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Cryopreservation method for adherent culture of cells

A technology of cryopreservation and adherent culture, which is applied to the preservation, application, and animal husbandry of human or animal bodies. It can solve the problems of insufficient cell numbers, changes in the test plan, and discarding, etc., and achieves simple steps and saves experiments. The effect of fees

Inactive Publication Date: 2013-09-11
INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in actual work, sometimes there are unexpected situations such as temporary power failure, problems in the incubator, or changes in the experimental plan. At this time, the cells have not yet adhered to 80-90%, which is not enough for the cells required for traditional cryopreservation. Quantity; however, cells are more precious, it is a pity to discard them or even lose this kind of qualitative resource forever

Method used

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  • Cryopreservation method for adherent culture of cells
  • Cryopreservation method for adherent culture of cells
  • Cryopreservation method for adherent culture of cells

Examples

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Effect test

Embodiment 1

[0023] The cell culture solutions in Example 1 and Example 2 are both DMEM / F12 solutions with 10% (V / V) FBS added. The reagents used were purchased from Sigma except Supercool X-1000 which was purchased from 21st Century Medicine Company. The culture bottles used were all purchased from Nunc Company, which can withstand low-temperature freezing at -80°C, and will not deform or break after thawing.

[0024] Example 1

[0025] On October 27, 2011, purebred Holstein cow mammary gland cells were cultured in vitro, and 30-40% of the cells adhered to the wall after 18 hours ( figure 1 -A, 100×), at this time, the power was cut off about 6 hours after receiving the notice from the power supply company. In order to preserve this batch of precious germplasm resources, we carried out cryopreservation.

[0026] 1. 2× freezing liquid preparation

[0027] Dissolve 2 ml of DMSO, 4 ml of FBS, and 0.4 ml of Supercool X-1000 in 3.6 ml of DMEM / F12 solution, and then dispense into sterilized ...

Embodiment 2

[0033] On December 8, 2011, pure Angus bovine subcutaneous adipocytes were cultured in vitro by tissue block method, and after 4 days, 20-30% of the cells were released and adhered to the wall ( figure 2 -A, 100×), at this time, the carbon dioxide incubator has an alarm signal, indicating that the concentration of carbon dioxide gas is not enough, and the problem has not been solved after ventilation. We suspect that there is a problem inside the incubator and needs to be repaired. In order to preserve this batch of precious germplasm resources, we have carried out cryopreservation treatment.

[0034] 1. 2× freezing liquid preparation

[0035] Dissolve 1.8 ml of DMSO, 3.6 ml of FBS, and 0.6 ml of Supercool X-1000 in 4.0 ml of DMEM / F12, then dispense into sterilized 1.5 ml centrifuge tubes, 0.5 ml in each tube.

[0036] 2. Cell cryopreservation

[0037] First, suck up the cell culture medium in the T25 culture bottle, then add 0.5ml of fresh cell culture medium, then slowly ...

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Abstract

The invention discloses a cryopreservation method for adherent culture of cells. The method comprises directly adding refrigerating fluid in a cell culture bottle, then cooling the culture bottle step by step, finally placing the culture bottle in an ultra-low-temperature refrigerator to store the culture bottle, storing the culture bottle for 2-3 months, unfreezing the cells when culture conditions permit, and continuing culture. Freezing and unfreezing steps are simple and rapid, the steps of cell dissociation and centrifugation and the like in a traditional freezing method are omitted, cell pollution and damage to cell viability are greatly reduced, the influence of adherence degree of the cells is avoided during freezing, the adherence degree of the cells can be 20-30%, limitation of traditional freezing to the number of the cells is avoided, and the cryopreservation method is extremely suitable for dealing with destructive damage which sudden conditions in a laboratory may cause to cell culture. A cell cryopreservation pipe does not need purchasing, the original culture bottle is directly utilized to carry out freezing, the cells do not need to be inoculated to a new culture bottle after unfreezing, culture fluid is added to the original culture bottle to continue culture, and the testing expenses are greatly reduced.

Description

technical field [0001] The invention relates to a biological cryopreservation technology, in particular to a cryopreservation method for adherent cultured cells. Background technique [0002] With the rapid development of biotechnology, cell culture technology has been widely used in various fields such as biology, medicine, and new drug research and development, and has become an indispensable technical means in scientific research or production. The laboratory generally arranges special personnel to carry out cell culture work, in order to provide cells with good growth shape and genetic stability for follow-up experimental research. However, long-term in vitro culture of cells will cause degeneration or transformation of cell growth and morphology, and cells will lose their original genetic characteristics; sometimes cell contamination will cause interruption of passage and loss of germplasm cells. Therefore, freezing methods are usually used to preserve cells in practic...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A01N1/02
Inventor 谭秀文赵红波宋恩亮刘桂芬成海建刘晓牧万发春
Owner INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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