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Serum-free medium for monkey embryonic stem cell

A technology of serum-free medium and embryonic stem cells, which is applied in the field of stem cell culture to achieve the effects of determining components, eliminating pathogen contamination, and ensuring safety

Active Publication Date: 2017-05-31
北京赛斯达生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the serum-free medium on the market is mainly aimed at the cultivation of human embryonic stem cells, and there is no commercial medium that can maintain the undifferentiated state and totipotency of monkey embryonic stem cells for a long time

Method used

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  • Serum-free medium for monkey embryonic stem cell
  • Serum-free medium for monkey embryonic stem cell
  • Serum-free medium for monkey embryonic stem cell

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1, the preparation of culture medium

[0031] This embodiment provides a serum-free medium for monkey embryonic stem cells, which uses DMEM-F12 as a basal medium, and adds the following additive components to the basal medium, the amount of each additive component in the basal medium Concentrations are also as follows:

[0032]

[0033]

[0034] The non-essential amino acids consist of equal concentrations of alanine, arginine, aspartic acid, cystine, proline, and tyrosine.

[0035] See the table below for the cell culture reagents and cytokine manufacturers and article numbers used in this example:

[0036]

[0037]

[0038] The complete medium prepared above was sterilized by filtration with a 0.22 μm filter, and stored at -20°C in the dark.

[0039] The parameters of the above medium are as follows:

[0040] pH:7.4

[0041] Osmotic pressure: 340mosm

[0042] Bacteria, fungus detection: negative

[0043] Chlamydia, mycoplasma detection: ne...

Embodiment 2

[0045] Embodiment 2, the cultivation of macaque embryonic stem cell line in feeder layer system

[0046] 1. Experimental material: macaque embryonic stem cell line.

[0047] 2. Test method: Divide the test materials into test group and control group, among which:

[0048] Experimental group: the P25 macaque embryonic stem cells were divided into 5×10 4 Planted at a density of / well into a 6-well plate pre-coated with feeder cells, which are a monolayer of mouse embryonic fibroblasts treated with 10 μg / ml mitogen-C for 2-2.5 hours cells, and the monkey embryonic stem cell serum-free medium obtained in Example 1 was added to the wells of the 6-well plate and placed at 37 ° C, 5% CO 2 The continuous culture was carried out in the incubator, the medium was replaced once a day, and the subculture was performed once every 5 days. Collagenase IV was used to digest for 3 minutes during subculture, and macaque embryonic stem cell colonies were cut into appropriate and uniform small ...

Embodiment 3

[0051] Embodiment 3, the cultivation of rhesus monkey embryonic stem cell line in feeder-free system

[0052]1. Experimental material: macaque embryonic stem cell line.

[0053] 2. Test method: Divide the test materials into test group and control group, among which:

[0054] Experimental group: the P25 macaque embryonic stem cells were divided into 5×10 4 The density of / well was planted into a 6-well plate pre-coated overnight with Matrigel, and the serum-free medium of monkey embryonic stem cells obtained in Example 1 was added and placed at 37 ° C, 5% CO 2 The incubator was used for continuous culture, the culture medium was replaced once a day, and the culture medium was subcultured once every 5 days. Subculture was digested with EDTA for 3 minutes, and the macaque embryonic stem cell line was cloned into uniform small pieces by gently pipetting with a 1mL gun, and then subcultured at a ratio of 1:5.

[0055] Control group: the method is the same as that of the test gr...

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Abstract

The invention discloses a serum-free medium for a monkey embryonic stem cell. The serum-free medium comprises a basic medium and additives, wherein the additives are dissolved in the basic medium; the basic medium comprises a DMEM-F12 medium; and the additive comprises L-glutamine, non-essential amino acid, L-ascorbic acid, sodium selenite, beta-mercaptoethanol, human serum albumin, heparin sodium, human transferring, insulin human, a basic fibroblast growth factor, a transforming growth factor beta 1 and an epidermal growth factor. The serum-free medium is capable of maintaining an undifferentiation state and totipotency of the monkey embryonic stem cell within a relatively long time, and is applicable to culture of the monkey embryonic stem cell in both a free-feeding layer system and a feeding layer system.

Description

technical field [0001] The invention relates to the field of stem cell culture, in particular to a serum-free medium for monkey embryonic stem cells. Background technique [0002] Embryonic stem cells (ESCs) are highly undifferentiated cells isolated from the inner cell mass (Inner Cell Mass, ICM) or primordial germ cells (PGCs) of early embryos and can be cultured in vitro. , which can differentiate into permanent cell lines of various tissues and cell types derived from the 3 germ layers. At present, it is generally believed that embryonic stem cells will have an important impact on the fields of in vitro research on animal and human embryogenesis and development, new gene discovery, and drug screening. [0003] Although human embryonic stem cells (hESC) have important research and application value in the fields of regenerative medicine, drug screening and developmental biology, the use of human embryonic stem cells and primoryos mainly involves moral and ethical issues....

Claims

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Application Information

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IPC IPC(8): C12N5/0735
CPCC12N5/0606C12N2500/24C12N2500/38C12N2500/44C12N2500/32C12N2501/11C12N2501/115C12N2501/15C12N2501/33
Inventor 穆小生盖丽云李刚毅
Owner 北京赛斯达生物技术有限公司
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