JPH04501806A - Method for promoting growth of substrate-dependent cells - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Method for promoting growth of substrate-dependent cells This application is filed under pending U.S. Application No. 07/355.590, dated May 23, 1989. This is a continuation application of No.

The present invention generally relates to substrate-dependent (an chorage-dependent) cell proliferation promotion method, and in particular The present invention relates to a method for promoting proliferation using seed polymer hollow fiber membranes.

A cell culture device for in vitro cell culture having a shell containing multiple hollow fiber membranes (recently known) This device uses oxygen, nutrients, and other chemical irritants to A containing medium is conveyed through the hollow fiber membrane. Nutrients and gases are diffused and externally convected transported through the hollow fiber membrane by The cell has a fluid flow between the fibers and the shell wall. Proliferate at pace.

Hollow fiber culture devices can maintain many types of cells at high densities in vitro. has been proven to be ideal for The material transport properties of the hollow fiber culture device are It is an effective means of supplying nutrients and gases and removing waste from the culture.

Semi-porous hollow fiber membranes with various porosity can be selected. Select appropriate porosity The selection maintains cell products on the outside of the fiber, eliminating waste and contaminating The waste material passes through the pores of the membrane and into the interior of the hollow fibers and is removed from the waste.

Examples of prior art cell culture devices include U.S. Pat. No. 4,804,628 and described therein. This is stated in the patent. Materials used in prior art hollow fiber membranes Examples include collagen-coated cellulose acetate, silicone carbonate capillary (U.S. Pat. No. 3.821.087); as well as polyacrylic resins such as polymethyl methacrylate. There are various natural and synthetic polymeric materials (U.S. Pat. No. 4,546,083).

U.S. Pat. No. 4,439,322 describes hollow fibers useful in blood purification or dialysis. ionic crosslinked polymethylmethane containing pendant sulfonate groups and quaternary nitrogen groups. mentions tacrylate copolymers. “Surface treatment” by Ramsay et al. Surface Treatment and Cell Attachment tach+aent) J, Invitro, Vol. 20, No. 10 (1984) Polymethacrylate sheets have relatively poor adhesion to substrate-dependent cells. Discloses that.

b2 substrate dependent cells Most mammalian cells have substrates to which they exhibit physical or chemical affinity. It is cultured as attached to. An exception to this format is notable: Usually belongs to hematopoietic cells or malignantly transformed cell lines. Release from adhesion suspended This condition greatly facilitates the culture of cells. However, possible or established A large group of cell lines of industrial value are predominantly substrate-dependent categories. Enter Golly. Most of the proteins with high market value are recombinant-transfected. recombinant-transfected cells and malignant cells They fall into this group as a result of transformed cell lines. Cell attachment and spreading are proliferation and provide fundamental functional benefits for differentiation. This increase in surface area is substrate dependent There are many receptor sites for mitogenic regulators required by cells. 5ite).

Brief description of the drawing The drawing is a photograph of a hollow fiber membrane. Figures 1 and 2 show 20x and 33x, respectively. This is a photograph of a cellulose membrane taken at magnification. 3 and 4 are manufactured according to the present invention. These are 20x and 50x photographs of ionically cross-linked PMMA membranes, respectively.

Summary of the invention The problems associated with substrate-dependent cell production in hollow fiber bioreactors are Identification of suitable materials to produce hollow fiber membranes that allow growth and product production.

Certain ionically cross-linked polymers support substrate-dependent cell growth in hollow fiber bioreactors. It has now surprisingly been found that this protein provides an excellent material for the optimal propagation of .

Therefore, the present invention provides anionic and cationic substituents and from about 0.5 to about 20 mole % of pendant groups selected from the group consisting of possible salts thereof; Substrate-dependent on hollow fiber membranes using hollow fiber membranes made of ionically cross-linked polymers containing The present invention relates to a method for promoting the proliferation of sex cells.

The present invention also provides a method for promoting growth of substrate-dependent cells on a hollow fiber membrane, the method comprising: Methyl methacrylate containing about 0.5 to 10 moles of pendant sulfonate group-containing monomer and about 0.5 to 10 mol% of monomers having pendant quaternary nitrogen-containing groups. and a methyl methacrylate copolymer comprising:

Disclosure of invention The polymer used as the hollow fiber in the present invention is ionizable at physiological pH. about 0.5 to about 20 mole %, preferably about 1 to about 5 mole %, pendant groups. A polymer, preferably an ionically crosslinked polymer. Ionizable groups are anions substituents and their salts, which are either cationic or cationic, as listed below. include. Additionally, copolymers containing anionic groups are copolymers containing cationic groups. can be combined with to produce an ionically crosslinked polymer. Preferred blends of the copolymers The groups preferably contain a slightly smaller proportion of either type of group, preferably in a ratio of about 5:1 to 1:5. including blends in excess, such as in a ratio of about 1.1:1.

A preferred polymer is a copolymer of polymethyl methacrylate (PMMA) . In addition, other conventional polymers may be used if they do not have ionizable pendant groups. However, they can be added to modify physical properties.

Cationic groups include common quaternary nitrogen-containing groups and salts thereof. The preferred salt is Nori chloride salts, especially chloride salts.

Anionic groups include sulfonate groups, such as sulfonic acid and carboxylic acid groups and their salts. including. Typical salts include metal cations, such as -valent, divalent or trivalent cations. , i.e. salts of sodium, potassium, calcium, aluminum, etc., especially It is a sodium salt.

Preferred polymethyl methacrylate (PMMA) membranes that can be used in the present invention and their The method of manufacturing these is described in detail in U.S. Pat. No. 4,439,322, The patents are incorporated herein by reference.

Preferred PMMA membranes have two types of polymers: (a) pendant sulfonate groups; a methyl methacrylate copolymer comprising about 0.5 to 10 mol% of monomers having (b) about 0.5 to 10 mol% of a monomer having a pendant quaternary nitrogen-containing group; methyl methacrylate copolymer.

One of the copolymers is a polyanionic type with sulfonic groups; Since the other polymer in the copolymer has quaternary nitrogen-containing groups, The membrane to be used is an ionically cross-linked multi-ion composite membrane.

The two copolymers can be mixed in a weight ratio of about 1=9 to 9:1.

This mixing ratio is such that the number of sulfonate groups and the number of quaternary nitrogen-containing groups in the copolymer is approximately 5=1. to 1=5, preferably from about 2:1 to 1:2. It is preferable to select the From a membrane strength standpoint, the ionic complex is essentially It should be formed from equal numbers of sulfonate groups and quaternary nitrogen-containing groups. However, The number of sulfonate groups and the number of quaternary nitrogen-containing groups in the polymer make the membrane cationic or anionic. It is also preferable to use a mixing ratio that ensures that the mixture has a ratio that makes it ionic. Delicious.

Hollow fiber membranes that can be used in the present invention have a substantially circular hollow cross section, about 5 to 500 microns. with a uniform wall thickness within the range of . The porosity of the membrane is within the molecular weight exclusive range of 6KD to approximately 3000KD. weightexclusion range).

Typical examples of substrate-dependent cell lines are common diploid cell lines, such as human fetal lung cells. cells, human foreskin cells, human embryonic kidney cells, chicken, rabbit, mouse and rat cells. Embryonic fibroblasts, chimpanzee embryonic fibroblasts, rat glial cells, feline embryonic lines Fibroblasts and secondary monkey kidney cells; secondary cells, mouse macrophages, rat pancreas cells, rat hepatocytes, chicken embryo fibroblasts, e.g. mouse fibroblasts, normal human liver cells, Chinese hamster ovary and lung cells, baby gnome star kidneys cells, Chinopan-fetal lung cells, African green monkey kidney cells, mouse L cells, H eLa, mouse macrophage cell line, transformed canine kidney cells, sarcoma line Transformed rat glial cells, mouse fibroblasts, human glioma cells, human bone sarcoma cells, Margin-Darby canine kidney cells, KB cells, rhesus monkey kidney cells, McCoy cells, human thyroid cancer cells, human transverse stroma muscle tumor cells, rat muscle-derived fibroblasts, and rabbit keratocytes.

Next, the present invention will be explained in more detail based on the following examples. are illustrative and do not define the scope of the invention as defined by the claims. It is not limited or limited.

Example 1 Culture of substrate-dependent human glioma cell lines in a hollow fiber bioreactor system Are suitable. Ionically crosslinked polymethyl methacrylate (PMMA) hollow fiber Toray R Filtryzer J Dialyzer Series Leeds] provides good access of cells into the special capillary space (ECS) of the bioreactor. Allows for adhesion. A cell-free stream flows through the lumen of the fiber, carrying nutrients and fine particles. Diffusion exchanges with metabolic waste products by cell mass. bioria The large surface area of the vector (2011) allows for effective mass transport and facilitates tissue-like cells. Density is obtained. Glioma mesenchymal special cell matrix protein (GMEM) , a 1000Kd glycoprotein is secreted by cells within the EC8 and in situ It is recovered by a 10 Kd molecular weight cutoff (NWCO) of empty fiber membranes.

A typical bioreactor has an integrating reservoir. ir) is formed as a recirculating flow loop starting and ending within the ir. 4 vibration pumps Drive this loop at 00vl/min. Above the bioreactor for oxygen supplementation An oxygenator was placed in the stream. The air pump controls the air flow to the oxygenator. Ru. A spiral infusion pump supplies fresh medium to the reservoir. Blood using another screw pump The supernatant is supplied to bioreactor EC3 and GMEM is collected.

The temperature and pH of the culture medium were monitored within the reservoir. The temperature was maintained constant at 37°C. The pH was maintained at 7.25 using a carbon dioxide/bicarbonate buffer system.

System assembly and sterilization Preassemble and sterilize hollow fiber autoclavable parts . Incorporate a sterile hollow fiber bioreactor and oxygenator into the system under aseptic conditions It will be done. Transfer the complete assembly to a benchtop room. All ancillary equipment, e.g. Carboys, bottles, gas lines and appliances are assembled into this assembly. Connect to.

- Expand in complete medium using a flask. Complete medium contains 10% fetal bovine serum ( FBS), modified Dulbecco with 2% L-glutamine It consists of Eagle medium (DMEN). Cells are flushed with buffered saline during transport. Hemocytometer (h) using water wash, trypsin addition and fresh medium Count viable cells on an emocuteometer. T-175 hula A suitable inoculum was obtained from Sco. Centrifuge the culture, resuspend in medium, and attach Transfer to seed jar. Development of the inoculum takes approximately 10 days.

■ After putting the inoculum into the bioreactor EC8, remove the appropriate amount from the EC8 supply line. Wash the remaining inoculum with a volume of FBS-containing DMEN.

Manufacturing After inoculation, the “proliferation phase” that contributes to the rapid development of cell populations is the most important stage for cell proliferation. started under suitable conditions. Adjust the injection rate to keep up with the proliferating cell population. Facilitate. Next, the cells transition from the proliferation phase to the maintenance state (week 3). in this case , the cells cease to proliferate, but their viability and activity are preserved. process The remainder of the (run) is the “manufacturing phase”, which is maintained at 2 mg/ml for the next 4 weeks. It contributes to the production of GMEM, which is retained and recovered at a rate of 7Q mg/day.

We show that substrate-dependent cell lines grow favorably on ionically cross-linked PMMA substrates. To further illustrate other such cell lines, ionically cross-linked PMMA and cellular cultured in vitro using in-hollow fibers. Cutanderfelain kidney cells (Crandell Fe1ine Hdney Ce1l) (CRFK) , which has epithelial properties used for this purpose. It is a substrate-dependent cell line. (1) Thin Substrate preference of cells and (2) hollow fiber cells. experiments with hollow fiber materials to confirm the possible use of substrate materials in cell cultures. In vitro cell culture methods are described below. Using this method, substrate-dependent cells can be We demonstrate the benefits of ionically cross-linked PMMA as a substrate for effective culture.

Materials and Methods Two types of hollow fiber material sources, i.e., ionically crosslinked PMMA and Cellulose was used. The fibers were washed, autoclaved, and loaded with CRFK cells. Equal cultures were placed in separate T-flasks inoculated. Allow the culture to grow undisturbed over a period of time. I let it grow. After this period, the cultures were observed and photographed.

1. Hollow fiber bioreactors, one containing PMMA and the other containing cellulose fibers The fibers were removed. Fiber source: PMMA:rfilt Riser” dialyzer series, tray cellulose: rC-DaKJ dialyzer series , C, D, Medical 2, put the fibers in a beaker of distilled water, then add a large amount of distilled water. and rinsed several times.

3. Each culture required a small number of fibers. Therefore, about 1-2C ■ 3 amount of fiber The bundle was placed in a beaker of distilled water. Autoclave the fibers to be used in a beaker. Sterilized (30 minutes, wet cycle).

4. After the sterilization cycle, the fibers were placed into independent T175 tissue culture flasks.

5. Both PMMA-containing flask and cellulose fiber-containing flask were filled with 40xlO Approximately 50.011 cells of CRFK culture were added at a cell concentration of 'cells/ml. The culture medium is , 10% bovine baby serum [Ilyclone] and] 2% L-G Is it DME high glucose supplemented with [Iruvin]? It was made up of several things.

6. Both flasks were heated to 37°C and 7.5% CO! into the incubator set below. It was.

7. Observe the cultures and their growth progress under microscopy at various times after the start of the experiment. Observed. Photographs were taken on the fourth day after the start of the experiment.

Cells proliferated well on both flask surfaces as expected. However, P MMA Only fibers showed cell attachment and proliferation. No cell proliferation was observed in set fibers. Ta. The difference in the ability of the two types of fibers to promote CRFK cell proliferation is clearly seen in the photograph.

Photo description 1. Cellulose (20x) 2. Cellulose (33x) No cell proliferation was observed in the cellulose fibers (Figures 1 and 2). fineness of fiber perception Although the presence of cells was observed, no interaction was observed between the two.

3. PMMA (20x) In Figure 3, cells proliferating on the entire fiber surface are observed, and the cell contrast in the image area is easily seen.

4. PMMA (50x) In the close-up of the PMMA fiber in Figure 4, cells adhere to the entire outer surface of the hollow fiber. is shown. This was common to all PMMA fibers in the culture.

Engraving (no changes to the content) FIG, f c Lurose Koku KRL rzox>FIG, 3 Io>4s P MMAv Sky J Lean-U Ig (20X) FIG, 4 / IK Lean v’1# PHHA +’lf$, H (50×) Procedural Amendment (Kun) February 1991//Kouen

Claims (10)

[Claims] 1. In a method for promoting substrate-dependent cell proliferation on hollow fiber membranes, anionic and and cationic substituents as well as those that are ionizable at physiological pH. an ionic bridge containing from about 0.5 to about 20 mole percent of pendant groups selected from the group consisting of salts; A method consisting of using a hollow fiber membrane consisting of a bridged polymer. 2. A claim that the cationic substituent is selected from the group consisting of quaternary nitrogen-containing groups and salts thereof. Method of item 1. 3. Anionic substituents consisting of sulfonate groups, carboxylic acid groups and salts thereof 2. The method of claim 1, wherein the method is selected from the group consisting of: 4. The polymer is an anionic substituent-containing copolymer and a cationic substituent-containing copolymer. 2. The method of claim 1, comprising a blend of: 5. In a method for promoting substrate-dependent cell growth on hollow fiber membranes; Methyl methacrylate containing about 0.5 to 10 mol% of monomers having tosulfonate groups rate copolymer and about 0.5 to 1 monomer having pendant quaternary nitrogen-containing groups. A method consisting of a mixture with a methyl methacrylate copolymer containing 0 mol%. 6. The ratio of sulfonate-containing groups to quaternary nitrogen-containing groups in the copolymer is about 5:1 to 1:5. 6. The method of claim 5, wherein: 7. 6. The method of claim 5 further comprising a vinyl monomer in the copolymer. 8. 2. The method of claim 1, wherein the substrate-dependent cells are human glioma cells. 9. 6. The method of claim 5, wherein the substrate-dependent cells are human glioma cells. 10. Substrate-dependent cell growth promotion on hollow fiber membranes in cell culture bioreactors 1 to about 5 moles of monomer having pendant sulfonate groups. % and from about 1 to about 5 mole % of monomer containing pendant quaternary nitrogen-containing groups; consists of a methyl methacrylate copolymer containing a slight excess of pendant groups of either A method consisting of using hollow fiber membranes.