Functional Comparison of HepaRG Cells and Primary Human Hepatocytes - - PowerPoint PPT Presentation

▶
functional comparison of heparg cells and primary human
SMART_READER_LITE
LIVE PREVIEW

Functional Comparison of HepaRG Cells and Primary Human Hepatocytes - - PowerPoint PPT Presentation

Dec 13, 2023 288 likes •470 views

Functional Comparison of HepaRG Cells and Primary Human Hepatocytes in Monolayer and Spheroid Culture as Repeated Exposure Models for Hepatotoxicity Jinpeng Li January 9, 2020 Background Predicting repeated exposure hepatotoxicity is of

slide-1
SLIDE 1

Functional Comparison of HepaRG Cells and Primary Human Hepatocytes in Monolayer and Spheroid Culture as Repeated Exposure Models for Hepatotoxicity

Jinpeng Li January 9, 2020

slide-2
SLIDE 2
  • Predicting repeated exposure hepatotoxicity is of critical importance for the safety assessment of

chemicals.

  • The advantage of human cell-based in vitro models:
  • 1) time- and resource-efficient
  • 2) genetically and physiologically relevant
  • The challenge of long-term in vitro culture of liver cells:
  • Rapid dedifferentiation in conventional monolayer culture.
  • Culture method

2D Sandwich culture Extracellular Matrix Hepatocytes Collagen ~one week

Background

3D spheroid culture Ultra low attachment >3 weeks

slide-3
SLIDE 3
  • Primary human hepatocytes (PHH)
  • Gold standard
  • Limitations: availability, variability/reproducibility
  • HepaRG cell
  • Derived from hepatocarcinoma
  • Highly stable cell line
  • Exhibit many characteristics similar to PHH:
  • major xenobiotic sensors (PXR, CAR and AHR)
  • drug transporters
  • phase I and II drug metabolizing enzymes
  • hepatic transcription factors involved in stress response pathways

Background

  • Objective: Characterize and compare models for hepatotoxicity assessment after repeated

exposure

2D sandwich culture 3D spheroid culture

slide-4
SLIDE 4
  • PHH
  • 2D:

Day0 Day 1 Day 4 Day7

  • 3D:

Day 1 Day 4 Day7 Day 14 Day 21

Morphology

Unpublished data

slide-5
SLIDE 5
  • HepaRG
  • 2D:

Day0 Day 1 Day 4 Day7

  • 3D:

Day 1 Day 4 Day7 Day 14 Day 21

Morphology

Unpublished data

slide-6
SLIDE 6

3 hr 4 day 7 day 4 day 7 day 14 day 21 day Cellular ATP (RLU per 103 cells) 3 hr 4 day 7 day 4 day 7 day 14 day 21 day Cellular ATP (RLU per 103 cells)

Viability (cellular ATP content)

Modified based on Li et al. (2019). Applied In Vitro Toxicology.

slide-7
SLIDE 7

3 hr 4 day 7 day 4 day 7 day 14 day 21 day Fold change relative to freshly thawed cells 3 hr 4 day 7 day 4 day 7 day 14 day 21 day Fold change relative to freshly thawed cells 3 hr 4 day 7 day 4 day 7 day 14 day 21 day Fold change relative to freshly thawed cells 2 4 6 8

CYP2B6

Time after seeding 3 hr 4 day 7 day 4 day 7 day 14 day 21 day Fold change relative to freshly thawed cells

PHH HepaRG

Metabolic activities

Modified based on Li et al. (2019). Applied In Vitro Toxicology.

slide-8
SLIDE 8

Spectrophotometry based measurement

Absorbance R square 0.9987 Equation Y = 0.01034*X - 0.0005075

Urea production

Amino acid NH3 (ammonia) Urea Kidney

Blood

Liver

Unpublished data

slide-9
SLIDE 9

3 hr 4 day 7 day 4 day 7 day 14 day 21 day Urea (mg/L/106 cells) 3 hr 4 day 7 day 4 day 7 day 14 day 21 day Urea (mg/L/106 cells)

Urea production

Modified based on Li et al. (2019). Applied In Vitro Toxicology.

slide-10
SLIDE 10

Days 1 2 3 4 5 6 7 8 9 10 11 12 13 14 2D_d7 (3) ↓ ↓ ↓

√

3D_d7 (3) ↓ ↓ ↓

√

3D_d14 (6) ↓ ↓ ↓ ↓ ↓ ↓

√

  • Chemical treatment regimen

↓ : Chemical treatment √ : Viability assessment (ATP content)

Acetaminophen Aflatoxin B1

Assessment of model sensitivity

slide-11
SLIDE 11

Assessment of model sensitivity

Viability (% of vehicle) EC50 2D_7d 2286 3D_7d 1613 3D_14d 1951 Viability (% of vehicle) EC50 2D_7d 4793 3D_7d 3917 3D_14d 1979 Modified based on Li et al. (2019). Applied In Vitro Toxicology. 2D_7d 3D_7d 3D_14d 2000 4000 6000

Ibufenac

EC50 ( M) PHH HepaRG Cmax 10X Cmax

slide-12
SLIDE 12

Assessment of model sensitivity

1 2 3 4 5 50 100 150

  • Conc. (log[ M])

2D_7d 3D_7d EC50 2D_7d 2958 3D_7d 1244 3D_14d 957.5

PHH

3D_14d 1 2 3 4 5 50 100 150

  • Conc. (log[ M])

Viability (% of vehicle) EC50 2D_7d 2972 3D_7d 1664 3D_14d 1109

HepaRG

2D_7d 3D_7d 3D_14d Modified based on Li et al. (2019). Applied In Vitro Toxicology. 2D_7d 3D_7d 3D_14d 1000 2000 3000 4000

Ibuprofen

EC50 ( M) PHH HepaRG Cmax 10X Cmax

slide-13
SLIDE 13

Assessment of model sensitivity

1 2 3 4 5 50 100 150

  • Conc. (log[ M])

Viability (% of vehicle) EC50 2D_7d 6038 3D_7d 1509 3D_14d 646.6

PHH

2D_7d 3D_7d 3D_14d 1 2 3 4 5 50 100 150

  • Conc. (log[ M])

Viability (% of vehicle) EC50 2D_7d 3981 3D_7d 2516 3D_14d 1472

HepaRG

2D_7d 3D_7d 3D_14d 2D_7d 3D_7d 3D_14d 2000 4000 6000 8000

Acetaminophen

EC50 ( M) PHH HepaRG

Acetaminophen

Modified based on Li et al. (2019). Applied In Vitro Toxicology. Action level

slide-14
SLIDE 14

Assessment of model sensitivity

  • 2
  • 1

1 2 50 100 150

  • Conc. (log[ M])

Viability (% of vehicle) EC50 2D_7d 0.8975 3D_7d 0.1475 3D_14d 0.07675

PHH

2D_7d 3D_7d 3D_14d

  • 2
  • 1

1 2 50 100 150

  • Conc. (log[ M])

Viability (% of vehicle) EC50 2D_7d 2.313 3D_7d 0.5606 3D_14d 0.1712

HepaRG

2D_7d 3D_7d 3D_14d 2D_7d 3D_7d 3D_14d 0.0 0.5 1.0 1.5 2.0 2.5

Aflatoxin B1

EC50 ( M) PHH HepaRG

Aflatoxin B1

Modified based on Li et al. (2019). Applied In Vitro Toxicology. Exposure level

slide-15
SLIDE 15

PHH HepaRG 2D 3D 2D 3D Viability * ** * *** Hepatic phenotype * *** * *** Sensitivity * *** * *** Reproducibility * *** Availability * ***

Summary

slide-16
SLIDE 16

Acknowledgement

  • Dr. Sue Marty
  • Dr. Matt LeBaron
  • Dr. Raja Settivari
  • CMT/TK Group
slide-17
SLIDE 17

Thank you for your attention!

?