Mesenchymal stem cells (MSC) expansion methods and materials
View Patent ↗The subject invention concerns materials and methods for growing and expanding MSC while maintaining their undifferentiated phenotype, self-renewal ability, and/or multi-lineage potential. In one embodiment, a method of the invention comprises i) seeding freshly isolated MSC on a planar surface or a 3-D scaffold and growing the cells under physiological or low O 2 tension for a period of time sufficient to support formation of 3-D ECM network; ii) decellularizing the planar surface or 3-D scaffold; and iii) reseeding the decellularized planar surface or 3-D scaffold with MSCs, whereby the reseeded MSCs can be grown on the scaffold and maintain an undifferentiated phenotype. In one embodiment, the 3-D scaffold comprises or is composed of PET. In one embodiment, the MSC are human MSC (hMSC).
1. A method for growing and/or expanding mammalian mesenchymal stem cell (MSC), the method comprising:
a) seeding freshly isolated mammalian MSC on a planar surface or a porous three-dimension (3-D) scaffold, wherein said surface or scaffold comprises one or more thermally responsive films or coatings;
b) growing said mammalian MSC under an O 2 tension that is lower than 20%;
c) decellularizing said surface or scaffold; and
d) reseeding said surface or scaffold with freshly isolated mammalian MSC, whereby said reseeded mammalian MSC grow and maintain an undifferentiated phenotype.
2. The method of claim 1 , wherein said O 2 tension is between about 1% and 10%; or about 1% and 5%; or about 1% and 3%; or is about 2%.
3. The method of claim 1 , wherein said decellularizing step is performed using a detergent and an enzyme that degrades nucleic acid.
4. The method of claim 1 , further comprising the step of harvesting said reseeded MSC from said surface or scaffold.
5. The method of claim 1 , wherein said mammalian MSC are human MSC.
6. The method of claim 1 , wherein said mammalian MSC are isolated from bone marrow or adipose tissue of a mammal.
7. The method of claim 1 , wherein said surface or scaffold is non-degradable.
8. The method of claim 1 , wherein said surface or scaffold is composed of one or more of poly(styrene), poly(carprolactone), nylon, or poly(ethylene terephthalate) (PET).
9. The method of claim 1 , wherein said reseeded cells are allowed to grow on said surface or scaffold for up to about three months.
10. The method of claim 1 , wherein said thermally responsive film or coating comprises one or more of N-isopropylacrylamide, poly(allylamine hydrochloride)-co-poly(N-isopropylacrylamide), or poly(styrene sulfonate)-co-poly(N-isopropylacrylamide).
11. The method of claim 1 , wherein said one or more thermally responsive films or coatings comprises a terminal layer of positively charged allylamine hydrochloride (PAH), or negatively charged styrene sulfonic acid (PSS), or serum.
12. The method of claim 1 , wherein said reseeded MSC are harvested from said surface by modulating the temperature of said thermally responsive film or coating to a temperature where cells adhered thereto are released.
13. The method of claim 12 , wherein said harvested MSC are further grown and cultivated in suspension culture.
14. The method of claim 1 , wherein said MSC are grown on said scaffold in a perfusion bioreactor.
15. The method of claim 1 , wherein said MSC are grown in cell culture media comprising an animal serum at a concentration less than about 5% v/v or in serum-free cell culture media.
16. A composition comprising a decellularized extracellular matrix (ECM) prepared in vitro by:
a) seeding freshly isolated mammalian MSC on a planar surface or a porous three-dimension (3-D) scaffold, wherein said surface or scaffold comprises one or more thermally responsive films or coatings;
b) growing said mammalian MSC under an O 2 tension that is lower than 20%; and
c) decellularizing said surface or scaffold.
17. The composition of claim 16 , wherein said O 2 tension is between about 1% and 10%; or about 1% and 5%; or about 1% and 3%; or is about 2%.
18. The composition of claim 16 , wherein said decellularizing step is performed using a detergent and an enzyme that degrades nucleic acid.
19. The composition of claim 16 , wherein said surface or scaffold is composed of one or more of poly(styrene), poly(carprolactone), nylon, or poly(ethylene terephthalate) (PET).
20. The composition of claim 16 , wherein said thermally responsive film or coating comprises one or more of N-isopropylacrylamide, poly(allylamine hydrochloride)-co-poly(N-isopropylacrylamide), or poly(styrene sulfonate)-co-poly(N-isopropylacrylamide).
21. The composition of claim 16 , wherein said one or more thermally responsive films or coatings comprises a terminal layer of positively charged allylamine hydrochloride (PAH), or negatively charged styrene sulfonic acid (PSS), or serum.
22. A composition comprising a decellularized extracellular matrix (ECM) prepared in vitro by:
a) seeding freshly isolated mammalian MSC on a planar surface or a porous three-dimension (3-D) scaffold, wherein said surface or scaffold is composed of one or more of poly(styrene), poly(carprolactone), nylon, or poly(ethylene terephthalate) (PET);
b) growing said mammalian MSC under an O 2 tension that is lower than 20%; and
c) decellularizing said surface or scaffold.