Patent EP0097907A2 successfully refreshed from Google Patents.
EP0097907A2
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EP
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Cell culture microcarriers
Assignee: Flow General Inc
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Filing: 1983-06-21
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Granted: —
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Expires: 2003-06-21
Abstract
Abstract Cell culture microcarriers made of water-insolubilized protein. The protein is substantially homogeneously distributed throughout the microcarrier. The cell culture microcarrier is made by forming droplets of an aqueous solution of a protein, suspending the droplets in a fluid, solidifying the suspended droplets into cell culture microcarriers and recovering the cell culture microcarriers. Preferably, prior to recovery, the cell culture microcarriers are hardened with a crosslinking agent, preferably glutaraldehyde and neutralized with a dilute solution of the protein. An apparatus for converting a water-based protein solution to microspheres. The apparatus has a heated, thermostatically controlled reservoir for the protein solution. The protein solution is supplied to a spray head. The protein solution is maintained at approximately at least the same temperature at the spray head as in the reservoir and a predetermined pressure is maintained on the protein solution at the spray head. A receptacle receives the droplets emitted by the spray head. The droplets are suspended and solidified into microspheres by flowing a low temperature, water immiscible suspension liquid across the receptacle and microspheres are collected. The cell culture microcarriers have been used to grow and harvest anchorage dependent cells and anchorage dependent cell products, such as interferon. The cell culture microcarriers produce outstanding cell growth and possess superior subcultivation ability.
Inventors
- 👤 Wolfgang K. Haller
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-25 20:02
CPC Classifications
C; C12; C12N; C12N5/00; C12N5/0068; C12N5/0075; C12N2533/00; C12N2533/50; C12N2533/54
Claims Analysis
Total Claims: 120
Claim 1
Independent Claim
17 words
A cell culture microcarrier comprising a water-insolubilized protein, said protein being substantially homogeneously distributed throughout said microcarrier.
Claim 2
Dependent Claim
17 words
A cell culture microcarrier comprising a water-insolubilized protein, said protein being substantially homogeneously distributed throughout said microcarrier.
Claim 3
Dependent Claim
26 words
The microcarrier of claim 1, wherein said protein is selected from the group consisting of collagen, casein, soybean protein, silk, keratin, egg proteins and blood proteins.
Claim 4
Dependent Claim
10 words
The microcarrier of claim 2, wherein said protein is collagen.
Claim 5
Dependent Claim
20 words
The microcarrier of claim 1, wherein said microcarrier comprises the reaction product of said protein and a cross- linking agent.
Claim 6
Dependent Claim
11 words
The microcarrier of claim 4, wherein said crosslinking agent is multifunctional.
Claim 7
Dependent Claim
39 words
The microcarrier of claim 5, wherein said multifunctional crosslinking agent contains a functional group selected from the group consisting of aldehydes, epoxies, haloalkyls, isothiocyanates, isocyanates, diazo, arylnitrenes, azides, acylhalides, acylanhydrides, diolcarbonates, chloroformates, imidoesters, N-hydroxysuccinimidylesters, maleinimides, carboxydiimidazols, formylesters and halotriazines.
Claim 8
Dependent Claim
12 words
The microcarrier of claim 6, wherein said multifunctional crosslinking agent is glutaraldehyde.
Claim 9
Dependent Claim
16 words
The microcarrier of claim 6, wherein said multifunctional crosslinking agent is 1, 4, butanediol diglycidyl ether.
Claim 10
Dependent Claim
22 words
The microcarrier of claim 4, wherein said crosslinking agent is selected for the group consisting of formaldehyde, epichlorohydrin, phosgene and aldehyde starch.
Claim 11
Dependent Claim
18 words
The microcarrier of claim 1, wherein said microcarrier has a diameter of about 50 to about 350 microns.
Claim 12
Independent Claim
44 words
A method for producing cell culture microcarriers comprising the steps of: (a) forming droplets of an aqueous solution of a protein; (b) suspending said droplets in a fluid; (c) solidifying said suspended droplets into cell culture microcarriers; and (d) recovering said cell culture microcarriers.
Claim 13
Independent Claim
44 words
A method for producing cell culture microcarriers comprising the steps of: (a) forming droplets of an aqueous solution of a protein; (b) suspending said droplets in a fluid; (c) solidifying said suspended droplets into cell culture microcarriers; and (d) recovering said cell culture microcarriers.
Claim 14
Dependent Claim
26 words
The method of claim 11, wherein said protein is selected from the group consisting of collagen, casein, soybean protein, silk, keratin, egg proteins and blood proteins.
Claim 15
Dependent Claim
10 words
The method of claim 12, wherein said protein is collagen.
Claim 16
Dependent Claim
18 words
The method of claim 11, wherein said droplets are formed by atomizing said solution from a pressure nozzle.
Claim 17
Dependent Claim
19 words
The method of claim 11, wherein said droplets are formed by atomizing said solution from a gas-propelled spray nozzle.
Claim 18
Dependent Claim
19 words
The method of claim 11, wherein said droplets are formed by atomizing said solution from a rapidly spinning surface.
Claim 19
Dependent Claim
19 words
The method of claim 11, wherein said droplets are formed by atomizing said solution from an ultrasonically driven nozzle.
Claim 20
Dependent Claim
14 words
The method of claim 11, wherein said droplets are formed by emulsifying said solution.
Claim 21
Dependent Claim
15 words
The method of claim 11, wherein said fluid contains a solidification agent for said droplets.
Claim 22
Dependent Claim
24 words
The method of claim 19, wherein said solidification agent is selected from the group consisting of ammonia, an aldehyde, an epoxy compound and epichlorohydrin.
Claim 23
Dependent Claim
12 words
The method of claim 20, wherein said aldehyde is a multifunctional aldehyde.
Claim 24
Dependent Claim
11 words
The method of claim 21, wherein said multifunctional aldehyde is glutaraldehyde.
Claim 25
Dependent Claim
14 words
The method of claim 20, wherein said epoxy compound is a multifunctional epoxy compound.
Claim 26
Dependent Claim
16 words
The method of claim 23, wherein said multifunctional epoxy compound is 1, 4 butanediol diglycidyl ether.
Claim 27
Dependent Claim
10 words
The method of claim 20, wherein said aldehyde is formaldehyde.
Claim 28
Dependent Claim
12 words
The method of claim 11, wherein said fluid is a gaseous medium.
Claim 29
Dependent Claim
11 words
The method of claim 26, wherein said gaseous medium is air.
Claim 30
Dependent Claim
24 words
The method of claim 26, wherein said gaseous medium contains a compound selected from the group consisting of hydrogen chloride, ammonia, formaldehyde and glutaraldehyde.
Claim 31
Dependent Claim
12 words
The method of claim 11, wherein said fluid is a liquid medium.
Claim 32
Dependent Claim
17 words
The method of claim 29, wherein said liquid medium is immiscible with said droplets of said solution.
Claim 33
Dependent Claim
13 words
The method of claim 30, wherein said liquid medium is a halogenated hydrocarbon.
Claim 34
Dependent Claim
23 words
The method of claim 31, wherein said halogenated hydrocarbon is selected from the group consisting of methylene chloride, 1,1,1-trichloroethane, trichloroethylene and Freon® T.F.
Claim 35
Dependent Claim
14 words
The method of claim 29, wherein said liquid medium is miscible with said droplets.
Claim 36
Dependent Claim
12 words
The method of claim 33, wherein said liquid medium is an acid.
Claim 37
Dependent Claim
13 words
35- The method of claim 34, wherein said acid is an inorganic acid.
Claim 38
Dependent Claim
25 words
The method of claim 35, wherein said inorganic acid is selected from the group consisting of nitric acid, hydrochloric acid, phosphoric acid and sulfuric acid.
Claim 39
Dependent Claim
12 words
The method of claim 34, wherein said acid is an organic acid.
Claim 40
Dependent Claim
21 words
The method of claim 37, wherein said organic acid is selected from the group consisting of oxalic acid and formic acid.
Claim 41
Dependent Claim
12 words
The method of claim 33, wherein said liquid medium is a base.
Claim 42
Dependent Claim
20 words
The method of claim 39, wherein said base is selected from the group consisting of ammonia, alkalihydroxide and an amine.
Claim 43
Dependent Claim
12 words
The method of claim 33, wherein said liquid medium is an aldehyde.
Claim 44
Dependent Claim
18 words
The method of claim 41, wherein said aldehyde is selected from the group consisting of formaldehyde and glutaraldehyde.
Claim 45
Dependent Claim
27 words
The method of claim 11, wherein said solidifying is accomplished by maintaining a temperature differential between said droplets of said solution of said protein and said fluid.
Claim 46
Dependent Claim
16 words
The method of claim 43, wherein said fluid is hotter than said droplets of said solution.
Claim 47
Dependent Claim
16 words
The method of claim 43, wherein said fluid is colder than said droplets of said solution.
Claim 48
Dependent Claim
31 words
The method of claim 43, wherein said temperature of said fluid is hotter than the temperature of said droplets of said solution and further, wherein said solution contains a solidification agent.
Claim 49
Dependent Claim
20 words
The method of claim 11, wherein said solidifying results from a solidification agent present in said solution of said protein.
Claim 50
Dependent Claim
23 words
The method of claim 47, wherein said agent is water soluble and is selected from the group consisting of aldehyde, acid and base.
Claim 51
Dependent Claim
23 words
The method of claim 11, further including, after the step of solidifying and prior to the step of recovering, the step of hardening.
Claim 52
Dependent Claim
14 words
The method of claim 49, wherein said hardening is accomplished by a crosslinking agent.
Claim 53
Dependent Claim
20 words
The method of claim 50, wherein the hardening effect of said crosslinking agent is accelerated by the application of heat.
Claim 54
Dependent Claim
28 words
The method of claim 51, wherein the hardening effect of said crosslinking agent is further accelerated by increasing the application of heat without liquifying said cell culture microcarriers.
Claim 55
Dependent Claim
54 words
The method of claim 52, wherein said protein is collagen, wherein said crosslinking agent is glutaraldehyde and wherein said heat is initially applied at 25°C for 1 to 25 hours, followed by increasing said heat to 70°C for 1 to 25 hours, followed by increasing said heat to 100°C for 1 to 25 hours.
Claim 56
Dependent Claim
42 words
The method of claim 52, wherein said heat is initially applied at 25°C for 15 to 20 hours, followed by applying said heat at 70°C for 6 to 10 hours, followed by applying said heat at 100°C for 15 to 20 hours.
Claim 57
Dependent Claim
11 words
The method of claim 50 wherein said crosslinking agent is multifunctional.
Claim 58
Dependent Claim
20 words
The method of claim 55, wherein said multifunctional crosslinking agent is selected from the group consisting of aldehyde and epoxy.
Claim 59
Dependent Claim
10 words
The method of claim 56, wherein said aldehyde is glutaraldehyde.
Claim 60
Dependent Claim
14 words
The method of claim 57, wherein said glutaraldehyde is combined with a reducing agent.
Claim 61
Dependent Claim
12 words
The method of claim 58, wherein said reducing agent is sodium dithionite.
Claim 62
Dependent Claim
11 words
The method of claim 50, wherein said crosslinking agent is formaldehyde.
Claim 63
Independent Claim
34 words
The method of any one of claims 19-25, 47-48 and 50-60, further including, prior to the step of recovering, the step of neutralizing excess solidification or cross- linking agent from said cell culture microcarrier.
Claim 64
Dependent Claim
31 words
The method of claim 61, wherein said neutralizing is effected by treating said microcarrier with a solution of said protein, said solution being sufficiently dilute to avoid solidification of said protein.
Claim 65
Dependent Claim
25 words
The method of claim 61, further including, after the step of neutralizing and prior to the step of recovering, the step of sterilizing said microcarriers.
Claim 66
Dependent Claim
18 words
The method of claim 61, further including, after the step of recovering, the step of sterilizing said microcarriers.
Claim 67
Independent Claim
29 words
The method of any one of claims 11 - 18, 26 - 46 and 49, further including, prior to the step of recovering, the step of sterilizing said microcarriers.
Claim 68
Independent Claim
24 words
The method of any one of claims 11-18, 26-46 and 49, further including, after the step of recovering, the step of sterilizing said microcarriers.
Claim 69
Dependent Claim
22 words
The method of claim 11, wherein said droplets are formed in a gaseous medium and wherein said fluid is a liquid medium.
Claim 70
Dependent Claim
14 words
The method of claim 67, wherein said liquid medium is immiscible with said droplets.
Claim 71
Independent Claim
55 words
A method of growing anchorage-dependent cells comprising the steps of (a) providing a suspension comprising cell culture microcarriers, an innoculum of said cells and a nutrient- containing growth medium, said microcarriers comprising a water-insolubilized protein, said protein being substantially homogeneously distributed throughout said microcarrier and (b) maintaining said suspension under conditions conducive to cell growth.
Claim 72
Independent Claim
55 words
A method of growing anchorage-dependent cells comprising the steps of (a) providing a suspension comprising cell culture microcarriers, an innoculum of said cells and a nutrient- containing growth medium, said microcarriers comprising a water-insolubilized protein, said protein being substantially homogeneously distributed throughout said microcarrier and (b) maintaining said suspension under conditions conducive to cell growth.
Claim 73
Independent Claim
61 words
A method of producing anchorage-dependent cells comprising the steps of (a) providing a suspension comprising cell culture microcarriers, an innoculum of said cells and a nutrient- containing growth medium, said cell microcarriers comprising a water-insolubilized protein, said protein being substan- ially homogeneously distributed throughout said microcarrier, (b) maintaining said suspension under conditions conducive to cell growth, and (c) harvesting said cells.
Claim 74
Independent Claim
61 words
A method of producing anchorage-dependent cells comprising the steps of (a) providing a suspension comprising cell culture microcarriers, an innoculum of said cells and a nutrient- containing growth medium, said cell microcarriers comprising a water-insolubilized protein, said protein being substan- ially homogeneously distributed throughout said microcarrier, (b) maintaining said suspension under conditions conducive to cell growth, and (c) harvesting said cells.
Claim 75
Independent Claim
69 words
A method of producing anchorage-dependent cell products comprising the steps of: (a) forming a suspension, in a suitable cell culture mediun, of cell culture microcarriers comprising a water-insolubilized protein, said protein being substantially homogeneously distributed throughout said microcarrier; (b) innoculating said culture with anchorage-dependent cells to form a cell culture; (c) maintaining said cell culture under conditions conducive to production of cell products; and (d) harvesting said cell products.
Claim 76
Independent Claim
69 words
A method of producing anchorage-dependent cell products comprising the steps of: (a) forming a suspension, in a suitable cell culture mediun, of cell culture microcarriers comprising a water-insolubilized protein, said protein being substantially homogeneously distributed throughout said microcarrier; (b) innoculating said culture with anchorage-dependent cells to form a cell culture; (c) maintaining said cell culture under conditions conducive to production of cell products; and (d) harvesting said cell products.
Claim 77
Dependent Claim
12 words
The method of claim 71, wherein said cell growth product is interferon.
Claim 78
Independent Claim
34 words
The method of any one of claims 69, 70 or 71, further including, immediately after the step of maintaining said suspension under conditions conducive to cell growth, the step of subcultivating said cell growth.
Claim 79
Dependent Claim
26 words
The method of claim 73, wherein said subcultivating is accomplished by separating said cells from said microcarriers and introducing additional cell culture microcarriers into said suspension
Claim 80
Dependent Claim
46 words
The method of claim 73, wherein said subcultivating is accomplished by separating said cells from said microcarriers, removing said cells from said medium and said microcarrier, introducing said cells into a new medium and adding new cell culture microcarriers to said new medium containing said cells.
Claim 81
Dependent Claim
144 words
Apparatus for converting a water-based protein solution to microspheres comprising: a heated, thermostatically controlled reservoir for the protein solution; at least one spray head; means for supplying the protein solution from the reservoir to said spray head; means for applying heat to said supplying means for maintaining the protein solution in said at least one spray head at approximately at least the same temperature as the protein solution in the reservoir; means for maintaining a predetermined pressure on the protein solution at the spray head; at least one receptacle positioned beneath the said spray head for receiving the droplets emitted by the spray head; means for flowing a low temperature, water immiscible suspension liquid across said at least one receptacle for suspending and solidifying said received droplets into discrete microspheres in said suspension liquid; and means for collecting said microspheres from said suspension liquid.
Claim 82
Dependent Claim
144 words
Apparatus for converting a water-based protein solution to microspheres comprising: a heated, thermostatically controlled reservoir for the protein solution; at least one spray head; means for supplying the protein solution from the reservoir to said spray head; means for applying heat to said supplying means for maintaining the protein solution in said at least one spray head at approximately at least the same temperature as the protein solution in the reservoir; means for maintaining a predetermined pressure on the protein solution at the spray head; at least one receptacle positioned beneath the said spray head for receiving the droplets emitted by the spray head; means for flowing a low temperature, water immiscible suspension liquid across said at least one receptacle for suspending and solidifying said received droplets into discrete microspheres in said suspension liquid; and means for collecting said microspheres from said suspension liquid.
Claim 83
Dependent Claim
65 words
The apparatus of claim 76 wherein said supplying means includes a loop from and back to said reservoir said loop including an elongated, substantially horizontal pipe, said apparatus includes a plurality of spray heads spaced along said elongated, horizontal pipe and a like plurality of receptacles for individually receiving the droplets emitted by the spray heads and means for maintaining predetermined temperatures throughout said loop.
Claim 84
Dependent Claim
38 words
The apparatus of claim 77, wherein said heated reservoir includes a first section for containing the water-based protein solution and a second section for containing . water for priming and flushing said circulating means and said spray heads.
Claim 85
Dependent Claim
25 words
The apparatus of claim 78, wherein said heated reservoir includes means for maintaining both said first and second sections at a temperature of approximately 50°C.
Claim 86
Dependent Claim
36 words
The apparatus of claim 77, wherein said supplying means includes a piston pump for forcing said protein solution through said loop and a buffer and restrictor in said loop for attenuating pulses created by said pump.
Claim 87
Dependent Claim
56 words
The apparatus of claim 78, wherein said loop includes a flexible member on the input of said loop and a flexible member on the output of said loop, wherein said flexible member may selectively be inserted either into said first section for circulating said protein solution or into said second section for circulating said heated water.
Claim 88
Dependent Claim
24 words
The apparatus of claim 81 also including a drain and wherein either or both of said flexible members may selectively discharge into said drain.
Claim 89
Dependent Claim
27 words
The apparatus of claim 81 including a screen in said first section for receiving the input end of said flexible member on the input of said loop.
Claim 90
Dependent Claim
30 words
The apparatus of claim 77, wherein each of said spray heads is dimensioned for discharging as atomized solution only a small fraction of the protein solution circulating through said loop.
Claim 91
Dependent Claim
25 words
The apparatus of claim 84, wherein each of said spray heads includes means for imparting a swirl to the jet emanating from the spray head.
Claim 92
Dependent Claim
21 words
The apparatus of claim 84, wherein each of said spray heads includes means for monitoring the temperature of the spray head.
Claim 93
Dependent Claim
16 words
The apparatus of claim 84, wherein each of said spray heads includes a noncircular elongated orifice.
Claim 94
Dependent Claim
17 words
The apparatus of claim 77, wherein said heat-applying means includes heating jackets selectively placed on said loop.
Claim 95
Dependent Claim
16 words
The apparatus of claim 88 including heating jackets on the upstream sides of said spray heads.
Claim 96
Dependent Claim
26 words
The apparatus of claim 77, wherein said pressure maintaining means includes an adjustable throttle for adjusting the pressure of said protein solution on said spray heads.
Claim 97
Dependent Claim
12 words
The apparatus of claim 90 including a pressure gauge in said loop.
Claim 98
Dependent Claim
19 words
The apparatus of claim 77, wherein said flowing means includes means for circulating said suspension liquid through a loop.
Claim 99
Dependent Claim
17 words
The apparatus of claim 92, wherein said suspension liquid circulating means includes a reservoir and a pump.
Claim 100
Dependent Claim
33 words
The apparatus of claim 92 also including means for maintaining said low temperature suspension liquid at a temperature of approximately 0° to 10°C but not below the freezing point of the protein solution.
Claim 101
Independent Claim
47 words
The apparatus of any one of claims 77, 92, 93 and 94, wherein each of said receptacles includes means for developing a pressure head of suspension liquid across an input section of each receptacle for increasing the speed of flow of the suspension liquid across the receptacle.
Claim 102
Dependent Claim
58 words
The apparatus of claim 95, wherein each of said receptacles is substantially rectangular, said pressure head developing means is a barrier across the input end of the receptacle and spaced slightly above the bottom of the receptacle and each receptacle includes a depressed area at the end opposite said barrier for producing a cascade across the rectangular receptacle.
Claim 103
Dependent Claim
30 words
The apparatus of claim 95, wherein said receptacles are dimensioned and positioned for receiving the droplets emanating from the individual spray heads only in the flow of the suspension liquid.
Claim 104
Dependent Claim
24 words
The apparatus of claim 77 also including closable gate means for selectively preventing entrance of the discharge from said spray heads into said receptacles.
Claim 105
Dependent Claim
13 words
The apparatus of claim 77, wherein said suspension liquid is a halogenated hydrocarbon.
Claim 106
Dependent Claim
27 words
The apparatus of claim 99, wherein said halogenated hydrocarbon is selected from the group consisting of CH 2 CL 2 , 1-1-1 trichloroethane, trichloroethylene and Freon® TF.
Claim 107
Dependent Claim
19 words
The apparatus of claim 77, wherein said collecting means includes means for separating said spheres from said suspension liquid.
Claim 108
Dependent Claim
25 words
The apparatus of claim 96 also including a common discharge for said receptacles and wherein said collecting means includes a filter in said common discharge.
Claim 109
Dependent Claim
19 words
The apparatus of claim 102 wherein said filter is a screen having a mesh size of about 500 um.
Claim 110
Independent Claim
91 words
A method for converting a water-based protein solution into microspheres comprising the steps of: spraying the protein solution as a liquid into a gaseous atmosphere to form discrete droplets; maintaining a temperature differential between the solution and the gaseous atmosphere wherein the gaseous atmosphere is at a lower temperature than the temperature of the solution before spraying; flowing a cooled water-immiscible suspension liquid across the path of fall of said discrete droplets to form microspheres suspended in said cooled suspension liquid; and collecting said cell culture microcarriers from said suspension liquid.
Claim 111
Independent Claim
91 words
A method for converting a water-based protein solution into microspheres comprising the steps of: spraying the protein solution as a liquid into a gaseous atmosphere to form discrete droplets; maintaining a temperature differential between the solution and the gaseous atmosphere wherein the gaseous atmosphere is at a lower temperature than the temperature of the solution before spraying; flowing a cooled water-immiscible suspension liquid across the path of fall of said discrete droplets to form microspheres suspended in said cooled suspension liquid; and collecting said cell culture microcarriers from said suspension liquid.
Claim 112
Dependent Claim
27 words
The method of claim 104, wherein the step of spraying includes the step of circulating an excess supply of protein solution across a plurality of spray heads.
Claim 113
Dependent Claim
30 words
The method of claim 105, wherein the step of circulating a supply of protein solution includes the step of maintaining the circulating protein solution at a temperature of about 50°C.
Claim 114
Dependent Claim
11 words
The method of claim 104, wherein said gaseous atmosphere is air.
Claim 115
Dependent Claim
33 words
The method of claim 104, wherein the step of flowing the cooled suspension liquid includes the step of maintaining the temperature of the suspension liquid slightly above the freezing temperature of the microspheres.
Claim 116
Dependent Claim
27 words
The method of claim 105, wherein the step of flowing includes the step of developing individual flow paths across the fall from the plurality of spray heads.
Claim 117
Dependent Claim
34 words
The method of claim 109 including the step of dimensioning the spray cones from the spray heads and the individual flow paths for receiving the spray cones individually and directly onto the flow paths.
Claim 118
Dependent Claim
32 words
The method of claim 109, wherein the step of developing individual flow paths includes the step of forming a cascade in each flow path for receiving each spray cone in a cascade.
Claim 119
Dependent Claim
37 words
The method of claim 111, wherein the step of forming a cascade includes the steps of raising a pressure head in each flow path and depressing a portion of each flow path downstream from the pressure head.
Claim 120
Dependent Claim
30 words
The method of claim 109, wherein the step of collecting the spheres includes the steps of combining the individual flow paths and separating the spheres from the combined flow path.
References Cited (Backward Citations)
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