Stem Cell Culture, Differentiation, and Scale Up · Stem Cell Culture, Differentiation, and...

Paula FlahertyTechnology ManagerDeepa Saxena, Ph.D.Senior Development ScientistISSCR 2015 June 26, 2015

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Corning Innovation ShowcaseStem Cell Culture, Differentiation, and Scale‐Up

Novel Technologies Enabling Research and Cell Processing Applications

2© 2015 Corning Incorporated

Today’s Topics

• Introduction: Corning® Life Sciences• Corning cell culture surfaces technologies• Industry trend: Animal-free • Corning PureCoat™ ECM mimetic cultureware

Collagen I peptide Fibronectin peptide

• Scale up for cell manufacturing: Examples

• Corning hPSC culture substrates for expansion and differentiation

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Corning Incorporated HighlightsFounded:1851

Headquarters:Corning, New York

Employees:~35,000 worldwide

2014 Sales:$10B

R&D Investment:~10% of sales

Fortune 500 Rank (2015): 297

• Corning is one of the world’s leading innovators in materials science. For more than160 years, Corning has applied its unparalleled expertise in specialty glass, ceramics, and optical physics to develop products that have created new industries and transformed people’s lives.

• Corning succeeds through sustained investment in R&D, a unique combination of material and process innovation, and close collaboration with customers to solve tough technology challenges.


Corning: Leader in Cell Culture Surface InnovationA Century of Surface Technology

1915 Heat-resistant Pyrex® glass

1907Glass Surfaces used to culture

mammalian cells

1910 1920 1930 1940 1950 1960 1970 1980 1990 20102000

2010Synthemax®

Synthetic VN peptide surface

2012

1959 TC treatment and gamma irradiation

TC-treated Polystyrene

1985Matrigel® matrix, defined ECMs

In vivo-like cell growth and differentiation

1987BioCoat™ surface

Convenient and consistent pre-coated cultureware

2012PureCoat™ ECM

Mimetic Fibronectin and Collagen I

Synthetic peptide surfaces

1990sPrimaria™

and CellBIND®

Charged-based modification of plastic surfaces

2000sPureCoat surfacesAmine and CarboxylUltra-Low AttachmentHydrogel-coated low bind surface

1953Development of

plastic 100 mm petri dish for U.S. Army

Glass + Plastic + Biological + Synthetic

2015

2014Corning

rLaminin-521Animal-free pluripotent

stem cell surface


Cell Manufacturers are Rapidly Progressing from Scientific Knowledge to Process Engineering

“Critical Product Quality and Process Controls” has been identified as a major technology need for cell manufacturers*

Cell manufacturing is moving towards less animal products in their processes − Reducing the risk of adventitious agent contamination − Enabling control of cell manufacturing process

• Reducing variability of raw materials• Reducing testing required lot-to-lot• Progressing from fundamental science to engineering

− Enabling optimization of process − Supporting predictive productivity− Facilitating downstream purification

*Cell Manufacturing Consortium, US National Institute of Standards and Technology (NIST), Advanced Manufacturing Technology, Identifying Industry Needs, 05.08.15

Science and TechnologyDevelop fundamental understanding

of product, process, and material

Engineering/ManufacturingDevelop and control advanced

product and process technology


Complete and Customizable Cell Culture Environments

• Corning® Matrigel® matrix• Purified biological, xeno-free, and

synthetic ECMs• ECM mimetic cultureware

• cGMP media manufacturing• Serum• Serum-free media supplements and

growth factorsCorning CellSTACK®

Multi-layer flasks

Coated microcarriers

Customizable solutions for all stages of cell and tissue culture research


Advanced Surfaces and Extracellular Matrices

Vialed Extracellular Matrices (ECMs)Corning® Matrigel® matrix Defined ECMs

Animal-derivedXeno-free

Recombinant ECMsrLaminin-521 (Human)

Synthetic ECMs SynthemaxCorning PuraMatrix™

Custom ECM formulations

Synthetic ECM Mimetic Pre-coated Cultureware

Corning PureCoat™Fibronectin peptideCollagen I peptiderLaminin-521 coming soon

• Synthemax (custom)Vitronectin peptide

Biological ECM Pre-coated CulturewareCorning BioCoat™

• Plates• Inserts• Flasks• Coverslips/slides• Dishes

•Custom coatings

Enhanced Tissue Culture-treatedCorning CellBIND® surface

(-) chargeCorning PureCoat

Amine (+) chargeCarboxyl (-) charge

Corning Primaria™(+) and (-) mixed charge

Ultra-Low Attachment Hydrogel-coated

Functional Attachment ChargeFunctional Attachment Functional Attachment

General cell culture, HTS, specialized applications HTS, general cell culture Specialized applications:

cell culture enablement General cell culture, HTS, specialized applications

Corning® PureCoat™ ECM Mimetic Cultureware

Synthetic peptide covalently attached on a surface

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Corning® PureCoat™ ECM Mimetic Cultureware

• Animal-free, synthetic peptide (Collagen I peptide or Fibronectin peptide) covalently attached on a surface

• Support attachment and expansion of cell types that require Collagen I or Fibronectin ECM coating

• Pre-coated, ready to use• Stable at room temperature for 18 months• Manufactured in a GMP compliant facility• US Class I medical device • Sterile by aseptic processing• Compatible with a wide range of dissociation reagents

and serum-free, xeno-free, and animal-free media• Available in 24-well, 6-well, T-75, T-175, 3-Layer or

5-Layer Multi-Flask format, and custom Corning CellSTACK®


Corning® PureCoat™ ECM Mimetic CulturewareManufacturing and Quality

• Manufactured under cGMP per the requirements of FDA 21 CFR 820

• Manufacturing facility ISO 9001, ISO 13485, and ISO 14001 certified

• Animal-free manufacturing process

• Quality control confirms the following: Nonpyrogenic Nontoxic Functional activity Sterile

• Regulatory documentation available ISO Certificates Certificate of Compliance Certificate of Analysis

− Lot-specific QC results

End-user

No animal components in final product

No animal components used in production of raw materials


Synthetic and Defined Cultureware Suitable for Expansion of a Variety of Cell Types

Corning® PureCoat™

ECM MimeticExamples of Cell Types Expanded in Defined Cell Culture Environments

Fibronectin Peptide Human BM-derived mesenchymal stem cells

Human endothelial progenitor cells

Human adipose-derived stem cells

CHO cells

Vero cells

Collagen I Peptide Human keratinocytes

Human corneal cells

Human adipose-derived stem cells

Human endothelial progenitor cells

Human cord blood-derived mesenchymal stem cellsVero cells

Corning® PureCoat™ ECM Mimetic CulturewareCollagen I peptideSynthetic surface for cell types that require Collagen I coating IS

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R 2015


Corning® PureCoat™ Collagen I Peptide Supported Human Keratinocyte Culture and Functionality

• Human neonatal keratinocytes

• Culture in animal-free medium EpiLife® with S7 supplements

• Control: Rat tail collagen, and ECM matrix recommended with the medium

Collagen I Mimetic Coating MatrixRat Tail Collagen I

Comparable growth on 3 surfaces

Cumulative population doubling

02468

101214

1 2 3 4

Passage number

Popu

latio

n do

ublin

g

Collagen I mimeticRat tail collagen ICoating matrix

Time 0 2 hr

3 hr 4 hr

Division-arrested cells migrated to heal scratch wound

Comparable cell morphology

Corning® PureCoat™ ECM Mimetic CulturewareFibronectin peptide Synthetic surface for cell types that require Fibronectin coating IS

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Xeno-free Human Mesenchymal Stem Cell Culture on Fibronectin Mimetic Surface

• Human bone marrow-derived MSCs in MesenCult™-XF medium

• Control: Human-origin coating matrix

Fibronectin Mimetic Human-origin matrix

AdipogenicOsteogenic

Comparable morphology and growth on Fibronectin mimetic and human-origin matrix.

Cells maintained multipotency after expansion on Fibronectin mimetic.

Cells expressed ISCT established marker profile.

Successfully tested with additional xeno-free media.


Human Endothelial Colony Forming Cell (ECFCs®) Culture on ECM Mimetic Collagen I and Fibronectin Peptide

• Culture for 4 passages in EGM™-2 BulletKit™containing 2% FBS (Lonza)− DiI-acetylated-LDL uptake− Differentiation on Corning® BioCoat™

angiogenesis system: endothelial cell tube formation

• Controls: Corning rat tail Collagen I, human Fibronectin

Cumulative population doubling of ECFCs

0

2

4

6

8

10

12

2 3 4Passage number

Pop

ulat

ion

doub

ling

Collagen I Mimetic Rat tail collagen IFibronectin Mimetic Human fibronectin

ECFCs demonstrated typical morphology, functionality and differentiation after expansion on Corning PureCoat™ ECM mimetic cultureware.

Collagen I Mimetic Fibronectin Mimetic

Mor

phol

ogy

DiI-

Ac-

LDL

upta

ke

Tube

form

atio

n

ECM mimetic Fibronectin and Collagen I both supported ECFC® expansion under low-serum conditions.

Corning® stemgro® hMSC Culture Environment

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Corning® stemgro® hMSC Culture Environment• Serum-free, xeno-free, defined medium• In combination with Corning CellBIND® surface

provides complete cell culture environment for hMSCs

• Enables efficient expansion of hMSCsCorning Cat . No.40-410-KIT

stemgro/CellBIND

10% FBS/TC‐treated Competitor/CellBIND


Surface Markers, Karyotype and Differentiation After 7 Passages in Corning® stemgro® hMSC Medium

Medium/Surface Passage # CD73 CD90 CD105 CD14 CD45

Input Cells p0 95.2 95.0 96.8 0.0 0.0

stemgro/CellBIND® p5 98.5 99.2 96.7 0.0 0.0

Competitor/CellBIND p5 88.4 93.0 95.1 0.0 0.0

Adipogenic- Oil Red O Osteogenic Alizarin Red Chondrogenic Alcian Blue

Scale-up and Manufacturing

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Cost-effective Scale-up in Corning®

PureCoat™ ECM Mimetic Multi-Flask• Easy and cost effective scale-up with

consistent performance

• Uniform cell growth between the layers results in equivalent cell yield/cm2 in T-175, 3-Layer and 5-Layer ECM mimetic Multi-Flasks

Scale-up

0

5000

10000

15000

20000

25000

30000

35000

T-175 3-Layer 5-Layer

Cel

l yie

ld /c

m s

q.

MSCsKeratinocytes

Cost/ sq. cm.

Cost effective

Equivalent cell yield

Uniform cell growth


Corning® PureCoat™ ECM Fibronectin Mimetic Closed System Corning CellSTACKs® Have Been Qualified to Manufacture MSC Treatment for Graft-versus-Host Disease

Typical MSC morphology was observed for the MSCs cultured on Fibronectin mimetic surface.

400 μm400 μm

020406080

100120

CS2L T175 T75

% Viability, 9 Days in Culture

0.00E+00

2.00E+07

4.00E+07

6.00E+07

8.00E+07

1.00E+08

T-75 T-175 ML5 CS2 CS10

Cell Yield/Vessel Type

Viability of MSCs cultured on Fibronectin mimetic Corning CellSTACK 2-layer, T-175, and T-75 flasks were comparable.

Cell yields from T-75, T-175, 5-Layer Multi-Flask (n=3), Corning CellSTACK (n=3), and 10-Layer demonstrated scalable yields.


MSCs Maintained Characteristic Immunophenotypeand Multi-lineage Differentiation Capability

MSCs were positive for CD13, CD29, CD44, CD73, CD90, CD105, and negative for CD45, CD80, and HLA-DR.

MSCs were differentiated into adipogenic, osteogenic, and chondrogenic lineages.

Adipogenic (Oil red O) Chondrogenic (Alcian Blue)Osteogenic (von kossa)

Poster Presentation IIT-10009 6/25/15


Xeno-free Culture of Human Adipose Stem Cells (hASC) in Corning®

stemgro® hMSC Medium and Corning CellBIND® Surface Cells exhibited a spindle-like morphology, and at a confluence higher than 75%, cell colonies formed a typical “river-like” pattern of growth.

Average doubling time (h) for two sub-culturing steps of expanded cells, for hASC061 and hASC064 was similar, 51 and 54h, respectively. For hASC057 a shorter doubling time was observed, i.e., an average time of 32h. Viability of expanded cells varied from 93% to 99%.


hASCs Maintained Characteristic Immunophenotypeand Multi-lineage Differentiation Capability

Poster Presentation IIT-10010 6/25/15

Concomitant expression of CD73, CD90 and CD105 in the absence of CD34 range between 90.0 to 97.6%. Cells expressed low hematopoietic and endothelial markers, i.e., expression of 1.3% for CD31, 2.5% for CD34 and 0.5%, for CD45.

Successful differentiation into the three mesenchymallineages


Summary

Corning® PureCoat™ ECM mimetic cultureware: • Synthetic, defined, and animal-free• Compatible with multiple media • Support xeno-free expansion of multiple cell types while maintaining functionality• Provide a ready to use alternative to ECM protein coating where animal-free and

defined conditions are desirable• Demonstrated to support cell manufacturing scale

Corning stemgro® hMSC culture environment:• Serum-free, xeno-free, defined medium• In combination with Corning CellBIND® surface provides complete cell culture

environment for hMSCs• Enables efficient expansion of hMSCs• Has demonstrated expansion of hASC derived from xeno-free cGMP cryopreserved

human stromal vascular fraction (hSVF)

For a listing of trademarks, visit us at www.corning.com/lifesciences/trademarks.All other trademarks in this document are the property of their respective owners.© 2015 Corning Incorporated. All rights reserved.

Stem Cell Culture, Differentiation, and Scale Up · Stem Cell Culture, Differentiation, and...

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