Improved isolation of murine hepatocytes for in vitro malaria liver stage studies.
BACKGROUND
Primary hepatocyte cultures are a valuable tool for the understanding of cellular and molecular phenomena occurring during malaria liver stage. This paper describes an improved perfusion/dissociation procedure to isolate hepatocytes from mouse liver that is suitable for malaria studies and allows reproducible preparation of primary hepatocytes with consistent cell yields and controlled purity.


RESULTS
This protocol is a detailed description of a technique to isolate and culture mouse hepatocytes and represents an improvement over previous descriptions of hepatocyte isolation for malaria studies, regarding three technical aspects: (1) dissociation reagents choice; (2) cell separation gradient and (3) cell purity control. Cell dissociation was optimized for a specific collagenase digestion media. The cell dissociation step was improved by using a three-layer discontinuous gradient. A cell purity check was introduced to monitor the expression of CD95 on hepatocytes using flow cytometry methods.


CONCLUSIONS
The procedure described allows reproducible recovery of one to three million hepatocytes per preparation with cell purity of about 90% as determined by FACS analysis. Completion of the protocol is usually achieved in about four hours per preparation and pooling is suggested for multiple preparations of larger number of cells.
Gonçalves
Lígia A
LA
Instituto Gulbenkian de Ciência, Rua da Quinta Grande, 6, 2781-901 Oeiras, Portugal. ligdeus@igc.gulbenkian.pt
Vigário
Ana M
AM
Penha-Gonçalves
Carlos
C
D000818:Animals; D018929:Cell Culture Techniques; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D002478:Cells, Cultured; D017364:Collagenases / Q000378:metabolism; D004210:Dissection; D005434:Flow Cytometry / Q000295:instrumentation; D022781:Hepatocytes / Q000166:cytology / Q000469:parasitology*; D008288:Malaria / Q000473:pathology*; D051379:Mice; D010477:Perfusion; D010961:Plasmodium; D011205:Povidone; D012822:Silicon Dioxide; D014162:Transfection
C016039:Percoll; D012822:Silicon Dioxide; D017364:Collagenases; D011205:Povidone