JP4445765B2 - Cell culture equipment - Google Patents

Description

The present invention relates to a cell culture apparatus capable of culturing cells seeded on a three-dimensional substrate of various shapes and sizes with high efficiency and high density.

Recent advances in cell engineering technology have enabled the culturing of numerous animal cells, including human cells, and rapid research on so-called regenerative medicine that uses these cells to reconstruct human tissues and organs. Is going on. In regenerative medicine, the point is whether cells can proliferate and differentiate to construct a three-dimensional biological tissue-like structure. Usually, when such a biological tissue-like structure is produced, cells are seeded and cultured on a three-dimensional substrate that serves as a scaffold for tissue or organ regeneration. If the biological tissue-like structure obtained in this way is transplanted into a patient, the biological tissue-like structure performs the same function as the tissue or organ, or the patient's own tissue using the biological tissue-like structure as a scaffold Or an organ regenerates.
In general, a human tissue or organ has a structure in which various cells are accumulated at a very high density via an intercellular substance, and a biological tissue-like structure used for regenerative medicine is also included in such a tissue or organ. It is important that a structure as close as possible is formed.

On the other hand, not only in the field of regenerative medicine, for example, a large amount of animal cells are also required for research such as cosmetics and pharmaceuticals. As a means for obtaining such a large amount of cells, seeding on a three-dimensional substrate is performed. Attempts have been made to detach and collect cells from the three-dimensional substrate after culturing such cells at high density.

In any of these uses, a method for culturing cells seeded on a three-dimensional substrate at high density is required.
As a conventional cell culture method, for example, a cell is adhered to the bottom of a cell culture flask, a predetermined amount of a culture solution is added to the cell culture flask, and the cell culture flask is left to stand in a CO ₂ incubator or the like. Incubation method is mentioned. However, the stationary culture method is supposed only to cultivate the number of cells up to the extent that the bottom of the cell culture flask is covered. When living tissue-like structures with extremely high cell density and containing a large number of cells are cultured by the conventional static culture method, nutrient components contained in the culture are insufficient or the cells are secreted in a very short time. The pH of the culture solution fluctuates due to the waste product, and the cell growth rate is remarkably restricted, or the cells may be killed in some cases.
On the other hand, it is conceivable to use a large amount of culture solution or shorten the exchange cycle of the culture solution. However, according to such a method, a large apparatus is required or contamination with microorganisms occurs. There was a problem that the probability became much higher.

In addition, cells seeded on a three-dimensional substrate often adhere to the surface of the substrate with the best environment and proliferate, and then secrete an extracellular matrix such as collagen to cover the surface of the three-dimensional substrate. . In the three-dimensional base material covered with the extracellular matrix in this manner, the culture solution is difficult to be supplied to the inside of the three-dimensional base material, so that the cells remain only on the surface of the base material and remain inside the base material. The cells that had penetrated uniformly to the inside of the three-dimensional substrate at the seeding stage could not receive nutrients and oxygen from the medium and could not grow sufficiently, so expected. There was a problem that high-density culture was not possible.

Furthermore, when manufacturing a biological tissue-like structure for use in regenerative medicine in particular, it is necessary to culture cells on a three-dimensional substrate of various shapes and sizes depending on the site and purpose of transplantation. At present, there is only a cell culture device capable of culturing uniform shapes and sizes. Therefore, when cells are cultured on three-dimensional substrates having different shapes and sizes, it is necessary to newly manufacture a cell culture device for each three-dimensional substrate, and the cost performance is extremely poor. It was.

An object of the present invention is to provide a cell culture apparatus capable of culturing cells seeded on a three-dimensional base material of various shapes and sizes with high efficiency and high density in view of the above-mentioned present situation. And

The present invention relates to a cell culture apparatus for culturing cells seeded on a three-dimensional substrate using a culture medium circulating in the system, means for supporting the three-dimensional substrate to which the cells are adhered, and circulating the culture liquid A means for defoaming bubbles in the culture solution, a means for adjusting the pH, dissolved oxygen concentration and temperature of the culture solution, and means for supporting the three-dimensional substrate to which the cells are attached A cell culture device in which a plurality of units can be attached and detached.
The present invention is described in detail below.

The cell culture apparatus of the present invention comprises at least means for supporting a three-dimensional substrate to which cells are adhered (hereinafter also referred to as substrate supporting means), means for circulating a culture solution (hereinafter also referred to as circulation means), culture solution Means for defoaming the bubbles therein (hereinafter also referred to as defoaming means) and means for adjusting the pH, dissolved oxygen concentration and temperature of the culture solution (hereinafter also referred to as culture solution adjusting means).

The substrate supporting means is not particularly limited as long as the three-dimensional substrate can be held so as not to lose its shape, but is substantially the same in shape and size as the three-dimensional substrate seeded with the target cells. What has the structure where the culture solution inflow port and the culture solution outflow part are arrange | positioned at the both ends which pinched | interposed the space | gap part with the space | gap part is preferable. If the shape and size of the gap between the substrate holding means and the three-dimensional substrate are not the same, the culture solution will selectively flow through the gap formed between the substrate holding means and the three-dimensional substrate. The culture solution may not circulate sufficiently in the three-dimensional substrate.
A schematic diagram showing a cross section of one embodiment of such a substrate supporting means is shown in FIG.

The substrate supporting means 1 shown in FIG. 1 has a gap 2 for arranging a three-dimensional substrate, and a culture solution inlet 3 and a culture solution outlet 4 are arranged on both sides of the gap 2, respectively. It is a holder-like thing. The base material carrying means shown in FIG. 1 has a structure that is fitted by a screw structure or the like, and a three-dimensional base material seeded with cells can be disposed in the gap portion 2 by opening and closing this. Further, in the gap 2, the three-dimensional substrate may be sandwiched between the two support filters 11 via the spacer 10 so that the arranged three-dimensional substrate does not lose its shape.
In the base material supporting means 1 shown in FIG. 1, if the shape and size of the gap 2 are the same as the target three-dimensional base material, the culture solution flowing in from the culture solution inlet 3 is forced to be tertiary. It passes through the inside of the original substrate and flows out from the culture solution outlet 4. Therefore, since the culture solution reliably circulates to the inside of the three-dimensional substrate, cells can be grown with high efficiency and high density.

In the cell culture apparatus of the present invention, the substrate supporting means may be arranged in any direction, but is arranged so that the culture solution inlet is at the bottom and the culture solution outlet is at the top. It is preferable. When it arrange | positions in this way, a culture solution can be made to pass through the inside of a three-dimensional base material more reliably.

In the cell culture apparatus of the present invention, the substrate supporting means is unitized, and a plurality of units can be attached and detached. Thereby, the cell adhere | attached on the several three-dimensional base material from which a shape and a magnitude | size differ can be culture | cultivated simultaneously with one apparatus. Even when cells are cultured on a specially-shaped three-dimensional substrate, if only the substrate supporting means is produced, it can be immediately cultured with the cell culture apparatus of the present invention, and the cost performance is extremely excellent.
A schematic diagram showing an example of the unitized substrate supporting means is shown in FIG.
The unit 5 shown in FIG. 2 includes a defoaming means 6 described later in addition to the base material carrying means 1. The culture solution is defoamed through the defoaming means 6 and then flows into the base material supporting means 1 through the culture solution inlet 3 and passes through the three-dimensional base material seeded with the cells placed in the gap 2. After that, it flows out from the culture solution outlet 4. Since the unit 5 shown in FIG. 2 can be mounted on the cell culture apparatus in a sliding manner, the mounting operation is extremely easy.
The cell culture device of the present invention can attach and detach a plurality of such units.

The circulating means is not particularly limited as long as it can circulate a certain amount of culture broth accurately, but a tube pump is suitable because it can circulate a low flow rate culture broth stably. As the tube pump, a commercially available one can be used. In addition, a programmable pump or a double plunger type pump can also be used to circulate the culture solution in a regular fluctuating flow such as a pulsating flow.
In addition, when circulating a culture solution using a tube pump, it is preferable that each tube is connected to the said base material carrying means 1 each. Thereby, the flow volume of the culture solution supplied to the said base material support means can be controlled more correctly.

The defoaming means is not particularly limited, and a conventionally known method can be used. For example, the culture solution is introduced into a liquid reservoir portion having an appropriate volume, and bubbles accumulated in the upper portion of the liquid reservoir are released to the outside. The method is simple and preferable.

The culture medium adjusting means is not particularly limited, but for example, means for supplying a gas whose carbon dioxide and oxygen concentrations are adjusted is preferable. Usually, since the culture solution is a buffer solution having a buffer function, even if the pH of the culture solution fluctuates due to waste products secreted by the cells, or oxygen in the culture solution is consumed by the cells The pH can be easily adjusted to a physiological pH by supplying carbon dioxide and a gas whose oxygen concentration is adjusted, and oxygen can be dissolved. In particular, it is preferable to use a humidified gas as the gas whose carbon dioxide and oxygen concentrations are adjusted, because evaporation of moisture from the culture solution can be suppressed.
In addition, as a means of adjusting the temperature of the culture solution, only the culture solution may be heated using a heater or the like. However, since the device itself can be made extremely compact, the entire device should be installed in a commercially available incubator. You may adjust temperature by.

When the culture medium adjusting means is a means for supplying a gas with adjusted carbon dioxide and oxygen concentration, the carbon dioxide and oxygen concentration may be directly adjusted and the humidified gas may be blown into the circulating culture medium. A large amount of bubbles may be generated in the culture solution. Therefore, if the cell culture apparatus of the present invention is provided with a culture solution storage tank, and a gas with adjusted carbon dioxide and oxygen concentration is blown into the gas phase of the culture solution storage tank, the gas-liquid interface of the culture solution It is preferable because the pH of the culture solution can be adjusted without generating gas bubbles by exchanging gas.

The cell culture device of the present invention further includes a flow meter for measuring the flow rate of the culture solution, a filter for removing solid waste in the culture solution, a collecting pipe and a distribution pipe for controlling the flow of the culture solution; Sensor for monitoring pH, dissolved oxygen concentration, temperature, etc .; Preliminary culture fluid storage tank for easy exchange of culture fluid and its flow path switching valve, culture fluid sampling port, substrate support means during operation You may have a bypass switching bubble etc. for exchange.

A conceptual diagram of the cell culture apparatus of the present invention is shown in FIG.
In the cell culture apparatus shown in FIG. 3, after the culture solution stored in the culture solution storage tank 8 is infused by the tube pump 7 and passed through the defoaming means 6, the cells in the substrate supporting means 1 are seeded. Supplied to a three-dimensional substrate. The culture fluid that has flowed out of the substrate supporting means 1 is returned again to the culture fluid storage tank 8. In the culture solution storage tank 8, carbon dioxide-containing gas is blown to adjust the pH of the culture solution that has fluctuated due to wastes and the like.

A schematic diagram showing one embodiment of the cell culture device of the present invention is shown in FIG.
In the cell culture apparatus of the present invention shown in FIG. 4, six units 5 each having a substrate carrying means and a defoaming means (filter) are mounted, and a culture solution is circulated by a tube pump 7. Since the entire cell culture apparatus is installed in the incubator 9, the entire temperature can be controlled very accurately.

According to the cell culture device of the present invention, cells seeded on a three-dimensional substrate can be cultured with extremely high efficiency and high density. Moreover, the cell seed | inoculated on the three-dimensional base material which has various shapes and magnitude | sizes can be culture | cultivated only by preparing a base material support means according to the shape and size of the three-dimensional base material to be used. Furthermore, the cells seeded on different three-dimensional substrates such as a plurality of shapes can be freely cultured simply by detaching the unitized substrate supporting means.
The cell culture device of the present invention can be sterilized or the like easily because it can be autoclaved as a whole or for each unit. Furthermore, the entire apparatus can be collected very compactly.

ADVANTAGE OF THE INVENTION According to this invention, the cell culture apparatus which can culture | cultivate the cell seed | inoculated on the three-dimensional base material of various shapes and sizes with high efficiency and high density can be provided.

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

Example 1
As shown in FIG. 2, the base material supporting means 1 as shown in FIG. 1 is prepared so that the gap 2 has a disk shape with a diameter of 15 mm and a thickness of 2 mm, and this is combined with a support filter with a hole diameter of 320 μm. 4 was prepared, and the cell culture apparatus shown in FIG. The entire cell culture apparatus was placed in an incubator and kept at a temperature of 37 ° C.

A non-woven fabric made of polyglycolic acid (PGA) having a fiber diameter of 12 μm is processed into a disk shape having a diameter of 15 mm and a thickness of 2 mm, and this is used as a three-dimensional culture substrate to shake about 880000 cells of rat undifferentiated mesenchymal stem cells. (12 hours). Five three-dimensional substrates seeded with such cells were prepared.
The cells were completely adhered to the substrate by the shaking seeding method, and then each cell was placed in the gap 2 of the substrate supporting means 1 and used for the cell culture apparatus.

A tertiary solution in which 125 mL of the culture solution (α-MEM medium (manufactured by Invitrogen) + 15% fetal calf serum (manufactured by Hyclone Laboratories)) stored in the culture solution storage tank 8 is divided by the infusion pump 7 and seeded with cells. The culture was carried out for 3 days while supplying the original substrate.

After completion of the culture, each three-dimensional substrate was taken out, and the number of cells in the three-dimensional substrate was measured for the four by a DNA quantification method. The results are shown in Table 1.
In addition, after fixing one of the three-dimensional substrates with formalin, a section was prepared by embedding in paraffin and cutting the central part. A hematoxylin-eosin stained image of this section is shown in FIG. 5a. From FIG. 5a, it was found that the cells were uniformly proliferating to the inside of the three-dimensional substrate.

(Comparative Example 1)
Five three-dimensional substrates (PGA non-woven fabrics) seeded with rat bone marrow undifferentiated mesenchymal stem cells were prepared in the same manner as in Example 1.
These three-dimensional substrates were individually placed in a 100 mm dish for cell culture containing 25 mL of a culture solution (α-MEM medium (manufactured by Invitrogen) + 15% fetal calf serum (manufactured by High Clone Laboratories)), and 5% The culture was stationary for 3 days in an incubator adjusted to CO ₂ and 37 ° C.

After completion of the culture, each three-dimensional substrate was taken out, and the number of cells in the three-dimensional substrate was measured for the four by a DNA quantification method. The results are shown in Table 1.
In addition, after fixing one of the three-dimensional substrates with formalin, a section was prepared by embedding in paraffin and cutting the central part. A hematoxylin-eosin stained image of this section is shown in FIG. 5b. From FIG. 5b, it was found that the cells grew only on the surface of the three-dimensional substrate and hardly penetrated into the substrate.

ADVANTAGE OF THE INVENTION According to this invention, the cell culture apparatus which can culture | cultivate the cell seed | inoculated on the three-dimensional base material of various shapes and sizes with high efficiency and high density can be provided.

It is a schematic diagram which shows the cross section of 1 aspect of a base material support means. It is a schematic diagram which shows an example of the base-material support means unitized. It is a conceptual diagram of the cell culture apparatus of this invention. It is a schematic diagram which shows one embodiment of the cell culture apparatus of this invention. It is the HE dyeing | staining image of the cross section of the three-dimensional base material which seed | inoculated the cell obtained in Example 1 (a) and Comparative Example 1 (b).

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Base material support means 2 Cavity part 3 Culture solution inlet 4 Culture solution outlet 5 Unit 6 Defoaming means 7 Tube pump 8 Culture solution storage tank 9 Incubator 10 Spacer 11 Support filter

Claims (2)

A cell culture device for culturing cells seeded on a three-dimensional substrate using a culture medium circulating in the system,
Means for supporting a three-dimensional substrate to which cells are adhered, means for circulating the culture solution, means for defoaming bubbles in the culture solution, and means for adjusting the pH, dissolved oxygen concentration and temperature of the culture solution ,
The means for supporting the three-dimensional substrate to which the cells are attached is unitized, and a plurality of units can be attached and detached, and
The means for supporting the three-dimensional substrate to which the cells are adhered is disposed at both ends with the gap portion having substantially the same shape and size as the three-dimensional substrate to which the target cell is adhered, sandwiching the gap portion. culture fluid inlet and the culture liquid outlet portion and a cell culture apparatus, comprising a. PH of the culture medium, means for adjusting the dissolved oxygen concentration, carbon dioxide and oxygen concentration is adjusted, according to claim 1 Symbol placement of the cell culture apparatus characterized in that it is a means for supplying a humidified gas.

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