JP2020080836A - Cell culture method using porous membrane-like cell culture substrate - Google Patents

Abstract

To provide cell culture methods capable of eliminating the risk of biological harmful effects and contamination, and producing cells with efficiency and large amount.SOLUTION: Provided is a cell culture method, in which cells are seeding on a porous membrane-like cell culture substrate in which a circular fine through hole with a diameter of 10 to 80 μm or a polygonal fine through hole with a side of 10 to 80 μm is formed on a biocompatible thin film material, subsequently, in a cell culture, by bringing the cell culture substrate into contact with a plate culture substrate, proliferated cells are transferred to the plate culture substrate through the through hole of the cell culture substrate.SELECTED DRAWING: None

Description

Detailed Description of the Invention

The present invention relates to a cell culture method for continuously and continuously increasing the number of cells without interrupting the cell culture.

In recent years, in many cell cultures, a plate culture method is generally adopted as seen in JP 2010-200745 A (Patent Document 1) and JP 2004-254674 A (Patent Document 2). That is, when an animal cell is seeded on a culture substrate, it first adheres to the substrate and stabilizes its position, then divides while migrating in the horizontal direction, and the number increases. Its migration distance is the minimum distance to secure the increased attachment space of cells to the substrate after division, except for the special case of jumping away from the substrate such as cancer cells. It is about to move. Since cells are destined to die once they leave the substrate and become in a floating state, it is typical of cell migration that the propagated cell group moves in plane on the culture substrate.

In planar culture, the cell population can grow and increase in number until it fills the area on the substrate surface, but if the culture is continued, the accumulation of cells becomes overcrowded, and sometimes the increased cells cover the original cells. Sometimes. This is called stratification, but when these conditions progress, it becomes difficult for the covered cells to take in nutrients from the culture solution, growth slows down, and eventually the adherence to the substrate is lost and the substrate is covered. There is a limit to the number of cells that can be grown by plate culture because handling is difficult because the entire cell group that has grown in the form of a membrane is separated from the substrate.

The state in which the flat plate substrate surface is almost completely covered with cells as described above in flat plate culture is called confluent. In order to continuously increase the number of cells, it is essential to keep the population of cells at an optimal density for growth. Therefore, it is necessary to divide the culture system and supply a new medium before the cells on the plane of the substrate become overcrowded. Therefore, the cells are passaged at the logarithmic growth stage slightly before the confluent state.

This passage is also called transplantation, and once the culture solution filling the substrate being cultured is removed, the cells are artificially detached from the substrate, and the culture substrate having a larger area than the original substrate (hereinafter, It is necessary to transfer it to a "culture dish") or a plurality of culture dishes. At this time, the cells must be separated from the dish, and the separated cells must be separated into single cells so that they can be evenly distributed and seeded on the substrate surface of the transfer destination dish.

Widely used to do this is an enzyme called trypsin, a type of proteolytic enzyme. It is customary to adjust the pH to 7.6 to 7.8 and further add EDTA in order to sufficiently exert the effect of this enzyme. However, some cells may be undesirably affected by this trypsin treatment. For example, when the cells are strongly adhered to the substrate, or when it takes time to disperse the cells separated from the substrate into a single cell, trypsin acts to cause adverse effects such as cell death.

In order to solve this, a method of enlarging the area of the flat portion of the culture dish can be considered, but this method requires the size of the dish to be increased, and the amount of the culture solution required increases accordingly. The culture medium needs to be changed regularly, but at the beginning of the culture, there is usually only a small number of cells on the vast culture dish, and most of the culture medium added until the number of cells reaches a considerable number. Is wasted. Moreover, as the size of the dish increases, so does the size of the incubator that stores it and assists in its growth. Until the number of cells increases, extra space has to be allocated.

Further, under such conditions, long-term culture is carried out on one culture dish, but in a large area, the distribution density of cell groups becomes non-uniform with the culture. Since the cell density becomes excessively high at the site where the cell growth is started from the early stage of culture and the growth is suppressed, the cell growth efficiency in the entire dish is reduced.

In the first place, cell culture basically mimics the growth mode of cells in the living body, so in the process, performing subculture operation by trypsin treatment, which cannot be performed in the living body, is essential for the basic living of the organism. It is a deviant process. Therefore, it can be said that the method of continuously increasing the number of cells without the trypsin treatment is the cell culture method based on the biological process.

JP, 2010-200745, A JP, 2004-254674, A

For the above reasons, when using the conventional plate culture method using a culture dish to determine the number of cells above the confluent state, the cells are once separated from the culture dish by enzyme treatment and then singulated, It is said that it is rational to disperse the seeds in the above culture dish and seed them. However, the application of the trypsin enzyme used at that time is an action that cannot be performed in the living body, and its harmful action cannot be denied because it is biological.

Also, the process of separating and unifying the cultured cells, which is essential in the current cell culture method, from the substrate and then repeating the reseeding is extremely complicated, and this process is externally contaminated with bacteria, which is a major enemy of culture. There was a problem of increased risk (hereinafter referred to as "contamination"), and once contaminated with bacteria, the culture had to be interrupted at that point.

As described above, the conventional substituting method using a cell-separating enzyme such as trypsin not only modifies the aspect of the cell proliferation that should be originally present, but also interferes with the culturing process due to contamination, and the labor required for it. It can be said that the cell culture process has many inconvenient problems.

In view of the above circumstances, the present invention, when growing cells by cell culture, culturing on a porous membrane in which through-holes perforated with nano-order accuracy in a membrane material having biocompatibility are regularly arranged. Then, the cells grown in the flat portion between the through holes migrate through the through holes to the vertically adjacent culture substrates, transfer the cells to a plurality of culture substrates, and the cells are transferred according to the number of culture substrates. It is an object to provide a method for amplifying numbers.

The present inventors, in order to solve the above problems, by utilizing the function of cells to migrate, by transferring a part of the cells grown on the cell culture substrate to a new culture substrate to grow, continuous, It succeeded in efficiently and efficiently amplifying the number of cells, and completed the present invention. This is due to the fact that the cells proliferate themselves and the number of cells increases, so that they migrate and move to another position. Specifically, the cells divide on the original culture substrate and move horizontally. New culture substrates are installed vertically adjacent to each other at the tip of the expanded volume, and some migrating cells are allowed to migrate to proliferate the cells on the new substrate.

That is, the present invention provides a cell culture substrate in the form of a porous film in which a thin film material having biocompatibility is formed with circular fine through holes having a diameter of 10 to 80 μm or polygonal fine through holes having a side of 10 to 80 μm. After seeding the cells in the cell culture medium, by bringing the cell culture substrate into contact with the plate culture substrate in the cell culture medium, it is possible to transfer cells grown through the through holes of the cell culture substrate to the plate culture substrate. A method for culturing cells according to the first aspect of the present invention, and a thin film material having biocompatibility, in which circular fine through holes having a diameter of 10 to 80 μm or polygonal fine through holes having a side of 10 to 80 μm are formed. After seeding the cells on the membranous cell culture substrate, in the cell culture medium, by continuing the culture in the form of contact lamination of a plurality of the cell culture substrate, the cells grown through the through holes of the cell culture substrate, The cell culture method according to the second aspect of the present invention is characterized in that the cells are sequentially transferred to the cell culture substrate groups that are in contact with each other in the vertical direction, and further, when the cells move in the porous membrane cell culture substrate according to the first or second aspect of the invention. A cell culture method of the third invention, wherein a burr-like guide path is provided at the edge of the fine through-hole serving as a passage to assist the cell to migrate to another culture substrate.

According to the above-described cell culture method of the present invention, by culturing on a porous membrane in which through holes perforated with nano-order accuracy are regularly arranged in a film material having biocompatibility, a plane between each through hole is obtained. The cells that have proliferated in a portion migrate through the through holes to the adjacent culture substrates, transfer the cells to multiple culture substrates, and the number of cells can be amplified according to the number of culture substrates. It is possible to efficiently and mass-produce, and it is expected to make a great contribution to the practical application to regeneration of a living body.

It is a conceptual diagram regarding the cell culture of the present invention. It is an enlarged view showing an example of a through-hole provided in a porous membrane-like cell culture substrate of the present invention. FIG. 3 is an enlarged view showing an example of a burr-shaped guide path provided at the hole edge on the back surface side of the through hole of the porous membrane cell culture substrate of the present invention. FIG. 3 is an enlarged view showing an example of a state in which a space is secured in two adjacent cell culture substrates of the present invention. FIG. 3 is an enlarged view of a blade-shaped press punching tool showing an example of a tool for forming a through hole. It is a conceptual diagram which shows the drilling example by the said through-hole formation tool. It is an electron micrograph which shows an example of the said through-hole. It is a conceptual diagram which shows an example of the cell culture process of this invention. FIG. 3 is an enlarged view showing a state of cells grown around a through hole of the porous membrane cell culture substrate of the present invention. It is a conceptual diagram which shows the application example which concerns on the mass production of cells based on the culture method of this invention.

In the present invention, first, a thin film having biocompatibility and capable of adhering, dividing and migrating cells is prepared in the same manner as a plate culture substrate. Then, fine through holes are formed in the thin film at a certain interval so that cells can pass through.

As a result of movement due to proliferation, some cells that have been cultivated on this porous membrane-shaped cell culture substrate are extruded toward the through holes, so that the cells enter the through holes. By arranging the through hole as a through hole so that a new culture substrate comes into contact with the outlet, cells that have entered the through hole will eventually adhere to the new culture substrate and start to grow on the substrate. This is the first mode which is one of the constituent features of the present invention.

In order to move cells between different substrates in this first mode, it is necessary to provide a gap of about 10 μm, which is close to the cell size, between the original substrate that is the source of cells and the new substrate that receives cells. In that case, considering the case where the substrates are not perfectly flat, it is desirable to arrange as many through holes as possible so that cells can be transferred when the mutually opposing convex portions approach each other in that situation. In order to increase the probability of moving to the new substrate through the through holes, it is necessary to consider the arrangement density of the through holes and the shape and size of the through holes.

The shape of human somatic cells is spherical in the suspended state, and the diameter is approximately 10 μm. The width diameter when this is split twice into four pieces is (radius×2)+[(radius×2)×√2]≈24 μm, and when it is split four times, (radius×2)+{ [(Radius×2)×√2]}×3≈52 μm. Therefore, when the number of cells is increased by 16 times by dividing four times, if there is a through hole within a range of about 50 μm from that, it is considered that the cells located at the outer edge of the cell group will reach the position where the through hole exists. .. Therefore, in the calculation, by providing the through holes at intervals of about 50 μm, many cells proceed to the position where the through holes are formed due to the progress of cell division.

Next, regarding the shape and size of the through hole, it is considered that a spherical cell body having a diameter of 10 μm can pass through, but the cell is rich in flexibility, and the shape can be freely changed. For example, a flat shape may have a diameter of 20 to 30 μm. Therefore, a through hole having a diameter of less than 10 μm will not enter and will straddle the hole. Therefore, in order to promote the entry into the through-hole, it is preferable to use a circle having a diameter of 20 μm or more or a square hole having a side of 20 μm or more.

The larger the size of the through hole, the easier it is for cells to enter the through hole. However, if the size of each through hole is increased, the area of the flat plate between the through holes decreases relatively and the cells attach. , There is less space to split. Therefore, it is necessary to adjust the shape and size of the through holes and the number of arrangements (arrangement density) per unit area to be optimum. This is the second form of the constituent features of the present invention.

Although the first and second modes allow some cells to migrate from the initially expanded cell group and move from the original culture substrate to another new culture substrate, new cells can be transferred through the through holes of the second mode. If cells adhere to a different culture substrate, the movement becomes more efficient if the distance between the exit of the through hole and the new substrate becomes short. The distance, or gap, must be about the distance cells can migrate and cross, ie, the size of the cell.

Judging from the human cell size, the gap that can be overcome on the culture substrate by migration is estimated to be about 10 μm. Therefore, in the present invention, in order to move the cells from the through holes of the original culture substrate to which the cultured cells are attached to the new substrate, cell migration assistance and gap formation are provided at the hole edges of each through hole of the original substrate. It is preferable to provide a desired burr-like guide path so that a gap with the new substrate is about 10 μm without being influenced by the unevenness of each substrate surface. This is the third mode of the constituent requirements of the cell culture method of the present invention.

In the cell culture method of the present invention, a plurality of the above-mentioned third-type culture substrates are superposed on each other, so that the gap between the edge of the through-hole of the original substrate on which the cells proliferate and the new culture substrate of the movement destination is about 10 μm. It maintains a close state and establishes a passageway for the migrated cells to grow on a new substrate, and a space around it, that is, between the original and new culture substrates, in which the culture solution perfuses abundantly. This makes it possible to mass-produce cells, which is the fourth mode of the constituent requirements of the cell culture method of the present invention.

Brief description of the invention with reference to the drawings

The conceptual diagram for satisfying the said main structural requirements in this invention is shown. [FIG. 1] is a conceptual view of the first mode, in which a through-hole 3 is provided in a thin-film cell culture substrate 1 having biocompatibility. In the present invention, the thin film material having biocompatibility is preferably pure titanium or titanium alloy, but it goes without saying that other biocompatible metals, ceramics, plastics, etc. are also applicable. Although the through hole 3 is circular or polygonal, it needs to have a shape that allows migrating cells to easily enter so that it does not cross the entrance of the hole. As an example, the thin film culture substrate 1 which is a material having biocompatibility has a thickness (1) of 1 to 50 μm, and the through hole 3 provided therein is a square having a side length (2) of 10 to 80 μm.

Some of the cells that have been cultured and propagated on the plane of the thin film culture substrate 1 enter the through holes 3 by migration as shown by the thick arrows, and eventually pass through the through holes to be located in close proximity to the culture substrate 1. Reach the culture substrate 2. The cells that have reached the new substrate 2 are provided with a space for proliferating and increasing the number of cells. [FIG. 1] is an example of a specific shape showing the first mode, and the form and size shown here are not limited thereto.

[FIG. 2] is a conceptual diagram of the second mode, and shows an example of dimensions when the through hole 3 is provided in the culture substrate 1 having biocompatibility. With reference to the example of FIG. 1, it is a through hole 3 in which square holes formed in the size of <2> are arranged at regular intervals in <4>. Here, the size of (2) and the distance of (4) are determined by the size of the cultured cells, the number of cells that can be cultured in the plate around the through hole, and the number of cells that can enter at once for each through hole. It is determined.

In this example, since a certain proportion of the cell group that has been cultured and propagated in the flat plate portion between the through holes 3 enters the through holes, it changes into the through holes 3 by changing the interval of the through holes 3 to be formed. The proportion of cells can be adjusted. Considering the size of the through hole, and considering the size of the through hole 3 through which cells can pass, the cell migration ability, and the like, it is appropriate to adjust the interval (4) between 30 and 500 μm.

Further, regarding the shape and size of each through hole 3, it is appropriate to make it a circle having a diameter of 10 to 80 μm or a polygon having a side of 10 to 80 μm depending on the number of cells that simultaneously enter. If the size of the through-hole 3 is less than 10 μm, it becomes difficult for the cultured cells to pass through, while if it is larger than 80 μm, there is a problem that the space for cells to attach and divide becomes relatively small, or the cell culture becomes uneven. The size and interval of the through holes 3 are set so as to be adapted to the type of cell and the purpose of the proliferation method thereof, and the size and interval thereof may be changed on one material surface depending on the situation. It is possible.

For example, in the center of one sheet of material, the interval between the formation of through-holes is lengthened in order to give priority to the plate-like cell growth ability, while when the number of cell groups increases and reaches the periphery, new In order to positively transfer to the substrate, it is effective to shorten the interval between the through holes in the peripheral portion of the material surface.

As described above, the specifications for forming the through-holes in the membranous cell culture substrate in the second mode are set according to the purpose and conditions of cell culture, and therefore the form and size thereof are preferably within the above range.

[FIG. 3] is a conceptual diagram of the third mode and shows an example of the shape of the through hole 3 provided in the thin film culture substrate having biocompatibility. With reference to the example of FIG. 1, its shape is represented by a wing-shaped burr-shaped guide path 4 that rises from a hole edge of a square hole formed in size (2) substantially perpendicularly to the substrate. In this process, cells that have entered the through-hole 3 first migrate from the edge of the through-hole to the burr-like guide path 4 and migrate. Since the tip portion of the burr-shaped guide path 4 is close to the new culture substrate 2 in FIG. 1 at a distance of about 10 μm, the cells that have migrated through the guide path 4 will eventually adhere to the new culture substrate 2.

In this way, the burr-shaped guide path 4 provided in the through hole 3 in the third mode has a part of about 10 μm with respect to the material to which the cells move, regardless of the unevenness of the opposing substrate surfaces. The form and size thereof are not particularly limited as long as the condition of being close to the distance is satisfied.

[FIG. 4] is a conceptual diagram of the fourth mode, in which cells are proliferated and a cell culture substrate 1 from which cells are supplied and a new culture substrate 2 from which cells move through through holes 3. An example of a state where a space (5) for growing cells at the moved destination is secured when the cells are brought close to each other is shown. With reference to the examples of [FIG. 1] and [FIG. 3], a cell is proliferated and a through-hole is provided to the original culture substrate 1 capable of migrating and supplying the cell to a new culture substrate 2. The new culture substrate 2 to which cells are supplied through 3 is located on the side of the burr-like guide passage 4 rising from the edge of the through hole 3 of the original substrate 1 and is in a state of being almost in contact with the tip of the guide passage 4.

In this state, the guide path 4 acts as a spacer, and a space (5) is secured between the substrates 1 and 2. The cells move from the original substrate 1 to the new substrate 2 via the void (5), pass through the void (5) between the substrates 1 and 2 formed by the guide path 4, and reach the surface of the new substrate 2. It travels widely and progresses. In addition, the void (5) formed by the guide path 4 also has an effect of perfusing a sufficient culture medium for the cells to grow.

The present invention has succeeded in extremely efficiently increasing the frequency with which cells can move between each substrate by the method constituted by the above first to fourth modes. It is a basic specification.

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

As an example of the present invention, an example in which a metal foil having a thickness of 20 μm and made of pure titanium having excellent biocompatibility is used as a material will be described. First, as a method of forming fine through holes, "Kenzan-shaped press punching tool 5" as disclosed in Japanese Patent Application Laid-Open No. 2014-8585 as shown in FIG. 5 of the present application is used. In this, a 2 mm square column made of a hard metal such as tungsten carbide steel was subjected to grid grinding with an ultrathin rotating grindstone for processing integrated circuits to form square protrusions 6 at intervals of 75 μm. It is a sword-shaped press punch.

When this through-hole is used to form the through hole 3 in the culture substrate 1 made of pure titanium foil by the method illustrated in FIG. 6, a burr-like guide path 4 is formed in the exit direction of the through-hole.

[FIG. 7] is an electron micrograph of a pure titanium foil in which through holes are actually formed. As shown in (a) of the drawing, 25 μm square through-holes are formed in a grid pattern with a distance of 75 μm between the centers of the holes. Further, an enlarged view of the through hole portion on the side where the punching tool 5 is pushed in, that is, on the inlet side is shown in (b). The through hole 3 is a square hole in which the shape of the protrusion 6 of the punch 5 is reflected, and an additive is not seen at the edge thereof. The shape of the through hole may be circular or polygonal such as square, hexagonal, octagonal, etc., but is preferably quadrangular, and more preferably square.

On the other hand, (c) shows an enlarged photograph of the side of the punch 5 on which the projection 6 is removed, that is, the punched portion on the exit side. At the edge of the square hole, a guide path 4 due to burrs that is considered to be derived from titanium foil is recognized. In the through hole formed by this processing method, the presence or absence of burrs on the edge of the formed through hole is clearly separated on the inlet side on which the punch tool is actuated and on the outlet side from which the punch tool is removed.

Usually, the "burr" which is necessarily formed after such a punching process is generally removed as a troublesome member which deteriorates the processing quality. However, according to the present invention, it is exactly this troublesome "burr". It is used as one of the core points of. That is, the "burr" imparted to the edge of the through-hole in the third mode, which is a constituent feature for realizing the present invention, is effectively used as a guide path for cell migration.

In the present invention, not only "burrs" due to such a punching tool, but also by-products called "dross", which are protrusions around the through-holes generated by laser drilling, fall under the third requirement of the present invention. You can use it.

The 25 μm square through-holes formed in the metal foil with a thickness of 20 μm shown in this example have a size of the through-holes of about 2 to 3 times that of human somatic cells with a diameter of 10 μm and can be infiltrated with a culture solution. Therefore, there is no hindrance to the passage of cells, and the present invention is effective in the first mode described above. Regarding the specification that these through-holes are arranged substantially evenly at intervals of 75 μm, it is a structure that allows cells to divide and proliferate four or more times in the flat plate portion between each through-hole. It is effective for the present invention.

In addition, in this example, the burr formed on the edge of the through hole can be used as the guide path 4 for moving the cells that have entered the through hole to the new adjacent culture substrate. It is effective for the present invention, and since the burr has not only a mechanically considerable strength but also exists on the entire one surface of the through holes formed at intervals of 75 μm, the burr and the culture substrate from which the cells are supplied, The present invention is effective in the present invention in the fourth mode, since it also acts as a spacer for maintaining a distance from a new culture substrate that receives the treated cells.

An example relating to cell culture using a cell culture substrate produced by satisfying the respective conditions of the above-described first to fourth modes based on the above-mentioned example is shown in FIG. (D) in the figure is a culture in which cells have already been sufficiently cultured on the substrate and a large number of proliferated cells adhere to the surface (a), and the same specifications as (a), but no cells adhere. Substrate (a) is placed under (a), and the same substrate (d) as (a) is placed over (a), both of which are outlets for through-hole drilling by the press punching tool, that is, through-holes. The state in which the surface with burrs at the hole edges is placed on the lower side and the layers are immersed in the culture solution in the culture dish (c) is shown.

When culturing is carried out in this way, as shown in (e) of FIG. 8, the cells attached to (a) pass through the through-holes of (a), and the burrs at the edges of the through-holes on the lower surface are formed. It moves through the guide path and reaches the surface of the adjacent culture substrate (a). The cells that have migrated from (a) to (a) can proliferate on the surface of (a) and increase the number of cells. The cells that grew in (a) eventually passed through the through-holes in (a), moved through the burr-shaped guideways at the edges of the through-holes on the lower surface, and reached the bottom surface of the culture dish (c) where the lower surface was close. The adhered cells continue to be cultured on the surface of (c), and the number of cells can be further increased.

In addition, on the substrate (d) having the same specifications as that of (a), which was adjacent to the culture substrate (a) to which the cells adhered, the burr-like guideway on the lower surface of (d) adhered to the cells (a) When the cells are sufficiently close to the upper surface of (a), the cells of (a) adhere to the burr-like guideway on the lower surface of (d) and migrate upward, so that the lower surface of the substrate (d) or the through hole of (d) It can pass through, reach the upper surface of (d), and grow on the surface of (d).

As described above, the above-described embodiments are typical examples of the present invention, but the present invention satisfies some or all of the above-mentioned first to fourth modes for the thin film material having biocompatibility. As long as the method allows cells to move between a plurality of porous membrane-like culture substrates and between a plurality of porous membrane-like culture substrates and a culture dish by stacking a plurality of porous membrane-like culture substrates to achieve the above structure, It can be within the scope of the invention.

Based on the above examples, the porous membrane-like culture substrate having the above-mentioned specifications was produced, and the cell culture was carried out. The results are as follows. The porous membrane-shaped culture substrate used was one in which a pure titanium foil having a thickness of 20 μm was perforated by a method according to Japanese Unexamined Patent Publication No. 2014-8585 using a sword-shaped press piercing tool to form fine through holes. ..

Porous film-like culture substrate was immersed in a culture dish filled with 10% FBS α-MEM as a culture solution, and the osteoblast-like cell line MC3T3-E1 isolated and established from the mouse calvaria was seeded, and the porous film-like culture substrate The colonization of the cells was confirmed.

The state of the attached cells is shown in [Fig. 9]. In this figure, (k) and (l) are porous membrane-shaped culture substrates on which cell culturing was carried out after the burr-like guideways formed at the edges of the fine through holes were located on the lower surface. Further, (m) is an immunostaining image of the substrate in that state, and in the color image (black and white image in FIG. 9), actin (green) showing cell nucleus (blue) and cell body skeleton is shown. Has been confirmed.

In (k), it can be seen that cells are attached to various places on the membrane. (L) is a partially enlarged image of (k), and it can be seen that some of the attached cells have entered the through-hole. Further, in (m), most of the cell nuclei are present in the through holes. This demonstrates that the cells were alive within the through-holes.

From the above findings, by the method of the present invention, it is possible to cultivate cells on the porous film-like culture substrate, and it is possible to realize the action of cells passing through the fine through holes designed by the present invention. Proved that there is.

Further, (n) and (o) in FIG. 9 are porous membranous culture substrates in which cell culture was performed after the burr-like guideways formed at the edges of the fine through holes were positioned on the upper surface. is there. Further, (p) shows an immunostained image of the substrate in that state, and in the color image (shown as a black and white image in FIG. 9), actin (green) showing cell nucleus (blue) and cell body skeleton is shown. Has been confirmed.

In (n), it can be seen that cells are attached everywhere on the membrane. (O) is an image obtained by partially enlarging (n) and shows that cells are present in the through holes. Furthermore, it is confirmed that cells are actively attached to the periphery of the burr-like guideway and the branch of the cell body is attached to the tip of the burr-like guideway. Further, in (p), cell nuclei are located everywhere on the surface of the membrane and in the through-holes, indicating that the culture and proliferation are extremely vigorous.

From the above findings, it is proved that the fine burr at the edge of the through-hole provided on the porous film-shaped culture substrate according to the method of the present invention does not hinder the cell culture, and the cells use this as a scaffold. , It was shown that the taxiway could be used as a support point.

Thus, by overlaying a new culture substrate in close proximity to the substrate on which cells have been cultured by the method of the present invention, or by stacking a plurality of new culture substrates in a sandwich form, [Fig. 8] is obtained. It was confirmed that the cells migrated between the substrates as shown, and the cells continuously and vigorously proliferated on the substrate of the migration destination.

The present invention is not limited to the above-mentioned embodiment in which cell culture is continuously carried out by stacking the above-mentioned porous membrane-like culture substrates, but using all or part of the principles of the first to fourth modes, Any method in which cell culture can be continued without substituting by conventional trypsin treatment can be considered to be according to the present invention.

In summary of the above examples, titanium foil, which has excellent biocompatibility and is also useful as a plate culture substrate, is used as a material, and a sufficient plate area is secured so that cells on the substrate can obtain a logarithmic growth phase. By arranging through holes for sending cells that proliferate and move to other substrates at an optimal size and spacing, it is possible to grow cells on their own substrate and send cells to other substrates. The structure is a main condition necessary for the present invention.

It should be noted that cells that have entered the through holes usually adhere to the back surface of the substrate and migrate after passing through the through holes, and therefore do not move to other substrates. On the other hand, in the present invention, the burr provided on the edge of the through hole acts as a guide path for transferring cells to another substrate, and if the distance to the substrate to which the cell is moved is further reduced to about 10 μm, this Cells that have migrated along the structure are accessible for migration to other substrates and the cells are transferred.

In this case, the surface on which the cells move should be as smooth as possible. The reason for this is that, generally, when cultured cells have dents, irregularities, undercuts, etc. that hold cells, a so-called scaffolding effect appears in which cells enter and do not move, and cell transfer is hindered. Therefore, it is effective to suppress the scaffold effect by using a smooth pure titanium foil as in the above-mentioned embodiment. Therefore, in the present invention, it is not appropriate to use the sponge-like porous material.

Industrial applications

If the cultured cells can move between the substrates by the process of the present invention as described above, it is possible to increase the number of cells efficiently and easily by repeating the operation of successively overlaying a new substrate on the culture substrate on which the cells have grown. Moreover, in that case, the subculture operation that has been indispensable every time the container is moved, which is seen in the conventional plate culture method, is completely unnecessary, and moreover, an enzyme drug such as trypsin that individually unifies the cells is used. There is no need to make it work. Therefore, the present invention can continuously utilize the natural cell growth process close to that in the living body, and can completely eliminate the adverse effects of enzyme drugs.

An example of [FIG. 10] is shown as a form in which the present invention is used for mass production of cells. By stacking porous membrane-shaped culture substrates formed according to the specifications of the present invention and aseptically storing the culture device (e), by appropriately supplying or perfusing the culture solution, cells are stored in all the stored culture substrates. Propagate so that it spreads.

When a sufficient number of cells have been obtained by culturing, it is possible to take out the culture substrate from the stored (e) and use the attached and proliferated cells one by one. In this case, a porous membrane-shaped culture substrate made of titanium foil can be introduced into the living body (f) by aseptic operation to perform cell therapy. Further, when carrying out a molecular biological test such as a genetic test or evaluation of the degree of differentiation of the proliferated cells, one culture substrate was taken out and the cells were transferred to an ordinary plate culture dish as shown in (K). After that, it is possible to carry out the inspection by a conventional operation.

1... Porous Membrane Cell Culture Substrate 2... New Culture Substrate 3... Through Hole 4... Burr-Like Guidance Path 5... Kenyama-shaped Press Punch Tool 6... Protrusion

Claims (3)

After seeding cells on a porous membrane-like cell culture substrate in which a circular fine through hole with a diameter of 10 to 80 μm or a polygonal fine through hole with a side of 10 to 80 μm is formed in a thin film material having biocompatibility A method for culturing cells, wherein the cells grown in the cell culture medium are brought into contact with the plate culture substrate to transfer the cells grown through the through holes of the cell culture substrate to the plate culture substrate. .. After seeding cells on a porous membrane-like cell culture substrate in which a circular fine through hole with a diameter of 10 to 80 μm or a polygonal fine through hole with a side of 10 to 80 μm is formed in a thin film material having biocompatibility , In the cell culture medium, by continuing the culture in the form of contacting and stacking a plurality of the cell culture substrates, the cells grown through the through holes of the cell culture substrate are sequentially transferred to each cell culture substrate group that contacts vertically. A method for culturing cells, which comprises: The above-mentioned porous membrane-shaped cell culture substrate is provided with a burr-shaped guide path at the edge of the fine through-hole that serves as a passage when cells move, which assists the behavior of cells migrating to other culture substrates. The method of culturing cells according to claim 1 or 2.