# US 2018/0020658 A1 · Enhancement of cell cryopreservation with glycolipids

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- Number: US 2018/0020658 A1
- Document ID: US20180020658A1
- Status: Published application
- Kind: A1
- Application number: 15/655,381
- Filed: 2017-07-20
- Published: 2018-01-25
- Earliest claimed: 2016-07-22
- Assignees: Tissue Testing Technologies LLC
- Applicants: Tissue Testing Technologies LLC
- Inventors: Lia H. CAMPBELL; Kelvin GM BROCKBANK
- Web page: https://patentgrep.com/patent/US20180020658A1

## CPC

- A01N 1/125: Freeze protecting agents, e.g. cryoprotectants or osmolarity regulators
- A01N 1/144: for temperature control, e.g. refrigerators or freeze-drying apparatus
- A01N 1/162: Temperature processes, e.g. following predefined temperature changes over time
- C07H 15/06: being a hydroxyalkyl group esterified by a fatty acid
- A01N: PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF (preservation of food or foodstuff A23); BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES (preparations for medical, dental or toiletry purposes which kill or prevent the growth or proliferation of unwanted organisms A61K); PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)

## Abstract

Living cellular material may be preserved by incubating the cellular material in a culture medium containing at least one glycolipid, and then subjecting the cellular material to a preservation protocol.

## Claims

### Claim 1

A method for preserving living cellular material, comprising:
exposure of the cellular material to medium containing at least one glycolipid and a cryoprotectant for a predetermined amount of time; and
after the exposure, subjecting the cellular material to a preservation protocol comprising cryopreservation at a cryopreservation temperature of about −80° C. or less.

### Claim 2 (depends on 1)

The method of claim 1, wherein the at least one glycolipid is an anti-freeze glycolipid isolated from one or more member selected from the group consisting of Tipula trivittat, Ceruchus piceus, Solanum dulcamara, Dendroides canadensis, and Cucujus clavipes.

### Claim 3 (depends on 1)

The method of claim 1, wherein the at least one glycolipid comprises a β-mannopyranosyl-(1→4) β-xylopyranose backbone and a lipophilic moiety selected from the group consisting of an alkyl chain substituted with one or more hydroxyl groups; an alkyl chain substituted with one or more carboxy groups; a fatty acid; a mono-glyceride; di-glyceride; tri-glyceride; a sterol, and a phospholipid.

### Claim 4 (depends on 1)

The method of claim 1, wherein the cellular material is exposed to the medium for 6-72 hours before subjecting the cellular material to the preservation protocol, and the preservation protocol comprises cooling the cellular material from 37° C. to a freezing temperature of the medium at a cooling rate in the range of from 10 to 30° C. per minute.

### Claim 5 (depends on 1)

The method of claim 1, wherein the preservation protocol comprises cooling the cellular material in the medium, where the medium contains at least one glycolipid at a concentration in the range of from 1 pM to 1 nM.

### Claim 6 (depends on 1)

The method of claim 1, wherein the medium is a cryoprotectant composition and the cryoprotectant is present in the cryoprotectant composition in an amount of from about 0.05 M to about 11 M, the cryoprotectant being selected from the group consisting of acetamide, agarose, alginate, alanine, albumin, ammonium acetate, anti-freeze proteins, butanediol, chondroitin sulfate, chloroform, choline, cyclohexanediols, cyclohexanediones, cyclohexanetriols, dextrans, diethylene glycol, dimethyl acetamide, dimethyl formamide, dimethyl sulfoxide, erythritol, ethanol, ethylene glycol, ethylene glycol monomethyl ether, formamide, glucose, glycerol, glycerophosphate, glyceryl monoacetate, glycine, glycoproteins, hydroxyethyl starch, inositol, lactose, magnesium chloride, magnesium sulfate, maltose, mannitol, mannose, methanol, methoxy propanediol, methyl acetamide, methyl formamide, methyl ureas, methyl glucose, methyl glycerol, phenol, pluronic polyols, polyethylene glycol, polyvinylpyrrolidone, proline, propanediol, pyridine N-oxide, raffinose, ribose, serine, sodium bromide, sodium chloride, sodium iodide, sodium nitrate, sodium nitrite, sodium sulfate, sorbitol, sucrose, trehalose, triethylene glycol, trimethylamine acetate, urea, valine and xylose.

### Claim 7 (depends on 6)

The method of claim 6, wherein an additional cryoprotectant selected from the group consisting of trehalose, glucose, glycerol, maltose, mannitol, mannose, methyl glucose, raffinose, ribose, sucrose, and xylose is added to the cryoprotectant composition.

### Claim 8 (depends on 1)

The method of claim 1, wherein the cellular material is selected from the group consisting of keratinocytes, hepatocytes, and cardiac myocytes.

### Claim 9 (depends on 1)

The method of claim 1, wherein a cell viability (%) of the cellular material after completion of the preservation protocol is at least 60%.

### Claim 10 (depends on 1)

The method of claim 1, wherein the medium does not contain an anti-freeze protein/peptide.

### Claim 11

Cryopreserved cells obtained by exposure of a living cellular material to medium containing at least one glycolipid and a cryoprotectant for a predetermined amount of time; and
after the exposure, subjecting the cellular material to a preservation protocol; wherein a cell viability (%) of the cellular material after the preservation protocol is at least 60%.

### Claim 12 (depends on 11)

A method comprising administering the cryopreserved cells of claim 11 to a patient.

### Claim 13

A method for increasing production yield of viable cryopreserved cellular material, comprising:
exposure of a cellular material to medium containing at least one glycolipid and a cryoprotectant for a predetermined amount of time to form a cryopreservation composition;
subjecting the cryopreservation composition to a preservation protocol comprising cryopreservation at a cryopreservation temperature of about −80° C. or less, wherein
the preservation protocol comprises cooling the cellular material at a cooling rate greater than about −6.0° C. per minute; and
after completion of the preservation protocol, recovering the cryopreserved cellular material; wherein a cell viability (%) of the recovered cryopreserved cellular material is at least 60%.

### Claim 14 (depends on 13)

The method of claim 13, wherein the recovered cryopreserved cellular material exhibits improved proliferative growth.

### Claim 15 (depends on 13)

The method of claim 13, wherein the cell viability (%) of the recovered cryopreserved cellular material is at least three orders of magnitude greater than a cell viability (%) achieved by performing a preservation protocol under identical conditions except where DMSO alone is used as the cryoprotectant.

### Claim 16 (depends on 13)

The method of claim 13, wherein the cooling rate is in the range of from about −6 to about −10° C. per minute.

### Claim 17 (depends on 13)

The method of claim 13, wherein the cryoprotectant is present in the cryoprotectant composition in an amount of from about 0.05 M to about 11 M, and the cryoprotectant is selected from the group consisting of acetamide, agarose, alginate, alanine, albumin, ammonium acetate, anti-freeze proteins, butanediol, chondroitin sulfate, chloroform, choline, cyclohexanediols, cyclohexanediones, cyclohexanetriols, dextrans, diethylene glycol, dimethyl acetamide, dimethyl formamide, dimethyl sulfoxide, erythritol, ethanol, ethylene glycol, ethylene glycol monomethyl ether, formamide, glucose, glycerol, glycerophosphate, glyceryl monoacetate, glycine, glycoproteins, hydroxyethyl starch, inositol, lactose, magnesium chloride, magnesium sulfate, maltose, mannitol, mannose, methanol, methoxy propanediol, methyl acetamide, methyl formamide, methyl ureas, methyl glucose, methyl glycerol, phenol, pluronic polyols, polyethylene glycol, polyvinylpyrrolidone, proline, propanediol, pyridine N-oxide, raffinose, ribose, serine, sodium bromide, sodium chloride, sodium iodide, sodium nitrate, sodium nitrite, sodium sulfate, sorbitol, sucrose, trehalose, triethylene glycol, trimethylamine acetate, urea, valine and xylose.

### Claim 18 (depends on 13)

The method of claim 13, wherein an additional cryoprotectant selected from the group consisting of trehalose, glucose, glycerol, maltose, mannitol, mannose, methyl glucose, raffinose, ribose, sucrose, and xylose is added to the cryoprotectant composition.

### Claim 19 (depends on 13)

The method of claim 13, wherein the cellular material is selected from the group consisting of keratinocytes, hepatocytes, and cardiac myocytes.

### Claim 20 (depends on 13)

The method of claim 13, wherein the cryopreservation composition does not contain an anti-freeze protein/peptide, and at least one glycolipid comprises a β-mannopyranosyl-(1→4) β-xylopyranose backbone and a lipophilic moiety selected from the group consisting of an alkyl chain substituted with one or more hydroxyl groups; an alkyl chain substituted with one or more carboxy groups; a fatty acid; a mono-glyceride; di-glyceride; tri-glyceride; a sterol, and a phospholipid.