JP4928831B2 - Automatic culture equipment - Google Patents

Description

  The present invention relates to an automatic culture apparatus, and more particularly to an automatic culture apparatus that separates stem cells from bone marrow fluid and safely and automatically realizes a culture protocol for inducing proliferation and differentiation.

Regenerative medicine is attracting attention as a method for repairing deficient tissue, for example. Such regenerative medicine is treatment using cells, and requires in vitro cell culture work including culture in an incubator and tests such as ALP (alkaline phosphatase) activity. For example, in the treatment of a subject lacking bone or cartilage, there is a method in which mesenchymal stem cells (hereinafter abbreviated as MSC (Mesenchymal Stem Cell) as appropriate) are cultured, differentiated into bone or cartilage, and transplanted to the subject. Proposed. This MSC is known to be contained in bone marrow fluid such as iliac bone. However, complicated culture work is required to separate and culture MSC contained in bone marrow fluid to differentiate into bone and cartilage. In addition, there may be a risk of contamination of MSC during the culture, so-called contamination. In general, MSC culture work is performed, for example, by the following procedure.
(1) Puncture the iliac bone and collect bone marrow fluid with a syringe containing a blood coagulation inhibitor such as heparin sodium.
(2) In order to eliminate blood cells such as erythrocytes contained in the bone marrow fluid and to separate MSC, the bone marrow fluid is put into a centrifuge tube, centrifuged, and the liquid in the MSC layer that is the target cell by density gradient method Is sucked out with a pipetter, diluted with a medium or the like, and then poured into a petri dish or other incubator for incubation (incubation).

In addition, MSC adheres to the petri dish in the process of culturing with a medium under the predetermined culture conditions. Therefore, by exchanging the medium after an appropriate period of time, blood cells that do not adhere to the petri dish are eliminated to separate the MSC. can do.
(3) Then, it culture | cultivates continuously on a predetermined culture condition. Here, the predetermined culture conditions are generally a temperature of around 37 ° C. and a CO ₂ concentration of 5%. The culture is continued by changing the medium every 3 days using a pipetter or the like.
(4) When cells grow and colonies with high cell density are formed locally, or when cells grow to about 60 to 90% of the culture area, subculture is performed by reseeding in a plurality of new dishes. This passage is carried out by putting proteolytic enzymes in a petri dish in culture, floating the cells, injecting a proteolytic enzyme inactivator after several minutes, and reseeding in a plurality of new petri dishes. Thus, the number of petri dishes increases by performing passage several times with cell proliferation. For example, when the area of the incubator is about 75 cm ² , about 1 to 1.5 × 10 ⁶ cells can be collected.
(5) After an appropriate number of days have passed and the cells have grown to a sufficient amount, the medium is switched to a medium containing an appropriate component according to the use, such as bone or cartilage, to induce differentiation into cells of the target tissue.
(6) To collect cultured cells of the target tissue, put proteolytic enzyme in a petri dish to float the cells, and after several minutes inject a proteolytic enzyme inactivator. The collected cultured cells are used for transplantation into a subject. For example, in the end, 1 × 10 ⁷ or more cells are often required. For example, in the case of a petri dish of about 75 cm ² , 10 or more cells are required.

  As described above, since the work of culturing cells used for regenerative medicine involves complicated work, an automatic culture apparatus that automatically performs a culture operation according to a culture protocol has been proposed in Patent Document 1 and the like. According to this, since complicated culture work can be automated and culture operation can be performed in an airtight and clean environment, contamination that contaminates cultured cells can be avoided and safety can be improved. be able to.

JP 2004-16194 A

  However, the automatic culture apparatus described in Patent Document 1 is basically an automated manual culture operation. For example, the culture medium in the petri dish is exchanged by opening the petri dish lid in the incubator and using a pipette. Like to do. Therefore, there is room for improvement in reducing the risk of contamination even if the inside of the incubator or the housing for housing the incubator is kept in a clean environment. Moreover, since the number of petri dishes increases, it becomes a very complicated operation | work and generation | occurrence | production of a work mistake cannot be denied. In particular, when applied to the culture of mesenchymal stem cells (MSC), no consideration is given to automating the operation of separating MSC from bone marrow fluid.

  The first object of the present invention is to reduce the risk of contamination, further improve the safety of cultured cells, and to automate the operation of separating stem cells from bone marrow fluid.

  Further, in addition to the first problem, the present invention has a second problem of separating a stem cell from bone marrow fluid and realizing a culture protocol for inducing proliferation and differentiation safely and automatically.

In order to solve the first problem, an automatic culture apparatus according to the present invention includes a cell incubator that is installed in an incubator and formed airtight, a medium container in which a medium is stored, and a protease that is stored. A protease container, a washing liquid container in which a washing liquid is stored, a bone marrow liquid container in which bone marrow fluid is stored, the medium container, the washing liquid container, and the like via a liquid feeding tube that is airtightly connected to the incubator. A liquid feeding means for selectively injecting a liquid in a bone marrow fluid container into the incubator; a draining means for discharging the liquid in the incubator through a drainage tube connected airtight to the incubator ; And a control means for controlling the liquid delivery means and the drainage means according to a predetermined culture protocol, the control means controlling the liquid delivery means and injecting the medium into the incubator. Bone marrow fluid into the incubator An initial operation to be allowed to stand for a set time, and after the initial operation, the draining means and the liquid feeding means are controlled, and the inside of the incubator is washed with the medium or the washing liquid and adhered to the incubator Controlling a separation operation for selectively separating stem cells to be performed and a culture operation for injecting the medium into the incubator after the separation operation and culturing the stem cells, and the culture operation is acquired in the incubator When it was confirmed that the growth rate of the stem cells was suppressed based on the image of, after the stem cells were suspended by controlling the liquid feeding means and injecting the proteolytic enzyme into the incubator, It comprises a subculture operation of injecting and suspending the medium .

  According to the present invention having such a configuration, the incubator is formed in an airtight manner, and a liquid such as a culture medium, a washing solution, or a bone marrow fluid is sent to the incubator through an airtightly connected liquid feeding tube. Therefore, it is possible to prevent contaminants from entering the incubator through a culture instrument such as a pipette or from the surrounding air. As a result, the risk of contamination can be reduced and the safety of the automatically cultured cells can be further improved. In particular, the discharge of the liquid in the incubator and the injection of the washing liquid are preferably performed repeatedly to selectively separate the stem cells adhering to the incubator, so that the stem cell separation operation can be automated.

In order to solve the second problem, an automatic culture apparatus according to another invention of the present invention includes a cell incubator that is installed in an incubator and formed airtight, and a component that inactivates a proteolytic enzyme. A first medium container storing a first medium, a second medium container storing a second medium different from the first medium, and a proteolytic enzyme container storing a proteolytic enzyme A cleaning liquid container in which a cleaning liquid is stored, a bone marrow liquid container in which bone marrow fluid is stored, and the first medium container and the second medium container via a liquid feeding tube that is airtightly connected to the incubator. The incubator via a liquid feeding means for selectively injecting the liquid in the proteolytic enzyme container, the washing liquid container and the bone marrow fluid container into the incubator, and a drain tube connected to the incubator in an airtight manner . Select the liquid in the drainage tank or cell recovery tank. A draining means for selectively discharging, and a control means for controlling the liquid feeding means and the draining means in accordance with a predetermined culture protocol, wherein the control means controls the liquid feeding means. An initial operation of injecting the bone marrow fluid into the incubator after injecting the first medium into the incubator and allowing it to stand for a set time; and controlling the drainage means and the liquid delivery means after the initial operation. Separating the stem cell adhering to the incubator by washing the inside of the incubator with the medium or the washing solution, and controlling the liquid feeding means after the separation operation to control the first medium. A culture operation for injecting the stem cell into the incubator, and confirming that the stem cells have sufficiently proliferated by the culture operation, and then controlling the liquid feeding means to culture the second medium. The stem cells are injected into the vessel A differentiation operation for differentiating into cells, and after the differentiation operation, the liquid feeding means is controlled to inject the proteolytic enzyme into the incubator to float the stem cells, and the first medium is injected into the incubator. The cell collection operation for discharging the stem cells floating in the cell recovery tank to the cell collection tank, and the culturing operation is performed such that the proliferation rate of the stem cells is suppressed based on the acquired image in the incubator. When confirmed, it comprises a subculture operation in which the liquid feeding means is controlled to inject the proteolytic enzyme into the incubator to suspend the stem cells, and then inject and suspend the medium .

  According to this, stem cells are separated from bone marrow fluid, and a culture protocol for inducing proliferation and differentiation can be realized safely and automatically.

  In the present invention, it further comprises rocking means for rocking the incubator, and the control means is configured to rock the incubator by controlling the rocking means during the separation operation. Can be improved.

  According to the present invention, the risk of contamination can be reduced, the safety of cultured cells can be further improved, and the operation of separating stem cells from bone marrow fluid can be automated.

  According to another invention of the present invention, stem cells can be separated from bone marrow fluid, and a culture protocol for inducing proliferation and differentiation can be realized safely and automatically.

  Hereinafter, the best mode for carrying out the present invention will be described. FIG. 1 is a block configuration diagram of an embodiment of the automatic culture apparatus of the present invention. FIG. 2 to FIG. 6 are flowcharts showing an operation procedure of a culture protocol according to an embodiment of the present invention suitable for culturing mesenchymal stem cells from bone marrow fluid.

  As shown in FIG. 1, the incubator 2 of the present embodiment is formed in a sealed state using a circular petri dish. The incubator 2 is airtightly connected with a tube 4 on the liquid feeding side, a tube 8 on the discharge side, and a tube 6 for air exchange. The incubator 2 may be any material that is non-cytotoxic, and for example, polystyrene and polyethylene terephthalate are preferable. In addition, the shape of the incubator 2 is not particularly limited, although it is preferable to use a circular petri dish as in the present embodiment.

Bag 10 was placed in a washing liquid, the bag 12 containing the proteolytic enzyme, bag 14 containing the first medium, it is provided with a bag 15 containing the second medium different components from the first medium . The first medium bag 14 sera inactivate the proteolytic enzyme is contained. In addition, a drug having a composition suitable for differentiating into target tissue cells is used for the second medium of the bag 15. Although not shown in the drawings, these bags are stored in a refrigerator that is kept cold (about 4 ° C.). Each bag 10, 12, 14, 15 is connected to a tube 4 on the liquid feeding side of the incubator 2. In addition, pinch valves 16, 18, 20, and 21 are provided on the branch tubes connected to the bags 10, 12, 14, and 15. The tube 4 is provided with a heater 26 for preheating the liquid to be injected from the bags 10, 12, 14, 15 into the incubator 2. The tube 4 between the heater 26 and the incubator 2 is provided with a pinch valve 22 and a pump 34. The pump 34 is preferably a type in which outside air does not enter, and a roller pump is preferable. The roller pump is configured to rotate, for example, three rollers integrally, and the tube 4 is wound around the outer periphery of the three rollers.

  A liquid reservoir 32 is connected to the tube 4 between the pump 34 and the pinch valve 22 via a tube 25. The opening of the liquid reservoir 32 is covered with the film 30. Bone marrow fluid collected by puncturing the syringe 30 with the syringe 28 and collected in the syringe 28 is injected into the liquid reservoir 32.

  Connected to the tube 8 on the discharge side of the incubator 2 are a cell collection bag 48 that is a cell collection container for containing the cultured cell solution and a bag 50 for discharging the waste solution in the incubator 2. . The tube 8 is provided with a pump 42 such as a roller pump, and the branch tubes connected to the bags 48 and 50 are provided with pinch valves 44 and 46, respectively. Here, the bags 10, 12, 14, 15, 48 and the tubes 4, 6, 8, 25, etc. may be any material that is non-toxic to cells. For example, vinyl chloride or silicon is preferable.

The tip of the air exchange tube 6 connected to the incubator 2 is opened in the incubator 36 through an air filter 38. This prevents outside air from directly entering the inside of the incubator 2 and reduces the risk of contamination. An air filter 40 is provided in the incubator 30, and the air filter 40 is connected to the tubes 4 between the bags 10, 12, 14, 15 and the heater 26 via the tubes 27. Although not shown, the incubator 36 is provided with means for adjusting the internal temperature and carbon dioxide concentration. For example, the temperature is controlled to 30 ° C. to 40 ° C., and the CO ₂ concentration is controlled to 5%. The incubator 2 is provided with image acquisition means 52 such as a microscope with a CCD camera for observing cultured cells. Further, rocking means 54 for rocking the incubator 2 is provided.

  The pinch valves 16, 18, 20, 21, 22, 23, 24, 44, 46, the pumps 34, 42, the image acquisition means 52, and the swinging means 54 are driven and controlled by a control device (not shown). It has become.

  The operation procedure of the control apparatus that controls the automatic culture apparatus of the present embodiment configured as described above according to a predetermined culture protocol will be described with reference to FIGS. FIG. 2 is a schematic flowchart of a culture protocol for culturing mesenchymal stem cells from bone marrow fluid using the automatic culture apparatus of FIG.

[Step S1: Initial / separation operation]
When an operator inputs a cell culture start command to the control device, a series of sequence operations starts according to the culture protocol. First, the first medium of the bag 14 is injected into the incubator 2, and then the bone marrow fluid is injected into the incubator 2 from the liquid reservoir 32. After resting for a while (for example, 30 minutes or more), the incubator 2 The medium is drained into the bag 50. Thereby, cells other than the mesenchymal stem cells that adhere to the incubator 2 can be discharged and the mesenchymal stem cells can be separated. In this case, the separation accuracy of the mesenchymal stem cells is improved by repeating the process of injecting the washing solution of the bag 10 or the first medium of the bag 14 into the incubator 2 and discharging again as necessary. Can do.

[Step S2: Culture Operation]
In step S2, the mesenchymal stem cells left in the incubator 2 are cultured in the first medium. In this step, if necessary, an operation of exchanging the medium and sending air into the medium is performed. This culture is performed until the number of cells in the incubator 2 reaches a predetermined sufficient number.

[Step S3: Passage operation]
The passage operation is usually a treatment included in the culture operation. That is, even if the number of cells has not increased sufficiently in the culturing operation in step S2, if a colony having a high cell density is locally formed in the incubator 2, the growth rate in that region is suppressed. Therefore, the subculture is performed by reseeding the cells in the incubator 2. This passaging be injected after by injecting proteolytic enzymes of the bag 12 is suspended cells incubator 2 in the culture, the first medium deactivator proteolytic enzyme is contained To do. Thus, the cell density in the incubator 2 is equalized by performing subculture a plurality of times with cell proliferation, and proliferation can be promoted.

[Step S4: Differentiation Operation]
When an appropriate number of days have passed and the number of cells has reached a sufficient number by the culturing operation, for example, when the image of the image acquisition means 52 is confirmed, the culturing operation is stopped and the cultured mesenchymal stem cells To induce differentiation into the desired tissue cells. This differentiation operation is performed by switching to a second medium containing an appropriate component according to the use such as bone or cartilage, and culturing the cells in a target tissue. This culture is the same as the culture operation in step S2.

[Step S5: Cell recovery operation]
When the differentiation of the target tissue into cells is completed by the operation in step S4, the cells in the incubator 2 are collected. This recovery, incubator 2 put proteolytic enzymes of the bag 12 is suspended cells, by injecting a first medium containing deactivator proteolytic enzyme after several minutes, collected in bag 48 To do. The collected cultured cells are used for transplantation into a subject.

  Here, the detailed processing procedure of each step S1-S5 is demonstrated using FIGS.

  FIG. 3 is a detailed flowchart of the initial / separation operation. First, when a cell culture start command is input, in step S11, the pinch valves 20 and 22 are opened, the pump 34 is operated, and the first medium is injected into the incubator 2 from the bag 14. At this time, the air inside the incubator 2 is exhausted from the air filter 38, so that the medium can be injected smoothly.

  Next, in step S <b> 12, bone marrow fluid is injected from the liquid reservoir 32 into the incubator 2. Bone marrow fluid is injected into the liquid reservoir 32 by puncturing the film 30 with a syringe 28 containing bone marrow fluid previously mixed with a medium or the like. Bone marrow fluid is injected into the incubator 2 by opening the pinch valve 24 and operating the pump 34. At this time, since the air inside the incubator 2 is exhausted from the air filter 38, the bone marrow fluid can be smoothly injected without clogging the tube.

  Next, in step S13, the bone marrow fluid is injected into the incubator 2 and left to stand for at least 30 minutes. Thereby, mesenchymal stem cells are adhered to the incubator 2.

  Next, in step S14, the pinch valve 46 is opened and the pump 42 is operated in order to remove other cells such as blood cells, leaving only the mesenchymal stem cells, and the culture medium in the incubator 2 is stored in the bag 50. To discharge. In this case, since the outside air is sucked into the incubator 2 from the air filter 38, the culture medium can be smoothly discharged without negative pressure in the incubator 2. Thereafter, the pinch valves 20 and 22 are opened, and the pump 34 is operated to inject the first medium.

  In this cleaning, instead of the pinch valve 20, the pinch valve 16 may be opened and the cleaning solution for the bag 10 may be used. Further, it is effective to perform this cleaning a plurality of times. Furthermore, after putting the culture medium (or washing solution), if the incubator 2 is shaken by driving the rocking means 54, a higher washing effect can be obtained. In this way, only mesenchymal stem cells are separated in the incubator 2.

  FIG. 4 is a detailed flowchart of the culturing operation and the passage process in FIG. As shown in the figure, the culture operation consists of steps S21 to S24, and the passaging process consists of steps S31 to S34. In the present embodiment, it is assumed that the first culture medium is injected into the incubator 2 at the end of the cleaning process in step S14 in FIG. 3, but when the cleaning liquid is used at the end of the cleaning process, In step S21, the first medium is injected. The culture is performed by leaving the incubator 2 in the incubator 36 for a predetermined period (for example, about 3 days). During this time, since the incubator 2 is in a sealed state, the inside of the incubator 2 is appropriately ventilated. In this ventilation, in step S22, the pinch valves 22 and 23 are opened, the pump 34 is operated, the air in the incubator 36 is sucked from the air filter 40 and injected into the incubator 2. In this ventilation, the air in the incubator 2 is exhausted through the air filter 38. This ventilation may be performed any number of times other than the medium exchange and cell recovery described later, or may be performed continuously all the time.

  As culture is continued, it is necessary to replace the medium regularly, in order to remove unnecessary metabolites of cells eluted in the medium and to supplement nutrients of the medium. Therefore, in step S23, the pinch valve 46 is opened and the pump 42 is operated to discharge the medium in the incubator 2 to the bag 50. At this time, since the outside air is sucked into the incubator 2 through the air filter 38, the medium can be discharged smoothly. Next, the pinch valves 20 and 22 are opened, the pump 34 is operated, and the first medium is injected. Since the air inside the incubator 2 is exhausted from the air filter 38, the medium can be injected smoothly. The medium exchange in step S23 is usually performed every three days, but this time can be set in advance or arbitrarily according to the state of cell proliferation. The number of times is not limited.

  After culturing in this way, an image in the incubator 2 is acquired by the image acquisition means 52 in step S24, and the state of cell proliferation is confirmed based on the acquired image. If the sufficient number of cells has not been reached with respect to the intended number of cells, the process proceeds to the passage operation of step S3.

  The passaging operation is performed according to the procedure of steps S31 to S34 in FIG. First, although not shown, the pinch valve 46 is opened and the pump 42 is operated to discharge the medium in the incubator 2 to the bag 50. Subsequently, the pinch valves 16 and 22 are opened, the pump 34 is operated, and the cleaning solution for the bag 10 is injected into the incubator 2. Further, the pinch valve 46 is opened, the pump 42 is operated, and the cleaning liquid in the incubator 2 is discharged. Next, the pinch valves 18 and 22 are opened, the pump 34 is operated, and the proteolytic enzyme in the bag 12 is injected into the incubator 2. Thereby, the cells adhering to the incubator 2 are detached and floated.

In step S32, in order to deactivate the proteolytic enzymes, to open the pinch valve 20 and 22, the pump 34 is operated, to inject a first medium bag 14 in the culture vessel 2. In the next step S33, the oscillating means 54 is operated to shake the incubator 2, and the cells are suspended and uniformly dispersed in the medium. And in step S34, after leaving still for a fixed time (for example, 30 minutes), the cell is made to adhere to the incubator 2, and then it returns to culture of step 21.

  Such culturing operation and subculture operation are repeated, and when a sufficient number of cells can be confirmed in step S24, the culturing operation is terminated, and the process proceeds to the differentiation operation of FIG. 5 in step S4. Note that the inside of the incubator 2 is sucked and exhausted through the air filter 38 when draining from the incubator 2 and when injecting the liquid into the incubator 2.

  The differentiation operation shown in FIG. 5 is an operation for inducing differentiation of cultured mesenchymal stem cells into target tissue cells, and switching to a second medium containing an appropriate component according to the use such as bone or cartilage. And then induced into cells of the target tissue. As shown in the figure, in step S41, the first medium used for the culture is replaced with a second medium. In this exchange, the pinch valve 46 is opened, the pump 42 is operated, and the first medium in the incubator 2 is discharged to the bag 50. Subsequently, the pinch valves 21 and 22 are opened, the pump 34 is operated, and the second medium is injected. Next, in step S42, the cells are cultured in the second medium to be differentiated into target cells. In this culturing process, the pinch valves 22 and 23 are opened and the pump 34 is operated to send air into the incubator 2 as necessary.

  When the differentiation operation is completed, the process proceeds to the cell recovery operation in steps S51 to S53 in FIG. First, although not shown, the pinch valve 46 is opened and the pump 42 is operated to discharge the second medium inside the incubator. Subsequently, the pinch valves 16 and 22 are opened, the pump 34 is operated, and the cleaning liquid is injected. Further, the pinch valve 46 is opened, the pump 42 is operated, and the cleaning liquid in the incubator 2 is discharged.

  In step S51, the pinch valves 18 and 22 are opened and the pump 34 is operated to inject the proteolytic enzyme injection into the incubator 2 to float the cells in the incubator 2. Next, in step S52, a culture medium (first or second?) Is injected into the incubator 2. In step S53, the pinch valve 44 is opened and the pump 42 is operated to discharge the cells in the incubator 2 to the collection bag 48. After discharging, the tube connected to the collection bag 48 is cut and sealed by heat sealing or the like. The collection bag 48 is carried to a department that uses cultured cells.

  Thus, when the cell recovery operation is completed, the end of the sequence control is displayed on the display device of the control device.

  In the operation flowchart described above, the medium 15 in FIG. 2 may be used as the medium used in steps 11 and 15. In this case, the operation of the pinch valve 20 that is operated when using the culture medium 12 is 21.

(Example)
Hereinafter, the results of culturing mesenchymal stem cells from human bone marrow fluid using the automatic culture apparatus of the above embodiment will be described specifically and in detail.
(1) Experimental conditions The control apparatus of the automatic culture apparatus of FIG. 1 was equipped with sequence control software for executing the culture protocol of FIGS. Moreover, the chemical | medical agent used for culture | cultivation is as Table 1.

  Surgically collected 29-year-old male bone marrow 10m1 diluted 3 times with DMEM (10% FBS, 1% PS) (total volume 30ml), put into the reservoir 32, and cultured with the culture protocol in Fig. 2 Started. For the incubator 2, a circular petri dish having a diameter of 25 cm was used.

(2) Culture by culture protocol The bone marrow put into the liquid reservoir 32 reached the incubator 2 without causing clogging, and was mixed in the culture vessel 2 with 150 ml of the first medium. Bone marrow fluid immediately after injection contains a large amount of blood cell components, and nothing appears in the microscopic image due to the light transmittance, but after standing and washing in steps S13 and S14 in FIG. As shown in FIG. 7, the floating cells were removed. In this example, seven washes were performed.

  On the 10th day after the start of the culture, the inside of the incubator 2 was observed, and while there was a portion with a small number of cells (FIG. 8A), colonies with dense cells were confirmed (FIG. 8B). . At this stage, in order to make the density distribution of the cells uniform in the incubator, the passage operation (steps S31 to S34 in FIG. 4) was performed. As a result, the cells were detached in the incubator 2 and were uniformly seeded again. (FIG. 8 (C)). Furthermore, as a result of culturing mesenchymal stem cells (MSC) by the culturing operation of steps S21 to S23 in FIG. 4 after passage, MSC reached confluence in the incubator 2 (FIG. 8D).

For MSCs that reached confluence in the incubator 2, the differentiation medium was added and cultured for about 1 week (21 days from the start of culture) by the procedures of steps S41 to S43 in FIG. And as a result of performing cell collection | recovery in the procedure of FIG.6 S51-S53, 3 * 10 < ⁷ > MSC was obtained in the collection | recovery bag 48. FIG.

(3) Discussion of experimental results The process of separation, purification, growth, differentiation, and recovery of MSC from bone marrow fluid can be automatically performed in a completely closed system environment. Also, there was no contamination and no fear of it. Furthermore, since an incubator having an area approximately 6.5 times as large as the commonly used 75 cm ² incubator was used, the expected value was about 1 × 10 ⁷ at most, but 3 × 10 ^7. Individual and very many cells could be obtained.

(4) Biochemical evaluation of cultured cells 1) Evaluation method In order to confirm the differentiation potential of the cells cultured by the method of this example, after measuring the total number of recovered cells, a part of them was collected and PNPP was collected. The alkaline phosphatase activity (ALP activity) was measured using this method. In addition, as controls, MSCs of 40-year-old women cultured by the conventional method and 40-year-old men cultured by the automatic culture apparatus of the present invention were also used for comparison. Note that p-Nitrophenyl phosphate tablets sets (manufactured by Sigma) were used as reagents for measuring ALP activity.

2) Evaluation results As a result of the measurement, MSCs derived from a 29-year-old man who had been subjected to differentiation induction treatment by the method of the present invention and MSCs derived from a 40-year-old woman performed by the method showed high ALP activity. It was shown that MSCs that had undergone differentiation induction treatment in FIG. Although the ALP activity of 40-year-old male MSC performed in the present invention is low, it is considered that the age and condition of the subject affected rather than the effect of the present invention.

3) Interpretation of results It was clarified that separation and purification, growth and differentiation of MSC from bone marrow fluid can be automatically realized by this example. In addition, MSCs from bone marrow fluid were shown to have the ability to differentiate into bone.

(5) Bone induction transplantation experiment using cultured cells 1) Method In vivo evaluation by bone differentiation using bone marrow cells cultured by the method of this example was performed by cell transplantation into nude mice. Specifically, 1 × 10 ⁶ cells collected were mixed with 50 mg of TCP (Tricalcium phosphate) -reliable granules, transplanted subcutaneously into nude mice, removed 4 weeks later, and subjected to HE staining (hematoxylin / eosin staining). Observed with a microscope.

2) Results As a result of the transplantation experiment, it was found that the cells mixed in the TCP grains had a portion differentiated into bone, and the obtained cells had bone differentiation ability (Fig. 10).

3) Interpretation of results MSCs separated, purified, grown and differentiated by the method of this example were shown to have bone forming ability.

  As described above, according to this embodiment, the incubator 2 is formed in an airtight manner, and a liquid such as a culture medium, a washing solution, or a bone marrow fluid is supplied to the incubator 2 through the tube 4 in an airtight manner. Therefore, it is possible to prevent contaminants from entering the incubator 2 through a culture device such as a pipette or from the surrounding air. Further, the cultured cells in the incubator 2 can be collected in the bag 48 through the tube 8 that is airtightly connected. As a result, the risk of contamination can be reduced and the safety of cultured cells can be further improved.

  In particular, the mesenchymal stem cells adhering to the incubator 2 are selectively separated by preferably repeating the washing process by discharging the liquid such as the medium in the incubator 2 and injecting the washing liquid. The mesenchymal stem cell separation operation can be automated. In addition, since the incubator 2 is operated to move the incubator 2 during the separation operation, the separation efficiency of the mesenchymal stem cells can be improved.

  Further, according to the present embodiment, stem cells are separated from bone marrow fluid, and a culture protocol for inducing proliferation and differentiation can be realized safely and automatically.

It is a block diagram of the automatic culture apparatus of one Embodiment of this invention. It is a flowchart which shows the outline | summary procedure of the culture protocol of one Embodiment of this invention. It is a flowchart which shows the detailed procedure of initial stage and isolation | separation operation. It is a flowchart which shows the detailed procedure of culture | cultivation operation and a subculture operation. It is a flowchart which shows the detailed procedure of differentiation operation. It is a flowchart which shows the detailed procedure of cell collection | recovery operation. It is an example of the cell image in initial stage and isolation | separation operation. It is an example of the cell image in culture | cultivation. It is a figure which compares and shows the ALP activity value of a cultured cell. It is a tissue specimen image after transplanting a cultured cell to a mouse.

Explanation of symbols

2 Incubator 4, 8 Tube 10, 12, 14, 15 Bag 16, 18, 20, 21, 22, 24, 23, 44, 46 Pinch valve 26 Heater 32 Liquid reservoir 34, 42 Pump 36 Incubator 48, 50 Bag 52 Image acquisition means 54 Oscillating means

Claims (3)

An incubator of cells that are installed in an incubator and formed airtight, a medium container in which a medium is stored, a proteolytic enzyme container in which a proteolytic enzyme is stored, a cleaning liquid container in which a cleaning liquid is stored, and a bone marrow fluid The culture medium container, the proteolytic enzyme container, the washing liquid container, and the bone marrow liquid container are selectively supplied with the liquid from the stored bone marrow liquid container and a liquid feeding tube connected to the incubator in an airtight manner. Liquid feeding means for injecting the liquid into the culture vessel, drainage means for discharging the liquid in the incubator via a drainage pipe connected airtight to the incubator, the liquid feeding means and the drainage means are predetermined. Control means for controlling according to the culture protocol,
The control means controls the liquid feeding means, injects the medium into the incubator, and then injects the bone marrow fluid into the incubator and allows it to stand for a set time; and
A separation operation for selectively separating stem cells that adhere to the incubator by controlling the draining means and the liquid feeding means after the initial operation, and washing the inside of the incubator with the medium or the washing liquid;
Controlling the culture operation of injecting the medium into the incubator after the separation operation and culturing the stem cells ,
In the culture operation, when it is confirmed that the growth rate of the stem cells is suppressed based on the acquired image in the incubator, the liquid feeding means is controlled to supply the proteolytic enzyme to the incubator. An automatic culture apparatus comprising a subculture operation of injecting and suspending the medium after injecting and suspending the stem cells . An incubator for cells that are installed in an incubator and formed airtight, a first medium container in which a first medium containing a component that inactivates a proteolytic enzyme is stored, and the first medium A second medium container in which a different second medium is stored; a proteolytic enzyme container in which a proteolytic enzyme is stored; a cleaning liquid container in which a cleaning liquid is stored; a bone marrow fluid container in which bone marrow fluid is stored; The liquids in the first medium container, the second medium container, the proteolytic enzyme container, the washing liquid container, and the bone marrow liquid container are selectively cultured through a liquid feeding tube that is airtightly connected to an incubator. Liquid feeding means for injecting into the incubator, drainage means for selectively discharging the liquid in the incubator to a drainage tank or a cell recovery tank via a drainage pipe connected airtight to the incubator, and The liquid feeding means and the draining means are predetermined. And control means for controlling in accordance with the culture protocol,
The control means controls the liquid feeding means, injects the first medium into the incubator, and then injects the bone marrow fluid into the incubator and allows it to stand for a set time;
A separation operation for selectively separating stem cells that adhere to the incubator by controlling the draining means and the liquid feeding means after the initial operation, and washing the inside of the incubator with the medium or the washing liquid;
A culture operation for controlling the liquid feeding means after the separation operation and injecting the first medium into the incubator to culture the stem cells;
After confirming that the stem cells have sufficiently proliferated by the culture operation, a differentiation operation for controlling the liquid feeding means and injecting the second medium into the incubator to differentiate the stem cells into tissue cells;
After the differentiation operation, the liquid feeding means is controlled to inject the proteolytic enzyme into the incubator to suspend the stem cells, and the first medium is injected into the incubator to float the stem cells Control the cell recovery operation to discharge to the cell recovery tank ,
In the culture operation, when it is confirmed that the growth rate of the stem cells is suppressed based on the acquired image in the incubator, the liquid feeding means is controlled to supply the proteolytic enzyme to the incubator. An automatic culture apparatus comprising a subculture operation of injecting and suspending the medium after injecting and suspending the stem cells . In the automatic culture apparatus according to claim 1 or 2 ,
The automatic culture apparatus further comprises a swinging means for swinging the incubator, and the control means swings the incubator by controlling the swinging means during the separation operation.

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