CPC C12N 5/0656 (2013.01) [C12N 5/0696 (2013.01); C12N 2501/10 (2013.01); C12N 2506/1307 (2013.01); C12N 2506/45 (2013.01)] | 26 Claims |
1. A method of manufacturing extracellular matrix (ECM), the method comprising:
(a) culturing first fibroblasts with one or more de-differentiation factors, thereby de-differentiating the first fibroblasts into induced Pluripotent Stem Cells (iPSCs);
(b) culturing the iPSCs with one or more fibroblast differentiation factors, thereby differentiating the iPSCs into second fibroblasts;
(c) culturing the second fibroblasts in a solution comprising serum at an initial concentration of 0.1% to 20% (v/v), until the second fibroblasts produce mature collagens;
(d) gradually reducing the concentration of serum in the fibroblast culture of step (c) by performing multiple rounds of replacing a portion of the serum-containing solution of step (c) with a serum-free solution over a period of at least 5 days such that there is an at least 95% reduction in the concentration of serum (v/v),
wherein gradually reducing the concentration of serum results in the second fibroblasts producing soluble mature ECM without substantially degrading the ECM; and
(e) isolating the soluble mature ECM from the second fibroblasts, wherein isolating the soluble mature ECM comprises contacting the second fibroblasts with dextranase,
thereby manufacturing the ECM.
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