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Composition for cryopreservation of bovine reproductive cells and cryopreservation method therefor

A technology for cryopreservation and cryopreservation, which is applied in the preservation of germ cells, human or animal bodies, animal cells, etc. It can solve problems such as strong cytotoxicity and achieve the effect of improving the productivity of offspring and livestock.

Active Publication Date: 2019-04-26
BERTELSMANN MUSIC GROUP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, any of the conventional cryoprotectants used for freezing bovine cells has a problem of strong cytotoxicity

Method used

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  • Composition for cryopreservation of bovine reproductive cells and cryopreservation method therefor
  • Composition for cryopreservation of bovine reproductive cells and cryopreservation method therefor
  • Composition for cryopreservation of bovine reproductive cells and cryopreservation method therefor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] [Example 1] Cryopreservation of bovine sperm using antifreeze polyamino acid

[0054] 1-1. Preparation of Cryopreservation Solution

[0055] 65% by mol% of succinic anhydride (manufactured by Wako Pure Chemical Industries)) was added to 25% aqueous solution of ε-polylysine (manufactured by Chisso, molecular weight: 4000) to prepare ε-polylysine An antifreeze polyamino acid in which 60 mol% of the amino groups in the acid molecule are carboxylated and blocked.

[0056] Secondly, the prepared antifreeze polyamino acid (CPLL) solution (about 33w / w% aqueous solution) and glycerol (Gly) were added to Dulbcco's modified medium (DMEM, Gibco (registered trademark) 11330-032, glucose amount: 3151mg / L) Medium to specified ratio (glycerol V / V%, CPLL w / w%). At this time, neutralize with 1N hydrochloric acid or sodium hydroxide aqueous solution so that pH may be in the range of 7.0-8.0.

[0057] 1-2. Cryopreservation method of bovine spermatozoa

[0058] After checking the numb...

Embodiment 2

[0103] [Example 2] cryopreservation of bovine embryos using antifreeze polyamino acids

[0104] 2-1. Preparation of Cryopreservation Solution

[0105] 65% by mol% of succinic anhydride (manufactured by Wako Pure Chemical Industries) was added to 25% aqueous solution of ε-polylysine (manufactured by Chisso, molecular weight: 4000) to prepare ε-polylysine An antifreeze polyamino acid in which 60 mol% of the amino groups in the molecule are carboxylated and blocked.

[0106] Next, the prepared solution of antifreeze polyamino acid (CPLL), ethylene glycol (EG), propylene glycol (PD) and fetal calf serum (FCS or CS) were added to phosphate-buffered saline (PBS, manufactured by Gibco) to the specified Proportion (w / w%). At this time, neutralize with 1N hydrochloric acid or sodium hydroxide aqueous solution so that pH may be in the range of 7.0-7.5.

[0107] 2-2. Cryopreservation method of bovine embryos

[0108] Bovine embryos grown from morula to blastocyst stage embryos are wa...

Embodiment 3

[0128] [Example 3] Cryopreservation of bovine somatic cells using antifreeze polyamino acids

[0129] 3-1. Preparation of Cryopreservation Solution

[0130] 65% by mol% of succinic anhydride (manufactured by Wako Pure Chemical Industries) was added to 25% aqueous solution of ε-polylysine (manufactured by Chisso, molecular weight: 4000) to prepare ε-polylysine An antifreeze polyamino acid in which 60 mol% of the amino groups in the amino acid molecule are carboxylated and blocked.

[0131] Secondly, the prepared antifreeze polyamino acid solution (CPLL), dimethyl sulfoxide (DMSO) and fetal calf serum (CS) were added to Dulbcco's modified medium (DMEM, manufactured by Sigma-Aldrich) to a specified ratio (w / w%). At this time, neutralize with 1N hydrochloric acid or sodium hydroxide aqueous solution so that pH may be in the range of 7.0-7.5.

[0132] 3-2. Cryopreservation method of bovine somatic cells

[0133] Bovine somatic cells cultured to confluence in tissue culture fla...

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Abstract

The present invention provides a liquid for cryopreservation of bovine reproductive cells such as bovine sperms and a cryopreservation method therefor. Use is made of a cryopreservation liquid wherein0.3-0.9 w / w% of an amphoteric polyelectrolyte (an antifreeze polyamino acid), said amphoteric polyelectrolyte being prepared by, relative to 50-99 mol% of amino groups in [epsilon]-poly-L-lysine having a number-average molecular weight of 1,000-20,000, reacting succinic anhydride and thus introducing an appropriate amount of carboxyl groups thereinto, and 2-4 w / w% of glycerol are dissolved in a physiological solution. A preferred embodiment of the cryopreservation method comprises: a first dilution step for diluting bovine semen 2.5-10 fold with a physiological solution and maintaining at 2-8DEG C; and a second dilution step for adding dropwise a physiological solution containing the amphoteric polyelectrolyte (antifreeze polyamino acid) and glycerol to the suspension obtained in the first dilution step while maintaining at 2-8 DEG C so as to further dilute the bovine semen 5-20 fold.

Description

technical field [0001] The present invention relates to a cryopreservation composition and cryopreservation method useful for cryopreservation of, for example, bovine germ cells. Background technique [0002] In order to preserve animal cells or tissues for a long period of time, a cryopreservation method at a temperature below 0° C. is routinely used. However, when animal cells or tissues are frozen containing water, water molecules crystallize while excluding the medium of solutes and impurities during freezing to form ice crystals composed of only water molecules, so solutes and impurities in the hydrated matter The medium of the substance diffuses unevenly, and freeze concentration occurs. [0003] In order to prevent such freeze concentration, methods of adding various low-molecular compounds have been performed. For example, when cryopreserving cells, in order to minimize the damage to the cells caused by intracellular crystallization during cryopreservation, low-mol...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02C12N5/073C12N5/076
CPCA01N1/0221A01N1/021A01N1/0284A01N1/02A01N1/0226A01N1/0231
Inventor 窪田力安藤贵朗玄丞烋玄优基今村修二月精悟
Owner BERTELSMANN MUSIC GROUP
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