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Human normal renal tubule primary cell and in-vitro isolated culture and application thereof

A primary cell, isolation and culture technology, applied in the field of cell biology, can solve the problems of limited number of progeny cells, aging and death, inability to accurately reflect clinical results, etc., to achieve sensitive cytotoxic/nephrotoxic response, sensitive and accurate response , the effect of proliferative vitality

Active Publication Date: 2019-09-17
武汉赛尔朗灵科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The Chinese patent CN109022350A has been published in the prior art, relying on high-purity primary renal tubular epithelial cells of mice to establish a cell model, although the purity of the primary renal tubular epithelial cells obtained in the experiment is as high as 92%, but it is used in the study of acute kidney injury or chronic renal failure The damage mechanism of renal tubular epithelial cells in diseased renal tubular epithelial cells, there are still species differences between primary renal tubular epithelial cells of mice and humans, which cannot accurately reflect the clinical results
[0007] The literature published in the prior art "Research on Primary Culture, Passage and Identification of Human Kidney Proximal Tubular Epithelial Cells" provides a method for the isolation and culture of human renal tubular epithelial cells to obtain human renal tubular epithelial cells, excluding species However, the number of subcultures of human renal tubular epithelial cells is limited, the number of progeny cells obtained is limited, and the morphology of cells obtained by continuous culture changes to senescence and death, which is not conducive to the study of drug cytotoxicity and nephrotoxicity

Method used

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  • Human normal renal tubule primary cell and in-vitro isolated culture and application thereof
  • Human normal renal tubule primary cell and in-vitro isolated culture and application thereof
  • Human normal renal tubule primary cell and in-vitro isolated culture and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0050] Example 1: In vitro isolation, culture and identification of human normal renal tubular primary cell XHRN-01

[0051] 1. In vitro isolation and culture of human normal renal tubular primary cells XHRN-01

[0052] (1) After approval by the hospital ethics committee, clear cell carcinoma patients or patient guardians agree and sign an informed consent form, fresh clinical paranephrectomy tissue specimens were obtained from Wuhan Union Medical College Hospital, and no cancer cell invasion or metastasis was verified by pathological results.

[0053] (2) Immediately put the obtained tissue specimens into pre-cooled sterile tissue preservation solution (containing 1000 U / mL penicillin, 1000 μg / mL streptomycin sulfate, 2.5 μg / mL amphotericin and 50 μg / mL gentamicin ) into a collection tube, and immediately placed in a sample transport box at 4°C, and transported to the laboratory within 4 hours for cell separation.

[0054] (3) In vitro isolation and culture:

[0055] S1. Th...

Embodiment 2

[0085] Example 2: Subculture and Identification of Human Normal Renal Tubular Primary Cell XHRN-01

[0086] 1. Subculture of primary human normal renal tubular cells XHRN-01

[0087] The human normal renal tubular primary cells obtained in Example 1 were subcultured, and the specific steps were as follows:

[0088] S7. When the cell abundance of human normal renal tubular primary cells XHRN-01 cultured in T25 culture flask reaches 80%, rinse the cells twice with 1xPBS (pH7.2-7.4), add 1mL Accuse digestion solution, and digest the monolayer Cells 3-5min.

[0089] S8. After the digestion reaction, centrifuge at 1000rpm for 5min, remove the supernatant, collect the cell suspension, resuspend with 1mL COMR complete medium, supplement the medium, and put it into a T25 culture bottle for cultivation according to the ratio of 1 to 2.

[0090] The COMR complete medium has the same composition as the COMR complete medium in Example 1, including: adding 1mM sodium pyruvate, 5% fetal c...

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Abstract

The invention discloses a human normal renal tubule primary cell and in-vitro isolated culture and application thereof. The cell is named human normal renal tubule primary cell XHRN-01, and the preservation number of the cell is CCTCC NO:C201970. An isolated culture method of the primary cell comprises the steps that the clinical postoperative excised tissue near the kidney cancer is acquired in vitro, no cancer cell infection is identified through the pathology, the blood vessels and adipose tissue in the tissue are removed, a tissue sample is subjected to collagenase / trypsin digestion, filtration and low-speed centrifugation to obtain cell pellets, after a red blood cell lysis buffer lyses red blood cells, a COMR complete medium is used for resuspending the cells, and a cell line is constructed through continuous cell culture. The cell can be applied in the physiological study on human normal cells, study on drug toxicity of in-vitro normal cells, study on the pathogenesis of the kidney cancer and related diseases including the renal cancer, chronic nephritis and infectious diseases and study on screening of therapeutic drugs.

Description

technical field [0001] The invention belongs to the field of cell biology, and more specifically relates to a primary human normal renal tubule cell and its in vitro isolation and application. Background technique [0002] The kidney is a functional organ with a large blood flow in the body. Toxic substances in the blood can quickly reach the kidney. After the drug enters the body, most drugs are filtered by the glomerulus, secreted by the proximal convoluted tubule, reabsorbed by the distal convoluted tubule, and the epithelial cells of the small closure. Metabolic processes such as degradation are excreted from the body, and this process can involve structural or functional changes in the kidneys, resulting in nephrotoxicity or renal injury. In addition, the kidney's function of concentrating urine further increases the concentration of toxic substances in kidney cells and renal tubules; at the same time, because the kidney tissue is in a state of high metabolism, requires...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12Q1/02C12R1/91
CPCC12N5/0686C12N2500/25C12N2500/38C12N2501/11C12N2509/00G01N33/5014G01N33/5044
Inventor 刘红亚章小平谌科王前进王鲜史健吕庆洋熊之勇
Owner 武汉赛尔朗灵科技有限公司
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