A culturing method of human embryonic stem cells and a special culturing medium thereof

Abstract

This invention discloses a method for culturing human embryonic stem cells and the special culture medium. The formula of the culture media comprises: N2 5-30mL, B27 10-60mL, glutamine 0.0146-0.73g, beta-mercaptoethanol 5-10uL, one or several non-essential amino acids in total 3-30mL, alkaline desmocyte growth factor 20-200mg, bovine serum albumin 0.2-0.8g, using DMEM / F12 to volume to 1000ml, pH7.2-7.4. The culture meida is characterized of determined ingredients without any elements from animal sources and high fail-safe reliability for application. The culturing method and clutue meidum of this invent can give the culturing of human embryonic stem cells beneficial effects like fast proliferation, low death rate, safety, stability and long generation time. This invention will play important rols in the culturing of human embryonic stem cells to satisfy the needs of clinical and medical research, with a bright prospect of broad application.

Description

technical field [0001] The present invention relates to a cell culture method and its special culture medium, in particular to a human embryonic stem cell culture method and its special culture medium. Background technique [0002] Stem cells are a group of special cells that exist in multicellular organs at the developmental or adult stage. Their most notable feature is the dual ability of unlimited self-renewal and differentiation to produce various daughter cells, collectively referred to as stemness (stemness). Embryonic stem cells (Embryonic Stem Cell, ES) come from the inner cell mass of the fertilized egg that develops into the blastocyst. The constituent cells of an organ or tissue. [0003] The first mammalian embryonic stem cell line was isolated from mice (Evans MJ, KaufmanMH.Nature, 292(5819):154-156.1981; Martin GR.Proc Natl Acad Sci USA, 78(12):7634-7638.1981 ). Researchers have proved that these cells can integrate into blastocysts and participate in normal...

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Problems solved by technology

However, MEF is a primary cell isolated from mouse embryos, which may contain components of mouse origin, which greatly limits the clinical application of human embryonic stem cells

An article published in Nature Medicine in January 2005 pointed out that: MEF cells and serum substitutes, two animal-derived components currently used to culture human embryonic stem cells, contain a common mammalian sialic acid—Neu5Gc, The human embryonic stem cells and their differentiation products (such as embryonic bodies) that are established and cultured by traditional methods are contaminated with a large amount of Neu5Gc. Since many people contain circulating antibodies against Neu5Gc in their bodies, when human embryonic stem cells or differentiated When the product is transplanted into the human body, it is easy to cause the human body to produce an immune response against the embryonic stem cell or its product, resulting in transplantation failure (Martin MJ.Nat Med, 11(2):228-232.2005)

Replacing Martrigel with laminin can only partially solve this problem, and the quality of laminin produced in different batches is difficult to control, which will affect the state of cultured cells (Xu RH.Nat Methods, 2:185-190.2005)

There are also some systems that use human-derived cells to cultivate human embryonic stem cells to avoid the use of mouse-derived MEF cells. Subprepared feeder layers were also qualitatively unstable

[0010] Recently, Professor Thomson developed a culture system with defined components and no animal sources (Tenneille EL. Nat Biotechnology, 24:185-187.2006). In this system, a mixture of collagenIV, fibronectin, vitaminectin, and laminin was used as a coating The matrix of the culture dish, five growth factors bFGF, LiCl, GABA, pipecolic acid and TGFb were added to the medium, but there were some problems in this system, such as the embryonic stem cells cultured with this system mutated after 4 months, Karyotype changed to XXY (Klinefelter syndrome)

Professor S.Ding and others have done similar research, but the composition of the culture system they established is not completely certain, and still contains some animal-derived components, because the extracellular matrix they use is still derived from mice matrigel (S. Yao. Proc Natl Acad Sci U S A, 103(18):6907-6912.2006)

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