Device for ensuring a sterile environment for the incubation of cell cultures

Abstract

A device for providing a sterile environment for culturing of cell cultures is presented, comprising a heating chamber (100) with heating elements, sensors for temperature, humidity and CO2 and a gas supply providing a gas connection inside. The device further has a culture vessel (110) located in the heating chamber (100). This has a sealable passage (130) for each sensor, and also has a sealable gas inlet and outlet (130) coupled to the gas connection inside the heating chamber (100). The sensors of the heating chamber (100) are arranged to fit into the respective passage (130) of the culture vessel (110). The device further has a water reservoir (120) arranged in the culture vessel (110). The three elements of the heating chamber (100), the culture vessel (110) and the water reservoir (120) can be sterilized using the best method for each. Since the sensors are not part of the culture vessel (110), the culture vessel can also be cleaned at high temperature. The sterilized heating chamber (100) ensures that the cell cultures in the culture vessel (110) are not contaminated from the outside, nor does it cause contamination in the laboratory.

Description

Technical Field

[0001] This invention relates to a device for ensuring a sterile environment for the incubation of cell cultures. This device is primarily used for incubating cell cultures, mainly for medical and research purposes, where the sterility of all elements in contact with the cell cultures and the avoidance of any contamination of these cell cultures are of paramount importance.

[0002] The invention of gene-editing scissors (CRISP / CAS) has opened up possibilities for targeted modification of genetic material. Against this backdrop, various research groups are already developing new cell therapy methods based on the manipulation of genetic material. In the field of human medical treatment, this will involve personalized medicine, where patient material (i.e., the patient's living cells) is incubated in cell cultures before and after gene manipulation. To strictly avoid contamination of this patient material, it is necessary to safely ensure a sterile environment for these cell cultures during the culture phase. Background Technology

[0003] Cell cultures are cultured in an incubator that provides a selectable constant temperature (typically 37°C), adjustable CO2 concentration (e.g., 5%), and high relative humidity. Therefore, the incubator provides ideal growth conditions for any type of cell, i.e., ideal culture conditions for both desirable and undesirable cultures.

[0004] A particularly important component of an incubator is the water supply. Essentially, the water in the incubator has the task of maintaining a relatively high humidity level close to 100%, ensuring that the aqueous nutrient medium containing the cells of the cell culture does not evaporate or evaporates very little. To prevent the growth of cellular biocontaminants in the water reservoir, substances toxic to protozoa are typically added.

[0005] The above issues raise two problems: First, the water can become contaminated and then multiply in the incubator, even if the water itself contains added toxic substances. Second, the combination of relatively high humidity and the optimal temperature for cell growth can stimulate any contamination that may be present in explosive growth, and this contamination can be amplified many times over if the humidity condenses in the incubator.

[0006] In reality, ideal growth conditions in an incubator can not only lead to the proliferation of patient cells, but also inadvertently introduce foreign cells, i.e., impurities or contaminants. This can result in contamination of the actual cell culture and patient materials, rendering the patient materials unusable for treatment, and in the worst case, causing the patient to die from contamination.

[0007] To reduce contamination of the incubator by foreign cells, the interior of a conventional incubator must be sterilized (decontaminated) periodically. This requires maintaining the incubator's internal temperature at approximately 200°C for several hours. In this way, the cell population is strongly suppressed, but not completely eliminated. For example, a 1000-fold reduction in contamination is quite common.

[0008] Another method to reduce the risk of contamination is to place the sample in a separate, sealed container. This container is typically equipped with a breathable membrane to allow for gas and moisture exchange with the atmosphere within the incubator. The membrane itself is usually designed as a sterile filter, which significantly reduces the entry of biological contaminants into the container. However, such a container does not prevent contamination within the incubator itself, and particularly does not prevent contamination on the outer surfaces of such a container. In particular, contaminants can preferentially deposit on the outer surfaces of the sterile filter of such a container, thus not preventing the carryover of these contaminants into the container's interior.

[0009] To monitor or regulate parameters such as temperature or CO2 concentration in an incubator, it is necessary to place sensors inside. However, many of these sensors are sensitive to high temperatures; for example, typical CO2 sensors cannot withstand temperatures above 160°C. Therefore, decontamination of the incubator requires effort, as at least some sensors must be removed beforehand. Furthermore, although these sensors themselves cannot be purified, they still need to be reinstalled inside the incubator, which at least partially negates the purification effect.

[0010] For many current applications in cell biology and medicine, and especially for potential future applications such as the treatment of genetic diseases (e.g., cancer, leukemia, or hereditary disorders), a completely sterile environment in the incubator is essential.

[0011] A system is known from the published document WO 2015 / 172882A1, which aims to improve laboratory safety through mutually self-sufficient microculturers, each microculturer designed for only one sample carrier, specifically to prevent sample mixing and potential contamination. However, this system cannot guarantee complete sterility.

[0012] Publication WO 2011 / 130865 A2 describes an incubator of a largely conventional design, in which, to prevent sample contamination, as many processing steps as possible are intended to be performed within the incubator by machine without human intervention. Specialized devices such as robotic arms are provided for this purpose. However, even under these conditions, complete sterility cannot be guaranteed. Invention Overview

[0014] The object of this invention is to provide a device that allows for improved sterility in the cultivation of cell cultures.

[0015] This objective is achieved through the subject matter of the independent claims. Further advantageous embodiments of the objective of the independent claims are indicated in the sub-claims. The wording of all claims, by reference, becomes part of this specification.

[0016] Unless otherwise disclosed, the use of the singular does not imply the exclusion of the plural, and vice versa.

[0017] To achieve this objective, an apparatus for ensuring a sterile environment for cell culture cultivation is proposed. The apparatus has a sealable and / or sealable heating chamber with at least one heating element. The heating chamber has sensors for temperature and / or humidity and / or CO2, and at least one connection for an external gas supply. The connection is connected to a conduit providing a gas connection inside the heating chamber. The gas supply is typically CO2, but other gases may be provided for specific cell cultures. The apparatus also has a sealable and / or sealable culture vessel located within the heating chamber for containing the cell culture. The culture vessel does not have its own heating system. The culture vessel has a sealable channel for each of the sensors, which communicates with the atmosphere within the culture vessel. The culture vessel also has at least one sealable gas inlet and / or outlet, which is coupled to at least one gas connection inside the heating chamber. The sensors of the heating chamber are arranged to be introduced into the respective sealable channels of the heating chamber when the culture vessel is inserted into the heating chamber. In this way, the sensors of the heating chamber can provide measurement data about the conditions within the culture vessel. The apparatus also has a water reservoir arranged in a culture tank.

[0018] Using this device significantly improves sterilization during cell culture because the three components—the heating chamber, culture vessel, and water reservoir—can be sterilized separately using their respective optimal processes. Since the sensor is not located within the culture vessel, it can be purified at high temperatures (e.g., above 200°C) if necessary. The sterilizing heating chamber ensures that the cell cultures within the culture vessels are not contaminated by external sources or during laboratory contamination.

[0019] A sealable gas inlet and / or outlet can be coupled to a gas port located inside the heating chamber, thereby enabling the maintenance of optimal conditions within the culture vessel. These conditions can be controlled by sensors installed in the sealable diffuser.

[0020] By covering each of the sensors in the heating chamber with a sterile filter, contamination of the cell cultures in the culture vessel by the sensors can be prevented.

[0021] Similarly, contamination of the gas supply can be avoided by the fact that at least one connection of the heating chamber for the gas supply, the pipeline connected thereto, or the gas connection via the pipeline and disposed inside the heating chamber has a sterile filter.

[0022] For the culture container to be inserted into the heating chamber, it is advantageous if the heating chamber has a tightly closed lid or door, wherein the opening of the lid or door has a cross-section larger than the width of the culture container.

[0023] Sensor contamination can also be prevented by ensuring that each sealable channel on the culture vessel has a sterile filter. This design is particularly preferred. It can serve as a replacement for or complement to a sterile filter directly mounted on the sensor in the heating chamber.

[0024] Preferably, contamination of the gas supply can be prevented by at least one sealable gas inlet and / or outlet on the culture vessel having a sterile filter. This can, in turn, serve as a substitute for or supplement to a sterile filter located directly at the gas connection to the heating chamber.

[0025] To ensure that the culture containers are sterilized before use, it is advantageous if the culture containers are disposable components. This can already be manufactured under sterile or cleanroom conditions.

[0026] If the culture vessel has a tightly closing lid or door, it facilitates the introduction of sample carriers along with the cell culture.

[0027] It is particularly advantageous to cover the water reservoir with a semi-permeable membrane. This membrane should be impermeable to water but allow water vapor to pass through. For example, nonwoven fabrics made of high-density polyethylene (PE-HD) have proven ideal for this application. In this way, water can already be added when the reservoir is formed.

[0028] If the semi-permeable membrane of the water reservoir is sealed with a membrane that is impermeable to water and water vapor (such as a plastic membrane) before use, water can be prevented from evaporating from the reservoir prematurely. The membrane is then removed for use.

[0029] If the reservoir contains sterile ultrapure water, then potential contamination of cell cultures via the reservoir becomes particularly unlikely.

[0030] If the water reservoir is a disposable component, the sterility of the water reservoir can be ensured particularly well.

[0031] If a control and / or regulation system exists that can influence conditions in the culture tank based on data from sensors in the heating chamber, the culture conditions in the culture tank can be specifically optimized. Data from the temperature sensor is used to control and / or regulate the heating element in the heating chamber, and data from the CO2 sensor is used to control and / or regulate at least one gas supply. Data from the humidity sensor is used to output warning messages; for example, if the humidity reaches a value that is too low, a warning message may prompt the user to replace the water tank.

[0032] This problem is further addressed by a sealed and / or sealable culture vessel for containing cell cultures. The culture vessel has at least one sealable channel for a sensor in communication with the atmosphere within the culture vessel. Furthermore, the culture vessel has at least one sealable gas inlet and / or outlet coupled to a gas port. Finally, a reservoir is located inside the culture vessel.

[0033] Such culture containers can be easily purified and / or even manufactured under aseptic conditions. Therefore, contamination-sensitive samples can be placed in such containers and then specially protected throughout the incubation process. The culture containers can then be placed in a heating chamber, as previously described. Sealable gas inlets and / or outlets can be coupled to gas connections located inside the heating chamber, thereby maintaining optimal conditions within the culture container. These can be controlled by sensors installed in the sealable channels.

[0034] Therefore, this culture vessel can be used in the apparatus described above.

[0035] Further details and features are provided in the following description of preferred embodiments in conjunction with the accompanying drawings. Various features may be implemented individually or in combination with each other. The possibilities for solving this problem are not limited to the embodiments. For example, the range specification always includes all (unmentioned) intermediate values ​​and all possible sub-intervals. Brief description of the attached diagram

[0037] The figures schematically illustrate examples of embodiments. The same reference numerals in the figures denote the same or functionally identical elements, or elements that correspond to each other in function. Detailed information:

[0038] Figure 1 This is a general schematic diagram of the device according to the present invention;

[0039] Figure 2 This is a schematic diagram of the heating chamber of the device according to the present invention;

[0040] Figure 3 This is a schematic diagram of the culture vessel of the device according to the present invention; and

[0041] Figure 4 This is a schematic diagram of the water storage device according to the present invention. Invention Details

[0043] Figure 1The overall view shows the apparatus used to ensure a sterile environment for the incubation of cell cultures, allowing the relationship between the three main components to be seen. The heating chamber 100 is an external element and contains, in normal operation, a culture container 110, which may vary in size depending on the culture to be cultured. Preferably, the culture container is designed to accommodate sample carriers (e.g., microplates or petri dishes). However, larger designs are, of course, also possible.

[0044] A water reservoir 120 is provided inside the culture container. In the selected embodiment, a connector or connecting element 130 is arranged at the rear of the culture container, connecting the culture container 110 to the heating chamber 100. The following is in conjunction with... Figure 2 and Figure 3 These connectors or connecting elements will be described in more detail.

[0045] Figure 2 Heating chamber 100 is shown. This forms a housing that ensures the required temperature. For this purpose, the heating chamber contains heating element 210. Required sensors, such as temperature, relative humidity, or CO2 content, are also located in heating chamber 100.

[0046] exist Figure 2 For clarity, only the CO2 sensor 220 is shown. This sensor is attached to a retainer 230, which positions the sensor so that it is in the correct position when the culture vessel is inserted. Additionally, at least one connector for gas supply is provided. This port connects to a conduit that provides a gas port 240 inside the heating chamber. Preferably, two such gas ports are provided, one providing, for example, a CO2 supply, and the other providing a gas extraction component.

[0047] Preferably, the door 250 is positioned opposite the sensor and the gas connection and is designed to be airtight. Alternatively, for example, a removable cap may be provided. The opening of the cap or door 250 should have a sufficiently large cross-section to allow the culture container 110 to be inserted without difficulty.

[0048] To avoid contaminating the laboratory environment by bringing the heating chamber 100 into the laboratory, it is advantageous if the heating chamber is cleaned and placed in a package that can be sterilized before being brought into the laboratory. This package consists at least partially of a semi-permeable membrane that is breathable but has sufficient pores to prevent bacteria from passing through. Sterilization can then be performed, for example, by venting with ethylene oxide in a known manner.

[0049] Figure 3A culture container 110 is shown, which is inserted into and can be removed from a heating chamber. The culture container has at least one sealable channel 310 for sensors, which communicates with the atmosphere within the culture container. This channel 310 is preferably covered internally by a sterile filter 320 to protect the culture container from external contamination. Heating chamber sensors are arranged to insert into the respective sealable channels of the culture container 110 when it is inserted into the heating chamber. As a result, they can provide data about the conditions within the culture container 110. The culture container also includes at least one sealable gas inlet and / or outlet 330 adapted to at least one gas port inside the heating chamber. Each of these gas inlets and / or outlets preferably has a sterile filter 340, thereby protecting the culture container 110 from external contamination but also preventing contamination from the culture container to the environment. This may be necessary, for example, if infectious material is present in the cell culture. Preferably, both a gas inlet and a gas outlet are provided in each case, which facilitates gas exchange within the culture container.

[0050] Furthermore, the culture container 110 has an opening through which a sample carrier containing cell culture can be inserted or removed. This opening can be closed by a lid or door 350. Closing the lid or door 350 should ensure that the culture container 110 is airtight relative to the environment. Inside the culture container, a water reservoir 120 is also provided.

[0051] To ensure a sterile environment for culturing cell cultures, the culture container 110 is cleaned and sterilized before use. Advantageously, the culture container 110 is disposable. Components particularly important to prevent contamination, namely the sensor channel 310, gas inlet or outlet 330, and associated sterile filters 320, 340, are part of the culture container. Therefore, they are included in delivery and are sterilized and processed.

[0052] It is also meaningful to provide special packaging (e.g., a semi-permeable membrane) for sterilizing the culture container 110. Sterilization can be performed using various methods, such as gamma irradiation, ethylene oxide venting, or heat treatment.

[0053] For the use of culture container 110 in a cleanroom environment, it is also advantageous if the entire production of the culture container (including aseptic packaging) is carried out in a cleanroom. Then, the finished product (i.e., the culture container 110 in aseptic packaging) can still be provided with cleanroom-compatible outer packaging (e.g., plastic bags).

[0054] exist Figure 4In the diagram, the water reservoir 120 is schematically shown as another component of the device according to the invention. In practice, it is not necessarily cubic. For example, a cylindrical or bubble-shaped design is also conceivable. The water reservoir 120 preferably has a semi-permeable membrane 410, such as a Tyvek membrane, on at least a portion of its surface. The semi-permeable membrane allows gaseous water vapor to escape from the water reservoir into the atmosphere within the culture vessel, but prevents liquid water from leaking from the water reservoir.

[0055] Before the water reservoir is inserted into the culture container, the semipermeable membrane is preferably sealed with membrane 420 (e.g., watertight and airtight plastic membrane). Figure 4 The arrow in the image indicates that the sealing membrane 420 should be removed before inserting the water reservoir.

[0056] Particularly advantageous is if the water reservoir 120 is designed to be inserted separately into the culture container 110 as a disposable water reservoir. In this case, the water reservoir 120 can be filled with water during manufacturing. Sterile ultrapure water is preferred.

[0057] To achieve the goal of providing a sterile environment for cultivation, it is advantageous to externally sterilize the reservoir 120 before use. For this purpose, the reservoir can be placed in packaging for sterilization, such as in a package with a semi-permeable membrane, and within the heating chamber 100 and other components of the culture container 110. Sterilization can be performed using various methods, such as gamma ray irradiation or ethylene oxide venting.

[0058] For the water reservoir 120 to be used in a cleanroom environment, it is also advantageous if the entire production of the water reservoir, including the water and aseptic packaging, is carried out in a cleanroom, and the entire assembly (i.e., the water reservoir 120, the water, the water vapor permeation membrane 410, the seal 420, and the aseptic packaging) is again packaged in a suitable cleanroom container (e.g., a plastic bag).

[0059] Compared with existing technologies, the above-mentioned device has the following advantages:

[0060] Because the gas inlet of the culture vessel is adapted to the heating chamber, there is no gas exchange between the heating chamber and the culture vessel, making it impossible for the sterile filter to be contaminated from the heating chamber area. The gas inlet or outlet only allows gas to flow into the culture tank from the gas connection point.

[0061] Similarly, the gas outlet of the culture vessel is adapted to the heating chamber to prevent gas exchange between the culture vessel and the heating chamber. In this way, it is impossible for the heating chamber to be contaminated from the area of ​​the culture vessel.

[0062] Furthermore, the device according to the invention offers the advantage that the sensors are located outside the culture container and are therefore not contaminated by the samples inside the culture container, as they are separated from the culture container by a sterile filter.

[0063] In addition, using sterile culture containers can prevent sample contamination. This is especially true if the culture containers are sterile, single-use items.

[0064] The use of ultrapure water also eliminates the risk of water contamination, as the ultrapure water is introduced into sterile culture containers within sterile, disposable containers. Even if the relative humidity is too high and water condenses in the culture container, there is no risk because all components are sterilized before use.

[0065] the term

[0066] Incubator

[0067] An incubator is a biological device used to create and maintain controlled outdoor conditions for various developmental and growth processes. It is used to create and maintain a microclimate with strictly controlled humidity and temperature conditions. Incubators are equipped with timers and temperature controllers, and in some cases, settings to regulate the fresh air supply. The set temperature is adjusted to the optimal temperature for the microorganisms to be cultured. CO2 incubators are used for culturing animal cells. (Based on de.wikipedia.org / wiki / Incubator_(Biology))

[0068] microplate

[0069] Microplates (or microplates) are multi-sample carriers. Typically rectangular, microplates are usually made of plastic, but for very specialized applications, they are also made of glass. They contain 6 (2×3) to 1536 (32×48) wells, isolated from each other in rows and columns. The precise dimensions (length × width × height) are 127.76 × 85.48 × 14.35 mm, according to ANSI standards and the Society for Biomolecular Screening (SBS) recommendations. Microplates are used for a wide variety of microbiological operations. Typical applications include screening cell cultures or technical bioreactors. Because of the consistent number of wells and the type of microplate used, microplates are suitable for parallel culture and testing of large numbers of different samples. Due to their standardized size, almost all operations can be automated using suitable robotics. (Based on de.wikipedia.org / wiki / Microtiterplate)

[0070] Sample carrier

[0071] Microbial samples or cell cultures are stored in containers called sample carriers. These containers can be of a wide variety depending on the type of sample. However, petri dishes or microplates are commonly used, and sometimes conical flasks or similar containers are used. Multiple sample carriers, such as microplates, are also referred to as sample carrier systems.

[0072] sterile filter

[0073] In aseptic filtration, microorganisms are separated from the material to be sterilized by filtration. Membranes with pore sizes of 0.1 to 0.22 μm are commonly used as filters. Aseptic filtration is often used for sterilizing heat-sensitive solutions, such as tissue culture solutions containing serum. Major applications include the aseptic filtration of aqueous solutions, heat-sensitive nutrient solutions, vitamin solutions, serum, viral vaccines, plasma fractions, and protein solutions. (Based on https: / / de.wikipedia.org / wiki / Sterilisation#Sterilfiltration)

[0074] Figure Labels

[0075] 100 heating chamber

[0076] 110 culture container

[0077] 120 water storage tank

[0078] 130 Connectors or connecting elements

[0079] 210 heating element

[0080] 220CO2 sensor

[0081] 230 sensor holder

[0082] 240 gas connector

[0083] Door to the 250 heating chamber

[0084] 310 sensor channels

[0085] 320 sensor sterile filter

[0086] 330 Gas Inlet or Outlet

[0087] 340 Sterile filters for gas inlet or outlet

[0088] 350 culture container door

[0089] 410 semi-permeable membrane

[0090] 420 membrane

Claims

1. An apparatus for providing a sterile environment for the culture of cell cultures, comprising: Sealed heating chamber (100); The heating chamber (100) includes at least one heating element (210); The heating chamber (100) includes sensors (220) for temperature and / or humidity and / or CO2. The heating chamber (100) has at least one connection for external gas supply, wherein the connection is connected to a conduit (240) that provides a gas connection inside the heating chamber. Sealed culture container (110) for containing cell cultures; The culture container (110) is located in the heating chamber (100); The culture container (110) has a sealable channel (310) that is in communication with the atmosphere in the culture container of each of the sensors for the heating chamber (100); The culture container (110) includes at least one sealable gas inlet and / or outlet (330), the at least one sealable gas outlet and / or inlet being coupled to at least one gas conduit (240) inside the heating chamber. The sensor (220) of the heating chamber (100) is arranged such that when the culture vessel (110) is inserted into the heating chamber (100), the sensor is inserted into a corresponding sealed channel (310) of the culture vessel (110); and Water storage tank (120); The water reservoir (120) is arranged in the culture container (110).

2. The apparatus according to claim 1, characterized in that, Each of the sensors in the heating chamber (100) is covered by a sterile filter.

3. The apparatus according to claim 1, characterized in that, At least one connector of the heating chamber for gas supply or a gas conduit (240) connected thereto and disposed inside the heating chamber includes a sterile filter.

4. The apparatus according to claim 1, characterized in that, The heating chamber (100) has a tightly closed cover or door (250); The opening of the cover or door (250) has a cross-section that is wider than the culture container (110).

5. The apparatus according to claim 1, characterized in that, Each sealable channel (310) on the culture container (110) includes a sterile filter (320).

6. The apparatus according to claim 1, characterized in that, At least one sealable gas inlet and / or outlet (330) on the culture container (110) includes a sterile filter (340).

7. The apparatus according to claim 1, characterized in that, The culture container (110) is a disposable component.

8. The apparatus according to claim 1, characterized in that, The culture container (110) has a tightly closed lid or a tightly closed door (350).

9. The apparatus according to claim 1, characterized in that, The water reservoir (120) is covered by a semi-permeable membrane (410).

10. The apparatus according to claim 9, characterized in that, The semi-permeable membrane (410) of the water reservoir (120) is sealed with a water- and water vapor-impermeable membrane (420) before use, and the membrane is removed for use.

11. The apparatus according to claim 1, characterized in that, The water reservoir (120) contains sterile ultrapure water.

12. The apparatus according to claim 1, characterized in that, The water storage device (120) is a disposable component.

13. The apparatus according to claim 1, characterized in that, Provide control and / or regulation systems; The control and / or regulation described therein can influence the conditions in the culture vessel (110) based on data from the sensor (220) of the heating chamber (100); The temperature sensor data is used to control and / or adjust the heating element (210) of the heating chamber (100). The data from the CO2 sensor (220) is used to control and / or regulate the at least one gas supply; and The data from the humidity sensor is used to output a warning message.

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