Human Hepatocyte ApplicationsRecommended Products

Use a collagenase supplemented with a neutral protease for human hepatocyte applications.

The recommended formulation for a purified enzyme mixture to isolate human hepatocytes was based on the biochemical analysis of a “good lot” of Sigma Type XI (Lot 044K8638) that the Stephen Strom’s laboratory at University of Pittsburgh used for human hepatocyte isolation. Further characterization of cells isolated with the purified enzyme mixture or the Lot 044K8638 showed there was no difference in the characteristics of the recovered hepatocytes.(1)

Modified Seglen’s Two-Step Isolation MethodRecommended Method

Human hepatocyte isolation uses the same steps that Seglen applied in his description of a two step process for rat hepatocyte isolation.(2) The primary difference between rat and human hepatocyte isolation is the handling of the liver tissue. The three common sources of tissue are:

RESECTED SURGICAL TISSUE

Surgically resected liver tissue which is discarded as part of a surgical procedure.

The use of surgically resected liver tissue requires two additional steps before following the Seglen cell isolation procedure.

LEFT LOBE

The left lobe of the unused liver from an organ donor which is smaller than the right lobe of the liver.

The same steps of the Seglen procedure are used for digesting the left liver lobe (i.e., the smallest lobe).

ENTIRE LIVER

The entire liver from an unused liver from an organ donor.

The same steps of the Seglen procedure are used for digesting the entire organ.

RESECTED SURGICAL TISSUE EXTRA STEPS

First, the resected tissue should only have one cut surface which should be sealed with medical glue to prevent uncontrolled leakage of the enzyme solution from the tissue.

Second, several blood vessels on the cut surface of the tissue must be evaluated for their ability to perfuse balanced salt solution to the entire tissue. After these vessels are selected, a cannula is surgically tied into each vessel. Any vessel that gave poor perfusion are surgically tied off. The enzyme solution delivered into the tissue via the cannula by using a syringe.

Several reports described human hepatocyte isolation in detail.(3-5) VitaCyte sponsored a webinar on “Collagenase selection and process optimization for high yield hepatocyte isolation” presented by Raf Witek. This webinar provides more insights into this procedure than found in the cited publications.

The recommended enzyme formulation for human hepatocyte isolation uses 2500 collagen degradation activity units (CDA U) of Collagenase MA and 2200 neutral protease activity (NPA) U per mL of enzyme solution.(1) This formulation has been routinely used by several commercial suppliers of cryopreserved human hepatocytes.

Expected Cell Yield



Human Adult Resected Tissue
30-40 million hepatocytes per g tissue



Human Pediatric Resected or Intact
50-75 million hepatocytes per g tissue

Source: Collagenase Selection and Process Optimization for High Yield Hepatocyte Isolation

Perspectives

Hepatocyte yields are impacted by the warm and cold ischemia time of the excised tissue or organ. The ideal source is to recover hepatocytes from resected human liver surgical tissue. However, this requires careful handling of the tissue once it is handed off to pathology for evaluation. Recovery of hepatocytes from unused donor livers is usually ≥ 24 hours. The lower viabilities of the recovered cells leads many end users to use a Percoll separation step to remove dead cells.(6)

References

Gramignoli R, Green M, Tahan V, Dorko K, Skvorak K, Marongiu F, et al. Development and application of purified tissue dissociation enzyme mixtures for human hepatocyte isolation. Cell Transplantation. 2012;21:1245-60.
Seglen PO. Preparation of isolated rat liver cells. Methods Cell Biology. 1976;13:29-83.
Gomez-Lechon MJ, Donato T, Ponsonda X, Fabra R, Trullenque R, Castell JV. Isolation, culture and use of human hepatocytes in drug research. In vitro Methods Pharmceutical Research. 1997:129-53.
Bayliss M, Somers G. Isolation and culture of human hepatocytes. Methods in Molecular Medicine (Human Cell Culture Protocols). 2004 2004;107:249-67.
LeCluyse EL, Alexandre E, Hamilton GA, Viollon-Abadie C, Coon DJ, Jolley S, et al. Isolation and culture of primary human hepatocytes. Methods Molecular Biology. 2005;290:207-29.
Horner R, Gassner J, Kluge M, Tang P, Lippert S, Hillebrandt K, et al. Impact of Percoll purification on isolation of primary human hepatocytes. Scientific Reports. 2019;9:6542.