Method for producing sterilized cell-containing containers, method for improving the sterilization efficiency of cell-containing containers, and recording media, computer and system used therein.

Abstract

This invention provides a novel technological method for efficiently sterilizing cell-containing containers using hydrogen peroxide gas. [Solution] A method for producing a sterilized cell-containing container, (A) A first chamber having a gas inlet and outlet, A second chamber having a culture preparation operation section is sealed and connected to the first chamber via an opening / closing port. A step of preparing an isolator equipped with, (B) A step of exposing a cell-containing container containing freeze-thawed cells in the closed first chamber to hydrogen peroxide gas. We provide a method that includes the following:

Description

[Technical Field]

[0001] This disclosure relates to a method for producing sterilized cell-containing containers, a method for improving the sterilization efficiency of cell-containing containers, and recording media, computers, and systems used therein.

[0002] (Background of the invention) Today, cell-based experiments and treatments are diverse and are performed by various institutions and organizations for a wide range of purposes. In particular, containers used for cell culture and cell storage must be free from contamination by microorganisms such as bacteria and viruses, making sterilization technology that guarantees sterility a crucial factor. Therefore, it is necessary to sterilize cell-containing containers and equipment used in cell culture efficiently, while ensuring that disinfectants do not harm workers.

[0003] Furthermore, cryopreservation of cell therapies contributes to the stable supply and long-term storage of therapeutic products, significantly impacting stem cell research and cell therapy. Cells, especially stem cells, are most vulnerable during the transition from a metabolically active state to a frozen state, and vice versa. Therefore, the addition of dimethyl sulfoxide (DMSO) is widely used to prevent ice crystallization within cells during cryopreservation, which can cause damage. However, high concentrations of DMSO can be cytotoxic, so to avoid damage due to cytotoxicity, it has been necessary to dilute the DMSO by rapidly adding culture medium in a sterile environment after thawing the cells.

[0004] Under these technological circumstances, various isolators have been developed that allow for cell culture, testing, and other operations to be performed in a sealed workroom that is nearly sterile by killing microorganisms and bacteria.

[0005] When working with cell-containing containers using an isolator, the work area is sterilized beforehand. Sterilization of the work area is performed by first supplying a sterilization gas such as hydrogen peroxide gas into the work area, and then aerating the air containing the sterilization gas inside the work area with outside air (see, for example, Patent Documents 1 to 4).

[0006] An isolator is a box-shaped structure that can create an airtight space inside, and it typically includes a window that allows visibility into the interior and work gloves. These windows and work gloves are usually made of resins such as acrylic, polycarbonate (PC), polyamide (PA), or polyethylene terephthalate (PET), and these resins are absorbent of hydrogen peroxide. Therefore, when hydrogen peroxide gas is supplied to sterilize the internal space, some of the hydrogen peroxide gas may be absorbed by the aforementioned window material. Furthermore, because the internal space of an isolator has a certain volume required for the work, hydrogen peroxide is not easily discharged even with aeration, which causes the treatment time with hydrogen peroxide gas to be prolonged. [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Japanese Patent Publication No. 2014-54229 [Patent Document 2] Japanese Patent Publication No. 2013-74862 [Patent Document 3] WO2012 / 132146 publication [Patent Document 4] Special Publication No. 1-502722 [Overview of the project]

[0008] The Disclosers have recently discovered, through diligent research, that cell-containing containers can be efficiently sterilized using hydrogen peroxide gas with a device having a specific configuration comprising multiple sealed, interconnected internal spaces. This disclosure is based on these findings.

[0009] One objective of this disclosure is to provide a novel technical means for efficiently sterilizing cell-containing containers using hydrogen peroxide gas.

[0010] According to one embodiment of the present disclosure, a method for producing a sterilized cell-containing container, (A) A first chamber having a gas inlet and outlet, A second chamber having a culture preparation operation section is sealed and connected to the first chamber via an opening / closing port. A step of preparing an isolator equipped with, (B) A step of exposing a cell-containing container containing freeze-thawed cells in the closed first chamber to hydrogen peroxide gas. A method is provided that includes the following:

[0011] According to another embodiment of the present disclosure, a method for improving the sterilization efficiency of a cell-containing container, (A) A first chamber having a gas inlet and outlet, A second chamber having a culture preparation operation section is sealed and connected to the first chamber via an opening / closing port. A step of preparing an isolator equipped with, (B) A step of exposing a cell-containing container containing freeze-thawed cells in the closed first chamber to hydrogen peroxide gas. A method that includes It will be provided.

[0012] According to another embodiment of the present disclosure, a recording medium for recording a computer program for carrying out any of the above methods, or a computer having such a program, is provided.

[0013] According to another embodiment of the present disclosure, a system for producing a sterilized cell-containing container, A first chamber having a gas inlet and outlet, The first chamber is sealed and connected to the second chamber via an opening / closing port, and the second chamber has a culture preparation operation section. A hydrogen peroxide gas inlet / outlet device is sealed and connected to the first chamber mentioned above. A sterilization control unit that is connected to the above-described device and controls the sterilization conditions of the cell-containing container with hydrogen peroxide gas in the above-described first chamber, and A system comprising is provided.

[0014] According to the present disclosure, a cell-containing container can be efficiently sterilized using hydrogen peroxide gas.

Brief Description of the Drawings

[0015] [Figure 1] It is a flowchart of a method for manufacturing a sterilized cell-containing container in one embodiment of the present disclosure. [Figure 2] It is a schematic diagram of an isolator in one embodiment of the present disclosure. [Figure 3] It is a schematic diagram of a method for manufacturing a sterilized cell-containing container in one embodiment of the present disclosure. [Figure 4] It is a schematic diagram for explaining an incubation step, a culture container sealing step, and a sterilization step of a sealed culture container in one embodiment of the present disclosure. [Figure 5] It is a block diagram of a system for manufacturing a sterilized cell-containing container according to one embodiment of the present disclosure. Detailed Description of the Invention

[0016] According to one embodiment of the present disclosure, a method for manufacturing a sterilized cell-containing container is (A) Preparing an isolator comprising a first chamber having a gas inlet / outlet and a second chamber having a culture preparation operation unit hermetically connected to the first chamber via an opening / closing port, and and (B) exposing a cell-containing container containing cells thawed by freezing and thawing to hydrogen peroxide gas in the closed first chamber comprising.

[0017] ​As described above, it is a surprising fact to those skilled in the art that the sterilization time for cell preparations can be significantly shortened by adjusting the hydrogen peroxide gas exposure conditions in the first chamber, which is sealed and connected to the second chamber containing the culture preparation operation section, in an isolator.

[0018] According to a preferred embodiment, the method of the present disclosure includes an isolator preparation step (A), a cell-containing container sterilization step (B) containing freeze-thawed cells, a culture preparation step (C), an incubation step (D), a culture container sealing step (E), and a sealed culture container sterilization step (F), as shown in the flowchart of Figure 1. Hereinafter, one embodiment of the present disclosure will be described step by step.

[0019] Isolator preparation process (A) According to one embodiment of the present disclosure, as an isolator preparation step (A), an isolator is prepared comprising a first chamber having a gas inlet and outlet, and a second chamber having a culture preparation operation section, which is sealed and connected to the first chamber via an opening and closing port.

[0020] Figure 2 is a schematic diagram of an isolator in one embodiment of the present disclosure. In Figure 2, the isolator 1 comprises a first chamber 2 in which the sterilization process (B) is carried out, and a second chamber 3 in which the culture preparation process is carried out. The first chamber 2 and the second chamber 3 are sealed and connected via an opening / closing port 4.

[0021] In the isolator 1, the first chamber 2 has another opening 4', and a sterilization target (such as a cell-containing container that has been frozen and thawed) can be placed in the first chamber 2 through the opening 4'. Therefore, according to one embodiment of the present disclosure, the first chamber is an inlet / outlet section that has the function of loading and unloading cell-containing containers (i.e., the first chamber can load and unload cell-containing containers).

[0022] Furthermore, in the isolator 1, the first chamber 2 has a gas inlet / outlet 5, and is sealed and connected to the sterilization condition control device 6 via the gas inlet / outlet 5. Although not shown in the figures, the sterilization condition control device 6, as shown in Figure 5 which will be described later, is configured as an integrated unit consisting of a hydrogen peroxide gas inlet / outlet device 6a and a sterilization control unit 6b that controls the sterilization conditions.

[0023] Therefore, according to a preferred embodiment of the present disclosure, the isolator can be configured, for example, by using the first chamber 2 as an inlet / outlet section and connecting the hydrogen peroxide gas inlet / outlet device 6a, which is part of the sterilization condition control device 6, to the first chamber 2. Alternatively, the sterilization control unit 6b, which is another part of the sterilization condition control device 6, may be connected to the first chamber via a sensor and configured to perform sterilization while sensing the hydrogen peroxide gas treatment conditions (gas concentration, time, etc.) in the first chamber.

[0024] Furthermore, in the isolator 1, the second chamber 3 has an internal space for performing culture preparation operations, and is equipped with a culture preparation operation section 7, 7' for performing culture preparation operations (injection of culture medium, dilution, sampling, etc.), a centrifuge 8 necessary for culture preparation operations, and a rack 9 for arranging instruments necessary for culture operation processing. Although not shown in the figures, the second chamber 3 may also be equipped with a window that allows the interior to be viewed and four work gloves necessary for two operators to perform culture preparation operations from the outside.

[0025] Furthermore, in the isolator 1, the second chamber 3 may be connected to or separated from the RTP (Rapid Transfer Port) container 10 by attachment or detachment. The cell-containing container obtained by the culture preparation procedure can be transported to the incubator using the RTP container 10 and incubated.

[0026] The volume of the first chamber is not particularly limited, but is preferably small from the viewpoint of rapid and efficient sterilization. According to one embodiment of the present disclosure, the volume of the first chamber is, for example, 50 to 400 L, preferably 100 to 300 L, and more preferably 120 to 250 L.

[0027] Furthermore, the volume of the second chamber is not particularly limited, but it is preferable to set it so that culture preparation operations can be carried out sufficiently. According to one embodiment of the present disclosure, the volume of the second chamber is, for example, 500 to 2500 L, preferably 700 to 2200 L, and more preferably 1000 to 1700 L.

[0028] Furthermore, according to one embodiment of the present disclosure, from the viewpoint of balancing rapid and efficient sterilization and smooth implementation of culture preparation operations, it is preferable that the volume of the first chamber is smaller than the volume of the second chamber. The volume ratio of the first chamber to the second chamber (volume of the first chamber:volume of the second chamber) is, for example, 1:2 to 1:20, preferably 1:3 to 1:15, and more preferably 1:5 to 1:12.

[0029] Sterilization process of cell-containing containers containing frozen and thawed cells (B ) Furthermore, according to one embodiment of the present disclosure, as a sterilization step (B), a cell-containing container containing cells that have been frozen and thawed in the first chamber described above in a closed state is exposed to hydrogen peroxide gas.

[0030] The type of cells used in the method disclosed herein is not particularly limited, but is, for example, stem cells, and more preferably adipose tissue-derived stem cells (also known as adipose tissue-derived stromal stem cells). Stem cells and other regenerative medicine materials require rapid sterilization to avoid contamination by microorganisms such as bacteria and viruses, and therefore are preferably used in the method disclosed herein.

[0031] Furthermore, according to one embodiment of this disclosure, as described above, the cell-containing container is a container obtained by freezing and thawing a cell-containing container that has been cryopreserved. Examples of such cell-containing containers include vials, ampoules, etc., made of glass or plastic (e.g., polypropylene). The cryopreservation and thawing of the cell-containing container may be carried out, for example, using a water bath. It is preferable to include DMSO in the cell-containing container from the viewpoint of avoiding damage during cell cryopreservation. The method of this disclosure can be used advantageously in avoiding cytotoxicity caused by DMSO because it can achieve rapid sterilization.

[0032] In a preferred embodiment of this disclosure, as shown in Figure 1, step (B1) is used to pre-set the sterilization conditions for the cell-containing container with hydrogen peroxide gas in the closed first chamber. It is more preferable to use a biological indicator to set the sterilization conditions. It is even more preferable to use spore-forming bacteria as the biological indicator. It is also even more preferable to use a measurement method such as confirming the death of spore-forming bacteria to set the sterilization conditions. It is more preferable that step (B1) is performed before step (B2) described later. Here, the sterilization conditions for the cell-containing container with hydrogen peroxide gas can be set by appropriately adjusting the operating conditions of the hydrogen peroxide gas inlet / outlet device (injection rate, discharge rate, maintenance time, etc.) according to the volume of the first chamber so as to achieve a desired sterilization level.

[0033] Furthermore, regarding the validity of the sterilization conditions for cell-containing containers after the sterilization process, it is preferable to perform validation using a biological indicator and ensure that the sterilization level is guaranteed under the conditions established by said validation. A criterion value for distinguishing the sterilization level of cell-containing containers after the sterilization process is, for example, a reduction of 6 log or more in indicator bacteria. The above indicator bacteria are preferably spore-forming bacteria. The above bacteria are preferably bacteria of the genus Geobacillus, and more preferably Geobacillus stearothermophilus. The above criterion values ​​may also be in accordance with the description in the Ministry of Health, Labour and Welfare's Pharmaceutical and Food Safety Bureau Monitoring and Guidance Division, Narcotics Control Division's administrative notice, "Guidelines for the manufacture of sterile pharmaceuticals by aseptic operation method" (revised edition) (April 20, 2011).

[0034] Furthermore, according to a preferred embodiment of this disclosure, as step (B2), a cell-containing container containing freeze-thawed cells is exposed to hydrogen peroxide gas in a closed first chamber. According to a preferred embodiment of this disclosure, the hydrogen peroxide gas exposure treatment includes an injection step, a maintenance step, and a discharge step of the hydrogen peroxide gas. Therefore, more specific sterilization conditions in the exposure treatment in the first chamber include the injection rate, gas concentration, maintenance time, and discharge rate of the hydrogen peroxide gas.

[0035] According to one embodiment of the present disclosure, the injection rate of hydrogen peroxide gas into the first chamber in step (B) is not particularly limited, but is, for example, 0.05 to 0.2 g / second, preferably 0.07 to 0.15 g / second, and more preferably 0.08 to 0.12 g / second.

[0036] Furthermore, according to one embodiment of the present disclosure, the hydrogen peroxide gas concentration in the first chamber after the hydrogen peroxide gas is injected in step (B) is not particularly limited, but for example, 0.001 to 0.4 g / m³ 3 The concentration is preferably 0.005 to 0.2 g / m³. 3 And more preferably 0.01~0.12 g / m 3 That is the case.

[0037] According to one embodiment of the present disclosure, the maintenance time of the hydrogen peroxide gas in the first chamber after the hydrogen peroxide gas is injected in step (B) is not particularly limited, but is, for example, 60 to 420 seconds, preferably 90 to 360 seconds, and more preferably 120 to 240 seconds.

[0038] According to preferred embodiments of the present disclosure, the discharge rate of hydrogen peroxide gas from the first chamber in step (B) is not particularly limited, but for example, 0.1 to 5 m 3 The interval is / second, preferably 0.2 to 4m 3 It is / second, more preferably 0.25~3m 3 It is per second.

[0039] Furthermore, the hydrogen peroxide gas exposure treatment may include, in addition to the injection, maintenance, and discharge processes of the hydrogen peroxide gas, a preconditioning process to prepare the system for immediate discharge of hydrogen peroxide gas, and a stabilization process to adjust the air pressure between the first and second discharge chambers to the same level.

[0040] According to one embodiment of the present disclosure, the time of the preconditioning step before the injection step in step (B) is not particularly limited, but is, for example, 20 to 120 seconds, preferably 25 to 90 seconds, and more preferably 20 to 80 seconds.

[0041] Furthermore, according to one embodiment of the present disclosure, the time of the stabilization step after the discharge step in step (B) is not particularly limited, but is, for example, 30 to 300 seconds, preferably 45 to 240 seconds, and more preferably 60 to 150 seconds.

[0042] Furthermore, according to one embodiment of the present disclosure, the total time of each step included in the hydrogen peroxide gas exposure treatment in step (B) (the execution time of step (B)) is not particularly limited, but is for example 180 to 3600 seconds, preferably 360 to 2400 seconds, and more preferably 450 to 1500 seconds. According to one embodiment of the present disclosure, the sterilization time with hydrogen peroxide gas can be significantly shortened by adjusting the sterilization conditions in the first chamber of the isolator.

[0043] Culture preparation operation process (C) Furthermore, according to one embodiment of the present disclosure, in step (C), the cell culture preparation operation of the cells in the cell-containing container obtained in step (B) is performed in a closed second chamber to obtain a cell culture container containing the cells after the culture preparation operation.

[0044] The cell-containing container obtained in step (B) can be transferred from the first chamber to the second chamber where culture preparation operations are performed via an opening / closing port. Figure 3 is a schematic diagram illustrating the culture preparation operation step (C) and the incubation step (D) described later.

[0045] According to one embodiment of the present disclosure, the culture preparation operation includes, as shown in the schematic diagram of Figure 3, a) a transfer step of moving a cell-containing container containing frozen and thawed cells into the second chamber of the isolator; b) a transfer step of transferring the cells, along with the culture medium, from the cell-containing container containing frozen and thawed cells to a container usable for centrifugation (e.g., a microtube such as a conical tube or Eppendorf tube); c) a centrifugation and supernatant removal step; d) a culture medium addition step of adding the culture medium to the container usable for centrifugation containing the cells after the supernatant has been removed; and e) a step of transferring the cells, along with the culture medium, from the container usable for centrifugation to a cell culture vessel. Furthermore, before and after the culture preparation operation, measurements of cell number, cell viability, sterilization level, etc., may be performed according to known methods.

[0046] According to one embodiment of this disclosure, from the viewpoint of achieving safe sterilization, it is preferable to perform sterilization in advance and to perform the culture preparation operation in the second room under the Grade A environment of the Ministry of Health, Labour and Welfare's Pharmaceutical and Food Safety Bureau Monitoring and Guidance Division, Narcotics Control Division's administrative notice "Guidelines for the manufacture of sterile pharmaceuticals by aseptic operation method" (revised edition) (dated April 20, 2011).

[0047] According to one embodiment of the present disclosure, the cell culture vessel used in step (C) is not particularly limited, but may be a commonly used known cell culture vessel such as a plastic tube or a cell culture flask. Furthermore, the lid of the cell culture vessel obtained in step (C) preferably has a ventilation filter, from the viewpoint of ensuring the ventilation necessary for cell survival in the subsequent incubation step. Examples of cell culture vessels having a ventilation filter include multi-stage culture devices, and specifically, known products such as CellFactory (Thermo Fisher Scientific) and CellStack (Corning) can be mentioned.

[0048] Incubation process (D) According to one embodiment of the present disclosure, the method comprises the step of incubating the cell culture vessel obtained in step (C) in an incubator.

[0049] When moving the cell culture vessel from the second chamber of the isolator to the incubator, an RTP or PB (Plant Box) as shown in Figure 2 may be used. The cell incubation conditions are not particularly limited and may be set appropriately using an incubator known to those skilled in the art, depending on the type and properties of the cells.

[0050] According to one embodiment of the present disclosure, from the viewpoint of maintaining the sterility of the product, it is preferable to perform the culture in an incubator under an environment of Grade C or higher as defined in the Ministry of Health, Labour and Welfare's Pharmaceutical and Food Safety Bureau Monitoring and Guidance Division, Narcotics Control Division's administrative notice "Guidelines for the manufacture of sterile pharmaceuticals by aseptic manipulation methods" (revised edition) (dated April 20, 2011).

[0051] Culture vessel sealing process (E) / Sterilization process of sealed culture vessels (F) According to one embodiment of the present disclosure, as step (E), the lid of the cell culture vessel obtained in step (D) is sealed with a sealing material that prevents the intrusion of hydrogen peroxide gas. In a preferred embodiment, as step (F), the cell culture vessel sealed with the sealing material in step (E) is exposed to hydrogen peroxide gas. Step (E) is not particularly limited, but for example, it may be carried out in an environment with a cleanliness level of grade C or higher, preferably in a room with a cleanliness level of grade C (also called a sterile room), and more preferably in a sterile room where a system or isolator for producing sterilized cell-containing vessels is located (or equipped). Step (F) is not particularly limited, but it is preferably carried out in the first room.

[0052] Figure 4 is a schematic diagram illustrating the culture vessel sealing step (E) and the sterilization step (F) of the sealed culture vessel after the incubation step in one embodiment of the present disclosure. It is preferable to further promote cell proliferation by performing a culture preparation procedure again in the cell culture vessel after incubation. However, if the cell culture vessel lid has a ventilation filter when exposure treatment with hydrogen peroxide gas is performed prior to the cell culture preparation procedure, there is a risk that hydrogen peroxide gas may enter the cell culture vessel and inhibit cell proliferation. Therefore, according to a preferred embodiment of this disclosure, as shown in Figure 4, it is preferable to seal the lid of the cell culture vessel with a sealing material that prevents the entry of hydrogen peroxide gas prior to exposure with hydrogen peroxide gas again after the incubation process.

[0053] The sealing material for the cell culture vessel is preferably shaped to cover, for example, the ventilation filter on the lid of the cell culture vessel. Furthermore, from the viewpoint of rapid sealing, the sealing material is preferably in the form of a seal that can be attached to and detached from the lid of the cell culture vessel. In addition, from the viewpoint of blocking hydrogen peroxide gas, the sealing material may be, for example, a laminate of a resin and a thin metal film. Examples of the resin include polyethylene terephthalate (PET), polyethylene, polypropylene, nylon, etc., with PET being preferred. The metal is not particularly limited, but is preferably aluminum (aluminum foil), etc. The resin layer and the metal layer may each be one layer or two or more layers.

[0054] Furthermore, according to one embodiment of the present disclosure, as shown in Figure 4, after exposure with hydrogen peroxide gas, the sealed cell culture vessel may be moved to the second chamber of the isolator, the sealed lid may be discarded, and the lid of a pre-prepared ventilation filter may be replaced to ensure ventilation to the cells, and the culture preparation procedure may be performed.

[0055] Method for improving the sterilization efficiency of cell-containing containers Furthermore, according to another embodiment of the present disclosure, a method for improving the sterilization efficiency of a cell-containing container, (A) A first chamber having a gas inlet and outlet, A second chamber having a culture preparation operation section is sealed and connected to the first chamber via an opening / closing port. A step of preparing an isolator equipped with, (B) A step of exposing a cell-containing container containing freeze-thawed cells in the closed first chamber to hydrogen peroxide gas. A method is provided that includes the following:

[0056] According to another preferred embodiment of the present disclosure, the above method is a method for shortening the sterilization time of a cell-containing container. Furthermore, according to yet another more preferred embodiment of the present disclosure, step (B) includes pre-setting sterilization conditions for the cell-containing container with hydrogen peroxide gas in the closed first chamber. It is more preferable to use a biological indicator for setting the sterilization conditions. It is even more preferable to use spore-forming bacteria as the biological indicator. It is also even more preferable to use a measurement method, such as confirming the death of spore-forming bacteria, for setting the sterilization conditions. A method for improving the sterilization efficiency of a cell-containing container can be carried out in accordance with the method for producing a sterilized cell-containing container according to the present disclosure.

[0057] A system / computer program, recording medium, or computer containing the same for manufacturing sterilized cell-containing containers. Furthermore, according to another embodiment of the present disclosure, a system for producing sterilized cell-containing containers is provided. An embodiment of the system of the present disclosure will be described below with reference to Figure 5. Figure 5 is a block diagram of the system for producing sterilized cell-containing containers. In Figure 5, the interfaces for inputting and outputting information are omitted from the illustration.

[0058] In Figure 5, the system 11 for producing sterilized cell-containing containers is: A first chamber 2 having a gas inlet and outlet, The first chamber is sealed and connected to the second chamber 3 via an opening and closing port, and has a culture preparation operation section. A hydrogen peroxide gas inlet / outlet device 6a is sealed and connected to the first chamber, The apparatus is equipped with a sterilization control unit 6b connected to it, which controls the sterilization conditions of the cell-containing container using hydrogen peroxide gas in the first chamber.

[0059] In the system 11 shown in Figure 5, the first chamber 2 and the second chamber 3 are located within the isolator 1 or are part of the isolator 1. In the system 11, the hydrogen peroxide gas inlet / outlet device 6a and the sterilization control unit 6b are integrated as a sterilization condition control unit 6.

[0060] The hydrogen peroxide gas inlet / outlet device 6a may include a tank for containing hydrogen peroxide gas and a pump for discharging hydrogen peroxide gas, and can be configured to operate in response to a signal from the sterilization control unit.

[0061] From the perspective of achieving safe sterilization, the system disclosed herein is preferably installed and used in an environment of Grade C or higher as defined in the Ministry of Health, Labour and Welfare's Pharmaceutical and Food Safety Bureau, Monitoring and Guidance Division, Narcotics Control Section's administrative notice, "Guidelines for the manufacture of sterile pharmaceuticals by aseptic techniques" (revised edition) (dated April 20, 2011).

[0062] In this disclosure, the series of processes for producing sterilized cell-containing containers can be performed by hardware, software, or a combination thereof. When performing the process by software, a program recording the processing sequence can be installed and executed in the memory of a computer embedded in dedicated hardware, or the program can be installed and executed on a general-purpose computer capable of performing various processes.

[0063] For example, programs can be pre-recorded on recording media such as hard disks or ROMs. Alternatively, programs can be temporarily or permanently stored (recorded) on removable recording media such as flexible disks, CD-ROMs, MO disks, DVDs, magnetic disks, and semiconductor memory.

[0064] In addition to installing programs from removable storage media as described above, programs can also be downloaded from download sites and transferred wirelessly to the computer, or transferred via a wired connection over a network such as a LAN or the internet. The computer can then receive the programs transferred in this way and install them onto its internal storage media, such as a hard disk.

[0065] The method disclosed herein can be suitably implemented on a computer that stores the above-mentioned computer program in its internal memory.

[0066] Furthermore, the various processes described herein may not only be executed sequentially as described, but may also be executed in parallel or individually as needed, depending on the processing capacity of the device performing the process. Also, in this specification, a system is a logical collection of multiple devices, and is not limited to devices in each configuration being located in the same enclosure. While embodiments of this disclosure have been described, this disclosure is not limited to the embodiments described above, and various modifications can be implemented within the scope of this disclosure.

[0067] Furthermore, according to one embodiment of the present disclosure, the following [1] to

[23] are provided. [1] A method for producing a sterilized cell-containing container, (A) A first chamber having a gas inlet and outlet, A second chamber having a culture preparation operation section is sealed and connected to the first chamber via an opening / closing port. A step of preparing an isolator equipped with, (B) A step of exposing a cell-containing container containing freeze-thawed cells in the closed first chamber to hydrogen peroxide gas. A method that includes the following: [2] The method according to [1], wherein the volume of the first chamber is smaller than the volume of the second chamber. [3] The method according to [1] or [2], wherein the volume ratio of the first chamber and the second chamber (volume of the first chamber:volume of the second chamber) is 1:2 to 1:20. [4] The method according to any one of [1] to [3], wherein step (B) above is set in advance for sterilization conditions of the cell-containing container with hydrogen peroxide gas in the closed first chamber. [5] The method according to any of [1] to [4], wherein the above cells are stem cells. [6] The method according to [5], wherein the stem cells are derived from adipose tissue. [7] The method according to any one of [1] to [6], wherein step (B) above comprises an injection step, a maintenance step and a discharge step of hydrogen peroxide gas. [8] After injecting hydrogen peroxide gas in step (B) above, the hydrogen peroxide gas concentration in the first chamber is 0.01 to 0.2 g / m³. 3 The method described in any of [1] to [7]. [9] The method according to any one of [1] to [8], wherein the injection rate of hydrogen peroxide gas into the first chamber in step (B) is 0.05 to 0.2 g / second.

[10] The discharge rate of hydrogen peroxide gas from the first chamber in the above process (B) is 0.1 to 5 m 3 The method used is one of the methods [1] to [9], which is per second.

[11] The method according to any of [1] to

[10] , wherein the execution time of step (B) above is 180 to 3600 seconds.

[12] The method according to any one of [1] to

[11] , wherein the first chamber is the loading and unloading section for the cell-containing container.

[13] (C) The method according to any one of [1] to

[12] , further comprising the step of performing a cell culture preparation operation in the cell-containing container obtained in step (B) above in the closed second chamber above to obtain a cell culture container containing the cells after the above operation.

[14] The method according to

[13] , wherein the lid of the cell culture vessel obtained in step (C) above has a ventilation filter.

[15] (D) The method according to any one of [1] to

[14] , further comprising the step of incubating the cell culture vessel obtained in step (C) in an incubator.

[16] (E) The method according to

[15] , further comprising the step of sealing the lid of the cell culture vessel obtained in step (D) with a sealing material that prevents hydrogen peroxide gas from entering.

[17] (F) The method according to

[16] , further comprising the step of exposing the cell culture vessel sealed with the sealing material in step (E) above to hydrogen peroxide gas.

[18] A method for improving the sterilization efficiency of cell-containing containers, (A) A first chamber having a gas inlet and outlet, A second chamber having a culture preparation operation section is sealed and connected to the first chamber via an opening / closing port. A step of preparing an isolator equipped with, (B) A step of exposing a cell-containing container containing freeze-thawed cells in the closed first chamber to hydrogen peroxide gas. A method that includes the following:

[19] The method according to

[18] , which is a method for shortening the sterilization time of cell-containing containers.

[20] The method according to

[18] or

[19] , wherein step (B) above comprises presetting sterilization conditions for the cell-containing container with hydrogen peroxide gas in the closed first chamber. A recording medium for recording a computer program for carrying out any of the methods described in

[21] [1] to

[17] , or a computer containing such a recording medium.

[22] A system for producing sterilized cell-containing containers, A first chamber having a gas inlet and outlet, The first chamber is sealed and connected to the second chamber via an opening / closing port, and the second chamber has a culture preparation operation section. A hydrogen peroxide gas inlet / outlet device is sealed and connected to the first chamber mentioned above. A sterilization control unit connected to the above-mentioned apparatus controls the sterilization conditions of the cell-containing container with hydrogen peroxide gas in the first chamber. A system that includes these features.

[23] The system according to

[22] , wherein the cell-containing container contains frozen and thawed cells. [Examples]

[0068] The following describes the disclosure in detail, but the disclosure is not limited to these examples. The measurement methods and units used in this disclosure conform to the provisions of JIS (Japanese Industrial Standards).

[0069] Test Example 1 1-1 The following tests were conducted in accordance with the "Guidelines for the Manufacture of Sterile Pharmaceuticals by Aseptic Techniques" (revised edition) (dated April 20, 2011) issued by the Pharmaceutical and Food Safety Bureau, Narcotics Control Division of the Ministry of Health, Labour and Welfare.

[0070] Preparation of frozen cell-containing containers Cell culture procedures were performed under cleanliness level A conditions as defined in the "Guidelines for the Manufacturing of Sterile Pharmaceuticals by Aseptic Techniques" (revised edition). Specifically, stromal vascular fractions were isolated from human subcutaneous adipocytes, and adipose tissue-derived stem cells (ASCs) were selected. The obtained adipose tissue-derived stem cells were seeded in tissue culture flasks.

[0071] Next, the tissue culture flasks were transferred to an environment meeting cleanliness level B or C as defined in the "Guidelines for the Manufacturing of Sterile Pharmaceuticals by Aseptic Techniques" (revised edition), and cell culture was performed at 37°C and 5% CO2.

[0072] The obtained cells were centrifuged, the supernatant was discarded, and a cell suspension was prepared by pipetting. This suspension was diluted with freezing medium (DMSO-containing medium), dispensed into 1 mL polypropylene ampoules, and frozen at -80°C to obtain frozen cell-containing containers. These were then stored under liquid nitrogen conditions.

[0073] Freezing and thawing of cell-containing containers / Exposure to hydrogen peroxide gas The frozen cell-containing containers were placed in 37°C warm water to quickly thaw them, thus performing a freeze-thaw procedure.

[0074] Next, as shown in FIG. 5, a sterilization test of the frozen cell-containing container was conducted using a system for manufacturing a sterilized cell-containing container. The above system is arranged in a room with a cleanliness level of grade C, and has a first chamber (volume 165 L, cleanliness level before sterilization: grade C, cleanliness level after sterilization: grade A) having an inlet / outlet for hydrogen peroxide gas, and a second chamber (volume 1486 L: cleanliness level grade A) having a culture preparation operation section hermetically connected to the first chamber via an opening / closing port, as part of an isolator. Further, the above system includes, as part of a sterilization control device, an inlet / outlet device for hydrogen peroxide gas hermetically connected to the first chamber, and a sterilization control section connected to the inlet / outlet device for hydrogen peroxide gas for controlling the sterilization conditions of the cell-containing container with hydrogen peroxide gas in the first chamber.

[0075] In the sterilization test, as a biological indicator (BI), prepared bacterial spores (10 6 pieces) of G. stearothermophilus (ATCC #7953) were used, and it was confirmed that sterilization was completed after the sterilization treatment in the first chamber.

[0076] The conditions and results of the sterilization test in the first chamber are shown in Table 1 below.

Table 1

[0077] It was confirmed that the sterilization treatment in the first chamber can be shortened to 9 minutes and 6 seconds. By shortening the sterilization treatment time to about 9 minutes, it is considered that the damage to cells by DMSO in the cell-containing container can be significantly suppressed. Although not shown in the table, when the first chamber was used simply as an inlet / outlet section and the sterilization treatment with hydrogen peroxide gas was carried out in the second chamber, it took 120 minutes. When the sterilization treatment time takes about 120 minutes, it is considered that the damage to cells by DMSO in the cell-containing container becomes larger compared to Test Section 1 and Test Section 2.

[0078] 1-2 After the sterilization treatment in 1-1, perform the culture preparation procedure according to the steps shown in Figure 3, and use a cell culture vessel with a ventilation filter (multi-stage culture device (cell stack) (culture area 3,180 cm²) 2 Cell culture was performed in an incubator using a Corning incubator (incubation conditions: 37°C, approximately 120-168 hours). Furthermore, after incubation, the cell culture vessels with aeration filters were sealed with a sealing agent (Easy Peel Film, manufactured by Micronics Co., Ltd., a laminated film consisting of PET, aluminum foil, and sealant) according to the procedure shown in Figure 4, and then exposed to hydrogen peroxide gas again. Sterilization was confirmed to be complete using the same method as in 1-1. The amount of hydrogen peroxide gas injected was 10 g, and the hydrogen peroxide gas injection concentration was 0.06 g / m³ 3 The total sterilization time was 12 minutes and 28 seconds. The sealing with the sealing agent (the sealing process of the culture vessels) was performed in a cleanliness level grade C room (also called a sterile room) where the system for producing the sterilized cell-containing vessels described above is located, and the second exposure with hydrogen peroxide gas (the sterilization process of the sealed culture vessels) was performed in the first room.

[0079] Cell viability in cell culture vessels sealed with a sealing agent and stored for 30 minutes, 1 hour, and 2 hours was confirmed by cell counting, and the cell viability was 99.0-99.3%.

[0080] Test Example 2 The same test as in Test Example 1, Section 1-1 was performed, except for changing the amount of hydrogen peroxide gas injected and the sterilization treatment time.

[0081] The results are shown in Table 2 below. [Table 2]

[0082] In all of the test plots 3 through 10, it was confirmed that sterilization was completed within 9 minutes. [Explanation of symbols]

[0083] Isolator 1 First room 2 Second room 3 Opening / closing 4, 4' Gas inlet / outlet 5 Sterilization condition control device 6 Hydrogen peroxide gas inlet / outlet device 6a Sterilization control unit 6b Culture preparation operation section 7,7' Centrifuge 8 Rack 9 RTP Container 10 System 11 for manufacturing sterilized cell-containing containers

Claims

1. A method for producing a sterilized cell-containing container, (A) A first chamber having a gas inlet and outlet, A second chamber having a culture preparation operation section is sealed and connected to the first chamber via an opening / closing port. A step of preparing an isolator equipped with, (B) A step of exposing a cell-containing container containing cells that have been frozen and thawed in the closed first chamber to hydrogen peroxide gas. A method that includes the following:

2. The method according to claim 1, wherein the volume of the first chamber is smaller than the volume of the second chamber.

3. The method according to claim 1, wherein the volume ratio of the first chamber to the second chamber (volume of the first chamber : volume of the second chamber) is 1:2 to 1:

20.

4. The method according to claim 1, wherein step (B) includes pre-setting sterilization conditions for a cell-containing container with hydrogen peroxide gas in the closed first chamber.

5. The method according to claim 1, wherein the cells are stem cells.

6. The method according to claim 5, wherein the stem cells are adipose tissue-derived stem cells.

7. The method according to claim 1, wherein step (B) comprises an injection step, a maintenance step and a discharge step of hydrogen peroxide gas.

8. In step (B) above, the hydrogen peroxide gas concentration in the first chamber after the injection of hydrogen peroxide gas is 0.01 to 0.2 g / m³. ３ The method according to claim 7.

9. The method according to claim 8, wherein the injection rate of hydrogen peroxide gas into the first chamber in step (B) is 0.05 to 0.2 g / second.

10. The discharge rate of hydrogen peroxide gas from the first chamber in step (B) is 0.1 to 5 m ３ The method according to claim 8, wherein the value is per second.

11. The method according to claim 8, wherein the time taken to perform step (B) is 180 to 3600 seconds.

12. The method according to claim 1, wherein the first chamber is an inlet / outlet section for cell-containing containers.

13. (C) The method according to claim 1, further comprising the step of performing a cell culture preparation operation on the cells in the cell-containing container obtained in step (B) in the closed second chamber to obtain a cell culture container containing the cells after the operation.

14. The method according to claim 13, wherein the lid of the cell culture vessel obtained in step (C) has a ventilation filter.

15. (D) The method according to claim 13, further comprising the step of incubating the cell culture vessel obtained in step (C) in an incubator.

16. (E) The method according to claim 15, further comprising the step of sealing the lid of the cell culture vessel obtained in step (D) with a sealing material that prevents the intrusion of hydrogen peroxide gas.

17. (F) The method according to claim 16, further comprising the step of exposing the cell culture vessel sealed with the sealing material in step (E) to hydrogen peroxide gas.

18. A method for improving the sterilization efficiency of cell-containing containers, (A) A first chamber having a gas inlet and outlet, A second chamber having a culture preparation operation section is sealed and connected to the first chamber via an opening / closing port. A step of preparing an isolator equipped with, (B) A step of exposing a cell-containing container containing cells that have been frozen and thawed in the closed first chamber to hydrogen peroxide gas. A method that includes the following:

19. The method according to claim 18, which is a method for shortening the sterilization time of a cell-containing container.

20. The method according to claim 18 or 19, wherein step (B) includes presetting sterilization conditions for the cell-containing container with hydrogen peroxide gas in the closed first chamber.

21. A recording medium for recording a computer program for carrying out the method described in any one of claims 1 to 17, or a computer having such a program.

22. A system for manufacturing sterilized cell-containing containers, A first chamber having a gas inlet and outlet, The first chamber is sealed and connected to the second chamber via an opening / closing port, and the second chamber has a culture preparation operation section. A hydrogen peroxide gas inlet / outlet device is sealed and connected to the first chamber, A sterilization control unit connected to the above apparatus controls the sterilization conditions of the cell-containing container with hydrogen peroxide gas in the first chamber. A system that includes these features.

23. The system according to claim 22, wherein the cell-containing container contains frozen and thawed cells.

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