Low-density-tolerant feeder-layer-free human pluripotency stem cell culture medium

Abstract

The present invention discloses a feeder-layer-free human pluripotency stem cell culture medium, which contains L-ascorbate-2 magnesium phosphate, sodium selenite, recombinant human insulin, human apotransferrin, basic fibroblast growth factor, transforming growth factor beta 1, heparin lithium and / or heparin sodium, a DMEM / F-12 base culture medium and an osmotic pressure regulator. According to the present invention, with the culture medium, when the hiPSC culture is performed in low density and normal density, the proliferation rate is high, and the cell morphology and the pluripotency are well maintained; the use of the expensive heparitin sulfate is not required so as to substantially reduce the cost; and almost all of the hiPSCs maintaining cultures at the current stage can be met, and various small molecule compounds are added to perform induction reprogramming culture (removal of TGFB1 and DM), such the culture medium is suitable for extensive researches of various basic scientific researches and clinical scientific research experiments.

Description

technical field [0001] The invention relates to a low-density culture medium for human pluripotent stem cells without a feeder layer. Background technique [0002] Pluripotent stem cells (Pluripotency stem cells, iPSC) have the ability of self-renewal and pluripotency, and they can differentiate into any tissue and cell of the three embryonic germ layers (ectoderm, mesoderm, endoderm). Developmental biology and other fields play an important role. iPSCs include embryonic stem cells (Embryonic stem cells, ESCs) and induced pluripotent stem cells (induced pluripotency stem cells, iPSCs). Both ESCs and iPSCs have similar gene expression profiles, self-renewal capabilities, pluripotency, and epigenetic Spectrum, the former comes from the inner cell mass of the blastocyst stage, and the latter comes from the pluripotent stem cells induced and purified by introducing reprogramming factors into adult cells. The most studied IPSC species at this stage include mice and humans, wher...

AI Technical Summary

Problems solved by technology

Although the birth of mTeSR1 has led to the rapid development of the hiPSC field, mTeSR1 still contains a large amount of BSA, which is not suitable for clinical research and is expensive. Therefore, StemCell uses human serum albumin (HSA) to replace the BSA of mTeSR1, and uses human-derived cytokines , developed TeSR TM 2. The medium does not contain any animal-derived substances and is suitable for clinical research related work. Many other companies have also developed their own unique IPSCs medium accordingly.

However, TeSR2 and other similar media still contain a large amount of cytokines and proteins, with complex components, and the use effect is worse than mTeSR1, which cannot widely meet the needs of basic scientific research and clinical research, and also increases the uncertainty of research development and differentiation. In 2011, the Thomson laboratory identified eight mediums that are the main components of mTeSR1, that is, the E8 medium, which can stably maintain the pluripotency and self-renewal ability of iPSCs for a long time (Nat Methods.2011May; 8(5):424- 9.)

With the widespread use of E8 in related fields around the world, people have gradually discovered that E8 has many shortcomings, which affect its further application. The shortcomings include: 1. Since there is no HSA or BSA, if the medium is added to promote reprogramming Cells such as small molecular compounds or β-mercaptoethanol will show obvious cytotoxicity, and cannot be used in the process of efficiently inducing reprogramming, especially in the process of extreme condition reprogramming, which will affect the differentiation experiments of adding different small molecular compounds, and also limit the low Cultivate hiPSCs under density conditions (our experiments found that less than 2,000 hiPSCs per well in a 96-well plate were cultured with E8, and the cells could hardly grow until the cells reached 4,000 to stabilize and healthy growth); 2. The cells contained in E8 The factor concentration is high, and the required cost is still high

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