Cryopreserving macrophages
The present invention relates to a method of improving the viability of macrophages subjected to cryopreservation, particularly for macrophages which are to be used in therapy, wherein the method comprises a step of maintaining the macrophages at a temperature of 2-12° C. for at least 30 minutes during either freezing or thawing procedures. Following the holding step during cooling, a cooling rate of 1 to 5° C. is used until the macrophages in a medium are frozen. For thawing the macrophages, a warming rate of 1 to 5° C. per minute is used until a temperature of 35-37° C. is reached. The present invention further relates to the cryopreserved macrophages, and the thawed macrophages produced by such methods. The technique may provide macrophages that are GMP-compliant and have a viability of at least 60%.
1 . A method of improving the viability of macrophages subjected to cryopreservation, wherein said method comprises the following steps during freezing of the macrophages:
(a) bringing the macrophages in a medium to a temperature of 2-12° C., and maintaining this temperature for at least 30 minutes; followed by:
(b) cooling of the macrophages in a medium at a rate of 1 to 5° C. per minute until the macrophages in the medium are frozen.
2 . The method according to claim 1 , wherein the method comprises thawing the cryopreserved macrophages produced according to claim 1 , wherein the cryopreserved macrophages are in a medium, the method comprising:
(c) warming cryopreserved macrophages to a temperature of between about 2-12° C. and maintaining the medium at a temperature of between about 2-12° C. for a period of at least 30 minutes; and
(d) warming the cryopreserved macrophages of step (c) at a warming rate of 1-5° C. per minute until a temperature of 35-37° C. is reached.
3 . The method of claim 2 , further comprising one or more additional steps:
(i) diluting the cryopreserved macrophages of step (a) in medium; and/or
(ii) maintaining the macrophages of step (b) at a temperature of about 37° C. for at least 30 minutes.
4 . The method of claim 1 , wherein the macrophages are isolated macrophages or macrophages produced in vitro.
5 . The method of claim 1 , wherein the macrophages are present in the medium at a concentration of at least 5×10 6 cells/mL.
6 . The method of claim 1 wherein the macrophages are present in a medium containing a cryoprotectant.
7 . The method of claim 3 , wherein the medium for diluting contains rhMCSF (recombinant human macrophage colony stimulating factor), optionally at a concentration of between 50-150 ng/mL.
8 . The method of claim 1 , wherein the method is GMP compliant.
9 . The method of claim 1 , wherein the macrophages are present in the medium at a concentration of at least 1×10 7 cells/mL.
10 . A population of cryopreserved macrophages produced according to the method of claim 1 .
11 . A population of thawed macrophages produced according to the method of claim 3 .
12 . A cryopreserved therapeutic composition comprising a population of cryopreserved macrophages according to claim 11 , wherein said macrophages have a viability of at least 60%.
13 . The composition of claim 12 , wherein said viability is at least 60% after at least 3 months of cryopreservation, at least 6 months of cryopreservation, or at least 1 year of cryopreservation.
14 . A cryopreserved therapeutic composition comprising a population of cryopreserved macrophages produced according to the method of claim 1 , wherein upon thawing, said macrophages have a viability of at least 60%, wherein the cryopreserved macrophages are thawed according to the method of claim 2 .
15 . A method of treating a subject with macrophages having a disease in need thereof, comprising administering to the subject an effective amount of a population of thawed macrophages cryopreserved according to claim 11 before administration to the subject.
16 . The method of claim 15 , wherein the disease is a liver disease.
17 . The method of claim 16 , wherein the liver disease is liver cirrhosis.