This poster can be viewed online byscanning the following QR code:

Figure 4. (A) Image of hT1 organoid plate from the Perkin Elmer ViewLux uponCellTiter-Glo reagent addition immediately after medium change. Center row effectobserved in No Nanoshuttle (NS) plate was not present in plates using m3Dbioprinting technology with NS labeling. (B) RLU decreased by ~20% after 3 timesof medium change, which suggests certain degree of cell loss for hT1. Note: Notall cells are efficiently labeled with NS particles in the flask. Thus portion of cellslost from medium removal are probably non-labeled or weakly labeled cells. (C)The residual volume measured by gravimetric method is 25 ± 7 nL (n=4 plates),which is only 0.1% of original medium volume.

Figure 2. (A) hT1-CAF spheroids cultured using m3D bioprinting technology in fullplate at 24 hrs post-seeding imaged by Scripps HIAPI imager and standardmicroscopy. Only 1 spheroid was lost after 3 times of medium change usingBlueWasher magnetic spin method. From the 10X objective images, hT1-CAFspheroids stayed intact after 3 times of magnetic spin. (B) Concentration-responsecurves of 5 known chemotherapeutics on hT1-CAF spheroids with and withoutmedium change using BlueWasher. Drug response of controls on hT1-CAF spheroidswas not affected by medium change using magnetic spin. Assay statistics (S/N, Z andZ’ ) are listed for each CRC plate.

Ø m3D bioprinting technology incorporating magnetic force coupled with GreinerBio-One cell repellent plate for 3D culture has been validated for HTS drugscreening.

Ø m3D bioprinting technology in combination with BlueWasher magnetic spin formedium removal has been evaluated using two representative 3D pancreaticcancer cultures.

Ø BlueWasher magnetic spin for liquid removal does not cause destruction to 3DhT1-CAF spheroids or barely cause spheroid loss.~20% of hT1 cells are lost after magnetic spin, which are probably non-labeledcells.§ Cell loss in m3D plates does not follow the Center Row pattern.

Ø Medium change does not affect CRC of control drugs on 3D pancreatic cancerculture.

Ø Magnetic spin effectively removes liquid in Greiner 384 GNF compatible plates.

Ø BlueWasher magnetic spin method for medium removal works well with n3Dbioprinting system.

Validation of Magnetic 3D Spheroid Bioprinting in Combination with a BlueWasher

Shurong Hou1, Pierre Baillargeon1, Glauco R. Souza2, Jan Seldin3, Frank Feist4, Louis Scampavia1 and Timothy Spicer1

1The Scripps Research Institute Molecular Screening Center, Scripps Florida, 130 Scripps Way, Jupiter, Florida, USA2Nano3D Biosciences, Inc. and University of Texas Health Science Center at Houston, Houston Texas, 7000 Fannin St., Houston TX, USA

3Greiner Bio-One North America, Inc., 4238 Capital Drive, Monroe, NC, USA4BlueCatBio MA, Inc., 58 Elsinore St., Concord, MA, USA

Abstract

Three-dimensional (3D) cell models are thought to better mimic thecomplexity of in vivo tumors. We have previously enabled an HTS-compatible method using cell-repellent plates combined with amagnetic force bioprinting technology that affords large scale testingof spheroids and organoids in flat-bottom 384 and 1536 well plates.This type of 3D biology requires tissue culture in suspension whichmakes feeding, media transfers, washing, etc., problematic. Toaddress this we combined the 384 well formatted n3D technology withthe utility of the BlueWasher equipped with magnetic spin features.We validated this system for rapid removal and replacement of mediato facilitate adaptation of a non-homogeneous format within 3D HTS.Our work demonstrates the effectiveness of both the magnetic 3D(m3D) bioprinting system and the BlueWasher in the followingaspects in 384 well plates: 1) Retention of 3D spheroids and theirintegrity 2) Precision and accuracy of liquid manipulation 3)Qualification in terms of compound controls, Z’ and S:B when usingphenotypic 3D primary pancreatic tumor cell based assays.

BlueWasher Magnetic Spin Evaluation: Spheroid loss or disruption?

BlueWasher Magnetic Spin Evaluation: Consistency across plate and dead volume

Conclusion

hT1-CAF Spheroids

10X10X10X

Figure 3. (A) hT1 organoids cultured using m3D bioprinting technology at 24hrs post-seeding imaged by standard microscopy. (B) Concentration-response curves of 5known chemotherapeutics on hT1-CAF spheroids with and without medium changeusing BlueWasher magnetic spin.

Dead volume by Gravimetric method25 ± 7 nL (n=4)

0.1% of original volume

hT1 Organoids

(A)

(B)

(A)

(B) (C)

Figure 1. (A) Greiner cell repellent plates readily form spheroids using cellscombined with Nanoshuttle. This can be done on the bottom side of the platesusing a magnetic driver. Note that the shape of the bottom of the wells iscompatible with automated confocal microscopy. Z-stack image of HT-29 andPANC-1 spheroids cultured using n3D technology is shown here as the examples.(B) Assay protocol for HTS drug testing in 384w format, in combination withBlueWasher for medium change. (C) Cells in 2D monolayer and in 3Dspheroid/organoid format demonstrated different responses to therapeuticcompounds. hT1 cells are the primary pancreatic cancer cells, and hT1-CAF cellsare the fibroblasts generated from the same tumor mass as hT1 cancer cells. Theyform two types of 3D structure with different morphology.

n3D Bioprinting Technology with BlueWasher

10X

(A)

(B)

(C)

2D

3D

Ø This project was a joint collaboration with n3D Biosciences, Greiner Bio-One,BlueCatBio and the Scripps Research Institute.

Ø This work was supported by NIH NCI IMAT Award R33 CA206949

Acknowledgement