at same concentration. The Smart Flare methodology used here to evaluate transcription level of Oct-4 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as housekeeping gene, offers a unique possibility to track single-cell gene expression profiling and provides information that cannot be derived from whole population data (2). While HL, 1 and 3, along with CDDP, induce statistically significant decrease in percentages of Oct-4-expressing cells as well as mean fluorescence intensity (MFI) in MCF-7 spheres, compound 3 is the single one which induces such response in AsPC-1 3D spheroids. A suppressed transcription of GAPDH, commonly used as reference in population analysis, is particularly impressive in MCF-7 spheroids. Current results are in accordance with previous discovery that cells within 3D tumors use tricarboxylic acid cycle as the main energy source instead of glycolysis (3). Aside from the conclusion that compound 3 presents a superior activity as compared to CDDP, observed differences in GAPDH expression profile between MCF-7 and AsPC-1 spheroids indicate that additional reference genes should be monitored in genetic analysis of 3D tumor models. Reference List 1. K. Hochedlinger, K. Plath, Development 136, 509 (2009). 2. A. Stahlberg, V. Rusnakova, M. Kubista, Brief. Funct. Genomics 12, 81 (2013). 3. M. Sato et al., Oncotarget. 7, 33297 (2016).

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Bogacka Iwona (1)

Paukszto, Ł (1); Kunicka, Z (1); Mierzejewski, K (1); Jastrzębski, J.P (1); Szydłowska, A (1); Kurzyńska, A (1) (1)University of Warmia and Mazury in Olsztyn, Poland

The effect of PPAR gamma ligands on the transcriptome profile of LPS-treated porcine endometrium during mid-luteal phase of the estrous cycle

Peroxisome proliferator-activated receptors belong to the nuclear receptors family and as transcription factors modulate the expression of many target genes regulating reproductive processes. In this study, gene expression patterns in the porcine endometrium treated in vitro with LPS and/or PPAR ligands, agonists: 15d-prostaglandin J2 (PGJ2) or pioglitazone and antagonist T0070907, was determined by high-throughput transcriptome sequencing. To identify expression profiles of treated tissues, cDNA libraries were created using TruSeq RNA Sample Prep Kit v2 with the appropriate pairs of indexing adapters and RNA-seq was performed on the NovaSeq 6000 Illumina platform. After checking the quality control, 2 × 100 bp paired-end reads were mapped to the pig reference genome (Sus_scrofa.Sscrofa11.1.98) using the STAR mapper. To identify differentially expressed genes (DEGs) we applied

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Cufflinks method. The final results constituted DEGs, significantly confirmed by statistical test (adjusted p-value < 0.05). Enrichment gene ontology and pathway analysis were performed with use of gProfileR based on Gene Ontology (GO), and Kyoto Encyclopaedia of Genes and Genomes (KEGG) databases. During preprocessing quality control procedure 1 031 782 380 of 1 118 471 092 raw reads were survived, of them, approximately 94,4 % were uniquely mapped to the reference porcine genome. Within all experimental comparisons we detected 3, 4, 62 and 239 DEGs for PGJ2 vs LPS, P1 vs LPS, T vs LPS and LPS vs control, respectively. DEGs, differ between T and LPS samples, were also annotated to 18 biological process (BP), one molecular function (MF) and 4 KEGG molecular pathways. Within BP category, the most interesting DEGs were enriched to ‘inflammatory response’, ‘response to interleukin-1’ and ‘granulocyte migration’. Enrichment analysis revealed that the antagonist of PPARg may modulate four genes: CCL3L1, LBP, CCL4, ENSSSCG00000039214, which are components of Toll-like receptor signaling pathway. Our results pave the way for further research into PPARγ-dependent changes in the porcine endometrium during inflammatory stage induced by LPS.

Supported by National Science Centre, Poland, grant no. 2015/17/B/NZ9/03596.

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Kistanova, Elena (1)

Abadjieva, D (1); Kestendjieva, S (1); Sotirov, R (1); Kostadinova, M (1); Pashova, S (1); Vinketova, K (1); Stoyanova, E (1); Oreshkova, T (1); Mourdjeva, M (1) (1)Institute of Biology and Immunology of Reproduction, BAS, Sofia, Bulgaria

Mitochondrial DNA content and Dnmts transcripts level in MSC cultured in 3D conditions with polyphenol oregonin

Spheroids are 3D cell culture model, which is well utilised for in vitro screening of new pharmaceutical compounds as well as for estimation of their effects on the epigenetic changes in stem cell. Recently scientific data provide evidences on the importance of mitochondria in the epigenetic events in the cells. Oregonin is a plant-derived phenolic compound with anti-oxidative and anti-inflammatory properties. Its effects on the DNA methylation enzymes have been reported in 2D cell cultures. This study explores the effects of oregonin on the Dnmts transcripts and mtDNA content in MSC spheroids. The spheroids were produced by method of the hanging drop from the MSC derived from the adipose tissue of three donors and cultivated with different concentrations of

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oregonin: 0, 10 μM, 50 μM и 100 μM. The genomic DNA and total RNA were isolated using TRIzol reagent after 48 hours of cultivation. The RNA was converted to cDNA and the samples were subjected to the qRT-PCR for the analysis of Dnmt1, mtDnmt1, Dnmt3a, Dnmt3b and Tfam transcripts. The DNA was used for an assessment of the mtDNA copy number per cell on the base of ratiometric assay of the levels of a mitochondrial gene Nd1 against a nuclear gene b-actin. The mtDNA copy number tended to increase in the samples cultivated with 100 μM oregonin. The changes of mtDNA content positively corresponded with changes of Tfam transcripts (r=0.78; p=0.21). The doses of 10 and 50 μM oregonin inhibited the expression of all Dnmts compared to the control samples. The dose of 100 μM provoked different response: Dnmt1 and Dnmt3a transcripts did not change significantly, Dnmt3b transcripts decreased, while mtDnmt1 transcripts increased. Close positive correlation was established between mtDnmt1 transcripts and mtDNA copy number (r=0.97; p=0.034). Analysis of samples in dependence on the donor showed clearly individual response to the oregonin treatment. In conclusion, oregonin affects copy number of mtDNA and provokes changes in the expression of Dnmts in MSC-spheroids. Acknowledgments: This work was supported by the Bulgarian National Science Fund, KP06 KOST-21.

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Kornhäuser, Shani (1)

Sanchez-Lopez, JA (1); Heifetz, Y (1) (1) The Hebrew University, Israel

The Accessory-glands of the Drosophila female as a source for secretions within the reproductive tract

Successful fertilization in the Drosophila melanogaster fruit fly is strongly dependent on both the male and female. The male, on one side, is transferring sperm and seminal fluid that cause changes in the activity of the female reproductive tract (RT). These changes can be observed as the increase in egg laying, ovulation, sperm storage and female behavioral changes. On the other hand, the female RT secretes components that are important for transporting the egg, fertilization and embryo development. The female reproductive tract (RT) environment is composed by a milieu derived from two major secretory centers, the Spermatheca with its secretory cells (SSC), and the female Accessory-glands (AGs); both sources of important secretions, affect other organs and secretory epithelial cells along the RT. The female AGs, that are connected to the uterus, are composed of a monolayer of secretory cells that border the AG lumen. Simultaneous disruption of

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Spermatheca and female AG development at early stages of the fly’s development resulted in female sterility.

Even though the female AGs are crucial for female fertility, very little is known about the composition and functions of their secretions. Our hypothesis is that the female AGs regulate processes at a distance in the RT via secretion of soluble factors and Extracellular Vesicles (EVs) into the RT environment. Here we focus on the mechanism of cell-cell communication via EVs within the AGs. Our results indicate that the AGs have a major secretory activity, that changes after mating and is influenced by the presence of both sperm and seminal fluid. Our observations imply, for the very first time, the importance of EVs within the AG as a regulator of the female RT activity.

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Kukolj, Tamara (1)

Kukolj, T(1); Trivanović, D (1); Okić Djordjević, I (1); Drvenica, I (1); Obradović, H (1); Mojsilović, S (1); Jauković, A (1); Bugarski, D (1) (1) Institute for Medical Research, University of Belgrade, Serbia

Adipose tissue extracellular matrix as in vitro biomimetic for studying breast cancer cells responsivness to doxorubicin

Tumor microenvironment (TME) has received significant attention as underlying factor in chemoresistance and cancer recurrence. However, conventional in vitro models could not recapitulate intricate interactions in TME. Recent data indicated that adipose tissue grafts can stimulate residual cancer cells growth, pointing to the important role of ECM composition in breast TME. To recapitulate more accurately ECM conditions of native breast cancer niche, we generated decellularized human adipose tissue ECM (AT-ECM) as biomimetic microenvironment for in vitro studies. We investigated the potential of AT-ECM to modulate the growth and epithelial to mesenchymal transition (EMT) of breast cancer cells (MCF-7 and MDA-MB-231 lines), as well as for

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doxorubicin therapeutic efficacy. Human adipose tissue from healthy patients, was decellularized, lyophilized, solubilized and used for pre-coating of standard cell culture dishes. As control groups tissue culture polystyrene (TCP) surfaces, collagen- and fibronectin-coated surfaces were used. Cellular metabolic activity and proliferation were determined by MTT or CFSE labeling, while the EMT was determined by epithelial (E-cadherin) and mesenchymal (Vimentin) markers expression. Growth rate and metabolic activity of both cell lines cultured on AT-ECM were unchanged in comparison to the cells cultured on the control TCP and collagen-treated surfaces, confirming that AT-ECM provides a viable environment for tested cancer cells. However, following the doxorubicin treatment, in the presence of AT-ECM, MCF-7 and MDA-MB-231 cell lines demonstrated higher metabolic activity in comparison to the other surfaces, indicating that the presence of AT-ECM significantly affects the antiproliferative effect of doxorubicin. Additionally, the AT-ECM microenvironment promoted the EMT of both cell lines, by decreasing E-cadherin and increasing Vimentin expression. In conclusion, AT-ECM can markedly affect cellular behavior of breast cancer cells in response to chemotherapeutics. More importantly, our results indicated that AT-ECM can provide in vitro biomimetic microenvironment for more accurate cancer cells growth determination and screening of cancer therapeutics.

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Mierzejewski, Karol (1) Paukszto, Ł (1); Kunicka, Z (1); Jastrzębski, JP (1); Szydłowska, A (1); Kurzyńska, A (1); Bogacka, I (1) University of Warmia and Mazury in Olsztyn, Poland Alternative splicing in the transcriptome of the porcine LPS-stimulated endometrium treated with PPARγ ligands during the mid-luteal phase of the estrous cycle Peroxisome proliferator-activated receptors (PPARs) belong to a ligand-dependent nuclear receptor family. Numerous studies have revealed the presence and significance of different PPAR isoforms in the reproductive system. In this study, we determine the effect of PPAR gamma ligands (agonists: 15d-prostaglandin J2 (PGJ2) or pioglitazone and antagonist T0070907) on a global transcriptome profile in the porcine LPS-stimulated endometrium, incubated in vitro. To enriched global transcriptome analysis, we predicted alternative splicing (AS) events by replicate multivariate analysis of transcript splicing (rMATS v.3.2.5) method. Using equal length (90 bp) paired-end reads and Binary Alignment Map (BAM) files, we calculated percent splicing inclusion (PSI) for all splicing events. Differential alternative splicing (DAS) events between experimental samples and controls

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were statistically tested (FDR < 0.05). Additionally, splicing sites with ΔPSI > 0.1 were classified as significant. Alternative events were divided into five main types: alternative 5′ splice site (A5SS), alternative 3′ splice site (A3SS), mutually exclusive exons (MXE), retention intron (RI) and skipping exon (SE). Within LPS vs control comparison we detect 218 genes differ in alternative splicing. However, LPS-treated samples with induction of PPARγ agonists indicate 227 and 270 DAS events for PGJ2 and pioglitazone, respectively. Summarizing, those events were located in range of 343 genes. Finally, the PPARγ antagonist (T0070907) were engaged in occurrence of 384 DAS events, in comparison with samples treated only by LPS. Gene ontology analysis of LPS vs control revealed that identified DAS genes may modulate intracellular transport, play crucial function in GTPase activator activity and are cellular components of endoplasmic reticulum exit site. In the other, LPS administration with PPARγ ligands exposure change expression within splicing sites of genes engaged in endopeptidase activity, RNA-dependent ATPase activity, transferase activity. Supported by National Science Centre, Poland, grant no. 2015/17/B/NZ9/03596.

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Petrova, Valeria (1)

Yonkova, P (1); Simeonova, G (1); Vachkova, E(1) (1)Trakia University, Stara Zagora, Bulgaria

Improvement of adipogenic protocol for equine subcutaneous ADSCs in vitro

Traumatic tendinitis is one of the most common injuries in athletic horses and the cell therapy with autologous transplanted cells seems to be promising. The subcutaneous adipose tissue is one of the best candidates as a source of mesenchymal stem cells (MSCs), possessing functional plasticity and ability to differentiate in various cell types. Therefore, the proper production of extracellular matrix (ECM) is crucial for further tendon recovery and prevention of reparative outcome. There are many difficulties related to the purification, identification and verification of the multipotent properties of the primary isolated adipose derived MSCs (ADSCs), and their subsequent clinical application. The samples were collected from subcutaneous adipose tissue depot in paracoccygeal region. The cells were cultured up to passage 3 and then several concentrations of Indomethacin alone (0.1 mM, 0.05 mM and 0.02 mM), IBMX(3-isobutyl-1-methylxanthine) alone (0.5 mM, 0.25 mM

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and 0.1 mM) and in combinations were applied. The number of applied inductions was also tested. Additional osteogenic and chondrogenic differentiations were performed for multypotency of the isolated. The IBMX alone is not able to induce intracellular lipid droplets (LDs) formation in any of the applied concentrations. The cellular monolayer was intact, homogenous and 100% confluent. The Indomethacin treatment caused gradient cytotoxic effect showing particular cellular detachment proportional to the increased concentrations. The best results were observed in the combined treatment with 1⁄2 of the lowest Indomethacin and the highest of IBMX concentrations. The formation of intracellular LDs droplets started during the second cycle and prolonged till the end of the third induction-maintains cycle. The cellular monolayer was confluent, intact, without any signs of cellular detachment during the whole experimental period of 21 days. Applying of the proper cocktail of inductors is crucial for revealing multipotent properties of MSCs during their tri-lineage differentiation. This could affect further exploration of ADSCs as allogenic transplants in veterinary medicine as a supportive therapy in tendon regeneration and ligament ECM remodeling in horses.

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Rizos, Dimitrios (1)

Leal, CLV (1,2); Cañón-Beltrán, K (1); Cajas, YN (1); Yaryes, A (1); Beltrán-Breña, P (1); Hamdi, M (1); Gutiérrez-Adán, A (1); González, ME (3); Rizos, D (1) (1) National Institute for Agriculture and Food Research and Technology (INIA), Madrid, Spain; (2) University of São Paulo (USP), Pirassununga, Brazil; (3) University of Madrid (UCM), Madrid, Spain.

Sequential in vitro culture with oviductal and uterine extracellular vesicles affects mitochondrial activity, lipid contents and lipid metabolism transcripts in bovine blastocysts Extracellular vesicles (EV) are released by cells and transport cargo that influence functions of other cells. Oviductal fluid (OF) and uterine fluid (UF) have been shown to improve quality of embryos during in vitro culture which may be due to their EV. Thus, the aim of this study was to evaluate the effect of EV from OF and UF on a sequential in vitro culture system on the development and quality of bovine embryos. Zygotes were cultured in SOF supplemented either with 3 mg/mL BSA or 5% EV-depleted FCS (dFCS) in the presence (BSAEV and dFCSEV) or absence of 3×105 EV/mL from OF (D1 to D4) and UF (D4 to D9), mimicking in vivo conditions. Size and concentration of EV were assessed by nanotracking analysis system and their morphology by transmission electron microscopy. Embryo development was recorded on Days 7–8. Day 7-8 blastocysts were assessed for quality by staining with Mitotracker Deep Red for

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mitochondrial activity (fluorescence intensity recorded in arbitrary units (a.u)) and Bodipy 493/503 for lipid content (lipid droplet area in μm2/cell). Relative mRNA abundance of lipid metabolism-related transcripts was assessed as well. Data were analysed by one-way ANOVA. Blastocyst yield on D8 was lower (p<0.05) in BSA compared to dFCS groups (BSA: 29.8±2.1; BSAEV: 27.0±1.8% vs dFCS: 39.4±2.4; dFCSEV: 39.5±2.8%) irrespective of EV supplementation. Mitochondrial activity was increased (p<0.05) by EV in dFCSEV compared to other groups. Lipid content was decreased (p<0.05) in the presence of EV only in dFCSEV (0.277±0.03 fiau) compared to BSA (0.374±0.02 fiau) and BSAEV (0.377±0.02 fiau) groups, while dFCS had highest contents (0.530±0.03 fiau, p<0.05). Regarding transcript abundance, PPARG was downregulated and ACC upregulated by EV irrespective of protein source in medium. In contrary, EV affected some transcripts depending on the medium (CD36 upregulated in dFCSEV, downregulated in BSAEV; PLIN2 downregulated in dFCSEV and ATGL in BSAEV, p<0.05). In conclusion, mimicking physiological conditions using EV from OF and UF in sequential IVC does not affect development but improves embryo quality by increasing blastocysts mitochondrial activity and decreasing lipid contents, while it also favours the expression of specific lipid metabolism transcripts. Functional effects of EV may be influenced by protein source in medium. Funding: MINECO-Spain AGL2015-70140-R; Cajas YN: SENESCYT-Ecuador; Leal CLV: FAPESP-Brazil 2017/20339-3, CNPq-Brazil 304276/2018-9.

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Rodrigues, Joana S (1)

Cipriano, M (1); Camões, SP (1), Miranda, JP (1) (1) Research Institute for Medicines (iMed.ULisboa), Faculdade de Farmácia, Universidade de Lisboa, Lisbon, Portugal

Hepatocyte-like cells in microfluidic devices regulate energy metabolism in response to insulin and glucagon

Liver has a unique role in energy homeostasis, providing glucose as fuel substrate for extrahepatic tissues during fasting whereas in the fed state, liver stores glycogen, fatty acids and amino acids. Microfluidic devices (MDs) are a complex culture system, mimicking more closely some aspects of the in vivo microenvironment, such as media flow. Therefore, the aim was to develop a liver model metabolically relevant and responsive. This work focused on the evaluation of human neonatal mesenchymal stem cells (hnMSCs)-derived hepatocyte-like cells (HLCs) response to insulin and glucagon in key pathways of the energy metabolism while cultured in double channel PDMS-based MDs. A three-step hepatic differentiation protocol was applied to hnMSCs. The differentiation process was initiated under 2D conditions; whereas at day 17 (D17), cells were

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transferred and seeded in MDs for the final hepatic maturation step. HLCs functionality was evaluated up to D34 regarding albumin and urea production. Additionally, expression of genes involved in glycolysis, gluconeogenesis, fatty acid metabolism, bile acid metabolism and mitochondrial function in response to 80 nM insulin, 100 mM of glucagon and 8h- and 10h-glucose fasting was assessed in HLCs cultured in MD and 2D and in primary human hepatocytes at D34. Functional HLCs were maintained up to two weeks in culture presenting stable morphology, urea and albumin production, over time. Most importantly, HLCs expressed genes regarding glycolysis and lipogenesis (Pdk4), gluconeogenesis (Pepck and G6pase), fatty acid oxidation (Ppara), bile acid metabolism (Fxr) and mitochondrial function and biogenesis (Pgc-1a) with similar trend to that observed in a physiologic context, in response to insulin and glucagon. This work resulted in functional and metabolic responsive HLCs in a novel culture system that may provide a relevant system for pre-clinical research.

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Sanchez-Lopez, Javier Arturo (1)

Shuchmacher, R (1); Heifetz, Y (1) (1)Department of Entomology, The Hebrew University, Rehovot, Israel

Macromolecular crowding alters the cell growth pattern and ECM deposition of endometrial cells in vitro

Modelling the interactions between the maternal endometrium and the embryo has challenged scientists to improve in vitro systems for its study. As the endometrium replenishes itself cyclically, new extracellular matrix (ECM) is assembled. Additionally, during decidualization the endometrial ECM is remodelled to create the adequate environment for embryo implantation. The ECM is an essential basic component of all tissues and organs which apart from giving structural support, promotes tissue functionality by inducing cell differentiation, proliferation and migration. To mimic the biological function of the ECM in vitro, artificial polymer scaffolds, gels or coatings with ECM components have been introduced to cell culture models. These artificial scaffolds, however, do not fully confer ECM biological activity. One possible way to fully confer ECM biological activity is using macromolecular crowding.

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Macromolecular crowding allows cells to recreate their microenvironments and improve cell functionality by concentrating the availability of substrates in the culture media, and enhancing the capacity of protein folding and ECM assembly. To test whether macromolecular crowding of endometrial epithelial cell (EEC) microenvironment would influence the capacity of ECM assembly and cellular function, we examined receptive and non- receptive EEC. We compared ECM deposition, expression profile of endometrial markers and interactions with trophoblast cells. We will describe the behaviour of the two different EEC lines under crowded environments. The ability to alter the cell molecular microenvironment will allow us to improve our understanding of the endometrium-embryo interactions during embryo implantation.

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Shuhmaher, Rita (1)

Sanchez-Lopez, JA (1); Heifetz, Y (1) (1) The Hebrew University of Jerusalem, Israel

Preparation for receptiveness: How the endometrial cells change through the menstrual cycle

Implantation is a critical stage in early gestation, when the blastocyst adheres and invades the uterus. Only a well-coordinated crosstalk between the receptive uterine endometrium and the blastocyst will lead to successful implantation. This crosstalk is mediated by a range of molecules such as cytokines, chemokines, integrins and miRNAs. In recent years, there are emerging evidence which suggests that extracellular vesicles (EVs) and lipid droplets (LDs) are also important mediators in the embryo-maternal crosstalk . To gain receptiveness, in each menstrual cycle, the uterine endometrium undergoes structural and transcriptional changes mediated by the ovarian hormones, estrogen and progesterone. Aside from the window of implantation, the time in which the endometrium is receptive, the endometrium is a non-receptive tissue, preventing invasion of the embryo. Failure in implantation poses one of the limiting factors in the assisted reproduction field, as such

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there is an extensive research on implantation and the endometrium-blastocyst crosstalk. One of the common models used in-vitro to study implantation is endometrial and trophoblast human cell lines. However, only few studies combined the endometrial cell lines and hormonal priming. Moreover, there is limited knowledge on how endometrial-derived EVs population and LDs secretion pattern change through the menstrual cycle, and how these changes affect the early interactions with the blastocyst. To examine and characterize hormone-induced changes in endometrial-derived EVs and LD population we used different hormonal combinations to prime the endometrium and mimic hormonal cyclicity. We will present hormonal cyclicity-depended profiles of endometrial-derived LDs and endometrial derived EV characterization. The results obtained will allow a better understanding of the changes that the endometrium undergoes and will guide future studies aimed at improving in-vitro model for implantation.

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Štampar Martina (1,2)

Breznik, B (1); Žabkar, S; Filipič, M (1); Žegura, B (1) (1) National Institute of Biology, Slovenia; (2) Jozef Stefan International Postgraduate School, Slovenia

3D cell model for the in vitro determination of induced genotoxic effects

For the purpose of safety evaluation, hepatic two-dimensional cell cultures are used, but they have several limitations, including low expression of metabolic enzymes. Thus, there is a high demand for development of physiologically more relevant in vitro cell-based systems that can restore highly complex microenvironment and can provide more predictive results for human exposure. In the present study, we developed hepatic in vitro 3D cell model from HepG2 cells with the forced floating method. The spheroids were cultured under static conditions for 3 days and validated for genotoxicity assessment by testing genotoxic activity of indirect acting compound, benzo(a)pyrene (BaP). Spheroids were exposed to BaP (0.1, 1, 10, 20 μM) for 24h. The influence of BaP on the spheroid growth was monitored by planimetry, while live/dead cells in spheroids were determied by FDA/PI staining and evaluated

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by confocal microscopy. The results revealed that BaP decreased spheroid surface area at ≥ 10 μM and affected cell viability at 20 μM. For further analyses the single cell suspension of viable cells was obtained by the combination of mechanical degradation and enzymatic (trypsin) digestion. The effect of BaP on cell proliferation (Ki67 marker) and cell cycle (Hoechst staining) alterations was assessed by flow cytometry, and its genotoxic activity was determined with the comet and ■H2AX assays. At applied conditions BaP (10 μM) reduced the number of Ki67 positive cells and affected cell division by arresting HepG2 cells in S phase of the cell cycle. BaP induced the formation of DNA single (comet) and double (■H2AX) strand breaks. On the mRNA level (qPCR) BaP deregulated the expression of phase I (CYP1A1, CYP1A2) and II (UGT1A1, SULT1B1 and NAT2) enzymes and DNA damage responsive genes (P53, GADD45α, CDKN1A). In summary, the newly developed HepG2 3D model due to its more complex structure and improved metabolic capacity provides a suitable experimental model for genotoxicity assessment.

Acknowledgements: This study was supported by the Slovenian research agency, Program P1-0245 and Grant to MŠ, and by the COST Action 16119.

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Vidakovic Melita (1)

Đorđević M (1); Sarić A (1); Tolić A (1); Arambašić Jovanović J (1); Uskoković A (1); Rajić J (1); Đorđević (1); Grdović N (1); Dinić S (1); Mihailović M (1); Jurkowski T (2); Si Chen (3); Dučić T (4) (1) Institute for Biological Research, National Institute of the Republic of Serbia, Serbia; (2) School of Biosciences, Sir Martin Evans Building, Cardiff, UK; (3) Argonne National laboratory, lemont, IL, USA; (4) CELLS − ALBA, Cerdanyola del Vallès, Spain

Insulin-based distinction between pancreatic alpha cells, epigenetic edited alpha cells that started producing insulin and beta cells using the X-ray fluorescence microscopy

Cell-replacement therapy in diabetes will become available only with the establishment of new sources of pancreatic beta cells. A few success stories have been reported after the transdiferentiation of pancreatic exocrine cells, human fibroblasts and liver cells into insulin-producing cells. We are developing a synthetic epigenetic tool that promises stable modification of gene expression profiles of cells by rewriting epigenetic signaling. This will provide a DIRECT ROUTE to cell transdifferentiation and establish a proof of

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concept of cellular transdifferentiation based on epigenetic reprogramming, instead of transfection with transcription factors.

Using X-ray fluorescence microscopy we confirmed the production and presence of insulin in the vesicles by direct imaging of Zn 2+ in (i) pancreatic beta cells (PBC) (positive control with natural ability to synthesize and storage insulin) and (ii) Epi-CRISPR transfected pancreatic alpha cells that due to Epi-CRISPR-induced targeted DNA methylation of certain genes are transdifferentiated to insulin-producing cells (Epi-CRISPR-targeted insulin-producing cells) (Epi-IPC), as well as (iii) the non-treated pancreatic alpha cells (PAC) - a negative control for insulin synthesis/storage/detection. The Epi-CRISPR-targeted insulin-producing cells are insulin/glucagon bihormonal cells that slowly stop producing glucagon and start to synthesize insulin which we detected by immunostaining and RT-qPCR in our laboratory.

Being able to set the X-ray fluorescence microscopy as a method of choice for making a clear distinction between pancreatic alpha cells and epigenetic edited pancreatic alpha cells that started producing insulin, will allow us to further extend our research from in cellulo to in vivo level. This could upgrade treatment of diabetic patients in future since this epigenetic cellular reprogramming could become a method of choice for beta cell replacement and thereby reduce the need for pancreatic transplantation.

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Zelinger, Einat (1)

Rechav, K (2); Brumfeld, V(2), Heifetz, Y (1) (1) Entomology, The Hebrew University of Jerusalem, (2) Chemical research support, Weizmann Institute, Israel

Drosophila sperm storage: new insights using 3D correlative microscopy

Females from diverse taxa store sperm in specialized storage organs. Most insect sperm storage organs have a tubular structure, typically consisting of a central lumen surrounded by epithelial cells. These specialized tubules perform the essential tasks of transporting sperm to and from the female reproductive tract and supporting sperm survival and function, often for days up to weeks or more.

How do the female sperm storage organs provide an environment favorable to sperm survival is mostly unknown. To address this, we have taken a triple correlative approach of confocal, micro-computed tomography (micro-CT), and focused ion beam scanning electron microscopy (FIB-SEM), to dissect sperm-female interactions in Drosophila melanogaster.

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Using this approach, we were able to scan the distal portion of the seminal receptacle at low magnification, and subsequently zoom in, for further analysis on an ultrastructural level, rendering valuable and detailed two- and three-dimensional information of the receptacle’s epithelial cells and the lumen’s wall. This triple correlative approach allows us to address questions related to sperm-female interactions. Furthermore, such an approach is suitable not only for Drosophila but also for other organisms with soft delicate tissues.

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Ziogas, Algirdas (1, 2)

Novikova, J (1, 2); Ziogiene, D (4); Denkovskij, J (1); Misiunas, A (3); Kielaite-Gula, A (5); Vitkus, D (5); Rackauskas, R (5); Sokolovas, V (5) ;Strupas, K (5). (1) Centre for Innovative Medicine, Lithuania; (2) Vilnius Gediminas Technical University, Lithuania; (3) Center for Physical Sciences and Technology, Lithuania; (4) Centre of Life Sciences of Vilnius University, Lithuania; (5) Vilnius University, Lithuania.

Improving mammalian cell protein production for 3D tissue engineering: the impact of signal peptides on recombinant protein synthesis and secretion

Protein expression in mammalian cell lines become standard for the manufacture of biomolecules in biopharmaceutical industry settings and is widely used for production of proteins for research. While it is relatively cheap to produce human proteins in prokaryotes, the lack of glycosylation, accumulation of protein in inclusion bodies or unfolding of some proteins results in undesirable protein characteristics that impact their application. Selecting a process for recombinant protein expression which relies on many variables such as cell line, culture media, and culture method

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all impact the yield, physical characteristics, and biologic activity of the proteins. We have developed mammalian expression vector system for transient expression that provides rapid, flexible and economical protein production alternative to time consuming and costly process of generating stable cell lines. In the first step have designed a DNA fragment toolbox where the individual DNA building blocks can be combined in a flexible manner for the construction of a multitude of vectors. The individual building blocks in the toolbox have been made either by PCR or by simple annealing of complementary DNA stretches. After the synthesis and secretion of mammalian proteins in Expi293 system they could be used later for multi-protein printing or three-dimensional (3D) matrix hydrogels with controllable properties. Our results show that the different signal peptides tested varied extensively in their relative abilities to generate functional proteins. Engineered proteins were tested in in-vitro 3D model systems of cell migration, morphogenesis and they are also instrumental in studying environment-dependent cell responses to soluble growth factors, hormones or drugs. Furthermore, engineered proteins could be applied in relevant in vivo models of tissue regeneration which require a precise spatio-temporal control of individual environmental parameters.

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Participants

Last name: First name: Country of Affiliation:

Email

Akgün Elif Ece Turkey t.a.t.u.e@hotmail.com

Alexandrova Radostina Bulgaria rialexandrova@hotmail.com

Alksne Milda Lithuania milda.peciukaityte@gf.vu.lt

Andronowska Aneta Poland a.andronowska@pan.olsztyn.pl

Apel Ido Israel ido.apel@mail.huji.ac.il

Artzy-Schnirman Arbel Israel arbel@bm.technion.ac.il

Avinoam Ori Israel ori.avinoam@weizmann.ac.il

Bar Assaf Israel assafb@post.bgu.ac.il

Bardoogo Yael Israel olive.yael@gmail.com

Baskin Aharon Israel ashbaskin@gmail.com

Bayuh Argaw Rachel Israel rachel.bayuh@bioind.com

Belleli Alon Israel alonb@avba.co.il

Benny Ofra Israel ofrab@ekmd.huji.ac.il

Bernys Andres Ecuador wladimir.bernys@mail.huji.ac.il

Bissoyi Akalabya Israel akalabya.bissoyi@mail.huji.ac.il

Bitan Amir Israel amir.bitan@ocean.org.il

Bjelogrlic Snezana Serbia snezanakb@gmail.com

Bogacka Iwona Poland iwonab@uwm.edu.pl

Braslavsky Ido Israel ido.braslavsky@mail.huji.ac.il

catane liora Israel liora.jacobs@mail.huji.ac.il

Duran Oyku ITALY oykudrn99@gmail.com

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Furtos Gabriel Romania gfurtos@yahoo.co.uk

Gadella Bart the Netherlands

b.m.gadella@uu.nl

Gadot Omer Israel omer.gt@gmail.com

García-Hevia Lorena Spain lgarcia@idival.org

Garini Yuval Israel yuval.garini@biu.ac.il

Gershon Eran Israel eran.gershon1@mail.huji.ac.il

Halevy Tomer Israel tomer@supermeat.com

Heifetz Yael Israel yael.heifetz@mail.huji.ac.il

Kalo Dorit Israel dorit.kalo@mail.huji.ac.il

Khnykin Denis Norway denisdnr@gmail.com

Kiri Mark Dan Jerusalem

Kistanova Elena Bulgaria kistanova@gmail.com

Komsky-Elbaz Alisa Israel alisakomsky@yahoo.com

Kornhäuser Shani Israel shani.kornhauser@mail.huji.ac.il

Koven Bill Israel koven@ocean.org.il

Krysiak Zuzanna Poland krysiak@agh.edu.pl

Kukolj Tamara Serbia kukoljtamara@gmail.com

Levavi-Sivan Berta Israel berta.sivan@mail.huji.ac.il

Levenberg Shulamit Israel shulamit@bm.technion.ac.il

Maoz Ben Israel bmaoz@tauex.tau.ac.il

Mierzejewski Karol Poland karol.mierzejewski@uwm.edu.pl

Mihailovic Mirjana Serbia mista@ibiss.bg.ac.rs

Miranda Joana P Portugal jmiranda@ff.ul.pt

Mirica Ioana-Codruta Romania mirica_codruta@yahoo.com

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Nahmias Yaakov Israel ynahmias@cs.huji.ac.il

naim may Israel maynaim@gmail.com

Navarrete Santos

Anne Germany a.navarrete-santos@medizin.uni-halle.de

Newman Abigail Israel abigail.m.newman@gmail.com

Ortega Nicolás Sweden nicolas.ortega@ki.se

Petrova Valeria Bulgaria valeria.petrova1075@gmail.com

Priscila Ramos Ibeas Spain priscilaramosibeas@gmail.com

Pušić Maja Croatia maja.pusic@biol.pmf.hr

Raya Angel Spain araya@cmrb.eu

Regev-Rudzki Neta Israel neta.regev-rudzki@weizmann.ac.il

Ribarski Chorev Ivana Israel ivana.ribarski-chorev@mail.huji.ac.il

Rizos Dimitrios Spain drizos@inia.es

Roelen Bernard Netherlands b.a.j.roelen@uu.nl

Rohrer Jack Switzerland jack.rohrer@zhaw.ch

Rosenfeld Hanna Israel hannarosenfeld@gmail.com

Sanchez-Lopez Javier Arturo Israel javier.sanchezlo@mail.huji.ac.il

Schlesinger Sharon Israel sharon.shle@mail.huji.ac.il

Shaked Natan T. Israel nshaked@tau.ac.il

Shalom Gothilf Amalia Israel amalia.shalomgoth@mail.huji.ac.il

Shechtman Yoav Israel yoavsh@bm.technion.ac.il

Shimoni Chen Israel chen.shimoni@mail.huji.ac.il

Shoseyov Oded Israel shoseyov@agri.huji.ac.il

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shpigel Nahum Israel nahum.shpigel@mail.huji.ac.il

Shuhmaher Rita Israel rita.shuhmaher@mail.huji.ac.il

Šimoliūnas Egidijus Lithuania egidijus.simoliunas@gmc.vu.lt

Soen Yoav Israel yoavs@weizmann.ac.il

Štampar Martina Slovenia martina.stampar@nib.si

Strauss Carmit Israel carmit.feliks@mail.huji.ac.il

Stryiński Robert Poland robert.stryinski@uwm.edu.pl

Szyf Moshe CA moshe.szyf@mcgill.ca

Tal Shay Israel shay.tal@ocean.org.il

Tal Ayellet Israel ayellet.tal@mail.huji.ac.il

Tandler Amos Israel tandleramos@gmail.com

Tavor- re'em Tali Israel talire@jce.ac.il

Tawil Shadi Israel swtawil@gmail.com

Thumfart Kristina Maria Switzerland thumfart@hifo.uzh.ch

Toto Nienguesso Alicia Germany alicia.toto-nienguesso@medizin.uni-halle.de

Twena Shai Israel shai.twena@mail.huji.ac.il

Vachkova Ekaterina Bulgaria katvach@gbg.bg

Vidakovic Melita Serbia melita@ibiss.bg.ac.rs

yaacobi artzi shira Israel shiraya21@gmail.com

Yissachar Nissan Israel nissany1@gmail.com

Zaritsky Assaf Israel assafzar@gmail.com

Zegura Bojana Slovenia bojana.zegura@nib.si

Zelinger Einat Israel einat.zelinger@mail.huji.ac.il

Ziogas Algirdas Lithuania algirdas.ziogas@imcentras.lt

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