Bioreactor system for cell culture

Abstract

The present invention relates to a bioreactor system for cell culture. More specifically, the present invention relates to a compact bioreactor system having multiple integrated functions and allowing both small scale static culturing and scale-up rocking culturing in the same bioreactor. The bioreactor system comprises a tray for placing cell culture bags having an adjustable volume, a lid covering the cell culture bags and provided with a heating function, an integrated perfusion unit, an integrated component loading unit, and an integrated unit for automated cell culture sampling, wherein the bioreactor system is controlled by a single control unit. The present invention also relates to a cell culture method for culturing therapeutic cells using the bioreactor system.

Description

Technical Field

[0001] This invention relates to a novel bioreactor system for cell culture. More specifically, this invention relates to a compact bioreactor system with multiple integrated functions, allowing for both small-scale static culture and scale-up rocking culture within the same bioreactor. Background Technology

[0002] Cell therapy is an emerging but rapidly expanding field in biotechnology that involves the administration of autologous or allogeneic cells to achieve therapeutic effects in vivo. The adoptive T-cell transfer protocol in allogeneic hematopoietic stem cell transplantation (HSCT) settings presupposes that the peripheral blood contains T cells capable of acting as a medium to induce antitumor and / or antiviral activity in HSCT receptors.

[0003] Hematopoietic stem cell transplantation (HSCT) is the transplantation of pluripotent hematopoietic stem cells, typically derived from bone marrow, peripheral blood, or umbilical cord blood. It can be autologous (using the patient's own stem cells) or allogeneic (using stem cells from a donor). This is a medical procedure in the field of hematology, most commonly used for patients with certain blood cancers or bone marrow cancers, such as multiple myeloma or leukemia. In these cases, the recipient's immune system is often damaged by radiation or chemotherapy before transplantation.

[0004] Adoptive cell transfer refers to the transfer of cells (most commonly immune-derived cells) back to the same patient or a new recipient host, with the aim of transferring immune function and characteristics to the new host. If possible, using autologous cells helps the recipient by reducing graft-versus-host disease (GVHD).

[0005] In T-cell-based therapies, these cells are expanded in vitro using cell culture methods heavily reliant on the immunomodulatory effects of interleukin-2, and then returned in large quantities intravenously to the patient in an activated state. Anti-CD3 antibodies are commonly used to promote the proliferation of cultured T cells. Studies on interleukin-21 have shown that it may also play an important role in enhancing the efficacy of in vitro-prepared T-cell-based therapies. An emerging therapeutic approach for various diseases is stem cell transfer to achieve therapeutic effects. Clinically, this method has been used to transfer immune-boosting or tolerance-inducing cells (often lymphocytes) to patients to enhance immunity against viruses and cancers, or to improve tolerance in cases of autoimmune diseases such as type 1 diabetes or rheumatoid arthritis. Cells used for adoptive therapy can be genetically altered using recombinant DNA technology to achieve any desired quantity. In the case of T-cell adoptive therapy, one example is the addition of chimeric antigen receptors (CARs) to redirect cytotoxicity and helper T cell specificity.

[0006] Adoptive transfer of autologous tumor-infiltrating lymphocytes (TILs) or genetically re-directed peripheral blood mononuclear cells has been used to successfully treat patients with advanced solid tumors (including melanoma and colorectal cancer) and patients with CD19-expressing hematologic malignancies.

[0007] Tumor-infiltrating lymphocytes (TILs) are white blood cells found in tumors. TILs are involved in killing tumor cells, and the presence of lymphocytes in tumors is generally associated with better clinical outcomes. Numerous clinical trials have used TILs to treat patients with metastatic melanoma (a deadly form of skin cancer). Tumor reduction of 50% or more has been observed in approximately half of melanoma patients treated with TILs. Some patients have experienced complete remission with no detectable tumor for the remaining years after TIL treatment.

[0008] Clinical trials are underway to use TILs to treat gastrointestinal cancers, such as colorectal cancer, and cancers associated with human papillomavirus (HPV), such as cervical cancer. Scientists are also investigating whether TILs can be used to treat other tumors, including those of the lungs, ovaries, bladder, and breast.

[0009] Adoptive T-cell therapy involves the isolation and in vitro expansion of tumor-specific T cells to achieve a greater number of T cells than can be obtained through simple inoculation. These tumor-specific T cells are then infused into a patient with cancer, attempting to enable their immune system to overwhelm the remaining tumor via T cells capable of attacking and killing cancer cells. Several forms of adoptive T-cell therapy are used for cancer treatment; culturing tumor-infiltrating lymphocytes or TILs, isolating and expanding a specific T cell or clone, and even using T cells designed to effectively recognize and attack tumors.

[0010] Recent years have seen signs of success in clinical trials, such as the regulatory approvals of products like Prochymal (Osiris Therapeutics Inc.) and ChondroCelect (TiGenix). However, many new cell therapies treat relatively small numbers of patients, so a universal system for delivering these products from the manufacturing site to clinics has not yet been established. The feasibility of a universal system and the options for delivering cell therapy products to clinics still need to be explored. This also highlights some challenges that remain for the industry in this area, challenges that need to be addressed if these therapies are to be more widely adopted.

[0011] Cell therapy involves many mandatory stages, from cell collection to cell injection into patients. Cell culture for cell therapy is performed in a cleanroom environment. Cell culture and cleanrooms have many mandatory regulations, such as particle size and quantity in the cleanroom, the number of patient samples processed at one time, and the sterile environment and the number of instruments per set.

[0012] Current cell culture systems are stand-alone, require large spaces, and cannot process multiple patient samples simultaneously. The current processes for cell culture used in cell therapy involve significant human intervention, which can contaminate cell cultures and impair cell growth, especially for small-sized cell cultures. Therefore, there is a need for better bioreactor systems. Summary of the Invention

[0013] This invention provides a compact bioreactor that includes a single system to control multiple instruments, which means optimal use of cleanroom space.

[0014] Therefore, in a first aspect, the present invention relates to a bioreactor system (1) for cell culture, comprising a tray (2) for placing a cell culture bag (3) having an adjustable volume, a cover (4) covering the cell culture bag and having a heating function, an integrated perfusion unit (5,6), an integrated element loading unit (8), and an integrated unit for automated cell culture sampling, wherein the bioreactor system is controlled by a single control unit.

[0015] The tray (2) is movable and allows for rocking cell culture at both static and desired rotational speeds. Optionally, multiple, for example, 2-5 trays (2) are present on the bioreactor system and stacked vertically thereon. Each tray can hold at least one cell culture bag. Optionally, the tray is equipped with a barcode reader, and the cell bag is equipped with a barcode.

[0016] The volume of the cell culture bag (3) can be increased from, for example, 50 mL to 3000 mL. This can be done by automatically clamping the cell bag to the desired volume. Clamping can also be done by the cap, as will be described in the details section.

[0017] In a preferred embodiment, a heating element is provided inside the top of the lid (4). When the lid is closed on top of the tray and surrounds the cell culture bag, this forms an incubator-like environment.

[0018] Preferably, the integrated infusion unit includes a media supply bag (5) and a waste bag (6), as well as corresponding conduits for connection to the cell bag (3), and a pump for driving the media into the bag and expelling waste from the bag. The integrated element load unit (8) preferably includes a platform assembled with a compressible pressure sensing element capable of bearing the full weight of the system.

[0019] In one embodiment, the bioreactor system is mobile and includes an attachable trolley (7), thereby making the entire compact bioreactor system mobile.

[0020] In another embodiment, multiple, for example 2-5, bioreactor systems are stacked on top of each other in a vertical direction.

[0021] Multiple bioreactor systems will be controlled by a single control unit, offering the flexibility to be remotely monitored and controlled from a personal computer or mobile device.

[0022] In a second aspect, the present invention relates to a method for cell culture, comprising initiating the culture of a cell culture in a bioreactor under quiescent conditions as described above, wherein the volume of the cell culture bag is set to 50-500 mL and the culture is heated to 37°C, and then, after 1-6 days, preferably 3-4 days, the bag volume is expanded to 1500-3000 mL, and culture is continued at room temperature with a shaking motion, wherein the initiation and continued culture are carried out in the same cell culture bag and on the same bioreactor system. Preferably, the initial cell culture volume is 30-350 mL, and the scale-up culture volume is 500-1500 mL, suitable for cell culture bags with a maximum volume of 2000 mL.

[0023] Preferably, the starting cell culture is selected from stem cells (e.g., human hematopoietic stem cells), immune-derived cells (e.g., T cells or NK cells), tumor-infiltrating cells (TILs), or any other cells suitable for cell therapy. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the bioreactor system of the present invention, wherein the cover or cover of the cell culture bag has been removed.

[0025] Figure 2 For example Figure 1 The diagram shows a bioreactor system, but it includes an attachable trolley.

[0026] Figure 3 For example Figure 2 A schematic diagram of a bioreactor system, in which a cover or shroud is placed on top of a cell culture bag.

[0027] Figure 4 This is a schematic diagram illustrating multiple trays of a bioreactor stacked on top of each other.

[0028] Figure 5 This is a schematic diagram of the integrated element load unit of a bioreactor system.

[0029] Figure 6 A lid is shown that covers a cell bag with a shape suitable for static culture of small cell cultures.

[0030] Figure 7 A cap is shown covering a cell bag with a shape suitable for swing culture of cell cultures on a scale-up basis. Detailed Implementation

[0031] The novel bioreactor of this invention provides both static and rocking cell culture, and users can select the most suitable option for a specific cell culture. For example, static culture typically requires a small amount of starting culture, while rocking culture requires a larger production volume.

[0032] This invention also allows for expansion from small to large volumes by increasing the cell bag volume; that is, a single bag is used for both small-scale and scale-up cultures. A certain volume of a single-size bag is sealed and separated from the remaining volume for small-volume static culture. Once the static culture phase is complete, the sealed small volume is opened, allowing the culture to occupy the entire bag volume for scale-up culture.

[0033] The bioreactor provides, in particular, integrated swinging, perfusion, and media addition, as well as controlled heating for cell culture. A novel heating method is provided that eliminates the loss of small-sized cultures due to evaporation and condensation. Environmental conditions such as temperature and gas within the cell culture bag are maintained and controlled by an incubator-like structure beneath the lid of the bioreactor of this invention. Unlike existing heating pad-type temperature controls that pose a potential threat of cell scalding during the static phase, the bioreactor of this invention provides incubator-like heating by utilizing hot air circulation for heating control, while maintaining air temperature within a closed environment enclosed by the lid covering the cell bags.

[0034] Furthermore, the possibility of remote monitoring and control will reduce the physical administration of the cell culture process and avoid contamination caused by entering the cleanroom.

[0035] Figures 1-2 The bioreactor system 1 is shown in the open state, with the cover or lid 4 removed, and the culture bag 3 and its connection to the medium feed 5 and waste bag 6 are shown. The lid 4 will be placed back on the culture bag 3 on the tray 2 to maintain temperature and limit light penetration onto the culture. The tray 2 is controlled to a stationary or racked state.

[0036] like Figure 2 As shown, the compact bioreactor system can be mounted on a trolley 7 for mobility. The bioreactor can be easily moved inside or outside a cleanroom with the aid of the trolley. The trolley can carry all the necessary accessories and consumables for cell culture.

[0037] Figure 3The diagram shows a bioreactor placed on a trolley with its lid 4 closed. The lid 4 surrounds the cell culture bag 3 and abuts against the tray 2 to form an enclosed space, such as an incubator for the cell culture bag 3. The interior of the lid 4 is equipped with controlled heating, for example, using hot air circulation.

[0038] The bioreactor of this invention can process multiple patient samples at once by stacking trays. For example... Figure 4 As shown, multiple trays (e.g., 2-5) can be arranged in parallel on a first tray, each tray having the same characteristics as the first tray in terms of, for example, rocking ability and cell bag placement. The stacked trays will isolate each sample under independent operating conditions. In the case of stacked trays, a common cap will be placed on all existing trays.

[0039] Preferably, the tray 2 is equipped with a barcode reader, and the cell bag 3 is equipped with a barcode. The cell bag barcode will record patient details, and the tray barcode reader will read the cell bag barcode for the traceability of the patient's cells.

[0040] Compact bioreactors can also be stacked on top of each other as independent instruments, thus taking advantage of vertical space to reduce footprint.

[0041] Furthermore, the bioreactor system of the present invention is equipped with, as follows Figure 5 The integrated element loading unit 8 shown is an example. The integrated element loading unit 8 reduces the overall size of the bioreactor system and facilitates monitoring of quantities during perfusion.

[0042] During cell expansion, cells must undergo an initial static phase of 3-4 days under controlled temperature and gas conditions. In the prior art, the static process is carried out in an incubator, followed by cell expansion during the static phase, where the cultured cells are removed from the incubator and transferred from T-flasks or small bags to larger-volume bags for scale-up. In this invention, static culture is carried out in the smaller volume of cell culture bag 3, and scale-up culture is carried out in the same bag, but a scaled-up version. During static culture, tray 2 remains stationary, and lid 4 is closed. During scale-up culture, tray 2 is in a rocking motion, and lid 4 can be opened or closed as needed.

[0043] exist Figure 6 and Figure 7 The diagram illustrates how the lid 4 controls the volume of the cell culture bag 3. The small volume of the cell culture bag can be controlled by the lid 4, which limits the volume of the cell culture bag 3. Figure 6 As shown, cover 4a can be compared to Figure 7The conventional lid 4 shown has a smaller size. The edge of the lid 4a restricts the volume of the uniformly sized (e.g., 2 liters) bag to a smaller volume, such as 300 mL suitable for static culture. When the lid 4a is closed and placed under pressure relative to the cell bag 3, it will restrict the volume of the cell bag to the desired size. Figure 7 In the above, lid 4 is a standard-sized lid without volume limiting function, suitable for use during the amplification of 2L cultures. If a smaller scale of culture is used, such as 500-1000mL, a lid suitable for that culture volume will be used, i.e., a lid that limits the required culture volume.

[0044] After the static culture phase, removing the cap 4a releases the restricted volume of the bag, allowing it to scale up to 2L. The medium bag 5 fills the cell bag 3 with medium via tubing, and the tray 2 is agitated for cell growth. The waste bag 6 collects waste from the cell bag 3 via tubing during perfusion.

[0045] Monitoring cell growth is crucial during cell culture, primarily based on pH, dissolved oxygen (DO), and cell density. While pH and DO can be monitored using sensors that do not contact the culture, such as traditional optical sensors, cell density requires periodic sample extraction from the culture. Current bioreactors integrate automated sampling subsystems to collect and separate individual samples.

Claims

1. A bioreactor system (1) for cell culture, comprising a tray (2) for holding a cell culture bag (3) having an adjustable volume, the cell culture bag having a removable lid having a top and an interior, an integrated perfusion unit, an integrated element loading unit (8), and an integrated unit for automated cell culture sampling, wherein the bioreactor system is controlled by a single control unit, and wherein the top of the lid has an enclosed heating control system inside, wherein hot air is circulated in the enclosed environment; in, During the static culture phase, the lid restricts the volume of the cell culture bag, and after the static culture phase, the restricted volume of the cell culture bag is released by removing the lid.

2. The bioreactor system according to claim 1, characterized in that, The tray (2) is movable and allows for rocking cell culture at both static and desired rpm.

3. The bioreactor system according to claim 1, characterized in that, Multiple trays (2) are present on the bioreactor system and stacked vertically thereon.

4. The bioreactor system according to claim 1, claim 2, or claim 3, characterized in that, The volume of the cell culture bag (3) can be increased from 50 mL to 3000 mL.

5. The bioreactor system according to any one of claims 1-3, characterized in that, The integrated infusion unit includes a media supply bag (5) and a waste bag (6), as well as corresponding pipes for connecting to the cell culture bag (3) and a pump for driving the media into the cell culture bag and expelling waste from the cell culture bag.

6. The bioreactor system according to any one of claims 1-3, characterized in that, The bioreactor system is mobile and includes an attachable trolley (7).

7. The bioreactor system according to any one of claims 1-3, characterized in that, The integrated element load unit (8) includes a platform assembled with a compression-type pressure measuring element that bears the entire weight of the system.

8. The bioreactor system according to any one of claims 1-3, characterized in that, The tray is equipped with a barcode reader, and the cell culture bag is equipped with a barcode.

9. The bioreactor system according to any one of claims 1-3, characterized in that, Multiple bioreactor systems are stacked on top of each other in a vertical direction.

10. The bioreactor system according to claim 9, characterized in that, The multiple bioreactor systems will be controlled by a single control unit, providing the flexibility to be remotely monitored and controlled from a personal computer or mobile device.

11. The bioreactor system according to claim 3, characterized in that, Two to five trays (2) are present on the bioreactor system and stacked vertically thereon.

12. The bioreactor system according to claim 9, characterized in that, Two to five bioreactor systems are stacked on top of each other vertically.

13. A method for cell culture, comprising initiating the culture of a cell culture in a bioreactor according to any one of claims 1-12 under quiescent conditions, wherein, The cell culture bag is set to a volume of 50-500 mL and the culture is heated to 37°C. Then, after 1-6 days, the bag volume is expanded to 1500-3000 mL and cultured at room temperature with a rocking motion for scale-up of the culture. The initial and scale-up cultures are carried out in the same cell culture bag and on the same bioreactor system.

14. The method according to claim 13, characterized in that, The starting cell culture is selected from stem cells, immune-derived cells, tumor-infiltrating cells (TILs), or any other cells suitable for cell therapy.

15. The method according to claim 13, characterized in that, Increase the bag volume to 1500-3000 mL after 3-4 days.

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