Method for isolating and culturing human primary hepatocytes
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A technique for the isolation and cultivation of primary hepatocytes, which is applied in the biological field of in vitro culture of hepatocytes, can solve the problems of long time required, easy pollution, and many operating procedures, and achieve the effects of saving cost, saving use, and low cost
Inactive Publication Date: 2011-05-18
ZHUJIANG HOSPITAL SOUTHERN MEDICAL UNIV
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However, this method is not only expensive for the perfusion device, but also has many operating links, high technical requirements, long time required, and easy contamination, and it is not suitable for human liver tissue obtained by surgical resection, so this method is limited in general laboratories. And the application of human primary hepatocytes in medicine
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Embodiment 1
[0023] Embodiment 1: The specific steps of the method for the isolation and culture of primary human hepatocytes from human liver tissue obtained by surgical resection are as follows:
[0024] 1. After informed consent, take the normal liver tissue (approximately 3cm × 2cm × 1cm, about 8 grams) removed from the patient undergoing hepatectomy, and quickly put it into the liver tissue preservation solution DMEM (high sugar) + 1% bismuth Anti-(penicillin, streptomycin, that is, the working concentration of penicillin is 100U / ml, and the working concentration of streptomycin is 100ug / ml) in a sterile centrifuge tube, put it in an ice box (temperature 0 ~ 4 ℃) and bring it back to the experiment room;
[0025] 2. Take out the centrifuge tube containing the liver tissue block, wipe the surface with alcohol cloth for disinfection, and put it into the ultra-clean bench; take out the tissue block with sterile forceps, put it in a petri dish, and clean the liver with D-Hank's solution a...
experiment example 1
[0037] Experimental Example 1: Trypan blue staining to detect freshly isolated primary human hepatocytes. Trypan blue is mainly used to identify the survival of primary cultured cells after separation. Stain directly with 0.4% trypan blue under a microscope Visual observation is enough, live cells are not stained.
[0038] First prepare 1.4% trypan blue mother solution, weigh 4g trypan blue, add a small amount of distilled water to grind, add double distilled water to 100ml, filter with filter paper, store at 4°C, and dilute to 0.4% with PBS when used; the staining steps are:
[0039] 1. Prepare a single-cell suspension from the freshly separated human primary hepatocytes obtained in the above step 6, suspend the cells with DMEM high-glucose solution, and dilute appropriately (10 5 cells / ml);
[0040] 2. Staining: Take 1 drop of cell suspension with a pipette and drop it on the glass slide, add 1 drop of 0.4% trypan blue solution, and mix for 3 minutes;
[0041] 3. Observe. ...
experiment example 2
[0043] Experimental Example 2: Calculation of cell viability in trypan blue exclusion experiment, take a drop of cell suspension and drop it on a glass slide, trypan blue exclusion, observe under a microscope, count 200 cells, cell viability = (number of live cells / 200)×100%, the result of counting in this experiment is that the number of live cells is 180, that is, the cell viability is 90%.
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Abstract
The invention provides a method for isolating and culturing human primary hepatocytes, comprising the following steps: cleaning blood clots on surfaces of hepatic tissues by using D-Hank's solution and cutting off connective tissues; using a needle syringe for puncturing on multipoints to perfuse anterior perfusion solution preheated to 38 DEG C so that a hepatic tissue block changes from dark red to gray and the effluent anterior perfusion solution becomes clear; using the needle for puncturing on multipoints to perfuse preheated recyclable type II collagenase solution until the hepatic tissue block is loosened and inelastic and has turtleback cracks on the surface; scissoring the hepatic tissue block, passively isolating the hepatic tissues, removing residual envelops and fibrillar connective tissues and continuously shaking and digesting in the type II collagenase solution at 37 DEG C; blowing and beating digest into hepatocyte suspension, filtering the suspension under ice bath condition, collecting filtered suspension, transferring the filtered suspension to a centrifuging tube and centrifuging at low speed; adding bottom precipitate produced from the primary low-speed centrifuging of the filtered suspension to erythrocyte lysate, blowing and beating, standing at room temperature, adding Dulbecco's modified eagle medium (DMEM), mixing the mixture uniformly, cleaning, centrifuging at low speed, adding DMEM again, mixing uniformly, centrifuging at low speed for 2-4 times and adding bottom precipitate produced from the last-time low-speed centrifuging to Williams' MediumE complete medium suspension; adjusting the hepatocytes density to 1*105/ml, inoculating the hepatocytes in a culture bottle laid with rat tropocollagen, carrying out constant culturing in a CO2 incubator at 37 DEG C, changing the suspension to remove died hepatocytes and non-adherent hepatocytes after the hepatocytes are adhered to the wall and continually culturing the hepatocytes.
Description
technical field [0001] The invention relates to the technical field of in vitro culture of hepatocytes, in particular to a stable and convenient method for isolating and culturing primary human hepatocytes. Background technique [0002] Hepatocytes have been widely used in medical related fields. For a long time, many scholars at home and abroad have conducted a lot of research on the isolation methods and culture conditions of primary hepatocytes. Difficult, especially for primary human hepatocytes. The early methods of separating hepatocytes were mainly non-enzymatic separation of hepatocytes, including mechanical methods (such as homogenization) and chelation methods. However, the mechanical method has great damage to the liver cells, and the viability of the isolated liver cells is low; the chelation method uses Ca 2+ , Mg 2+ The chelating agent (such as citrate or EDTA) is used to separate hepatocytes, but the effect of chelating agent alone is not good, and it is of...
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