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Cell freezing medium

A cryopreservation, cell technology, applied in the field of cell biology, can solve problems such as unknown virus risks, and achieve the effects of low cost, simple components, and high resuscitation rate.

Inactive Publication Date: 2018-11-06
北京常箐藤生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, bovine serum may contain mad cow disease virus, endotoxin, exotoxin, and potential risks of unknown viruses

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] A cell cryopreservation solution, comprising: trehalose, hydroxyethyl starch 130, sodium chloride, human serum albumin and distilled water.

[0028] Freezing solution 1, trehalose content 5%, hydroxyethyl starch 130 content 2%, sodium chloride 0.9%, human serum albumin 3% and distilled water.

[0029] Freezing liquid 2, trehalose content 6%, hydroxyethyl starch 130 content 3%, sodium chloride 0.9%, human serum albumin 4% and distilled water.

[0030] Freezing liquid 3, trehalose content 7%, hydroxyethyl starch 130 content 4%, sodium chloride 0.9%, human serum albumin 6% and distilled water.

[0031] Freezing solution 4, containing 8% trehalose, 5% hydroxyethyl starch 130, 0.9% sodium chloride, 5% human serum albumin and distilled water.

[0032] Follow the steps below to configure:

[0033] 1) Weigh trehalose and dissolve it in 10mL of distilled water;

[0034] 2) adding hydroxyethyl starch 130, sodium chloride and human serum albumin to the step 1);

[0035] 3) The...

Embodiment 2

[0038] Human umbilical cord mesenchymal stem cells were used for experiments, and the cell cryopreservation solution configured in Example 1 was tested. Cell culture and cell cryopreservation use the following steps:

[0039] 1) Microscopically observe the cells grow to a density of about 95% and freeze them;

[0040] 2) Aspirate the culture medium, add 15mL of normal saline, shake gently, and discard the normal saline;

[0041] 3) Add 3 mL of trypsin to the culture bottle, digest at room temperature for 5 min, observe under a microscope that all cells are suspended, add 5 mL of α-MEM medium (containing 10% serum substitute), and terminate the digestion;

[0042] 4) Draw the cell suspension into a centrifuge tube, and centrifuge at 1100rpm for 5min;

[0043] 5) After centrifugation, discard the supernatant, add 45 mL of normal saline, absorb a drop of cells and count 9×10 6 cells, the cell viability rate is 97%, centrifuged at 1100rpm for 5min;

[0044] 6) Discard the norm...

Embodiment 3

[0070] Use human umbilical cord blood to extract hematopoietic stem cells for experiments. Hematopoietic stem cells and mesenchymal stem cells belong to different types of cells. Hematopoietic stem cells are the progenitor cells of immune cells. The cell diameter, cell shape, and cell characteristics are similar to those of suspension cultured immune cells. Stem cells are used as an example to represent suspension cells and test novel stocks. The No. 1-4 cell cryopreservation solution configured in Example 1 was tested. Cell isolation and cell cryopreservation use the following steps:

[0071] 1) Take 15mL of qualified umbilical cord blood and mix with 15mL of PBS (pH7.0)

[0072] 2) Add 15mL lymphocyte separation medium to a 50mL centrifuge tube, and slowly add the mixed PBS and cord blood solution

[0073] 3) Put it in a centrifuge and centrifuge at 400g for 30min

[0074] 4) Carefully absorb the buffy coat layer in the middle of the centrifuge tube, add PBS to rinse, cen...

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Abstract

The invention provides a cell freezing medium without containing dimethyl sulfoxide and animal source components. The freezing medium is prepared from trehalose, hydroxyethyl starch 130, sodium chloride, human serum albumin and distilled water. The cell freezing medium disclosed by the invention has the advantages of high thawing rate, simple component, good stability and the like.

Description

technical field [0001] The invention relates to the technical field of cell biology, in particular to a cell cryopreservation solution. Background technique [0002] At present, stem cell and immune cell technologies are widely used, and cell storage is an important link in the entire cell industry chain. Cell cryopreservation requires the use of protective agents to prevent cells from being damaged by ice crystals, dehydration and water absorption during cryopreservation and recovery. The most widely used protective agent is dimethyl sulfoxide (DMSO), which is a permeable protective agent that can lower the freezing point of cells, reduce the formation of ice crystals, reduce the damage of free radicals to cells, and change the biofilm’s response to electrolytes, drugs, Permeability of toxicants and metabolites. However, studies have shown that DMSO has certain toxic effects, interacting with protein hydrophobic groups, leading to protein denaturation, vascular toxicity, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02
CPCA01N1/0221
Inventor 董健伸孙永沛常二凤吴思
Owner 北京常箐藤生物科技有限公司
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