New cell culture methods

Abstract

In cell culture, the microcarriers are brought into contact with cells adhering to the bottom surface of a first culture substrate having a bottom surface treated for cell adhesion.

Description

[Technical Field]

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

[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 known as a mass cultivation technique for cells and the like. 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 proliferate 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 proliferation 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 consecutive 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 by gradually scaling up a culture medium containing adherent cells, microcarriers, and a culture medium, in which 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 the 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 in another culture substrate, such as a bioreactor, along with the microcarriers. The free (non-adherent) cells are cultured with the microcarriers 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 the culture conditions that ensure adequate contact requires considerable effort. In particular, efficient cell culture on the surface of microcarriers requires as uniform cell adhesion as possible. Therefore, determining the culture conditions that ensure adequate contact is challenging, resulting in a problem that hinders the efficiency of cell culture. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2010 / 138702 [Patent Document 2] Special Publication No. 2013-515473 [Patent Document 3] International Publication No. WO2021 / 181819 Brochure Summary of the Invention [Problem to be solved by the invention]

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

[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, A method for culturing cells is provided, which includes 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. [Effects of the Invention]

[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. [Brief explanation of the drawings]

[0010] [Figure 1] Figure 1A shows a plan view of a phase-contrast image obtained by adding microcarriers to a first culture substrate with cells attached to its bottom surface in the contact step, and Figure 1B shows a color-coded image of the microcarriers (black area) and the bottom surface of the first culture substrate (gray area) after the microcarriers were brought into contact with the cells. [Figure 2] 1 shows a phase contrast microscope image of a microcarrier for cell culture. [Figure 3] 1 shows a phase contrast microscope image of a dry microcarrier for cell culture. [Figure 4]Figure 4A shows a phase-contrast microscope image of the inside of a well immediately after the microcarriers were added. Figure 4B shows a phase-contrast microscope image of the well (the well that contained the attached microcarriers) immediately after the microcarriers with human adipose-derived stem cells attached were transferred to another well. Figure 4C shows a fluorescence microscope image of the well to which the microcarriers with human adipose-derived stem cells attached were transferred. [Figure 5] Figure 5A shows a fluorescence microscopy image of microcarriers collected from a culture medium containing microcarriers into which human adipose-derived stem cells were migrated. Figure 5B shows a fluorescence microscopy image of microcarriers collected from a culture medium containing microcarriers into which human adipose-derived stem cells were migrated and after culturing. Figure 5C shows a fluorescence microscopy image of microcarriers immediately after adding new swollen microcarriers to a culture medium containing microcarriers into which human adipose-derived stem cells were migrated. Figure 5D shows a fluorescence microscopy image of microcarriers after adding new swollen microcarriers to a culture medium containing microcarriers into which human adipose-derived stem cells were migrated and culturing. Note that the 200 μm scale on the left of Figure 5A corresponds to both Figures 5A and 5B. The 200 μm scale on the left of Figure 5C corresponds to both Figures 5C and 5D. [Figure 6] FIG. 6 shows a phase-contrast microscope image of the microcarrier to which ES cells were attached. [Figure 7]Figure 7A shows a phase-contrast microscope image of a well of a 24-well plate immediately after microcarriers with human adipose-derived stem cells attached thereto were transferred together with culture medium into the wells of a 24-well plate coated with a 1 mg / g aqueous solution of gelatin. Figure 7B shows a phase-contrast microscope image of the well bottom of a 24-well plate immediately after the microcarriers were removed after the human adipose-derived stem cells had been transferred from the microcarriers to the bottom of the wells. Figure 7C shows a phase-contrast microscope image of the well bottom of a 24-well plate after the microcarriers were removed and the human adipose-derived stem cells were cultured for one day at a temperature of 37°C and a CO2 concentration of 5%. Figure 7D shows a phase-contrast microscope image of the well bottom immediately after the removed microcarriers were transferred together with culture medium into the wells of a 24-well plate coated with a 1 mg / g aqueous solution of gelatin. [Figure 8] FIG. 8 is a schematic diagram showing the behavior of cells and microcarriers over time after they are both placed in a culture substrate when cells are cultured using microcarriers. [Figure 9] Figure 9 is a schematic diagram showing the behavior of cells and microcarriers over time after they are both placed into a culture substrate when cells are brought into contact with microcarriers using a culture substrate that has not been treated for cell adhesion or has been treated for non-cell adhesion and has an uneven bottom. [Figure 10] Figure 10 is a schematic diagram showing the behavior of cells and microcarriers over time after they are both placed into a culture substrate when cells are brought into contact with the microcarriers using oblate spherical microcarriers. [Figure 11] 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. DETAILED DESCRIPTION OF THE INVENTION

[0011] [Cell culture method] According to one aspect of the present disclosure, a method for culturing cells (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 proliferating cells but also keeping cells alive (maintaining them) without substantially dividing and proliferating them. 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 by treatment with 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 or mutation, or reduce the cell yield. The culture method of the present disclosure can improve the efficiency of cell culture by reducing the time required for such treatment with cell dissociation enzymes and further suppressing a decrease in cell yield due to cell damage, mutation, etc.

[0014] Furthermore, the culture method of the present disclosure involves bringing cells adhered to the bottom surface of a culture substrate (i.e., in an immobilized state) into contact with microcarriers to transfer the cells to the microcarriers. Therefore, it is substantially unnecessary to consider the culture conditions required to ensure appropriate contact between free cells and microcarriers, which also provides the further advantage of improving the efficiency of cell culture.

[0015] Furthermore, the culture method of the present disclosure can efficiently transfer cells to microcarriers by bringing cells adhered to the bottom surface of a culture substrate into contact with the microcarriers. Conventionally, cell culture methods involving transferring cells from one carrier to another are known. This method requires the initial preparation of carriers with cells attached thereto (i.e., carriers with cells attached thereto that serve as the starting point for cell culture), but it is known that preparing such carriers with cells attached thereto takes a considerable amount of time.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] <Contact process> 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 its bottom surface. In the contacting step, the cells adhering to the bottom surface of the first culture substrate are brought into contact with the microcarriers, which is thought to result in the cells migrating 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 independently of the contacting step. 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. In other words, "cells migrate from the bottom surface of the first culture substrate to the microcarriers" not only means that a cell itself migrates from the bottom surface of the first culture substrate toward the microcarriers, but also means that a cell divides and proliferates from the bottom surface of the first culture substrate toward the microcarriers. The reason why cells migrate to the microcarriers is unclear, but it is thought that cells migrate from a densely packed region (i.e., the bottom surface of the first culture substrate) toward a sparsely packed region (i.e., the microcarriers) 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 microcarriers, but also divide simultaneously, causing the cells to migrate from the densely packed region (i.e., the bottom surface of the first culture substrate) toward the sparsely packed region (i.e., the microcarriers) by dividing and proliferating. 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 contacted with the cells. For example, if passaging becomes necessary during a vacation, adding microcarriers before the vacation will cause cells adhering to the culture substrate surface (e.g., a petri dish or flask) to migrate toward the microcarrier, thereby reducing the number of cells remaining on the culture substrate surface 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 that have 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 cell adhesion treatment described above, 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 More than 60,000μm 2The 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, the microcarrier is a microcarrier containing a gel that swells with liquid. Microcarriers containing a gel that swells with liquid are more flexible and lightweight than microcarriers made of plastic commonly used in cell culture. When added to a culture substrate, they can reduce the load on cells adhering to the bottom surface. Furthermore, the flexibility of the microcarrier makes it more easily deformable, which increases the contact area between the microcarrier and the bottom surface of the culture substrate, thereby facilitating the migration of cells adhering to the bottom surface of the culture substrate. 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" in International Publication WO 2022 / 239810. 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 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 at least 2 hours to allow the gelatin to infiltrate into the microparticle gel. Next, the gelatin-infiltrated microparticle gel is vacuum-dried and then dry-heat dried 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, as determined by the following formula: S>Nπ(D / 2) 2 By 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 or not 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 adding microcarriers to a first culture substrate with cells adhering to its bottom surface to bring the cells and microcarriers into contact 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 exist 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 the gray areas in Figure 1B do not exist), 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 of ​​the microcarriers Nπ(D / 2) is smaller than the total area S of the bottom surface of the first culture substrate. 2Due to its small size, the overlap between microcarriers can be suppressed. 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, measure the total mass W of the microcarriers used, swell it to a known volume L with a culture solution to prepare a microcarrier suspension, sample the obtained suspension with a volume X (<L), and add it to an observation container with a flat bottom and transparent bottom. Then, measure the number Y of all microcarriers observed by phase-contrast microscopy, grasp the number of microcarriers per unit weight based on the formula (YL / X) / W, and determine the total number N of microcarriers from the total mass of the microcarriers used. When adding microcarriers to the observation container, adjust the bottom area of the observation container and the addition amount of microcarriers so that 10 or more microcarriers can be observed without overlap and perform the measurement. The average particle diameter D of the microcarriers is observed by a phase-contrast microscope, and the equivalent diameter of a sphere of equal volume is measured for all observed microcarriers, and the average value is taken as the average particle diameter D. 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 2 Due to its small size, most of the microcarriers will come into contact with the bottom surface of the first culture substrate, and as a result, the cells adhering to the bottom surface of the culture substrate can be efficiently transferred to the microcarriers for the added microcarriers.

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

[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 and 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, and other cells widely used as cell substrates for the production of biopharmaceuticals and viral vectors, as well as 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 incubation step> The culture method of the present disclosure may include a pre-culture step, prior to the contact step, in which cells are seeded on a first culture substrate having a bottom surface treated for cell adhesion and the cells are cultured 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 culture process> The culture method of the present disclosure may include a first culture step of culturing the cells that have been transferred from the bottom surface of the first culture substrate to the microcarriers in the contact step and transfer 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 cells, the composition of the culture medium, the type, shape, volume, etc. The first culture step may be performed in a static 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 process> In the culture method of the present disclosure, after the contacting step described above, microcarriers bearing cells that migrated from the first culture substrate during the contacting step are collected from the first culture substrate. Microcarrier collection can be performed using a method commonly used in cell culture using microcarriers. In one embodiment, the collection can be performed by shaking the first culture substrate sufficiently to move the microcarriers in the culture medium, tapping (lightly tapping) the first culture substrate and / or pipetting the microcarriers, and then aspirating the microcarriers. From the perspective 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 capable of changing the detachment properties of adherent cells depending on the temperature (i.e., a temperature-responsive culture substrate) can facilitate the migration of cells from the culture substrate to microcarriers and the dispersion of the cell-adhered microcarriers into the culture medium. Examples of temperature-responsive culture substrates include Cepallet® manufactured by DIC Corporation and UpCell® manufactured by CellSeed Co., Ltd. When these temperature-responsive culture substrates are used as the culture substrate, for example, by keeping the temperature at 20°C or below 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 carried out in a closed system.

[0040] In the collection step, the 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 the same culture medium is preferably used. The collection step may further include, as necessary, adding a cell dissociation enzyme or the like to the culture substrate after the cell-adhered microcarriers have been collected, thereby detaching and collecting the cells remaining on the culture substrate. The detached and collected cells (i.e., free cells not attached to the microcarriers) can be subjected to a further culture step together with the cells attached to the microcarriers and collected. Therefore, detaching and collecting the free cells remaining on the culture substrate can further improve the cell culture efficiency. 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 attach and grow 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 culture, 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 culture efficiency. In particular, when the further culture step is performed using a bioreactor, introducing new microcarriers into the bioreactor can further improve cell culture 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 culture process> The culture method of the present disclosure may include a second culture step of culturing the cells adhered to the microcarriers collected in the collection step described above.

[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 or different culture mediums may 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 carried out in a static state, or in a state where the microcarriers with cells attached to their surfaces are suspended in the culture medium (i.e., in a floating state), but is preferably carried out in a floating state.

[0047] The further culture step is not particularly limited as long as it is a step that is normally carried out in cell culture, and for example, a re-contact step, a removal step, a recovery step, etc., which will be described later, can be carried out. <Re-contact process> In one embodiment, when the culture method of the present disclosure includes the above-mentioned collection step, the culture method of the present disclosure may further include a re-contacting step as a 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 the 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 (size, shape, growth rate, etc.) of the cells, 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 process> 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 the microcarriers 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 alginate is cross-linked with divalent or higher cations, the alginate can be de-crosslinked by using a substance that inhibits the cross-linking by competing with the divalent or higher cations that contribute to the cross-linking, thereby dissolving and removing the microcarrier. Examples of such substances that inhibit cross-linking include chelating agents and monovalent cations. These cross-linking 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 the monovalent cations, or in a solvent that contains the monovalent cations. The monovalent cations may also be used in the form of a buffer solution (e.g., PBS) that contains the 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, and the like.

[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 process> 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. Microcarrier solubilization can be carried out, 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. [Example]

[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 the culture substrate to the microcarrier 1 Microcarriers used to confirm cell migration from the bottom surface of the culture substrate to the microcarriers 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, sodium alginate solution was added dropwise from a 32G syringe needle to the calcium chloride solution to produce calcium alginate microparticle gels (alginic acid cross-linked with calcium ions) with an average particle size of approximately 200 μm (measured by optical microscopy) (see Figure 2). The microparticle gels were washed with 70% ethanol and water and collected using a cell strainer (352340, Falcon). The microparticle gels were immersed in a 4% by weight aqueous solution of autoclaved alkali-treated gelatin (G9391-100G, Sigma-Aldrich) and left at 20°C for at least 2 hours to allow gelatin to infiltrate into the microparticle gels. Optical microscopy of the gelatin-infiltrated microparticle gels revealed that the average particle size of the microparticle gels remained 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 dry microcarriers (see Figure 3). Note that the powder was white immediately after vacuum drying, but turned brown after dry heat drying.

[0062] Using the microcarriers prepared according to the above-mentioned procedure, the migration of cells from the bottom surface 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. Culture medium 1 was prepared by adding b-FGF (basic fibroblast growth factor, Fibroblast Spray 500, manufactured by Kaken Pharmaceutical Co., Ltd.) to 20 ng / mL of α-MEM containing 20% ​​FBS, and then adding a 5.6% by mass calcium chloride aqueous solution at 1 / 100 the volume after b-FGF addition. 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 allowed to stand at 37 °C for 1 hour. After the gelatin solution was absorbed and washed once with PBS, a gelatin-coated culture substrate was obtained. 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, followed by culture for 3 days at a temperature of 37°C and a CO2 concentration of 5%. After culturing for 3 days, 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 (corresponding 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 below), and then subjected to 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 with the cells attached together with 1 mL of culture solution 1, and the wells were left to stand for another 3 days at a temperature of 37°C and a CO2 concentration of 5%, allowing the cells to come into contact with the microcarriers and migrate 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) 2It was confirmed that the above requirement was satisfied. Furthermore, the gaps between the microcarriers observed in the photographed area of ​​the phase contrast microscope (plan view) were 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 microcarrier side, causing cell migration.

[0065] Furthermore, three days after the microcarriers were added, the wells were pipetted (corresponding to step (B) described below), and 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 the wells were pipetted and the cell-adhered microcarriers were transferred to another well three days after the microcarriers were added. 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, 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 attached to 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 that had been attached 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 culture substrates such as petri dishes or flasks, conventional methods require at least 13 steps: (1) removal of culture medium, (2) washing with buffer solution, (3) removal of buffer solution, (4) addition of cell dissociation enzyme, (5) cell detachment by incubation, (6) complete cell detachment by tapping or pipetting, (7) inactivation of cell dissociation enzyme by addition of culture medium, (8) transfer of cells to a centrifuge tube, (9) separation of cells from culture medium by centrifugation, (10) removal of culture medium, (11) resuspension in culture medium, (12) preparation of the destination culture substrate, and (13) seeding of cells onto the destination culture substrate. Furthermore, when the above-mentioned step (13) "seeding cells onto a culture substrate for passage" 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 passage adherent cells. In contrast, in this test example, adherent cells can be passaged in a total of five steps: step (A) transferring cells adhered to the bottom of the culture substrate to swollen microcarriers, and step (B) detaching the cell-adhered microcarriers from the culture substrate, in addition to the above-mentioned steps (1), (12-1), and (12-2). 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 labor required for cell passaging.

[0068] Test Example 2: Confirmation of cell migration from the bottom of the culture substrate to the microcarrier 2 The culture vessel was changed to a 6-well plate coated with 1 mg / g gelatin 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 6.25cm 2 Therefore, S>Nπ(D / 2) 2 It was confirmed that the above requirement 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 after that, 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 cells adhering to the bottom of the wells can be migrated from the bottom of the culture substrate to the microcarriers without treatment with cell-dissociating enzymes such as trypsin, even when the scale of the culture vessel is enlarged or the contact period between the cells and the microcarriers is shortened.

[0069] Test Example 3-1: Confirmation of cell migration from the bottom of the culture substrate to the microcarrier and from one microcarrier to another 1 Using microcarriers prepared according to the procedure shown in Test Example 1, the migration of cells from the bottom surface of the culture substrate to the microcarriers and the migration of cells from one microcarrier to another was confirmed. First, human adipose-derived stem cells identical to those used in Test Example 1 were prepared. 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 at a concentration of 20 ng / mL, and then adding a 5.6% by mass calcium chloride aqueous solution at 1 / 200 the volume after b-FGF addition. 250,000 human adipose-derived stem cells were seeded onto 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 cells were cultured for 3 days at 37°C and 5% CO2. After 3 days of culture, the cells adhered to the surface of the petri dish, spread, and proliferated. After reaching approximately 90% confluence, culture medium 2 was removed from the petri dish (corresponding to "step (1)" described above).

[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 dish, D: average particle diameter of the microcarriers) was 35.4 cm 2 The microcarriers were placed into the petri dish with the cells attached together with 10 mL of culture medium 2, and left to stand for another day under conditions of a temperature of 37°C and a CO2 concentration of 5%, allowing the cells to come into contact with the microcarriers and transfer the cells from the petri dish to the microcarriers (corresponding to "Step A" described above). The area S of the bottom of the petri dish was 56.7 cm 2 Therefore, S>Nπ(D / 2) 2 It was confirmed that the above requirement was satisfied. Furthermore, the gaps between the microcarriers observed in the photographed range of the phase contrast microscope (plan view) were 35%.

[0071] Next, the side of the dish was gently tapped to vibrate (corresponding to "Step B" described above), and culture medium 2 containing the microcarriers into 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. The collected microcarriers were then placed in a spinner flask (product number: 3152, manufactured by Corning Incorporated), and culture medium 2 was added to a total volume of 45 mL (corresponding to "Step 12-2" described above). The microcarriers were then cultured with continuous stirring for 3 days at a temperature of 37°C and a CO2 concentration of 5%. A portion of the microcarriers after culture 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 microcarriers after culture is shown in Figure 5B. 5A and 5B correspond to the scale of 200 μm shown on the left side of FIG. 5A.

[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 proliferated until they covered the entire surface of the microcarrier.

[0073] Next, new microcarriers prepared according to the procedure described 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 collected microcarriers containing the collected culture solution 2 so that the total volume of the swollen new microcarriers and culture solution 2 was 60 mL. The microcarriers were cultured with intermittent agitation at 37°C and a CO2 concentration of 5% for 1 day, and then cultured with continuous agitation for an additional 2 days. The swollen new microcarriers and some of the microcarriers immediately after the addition of culture solution 2 were observed under a fluorescence microscope (stained with calcein-AM solution (product number: 19177-14, manufactured by Nacalai Tesque, Inc.)). Figure 5C shows fluorescence microscopic images of the swollen new microcarriers and the microcarriers immediately after the addition of culture solution 2. After culturing, some of the microcarriers were 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 microcarriers after culturing is shown in Figure 5D. Note that the scale of 200 μm indicated 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 attached and proliferated cells (i.e., microcarriers that have been cultured with cells) to microcarriers with few cells attached (i.e., newly added microcarriers).

[0075] Furthermore, this test example, like Test Example 1, 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-dissociating enzyme such as trypsin, and can significantly reduce the number of steps required in conventional culture methods (i.e., can significantly reduce the time and labor required for cell passaging). 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 the microcarrier and from one microcarrier to another 2 Using microcarriers prepared according to the procedure shown in Test Example 1, cell migration from the bottom of the culture substrate to the microcarriers and cell migration from one microcarrier to another were confirmed according to the same procedure as in 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 10% FBS-containing α-MEM at 20 ng / mL. Cell migration was confirmed in the same manner as in 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 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, the migration of cells from the bottom surface of the culture substrate to the microcarriers was confirmed. First, embryonic stem cells (ES cells) were prepared as cells. Culture medium 3 was prepared by adding 5.6% by mass of calcium chloride aqueous solution to mTeSR™ 1 (manufactured by Veritas Corporation) in an amount of 1 / 400 of the volume of mTeSR™ 1. Next, feeder cells were cultured at a density of 5.0 × 10 cells / mL using 10 mL of serum-containing DMEM on a 10 cm dish (flat bottom) coated with a 1 mg / g gelatin aqueous solution. 5 One day later, the serum-containing DMEM was removed from the dish, and 2.5 × 10 ES cells were seeded. 5 After seeding, 10 mL of culture medium 3 was added and cultured for 5 days. After 5 days of culture, it was confirmed that the cells had adhered to the surface of the dish, spread, and proliferated, and 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 Nπ(D / 2) 2 (N: total number of microcarriers placed in the dish, D: average particle diameter of the microcarriers) was 35.4 cm 2 The microcarriers were placed into the dish with the ES cells attached together with 10 mL of culture medium 3, and left to stand for another 2 days at a temperature of 37°C and a CO2 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 requirement was satisfied. Furthermore, the gaps between the microcarriers observed in the photographed range of the phase contrast microscope (plan view) were 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. Figure 6 shows a phase-contrast microscope image of the collected microcarriers. 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, microcarriers onto which cells had migrated from the bottom of a 6-well plate were transferred, together with 2 mL of culture medium 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-attached microcarriers. The cells were then contacted with the bottom of the 24-well plate at 37°C and 5% CO2 for one day, allowing the cells 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 per well. The cells were then further cultured at 37°C and 5% CO2. Figure 7B shows a phase-contrast microscope image of the bottom of the wells of the 24-well plate immediately after the microcarriers were removed. Figure 7C shows a phase-contrast microscope image of the bottom of the wells after the cells had been cultured at 37°C and 5% CO2 for one day. Meanwhile, the removed microcarriers were transferred together with 2 mL of culture medium 1 to wells of a 24-well plate coated with a separately prepared 1 mg / g gelatin aqueous solution. A phase-contrast microscopic image of the well bottom immediately after transfer is shown in Figure 7D. The microcarriers were then left in contact with the well bottom of the 24-well plate for one day at 37°C and 5% CO2, allowing the cells to migrate from the microcarriers to the well bottom.

[0081] Figures 7A and 7B show that cells attached to the microcarriers 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 maintained their proliferation potential. These results demonstrate that cells can be transferred (i.e., passaged) from one culture substrate bottom to another without treatment with cell dissociation enzymes such as trypsin. Furthermore, Figure 7D shows that cells adhered to the microcarriers removed after cell transfer into the wells of a 24-well plate. Furthermore, when the cells attached 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 transfer from microcarriers to the bottom of a culture substrate can be repeated.

[0082] Other aspects of the present disclosure relate to the following [1] to [5]. [1] 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. [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 When the total area of ​​the bottom surface of the first culture substrate is S, the total number of the microcarriers is N, and the average particle diameter of the microcarriers is D, the following formula is used: S>Nπ(D / 2) 2 The method according to [1], wherein the cells are contacted with the microcarriers so as to satisfy the above. [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 one of [1] to [3], wherein the microcarrier comprises a gel that swells with a liquid. [5] The method according to any one of [1] to [4], further comprising a transfer step of transferring the cells that have come into contact with the microcarriers in the contact step to the surface of the microcarriers. [6] The method according to [5], further comprising a first culturing step of culturing the cells that have been migrated to the surface of the microcarriers in the migration step. [7] The method according to [6], wherein the culture in the first culture step is carried out by suspending the microcarriers having the cells attached to their surfaces in a culture medium. [8] The method according to any one 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 one of [1] to [8], 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 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 the activities of these.

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

[10] , 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 according to

[11] , wherein the microcarriers contain gelatin and further contain calcium alginate.

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

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

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

[14] , further comprising a collecting step of collecting the microcarriers that have been brought into contact 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 suspending the cells adhered to the microcarriers collected in the collecting step in a culture medium and culturing the cells.

[17] a re-contacting step in which the microcarriers collected in the collecting step are placed on a second culture substrate having a bottom surface that has been treated for cell adhesion, and the cells in contact with the collected microcarriers are brought into contact with the bottom surface of the second culture substrate, thereby adhering the cells to the bottom surface of the second culture substrate; and a removing step of removing the microcarriers from the second culture substrate; The method according to

[15] , further comprising:

[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 recovery step of recovering the cells cultured in the second culture step.

[20] A method for producing a cell-containing pharmaceutical composition, comprising a step of mixing cells obtained by the method described in 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] Yet another aspect of the present disclosure provides a method for culturing cells, including a contact step of contacting cells with microcarriers on a culture substrate that has not been treated for cell adhesion or that has been treated for cell non-adhesion and that has an uneven bottom. According to this culture method, contacting cells with microcarriers on a culture substrate with an uneven bottom increases the contact opportunity (contact rate) between the microcarriers and cells, particularly on the bottom surface of the culture substrate, thereby improving the adhesion rate between cells and microcarriers. While the reason for the improved adhesion rate between cells and microcarriers in this culture method is unclear, it is hypothesized as follows: Typically, when culturing cells using microcarriers, both the cells and microcarriers are placed on 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 (see Figure 8A). Furthermore, cells, particularly adhesive cells, that contact and adhere to microcarriers have higher survival and proliferation rates, while cells that do not contact microcarriers either die or survive but experience a low 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 is within a specific range. Specifically, the culture substrate is adjusted so that the water contact angle of the bottom surface is less than 60° or more than 70°. By ensuring that the water contact angle of the bottom surface of the culture substrate is 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 comprising a contact step of contacting cells with oblate microcarriers in a culture substrate. According to this culture method, contacting the microcarriers with the cells in the culture substrate increases the chance of contact between the microcarriers (contact rate), thereby improving the 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 generally 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, if they survive, have a low proliferation rate. Even if some cells sink after the microcarriers, come into contact with the microcarriers, and adhere, the chance of contact 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 will contact and adhere to the upper part of the spherical microcarriers that sank earlier, and grow there. Furthermore, since the microcarriers have an oblate spherical shape, the area that is in close proximity to the bottom surface of the culture substrate will increase, which is thought to increase the opportunity for contact and adhesion between the cells that sank earlier and / or cells that sank without coming into contact with 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] Another aspect of the present disclosure provides a method for culturing cells, including a contact step of contacting cells with microcarriers on a culture substrate having a cell adhesion-treated, flat bottom. As shown in FIG. 11 , by using a culture substrate with a cell adhesion-treated bottom, cells in contact with the bottom adhere and proliferate (spread) on the bottom, increasing their chances of contact with microcarriers that are in contact with the bottom or are nearby but not adhered to the bottom. This is believed to result in increased cell culture efficiency. Meanwhile, as shown in FIG. 11 , cells not in contact with the bottom also adhere and proliferate on the surface of microcarriers upon contact, which is believed to increase cell culture efficiency. Thus, according to the culture method of this aspect, not only cells in contact with the bottom of the culture substrate but also cells not in contact with the bottom of the culture substrate can be contacted and proliferated by microcarriers. Therefore, according to the method of this aspect, efficient cell culture is possible without any particular restrictions on the order of seeding cells and microcarriers 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 form of the cell sheet. 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 (for example, until they reach confluence).

[0095] A cell sheet can be obtained by detaching a cell sheet cultured on a second culture substrate from the second culture substrate. Known methods can be used to detach a 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, 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 When the total area of ​​the bottom surface of the first culture substrate is S, the total number of the microcarriers is N, and the average particle diameter of the microcarriers is D, the following formula is used: S>Nπ(D / 2) 2 contacting the cells with the microcarriers so as to satisfy The method, wherein the microcarriers comprise a gel that swells with a liquid.

2. The method of claim 1 , wherein the bottom surface of the first culture substrate has a recess and a protrusion.

3. The method of 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.

4. The method according to claim 3, further comprising a first culturing step of culturing the cells that have been transferred to the surface of the microcarriers in the transferring step.

5. The method according to claim 4, wherein the culturing in the first culturing step is carried out by suspending the microcarriers having the cells attached to their surfaces in a culture medium.

6. 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.

7. 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.

8. 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.

9. 2. 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.

10. 10. The method of claim 9, wherein the microcarriers comprise gelatin and further comprise calcium alginate.

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

12. The method of claim 2 , 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.

13. 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.

14. The method according to claim 13, 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.

15. a re-contacting step in which the microcarriers collected in the collecting step are placed on a second culture substrate having a bottom surface that has been treated for cell adhesion, and the cells in contact with the collected microcarriers are brought into contact with the bottom surface of the second culture substrate, thereby adhering the cells to the bottom surface of the second culture substrate; and a removing step of removing the microcarriers from the second culture substrate; 14. The method of claim 13, further comprising:

16. The method of claim 15 , wherein the bottom surface of the second culture substrate has cell adhesive properties.

17. The method according to claim 14, further comprising a recovery step of recovering the cells cultured in the second culture step.

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

19. 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 claim 15 to form a cell sheet, and detaching the cell sheet from the second culture substrate.

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