Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

• Microcarrier culture is known as a mass culture technique for cells and other materials.
• cells, culture medium, and microcarriers that serve as a scaffold for the cells are introduced into a culture vessel, the culture medium is intermittently stirred to suspend the cells and microcarriers, and the suspended cells come into contact with the microcarriers as they descend, thereby adhering to the surface of the microcarriers and growing (see, for example, Patent Document 1).
• Microcarriers are capable of providing an extremely large adhesion and growth surface area relative to their volume, making them advantageous for mass cell cultivation.
• the inventors have discovered that in cell culture, by carrying out a contact step in which cells adhering to the bottom surface of a first culture substrate having a bottom surface treated for cell adhesion are brought into contact with microcarriers, cells can be transferred to microcarriers and easily passaged without treatment using a cell dissociation enzyme such as trypsin, thereby improving the efficiency of cell culture.
• a cell dissociation enzyme such as trypsin
• a method for culturing cells which includes a contacting step of contacting cells adhered to the bottom surface of a first culture substrate having a bottom surface treated for cell adhesion with microcarriers.
• Figure 5B shows a fluorescence microscope image of a microcarrier collected from a culture medium containing a microcarrier into which human adipose-derived stem cells were migrated and after culturing.
• Figure 5C shows a fluorescence microscope image of a microcarrier immediately after adding a new swollen microcarrier to a culture medium containing a microcarrier into which human adipose-derived stem cells were migrated.
• Figure 5D shows a fluorescence microscope image of a microcarrier after adding a new swollen microcarrier to a culture medium containing a microcarrier into which human adipose-derived stem cells were migrated and culturing.
• FIG. 6 shows a phase contrast microscope image of the microcarrier to which ES cells were attached.
• Figure 7A shows a phase-contrast microscope image of a well of a 24-well plate immediately after microcarriers with human adipose-derived stem cells attached thereto were transferred together with culture medium into the wells of a 24-well plate coated with a 1 mg/g aqueous gelatin solution.
• Figure 7B shows a phase-contrast microscope image of the well bottom of a 24-well plate immediately after the microcarriers were removed after the human adipose-derived stem cells had been transferred from the microcarriers to the well bottom.
• Figure 7C shows a phase-contrast microscope image of the well bottom of a 24-well plate after the microcarriers were removed and the human adipose-derived stem cells were cultured at 37°C and 5% CO2 for one day.
• Figure 7D shows a phase-contrast microscope image of the well bottom of a 24-well plate immediately after the removed microcarriers were transferred together with culture medium into the wells of a 24-well plate coated with a 1 mg/g aqueous gelatin solution.
• FIG. 8 is a schematic diagram showing the behavior of cells and microcarriers over time after they are both placed in a culture substrate when cells are cultured using microcarriers.
• Figure 9 is a schematic diagram showing the behavior of cells and microcarriers over time after they are both placed into a culture substrate when cells are brought into contact with microcarriers using a culture substrate that has not been treated for cell adhesion or has been treated for non-cell adhesion and has an uneven bottom.
• Figure 10 is a schematic diagram showing the behavior of cells and microcarriers over time after they are both placed into a culture substrate when cells are brought into contact with the microcarriers using oblate spherical microcarriers.
• Figure 11 is a schematic diagram showing the behavior of cells and microcarriers over time after they are both placed into a culture substrate when cells are brought into contact with microcarriers using a culture substrate with a bottom that has been treated with cell adhesion and does not have an uneven surface.
• a method for culturing cells (also referred to herein as the "culturing method of the present disclosure”) is provided, which includes a contacting step, as an essential step, as described below.
• “culturing” cells encompasses not only dividing and growing cells, but also keeping cells alive (maintaining them) without substantial cell division and growth.
• the culturing method of the present disclosure is advantageous in that it can improve the efficiency of cell culture by easily transferring cells fixed in contact with the bottom surface of a culture substrate to a microcarrier for cell passage without treatment with a cell dissociation enzyme such as trypsin.
• treatment with a cell dissociation enzyme such as trypsin is commonly used to collect cells adhered to a culture substrate, but such cell dissociation enzymes may damage the cells.
• proteolytic enzymes may non-selectively remove receptors on the cell surface.
• treatment with a cell dissociation enzyme requires a certain amount of time and centrifugation, which may cause cell damage, mutation, or reduce the cell yield.
• the culture method of the present disclosure can improve cell culture efficiency by reducing the time required for such cell-dissociating enzyme treatment and further suppressing a decrease in cell yield due to cell damage, mutation, etc.
• the culture method of the present disclosure involves contacting cells adhered to the bottom surface of a culture substrate (i.e., immobilized) with microcarriers to transfer the cells to the microcarriers. Therefore, it is essentially unnecessary to consider the culture conditions required to ensure appropriate contact between free cells and microcarriers, which also provides the further advantage of improving cell culture efficiency.
• the culture method of the present disclosure also allows cells adhered to the bottom surface of a culture substrate to be contacted with microcarriers, allowing efficient cell transfer to the microcarriers. Conventionally, cell culture methods involving transferring cells from one carrier to another are known.
• the culture method of the present disclosure allows cells adhered to the bottom surface of a culture substrate to come into contact with the microcarriers, thereby efficiently transferring the cells to the microcarriers, and therefore allows efficient production of microcarriers to which such cells are adhered.
• Each step of the culture method of the present disclosure is described in detail below.
• ⁇ Contact process> cells adhering to the bottom surface of a first culture substrate having a bottom surface treated for cell adhesion are brought into contact with microcarriers. Specifically, the cells are brought into contact with the microcarriers by adding the microcarriers to the first culture substrate having cells adhering to its bottom surface. In the contacting step, the cells adhering to the bottom surface of the first culture substrate are brought into contact with the microcarriers, which is thought to cause the cells to migrate from the bottom surface of the first culture substrate to the microcarriers (particularly, to the surface of the microcarriers).
• This step of migrating cells from the bottom surface of the first culture substrate to the microcarriers may be performed in parallel with the contacting step or as a step independent of the contacting step.
• migration of cells refers not only to the migration of a cell itself from one location to another, but also to the division and proliferation of a cell from one location to another.
• “cells migrate from the bottom surface of the first culture substrate to the microcarriers” not only means that a cell itself migrates from the bottom surface of the first culture substrate toward the microcarriers, but also means that a cell divides and proliferates from the bottom surface of the first culture substrate toward the microcarriers.
• cells migrate to the microcarriers are unclear, but it is thought that they migrate from a densely packed region (i.e., the bottom surface of the first culture substrate) toward a sparsely packed region (i.e., the microcarriers) to avoid the crowding.
• cells during the contacting step, cells not only migrate from the bottom surface of the first culture substrate to the microcarriers, but also divide and proliferate simultaneously, resulting in cell migration from a densely packed region (i.e., the bottom surface of the first culture substrate) toward a sparsely packed region (i.e., the microcarriers).
• the contacting step is thought to be capable of transferring cells from the bottom surface of the first culture substrate to the microcarriers, it is thought that the period during which cells in the culture substrate become confluent can be controlled by appropriately adjusting the timing of contacting the cells with the microcarriers and the amount of microcarriers contacted with the cells during the contacting step. For example, if cell passage becomes necessary while an experimenter is away, excessive cell proliferation (overconfluence) in the culture substrate can be suppressed by placing microcarriers in the culture substrate and transferring the cells to the microcarriers before the experimenter leaves, making it easy to adjust the timing of cell passage.
• the period during which cells in the culture substrate become confluent can be controlled by appropriately adjusting the timing of contacting the cells with the microcarriers and the amount of microcarriers contacted with the cells. For example, if passaging becomes necessary during a vacation, adding microcarriers before the vacation will cause cells adhering to the culture substrate surface (e.g., a petri dish or flask) to migrate toward the microcarrier side, reducing the number of cells remaining on the culture substrate surface and preventing excessive confluence, and allowing the cell passaging period to be easily extended beyond the vacation date.
• the culture substrate surface e.g., a petri dish or flask
• the culture substrate for contacting cells with microcarriers is not particularly limited as long as it is a container-shaped culture substrate with a bottom, and examples that can be used include petri dishes and flasks.
• Cell adhesion treatment refers to a treatment that improves the cell adhesiveness of the culture substrate, allowing cells to be cultured while adhering to the culture substrate.
• this treatment may involve oxidizing the substrate surface to make it hydrophilic (hydrophilization treatment).
• hydrophilization treatments include plasma treatment, corona discharge treatment, oxidizing agent treatment, hydrophilic substance coating treatment, and radiation treatment.
• 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 spreadability.
• matrices include type I collagen, type IV collagen, fibronectin, laminin, Matrigel, gelatin, elastin, proteoglycan, vitronectin, and peptides and protein domains that possess these activities (e.g., RGD peptide, laminin E8, etc.).
• the first culture substrate may be a culture substrate prepared by subjecting a non-cell-adhesion-treated culture substrate to the cell adhesion treatment described above, or a culture substrate that has already been subjected to a cell adhesion treatment, such as a commercially available culture substrate that has already been subjected to a cell adhesion treatment, may be purchased and used.
• the first culture substrate is subjected to a cell adhesion treatment so that the water contact angle of its bottom surface is within a specific range. Specifically, the first culture substrate is subjected to a cell adhesion treatment so that the water contact angle of its bottom surface is between 60 degrees and 70 degrees. By having the water contact angle of the bottom surface of the first culture substrate within this range, the cell adhesion rate becomes appropriate, and the efficiency of cell culture can be further improved.
• the first culture substrate has at least a portion, preferably the entire bottom surface, including concave and convex portions (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 contact opportunity between the microcarriers and cells adhered to the surface of the culture substrate and further improving the efficiency of cell culture.
• the concave portions on the bottom surface of the first culture substrate are preferably hemispherical or approximately hemispherical, with a width greater than the average particle diameter of the microcarriers used and a depth equal to or less than half the average particle diameter of the microcarriers used.
• the first culture substrate used in the contacting step, having cells adhered to its cell adhesion-treated bottom surface may be prepared by subjecting an appropriate culture substrate to cell adhesion treatment as necessary, seeding the cells, and culturing them, or it may be a commercially available or assigned product.
• the first culture substrate having cells adhered to its cell adhesion-treated bottom surface can be prepared by the method described in the culturing step below.
• microcarrier containing a gel that expands in response to liquid is the dry microcarrier described in "Test Example 1" of International Publication WO 2022/239810.
• This dry microcarrier can be prepared according to the following procedure. First, a 1% by weight aqueous solution of sodium alginate and a 1% by weight aqueous solution of calcium chloride are prepared. Next, the sodium alginate solution is dripped into the calcium chloride solution using a 32G syringe needle to produce a calcium alginate microparticle gel. In the microparticle gel, the alginate is crosslinked by calcium ions. The microparticle gel is then washed with 70% ethanol and water and collected using a cell strainer.
• microparticle gel is then immersed in a 4% by weight aqueous solution of autoclaved alkali-treated gelatin and left at 20°C for at least two hours to allow the gelatin to penetrate the interior of the microparticle gel.
• the gelatin-soaked microparticle gel is then vacuum-dried, followed by dry-heat drying at 150°C for two hours to obtain a powdered dry microcarrier.
• the microcarrier may contain a cell adhesive substance to improve its cell adhesiveness.
• the cell adhesive substance is not particularly limited as long as the effects of the present disclosure are achieved, but examples include type I collagen, type IV collagen, fibronectin, laminin, Matrigel, gelatin, elastin, proteoglycan, vitronectin, and peptides and protein domains having the activities of these substances (e.g., RGD peptide, laminin E8, etc.).
• the cell adhesive substance preferably contains at least one substance selected from the group consisting of type I collagen, type IV collagen, fibronectin, laminin, Matrigel, and gelatin.
• the number Y of all microcarriers observed using a phase-contrast microscope is measured, and the number of microcarriers per weight is determined based on the formula (YL/X)/W.
• the total number N of microcarriers is determined from the total mass of the microcarriers used. Note that when adding microcarriers to the observation container, the bottom area of the observation container and the amount of microcarriers added are adjusted so that at least 10 microcarriers can be observed without overlapping.
• the average particle diameter D of the microcarriers is determined by observing the microcarriers with a phase-contrast microscope, measuring the equivalent sphere diameter of all observed microcarriers, and averaging the measured diameters to determine the average particle diameter D.
• the total area N ⁇ (D/2) 2 of the microcarriers is smaller than the total area S of the bottom surfaces of the first culture substrate, most of the microcarriers come into contact with the bottom surface of the first culture substrate, and as a result, cells adhering to the bottom surface of the culture substrate can be efficiently transferred to the microcarriers when they are added.
• the cells preferably used are adherent cells that can be cultured on the bottom surface of the first culture substrate or on the surface of microcarriers.
• examples of such cells include hepatocytes (liver parenchymal cells), Kupffer cells, endothelial cells such as vascular endothelial cells and corneal endothelial cells, fibroblasts, osteoblasts, osteoclasts, periodontal ligament-derived cells, epidermal cells such as epidermal keratinocytes, epithelial cells such as tracheal epithelial cells, gastrointestinal epithelial cells, cervical epithelial cells, and corneal epithelial cells, mammary gland cells, pericytes, myoblasts, myotubes, satellite cells, muscle cells such as smooth muscle cells and cardiac muscle cells, kidney cells, pancreatic islet cells of Langerhans, nerve cells such as peripheral nerve cells and optic nerve cells, chondrocytes, and bone cells.
• hepatocytes liver parenchymal cells
• These cells may be primary cells directly collected from tissues or organs, or may be cells that have been passaged for several generations. Furthermore, these cells may be undifferentiated cells such as embryonic stem cells or iPS cells, somatic stem cells such as mesenchymal stem cells with differentiation potential, unipotent stem cells such as vascular endothelial progenitor cells with unidifferentiation potential, or cells that have completed differentiation.
• the cells may also be CHO cells, 293 cells, 3T3 cells, Vero cells, MRC5 cells, HeLa cells, HEK293 cells, hybridoma cells, etc., which are widely used as cell substrates for the production of biopharmaceuticals and viral vectors, as well as established cell lines derived from these cells.
• the cells may be a single type of cell, or two or more types of cells.
• the culture method of the present disclosure may include a pre-culture step, prior to the contact step, in which cells are seeded on a first culture substrate having a bottom surface treated for cell adhesion and the cells are cultured on the first culture substrate.
• the amount of cells to be seeded on the first culture substrate can be appropriately determined depending on the type and characteristics of the cells, the area of the bottom surface of the first culture substrate, etc., and can be, for example, 100 to 100,000 cells/cm 2 , preferably 1,000 to 10,000 cells/cm 2 , as the amount per area of the bottom surface of the first culture substrate.
• the culture medium used to culture the cells can be the same as that described in the contact step above.
• the same or different culture medium may be used in the contact step and preliminary culture step, but it is preferable to use the same culture medium.
• the culture conditions for the preliminary culture step can be set appropriately depending on the type and characteristics of the cells, the composition of the culture medium, and the type, shape, and volume of the culture substrate.
• the preliminary culture step may be performed in a static or agitated state, but is preferably performed in a static state so that the cells adhere to the bottom surface of the first culture substrate.
• the culture method of the present disclosure may include a first culture step of culturing the cells that have been transferred from the bottom surface of the first culture substrate to the microcarriers in the contact step and transfer step described above.
• the culture medium used to culture the cells can be the same as that described in the contact step above.
• the same or different culture medium may be used in the contact step and the first culture step, but it is preferable to use the same culture medium.
• the culture conditions for the first culture step can be set appropriately depending on the type and characteristics of the cells, the composition of the culture medium, and the type, shape, and volume of the culture substrate.
• the first culture step may be performed in a static state, or in a state where microcarriers with cells attached to their surface are suspended in the culture medium (i.e., in a floating state), but is preferably performed in a floating state.
• microcarriers to which cells that migrated from the first culture substrate in the contacting step have adhered are collected from the first culture substrate.
• the collection of microcarriers can be performed by a method commonly used in cell culture using microcarriers.
• the collection can be performed by shaking the first culture substrate to the extent that the microcarriers move in the culture medium, tapping (lightly tapping) the first culture substrate and/or pipetting the microcarriers, and then aspirating the microcarriers. From the viewpoint of performing the collection step in a closed system, it is preferable to shake the first culture substrate and/or tap the first culture substrate (preferably from the side), and then aspirate the microcarriers.
• a culture substrate that can change the detachment property of adherent cells depending on the temperature i.e., a temperature-responsive culture substrate
• a temperature-responsive culture substrate can more easily migrate cells from the culture substrate to the microcarriers and disperse the microcarriers to which the cells have adhered into the culture medium.
• temperature-responsive culture substrates include Cepallet (registered trademark) manufactured by DIC Corporation and UpCell (registered trademark) manufactured by CellSeed Co., Ltd.
• temperature-responsive culture substrates When using these temperature-responsive culture substrates as culture substrates, for example, by keeping the temperature below 20°C during the process of transferring cells from the culture substrate to microcarriers, the cells become more easily detached from the culture substrate, thereby facilitating the transfer of cells from the culture substrate to the microcarriers.
• temperature-responsive culture substrates allow cells to be transferred without the need for chemical cell treatment using cell dissociation enzymes or physical cell treatment such as tapping or pipetting, thereby suppressing chemical and physical invasion of cells.
• the use of a temperature-responsive culture substrate allows the collection process to be performed in a closed system.
• collection of the cell-adhered microcarriers is preferably carried out in the presence of a culture medium.
• the culture medium described in the contact step above can be used.
• the same or different culture mediums may be used in the contact and collection steps, but the same culture medium is preferably used.
• the collection step may further include, as necessary, adding a cell dissociation enzyme or the like to the culture substrate after collection of the cell-adhered microcarriers, thereby detaching and collecting the cells remaining on the culture substrate.
• the detached and collected cells i.e., free cells not attached to the microcarriers
• the cell culture efficiency can be further improved.
• the free cells may be cultured together with new microcarriers in advance to allow them to attach and proliferate on the microcarriers before being subjected to the further culture step.
• the cell-adhered microcarriers and free cells collected in the collection step can be subjected to a further culture step, if necessary.
• the cell-adhered microcarriers, and optionally the free cells can be placed on a second culture substrate and further cultured.
• the second culture substrate used in the further culture step is not particularly limited, but examples include petri dishes, flasks, bioreactors, etc.
• the cell culture efficiency can be further improved by placing the free cells in addition to the cell-adhered microcarriers into the bioreactor.
• new microcarriers i.e., microcarriers without cells attached
• the space available for cell attachment and proliferation is increased, improving cell culture efficiency.
• adding new microcarriers to the bioreactor can further improve cell culture efficiency.
• the bottom surface of the second culture substrate may be treated for cell adhesion.
• the cell adhesion treatment on the bottom surface of the second culture substrate may be the same as the cell adhesion treatment on the bottom surface of the first culture substrate described above.
• the culture method of the present disclosure may include a second culture step of culturing the cells adhered to the microcarriers collected in the collection step described above.
• the culture medium used to culture the cells can be the same as that described in the contact step above.
• the same or different culture medium may be used in the contact step and the second culture step, but it is preferable to use the same culture medium.
• the culture conditions for the second culture step can be set appropriately depending on the type and characteristics of the cells, the composition of the culture medium, and the type, shape, and volume of the culture substrate.
• the second culture step may be performed in a static state, or in a state where microcarriers with cells attached to their surfaces are suspended in the culture medium (i.e., in a floating state), but is preferably performed in a floating state.
• the further culture step is not particularly limited as long as it is a step that is normally carried out in cell culture, and for example, a re-contact step, a removal step, a recovery step, etc., which will be described later, can be carried out.
• the culture method of the present disclosure may further include a re-contacting step as a culture step.
• the re-contacting step the microcarriers collected in the collection step are placed on a second culture substrate having a bottom surface treated for cell adhesion, and the cells adhering to the collected microcarriers are brought into contact with the bottom surface of the second culture substrate.
• the microcarriers to which cells have adhered are added to the second culture substrate having a bottom surface treated for cell adhesion, thereby bringing the cells adhering to the microcarriers into contact with the bottom surface of the second culture substrate. It is believed that contacting the microcarriers to which cells have adhered with the bottom surface of the second culture substrate causes the cells to migrate from the microcarriers to the bottom surface of the second culture substrate.
• the amount of microcarriers to be brought into contact with the second culture substrate can be appropriately determined depending on the area of the bottom surface of the second culture substrate, the size of the microcarriers, the number of cells adhering to the microcarriers, the type and characteristics (size, shape, growth rate, etc.) of the cells, and the amount per area of the bottom surface of the second culture substrate can be, for example, 1 to 2,000,000 cells/ cm2 .
• contact between the cells adhering to the microcarriers and the bottom surface of the second culture substrate is preferably carried out in the presence of a culture medium.
• the second culture substrate may or may not have culture medium pre-filled.
• the culture medium poured into the second culture substrate can be the same as that described above for the contacting step.
• the same or different culture medium may be used in the contacting step, collecting step, and re-contacting step, but preferably the same culture medium is used.
• the culture method of the present disclosure may also include a removal step as a further culture step.
• the microcarriers placed on the second culture substrate in the re-contacting step are removed.
• the microcarriers can be removed by a method commonly used in cell culture using microcarriers.
• the microcarriers can be removed by shaking the second culture substrate to an extent that the microcarriers move in the culture medium, tapping (lightly tapping) the second culture substrate and/or pipetting the microcarriers, and then aspirating the microcarriers. From the viewpoint of performing the removal step in a closed system, it is preferable to shake the second culture substrate and/or tap the second culture substrate (preferably from the side), and then aspirate the microcarriers.
• Microcarriers can be removed from the second culture substrate by methods commonly used in cell culture using microcarriers.
• the alginate when using microcarriers with a structure in which alginate is cross-linked with divalent or higher cations, the alginate can be de-crosslinked by using a substance that inhibits the cross-linking by competing with the divalent or higher cations that contribute to the cross-linking, resulting in the dissolution and removal of the microcarrier.
• substances that inhibit cross-linking include chelating agents and monovalent cations. These cross-linking inhibitors may be used alone or in combination of two or more.
• chelating agents commonly used in cell culture include ethylenediaminetetraacetic acid (EDTA), glycol ether diaminetetraacetic acid (EGTA), 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), N'-(2-hydroxyethyl)ethylenediamine-N,N,N'-triacetic acid (HEDTA), and nitrilotriacetic acid (NTA).
• EDTA ethylenediaminetetraacetic acid
• EGTA glycol ether diaminetetraacetic acid
• BAPTA 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
• HEDTA N'-(2-hydroxyethyl)ethylenediamine-N,N,N'-triacetic acid
• NTA nitrilotriacetic acid
• EDTA is particularly preferred.
• the concentration of the chelating agent is not particularly limited and can be set appropriately depending on the type of chelating agent, the type of solvent in which the chelating agent is dissolved, and other factors.
• the concentration of the chelating agent can be, for example, 0.5 to 20 mM, 0.75 to 15 mM, or 1 to 10 mM.
• Examples of monovalent cations include sodium ions and potassium ions.
• the monovalent cations may be dissolved in a solvent that does not contain the monovalent cations, or in a solvent that contains the monovalent cations.
• the monovalent cations may also be used in the form of a buffer solution (e.g., PBS) that contains the monovalent cations.
• PBS buffer solution
• the concentration of the monovalent cations is not particularly limited and can be set appropriately depending on the type of monovalent cation, the type of solvent in which the monovalent cations are dissolved, and other factors.
• the concentration of the monovalent cations can be, for example, 10 to 500 mM, 30 to 300 mM, 50 to 200 mM, etc.
• microcarriers are preferably removed in the presence of culture medium. If the culture medium used in the re-contact step is present in the second culture substrate, microcarriers may be removed in its presence. If the culture medium used in the re-contact step is not present in the second culture substrate, microcarriers may be removed by adding a separate culture medium to the second culture substrate.
• the culture medium added to the second culture substrate can be the same as that described above for the contact step. The same culture medium may be used in the contact step, collection step, or re-contact step and the removal step, or different culture mediums may be used, but the same culture medium is preferably used.
• the method of the present disclosure may further include a recovery step of recovering the cultured cells after the first culture step and/or the second culture step.
• the recovery step may be carried out, for example, by solubilizing the microcarriers used in each culture step.
• the microcarriers may be solubilized, for example, by adding a chelating agent to the culture medium.
• adding a chelating agent to the culture medium causes the microcarriers for cell culture to react with the chelating agent, removing the divalent or higher cations cross-linking the alginic acid from the alginic acid, thereby solubilizing the microcarriers for cell culture.
• the microparticle gel was immersed in ethanol and collected with a cell strainer, eliminating the gelatin outside the microparticle gel and trapping the gelatin within the microparticle gel.
• Optical microscope observation revealed that the calcium alginate microparticle gel was colorless before the gelatin was trapped, and the gelatin aqueous solution was slightly yellowish.
• the inside of the microparticle gel was slightly yellowish and the outside of the microparticle gel was colorless, confirming that gelatin had been trapped inside the microparticle gel.
• microcarriers prepared according to the above-mentioned procedure, the migration of cells from the bottom surface of the culture substrate to the microcarriers was confirmed.
• human adipose-derived stem cells (PT-5006, manufactured by Lonza Inc.) were prepared as cells.
• Culture solution 1 was prepared by adding b-FGF (basic fibroblast growth factor, Fibroblast Spray 500, manufactured by Kaken Pharmaceutical Co., Ltd.) to ⁇ -MEM containing 20% FBS to a concentration of 20 ng/mL, and further adding a 5.6% by mass aqueous calcium chloride solution in an amount of 1/100 of the volume after addition of b-FGF.
• b-FGF basic fibroblast growth factor, Fibroblast Spray 500, manufactured by Kaken Pharmaceutical Co., Ltd.
• microcarriers prepared according to the procedure described above were immersed in the same culture medium 1 prepared separately and allowed to swell at 37°C for 30 minutes (corresponding to "Step (12-1)” described below).
• the microcarriers were then placed into the wells containing the cells together with 1 mL of culture medium 1 so that N ⁇ (D/2) 2 (N: total number of microcarriers placed in the well, D: average particle size of the microcarriers) was 1.25 cm 2.
• N total number of microcarriers placed in the well
• D average particle size of the microcarriers
• FIGS. 4A and 4B show phase-contrast microscope images of the wells immediately after the microcarriers were added, and of the wells (which contained microcarriers with cells attached) immediately after the wells were pipetted three days after the microcarriers were added and the cell-adhered microcarriers were transferred to another well.
• Figure 4C also shows a fluorescence microscope image (stained with calcein-AM solution (product number: 19177-14, manufactured by Nacalai Tesque, Inc.)) of the wells to which the cell-adhered microcarriers were transferred.
• the black arrow in Figure 4B indicates the position in the well where the cell-adhered microcarriers are presumed to have been present.
• Figure 4B shows that cells are no longer present in the location where the microcarriers with cells attached were assumed to have been present.
• Figures 4A and 4C show that cells have migrated from the bottom of the well to the microcarriers. These results demonstrate that cells that had been attached to the bottom of the well can be migrated to the microcarriers without treatment with cell dissociation enzymes such as trypsin.
• Test Example 2 Confirmation of cell migration from the bottom 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 seeding number of cells was changed to 20,000 cells/well, the culture period was changed to 5 days, the microcarriers were added to the wells together with 6 mL of culture medium 1, and the contact period between the cells and the microcarriers was changed to 1 day.
• the total area S of the bottom surface of each well of the 6-well plate was 9 cm2 , and N ⁇ (D/2) 2 was 6.25 cm2 , confirming that S > N ⁇ (D/2) 2 .
• the gap between the microcarriers observed in the photographed area (planar view) using a phase-contrast microscope was 36%.
• the 6-well plate was shaken and observed under a phase-contrast microscope, confirming that the microcarriers did not move at all.
• the same procedure as in Test Example 1 performed three days after adding the microcarriers, yielded observation results equivalent to those shown in Figures 4A-C. That is, it was confirmed that cells had migrated from the bottom of the well to the microcarriers.
• Test Example 3-1 Confirmation of cell migration from the bottom of the culture substrate to the microcarrier and from one microcarrier to another 1 Using microcarriers prepared according to the procedure shown in Test Example 1, the migration of cells from the bottom surface of the culture substrate to the microcarriers and the migration of cells from one microcarrier to another were confirmed. First, human adipose-derived stem cells identical to those used in Test Example 1 were prepared.
• Culture medium 2 was prepared by adding b-FGF (basic fibroblast growth factor, Fibroblast Spray 500, manufactured by Kaken Pharmaceutical Co., Ltd.) to ⁇ -MEM containing 10% FBS at a concentration of 20 ng/mL, and then adding a 5.6% by mass calcium chloride aqueous solution at 1/200 the volume after b-FGF addition.
• 250,000 human adipose-derived stem cells were seeded onto a 10 cm dish (flat bottom) coated with a 1 mg/g gelatin aqueous solution, and 10 mL of culture medium 2 was added. The cells were cultured for three days at 37°C and 5% CO2 . After three days of culture, the cells adhered to the surface of the dish, spread, and proliferated. After confirming that the cells had reached approximately 90% confluence, culture medium 2 was removed from the dish (corresponding to the above-mentioned "step (1)").
• b-FGF basic fibroblast growth factor, Fibroblast Spray 500, manufactured by Kaken Pharmaceutical Co
• microcarriers prepared according to the procedure shown in Test Example 1 were prepared, immersed in separately prepared culture solution 2, and allowed to swell at 37°C for 30 minutes (corresponding to the above-mentioned "step (12-1)").
• the microcarriers were then placed into the cell-adhered dish together with 10 mL of culture solution 2 so that N ⁇ (D/2) 2 (N: total number of microcarriers placed in the dish, D: average particle size of the microcarriers) was 35.4 cm 2.
• microcarriers were then left to stand for another day at 37°C and a CO 2 concentration of 5% to allow contact between the cells and the microcarriers, and the cells were transferred from the dish to the microcarriers (corresponding to the above-mentioned "step A").
• the area S of the bottom of the dish was 56.7 cm 2 , confirming that S > N ⁇ (D/2) 2 was satisfied.
• the gap between the microcarriers observed within the imaging range of phase-contrast microscopy (planar view) was 35%.
• Step 12-2 the collected microcarriers were placed in a spinner flask (product number: 3152, manufactured by Corning Incorporated), and culture medium 2 was added so that the total volume became 45 mL (corresponding to the above-mentioned "Step 12-2"), and the microcarriers were cultured with continuous stirring for 3 days under conditions of a temperature of 37°C and a CO2 concentration of 5%.
• a portion of the microcarriers after culture was observed under a fluorescence microscope (stained with calcein-AM solution (product number: 19177-14, manufactured by Nacalai Tesque, Inc.)).
• a fluorescence microscope image of the microcarriers after culture is shown in Figure 5B.
• 5A and 5B correspond to the scale of 200 ⁇ m shown on the left side of FIG. 5A.
• Figure 5A shows that cells adhered to the bottom of a petri dish can be transferred to a microcarrier without treatment with a cell dissociation enzyme such as trypsin.
• Figure 5B also shows that the cells transferred to the microcarrier proliferated until they covered the entire surface of the microcarrier.
• new microcarriers prepared according to the procedure shown in Test Example 1 were immersed in separately prepared culture solution 2 and allowed to swell at 37°C for 30 minutes to prepare swollen new microcarriers.
• Culture solution 2 was added to the above culture solution 2 containing the collected microcarriers so that the total volume of the swollen new microcarriers and culture solution 2 was 60 mL, and the microcarriers were cultured with intermittent agitation at 37°C and 5% CO2 for 1 day, followed by continuous agitation for an additional 2 days.
• Figure 5C shows that there are microcarriers with cells attached over most of their surface, and microcarriers with few cells attached. It is expected that the microcarriers with cells attached over most of their surface are microcarriers that have been cultured with cells, while the microcarriers with few cells attached are newly added microcarriers. On the other hand, Figure 5D shows that cells are attached over most of the microcarriers. These results suggest that cells are migrating from microcarriers with attached and proliferating cells (i.e., microcarriers that have been cultured with cells) to microcarriers with few cells attached (i.e., newly added microcarriers).
• this Test Example also suggests that the culture method of the present disclosure can transfer cells adhered to the bottom of a petri dish to a microcarrier without treatment with a cell-dissociating enzyme such as trypsin, and that the number of steps required in conventional culture methods can be significantly reduced (i.e., the time and labor required for cell passaging can be significantly reduced). Furthermore, since culture was performed on a larger scale in this Test Example than in Test Example 1, it suggests that the culture method of the present disclosure can also be performed on a large scale. It also suggests that the culture method can be easily performed in a closed system by replacing the petri dishes and spinner flasks used in this Test Example with containers connected to tubing that can be sterilely connected.
• a cell-dissociating enzyme such as trypsin
• Test Example 3-2 Confirmation of cell migration from the bottom of the culture substrate to the microcarriers and from one microcarrier to another 2 Using microcarriers prepared according to the procedure shown in Test Example 1, cell migration from the bottom of the culture substrate to the microcarriers and cell migration from one microcarrier to another were confirmed following the same procedure as in Test Example 3-1, except that the composition of the culture medium was changed. Specifically, culture medium 2' was prepared by adding b-FGF (basic fibroblast growth factor, Fibroblast Spray 500, manufactured by Kaken Pharmaceutical Co., Ltd.) to ⁇ -MEM containing 10% FBS at 20 ng/mL.
• b-FGF basic fibroblast growth factor, Fibroblast Spray 500, manufactured by Kaken Pharmaceutical Co., Ltd.
• Test Example 4 Confirmation of cell migration from the bottom of the culture substrate to the microcarrier 3 Using microcarriers prepared according to the procedure shown in Test Example 1, the migration of cells from the bottom surface of the culture substrate to the microcarriers was confirmed.
• ES cells embryonic stem cells
• Culture medium 3 was prepared by adding 5.6% by mass of calcium chloride aqueous solution to mTeSRTM 1 (manufactured by Veritas Corporation) in an amount of 1/400 of the volume of mTeSRTM 1.
• 5.0 x 10 5 feeder cells were seeded using 10 mL of serum-containing DMEM onto a 10 cm dish (flat bottom) coated with a 1 mg/g gelatin aqueous solution.
• the serum-containing DMEM was removed from the dish, and 2.5 x 10 5 ES cells were seeded. 10 mL of culture medium 3 was added and the cells were cultured for five days. After five days of culture, it was confirmed that the cells had adhered to the surface of the dish, spread, and proliferated, and 10 mL of culture medium 3 was removed from the dish.
• microcarriers prepared according to the procedure shown in Test Example 1 were prepared, immersed in separately prepared culture solution 3, and allowed to swell at 37°C for 30 minutes. After that, the microcarriers were placed into the petri dish with the adhered ES cells together with 10 mL of culture solution 3 so that N ⁇ (D/2) 2 (N: total number of microcarriers placed in the petri dish, D: average particle diameter of the microcarriers) was 35.4 cm2 . The microcarriers were then left to stand for two more days at a temperature of 37°C and a CO2 concentration of 5% to allow contact between the cells and the microcarriers and to allow the cells to migrate from the petri dish to the microcarriers.
• the area S of the bottom of the petri dish was 56.7 cm2 , confirming that S > N ⁇ (D/2) 2 was satisfied. Furthermore, the gap between the microcarriers observed within the imaging range of phase-contrast microscopy (planar view) was 35%.
• FIG. 6 shows a phase-contrast microscope image of the collected microcarriers.
• Figure 6 shows that ES cells had adhered to the collected microcarriers. This demonstrates that ES cells that had adhered to the bottom of the dish can be transferred to the microcarriers without treatment with a cell-dissociating enzyme such as trypsin.
• Test Example 5 Confirmation of cell migration from cell-adhered microcarriers to a culture substrate
• the microcarriers to which cells had migrated from the bottom of the wells of a 6-well plate were transferred, together with 2 mL of culture medium 1, to the wells of a 24-well plate coated with a 1 mg/g gelatin aqueous solution.
• a phase-contrast microscope image of the wells of the 24-well plate immediately after the transfer of the cell-adhered microcarriers is shown in Figure 7A.
• the cells were then contacted with the bottom of the wells of the 24-well plate for one day at a temperature of 37°C and a CO2 concentration of 5%, allowing the cells to migrate from the microcarriers to the bottom of the wells.
• the microcarriers were removed, and culture medium 1 was added to a volume of 2 mL/well. The cells were further cultured at a temperature of 37°C and a CO2 concentration of 5%.
• a phase-contrast microscope image of the bottom of the wells of the 24-well plate immediately after the microcarriers were removed is shown in Figure 7B.
• the cells on the bottom of the wells of the 24-well plate were cultured for one day at 37°C and 5% CO2 , and a phase-contrast microscope image of the well bottom is shown in Figure 7C.
• microcarriers along with 2 mL of Culture Solution 1, were transferred to wells of a 24-well plate coated with a separately prepared 1 mg/g aqueous gelatin solution.
• a phase-contrast microscope image of the well bottom immediately after transfer is shown in Figure 7D.
• the microcarriers were then left in contact with the bottom of the wells of the 24-well plate for one day at 37°C and 5% CO2 , allowing the cells to migrate from the microcarriers to the bottom of the wells.
• Figures 7A and 7B show that cells attached to the microcarriers have migrated to the bottom of the wells of a 24-well plate.
• Figures 7B and 7C also confirm that the cells are proliferating, demonstrating that the migrated cells maintain their proliferation ability. These results demonstrate that cells can be migrated (i.e., passaged) from the bottom of a culture substrate to the bottom of another culture substrate without treatment with a cell-dissociating enzyme such as trypsin.
• Figure 7D shows that cells adhere to the microcarriers after they have been migrated to the wells of a 24-well plate (microcarriers removed after cell migration).
• a method for culturing cells comprising a contacting step of contacting cells adhering to the bottom surface of a first culture substrate having a bottom surface treated for cell adhesion with microcarriers.
• a contacting step when the bottom surface of the first culture substrate is viewed from above, gaps exist between at least some of the microcarriers, and the total area of the bottom surface of the first culture substrate is S, the total number of the microcarriers is N, and the average particle diameter of the microcarriers is D, the following formula: S>N ⁇ (D/2) 2
• the method according to [1] wherein the cells are contacted with the microcarriers so as to satisfy the above.
• a method for producing a cell-containing pharmaceutical composition comprising a step of mixing cells obtained by the method according to any one of [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 re-contacting step according to [17] or [18] to form a cell sheet, and detaching the cell sheet from the second culture substrate.
• the culture method of this embodiment uses a culture substrate that has not been treated for cell adhesion or has been treated for non-cell adhesion and has an uneven bottom to bring cells and microcarriers into contact with each other, which is thought to increase the rate of contact between the two and result in increased cell culture efficiency.
• the cell-non-adhesive treatment of the culture substrate is not particularly limited as long as it makes it difficult for cells to adhere to the culture substrate, and treatments commonly used in cell culture can be used.
• the cell-non-adhesive treatment of the culture substrate is performed so that the water contact angle of the bottom surface is within a specific range. Specifically, the culture substrate is adjusted so that the water contact angle of the bottom surface is less than 60° or greater than 70°. Having the water contact angle of the bottom surface of the culture substrate within this range allows for a more appropriate cell adhesion rate, further improving the efficiency of cell culture.
• the culture substrate used in the culture method of this embodiment may be not only a culture substrate prepared by the above-mentioned cell-non-adhesive treatment, but also a culture substrate having a bottom surface in a cell-non-adhesive state as described above, even without the cell-non-adhesive treatment.
• the culture substrate may be a culture substrate prepared by subjecting a culture substrate that has not been subjected to the cell-non-adhesive treatment to the above-mentioned cell-non-adhesive treatment, or a culture substrate that has been subjected to the cell-non-adhesive treatment in advance, such as a commercially available culture substrate that has been subjected to the cell-non-adhesive treatment, may be purchased and used.
• the culture substrate may be coated with a material that has low affinity for cells.
• materials include 2-methacryloyloxyethyl phosphorylcholine (MPC) and polyethylene glycol (PEG).
• the culture method of this embodiment can be carried out in the same manner as the culture method of the present disclosure, except that the first culture substrate in the culture method of the present disclosure described above is replaced with a culture substrate that has not been treated for cell adhesion or has been treated for non-cell adhesion and has a bottom with an uneven surface. Therefore, the culture method of this embodiment can be carried out in the same manner as the various steps described in the culture method of the present disclosure described above.
• a cell culture method includes a contact step in which cells are brought into contact with oblate microcarriers in a culture substrate.
• contact between the microcarriers and cells in the culture substrate increases the contact opportunity (contact rate) between the microcarriers and cells, resulting in an improved adhesion rate between the cells and the microcarriers.
• contact rate contact opportunity
• the reason why this aspect of the culture method improves the adhesion rate between cells and the microcarriers is unclear, but it is hypothesized as follows: Typically, when culturing cells using microcarriers, both the cells and the microcarriers are placed in the culture substrate.
• the microcarriers generally have a larger mass, so the microcarriers sink to the bottom of the culture substrate first, followed by the cells.
• cells, particularly adhesive cells, that come into contact with and adhere to the microcarriers have higher survival and proliferation rates, while cells that do not come into contact with the microcarriers either die or, if they survive, experience a low proliferation rate. Even if some cells sink after the microcarriers, come into contact with the microcarriers, and adhere, the contact opportunity is limited, which is thought to result in limited cell culture efficiency.
• microcarriers that have been formed into an oblate spherical shape may be used, or the shape of the microcarriers 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 for applying an external force to the microcarriers is not particularly limited as long as it is a method commonly used in cell culture, and examples include applying centrifugal force using a centrifuge, or applying magnetic force if the microcarriers contain magnetic particles.
• the culture method of this embodiment can be carried out in the same manner as the culture method of the present disclosure, except that oblate spherical microcarriers are used as the microcarriers in the culture method of the present disclosure described above. Therefore, the culture method of this embodiment can be carried out in the same manner as the various steps described in the culture method of the present disclosure described above.
• a method for culturing cells comprising a contact step of bringing cells into contact with microcarriers in a culture substrate having a cell adhesion-treated, flat bottom surface.
• a culture substrate having a cell adhesion-treated bottom surface by using a culture substrate having a cell adhesion-treated bottom surface, cells in contact with the bottom surface adhere and proliferate (spread) to the bottom surface, increasing their chances of contact with microcarriers that are in contact with the bottom surface or that are nearby but not adhered to the bottom surface. This is believed to result in increased cell culture efficiency.
• FIG. 11 by using a culture substrate having a cell adhesion-treated bottom surface, cells in contact with the bottom surface adhere and proliferate (spread) to the bottom surface, increasing their chances of contact with microcarriers that are in contact with the bottom surface or that are nearby but not adhered to the bottom surface. This is believed to result in increased cell culture efficiency.
• FIG. 11 by using a culture substrate having a cell adhesion-treated bottom surface,
• cells that are not in contact with the bottom surface also adhere and proliferate on the surface of microcarriers upon contact with the microcarriers, which is believed to increase cell culture efficiency.
• 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 the microcarriers and proliferated. Therefore, according to the method of this aspect, efficient cell culture is possible without any particular restrictions on the order in which cells and microcarriers are seeded on the culture substrate.
• the cells obtained by the method of the present disclosure can be used as a pharmaceutical composition (i.e., a cell-containing pharmaceutical composition).
• a pharmaceutical composition can be produced using conventional techniques for producing pharmaceutical compositions, for example, using the cells recovered by the recovery step described above and, if necessary, pharmaceutically acceptable additives (e.g., excipients, solvents, cryopreservation solutions, etc.).
• a cryopreservation solution containing a cell protective agent can be used.
• cell protective agents include dimethyl sulfoxide (DMSO), ethylene glycol, propylene glycol, sericin, and glycerol.
• DMSO dimethyl sulfoxide
• a pharmaceutical composition can be produced by suspending and mixing the cells recovered by the recovery process described above in a cryopreservation solution.
• a cell sheet can be produced by culturing the cells transferred to the second culture substrate by the above-described re-contacting step to form a cell sheet.
• the culture medium and culture conditions used to culture the cells transferred to the second culture substrate can be appropriately set according to the cell type and the desired form of the cell sheet.
• the cells transferred to the second culture substrate can be cultured to an extent that a cell sheet can be formed on the second culture substrate (for example, until they reach confluence).
• 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 a method using an enzyme such as dispase to detach the cell sheet, and a method using a culture substrate that can change the detachability of adherent cells depending on the temperature (i.e., a temperature-responsive culture substrate) as the second culture substrate.
• temperature-responsive culture substrates include the aforementioned Cepallet (registered trademark) manufactured by DIC Corporation and UpCell (registered trademark) manufactured by CellSeed Co., Ltd.
• the cell sheet detached from the second culture substrate can be used, for example, as a transplant material to reconstruct tissue that has lost function due to illness or injury. It is expected that producing cell sheets using the method disclosed herein will enable the mass cultivation of cell sheets.

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