JPS61242576A - Spherule for cultivating cell - Google Patents

Abstract

PURPOSE:The titled spherule, consisting essentially of a silicone rubber spherule, capable of high-temperature steam sterilization, and floating in a container by gentle stirring, and cultivating a large amount of cells with safety. CONSTITUTION:A spherule obtained by dropping a composition of a room temperature curing or cold curing liquid silicone rubber which is liquid at ordinary temperature, e.g. organopolysiloxane having >=2 vinyl groups linked to the silicon atom in the molecule thereof and an organoydrgenpolysiloxane having >=3 hydrogen atoms linked to the silicon atom and a platinum based curing catalyst, into an incompatible liquid, e.g. water, and heating the liquid at for example 40-80 deg.C, or irradiating the liquid with ultraviolet light using a high- pressure mercury lamp to cure the composition. The diameter of the spherule can be set at 100mum-5mm having a curvature of 10 times or more based on the diameter of the cell to be multiplied.

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention relates to small particles for cell culture, particularly silicone rubber as an artificial substrate useful for mass culture of cells by microcarrier method in biotechnology technology. This invention relates to small particles for cell culture, which are mainly made of small spheres.

(Prior Art) Regarding mass culturing of cells in the field of biotechnology, since cells cannot proliferate in a suspended state, it is necessary to attach them to a vessel wall or the like and grow them in a monolayer state.

Therefore, for this proliferation, it is necessary to increase the culture area, that is, the area of the medium, and various methods have been proposed for this purpose.

Cell culture methods that increase the area of this medium include the large culture bottle method, the roller bottle method, the stearin type culture method, the multilayer plate method, the multilayer monolayer method, the glass fiber column method, the holofiber method, the spinner method, and the super piece method. Among these methods, the super piece method, also known as the microcarrier method, is said to have the highest ratio of culture area to culture volume. This microcarrier method releases a large amount of spherical pieces with a curvature that allows cells to adhere to them in a culture container such as a flask, and suspends the cells by gently stirring them together with the culture medium. It is said that this allows for cell proliferation of several hundred blades per millimeter. Note that the spherical piece used for this purpose is usually 10 times the diameter of the cell itself, since cells can stick to it if it has a curvature of approximately 10 times the diameter of the cell itself.
Microspheres with a size of 0 JLm or more are used, and these include inorganic beads such as glass beads and hollow glass beads, and synthetic polymer beads such as polypropylene, polyethylene, and polystyrene. Its surface is said to be plasma-treated or coated with a polymeric material so that cells can easily attach to it.

(Disadvantages of the prior art) However, since these microbeads have a hard surface and a specific gravity of 2 or higher, the cells may be destroyed by collisions of the beads during stirring, or they may settle. Hollow beads that solve these problems have the drawbacks of being difficult to manufacture, difficult to adjust density, and easily damaged. Furthermore, beads made of synthetic polymer materials have a light specific gravity and are relatively easy to control, but they have the disadvantage of lacking heat resistance to heat treatments such as washing and sterilization. There is also a mechanical problem in that it is easily damaged.

(Structure of the Invention) The present invention relates to a cell culture microsphere that solves the above-mentioned disadvantages, and is characterized in that it is mainly made of silicone rubber microspheres.

In other words, the present inventors have conducted various studies on the development of small spheres for cell culture that do not have the disadvantages of ``double-cut'', and found that if they were made of silicone rubber, silicone rubber would It must be heat resistant to withstand high temperatures and non-toxic, with a JIS hardness of 20 to 80°H and a specific gravity of 0.9 to 1.2.
Because it is an elastic body, it can be sterilized by boiling or high-temperature steam sterilization at 120°C, and it can also be suspended in a container by gentle stirring, and this collision will also destroy cells. They found that this method allows for easy and safe mass culture of cells, and completed research on the material of this silicone rubber, its manufacturing method, surface treatment method, etc., and completed the present invention. .

As mentioned above, the small spheres for cell culture of the present invention are mainly made of silicone rubber. Since the silicone rubber is made by dropping it into a liquid bath and curing it while suspended in the liquid bath, it is preferable that this silicone rubber exhibits a liquid state at room temperature. The liquid silicone rubber may be one commercially available as a room-temperature curing type or a low-temperature curing type. A silicone rubber composition called an addition reaction type, in which an organohydrodiene polysiloxane containing at least three hydrogen atoms bonded to silicon atoms and a platinum-based curing catalyst is added, or a silicone rubber composition containing at least three hydrogen atoms bonded to silicon atoms in the molecule. A dealcoholization type consisting of an organopolysiloxane having a bonded hydroxyl group and an alkoxysilane bonded to a silicon atom in the molecule or a partial hydrolysis thereof, or a silane having an oxime group bonded to a silicon atom or a partial hydrolysis product thereof. Alternatively, a ketone-free two-component room temperature curable silicone rubber composition is exemplified. Note that if the viscosity of these liquid silicone rubbers is too high, it will be difficult to form droplets for producing spherules, and if the viscosity is too low, the shape of the spherules obtained will be unstable. So 2.0
00 to 300,000 cp, preferably 3°00
0~100. ．． It is better to use 0OOcp, but
This may be freely selected depending on the shape and size of the target small sphere.

The small spheres for cell culture of the present invention are obtained by dropping this liquid silicone rubber as droplets into a liquid bath that is incompatible with silicone rubber from a cock or the like that allows the diameter of the droplets to be controlled, and then floating and hardening the particles. be able to. Examples of liquids that are incompatible with this silicone rubber include water, water-methyl alcohol, and wood-ethyl alcohol. The droplets may be heated to a temperature in the range of 40 to 80°C, or the droplets may be irradiated with ultraviolet rays from a high-pressure mercury lamp or the like. The size of this droplet is at least 10 times the diameter of the target cell, as it is necessary for the target cell to attach to the spherule made from the droplet and grow as a monolayer.
It is necessary to have a curvature of 10011. The thickness may be in the range of ~5 mm.

The small spheres for cell culture of the present invention may be placed in a cell culture container, and have a specific gravity close to 1 and a BME
It floats in culture medium such as medium, MEM medium, 199 medium, Grace's insect medium under gentle stirring, so if the temperature of this liquid is maintained at the optimal temperature for cell growth of 20°C ± 10°C, this Cells attached to the spherules can proliferate on the spherule surface and accomplish their initial goal.

In addition, this small sphere is made of silicone rubber elastic material, so even if it collides with the object under stirring, it will not be destroyed, and it is an elastic material. Therefore, there is no fear that cells will be destroyed by this collision, and since it is made of silicone rubber with excellent heat resistance, its properties will be maintained even if it is sterilized by boiling or treated with high-temperature steam before repeated use. Since it does not change and is highly transparent, samples can be easily observed under a microscope to check the state of cell proliferation.

Note that since the small spheres are made of silicone rubber, their surfaces are usually water repellent and highly hydrophobic. Therefore, if the target cell easily adheres to a hydrophobic substrate, it can be used as is, but if the cell easily adheres to a hydrophilic substance, the surface can be covered with a known silane cup. Ring agent, e.g. aminosilane type coupling agent KBM-603, K
It is better to make the surface hydrophilic by coating it with BM-602, KBM-903, KBM-573 (part name manufactured by Shin-Etsu Chemical Co., Ltd.), or to make the surface hydrophilic by irradiating it with ultraviolet rays. .

Next, examples of the present invention will be given, and the viscosity in the examples is 25°C.
This shows the measured values at .

Example 1 An addition reaction type two-component silicone rubber composition having a viscosity of 50,000 cp - KE-X-32-389A [manufactured by Shin-Etsu Chemical Co., Ltd., part name] was poured into a glass container with a diameter of 0.0 cp.
A 2 mm droplet is dropped into warm water maintained at 65°C and suspended under stirring, and the suspension is heated and vulcanized to give a 0.
When we made microspheres of 2 mm in diameter, we found that they had a rubber hardness of 55°, H1 specific gravity of 1.12, and good transparency.
It has a heat resistance that allows regular use at 0''C.

Next, in a 500 mfL spinner flask, add 50 mJL of Daribetsko MEM supplemented with 10% tallow calf serum to the culture solution, and add 2 g of the sterilized silicone microparticles (approximately 400,000 pieces) to this. After addition, diploid cells of the human-derived cell line Detroit551 and MEM
Add culture medium to make the total volume 100mM, and
551 was set to 3-4X 104 th/rnl.

This culture system was then transferred to a CO2 incubator at 37° C. and cultured for 6 days with slow stirring at a rotation speed of 7 rpm, and it was confirmed that the cells had multiplied 4 to 5 times.

Example 2 Silane coupling agent KBM603 (part name manufactured by Shin-Etsu Chemical Co., Ltd.) in the hot water for producing small spheres in Example 1
A diluted solution of ethyl alcohol was added dropwise, and the surface of the resulting silicone rubber particles was coated with the silane coupling agent. Since it was shown to exhibit lignophilic properties, it became suitable for propagating cells that have the physical property of adhering to lignophilic substances.

Example 3 Addition reaction type low temperature curing liquid silicone rubber with a viscosity of 50,000 cp - KE-106LTV [part name manufactured by Shin-Etsu Chemical Co., Ltd.] was used in the same manner as in Example 1.
0 to 300 jL of silicone rubber particles are made, and then added to the vulcanized small spheres in hot water for 2 hours with stirring.
The wetting properties of the surface were improved by irradiating it with ultraviolet light from KW's high-pressure mercury lamp for 20 to 60 seconds, and the material was found to be suitable for the growth of cells that have the physical properties of adhering to parent wood materials. became.

Claims (1)

[Scope of Claims] 1. Small spheres for cell culture, characterized in that the main material is silicone rubber small spheres. 2. The small sphere for cell culture according to claim 1, wherein the small sphere has a diameter of 100 μm to 5 mm. 3. A small sphere for cell culture according to claim 1, wherein the surface of the small sphere is coated with a silane coupling agent. 4. A small sphere for cell culture according to claim 1, wherein the surface of the small sphere has been modified by plasma or ultraviolet irradiation.