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Addition agent, preparation, and culture medium

A medium and additive technology, applied in the field of medium, can solve the problems of lack of components and unsatisfactory effects in the medium

Inactive Publication Date: 2006-10-18
维特罗莱夫公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, since serum is a large and undefined chemical substance, removing serum from culture media and attempting to produce media with only macromolecular substitutions is not ideal or not effective at all for a number of reasons as the media lacks the necessary components

Method used

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Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0020] There are two ways to prepare the medium additive. One is to prepare the medium additive independently and add it after the medium is made. The other method is to directly add the components of the medium additive to the medium during the medium preparation process. medium.

[0021] For example, the medium supplement of the present invention can be independently prepared as follows. The medium additive rHA can be made into a stock solution, which can be made into a concentrated stock solution with a concentration of 50-500 mg / ml, usually 250 mg / ml, by adding water, saline or culture medium. Alternatively, make a 250 mg / ml stock solution. Fermentation of HYN can be made into a concentrated stock solution of 10-500 mg / ml, usually 500 mg / ml, in water, saline or culture medium. Prepared by adding water, saline or medium to the bottle and adding appropriate amount of HYN to the solution. Then shake vigorously or mix with a stir bar to dissolve the HYN. 500mg / ml solution ...

Embodiment 1

[0031] Medium G1.2 / G2.2 was prepared from the concentrated stock solutions shown in Table 1 below. Add 200 μl of rHA stock solution at 250 mg / ml to 9.8 ml medium. Preliminary experiments investigated the effect of replacing albumin purified from blood with rHA on the development of outbred mouse embryos in culture medium. Fertilized eggs were cultured for 4 days at one of 3 different rHA concentrations. Embryos were incubated at 37°C, 6% CO 2 : 5% O 2 : 89%N 2 For culture, culture volume 10 embryos: 20 μl medium. Embryos were cultured for 48 hours in G2.2 medium after 48 hours in G1.2 medium. The negative control group was not treated with protein, and the positive control group was treated with 5 mg / ml HSA (blood product). The results are shown in Table 2 below.

[0032] stock solution

validity period

Element

G1.2

(g / L)

G2.2

(g / L)

FG1

(g / L)

A

x10 concentrated solution

3 months

NaCl

5.26

...

Embodiment 2

[0063] Medium G1.2 / G2.2 was prepared as described in Example 1 from concentrated stock solutions. Add 10ml culture medium with 100μl of ×100 stock solution to ferment HYN.

[0064] Preliminary experiments investigated the effect of substituting HYN for albumin purified from blood on the development of cultured outbred mouse embryos. Fertilized eggs were cultured for 4 days at one of 4 different HYN concentrations. Embryos were incubated at 37°C, 6% CO 2 : 5% O 2 : 89%N 2 For culture, culture volume 10 embryos: 20 μl medium. Embryos were cultured for 48 hours in G2.2 medium after 48 hours in G1.2 medium. The negative control group was not treated with protein. The results are shown in Table 3 below.

[0065] HYN(mg / ml)

Blastocyst

to hatch

cell number

ICM number

TE number

%ICM / Total

0

82.4 a

38.3 a

67.3±2.8 a

16.1±0.7 a

50.3±2.3 a

24.6±0.8 a

0.125

88.6 a

57.1b c

...

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PUM

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Abstract

The present invention provides a supplement and a culture media useful for culturing mammalian gametes and embryonic tissue. The culture media comprises at least one of recombinant human albumin, fermented hyaluronan, and citrate. Because the constituents are produced from non-conventional sources, the culture medium is free from contaminants such as viruses, prions and endotoxins. Additionally, because the medium is completely defined, the medium is not subject to variations which can impair the development of mammalian cells and prevent meaningful comparisons of empirical studies.

Description

[0001] This application is a divisional application of the parent application whose application number is CN01813807.1. The application date of the parent case is June 9, 2001; the title of the invention is "Mammalian Gamete and Embryo Medium Additive and Its Application Method". technical field [0002] The invention relates to a culture medium which can provide an effective environment for cell culture. More specifically, the present invention relates to refined media supplements containing ingredients obtained from unconventional sources which, when added to media, avoid the problems of existing media. Background technique [0003] For many years, mammalian embryonic cell culture medium supplements have been derived from animal body fluids, especially serum. Although serotype media supplements are effective for culturing specific types of cells and tissues, they also have disadvantages. Serotype medium supplements are not ideal for cell culture, one of the main reasons ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N5/073
Inventor D·K·加纳M·T·拉尼
Owner 维特罗莱夫公司
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