US6238922B1
Expired / Lapsed
US
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Use of collagenase in the preparation of neural stem cell cultures
Assignee: StemCells California Inc
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Filing: 1999-02-26
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Granted: 2001-05-29
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Expires: 2019-02-26
Abstract
Abstract The invention provides a method for using collagenase to dissociate neural stem cells in neural stem cell cultures when passaging aggregated neural stem cells. The collagenase treatment results in an increased cell viability and an increased number of proliferated neural stem cells over time.
Inventors
- 👤 Nobuko Uchida
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-03 05:58
CPC Classifications
C; C12; C12N; C12N5/00; C12N5/06; C12N5/0602; C12N5/0618; C12N5/0623; C12N2509/00
Claims Analysis
Total Claims: 38
Claim 1
Independent Claim
138 words
A method for the in vitro proliferation of a neural stem cell culture comprising the steps of: (a) obtaining dissociated neural tissue in cell suspension, the suspension containing one or more multipotent neural stem cells capable of producing progeny that are capable of differentiating into neurons and glia; (b) culturing the cell suspension in a culture medium containing at least one proliferation-inducing growth factor to proliferate the neural stem cells in (a) to generate a neural stem cell culture comprising aggregated neural stem cells; and (c) passaging the cell culture in (b) by treating the culture with an amount of a collagenase preparation effective to dissociate the aggregated neural stem cells in the culture and passing the cell culture to a culture medium containing at least one proliferation-inducing growth factor to further proliferate the neural stem cell culture.
Claim 2
Independent Claim
138 words
A method for the in vitro proliferation of a neural stem cell culture comprising the steps of: (a) obtaining dissociated neural tissue in cell suspension, the suspension containing one or more multipotent neural stem cells capable of producing progeny that are capable of differentiating into neurons and glia; (b) culturing the cell suspension in a culture medium containing at least one proliferation-inducing growth factor to proliferate the neural stem cells in (a) to generate a neural stem cell culture comprising aggregated neural stem cells; and (c) passaging the cell culture in (b) by treating the culture with an amount of a collagenase preparation effective to dissociate the aggregated neural stem cells in the culture and passing the cell culture to a culture medium containing at least one proliferation-inducing growth factor to further proliferate the neural stem cell culture.
Claim 3
Dependent Claim
16 words
The method of claim 1 wherein the amount of the collagenase preparation is between 18-180 mU/ml.
Claim 4
Dependent Claim
16 words
The method of claim 1 wherein the amount of the collagenase preparation is between 54-126 mU/ml.
Claim 5
Dependent Claim
28 words
The method of claim 1 wherein the collagenase preparation further comprises at least one molecule selected from the group consisting of sulfhydryl protease, clostripain, aminopeptidase, or combinations thereof.
Claim 6
Dependent Claim
18 words
The method of claim 1 wherein the collagenase preparation is substantially pure, and contains minimal secondary proteolytic activity.
Claim 7
Dependent Claim
43 words
The method of claim 1 , wherein the proliferation-inducing growth factor is selected from the group consisting of epidermal growth factor, amphiregulin, acidic fibroblast growth factor, basic fibroblast growth factor, transforming growth factor alpha, leukocyte inhibitory factor (LIF), glycostatin C and combinations thereof.
Claim 8
Dependent Claim
17 words
The method of claim 1 wherein the neural stem cell culture comprises genetically modified neural stem cells.
Claim 9
Dependent Claim
40 words
The method of claim 1 , further comprising the step of differentiating the neural stem cell culture of (c) to produce a cell culture comprising differentiated neural cells selected from the group consisting of astrocytes, neurons, oligodendrocytes, and combinations thereof.
Claim 10
Dependent Claim
34 words
The method of claim 1 or claim 8 , further comprising contacting the neural stem cell culture with a biological agent, and determining the effects of the biological agent on cells in the culture.
Claim 11
Dependent Claim
15 words
The method of claim 1 wherein the neural stem cell culture is a suspension culture.
Claim 12
Dependent Claim
15 words
The method of claim 1 wherein the neural stem cell culture is an adhesion culture.
Claim 13
Dependent Claim
16 words
The method of claim 1 wherein the neural stem cell culture comprises human neural stem cells.
Claim 14
Independent Claim
155 words
A method for the in vitro proliferation of a neural stem cell culture comprising the steps of: (a) obtaining dissociated neural tissue in cell suspension, the suspension containing one or more multipotent neural stem cells capable of producing progeny that are capable of differentiating into neurons and glia; (b) culturing the cell suspension in a culture medium containing at least one proliferation-inducing growth factor to proliferate the neural stem cells in (a) to generate a neural stem cell culture comprising aggregated neural stem cells; and (c) passaging the cell culture in (b) by treating the culture with an amount of a collagenase preparation effective to dissociate the aggregated neural stem cells in the culture and passing the cell culture to a culture medium containing at least one proliferation-inducing growth factor to further proliferate the neural stem cell culture, wherein the percent viability of the cells in the culture is at least 60% after being passaged.
Claim 15
Independent Claim
155 words
A method for the in vitro proliferation of a neural stem cell culture comprising the steps of: (a) obtaining dissociated neural tissue in cell suspension, the suspension containing one or more multipotent neural stem cells capable of producing progeny that are capable of differentiating into neurons and glia; (b) culturing the cell suspension in a culture medium containing at least one proliferation-inducing growth factor to proliferate the neural stem cells in (a) to generate a neural stem cell culture comprising aggregated neural stem cells; and (c) passaging the cell culture in (b) by treating the culture with an amount of a collagenase preparation effective to dissociate the aggregated neural stem cells in the culture and passing the cell culture to a culture medium containing at least one proliferation-inducing growth factor to further proliferate the neural stem cell culture, wherein the percent viability of the cells in the culture is at least 60% after being passaged.
Claim 16
Dependent Claim
23 words
The method of claim 13 , wherein the percent viability of the cells in the culture is at least 75% after being passaged.
Claim 17
Dependent Claim
23 words
The method of claim 13 , wherein the percent viability of the cells in the culture is at least 85% after being passaged.
Claim 18
Dependent Claim
17 words
The method of claim 13 , wherein the amount of the collagenase preparation is between 18-180 mU/ml.
Claim 19
Dependent Claim
17 words
The method of claim 13 , wherein the amount of the collagenase preparation is between 54-126 mU/ml.
Claim 20
Dependent Claim
29 words
The method of claim 13 , wherein the collagenase preparation further comprises at least one molecule selected from the group consisting of sulfhydryl protease, clostripain, aminopeptidase, or combinations thereof.
Claim 21
Dependent Claim
19 words
The method of claim 13 , wherein the collagenase preparation is substantially pure, and contains minimal secondary proteolytic activity.
Claim 22
Dependent Claim
18 words
The method of claim 13 , wherein the neural stem cell culture comprises genetically modified neural stem cells.
Claim 23
Dependent Claim
40 words
The method of claim 13 , further comprising the step of differentiating the neural stem cell culture of (c) to produce a cell culture comprising differentiated neural cells selected from the group consisting of astrocytes, neurons, oligodendrocytes, and combinations thereof.
Claim 24
Dependent Claim
15 words
The method of claim 13 wherein the neural stem cell culture is a suspension culture.
Claim 25
Dependent Claim
15 words
The method of claim 13 wherein the neural stem cell culture is an adhesion culture.
Claim 26
Dependent Claim
16 words
The method of claim 13 wherein the neural stem cell culture comprises human neural stem cells.
Claim 27
Independent Claim
123 words
A method for expanding a neural stem cell culture comprising the steps of: (a) culturing a neural stem cell culture that contains aggregated multipotent neural stem cells capable of producing progeny that are capable of differentiating into neurons and glia in a culture medium containing at least one proliferation-inducing growth factor to proliferate the culture; and (b) passaging the cell culture in (a) by treating the culture with an amount of a collagenase preparation effective to dissociate the aggregated neural stem cells in the culture and passing the cell culture to a culture medium containing at least one proliferation-inducing growth factor to further proliferate the neural stem cell culture, wherein there is at least a three-fold increase in cell number between weekly passages.
Claim 28
Independent Claim
123 words
A method for expanding a neural stem cell culture comprising the steps of: (a) culturing a neural stem cell culture that contains aggregated multipotent neural stem cells capable of producing progeny that are capable of differentiating into neurons and glia in a culture medium containing at least one proliferation-inducing growth factor to proliferate the culture; and (b) passaging the cell culture in (a) by treating the culture with an amount of a collagenase preparation effective to dissociate the aggregated neural stem cells in the culture and passing the cell culture to a culture medium containing at least one proliferation-inducing growth factor to further proliferate the neural stem cell culture, wherein there is at least a three-fold increase in cell number between weekly passages.
Claim 29
Dependent Claim
19 words
The method of claim 25 wherein there is at least a five-fold increase in cell number between weekly passages.
Claim 30
Dependent Claim
15 words
The method of claim 25 wherein the neural stem cell culture is a suspension culture.
Claim 31
Dependent Claim
15 words
The method of claim 25 wherein the neural stem cell culture is an adhesion culture.
Claim 32
Dependent Claim
16 words
The method of claim 25 wherein the neural stem cell culture comprises human neural stem cells.
Claim 33
Independent Claim
132 words
A method for expanding a neural stem cell culture comprising the steps of: (a) culturing a neural stem cell culture that contains aggregated multipotent neural stem cells capable of producing progeny that are capable of differentiating into neurons and glia in a culture medium containing at least one proliferation-inducing growth factor to proliferate the culture; and (b) passaging the cell culture in (a) by treating the culture with an amount of a collagenase preparation effective to dissociate the agregated neural stem cells in the culture and passing the cell culture to a culture medium containing at least one proliferation-inducing growth factor to further proliferate the neural stem cell culture, wherein the accumulated cell number can be expanded from 10 6 cells to at least 10 9 cells in less than 49 days.
Claim 34
Independent Claim
132 words
A method for expanding a neural stem cell culture comprising the steps of: (a) culturing a neural stem cell culture that contains aggregated multipotent neural stem cells capable of producing progeny that are capable of differentiating into neurons and glia in a culture medium containing at least one proliferation-inducing growth factor to proliferate the culture; and (b) passaging the cell culture in (a) by treating the culture with an amount of a collagenase preparation effective to dissociate the agregated neural stem cells in the culture and passing the cell culture to a culture medium containing at least one proliferation-inducing growth factor to further proliferate the neural stem cell culture, wherein the accumulated cell number can be expanded from 10 6 cells to at least 10 9 cells in less than 49 days.
Claim 35
Dependent Claim
28 words
The method of claim 30 wherein the accumulated cell number can be expanded from 10 6 cells to at least 10 9 cells in less than 42 days.
Claim 36
Dependent Claim
15 words
The method of claim 30 wherein the neural stem cell culture is a suspension culture.
Claim 37
Dependent Claim
15 words
The method of claim 30 wherein the neural stem cell culture is an adhesion culture.
Claim 38
Dependent Claim
17 words
The method of claim 30 , wherein the neural stem cell culture comprises human neural stem cells.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 2004-05-28 | 2004-11-27 | 2005-05-29 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2008-05-28 | 2008-11-27 | 2009-05-29 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2012-05-28 | 2012-11-27 | 2013-05-29 | $7700.00 | Window Closed / Lapsed |