US 11,718,829 B2
Methods and compositions for manufacturing extracellular matrix
Francis Christopher Zeigler, Carlsbad, CA (US)
Assigned to Breakthrough Tech LLC, Aliso Viejo, CA (US)
Filed by Breakthrough Tech LLC, Aliso Viejo, CA (US)
Filed on Jul. 28, 2017, as Appl. No. 15/662,901.
Prior Publication US 2019/0032017 A1, Jan. 31, 2019
Int. Cl. C12N 5/077 (2010.01); C12N 5/074 (2010.01)
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.