ADME/Tox Study and a New Hepatocyte Cell Line – Corning ......• Minimal drug inducibility, i.e.,
Transcript of ADME/Tox Study and a New Hepatocyte Cell Line – Corning ......• Minimal drug inducibility, i.e.,
Alternative Hepatocyte Models for In Vitro ADME/Tox Study and a New Hepatocyte Cell Line – Corning® HepatoCells
Rongjun Zuo, Ph.D.Senior ScientistCorning Life Sciences
May 12, 2015
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Presentation Outline
• Hepatocyte models – Primary human hepatocytes– Renewable hepatocyte sources– Novel hepatocyte culture technology
• Corning® HepatoCells and characterization• Summary
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Current Models for Drug ADME Studies
EODMT 2014 Costa. e al
• Easy to use; amenable to high throughput platform
• Used for toxicity & DDI studies
• Co-culture with other cells• Able to be cryopreserved• Available for multiple
species• De-differentiate in culture• Lack representation of
intact liver structure
• Cell line: lower cost, renewable source.
• Need to be evaluated for target applications
• Easy to use; amenable to high throughput platform
• Express phase I & II enzymes
• Used for clearance, inhibition, covalent binding
• Pool of large number of donors
• Can be recovered from frozen tissue
• Low cost• Available for multiple
species• Need co-factors• Lack a set of liver functions• Limited assay time
• Allow better cell-cell & cell-matrix interactions
• Represent the in vivo-like conditions in terms of cell function & morphology, nutrition, oxygenation, configuration.
• Enabling better prediction of drug toxicity
• Maintain long term culture; good for chronic drug treatment
• Low to medium throughput• Some 3D formats difficult
for imaging
• In vivo architecture reserved
• In vivo-like expression of drug metabolizing enzymes, transporters, and functional bile canaliculi
• Zone specific metabolism and toxicity may be studied
• Hepatic function reserved for <24 hrs.
• Complicated to use• Not a high throughput
system• Difficult to obtain human
tissue
• Most suitable model to study different organ-interaction
• In vivo studies are a requirement for drug approval
• Have inter-species differences
• High cost• Present ethical issues
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Primary Human Hepatocytes
• Primary human hepatocytes are considered the gold standard model for in vitro drug metabolism/toxicity studies
• Contain all the hepatic enzymes, co-factors (NADPH, UDPGA, GSH, PAPS, etc.), transporter proteins needed for drug metabolism studies and hepatotoxicity studies.
• Eliminate species difference for IVIVE compared to animal hepatocytes. • Contain full machinery for enzyme regulation, important for CYP induction
and inhibition study.• Can form in vivo-like hepatobiliary network for drug transport study
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Need for Renewable Hepatocyte Sources• Inherent limitations of primary human hepatocytes
– Large lot-to-lot variability – Need to qualify every lot (time consuming)– Limited supply of high quality lots/limited lot sizes– Tendency to de-differentiate in culture– High cost
• Increasing need for renewable hepatocytes sources– Human hepatoma cell lines
• HepG2• HepaRG
– Stem cell or induced pluripotent stem cells derived hepatocyte-like cells– Immortalized hepatocyte-like cell lines
• Corning® HepatoCells
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Renewable Hepatocyte SourcesHuman Hepatoma Cell Line
• HepG2– Widely used for toxicity screening– Low metabolic capability
• HepaRG– Pros:
• Possessing most hepatocyte-specific functions, compared to other hepatoma cell lines
• Metabolism competent• Show comparable sensitivity to a group of
hepatotoxic compounds similar to primary human hepatocytes
– Cons:• Mixture of 2 cell types; difficult to control the ratio• Large lot-to-lot variation observed• Need high concentration of DMSO to maintain
differentiated status• Relatively low expression of some uptake
transporters
Arch Toxicol(2012) Lin, et al.
Cell Biol Toxicol (2012) Gerets, et al.
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Renewable Hepatocyte SourceHepatocyte-like Cells Differentiated from Human ESC/iPSC
Protocol
Gene & Protein Expression
Cellular Morphology
Functional Characterization: Albumin secretion, urea synthesis, glycogen storage, indocyanine green uptake, low density lipoprotein uptake, gene expression of hepatocyte specific genes, drug metabolism, etc.
Differentiation driven by viral transfection of transcription factors, recombinant growth factors, or small molecules (or transdifferentiation from fibroblast cells)
Characterization of Differentiation Efficiency
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Hepatocyte-like Cells Differentiated from hESC/iPSCExpression of Hepatocyte-specific Genes/Proteins
Efficiency of Phase III Differentiation
• High expression level of fetal genes, e.g., AFP.
• Low expression of adult genes, i.e., CYP3A4.
Stem Cell Reports (2015) Siller R, et al.
Plos One (2015) Ishikawa, T., et al.
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Hepatocyte-like Cells Derived from hESC/iPSCFunctional Characterization for Drug Metabolism
Metabolism capability, drug inducibility, and transporter activity
• Significant metabolism activity compared to pluripotent cell control • Minimal drug inducibility, i.e., <3 fold for CYP3A4. Not suitable for induction assay• Transporter activity shown in the lower range of primary human hepatocytes
Black: hESCpluripotent control
Blue: basal activity
Orange: induced activity with either omeprazole (1A2) or rifampicin (3A4)
Stem Cell Report (2015) Siller et al
Cell Stem Cell (2014) Huang, et al.
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Considerations in Using hESC/iPSC Derived Hepatocytes• Majority of the improved differentiation protocols can achieve significant expression of
hepatocyte-specific genes/proteins compared to their parental cells; however the differentiated cells are far different from mature hepatocytes in terms of important drug metabolism relevant functions
– Need better understanding of development of an adult phenotype, the mechanisms of cell reprogramming, and the role of the tissue culture microenvironment.
• Although much higher differentiation efficiency obtained with improved protocols (e.g., using small molecules), technical challenges still remain
– Scalability, fully defined conditions, cost, and reproducibility • In order to validate hepatocytes derived from hESC/iPSC for drug metabolism application, it
is critical to use:– The right assays, e.g., LCMS or HPLC (vs. P450-Glo™) for metabolism capacity– The right reference control cells, e.g., primary adult hepatocytes (vs. cell line such as
HepG2) with culture format appropriate for target assays
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Novel Hepatocyte Culture Technology- 3D Hepatocyte Culture
• Primary hepatocytes tend to dedifferentiate in 2D in vitro culture, which results in limited life span, greatly reduced hepatic specific functions, such as metabolic activity and transporter activity.
• There is an increasing need for a more in vivo-like hepatocyte culture format for better in vitro to in vivo prediction of drug ADME features.
• 3D hepatocyte culture can maintain hepatocyte polarization and functionality better than conventional 2D model due to a more in vivo-like environment created by optimal cell-ECM and cell-cell contacts
Altered cytoskeleton structure
Reduced cell-cell & cell-ECM contact
Reduced polarization & signal pathways
De-differentiation over culture time
Deterioration of function
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Static 3D Cultures - Examples
Adopted from Exp Biol Med (2014) Bale et al
RegeneMed InSphere HepreGen• Co-culture of non-parenchymal cells
and hepatocytes on nylon scaffold Transwell® seeded at near physiological ratio.
• Co-culture induces ECM/GF production to form tissue structure that supports long-term function.
• Maintain up to 3 months hepatic functions of both rat & human hepatocytes, including, albumin, transferrin, fibrinogen secretion, and urea synthesis; maintain stable CYP3A4, 1A1 and 2C9 activity.
• Demonstrated inflammatory response of the liver tissue upon exposure to LPS and release of pro-inflammatory cytokines measured.
• Human 3D liver tissue has been used to test drug toxicity.
• Expensive
• Spheroids are formed using hanging drop technology with hepatocytes and non-parenchymal cells introduced into the drop, transferred into a 96-well microplate and cultured.
• Maintain stable function for up to 5 weeks. • Cellular phenotype of endothelial and
Kupffer cells are maintained within the spheroids.
• Better acetaminophen and diclofenac TC50values in toxicity assays when compared with 2D cultures
• High reproducibility in spheroid size, dimension and density.
• Forming spheroids is time consuming without robot
• Instrument sensitivity may be an issue with analysis of small sample amounts.
• Micro-patterned plate with hepatocytes surrounded by stromal cells (3T3-J2 mouse fibroblast cells).
• Maintain stable function for up to 6 weeks, including albumin secretion, urea synthesis, Phase I and II drug metabolism, and formation of bile canaliculi.
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3D Perfusion Cultures - Examples
CellAsic Zyoxel Hurel• A pseudo-3D culture in microfluidic plate• Micro-structure pattern mimics sinusoidal
barrier function of liver, shielding the hepatocytes from shear stress of medium.
• Nutrient exchange achieved with media flow.
• Showed high cell viability up to 7 days and response to drug.
• In vivo structure not maintained.• Can not control flow rate; need extensive
training on cell loading.• Need to establish protein synthesis and
drug metabolism.• Expensive system
• Consists of two reservoirs with one for cells and the other for media for recirculation
• Perfusion using pump to provide nutrient exchange; oxygen concentration similar to a sinusoid.
• Maintained high viability and phenotype for up to 13 days in co-culture of hepatocytes and NPC.
• Not amenable to imaging due to presence of scaffold .
• Further characterization are needed on NPCs since they are liver sinusoidal endothelial cell-enriched fraction.
• Cell Culture Analog (CCA) with cell seeding area and multiple devices in parallel
• Able to achieve high-density seeding of hepatocytes and maintain viability and metabolic functions under flow condition for 24 hours in initial short duration studies.
• Co-culture of hepatocyte with non-parenchymal cells shows high-viability and in vivo-like clearance for various drugs up to 8 days.
Additional studies to evaluate important liver functions (albumin secretion, urea excretion, drug toxicity) are needed to realize their full potential.
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Corning Solutions to Provide Renewable HepatocytesCorning Solution…..• Develop a “renewable” hepatocyte-like product with hepatocyte morphology and functions
similar to primary human hepatocytes Consistent lot-to-lot performance (eliminates need to pre-qualify lots) Large lot sizes for long-term comparative studies (500 to 1,000 vials) Easy to use format: “Thaw and Go” reagent
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• Fresh plated human hepatocytes, with or without Corning® Matrigel®matrix overlay
• Induction-qualified cryopreserved human hepatocytes
• Metabolism-qualified cryopreserved human hepatocytes
• Transporter-qualified cryopreserved human hepatocytes
• Metabolism-qualified cryopreserved human kepatocyte suspension
• Transporter-qualified Cryopreserved human hepatocyte suspension
• Cryopreserved animal hepatocyte suspension (rat, mouse, dog, monkey)
Corning Hepatocyte Portfolio to Support ADME Studies
Suspension Hepatocytes Plated Hepatocytes
Corning HepatoCellsA Renewable Hepatocyte Product
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• Mature hepatocyte-like morphology; homogeneous population • Wild type genotype for important Cytochrome P450s
− CYP2D6, CYP2C9, CYP2C19
• >8 million cells per vial
• >80% post thaw viability
• No percoll density gradient required
Product Feature
• Robust CYP3A4 induction (>10 fold), as well as CYP1A2 and 2B6 induction
• Better than current alternative models and some lots of PHH
• Consistency− Large lot size (500 to 1,000 vials) helps to ensure low lot-to-lot
variation, and no back orders
• Convenience/easy to use− No special additives required for differentiation− One medium for all phases of culturing (plating, maintenance,
induction)
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Mature Hepatocyte-Like Morphology
100% confluence Pure population Typical cuboidal
morphology Double/Multi
Nucleation Distinct nucleoli Distinct cell‐cell
contact Bile canaliculi
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Application: CYP Induction
• Regulatory agencies recommend to use primary human hepatocytes in in vitro test for CYP3A4, 1A2, and 2B6 induction.
• Any alternative hepatic cell models are expected – To possess all 3 induction regulation pathways – To respond to prototypical inducers in a similar pattern to primary human
hepatocytes– To perform consistently to save time and cost on screening
Corning® HepatoCells are evaluated against primary human hepatocytes for their performance in induction applications.
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EYFP-hCAR Expression and LocalizationData courtesy from Prof. Hongbing Wang, University of Maryland
Phenobarbital-responsive CAR Nuclear Translocation
Corning HepatoCellsNon-treated
PB treated
Human Hepatocytes
PB treated
Non treatedNo nucleus expression
Translocated to nucleus after PB treatment
No nucleus expression
Translocated to nucleus after PB treatment
CAR translocation upon PB treatment in Corning HepatoCells was similar to primary hepatocytes; not seen in many cell lines.
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Induction Assay in 96-well Microplate
Comparable CYP induction response to primary human hepatocytes with much smaller lot-to-lot variability.
All data are mean of several lots for each cell type.
18.5(n=15)
28.7(n=15)
7.8(n=15)
19.6(n=6)
32.4(n=3)
5.0(n=3)
0
10
20
30
40
50
CYP3A4 CYP1A2 CYP2B6
Fold In
duction
Primary humanhepatocytes
Corning HepatoCells
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FDA and EMA recommend using PHH to evaluate induction potential• To predict in vivo induction potential:
− Primary hepatocytes need to be previously characterized with a sufficient number of known clinical inducers and non-inducers to determine a threshold.
Corning® HepatoCells were evaluated − As an in vitro screening tool for predicting clinical CYP3A4
• 3 batches of HepatoCells• 13 known clinical CYP3A4 inducers• Enzymatic activity and mRNA expression
− Using Relative Induction Score (RIS) approach
− The prediction was compared with that of primary human hepatocytes
Application: Induction Prediction with the RIS Approach
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R2 = 0.99
Phenytoin, M0.1 1 10 100 1000
Fold
Indu
ctio
n
0
2
4
6
8
10
12
R2 = 0.95
Rifampicin, M
0.1 1 10 100
Fold
Indu
ctio
n
5
10
15
20
R2 = 0.96
Carbamazepine, M0.1 1 10 100
Fold
Indu
ctio
n
0
1
2
3
4
5
6
Examples of Concentration-dependent Induction Response Curve
Concentration‐dependent induction response curves were generated for 13 compounds All curves showed good correlation with R2 >0.9 3 pilot lots showed similar concentration‐dependent response curve
Probenecid, M
0.01 0.1 1 10 100
CYP
3A4
Fold
0
5
10
15
20
25
30
35
R2 = 0.98
EC50 = 42 MEmax = 31
Probenecid, M0.01 0.1 1 10 100
CYP
3A4
Fold
0
10
20
30
40
R2 = 0.9
EC50 = 51 M Emax = 39
Probenecid, M
0.01 0.1 1 10 100
CYP
3A4
Fold
0
10
20
30
40
R2 = 0.98
EC50 = 44 MEmax = 36
PR Lot 2 PR Lot 3A PR Lot 3B
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RIS Comparison between Corning® HepatoCells and Primary Hepatocytes
Similar RIS data obtained from 3 prototype lots of Corning HepatoCells. HepatoCells showed similar RIS ranking as primary human hepatocytes. HepatoCells showed much smaller variation than primary human hepatocytes
(average % CV is 18.2% for HepatoCells, and 53.3% for primary human hepatocytes).
Compounds Observed AUC change
RIS from Corning HepatoCells RIS from Primary Human Hepatocyte Average RIS %CV
Lot PR2 Lot PR3A Lot PR3B Lot 295 Lot 312 Lot 318 HepatoCells PHH HepatoCells PHH
Dexamethasone 19 0.0042 0.0053 0.0053 0.001 0.001 0.0004 0.0050 0.0008 12.7% 43.3%
Terbinafine* 25 0.047 0.059 0.040 0.027 0.024 0.007 0.0485 0.0193 20.1% 55.8%
Nifedipine* 4 0.048 0.052 0.045 0.019 0.007 0.011 0.0484 0.0123 7.4% 49.5%
Pleconaril* 35 0.087 0.095 0.101 0.025 0.029 0.011 0.0944 0.0217 7.5% 43.6%
Omeprazole ‐25 0.189 0.139 0.154 0.008 0.019 0.019 0.161 0.0153 16.0% 41.4%
Pioglitazone* 26 0.190 0.199 0.178 0.012 0.011 0.025 0.189 0.0160 5.5% 48.8%
Troglitazone* 65 0.513 0.355 0.650 0.12 0.2 0.03 0.506 0.1167 29.2% 72.9%
Phenobarbitol 61 0.902 0.729 1.158 0.88 2.4 1.6 0.930 1.63 23.2% 46.7%
Carbamazepine* 94 1.25 1.074 0.723 1.1 1.1 9.3 1.02 3.83 26.5% 123.5%
Phenytoin* 94 1.50 1.381 1.908 1 1.3 1.8 1.60 1.37 17.3% 29.6%
Rifampicin* 97 10.93 8.175 16.201 7 11 6.4 11.77 8.13 34.7% 30.7%
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Prediction of In Vivo DDI: RIS Model
RIS values (generated using EC50 and Emax data from a group of clinical inducers and non‐inducers) correlated well with observed AUC decrease.
All 3 pilot lots generated similar RIS cut‐off value at 20% AUC decrease, suggesting no calibration necessary for each individual lot of Corning® HepatoCells.
CorningHepatoCells
Primary Hepatocytes
PR2 0.116 Lot 295 0.21PR3 0.135 Lot 312 0.054PR4 0.121 Lot 318 0.23Mean 0.12 Mean 0.16%CV 7.5% %CV 58.5%
RIS cut-off at 20% AUC decrease
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Prediction Accuracy: Predicted AUC Change vs. Observed AUC Change
Comparable prediction accuracy between Corning® HepatoCells and primary human hepatocytes (all within 20% of observed value)
R2 = 0.96R2 = 0.91
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Application: Transporter Characterization
• Drug transporters play an important role in drug-induced hepatotoxicity and adverse drug-drug interactions.
• Many hepatic cell lines are found to express low level of uptake transporters.
Corning® HepatoCells were evaluated for transport of prototypical substrates of important hepatic uptake transporters.
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Uptake Transporter Study: Time-dependent and Concentration-dependent Uptake of OCT1 Substrates
Time, min 5 10 20 30
Substrate Lot Uptake Ratio (37C/4°C)
MPP+PR2 12.5 12.5 12.0 11.3PR3A 11.6 11.0 14.1 10.7
TEAPR2 2.3 3.3 2.0 5.2PR3A 1.8 2.3 3.7 2.7
MetforminPR2 2.4 6.0 3.8 3.4PR3A 3.2 2.5 3.1 2.7
0
5
10
15
20
25
30
35
0 10 20 30
MPP
+ uptake (pmol/m
g)
Incubation Time, min
37C_PR2 4C_PR237C_PR3A 4C_PR3A
Time-dependent MPP+ Uptake
[MPP+], M
0 200 400 600 800 1000 1200
Upt
ake
activ
ity, p
mol
/min
/mg
0
50
100
150
200
250
300
Vmax = 266 pmol/min/mgKm = 75 M
Concentration‐dependent MPP+ UptakeTransporter Substrate
Km, µM
CorningHepatoCells
1CorningTransportoCells™
2Human Hepatocytes
3Human Hepatocytes
OCT1MPP+ 75 n.a 101 N/A
TEA 1940 713 N/A 407
Reference data: 1,3 In house data (3suspension hepatocytes); 2Chem Biol Interact. 190:165-70, 2011.
Corning® HepatoCells demonstrated time- and concentration-dependent uptake of 3 prototypic substrates of hepatic transporter OCT1 with good signal to noise ratio
Km values align with literature data.
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Uptake Transporter Study: Inhibition of Uptake of OCT1 Substrates
1Zhang L. et al. JPET 51:913-921, 1997 2Zhang L. et al. JPET 286:354-61, 1998
Test system Substrate IC50
Corning® HepatoCells MPP+ 0.27+/-0.046Corning TransportoCells™ MPP+ 2.2
X. laevis oocyte1 MPP+ 4.7HeLa cell line2 TEA 2.7
Inhibition of MPP+ Uptake by Decynium‐22 Lot # IC50, µM
Lot 1 0.26Lot 2 0.34Lot 3 0.22Lot 4 0.24Lot 5 0.27Mean 0.27%CV 17%
Five different lots of Corning HepatoCells showed similar Decynium-22 inhibition of MPP+ uptake with small lot-to-lot variation.
The IC50 values from Corning HepatoCells are comparable with literature values from other cell systems.
IC50 from 5 lots
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Uptake Transporter Study: Concentration-dependent Uptake of OATP1B1/1B3 and NTCP Substrates
Corning® HepatoCells demonstrated concentration-dependent uptake of Rosuvastatin and TCA.
Km values are comparable to literature data.
High signal-to-noise ratio suggests active expression of functional uptake transporter proteins.
Transporter Substrate Km, M Test system ReferenceNTCP Rosuvastatin 6.5 HeLa
1OATP1B1 Rosuvastatin 7.3 HeLaOATP1B3 Rosuvastatin 9.8 HeLaOATP1B1 Rosuvastatin 0.8 HEK 2OATP1B3 Rosuvastatin 14.2 HEKNTCP TCA 2.1 HEK 3NTCP TCA 7.5 HeLa 4
OATP1B3 TCA 5.8 Oocyte 5NTCP TCA 14 Corning TransportoCells™
6TCA 8.2 Human hepatocytes
Uptake Ratio
Substrate, µM Rosuvastatin TCA
2 8.8 9.910 7.1 8.620 3.6 6.0
[Rosuvastatin], M
0 5 10 15 20 25 30
Upta
ke a
ctiv
ity, p
mol
/min
/mg
0
10
20
30
40
Km = 6.7 MVmax = 36.5 pmol/min/mg
Rosuvastatin Uptake
[TCA], M0 5 10 15 20 25
Upta
ke a
ctiv
ity, p
mol
/min
/mg
0
2
4
6
8
10
12
14
16
18
20
Km = 8.5 MVmax = 23.6 pmol/min/mg
TCA Uptake
1Ho RH et al. Gastro. 130:1793-1806, 2006; 2Kitamura S. et al DMD 36:2014-23, 2008; 3Leslie EM et al. JPET 321:1170-8, 2007; 4Ho RH et al. JBC 279:7213-22, 2004; 5Abe T et al. Gastroenterology 120:1689-99, 2001; 6 Corning in-house data
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Applications: Toxicity, Spheroid, and Others
• In vitro assessment of drug induced hepatotoxicity requires using metabolism-competent cell models.
• Primary hepatocytes are not amenable to chronic hepatotoxicity study due to short life span and tendency to de-differentiate in culture.
Corning® HepatoCells are evaluated for assessing metabolism-dependent toxicity, 3D spheroid formation, and other potential applications.
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Aflatoxin B1 Toxicity Test
Corning HepatoCells showed dose‐dependent response to Aflatoxin B1. Ketoconazole inhibited toxicity caused by Aflatoxin B1.
Day 0 Day 1 Day 2 Day 3 Day 4 Day 5
Seed Corning® Matrigel® matrix overlay
Media change
24‐hr Aflatoxin B1 exposure, with or without Ketoconazole
Viability measurement
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Uniform-sized Spheroids from Corning® HepatoCells in 96-well Ultra-Low Attachment Surface Microplates
Potential applications include:• 3D modeling and co-culture with non-parenchymal cells, e.g. Kupffer cells• Extended culture while maintaining functionality• Adaptation of high content imaging assay
6.25K3.13K
1.56K0.78K
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Other Potential Applications
• Current beta testing showed better or similar performance of Corning®
HepatoCells compared with primary human hepatocytes in the following applications
– Viral infection– Lipid metabolism
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Summary Corning® HepatoCells demonstrated typical mature hepatocyte morphology. Fold induction for 3 CYPs are comparable to PHH. Much smaller lot-to-lot variation than PHH. HepatoCells predicted in vivo CYP3A4 inducers similarly as PHH. HepatoCells showed consistent RIS cut-off values among 3 pilot lots, suggesting a
1-time calibration may be enough, saving time and cost for lot qualification. HepatoCells actively express functional uptake transporters as shown by uptake
of prototypical substrates of OCT1, OATP1B1/1B3, and NTCP. HepatoCells showed dose-dependent response to metabolism-based toxic
compound which can be reversed by CYP450 inhibitor. HepatoCells can form 3D spheroids.
In conclusion, Corning HepatoCells are a very promising renewable hepatocyte model system for ADME/Tox research.
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Future/Ongoing Studies
• Gene expression profiling
• CYP, FMO, and UGT (Phase 2) metabolism
• ABC transporters (efflux)
• Tox studies (DILI)