EP0426830A1 - Method of enhancing growth of anchorage dependent cells - Google Patents
Method of enhancing growth of anchorage dependent cellsInfo
- Publication number
- EP0426830A1 EP0426830A1 EP90908822A EP90908822A EP0426830A1 EP 0426830 A1 EP0426830 A1 EP 0426830A1 EP 90908822 A EP90908822 A EP 90908822A EP 90908822 A EP90908822 A EP 90908822A EP 0426830 A1 EP0426830 A1 EP 0426830A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- groups
- hollow fiber
- cells
- anchorage
- pendent
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0068—General culture methods using substrates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/30—Synthetic polymers
Definitions
- 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.
- 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.
- FIGS. 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.
- 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.
- 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.
- 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.
- 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) .
- PMMA polymethylmethacrylate
- 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.
- 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.
- 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.
- an ion complex should be formed of substantially equal numbers of sulfonate and quaternary nitrogren-containing groups.
- 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.
- 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
- System Configuration - Hollow fiber bioreactors are well suited to the culture of a human glioma cell line.
- the ionic crosslinked polymethylmethacrylate (PMMA) hollow fibers allow excellent attachment of cells in the extra capillary space (ECS) of the bioreactor.
- ECS extra capillary space
- 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
- MWCO molecular weight cut off
- 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 ⁇ 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.
- DMEM Dulbecco's Modified Eagle's Medium
- FBS Fetal Bovine Serum
- 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.
- the ECS feed line is flushed of residual inoculum with an appropriate volume of DMEM containing FBS.
- 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.
- CRFK Crandell Feline Kidney Cells
- 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.
- 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.
- 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.
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
L'invention concerne un procédé améliorant la croissance de lignées cellulaires dépendant de la fixation dans des bioréacteurs de culture de cellules de membranes à fibres creuses en utilisant des membranes à fibres creuses constituées d'un polymère ionique réticulé, de préférence du polyméthylméthacrylate, contenant des substituants cationiques et/ou anioniques qui sont ionisables à un pH physiologique.The invention relates to a method for improving the growth of attachment-dependent cell lines in hollow fiber membrane cell culture bioreactors using hollow fiber membranes made of a cross-linked ionic polymer, preferably polymethylmethacrylate, containing cationic and/or anionic substituents which are ionizable at physiological pH.
Description
Claims
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US35559089A | 1989-05-23 | 1989-05-23 | |
US355590 | 1989-05-23 |
Publications (2)
Publication Number | Publication Date |
---|---|
EP0426830A1 true EP0426830A1 (en) | 1991-05-15 |
EP0426830A4 EP0426830A4 (en) | 1993-09-08 |
Family
ID=23398016
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP19900908822 Withdrawn EP0426830A4 (en) | 1989-05-23 | 1990-05-23 | Method of enhancing growth of anchorage dependent cells |
Country Status (3)
Country | Link |
---|---|
EP (1) | EP0426830A4 (en) |
JP (1) | JPH04501806A (en) |
WO (1) | WO1990014417A1 (en) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0567886A3 (en) * | 1992-04-21 | 1994-11-02 | Kurashiki Boseki Kk | Coating composition for culturing animal adhesive cells and method for culturing of the cells in serum-free condition. |
BE1008955A3 (en) * | 1994-11-14 | 1996-10-01 | Univ Catholique Louvain | Process for obtaining and products obtained biomaterials. |
US5618718A (en) * | 1994-12-30 | 1997-04-08 | Universite Laval | Production of a contractile smooth muscle |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US3883393A (en) * | 1972-05-18 | 1975-05-13 | Us Health Education & Welfare | Cell culture on semi-permeable tubular membranes |
US3910819A (en) * | 1974-02-19 | 1975-10-07 | California Inst Of Techn | Treatment of surfaces to stimulate biological cell adhesion and growth |
JPS5714640A (en) * | 1980-07-02 | 1982-01-25 | Toray Ind Inc | Separating membrane of methyl methacrylate type |
JPS5889179A (en) * | 1981-11-24 | 1983-05-27 | Japan Synthetic Rubber Co Ltd | Cell culture bed |
-
1990
- 1990-05-23 EP EP19900908822 patent/EP0426830A4/en not_active Withdrawn
- 1990-05-23 WO PCT/US1990/002778 patent/WO1990014417A1/en not_active Application Discontinuation
- 1990-05-23 JP JP2508516A patent/JPH04501806A/en active Pending
Non-Patent Citations (1)
Title |
---|
See references of WO9014417A1 * |
Also Published As
Publication number | Publication date |
---|---|
EP0426830A4 (en) | 1993-09-08 |
WO1990014417A1 (en) | 1990-11-29 |
JPH04501806A (en) | 1992-04-02 |
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Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
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18D | Application deemed to be withdrawn |
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