RU2772731C2 - Bioreactor for adhesive cell culture, device for aseptic sampling and method for obtaining cell culture product (options) - Google Patents

Abstract

FIELD: cell biology.

SUBSTANCE: group of inventions relates to a bioreactor, a device and a method for aseptic sampling of solid substrate, and a method for aseptic sampling of substrate for an adhesive cell culture. The bioreactor contains a bioreactor vessel containing a wall separating the space inside and outside the bioreactor vessel. A liquid cultural medium and solid substrate are contained in aseptic state inside the bioreactor vessel. The wall of the bioreactor vessel has a port passing through it, aseptically connected to a case of the sampling device, separating the space outside and inside the case of the sampling device. The case of the sampling device is located on the port so that the space inside the case of the sampling device is in aseptic communication with the space inside the bioreactor vessel. The device for sampling of solid substrate is placed in the space inside the case of the sampling device so that the device for sampling of solid substrate provides the possibility of aseptic sampling of solid substrate contained in the space inside the bioreactor vessel.

EFFECT: device for aseptic sampling of substrate for the adhesive cell culture from the bioreactor allows for an operator to directly assess the density or confluence of cells on substrate without cell culture contamination.

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Description

State of the art

As is well known in large scale cell culture, either suspension cell culture or adherent cell culture is used, see Merten, Otto-Wilhelm et al., Manufacturing Of Viral Vectors For Gene Therapy: Part I. Upstream Processing. Therapy: Part I. Pretreatment). – Pharmaceutical Bioprocessing, 2014, pp.183–203. Suspension cell culture refers to the cultivation of free-floating cells in a liquid medium intended for cell culture. Alternatively, it is possible to culture cells attached to microbeads, which in turn float freely in the medium. In the description of this invention, the term "suspension" cell culture covers both of these approaches to the cultivation of cell cultures.

Alternatively, it is possible to culture cells attached to a solid substrate which is so bulky or dense that it cannot be freely suspended in the culture medium. Such substrates are, for example, ordered synthetic fibers. Alternatively, paper-like fiber sheets can be used, which can be cut into smaller pieces.

Commercial cell culture often requires periodic sampling of the cell culture for analysis. Sampling a suspension cell culture is a relatively simple operation. Since the cells (either by themselves or on microbeads) are suspended in the liquid culture medium, it is sufficient to take a sample of the culture medium with the cells suspended in it. This can be done with a pipette through the sampling port of the bioreactor.

When the cells are cultured attached to a substrate that is not to be suspended, a sample of the liquid culture medium is obtained using a pipette, and not the substrate to which the cells are attached. This makes it possible to evaluate the culture medium by the metabolites of the cell culture, but does not make it possible to directly examine the cultured cells.

A known method of sampling adherent cells by adding protease to the culture medium. It provides separation of adherent cells from the substrate, allowing the cells to float freely in the liquid medium. Thus, a pipette can be used to sample the culture medium and obtain a cell sample. However, the protease affects the cell culture. In addition, the cells released by the protease do not allow obtaining some important data, for example, to determine the degree of confluence of cells attached to a solid substrate. Thus, the protease provides efficient release of adherent cells upon completion of their cultivation, but the use of the protease is unacceptable for sampling adherent cells during their cultivation.

Therefore, there is a need in the art for a method for sampling cells attached to a non-suspendable substrate in an adherent cell culture system that would allow cells to be sampled without significantly affecting and altering remaining (after sampling) cells.

The author found a simple and technological solution to this problem. At the same time, it allows you to maintain an aseptic environment even in large industrial bioreactors for adherent cells. In this document, the term "aseptic" is used to mean "free of unwanted microorganisms".

This application claims priority to U.S. Provisional Application No. 62/419,613, filed November 09, 2016, the contents of which are incorporated herein by reference.

Disclosure of invention

The device comprises an aseptic sampling container or housing, such as a sterile sampling tube, aseptically attached to a port on a cell culture bioreactor vessel. The vessel contains the culture medium and the solid substrate for the adherent cell culture. The aseptic housing contains a device for obtaining a sample of the cell culture substrate with adherent cells attached to it. After sampling, the aseptic body is sealed and detached from the cell culture vessel, so that the vessel remains in an aseptic state after detaching the aseptic body. The cell culture substrate sample contained in the aseptic housing can then be analyzed.

Brief description of the drawings

In FIG. 1 depicts an embodiment of a sampling device, namely a forceps placed under aseptic conditions in a sampling tube from a bioreactor, and illustrates its use.

In FIG. 2 shows another embodiment of the sampling device, namely forceps placed under aseptic conditions in a sterile sample bag.

In FIG. 3 is a photograph of an embodiment in which the tweezers are placed in the sampling tube as shown in FIG. one.

In FIG. 4 is a photograph of tweezers immersed in a bioreactor vessel to access and retrieve solid cell culture substrate.

In FIG. 5 is a photograph of the use of the tweezers placed in the sampling tube by the operator.

In FIG. Figure 6 shows a photograph of the tweezers used to remove a fragment of the substrate for cell culture from the bioreactor.

Implementation of the invention

In this specification, bioreactors refer not only to biological reactors per se, but also to fermenters and tissue culture containers. Bioreactors provide suitable growth conditions for various cultured microorganisms, referred to here as cells. In most cases, it is desirable that only certain types of cells grow in the bioreactor, and the growth of any other cells, such as bacteria, is highly undesirable. Therefore, quite complex measures are taken to protect bioreactors and all used solutions from contamination by cells from the environment.

The bioreactor vessel for culturing an adherent cell culture contains a liquid culture medium in which a solid substrate is located, forming a surface to which cultured cells can attach and grow. Thus, a closed aseptic environment for adherent cell culture is created.

Many different suitable solid substrates are known that are capable of providing a solid support to which cultured cells can attach. Typically, the substrate is a fiber because fibers are inexpensive homogeneous materials and provide a high surface area to volume ratio. In the art, the fiber may be a polymeric fiber such as a polyester or polypropylene fiber. Other fibers can also be used, such as silicate or lactate electrospun nanofibers.

The substrate can be used in the form of ordered or loose fibers. The substrate may also be in the form of a sheet. The leaf substrate may be monolithic. Sheets can be made from fibers in the same way that a sheet of paper is made from cellulose fibers. A variety of substrates and carriers are known in the art to provide a "fixed layer" for culturing an adherent cell culture. In the present invention, they are all largely interchangeable equivalents.

Sampling from bioreactors for cell counting and analysis is of paramount importance in biotechnology. Aseptic collection of cells (or carriers on which cells grow) from fixed bed bioreactors is known to be difficult or impossible, see reference above: Merten et al. (2014). Therefore, cell growth is monitored and evaluated using a biomass sample or by measuring the rate of nutrient uptake or the rate of formation of metabolic products dissolved in the liquid culture medium. However, the distribution of cells along the carrier substrate remains unknown. Thus, it is not possible to determine the confluence of cells and analyze the adherent cells themselves.

This poses a serious problem for some types of cell cultures. For example, when transfecting plasmids, the main factor determining the efficiency and effectiveness of transfection is the local density or confluence of the cells. Instead of actual observations of confluence, the art uses an estimated average number of cells. However, the average number of cells may not reflect the actual pattern of cell growth. Cells, instead of evenly distributed, may grow in dense clusters in one area, but are completely absent in another area.

Thus, there is a need in the art for sampling the solid carrier substrate to which the cells are attached. This would allow a more successful optimization of the process. In particular, this would allow accurate analysis of transfection efficiency, showing where and how successfully the transfection mixture was internalized by the cells. In addition, it would allow a range of cellular assays such as proteomic and gene expression assays to be applied. Thus, the lack of options for sampling from fixed bed bioreactors is a significant disadvantage of industrial biotechnology.

A simple, cost-effective solution was found, while maintaining high accuracy. The essence of the invention is further explained with reference to FIG. one.

In FIG. 1 shows the structure and step-by-step operation of one embodiment of the invention. In FIG. 1 shows a bioreactor vessel (1) for cultivating an adhesive culture filled with a liquid culture medium. In this liquid medium is a solid substrate (2) for cell culture (Fig. 1 shows small strips of fibrous material). Bioreactor vessel (1) contains one or more aseptic ports (3). Commercially available bioreactors typically have multiple ports for, for example, fresh medium inlet, used media outlet, fresh oxygen or other gas supply, and the like.

A housing (4) for the sampling device is aseptically attached to at least one port. As shown in FIG. 1, the body for the sampling device is a flexible tube. It is also possible to use a sterile bag, as shown in Fig. 2, with a sealable opening at the end (8) through which a sampling device (5) can be loaded into the bag, such as forceps, and then the bag (4) can be aseptically sealed. Alternatively, for example, a rigid plastic or glass tube can be used.

The housing (4) for the sampling device contains a device (5) for sampling the solid substrate, which makes it possible to obtain a sample of the solid substrate (2) for adherent cell culture. For example, in FIG. 1 sampling device is a tweezers. In FIG. 2 this device is a tongs. You can also use a rod with a hook on the end, similar to a fishhook. A suction device may also be used to obtain a sample of a solid substrate for an adherent cell culture.

When using tweezers or tongs, it is desirable that the body (4) be flexible enough to allow the operator to manually manipulate the tweezers or tongs by manipulating the body. If the sampling device (5) is made of ferrous metal, then a magnet (7) can be used to move the sampling device (5) inside the housing (4).

The body (4) can be sealed and then detached from the bioreactor. In this case, it is desirable that the seal at the end of the housing that is connected to the bioreactor is easily opened, allowing the operator to withdraw the tweezers through the resulting opening. If necessary, the body (4) may contain a package that provides more space for working with large tools, such as nippers (Fig. 2).

To prepare the device for operation, the housing (4) must be assembled, the sampling device (5) placed in it, the assembly sealed, and then the assembly sterilized, for example, by autoclaving or irradiation. After that, the sterilized hermetically sealed body (4) with the sampling device (5) should be aseptically connected to the port (3) of the bioreactor. This port may be a conventional port provided on a conventional commercially available bioreactor. Alternatively, the bioreactor can be provided with a new port dedicated to sampling the substrate, or more than one such port. This allows the operator to continue to use the available conventional ports for their intended purpose, such as inlet and outlet of culture medium, gas supply, and the like. It is desirable to provide the bioreactor with several ports so that the ports can be tightly closed after use and thereby minimize the risk of contamination. Alternatively, the same port (3) can be used multiple times.

In FIG. 1 also illustrates a method for sampling a solid substrate. To obtain a substrate sample, first attach the housing (4) to the port (A). The port (B) is then opened (if necessary) and the sampling device is inserted through the open port (B) into the bioreactor containing the solid substrate (2) for cell culture. This procedure can be carried out using the magnet (7). Then get a sample of the solid substrate (2) for cell culture using the device (5) for sampling; if tweezers in a flexible tube are used, the sample can be taken by simply squeezing the flexible tube to manipulate the tweezers. Thereafter, the sampling device (5) with the retained sample of the solid substrate (2) for cell culture is returned to the housing through the open port (B), for example by means of a magnet or by manipulating the housing. The body is then isolated (6) from the bioreactor. After that, the device (5) for sampling can be removed from the housing (4) without polluting the bioreactor (1).

Biosecurity is an important aspect of the invention. After isolation (6) of the body (4) from the bioreactor (1), that part of the body (4) in which the device (5) for sampling and the sample (2) is located can be disconnected from the port (3), placed in a fume hood with laminar flow and open in it, providing aseptic 5 extraction of the sample (2). Due to this, the selected material is not subjected to open processing in the laboratory. This eliminates the risk of contamination associated with open processing of the selected material. In addition, open handling of samples is not permitted if the material in the bioreactor poses a health hazard to the operators.

In a clinical production run where the bioreactor produces a biological product intended for human use, antibiotics cannot be used in the culture medium, the bioreactor body must not have a hatch, and regulations prohibit opening the bioreactor. The premise of this invention was that the author ran into these limitations when trying to figure out what was going on with the adherent cell culture in the bioreactor and was forced to find a solution when it was forbidden to open the bioreactor or use antibiotics in its environment to prevent accidental contamination. The author began to work on this idea, intending to use the ports available in the body of a commercially available bioreactor for sterile sampling of the substrate from the reactor. However, these principles also apply to a custom bioreactor with an additional port dedicated to sampling the substrate, or more than one port. The main idea of the invention is to provide a mechanism for aseptic sampling of a solid cell culture substrate without affecting the viability of the entire cell culture.

Given the proposed basic idea, you can easily create other equivalent embodiments of the invention. For example, instead of tweezers, a serrated metal rod can be used, where the serrations allow the sample of fibrous substrate to be hooked and held. Such a serrated tool can be raised and lowered using an external magnet. Alternatively, the body may be a flexible plastic film or bag, with the operator able to hand hold the rod at the distal end through the plastic film, manipulating the rod as needed.

In some bioreactors, the carrier is enclosed within an annular basket. On top of the media layer is covered with a grid and a top grate that holds the bulk of the carrier inside the basket. However, there is a gap between the wall of the basket and the top grate through which samples can be taken from the carrier layer using the tweezers or the hook described above. If such a gap is not available, nippers can be used to create a hole for sampling the media. In this application, it is most practical to use the sampling device housing, which is a tube and bag kit as shown in FIG. 2.

Example 1

Device testing was performed on a conventional iCELLis™ 500 disposable bioreactor. In FIG. Figures 3–6 show photographs taken at different stages of testing. During preparation for testing, the sampling device (in this example, tweezers) was inserted tip first through the first end of the sampling device housing, the diameter of which corresponded to that of the bioreactor port, as shown in FIG. 3. Used ¾" tubing to fit the ¾" vent port. In this example, an MPX adapter was used at the first end of the tube. The second end of the tube may be sealed or provided with an adapter. The sampling device was then autoclaved, fitting the adapters with suitable caps (in this case MPX caps for MPX adapters). After autoclaving the device, the MPX caps were used to aseptically close the housing in a laminar flow fume hood.

During testing, the gas supply to the reactors was stopped for the duration of sampling. The second end of the housing for the sampling device was welded to the vent port. The sampling device was then inserted through the tube into the port as shown in FIG. 4. In the iCELLis™ 500 bioreactor, the sampling device can be guided through the gap between the fixed bed basket and the top grid of the bioreactor and pick up one or more fixed bed carriers. A strong magnet can be used to make picking up the media easier. Lubricant or ethanol can be injected into the carrier container to reduce friction if these are not harmful to the process. The use of a sampling device container made of a thin and flexible material allows the operator to manually move the forceps inside the container as shown in FIG. 5. Successful capture of an adherent cell culture substrate fragment is shown in FIG. 6.

The sampling device, along with the captured carrier or substrate, was then moved from the bioreactor to the housing. In this example, a body of sufficiently long tube was used to allow re-welding of the exhaust tube. In general, it is preferable to retract the sampling device into the housing in which it was previously located. The tube was then sealed and/or a new tube welded into the vent line.

Thus, the tweezers and carrier samples can be aseptically isolated or sealed in the sampling device's holding tube. The design of the device allows this to be done without risk to the biological safety of the operator and without the risk of contamination of the reactor.

The sampler case tube can then be transferred to a laminar flow fume hood and opened there for carrier sample preparation and analysis.

Claims (38)

1. A bioreactor for adherent cell culture, containing a bioreactor vessel having a wall of the bioreactor vessel separating the space outside the bioreactor vessel and the space inside the bioreactor vessel, while: - the space inside the bioreactor vessel provides aseptic maintenance of the liquid culture medium and the solid substrate for the adherent cell culture; - the wall of the bioreactor vessel has a port passing through it, providing communication between the space outside the bioreactor vessel and the space inside the bioreactor vessel; - the port is aseptically connected to the sampler housing dividing the space outside the sampler housing and the space inside the sampler housing, the sampler housing being positioned on the port such that the space inside the sampler housing sampling is in aseptic communication with the space inside the bioreactor vessel; - the space inside the housing for the sampling device allows aseptic placement of the device for sampling the solid substrate, providing a sample of the solid substrate for adherent cell culture contained in the space inside the bioreactor vessel; the solid substrate sampler is positioned in the space within the sampler housing such that the solid substrate sampler enables aseptically obtaining a sample of the adherent cell culture solid substrate contained in the space within the bioreactor vessel. 2. Bioreactor according to claim. 1, in which the sampling device is selected from the group consisting of tweezers, tongs, a suction device and a hook. 3. The bioreactor of claim 2, wherein the sampling device housing comprises a tube and the sampling device comprises tweezers. 4. The bioreactor of claim. 2, in which the case for the sampling device contains a package, and the sampling device contains tongs for removing foreign bodies. 5. The bioreactor of claim. 1, further comprising a magnet located in the space outside the housing for the sampling device and allowing you to move the device for sampling located in the space inside the housing for the sampling device. 6. The bioreactor according to claim 1, further comprising a membrane located between the space inside the bioreactor vessel and the space inside the housing for the sampling device. 7. The bioreactor of claim. 1, in which the housing for the device for sampling provides the possibility of aseptic detachment from the port. 8. Bioreactor according to claim 2, wherein the sampling device comprises a suction device. 9. A device for aseptically obtaining a sample of a solid substrate for an adherent cell culture contained in the interior of the bioreactor vessel, comprising a housing for the sampling device separating the space outside the housing for the sampling device and the space inside the housing for the sampling device, while : - the body for the sampling device allows it to be installed on the port of the bioreactor for adherent cell culture so that the space inside the body for the sampling device can have aseptic communication through the port with the interior of the bioreactor vessel; the space within the body for the sampling device accommodates the solid substrate sampling device to obtain a sample of the solid substrate for the adherent cell culture contained in the space inside the bioreactor vessel; the solid substrate sampler is placed in the space within the sampler housing so that the solid substrate sampler allows aseptic sampling of the solid substrate for the adherent cell culture contained in the space inside the bioreactor vessel. 10. The device of claim 9, wherein the sampling device is selected from the group consisting of tweezers, tongs, a suction device, and a hook. 11. The apparatus of claim. 10, wherein the sampling device housing comprises a tube and the sampling device comprises tweezers. 12. The apparatus of claim. 10, wherein the sampling device housing comprises a bag and the sampling device comprises foreign body forceps. 13. The apparatus of claim. 9, wherein the sampling device comprises a suction device. 14. The method of aseptic sampling of the substrate for adhesive cell culture, including: - providing a bioreactor as described in claim 1, wherein the space within the bioreactor vessel provides aseptic containment of the liquid culture medium and solid substrate for the adherent cell culture, and then - use of a sampling device to aseptically obtain a sample of a solid substrate for adherent cell culture. 15. A method for aseptic sampling of a substrate for an adhesive cell culture, comprising: - providing a bioreactor as described in claim 1, wherein the space within the bioreactor vessel provides aseptic containment of the liquid culture medium and solid substrate for the adherent cell culture, then - use of a sampling device to aseptically obtain a sample of a solid substrate for adherent cell culture, and then - aseptic detachment of the housing for the sampling device from the port. 16. A method for obtaining a cell culture product, including: - providing a bioreactor as described in claim 1, wherein the space within the bioreactor vessel provides aseptic containment of the liquid culture medium and solid substrate for the adherent cell culture, then - culturing the cell culture in a liquid culture medium, then - using a sampling device to aseptically obtain a sample of a solid substrate for adherent cell culture, and then - analysis of the sample for the presence of a cell culture product. 17. A method for obtaining a cell culture product, including: - providing a bioreactor as described in claim 7, wherein the space within the bioreactor vessel provides aseptic containment of the liquid culture medium and solid substrate for the adherent cell culture, then - culturing the cell culture in a liquid culture medium, then - using a sampling device to aseptically obtain a sample of a solid substrate for adherent cell culture, and then - analysis of the sample for the presence of a cell culture product.

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