Z Gastroenterol 2005; 43 - P232
DOI: 10.1055/s-2005-920012

Evaluation of cryopreserved human hepatocytes for functional studies in vitro and in a small animal model

KL Streetz 1, M Elazar 2, S Levenberg 3, MS Chua 4, D Wang 5, J Glenn 2, S So 4, J Ludlow 6, M Kay 1
  • 1Dept. of Pediatrics & Genetics, Stanford University, Stanford, Ca, USA
  • 2Dept. of Gastroenterology and Hepatology, Stanford University, Palo Alto, California, USA
  • 3Department of Chemical Engineering, MIT, Cambridge, MA, USA
  • 4Dept. of Surgery, Stanford University, Stanford, CA, USA
  • 5Dept. of Mechanical Engineering, Stanford University, Stanford, Ca, USA
  • 6Vesta Therapeutics, Inc., Durham, NC, USA

Cryopreserved human hepatocytes (CHH) are an attractive cell source for hepatocyte transplantations as they can be used on demand. Here we evaluated these cells for research purposes in vitro and in a small animal model.

Plating efficiency varied greatly between analysed batches, however succesfully attached cells then stayed alive and expressed hepatocyte specific markers for up to 7 days. To test infectability with human hepatotrophic viruses we incubated the CHH with infectious Hepatitis-D positive serum. Whereas freshly isolated human hepatocytes showed nuclear staining of HDV-core antigen in >50% of the cells, only a single cell in all tested CHH displayed a positive anti-HD staining.

Next we transplanted the cryopreserved hepatocytes into NOD-SCID mice whether directly intraspleenically or into extrahepatic sites. Additionally CHH were seeded onto biodegradable scaffolds before transplantation. To monitor hepatocyte survival we measured human-alpha1antitrypsin level (hAAT) in the serum of the transplanted mice over time. HAAT became undetectable as soon as 10d after intraspleenic injection in untreated mice (15d in mice pretreated with an liver injuring Adenovirus (Ad-uPA). In contrast to this we obtained long term survival (up to 4 month) of matrigel embedded cells transplanted into subcutaneous pockets and of the scaffold-pre-seeded cells, althought hAAT levels initially declined rapidly as well until reaching a plateau at around 50–100ng/ml. We confirmed the longterm survival of the transplanted hepatocytes immunhistochemically by hAAT staining.

Summary: CHH stably expressed markers of human hepatocytes in vitro and in vivo. Surprisingly we have not been able to detect significant infectability with HDV, thus potentially limiting the use CHH to investigate human hepatotrophic viruses. Interestingly we obtained significant better long term survival of cryopreseved human hepatocytes transplanted at extrahepatic sites compared to the direct intraspleenic injections. Transplantation at ectopic sites might be an alternative approach in creating needed xenotransplant models for human hepatocytes if transplant conditions can be improved by things as local delivery of growth factors and further modification of the co-transplanted biomatrices.

Keywords: hepatocyte transplantation