Duty Cycle for Cell Culture Systems

Abstract

An automated cell culture system with one or more pumps configured to operate on a duty cycle prevents excessive heat generation, allowing the cell culture system to operate inside a conventional incubator without overheating. Duty cycling involves switching the pump between on and off modes. By starting the pumps for short periods and then stopping them, less heat is generated. To account for reduced pumping time during a cycle, the pump can be started at a higher flow rate while it is on, so that the average flow rate over the course of the cycle is not reduced. The system of the present invention employs a duty cycle in which the on cycle is shorter than the off cycle, and in particular, the on cycle is less than 20% of the duration of the entire duty cycle.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of and priority to U.S. patent application Ser. No. 16 / 539,916, filed Aug. 13, 2019, the contents of which are incorporated by reference.

[0002] FIELD OF THEINVENTION The present invention relates generally to cell culture chambers, and more particularly to systems and methods for pumping fluids to and from cell culture chambers. [Background technology]

[0003] Many areas of clinical research and therapy require the isolation, preparation, and propagation of cell lines. To recreate 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 slight environmental fluctuations. For example, even a slight increase in temperature from 37°C to 39°C (i.e., the equivalent of a 102°F fever) can rapidly kill some cells. Cell culture processes can require culture for several days, meaning that an effective cell culture system needs to be capable of maintaining these precise conditions over long periods of time. Standalone cell culture systems can maintain temperature and gas levels, but are expensive and impractical for many laboratories. An alternative is to grow cells in a cell culture chamber inside a conventional incubator. However, while these arrangements are less expensive, they are less effective at maintaining precise temperature conditions because the equipment inside the incubator generates heat that cannot be easily dissipated. Summary of the Invention [Means for solving the problem]

[0004] The present disclosure provides an automated cell culture system with pumps configured to operate on a duty cycle, which prevents excessive heat generation and thereby allows the cell culture system to maintain a proper 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. While in conventional cell culture systems, much of the heat is generated by the pumping system used to pump medium and remove waste from the cell culture vessel or chamber, in the disclosed system, the pumps operate on a duty cycle, where the pumps switch between on and off modes. By starting the pumps for short periods of time and then stopping them, less heat is generated.

[0005] The present invention recognizes that simply starting a standard pump for a short period of time and then turning it off may not be sufficient to effectively deliver nutrients and remove waste products. To maintain the overall flow rate, the pump of the present invention operates at a higher flow rate while it is on to compensate for the reduced overall pumping time. However, to emulate static cell culture and avoid pulsatile flow that may stimulate certain biological processes and reactions, the disclosed cell culture system can be operated with a low level of perfusion flow within the laminar flow range. The optimal duty cycle for delivering nutrients and extracting waste products at low flow rates may therefore be less than 20%, meaning that the pump is 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 turned off completely for the next 48 seconds. Thus, the overall average flow rate is 10 microliters per minute, and the relatively long power off times significantly reduce the amount of heat generated over the course of the cycle.

[0006] Aspects of the 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 pump. The processor is configured to operate the pump with a rotating duty cycle, each cycle of the duty 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 other cycle of the duty cycle.

[0007] In embodiments, the cell culture system is sized and configured to fit inside the 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 medium to the cell culture chamber via the inlet and remove waste from the cell culture chamber via the outlet. During an 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 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 with a processor configured to implement a cycling duty cycle. Each cycle of the duty cycle has the same average flow rate and includes an on cycle during which fluid flows to and from the cell culture chamber, and an off cycle during which fluid flow is stopped. The off cycle is longer than the on cycle.

[0010] In some embodiments of the method, the cell culture system is sized and configured to fit inside the incubator. The cell culture chamber may include an inlet, an outlet, and a fluid reservoir in fluid communication with the inlet. In embodiments, operating the pump includes delivering cell medium to the cell culture chamber via the inlet and removing waste from the cell culture chamber via the outlet. During an 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. The present specification also provides, for example, the following items: (Item 1) 1. A cell culture system comprising: a cell culture chamber; one or more pumps in fluid communication with the cell culture chamber; a processor operatively connected to the one or more pumps, the processor configured to operate the one or more pumps with a cycling duty cycle, each cycle of the duty cycle comprising an on cycle and an off cycle that is 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) 2. The cell culture system of claim 1, wherein the cell culture system is sized and configured to fit inside an incubator. (Item 3) 2. The cell culture system of claim 1, wherein the cell culture chamber comprises an inlet and an outlet. (Item 4) 4. The cell culture system of item 3, further comprising a fluid reservoir in fluid communication with the inlet of the cell culture chamber. (Item 5) 4. The cell culture system of claim 3, wherein the pump is configured to deliver cell culture medium to the cell culture chamber via the inlet and remove waste material from the cell culture chamber via the outlet. (Item 6) 2. The cell culture system of claim 1, wherein during the on cycle, the pump forces the flow of fluid into and out of the cell culture chamber. (Item 7) 7. The cell culture system of claim 6, wherein the fluid comprises a cell culture medium. (Item 8) 2. The cell culture system of claim 1, wherein the duty cycle has a duration of about 60 seconds. (Item 9) 2. The cell culture system of claim 1, wherein the on cycle lasts for less than 20% of the duration of the duty cycle. (Item 10) 2. The cell culture system of claim 1, wherein the average flow rate is less than 1,000 μL of fluid per minute. (Item 11) 1. A method for culturing cells, the method comprising: providing a cell culture chamber in fluid communication with a pump; operating the pump with a processor configured to implement a revolving duty cycle, each revolving duty cycle having the same average flow rate and comprising an on cycle during which fluid flows to and from the cell culture chamber and an off cycle during which fluid flow is stopped, the off cycle being longer than the on cycle; A method comprising: (Item 12) 12. The method of claim 11, wherein the cell culture system is sized and configured to fit inside an incubator. (Item 13) 12. The method of claim 11, wherein the cell culture chamber comprises an inlet and an outlet. (Item 14) 14. The method of claim 13, further comprising a fluid reservoir in fluid communication with the inlet of the cell culture chamber. (Item 15) 14. The method of claim 13, wherein operating the pump comprises delivering cell culture medium to the cell culture chamber via the inlet and removing waste material from the cell culture chamber via the outlet. (Item 16) 12. The method of claim 11, wherein during the on cycle, the pump forces the flow of fluid into and out of the cell culture chamber. (Item 17) 17. The method of claim 16, wherein the fluid comprises a cell culture medium. (Item 18) Item 12. The method of item 11, wherein the duty cycle has a duration of about 60 seconds. (Item 19) Item 12. The method of item 11, wherein the on cycle lasts for less than 20% of the duration of the duty cycle. (Item 20) 12. The method of claim 11, wherein the average flow rate is less than 1,000 μL of fluid per minute. [Brief description of the drawings]

[0012] [Figure 1A] 1A and 1B show a cell culture system for use with the present invention. [Figure 1B] 1A and 1B show a cell culture system for use with the present invention. [Figure 2A] 2A and 2B show the cell differentiation cassette. [Figure 2B] 2A and 2B show the cell differentiation cassette. [Diagram 3] FIG. 3 shows a cell culture system using the cell differentiation cassette of FIGS. 2A and 2B. [Figure 4]FIG. 4 shows a system of the present invention with one cell culture chamber. [Diagram 5] FIG. 5 shows a process for connecting cell culture chambers and transferring fluids between them. [Figure 6] FIG. 6 shows a system of the present invention with multiple cell culture chambers. [Figure 7] FIG. 7 illustrates a system of the present invention, according to one embodiment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] (Detailed Description) The present disclosure provides a cell culture system including a pump that can be used inside a conventional incubator and is configured to run on a duty cycle to effectively maintain fluid flow while avoiding excessive heat generation. The disclosed system and related methods address the challenges with automated cell culture systems that sit inside a conventional incubator that need to dissipate heat. The most common source of heat in automated cell culture systems is the pumping system, which pumps medium and removes waste from the cell culture vessel / chamber. The pumping system according to the present disclosure generates less heat. The disclosed system and method are particularly useful in perfusion systems where pumps must be run continuously over a period of days. Perfusion systems are highly desirable in automated cell culture because perfusion (unlike static culture, where cells sit in a medium that becomes depleted of nutrients without replenishment, along with a continuous accumulation of waste) is a way to ensure that cultured cells are not only exposed to a defined concentration of nutrients, but also to ensure the continuous removal of metabolic waste.

[0014] The disclosed system can also be used with cell 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 incubation temperature is physiological temperature or 37° C. Because heat transfers from hot to cold areas along a temperature gradient, most incubators only have the ability to heat their interiors, not cool them. Furthermore, many incubators are designed with 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 would generally cause the interior temperature to rise rapidly. Once this occurs, the temperature control system would stop the heating. However, the insulating characteristics of the incubator would prevent the temperature from decreasing to the desired level in a timely manner. The present invention solves this problem by operating its pumps at a duty cycle that achieves the desired fluid flow levels while generating much less heat inside the incubator.

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

[0016] The optimal duty cycle for use with the present invention to deliver nutrients and extract waste products at low flow rates is for the pump or pumps to 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 turned off completely for the next 48 seconds. Thus, the overall average flow rate is still 10 microliters per minute, and the relatively long off time significantly reduces the amount of heat generated. In a conventional incubator, if the heat generation from the instrument is kept at less than 2 watts, undesirable temperature rise can be avoided.

[0017] In other embodiments, the duty cycle may 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 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 more. 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 with a duty cycle is useful for a variety of different cell culture systems and settings. Several embodiments of cell culture systems employing duty cycles to control pump activity according to the present disclosure are described below and illustrated in the figures.

[0019] FIG. 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 into and out of a cell culture cartridge 120. The pump 110 is operably connected to a processor 199 configured to receive instructions from a 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 during which the pump is on and the duration of the off cycle during which 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 as one duty cycle ends, another begins, which has the effect of turning the pump 110 on and off intermittently.

[0020] The cell culture cartridge 120 has a bottom surface 125 to which the 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 tubing 140 connects the fluid inlets with a differentiation medium reservoir (perfusion source) 180 that contains differentiation medium 182. The differentiation medium reservoir 180 contains differentiation medium 182 that will be pumped into the cell culture cartridge 120. The connecting tubing 140 also connects the fluid outlet 135 with a waste reservoir 184. 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 a sterile system. In other embodiments, the lids 170 and 175 are removable. Stopcocks and / or LAVs 160 and 165 on the reservoir bottles 180 and 184 allow for sterile transfer of differentiation medium, filling the inlet bottles and removing waste from the outlet bottles. The console 190 provides a designated space for the arrangement of the components mentioned above and also provides a display / user interface 192, connections 194, and an on / off switch 196.

[0021] FIG. 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, which is configured to receive instructions from a memory and 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 during which the pump is on and the duration of the off cycle during which 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 as one duty cycle ends, another begins, which has the effect of turning the pump 710 on and off intermittently.

[0022] The cell culture cartridge 720 has a bottom surface 725 to which the 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 with 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 that will be pumped into the cell culture cartridge 720. The connecting tubing 740 also connects the fluid outlet 735 with a waste reservoir 784, which is another bag. The decomposition medium reservoir 780 and the waste reservoir 784 are supported by struts 789. The depleted medium 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 above-mentioned components, and also provides a display / user interface 792 , connections 794 , and an on / off switch 796 .

[0023] Although system 100 is shown with a single pump, it is 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 cartridge and into waste reservoir 184. In embodiments having more than one pump, each pump may be connected to the same or different processors, each configured to activate its respective pump with a duty cycle that may have the same or different parameters as one another.

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

[0025] In certain embodiments, one or more pumps are operably coupled to the 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 cartridge can also include one or more fluid reservoirs. The fluid reservoirs can be in fluid communication with the cell culture chamber and operably coupled to the one or more pumps. One or more tubes for connecting the fluid reservoirs to the pumps and the cell culture chamber are also provided. In certain aspects, the one or more pumps are configured for pumping fluid from the fluid reservoirs, through the cell culture chamber, and into a waste collection reservoir. The one or more pumps are operably coupled to a processor that turns the 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 in communication with the processor. The processor, memory, and computer configuration are described in more detail below. In certain embodiments, fluid travels from a fluid reservoir, through tubing to a pump and into a cell culture chamber via an inlet, back out of the cell culture chamber via an outlet, and through tubing into a waste collection reservoir.

[0026] In some embodiments, the fluid reservoir and / or waste collection reservoir can be provided as one or more capped bottles, 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 an outlet that are fluidly coupled to the inlet of one or more cell culture chambers. In some aspects, 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 inlets or outlets.

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

[0028] Thus, in one embodiment, the pump is housed separately from the cell culture cartridge, but is still in fluid and operative 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. While the duty cycle operation of the pump is sufficient by itself to prevent overheating, in some embodiments, the system may be operatively connected to a heat sink and / or fan for additional heat dissipation. Regardless of the configuration, the pump is operatively coupled to a processor for implementing the duty cycle, and the pump is also operatively coupled to the cell culture cartridge, and thus the cell culture chamber. Additional details regarding perfusion-based automated cell culture systems, such as a small-scale culture system for endothelial cell culture with on-board reagent storage and perfusion enabled by an on-board disposable peristaltic pump and a larger scale culture system for dendritic cell generation from monocytes using chambers with polystyrene bottoms, 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 yet another aspect, the cell culture chamber includes one or more sensors operably coupled to the cell culture chamber. The sensors may be capable of measuring any suitable parameter. For example, the sensors may be capable of measuring one or more parameters in 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, the one or more sensors can be coupled to one or more of the cell culture chambers. In an embodiment, the one or more sensors are coupled to one or more cell culture chambers, but not to all of the chambers in the system. In other embodiments, the one or more sensors are coupled to all of the cell culture chambers in the system. In a system having multiple chambers operably coupled to one or more sensors, the sensors may be the same in each of the chambers to which they are coupled, they may all be different, or some sensors may be the same and some may be different. In one aspect, the one or more sensors are operatively 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 computer systems for implementing the methods of the invention using cell culture chambers are provided below.

[0030] In some embodiments, one or more sensors can measure the temperature in one or more cell culture chambers, fluid reservoirs, tubing, and / or incubator in general. The temperature sensor can provide feedback to the processor controlling the duty cycle indicating whether the on cycle 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 increasing, 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 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 a fluid connector. In some embodiments, the additional container includes 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 greater than 100 cell culture chambers that are fluidly connected to each other in series and configured to generate immunotherapy products. Alternatively or in addition, one or more cell culture chambers can be arranged in parallel with each other to allow for the generation 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 medium and cytokines can be provided to the cell mixture in the cell culture chamber to aid in the formation of cell-based immunotherapy products. In the plate-based protocol for stimulation of T cells with DCs, a culture volume of about 2 mL is maintained from the start, and injection of cytokines is performed twice within each 7-day stimulation period. The major advantage of perfusion is the ability to maintain a consistent local concentration profile of medium and cytokines, which ensures higher yields and the potential to speed up 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, along with the high sensitivity of DCs to mechanical forces, poses challenges for the stimulation and proliferation of antigen-specific T cells, especially with regard to the flow of fluids through the cell culture chamber. Thus, in those embodiments in which medium and cytokines are provided via perfusion, the system of the present invention must be capable of supplying nutrients and cytokines to the cells without removing them from the cell culture cartridge, while also taking into account the shear sensitivity of certain antigen-presenting cells such as DCs. Essentially, the system and method of some embodiments of the present invention aim to optimize the retention of autocrine / paracrine signals favoring T cell proliferation while renewing growth factors and maintaining minimal physical stimulation of 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 medium flow arrangements other than unidirectional flow, such as countercurrent medium flow arrangements.

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

[0034] The systems of the invention can also include or be operatively coupled to one or more control systems for controlling the movement of fluids through the system, monitoring and controlling various parameters within the system, such as temperature, as well as detecting the presence of cell-based immunotherapy product, product quantity (directly or indirectly), conversion rate, etc. The systems may also be equipped with numerous classes of software, such as advanced real-time process monitoring and control processes that allow feedback control, and processes that allow integration and scale-up given reaction and purification results obtained using the systems.

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

[0036] 2A-2B show the design of a dendritic cell differentiation cassette compatible with the present invention. The cassette 200 is constructed from the layers shown on the left side of FIG. 2A, assembled with 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, which forms a central open fluid space within the cassette. The floor of the chamber is, or contains as part of, the MC binding surface. The preferred geometry of the cell culture chamber is that of a flat thin space, with all its interior rounded, with no corners or vertices. An oval or rounded rectangular profile of the chamber is preferred. The flat surfaces and low height help to avoid turbulence that would lead to fluid shear stresses that could disturb 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 exposure of the cells therein to shear stresses. This is accomplished by the use of flat surfaces with little protrusions or surface roughness, by the avoidance of sharp boundaries in the fluid paths 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), where possible, and by the inclusion of a bubble trap or venting mechanism for the elimination of gas bubbles during perfusion of the cell culture chamber. The achievement of both laminar flow and the elimination of gas bubbles is aided by the positioning of the inlet and outlet ports on opposite sides of the cell growth chamber, as shown in FIG. 2A. Additionally, the cassette can be mounted at an angle, and the outlet port positioned above the level of the inlet port, to ensure that any air bubbles entering the cell growth chamber through the inlet port are rapidly eliminated at the outlet port by rising to the outlet port, assisted by their buoyancy.

[0037] The fluidic devices of the present invention, including the dendritic cell differentiation cassette or any cell growth or culture chamber, can be provided in either microfluidic embodiments (i.e., one or more channels or chambers therein have dimensions in the range of about 1 μm to about 999 μm) or macrofluidic embodiments (all of the channels or chambers therein have dimensions of about 1 mm or greater). The fluidic devices can further include fluid reservoirs, additional fluidic channels or compartments, gaskets or seals, mixing areas, valves, pumps, outlets, channels for pressurized gases, electrical conductors, reagents, ports, and tubing as required by the particular design. They may also contain one or more control modules, transmitters, receivers, processors, memory chips, batteries, displays, buttons, controls, motors, pneumatic actuators, antennas, electrical connectors, and the like. The devices preferably contain 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, chemical functionalized coatings, proteins, antibodies, glycoproteins, lipids, glycolipids, nucleic acids, proteoglycans, glycosaminoglycans, cytokines, or cells. The device is also preferably compatible with use in standard mammalian cell culture vessels, and in some embodiments does not allow diffusion of gases through the material, as this may alter the composition of the medium within the device. The fluidic devices of the present invention are also preferably modular, capable of fluid connection to other similar devices, either in series (i.e., fluid flows from one device into another) or in parallel, and may be configured to physically stack with one another, or physically arranged within related devices such as culture vessels, pumps, or dendritic cell development systems. The fluidic devices of the present invention are preferably free of fluid leakage under operating conditions and capable of sterile operation over periods 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] The dendritic cell generation system employing the cassette of FIGS. 2A-2B includes at least a cell culture chamber, a pump, a media reservoir, and a fluid connection between the media 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 media 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 media 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 the fluid inlet port of one or more dendritic cell differentiation cassettes, a fluid collection reservoir fluidly coupled to the fluid outlet port of one or more dendritic cell differentiation cassettes, and provided as a pump configured to pump fluid from the media reservoir, through the cell culture chamber of one or more dendritic cell differentiation cassettes, into the fluid collection reservoir.

[0040] An embodiment of a DC generation system 300 is depicted in Figure 3. The system includes a housing 310 with space to contain a medium reservoir 340 and a waste reservoir 350 (the size and shape of commercially available glass or plastic medium bottles with plastic caps, respectively), a mounting area for a DC differentiation cassette 200, an exposed peristaltic pump head configured to receive peristaltic pump tubing leading from the medium bottles to the inlet port of the cassette (separate tubing leading from the outlet port of the cassette to the waste bottle does not need to pass through the pump head), a display 330, and a control button, knob, or switch. The pump is controlled by a processor 299, which is configured to activate the pump with 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., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more cassettes and pump heads, one per cassette, etc.) are also envisioned. In such multi-cassette systems, the processor, control electronics, displays, and buttons, knobs, or switches may either be shared among the different cassettes or duplicated, one set per cassette.

[0042] In another exemplary embodiment, as shown in FIG. 4, a bioreactor 410 is provided that includes a cell culture chamber 420 that includes 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 an embodiment, the bioreactor 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 the chamber 420, and associated inlets 436 and outlets 438 for transporting fluids to and from the reservoirs 432, 434 through the chamber 420. The bioreactor 410 will also include one or more pumps 440 operably 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. The fluid reservoirs 432 can be in fluid communication with the cell culture chamber 410 and operably coupled to the one or more pumps 440. One or more tubes for connecting the fluid reservoirs to the pumps and the cell culture chambers are also provided. In certain aspects, the one or more pumps are configured to pump fluid from the fluid reservoirs, through the cell culture chamber, and into a waste collection reservoir. In the exemplary embodiment shown in FIG. 4, fluid travels from the fluid reservoirs 432 through tubing 452 to the pump 440, into the cell culture chamber 420 via the inlet 436, back out of the cell culture chamber 420 via the outlet 438, through tubing 454, and into the waste collection reservoir 434. One or more pumps are connected to a processor 499 that activates the pumps with a duty cycle as disclosed herein.

[0043] The inlets and outlets of the reactor 410 can be used to fluidly couple the chamber with one or more additional containers via fluid connectors. In an embodiment, the additional containers include one or more additional cell culture chambers, as will be described in more detail below. 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 greater than 100 cell culture chambers fluidly connected with each other in series and configured to generate immunotherapy products. Alternatively or in addition, one or more cell culture chambers can be arranged in parallel with each other to allow for the generation 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 sterile connections.

[0044] An exemplary configuration of a multiple bioreactor system can be found in panels B and C of FIG. 5, with additional details regarding the processes carried out using this configuration provided below. As shown in FIG. 5, in the case where a second bioreactor 510 is involved, the second cell culture chamber 520 is moved into position 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, transferring 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 reservoirs, pumps, and associated tubing. However, it should be understood that reservoirs and pumps may be shared between bioreactors. The pumps can be connected to the same processor or different processors for controlling their duty cycles.

[0046] In an embodiment, such as when one or more bioreactors are arranged in series with one another as shown in FIG. 5, there is a 1:1 ratio of cell culture inlets to cell culture outlets. In other embodiments, the ratio of outlets to inlets for at least some of the bioreactors 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 streams, one stream being delivered into the second cell culture chamber and the second stream being delivered into the third cell culture chamber. In this configuration, both the second and third cell culture chambers can be used to further stimulate and expand 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. In this way, one of the chambers is left without additional sensors, some of which may need to penetrate the walls of the cell culture chamber, which may add risk of leakage and / or contamination.

[0047] 6 shows another embodiment of a multiple 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, which implements a duty cycle as described herein.

[0048] In certain embodiments, one or more bioreactors can be provided in a system that contains modules for effecting various other processes prior to, in parallel with, or subsequent to the processes carried out in the cell culture chambers of the bioreactors. Other configurations of multiple bioreactor systems 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 various embodiments disclosed herein, the systems and methods involve computer components such as a memory for storing instructions related to duty cycles and a processor for executing instructions and thereby controlling the pump. Aspects of the disclosure described herein, such as controlling the movement of fluids through the system and monitoring and controlling various parameters as described above, can be implemented using any type of computing device, such as a computer or programmable logic controller (PLC), including a processor, e.g., a central processing unit, or any combination of computing devices, each device performing 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, e.g., a smart tablet, a smartphone, or a specialized device produced for the system.

[0050] The methods of the present disclosure can be implemented using software, hardware, firmware, hardwiring, or any combination of these. Features implementing functionality can also be physically located in various locations, including being distributed such that some of the functionality is implemented in different physical locations (e.g., an imaging device in one room and a host workstation in another room or in a separate building, with wireless or wired connections).

[0051] Processors suitable for the execution of a computer program include, by way of example, both general purpose 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, the computer will also include one or more non-transient mass storage devices for storing data, such as magnetic, magneto-optical, or optical disks, or be operatively coupled to receive data from or transfer data to, or both. In some embodiments, the sensors on the system transmit process data via Bluetooth to a central data collection unit located outside the incubator. In some embodiments, the 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 all forms of non-volatile memory, including, by way of example, 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 an I / O device, e.g., a CRT, LCD, LED, or projection device, for displaying information to the user, and input or output devices, such as a keyboard and pointing device (e.g., a mouse or trackball), by which the user may provide input to the computer. Other types of devices can be used to provide 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 from the user can be received in any form, including acoustic, speech, or tactile input.

[0054] The subject matter described herein can be implemented in a computing system that includes a back-end component (e.g., a data server), a middleware component (e.g., an application server), or a front-end component (e.g., a client computer having a graphical user interface or web browser through which a user may 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 through a network, e.g., a communications network, by any form or medium of digital data communication. Examples of communications networks include a cellular network (e.g., 3G or 4G), a local area network (LAN), and a wide area network (WAN), e.g., the Internet.

[0055] The subject matter described herein 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 device (e.g., a programmable processor, computer, or multiple computers). The computer programs (also known as programs, software, software applications, apps, macros, or code) can be written in any form of programming language, including compiled or interpreted languages ​​(e.g., C, C++, Perl), which can be deployed in any form, including as a stand-alone program or as a module, component, subroutine, or other unit 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. A program can be stored among files or portions of files that hold other programs or data, in a single file dedicated to the program, or in multiple associated files (e.g., a file that stores one or more modules, subprograms, or portions of code). A computer program can be deployed to be executed on one computer or multiple computers at one site, or distributed across multiple sites and interconnected by a communications network.

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

[0058] Writing a file according to embodiments of the present invention involves transforming a tangible non-transitory computer readable medium, for example by adding, removing, or rearranging particles (e.g., involving net charge or dipole moment into a pattern of magnetization by a read / write head), which then represents a new collocation of information about an objective physical phenomenon desired by and useful to a user. In some embodiments, writing involves a physical transformation of matter in the tangible non-transitory computer readable medium (e.g., involving certain optical properties, such that an optical read / write device can then read the new useful collocation of information, e.g., burning a CD-ROM). In some embodiments, writing a file involves transforming a physical flash memory device, such as a NAND flash memory device, to store 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 invoked manually, or programmatically, or automatically, for example, by a save command from software or a write command from a programming language.

[0059] A suitable computing device typically includes mass memory, at least one graphical user interface, at least one display device, and typically includes communication between devices. Mass memory illustrates a type of computer-readable medium, i.e., computer storage media. Computer storage media may include volatile, non-volatile, removable, and non-removable media implemented in any method or technology for storage of 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 technology, CD-ROM, digital versatile disks (DVDs), 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 that can be accessed by a computing device.

[0060] As those skilled in the art will recognize as necessary or optimal for the practice of the methods of the present invention, a 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), a main memory, and a static memory, in communication with each other via a bus.

[0061] In the exemplary embodiment shown in FIG. 7, the system 600 may include a computer 649 (e.g., a laptop, desktop, or tablet). The computer 649 may be configured to communicate across a network 609. The computer 649 includes one or more processors 659 and memory 663, and input / output mechanisms 654. When the method of the present invention employs a client / server architecture, the operations of the method of the present invention may be implemented using a server 613, including one or more of a processor 621 and memory 629, which may obtain data, instructions, etc., or provide results via an interface module 625, or provide results as a file 617. The server 613 may engage via the computer 649 or a terminal 667 via the network 609, or the server 613 may be directly connected to the terminal 667, including one or more processors 675 and memory 679, and input / output mechanisms 671.

[0062] System 600 or a machine according to an exemplary embodiment of the 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. Computer systems or machines 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 generation device (e.g., a speaker), a touch screen, 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.

[0063] Memory 663, 679, or 629 according to an exemplary embodiment of the invention may include a machine-readable medium on which one or more sets of instructions (e.g., software) are stored that embody any one or more of the methodologies or functions described herein. The software may also reside, completely or at least partially, within the primary memory and / or within the processor during its execution by the computer system, with the primary memory and processor also constituting machine-readable media. The software may further be transmitted or received over a network via a network interface device.

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

[0065] (Equivalent) While the present invention has been described in conjunction with certain embodiments, those skilled in the art, after perusal of the foregoing specification, will be able to effect various modifications of the compositions and methods described herein, substitutions of equivalents thereof, and other alterations.

Claims

1. 1. A cell culture system comprising: a cell culture chamber; one or more pumps in fluid communication with the cell culture chamber, the one or more pumps operably coupled to one or more perfusion fluid reservoirs and configured to pump medium into and out of the cell culture chamber via inlets and outlets disposed on the cell culture chamber; a processor operatively connected to the one or more pumps, the processor configured to operate the one or more pumps in a continuous loop of recurring duty cycles such that as one duty cycle ends another duty cycle begins, each recurring duty cycle comprising an on cycle during which fluid flows to and from the cell culture chamber and an off cycle during which fluid flow is stopped, the off cycle being longer than the on cycle; a memory in communication with the processor, the memory including one or more calculated duty cycle parameters stored as a set of instructions executable by the processor to operate the one or more pumps in a continuous loop of the cycling duty cycles, the set of instructions defining, for each cycling duty cycle, an overall duration of the cycling duty cycle, a duration of an off cycle, and a duration of an on cycle, such that each cycling duty cycle has the same average flow rate; and A cell culture system comprising:

2. 10. The cell culture system of claim 1, wherein the cell culture system is sized and configured to fit inside 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 on cycle, the one or more pumps are 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.

5. 10. The cell culture system of claim 1, wherein during the on cycle, the pump forces a flow of fluid into and out of the cell culture chamber.

6. The cell culture system of claim 5 , wherein the fluid comprises a cell culture medium.

7. 10. The cell culture system of claim 1, wherein the duty cycle has a duration of 60 seconds.

8. 2. The cell culture system of claim 1, wherein the on cycle lasts for less than 20% of the duration of the duty cycle.

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

10. 1. A method for culturing cells, the method comprising: providing a cell culture chamber in fluid communication with a pump; operating the pump with a processor configured to implement a cycling duty cycle, the processor comprising a memory in communication with the processor, the memory including one or more calculated duty cycle parameters stored as a set of instructions executable by the processor to operate the pump with the cycling duty cycle, the set of instructions defining an overall duration of the cycling duty cycle, a duration of an off cycle, and a duration of an on cycle, each cycle of the duty cycle comprising the same average flow rate, comprising an on cycle during which fluid flows to and from the cell culture chamber and an off cycle during which fluid flow is stopped, the off cycle being longer than the on cycle, the cell culture chamber comprising an inlet and an outlet; A method comprising:

11. The method of claim 10, wherein the method is performed by a cell culture system comprising the cell culture chamber, the pump and the processor, the cell culture system being sized and configured to fit inside an incubator.

12. The method of claim 10 , further comprising a fluid reservoir in fluid communication with the inlet of the cell culture chamber.

13. 11. The method of claim 10, wherein operating the pump comprises delivering cell culture medium to the cell culture chamber via the inlet and removing waste material from the cell culture chamber via the outlet.

14. The method of claim 10, wherein during the on cycle, the pump forces fluid flow into and out of the cell culture chamber.

15. The method of claim 14 , wherein the fluid comprises a cell culture medium.

16. The method of claim 10 , wherein the duty cycle has a duration of 60 seconds.

17. The method of claim 10 , wherein the on cycle lasts for less than 20% of the duration of the duty cycle.

18. 11. The method of claim 10, wherein the average flow rate is less than 1,000 μL of fluid per minute.

19. The cell culture system of claim 1, wherein the system calculates the one or more duty cycle parameters based on a desired average flow rate, a thermal output of the system, and a desired cell culture temperature inside the incubator.

20. 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.

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