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Long-term cryopreservation and resuscitation method for animal tissues, clinical tissues and biopsy samples

A technology for animal tissue and biopsy samples, which is applied to the preservation of human or animal bodies, animal cells, vertebrate cells, etc., can solve problems such as inability to recover 100%, decline in the integrity of recovered cells, and inability to effectively pass on long-term passages. The effect of good vitality and subculture activity

Active Publication Date: 2022-05-13
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

More importantly, some of these cryopreservation and resuscitation techniques have an optimal protection period of only about 30 days, and the integrity of the resuscitated cells will be significantly reduced if it exceeds 30 days; some resuscitated cells cannot recover 100% of their activity and cannot be effective for a long time Pass down

Method used

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  • Long-term cryopreservation and resuscitation method for animal tissues, clinical tissues and biopsy samples
  • Long-term cryopreservation and resuscitation method for animal tissues, clinical tissues and biopsy samples
  • Long-term cryopreservation and resuscitation method for animal tissues, clinical tissues and biopsy samples

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Primary isolation culture, cryopreservation, resuscitation of human high-grade glioma primary cells, isolation culture and subculture of resuscitated primary cells

[0029] 1. Primary isolation and culture of human high-grade glioma primary cells

[0030] (1) Through the hospital ethics committee, with the consent of the patient or the patient's guardian and after signing the informed consent, fresh clinical glioma resection specimens were obtained from Wuhan Union Medical College Hospital. The specimens were WHO grade glioblastoma.

[0031] (2) Immediately put the resected specimen 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 the collection tube, and immediately put into a 4 ℃ sample transport box, transported to the laboratory within 4 hours for cell separation.

[0032] (3) Primary isolation culture: Obtain the tissue in a biolog...

Embodiment 2

[0047] Example 2: Primary isolation culture, cryopreservation, resuscitation of human prostate cancer adenocarcinoma primary cells, isolation culture and subculture of resuscitated primary cells

[0048] 1. Primary isolation and culture of primary human prostate cancer adenocarcinoma cells

[0049] (1) After passing the hospital ethics committee, obtaining the consent of the patient or the guardian of the patient and signing the informed consent, a fresh clinical prostate cancer resection specimen was obtained from Wuhan Tongji Hospital, which was prostate adenocarcinoma (Gleason score 4+4=8).

[0050] (2) Immediately put the resected specimen 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 the collection tube, and immediately put into a 4 ℃ sample transport box, transported to the laboratory within 4 hours for cell separation.

[0051] (3) Primary isol...

Embodiment 3

[0066] Example 3: Primary isolation culture, frozen preservation, recovery of dog penile carcinoma primary cells, isolation culture and subculture of recovered primary cells

[0067] 1. Primary isolation and culture of dog penile carcinoma primary cells

[0068] (1) Immediately put the resected specimen into the 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 the collection tube, and immediately put into a 4 ℃ sample transport box, transported to the laboratory within 4 hours for cell separation.

[0069] (2) Primary isolation culture: Obtain the tissue in a biological safety cabinet, rinse it once with absolute ethanol, and rinse it twice with 1×PBS (pH 7.2-7.4), and remove blood vessels, For fat and necrotic tissue, use dissecting scissors to cut the tissue until minced; add 20ml of digestive solution containing 1×type I collagenase and 0.25% trypsin-0.2% ...

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Abstract

The invention discloses a long-term cryopreservation and recovery method for animal tissues, clinical tissues and biopsy samples, and belongs to the technical field of animal cell cryopreservation. The cryopreservation method comprises the following steps: cleaning fresh tissues, and cryopreserving with a special cryopreservation solution; the resuscitation method comprises the following steps: thawing cryopreserved tissues in a water bath, rinsing with a special resuscitation solution, treating with a red blood cell lysis solution, centrifuging, rinsing with a resuscitation solution, and finally obtaining resuscitated tissue cells. According to the method provided by the invention, very good cryopreservation and resuscitation effects can be realized aiming at different types of animal tissue cells; after being cryopreserved for at most two years, the strain still has very good passage activity after resuscitation.

Description

technical field [0001] The invention belongs to the technical field of cryopreservation of animal cells, in particular to a method for long-term cryopreservation and resuscitation of animal tissues, clinical tissues and biopsy samples. Background technique [0002] The cryopreservation of animal tissues, clinical tissues and biopsy samples is a method of cleaning and trimming fresh tissues, placing them in a cryopreservation solution, and then transferring them to liquid nitrogen for long-term storage. The recovery of animal tissue, clinical tissue and biopsy samples is to re-obtain active tissue cells by subjecting the frozen tissue in the cryopreservation tube to a special treatment method. [0003] At present, many animal tissues undergo the existing cryopreservation and resuscitation techniques, and the viability of the tissue cells obtained after resuscitation is significantly lower than that before cryopreservation, and cannot be stably passaged or even completely pass...

Claims

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Application Information

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IPC IPC(8): A01N1/02C12N5/09
CPCA01N1/0221A01N1/0284C12N5/0693
Inventor 刘红亚王前进刘霞李继新
Owner 武汉赛尔朗灵科技有限公司
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