The Center for Alternatives to Animal Testing is an academic center affiliated with the Division of Toxicological Sciences in the Department of Environmental Health Sciences of the Johns Hopkins University Bloomberg School of Public Health.
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TestSmart HPV
April 26-27, 1999
Hyatt Fair Lakes
12777 Fair Lakes Circle
Fairfax, VA 22033
A workshop of The Johns Hopkins Center for Alternatives to Animal Testing
TestSmart is a program of the Vision 20/20 forum
This workshop is partially funded through a grant by the Vira I. Heinz Endowment
Abstract for TestSmart--A Humane and Efficient Approach to Screening Information Data Sets (SIDS) Data
Use of Human Cells in High Throughput Screening
Paul M. Silber, Ph. D.
In Vitro Technologies, Inc.
Why is the Liver a Primary Target for Chemical Toxicity?
- Bioactivation capacity.
- First systemic organ in contact with orally ingested compounds.
- High perfusion rate.
Why is the Kidney a Secondary Target for Chemical Toxicity?
- Concentrating ability.
- High blood flow.
- Bioactivation.
Observation
- The liver is the primary target for chemical toxicity.
- Kidney is an important secondary target for chemical toxicity.
In Vitro Toxicity Models
- Isolated perfused organs
- Tissue slices
- "Engineered" cells
- Isolated cells
Isolated Perfused Organs
- Time consuming, labor intensive.
- Does not spare animals.
- Considers intact organ interaction.
Tissue Slices
- Many can be prepared from any soft tissue.
- Animal sparing.
- Integrated cell function.
- Difficult to cryopreserve.
"Engineered" Cells
- Convenient and reproducible.
- Standardized commercial reagent.
- Never a complete model of the entire human cell - they only contain what you "put into them".
Isolated Human Cells
- High throughput assays possible.
- Interaction of all cellular processes and components (intact plasma membrane).
- Many possible endpoints of toxicity.
Isolated Human Cells
- Complete bioactivation system present.
- Human-specific.
- Standardized commercial reagent.
USFDA Guidance for Industry
Cryopreserved Human Hepatocytes and HTS
- Isolation, culturing, cryopreservation
- Characterization
- High-throughput screening protocol
Isolation of Human Hepatocytes
- Source: Human livers procured but not used for transplantation
- Two-step collagenase digestion
- Viability: >80%
- Yield: 10-30 billion viable cells per isolation.
Human Hepatocyte Isolation:
- Viability and Yield
Advantages of Cryopreserved Human Hepatocytes
- Experiments can be planned without the need for a fresh human liver.
- Multiple experiments can be performed at different times with hepatocytes from the same donors.
- Studies can be performed with characterized (genotyped and phenotyped) hepatocytes.
Advantages of Cryopreserved Human Hepatocytes (Cont.)
- Studies can be performed with pooled hepatocytes from different donors.
- Hepatocytes from a specific donor can be shipped to multiple laboratories for experimentation.
- Allows HTS with human hepatocytes.
Human Hepatocytes and Toxicology
- Human-specific hepatotoxicity model.
- Dose-response and mechanistic evaluation to aid safety assessment. Facilitates screening/detailed dose-response studies of chemicals and drugs.
HHHTS: Cytotoxicity
Sample Protocol
- 96-well plates
- 50,000 cells per well
- 4 hour incubation
- 1 mM of test chemical (single conc.) or multiple concentrations for LC-50
- MTT assay for viability
- Direct addition of acidified isopropanol for extraction
- 96-well plate reader at 570 nm
HHHTS for Cytotoxicity
Cryopreserved Human Hepatocytes and HTS
- Procedures developed for isolation, culturing, and cryopreservation.
- Phase I and II metabolism activities retained in cryopreserved human hepatocytes.
- A cryopreserved human hepatocyte bank has been initiated.
- HTS assays have been developed for early screening of toxicity.
Cryopreserved Human Renal Proximal Tubules (RPT)
- Procedures developed for isolation, culturing, and cryopreservation.
- Active transport and cell polarity retained in cryopreserved human RPT.
- A cryopreserved human RPT bank has been initiated.
- HTS assays being developed for early screening of potential nephrotoxicity.