Cell culture systems and uses thereof

Abstract

To provide cell culture systems and uses thereof.SOLUTION: Systems for monitoring and controlling cell culture comprise a cell culture apparatus operably associated with a controller. The controller comprises a hardware processor coupled to memory containing instructions executable by the processor to cause the controller to receive data associated with cells to be cultured; connect to one or more databases to receive cell culture protocol data; and determine a cell culture protocol for the cells to be cultured. Methods of determining a cell culture protocol comprise receiving data associated with cells to be cultured; connecting to one or more databases to receive data about cell culture protocols; and determining a cell culture protocol for the cells to be cultured.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of and priority to U.S. Provisional Patent Application No. 62 / 828,696, filed April 3, 2019, the contents of each of which are incorporated by reference.

[0002] The present invention relates generally to cell culture methods and systems. [Background technology]

[0003] Cell culture is an important tool in biological research and is used in studies related to cancer, vaccines, and protein therapeutics. The process of cell culture involves maintaining cells outside their native body under precise conditions.

[0004] Typically, laboratory technicians follow existing protocols for a particular cell type when performing cell culture procedures. However, existing cell culture procedures involve many physical steps, extensive monitoring, and are laborious and time-consuming, performed by laboratory technicians. The lack of automation and biases caused by laboratory technicians, i.e., the use of introduced cell culture protocols without any additional input, hinder the development and optimization of cell culture procedures. Summary of the Invention [Means for solving the problem]

[0005] The present invention provides a method and system for determining a cell culture protocol to provide an adjusted cell culture procedure. The device according to the present invention is equipped with sensors and controllers to enable precise cell culture condition monitoring and control. The system of the present invention is also configured to communicate with a database containing data related to the cell culture procedure. The system and method of the present invention uses data obtained from the database, real-time feedback from the sensors, or a combination thereof to determine and, optionally, optimize the current cell culture procedure to provide an adjusted cell culture procedure. Data from the adjusted cell culture procedure may in turn be stored in the database and used for future cell culture procedures.

[0006] By communicating with one or more databases, cell culture procedure data from the databases can be reviewed, analyzed, and considered with respect to using the data as input for adjustment of the current cell culture procedure. For example, the database may be a publicly available database with an unlimited number of cell culture protocol data available, or the database may alternatively be an internal database, such as a database containing information on cell culture procedures already performed for that cell type. In some cases, a combination of public and internal databases is accessed and information is pulled from both databases to create an adjusted cell culture protocol. The systems and methods of the present invention then use that input, optionally together with real-time feedback data from sensors, to create, execute, and optionally optimize a cell culture procedure, thereby performing an adjusted or personalized cell culture procedure. Among other things, the present invention reviews data from the databases and provides customized cell culture procedures in a timely manner. If a laboratory technician were to review even a portion of the data from the unlimited number of cell culture protocol data available from public databases, the duration of determining the current cell culture procedure would increase exponentially.

[0007] In some embodiments, the process is fully automated without any interference or input from a laboratory technician. In other embodiments, input from a laboratory technician may be useful or required. In such embodiments, the system of the present invention may be designed to have alerting, monitoring, and / or decision-making capabilities. By providing such capabilities to the system of the present invention, user (e.g., laboratory technician) input is kept to a minimum, saving countless hours in determining cell culture procedures and eliminating any bias the user may have, such as from past cell culture experiments.

[0008] In some embodiments of the present invention, the cell culture systems, devices, and methods have alert capabilities. For example, if the levels of pH, dissolved oxygen, total biomass, cell diameter, or temperature fall outside of user-defined or system-learned ranges, the system will send an alert to the user. In some cases, the alert may have the terminal form of an email alert, an audio alert, a text alert, or a combination thereof.

[0009] In some embodiments of the present invention, the systems, devices, and methods have monitoring capabilities. For example, profiles of pH, dissolved oxygen, total biomass, cell diameter, and temperature are read from the system. The profiles may be transmitted to a network such as the cloud, where they may be read by any compatible device (e.g., a smartphone) in a continuous readout format.

[0010] In some embodiments of the present invention, the systems, devices, and methods have decision-making capabilities. For example, if the levels of pH, dissolved oxygen, total biomass, cell diameter, or temperature fall outside user-defined or system-learned thresholds, the system will make a decision. Examples of decisions include deciding to terminate the culture process, to stop further reagent use, to alert the user, and to shut down the system.

[0011] One aspect of the invention is directed to a system for monitoring and controlling cell culture, the system comprising a cell culture device operatively associated with a controller comprising a hardware processor coupled to a memory containing instructions executable by the processor to cause the controller to receive data associated with cells to be cultured, connect to one or more databases to receive cell culture protocol data, and determine a cell culture protocol for the cells to be cultured.

[0012] The controller may be any suitable controller. In some embodiments of the invention, the controller is integrated. In other embodiments, the controller is distributed.

[0013] Some embodiments of the invention are directed to single-use components. In some examples, the cell culture device is a single-use cell culture device. In some examples, the cell culture device includes one or more sensors communicatively coupled to the controller to provide data regarding the cells. In some examples of the invention, the one or more sensors are single-use sensors.

[0014] In an embodiment of the present invention, the controller is further configured to update the cell culture protocol based on feedback from one or more sensors during cell culture. The feedback may be any suitable feedback from the sensor. In an embodiment, the feedback is related to at least one of pH, glucose concentration, lactate concentration, dissolved oxygen, total biomass, cell diameter, temperature, cell type, medium type, and fluid flow rate.

[0015] Any suitable database may be used in the system of the present invention to be connected to receive cell culture protocol data. In an embodiment, the one or more databases are databases that comprise one or more cell culture protocols previously developed by the system. In an embodiment, the one or more databases are publicly available databases that comprise one or more cell culture protocols. Those skilled in the art will recognize databases that are suitable for use with the present invention. For example, those skilled in the art may use the cell culture database described in Cell-culture Database: Literature-based reference tool for human and mammalian experimentally based cell culture applications; Amirkia and Qiubao, Bioinformation, 2012, 8(5): 237-238 (incorporated herein by reference in its entirety).

[0016] One aspect of the invention is directed to a method of determining a cell culture protocol, the method including receiving data associated with cells to be cultured, connecting to one or more databases to receive the data regarding the cell culture protocol, and determining a cell culture protocol for the cells to be cultured.

[0017] In some embodiments of the present invention, the method further includes updating the cell culture protocol based on feedback during cell culture. The feedback is from one or more sensors disposed on the cell culture device and communicatively coupled to the controller. In some embodiments, the feedback is associated with at least one of pH, glucose concentration, lactate concentration, dissolved oxygen, total biomass, cell diameter, temperature, cell type, medium type, and fluid flow rate.

[0018] Any suitable database may be used in the method of the present invention.In some embodiments, one or more databases are databases that comprise one or more cell culture protocols that have been previously developed by the system for monitoring and controlling cell culture.In some embodiments, one or more databases are publicly available databases that comprise one or more cell culture protocols.

[0019] In some embodiments of the invention, the determined cell culture protocol is personalized based on the received data associated with the cells to be cultured. In some embodiments of the invention, the determined personalized cell culture protocol is personalized based on the received data associated with the cells to be cultured for the human subject.

[0020] An aspect of the present invention is directed to a method of determining a personalized cell culture protocol. The method includes receiving data associated with cells to be cultured for a human subject, connecting to one or more databases to receive data about the cell culture protocol, and determining a personalized cell culture protocol for the cells to be cultured for the human subject. In some embodiments, the method of the present invention further includes updating the personalized cell culture protocol based on feedback during cell culture. The feedback is from one or more sensors disposed on the cell culture device and communicatively coupled to the controller. In some embodiments, the feedback is associated with at least one of pH, glucose concentration, lactate concentration, dissolved oxygen, total biomass, cell diameter, temperature, cell type, medium type, and fluid flow rate.

[0021] The method of the present invention further includes a step of reporting the determined cell culture protocol. The report includes information about the steps performed in the adjusted cell culture procedure, including non-limiting examples of temperature, pH, media type, fluid flow rates, and duration of each step of the procedure. In some examples, the report is a printed report or is presented on a user display screen of the system, such as a cell phone, tablet, or laptop.

[0022] In some embodiments, the systems and methods of the present invention use data from a public database for use in determining a cell culture protocol. A suitable public database comprises data regarding one or more cell culture protocols. In an embodiment, the systems and methods of the present invention use data from an internal database for use in determining a cell culture protocol. The internal database may include information regarding cell protocols previously used in a laboratory setting. For example, the database may include information obtained from a cell device setting and information from a lab notebook. The information in the internal database may include any relevant information regarding the cell culture protocol, such as the cell type, media type, pH, temperature, duration of the culture step, and fluid flow rate used during culture. In other embodiments, the systems and methods of the present invention use data from a combination of databases for use in determining a cell culture protocol. The databases may be publicly available databases, internal databases, or a combination thereof. In an embodiment, the systems and methods of the present invention use data from one or more databases and also include feedback data from sensors for use in determining a cell culture protocol. The feedback data includes data from a plurality of sensors monitoring the conditions of the cell culture procedure.

[0023] In an embodiment, a controller operatively associated with the cell culture device receives data associated with the cells to be cultured, such as cell type. The controller then connects to a database, which may be any suitable public or internal database comprising one or more cell culture protocols. The controller receives the cell culture protocol data from the database and uses the data to determine the current cell culture protocol. In some cases, the determined cell culture protocol comprises a protocol pulled directly from a public or internal database. In some cases, the determined cell culture protocol may be used immediately for cell culture. The determined cell culture protocol may also be stored for future use, such as stored in an internal database.

[0024] In some cases, the controller may also receive data from multiple sensors on the cell culture device, such as temperature, pressure, pH, temperature, and fluid flow rates. The data obtained from the sensors is used to modify the cell culture protocol obtained from the database, thereby determining the cell culture protocol based on the data obtained from the database and the feedback data. Such determined cell culture protocol may be used immediately for cell culture. The determined cell culture protocol may also be stored for future use, such as in an internal database. The present invention provides, for example, the following: (Item 1) 1. A system for monitoring and controlling a cell culture, the system comprising: 1. A cell culture device operatively associated with a controller, the controller comprising a hardware processor coupled to a memory, the memory containing instructions that cause the controller to: Receiving data associated with the cells to be cultured; connecting to one or more databases to receive cell culture protocol data; determining a cell culture protocol for the cells to be cultured; a cell culture device operable by a processor to cause the cell culture device to A system comprising: (Item 2) 2. The system of claim 1, wherein the controller is integrated. (Item 3) 2. The system of claim 1, wherein the controller is distributed. (Item 4) 2. The system of claim 1, wherein the cell culture device is a single-use cell culture device. (Item 5) 2. The system of claim 1, wherein the cell culture device comprises one or more sensors communicatively coupled to the controller to provide data regarding the cells. (Item 6) Item 6. The system of item 5, wherein the one or more sensors are single-use sensors. (Item 7) 2. The system of claim 1, wherein the controller is further configured to update the cell culture protocol based on feedback from the one or more sensors during cell culture. (Item 8) 8. The system of claim 7, wherein the feedback is related to at least one of pH, glucose concentration, lactate concentration, dissolved oxygen, total biomass, cell diameter, temperature, cell type, medium type, and fluid flow rate. (Item 9) 2. The system of claim 1, wherein the one or more databases are a database comprising one or more cell culture protocols previously developed by the system. (Item 10) 2. The system of claim 1, wherein the one or more databases are publicly available databases comprising one or more cell culture protocols. (Item 11) 2. The system of claim 1, wherein the determined cell culture protocol is personalized based on the received data associated with the cells to be cultured. (Item 12) 1. A method for determining a cell culture protocol, comprising: Receiving data associated with the cells to be cultured; connecting to one or more databases to receive data about a cell culture protocol; determining a cell culture protocol for the cells to be cultured; A method comprising: (Item 13) 13. The method of claim 12, further comprising updating the cell culture protocol based on feedback during cell culture, the feedback being from one or more sensors disposed on the cell culture device and communicatively coupled to a controller. (Item 14) 14. The method of claim 13, wherein the feedback is associated with at least one of pH, glucose concentration, lactate concentration, dissolved oxygen, total biomass, cell diameter, temperature, cell type, medium type, and fluid flow rate. (Item 15) 13. The method of claim 12, wherein the one or more databases are a database comprising one or more cell culture protocols previously developed by a system for monitoring and controlling cell culture. (Item 16) 13. The method of claim 12, wherein the one or more databases are publicly available databases comprising one or more cell culture protocols. (Item 17) 13. The method of claim 12, wherein the determined cell culture protocol is personalized and optimized based on the received data associated with the cells to be cultured. (Item 18) 13. The method of claim 12, further comprising reporting the determined cell culture protocol. (Item 19) 20. The method of claim 18, wherein reporting includes providing an alert when a level falls outside a defined range. (Item 20) 20. The method of claim 19, wherein the alert comprises an email alert, an audio alert, a text alert, or a combination thereof. (Item 21) 20. The method of claim 19, wherein the level comprises a pH level, a dissolved oxygen level, a total biomass level, a cell diameter level, or a temperature level. (Item 22) 20. The method of claim 18, wherein reporting further comprises providing the monitoring information to a user. (Item 23) 23. The method of claim 22, wherein the monitoring information comprises profiles of pH, dissolved oxygen, total biomass, cell diameter, and temperature. (Item 24) 13. The method of claim 12, wherein determining the cell culture protocol further comprises deciding to terminate the culture process, stop using additional reagents, alert the user, or shut down the system. (Item 25) 1. A method for determining a personalized cell culture protocol, comprising: Receiving data associated with the cells to be cultured regarding the human subject; connecting to one or more databases to receive data about a cell culture protocol; determining a personalized cell culture protocol for cells to be cultured for said human subject; A method comprising: (Item 26) 26. The method of claim 25, further comprising updating the personalized cell culture protocol based on feedback during cell culture, the feedback being from one or more sensors disposed on the cell culture device and communicatively coupled to a controller. (Item 27) 27. The method of claim 26, wherein the feedback is associated with at least one of pH, glucose concentration, lactate concentration, dissolved oxygen, total biomass, cell diameter, temperature, cell type, medium type, and fluid flow rate. (Item 28) 26. The method of claim 25, wherein the one or more databases are a database comprising one or more cell culture protocols previously developed by the system for monitoring and controlling cell culture. (Item 29) 26. The method of claim 25, wherein the one or more databases are publicly available databases comprising one or more cell culture protocols. (Item 30) 26. The method of claim 25, wherein the determined personalized cell culture protocol is personalized based on the received data associated with the cells to be cultured for the human subject. (Item 31) 26. The method of claim 25, further comprising reporting the determined personalized cell culture protocol. (Item 32) 1. A method for optimizing a cell culture protocol, comprising: Receiving data associated with the cells to be cultured; setting user-defined parameters at levels to be maintained during cell culture; Implementing a cell culture protocol; measuring a level of said user-defined parameter during cell culture; optimizing the cell culture protocol by determining whether to modify cell culture conditions to maintain levels of the user-defined parameters; A method comprising: (Item 33) 33. The method of claim 32, further comprising periodically measuring the level of said parameter during said cell culture protocol. (Item 34) 33. The method of claim 32, wherein the user-defined parameters comprise pH, turbidity, glucose concentration, lactate concentration, other measures of cell health or identity, or combinations thereof. (Item 35) 33. The method of claim 32, further comprising altering cell culture conditions. (Item 36) 36. The method of claim 35, wherein altering the cell culture conditions comprises manipulating the medium flow rate to alter the glucose concentration or lactate concentration. (Item 37) 36. The method of claim 35, wherein altering the cell culture conditions comprises adding a supplement. (Item 38) 38. The method of claim 37, wherein the supplement comprises cytokines, growth factors, and serum. (Item 39) 33. The method of claim 32, further comprising storing the optimized cellular protocol in a database. [Brief description of the drawings]

[0025] [Figure 1] FIG. 1 illustrates a method for cell culture according to one embodiment of the present invention.

[0026] [Diagram 2] FIG. 2 shows an embodiment of the system of the present invention with an integrated controller.

[0027] [Diagram 3] FIG. 3 illustrates an embodiment of the system of the present invention with a distributed controller.

[0028] [Figure 4] FIG. 4 shows a block diagram of a system for cell culture according to the method of the present invention.

[0029] [Diagram 5] FIG. 5 illustrates an embodiment of a machine learning system of the present invention.

[0030] [Figure 6] FIG. 6 shows a front view of an embodiment of a cell culture cartridge and system for use in the present invention.

[0031] [Figure 7] FIG. 7 shows a top view of an embodiment of a cell culture cartridge and system for use in the present invention.

[0032] [Figure 8] FIG. 8 shows a left side view of an embodiment of a cell culture cartridge and system for use in the present invention.

[0033] [Figure 9] FIG. 9 shows a right side view of an embodiment of a cell culture cartridge and system for use in the present invention.

[0034] [Figure 10] FIG. 10 shows an embodiment of a system for use in the present invention.

[0035] [Figure 11] FIG. 11 shows an embodiment of a two-cartridge system for use in the present invention.

[0036] [Figure 12] FIG. 12 shows an embodiment illustrating the transfer of a smaller cartridge to an infusion bag for use in the present invention.

[0037] [Figure 13] FIG. 13 illustrates embodiments of disposable and non-disposable components for use in the present invention.

[0038] [Figure 14] FIG. 14 shows an embodiment of an automated fluidic system for use in the present invention.

[0039] [Figure 15] FIG. 15 shows an embodiment of a system with one cell culture chamber for use in the present invention.

[0040] [Figure 16] FIG. 16 shows an embodiment of a dendritic cell generation system for use in the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] The present invention provides a method and system for cell culture that can provide a tailored or personalized cell culture procedure. The method of the present invention includes a step of determining a cell culture protocol. In the method of the present invention, data associated with the cells to be cultured is received. The system of the present invention then connects to one or more databases to receive data about the cell culture protocol. In addition, the device used for the cell culture procedure may optionally be equipped with a plurality of sensors. The sensors are communicatively coupled to the controller. The sensors provide real-time data related to the cell culture conditions. The data obtained from the one or more databases is used to determine a cell culture protocol for the cells to be cultured, and optionally, the data obtained from the real-time feedback from the sensors may be used to optimize or adjust the cell culture protocol being performed. The protocol adjusted by the sensor feedback may then be stored as a new cell culture protocol for future cell cultures.

[0042] By providing such devices, systems, and methods, the present invention allows for culture procedures to be tailored, customized, and optionally optimized. Such an approach avoids extensive interaction and input from laboratory technicians in determining cell culture protocols. In turn, data related to such tailored cell culture procedures may be stored in a database, such as an internal database, for use in performing, developing, and determining future cell culture procedures.

[0043] 1 illustrates a method for determining a cell culture protocol. The method includes receiving (510) data associated with cells to be cultured. The data may include any suitable data, such as non-limiting examples of cell type, number of cells, pH, temperature, and type of media.

[0044] The method further includes connecting to one or more databases (520) to receive data about the cell culture protocols. Any suitable database may be used in the methods of the present invention. In some embodiments, the one or more databases are databases comprising one or more cell culture protocols previously developed by the system for monitoring and controlling cell culture. In some embodiments, the one or more databases are publicly available databases comprising one or more cell culture protocols.

[0045] The method further includes determining (530) a cell culture protocol for the cells to be cultured. In an embodiment of the invention, machine learning is used to determine the cell culture protocol. Initial data about the cells is provided and machine learning is used to analyze data from one or more databases, correlate the data from the databases to the initial data, and determine, adjust, and optionally optimize the cell culture protocol.

[0046] In some embodiments of the invention, the method further comprises updating (540) the cell culture protocol based on feedback during cell culture. The feedback is from one or more sensors disposed on the cell culture device and communicatively coupled to the controller. In some embodiments, the feedback is associated with at least one of pH, glucose concentration, lactate concentration, dissolved oxygen, total biomass, cell diameter, temperature, cell type, medium type, and fluid flow rate. In some embodiments of the invention, the determined cell culture protocol is personalized based on the received data associated with the cells to be cultured.

[0047] The method of the present invention further includes a step of reporting (550) the determined cell culture protocol. Any suitable reporting method may be used. In some embodiments, the cell culture system has an alerting capability. For example, if the levels of pH, dissolved oxygen, total biomass, cell diameter, or temperature fall outside of a user-defined or system-learned range, the system sends an alert to the user. In some cases, the alert may have a terminal form of an email alert, a voice alert, a text alert, or a combination thereof. In some embodiments of the present invention, the system and method have a monitoring capability. For example, profiles of pH, dissolved oxygen, total biomass, cell diameter, and temperature are read from the system. The profiles may be transmitted to a network such as the cloud, where they may be read by any compatible device (e.g., a smartphone) in a continuous readout format. In some embodiments of the present invention, the system and method have a decision-making capability. For example, if the levels of pH, dissolved oxygen, total biomass, cell diameter, or temperature fall outside of a user-defined or system-learned threshold, the system makes a decision. Examples of decisions include deciding to terminate the incubation process, to stop further reagent use, to alert the user, and to shut down the system.

[0048] Certain aspects of the invention are directed to a method of determining a personalized cell culture protocol. The method includes receiving data associated with cells to be cultured for a human subject, connecting to one or more databases to receive data about the cell culture protocol, and determining a personalized cell culture protocol for the cells to be cultured for the human subject. In some embodiments, the method of the invention further includes updating the personalized cell culture protocol based on feedback during cell culture. The feedback is from one or more sensors disposed on the cell culture device and communicatively coupled to the controller. In some embodiments, the feedback is associated with at least one of pH, glucose concentration, lactate concentration, dissolved oxygen, total biomass, cell diameter, temperature, cell type, medium type, and fluid flow rate. In some embodiments of the invention, the determined personalized cell culture protocol is personalized based on the received data associated with cells to be cultured for the human subject. The method of the invention further includes reporting the determined personalized cell culture protocol.

[0049] For example, the systems and methods of the present invention may be used for the generation of cell-based immunotherapy products. A step in generating a cell therapy product includes co-culture of stimulated antigen-presenting cells with T-cell-containing cells in a biological reactor containing a cell culture chamber. A supernatant containing an expanded therapeutic T-cell product is generated during the culture. In some aspects, to generate a sufficient amount of antigen-specific T cells to induce a therapeutic response in a patient, the T cells must undergo additional culture in one or more additional cell culture chambers. To effect this additional culture, a transfer of the supernatant from the culture chamber in which it was generated to a subsequent cell culture chamber containing a fresh supply of antigen-presenting cells must be performed. The transfer of the supernatant between cell culture chambers may involve the introduction of a gas flow into the first cell culture chamber, which transfers the supernatant comprising the first cell product through a fluid connector into the new cell culture chamber. Additionally, during each of the culture steps, a perfusion fluid containing, for example, medium and cytokines, can be perfused into the chamber. In one aspect, the perfusion fluid flows through the chamber along a vertical flow path to ensure that the cells remain within the chamber during culture. In one embodiment of the invention, the cells are harvested. Cell harvesting is typically accomplished by injecting a cold buffer into the cartridge. In some embodiments of the invention, a Peltier device may be integrated below the cartridge to cool the cartridge to anywhere from about 20° C. to about 30° C., which allows for release without the need to dilute the cells in more fluid volumes.

[0050] Certain aspects of the invention are directed to a system for monitoring and controlling cell culture, such as the non-limiting embodiments shown in Figures 2 and 3. The system comprises a cell culture device operatively associated with a controller. The controller comprises a hardware processor coupled to a memory containing instructions executable by the processor to cause the controller to receive data associated with the cells to be cultured, connect to one or more databases to receive cell culture protocol data, and determine a cell culture protocol for the cells to be cultured. The controller may be any suitable controller. In certain embodiments of the invention, the controller is integrated. In other embodiments, the controller is distributed.

[0051] Some embodiments of the present invention are directed to single-use components. By providing single-use components, the sterility of the system may be maintained and the system may be customized to a desired cell culture procedure for a defined cell. In some examples, the cell culture device is a single-use cell culture device or a cell culture cartridge. In some examples, the cell culture device comprises one or more sensors communicatively coupled to the controller to provide data regarding the cells. In some examples, the one or more sensors are single-use sensors.

[0052] In an embodiment of the invention, the controller is further configured to update the cell culture protocol based on feedback from one or more sensors during cell culture. The feedback may be any suitable feedback from the sensor. In an embodiment, the feedback is associated with at least one of pH, glucose concentration, lactate concentration, dissolved oxygen, total biomass, cell diameter, temperature, cell type, medium type, and fluid flow rate. In some embodiments of the invention, the determined cell culture protocol is personalized based on the received data associated with the cells to be cultured.

[0053] Any suitable database may be used in the system of the present invention to be connected to receive cell culture protocol data, including cell type, effective media and antibiotics, media and antibiotic concentrations, and culture conditions such as temperature, pH, fluid flow rates, pressure, etc. Those skilled in the art will recognize databases that are suitable for use with the present invention.

[0054] In an embodiment, the one or more databases are databases that comprise one or more cell culture protocols previously developed by the system. Such databases may be described as internal databases. The information contained in the database may be obtained from a lab notebook or settings entered in a cell culture device. The database may contain information about the cell culture protocols, such as cell type, medium type, temperature, pH, pressure, fluid flow rate, and duration of the culture steps.

[0055] In certain embodiments, the one or more databases are publicly available databases that comprise one or more cell culture protocols. In some embodiments, one of skill in the art may use the cell culture database described in Amirkia and Qiubao, Cell-culture Database: Literature-based reference tool for human and mammalian experimentally based cell culture applications; Bioinformation, 2012; 8(5): 237-238, which is incorporated herein by reference in its entirety. The Cell-culture Database is publicly available at http: / / cell-lines.toku-e.com and is useful for selecting the most effective media and antibiotics for cells, determining antibiotic concentrations and combinations for selection and transfection experiments, and searching literature related to the cell line or plasmid or vector of interest. To use the Cell-culture Database, the name of the cell line, plasmid, or vector is typed into the search box and the relevant data is viewed. The database provides information about other experiments that used the same cell line or plasmid, such as other media used to grow the cells.

[0056] In some embodiments, data from the database is not available for use, such as when an experiment is being run for the first time, or when a type of cell is being cultured for the first time. In such embodiments, the methods and systems of the present invention optimize cell culture protocols by sensing user-defined parameters throughout the cell culture process, and implement changes to the protocol to maintain set levels of the user-defined parameters.

[0057] In an embodiment, a method of optimizing a cell culture protocol includes receiving data associated with cells to be cultured. A user-defined parameter is set to a level to be maintained during cell culture. The user-defined parameter comprises pH, turbidity, glucose concentration, lactate concentration, other measurements of cell health or identity, or a combination thereof. A cell culture protocol is implemented and a level of the user-defined parameter is measured during cell culture. The level of the parameter may be measured periodically during the cell culture protocol. The cell culture protocol is optimized by determining whether to alter the cell culture conditions to maintain the level of the user-defined parameter. In some instances, the method includes altering the cell culture conditions. In one example, altering the cell culture conditions includes manipulating a flow rate of a medium to alter a glucose concentration or a lactate concentration. In another example, altering the cell culture conditions includes adding a supplement. The supplement comprises a cytokine, a growth factor, and a serum. The method further includes storing the optimized cell protocol in a database for future use.

[0058] FIG. 2 shows an embodiment of a system 300 of the present invention. A controller 305 is integrated. The controller 305 and cell culture cartridge 310 are shown arranged on a console 315. A sensor 340 is placed on the cell culture cartridge 310 for monitoring conditions. The controller 305 is communicatively coupled to one or more sensors 340. The controller 305 is communicatively coupled to a peristaltic pump 335, which is used to pump fluids in and out of the cell culture cartridge 310. The cell culture cartridge 310 has a bottom surface to which cells adhere. In other embodiments, the cells do not adhere to the bottom surface. The cell culture cartridge 310 has one or more fluid inlets and one or more fluid outlets. Connecting tubing (not shown) connects the fluid inlets to a differentiation medium reservoir (perfusion source) 325 that contains differentiation medium. The differentiation medium reservoir 325 contains the differentiation medium that will be pumped into the cell culture cartridge 310. Connecting tubing also connects the fluid outlet with waste reservoir 330. Depleted medium will be pumped out of cell culture cartridge 310, through the outlet, and into waste reservoir 330. In some cases, the lids on differentiation medium reservoir 325 and waste reservoir 330 are not removable, thereby maintaining a sterile system. In other embodiments, the lids are removable. Stopcocks and / or Luer-activated valves (LAVs) on reservoir bottles 325 and 330 allow for sterile transfer of differentiation medium, filling the inlet bottle, and removing waste from the outlet bottle. Console 315 provides a designated space for the arrangement of the components mentioned above, and also provides a display / user surface 320, connections, and on / off switches.

[0059] FIG. 3 shows an embodiment of a system 400 of the present invention. A controller 405 is distributed. The controller 405 and cell culture cartridge 410 are shown arranged on a console 415. A sensor 440 is disposed on the cell culture cartridge 410 for condition monitoring. The controller 405 is communicatively coupled to one or more sensors 440. The controller 405 is communicatively coupled to a peristaltic pump 435, which is used to pump fluid in and out of the cell culture cartridge 410. The cell culture cartridge 410 has a bottom surface to which cells adhere. In other embodiments, the cells do not adhere to the bottom surface. The cell culture cartridge 410 has one or more fluid inlets and one or more fluid outlets. Connecting tubing (not shown) connects the fluid inlets to a differentiation medium reservoir (perfusion source) 425 that contains differentiation medium. The differentiation medium reservoir 425 contains differentiation medium that will be pumped into the cell culture cartridge 410. Connecting tubing also connects the fluid outlet with the waste reservoir 430. Depleted medium will be pumped out of the cell culture cartridge 410, through the outlet, and into the waste reservoir 430. In some cases, the lids on the differentiation medium reservoir 425 and waste reservoir 430 are not removable, thereby maintaining a sterile system. In other embodiments, the lids are removable. Stopcocks and / or Luer-activated valves (LAVs) on the reservoir bottles 425 and 430 allow for sterile transfer of differentiation medium, filling the inlet bottle, and removing waste from the outlet bottle. The console 415 provides a designated space for the arrangement of the components mentioned above, and also provides a display / user surface 420, connections, and on / off switches.

[0060] The cartridge may be constructed from any suitable material. In some cases, the cartridge is constructed from polystyrene, acrylate, or a combination thereof. As one example, the base or bottom is made of polystyrene and the top and sides are acrylate. As another example, for mass production, the cartridge may be made entirely from polystyrene.

[0061] In one exemplary embodiment, the bottom surface is comprised of polystyrene and / or acrylate. The use of the same polystyrene surface for dendritic cell (DC) generation throughout one cycle of T cell stimulation is highly beneficial from a bioprocessing perspective, as it eliminates multiple transfer steps that would otherwise be necessary, thereby enabling a closed system for T cell manufacturing for DC stimulation therapy.

[0062] Additionally, any suitable material treatment may be performed on the cartridge. In some embodiments, the bottom polystyrene surface may be modified to promote cell adhesion. For example, the bottom polystyrene surface may be treated with air or oxygen plasma, also known as glow or corona discharge. For example, the bottom polystyrene surface may be modified with proteins or polyamino acids known to promote cell adhesion, including, but not limited to, fibronectin, laminin, and collagen.

[0063] The bottom is made of 6 and 24 well plates (9.5 cm 2 and 1.9 cm 2 ) or T-flasks (25 cm 2 ~225cm 2 ) and the surface area can be approximately 2.0 cm 2 ~about 500cm 2 , for example, about 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0, 16.0, 17.0, 18.0, 19.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 55.0, 60.0, 65.0, 70.0, 75.0, 100.0, 125.0, 150.0, 175.0, 200.0, 400.0, 500.0 cm 2It should be understood that the well plates may be smaller than conventional well plates (e.g., having a surface area comparable to standard cell culture dishes and flasks) or even much larger, such as having a surface area of ​​100 μm to 100 μm, and any surface area in between, and the surfaces may be rigid (flasks) or flexible (bags).

[0064] The surfaces of the cell culture cartridge can be joined together using any method known in the art, such as mechanical fastening, adhesive and solvent bonding, and welding. However, given that the cellular immunotherapy products produced using the systems and methods of the present invention will be administered to human patients, regulatory issues may prevent the use of some or all adhesives in assembling the cell culture chamber. Thus, in certain embodiments, the surfaces are joined together without the use of adhesives. In one embodiment, all surfaces of the cell culture chamber, such as the bottom wall, side wall, and top wall, are made of a first material (e.g., polystyrene) and are joined together using ultrasonic welding. It should be understood that the above configurations are only examples and other configurations for joining the surfaces are also contemplated embodiments of the present invention.

[0065] The height of one or more cell culture chambers may vary. For example, but not limited to, exemplary ranges of cell culture chamber heights include any height between 0.5 mm and 100 mm, such as 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 55.0, 60.0, 65.0, 70.0, 75.0, 80.0, 85.0, 90.0, 95.0, 100.0 mm, or any height therebetween. In certain embodiments, the chamber height may be comparable to the liquid height in cultures typically performed in 6- and 24-well plates, such as 2-6 mm, with volumetric capacity of about 0.8 mL to 6 mL. In other embodiments, the cell culture chamber may be approximately 50 cm 2 The size may be large, such as 10mm to 50mm, with a culture surface of

[0066] In some embodiments of the present invention, the cartridge is optically clear or transparent. Such optical transparency, combined with the fluid ports being properly isolated, allows the user to view the cells in any vertical plane within the cartridge. Additionally, stopcocks may be installed on the cartridge or on the reservoir bottle. In particular, stopcocks may be installed on specific ports on the cartridge, each performing a specific function. The installation is specific to each function, and work was performed to determine the optimal location to ensure the process is successful and the workflow is easy. For example, a stopcock may be used for seeding and harvesting, and a Luer-activated valve (LAV) on the stopcock allows a syringe to be connected aseptically. A stopcock may be used for seeding and harvesting (adding cold buffer for washing), and air inside the cartridge will flow out through a filter in this stopcock when the cell solution is seeded into the cartridge. As another example, a stopcock may be used for harvesting, and air inside the cartridge will flow into the cartridge when the cell solution is removed. The filter attached to the stopcock avoids pressure or vacuum build-up in the cartridge when liquid is being added to or removed from the cartridge. In the present invention, the LAV may be used on the bottle to add and / or remove medium. Traditionally, LAVs are commercially available for use for anesthesia and IV lines. Therefore, using the LAV for adding or removing medium deviates from traditional use.

[0067] 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 smart phone, or a specialized device produced for the system.

[0068] 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).

[0069] Processors suitable for executing computer programs include, by way of example, both general-purpose and special-purpose microprocessors, and any one or more processors of any type of digital computer. Generally, the processor will receive instructions and data from a read-only memory or a random access memory or both. The elements of a computer are a processor for executing instructions and one or more memory devices for storing instructions and data. Generally, a computer will also include, or be operatively coupled to receive data from, or transfer data to, one or more non-transitory mass storage devices, such as magnetic, magneto-optical, or optical disks, for storing data, or both. In some embodiments, sensors on the system transmit process data via Bluetooth to a central data collection unit located outside the incubator. In some embodiments, data is transmitted directly to the cloud, rather than to a physical storage device. Suitable information carriers 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.

[0070] 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.

[0071] 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, 4G, or 5G), a local area network (LAN), and a wide area network (WAN), e.g., the Internet.

[0072] 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, a 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.

[0073] 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.

[0074] 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).

[0075] 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., with 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., with 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, programmatically, or automatically, for example, by a save command from software or a write command from a programming language.

[0076] 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 (RFID) tags or chips, or any other medium that can be used to store desired information and that can be accessed by a computing device.

[0077] 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.

[0078] In the exemplary embodiment shown in FIG. 4, 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 be engaged 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.

[0079] 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 cart, or a cellular modem.

[0080] 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 also be transmitted or received over a network via a network interface device.

[0081] 5 illustrates a machine learning system 201, according to one embodiment. The machine learning system 201 has access to data from multiple sources 205. Any suitable source of data 205 may be provided to the machine learning system 201.

[0082] In a preferred embodiment, multiple data sources 205 are fed into a machine learning system 201. Any suitable machine learning system 201 may be used. For example, the machine learning system 201 may include one or more of a random forest, a support vector machine, a Bayesian classifier, and a neural network. In the depicted embodiment, the machine learning system 201 includes a random forest 209. In some embodiments, the computer system comprises an autonomous machine learning system that associates functional biomarker measurements with known cancer status in an unsupervised manner. The autonomous machine learning system may include a deep learning neural network including an input layer, multiple hidden layers, and an output layer. The autonomous machine learning system may represent a training dataset using multiple features, each feature comprising a feature vector.

[0083] The machine learning system 201 may access the data from multiple sources 205 in any suitable format, including, for example, as a summary table (e.g., formatted as comma separated values) or as a whole (e.g., parsed by a script such as Perl or SQL in the machine learning system 201). However, in the initial format, the data can ultimately be understood to include multiple entries 213. Each entry preferably includes data or a value that provides information to the system 201. The value may be a numeric value, or it may be a string, such as a disease classification code (e.g., an ICD-9 code or an ICD-10 code), which may be aggregated from different sources.

[0084] Most preferably, each entry 213 in the data is specific to one data point from the protocol and assigned to a predefined category. In the case of providing a personalized cell culture protocol, it is understood that the data source 205 may provide anonymous data. In such a case, each entry 213 is preferably specific to the patient and is tracked to that patient by a patient ID value, which may be a random string or code. The external data source 205 may provide the patient ID, or the machine learning system 201 may assign a patient ID to each entry 213. Each entry 213 also preferably has a category. For example, if the data entry 213 is information or data about an initial cell, the category may be "initial" (and the value for the entry 213 is a specific data point). In another example, if the data source 205 is information or data from a publicly available cell culture protocol database, the data entry 213 may be categorized as a database entry, and the value may be a specific condition for that particular protocol, such as time, medium, temperature, pH, etc. The machine learning system 201 accesses multiple data sources 205 and discovers associations therein.

[0085] The devices and methods of the present disclosure may provide a user interface, for example in the form of a portal or dashboard. Any suitable information may be provided on the dashboard, such as data associated with the running conditions of a cell culture procedure, data imported from one or more publicly available databases, and / or feedback from the running cell culture procedure.

[0086] Discovering the association may include observing a co-occurrence of event categories in multiple cell culture procedures that is significantly different from the expected number of co-occurrences. In an embodiment of the present invention, the input to the machine learning algorithm is scaled or normalized to facilitate meaningful comparison across categorically different input types. Scaling and normalization methods are included. Scaling is used to achieve a goal, for example, to divide each individual's data by a number so that the range of values ​​for all data lies within a certain interval, such as [0,1].

[0087] Scaling details may include options such as "none", "centering", "autoscaling", "range scaling", "palette scaling" (default="autoscaling"). Several different scaling methods are provided: "none", which provides no scaling method; "centering", which centers the mean at zero; "autoscaling", which scales the data by centering the mean at zero and dividing each variable by the variance; "range scaling", which scales the data by centering the mean at zero and dividing each variable by the difference between the minimum and maximum values; and "palette scaling", which scales the data by centering the mean at zero and dividing each variable by the square root of the standard deviation. Unit scaling divides each variable by the standard deviation so that each variance is equal to 1.

[0088] Normalization details may be included and used. Like scaling, normalization may be used to divide or shift the total data set, for example to facilitate comparison of data from different sources or in different formats. For example, the z-score of the data points, i.e., (z-μ) / σ, may be used. This normalization is determined by the mean of the data and its variance.

[0089] Several different normalization methods are provided: "none" applies no normalization method, and "pqn" applies a probabilistic quotient normalization (Dieterle, 2006, Probabilistic quotient normalization as robust method to account for dilution of complex biological mixtures: application in 1 H NMR metabonomics, Anal Chem 78(13):4281-90 (incorporated by reference), where "sum" normalizes the sample to the sum of the absolute values ​​of all variables for a given sample, "median" normalizes the sample to the median value of all variables for a given sample, and "square root" normalizes the sample to the root of the sum of the squared values ​​of all variables for a given sample.

[0090] The systems and methods of the present disclosure include a machine learning system 201. The machine learning system 201 is preferably implemented in a tangible computer system constructed to implement the methods described herein. Any machine learning algorithm may be used to analyze the data, including, for example, random forests, support vector machines (SVMs), or boosting algorithms (e.g., adaptive boosting (AdaBoost), gradient boosting (GBM), or extreme gradient boosting (XGBoost)), or neural networks such as H2O.

[0091] Machine learning algorithms are generally one of the following types: (1) bagging (to reduce variance), (2) boosting (to reduce bias), or (3) stacking (to improve predictive power). In bagging, multiple predictive models (generally of the same type) are constructed from subsets of the classification data (classes and features) and then combined into a single classifier. Random forest classifiers are of this type. In boosting, an initial predictive model is iteratively improved by examining the prediction error. Adaboost and extreme gradient boosting are of this type. In stacking models, multiple predictive models (generally of different types) are combined to form a final classifier. These methods are called ensemble methods. The basic or starting method in ensemble methods is often a decision tree. A decision tree is a non-parametric supervised learning method that uses simple decision rules to infer classifications from features in the data. They have several advantages in that they are simple to understand and can be visualized as a tree starting at a root (usually a single node) and branching repeatedly to leaves (multiple nodes) that are associated with classifications.

[0092] In some embodiments, the methods and systems of the present invention use a machine learning system 201 that uses a random forest 209. A random forest uses decision tree learning, where a model is built to predict the value of a target variable based on several input variables. Decision trees can be broadly divided into two types. In classification trees, the target variable takes on a finite set of values ​​or classes, while in regression trees, the target variable can take on continuous values, such as real numbers. Examples of decision tree learning include classification trees, regression trees, boosted trees, bootstrap aggregation trees, random forests, and rotation forests. In decision trees, decisions are made sequentially at a series of nodes that correspond to input variables. A random forest includes multiple decision trees to improve the accuracy of predictions. See Breiman, 2001, Random Forests, Machine Learning 45:5-32, incorporated herein by reference. In random forests, bootstrap aggregation or bagging is used to average predictions by multiple trees given different sets of training data. In addition, a random subset of features is selected at each branch point in the learning process, which reduces spurious correlations that may result from the presence of individual features that are strong predictors of the response variable.

[0093] SVMs can be used for classification and regression. When used to classify new data into one of two categories, such as diseased or not diseased, SVMs create a hyperplane in a multidimensional space that separates the data points into one category or the other. Although the original problem can be expressed in terms that require only a finite dimensional space, a linear separation of the data between categories may not be possible in a finite dimensional space. As a result, a multidimensional space is selected to allow the construction of a hyperplane that results in a clean separation of the data points. See Press, WH et al., Section 16.5. Support Vector Machines. Numerical Recipes: The Art of Scientific Computing (3rd ed.). New York: Cambridge University (2007), incorporated herein by reference. SVMs can also be used in support vector clustering. See Ben-Hur, 2001, Support Vector Clustering, J Mach Learning Res 2:125-137, incorporated herein by reference.

[0094] Boosting algorithms are machine learning ensemble meta-algorithms for reducing bias and variance. Boosting focuses on turning weak learners into strong learners, where weak learners are defined as classifiers that are only weakly correlated with the true classification, while strong learners are classifiers that are positively correlated with the true classification. Boosting algorithms consist of iteratively learning weak classifiers over a distribution and adding them to a final strong classifier. The added classifiers are typically weighted based on their accuracy. Boosting algorithms include Adaboost, Gradient Boosting, and XGBoost. See Freund, 1997, A decision-theoretic generalization of on-line learning and an application to boosting, J Comp Sys Sci 55:119; and Chen, 2016, XGBoost: A Scalable Tree Boosting System, arXiv:1603.02754, both of which are incorporated herein by reference.

[0095] Neural networks modeled on the human brain enable information processing and machine learning. Neural networks include nodes that mimic the functions of individual neurons, and the nodes are organized into layers. Neural networks include an input layer, an output layer, and one or more hidden layers that define the connections from the input layer to the output layer. The systems and methods of the present invention may include any neural network that facilitates machine learning. The system may include known neural network architectures such as GoogLeNet (Szegedy, et al. Going deeper with convolutions, in CVPR 2015, 2015); AlexNet (Krizhevsky, et al. Imagenet classification with deep convolutional neural networks, in Pereira, et al. Eds., Advances in Neural Information Processing Systems 25, pages 1097-3105, Curran Associates, Inc., 2012); VGG16 (Simonyan & Zisserman, Very deep convolutional networks for large-scale image recognition, CoRR, abs / 3409.1556, 2014); or FaceNet (Wang et al., Face Search at Scale: 80 Million Gallery, 2015) (each of the foregoing references is incorporated herein by reference).

[0096] Deep learning neural networks (also known as deep structured learning, hierarchical learning, or deep machine learning) include a class of machine learning operations that use a cascade of many layers of nonlinear processing units for feature extraction and transformation. Each successive layer uses the output from the previous layer as input. The algorithms may be supervised or unsupervised, and applications include pattern analysis (unsupervised) and classification (supervised). Some embodiments are based on unsupervised learning of multiple levels of features or representations of data. Higher level features are derived from lower level features to form a hierarchical representation. The features are preferably represented in nodes as feature vectors. Deep learning with neural networks involves learning multiple levels of representations that correspond to different levels of abstraction, the levels forming a hierarchy of concepts. In some embodiments, the neural network includes at least five hidden layers, preferably more than ten. The many layers between the input and the output allow the system to operate through multiple processing layers.

[0097] Deep learning is part of a broad family of machine learning methods based on learning representations of data. Observations can be represented in many ways, such as vectors of intensity values ​​per pixel, or in more abstract ways as sets of edges, regions of particular shapes, etc. Their features are represented at nodes in the network. Preferably, each feature is structured as a feature vector, a multidimensional vector of numerical features that represent an object. Features provide such a representation of an object because the numerical representation facilitates processing and statistical analysis. Feature vectors are similar to vectors of explanatory variables used in statistical procedures such as linear regression. Feature vectors are often combined with weights using dot products to construct a linear predictor function that is used to determine a score to make a prediction.

[0098] The vector space associated with these vectors may be referred to as the feature space. Dimensionality reduction may be employed to reduce the dimensionality of the feature space. Higher level features can be obtained from already available features and added to the feature vector in a process called feature construction. Feature construction is the application of a set of structural operators to a set of existing features, resulting in the construction of new features.

[0099] In the network, nodes are connected in layers and signals proceed from the input layer to the output layer. In one embodiment, each node in the input layer corresponds to a distinct one of the features from the training data. The nodes of the hidden layer are calculated as a function of bias terms and weighted sums of the nodes of the input layer, and a distinct weight is assigned to each connection between a node of the input layer and a node in the hidden layer. The bias terms and weights between the input layer and the hidden layer are learned autonomously in the training of the neural network. The network may contain thousands or hundreds of nodes and connections. Typically, the signals and states of an artificial neuron are typically real numbers between 0 and 1. Optionally, there may be a threshold or limit function for each connection and the unit itself, so that the signal must exceed the limit before it is propagated. Backpropagation is the use of forward stimuli to modify the connection weights, and is sometimes performed to train the network using known correct outputs. See WO 2016 / 182551, U.S. Publication No. 2016 / 0174902, U.S. Patent No. 8,639,043, and U.S. Publication No. 2017 / 0053398, each of which is incorporated by reference herein.

[0100] In some embodiments, the data set is used to cluster the training set. Specific exemplary clustering techniques that can be used in the present invention include, but are not limited to, hierarchical clustering (agglomerative clustering using nearest neighbor, farthest neighbor, average linkage, centroid, or sum of squares algorithms), k-means clustering, fuzzy k-means clustering algorithm, and Jarvis-Patrick clustering.

[0101] A Bayesian network is a probabilistic graphical model that represents a set of random variables and their conditional dependencies via a directed acyclic graph (DAG). The DAG has nodes that represent random variables, which can be observable quantities, latent variables, unknown parameters, or hypotheses. Edges represent conditional dependencies, and unconnected nodes represent variables that are conditionally independent of each other. Each node is associated with a probability function that takes as input a particular set of values ​​for the node's parent variables and gives (as output) the probability (or probability distribution, if applicable) of the variable represented by the node.

[0102] Regression analysis is a statistical process for estimating relationships between variables, such as traits and outcomes. It includes techniques for modeling and analyzing relationships between multiple variables. Specifically, regression analysis focuses on the change in a dependent variable in response to a change in a single independent variable. Regression analysis can be used to estimate the conditional expectation of the dependent variable given the independent variable. The variation of the dependent variable can be characterized around the regression function and described by a probability distribution. The parameters of the regression model may be estimated using, for example, least squares, Bayesian methods, percentage regression, least absolute deviations, non-parametric regression, or distance metric learning.

[0103] Any suitable machine learning algorithm may be included. In some embodiments, the machine learning system 201 includes a random forest 209. The machine learning system may learn in a supervised or unsupervised manner. A machine learning system that learns in an unsupervised manner may be referred to as an autonomous machine learning system. The autonomous machine learning system may employ both supervised and unsupervised learning cycles, although other versions are within the scope of the present invention. The random forest 209 may be operated autonomously and may include both supervised and unsupervised learning cycles. See Criminisi, 2012, Decision Forests: A unified framework for classification, regression, density estimation, manifold learning and semi-supervised learning, Foundations and Trends in Computer Graphics and Vision 7(2-3):81-227, incorporated herein by reference. In some embodiments, the autonomous machine learning system 201 comprises a random forest 209. In some embodiments, the autonomous machine learning system 201 discovers associations through operations that include at least a cycle of unsupervised learning. Cell culture device architecture

[0104] In some embodiments of the invention, the systems and methods of the invention may use cell culture apparatus devices such as those described in U.S. Application No. 16 / 192,062, U.S. Application No. 16 / 310,680, U.S. Application No. 15 / 970,664, U.S. Application No. 15 / 736,257, International Application No. PCT / US2017 / 039538, International Application No. PCT / US2016 / 060701, and International Application No. PCT / US2016 / 040042, all of which are incorporated herein in their entirety. Such devices may be equipped with sensors and controllers according to the invention.

[0105] In an embodiment, the device used in the present invention may be an automated cell culture cartridge and system for the generation of dendritic cells with uniform symmetric flow within the cell culture cartridge. The device may be a fully enclosed sterile immature DC (iDC) generation system to generate iDCs at a clinical scale, virtually eliminating the need for multiple well plates (or T-flasks / bags), ensuring a sterile and particulate-free culture system, and reducing technician time in maintaining the cell culture. In an embodiment, the device is an automated cell culture system for the sterile generation of therapeutically relevant numbers of iDCs within a single cell culture cartridge. The system is also capable of further processing of iDCs to mature them via the addition of maturation reagents, and stimulation via the addition of one or more antigens to the cell culture chamber.

[0106] The cell culture system includes a cell culture cartridge that includes multiple zones that are geometrically configured to provide symmetric fluid flow channels within the cell culture chamber and avoid dead areas in the flow within the cell culture chamber. In some cases, the cartridge for the cell culture device is optically clear or transparent. Such optical transparency, in combination with appropriate isolation of the fluid ports, allows the user to view the cells at any vertical plane within the cartridge. As shown in Figures 6-9, the embodiment includes an optically clear or transparent cell culture cartridge for use with the present invention. Figure 6 shows a front view of a cell culture cartridge and system for use with the present invention. Figure 7 shows a top view of a cell culture cartridge and system for use with the present invention. Figure 8 shows a left side view of a cell culture cartridge and system for use with the present invention. Figure 9 shows a right side view of a cell culture cartridge and system for use with the present invention.

[0107] Furthermore, as shown in Figures 6-9, stopcocks may be installed on the cartridge or on the reservoir bottle. In particular, stopcocks are installed on specific ports on the cartridge, each performing a specific function. The installation is specific to each function, and work was performed to determine the optimal location to ensure the process is successful and the workflow is easy. For example, the stopcock at the front is for seeding and harvesting, and the Luer-activated valve (LAV) above the stopcock allows the syringe to be connected aseptically. A filter attached to the stopcock avoids pressure or vacuum build-up in the cartridge when liquid is being added to or removed from the cartridge. In the present invention, the LAV may be used on the bottle to add and / or remove medium.

[0108] FIG. 10 shows an embodiment of a system 100 for use with the present invention. A peristaltic pump 110 is provided. The pump 110 is used to pump fluids in and out of a cell culture cartridge 120. 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 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 the 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. 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 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 provides a display / user interface 192, connections 194, and an on / off switch 196.

[0109] Figure 11 shows an embodiment of a device with two cartridges for use with the present invention. A cell culture cartridge 1200 is provided for differentiation of monocytes to dendritic cells. A smaller cartridge 1220 is provided for maturation and antigen pulsing. In other embodiments, maturation and antigen pulsing may be performed in the main cell culture cartridge without the use of a second cartridge.

[0110] 12 shows an embodiment of a device for use with the present invention having a smaller cartridge 1320 for maturation and antigen pulsing. The smaller cartridge 1320 is fluidly connected to an infusion bag 1330, which contains the final product transferred from the smaller cartridge 1320.

[0111] FIG. 13 shows the disposable and non-disposable components of a device for use with the present invention. The EDEN console 1410 is non-disposable and has a length L. In this embodiment, the length L is 14 inches. The smaller cartridge 1420 is for maturation and antigen pulsing. Connecting tubing 1430 connects the inlet and outlet with the reservoir and cartridge. The smaller cartridge 1420 and connecting tubing 1430 are single use and disposable.

[0112] FIG. 14 shows an embodiment of the EDEN automated fluidic system that can be used with the present invention. The EDEN system generates monocyte-derived immature dendritic cells (iDCs) while continuously perfusing fresh differentiation medium into a cell culture cartridge. EDEN was developed to generate therapeutically relevant numbers of iDCs in a single cell culture cartridge that is fully enclosed and not open to the outside environment. Fresh differentiation medium was perfusing into the cartridge and depleted medium was removed. EDEN-generated iDCs exhibited phenotypic expression and iDC yields similar to 6-well plate-generated iDCs. iDCs matured in the cartridge according to the present invention exhibited canonical CD80 / 83 / 86 upregulation and CD209 downregulation.

[0113] In some embodiments of the invention, a device such as the bioreactor 1110 shown in Figure 15 is used. The bioreactor 1110 includes a cell culture chamber 1120 including a bottom surface 1122 and at least one additional surface 1124. The bottom surface 1122 is made of a first material to which cells adhere, and the at least one additional surface 1124 is made of a second material that is gas permeable. The cell culture chamber also includes one or more inlets 1126, 1136 and one or more outlets 1128, 1138. In certain embodiments, the bioreactor also includes at least one perfusion fluid reservoir 1132, at least one waste fluid reservoir 1134, at least one pump 1140 for moving perfusion fluid through the chamber 1120, and associated inlets 1136 and outlets 1138 for transporting fluids to and from the reservoirs 1132, 1134 and through the chamber 1120.

[0114] With respect to the cell culture chamber 1120, the first material can be any material that is biocompatible and to which antigen presenting cells (APCs), such as dendritic cells (DCs), will adhere. During the T cell stimulation and expansion process carried out in the cell culture chamber 1120, mature APCs will develop and preferably adhere to the bottom surface 1122, while the T cells will remain in the supernatant above the bottom surface, making it easier to obtain the expanded T cells separately.

[0115] In one exemplary embodiment, the first material is made of polystyrene. One benefit of using polystyrene for the bottom surface on which the culture will take place is the useful role that this material plays in the process of generating dendritic cells from PBMCs. Specifically, the polystyrene surface can be used to enrich monocytes from a heterogeneous suspension of PBMCs. This is the first step in the culture process utilized to generate DCs by differentiation of monocytes via culture in a medium containing IL4 and GM-CSF, for example. The use of the same polystyrene surface for dendritic cell generation throughout one cycle of T cell stimulation is highly beneficial from a bioprocessing perspective, as it eliminates multiple transfer steps that would otherwise be necessary, thereby enabling a closed system for T cell manufacturing for DC stimulation therapy.

[0116] In another embodiment, at least one additional surface 1124 includes a second material that is gas permeable to provide for gas exchange to occur within the cell culture chamber. By fabricating the cell culture chamber such that the bottom surface is made of a material to which cells adhere, such as polystyrene, and at least one additional surface, such as a side wall and / or a top wall, is made, at least in part, from a gas permeable material, high surface area gas exchange is achieved in the system of the present embodiment. Having a large surface with high permeability other than the bottom surface provides the ability to achieve a higher level of gas exchange without the need to sacrifice the adhesive properties of the bottom surface, relative to prior art culture systems that were limited in the amount of culture medium that could be contained and / or lacked a culture-friendly surface to which cells could adhere.

[0117] In certain embodiments, the second material comprises one or more materials having a permeability to oxygen of a permeability coefficient of 350 or greater and a permeability to carbon dioxide of a permeability coefficient of 2,000 or greater, the permeability coefficient being expressed in units of [cm 3 ][cm] / [cm 2

[0023] [s][cm Hg]. Exemplary materials include poly(dimethylsiloxane) (PDMS), which is well known for its high oxygen and carbon dioxide permeability (up to three orders of magnitude higher than materials such as polystyrene and PMMA), and silicone-containing materials such as polymethylpentene. In one exemplary embodiment, the cell culture chamber comprises a polystyrene floor and silicone side and top walls.

[0118] In certain aspects, in addition to the second material, at least one additional surface 1124 can also be comprised of the first material. For example, but not limited to, the additional surface 1124, such as one or more side walls and / or a top wall, can incorporate a second material (e.g., a highly permeable polymer such as silicone) within a frame made of a first material (e.g., polystyrene). It is also envisioned that the bottom surface can also be comprised of the second material. However, in some embodiments, the second material is only intermittently distributed throughout the bottom surface to ensure that the first material covers a sufficient surface area so that cells can adhere to the surface.

[0119] In an embodiment, the bioreactor 1110 will also include one or more pumps 1140 operably coupled to the cell culture chamber 1120 for perfusing perfusion medium into the cell culture chamber. The bioreactor 1110 may also include one or more fluid reservoirs 1132. The fluid reservoirs 1132 may be in fluid communication with the cell culture chamber 1110 and operably coupled to the one or more pumps 1140. One or more tubes for connecting the fluid reservoirs to the pumps and the cell culture chambers are also provided. In an aspect, the one or more pumps are configured to pump fluid from the fluid reservoirs, through the cell culture chambers, and into a waste collection reservoir. In the exemplary embodiment shown in FIG. 15, fluid travels from fluid reservoir 1132, through tubing 1152 to pump 1140, into cell culture chamber 1120 via inlet 1136, back out of cell culture chamber 1120 via outlet 1138, and through tubing 1154 into waste collection reservoir 1134.

[0120] In some embodiments, the fluid reservoirs and / or waste collection reservoirs can each 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.

[0121] In certain embodiments, one or more bioreactors 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 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.

[0122] Furthermore, in some situations, the conditions within the incubator remain stable and automated recording of temperature is possible in order to have knowledge of temperature fluctuations to correlate with any anomalies in the reactions carried out within the incubator. Thus, the supply of any power should not alter the environment within the incubator. For example, some pumps generate heat. Thus, in one embodiment, the pump is housed separately from the bioreactor but still in fluid and operative communication with the reactor. In another embodiment, the pump is attached directly to the bioreactor and located within the incubator but is heat-free or operatively connected to a heat sink and / or fan to dissipate heat. Regardless of the configuration, the pump is operatively coupled to the bioreactor, which in turn is operatively coupled to the cell culture chamber.

[0123] The system may also include a cell culture reservoir and, optionally, a heater to control the temperature of the fluid reservoir. In such a configuration, no incubator is required and the system may operate autonomously using only a source of power. If the system lacks a heater, it may be operated inside the cell incubator.

[0124] In other aspects, the cell culture chamber includes one or more sensors (not shown) operably coupled to the cell culture chamber. 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 certain embodiments, 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 systems having multiple chambers operably coupled to one or more sensors, the sensors can be the same in 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 certain aspects, the one or more sensors are operably coupled to a computer system (not shown in FIG. 15) having a central processing unit for executing instructions such that automatic monitoring and adjustment of the parameters is possible.

[0125] 16 shows an embodiment of a dendritic cell (DC) generation system 2300 described in International Application No. PCT / US2016 / 040042, the contents of which are incorporated herein by reference. Such a device may be used in conjunction with the systems and methods of the present invention. The system includes a housing 2310 with space to contain a culture medium reservoir 2340 and a waste reservoir 2350 (the size and shape of a commercially available glass or plastic culture medium bottle with a plastic cap, respectively), a mounting area for a DC differentiation cassette or chip 2200, an exposed peristaltic pump head configured to receive peristaltic pump tubing leading from the culture medium bottle 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 2330, a luer lock fitting 2278, and a control button, knob, or switch. The system may also include a heater (not shown) to control the temperature of the cassette and, optionally, the culture medium reservoir; in such a configuration, no incubator is required and the system may operate autonomously using only a source of power. If the system lacks a heater, it may be operated inside the cell incubator. Similar systems including two or more cassettes and pump heads (e.g., one per cassette, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more cassettes and pump heads) are also envisioned. In such multi-cassette systems, the control electronics, displays, and buttons, knobs, or switches may either be shared between different cassettes or duplicated, one set per cassette. (Example) EXAMPLES

[0126] Public Database In certain embodiments, the systems and methods of the invention pull data from a public database for use in determining cell culture protocols. Any suitable public database may comprise data regarding one or more cell culture protocols, and the systems of the invention may connect to the database to receive the cell culture protocol data. For example, the invention is directed to Amirkia and Qiubao, Cell-culture Database: Literature-based reference tool for human and mammalian experimentally based Data may be drawn from the Cell-culture Database described in cell culture applications; Bioinformation, 2012; 8(5): 237-238, incorporated herein by reference in its entirety. The Cell-culture Database is publicly available at http: / / cell-lines.toku-e.com and is useful for selecting the most effective media, supplements, and antibiotics for cells, determining antibiotic concentrations and combinations for selection and transfection experiments, and searching literature related to the cell line or plasmid or vector of interest. To use the Cell-culture Database, the name of the cell line, plasmid, or vector is typed into the search box and the relevant data is viewed. The database provides information about other experiments that have used the same cell line or plasmid, such as other media used to grow the cells.

[0127] In such an embodiment, a controller operatively associated with the cell culture device receives initial data associated with the cells to be cultured. For example, a user or laboratory technician inputs data regarding the cell line. The controller then connects to a publicly available database, such as the Cell-culture Database, which provides various information about the cell culture protocol once the relevant input data is provided. The controller provides the data regarding the cell line as an "entry" in the Cell-culture Database. The method of the invention includes the steps of viewing results obtained from such inputs, such as the medium used to grow the cells, and using the results to determine the cell culture protocol.

[0128] In some cases, the determined cell culture protocol comprises a protocol pulled directly from a public database. In some cases, the determined cell culture protocol may be used immediately for cell culture. The determined cell culture protocol may also be stored for future use, such as stored in an internal database. EXAMPLES

[0129] Internal Database In an embodiment, the systems and methods of the present invention pull data from an internal database for use in determining a cell culture protocol. The internal database may include information regarding cell culture protocols previously used in a laboratory setting. For example, the database may include information obtained from a cell device installation and information from a lab notebook. The information in the internal database may include any relevant information regarding the cell culture protocol, such as cell type, media type, pH, temperature, duration of culture steps, and fluid flow rates used during culture.

[0130] In such an embodiment, a controller operatively associated with the cell culture device receives data associated with the cells to be cultured. For example, a user or laboratory technician inputs data regarding a cell line. The controller then connects to an internal database, such as a database that documents all previous cell culture protocols used in a laboratory. Based on the input, the database provides information related to past cell culture protocols used with that cell type. For example, the information may include the type of medium used during culture, pH, temperature, duration of steps, and fluid flow rates. The method of the present invention includes the steps of viewing the results obtained from such inputs, such as the medium used to grow the cells, and using the results to determine the cell culture protocol.

[0131] In some cases, the determined cell culture protocol comprises a protocol retrieved directly from an internal database. In some cases, the determined cell culture protocol may be used immediately for cell culture. The determined cell culture protocol may also be stored for future use, such as stored in an internal database. EXAMPLES

[0132] Combining Data Sets In an embodiment, the systems and methods of the present invention pull data from a combination of databases for use in determining cell culture protocols. The databases may be any suitable database that comprises one or more cell culture protocols. For example, the databases may be a combination of publicly available databases. In another example, the databases may be a combination of publicly available databases and internal databases.

[0133] In such an embodiment, a controller operatively associated with the cell culture device receives data associated with the cells to be cultured. The controller then connects to a first database, such as a public database, to receive the cell culture protocol data. The controller then connects to another database, such as an internal database, to receive the cell culture protocol data. The controller then determines a cell culture protocol for the cells to be cultured based on the data obtained from the public database and the internal database.

[0134] In some cases, the determined cell culture protocol comprises a protocol pulled directly from an internal database and modified based on data from a public database. In some cases, the determined cell culture protocol comprises a protocol pulled directly from a public database and modified based on data from an internal database. In some cases, the determined cell culture protocol comprises a protocol pulled directly from a first public database and modified based on data from a second public database. In some cases, the determined cell culture protocol may be used immediately for cell culture. The determined cell culture protocol may also be stored for future use, such as stored in an internal database. EXAMPLES

[0135] Database and Feedback In certain embodiments, the systems and methods of the present invention draw data from one or more databases for use in determining a cell culture protocol and also include feedback data from sensors, including data from a plurality of sensors monitoring conditions of the cell culture procedure.

[0136] In such an example, a controller operatively associated with the cell culture device receives data associated with the cells to be cultured, such as cell type. The controller then connects to a database, which may be any suitable public or internal database, comprising one or more cell culture protocols. The controller receives the cell culture protocol data from the database. The controller receives data from a number of sensors on the cell culture device, such as temperature, pressure, pH, temperature, and fluid flow rates. The data obtained from the sensors is used to modify the cell culture protocol obtained from the database, thereby determining the cell culture protocol based on the data obtained from the database and the feedback data. The determined cell culture protocol may be used immediately for cell culture. The determined cell culture protocol may also be stored for future use, such as in an internal database. EXAMPLES

[0137] User-defined parameter optimization In an embodiment, the system and method of the present invention may be used to optimize a cell culture procedure based on user-defined parameters. In some cases, the user-defined parameters are selected from pH, turbidity (reflecting cell growth), glucose, lactate, or any other measurement of cell health or identity. A user would input the desired parameters and load the system with cells and base medium. The method of the present invention is then used to self-optimize the cell culture procedure in the system to maintain the user-defined set of parameters. In such an embodiment, the method and system of the present invention senses the level of the parameter or parameters of interest at least once during the cell culture process. Optionally, the parameter of interest may be sensed multiple times throughout the cell culture process.

[0138] The present invention then optimizes the user-defined parameters by determining whether to change the culture conditions. For example, the system and method of the present invention includes making a decision to change the culture conditions based on the sensed parameter levels. In some situations, information regarding parameter optimization may not be available from a database, such as when a new experiment or protocol is being run for the first time. In some cases, the system and method of the present invention then changes the culture conditions based on the decision. In some cases, the system and method of the present invention manipulates flow rates to change glucose or lactate concentrations. In some cases, the system and method of the present invention adds supplements, such as cytokines, growth factors, and serum, from a reservoir. The reservoir may be included within the system (or on-board) or may be external to the incubator and connected to the culture vessel via a pump. Following completion of the cell culture procedure, the method and system of the present invention stores the optimized protocol in a database, such as an internal database, to serve as a reference for future use. (Incorporated by reference)

[0139] 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. Equivalent

[0140] 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. A method for determining a personalized cell culture protocol, the method comprising: A cell culture system is provided, the cell culture system comprising: A cell culture device, the cell culture device comprising one or more fluid inlets and one or more fluid outlets; a fluid source fluidly connected to one of the one or more fluid inlets; a controller configured to communicate with one or more of a publicly available database and an internal database, each of the publicly available database and the internal database including cell culture protocol data; one or more sensors disposed on the cell culture device communicatively coupled to the controller, the one or more sensors configured to measure one or more parameters within the cell culture device, the one or more parameters including one or more of pH, glucose concentration, lactate concentration, dissolved oxygen, total biomass, cell diameter, temperature, cell type, medium type, cell metabolite concentration, and fluid flow rate; one or more pumps communicatively coupled to the controller, the one or more pumps configured to pump fluid into the cell culture device through the one or more fluid inlets and out of the cell culture device through the one or more fluid outlets; an interface by means of which one or more users can interact, via associated computing devices, to determine and monitor cell culture protocols; and receiving, as input from the one or more users, data associated with cells to be cultured for a human subject; communicating with the one or more of the publicly available database and the internal database to receive the cell culture protocol data; determining a personalized cell culture protocol for the cells to be cultured for the human subject by analyzing one or more of the cell culture protocol data, the data associated with the cells, and the one or more parameters using a machine learning system; updating the personalized cell culture protocol based on feedback during cell culture from the one or more sensors; A method comprising:

2. The method of claim 1, wherein the feedback is associated with parameters including at least one of pH, glucose concentration, lactate concentration, dissolved oxygen, total biomass, cell diameter, temperature, cell type, medium type, and fluid flow rate.

3. The method of claim 1, wherein the internal database is a database containing one or more cell culture protocols previously developed by a system for monitoring and controlling cell culture.

4. The method described in claim 1, wherein the determined personalized cell culture protocol is personalized based on the received data associated with the cells to be cultured for the human subject.

5. The method of claim 1, further comprising reporting the determined personalized cell culture protocol.

6. The method of claim 5, wherein reporting includes providing an alert when the level is outside a specified range.

7. The method of claim 6, wherein the alert includes an email alert, an audio alert, a text alert, or a combination thereof.

8. The method of claim 6, wherein the level includes a pH level, a dissolved oxygen level, a total biomass level, a cell diameter level, or a temperature level.

9. The method of claim 1, wherein the cell culture device is a single-use cell culture device.

10. A method for optimizing a cell culture protocol, the method comprising: A cell culture system is provided, the cell culture system comprising: A cell culture device, the cell culture device comprising one or more fluid inlets and one or more fluid outlets; a fluid source fluidly connected to one of the one or more fluid inlets; a controller configured to communicate with one or more of a publicly available database and an internal database, each of the publicly available database and the internal database including cell culture protocol data; one or more sensors disposed on the cell culture device communicatively coupled to the controller, the one or more sensors configured to measure one or more parameters within the cell culture device, the one or more parameters including one or more of pH, glucose concentration, lactate concentration, dissolved oxygen, total biomass, cell diameter, temperature, cell type, medium type, cell metabolite concentration, and fluid flow rate; one or more pumps communicatively coupled to the controller, the one or more pumps configured to pump fluid into the cell culture device through the one or more fluid inlets and out of the cell culture device through the one or more fluid outlets; an interface by means of which one or more users can interact, via associated computing devices, to determine and monitor cell culture protocols; and receiving data associated with the cells to be cultured as input from the one or more users; setting user-defined parameters at levels to be maintained during cell culture; Implementing a cell culture protocol; measuring the level of the user-defined parameter during cell culture via the one or more sensors; optimizing the cell culture protocol by determining whether to modify cell culture conditions to maintain the level of the user-defined parameter; A method comprising:

11. The method of claim 10, further comprising periodically measuring the level of the parameter during the cell culture protocol.

12. The method of claim 10, wherein the user-defined parameters include pH, turbidity, glucose concentration, lactate concentration, other measurements of cell health or identity, or combinations thereof.

13. The method of claim 10, further comprising altering cell culture conditions.

14. The method described in claim 13, wherein altering the cell culture conditions includes altering the glucose concentration or lactate concentration by manipulating the flow rate of the culture medium.

15. The method of claim 13, wherein altering the cell culture conditions comprises adding a supplementary substance.

16. The method of claim 15, wherein the supplemental substances include cytokines, growth factors and serum.

17. The method of claim 10, further comprising storing the optimized cell culture protocol in a database.

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