Method of Cryopreservation of Stem Cell-Derived Retinal Pigment Epithelial Cells on Polymeric Substrate

Patent metadata

Field	Value
Publication number	US-2020045957-A1
Application number	US-201916658457-A
Country	US
Kind code	A1
Filing date	Oct 21, 2019
Priority date	Apr 29, 2011
Publication date	Feb 13, 2020
Grant date	—

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 MATRIGEL® biological cell culture substrate, vitronectin, and RETRONECTIN® 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 configured for implantation into a target site, the polymer substrate comprising:
a non-porous parylene substrate; 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 substrate of claim 14 , wherein the substrate has a permeability configured to permit adequate nutrient passage to the cells or adequate passage of cellular waste material away from the substrate.

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

17 . The substrate of claim 16 , wherein the coating comprises one or more of MATRIGEL® biological cell culture substrate, vitronectin, and RETRONECTIN® reagent.

18 . The substrate of claim 14 , wherein said parylene substrate comprises parylene C.

19 . The substrate of claim 14 , wherein the seeded cells comprise human embryonic stem cell-retinal pigment epithelium (hESC-RPE) cells.

20 . The substrate of claim 19 , wherein the seeded cells comprise a monolayer of fully differentiated hESC-RPE cells.

Assignees

• Univ Southern California

Inventors

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

Classifications

• 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

• A61P9/00

  Drugs for disorders of the cardiovascular system · CPC title

• A61P43/00

  Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 · CPC title

• A61P27/02

  Ophthalmic agents · CPC title

• A61P11/00

  Drugs for disorders of the respiratory system · CPC title