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Method for separating primary adult hepatocytes, and special sterile apparatus box thereof

A hepatocyte and instrument box technology, applied in artificial cell constructs, animal cells, vertebrate cells, etc., can solve the problems of unable to meet the huge demand of human hepatocytes, unable to implement liver perfusion, and difficult to find perfusion blood vessels, etc. The effect of reducing tedious preparation work, simple structure and convenient use

Inactive Publication Date: 2011-10-19
SOUTHERN MEDICAL UNIVERSITY
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  • Summary
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  • Application Information

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Problems solved by technology

However, at present, the donor livers for liver transplantation are extremely scarce, and the donor livers discarded due to incompatible types are even scarcer, completely unable to meet the huge demand for human hepatocytes in basic research
With the development of surgical techniques, the normal liver tissue resected along with the lesion has gradually become smaller, and most of the obtained normal liver tissue blocks are extremely irregular. cell isolation

Method used

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  • Method for separating primary adult hepatocytes, and special sterile apparatus box thereof
  • Method for separating primary adult hepatocytes, and special sterile apparatus box thereof
  • Method for separating primary adult hepatocytes, and special sterile apparatus box thereof

Examples

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Effect test

Embodiment 1

[0037] The setting of embodiment one disposable aseptic instrument box

[0038] figure 1 An embodiment of the disposable kit for isolating primary human hepatocytes shown is composed of an outer packaging bag 1 , a box body 4 and operating instruments, and the box body 4 is set in the outer packaging bag 1 . Operating equipment includes 10cm sterile Petri dish 2, 100ml sterile beaker 7, 20ml sterile beaker 8, 25ml sterile serum bottle 3, 50ml sterile triangular beaker 9 and 50ml sterile triangular beaker 10, 200 mesh sterile nylon filter 14 and 100 mesh sterile nylon filters 5, 10ml sterile syringes 15 and 20ml sterile syringes 11, sterile forceps 6 and sterile scissors 13, two 15ml centrifuge tubes 12. The box body 4 is provided with a plurality of grooves matching the shape and volume of the above-mentioned operating instruments, and each of the above-mentioned operating instruments is correspondingly embedded in the grooves in the corresponding box body 4 .

[0039] These...

Embodiment 2

[0040] Example 2 Isolation and Culture of Primary Adult Hepatocytes

[0041] DMEM medium: prepared according to the formula in "Cell Culture" (Situ Zhenqiang, Wu Junzheng editor-in-chief. Xi'an: World Book Publishing Company, 2007. pp. 50-52).

[0042] The composition of preperfusate contains NaCl 8g / L, KCl 0.4g / L, NaCl 2 HPO 4 12H 2 O 0.12g / L, KH 2 PO 4 0.06g / L, NaHCO 3 0.35g / L, glucose 1.0g / L, HEPES 2.38g / L, EDTA 0.74g / L.

[0043] The type IV collagenase solution is prepared by dissolving the type IV collagenase in DMEM medium, and the concentration of the type IV collagenase is 0.05-0.1%. The amount of type IV collagenase solution can be determined according to the size and weight of liver tissue. However, in order to save resources, it has been proved by experiments that 15ml of type IV collagenase solution is used when the weight of liver tissue is 1.0-3.0g, and 20ml of IV collagenase solution is used when the weight of liver tissue is 3.1g-4.0g. Type IV collagena...

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Abstract

The invention discloses a method for separating primary adult hepatocytes. The method comprises the following steps: (1) carrying out multi-point puncture on surface of isolated adult hepatic tissue through a needle syringe, and injecting a preperfusate, wherein connective tissues are removed from the isolated adult hepatic tissue; (2) carrying out the multi-point puncture on the surface of the isolated adult hepatic tissue through the needle syringe again, and injecting a IV collagenase solution; (3) separating the isolated adult hepatic tissue, followed by adding the IV collagenase solution and carrying out digesting through vibration at a temperature of 37 DEG C to obtain digest; (4) carrying out filtering for the digest, followed by centrifuging and collecting cell aggregate in the underlayer, then resuspending the adult hepatocyte aggregate through a hepatocyte wash buffer, followed by filtering and centrifuging, then abandoning supernatant and collecting the adult hepatocyte aggregate in the underlayer; (5) washing the adult hepatocyte aggregate in the underlayer from the step (4) through a serum-free DMEM medium to obtain the primary adult hepatocytes. The invention further discloses a disposable special sterile apparatus box for separating the primary adult hepatocytes. With the present invention, the disposable special sterile apparatus box is adopted, the primary adult hepatocytes are separated through the multi-point puncture on the surface of the tissue and the injection, such that operation is simplified, cost is reduced, and the method and the apparatus box are applicable for extracting the hepatocytes from small pieces of the irregular isolated adult hepatic tissues of recovery of liver resection.

Description

technical field [0001] The invention relates to a method for isolating primary cells, in particular to a method for isolating primary adult hepatocytes, and also relates to a special aseptic instrument box for isolating primary adult hepatocytes. Background technique [0002] There are many methods for separating hepatocytes, such as mechanical separation, chelation and enzymatic digestion. In 1956, Sorrentino used the mechanical method to separate fresh liver tissue for the first time, but the hepatocytes obtained by the mechanical separation method had a small number of cells and poor activity, so they were gradually abandoned. In 1967, Howard created the collagenase perfusion method (Howard RB, Pesch LA. Preparation and partial characterization of intact isolated parenchymal cells from rat liver. Biol Chem, 1968, 243: 3105-3114), and in 1972, Seglen further developed this method It is a two-step perfusion method (Seglen PO. Preparation of isolated rat liver cells. Method...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12M1/00
Inventor 刘思德李爱民林建华赵芯梅王亚东匡雷
Owner SOUTHERN MEDICAL UNIVERSITY
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