An Improved Time- and Labor- Efficient Protocol for Mouse Primary Hepatocyte Isolation

Summary

Primary hepatocytes are a valuable tool to study liver response and metabolism in vitro. Utilizing commercially available reagents, an improved time- and labor-efficient protocol for mouse primary hepatocyte isolation was developed.

Abstract

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on different needs, like time, labor, cost, and primary hepatocyte usage, resulting in various primary hepatocyte isolation protocols. However, the numerous steps and time-consuming reagent preparations in primary hepatocyte isolation are major drawbacks for efficiency. After comparing different protocols for their pros and cons, the advantages of each were combined, and a rapid and efficient primary hepatocyte isolation protocol was formulated. Within only ~35 min, this protocol could yield as much, if not more, healthy primary hepatocytes as other protocols. Further, glucose metabolism experiments performed using the isolated primary hepatocytes validated the usefulness of this protocol in in vitro liver metabolism studies. We also extensively reviewed and analyzed the significance and purpose of each step in this study so that future researchers can further optimize this protocol based on needs.

Introduction

The liver serves as one of the most important organs in the vertebrate body due to the vital role it plays in numerous life-supporting functions like food digestion, blood circulation, and detoxification. Usage of mouse primary hepatocyte in vitro culture is increasingly popular in studies of carbohydrate metabolism and hepatic carcinoma. Therefore, it is important to develop a convenient method for mouse primary hepatocyte isolation while maintaining its innate physiological function. Due to its function as a hub of glucose metabolism, the liver is also central to glucose production and storage¹. Experiments with primary hepatocytes <....

Protocol

All procedures were approved by the Johns Hopkins Animal Care and Use Committee. C57BL/6 female mice (8-week old) were used in this study.

1. Preparation:

1. Mix William's E Medium (GlutaMAX Supplement) with 10% FBS and 1% antibiotic-antimycotic solution to make the culture medium.
2. Filter 25 mL of collagenase-dispase medium (e.g., Liver Digest Medium) through a 0.45 µm syringe filter to remove particle debris.
3. Warm 50 mL of double-distilled H₂O (ddH₂O), 35 mL of perfusion Medium (e.g., Liver Perfusion Medium) (or 50 mL at the first time using this protocol), and 25....

Results

In order to test the efficiency, the present primary hepatocyte isolation protocol was performed on 8-week old female C57BL/6 mice. The attachment and purity of isolated primary hepatocytes were tested. Primary hepatocyte isolation is used for a wide variety of experiments on liver physiology, such as hepatic drug effects and glucose metabolism, pharmaceutical biomarker activity², insulin sensitivity, and glucose production. Therefore, the activities of the primary hepatocytes isolated with this p.......

Discussion

Various primary hepatocyte isolation protocols have been developed. They also have been kept optimized and adapted based on different needs (Table 1). Isolation protocols are generally composed of two parts: perfusion (including enzyme digestion) and purification.

The perfusion can be performed with the entire liver in vivo^2,20,21,22,

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1x PBS	Gibco	10010023
10x HBSS	Gibco	14065-056
12-well Plate	FALCON	353043	Coating not required
6-well Plate	FALCON	353046	Coating not required
anti-AKT	Cell Signaling	2920S
Antibiotic Antimycotic Solution (100x), Stabilized	Sigma-Aldrich	A5955
anti-FOXO1	Cell Signaling	97635S
anti-GAPDH	Cell Signaling	2118S
anti-p-AKT (S473)	Cell Signaling	9271L
anti-PEPCK	Santa Cruz	SC-166778
anti-p-FOXO1 (S256)	Cell Signaling	84192S
Cell Strainer, 70 µm	CELLTREAT	229483
Closed IV Catheter, 24 Gauge 0.75 IN	Becton Dickinson	383511
DMEM, no glucose, no glutamine, no phenol red	ThermoFisher Scientific	A1443001
EnzyChrom Glucose Assay Kit	BioAssay Systems	EBGL-100
Fetal Bovine Serum (FBS)	Hyclone	SH30071.03
Forskolin	MilliporeSigma	F3917-10MG
Glucagon	Sigma-Aldrich	G2044
Goat Anti-mouse IgG Secondary Antibody	LI-COR	926-68070
Goat Anti-rabbit IgG Secondary Antibody	LI-COR	926-32211
GraphPad Prism 8	GraphPad Software	NA
Hepatocyte Wash Medium	Gibco	17704-024
IBMX	Cell Signaling	13630S
Insulin	Lilly	NDC 0002-8215-01
Ketamine HCL (100 mg/mL)	Hospira Inc	NDC 0409-2051-05
L-Glutamine	Gibco	25030081
Liver Digest Medium	Gibco	17703-034	Aliquot within tissue culture hood to 25 mL each in 50 mL tube, and keep in -20 °C freezer
Liver Perfusion Medium	Gibco	17701-038
Pen Strep	Gibco	15140122
Percoll	GE Healthcare	17-0891-01
Peristaltic Pump	Gilson	Minipuls 2	Capable of pumping at 4 mL/min
Petri Dish	Fisherbrand	08-757-12
Refrigerated Centrifuge	Sorvall	Legend RT	Capable to centrifuge 50 mL tube at 4 °C
Sodium L-Lactate	Sigma-Aldrich	L7022
Sodium Pyruvate	Gibco	11360070
Syringe Filter, PVDF 0.45 µm 30mm diameter	CELLTREAT	229745
Syringe, 0.5 mL	Becton Dickinson	329461
Syringe, 60 mL	Becton Dickinson	309653
Trypan Blue Solution, 0.4%	Gibco	15250061
Tube, 15 mL	Corning	430052
Tube, 50 mL	Corning	430290
Water Bath Tank	Corning	CLS6783	Or any water bath tank capable of heating up to 45 °C
William’s E Medium (GlutaMAX Supplement)	Gibco	32551020
Xylozine (100 mg/mL)	Vetone Anased LA	NDC13985-704-10