Novel cell culturing method

Abstract

In cell culture in the present invention, cells adhered to the bottom surface of a first culture substrate having a bottom surface that has been treated for cell adhesion are brought into contact with a microcarrier.

Description

New cell culture method

[0001] The present disclosure relates to novel cell culture methods.

[0002] Efficient mass cultivation of cells and tissues is required in fields such as pharmaceutical production, gene therapy, regenerative medicine, and immunotherapy. Microcarrier culture is a well-known mass-cultivation technique for cells and other tissues. Microcarrier culture involves, for example, introducing cells, a culture medium, and microcarriers that serve as a scaffold for cell adhesion into a culture vessel, intermittently stirring the culture medium to suspend the cells and microcarriers, and allowing the suspended cells to adhere to and grow on the surface of the microcarriers as they descend and come into contact with the microcarriers (see, for example, Patent Document 1). Microcarriers are advantageous for mass cultivation of cells because they can provide a very large adhesion and growth surface area relative to their volume.

[0003] Various culture methods using microcarriers have been known. For example, a method for producing adherent cells is known, which involves placing adherent cells in a culture vessel containing a culture medium and microcarriers and performing several serial cell passages in the same vessel. In this method, all or part of the cell population from the previous generation is used to passage the cells to the next generation (see, for example, Patent Document 2). Another known method involves culturing cells in a culture medium containing adherent cells, microcarriers, and a culture medium while gradually scaling up the culture medium. In this method, cells adhered to the microcarriers are migrated to and attached to another fresh microcarrier (see, for example, Patent Document 3). However, all of these culture methods require a treatment step using a cell-dissociating enzyme such as trypsin, which reduces the efficiency of cell culture. Furthermore, conventionally, when adherent cells are passaged from a culture substrate such as a petri dish or flask, many steps are required, such as removal of the culture medium, washing with a buffer solution, removal of the buffer solution, addition of a cell-dissociating enzyme, cell detachment by incubation, complete cell detachment by tapping or pipetting, inactivation of the cell-dissociating enzyme by adding culture medium, transfer of the cells to a centrifuge tube, separation of the cells from the culture medium by centrifugation, removal of the culture medium, resuspension in culture medium, preparation of a destination culture substrate, and seeding of the cells onto the destination culture substrate. This requires a great deal of time and effort for cell passage, and these steps are difficult to perform in a closed system. In particular, when cells are transferred from the bottom of a culture substrate such as a petri dish or flask to microcarriers, the aforementioned "preparation of the destination culture substrate" requires steps of suspending and / or swelling microcarriers in culture medium and adding the suspended and / or swollen microcarriers and culture medium to the culture substrate, which requires additional time and effort.

[0004] Furthermore, when cells are passaged by transferring them from the bottom of a culture substrate, such as a petri dish or flask, to a microcarrier, the cells are typically detached and collected from the bottom of the culture substrate using a cell-dissociating enzyme, then placed together with the microcarriers in another culture substrate, such as a bioreactor, where they are cultured together with the microcarriers in a free (non-adherent) state and then continuously or intermittently stirred to adhere to the microcarriers. However, adhesion between the free cells and the microcarriers requires contact between the cells and the microcarriers, which move relative to each other in the culture medium. Determining culture conditions that ensure adequate contact requires considerable effort. In particular, efficient cell culture on the surface of microcarriers requires that cells adhere as evenly as possible to the microcarrier surface. Therefore, determining culture conditions that ensure adequate contact is challenging, resulting in a problem of impaired cell culture efficiency.

[0005] International Publication No. 2010 / 138702 Special Publication No. 2013-515473 International Publication No. WO2021 / 181819 Pamphlet

[0006] Under these circumstances, there is a need for more efficient cell culture methods.

[0007] The present inventors have found that in cell culture, by carrying out a contact step in which cells adhering to the bottom surface of a first culture substrate having a bottom surface treated for cell adhesion are brought into contact with microcarriers, cells can be transferred to microcarriers and easily passaged without treatment with a cell dissociation enzyme such as trypsin, thereby improving the efficiency of cell culture. The present disclosure is based on these findings.

[0008] That is, according to one aspect of the present disclosure, there is provided a method for culturing cells, comprising a contacting step of contacting cells adhering to the bottom surface of a first culture substrate having a bottom surface treated for cell adhesion with a microcarrier.

[0009] The culture method of the present disclosure is advantageous in that it can improve the efficiency of cell culture because cells that are adhered and fixed to the bottom surface of the culture substrate can be easily transferred to microcarriers for cell passage without treatment with a cell-dissociating enzyme such as trypsin.

[0010] FIG. 1A shows an image (plan view) observed with a phase-contrast microscope after contacting cells with microcarriers by adding the microcarriers to a first culture substrate with cells adhering to its bottom surface in the contacting step. FIG. 1B shows an image obtained by color-coding the image observed with a phase-contrast microscope into the microcarrier portion (black portion) and the bottom surface of the first culture substrate (gray portion). This shows a phase-contrast microscope image of a microcarrier for cell culture. This shows a phase-contrast microscope image of a dried microcarrier for cell culture. FIG. 4A shows a phase-contrast microscope image of the inside of a well immediately after the microcarriers were added. FIG. 4B shows a phase-contrast microscope image of a well (which contained the adhered microcarriers) immediately after the microcarriers with human adipose-derived stem cells adhered thereto were transferred to another well. FIG. 4C shows a fluorescence microscope image of the well to which the microcarriers with human adipose-derived stem cells adhered thereto were transferred. FIG. 5A shows a fluorescence microscope image of microcarriers collected from a culture medium containing microcarriers into which human adipose-derived stem cells were transferred. Figure 5B shows a fluorescence microscope image of microcarriers collected from a culture medium containing microcarriers onto which human adipose-derived stem cells had been migrated, after culturing. Figure 5C shows a fluorescence microscope image of microcarriers immediately after adding new swollen microcarriers to a culture medium containing microcarriers onto which human adipose-derived stem cells had been migrated. Figure 5D shows a fluorescence microscope image of microcarriers after adding new swollen microcarriers to a culture medium containing microcarriers onto which human adipose-derived stem cells had been migrated, followed by culturing. Note that the 200 μm scale on the left of Figure 5A corresponds to both Figures 5A and 5B. Furthermore, the 200 μm scale on the left of Figure 5C corresponds to both Figures 5C and 5D. Figure 6 shows a phase-contrast microscope image of microcarriers onto which ES cells had adhered. Figure 7A shows a phase-contrast microscope image of a well of a 24-well plate immediately after transferring the microcarriers onto which human adipose-derived stem cells had adhered, together with the culture medium, into the well of the plate coated with a 1 mg / g gelatin aqueous solution. FIG. 7B shows a phase-contrast microscope image of the bottom surface of a well of a 24-well plate immediately after the microcarriers were removed after human adipose-derived stem cells had been transferred from the microcarriers to the bottom surface of the wells.FIG. 7C shows human adipose-derived stem cells on the bottom of the wells of a 24-well plate after the microcarriers were removed, cultured at a temperature of 37° C. and CO. ２ Figure 7 shows a phase-contrast microscope image of the well bottom after one day of culture under conditions of a gelatin concentration of 5%. Figure 7D shows a phase-contrast microscope image of the well bottom immediately after transferring the removed microcarriers, together with the culture medium, to wells of a 24-well plate coated with a 1 mg / g gelatin aqueous solution. Figure 8 is a schematic diagram showing the behavior of cells and microcarriers over time after they are both placed on a culture substrate when cells are cultured using microcarriers. Figure 9 is a schematic diagram showing the behavior of cells and microcarriers over time after they are both placed on a culture substrate when cells are contacted with microcarriers using a culture substrate that is not cell-adhesive treated or has been treated for non-cell adhesion and has an uneven bottom. Figure 10 is a schematic diagram showing the behavior of cells and microcarriers over time after they are both placed on a culture substrate when cells are contacted with microcarriers using oblate spherical microcarriers. Figure 11 is a schematic diagram showing the behavior of cells and microcarriers over time after they are both placed into a culture substrate having a cell adhesion-treated, non-textured bottom when the cells and microcarriers are brought into contact with each other using a culture substrate having a cell adhesion-treated, non-textured bottom.

[0011] [Cell Culturing Method] According to one aspect of the present disclosure, a cell culturing method (also referred to herein as the "culturing method of the present disclosure") is provided, which includes a contacting step, as an essential step, as described below. In this disclosure, "culturing" cells encompasses not only dividing and growing cells, but also keeping cells alive (maintaining them) without substantial cell division and growth. The culturing method of the present disclosure is advantageous in that it can improve the efficiency of cell culture by easily transferring cells fixed in contact with the bottom surface of a culture substrate to a microcarrier for cell passage without treatment with a cell dissociation enzyme such as trypsin. Specifically, in cell culture, cells adhered to a culture substrate are typically collected using a cell dissociation enzyme such as trypsin, but such cell dissociation enzymes may damage the cells. For example, proteolytic enzymes may non-selectively remove receptors on the cell surface. Furthermore, in many cases, treatment with a cell dissociation enzyme requires a certain amount of time and centrifugation, which may cause cell damage, mutation, or reduce the cell yield. The culture method of the present disclosure can improve cell culture efficiency by reducing the time required for such cell-dissociating enzyme treatment and further suppressing a decrease in cell yield due to cell damage, mutation, etc.

[0014] Furthermore, the culture method of the present disclosure involves contacting cells adhered to the bottom surface of a culture substrate (i.e., immobilized) with microcarriers to transfer the cells to the microcarriers. Therefore, it is essentially unnecessary to consider the culture conditions required to ensure appropriate contact between free cells and microcarriers, which also provides the further advantage of improving cell culture efficiency. The culture method of the present disclosure also allows cells adhered to the bottom surface of a culture substrate to be contacted with microcarriers, allowing efficient cell transfer to the microcarriers. Conventionally, cell culture methods involving transferring cells from one carrier to another are known. This method requires the initial preparation of carriers bearing cells (i.e., carriers bearing cells that serve as the starting point for cell culture), but it is known that preparing such carriers bearing cells is time-consuming.The culture method of the present disclosure allows cells adhered to the bottom surface of a culture substrate to come into contact with the microcarriers, thereby efficiently transferring the cells to the microcarriers, and therefore allows efficient production of microcarriers to which such cells are adhered. Each step of the culture method of the present disclosure is described in detail below.

[0012] <Contacting Step> In the contacting step, cells adhering to the bottom surface of a first culture substrate having a bottom surface treated for cell adhesion are brought into contact with microcarriers. Specifically, the cells are brought into contact with the microcarriers by adding the microcarriers to the first culture substrate having cells adhering to the bottom surface. In the contacting step, it is believed that contact between the cells adhering to the bottom surface of the first culture substrate and the microcarriers causes the cells to migrate from the bottom surface of the first culture substrate to the microcarriers (particularly, to the surface of the microcarriers). This step of migrating cells from the bottom surface of the first culture substrate to the microcarriers (also referred to herein as the "migration step") may be performed in parallel with the contacting step or as a step independent of the contacting step. Note that in the present disclosure, "migration" of cells refers not only to the migration of a cell itself from one location to another, but also to the division and proliferation of a cell from one location to another. That is, "cells migrate from the bottom surface of the first culture substrate to the microcarrier" not only means that a cell itself migrates from the bottom surface of the first culture substrate to the microcarrier, but also means that a cell divides and proliferates from the bottom surface of the first culture substrate to the microcarrier. The reason why cells migrate to the microcarrier is unclear, but it is thought that cells migrate from a densely packed region (i.e., the bottom surface of the first culture substrate) to a sparsely packed region (i.e., the microcarrier) to avoid the densely packed region. In one embodiment, during the contacting step, cells not only migrate from the bottom surface of the first culture substrate to the microcarrier, but also divide and proliferate simultaneously, resulting in cell migration from a densely packed region (i.e., the bottom surface of the first culture substrate) to a sparsely packed region (i.e., the microcarrier). Thus, it is believed that the contact step can move cells from the bottom surface of the first culture substrate to the microcarriers. Therefore, it is believed that the period during which cells in the culture substrate become confluent can be controlled by appropriately adjusting the timing of contacting the cells with the microcarriers and the amount of microcarriers that are contacted with the cells in the contact step.For example, if cell passage becomes necessary while an experimenter is away, excessive cell proliferation (overconfluence) in the culture substrate can be suppressed by placing microcarriers in the culture substrate and transferring the cells to the microcarriers before the experimenter leaves, making it easy to adjust the timing of cell passage.

[0013] Furthermore, in the contacting step, the period during which cells in the culture substrate become confluent can be controlled by appropriately adjusting the timing of contacting the cells with the microcarriers and the amount of microcarriers to be contacted with the cells. For example, if passaging becomes necessary during a vacation, adding microcarriers before the vacation will cause cells adhering to the surface of the culture substrate (e.g., a petri dish or flask) to migrate toward the microcarrier, thereby reducing the number of cells remaining on the surface of the culture substrate and preventing excessive confluence, and the time for cell passaging can be easily extended beyond the vacation date.

[0014] The culture substrate for contacting cells with microcarriers (i.e., the first culture substrate) is not particularly limited as long as it is a container-shaped culture substrate with a bottom, and for example, a petri dish, a flask, etc. can be used.

[0015] At least the bottom surface of the first culture substrate is subjected to a cell adhesion treatment. The cell adhesion treatment refers to a treatment that improves the cell adhesiveness of the culture substrate so that cells can be cultured while adhering to the culture substrate, and examples of such treatments include a treatment that oxidizes the substrate surface to make it hydrophilic (hydrophilization treatment). Examples of hydrophilization treatments include plasma treatment, corona discharge treatment, oxidizing agent treatment, hydrophilic substance coating treatment, and radiation treatment.

[0016] Alternatively, cell adhesion treatment can be performed by coating the culture substrate with a matrix that has high affinity for cells to improve cell adhesion and spreadability. Examples of such matrices include type I collagen, type IV collagen, fibronectin, laminin, Matrigel, gelatin, elastin, proteoglycan, vitronectin, and peptides and protein domains having these activities (e.g., RGD peptide, laminin E8, etc.).

[0017] The first culture substrate may be a culture substrate prepared by subjecting a culture substrate that has not been subjected to a cell adhesion treatment to the above-described cell adhesion treatment, or a culture substrate that has been subjected to a cell adhesion treatment in advance, such as a commercially available culture substrate that has been subjected to a cell adhesion treatment, may be purchased and used.

[0018] In a preferred embodiment, the first culture substrate is subjected to a cell adhesion treatment so that the water contact angle of its bottom surface falls within a specific range. Specifically, the first culture substrate is subjected to a cell adhesion treatment so that the water contact angle of its bottom surface falls within a range of 60° to 70°. When the water contact angle of the bottom surface of the first culture substrate falls within this range, the cell adhesion rate becomes appropriate, and the efficiency of cell culture can be further improved.

[0019] In a preferred embodiment, the first culture substrate has a portion including concave and convex portions (i.e., unevenness) on at least a portion, preferably the entire bottom surface. The unevenness of the first culture substrate can increase the contact area between the microcarriers and the surface of the culture substrate, thereby increasing the contact opportunity between the microcarriers and the cells adhered to the surface of the culture substrate, and further improving the efficiency of cell culture. Particularly preferably, the concave portions on the bottom surface of the first culture substrate are preferably hemispherical or approximately hemispherical, and have a width greater than the average particle diameter of the microcarriers used and a depth equal to or less than half the average particle diameter of the microcarriers used. For example, the width of the concave portions is preferably such that the area of ​​the concave portions when viewed in plan is 40 μm. ２ 60,000 μm or more ２ The depth of the recess is preferably set to be 5 μm or more and 200 μm or less.

[0020] The first culture substrate having cells adhered to its bottom surface, which has been subjected to a cell adhesion treatment and which is used in the contacting step, may be prepared by subjecting an appropriate culture substrate to a cell adhesion treatment as necessary, seeding the cells, and culturing them, or may be a commercially available or assigned product. In one embodiment, the first culture substrate having cells adhered to its bottom surface, which has been subjected to a cell adhesion treatment, may be prepared by the method described in the culturing step below.

[0021] The microcarrier is not particularly limited as long as it achieves the effects of the present invention, and microcarriers commonly used in cell culture can be used. In a preferred embodiment, a microcarrier containing a gel that swells with liquid is used as the microcarrier. Microcarriers containing a gel that swells with liquid are more flexible and lightweight than microcarriers made of plastic commonly used in cell culture, and therefore, when added to a culture substrate, can reduce the load on cells adhered to the bottom surface. Furthermore, the flexibility of the microcarrier makes it more easily deformable, which can increase the contact area between the microcarrier and the bottom surface of the culture substrate, thereby facilitating the migration of cells adhered to the bottom surface of the culture substrate to the microcarrier. Examples of gels that swell with liquid include alginate gel, galacturonic acid gel, dextran gel, Tetra-PEG gel, and gelatin gel.

[0022] An example of a microcarrier containing a gel that swells with liquid is the dry microcarrier described in "Test Example 1" of International Publication WO 2022 / 239810. Specifically, this dry microcarrier can be prepared according to the following procedure. First, a 1% by mass aqueous solution of sodium alginate and a 1% by mass aqueous solution of calcium chloride are prepared. Next, the sodium alginate aqueous solution is dropped into the calcium chloride aqueous solution using a 32G syringe needle to prepare a calcium alginate microparticle gel. In the microparticle gel, alginic acid is crosslinked by calcium ions. Next, the microparticle gel is washed with 70% ethanol and water, and the microparticle gel is collected using a cell strainer. Next, the microparticle gel is immersed in a 4% by mass aqueous solution of autoclaved alkali-treated gelatin and left at 20°C for 2 hours or more to allow the gelatin to infiltrate into the microparticle gel. Next, the gelatin-infiltrated microparticle gel is vacuum-dried, followed by dry heat drying at 150°C for 2 hours to obtain a powdered dry microcarrier.

[0023] Furthermore, the microcarrier may contain a cell adhesive substance to improve its cell adhesiveness. The cell adhesive substance is not particularly limited as long as the effects of the present disclosure are achieved, but examples include type I collagen, type IV collagen, fibronectin, laminin, Matrigel, gelatin, elastin, proteoglycan, vitronectin, and peptides and protein domains having the activities of these substances (e.g., RGD peptide, laminin E8, etc.). The cell adhesive substance preferably contains at least one selected from the group consisting of type I collagen, type IV collagen, fibronectin, laminin, Matrigel, and gelatin.

[0024] The average particle size of the microcarriers is not particularly limited as long as it is larger than the cells to be cultured. For example, if the cells to be cultured are of a typical size (diameter of about 10 μm), the average particle size of the microcarriers (average particle size after expansion) can be, for example, 10 μm or more and 1 mm or less.

[0025] The amount of microcarriers to be brought into contact with cells can be appropriately determined depending on the type and characteristics (size, shape, growth rate, etc.) of the cells, the area of ​​the bottom surface of the first culture substrate, etc., and the amount per area of ​​the bottom surface of the first culture substrate is, for example, 1 to 2,000,000 cells / cm. ２ It can be said that:

[0026] In a preferred embodiment, contact between cells and microcarriers is achieved by adjusting the addition of microcarriers to achieve a specific condition described below. Specifically, when the bottom surface of the first culture substrate is viewed from above, gaps exist between at least some of the microcarriers, and the total area of ​​the bottom surface of the first culture substrate is S, the total number of added microcarriers is N, and the average particle diameter of the microcarriers is D, and the following formula is satisfied: S>Nπ(D / 2) ２By adding microcarriers to the first culture substrate so as to satisfy the above formula and contacting the cells with the microcarriers, cells adhering to the bottom surface of the culture substrate can be efficiently transferred to the added microcarriers.

[0027] A method for determining whether gaps exist between at least some of the microcarriers when the bottom surface of the first culture substrate is viewed in plan during the contacting step will be described with reference to FIG. 1 . FIG. 1A shows an image (plan view) observed with a phase-contrast microscope after contacting the cells with the microcarriers by adding microcarriers to a first culture substrate with cells adhering to its bottom surface during the contacting step. FIG. 1B shows an image obtained by color-coding the image observed with the phase-contrast microscope into microcarrier portions (black portions) and the bottom surface of the first culture substrate (gray portions). In FIG. 1B, it is determined that gaps exist between at least some of the microcarriers when gray portions are present between at least some of the black portions. The amount of microcarriers added is determined so that the ratio of gaps between the microcarriers to the total area of ​​the bottom surface of the first culture substrate is preferably 50% or less, more preferably 30% or less, and even more preferably 10% or less. On the other hand, if there are no gaps between the microcarriers (i.e., if there are no gray areas in Figure 1B), there will be many microcarriers that do not come into contact with the bottom surface of the first culture substrate (the microcarriers will be arranged overlapping each other), and since cells cannot migrate to the microcarriers that do not come into contact with the bottom surface of the first culture substrate, some of the microcarriers will be wasted.

[0028] Furthermore, the total area Nπ(D / 2) of the microcarriers is smaller than the total area S of the bottom surface of the first culture substrate. ２The small size of the microcarriers suppresses overlapping of the microcarriers. The total number N of microcarriers added to the first culture substrate can be calculated from the total mass W of the microcarriers used. Specifically, the total mass W of the microcarriers used is measured, and the microcarriers are swelled with culture medium to a known volume L to prepare a microcarrier suspension. The resulting suspension is sampled at volume X (<L) and added to a transparent observation container with a flat bottom. Next, the number Y of all microcarriers observed under a phase-contrast microscope is measured, and the number of microcarriers per weight is determined based on the formula (YL / X) / W. The total number N of microcarriers is determined from the total mass of the microcarriers used. Note that when adding microcarriers to the observation container, the bottom area of ​​the observation container and the amount of microcarriers added are adjusted so that at least 10 microcarriers can be observed without overlapping. The average particle diameter D of the microcarriers is determined by observing the microcarriers under a phase-contrast microscope, measuring the equivalent sphere diameter of all observed microcarriers, and averaging the results to obtain the average particle diameter D. The total area of ​​the microcarriers Nπ(D / 2) is smaller than the total area S of the bottom surface of the first culture substrate. ２ Because the distance is small, most of the microcarriers come into contact with the bottom surface of the first culture substrate, and as a result, cells adhering to the bottom surface of the culture substrate can be efficiently transferred to the microcarriers that have been added.

[0029] In the contacting step, the contact between the cells and the microcarriers is preferably carried out in the presence of a culture medium. The culture medium may be a so-called basal medium as its base, or may be a medium prepared appropriately depending on the cells to be cultured. The basal medium is not particularly limited as long as it is a commercially available basal medium, and examples thereof include MEM, α-MEM, DMEM, RPMI-1640, and Hams F12.

[0030] The contact conditions between cells and microcarriers can be appropriately set depending on the type, characteristics (size, shape, growth rate, etc.) and number of cells, the type, shape and volume of the culture substrate, the shape, size and number of microcarriers, etc. The contact step may be carried out in a static state or in an agitated state, but is preferably carried out in a static state.

[0031] The cells preferably used are adherent cells that can be cultured on the bottom surface of the first culture substrate or the surface of a microcarrier. Examples of such cells include hepatocytes (liver parenchymal cells), Kupffer cells, endothelial cells such as vascular endothelial cells and corneal endothelial cells, fibroblasts, osteoblasts, osteoclasts, periodontal ligament-derived cells, epidermal cells such as epidermal keratinocytes, epithelial cells such as tracheal epithelial cells, gastrointestinal epithelial cells, cervical epithelial cells, and corneal epithelial cells, mammary gland cells, pericytes, myoblasts, myotubes, satellite cells, muscle cells such as smooth muscle cells and cardiac muscle cells, kidney cells, pancreatic islet cells of Langerhans, nerve cells such as peripheral nerve cells and optic nerve cells, chondrocytes, and bone cells. These cells may be primary cells directly collected from tissues or organs, or may be cells that have been passaged for several generations. Furthermore, these cells may be undifferentiated cells such as embryonic stem cells or iPS cells, somatic stem cells such as mesenchymal stem cells with differentiation potential, unipotent stem cells such as vascular endothelial progenitor cells with unidifferentiation potential, or cells that have completed differentiation. The cells may also be CHO cells, 293 cells, 3T3 cells, Vero cells, MRC5 cells, HeLa cells, HEK293 cells, hybridomas, etc., which are widely used as cell substrates for the production of biopharmaceuticals and viral vectors, or established cell lines derived from these cells. Furthermore, the cells may be a single type of cell, or two or more types of cells. By applying the culture method of the present disclosure to such cells, medical products and foods that are expected to have therapeutic effects can be produced.

[0032] <Preliminary culture step> The culture method of the present disclosure may include a preliminary culture step, prior to the above-described contact step, of seeding cells on a first culture substrate having a bottom surface that has been treated for cell adhesion, and culturing the cells on the first culture substrate.

[0033] The amount of cells seeded on the first culture substrate can be appropriately determined depending on the type and characteristics of the cells, the area of ​​the bottom surface of the first culture substrate, etc., and the amount per area of ​​the bottom surface of the first culture substrate is, for example, 100 to 100,000 cells / cm. ２ , preferably 1,000 to 10,000 particles / cm ２ It can be said that:

[0034] In the preliminary culture step, the culture medium used for culturing the cells can be the same as that described in the contact step. The same or different culture mediums may be used in the contact step and the preliminary culture step, but it is preferable to use the same culture medium.

[0035] The culture conditions in the preliminary culture step can be appropriately set depending on the type and characteristics of the cells, the composition of the culture solution, the type, shape, volume, etc. The preliminary culture step may be performed in a static state or in an agitated state, but is preferably performed in a static state so that the cells adhere to the bottom surface of the first culture substrate.

[0036] <First Culturing Step> The culturing method of the present disclosure may include a first culturing step of culturing the cells that have been transferred from the bottom surface of the first culture substrate to the microcarriers in the contacting step and transferring step described above.

[0037] In the first culture step, the culture medium used for culturing the cells can be the same as that described in the contact step. The same or different culture medium may be used in the contact step and the first culture step, but it is preferable to use the same culture medium.

[0038] The culture conditions in the first culture step can be appropriately set depending on the type and characteristics of the cells, the composition of the culture medium, the type, shape, volume, etc. The first culture step may be performed in a stationary state, or in a state where microcarriers with cells attached to their surfaces are suspended in a culture medium (i.e., in a floating state), but is preferably performed in a floating state.

[0039] <Collection Step> In the culture method of the present disclosure, after the contact step described above, microcarriers to which cells migrated from the first culture substrate in the contact step are attached are collected from the first culture substrate. Microcarrier collection can be performed by a method commonly used in cell culture using microcarriers. In one embodiment, the collection can be performed by shaking the first culture substrate to the extent that the microcarriers move in the culture medium, tapping (lightly tapping) the first culture substrate and / or pipetting the microcarriers, and then aspirating the microcarriers. From the viewpoint of performing the collection step in a closed system, it is preferable to shake the first culture substrate and / or tap the first culture substrate (preferably from the side), and then aspirate the microcarriers. In another embodiment, the use of a culture substrate that can change the detachment property of adherent cells depending on the temperature (i.e., a temperature-responsive culture substrate) can more easily migrate cells from the culture substrate to the microcarriers and disperse the cell-adhered microcarriers into the culture medium. Examples of temperature-responsive culture substrates include Cepallet (registered trademark) manufactured by DIC Corporation and UpCell (registered trademark) manufactured by CellSeed Co., Ltd. When using these temperature-responsive culture substrates as culture substrates, for example, by keeping the temperature below 20°C during the process of transferring cells from the culture substrate to microcarriers, the cells become more easily detached from the culture substrate, thereby facilitating the transfer of cells from the culture substrate to the microcarriers. In particular, temperature-responsive culture substrates allow cells to be transferred without the need for chemical cell treatment using cell dissociation enzymes or physical cell treatment such as tapping or pipetting, thereby suppressing chemical and physical invasion of cells. Furthermore, the use of a temperature-responsive culture substrate allows the collection process to be performed in a closed system.

[0040] In the collection step, collection of the cell-adhered microcarriers is preferably carried out in the presence of a culture medium. The culture medium described in the contact step above can be used. The same or different culture mediums may be used in the contact step and collection step, but preferably the same culture medium is used. Furthermore, the collection step may further include, as necessary, adding a cell dissociation enzyme or the like to the culture substrate after collection of the cell-adhered microcarriers to detach and collect the cells remaining on the culture substrate. The detached and collected cells (i.e., free cells not adhering to the microcarriers) can be subjected to a further culture step together with the cells that were collected and adhered to the microcarriers. Therefore, by detaching and collecting the free cells remaining on the culture substrate, the cell culture efficiency can be further improved. When subjecting the free cells to a further culture step, if necessary, the free cells may be cultured together with new microcarriers in advance to allow them to adhere and proliferate on the microcarriers before being subjected to the further culture step.

[0041] As described above, the cell-adhered microcarriers and free cells collected in the collection step can be subjected to a further culture step, if necessary. For example, the cell-adhered microcarriers, and optionally the free cells, can be placed on a second culture substrate and further cultured. The second culture substrate used in the further culture step is not particularly limited, but examples include petri dishes, flasks, and bioreactors. In particular, when the further culture step is carried out using a bioreactor, the cell culture efficiency can be further improved by placing the free cells in addition to the cell-adhered microcarriers into the bioreactor.

[0042] When the cell-adhered microcarriers, and optionally the free cells, are introduced into a second culture substrate for further culturing, new microcarriers (i.e., microcarriers without cells attached thereto) may be introduced into the second culture substrate in addition to the cell-adhered microcarriers and free cells. Introducing new microcarriers in addition to the cell-adhered microcarriers and free cells increases the space available for cell adhesion and proliferation, thereby improving cell culturing efficiency. In particular, when the further culturing step is performed using a bioreactor, introducing new microcarriers into the bioreactor can further improve cell culturing efficiency.

[0043] The bottom surface of the second culture substrate may be subjected to a cell adhesion treatment. The cell adhesion treatment of the bottom surface of the second culture substrate may be the same as the cell adhesion treatment of the bottom surface of the first culture substrate described above.

[0044] <Second Culturing Step> The culturing method of the present disclosure may include a second culturing step of culturing the cells adhered to the microcarriers collected in the above-mentioned collecting step.

[0045] In the second culture step, the culture medium used for culturing the cells can be the same as that described in the contact step. The same culture medium or different culture mediums can be used in the contact step and the second culture step, but it is preferable to use the same culture medium.

[0046] The culture conditions in the second culture step can be appropriately set depending on the type and characteristics of the cells, the composition of the culture medium, the type, shape, volume, etc. The second culture step may be performed in a stationary state, or in a state where microcarriers with cells attached to their surfaces are suspended in the culture medium (i.e., in a floating state), but is preferably performed in a floating state.

[0047] The further culture step is not particularly limited as long as it is a step typically performed in cell culture, and examples thereof include a re-contacting step, a removing step, and a recovering step, which will be described later. <Re-contacting Step> In one embodiment, when the culture method of the present disclosure includes the collection step described above, the culture method of the present disclosure may also include a re-contacting step as a further culture step. In the re-contacting step, the microcarriers collected in the collection step are placed on a second culture substrate having a bottom surface treated for cell adhesion, and the cells adhering to the collected microcarriers are brought into contact with the bottom surface of the second culture substrate. Specifically, the microcarriers to which cells have adhered are added to a second culture substrate having a bottom surface treated for cell adhesion, thereby bringing the cells adhering to the microcarriers into contact with the bottom surface of the second culture substrate. It is believed that contacting the microcarriers to which cells have adhered with the bottom surface of the second culture substrate causes the cells to migrate from the microcarriers to the bottom surface of the second culture substrate.

[0048] The amount of microcarriers to be brought into contact with the second culture substrate can be appropriately determined depending on the area of ​​the bottom surface of the second culture substrate, the size of the microcarriers, the number of cells adhering to the microcarriers, the type and characteristics of the cells (size, shape, growth rate, etc.), etc., and the amount per area of ​​the bottom surface of the second culture substrate is, for example, 1 to 2,000,000 cells / cm. ２ It can be said that:

[0049] In the re-contacting step, contact between the cells adhering to the microcarriers and the bottom surface of the second culture substrate is preferably carried out in the presence of a culture medium. The second culture substrate may or may not be pre-filled with a culture medium. The culture medium to be filled into the second culture substrate can be the same as that described in the contacting step. The same or different culture mediums may be used in the contacting step, collecting step, and re-contacting step, but preferably the same culture medium is used.

[0050] <Removal Step> In one embodiment, when the culture method of the present disclosure includes the re-contacting step described above, the culture method of the present disclosure may also include a removal step as a further culture step. In the removal step, the microcarriers placed on the second culture substrate in the re-contacting step are removed. The microcarriers can be removed by a method commonly used in cell culture using microcarriers. For example, the microcarriers can be removed by shaking the second culture substrate to an extent that they move in the culture medium, tapping (lightly tapping) the second culture substrate, and / or pipetting the microcarriers, followed by aspirating the microcarriers. From the viewpoint of performing the removal step in a closed system, it is preferable to shake the second culture substrate and / or tap the second culture substrate (preferably from the side), followed by aspirating the microcarriers.

[0051] The method for removing the microcarriers from the second culture substrate can be carried out by a method commonly used in cell culture using microcarriers. For example, when using microcarriers having a structure in which alginic acid is crosslinked with divalent or higher cations as the microcarrier, the alginic acid can be decrosslinked by using a substance that inhibits crosslinking by competing with the divalent or higher cations that contribute to the crosslinking, thereby dissolving and removing the microcarrier. Examples of such crosslinking inhibitors include chelating agents and monovalent cations. These crosslinking inhibitors may be used alone or in combination of two or more.

[0052] Examples of chelating agents commonly used in cell culture include ethylenediaminetetraacetic acid (EDTA), glycol ether diaminetetraacetic acid (EGTA), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), N'-(2-hydroxyethyl)ethylenediamine-N,N,N'-triacetic acid (HEDTA), and nitrilotriacetic acid (NTA). Of the above-mentioned chelating agents, EDTA is particularly preferred. The concentration of the chelating agent is not particularly limited and can be appropriately determined depending on the type of chelating agent, the type of solvent in which the chelating agent is dissolved, and other factors. The concentration of the chelating agent can be, for example, 0.5 to 20 mM, 0.75 to 15 mM, or 1 to 10 mM.

[0053] Examples of monovalent cations include sodium ions and potassium ions. The monovalent cations may be used by dissolving them in a solvent that does not contain monovalent cations, or by dissolving them in a solvent that contains monovalent cations. The monovalent cations may also be used in the form of a buffer solution (e.g., PBS) that contains monovalent cations. The concentration of the monovalent cations is not particularly limited and can be appropriately set depending on the type of monovalent cation, the type of solvent in which the monovalent cations are dissolved, and the like. The concentration of the monovalent cations can be, for example, 10 to 500 mM, 30 to 300 mM, 50 to 200 mM, etc.

[0054] In the removal step, the removal of the microcarriers is preferably carried out in the presence of a culture medium. If the culture medium used in the re-contacting step is present in the second culture substrate, the microcarriers may be removed in the presence of the culture medium. If the culture medium used in the re-contacting step is not present in the second culture substrate, the microcarriers may be removed by adding a separate culture medium to the second culture substrate. The culture medium added to the second culture substrate can be the same as that described in the contacting step above. The same culture medium may be used in the contacting step, collecting step, or re-contacting step and the removing step, or different culture mediums may be used, but the same culture medium is preferably used.

[0055] <Recovery Step> In one embodiment, the method of the present disclosure may further include a recovery step of recovering the cultured cells after the first culture step and / or the second culture step. The recovery step is carried out, for example, by solubilizing the microcarriers used in each culture step. The microcarriers can be solubilized, for example, by adding a chelating agent to the culture medium. For example, when the culture step is carried out using microcarriers for cell culture containing alginic acid cross-linked with divalent or higher cations, adding a chelating agent to the culture medium causes the microcarriers for cell culture to react with the chelating agent, removing the divalent or higher cations cross-linking the alginic acid from the alginic acid, thereby solubilizing the microcarriers for cell culture.

[0056] EDTA is preferably used as a chelating agent in the recovery process. Furthermore, adding an alginate-degrading enzyme or a protease in addition to the chelating agent can hydrolyze alginic acid and gelatin, allowing for faster solubilization of the cell culture microcarriers. On the other hand, from the viewpoint of minimizing damage to cells, it is preferable not to use a protease. Furthermore, from the viewpoints of safety management and cost, it is also preferable not to use an alginate-degrading enzyme in order to minimize the amount of added substances. For these reasons, it is most preferable to solubilize the cell culture microcarriers using a chelating agent alone.

[0057] The present disclosure will be described in more detail below using examples, but the present disclosure is not limited to the following examples.

[0058] Test Example 1: Confirmation of cell migration from the bottom of a culture substrate to a microcarrier 1 Microcarriers used to confirm cell migration from the bottom of a culture substrate to a microcarrier were prepared according to the following procedure: First, a 1% by mass aqueous solution of sodium alginate (194-13321, manufactured by Fujifilm Corporation) and a 1% by mass aqueous solution of calcium chloride (038-24985, manufactured by Fujifilm Corporation) were prepared.

[0059] Next, a sodium alginate solution was dropped into the calcium chloride solution using a 32G syringe needle to produce a calcium alginate microparticle gel (alginic acid cross-linked with calcium ions) with an average particle diameter of approximately 200 μm (measured by optical microscopy) (see Figure 2). The microparticle gel was washed with 70% ethanol and water and collected using a cell strainer (352340, manufactured by Falcon). The microparticle gel was immersed in a 4% by weight aqueous solution of autoclaved alkali-treated gelatin (G9391-100G, manufactured by Sigma-Aldrich) and left at 20°C for at least 2 hours to allow gelatin to infiltrate into the microparticle gel. When the gelatin-infiltrated microparticle gel was observed under an optical microscope, the size of the microparticle gel remained at an average particle diameter of approximately 200 μm, and no change in shape was observed.

[0060] After collecting the microparticle gel with a cell strainer, the microparticle gel was immersed in ethanol and collected with a cell strainer to remove the gelatin outside the microparticle gel and confine the gelatin within the microparticle gel. Here, optical microscope observation showed that the calcium alginate microparticle gel before encapsulating the gelatin was colorless and the gelatin aqueous solution was slightly yellowish, but after surrounding it with ethanol to encapsulate the gelatin, the inside of the microparticle gel was slightly yellowish and the outside of the microparticle gel was colorless, confirming that the gelatin was encapsulated within the microparticle gel.

[0061] After vacuum drying in this state, the microcarriers were subjected to dry heat drying at 150°C for 2 hours, which removed the water and reduced the size to approximately 90 μm (measured by optical microscope observation), yielding powdered dried microcarriers (see Figure 3). The powder was white immediately after vacuum drying, but turned brown after dry heat drying.

[0062] Using microcarriers prepared according to the procedure described above, cell migration from the bottom of the culture substrate to the microcarriers was confirmed. First, human adipose-derived stem cells (PT-5006, manufactured by Lonza Inc.) were prepared as cells. Furthermore, culture medium 1 was prepared by adding b-FGF (basic fibroblast growth factor, Fibroblast Spray 500, manufactured by Kaken Pharmaceutical Co., Ltd.) to α-MEM containing 20% ​​FBS at a concentration of 20 ng / mL, and further adding a 5.6% by mass calcium chloride aqueous solution in an amount of 1 / 100 of the volume after addition of b-FGF. Next, a 1 mg / g gelatin aqueous solution (GLS250 gelatin solution, manufactured by Nitta Gelatin Co., Ltd.) was spread over the entire bottom surface of a 24-well plate (Nunc™ Cell-Culture Treated Multidishes, manufactured by Thermo Fisher Scientific, product number: 142475), and the plate was left to stand at 37°C for 1 hour. After that, the gelatin solution was absorbed and the plate was washed once with PBS to obtain a gelatin-coated culture substrate. Human adipose-derived stem cells were seeded into the wells of the obtained culture substrate at 5,000 cells / well, and culture medium 1 was added at 2 mL / well. The plate was then incubated at 37°C and CO ２ After the 3-day culture, it was confirmed that the cells had adhered to the well surface, spread, and proliferated, and had reached approximately 70% confluence, and 1 mL of culture medium 1 was removed from the well (this corresponds to "step (1)" described below).

[0063] Next, microcarriers prepared according to the above-mentioned procedure were prepared, immersed in the same culture solution 1 as above, separately prepared, and allowed to swell at 37°C for 30 minutes (corresponding to "step (12-1)" described later), and then subjected to a swell test using Nπ(D / 2) ２ (N: total number of microcarriers placed in the well, D: average particle diameter of the microcarriers) was 1.25 cm ２ The microcarriers were placed into the wells to which the cells had adhered together with 1 mL of culture medium 1, and the cells were incubated at 37°C and CO ２The cells were allowed to contact the microcarriers at a concentration of 5% for another 3 days, and the cells were transferred from the wells to the microcarriers (corresponding to "Step (A)" described below). The total area S of the bottom of each well of the 24-well plate was 1.9 cm ２ Therefore, S>Nπ(D / 2) ２ It was confirmed that the above condition was satisfied. Furthermore, the gap between the microcarriers observed in the photographed range of observation (plan view) using a phase contrast microscope was 34%.

[0064] Next, one day after the microcarriers were added, the 24-well plate was shaken and observed under a phase-contrast microscope. It was confirmed that the microcarriers did not move at all. This suggests that the cells adhering to the bottom of the culture substrate were also adhering to the microcarriers, causing cell migration.

[0065] Furthermore, after pipetting the wells three days after the microcarriers were added (corresponding to step (B) described below), the entire 2 mL of culture medium 1 containing the cell-adhered microcarriers was collected and transferred to another well to which no cells had been added (corresponding to "step (12-2)" described below). Figures 4A and 4B show phase-contrast microscopic images of the wells immediately after the microcarriers were added, and of the wells (wells containing microcarriers with cells attached) immediately after pipetting the wells three days after the microcarriers were added and the microcarriers with cells attached were transferred to another well. Figure 4C also shows a fluorescence microscopic image of the wells to which the cell-adhered microcarriers were transferred (stained with calcein-AM solution (product number: 19177-14, manufactured by Nacalai Tesque, Inc.)). The black arrow in Figure 4B indicates the position in the well where the cell-adhered microcarriers are presumed to have been present.

[0066] Figure 4B shows that cells are no longer present in the area where the cells were previously present on the microcarriers. Figures 4A and 4C also show that cells have migrated from the bottom of the well to the microcarriers. These results demonstrate that cells adhering to the bottom of the well can be migrated to the microcarriers without treatment with cell-dissociating enzymes such as trypsin.

[0067] Furthermore, when passaging adherent cells from a culture substrate such as a petri dish or a flask, conventional methods require at least 13 steps: (1) removal of the culture medium, (2) washing with a buffer solution, (3) removal of the buffer solution, (4) addition of a cell-dissociating enzyme, (5) detachment of the cells by incubation, (6) complete detachment of the cells by tapping, pipetting, etc., (7) inactivation of the cell-dissociating enzyme by adding the culture medium, (8) transfer of the cells to a centrifuge tube, (9) separation of the cells and the culture medium by centrifugation, (10) removal of the culture medium, (11) resuspension in the culture medium, (12) preparation of the culture substrate to be passed on, and (13) seeding of the cells on the culture substrate to be passed on. Furthermore, when the above-described step (13) "seeding cells onto a culture substrate to be subcultured" involves transferring cells from the bottom of a culture substrate, such as a petri dish or flask, to microcarriers, step (12) requires two steps: (12-1) suspending and / or swelling microcarriers in culture medium, and (12-2) adding the suspended and / or swollen microcarriers and culture medium to the culture substrate. In other words, conventional methods typically require at least 14 steps to subculture adherent cells. In contrast, in this test example, adherent cells can be subcultured in a total of five steps, including steps (1), (12-1), and (12-2) above, as well as step (A) transferring cells adhered to the bottom of the culture substrate to swollen microcarriers and step (B) detaching the microcarriers with cells attached from the culture substrate. This suggests that the culture method of the present disclosure can significantly reduce the number of steps required in conventional methods, thereby significantly reducing the time and effort required for cell subculture.

[0068] Test Example 2: Confirmation of cell migration from the bottom of the culture substrate to the microcarriers 2 Cells were cultured and migrated in the same manner as in Test Example 1, except that the culture vessel was changed to a 6-well plate coated with a 1 mg / g gelatin aqueous solution, the seeding number of cells was changed to 20,000 cells / well, the culture period was changed to 5 days, the microcarriers were placed into the wells together with 6 mL of culture solution 1, and the contact period between the cells and the microcarriers was changed to 1 day. The total area S of the bottom of each well of the 6-well plate was 9 cm ２ and Nπ(D / 2) ２ is 6.25 cm ２ Therefore, S>Nπ(D / 2) ２ It was confirmed that the above condition was satisfied. Furthermore, the gap between microcarriers observed within the photographed area in the phase-contrast microscope (plan view) was 36%. As in Test Example 1, when the 6-well plate was shaken and observed under a phase-contrast microscope, it was confirmed that the microcarriers did not move at all. Immediately thereafter, the same procedure as in Test Example 1, performed three days after the microcarriers were added, yielding observation results equivalent to those shown in Figures 4A to 4C. That is, it was confirmed that cells had migrated from the bottom of the wells to the microcarriers. These results demonstrate that even when the scale of the culture vessel is increased or the contact period between the cells and the microcarriers is shortened, cells adhered to the bottom of the wells can be migrated from the bottom of the culture substrate to the microcarriers without treatment with a cell-dissociating enzyme such as trypsin.

[0069] Test Example 3-1: Confirmation of Cell Migration from the Bottom of a Culture Substrate to Microcarriers and from Microcarriers to Microcarriers 1 Using microcarriers prepared according to the procedure shown in Test Example 1, cell migration from the bottom of a culture substrate to microcarriers and from microcarriers to microcarriers were confirmed. First, the same human adipose-derived stem cells as in Test Example 1 were prepared as cells. Furthermore, culture medium 2 was prepared by adding b-FGF (basic fibroblast growth factor, Fibroblast Spray 500, manufactured by Kaken Pharmaceutical Co., Ltd.) to α-MEM containing 10% FBS to a concentration of 20 ng / mL, and further adding a 5.6% by mass calcium chloride aqueous solution in an amount of 1 / 200 of the volume after the addition of b-FGF. 250,000 human adipose-derived stem cells were seeded on a 10 cm Petri dish (flat bottom) coated with a 1 mg / g gelatin aqueous solution, and 10 mL of culture medium 2 was added. The mixture was incubated at 37°C and CO ２ After the 3-day culture, it was confirmed that the cells had adhered to the surface of the dish, spread, and proliferated, and had reached approximately 90% confluence, and then the culture medium 2 was removed from the dish (corresponding to the above-mentioned "step (1)").

[0070] Next, microcarriers prepared according to the procedure shown in Test Example 1 were immersed in separately prepared culture solution 2 and allowed to swell at 37°C for 30 minutes (corresponding to the above-mentioned "step (12-1)"), and then Nπ(D / 2) ２ (N: total number of microcarriers placed in the petri dish, D: average particle diameter of the microcarriers) was 35.4 cm ２ The microcarriers were placed in the petri dish with the cells attached together with 10 mL of culture medium 2, and the resulting mixture was incubated at 37°C and CO ２ The cells were allowed to stand for another day at a concentration of 5% to contact the microcarriers, and the cells were transferred from the dish to the microcarriers (corresponding to the above-mentioned "Step A"). The area S of the bottom of the dish was 56.7 cm ２ Therefore, S>Nπ(D / 2) ２It was confirmed that the above condition was satisfied. Furthermore, the gap between the microcarriers observed in the photographed range of observation (plan view) using a phase contrast microscope was 35%.

[0071] Next, the side of the dish was lightly tapped to vibrate (corresponding to the above-mentioned "Step B"), and culture medium 2 containing the microcarriers onto which the human adipose-derived stem cells had been transferred was collected. A portion of the collected microcarriers was observed under a fluorescence microscope (stained with calcein-AM solution (product number: 19177-14, manufactured by Nacalai Tesque, Inc.)). A fluorescence microscope image of the collected microcarriers is shown in Figure 5A. Next, the collected microcarriers were placed in a spinner flask (product number: 3152, manufactured by Corning Incorporated), and culture medium 2 was added so that the total volume was 45 mL (corresponding to the above-mentioned "Step 12-2"), and the mixture was incubated at 37°C and CO ２ The microcarriers were cultured under continuous stirring for 3 days at a concentration of 5%. After the culture, a portion of the microcarriers was observed under a fluorescent microscope (stained with calcein-AM solution (product number: 19177-14, manufactured by Nacalai Tesque, Inc.)). The fluorescent microscope image of the microcarriers after the culture is shown in Figure 5B. Note that the 200 μm scale on the left of Figure 5A corresponds to both Figures 5A and 5B.

[0072] Figure 5A shows that cells adhering to the bottom of a petri dish can be transferred to a microcarrier without treatment with a cell dissociation enzyme such as trypsin, and Figure 5B shows that the cells transferred to the microcarrier proliferated until they covered the entire surface of the microcarrier.

[0073] Next, new microcarriers prepared according to the procedure shown in Test Example 1 were immersed in separately prepared culture solution 2 and allowed to swell at 37°C for 30 minutes to prepare swollen new microcarriers. Culture solution 2 was added to the above culture solution 2 containing the collected microcarriers so that the total volume of the swollen new microcarriers and culture solution 2 was 60 mL, and the mixture was incubated at 37°C and CO ２The microcarriers were cultured under intermittent agitation at a concentration of 5% for one day, and then cultured with continuous agitation for two more days. The swollen new microcarriers and portions of the microcarriers immediately after the addition of Culture Solution 2 were observed under a fluorescent microscope (stained with calcein-AM solution (product number: 19177-14, manufactured by Nacalai Tesque, Inc.)). Fluorescence microscope images of the swollen new microcarriers and microcarriers immediately after the addition of Culture Solution 2 are shown in Figure 5C. After culture, portions of the microcarriers were observed under a fluorescent microscope (stained with calcein-AM solution (product number: 19177-14, manufactured by Nacalai Tesque, Inc.)). Fluorescence microscope images of the microcarriers after culture are shown in Figure 5D. The 200 μm scale on the left of Figure 5C corresponds to both Figures 5C and 5D.

[0074] Figure 5C shows that there are microcarriers with cells attached over most of the surface and microcarriers with few cells attached. It is expected that the microcarriers with cells attached over most of the surface are microcarriers that have been cultured with cells, while the microcarriers with few cells attached are newly added microcarriers. On the other hand, Figure 5D shows that cells are attached over most of the surface of most microcarriers. These results suggest that cells are migrating from microcarriers with cells attached and proliferating (i.e., microcarriers that have been cultured with cells) to microcarriers with few cells attached (i.e., newly added microcarriers).

[0075] Furthermore, as in Test Example 1, this Test Example also suggests that the culture method of the present disclosure can transfer cells adhered to the bottom of a Petri dish to a microcarrier without treatment with a cell dissociation enzyme such as trypsin, and that the number of steps required in conventional culture methods can be significantly reduced (i.e., the time and labor required for cell passaging can be significantly reduced). Furthermore, in this Test Example, culture was performed on a larger scale than in Test Example 1, suggesting that the culture method of the present disclosure can also be performed on a large scale. Furthermore, by replacing the Petri dishes and spinner flasks used in this Test Example with containers connected to tubing that can be sterilely connected, it is suggested that the culture method can be easily performed in a closed system.

[0076] Test Example 3-2: Confirmation of Cell Migration from the Bottom of the Culture Substrate to Microcarriers and from Microcarriers to Microcarriers 2 Using microcarriers prepared according to the procedure shown in Test Example 1, cell migration from the bottom of the culture substrate to microcarriers and from microcarriers to microcarriers was confirmed following the same procedure as Test Example 3-1, except that the composition of the culture medium was changed. Specifically, culture medium 2' was prepared by adding b-FGF (basic fibroblast growth factor, Fibroblast Spray 500, manufactured by Kaken Pharmaceutical Co., Ltd.) to α-MEM containing 10% FBS to a concentration of 20 ng / mL. Cell migration was confirmed in the same manner as Test Example 3-1, except that culture medium 2' was used instead of culture medium 2 when culturing on a 10 cm Petri dish (plate culture) in Test Example 3-1. As a result, although not shown, results similar to those of Test Example 3-1 described above were obtained. Therefore, it can be seen that the effects of the present disclosure are similarly achieved even when the composition of the culture medium is changed.

[0077] Test Example 4: Confirmation of Cell Migration from the Bottom of the Culture Substrate to the Microcarrier 3 Using microcarriers prepared according to the procedure shown in Test Example 1, cell migration from the bottom of the culture substrate to the microcarrier was confirmed. First, embryonic stem cells (ES cells) were prepared as cells. Furthermore, as culture medium 3, mTeSR™ 1 (manufactured by Veritas Corporation) was prepared by adding a 5.6% by mass calcium chloride aqueous solution in an amount of 1 / 400 of the volume of mTeSR™ 1. Next, 5.0 x 10 feeder cells were added to a 10 cm Petri dish (flat bottom) coated with a 1 mg / g gelatin aqueous solution using 10 mL of serum-containing DMEM. ５ One day later, the serum-containing DMEM was removed from the dish, and 2.5 × 10 ES cells were seeded. ５ After 5 days of culture, it was confirmed that the cells had adhered to the surface of the dish, spread, and proliferated, and then 10 mL of culture medium 3 was removed from the dish.

[0078] Next, microcarriers prepared according to the procedure shown in Test Example 1 were immersed in separately prepared culture solution 3, allowed to swell at 37°C for 30 minutes, and then subjected to a swell test using a culture solution containing Nπ(D / 2) ２ (N: total number of microcarriers placed in the petri dish, D: average particle diameter of the microcarriers) was 35.4 cm ２ The microcarriers were placed into the petri dish to which the ES cells had adhered together with 10 mL of culture medium 3, and the resulting mixture was incubated at 37°C and CO ２ The dish was left standing for another 2 days at a concentration of 5% to allow contact between the cells and the microcarriers, and the cells were transferred from the dish to the microcarriers. The area S of the bottom of the dish was 56.7 cm ２ Therefore, S>Nπ(D / 2) ２ It was confirmed that the above condition was satisfied. Furthermore, the gap between the microcarriers observed in the photographed range of observation (plan view) using a phase contrast microscope was 35%.

[0079] The side of the dish was then gently tapped to vibrate, and the culture medium 3 containing the microcarriers to which the ES cells had been transferred was collected. A phase-contrast microscope image of the collected microcarriers is shown in Figure 6. Figure 6 shows that ES cells had adhered to the collected microcarriers. This demonstrates that ES cells adhering to the bottom of the dish can be transferred to the microcarriers without treatment with a cell-dissociating enzyme such as trypsin.

[0080] Test Example 5: Confirmation of cell migration from cell-adhered microcarriers to a culture substrate In Test Example 2, the microcarriers to which cells had migrated from the bottom of the wells of a 6-well plate were transferred, together with 2 mL of culture solution 1, to wells of a 24-well plate coated with a 1 mg / g gelatin aqueous solution. Figure 7A shows a phase-contrast microscope image of the wells of the 24-well plate immediately after the transfer of the cell-adhered microcarriers. Next, the cells were cultured at a temperature of 37°C and CO ２ The cells were allowed to contact the microcarriers with the bottom of the wells of a 24-well plate for 1 day at a concentration of 5% and allowed to migrate from the microcarriers to the bottom of the wells. After the cells had migrated from the microcarriers to the bottom of the wells, the microcarriers were removed, and 2 mL of culture medium 1 was added to each well. The cells were incubated at 37°C and CO ２ The cells were further cultured under conditions of a 5% concentration. A phase-contrast microscope image of the bottom of the wells of the 24-well plate immediately after the microcarriers were removed is shown in Figure 7B. Next, the cells on the bottom of the wells of the 24-well plate after the microcarriers were removed were incubated at a temperature of 37°C and CO ２ Figure 7C shows a phase-contrast microscope image of the well bottom after one day of culture at a concentration of 5%. Meanwhile, the removed microcarriers, together with 2 mL of culture solution 1, were transferred to wells of a 24-well plate coated with a separately prepared 1 mg / g gelatin aqueous solution. Figure 7D shows a phase-contrast microscope image of the well bottom immediately after transfer. Then, the microcarriers were incubated at 37°C and CO ２ The microcarriers were contacted with the bottom of the wells of a 24-well plate at a concentration of 5% for one day to allow the cells to migrate from the microcarriers to the bottom of the wells.

[0081] Figures 7A and 7B show that cells adhering to the microcarriers have migrated to the bottom of the wells of a 24-well plate. Figures 7B and 7C also confirm cell proliferation, indicating that the migrated cells maintain their proliferation potential. These results demonstrate that cells can be migrated (i.e., passaged) from the bottom of a culture substrate to the bottom of another culture substrate without treatment with cell dissociation enzymes such as trypsin. Furthermore, Figure 7D shows that cells adhere to the microcarriers removed after cell migration. Furthermore, when the cells adhering to the microcarriers shown in Figure 7D were transferred to the wells of a 24-well plate for one day, the results were similar to those shown in Figures 7B and 7C, demonstrating that cell migration from microcarriers to the bottom of a culture substrate can be repeated.

[0082] Another aspect of the present disclosure relates to the following [1] to [5]: [1] A method for culturing cells, comprising a contacting step of contacting cells adhered to the bottom surface of a first culture substrate having a bottom surface that has been subjected to a cell adhesion treatment with microcarriers. [2] In the contacting step, when the bottom surface of the first culture substrate is viewed from above, gaps exist between at least some of the microcarriers, and the following formula is satisfied: S>Nπ(D / 2), where S is the total area of ​​the bottom surface of the first culture substrate, N is the total number of microcarriers, and D is the average particle diameter of the microcarriers. ２[1] The method according to [1], wherein the cells are contacted with the microcarriers so as to satisfy the condition. [3] The method according to [1] or [2], wherein the bottom surface of the first culture substrate has an uneven surface. [4] The method according to any of [1] to [3], wherein the microcarriers comprise a gel that swells with a liquid. [5] The method according to any of [1] to [4], further comprising a transfer step of transferring the cells that have contacted the microcarriers in the contact step to the surface of the microcarriers. [6] The method according to [5], further comprising a first culture step of culturing the cells that have migrated to the surface of the microcarriers in the transfer step. [7] The method according to [6], wherein the culture in the first culture step is carried out by suspending the microcarriers, to the surface of which the cells have adhered, in a culture solution. [8] The method according to any of [1] to [7], further comprising a pre-culture step of culturing the cells on the first culture substrate before the contact step. [9] The method according to any of [1] to [8], wherein the bottom surface of the first culture substrate has a water contact angle of 60 degrees or more and 70 degrees or less.

[10] The method according to any one of [1] to [9], wherein the bottom surface of the first culture substrate has a coating containing at least one selected from the group consisting of type I collagen, type IV collagen, fibronectin, laminin, Matrigel, gelatin, elastin, proteoglycan, vitronectin, and peptides and protein domains having their activities.

[11] The method according to any one of [1] to

[10] , wherein the microcarrier contains at least one selected from the group consisting of type I collagen, type IV collagen, fibronectin, laminin, Matrigel, and gelatin.

[12] The method according to

[11] , wherein the microcarrier contains gelatin and further contains calcium alginate.

[13] The method according to any one of [1] to

[12] , wherein the average particle size of the microcarrier is 10 μm or more and 1 mm or less.

[14] The method according to [3], wherein the average particle size of the microcarrier is smaller than the diameter of the recesses on the bottom surface of the first culture substrate.

[15] The method according to any one of [1] to

[14] , further comprising a collecting step of collecting the microcarriers that have been contacted with the cells in the contacting step from the first culture substrate.

[16] The method according to

[15] , further comprising a second culturing step of culturing the cells adhered to the microcarriers collected in the collecting step by suspending them in a culture medium.

[17] The method according to

[15] , further comprising a re-contacting step of placing the microcarriers collected in the collecting step on a second culture substrate having a bottom surface treated for cell adhesion, bringing the cells in contact with the bottom surface of the second culture substrate into contact with the cells in contact with the collected microcarriers, and a removing step of removing the microcarriers from the second culture substrate.

[18] The method according to

[17] , wherein the bottom surface of the second culture substrate has cell adhesive properties.

[19] The method according to any one of

[16] to

[18] , further comprising a recovering step of recovering the cells cultured in the second culturing step.

[20] A method for producing a cell-containing pharmaceutical composition, comprising a step of mixing the cells obtained by the method according to any one of [1] to

[19] with a pharmaceutically acceptable excipient, solvent, or cryopreservation solution.

[21] A method for producing a cell sheet, comprising the steps of culturing the cells adhered to the bottom surface of the second culture substrate in the re-contacting step according to

[17] or

[18] to form a cell sheet, and detaching the cell sheet from the second culture substrate.

[0083] According to yet another aspect of the present disclosure, there is provided a method for culturing cells, comprising a contact step of contacting cells with microcarriers on a culture substrate that has not been subjected to cell adhesion treatment or that has been subjected to cell non-adhesion treatment and has a textured bottom. According to this culture method, contacting the cells with the microcarriers on a culture substrate with a textured bottom increases the contact opportunity (contact rate) between the microcarriers and the cells, particularly on the bottom surface of the culture substrate, thereby improving the adhesion rate between the cells and the microcarriers. While the reason for the improved adhesion rate between the cells and the microcarriers in this culture method is unclear, it is hypothesized as follows: Typically, when culturing cells using microcarriers, both the cells and the microcarriers are introduced into the culture substrate. However, in this case, the microcarriers typically have a larger mass, so the microcarriers sink to the bottom of the culture substrate first, followed by the cells (see FIG. 8A ). Furthermore, cells, particularly adhesive cells, that contact and adhere to the microcarriers have higher survival and proliferation rates, while cells that do not contact the microcarriers either die or survive but experience a lower proliferation rate. Furthermore, even if some cells sink behind the microcarriers and come into contact with and adhere to them, the contact opportunity is limited, which is thought to result in limited cell culture efficiency (see Figures 8B and 7C).In contrast, as shown in Figure 9, the culture method of this embodiment uses a culture substrate that has not been treated for cell adhesion or has been treated for non-cell adhesion and has an uneven bottom to bring cells and microcarriers into contact with each other, which is thought to increase the contact rate between the two and result in increased cell culture efficiency.

[0084] The cell-non-adhesive treatment of the culture substrate is not particularly limited as long as it makes cells less likely to adhere to the culture substrate, and any treatment commonly used in cell culture can be used. In one embodiment, the cell-non-adhesive treatment of the culture substrate is performed so that the water contact angle of the bottom surface of the culture substrate falls within a specific range. Specifically, the culture substrate is adjusted so that the water contact angle of the bottom surface of the culture substrate is less than 60° or more than 70°. By ensuring that the water contact angle of the bottom surface of the culture substrate falls within this range, the cell adhesion rate becomes more appropriate, and the efficiency of cell culture can be further improved.

[0085] The culture substrate used in the culture method of this embodiment may be not only a culture substrate prepared by the above-mentioned cell-nonadhesive treatment, but also a culture substrate having a bottom surface in a cell-nonadhesive state as described above without the cell-nonadhesive treatment. The culture substrate may be a culture substrate prepared by subjecting a culture substrate that has not been subjected to the cell-nonadhesive treatment to the above-mentioned cell-nonadhesive treatment, or a culture substrate that has been subjected to the cell-nonadhesive treatment in advance, such as a commercially available culture substrate that has been subjected to the cell-nonadhesive treatment, may be purchased and used.

[0086] In one embodiment, in addition to the cell-free treatment described above, the culture substrate may be coated with a material that has low affinity for cells, such as 2-methacryloyloxyethyl phosphorylcholine (MPC) or polyethylene glycol (PEG).

[0087] The culture method of this embodiment can be carried out in the same manner as the culture method of the present disclosure, except that the first culture substrate in the culture method of the present disclosure described above is replaced with a culture substrate that has not been treated for cell adhesion or has been treated for non-cell adhesion and has a bottom with an uneven surface. Thus, the culture method of this embodiment can be carried out in the same manner as the various steps described in the culture method of the present disclosure described above.

[0088] Another aspect of the present disclosure provides a cell culture method including a contact step of contacting cells with oblate spherical microcarriers in a culture substrate. According to this culture method, contacting the microcarriers with the cells in the culture substrate increases the contact opportunity (contact rate) between the microcarriers and the cells, resulting in an improved adhesion rate between the cells and the microcarriers. The reason why this culture method improves the adhesion rate between the cells and the microcarriers is unclear, but it is hypothesized as follows: Typically, when culturing cells using microcarriers, both the cells and the microcarriers are placed in the culture substrate. In this case, the microcarriers typically have a larger mass, so the microcarriers sink to the bottom of the culture substrate first, followed by the cells. Cells, particularly adherent cells, that contact and adhere to the microcarriers have higher survival and proliferation rates, while cells that do not contact the microcarriers either die or survive with a low proliferation rate. Even if some cells sink after the microcarriers, come into contact with the microcarriers, and adhere, the contact opportunity is limited, which is thought to limit the efficiency of cell culture. In contrast, as shown in Figure 10, according to the culture method of this embodiment, by making the microcarriers that come into contact with the cells spherical, it is possible to increase the contact rate between the two, and as a result, it is thought that the efficiency of cell culture can be improved. That is, it is thought that the cells that sink later contact and adhere to the upper part of the spherical microcarriers that sank earlier, resulting in proliferation. Furthermore, since the microcarriers have an oblate spherical shape, the area that is in close proximity to the bottom surface of the culture substrate is increased, which is thought to increase the opportunity for contact and adhesion between the cells that sank earlier and / or cells that sank without contacting the upper part of the microcarriers.

[0089] In the culture method of this embodiment, microcarriers that have been formed into an oblate spheroid shape may be used, or the shape of the microcarriers may be changed to an oblate spheroid shape by temporarily applying an external force to the microcarriers at least during the contact step. The method for applying an external force to the microcarriers is not particularly limited as long as it is a method commonly used in cell culture, and examples include applying centrifugal force using a centrifuge or the like, or applying magnetic force when the microcarriers contain magnetic particles.

[0090] The culture method of this embodiment can be carried out in the same manner as the culture method of the present disclosure, except that oblate spherical microcarriers are used as the microcarriers in the culture method of the present disclosure described above. Therefore, the culture method of this embodiment can be carried out in the same manner as the various steps described in the culture method of the present disclosure described above.

[0091] According to yet another aspect of the present disclosure, a method for culturing cells is provided, comprising a contacting step of contacting cells with microcarriers on a culture substrate having a cell adhesion-treated, flat bottom surface. As shown in FIG. 11 , by using a culture substrate having a cell adhesion-treated bottom surface, cells in contact with the bottom surface adhere and proliferate (spread) on the bottom surface, increasing their chances of contact with microcarriers that are in contact with the bottom surface or are nearby but not adhered to the bottom surface, which is believed to result in increased cell culture efficiency. On the other hand, as shown in FIG. 11 , cells not in contact with the bottom surface also adhere and proliferate on the surface of microcarriers upon contact, which is believed to increase cell culture efficiency. Thus, according to this culture method, not only cells in contact with the bottom surface of the culture substrate but also cells not in contact with the bottom surface of the culture substrate can be contacted and proliferated by microcarriers. Therefore, according to this method, efficient cell culture can be achieved without any particular restrictions on the order in which cells and microcarriers are seeded on the culture substrate.

[0092] Furthermore, the cells obtained by the method of the present disclosure can be used as a pharmaceutical composition (i.e., a cell-containing pharmaceutical composition). Such a pharmaceutical composition can be produced, for example, by using the cells recovered by the above-described recovery step and, if necessary, pharmaceutically acceptable additives (e.g., excipients, solvents, cryopreservation solutions, etc.) using conventional techniques for producing pharmaceutical compositions.

[0093] The cryopreservation solution may contain a cell protective agent, such as dimethyl sulfoxide (DMSO), ethylene glycol, propylene glycol, sericin, or glycerol. The cells recovered by the recovery step described above can be suspended in a cryopreservation solution and mixed to produce a pharmaceutical composition.

[0094] According to yet another aspect of the present disclosure, a cell sheet can be produced by culturing the cells transferred to the second culture substrate by the above-described re-contacting step to form a cell sheet. The culture medium and culture conditions used to culture the cells transferred to the second culture substrate can be appropriately set according to the cell type and the desired cell sheet form. The cells transferred to the second culture substrate can be cultured to an extent that a cell sheet can be formed on the second culture substrate (e.g., until confluence is reached).

[0095] A cell sheet can be obtained by detaching a cell sheet cultured on the second culture substrate from the second culture substrate. Known methods can be used to detach the cell sheet from the second culture substrate. Examples include a method of detachment using an enzyme such as dispase, and a method using a culture substrate that can change the detachability of adherent cells depending on the temperature (i.e., a temperature-responsive culture substrate) as the second culture substrate. Examples of temperature-responsive culture substrates include the aforementioned Cepallet (registered trademark) manufactured by DIC Corporation and UpCell (registered trademark) manufactured by CellSeed Co., Ltd.

[0096] The cell sheet detached from the second culture substrate can be used, for example, as a transplant material for reconstructing defective tissue that has lost function due to disease, injury, etc. It is expected that by producing cell sheets using the method of the present disclosure, it will be possible to culture cell sheets in large quantities.

Claims

1. A method for culturing cells, comprising a contacting step of contacting cells adhered to the bottom surface of a first culture substrate having a bottom surface treated for cell adhesion with microcarriers.

2. In the contacting step, when the bottom surface of the first culture substrate is viewed in plan, there are gaps between at least some of the microcarriers, and the following formula is satisfied, where S is the total area of ​​the bottom surface of the first culture substrate, N is the total number of the microcarriers, and D is the average particle diameter of the microcarriers: S>Nπ(D / 2) ２ The method of claim 1, wherein the cells are contacted with the microcarriers so that 3. The method of claim 1, wherein the bottom surface of the first culture substrate has recesses and protrusions.

4. The method of claim 1, wherein the microcarriers comprise a gel that swells with a liquid.

5. The method according to claim 1, further comprising a transfer step of transferring the cells that have contacted the microcarriers in the contact step to the surface of the microcarriers.

6. The method according to claim 5, further comprising a first culturing step of culturing the cells that have migrated to the surface of the microcarriers in the transferring step.

7. The method according to claim 6, wherein the culturing in the first culturing step is carried out by suspending the microcarriers with the cells attached to their surfaces in a culture medium.

8. The method of claim 1, further comprising a pre-culture step of culturing the cells on the first culture substrate prior to the contacting step.

9. The method according to claim 1, wherein the water contact angle of the bottom surface of the first culture substrate is 60 degrees or more and 70 degrees or less.

10. The method of claim 1, wherein the bottom surface of the first culture substrate has a coating containing at least one selected from the group consisting of type I collagen, type IV collagen, fibronectin, laminin, Matrigel, gelatin, elastin, proteoglycan, vitronectin, and peptides and protein domains having the activities thereof.

11. The method of claim 1, wherein the microcarrier comprises at least one selected from the group consisting of type I collagen, type IV collagen, fibronectin, laminin, matrigel, and gelatin.

12. The method of claim 11, wherein the microcarriers comprise gelatin and further comprise calcium alginate.

13. The method according to claim 1, wherein the average particle size of the microcarriers is 10 μm or more and 1 mm or less.

14. The method of claim 3, wherein the average particle size of the microcarriers is smaller than the diameter of the recess in the bottom surface of the first culture substrate.

15. The method of claim 1, further comprising a collecting step of collecting the microcarriers that have been contacted with the cells in the contacting step from the first culture substrate.

16. The method according to claim 15, further comprising a second culturing step of culturing the cells adhered to the microcarriers collected in the collecting step by suspending the cells in a culture medium.

17. The method according to claim 15, further comprising a re-contacting step of placing the microcarriers collected in the collecting step on a second culture substrate having a bottom surface treated for cell adhesion, bringing the cells in contact with the collected microcarriers into contact with the bottom surface of the second culture substrate, and allowing the cells to adhere to the bottom surface of the second culture substrate; and a removing step of removing the microcarriers from the second culture substrate.

18. The method of claim 17, wherein the bottom surface of the second culture substrate has cell adhesive properties.

19. The method according to claim 16, further comprising a recovery step of recovering the cells cultured in the second culture step.

20. A method for producing a cell-containing pharmaceutical composition, comprising the step of mixing cells obtained by the method of claim 1 with a pharmaceutically acceptable excipient, solvent, or cryopreservation solution.

21. A method for producing a cell sheet, comprising the steps of culturing the cells adhered to the bottom surface of the second culture substrate in the re-contacting step described in claim 17 to form a cell sheet, and detaching the cell sheet from the second culture substrate.

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