Cell culture substrate comprising a laminin and a cadherin
The present disclosure related to isolated laminin-521, methods for making recombinant laminin-521, host cells that express recombinant laminin-521, and compositions containing laminin-521. Laminin-521 can maintain stem cells in vitro pluripotency, enable self-renewal, and enable single cell survival of human embryonic stem cells. When pluripotent human embryonic stem cells are cultured on plates coated with recombinant laminin-521 (laminin-11), in the absence of differentiation inhibitors or feeder cells, the embryonic stem cells proliferate and maintain their pluripotency. It has also been discovered that human recombinant laminin-521 (laminin-11) provides single cell survival of stem cells after complete dissociation into a single cell suspension. Useful cell culture mediums containing at most 3.9 ng/ml of beta fibroblast growth factor (bFGF) are also described herein.
1. A method for maintaining stem cells, comprising:
plating the stem cells on a substrate comprising a laminin and a cadherin,
wherein the laminin is an intact protein; and
exposing the stem cells to a cell culture medium comprising at least 0.3 mM of albumin;
wherein the laminin is laminin-521;
wherein the cadherin is e-cadherin; and
wherein the weight ratio of the laminin-521 to the e-cadherin is from 5:1 to 10:1.
2. The method of claim 1 , wherein the cell culture medium is mTeSR1 medium with additional albumin to arrive at an albumin concentration of at least 0.3 mM.
3. The method of claim 1 , wherein the substrate and the medium do not contain any differentiation inhibitors, feeder cells, differentiation inductors, or apoptosis inhibitors.
4. The method of claim 1 , wherein the stem cells are plated at a density of 200 cells/mm 2 or less.
5. The method of claim 1 , wherein the stem cells are plated at a density of 200 cells/mm 2 or more.
6. The method of claim 1 , wherein the stem cells are plated such that no two stem cells contact each other.
7. The method of claim 1 , wherein the cell culture medium is mTeSR1 medium with about 0.39 mM albumin.
8. The method of claim 1 , wherein the albumin is human serum albumin.
9. The method of claim 1 , wherein the human serum albumin is recombinant human serum albumin.
10. The method of claim 1 , wherein the laminin is an effective recombinant laminin.