Cryopreservation of primate embryonic stem cells
Primate embryonic stem cells are cryopreserved by resuspension in a freezing medium and slow cooling at a controlled rate. In some embodiments, prior to the controlled freezing step, the suspension of cells is cooled to a temperature just below freezing, and ice crystal formation is induced. The cryopreserved cell aggregates are useful in transplantation, for experimental evaluation, and as a source of lineage and cell specific products, and as targets for the discovery of factors or molecules that can affect them.
1. A method for the cryopreservation of human embryonic stem cells, the method comprising:
partially dissociating an in vitro culture of human embryonic stem cells to provide a population of cell aggregates;
resuspending said cell aggregates in a freezing medium comprising from about 5% to about 15% cyroprotectant;
placing said resuspended cell aggregates in a closed container for freezing;
cooling the container to a temperature just below freezing and inducing ice crystal formation of the freezing medium;
slow cooling said resuspended cell aggregates at a rate not faster than about −2° C./min to a temperature of not higher than about −30° C., followed by a rapid cooling to not higher than about −80° C., to provide a cryopreserved sample;
wherein upon thawing, the viability of cell aggregates in said cryopreserved sample is at least about 50%.
2. The method according to claim 1 , further comprising storing said cryopreserved sample at a temperature of not higher than about −80° C.
3. The method according to claim 2 , wherein said storing is in liquid nitrogen.
4. The method according to claim 1 , wherein said freezing medium is free of added type IV collagen or laminin.
5. The method according to claim 1 , further comprising the step of thawing said cryopreserved sample.
6. The method according to claim 5 , wherein said thawing comprising contacting said cryopreserved sample with a bath at a temperature from about 15° C. to 37° C.
7. The method according to claim 1 , wherein the ES cell aggregates are prepared by enzymatic digestion of adherent cells to yield aggregates of from about 25 cells, to about 200 cells.
8. A cryopreserved human embryonic stem (ES) cell composition, comprising:
a suspension of human ES cell aggregates in a freezing medium, prepared by the method according to claim 1 , wherein said composition is maintained at a temperature of not higher than about −80° C., wherein upon thawing, the viability of cell aggregates in said cryopreserved sample is at least about 50%.
9. The composition according to claim 8 , wherein said freezing medium is free of added type IV collagen or laminin.
10. The composition according to claim 8 , wherein the ES cell aggregates are prepared by enzymatic digestion of adherent cells to yield aggregates of from about 25 cells, to about 200 cells.
11. A method for the cryopreservation of primate embryonic stem cells, the method comprising:
partially dissociating an in vitro culture of primate embryonic stem cells to provide a population of cell aggregates;
resuspending said cell aggregates in a freezing medium comprising from about 5% to about 15% cyroprotectant;
placing said resuspended cell aggregates in a closed container for freezing;
cooling the container to a temperature just below freezing and inducing ice crystal formation of the freezing medium;
slow cooling said resuspended cell aggregates at a rate not faster than about −2° C./min to a temperature of not higher than about −30° C., followed by a rapid cooling to not higher than about −80° C., to provide a cryopreserved sample;
wherein upon thawing, the viability of cell aggregates in said cryopreserved sample is at least about 50%.