Temperature calibration method and device for use in automated bioreactors

Abstract

The present disclosure provides devices and related methods for monitoring and controlling temperature in automated biomaterials engineering systems, including tissue engineering systems, that utilize internal temperature measurements within the automated system to map temperatures during various processes performed within the system.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 089,840, entitled "Temperature Calibration Methods and Devices for Use in Automated Bioreactors," filed October 9, 2020, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure provides devices and related methods for temperature monitoring and control in automated biomaterials engineering systems, including tissue engineering systems, that utilize measurements of the internal temperature of the automated system to map temperatures during various processes performed within the system. [Background technology]

[0003] As the clinical adoption of advanced cell and biomaterial therapies is expected to accelerate, increasing attention is being paid to the underlying manufacturing strategies that will enable these therapies to benefit patients worldwide. While cell therapies hold great clinical potential, high manufacturing costs relative to reimbursement present a significant barrier to commercialization. Therefore, the need for cost-effectiveness, process efficiency, and product consistency is driving automation efforts across many cell therapy fields.

[0004] The production of therapeutic cell populations involves the automation of various processes, including the integration of cell activation, transduction, and expansion into commercial manufacturing platforms to translate these important therapies to broad patient populations.

[0005] During the various processes of manufacturing biological materials, including cell production, there is a need to monitor temperature changes and gradients in such automated systems to ensure that biological processes are carried out at the correct temperature and that materials for use are stored at the appropriate temperature. The present invention meets these needs. Summary of the Invention

[0006] One aspect of the present disclosure relates to a calibration cassette for use in an automated biomaterials engineering system. The calibration cassette includes a cryo-chamber, a high-temperature chamber, one or more fluidics pathways, and an electrical connection element. The cryo-chamber includes a media reservoir and a first array of sealed temperature probes within the media reservoir. The high-temperature chamber is separated from the cryo-chamber by a thermal barrier and includes a cell culture chamber and a second array of sealed temperature probes within the cell culture chamber. The one or more fluidics pathways are connected to the cell culture chamber and the media reservoir and include a third array of sealed temperature probes within the one or more fluidics pathways. The electrical connection element is electrically connected to each of the first, second, and third arrays of sealed temperature probes.

[0007] One aspect of the present disclosure relates to a production cassette for use in an automated cell engineering system, the production cassette including: a low temperature chamber including a cell culture medium reservoir and a first array of sealed temperature probes within the cell culture medium reservoir; a high temperature chamber for performing cell culture activation, transduction, and / or expansion, the high temperature chamber being separated from the low temperature chamber by a thermal barrier, the high temperature chamber including a cell culture chamber and a second array of sealed temperature probes within the cell culture chamber; one or more fluidics pathways connected to the cell culture chamber and the cell culture medium reservoir, the one or more fluidics pathways including a third array of sealed temperature probes within the one or more fluidics pathways; and an electrical connection element electrically connected to each of the first, second, and third arrays of sealed temperature probes, the one or more fluidics pathways providing recirculation, waste removal, and homogenous gas exchange and nutrient distribution to the cell culture chamber.

[0008] One aspect of the present disclosure relates to a method for monitoring temperature in an automated biomaterials engineering system, the method including: receiving, by a control circuit, a set of internal temperature measurements while a first cassette is housed within the automated biomaterials engineering system, the set of internal temperature measurements indicative of a temperature within the first cassette and generated during the time period by an array of temperature probes disposed within the first cassette; receiving, by the control circuit, ambient temperature measurements while the first cassette is housed within the automated biomaterials engineering system, the ambient temperature measurements indicative of a temperature outside the first cassette and generated during the time period by a system temperature probe of the automated biomaterials engineering system disposed outside the first cassette; and determining, by the control circuit, a set of temperature offset values ​​indicative of respective differences between the set of internal temperature measurements and the ambient temperature measurements.

[0009] One aspect of the present disclosure relates to a method of temperature control implemented in an automated biomaterials engineering system, the method including receiving, by a control circuit, a set of internal temperature measurements during a first time period while a first cassette is housed within the automated biomaterials engineering system, the set of internal temperature measurements being indicative of a temperature within the first cassette and generated during the first time period by an array of temperature probes disposed within the first cassette, and receiving, by the control circuit, a first ambient temperature measurement while the first cassette is housed within the automated biomaterials engineering system, the first ambient temperature measurement being indicative of a temperature outside the first cassette and generated during the first time period by a system temperature sensor of the automated biomaterials engineering system disposed outside the first cassette. determining, by the control circuitry, a set of temperature offset values ​​indicative of respective differences between the set of internal temperature measurements and the first ambient temperature measurement; determining, by the control circuitry, a target internal temperature value for a location within the second cassette; and controlling, by the control circuitry, a heating or cooling device of the automated biomaterials engineering system during a second time period that the second cassette is housed within the automated biomaterials engineering system based on the target internal temperature value, the set of temperature offset values, and one or more additional ambient temperature measurements generated by a system temperature sensor during the second time period, wherein the system temperature sensor is disposed outside the second cassette. [Brief explanation of the drawings]

[0010] [Figure 1] 1 illustrates various steps that may be performed using a cassette of an automated biomaterials engineering system as described in embodiments herein. [Figure 2A] 1 illustrates an exemplary cassette according to embodiments herein. [Figure 2B] 1 illustrates an exemplary cassette for use in temperature measurement and / or calibration as described herein. [Figure 2C]1 illustrates an exemplary cassette for use in temperature measurement and / or calibration as described herein. [Figure 2D] 1 illustrates an exemplary cassette for use in temperature measurement and / or calibration as described herein. [Figure 2E] 1 illustrates a sealed temperature probe according to an embodiment herein. [Figure 3A] 1 shows an image of an automated biomaterials engineering system according to embodiments herein. [Figure 3B] 1 shows an image of an automated biomaterials engineering system according to embodiments herein. [Figure 3C] 1 shows an image of an automated biomaterials engineering system and cassette, according to embodiments herein. [Figure 3D] 1 shows an image of an automated biomaterials engineering system and cassette, according to embodiments herein. [Figure 3E] 1 shows an image of a cassette according to an embodiment of the present disclosure. [Figure 3F] 1 shows images of an automated biomaterials engineering system, cassette, and computing device according to one embodiment of the present disclosure. [Figure 3G] 1 shows an image of an automated biomaterials engineering system capable of receiving a first cassette and a second cassette, according to an embodiment of the present disclosure. [Figure 4] 1 illustrates a laboratory space housing an exemplary biomaterials engineering system as described in embodiments herein. [Figure 5] 1 illustrates a flow path of an automated biomaterials engineering system as described in embodiments herein. [Figure 6] 1 depicts a flow diagram of an exemplary method for determining a temperature offset, according to an embodiment of the present disclosure. [Figure 7A] 1 shows a cassette for use in temperature measurement and / or calibration as described herein. [Figure 7B]1 shows a cassette for use in temperature measurement and / or calibration as described herein. [Figure 8A] 1 illustrates a temperature offset value according to an embodiment of the present disclosure. [Figure 8B] 1 illustrates a temperature offset value according to an embodiment of the present disclosure. [Figure 9] 1 depicts a cassette that may be disposed in an automated biomaterials engineering system, according to one embodiment of the present disclosure. [Figure 10A] 1 depicts a control temperature and a medium temperature being affected by operation of a heating device according to one embodiment herein. [Figure 10B] 1 depicts a control temperature and a medium temperature being affected by operation of a heating device according to one embodiment herein. DETAILED DESCRIPTION OF THE INVENTION

[0011] It should be understood that the specific implementations shown and described herein are examples and are not intended to otherwise limit the scope of the present application in any way.

[0012] Published patents, patent applications, websites, company names, and scientific literature referred to herein are incorporated by reference in their entirety to the same extent as if each were specifically and individually indicated to be incorporated by reference. Any conflict between any reference cited herein and the specific teachings of this specification shall be resolved in favor of the latter. Similarly, any conflict between an art-understood definition of a word or phrase and a definition of a word or phrase specifically taught herein shall be resolved in favor of the latter.

[0013] As used herein, the singular forms "a," "an," and "the" specifically include the plural of the term they refer to, unless the content clearly dictates otherwise. The term "about" is used herein to mean approximately, within the region of, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the stated numerical values. In general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of 20%.

[0014] Unless otherwise defined, technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this application pertains. Reference is made herein to various methodologies and materials known to those skilled in the art.

[0015] The methods, devices, and systems provided herein are described with reference to their application using cassettes for use in automated biomaterials engineering systems. Figure 1 shows an exemplary cassette 102 in which various processes may be performed in a sealed, automated system that enables the production of various samples and populations, particularly "biological materials," including proteins, peptides, antibodies, antibody fragments, and cells. Such processes may include steps for activating, transducing, expanding, concentrating, washing, and collecting / harvesting proteins and / or cells.

[0016] As described herein, the cassettes and methods are suitably utilized and implemented in a fully enclosed automated biomaterials engineering system 300 (see Figures 3A and 3B), including an automated cell engineering system, which preferably has instructions for performing steps such as activation, transduction, expansion, enrichment, and harvesting. For example, a cell engineering system for the automated production of genetically modified immune cells, including CAR T cells, is described in U.S. Patent Application No. 16 / 119,618, filed August 31, 2018 (the disclosure of which is incorporated herein by reference in its entirety), and is also referred to herein as the automated cell engineering system, COCOON®, or COCOON™ system.

[0017] For example, a user can provide an automated cell engineering system pre-filled with cell culture materials and reagents (e.g., activation reagents, vectors, cell culture media, nutrients, and selection reagents, etc.), as well as parameters for cell production (e.g., starting number of cells, type of media, type of activation reagent, type of vector, and number or quantity of cells to be produced, etc.). The automated cell engineering system can perform various automated methods, including methods for producing genetically modified immune cell cultures, including CAR T cells, without further input from the user. In some embodiments, a fully enclosed automated cell engineering system minimizes contamination of the cell cultures by reducing exposure of the cell cultures to non-sterile environments. In additional embodiments, a fully enclosed automated cell engineering system minimizes contamination of the cell cultures by reducing the user's handling of the cells.

[0018] The automated engineering system can also be used to prepare other biological materials, including various proteins, peptides, antibodies, antibody fragments, and the like.

[0019] As described herein, the automated biomaterials engineering system 300 preferably includes a cassette 102. As used herein, "cassette" refers to a largely self-contained, removable, and replaceable element of the automated biomaterials engineering system that includes one or more chambers for carrying out the various elements of the methods described herein, and preferably also includes one or more of cell culture media, activation reagents, wash media, etc.

[0020] 2A shows an exemplary cassette 102 for use in an automated biomaterials engineering system, including an automated cell engineering system. In an embodiment, the cassette 102 includes a cell sample input 202. The cell sample input 202 is shown in FIG. 2A as a vial or chamber into which a cell sample can be placed prior to introduction or loading into the cassette 102. In other embodiments, the cell sample input 202 may simply be sterile locking tubing (e.g., a Luer lock tubing connection, etc.) to which a syringe or cell-containing bag, such as a blood bag, can be connected.

[0021] Cassette 102 further includes a cell culture chamber 206. Examples of features and uses of cell culture chamber 206 are described herein. Cassette 102 also includes a pumping system 520 (see FIG. 5 for an exemplary location within the flow path) fluidly connected to cell culture chamber 206.

[0022] As used herein, "fluidically connected" means that one or more components of a system, such as the components of cassette 102, are connected via suitable elements that allow fluids (including gases and liquids) to pass between the components without leaking or losing volume. Exemplary fluid connections include various tubing, channels, and connections known in the art, such as silicone or rubber tubing, luer lock connections, and the like. It should also be understood that fluidly connected components can include additional elements between each of the components while still maintaining a fluid connection. That is, fluidly connected components can include additional elements such that fluid passing between the components can, but is not required to, pass through these additional elements.

[0023] The pumping system 520 is preferably a peristaltic pump system, although other pumping systems may be utilized. A peristaltic pump refers to a type of positive displacement pump for pumping fluid. The fluid is preferably contained within a flexible tube mounted inside a pump casing, which is often circular. A rotor, with multiple "rollers," "shoes," "wipers," or "lobes" attached to its periphery, compresses the flexible tube. As the rotor rotates, it pinches closed (i.e., "occludes") the portion of the tube under pressure, thereby pumping the fluid through the tube. Furthermore, when the tube opens ("recovers" or "rebounds") after a cam passes, fluid flow is induced through the pump. This process, called peristalsis, is used to move fluid through flexible tubes. Typically, there are two or more rollers, or wipers, that occlude the tube and trap the body of fluid between them. The body of fluid is then transported toward the pump outlet.

[0024] In embodiments, cassette 102 further includes one or more fluidics pathways suitably connected to the cell culture chamber (see 232 inside cassette 102 in FIG. 2A ). Cassette 102 also includes a cell sample output 208 fluidly connected to the cell culture chamber. As described herein, cell sample output 208 can be utilized to harvest cells according to various automated procedures, either for further processing, storage, or potential use in a patient, or for further processing to isolate a desired protein or peptide produced by the cells. Cell sample output 208 can also be a sample port 220, as described herein, that allows a cell sample to be removed from the cassette for transduction, such as electroporation, and then returned to the cassette for further automated processing. Examples of fluidics pathways 232 include various tubing, channels, capillaries, microfluidic elements, etc., that provide nutrients, solutions, etc. to elements of the cassette, as described herein.

[0025] In an exemplary embodiment, provided herein is a cassette for temperature measurement and / or calibration 240 for use in an automated biomaterials engineering system. Cassette 240 may be used as a calibration cassette or a production cassette. A "calibration" cassette refers to a cassette that is not utilized during the production of a biomaterial and, therefore, serves merely as a trial or dummy cassette for measurement of temperature fluctuations and gradients during the process. A "production" cassette refers to a cassette that may be utilized to perform the production of a biomaterial, including cells. Both calibration cassettes and production cassettes are discussed herein with respect to cassette 240, as shown in the figure.

[0026] As shown in FIG. 2B, cassette 240 preferably includes a cryogenic chamber 250 including a media reservoir 228 (see FIG. 2A) and a first array 262 of sealed temperature probes 252 within media reservoir 228. Sealed temperature probes 252 are represented in FIGS. 2B-2D as short solid lines. As used herein, an "array" 262 of sealed temperature probes 252 refers to an arrangement of a plurality of sealed temperature probes 252 that allows the probes to measure temperatures at multiple different points within a structure for ultimate use in mapping or describing the temperature profile of a surface, structure, container, body, etc. A "plurality" includes two or more of an item including a sealed temperature probe, and preferably includes three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, fifteen or more, twenty or more, twenty-five or more, thirty or more, etc., of items such as sealed temperature probes 252.

[0027] In an exemplary embodiment, each of the first, second, and third arrays of sealed temperature probes includes at least two sealed temperature probes, i.e., each array configured to provide temperature information for a particular section of cassette 240, including, for example, hot chamber 254, cold chamber 250, and fluidics pathway 232. In a preferred embodiment, each of the different arrays 262 of sealed temperature probes 252 includes between 2 and 20 sealed temperature probes within each array, more preferably between 2 and 15 sealed temperature probes, 2 and 10, 2 and 9, 2 and 8, 2 and 7, 2 and 6, 2 and 5, 2 and 4, or 2 and 3 sealed temperature probes. In an embodiment, the total number of sealed temperature probes 252 in all of the arrays 262 is preferably between 10 and 15 probes, more preferably 10, 11, 12, 13, 14, or 15 total probes.

[0028] As shown in FIG. 2B, cassette 240 also preferably includes a high temperature chamber 254 separated from low temperature chamber 250 by a thermal barrier 256. Preferably, high temperature chamber 254 includes cell culture chamber 206 and a second array 262 of sealed temperature probes 252 within cell culture chamber 206 (see FIG. 2C, which shows a side view of cassette 240; FIG. 2D shows a top view of cassette 240).

[0029] In embodiments, the cold chamber 250 may include a refrigerated area 226 suitable for storing cell culture media. For example, a hot chamber 254 suitable for performing cell culture activation, transduction, and / or expansion within the cell culture chamber 206. Preferably, the hot chamber is separated from the cold chamber by a thermal barrier 256, which may be an insulating layer, section, or chamber that maintains a distinct temperature between the hot and cold regions. As used herein, "cold chamber" refers to a chamber that is preferably maintained below room temperature, more preferably between about 4°C and about 8°C, to maintain cell culture media or the like at refrigerated temperatures. The cold chamber may include a medium bag or other holder containing about 1 L, about 2 L, about 3 L, about 4 L, or about 5 L of fluid. Additional medium bags or other fluid sources may be externally connected to the cassette and connected to the cassette via an access port. The brackets shown in Figures 2B and 2C indicating the locations of the cold chamber 250 and hot chamber 254 are representative and not limiting as to the overall dimensions of either chamber, but instead are provided to indicate the preferred location and nominal dimensions of each chamber.

[0030] As used herein, "high temperature chamber" refers to a chamber that is preferably maintained above room temperature, and more preferably at a temperature that permits cell proliferation and growth, i.e., about 35-40° C., more preferably about 37° C. In embodiments, the high temperature chamber preferably comprises a cell culture chamber 206 (also referred to throughout as a proliferation chamber or cell growth chamber).

[0031] In a further embodiment of cassette 240, one or more fluidics paths contained within cassette 240 and connected to the cell culture chamber and media storage vessel also preferably include a third array 262 of temperature probes 252 sealed within one or more fluidics paths 232 (see Figures 2B and 2C).

[0032] As shown in Figures 2B-2D, the sealed temperature probes 252 also preferably include electrical connection elements 258 electrically connected to each of the first, second, and third arrays of sealed temperature probes. For clarity, not all electrical connection elements 258 are shown in Figures 2B-2D. Note that preferably, each of the sealed temperature probes 252 includes an electrical connection element 258 to enable electrical connection of the sealed temperature probe to a power source and / or to enable communication of a measurement signal.

[0033] As used herein, a "sealed temperature probe" refers to a device capable of measuring the temperature of a surface, solution, or gas and includes a cover that seals the probe and limits or preferably prevents the transfer of liquid and / or gas beyond the cover. An exemplary sealed temperature probe 252 is shown in FIG. 2E , showing a temperature probe 282 (for measuring temperature) and a cover (or seal) 284 (only the cover is visible because the probe is within the cover), as well as electrical connection elements 258. Suitable covers that may be utilized include various polymers, and preferably the cover provides a hermetic or gas-tight seal around the temperature probe. An exemplary temperature probe included a resistance temperature detector (RTD) hermetically sealed by a polymer cover. Additional temperature probes that may be utilized include thermocouples and thermistors, which also preferably include a seal or cover to reduce or eliminate contamination of the probe by fluids, gases, or the like.

[0034] As described herein, in an exemplary embodiment, the medium storage vessel 228 is a bag and the array 262 of sealed temperature probes 252 is attached to the interior surface of the bag. As described herein, the cell culture chamber 206 is preferably flat and substantially inflexible. It has been found that such a cell culture chamber allows for increased cell yield during a cell or biomaterial production process. In an embodiment, the array 262 of sealed temperature 252 probes is attached to the bottom and / or sides of the cell culture chamber.

[0035] As described herein, the fluidics pathway within the cassette preferably includes various tubing and connectors, and in embodiments, also includes a third array 262 of sealed temperature probes 252, located within the tubing. See FIG. 2B for a central section of cassette 240, showing potential exemplary locations of sealed temperature probes 252 within the tubing. The locations of the temperature probes within the tubing can be designed so that temperature profiles and gradients can be measured at various sections of the fluid pathway, including, for example, within tubing configured to deliver cell culture medium to cell culture chamber 206, within tubing configured to remove cells from cell culture chamber 206, within tubing configured to mix cell culture medium to regulate gas content, within tubing configured to allow sampling of a cell culture process or other biomaterial process, within tubing configured to provide an input pathway to cell culture chamber 206, and other configurations. Methods for attaching the sealed temperature probe 252 to one or more sections or elements of the cassettes described herein are known in the art and include, for example, the use of various adhesives, tapes, glues, heat sealing methods, soldering methods, connection by mechanical fasteners, and direct integration into the cassette elements, such as during forming, molding, or manufacturing.

[0036] Preferably, the cassette 240 described herein further includes a control circuit 270 electrically connected to the electrical connection elements 258 and thus connected to the sealed temperature probe 252. FIG. 2B shows an exemplary location of the control circuit 270 and its electrical connection 272 to one of the electrical connection elements 258. It should be understood that the other electrical connection elements 258, and thus the probe 252, may be connected to the control circuit 270 in a similar manner but are not shown in the figure for ease of visualization. The control circuit 270 may also be located in any desired location on the cassette 240, including, for example, along the side, bottom, or top of the cassette 240 (see FIG. 2C for an exemplary side location and FIG. 2D for an exemplary top location), and may be located internal (i.e., inside the housing) or external to the cassette. The location of the control circuit 270 shown in the figures is for illustrative purposes only, and one of ordinary skill in the art will readily understand that other locations and configurations may also be used. While the control circuitry is preferably electrically wired to the temperature probe, some embodiments may involve a wireless connection (e.g., radio frequency, Bluetooth, etc.) for communicating with the temperature probe. In such embodiments, another device may be electrically wired to the temperature probe via a wired connection and may further have a wireless connection with the control circuitry; that is, the device may be configured to communicate wirelessly with the control circuitry. Such a device may, for example, include data acquisition circuitry having a wired connection to the temperature probe and configured to receive measurements or other data generated by the temperature probe, and may further include a wireless module (also referred to as wireless communication circuitry) that may relay such data to the control circuitry via a wireless connection.

[0037] As used herein, “control circuitry” refers to electronic circuitry that provides functionality related to temperature control and / or temperature measurement. Control circuitry 270 may be configured, for example, to receive and process temperature measurements made by temperature sensor 252 or any other temperature sensor. In some embodiments, the control circuitry may be configured to control a communications module (e.g., a wireless module) to communicate the temperature measurements to a computer system for recording the temperatures. In some cases, the control circuitry may be configured to control or otherwise affect temperature correction (i.e., increasing or decreasing the temperature), such as by controlling a heating or cooling device. In embodiments, control circuitry 270 provides a simple measurement and recording function of the temperature of cassette 240 to enable temperature mapping, as described herein. In some embodiments, control circuitry 270 may also be utilized to record or log temperature measurements over a set period of time for later use or retrieval. In such embodiments, if control circuitry 270 is external to a computer, the control circuitry need not be immediately connected to the computer, but instead can connect (either wirelessly or via a direct connection) and transfer data at a later time. In an embodiment, control circuitry 270 may be programmed to turn off or enter a low power state between temperature measurements to reduce unintentional heating of the cassette. In one embodiment, control circuitry 270 may include processing circuitry such as one or more microprocessors, microprocessor cores, programmable logic circuits (PLCs), field programmable gate array (FPGA) circuits, application specific integrated circuits (ASICs), microcontroller units (MCUs), and / or any other control circuitry.

[0038] As discussed in more detail below, control circuitry 270 may be located within or otherwise associated with the cassette, the automated biomaterials engineering system, or some other device, such as a desktop computer or laptop, that communicates with the automated biomaterials engineering system. For example, control circuitry 270 may be associated with cassette 240, and thus may be directly connected to, contained within, or part of cassette 240. In other embodiments, control circuitry 270 may be associated with automated biomaterials engineering system 300. For example, as shown in FIG. 3B, control circuitry 270 may be connected to, contained within, or otherwise part of biomaterials engineering system 300. When cassette 240 is inserted into system 300, cassette 240 may communicate with the control circuitry via a wired electrical connection or a wireless connection. For example, if the cassette has one or more electrical contacts or other electrical conductors (e.g., wires) extending from locations inside the cassette to locations outside the cassette or on the outer surface of the cassette, the electrical conductors may provide a wired electrical connection to provide communication and / or power to various components of the cassette. In such an example, the electrical connector may include or be electrically connected to electrical connection element 258, which may be electrically connected to temperature probe 252 within cassette 240. If the electrical conductors are also electrically connected (directly or indirectly) to control circuitry 270, they may provide an electrical connection through which communication may occur between control circuitry 270 and temperature probe 252. For example, this may be done by plugging electrical connection element 258 into a connector electrically connected to control circuitry 270, either when cassette 240 is being inserted into system 300 (i.e., as a plug-and-play connection) or after cassette 240 is inserted (i.e., as an additional connection, perhaps used only if desired).As described above, communication (also referred to as a communication connection or communication coupling) between cassette 240 and control circuit 270 may be performed wirelessly, particularly if cassette 240 does not have conductors capable of providing a wired connection between a location inside cassette 240 and a location outside cassette 240.

[0039] Various filters or separation devices can be utilized in the cassettes and methods described herein. For example, a magnetic separation process can be utilized to remove and separate undesired cells and debris from a cell population. In such embodiments, magnetic beads or other structures to which biomolecules (e.g., antibodies, antibody fragments, etc.) are attached can interact with the target cells. Various magnetic separation methods, including the use of filters, columns, flow tubes, or channels with magnetic fields, can then be used to separate the target cell population from undesired cells, debris, etc. that may be present in the cell sample. For example, the target cell population can flow through a tube or other structure and be exposed to a magnetic field, whereby the target cell population is retained or retained by the magnetic field, allowing the undesired cells and debris to pass through the tube. The magnetic field can then be turned off, allowing the target cell population to pass over an additional retention chamber or other area of ​​the cassette for further automated processing. Additional filtration can include traditional column filtration or the use of other filtration membranes and structures.

[0040] In embodiments where a magnetic separation process is utilized, the cassettes described herein may also further include a magnetic probe for measuring and mapping the magnetic flux within and surrounding the cassette, which provides information that can be utilized to calibrate, verify, and / or control the magnetic field during the separation process.

[0041] In a further embodiment, cassette 240 further includes a waste collection chamber 510. In additional embodiments, satellite volumes 550, which can provide additional storage capabilities to the cassette, increase the overall volume of the automated process. Exemplary locations of satellite volumes 550 are shown in the flow paths of FIG. 5. The cassette may also include one or more fluidics paths (collectively 232) that provide recirculation, waste removal, and uniform gas exchange, as well as distribution of nutrients to various portions of the cassette, including the cell culture chambers, without disturbing the cells within the cell culture chambers. Cassette 240 also includes one or more valves 522 or 552 for controlling flow through the various fluidics paths (see FIG. 5 for exemplary locations within the flow paths).

[0042] In an exemplary embodiment, as shown in FIGS. 2A and 2B, the cell culture chamber 206 is a flat, non-flexible chamber (i.e., made of a substantially non-flexible material, such as plastic) that does not easily bend or flex. The use of a non-flexible chamber allows the cells to be maintained in a substantially undisturbed state. As shown in FIG. 2A, the cell culture chamber 206 is oriented to allow the cell culture to spread across the bottom of the cell culture chamber. As shown in FIG. 2A, the cell culture chamber 206 is preferably maintained in a position parallel to the floor or table, maintaining the cell culture in an undisturbed state and allowing the cell culture to spread across a wide area at the bottom of the cell culture chamber. In an embodiment, the overall thickness (i.e., chamber height) of the cell culture chamber 206 is low, approximately 0.5 cm to approximately 5 cm. Preferably, the cell culture chamber has a volume of approximately 0.50 ml to approximately 300 ml, more preferably approximately 50 ml to approximately 200 ml, or the cell culture chamber has a volume of approximately 180 ml. Using a low chamber height (less than 5 cm, preferably less than 4 cm, less than 3 cm, or less than 2 cm) allows for effective medium and gas exchange in close proximity to the cells. Ports are configured to allow mixing through fluid recirculation without disturbing the cells. Static vessels of greater height can create concentration gradients, limiting oxygen and fresh nutrients to areas near the cells. Through controlled flow dynamics, medium exchange can be performed without disturbing the cells. Medium can be removed from additional chambers (where no cells are present) without risk of cell loss.

[0043] As described herein, in exemplary embodiments, the cassette is pre-filled with one or more of cell culture, culture medium, if desired, cell wash medium, activation reagents, and / or vectors, and any combination thereof. In further embodiments, these various elements may be added later via suitable injection ports, etc.

[0044] As described herein, in embodiments, the cassette preferably further includes one or more of a pH sensor 524, a glucose sensor (not shown), an oxygen sensor 526, a carbon dioxide sensor (not shown), a lactate sensor / monitor (not shown), and / or an optical density sensor (not shown). See FIG. 5 for exemplary locations within the flow path. The cassette may also include one or more sampling and / or injection ports. Examples of such sampling and injection ports 220 and 222 are shown in FIG. 2A and are exemplary locations within the flow path shown in FIG. 5, which may include access ports for connecting the cartridge to external devices, such as an electroporation unit or additional media source. FIG. 2A also shows the locations of the input 202, a preheated warming bag 224 that can be used to warm cell culture media, etc., and a secondary chamber 230.

[0045] In embodiments, cassette 240 may also include a cell wash system 512 that is suitably contained within the cassette (i.e., within the structure shown in FIG. 2A ) and fluidly connected to the cassette's fluidics system. In embodiments, cell wash system 512 is a container or bag contained within cassette 240 that suitably contains a cell wash medium. The cell wash medium is suitably used to wash the desired cell population to remove any unwanted waste cells or contamination prior to transferring the cell population into or out of the cassette for further processing or use. Cell wash system 512 may also be contained outside of cassette 102.

[0046] Cassette 102 may also optionally further include a cell-retaining chamber 516 (not visible in FIG. 2 because it is located inside cassette 102). FIG. 5 shows an exemplary location of cell-retaining chamber 516 within the flow path of the cassette. Cell-retaining chamber 516 is preferably a reservoir or suitable chamber located within the cassette that can retain a population of cells therein either before or after various stages of processing, as described herein.

[0047] In a further embodiment, provided herein is a production cassette for use in an automated cell engineering system, comprising: a low temperature chamber comprising a cell culture medium reservoir and a first array of sealed temperature probes within the cell culture medium reservoir; a high temperature chamber for performing cell culture activation, transduction, and / or expansion, the high temperature chamber being separated from the low temperature chamber by a thermal barrier, the high temperature chamber comprising a cell culture chamber and a second array of sealed temperature probes within the cell culture chamber; one or more fluidics pathways connected to the cell culture chamber and the cell culture medium reservoir, the one or more fluidics pathways comprising a third array of sealed temperature probes within the one or more fluidics pathways; and an electrical connection element electrically connected to each of the first, second, and third arrays of sealed temperature probes, wherein the one or more fluidics pathways provide recirculation, waste removal, and homogenous gas exchange, as well as nutrient distribution to the cell culture chamber.

[0048] As previously mentioned, the cassette 240 described herein, including the sealed temperature probe 252, can be utilized as a calibration cassette and / or a production cassette. In embodiments in which the cassette is utilized as a calibration cassette, the cassette is designed and implemented as if it were to produce a cell or biomaterial product, but no product is actually produced. Instead, the cassette simply provides calibration of the associated system / platform, production cassette design, and temperature of various sections and regions of the production cassette or process design for use with the production cassette. In embodiments in which the cassette is a production cassette, in addition to providing information regarding the temperature profile within the cassette, the system is also configured to prepare the desired cells and / or biomaterial for eventual use in a patient or research setting. In embodiments in which the cassette is a production cassette, the temperature probe is suitably removed and cleaned / sterilized or replaced between uses, especially if each different use is for a different patient.

[0049] As described herein, in an embodiment of the production cassette, the cell culture medium reservoir is a bag and a first array of sealed temperature probes is attached to the interior surface of the bag. Preferably, the cell culture chamber is flat and substantially non-flexible, and a second array of sealed temperature probes within the cell culture chamber is attached to the bottom and / or sides of the cell culture chamber. In an additional embodiment, the one or more fluidics paths include tubing and connectors, and a third array of sealed temperature probes is located within the tubing.

[0050] In embodiments, the first, second, and third arrays of sealed temperature probes include resistance temperature detectors (RTDs) hermetically sealed by polymer covers. Additional examples of temperature probes are described herein. Suitable numbers of probes for use in the various arrays are described herein, and in embodiments, the first, second, and third arrays of sealed temperature probes each include at least two sealed temperature probes, preferably two to four sealed temperature probes, and in embodiments, the first, second, and third arrays of sealed temperature probes include a total of 12 sealed temperature probes.

[0051] As described herein, the production cassette also preferably includes control circuitry electrically connected to the electrical connection elements for interaction with the temperature probe (i.e., for measuring, recording, correcting, etc.). The control circuitry may be associated with the production cassette (i.e., connected inside or outside the cassette), or may be associated with an automated cell engineering system.

[0052] The devices, systems, and methods described herein are suitable for use in monitoring, mapping, and / or controlling temperature within a cassette of a biomaterials engineering system. However, similar approaches can be utilized with probes that measure other variables within the cassette, including, for example, pH, dissolved oxygen, fluid flow, magnetic fields, etc. Probes for measuring such variables are known in the art and, like temperature probes, can be arranged within the cassette in an array format, allowing these variables to be measured across all or a portion of the cassette in order to map and monitor the variables during various process steps of a tissue or biomaterials engineering method.

[0053] In a further embodiment, a remote temperature probe can be used to monitor, record, and provide feedback on the temperature of a cassette in an automated biomaterials engineering system. Such a remote temperature probe can include, for example, an infrared temperature sensing device that can be installed within the automated engineering system and record one or more temperatures within the cassette as the automated process is performed.

[0054] 3A-3B show a COCOON® automated cell engineering system 300 with a cassette 240 positioned therein (the cover of the automated cell engineering system is open in FIG. 3B). Also shown is an exemplary user interface 304, which may include the ability to use and receive input via a barcode reader and a touchpad or other similar device.

[0055] The automated cell engineering systems and cassettes described herein preferably have three related volumes: cell culture chamber volume, working volume, and total volume. Preferably, the working volume used within the cassette ranges from 180 mL to 460 mL based on the process step, and can be increased to about 500 mL, about 600 mL, about 700 mL, about 800 mL, about 900 mL, or about 1 L. In embodiments, the cassette has a 4*10 9 Cells-10*10 9The cell concentration during the process can be easily achieved at 0.3*10 6 cells / ml to approximately 10*10 6 The working volume varies from 100 to 1000 cells / ml. Cells are placed in the cell culture chamber while the medium is continuously recirculated through additional chambers (e.g., cross-flow reservoirs and satellite volumes) to increase the working volume, as described herein.

[0056] The fluidics pathways, including the gas exchange lines, may be made from a gas-permeable material, such as silicone. In some embodiments, the automated cell engineering system recirculates oxygen throughout the chamber substantially free of yield during the cell production method. Thus, in some embodiments, the oxygen level of the cell culture in the automated cell engineering system is higher than the oxygen level of the cell culture in the flexible gas-permeable bag. Higher oxygen levels may be important during the cell culture expansion step, as the increased oxygen levels may support increased cell growth and proliferation.

[0057] 3C and 3D depict embodiments of an automated biomaterials engineering system, such as the COCOON® automated tissue engineering system or any other biomaterials engineering system, and a cassette that may be disposed within the automated biomaterials engineering system. More specifically, FIG. 3C depicts a cassette 340 having multiple temperature probes 352 and having control circuitry 270. Temperature probes 352 may be configured to measure an internal temperature within cassette 340. The figure also depicts an automated biomaterials engineering system 301 that may receive cassette 340. Automated biomaterials engineering system 301 may include a system temperature probe 242 and a heating or cooling device 250. System temperature probe 242 may be configured to measure a temperature outside cassette 340 (which may be referred to as ambient temperature). In some cases, automated biomaterials engineering system 301 may have its own control circuitry, which may be separate from control circuitry 270.

[0058] 3D depicts control circuitry 270 being located within or otherwise associated with an automated biomaterials engineering system 301. In this example, system 301 may be configured to receive cassette 341 having wireless module 362, and control circuitry 270 of system 301 may be configured to receive internal temperature measurements from within cassette 341 via wireless module 362.

[0059] FIG. 3E depicts an example of cassette 340 / 341. More specifically, cassette 340 in this example may have a temperature probe 352 and a printed circuit board for processing temperature measurements made by temperature probe 352 and / or for communicating the temperature measurements to an external device, such as control circuit 270 of FIG. 3D. For example, the printed circuit board may include a frequency filter, an analog front end (AFE), an analog-to-digital converter, and control circuitry (e.g., a microcontroller unit (MCU)) for processing the temperature measurements, as well as a wireless module 362 for transmitting the temperature measurements to another device. In one embodiment, the MCU may be configured to store the temperature measurements on a removable memory device or some other non-transitory computer-readable or circuit-readable medium. In some cases, in-circuit programming on the removable memory device and / or printed circuit board may store instructions that can be executed by the MCU.

[0060] 3F depicts an example in which control circuitry 270 is located within or otherwise associated with a computing device 303, such as a laptop or desktop computer or other personal computer (PC), in communication with cassettes 340 / 341 and / or automated biomaterials engineering system 301. Control circuitry 270 may be configured, for example, to receive temperature measurements or other data from cassettes 340 / 341 and / or from automated biomaterials engineering system 301 and / or may be configured to control one or more components of automated biomaterials engineering system 301. In one embodiment, cassettes 340 / 341 / 342, automated biomaterials engineering system 301, and / or computing device 302 may include non-transitory computer-readable media such as a hard disk drive (HDD), solid-state drive (SSD), flash memory, or any other storage device. The non-transitory computer-readable media may store data, such as temperature measurements, and / or instructions that may be executed by control circuitry 270. These instructions may be used to perform one or more of the methods discussed herein, such as, for example, method 600 discussed below.

[0061] 3G illustrates an example in which automated biomaterials engineering system 301 may be configured to receive a first cassette or a first type of cassette, such as cassette 340 / 341, and, either at a different time or simultaneously, receive a second cassette or a second type of cassette, such as cassette 342. In one embodiment, cassette 342 may have fewer temperature probes than cassette 340 / 341, or may not have a temperature probe within cassette 342. In some cases, cassette 340 / 341 may be a calibration cassette and cassette 342 may be a production cassette.

[0062] In embodiments, the methods and cartridges described herein are utilized with the COCOON® platform (Octane Biotech, Kingston, ON), which integrates multiple unit operations on a single turnkey platform. Multiple cell protocols are provided, tailored to very specific cell processing objectives. To provide an efficient and effective automation transition, the described methods utilize the concept of application-specific / sponsor-specific disposable cassettes that combine multiple unit operations, all focused on the core requirements of the final cell therapy product. Multiple automated cell engineering systems 300 can be integrated together into large-scale, multiple unit operations for the production of large quantities of cells or multiple different cell samples for individual patients (see Figure 4).

[0063] Also shown in Figure 5 are exemplary positioning of various sensors (e.g., pH sensor 524, dissolved oxygen sensor 526), ​​as well as one or more fluidics pathways 540, preferably comprising silicone-based tubing components, connecting various sensors, sampling / sample ports, and valves (including bypass check valve 552). As described herein, the use of silicone-based tubing components allows for oxygen delivery through the tubing components to facilitate gas transfer and optimal oxygenation for the cell culture. Also shown in Figure 5 is the use of one or more hydrophobic filters 554 or hydrophilic filters 556 within the flow paths of the cassette.

[0064] In an additional embodiment, provided herein is an automated cell engineering system 300. As shown in Figures 3A and 3B, the automated cell engineering system 300 preferably includes a sealable housing 302 and a cassette 240 contained within the sealable housing. As used herein, "sealable housing" refers to a structure that can be opened and closed, into which a cassette 240 as described herein can be placed, and which can be integrated with various components such as fluid supply lines, gas supply lines, power, cooling connections, heating connections, etc. As shown in Figures 3A and 3B, the sealable housing can be opened (Figure 3B) to allow insertion of the cassette, and closed (Figure 3A) to maintain a closed and sealed environment to enable the cassette to be utilized to perform the various automated processes described herein.

[0065] Automation of unit operations in cell therapy production offers opportunities for universal benefits across allogeneic and autologous cell therapy applications. For autologous cell products, and the unique patient-specific scenarios further highlighted by the clinical success of these therapies, the benefits of automation are particularly attractive due to the significant microlot complexity associated with small-batch GMP compliance, economics, patient traceability, and early identification of process deviations. With the emergence of complex manufacturing protocols, the value of end-to-end integration of automated unit operations in microlot cell production has been highlighted, a fact that has been largely underexplored to date. However, with the anticipated demand for these therapies following imminent approval, implementing a fully closed, end-to-end system could provide a much-needed solution to manufacturing bottlenecks such as hands-on time and footprint.

[0066] Developers of advanced therapies are encouraged to consider automation early in the clinical transition deployment and scale-up of clinical trial protocols. Early automation can impact protocol development, avoid the need for comparison studies when switching from manual to automated processes at a later stage, and better understand the longer-term route to commercialization.

[0067] Show me how One aspect of the present disclosure relates to a method for performing temperature monitoring and / or temperature control in an automated biomaterials engineering system, such as system 300 / 301 discussed above. The method may be performed by control circuitry, such as control circuitry 270 discussed above. As mentioned above, control circuitry 270 may be located within or otherwise associated with a cassette (e.g., cassettes 240, 340, or 341 of FIGS. 2B, 2C, 3C, and 3D), located within or otherwise associated with the automated biomaterials engineering system (e.g., 300 / 301 of FIGS. 3A-3F), or associated with some other device (e.g., computing device 303).

[0068] FIG. 6 shows a flow diagram of an exemplary method 600 in which temperature monitoring is performed. The method may be performed, for example, by control circuit 270. In one embodiment, method 600 may begin with or otherwise include step 602, in which the control circuit receives a set of internal temperature measurements indicative of the temperature within a first cassette, such as cassette 240 of FIGS. 2B and 3A or cassette 340 / 341 of FIGS. 3D-3F. In one embodiment, the set of internal temperature measurements may be received from an array of temperature probes disposed within the first cassette, such as the array of temperature probes 252 of FIG. 2B or the array of temperature probes 352 (also referred to as temperature sensors) of FIGS. 3D-3E. In such an embodiment, the control circuit may receive the set of internal temperature measurements from the array of temperature probes via a wired connection, such as provided by electrical connection element 258 of FIG. 2B, or via a wireless connection, such as provided by the wireless module of FIGS. 3D / 3E. For example, as shown in FIG. 3C , if the control circuitry (e.g., 270) is disposed within the first cassette, the control circuitry may receive the temperature measurements via a wired connection, such as via electrical connection element 258 of FIG. 2A or 2B in some implementations. As shown in FIGS. 3D and 3F , if the control circuitry (e.g., 270) is disposed outside the first cassette, the control circuitry may receive the temperature measurements via wireless module 362 or some other communication circuitry, or via a wired electrical connection (if present). As described above, wireless module 362 may relay data collected by data acquisition circuit 361 from temperature probe 352. In some cases, if temperature probe 352 has an impedance that varies based on temperature, data acquisition circuit 361 may be configured to determine an impedance value of temperature probe 352 and calculate a temperature value using the impedance value. Wireless module 362 may then be configured to wirelessly communicate the temperature value to control circuit 270. In some cases, if the control circuit 270 has a wired connection to a temperature probe, the control circuit 270 may be configured to calculate a temperature value based on the impedance value of the temperature probe.

[0069] In some scenarios, the set of internal temperature measurements may be generated by a temperature probe and / or received by a control circuit during a first period when a first cassette (e.g., 240 or 340 / 341) is housed in an automated biomaterials engineering system (e.g., 300 / 301), such as the scenario shown in FIG. 3B. In some implementations, the first cassette may be a calibration cassette, as discussed above, and the first period may be part of a calibration phase. In some implementations, the first cassette may be a production cassette, also discussed above.

[0070] In one embodiment, an array of temperature probes may be disposed at multiple respective locations within the first cassette, as depicted in Figures 2B and 2C. In such an embodiment, the set of internal temperature measurements may correspond to multiple respective locations. For example, Figure 7A shows 、 The set of temperature measurements made at time t1 (also called Time 1) 場所1,時間1 ~Temp 場所11,時間1 Figure 7B shows 、 The set of temperature measurements made at time t2 or time 2. 場所1,時間2 ~Temp 場所11,時間2 As depicted in FIGS. 7A and 7B, a set of temperature measurements, Temp 場所1,時間1 ~Temp 場所11,時間1 may indicate the temperature at locations 1-11 at time t1 or time 1, and the set of temperature measurements Temp 場所1,時間2 ~Temp 場所11,時間2 may represent the temperature at the same location at time t2 (also referred to as Time 2). In some cases, Time 1 and Time 2 may each be a point in time during a first period in which the first cassette is disposed within the automated biomaterials engineering system. The two points in time may correspond, for example, to two different stages of a biomaterials production process (e.g., two different steps in the production process) being performed by the biomaterials engineering system.

[0071] In one embodiment, the set of internal temperature measurements may be used by the control circuit to generate a temperature map showing how the temperature varies spatially throughout the first cassette. For example, the temperature measurements Temp 場所1,時間1 ~Temp 場所11,時間1 teeth 、 The temperature measurement Temp may be used to generate a first temperature map showing how the temperature in the first cassette at a first time varies spatially. 場所1,時間2 ~Temp 場所11,時間2 teeth 、 It can be used to generate a second temperature map showing how the temperature within the first cassette at a second time varies spatially.

[0072] In one embodiment, the control circuitry (e.g., 270) may wirelessly transmit the set of internal temperature measurements to a computing device, such as computing device 303 of Figure 3D. Computing device 303 may be, for example, a desktop computer or laptop used to log the temperature within the first cassette or any other cassette compatible with the automated biomaterials engineering system.

[0073] Returning to FIG. 6, method 600, in one embodiment, may include step 604 in which the control circuitry receives an ambient temperature measurement during the period in which the first cassette is housed within the automated biomaterials engineering system. In one embodiment, the ambient temperature measurement may be generated by a system temperature probe (also referred to as a system temperature sensor) of the automated biomaterials engineering system, such as system temperature probe 253 shown in FIGS. 3C-3G or system temperature probe 753 shown in FIGS. 7A and 7B. The system temperature probe may be disposed outside the first cassette, and the ambient temperature measurement may indicate a temperature outside the first cassette (e.g., outside cassette 240, 340, or 341). As an example, FIG. 7A shows 、 The ambient temperature measurement Temp generated by the system temperature probe 753 at time 1 and / or received by the control circuit at a first point in time, i.e., time 1 周囲,時間17B depicts the signal generated by probe 753. 、 and / or an ambient temperature measurement Temp received by the control circuit at a second time point, i.e., Time 2. 周囲,時間2 It depicts the following.

[0074] 7B, method 600, in one embodiment, may include step 606, in which the control circuit determines a set of temperature offset values ​​indicative of respective differences between the set of internal temperature measurements and the ambient temperature measurements. For example, FIG. 8A illustrates a first set of internal temperature measurements, Temp, corresponding to a first time point. 場所1,時間1 ~Temp 場所11,時間1 and 、 Ambient temperature measurement Temp corresponding to the first time point 周囲,時間1 Show the difference between 、 The figure provides an example of a first set of temperature offset values. The figure also provides a second set of internal temperature measurements, Temp. 場所1,時間2 ~Temp 場所11,時間2 and 、 Ambient temperature measurement Temp corresponding to the second point in time 周囲,時間2 Show the difference between 、 8A and 8B provide an example of a second set of temperature offset values. In some cases, the first time point may correspond to a first stage of a biological material production process (e.g., a first biological protocol), while the second time point may correspond to a second stage of the biological material production process (e.g., a second biological protocol), as shown in FIG. 8B. As shown in FIG. 8A and 8B, the temperature offset values ​​determined in step 606, in one embodiment, may be dynamic offset values ​​covering different biological protocols, or more generally, covering different time points in the first period of time.

[0075] In one embodiment, the temperature offset value may be used to facilitate temperature control. Temperature control may involve, for example, controlling a heating or cooling device, such as device 250 of FIGS. 3C and 3D, to cause a location within a cassette, such as cassette 342 of FIG. 3G or cassette 742 of FIG. 9, to reach a desired temperature, such as a target temperature value. In some cases, the cassette (e.g., 240 or 340 / 341) used to determine the temperature offset value discussed above may be a first cassette, such as a calibration cassette. In this embodiment, the cassette (e.g., 342 / 742) on which temperature control is performed may be a second cassette, such as a production cassette. In such a case, the temperature offset value may have been determined during a first period when the first cassette is disposed within the automated biotechnology system, and temperature control may be performed during a second period when the second cassette is disposed within the automated biotechnology system. During the second period, the first cassette can be removed from, for example, an automated biomaterials engineering system, while the second cassette can be placed into the system. In some implementations, the second cassette can have no temperature probes disposed therein or can have fewer temperature probes than the number of temperature probes disposed in the first cassette.

[0076] In one embodiment, the determination of the temperature offset values ​​and the control of the temperature may be performed by the same control circuit or by two separate control circuits, for example, a first control circuit may determine the temperature offset values ​​and store them in a storage device, while the same control circuit or another control circuit may later retrieve the temperature offset values ​​from the storage device and perform temperature control based on the temperature offset values.

[0077] In one embodiment, performing temperature control may involve determining a relationship between ambient and internal temperature values ​​at locations within the cassette (e.g., 342 / 742) using the temperature offset value determined in step 606. In some cases, an automated biomaterials engineering system may have a system temperature probe, such as probe 753 of FIG. 9 , to determine ambient temperature values, but a cassette (e.g., 342 / 742) may have no temperature probes or only a few temperature probes. Thus, the control circuitry may not have direct measurements regarding the internal temperature within the cassette (e.g., 342 / 742) or may have only a few direct measurements. In such cases, the control circuitry (e.g., 270) may rely on the temperature offset value to infer information regarding the temperature within the cassette (e.g., 342 / 742) based on the ambient temperature values ​​measured by the system temperature probes and / or may determine which ambient temperature to target to provide a desired internal temperature for the locations within the cassette (e.g., 342 / 742).

[0078] In one embodiment, the control circuitry (e.g., control circuit 270) running the temperature control may determine a target internal temperature value for a location within a cassette, such as the second cassette (e.g., 342 / 724) discussed above. The determination may be performed before or after the cassette is placed within the automated biomaterials engineering system. In some cases, the location may be one of locations 1-11 within cassette 742 of FIG. 9. These locations may be the same as the Temp. 0.11 locations within cassette 240 of FIGS. 7A and 7B. 場所1 ~Temp 場所11 may be identical to or otherwise correspond to the respective locations where the temperature values ​​were measured. In some cases, the locations associated with the target internal temperature values ​​may be between two or more of locations 1-11 in FIG. 9. In some cases, if the automated biomaterials engineering system is an automated cell engineering system, the target internal temperature value may be a desired cell culture temperature value for the cell culture in the cassette (e.g., 342 / 742). The desired cell culture temperature value may be, for example, a value that promotes cell growth.

[0079] In one embodiment, the control circuit (e.g., 270) may control the temperature by controlling a heating or cooling device of the automated biomaterials engineering system. This operation may occur during the time period during which the cassette (e.g., 342 / 742) is disposed within the automated biomaterials engineering system. As noted above, this time period may be a second time period, although a set of temperature offset values ​​may have been determined during a first time period during which another cassette (e.g., 240) is disposed within the automated biomaterials engineering system, as discussed above.

[0080] In one embodiment, the control circuitry may control a heating or cooling device based on a target internal temperature value, a set of temperature offset values, and one or more ambient temperature measurements generated by a system temperature probe (e.g., 753). More specifically, the control circuitry may control a heating or cooling device to cause an estimated internal temperature value to reach a target internal temperature value and / or cause an ambient temperature value to reach a target ambient temperature value. The estimated internal temperature value and / or the target ambient temperature value may be determined based on a set of temperature offset values, as discussed in more detail below.

[0081] In one embodiment, the control circuitry may perform temperature control by estimating an internal temperature value for a location within the cassette (e.g., 342 / 742), as described above, such that the control circuitry can control a heating or cooling device based on the difference between the estimated internal temperature value and the target internal temperature value, or more specifically, to reduce the difference so that the estimated internal temperature value approaches the target internal temperature value. In some cases, the estimated internal temperature value for a location within the cassette (e.g., 342 / 742) may be estimated based on an ambient temperature value measured by a system temperature probe (e.g., 753) of FIG. 9 and based on a temperature offset value corresponding to that location within the cassette (e.g., 342 / 742). A temperature offset value for a location within a cassette (e.g., 342 / 742), such as the second cassette discussed above, may be equal to or based on one of the set of temperature offset values ​​discussed above with respect to step 606. The set of temperature offset values ​​may have been determined using another cassette, such as the first cassette (e.g., 240 or 340 / 341) discussed above. More specifically, a temperature offset value for a location in the second cassette can be equal to or based on a temperature offset value for a corresponding location in the first cassette. In some implementations, the first cassette and the second cassette can have the same or similar shape or layout, and a location in the first cassette can correspond to a location in the second cassette if the two locations are spatially located in the same or similar locations relative to their respective cassettes. That is, two corresponding locations can be the same or similar locations relative to their respective cassettes.

[0082] As an example, location 1 (Temp. 1) in cassette 240 of FIGS. 7A and 7B 場所1,時間1 and Temp 場所1,時間2 ) may correspond to location 1 in cassette 742 in FIG. 9. In such an example, the temperature offset value for location 1 in cassette 742 may be the Offset 場所1,時間1 or Offset 場所1,時間29. The temperature offset value may be equal to or based on a temperature offset value for a corresponding location in cassette 240, such as Temp. 周囲 Based on the ambient temperature value, such as Offset 場所1,時間1 or Offset 場所1,時間2 For example, the control circuit may determine an estimated internal temperature value for location 1 within cassette 742 based on an Offset 場所1,時間1 or Offset 場所1,時間2 An estimated internal temperature value for location 1 within cassette 742 may be determined by subtracting

[0083] As mentioned above, the temperature offset values ​​determined in step 606 may, in one embodiment, be dynamic offset values ​​that account for various points in time (e.g., various biological protocols) within a period of time, such as the first period of time discussed above, during which the first cassette (e.g., 240) is disposed within the automated biomaterials engineering system. For example, as depicted in FIGS. 8A and 8B, the dynamic temperature offset values ​​determined during the first period of time may include multiple sets of temperature offset values ​​corresponding to different points in time during the first period of time, e.g., Time 1 (e.g., Time 2), Time 3 (e.g., Time 4), Time 5 (e.g., Time 6), Time 7 (e.g., Time 8), Time 9 (e.g., Time 10), Time 11 (e.g., Time 12), Time 13 (e.g., Time 14), Time 15 (e.g., Time 16), Time 17 (e.g., Time 18), Time 19 (e.g., Time 20), Time 21 (e.g., Time 22), Time 23 (e.g., Time 24), Time 25 (e.g., Time 26), Time 28 (e.g., Time 29), Time 30 (e.g., Time 31), Time 32 (e.g., Time 33), Time 34 (e.g., Time 35), Time 36 (e.g., Time 37), Time 38 (e.g., Time 39), Time 40 (e.g., Time 41), Time 42 (e.g., Time 43), Time 44 (e.g., Time 45), Time 46 (e.g., Time 46), Time 47 (e.g., Time 48), Time 49 (e.g., Time 49), Time 50 (e 、 The temperature offset value corresponding to the first biological protocol 場所1,時間1 ~Offset 場所11,時間1 The first set of, and time 2 (e.g. 、 The temperature offset value corresponding to the second biological protocol (Offset 場所1,時間2 ~Offset 場所11,時間2 In this embodiment, determining an estimated internal temperature value for a location within the second cassette (e.g., 342 / 742) at a time during the second period (the second cassette is installed in the automated biomaterials engineering system) may involve determining a temperature offset value corresponding to that time and that location. The time may be when the internal temperature value is being estimated or when the estimated value is to be used.

[0084] In some examples, the corresponding temperature offset value may be a value estimated at a corresponding time during the first time period discussed above (when multiple sets of temperature offset values ​​are determined). In these examples, a time point within the first time period may correspond to a time point within the second time period, for example, if the two time points belong to the same stage of the biological material production process (e.g., belong to the same biological protocol) and / or have the same temporal offset relative to the start or end of the respective time periods. As an example, if the control circuitry determines an estimated internal temperature value for Location 1 during the first biological protocol within the second time period, the control circuitry may determine that the corresponding temperature offset value corresponds to Location 1. 、 and the first biological protocol corresponds to Offset 場所1,時間1 If the control circuitry determines an estimated internal temperature value for the same location during a second biological protocol in a second time period, the control circuitry may determine that the corresponding temperature offset value is Offset 場所1,時間2 It can be determined that:

[0085] In some cases, the control circuitry may perform temperature control for the cassette (e.g., 342 / 742) discussed above by estimating a temperature map that indicates how the temperature varies spatially throughout the cassette. The temperature map may be generated based on temperature offset values, such as the first and / or second set of temperature offset values ​​discussed above, and one or more temperature ambient measurements generated during a second time period. In some implementations, the control circuitry may generate the temperature map by estimating internal temperature values ​​at various locations within the cassette, as discussed above. For example, the control circuitry may subtract from the ambient temperature measurements (or more specifically, from the ambient temperature values) temperature offset values ​​that correspond to those locations and to the time the ambient temperature measurements were made.

[0086] In some implementations, the control circuitry may determine a temperature map for the cassette (e.g., 342 / 742) within a second time period based on the temperature map generated within the first time period. In such implementations, the temperature map may indicate estimated internal temperature values ​​at various locations within the cassette (e.g., 342 / 742) at a time point, and the control circuitry may control a heating or cooling device based on the temperature map. The control circuitry may be configured to generate a single temperature map, or may generate multiple temperature maps corresponding to multiple time points within the second time period (e.g., corresponding to multiple biological protocols). When the control circuitry is determining a temperature map for a first time point within the second time period, the control circuitry may generate a first set of temperature offset values, Offset 場所1,時間1 ~Offset 場所11,時間1 Select 、 When the control circuit is determining the temperature map for a second time point within the second time period, the control circuit may determine a second set of temperature offset values. 場所1,時間2 ~Offset 場所11,時間2 Select 、 A temperature map may be determined.

[0087] As described above, the control circuitry may perform heating control by causing the estimated internal temperature value to approach the target internal temperature value and / or by causing the measured ambient temperature value to approach the target ambient temperature value. In one embodiment, the control circuitry may determine the target ambient temperature value based on the target internal temperature value and / or the estimated internal temperature value. For example, the control circuitry may determine the target ambient temperature value by adding the target internal temperature value for a particular location within the cassette to a temperature offset value corresponding to that location. As an example, the control circuitry may determine the target internal temperature value for location 7 of FIG. 9 as the Offset 場所7,時間1 or Offset 場所7,時間2 The target ambient temperature value may be determined by adding

[0088] In one embodiment, the control circuit may determine multiple target ambient temperature values ​​for multiple points within the second time period. For example, to cause location 7 within the cassette (e.g., 742) to reach a target internal temperature value at various times, the control circuit may determine an Offset corresponding to the first time point. 場所7,時間1 By using 、 The control circuit may determine a first target ambient temperature value for a first time point within the second time period. The control circuit may determine an Offset corresponding to the second time point. 場所7,時間2 By using 、 A second target ambient temperature value for a second point within the second time period may be determined. The two time points may be part of two different biological protocols within the second time period or may be part of the same biological protocol. The control circuit may control (e.g., activate or deactivate) a heating or cooling device to transition a measured ambient temperature, which may be measured, for example, by a system temperature probe (e.g., 753), from a first target ambient temperature value to a second target ambient temperature value. For example, FIG. 10A depicts an example of a target ambient temperature and / or measured ambient temperature, which may be referred to as a control temperature because it is used to control a heating or cooling device. The figure further depicts an internal temperature value, which may be referred to as a medium temperature because the cassette in this example may contain cell culture medium. In this example, the control circuit may control the heating device to transition the control temperature from a first target ambient temperature value to a second ambient temperature value, or more specifically, from a higher target ambient temperature value to a lower ambient temperature value. Using at least two different target ambient temperature values ​​may accelerate heating or cooling of the medium in the cassette. For example, the use of a higher initial ambient temperature value in Figure 10A may cause the internal temperature value (e.g., medium temperature) to rise more quickly compared to implementations in which the control circuitry simply attempts to maintain a single target ambient or control temperature over a particular period of time. An example of the latter, in which a single control temperature is used, is depicted in Figure 10B. Thus, the example of Figure 10A may use the heating device in a more aggressive and / or dynamic manner to better compensate for the thermal lag between the ambient temperature and the internal temperature (e.g., medium temperature) and to bring the internal temperature to the target value more quickly.

[0089] In one embodiment, the temperature offset value may facilitate a more aggressive and / or dynamic manner of controlling a heating or cooling device. More specifically, while increasing the ambient temperature may help compensate for thermal lag between the ambient temperature and the internal temperature, the control circuit may need to ensure that the internal temperature does not become too high or too low, which may damage the cell culture medium in the cassette (e.g., 342 / 742) discussed above, for example. The control circuit may use the temperature offset value to more accurately determine an ambient temperature value at which the internal temperature value is less likely to be too high or too low, and / or to more accurately estimate the internal temperature value to confirm that the internal temperature value is too high or too low, even if the control circuit cannot directly measure the internal temperature value. In one embodiment, if the temperature offset value is dynamic by corresponding to multiple points in time, the control circuit may use the dynamic temperature offset value to adjust how the ambient temperature is being controlled. For example, the dynamic temperature offset value may assist the control circuit in determining when to transition from a higher initial ambient temperature value (e.g., a target ambient temperature value) to the lower ambient temperature of FIG. 10A so as to quickly increase the internal temperature but avoid the internal temperature exceeding its target value.

[0090] In one embodiment, the automated materials engineering system has multiple system temperature probes measuring multiple ambient temperature values ​​at multiple respective locations outside the cassette (e.g., 342 / 742), and the control circuitry can be configured to determine multiple target ambient temperature values ​​corresponding to the multiple system temperature probes and / or corresponding to the multiple locations.

[0091] Additional Exemplary Embodiments Embodiment 1 relates to a calibration cassette for use in an automated biomaterials engineering system, comprising: a low-temperature chamber including a medium reservoir and a first array of sealed temperature probes within the medium reservoir; a high-temperature chamber separated from the low-temperature chamber by a thermal barrier, the high-temperature chamber including a cell culture chamber and a second array of sealed temperature probes within the cell culture chamber; one or more fluidics pathways connected to the cell culture chamber and the medium reservoir, the one or more fluidics pathways including a third array of sealed temperature probes within the one or more fluidics pathways; and an electrical connection element electrically connected to each of the first, second, and third arrays of sealed temperature probes. Embodiment 2 includes the calibration cassette of embodiment 1, wherein the medium storage container is a bag and the first array of sealed temperature probes is attached to the interior surface of the bag. Embodiment 3 includes the calibration cassette of embodiment 1 or 2, wherein the cell culture chamber is flat and substantially non-flexible, and a second array of sealed temperature probes is attached to the bottom and / or sides of the cell culture chamber. Embodiment 4 includes a calibration cassette of any of embodiments 1-3, wherein one or more fluidics paths include tubing and connectors, and a third array of sealed temperature probes is located within the tubing. Embodiment 5 includes the calibration cassette of any of embodiments 1-4, wherein the first, second, and third arrays of sealed temperature probes include resistance temperature detectors (RTDs) hermetically sealed by a polymer cover. Embodiment 6 includes the calibration cassette of any of embodiments 1-5, wherein the first, second, and third arrays of sealed temperature probes each include at least two sealed temperature probes. Embodiment 7 includes the calibration cassette of embodiment 6, wherein the first, second, and third arrays of sealed temperature probes each include two to four sealed temperature probes. Embodiment 8 includes the calibration cassette of embodiment 7, wherein the first, second, and third arrays of sealed temperature probes include a total of 12 sealed temperature probes. A ninth embodiment includes the calibration cassette of any of the first to eighth embodiments, wherein the electrical connection element is electrically connected to a control circuit associated with the calibration cassette. Embodiment 10 includes a calibration cassette according to any of embodiments 1 to 8, wherein the electrical connection element is configured to be electrically connected to a control circuit associated with an automated biomaterials engineering system.

[0033] Embodiment 11 relates to a production cassette for use in an automated cell engineering system, comprising: a low-temperature chamber comprising a cell culture medium reservoir and a first array of sealed temperature probes within the cell culture medium reservoir; a high-temperature chamber for performing cell culture activation, transduction, and / or growth, the high-temperature chamber being separated from the low-temperature chamber by a thermal barrier and comprising a cell culture chamber and a second array of sealed temperature probes within the cell culture chamber; one or more fluidics pathways connected to the cell culture chamber and the cell culture medium reservoir, the one or more fluidics pathways comprising a third array of sealed temperature probes within the one or more fluidics pathways; and an electrical connection element electrically connected to each of the first, second, and third arrays of sealed temperature probes, wherein the one or more fluidics pathways provide recirculation, waste removal, and homogenous gas exchange, as well as nutrient distribution to the cell culture chamber. Embodiment 12 includes the production cassette of embodiment 11, wherein the cell culture medium storage container is a bag and the first array of sealed temperature probes is attached to the interior surface of the bag. Embodiment 13 includes the production cassette of embodiment 11 or 12, wherein the cell culture chamber is flat and substantially non-flexible, and wherein a second array of sealed temperature probes within the cell culture chamber is attached to the bottom and / or sides of the cell culture chamber. Embodiment 14 includes a production cassette described in any of embodiments 11 to 13, wherein one or more fluidics paths include tubing and connectors, and a third array of sealed temperature probes is located within the tubing. Embodiment 15 includes a production cassette described in any of embodiments 11 to 14, wherein the first, second, and third arrays of sealed temperature probes include resistance temperature detectors (RTDs) hermetically sealed by a polymer cover. Embodiment 16 includes the production cassette of any of embodiments 11-15, wherein the first, second, and third arrays of sealed temperature probes each include at least two sealed temperature probes. Embodiment 17 includes the production cassette of embodiment 16, wherein the first, second, and third arrays of sealed temperature probes each include two to four sealed temperature probes. Embodiment 18 includes the production cassette of embodiment 17, wherein the first, second, and third arrays of sealed temperature probes include a total of 12 sealed temperature probes. Embodiment 19 includes a production cassette according to any of embodiments 11-18, wherein the electrical connection element is electrically connected to control circuitry associated with the production cassette. Embodiment 20 includes a production cassette according to any of embodiments 11-18, wherein the electrical connection element is configured to be connected to control circuitry associated with an automated cell engineering system. Embodiment 21 includes a method for monitoring temperature in an automated biomaterials engineering system. The method of this embodiment includes receiving, by a control circuit, a set of internal temperature measurements while a first cassette is housed within the automated biomaterials engineering system, the set of internal temperature measurements indicative of a temperature within the first cassette and generated during the period by an array of temperature probes disposed within the first cassette; receiving, by the control circuit, ambient temperature measurements while the first cassette is housed within the automated biomaterials engineering system, the ambient temperature measurements indicative of a temperature outside the first cassette and generated during the period by a system temperature probe of the automated biomaterials engineering system disposed outside the first cassette; and determining, by the control circuit, a set of temperature offset values ​​indicative of respective differences between the set of internal temperature measurements and the ambient temperature measurements. Example 22 includes the method of example 21, in which the control circuitry is associated with the first cassette. Example 23 includes the method of example 21, in which the control circuitry is associated with an automated biomaterials engineering system. Embodiment 24 includes the method of any one of Embodiments 21 to 23, wherein the automated biomaterials engineering system is an automated cell engineering system. Embodiment 25 includes a method according to any one of embodiments 21 to 24, wherein an array of temperature probes is disposed at a plurality of respective locations within the first cassette, the set of internal temperature measurements corresponds to a plurality of respective locations within the first cassette, and the method further includes generating a temperature map showing how temperature varies spatially throughout the first cassette based on the set of internal temperature measurements. Embodiment 26 includes the method of embodiment 25, wherein the set of internal temperature measurements is a first set of internal temperature measurements corresponding to a first time point within the period of time, and the temperature map is a first temperature map showing how temperature varies spatially throughout the first cassette at the first time point within the period of time, and the method further includes receiving a second set of internal temperature measurements generated by an array of temperature probes in the first cassette, the second set of internal temperature measurements indicating temperatures at multiple respective locations at the second time point within the period of time, and generating a second temperature map showing how temperature varies spatially throughout the first cassette at the second time point within the period of time based on the second set of internal temperature measurements. Embodiment 27 includes the method of embodiment 26, wherein the ambient temperature measurements are first ambient temperature measurements corresponding to a first time point within the period of time, and the set of temperature offset values ​​are a first set of temperature offset values ​​that also correspond to the first time point, and the method further includes receiving a second ambient temperature measurement indicative of a temperature outside the first cassette at a second time point within the period of time, and determining a second set of temperature offset values ​​indicative of respective differences between the second ambient temperature measurement and the second set of internal temperature measurements, the second set of temperature offset values ​​corresponding to the second time point, wherein the first time point belongs to a first stage of a biological material production process, and the second time point belongs to a second stage of the biological material production process. Example 28 includes the method of any one of Examples 21 to 27, further including wirelessly transmitting the set of internal temperature measurements to a computing device. Embodiment 29 includes and relates to a method of temperature control performed in an automated biomaterials engineering system, the method including receiving, by a control circuit, a set of internal temperature measurements during a first period when a first cassette is housed within the automated biomaterials engineering system, the set of internal temperature measurements being indicative of a temperature within the first cassette and being generated during the first period by an array of temperature probes disposed within the first cassette; and receiving, by the control circuit, a first ambient temperature measurement when the first cassette is housed within the automated biomaterials engineering system, the first ambient temperature measurement being indicative of a temperature outside the first cassette and being generated during the first period by an array of temperature probes disposed outside the first cassette. The method includes receiving, during a first period of time, temperature measurements generated by the temperature sensor; determining, by a control circuit, a set of temperature offset values ​​indicative of respective differences between the set of internal temperature measurements and a first ambient temperature measurement; determining, by the control circuit, a target internal temperature value for a location within the second cassette; and controlling, by the control circuit, a heating or cooling device of the automated biomaterials engineering system during a second period of time during which the second cassette is housed within the automated biomaterials engineering system based on the target internal temperature value, the set of temperature offset values, and one or more additional ambient temperature measurements generated by the system temperature sensor during the second period of time, wherein the system temperature sensor is disposed outside the second cassette. Embodiment 30 includes the method of embodiment 29, wherein the automated biomaterials engineering system is an automated cell engineering system, and the target internal temperature value is a desired cell culture temperature value for the cell culture in the second cassette. Embodiment 31 includes a method according to embodiment 29 or 30, further comprising generating a temperature map showing how the temperature varies spatially throughout the second cassette based on a set of temperature offset values ​​and one or more additional ambient temperature measurements generated during the second period, and wherein the heating or cooling device is controlled based on the temperature map. Embodiment 32 includes the method of embodiment 31, in which controlling the heating or cooling device includes determining an estimated internal temperature value for a location within the second cassette, and the heating or cooling device is controlled based on the difference between the estimated internal temperature value and the desired internal temperature value. Embodiment 33 includes a method according to embodiment 32, in which controlling the heating or cooling device includes determining a target ambient temperature value based on an estimated internal temperature value and / or a target internal temperature value, and the heating or cooling device is controlled to bring the temperature measured by the system temperature probe closer to the target ambient temperature value. Embodiment 34 includes a method according to embodiment 33, in which the set of temperature offset values ​​is one of a plurality of sets of temperature offset values ​​determined during a first period of time, the plurality of sets corresponding to different points in time within the first period of time, and is generated by selecting a set of temperature offset values ​​from among the plurality of sets of temperature offset values ​​based on a determination that the determined temperature map corresponds to a point in time within a second period of time and that the set of temperature offset values ​​also corresponds to a point in time within the second period of time. Embodiment 35 includes the method of embodiment 34, wherein the set of temperature offset values ​​is associated with a time point within a first time period, and both the time point within the first time period and the time point within the second time period belong to the same stage of a biological material production process having multiple stages. Embodiment 36 includes the method of embodiment 34 or 35, wherein the temperature map is a first temperature map, and the method further includes selecting a second set of temperature offset values ​​from among a plurality of sets of temperature offset values ​​at a second time point within a second period of time, wherein the selected second set of temperature offset values ​​corresponds to the second time point within the second period of time, and generating a second temperature map based on the second set of temperature offset values, wherein the second temperature map is associated with the second time point within the second period of time, and wherein the heating device or the cooling device is controlled based on the second temperature map. Embodiment 37 includes the method of embodiment 36, in which the target ambient temperature value is a first target ambient temperature value, and the method further includes determining a second estimated internal temperature value, determining a second target ambient temperature value based on the second estimated internal temperature value and / or the target internal temperature value, and controlling a heating device or a cooling device to transition the temperature measured by the system temperature probe from the first target ambient temperature value to the second target ambient temperature value. Example 38 includes the method of example 37, in which the first target ambient temperature value is higher than the second target ambient temperature value. Example 39 includes the method of any of Examples 21-38, wherein the second cassette does not have a temperature sensor disposed therein.

[0092] It will be readily apparent to those skilled in the relevant art that other suitable modifications and adaptations to the methods and applications described herein can be made without departing from the scope of any of the embodiments.

[0093] While certain embodiments have been illustrated and described herein, it is to be understood that the claims should not be limited to the specific forms or arrangements of parts described and shown. Although exemplary embodiments have been disclosed herein, and specific terms are employed, they are used in a generic and descriptive sense only, and not for purposes of limitation. Modifications and variations of the embodiments are possible in light of the above teachings. It is therefore to be understood that the embodiments may be practiced otherwise than as specifically described.

[0094] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. 1. A calibration cassette for use in an automated biomaterials engineering system, comprising: a cryogenic chamber including a media reservoir and a first array of sealed temperature probes within the media reservoir; a high temperature chamber separated from the low temperature chamber by a thermal barrier, the high temperature chamber including a cell culture chamber and a second array of sealed temperature probes within the cell culture chamber; one or more fluidics pathways connected to the cell culture chamber and the media reservoir, the one or more fluidics pathways including a third array of temperature probes sealed within the one or more fluidics pathways; an electrical connection element electrically connected to each of the first, second, and third arrays of sealed temperature probes.

2. 2. The calibration cassette of claim 1, wherein the medium storage container is a bag and the first array of sealed temperature probes is attached to an interior surface of the bag.

3. 3. The calibration cassette of claim 1, wherein the cell culture chamber is flat and substantially non-flexible, and the second array of sealed temperature probes is attached to the bottom and / or sides of the cell culture chamber.

4. 4. A calibration cassette as described in any one of claims 1 to 3, wherein the one or more fluidics paths include tubing and connectors, and the third array of sealed temperature probes is located within the tubing.

5. 5. A calibration cassette according to any preceding claim, wherein the first, second and third arrays of sealed temperature probes comprise resistance temperature detectors (RTDs) hermetically sealed by polymer covers.

6. A calibration cassette according to any preceding claim, wherein the first, second and third arrays of sealed temperature probes each comprise at least two sealed temperature probes.

7. 7. The calibration cassette of claim 6, wherein the first, second, and third arrays of sealed temperature probes each include two to four sealed temperature probes.

8. 8. The calibration cassette of claim 7, wherein the first, second, and third arrays of sealed temperature probes include a total of 12 sealed temperature probes.

9. A calibration cassette according to any preceding claim, wherein the electrical connection element is electrically connected to a control circuit associated with the calibration cassette.

10. 9. The calibration cassette of claim 1, wherein the electrical connection element is configured to be electrically connected to a control circuit associated with the automated biomaterials engineering system.

11. 1. A production cassette for use in an automated cell engineering system, comprising: a cryogenic chamber including a cell culture medium reservoir and a first array of sealed temperature probes within the cell culture medium reservoir; a high temperature chamber for performing activation, transduction, and / or expansion of cell cultures, said high temperature chamber being separated from said low temperature chamber by a thermal barrier, said high temperature chamber comprising a cell culture chamber and a second array of sealed temperature probes within said cell culture chamber; one or more fluidics pathways connected to the cell culture chamber and the cell culture medium reservoir, the one or more fluidics pathways including a third array of temperature probes sealed within the one or more fluidics pathways; an electrical connection element electrically connected to each of the first, second, and third arrays of sealed temperature probes; A production cassette, wherein the one or more fluidics pathways provide recirculation, waste removal, and homogenous gas exchange, as well as nutrient distribution to the cell culture chambers.

12. 12. The production cassette of claim 11, wherein the cell culture medium storage container is a bag and the first array of sealed temperature probes is attached to an interior surface of the bag.

13. 13. The production cassette of claim 11 or 12, wherein the cell culture chamber is flat and substantially non-flexible, and the second array of sealed temperature probes within the cell culture chamber is attached to the bottom and / or sides of the cell culture chamber.

14. 14. A production cassette according to any one of claims 11 to 13, wherein the one or more fluidics paths comprise tubing and connectors, and the third array of sealed temperature probes is located within the tubing.

15. 15. The production cassette of any one of claims 11 to 14, wherein the first, second, and third arrays of sealed temperature probes comprise resistance temperature detectors (RTDs) hermetically sealed by a polymer cover.

16. 16. The production cassette of any one of claims 11 to 15, wherein the first, second, and third arrays of sealed temperature probes each comprise at least two sealed temperature probes.

17. 17. The production cassette of claim 16, wherein the first, second, and third arrays of sealed temperature probes each comprise from two to four sealed temperature probes.

18. 20. The production cassette of claim 17, wherein the first, second, and third arrays of sealed temperature probes comprise a total of 12 sealed temperature probes.

19. The production cassette of any one of claims 11 to 18, wherein the electrical connection element is electrically connected to control circuitry associated with the production cassette.

20. 19. The production cassette of any one of claims 11 to 18, wherein the electrical connection element is configured to be connected to control circuitry associated with the automated cell engineering system.

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