Patent US4996154A successfully refreshed from Google Patents.
US4996154A
Expired / Lapsed
US
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Method for growing cellular tissue
Assignee: EMD Millipore Corp
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Filing: 1989-05-04
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Granted: 1991-02-26
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Expires: 2009-05-04
Abstract
Abstract A method is described for producing tissue from cells, such as keratinocyte, epithelial or endothelial cells, in virto. Cells are grown on a porous cell-growth substrate having specific growth factors therein e.g., on a collagen activated coated microporous substrate. Cells can be grown to form a confluent monolayer or differentiated tissue which can be used for studying cell toxicology.
Inventors
- 👤 Joseph E. Gabriels, Jr.
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-24 20:08
CPC Classifications
C; C12; C12N; C12N5/00; C12N5/06; C12N5/0602; C12N5/0625; C12N5/0629; A; A61; A61L; A61L27/00; A61L27/50; A61L27/60; C12N5/0684; C12N5/0686; C12N2533/00; C12N2533/50; C12N2533/54
Claims Analysis
Total Claims: 20
Claim 1
Independent Claim
83 words
A method of producing tissue from keratinocyte cells, comprising the steps of: a. contacting a collagen coated polymeric microporous substrate suitable for growing cells with growth factor specific for growth of said keratinocyte cells in vitro, wherein the collagen comprises activated sites for attaching the growth factors thereto; b. subsequently seeding keratinocyte cells onto the microporous substrate; and c. maintaining the seeded microporous substrate under conditions suitable for cell growth to thereby produce a confluent monolayer of tissue or uniformly differentiated multilayer tissue.
Claim 2
Independent Claim
83 words
A method of producing tissue from keratinocyte cells, comprising the steps of: a. contacting a collagen coated polymeric microporous substrate suitable for growing cells with growth factor specific for growth of said keratinocyte cells in vitro, wherein the collagen comprises activated sites for attaching the growth factors thereto; b. subsequently seeding keratinocyte cells onto the microporous substrate; and c. maintaining the seeded microporous substrate under conditions suitable for cell growth to thereby produce a confluent monolayer of tissue or uniformly differentiated multilayer tissue.
Claim 3
Dependent Claim
25 words
The method of claim 1, wherein tissue grown on the microporous substrate is maintained as a submerged culture or raised to the air liquid interface.
Claim 4
Dependent Claim
8 words
Tissue produced by the method of claim 1.
Claim 5
Independent Claim
151 words
A method of producing tissue from keratinocyte cells, comprising the steps of: a. treating collagen coated on a polymeric microporous substrate suitable for growing cells, to thereby provide activated sites in the collagen for attaching growth factors thereto; b. contacting the collagen-coated microporous substrate with cell culture medium comprising epidermal growth factor and pituitary extract containing growth factor therein, under conditions whereby the growth factors are dispersed within the substrate and attached thereto; c. removing the medium from the substrate to provide a medium-free substrate; d. contacting the medium-free microporous substrate with a sufficient amount of cell culture medium comprising serum, epidermal growth factor and pituitary extract containing growth factor therein, to saturate remaining activated sites; e. subsequently seeding keratinocyte cells onto the microporous substrate; and f. maintaining the seeded microporous substrate under conditions suitable for cell growth to thereby produce a confluent monolayer of tissue or uniformly differentiated multilayer tissue.
Claim 6
Independent Claim
151 words
A method of producing tissue from keratinocyte cells, comprising the steps of: a. treating collagen coated on a polymeric microporous substrate suitable for growing cells, to thereby provide activated sites in the collagen for attaching growth factors thereto; b. contacting the collagen-coated microporous substrate with cell culture medium comprising epidermal growth factor and pituitary extract containing growth factor therein, under conditions whereby the growth factors are dispersed within the substrate and attached thereto; c. removing the medium from the substrate to provide a medium-free substrate; d. contacting the medium-free microporous substrate with a sufficient amount of cell culture medium comprising serum, epidermal growth factor and pituitary extract containing growth factor therein, to saturate remaining activated sites; e. subsequently seeding keratinocyte cells onto the microporous substrate; and f. maintaining the seeded microporous substrate under conditions suitable for cell growth to thereby produce a confluent monolayer of tissue or uniformly differentiated multilayer tissue.
Claim 7
Independent Claim
128 words
A method of preparing a substrate for growing tissue from one cell type thereon, comprising the steps of: a. treating a collagen coated polymeric microporous substrate to thereby provide activated sites in the collagen for attaching growth factor thereto; b. contacting the collagen coated microporous substrate with cell culture medium comprising growth factor specific for growth of said cells under conditions whereby the growth factor is dispersed within the substrate and attached thereto; c. removing the medium from the substrate to provide a medium-free substrate; and d. contacting the medium-free substrate with an amount of cell culture medium comprising growth factor and non-specific protein sufficient to saturate remaining activated sites, to thereby produce a substrate for growing a confluent monolayer of tissue or uniformly differentiated multilayer tissue thereon.
Claim 8
Independent Claim
128 words
A method of preparing a substrate for growing tissue from one cell type thereon, comprising the steps of: a. treating a collagen coated polymeric microporous substrate to thereby provide activated sites in the collagen for attaching growth factor thereto; b. contacting the collagen coated microporous substrate with cell culture medium comprising growth factor specific for growth of said cells under conditions whereby the growth factor is dispersed within the substrate and attached thereto; c. removing the medium from the substrate to provide a medium-free substrate; and d. contacting the medium-free substrate with an amount of cell culture medium comprising growth factor and non-specific protein sufficient to saturate remaining activated sites, to thereby produce a substrate for growing a confluent monolayer of tissue or uniformly differentiated multilayer tissue thereon.
Claim 9
Dependent Claim
14 words
The method of claim 5 wherein the microporous polymeric substrate is a microporous membrane.
Claim 10
Dependent Claim
14 words
A substrate for growing tissue from cells produced by the method of claim 5.
Claim 11
Independent Claim
104 words
A method of producing tissue from one cell type in vitro, comprising the steps of: (a) treating a collagen coated porous substrate suitable for growing cells, to thereby provide activated sites in the collagen for attaching growth factor thereto; (b) contacting the collagen coated porous substrate with growth factor specific for growth of said cells in vitro whereby the growth factor is dispersed within the substrate and attached thereto; (c) subsequently seeding the cells onto the porous substrate; and (d) maintaining the seeded porous substrate under conditions suitable for cell growth to thereby produce a confluent monolayer of tissue or uniformly differentiated multilayer tissue.
Claim 12
Independent Claim
104 words
A method of producing tissue from one cell type in vitro, comprising the steps of: (a) treating a collagen coated porous substrate suitable for growing cells, to thereby provide activated sites in the collagen for attaching growth factor thereto; (b) contacting the collagen coated porous substrate with growth factor specific for growth of said cells in vitro whereby the growth factor is dispersed within the substrate and attached thereto; (c) subsequently seeding the cells onto the porous substrate; and (d) maintaining the seeded porous substrate under conditions suitable for cell growth to thereby produce a confluent monolayer of tissue or uniformly differentiated multilayer tissue.
Claim 13
Dependent Claim
14 words
The method of claim 8 wherein the porous substrate is a microporous polymeric membrane.
Claim 14
Dependent Claim
8 words
Tissue produced by the method of claim 8.
Claim 15
Dependent Claim
25 words
The method of claim 8, wherein tissue grown on the porous substrate is maintained as a submerged culture or raised to the air liquid interface.
Claim 16
Independent Claim
144 words
A method of producing tissue from one cell type in vitro, comprising the steps of: a. treating a collagen coated porous substrate suitable for growing cells to thereby provide activated sites in the collagen for attaching growth factor thereto; b. contacting the collagen coated porous substrate with cell culture medium comprising growth factor specific for growth of said cells under conditions whereby the growth factor is dispersed within the substrate and attached thereto; c. removing the medium from the substrate to provide a medium-free substrate; d. contacting the medium-free substrate with an amount of cell culture medium comprising growth factor and non-specific protein, sufficient to saturate remaining activated sites; e. subsequently seeding the cells onto the porous substrate; and f. maintaining the seeded porous substrate under conditions suitable for cell growth to thereby produce a confluent monolayer of tissue or uniformly differentiated multilayer tissue.
Claim 17
Independent Claim
144 words
A method of producing tissue from one cell type in vitro, comprising the steps of: a. treating a collagen coated porous substrate suitable for growing cells to thereby provide activated sites in the collagen for attaching growth factor thereto; b. contacting the collagen coated porous substrate with cell culture medium comprising growth factor specific for growth of said cells under conditions whereby the growth factor is dispersed within the substrate and attached thereto; c. removing the medium from the substrate to provide a medium-free substrate; d. contacting the medium-free substrate with an amount of cell culture medium comprising growth factor and non-specific protein, sufficient to saturate remaining activated sites; e. subsequently seeding the cells onto the porous substrate; and f. maintaining the seeded porous substrate under conditions suitable for cell growth to thereby produce a confluent monolayer of tissue or uniformly differentiated multilayer tissue.
Claim 18
Dependent Claim
14 words
The method of claim 12 wherein the porous substrate is a microporous polymeric membrane.
Claim 19
Dependent Claim
11 words
The method of claim 12 wherein the cells are keratinocyte cells.
Claim 20
Dependent Claim
8 words
Tissue produced by the method of claim 12.
References Cited (Backward Citations)
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1994-02-25 | 1994-08-27 | 1995-02-26 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1998-02-25 | 1998-08-27 | 1999-02-26 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2002-02-25 | 2002-08-27 | 2003-02-26 | $7700.00 | Window Closed / Lapsed |