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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 in vitro are a must to most glucose metabolism studies. Therefore, for years, various research groups have developed protocols for mouse primary hepatocyte isolation.

The general procedure of mouse hepatocyte isolation is to first flush out blood in the liver with an isosmotic liquid such as phosphate-buffered saline (PBS) or Hanks' Balanced Salt Solution (HBSS) and then use collagenase-containing solution to dissociate hepatocytes. These protocols share a general procedure but differ in reagents and steps based on different needs. However, preparing required reagents and performing isolation steps take time. In developing the present protocol, efficiency was set as a priority, with all reagents ready-to-use and available from the market, and as few steps as possible. The overall goal of this protocol is to provide a fast and labor-efficient method to isolate primary hepatocytes from mouse, without jeopardizing the isolated primary hepatocyte purity and viability.

Protokół

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 mL of filtered collagenase-dispase medium in a 45 °C water bath for 30 min.
4. Within a sterile tissue culture hood, mix 2 mL of 10x HBSS and 18 mL of Percoll in a 50 mL tube to make 20 mL 1x Percoll-HBSS and keep on ice or 4 °C.
   NOTE: 1x Percoll-HBSS can be kept at 4 °C for at least 6 months.
5. Within a sterile tissue culture hood, pour 30 mL of wash medium (e.g., Hepatocyte Wash Medium) into a clean Petri dish, and keep on ice.
6. Submerge the pumping tube in the water of a 45 °C water bath. Results are most reliable if the room temperature is at 25 °C.
7. Prepare 2 mL of 1x anesthetics by mixing 225 µL of Ketamine HCL, 93.75 µL of Xylazine, and 1681 µL of 1x PBS.
8. Anesthetize one mouse using an approved method. Here the mouse was intraperitoneally injected with 150 µL of 1x anesthetics. Perform the tests for loss of reflexes such as reaction to toe pinching to ensure full anesthesia.
9. Secure the mouse on its back onto the dissection pad by four limbs, by either pining or using water-proof tape or other methods approved by the institution's Animal Care and Use Committee (or equivalents).
10. Prepare sterilized forceps and scissors for dissection. To avoid possible contamination, conduct all steps within a sterile hood.

2. Procedure:

1. Using a peristaltic pump, start pumping warmed-up ddH₂O at a speed of 4 mL/min for 5 min. Change the pumping tube from water to a warmed-up perfusion medium.
2. Disinfect the anesthetized mouse's abdomen with 70% EtOH and cut open with a scissor to expose the liver, portal vein, and inferior vena cava (IVC).
3. Stop the peristaltic pump. Insert a 24 G catheter (e.g., Closed IV Catheter, 24 G, 0.75 IN) into IVC. Start pumping and cut the portal vein open.
4. Continue pumping until the flushed-out liquid is clear (around 3-5 min). Press the portal vein every minute to let liquid reach every corner of the liver. Be careful not to let air bubbles enter the IVC.
   NOTE: This step is to flush out as much blood as possible from the liver.
5. Change the pumping tube from the perfusion medium to the collagenase-dispase medium. Continue pumping until all 25 mL of the collagenase-dispase medium is depleted while doing step 2.6.
6. Press the portal vein every minute to let liquid reach every corner of the liver.
   NOTE: At this stage, the complete loss of blood is fatal to the mouse. The death of the mouse can be confirmed by a lack of heartbeat after the experiment. Dispose of the carcass as per facility policies.
7. Isolate the whole liver out without gallbladder to the 30 mL wash medium in the Petri dish on ice.
8. Tear it up into pieces with forceps to release primary hepatocytes into solution. This step would turn the wash medium into a cloudy solution full of released primary hepatocytes and small liver pieces.
9. Filter the cloudy solution in step 2.8 through a 70 µm cell strainer into the 20 mL 1x Percoll-HBSS in a 50 mL tube on ice. Mix by inverting the tube 20 times.
10. Centrifuge at 300 x g for 10 min at 4 °C.
11. Within the tissue culture hood, aspirate the supernatant. Wash the pellet with cold 30 mL of wash medium.
12. Centrifuge at 50 x g for 5 min at 4 °C.
13. Remove the supernatant and resuspend the pellet in 25 mL of the culture medium (or appropriate other volumes) within the tissue culture hood.
14. Count the cell number and plate the cells on desired culture plates according to the experimental design.
    NOTE: Primary hepatocytes properly isolated from one 8-week-old mouse are usually sufficient to be plated on four 6-well plates or four 12-well plates.

Wyniki

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...

Dyskusje

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,

Materiały

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