Protocol for Primary Mouse Hepatocyte Isolation

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Associated Data

Methods Video S1. Mouse Positioning and Preparations for Cannulation The anesthetized mouse is positioned on the dissection tray. The mouse fur and skin are cut in a “U” shape. Mouse intestine and the rest of viscera are moved to the right and both the portal vein and vena cava are revealed. Steps: 7–10.

Methods Video S2. Liver Cannulation and Perfusion The inferior vena cava is cannulated and the liver is perfused to wash out blood and circulating cells as well as to eliminate calcium via EDTA. Steps: 11–15.

Methods Video S3. Liver Digestion Collagenase (Liberase) is perfused to the liver to facilitate hepatocyte dispersion. Steps 16–19.

Methods Video S4. Liver Dissection The liver is dissected out gently by removing all connections to other organs. Step 20.

Methods Video S5. Hepatocyte Purification The liver is removed to a 10 cm plate and is punctured repeatedly. Liver cells are released to the media and filtered. Steps 22–24.

This study did not generate any unique datasets or code.

Summary

Primary hepatocytes are a vital tool in various biomedical research disciplines, serving as an ex vivo model for liver physiology. Obtaining high yields of viable primary mouse hepatocytes is technically challenging, limiting their use. Here, we present an improved protocol based on the classic two-step collagenase perfusion technique. The liver is washed by perfusion, hepatocytes are dissociated by collagenase, separated from other cells, and cultured. This protocol was optimized to significantly reduce procedure duration and improve hepatocyte yield and viability.

Graphical Abstract

Highlights

Primary hepatocytes are a vital tool in various biomedical research disciplines, serving as an ex vivo model for liver physiology. Obtaining high yields of viable primary mouse hepatocytes is technically challenging, thereby limiting their use. Here, we present an improved protocol based on the classic two-step collagenase perfusion technique. The liver is washed by perfusion, hepatocytes are dissociated by collagenase, separated from other cells, and cultured. This protocol was optimized to significantly reduce procedure duration and improve hepatocyte yield and viability.

Before You Begin

Timing: 20 min. (plus incubation for 4–16 h)

Note: Steps 1–4 should be done in sterile conditions, in a biological hood (i.e., biosafety cabinet)

Prepare cell culture plates; 12-well, 6-well, 10 cm, and 15 cm plates are suitable for hepatocyte plating.

Note: Lower diameter plates may be suitable but are less optimal due to decreased efficiency of cell dispersion across the well surface.

Note: Collagen-coated plates can be prepared days in advance provided they are stored under sterile conditions.

Key Resources Table

Materials and Equipment

Collagen Solution (50 mL)

Note: Due to viscosity of collagen, we recommend doing a serial dilution (e.g., diluting collagen 1:100 and then diluting it again 1:100)

EGTA can be used as alternative to EDTA. We have found no difference in yield between the two chelating agents.

Note: The final pH at 37°C should be 7.4

Note: Many protocols add dexamethasone, insulin, transferrin and selenium to maintenance media. We found that this is not needed in short-term culturing. Importantly, these reagents profoundly affect hepatocyte biology (Batista et al., 2019; Goldstein et al., 2013; Lin et al., 2007; Weiller et al., 2004) and thus may affect experiment outcome.

Liberase Stock Solution

The preparation of Liberase solution detailed here relates to preparation of stock concentration from powder. The stock is further diluted to a final concentration of 25 μg/mL during the procedure (step 5).

We found that Liberase, a specific type of collagenase is significantly more reproducible than other commercial collagenases.

Aliquot and store at −80°C. We have found that using aliquoted and frozen Liberase (with one or two freeze-thaw cycles) does not substantially affect enzyme activity (in contrast to other types of collagenase). Thus, there is no need to freshly prepare a Liberase solution.

Note: Percoll solution should be prepared fresh during the procedure.

Step-By-Step Method Details

This protocol is aimed at isolating hepatocytes from mouse liver. Following anesthesia, the vena cava is cannulated and the liver is perfused to chelate calcium and wash out blood. Then, collagenase is perfused to the liver in order to dissociate extracellular matrix. Finally, the liver is dissected and hepatocytes are purified by density-based separation. This protocol presents several advances over similar protocols (Berry and Friend, 1969; Casciano, 2000; Klaunig et al., 1981; Li et al., 2010; Renton et al., 1978; Seglen, 1976; Severgnini et al., 2012). The main improvements of this protocol are better reproducibility, shortened duration, reduced technical challenge, increased yield and higher viability. These are achieved by several steps we altered or optimized. For example: (a) We found that retrograde perfusion through the vena cava permits easier cannulation as opposed to portal vein cannulation. (b) Periodical clamping of the portal vein provides a visible checkpoint for proper perfusion and greatly facilitates efficient washing and digestion. (c) The type of collagenase used (Liberase) shows quicker digestion and better reproducibility compared to other collagenases. (d) Percoll-based density separation results in a population of purified hepatocytes of high viability. Some of these protocol improvements were already implemented in our previous publications (Goldstein et al., 2017a; Goldstein et al., 2017b) where we isolated hepatocytes for experiments demanding a high yield of cells (such as chromatin immunoprecipitation sequencing – ChIP-seq).

Pump Preparation and Mouse Anesthesia

Here, the pump is washed and primed with perfusion buffer. The mouse is anesthetized and positioned on the dissection tray.

This section is shown in Methods Video S1.