Patent US5175093A successfully refreshed from Google Patents.
US5175093A
Expired / Lapsed
US
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Bioactive cells immobilized in alginate beads containing voids formed with polyethylene glycol
Assignee: Lehigh University
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Filing: 1989-11-07
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Granted: 1992-12-29
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Expires: 2009-11-07
Abstract
Abstract Bioactive cells are immobilized by suspending bioactive cells in an aqueous solution of a salt-free osmolarity adjuster, alginate and polyethylene glycol, dividing the suspension into bead-sized globules, contacting the globules with a solution of divalent cations to gel the alginate to form beads containing polyethylene glycol-filled areas and removing the polyethylene glycol to form voids in the beads entrapping the bioactive cells. By perfusing the beads with a nutrient medium, the cells can proliferate in the voids to produce high cell densities and produce a product which can be separated from the medium.
Inventors
- 👤 Douglas B. Seifert
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-06 04:27
CPC Classifications
C; C12; C12N; C12N1/00; C12N1/02; C12N11/00; C12N11/02; C12N11/04
Claims Analysis
Total Claims: 22
Claim 1
Independent Claim
136 words
Method of immobilizing bioactive cells comprising forming a suspension of said cells in a solution of a salt-free osmolarity adjuster which prevents precipitation of alginate in said solution, between 0.25 wt % and 5 wt % alginate and between 10 wt T and 30 wt % polyethylene glycol in water, dividing said suspension into bead-sized globules, between 20 μ and 5 mm in diameter, contacting said globules with a solution of divalent cations to cause said alginate to gel into gelled alginate beads, and removing said polyethylene glycol from said gelled alginate beads, to produce alginate gel beads of between 20 μ and 5 mm in diameter containing said cells, said beads containing a multiplicity of enclosed void spaces projecting radially from the center of the beads, and said cells being entrapped in said void spaces.
Claim 2
Dependent Claim
136 words
Method of immobilizing bioactive cells comprising forming a suspension of said cells in a solution of a salt-free osmolarity adjuster which prevents precipitation of alginate in said solution, between 0.25 wt % and 5 wt % alginate and between 10 wt T and 30 wt % polyethylene glycol in water, dividing said suspension into bead-sized globules, between 20 μ and 5 mm in diameter, contacting said globules with a solution of divalent cations to cause said alginate to gel into gelled alginate beads, and removing said polyethylene glycol from said gelled alginate beads, to produce alginate gel beads of between 20 μ and 5 mm in diameter containing said cells, said beads containing a multiplicity of enclosed void spaces projecting radially from the center of the beads, and said cells being entrapped in said void spaces.
Claim 3
Dependent Claim
16 words
The method of claim 1 wherein said cells are 1/2 μ to 30 μ in diameter.
Claim 4
Dependent Claim
15 words
The method of claim 1 wherein said suspension is divided by extrusion of said suspension.
Claim 5
Dependent Claim
15 words
A celled alginate bead containing immobilized bioactive cells produced by the method of claim 1.
Claim 6
Dependent Claim
38 words
A gelled alginate bead containing immobilized bioactive cells produced by the method of claim 1 wherein said void spaces have a width from 5 μ to 200 μ, and a length up to the radius of the bead.
Claim 7
Dependent Claim
38 words
A gelled alginate bead containing immobilized bioactive cells produced by the method of claim 2 wherein said void spaces have a width from 5 μ to 200 μ, and a length up to the radius of the bead.
Claim 8
Dependent Claim
30 words
The gelled alginate bead containing immobilized bioactive cells of claim 4 or 5 or 6 wherein said bead generally spherical and is between 0.5 mm and 5 mm in diameter.
Claim 9
Dependent Claim
24 words
The gelled alginate bead containing immobilized bioactive cells of claim 7 wherein said void spaces have a width from 10 μ to 200 μ.
Claim 10
Dependent Claim
22 words
The gelled alginate bead containing immobilized bioactive cells of claim 4 or 5 or 6 wherein said bioactive cells comprise insect cells.
Claim 11
Dependent Claim
22 words
The gelled alginate bead containing immobilized bioactive cells of claim 4 or 5 or 6 wherein said bioactive cells comprise mammalian cells.
Claim 12
Dependent Claim
21 words
The gelled alginate bead containing immobilized bioactive cells of claim 4 or 5 or 6 wherein said bioactive cells comprise hybridomas.
Claim 13
Dependent Claim
37 words
The gelled alginate bead containing immobilized bioactive cells of claim 4 or 5 or 6 wherein said bioactive cells are anchorage dependent cells and are combined with a compatible anchorage substrate, also included within said void spaces.
Claim 14
Independent Claim
169 words
A method for continuously producing and removing the product of bioactive cells comprising first immobilizing said bioactive cells by forming a suspension of said cells in an aqueous solution of a salt-free osmolarity adjuster which prevents precipitation of alginate in said solution, between 0.25 wt % and 5 wt % alginate and between 10 wt % and 30 wt T polyethylene glycol, dividing said suspension into bead-sized globules of 20 μ to 5 mm in diameter and contacting said globules with a solution of divalent cations to cause said alginate to form gel beads, removing said polyethylene glycol from said gel beads to produce alginate gel beads of between 20 μ and 5 mm in diameter containing said cells, said beads containing a multiplicity of enclosed void spaces projecting radially from the center of the beads, and said cells being entrapped in said void spaces and thereafter perfusing a bed of said beads containing said cells with a medium and removing products produced by said cells from the medium.
Claim 15
Independent Claim
169 words
A method for continuously producing and removing the product of bioactive cells comprising first immobilizing said bioactive cells by forming a suspension of said cells in an aqueous solution of a salt-free osmolarity adjuster which prevents precipitation of alginate in said solution, between 0.25 wt % and 5 wt % alginate and between 10 wt % and 30 wt T polyethylene glycol, dividing said suspension into bead-sized globules of 20 μ to 5 mm in diameter and contacting said globules with a solution of divalent cations to cause said alginate to form gel beads, removing said polyethylene glycol from said gel beads to produce alginate gel beads of between 20 μ and 5 mm in diameter containing said cells, said beads containing a multiplicity of enclosed void spaces projecting radially from the center of the beads, and said cells being entrapped in said void spaces and thereafter perfusing a bed of said beads containing said cells with a medium and removing products produced by said cells from the medium.
Claim 16
Dependent Claim
22 words
The method of claim 13 wherein said cells are hybridoma ATCC HB 121 cells and said medium is Dulbecco's Modified Eagle Medium.
Claim 17
Dependent Claim
18 words
A method as recited in claim 13 wherein said medium also includes cell nutrients for said bioactive cells.
Claim 18
Independent Claim
157 words
A method for obtaining the products of bioactive cells comprising: mixing said bioactive cells with an aqueous solution of a salt-free osmolarity adjuster which prevents precipitation of alginate in said solution, between 0.25 wt % and 5 wt % alginate and between 10 wt % and 30 wt % polyethylene glycol; forming porous beads of said mixture by adding bead size globules of said mixture to a divalent ion-containing solution; removing said polyethylene glycol from said beads to produce alginate gel beads of between 20 μ and 5 mm in diameter containing said cells, said beads containing a multiplicity of enclosed void spaces projecting radially from the center of the beads, and said cells being entrapped in said void spaces; placing said beads in a medium including a nutrient for said cells; allowing said cells to grow and produce cell products in said medium; and draining at least part of said medium, and separating therefrom cell products.
Claim 19
Independent Claim
157 words
A method for obtaining the products of bioactive cells comprising: mixing said bioactive cells with an aqueous solution of a salt-free osmolarity adjuster which prevents precipitation of alginate in said solution, between 0.25 wt % and 5 wt % alginate and between 10 wt % and 30 wt % polyethylene glycol; forming porous beads of said mixture by adding bead size globules of said mixture to a divalent ion-containing solution; removing said polyethylene glycol from said beads to produce alginate gel beads of between 20 μ and 5 mm in diameter containing said cells, said beads containing a multiplicity of enclosed void spaces projecting radially from the center of the beads, and said cells being entrapped in said void spaces; placing said beads in a medium including a nutrient for said cells; allowing said cells to grow and produce cell products in said medium; and draining at least part of said medium, and separating therefrom cell products.
Claim 20
Dependent Claim
22 words
The method of claim 16 wherein said cells are hybridoma ATCC HB 121 cells and said medium is Dulbecco's Modified Eagle Medium.
Claim 21
Dependent Claim
21 words
The method of claim 17 wherein said beads are generally spherical and are between 0.5 mm and 5 mm in diameter.
Claim 22
Dependent Claim
35 words
The method of claim 16, wherein: said bead-sized globules are between 0.5 mm and 5 mm in diameter; said polyethylene glycol has a molecular weight of 8000; and said divalent ion is Ca 2+ .
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) | 1995-12-29 | 1996-06-29 | 1996-12-29 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1999-12-29 | 2000-06-29 | 2000-12-29 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2003-12-29 | 2004-06-29 | 2004-12-29 | $7700.00 | Window Closed / Lapsed |