3D Cell Clusters for Predictive Toxicity AnalysisLindsey Ott1, Janette Williams1,2, Karthik Ramachandran1, Lisa Stehno-Bittel1,2

1Likarda LLC, Kansas City, KS; 2School of Health Professions, University of Kansas Medical Center, Kansas City, KS

1. Herper, M. The truly staggering cost of inventing new drugs. (2012) Forbes, 2/10/12.

2. Ramachandran K, Peng X, Bokvist K, Stehno-Bittel L. (2014) Assessment of reaggregated human pancreatic islets for secondary drug screening. Br. J. Pharm. 171(12):3010-3022.

3. Williams SJ, Schwasinger-Schmidt T, Zamierowski D, Stehno-Bittel L. (2012) Diffusion into human islets is limited to molecules below 10 kDa. Tissue and Cell. 44(5):332-341.

4. MacGregor RR, Williams SJ, Tong PY, Kover K, Moore WV, Stehno-Bittel, L. (2006) Small rat islets are superior to large islets in vitro function and in transplantation outcomes. Am J. Physiol Endocrinol. Metabl. 295(5):E771-9.

5. Ramachandran K, Williams SJ, Huang HH, Novikova L, Stehno-Bittel L. (2013) Engineering islets for improved performance by optimized reaggregation in a micromold. Tiss Eng. 19(5):604-612.

Figure 4: Spheroid formation of primary and culturedhepatocytes.A) Primary hepatocytes cultured in 2D. B) Same cellsclustering in the micromold. C) Cells of the transformedhepatic cell line (HepaRG) were tested in standard 2Dmonolayers and in 3D cell clusters. D) HepaRG clusterswere formed by loading the cells into micromolds thatresulted in formation of 3D cluster in 7 days.

• While pancreatic endocrine work indicated thatspheroids under 100 microns in diameter wereoptimal for drug screening2, the results presentedhere show that pancreatic acinar spheroids optimallyresponded toxin exposure when they were 20-40microns in diameter.

• Cultured HepaRG cells tested in 2D and 3D,demonstrated greater sensitivity to a toxin,chlorpromazine in 2D rather than 3D.

• HepaRG clusters that are smaller in diameter (under40 microns) will be retested for sensitivity to toxins.

Summary – The drug sensitivity of 3D spheroidscompared to 2D cell culture, is dependent on the celltype, and likely on the size of the spheroids.

Results

References

Conclusions

Figure 2: Purity and Viability of Exocrine Spheroids.A) Canine pancreatic exocrine spheroids were tested for purityby staining for endocrine cells. In over 450 acinar spheroidstested , total purity was 99.6%.B) Viability of the 3D exocrine spheroids was high (green =calcein green staining), with few dead cells within the spheroids(red).

The ability of in vitro assays to produce reliable and medically-relevant information is essential to new drug discovery. Cell culturelines grown in monolayers are the standard tool for toxicity screens,yet there are significant limitations with this model. Namely theresults often do not predict what will happen in the body. 3D cellculture has quickly become the new standard for drug screening,especially in the cancer field, yet it has been slow to take hold for invitro toxicology testing. Comparing the same cell types grown in 2Dversus 3D, researchers have shown differences in the proteinprofiles, gene expression, signal transduction, cellular growth,function and morphology. We have published studies with knownanti-cancer and anti-diabetes drugs on cells cultured in 3D showingthat they were more predictive of the known in vivo response thanthe 2D outcomes. The goal of this study was to optimize the use of3D hepatocyte cell clusters to standard in vitro toxicology assays anddetermine their predictive abilities. Cells of the transformed hepaticcell line (HepaRG) were tested in standard 2D monolayers and in 3Dcell clusters. HepaRG clusters were formed by loading the cells intoglass micromolds that resulted in formation of 3D cluster in 2 days.Clusters were washed from the molds and measured for size. Theaverage diameter was between 75-100 μm. We have shown fromprevious work that cell clusters with a diameter over 125 μm havesignificant diffusion limitations that reduce the exposure of the corecells to test products. Viability assays of the 3D HepaRG clusters ondays 5-10 showed high viability with 99% live cells. CytochromeP450 (as measured by P450-Glo, Promega) was 20% greater in the3D cell clusters than the 2D monolayer. Surprisingly, the size of thecell cluster appears to affect the sensitivity of the cells to test articles.

Abstract

IntroductionThe current cost to bring a single, new drug to market is estimated atjust over $1 billion, but can be as high as $4-11 billion when includingdrug portfolios and R&D pipelines.1 At this high cost, generating onenew drug/year is not enough to recoup development costs. The highfailure rate, particularly when drugs have reached human clinicaltrials, accounts for a major portion of these costs. Currently, fewerthan 1 in 10 drugs tested in human clinical trials makes it throughphase II clinical trials.1 That means that the developmental andpreclinical drug testing failed to predict the in vivo human response.The expenditure loss due to these high failure rate needs to bereduced. By creating a better method to conduct early drugscreening on high quantities of 3D spheroids, we can potentiallyimprove the success rate of new drugs under development.

We started our work screening diabetes drugs using intact islets, andfound that the size of the islet made a dramatic difference in theresponse of the islet to drug applications2. Large islets had too greatof a diffusional barrier to allow the majority of the cells in the islet tocome in contact with the test article3. Through extensive research,we determined that islets > 100 micron in diameter lacked thediffusion to maintain viability in culture and did not respond well to invitro drug testing4. To correct this issue, we invented the micromold,a device to create uniform 3D spheroids of under 100 microns indiameter5. Subsequently, we utilized the micromold to create 3Dspheroids of a variety of cell types to screen for anti-cancer drugs,oral health products, and anti-diabetes drugs.

The purpose of this study was to optimize and test the experimentalconditions for 3D toxicity screening using spheroids produced in themicromold.

Figure 1: Microplate design.A) The version 1 plate design allows easy cluster retrieval forassays completed in standard microwell plates5. B) Version 2plate design for complete in-well assays.

• Canine pancreata were digested with enzymes usingstandard procedures. Purity of the preparation was testedusing dithizone staining. Viability was tested using calceingreen (Invitrogen) and IP.

• The use of HepaRG cells in a 3D in vitro cell-based assayfor predicting drug induced liver injury (DILI) wereinvestigated. Cells were grown in 2D and 3D and exposedto a DILI positive drug (chlorpromazine) for 3 days.

• Chlorpromazine concentrations were based on thetherapeutic maximum plasma concentration (Cmax) anddosed at 1.6, 6.3, 25, and 100-fold Cmax. After treatment,the cell viability, caspase 3/7 activity, P450 CYP3A4 enzymeactivity, and cell membrane integrity were assessed.

Methods

Figure 6: 2D versus 3D HepaRG response to toxin.A) Exposure to increasing doses of chlorpromazineresulted in a dose-dependent decrease in ATP in theHepaRG cells grown in 2D, but not in 3D. B) Likewise,there was a dose-dependent increase in released LDHlevels in the cells cultured in 2D, but not 3D. At 100-foldconcentrations, the cell viability of 2D and 3D culturesdeclined dramatically, indicating a positive response tothe drug induced liver injury. 3D HepaRG spheroidsused for this assay were between 75-100 microns.

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Figure 3: Pancreatic toxicity assay optimal in small 3Dclusters (20-40 micron diameter).Primary canine pancreatic acinar cells plated in 2D (closedcircles) failed to respond to chlorpromazine at all dosestested. Likewise, the same primary cells in 3D failed torespond to the cytotoxins, when in clusters ranging from 40-100 microns in diameter (open circles). However, whensmaller clusters were separated and used for toxicityscreening, they were sensitive to chlorpromazine (closedtriangles).

Table 1: Viability was measured for 7 days following isolation.Overall viability was > 90%, until day 7. However, it was notedthat the larger clusters (near 100 microns in diameter) hadpoorer cell viability. Compared to the canine acinar cellscultured in 2D, which had a cell viability value of 60%, all 3Dspheroid showed consistently improved viability.

Figure 5: 3D HepaRG spheroids.A) HepaRG clusters were washed from the molds andmeasured for size. The average diameter was between 75-100 μm. Viability assays of the 3D HepaRG clusters ondays 5-10 showed high viability with 99% live cells.

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