Method and special culture medium for subculturing chicken embryonic stem cells for long time

Abstract

The invention discloses a method and a special culture medium for subculturing chicken embryonic stem cells for a long time. The method is characterized by comprising the following steps of: isolating cells of area pellucida of X-stage blastoderm, dispersing into single cells or small cell masses through mechanical blowing and beating, inoculating on an STO feeder layer, culturing the chicken embryonic stem cells in the special culture medium at the temperature of between 37 and 38 DEG C, and subculturing once every 3 to 5 days, wherein the special culture medium comprises 600 to 900mL of conditioned medium, 50 to 150mL of fetal calf serum, 5 to 20mL of chicken serum, 5 to 25mL of one or a mixture of more of non-essential amino acids, 0.146 to 0.292g of L-glutamine, 6 to 14mu L of beta-mercaptoethanol, 1 to 2*10<6> IU of mouse leukemia inhibitory factor, 10 to 50mu g of alkaline fibroblastic growth factor and 5 to 20mu g of stem cell growth factor; and fixing the volume of the ingredients to 1,000mL by using a dulbecco's modified eagle medium (DMEM) (high glucose), and regulating the pH value to 7.2 to 7.5. The conditioned medium is obtained by the steps of: culturing BRL-3A cells until the cells are converged, collecting supernatant of the cultured cells, performing centrifugal separation, and filtering by using a filter membrane to obtain the conditioned medium. Compared with the prior art, the method has the advantages that: the long-term subculture of the chicken embryonic stem cells can be realized, the proliferation of the cells is quick, and the cloning yield is high.

Description

technical field The invention relates to the field of animal cell engineering, in particular to a method for cultivating chicken embryonic stem cells and a special culture medium thereof. Background technique Embryonic stem cells are highly undifferentiated cells with developmental pluripotency isolated from the inner cell mass or primordial germ cells of early animal embryos. It has broad application prospects in tissue and organ engineering, cell therapy, and animal genetic modification and manipulation. Embryonic stem cells have a tendency to differentiate spontaneously. The key technology for culturing embryonic stem cells is to select a suitable culture system so that the cells can proliferate rapidly and indefinitely while maintaining an undifferentiated state. Commonly used methods include: (1) feeder layer cells are used to inhibit the differentiation of embryonic stem cells through a mechanism that is not yet fully understood; (2) conditioned medium: conditioned m...

AI Technical Summary

Problems solved by technology

An important reason why chicken embryonic stem cells cannot be subcultured in vitro for a long time is that the current embryonic stem cell culture system, including the medium formula, is designed based on mammals such as mice or humans, and there are great differences between avian cells and mammalian cells , some culture conditions or medium formulations suitable for mammalian embryonic stem cells may not fully meet the needs of chicken embryonic stem cell culture in vitro

Therefore, although these culture conditions or medium formulations can maintain chicken embryonic stem cells in an undifferentiated state for a period of time, they will not be able to maintain this state during long-term subculture, resulting in rapid differentiation of cells

Images