The UK Human Tissue Bank provides fresh hepatocytes - plated or in suspension and characterisaed crypreserved hepatocytes for metabolism or induction studies and protein expression.
Fresh Human Hepatocytes
Human tissue is inherently variable and there is additional variation introduced by disease, age, medical history, treatment immediately prior to liver tissue donation and other factors. These may affect the viability and yield of hepatocytes obtained. The availability of fresh hepatocytes is dependant on the supply of fresh liver tissue; UKHTB undertake isolations two or three times a week.
This document is aimed at giving a brief general overview of how tissue is obtained and processed to isolate cells.
Patient sources
The tissue is available from two sources; non-transplantable liver tissue from cadaveric organ donors, and tissue that would otherwise be discarded from patients undergoing liver resections, mainly for primary or secondary cancer.
Consent
Patient consent in writing is gained prior to donation of surplus surgical tissues in all cases. Patients are given a patient information booklet explaining how the tissue will be used and it is explained that donors have no right to IP or commercial gain from their donation. We receive medical and drug history data as well as confirmation of consent via the surgical operating teams. UKHTB does not receive identifiable donor data.
Isolation
Hepatocyte isolation is undertaken using a modified two-step Seglen technique (1). Once isolated, the cell number and viability are determined and researchers contacted. The required cells will then be despatched by courier as soon as possible. Despatch times for fresh cells are normally towards the end of the working day.
Cell quality
It is difficult to predict the yield and viability of cells from a tissue donation until the isolation procedure has been completed (2). Average hepatocyte viability over the past 12 months has been 83% and therefore we do not use Percoll unless requested.
Continuing Research
Although the isolation process is well established, UKHTB is continuing to optimise and improve the quality of the hepatocytes delivered to researchers in an effort to maximise all tissue donated.
References
1. Seglen PO. Preparation of isolated rat liver cells. Methods Cell Biol. 1976;XIII:201
2. Lloyd TD, Orr S, Patel R, Crees G, Chavda S, Vadyar H, Berry DP, Sherlock D, Dennison AR. Effect of patient, operative and isolation factors on subsequent yield and viability of human hepatocytes for research use. Cell Tissue Bank. 2004; 5(2):81-7.
Cryopreserved Hepatocytes
To optimise the use of human tissue any remaining cells that are of suitable viability that are not sent out fresh to researchers are cryopreserved for future use.
Characterisation data is now available on several batches of cryopreserved hepatocytes suitable for induction and metabolism studies.
Induction - click here to view data sheet
Hepatocytes are cryopreserved by UKHTB at -80 using a cryoprotectant. They are stored at UKHTB in liquidnitrogen.Hepatocytes are thawed and a Trypan Blue viability check is carried out. Cells are plated in 24 well plates and incubated overnight; the monolayer is then assessed for confluence.
If the monolayer is satisfactory, induction studies are carried out.Following 72 hours exposure withB-naphthoflavone(BNF: a prototypical CYP1A enzyme inducer) and rifampicin (RIF: a prototypical CYP3A enzyme inducer)cells are assayed for induction with Ethoxyresorufin (BNF) and Testosterone (RIF).
Metabolism - click here to view data sheet
Hepatocytes are cryopreserved by UKHTB at -80 using a cryoprotectant. They are stored at UKHTB in liquid nitrogen.
Cells are assayed for metabolism activity. Cells are incubated with probe substrates and samples removed at appropriate timepoints and analysed.
Probe substrates for estimation metabolic activity
- 7-Ethoxy resorufin - CYP1A
- Diclofenac - CYP2C9
- Bufuralol - CYP2D6
- Testosterone - CYP3A4
- 7-Hydroxycoumarin - UGT (s)
Protein Expresion
UKHTB can also provide cryopreserved hepatocytes fro use in protein expression studies. Please contact us for more information.
All researchers wishing to receive human tissue from UKHTB must undergo approval through an application to UKHTB and our Scientific Review Board and a site visit and a Tissue Transfer Agreement must be in place. Please contact us for an application form and more details on this process.
