JP2014054229A - Method and device of culturing cells - Google Patents

Abstract

PROBLEM TO BE SOLVED: To sterilize a cell culture device efficiently.SOLUTION: Hydrogen peroxide solution and compressed air are sprayed into a vaporization chamber (16c), sprayed hydrogen peroxide solution is heated to boiling point or higher and completely vaporized by heating means arranged in a vaporization chamber (16c). Then, after at least an operation section (3) which performs a culture operation of cells, culture sections (4a,4b) which culture the cells, and a carry in/out storage section (2) for carrying in/out materials and tools are sterilized by spraying the solution, cell culture is started.

Description

  The present invention relates to a cell culture method and apparatus utilizing sterilization with hydrogen peroxide.

  It is being put into practical use that cell culture is performed using tissues, cells, fertilized eggs and the like of various parts of the human body and used for regenerative medicine. In this cell culture, it is important that cells and the like are not contaminated by bacteria during the culture. Therefore, sterilization is performed to make the working atmosphere aseptic before cell culture.

  A bactericidal agent is generally used for the bactericidal treatment, and use of ozone gas (for example, refer to Patent Document 1), hydrogen peroxide (for example, refer to Patent Documents 3 and 4), and the like has been proposed. Moreover, although it is not a disinfectant, using the heated water vapor | steam for disinfection is also proposed (for example, refer patent document 2).

International Publication No. 2004/011593 JP 2006-115798 A JP 2006-68122 A International Publication No. 2012/043682

  A certain sterile atmosphere must be ensured when culturing cells. Furthermore, in order to spread regenerative medicine, culture productivity must be increased.

  However, the sterilization method using a sterilizing gas such as ozone gas as a sterilization technique has a risk that the gas remains in a sterile atmosphere and kills cultured cells.

  In addition, heat sterilization with steam has a problem that sterilization takes a long time and sterilization tends to be uncertain.

  When hydrogen peroxide is used, it is highly reliable for ensuring sterility, but the conventional spraying method of hydrogen peroxide solution can be dripped onto a heating plate to vaporize the hydrogen peroxide solution, or an ultrasonic vibration device However, since it is difficult to vaporize a large amount in a short time, the sterilization treatment takes a long time, and the productivity of cell culture is inferior.

  An object of the present invention is to solve the above-mentioned problems and to ensure reliable sterility and high productivity for cell culture.

  In order to solve the above problems, the present invention employs the following configuration.

  That is, in the invention according to claim 1, the hydrogen peroxide solution and the compressed air are sprayed into the vaporizing chamber (16c), and the sprayed hydrogen peroxide solution is heated in the vaporizing chamber (16c). In order to completely evaporate by heating above the boiling point by the above), the operation unit (3) for culturing cells, the culture unit (4a, 4b) for culturing cells, and materials and instruments are taken in and out. A cell culture method is adopted in which cell culture is started after spraying and sterilizing at least three of the storage / exit section (2).

  As described in claim 2, in the cell culture method according to claim 1, the hydrogen peroxide solution and the compressed air are sprayed into the vaporizing chamber (16c) by the two-fluid spray nozzle (15) to be vaporized, It is also possible to discharge the vaporized product from the discharge nozzle (16b) and spray it on the three parties.

  As described in claim 3, in the cell culture method according to claim 1, the vaporized hydrogen peroxide solution is mixed with hot air so that the operation unit (3), the culture unit (4a, 4b), and loading / unloading. It is also possible to spray each part (2).

  As described in claim 4, in the cell culturing method according to any one of claims 1 to 3, the operation unit (3), the culture unit (4a, 4b) and the loading / unloading unit (2) It is also possible to vaporize excess hydrogen peroxide solution by spraying hydrogen peroxide solution on each of them and then blowing sterile air at room temperature or higher.

  The invention according to claim 5 includes at least an operation unit (3) for performing cell culture operation, a culture unit (4a, 4b) for cell culture, and a loading / unloading unit (2) for loading and unloading materials and instruments. Sealed doors (9, 23a, 23b) that are provided with three parties and can be opened and closed between the loading / unloading unit (2) and the operation unit (3) and between the operation unit (3) and the culture unit (4a, 4b). In the cell culture apparatus isolated by (1), hydrogen peroxide solution and compressed air are sprayed into the vaporizing chamber (16c), and the sprayed hydrogen peroxide solution is heated in the vaporizing chamber (16c). The cell culture apparatus provided with the sprayer (13) for spraying and sterilizing the above three components after being heated to the boiling point or higher and completely vaporized by the above.

  As described in claim 6, in the cell culture device according to claim 5, the nebulizer (13) includes a two-fluid spray nozzle (15) for spraying hydrogen peroxide and compressed air, and a two-fluid spray. It is also possible to have a vaporizing chamber (16c) for vaporizing what is sprayed by the nozzle (15) and a discharge nozzle (16b) for discharging what is vaporized from the vaporizing chamber (16c).

  As described in claim 7, in the cell culture device according to claim 5 or 6, a hot air supply unit (19) is provided, and the hot air from the hot air supply unit (19) is supplied to the nebulizer (13). ) And mixed with hydrogen peroxide gas discharged from the operation unit (3), the culture unit (4a, 4b), and the loading / unloading unit (2). It is.

  According to the present invention, the hydrogen peroxide solution and the compressed air are sprayed into the vaporization chamber (16c), and the sprayed hydrogen peroxide solution is heated above the boiling point by the heating means (16a) disposed in the vaporization chamber (16c). After being completely vaporized by heating, an operation part (3) for culturing cells, a culture part (4a, 4b) for culturing cells, and a storage / exit part for taking in and out materials and instruments (2) Since it is a cell culture method in which cell culture is started after spraying and sterilizing at least three parties, contamination by bacteria and the like can be reliably prevented during cell culture, and sterilization should be performed in a short time. Can do. Therefore, the productivity of cell culture is improved.

  Further, in the present invention, hydrogen peroxide solution and compressed air are sprayed into the vaporizing chamber (16c) by the two-fluid spray nozzle (15) and vaporized, and the vaporized one is discharged from the discharge nozzle (16b). When this discharged material is sprayed on the above three parties, a large amount of hydrogen peroxide solution can be easily vaporized, so that the sterilization time can be shortened.

  In the present invention, the vaporized hydrogen peroxide solution is mixed with hot air and sent to each of the operation unit (3), the culture unit (4a, 4b), and the loading / unloading unit (2) for spraying. Can spray the hydrogen peroxide spray to the details of the cell culture device to sterilize every corner.

  Further, in the present invention, after the hydrogen peroxide solution is sprayed on each of the operation unit (3), the culture unit (4a, 4b), and the loading / unloading unit (2), excess excess is generated by blowing sterile air at room temperature or higher. When hydrogen oxide water is vaporized, the hydrogen peroxide solution remaining slightly in the cell culture apparatus can be completely extinguished.

1 is a schematic view of a cell culture device according to the present invention. This is a hydrogen peroxide sprayer.

  Hereinafter, although the cell culture method and apparatus for implementing this invention are demonstrated, this invention is not limited to the following description.

  As shown in FIG. 1, the cell culture device according to the present invention includes an entry / exit unit 2, an operation unit 3, and culture units 4 a and 4 b in its outer shell 1.

  Partitions 5 and 6 partition between the loading / unloading unit 2 and the operation unit 3 and between the operation unit 3 and the culture units 4a and 4b, respectively. Moreover, the culture parts 4a and 4b are divided into a plurality by the partition walls 7.

  The loading / unloading unit 2 carries in specimens such as cells and tissues necessary for cell culture, media such as culture medium, trypsin, trypsin inhibitor, buffer, physiological saline, and other equipment, petri dishes, pipettes, centrifuge tubes, It is a room for carrying out cultured cells, waste chemicals, equipment, etc.

  Sealing doors 8 and 9 are provided on the outer shell 1 and the partition wall 5 of the loading / unloading unit 2, respectively. The entry / exit part 2 is isolated from the outside by the sealed door 8 of the outer shell 1, and the entry / exit part 2 is isolated from the operation part 3 by the sealed door 9 of the partition wall 5. Each of the sealed doors 8 and 9 can be opened and closed.

  In addition, a fan 10, HEPA filters 11 and 12 for introducing and exhausting air from the fan 10, and a hydrogen peroxide sprayer 13 are attached to the outer shell 1 of the warehouse 2.

  The fan 10 is for pumping air into the loading / unloading unit 2. This air is sterilized and sterilized by the HEPA filter 11, then flows into the loading / unloading unit 2, and a part of the air flows into the HEPA filter 12. Is discharged out of the loading / unloading unit 2. Thereby, the inside of the storage / exit part 2 is positively pressurized by aseptic air.

  In addition, an electric heater 14 which is a heating means for aseptic air is provided on the downstream side of the HEPA filter 11. Aseptic air that has passed through the HEPA filter 11 is heated by the electric heater 14 as necessary, and then supplied into the storage / exit section 2 as sterile hot air. For example, if a spray of hydrogen peroxide from the sprayer 13 is blown into the loading / unloading unit 2 and then the sterile hot air is blown into the loading / unloading unit 2, excess excess in the loading / unloading unit 2 can be obtained. Hydrogen oxide water is quickly vaporized and reliably discharged out of the loading / unloading unit 2. When aseptic air is heated, the temperature is desirably 120 ° C. or lower.

  As shown in FIG. 2, the sprayer 13 includes a hydrogen peroxide solution supply unit 15 that is a two-fluid spray nozzle that supplies an aqueous solution of hydrogen peroxide that is a sterilizing agent in the form of mist droplets, and the hydrogen peroxide solution supply unit. And a vaporizing section 16 that heats and vaporizes the mist droplets of the hydrogen peroxide solution supplied from 15 to a non-decomposition temperature equal to or higher than its boiling point.

  The hydrogen peroxide solution supply section 15 introduces an aqueous solution of hydrogen peroxide and compressed air from the hydrogen peroxide solution supply path 15a and the compressed air supply path 15b, respectively, and sprays the aqueous solution of hydrogen peroxide into the vaporization section 16. It has become.

  The vaporizing unit 16 is a pipe having a heater 16a sandwiched between inner and outer walls, and heats and vaporizes the mist of hydrogen peroxide water blown into the vaporizing chamber 16c in the pipe. The vaporized hydrogen peroxide gas is ejected as condensed mist M from the discharge nozzle 16b to the outside of the vaporizing section 16. The heater 16a is a band heater in this embodiment, but may be replaced with other heating means such as an induction heater.

  The hydrogen peroxide solution sent to the hydrogen peroxide solution supply unit 15 is an aqueous solution containing 10 to 60% by weight, preferably 30 to 40% by weight of hydrogen peroxide, and if necessary, a stabilizer or the like. The inclusion is used.

  The hydrogen peroxide solution is stored in a tank 17 shown in FIG. 1, and is supplied from here to a two-fluid spray nozzle which is a hydrogen peroxide solution supply unit 15. Compressed air sent from a compressor (not shown) to the hydrogen peroxide solution supply unit 15 is preferably at a pressure of 0.2 to 0.4 MPa, and is supplied to the hydrogen peroxide solution supply unit 15 through the HEPA filter 18. Moreover, the temperature in the vaporization part 16 is hold | maintained at 120 to 160 degreeC.

  The hydrogen peroxide water gas generated in the sprayer 13 flows into the loading / unloading section 2 from the discharge nozzle 16b as a mist M of hydrogen peroxide water, and the inner surface of the outer shell 1 of the loading / unloading section 2; Sterilize by touching the bulkhead 5 and others without any spots.

  As shown in FIG. 1, an auxiliary nozzle 19 that discharges aseptic heated air toward the mist M of the hydrogen peroxide solution is provided in the vicinity of the discharge nozzle 16b of the sprayer 13 as necessary. This heated air can be obtained by sterilizing the air blown from the fan 20 with the filter 21 and heating it with the electric heater 22. When this heated air is ejected from the auxiliary nozzle 19, it mixes with the hydrogen peroxide mist M discharged from the discharge nozzle 16 b, thereby accelerating the hydrogen peroxide mist M and corners in the loading / unloading unit 2. Go around. The heated air ejected from the auxiliary nozzle 19 is desirably 120 ° C. or higher.

  The operation unit 3 takes out the petri dish after culturing from the culture units 4a and 4b, or performs a subculture operation for exchanging the medium or separating cells from the petri dish and distributing them to a plurality of different petri dishes. The room. These operations are performed manually from the outside by a glove (not shown) or by a robot (not shown) installed in the operation unit 3.

  As in the case of the loading / unloading unit 2, the operation unit 3 includes a fan 10, HEPA filters 11 and 12 for introducing and discharging air, an electric heater 14, a hydrogen peroxide sprayer 13, an auxiliary nozzle 19, and the like. Is provided.

  The partition 6 between the culture units 4a and 4b is provided with sealed doors 23a and 23b.

  As in the case of the loading / unloading unit 2, a mist M of hydrogen peroxide water is blown into the operation unit 3 to sterilize the operation unit 3, and then aseptic air is blown to positive pressure.

  The culture units 4a and 4b are rooms for culturing specimens, cells, and the like. Although described as two chambers in FIG. 1, it can be increased or decreased as appropriate. When divided into a plurality of sections, different specimens can be cultured simultaneously.

  HEPA filters 24a and 24b are provided on the outer shell 1 of the culture units 4a and 4b.

  The sterilization of the culture units 4a and 4b is performed simultaneously with the sterilization in the operation unit 3 as will be described later.

  Next, the operation of the cell culture device will be described.

  The chemicals, equipment and the like carried into the storage / exit section 2 are packaged and sealed with a film or the like, and are sterilized by gamma ray sterilization or the like. The specimen is sealed in a sterile state.

  After these are loaded into the loading / unloading unit 2 from the opened closed door 8 and the closed door 8 is closed, the hydrogen peroxide mist M vaporized by the hydrogen peroxide sprayer 13 is loaded / unloaded unit 2. Sprayed inside. At this time, aseptic heated air is blown from the auxiliary nozzle 19 and mixed with the mist M of the hydrogen peroxide solution blown from the discharge nozzle 16b of the sprayer 13 to accelerate the mist M. As a result, the mist M quickly reaches every corner in the loading / unloading unit 2.

  After the hydrogen peroxide mist M is sprayed for a certain period of time, aseptic air sent from the fan 10 through the HEPA filter 11 flows into the loading / unloading unit 2, and thereby the inside of the loading / unloading unit 2 is positively pressurized. At this time, the electric heater 14 is turned ON, and the aseptic air sent through the HEPA filter 11 is heated by the electric heater 14. As a result, hot air is blown into the loading / unloading unit 2 for a certain period of time, and the hydrogen peroxide solution slightly remaining in the loading / unloading unit 2 is quickly vaporized and discharged from the HEPA filter 12 to the outside of the loading / unloading unit 2. Is done.

  Thereafter, the sealed wrapping material of the specimen, medicine and equipment is opened in the loading / unloading unit 2 and the contents are exposed.

  On the other hand, the inside of the operation unit 3 is sterilized by the mist M of the hydrogen peroxide solution discharged from the discharge nozzle 16 b of the sprayer 13 and the hot air discharged from the auxiliary nozzle 19. Then, the inside of the operation unit 3 is positively pressurized by aseptic air sent from the fan 10 through the HEPA filter 11.

  Thereafter, the sealing door 9 in the partition wall 5 between the loading / unloading unit 2 and the operation unit 3 is opened, and the contents are transferred from the loading / unloading unit 2 to the operation unit 3.

  A specimen which is a kind of contents carried into the operation unit 3 is transferred to a petri dish together with a medium after centrifugation as necessary.

  Then, after the sealing door 9 is closed, the sealing door 8 is opened, and the sealed packaging material is discharged from the storage / removal unit 2 to the outside of the outer shell 1. Furthermore, after the sealing door 8 is closed, the inside / outlet part 2 is sterilized and positively pressurized.

  As described above, the operation unit 3 is sterilized with the mist M of hydrogen peroxide and positively pressurized with aseptic air, but the operation unit 3 and the culture units 4a and 4b are sterilized in the operation unit 3. When the closed doors 23a and 23b of the partition wall 6 between the two are opened, the inside of the culture units 4a and 4b is simultaneously sterilized by the mist M of the hydrogen peroxide solution from the sprayer 13. In addition, air sterilized from the fan 10 and sterilized through the HEPA filter 11 is blown for a certain period of time, so that hydrogen peroxide remaining in the culture units 4a and 4b is completely removed from the HEPA filters 24a and 24b. Discharged.

  The specimen transferred to the petri dish together with the culture medium in the operation unit 3 is put into a desired room of the culture units 4a and 4b from the sealed doors 23a and 23b by a glove or a robot and cultured.

  The petri dish cultured in the culture units 4a and 4b is taken out into the operation unit 3 from the open sealed doors 23a and 23b. Then, in the operation unit 4, a culture medium exchange, a subculture operation for separating the cells from the petri dish, distributing them to a plurality of different petri dishes, and the like are performed. These operations can be performed manually by a glove from the outside, but can also be performed by operating a robot installed in the operation unit.

  The operation unit 3 performs the sterilization process every time a different specimen is operated. The culture units 4a and 4b are also sterilized each time the sample is changed. This treatment completely prevents cross contamination.

  Cells after culturing, unnecessary chemicals, equipment and the like are carried out from the sealed door 8 of the loading / unloading unit 2 to the outside.

  In addition, each part of the loading / unloading unit 2, the operation unit 3, and the culture units 4 a and 4 b is blown by the fan 10 in order to maintain sterility after the sterilization process, and is sterilized by passing through the HEPA filter 11. Although positive pressure is applied, the electric heater 14 is preferably turned off during blowing to maintain the positive pressure. In addition, sterility is maintained while the inside of each of the storage / removal unit 2, the operation unit 3, and the culture units 4a and 4b is isolated from the outside by the HEPA filters 11 and 12. In addition, excess hydrogen peroxide sprayed in each of the storage / removal unit 2, the operation unit 3, and the culture units 4a and 4b is discharged to the outside through the HEPA filters 12, 24a and 24b. It is desirable to decompose the hydrogen through a catalyst.

  Although the present invention is configured as described above, the present invention is not limited to the above-described embodiment, and various modifications can be made within the scope of the gist of the present invention.

DESCRIPTION OF SYMBOLS 2 ... Entry / exit part 3 ... Operation part 4a, 4b ... Culture | cultivation part 9, 23a, 23b ... Sealing door 13 ... Nebulizer 15 ... Two-fluid spray nozzle 16a ... Heater 16b ... Discharge nozzle 16c ... Vaporization chamber 19 ... Auxiliary nozzle

Claims (7)

  After spraying hydrogen peroxide solution and compressed air into the vaporization chamber, and heating the sprayed hydrogen peroxide solution above the boiling point by the heating means placed in the vaporization chamber, it is completely vaporized, and this is then cultured A cell culture method characterized by starting cell culture after spraying and sterilizing at least three of an operation unit for performing operations, a culture unit for culturing cells, and a loading / unloading unit for loading and unloading materials and instruments.   2. The cell culture method according to claim 1, wherein the hydrogen peroxide solution and the compressed air are sprayed into a vaporizing chamber by a two-fluid spray nozzle to be vaporized, and the vaporized product is discharged from the discharge nozzle and sprayed to the three parties. A cell culture method comprising the steps of:   2. The cell culturing method according to claim 1, wherein the vaporized hydrogen peroxide solution is mixed with hot air, sent to each of the operation unit, the culture unit, and the loading / unloading unit for spraying.   In the cell culture method in any one of Claims 1 thru | or 3, after spraying hydrogen peroxide water to each of the said operation part, a culture part, and a storage / exiting part, it is surplus by blowing in sterile air more than normal temperature A cell culture method comprising vaporizing hydrogen peroxide water.   It is equipped with at least three of an operation unit for culturing cells, a culture unit for culturing cells, and a loading / unloading unit for loading and unloading materials and instruments. In a cell culture device that is isolated from each other by a closed door that can be opened and closed, spray hydrogen peroxide solution and compressed air into the vaporization chamber, and heat the sprayed hydrogen peroxide solution placed in the vaporization chamber A cell culturing apparatus provided with a sprayer for spraying and sterilizing the above three components after heating them to a boiling point or higher by means and completely evaporating them.   The cell culture apparatus according to claim 5, wherein the nebulizer includes a two-fluid spray nozzle that sprays hydrogen peroxide and compressed air, a vaporization chamber that vaporizes what is sprayed by the two-fluid spray nozzle, and a vaporization chamber. A cell culture device comprising a discharge nozzle for discharging vaporized material.   The cell culture apparatus according to claim 5 or 6, wherein a hot air supply unit is provided, and the hot air from the hot air supply unit is mixed with hydrogen peroxide gas discharged from the sprayer, and the operation unit, A cell culture device, wherein the cell culture device is sent to each of a culture unit and an entry / exit unit.

Images