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Preparation method of human renal podocytes

A technology of podocytes and cells, applied in cell dissociation methods, urinary tract/kidney cells, artificial cell constructs, etc., can solve the problems of small glomerular yield, incapable of subculture or bioassay, and achieve value-added rate High, improved yield, high output effect

Active Publication Date: 2021-11-23
山西省人民医院 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Yield of glomeruli isolated manually is too small for subculture or bioassay

Method used

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  • Preparation method of human renal podocytes
  • Preparation method of human renal podocytes
  • Preparation method of human renal podocytes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 Preparation Example 1 of Human Kidney Podocytes

[0055] 1) Digestion and isolation of human kidney single cells

[0056] Cut up the kidney tissue obtained in nephrectomy, weigh it, and add the corresponding volume of mixed enzyme (0.5 mg / mL colⅡ+0.0625 mg / mL ColIV+0.125 mg / mL colⅠ+0.025 mg / mL colⅠ+0.025 mg / mL DNaseⅠ+0.025mg / mL Hyaluronidase) for 20min, stop the digestion with 10% FBS, pass through a 70μm cell sieve, centrifuge, remove the supernatant to obtain single cells, and grind the remaining tissue on the cell sieve with a syringe tip , wash the cell sieve, and obtain the total single-cell suspension of the tissue after filtration. Resuspend the single cells with EGM-MV medium + 20% FBS, change the medium in time, and remove dead cells.

[0057] 2) Extraction of human kidney podocytes—magnetic bead sorting

[0058] After culturing the culture medium for one week, when the cells adhere to the wall and the number of cells is large, digest with 0.25% tr...

Embodiment 2

[0062] Example 2 Preparation Example 2 of Human Kidney Podocytes

[0063] 1) Digestion and separation of human kidney podocytes

[0064] Cut up the kidney tissue obtained in nephrectomy, weigh it, and add the corresponding volume of mixed enzyme (1mg / mL colⅡ+0.125mg / mL ColIV+0.25mg / mL colⅠ+0.05mg / mL DNaseⅠ+0.05mg / mL Hyaluronidase) to digest for 20min, stop the digestion with 10% FBS, pass through a 70μm cell sieve, centrifuge, remove the supernatant to obtain single cells, and put the remaining tissue on the cell sieve with the rubber soft tip of the syringe after filtration Grind, clean the cell sieve, and obtain a single cell suspension after filtration; resuspend the single cell with EGM-MV medium + 20% FBS, change the medium in time, and remove the dead cells;

[0065]2) Human kidney podocyte magnetic bead sorting

[0066] After culturing the culture medium for one week, when the cells adhere to the wall and the number of cells is large, digest with 0.25% trypsin, stop ...

Embodiment 3

[0071] Example 3 Preparation Example 3 of Human Kidney Podocytes

[0072] 1) Digestion and separation of human kidney podocytes

[0073] Cut up the kidney tissue obtained in nephrectomy, weigh it, and add a corresponding volume of mixed enzyme (2 mg / mL col II + 0.25 mg / mL Col IV + 0.5 mg / mL col I + 0.1 mg / mL DNaseⅠ+0.1mg / mLHyaluronidase) for 20min, stop the digestion with 10% FBS, pass through a 70μm cell sieve, centrifuge, remove the supernatant to obtain single cells, grind the remaining tissue on the cell sieve with a syringe tip, and wash the cells Sieve and filter to obtain a single cell suspension; resuspend the single cells with EGM-MV medium + 20% FBS, change the medium in time, and remove dead cells;

[0074] 2) Human kidney podocyte magnetic bead purification

[0075] After culturing the culture medium for one week, when the cells adhere to the wall and the number of cells is large, digest with 0.25% trypsin, stop digestion with 10% FBS, and count the cell suspens...

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Abstract

The invention belongs to the technical field of biochemistry, and provides a preparation method of human renal podocytes. The human renal podocytes conforms to an expression result of a human renal podocyte marker; and meanwhile, the preparation method is good in repeatability and simple to operate, and the human renal podocytes with high yield, higher value-added rate and high purity can be obtained.

Description

technical field [0001] The invention belongs to the technical field of biochemistry, and in particular relates to a preparation method of human kidney podocytes. Background technique [0002] The kidney is an important organ of the human body, which is used to remove metabolites and reabsorb functional substances in the body, so as to ensure the stability of the internal environment of the body. Podocytes, the visceral epithelial cells of the renal capsule, attach to the outside of the glomerular basement membrane (GBM), together with vascular endothelial cells and the glomerular basement membrane, constitute the glomerular hemofiltration barrier. Podocytes can promote glomerular development, resist glomerular pressure, maintain vascular loop shape, regulate glomerular filtration rate, produce VEGF to regulate endothelial cells, participate in inflammation and immune response, synthesize and decompose glomerular basement membrane and other functions. Common podocyte diseas...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0686C12N2509/00C12N2529/00C12N2501/24C12N2501/165C12N2500/25Y02A50/30
Inventor 李亚峰支文强史淑红王倩韩重阳李荣山
Owner 山西省人民医院
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