JP2019154277A - Cell culture vessel - Google Patents

Abstract

To provide a cell culture vessel that performs efficiently culture, clean, and recover cells.SOLUTION: A cell culture vessel contains a porous body having one or more through holes in longitudinal direction.SELECTED DRAWING: None

Description

  The present invention relates to a cell culture container including a porous body having one or more through holes.

  In recent years, cancer immunotherapy has attracted attention as a fourth cancer treatment method after surgery, chemotherapy, and radiation therapy. Cancer immunotherapy is a general term for cancer treatments that utilize the power of immunity inherently in humans, and one of them is immune cell therapy. This is because immune system cells such as lymphocytes in the blood of cancer patients and dendritic cells that present antigens of cancer cells to lymphocytes are taken out of the body, cultured and strengthened, and then returned to the body. It is a way to try to treat cancer by increasing In recent years, with the advent of immune checkpoint inhibitors, it can be said that the importance of immune cell therapy for cancer has increased again.

  Currently, cells used for immune cell therapy, such as T lymphocytes, are cultured in flasks or culture bags that simulate flasks. Cell culture using flasks and culture bags is simple and easy to operate, but requires a large area and volume, requires a large amount of culture space, and increases the size of automatic culture devices. There is a problem such as.

  Patent Document 1 discloses a hollow fiber membrane type bioreactor for cell proliferation and growth, which is a flow for circulating different media in a cell space (hollow fiber hollow part) and a cell-free space (hollow fiber outer side). A bioreactor system having a channel is disclosed. However, in order to modularize the hollow fiber membrane, it is necessary to go through complicated steps, and it is difficult to simplify and reduce the cost.

Special table 2003-510068 gazette

  An object of the present invention is to provide a cell culture container capable of efficiently culturing, washing, and collecting cells.

  As a result of intensive studies to solve the above problems, the present inventor has found that the above problems can be solved by the following means, and has reached the present invention.

That is, this invention consists of the following structures.
1. A cell culture vessel comprising a porous body having one or more through-holes in the longitudinal direction.
2. 2. The cell culture container according to 1, wherein the porous body is made of a material selected from the group consisting of aluminum oxide, titanium oxide, and zirconium oxide.
3. 3. The cell culture container according to 1 or 2, wherein the porous body is a multi-capillary filter.
4). The cell culture container according to any one of 1 to 3, wherein the porous body has a separation layer having a pore diameter of 0.1 μm to 1.5 μm.

  According to the present invention, efficient cell culture and simple removal of a culture solution are possible, and therefore, application to immune cell therapy can be expected.

It is a schematic diagram which shows an example of the porous body which has a through-hole (capillary). It is a schematic diagram which shows the structure around the through-hole of a porous body. It is a schematic diagram which shows an example of a cell culture container. It is a schematic diagram which shows an example of the cell culture apparatus using the cell culture container of this invention.

(Porous body)
In the present invention, the porous body used as the cell culture substrate is not particularly limited as long as the cells can be held in the lumen (capillary) of the porous body and can penetrate a solution or a low-molecular substance. Examples thereof include ceramics such as titanium oxide and zirconium oxide, 2-hydroxyethyl methacrylate, polyurethane, polymethyl methacrylate, polylactic acid, polyhydroxyalkanoate, polyacrylonitrile, polyvinylidene fluoride, silk fibroin and the like. . These derivatives may be the main component. Further, the liquid-permeable porous material may be a material obtained by chemically modifying these materials, for example, a material subjected to a hydrophilic treatment. By performing the hydrophilic treatment, it becomes easy to supply a liquid component such as a culture solution to the cultured cells. Examples of the method for hydrophilizing the surface of the porous body include a method of treating the porous body with a hydrophilic polymer such as polyvinyl pyrrolidone or ethylene-vinyl alcohol copolymer, glycerin or ethanol. In addition, a coating agent such as collagen or fibronectin may be used in order to give light adhesion to the porous body of cells depending on the application.

  In the present invention, the porous body is preferably cylindrical, but may be in the shape of an elliptical cylinder or a polygonal cylinder. The porous body is preferably a multi-capillary type having a plurality of through holes. The cross-sectional shape of the through hole is not particularly limited, and for example, porous bodies having various cross-sectional shapes as shown in Table 1 are available, and any of them may be used. Moreover, the diameter of each through-hole should just ensure the space where the movement of the cell aggregate to culture | cultivate is performed freely, and should just be 1 mm-6 mm. Table 1 shows a schematic cross-sectional view of a multi-capillary porous body having an outer diameter of 25 mmφ. For example, (a) in Table 1 has 7 cylindrical through-holes substantially parallel to the longitudinal direction. Each of the porous bodies is provided, and the average diameter of the through holes is about 6 mm. Moreover, (g) of Table 1 is a porous body provided with 93 through-holes substantially parallel to the longitudinal direction, and its average diameter is about 1.6 mm. The porous body preferably has an outer diameter of 10 mm to 50 mm and a length of 50 mm to 300 mm from the viewpoint of handleability.

  In the present invention, the porous body preferably has a structure composed of a separation layer and a support layer, and may have an intermediate layer between the separation layer and the support layer. Further, the separation layer and the support layer may be made of the same material, or may be a combination of different materials. The separation layer preferably has a co-continuous structure with an average pore size of 0.1 μm to 1.5 μm. FIG. 1 is a schematic view showing an example of a cell culture substrate (porous body) used in the cell culture container of the present invention. 2 is a support layer, 3 is a through-hole, and 4 is a separation layer. FIG. 2 is an enlarged view of the periphery of the through hole. The support layer shown in the figure is made of titanium oxide (titania) and has a co-continuous structure with a pore diameter of 4.5 μm. The separation layer is made of titanium oxide (titania) + zirconium oxide (zirconia) and has a co-continuous structure with a pore diameter of 0.1 μm. By using a porous body having such a co-continuous structure, that is, a structure that does not have voids (bubbles) that do not penetrate in the cross-sectional direction of the porous body as a cell culture substrate, Substance exchange such as cell metabolites (waste products) can be performed more smoothly. Further, by providing a separation layer having a relatively small pore diameter on the through-hole side, it is possible to prevent cells from dropping into the pores and damaging the cells when culturing the cells in the through-holes.

(Cell culture vessel)
In the present invention, the cell culture container may be of a configuration in which the inside is partitioned into a first chamber and a second chamber by a porous body. For example, the cell culture vessel has a porous structure having a through-hole (capillary) as shown in FIG. An example is one in which the internal space of the container 6 is partitioned by the material, and one space constitutes the first chamber and the other space constitutes the second chamber. In this case, the through hole of the porous body 1 may be the first chamber, the external space of the porous body may be the second chamber, and conversely, the external space may be the first chamber and the through hole may be the second chamber. . In addition, when using the porous body which has a separation layer in the through-hole side, it is preferable to make a through-hole into a 1st chamber. In FIG. 3, the cell culture container is provided with inlets 7a and 8a and outlets 7b and 8b communicating with the first chamber and the second chamber, respectively.

  When the cell culture vessel is used, for example, when culturing cells in the through-hole, the cells are seeded by injecting a cell suspension from the end conduit. After cell seeding, culture is preferably performed by introducing a medium from the side conduit and perfusing. By using such a cell culture container, a fresh medium can be always supplied to the cells, and the replacement work required when using a petri dish or a flask as a culture substrate becomes unnecessary, and the operator's restraint time is reduced. Can be reduced.

(Cell culture device)
FIG. 4 shows an example of a cell culture apparatus using the cell culture container of the present invention. FIG. 4 exemplifies a cell culture container that houses the multi-capillary porous body shown in FIG. Into the introduction port 7a communicating with the through hole (first chamber) of the cell culture container, the cell washing solution is supplied from the introduction port 40 and the cell washing solution container 10 through the flow path for feeding a cell suspension containing floating cells. A flow path for feeding liquid is connected. A valve 20 is provided in the middle of the flow path so that the flow path of the cell suspension and the cell washing solution can be switched. In addition, a flow path for discharging the cell recovery liquid after culturing is connected to the discharge port 7b communicating with the through hole (first chamber) of the cell culture container, and the flow rate is adjusted in the middle of the flow path. And a valve 22 for switching the flow path to the liquid delivery pump 31, the cell collection container 12, or the discharge port 50. On the other hand, a flow path for feeding the culture solution from the culture solution storage container 9 to the outside of the porous body (second chamber) is introduced into the introduction port 8a communicating with the outside of the porous body of the cell culture vessel (second chamber). Is connected. The discharge port 8b communicating with the second chamber is connected to a flow path for discharging the cell culture solution or the cell washing solution, and a liquid feed pump 30 is provided in the middle of the flow path. The collected culture solution or washing solution is connected to a collection container 11 for collecting.

(Cells to be cultured)
In the present invention, the cells to be cultured are not particularly limited, but floating / non-adhesive animal cells are preferable. The origin of the cells is not particularly limited, and those derived from any animal such as humans, pigs, dogs and mice can be used. In addition, floating animal cells can target both primary cultured cells and established cells. Stem cells such as blood stem cells, progenitor cells, blood cell T lymphocytes, natural killer cells, and the like may be used. In addition, these cells may be cells into which a foreign gene has been introduced before culturing, or may be cells that have been previously stimulated and processed with stimulating factors such as antibodies and ligands.

(Cell culture)
When culturing cells, the cell suspension filled in the through-hole (first chamber) of the cell culture container is brought into contact with the cell culture fluid flowing outside the porous body (second chamber) via the porous body. By using the diffusion phenomenon, the culture solution components are transferred (permeated) from the cell culture solution to the first chamber, and cell metabolites (waste products) accompanying the culture are transferred (permeated) to the second chamber. The cells are cultured while preparing the culture environment.

Referring to FIG. 4, cell suspension in which cells are suspended is introduced from inlet 40 with valve 21 closed, and the cell suspension is filled into the through-hole (first chamber). After the cell suspension is filled, the valve 20 is closed. When the cell suspension is filled into the through-hole (first chamber), the pump 30 is started at the same time or before and after, and the culture medium is cultured from the culture solution storage container 9 toward the collection container 11 via the porous exterior (second chamber). Pump the liquid. At this time, the flow rate of the culture solution is preferably adjusted according to the degree of cell growth and the environment. Moreover, it is preferable to install at least the cell culture container, the culture solution storage container, and the flow path connecting them in an incubator equipped with a temperature and CO ₂ concentration control mechanism.

In the present invention, the cell suspension refers to a suspension of suspension cells in a cell culture solution so as to be 1 × 10 ⁵ to 1 × 10 ⁷ cells / mL.

(Cell culture medium)
In the present invention, the culture medium (medium) is a so-called basal medium for growing and proliferating cells and containing amino acids, vitamins, inorganic salts, sugars and the like as nutrients. Examples of such a medium include Minimum Essential Medium (MEM), Basal Medium Eagle (BME), Media199, Dulbecco's Modified Eagle Medium (D-MEM), α-Minimum Essential Medium (α-MEM), and Ham's F-10. Nutrient Mixture (Ham's F-10), Ham's F-12 Nutrient Mixture (Ham's F-12), RPMI 1640, L-15, Iscove's Modified Dulbecco's Medium (IMDM), ES medium, MCDB 131 Medium, CMRL 1066 Media, DM- 160 Medium, Fisher's Medium, StemSpan Medium, StemPro Medium, Hybridoma Serum Free Medium (Hybridoma SFM), mTeSR1 (modified Tenneille Serum Replacer 1), Essential 8, Repro FF / FF2 / XF, StemSure (registered trademark), CELRENA, S- Examples include, but are not limited to, commercially available cell culture media such as Medium and serum-free culture media for human lymphocytes, and mixtures thereof.

(Floating cell washing process)
The floating cell washing step is a step of washing with a cell washing solution in a state where the floating cells after culturing are held in the through hole (first chamber).

  Referring to FIG. 4, after culturing is completed, valve 23 is closed, and then valve 23 is operated so that the cell washing solution can be sent from cell washing solution container 10 to the through-hole (first chamber) of the cell culture vessel. Then, the pump 30 is started. Since the porous body 1 has a pore size that allows the culture solution components and cell metabolites (waste products) to permeate but does not permeate the cells, the culture solution components and metabolites (waste products) pass through the porous body together with the washing solution. (Filtered) and collected in the collection container 11. Note that if the filtration pressure is too high, the cells may be damaged, so the pump output must be adjusted accordingly.

  In the present invention, it is preferable to use physiological saline or phosphate buffered saline (PBS) as the cell washing solution.

(Recovery of cells)
For cell collection, the cultured cells that have been washed are discharged from the through-hole (first chamber) together with the washing solution and the culture solution and collected in the cell collection container 12.

  Referring to FIG. 4, after the cells are washed, in order to collect the cells into the cell collection container 12 from the through-hole (first chamber) of the cell culture container, the valves 21 and 22 are operated to operate the flow path. To communicate. Thereafter, the cleaning liquid containing cells can be collected in the collection container 12 by starting the pump 31. In the case where the cells are adherent cells, after the perfusion of the culture solution is stopped or after the washing of the cells is completed, a divalent cation-free phosphate buffered saline (PBS) is perfused for a certain period of time. Next, PBS is removed, protease such as trypsin is filled outside the through-hole and the porous body, and incubated for a certain time. After the cells are detached from the porous body, the cell suspension is collected in the collection container 12.

(Flow rate of culture solution)
In the present invention, the culture solution may flow continuously or intermittently. However, if the flow rate is too small, the nutrient supply to the cells is not sufficient, and the cells are difficult to grow. On the other hand, even if the flow rate is too high, the environmental change around the cell is drastic, the cell is not adapted to the surrounding environment, and the cell is difficult to proliferate. Thus, it is preferable to adjust the flow rate of the culture solution flowing or circulating in the cell culture vessel according to the degree of cell growth and the environment. The method for examining the degree of cell proliferation is not particularly limited, but can be performed based on the measurement results such as the concentration of glucose and lactate in the culture solution.

  Hereinafter, the effectiveness of the present invention will be described with reference to examples, but the present invention is not limited thereto. The following describes a typical implementation of the cell culture method of the present invention.

[Example 1]
(Porous material having through holes)
As a porous body having through holes, a ceramic filter was prepared as follows. That is, the porous body of (a) shown in Table 1 (Techno Alpha Co., Ltd., outer diameter 25 mm, number of through-holes 7, through-hole diameter 6 mm, through-hole volume 49.5 cm ³ , support layer: titania, separation layer: titania + zirconia ) Was cut to a length of 250 mm, thoroughly washed with ultrapure water, and then sterilized in an autoclave. The pore size of the separation layer was 0.1 μm, and the pore size of the support layer was 4.5 μm.

(Preparation of cell culture container)
A test cell culture vessel was prepared as follows. The porous body is placed in a cylindrical polycarbonate case having an inner diameter of 34 mm and a length of 254 mm, and both ends are fixed to the end of the case so as not to block the through holes. A cell culture vessel was prepared.

(Separation of lymphocytes)
Human blood was collected in a tube containing heparin, and 3 ml of a blood solution prepared by mixing this with an equal volume of PBS was dispensed into a 15 ml centrifuge tube. Next, 6 ml of a density gradient separation medium (Lymphoryte (registered trademark), Cosmo Bio, model number: CL5010) was gently layered on the dispensed blood solution, taking care not to disturb the liquid level. A lymphocyte layer formed by centrifugation at 800 × g for 20 minutes was collected and suspended in PBS. Again, centrifugation was performed at 800 × g for 20 minutes to obtain a lymphocyte pellet. The above operation was repeated to obtain about 2.0 × 10 ⁷ lymphocytes.

(Cell culture experiment)
The cell culture experiment was performed using the cell culture apparatus shown in FIG. In the cell culture container, the through hole of the porous body was the first chamber, and the outside of the porous body was the second chamber. A cell suspension obtained by suspending 3 × 10 ⁶ of the obtained lymphocytes in 100 mL of a serum-free culture solution for human lymphocytes (Cell Science Laboratory Co., Ltd., code: 1020P10) is introduced from the inlet 40 of the cell culture apparatus. The solution was fed and filled into the first chamber of the porous body. After filling, the valves 20 and 21 were closed, the pump 30 was started, and the culture solution was allowed to flow outside the porous body (second chamber), and then cultured at 37 ° C. for 4 days in a CO ₂ incubator. The flow rate of the culture solution was 0.5 mL / min.

(Cell washing)
After culturing for 4 days, the culture medium perfusion was stopped, the valve 20 was switched, and the cells were washed using phosphate buffered saline (PBS) as a cell washing solution.

(Recovery of cells)
After washing the cultured cells, the pump 30 was stopped and the valve 20 was closed. Subsequently, after switching the valves 21 and 22 to connect the discharge port 7b of the cell culture container 5 and the cell collection container 12, the pump 31 was started and the cultured cells were collected in the cell collection container 12 together with the cell culture solution. . The number of cells was counted for the collected cells. The results are shown in Table 2.

(Measurement of cell recovery)
The collected liquid containing the cells from the cell culture container was finally suspended in 10 ml of the culture liquid by centrifugation. A solution obtained by mixing the suspension and trypan blue staining solution at 1: 1 was added to a hemocytometer, and the number of cells (cells) and cell viability (%) were measured under a microscope according to the following procedure.
1. The surface of the hemocytometer and cover glass was washed with 70% isopropanol, and excess isopropanol was wiped off and air-dried.
2. The side surface of the cover glass was wetted with a reagent grade water and attached to a hemocytometer.
3. The cell suspension was thoroughly stirred with a Pasteur pipette or the like, and immediately poured into a hemocytometer to fill the groove.
4.1 to 3 were performed using another hemocytometer (measured twice and averaged).
5). A hemocytometer was placed on the microscope and focused on the grid lines (10 × objective).
6). Using a counter, the number of cells in 1 mm ² area was quickly measured.
* Since errors are likely to occur, at least 100-500 cells are counted for accurate counting.
Calculation method:
C = N × 10 ⁴
C: Number of cells per ml N: Average number of measured cells 10 ⁴ : Conversion value of volume for 1 mm ² Total number = C × V
V = volume of the liquid in which the cells are suspended

[Example 2]
Cell culture was performed in the same manner as in Example 1 except that (d) in Table 1 was used as the porous body. The results are shown in Table 2. The pore size of the separation layer was 0.8 μm, and the pore size of the support layer was 4.5 μm.

[Example 3]
Cell culture was performed in the same manner as in Example 1 except that (g) in Table 1 was used as the porous body. The results are shown in Table 2. The pore size of the separation layer was 1.4 μm, and the pore size of the support layer was 4.5 μm.

[Comparative Example 1]
Five T-225 flasks (cell culture area: 225 cm ² ) were seeded with 0.6 × 10 ⁶ human lymphocytes per flask (total of 3.0 × 10 ⁶ ), and for human lymphocytes as in Example 1. Using a serum-free culture solution (Cell Science Laboratory Co., Ltd., code: 1020P10), the cells were cultured at 37 ° C. for 4 days in a CO ₂ incubator. After completion of the culture, cells were collected from all flasks together with the culture solution. The collected cells were dispensed into a 50 ml centrifuge tube and centrifuged at 1000 rpm for 10 minutes, and then the supernatant culture was aspirated and the cells were suspended in 20 ml of PBS. This operation was repeated 4 times in total and finally suspended in 10 ml of PBS, and the number of cells and the cell viability were measured.

  For the examples and comparative examples, the same experiment was repeated 8 times, and the number of recovered cells and the cell viability were determined (Table 1). As a result, the Example showed good results in both the number of recovered cells and the cell viability. On the other hand, in the comparative example, both the number of recovered cells and the cell viability were lower than in the example. Moreover, the error between experiments was large, and it was found that there was a problem in reproducibility. From this, it can be said that the present invention can provide a cell culture vessel that can not only cultivate cells efficiently at high density, but also does not damage the cells and causes little loss of cell recovery.

* Number of cells (× 10 ⁶ )

  The present invention enables efficient suspension cell culture and simple removal of the culture solution, and can be expected to be applied to immune cell therapy.

1 Porous body 2 Through hole (capillary)
3 Support layer 4 Separation layer 5 Cell culture container 6 Cylindrical containers 7a, 8a Inlet 7b, 8b Discharge port 9 Culture liquid container 10 Washing liquid container 11 Recovery container 12 Cell recovery container 20, 21, 22, 23 Valve 30, 31 Liquid pump

Claims (4)

  A cell culture vessel comprising a porous body having one or more through-holes in the longitudinal direction.   The cell culture container according to claim 1, wherein the porous body is made of a material selected from the group consisting of aluminum oxide, titanium oxide, and zirconium oxide.   The cell culture container according to claim 1 or 2, wherein the porous body is a multi-capillary filter.   The cell culture container according to claim 1, wherein the porous body has a pore diameter of 0.1 μm to 1.5 μm.