JPH0761253B2 - Cell culture carrier and method for producing the same, and method for producing cell culture carrier having anionic hydrophilic property - Google Patents

Description

TECHNICAL FIELD The present invention relates to a cell culture carrier with high culture efficiency for tissue culture of a living body and a method for producing the same.

[Prior Art] Cell culture depends on the type of support carrier, 1) monolayer culture, 2)
Suspension culture, 3) embedded culture. The monolayer culture method is a method of culturing cells that can grow only after being adhered to a carrier, and is a method that is widely adopted as a surface culture. However, in such a case, there is a problem that the cell growth is performed on a flat surface, the cell morphology is different from that of the living body, and the cell growth stops when the surface is completely covered. In addition, the suspension culture method is a method of suspending and culturing in a culture medium, and is limited only to cells such as blood cells and cancer cells that do not require adhesion to a carrier. However, it is difficult for many kinds of cells, especially epithelial cells, to express a proliferative function by such a simple method, and vigorous studies have been made for culturing in a more similar environment in the living body. Therefore, attempts have been made to remove waste products from cells, use hollow fiber membranes for the purpose of supplying nutrients to cells, and three-dimensional culture with collagen sponge as an embedding culture method.

The hollow fiber membrane is basically a two-dimensional culture, which is naturally limited. In the case of a collagen gel sponge, a cell morphology very similar to that of a living body is obtained, and it is possible to continue to grow without changing the morphology of the living body.

[Problems to be Solved by the Present Invention]

As a problem of the conventional culture method using collagen gel, the size of the pores of the gel sponge cannot be uniformly and well processed, and the mechanical strength is weak, so that it cannot be processed into various forms and it is a major factor in actual mass culture. It was a problem. The present invention provides a highly practical, high-performance cell culture carrier that solves the problems of reproducibility of culture characteristics, moldability, mechanical strength, and ensures excellent tissue growth, and a method for producing the same.

[Means for solving problems]

 The carrier for cell culture of the present invention has the following constitution.

That is, it is a cell culture carrier comprising ultrafine fibers of 1.0 dtex or less having anionic hydrophilicity.

The method for producing the cell culture carrier of the present invention has the following constitution.

That is, when the polymer is spun to obtain a cell culture carrier composed of ultrafine fibers of 1.0 dtex or less, the polymer stage,
The method for producing a carrier for cell culture is characterized in that an anionic hydrophilic group is introduced at either the fiber stage or the carrier stage.

Furthermore, the method for producing a cell culture carrier having an anionic hydrophilic property of the present invention has the following constitution.

That is, it is a method for producing a cell culture carrier having an anionic hydrophilic property, which is characterized by forming a substrate made of ultrafine fibers of 1.0 dtex or less and then performing plasma treatment.

The polymer constituting the ultrafine fibers of 1.0 dtex or less of the present invention is polyester, polyamide, polyolefin, polytetrafluoroethylene, rayon, vinyl polymers such as polystyrene and methyl methacrylate, polyamino acid, collagen, polyurethane, polysulfone, polyphenylene sulfide. , Polyphenylene sulfoxide, polycarbonate, polyphosphazene, polyacetal, and the like, and copolymers thereof can be appropriately selected. Of these, polyester is particularly preferable. These polymers are subjected to plasma treatment or imparted with a sulfone group or a carboxyl group to impart anionic hydrophilicity. In the present invention, a sulfone group is particularly preferable.

The anionic hydrophilic property in the present invention means a state in which at least the fiber surface has anionic property and exhibits hydrophilic property. The feature of the present invention lies in the anionic property on the fiber surface and is not related to the anion density of the entire fiber. That is, the anion does not necessarily exist inside the fiber. Therefore, the average ion concentration as a whole does not make much sense. In the present invention, a measure of the effect of the anionic property is the hydrophilic property of the fiber surface forming the carrier. This quantitative expression of hydrophilicity is generally extremely difficult due to the involvement of form factors. As a convenience,
The following method is recommended here. This method has a capacity of about 1c
Fix the syringe of c vertically with the holder, and use it as an injection needle.
Attach a drop of 0.005cc. Set the base material (carrier) horizontally 5 cm below this. A drop of distilled water is dropped onto the base material (carrier) from a syringe, and the time from when the water droplets are dropped on the base material to when there is no special reflection of water drops on the base material is read up to 0.1 seconds. This is performed 5 times at different places, and the average value is defined as hydrophilicity. In the present invention, this value is 50 seconds or less, preferably 30 seconds or less, and particularly preferably 10 seconds or less.

In the present invention, the introduction of the anionic hydrophilic property can be achieved by means of copolymerization of those having such an ionic group, direct sulfonation or carboxylation of the polymer, or graft polymerization.

Such treatment may be performed at any of the polymer stage, fiber stage and carrier stage.

When it is carried out at the polymerization stage, for example in the case of polyester
It is also possible to mix and add sodium sulfo-dimethylisophthalic acid as a part of the acid component, or to sulfonate the benzene ring like polystyrene. Particularly in the case of polystyrene, it is preferable to carry out after fiberizing. Further, in the case of graft polymerization, it is premised that it is formed into fibers, but it may be formed as it is into fibers or a carrier and then, as it is, or in order to facilitate the reaction, it may be subjected to plasma treatment and then grafting treatment. A known method can be applied to such grafting.

Plasma treatment is another physical means. Even if the ion density of the whole carrier is not high, such a method is likely to obtain a remarkable effect because the outermost surface of the fiber is ionized. The combination of such physical means and the above-mentioned chemical means may sometimes be able to obtain an even greater effect than in the case of using it alone, and such means may be appropriately combined and used.

The ultrafine fibers in the present invention require 1.0 dtex or less, but a particularly preferred range is 0.5 dtex or less, and more preferably
It is less than 0.1 dtex. By using such ultrafine fibers, the familiarity with cells is remarkably increased, and a remarkable proliferative effect of cells appears in combination with hydrophilicity or anionization.

In obtaining ultrafine fibers, it is possible to use ordinary melting, wet, dry, and spinning methods, but especially when ultrafine fibers are required, multicomponent fibers as suggested in USP 3,531,368, USP 3,350,488 etc. It is recommended to spin ultrafine fibers and remove one component to form ultrafine fibers. The fibers are processed into a carrier using techniques such as woven, knitted, non-woven fabric, and braid.

For more direct processing, high speed yarns can be accumulated and formed into a carrier, as represented by melt blowing. It is also possible to form fibrils into ultrafine fibers by forming a film and then stretching. Such a phenomenon is a phenomenon that is remarkably observed in polyolefins, polytetrafluoroethylene, and the like, and can be effectively used for such substances that are easily fibrillated.

In the case of a non-woven fabric, in addition to the direct spinning method, a web may be formed by a card machine, and then needle punching, water punching, binder treatment and the like may be appropriately performed. Any binder may be used as long as it does not have biotoxicity and does not interfere with the function of ultrafine fibers. Specifically, for example, polyesters, polyamides, polyolefins, polyurethanes, etc. are particularly preferable as the heat-fusion type which is adhered by heat. In the case of a liquid, emulsion, or gel, for example, polyurethane, collagen, polyamino acid, or polystyrene is applied in a liquid state, and then dried or directly placed in a coagulation bath to be solidified. This is just an example, and the present invention is not limited to this and should be understood more expansively.

In forming the carrier, any shape such as a film shape, a rod shape, a spherical shape (marimo shape), a tanzaque shape, a bellows shape, a spiral shape, a belt shape, a dice shape can be used. Of these, marimo-shaped ones are particularly preferable. This marimo-like formation may be achieved by spraying fibers onto a hemispherical net mold and hardening with a binder, or by using a short tow-like product with the tips bonded (fused),
It can be formed by applying it to a card machine and entangled the fibers in the tow. Further, in order to obtain substantially the same effect, thick non-woven fabric may be cut into dice. As described above, the present invention is excellent in moldability and mechanical strength can be sufficiently enhanced to the same level as that formed by conventional weaving and knitting means. Such a carrier can be applied as a microcarrier for suspension culture, which is located between the single culture and the suspension culture by making it fine. Further, in the carrier of the present invention, the effect is further exhibited by having the napped hair. The napped hair here means a loop or fluffy ultrafine fiber. In this formation, weaving, formation of loop structure at the time of knitting,
Alternatively, it can be achieved by the use of chenille yarn, or by processing with a raising machine, a buffing machine, a needle punch machine, or a water jet punch machine after forming the carrier.

By having the naps of such ultrafine fibers, it is possible to increase the contact area with cells, improve the flexibility of tissues that can follow the morphological changes of cells, and adjust the pores involved in nutritional supplementation and waste removal. Therefore, a good carrier function is added.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

〔Example〕

Example 1 In the composite fiber shown in USP 3,531,368, the island component was
80 parts of polyethylene terephthalate copolymerized with 8 mol% of sodium sulfoisophthalic acid, 20 parts of polystyrene as sea component, 36 islands / filament, 83dt
A yarn of ex-24f was obtained (equivalent to 0.078 dtex of single yarn). Using this, a 5-sheet satin fabric was formed. After scouring, it was immersed in trichloroethylene to remove polystyrene and then dried. Further, it was thoroughly washed with ethanol, dried and then plasma treated. Sterilize this with ethylene oxide gas,
After degassing for 3 days, the cells were placed on a plastic petri dish and rat hepatocytes were cultured. RPMI1640 was added to the culture medium at this time.
The cells (containing 10% FCS) were used without serum and cultured in a carbon dioxide gas incubator. As a control, the one using polyethylene terephthalate as the island component of the plastic petri dish and the composite fiber was treated in exactly the same manner and tested in the same manner.

As a result, in the case of the plastic petri dish, full growth covering the entire petri dish did not occur even after 7 days, but when this value was set to 1 and relative evaluation was carried out, 50% was obtained by copolymerizing sodium isophthalic acid with polyethylene terephthalate. In the case of only, a clear effect was recognized with 10 times.

Comparative Example 1 A test was conducted in the same manner as in Example 1 except that polyethylene terephthalate was used as the island component.

As a result, the relative value of proliferation was 5.

Example 2 Using a copolymer of sodium isophthalic acid (7 mol%) and butylene terephthalate, the number of islands was changed so that the single yarn thicknesses were 1.4 dtex, 0.5 dtex and 0.009 dtex, respectively. A composite fiber similar to the above was obtained. Each of the fibers was crimped and cut into a staple having a length of 51 mm. This was put on a card and a cross wrapper, and then needle punched to create a felt with a basis weight of 250 g / m ² .
The felt was soaked in hot water at 98 ° C. to shrink it, dried and then soaked in trichlorethylene to remove polystyrene. These were subjected to plasma treatment, then individually placed in a petri dish and injected into the culture solution. Then, canine fibroblasts were injected at a concentration of 1 × 10 ⁵ cells / ml, and cultured in a carbon dioxide incubator at 37 ° C. for 7 days.

In addition, thymidine labeled with tritium was mixed in the culture solution. This is a labeler of thymidine, which requires thymidine to form nuclei when cell division occurs, and utilizes this uptake of thymidine in cells to measure the degree of cell division. The degree of division can be quantified by measuring the tritium concentration with a scintillation counter.

As a result of this measurement, when the number of cultured cells when the growth stopped when the cells grew to full growth by culturing only in a plastic petri dish, the number of cultured cells of 1.4 dtex was about 5 times, 0.5
The value of dtex was about 10 times, and that of 0.009 dtex was 70 times. This result shows that a remarkable effect can be obtained by making the size extremely small.

[Effects of the Invention] As in the present invention, the surface of the ultrafine fibers having anions has a very good compatibility with cells, and the use of a carrier made of such fibers enables stable cell growth over a long period of time. And exhibits an extremely high-performance carrier function.

Also, as a three-dimensional culture carrier, cells differentiated close to the living body and their functions were maintained, enabling long-term survival.

In particular, by using ultrafine fibers, the voids between the fibers become uniform, and due to the capillary phenomenon due to this micro passage,
It is presumed that the diffusion and transport effect is increased, and as a result, the nutrient supply to the cells and the removal of waste products, which are indispensable for cell culture, are carried out very smoothly.

Claims (6)

[Claims] 1. A cell culture carrier comprising ultrafine fibers of 1.0 dtex or less having anionic hydrophilicity. 2. A hydrophilic group having a sulfone group, a carboxyl group,
The cell culture carrier according to claim 1, wherein the carrier is provided with at least one of the above. 3. When a polymer is spun to obtain a cell culture carrier comprising ultrafine fibers of 1.0 dtex or less, it is possible to introduce an anionic hydrophilic group at any of the polymer stage, fiber stage and carrier stage. A method for producing a characteristic cell culture carrier. 4. A method for producing a cell culture carrier having an anionic hydrophilic property, which comprises subjecting a substrate made of ultrafine fibers having a dtex of 1.0 dtex or less to plasma treatment. 5. When forming a carrier, a non-woven web is formed by using multi-component fibers, and the fibers are entangled with each other by a physical means such as needles or water jets, and then finely divided,
The method for producing a cell culture carrier according to claim (3), further comprising cutting into chips. 6. When forming a carrier, a short tow-shaped product obtained by adhering (fusing) the tips of multi-component fibers is applied to a card machine to entangle the fibers in the tow-shaped product and then made into ultrafine fibers. A method for producing the cell culture carrier according to claim (3).