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Cryopreservation method for tissue block used for cell culture

A technology of cryopreservation and cell culture, applied in the preservation of human or animal body, animal cells, vertebrate cells, etc., can solve the problems of tissue sampling, etc., to prevent the formation of ice crystals and reduce the effect of damage.

Active Publication Date: 2014-11-26
INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, if the tissue is too large, it is not conducive to the penetration of the cryoprotectant, which will affect the effect of cryopreservation
Although there are currently reports of successful cryopreservation of tissue blocks (Sun Sujun, An Zhixing, Peng Xinrong, Zhang Yong. Experiments on cryopreservation of goat mammary gland tissue blocks, Journal of Northwest A&F University (Natural Science Edition), 2005,33(4):5-8 ), but the operation process of this freezing method is very cumbersome and time-consuming. The specific performance is: add the base liquid first, pre-cool on ice for 15 minutes, put the tissue block in and balance it on a shaker for 6 hours; add DMSO twice; balance at 0°C during freezing 1h, equilibrate at -20°C for 2h

Method used

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  • Cryopreservation method for tissue block used for cell culture
  • Cryopreservation method for tissue block used for cell culture

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Experimental program
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Effect test

Embodiment 1

[0022] 1. Refrigerant preparation

[0023] Dissolve 9 ml of DMSO, 1 ml of Supercool X-1000, 18 ml of FBS, 0.2 g of glutathione, and 1.2 g of sucrose in 72 ml of DMEM / F12, filter through a 0.22 micron membrane filter and store at 4°C for use.

[0024] 2. Preparation of thawing solution

[0025] Dissolve 12 ml of FBS, 0.2 g of glutathione, and 3 g of sucrose in 88 ml of DMEM / F12 solution, filter through a 0.22-micron membrane filter, and store at 4°C for use.

[0026] 3. Muscle tissue sample processing

[0027] Cut the lateral muscle tissue of the hindlimb of adult Luxi yellow cattle from the slaughterhouse, put it in PBS solution containing 1% penicillin and streptomycin solution, and bring it back to the laboratory within 2 hours. In the sterile ultra-clean bench, the muscle tissue was first placed in a plate and trimmed to 1 cm 3 blocks, and then washed 8 times with PBS solution. During washing, the operator held the curved ophthalmic forceps in the left hand to hold the t...

Embodiment 2

[0034]1. Refrigerant preparation

[0035] Dissolve 11 ml of DMSO, 1.2 ml of Supercool X-1000, 20 ml of FBS, 0.15 g of glutathione, and 1 g of sucrose in 67.8 ml of DMEM / F12, filter through a 0.22-micron membrane filter and store at 4°C for later use.

[0036] 2. Preparation of thawing solution

[0037] Dissolve 12 ml of FBS, 0.15 g of glutathione, and 2 g of sucrose in 88 ml of DMEM / F12, filter through a 0.22-micron membrane filter, and store at 4°C for use.

[0038] 3. Breast tissue sample processing

[0039] The mammary gland tissue of Holstein cows was cut from the slaughterhouse, placed in PBS solution containing 1% penicillin and streptomycin solution and brought back to the laboratory within 3 hours. In a sterile ultra-clean bench, first place the breast tissue in a plate and trim it to 1.2 cm 3 blocks, and then washed 6 times with PBS solution. During the washing, the operator held the curved ophthalmic forceps in the left hand to clamp the tissue block, and held the...

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Abstract

The invention discloses a cryopreservation method for a tissue block used for cell culture. The method includes: firstly performing homogenate treatment on a tissue into a chyle state, then adding a refrigerating fluid and mixing them uniformly, subpackaging the mixture into a freezing tube containing sucrose, DMSO and glutathione, then placing the freezing tube in a cryopreservation box and letting the box stay overnight in a -80DEG ultra low temperature refrigerator, and finally putting the freezing tube into liquid nitrogen for long-term preservation. During unfreezing, centrifugal washing by an unfreezing solution and a culture solution is performed in order, then the tissue block can be directly subjected to inoculated culture or can be digested into a single cell and is then subjected to culture. The method provided in the invention firstly employs a homogenizer to treat the tissue into a chyle state, thus facilitating permeation of a cryoprotectant into tissue cells and removal of intracellular water. At the same time, the impermeable protective agent sucrose added in the refrigerating fluid is also conducive to removal the water from the tissue cells. The adding of the antioxidant substance glutathione helps alleviate the damage of oxygen free radicals on intracellular protein. After unfreezing, the tissue block is directly inoculated in a culture bottle, and the adherent survival rate is over 96%.

Description

technical field [0001] The invention relates to a biological cryopreservation technology, in particular to a cryopreservation method for tissue blocks used for cell culture. Background technique [0002] With the rapid development of biotechnology, cell culture technology has been widely used in biology, medicine, animal husbandry and other life science fields, and has become an indispensable technical means in scientific research or production. At present, almost all tissues of the animal body are inoculated into culture flasks after simple mechanical or protease treatment, and can proliferate under suitable culture conditions (Spier RE. Large-scale mammalian cell culture: methods, applications and products. Curr Opin Biotechnol. 1991 Jun;2(3):375-9). However, studies have shown that these cells should not be cultured for too many passages in vitro, otherwise adverse phenomena such as slow cell growth, aging, and gradual loss of biological functions will occur. Therefore,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A01N1/02C12N5/071
Inventor 谭秀文游伟靳青刘桂芬刘晓牧宋恩亮万发春
Owner INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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