US6485959B1 — Cell preconditioning and cryopresevation medium

Claim 1

Independent Claim 133 words

An aqueous medium for preconditioning and cryopreservation of cells harvested from a donor comprising: water; adenosine; a calcium channel blocker; and a cell nutrient matrix comprising a sufficient amount of nutrients to sustain the metabolic needs of harvested cells during an incubation period of at least 10 minutes without producing detectable levels of lactate or substantially depleting the nutrients so as to maintain viability of the harvested cells, wherein the cell nutrient matrix contains a carbon and energy source; at least one vitamin selected from the group consisting of pantothenate, choline chloride, folic acid, inositol, niacinamide, pyridoxal, riboflavin and thiamine; and at least one amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.

Claim 2

Dependent Claim 133 words

An aqueous medium for preconditioning and cryopreservation of cells harvested from a donor comprising: water; adenosine; a calcium channel blocker; and a cell nutrient matrix comprising a sufficient amount of nutrients to sustain the metabolic needs of harvested cells during an incubation period of at least 10 minutes without producing detectable levels of lactate or substantially depleting the nutrients so as to maintain viability of the harvested cells, wherein the cell nutrient matrix contains a carbon and energy source; at least one vitamin selected from the group consisting of pantothenate, choline chloride, folic acid, inositol, niacinamide, pyridoxal, riboflavin and thiamine; and at least one amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.

Claim 3

Dependent Claim 133 words

A cell culture suspension comprising: eukaryotic or aerobic prokaryotic cells suspended in an aqueous medium containing: adenosine, a calcium channel blocker; and a cell nutrient matrix comprising a sufficient amount of nutrients to sustain the metabolic needs of the cells during an incubation period of at least 10 minutes without producing detectable levels of lactate or substantially depleting the nutrients so as to maintain viability of the cells, wherein the cell nutrient matrix contains a carbon and energy source, at least one vitamin selected from the group consisting of pantothenate, choline chloride, folic acid, inositol, niacinamide, pyridoxal, riboflavin and thiamine; and at least one amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.

Claim 4

Dependent Claim 133 words

A cell culture suspension comprising: eukaryotic or aerobic prokaryotic cells suspended in an aqueous medium containing: adenosine, a calcium channel blocker; and a cell nutrient matrix comprising a sufficient amount of nutrients to sustain the metabolic needs of the cells during an incubation period of at least 10 minutes without producing detectable levels of lactate or substantially depleting the nutrients so as to maintain viability of the cells, wherein the cell nutrient matrix contains a carbon and energy source, at least one vitamin selected from the group consisting of pantothenate, choline chloride, folic acid, inositol, niacinamide, pyridoxal, riboflavin and thiamine; and at least one amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.

Claim 5

Dependent Claim 132 words

An aqueous medium for preconditioning and cryopreservation of cells harvested from a donor comprising: water; adenosine; a calcium channel blocker; and a cell nutrient matrix comprising a sufficient amount of nutrients to sustain the metabolic needs of the harvested cells during an incubation period of at least 10 minutes without producing detectable levels of lactate or substantially depleting the nutrients so as to maintain viability of the harvested cells, wherein the cell nutrient matrix contains 18-24 mM glucose; at least one vitamin selected from the group consisting of pantothenate, choline chloride, folic acid, inositol, niacinamide, pyridoxal, riboflavin and thiamine; and at least one amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.

Claim 6

Dependent Claim 132 words

An aqueous medium for preconditioning and cryopreservation of cells harvested from a donor comprising: water; adenosine; a calcium channel blocker; and a cell nutrient matrix comprising a sufficient amount of nutrients to sustain the metabolic needs of the harvested cells during an incubation period of at least 10 minutes without producing detectable levels of lactate or substantially depleting the nutrients so as to maintain viability of the harvested cells, wherein the cell nutrient matrix contains 18-24 mM glucose; at least one vitamin selected from the group consisting of pantothenate, choline chloride, folic acid, inositol, niacinamide, pyridoxal, riboflavin and thiamine; and at least one amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.

Claim 7

Dependent Claim 130 words

A cell suspension comprising: eukaryotic or aerobic prokaryotic cells suspended in an aqueous medium containing: adenosine; a calcium channel blocker; and a cell nutrient matrix comprising a sufficient amount of nutrients to sustain the metabolic needs of the cells during an incubation period of at least 10 minutes without producing detectable levels of lactate or substantially depleting the nutrients so as to maintain viability of the cells, wherein the cell nutrient matrix contains 18-24 mM glucose, at least one vitamin selected from the group consisting of pantothenate, choline chloride, folic acid, inositol, niacinamide, pyridoxal, riboflavin and thiamine, and at least one amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, metonine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.

Claim 8

Dependent Claim 130 words

A cell suspension comprising: eukaryotic or aerobic prokaryotic cells suspended in an aqueous medium containing: adenosine; a calcium channel blocker; and a cell nutrient matrix comprising a sufficient amount of nutrients to sustain the metabolic needs of the cells during an incubation period of at least 10 minutes without producing detectable levels of lactate or substantially depleting the nutrients so as to maintain viability of the cells, wherein the cell nutrient matrix contains 18-24 mM glucose, at least one vitamin selected from the group consisting of pantothenate, choline chloride, folic acid, inositol, niacinamide, pyridoxal, riboflavin and thiamine, and at least one amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, metonine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine.

Claim 9

Dependent Claim 139 words

An aqueous medium for preconditioning and cryopreservation of cells harvested from a donor comprising: water; from about 2.7 mM to about 3.6 mM adenosine; from about 0.04 mM to about 0.07 mM verapamil; a cryoprotectant; and a cell nutrient matrix comprising a sufficient amount of nutrients to sustain the metabolic needs of harvested cells when the harvested cells are maintained in oxygenated aqueous medium at a temperature of from about 35° to about 40° C. for a period of from about ten minutes to about 2 hours without producing detectable levels of lactate or substantially depleting the nutrients so as to maintain the viability of the harvested cells, wherein the cell nutrient matrix contains an energy source and at least one vitamin selected from the group consisting of pantothenate, choline chloride, folic acid, inositol, niacinamide, pyridoxal, riboflavin, and thiamine.

Claim 10

Dependent Claim 139 words

An aqueous medium for preconditioning and cryopreservation of cells harvested from a donor comprising: water; from about 2.7 mM to about 3.6 mM adenosine; from about 0.04 mM to about 0.07 mM verapamil; a cryoprotectant; and a cell nutrient matrix comprising a sufficient amount of nutrients to sustain the metabolic needs of harvested cells when the harvested cells are maintained in oxygenated aqueous medium at a temperature of from about 35° to about 40° C. for a period of from about ten minutes to about 2 hours without producing detectable levels of lactate or substantially depleting the nutrients so as to maintain the viability of the harvested cells, wherein the cell nutrient matrix contains an energy source and at least one vitamin selected from the group consisting of pantothenate, choline chloride, folic acid, inositol, niacinamide, pyridoxal, riboflavin, and thiamine.

Claim 11

Dependent Claim 36 words

The aqueous medium for preconditioning and cryopreservation of cells harvested from a donor according to claim 5 , wherein the concentration of adenosine in the aqueous medium is from about 2.9 mM to about 3.1 mM.

Claim 12

Dependent Claim 36 words

The aqueous medium for preconditioning and cryopreservation of cells harvested from a donor according to claim 6 , wherein the concentration of verapamil in the aqueous medium is from about 0.05 mM to about 0.06 mM.

Claim 13

Dependent Claim 55 words

The aqueous medium for preconditioning and cryopreservation of cells harvested from a donor according to claim 5 wherein the cell nutrient matrix contains at least one amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

Claim 14

Dependent Claim 39 words

The aqueous medium for preconditioning and cryopreservation of cells harvested from a donor according to claim 8 , further comprising at least one inorganic salt in a concentration equal to the concentration of the inorganic salts in the cells.

Claim 15

Dependent Claim 37 words

The aqueous medium for preconditioning and cryopreservation of cells harvested from a donor according to claim 9 , wherein the inorganic salt includes chloride, phosphate, sulfate or selenile anions and sodium, potassium, magnesium, copper or zinc cations.

Claim 16

Dependent Claim 25 words

The aqueous medium for preconditioning and cryopreservation of cells harvested from a donor according to claim 10 , wherein the aqueous medium is slightly hypertonic.

Claim 17

Dependent Claim 29 words

The aqueous medium for preconditioning and cryopreservation of cells harvested from a donor according to claim 10 , wherein the aqueous medium has an osmolarity of approximately 330 mOsml/L.

Claim 18

Dependent Claim 22 words

The aqueous medium for preconditioning and cryopreservation of cells harvested from a donor according to claim 10 , further comprising a saccharide.

Claim 19

Dependent Claim 23 words

The aqueous medium for preconditioning and cryopreservation of cells harvested from a donor according to claim 13 , wherein the saccharide is glucose.

Claim 20

Dependent Claim 33 words

The aqueous medium for preconditioning and cryopreservation of cells harvested from a donor according to claim 10 , further comprising pyruvate in an amount ranging from about 0.9 mM to about 1.1 mM.

Claim 21

Dependent Claim 22 words

The aqueous medium for preconditioning and cryopreservation of cells harvested from a donor according to claim 10 , further comprising a hormone.

Claim 22

Dependent Claim 34 words

The aqueous medium for preconditioning and cryopreservation of cells harvested from a donor according to claim 16 , wherein the hormone is insulin, dexamethasone, leutropic hormone, transferrin, somatropin, linoleic acid, or fetal bovine serum.

Claim 23

Dependent Claim 30 words

The aqueous medium for preconditioning and cryopreservation of cells harvested from a donor according to claim 17 , wherein the hormone bovine pancreas insulin, sheep leutropic hormone, or human transferrin.

Claim 24

Dependent Claim 25 words

The aqueous medium for preconditioning and cryopreservation of cells harvested from a donor according to claim 10 , further comprising an oxygen free radical scavenger.

Claim 25

Dependent Claim 35 words

The cell medium for preconditioning and cryopreservation of cells harvested from a donor according to claim 19 , wherein the oxygen free radical scavenger is allopurinol, glutathione or a combination of glycine, glutamate, and cysteine.

Claim 26

Dependent Claim 38 words

The aqueous medium for preconditioning and cryopreservation of cells harvested from a donor according to claim 19 , wherein the aqueous medium is saturated with a gas having an oxygen content of no less than 80 volume %.

Claim 27

Dependent Claim 24 words

The aqueous medium for preconditioning and cryopreservation of cells harvested from a donor according to claim 19 , wherein the cryoprotectant is dimethyl sulfoxide.

Claim 28

Dependent Claim 72 words

The aqueous medium for preconditioning and cryopreservation of cells harvested from a donor in accordance with claim 19 , further comprising a mild buffer solution having a content and concentration such that the aqueous medium has a first pH that ranges from about 7.3 to about 7.5 at a temperature above 35° C. and has a second pH that ranges from about 6.4 to about 6.6 at a temperature below 4° C.

Claim 29

Dependent Claim 28 words

The aqueous medium for preconditioning and cryopreservation of cells harvested from a donor of claim 23 , wherein the first pH of the aqueous medium is about 7.4.

Claim 30

Dependent Claim 28 words

The aqueous medium for preconditioning and cryopreservation of cells harvested from a donor of claim 24 , wherein the second pH of the aqueous medium is about 6.5.

Claim 31

Dependent Claim 34 words

The aqueous medium for preconditioning and cryopreservation of cells harvested from a donor of claim 23 , wherein the buffer comprises a sodium carbonate buffer, a HEPES buffer or a combination of the two.

Annuity Stage	Window Opens	Payment Deadline	Surcharge Period Until	Estimated Fee	Status
3.5 Years (Year 4)	2005-11-25	2006-05-27	2006-11-26	$2000.00	Window Closed / Lapsed
7.5 Years (Year 8)	2009-11-25	2010-05-27	2010-11-26	$3760.00	Window Closed / Lapsed
11.5 Years (Year 12)	2013-11-25	2014-05-27	2014-11-26	$7700.00	Window Closed / Lapsed

Cell preconditioning and cryopresevation medium

Abstract

Inventors

Bibliographic Details

Claims Analysis

References Cited (Backward Citations)

USPTO Annuity & Maintenance Windows

Assigned Categories & Tags

Assign Category Tag