WO2013136372A1

WO2013136372A1 - Cell sheet, cell culture method, and cell culture apparatus

Info

Publication number: WO2013136372A1
Application number: PCT/JP2012/001834
Authority: WO
Inventors: 亮太中嶌; 志津松岡; 豊茂小林
Original assignee: 株式会社日立製作所
Priority date: 2012-03-16
Filing date: 2012-03-16
Publication date: 2013-09-19
Also published as: JP5991368B2; JPWO2013136372A1

Abstract

A cell culture method characterized by culturing a stem cell or a progenitor cell that can form a tissue for a first period, which is a portion of a period for causing the self-replication of the stem cell or the progenitor cell and included in a period for culturing of the stem cell or the progenitor cell, while supplying oxygen in a first supply amount, then changing the amount of oxygen to be supplied from the first supply amount to a second supply amount which is higher than the first supply amount on the basis of the degree of the proliferation of the stem cell or the progenitor cell by self-replication, and then culturing the stem cell or the progenitor cell for a second period, which is a period including a period for causing the differentiation of the stem cell, while supplying oxygen in the above-mentioned second supply amount, wherein no feeder cell is used during the first period and the second period.

Description

Cell sheet, cell culture method and cell culture apparatus

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.

Among the regenerated tissues, vegetative cells often called feeder cells are used when producing epithelial cell sheets. As this vegetative cell, a cell called NIH / 3T3 or 3T3-J2 derived from a mouse is generally used. In regenerative medicine, 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”). When using 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.

In order to solve these problems, 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.

However, 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 (see Non-Patent Document 1). In the treatment of patients with corneal limbal stem cell exhaustion, 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). There is a problem that a cell sheet produced without cells and having reduced stem cells cannot be applied to treatment.

In view of the above problems, 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.

From 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%).

According to the cell culturing method and the cell culturing apparatus of the present invention, 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.

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. The figure which shows the days required for cell culture in the comparative examples 2 and 3 and the examples 2-6. The figure which shows the phase-contrast microscope image of the corneal limbal epithelial cell in the comparative examples 2 and 3 and the example 4. FIG. The figure which shows the external appearance of the corneal epithelial cell sheet | seat in the comparative examples 2 and 3 and the examples 2-6. The figure which shows the cell number in a corneal epithelial cell sheet | seat in the comparative examples 2 and 3 and the examples 2-6. The figure which shows the colony formation rate of the stem cell and progenitor cell in a corneal epithelial cell sheet | seat in the comparative examples 2 and 3 and the examples 2-6. The figure which shows colony formation of the stem cell and progenitor cell in a corneal epithelial cell sheet | seat in the comparative examples 2 and 3 and the examples 2-6 The figure which shows the hematoxylin-eosin dyeing | staining image of the corneal epithelial cell sheet section | slice in the comparative examples 2 and 3 and the examples 2-6. The figure which shows the CK3 and cell nucleus dyeing | staining image of a corneal epithelial cell sheet | seat section in Comparative Examples 2 and 3 and Examples 2-5. 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 when adding an optical coherence tomography to the structure of FIG. The apparatus block diagram at the time of adding an electrical resistance measuring apparatus to the structure of FIG. The figure which shows the relationship between a culture | cultivation day and the magnitude | size of a cell. The figure showing the distribution of the magnitude | size of the cell in the time of confluence and a paving stone form. The figure which shows confluent and a paving stone form. 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 in the case of switching directly the oxygen concentration in the culture container which can change a gas permeable membrane. 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 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 figure which shows the culture container which can replace | exchange gas permeable membrane. The figure which shows the CK3 and cell nucleus dyeing | staining image of a corneal epithelial cell sheet section | slice in the comparative examples 2 and 3 and the examples 7-10. The figure which shows a culture container. The modification of FIG.

The form for carrying out the present invention will be described with reference to examples. However, these forms and examples are merely examples for realizing the present invention, and do not limit the technical scope of the present invention. In each drawing, the same reference numerals are assigned to common components.

In this example, the principle and method of shortening the regenerative tissue production period by controlling the oxygen concentration will be described. The following is an example using a rabbit corneal epithelial cell sheet as a model. 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. Hereinafter, the principle of shortening the culture period will be described using 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. Hereinafter, the differentiation phase is referred to as the stratification phase.

First, the principle of producing a stratified epithelial cell sheet retaining stem cells without using feeder cells by culturing at a low oxygen concentration in the self-renewal phase and culturing at a normal oxygen concentration in the stratification phase is as follows. This will be explained using demonstration.

First, in the colony formation test using cells collected from the rabbit cornea ring, the case of culturing at 20% O ₂ without using feeder cells is compared with Comparative Example 1, and culturing at 5% O ₂ without using feeder cells. The case was taken as Example 1 and the degree of colony formation was determined. FIG. 1 is a diagram showing the results. In 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.

Subsequently, in the formation of a corneal epithelial cell sheet, 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 cultured at 2% O ₂ without using feeder cells was referred to as Comparative Example 3. 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. In Comparative Example 2, on the 11th day of culture, and in Comparative Example 3 on the 12th day of culture, 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. In both the comparative example and the actual example, 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.

The results shown in 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 . In addition, it was found that in the stratification period, differentiation is suppressed at low oxygen, but differentiation occurs at O _{2 of} 15% or more. Therefore, 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.

(Rabbit corneal epithelial cell culture method)
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 μg / 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. Sowing. As the culture solution, KCM medium containing 5% FBS used for culturing epithelial cells was used. 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.

(Appearance inspection and cell number measurement method of corneal epithelial cell sheet)
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. A method for calculating the number of cells contained in the prepared cell sheet is described below. First, dispase (200 U / ml) was injected into the lower culture layer and treated at 37 ° C. for 60 minutes. After the treatment, the cell sheet was peeled from the culture surface with tweezers, and the appearance was examined. The detached cell sheet was treated with 0.25% trypsin at 37 ° C. for 10 minutes to determine the number of cells contained in the cell sheet as a cell suspension (TC10 bio-rad) (n = 4).

(Calculation method of colony formation rate)
In order to determine the colony formation rate, the cell sheet detached with dispase was used as a cell suspension using a 0.25% trypsin solution, and 2,000 cells of which were treated with mitomycin C-treated NIH-3T3 cells. It seed | inoculated to the 10cm dish seed | inoculated so that it might become * 10 < ⁴ > / cm < ² >, and it culture | cultivated for about 10 days in the KCM culture medium. The colony formation rate was calculated by calculating the ratio of the number of colonies that appeared (diameter of about 2 mm or more) / 2000.

(Tissue section preparation of corneal epithelial tissue, tissue section staining method)
In order to prepare a tissue section, the cell sheet was frozen and embedded according to a conventional method. A 10 μm thick section was prepared from the frozen embedded tissue with a microtome. Using the prepared section, hematoxylin-eosin staining and immunohistochemical staining were performed according to a conventional method. Anti-CK3 antibody (AE5) was used for immunohistochemical staining.

The range of cell types to which the above method can be applied will be described with reference to FIG. 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.

Next, 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. First, cells seeded in a culture space such as a culture vessel are cultured so that the culture space has a low oxygen concentration. In the self-renewal phase, the cells repeat proliferation by replication more rapidly than the normoxic concentration. Depending on the type of stem cells to be cultured, 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. When the cell occupancy with respect to the culture surface becomes 100%, as described in the above principle, the cell is positioned as confluent, so the oxygen concentration is switched to the normal oxygen concentration (20%). Note that the occupation ratio for determining the switching timing can be arbitrarily set to 80% or 90% depending on the characteristics of the cell type.

In addition, regarding the point of time when the oxygen concentration is switched, it is effective to switch not only in a state where the cells are confluent as described above but also in a cobblestone form. As shown in FIG. 11, the cobblestone form is a state that occurs from the time when cells become confluent until the start of the differentiation phase. By switching oxygen in the cobblestone form, the oxygen level is reduced until just before the differentiation phase. Can be cultured in a shorter period of time.

Furthermore, after switching the oxygen concentration, as shown in FIG. 11, by confirming whether a phenomenon called tight junction that occurs in the differentiation period after the paving stone shape occurs, the cells after the oxygen concentration switching It is possible to evaluate the quality of normal culture. 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. For example, 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.

In this example, 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. In the following description of the present embodiment, 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.

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. However, 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. In addition, the display screen 13 may not be a visual display, but may prompt an instruction for hearing such as a buzzer.

In order to automatically control the oxygen concentration, it is desirable to automatically recognize the cell state and determine the appropriate time for changing the oxygen concentration. First, 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.

Since 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. However, 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.

Also, as another method for determining confluence, it is possible to determine from the average cell size. As shown in FIG. 15, since the cells adhered to the culture surface usually grow while stretching the temporary foot, the cell size becomes larger than that immediately after seeding, and the average 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.

Further, the control device 2 changes the oxygen concentration after the specified maximum value, that is, after the confluent timing. At this time, 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. Further, instead of 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. Alternatively, a dispersion value for switching the oxygen concentration may be set in advance.

In addition to the method for discriminating the oxygen concentration switching time from the cell image as described above, it is possible to determine the oxygen concentration switching by analyzing the cell state with the optical coherence tomometer 14 as shown in FIG. In 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. On the other hand, in 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. . 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. 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. In addition, since the size of the cell can be analyzed by an image analysis method described later, the interval at which the cross-sectional image is acquired may be determined depending on the degree of cell proliferation. Further, 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.

As described above, it is possible to determine whether the state is confluent and to switch the oxygen concentration. However, it is also possible to set the switching point to be after a predetermined time from the point when it becomes confluent. It is effective for. When a predetermined time elapses, a paving stone-like form, which will be described in detail in Example 3, 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.

In the present embodiment, the case where the control device 2 has an arithmetic means for switching the oxygen concentration has been described. However, 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. For example, 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. First, according to the cell analysis result described in Example 2, 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. From this signal distribution, the cobblestone shape is discriminated based on whether or not the signal is regularly detected every 5 to 15 μm, 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.

As another method for determining whether or not it is a cobblestone shape, there is a method for determining from the distribution of the signal of the image of the cell imaged 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 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.

After the culture surface becomes confluent, 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.

It is also possible to determine the cobblestone form by using the average cell size described in Example 2. As shown in FIG. 15, in the cobblestone form, the time series change of the cells is condensed from the confluent state and stays at a constant size. Therefore, the oxygen concentration is switched during the period of the constant size. The calculation method in the control device 2 until the period is obtained is the same as the method described in the second embodiment.

It is also possible to determine the cobblestone form from the cell size distribution analyzed by the control device 2. As shown in FIG. 16, 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.

Therefore, when 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.

In this example, a method for switching the oxygen concentration in the entire culture tank, which is a culture space, by the cell culture method described in Example 1 and the oxygen concentration automatic control method shown in Examples 2 and 3 will be described.

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. In the case of the above apparatus configuration, 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. At this time, 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.

提示 In addition to the method using optical coherence tomography, 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, and 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.

In Example 4, the apparatus configuration for controlling the oxygen concentration in the culture tank was used. However, as in the configuration of FIG. 19, 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.

With this configuration, water vapor can also flow into the culture vessel using the gas supply port in the culture vessel. Moreover, when the whole culture tank is a constant temperature and humidity environment, since the culture tank itself is not a sterile space, there is a risk of mold and bacteria breeding. On the other hand, when the inside of the culture container is a constant temperature and high humidity environment, the inside of the culture container is an aseptic space, so there is an advantage that the risk of mold and bacteria breeding is low. Further, since it is not necessary to provide a special film on the culture container, the culture container preparation process can be simplified. 20 to 22 show modified examples of the device configuration as in the fourth embodiment.

In Examples 4 and 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. With 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. For example, as shown in FIG. 23, 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.

In this 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. The suppression film | membrane 16 has used what is used as pharmaceuticals and food packaging materials, such as a polyethylene terephthalate, PVA, nylon, nylon type | system | group, and a silica vapor deposition type | system | group film. Moreover, 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. In the self-replication period, the cells are cultured while holding the suppression film 16. This realizes a low oxygen culture environment. In order to obtain a normal oxygen concentration after the completion of self-replication, 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.

After the suppression membrane is removed, cell differentiation culture is performed. At this time, the culture solution may evaporate because the inside of the culture tank is not a humid environment. As a method for avoiding this, 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.

When feeder cells are used in the lower layer of the culture vessel, components secreted from the feeder cells are required, so it is not preferable to reflux or constantly pass the culture solution in the lower layer. However, when feeder cells are not used, the culture medium in the lower layer can be refluxed or constantly passed. For example, in a 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.

When the culture solution is refluxed or constantly passed, the culture solution can be circulated using, for example, 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. Specifically, 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.

It should be noted that 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).

In the above, an example in which the height of the liquid injection port and the discharge port is changed is shown, but not limited to this, 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.

29 and 30 show an example of a configuration for constantly passing liquids, but 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. For example, 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.

Because the culture medium is refluxed and constantly passed as described above, 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. Alternatively, improved cell culture can be performed.

The present invention is useful as a cell culture method and a cell culture apparatus.

DESCRIPTION OF SYMBOLS 1 ... Cell culture apparatus 2 ... Control apparatus 3 ... Constant temperature bath 4 ... Culture container 5 ... Temperature control part 6 ... Humidity control part 7 ... Gas supply part 8 ... Gas concentration control part 9 ... Culture solution / waste liquid tank 10 ... Culture solution supply Pump 11 ... Temperature / humidity / gas sensor 12 ... CCD camera 13 for cell observation ... Display device 14 ... Optical coherence tomometer 15 ... Electrical resistance measuring device 16 ... Suppression membrane 17 ... Porous membrane 18 ... Frame 19 ... Temperature sensor 20 ... Insert Container 21 ... Culture container 22 ... Culture container

Claims

During the period of culturing the stem cells or progenitor cells that form the tissue,
Culturing a first period of time within which the stem cells or progenitor cells self-replicate with a first oxygen supply,
Based on the degree of proliferation due to self-proliferation of the stem cells or progenitor cells, change to a second oxygen supply amount of oxygen supply amount higher than the first oxygen supply amount,
Culturing a second period including a period in which the stem cells differentiate with the second oxygen supply amount;
A cell culture method, wherein no feeder cells are used in the first period and the second period.
During the period of culturing stem cells or progenitor cells that form epithelial tissue,
Culturing a first period of time within which the stem cells or progenitor cells self-replicate with a first oxygen supply,
Based on the degree of proliferation due to self-proliferation of the stem cells or progenitor cells, change to a second oxygen supply amount of oxygen supply amount higher than the first oxygen supply amount,
Culturing a second period including a period in which the stem cells differentiate with the second oxygen supply amount;
A cell culture method, wherein no feeder cells are used in the first period and the second period.
The first oxygen supply amount is 1% or more and less than 15%,
The cell culture method according to claim 1, wherein the second oxygen supply amount is 15% or more.
A cell sheet produced by the cell culture method according to claim 1.
A cell culture device having a culture region for culturing stem cells or progenitor cells forming a tissue,
An oxygen regulator that regulates the amount of oxygen supplied in the culture region;
A control unit for controlling the oxygen control unit;
Of the cell culture period,
A first oxygen supply in a first period within which the stem cells or progenitor cells self-replicate;
A second oxygen supply amount of an oxygen supply amount greater than the first oxygen supply amount in a second period including a period in which the stem cells or progenitor cells differentiate; and
Control
A controller that controls the oxygen regulator to change the first oxygen supply amount to the second oxygen supply amount based on the degree of proliferation of the stem cells or progenitor cells by self-replication; and
Have
A cell culture device, wherein no feeder cells are used in the first period and the second period.
A container holding part for holding a culture container having a culture region in which stem cells or progenitor cells forming epithelial tissue are cultured;
An oxygen control unit for adjusting the oxygen supply amount in the culture vessel;
A control unit for controlling the oxygen control unit;
Of the stem cell or progenitor cell culture period,
A first oxygen supply amount in a first period within a period in which the stem cells or progenitor cells grow so as to be in contact with a culture surface formed in the culture region;
A second oxygen supply having a higher oxygen supply amount than the first oxygen supply amount in a second period including a period in which the stem cells or progenitor cells grown on the culture surface grow so as to be layered on the culture surface Quantity,
Control
A control unit that controls the oxygen adjusting unit to change the first oxygen supply amount to the second oxygen supply amount based on the degree of growth in a period of growing so as to be in contact with the culture surface;
Have
A cell culture device, wherein no feeder cells are used in the first period and the second period.
The first oxygen supply amount is 1% or more and less than 15%,
6. The cell culture device according to claim 5, wherein the second oxygen supply amount is 15% or more.
The cell culture device comprises:
Further comprising an imaging unit for imaging the stem cells or progenitor cells,
The controller is
The cell culture device according to claim 5, wherein the degree is calculated from an image acquired from the imaging unit.
The controller is
During the self-replication period, when the stem cells or progenitor cells proliferate by self-replication up to a predetermined region in the culture region, the first oxygen supply amount is changed to the second oxygen supply amount, The cell culture device according to claim 8, wherein the oxygen control unit is controlled.
The controller is
In the self-replicating period, after the stem cells or progenitor cells proliferate by self-replication up to a predetermined region in the culture region, the first oxygen supply amount is set to the second oxygen when a predetermined time has elapsed. The cell culture device according to claim 8, wherein the oxygen control unit is controlled so as to change to an oxygen supply amount.
The cell culture device comprises:
The culture region has a first surface for culturing stem cells or progenitor cells,
The cell culture device according to claim 5, wherein the degree of proliferation is an occupation ratio of the stem cells or progenitor cells to the first surface.
The cell culture device comprises:
Further comprising an imaging unit for imaging the stem cells or progenitor cells,
The controller is
The cell culture device according to claim 11, wherein the occupation ratio is calculated from an image acquired from the imaging unit.
The cell culture device comprises:
The cell culture device according to claim 12, further comprising a display unit that displays the occupation ratio.
The controller is
13. The cell culture device according to claim 12, wherein the oxygen control unit is controlled to change the first oxygen supply amount to the second oxygen supply amount based on the occupation rate.
The cell culture device comprises:
Further comprising an imaging unit for imaging the stem cells or progenitor cells,
The controller is
6. The cell culture device according to claim 5, wherein an average time-series change in the size of the stem cell or progenitor cell is calculated based on an image acquired from the imaging unit.