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Cell vitrification freezing liquid and freezing method

A vitrification and vitrification technique applied to the vitrification of cells. It can solve the problem of long time consumption, and achieve the effect of reducing chemical toxicity, improving stability, and resisting osmotic damage.

Inactive Publication Date: 2019-04-19
陈子江 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the operation of this kind of freezing liquid, the balance liquid needs to be balanced for 5-10 minutes, and the vitrification liquid needs to be placed for 1-2 minutes. The whole freezing operation takes a long time

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1: the preparation method of vitrification liquid:

[0027] 1. Take 10ml as an example to prepare rinsing solution (BS), the improved human fallopian tube fluid (mHTF) containing 5% (V / V) human serum albumin (HSA), that is, take 9.5ml of mHTF, and then add 0.5 ml of HSA.

[0028] 2. Take 10ml as an example to prepare a balance solution (BV1), which contains 7.5% (V / V) ethylene glycol (EG) + 7.5% (V / V) dimethyl sulfoxide (DMSO), and the rest is rinse solution ( BS), that is, take 8.5ml of BS, then add 0.75ml of EG and 0.75ml of DMSO.

[0029] 3. Take 10ml as an example to prepare vitrification solution (BV2), containing 15% (V / V) EG + 15% (V / V) DMSO + 0.65mol / L trehalose + 10mg / ml polysucrose (Ficoll). The amount is rinse solution (BS), that is, take 10ml of BS to dissolve 3.783g of trehalose and 0.143g of polysucrose, after they are completely dissolved, take 7.0ml and add 1.5ml of EG and 1.5ml of DMSO.

Embodiment 2

[0030] Example 2: Vitrification of stage four blastocysts

[0031] This embodiment uses the vitrification solution prepared in Example 1.

[0032] 1. Under an inverted microscope, use 1480nm infrared laser to perforate the junction of trophoblast cells in the high-quality rabbit blastocysts that have developed to the fourth stage, discharge the cystic fluid in the cyst cavity, and observe obvious shrinkage.

[0033] 2. Transfer the four-stage blastocysts after auxiliary shrinkage to the washing solution (BS) for 5 seconds on a temperature platform at 37°C.

[0034] 3. Transfer the washed four-stage blastocysts to the balance solution (BV1), and balance for 2 minutes.

[0035] 4. Transfer the equilibrated stage IV blastocysts to vitrification solution (BV2) and let stand for 30 seconds.

[0036] 5. Put the fourth-stage blastocysts in the vitrification solution (BV2) into the vitrification carrier and store them in liquid nitrogen.

[0037] The overall flow is 2 minutes and 3...

Embodiment 3

[0039] Example 3: Vitrification of stage five blastocysts

[0040] This embodiment uses the vitrification solution prepared in Example 1.

[0041] 1. Under an inverted microscope, use a 1480nm infrared laser to penetrate the trophoblast cell junction of the high-quality mouse blastocysts that have developed to the fifth stage, discharge the cystic fluid in the cyst cavity, and observe obvious shrinkage .

[0042] 2. Transfer the fifth-stage blastocysts after the auxiliary shrinkage to the washing solution (BS) for 8 seconds on a temperature platform at 37°C.

[0043] 3. Transfer the washed stage five blastocysts to the balance solution (BV1) and balance for 3 minutes.

[0044] 4. Transfer the equilibrated stage five blastocysts to vitrification solution (BV2) and let stand for 20 seconds.

[0045] 5. Load the fifth-stage blastocysts in the vitrification solution (BV2) into the vitrification carrier and put them into liquid nitrogen for storage.

[0046] The overall process...

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PUM

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Abstract

The invention belongs to the field of freezing and provides a cell vitrification freezing liquid and a freezing method. The freezing liquid comprises a washing liquid, an equilibrium liquid and a vitrifying liquid. With the freezing fluid, the action time in the equilibrium liquid and the vitrifying liquid is shortened, and chemical toxicity of an osmosis protective agent is reduced. In addition,the operation is simple, and the cell survival rate is high.

Description

technical field [0001] The invention belongs to the freezing field and relates to vitrification of cells. Background technique [0002] The concept of "vitrification" refers to the formation of a high-viscosity, amorphous, transparent glass state between liquid and solid when the temperature of water or solution is rapidly cooled to or below the temperature range of -110 to -100 °C . therefore. The basic principle of vitrification technology is to use a high-concentration cryoprotectant solution to replace the water in the cells, and after rapid cooling, it will be transformed from a liquid state into a glass-like amorphous solid state. There is no ice crystal formation in this state change. Avoid ice crystal formation damage to cell lipid membrane and cytoskeleton structure. [0003] In 1998, Mukaida et al. used vitrification technology for the first time to freeze and store embryos and transfer them successfully. Later, vitrification technology was successfully used to...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02
CPCA01N1/0221A01N1/0284
Inventor 陈子江吴克良李城马金龙
Owner 陈子江
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