Method of cryopreservation of stem cell-derived retinal pigment epithelial cells on polymeric substrate

Patent metadata

Field	Value
Publication number	US-11533908-B2
Application number	US-201916658457-A
Country	US
Kind code	B2
Filing date	Oct 21, 2019
Priority date	Apr 29, 2011
Publication date	Dec 27, 2022
Grant date	Dec 27, 2022

Abstract

Disclosed herein are methods and compositions for the cryopreservation of stem cells, such as stem-cell derived retinal pigment epithelial cells, that have been seeded onto and cultured on a substrate, such as a polymeric substrate. Such cryopreserved stem cells are useful for cell therapies, such as treatment of ocular damage or disease.

First claim

What is claimed is:

1. A method of cryopreserving a monolayer of seeded cells, stem cells, stem cell derived cells, or combinations thereof on a substrate, the method comprising:
exposing a parylene substrate seeded with a monolayer of cells to a temperature ramp-down phase with a temperature reduction rate between 10° C. per 10 seconds to 10° C. per 120 seconds to a final temperature between −20° C. and −100° C., wherein the substrate has one or more characteristics that enhance a viability of the seeded cells, the characteristics selected from the group consisting of:
(i) a coefficient of thermal expansion of the substrate;
(ii) a substrate elasticity parameter; and
(iii) a substrate thickness,

transferring the cell-seeded substrate to an intermediate temperature between −20° C. and −100° C. for a first period of time; and
maintaining the cells at a storage temperature that is lower than the intermediate temperature for a second period of time, thereby obtaining cryopreserved cells.

2. The method of claim 1 , wherein said first period of time is between 6 and 48 hours.

3. The method of claim 1 , wherein said storage temperature is achieved using liquid nitrogen.

4. The method of claim 1 , wherein the substrate further comprises a coating to enhance adhesion of the cells to the substrate, wherein the coating comprises one or more of a solubilized basement membrane matrix biological cell culture substrate, vitronectin, and a recombinant human fibronectin (CH-296) reagent, and wherein said coating enhances the viability of the cells during cryopreservation, after cryopreservation, or both.

5. The method of claim 1 , wherein said parylene substrate comprises parylene C.

6. The method of claim 1 , wherein the seeded cells comprise human embryonic stem cell-retinal pigment epithelium (hESC-RPE) cells.

7. The method of claim 6 , wherein the seeded cells comprise a monolayer of fully differentiated hESC-RPE cells.

8. The method of claim 1 , wherein the parylene substrate is non-porous and permeable.

9. The method of claim 1 , further comprising thawing the cryopreserved cells on said substrate by a method comprising:
warming the cell-seeded substrate to a target temperature using a temperature ramp-up heating rate to obtain thawed cells seeded on the substrate, wherein the thawed cells retain viability and/or functionality post-thaw.

10. The method of claim 9 , wherein the thawed cells are cultured in a media comprising one or more of a fibroblast growth factor (FGF) and an FGF supplement.

11. The method of claim 9 , wherein the thawed cells are cultured in a media without growth supplements.

12. The method of claim 1 , wherein the temperature ramp-down phase comprises a temperature reduction rate that is within a range of 7° C. per minute to 17° C. per minute.

13. The method of claim 1 , wherein the exposing is to a final temperature of about −90° C.

14. A cell-seeded polymer substrate apparatus, the polymer substrate comprising:
a cryovial;
a non-porous parylene substrate within the cryovial;
a monolayer of seeded cells, stem cells, stem cell derived cells, or combinations thereof on the substrate,
wherein the substrate is configured to withstand cryopreservation at a temperature ramp-down rate between 10° C. per 10 seconds to 10° C. per 120 seconds to a final temperature between −20° C. per and −100° C. and a lower storage temperature thereafter;
wherein the substrate has one or more characteristics that enhance a viability of the seeded cells, the characteristics selected from the group consisting of:
(i) a coefficient of thermal expansion;
(ii) a substrate elasticity parameter; and
(iii) a substrate thickness.

15. The apparatus of claim 14 , wherein the substrate further comprises:
a coating to enhance adhesion and viability during and after cryopreservation.

16. The apparatus of claim 15 , wherein the coating comprises one or more of a solubilized basement membrane matrix biological cell culture substrate, vitronectin, and a recombinant human fibronectin (CH-296) reagent.

17. The apparatus of claim 14 , further comprising:
a protein free freezing medium surrounding the substrate in the cryovial.

18. The apparatus of claim 14 , wherein the monolayer of seeded cells on the substrate in the cryovial is held at an intermediate temperature between −20° C. and −100° C.

19. The apparatus of claim 18 , wherein the monolayer of seeded cells on the substrate in the cryovial is held at an intermediate temperature of −90° C.

20. The apparatus of claim 14 , wherein the monolayer of seeded cells on the substrate in the cryovial is stored in liquid nitrogen.

Assignees

• Univ Southern California

Inventors

• Zhu Danhong
• Hinton David R
• Ahuja Ashish
• Humayun Mark S

Classifications

• A61P13/12

  of the kidneys · CPC title

• A61L27/58

  Materials at least partially resorbable by the body · CPC title

• A61P9/10

  for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis · CPC title

• A61L27/3813

  Epithelial cells, e.g. keratinocytes, urothelial cells · CPC title

• A61P27/02

  Ophthalmic agents · CPC title