Media for stem cells

Patent metadata

Field	Value
Publication number	US-10150946-B2
Application number	US-201414769724-A
Country	US
Kind code	B2
Filing date	Feb 21, 2014
Priority date	Feb 21, 2013
Publication date	Dec 11, 2018
Grant date	Dec 11, 2018

Abstract

The present invention relates to a medium for storing, preserving, culturing, and/or differentiating stem cells. The medium employs a synthetic thermo-responsive copolymer, dispersed in an aqueous vehicle, which allows the medium to be readily “switched” between a non-gelatinous (fluid) form and a gelatinous form by simply adjusting temperature across the gelling temperature of the medium. As such, stem cells can be easily captured within a 3D gel matrix by simply mixing the stem cells with the non-gelatinous/fluid form of the medium and then gelling the medium by adjusting the temperature across the gelling temperature of the medium. The stem cells encapsulated within the gelatinous form of the medium may then be preserved for a significant time period before either using the stem cells directly within the medium (e.g. in therapy, or in culturing, differentiation, and such like), or after their extraction from the medium.

First claim

The invention claimed is:

1. A medium for stem cells, the medium comprising: 1-30% w/w particles of a synthetic thermo-responsive co-polymer dispersed in 70-99% w/w of an aqueous vehicle;
wherein the medium is capable of undergoing a change between a non-gelatinous form and a gelatinous form in response to a change in temperature and, in the non-gelatinous form, the medium comprises a colloidally stable aqueous dispersion of the particles of the synthetic thermo-responsive copolymer and, in the gelatinous form, the medium is in the form of a gel that provides a scaffold or matrix capable of accommodating stem cells;
and wherein the aqueous vehicle further comprises nutrients for maintaining the viability of stem cells;
wherein the thermo-responsive copolymer comprises at least one hydrophilic polymeric block P 1 , derived from a monomer M 1 , and at least one hydrophobic polymeric block P 2 which represents a substantially aqueously insoluble polymeric component derived from an aqueously soluble RAFT monomer M 2 ; wherein the RAFT monomer M 2 is a monomer capable of undergoing a Reversible Addition-Fragmentation chain Transfer (RAFT)-type polymerization process via RAFT polymerization.

2. The medium of claim 1 , wherein the medium exists in the non-gelatinous form at temperatures below a gelling temperature and in a gelatinous form at temperatures above the gelling temperature of the medium, and the medium is in the gelatinous form at temperatures greater than or equal to 20° C.

3. The medium of claim 1 , wherein the particles of the synthetic thermo-responsive co-polymer are of a substantially spherical shape when the medium is in the non-gelatinous form, and are substantially anisotropic worms or worm-like particles when the medium is in the gelatinous form.

4. The medium of claim 1 , wherein the thermo-responsive copolymer is defined by Formula B:

wherein P 1 represents a polymeric component derived from a monomer M 1 and P 2 represents a substantially aqueously insoluble polymeric component derived from an aqueously soluble monomer M 2 .

5. The medium of claim 4 , wherein:
Each monomer M 1 is selected from a monomer of the Formula M 1A , M 1B , and/or M 1c :

where R 1 , R 10 and R 11 represent a substituent of M 1A or M 1C which allows P 1 to be at least partially aqueously soluble,
R 2 represents H, CH 3 or CN,
R S represents one or more substituents of the aromatic ring effective to allow P 1 to be at least partially aqueously soluble, and
each monomer M 2 is selected from a monomer of the Formulae

M 2A and/or M 2B ;
Where R 3 is a substituent of M 2 which allows P 2 to be substantially aqueously insoluble, and R 4 and R 6 independently represent H or methyl;
or P 1 is a copolymer comprising monomer M 2 with a monomer M 2 provided that the polymer P 1 remains at least partially aqueously soluble.

6. The medium of any of claim 4 or 5 , wherein P 1 is a polymer or co-polymer comprising glycerol monomethacrylate (GMA) monomeric units, and P 2 is a polymer or copolymer comprising 2-hydroxypropyl methacrylate (HPMA) monomeric units.

7. The medium of claim 6 , wherein the thermo-responsive copolymer is a block copolymer of Formula B [P 1 -P 2 ] selected from the group consisting of:
PGMA-PHPMA, wherein the PGMA block has a degree of polymerization between 20 and 200; and the PHPMA block has a degree of polymerization between 50 and 300;
PGMA-P (GMA-HPMA), wherein the PGMA block has a degree of polymerisation (DP) of 10 to 120, and the P (GMA-HPMA) block may suitably have a degree of polymerisation (DP) of between 20 and 200; and
PGMA-P (DEGMA-HPMA), wherein the PGMA block has a degree of polymerisation between 20 and 200; the P (DEGMA-HPMA) block has a degree of polymerisation (DP) between 20 and 200; and the ratio of DEGMA to HPMA monomer units within the P (DEGMA-HPMA) block is between 10: and 1:10.

8. The medium of claim 1 , wherein the stem cells are mammalian stem cells.

9. A kit for preparing the medium of claim 1 , the kit comprising:
particles of a synthetic thermo-responsive copolymer; and an aqueous vehicle for maintaining the viability of stem cells.

10. A method of storing or preserving one or more stem cells, the method comprising:
contacting a non-gelatinous form of the medium of claim 1 with one or more stem cells to produce a fluid stem cell composition;
changing the temperature of the medium to cause the medium to change from the non-gelatinous form to the gelatinous form and thereby produce a gelled stem cell composition; and
storing the gelled stem cell composition.

11. The method of claim 10 , wherein step (i) is performed with the stem cell medium at a temperature of at or below 10° C.; and step (ii) comprises adjusting the temperature of the stem cell matrix from at or below 10° C. up to or above 18° C.

12. A stem cell composition comprising one or more stem cells dispersed within the medium of claim 1 .

13. The stem cell composition of claim 12 , wherein the stem cells are mammalian stem cells.

14. A method of releasing one or more stem cells from a gelled stem cell composition, the method comprising:
providing a gelled stem cell composition comprising one or more stem cells dispersed within a gelatinous form of the medium of claim 1 ;
changing the temperature of the stem cell medium to cause the medium to change from the gelatinous form to the non-gelatinous form to thereby produce a fluid stem cell composition; and
optionally thereafter isolating said stem cells from the fluid stem cell composition.

15. The medium as claimed in claim 1 , wherein transition between the non-gelatinous form and the gelatinous form, and vice versa, of the medium is a phase transition due to a temperature-dependent change in the morphology of the particles of a synthetic thermo-responsive co-polymer.

16. The medium as claimed in claim 1 , wherein the particles of synthetic thermo-responsive co-polymer are micellar particles within the aqueous vehicle, and the synthetic thermo-responsive co-polymer is defined by Formula B:

wherein P 1 and P 2 are distinct polymeric chains within the co-polymer, P 1 being hydrophilic relative to P 2 such that P 1 is a stabilizer chain and P 2 is a core-forming chain, wherein P 2 is thermo-responsive.

17. The medium as claimed in claim 1 , wherein in the non-gelatinous form of the medium, the particles of the thermo-responsive copolymer are nanoparticles that are less than 500 nm in size.

18. The medium as claimed in claim 1 , wherein the medium is an ultra-filtrated medium filtered via a 0.01-1 μm filter.

19. The medium as claimed in claim 1 , wherein the particles of the synthetic thermo-responsive co-polymer are present within the aqueous vehicle at a concentration of 2-12 wt %.

20. The medium as claimed in claim 1 , wherein the particles of the synthetic thermo-responsive co-polymer are present within the aqueous vehicle at a concentration of 3-7 wt %.

21. The medium of claim 1 , wherein P 1 is a copolymer, which comprises two or more m

Assignees

• Univ Sheffield

Inventors

• Canton Irene
• Armes Steven Peter
• Warren Nicholas John
• Moore Harry
• Battaglia Giuseppe
• Cecchin Denis

Classifications

• C12N2539/10

  Coating allowing for selective detachment of cells, e.g. thermoreactive coating · CPC title

• C12N2533/40

  Polyhydroxyacids, e.g. polymers of glycolic or lactic acid (PGA, PLA, PLGA); Bioresorbable polymers · CPC title

• C12N5/0068 Primary

  General culture methods using substrates (for specific animal cell type C12N5/06) · CPC title