JP2005110676A - Living cell culture substrate, method for producing the substrate, etching treatment apparatus used in the method for producing the same, and method for culturing living cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a living cell culture substrate capable of making a living cell adhere to/proliferate in the inside of a polymer material, to say nothing of on a surface of the polymer material, and capable of being contacted with an organism or positioned inside the organism, to provide a method for producing the living cell culture substrate, and to provide an etching treatment apparatus using low-temperature plasma, capable of being easily operated in a medical site, or the like. <P>SOLUTION: The living cell culture substrate is obtained by molding any one or two kinds of polylactic acid and a polylactic acid copolymer into a porous material, then subjecting a molded article comprising the porous material, or a molded article which is obtained by heat-treating the porous material, to etching treatment using the low-temperature plasma under atmospheric pressure. The etching treatment is conducted by using the etching treatment apparatus, wherein the etching treatment apparatus is equipped with a holding base for holding the molded article and a plasma generating unit for irradiating the molded article held on the holding base with a flow of the low-temperature plasma in a casing in which the atmospheric pressure is maintained, and further with a gas supply unit for supplying one, two or more kinds of plasma working gases selected from air, nitrogen, argon, carbon dioxide, and octafluoropropane, so that the molded article is subjected to the etching treatment with the low-temperature plasma under the atmospheric pressure. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a living cell culture substrate obtained by etching a porous molded article made of polylactic acid and a polylactic acid copolymer with low-temperature plasma, a method for producing the living cell culture substrate, and an etching apparatus used in the production method The present invention also relates to a method for culturing living cells using the living cell culture substrate.

  Conventionally, as a material for culturing live animal cells, in order to modify the surface of the polymer material and improve its adhesion, the method of coating the surface of the polymer material, the method of chemical treatment, the method of etching treatment, ion A method of irradiating is proposed. In particular, it is said that a method of depositing carbon on the surface of a polymer material and culturing cells on the deposited carbon is useful (see, for example, Patent Document 1).

However, in these methods, the polymer material itself is poorly degradable, and therefore cannot be placed in the living body, and since it is a mere surface treatment, living cells cannot grow inside the polymer material. Have the disadvantages.
In addition, there is known a method of surface modification by irradiating a plasma flow in a stable gas atmosphere by diluting oxygen or helium gas in a vacuum chamber, but such equipment is expensive and the installation location is limited. However, the operation is difficult, and it takes time and effort to prepare, resulting in high costs.

Japanese Patent Laid-Open No. 7-23768

  The present invention develops a living cell culture substrate that allows living cells to attach and multiply not only on the surface of a polymer material but also to the inside thereof, and to be brought into contact with or placed in a living body, and a method for producing the same. In addition, the present invention aims to develop an etching processing apparatus, particularly an etching processing apparatus using low-temperature plasma that does not have a large-scale apparatus configuration and can be easily operated in a medical field.

As a result of intensive studies to achieve the above object, the present inventors have achieved the object of the present invention by using polylactic acid and a polylactic acid copolymer as a polymer material.
That is, a living cell obtained by forming one or two of polylactic acid and a polylactic acid copolymer into a porous body, and etching the molded article of the porous body with low-temperature plasma under atmospheric pressure It is a culture substrate, and the plasma working gas used for the etching treatment is preferably one or more of air, nitrogen, argon, carbon dioxide and octafluoropropane (C ₃ F ₈ ), The polylactic acid copolymer is preferably one or two of a polylactic acid-glycolic acid copolymer and a polylactic acid-caprolactone copolymer, and a living cell culture substrate is used as a tissue regeneration scaffold material. Is particularly preferred.

In the second aspect of the present invention, any one or two of polylactic acid and polylactic acid copolymer is made into a porous body, and the molded product of the porous body is crystallized by heat treatment, and is then subjected to atmospheric pressure. A living cell culture substrate characterized by being etched by low-temperature plasma, wherein the plasma working gas used for the etching treatment is air, nitrogen, argon, carbon dioxide and octafluoropropane (C ₃ F ₈ ) Preferably, the polylactic acid copolymer is any one or two of a polylactic acid-glycolic acid copolymer and a polylactic acid-caprolactone copolymer. It is particularly preferable to use a living cell culture substrate as a tissue regeneration scaffold material.

In the third aspect of the present invention, any one or two of polylactic acid and polylactic acid copolymer are freeze-ground, and the resulting powder is used as a porous molded article. The porous molded article is subjected to atmospheric pressure. A method for producing a living cell culture substrate characterized by etching with low-temperature plasma in which a method of forming a porous body is freeze-pulverized and the resulting powder is sintered to form a porous body The method is preferable, and it is preferable that the etching process is performed 2 to 4 times by low-temperature plasma under atmospheric pressure, and the plasma working gas used for the etching process is air, nitrogen, argon, carbon dioxide and octafluoropropane (C ₃ is preferably any one or more of F _8), in the etching process, reducing the pressure space facing the opposite surface to the plasma irradiated surface of the porous molded article, thereby cold plasma Preferably, the flow is sucked into the porous molded article, and the polylactic acid copolymer is one or two of a polylactic acid-glycolic acid copolymer and a polylactic acid-caprolactone copolymer. It is particularly preferred.

In the fourth aspect of the present invention, either one or two of polylactic acid and polylactic acid copolymer are freeze-ground, and the resulting powder is used as a porous molded article, and the porous molded article is heat-treated. A method for producing a living cell culture substrate characterized in that it is crystallized by a low temperature plasma under atmospheric pressure, and a method for producing a porous article is freeze-pulverized and obtained powder The method is preferably a method of sintering a porous molded article, preferably crystallized by heat treatment of the porous molded article, and particularly preferably heat treatment at 80 ° C to 140 ° C. Further, it is preferable that the etching process is performed 2 to 4 times with low-temperature plasma under atmospheric pressure, and the plasma working gas used for the etching process is air, nitrogen, argon, carbon dioxide, and octafluoropropane (C ₃ F ₈ ) Any one or more of ₈ ) is preferable, and in the etching process, the space facing the surface opposite to the plasma irradiation surface of the porous body molded product is decompressed, whereby a low temperature plasma flow is formed into the porous body. The product is preferably sucked into the product, and the polylactic acid copolymer is preferably one or two of a polylactic acid-glycolic acid copolymer and a polylactic acid-caprolactone copolymer.

5th of this invention is an apparatus used for the manufacturing method of a living cell culture base material, Comprising: The holding stand holding the said porous body molded product in the housing | casing by which atmospheric pressure is maintained, It hold | maintained at the said holding stand. And a plasma generator for irradiating the porous molded article with a low-temperature plasma flow, and the plasma generator is provided with any one of air, nitrogen, argon, carbon dioxide, and octafluoropropane (C ₃ F ₈ ). Gas supply means for supplying one or more plasma working gases is provided, and the porous molded product held on the holding table is etched by the low temperature plasma under atmospheric pressure. An apparatus for etching a living cell culture substrate, wherein the casing has a hermetically sealed structure and is provided with a gas supply means for filling the casing with the same gas as the plasma working gas. In addition, it is preferable that a moving means for relatively moving the holding table and the plasma generating device is provided, and a position opposite to the plasma irradiation surface of the porous molded article to be held faces the holding table. An internal space that opens to the inside of the porous molded article is provided with gas exhaust means for exhausting the gas in the internal space to the outside of the housing, and the internal space is depressurized by the gas exhaust to generate a low-temperature plasma flow Preferably, a temperature sensor for detecting a temperature in the vicinity of the low-temperature plasma flow of the plasma generator is particularly preferably provided in the casing.

The sixth aspect of the present invention is a living cell obtained by etching any one or two of polylactic acid and a polylactic acid copolymer into a porous body, and subjecting the molded article of the porous body to low temperature plasma under atmospheric pressure. A method for culturing a living cell, characterized by seeding and culturing a living cell on a culture substrate, wherein plasma working gas used for etching treatment is air, nitrogen, argon, carbon dioxide and octafluoride Any one or more of propane (C ₃ F ₈ ) is preferable, and the polylactic acid copolymer is any one of a polylactic acid-glycolic acid copolymer and a polylactic acid-caprolactone copolymer. Or it is especially preferable that they are 2 types.

According to the seventh aspect of the present invention, any one or two of polylactic acid and polylactic acid copolymer is made into a porous body, and after crystallization is performed by heat-treating the molded article of the porous body, the temperature is lowered under atmospheric pressure. A living cell culturing method characterized by seeding and culturing a living cell on a living cell culture substrate formed by etching with plasma, wherein the plasma working gas used for the etching treatment is air, nitrogen , Argon, carbon dioxide and octafluoropropane (C ₃ F ₈ ) are preferably one or more, and the polylactic acid copolymer is a polylactic acid-glycolic acid copolymer and polylactic acid- It is particularly preferable that one or two of the caprolactone copolymers be used.

Eighth of the present invention is a living cell obtained by subjecting one or two of polylactic acid and polylactic acid copolymer to a porous body, and etching the molded article of the porous body with low-temperature plasma under atmospheric pressure. A living cell culturing method characterized by culturing a living cell by contacting the living cell as a tissue regeneration scaffold material, wherein the plasma working gas used for the etching treatment is air, nitrogen , Argon, carbon dioxide and octafluoropropane (C ₃ F ₈ ) are preferably one or more, and the polylactic acid copolymer is a polylactic acid-glycolic acid copolymer and polylactic acid- It is particularly preferable that one or two of the caprolactone copolymers be used.

In the ninth aspect of the present invention, any one or two of polylactic acid and a polylactic acid copolymer are made into a porous body, and the molded product of the porous body is crystallized by heat treatment, and then cooled at a low temperature under atmospheric pressure. A method for culturing a living cell, characterized in that a living cell culture substrate formed by etching with plasma is used as a tissue regeneration scaffold material and brought into contact with the living cell to culture the living cell. The plasma working gas used is preferably one or more of air, nitrogen, argon, carbon dioxide and octafluoropropane (C ₃ F ₈ ), and the polylactic acid copolymer is polylactic acid- It is particularly preferable that one or two of a glycolic acid copolymer and a polylactic acid-caprolactone copolymer are used.

  The living cell culture substrate of the present invention is a porous molded article made of polylactic acid and a polylactic acid copolymer. Since the surface of the porous body inside the molded article is etched, Cells can proliferate. Moreover, even when the molded product is placed in a living body, the molded product is decomposed in the body, and thus is a living cell culture substrate suitable for transplantation in the body. In particular, when osteoblasts are proliferated, the molded article and bone cells are replaced in the body, which is suitable as a tissue regeneration scaffold material without side effects. Further, when the porous molded article is crystallized by heat treatment, the surface becomes smooth and durability is imparted, so that a treatment method corresponding to the degree of bone damage can be provided.

  In the method for producing a living cell culture substrate of the present invention, polylactic acid and a polylactic acid copolymer are made into a porous body, and after the porous body is formed into a molded product, the surface of the porous body is etched by low-temperature plasma. The etching process could be performed. In particular, polylactic acid and polylactic acid copolymer can be made into a powder by freeze pulverization, and the obtained powder can be processed to obtain the porous body. It became possible to produce cell culture substrates. In addition, when the porous body molded product is crystallized by heat treatment, the surface becomes smooth and durable, so that it is possible to produce a living cell culture substrate having arbitrary properties. .

  The live cell culture substrate etching apparatus of the present invention is an inexpensive and large-scale apparatus configuration, can be easily operated without taking time and effort, and can be easily processed at medical sites, etc. By performing etching treatment by selecting a predetermined gas with respect to the molded article using the apparatus, the wettability can be increased, and as a result, the cell adhesion ability and the cell growth ability of the living cell culture substrate can be improved. In addition, since the housing has a sealed structure, and the gas supply means for filling the same gas as the plasma working gas is provided in the housing, the inflow of gas from the outside of the housing is prevented and the internal gas distribution is disturbed. Can be prevented and plasma irradiation can be performed efficiently. Further, the holding table is formed with an internal space that opens to a position facing the surface opposite to the plasma irradiation surface of the molded product to be held, and is provided with gas discharge means for discharging the gas in the internal space to the outside of the housing, By exhausting the gas, the internal space can be decompressed and a low-temperature plasma flow can be sucked into the molded product to reliably perform the etching process.

  The living cell culture method of the present invention uses the above-mentioned living cell culture substrate of the present invention, as well as tumor cells such as HeLa cells, mouse osteoblast-like cells (MC3T3-E1 cells), human osteoblasts ( HOB cells) and other live cells that are usually said to be difficult to culture can be cultured.

  Next, embodiments of the present invention will be described in detail with reference to the accompanying drawings.

  The living cell culture substrate of the present invention is formed by using any one or two of polylactic acid and polylactic acid copolymer as a porous material, and etching the molded product of the porous material with low-temperature plasma under atmospheric pressure. This is the gist.

  The polylactic acid and polylactic acid copolymer used in the present invention are biodegradable synthetic resins, and mainly include polylactides, polylactones, and glycol / dicarboxylic acid polycondensation compounds that are aliphatic polyesters. be able to. Examples of polylactides include polylactic acid, polyglycolic acid, and copolymers thereof, examples of polylactones include poly ε-caprolactone, and examples of glycol / dicarboxylic acid polycondensation compounds , Polyethylene succinate, polybutylene succinate and the like.

  As a method of obtaining one or two porous bodies of the polylactic acid and the polylactic acid copolymer synthetic resin, a foaming agent decomposition method in which a foaming agent is decomposed to generate nitrogen gas or carbon dioxide gas, or evaporation Solvent diffusion method that adds mold solvent to matrix, gas mixing method that mechanically mixes gaseous foaming agent with matrix, chemical reaction method that uses gas generated in resin polymerization process, and soluble substances are premixed In addition, an elution method that dissolves in water or a solvent after molding, a sintering method in which powders are bonded to each other by a sintering action at a melting temperature or lower, and the like, in particular, the sintering method is excellent in communication, Since it is strong and does not use a foaming agent or a solvent, it is preferable from the viewpoint of safety when it is installed in a living body or brought into contact with a living body.

  When the above sintering method is employed, it is necessary to pulverize the synthetic resin. However, the synthetic resin used in the present invention cannot be pulverized by a normal pulverization method, and thus a freeze pulverization method is employed. Is preferred. The synthetic resin uses pellets which are irregular particles having a particle size of 4 mm or less, and this is cooled with liquid nitrogen and pulverized. Although the powder has an irregular shape, the particle size is preferably 100 to 500 μm, and after pulverization, the powder is collected by screening.

  The molded product of the present invention can be molded into an arbitrary shape depending on the application, but it is preferable to carry out the etching process after processing into a plate shape with a mold because an etching process using low temperature plasma is considered. .

  1 and 2 are a front view and a side view showing a schematic configuration of an etching processing apparatus for etching the molded article, FIGS. 1 to 4 show typical embodiments, and reference numeral 1 denotes an etching processing apparatus. Reference numeral 2 denotes a holding table, and 3 denotes a plasma generator.

The living cell culture substrate etching apparatus 1 according to the present invention includes a holding table 2 for holding the molded product and a molded product held on the holding table 2 in a casing 10 in which atmospheric pressure is maintained. A plasma generator 3 that irradiates a low-temperature plasma flow is provided, and the plasma generator 3 is provided with one or more plasma working gases of air, nitrogen, argon, carbon dioxide, and C ₃ F _8. A gas supply means 4 is provided to supply the molded product held by the holding table 2 with the low-temperature plasma under atmospheric pressure.

  The plasma generator 1 is an arc plasma apparatus similar to the conventional one that generates a low-temperature plasma flame by applying a voltage to a pair of opposed electrodes to perform arc discharge and supplying a plasma working gas between the electrodes. In this embodiment, 3D plasma irradiation apparatus PS-601C manufactured by Sink Engineering Co., Ltd. is used.

  The casing 10 is not necessarily required to be a sealed structure as long as it can be safe against the low-temperature plasma flow and can maintain a static pressure around the molded product, but in this embodiment, a sealed structure is adopted. A gas supply means 4 for filling the casing 10 with the same gas as the plasma working gas is provided. Specifically, as shown in FIG. 2, apart from the plasma working gas pipe 40 that penetrates the housing wall surface 11 and is connected to the plasma generating device 3, it projects from the housing wall surface to the inside. A second pipe 41 having a filling gas supply port 41a for releasing the same gas as the plasma working gas is provided in the internal space. When the plasma working gas is a plurality of mixed gases, the gas supplied by the gas supply unit 4 may be a part of the gas included in the working gas.

  The plasma generator 3 is supported in a cantilevered manner forward with respect to the shaft body 50 via a bracket 31 screwed into a thread groove on the outer periphery of the shaft body 50 erected vertically on the base 5. The shaft body 50 is configured to be movable up and down along the thread groove.

  In addition, the holding table 2 is provided at a position directly below the base 5 corresponding to the plasma generator 3. In this example, the holding table 2 is arranged along the left and right directions including the position on the base 5. The guide rails 51 and 51 of the book are extended in parallel, and the corresponding groove portions 20 and 20 on the lower surface of the holding base 2 are fitted into the guide rails 51 and 51, respectively. 52 is configured to be movable in the left-right direction.

  Note that the number of the guide rails is not limited to two, but may be one or three or more, and as shown in FIGS. 5 and 6, the guide rails 53 and 53 provided on the base 5 have groove portions 60. , 60 is provided and an intermediate base 6 is provided so as to be movable in the rail direction, and guide rails 54, 54 are provided on the intermediate base 6 in a direction perpendicular to the guide rail 53, 2 A structure in which the groove portions 20 and 20 on the lower surface are fitted to the guide rails 53 and 53 is also a preferred modification. By providing a driving mechanism, the holding base 2 and the intermediate base 6 are combined with the base 5. On the other hand, it can move in the depth direction along the guide rail 53, and can also move in the left and right directions along the guide rail 54 of the intermediate base 6.

  As described above, the holding table 5 and the plasma generator 3 are configured to be relatively movable in the height direction along the shaft body 50, and the holding table 2 is moved with respect to the base 5. It is configured to be relatively movable in the horizontal direction.

  The holding base 2 is formed with a hollow internal space 21 opened on the upper surface. In this example, a disk-shaped attachment jig 7 is attached so as to cover the upper surface opening 21a of the cylindrical internal space 21. It has been. As shown in FIGS. 3 and 4, the mounting jig 7 has a stepped portion 71 having a reduced diameter for supporting the above-described molded product P formed in a disk shape so that the upper surface is flush with the upper surface. A plurality of holes 70 are formed, and each molded product P is attached to the upper surface opening 21 a of the holding table 2 through the attachment jig 7 while being held in the through holes 70.

A communication hole 22 communicating with the outside is formed in the side wall of the internal space 21, and the gas in the internal space 21 is discharged outside the casing by the pump 81 through the discharge pipe 80 attached to the communication hole 22. A gas discharge means 8 is configured to discharge and thereby suck down a low-temperature plasma flow applied to the molded product by decompressing the internal space 21 to the inside of the molded product.
Such a suction structure by the gas discharge means is not necessarily required. As shown in FIGS. 5 to 8, a disk in which the molded product P is held in the recess 72 that does not pass through the holding base 2 having no internal space. It may be formed by attaching a mounting jig 7 in the shape of a ring.

  In this embodiment, a temperature sensor 9 for detecting the temperature in the vicinity of the low-temperature plasma flow of the plasma generator is provided in the casing 10 near the low-temperature plasma outlet of the plasma generator 3. However, such a temperature sensor may be provided at another position.

  The living cell culture method according to the present invention uses the above-mentioned living cell culture substrate etched as described above, as well as tumor cells such as HeLa cells, mouse osteoblast-like cells (MC3T3-E1 cells) In addition, living cells such as human osteoblasts (HOB cells) which are usually difficult to culture can be cultured. That is, since the living cell culture substrate of the present invention has good wettability, the living cell culture substrate is put on the surface of the culture substrate by introducing the living cell culture substrate into a liquid medium in which the living cells are floating. Adhere to and start to grow.

(Example 1)
Pellets of polylactic acid having a particle size of about 4 mm (manufactured by Shimadzu Corporation, Lacty 5000) were cooled with liquid nitrogen and then pulverized and sieved to obtain powders having average particle sizes of 350 μm, 250 μm and 125 μm. The obtained powder was heated at 180 to 190 ° C. for 20 to 30 minutes under no pressure by a small heating press (manufactured by Imoto Seisakusho) to obtain a porous body. Next, a disc having a diameter of 12 mm and a thickness of 3 to 10 mm and a flat plate having a size of 6 × 60 × 3 mm were prepared. The obtained circular plate and flat plate are etched by low temperature plasma in the etching processing apparatus 1. The etching process is performed by maintaining the distance between the electrode of the plasma generator 3 and the surface of the molded product at 20 mm, generating an arc discharge between the electrodes at AC 10 kV and 60 mA, and supplying each selected plasma working gas. The substrate 2 is processed by reciprocating left and right once at a moving speed of 5 mm / sec.

Regarding the disc manufactured in Example 1, the disc that was not etched with the low temperature plasma was “untreated”, the disc that was etched with the low temperature plasma in air was “Air”, and the etching was performed in carbon dioxide gas. The treated disc was “CO ₂ ”, the disc that was etched in nitrogen gas was “N ₂ ”, the disc that was etched in argon was “Ar”, and was etched in octafluoropropane. The disc was designated as “C ₃ F ₈ ”, and the contact angle with water, the surface free energy, and the zeta potential were measured for each disc.

  Table 1 shows the contact angle with water and surface free energy of each disk including untreated disks. As can be seen from Table 1, the contact angle with water was found to be large in all the discs etched by low-temperature plasma compared to the untreated discs and excellent in wettability. Also, as can be seen from Table 1, the surface free energy is higher than that of the untreated disk except for the etched disk in octafluoropropane. A large value was shown. As shown in FIG. 9, the zeta potential showed a larger value in the etched disc than in the untreated disc.

(Example 2)
Using mouse osteoblast-like cells (MC3T3-E1 cells), human osteoblasts (HOB cells) and HeLa cells, etching with 46-well multiwell wells for cell culture and sterilized low-temperature plasma The treated porous body was placed in a well. 1 ml of a medium containing 1 × 10 ⁴ cells / ml of cells was added to 1 well, and cells were seeded. The culture was performed in an incubator at 37 ° C. for 1 to 7 days. Cell adhesion was measured by absorbance using MC3T3-E1 cells and HeLa cells, and the results are shown in Table 2. Further, the cell proliferation ability was measured by absorbance and is shown in Table 3. As can be seen from Tables 2 and 3, discs etched with low-temperature plasma both have higher cell adhesion and cell growth ability than untreated discs, and are superior in cell culture. I understood.

  The cultured cells were observed by fixing the cells, drying them, depositing gold, and observing them with a scanning electron microscope. In addition, in the evaluation of cell adhesion to the porous body, adhesion / proliferation / extension was observed only in the etched porous body for both mouse osteoblast-like cells (MC3T3-E1 cells) and human osteoblasts (HOB cells). .

(Example 3)
Polylactic acid pellets (manufactured by Shimadzu Corporation, Lacty 5000) are heated and melted at 215 ° C. for 30 minutes, and injected into a 4 ° C. mold at 15 MPa using an injection molding machine (sulfone jet 3000, Hydental Japan). Was held for 30 seconds to mold a disk sample of φ16 × 2 mm. In the plasma treatment, the gap between the electrode and the sample was set to 20 mm in the atmosphere using the plasma irradiation apparatus of the present invention, and the sample was untreated, 1, 2, and 4 reciprocated at a speed of 5 mm / sec. A scanning probe microscope image of the untreated sample is shown in FIG. 10, and a scanning probe microscope image of the one-way processed sample is shown in FIG. As can be seen from FIG. 10, the injection-molded polylactic acid sample was amorphous, and irregular streaks with the mold surface transferred were observed, and the surface roughness (Ra) determined from the scanning probe microscope image was 7. It was 5 ± 2.1 nm. As can be seen from FIG. 11, the trace of the mold surface disappears on the surface of the sample after one round of the plasma treatment, and the unevenness of the uniform shape is distributed throughout the sample, and the surface roughness obtained from the scanning probe microscope image (Ra) was 10.1 ± 2.5 nm. As the number of plasma treatments increased, the height of the convex portion decreased.

Example 4
A disc sample of φ16 × 2 mm of polylactic acid was molded by the method of Example 3. The molded sample was treated at 80 ° C., 100 ° C. and 140 ° C. for 1 hour in a hot press, and the crystallinity (Xc) was obtained from Equation 1 using a thermal analyzer. The results were 21.7%, 41.7% and 62.9%, respectively. When the crystallinity of the sample increases, the unevenness of the sample surface due to the plasma treatment decreases, and it is considered that the etching effect of the plasma treatment affects the amorphous part of the sample surface.

  Xc = 100 · (ΔHm + ΔHc) / 93 (Formula 1)

  By placing a porous body in which desired living cells are adhered, proliferated, and extended with a living cell culture substrate according to the present invention at a required location in the living body, the desired living cells proliferate, but the porous body is decomposed. And disappear. For example, when osteoblasts cultured in a porous body are placed in a bone defect site, the osteoblasts proliferate and form bone, but the porous body is decomposed and discharged out of the body. In addition, by putting a culture of gingival cells in a porous body into a periodontal pocket, a therapeutic effect can be expected such that the gingival cells proliferate, the periodontal pocket is filled with the gum, and the periodontal pocket disappears.

The front view which shows typical embodiment of the etching processing apparatus which concerns on this invention. Similarly side view. The perspective view which shows a mode that a molded article is mounted | worn with a mounting jig. Sectional drawing which shows the attachment jig holding the molded article. The front view which shows the other example of the etching processing apparatus which concerns on this invention. Similarly side view. The perspective view which shows a mode that a molded article is mounted | worn with the attachment jig which concerns on a modification. Sectional drawing which shows the attachment jig which concerns on the modification which hold | maintained the molded article. The graph which shows the zeta potential of each disk. Scanning probe microscope image of a plasma untreated disk sample. The scanning probe microscope image of the disk sample of the plasma 1 round-trip process.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Etching processing apparatus 2 Holding stand 3 Plasma generator 4 Gas supply means 5 Base 6 Intermediate base 7 Mounting jig 8 Gas discharge means 9 Temperature sensor 10 Housing 11 Wall surface 20 Groove portion 21 Internal space 21a Opening portion 22 Communication hole 31 Bracket 40 piping 41a supply port 41 piping 50 shaft body 51 guide rail 52 motor 53 guide rail 54 guide rail 60 groove portion 70 through hole 71 step portion 72 recess portion 80 discharge pipe 81 pump P porous body molded product

Claims (26)

  A living cell culture medium characterized by comprising any one or two of polylactic acid and a polylactic acid copolymer as a porous material, and a molded product of the porous material is etched with low-temperature plasma under atmospheric pressure. Wood.   Either one or two of polylactic acid and polylactic acid copolymer is made into a porous body, and the molded product of the porous body is crystallized by heat treatment, and then etched by low-temperature plasma under atmospheric pressure. A living cell culture substrate characterized by comprising: The plasma working gas used for the etching treatment is any one or more of air, nitrogen, argon, carbon dioxide, and octafluoropropane (C ₃ F ₈ ). The living cell culture substrate described in 1.   4. The method according to claim 1, wherein the polylactic acid copolymer is one or two of a polylactic acid-glycolic acid copolymer and a polylactic acid-caprolactone copolymer. 5. The living cell culture substrate as described.   The living cell culture substrate according to any one of claims 1 to 4, which is used as a tissue regeneration scaffold material.   Either one or two of polylactic acid and polylactic acid copolymer are freeze-pulverized, and the resulting powder is made into a porous molded article, and the porous molded article is etched with low-temperature plasma under atmospheric pressure. A method for producing a living cell culture substrate.   Either one or two of polylactic acid and a polylactic acid copolymer are freeze-pulverized, and the resulting powder is made into a porous molded article, and the porous molded article is crystallized by heat treatment. A method for producing a living cell culture substrate, characterized by etching with low-temperature plasma under atmospheric pressure.   The method of forming the porous molded article is a method of freeze-pulverizing any one or two of the polylactic acid and the polylactic acid copolymer and sintering the obtained powder to form a porous body. The method for producing a living cell culture substrate according to claim 6 or 7, characterized in that   The method for producing a living cell culture substrate according to any one of claims 6 to 8, wherein the etching treatment with low-temperature plasma under the atmospheric pressure is performed 2 to 4 times.   The method for producing a living cell culture substrate according to any one of claims 7 to 9, wherein the heat treatment is performed at 80 ° C to 140 ° C. 11. The plasma working gas used for the etching process is any one or more of air, nitrogen, argon, carbon dioxide, and octafluoropropane (C ₃ F ₈ ). The method for producing a living cell culture substrate according to any one of the above.   In the etching process, the space where the surface opposite to the plasma irradiation surface of the porous molded article faces is decompressed, and thereby a low-temperature plasma flow is sucked into the porous molded article. Item 12. The method for producing a living cell culture substrate according to any one of Items 6 to 11.   The polylactic acid copolymer is any one or two of a polylactic acid-glycolic acid copolymer and a polylactic acid-caprolactone copolymer, according to any one of claims 6 to 12. The manufacturing method of the living cell culture base material of description. An apparatus for use in the method for producing a living cell culture substrate according to any one of claims 6 to 13, wherein a holding table for holding the porous body molded product in a casing in which atmospheric pressure is maintained; A plasma generator for irradiating the porous molded article held on the holding table with a low-temperature plasma flow is provided, and the plasma generator is provided with air, nitrogen, argon, carbon dioxide, and octafluoropropane (C _3). F ₈ ) is provided with a gas supply means for supplying one or more plasma working gases, and the porous molded product held on the holding table is subjected to the low-temperature plasma under atmospheric pressure. An etching apparatus for a living cell culture substrate, characterized by performing an etching process.   15. The apparatus for etching a living cell culture substrate according to claim 14, wherein the casing has a sealed structure, and gas supply means for filling the casing with the same gas as the plasma working gas is provided.   16. The apparatus for etching a living cell culture substrate according to claim 14, further comprising a moving means for moving the holding table and the plasma generator relative to each other.   In the holding table, an internal space that opens to a position facing the surface opposite to the plasma irradiation surface of the molded product to be held is formed, and a gas discharge means for discharging the gas in the internal space to the outside of the housing is provided. The apparatus for etching a living cell culture substrate according to any one of claims 14 to 16, wherein the internal space is decompressed by discharging the gas and a low-temperature plasma flow is sucked into the porous molded article.   The living cell culture substrate etching apparatus according to any one of claims 14 to 17, wherein a temperature sensor for detecting a temperature in the vicinity of a low-temperature plasma flow of the plasma generator is provided in the casing.   With respect to a living cell culture substrate formed by etching one or two of polylactic acid and a polylactic acid copolymer into a porous body, and etching the molded article of the porous body with low-temperature plasma under atmospheric pressure, A method for culturing a living cell, wherein the cell is seeded and cultured.   One or two types of polylactic acid and polylactic acid copolymer are made into a porous body, crystallized by heat-treating the molded article of the porous body, and then etched by low-temperature plasma under atmospheric pressure A method for culturing a living cell, comprising cultivating a living cell culture substrate by seeding the living cell. 21. The plasma working gas used for the etching treatment is any one or more of air, nitrogen, argon, carbon dioxide, and octafluoropropane (C ₃ F ₈ ). A method for culturing a living cell according to 1.   The polylactic acid copolymer is any one or two of a polylactic acid-glycolic acid copolymer and a polylactic acid-caprolactone copolymer according to any one of claims 19 to 21. The method for culturing a living cell according to the description.   A tissue regeneration scaffold is a living cell culture substrate obtained by etching any one or two of polylactic acid and polylactic acid copolymer into a porous material, and etching the molded article of the porous material with low-temperature plasma under atmospheric pressure. A method of culturing a living cell, comprising culturing the living cell as a material by contacting the living cell.   One or two types of polylactic acid and polylactic acid copolymer are made into a porous body, crystallized by heat-treating the molded article of the porous body, and then etched by low-temperature plasma under atmospheric pressure A method for culturing a living cell, comprising culturing the living cell by using the living cell culture substrate as a tissue regeneration scaffold material and contacting the living cell. 25. The plasma working gas used for the etching process is any one or more of air, nitrogen, argon, carbon dioxide, and octafluoropropane (C ₃ F ₈ ). A method for culturing a living cell according to 1. The polylactic acid copolymer is any one or two of a polylactic acid-glycolic acid copolymer and a polylactic acid-caprolactone copolymer according to any one of claims 23 to 25. The method for culturing a living cell according to the description.

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