Construction method and culture system of urine-derived renal stem cell three-dimensional differentiation model

A technique for culturing and constructing stem cells, which is applied in the field of the construction method and culture system of a three-dimensional differentiation model of urine-derived renal stem cells, can solve the problem of low differentiation efficiency of urine-derived renal stem cells, difficult repair of damaged kidney tissue, and inability to obtain urine-derived renal stem cells. The problems of isolation and culture preparation, etc., to achieve the effect of maintaining proliferation activity, maintaining characteristics, and simple composition

Active Publication Date: 2020-11-20
TONGJI UNIV +1
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Problems solved by technology

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the difficulty in repairing damaged kidney tissue for transplantation treatment due to the inability to separate and culture kidney stem cells with the ability to repair kidney damage in the prior art and the use of two-dimensional culture to induce The defect of low differentiation efficiency of urine-derived re

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  • Construction method and culture system of urine-derived renal stem cell three-dimensional differentiation model
  • Construction method and culture system of urine-derived renal stem cell three-dimensional differentiation model
  • Construction method and culture system of urine-derived renal stem cell three-dimensional differentiation model

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Embodiment 1

[0038] This embodiment provides a method for constructing a three-dimensional differentiation model of urine-derived renal stem cells, comprising the following steps:

[0039] (1) Separation and two-dimensional cell culture steps: Take 3 centrifuge tubes, add 500 μl of penicillin / streptomycin double antibody solution (100X) and 50 μl of 2.5 mg / ml amphotericin solution to each of them, and take urine from patients with chronic kidney disease 150ml, add the urine to the above three centrifuge tubes on average, centrifuge at 420g for 10 minutes, discard the supernatant, keep about 2ml of urine at the bottom of the centrifuge tube, and combine the urine in all the centrifuge tubes into one centrifuge tube , add washing buffer to 50ml, blow and resuspend the bottom cell pellet, centrifuge again at 380g for 15 minutes, discard the supernatant and add washing buffer, repeat this process 3 times, after the last centrifugation, discard the supernatant completely, Only the bottom cell p...

Embodiment 2

[0048] This embodiment provides a method for constructing a three-dimensional differentiation model of urine-derived renal stem cells, comprising the following steps:

[0049] (1) Separation and two-dimensional cell culture steps: Take 3 centrifuge tubes, add 500 μl of penicillin / streptomycin double antibody solution (100X) as the extraction solution, take 150ml of urine from patients with chronic kidney disease, and add the urine to the above 3 centrifuge at a speed of 380g for 15 minutes, discard the supernatant, keep about 1ml of urine at the bottom of the centrifuge tube, combine the urine in all centrifuge tubes into one centrifuge tube, add washing buffer to 50ml, and pipette Resuspend the bottom cell pellet, centrifuge again at 420g for 10 minutes, discard the supernatant and add washing buffer, repeat this process 2-3 times, after the last centrifugation, completely discard the supernatant, leaving only the bottom cell pellet, add 1ml of kidney stem cell culture medium...

experiment example 1

[0054] Experimental Example 1 Identification of Urine-derived Kidney Stem Cells

[0055] This experimental example provides a kind of identification of human urine-derived renal stem cells, including the following steps:

[0056]The P3 generation urine-derived renal stem cells prepared in step (1) in Example 1 were taken for cell immunofluorescence staining to identify renal stem cell markers SOX9 and PAX2. When the stem cell clone grows to a size of 20-30 cells, fix the cells with 4% paraformaldehyde for 10 minutes. After fixation, wash 3 times with PBS for 5 minutes each time to remove residual paraformaldehyde. Add 2.5% Triton X-100 to permeabilize the cells for 5-8 minutes. Wash 3 times with PBS, 5 minutes each. Add PBS solution containing 5%-10% donkey serum, and block the cells for 30-60 minutes. Remove donkey serum PBS solution, add PBS solution containing primary antibodies (SOX9 and PAX2), and incubate overnight at 4°C. Remove the PBS solution containing the prim...

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Abstract

The invention provides a construction method and a culture system of a urine-derived renal stem cell three-dimensional differentiation model. The construction method comprises the following steps: extraction and two-dimensional cell culture: extracting stem cells from urine, and performing subculturing with a urine-derived renal stem cell culture solution on trophoblastic cells to obtain urine-derived renal stem cells; and three-dimensional cell differentiation: adding the urine-derived renal stem cells into the urine-derived renal stem cell culture solution, performing re-suspending to obtaina urine-derived renal stem cell suspension, taking a scaffold material, and adding the scaffold material into the urine-derived renal stem cell suspension for culturing to obtain the three-dimensional differentiation model. The trophoblastic cells are fibroblasts, through co-culture with the trophoblastic cells, a large amount of renal stem cells with high activity and purity can be effectively obtained, meanwhile, the characteristics, namely self-renewal capacity and differentiation potential, of the renal stem cells can be well maintained, and the renal stem cells can form regenerated tissues and micro-organs in three-dimensional differentiation within a short period of time for transplantation treatment and drug screening.

Description

technical field [0001] The invention relates to the field of cell biology, in particular to a method for constructing a three-dimensional differentiation model of urine-derived kidney stem cells and a culture system. Background technique [0002] End-stage renal failure caused by various kidney-related diseases or toxic substances is a major problem in the medical field. The only effective radical treatment for renal failure at present is orthotopic kidney transplantation. Due to the shortage of donor organs, complex surgery, high cost, complicated complications and other factors, kidney transplantation is not suitable for all patients with end-stage renal failure. In recent years, as an alternative treatment for organ transplantation, the role of cell transplantation in medical fields such as blood system diseases, autoimmune diseases, and functional disorders of important organs such as the heart, liver, and kidney has been paid more and more attention. Its potential appl...

Claims

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Application Information

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IPC IPC(8): C12N5/071
CPCC12N5/0687C12N2513/00C12N2502/1323
Inventor 左为张婷
Owner TONGJI UNIV
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