System, device, and method for cell cryopreservation via sand-mediated ice seeding

Patent metadata

Field	Value
Publication number	US-12382953-B2
Application number	US-202217659432-A
Country	US
Kind code	B2
Filing date	Apr 15, 2022
Priority date	Apr 16, 2021
Publication date	Aug 12, 2025
Grant date	Aug 12, 2025

Abstract

Human induced pluripotent stem cells (hiPSCs) possess tremendous potential for tissue regeneration and banking hiPSCs by cryopreservation for their ready availability is crucial to their widespread use. However, contemporary methods for hiPSC cryopreservation are associated with both limited cell survival and high concentration of toxic cryoprotectants and/or serum. The latter may cause spontaneous differentiation and introduce xenogeneic factors, which may compromise the quality of hiPSCs. Here, sand from nature is discovered to be capable of seeding ice above −10° C., which enables cryopreservation of hiPSCs with no serum, minimized cryoprotectant, and high cell survival. Furthermore, the cryopreserved hiPSCs retain high pluripotency and functions judged by the pluripotency marker expression, cell cycle analysis, and capability of differentiation into the three germ layers. This unique sand-mediated cryopreservation method may greatly facilitate the convenient and ready availability of high-quality hiPSCs and probably many other types of cells/tissues for the emerging cell-based translational medicine.

First claim

What is claimed is:

1. A method of cell/tissue cryopreservation comprising:
immobilizing a silicon dioxide (SiO 2 ) based sand on a polydimethylsiloxane (PDMS) film to form a sand-PDMS film, wherein the SiO 2 based sand is non-toxic and partially embedded in the PDMS film such that at least a portion of sand surface is exposed, adhering the sand-PDMS film to a portion of an inner plastic surface of a container;
suspending the cells/tissues in a solution within the container with the sand surface being exposed to the cell/tissue suspension, wherein the sand-PDMS film prevents the SiO 2 based sand from mixing with the solution; and
cooling the solution to a temperature at least negative twenty degrees Celsius (−20° C.), wherein the sand-PDMS film seeds ice into the solution at a temperature warmer than negative fifteen degrees Celsius (−15° C.).

2. The method of claim 1 , wherein the method is free from vitrification and/or use of a serum.

3. The method of claim 1 , further comprising using a serum.

4. The method of claim 1 , further comprising a cryoprotectant at a concentration of no more than 15%.

5. The method of claim 1 , wherein the cells/tissues are human induced pluripotent stem cells (hiPSCs).

6. The method of claim 1 , further comprising storing the cells/tissues in a frozen state.

7. The method of claim 6 , further comprising thawing the cells/tissues with a survival rate of at least seventy percent (70%).

8. The method of claim 7 , wherein the cells/tissues are human induced pluripotent stem cells (hiPSCs) and wherein the method further comprises retaining (i) pluripotency in the hiPSCs after thawing the hiPSCs and (ii) a capability of the hiPSCs to differentiate into the three germ layers.

9. The method of claim 7 , further comprising, after thawing, practicing and/or researching cell-based translational medicine with the thawed cells/tissues.

10. The method of claim 1 , wherein the cells/tissues are selected from the group consisting of stem cells, immune cells, single somatic cells, somatic cell aggregates, pancreatic islets and ovarian follicles.

11. The method of claim 1 , wherein the sand-PDMS film comprises a cured combination of the SiO 2 based sand, a PDMS prepolymer, and a curing agent.

12. The method of claim 11 , wherein the PDMS prepolymer and the curing agent form a layer having a thickness between one hundredth millimeters (0.01 mm) and one hundred millimeters (100 mm).

13. The method of claim 11 , wherein the PDMS prepolymer and the curing agent form a layer having a thickness between one tenth millimeters (0.1 mm) and ten millimeters (10 mm).

14. The method of claim 11 , wherein the PDMS prepolymer and the curing agent form a layer having a thickness between one half millimeters (0.5 mm) and one and one half millimeters (1.5 mm).

15. The method of claim 1 , wherein the container is a cryovial having at least one opening and wherein the container further comprises a removable cap or plug for opening and closing the at least one opening.

16. The method of claim 15 , wherein the cryovial further comprises a base for supporting the cryovial such that the cryovial can rest upright on a flat surface without tipping or rolling.

17. The method of claim 1 , wherein the sand-PDMS film seeds ice into the solution at a temperature warmer than negative ten degrees Celsius (−10° C.).

Assignees

• Univ Maryland

Inventors

• He Xiaoming
• Li Weijie
• Jiang Bin

Classifications

• A01N1/147

  Carriers for immersion in cryogenic fluid for slow freezing or vitrification · CPC title

• A01N1/125

  Freeze protecting agents, e.g. cryoprotectants or osmolarity regulators · CPC title

• C12N5/0696

  Artificially induced pluripotent stem cells, e.g. iPS · CPC title

• A01N1/10

  Preservation of living parts · CPC title

• C12N2533/10

  Mineral substrates · CPC title