WO2015004762A1 - Méthode de culture cellulaire, dispositif et feuille cellulaire - Google Patents

Méthode de culture cellulaire, dispositif et feuille cellulaire Download PDF

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WO2015004762A1
WO2015004762A1 PCT/JP2013/068924 JP2013068924W WO2015004762A1 WO 2015004762 A1 WO2015004762 A1 WO 2015004762A1 JP 2013068924 W JP2013068924 W JP 2013068924W WO 2015004762 A1 WO2015004762 A1 WO 2015004762A1
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cells
cell
compound
period
cell culture
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Japanese (ja)
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亮太 中嶌
志津 松岡
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株式会社日立製作所
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/02—Membranes; Filters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/08—Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue

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  • the present invention relates to a cell culture technique, and in particular, to a cell culture method, a cell culture apparatus, and a cell sheet to be produced.
  • a sheet is used.
  • the reduction of regenerative tissue manufacturing cost is the most important issue.
  • development of a technique for shortening the cell sheet production process which usually takes several weeks, is expected.
  • Patent Document 1 culture is performed at an oxygen concentration lower than the oxygen concentration (O 2 of about 20%) which is a normal cell culture environment, and the proliferation of epithelial stem / progenitor cells, which are cells forming stratified epithelial tissue, is promoted.
  • a means for shortening the production period of the stratified epithelial cell sheet by setting the oxygen concentration to about 20% at the stratification stage is disclosed.
  • Non-Patent Document 1 discloses a means for efficiently producing an epidermal cell sheet by adding an IL-1 receptor antagonist or an IL-1 ⁇ neutralizing antibody to promote cell proliferation.
  • Non-Patent Document 2 discloses that epidermal cell proliferation is promoted by adding a low molecular weight compound that suppresses Notch signaling.
  • Patent Document 1 requires an expensive apparatus for controlling the oxygen concentration, and Non-Patent Document 1 requires expensive reagents such as cytokines and antibodies, thereby reducing the production cost of the cell sheet. The problem of doing was not solved.
  • Non-Patent Document 2 how to shorten the culture period as a whole culture period including the period for forming the three-dimensional structure of the cell sheet after culturing using a low molecular weight compound that suppresses Notch signaling. This problem has not been solved.
  • the object of the present invention is to solve such problems, shorten the production period, and produce a multi-layered epithelial tissue using a compound that suppresses differentiation, which can be produced in large quantities at low cost, and produced by the method
  • An object of the present invention is to provide a cell sheet and a cell culture apparatus.
  • the first period of the self-replicating period of the cells is the first period of the compound that suppresses differentiation.
  • the compound that suppresses differentiation is changed to a second supply amount that is less than the first supply amount, and a second period including a period in which the cells are stratified is A cell culture method for culturing with a second supply amount is provided.
  • a cell culture apparatus a culture solution supply unit for supplying a culture solution to a culture region for culturing cells forming a stratified epithelial tissue
  • the control unit includes a compound concentration adjusting unit that adjusts the supply amount of the compound that suppresses differentiation into the culture solution supplying unit, and a control unit
  • the control unit is a cell in a period of culturing the cells that form the stratified epithelial tissue.
  • the first period within the period of self-replication is cultured with the first supply amount of the compound that suppresses differentiation, and the compound that suppresses differentiation is determined from the first supply amount based on the degree of cell proliferation.
  • a cell culture device configured to control a compound concentration control unit so that a second supply amount is changed to a small second supply amount and a second period including a period in which cells are layered is cultured at a second supply amount.
  • the cell culture sheet among the period of culturing the cells forming the stratified epithelial tissue, the first period within the period of self-replication of the cells,
  • the compound is cultured with the first supply amount of the compound that suppresses differentiation, and the compound that suppresses differentiation is changed to a second supply amount that is less than the first supply amount based on the degree of cell proliferation,
  • the cell culture method, the cell culture apparatus, and the cell sheet according to the present invention it is possible to culture cells forming a tissue in a short time.
  • FIG. 1 The figure which shows the experimental condition based on Example 1.
  • FIG. The figure which shows the flow of cell sheet formation based on Example 1.
  • FIG. The figure which shows the phase-contrast microscope image from the 1st day to the 6th day of human epidermal cell culture based on Example 1.
  • FIG. The figure which shows the phase-contrast microscope image from the human epidermis cell culture based on Example 1 from the 7th day to the 14th day.
  • FIG. 1 The figure which shows the result of having quantified the intracellular DNA of the culture
  • FIG. The figure which shows the culture result list based on Example 1.
  • FIG. The figure which shows the relationship between the culture
  • FIG. The block diagram of the automatic cell culture apparatus in the case of switching the oxygen concentration of the whole culture tank based on Example 2.
  • FIG. The block diagram of an apparatus when an optical coherence tomography is added to the structure of the automatic cell culture apparatus based on Example 2.
  • FIG. 1 The block diagram of the apparatus at the time of adding an optical coherence tomography and a transepithelial electrical resistance measuring apparatus to the structure of the automatic cell culture apparatus based on Example 2.
  • FIG. 4 The block diagram of the apparatus in the case of switching the oxygen concentration in the culture container based on Example 4 directly.
  • FIG. 4 The apparatus block diagram at the time of adding an optical coherence tomometer and a transepithelial electrical resistance measuring apparatus to the structure of the automatic cell culture apparatus based on Example 4.
  • the period of culturing with the first supply amount of the compound that suppresses differentiation is the first period
  • the second period of the compound that suppresses differentiation The period of culturing with the supplied amount will be referred to as the second period.
  • This first period is a period within a period in which the cells forming the stratified epithelial tissue are self-replicating
  • the second period is a period including a period in which the cells forming the stratified epithelial tissue are stratified.
  • the cell species is not limited to humans, and may be mammalian cells such as mice, rats, rabbits, dogs, and pigs.
  • the cell type is not limited to epidermal cells, and may be other stratified epithelial cells such as oral cavity and cornea.
  • This stratified epithelial cell means a cell that forms stratified epithelial tissue, and includes, for example, a stem cell or a progenitor cell.
  • the culture process of the stratified epithelial cell sheet is divided into a self-replication period in which proliferating cells such as stem cells and progenitor cells self-replicate, and a differentiation period in which cell differentiation is performed after occupying a certain amount of the culture surface by self-replication.
  • proliferating cells such as stem cells and progenitor cells self-replicate
  • differentiation period in which cell differentiation is performed after occupying a certain amount of the culture surface by self-replication.
  • ⁇ -secretase inhibitor DAPT that suppresses Notch signaling was used at 10 ⁇ M.
  • Commercially available human epidermal cells were used as the stratified epithelial cells.
  • NIH / 3T3 cells which are mouse fibroblasts, are used as feeder cells
  • Comparative Example 1 shows the case where DAPT dissolution reagent DMSO is added during the entire culture period
  • Comparative Example 2 shows the case where DAPT 10 ⁇ M is added during the whole culture period
  • Self Example 1 was the case where DAPT 10 ⁇ M was added during the replication phase and only DMSO was added during the stratification phase.
  • FIG. 1 is a diagram showing a list of experimental conditions according to the present embodiment described above.
  • FIG. 2 is a diagram schematically showing the flow of cell sheet formation according to this example.
  • Proliferating cells such as stem cells and progenitor cells seeded on the culture surface adhere, proliferate, and are cultured closely without any gaps in the culture area (hereinafter referred to as confluent), and then by self-replication.
  • the cells are condensed by the proliferation of the cells, and the volume of each cell is reduced to be spread (hereinafter referred to as a paving stone shape).
  • the differentiated cells are then stratified, completing the stratified epithelial cell sheet.
  • FIG. 3 is a diagram showing phase contrast microscopic images of Comparative Example 1, Comparative Example 3, and Examples 1 and 2 up to the sixth day of culture.
  • Comparative Example 1 cells were in the process of cell growth on the 6th day of culture, whereas in Example 1, confluence was passed on the 6th day of culture and showed a paving stone form.
  • Comparative Example 3 cells were in the process of cell growth on the 6th day of culture, whereas in Example 2, confluence was passed on the 6th day of culture and showed a paving stone-like form.
  • FIG. 4 is a diagram showing phase contrast microscopic images of Comparative Examples 1, 2, 3, 4, and Examples 1 and 2 after the seventh day of culture according to the present example.
  • Comparative Example 1 confluence occurred on the 12th day of culture and a paving stone-like form was exhibited, and the culture was terminated on the 18th day of culture, whereas in Comparative Example 2 and Example 1, the 12th day of culture. The culture was terminated on the eyes.
  • Comparative Example 2 the paving stone-like form was not maintained after the seventh day of culture, whereas in Example 1, a clean paving stone-like form was maintained.
  • Comparative Example 3 In the case where no feeder cells were used, in Comparative Example 3, cells did not proliferate even after 18 days of culture, whereas in Comparative Example 4 and Example 2, the culture was terminated on the 12th day of culture. In Comparative Example 4, the paving stone-like form was not maintained after the seventh day of culture, whereas in Example 2, a clean paving stone-like form was maintained.
  • FIG. 5 is a view showing the appearance of the cell sheet before peeling from the culture surface after the cultivation of Comparative Examples 1, 2, 3, 4, and Examples 1 and 2 according to this example and the appearance after peeling.
  • the cell sheet was completed in 18 days of culture in Comparative Example 1, whereas the cell sheet was thin in 12 days in Comparative Example 2, but the cell surface was thin. The cell sheet contracted significantly after peeling.
  • Example 1 a thick cell sheet could be prepared in 12 days of culture, and the cell sheet was kept clean after being detached from the culture surface.
  • Example 2 shows that when feeder cells are used, in Example 1, the cell sheet preparation period can be shortened by about 6 days. Surprisingly, when feeder cells are not used, it is difficult to prepare conventional cell sheets. On the other hand, Example 2 shows that a cell sheet can be produced in a period equivalent to that when feeder cells are used and in a period shorter by about 6 days than Comparative Example 1.
  • FIG. 6 shows the end of the culturing according to this example, that is, the 18th day of culturing of Comparative Example 1, the 12th day of culturing of Comparative Example 2, the 12th day of culturing of Comparative Example 4, the 12th day of culturing of Example 1, and the culturing of Example 2.
  • the analyzed genes are epithelial stem / progenitor cell markers TP63, KRT15, ITGA6, ITGB1, epidermal spinal layer cell markers KRT1, KRT10, IVL, and epidermal granule cell marker FLG, LOR.
  • the housekeeping gene for correction was TBP.
  • the gene expression levels of TP63 and ITGA6 were about twice that of Comparative Example 1 in Examples 1 and 2 (p ⁇ 0.05).
  • the expression level of KRT15 was not significantly different between Example 2 and Comparative Example 1, but was significantly lower than Example 1 and was about 0.3 times (p ⁇ 0.05).
  • the expression level of ITGB1 was not significantly different from Comparative Example 1 in Examples 1 and 2.
  • Example 1 The expression levels of KRT1, KRT10, FLG and LOR were 3-8 times higher in Example 1 than in Comparative Example 1 (p ⁇ 0.05).
  • Example 2 was 0.3-0.01 times that of Comparative Example 1 (p ⁇ 0.05).
  • the expression level of IVL was not significantly different from Comparative Example 1 in Examples 1 and 2.
  • FIG. 7 is a diagram showing the results of quantitative analysis of the number of cells on the 6th day of culture in Comparative Examples 1 and 3 and Examples 1 and 2, according to this example.
  • Example 1 the amount of DNA contained in the cells was significantly larger than that in Comparative Example 1, and in Example 2, the amount of DNA contained in the cells was significantly larger than that in Comparative Example 3. This result supports the result of the present embodiment shown in FIG.
  • FIG. 8 is a table summarizing the results of FIGS. 3 to 6 according to the present embodiment.
  • the results shown in FIG. 3 to FIG. 8 show that in Example 1, a regenerated tissue can be produced in a shorter time than Comparative Example 1 which is a conventional method, and the produced tissue is the same structure as Comparative Example 1 of the conventional method. It shows that. Furthermore, in Example 2, the regenerated tissue can be produced with good reproducibility compared to Comparative Example 3 that has been difficult to produce in the past, indicating that the tissue is equivalent to Comparative Example 1.
  • the feeder cells described above have a role of assisting the proliferation and stratification of stratified epithelial cells, and are frequently used in this field.
  • many different types of cells are used as feeder cells. It is not preferable when the cell sheet is used for treatment. Therefore, it is better to be able to produce a cell sheet free of feeder cells.
  • the method described in the above-described example enables the production of a material that has been difficult to produce under the feeder cell-free condition, which is more preferable in regenerative medicine.
  • the cell sheet production period has been shortened by about 6 days. This is a surprising result and data showing the usefulness of the method.
  • the example described above is an example showing a method for producing a stratified epithelial cell sheet in a short period of time.
  • a compound that suppresses differentiation in addition to the above-mentioned DAPT, a ⁇ -secretase inhibitor that suppresses Notch signaling is used.
  • Use of certain L-685 458, DibenzazepineLY (LY411575), LY450136, MK-0752, etc. can be considered, but is not limited thereto.
  • any protein involved in stratification / differentiation of the stratified epithelium may be used, and cells can be cultured in a shorter period than the time required for conventional culture methods.
  • the concentration of the compound to be added during the self-replication period may be a concentration that can suppress the activity of the differentiation-related protein, and LC50 that is a concentration that suppresses the activity of the protein by half is preferable, but preferably the activity of the protein is completely
  • the concentration that can be suppressed is good.
  • the concentration of the compound in the stratification phase may be a concentration that can cancel the inhibition of the activity of the differentiation-related protein, and is less than LC50 that is a concentration that suppresses the activity of the protein by half, but preferably the activity of the protein is completely suppressed. It is better not to add a compound so that it can be released. Specific methods for the various experiments described above are shown below.
  • ⁇ Human epidermal cell culture method As the epidermal cells, commercially available normal human epidermal keratinocytes (DS Pharma) were used. The thawed and frozen cells were cultured as P1 in a dedicated serum-free medium (DS Pharma), and the cells (P3) that had been subcultured twice and proliferated were stored frozen and used as a cell source for cell sheet preparation. At the time of cell sheet preparation, cryopreserved P3 cells cultured in a dedicated serum-free medium were used for experiments.
  • DS Pharma normal human epidermal keratinocytes
  • a 6-well plate insert (BD bioscience, pore size 0.4 ⁇ m) and a 6-well plate (Asahi Techno Glass) were used as the culture container, and 5% FBS used for culturing epithelial cells was used as the culture medium.
  • the containing KCM medium was used.
  • NIH / 3T3 mouse-derived fibroblasts were used as feeder cells.
  • 3T3 was treated with mitomycin C for 2 hours (10 ⁇ g / ml) to stop cell growth before culturing.
  • DAPT was manufactured by Calbiochem, and DMSO was manufactured by Nacalai Tesque.
  • Comparative Example 1 was cultured on the 18th day, Comparative Example 2 was cultured on the 12th day, Comparative Example 4 was cultured on the 12th day, Example 1 was cultured on the 12th day, and Example 2 was cultured from the culture surface after culturing on the 12th day.
  • the sheet was peeled off, washed with PBS, and stored frozen as a sample for RNA extraction. Thereafter, cDNA was prepared from the cryopreserved cells using RNeasy plus mini kit (Qiagen) and used as a PCR template.
  • the inter-sample correction gene is TATA-box binding protein, ⁇ Np63 (TP63) and Cytokeratin15 (KRT15), Integrin ⁇ 6 (ITGA6), Integrin ⁇ 1 (ITGB1), and epitoid differentiation marker Cytokeratin1 (KRT1) ), Cytokeratin10 (KRT10), Involucrin (IVL), Filaggrin (FLG), Loricrin (LOR), using a total of nine Taqman probes, the amount of gene expression in each sample was quantified with a real-time PCR device (Takara Bio Inc.) did.
  • a 15 ⁇ m-thick section was prepared with a microtome from the frozen embedded tissue. Using the prepared sections, hematoxylin-eosin staining was performed according to a conventional method.
  • the concentration of the compound that suppresses differentiation is changed, in other words, the supply amount of the compound that suppresses differentiation is the first supply amount.
  • a specific method for changing from the first supply amount to the second supply amount will be described.
  • cells seeded in a culture space such as a culture vessel are cultured in a culture solution to which a predetermined amount of a compound that suppresses differentiation is added.
  • a culture solution to which a predetermined amount of a compound that suppresses differentiation is added.
  • the self-replication phase cells repeat proliferation by replication more rapidly than when no compound that suppresses differentiation is added.
  • the overall size of cells that have grown and bound, or the size of a single cell, the rate of growth, and the like may vary. Therefore, it is desirable that the cell concentration obtained by self-replication is arbitrarily determined depending on the cell type, and the concentration of the compound that suppresses differentiation is switched from the first concentration to the second concentration.
  • the cells from which differentiation suppression has been released start differentiation and stratify.
  • the degree of cell proliferation can also be determined by the occupation ratio with respect to the culture surface (first surface) to be cultured.
  • the cells start to grow by self-replication so as to spread over the culture surface of the seeded culture space.
  • the cell occupancy with respect to the culture surface, which is the first surface becomes 100%, as described in the above principle, it is positioned as a confluent state.
  • the supply amount is switched by switching from the first supply amount to the second supply amount.
  • the occupation ratio for determining the switching timing can be arbitrarily set to 80% or 90% depending on the characteristics of the cell type.
  • the cobblestone form is a state that occurs from the time when cells become confluent until the start of the differentiation phase, and by switching the concentration of the compound that suppresses differentiation in the cobblestone form, the differentiation phase is compared to the confluent state. Since it can culture
  • the cells after switching the oxygen concentration are normally cultured by checking whether a phenomenon called tight junction that occurs in the differentiation period after the paving stone-like form occurs.
  • Tight junction is a transmembrane protein, a structure in which the cell gap is closed by claudin and occludin, and the cultured cells become tightly bound, thereby causing a paracellular pathway of dissolved substances, ions, and water. Is controlling. In other words, it can be determined by confirming the occurrence of tight junction that the cells are cultured in the absence of externally dissolved substances or contaminants.
  • the method of controlling the concentration of the compound that suppresses differentiation can be controlled by providing a plurality of culture solution tanks containing a culture solution having a predetermined compound concentration. Alternatively, it can be controlled by providing a mechanism for adjusting the compound concentration to an arbitrary concentration and adding it to the culture solution.
  • an example of an automatic cell culture device equipped with a function of automatically controlling the concentration of a compound that suppresses differentiation based on the principle and method described in Example 1 with reference to FIGS. Will be described.
  • the calculation means and the calculation function related to the control of the compound concentration will be described as a configuration example built in the control device in the automatic cell culture apparatus after this example, but the calculation means and the calculation function related to the control of the compound concentration are
  • the CPU is not limited to software executed by a central processing unit (CPU) that constitutes the control device 2 that functions as a control unit, but is a CPU built in an external computer of an automatic cell culture device or the like
  • the software may be executed by software or external dedicated hardware.
  • the automatic cell culture apparatus of this example will be outlined using the example of the overall configuration shown in FIG.
  • various components such as various control units controlled by the control device 2, pumps, sensors, and the like are connected to a thermostatic chamber 3 including a culture vessel 4. That is, a temperature adjusting unit 5 for controlling the temperature in the thermostatic chamber 3, a humidity adjusting unit 6 for controlling the humidity, and a gas concentration adjusting unit 8 connected to the gas supply unit 7 for controlling the gas concentration.
  • a temperature adjusting unit 5 for controlling the temperature in the thermostatic chamber 3
  • a humidity adjusting unit 6 for controlling the humidity
  • a gas concentration adjusting unit 8 connected to the gas supply unit 7 for controlling the gas concentration.
  • control device 2 includes a culture solution supply pump 10 having a liquid feeding tube connected to a culture solution / drainage tank 9 that holds the culture solution and waste solution for automatically replacing the culture solution in the culture vessel. And a compound supply unit 20 that supplies the compound to the culture vessel 4 or the culture solution tank 9 and a compound concentration adjustment unit 21 that adjusts the compound concentration, and controls the operation of each component. Furthermore, the control device 2 is connected with a temperature / humidity / CO 2 / O 2 sensor 11, a cell observation CCD camera 12, and a display unit 13 for the purpose of controlling the operation of these components. Yes. A value in the thermostat 3 is detected by the temperature / humidity / CO 2 / O 2 sensor 11.
  • oxygen is supplied into the thermostat 3, so that the closed culture vessel 4 can be supplied with a gas, such as polystyrene, polycarbonate, polyethylene terephthalate, polymethylpentene, or the like, preferably It is better to provide a porous film made of polycarbonate, polyethylene terephthalate, or polyimide.
  • the porous diameter is preferably less than 20 nm in order to avoid invasion of viruses and bacteria into the culture vessel.
  • the control device 2 controls the concentration of the compound that suppresses differentiation
  • the user can directly observe the cells in the automatic culture device 1 to switch the compound concentration.
  • An image captured by an imaging unit that observes and images cells such as the camera 12 may be displayed on the display unit 13 for a display screen, and the user may switch the compound concentration based on the captured image.
  • the display unit 13 may use an output unit that prompts an instruction for hearing such as a buzzer instead of a visual display.
  • the control device 2 that has captured cells with the CCD camera 12 during culture and has acquired the cell images performs a process of detecting cells from the acquired image data by executing a predetermined function program.
  • the binarization based on the black and white or gray scale of the image is performed based on the luminance, and the cell occupation area in the image is calculated.
  • the compound concentration adjusting unit 21 is controlled to change the compound concentration.
  • the compound concentration is not switched, and the culture at the initial compound concentration state is continued, and the above operation is repeated at a predetermined timing, so that the cell occupation area becomes 100%. When this is reached, switch the compound concentration.
  • the compound concentration Since the cell occupancy increases as the self-replication of cells progresses, it is desirable to switch the compound concentration at a confluence of about 100% of the cell occupancy near the final process of self-replication. However, 80% or 90% can be arbitrarily set in accordance with the specifications of the CCD camera 12, the state of cell culture, the area where cells are actually cultured on the culture surface, and the like. If the cells do not reach confluence depending on the cell type, the timing for switching the compound concentration may be set according to the size and occupied area until the self-replication of the cells is completed.
  • the horizontal axis represents the number of days of culture and the vertical axis represents the average cell size ( ⁇ m).
  • the size is larger than that immediately after seeding, and the average cell size is the maximum near the confluence.
  • the cobblestone shape shown in FIG. 2 is reached, the number of cells per area increases and the cells shrink, so that the average cell size gradually decreases. Is fixed, so the cell size is constant.
  • the CCD camera 12 of the automatic cell culture device 1 in FIG. 10 takes a plurality of images in time series, and the control device 2 calculates cell size statistics using a plot file or the like based on the plurality of images. Furthermore, the average cell size for each image is calculated from the calculated statistical data, and compared with the average cell size included in the preceding and succeeding images in time series. By comparison, the average time-series change of the cell size can be calculated, and the timing when the cell size becomes the maximum value can be specified.
  • control device 2 changes the compound concentration from the first concentration to the second concentration after the specified maximum value, that is, after the confluent timing.
  • the maximum cell size or the specified switching timing period may be displayed on the display screen of the display unit 13 to prompt the user to switch the compound concentration.
  • the method of specifying the maximum cell size is not limited to this, and a specific value is set in advance according to the cell type, and the cells self-replicate to an average size exceeding this value. You may make it switch a compound concentration at the timing which performed.
  • the horizontal axis is the cell size
  • the vertical axis is the cell number, as in the case of confluence and the cobblestone form shown in FIGS. 18A and 18B, not the average cell size.
  • the time series change in the distribution of the graph may be analyzed from the image, and the point at which the distribution peak becomes the maximum may be set as the maximum value, or the dispersion value for switching the compound concentration may be set in advance.
  • the compound is obtained by analyzing the cell state with an optical coherence tomometer 14 as shown in FIG. Density switching can be determined.
  • Density switching can be determined.
  • the method of judging from the cell image since the judgment is made by observing several points on the culture surface, there is a possibility that it is not possible to know whether or not the part that was not observed is confluent.
  • the method of analyzing the cell state with the optical coherence tomography 14 it is possible to detect a cell defect location on the entire culture surface.
  • the optical coherence tomography 14 can irradiate the sample with one of the two divided infrared lights and cause the reflected light and the other light to interfere with each other, thereby imaging the surface and cross section of the tissue on the entire medium surface. . Furthermore, since the optical coherence tomography 14 can measure the thickness of the cross section, it can be applied to non-invasively evaluate the quality of whether or not the produced cell sheet is differentiated.
  • the light source installed in the optical coherence tomography 14 can be provided with a drive unit that can change the irradiation position of the infrared light emitted from the light source.
  • a drive unit that can change the irradiation position of the infrared light emitted from the light source.
  • an image of a cross section in one direction of the culture surface is acquired.
  • a cross-sectional image of the entire culture surface can be obtained by acquiring a plurality of cross-sectional images while moving the drive means in a direction perpendicular to the one direction. It is desirable that the vertical interval of the acquired cross-sectional images be set to be narrower than the cell size so that there is no leakage at the cell defect site.
  • the interval at which the cross-sectional image is acquired may be determined depending on the degree of cell proliferation.
  • the acquired image may be displayed on the display unit 13.
  • a display method only an image with a cell defect may be displayed, or only a cell defect may be warned with a buzzer. The presence or absence of cells can be determined.
  • the control device 2 of the automatic cell culture device 1 of the present embodiment switches the compound concentration if it is confluent.
  • the switching time it is possible to determine whether the state is confluent and switch the compound concentration, but it is also possible to set the switching time to be after a predetermined time from the time when it became confluent. It is effective for. When a predetermined time elapses, a paving stone shape which will be described later is generated. Therefore, if a switching time point in consideration of the occurrence of this paving stone shape is set in advance, culture can be performed in a shorter time.
  • the above-described cell image method of FIG. 10 and the optical coherence tomography method of FIG. 11 can be used in combination, and the compound concentration can be automatically controlled more reliably. For example, when it is determined that the cell occupation area with respect to the culture surface is equal to or greater than a set value (for example, 100%) and the entire culture surface (for example, 100%) has a thickness of cells, the compound concentration is switched. As a result, the accuracy of determining confluence can be increased.
  • FIG. 13 shows another modification of the second embodiment to which the electrical resistance measuring device 15 is attached.
  • Epithelial cells form tight junctions when the cells are tightly coupled. When tight junctions are formed between cells, exchange of ions between cells is blocked, and thus resistance occurs when a voltage is applied between cells. That is, it can be determined from the electric resistance value whether the cells are dense and have a paving stone shape and a tight bond is formed.
  • control device 2 calculates the time series change of the resistance value by the electrical resistance measuring device 15, and analyzes whether or not the resistance value changes in the form of an exponential function from the calculation result. If the resistance value changes with an exponential function, it is determined that tight coupling has occurred.
  • the electrical resistance measuring device 15 may be used in combination with the optical coherence tomography 14 as shown in FIG. 13 to check the quality of the cultured cells.
  • the control device 2 has calculation means for switching the compound concentration.
  • the compound concentration adjustment section 21 is provided with calculation means, and the compound concentration adjustment section 21 independent of the control apparatus 2 is used. It is also possible to control changes in the compound concentration.
  • the method of controlling the compound concentration is adjusted by the compound concentration adjusting unit 21 in order to change the compound supply amount supplied from the compound supply unit 20 from the first supply amount to the second supply amount. Control may be performed by the control device 2.
  • Example 3 an example of a cell culturing method and an automatic cell culturing apparatus when the timing of switching the concentration of a compound that suppresses differentiation is set to a point in time when a cobblestone form is shown will be described. That is, the concentration switching time is not a confluence but a cobblestone-like state in which the cell density is increased and the volume of each cell is reduced and spread after confluence.
  • differentiation such as stratification is an event that occurs after passing through a paving stone-like form, so that it is possible to produce a desired tissue earlier by switching the compound concentration when the paving stone-like form is shown. .
  • the overlapping description is abbreviate
  • the control device 2 of the automatic cell culture device 1 in FIG. 10 processes the image so that the cell space in the cell image becomes clear after the cell occupancy reaches 100% based on the cell analysis result described in the second embodiment. To do. After that, as shown in FIG. 19, a change in luminance on one line at an arbitrary point of an arbitrarily set image is calculated as a signal. From the plot profile which is the distribution of this signal, the paving stone shape is discriminated based on whether or not the signal is regularly detected at intervals of about 5 to 20 ⁇ m, which is the cell size in the paving stone shape. In this case, the compound concentration is changed by the method described in Example 2.
  • control device 2 of the present embodiment does not show a paving stone shape, that is, when the cell is not the size in the paving stone shape as described above in the detection result of the signal indicating the length between cells, Continue culturing at the compound concentration and repeat the procedure shown above.
  • the CCD camera 12 determines from the distribution of the signal of the cell image captured by the CCD camera 12 using the average or distribution of the cell size.
  • the peripheral portion of the outline of each cell in the captured image is shown with a relatively low luminance in the image and a high luminance between the cell itself and each cell. That is, since the number of cells is small at the early stage of culture, there are many portions where the luminance is high, and the average luminance of the image is high. Furthermore, as the number of cells increases, the portion around the cells having a low luminance also increases, so the average luminance of the image gradually decreases.
  • the control apparatus 2 can discriminate
  • the size of the cell in the self-renewal phase is larger than the paving stone shape as described above. Furthermore, since the culture progresses in each region of the culture surface, the region of the dispersion value of the distribution is widened. The cell size and the size distribution gradually decrease as the cobblestone morphology is approached, and when the cobblestone morphology is formed, the cell size during the self-renewal phase is a minimum and constant value. Become.
  • the control device 2 shifts to a region where the cell size distribution is the smallest or when the width of the dispersion value due to the distribution is the narrowest, the time series change of the size distribution is a constant value. In this case, or by a combination of these, it is possible to discriminate the cobblestone form and switch the compound concentration.
  • the apparatus configuration is configured to control the oxygen concentration in the culture tank.
  • the humidity control unit, the gas concentration control unit, and the humidity / CO 2 / O 2 sensor are connected to the culture vessel. This is an example in the case of supplying gas into the culture vessel.
  • the humidity controller 6, the gas concentration controller 6, and the humidity / CO 2 / O 2 sensor 11 are connected to the culture container 4. Gas is supplied directly into 4. With this configuration, water vapor can also flow into the culture vessel 4 using the gas supply port in the culture vessel 4. Further, when the entire culture tank 3 is in a constant temperature and high humidity environment, the culture tank 3 itself is not a sterile space, and thus there is a risk of mold and bacteria breeding. On the other hand, when the inside of the culture container 4 is a constant temperature and high humidity environment, since the inside of the culture container 4 is an aseptic space, there is an advantage that the risk of mold and bacteria breeding is low.
  • FIGS. 15 to 17 show a modification of the apparatus configuration of FIG. 14, and a modification of the apparatus configuration of FIG. 10 in the second embodiment except that the gas concentration adjusting unit 8 is connected to the culture vessel 4. This corresponds to the configuration of FIG. 11 to FIG. 13 and will not be described here.
  • the present invention described in detail above is capable of culturing cells forming a tissue in a short time, and is extremely useful as a cell culture method, a cell culture device, and a cell sheet.
  • this invention is not limited to the above-mentioned Example, Various modifications are included.
  • the above-described embodiments have been described in detail for better understanding of the present invention, and are not necessarily limited to those having all the configurations described.
  • a part of the configuration of one embodiment can be replaced with the configuration of another embodiment, and the configuration of another embodiment can be added to the configuration of one embodiment.
  • a culture solution supply unit for supplying a culture solution to a culture region for culturing cells forming a stratified epithelial tissue;
  • a compound concentration adjusting unit for adjusting a supply amount of a compound that suppresses differentiation into the culture region or the culture solution supply unit;
  • a control unit An optical coherence tomography that irradiates the first light that transmits the cell and the second light that is reflected by the surface of the cell;
  • the controller is During the period of culturing cells that form stratified epithelial tissue, Culturing a first period within a period of self-replication of the cells with a first supply of a compound that inhibits differentiation; Based on the degree of proliferation of the cells, the compound that inhibits the differentiation is changed to a second supply amount that is less than the first supply amount, Controlling the compound concentration regulator so that the second period including the period in which the cells are stratified is cultured in the second supply amount; A cell culture device.
  • a culture solution supply unit for supplying a culture solution to a culture region for culturing cells forming a stratified epithelial tissue;
  • a compound concentration adjusting unit for adjusting a supply amount of a compound that suppresses differentiation into the culture region or the culture solution supply unit;
  • a control unit An electrical resistance measurement unit for measuring the electrical resistance value of the cell,
  • the controller is During the period of culturing cells that form stratified epithelial tissue, Culturing a first period within a period of self-replication of the cells with a first supply of a compound that inhibits differentiation; Based on the degree of proliferation of the cells, the compound that inhibits the differentiation is changed to a second supply amount that is less than the first supply amount, Controlling the compound concentration regulator so that the second period including the period in which the cells are stratified is cultured in the second supply amount; A cell culture device.
  • a culture solution supply unit for supplying a culture solution to a culture region for culturing cells forming a stratified epithelial tissue;
  • a compound concentration adjusting unit for adjusting a supply amount of a compound that suppresses differentiation into the culture region or the culture solution supply unit;
  • a control unit An electrical resistance measurement unit for measuring the electrical resistance value of the cells;
  • An optical coherence tomography that irradiates the first light that transmits the cell and the second light that is reflected by the surface of the cell;
  • the controller is During the period of culturing cells that form stratified epithelial tissue, Culturing a first period within a period of self-replication of the cells with a first supply of a compound that inhibits differentiation; Based on the degree of proliferation of the cells, the compound that inhibits the differentiation is changed to a second supply amount that is less than the first supply amount, Controlling the compound concentration regulator so that the second period including the period in which the cells are stratified is cultured in the second supply amount;
  • a cell culture device according to disclosure example 3, Further comprising an imaging unit for imaging the cells, The controller is From the image acquired from the imaging unit, or the output of the electrical resistance measuring unit or the optical coherence tomography, the degree of proliferation of the cells is calculated, A cell culture device.
  • a culture vessel for culturing cells forming the stratified epithelial tissue A thermostat for controlling the culture vessel to a predetermined temperature; A culture solution supply unit for supplying a culture solution to the culture vessel; A compound concentration adjusting unit for adjusting a supply amount of a compound that suppresses differentiation into the culture region or the culture solution supply unit; A control unit, The controller is During the period of culturing cells that form stratified epithelial tissue, Culturing a first period within a period of self-replication of the cells with a first supply of a compound that inhibits differentiation; Based on the degree of proliferation of the cells, the compound that inhibits the differentiation is changed to a second supply amount that is less than the first supply amount, Controlling the compound concentration regulator so that the second period including the period in which the cells are stratified is cultured in the second supply amount; A cell culture device.
  • a cell culture device according to disclosure example 5, A temperature adjusting unit for adjusting the temperature of the thermostat; A humidity adjusting unit for adjusting the humidity of the thermostatic chamber; A gas concentration adjusting unit for adjusting the gas concentration of the thermostatic chamber, The controller is Controlling the temperature adjusting unit, the humidity adjusting unit, and the gas concentration adjusting unit; A cell culture device.
  • a cell culture device according to disclosure example 5, A temperature adjusting unit for adjusting the temperature of the thermostat; A humidity adjusting unit for adjusting the humidity of the culture vessel; A gas concentration adjusting unit for adjusting the gas concentration of the culture vessel, The controller is Controlling the temperature adjusting unit, the humidity adjusting unit, and the gas concentration adjusting unit; A cell culture device.

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Abstract

La présente invention concerne un dispositif de culture cellulaire et analogue, permettant de former rapidement un tissu, un dispositif de régulation (2) régule une unité d'ajustement de la concentration de composé (21) afin de réguler une quantité de composé pour inhiber une première différentiation pendant une première période parmi des périodes de culture cellulaire pour former un tissu épithélial stratifié, pendant une période pendant laquelle les cellules s'autorépliquent, et une quantité de composé pour inhiber une deuxième différentiation, cette deuxième différentiation étant moins importante que la quantité de composé pour l'inhibition de la première différentiation, au cours d'une deuxième période comprenant une période pendant laquelle les cellules se différencient et se stratifient. Le dispositif de régulation régule l'unité d'ajustement de la concentration de composé afin de modifier une première quantité administrée en une deuxième quantité administrée en fonction du degré de prolifération de cellules par autoréplication.
PCT/JP2013/068924 2013-07-10 2013-07-10 Méthode de culture cellulaire, dispositif et feuille cellulaire WO2015004762A1 (fr)

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JP2019000038A (ja) * 2017-06-15 2019-01-10 国立大学法人山口大学 積層化細胞シートの作製方法及び積層化細胞シート
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US10846849B2 (en) 2016-06-16 2020-11-24 Hitachi High-Tech Corporation Method for analyzing state of cells in spheroid
JP2019000038A (ja) * 2017-06-15 2019-01-10 国立大学法人山口大学 積層化細胞シートの作製方法及び積層化細胞シート

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