WO2015107667A1 - Dispositif de culture de cellules - Google Patents

Dispositif de culture de cellules Download PDF

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Publication number
WO2015107667A1
WO2015107667A1 PCT/JP2014/050735 JP2014050735W WO2015107667A1 WO 2015107667 A1 WO2015107667 A1 WO 2015107667A1 JP 2014050735 W JP2014050735 W JP 2014050735W WO 2015107667 A1 WO2015107667 A1 WO 2015107667A1
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Prior art keywords
cell
cell culture
culture
culture device
quality
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PCT/JP2014/050735
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English (en)
Japanese (ja)
Inventor
貴之 野崎
中村　拓
広斌 周
政晴 木山
鈴木　大介
由美子 五十嵐
志津 武田
菅谷　昌和
光一 寺田
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株式会社日立製作所
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Priority to PCT/JP2014/050735 priority Critical patent/WO2015107667A1/fr
Publication of WO2015107667A1 publication Critical patent/WO2015107667A1/fr

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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
    • 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
    • C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
    • 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
    • C12M41/48—Automatic or computerized control

Definitions

  • the present invention relates to a culture apparatus, and relates to a technique for culturing cells or tissues by automatic operation and determining the quality of the cultured cells or tissues.
  • Regenerative medicine that uses regenerated tissue produced from cells as a raw material to restore the function of organs and the like is expected as a radical treatment method for diseases for which there has been no conventional treatment method.
  • the subjects of treatment range from skin, cornea, esophagus, heart, bone, cartilage, etc., and their clinical applications are rapidly increasing.
  • SOP Standard Procedure
  • GMP Good Manufacturing Practice
  • CPC Cell Processing Center
  • regenerated tissue is derived from cells, it is impossible to produce completely the same regenerated tissue for all indicators.
  • the distribution of cells, proteins, etc. constituting the regenerated tissue after production cannot be completely spatially uniform.
  • the distribution of stem cells in one regenerative tissue, the thickness of the regenerated tissue, and the presence or absence of irregularities differ depending on the location of the regenerative tissue.
  • Such information is obtained at different culturing periods, for example, when noninvasive observation using a microscope is used.
  • transplanting a regenerated tissue after manufacture it is possible to select and transplant a good one from a plurality of regenerated tissues manufactured at the same time, or to trim and reshape a single regenerated tissue for transplantation.
  • the distribution of cells, proteins, etc. that make up the regenerated tissue after production is not spatially uniform, and there are parts that are more suitable for transplantation and parts that are not, but the regenerated tissue is trimmed and transplanted. In doing so, it is desirable to be able to determine which part of the regenerated tissue is suitable for transplantation.
  • Patent Documents 1 and 2 an apparatus capable of determining the quality of cells or tissues with respect to a spatial position by acquiring and analyzing a microscope image or the like during culture is disclosed. Has been. It is also possible to obtain global information on the entire culture container by superimposing microscopic images as local information on the culture container.
  • information used as an index for determining the quality of the regenerated tissue can be obtained at different culture periods, particularly when non-invasive observation using a microscope is used.
  • the distribution of stem cells is information obtained in the early stage of culture, while the thickness of the regenerated tissue and the presence or absence of irregularities are obtained in the later stage of culture.
  • the place where the regenerated tissue after production is taken out is the operating room or a place close to it, while the culture or observation is performed on the CPC or a production place equivalent thereto.
  • Patent Documents 1 and 2 are apparatuses that can determine the quality of a regenerated tissue by analyzing each of a plurality of pieces of information acquired at various times in a culture process. Therefore, for the above-mentioned purpose, a function for determining pass / fail by integrating a plurality of pieces of information at various times in the culture process is necessary. In addition, when selecting a good one from multiple regenerative tissues or selecting and trimming a good part from a single regenerative structure, a function is required to present that information to the operator. It is.
  • An object of the present invention is to solve such problems, and to provide a cell culture device capable of determining the quality of a regenerated tissue based on integrated information on different culture periods and providing the information at the time of transplantation. .
  • a culture container in which cells are held a photographing unit for photographing a cell image of cells held in the culture container, and a plurality of divisions of the cell image photographed by the photographing unit
  • a cell culture apparatus having a configuration including a control unit that can evaluate the quality of regeneration based on information of cell images of different culture periods in a region.
  • the cell culture device of the present invention it is possible to select a plurality of regenerated tissues suitable for transplantation from among a plurality of regenerated tissues, or to select a part suitable for transplantation from one regenerated tissue. . Furthermore, it is possible to carry out the work in a state where the information is reflected in the regenerated tissue at the time of transplantation.
  • Example 1 is a culture container in which cells are held, a photographing unit that photographs a cell image of the cells held in the culture container, and a plurality of divided regions of the cell image photographed by the photographing unit, cells in different culture periods
  • a plurality of pieces of information on different culture periods obtained from a photographing unit such as a microscope are integrated, and an image that can be used to determine pass / fail is provided. Judgment results can be presented to workers at the time of transplantation.
  • the control unit of the automatic culture apparatus accumulates the cell images acquired using the imaging unit, and records information such as stem cell distribution, thickness, unevenness and the like along with the coordinates in the culture container. A plurality of acquired images are overlapped to create an image that can be comprehensively determined as good or bad for the regenerated tissue. Using this, the control unit determines the quality of the regenerated tissue, that is, selects a part suitable for transplantation.
  • the determination result is presented to the operator at the time of transplantation. Coordinates are described in the culture vessel main body in which the regenerated tissue is stored, and based on the coordinates, the operator can reflect the determination result in the regenerated tissue. Or the display part which can display a determination result is used, and an operator enables reflection of a determination result to a reproduction
  • the automatic culture apparatus is an incubator section 100 that is a space for culturing cells at a culture temperature of 37 ° C., and a control terminal used by an operator / user for operation control of the automatic culture apparatus.
  • 101 a gas supply unit 102 for supplying CO 2 gas or the like to the incubator unit 100, a medium bottle 114 containing a medium inside, a refrigerator 103 for storing a culture supernatant bag 115 for collecting the culture supernatant, and a power supply box included
  • a control unit 104 for controlling the automatic culture apparatus.
  • Reference numeral 116 denotes a gas supply unit 102, a tube for supplying necessary gas, medium, and the like from various bottles.
  • the control terminal 101 and the control unit 104 are collectively referred to as a control unit.
  • the incubator unit 100 includes a culture vessel unit 106 that accommodates a plurality of culture vessels 105 for culturing cells.
  • the cells in the culture vessel 105 are observed with the microscope 107.
  • the microscope 107 is provided with a camera (not shown) for capturing a cell image observed with the microscope as an image, and the microscope 107 and the camera function as a photographing unit.
  • a rotary valve mechanism 108 that switches the supply of the culture medium and the like to each culture vessel 105, an electromagnetic valve that feeds the culture medium and the like to each culture vessel 105, a tube pump, etc.
  • a flow path portion 109 having a drive system is installed.
  • an upper layer cell bottle 110, a lower layer cell bottle 111, a humidification bottle unit 112, a preheating bottle unit 113, and the like are installed and connected by a tube (not shown).
  • the automatic culturing apparatus of the present embodiment is a cell seeding by feeding a cell suspension to the culture vessel 105, a culture for maintaining the temperature at 37 ° C. while appropriately performing gas exchange, an old medium is discharged, and a new medium is supplied. Replace the medium, observe the cells with a microscope, etc.
  • the steps performed by the automatic culture apparatus are cell seeding, medium exchange, culture, and microscopic observation. However, it goes without saying that some steps can be replaced manually.
  • a microscope image is acquired by a photographing unit including a camera. Coordinates are set in advance for each culture vessel displayed on the display of the control terminal 101.
  • the coordinates can be uniquely determined for the culture vessel by an image processing program operating on the control terminal 101 or a central processing unit (CPU) in the control unit 104. That is, even if the direction of the culture vessel changes, the coordinate information is displayed on the display so as not to change. Further, the entire culture surface of the culture container can be divided based on the coordinates set for each culture container.
  • Each of the divided areas corresponds to one microscope image that can be acquired at the time of observation with a microscope. That is, when a microscopic image of each divided region is taken and all the microscopic images are superimposed, the entire culture surface of the culture vessel is reconstructed. This makes it possible to obtain global information for the entire culture vessel.
  • FIG. 3 shows an example of a cell image acquired by the imaging unit including the microscope 107 of the automatic culture apparatus of this example.
  • an index example is shown on the left side
  • a cell image example corresponding to the index is shown on the right side.
  • the presence / absence of colony formation, the arrival / non-arrival of a cell dense state, and the presence / absence of unevenness are exemplified.
  • epidermal cells such as oral mucosa cells, corneal epithelial cells, and epidermal cells
  • the cells that form colonies are often derived from somatic stem cells. By distinguishing the divided areas where colonies are formed on the culture surface from the divided areas where the colonies are not formed, it is possible to serve as an index for determining the distribution of somatic stem cells on the culture surface.
  • the cells cover the surface of the culture and become confluent.
  • the divided regions that are in the cell dense state indicate that the number of cells is large. Therefore, it can be used as an index for determining the number of cells using the arrival or non-arrival of the cell dense state at a certain time as an index.
  • As an index other than the date and time to reach the cell confluence state it is possible to calculate the cell occupancy rate in each divided region and evaluate the cell number over time based on the value before the date and time to reach the cell confluence state It is.
  • the later stage of the culture that has undergone a cell-packed state there may be a mixture of a divided region where unevenness as shown by the concave portion 302 in FIG. 3 occurs in the regenerated tissue and a smooth divided region.
  • a thick divided region and a thin divided region can be mixed.
  • the presence / absence of unevenness and the thickness can be used as an index for determining the quality.
  • the criteria for determining whether or not colonies are formed, whether cells are densely reached / not reached, and whether there are irregularities are as follows. Cell types to be cultured and organ types, autologous cells or allogeneic cells to be subjected to regenerative medical treatment It may change depending on the selection of
  • somatic stem cells for corneal epithelial stem cell exhaustion using autologous corneal epithelial cells or oral mucosal cells collected from the patient himself
  • adopt a criterion that makes the content of somatic stem cells different from that of self-derived cells. can also be considered.
  • esophageal epithelium regeneration in the case of esophageal regeneration aimed at avoiding esophageal stricture by transplanting into the esophagus after resection of esophageal cancer using autologous oral mucosal cells or epidermal cells collected from the patient himself Therefore, for the demand for unevenness and smoothness in the regenerated tissue, for example, it is conceivable to adopt a judgment criterion different from the above-mentioned corneal epithelial regeneration.
  • the criteria for the somatic stem cell content, the presence / absence of unevenness, and smoothness may employ different criteria from the aforementioned corneal epithelial regeneration.
  • the condition that the thickness of the regenerated tissue is within a certain range may be a condition, and in that case, the judgment criteria may be determined using the date and time when the cell is congested as an index. Conceivable. Note that the judgment criteria described here are merely examples, and the judgment criteria for each index may be changed in accordance with an increase in future scientific knowledge.
  • FIG. 4 a flow of integrating a plurality of pieces of information at different culture periods in the control unit of the automatic culture apparatus of the present embodiment using cell images in each divided region acquired by the microscope, and performing pass / fail judgment using the information. Indicates.
  • the culture container used in the automatic culture apparatus of the present example has a notch or the like on the outer surface thereof, so that the direction of the culture container can be visually recognized by the culture container alone.
  • the position of the culture vessel is uniquely determined by cutting or the like.
  • the orientation of the culture container is always constant, especially during cell observation in the automatic culture apparatus.
  • coordinates are provided with respect to the culture surface in the culture container, based on the notch of the culture container.
  • the relative position of each part is always constant, and as a result, the coordinates are also constant. These coordinates are also inputted in advance in the control unit.
  • Step 1 After the operator installs the flow path including the culture vessel to the automatic culture apparatus, the automatic culture apparatus is started (Step 1, hereinafter abbreviated as S1).
  • the culture container is installed in a fixed direction with respect to the culture container base of the culture container unit 106 by cutting or the like.
  • the coordinates of the culture container relative to the direction of the culture container that is, the coordinate axis and the scale of the coordinates are input in advance in the control unit such as the control terminals 104 and 401.
  • the control terminal 104 obtains the size of the cell image according to the input information, and divides the culture surface into divided regions having the same size (S2). Specifically, for example, the control terminal 104 determines the size of the divided region by selecting an objective lens of a microscope. Since each divided area has the same area as the cell image at the time of observation, the entire culture surface can be reconstructed by superimposing the cell images for all the divided areas. As another method, after reconstructing the entire culture surface using the image data of the cell image taken at an arbitrary microscope magnification, by re-dividing the reconstructed culture surface into divided areas of the size desired by the operator, A divided region having an arbitrary size may be used.
  • the operator selects and inputs an index and criteria for determining the quality of the regenerated tissue based on information such as the cell type to be cultured and the organ type to be transplanted.
  • information such as the cell type to be cultured and the organ type to be transplanted.
  • automatic culture is started (S4).
  • the criteria for pass / fail judgment are specifically information on the distribution of stem cells, reaching and non-arrival of the densely packed state, the thickness of the regenerated tissue, and unevenness information. These indicators can also be classified into the distribution of stem cells used before reaching the cell congested state, the thickness of the regenerated tissue used after reaching the cell confluent state, and unevenness information. It is good also as using the index which uses time as a boundary properly mechanically.
  • the quality determination criteria may be determined in advance from a combination of cell types to be cultured, organ types to be transplanted, and the like.
  • the operator inputs information such as the cell type to be cultured and the organ type to be transplanted, and the control unit selects an appropriate quality determination criterion based on the information.
  • the control terminals 101 and 401 recognize the colonies, the cell occupying portions, the unevenness, and the like based on the indexes set in advance for each divided region with respect to the acquired cell images of all the divided regions. Specifically, in the case of a colony, in the cell image in each divided region, the state where the cells that are colonies are dense is recognized by image analysis of the control terminals 101 and 401, and the number and size thereof are measured (S6). Also, as an image process, a color is given to the recognized colony so that the colony can be easily recognized when the operator views the image. In an operation described later, an operation for superimposing images of different culture periods is performed. At this time, different colors can be simultaneously analyzed by adding colors.
  • means such as three-dimensional display can be used instead of or together with this color display.
  • this means such as three-dimensional display it is possible to display each position on the culture surface with XY coordinates and display the evaluation result at each position as a numerical value with Z coordinates. For example, in the case of an indicator of the presence or absence of a colony, 1 may be set when there is a colony, and 0 may be set when there is no colony. As a result, it is possible to display a three-dimensional property corresponding to the position of the culture surface.
  • the cells are recognized by image analysis of the control unit, and the ratio of the total area occupied by all cells in the divided region is obtained as the cell occupation area. If it is 100%, it has reached the cell dense state, and if it is less than 100%, it has not reached the cell dense state.
  • the recognized cells are colored to make it easier to recognize the degree of cell congestion when an operator views the image.
  • the thickness of the regenerated tissue at the later stage of culture the thickness of the regenerated tissue is measured using the position where the microscope is focused in each divided region as an index.
  • a color corresponding to the height of the regenerative tissue makes it easy to recognize the height according to the location of the regenerative tissue when the operator views the image.
  • the size and shape of the concave portions are recognized by image analysis.
  • tissue is measured by making into a parameter
  • colors are given according to the unevenness of the regenerated tissue, so that the presence or absence of unevenness can be easily recognized when the operator views the image.
  • pass / fail is determined for each index in each divided area.
  • the determination criteria for pass / fail are determined based on the index and standard input by the operator before the start of culture, or the index and standard determined by the control unit based on the input cell type and target organ information.
  • the colony distribution for example, in the case of corneal regeneration for stem cell exhaustion using autologous cells, it is desirable that the colony distribution be large.
  • the number and size of colonies when the culture is good in advance and the respective values when the culture is not good are obtained, and the value therebetween (for example, the average value) is set as a threshold value for the pass / fail judgment criterion.
  • the other indicators are also determined by using the values when they are good and bad.
  • the index that can be determined depends on the time when the observation is performed, the index that is determined according to the culture time may be narrowed down.
  • the colony index is generally only in the initial stage of culture, and the presence or absence of irregularities is in the late stage of culture. Therefore, when narrowing down the index to be implemented, the index of presence / absence of unevenness is not adopted in the early stage of culture, and the colony index is not adopted in the later stage of culture.
  • By selecting an index according to the culture period it is possible to reduce the processing amount of the control unit. Or conversely, it may be determined by all indicators in any culture period. Thereby, even when the cell growth situation is different from a general result by chance, an appropriate determination can be made.
  • a table 601 in FIG. 6 shows an example of specific pass / fail determination according to the cell type and treatment target organ used in the automatic culture apparatus of the present embodiment.
  • the cell images of the divided regions after the image processing are superimposed to form an image of the entire culture vessel (S7).
  • the information obtained by photographing the cell image is presented to the worker so that the worker can grasp the growth state of the cell globally.
  • a cell image of each divided region, that is, an enlarged part of the inside of the culture container is presented to the worker so that the worker can locally grasp the cell growth state. All of these information is stored in a database in the control unit.
  • the culture continues until the culture period ends (S8).
  • the analysis results at various times are accumulated in the database, the analysis results at different times in each micro area are first integrated (S9), and the quality of the regenerated tissue is judged (S10). ).
  • S9 the quality of the regenerated tissue is judged
  • S10 the quality of the regenerated tissue is judged.
  • an image of the entire culture container evaluated for the colony distribution performed in the early stage of culture and an image evaluated for the thickness and the presence / absence of irregularities of the regenerated tissue performed in the late stage of culture are superimposed. By superimposing images, it is possible to simultaneously evaluate the colony distribution, thickness, and unevenness obtained at different culture periods in each divided region in the culture vessel.
  • the control unit selects a divided region suitable for transplantation from the classified contents and displays it on the screens of the control terminals 101 and 401.
  • index examples include colony distribution, reaching or not reaching a cell dense state, thickness of regenerated tissue, and unevenness of regenerated tissue.
  • One or more of these indexes may be selected and combined.
  • One may be evaluated alone, or all four may be evaluated simultaneously.
  • select colony-distributed, stem-celled cells that have good cell growth early in the time when the cells have reached a dense state, thick regenerative tissue, and flat areas with no irregularities. An example is to do.
  • the control unit presents a cut portion for the regenerated tissue in consideration of the overall quality information of the entire culture vessel and the ease of trimming using a scissors and scissors by hand (S13). This is because trimming is performed manually and it is difficult to process into a complicated shape. And after completion
  • FIG. 5 schematically shows an example when the control terminal 401 which is a control unit in the flow in FIG. 4 integrates information of a plurality of cell images.
  • Each of (A), (B), (C), and (D) of FIG. 5 performs image processing by the control unit based on indices such as colony distribution and cell occupancy for the cell images taken for each divided region. Then, a macro image of the entire culture vessel is obtained by superimposing the processed cell images.
  • (A) in FIG. 5 is the time when the colony 501 is formed. Generally obtained early in the culture. The portion forming the colony 501 corresponds to a place where somatic stem cells are distributed, for example.
  • (B) in FIG. 5 is the time when the cells are stratified. Generally obtained late in culture. There are thick and thin portions depending on the location, and also there are uneven portions and smooth portions depending on the location, and the concave portion 502, the thin portion 503 with a small number of cells, and the thick portion 504 with a large number of cells are shown schematically.
  • FIG. 5 (C) in FIG. 5 is a superposition of the images of FIG. 5 (A) and FIG. 5 (B), which are information obtained at different culture periods.
  • FIG. 5 (A) and FIG. 5 (B) are information obtained at different culture periods.
  • the distribution of colonies 501 obtained in the early stage of culture, and the thickness and unevenness of the regenerated tissue obtained in the late stage of culture such as concave parts, thin parts with few cells and thick parts with many cells, etc.
  • Information based on a plurality of indices obtained at different culture periods can be shown in an integrated manner.
  • this image consists of divided areas based on the coordinates assigned in advance to the culture surface, the quality determination of each divided area is controlled based on each index for the image superimposed in each divided area. Department will judge. Based on the result of pass / fail judgment, it is judged whether the tissue is a regenerative tissue that can be used for transplantation based on the information of the entire culture container, or the portion that can be used for transplantation is determined based on the pass / fail information for each location in the regenerative tissue. Sometimes it is used for transplantation by trimming that part.
  • the pass / fail judgment result obtained for each divided region and the trimming of the regenerated tissue using the operator's scissors and scissors Considering the ease of operation, a cutting position to be trimmed is determined, and a cutting position 507 is set, and a region 505 used for transplantation and a region 506 not used for transplantation are separated. Then, the transplanter or the operator can grasp the spatial positional relationship of the pass / fail judgment result in the regenerated tissue based on the coordinates in the culture container by browsing the superimposed images on the screen. .
  • the divided area shown in FIG. 5D is an example.
  • the pass / fail judgment information in the obtained database 402 is presented to the operator at the time of transplantation.
  • the output method of the pass / fail judgment information is, for example, a method of printing with a printer or the like from the control unit of the automatic culture apparatus, the operator transporting it together with the culture container, and referring to it in the operating room where transplantation and trimming are performed as necessary. is there.
  • an automatic culture device is connected to an external device via a line, the quality determination information is transferred to the external device via the line, and a terminal device display installed in an operating room for transplantation and trimming as necessary, etc. The worker performs the work while referring to it.
  • the support membrane used when transplanting the regenerative tissue that is, a layer that is superimposed on the regenerative tissue to maintain the shape of the regenerative tissue and can be easily lifted by overlapping with the regenerative tissue Typical pass / fail judgment information may be printed.
  • the printed support is sterilized by an autoclave or the like before use.
  • the content of the pass / fail judgment information output from the automatic culture apparatus of the present embodiment is, for example, a culture container number containing a regenerated tissue suitable for transplantation.
  • the regenerated tissue in the culture vessel that was not good is not used for transplantation.
  • data on the cutting position for trimming reflecting a portion suitable for transplantation in the regenerated tissue of each culture container is output. The operator performs trimming while referring to the cutting position, and uses a good portion for transplantation.
  • information on the orientation of the regenerated tissue that is, the orientation of the culture vessel, is essential. The direction of the culture vessel can be visually confirmed.
  • the output quality information also includes position information including a position such as a scraper, thereby avoiding erroneous recognition of the orientation of the regenerated tissue.
  • Step M1 flow path installation> After the operator installs the flow path including the culture vessel in the automatic culture apparatus, the automatic culture apparatus is started (S1 in FIG. 4; the same applies to the following).
  • the flow path includes a culture vessel, a cell bottle containing a cell suspension, a medium bottle containing a medium, a culture supernatant bag for collecting the culture supernatant, and a flow path tube connecting them.
  • the culture container is sequentially accommodated in a pre-installed heat storage unit.
  • the culture container is provided with coordinates. After installing the flow path, check the installation normality.
  • Activation> Activate the automatic culture device.
  • An operator starts the operation by pressing the start switch of the operation unit in the control device.
  • the inside of the apparatus is in a clean environment by performing disinfection or sterilization in advance.
  • the operation screen of the display of the control unit it is confirmed that the internal environment of the automatic culture apparatus is appropriate.
  • the temperature of the incubator is 37 ° C.
  • Step M3 Determination of micro area and culture schedule during evaluation> An automatic culture schedule carried out by the automatic culture apparatus and a micro area for determining the quality of the regenerated tissue are determined (S2 and S3). Conditions such as date and time for performing operations such as cell seeding, medium exchange, culture supernatant collection, gas exchange, microscopic observation, tissue collection for examination, and tissue collection for transplantation are input from the operation unit of the control unit. In addition, the operator selects the criteria for determining the quality of the regenerated tissue based on information such as the type of cell to be cultured and the type of organ to be transplanted. Based on the magnification by the microscope at the time of observation, the culture surface is divided into a plurality of divided regions using coordinates provided in the culture vessel. Each divided area has the same area as the cell image at the time of observation, and the whole culture surface can be reconstructed by superimposing the cell images for all the divided areas.
  • Step M4 Cell seeding> Cell seeding corresponding to the start of automatic culture is performed (S4). After opening and closing the appropriate solenoid valve, operate the tube pump to aspirate the cell suspension from the cell bottle. Deliver the cell suspension to the culture vessel. After the seeding of all the culture vessels is completed, an actuator attached to the culture vessel base where the culture vessels are installed is operated to tilt and shake the culture vessel base to make the cell distribution uniform.
  • Step M5 Gas exchange> Immediately after cell seeding, gas exchange is performed by feeding a predetermined amount of gas into each culture vessel. Gas exchange is also performed several times a day during the culture period.
  • gas to be supplied air containing a CO2 concentration of 5% is used as an example.
  • the gas is supplied to each culture vessel from a gas cylinder through a humidification bottle with a flow rate controlled by a gas flow meter in a state where water molecules are saturated. Unnecessary gas after being supplied to the culture vessel is discharged out of the flow path through the filter.
  • the filter adjusts the pressure in the flow path as necessary. For example, a filter having a quality that does not pass particles of 0.22 ⁇ m or more is used.
  • ⁇ Step M6 Culture> Culturing is performed for a predetermined time in a state where the culture vessel is left still horizontally. During the culture, the temperature is maintained at 37 ° C. with an incubator. The air in the apparatus is constantly stirred by a fan so that the temperature distribution is always uniform. Although not shown in this example, it is possible to improve the production safety by attaching a particle counter or a viable count device to the apparatus and monitoring the cleanliness. In addition, it also serves as the process of S8.
  • ⁇ Step M7 Observation with a microscope>
  • a cell image is acquired using a microscope installed in the automatic culture apparatus (S5).
  • the light source installed in the automatic culture apparatus is appropriately illuminated to focus on the cell and image.
  • Cell images of all the divided areas are acquired based on the preset coordinates and the divided areas.
  • the acquired cell image is stored in a database in the control unit, viewed on the control terminal of the automatic culture apparatus, and the operator confirms the state of the cell as appropriate.
  • the operator manually operates the microscope as necessary to observe and photograph the cells.
  • the control unit For the acquired cell images of all divided regions, the control unit recognizes colonies, cell occupying portions, irregularities and the like based on indexes set in advance for each divided region, and determines pass / fail based on those indexes (S6). Since the index that can be determined differs depending on the time when the observation is performed, the determination may be made using only a specific index based on the culture time. This can reduce the processing amount of the control unit. Alternatively, the determination may be made by all indicators at any culture time. Thereby, even when the cell growth situation is different from a general result by chance, an appropriate determination can be made.
  • ⁇ Step M8 Medium exchange> Medium exchange is performed once every few days during the culture period.
  • the medium stored at 4 ° C. in the refrigerator is fed to the preheating bottle and preheated.
  • the culture container is tilted by the actuator to improve the discharge efficiency.
  • a new medium is immediately supplied into the culture vessel.
  • the old medium is finally discharged into the culture supernatant bag. If necessary, the culture supernatant in the culture supernatant bag is collected, and the growth state of the cells is evaluated by medium component analysis.
  • Step M9 Collection of tissue for examination> On the day before the scheduled date of transplantation, a part of the culture container being cultured is collected for examination. The door of the automatic culture apparatus is opened, and the flow path tube of the culture container for inspection is aseptically cut and removed by means such as heat welding. The removed culture container is transported outside the safety cabinet or CPC and promptly inspected. For example, the number of cells in a biological sample, survival rate, expression of a specific protein, etc. are evaluated.
  • Step M10 Culture and medium exchange just before transplantation> Culture is performed by the same operation as in step S6. And just before implementing step S11, culture medium exchange by the same operation as step S8 is performed.
  • Step M11 Collection of transplanted tissue>
  • a biological sample is collected and used for regenerative medical treatment.
  • the quality of the entire culture container is determined using information on different culture periods (S12).
  • the cut region of the regenerated tissue is determined.
  • the culture vessel is aseptically separated from the flow path and removed from the incubator. Carry it into the safety cabinet as needed.
  • Step M12 Output of pass / fail judgment information from control unit>
  • the quality determination information is printed on paper or the like from the control unit of the automatic culture apparatus.
  • the automatic culture apparatus may be connected to an external device via a line, and pass / fail judgment information may be transferred to the external device and printed from the external device. Further, the transferred pass / fail judgment information may be displayed on a terminal prepared at the transplantation site at the transplantation stage performed at M15. In addition, when printing on paper etc., it conveys with a culture container.
  • ⁇ Step M13 Accommodation in transport container and transport>
  • the culture container is accommodated in a transport container for short distance or long distance in the shipping room.
  • the transport container is taken out of the CPC and transported to the operating room by means of vehicles, railroads, airplanes, hand-carrying etc. as necessary.
  • Step M14 Acceptance inspection after transportation> Before treatment in the operating room, cell observation with a microscope is performed as an acceptance test if necessary. In the case of short-distance transportation, it is assumed that the state is almost the same as that immediately before transportation.
  • Step M15 Transplant> After reaching the operating room, the regenerated tissue is removed from the culture vessel. At the time of opening, organisms and particles such as bacteria may be attached to the outside of the culture container, so the culture container is opened aseptically so as to maintain cleanliness.
  • the operator refers to the pass / fail judgment information displayed on the paper output from the automatic culture apparatus or the like and transported with the culture container, or the terminal installed in the operating room. Then, the regenerated tissue in the culture vessel that was good is transplanted. Or it trims, referring a cutting position, and uses a favorable part for transplant.
  • Step M16 End> After the device is cleaned, it shuts down and ends (S14).
  • Example 2 an example in which the information output method at the time of transplantation is different in the pass / fail judgment flow shown in FIG. 4 will be described.
  • Electronic identification means such as an IC tag or a two-dimensional barcode, that is, an individual identification number (ID) is attached to the culture container in advance. These are read by an electronic reader, and have an individual identification number of the culture vessel, a URL that allows access to a site where pass / fail judgment information of the control unit is placed, and the like. After completion of production by the automatic culture apparatus, the culture container is transported to the operating room. In the operating room, IC tags, two-dimensional barcodes, etc. are read to prevent misplacement.
  • ID individual identification number
  • the information storage location of the pass / fail judgment information is accessed from a URL that can be read, and the pass / fail judgment information is displayed on a terminal installed in the operating room in advance.

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Abstract

La présente invention permet de déterminer la qualité de la régénération tissulaire et d'informations courantes au moment de la transplantation dans un dispositif de culture pour mettre en culture des cellules ou des tissus. L'invention concerne également un dispositif de culture automatique comprenant un récipient de culture dans lequel sont retenues des cellules, une unité d'imagerie pour capturer des images des cellules retenues dans le récipient de culture et une unité de commande telle qu'un terminal de commande (401) pour pouvoir accéder à la qualité de la régénération sur la base des informations relatives aux images de cellules d'une pluralité de régions divisées des images de cellules capturées par l'unité d'imagerie et de différentes périodes de culture. Du fait de cette configuration, la présente invention permet d'intégrer une pluralité d'ensembles d'informations obtenues à différentes périodes de culture, d'utiliser les informations intégrées afin de déterminer la qualité et de présenter le résultat de la détermination à un professionnel au cours de la transplantation.
PCT/JP2014/050735 2014-01-17 2014-01-17 Dispositif de culture de cellules WO2015107667A1 (fr)

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CN107831084A (zh) * 2017-12-08 2018-03-23 山东商业职业技术学院 一种生鲜水产品品质综合测定装置及测定方法
JPWO2019003271A1 (ja) * 2017-06-26 2020-04-23 オリンパス株式会社 細胞観察システム
US11493745B2 (en) 2017-06-26 2022-11-08 Evident Corporation Cell observation system
US11579063B2 (en) 2017-01-31 2023-02-14 Fujifilm Corporation Cell culture apparatus, imaging unit, and culture monitoring method

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Publication number Priority date Publication date Assignee Title
JP2008092882A (ja) * 2006-10-13 2008-04-24 Hitachi Medical Corp 自動培養装置
JP2011193741A (ja) * 2010-03-17 2011-10-06 Asahi Glass Co Ltd 培養容器
WO2012020458A1 (fr) * 2010-08-12 2012-02-16 株式会社日立製作所 Dispositif de culture automatique
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US11579063B2 (en) 2017-01-31 2023-02-14 Fujifilm Corporation Cell culture apparatus, imaging unit, and culture monitoring method
JPWO2019003271A1 (ja) * 2017-06-26 2020-04-23 オリンパス株式会社 細胞観察システム
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