JPH0577390B2 - - Google Patents

Description

Claim 1: A weight-bearing microsponge for immobilizing bioactive materials in a driven bioreactor system, the microsponge comprising a porous biostable matrix of a biocompatible polymer comprising an inert weight-bearing material; Matrix is about 1 to about 150
having an opening to a surface pore structure having an average pore size in the micron range, the pores of the matrix occupying about 70 to about 98% by volume of the microsponge;
The above microsponge, wherein the microsponge is also characterized in that it has an average particle size of about 100 to about 1000 microns and a specific gravity of about 1.05 or more. 2. The microsponge according to claim 1, on which a bioactive material selected from the group consisting of enzymes, microorganisms, dead cells and living cells is immobilized. 3 Biostable, biocompatible polymers include collagen, cellulose, dextran, dextrin,
2. The microsponge of claim 1, which is selected from the group consisting of polyamide, polyester, starch, agarose, carrageenan, polyurethane, polyvinyl alcohol, polyacrylate, polymethacrylate, and polyacrylamide. 4. The microsponge of claim 1, wherein the inert weight-giving material is selected from the group consisting of metals, metal alloys, metal oxides, and ceramics. 5. The weight-imparting material has a specific gravity of about 4.0 or more,
5. The microsponge of claim 4, wherein the microsponge has a specific gravity of about 1.3 or greater. 6. The microsponge of claim 5, wherein the inert weight-imparting material is dispersed in the porous matrix as a finely divided powder. 7. The microsponge according to claim 5, wherein the weight-imparting material is centrally disposed as a solid core material, around which a porous matrix is formed. 8. The microsponge of claim 5, wherein the inert weight-giving material is selected from the group consisting of chromium, tungsten, cobalt, molybdenum, titanium, nickel, and alloys. 9. The microsponge of claim 8, wherein the weight-imparting material is titanium and the microsponge has immobilized hybridoma cells. 10. The microsponge of claim 1, wherein the bioactive, biocompatible polymer is highly crosslinked collagen. 11. A highly cross-linked collagen matrix is prepared by: (a) pulverizing a collagen source selected from the group consisting of collagen type and collagen; (b) mixing the pulverized collagen with an acidic liquid medium; and (c) a collagen-liquid medium. lyophilizing the mixture into a dry matrix; and (d) contacting the collagen in the dry matrix with a crosslinking agent selected from the group consisting of (i) carbodiimides and difunctional succinimidyl active esters; (ii) 2. The microsponge of claim 1, prepared by subjecting the solid droplets to elevated temperatures under vacuum, and (iii) crosslinking by a treatment selected from the group consisting of: a combination thereof. 12. The microsponge of claim 11, wherein the inert weight-imparting material is added as a finely divided powder to the mixture of collagen and acidic liquid medium prior to lyophilization. 13 Collagen is a type of tendon collagen.
The micro sponge according to claim 12. Description This invention was made on or under contract with the NIH. The Government has rights to this invention under SBIR Grant No. CA37430. BACKGROUND OF THE INVENTION Field of the Invention This invention is in the field of immobilizing bioactive materials, and in particular relates to improved microsponges for use in driven bioreactor systems. The present invention also relates to microorganisms and cells (hereinafter collectively referred to as organisms)
The present invention relates to the field of culturing organisms immobilized on and/or in microsponges in a driven reactor format as an underwater floating system. Description of the Prior Art A wide variety of chemical products are produced by culturing living organisms. For example, there are many fermentation methods for producing antibiotics and other drugs, alcoholic beverages, cheese, etc. However, most fermentation methods are carried out commercially using batch reaction procedures, despite the recognized value of continuous culture techniques. Recent advances in the fields of genetic engineering and medicine have ushered in a new era in the production of chemical products by biological culture. For example, mammalian hybrid cells or hybridomas have been developed that secrete antibodies specific for known antigens, such as tetanus toxoid. E.
Bacteria such as coli have been genetically engineered to produce specific proteins, such as insulin. However, to best utilize this new technology, batch fermentation methods are used to continuously cultivate such organisms at high concentrations and under optimal growth conditions to maximize the production of such products. We must develop technology that surpasses the Various devices for immobilizing bioactive materials are known. Solid supports have long been used for the immobilization of microorganisms in wastewater treatment and related fermentation processes.
More recently, solid microcarriers have been used to obtain high cell densities for culturing attachment-dependent cells. For example, microporous polymeric supports fabricated from dextran have been used for cell culture.
Such supports are available from Pharmacia Fine Chemicals
It can be obtained commercially under the trade name Cytodex. However, such solid biological supports are unsuitable for driven reactor systems such as vigorously agitated tanks and fluidized beds, where virtually all of the cells adhere to the surface of the support and This is because they are exposed to impact stress and trauma during operation. Porous inorganic microcarriers are also known and such supports potentially offer protection to cells in drive applications since the cells reside inside the microcarrier. Unfortunately, inorganic microcarriers cannot be made to have the proper combination of permeability and specific gravity to function satisfactorily in all drive applications. For example, Messing et al.
The porous fritted glass or cordierite supports described in U.S. Pat. No. 4,153,510 generally exhibit a specific gravity of less than about 1.3 in aqueous suspension systems if their void fraction is greater than about 80%. wax (note that the void fraction for the Messing support is not disclosed). It is understandable that these supports are not suitable for any driving reaction system where high specific gravity is generally required to ensure high relative velocities to the maximum rates of mass and energy transfer. Accordingly, these supports have generally been assigned for use in packed bed applications. Furthermore, it is unknown how suitable these prior art microcarriers are for culturing attachment-dependent organisms such as hybridomas. One object of the present invention is to provide a microsponge containing immobilized bioactive material suitable for use in a driven reaction system. Another object of the invention is to provide a microsponge suitable for immobilizing a wide variety of organisms characterized by widely varying sizes and degrees of attachment to solid supports. . It is a further object of the present invention to provide a microsponge suitable for use in a driven reactor system for continued growth and regeneration of immobilized organisms. It is also an object of the present invention to provide a microsponge suitable for a driven reactor system that helps maximize the metabolic activity of immobilized organisms. Yet another object of the present invention is to provide a method for continuously culturing organisms at high concentrations. Yet another object of the present invention is to provide a microsponge suitable for a driven reactor system that allows organisms to be cultured at high concentrations with maximum growth rate or maximum metabolic activity. Another object of the invention is to provide a method for culturing both attachment-dependent and attachment-independent organisms in vitro. These and other objects of the invention will become apparent from a consideration of the specification and claims. SUMMARY OF THE INVENTION The present invention relates to a weighted microsponge (preferably a collagen microsponge) for immobilizing bioactive materials in a driving bioreactor system, said microsponge comprising an inert weightable material. It consists of a porous, biostable, highly cross-linked collagen matrix, said collagen matrix having a size from about 1 micron to about
Open to a surface pore structure with an average pore size in the range of 150 microns, the pores of the matrix account for about 70 to about 98 volume percent of the microsponge, and the microsponge also has about 100 microns. It has an average particle size of from microns to about 1000 microns and a specific gravity of about 1.05 or more. The present invention also relates to a continuous in vitro culture method of an organism for the production of biochemical products, which method comprises: (a) pulverizing a collagen source selected from the group consisting of type (i); (iii) lyophilizing the collagen-liquid medium mixture into a dry sponge matrix; and (iv) combining the collagen of the sponge matrix with (1) a carbodiimide and a difunctional Contacting said collagen with a crosslinking agent selected from the group consisting of succinimidyl active esters, (2) exposing said dry sponge matrix to elevated temperatures under vacuum, and (3) combinations thereof. (b) inoculating the highly cross-linked collagen microsponge of step (a) with a culture of said organism; (c) inoculating The process consists of the following steps: (d) supplying a nutrient medium in direct contact with highly cross-linked collagen microsponges; and (d) recovering biochemical products along with the nutrient medium effluent. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a photomicrograph showing a suitable collagen microsponge matrix of the present invention illustrating the fibrous structure. FIG. 2 is a photomicrograph of one collagen microsponge matrix according to the invention illustrating the fiber structure. DETAILED DESCRIPTION The present invention provides immobilizable active materials suitable for use in driven bioreactor systems, particularly weight-imparting microsponges containing biological organisms, preferably collagen microsponges, as well as biological organisms for the production of biochemical products. Directed to the in vitro continuous culture method of the body. As used throughout this specification and claims, the term "bioactive material" broadly encompasses enzymes and other chemical agents, such as chelating agents, hormones, antibodies, etc., and cells of living organisms, ie, microorganisms and higher organisms. As used throughout this specification and claims, the term "organism" broadly encompasses both microorganisms and cells of higher organisms. Organisms can be derived from an unlimited number of sources including bacteria, fungi, viruses, algae, yeast, animal cells (tissues), such as mammalian, insect and fish and plant cells. The terms "organism" and "cell" will be used interchangeably throughout this specification and claims. Also, the term "biochemical product" used throughout this specification and claims.
refers not only to the primary and secondary metabolites produced by such an organism, but also to the cellular material or the organism's own biomass, including, for example, non-secreted products. Since the weighted microsponges of the present invention are particularly effective when used in biological culture, they will generally be described in terms of such embodiments, but are not intended to be so limited. Microsponges according to the present invention are made from biocompatible (eg, non-toxic) aggregates that are stable during use for a reasonable period of time, for example on the order of many months. A particularly suitable mix sponge according to the invention is formed from highly cross-linked collagen. Biocompatibility refers to the ability of a polymeric material, e.g. ) refers to the ability of a polymeric (e.g., collagen matrix) material to support a viable culture of an organism. Stability or biostability of a matrix material refers to its ability to maintain its strength and integrity under in vitro conditions for a period of time appropriate for culturing the organism of interest. For example, in the case of hybridoma culture for monoclonal antibody production, the driving bioreactor may be 3 to 6
It is expected that it will be operated continuously for several months or more. Therefore, the matrix material must be biostable for this period. Both natural and synthetic polymeric materials can be used as matrix materials. Examples of suitable polymers include polysaccharides such as dextran, dextrin, starch, cellulose, agarose, carrageenan, etc.; proteins such as collagen; and synthetic polymers such as polyvinyl alcohol, polyacrylates, polymethacrylates, etc.
Includes polyacrylamide, polyester, polyurethane, polyamide, and the like. Generally, biocompatibility and biostability of a material can be demonstrated using routine experimentation. Based on its biocompatibility and strength, collagen is currently the material of choice. Collagen is a biodegradable polymer found in animals, including humans. It has numerous uses in the medical field and, in most applications, various crosslinking agents such as aldehydes such as glutaraldehyde and formaldehyde; ethyl chloroformate; dimethyl adipimidate; N,N-methylenethi Acrylamide; 1,2-diacrylamide ethylene glycol; Cyanamide; N,N'-
Diallyltartaric acid diamide; cyanogen bromide; concanavalin A; 6-aminohexanoic acid; 1,6-diaminohexane; succinimidyl active ester;
insoluble forms using carbodiimides and compounds with similar cross-linking groups and/or physical processing techniques such as lyophilization and intensive dehydration at vacuums above about 50 mTorr and temperatures ranging from 50°C to 200°C. reconstituted and crosslinked. Unfortunately, many if not all of the conventional cross-linking agents that are unavoidably present in cross-linked collagen, especially glutaraldehyde, cause adverse biological effects and are therefore cytotoxic. Recently, new collagen-based matrices with improved biostability have been discovered. This collagen material is made without conventional crosslinking agents. In this matrix type I, or collagen, carbodiimide or succinimidyl active ester and/or
Cross-linked using severe dehydration conditions at temperatures ranging from Such cross-linked collagen generally has a molecular weight of about 1×10 ⁶ to greater than 50×10 ⁶ . The molecular weight of collagen between adjacent crosslinks varies from about 1000 to 100000 due to the formation of covalent bonds.
Because of its resistance to degradation by collagenases and other proteinases, this cross-linked collagen has been found to be particularly suitable as a porous matrix for microsponges. In fact, microsponges made from this collagen material exhibited several surprising properties when used in continuous culture of immobilized organisms, particularly hybridoma cells leaching monoclonal antibodies. For example, hybridomas cultured in essentially protein-free media produce more monoclonal antibodies when immobilized on and/or in such collagen matrix microsponges than in the absence of such matrices. It is much more effective. Furthermore, microsponges made from the cross-linked collagen materials specifically mentioned here appear to preferentially retain living (viable) cells at high concentrations and eliminate non-viable cells. . Particularly suitable crosslinked collagen can be made from both soluble and insoluble collagen of type I and. Soluble collagen is produced by restriction enzyme digestion and/or extraction of tissues rich in these types of collagen. Insoluble collagen is derived from the following typical sources: Type I collagen: cow, pig, chicken and fish skin, cow and chicken tendon, and cow and chicken bone (including fetal tissue); : bovine articular cartilage, nasal septum, sternum cartilage; and types of collagen: bovine and human aorta and skin. For example, type I collagen obtained from bovine dermis can be used. Preferably, type I tendon collagen is used. In the broad practice of the present invention, there are no particular limitations on the physical form or macroscopic profile of the crosslinked collagen microsponges. Microsponges can be used in a variety of physical forms including beads, flakes, discs, fibers, films, coatings (eg, on extended surface indicators), and sheets. For example, the microsponge can be provided as a coating on conventional indicators such as pellets, stars, helical rings, cross-partition rings, Laschichi rings, Burl saddles, interox saddles, cap rings, Tellerette rings, and the like. Microsponges can also be provided in sheet form, cylindrical form, or some other undefined structure. In the wide implementation of the method of the invention, there are no particular restrictions on the size of the microsponges. For example, for packed or fixed bed applications (eg, plug flow reactors), microsponges can be provided in dimensions typical of such applications. In order to be adapted to culturing high concentrations of organisms in a driven reactor system and to allow the transfer of nutrients to the immobilized organisms and the desired products from the microsponge, the weight-imparting microspheres of the present invention The sponge, preferably collagen, must meet several functional requirements. Microsponges are typically in the form of beads and should have a particle size in the range of about 100 microns to 1000 microns, preferably about 200 microns to 500 microns. At larger particle sizes, production of the desired product by reaction between the immobilized bioactive material and the liquid medium in contact with it is
The entire internal volume of the porous structure is not utilized effectively;
Therefore, the volumetric productivity of the drive reactor using such microsponges is impaired. Smaller particle sizes present practical problems in microsponge production and drive reactor operation. The permeability of microsponges is another important issue. The permeability of a microsponge is determined by the interaction between its porosity or void fraction and its pore structure. Void fraction is defined as the ratio of the interstitial volume of a material to the total volume occupied by the material, and is often expressed as a percentage. To allow operation at high biological concentrations, microsponges must be approximately 70% to 98%
% void fraction. Preferably, the void fraction of the collagen microsponge is greater than 85%, and most preferably greater than about 90%. The microsponge must also have openings to the surface pore structure. This facilitates cell entry, cell retention, and subsequent cell development without excessive shear forces.
and allowing evacuation of excess cell populations. For example, if the desired product is not secreted by the organism (e.g., a genetically engineered E. coll with a non-leachable rDNA product like insulin), the organism will You have to be able to escape from the sponge. If this process is to proceed in a continuous manner without rupturing the microsponge structure, an open pore structure is absolutely necessary. The desired biological product is recovered as a component of the culture harvest fluid. The microsponge should contain pores with an average size ranging from about 1 micron for the smallest microorganisms and viruses to about 150 microns for large mammalian and plant cells. Generally, the pores of the microsponge should be at least as large as the smallest major dimension of the immobilized bioactive material and less than about five times the largest major dimension. Preferably, the pore size of the matrix is on the order of 1.5 to 3 times the average diameter of the organism or cell. If unknown, the minimum and maximum major dimensions of an organism can be determined using known techniques. Applicants believe that the combinations of particle size and pore size described herein provide sufficient mass transfer of constituents, e.g. nutrients, to the immobilized organism, as well as desired metabolites from the constituents, e.g. the immobilized organism. It has been found that sufficient mass transfer is ensured. For use in driven reactor systems, microsponges (eg collagen) must also be weighted. The polymeric materials (eg, crosslinked collagen) used as matrix materials in the present invention generally have a specific gravity of about 1.0 or less. For proper operation in a driven reactor, a specific gravity of about 1.05 or greater, preferably about 1.3 or greater, and most preferably about 1.6 to 2.0 is desired. It is surprising that by introducing certain weight-imparting additives into the microsponge, it is possible to obtain collagen microsponges with appropriate specific gravity without undesirably reducing their void fraction. I also understood. The weight-imparting additive must be substantially inert in the reactor environment and must be non-toxic to the immobilized organism, or the additive must be suitably treated to render it non-toxic. There must be. Also, the weight-imparting additive must not adversely affect the productivity of the immobilized organism. Generally, materials such as metals and their alloys and oxides, and ceramics having a specific gravity of about 4 or higher, and preferably about 7 or higher, are used. Examples of weight-bearing additives suitable for use in the broad practice of this invention include chromium, tungsten, molybdenum,
Cobalt, nickel, titanium and their alloys, such as Monel, 316 stainless steel, Vitalium (cobalt alloy containing chromium and molybdenum),
Titanium 6A1-4V (a titanium alloy containing aluminum and vanadium), and Highness Stellite Alloy 25 (a cobalt alloy containing chromium, nickel, tungsten and manganese). However, many of these materials may be incompatible with certain organisms, and routine experimentation may be required to assess toxicity for certain applications. For example, titanium is the weight-giving material of choice for hybridomas because most other metals are cytotoxic. The weight-imparting additive can be thoroughly introduced and dispersed into the microsponge as a finely pulverized powder.
Most particles have a particle size of about 10 to 40 microns. However, to minimize the surface area of the weight-imparting additive, it is desirable to use it as a solid core in the microsponge. Sufficient weighting material is added to obtain a microsponge with the desired specific gravity. A 50 micron diameter weight-imparting additive core with a specific gravity of about 7.0 is coated with a 50 micron thick layer of collagen with an average pore size of 20 to 40 microns and a void fraction of about 99%.
This results in a microsponge with a specific gravity of about 1.7 and a total void fraction of about 85%. Such microsponges are particularly suitable for use in aerobically driven reactor systems. Finally, for driving applications the collagen microsponge must exhibit adequate resistance to abrasion. A single dose of microsponge should preferably have a useful life on the order of 3 to 6 months or more. Generally, the microsponge should exhibit no more than about 10% volume loss after three months of operation. Typically, living organisms exhibit wide variation in their degree of attachment to solid supports. For example, some organisms readily attach or attach to a wide variety of supports, including both organic and inorganic materials, whereas others attach only to supports of biological origin. (adhesion-dependent organisms). Other organisms show little direct attachment to any support material (adhesion-independent organisms). The microsponge according to the invention is suitable for the immobilization of virtually any type of organism due to its construction from a polymeric (organic) material and its permeability (porosity and pore structure). There must be. In fact, as discussed in more detail below, it is even possible to tailor the microstructure or profile of the microsponge to best accommodate the adhesion tendencies of immobilized organisms. For example, a microsponge with a wire mesh structure (FIG. 1) can be used with attachment-independent organisms, whereas a microsponge with a lobed structure (FIG. 2) can be used with attachment-dependent organisms. Any suitable procedure used by the prior art to immobilize such organisms on microsponges can be used in the present invention, including techniques such as adsorption and chemical coupling. For example, in the case of some organisms, it may be sufficient to mix collagen microsponges into the fermentation liquid inoculated with the particular organism. After a short time, organisms colonize the microsponge and become trapped within its pores. In the case of some organisms, such as fibroblasts and hybridomas, it may be desirable to coat the microsponges with adhesion-promoting materials such as fibronectin, polylysine, and anti-hybridoma antibodies prior to inoculation. Other techniques, such as applying a net charge to the surface of the microsponge, can also be used to promote immobilization. In the broad practice of the present invention, there is no particular limitation on the procedure used to directly contact the immobilized bioactive material with a liquid reagent stream, such as a growth support medium for the culture of immobilized organisms, in a stirred tank. It will be recognized by those skilled in the art that any of the numerous devices available in the prior art may be used, including such known devices as reactors, fixed bed reactors, fluidized bed reactors, moving bed reactors, and the like. Dew. Generally, when culturing an organism, the microsponge is placed in a suitable reactor in which it is mixed with the nutrient fermentation liquid and the organism inoculum. The microsponge should be completely immersed. micro sponge,
Incubation is performed so that organisms grow and colonize the porous matrix of the microsponge. Other substances necessary for the development of new nutritional fermentation liquid,
For example, in the case of aerobic organisms, the reactor is continuously fed with oxygen and a harvest liquor containing biochemical products is collected. The biochemical product may consist of excess biomass including primary or secondary metabolites of the immobilized organism, eg non-secreted products produced by the immobilized organism, reaction products catalyzed by immobilized enzymes, and the like. One special advantage of the microsponge of the present invention is that
The advantage is that it can be used in mixing or drive systems such as fluidized bed reactors. As used herein, the term "driven reactor" refers to a reactor system in which the relative motion between the microsponge and the fluid medium is imparted in part by imparting motion to the microsponge itself. refers to Such reactor systems substantially enhance mass and energy transfer. A particularly suitable drive reactor system is co-filed U.S. Patent No. 706,872.
Specification (Robert C. Dean, February 28, 1985)
Jr., Peter V. Grela and Subhash B. Karkare.
(filed under the name of). To create highly cross-linked collagen microsponges, a suitable collagen source is first ground into small "fiber" sizes. In general, collagen is a particle (fiber) with a maximum dimension of about 200 microns.
For example, it is ground using a Wiley mill to obtain . If possible, use collagen source materials from 1 to 50.
Grind (ie, dry mill) to produce fibers with a diameter on the order of microns and a length of no more than about 200 microns. Proper comminution of the collagen source material is important to obtain microsponges with the desired structure. The ground collagen is then made into a collagen-based solution or dispersion, i.e. soluble collagen is dissolved in a solvent, or insoluble collagen is dispersed in a solvent by mixing with a suitable solvent, especially an acid, such as dilute hydrochloric acid, dilute acetic acid, etc. . Organic acids are particularly suitable in the present invention, including acetic acid, lactic acid, propionic acid, butyric acid, and the like. Some long chain fatty acids can also be used. Mix the collagen into the liquid (solvent) using standard mixing equipment. Preferably, in the case of collagen dispersions, mixing is achieved by high level agitation, for example using a Waring blender, so as to create microfibrils of collagen. The collagen and solvent mixture typically contains 0.5% to about 1.5% collagen by weight. The mixture should preferably be about 2.0 to about 4.0
It is preferable to exhibit a pH within the range of . A pH in the range of 1.0 to 2.0 can also be used as long as the temperature of the mixture is lowered sufficiently (eg, about 4° C.) to avoid denaturation of the collagen. The weight-imparting additive is then combined with the collagen-liquid mixture, and the composite mixture is formed into small droplets and rapidly solidified by freezing at a temperature below about 0°C, preferably below about -30°C. to create particles of desired size. Any known technique for producing small particles can be used in the practice of this invention.
Suitable techniques include, among others, pressure or air shear atomization, emulsification techniques, droplet formation using Raleigh liquid jet instability techniques, extrusion techniques, droplet formation using gravity or centrifugal force, electrostatic droplet formation, and Includes droplet formation methods that use inertial forces. For example, appropriately sized particles can be prepared using inertial forces to form small droplets at the orifice of a vibrating needle. The droplets can be frozen by dropping them into a cold bath of liquid nitrogen. Obviously other cooling baths for freezing droplets can also be used, such as chilled ethanol. It may also be possible to reduce the extremely large particles formed by freezing to the desired particle size by destructive techniques such as grinding. Additional techniques, as well as various coating methodologies, can also be used to form microsponges containing solid cores of weight-imparting additives. In this case, a shell of collagen matrix would surround the weighting core. Those skilled in the art will recognize other techniques suitable for forming small particles of the type described above, and the invention is not intended to be limited to any particular technique. The pore size and structure of collagen microsponges are influenced by various factors. For example, changes in collagen concentration appear to affect pore size, with higher collagen concentrations tending to produce smaller pore sizes. The PH of the mixture and the specific acid used to prepare the mixture also influences the pore size and structure of the resulting microsponges. For example, a PH that is too low tends to significantly limit the pore size of the microsponge, whereas a PH that is too high can cause the bulk collagen phase to separate from the original solution or dispersion.
This prevents the formation of porous structures and tends to remain in the dispersed phase when using finely divided weight imparting additives. Freezing rate also appears to affect the structure of the microsponges, and the structure will also vary with changes in collagen type. The frozen composite is then vacuum lyophilized using conventional equipment preferably operated at a vacuum of about 50 mTorr or more and at a temperature in the range of about 22°C to 100°C. The combination of freezing and drying is called freeze-drying. The lyophilized collagen matrix composite is then treated to crosslink the collagen. Collagen is preferably carbodiimide or N
-Hydroxysuccinimide-derived active esters (succinimidyl active esters) or by intensive dehydration at elevated temperatures or by a combination of these treatments. The strength and biostability of collagen matrices thus produced are influenced by the degree of crosslinking introduced by such treatment. These crosslinking methods provide collagen matrices that are surprisingly resistant to degradation by collagenases and other enzymes, thereby making these materials particularly suitable for biological culture. Examples of carbodiimides that can be used in chemical treatments are cyanamide and 1-ethyl-3-(3
-dimethylaminopropyl)-carbodiimide hydrochloride. Suitable difunctional succinimidyl active esters include difunctional N-hydroxysuccinimide, 3,3'-dithio(sulfosuccinimidyl) propionate and bis(sulfosuccinimidyl) suberate. When using such chemical crosslinkers, the dried collagen matrix material is soaked in the crosslinker solution at about room temperature for a period of about 2 to 96 hours. The crosslinker solution may contain from about 0.1 to about 1.5% (weight/volume) crosslinker. Alternatively, the crosslinking agent may be added to the original solution or dispersion of the collagen source. To crosslink the collagen matrix using intensive dehydration, the microsponge is subjected to a vacuum of about 50 mTorr or more at temperatures ranging from about 50°C to about 200°C, such as about 100° to 110°C, for about 2 to about 96 hours. Expose at a temperature of As mentioned above, these two treatments can be used in combination, and it is preferred to use an intensive dehydration treatment followed by a chemical treatment to crosslink the collagen matrix. Generally, however, if the chemical treatment precedes the dehydration treatment, the crosslinking agent is added to the original collagen solution or dispersion prior to matrix particle preparation and lyophilization to facilitate the subsequent vacuum dehydration treatment. should be added directly. Also, as previously mentioned, the strength and biostability of collagen matrices are influenced by the degree of crosslinking introduced by such treatments. These crosslinking methods provide collagen matrices that are surprisingly resistant to collagenase and other enzymatic degradation, thereby making these materials particularly suitable for biological culture. Whenever chemical treatments are used, the collagen matrix should be thoroughly washed prior to further use to remove excess crosslinking agent. Further information on procedures for producing highly cross-linked collagen can be found in Frederick H. Silver, Richard A. Berg,
David E. Birk, Kevin Weadock and Conrad
Whyne, U.S. Patent Application No. 593,733, filed March 17, 1984 and entitled "Biodegradable Matrices and Method of Making the Same," the disclosure of which is incorporated herein by reference. There is. After thoroughly washing the crosslinked collagen matrix in ultrapure water, the microsponge can then be sterilized using conventional sterilization techniques. One particular advantage of collagen microsponges is that they can be manufactured separately from the bioimmobilization process, so that they can be properly sterilized or stored before inoculation. Microsponges are preferably sterilized using gamma irradiation. Ethylene oxide may alternatively be used, as long as important features of the microsponge are not compromised, and this may be an additional sterilization procedure known to those skilled in the art. When sterilizing microsponges with ethylene oxide, obviously the particles must be aerated well to remove all traces of this sterilizing agent before later use of the microsponges for biological culture. . It has also been discovered that the high temperature and prolonged intensive dehydration treatment used to cross-link the collagen satisfactorily sterilizes the microsponges, thus eliminating the need for additional treatments. Sterilized microsponges may be packaged aseptically for delivery to the ultimate consumer. The user simply places the microsponge in a pre-sterilized reactor, adds the appropriate nutrients and inoculum, and begins operation. In particularly suitable embodiments, this packaging actually provides the necessary connections for supplying the nutrient flow, removing the harvest liquid, and optionally ancillary operations, such as heat exchange, oxygen supply and process control. Consists of a disposable reaction vessel with a For fluidized bed reactions, the vessel will contain suitably designed distribution plates. Such pre-packed disposable reaction vessels are approximately 0.1 to 10
It can have a capacity of In this case, the reactor user simply bundles the process equipment, consisting of pumps, valves, piping, heat and gas exchangers, and various instruments and probes, and begins operation. Providing a disposable reactor pre-packed with a sterilizing agent ready-to-use microsponge significantly simplifies the start-up of a biological culture procedure, especially when changing from one culture to another. Although not completely understood, changes in process parameters lead to significant changes in the structure or profile of the highly crosslinked collagen matrix. Figures 1 and 2, which are micrographs of collagen matrices made using the technique described above, illustrate these different structures. The micrographs in Figures 1 and 2 were obtained using a scanning electron microscope. FIG. 1 shows a collagen matrix having a substantially wire mesh structure. In this structure, the diameter of the fiber network is typically about 1
It is on the order of microns. This structure is particularly desirable for attachment-independent organisms such as hybridomas. In this matrix, such organisms become entrapped in and on the matrix structure. Figure 2 shows a leaf-like matrix structure. The leaves of this structure typically have a thickness on the order of about 1 micron. This structure is particularly suitable for attachment-dependent cells such as fibroblast cells. It is now believed that the rate of cooling (freezing) of the droplets during the bead manufacturing process influences the morphology of the collagen microsponges. The following examples are intended to explain the invention in more detail and are not intended to limit the scope of the invention. Example 1 This example describes the production of microsponges of highly cross-linked collagen matrix material. Partially purified tendon collagen was purified by VWR.
A Wiley mill available from Scientific was used to obtain fibers having a length of less than about 200 microns and a diameter of about 5 to 50 microns. Next, a certain amount of crushed collagen material was mixed with an acetic acid solution using a Waring blender to prepare a collagen-based dispersion having a pH of about 3.0 and about 1.0% collagen (by weight). Titanium, an inert weighting material, was then added to the collagen-based dispersion as a fine powder with a particle size ranging from about 5 to about 180 microns. The composite mixture was then shaped into solid particles by first forming droplets of the composite mixture. Droplets are approximately 90
It was created by flowing the composite mixture through a vibrating hollow needle with an inner diameter of approximately 1.3 mm that was vibrated at a frequency of Hz. The droplets fell into a cooling bath of liquid nitrogen and quickly froze. Then freeze droplets of this complex mixture in Virtis at a vacuum of about 10 mTorr for about 48 hours.
Vacuum drying was performed using Freezemobile Lyophilizer Model 6. After this freeze-drying, the dried microsponge is VWR
Using a Scientific drying oven at a temperature of approximately 100℃
The sample was subjected to strong dehydration (dehydration heat treatment) under a vacuum of about 10 mTorr for about 72 hours. The microsponges were then treated with a 1.0% (by weight) solution of cyanamide as a chemical crosslinker at a pH of about 5.5 and about 20° C. for about 24 hours. The highly crosslinked microsponges were then thoroughly washed using ultrapure water for about 24 hours, dried, and then sterilized by gamma irradiation. This micro sponge has a particle size within the range of approximately 200 to 800 microns,
It had a void fraction of about 77%, a pore size of about 20 microns, and a specific gravity of about 1.1. This microsponge had a wire mesh microstructure. Example 2 The microsponges of Example 1 were able to support the growth of hybridoma cells. In particular, about 300
ml of microsponge could be included in a 600 ml reaction vessel. Microsponges could be inoculated with hybridoma cells and cultured using appropriate nutrient media. The reactor could be operated at solids concentrations of about 25-40% while vigorously stirring the reactor contents. A nutrient medium containing 10% fetal calf serum, such as Delbecko Modified Eagle medium, was fed continuously to the reactor and a product stream containing monoclonal antibodies could be recovered at substantially equivalent flow rates. It will be apparent to those skilled in the art that many modifications may be made without departing from the true spirit and scope of the invention. It is intended that the spirit and scope of the invention be limited only by the scope of the claims.