No-spin cryopreservation technique and resulting products
Methods and processes for cryopreservation and direct cell thawing and seeding or suspension after cryopreservation, including methods that eliminate the necessity of post-thaw wash, spin, and frequent practice of performing a cell count. Cell compositions and no-spin cell products produced using the methods are also described.
1. A method for thawing and recovery of cryopreserved hepatocytes, said method comprising:
providing a concentrated composition of cryopreserved hepatocytes comprising a first amount of hepatocytes dispersed in a freezing solution, said freezing solution comprising a cryoprotectant dispersed in medium;
thawing said concentrated composition of cryopreserved hepatocytes to yield a thawed composition of hepatocytes, wherein said thawed composition of hepatocytes comprises thawed hepatocytes and cryoprotectant dispersed in a medium; and
directly plating said thawed composition of hepatocytes or directly suspending said thawed composition of hepatocytes in a suspension assay, without removal of said cryoprotectant.
2. The method of claim 1 , wherein said directly plating or directly suspending are carried out without subjecting said thawed composition of hepatocytes to centrifugation.
3. The method of claim 1 , wherein said directly plating or directly suspending are carried out without subjecting said thawed composition of hepatocytes to washing with fresh medium or thawing medium.
4. The method of claim 1 , wherein said directly plating or directly suspending are carried out without plating and counting said hepatocytes in said thawed composition of hepatocytes.
5. The method of claim 1 , wherein said concentrated composition of cryopreserved hepatocytes is provided in a cryopreservation container, said thawing comprising:
warming said cryopreservation container until said concentrated composition of cryopreserved hepatocytes is at least partially thawed; and
diluting said at least partially-thawed concentrated composition of hepatocytes with prewarmed thawing medium to achieve a desired hepatocytes concentration for said plating or said suspending.
6. The method of claim 5 , wherein said diluting comprises adding said thawing medium to said cryopreservation container.
7. The method of claim 5 , wherein said diluting comprises transferring said at least partially-thawed concentrated composition of hepatocytes to a second container containing said prewarmed thawing medium.
8. The method of claim 5 , wherein said diluting comprises transferring said at least partially-thawed concentrated composition of hepatocytes to a second container and adding said prewarmed thawing medium to said second container.
9. The method of claim 1 , wherein said concentrated composition of cryopreserved hepatocytes is provided in a cryopreservation container, said thawing comprising adding prewarmed thawing medium to said cryopreservation container to simultaneously thaw and dilute the hepatocyte composition to achieve a desired hepatocyte concentration for said plating or said suspending.
10. The method of claim 1 , wherein said thawing comprises mixing said concentrated composition of cryopreserved hepatocytes with pre-warmed suspension assay buffer or medium.
11. The method of claim 10 , wherein said concentrated composition of cryopreserved hepatocytes is at least partially thawed using a warm water bath before mixing with said pre-warmed suspension assay buffer.
12. The method of claim 1 , wherein said directly plating comprises seeding said thawed composition of hepatocytes on a culture plate or culture support as a cell monolayer.
13. The method of claim 1 , wherein said first amount of hepatocytes is a concentrated cell suspension comprising from about 2 million hepatocytes/mL to about 200 million hepatocytes/mL of said cell suspension.
14. The method of claim 13 , wherein said concentrated cell suspension and freezing solution are mixed in a range of from about 0.1:1.9 to about 1.9:0.1 by volume in said concentrated composition of cryopreserved hepatocytes.
15. The method of claim 1 , wherein said cryoprotectant is ethylene glycol, propylene glycol, dimethyl sulfoxide (DMSO), fetal bovine serum (FBS), propanediol, glycerol, or a mixture of the foregoing.
16. The method of claim 15 , wherein said cryoprotectant is DMSO, said freezing solution comprising from about 5% to about 20% (v/v) DMSO.