Cell culture chamber having an improved cell contact surface

Abstract

The present disclosure provides cell culture chambers for use in automated cell engineering systems, particularly cell culture chambers comprising improved cell contact surfaces. The improved cell contact surfaces may include surface coatings that promote improved cell growth, adhesion, differentiation, phenotypic maintenance, and / or transduction, cell contact surfaces comprising non-porous, gas-permeable materials, and other modifications to the cell contact surfaces. Cassettes comprising the cell culture chambers are also provided.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 925,392, filed Oct. 24, 2019, the disclosure of which is hereby incorporated by reference in its entirety.

[0002] [Technical Field] The present disclosure provides a cell culture chamber for use in an automated cell engineering system, specifically, a cell culture chamber including an improved cell contact surface. The improved cell contact surface may include a surface coating that promotes improved cell growth, adhesion, differentiation, phenotype maintenance, and / or transduction, a cell contact surface including a non - porous gas - permeable material, and other modifications to the cell contact surface. A cassette including the cell culture chamber is also provided.

[0003] [Background Art] As the clinical introduction of advanced cell therapies is expected to accelerate, attention has been focused on the fundamental manufacturing strategies for these therapies to benefit patients worldwide. Cell therapies have great clinical potential, but due to high manufacturing costs compared to reimbursement, they pose high barriers to commercialization. For this reason, due to the need for cost - effectiveness, process efficiency, and product consistency, efforts for automation in many cell therapy fields, particularly in T - cell immunotherapy, are being promoted.

[0004] To transfer these important immunotherapies to a broad patient population, it is essential to incorporate cell activation, transduction, and expansion into a commercial manufacturing platform. To make these life-saving treatments applicable to patient populations worldwide, it is necessary to effect changes in manufacturing technology to support made-to-order medicine. The advantages of automation include labor time savings associated with automation, as well as improvements in product consistency, reduced room partitioning, reduced cleanroom footprint, reduced complex training, and improved scale-up and logistics tracking. Furthermore, by using automatically generated electronic batch records to provide a history of all processing equipment, reagents, patient identification, operator identification, in-process sensor data, etc., the documentation process can be rationalized using software.

[0005] What is needed to advance these therapies and automation systems are components of a cell expansion system, such as a cell culture chamber, that increase cell output or provide desired cell characteristics. This application meets these needs.

[0006] [Summary of the Invention] In some embodiments, provided herein is a cell culture chamber for use in an automated cell engineering system, the cell culture chamber comprising a flat, non-flexible chamber having a low chamber height and a cell contact surface, at least a portion of the cell contact surface comprising a non-porous gas-permeable material.

[0007] In a further embodiment, a cell culture chamber for use in an automated cell engineering system, the cell culture chamber comprising a flat, non-flexible chamber having a chamber height of from about 0.5 cm to about 4 cm and a cell contact surface, at least 50% of the cell contact surface comprising a non-porous gas permeable material comprising silicone, fluorinated ethylene propylene (FEP), or ethylene vinyl olefin (EVO), the cell culture chamber further comprising at least one of a distal port configured to allow removal of air bubbles from the cell culture chamber and / or configured as a recirculation port, a central port configured to function as a recirculation inlet port, and a proximal port configured to function as a drain port for cell removal. The cell culture chamber is provided herein.

[0008] Also provided herein is a cassette for use in an automated cell engineering system, the cassette comprising a high temperature chamber for performing activation, transduction, and / or expansion of cell cultures, the high temperature chamber comprising a cell culture chamber, and one or more fluidic pathways connected to the cell culture chamber, the fluidic pathways providing recirculation, waste removal, and homogeneous gas exchange and nutrient distribution to the cell culture chamber without disturbing the cells within the cell culture chamber, the cell culture chamber being a flat, non-flexible chamber having a low chamber height and a cell contact surface, the cell culture chamber being maintained in a substantially planar orientation within the cassette, and at least a portion of the cell contact surface comprising a non-porous gas permeable material.

[0009] In additional embodiments, there is provided a cell culture chamber for use in an automated cell engineering system, the cell culture chamber comprising: a flat and non-flexible chamber having a low chamber height; and a surface coating on the chamber, the surface coating on the chamber being selected from the group consisting of a surface coating that activates cells, a surface coating that modulates biological pathways within cells, a surface coating that enhances cell growth, a surface coating that improves cell adhesion, a surface coating that inhibits cells, a surface coating that responds to media conditions, and a surface coating having a controlled solubility.

[0010] In further embodiments, there is provided a cassette for use in an automated cell engineering system, the cassette comprising: a high-temperature chamber for performing activation, transduction, and / or expansion of cell cultures, the high-temperature chamber comprising a cell culture chamber; and one or more fluid pathways connected to the cell culture chamber, the one or more fluid pathways providing recirculation, waste removal, and homogeneous gas exchange and nutrient distribution to the cell culture chamber without disturbing the cells within the cell culture chamber, wherein the cell culture chamber is a flat and non-flexible chamber having a low chamber height, the cell culture chamber is maintained in a substantially planar orientation within the cassette, and the cell culture chamber has a surface coating selected from the group consisting of a surface coating that activates cells, a surface coating that modulates biological pathways within cells, a surface coating that enhances cell growth, a surface coating that improves cell adhesion, a surface coating that inhibits cells, a surface coating that responds to media conditions, and a surface coating having a controlled solubility.

[0011] Also provided herein is a cassette for use in an automated cell engineering system, the cassette comprising: a high temperature chamber for performing activation, transduction, and / or expansion of cell culture, the high temperature chamber comprising a cell culture chamber, the cell culture chamber being a flat and non-flexible chamber having a low chamber height, the cell culture chamber being maintained in a substantially planar orientation within the cassette; one or more fluid pathways connected to the cell culture chamber, the fluid pathways providing recirculation, waste removal, and homogeneous gas exchange and nutrient distribution to the cell culture chamber without disturbing the cells within the cell culture chamber; and a fluid pathway connected to the cell culture chamber configured to introduce a surface coating material to the cell culture chamber, the surface coating material being selected from the group consisting of a surface coating material that activates cells, a surface coating material that modulates biological pathways within cells, a surface coating material that enhances cell growth, a surface coating material that improves cell adhesion, a surface coating material that inhibits cells, a surface coating material that responds to media conditions, and a surface coating material having a controlled solubility. BRIEF DESCRIPTION OF THE DRAWINGS

[0012]

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[0013] [Mode for Carrying Out the Invention] The production of genetically engineered cells containing CAR T cells typically requires manual involvement due to the product being patient - specific. The automation of CAR T cell culture has been particularly difficult due to multiple sensitive unit operations including cell activation, transduction, and expansion. For this reason, an automated method for producing CAR T cells using a fully - enclosed cell engineering system, as well as components for use in such a cell engineering system, particularly a cell culture chamber, are described herein.

[0014] Automated Cell Processing For the production of genetically engineered cells, including autologous cell therapies such as T cell therapy, the need for cost - effectiveness, process efficiency, and product consistency is particularly acute, as the production of micro - lot (1 patient per lot) batches lacks the economies of scale available in allogeneic (multiple patients per lot) processes. The more extensive and localized labor force and facilities required for micro - lots pose significant demands on logistics for manual production and compliance with GMP, particularly with regard to personnel availability and training. Additionally, the potential for variability in techniques between operators can introduce undesirable risks in consistently meeting release criteria and ensuring safe and reliable products.

[0015] As described herein, the installation and comprehensive validation of automated manufacturing provide solutions to these logistics and operational challenges. An important approach for introducing automation into the production process is for the operator to identify the major modular steps that apply physical or chemical changes to the production materials, termed "unit operations". In the case of cell manufacturing, this includes steps such as cell separation, genetic engineering, expansion, washing, concentration, and cell harvesting. Manufacturers often view the bottlenecks of the focal process as immediate opportunities for introducing automation. This is reflected in the technical operating spectra of most commercially available bioreactors, which tend to focus on individual process steps. The process challenges in cell manufacturing, from maintaining sterility to sample tracking, are addressed herein by full automation that produces consistent cell output while reducing inevitable process variability. The methods described herein also provide simplification and support compliance with GMP standards through associated electronic records.

[0016] Although specific protocols can vary with respect to the production of T cells, a generalized chimeric antigen receptor T cell (CAR T) process is shown in FIG. 1A - 1B as follows. FIG. 1A - 1B illustrates the unit operations of CAR T cell manufacturing from the initial processing of a patient blood sample to the formulation of the output cells for autologous T cell therapy.

[0017] As described herein, to achieve cell manufacturing automation, an understanding of the cell state at each transition point and how they are affected by specific unit operations is obtained by the methods described herein. Micro-lot production for patient-specific therapies must respect the major process sensitivities that affect the feasibility of automation. The automation described herein successfully accepts various process steps.

[0018] Table 1 below highlights the challenges of some of the process steps identified for T cell automation and describes the impact of sensitivity on the automation strategy. For all unit operations, due to the risk of contamination, the open movement of cells between respective devices is an important sensitivity.

[0019]

Table 1-1

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[0020] By adjusting the automation of manual processes around the sensitivities listed in Table 1, the normal conversion, maintenance, or improvement of cell therapy performance can be assisted.

[0021] A single all-in-one system provides significantly higher space efficiency and can minimize the installation area required in an expensive GMP cleanroom. For example, as shown in Figure 2, a fully integrated automation system is designed to reduce the expensive GMP cleanroom space by making the most of the required installation area. Figure 2 shows 96 patient-specific full units executed in a standard laboratory space.

[0022] Methods are described herein for sensing culture conditions and responding as a high-performance bioreactor by controlling factors such as physical agitation, pH, feeding, and gas handling. Further, compared to allogeneic therapy, there are significantly different challenges associated with autologous therapy technology transfer. Autologous products may have more restrictions on stability between the manufacturing process and patient treatment. The site can be located on a global scale rather than a single center. By locking down (e.g., fully enclosing) the all-in-one system, the technology transfer process between sites is significantly improved.

[0023] As described herein, in embodiments, the methods and components described herein utilize the COCOON platform (Octane Biotech (Kingston, ON)) that integrates multiple unit operations in a single turnkey platform (see, e.g., U.S. Patent Application Publication No. 2019 / 0169572, which is hereby incorporated by reference in its entirety). However, other fully or partially automated cell culture devices may be used in accordance with the embodiments herein, and these devices include commercially available ones such as PRODIGY available from Miltenyi Biotech, Inc., XURI and SEFIA from General Electric Healthcare, and systems available from Atvio Biotech Ltd.

[0024] CAR T cells were expanded (including activation, viral transduction and expansion, concentration, and washing) within a fully integrated closed automated system using the methods and devices described herein (Figure 3).

[0025] In an exemplary embodiment, the methods and systems described herein produce at least about 50 million viable genetically modified immune cells. In a preferred embodiment, the methods described produce at least about 100 million viable genetically modified immune cells, or at least about 200 million cells, at least about 300 million cells, at least about 400 million cells, at least about 500 million cells, at least about 600 million cells, at least about 700 million cells, at least about 800 million cells, at least about 1 billion cells, at least about 1.1 billion cells, at least about 1.2 billion cells, at least about 1.3 billion cells, at least about 1.4 billion cells, at least about 1.5 billion cells, at least about 1.6 billion cells, at least about 1.7 billion cells, at least about 1.8 billion cells, at least about 1.9 billion cells, at least about 2 billion cells, at least about 2.1 billion, at least about 2.2 billion, at least about 2.3 billion, at least about 2.4 billion, at least about 2.5 billion, at least about 2.6 billion, at least about 2.7 billion, at least about 2.8 billion, at least about 2.9 billion, or at least about 3 billion genetically modified immune cells.

[0026] The methods and systems described herein can also be used for the production of stem cells, including pluripotent stem cells, hematopoietic stem cells, or mesenchymal stem cells. In an exemplary embodiment, the methods and systems described herein produce at least about 50 million viable stem cells. In a preferred embodiment, the described methods produce at least about 100 million viable stem cells, or at least about 200 million cells, at least about 300 million cells, at least about 400 million cells, at least about 500 million cells, at least about 600 million cells, at least about 700 million cells, at least about 800 million cells, at least about 1 billion cells, at least about 1.1 billion cells, at least about 1.2 billion cells, at least about 1.3 billion cells, at least about 1.4 billion cells, at least about 1.5 billion cells, at least about 1.6 billion cells, at least about 1.7 billion cells, at least about 1.8 billion cells, at least about 1.9 billion cells, at least about 2 billion cells, at least about 2.1 billion, at least about 2.2 billion, at least about 2.3 billion, at least about 2.4 billion, at least about 2.5 billion, at least about 2.6 billion, at least about 2.7 billion, at least about 2.8 billion, at least about 2.9 billion, or at least about 3 billion stem cells.

[0027] Cell engineering system The components of a fully enclosed cell engineering system 400 (also referred to as an automated cell engineering system) (see FIGS. 4A and 4B) are described herein, and preferably, thereon, there is an explanation for performing activation, transduction, expansion, concentration, and collection steps. A cell engineering system for the automated production of genetically modified immune cells including CAR T cells is described herein, which is also referred to throughout as an automated cell engineering system, COCOON, or COCOON system. For example, a user can provide a cell engineering system pre-filled with cell culture and reagents (such as activation reagents, vectors, cell culture media, nutrients, selection reagents, etc.) and parameters for cell production (such as the number of starting cells, type of medium, type of activation reagent, type of vector, number of cells or doses produced, etc.), and this cell engineering system can execute a method for producing various cells including stem cells and genetically modified immune cell cultures including CAR T cells without further input from the user. At the end of the automated production process, this cell engineering system may notify the user to collect the produced cells (for example, by playing a notification message or sending a mobile app notification). In some embodiments, this fully enclosed cell engineering system includes a sterile cell culture chamber. In some embodiments, this fully enclosed cell engineering system minimizes contamination of the cell culture by reducing exposure of the cell culture to a non-sterile environment. In additional embodiments, this fully enclosed cell engineering system minimizes contamination of the cell culture by reducing handling of the cells by the user.

[0028] As described herein, this cell engineering system preferably includes a cassette 402. For this reason, in embodiments, a cassette for use in an automated cell engineering system is provided herein. As used herein, "cassette" refers to a substantially self-contained, removable, and replaceable element of a cell engineering system that includes one or more chambers for performing various elements of the methods described herein and preferably also includes one or more of cell culture media, activation reagents, vectors, etc.

[0029] Figure 4B shows an embodiment of the cassette 402 according to an embodiment of the present invention. In an embodiment, the cassette 402 optionally includes a cryogenic chamber 404 for storage of cell culture medium and preferably includes a high-temperature chamber 406 for performing activation, transduction, and / or expansion of immune cell cultures. Preferably, the high-temperature chamber 406 is separated from the cryogenic chamber 404 by a thermal barrier. As used herein, a "cryogenic chamber" refers to a chamber that is maintained at a refrigeration temperature, lower than room temperature, more preferably at about 4°C to about 8°C, preferably for maintaining cell culture medium. The cryogenic chamber may include a bag or other holder for the medium containing about 1L, about 2L, about 3L, about 4L, or about 5L of fluid. Additional medium bags or other fluid sources may be externally connected to the cassette and connected to the cassette via an access port.

[0030] As used herein, a "high-temperature chamber" refers to a chamber that is preferably maintained at a temperature higher than room temperature, more preferably at a temperature that enables cell proliferation and growth, i.e., about 35 - 40°C, more preferably at about 37°C.

[0031] In an embodiment, the high-temperature chamber 406 preferably includes a cell culture chamber 410 (also referred to throughout as a growth chamber or cell growth chamber) as shown in FIGS. 4D and 4E.

[0032] This cassette further includes one or more fluid pathways connected to the cell culture chamber, and this fluid pathway (fluid pathway 408 in the cartridge 402 of FIG. 4B) provides recirculation, waste removal, and homogeneous gas exchange and nutrient distribution to the cell culture chamber without disturbing the cells within the cell culture chamber. The cassette 402 also further includes one or more pumps 405 including a peristaltic pump for driving fluid through the cassette as described herein, and one or more valves 407 for controlling the flow through the various fluid pathways.

[0033] In an exemplary embodiment, as shown in FIG. 4D, the cell culture chamber 410 is a flat and non-flexible chamber 470 that does not easily bend or flex (i.e., made of a substantially non-flexible material such as plastic). The use of a non-flexible chamber makes it possible to maintain the cells in a substantially undisturbed state. As shown in FIG. 4E, the cell culture chamber 410 is oriented such that the cell culture (preferably, an immune cell culture) can spread across the entire bottom 412 of the cell culture chamber. As shown in FIG. 4E, the cell culture chamber 410 is preferably maintained in a substantially flat position, i.e., parallel to the floor or table, to keep the cell culture undisturbed and allow the cell culture to spread over a wide area of the bottom 412 of the cell culture chamber. As used herein with respect to the cell culture chamber, "flat" means that the bottom 412 (as well as the top and sides) of the chamber has a deflection or warp of less than about 5°, preferably about 0° to about 4°, or about 3° in the shape of the chamber (from a completely flat surface or plane). This slight warp allows for uniform seeding and growth of cells on the bottom 412, but provides an angle sufficient to raise and remove air bubbles to the highest point.

[0034] In an embodiment, the total thickness of the cell culture chamber 410 (i.e., the chamber height 442) is low, on the order of about 0.5 cm to about 5 cm. Preferably, the cell culture chamber has a volume of about 0.50 ml to about 500 ml, about 0.50 ml to about 300 ml, more preferably about 50 ml to about 200 ml, or the cell culture chamber has a volume of about 180 ml. The use of a low chamber height 442 (less than 5 cm, preferably less than 4 cm, less than 3 cm, or less than 2 cm, or about 0.5 cm to about 4 cm, about 0.5 cm to about 3 cm, about 0.5 cm to about 2 cm, or about 1 cm to about 2 cm, or about 1 cm to about 3 cm, or about 2 cm to about 3 cm) allows for effective medium and gas exchange close to the cells. The ports are configured to allow mixing via fluid recirculation without disturbing the cells. Taller static vessels can create concentration gradients, which can limit oxygen and fresh nutrients in the area near the cells. By controlling the hydrodynamics, medium exchange can be performed without disturbing the cells. The medium can be removed from an additional chamber (where there are no cells) without risk of cell loss.

[0035] In an embodiment, a cell culture chamber 410 for use in an automated cell engineering system 400 is provided herein, the cell culture chamber comprising a flat and non-flexible chamber 470 having a low chamber height 442 and a cell contact surface 480, at least a portion of the cell contact surface including a non-porous gas permeable material 482.

[0036] In an exemplary embodiment, the cell contact surface 480 refers to the bottom 412 of the cell culture chamber 410. However, the "cell contact surface" can also include additional elements within the cell culture chamber 410, such as various scaffolds, supports, growth platforms, etc. The "cell contact surface" can also refer to one or more sides 416 of the cell culture chamber 410. The cell engineering system 400 described herein has the ability to tilt and rotate the cell culture chamber 410, during which additional components of the chamber can become the cell contact surface as the cells move within the chamber. The cell contact surface can also include a plurality of structures that are cut or fabricated into the surface to create channels, waves, or other structures to increase cell adhesiveness and add surface area.

[0037] In a further embodiment, the non-cell contact surface of the culture chamber 410 can also include a non-porous gas permeable material 482. Preferably, in addition to the bottom 412 of the cell culture chamber 410 that includes the non-porous gas permeable material 482, the top 414 of the cell culture chamber 410 also includes the non-porous gas permeable material 482.

[0038] As used herein, a "non-porous gas permeable material" means any composition, film, or material used in a gas permeable cell culture device that allows gases to enter through the cell culture chamber 410 but does not contain pores or holes that would allow the passage or leakage of liquids (e.g., cell culture medium). Exemplary non-porous gas permeable materials include, but are not limited to, silicone, fluorinated ethylene propylene (FEP), polyolefin, ethyl vinyl olefin (EVO), and ethylene vinyl acetate copolymer. The non-porous gas permeable materials described herein are preferably useful for delivering one or more gases, including oxygen, nitrogen, CO2, etc., to the cells within the cell culture chamber 410.

[0039] In an embodiment, the non-porous gas-permeable material 482 does not permit evaporation of water from the cell culture chamber 410. This can be achieved either by selection of a suitable material that restricts or eliminates evaporation of water, or by use of a water layer that is above the cell culture medium within the cell culture chamber 410 but below the gas-permeable material 482 included in the upper portion 414 of the cell culture chamber 410. In other embodiments, two or more different gas-permeable materials 482 can be utilized, one material at the upper portion 414 of the cell culture chamber 410 and a different material at the bottom 412.

[0040] In an exemplary embodiment, “a portion” of a non-cell contact surface such as the cell contact surface 480 or the upper portion 414 includes a non-porous gas-permeable material. As used herein, “a portion” refers to at least about 20% of the cell contact surface or non-cell contact surface that is composed of the non-porous gas-permeable material. That is, at least about 20% of the surface designed to contact cells (preferably the bottom 412 of the cell culture chamber), or other scaffolds, supports, or structures, or non-cell contact surfaces such as the upper portion 414, is made from a non-porous gas-permeable material as a major structural element of the surface. In embodiments where less than 100% of the cell contact surface is made from a non-porous gas-permeable material, the remainder of the cell contact surface can include other suitable materials including various plastics (e.g., polypropylene, polystyrene, etc.) that promote cell adhesion and growth. In embodiments where less than 100% of the non-cell contact surface is made from a non-porous gas-permeable material, the remainder of the non-cell contact surface can include other suitable materials including various plastics (e.g., polypropylene, polystyrene, etc.) that promote structural support.

[0041] In an embodiment, at least about 30% of the cell contact surface is composed of a non-porous gas-permeable material, more preferably at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% (i.e., the entirety) includes a non-porous gas-permeable material.

[0042] In an embodiment, at least about 30% of the non-cell contact surface is composed of a non-porous gas permeable material, more preferably at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% (i.e., the whole) of the non-cell contact surface comprises a non-porous gas permeable material.

[0043] Figures 4G - 4H show schematic exemplary structures where less than the entire cell contact surface (in this case, the bottom 412 of the cell culture chamber 410) comprises a non-porous gas permeable material. In such an embodiment, the cell culture chamber comprises a cell contact surface 480 that includes a plurality of distinct sections, and each section comprises a non-porous gas permeable material 482. For example, the holes or distinct sections of the cell contact surface 480 can comprise the non-porous gas permeable material 482, while other portions of the bottom 412 comprise other suitable materials, including various plastics that promote cell adhesion and growth. These holes or sections can be regular or irregular in shape and can be of any size or number, depending on the desired coverage of the cell contact surface 480 by the non-porous gas permeable material 482. The chamber 470 is depicted as having a cylindrical shape, but other shapes can be readily envisioned, and the shapes in Figures 4G - 4H are provided for illustrative purposes only.

[0044] In a further embodiment, one or more sides of the cell culture chamber 410 can be provided with at least a portion comprising the non-porous gas permeable material 482.

[0045] In yet a further embodiment, additional components of the cassette 402, such as, for example, the pump 405, the valve 407, and the fluid path 408, can also comprise the non-porous gas permeable material 482 that can assist in transferring gas to the cells as it passes through the cassette 402.

[0046] The silicone used herein is a synthetic polymer made of repeating units of siloxane, combined with carbon, hydrogen, and sometimes other elements. As detailed herein, using silicone as a non-porous gas-permeable material in a cell culture chamber increases gas exchange. Silicone has excellent oxygen permeability, enables optical observation, is not easily punctured, and can be easily processed into a wide variety of shapes.

[0047] In an exemplary embodiment, the thickness of the non-porous gas-permeable material is less than about 0.5 inches, more preferably less than about 0.2 inches, less than about 0.1 inches, less than about 0.05 inches, or about 0.010 - 0.050 inches, about 0.010 inches, about 0.020 inches, about 0.030 inches, about 0.040 inches, or about 0.050 inches.

[0048] In a further embodiment, the non-porous gas-permeable material can be stretched to make it thinner, thereby increasing gas exchange through the material. This stretching can be performed before the material is integrated into the structure of the cell culture chamber 410, or the material can be placed under tension during the preparation of the cell culture chamber 410 or while the material is being included in the cell culture chamber, as a result of which the non-porous gas-permeable material is stretched.

[0049] Exemplary sources of silicone and silicone-based materials include TEFLON®, as well as WO01 / 92462, U.S. Patent Nos. 4,939,151, 6,297,046, and 9,255,243, the disclosures of each of which are hereby incorporated by reference in their entirety.

[0050] In an exemplary embodiment, the cell contact surface may further include a surface coating on the surface selected from the group consisting of a surface coating that activates cells, a surface coating that regulates biological pathways within cells, a surface coating that enhances cell growth, a surface coating that increases the transduction efficiency of cells, a surface coating that improves the selection for a particular type of cell, a surface coating that improves cell adhesion, a surface coating that inhibits cells, a surface coating that responds to media conditions (e.g., color change as an aid for monitoring), and a surface coating that has a controlled solubility (e.g., for obtaining a controlled release of the coating content). Thus, in an embodiment, the non-porous gas-permeable material that preferably constitutes part or all of the cell contact surface may further include the surface coating described herein. In other embodiments, the remainder of the cell contact surface that does not include the non-porous gas-permeable material may also include the surface coating described herein. In other embodiments, the entire cell contact surface does not include the non-porous gas-permeable material but still includes the surface coating described herein. Cell adhesion can also provide an opportunity for cell selection that promotes the growth of the desired population while not allowing the adhesion of the undesired population.

[0051] As used herein, "surface coating" refers to a material that forms a film, layer, or coating on one or more surfaces of the cell culture chamber 410. Methods for applying the surface coating to the surface of the cell culture chamber may include, for example, deposition methods such as painting, dip coating, layering, flowing, spraying, spin coating, and the like.

[0052] In an exemplary embodiment, the surface coating is preferably provided on the bottom 412 of the cell culture chamber 410, which preferably includes the cell contact surface 480 and the non-porous gas-permeable material 482. However, the surface coating can also be provided on the top 414 and / or the side 416 of the cell culture chamber (see FIG. 4F).

[0053] As used herein, "a surface coating that activates cells" refers to a material, substrate, or component that causes cells to proliferate and / or differentiate.

[0054] As used herein, "a surface coating that modulates a biological pathway in a cell" refers to a material, substrate, or component that causes one or more actions among molecules in a cell, resulting in a particular product or change in the cell. For example, such a surface coating can induce the assembly of new molecules such as fats or proteins, turn genes on and off, or cause cells to move.

[0055] As used herein, "a surface coating that enhances cell growth" refers to a material, substrate, or component that causes cell growth to occur faster or in greater numbers than in the absence of the material.

[0056] As used herein, "a surface coating that improves cell adhesion" refers to a material, substrate, or component that causes cells to interact better with the surface, adhere to the surface, and interact with other cells as the cells adhere to the surface.

[0057] As used herein, "a surface coating that inhibits cells" refers to a material, substrate, or component that does not allow cells to grow and / or does not allow cells to adhere to the cell contact surface.

[0058] As used herein, "a surface coating that responds to media conditions" refers to a material, substrate, or component that changes when the media conditions change. Exemplary changes include changes in temperature, pH, oxygen level or concentration, level of toxic gas, presence of toxic substances, and this includes, for example, color change as a form of monitoring.

[0059] As used herein, "surface coating having controlled solubility" refers to a material, substrate, or component that is released from a surface at a specific time or in response to a specific temperature or pH, for example, to obtain controlled release of the coating contents.

[0060] Exemplary surface coatings that can be applied to one or more surfaces of a cell culture chamber include polycationic reagents (polybrene, protamine sulfate, poly-L-lysine, peptides having a net positive charge, amphiphilic cationic peptides), poloxamers, adhesion molecules such as fibronectin or modified fibronectin (RETRONECTIN®), antibodies, antibody conjugates, protein targeting domains (including DNA and RNA) such as nucleic acids, polylactic acid, polyvinyl alcohol, polysaccharides or dextran or derivatives thereof, collagen types (I-VIII), polyethylene glycol (PEG), fibrin, vitronectin, laminin, elastin, gelatin, hyaluronic acid, keratan sulfate, chondroitin sulfate, heparan sulfate, proteoglycan, poly-d-lysine, avidin, streptavidin, biotin, antibodies against biotin or protein tags, Ilsopeptag, BCCP, Myc tag, calmodulin tag, FLAG tag, HA tag, His tag, maltose binding protein tag, Nus tag, glutathione-S-transferase tag, green fluorescent protein tag, thioredoxin tag, S tag, Sof tag 1, Sof tag 3, Strep tag, SBP tag, Ty tag, certia, polylactic acid, polyvinyl alcohol, polysaccharides and dextran.

[0061] Additional surface coatings can include diagnostic agents that undergo a change in color or luminescence / fluorescence upon binding to a specific cell type or cell surface receptor. In such embodiments, the surface coating can function as a signal that a cell has reached a desired stage or that a cell has reached a desired confluence or other characteristic.

[0062] In additional embodiments, "surface treatment" can also be utilized on one or more surfaces of the cell culture chamber 410 that contact the cells, such as, for example, the pump 405, valve 407, and fluid path 408, and / or on one or more surfaces within the cartridge 402, and can also be applied to the non-porous gas-permeable material 482. Exemplary surface treatments include chemical treatment, etching, micro-etching, electrochemical treatment, etc. Additional surface treatments include gas plasma etching for modifying the surface structure of the cell culture chamber, including the cell contact surface such as a polystyrene surface. Exemplary gas plasma treatments include using air, oxygen, amine, etc. to generate desired functional groups on the surface of the cell culture chamber. These surface treatments can result in changes in the hydrophobicity of the surface and cause a series of changes in biological effects, including an increase in activation, an increase in transduction efficiency, an increase or decrease in cell binding, an acceleration of proliferation, the selection of certain cell types, the prevention or increase of cell adhesion, changes in cell biological pathways, cell differentiation, etc.

[0063] In addition, gas plasma treatment can be used to increase the ability of the non-porous gas-permeable material 482 to adhere, glue, or otherwise attach to surfaces such as the bottom 412 or top 414 or the cell culture chamber 410, or other surfaces including the polystyrene surface. Since many non-porous gas-permeable materials 482 can be hydrophobic, it can be difficult to adhere them to a rigid surface such as polystyrene with an adhesive or other bonding agent. Plasma oxidation of both the cell culture chamber 410 and the non-porous gas-permeable material 482 increases the surface energy of both surfaces of the structure. Since a return to hydrophobic characteristics can occur quickly, it is desirable to quickly adhere, for example, glue, the non-porous gas-permeable material 482 to the cell culture chamber 410 in order to achieve a strong bond and a lasting structural integrity.

[0064] Considerations of micro- and nano-etching regarding cell behavior in a cell culture environment are provided, for example, in Martinez, et al., "Effects of artificial micro-and nano-structure surfaces on cell behaviour," Annals of Anatomy 191:126-135(2009), the disclosure of which is hereby incorporated by reference in its entirety.

[0065] The non-porous gas permeable material 482 can also be cast onto or within a material such as the cell culture chamber 410 to provide the gas permeable characteristics described herein. Casting of the non-porous gas permeable membrane material 482 can be performed by various methods known in the art. By casting the non-porous gas permeable membrane material 482 onto or within a cell culture chamber 410 such as plastic, polystyrene, or a chamber, the structural strength and integrity of the gas permeable membrane can also be increased while still maintaining the gas permeable characteristics.

[0066] In other embodiments, the non-porous gas permeable material 482 can be injected into a material such as the cell culture chamber 410 to provide the gas permeable characteristics described herein. Injecting the non-porous gas permeable membrane material 482 into a cell culture chamber can be performed by various methods known in the art. By injecting the non-porous gas permeable membrane material 482 into a cell culture chamber 410 such as plastic, polystyrene, or a chamber, the structural strength and integrity of the gas permeable membrane can also be increased while still maintaining the gas permeable characteristics.

[0067] As described herein, the surface coatings or surface treatments described herein preferably activate immune cells (e.g., T cells), and in embodiments, stem cells or progenitor cells, or modulate their biological pathways, promote their proliferation, or improve their adhesiveness. In preferred embodiments, the stem cells are pluripotent stem cells, hematopoietic stem cells, or mesenchymal stem cells. In additional embodiments, cells that can be grown in the automated cell engineering systems described herein and using the methods described include connective tissue cells, heart cells, retinal cells, muscle cells, skin cells, and the like.

[0068] In an exemplary embodiment, as shown in FIG. 4F, the cell culture chamber 410 further includes at least one of a distal port 420 configured to allow removal of air bubbles from the cell culture chamber and / or configured as a recirculation port, a central port 422 configured to function as a recirculation inlet port, and a proximal port 424 configured to function as a drain port for cell removal.

[0069] In a further embodiment, a cell culture chamber 410 for use in an automated cell engineering system is provided herein, the cell culture chamber comprising a flat and non-flexible chamber having a chamber height of about 0.5 cm to about 4 cm and a cell contact surface 480. Preferably, at least 50% of the cell contact surface comprises a non-porous gas permeable material 482 comprising silicone.

[0070] In additional embodiments, the cell culture chamber further includes at least one of a distal port configured to allow removal of air bubbles from the cell culture chamber and / or configured as a recirculation port, a central port configured to function as a recirculation inlet port, and a proximal port configured to function as a drain port for cell removal. Preferably, the cell culture chamber has a volume of about 50 ml to about 200 ml.

[0071] As described herein, in an exemplary embodiment, the cell contact surface includes a plurality of discrete sections, each section including a non-porous gas permeable material. In a preferred embodiment, the entire cell contact surface includes a non-porous gas permeable material.

[0072] In a further embodiment, the cell contact surface further includes a surface coating on the cell contact surface selected from the group consisting of a surface coating that activates cells, a surface coating that modulates biological pathways within cells, a surface coating that enhances cell growth, and a surface coating that improves cell adhesion. Exemplary surface coatings are described herein.

[0073] In yet a further embodiment, a cassette 402 for use in an automated cell engineering system 400 is provided herein, the cassette comprising a chamber volume configured to contain a cell culture and a satellite volume 430 (i.e., the satellite volume contains no cells) for increasing the working volume of the cell culture chamber by providing an additional volume for media and other working fluids without containing the cell culture. The cassette includes a cell culture chamber 410 for performing activation, transduction, and / or expansion of a cell culture, preferably an immune cell culture. Preferably, the satellite volume is in fluid connection with the cell culture chamber such that media is exchanged with the culture chamber without disturbing the cell culture. In an exemplary embodiment, the satellite volume is a bag, and in other embodiments, the satellite volume is a non-deformable chamber. In an embodiment, the satellite volume is from about 0.50 ml to about 300 ml, and more preferably from about 150 ml to about 200 ml. FIGS. 4D - 4E show the location of the satellite volume 430 within the cassette 602.

[0074] As described herein, the cell culture chamber 410 is preferably not a centrifuge chamber. That is, in an embodiment, the cell culture chamber does not include a portion or configuration that enables separation of cells by rotating the chamber to generate a centripetal force. Preferably, the cell culture chamber described herein remains substantially stationary within the cassette.

[0075] Figures 5A - 5B show additional examples of the cell culture chamber 410, including the structural section 502, and sections or portions of the non - porous gas - permeable material 482 across the bottom 412 and / or top 414 of the cell culture chamber 410. Figure 5B shows the bottom 412 of the cell culture chamber 410 that constitutes the cell contact surface 480 and includes a section or portion of the non - porous gas - permeable material 482 across the surface 480. These cross - sections or portions can be of any shape, such as circular, square, rectangular, triangular, other polygonal, or random shapes, or combinations of such shapes. The structural section 502 provides support to the bottom 412 while still allowing a significant number of sections or portions of the non - porous gas - permeable material 482. Figure 5C shows the incorporation of the cell culture chamber 410, including the structural section 502 and sections or portions of the non - porous gas - permeable material 482, integrated into the cassette 402 of the automated cell engineering system 400.

[0076] Figures 5D - 5E show still further examples of the cell culture chamber 410, including the structural section 520 and the non - porous gas - permeable material 482 across the bottom 412 and / or top 414 of the cell culture chamber 410. The structural section 520 of the embodiment shown in Figures 5D - 5E can be a mesh, honeycomb, or similar structure that has open sections that increase the use of the non - porous gas - permeable material 482 while still maintaining structural stability. In an embodiment, the structural section 520 is sandwiched between sheets of the non - porous gas - permeable material 482 to provide structural rigidity while still being able to increase gas permeability.

[0077] Figures 5F - 5I show various manufacturing techniques that can be used to create a cell culture chamber containing a non - porous gas - permeable material 482. For example, in Figure 5F, the thickness of the non - porous gas - permeable material 482 can be increased such that it provides sufficient structural support for the cell layer 530 and the required amount of medium 540 by itself. In Figure 5G, a structural section 520 of a support material (e.g., the polystyrene of the cell culture chamber 410) can be used to provide sufficient support. In Figure 5H, this structural support can take the form of a mesh or honeycomb structure 550 that still limits the flexure of the non - porous gas - permeable material 482. As described herein, in Figure 5I, the support structure and the non - porous gas - permeable material 482 can be cast or injected together to create a non - porous gas - permeable structural material 560. For example, silicone - embedded plastics or custom EVO configurations can be utilized.

[0078] In yet a further embodiment, a cassette 402 for use in an automated cell engineering system 400 is provided herein, the cassette optionally including a cryogenic chamber 404 suitable for storage of cell culture medium and optionally a thermophilic chamber 406 suitable for performing activation, transduction, and / or expansion of cell culture, the thermophilic chamber including the cell culture chamber 410, and one or more fluid paths connected to the cell culture chamber, the fluid paths providing recirculation, waste removal, and homogeneous gas exchange and nutrient distribution to the cell culture chamber without disturbing the cells within the cell culture chamber.

[0079] As described herein, preferably, the cell culture chamber 410 is a flat and non - flexible chamber having a low chamber height and a cell contact surface, and the cell culture chamber is maintained in a substantially planar orientation within the cassette. As described herein, "substantially planar" means that the cell culture chamber is maintained within about 15°, more preferably within about 10°, or within about 5° of the horizontal line (i.e., substantially parallel to the flat ground).

[0080] In yet further embodiments, the cell culture chamber 410 may be oriented in a non-planar orientation (e.g., ±25 to 45°, or about ±30° from a horizontal planar orientation), whereby a sedimented cell population having different cell densities in different regions of the chamber can result.

[0081] Preferably, at least a portion of the cell contact surface comprises the non-porous gas permeable membrane described herein.

[0082] In embodiments, the cell culture chamber 410, and preferably the cell contact surface 480 and the non-porous gas permeable material 482 (when utilized), have a surface coating selected from the group consisting of a surface coating that activates cells, a surface coating that modulates intracellular biological pathways, a surface coating that enhances cell growth, and a surface coating that improves cell adhesion. Exemplary surface coatings are described herein. In embodiments, the cell culture chamber 410, and preferably the cell contact surface 480 and the non-porous gas permeable material 482 (when utilized), include the surface treatments described herein.

[0083] In yet further embodiments, a cassette 402 for use in the automated cell engineering system 400 is provided herein, the cassette optionally including a cryogenic chamber 404 for storage of, for example, cell culture medium, and optionally a warm chamber 406 for performing activation, transduction, and / or expansion of cell cultures, the warm chamber including the cell culture chamber 410.

[0084] Preferably, the cassette comprises one or more fluidic pathways connected to the cell culture chamber, which provide recirculation, waste removal, and homogeneous gas exchange and nutrient distribution to the cells within the cell culture chamber without disturbing the cells. Cartridge 402 preferably further includes a fluidic pathway 460 connected to the cell culture chamber configured to introduce a surface coating material into the cell culture chamber. In such embodiments, the fluidic pathway for introducing the surface coating can be an internal component (e.g., a bag, reservoir, or container) within the cassette that contains the coating material to be coated onto the cell culture chamber. In other embodiments, the fluidic pathway can be a tube or similar connection to an external valve for attachment to a syringe, bag, etc. for delivering the surface coating material.

[0085] In a preferred embodiment, a cell culture chamber for use in the automated cell engineering system described herein includes a surface coating material already coated on one or more surfaces of the cell culture chamber.

[0086] However, in further embodiments, the surface coating material can be provided via one or more fluidic pathways within (or external to) the automated cell engineering system and then added to the cell culture chamber to form a surface coating on the cell culture chamber. For example, an adhesion molecule or other coating material can be contained within a bag or chamber within the cassette / automated cell engineering system and then pumped into the cell culture chamber. Alternatively, the adhesion molecule can be added to the cell culture chamber via one or more ports using a syringe, bag, or similar device. Next, after drying the coating on the cell culture chamber, the excess can be removed and then optionally washed with cell culture medium or other means to remove unbound surface coating material.

[0087] As described herein, in an exemplary embodiment, the cassette is pre-filled with one or more of a cell culture, a culture medium, an activation reagent, and / or a vector (including any combination thereof). In further embodiments, these various elements can be added later via a suitable injection port or the like.

[0088] As described herein, in an embodiment, the cassette preferably further includes one or more of a pH sensor, a glucose sensor, an oxygen sensor, a lactate sensor, a cell counting module, a carbon dioxide sensor, a lactic acid sensor / monitor, and / or an optical density sensor. The cassette may also include one or more sampling ports and / or injection ports. Examples of such sampling ports and injection ports can include access ports for connecting the cartridge to an external device such as an electroporation unit or an additional media source.

[0089] Exemplary components of a cell engineering system can include a gas control seal, a heating zone, an actuator, a pivot for rocking or tilting the cell engineering system if desired, and a cryogenic zone for holding a cryogenic chamber. A user interface that may include a barcode reader, and the ability to receive inputs using a touchpad or other similar device may also be included.

[0090] One or more fluid paths preferably including various sensors (e.g., a pH sensor, a dissolved oxygen sensor), as well as sampling / sample ports and various valves (control valves, bypass check valves), and silicone-based tube components for connecting the components, can be arranged as needed. As described herein, the use of silicone-based tube components enables oxygenation through the tube components, facilitating gas transfer and optimal oxygenation of the cell culture.

[0091] In some embodiments, the present cell engineering system includes a plurality of chambers. In further embodiments, each of the activation step, transduction step, expansion step, concentration step, and harvesting step of the methods for the cells described herein is performed in a different chamber of the plurality of chambers of the present cell engineering system. In some embodiments, the cells are not substantially disturbed during transfer from one chamber to another. In other embodiments, the steps of the method are performed in the same chamber of the present cell engineering system, and the chamber environment is automatically adjusted by the present cell engineering system to be as required for each step of the method. Thereby, the cells can be prevented from being disturbed during various steps.

[0092] In some embodiments, the present cell engineering system has improved gas exchange compared to a flexible gas-permeable bag for cell culture. In some embodiments, the present cell engineering system includes a gas exchange line. The gas exchange line may be made of a gas-permeable material such as silicone, for example. In some embodiments, the gas permeability coefficient of the gas exchange line is higher than the permeability coefficient of the material used for the flexible gas-permeable bag. In some embodiments, the present cell engineering system recirculates oxygen across a substantially non-deformable chamber during the cell production method. For this reason, in some embodiments, the oxygen level of the cell culture in the present cell engineering system is higher than the oxygen level of the cell culture in the flexible gas-permeable bag. An increase in the oxygen level can support an increase in cell growth and proliferation, so a higher oxygen level can be important in the cell culture expansion step.

[0093] In some embodiments, the present cell engineering system continuously recirculates the culture medium across the chamber without disturbing the cells. For example, the present cell engineering system can continuously replenish nutrients, remove waste products, and circulate released cytokines and dissolved gases across the chamber while keeping the cells in the same region of the chamber. Continuous circulation can improve the uniform distribution of good materials and the uniform removal of bad materials without disturbing the cells, thereby reducing local effects caused by non-uniform distribution.

[0094] In some embodiments, the present cell engineering system provides carbon dioxide across the chamber during a cell production method (including CAR T production). CO2 can help maintain the target pH in the cell culture, which may be important for cell growth and proliferation. In some embodiments, the present cell engineering system monitors the CO2 level of the cell culture and adjusts the amount of CO2 provided based on the measured CO2 level. For example, as the cell culture increases, the amount of CO2 produced by the cells correspondingly increases. The present cell engineering system reduces the amount of CO2 provided. The desired CO2 level of the cell culture can be defined by the user, for example, at about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% CO2. Since the present cell engineering system constantly adjusts the amount of CO2 provided based on the measured CO2 level of the cell culture, the present cell engineering system can maintain the desired CO2 level throughout the manufacturing process. The amount of CO2 in the cell culture can also affect the pH of the culture, as dissolved CO2 generally acidifies the solution (by reacting with water to form carbonic acid). Therefore, a more stable pH can be obtained by maintaining a constant CO2 level in the cell culture. Thus, in embodiments, the pH level of the cell culture remains substantially constant during the production process. In further embodiments, the pH level of the transduced cell culture remains substantially constant during the growth step.

[0095] The yield from the production of genetically modified immune cells, including CAR T cell production, can be affected by activation and transduction efficiency, as well as cell growth conditions. The activation efficiency can be improved by more stable contact between the cells and the activation reagent. When the activation reagent is added to the cell culture chamber, a local effect is created due to the non-uniform distribution of cells caused by the movement of cells across the culture vessel. In contrast to flexible culture bags, cells grown in non-deformable chambers remain undisturbed during the activation process, which can contribute to higher activation efficiency.

[0096] The growth conditions of cell cultures can also improve cell yield. For example, higher oxygen levels in this cell engineering system, facilitated by highly gas-permeable tubes and continuous recirculation of oxygen within the cell culture chamber, may increase cell growth. The ability of this cell engineering system to constantly monitor the state of the cell culture and make appropriate adjustments can also be advantageous. For example, this cell engineering system can monitor the CO2, O2, N2, and / or pH levels of the cell culture and adjust the levels of CO2, O2, or N2. Nutrients can be provided in a timely and consistent manner and evenly distributed to the cell culture. Thus, an automated method for producing various cell types, including connective tissue cells, heart cells, retinal cells, stem cells, and genetically modified immune cells including CAR T cells, as described herein, provides a more advantageous higher cell yield compared to manual methods or methods utilizing flexible culture bags. Accordingly, in some embodiments, the method for automatically producing the various cells described herein using the cell engineering system described herein produces at least 10% more, at least 15% more, at least 20% more, at least 25% more, at least 30% more, at least 35% more, at least 40% more, at least 45% more, at least 50% more, at least 55% more, at least 60% more, at least 65% more, at least 70% more, at least 75% more, at least 80% more, at least 85% more, at least 90% more, at least 95% more, or at least 100% more cells than a method utilizing a flexible gas-permeable bag for cell culture. In an embodiment, the number of cells produced by the method described herein is at least about 2 billion (i.e., 2×10 9 ) cells, which includes at least about 2.1 billion, at least about 2.2 billion, at least about 2.3 billion, at least about 2.4 billion, at least about 2.5 billion, at least about 2.6 billion, at least about 2.7 billion, at least about 2.8 billion, at least about 2.9 billion, or at least about 3 billion cells.

[0097] Figures 6A-6B show a comparison between the dissolved oxygen concentration measured in a cell culture chamber (Figure 6A) containing the non-porous gas permeable membrane described herein, and a chamber without the gas permeable membrane (Figure 6B). As shown, the dissolved oxygen concentration was higher with the gas permeable membrane incorporated.

[0098] Figures 6C-6D show a comparison between the pH measured in a cell culture chamber (Figure 6C) containing the non-porous gas permeable membrane described herein, and a chamber without the gas permeable membrane (Figure 6D). As shown, the pH remained more constant with the gas permeable membrane incorporated.

[0099] Figure 7 shows the effect of the thickness of the non-porous gas permeable material 482 on oxygen transport. As shown, as the thickness of the gas permeable material decreases (i.e., thinner material), the oxygen permeability increases.

[0100] Figure 8 shows the effect of increased gas exchange on cell growth. For both a cell culture chamber of only polystyrene and a cell culture chamber containing a non-porous gas permeable material (silicone), the surface area, fluid height, and supply profile were kept the same. In the case of the cell culture chamber of only polystyrene, gas exchange was provided only through the headspace above the cell culture medium in the cell culture chamber. The chamber containing the non-porous gas permeable material also enabled gas exchange through the silicone. The final cell density of the cell culture chamber of only polystyrene was 15M cells / cm2, while the cell density with increased gas permeability was 55M cells / cm2, an increase of about 277%.

[0101] Additional Exemplary Embodiments Embodiment 1 is a cell culture chamber for use in an automated cell engineering system, the cell culture chamber comprising a flat and non-flexible chamber having a low chamber height and a cell contact surface, at least a portion of the cell contact surface comprising a non-porous gas permeable material.

[0102] Embodiment 2 includes the cell culture chamber according to Embodiment 1, further comprising at least one of a distal port configured to enable removal of bubbles from the cell culture chamber and / or configured as a recirculation port, a central port configured to function as a recirculation inlet port, and a proximal port configured to function as a drain port for cell removal.

[0103] Embodiment 3 includes the cell culture chamber according to Embodiment 1 or 2, wherein the cell contact surface includes a plurality of separate sections, and each section includes a non-porous gas-permeable material.

[0104] Embodiment 4 includes the cell culture chamber according to Embodiment 1 or 2, wherein at least about 50% of the cell contact surface includes a non-porous gas-permeable material.

[0105] Embodiment 5 includes the cell culture chamber according to Embodiment 1 or 2, wherein the entire cell contact surface includes a non-porous gas-permeable material.

[0106] Embodiment 6 includes the cell culture chamber according to any one of Embodiments 1 to 5, wherein the non-porous gas-permeable material includes silicone, fluorinated ethylene propylene (FEP), or ethyl vinyl olefin (EVO).

[0107] Embodiment 7 includes the cell culture chamber according to any one of Embodiments 1 to 6, having a chamber height of about 0.5 cm to about 4 cm.

[0108] Embodiment 8 includes the cell culture chamber according to any one of Embodiments 1 to 7, having a volume of about 50 ml to about 200 ml.

[0109] Embodiment 9 includes the cell culture chamber according to any one of Embodiments 1 to 8, wherein the cell culture chamber is not a centrifuge chamber.

[0110] Embodiment 10 includes the cell culture chamber according to any one of Embodiments 1 to 9, further comprising a surface coating on the cell contact surface selected from the group consisting of a surface coating that activates cells, a surface coating that regulates biological pathways within cells, a surface coating that enhances cell growth, a surface coating that improves cell adhesiveness, a surface coating that inhibits cells, a surface coating that responds to culture conditions, and a surface coating having a controlled solubility.

[0111] Embodiment 11 includes the cell culture chamber according to Embodiment 10, wherein the surface coating contains an adhesion molecule.

[0112] Embodiment 12 includes the cell culture chamber according to Embodiment 11, wherein the adhesion molecule is fibronectin or modified fibronectin.

[0113] Embodiment 13 includes the cell culture chamber according to any one of Embodiments 1 to 12, wherein a part of the cell contact surface further includes a surface treatment.

[0114] Embodiment 14 is a cell culture chamber for use in an automated cell engineering system, the cell culture chamber comprising a flat and non-flexible chamber having a chamber height of about 0.5 cm to about 4 cm and a cell contact surface, at least 50% of the cell contact surface comprising a non-porous gas-permeable material including silicone, fluorinated ethylene propylene (FEP), or ethylene vinyl olefin (EVO), and the cell culture chamber further comprising at least one of a distal port configured to allow removal of air bubbles from the cell culture chamber and / or configured as a recirculation port, a central port configured to function as a recirculation inlet port, and a proximal port configured to function as a drain port for cell removal.

[0115] Embodiment 15 includes the cell culture chamber according to Embodiment 14, wherein the cell contact surface includes a plurality of separate sections, each section comprising a non-porous gas-permeable material.

[0116] Embodiment 16 includes the cell culture chamber according to Embodiment 14, wherein the entire cell contact surface contains a non-porous gas-permeable material.

[0117] Embodiment 17 includes the cell culture chamber according to any one of Embodiments 14 to 16, which has a volume of about 50 ml to about 200 ml.

[0118] Embodiment 18 includes the cell culture chamber according to any one of Embodiments 14 to 17, wherein the cell culture chamber is not a centrifuge chamber.

[0119] Embodiment 19 includes the cell culture chamber according to any one of Embodiments 14 to 18, wherein the cell contact surface further includes a surface coating selected from the group consisting of a surface coating that activates cells, a surface coating that regulates biological pathways within cells, a surface coating that enhances cell growth, a surface coating that improves cell adhesion, a surface coating that inhibits cells, a surface coating that responds to medium conditions, and a surface coating having a controlled solubility.

[0120] Embodiment 20 includes the cell culture chamber according to Embodiment 19, wherein the surface coating contains an adhesion molecule.

[0121] Embodiment 21 includes the cell culture chamber according to Embodiment 20, wherein the adhesion molecule is fibronectin or modified fibronectin.

[0122] Embodiment 22 includes the cell culture chamber according to any one of Embodiments 14 to 21, wherein a part of the cell contact surface further includes a surface treatment.

[0123] Embodiment 23 is a cassette for use in an automated cell engineering system, the cassette being a high-temperature chamber for performing activation, transduction, and / or expansion of cell culture, the high-temperature chamber comprising a high-temperature chamber including a cell culture chamber, and one or more fluid paths connected to the cell culture chamber, the fluid paths providing recirculation, waste removal, and homogeneous gas exchange and nutrient distribution to the cell culture chamber without disturbing the cells within the cell culture chamber, the cell culture chamber being a flat, non-flexible chamber having a low chamber height and a cell contact surface, the cell culture chamber being maintained in a substantially planar orientation within the cassette, and at least a portion of the cell contact surface including a non-porous gas-permeable material.

[0124] Embodiment 24 includes the cassette according to Embodiment 23, further comprising at least one of a distal port configured to allow removal of air bubbles from the cell culture chamber and / or configured as a recirculation port, a central port configured to function as a recirculation inlet port, and a proximal port configured to function as a drain port for cell removal.

[0125] Embodiment 25 includes the cassette according to Embodiment 23 or 24, wherein the cell contact surface includes a plurality of discrete sections, each section including a non-porous gas-permeable material.

[0126] Embodiment 26 includes the cassette according to Embodiment 23 or 24, wherein at least about 50% of the cell contact surface includes a non-porous gas-permeable material.

[0127] Embodiment 27 includes the cassette according to Embodiment 23 or Claim 24, wherein the entire cell contact surface includes a non-porous gas-permeable material.

[0128] Embodiment 28 includes the cassette according to any one of Embodiments 23 to 27, wherein the non-porous gas-permeable material includes silicone, fluorinated ethylene propylene (FEP), or ethyl vinyl olefin (EVO).

[0129] Embodiment 29 includes the cassette according to any one of Embodiments 23 to 28, having a chamber height of about 0.5 cm to about 4 cm.

[0130] Embodiment 30 includes the cassette according to any one of Embodiments 23 to 29, having a volume of about 50 ml to about 200 ml.

[0131] Embodiment 31 includes the cassette according to any one of Embodiments 23 to 29, wherein the cassette does not include a centrifuge chamber.

[0132] Embodiment 32 includes the cassette according to any one of Embodiments 23 to 31, further including a surface coating on the cell contact surface selected from the group consisting of a surface coating that activates cells, a surface coating that regulates biological pathways within cells, a surface coating that enhances cell growth, a surface coating that improves cell adhesion, a surface coating that inhibits cells, a surface coating that responds to culture medium conditions, and a surface coating having a controlled solubility.

[0133] Embodiment 33 includes the cassette according to Embodiment 32, wherein the surface coating includes an adhesion molecule.

[0134] Embodiment 34 includes the cassette according to Embodiment 33, wherein the adhesion molecule is fibronectin or modified fibronectin.

[0135] Embodiment 35 includes the cassette according to any one of Embodiments 23 to 34, wherein the cassette is pre-filled with a culture medium, an activation reagent, and optionally a vector.

[0136] Embodiment 36 includes the cassette according to any one of Embodiments 23 to 35, further comprising one or more of a pH sensor, a glucose sensor, an oxygen sensor, a lactate sensor, a cell counting module, a carbon dioxide sensor, and / or an optical density sensor.

[0137] Embodiment 37 includes the cassette according to any one of Embodiments 23 to 36, further comprising one or more sampling ports and / or injection ports.

[0138] Embodiment 38 includes the cassette according to any one of Embodiments 23 to 37, further comprising an access port for connecting the cassette to an external device.

[0139] Embodiment 39 includes the cassette according to Embodiment 38, wherein the external device includes an electroporation unit or an additional media source.

[0140] Embodiment 40 includes the cassette according to any one of Embodiments 23 to 39, wherein one or more of the fluid paths are silicone-based tube components, and the silicone-based tube components enable oxygen supply through the tube components.

[0141] Embodiment 41 includes the cassette according to any one of Embodiments 23 to 40, wherein a part of the cell contact surface further includes a surface treatment.

[0142] Embodiment 42 includes the cassette according to any one of Embodiments 23 to 41, further comprising a cryogenic chamber for storing a cell culture medium.

[0143] Embodiment 43 is a cell culture chamber for use in an automated cell engineering system. This cell culture chamber includes a flat and non-flexible chamber having a low chamber height, and a surface coating on the chamber, the surface coating being selected from the group consisting of a surface coating that activates cells, a surface coating that regulates biological pathways within cells, a surface coating that enhances cell growth, a surface coating that improves cell adhesion, a surface coating that inhibits cells, a surface coating that responds to medium conditions, and a surface coating having a controlled solubility.

[0144] Embodiment 44 is the cell culture chamber according to Embodiment 43, wherein the cells are immune cells.

[0145] Embodiment 45 is the cell culture chamber according to Embodiment 43, wherein the cells are stem cells or progenitor cells.

[0146] Embodiment 46 is the cell culture chamber according to Embodiment 45, wherein the stem cells are pluripotent stem cells, hematopoietic stem cells, or mesenchymal stem cells.

[0147] Embodiment 47 is the cell culture chamber according to Embodiment 43, wherein the cells are connective tissue cells, heart cells, or retinal cells.

[0148] Embodiment 48 is the cell culture chamber according to any one of Embodiments 43 to 47, further comprising at least one of a distal port configured to allow removal of air bubbles from the cell culture chamber and / or configured as a recirculation port, a central port configured to function as a recirculation inlet port, and a proximal port configured to function as a drain port for cell removal.

[0149] Embodiment 49 is the cell culture chamber according to any one of Embodiments 43 to 48, wherein the surface coating contains an adhesion molecule.

[0150] Embodiment 50 is the cell culture chamber according to Embodiment 49, wherein the adhesion molecule is fibronectin or modified fibronectin.

[0151] Embodiment 51 is the cell culture chamber according to any one of Embodiments 43 to 50, having a height of about 0.5 cm to about 4 cm.

[0152] Embodiment 52 is the cell culture chamber according to any one of Embodiments 43 to 51, having a volume of about 50 ml to about 200 ml.

[0153] Embodiment 53 is the cell culture chamber according to any one of Embodiments 43 to 52, wherein the cell culture chamber is not a centrifuge chamber.

[0154] Embodiment 54 is the cell culture chamber according to any one of Embodiments 43 to 53, wherein a part of the chamber further includes a surface treatment.

[0155] Embodiment 55 is a cassette for use in an automated cell engineering system. The cassette includes a high-temperature chamber for performing activation, transduction, and / or expansion of cell culture. The high-temperature chamber includes a cell culture chamber, and one or more fluid paths connected to the cell culture chamber. The fluid paths provide recirculation, removal of waste products, and homogeneous gas exchange and nutrient distribution to the cell culture chamber without disturbing the cells in the cell culture chamber. The cell culture chamber is a flat and non-flexible chamber having a low chamber height. The cell culture chamber is maintained in a substantially planar orientation within the cassette. The cell culture chamber has a surface coating selected from the group consisting of a surface coating that activates cells, a surface coating that regulates biological pathways within cells, a surface coating that enhances cell growth, a surface coating that improves cell adhesion, a surface coating that inhibits cells, a surface coating that responds to medium conditions, and a surface coating having a controlled solubility.

[0156] Embodiment 56 is a cassette for use in an automated cell engineering system, the cassette comprising a high-temperature chamber for performing activation, transduction, and / or expansion of cell culture, the high-temperature chamber including a cell culture chamber, the cell culture chamber being a flat and non-flexible chamber having a low chamber height, the cell culture chamber being maintained in a substantially planar orientation within the cassette, a high-temperature chamber; one or more fluid paths connected to the cell culture chamber, the fluid paths providing recirculation, waste removal, and homogeneous gas exchange and nutrient distribution to the cell culture chamber without disturbing the cells within the cell culture chamber; and a fluid path connected to the cell culture chamber configured to introduce a surface coating material into the cell culture chamber, the surface coating material being selected from the group consisting of a surface coating material that activates cells, a surface coating material that modulates biological pathways within cells, a surface coating material that enhances cell growth, a surface coating material that improves cell adhesion, a surface coating material that inhibits cells, a surface coating material that responds to medium conditions, and a surface coating material having a controlled solubility.

[0157] Embodiment 57 is the cassette according to claim 55 or 56, further comprising a low-temperature chamber for storing a cell culture medium.

[0158] Embodiment 58 is the cassette according to embodiment 55 or 56, wherein the cells are immune cells.

[0159] Embodiment 59 is the cassette according to embodiment 55 or 56, wherein the cells are stem cells.

[0160] Embodiment 60 is the cassette according to embodiment 59, wherein the stem cells are pluripotent stem cells or mesenchymal stem cells.

[0161] Embodiment 61 is the cassette according to embodiment 55 or 56, wherein the cells are connective tissue cells, heart cells, or retinal cells.

[0162] Embodiment 62 is the cassette according to any one of Embodiments 55 to 61, further comprising at least one of a distal port configured to allow removal of bubbles from the cell culture chamber and / or configured as a recirculation port, a central port configured to function as a recirculation inlet port, and a proximal port configured to function as a drain port for cell removal.

[0163] Embodiment 63 is the cassette according to any one of Embodiments 55 to 62, wherein the surface coating material contains an adhesion molecule.

[0164] Embodiment 64 is the cassette according to Embodiment 63, wherein the adhesion molecule is fibronectin or modified fibronectin.

[0165] Embodiment 65 is the cassette according to any one of Embodiments 55 to 64, having a height of about 0.5 cm to about 4 cm.

[0166] Embodiment 66 is the cassette according to any one of Embodiments 55 to 65, having a volume of about 50 ml to about 200 ml.

[0167] Embodiment 67 is the cassette according to any one of Embodiments 55 to 66, wherein the cell culture chamber is not a centrifugation chamber and the cassette does not include a centrifugation chamber.

[0168] Embodiment 68 is the cassette according to any one of Embodiments 55 to 67, wherein the cassette is pre-filled with a culture medium, an activation reagent, a surface coating material, and optionally a vector.

[0169] Embodiment 69 is the cassette according to any one of Embodiments 55 to 68, further comprising one or more of a pH sensor, a glucose sensor, an oxygen sensor, a lactate sensor, a cell counting module, a carbon dioxide sensor, and / or an optical density sensor.

[0170] Embodiment 70 is the cassette according to any one of Embodiments 55 to 69, further comprising one or more sampling ports and / or injection ports.

[0171] Embodiment 71 is the cassette according to any one of Embodiments 55 to 70, further comprising an access port for connecting the cassette to an external device.

[0172] Embodiment 72 is the cassette according to Embodiment 71, wherein the external device includes an electroporation unit or an additional media source.

[0173] Embodiment 73 is the cassette according to any one of Embodiments 55 to 72, wherein one or more of the fluid paths are silicone-based tube components, and the silicone-based tube components enable oxygen supply through the tube components.

[0174] Embodiment 74 is the cassette according to any one of Embodiments 55 to 73, wherein a part of the cell contact surface further includes a surface treatment.

[0175] It will be readily apparent to those skilled in the relevant art that other suitable modifications and adaptations to the methods and uses described herein can be made without departing from the scope of any of the embodiments.

[0176] Although certain embodiments are illustrated and described herein, it is understood that the claims should not be limited to the specific forms or arrangements of the parts shown and described. In this specification, exemplary embodiments are disclosed and certain terms are used, but they are used only in a general and descriptive sense and not for purposes of limitation. Modifications and variations of the embodiments are possible in light of the above teachings. Thus, it is understood that the embodiments may be practiced otherwise than as specifically described.

[0177] All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. The present invention may also be in the following aspects. Item 1. A cell culture chamber for use in an automated cell engineering system, comprising a flat and non - flexible chamber having a low chamber height and a cell contact surface, wherein at least a part of the cell contact surface comprises a non - porous gas - permeable material, the cell culture chamber. Item 2. The cell culture chamber, (a) a distal port configured to enable removal of air bubbles from the cell culture chamber and / or configured as a recirculation port, (b) a central port configured to function as a recirculation inlet port, and (c) a proximal port configured to function as a drain port for cell removal, of which at least one is further provided, the cell culture chamber according to Item 1. Item 3. The cell contact surface comprises a plurality of separate sections, each section comprising the non - porous gas - permeable material, the cell culture chamber according to Item 1 or 2. Item 4. At least about 50% of the cell contact surface comprises the non - porous gas - permeable material, the cell culture chamber according to Item 1 or 2. Item 5. The entire cell contact surface comprises the non - porous gas - permeable material, the cell culture chamber according to Item 1 or 2. Item 6. The non - porous gas - permeable material comprises silicone, fluorinated ethylene propylene (FEP), or ethyl vinyl olefin (EVO), the cell culture chamber according to any one of Items 1 - 5. Item 7. Having a chamber height of about 0.5 cm to about 4 cm, the cell culture chamber according to any one of Items 1 - 6. Item 8. Having a volume of about 50 ml to about 200 ml, the cell culture chamber according to any one of Items 1 - 7. Item 9. The cell culture chamber is not a centrifuge chamber, the cell culture chamber according to any one of Items 1 - 8. Item 10. The cell contact surface, i. a surface coating that activates cells, ii. a surface coating that regulates biological pathways within cells, iii. a surface coating that enhances cell growth, iv. a surface coating that improves cell adhesion, v. a surface coating that inhibits cells, vi. a surface coating that responds to culture conditions, and vii. a surface coating having a controlled solubility, further comprising a surface coating on the cell contact surface selected from the group consisting of, the cell culture chamber according to any one of items 1 to 9. Item 11. The cell culture chamber according to item 10, wherein the surface coating contains an adhesion molecule. Item 12. The cell culture chamber according to item 11, wherein the adhesion molecule is fibronectin or modified fibronectin. Item 13. The cell culture chamber according to any one of items 1 to 12, wherein a part of the cell contact surface further comprises a surface treatment. Item 14. A cell culture chamber for use in an automated cell engineering system, comprising a flat and non-flexible chamber having a chamber height of about 0.5 cm to about 4 cm and a cell contact surface, at least 50% of the cell contact surface comprises a non-porous gas-permeable material comprising silicone, fluorinated ethylene propylene (FEP), or ethyl vinyl olefin (EVO), the cell culture chamber is (a) a distal port configured to allow removal of bubbles from the cell culture chamber and / or as a recirculation port, (b) a central port configured to function as a recirculation inlet port, and (c) further comprising at least one of a proximal port configured to function as a drain port for cell removal, the cell culture chamber. Item 15. The cell culture chamber according to item 14, wherein the cell contact surface comprises a plurality of separate sections, each section comprising the non-porous gas-permeable material. Item 16. The cell culture chamber according to item 14, wherein the entire cell contact surface comprises the non-porous gas-permeable material. Item 17. The cell culture chamber according to any one of items 14 to 16, having a volume of about 50 ml to about 200 ml. Item 18. The cell culture chamber according to any one of items 14 to 17, wherein the cell culture chamber is not a centrifuge chamber. Item 19. The cell contact surface is i. a surface coating that activates cells, ii. a surface coating that regulates biological pathways within cells, iii. a surface coating that enhances cell growth, iv. a surface coating that improves cell adhesion, v. a surface coating that inhibits cells, vi. a surface coating that responds to culture conditions, and vii. a surface coating having a controlled solubility, the cell culture chamber according to any one of items 14 to 18, further comprising a surface coating on the cell contact surface selected from the group consisting of. Item 20. The cell culture chamber according to item 19, wherein the surface coating contains an adhesion molecule. Item 21. The cell culture chamber according to item 20, wherein the adhesion molecule is fibronectin or modified fibronectin. Item 22. The cell culture chamber according to any one of items 14 to 21, wherein a part of the cell contact surface further comprises a surface treatment. Item 23. A cassette for use in an automated cell engineering system, comprising: (a) a high-temperature chamber for performing activation, transduction, and / or expansion of cell culture, the high-temperature chamber comprising a cell culture chamber; (b) one or more fluid paths connected to the cell culture chamber, the fluid paths providing recirculation, waste removal, and homogeneous gas exchange and nutrient distribution to the cell culture chamber without disturbing the cells within the cell culture chamber. The cell culture chamber is a flat and non-flexible chamber having a low chamber height and a cell contact surface, and the cell culture chamber is maintained in a substantially planar orientation within the cassette. A cassette, wherein at least a part of the cell contact surface comprises a non-porous gas-permeable material. Item 24. The cell culture chamber is i. a distal port configured to allow removal of bubbles from the cell culture chamber and / or as a recirculation port; ii. a central port configured to function as a recirculation inlet port; and iii. a proximal port configured to function as a drain port for cell removal. The cassette according to item 23, further comprising at least one of the above. Item 25. The cassette according to item 23 or 24, wherein the cell contact surface comprises a plurality of separate sections, each section comprising the non-porous gas-permeable material. Item 26. The cassette according to item 23 or 24, wherein at least about 50% of the cell contact surface comprises the non-porous gas-permeable material. Item 27. The cassette according to item 23 or 24, wherein the entire cell contact surface comprises the non-porous gas-permeable material. Item 28. The cassette according to any one of items 23 to 27, wherein the non-porous gas-permeable material comprises silicone, fluorinated ethylene propylene (FEP), or ethyl vinyl olefin (EVO). Item 29. The cassette according to any one of items 23 to 28, having a chamber height of about 0.5 cm to about 4 cm. Item 30. The cassette according to any one of items 23 to 29, having a volume of about 50 ml to about 200 ml. Item 31. The cassette according to any one of items 23 to 29, wherein the cassette does not include a centrifugation chamber. Item 32. The cell contact surface is i. a surface coating that activates cells, ii. a surface coating that regulates biological pathways within cells, iii. a surface coating that enhances cell growth, iv. a surface coating that improves cell adhesion, v. a surface coating that inhibits cells, vi. a surface coating that responds to culture medium conditions, and vii. a surface coating having a controlled solubility, and further includes a surface coating on the cell contact surface selected from the group consisting of: The cassette according to any one of items 23 to 31. Item 33. The cassette according to item 32, wherein the surface coating comprises an adhesion molecule. Item 34. The cassette according to item 33, wherein the adhesion molecule is fibronectin or modified fibronectin. Item 35. The cassette according to any one of items 23 to 34, wherein the cassette is pre-filled with a culture medium, an activation reagent, and optionally a vector. Item 36. The cassette according to any one of items 23 to 35, further comprising one or more of a pH sensor, a glucose sensor, an oxygen sensor, a lactate sensor, a cell counting module, a carbon dioxide sensor, and / or an optical density sensor. Item 37. The cassette according to any one of items 23 to 36, further comprising one or more sampling ports and / or injection ports. Item 38. The cassette according to any one of items 23 to 37, further comprising an access port for connecting the cassette to an external device. Item 39. The cassette according to item 38, wherein the external device comprises an electroporation unit or an additional medium source. Item 40. One or more of the fluid paths are silicone-based tube components, and the cassette according to any one of items 23 to 39 includes the silicone-based tube components that enable oxygen supply through the tube components. Item 41. The cassette according to any one of items 23 to 40, wherein a part of the cell contact surface further includes a surface treatment. Item 42. The cassette according to any one of items 23 to 41, further comprising a low-temperature chamber for storing a cell culture medium. Item 43. A cell culture chamber for use in an automated cell engineering system, (a) a flat and non-flexible chamber having a low chamber height, and (b) a surface coating on the chamber, i. a surface coating that activates cells, ii. a surface coating that regulates biological pathways within cells, iii. a surface coating that enhances cell growth, iv. a surface coating that improves cell adhesion, v. a surface coating that inhibits cells, vi. a surface coating that responds to medium conditions, and vii. a surface coating having a controlled solubility, the surface coating on the chamber selected from the group consisting of, a cell culture chamber. Item 44. The cell culture chamber according to item 43, wherein the cells are immune cells. Item 45. The cell culture chamber according to item 43, wherein the cells are stem cells or progenitor cells. Item 46. The cell culture chamber according to item 45, wherein the stem cells are pluripotent stem cells, hematopoietic stem cells, or mesenchymal stem cells. Item 47. The cell culture chamber according to item 43, wherein the cells are connective tissue cells, heart cells, or retinal cells. Item 48. The cell culture chamber, (a) a distal port configured to enable removal of bubbles from the cell culture chamber and / or as a recirculation port, (b) a central port configured to function as a recirculation inlet port, and (c) a proximal port configured to function as a drain port for cell removal, the cell culture chamber according to any one of items 43 to 47 further comprising at least one of. Item 49. The cell culture chamber according to any one of items 43 to 48, wherein the surface coating includes an adhesion molecule. Item 50. The cell culture chamber according to item 49, wherein the adhesion molecule is fibronectin or modified fibronectin. Item 51. A cell culture chamber according to any one of items 53 to 50, having a height of about 0.5 cm to about 4 cm. Item 52. A cell culture chamber according to any one of items 43 to 51, having a volume of about 50 ml to about 200 ml. Item 53. The cell culture chamber according to any one of items 43 to 52, wherein the cell culture chamber is not a centrifugation chamber. Item 54. The cell culture chamber according to any one of items 43 to 53, wherein a part of the chamber further includes a surface treatment. Item 55. A cassette for use in an automated cell engineering system, (a) A high-temperature chamber for performing activation, transduction, and / or expansion of cell culture, the high-temperature chamber including a cell culture chamber, and (b) One or more fluid paths connected to the cell culture chamber, the fluid paths providing recirculation, removal of waste, and homogeneous gas exchange and nutrient distribution to the cell culture chamber without disturbing the cells in the cell culture chamber. The cell culture chamber is a flat and non-flexible chamber having a low chamber height, and the cell culture chamber is maintained in a substantially planar orientation within the cassette. The cell culture chamber is i. A surface coating that activates cells, ii. A surface coating that regulates biological pathways within cells, iii. A surface coating that enhances cell growth, iv. A surface coating that improves cell adhesion, v. A surface coating that inhibits cells, vi. A surface coating that responds to medium conditions, and vii. A cassette having a surface coating selected from the group consisting of surface coatings having a controlled solubility. Item 56. A cassette for use in an automated cell engineering system, (a) A high-temperature chamber for performing activation, transduction, and / or expansion of cell culture, the high-temperature chamber including a cell culture chamber, The cell culture chamber is a flat and non-flexible chamber having a low chamber height, and the cell culture chamber is maintained in a substantially planar orientation within the cassette. (b) One or more fluid pathways connected to the cell culture chamber, wherein the fluid pathways provide recirculation, waste removal, and homogeneous gas exchange and nutrient distribution to the cell culture chamber without disturbing the cells within the cell culture chamber. (c) A fluid pathway connected to the cell culture chamber configured to introduce a surface coating material into the cell culture chamber, wherein the surface coating material i. A surface coating material that activates cells, ii. A surface coating material that modulates biological pathways within cells, iii. A surface coating material that enhances cell growth, iv. A surface coating material that improves cell adhesion, v. A surface coating material that inhibits cells, vi. A surface coating material that responds to media conditions, and vii. A surface coating material having a controlled solubility, and is selected from the group consisting of, a fluid pathway. A cassette comprising. Item 57. The cassette according to item 55 or 56, further comprising a cryogenic chamber for storing a cell culture medium. Item 58. The cassette according to item 55 or 56, wherein the cells are immune cells. Item 59. The cassette according to item 55 or 56, wherein the cells are stem cells. Item 60. The cassette according to item 59, wherein the stem cells are pluripotent stem cells or mesenchymal stem cells. Item 61. The cassette according to item 55 or 56, wherein the cells are connective tissue cells, heart cells, or retinal cells. Item 62. The cell culture chamber is (a) A distal port configured to allow removal of air bubbles from the cell culture chamber and / or configured as a recirculation port, (b) A central port configured to function as a recirculation inlet port, and (c) The cassette according to any one of items 55 to 61, further comprising at least one of a proximal port configured to function as a drain port for cell removal. Item 63. The cassette according to any one of items 55 to 62, wherein the surface coating material comprises an adhesion molecule. Item 64. The cassette according to item 63, wherein the adhesion molecule is fibronectin or modified fibronectin. Item 65. The cassette according to any one of items 55 to 64, having a height of about 0.5 cm to about 4 cm. Item 66. The cassette according to any one of items 55 to 65, having a volume of about 50 ml to about 200 ml. Item 67. The cassette according to any one of items 55 to 66, wherein the cell culture chamber is not a centrifugation chamber and the cassette does not include a centrifugation chamber. Item 68. The cassette according to any one of items 55 to 67, wherein the cassette is pre-filled with a culture medium, an activation reagent, the surface coating material, and optionally a vector. Item 69. The cassette according to any one of items 55 to 68, further comprising one or more of a pH sensor, a glucose sensor, an oxygen sensor, a lactate sensor, a cell counting module, a carbon dioxide sensor, and / or an optical density sensor. Item 70. The cassette according to any one of items 55 to 69, further comprising one or more sampling ports and / or injection ports. Item 71. The cassette according to any one of items 55 to 70, further comprising an access port for connecting the cassette to an external device. Item 72. The cassette according to item 71, wherein the external device includes an electroporation unit or an additional medium source. Item 73. The cassette according to any one of items 55 to 72, wherein one or more of the fluid paths are silicone-based tube components, and the silicone-based tube components enable oxygen supply through the tube components. Item 74. The cassette according to any one of items 55 to 73, wherein a part of the cell contact surface further includes a surface treatment.

Claims

1. A cassette as a removable and replaceable element for use in an automated cell engineering system, (a) A high-temperature chamber configured to perform activation, transduction, and / or expansion of cell culture, wherein the high-temperature chamber includes a cell culture chamber, a high-temperature chamber, (b) A low-temperature chamber configured to store reagents, and (c) One or more fluid paths connected to the cell culture chamber, wherein the fluid paths provide recirculation, waste removal, and homogeneous gas exchange and nutrient distribution to the cell culture chamber without disturbing the cells in the cell culture chamber. One or more fluid paths, comprising: The cell culture chamber is a flat and non-flexible chamber having a low chamber height and a cell contact surface, and the cell culture chamber is maintained in a substantially planar orientation within the cassette, A cassette, wherein at least a part of the cell contact surface includes a non-porous gas-permeable material having a thickness sufficient to provide structural support to a cell layer received on the cell contact surface.

2. The cell culture chamber is i. A distal port configured to allow removal of air bubbles from the cell culture chamber and / or configured as a recirculation port, ii. A central port configured to function as a recirculation inlet port, and iii. The cassette according to claim 1, further comprising at least one of a proximal port configured to function as a drain port for cell removal.

3. The cassette according to claim 1 or 2, wherein the non-porous gas-permeable material includes silicone, fluorinated ethylene propylene (FEP), or ethyl vinyl olefin (EVO).

4. The cassette according to any one of claims 1 to 3, wherein the cassette does not include a centrifugation chamber.

5. wherein the cell contact surface is i. a surface coating that activates cells, ii. a surface coating that regulates intracellular biological pathways, iii. a surface coating that enhances cell growth, iv. a surface coating that improves cell adhesion, v. a surface coating that inhibits cells, vi. a surface coating that responds to culture medium conditions, and vii. a surface coating having a controlled solubility, and further comprising a surface coating on the cell contact surface selected from the group consisting of: The cassette according to any one of claims 1 to 4.

6. The cassette according to claim 5, wherein the surface coating comprises an adhesion molecule.

7. The cassette according to claim 6, wherein the adhesion molecule is fibronectin or modified fibronectin.

8. The cassette according to any one of claims 1 to 7, wherein the cassette is pre-filled with a culture medium, an activation reagent, and optionally a vector.

9. The cassette according to any one of claims 1 to 8, further comprising one or more of a pH sensor, a glucose sensor, an oxygen sensor, a lactate sensor, a cell counting module, a carbon dioxide sensor, and / or an optical density sensor.

10. The cassette according to any one of claims 1 to 9, further comprising an access port for connecting the cassette to an external device.

11. The cassette according to claim 10, wherein the external device comprises an electroporation unit or an additional medium source.

12. The cassette according to any one of claims 1 to 11, wherein one or more of the fluid paths include a silicone-based tube component that enables oxygen supply through the tube component.

13. The cassette according to any one of claims 1 to 12, wherein a part of the cell contact surface further includes a surface treatment.

14. The cassette according to any one of claims 1 to 13, wherein the low-temperature chamber is further configured to store a cell culture medium.

15. The cell culture chamber has a chamber height of about 0.5 cm to about 4 cm, at least 50% of the cell contact surface includes a non-porous gas-permeable material containing silicone, fluorinated ethylene propylene (FEP), or ethyl vinyl olefin (EVO), and the cell culture chamber, (a) a distal port configured to enable removal of air bubbles, (b) a central port configured to function as a recirculation inlet port, and (c) The cassette according to any one of claims 1 to 14, further comprising at least one of a proximal port configured to function as a drain port for cell removal.

16. The cell contact surface, i. a surface coating that activates cells, ii. a surface coating that regulates biological pathways within cells, iii. a surface coating that enhances cell growth, iv. a surface coating that improves cell adhesion, v. a surface coating that inhibits cells, vi. a surface coating that responds to medium conditions, and vii. The cassette according to claim 15, further comprising a surface coating on the cell contact surface selected from the group consisting of surface coatings having a controlled solubility. **Claim 17**: The cassette according to claim 16, wherein the surface coating contains adhesion molecules. **Claim 18**: The cassette according to claim 17, wherein the adhesion molecules are fibronectin or modified fibronectin. **Claim 19**: The cassette according to any one of claims 15 to 18, wherein a part of the cell contact surface further comprises a surface treatment.

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