Duty cycle for cell culture systems

Abstract

To provide a duty cycle for suitable cell culture systems.SOLUTION: An automated cell culture system with one or more pumps configured to operate on a duty cycle prevents excess heat generation, allowing the cell culture system to operate inside a conventional incubator without overheating. The duty cycle involves switching the pump between on and off modes. By running pumps for a short period of time and then shutting them off, less heat is produced. To account for the reduced pumping time during the cycle, the pump can be run at a higher flow rate while it is on, so that an average flow rate over the course of the cycle is not reduced. Systems of the invention employ duty cycles in which the on-cycle is shorter than the off-cycle, and particularly where the on-cycle is less than 20% of the duration of the entire duty cycle.SELECTED DRAWING: Figure 1A

Description

Technical Field

[0001] (Cross - Reference to Related Applications) This application claims the benefit and priority of U.S. Patent Application No. 16 / 539,916, filed on August 13, 2019, the content of which is incorporated by reference herein.

[0002] (Field of the Invention) The present invention generally relates to cell culture chambers, and more specifically, to systems and methods for pumping fluid into and out of cell culture chambers.

Background Art

[0003] Many fields of clinical research and therapy require the isolation, preparation, and growth of cell lines. To reproduce the desired growth conditions for the cells of interest, the culture medium needs to be controlled with respect to variables such as temperature, gas levels, and nutrient concentrations. Cells can be extremely sensitive to minor environmental fluctuations. For example, even a slight increase in temperature from 37°C to 39°C (i.e., equivalent to a fever of 102°F) can rapidly kill certain cells. The cell culture process can require culturing over several days, which means that an effective cell culture system needs to be able to maintain these precise conditions over long periods. Stand - alone cell culture systems can maintain temperature and gas levels, but they are expensive and not practical for many laboratories. An alternative is to grow cells within a cell culture chamber inside a conventional incubator. However, these setups are less expensive, but because the equipment inside the incubator generates heat that cannot be easily dissipated, they are not very effective at maintaining precise temperature conditions.

Summary of the Invention

Means for Solving the Problems

[0004] The present disclosure provides an automated cell culture system with a pump configured to operate in a duty cycle, which prevents excessive heat generation, thereby enabling the cell culture system to maintain an appropriate cell culture temperature while operating inside a conventional incubator. By generating less heat, the system of the present invention reduces the amount of heat that needs to be dissipated and avoids conditions that would lead to overheating of the system inside the incubator. In conventional cell culture systems, much of the heat is generated by the pumping system used to inject the medium and remove waste from the cell culture vessel or chamber, while in the disclosed system, the pump operates in a duty cycle where the pump switches between on and off modes. By activating the pumps for short periods and then stopping them, less heat is generated.

[0005] The present invention recognizes that simply activating a standard pump for a short period and then turning it off may not be sufficient to effectively deliver nutrients and remove waste. To maintain the overall flow rate, the pumps of the present invention operate at a higher flow rate while they are on to compensate for the reduced overall pumping time. However, to avoid pulsatile flow that can emulate static cell culture and stimulate certain biological processes and reactions, the disclosed cell culture system can be operated using low-level perfusion flow within the laminar flow regime. The optimal duty cycle for delivering nutrients and extracting waste at low flow rates is thus less than 20%, meaning that the pump can be on for less than 20% of the time. For example, if the desired overall flow rate is 10 microliters per minute, for a 20% duty cycle, the pump delivers 10 microliters within the first 12 seconds (20% of 60 seconds) and then is completely powered off for the next 48 seconds. Thus, the overall average flow rate is 10 microliters per minute, and the relatively long power-off time significantly reduces the amount of heat generated over the course of the cycle.

[0006] Aspects of the present invention involve a cell culture system including a cell culture chamber, one or more pumps in fluid communication with the cell culture chamber, and a processor operably connected to the pumps. The processor is configured to operate the pumps in a circulating duty cycle, and each cycle of the duty cycle comprises 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 other cycle of the duty cycle.

[0007] In an embodiment, the cell culture system is sized and configured to fit inside an incubator. The cell culture chamber may include an inlet, an outlet, and a fluid reservoir in fluid communication with the inlet. The pump may be configured to deliver cell culture medium to the cell culture chamber via the inlet and remove waste from the cell culture chamber via the outlet. During the on cycle, the pump forces the flow of fluid into and out of the cell culture chamber.

[0008] In some embodiments, the duty cycle has a duration of about 60 seconds. The on cycle preferably lasts for less than 20% of the duration of the duty cycle. In some embodiments, the average flow rate is less than 1,000 μL of fluid per minute.

[0009] In a related aspect, the present invention involves 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 execute a circulating duty cycle. Each cycle of the duty 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 an off cycle in which fluid flow is stopped. The off cycle is longer than the on cycle.

[0010] In some embodiments of the present method, the cell culture system is sized and configured to fit inside an incubator. The cell culture chamber may include an inlet, an outlet, and a fluid reservoir in fluid communication with the inlet. In an embodiment, operating the pump includes delivering cell culture medium to the cell culture chamber through the inlet and removing waste from the cell culture chamber through the outlet. During the on-cycle, the pump forces the flow of fluid into and out of the cell culture chamber.

[0011] In some embodiments, the duty cycle has a duration of about 60 seconds. The on-cycle preferably lasts for less than 20% of the duration of the duty cycle. In some embodiments, the average flow rate is less than 1,000 μL of fluid per minute. This specification also provides, for example, the following items. (Item 1) A cell culture system, a cell culture chamber, one or more pumps in fluid communication with the cell culture chamber, a processor operably connected to the one or more pumps, the processor being configured to operate the one or more pumps in a circulating duty cycle, each cycle of the duty cycle comprising an on-cycle and an off-cycle longer than the on-cycle, the processor operating the duty cycle such that each cycle of the duty cycle has the same average flow rate. A cell culture system comprising. (Item 2) The cell culture system according to item 1, wherein the cell culture system is sized and configured to fit inside an incubator. (Item 3) The cell culture system according to item 1, wherein the cell culture chamber comprises an inlet and an outlet. (Item 4) The cell culture system according to item 3, further comprising a fluid reservoir in fluid communication with an inlet of the cell culture chamber. (Item 5) The cell culture system according to item 3, wherein the pump is configured to deliver a cell culture medium to the cell culture chamber through the inlet and remove waste from the cell culture chamber through the outlet. (Item 6) The cell culture system according to item 1, wherein during the on-cycle, the pump forces the flow of fluid to and from the cell culture chamber. (Item 7) The cell culture system according to item 6, wherein the fluid comprises a cell culture medium. (Item 8) The cell culture system according to item 1, wherein the duty cycle has a duration of about 60 seconds. (Item 9) The cell culture system according to item 1, wherein the on-cycle lasts for less than 20% of the duration of the duty cycle. (Item 10) The cell culture system according to item 1, wherein the average flow rate is less than 1,000 μL of fluid per minute. (Item 11) A method for culturing cells, the method comprising: providing a cell culture chamber in fluid communication with a pump; and operating the pump using a processor configured to execute a circulating duty cycle, each cycle of the duty cycle having the same average flow rate and comprising an on-cycle in which fluid flows to and from the cell culture chamber and an off-cycle in which fluid flow is stopped, the off-cycle being longer than the on-cycle. comprising. (Item 12) The cell culture system is sized and configured to fit inside an incubator, the method according to item 11. (Item 13) The method according to item 11, wherein the cell culture chamber comprises an inlet and an outlet. (Item 14) The method according to item 13, further comprising a fluid reservoir in fluid communication with the inlet of the cell culture chamber. (Item 15) Operating the pump includes delivering cell culture medium to the cell culture chamber through the inlet and removing waste from the cell culture chamber through the outlet, according to the method of item 13. (Item 16) During the on-cycle, the pump forces the flow of fluid into and out of the cell culture chamber, according to the method of item 11. (Item 17) The fluid includes cell culture medium, according to the method of item 16. (Item 18) The duty cycle has a duration of about 60 seconds, according to the method of item 11. (Item 19) The on-cycle lasts for less than 20% of the duration of the duty cycle, according to the method of item 11. (Item 20) The average flow rate is less than 1,000 μL of fluid per minute, according to the method of item 11.

Brief Description of the Drawings

[0012]

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Mode for Carrying Out the Invention

[0013] (Detailed Description) The present disclosure provides a cell culture system including a pump that can be used inside a conventional incubator, is activated in a duty cycle, and is configured to effectively maintain fluid flow while avoiding excessive heat generation. The disclosed system and related methods address the problems associated with automated cell culture systems that sit inside conventional incubators that need to dissipate heat. The most common source of heat in an automated cell culture system is the pumping system, which injects media and removes waste from the cell culture vessel / chamber. The pumping system according to the present disclosure generates less heat. The disclosed system and methods are particularly useful in perfusion systems where the pump has to be continuously activated over a period of several days. A perfusion system ensures that perfusion not only ensures that the cultured cells are exposed to a defined concentration of nutrients (unlike static cultures where cells sit in a medium that depletes of nutrients without replenishment along with the continuous accumulation of waste), but is also a method for ensuring the continuous removal of metabolic waste, and is thus highly desirable in automated cell culture.

[0014] The disclosed system can also be used in combination with cell culture incubators that are not designed to dissipate heat. Such incubators typically operate in a room temperature environment where the ambient temperature is less than 30°C. The most common desired culture temperature is physiological temperature or 37°C. Since heat is transferred from a higher temperature region to a lower temperature region along a temperature gradient, most incubators have only the ability to heat their interiors and do not cool them. Additionally, many incubators are designed using insulation-like components such as water jackets that are designed to effectively retain heat. Given those conditions, the presence of a heat source within a conventional incubator will generally cause the internal temperature to rise rapidly. Once this occurs, the temperature control system will stop heating. However, the insulation characteristics of the incubator will prevent the temperature from decreasing to the desired level in a timely manner. The present invention solves this problem by operating the pump in a duty cycle that achieves the desired fluid flow level while generating much less heat inside the incubator.

[0015] A duty cycle is the cycle of operation of a device that operates intermittently rather than continuously. The duty cycle can be defined by the amount of time a device is on and off over the course of one cycle of operation, or it can be defined by the percentage of available time that the device is active. Operating one or more pumps within a cell culture system in a duty cycle solves problems associated with the incubator (or any other warm environment). However, starting the pumps for a short period and then turning them off reduces the heat output, but simply starting the pumps in a discontinuous manner may not be sufficient to provide the desired fluid flow. The flow rate in the duty cycle must be carefully adjusted. For one, the overall flow rate must be sufficient to effectively deliver nutrients and remove waste. However, pulsatile flow is similar to the natural pulsatile rhythm in the human body, and many cell types are sensitive to this. Pulsatile flow can cause certain biological processes and reactions, or it can cause shear in many cell types that have increased shear sensitivity. Thus, in the desire to closely emulate static culturing widely used in biological research settings, it is desirable to operate the cell culture system using low-level perfusion flow. "Low" in this context is clearly within the region of laminar flow (wall shear stress levels clearly below the physiological level of 15 dynes / cm 2 ), and can typically mean fluid flow at a magnitude of less than 1,000 μL per minute. In some embodiments, the average flow is less than 100 μL per minute. In other embodiments, the average flow is less than 10 μL per minute.

[0016] The optimal duty cycle for use in combination with the present invention to deliver nutrients and extract waste at low flow rates is that the pump or pumps operate for less than 20% of the available time. For example, if the desired overall flow rate is 10 microliters per minute, for a 20% duty cycle, the pump delivers 10 microliters within the first 12 seconds (20% of 60 seconds) and then is completely powered off for the next 48 seconds. Thus, the overall average flow rate remains 10 microliters per minute, and the relatively long off time significantly reduces the amount of heat generated. In a conventional incubator, if heat generation from the instrument is maintained at less than 2 watts, an undesirable temperature increase can be avoided.

[0017] In other embodiments, the duty cycle can be about 1%, about 2%, about 5%, about 10%, about 25%, about 30%, about 40%, about 50%, or more. The duration of the duty cycle is preferably about 1 minute, but in various embodiments can 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 over the course of the duty cycle is generally less than 1,000 μL per minute. In embodiments, this is less than 100 μL per minute or less than 10 μL per minute.

[0018] Operating one or more pumps in a duty cycle is useful for various different cell culture systems and settings. Some embodiments of cell culture systems that employ a duty cycle to control pump activity according to the present disclosure are described below and shown in the figures.

[0019] Figure 1A shows an embodiment of a system 100 for generating dendritic cells. A peristaltic pump 110 is provided. The pump 110 is used to pump fluid in and out of the cell culture cartridge 120. The pump 110 is operably connected to a processor 199 configured to receive instructions from memory and operate the pump 110 in a duty cycle as described herein. The instructions define the duration of the duty cycle and the duration of the on-cycle when the pump is on and the duration of the off-cycle when the pump is off. The on-cycle and / or the off-cycle can be defined as an amount of time or as a percentage of the available time in the duty cycle. Generally, the duty cycle is repeated in a continuous loop such that when one duty cycle ends, another one begins, which has the effect of intermittently turning the pump 110 on and off.

[0020] The cell culture cartridge 120 has a bottom surface 125 to which cells adhere. In other embodiments, the cells do not adhere to the bottom surface. The cell culture cartridge 120 has eight fluid inlets 145 arranged at the corners of the cell culture cartridge 120. One fluid outlet 135 is arranged at the center of the cell culture cartridge 120. Connecting tubes 140 connect the fluid inlets to a differentiation medium reservoir (perfusion source) 180 containing a differentiation medium 182. The differentiation medium reservoir 180 contains the differentiation medium 182 that will be pumped into the cell culture cartridge 120. The connecting tubes 140 also connect the fluid outlet 135 to a waste reservoir 184. The depleted medium will be pumped out of the cell culture cartridge 120, through the outlet 135, and into the waste reservoir 184. The lids 170 and 175 on the differentiation medium reservoir 180 and the waste reservoir 184 are not removable, thereby maintaining the sterilization system. In other embodiments, the lids 170 and 175 are removable. Valves and / or LAVs 160 and 165 on the reservoir bottles 180 and 184 enable the sterile transfer of the differentiation medium, fill the inlet bottle, and remove waste from the outlet bottle. The console 190 provides a designated space for the arrangement of the components mentioned above, and also provides a display / user interface 192, a connection 194, and an on / off switch 196.

[0021] Figure 1B shows another embodiment of a system 700 for generating dendritic cells. A peristaltic pump 710 is provided. The pump 710 is used to pump fluid in and out of a cell culture cartridge 720. The pump 710 is operably connected to a processor 799 that receives instructions from memory and is configured to operate the pump 710 in a duty cycle as described herein. The instructions define the duration of the duty cycle and the duration of the on-cycle when the pump is on and the duration of the off-cycle when the pump is off. The on-cycle and / or off-cycle can be defined as an amount of time or as a percentage of the available time in the duty cycle. Generally, the duty cycle is repeated in a continuous loop such that when one duty cycle ends, another one begins, which has the effect of intermittently turning the pump 710 on and off.

[0022] The cell culture cartridge 720 has a bottom surface 725 to which cells adhere. In other embodiments, the cells do not adhere to the bottom surface. The cell culture cartridge 720 has eight fluid inlets 745 arranged at the corners of the cell culture cartridge 720. One fluid outlet 735 is arranged at the center of the cell culture cartridge 720. Connecting tubing 740 connects the fluid inlets to a differentiation media reservoir (perfusion source) 780 containing differentiation media 782. The differentiation media reservoir 780 is in the form of a sterile bag containing the differentiation media 782 that will be pumped into the cell culture cartridge 720. The connecting tubing 740 also connects the fluid outlet 735 to a waste reservoir 784, which is another bag. The differentiation media reservoir 780 and the waste reservoir 784 are supported by a support post 789. Depleted media will be pumped out of the cell culture cartridge 720, through the outlet 735, and into the waste reservoir 784. The console 790 provides a designated space for the arrangement of the components mentioned above, and also provides a display / user interface 792, a connection 794, and an on / off switch 796.

[0023] System 100 is shown with a single pump, but it should be understood that the cell culture system of the present invention can 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 into waste reservoir 184 away from the cell culture cartridge. In embodiments having more than one pump, each pump may be connected to the same processor or different processors that are configured to activate their individual pumps in a duty cycle, which may have the same or different parameters from each other.

[0024] System 100 is sized and configured to be installed inside a conventional incubator where it can operate for a sufficient period of time to generate a desired population of dendritic cells. The pump or pumps function according to processor instructions in a duty cycle, which provides desired fluid flow conditions and prevents overheating of the system. Additional features and configurations of the system for generating dendritic cells compatible with the present disclosure are described in U.S. Application No. 16 / 192,062, filed November 15, 2018, the content of which is incorporated herein by reference.

[0025] In one embodiment, one or more pumps are operably coupled to a cell culture chamber to perfuse a perfusion medium into the cell culture chamber. The perfusion medium includes any suitable medium. In some embodiments, the perfusion medium is a differentiation medium. The cell culture cartridge can also include one or more fluid reservoirs. The fluid reservoir is in fluid communication with the cell culture chamber and can be operably coupled to one or more pumps. One or more tubes are also provided for connecting the fluid reservoir to the pump and the cell culture chamber. In one aspect, one or more pumps are configured to pump fluid from the fluid reservoir, through the cell culture chamber, and into a waste collection reservoir. One or more pumps are operably connected to a processor that turns one or more pumps on and off over the course of a duty cycle. The parameters of the duty cycle are defined by a set of instructions stored in a memory that communicates with the processor. The processor, memory, and computer configuration are described in more detail below. In one embodiment, fluid moves from the fluid reservoir, through tubing, to the pump, and into the cell culture chamber through an inlet, back out of the cell culture chamber through an outlet, and through tubing into the waste collection reservoir.

[0026] In one embodiment, the fluid reservoir and / or the waste collection reservoir can each be provided as one or more capped bottles that are either contained within the cell culture chamber or fluidly coupled to the chamber. Each reservoir contains an inlet port and an outlet port, or an outlet port and a drain that is fluidly coupled to an inlet of one or more cell culture chambers. In one aspect, for example, a luer connector and a silicone gasket cut to fit around the luer connector can be used to prevent leakage through one or both of the inlet or outlet.

[0027] In one embodiment, one or more cell culture cartridges are sized and configured to fit within an incubator such that the process will be carried out within the incubator. Conditions within the incubator include a sustained temperature of 37°C and a relative humidity of 95 - 100%. Thus, the materials selected must have integrity to withstand these conditions on the premise that materials (including fluids and biologics) tend to expand under such conditions. Further, in some situations, the conditions within the incubator remain stable and automated temperature recording is possible to have knowledge of temperature fluctuations to correlate with any abnormalities in the reactions carried out within the incubator. According to the present disclosure, any power sources and pumps are configured such that their duty cycles do not change the environment within the incubator so as to prevent them from generating excessive heat.

[0028] Thus, in one embodiment, the pump is stored separately from the cell culture cartridge but is still in fluid and operable communication with the cell culture cartridge. In another embodiment, the pump is directly attached to the cell culture cartridge. In all embodiments, the pump is configured to be located within the incubator. The duty cycle operation of the pump is sufficient on its own to prevent overheating, but in some embodiments, the system may be operably connected to a heat sink and / or fan for additional heat dissipation. Regardless of the configuration, the pump is operably coupled to a processor for executing the duty cycle and the pump is also operably coupled to the cell culture cartridge and thus 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 on-board reagent storage and perfusion enabled by an on-board disposable peristaltic pump and larger-scale culture systems for dendritic cell generation from monocytes using a chamber with a polystyrene bottom can be found in US 2018 / 0171296, US20180251723, and WO 2018 / 005521, each of which is incorporated herein by reference in its entirety.

[0029] In still other aspects, the cell culture chamber includes one or more sensors operably coupled to the cell culture chamber. The sensors can be capable of measuring any suitable parameter. For example, the sensors can be 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 can be coupled to one or more of the cell culture chambers. In certain embodiments, one or more sensors are coupled to one or more cell culture chambers, but not to all of the chambers within the system. In other embodiments, one or more sensors are coupled to all of the cell culture chambers within the system. In a system having multiple chambers operably coupled to one or more sensors, the sensors can be the same for each of the chambers to which they are coupled, they can all be different, or some sensors can be the same and some can be different. In one aspect, one or more sensors are operably coupled to a computer system having a central processing unit for executing instructions such that automatic monitoring and adjustment of the parameters is possible. Additional details regarding the computer system for implementing the methods of the present invention using the cell culture chamber are provided below.

[0030] In some embodiments, one or more sensors can measure the temperature within one or more cell culture chambers, fluid reservoirs, tubes, and / or generally within the incubator. The temperature sensor can provide feedback indicating whether the on-cycle should be increased or decreased to finely tune the temperature within the system to a processor that controls the duty cycle. If the sensor detects that the temperature of the cell culture is rising, the duty cycle can be adjusted so that the pump is on for a shorter period to generate less heat. If the on-cycle is shortened, the pump will generally increase the fluid flow so that the average fluid flow over the course of the duty cycle remains the same.

[0031] In some embodiments, the cell culture chamber has an inlet and an outlet, both of which can be used to fluidly couple 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 can include, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or any number between or above 100 cell culture chambers that are fluidly connected to each other in series and configured to produce immunotherapy products. Alternatively, or in addition, one or more cell culture chambers can be arranged in parallel to each other to enable the production of immunotherapy products for more than one individual at a time. In a preferred embodiment, the cell culture chambers of the cell culture cartridge are connected via a sterile connection.

[0032] In one embodiment, perfusion of the medium and cytokines can be provided to the cell mixture within the cell culture chamber to assist in the formation of cell-based immunotherapy products. In a plate-based protocol for stimulation of T cells by DCs, a culture volume of approximately 2 mL is maintained from initiation, and cytokine injection is performed twice within each 7-day stimulation period. The main advantage of perfusion is the ability to maintain a consistent local concentration profile of the medium and cytokines, which ensures a potentially higher yield and the ability to accelerate the process of monocyte differentiation into DCs compared to prior art plate-based protocols. However, the combination of adherent (DC) and non-adherent (T cell) types poses challenges to the stimulation and proliferation of antigen-specific T cells, particularly with respect to the flow of fluid through the cell culture chamber, along with the high sensitivity of DCs to mechanical forces. Thus, in those embodiments where the medium and cytokines are provided via perfusion, the system of the present invention must be able to supply nutrients and cytokines to the cells without removing the cells from the cell culture cartridge, while also taking into account the shear sensitivity of certain antigen-presenting cells such as DCs. In essence, the systems and methods of some embodiments of the present invention aim to replenish growth factors and optimize the retention of autocrine / paracrine signals favorable for T cell proliferation while maintaining minimal physical stimulation of the DCs. To account for this, both the direction and rate of perfusion flow through the cell culture chamber must be considered. For example, some embodiments of the present invention may comprise a medium flow arrangement other than a unidirectional flow such as a countercurrent medium flow arrangement.

[0033] In an embodiment, a cell culture system is provided that includes a cell culture chamber and a central processing unit having a memory containing instructions executable by the central processing unit. In one aspect, the instructions cause the system to receive, as a first input, data comprising the size of the cell culture chamber, and as a second input, data comprising a first concentration of a first cell type and a second concentration of a second cell type in one or more fluids that will be introduced into the cell culture chamber, and based on the first and second inputs, calculate a perfusion rate of a perfusion fluid that will be introduced into the cell culture chamber that maximizes the probability that the first cell type and the second cell type will contact each other within the cell culture chamber. The system may further calculate desired duty cycle parameters (e.g., duration of the duty cycle, percentage of time with respect to the on cycle, and flow rate during the on cycle) based on a desired average flow rate, a heat output of the system, and a desired cell culture temperature inside the incubator. The system also includes one or more pumps operably coupled to one or more perfusion fluid reservoirs and operably coupled to the central processing unit such that the central processing unit also controls the perfusion rate of the perfusion fluid by executing a duty cycle to control one or more pumps.

[0034] The system of the present invention may also include or be operably coupled to one or more control systems for controlling the movement of fluid through the system, monitoring and controlling various parameters such as temperature within the system, and additionally detecting the presence, quantity (directly or indirectly), conversion rate, etc. of cell-based immunotherapy products. The system may also be equipped with a number of classes of software such as an advanced real-time process monitoring and control process that enables feedback control, and a process that enables integration and scale-up based on reaction and purification results obtained using the system.

[0035] A system for the automated generation of dendritic cells from monocytes (MC) obtained from peripheral blood is shown in FIGS. 2A-B and 3. The system described incorporates the disclosed duty cycle pump operation and can enable the present system to be used inside a standard incubator.

[0036] Figures 2A-2B show the design of a dendritic cell differentiation cassette that is compatible with the present invention. Cassette 200 is constructed from the layers shown on the left side of Figure 2A, assembled using a double-sided adhesive film. The design of the cassette allows it to receive a suitable volume of whole blood or another fluid sample containing MCs and bind essentially all of the MCs contained in the sample. The cassette contains a cell culture chamber that forms a central open fluid space within the cassette. The floor of the chamber is, or contains as part of it, an MC binding surface. The preferred geometry of the cell culture chamber is that of a flat, thin space with all its inner surfaces rounded and no corners or vertices. An oval or rounded rectangular profile of the chamber is preferred. The flat surface and low height help avoid turbulent flow that would lead to fluid shear stress that could disrupt the cells within the chamber and reduce both cell viability and yield. Thus, an important feature of the cassette is that it avoids or minimizes the exposure of the cells within it to shear stress. This is accomplished by the use of flat surfaces with little or no protrusions or surface roughness, by the avoidance of sharp boundaries within the fluid path and within the cell culture chamber, by the use of laminar flow (enhanced by keeping the cell culture chamber thin, such as about 0.1 mm to about 2 mm in height), and by the inclusion of a bubble trap or degassing mechanism for the removal of gas bubbles during perfusion of the cell culture chamber. The achievement of both laminar flow and gas bubble removal is facilitated by the positioning of the inlet and outlet ports on opposite sides of the cell growth chamber, as shown in Figure 2A. Further, the cassette is mounted at an angle to ensure that any air bubbles entering the cell growth chamber through the inlet port are quickly removed at the outlet port by rising to the outlet port by virtue of their buoyancy, and the outlet port can be positioned above the level of the inlet port.

[0037] The fluid device of the present invention, which includes a dendritic cell differentiation cassette or any cell growth or culture chamber, can be provided in either a microfluidic embodiment (i.e., one or more channels or chambers therein have dimensions in the range of about 1 μm to about 999 μm) or a macrofluidic embodiment (wherein all of the channels or chambers therein have dimensions of about 1 mm or greater). The fluid device can further include a fluid reservoir, additional fluid channels or compartments, gaskets or seals, mixing zones, valves, pumps, outlets, channels for pressurized gas, electrical conductors, reagents, ports, and tubing, as required by a particular design. They can also contain one or more control modules, transmitters, receivers, processors, memory chips, batteries, displays, buttons, control devices, motors, pneumatic actuators, antennas, electrical connectors, and the like. The device preferably contains only materials that are non-toxic to mammalian cells and compatible with sterilization by the use of alcohol and / or heat. If required, the surface of the device can be made more hydrophilic, such as 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 also preferably is compatible with use within a standard mammalian cell culture incubator, and in some embodiments, it does not allow diffusion of gas through the materials because it can modify the composition of the medium within the device. The fluid device of the present invention also preferably is modular and is fluidly connectable to other similar devices either in series (i.e., fluid flows from one device into another) or in parallel, and is configured to physically stack with each other, or can be physically arranged within a related device such as an incubator, pump, or dendritic cell generation system. The fluid device of the present invention preferably has no fluid leakage under operating conditions and enables sterilized operation over a period of days to weeks.Other configurations of the dendritic cell differentiation cassette are also envisioned and are described in more detail in US 2018 / 0171296, the contents of which are incorporated herein by reference.

[0038] A dendritic cell generation system employing the cassette of FIGS. 2A-2B includes at least a cell culture chamber, a pump, a medium reservoir, and a fluid connection between the medium reservoir, the pump, and the cell culture chamber. The system includes a processor operably connected to the pump to execute instructions for activating the pump in a duty cycle as described herein.

[0039] The system can also be provided without a cell culture chamber, which can optionally be added to the system by the user, along with one or more tubes for connecting the medium reservoir to the pump and the DC differentiation cassette. The cell culture chamber can be provided as part of one or more dendritic cell differentiation cassettes as described above, or as one or more different structures. The medium reservoir can be one or more capped bottles each containing an inlet port and an outlet port or an outlet port and a drain port, fluidly coupled to a fluid inlet port of one or more dendritic cell differentiation cassettes, a fluid collection reservoir fluidly coupled to a fluid outlet port of one or more dendritic cell differentiation cassettes, and a pump configured to pump fluid from the medium reservoir, through the cell culture chamber of one or more dendritic cell differentiation cassettes, into the fluid collection reservoir.

[0040] An embodiment of the DC generation system 300 is depicted in FIG. 3. The system includes a housing 310 with a space for containing a media reservoir 340 and a waste reservoir 350 (each of the size and shape of a commercially available glass or plastic media bottle with a plastic cap), a mounting area for the DC differentiation cassette 200, an exposed peristaltic pump head configured to receive peristaltic pump tubing connecting from the media bottle to the inlet port of the cassette (separate tubing connecting from the outlet port of the cassette to the waste bottle need not pass through the pump head), a display 330, and control buttons, knobs, or switches. The pump is controlled by a processor 299 configured to activate the pump in a duty cycle.

[0041] System 300 is sized and configured to be positioned and operated within a conventional incubator. Similar systems including two or more cassettes and pump heads (e.g., one per cassette such as two, three, four, five, six, seven, eight, nine, ten, or more cassettes and pump heads) are also envisioned. In such a multi-cassette system, the processor, control electronics, display, and buttons, knobs, or switches may either be shared among different cassettes or replicated one set per cassette.

[0042] In another exemplary embodiment, as shown in FIG. 4, a biological reactor 410 is provided that includes a cell culture chamber 420 having a bottom surface 422 and at least one additional surface 424. The bottom surface 422 is made of a first material to which cells adhere, and the at least one additional surface 424 is made of a second material that is gas permeable. The cell culture chamber also includes one or more inlets 426, 436 and one or more outlets 428, 438. In some embodiments, the biological reactor also 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 chamber 420, and associated inlets 436 and outlets 438 for transporting fluid to and from reservoirs 432, 434 through chamber 420. The biological reactor 410 will also include one or more pumps 440 operably coupled to the cell culture chamber 420 to perfuse perfusion media into the cell culture chamber. The biological reactor 410 can also include one or more fluid reservoirs 432. The fluid reservoir 432 is in fluid communication with the cell culture chamber 410 and can be operably coupled to one or more pumps 440. One or more tubes are also provided for connecting the fluid reservoir to the pump and the cell culture chamber. In some aspects, one or more pumps are configured to pump fluid from the fluid reservoir, through the cell culture chamber, and into the waste collection reservoir. In the exemplary embodiment shown in FIG. 4, fluid moves from fluid reservoir 432, through tubing 452, to pump 440, into cell culture chamber 420 via inlet 436, back out of cell culture chamber 420 via outlet 438, and through tubing 454 into waste collection reservoir 434. One or more pumps are connected to a processor 499 that activates the pumps in a duty cycle as disclosed herein.

[0043] The inlets and outlets of reactor 410 can be used to fluidly couple the chamber with one or more additional vessels via fluid connectors. In certain embodiments, the additional vessels include one or more additional cell culture chambers, as will be described in more detail below. The systems of the present invention can include, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or any number between or above 100 cell culture chambers that are fluidly connected to each other in series and configured to produce immunotherapy products. Alternatively, or in addition, one or more cell culture chambers can be arranged in parallel to each other to enable the production of immunotherapy products for more than one individual at a time. In a preferred embodiment, the cell culture chambers of the bioreactor are connected via a sterile connection.

[0044] An exemplary configuration of the multi-bioreactor system can be found in Panels B and C of FIG. 5, along with additional details regarding the processes implemented using the present configuration provided below. As shown in FIG. 5, when a second bioreactor 510 is involved, the second cell culture chamber 520 is positioned to connect with the first cell culture chamber 420 via the outlet of the first chamber and the inlet of the second chamber. The connection is preferably a sterile connection. The connection allows for the injection of sterile air into the first cell culture chamber 420 and transfers the supernatant containing the expanded T cells into the second cell culture chamber 520. Alternative techniques known in the art of fluid flow may be employed to transfer the supernatant from the first cell culture chamber 420 to the second cell culture chamber 520.

[0045] Also, as shown, each bioreactor includes its own fluid and waste collection reservoir, pump, and associated tubing. However, it should be understood that the reservoirs and pumps can be shared among the bioreactors. The pumps can be connected to the same processor or different processors for controlling their duty cycles.

[0046] In certain embodiments, as shown in FIG. 5, a 1:1 ratio of cell culture inlet to cell culture outlet exists, such as when one or more bioreactors are arranged in series with each other. In other embodiments, the outlet to inlet ratio for at least a portion of the bioreactor is 1:2. For example, the outlet of one cell culture chamber 420 can be fluidly connected to the inlets of two cell culture chambers (not shown) such that the fluid flowing out of the first cell culture chamber 420 is split into two flows, one flow being sent into the second cell culture chamber and the second flow being sent into the third cell culture chamber. In this configuration, both the second and third cell culture chambers can be used to further stimulate and grow T cells. Additionally, or alternatively, one of the second or third cell culture chambers can be configured to enable monitoring of reaction and flow parameters using one or more sensors (e.g., for measuring temperature) operably coupled to the chamber. Thus, one of the chambers remains without additional sensors, and some of them may need to penetrate the walls of the cell culture chamber, which can add a risk of leakage and / or contamination.

[0047] FIG. 6 shows another embodiment of the multi-bioreactor system 900. The system 900 includes a first cell culture chamber 820 and a second cell culture chamber 920 having inlets 845 and 945 connected to tubing 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 pumping of fluid from the fluid reservoir 980 to the cell culture chambers 820 and 920. The pumps are controlled by a processor 999 that executes a duty cycle as described herein.

[0048] In certain embodiments, one or more bioreactors can be provided in a system containing modules for effecting various other processes prior to, in parallel with, or subsequent to the processes occurring within the cell culture chambers of the bioreactor. Other configurations of the multi-bioreactor system are also envisioned and are described in further detail in WO 2018 / 005521, the contents of which are incorporated herein by reference.

[0049] As described with respect to the various embodiments disclosed herein, the systems and methods involve computer components such as a memory for storing instructions related to a duty cycle and a processor for executing the instructions to thereby control a pump. Aspects of the present disclosure such as control of fluid movement through the system as described above and monitoring and control of various parameters can be implemented using any type of computing device such as a computer or programmable logic controller (PLC) including, for example, a central processing unit, or any combination of computing devices where each device implements at least a portion of the process or method. In some embodiments, the systems and methods described herein may be implemented using a handheld device such as a smart tablet, a smartphone, or a special device produced for the system.

[0050] The methods of the present disclosure can be implemented using software, hardware, firmware, hardwiring, or any combination thereof. Features implementing the functions can also be physically located in various positions, including where parts of the functions are distributed so as to be implemented in different physical locations (e.g., with wireless or wired connections, such as an imaging device in one room and a host workstation in another room or in a separate building).

[0051] Processors suitable for the execution of a computer program include, by way of example, both general and special purpose microprocessors, and any one or more processors of any kind of digital computer. Generally, a processor will receive instructions and data from a read only memory or a random access memory or both. Elements of a computer are a processor for executing instructions and one or more memory devices for storing instructions and data.

[0052] Generally, a computer is also operatively coupled to, or includes, or both, one or more non-transitory mass storage devices for storing data, such as magnetic, magneto-optical disks, or optical disks, or receives data therefrom, or transfers data thereto. 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 transmitted directly to the cloud rather than to a physical storage device. Information carriers suitable for embodying computer program instructions and data include, by way of example, all forms of non-volatile memory, including semiconductor memory devices (e.g., EPROM, EEPROM, solid state drives (SSD), and flash memory devices), magnetic disks (e.g., internal hard disks or removable disks), magneto-optical disks, and optical disks (e.g., CD and DVD disks). The processor and memory can be supplemented by, or incorporated in, dedicated logic circuitry.

[0053] To provide for interaction with a user, the subject matter described herein can be implemented on a computer having I / O devices, such as a CRT, LCD, LED, or projection device for displaying information to the user, and input or output devices such as a keyboard and a pointing device (e.g., a mouse or trackball) by which the user can provide input to the computer. Other kinds of devices can also be used to provide for interaction with a user as well. For example, feedback provided to the user can be any form of sensory feedback (e.g., visual feedback, auditory feedback, or tactile feedback), and input received from the user can be received in any form including acoustic, speech, or tactile input.

[0054] The subject matter described in this specification can be implemented in a computing system that includes back-end components (such as data servers), middleware components (such as application servers), or front-end components (such as client computers having a graphical user interface or web browsers through which a user can interact with an implementation of the subject matter described in this specification), or any combination of such back-end, middleware, and front-end components. The components of the system can be interconnected through a network, such as a communication network, by digital data communication in any form or medium. Examples of communication networks include cellular networks (such as 3G or 4G), local area networks (LANs), and wide area networks (WANs), such as the Internet.

[0055] The subject matter described in this specification can be implemented as one or more computer program products, such as one or more computer programs tangibly embodied in an information carrier (e.g., in a non-transitory computer-readable medium) for execution by, or to control the operation of, a data processing apparatus (e.g., a programmable processor, a computer, or multiple computers). A computer program (also known as a program, software, software application, app, macro, or code) can be written in any form of programming language, including compiled or interpreted languages (e.g., C, C++, Perl), and it can be deployed in any form, including as a stand-alone program or as modules, components, subroutines, or other units suitable for use in a computing environment. The systems and methods of the present invention can 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®.

[0056] A computer program does not necessarily correspond to a file. The program can be stored in a file or a part of a file that holds other programs or data, in a single file dedicated to the program, or in multiple coordinated files (e.g., files that store one or more modules, subprograms, or parts of code). A computer program can be executed on one computer or multiple computers at one location, or can be distributed across multiple locations and deployed to be interconnected by a communication network.

[0057] The file can be a digital file stored, for example, on a hard drive, SSD, CD, or other tangible non-transitory medium. The file can be transmitted from one device to another via a network (e.g., as packets transmitted from a server to a client through, for example, a network interface card, modem, wireless card, or the like).

[0058] Writing a file according to an embodiment of the present invention involves transforming a tangible non-transitory computer-readable medium, for example, by adding, removing, or rearranging particles (e.g., with a net charge or dipole moment to a magnetization pattern by a read / write head), where the pattern then represents a new collocation of information about an objective physical phenomenon desired by and useful to the user. In some embodiments, writing involves a physical transformation of the substance in the tangible non-transitory computer-readable medium (e.g., burning a CD-ROM, with certain optical properties so that an optical read / write device can then read a new useful collocation of information). In some embodiments, writing a file involves converting a physical flash memory device such as a NAND flash memory device and storing information by transforming physical elements in an array of memory cells made from floating gate transistors. Methods of writing files are well known in the art and can be exercised manually, or by a program, or automatically by a save command from software or a write command from a programming language.

[0059] Suitable computing devices typically include a large-capacity memory, at least one graphical user interface, and at least one display device, and typically include communication between devices. The large-capacity memory exemplifies a certain type of computer-readable medium, namely, a computer storage medium. A computer storage medium may 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 disk (DVD), or other optical storage devices, magnetic cassettes, magnetic tapes, magnetic disk storage devices, or other magnetic storage devices, radio frequency identification tags or chips, or any other medium that can be used to store desired information and can be accessed by a computing device.

[0060] Since those skilled in the art will recognize what is necessary or optimal for the implementation of the method of the present invention, the computer system or machine employed in 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) that communicate with each other via a bus, a main memory, and a static memory.

[0061] In the exemplary embodiment shown in FIG. 7, system 600 can include a computer 649 (e.g., a laptop, desktop, or tablet). Computer 649 may be configured to communicate across network 609. Computer 649 includes one or more processors 659, memory 663, and input / output mechanism 654. When the method of the present invention employs a client / server architecture, the operation of the method of the present invention can be implemented using a server 613 that includes one or more of processors 621 and memory 629 that can obtain data, instructions, etc., or provide results via interface module 625, or provide results as file 617. Server 613 may engage 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, memory 679, and input / output mechanism 671.

[0062] 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)) for any of I / O 649, 637, or 671. A 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 touch screen, an accelerometer, a microphone, a cellular radio frequency antenna, and a network interface device, which can be, for example, a network interface card (NIC), a Wi-Fi card, or a cellular modem.

[0063] Memory 663, 679, or 629 according to an exemplary embodiment of the present invention can include a machine-readable medium having stored thereon one or more sets of instructions (e.g., software) that embody any one or more of the methodologies or functions described herein. The software may also reside, in whole or in part, within main memory and / or within a processor during execution thereof by a computer system, and the main memory and the processor also constitute a machine-readable medium. The software may further be transmitted or received via a network through a network interface device.

[0064] (Incorporation by reference) References and citations to other documents such as patents, patent applications, patent publications, magazines, books, papers, web content, etc. have been made throughout this disclosure. All such documents are hereby incorporated by reference in their entirety for all purposes.

[0065] (Equivalents) Although the present invention has been described in conjunction with certain embodiments, those skilled in the art will, after reading the foregoing specification, be able to effect various changes in the compositions and methods described herein, substitution of their equivalents, and other modifications.

Claims

1. A cell culture system comprising: a cell culture chamber in fluid communication with the pump; A processor in communication with the pump, the processor coupled to a non-transitory computer readable memory including instructions, the instructions comprising: receiving as input data comprising a size of the cell culture chamber; Calculating a desired average fluid flow rate that will be introduced into the cell culture chamber; calculating one or more duty cycle parameters based on the desired average fluid flow rate; operating said pump in a loop of recurring duty cycles such that as one duty cycle ends another duty cycle begins, each recurring duty cycle having the same average flow rate; a processor and a program executable by the processor to cause the computer system to execute A cell culture system comprising:

2. The duty cycle is: an on cycle in which fluid flows into and out of the cell culture chamber; An off cycle in which fluid flow is stopped; 2. The system of claim 1, wherein the off cycle is longer than the on cycle.

3. The cell culture system of claim 2, wherein the duty cycle has a duration of from 1 second to 20 minutes.

4. The cell culture system of claim 3, wherein the duty cycle has a duration of 60 seconds.

5. The cell culture system of claim 2, wherein the on cycle lasts for less than 20% of the duration of the duty cycle.

6. The cell culture system of claim 1, wherein the one or more duty cycle parameters are further calculated based on one or more of the thermal output of the system and a desired cell culture temperature inside the incubator.

7. The cell culture system of claim 1, wherein the calculated duty cycle parameters are stored as a set of instructions executable by the processor to operate the pump.

8. The cell culture system of claim 7, wherein the set of instructions specifies, for each cycling duty cycle, the overall duration of the cycling duty cycle, the duration of an off cycle, and the duration of an on cycle.

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

10. The cell culture system of claim 1, wherein calculating the desired average flow rate further includes receiving as input data comprising a first concentration of a first cell type and a second concentration of a second cell type in one or more fluids that will be introduced into the cell culture chamber.

11. The cell culture system of claim 10, wherein the calculated desired average flow rate maximizes the probability that the first cell type and the second cell type will contact each other within the cell culture chamber.

12. The cell culture system of claim 1, wherein the same average flow rate is within the laminar flow range.

13. The cell culture system of claim 1, wherein the average flow rate is less than 1,000 μL of fluid per minute.

14. The cell culture system of claim 13, wherein the average flow rate is less than 10 μL per minute.

15. The cell culture system of claim 1, wherein the cell culture system is sized and configured to fit inside an incubator.

16. The cell culture system of claim 1, wherein the cell culture chamber has an inlet and an outlet.

17. The cell culture system of claim 16, further comprising a fluid reservoir in fluid communication with the inlet of the cell culture chamber.

18. The cell culture system of claim 16, wherein the pump is operably coupled to the fluid reservoir, the pump configured to pump fluid into the cell culture chamber via the inlet and remove waste from the cell culture chamber via the outlet.

19. The cell culture system of claim 1, wherein the duty cycle emulates static cell culture and avoids pulsating flow.

20. The cell culture system of claim 1, wherein the duty cycle exposes cultured cells in the cell culture chamber to a defined concentration of nutrients.

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