US6140123A — Method for conditioning and cryopreserving cells

Claim 1

Independent Claim 103 words

A method for preconditioning and cryopreservation of cells harvested from a donor comprising the steps of: suspending cells harvested from a donor in a cell conditioning and cryopreservation medium; incubating the cell suspension for a period of at least 10 minutes; and then freezing the cells; where the cell conditioning and cryopreservation medium includes adenosine, a calcium channel blocker, and a cell nutrient matrix comprising a sufficient amount of nutrients to sustain metabolic needs of the harvested cells during the incubation period without producing detectable levels of lactate or substantially depleting the nutrients, so as to maintain the viability of the harvested cell.

Claim 2

Independent Claim 103 words

A method for preconditioning and cryopreservation of cells harvested from a donor comprising the steps of: suspending cells harvested from a donor in a cell conditioning and cryopreservation medium; incubating the cell suspension for a period of at least 10 minutes; and then freezing the cells; where the cell conditioning and cryopreservation medium includes adenosine, a calcium channel blocker, and a cell nutrient matrix comprising a sufficient amount of nutrients to sustain metabolic needs of the harvested cells during the incubation period without producing detectable levels of lactate or substantially depleting the nutrients, so as to maintain the viability of the harvested cell.

Claim 3

Dependent Claim 16 words

The method in accordance with claim 1 wherein the cells are eukaryote or aerobic prokaryote cells.

Claim 4

Dependent Claim 21 words

The method in accordance with claim 1, wherein the concentration of adenosine is from about 2.7 mM to about 3.6 mM.

Claim 5

Dependent Claim 14 words

The method in accordance with claim 1, wherein the calcium channel blocker is verapamil.

Claim 6

Dependent Claim 21 words

The method in accordance with claim 4, wherein the concentration of verapamil is from about 0.04 mM to about 0.07 mM.

Claim 7

Dependent Claim 44 words

The method in accordance with claim 1 wherein the cell nutrient matrix contains at least one amino acid selected from the group comprising alanine, arginine, asparagine, aspartic acid, cystine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

Claim 8

Dependent Claim 36 words

The method in accordance with claim 1 wherein the cell nutrient matrix contains at least one vitamin selected from the group comprising pantothenate, choline chloride, folic acid, inositol, niacinamide, pyridoxal, riboflavin, and thiamine, and a saccharide.

Claim 9

Dependent Claim 24 words

The method in accordance with claim 1 wherein the cells are incubated for a period of from about 15 minutes to about 2 hours.

Claim 10

Dependent Claim 24 words

The method in accordance with claim 1 wherein the cells are incubated at a temperature of from about 35° C. and about 38° C.

Claim 11

Dependent Claim 20 words

The method in accordance with claim 1 further comprising the step of oxygenating the cell suspension during the incubation period.

Claim 12

Dependent Claim 33 words

The method in accordance with claim 10 wherein the cell suspension is oxygenated by bubbling oxygen through the suspension using a gas having an oxygen content of no less than 80 vol. %.

Claim 13

Dependent Claim 19 words

The method in accordance with claim 11 further comprising adding a oxygen free radical scavenger to the cell suspension.

Claim 14

Dependent Claim 19 words

The method in accordance with claim 12 wherein the oxygen free radical scavenger is allopurinol, glutathione or combinations thereof.

Claim 15

Dependent Claim 24 words

The method in accordance with claim 1 further comprising the step of adding a cryopreservative to the cell suspension after incubating and before freezing.

Claim 16

Dependent Claim 14 words

The method in accordance with claim 14 wherein the cryopreservative is Dimethyl Sulfoxide (DMSO).

Claim 17

Dependent Claim 32 words

The method in accordance with claim 15 further comprising cooling the cell suspension to a temperature of from about 2° C. to about 6° C. after incubation and before adding the DMSO.

Claim 18

Dependent Claim 27 words

The method in accordance with claim 1 wherein the cells are harvested from the donor at a temperature of from about 35° C. to about 38° C.

Claim 19

Independent Claim 178 words

A method for preconditioning and cryopreservation of cells harvested from a donor comprising the steps of: suspending eukaryote or aerobic prokaryote cells harvested from a donor in a cell conditioning and cryopreservation medium; incubating the cell suspension for a period of from about 15 minutes to about 2 hours at a temperature of from about 35° C. and about 38° C.; cooling the cell suspension to a temperature of from about 2° C. to about 6° C.; adding sufficient Dimethyl Sulfoxide (DMSO) to the cell suspension to cause a DMSO concentration in the cells of from about 11.0% to about 16.4% by volume; and then freezing the cells; where the cell conditioning and cryopreservative medium includes from about 2.7 mM to about 3.6 mM adenosine, from about 0.04 mM to about 0.07 mM verapamil, and a cell nutrient matrix comprising a sufficient amount of nutrients sustain the metabolic needs of the harvested cells during the incubation period without producing detectable levels of lactate or substantially depleting the nutrients, so as to maintain the viability of the harvested cells.

Claim 20

Independent Claim 178 words

A method for preconditioning and cryopreservation of cells harvested from a donor comprising the steps of: suspending eukaryote or aerobic prokaryote cells harvested from a donor in a cell conditioning and cryopreservation medium; incubating the cell suspension for a period of from about 15 minutes to about 2 hours at a temperature of from about 35° C. and about 38° C.; cooling the cell suspension to a temperature of from about 2° C. to about 6° C.; adding sufficient Dimethyl Sulfoxide (DMSO) to the cell suspension to cause a DMSO concentration in the cells of from about 11.0% to about 16.4% by volume; and then freezing the cells; where the cell conditioning and cryopreservative medium includes from about 2.7 mM to about 3.6 mM adenosine, from about 0.04 mM to about 0.07 mM verapamil, and a cell nutrient matrix comprising a sufficient amount of nutrients sustain the metabolic needs of the harvested cells during the incubation period without producing detectable levels of lactate or substantially depleting the nutrients, so as to maintain the viability of the harvested cells.

Claim 21

Dependent Claim 27 words

The method in accordance with claim 18 wherein the cells are harvested from the donor at a temperature of from about 35° C. and about 38° C.

Claim 22

Dependent Claim 44 words

The method in accordance with claim 18 wherein the cell nutrient matrix contains at least one amino acid selected from the group comprising alanine, arginine, asparagine, aspartic acid, cystine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

Claim 23

Dependent Claim 36 words

The method in accordance with claim 20 wherein the cell nutrient matrix contains at least one vitamin selected from the group comprising pantothenate, choline chloride, folic acid, inositol, niacinamide, pyridoxal, riboflavin, and thiamine, and a saccharide.

Claim 24

Dependent Claim 20 words

The method in accordance with claim 21 further comprising the step of oxygenating the cell suspension during the incubation period.

Claim 25

Dependent Claim 33 words

The method in accordance with claim 22 wherein the cell suspension is oxygenated by bubbling oxygen through the suspension using a gas having an oxygen content of no less than 80 vol. %.

Claim 26

Dependent Claim 19 words

The method in accordance with claim 23 further comprising adding a oxygen free radical scavenger to the cell suspension.

Claim 27

Dependent Claim 19 words

The method in accordance with claim 24 wherein the oxygen free radical scavenger is allopurinol, glutathione or combinations thereof.

Claim 28

Independent Claim 68 words

A method for freezing cells comprising the steps of: suspending harvested cells in a storage solution; adding a cryopreservative solution to the cells in an initial addition step and in at least on subsequent addition step, where the concentration of cryopreservative in solution is increased for each subsequent addition step, until a predetermined amount of cryopreservative has been absorbed by the cells; and then freezing the cell suspension.

Claim 29

Independent Claim 68 words

A method for freezing cells comprising the steps of: suspending harvested cells in a storage solution; adding a cryopreservative solution to the cells in an initial addition step and in at least on subsequent addition step, where the concentration of cryopreservative in solution is increased for each subsequent addition step, until a predetermined amount of cryopreservative has been absorbed by the cells; and then freezing the cell suspension.

Claim 30

Dependent Claim 16 words

The method in accordance with claim 26 wherein the cells are eukaryote or aerobic prokaryote cells.

Claim 31

Dependent Claim 33 words

The method in accordance with claim 26 further comprising precipitating the cells and removing at least some of the resulting supernatant, then resuspending the cells in the course of the subsequent addition step.

Claim 32

Dependent Claim 22 words

The method in accordance with claim 26 wherein the concentration of cryopreservative in solution is approximately doubled for each subsequent addition step.

Claim 33

Dependent Claim 22 words

The method in accordance with claim 26 further comprising establishing osmotic equilibrium inside and outside of the cells after an addition step.

Claim 34

Dependent Claim 52 words

The method in accordance with claim 26 wherein the cryopreservative is Dimethyl Sulfoxide (DMSO) the cells are maintained at a temperature of from about 2° C. to about 6° C. during each addition step, and a concentration of DMSO absorbed in the cells is from about 11.0% to about 16.4% by volume.

Claim 35

Dependent Claim 32 words

The method in accordance with claim 31 wherein the concentration of cryopreservative in the initial cryopreservative solution is from about 2.7% to about 3.9%, based on the volume of the cryopreservative solution.

Claim 36

Independent Claim 109 words

A method for freezing cells comprising the steps of: suspending harvested eukaryote or aerobic prokaryote cells in a storage solution maintained at a temperature of from about 2° C. to about 6° C.; adding a solution containing Dimethyl Sulfoxide (DMSO) to the cells in an initial addition step and in at least one subsequent addition step, where the concentration of DMSO is approximately doubled for each subsequent addition step; establishing osmotic equilibrium inside and outside of the cells after at least one of the subsequent addition steps, until the concentration of DMSO in the cells is from about 11.0% to about 16.4% by volume; and then freezing the cells.

Claim 37

Independent Claim 109 words

A method for freezing cells comprising the steps of: suspending harvested eukaryote or aerobic prokaryote cells in a storage solution maintained at a temperature of from about 2° C. to about 6° C.; adding a solution containing Dimethyl Sulfoxide (DMSO) to the cells in an initial addition step and in at least one subsequent addition step, where the concentration of DMSO is approximately doubled for each subsequent addition step; establishing osmotic equilibrium inside and outside of the cells after at least one of the subsequent addition steps, until the concentration of DMSO in the cells is from about 11.0% to about 16.4% by volume; and then freezing the cells.

Claim 38

Dependent Claim 33 words

The method in accordance with claim 33 further comprising precipitating the cells and removing at least some of the resulting supernatant, then resuspending the cells in the course of the subsequent addition step.

Claim 39

Dependent Claim 32 words

The method in accordance with claim 34 wherein the concentration of cryopreservative in the initial cryopreservative solution is from about 2.7% to about 3.9%, based on the volume of the cryopreservative solution.

Claim 40

Independent Claim 101 words

A method for removing a cryopreservative from harvested cells containing the cryopreservative comprising the steps of: adding an initial cryopreservative solution to a suspension of the harvested cells containing the cryopreservative, where the concentration of cryopreservative in the initial cryopreservative solution is less than the concentration of cryopreservative in the cells; adding at least one subsequent cryopreservative solution to the cells where the concentration of cryopreservative in the solution is further decreased for each subsequent cryopreservative solution; then adding an aqueous liquid that contains no cryopreservative to the cells so that the final concentration of cryopreservative in the cells is negligible.

Claim 41

Independent Claim 101 words

A method for removing a cryopreservative from harvested cells containing the cryopreservative comprising the steps of: adding an initial cryopreservative solution to a suspension of the harvested cells containing the cryopreservative, where the concentration of cryopreservative in the initial cryopreservative solution is less than the concentration of cryopreservative in the cells; adding at least one subsequent cryopreservative solution to the cells where the concentration of cryopreservative in the solution is further decreased for each subsequent cryopreservative solution; then adding an aqueous liquid that contains no cryopreservative to the cells so that the final concentration of cryopreservative in the cells is negligible.

Claim 42

Dependent Claim 16 words

The method in accordance with claim 36 wherein the cells are eukaryote or aerobic prokaryote cells.

Claim 43

Dependent Claim 36 words

The method in accordance with claim 36 further comprising precipitating the cells and removing at least some of the resulting supernatant, then resuspending the cells in the course of adding at least one subsequent cryopreservative solution.

Claim 44

Dependent Claim 34 words

The method in accordance with claim 36 further comprising precipitating the cells and removing at least some of the resulting supernatant, then resuspending the cells in the course of the adding the aqueous liquid.

Claim 45

Dependent Claim 24 words

The method in accordance with claim 36 wherein the concentration of cryopreservative in solution is approximately halved for each subsequent addition of cryopreservative solution.

Claim 46

Dependent Claim 24 words

The method in accordance with claim 36 further comprising establishing osmotic equilibrium inside and outside of the cells after adding a subsequent cryopreservative solution.

Claim 47

Dependent Claim 14 words

The method in accordance with claim 36 wherein the cryopreservative is Dimethyl Sulfoxide (DMSO).

Claim 48

Independent Claim 107 words

A method for removing Dimethyl Sulfoxide (DMSO) from harvested cells containing DMSO comprising the steps of: adding an initial DMSO solution to a suspension of harvested eukaryote or aerobic prokaryote cells containing DMSO, where the concentration of DMSO in the initial solution is approximately one-half of the concentration of DMSO in the cell; adding at least one subsequent DMSO solution to the cells where the concentration of DMSO in the solution is decreased by approximately one-half for each subsequent DMSO cryopreservative solution; then adding an aqueous liquid that does not contain DMSO to the cells so that the final concentration of DMSO in the cells is negligible.

Claim 49

Independent Claim 107 words

A method for removing Dimethyl Sulfoxide (DMSO) from harvested cells containing DMSO comprising the steps of: adding an initial DMSO solution to a suspension of harvested eukaryote or aerobic prokaryote cells containing DMSO, where the concentration of DMSO in the initial solution is approximately one-half of the concentration of DMSO in the cell; adding at least one subsequent DMSO solution to the cells where the concentration of DMSO in the solution is decreased by approximately one-half for each subsequent DMSO cryopreservative solution; then adding an aqueous liquid that does not contain DMSO to the cells so that the final concentration of DMSO in the cells is negligible.

Claim 50

Dependent Claim 36 words

The method in accordance with claim 43 further comprising precipitating the cells and removing at least some of the resulting supernatant, then resuspending the cells in the course of adding at least one subsequent DMSO solution.

Claim 51

Dependent Claim 33 words

The method in accordance with claim 44 further comprising precipitating the cells and removing at least some of the resulting supernatant, then resuspending the cells in the course of adding the aqueous liquid.

Claim 52

Dependent Claim 24 words

The method in accordance with claim 45 further comprising establishing osmotic equilibrium inside and outside of the cells after adding a subsequent DMSO solution.

Annuity Stage	Window Opens	Payment Deadline	Surcharge Period Until	Estimated Fee	Status
3.5 Years (Year 4)	2003-10-31	2004-05-01	2004-10-31	$2000.00	Window Closed / Lapsed
7.5 Years (Year 8)	2007-10-31	2008-05-01	2008-10-31	$3760.00	Window Closed / Lapsed
11.5 Years (Year 12)	2011-10-31	2012-05-01	2012-10-31	$7700.00	Window Closed / Lapsed

Method for conditioning and cryopreserving cells

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