Work cycle for a cell culture system

Abstract

An automated cell culture system having one or more pumps configured to operate in a duty cycle prevents excessive heat generation, allowing the cell culture system to operate within a conventional incubator without overheating. The duty cycle includes switching the pumps between an on mode and an off mode. By running the pumps for short periods of time and then turning them off, the heat generated is reduced. In view of the reduction in pumping time during the cycle, the pumps can be run at a higher flow rate while they are on, such that the average flow rate over the course of the cycle is not reduced. Systems of the invention employ a duty cycle in which the on period is shorter than the off period, particularly in which the on period is less than 20% of the duration of the overall duty cycle.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit and priority of U.S. Patent Application No. 16 / 539,916, filed August 13, 2019, the contents of which are incorporated herein by reference. Technical Field

[0003] The present invention relates generally to cell culture chambers, and more particularly to systems and methods for pumping fluids into and out of cell culture chambers. Background Technology

[0004] Many areas of clinical research and therapy require the isolation, preparation, and expansion of cell lines. To replicate the growth conditions needed for the cells of interest, variables in the culture medium, such as temperature, gas levels, and nutrient concentrations, need to be controlled. Cells are highly sensitive to minute environmental fluctuations. For example, even a small increase in temperature from 37°C to 39°C (i.e., the equivalent of 102°F) can rapidly kill some cells. Cell culture processes can often take several days, meaning that an effective cell culture system needs to be able to maintain these precise conditions for extended periods. Standalone cell culture systems can maintain temperature and gas levels, but this is expensive and impractical for many laboratories. Another alternative is to culture cells in a cell culture chamber within a conventional incubator. However, while these setups are less expensive, they are less effective at maintaining precise temperature conditions because the equipment within the incubator generates heat that is not easily dissipated. Summary of the Invention

[0005] This disclosure provides an automated cell culture system with pumps configured to operate during a work cycle, preventing excessive heat generation and thus allowing the cell culture system to maintain an appropriate cell culture temperature when operating within a conventional incubator. By generating less heat, the system of the present invention reduces the heat that needs to be dissipated, avoiding situations that could lead to overheating of the system within the incubator. In contrast, in conventional cell culture systems, the pumping systems used for injecting culture medium and removing waste products from cell culture containers or chambers generate significant amounts of heat. In the disclosed system, the pumps operate during a work cycle, switching between on and off modes. By running the pumps for short periods and then turning them off, the heat generated is reduced.

[0006] The present invention recognizes that simply running standard pumps for short periods and then shutting them off may not be sufficient for effectively delivering nutrients and removing waste. To maintain the overall flow rate, the pumps of the present invention operate at a higher flow rate when started to compensate for the reduced total pumping time. However, to simulate static cell culture and to avoid pulsed flow that could stimulate certain biological processes and reactions, the disclosed cell culture system can operate at a low level of perfusion flow within a laminar flow range. Therefore, the optimal duty cycle for delivering nutrients and removing waste products at low flow rates can be less than 20%, meaning the pump is on for less than 20% of the time. For example, if the desired overall flow rate is 10 μL / min, for a 20% duty cycle, the pump delivers 10 μL in the first 12 seconds (20% of 60 seconds) and then is completely shut off for the next 48 seconds. Thus, the overall average flow rate is 10 μL / min, and the relatively long shutdown time significantly reduces the heat generated during the cycle.

[0007] Aspects of the present invention include a cell culture system comprising a cell culture chamber, one or more pumps in fluid communication with the cell culture chamber, and a processor operatively connected to the pumps. The processor is configured to operate the pumps in a cyclic duty cycle, each cycle comprising an on-cycle and an off-cycle. The off-cycle is longer than the on-cycle. Each cycle of the duty cycle has the same average flow rate as each subsequent cycle of the duty cycle.

[0008] In this embodiment, the cell culture system is sized to fit within an incubator. The cell culture chamber may include an inlet and an outlet, and a fluid reservoir in fluid communication with the inlet. A pump may be configured to deliver cell culture medium to the cell culture chamber via the inlet and remove waste products from the cell culture chamber via the outlet. During an operating cycle, the pump forces fluid into and out of the cell culture chamber.

[0009] In some embodiments, the duty cycle has a duration of approximately 60 seconds. The on-time period preferably lasts less than 20% of the duty cycle duration. In some embodiments, the average flow rate is less than 1000 μL of fluid per minute.

[0010] In a related aspect, the present invention relates to a method for culturing cells. The method includes a first step of providing a cell culture chamber in fluid communication with a pump, and a second step of operating the pump using a processor configured to run a work cycle. Each cycle of the work cycle has the same average flow rate and includes an on-cycle in which fluid flows into and out of the cell culture chamber and a off-cycle in which fluid flow ceases. The off-cycle is longer than the on-cycle.

[0011] In some embodiments of the method, the cell culture system is sized to fit within an incubator. The cell culture chamber may include an inlet and an outlet, and a fluid reservoir in fluid communication with the inlet. In embodiments, operating the pump includes delivering cell culture medium to the cell culture chamber via the inlet and removing waste products from the cell culture chamber via the outlet. During an operating cycle, the pump forces fluid into and out of the cell culture chamber.

[0012] In some embodiments, the duty cycle has a duration of approximately 60 seconds. The on-time period preferably lasts less than 20% of the duty cycle duration. In some embodiments, the average flow rate is less than 1000 μL of fluid per minute. Attached Figure Description

[0013] Figure 1A and Figure 1B A cell culture system for use with the present invention is shown.

[0014] Figure 2A and Figure 2B A cell differentiation cassette is shown.

[0015] Figure 3 It shows the use of Figure 2A and Figure 2B Cell culture system with cell differentiation cassette.

[0016] Figure 4 The present invention illustrates a system having a cell culture chamber.

[0017] Figure 5 The process for connecting cell culture chambers and transferring fluids between these cell culture chambers is shown.

[0018] Figure 6 The system of the present invention having multiple cell culture chambers is shown.

[0019] Figure 7 A system according to certain embodiments of the present invention is shown. Detailed Implementation

[0020] This disclosure provides cell culture systems that can be used within conventional incubators and include pumps configured to operate during a work cycle to efficiently maintain fluid flow while avoiding excessive heat generation. The disclosed systems and related methods address the challenges of automating cell culture systems located within conventional incubators where heat dissipation is required. The most common heat source in automated cell culture systems is the pumping system, which injects culture medium and removes waste products from cell culture containers / chambers. The pumping system according to this disclosure generates less heat. The disclosed systems and methods are particularly useful in perfusion systems where the pumps must run continuously for several days. Perfusion systems are highly desirable in automated cell culture because perfusion is not only a method to ensure that cultured cells are exposed to a defined concentration of nutrients, but also a method to continuously remove metabolic waste products (unlike static culture, where cells are in a nutrient-depleted medium without replenishment, while waste products continuously accumulate).

[0021] The disclosed system can even be used in cell culture incubators that are not designed for heat dissipation. Such incubators typically operate at room temperature, below 30°C. The most common desired culture temperature is physiological temperature, or 37°C. Because heat is transferred along a temperature gradient from high-temperature to low-temperature regions, most incubators can only heat their interior, not cool it. Furthermore, many incubators are designed with insulator-like components, such as water jackets, designed to effectively retain heat. Under these conditions, the presence of heat sources in conventional incubators typically causes a rapid rise in internal temperature. Once this occurs, the temperature control system cuts off heating. However, the insulation features of the incubator prevent the temperature from dropping to the desired level in a timely manner. This invention addresses this problem by operating its pump in a cycle that achieves the desired fluid flow rate while generating significantly less heat within the incubator.

[0022] A duty cycle is the period during which equipment is operated intermittently rather than continuously. A duty cycle can be defined by the amount of time the equipment is turned on and off during a single operation cycle, or by the percentage of available time the equipment is running. Operating one or more pumps in a cell culture system during a duty cycle addresses the problems associated with an incubator (or any other warm environment). However, while running pumps for short periods and then shutting them off reduces heat output, simply operating pumps intermittently may not be sufficient to provide the desired fluid flow rate. Flow rates during a duty cycle must be carefully tuned. First, the total flow rate must be sufficient to deliver nutrients and effectively remove waste. However, pulsed flow mimics the natural pulsating rhythms in the human body, and many cell types are sensitive to this. Pulsed flow may cause certain biological processes and reactions to occur, or it may cause shearing in many cell types with increased shear sensitivity. Therefore, to closely simulate static cultures widely used in biological research environments, it is preferable to operate cell culture systems at low perfusion flow rates. In this context, "low" means completely laminar flow (wall shear stress levels well below 15 dynes / cm). 2 The average flow rate is below 1000 μL / min (at physiological levels). In some embodiments, the average flow rate is below 100 μL / min. In other embodiments, the average flow rate is below 10 μL / min.

[0023] The optimal duty cycle for delivering nutrients and extracting waste products at low flow rates for use with this invention is to operate one or more pumps for less than 20% of the available time. For example, if the desired total flow rate is 10 μL / min, for a 20% duty cycle, the pump delivers 10 μL over the first 12 seconds (20% of 60 seconds) and then is completely de-energized for the next 48 seconds. Thus, the overall average flow rate remains 10 μL / min, and the relatively long power-off time significantly reduces the amount of heat generated. In a conventional incubator, undesirable temperature rises can be avoided if the heat generated by the instrument is kept below 2 watts.

[0024] In other embodiments, the work cycle may be about 1%, about 2%, about 5%, about 10%, about 25%, about 30%, about 40%, about 50%, or greater. The duration of the work cycle is preferably about one minute, but in various embodiments it may be about 1 second, about 2 seconds, about 5 seconds, about 10 seconds, about 15 seconds, about 20 seconds, about 30 seconds, about 45 seconds, about 90 seconds, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 10 minutes, about 20 minutes, or longer. The average flow rate during the work cycle is typically below 1000 μL / min. In embodiments, the flow rate is below 100 μL / min, or below 10 μL / min.

[0025] Operating one or more pumps during a duty cycle is useful for a wide variety of cell culture systems and setups. Several embodiments of cell culture systems that employ a duty cycle to control pump effectiveness, according to this disclosure, are described below and illustrated in the accompanying drawings.

[0026] Figure 1A An embodiment of a system 100 for generating dendritic cells is shown. A peristaltic pump 110 is provided. Pump 110 is used to pump fluid into and out of cell culture cartridge 120. Pump 110 is operatively connected to a processor 199 configured to receive instructions from memory to operate pump 110 in a work cycle as described herein. These instructions specify the duration of the work cycle, as well as the duration of an on-cycle when the pump is on and the duration of a off-cycle when the pump is off. The on-cycle and / or off-cycle can be defined as an amount of time or a percentage of available time in the work cycle. Typically, the work cycle is repeated on a continuous loop such that when one work cycle ends, another work cycle begins, which has the effect of intermittently turning pump 110 on and off.

[0027] Cells are attached to the bottom surface 125 of the cell culture container 120. In other embodiments, cells are not attached to the bottom surface. The cell culture container 120 has eight fluid inlets 145 arranged at the corners of the cell culture container 120. A fluid outlet 135 is arranged at the center of the cell culture container 120. Connecting conduits 140 connect the fluid inlets to a differentiation medium reservoir (perfusion source) 180 containing differentiation medium 182. The differentiation medium reservoir 180 contains differentiation medium 182 to be pumped into the cell culture container 120. Connecting conduits 140 also connect the fluid outlet 135 to a waste reservoir 184. Depleted culture medium is pumped out of the cell culture container 120 through the outlet 135 and into the waste reservoir 184. The caps 170 and 175 on the differentiation medium reservoir 180 and the waste reservoir 184 are non-removable to maintain a sterile system. In other embodiments, the caps 170 and 175 are removable. The stopcocks on storage bottles 180 and 184 and / or LAVs 160 and 165 allow for the aseptic transfer of differentiation media to fill inlet bottles and remove waste from outlet bottles. Control console 190 provides designated space for the arrangement of the aforementioned components and also provides a display / user face 192, connectors 194, and an on / off switch 196.

[0028] Figure 1BAnother embodiment of a system 700 for generating dendritic cells is shown. A peristaltic pump 710 is provided. Pump 710 is used to pump fluid into and out of a cell culture cartridge 720. Pump 710 is operatively connected to a processor 799 configured to receive instructions from memory to operate pump 710 in a work cycle as described herein. These instructions specify the duration of the work cycle, as well as the duration of an on-cycle in which the pump is on and the duration of a off-cycle in which the pump is off. On-cycle and / or off-cycle can be defined as an amount of time or a percentage of available time in the work cycle. Typically, the work cycle is repeated on a continuous loop such that when one work cycle ends, another work cycle begins, which has the effect of intermittently turning pump 710 on and off.

[0029] Cells are attached to the bottom surface 725 of the cell culture container 720. In other embodiments, cells are not attached to the bottom surface. The cell culture container 720 has eight fluid inlets 745 arranged at the corners of the cell culture container 720. A fluid outlet 735 is arranged at the center of the cell culture container 720. A connecting conduit 740 connects the fluid inlets to a differentiation medium reservoir (perfusion source) 780 containing differentiation medium 782. The differentiation medium reservoir 780 is in the form of a sterile bag containing differentiation medium 782, which will be pumped into the cell culture container 720. The connecting conduit 740 also connects the fluid outlet 735 to a waste reservoir 784, which is another bag. The differentiation medium reservoir 780 and the waste reservoir 784 are supported by a rod 789. Depleted culture medium will be pumped out of the cell culture container 720 through the outlet 735 and into the waste reservoir 784. The console 790 provides designated space for the arrangement of the aforementioned components and also provides a display / user face 792, a connector 794, and an on / off switch 796.

[0030] System 100 is shown as having a single pump; however, it should be understood that the cell culture system of the present invention may have more than one pump. For example, one pump may be configured to pump fluid from reservoir 180, while another pump may be configured to pump waste away from the cell culture cassette to waste reservoir 184. In embodiments with more than one pump, each pump may be connected to the same processor or different processors, each processor being configured to operate its respective pump in a duty cycle, which may have the same or different parameters.

[0031] System 100 is sized to be placed within a conventional incubator, where it can operate for a sufficient period of time to produce the desired number of dendritic cells. One or more pumps operate in a duty cycle according to processor instructions, providing desirable fluid flow conditions and preventing the system from overheating. Additional features and configurations of a system for producing dendritic cells, compatible with this disclosure, are described in U.S. Application Serial No. 16 / 192,062, filed November 15, 2018, the contents of which are incorporated herein by reference.

[0032] In some embodiments, one or more pumps are operatively coupled to a cell culture chamber for perfusing a perfusion medium into the cell culture chamber. The perfusion medium includes any suitable culture medium. In some embodiments, the perfusion medium is a differentiation medium. The cell culture chamber may also include one or more fluid reservoirs. The fluid reservoirs are in fluid communication with the cell culture chamber and are operatively coupled to one or more pumps. One or more conduits are also provided for connecting the fluid reservoirs to the pumps and the cell culture chamber. In some aspects, one or more pumps are configured to pump fluid from the fluid reservoirs through the cell culture chamber and into a waste collection reservoir. One or more pumps are operatively coupled to a processor that turns the one or more pumps on and off during a duty cycle. The parameters of the duty cycle are defined by a set of instructions stored in a memory in communication with the processor. The processor, memory, and computer configuration will be described in detail below. In an embodiment, fluid moves from the fluid reservoir through conduits to the pumps, enters the cell culture chamber via an inlet, returns outside the cell culture chamber via an outlet, through conduits, and into the waste collection reservoir.

[0033] In some embodiments, fluid reservoirs and / or waste collection reservoirs may each be configured as one or more capped bottles contained within or fluidly coupled to a cell culture chamber. Each reservoir includes an inlet port and an outlet port, or an outlet port and a vent, which is fluidly coupled to the inlet of one or more cell culture chambers. In some aspects, for example, Luer connectors and silicone gaskets cut to fit around the Luer connectors may be used to prevent leakage through one or both of the inlet or outlet.

[0034] In some embodiments, one or more cell culture cassettes are sized to fit within an incubator, such that the process takes place inside the incubator. Conditions within the incubator include a sustained temperature of 37°C and a relative humidity of 95%–100%. Therefore, considering the tendency of materials (including fluids and biological materials) to expand under these conditions, the selected materials must possess the integrity to withstand these conditions. Furthermore, in some cases, conditions within the incubator are kept stable, and automated temperature recording allows for the monitoring of temperature fluctuations to correlate with any anomalies in the reaction conducted within the incubator. According to this disclosure, any power supplies and pumps are configured not to alter the environment within the incubator, as the duty cycle prevents them from generating excessive heat.

[0035] Accordingly, in one embodiment, the pump is housed separately from the cell culture chamber, but remains in fluid and operative communication with it. In another embodiment, the pump is directly attached to the cell culture chamber. In all embodiments, the pump is configured to be positioned within the incubator. The pump's duty cycle operation is sufficient to prevent overheating, but in some embodiments, the system may be operatively connected to a heat sink and / or fan for additional cooling. Regardless of the configuration, the pump is operatively coupled to a processor for running the duty cycle, and also operatively coupled to the cell culture chamber, and further to the cell culture chamber. Additional details regarding perfusion-based automated cell culture systems (such as small-scale culture systems for endothelial cell culture using onboard reagent storage devices and perfusion via onboard disposable peristaltic pumps; and larger-scale culture systems for generating dendritic cells from monocytes using chambers with polystyrene bottom surfaces) can be found in US 2018 / 0171296, US 20180251723 and WO 2018 / 005521, each of which is incorporated herein by reference in its entirety.

[0036] In other aspects, the cell culture chamber includes one or more sensors operatively coupled to the cell culture chamber. The sensors are capable of measuring any suitable parameter. For example, the sensors are capable of measuring one or more parameters within the cell culture chamber, such as pH, dissolved oxygen, total biomass, cell diameter, glucose concentration, lactate concentration, and cell metabolite concentration. In embodiments where the system includes multiple cell culture chambers, one or more sensors may be coupled to one or more of the cell culture chambers. In some embodiments, one or more sensors are coupled to one or more cell culture chambers, but not to all chambers in the system. In other embodiments, one or more sensors are coupled to all cell culture chambers in the system. In a system with multiple chambers operatively coupled to one or more sensors, in each chamber to which the sensors are coupled, the sensors may be the same, may all be different, or some sensors may be the same while some sensors may be different. In some aspects, one or more sensors are operatively coupled to a computer system having a central processing unit for executing instructions, enabling automatic monitoring and adjustment of parameters. Additional details regarding a computer system for implementing the methods of the present invention using a cell culture chamber are provided below.

[0037] In some embodiments, one or more sensors typically measure the temperature in one or more cell culture chambers, fluid reservoirs, tubing, and / or incubators. The temperature sensors can provide feedback to a processor that controls the duty cycle, indicating whether the on-time should be increased or decreased to fine-tune the temperature in the system. If the sensor detects that the temperature of the cell culture is too high, the duty cycle can be adjusted so that the pump is on for a shorter period of time to generate less heat. If the on-time is shortened, the pump will typically increase the fluid flow rate so that the average fluid flow rate remains constant throughout the duty cycle.

[0038] In some embodiments, the cell culture chamber has an inlet and an outlet, both of which can be used to fluidly connect the chamber to one or more additional containers via fluid connectors. In some embodiments, the additional containers include one or more additional cell culture chambers. The system of the present invention may include, for example, any number of cell culture chambers, between 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more than one hundred, configured to be fluidly connected in series to produce an immunotherapeutic product. Alternatively or additionally, one or more cell culture chambers may be arranged in parallel to allow for the production of immunotherapeutic products for more than one individual at a time. In a preferred embodiment, the cell culture chambers of the cell culture cassette are connected via aseptic connections.

[0039] In one embodiment, culture medium and cytokines can be perfused into a mixture of cells within one or more cell culture chambers to aid in the formation of cell-based immunotherapeutic products. In plate-based protocols for stimulating T cells with dendritic cells (DCs), a culture volume of approximately 2 mL is maintained from the outset, with cytokines infused twice during stimulation periods every 7 days. A key advantage of perfusion is the ability to maintain a consistent local concentration distribution of culture medium and cytokines, which ensures higher yields and the potential to accelerate the differentiation of monocytes into DCs compared to prior art plate-based protocols. However, the combination of attached (DCs) and non-attached (T cells), and the high sensitivity of DCs to mechanical forces, pose challenges to the stimulation and expansion of antigen-specific T cells, particularly regarding fluid flow through the cell culture chamber. Therefore, in embodiments where culture medium and cytokines are provided via perfusion, the system of the present invention must be able to deliver nutrients and cytokines to cells without removing them from the cell culture chamber, while also taking into account the shear sensitivity of certain antigen-presenting cells (such as DCs). Essentially, some embodiments of the systems and methods of this invention aim to optimize the retention of autocrine / paracrine signals that promote T cell proliferation, while renewing growth factors and maintaining minimal physical stimulation of DCs. To illustrate this, the direction and rate of perfusion flow through the cell culture chamber must be considered. For example, some embodiments of the invention may include a culture medium flow arrangement different from unidirectional flow, such as a countercurrent culture medium flow arrangement.

[0040] In one embodiment, a cell culture system is provided, comprising a cell culture chamber and a central processing unit (CPU). The CPU includes a memory containing instructions executable by the CPU. In some aspects, the instructions cause the system to receive first input data (including cell culture chamber dimensions), receive second input data (including a first concentration of a first cell type and a second concentration of a second cell type in one or more fluids to be introduced into the cell culture chamber), and calculate, based on the first and second inputs, a perfusion rate of perfusion fluid to be introduced into the cell culture chamber, the perfusion rate maximizing the probability of contact between the first and second cell types within the cell culture chamber. The system can further calculate desired duty cycle parameters (e.g., duty cycle duration, percentage of on-time, and flow rate during on-time) based on a desired average flow rate, the system's heat output, and a desired cell culture temperature within the incubator. The system also includes one or more pumps operatively coupled to one or more perfusion fluid reservoirs and operatively coupled to the CPU, such that the CPU also controls the one or more pumps by running a duty cycle, thereby controlling the perfusion rate of the perfusion fluid.

[0041] The system of the present invention may also include one or more control systems or be operatively coupled to such control systems for controlling the movement of fluid through the system; monitoring and controlling various parameters within the system, such as temperature; and detecting the presence, quantity (directly or indirectly), conversion rate, etc., of cell-based immunotherapy products. The system may also be equipped with various types of software, such as advanced real-time process monitoring and control processes that allow feedback control, and methods that allow the integration and scale-up of given reaction and purification results obtained using the system.

[0042] Systems that automatically generate dendritic cells from monocytes (MCs) obtained from peripheral blood, such as... Figures 2A to 2B and Figure 3 As shown. The described system can be operated in conjunction with the disclosed cycle pump to allow these systems to be used within a standard incubator.

[0043] Figures 2A to 2B A design for a dendritic cell differentiation cassette compatible with this invention is shown. Cassette 200 consists of… Figure 2A The layers shown on the left are assembled using double-sided adhesive film. The box is designed to receive a suitable volume of whole blood or another fluid sample containing MCs, essentially binding all of the MCs contained in the sample. The box contains a cell culture chamber that forms a centrally open fluid space within the box. The bottom plate of the chamber is the MC-binding surface or contains a portion of it. The preferred geometry of the cell culture chamber is a flat, thin space with rounded inner sides and no corners or vertices. An oval or rounded rectangular profile of the cell culture chamber is preferred. A flat surface and a low height help avoid turbulence, which can cause fluid shear stress, damaging cells within the cell culture chamber and potentially reducing cell viability and yield. Therefore, an important feature of the box is that it avoids exposing cells to shear stress or minimizes the degree to which cells are exposed to shear stress. This is achieved by using a flat surface with minimal protrusions or surface roughness, by avoiding sharp boundaries within the fluid path and cell culture chamber, by using laminar flow where possible (enhanced by maintaining a thin cell culture chamber, for example, a height of about 0.1 mm to about 2 mm), and by including a bubble collector or gas venting mechanism for eliminating air bubbles during cell culture chamber perfusion. Both the achievement of laminar flow and the elimination of air bubbles are facilitated by positioning the inlet and outlet ports on opposite sides of the cell growth chamber, such as... Figure 2A As shown. Furthermore, the box can be installed at an angle, with the outlet port positioned above the horizontal plane of the inlet port, to ensure that any air bubbles entering the cell growth chamber through the inlet port rise to the outlet port with the help of its buoyancy, and are thus quickly eliminated at the outlet port.

[0044] The fluid devices of the present invention (including dendritic cell differentiation cassettes or any cell growth or culture chambers) can be provided in microfluidic embodiments (i.e., where the size of one or more channels or chambers ranges from about 1 μm to about 999 μm) or large-fluidic embodiments (where the size of all channels or chambers is about 1 mm or greater). Depending on the specific design requirements, the fluid devices may further include fluid reservoirs, additional fluid channels or compartments, gaskets or seals, mixing zones, valves, pumps, vents, pressurized gas channels, electrical conductors, reagents, ports, and conduits. These fluid devices may also include one or more control modules, transmitters, receivers, processors, memory chips, batteries, displays, buttons, controls, motors, pneumatic actuators, antennas, electrical connectors, etc. These devices preferably contain only materials that are non-toxic to mammalian cells and compatible with sterilization by using alcohol and / or heat. Where desired, the surface of the device can be made more hydrophilic, for example by exposure to plasma, or can be coated with one or more gels, chemically functionalized coatings, proteins, antibodies, glycoproteins, lipids, glycolipids, nucleic acids, proteoglycans, glycosaminoglycans, cytokines, or cells. The device is also preferably compatible with use within a standard mammalian cell culture incubator, and in some embodiments, gas diffusion through the material is not permitted, as this could alter the composition of the culture medium within the device. The fluid device of the present invention is also preferably modular and capable of being fluidly connected in series (i.e., fluid flows from one device to another) or in parallel to other similar devices, and can also be configured to be physically stacked on top of each other, or physically arranged within related devices (e.g., incubators, pumps, or dendritic cell generation systems). The fluid device of the present invention is preferably leak-free under operating conditions and capable of aseptic operation over periods ranging from days to weeks. Other configurations of the dendritic cell differentiation cassette are also contemplated and are described in further detail in US 2018 / 0171296, the contents of which are incorporated herein by reference.

[0045] use Figures 2A to 2B The dendritic cell production system includes at least a cell culture chamber, a pump, a culture medium reservoir, and a fluid connection between the culture medium reservoir, the pump, and the cell culture chamber. The system includes a processor operatively connected to the pump for executing instructions to operate the pump in a duty cycle as described herein.

[0046] The system can also be configured without a cell culture chamber, which can be added by a user (optionally with one or more pipes for connecting a culture medium reservoir to a pump and a dendritic cell differentiation box). The cell culture chamber can be part of one or more dendritic cell differentiation boxes as described above, or as one or more distinct structures. The culture medium reservoir can be configured as one or more capped bottles, each containing an inlet port and an outlet port, or an outlet port and a vent, the vent being fluidly connected to a fluid inlet port of one or more dendritic cell differentiation boxes; a fluid collection reservoir fluidly connected to a fluid outlet port of one or more dendritic cell differentiation boxes; and a pump configured to pump fluid from the culture medium reservoir through the cell culture chamber of one or more dendritic cell differentiation boxes and into the fluid collection reservoir.

[0047] Figure 3 An embodiment of a DC generation system 300 is depicted. The system includes: a housing 310 having space for housing a culture medium reservoir 340 and a waste reservoir 350 (each being a commercially available glass or plastic culture medium bottle of the same size and shape with a plastic cap); a mounting area for a DC differentiation cassette 200; an exposed peristaltic pump head configured to receive peristaltic pump tubing from the culture medium bottle to the cassette's inlet port (an additional tubing from the cassette's outlet port to the waste bottle does not need to pass through the pump head); a display 330; and control buttons, knobs, or switches. The pump is controlled by a processor 299 configured to operate the pump during a duty cycle.

[0048] System 300 is configured to be positioned and operated within a conventional incubator. Similar systems comprising two or more boxes and pump heads (e.g., one per box, such as 2, 3, 4, 5, 6, 7, 8, 9, 10 or more boxes and pump heads) are also envisioned. In such multi-box systems, the processor, control electronics, display, and buttons, knobs, or switches can be shared between different boxes, or a set can be replicated for each box.

[0049] In another example embodiment, such as Figure 4As shown, a bioreactor 410 is provided, comprising a cell culture chamber 420, which includes a bottom surface 422 and at least one additional surface 424. The bottom surface 422 contains a first material to which cells attach, wherein the at least one additional surface 424 contains a breathable second material. The cell culture chamber also includes one or more inlets 426, 436 and one or more outlets 428, 438. In some embodiments, the bioreactor further includes at least one perfusion fluid reservoir 432, at least one waste fluid reservoir 434, at least one pump 440 for moving perfusion fluid through the chamber 420, and associated inlets 436 and outlets 438 for conveying fluid to and from the reservoirs 432, 434 and through the chamber 420. The bioreactor 410 will also include one or more pumps 440 operatively coupled to the cell culture chamber 420 for perfusing perfusion medium into the cell culture chamber. The bioreactor 410 may also include one or more fluid reservoirs 432. Fluid reservoir 432 is in fluid communication with cell culture chamber 410 and is operatively coupled to one or more pumps 440. One or more pipes are also provided for connecting the fluid reservoir to the pumps and the cell culture chamber. In some respects, one or more pumps are configured to pump fluid from the fluid reservoir through the cell culture chamber and into a waste collection reservoir. Figure 4 In the example embodiment shown, fluid moves from fluid reservoir 432, through conduit 452 to pump 440, and enters cell culture chamber 420 via inlet 436, returns outside cell culture chamber 420 via outlet 438, through conduit 454, and enters waste collection reservoir 434. One or more pumps are connected to processor 499, which operates the pumps in a duty cycle as disclosed herein.

[0050] The inlet and outlet of reactor 410 can be used to fluidly connect the chamber to one or more additional containers via fluid connectors. In some embodiments, the additional containers include one or more additional cell culture chambers, which will be described in more detail below. The system of the present invention may include, for example, any number of 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more than one hundred cell culture chambers configured to be fluidly connected in series with each other to produce immunotherapeutic products. Alternatively or additionally, one or more cell culture chambers may be arranged in parallel with each other to allow for the production of immunotherapeutic products for more than one individual at a time. In a preferred embodiment, the cell culture chambers of the bioreactor are connected via aseptic connections.

[0051] Example configurations of multi-bioreactor systems can be found in Figure 5The images B and C are provided below, along with additional details about the process using this configuration. Figure 5 As shown, in the case including the second bioreactor 510, the second cell culture chamber 520 is moved to a position to connect with the first cell culture chamber 420 via the outlet of the first chamber and the inlet of the second chamber. This connection is preferably a sterile connection. This connection allows sterile air to be injected into the first cell culture chamber 420 to transfer the supernatant containing expanded T cells into the second cell culture chamber 520. Alternative techniques known in the field of fluid flow can be used to transfer the supernatant from the first cell culture chamber 420 to the second cell culture chamber 520.

[0052] As also shown in the figure, each bioreactor includes its own fluid and waste collection reservoir, pumps, and associated piping. However, it should be understood that reservoirs and pumps can be shared between bioreactors. Pumps can be connected to the same processor or different processors to control the operating cycle of these pumps.

[0053] In some embodiments, the ratio of cell culture inlet to cell culture outlet is 1:1, such as when one or more bioreactors are arranged in series. Figure 5 As shown. In other embodiments, the outlet-to-inlet ratio of at least a portion of the bioreactor is 1:2. For example, the outlet of a cell culture chamber 420 may be fluidly connected to the inlet of two cell culture chambers (not shown), such that the fluid flowing out of the first cell culture chamber 420 is split into two streams, one stream being sent to a second cell culture chamber and the second stream to a third cell culture chamber. In this configuration, both the second and third cell culture chambers can be used to further stimulate T cells and promote their expansion. Additionally or alternatively, one of the second or third cell culture chambers may be configured to allow the use of one or more sensors operatively coupled to the chamber (e.g., for measuring temperature) to monitor reaction and flow parameters. In this way, one of the chambers has no additional sensors, and some sensors may need to penetrate the walls of the cell culture chamber, which may increase the risk of leakage and / or contamination.

[0054] Figure 6Another example of a multi-bioreactor system 900 is shown. System 900 includes a first cell culture chamber 820 and a second cell culture chamber 920, the first cell culture chamber having an inlet 845 and the second cell culture chamber having an inlet 945, these inlets being connected to conduits 940 in fluid communication with a fluid reservoir 980. The cell culture chambers have outlets 835 and 935 in fluid communication with a waste reservoir 984. Pumps 910a and 910b facilitate the pumping of fluid from the fluid reservoir 980 to the cell culture chambers 820 and 920. The pumps are controlled by a processor 999, which operates a duty cycle as described herein.

[0055] In some embodiments, one or more bioreactors may be arranged in a system comprising modules for performing various other processes before, simultaneously with, or after the processes occurring within the cell culture chamber of the bioreactor. Other configurations of multi-bioreactor systems are also contemplated and are described in further detail in WO 2018 / 005521, the contents of which are incorporated herein by reference.

[0056] As described in conjunction with the various embodiments disclosed herein, the systems and methods include computer components, such as memory for storing instructions related to a work cycle and a processor for executing the instructions to control the pump. Aspects of this disclosure described herein (such as controlling the movement of fluid through the system as described above, and monitoring and controlling various parameters) can be performed using any type of computing device (such as a computer or programmable logic controller (PLC)) including a processor, such as a central processing unit, or any combination of computing devices, wherein each device performs at least a portion of the process or method. In some embodiments, the systems and methods described herein can be performed using a handheld device, such as a smart tablet, smartphone, or a dedicated device manufactured for the system.

[0057] The methods disclosed herein can be implemented using software, hardware, firmware, hardwired, or any combination thereof. Features implementing the functionality can also be physically located in different places, including being distributed such that some functions are implemented in different physical locations (e.g., the imaging device is in one room and the host workstation is in another room, or in a separate building, for example, using wireless or wired connections).

[0058] For example, processors suitable for executing computer programs include general-purpose and special-purpose microprocessors, as well as any one or more processors in any type of digital computer. Typically, the processor receives instructions and data from read-only memory or random access memory, or both. The components of a computer are the processor for executing instructions and one or more memory devices for storing instructions and data.

[0059] Typically, the computer will also include one or more non-transitory mass storage devices (e.g., disks, magneto-optical disks, or optical disks) for storing data, or be operatively coupled to such one or more mass storage devices to receive data from or transmit data to them, or both. In some embodiments, sensors on the system transmit process data via Bluetooth to a central data collection unit located outside the incubator. In some embodiments, data is sent directly to the cloud rather than to physical storage devices. Information carriers suitable for implementing computer program instructions and data include all forms of non-volatile memory, including, for example, semiconductor memory devices (e.g., EPROM, EEPROM, solid-state drives (SSDs), and flash memory devices); disks (e.g., internal hard disks or removable disks); magneto-optical disks; and optical disks (e.g., CDs and DVDs). The processor and memory may be complemented or integrated therein by dedicated logic circuitry.

[0060] To provide interaction with the user, the subjects described herein can be implemented on a computer having I / O devices for displaying information to the user (e.g., CRT, LCD, LED, or projection devices) and input or output devices through which the user can provide input to the computer (e.g., keyboard and pointing devices (e.g., mouse or trackball)). Other types of devices can also be used to provide interaction with the user. For example, the feedback provided to the user can be any form of sensory feedback (e.g., visual feedback, auditory feedback, or tactile feedback), and input from the user can be received in any form, including sound, speech, or tactile input.

[0061] The subject matter described herein can be implemented in a computing system that includes back-end components (e.g., a data server), middleware components (e.g., an application server), or front-end components (e.g., a client computer with a graphical user interface or web browser through which a user can interact with an implementation of the subject matter described herein), or any combination of such back-end, middleware, and front-end components. The components of the system can be interconnected via a network through digital data communication (e.g., a communication network) of any form or medium. Examples of communication networks include cellular networks (e.g., 3G or 4G), local area networks (LANs), and wide area networks (WANs, e.g., the Internet).

[0062] The subject matter described herein can be implemented as one or more computer program products, such as one or more computer programs tangibly implemented on an information carrier (e.g., a non-transitory computer-readable medium) that are executed by or used to control the operation of a data processing apparatus (e.g., a programmable processor, a computer, or multiple computers). Computer programs (also referred to as programs, software, software applications, applications, macros, or code) can be written in any form of programming language (including compiled or interpreted languages ​​(e.g., C, C++, Perl)) and can be deployed in any form, including as standalone programs or as modules, components, subroutines, or other units suitable for use in a computing environment. The systems and methods of the present invention may include instructions written in any suitable programming language known in the art, including but not limited to C, C++, Perl, Java, ActiveX, HTML5, Visual Basic, or JavaScript.

[0063] A computer program does not necessarily correspond to a file. A program can be stored in a file or a portion of a file that holds other programs or data, in a single file dedicated to the program in question, or in multiple co-located files (e.g., a file that stores one or more modules, subroutines, or portions of code). A computer program can be deployed to execute on a single computer or multiple computers at a single site, or distributed across multiple sites and interconnected via a communication network.

[0064] Files can be digital files, for example, stored on hard drives, SSDs, CDs, or other tangible, non-transitory media. Files can be sent from one device to another over a network (e.g., via a network interface card, modem, wireless card, etc., as packets sent from a server to a client).

[0065] Writing a file according to embodiments of the invention includes, for example, transforming a tangible, non-transitory computer-readable medium by adding, removing, or rearranging particles (e.g., converting net charge or dipole moment into a magnetization mode via a read / write head), and these modes then represent a new arrangement of information about objective physical phenomena that are desired and useful to the user. In some embodiments, writing involves a physical transformation of the material in the tangible, non-transitory computer-readable medium (e.g., having certain optical properties such that an optical read / write device can then read the new and useful information arrangement, e.g., burning a CD-ROM). In some embodiments, writing a file includes transforming a physical flash memory device (such as a NAND flash memory device) and storing information by transforming the physical elements in a memory cell array made of floating gate transistors. Methods of writing files are well known in the art and can be invoked, for example, manually or automatically by a program, or by a save command in software or a write command in a programming language.

[0066] Suitable computing devices typically include mass storage, at least one graphical user interface, at least one display device, and often include communication between devices. Mass storage exemplifies a type of computer-readable medium, namely computer storage medium. Computer storage medium can include volatile, non-volatile, removable, and non-removable media implemented in any method or technology for storing information (such as computer-readable instructions, data structures, program modules, or other data). Examples of computer storage media include RAM, ROM, EEPROM, flash memory or other memory technologies, CD-ROM, digital versatile disc (DVD) or other optical storage devices, magnetic tape cassettes, magnetic tape, disk storage devices or other magnetic storage devices, RFID tags or chips, or any other medium that can be used to store desired information and is accessible by a computing device.

[0067] As those skilled in the art will recognize, for the execution of the methods of the present invention, the computer system or machine employed in the embodiments of the present invention may include one or more processors (e.g., a central processing unit (CPU), a graphics processing unit (GPU), or both), main memory, and static memory, which communicate with each other via a bus.

[0068] exist Figure 7In the illustrated example embodiment, system 600 may include computer 649 (e.g., laptop, desktop, or tablet computer). Computer 649 may be configured to communicate across network 609. Computer 649 includes one or more processors 659 and memory 663, as well as input / output mechanisms 654. In cases where the method of the present invention employs a client / server architecture, server 613 may be used to perform the operation of the method of the present invention. This server includes one or more of processors 621 and memory 629 capable of obtaining data, instructions, etc., or providing results via interface module 625 or as a file 617. Server 613 may be used via network 609 through computer 649 or terminal 667, or server 613 may be directly connected to terminal 667, which includes one or more processors 675 and memory 679, as well as input / output mechanisms 671.

[0069] For any of I / O 649, 637, or 671, the system 600 or machine according to an exemplary embodiment of the present invention may further include a video display unit (e.g., a liquid crystal display (LCD) or a cathode ray tube (CRT)). The computer system or machine according to some embodiments may also include an alphanumeric input device (e.g., a keyboard), a cursor control device (e.g., a mouse), a disk drive unit, a signal generating device (e.g., a speaker), a touchscreen, an accelerometer, a microphone, a cellular radio frequency antenna, and a network interface device, which may be, for example, a network interface card (NIC), a Wi-Fi card, or a cellular modem.

[0070] Memory 663, 679, or 629 according to exemplary embodiments of the present invention may include a machine-readable medium on which one or more sets of instructions (e.g., software) are stored, which implement any one or more methods or functions described herein. During execution of the software by a computer system, the software may also reside wholly or at least partially in main memory and / or a processor, which also constitute a machine-readable medium. The software may further be transmitted or received over a network via a network interface device.

[0071] Incorporation

[0072] Throughout this disclosure, other documents, such as patents, patent applications, patent publications, magazines, books, papers, and web content, have been referenced and cited. All of these documents are hereby incorporated in their entirety by reference for all purposes.

[0073] equivalent

[0074] Although the invention has been described in conjunction with certain embodiments, those skilled in the art will be able to make various changes, substitutions of equivalents, and other modifications to the compositions and methods set forth herein after reading the foregoing specification.

Claims

1. A cell culture system, comprising: Cell culture room; One or more pumps, the one or more pumps being in fluid communication with the cell culture chamber; as well as A processor operatively connected to the one or more pumps, the processor being configured to operate the one or more pumps in a continuous cycle of repeated duty, each cycle including an on-off cycle in which fluid flows into and out of the cell culture chamber and a off-off cycle in which fluid flow ceases, the off-off cycle being longer than the on-off cycle; as well as A memory communicating with the processor, wherein the memory contains one or more calculated duty cycle parameters stored as a set of instructions executable by the processor for operating the one or more pumps on a continuous cyclic repeating duty cycle, wherein the set of instructions specifies for each repeating duty cycle a duration of an on cycle, a duration of an off cycle, and a total duration of the repeating duty cycle such that each repeating duty cycle has the same average flow rate.

2. The cell culture system as described in claim 1, wherein, The cell culture system is sized to fit within an incubator.

3. The cell culture system of claim 1, further comprising a fluid reservoir in fluid communication with the inlet of the cell culture chamber.

4. The cell culture system of claim 1, wherein, During the start-up cycle, the one or more pumps are configured to deliver cell culture medium to the cell culture chamber via the inlet of the cell culture chamber and remove waste products from the cell culture chamber via the outlet of the cell culture chamber.

5. The cell culture system as described in claim 1, wherein, During the start-up cycle, the one or more pumps force fluid into and out of the cell culture chamber.

6. The cell culture system of claim 5, wherein, The fluid includes cell culture medium.

7. The cell culture system of claim 1, wherein, The work cycle lasts for approximately 60 seconds.

8. The cell culture system of claim 1, wherein, The start-up cycle lasted for less than 20% of the duration of the working cycle.

9. The cell culture system of claim 1, wherein, The average flow rate is less than 1000 μL of fluid per minute.

10. The cell culture system of claim 1, wherein, The system calculates one or more cycle parameters based on the desired average flow rate, the system's heat output, and the desired cell culture temperature within the incubator.

11. The cell culture system of claim 1, wherein, The one or more cycle parameters include one or more of the cycle duration, the percentage of the on-time cycle, and the flow rate during the on-time cycle.

12. A method for culturing cells, the method comprising: A cell culture chamber is provided in fluid communication with one or more pumps, wherein the cell culture chamber includes an inlet and an outlet; and The processor operates the one or more pumps, the processor being configured to run repeated duty cycles, and further includes a memory in communication with the processor containing one or more calculated duty cycle parameters stored as a set of instructions executable by the processor to operate the one or more pumps on the repeated duty cycles, wherein the set of instructions specifies the duration of an on-cycle, the duration of a off-cycle, and the total duration of the repeated duty cycles, wherein each repetition of the duty cycle includes having the same average flow rate and includes: an on-cycle in which fluid flows into and out of the cell culture chamber; and a off-cycle in which fluid flow is blocked, the off-cycle being longer than the on-cycle.

13. The method of claim 12, further comprising a fluid reservoir in fluid communication with the inlet of the cell culture chamber.

14. The method of claim 12, wherein, Operating the one or more pumps includes delivering cell culture medium to the cell culture chamber via the inlet and removing waste products from the cell culture chamber via the outlet.

15. The method of claim 12, wherein, During the start-up cycle, the one or more pumps force fluid into and out of the cell culture chamber.

16. The method of claim 15, wherein, The fluid includes cell culture medium.

17. The method of claim 12, wherein, The work cycle lasts for approximately 60 seconds.

18. The method of claim 12, wherein, The start-up cycle lasted for less than 20% of the duration of the working cycle.

19. The method of claim 12, wherein, The average flow rate is less than 1000 μL of fluid per minute.

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