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Renal tubular epithelial cell separation and culture method

A culture method, epithelial cell technology, applied in cell dissociation methods, urinary tract/kidney cells, tissue culture, etc., can solve the problems of decreased cell viability, cell injury, unevenness, etc., and achieve high cell survival rate and proliferation rate. Fast, high passaging effect

Pending Publication Date: 2022-02-15
广州华越肾科再生医学科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Enzymes with strong digestive ability, such as trypsin, can easily damage cells (uneven digestion)
During the digestion process of trypsin, some cells have not been digested, and a small number of cells are overdigested, but when the concentration of trypsin does not reach the effective concentration, the tissue cells cannot be effectively digested
The period from complete digestion to over-digestion of trypsin-digested tissue cells is very short, and it is difficult to control the digestion time. If the digestion is not complete, the yield will be small. Excessive digestion will lead to cell injury and decrease in cell viability, resulting in the inability to obtain a stable state. same cell

Method used

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  • Renal tubular epithelial cell separation and culture method
  • Renal tubular epithelial cell separation and culture method
  • Renal tubular epithelial cell separation and culture method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 The method for the isolation and culture of renal primary tubular epithelial cells

[0057] (1) Pretreatment of renal cortex: put the washed human kidney tissue into a stainless steel tray filled with 4°C PBS, use tissue scissors and tweezers to remove the capsule and fat, cut the kidney from the middle with a scalpel, and cut it along the cortex and For the color boundary line of the medulla, cut the cortex and separate the cortex completely. Put the separated cortex into a 50mL centrifuge tube filled with 4°C PBS, wash it thoroughly, suck the supernatant with a pipette gun, and repeat it several times to reach the solution. There is no blood in the tissue (if it is cortical tissue, only the cortex needs to be shredded). After weighing and recording the remaining tissue blocks, cut the remaining renal cortex with surgical scissors into 1-3 mm 3 , cut into the smallest piece possible. Soak in a petri dish with 4°C PBS, put an ice pack on the bottom of the pe...

Embodiment 2

[0066] The method for isolating and culturing renal primary tubular epithelial cells in Example 2 is the same as Example 1 except for the enzymatic digestion operation.

[0067] Enzymatic digestion operation: Dispase (1mg / mL) and type II collagenase (2mg / mL) were used for the first digestion to digest the fragments of renal cortex tissue for 60 minutes, then centrifuged to remove the supernatant, and for the second digestion, type IV collagenase ( 1 mg / mL) for 45 min; the DNA removal step was not included, and other operations were as in Example 1.

[0068] Result: see after tissue fragment digestion Figure 4 , can effectively and completely separate single cells, and the number of cells is large. The resulting renal tubular epithelial cells have a high survival rate on the complete medium, the cell viability can reach 60%, and the number of extracted cells can reach at least 3×10 6 / g; 1×10 6 Cells are seeded in T75 flasks, and they can reach complete confluence in 2-3 da...

Embodiment 3

[0070] The method for isolating and culturing renal primary tubular epithelial cells in Example 3 is the same as in Example 1 except for the enzymatic digestion operation.

[0071] Enzymatic digestion operation: for the first digestion, use a mixture of dispase (1mg / mL) and type IV collagenase (1mg / mL) to digest for 70min, then centrifuge to remove the supernatant, and add type II collagenase (2mg / mL) for the second digestion ) was digested for 45min; the DNA removal step was not included, and other operations were as in Example 1.

[0072] Result: results like Figure 6 As shown, it can be seen that single cells can be effectively and completely isolated, and the number of cells is large. The obtained cells have a high survival rate on the complete medium, the cell activity can reach 57%, and the cell number reaches at least 2×10 6 pcs / g; 1×10 6 When a cell is seeded in a T75 flask, it can reach complete confluence within 72 hours, and the cells still maintain a good shape...

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Abstract

The invention discloses a renal tubular epithelial cell separation and culture method which comprises the following steps: S1, mechanical tissue separation: taking a renal tissue sample, separating a cortex part, and then cutting into pieces; s2, enzymolysis: digesting with a mixed enzyme, and centrifuging; and S3, cell culture: resuspending the cell precipitate by using a complete culture medium, and inoculating the cell precipitate into a culture bottle. Wherein mechanical separation is simple, and mechanical damage is reduced to the maximum extent. The best digestion efficiency is achieved through mild enzymolysis, the obtained cells are high in activity, the activity of the obtained cells can reach 50% or above, the number of the cells at least reaches 1 * 10 < 6 > / g, the cells have high proliferation performance and high initial inoculation density, the cell proliferation environment pressure is small, a simple and convenient nutrient medium rich in nutrition is added, substrate layer planking is not needed, and the cost is low. The cell proliferation speed is at least one time faster than that reported in the literature; and if 1 * 10<6> cells are inoculated in a T75 bottle, complete convergence can be achieved within 72 hours.

Description

technical field [0001] The invention belongs to the technical field of cell extraction, and in particular relates to a method for separating and culturing renal tubular epithelial cells. Background technique [0002] The kidney is a complex organ mainly composed of glomeruli, tubules, mesangial cells, endothelial cells and podocytes. Separating and purifying these tissues and cells from organs, and then culturing these primary cells in vitro can truly reflect the state of cells in vivo, and the expression of functional proteins and markers is relatively complete, which is an indispensable part of basic research. In addition, with the rise of various therapeutic drugs related to kidney disease, the development of in vitro cultured cell system should be considered as the most direct and convenient platform for drug safety testing. There are several protocols for isolating the above-mentioned cells, among which the proximal renal tubular epithelial cells are isolated the most....

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/0686C12N2509/10C12N2509/00
Inventor 樊琛语刘小燕邱江王恒郑立新
Owner 广州华越肾科再生医学科技有限公司
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