METHOD OF ENHANCING GROWTH OF ANCHORAGE DEPENDENT CELLS

Abstract: There is disclosed a method of enhancing growth of anchorage-dependent cell lines in hollow fiber membrane cell culture bioreactors, through the use of hollow fiber membranes made of an ionic crosslinked polymer, preferably polymethylmethacrylate, containing cationic and/or anionic substituents which are ionizable at physiological pH.

Description: METHOD OF ENHANCING GROWTH OF ANCHORAGE DEPENDENT CELLS This application is a continuation of United States application Serial No. 07/355,590 filed 23 May 1989, which is still pending. BACKGROUND OF THE INVENTION 1. Field of the Invention The invention generally relates to a method of enhancing growth of anchorage-dependent cells in hollow fiber membrane cell culture bioreactors, and in particular, to obtaining such enhanced growth through the use of certain polymeric hollow fiber membranes. 2. Description of the Prior Art a. Cell Culture Devices Cell culture devices for culturing cells .in vitro having a shell with a plurality of hollow fiber membranes have been known for quite some time. Medium containing oxygen, nutrients and other chemical stimuli is transported through the lumen of the hollow fiber membranes. Nutrients and gases are carried across the hollow fiber membranes by diffusion and outward convective flow. Cells are grown in the fluid space between the fibers and the shell wall. Hollow fiber culture devices have been proven to be ideal for the maintenance of many types of cells at high densities in vitro. The mass transfer characteristics of hollow fiber culture devices provide an efficient means of delivering nutrients and gases and removing waste products from a culture. The semi-porous hollow fiber membranes can be selected with various pore sizes. With proper pore size selection, the cellular product can be maintained on the outside of the fibers, while waste products and contaminating proteins will pass through the membrane pores into the lumen of the hollow fibers where they can be subsequently removed from the culture. Examples of prior art cell culturing devices include U.S. Patent No. 4,804,628 and patents cited therein. Examples of materials used in prior art hollow fiber membranes include cellulose acetate, silicone carbonate and capillaries coated with collagen (U.S. Patent No. 3,821,087) and a great variety of natural and synthetic polymeric materials including polyacrylics such as polymethylmethacrylate (U.S. Patent No. 4,546,083). U.S. Patent No. 4,439,322 describes ionic crosslinked polymethylmethacrylate copolymers containing pendent sulfonate and quaternary nitrogen groups in the form of hollow fibers useful in blood purification, i.e., dialysis. Ramsay, et.al. , "Surface Treatments and Cell Attachment," In Vitro, Vol. 20, No. 10 (1984) discloses that untreated polymethylmethacrylate sheets have relatively poor adhesiveness to anchorage-dependent cells. b. Anchorage-Dependent Cells Most mammalian cells are cultured as attached to a substrate for which they exhibit a physical or chemical affinity. Exceptions to this mode are notable and usually belong to hemapoietic cell groups or malignantly transformed cell lines. Freedom from attachment greatly facilitates the culture of such cells in suspension. However, a large group of cell lines with potential or established industrial value fall into the category of dedicated anchorage-dependence. The bulk of high market value proteins fall into this group as a result of recombinant-transfected cells and malignantly transformed cell lines. Cell attachment and spreading offer functional advantages fundamental for growth and differentiation. Attached and spread cells have a significantly higher surface area to volume ratio than do spherical cells. This increase in surface area results in more receptor sites for mitotic stimulators/regulators required by anchorage-dependent cells. BRIEF DESCRIPTION OF THE DRAWINGS The drawings are photomicrographs of hollow fiber membranes. Figures 1 and 2 are photomicrographs of cellulose membranes taken at 2OX and 33X magnification, respectively. Figures 3 and 4 are photomiocrographs of ionic crosslinked PMMA membranes made according to the invention at 2OX and SOX magnification, respectively. SUMMARY OF THE INVENTION A problem with anchorage-dependent cell production in hollow fiber bioreactors is identifying suitable materials from which to manufacture hollow fiber membranes which allow optimum cell growth and product production. We have now discovered that certain ionic, crosslinked polymers unexpectedly provide an excellent material for optimum growth of anchorage-dependent cells in hollow fiber bioreactors. The invention therefore relates to a method of enhancing growth of anchorage-dependent cells on hollow fiber membranes comprising employing hollow fiber membranes comprising an ionic crosslinked polymer containing from about 0.5 to about 20 mol% of pendent groups selected from the group consisting of anionic and cationic substituents and salts thereof which are ionizable at physiological pH. The invention also relates to a method of enhancing growth of anchorage-dependent cells on hollow fiber membranes, wherein the membranes comprise a mixture of methylmethacrylate copolymer containing about 0.5 to 10 mol% of a monomer having pendent sulfonate groups, and a methylmethacrylate copolymer containing about 0.5 to 10 mol% of a monomer having pendent quaternary nitrogen-containing groups. DESCRIPTION OF THE INVENTION The polymers which may be used in this invention in the form of hollow fibers are polymers, preferably ionic, crosslinked polymers, having from about 0.5 to about 20 mol% and preferably from about 1 to about 5 mol% and especially from..about 2 to about 4 mol% of pendant groups that are ionizable at physiological pH. The ionizable groups may be either anionic or cationic and include the substituents and salts thereof listed below. Further, copolymers containing anionic groups can be combined with copolymers containing cationic groups to yield ionic crosslinked polymers. Preferred blends of the foregoing copolymers include those blends resulting in a ratio between about 5:1 and 1:5 and preferably a slight excess of ionic groups of either type, e.g. , about 1.1:1. Preferred polymers are copolymers of polymethylmethacrylate (PMMA) . In addition, other conventional polymers may be added for modification of physical properties even though they may not have pendent ionizable groups. Cationic groups include conventional quaternary nitrogen-containing groups and their salts. Preferred salts include halide and sulfate salts and especially chloride salts. Anionic groups include sulfonate groups, such as, for example, sulfonic acid; and carboxylic acid groups and their salts. Typical salts are metal cations, e.g., mono-, di- or tri- valent cations, i.e., sodium, potassium, calcium, aluminum, etc., and especially sodium salts. Preferred polymethylmethacrylate (PMMA) membranes which may be used in this invention and their method of manufacture are described in detail in U.S. Patent No. 4,439,322, which patent is incorporated by this reference. A preferred PMMA membrane consists of two kinds of polymers: a) a methylmethacrylate copolymer containing about 0.5 to 10 mol% of a monomer having pendent sulfonate groups, and b) a methylmethacrylate copolymer containing about 0.5 to 10 mol% of a monomer having pendent quaternary nitrogen-containing groups. As one of the copolymers is of the polyanionic type having sulfonate groups, and the other of the polycationic type having quaternary nitrogen- containing groups, the membrane which is manufactured therefrom is an ionically crosslinked polyion complex membrane. The two copolymers may be mixed in a weight ratio of about 1:9 to 9:1. This mix ratio is preferably selected to ensure that the numbers of the sulfonate and quaternary nitrogen-containing groups in the copolymers have a ratio of about 5:1 to 1:5, preferably about 2:1 to 1:2. From the standpoint of membrane strength, an ion complex should be formed of substantially equal numbers of sulfonate and quaternary nitrogren-containing groups. However, it is preferable to employ a mix ratio which ensures that the numbers of the sulfonate and quaternary nitrogen-containing groups in the copolymers have a ratio so that the membrane is either anionic or cationic. The hollow fiber membrane which may be used in the invention has a substantially circular hollow cross section, a uniform wall thickness in the range of about 5 to 500 microns, and an inside diameter of about 70 to 1,000 microns. Porosity of the membrane is engineered to provide a molecular weight exclusion range of 6 Kd to about 3000 Kd. Typical examples of anchorage-dependent cell lines include normal diploid cell strains, such as human embryonic lung, human foreskin, human embryonic kidney, chicken, rabbit, mouse and rat embryo fibroblasts, chimpanzee liver fibroblasts, rat glial cells, feline lung fibroblasts and secondary monkey kidney cells; primary cells, such as monkey, dog and rabbit kidney cells, mouse macrophages, rat pancreas cells, rat hepatocytes, chicken embryo fibroblasts, rat pituitary cells and amniotic fluid cells; established and transformed cell lines, such as mouse fibroblasts, normal rat kidney, Chinese hamster ovary and lung, baby hamster kidney, chimpanzee embryo lung, African green monkey kidney, mouse L cells, HeLa, mouse macrophage cell line, transformed dog kidney, sarcoma virus transformed rat kidney and mouse fibroblasts, human glioma, human osteosarcoma, Madin-Darby canine kidney, KB cells, rhesus monkey kidney, McCoy cells, human thyroid carcinoma, human rhabdomyosarcoma, rat muscle derived fibroblasts and rabbit cornea cells. The invention will now be described more specifically with reference to the following Examples, which are intended to be illustrative, but not to define or to limit the scope of the invention, which is defined in the appended claims. EXAMPLE I Culture of an Anchorage-Dependent Human Glioma Cell Line in a Hollow Fiber Bioreactor System System Configuration - Hollow fiber bioreactors are well suited to the culture of a human glioma cell line. The ionic crosslinked polymethylmethacrylate (PMMA) hollow fibers (Toray "Filtryzer" dialyzer series) allow excellent attachment of cells in the extra capillary space (ECS) of the bioreactor. A cell-free stream flows through the lumen of the fibers and undergoes diffusive exchange of nutrients and metabolic wastes with the cell mass. The large surface area (2.0 m 2 ) in the bioreactor provides efficient mass transfer, and tissue like cell densities are achieved. Glioma-mesenchymal extra cellular matrix protein (GMEM) , a 1000 Kd glycoprotein, is secreted by the cells in the ECS and recovered in situ by virtue of the 10 Kd molecular weight cut off (MWCO) of the hollow fiber membranes. A conventional bioreactor was configured in a recirculation flow loop that starts and ends in an integrating reservoir. An oscillating pump drives the loop at 400 ml/min. An oxygenator was located upstream of the bioreactor for oxygen replenishment. An air pump provides a controlled air flow rate to the oxygenator. A peristaltic infusion pump feeds fresh medium into the reservoir. A separate peristaltic pump was used to deliver serum to the bioreactor ECS, as well as harvest GMEM. The temperature and pH of the culture medium were monitored in the reservoir. Temperature was maintained constant at 37 β C and the pH was maintained at 7.25 using a carbon dioxide/bicarbonate buffering system. System Assembly and Sterilization - The auto- clavable portion of the hollow fiber system is pre-assembled and sterilized. The sterile-packed hollow fiber bioreactor and oxygenator are incorporated sterilely into the system. The completed assembly is transferred into a benchtop system chamber. All accessories, e.g., carboys, bottles, gas lines and instrumentation are connected to the assembly. Inoculum Generation - A single vial of glioma cells (about 2.5xl0 6 ) is reconstituted and expanded in complete medium using T-flasks. The complete medium consists of Dulbecco's Modified Eagle's Medium (DMEM) with 10% Fetal Bovine Serum (FBS) , 2% L-glutamine. During flash transfers, the cells are recovered by washing with buffered saline, trypsinization and resuspension in fresh medium. Viable cells are counted on a hemocytometer using the trypan blue exclusion method. A suitable inoculum was obtained from T-175 flasks. The culture was centrifuged, resuspended in the medium, and transferred into an inoculation bottle. Generation of inoculum takes about 10 days. Inoculation - After the inoculum is loaded into the bioreactor ECS, the ECS feed line is flushed of residual inoculum with an appropriate volume of DMEM containing FBS. Production - After inoculation, the "growth phase", dedicated to rapid generation of cell mass, was initiated by conditions optimal for cell growth. The infusion rate is stepped up to keep up with the proliferating cell mass. The cells then shift from a proliferative to a maintenance state (third week) . The growth of cells is then halted, but their viability and activity is preserved. The remainder of the run is the "production phase", dedicated to the production of GMEM which is maintained at 2 mg/ml for the next 4 weeks, and harvested at a rate of 70 mg/day. EXAMPLE II Anchorage-Dependent Cell Growth with Ionic Crosslinked PMMA and Cellulose Hollow Fibers To further illustrate that anchorage-dependent cell lines grow favorably on ionic crosslinked PMMA substrates, another such cell line was cultured in vitro with ionic crosslinked PMMA and with cellulose hollow fibers. Crandell Feline Kidney Cells (CRFK) are an anchorage-dependent cell line of an epithelial nature that were used for this purpose. Described below is a method for culturing cells with hollow fiber materials in vitro to determine 1) the cell's substrate preference and 2) the potential application of the substrate material in hollow fiber cell culture. This procedure was used to again demonstrate the advantage of ionic crosslinked PMMA as a substrate for the effective culturing of anchorage-dependent cells. Materials and Methods - Two different sources of hollow fiber materials were used; ionic crosslinked PMMA and Cellulose. The fibers were washed, autoclaved sterile, and placed into independent T-flasks seeded with equal cultures of CRFK cells. The cultures were allowed to grow undisturbed for a period of time. After that time period, observations and photomicrographs of the culture were taken. 1. Hollow fiber bioreactors, one containing the PMMA and another containing the Cellulose fibers, were opened and extracted of their fibers. Source of fibers: PMMA: "Filtryzer" dialyzer series, Toray Cellulose: "C-Dak" dialyzer series, CD. Medical. 2. The fibers were placed into a beaker of distilled water and subsequently rinsed several times with large amounts of distilled water. 3. A small number of fibers were needed for each culture; therefore, a bunch of fibers approximately 1-2 cm 3 in volume were placed in a beaker of distilled water. The fibers to be used were autoclaved sterile in the beaker (thirty minutes, wet cycle) . 4. After sterilization, the fibers were placed into independent T175 tissue culture flasks. 5. Into both the PMMA and Cellulose fiber containing flasks, 50.0 mis of a CRFK culture at a cell concentration of 4.0xl0 5 cells/ml was added. The culture media- consisted of DME High Glucose (Irvine) supplemented with 10% fetal bovine serum (Hyclone) and 2% L-glutamine (Whittaker) . 6. Both flasks were placed into an incubator set at 37 β C, under 7.5% C0 2 . 7. Observations of the cultures and their growth progress were made under microscopic examination at various times after initiating the experiment. On day 4 of the experiment, photomicrographs were taken. Observations - The cell grew well on both flasks' surface as would be expected; however, only the PMMA fibers displayed cell attachment and growth. No cell growth was observed on the Cellulose fibers. The difference between the two fibers' ability to promote growth of the CRFK cells is clear in the photomicrographs. Photomicrograph Legend: 1. Cellulose (20x) No cell growth is 2. Cellulose (33x) observed on the cellulose fibers (Figs. 1 and 2) . Note the presence of cells nearby the fibers, yet no interaction between the two is seen. 3. PMMA (20X) In Fig. 3, cells are observed growing all over the fiber surface and cell contrast can be seen easily on the edges. 4. PMMA (50X) In Fig. 4, a close up of the PMMA fiber, cells are shown adhering to the entire outer surface of the hollow fiber. This was common on all PMMA fibers in the culture.

Claims: WE CLAIM: 1. A method of enhancing growth of anchorage-dependent cells on hollow fiber membranes comprising employing hollow fiber membranes comprising an ionic crosslinked polymer containing from about 0.5 to about 20 ol percent of pendent groups selected from the group consisting of anionic and cationic substituents and salts thereof which are ionizable at physiological pH. 2. The method of Claim 1 wherein the cationic substituent is selected from the group consisting of quaternary nitrogen-containing groups and salts thereof. 3. The method of Claim 1 wherein the anionic substituent is selected from the group consisting of sulfonate groups, carboxylic acid groups and salts thereof. 4. The method of Claim 1 wherein the polymer comprises copolymers containing the anionic substituents blended with copolymers containing the cationic substituents. 5. A method of enhancing growth of anchorage- dependent cells on hollow fiber membranes, wherein the membranes comprise a mixture of a methylmethacrylate copolymer containing about 0.5 to 10 mol% of a monomer having pendent sulfonate groups, and a methylmethacrylate copolymer containing about 0.5 to 10 mol% of a monomer having pendent quaternary nitrogen-containing groups. 6. The method of Claim 5 wherein the sulfonate- containing groups and the quaternary nitrogen- containing groups in the copolymers are in a ratio between about 5:1 and 1:5. 7. The method of Claim 5 further including in the copolymer a vinyl monomer. 8. The method of Claim 1 wherein the anchorage- dependent cells are human glioma cells. 9. A method of Claim 5 wherein the anchorage- dependent cells are human glioma cells. 10. A method of enhancing growth of anchorage- dependent cells on hollow fiber membranes in cell culture bioreactors comprising employing hollow fiber membranes comprising a methylmethacrylate copolymer containing about 1 to about 5 mol percent of a monomer containing pendent sulfonate groups and about 1 to about 5 mol percent of a monomer containing pendent quaternary nitrogen-containing groups, the copolymer containing a slight excess of either of the pendent groups.

Inventors: GEBHARD TIMOTHY CONRAD, VEERAMALLU UDAY KUMAR, GEBHARD, TIMOTHY, CONRAD, VEERAMALLU, UDAY, KUMAR
Applicants: SYNBIOTICS CORP, SYNBIOTICS CORPORATION
Application year: 1990
Main classification: C12N5
IPC codes: C12M3, C12N5