Adeno-associated virus and cell maintenance and transfection

Abstract

A transfection robot that combines bio informatics, robotics and artificial intelligence (AI) in a single package. The transfection robot replaces a large component of the mundane work and increases the quality thereof.

Description

ADENO-ASSOCIATED VIRUS AND CELL MAINTENANCE AND TRANSFECTIONCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 562,944, filed March 8, 2024, and U.S. Provisional Patent Application No. 63 / 567,666, filed March 20, 2024, and U.S. Provisional Patent Application No. 63 / 665,843, filed June 28, 2024, the entire disclosures of which are incorporated herein by reference.FIELD

[0002] The present disclosure relates to automating and driving quality and efficiencies in cell and gene research and manufacturing.BACKGROUND

[0003] Cell and gene therapies have shown great promise, and personalized medicine can drastically and permanently solve some of the most debilitating and life-threatening diseases. The high cost of treatments limits the use and development of these therapies to only the wealthiest countries and individuals. In most countries, medical insurance is unlikely to cover costs associated with cell and gene therapies. The elevated cost to develop these treatments compared to traditional treatments is in large part due to challenges with logistics and manufacturing processes. A large part of cell / gene research and manufacturing is spent on time-intensive cell maintenance. This is often done by highly qualified scientists, and thus demands significant resources.

[0004] The nature of the biology of cell / gene research also makes the repeatability and consistency of batches difficult, as the input product (donor cells) exhibits highly varied cell behavior regardless of the source. Most pharmaceutical manufacturing caters to very large production batches intended to cover large portions of the general population. Thus, personalized medicine — which entails smaller patient groups — is significantly disadvantaged.

[0005] Unlike chemical compounds, cells exhibit varied characteristics from batch to batch, and therefore require careful attention. This makes it difficult to produce a precise, replicable procedure for optimally manufacturing cell batches, in part resulting in the low yields experienced in the industry.SUMMARY

[0006] In one embodiment, a closed-system automated incubator for cell and gene therapy (CGT) vector production is disclosed. The automated incubator includes an incubator module containing a plurality of multi-layer roller bottles. Each of the multi-layer roller bottles define a bottle interior configured for incubating a cell cluster, and are assigned an independent micro-environment configured to allow simultaneous production of distinct cell and vector serotypes within a single incubator module. The independent micro-environment includes one or more of programmable temperature, gas composition, or fluidic exchange parameters. The automated incubator also includes a plurality of port caps corresponding to the plurality of multi-layer roller bottles, each of the port caps rotatably coupled to a corresponding one of the multi-layer roller bottles, a vision system configured to generate image data representative of the cell cluster within each of the multi-layer roller bottles and a CGT processor receiving and responsive to the image data generated by the vision system. Also included is a memory storing one or more processor-executable instructions that, when executed, configure the CGT processor for executing an artificial intelligence (Al) engine to autonomously optimize growth conditions per bottle by adjusting media replenishment, gas exchange, and transfection agent delivery in real time, eliminating the need for external fluorescence-based assessment.

[0007] In another embodiment, a multi-layer concentric roller bottle for incubating a cell cluster is disclosed. The roller bottle comprises a port cap having a disk and a cap body, the disk defining at least one fluid exchange port, an outer bottle layer coupled to the cap body and having an inner face configured to be coated with a cell solution, and an inner bottle layer nested within the outer bottle layer. The inner bottle layer is independently rotatable with respect to the outer bottle layer and has an inner face configured to be coated with the cell solution.

[0008] In yet another embodiment, a method of growing cells is disclosed. The method includes injecting a cell culture into a multi-layer roller bottle and rotating the multi-layer bottle to evenly coat the nested layers with cell culture. The multi-layer roller bottle comprises an inner bottle layer nested within the outer bottle layer, the inner bottle layer being independently rotatable with respect to the outer bottle layer and having an inner face configured to be coated with the cell culture. The rotating and injecting are performed simultaneously.

[0009] Other objects and features of the present invention will be in part apparent and in part pointed out herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG. 1 is a block diagram of an automated incubator system according to an embodiment.

[0011] FIG. 2 is a perspective view of the incubator system of FIG. 1.

[0012] FIG. 3 is a front elevation of the incubator system of FIG. 1.

[0013] FIG. 4 is a rear elevation of the incubator system of FIG. 1.

[0014] FIG. 5 is a rear perspective of the incubator system of FIG. 1.

[0015] FIG. 6 is a side view of a vision system and roller bottle according to an embodiment.

[0016] FIG. 7 shows the aggregation of image slices into an aggregate image according to an embodiment.

[0017] FIG. 8A is a perspective of a roller bottle according to an embodiment.

[0018] FIG. 8B is an exploded view of the roller bottle of FIG. 8A.

[0019] FIG. 9A is an exploded view a port cap according to an embodiment.

[0020] FIG. 9B is a perspective view of FIG. 9A.

[0021] FIG. 10 is a flow chart showing a method according to an embodiment.

[0022] FIG. 11 A is an example image of a low-confluency cell cluster grown in accordance with the method of FIG. 11.

[0023] FIG. 1 IB is an example image of a moderate-confluency cell cluster grown in accordance with the method of FIG. 10.

[0024] FIG. 11C is an example image of a high-confluency cell cluster grown in accordance with the method of FIG. 11.

[0025] Corresponding reference characters indicate corresponding parts throughout the drawings.DETAILED DESCRIPTION

[0026] Term Definition: Cell Culture, Cell Cluster, and Culture Media. In the context of the present disclosure, the term "cell culture" is used to refer a liquid solution containing cells to be grown (e.g., cultured), as well as liquids for promoting cell growth. In the context of the present disclosure, the term "cell cluster" is used to refer specifically to some or all of the cells within a cell culture. In the context of the present disclosure, the term "culture media is used to refer to liquid nutrients for a cell cluster. Thus, a cell culture is a mixture of culture media and a cell cluster or clusters.

[0027] Referring now to FIGS. 1-5, an exemplary embodiment of a closed- system automated incubator for cell and gene therapy (CGT) vector production (broadly, automated incubator) in accordance with the present disclosure is illustrated, and is generally indicated at reference number 100. The incubator broadly comprises an incubator module 102, a plurality of multi-layer roller bottles 104 (broadly, roller bottles) for growing cell clusters, a vision system 106, a CGT processor 108, memory 110 storing an artificial intelligence (Al) engine 112, fluid reservoirs 114, and fluid tubing 116. The roller bottles 104 are fluidly connected to the fluid reservoirs 114 via the fluid tubing 116, and receive cell culture therefrom. In use, the cell culture is deposited in the roller bottles 104, which provide optimal conditions for cell growth. The roller bottles 104 are configured to rotate about a longitudinal bottle axis LB, coating the interior with cell culture. As the cell clusters within the cell culture develop (e.g., grow, reproduce, etc.), the vision system 106 images the interior of the roller bottles 104, thereby collecting information (e.g., data) regarding the development of the cells. The artificial intelligence engine 112 processes (e.g., interprets) this information to assess a cell cluster's health and predict the probability that it will yield a successful cell batch. Using these predictions, the artificial intelligence engine 112 promotes the growth of higher-potential batches by allocating resources thereto (e.g., cell culture or culture media), and prevents the allocation of resources to low-potential batches. As will be explained in further detail below, the incubator 100 of the present invention increases the production of viable batches with respect to both absolute value and percentage success, decreases labor costs through automation, and improves industry capacity for personalized CGT research.

[0028] The incubator module 102 includes a roller bottle sub-module 118 in which a plurality of roller bottles 104 is maintained. The roller bottles 104 of the illustrated embodiment are arranged in array to improve function through organization and optimization of space, contributing to the small form factor of the incubator module 102. The illustrated embodiment includes three rows of three roller bottles 104; however, the array may be configured in alternate permutations in alternative embodiments (e.g., five rows of eight roller bottles 104, one row of five roller bottles, etc.). The roller bottle sub-module 118 includes a housing 120 comprising a top wall and four side walls. The front-facing and rear-facing side walls 120A, 120B are constructed from a transparent material (e.g., glass, plastic) to allow an operator (e.g., a lab technician) to inspect the interior of the roller bottle sub-module 118 without opening (e.g., exposing) the roller bottle sub-module to ambient, thereby increasing the risk of contamination. This facilitates simplified monitoring of devices processes.

[0029] In an embodiment, incubator module 102 is configured to support modular expansion, allowing additional arrays of roller bottle 104 to be integrated for increased throughput without compromising sterility or automation efficiency.

[0030] The roller bottle sub-module 118 is configured to maintain optimal conditions for cell growth (e.g., reproduction) within the roller bottles 104. Conditions which affect cellular growth, such as temperature, humidity, pressure, gas composition, and light exposure, are controlled by various devices located within and / or connected to the roller bottle sub-module 118 (e.g., thermostatic devices, humidity controllers, lights, etc.). These devices are communicatively connected to the CGT processor 108 and are controlled thereby (e.g., via the artificial intelligence engine 112).

[0031] In the illustrated embodiment, the roller bottles 104 are held by a bottle rack 122. The bottle rack 122 includes a plurality of cross members 124 which extend transversely across the rows of roller bottles 104, as well as a plurality of rollers 126 which extend longitudinally along the length of the roller bottles 104. In the illustrated embodiment, each roller bottle 104 is maintained on two rollers 126. The rollers 126 are configured to rotate about an axis parallel to the length of the roller bottle 104 which they support (e.g., the longitudinal bottle axis LB).

[0032] In certain embodiments, the rollers 126 are mechanically connected to one or more electric motors which drive their rotation. For example, the rollers of a row may be connected to an electric motor at the end of the row via a belt or chain; upon activation of the motor, each of the rollers 126 rotate in unison, in turn rotating the roller bottles 104 which they support. In another embodiment, each of the rollers 126 are driven by their own motor, allowing for varied (e.g., independent) rotation. In yet another embodiment, the rollers 126 are not connected to a power source at all, and are merely supports for the roller bottles 104. In this embodiment, the roller bottles 104 themselves are driven by a motor.

[0033] In addition to longitudinal rotation, the incubator module 102 is configured to tilt (e.g., rotate) the roller bottles 104 about an axis perpendicular to then- respective longitudinal bottle axes LB. In the illustrated embodiment, this is performed by tilting the rollers 126 by raising and / or lowering the cross members 124. For example, the front cross members 124 (e.g., the cross members closest to the front side-wall 120A) may be lowered relative to the rear cross members (e.g., the cross members farthest from the front side wall 120A).

[0034] The incubator module 102 may also include partitions configured to define thermally isolated incubation stations within the roller bottle sub-module 118 (notshown). For example, with reference to the illustrated embodiments, the uppermost (e.g., third) bottle row may be thermally isolated from the bottom and center (e.g., first and second) bottle rows via a partition. Transportation of a roller bottle 104 from one incubation station to an alternate incubation station (e.g., from a heating incubation station to a cooling incubation station) may be performed manually by the operator, or automatically by a robotic arm (not shown).

[0035] Referring to FIG. 6, the incubator module 102 includes a vision system 106 configured to image cellular growth within the roller bottles 104. These are transmitted to the CGT processor 108, to which the vision system 106 is communicatively connected. In the illustrated embodiment, the vision system 106 includes a slide-rack 128 and an imaging device 130 (e.g., a camera, a bright-field microscope, a fluorescent microscope). Like the rollers 126, the slide rack 128 extends along the longitudinal bottle axis LB, perpendicular to the cross members 124.

[0036] The imaging device 130 is slidably connected to the slide rack 128, such that it may move longitudinally therealong while imaging the roller bottles 104. In the illustrated embodiment, a slide rack 128 and imaging device 130 are present for each of the roller bottles 104, however in alternative embodiments a single slide rack and imaging device may be used for each row of roller bottles. In this embodiment, the slide rack 128 and imaging device 130 are further configured to move transversely along the rows, and thus have 2 degrees of freedom (e.g., using a gantry- like system).

[0037] The vision system 106 collects many image slices 132 to be formed into a single aggregate image 134. This is generally depicted at FIG. 7. The imaging device 130 records a first image slice 132 by imaging the interior of the roller bottle 104 (e.g., a cell cluster) as it moves along the slide rack 128. This may be performed smoothly, wherein the imaging device 130 records continuously while in motion along the slide rack 128, or while the roller bottle 104 rotates, but may also be performed stepwise. When performed stepwise, the imaging device 130 takes a first image, moves, takes a second image, moves, etc. In both of these configurations, an image slice 132 is produced for a narrow portion of bottle interior (e.g.., a portion of the lining on an inner face of the roller bottle 104). The imaging device 130 may adjust the focus distance (i.e., the distance from the imaging device 130 at which a subject will be in focus) in order to more accurately image inner bottle layers.

[0038] After recording the first image slice 132, the bottle 104 is rotated such that a different portion of the bottle interior is in view for the imaging device 130, and a second image slice subsequently recorded. The amount by which the roller bottle 104 rotates after eachimage slice 132 is dependent on the field of view of the imaging device 130 (e.g., the width of the image slice). A narrow field of view requires more image slices 132 to be recorded in order to image the entire roller bottle interior, and a wide field of view requires fewer image slices to be recorded in order to image the entire roller bottle interior. The multiple image slices 132 are combined (e.g., aggregated) by the CGT processor 108 to yield the aggregate image, which includes all cell clusters within the roller bottle 104.

[0039] Referring again to FIGS. 1-5, the incubator module 102 also includes a reservoir sub-module 136 housing a plurality of fluid reservoirs 114. The fluid reservoirs 114 are configured to hold fluid to be deposited into the roller bottles 104 (e.g., cell culture, culture media, cleaning solution, water, gas, etc.), as well as fluid to be removed from the roller bottles (e.g., non-viable or undesirable cell cultures, excess moisture, etc.). The fluid reservoirs 114 are constructed from a durable, sterile material fit for prolonged exposure to biological substances (e.g., polyurethane, glass). The illustrated embodiment includes a cell culture reservoir 114A and a waste reservoir 114B, each in fluid connection with the roller bottles 104 via a plurality of fluid tubes 116. The fluids are pumped to and / or from the roller bottles 104 via a fluid pump 138 located in proximity to the fluid reservoirs 114. The illustrated embodiment shows a single fluid pump 138 for both reservoirs 114, however individual fluid pumps may be employed for each reservoir.

[0040] The incubator module 102 further comprises a user interface 140 for facilitating interaction between the CGT processor 108 and the operator. The user interface 140 of the illustrated embodiment includes a touch-screen display, however alternative devices may be employed to similar effect (e.g., keyboard and mouse). The user interface 140 facilitates a number of processes, such as machine calibration, process initiation and cancellation, and security authentication (e.g., asking for a password). For example, upon installation of the incubator module 102 in a lab, the user interface 140 may be used to connect the CGT processor 108 to the internet via Wi-Fi to allow for remote examination and / or control of incubator functions. The user interface 140 of the illustrated embodiment is located on the housing of the reservoir sub-module 136, but may be located elsewhere on the incubator module 102. It is to be understood that one or more of the CGT processor 108, memory 110, and vision system 106 may be housed within the incubator 100 or located externally.

[0041] Referring now to FIGS. 8A-9B, roller bottles 104 and port caps 142 are shown, and will now be described in detail. The roller bottles 104 include a plurality of concentrically nested bottle layers 144 which rotate independently (e.g., separately) along the longitudinal bottle axis LB, as well as a port cap 142 which forms a hermetically sealedconnection with the outermost bottle layer. The roller bottle layers 144 are formed from a durable material which facilitates a sterile environment for cell growth (e.g., metals such as stainless steel, polymers such as polyethylene or silicon elastomers, glasses or other ceramics, etc.). Each bottle layer 144 has an inner face 146 configured to be coated with cell culture. As cell solution is injected into the roller bottle 104, the roller bottle layers 144 rotate, distributing the cell solution evenly across the inner faces 146 thereof. The roller bottle 104 in its entirety may be tilted during this coating process to further improve the distribution of cell solution throughout the interior thereof.

[0042] The multiple nested bottle layers 144 provide increased surface area for cell growth in a compact form factor, increasing the potential yield of each roller bottle 104. Given that each subsequent nested bottle layer 144 is marginally smaller than the bottle layer which encompasses it, the dimensions do not interfere with their independent rotation. This avoids disruption to the imaging device's 130 visual fidelity. Each of the roller bottle layers 144 are connected at a first end 148 of the roller bottle.

[0043] The bottle layers 144 provide a 2D platform, such that cells can attach (e.g., adhere) to the inner faces 146 thereof. Generally, cells prefer to reproduce in an adherent manner. Existing cell growth systems suspend cells (e.g., float) in a large batch of culture media. The roller bottles 104 of the present disclosure allow existing cultures (e.g., those intended for use in a petri dish) to be used in the automated incubator 100, requiring no change to known culture designs.

[0044] The roller bottles 104 are specifically designed to maintain optimal conditions for cell growth. Because the roller bottle interior is hermetically sealed from the rest of the environment within the roller bottle sub-module 118, each roller bottle may be filled with a gas that optimally promotes cell growth for the specific cell type being grown therein. For example, certain cells may benefit from an oxygen rich environment, and may therefore be filled with a highly oxygenated gaseous mixture. The hermetically sealed environment also allows for customizable pressure (e.g., 0.5 atm., 1.0 atm., 2.0 atm., 10.0 atm., etc.).

[0045] Each port cap 142 generally comprise a disk 150 and a cap body 152, the cap body including a bottle clasp 154 and a disk retainer 156. In the illustrated embodiment, the bottle clasp 154 interfaces with the roller bottle 104 at a narrowed end region thereof (e.g., at an end region of the outer bottle layer), and makes a hermetic seal therewith. The seal may be achieved through various means. The bottle clasp 154 may include threads along its inner face which correspond to threads along the outer face of one of the end regions of the roller bottle 104. Thus, the bottle clasp 154 is screwed onto the bottle 104 and tightened. To improvethe seal's ability to withstand pressure, an O-ring may be included. In alternative embodiments, the bottle clasp 154 may be permanently coupled to the roller bottle 104. The bottle clasp 154 does not rotate with respect to the outer bottle layer 144, but does rotate with respect to the disk 150 and the disk retainer 156. In this way, the roller bottle 104 is free to rotate about the longitudinal bottle axis LB thereof without moving the disk 150 or disk retainer 156. In this manner, port cap 142 comprises a rotatable sealing mechanism, enabling fluid exchange without disrupting roller bottle rotation, reducing shear forces on the culture while preventing contamination.

[0046] The disk retainer 156 is a two-part retainer with first and second rings 156A, 156B spaced apart longitudinally on both sides (e.g., faces) of the disk 150. The rings 156A, 156B sandwich the diskl50, and may define elongate channels therethrough to allow for fasteners (not shown). The first ring 156A (farthest from the roller bottle 104) may include indentation to facilitate improved grip from a robotic handler (e.g., a robotic arm).

[0047] The disk retainer 156 and the bottle clasp 154 form a rotatable connection via a flange-and-channel system. The bottle clasp 154 defines an interior annular channel 158 which extends circumferentially about the interior thereof, having a channel width and a channel depth. The second ring 156B of the disk retainer 156 includes an annular flange 160 which extends circumferentially about the exterior face thereof, having a flange width and a flange height. The flange height and flange width are marginally less than the channel depth and channel width, respectively, allowing the bottle clasp 154 and disk retainer 156 to move relative to one another when nested.

[0048] The disk 150 defines at least one fluid exchange port 162. The fluid exchange port 162 makes a fluid connection between an area outside the roller bottle 104 (e.g., the interior of the roller bottle sub-module 118) and the interior of the roller bottle. This fluid connection is bridged by a fluid tube 116 configured to deposit and / or withdrawal / remove fluid from the roller bottle 104. These are the same fluid tubes 116 described above which, through the fluid pump 138, are fluidly connected to at least one fluid reservoir 114.

[0049] The illustrated embodiment includes three individual fluid exchange ports 162. The fluid exchange ports 162 have a perimeter shaped and sized to correspond with the shape and size of the fluid tubes 116. Like the joint between the bottle clasp 154 and the outer bottle layer of the roller bottle 104, the joint between each fluid exchange port 162 and its respective fluid tube 116 forms a hermetic seal, restricting the entrance or exit of material from the roller bottle 104 except through the fluid tubes. The fluid exchange ports 162 releasably retain the fluid tubes 116 such that they may be removed (e.g., when a roller bottle104 is inserted into or removed from the roller bottle sub-module 118). While not illustrated, the fluid tubes 116 may include a one-way valve to prevent backflow in a pressurized system. In certain embodiments, multiple fluid exchange tubes 116 may be fitted through a single fluid exchange port.

[0050] The overall effect achieved through the strategic design of the port cap 142 is a secure, sterile, and mobile junction between the fluid tubes 116 and the roller bottle 104. Because the roller bottles 104 are frequently rotated, tilted, or generally moved (e.g., laterally or longitudinally), there is risk of dislodgement of the fluid tubes 116 therefrom. The ability of the disk 150 (and disk retainer 156) to rotate independently from the bottle 104 (and bottle clasp 154) prevents the fluid tubes 116 from twisting, either individually or around one another. In this way the port caps 142 significantly improve the reliability of the automated incubator 100.

[0051] Referring now to FIG. 10, a system process is depicted in the form of a flow chart, and is generally indicated at reference number 1000.

[0052] At operation 1002, the automated incubator undergoes a preparation / calibration phase. This includes checking the fluid levels in the fluid reservoirs and rectifying any issues (e.g., filling an empty cell culture reservoir or culture agent reservoir, emptying a waste reservoir, etc.), and selecting environment conditions for the cells (e.g., temperature, humidity, exposure to light, etc.). Desired environment conditions for different cell types may be stored in memory and selected by the operator. For example, the operator may communicate to the CGT processor via the user interface that the bottles in the upper row are of cell type A and the cells in the bottom and center rows are cell type B. The CGT processor, having access to information in memory regarding the optimal environment conditions for cells of type A and type B, may prepare the roller bottle sub-module for these cell types (increase the temperature, decrease the temperature, etc.). If the roller bottle sub-module is loaded with at least one partition to define multiple incubation stations, such incubation stations may be adjusted to have independent environmental conditions. In certain embodiments, temperature may be controlled via the injection of heated / cooled gas through a fluid tube. In this embodiment, each roller bottle may have its temperature controlled independently, allowing for many different cell types to be grown simultaneously.

[0053] At operation 1004, the roller bottles are populated with cell culture. At this operation, the pump draws cell culture from the cell culture reservoir, and deposits it in the roller bottles. If cells of different type are desired, the pump(s) may draw cell cultures from multiple cell culture reservoirs to deposit them at different roller bottles. The roller bottles arerolled and tilted while the cell culture is deposited therein, coating the inner faces of each of the bottle layers. The vision system may be optionally engaged to provide real-time information to the CGT processor about the state of an injection process. For example, the vision system may image a roller bottle as it turns during injection to verify that the solution is being deposited in great enough quantity and spread evenly. If the CGT processor, having received such information, determines that the roller bottle in question has been inadequately impregnated (e.g., injected, filled, lined), it commands the pump to inject more cell culture and / or one or more of the DC motors to further rotate the bottle. The vision system can repeatedly or continuously report the status of the injection.

[0054] At operation 1006, the Al-driven vision system scans (e.g., images) the roller bottles to provide the CGT processor with data regarding the development (e.g., growth) of the cells therein. This step may be performed uniformly (e.g., across all roller bottles at the same time) and / or at fixed intervals (e.g., once an hour, once a day, once a week, etc.), but may also be performed in a customized fashion for each roller bottle, and therefore each cell cluster. For example, one roller bottle may be populated with cell type A and another with cell type B, wherein cell type A grows (e.g., reproduces) twice as fast as cell type B. The vision system, controlled by the CGT processor, may image cell type A more frequently (e.g., once a day) than cell type B (e.g., once every other day).

[0055] At operations 1008 and 1010, the CGT processor analyzes the images (e.g., cell data) provided by the vision system, and predicts cell cluster viability (e.g., viable or non-viable), as well as optimal conditions for continued cell growth. This is preferentially performed by an Al engine (e.g., a machine learning algorithm) including a modeling mechanism. The Al engine is trained using a training data set comprising a plurality of cellular image data and a plurality of transfection data or confluency data, such that the Al engine can receive an image from the vision system and automatically determine the confluency and / or transfection levels of the imaged cell cluster. The modeling mechanism may include differential expression, cell type composition, perturbation modeling, trajectory inference, or cell-cell communication.

[0056] At operation 1012, if the Al engine finds that a cell cluster is non-viable, it extracts the cell solution using a fluid tube, and deposits it in the waste reservoir. The conclusion that a cell cluster is non-viable does not necessarily entail a determination that the cells cannot yield a successful batch, but rather that the cost (monetary cost or other) of the resources being used by the cell cluster in question (e.g., more cell culture or culture agent,time in the incubator module, etc.) are not worth risking given the predicted outcome of the cell cluster.

[0057] At optional operation 1012', the automated incubator transmits, via a transmitter, a signal indicating that a cell cluster has been found to be non-viable and therefore disposed of. This signal can be communicated to a remote device (e.g., a cell phone through text or an app, a computer, an email inbox, etc.) associated with the operator (e.g., a lab technician outside of the lab). The transmitter may send the signal before disposing of the cell cluster, requesting permission from the operator to do so. The signal may include the image of the cell cluster provided by the vision system. In this way, the human operator can verify that the Al engine has correctly assessed cell viability. This is particularly useful when culturing cells which are difficult or costly to procure.

[0058] At operation 1014, the CGT processor (e.g., the Al engine) determines if the cell cluster is ready for removal (e.g., a complete batch). For example, the CGT processor may compare the confluency determined by the Al engine (e.g., 20%, 50%, 99%, 100%, etc.) to a threshold confluency (e.g., 90%, 95%, 100%). If the measured confluency is lower than the threshold confluency, the cell cluster is not ready for removal (e.g., harvesting, extraction). If the measured confluency is greater than or equal to the threshold confluency, the cell cluster is ready for removal. Referring briefly to FIG.S 11A-11C, aggregate images of cell clusters having different confluency levels are shown. FIG. 12A shows a low-confluency cell cluster which is not ready to harvest. FIG. 12B shows a moderate-confluency cell cluster which may or may not be ready to harvest. FIG. 12C shows a high-confluency cell cluster which is ready to harvest. Operation 1014 may be performed concurrently with operation 1008.

[0059] Referring again to FIG. 11, if it has been determined that the cell cluster is ready for harvesting, the CGT processor may proceed to operation 1012' to notify the operator. The CGT processor may also move the roller bottle holding the cell cluster in question to a cool incubator station for refrigeration, or cool the interior of the roller bottle directly with cool air via a fluid tube. At this operation, the CGT processor transmits (e.g., via the transmitter) a signal to the operator indicating that one or more of the cell clusters are ready for extraction. This signal can be communicated to a remote device (e.g., a cell phone through text or an app, a computer, an email inbox, etc.) associated with the operator (e.g., the lab tech outside of the lab). The transmitter may send the signal before cooling the cell cluster, requesting permission from the user to do so. The signal may include the image of the cell cluster provided by the vision system. In this way, the human operator can verify that the Al engine has correctly determined that the cell cluster is ready for harvesting, thereby prevent premature cooling.

[0060] At operation 1014', it has been determined that the cell cluster is not yet ready for extraction (e.g., harvesting, removal). At this operation, the CGT processor is configured to control a number of incubator functions to maximally increase the growth of the cell cluster. These function include, but are not limited to, waste removal, cell culture injection, culture agent injection, roller bottle rotation, roller bottle tilting, roller bottle transportation, temperature control, gas exchange, and sterility maintenance (e.g., cleaning). These functions are based on the growth conditions determined by the CGT processor (e.g., the Al engine) at operation 1008. Once the CGT processor has controlled such functions to promote cell growth, the vision system again scans the cell cluster (after a period of waiting for cell growth, e.g., one day), and transmits the image data to the CGT processor to repeat operation 1008.

[0061] While the systems and methods above have been described and disclosed in certain terms and have disclosed certain embodiments or modifications, persons skilled in the art who have acquainted themselves with the disclosure, will appreciate that it is not necessarily limited by such terms, nor to the specific embodiments and modification disclosed herein. Thus, a wide variety of alternatives, suggested by the teachings herein, can be practiced without departing from the spirit of the disclosure, and rights to such alternatives are particularly reserved and considered within the scope of the disclosure.

[0062] When introducing elements of the invention or embodiments thereof, the articles “a,” “an,” “the,” and “said” are intended to mean that there are one or more of the elements. The terms “comprising,” “including,” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0063] Not all of the depicted components illustrated or described may be required. In addition, some implementations and embodiments may include additional components. Variations in the arrangement and type of the components may be made without departing from the spirit or scope of the claims as set forth herein. Additional, different or fewer components may be provided and components may be combined. Alternatively, or in addition, a component may be implemented by several components.

[0064] The above description illustrates embodiments by way of example and not by way of limitation. This description enables one skilled in the art to make and use aspects of the invention, and describes several embodiments, adaptations, variations, alternatives and uses of the aspects of the invention, including what is presently believed to be the best mode of carrying out the aspects of the invention. Additionally, it is to be understood that the aspects of the invention are not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings.The aspects of the invention are capable of other embodiments and of being practiced or carried out in various ways. Also, it will be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.

[0065] It will be apparent that modifications and variations are possible without departing from the scope of the invention defined in the appended claims. As various changes could be made in the above constructions and methods without departing from the scope of the invention, it is intended that all matter contained in the above description and shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.

[0066] In view of the above, it will be seen that several advantages of the aspects of the invention are achieved and other advantageous results attained.

[0067] The Abstract and Summary are provided to help the reader quickly ascertain the nature of the technical disclosure. They are submitted with the understanding that they will not be used to interpret or limit the scope or meaning of the claims. The Summary is provided to introduce a selection of concepts in simplified form that are further described in the Detailed Description. The Summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the claimed subject matter.

Claims

WHAT IS CLAIMED IS:

1. A closed-system automated incubator for cell and gene therapy (CGT) vector production, comprising: an incubator module containing a plurality of multi-layer roller bottles, each of the multi-layer roller bottles defining a bottle interior configured for incubating a cell cluster, wherein each roller bottle is assigned an independent micro-environment configured to allow simultaneous production of distinct cell and vector serotypes types within a single incubator module, the independent micro-environment comprising one or more of programmable temperature, gas composition, or fluidic exchange parameters; a plurality of port caps corresponding to the plurality of multi-layer roller bottles, each of the port caps rotatably coupled to a corresponding one of the multilayer roller bottles; a vision system configured to generate image data representative of the cell cluster within each of the multi-layer roller bottles; an CGT processor receiving and responsive to the image data generated by the vision system; and a memory storing one or more processor-executable instructions that, when executed, configure the CGT processor for: executing an artificial intelligence (Al) engine to autonomously optimize growth conditions per bottle by adjusting media replenishment, gas exchange, and transfection agent delivery in real time, eliminating the need for external fluorescence-based assessment.

2. The automated incubator of claim 1, wherein the incubator module is configured to rotate each of the multi-layer bottles about a longitudinal bottle axis thereof.

3. The automated incubator of claim 2, wherein the incubator module is configured to tilt each of the multi-layer roller bottles about an axis orthogonal to the longitudinal bottle axis.

4. The automated incubator of claim 2 or claim 3, wherein each of the multilayer roller bottles comprises at least two concentric bottle layers configured to rotate independently about the longitudinal bottle axis.

5. The automated incubator of any one of claims 1-4, wherein each of the port caps comprises a disc and a cap body, the cap body having a bottle clasp and a disk retainer rotatably coupled to one another, and wherein the disc defines a fluid exchange port.

6. The automated incubator of claim 5, further comprising a plurality of fluid tubes, wherein fluid exchange port is configured to releasably and hermetically maintain a fluid tube therein.

7. The automated incubator of any one of claims 1-6, wherein the vision system generates the image data using at least one of bright-field microscopy or fluorescent microscopy.

8. The automated incubator of any one of claims 1-7, wherein the vision system comprises a slide-rack and an imaging device slidably coupled thereto, and wherein the imaging device is configured to image the cell cluster within each of the multi-layer roller bottles as the imaging device moves along the slide-rack.

9. The automated incubator of claim 8, wherein the multi-layer rollers bottles are arranged in an array, and wherein the slide rack is positioned below the array and the imaging device is configured to continuously image the cell cluster within each of the multi-layer roller bottles as the multi-layer rollers bottles rotate.

10. The automated incubator of any one of claims 1-9, further comprising a cold incubator station and a hot incubator station.

11. The automated incubator of any one of claims 1-10, wherein the Al engine comprises a modeling mechanism configured for automatically determining the at least one of confhiency or transfection levels, the modeling mechanism comprising at least one of differential expression, cell type composition, perturbation modeling, trajectory inference, or cell-cell communication.

12. The automated incubator of any one of claims 1-11, wherein the artificial intelligence engine is trained using a training data set comprising a plurality of cellular image data and a plurality of transfection data or confluency data.

13. The automated incubator of claim 12, wherein the artificial intelligence engine is configured to predict growth conditions using the image data.

14. The automated incubator of claim 13, wherein the CGT processor is configured to control a plurality of incubator module functions based on the predicted growthconditions, the plurality of incubator module functions comprising waste removal, culture injection, bottle rotation, bottle transportation, temperature control, gas exchange, and sterility maintenance.

15. A multi-layer concentric roller bottle for incubating a cell cluster comprising: a port cap comprising a disk and a cap body, the disk defining at least one fluid exchange port; an outer bottle layer coupled to the cap body and having an inner face configured to be coated with a cell solution; and an inner bottle layer nested within the outer bottle layer, the inner bottle layer being independently rotatable with respect to the outer bottle layer and having an inner face configured to be coated with the cell solution.

16. The multi-layer roller bottle of claim 16, wherein the outer bottle layer and the inner bottle layer are two of a plurality of nested bottle layers, and wherein each of the nested bottle layers is configured to rotate independently from one another about a longitudinal axis.

17. The multi-layer roller bottle of claim 17, wherein the disk defines a first fluid exchange port, a second fluid exchange port, and a third fluid exchange port, and wherein each of the fluid exchange ports are configured to releasably and hermetically maintain a fluid tube.

18. The multi-layer bottle of claim 18, wherein the cap body comprises a bottle clasp configured to interface with the multi-layer bottle and a disk retainer configured to secure the disk, and wherein the bottle clasp and disk retainer are rotatably coupled to each other.

19. The multi-layer bottle of claim 19, wherein the bottle clasp defines an interior annular channel extending circumferentially about an interior thereof, and wherein the disk retainer includes an annular flange configured to be retained by the interior annular channel.

20. A method of growing cells, comprising: injecting a cell culture into a multi-layer roller bottle, wherein the multi-layer roller bottle comprises an inner bottle layer nested within the outer bottle layer, the inner bottle layer being independently rotatable with respect to the outer bottle layer and having an inner face configured to be coated with the cell culture; and rotating the multi-layer bottle to evenly coat the nested bottle layers with the cell culture;wherein the rotating and injecting are performed simultaneously.

Images