Vitrification freezing and storing method of a little somatic cells for cloning domestic animal

A vitrification and preservation method technology, applied in the preservation of human or animal bodies, biochemical equipment and methods, applications, etc., can solve the problems of easy loss of small particles, large equipment costs, waste of cells, etc., and achieve the effect of cryopreservation Good, low cost, high survival rate

Inactive Publication Date: 2009-01-07
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, the problem with vitrification is that high-concentration vitrification solution will inevitably cause chemical toxicity and osmotic damage to oocytes.
However, the electron microscope used in the electron microscope copper mesh method is expensive, and the purchase of the instrument is expensive; in the OPS method, since the OPS tubes are drawn by hand with 0.25 ml plastic tubes, the diameters of the tubes are not very consistent, resulting in repeatable freezing effects. The stability is poor; the small particles frozen in the SSV method are easy to lose; the problem with the CLV method is that it is difficult to operate
In current cell freezing, each cryovial contains approximately 1×10 6 -5×10 6 If you directly use the cells after thawing, it will cause a lot of waste of cells; if you re-inoculate and culture after thawing, it will increase the workload.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] The steps for vitrification and cryopreservation of bovine ear fibroblasts are as follows:

[0020] (1), configuration liquid:

[0021] Base solution: add 20ml fetal bovine serum (V / V) and 0.5ml antibiotic solution (Antibiotic-Antimycotic) to 100ml cell culture medium (DMEM solution);

[0022] Vitrification solution I: add 2ml ethylene glycol (V / V) and 2ml dimethyl sulfoxide (V / V) to 100ml base solution;

[0023] Vitrification solution II: Add 30ml of ethylene glycol (V / V), 30ml of dimethyl sulfoxide (V / V) and 0.2 mole of trehalose to 100ml of base solution;

[0024] Thawing solution: Add 0.25 moles of sucrose to 100ml base solution;

[0025] (2), Freezing: Cultivate cells with 6-well culture plate, digest cells in 1 hole when the confluence of cells is 85%, collect cell suspension with 1.5 ml centrifuge tube, centrifuge at 1000g for 5 minutes, and remove supernatant. Then add 1 ml of Vitrification Solution I, centrifuge at 1000 g for 5 minutes, and remove the supern...

Embodiment 2

[0029] The steps of vitrification cryopreservation of frozen bovine fallopian tube epithelial cells are as follows:

[0030] (1), configuration liquid:

[0031] Base solution: add 30ml fetal bovine serum (V / V) and 1.5ml antibiotic solution (Antibiotic-Antimycotic) to 100ml cell culture medium (DMEM solution);

[0032] Vitrification solution I: add 4ml ethylene glycol (V / V) and 4ml dimethyl sulfoxide (V / V) to 100ml base solution;

[0033] Vitrification solution II: add 35ml of ethylene glycol (V / V), 35ml of dimethyl sulfoxide (V / V) and 0.3 mole of trehalose to 100ml of base solution;

[0034] Thawing solution: Add 0.35 moles of sucrose to 100ml base solution;

[0035] (2), Freezing: Cultivate cells with 6-well culture plates, digest cells in 1 hole when the confluence of cells is 80%, collect cell suspension with 1.5 ml centrifuge tube, centrifuge at 800g for 5 minutes, and remove supernatant. Then add 1 ml of Vitrification Solution I, centrifuge at 800 g for 5 minutes, and ...

Embodiment 3

[0039] The steps of vitrification cryopreservation of frozen goat fallopian tube epithelial cells are as follows:

[0040] (1), configuration liquid:

[0041] Base solution: Add 25ml of fetal bovine serum (V / V) and 1ml of antibiotic solution (Antibiotic-Antimycotic) to 100ml of cell culture medium (DMEM solution);

[0042] Vitrification solution I: add 3ml ethylene glycol (V / V) and 3ml dimethyl sulfoxide (V / V) to 100ml base solution;

[0043] Vitrification solution II: Add 30ml of ethylene glycol (V / V), 30ml of dimethyl sulfoxide (V / V) and 0.25 mole of trehalose to 100ml of base solution;

[0044] Thawing solution: add 0.3 mole of sucrose to 100ml base solution;

[0045] (2), Freezing: Cultivate cells with 6-well culture plate, digest cells in 1 hole when the degree of cell confluence is 80%, collect cell suspension with 1.5 ml centrifuge tube, centrifuge at 900g for 7 minutes, and remove supernatant. Then add 1 ml of Vitrification Solution I, centrifuge at 900 g for 7 minu...

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PUM

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Abstract

The invention discloses a vitrification freezing preservation method of a small aqueous cell used for cloning domestic animals and belongs to the field of vitrification freezing preservation. The operation steps of the method are as follows: (I) liquid preparation; (II) vitrification freezing; (III) thawing. Compared with the prior art, the vitrification freezing preservation method of a small aqueous cell used for cloning domestic animals of the invention has the characteristics of low cost expense, simple method, needing no expensive instruments, good vitrification freezing preservation effect and high survival rate after thawing.

Description

technical field [0001] The invention relates to a vitrification cryopreservation method, in particular to a vitrification cryopreservation method for a small amount of somatic cells of livestock clones. Background technique [0002] Since the birth of somatic cell cloned sheep "Dolly" in 1997, many kinds of somatic cell cloned animals have come out one after another. As we all know, in the study of somatic cell cloning, the reconstructed embryo is a nucleoplasmic hybrid, the nucleus is derived from the donor cell, and the cytoplasm is derived from the enucleated oocyte. Only after the donor cells carrying normal genetic material are integrated into the enucleated oocyte, the donor nucleus can initiate the cleavage of the reconstituted embryo under the action of cytoplasmic factors and then guide its normal development. If the genetic material of the donor cell is abnormal, the fusion will lead to abnormal cleavage of the reconstructed embryo, and then its development will b...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/06A01N1/02
Inventor 谭秀文万发春刘晓牧宋恩亮游伟吴乃科
Owner INST OF ANIMAL SCI & VETERINARY MEDICINE SHANDONG ACADEMY OF AGRI SCI
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