New cell culture methods

Abstract

To provide a more efficient method for cell culture. [Solution] A method for culturing cells, comprising a contact step of bringing cells adhering to the bottom surface of a first culture substrate having a cell-adhesion treated bottom surface into contact with microcarriers.

Description

[Technical Field]

[0001] This disclosure relates to a novel cell culture method. [Background technology]

[0002] In fields such as pharmaceutical production, gene therapy, regenerative medicine, and immunotherapy, there is a need to efficiently culture cells and tissues in large quantities. Microcarrier culture is a known technique for mass culture of cells and other tissues. Microcarrier culture involves, for example, introducing cells, culture medium, and microcarriers that serve as a cell adhesion scaffold into a culture vessel, intermittently agitating the culture medium to suspend the cells and microcarriers, and allowing the suspended cells to descend and come into contact with the microcarriers, thereby adhering to the surface of the microcarriers and proliferating (see, for example, Patent Document 1). Microcarriers can provide a very large adhesion and proliferation surface area relative to their volume, which is advantageous for mass culture of cells.

[0003] Various cell culture methods using microcarriers have been known to date. For example, a method for producing adherent cells is known that involves placing adherent cells in a culture vessel containing a culture medium and microcarriers, and performing several successive cell passages in the same vessel, with all or part of the cell population of the previous generation being used for cell passage to the next generation (see, for example, Patent Document 2). In addition, a method is known in which adherent cells, microcarriers, and a culture medium are cultured while gradually scaling up the culture medium, and cells adhered to microcarriers are made to migrate and attach to other fresh microcarriers (see, for example, Patent Document 3). However, all of these culture methods require a step of treatment using cell dissociation enzymes such as trypsin, which has the problem of reducing the efficiency of cell culture. Furthermore, conventionally, when subculturing adherent cells from culture substrates such as petri dishes or flasks, it is generally necessary to go through many steps, including removal of the culture medium, washing with buffer solution, removal of the buffer solution, addition of cell dissociation enzymes, cell detachment by incubation, complete cell detachment by tapping or pipetting, inactivation of cell dissociation enzymes by adding culture medium, transfer of cells to a centrifuge tube, separation of cells and culture medium by centrifugation, removal of the culture medium, resuspension in the culture medium, preparation of the subculturing substrate, and seeding of cells onto the subculturing substrate. This requires a great deal of time and effort for cell subculturing, and it is also difficult to perform these steps in a closed system. In particular, when transferring cells from the bottom of a culture substrate such as a petri dish or flask to a microcarrier, the "preparation of the subculturing substrate" requires the suspension and / or swelling of the microcarrier in the culture medium, and the addition of the suspended and / or swollen microcarrier and culture medium to the culture substrate, which requires even more time and effort.

[0004] Furthermore, when subculturing cells by transferring them from the bottom of a culture substrate such as a petri dish or flask to a microcarrier, it is common practice to detach and collect the cells from the bottom of the culture substrate using a cell dissociation enzyme, then transfer them together with the microcarrier to another culture substrate such as a bioreactor, culture the free (non-adherent) cells together with the microcarrier, and continuously or intermittently agitate them to adhere the cells to the microcarrier. However, in order to adhere free cells to a microcarrier, it is necessary to bring the cells, which are moving with each other in the culture medium, into contact with the microcarrier, and considerable consideration is required to derive culture conditions that provide appropriate contact opportunities. In particular, in order to efficiently culture cells on the surface of a microcarrier, it is necessary to adhere the cells as evenly as possible to the surface of the microcarrier, so it is not easy to derive culture conditions that provide appropriate contact opportunities, and as a result, there has been 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 Open Brochure WO2021 / 181819 [Overview of the project] [Problems that the invention aims to solve]

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

[0007] The present inventors have discovered that in cell culture, by bringing cells adhering to the bottom surface of a first culture substrate having a cell-adhesion treated bottom surface into contact with a microcarrier, cells can be easily transferred to the microcarrier without treatment using cell-dissociating enzymes such as trypsin, thereby improving the efficiency of cell culture. This disclosure is based on these findings.

[0008] In other words, according to one aspect of this disclosure, A method for culturing cells is provided, comprising a contact step of bringing cells adhering to the bottom surface of a first culture substrate having a cell-adhesion treated bottom surface into contact with microcarriers. [Effects of the Invention]

[0009] The culture method described herein is advantageous because it allows cells, which are fixed and adhered to the bottom surface of the culture substrate, to be easily transferred to a microcarrier without treatment using cell-dissociating enzymes such as trypsin, thereby improving the efficiency of cell culture. [Brief explanation of the drawing]

[0010] [Figure 1] Figure 1A shows an image (plan view) observed with a phase-contrast microscope after the cells and microcarriers have come into contact by adding microcarriers to the first culture substrate to which cells are adhered during the contact process. Figure 1B shows an image edited with the phase-contrast microscope, with the microcarrier area (black area) and the bottom surface of the first culture substrate (gray area) color-coded. [Figure 2] This image shows a phase-contrast microscope image of a microcarrier used for cell culture. [Figure 3] This image shows a phase-contrast microscope image of a dried microcarrier for cell culture. [Figure 4]Figure 4A shows a phase-contrast microscopic image in the well immediately after the microcarriers are introduced. Figure 4B shows a phase-contrast microscopic image of the well (the well containing the adhered microcarriers) immediately after the microcarriers adhered with human adipose-derived stem cells are transferred to another well. Figure 4C shows a fluorescence microscopic image of the well to which the microcarriers adhered with human adipose-derived stem cells are transferred. [Figure 5] Figure 5A shows a fluorescence microscopic image of the microcarriers collected from the culture solution containing the microcarriers to which human adipose-derived stem cells are transferred. Figure 5B shows a fluorescence microscopic image of the microcarriers after culturing the microcarriers collected from the culture solution containing the microcarriers to which human adipose-derived stem cells are transferred. Figure 5C shows a fluorescence microscopic image of the microcarriers immediately after adding swollen new microcarriers to the culture solution containing the microcarriers to which human adipose-derived stem cells are transferred. Figure 5D shows a fluorescence microscopic image of the microcarriers after adding swollen new microcarriers to the culture solution containing the microcarriers to which human adipose-derived stem cells are transferred and culturing. Note that in both Figure 5A and 5B, the scale of 200 μm attached to the left of Figure 5A corresponds. Also, in both Figure 5C and 5D, the scale of 200 μm attached to the left of Figure 5C corresponds. [Figure 6] Figure 6 shows a phase-contrast microscopic image of the microcarriers adhered with ES cells. [Figure 7]FIG. 7A shows a phase-contrast microscopic image of the well of a 24-well plate immediately after transferring microcarriers adhered with human adipose-derived stem cells together with a culture solution into the well of a 24-well plate coated with an aqueous gelatin solution of 1 mg / g. FIG. 7B shows a phase-contrast microscopic image of the bottom surface of the well of a 24-well plate immediately after removing the microcarriers after transferring human adipose-derived stem cells from the microcarriers to the bottom surface of the well. FIG. 7C shows a phase-contrast microscopic image of the bottom surface of the well after culturing human adipose-derived stem cells on the bottom surface of the well of a 24-well plate for 1 day under conditions of a temperature of 37° C. and a CO2 concentration of 5% after removing the microcarriers. FIG. 7D shows a phase-contrast microscopic image of the bottom surface of the well immediately after transferring the removed microcarriers together with a culture solution into the well of a 24-well plate coated with an aqueous gelatin solution of 1 mg / g. [Figure 8] FIG. 8 is a schematic diagram showing the behavior of cells and microcarriers over time after cells and microcarriers are both introduced into a culture substrate when culturing cells using microcarriers. [Figure 9] FIG. 9 is a schematic diagram showing the behavior of cells and microcarriers over time after cells and microcarriers are both introduced into a culture substrate when contacting cells and microcarriers using a culture substrate having a bottom surface with irregularities that has not been subjected to cell adhesion treatment or has been subjected to non-cell adhesion treatment. [Figure 10] FIG. 10 is a schematic diagram showing the behavior of cells and microcarriers over time after cells and microcarriers are both introduced into a culture substrate when contacting cells and microcarriers using flat spherical microcarriers. [Figure 11] FIG. 11 is a schematic diagram showing the behavior of cells and microcarriers over time after cells and microcarriers are both introduced into a culture substrate when contacting cells and microcarriers using a culture substrate having a bottom surface without irregularities that has been subjected to cell adhesion treatment.

MODE FOR CARRYING OUT THE INVENTION

[0011] [Methods for culturing cells] One aspect of this disclosure provides a method for culturing cells (hereinafter also referred to as the "Culture Method of this Disclosure"), which includes a contact step as an essential step, as described below. In this disclosure, "culturing" cells includes not only causing cells to divide and proliferate, but also keeping them alive (maintaining them) without substantially causing division and proliferation. The Culture Method of this Disclosure is advantageous in that it can improve the efficiency of cell culture because it allows cells fixed in contact with the bottom surface of a culture substrate to be easily transferred to a microcarrier for cell subculturing without treatment using cell-dissociating enzymes such as trypsin. Specifically, in cell culture, it is common to treat cells adhered to a culture substrate with cell-dissociating enzymes such as trypsin, but such cell-dissociating enzymes can damage cells. For example, proteolytic enzymes can non-selectively remove receptors on the cell surface. Furthermore, in many cases, treatment with cell-dissociating enzymes requires a certain processing time and also requires centrifugation, which can cause cell damage or mutation, or reduce cell yield. The culture method disclosed herein can improve the efficiency of cell culture by reducing the time required for treatment with such cell dissociation enzymes and further suppressing the decrease in cell yield due to cell damage or mutation. Furthermore, the culture method disclosed herein moves cells to microcarriers by bringing cells that are adhered to the bottom surface of the culture substrate (i.e., fixed) into contact with the microcarriers. Therefore, it is substantially unnecessary to consider the culture conditions required to obtain an appropriate contact opportunity when bringing free cells into contact with microcarriers, which further improves the efficiency of cell culture. Moreover, the culture method disclosed herein can efficiently move cells to microcarriers by bringing cells that are adhered to the bottom surface of the culture substrate into contact with the microcarriers. Conventionally, there is a known method of performing cell culture by moving cells from one carrier to another. In this method, it is necessary to first prepare a carrier to which cells are adhered (i.e., a carrier to which cells that serve as the starting point for cell culture are adhered), but it is known that preparing such a carrier to which cells are adhered takes a considerable amount of time.The culture method disclosed herein allows for the efficient transfer of cells to microcarriers by bringing cells, which are adhered to the bottom surface of the culture substrate, into contact with the microcarriers. Thus, microcarriers to which such cells are adhered can be efficiently obtained. The steps of the culture method disclosed herein are described in detail below.

[0012] <Contact process> In the contact step, cells adhering to the bottom surface of a first culture substrate having a cell-adhesion treated bottom surface are brought into contact with the microcarrier. Specifically, the cells and microcarrier are brought into contact by adding the microcarrier to the first culture substrate to which cells are adhering. In the contact step, it is thought that the cells move from the bottom surface of the first culture substrate to the microcarrier (particularly the surface of the microcarrier) by bringing the cells adhering to the bottom surface of the first culture substrate into contact with the microcarrier. This step of moving cells from the bottom surface of the first culture substrate to the microcarrier (also referred to as the "movement step" in this specification) may be performed in parallel with the contact step or as a step independent of the contact step. In this disclosure, "movement" of cells means not only that a cell itself migrates from one place to another, but also that a cell divides and proliferates from one place to another. In other words, "cells moving from the bottom surface of the first culture substrate to the microcarrier" means not only that a cell itself migrates from the bottom surface of the first culture substrate towards the microcarrier, but also that a cell divides and proliferates from the bottom surface of the first culture substrate towards the microcarrier. The reason why cells move to the microcarrier is not clear, but it is thought that they move from areas where cells are densely packed (i.e., the bottom surface of the first culture substrate) to areas where they are less dense (i.e., the microcarrier) in order to avoid that density. In one embodiment, during the contact process, cells do not simply migrate from the bottom surface of the first culture substrate to the microcarrier, but cell division also occurs in parallel, and cells move by dividing and proliferating from areas where cells are densely packed (i.e., the bottom surface of the first culture substrate) towards areas where they are less dense (i.e., the microcarrier). Thus, since the contact process allows cells to be moved from the bottom surface of the first culture substrate to the microcarrier, it is thought that the period during which cells in the culture substrate become confluent can be controlled by appropriately adjusting the timing of contact between cells and microcarriers and the amount of microcarriers that come into contact with the cells during the contact process.For example, if cell subculturing becomes necessary while the experimenter is absent, introducing microcarriers into the culture substrate before the absence allows the cells to move to the microcarriers, thereby suppressing excessive cell proliferation (overconfluence) in the culture substrate and easily adjusting the timing of cell subculturing.

[0013] Furthermore, by appropriately adjusting the timing of contact between cells and microcarriers and the amount of microcarriers brought into contact with the cells during the contact process, the period during which cells in the culture substrate become confluent can be controlled. For example, if subculturing is required during a vacation, by adding microcarriers before the vacation, cells adhering to the culture substrate surface (e.g., petri dishes or flasks) will move towards the microcarriers. This reduces the number of cells remaining on the culture substrate surface, suppressing excessive confluence and easily extending the cell subculturing period beyond the vacation schedule.

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

[0015] The first culture substrate has at least its bottom surface treated for cell adhesion. Cell adhesion treatment refers to a treatment that improves the cell adhesion properties of the culture substrate, enabling cells to be cultured while adhering to the culture substrate. Examples include treatments that oxidize and hydrophilize the substrate surface (hydrophilization treatment). Examples of hydrophilization treatments include plasma treatment, corona discharge treatment, oxidizing agent treatment, hydrophilic substance coating treatment, and radiation treatment.

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

[0017] As the first culture substrate, a culture substrate prepared by applying the above-described cell adhesion treatment to a culture substrate that has not undergone cell adhesion treatment may be used, or a culture substrate that has been pre-treated with cell adhesion treatment may be purchased and used.

[0018] In a preferred embodiment, the first culture substrate is treated to adhere cells such that the water contact angle of its bottom surface falls within a specific range. Specifically, the first culture substrate is treated to adhere cells such that the water contact angle of its bottom surface is between 60 and 70 degrees. Having the water contact angle of the bottom surface of the first culture substrate within this range results in an appropriate cell adhesion rate, thereby further improving the efficiency of cell culture.

[0019] In a preferred embodiment, the first culture substrate has a portion of its bottom surface, preferably all of it, that includes recesses and protrusions (i.e., unevenness). The unevenness of the first culture substrate increases the contact area between the microcarriers and the surface of the culture substrate, thereby increasing the opportunities for contact between the microcarriers and cells adhering to the surface of the culture substrate, and further improving the efficiency of cell culture. Particularly preferably, the recesses on the bottom surface of the first culture substrate are hemispherical or substantially hemispherical, have a width greater than the average particle diameter of the microcarriers used, and have a depth of less than or equal to half the average particle diameter of the microcarriers used. For example, the width of the recesses is preferably such that the area of ​​the recesses when viewed from above is 40 μm². 2 More than 60,000μm 2The following settings are configured. Furthermore, the depth of the recess is preferably set to 5 μm or more and 200 μm or less.

[0020] The first culture substrate, used in the contact process, has cells attached to a cell-adhesion treated bottom surface. This substrate may be prepared by applying cell-adhesion treatment to a suitable culture substrate as needed, seeding and culturing cells, or it may be obtained from a commercially available or transferable source. In one embodiment, the first culture substrate has cells attached to a cell-adhesion treated bottom surface.

[0021] The microcarrier is not particularly limited as long as the effects of the invention of this disclosure are achieved, and microcarriers commonly used in cell culture can be used. In a preferred embodiment, the microcarrier is a microcarrier containing a gel that expands when exposed to liquid. Microcarriers containing a gel that expands when exposed to liquid are more flexible and lighter than microcarriers made of plastic that are commonly used in cell culture, and therefore, when added to a culture substrate, they can reduce the load on cells adhering to the bottom surface of the substrate. Furthermore, because the microcarrier is flexible, it is easily deformable, which can increase the contact area between the microcarrier and the bottom surface of the culture substrate, and as a result, cells adhering to the bottom surface of the culture substrate can easily move to the microcarrier. Examples of gels that expand when exposed to liquid include alginate gel, galacturonic acid gel, dextran gel, Tetra-PEG gel, and gelatin gel.

[0022] Microcarriers containing gels that expand with liquid include, for example, the dry microcarriers described in "Test Example 1" of the international publication WO2022 / 239810. Specifically, these dry microcarriers can be prepared by 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 added dropwise to the calcium chloride aqueous solution using a 32G syringe needle to prepare a calcium alginate microparticle gel. In the microparticle gel, the alginate is crosslinked by calcium ions. Next, the microparticle gel is washed with 70% ethanol and water, and the microparticle gel is collected using a cell strainer. Next, the microparticle gel is immersed in a 4% by mass aqueous solution of autoclaved alkali-treated gelatin and left at 20°C for 2 hours or more to allow the gelatin to permeate into the microparticle gel. Next, the gelatin-permeated 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 adhesion substance to improve its cell adhesion. The cell adhesion substance is not particularly limited as long as the effects of this 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 activity thereof (e.g., RGD peptide, laminin E8, etc.). The cell adhesion substance preferably includes 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 microcarriers can be set without particular limitations, as long as it is larger than the cells being cultured. For example, if the cells being cultured are of a typical size (diameter approximately 10 μm), the average particle size of the microcarriers (average particle size after expansion) can be, for example, between 10 μm and 1 mm.

[0025] The amount of microcarriers to be brought into contact with the cells can be appropriately set depending on the type and characteristics of the cells (size, shape, proliferation rate, etc.), the area of ​​the bottom surface of the first culture substrate, etc. For example, the amount per unit area of ​​the bottom surface of the first culture substrate may be 1 to 2,000,000 cells / cm². 2 It can be done this way.

[0026] In a preferred embodiment, contact between cells and microcarriers is achieved by adjusting the addition of microcarriers to achieve the specific conditions described below. Specifically, when the bottom surface of the first culture substrate is viewed from above, there are gaps 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 size of the microcarriers is D, then the following formula applies: S > Nπ(D / 2) 2 Microcarriers are added to the first culture substrate in such a manner that the above equation is satisfied, and the cells are brought into contact with the microcarriers. By adding microcarriers to the first culture substrate in such a manner that the above equation is satisfied, and bringing the cells into contact with the microcarriers, cells adhering to the bottom surface of the culture substrate can be efficiently moved to the microcarriers.

[0027] In the contact process, a method for determining whether or not gaps exist between at least some microcarriers when viewing the bottom surface of the first culture substrate in plan view will be explained with reference to Figure 1. Figure 1A is an image (plan view) observed with a phase-contrast microscope after the cells and microcarriers have been brought into contact by adding microcarriers to the first culture substrate to which cells are adhered in the bottom surface during the contact process. Figure 1B is an image edited by color-coding the image observed with a phase-contrast microscope into microcarrier areas (black areas) and the bottom surface of the first culture substrate (gray areas). In Figure 1B, if gray areas exist between at least some black areas, it is determined that gaps exist between at least some microcarriers. The amount of microcarriers to be added is determined such that the ratio of gaps between 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 microcarriers (i.e., no gray areas in Figure 1B), a large number of microcarriers will not be in contact with the bottom surface of the first culture substrate (the microcarriers will be arranged overlapping each other). Since cells cannot move to the microcarriers that are not in contact with the bottom surface of the first culture substrate, some of the microcarriers will be wasted.

[0028] Furthermore, the total area Nπ(D / 2) of the microcarriers is greater 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. Next, 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 overlapping for measurement. The average particle diameter D of the microcarriers is observed by a phase-contrast microscope, and the equivalent diameter of a sphere with the same 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. As a result, for the added microcarriers, the cells adhered to the bottom surface of the culture substrate can be efficiently transferred to the 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. As the culture solution, one based on a so-called basal medium may be used, or one based on a medium appropriately prepared according to the cells to be cultured, etc. may be used. The basal medium is not particularly limited as long as it is a commercially available basal medium. For example, MEM, α-MEM, DMEM, RPMI-1640, Hams F12, etc. can be mentioned.

[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, and the shape, size, and number of microcarriers. The contact process may be carried out under static conditions or under agitation, but it is preferably carried out under static conditions.

[0031] As cells, adherent cells that can be cultured on the bottom surface of the first culture substrate or on the surface of the microcarrier are preferably used. Examples of such cells include hepatocytes (parenchymal cells of the liver), Kupffer cells, endothelial cells such as vascular endothelial cells and corneal endothelial cells, epidermal cells such as fibroblasts, osteoblasts, osteoclasts, periodontal ligament-derived cells and 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 cardiomyocytes, renal cells, pancreatic islet cells, nerve cells such as peripheral nerve cells and optic nerve cells, chondrocytes, and osteocytes. These cells may be primary cells directly collected from tissues or organs, or they may be cells that have been passaged several times. Furthermore, these cells may be undifferentiated cells such as embryonic stem cells or iPS cells, somatic stem cells such as mesenchymal stem cells with differentiation potential, unipotent stem cells such as vascular endothelial progenitor cells with single differentiation potential, or cells whose differentiation has been completed. The cells may also be CHO cells, 293 cells, 3T3 cells, Vero cells, MRC5 cells, HeLa cells, HEK293 cells, hybridomas, etc., which are widely used as cell substrates for the production of biopharmaceuticals and viral vectors, or cell lines derived from these. In addition, one type of cell may be used, or two or more types of cells may be used. By applying the culture method disclosed herein to such cells, medical products and food products that are expected to have therapeutic effects can be manufactured.

[0032] <Preliminary culture process> The culture method of this disclosure may include a preliminary culture step in which, prior to the contact step described above, cells are seeded on a first culture substrate having a cell-adhesion treated bottom surface, and the cells are cultured on the first culture substrate.

[0033] The amount of cells seeded on the first culture substrate can be appropriately set depending on the type and characteristics of the cells, the area of ​​the bottom surface of the first culture substrate, etc. For example, the amount per unit area of ​​the bottom surface of the first culture substrate may be 100 to 100,000 cells / cm². 2 Preferably 1,000 to 10,000 pieces / cm 2 It can be done this way.

[0034] In the preliminary culture step, the culture medium used for culturing cells can be the one described in the contact step above. The same culture medium may be used in the contact step and the preliminary culture step, or different culture media may be used, 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 medium, the type, shape, and volume of the culture substrate, etc. The preliminary culture step may be carried out under static conditions or under agitation, but it is preferably carried out under static conditions so that the cells adhere to the bottom surface of the first culture substrate.

[0036] <First culture process> The culture method of this disclosure may include a first culture step of culturing cells that have moved from the bottom surface of the first culture substrate to the microcarrier in the contact step and transfer step described above.

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

[0038] The culture conditions in the first culture step can be appropriately set depending on the type and characteristics of the cells, the composition of the culture medium, the type, shape, and volume of the culture substrate, etc. The first culture step may be carried out in a static state, or in a state in which microcarriers with cells attached to their surface are suspended in the culture medium (i.e., in a suspension state), but it is preferably carried out in a suspension state.

[0039] <Collection Process> In the culture method of this disclosure, after the contact step described above, the microcarriers to which cells that have moved from the first culture substrate in the contact step have adhered are collected from the first culture substrate. The collection of microcarriers can be carried out by a method commonly used in cell culture using microcarriers. In one embodiment, the first culture substrate can be shaken to the extent that the microcarriers move in the culture medium, the first culture substrate can be tapped (lightly struck), and / or the microcarriers can be pipettered, and then the microcarriers can be aspirated to collect them. From the viewpoint of performing the collection step in a closed system, it is preferable to shake the first culture substrate and / or tap the first culture substrate (preferably from the side), and then aspirate the microcarriers. In another embodiment, by using a culture substrate that can change the detachability of adherent cells in response to temperature (i.e., a temperature-responsive culture substrate), the movement of cells from the culture substrate to the microcarriers and the dispersion of the microcarriers to which cells have adhered into the culture medium can be made easier. Examples of temperature-responsive culture substrates include Cepallet® from DIC Corporation and UpCell® from Cellseed Co., Ltd. When using these temperature-responsive culture substrates as culture media, for example, by lowering the temperature to 20°C or below during the process of transferring cells from the culture substrate to the microcarrier, the cells become more easily detached from the culture substrate, and as a result, cell transfer from the culture substrate to the microcarrier can be facilitated. In particular, temperature-responsive culture substrates allow cells to be transferred without the need for chemical cell treatment with cell dissociation enzymes or physical cell treatment such as tapping or pipetting, thus suppressing chemical and physical damage to the cells. Furthermore, by using temperature-responsive culture substrates, the collection process can be carried out in a closed system.

[0040] In the collection step, the collection of microcarriers to which cells are attached is preferably carried out in the presence of a culture medium. The culture medium can be the one described in the contact step above. The same culture medium may be used in the contact step and the collection step, or different culture media may be used, but it is preferable to use the same culture medium. The collection step may also further include adding cell dissociation enzymes, etc., to the culture substrate after the collection of microcarriers to which cells are attached, to detach and collect any cells remaining on the culture substrate, as needed. The cells detached and collected in this way (i.e., free cells not attached to microcarriers) can be used in further culture steps together with the cells that were attached to microcarriers and collected. Therefore, by detaching and collecting the free cells remaining on the culture substrate, the cell culture efficiency can be further improved. When using free cells in further culture steps, if necessary, the free cells and new microcarriers may be cultured together beforehand to allow the free cells to attach to the microcarriers and proliferate before being used in further culture steps.

[0041] As described above, the cell-bound microcarriers and free cells collected in the collection step can be used in further culture steps as needed. For example, the cell-bound microcarriers, and optionally the free cells, can be added to a second culture substrate and cultured further. 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 adding free cells to the bioreactor in addition to the cell-bound microcarriers.

[0042] When further culturing cell-bound microcarriers, and optionally free cells, by introducing them into a second culture substrate, new microcarriers (i.e., microcarriers without cell attachments) may be introduced into the second culture substrate in addition to the cell-bound and free cells. Introducing new microcarriers in addition to the cell-bound and free cells increases the number of sites where cells can adhere and proliferate, thereby improving the efficiency of cell culture. In particular, if the further culture process is carried out using a bioreactor, introducing new microcarriers into the bioreactor can further improve the efficiency of cell culture.

[0043] The bottom surface of the second culture substrate may be treated for cell adhesion. The cell adhesion treatment of the bottom surface of the second culture substrate can 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 this disclosure may include a second culture step of culturing cells attached to 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 one described in the contact step above. The same culture medium may be used in the contact step and the second culture step, or different culture media may be used, 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, and volume of the culture substrate, etc. The second culture step may be carried out in a static state, or in a state in which microcarriers with cells attached to their surface are suspended in the culture medium (i.e., in a suspension state), but it is preferably carried out in a suspension state.

[0047] As a further culture step, there is no particular limitation as long as it is a step normally performed in cell culture. For example, the re-contact step, removal step, recovery step, etc., described later can be performed. <Re-contact step> In one embodiment, when the culture method of the present disclosure includes the above-described collection step, the culture method of the present disclosure may further include a re-contact step as a further culture step. In the re-contact step, the microcarriers collected in the collection step are placed on a second culture substrate having a bottom surface subjected to cell adhesion treatment, and the cells adhered to the collected microcarriers are brought into contact with the bottom surface of the second culture substrate. Specifically, by adding microcarriers to which cells are adhered to a second culture substrate having a bottom surface subjected to cell adhesion treatment, the cells adhered to the microcarriers are brought into contact with the bottom surface of the second culture substrate. By bringing the microcarriers to which cells are adhered into contact with the bottom surface of the second culture substrate, it is considered that the cells move 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 set according to the area of the bottom surface of the second culture substrate, the size of the microcarriers, the number of cells adhered to the microcarriers, the type of cells, characteristics (size, shape, growth rate, etc.), etc. As the amount per unit area of the bottom surface of the second culture substrate, for example, it can be 1 to 2,000,000 pieces / cm 2 can be set.

[0049] In the re-contact step, the contact between the cells adhered to the microcarriers and the bottom surface of the second culture substrate is preferably performed in the presence of a culture solution. The second culture substrate may or may not be pre-filled with a culture solution. As the culture solution to be introduced into the second culture substrate, those described in the above-described contact step can be used. The same culture solution may be used in the contact step, collection step, and re-contact step, or different culture solutions may be used, but preferably the same culture solution is used.

[0050] <Removal step> In one embodiment, if the culture method of the present disclosure includes the recontact step described above, the culture method of the present disclosure may include a removal step as a further culture step. In the removal step, the microcarriers placed on the second culture substrate in the recontact step are removed. The removal of microcarriers can be carried out by methods commonly used in cell culture using microcarriers. For example, the removal can be carried out by shaking the second culture substrate so that the microcarriers move in the culture medium, tapping (lightly hitting) the second culture substrate, and / or pipetting the microcarriers, and then aspirating the microcarriers. From the viewpoint of carrying out 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), and then aspirate the microcarriers.

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

[0052] Examples of chelating agents commonly used in cell culture include ethylenediaminetetraacetic acid (EDTA), glycol etherdiaminetetraacetic 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. Furthermore, the concentration of the chelating agent is not particularly limited and can be appropriately set depending on the type of chelating agent, the type of solvent in which the chelating agent is dissolved, etc. For example, the concentration of the chelating agent can be 0.5 to 20 mM, 0.75 to 15 mM, 1 to 10 mM, etc.

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

[0054] In the removal step, the removal of microcarriers is preferably carried out in the presence of a culture medium. If the culture medium used in the recontact step is present in the second culture substrate, the removal of microcarriers may be carried out in its presence. If the culture medium used in the recontact step is not present in the second culture substrate, a separate culture medium may be added to the second culture substrate to remove the microcarriers. The culture medium added to the second culture substrate can be the one described in the contact step above. The same culture medium may be used in the contact step, collection step, recontact step and removal step, or different culture media may be used, but it is preferable to use the same culture medium.

[0055] <Recovery Process> In one embodiment, the method of the present disclosure may further include a recovery step for recovering cultured cells after the first culture step and / or second culture step described above. The recovery step is carried out, for example, by solubilizing the microcarriers used in each culture step. Solubilization of microcarriers can be carried out, for example, by adding a chelating agent to the culture medium. For example, if the culture step is carried out using a cell culture microcarrier containing alginate crosslinked with divalent or higher cations, adding a chelating agent to the culture medium allows the cell culture microcarrier and the chelating agent to react, removing the divalent or higher cations crosslinking the alginate from the alginate and solubilizing the cell culture microcarrier.

[0056] EDTA is preferably used as a chelating agent in the recovery process. Furthermore, adding alginate-degrading enzymes or proteolytic enzymes in addition to the chelating agent can hydrolyze alginic acid and gelatin, allowing for faster solubilization of microcarriers for cell culture. On the other hand, from the viewpoint of minimizing damage to cells, it is preferable not to use proteolytic enzymes. Also, from the viewpoint of safety management and cost, it is preferable not to use alginate-degrading enzymes to minimize the number of added substances. Therefore, solubilizing the microcarriers for cell culture using only a chelating agent is the most preferable method. [Examples]

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

[0058] Test Example 1: Confirmation of cell migration from the bottom surface 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, an aqueous solution of sodium alginate was added dropwise to an aqueous solution of calcium chloride using a 32G syringe needle to prepare a microparticle gel of calcium alginate with an average particle size of approximately 200 μm (measured by optical microscopy) (see Figure 2). The microparticle gel was washed with 70% ethanol and water, and collected using a cell strainer (352340, Falcon). The microparticle gel was immersed in a 4% by mass aqueous solution of autoclaved alkali-treated gelatin (G9391-100G, Sigma-Aldrich) and left at 20°C for more than 2 hours to allow the gelatin to permeate the inside of the microparticle gel. When the microparticle gel with gelatin permeation was observed with an optical microscope, the size of the microparticle gel remained at an average particle size of approximately 200 μm, and no change in shape was observed.

[0060] After collecting the microparticle gel with a cell strainer, the microparticle gel was immersed in ethanol and collected again with the cell strainer to remove the gelatin from the outside of the microparticle gel and encapsulate the gelatin within the microparticle gel. Optical microscope observation revealed that the calcium alginate microparticle gel before gelatin encapsulation was colorless, and the gelatin aqueous solution was slightly yellowish. However, after surrounding the microparticle gel with ethanol and encapsulating the gelatin, the inside of the microparticle gel was slightly yellowish, while 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, dry heat drying at 150°C for 2 hours removed the water, reducing the size to approximately 90 μm (measured by optical microscopy) and yielding powdered dried microcarriers (see Figure 3). Immediately after vacuum drying, the powder was white, but it changed to brown after dry heat drying.

[0062] Using microcarriers prepared according to the procedure described above, we confirmed the movement of cells from the bottom surface of the culture substrate to the microcarriers. First, human adipose-derived stem cells (PT-5006, manufactured by Lonza Co., Ltd.) were prepared as the cells. As culture medium 1, b-FGF (basic fibroblast growth factor, Fibroblast Spray 500, manufactured by Kaken Pharmaceutical Co., Ltd.) was added to α-MEM containing 20% ​​FBS to a concentration of 20 ng / mL, and then 5.6% by mass calcium chloride aqueous solution was added in an amount equal to 1 / 100 of 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(trademark) Cell-Culture Treated Multidishes, manufactured by Thermo Fisher SCIENTIFIC, product number: 142475), and after standing at 37°C for 1 hour, the gelatin solution was aspirated, washed once with PBS, and a gelatin-coated culture substrate was obtained. Human adipose-derived stem cells were seeded at a density of 5,000 cells / well in the wells of the obtained culture substrate, and culture medium 1 was added at a density of 2 mL / well. The cultures were then incubated at a temperature of 37°C and a CO2 concentration of 5% for 3 days. After 3 days of incubation, it was confirmed that the cells had adhered to the well surface, spread, and proliferated, reaching approximately 70% confluence. 1 mL of culture medium 1 was then removed from each well (corresponding to "Step (1)" described later).

[0063] Next, prepare the microcarriers manufactured according to the procedure described above, immerse them in the same culture medium 1 prepared separately, swell them at 37°C for 30 minutes (corresponding to "Step (12-1)" described later), and then Nπ(D / 2) 2 (N: Total number of microcarriers introduced into the well, D: Average particle size of microcarriers) is 1.25 cm 2 To achieve this, the microcarriers were placed in the wells to which the cells were attached, along with 1 mL of culture medium 1. The mixture was then left to stand for another 3 days under conditions of 37°C and 5% CO2 concentration to allow the cells to come into contact with the microcarriers and to transfer the cells from the wells to the microcarriers (corresponding to "Step (A)" described later). The total bottom surface area S of each well in the 24-well plate is 1.9 cm². 2 Therefore, S > Nπ(D / 2) 2We confirmed that the conditions were met. In addition, the gap between microcarriers observed within the imaging range of the phase-contrast microscope (planar view) was 34%.

[0064] Next, on the first day after introducing the microcarriers, we observed the 24-well plate under a phase-contrast microscope while shaking it and confirmed that the microcarriers did not move at all. This suggests that the cells adhering to the bottom surface of the culture substrate also adhered to the microcarriers, and that the cells were migrating.

[0065] Furthermore, on the third day after introducing the microcarriers, after pipetting through the well (corresponding to step (B) described later), the entire volume of 2 mL of culture medium containing the cell-attached microcarriers was collected and transferred to another well that did not contain cells (corresponding to "step (12-2)" described later). Figures 4A and 4B show phase-contrast microscope images of the well immediately after introducing the microcarriers, and the well immediately after pipetting through the well on the third day after introducing the microcarriers and transferring the cell-attached microcarriers to another well (the well that contained the cell-attached microcarriers). Figure 4C shows a fluorescence microscope image of the well to which the cell-attached microcarriers were transferred (stained using calcein-AM solution (product number: 19177-14, manufactured by Nacalai Tesque Co., Ltd.)). The black arrows in Figure 4B indicate the location in the well where the cell-attached microcarriers are presumed to have been located.

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

[0067] Furthermore, when subculturing adhesive 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) detachment of cells by incubation, (6) complete detachment of cells by tapping or pipetting, (7) inactivation of cell dissociation enzyme by adding culture medium, (8) transfer of cells to a centrifuge tube, (9) separation of cells and culture medium by centrifugation, (10) removal of culture medium, (11) resuspension in culture medium, (12) preparation of the subculturing substrate, and (13) seeding of cells into the subculturing substrate. Furthermore, if step (13) described above, "seeding cells onto the culture substrate for subculturing," involves moving cells from the bottom of a culture substrate such as a petri dish or flask to a microcarrier, then step (12) described above requires two additional steps: (12-1) suspending and / or swelling the microcarrier in the culture medium, and (12-2) adding the suspended and / or swollen microcarrier and culture medium to the culture substrate. In other words, conventional methods typically require at least 14 steps to subculture adherent cells. On the other hand, in this test example, in addition to steps (1), (12-1), and (12-2) described above, adherent cells can be subculturised in a total of five steps: step (A) moving cells adhered to the bottom of the culture substrate to a swollen microcarrier, and step (B) detaching the microcarrier to which the cells are adhered from the culture substrate. This suggests that the culture method of this disclosure can significantly reduce the number of steps required by conventional methods, and as a result, can significantly reduce the time and effort required for cell subculturing.

[0068] Test Example 2: Confirmation of cell migration from the bottom surface of the culture substrate to the microcarrier 2 Cell culture and transfer were performed in the same manner as in Test Example 1, except that the culture vessel was changed to a 6-well plate coated with a 1 mg / g gelatin aqueous solution, the number of cells seeded was changed to 20,000 cells / well, the culture period was changed to 5 days, microcarriers were added to the wells together with 6 mL of culture medium 1, and the contact period between cells and microcarriers was changed to 1 day. The total bottom surface area S of each well in the 6-well plate was 9 cm². 2 And Nπ(D / 2) 2 It is 6.25cm 2 Therefore, S > Nπ(D / 2) 2 The conditions were confirmed to be met. In addition, the gap between microcarriers observed within the imaging range of the phase-contrast microscope (planar view) was 36%. Similar to Test Example 1, when the 6-well plate was observed with a phase-contrast microscope while being shaken, it was confirmed that the microcarriers did not move at all. Immediately afterward, the same procedure as on the 3rd day after microcarrier introduction in Test Example 1 was performed, and observation results equivalent to those in Figures 4A-C were obtained. That is, it was confirmed that cells had moved from the bottom of the wells to the microcarriers. From these results, it can be seen that even when the scale of the culture vessel is increased or the contact period between cells and microcarriers is shortened, cells that were adhered to the bottom of the wells can be moved from the bottom of the culture substrate to the microcarriers without treatment with cell dissociation enzymes such as trypsin.

[0069] Test Example 3-1: Confirmation of cell migration from the bottom surface of the culture substrate to the microcarrier, and cell migration from one microcarrier to another. Using microcarriers prepared according to the procedure shown in Test Example 1, we confirmed 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. First, human adipose-derived stem cells, the same as 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 5.6% by mass calcium chloride aqueous solution at a volume of 1 / 200 of the volume after b-FGF addition. 250,000 human adipose-derived stem cells were seeded on a 10 cm petri dish (with a flat bottom) coated with 1 mg / g gelatin aqueous solution. 10 mL of culture medium 2 was added, and the cells were cultured for 3 days at a temperature of 37°C and a CO2 concentration of 5%. After 3 days of culture, it was confirmed that the cells had adhered to, spread, and proliferated on the surface of the petri dish, reaching approximately 90% confluence, and then culture medium 2 was removed from the petri dish (corresponding to "Step (1)" described above).

[0070] Next, the microcarriers prepared according to the procedure shown in Test Example 1 were immersed in the separately prepared culture medium 2 and swelled at 37°C for 30 minutes (corresponding to "Step (12-1)" described above), and Nπ(D / 2) 2 (N: Total number of microcarriers placed in the petri dish, D: Average particle size of microcarriers) is 35.4 cm 2 To achieve this, the microcarriers were placed in a petri dish containing 10 mL of culture medium 2, and left to stand for another day under conditions of 37°C and 5% CO2 concentration to allow the cells to come into contact with the microcarriers, thereby transferring the cells from the petri dish to the microcarriers (corresponding to "Step A" described above). The bottom surface area S of the petri dish was 56.7 cm². 2 Therefore, S > Nπ(D / 2) 2 We confirmed that the conditions were met. In addition, the gap between microcarriers observed within the imaging range of the phase-contrast microscope (planar view) was 35%.

[0071] Next, the sides of the petri dish were lightly tapped to create vibrations (corresponding to "Step B" described above), and culture medium 2 containing the migrated human adipose-derived stem cells was collected. A portion of the collected microcarriers was observed using a fluorescence microscope (stained with calcein-AM solution (product number: 19177-14, manufactured by Nacalai Tesque Co., Ltd.)). The fluorescence microscope image of the collected microcarriers is shown in Figure 5A. Next, the collected microcarriers were placed in a spinner flask (product number: 3152, manufactured by Corning), and culture medium 2 was added to a total volume of 45 mL (corresponding to "Step 12-2" described above). The mixture was then cultured with continuous stirring for 3 days under conditions of 37°C and a CO2 concentration of 5%. A portion of the microcarriers after culture was observed using a fluorescence microscope (stained with calcein-AM solution (product number: 19177-14, manufactured by Nacalai Tesque Co., Ltd.)). The fluorescence microscope image of the microcarriers after culture is shown in Figure 5B. Note that both Figure 5A and 5B correspond to the 200 μm scale shown to the left of Figure 5A.

[0072] Figure 5A shows that cells adhering to the bottom of the petri dish can be moved to the microcarrier without treatment with cell-dissociating enzymes such as trypsin. Figure 5B shows that the cells that moved to the microcarrier proliferated until they covered the entire surface of the microcarrier.

[0073] Next, the new microcarriers prepared according to the procedure shown in Test Example 1 were immersed in a separately prepared culture medium 2 and swollen at 37°C for 30 minutes to prepare the swollen new microcarriers. To the culture medium 2 containing the collected microcarriers, culture medium 2 was added so that the total volume of the swollen new microcarriers and culture medium 2 reached 60 mL. The culture was incubated intermittently with stirring for 1 day under conditions of 37°C and a CO2 concentration of 5%, followed by continuous incubation with stirring for another 2 days. Immediately after the addition of the swollen new microcarriers and culture medium 2, a portion of the microcarriers was observed using a fluorescence microscope (stained with calcein-AM solution (product number: 19177-14, manufactured by Nacalai Tesque Co., Ltd.)). Figure 5C shows the fluorescence microscope image of the microcarriers immediately after the addition of the swollen new microcarriers and culture medium 2. After incubation, a portion of the microcarriers was observed using a fluorescence microscope (stained with calcein-AM solution (product number: 19177-14, manufactured by Nacalai Tesque Co., Ltd.)). Fluorescence microscope images of the microcarriers after culture are shown in Figure 5D. Note that the 200 μm scale on the left of Figure 5C corresponds to both Figures 5C and 5D.

[0074] Figure 5C shows that there are microcarriers to which cells are mostly attached, and microcarriers to which cells are mostly not attached. It is presumed that the microcarriers to which cells are mostly attached are those that have been cultured together with cells, while the microcarriers to which cells are mostly not attached are newly added microcarriers. On the other hand, Figure 5D shows that in most microcarriers, cells are mostly attached to which cells are attached. These results suggest that cells are migrating from microcarriers to which cells are mostly not attached (i.e., newly added microcarriers).

[0075] Furthermore, similar to Test Example 1, this test example suggests that the culture method disclosed herein can transfer cells adhering to the bottom of a petri dish to a microcarrier without treatment with cell-dissociating enzymes such as trypsin, and that it can significantly reduce the number of steps required in conventional culture methods (i.e., significantly reduce the time and effort required for cell subculturing). In addition, since this test example involves culturing on a larger scale than Test Example 1, it suggests that the culture method disclosed herein can be implemented on a large scale. Moreover, it suggests that the method can be easily implemented in a closed system by replacing the petri dishes and spinner flasks used in this test example with containers connected to sterile-connectable tubes.

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

[0077] Test Example 4: Confirmation of cell migration from the bottom surface of the culture substrate to the microcarrier 3 Using microcarriers prepared according to the procedure shown in Test Example 1, we confirmed the migration of cells from the bottom surface of the culture substrate to the microcarriers. First, embryonic stem cells (ES cells) were prepared as the cells. Culture medium 3 was prepared by adding 5.6% by mass calcium chloride aqueous solution to mTeSR® 1 (manufactured by Veritas Corporation) at a volume of 1 / 400 of the volume of mTeSR® 1. Next, feeder cells were placed in a 10cm petri dish (flat bottom) coated with 1mg / g gelatin aqueous solution using 10mL of serum-containing DMEM, resulting in a total of 5.0 × 10⁶ cells. 5 Individual cells were seeded. After one day, the serum-containing DMEM was removed from the petri dish, and the ES cells were separated into 2.5 × 10⁶ cells. 5 Individual seeds were seeded, 10 mL of culture medium 3 was added, and the cells were cultured for 5 days. After 5 days of culture, it was confirmed that the cells had adhered to the surface of the petri dish, spread, and proliferated, and 10 mL of culture medium 3 was removed from the petri dish.

[0078] Next, microcarriers prepared according to the procedure shown in Test Example 1 were immersed in culture medium 3 prepared separately, swelled at 37°C for 30 minutes, and then Nπ(D / 2) 2 (N: Total number of microcarriers placed in the petri dish, D: Average particle size of microcarriers) is 35.4 cm 2 To achieve this, the microcarriers were placed in a petri dish containing ES cells along with 10 mL of culture medium 3. The dish was then left to stand for another 2 days at a temperature of 37°C and a CO2 concentration of 5% to allow the cells to come into contact with the microcarriers and to transfer the cells from the petri dish to the microcarriers. The bottom surface area S of the petri dish was 56.7 cm². 2 Therefore, S > Nπ(D / 2) 2 We confirmed that the conditions were met. In addition, the gap between microcarriers observed within the imaging range of the phase-contrast microscope (planar view) was 35%.

[0079] Next, the sides of the petri dish were lightly tapped to create vibrations, and culture medium 3 containing the microcarriers from which the ES cells had moved was collected. A phase-contrast microscope image of the collected microcarriers is shown in Figure 6. From Figure 6, it can be seen that the ES cells were adhered to the collected microcarriers. Therefore, it can be seen that ES cells that were adhered to the bottom of the petri dish can be moved to the microcarriers without treatment with cell-dissociating enzymes such as trypsin.

[0080] Test Example 5: Confirmation of cell migration from cell-attached microcarriers to culture substrate. In Test Example 2, microcarriers from which cells had migrated from the well bottoms of a 6-well plate were transferred to the wells of a 24-well plate coated with a 1 mg / g gelatin aqueous solution, along with 2 mL of culture medium 1. 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. Next, the cells were brought into contact with the well bottoms of the 24-well plate for 1 day under conditions of 37°C and 5% CO2 concentration to allow the cells to migrate from the microcarriers to the well bottoms. After the cells had migrated from the microcarriers to the well bottoms, the microcarriers were removed, culture medium 1 was added to a volume of 2 mL / well, and the cells were further cultured under conditions of 37°C and 5% CO2 concentration. Figure 7B shows a phase-contrast microscope image of the well bottoms of the 24-well plate immediately after the removal of the microcarriers. Next, Figure 7C shows a phase-contrast microscope image of the well bottoms of the 24-well plate after the cells on the well bottoms of the 24-well plate, after the microcarriers had been removed, had been cultured for 1 day under conditions of 37°C and 5% CO2 concentration. Meanwhile, the removed microcarriers were transferred to the wells of a 24-well plate coated with a separately prepared 1 mg / g gelatin aqueous solution, along with 2 mL of culture medium 1. A phase-contrast microscope image of the well bottom immediately after transfer is shown in Figure 7D. Next, the microcarriers and the well bottoms of the 24-well plate were kept in contact for 1 day under conditions of 37°C and 5% CO2 concentration to transfer the cells from the microcarriers to the well bottoms.

[0081] Figures 7A and 7B show that cells attached to microcarriers migrate to the bottom of the wells in a 24-well plate. Figures 7B and 7C confirm that the cells are proliferating, indicating that the migrated cells maintain their proliferative capacity during migration. These results demonstrate that cells can be moved from one culture substrate bottom to another (i.e., subcultured) without treatment with cell-dissociating enzymes such as trypsin. Furthermore, Figure 7D shows that cells remain attached to the microcarriers after they have been moved to the wells of a 24-well plate (microcarriers removed after cell migration). When the cells attached to the microcarriers shown in Figure 7D were moved to the wells of a 24-well plate for one day, the same results as those shown in Figures 7B and 7C were observed, indicating that the migration of cells from microcarriers to the bottom of the culture substrate can be repeated.

[0082] Other aspects of this disclosure relate to the following [1] to [5]. [1] A method for culturing cells, comprising a contact step of bringing cells adhering to the bottom surface of a first culture substrate having a cell-adhesion treated bottom surface into contact with microcarriers. [2] In the contact step, when the bottom surface of the first culture substrate is viewed in plan, there is a gap between at least some of the microcarriers, When S is the total surface area of ​​the bottom of the first culture substrate, N is the total number of microcarriers, and D is the average particle size of the microcarriers, the following formula is used: S > Nπ(D / 2) 2 The method according to [1], wherein the cells and the microcarriers are brought into contact such that the following conditions are met. [3] The method according to [1] or [2], wherein the bottom surface of the first culture substrate is uneven. [4] The method according to any one of [1] to [3], wherein the microcarriers include a gel that swells with a liquid. [5] The method according to any one of [1] to [4], further comprising a transfer step of moving 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 culture step of culturing the cells that have moved onto the surface of the microcarrier in the transfer step. [7] The method according to [6], wherein the culture in the first culture step is carried out by suspending the microcarriers on which the cells are attached to the surface 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 is coated with 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 activity thereof.

[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 microcarrier comprises gelatin and further comprises 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 on the bottom surface of the first culture substrate.

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

[14] , further comprising a collection step of collecting the microcarriers that have come into contact with the cells in the contact step from the first culture substrate.

[16] The method according to

[15] , further comprising a second culture step of suspending cells attached to microcarriers collected in the collection step in a culture medium and culturing the cells.

[17] A re-contact step in which the microcarriers collected in the collection step are placed on a second culture substrate having a cell-adhesion treated bottom surface, 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 Removal step of removing the microcarriers from the second culture substrate. The method described in

[15] , further including the method described in

[15] .

[18] The method according to

[17] , wherein the bottom surface of the second culture substrate is cell-adherent.

[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. A method for producing a cell-containing pharmaceutical composition, comprising the step of mixing cells obtained by any of the methods described in

[20] [1] to

[19] with a pharmaceutically acceptable excipient, solvent, or cryopreservation solution. 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 recontact step described in

[21] ,

[17] or

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

[0083] Furthermore, according to yet another aspect of this disclosure, a method for culturing cells is provided, which includes a contact step of bringing cells and microcarriers into contact on a culture substrate that is either not treated for cell adhesion or has been treated for cell non-adhesion, and has a bottom surface with irregularities. According to the culture method of this aspect, by bringing the microcarriers and cells into contact on a culture substrate having a bottom surface with irregularities, the opportunity for contact (contact rate) between the microcarriers and cells, particularly on the bottom surface of the culture substrate, can be increased, and as a result, the adhesion rate between cells and microcarriers can be improved. The reason why the adhesion rate between cells and microcarriers can be improved in the culture method of this aspect is not clear, but it can be inferred as follows. Normally, when culturing cells using microcarriers, the cells and microcarriers are introduced together into the culture substrate. In this case, since the microcarriers generally have a larger mass, the microcarriers sink to the bottom of the culture substrate first, followed by the cells (see Figure 8A). Then, for cells, especially adherent cells, the survival rate and proliferation rate are higher when they are in contact with and adhere to the microcarriers, and cells that do not come into contact with the microcarriers either die or have a low proliferation rate even if they survive. Furthermore, even if some cells sink after the microcarriers and come into contact with and adhere to them, the opportunities for contact are limited, and as a result, the efficiency of cell culture is also limited (see Figures 8B and 7C). In contrast, as shown in Figure 9, according to the culture method of this embodiment, by using a culture substrate that is not treated for cell adhesion or is treated for cell non-adhesion, and has an uneven bottom, the contact rate between the cells and microcarriers can be increased, and as a result, the efficiency of cell culture can be increased.

[0084] The cell-non-adhesion treatment of the culture substrate is not particularly limited as long as it is a treatment that makes it difficult for cells to adhere to the culture substrate, and treatments commonly used in cell culture can be used. In one embodiment, the cell-non-adhesion treatment of the culture substrate is performed so that the water contact angle of its bottom surface is within a specific range. Specifically, the culture substrate is adjusted so that the water contact angle of its bottom surface is less than 60° or greater than 70°. Having the water contact angle of the bottom surface of the culture substrate within this range results in a more appropriate cell adhesion rate, which can further improve the efficiency of cell culture.

[0085] The culture substrate used in the culture method of this embodiment may be a culture substrate prepared by applying the cell non-adhesion treatment described above, or a culture substrate having a cell non-adhesion bottom surface as described above without applying the cell non-adhesion treatment. The culture substrate may be a culture substrate prepared by applying the cell non-adhesion treatment described above to a culture substrate that has not been treated with cell non-adhesion treatment, or a culture substrate that has been treated with cell non-adhesion treatment in advance may be purchased and used, for example, a commercially available cell non-adhesion treated culture substrate.

[0086] In one embodiment, the culture substrate may be coated with a material that has low affinity to cells, in addition to the cell-non-contact treatment described above. Examples of such materials include 2-methacryloyloxyethyl phosphorylcholine (MPC) and polyethylene glycol (PEG).

[0087] The culture method of this embodiment can be carried out in the same manner as the culture method of this disclosure, except that the first culture substrate in the culture method of this 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 surface with irregularities.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 this disclosure described above.

[0088] Furthermore, according to yet another aspect of this disclosure, a method for culturing cells is provided, which includes a contact step of bringing cells into contact with flattened spherical microcarriers in a culture substrate. According to the culture method of this aspect, by bringing the microcarriers and cells into contact in the culture substrate, the opportunity for contact between the microcarriers and cells (contact rate) can be increased, and as a result, the adhesion rate between cells and microcarriers can be improved. The reason why the adhesion rate between cells and microcarriers can be improved in the culture method of this aspect is not clear, but it can be inferred as follows. Normally, when culturing cells using microcarriers, the cells and microcarriers are introduced together into the culture substrate. In this case, since the microcarriers generally have a larger mass, the microcarriers sink to the bottom of the culture substrate first, followed by the cells. Then, cells, especially adherent cells, have a higher survival rate and proliferation rate if they come into contact with and adhere to the microcarriers, while cells that do not come into contact with the microcarriers either die or have a lower proliferation rate even if they survive. In addition, although there may be some cells that sink after the microcarriers and come into contact with and adhere to the microcarriers, the opportunity for contact is limited, and as a result, the efficiency of cell culture is also limited. 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 into a flattened spherical shape, the contact rate between the two can be increased, and as a result, the efficiency of cell culture can be increased. That is, it is thought that cells that sink later come into contact with and adhere to the upper part of the flattened spherical microcarriers that sink first and proliferate. Furthermore, because the microcarriers are flattened spherical, the portion that is in close proximity to the bottom surface of the culture substrate is increased, which is thought to increase the opportunities for contact and adhesion between cells that sink first and / or cells that sink 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 spherical shape in advance may be used, or their shape may be changed to an oblate spherical shape by temporarily applying an external force to the microcarriers at least during the contact step. The method of applying an external force to the microcarriers is not particularly limited as long as it is a method that is normally used in cell culture, and examples include applying centrifugal force using a centrifuge, or applying magnetic force if 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 this disclosure, except that a flattened spherical microcarrier is used as the microcarrier in the culture method of this 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 this disclosure described above.

[0091] Furthermore, according to yet another aspect of this disclosure, a method for culturing cells is provided, which includes a contact step of bringing cells into contact with microcarriers on a culture substrate having a cell-adhesion treated bottom surface that is smooth and free of irregularities. As shown in Figure 11, by using a culture substrate having a cell-adhesion treated bottom surface, cells in contact with the bottom surface adhere to and proliferate (spread) on the bottom surface, thus increasing the opportunity for contact with microcarriers that are present in contact with the bottom surface or nearby without being attached to the bottom surface, and as a result, it is thought that the efficiency of cell culture can be increased. On the other hand, as shown in Figure 11, even cells that are not in contact with the bottom surface adhere to and proliferate on the surface by coming into contact with microcarriers, thus increasing the efficiency of cell culture. Thus, according to the culture method of this aspect, not only cells in contact with the bottom surface of the culture substrate but also cells that are not in contact with the bottom surface of the culture substrate can be brought into contact with microcarriers and proliferated. Therefore, according to the method of this aspect, it is possible to perform efficient cell culture without particularly limiting the order of seeding when bringing cells and microcarriers into contact on the culture substrate.

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

[0093] A cryopreservation solution containing a cytoprotective agent can be used. Examples of cytoprotective agents include dimethyl sulfoxide (DMSO), ethylene glycol, propylene glycol, sericin, and glycerol. The cells recovered by the recovery process described above can be suspended in the cryopreservation solution and mixed to produce a pharmaceutical composition.

[0094] Furthermore, according to yet another aspect of this disclosure, a cell sheet can be manufactured by culturing the cells that have been moved to a second culture substrate by the recontact step described above to form a cell sheet. The culture medium and culture conditions when culturing the cells that have been moved to the second culture substrate can be appropriately set according to the cell type and the desired cell sheet morphology. The cells that have been moved to the second culture substrate can be cultured on the second culture substrate until they are able to form a cell sheet (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 the cell sheet from the second culture substrate. Examples include detachment using enzymes such as dispase, or using a culture substrate that can change the detachability of adherent cells according to temperature (i.e., a temperature-responsive culture substrate) as the second culture substrate. Examples of temperature-responsive culture substrates include Cepallet® from DIC Corporation and UpCell® from CellSeed Co., Ltd.

[0096] The cell sheet detached from the second culture substrate can be used, for example, as a transplant material to reconstruct defective tissue that has lost function due to disease or injury. It is expected that the cell sheet can be cultured in large quantities by producing it using the method disclosed herein.

Claims

1. A method for culturing cells, comprising a contact step of bringing cells adhering to the bottom surface of a first culture substrate having a cell-adhesion treated bottom surface into contact with microcarriers.

2. In the contact step, when the bottom surface of the first culture substrate is viewed from above, there is a gap between at least some of the microcarriers, When S is the total surface area of ​​the bottom of the first culture substrate, N is the total number of microcarriers, and D is the average particle size of the microcarriers, the following formula is used: S>Nπ(D / 2) ２ The method according to claim 1, wherein the cells and the microcarriers are brought into contact such that the condition is met.

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

4. The method according to claim 1, wherein the microcarriers include a gel that swells with a liquid.

5. The method according to claim 1, further comprising a transfer step of moving the cells that have come into contact with the microcarrier in the contact step to the surface of the microcarrier.

6. The method according to claim 5, further comprising a first culture step of culturing the cells that have moved onto the surface of the microcarrier in the transfer step.

7. The method according to claim 6, wherein the culture in the first culture step is carried out by suspending the microcarriers, on which the cells are attached to the surface, in a culture medium.

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

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

10. The method according to claim 1, wherein the bottom surface of the first culture substrate has a coating comprising 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 activity thereof.

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

12. The method according to claim 11, wherein the microcarrier comprises gelatin and further comprises calcium alginate.

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

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

15. The method according to claim 1, further comprising a collection step of collecting the microcarriers that have come into contact with the cells in the contact step from the first culture substrate.

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

17. A re-contact step in which the microcarriers collected in the collection step are placed on a second culture substrate having a cell-adhesion treated bottom surface, 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 Removal step of removing the microcarriers from the second culture substrate. The method according to claim 15, further comprising:

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

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

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

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

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