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WO2013136372A1 - Feuille cellulaire, procédé de culture cellulaire, et appareil de culture cellulaire - Google Patents

Feuille cellulaire, procédé de culture cellulaire, et appareil de culture cellulaire Download PDF

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Publication number
WO2013136372A1
WO2013136372A1 PCT/JP2012/001834 JP2012001834W WO2013136372A1 WO 2013136372 A1 WO2013136372 A1 WO 2013136372A1 JP 2012001834 W JP2012001834 W JP 2012001834W WO 2013136372 A1 WO2013136372 A1 WO 2013136372A1
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period
oxygen supply
cells
cell
supply amount
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English (en)
Japanese (ja)
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亮太 中嶌
志津 松岡
豊茂 小林
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Hitachi Ltd
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Hitachi Ltd
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Priority to PCT/JP2012/001834 priority Critical patent/WO2013136372A1/fr
Priority to JP2014504465A priority patent/JP5991368B2/ja
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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
    • 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
    • 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
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/34—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of gas
    • 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
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/02—Atmosphere, e.g. low oxygen conditions

Definitions

  • the present invention relates to a cell culture method, a cell sheet produced by the method, and an automatic culture apparatus.
  • Reconstructed tissue with a three-dimensional shape reconstructed outside the body has properties close to that of the living body, improving therapeutic effects in regenerative medicine that treats diseases using cells, and animal experiments in the development of pharmaceuticals and cosmetics. In recent years, it has become important from the standpoint of improving development efficiency with alternative cells and human-derived cells.
  • vegetative cells often called feeder cells are used when producing epithelial cell sheets.
  • a vegetative cell a cell called NIH / 3T3 or 3T3-J2 derived from a mouse is generally used.
  • the regenerative tissue produced using the above-mentioned feeder cells is xenogeneic, and the act of transplanting it into a living body corresponds to xenogeneic transplantation (Ministry of Health, Labor and Welfare: “Public with implementation of xenotransplantation” Guideline for regenerative medicine of epithelial system using 3T3J2 strain and 3T3NIH strain as feeder cells based on “Guidelines on sanitary infectious diseases”).
  • heterologous feeder cells for the production of regenerative tissues, there are major problems such as the inability to deny heterogeneous infectious diseases and the burden of strict quality control of feeder cells.
  • Non-Patent Document 1 uses a feeder cell because a nutrient for culture medium can be supplied from the basal side of the epithelial cell sheet by using a culture container called a cell culture insert. At least, it is disclosed that a stratified epithelial cell sheet can be obtained.
  • D.Murakami et al . The effect of micropores in the surface of temperature-responsive culture inserts on the fabrication of transplantable canine oral mucosal epithelial cell sheets: Biomaterials, 27, 5518-5523, 2006 Rama P. et al .: Limbal stem-cell therapy and long-term corneal regeneration: N Engl J Med, 363, 147-155, 2010
  • Non-Patent Document 1 it is known that the expression of p63, which is an epithelial stem cell marker, decreases in the cell sheet prepared by such a method.
  • p63 which is an epithelial stem cell marker
  • the presence of stem cells in the cell sheet (p63 strong positive rate of more than 3%) is an important factor for determining the prognosis of treatment (see Non-Patent Document 2).
  • a cell sheet produced without cells and having reduced stem cells cannot be applied to treatment.
  • the present invention automates a method for producing a cell sheet that has a colony formation rate of 3% or more indicating the presence of stem cells, which is close to the case where feeder cells are used even when feeder cells are free. It aims at providing the culture device which performs.
  • the first oxygen supply amount in the first period in which the stem cells or progenitor cells forming the tissue self-replicate and the first oxygen supply amount in the second period including the period in which the stem cells or progenitor cells differentiate A cell culture method characterized by controlling a second oxygen supply amount of a larger oxygen supply amount, and a cell culture device having a culture region for culturing stem cells or progenitor cells forming a tissue, wherein the culture region
  • a first oxygen supply amount in a first period within a period in which the stem cell or progenitor cell self-replicates, and an oxygen control unit that controls an oxygen concentration in the cell and a control unit that controls the oxygen control unit Is a cell culture device characterized by having a low oxygen concentration (less than 20%).
  • an epithelial cell sheet having a colony formation rate of 3% or more indicating the presence of stem cells is close to the case of using feeder cells without using feeder cells. It can be produced.
  • FIG. 1 The figure which shows colony formation of the corneal epithelial stem cell and progenitor cell in the comparative example 1 and the example 1.
  • FIG. 2 The figure which shows the days required for cell culture in the comparative examples 2 and 3 and the examples 2-6.
  • FIG. The figure which shows the external appearance of the corneal epithelial cell sheet
  • the figure which shows the cell type which can apply a present Example Schematic showing the confluence, the paving stone form, the time of tight bonding.
  • the apparatus block diagram in the case of switching the oxygen concentration of the whole culture tank.
  • the apparatus block diagram at the time of adding an optical coherence tomography and a transepithelial electrical resistance measuring apparatus to the structure of FIG. The apparatus block diagram in the case of switching the oxygen concentration in a culture container directly.
  • the apparatus block diagram at the time of adding an electrical resistance measuring apparatus to the structure of FIG. The apparatus block diagram at the time of adding an optical coherence tomography to the structure of FIG.
  • the apparatus block diagram at the time of adding an optical coherence tomography and a transepithelial electrical resistance measuring apparatus to the structure of FIG. The apparatus block diagram at the time of adding an optical coherence tomometer and an electrical resistance measuring apparatus to the structure of FIG.
  • the cell species is not limited to rabbits, and may be mammalian cells such as mice, rats, dogs, pigs, and humans.
  • the cell type is not limited to corneal epithelial cells, and may be stratified epithelial cells such as skin and oral cavity.
  • the culture process of the cells forming the tissue can be divided into a self-replication period in which stem cells / 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.
  • a corneal epithelial cell sheet as an example.
  • the differentiation in the corneal epithelial cell sheet is more specifically referred to as stratification in which cells form a laminated structure.
  • the differentiation phase is referred to as the stratification phase.
  • FIG. 1 is a diagram showing the results.
  • Comparative Example 1 almost no colonies were observed, whereas in Example 1, many colonies were observed despite the absence of feeder cells. This indicates that at low oxygen concentrations, stem cells and progenitor cells adhere and proliferate even without feeder cells. This result suggests that the activity and properties as stem cells may be maintained at low oxygen concentrations.
  • Example 2 shows a case where the self-replication phase is cultured at 1% O 2 and the stratification phase at 20% O 2, which is a normal oxygen concentration, without using feeder cells.
  • Example 3 when culturing at O 2 2%, stratification period at normal oxygen concentration of 20% O 2 , self-replication period at O 2 5%, stratification period at normal oxygen concentration of O 2 20% in the case of culturing example 4, the self-replicating life O 2 10%, examples 5 a case of culturing with O 2 20% normal oxygen concentration stratification period, the self-replicating life O 2 15%, stratified-life Example 6 when cultivated with 20% O 2 at normal oxygen concentration, Example 7 when culturing at 1% O 2 in both the stratification and self-replication phases, both in the stratification and self-replication phases illustrative case cultured in O 2 2% 8, stratification period, excessive self-replicating life both Illustrative 9 when cultured in O 2 5% both stratified phase, examples of the case where cultured in O 2 10% self-replicating life both process both 10, using feeder cells, cultured in O 2 20% in both processes both The case in which this was performed was Comparative Example 2, and the case in which both processes were culture
  • FIG. 2 is a graph showing the number of days required for cell sheet production in Comparative Examples 2 and 3 and Examples 2 to 6.
  • FIG. 3 is a diagram showing phase contrast microscopic images of cultures 5, 7, 9, 11, and 12 in Comparative Examples 2 and 3 and Example 4.
  • the cells are in a state of being densely cultured without any gaps (hereinafter referred to as “confluent”), and then cells by self-replication
  • the cells were condensed as a result of the growth of the cells, and the volume of each cell was reduced and spread (hereinafter referred to as a cobblestone shape), whereas in Examples 2 to 6, the cobblestone shape was observed on the ninth day.
  • the form was shown.
  • the stratification period was set to 5 days.
  • FIG. 4 is a diagram showing the appearance of the cell sheet in Comparative Examples 2 and 3 and Examples 2-6. All the cell sheets had a thickness that could withstand the tweezers operation.
  • FIG. 5 is a diagram showing the number of cells contained in the cell sheet in Comparative Examples 2 and 3 and Examples 2 to 6. From this result, it can be seen that any cell sheet has almost the same number of cells. From this result, it can be inferred that the cell sheet prepared without feeder cells and the cell sheet prepared with feeder cells are layered to the same extent.
  • FIG. 6 is a graph showing the colony formation rate indicating the proportion of stem cells contained in the cell sheet in Comparative Examples 2 and 3 and Examples 2 to 6. Comparing Comparative Example 2 and Comparative Example 3, it can be seen that the colony formation rate is reduced to about 1/4 due to the absence of feeder cells. On the other hand, in Examples 2 to 5, despite the absence of feeder cells, the colony formation rate was significantly higher than that of Comparative Example 3, which was about 3/4 of Comparative Example 2, indicating 3% or more. However, although Example 6 was significantly higher than Comparative Example 3, it was about half of Examples 2-5.
  • FIG. 7 is a diagram showing colony formation in Comparative Examples 2 and 3 and Example 4.
  • FIG. 8 shows tissue stained images in Comparative Examples 2 and 3 and Examples 2 to 6, and is intended for evaluation of a corneal epithelial cell sheet. According to this, it can be seen that the film is laminated to about 3 to 6 layers under any condition.
  • FIG. 9 is a diagram showing an immunostained image and a cell nucleus stained image of CK3, which is a corneal epithelial cell differentiation marker, in Comparative Examples 2 and 3 and Examples 2 to 5. According to this, it can be seen that cells layered in 3 to 6 layers are differentiated as corneal epithelial cells.
  • FIG. 28 is a diagram showing an immunostained image and a cell nucleus stained image of CK3, which is a corneal epithelial cell differentiation marker, in Comparative Examples 2 and 3 and Examples 7 to 10. According to this, it is understood that the stratification does not proceed sufficiently depending on the oxygen concentration in those cultured at a low oxygen concentration in both the stratification phase and the self-replication phase.
  • FIGS. 1 to 9 and 28 are all inferior to those of Comparative Example 2, which is a conventional method, by culturing at a low oxygen concentration in the self-renewal period even in the condition without feeder cells. This shows that a stratified epithelial cell sheet retaining a certain amount of stem cells can be produced. Result of verification by the above-mentioned principle, in conditions without feeder cells, self-replicating life with O 2 less than 1% to 15% without feeder cells, O 2 20% cultured from significantly colonization rate when in It was revealed that the colony formation rate was high, when feeder cells were present, and when the cells were cultured at 20% O 2 .
  • the self-replication period is O 2 1% or more and less than 15%, and the differentiation period O 2 15% or more is higher than the colony formation rate of the cell sheet prepared in Comparative Example 2, and retains the colony formation rate 3% or more.
  • the oxygen concentration was desirable for obtaining a multilayered epithelial cell sheet. The specific method of the above experiment is shown below.
  • the culture vessel was a 6-well cell culture insert and 6-well plate, or a 12-well cell culture insert and 12-well plate.
  • the day before culturing corneal epithelial cells NIH-3T3 cells treated with mitomycin C (10 ⁇ g / ml) for 2 hours at 37 ° C. were seeded as feeder cells in a 6-well plate at 2 ⁇ 10 4 / cm 2 . .
  • the day after the seeding of NIH-3T3 cells corneal epithelial cells were collected from the corneal limbus of a rabbit eyeball purchased from Funakoshi in accordance with a conventional method, and placed in a 6-well cell culture insert so as to be 1 ⁇ 10 4 / cm 2.
  • KCM medium containing 5% FBS used for culturing epithelial cells was used as the culture solution.
  • the culture solution was exchanged once on the 5th, 7th, and 9-16th days after the start of the culture for both the upper and lower layers of the cell culture container. During the culture period, the state of cell proliferation was confirmed with a phase contrast microscope.
  • the number of cells in the growth process was quantified from the number of cells contained in the cell sheet prepared under each culture condition and the amount of DNA.
  • tissue section preparation of corneal epithelial tissue tissue section staining method
  • the cell sheet was frozen and embedded according to a conventional method.
  • a 10 ⁇ m thick section was prepared from the frozen embedded tissue with a microtome.
  • hematoxylin-eosin staining and immunohistochemical staining were performed according to a conventional method.
  • Anti-CK3 antibody AE5 was used for immunohistochemical staining.
  • a cell to which the above method can be applied is a stem cell forming a tissue or a progenitor cell generated by differentiation of the stem cell. They first self-replicate and grow to a predetermined degree, and then start to differentiate at a general oxygen concentration. In general, differentiation tends to be suppressed in a hypoxic state. That is, switching from the hypoxic state to the normoxic state means the start of differentiation of the stem cells and progenitor cells that form the tissue.
  • a specific method for changing the oxygen concentration in the culture process of the stem cells and progenitor cells forming the tissue will be described.
  • cells seeded in a culture space such as a culture vessel are cultured so that the culture space has a low oxygen concentration.
  • the cells repeat proliferation by replication more rapidly than the normoxic concentration.
  • the overall size of proliferated and bound stem cells / progenitor cells, or the size of single cells, the rate of proliferation, etc. may vary. Therefore, it is desirable to arbitrarily determine the degree of size of cells obtained by self-replication depending on the cell type and switch from a low oxygen concentration to a normal oxygen concentration.
  • Cells exposed to normoxia start to differentiate and form tissues.
  • the degree of cell proliferation can also be determined by the occupation ratio with respect to the culture 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 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.
  • 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 for controlling the oxygen concentration can be controlled by the amount of oxygen supplied to the cells to be cultured.
  • the oxygen concentration of the gas supplied to the cells can be increased by supplying more oxygen or reducing the supply of nitrogen or carbon dioxide.
  • to reduce the oxygen concentration less oxygen is supplied.
  • it can be achieved by increasing the amount of nitrogen or carbon dioxide.
  • an automatic cell culture apparatus equipped with a function of automatically controlling the oxygen concentration based on the principle and method described in Example 1 will be described with reference to FIGS.
  • the calculation unit related to the control of the oxygen concentration will be described as an example incorporated in the control device 2, but the software and CPU related to the calculation unit are not limited to this, and an external computer, It may be built in the gas concentration adjusting unit 8 or the like.
  • the user When controlling the oxygen concentration, it is possible for the user to directly observe the cells in the cell culture apparatus and switch the oxygen concentration.
  • an imaging means for observing and imaging the cells such as the CCD camera 12 is provided.
  • the captured image may be displayed on the display screen 13 or the like, and the user may switch the oxygen concentration based on the captured image.
  • the display screen 13 may not be a visual display, but may prompt an instruction for hearing such as a buzzer.
  • 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. And binarizing based on the image, and calculating the cell occupation area in the image. After acquiring several points of data on the culture surface, if the cell occupation area is 100%, the oxygen concentration is changed from the low oxygen concentration to the normal oxygen concentration. When the cell occupation area does not reach the predetermined area, the oxygen concentration is not switched, the culture in the low oxygen state is continued, and the above operation is repeated at a predetermined timing, and the cell occupation area reaches 100%. Execute oxygen concentration switching at the time.
  • the cell occupancy increases as the self-replication of cells progresses, it is desirable to switch at a confluence near 100% of the cell occupancy near the final process of self-replication.
  • it can be arbitrarily set to 80% or 90% according to the specifications of the CCD camera, 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 oxygen concentration switching timing may be set in accordance with the size and occupied area until the self-replication of the cells is completed.
  • the cell size is the maximum value near the confluence. It becomes. After that, the number of cells per area increases and the cells shrink as it goes to the cobblestone form, so the average cell size gradually decreases, and after the cobblestone form, the cell shape is fixed The cell size is constant.
  • the CCD camera 12 captures 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. The average time-series change of the cell size is calculated by comparison, and the timing when the cell size becomes the maximum value is specified.
  • control device 2 changes the oxygen concentration after the specified maximum value, that is, after the confluent timing.
  • the display screen 13 may display the maximum cell size, the specified switching timing period, and the like to prompt the user to switch the oxygen concentration.
  • the method of specifying the maximum value of the cell size is not limited to this, and a specific value is set in advance according to the cell type, and the cell self-replicates to an average size exceeding this value.
  • the oxygen concentration may be switched at different times.
  • the average cell size as shown in FIG. 16 (a)
  • the time-series change of the size distribution is analyzed from the image, and the point at which the distribution peak is maximized is set as the maximum value.
  • a dispersion value for switching the oxygen concentration may be set in advance.
  • 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.
  • 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. Using this driving means, an image of a cross section in one direction of the culture surface is acquired. Furthermore, 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.
  • 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 16. As a display method, only an image with a cell defect may be displayed, or an image may not be displayed and a buzzer may warn that a cell defect has occurred. The presence or absence of cells can be determined. Based on the detection result of the optical coherence tomography 14, the control device 2 switches from a low oxygen concentration to a normal oxygen concentration if confluent.
  • the above-described cell image method and optical coherence tomography method can be used in combination, and the oxygen 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 cell thickness, By switching, the accuracy of determining confluence can be increased.
  • a set value for example, 100%
  • the entire culture surface for example, 100%
  • the control device 2 has an arithmetic means for switching the oxygen concentration.
  • the gas concentration adjusting section 8 is provided with an arithmetic means, and the gas concentration adjusting section 8 independent of the control apparatus 2 is used. It is also possible to control the change of the oxygen concentration.
  • the method for controlling the oxygen concentration may be controlled by the control device 2 so that the supply amount of each gas supplied from the gas supply unit is adjusted by the gas concentration adjustment unit 8.
  • the oxygen concentration of the gas supplied to the cells can be increased by supplying more oxygen or reducing the supply of nitrogen or carbon dioxide. Conversely, to reduce the oxygen concentration, less oxygen is supplied. Alternatively, it can be achieved by increasing the amount of nitrogen or carbon dioxide.
  • Oxygen concentration switching time is not at confluence, but after confluence, the cell density becomes high, and the volume of each cell becomes small, and it can be in a paving stone-like state. Since differentiation such as stratification is an event that occurs after passing through the cobblestone form, the desired tissue can be produced earlier by switching the oxygen concentration when the cobblestone form is shown.
  • the control device 2 processes the image so that the cell space in the cell image becomes clear after the cell occupancy reaches 100%. Thereafter, as shown in FIG. 17, a change in luminance on one line of an arbitrarily set image is calculated as a signal.
  • the cobblestone shape is discriminated based on whether or not the signal is regularly detected every 5 to 15 ⁇ m, which is the size of the cell in the cobblestone shape. Change the concentration from low oxygen concentration to normal oxygen concentration. If it does not show a cobblestone form, that is, if the cell is not the size in the cobblestone form as described above, in the detection result of the signal indicating the length between cells, continue the culture at a low oxygen concentration, Perform the above procedure again.
  • the peripheral portion of the outline of each cell in the imaged 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 cell peripheral portion (the portion with low luminance) also increases, so the average luminance of the image gradually decreases.
  • the number of cells increases because the cells are densely spread by the growth of the cells as the cobblestone shape is approached. That is, since the area around the outline of the cell in the image increases, the average luminance continues to decrease.
  • the paving stone-like form that is finally formed is a state in which the self-replication of cells on the culture surface is saturated, so the average brightness of the image is constant from the formation of the paving stone-like form until differentiation begins. It becomes the state of. Therefore, the control device 2 can determine the time when the average luminance of the image has a constant value as a paving stone shape, and can switch the oxygen concentration.
  • the size of the cell in the self-replication 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 determine the cobblestone form and switch the oxygen concentration.
  • FIG. 12 is a diagram schematically showing the configuration of the cell culture device 1, and each element controlled by the control device 2 is connected to the thermostatic chamber 3 and the culture vessel 4 disposed inside the thermostatic chamber 3. .
  • the control device 2 includes a temperature adjusting unit 5 for controlling the temperature of the thermostat 3, a humidity adjusting unit 6 for controlling the humidity in the culture vessel, and a gas concentration in the culture vessel,
  • a gas concentration adjusting unit 8 having a gas supply unit 7 and a culture solution supply pump having a liquid feeding tube connected to a tank 9 for holding the culture solution and waste solution for automatically exchanging the culture solution in the culture vessel 10, a temperature / humidity / CO 2 / O 2 sensor 11, a cell observation CCD camera 12, and a display screen 13 are connected for the purpose of controlling the operation of each component.
  • the temperature adjusting unit 4, the humidity adjusting unit 5, and the gas concentration adjusting unit 7 are connected to the thermostatic chamber 2, and the culture solution supply pump 8 is connected to the cell culture vessel 3.
  • oxygen is supplied into the thermostatic chamber, so that the closed culture vessel 3 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. This is a setting based on a parvovirus diameter of about 20 nm, the smallest virus currently known.
  • FIG. 13 shows a modified example of the apparatus configuration of Example 4 to which an optical coherence tomography 14 is attached based on Example 3. Since the optical coherence tomography 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.
  • a method for evaluating based on electrical resistance is presented as a non-invasive quality evaluation method for cell sheets.
  • 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.
  • FIG. 13 shows a modification of the second embodiment with the optical coherence tomography 14 attached
  • FIG. 14 shows a modification of the second embodiment with the electrical resistance measuring device 15 attached.
  • the 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 an exponential function shape 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 may be used in combination with the optical coherence tomography 14 as shown in FIG. 18 to check the quality of the cultured cells.
  • Example 4 the apparatus configuration for controlling the oxygen concentration in the culture tank was used.
  • the humidity control unit, the gas control unit, and the temperature / humidity / CO 2 / O 2 sensor are culture vessels.
  • An example in which the oxygen concentration in the culture vessel is controlled is shown as Example 5.
  • the configuration of the apparatus controls the oxygen concentration in the culture tank or the culture vessel, but the permeability of the gas permeable membrane installed in the culture vessel can be changed.
  • this configuration it is possible to control the oxygen concentration in the culture vessel without having to flow low oxygen gas or water vapor into the culture vessel.
  • the humidity controller, the gas controller, the CO 2, and the O 2 sensor are not required, and the apparatus configuration can be simplified.
  • 24 to 26 show modified examples of the device configuration.
  • the shape of the culture vessel is important.
  • An example of the culture container in the said apparatus structure is shown in FIG.
  • Two gas permeable membranes are provided on the frame 18 of the culture vessel 4, and the outermost layer suppresses the permeation of oxygen to form a suppression membrane 16 that can reduce the oxygen concentration in the culture vessel to a removable structure.
  • membrane 16 has used what is used as pharmaceuticals and food packaging materials, such as a polyethylene terephthalate, PVA, nylon, nylon type
  • the film etc. with a special layer called the super barrier film made from the Fuji Film company used by organic EL, electronic paper, a solar cell, etc. are mentioned.
  • the inner membrane is the porous membrane 17 having pores with a diameter of less than 20 nm as described in Example 2.
  • the cells are cultured while holding the suppression film 16. This realizes a low oxygen culture environment.
  • a mechanism capable of automatically removing the suppression film 16 is provided in the apparatus, and the mechanism is operated by the control device 2 so as to be in a period such as confluent or cobblestone form. The oxygen concentration in the culture vessel is changed by using the suppression film 16.
  • the method of removing the suppression film 16 is such that a manipulator having a driving means is installed in the cell culture device, and the control device 2 drives the manipulator to remove the suppression film 16.
  • the culture solution may evaporate because the inside of the culture tank is not a humid environment.
  • it is effective to provide a device for automatically injecting the evaporating culture solution into the cell container so that the amount of the culture solution is always kept constant.
  • the culture medium in the lower layer can be refluxed or constantly passed.
  • the culture container 21 containing one insert container 20 having a porous membrane as shown in FIG. 29 or a culture container 22 containing a plurality of insert containers 20 the culture container is circulated by refluxing the culture medium or constantly passing the culture container. Since the lower layer is always filled with a fresh culture solution, a cell sheet with a more stable or improved quality can be produced as compared with the case where reflux or constant flow is not performed.
  • the culture solution When the culture solution is refluxed or constantly passed, the culture solution can be circulated using, for example, a peristaltic pump.
  • a peristaltic pump In that case, for example, as shown in FIG. 30, by changing the height of the injection port and the discharge port, it is not necessary to block the flow path with a solenoid valve or the like, and after the culture container is filled with a certain amount of liquid, It is also possible to drain the liquid.
  • the discharge port may be made higher than the injection port. The height varies depending on the amount of liquid to be filled in the culture vessel.
  • the flow rate of the culture solution is desirably a flow rate that does not cause turbulence in order to prevent unintended damage to the cells.
  • the flow rate of the culture solution may be adjusted by controlling the operation of the peristaltic pump or the like by a control unit (not shown).
  • liquid injection and waste liquid is controlled by the control unit using an openable / closable member that shuts off the flow path with a solenoid valve or the like You may make it adjust.
  • the present invention is not limited to this, and the arrangement form of the insert container may be changed according to the purpose and application.
  • the insert may be arranged in a circular shape, or the installation height of the insert may be changed for each insert. At that time, a height adjusting mechanism may be provided.
  • the lower layer of the culture vessel is always filled with fresh culture liquid, so the quality is more stable than when refluxing and not constantly flowing.
  • improved cell culture can be performed.
  • the present invention is useful as a cell culture method and a cell culture apparatus.

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WO2015152025A1 (fr) * 2014-03-31 2015-10-08 テルモ株式会社 Procédé permettant d'évaluer la qualité d'une culture cellulaire sous forme de feuille
JPWO2015152025A1 (ja) * 2014-03-31 2017-04-13 テルモ株式会社 シート状細胞培養物の品質評価方法
JP2017522016A (ja) * 2014-06-27 2017-08-10 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア 培養哺乳動物輪部幹細胞、その産生方法及びその使用
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WO2016009789A1 (fr) * 2014-07-18 2016-01-21 株式会社日立ハイテクノロジーズ Dispositif de culture cellulaire et dispositif d'analyse d'images
JP2016021915A (ja) * 2014-07-18 2016-02-08 株式会社日立ハイテクノロジーズ 細胞培養装置および画像解析装置
JP2016111975A (ja) * 2014-12-16 2016-06-23 大日本印刷株式会社 細胞培養容器及び細胞積層体の作製方法
JP7465294B2 (ja) 2015-06-25 2024-04-10 オークランド ユニサービシズ リミテッド 組織を培養する装置及び方法
JP2018518190A (ja) * 2015-06-25 2018-07-12 オークランド ユニサービシズ リミテッド 組織を培養する装置及び方法
JP2022068312A (ja) * 2015-06-25 2022-05-09 オークランド ユニサービシズ リミテッド 組織を培養する装置及び方法
WO2017038887A1 (fr) * 2015-08-31 2017-03-09 アイ・ピース株式会社 Système de production de cellules souches pluripotentes
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JPWO2017038887A1 (ja) * 2015-08-31 2017-08-31 剛士 田邊 多能性幹細胞製造システム
JP2020089304A (ja) * 2018-12-05 2020-06-11 株式会社日立ハイテク 細胞培養装置および培養液の気体濃度制御方法
JP2021073942A (ja) * 2019-11-12 2021-05-20 株式会社日立製作所 細胞シート評価方法
JP7316912B2 (ja) 2019-11-12 2023-07-28 株式会社日立製作所 細胞シート評価方法

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