USES OF TREHALOSE IN CELL SUSPENSIONS
Disclosed are cellular compositions and methods relating to the use of aqueous trehalose media to suspend cells. A trehalose-containing medium can be used to inhibit cellular clumping, for example upon dilution of more concentrated cellular preparations into the trehalose-containing medium. In certain embodiments cells, after cryopreservation and thawing, are combined with a trehalose-containing medium to prepare a clumping-inhibited cell suspension.
1 . A method for preparing a cellular composition, comprising:
(a) thawing cryopreserved cells to provide thawed, viable cells; and
(b) combining the thawed, viable cells with an aqueous medium containing trehalose to provide a trehalose-containing cell suspension.
2 . A method, comprising:
(a) suspending cells in a cryoprotectant-containing medium to form a cryopreservable cell composition;
(b) subjecting the cryopreservable cell composition to cryopreservation conditions to form cryopreserved cells;
(c) thawing the cryopreserved cells to provide a thawed, viable cells; and
(d) combining the thawed, viable cells with an aqueous medium containing trehalose to provide a trehalose-containing cell suspension.
3 . A method for preparing a diluted cell suspension from a cell concentrate, comprising:
(a) thawing a cryopreserved cell composition having a first viable cell density of at least 3 million cells/mL to provide thawed, viable cells; and
(b) combining the thawed, viable cells with an aqueous medium containing trehalose to form a trehalose-containing cell suspension having a second viable cell density less than the first cell density.
4 . The method of any preceding claim, wherein the aqueous medium has a trehalose concentration in the range of 1% to 10% by weight.
5 . The method of any preceding claim, wherein the aqueous medium also contains about 0.9% by weight sodium chloride.
6 . The method of any preceding claim, wherein the aqueous medium has an osmolarity in the range of 200 to 600 mosmol/kg.
7 . The method of any preceding claim, wherein the cells are mammalian cells.
8 . The method of any preceding claim, wherein the cells are human cells.
9 . The method of any preceding claim, wherein the cells are stem cells.
10 . The method of any preceding claim, wherein the cells are mesenchymal stem cells.
11 . The method of claim 10 , wherein the mesenchymal stem cells are derived from dental tissue, testicle tissue, bone marrow; peripheral blood, placental tissue, uterine tissue, umbilical cord blood, umbilical cord tissue, or skin tissue.
12 . The method of any preceding claim, wherein the trehalose is present in an amount effective to reduce clumping of the cells in the trehalo se-containing cell suspension.
13 . The method of any preceding claim, wherein said combining is conducted so as to provide a trehalose concentration in the range of about 1.5% to about 7% by weight in the trehalose-containing cell suspension.
14 . The method of any preceding claim, wherein said combining includes the steps of:
removing the thawed cells from a cryopreservation container; and
combining the thawed cells with the aqueous medium containing trehalose in a second container.
15 . The method of any preceding claim, also comprising administering the trehalose-containing cell suspension to a human or animal patient.
16 . The method of any preceding claim, wherein the cells are canine, feline, equine, ovine, bovine, porcine, or human cells.
17 . The method of any preceding claim, wherein said combining comprises combining a liquid medium containing the thawed, viable cells and also containing DNA released from dead cells with the aqueous medium containing trehalose.
18 . The method of claim 17 wherein the liquid medium is a medium in which the cells had been cryopreserved.
19 . A cellular composition prepared by or preparable by a method according to any preceding claim.
20 . A cellular composition, comprising:
cells suspended in a liquid aqueous medium including trehalose; wherein the concentration of trehalose is at least 0.5% by weight of the composition.
21 . The cellular composition of claim 20 , wherein the trehalose is effective to inhibit clumping of the cells.
22 . The cellular composition of claim 20 or 21 , wherein the cells comprise mesenchymal stem cells.
23 . A method according to any one of claims 1 to 18 , also comprising filtering the trehalose-containing cell suspension.
24 . A kit, comprising:
a liquid trehalose composition; and
a container for example combining the liquid trehalose composition with cells.
25 . The kit of claim 24 , also comprising a container containing cells to be combined with the liquid trehalose composition.
26 . The kit of claim 24 or 25 , also comprising a filter.
27 . The method of any of claims 1 to 3 , wherein the aqueous medium also contains about 0.9% by weight sodium chloride.
28 . The method of any of claims 1 to 3 , wherein the aqueous medium has an osmolarity in the range of 200 to 600 mosmol/kg.
29 . The method of any of claims 1 to 3 , wherein the cells are mammalian cells.
30 . The method of any of claims 1 to 3 , wherein the cells are human cells.
31 . The method of any of claims 1 to 3 , wherein the cells are stem cells.
32 . The method of any of claims 1 to 3 , wherein the cells are mesenchymal stem cells.
33 . The method of claim 32 , wherein the mesenchymal stem cells are derived from dental tissue, testicle tissue, bone marrow; peripheral blood, placental tissue, uterine tissue, umbilical cord blood, umbilical cord tissue, or skin tissue.
34 . The method of any of claims 1 to 3 , wherein the trehalose is present in an amount effective to reduce clumping of the cells in the trehalose-containing cell suspension.
35 . The method of any of claims 1 to 3 , wherein said combining is conducted so as to provide a trehalose concentration in the range of about 1.5% to about 7% by weight in the trehalose-containing cell suspension.
36 . The method of any of claims 1 to 3 , wherein said combining includes the steps of:
removing the thawed cells from a cryopreservation container; and
combining the thawed cells with the aqueous medium containing trehalose in a second container.
37 . The method of any of claims 1 to 3 , wherein said combining comprises combining a liquid medium containing the thawed, viable cells and also containing DNA released from dead cells with the aqueous medium containing trehalose.
38 . The method of claim 37 wherein the liquid medium is a medium in which the cells had been cryopreserved.