Rat liver stellate cell culture experiment

Summary

Primary cultures of rat hepatic stellate cells can be (1) used for cell preservation, (2) used for molecular biology research, and (3) used for gene therapy research.

Operation method

Cell Culture Technology

Principle

Hepatocytes from mice are removed from the organism, treated with trypsin, chelating agent (commonly used EDTA), dispersed into single cells, and cultured in a suitable medium to allow the cells to survive, grow, and multiply.

Materials and Instruments

Male SD rats
Sodium pentobarbital Calf serum DMEM Enzyme digest I and II Nycodenz D-Hanks' solution HBSS Taipanlan
Surgical instruments Nylon mesh Phase contrast microscope Fluorescence microscope

Move

I. Experimental steps

1. Rats were dissected on an ultra-clean table after intraperitoneal anesthesia, and portal vein cannulation was performed.2. D-Hanks' solution was used for perfusion, while the inferior vena cava was clipped and washed out of blood, and then 100 ml of enzyme digest I (0.05% type IV collagenase) was used instead.3. After the liver was softened, the liver was isolated and minced, and then continued to be digested with enzyme digestion solution II (0.05% collagenase IV containing 20U/ml DNase I) for 15 min, filtered through a nylon mesh, and then centrifuged at 450 g for 5 min (4℃, the same hereinafter).4. Resuspend the precipitate with HBSS to 10 ml, mix with 20 ml of Nycodenz solution, place in centrifuge tubes and cover with 2-3 ml of HBSS, and centrifuge at 1450 g for 16 min before removing the cells from the interface.5. Collect the cells by centrifugation again after resuspension with HBSS, and resuspend them with DMEM for cell counting and determining the survival rate and yield, and finally inoculate them according to the conventional method ( ^{105 cells/cm2 )} , change the solution on the next day, and change the solution every 2-3 days thereafter.6. Succession method: after discarding the culture solution, wash twice with D-Hanks' solution, add 0.125% trypsin (0.05% EDTA is preferred for the original generation) to digest, observe the retraction of cell protrusions under the microscope (about 2-5 min), terminate the reaction with complete culture solution (DMEM+10% NBS) (if EDTA is not used, centrifugation can be dispensed with), and then blow the cells sufficiently (1:2-1:3). The reaction was terminated with complete culture solution (DMEM+10% NBS) (if EDTA was not used, centrifugation could be dispensed with), and then the cells were inoculated with 1:2-1:3 after sufficient blowing, and then continued to be cultured (5% _CO2, 37℃).

Results

On the next day of inoculation, the cells were stellate with lipid droplets in the cytoplasm under light microscope, and autofluorescence was seen at 328 nm under fluorescence microscope. Photographs were attached (Figure 1).

Figure 1. Rat hepatic stellate cells on the 2nd day of primary culture.

Caveat

1. Immunohistochemistry is required for further identification: Desmin and α-SMA; the latter is expressed after cell activation.

2. using a protocol that does not require in situ digestion, which is perfectly feasible, except that the digestion time is more difficult to control! Experiments were performed using cells cultured in situ or within 9 generations after passaging (preferably within 5 generations).

Common Problems

Electron microscopy: on the second day of inoculation, the cells were seen to be stellate with lipid droplets in the cytoplasm under light microscopy and autofluorescence at 328 nm under fluorescence microscopy.

I. DISCUSSION

Immunohistochemistry is required for further identification: desmin and α-SMA; the latter is expressed after cell activation. A protocol without in situ digestion has also been used, which is perfectly feasible, except that the digestion time is more difficult to control! Experiments are performed on primary cultured cells or on cells within 9 generations after passaging (preferably within 5 generations).

Literature

1. Yuan Taohua, Zhang Jinsheng, Zhang Yue'e, et al. In vitro culture of rat liver Ito cells and the inhibitory effect of heparin on them. Journal of Shanghai Medical University,1996,23(2):90-93.

2. Huang GC, Zhang JS, Tang QQ. Involvement of C/EBP-α; gene in in vitro activation of rat hepatic stellate cells. Biochem Biophys Res Commun,2004,324(4) :1309-1318. :1309-1318.

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