CRYOPRESERVATION

1 . A method of freezing NK-92®cells, the method comprising:

contacting NK-92® cells with a cell freezing medium, wherein the cell freezing medium comprises a first composition and a second composition, wherein the first composition comprises:

(a) one or more electrolytes selected from the group consisting of potassium ions, sodium ions, magnesium ions, and calcium ions;

(b) one or more macromolecular agents selected from the group consisting of human albumin, polysaccharide and colloidal starch;

(c) a pH buffer;

(d) at least one sugar;

(e) at least one sugar alcohol;

(f) an impermeant agent selected from the group consisting of lactobionate, gluconate, citrate and glycerophosphate; and

(g) DMSO;

and wherein the second composition comprises albumin.

2 . The method of claim 1 , wherein the NK-92® cells are contacted with the second composition before adding the first composition.

3 . The method of claim 1 , wherein the sugar alcohol is a 6-carbon sugar alcohol.

4 . The method of claim 1 , wherein the sugar is a simple sugar.

5 . (canceled)

6 . The method of claim 1 , wherein the albumin in the second composition is human albumin.

7 - 8 . (canceled)

9 . The method of claim 1 , wherein first composition comprises one or more of the following: 10% DMSO, one or more substrates effective for regeneration of ATP, one or more impermeant agents that can maintain ionic and osmotic balance during hypothermia, and glutathione.

10 . (canceled)

11 . The method of claim 10 , wherein the one or more substrates are selected from the group consisting of adenosine, fructose, ribose and adenine.

12 - 13 . (canceled)

14 . The method of claim 1 , wherein the first composition and second composition is mixed at a volume ratio between 1:5 and 5:1.

15 . The method of claim 1 , wherein a cell freezing medium comprises less than 10% DMSO.

16 . The method of claim 1 , wherein the method further comprises freezing the cells using a controlled rate freeze to reach a final temperature of −80° C. or lower.

17 . The method of claim 1 , wherein the method further comprises storing the cells at −80° C. to −196° C.

18 . The method of claim 16 , wherein the method further comprises thawing the preserved cells and administering the thawed cells to a patient in need via infusion.

19 . (canceled)

20 . The method of claim 9 , wherein more than 70% of the cells are viable at the time of thawing.

21 . (canceled)

22 . The method of claim 18 , wherein the thawed cells

i) have a direct cytoxicity of at least 80% against K562 cells at an effector to target ratio of 10:1;

ii) have a direct cytotoxicity that is 70-100% of that of the NK-92® cells before the cells are frozen;

iii) have a ADCC activity of at least 80-120% against Ramos cells at an effector to target ratio of 10:1, in the presence of an antibody targeting the Ramos cells; or

iv) have a ADCC activity that is 80-100% of that of the cells before the cells are frozen.

23 - 26 . (canceled)

27 . The method of claim 1 , wherein the NK-92® cells express a cytokine, Fc Receptor, chimeric antigen receptor, or any combination thereof.

28 . A NK-92® cell culture comprising:

NK-92® cells and a cell freezing medium, wherein the cell freezing medium comprises a first composition and a second composition, wherein the first composition comprises:

(a) one or more electrolytes selected from the group consisting of potassium ions, sodium ions, magnesium ions, and calcium ions;

(b) one or more macromolecular agents selected from the group consisting of human albumin, polysaccharide and colloidal starch;

(c) a pH buffer;

(d) at least one sugar;

(e) at least one sugar alcohol;

(f) an impermeant agent being at least one member selected from the group consisting of lactobionate, gluconate, citrate and glycerophosphate; and

(g) DMSO; and

wherein the second composition comprises albumin.

29 . The NK-92® cell culture of claim 28 , wherein the sugar alcohol is a 6-carbon sugar alcohol.

30 . The NK-92® cell culture of claim 28 , wherein the sugar is a simple sugar.

31 . (canceled)

32 . The NK-92® cell culture of claim 28 , wherein the second composition is human albumin.

33 . (canceled)

34 . The NK-92® cell culture of claim 28 , wherein the first composition and second composition is mixed at a volume ratio ranging from 5:1 to 1:5.

35 . The NK-92® cell culture of claim 28 , wherein the first composition comprises one or more of the following: 10% DMSO, a substrate effective for the regeneration of ATP, 20-60 mM potassium ions, 50-150 mM sodium ions, 0.001-1 mM magnesium ions, glutathione.

36 . (canceled)

37 . The NK-92® cell culture of claim 36 , wherein the substrate in the first composition is selected from the group consisting of adenosine, fructose, ribose and adenine.

38 - 41 . (canceled)

42 . The NK-92® cell culture of claim 28 , wherein the cell culture is kept at 80° C. to −196° C.

43 . The NK-92® cell culture of claim 42 , wherein at least 70% of NK-92® cells in the cell culture that has been kept at −80° C. to −196° C. are viable after cell culture is thawed.

44 . The NK-92® cell culture of claim 28 , wherein the NK-92® cells comprise a cytokine, Fc Receptor, chimeric antigen receptor, or a combination thereof.

45 . A cell freezing medium produced by mixing a first composition and a second composition at a one to one ratio, wherein the first composition comprises:

(a) one or more electrolytes selected from the group consisting of potassium ions, sodium ions, magnesium ions, and calcium ions;

(b) one or more macromolecular agents selected from the group consisting of human albumin, polysaccharide and colloidal starch;

(c) a pH buffer;

(d) at least one sugar;

(e) at least one sugar alcohol;

(f) an impermeant agent selected from the group consisting of lactobionate, gluconate, citrate and glycerophosphate; and

(g) DMSO;

and wherein the second composition comprises albumin.