Functionally closed cell processing system

Abstract

A functionally closed cell processing platform designed to minimize contamination and automate cell processing. The platform integrates multiple specialized cassettes, including a reagent sample cassette (RSC) for reagent delivery and sample collection, a process fluids cassette (PFC) for waste and buffer management, and a cell processing cassette (CPC) for cell handling, along with a transfer syringe cassette (TSC) for precise fluid transfer. The system features a centrifuge capable of multi-mode operation for cell concentration and mixing, pneumatic controls for fluid and microbubble manipulation, and UV sanitizers to maintain aseptic conditions. Advanced functionalities include optical and thermal sensors for real-time monitoring, automated control of fluid and reagent handling, and a microbubble cell selection system for precise target cell isolation. This innovative platform ensures efficient, contamination-free cell processing, supporting applications in gene therapy, cell manufacturing, and biomedical research.

Description

RELATED APPLICATIONS

[0001] This application claims priority from the United States Provisional Application withSerial Number 63 / 616710 which was filed on December 31, 2023. The Provisional Applicationis hereby incorporated by reference in its entirety.TECHNICAL FIELD OF THE DISCLOSURE

[0002] This disclosure is generally related to cell therapies, and more particularly a devicecapable of separating target cells from non-target cells in blood or blood products andgenetically modifying the target cells for clinical use in the field of cell and gene therapy.BACKGROUND

[0003] Gene-engineered autologous cell therapies, which utilize a patient's own cells re-engineered and expanded ex vivo, represent a revolutionary approach to addressing complexdiseases with high mortality and morbidity rates. These therapies leverage geneticmodifications to correct underlying defects, offering transformative health outcomes forconditions where conventional treatments are limited or ineffective. Such gene-modified celltherapies, including those targeting hematopoietic cells such as T-cells, NK (Natural Killer)cells, and stem cells, have become a cornerstone of modern precision medicine, enablingtargeted and durable interventions.

[0004] The field has witnessed rapid advancements, propelled by recent regulatory approvalsand a robust development pipeline. Since 2017, multiple CAR-T cell therapies have receivedFDA approval for hematologic cancers, with over 1000 additional gene-modified therapies inU.S. clinical trials by late 2019. Beyond CAR-T therapies, CD34+ hematopoietic stem cells(HSCs) have shown similar promise for inherited disorders, including beta-thalassemia andsevere combined immunodeficiency (SCID), with two therapies approved in the EU and over31 pediatric trials underway in the U.S. as of 2019. Market forecasts predict a substantialexpansion, with the global gene therapy market projected to grow from $18 billion in 2023 toover $97 billion by 2033. This growth reflects the increasing potential and demand for gene-modified cell therapies.

[0005] Originally developed to combat blood cancers, CAR-T therapies are now beingexplored for a broader spectrum of conditions, significantly expanding the pool of potentialpatients. These therapies have demonstrated notable promise in addressing a wide range ofindications:

[0006] Gene-modified immune cells, particularly CAR-T cells and NK cells, provide precisetargeting and elimination of cancer cells in blood and solid tumor cancers, offering a superioralternative to traditional treatments such as chemotherapy, radiation stem cell transplants forhematopoietic reconstitution, which are often associated with severe side effects and limitedefficacy.

[0007] Available hemoglobinopathies include the use of gene-modified hematopoietic stemcells that present potential curative treatments for genetic conditions such as sickle cell diseaseand beta-thalassemia by addressing the fundamental defects in hemoglobin production. Thereare also treatments available for autoimmune diseases through the engineering of immune cellsto regulate or eliminate malfunctioning immune responses, gene-modified therapies offer newhope for chronic autoimmune conditions like lupus, rheumatoid arthritis, and multiplesclerosis, which currently lack definitive cures.

[0008] There is emerging research showing the role of senescent cells in aging and associateddiseases. Gene-modified cell therapies can selectively target and clear these cells, mitigatingage-related conditions, improving quality of life, and potentially extending healthy lifespan.

[0009] These advancements signify a paradigm shift in medicine, emphasizing the precisionand adaptability of gene modification to address the root causes of complex diseases ratherthan merely managing symptoms. However, the potential of gene-modified cell therapies ishindered by significant challenges in manufacturing, scalability, and accessibility.

[0010] Challenges in Current Manufacturing Processes - Despite their clinical promise, theproduction of gene-modified cell therapies is constrained by inefficiencies, high costs, andlimited scalability. The reliance on outdated technologies and labor-intensive workflowsexacerbates these issues, making it difficult to meet the growing demand for these therapies.

[0011] Obsolete and Complex Technologies - Conventional manufacturing relies on multipledifferent antiquated equipment and processes that are inefficient, prone to cell loss, and at riskof contamination that are reported to result in up to 92.1% T-cell loss, requiring additional ex-vivo expansion steps to recover cell populations. These steps increase production time,variability, cell exhaustion, and contamination risks. Traditional bead-based systems, whichpermanently attach magnetic beads to cells, are inefficient and lack the flexibility required forsequential selections, limiting their utility for producing specialized cell subsets like memoryT-cells. Similarly, flow sorters, while capable of achieving high purity through multi-parametric sorting, are unsuitable for clinical-scale production due to their low throughput.

[0012] Lengthy and Inefficient Processing - The current "vein-to-vein" timeline for CAR-Tproduction is 30-40 days, much of which is spent on ex-vivo expansion to compensate for celllosses. These extended timelines increase the risk of cell exhaustion and depletion of criticalsubpopulations, such as memory T-cells, which are essential for sustained therapeutic efficacy.Centralized manufacturing further exacerbates delays, as cells must be frozen and thentransported between facilities for different processing stages, reducing cell viability, causingmanufacturing challenges due to tumor cell lysis, and increasing logistical complexity.

[0013] High Costs and Limited Accessibility - The inefficiencies of traditional methodstranslate into prohibitively high costs, with therapies often priced between $350,000 and$500,000 per patient, and some gene therapies exceeding $4 million per dose. These costs,coupled with the slow production process, restrict access to these treatments for many patientsand limit their adoption within healthcare systems.

[0014] Limited Capacity for Clinical and Research Applications - Existing productionsystems are not equipped to handle the demands of large-scale clinical applications or rapiditeration for research purposes. This limited capacity slows the development and testing ofnext-generation therapies, hindering innovation and the ability to serve broader patientpopulations.

[0015] Worsening Over Time - As gene-modified cell therapies are adapted to treat morediverse conditions—such as solid tumors, autoimmune diseases, neuromuscular disorders, andage-related conditions the shortfalls of current manufacturing approaches will becomeincreasingly pronounced. This growing demand highlights the urgency for scalable, efficient,and cost-effective production solutions.

[0016] Emerging Solutions and Remaining Challenges - Innovative approaches are beingdeveloped to address these challenges. Alternatives such as automation, non-viral vectors, andstable cell line platforms for recombinant adeno-associated virus (rAAV) production showpromise in enhancing scalability, consistency, and cost-efficiency. However, these methods areoften in the early stages and face hurdles in regulatory approval and commercial scalability.For instance, while stable rAAV producer cell lines can streamline production timelines andimprove reliability, they must overcome difficulties in balancing cell viability with productivityand stability, particularly with toxic rep / helper genes.

[0017] The Present Invention

[0018] The present invention seeks to address the critical challenges faced by current gene-modified cell therapy manufacturing processes, offering a streamlined, reliable, andeconomically viable platform to produce these transformative therapies. The present inventionintroduces a transformative platform designed to overcome the limitations of conventionalmanufacturing processes. Sometimes referred to herein as an AutoCell Platform (ACP), theplatform integrates advanced automation, closed-loop processing, and innovative technologiesto streamline workflows, reduce cell loss, and compress production timelines, reducing costsby an order of magnitude. The key objectives of the invention are as follows:

[0019] It is a first objective of the invention to integrate advanced automation with afunctionally closed-system technology, enabling a highly efficient and controlled productionprocess. By minimizing human intervention, this closed-loop design significantly reducescontamination risks and manual errors, enhancing the safety, consistency, and scalability ofgene-modified cell therapy manufacturing. Traditional methods, which often require 30-40days to complete a batch due to labor-intensive steps like washing, selection, activation,transduction, and formulation, are compressed to less than 3 days using this platform. Theelimination of the requirement for ex-vivo expansion to replace cells lost in manufacturing,improves efficiency, and lowers manufacturing costs to approximately one-tenth ofconventional levels, making these therapies more affordable and accessible.

[0020] Another objective of the invention is to provide a platform that introduces an automatedspinoculation process, a novel centrifuge-based method that combines spinning andinoculation (i.e., Spinoculation) to significantly improve genetic material uptake by target cells.Conventional methods typically achieve only 25 -35% efficiency. The invention's automatedspinoculation consistently achieves 70% efficiency, improving the quality and reliability ofgene-modified cells. This technique is particularly critical for the gene modification of targetcells within a single vessel, ensuring that high levels of genetic material are successfullydelivered into the target cells with minimal variability.

[0021] Another objective of the invention is to provide a scalable platform having a modular,standardized design that allows for standardized manufacturing across a wide variety of clinicaland research settings. Unlike traditional bespoke systems, which are tailored to specificfacilities and therapies, the platform can be replicated and deployed in diverse locations,including smaller or remote healthcare centers. This adaptability ensures that patients canreceive treatment directly at their treatment center, reducing logistical challenges liketransportation, freezing, and storage that often compromise cell viability. By enabling bothcentralized and decentralized manufacturing models, the invention expands the availability ofadvanced therapies while maintaining high levels of consistency and quality.

[0222] Another objective of the invention is to provide precision in cell selection andmodification, such as microbubble-assisted cell selection (MBCSA) and aptamer-guidedtargeting, to achieve unparalleled precision in the isolation and activation of specific cellpopulations. These advanced methods enable the production of high-quality CAR-T cells, NK(Natural Killer) cells, and gene-modified stem cells by focusing on key subpopulations thatmaximize therapeutic efficacy. Unlike conventional techniques that often result in high levelsof cell loss and contamination, the ACP's precise selection and modification processes ensureoptimal purity and functionality of the final therapeutic product.

[0023] Microbubble-assisted cell selection (MBCSA) is a critical innovation within the ACP,allowing for sequential and highly specific isolation of target cells. By utilizing microbubblesthat can attach and provide buoyancy to target cells, the platform separates selected cells fromthe bulk population, enabling multi-step enrichment without compromising cell viability. Thenafter separation has occurred, and non-target cells removed, the buoyancy can be removed byimploding the microbubbles with modest pressure increase in the container to then allow thetarget cells to undergo further processing steps. Similarly, aptamer-guided targeting employsmolecular binding agents with high specificity for particular cell surface markers, ensuringprecise activation and retention of the desired cell subsets.

[0024] The prevision of the ACP is particularly significant for the development of CAR-T celltherapies, where the composition and quality of T-cell subsets directly influence clinicaloutcomes. CAR-T products derived from carefully enriched subsets, such as CD8+ cytotoxicT-cells and CD4+ helper T-cells, are known to exhibit superior antitumor effects. The ACP hasdemonstrated its ability to achieve successful enrichment of CD8+ T-cells from a general T-cell population, highlighting its capacity to enhance the therapeutic potency of gene-modifiedcell products.

[0025] Another objective of the invention is to provide a versatile and customizable platformthat may accommodate a variety of cell types and therapies, including hematopoietic stem cells,T-cells, and NK cells. Customizable protocols enable the system to adapt to the uniquerequirements of different therapies, from CAR-T treatments for cancer to regenerativetherapies for age-related conditions. This versatility makes the platform a valuable tool acrossa spectrum of gene-modified therapies.

[0026] Another objective of the invention is to provide a compact and automated platform,enabling its deployment in FDA-licensed transplant centers or other localized healthcarefacilities. This proximity to patients reduces patient accessibility and logistical delays,minimizes cell viability loss due to freezing and thawing, and accelerates treatment availability.By facilitating point-of-care (POC) manufacturing, the platform enhances access to advancedtherapies for underserved populations and eliminates many of the inefficiencies inherent incentralized production models.

[0027] Another objective of the invention is to provide research and clinical support. Theplatform's automated and standardized processes free researchers to focus on developingeffective gene constructs rather than labor-intensive manufacturing tasks. This simplificationaccelerates translational research, allowing scientists to rapidly iterate on therapeutic vectordesigns and bring innovative therapies to clinical trials faster. By supporting the 592 cellbiology research facilities in the United States, the invention fosters a more dynamic andproductive research environment.

[0028] Another objective of the invention is to provide a platform incorporating robust andautomated quality control measures, including automated pressure decay testing for filterintegrity checks,, and septa disinfection modules for aseptic transfer operations, ensuringcompliance with stringent FDA standards. These built-in features reduce the risk ofcontamination, batch failure, and regulatory delays. The platform's integrated data trackingand lot release records collected real-time and provided concurrent to harvest of gene-modifiedtarget cells, further streamline regulatory filings, expediting approvals for new therapies andensuring that each batch meets rigorous quality standards.

[0029] It is yet another objective of the invention to address the high costs, inefficiencies, andlogistical challenges that have historically limited the adoption of gene-modified therapies. Byreducing production costs and compressing timelines, the platform significantly enhances thescalability of these therapies, making them viable options for a broader patient population. Thistransformation expands market opportunities and positions the platform as a foundationaltechnology for next-generation cell therapies.

[0030] It is yet another objective of the invention to provide innovative capabilities to supportthe development of therapies for diseases previously considered untreatable, including solidtumors, autoimmune conditions, and age-related degenerative disorders. Faster productiontimelines enable patients with aggressive diseases to receive timely treatments, including attime of initial diagnosis, while reduced costs make these therapies a sustainable option forchronic conditions. The platform's flexibility and precision unlock new therapeuticpossibilities, opening the door to lifesaving and life-extending treatments.

[0031] It is a still further objective of the invention is to provide a platform supporting bothcentralized and decentralized manufacturing approaches, providing flexibility to meet diverseclinical and logistical needs. Centralized facilities benefit from streamlined, high-throughputproduction, while decentralized setups enable faster vein-to-vein times, reducing the overalltimeline from apheresis to reinfusion and improving patient outcomes. This dual capabilitytransforms the logistics of cell therapy manufacturing, ensuring that therapies can be deliveredwhere and when they are needed.

[0032] The present invention represents a transformational approach to the production of gene-modified cell therapies. By addressing the inefficiencies, high costs, and scalability challengesof traditional methods, the platform improves access to these life-saving treatments for abroader range of patients. Its innovative design enhances research, accelerates regulatoryapprovals, and enables the development of new therapeutic options, ultimately advancing thefrontiers of precision medicine and expanding the global impact of gene-modified therapies.SUMMARY OF THE INVENTION

[0033] The AutoCell Platform (ACP) is a fully automated, functionally closed cell processingsystem engineered to facilitate aseptic, FDA-compliant transfers of reagents, buffers, culturemedia, and gases through various processing cassettes. The platform employs advanced roboticcontrols to coordinate precise movements and interactions among the cassettes. Thisintegration of robotic precision with a sterile, functionally closed environment makes the ACPhighly effective for manufacturing CAR T-cells and other cell-based therapies. By minimizingcontamination risks and optimizing automation, the ACP ensures the production of high-quality cell products suitable for clinical applications.

[0034] The system employs a series of cassettes including among others a Cell ProcessingCassette (CPC) in which target cells are introduced, selected, enriched, genetically modified,washed and concentrated.

[0035] A robotic control system ensures that each cassette operates in a coordinated sequence,maintaining aseptic conditions and eliminating the need for manual handling. This systemminimizes contamination risk while enhancing precision and efficiency in reagent and fluidmanagement. In one embodiment, the ACP's robotic mechanism precisely controls thepositioning and operation of the supporting cassettes:

[0036] Transfer Syringe Cassette (TSC): This cassette manages the precise transfer of reagentsand fluids into the CPC, enabling accurate volumetric dosing for processes such as washing,activation, and transduction.

[0037] Reagent / Sample Cassette (RSC): The RSC houses a plurality of pre-filled reagent vials,preferably six and preferably with a volume capacity of up to 20 mL. In some embodiments,these vials contain essential components such as microbubbles, vectors, and linkers.Additionally, the RSC contains designated sample vials, ready to receive cell samples forquality control (QC) analysis throughout the processing cycle.

[0038] Process Fluids Cassette (PFC): The PFC stores various process fluids, such as buffersand culture media, required at different stages of cell processing. These fluids are automaticallytransferred to the CPC as needed, supporting cell washing, sedimentation, and formulation.

[0039] The CPC incorporates a tapered geometry to optimize sedimentation and target cellrecovery during centrifugation. This is enhanced by pneumatic controls that manage wasteexpulsion, pressure decay testing of integrated hydrophobic and hydrophilic filters, andselective manipulation of buoyant microbubbles that aid in isolating target cells. The platformfurther features a centrifuge mechanism capable of operating in both centrifugation and mixingmodes, with automatic transitions between modes to support homogeneous cell distributionand reagent interaction. A unique multi-axis rotation capability enhances fluid mixing whilepreventing unintended sedimentation.

[0040] The platform includes UV sanitization modules positioned to disinfect fluid pathwaysand interfaces, ensuring aseptic conditions during all stages of processing. Optical and thermalsensors integrated into the system provide real-time feedback on critical parameters such ascell movement, temperature, and fluid levels. These sensors enable automated adjustments tocentrifuge speed, fluid flow rates, and reagent dispensing, ensuring precise control over cellprocessing steps.

[0041] To further enhance functionality, the invention incorporates a microbubble cellselection system. Functionalized microbubbles selectively bind target cells and, throughpneumatic manipulation, enable efficient isolation of these cells from non-target populations.The TSC facilitates precise fluid transfers and includes automated docking and disinfectionmodules to maintain sterility throughout operations. An advanced control module integratesthese features, dynamically managing fluid transfer, monitoring cell behavior, and ensuringconsistent processing outcomes.

[0042] The platform supports a wide range of applications, including gene therapy, cellmanufacturing, and biomedical research. By combining closed-system operations, automatedquality control, and modular scalability, this invention addresses critical challenges in cellprocessing, offering a cost-effective and contamination-free solution for advanced therapeuticand research purposes.

[0043] The ACP preserves more cells than traditional centralized manufacturing, where cellsoften experience losses due to freezing, transport, and multiple transfers. A central air pressuresystem enables precise fluid movement and sterile transfer, further ensuring that cells remainuncontaminated.

[0044] Upon completion of manufacturing, the ACP automatically compiles and publishes aQC batch record that includes all tracked parameters and relevant data for that batch. Thisstreamlined record enables rapid access and review, ensuring compliance with regulatoryrequirements while minimizing the time required for documentation. Pharmaceutical celltherapy facilities, particularly those specializing in CAR T-cell production, often employ moreQC staff than manufacturing staff due to the traditional approach of exhaustive record review.The ACP's integrated QC automation and release by exception capabilities address thisimbalance, allowing facilities to allocate resources more efficiently and reduce costs associatedwith cell therapy manufacturing.BRIEF DESCRIPTION OF THE FIGURES

[0045] In order to enhance the clarity and improve the understanding of the various elementsand embodiments shown herein, the figures have not necessarily been drawn to scale.Furthermore, elements that are commonly known and well understood to those in the industryare not depicted in order to provide a clear view of the various embodiments of the invention;thus, the drawings are generalized in form for the purpose of clarity and conciseness.

[0046] FIG. 1 shows a perspective view of a cell processing device in a closed mode inaccordance with the preferred embodiment of the present invention;

[0047] FIG. 2 shows perspective views of the cell processing device in an open mode;

[0048] FIG. 3 shows a cross-sectional side view of the cell processing device depicted inFIGS. 1 and 2 illustrating a centrifuge bucket in accordance with the preferred embodiment ofthe present invention;

[0049] FIG. 4 shows a perspective view of multiple cassettes relative to a lower housingassembly of the cell processing device depicted in FIGS. 1-3 in accordance with the preferredembodiment of the present invention;

[0050] FIG. 5 shows a perspective view of a flask and multiple cassettes wherein the depictionof the upper housing assembly and the lower housing assembly is removed in accordance withthe preferred embodiment of the present invention;

[0051] FIGS. 6 and 7 show two operating modes of a centrifuge of the cell processing devicein accordance with the preferred embodiment of the present invention;

[0052] FIG. 8 depicts a perspective view of the top surface of the CPC according to a preferredembodiment;

[0053] FIG. 9 is a perspective view of the top surface of the CPC according to an alternativeembodiment;

[0054] FIGS. 10A and 10B are an exploded perspective view of three filters according to anembodiment of the invention;

[0055] FIG. 11 shows a partial exploded view of an alternative embodiment of a centrifugebucket;

[0056] FIG. 12 shows an exploded view of a harvest valve assembly;

[0057] FIGS. 13-15 show exploded views of the CPC according to an embodiment of theinvention;

[0058] FIGS. 16 and 17 are cross-sectional views of the CPC;

[0059] FIG. 18 is a cross-sectional side view of the CPC according to an alternativeembodiment of the invention;

[0060] FIG. 19 shows a plan view of the CPC according to an embodiment of the invention;

[0061] FIG. 20 shows a side view of the CPC according to an embodiment of the inventionand with section lines D-D, E-E and F-F shown in more detail in FIGS. 26, 27 and 28,respectively;

[0062] FIGS. 21-25 show cross-sectional views of CPC according to embodiments of theinvention;

[0063] FIG. 26 shows the top view of the filters of the CPC according to an embodiment ofthe invention shown in cutline D-D in FIG. 20;

[0064] FIG. 27 depicts a top view of the main chamber of the CPC according to anembodiment of the invention shown in cutline E-E in FIG. 20;

[0065] FIG. 28 depicts a top view of the rotation valve of the CPC according to an embodimentof the invention shown in cutline F-F in FIG. 20;

[0066] FIG. 29 depicts a cross-sectional view of the rotation valve of the CPC showing asection of recovery tubing;

[0067] FIG. 30 is a top perspective view of the CPC;

[0068] FIG. 31 is a top cross-sectional view of the CPC showing section lines A-A, B-B andC-C, which define the views provided in FIGS. 32, 33 and 34;

[0069] FIG. 32 is a side view of the CPC showing section lines D1-D1, E1-E1 and F1-F1;

[0070] FIG. 33 is a cross-sectional view from FIG. 31 section line B-B;

[0071] FIG. 34 is a cross-sectional view from FIG. 21 section line C-C;

[0072] FIG. 35 is a cross-sectional view from FIG. 32 section line D1-D1;

[0073] FIG. 36 is a cross-sectional view from FIG. 32 section line E1-E1;

[0074] FIG. 37 is a cross-sectional view from FIG. 32 section line F1-F1;

[0075] FIG. 38 shows a plan view of the CPC according to an embodiment of the invention;

[0076] FIG. 39 shows is a cross-sectional of the CPC;

[0077] FIG. 40A shows a perspective view of a process fluids cassette (PFC) in accordancewith the preferred embodiment of the present invention;

[0078] FIG. 40B shows a plan view of the PFC;

[0079] FIG. 40C shows a first cross section view of the PFC;

[0080] FIGS. 42A shows a perspective view of a Reagent / Sample Cassette (RSC) inaccordance with the preferred embodiment of the present invention;

[0081] FIG. 42B shows a plan view of the Reagent / Sample Cassette (RSC);

[0082] FIGS. 43A 43C show various views of a Transfer Syringe Cassette (TSC) inaccordance with the preferred embodiment of the present invention;

[0083] FIGS. 44A – 44B show cross-sectional side views of reagent syringes of the TSCinteracting with the vial cassette in accordance with the preferred embodiment of the presentinvention;

[0084] FIGS. 44C – 44D show cross-sectional side views of sampling syringes of the TSCinteracting with the processing and vial cassettes in accordance with the preferred embodimentof the present invention;

[0085] FIG. 45A shows an actuator including a first gripping mechanism and a secondgripping mechanism in accordance with the preferred embodiment of the present invention;

[0086] FIG. 45B shows the second gripping mechanism of the actuator that moves downwardfrom the position illustrated in FIG. 45A in accordance with the preferred embodiment of thepresent invention;

[0087] FIG. 45C shows the first gripping mechanism of the actuator having moved downwardand fingers of the first gripping mechanism having moved to a closed state in accordance withthe preferred embodiment of the present invention;

[0088] FIG. 45D shows the movement of the actuator towards downward to push a needle ofthe reagent syringe out of a bottom end of the TSC in accordance with the preferredembodiment of the present invention;

[0089] FIG. 45E shows the movement of the second gripping mechanism towards upwarddirection while the first gripping mechanism remains stationary in accordance with thepreferred embodiment of the present invention;

[0090] FIG. 45F shows the movement of the actuator towards upward direction to draw theneedle in accordance with the preferred embodiment of the present invention;

[0091] FIG. 45G shows a configuration of the actuator to push the reagent syringe backdownward to engage the needle into another cassette in accordance with the preferredembodiment of the present invention;

[0092] FIG. 45H shows the second gripping mechanism that being moved downward to pressa plunger downward to expel fluids into other cassette in accordance with the preferredembodiment of the present invention;

[0093] FIG. 451 shows the actuator after moving upward to retract the needle into the TSC inaccordance with the preferred embodiment of the present invention;

[0094] FIGS. 45J – 45K show the actuator that being disengaging the first and the secondgripping mechanisms and from the reagent syringe in accordance with the preferredembodiment of the present invention;

[0095] FIG. 46 shows a cross-sectional side view illustrating how the Process Fluids Cassette(PFC) transfers fluid to the CPC in accordance with the preferred embodiment of the invention;

[0096] FIGS. 47 - 48 are a perspective view of exemplary CAN-bus cabling according to analternative embodiment of the invention;

[0097] FIGS. 49A – 49B are cross sectional views of a centrifuge wherein the centrifugebucket is in its mixing mode in accordance with the preferred embodiment of the presentinvention;

[0098] FIG. 49C is a cross-sectional view of a centrifuge when in rotation in accordance withthe preferred embodiment of the present invention;

[0099] FIG. 49D is a cross-sectional view of a centrifuge when stationary in accordance withthe preferred embodiment of the present invention;

[0100] FIG. 50 shows features of the heating mechanism of the centrifuge bucket inaccordance with the preferred embodiment of the present invention;

[0101] FIGS. 51A and 51B show utilization of microbeads to facilitate a unique compressionand release cycle for cell selection and concentration in accordance with the preferredembodiment of the present invention;

[0102] FIG. 52 shows an optical determination of valve rotation utilizing an optical encoderin accordance with the preferred embodiment of the present invention;

[0103] FIGS. 53A-53B show four primary rotational positions for CPC valves in accordancewith the preferred embodiment of the present invention;

[0104] FIG. 54 shows the cell processing cassette in an active mode in accordance with thepreferred embodiment of the present invention;

[0105] FIGS. 55A-55J show the CPC valve rotating through a series of positions to facilitatethe step-by-step processing involved in CAR T-cell production in accordance with thepreferred embodiment of the present invention;

[0106] FIG. 56 shows plan view of a portion of the CPC with components highlighted as partof the aseptic transfer process;

[0107] FIG. 57A is a perspective view of a portion of the ACP with a waste container;

[0108] FIG. 57B shows a carbon dioxide enriched container used as part of the aseptic transferprocess;

[0109] FIGS. 58A – 58D show the cell suspension when the CPC is being tilted in accordancewith the preferred embodiment of the present invention;

[0110] FIG. 59A a shows cutline H-H through the CPC in accordance with the preferredembodiment of the present invention;

[0111] FIG. 59B shows a cross section along H-H illustrating a cell suspension fluid in themain chamber in accordance with the preferred embodiment of the present invention;

[0112] FIG. 59C shows movement of fluid toother chamber in accordance with the preferredembodiment of the present invention;

[0113] FIG. 59D shows the fluid from the main chamber that continues to urge the fluidupwards in the standpipe in accordance with the preferred embodiment of the presentinvention;

[0114] FIG. 60A shows a radar system in use with the CPC in accordance with the preferredembodiment of the present invention

[0115] FIG. 60B shows a S shaped channel that directs radar from a radar emitter positionedabove and centered on the CPC cover in accordance with the preferred embodiment of thepresent invention;

[0116] FIG. 60C shows a rod for use with the radar system shown in FIGS. 60A and 60B inaccordance with the preferred embodiment of the present invention; and

[0117] FIGS. 61A and 61B show a perspective view of a CPC with a sealed stub of tubingcoiled in FIG. 61A and in uncoiled form in FIG. 61B.DETAILED DESCRIPTION OF THE DRAWINGS

[0118] In the following discussion that addresses a number of embodiments and applicationsof the present invention, reference is made to the accompanying drawings that form a parthereof, and in which is shown by way of illustration specific embodiments in which theinvention may be practiced. It is to be understood that other embodiments may be utilized, andchanges may be made without departing from the scope of the present invention.

[0119] Various inventive features are described below that can each be used independently ofone another or in combination with other features. However, any single inventive feature maynot address all or any of the problems discussed above. Further, one or more of the problemsdiscussed above may not be fully addressed by any of the features described below.

[0120] As used herein, the singular forms "a", "an" and "the" include plural referents unlessthe context clearly dictates otherwise. “And” as used herein is interchangeably used with “or”unless expressly stated otherwise. As used herein, the term "about" means + / - 5% of the recitedparameter. All embodiments of any aspect of the invention can be used in combination, unlessthe context clearly dictates otherwise.

[0121] Unless the context clearly requires otherwise, throughout the description and theclaims, the words 'comprise', 'comprising', and the like are to be construed in an inclusivesense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including,but not limited to". Words using the singular or plural number also include the plural andsingular number, respectively. Additionally, the words "herein,” “wherein”, “whereas""above,”, “below", and words of similar import, when used in this application, shall refer tothis application as a whole and not to any particular portions of the application.

[0122] The description of embodiments of the disclosure is not intended to be exhaustive or tolimit the disclosure to the precise form disclosed. While the specific embodiments of, andexamples for, the disclosure are described herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as those skilled in the relevantart will recognize.

[0123] This application describes an AutoCell Platform (ACP), an advanced, fully automated,and functionally closed cell processing system specifically designed to perform aseptic andFDA-compliant transfers of critical reagents, buffers, culture media, and gases through the lidof the CPC. Leveraging robotic controls, the platform orchestrates precise movements andcoordinated interactions between its integral components, including the Transfer SyringeCassette (TSC), Reagent / Sample Cassette (RSC), and Process Fluids Cassette (PFC). Byseamlessly integrating robotic precision with a sterile, functionally closed processingenvironment, the ACP ensures high-quality, contamination-free production of CAR T-cells andother cell-based therapies. This innovative system not only mitigates contamination risks butalso streamlines the manufacturing workflow, enabling efficient and consistent production oftherapeutic-grade cell products for clinical applications.

[0124] The ACP works as a functionally closed system. A functionally closed system refers toa controlled environment that operates without exposure to external contamination. Allmaterial transfers into or out of the system are performed aseptically or through sealedmechanisms designed to maintain the sterile integrity of the internal processes. SterileMaintenance of the CPC Interior through the use of aseptic connectors, sterile transfer ports,and sealed pathways ensures sterility during all operations. The internal environment is alsocompletely isolated from external contamination risks. Finally, the system is designed tosupport material exchange without compromising sterility, ensuring consistent and reliablefunctionality.

[0125] The several embodiments of the invention described herein solve at least the technicalproblem of applying multiple cell processing steps autonomously without the need for manualintervention by lab personnel. In particular, a cell processing device is described that may beconfigured to separate target cells from non-target cells and then apply changes to the separatedtarget cells. The ACP may be configured to operate with many different types of target cellsincluding at least but not limited to T cells, NK cells and CD34+ HPSCs.

[0126] Turning now to the figures, FIGS. 1 – 2 show perspective views of a cell processingdevice 100. In particular, FIG. 1 shows a perspective view of cell processing device 100 in aclosed state. Cell processing device 100 includes an upper housing assembly 102 hingedlyattached to a lower housing assembly 104. Upper housing assembly 102 includes a first door106 that allows for the insertion or removal of one or more cassettes into or out of upperhousing assembly 102. First door 106 may be configured to slide or swing between its openand closed states. Lower housing assembly 104 can include one or more controls formanipulating settings of cell processing device 100. In particular, these one or more controlscan include an on / off button 108, a touch sensitive electronic display 110 and an auxiliarycontrol 112. In some embodiments, auxiliary control 112 may be configured to initiate a shutdown or termination in operation of cell processing device 100. Touch sensitive electronicdisplay 110 may also be configured to display multiple operating parameters associated withcell processing device 100 and may be configured to allow an operator to make changes tovarious operating parameters of cell processing device 100. Lower housing assembly 104further comprises a second door 113 that allows for the insertion or removal of one or morevials from lower housing assembly 104 without separating upper housing assembly 102 fromthe lower housing assembly 104.

[0127] FIG. 2 shows upper housing assembly 102 rotated away from lower housing assembly104 to allow for the removal or insertion of one or more disposable cassettes into or out oflower housing assembly 104. Lower housing assembly 104 further defines a centrifuge well114 for accommodating operation of a processing device taking the form of a centrifuge 116.As depicted, centrifuge 116 may be a horizontal or swinging bucket type centrifuge thatincludes a rack or centrifuge bucket 120 for holding a sample container. Bucket 120 may berotatably coupled to opposing ends of rotor yoke 122, as depicted. Rotor yoke 122 is rotatedabout an axis of rotation 124 during some operating modes of centrifuge 116. In someembodiments, a cowling or aerodynamic covering may be employed around the rotatingcomponents to minimize unnecessary air drag during centrifugation, enhance the operationalefficiency of the system and optimizing energy usage during high-speed rotations An additionalcross-sectional view is shown in FIGS. 49A-49D.

[0128] It should be appreciated that in some embodiments, the processing device may includea larger number of buckets for accommodating additional cassettes (e.g. processing cassettes212 described below). For example, rotor yoke 122 could include additional buckets with anadditional bucket that would allow for cell processing device to perform a larger number ofconcurrent operations. In an alternative embodiment, the platform could operate with twobuckets and two counterweights, effectively running two independent processessimultaneously. For example, if the platform processes two leukapheresis collections-onecontaining 150 mL and the other 200 mL an additional 50 mL could be added to the smallercollection to equalize the volumes at 200 mL each. This approach eliminates the need for acounterweight, as the identical processes running concurrently in the balanced buckets ensuresystem stability without additional adjustments.

[0129] Lower housing assembly 104 further comprises a vial cassette recess 126 toaccommodate a cassette configured to hold multiple vials that can contain different reagents toassist in operation of cell processing device 100. In some embodiments, the cassetteaccommodated by vial cassette recess 126 may also include empty vials configured to receivematerial samples during operation of cell processing device 100. The material samples can helpto confirm proper operation of cell processing device 100 and / or to calibrate subsequentoperation of cell processing device 100. Upper housing assembly 102 includes through holes128 and 130 through which cassettes positioned within upper housing assembly 102 can extendthrough to interact with cassettes positioned within vial cassette recess 126 and bucket 120.Lower housing assembly 104 also defines flask recess 127, which is configured toaccommodate a flask for collecting waste materials.

[0130] In some embodiments, cell processing device 100 may include rubberized feet 101 thathelp to dissipate any vibrations transmitted through lower housing assembly 104 by rotation ofcentrifuge 116. In some embodiments the motor may be a stepper motor. In other embodiments,a customized centrifuge is utilized wherein the space between buckets is substantially filled,thereby minimizing air and therefore air resistance and noise as the centrifuge turns. In thisembodiment, the shape may be contoured to allow the swiveling of the bucket and may largelyfill the air space when the bucket is near horizonal, that is, when the centrifuge is at a highRPM.

[0131] FIG. 3 shows a cross-sectional side view of cell processing device 100. Upper housingassembly 102 is shown including a cassette positioning assembly 132 that defines a syringecassette recess 134 and a fluid cassette recess 136. Cassette positioning assembly 132 isconfigured to swap the position of cassettes positioned within recesses 134 and 136 in a LazySusan turntable manner. In addition to repositioning the cassettes positioned within recesses134 and 136, cassette positioning assembly 132 may be further configured to affect rotation ofcassettes around their own axis of rotation (not shown) within their respective recesses and tolower the cassettes at least partially into through holes 128 or 130 to allow for cassettes locatedwithin recesses 134 and 136 to interact with cassettes located in vial cassette recess 126 orbucket 120. In some embodiments, recesses 134 and 136 of cassette positioning assembly 132may each include an iris allowing the cassettes to be lowered through holes 128 or 130,respectively. It should be noted that in some implementations cassette positioning assembly132 may be configured to carry more than two cassettes. For example, a third cassette couldtake the form of a testing cassette used to confirm proper operation of a cassette disposed withincentrifuge well 114. The testing cassette can allow for the cell processing device 100 to confirmproper operation of various consumables such as filters during and / or after various cellprocessing operations are performed.

[0132] FIG. 4 is a perspective view showing the relative positioning of cassettes to lowerhousing assembly 104. Upper housing assembly 102 has been removed to more clearly showthe position of the cassettes relative to lower housing assembly 104. In particular, RSC 202 isshown positioned within vial cassette recess 126 directly below PFC 204. A waste tube 208 isshown and configured to receive waste produced during operation of the cell processing device.Waste tube 208 may be attached to PFC 204 and / or a TSC 210, which may each include awaste channel that allows for the extraction of waste material through a respective one of thecassettes. As depicted in FIG. 4, only PFC 204 is attached to waste tube 208. The PFC 204stores various process fluids, such as buffers and culture media, required at different stages ofcell processing. These fluids are automatically transferred to a Cell Processing Cassette (CPC)212 as needed, supporting cell washing, sedimentation, and formulation. Removal of wastematerial from CPC 212 may be affected by routing pressurized air through a pneumatic channelthat extends through PFC 204 and then into processing cassette 212, which in turn forces wastefluid out of CPC 212.

[0133] The waste-water port and fluid waste disposal system control liquid removal whilemaintaining target cell integrity and minimizing cell loss during cell processing. The waste-water port serves as a one-way outlet for fluid removal and is positioned centrally in the CPClid. Its height and placement are configured to ensure that fluid can be removed efficientlywhile leaving behind a precise, minimum volume of liquid—15 mL at the bottom of thesedimentation chamber when the CPC is upright and motionless. This design guarantees thattarget cells, which settle safely and are sequestered at the bottom of the sedimentation chamber,remain undisturbed during fluid extraction. The 15 mL volume left behind is consistent withthe design intent to protect the target cell population during processing. The exact volume ofresidual fluid may vary slightly across multiple runs due to variations in initial fluid volumesor operational conditions. The fluid waste disposal tube is centrally located within the CPC andextends down into the cone to the 15 mL fluid level when the CPC is upright and static.

[0134] The waste disposal tube serves as the primary exit path for fluid removal, extendingupward through the hollow center tube of the docked PFC and into an expandable, sealed wastecontainer. Positive air pressure, introduced into the CPC through a hydrophobic filter in thelid, propels the fluid upward through the waste disposal tube. During this process, the CPCvalve is rotated to one of the four park positions in the stator. This ensures that no otherpathways exist for fluid to exit the CPC except through the centrally placed waste disposaltube. The central positioning of the tube also ensures even and controlled fluid movement,preventing turbulence or disruption of the cell sediment. The flow rate is regulated to avoidwicking up a clinically significant number of cells-99.9% of target cells remain undisturbed.This precision protects the integrity of the sequestered target cells while ensuring efficientwaste fluid removal.

[0135] FIG. 5 shows a perspective view of all cassettes in the system according to a preferredembodiment, specifically showing RSC 202, PFC 204, TSC 210 and CPC 212. FIG. 5 alsoillustrates how bucket 120 is configured to rotate about axis of rotation 218 during operationof centrifuge 116.

[0136] FIGS. 6-7 show two operating modes of centrifuge 116. Specifically, FIG. 6 showsa first operating mode in which centrifuge 116 operates in a centrifuge mode and FIG. 7 showsa second operating mode in which centrifuge 116 operates in a mixing mode. In the firstoperating mode shown in FIG. 6, a mixing motor associated with bucket 120 may beconfigured to tilt bucket 120 further so that a top face of processing cassette 212 faces directlytoward axis of rotation 124. The depicted angle is typical of the angle achieved when exertinga force of about 50Gs on the sample contained within processing cassette 212.

[0137] FIG. 7 depicts centrifuge 116 operating in the second operating mode and consequentlya bottom surface of bucket 120 is shown tilted toward axis of rotation 124. An angle of rotationas well as a speed of rotation of bucket 120 may vary in accordance with a desired mixingmotion of sample material within processing cassette 212. Furthermore, bucket 120 willgenerally tilt toward and away from axis of rotation 124 while actively in a mixing operatingmode. Generally, rotor yoke 122 will not turn around axis of rotation 124 and mixing will belimited to rotation of bucket 120 about axis of rotation 124. In some embodiments, rotor yoke122 may be configured to turn much more slowly in the second operating mode than it doesduring the first operating mode. For example, a rotational speed of 10-20 RPM about axis ofrotation 124 can in cooperation with rotation about axis of rotation 124 provide a more complexmixing mode.

[0138] FIG. 8 is a perspective view of CPC 212. In particular, processing cassette generallyincludes a cover 402, upper housing component 404 and lower housing component 406. Thecover 402 includes multiple ports for assisting in introducing and withdrawing samplematerials from processing cassette 212.

[0139] The CPC ports include a fluid port 408 (FIT Receptacle 3) for receiving culture mediaand buffer solution from PFC 204. Fluid port 408 may include a one-way valve, taking theform of a check valve, that allows fluid to enter into processing cassette 212 through fluid port408 only when fluid port 408 mates with a port on PFC 204. Cover 402 further comprisespneumatic ports 410 and 412. Pneumatic outlet port 412 (FIT Receptacle 1) allows for theexpulsion of pressurized air from processing cassette 212 to reduce or equalize air pressurewithin processing cassette 212. Pneumatic inlet port 410 (FIT Receptacle 2) allows for theintroduction of air into processing cassette 212 to increase air pressure within a main chamber424 (see FIG. 16) of processing cassette 212. The details of the air pressure control systemwill be described below. Pneumatic ports 410 and 412 can also include a check valve and thecheck valve within pneumatic outlet port 412 is oriented in an opposite direction from the checkvalve within pneumatic inlet port 410. An alignment fin 427 for aligning processing cassette212 within bucket 120 is shown at bottom.

[0140] Cover 402 further comprises septa 414 and 416 (see for instance FIG. 24), which areconfigured to interact with and receive fluid supplied by injectors of TSC 210 or allow forcollection of fluid by extractors of TSC 210. Specifically, the septa include harvest septum 414and dissolved O2 well septum 416. All syringes within TSC 210 are sanitized prior to use andonly used for one fluid transference operation each. In some embodiments, the syringes withinTSC 210 may be concurrently sanitized by radiating the entire TSC with the syringes disposedwithin TSC 210. Upward facing surfaces or septa may be aligned in a single plane to reduce acomplexity of sanitizing septa 414 and 416 prior to engaging septa 414 and 416 with a needleof an injector. Cover 402 includes a waste port 418 through which waste fluids can exitprocessing cassette 212 when pressurized gas (e.g. air, nitrogen, or carbon dioxide) isintroduced into processing cassette 212 using pneumatic inlet port 410 and all other exits arerestricted. Cover 402 further comprises an input tubing port 420 that facilitates the introductionof a blood sample into processing cassette 212 prior to, or after insertion of processing cassette212 into cell processing device 100. Finally, cover 402 may include emergency recovery tubing490 and a fluid level laser window 430 that detects the fluid level inside, preferably to anaccuracy of 1mm. The recovery tubing 490, shown again in FIG. 29, provides a backup meansfor recovering fluids or materials from the CPC during processing. The fluid level laser window430 allows fluid volume and weight sensing that allows for real-time monitoring of fluid levelsand weight, aiding in accurate volume management and quality control.

[0141] The following features describe this multiple port configuration in detail:

[0142] The CPC 212 includes multiple ports including the fluid port 408 and the pneumaticports 410 and 412, at the top section of the cover 402 for fluid and gas transfer. The internalopenings of these ports are positioned in a straight line along the uppermost region of theinterior of the CPC 212. This in-line alignment ensures that when the CPC 212 tilts or rotates,the fluid level can rise up the walls of the tapered structure of main chamber 424 to itsmaximum allowable angle without reaching any port opening, preventing unwanted fluid entryinto any of the ports.

[0143] To facilitate fluid injection, removal, and gas exchange, the CPC requires access pointsthat may not align with the in-line configuration on the inside of the CPC lid. To achieve theirpurpose, the external locations of the ports are located radially in precise distances from thegeometric center of the lid to be compatible and dockable with the automated TSC or PFC.Collectively the external ports are positioned to allow docking by either the TSC or the PFCby positioning either cassette directly over, and centered, on the CPC lid needing only a preciserotation and lowering to dock their exit ports with the appropriate outlet port of the CPC lid.

[0144] Between the external ports and their respective in-line internal openings, fluid pathwaysare constructed to direct each port to its designated in-line position inside the CPC. Thesepathways bridge the offset positions of the external ports with the uniform, in-lineconfiguration on the inside. This arrangement allows the system to access each port externallywithout disrupting the internal alignment that prevents fluid from contacting the port openingsduring cell processing movements.

[0145] This in-line internal port configuration provides several functional advantages. First,by preventing fluid from reaching the ports, the CPC can tilt to a wider range of angles,maximizing mixing and fluid dynamics without the risk of unwanted fluid entry. Second, itprovides sterile access for syringe mechanisms. The offset external ports enable syringe orother transfer mechanisms to access the CPC without compromising the safety of the internalin-line configuration. Third the fluid pathways ensure that only designated fluids or gases canenter the CPC through controlled pathways, maintaining aseptic conditions and preventingunintended mixing

[0146] FIG. 8 and 9 also show a vent assembly 425 located on an exterior surface of upperhousing component 404. Vent assembly 425 includes vents that may be configured to facilitateventing of air located within fraction chambers disposed within lower housing component 406.This prevents the addition of solution from main chamber 424 into the fraction chambers fromcausing unwanted pressure buildup within the fraction chambers. Alternatively, processingcassette 212 may comprise vents that empty out into an area between cap 402 and filter 426 or428 that prevent any contaminates traveling through the vents from entering into main chamber424.

[0147] FIGS and 9 show an alternative embodiment partial exploded view of processingcassette 212 with cover 402 removed from upper housing component 404 to reveal multiplefilters disposed in a top region of upper housing component 404. In particular, a hydrophilicfilter 422 is a largest one of the filters. Hydrophilic filter 422 is positioned beneath fluid port408 and configured to prevent any contamination present in the fluid introduced through fluidport 408 from getting into and contaminating a main chamber 424 of processing cassette 212located just beneath hydrophilic filter 422. The large area and horizontal orientation ofhydrophilic filter 422 allows fluid introduced into processing cassette 212 through fluid port408 to spread evenly across hydrophilic filter 422 in order to pass through it. In someembodiments, hydrophilic filter 422 may have a pore size of about 0.2 microns, which isoperable to clean impurities from buffer or culture media solution being introduced throughfluid port 408. Processing cassette 212 further comprises hydrophobic filters 426 and 428 thatallow for the passage of air and / or other gases into and out of processing cassette 212 usingpneumatic ports 410 and 412. While filters 422, 426 and 428 are shown positioned within upperhousing component 404 to represent their position when processing cassette 212 is closed up,these filters are secured to an interior facing surface of cover 402 to prevent escape ofcontaminates located between filters 422, 426 and 428 and the interior facing surface of cover402. Alternatively, filters 422, 426 and 428 could be attached and / or sealed to an exterior-facing surface of cover 402 in which case ports 408, 410 and 412 could be packaged withrespective filters 422, 426 and 428 to form exterior filter assemblies.

[0148] Cover 402 is also depicted including multiple indentations. In particular, fluid levellaser window 430 is positioned directly below a laser sensor positioned on an exterior facingsurface of cover 402 and configured to measure a volume of fluid within a main chamber 424of processing cassette 212. Indentation 432 is directly above a pH probe 434 that is configuredto monitor a pH of sample material disposed in said main chamber 424 of processing cassette212. Indentation 436 is directly above a dissolved oxygen sensor that is helpful in confirmingan amount of dissolved oxygen within the main chamber of processing cassette 212 ismaintained at a desired level during operation of cell processing device 100. Cover 402 furthercomprises a waste standpipe 438, which provides a path by which waste solution within CPC212 may be evacuated through waste port 418.

[0149] Alignment fins 427 are distributed around a periphery of upper housing component 404and interact with grooves in bucket 120 to radially align processing cassette 212 with bucket120. This ensures proper alignment of openings in a downward facing surface of processingcassette 212. Lower housing component 406 further comprises drains 429-1 and 429-2 thataccommodate removal of material from fraction chambers located within lower housingcomponent 406.

[0150] FIG. 10A and FIG. 10B show the three filters 422, 426 and 428 disposed in the topregion of upper housing component 404.

[0151] FIG. 11 shows a partial exploded view of an alternative embodiment of bucket 120. Inparticular, FIG. 11 shows a magnetic valve actuator. The magnetic valve actuator includes amotor 440 configured to turn a shaft having a distal end attached to a first gear 442. First gear442 is configured to engage teeth on second gear 444, which is oriented orthogonally relativeto first gear 442 forming what is sometimes referred to as a miter gear configuration. It shouldbe noted that straight, spiral, zerol, miter or hypoid gear configurations are also possible.Consequently, rotation of first gear 442 by motor 440 results in rotation of second gear 444.Second gear 444 includes multiple recesses for accommodating the placement of magnets 446.Magnets 446 are generally rare-earth magnets formed from materials such as Neodymium orSamarium Cobalt. After placing magnets 446 within recesses of second gear 444, rotation ofsecond gear 444 generates a rotating magnetic field capable of rotating a valve positioned at abottom end of CPC 212. Other means of rotating the valve may be employed.

[0152] FIG. 12 shows an exploded view of a harvest valve assembly that includes a first valvesegment 454 and a second valve segment 456. First valve segment 454 may be configured torotate about its longitudinal axis relative to second valve segment 456, which is configured toremain stationary. First valve segment 454 defines a first channel segment 458 configured toguide solution received in a tapered recess 460 arranged at a first end of valve segment 454into one of multiple channel segments defined by second valve segment 456. While only asecond channel segment 462 is depicted in FIG. 12 it should be appreciated that second valvesegment 456 can include multiple channel segments with at least one channel segment forguiding sample materials to each of multiple fraction chambers arranged around a lowerperiphery of processing cassette 212. First valve segment 454 further comprises a non-circularsecond end, opposite the first end that is configured to engage a central opening in a magnetcarrier 466. The central opening can have a shape and size matching the shape and size of non-circular second end 464 of first valve segment 454. While second end 464 is depicted as beingroughly square in shape, the important aspect is that the geometry allows the central openingof magnet carrier 466 to impart a force on second end 464 sufficient to rotate first valvesegment 454 relative to second valve segment 456.

[0153] Magnet carrier 466 is configured with peripheral recesses sized to accept multiplemagnets 468 and formed from a non-magnetic material. In this context non-magnetic materialrefers to materials such as plastic or ceramic and mostly magnetically neutral metals such asaluminum. Use of a non-magnetic material prevents interference with a magnetic couplingbetween magnets 468 and magnets 446. In this way, rotation of magnets 446 in second gear444 causes rotation of the magnets 468, which then imparts a force to first valve segment 454sufficient to align channel segment 458 with a channel segment defined by second valvesegment 456 when it is time to drain sample material from main chamber 424 of processingcassette 212. While magnets 468 are described as magnets it should be appreciated that in someembodiments, magnet carrier 466 could instead carry magnetically attractable elements formedfrom ferritic material that would still be capable of responding to a shifting magnetic fieldgenerated by rotation of the magnets carried by second gear 444. Magnet carrier 466 sits in acircular opening defined by a base of a valve segment carrier 470. Valve segment carrier 470is sized to also receive second valve segment 456 and includes vertical walls 472 that preventrotation of second valve segment 456. The harvest valve assembly further comprises a non-magnetic cover 474 that prevents scratching of an interior of bucket 120 during rotation ofmagnet carrier 466.

[0154] FIGS. 13-15 show exploded views of the centrifuge bucket 120 of the preferredembodiment. where in particular, bucket 120 could instead include a modified second gear 444that is coupled to a drive shaft 445 by a plurality of fasteners 450. Bearing elements 447-1 and447-2 are positioned within a through hole extending through a bottom wall of bucket 120 andassist with rotation of second gear 444 and drive shaft 445 within the through hole. Any liquidinadvertently escaping the processing cassette may be prevented from entering into the recesshousing motor 440 by environmental seal 449, which fits around a base of drive shaft 445.FIG. 13 also illustrates how a distal end of drive shaft 445 is non-circular. In particular, thedistal end of drive shaft 445 can have a D-shaped or crescent-shaped distal end.

[0155] As shown in FIGS. 16 and 17, the top of the CPC 212 is configured with multiple inputand output ports, each serving a critical role in maintaining a sterile and controlled environmentfor CAR T-cell manufacturing. These ports allow for the efficient, aseptic transfer of variousfluids, gases, and samples thereby supporting the entire cell processing cycle, from initial inputto final harvest. Each input port is equipped with sterile filters or needle septa which are UVdisinfected before each use to ensure aseptic entry or extraction and fluids to preventcontamination and to maintain the purity of the internal environment. This strategicarrangement of input and output ports, along with integrated filtration systems, enables theACP to conduct complex cell processing operations in a closed, sterile environment, supportinghigh-purity CAR T-cell production with minimized contamination risk. As shown in FIG. 17,there is a sealed stub of tubing 1114 from blood or leukapheresis bag.

[0156] The CPC 212 includes dedicated input ports for gases-in 1104, fluids-in 1118, reagents-in 1116. Gases in extend through hydrophobic filters 426 and 428, which allow the introductionof sterile gases as needed for maintaining cell culture conditions or controlling internal pressureduring certain processing stages. Fluids port 1118 enables the addition of various fluidsrequired for cell processing, such as washing solutions, culture media, or transduction agentsinto the CPC. Fluids in are through hydrophilic filters 422. Reagents-in 1116 provides accessfor the controlled introduction of specific reagents, including those needed for cell activationor genetic modification steps.

[0157] The CPC 212 includes a sealed stub of tubing 1114 from a blood or leukapheresis bag.The tube is shown again in coiled and uncoiled form in FIGS. 61A and 61B, respectively. Thepatient's cells enter through this port for processing. A clot filter in this tube prevents clotsfrom passing. The bag with the cell sample from the patient is tube-sealed to this port.

[0158] The CPC 212 also features output ports for harvesting CAR T-cells-out 1110, Sterileair-access port 1106, gases-out 1102 and fluids-out 1108 and samples-out 1112. The outputport 1110 is dedicated to the final extraction of the resulting CAR T-cell product followingcompletion of the cell processing steps. The sterile air-out 1102 facilitates controlled releaseof gases to maintain internal pressure and prevent unwanted accumulation of air within thecassette 212. Through gases-out 1102, air is released through a hydrophobic filter 426 ensuringany gas leaving the system is sterile. Gases-out 1102 and fluids-out 1108 allow for the saferemoval of gases and fluids from the CPC 212 during washing, transduction, and otherintermediate stages. CAR T-cells-out port 1110 is dedicated to the final extraction of the CART-cell product following completion of the cell processing steps. Samples-out 1112 provides apathway for collecting cell samples or aliquots at different stages, enabling quality controltesting or process monitoring without disturbing the internal environment.

[0159] The ports of the CPC 212 are equipped with specialized filters such as 0.2\mu mhydrophobic filters 426, 428 and 0.2~\mu m hydrophilic filter 422 to support sterility. The 0.2~\mu mhydrophobic filters 426, 428 are used at various gas ports to ensure sterile air entry or exitthereby preventing contamination from external particles. The 0.2\mu m hydrophilic filter 422positioned beneath the coiled tubing allows for sterile fluid flow and ensures aseptic conditionsare maintained across all fluid transfers.

[0160] The wider top section of the CPC accommodates multiple ports for fluid and gastransfer. These ports, collectively, allow for the aseptic introduction of fluids, such as cellsuspensions, reagents, or culture media, and the removal of waste fluids and retrieval offormulated volume of gene-modified cells. The inclusion of gas exchange ports also providesaseptic introduction and removal of gases within the CPC, maintaining sterile conditions andsupporting processes that may require different levels of oxygen or carbon dioxide.

[0161] The CPC's circular lid design enables precise docking alignment through use of anumber of alignment features 1100 to mate with other system components, such as the PFCand the TSC. The lid of the CPC features multiple liquid transfer ports (septa) designed toenable precise, aseptic fluid handling and improve overall cell processing efficiency

[0162] FIG. 18 shows a cross-sectional side view of an alternative embodiment of theinvention of CPC 212 disposed within bucket 120. In particular, FIG. 18 shows how in thisembodiment, fastener 450 couples second gear 444 directly to drive shaft 445 and howenvironmental seal 449 prevents the intrusion of any fluids in bucket 120 from passing intoand disrupting operation of motor 440. The distal end of drive shaft 445 is shown protrudinginto a recess defined by first valve segment 454.

[0163] FIG. 19 shows a plan view of CPC 212 according to a preferred embodiment of theinvention.

[0164] FIG. 20 shows a side view of CPC 212 as well as section lines D-D, E-E, and F-F,which are shown in more detail in FIGS. 26, 27 and 28, respectively;

[0165] Section line D-D is a view upwards to the filter configuration and sensor configurationarranged directly beneath cover 402. Section line E-E is a downward view into the mainchamber 424 of CPC cassette 212 and section line F-F cuts through fractional chambersdefined by a lower periphery of CPC 212.

[0166] FIG. 21 shows a cross-sectional side view of CPC 212. Main chamber 424 may beconfigured to hold about 350mL of fluids, however, it should be appreciated that the size ofmain chamber 424 may vary depending on how large a blood sample is being processed andwhat dilution level for the blood sample is desired. FIG. 21 shows waste port 418 including afirst check valve that allows for the passage of fluid out of main chamber 424 and preventsfluids from entering into main chamber 424. FIG. 21 also depicts how fluid port 408 includesa second check valve oriented in an opposite direction from the first check valve that allowsfor fluids to enter into fluid port 408 but prevents fluids from exiting fluid port 408.

[0167] The CPC 212 features a tapered, cone or elliptic cone shape that narrows progressivelyfrom a wider diameter at the top to a narrower diameter at the bottom, wherein the ratio ofdiameters is preferably between 4 and 5, in less preferred embodiments lower than 4. Thisdesign provides a range of functional advantages: The tapered, conical or elliptical interiorgeometry of the CPC plays a critical role in achieving precise cell concentration, sedimentation,and mixing.

[0168] During centrifugation or sedimentation processes, the tapered shape of main chamber424 naturally directs sediment such as target cells, including hematopoietic stem andprogenitor cells, T-cells, or NK cells—toward the narrow bottom section of the CPC 212,referred to as the sedimentation column 431 (see FIG. 18). The sedimentation column featuressubstantially vertical, cylindrical walls that enhance measurement accuracy. This geometryfocuses sediment (preferably cells) into a narrow area, improving sensitivity for opticaldetection. In one embodiment, the sedimentation column 431 is designed with flat verticalsurfaces wide enough to accommodate the light beam emitted from an optical emitter to asensor.

[0169] The optical detection system for cell monitoring in the CPC 212 utilizes a plurality ofoptical emitter / detector pairs positioned along the sedimentation column to measure variationsin light transmission caused by the presence, movement, and distribution of cells within thesuspension. The system supports light across visible and non-visible wavelengths (e.g., infraredor ultraviolet), with visible light currently in use. The system is adaptable to accommodate non-visible wavelengths, depending on specific requirements for improved sensitivity, reducedinterference, or the optical properties of the sample or reagents. The detection system operatesthrough a configurable sequencing scheme to optimize cell detection, sedimentationmonitoring, and real-time feedback during cell processing operations. The sequencingalgorithm includes the following features.

[0170] The plurality of optical emitter / sensor pairs is at least two, preferably three, and insome embodiments more than three pairs to detect light signals through the sedimentationcolumn of the main chamber of the CPC. Although most of the main chamber of CPC is funnel-like and with a curved wall, the sedimentation column walls are essentially vertical and paralleland include flat portions through which the emitter-sensor pairs operate, the flat portionsminimizing disrupting reflections that would be caused were those surfaces curved. The flatsection of the sedimentation column is on both the emitter and sensor side. Preferably, thewavelength is an adjustable white light to allow it to best detect all cells and particularly whitecells during various stages of system operation.

[0171] The intensity of each emitter is configurable to optimize sensitivity and detectionperformance. The system can operate at absolute intensities (comparing detected light intensityagainst predefined thresholds to identify cell presence or movement) or utilize relativeintensities (monitoring changes across the array to determine sedimentation dynamics, celllayering, and distribution), accounting for fluid dynamics, optical path lengths, and variationsin cell density. The system may also utilize changes in the color of the transmitted or receivedlight as an additional parameter to measure cell density or identify specific characteristics ofthe cell suspension. It is known that cells or particles in a suspension can scatter, absorb, orreflect light differently depending on their density, size, or type. The color (wavelength) of thelight that passes through or is detected by the optical emitter / receiver pairs may change basedon how the light interacts with the suspension. As cell density increases, the fluid's opacitychanges, but the spectrum (color) of transmitted or scattered light can also shift. For example,densely packed cells may absorb more light in specific wavelength ranges (e.g., red or blue),causing a measurable color shift in the detected signal. By analyzing both light intensity(opacity) and color variation, the system can enhance sensitivity to small changes in celldensity; and potentially differentiate between cell types (e.g., red blood cells, T-cells, or NKcells) based on unique optical absorption characteristics. This provides a more robust andnuanced detection mechanism for cell concentration or composition. In summary the opticalemitter could use multi-wavelength light sources (e.g., white light or specific RGB LEDs), thereceivers could detect intensity, and wavelength shifts to measure color variation caused bychanges in the suspension. The system's algorithm may incorporate these readings into its cellcount or concentration calculations.

[0172] The plurality of optical emitter-sensor pairs allows for real-time monitoring and controlof cell layering and density, which is essential for precise cell recovery and distribution. Forinstance, the data from the optical emitter-sensor pairs may be used to control centrifuge RPM,for instance the speed of the centrifuge could be correlated to the level of packing of the cells.Once the cells are injected, they are allowed to settle to an empirically determined level ofpacking. This controlled centrifugation ensures the cells are optimally packed to receive thevector or reagent being delivered from the reagent septa 1116 and liquid transfer port 1170.When the valve is configured to allow so, that allows for precise introduction of reagentsdirectly to the centrifugally concentrated cell population located at the bottom of thesedimentation column.

[0173] In one example, if the bottommost sensor detects that cells are being packed toodensely, this information could be sent to the platform which then reduces the RPM of thecentrifuge. The emitters may be activated in sequential or parallel configurations, dependingon system requirements. For sequential activation, emitters may be enabled and disabled in acontrolled, top-to-bottom manner along the vertical array of emitter / detector pairs. The delaybetween enabling sequential emitters is configurable, allowing precise adjustment to optimizedetection sensitivity, resolution, or the dynamic properties of the cell suspension. Aconfigurable delay may also be introduced before rerunning the sequence.

[0174] In use, eventually all the cells pass by the top emitter-sensor pair, and then after someamount of time based on particle density, fluid dynamics, and centrifugal forces, they will passthe middle emitter-sensor pair, and finally the bottom emitter-sensor pair. In practice, thesystem may slow or stop centrifugation after they pass the top pair of optical emitter-sensors,because once the cells have passed this level above the bottom of the sedimentation column,they are protected from being removed from the CPC during the liquid removal process, whichis preferably performed via the application of positive pressure. The system is scalable toaccommodate additional emitter / detector pairs, enabling flexibility for varying sizes ofcassettes or similar apparatus. The number of emitter / detector pairs may be proportional to thesize of the sedimentation column used for cell detection and sedimentation. The system mayalso include multiple arrays of emitter / detector pairs positioned at 90-degree offsets to allowfor detection from two orthogonal angles, improving measurement accuracy and reliability.

[0175] In embodiments where the entire main chamber 424 or sedimentation column are notconstructed of transparent or semi-transparent materials, at least one optically clear window inthe sedimentation column 431 allows the sensor-emitter pairs 1062 to operate therethrough.

[0176] Real-time total cell concentration calculations are achieved by combining opticalsensor data with the known cell suspension volume. The total volume of the suspension isidentified either by weight (via strain gauges) or vertical height measurements (using laser orradar systems). The sedimentation column's narrow geometry significantly improvesmeasurement sensitivity, facilitating detection of subtle changes in light transmission thatcorrelate with the presence of target cells, such as hematopoietic stem and progenitor cells, T-cells, or NK cells.

[0177] To mitigate the limitation that the optical sensors only detect sediment within thesedimentation column, the system employs a swift but gentle mixing process immediatelybefore measurement. This ensures cells are evenly distributed throughout the cell suspensionvolume. Once mixing is complete, the system tracks the elapsed time as the CPC rotates to avertical, motionless position. This timing factor is critical for accurate algorithmic calculationsof cell counts.

[0178] The system uses an empirically determined algorithm that may account for one or moreof the following to refine cell count estimations, ensuring high accuracy even as sedimentationbegins shortly after mixing ceases, the determination considering the elapsed time since mixingceased; the light signal occlusion measured at each of the three optical sensor pairs; andsedimentation rates, which are essentially uniform across the sedimentation column and thebulk cell suspension above it.

[0179] The tapered geometry of the main chamber 424, which narrows from a wider top to anarrower bottom, ensures controlled movement of cells in suspension during cassette tilting.This motion, guided by gravity and the internal shape, can create uniform laminar flow mixingoptimized for different solution volumes by adjusting rotation rates while minimizing sheerstress on cells. The tapered geometry, which narrows from a wider top to a narrower bottom,also ensures controlled movement of cells in suspension when the cassette rotates on its axis.This motion, guided by gravity and the internal shape, can create uniform laminar flow mixingoptimized for different solution volumes by adjusting rotation rates.

[0180] To optimize performance, the interior walls of the CPC are constructed using a low-adhesion material, such as a biocompatible polymer with a smooth, diamond-polished finish.This surface treatment minimizes the likelihood of cell adhesion, ensuring that cells remainsuspended in the fluid and can be effectively concentrated, sequestered, or removed as required.The low-adhesion material significantly reduces cell loss caused by sticking, thereby enhancingrecovery rates and improving overall process efficiency. By carefully selecting materials andfinishes that prevent adhesion, the CPC maximizes the yield of viable cells at every stage ofthe processing cycle, supporting consistent and high-quality results.

[0181] Effective and thorough mixing is essential for the cell manufacturing process. Theprovided geometry enables the CPC to perform essential steps such as cell washing, activation,gene modification, and harvesting within a closed, sterile, and automated environment. Duringthese processes, cells must remain evenly distributed to ensure reliable cell selection,activation, and transduction. Uniform mixing also guarantees that all cells have equalinteraction with reagents and vectors while maintaining a representative distribution for qualitycontrol sampling. Without this optimized mixing, cells would settle unevenly, leading toinconsistent recoveries, reduced activation efficiency, and lower transduction rates.

[0182] The natural tendency of cells to sediment and concentrate is overcome through thecombination of tapered or conical geometry, precision rotation, and optimized fluid motion.During centrifugation or sedimentation processes, the tapered shape naturally directssediment such as target cells, including hematopoietic stem and progenitor cells, T-cells, orNK cells-toward the narrow bottom section, referred to as the sedimentation column.

[0183] By ensuring swift and thorough mixing, the CPC's design prevents premature cellsettling and maximizes exposure to reagents and genetic materials, significantly improvingoverall processing outcomes.

[0184] FIG. 22 depicts an alternative embodiment of the invention wherein a relative locationof a worm gear-driven valve assembly is shown. The worm gear-driven valve actuator includesmotor 440 which is positioned upon and electrically coupled to a printed circuit board assembly476. While details of individual circuit components are not provided herein it should be notedthat one of ordinary skill in the art would appreciate how the components on printed circuitboard assembly 476 may be configured to provide power and control commands for operationof motor 440 when actuation of the valve assembly is required. Alternative embodiment FIG.22 also depicts second gear 444 positioned within a recess defined by a downward-facingsurface of bucket 120 that is shaped to receive second gear 444. First valve segment 454 isshown extending into an opening defined by lower housing component 406 of CPC 212.

[0185] Hydrophilic filter 422 is shown directly beneath fluid port 408 and is responsible forscreening out any impurities in fluid being introduced through fluid port 408. Septa 414 and416 have a tapered geometry to guide received syringe needles.

[0186] FIG. 23 shows a cross-sectional side view of CPC 212 taken along section line C-Cand in accordance with a preferred embodiment of the invention.

[0187] As shown in FIG. 24, center of gravity (CG) of the bucket 120 varies based on liquidlevel. The pivot axis 1092 mixes cell suspension in CPC 212. FIG. 24 shows the changes inthe CG at 350ml as indicated at 1094 and 25ml as indicated at 1096. The vertical, centralpositioning of the CG for the centrifuge bucket and the CPC 212 is critical for ensuring efficientoperation during both mixing and centrifugation processes. Mixing of the cell suspensionoccurs only when the centrifuge bucket is not spinning centrifugally around the rotor. Tominimize the torque required by the mixing motor, the pivot axis of the bucket / CPC assemblymust be positioned very close to its CG. This ensures efficient mixing whether the cellsuspension volume is as low as 15 mL or as high as 350 mL. By aligning the pivot axis nearthe CG, the system achieves smooth, controlled oscillation with minimal energy expenditure.When the bucket and CPC are rotating around the centrifuge rotor, the bucket rotates upward,approaching a 90^{\circ} tilt. This occurs because the geometric center of the mass aligns closely withthat of the counterbalance, ensuring near-perfect balance. This alignment provides the mostuniform sedimentation rates for the cells, which move consistently away from the axis ofrotation under centrifugal forces. If a perfect 90^{\circ} rotation of the bucket / CPC assembly isrequired during centrifugal operation, the mixing motor can make precise adjustments due tothe minimal torque demands in this state. Such fine control ensures optimized conditions forcell sedimentation and subsequent processing.

[0188] In one embodiment, The system uses a Relative Centrifugal Force (RCF) compensationmechanism designed to regulate the sedimentation rates of cells within the cell suspension asthey enter the sedimentation column during centrifugation. If the sedimentation rate deviatesfrom the empirically determined optimal rate-either being too fast or too slow-the RCF isdynamically adjusted to achieve ideal packing of cells at the bottom of the sedimentationcolumn. This mechanism is particularly critical during spinoculation, where cells are co-mingled with vectors, to ensure efficient and uniform interaction between cells and vectors.

[0189] As shown in FIG. 25, for small volumes such as 100ml the bucket 120 can rotate asmuch as 135 degrees in either direction for maximum mixing without liquid entering the fluidpassage ports of the plurality of filters. The degree of possible tilt is thus inversely proportionalto the amount of liquid in the main chamber. For instance, with 115ml volume it may be ableto tilt to about 120 degrees in either direction, or with 160 ml it may only be able to rotate to110 degrees in either direction. The maximum capacity of liquid the CPC 212 can hold is 350mland the least capacity is 25 ml. The motor can rotate the bucket 120 to within one tenth of adegree of rotational accuracy. Thus, it is clear that the gravitational force is not the only causefor the rotation of the bucket 120. The pivot point of the bucket 120 is designed to maximizebucket 120 clearance during rotation.

[0190] FIG. 26 shows a view along cutline D-D of FIG. 20, looking upwards from inside theCPC to the underside of the filters 422, 426 and 428 of CPC 212. Filters 426 and 428 arepreferably hydrophobic filters that allow sterile gas entry and exit, with structural integrityconfirmed through automated pressure / decay testing, as well as hydrophilic filter 422positioned under coiled tubing on the CPC lid, this filter allows sterile fluid transfer into theCPC, also verified by pressure / decay testing. Preferably three 0.2-micron pore size filters areintegrated into the top of the CPC lid. These filters facilitate the aseptic transfer of fluids intothe CPC and the controlled exchange of gases (entry and exit) while maintaining a sterileenvironment. The filters ensure that all fluid and gas pathways remain uncontaminated duringcell processing.

[0191] FIG. 27 shows a view along cutline E-E of FIG. 20, looking down into the mainchamber 424 and the waste standpipe 438, which provides a path by which waste solutionwithin CPC may be evacuated through waste port 418. Also shown is a plurality of ribsdesigned to improve thermal transmission from the heater in the bucket 120 to the fluid in theCPC 212.

[0192] FIG. 28 shows a view along cutline F-F of FIG. 20, showing the four rotationalpositions of the valves. The valve rotates to four positions, each creating a different fluidconnection for a different process. Positioning between these four positions create no fluidconnections and here the CPC valve is said to be parked or closed. When the valve is at position1070, a fluid connection is open allowing the CPC to receive reagents from a reagent septum,position 1072 to create a fluid connection for the sequestration of non-target cells, location1074 creating a fluid connection with a compartment for the sequestration of 2^{nd} fraction T-cells or non-target cells preferably into a 30mL volume limited compartment, and location1076 which creates a fluid connection out of the CPC chamber to a harvest septum andultimately to a harvest vessel. When the valve is configured to allow so, reagent septa 1116connects to valve position 1070, a liquid transfer port, with a conduit tube that allows forprecise introduction of reagents directly to the centrifugally concentrated cell populationlocated at the bottom of the sedimentation column 431. This targeted delivery maximizesreagent efficiency and enhances cell processing outcomes.

[0193] The top of the CPC in the ACP is equipped with multiple input and output ports, eachplaying a critical role in maintaining a sterile and controlled environment for CAR T-cellmanufacturing. These ports enable the efficient, aseptic transfer of fluids, gases, and samplesthrough the lid, supporting the entire cell processing workflow—from the initial introductionof cells to the final harvest of gene-modified cells. Each input / output septa port is integratedwith a UV mechanism that disinfects opposing septa during docking. There is a septum foreach syringe located on the bottom of the TSC which will be one of the two opposing septa tobe disinfected by the UV disinfection module during any transfer of fluids. The opposing septathat will be simultaneously disinfected will be on either the RSC containers or the lid of theCPC. Specifically, the UV mechanism ensures that both the TSC septa and the CPC lid septa,or the RSC septa, are disinfected during reagent retrieval or sample deposition. Also,Hydrophobic filters allow sterile gas exchange, while hydrophilic filters manage the aseptictransfer of fluids. Both filter types undergo pressure / decay testing to confirm their structuralintegrity before and after processing clinical populations of gene-modified cells.

[0194] The aseptic transfer ports on the CPC lid preferably include both septa ports and filterports. Input and output septa are positioned on the CPC lid to align precisely with the TSCsyringes, enabling aseptic transfer of reagents, samples, and harvested cells.

[0195] This strategic combination of port design, UV disinfection, and filtration systemsensures aseptic entry and exit of gases and fluids, effectively preventing contamination andpreserving the purity of the CPC's internal environment. By maintaining these sterileconditions, the ACP enables the precise and automated execution of complex cell processingtasks, ensuring high-purity CAR T-cell production with minimal risk of contamination.

[0196] The CPC 212 is designed with dedicated input and output ports, ensuring sterile andefficient handling of gases, fluids, reagents, and samples throughout the cell processing cycle.Beginning first with the input ports, these may be categorized into four groupings. (1) Gases-in: Hydrophobic filters on the CPC lid allow sterile gas entry without contaminating the interiorenvironment. These filters also enable aseptic release of displaced air when fluids areintroduced into the CPC, maintaining cell culture conditions. (2) Fluids-in: Fluids required forcell processing, such as washing solutions, culture media, or transduction agents, pass throughhydrophilic filters. The filters have a larger surface area to accommodate the higher fluid flowrates necessary for efficient processing. (3) Reagents-in: A dedicated input port providescontrolled access for introducing specific reagents, such as those for cell selection, activation,or genetic modification. (4) Sealed Tubing with Clot Filter: Tubing connected to blood orleukapheresis bags enables sterile docking and entry of the patient's cells. The tubing includesa clot filter to remove unwanted clots before cells enter the CPC. After transfer, the tubing issealed one inch from the CPC lid entrance and secured in a nearby plastic clip to ensure safetyand avoid obstruction.

[0197] Turning now to the output ports, these may also be categorized into four groupings. (1)Gases out: A hydrophobic filter ensures that gas leaving the CPC is sterile and free ofcontaminants. (2) Harvested Gene-Modified Cells-out: dedicated septum allows for the asepticfinal harvest of the gene-modified cell product after processing steps are complete. (3) WasteFluids-out: This port, equipped with a one-way valve, safely expels waste fluids after cells arecentrifugally sequestered at the bottom of the sedimentation column. Optical sensors confirmcell positioning before waste removal, and positive air pressure propels the waste fluid into anexpandable, sealed container at a controlled rate, preventing the unintended removal ofsequestered cells. (4) Samples-out: A septum port allows the TSC to aseptically collect cellsuspension samples at various stages of the process for quality control or monitoring, withoutdisrupting the sterile internal environment.

[0198] FIG. 30 show the top perspective view of the CPC 212. The septums 414 and 416, areconfigured to interact with and receive fluid supplied by injectors of TSC 210 or allow forcollection of fluid by extractors of TSC 210. Specifically, the septums include harvest septum414 and dissolved O2 well septum 416.

[0199] FIG. 31 also shows side view lines A-A, and section lines B-B and C-C, which definethe views provided in FIGS. 32, 33 and 34, respectively. FIGS 33-34 are cross sectional viewswhile FIG. 32 is a side view with section lines D1-D1, E1-E1 and F1-F1, which are depictedas FIGS. 35, 36, and 37, respectively.

[0200] FIG. 38 shows the top of the CPC 212. The figure also shows view line A-A and sectionlines B-B, C-C, and G-G, which define the views provided in FIGS. 20, 21, and 59B-59D.View A-A is shown in FIG. 20, Section line B-B extends through fluid port 408, waste port418 and septum 414 and is shown as FIG. 21.

[0201] The pneumatic system within the ACP leverages precise gas pressure control to executemultiple critical functions throughout the cell processing workflow. First, it conducts pressuredecay tests to verify the integrity of hydrophobic and hydrophilic filters within the CPC 212,ensuring the sterility and reliability of fluid and gas transfers. Second, it utilizes pressurizedgas to implode microbubbles, facilitating cell release during certain processing steps. Third,the system applies pressure to the top of the PFC, enabling fluid transfer through a pie-shapedfilter exit receptacle into the CPC chamber. During this process, radar or laser sensors monitorthe fluid volume entering the chamber to ensure accuracy. Fourth, the pneumatic systempressurizes waste liquid lines to expel waste fluids out of the CPC while simultaneouslypreventing contamination. Once the liquid is removed, gas is applied to clear any residual liquidfrom the lines. Lastly, the pneumatic system propels genetically modified cells into the harvestsyringe, ensuring precise and aseptic transfer of the final cell product for collection. Theseversatile capabilities enhance the ACP's efficiency and reliability in delivering high-qualitycell therapies.

[0202] FIG. 39 is a cross-sectional view showing CPC 212 undergoing a centrifugationoperation in which target cell solution 1022 is pressed toward the bottom of main chamber 424and then first valve segment 454 is rotated to align first channel segment 458 with secondchannel segment 482, as depicted.

[0203] A port enables the aseptic passage of the gene-modified and formulated target cellsolution. The thin tube connects to a TSC syringe, which completes the transfer of the cellsolution to the RSC final harvest container with minimal to no loss of cells. This allows targetcell solution 1022 to be collected in target cell primary fraction chamber 480. Once a desiredamount of target cell solution 1022 is added to target cell primary fraction chamber 480additional target cell solution 1022 is added to target cell secondary fraction chamber 488 (seeFIG. 22) by rotating first valve segment 454 such that first channel segment 458 aligns with achannel segment associated with target cell secondary fraction chamber 488.

[0204] The centrifugation operation may be terminated once the primary and secondaryfraction chambers 480, 488 have received target cell solution 1022. Prior to removal ofprocessing cassette 212 from cell processing device 100 and retrieval of the final therapeuticdose or doses, cell processing device 100 may report on any irregularities noted during theprocess as evidenced by the sensor readings monitored throughout the cell processingoperation. Filters 422, 426 and 428 of processing cassette 212 may also be checked for integrityand any irregularities with the filters can also be reported. The pre-processing and post-processing filter integrity may be verified using a pressure decay method to individually checkeach filter. Pre-processing and post-processing filter integrity checks may be performed withincell processing device 100 by a testing cassette positioned within cassette positioning assembly132. The testing cassette may be configured to apply a known gas pressure to the inlet port ofeach filter (422, 426 or 428) while blocking all the exit ports. Once pressurized, a sterile ventingport (i.e. 412 or equivalent) is opened allowing gas flow only through the filter being tested.Measuring the pressure decay time in the inlet port volume against known limits determinesthe integrity of the filter. Filter integrity testing results are incorporated in the lot release report.

[0205] FIGS. 40A – 40C show various views of PFC 204. FIG. 40A shows a perspective viewof PFC 204 and how it has an overall cylindrical geometry. An upper surface of PFC 204includes multiple needleless entry ports 502 for adding solution into PFC 204. Addition ofsolution into PFC 204 is typically performed prior to placing PFC 204 into cell processingdevice 100. While four needleless entry ports 502 are depicted and correspond to four pieshaped chambers within PFC 204, it should be appreciated that PFC 204 could be divided upinto a larger or smaller number of pie-shaped chambers. PFC 204 can alternatively includeonly a single chamber holding a single type of fluid in which case cassette positioning assembly132 could include multiple fluid cassettes with one fluid cassette for each type of liquid needed.Each chamber of PFC 204 further comprises a corresponding pneumatic port 504 configuredto receive a fixed amount of compressed air for ejecting precise amounts of fluid from acorresponding chamber of PFC 204.

[0206] Pneumatic ports 504 may each include a filter for preventing the unintentional additionof contaminates to PFC 204 when pressurizing a particular chamber of PFC 204. The PFC lidpreferably contains four filters, one for each of its four pie-shaped compartments. Eachcompartment can hold up to 500 mL of buffer or other fluids. The 0.2-micron filters allowaseptic passage of air under controlled pressure (1 to 5 psi), which regulates the flow rate offluids through the docking port and the hydrophilic filter on the CPC lid.

[0207] In some embodiments, an amount of fluid ejected by PFC 204 into processing cassette212 can also be controlled based on feedback from the radar or laser level sensor. FIG. 40Aalso shows an upper end of a waste pass through 506 that extends along a longitudinal axis ofPFC 204 that allows waste sample material exiting waste port 418 of processing cassette 212to continue upward and into waste tube 208 and into a flask for collection. An upper end ofpneumatic pass through 508 is also shown. Pneumatic pass through 508 allows for air to beadded to or removed from processing cassette 212 by applying positive or negative pressure topneumatic pass through 508 while pneumatic pass through 508 is coupled to a pneumatic portof processing cassette 212. Any applied air pressure in each pie-shaped compartment iscarefully managed to ensure precise fluid transfer into the CPC, supporting efficient and sterileprocessing.

[0208] FIG. 40B shows a top view of PFC 204 showing how pneumatic ports 504 arepositioned along a periphery of PFC 204 and needleless entry ports 502 are arranged in a centralregion of PFC 204. Waste pass through 506 is located at the center of PFC 204 and pneumaticpass through 508 is shown outboard of needleless entry ports 502 and inboard of pneumaticports 504. FIG. 40B also shows section line F-F, which corresponds to FIG. 41.

[0209] FIG. 40C shows a bottom view of PFC 204 that includes another end of waste passthrough 506. The opposing ends of waste pass through 506 are generally connected by aflexible or rigid tube that extends along a longitudinal axis of PFC 204. FIG. 40C also showsanother end of pneumatic pass through 508, which is configured to engage pneumatic ports410 and 412 on processing cassette 212. Fluid exit ports 510 are also shown and are configuredto engage fluid port 408 on processing cassette 212.

[0210] FIG. 41 shows a cross-sectional side view of PFC 204 in accordance with section lineF-F from FIG. 40B. In particular, FIG. 41 shows two filled fluid chambers of PFC 204 as wellas internal components of needleless entry ports 502, pneumatic ports 504, waste pass through506 and fluid exit ports 510. In particular, waste pass through 506 and fluid exit ports 510include check valves to prevent contamination of the fluid stored within PFC 204 when PFC204 is not in use. For example, the check valve of waste pass through 506 is configured tocompress the spring when pressurized sample material exits processing cassette 212 and intowaste pass through 506.

[0211] FIGS. 42A – 42B show different views of RSC 202. FIG. 42A shows a perspectiveview of RSC 202 and FIG. 42B shows a top view of RSC 202. A total of six reagent vials 602and six prelabeled sample vials 604 are depicted in FIGS. 42A – 42B, however, it should beappreciated that a smaller or larger number of vials is possible and considered to be within thescope of the invention. The RSC 202 houses up to six pre-filled reagent vials that will be usedthrough the cell processing, preferably each with a volume capacity of up to 20 mL. These vialsmay contain essential components such as microbubbles, vectors, and linkers. Additionally,the RSC 202 contains designated sample vials, ready to receive cell samples for quality controlanalysis throughout the processing cycle. Sample vials 604 are positioned on stands 606 so thatupper surfaces of sample vials 604 are flush with upper surfaces of reagent vials 602. Each ofthe vials may be slid into place on RSC 202 by sliding the respective vial into clips 608, whichin combination with collars 610, prevents lateral and vertical movement of the vials whileinstalled on RSC 202. Each of vials 602 and 604 include a cap with a region 612 configured tobe pierced by a needle that allows for insertion or extraction of fluids from the vial. The systemmay include an automated quality control in the form of integrated sensors monitoring syringevolume and flow rates to detect any deviations, triggering alerts and adjustments as necessary.

[0212] The ACP features a UV disinfection module specifically designed to disinfect port septaprior to each fluid transfer, thus each needle transfer occurs under aseptic conditions, as theDisinfection Module (DM) uses intense UV radiation or light to disinfect the surfaces of boththe TSC septa and the opposing RSC or CPC septa. The UV light is focused on two opposingsepta preceding each needle penetration of either septa. A needle will emerge from a bottomsepta of the TSC and down into either a septa of the CPC lid or a septa of the RSC only afterboth septa have been disinfected. The UV disinfection module is mobile and will always beprecisely located to assure aseptic transfers involving the TSC and the RSC or CPC. No needlewill ever be extended outside of the TSC except when it is docked with the UV component andmechanically operated and under automated control by the system software. Preferably, asyringe will penetrate through a septum on the CPC lid and a septum on the sample vial 604,or a septum on the CPC lid and a septum on the reagent container. Septa may preferably befound on top of every sample vial 604 or reagent vial 602, the lid of the CPC, and the TSCitself has septa. Key components include:

[0213] UV-C Source: The module utilizes a UV-C wavelength light or radiation source (254-280 nm), which is optimal for microbial inactivation, effectively eliminating bacteria, viruses,and other contaminants on the septa surfaces. The UV light source is positioned to provide fullexposure to the septa.

[0214] Reflective Interior Coating: The UV sterilization chamber utilizes UV reflectivematerials that directs the UV-C light, ensuring complete and uniform exposure to the septafrom multiple angles. This design minimizes the risk of any shadowed or missed areas on thesepta, achieving comprehensive disinfection before the syringe needle pierces any septa.

[0215] Automated Detection and Activation System: The module includes an optical sensorthat detects the approach of the syringe toward the entry port. Once detected, the control systemautomatically initiates a disinfection cycle, activating the UV radiation source for a preciseduration. This automated activation eliminates the need for manual operation, ensuring thateach septa is sanitized just before use, reducing the possibility of contaminants settling on thesepta post-disinfection.

[0216] Timing and Control System: The UV module is programmed to deliver a controlled,timed exposure (typically between 5 to 15 seconds) to achieve effective disinfection withoutdegrading the septa material. Integrated with the ACP's central control system, the timingmechanism is calibrated to match processing needs and UV exposure requirements, allowingfor customized cycles depending on usage needs and material specifications.

[0217] In use, the UV sterilization module follows a streamlined process for disinfecting septaas part of the automated fluid handling sequence: Syringe Detection: The optical sensor detectsthe approach of a syringe as it nears the CPC entry port, triggering the disinfection cycle. UVExposure: The UV radiation source is activated to disinfect the surfaces of the opposed septaprior to the penetration of both septa by the TSC syringe needle and continues until the transferhas taken place and the TSC needle has withdrawn above its septa. The reflective interiormaterial ensures even UV exposure, effectively inactivating microbial contaminants on allexposed surfaces within the UV disinfection module. Completion and Shut-Off: Following thedisinfection cycle, the UV radiation source is deactivated.

[0218] The UV disinfection module is validated to achieve a preferably 6-log reduction inmicrobial load on the septa surfaces, ensuring aseptic conditions during fluid transfers. Thesterile integrity of the TSC syringe needles, originally achieved through gamma radiation ofthe packaged cassette prior to use with the ACP, remains uncompromised during the UVsterilization process, meeting all industry standards for aseptic processing environments. Themodule delivers approximately 750~mJ / cm^{2} of UV energy to achieve this high sterilizationefficacy, utilizing 170 mW of continuous direct current over 4.4 seconds. While direct currentis currently standard, future designs may incorporate pulsed electromagnetic energy to enhanceefficiency further.

[0219] Sample vials 604 are generally empty at the beginning of an operation being performedby cell processing device 100 and are gradually filled with samples taken from processingcassette 212 over a course of the operation being performed. This allows lab techniciansoperating the apparatus to check the samples over the course of the operation to validate thatthe operation is proceeding as expected. Reagent vials 602 generally contain fluids at the startof the operation, which are extracted by TSC 210 and transferred to processing cassette 212over the course of the operation. Exemplary fluids contained within reagent vials 602 includemicrobubbles, antibody linkers and disease vectors. In one embodiment each of reagent vials602 carries up to 20mL of fluid and each of sample vials 604 carries up to 5ml of fluid.

[0220] FIGS. 43A – 43C show various views of TSC 210. FIG. 43A shows a perspective viewof TSC 210 and how it can include multiple reagent syringes 702 arranged in a first circularconfiguration and multiple sampling syringes 704 arranged in a second circular configurationdisposed within the first circular configuration. A number of reagent and sampling syringes702 and 704 on PFC 204 will generally correspond to a number of reagent and sample vials602 and 604 contained on RSC 202. While only a plunger portion of each of syringes 702 and704 are visible in FIGS. 43A – 43B, it should be appreciated that each of syringes 702 and 704further comprises at least a needle and a solution receptacle.

[0221] FIGS. 43B – 43C show respective top and bottom views of TSC 210, a key componentof the TSC, designed to manage precise fluid transfers between other platform cassettes,preferably at least the CPC and RSC. The TSC houses multiple independently controlledsyringes that transfer fluids, such as reagents, buffers, and cell suspensions, with a high degreeof accuracy, maintaining sterility and consistency throughout various stages of cell processing.By integrating independent syringe control, pressure regulation, and a rotational dockingmechanism, the TSC provides a reliable and effective means of managing fluids in closed-loopcell processing, ensuring consistency, safety, and scalability in cell therapy manufacturing andother biological applications.

[0222] FIG. 43B shows how in some embodiments the syringe cassette may include a wastepass through 706 for removing waste sampling materials from CPC 212. In the event wastepass through 706 was applied to TSC 210, it would also generally include a pneumatic passthrough for supplying positive pressure to TSC 210 sufficient to drive waste sample materialthrough waste pass through 706. FIG. 43C shows how each of syringes 702 and 704 can havea corresponding port 708 through which their needle extends through septa to add or subtractfluid from a main chamber of processing cassette 212 or from vials on RSC 202. A first subsetof ports 708 are arranged along a periphery of PFC 204 correspond to reagent syringes 702 anda second subset of ports 708 arranged within a central portion of PFC 204 correspond tosampling syringes 704.

[0223] The TSC 210 manages the precise transfer of reagents and fluids into the CPC 212thereby enabling accurate volumetric dosing for processes such as washing, activation, andtransduction.

[0224] The TSC 210 is a key component of the ACP, designed to manage precise fluid transfersbetween other platform cassettes, such as the CPC 212, and RSC 202. The TSC 210 housesmultiple independently controlled syringes that transfer fluids, such as reagents, buffers, andcell suspensions, with a high degree of accuracy thereby maintaining sterility and consistencythroughout various stages of cell processing.

[0225] The structure of the TSC 210 may comprise a TSC housing, syringes, rotationaldocking mechanism, fluid transfer channels and ports, pressure control system and feedbacksensors. The cassette housing is a structurally reinforced, closed housing containing a series ofsyringes arranged in parallel, each within a designated compartment. The housing providessterile containment and is designed to prevent cross-contamination between fluids handled bydifferent syringes. Each syringe within the TSC 210 is configured to hold specific fluidvolumes, enabling independent control of fluid types such as culture media, buffer solutions,or processed cell suspensions. The syringes are equipped with adjustable plungers forvolumetric accuracy.

[0226] The rotational docking interface enables alignment with other platform cassettes. Thismechanism allows the TSC 210 to pivot between docking positions for each cassette,specifically the CPC 212 and RSC 202, ensuring precise fluid transfer across different stagesof cell processing. TSC 210 is integrated with fluid transfer pathways and docking ports thatalign with corresponding ports on the CPC 212 and RSC 202. These channels are sterile andsealed with automatic check valves that open only when the TSC 210 is docked with a specificcassette thereby preventing contamination and ensuring fluid containment. Each syringe isequipped with a pressure control system that enables fluid dispensation at a range of pressuresfrom approximately 1.5 to 45 PSI. This control is critical for managing various fluid types andviscosities, ensuring precise flow rates during dispensing into the CPC 212 or collection fromthe CPC 212 into the RSC 202. The TSC 210 includes feedback sensors positioned to monitorfluid levels within each syringe, providing real-time feedback on dispensed volumes and fluidlevels. These sensors communicate with the ACP's central control system to adjust fluidvolumes and pressures dynamically.

[0227] The TSC 210 aligns with the CPC 212 during stages requiring fluid transfer into or outof the cell processing environment. For instance, during washing and activation stages, theTSC 210 dispenses reagents, buffers, and activation solutions into the CPC 212 at precisevolumes and pressures to achieve optimal cell washing, isolation, or activation. During cellmodification stages, during gene insertion or modification stages, the TSC 210 transfers viralvectors or genetic reagents to the CPC 212, ensuring even distribution through controlledsyringe actuation.

[0228] The TSC 210 can also dock with the PFC 204 to draw specific fluids required for cellprocessing. The PFC 204 serves as a reservoir for various buffers, media, and reagents, whichthe TSC 210 transfers to the CPC 212 as needed. The TSC 210 syringes draw precise amountsof fluids from the PFC 204 to inject into the CPC 212 for specific processing steps, includingcell separation, incubation, or buffer exchanges.

[0229] The TSC interacts with the RSC. Regents may be obtained by the TSC 210 from theRSC and then transferred to the CPC212 via at least one syringe, wherein each large volumesyringe in the TSC 210 selectively draws fluid from the RSC and injects it into the CPC 212and each small syringe in the TSC 210 draws cell suspension samples from the CPC and injectsthem into a sample vial in the RSC 202 septa. After completing the cell processing steps, byaligning the TSC 210 with the CPC 212, the TSC 210 selectively retrieves gene modified andformulated cell suspensions from the CPC and dispenses them into the harvest container of theRSC for final storage or analysis. The TSC 210 transfers the final cell product, such as activatedor genetically modified cells, from the CPC 212 to the RSC 202, ensuring sterile containmentand minimal cell loss during transfer By aligning the TSC with the CPC, wherein the TSCselectively retrieves gene modified cell suspensions from the CPC and dispenses them into theRSC.

[0230] FIG. 44A shows a cross-sectional side view of TSC 210 interacting with RSC 202. Inparticular, TSC 210 is shown engaging RSC 202 such that syringe 702 is aligned with vial 602.Syringe 702 is held in position at the top end of TSC 210 by spring 802. A UV sanitizer 800 ispositioned between RSC 202 and TSC 210, which is configured to sanitize exterior surfaces ofthe septa of the TSC 210 and RSC 202 in order to prevent contamination of a needle of syringe702. While UV sanitizer 800 is not shown attached to another device, it should be appreciatedthat UV sanitizer 800 may be positioned between RSC 202 and TSC 210 by a swing armcapable of precise positioning of UV sanitizer 800. Since the top surfaces of vials 602 and 604are flush with one another, the swing arm could maintain UV sanitizer 800 at a constant heightand would only be responsible for maneuvering UV sanitizer 800 in x and y directions. Theclose-up view of UV sanitizer 800 shows an internal structure of UV sanitizer 800 and how itincludes a channel allowing needle 804 to extend through UV sanitizer 800 to engage andwithdraw fluid from vial 602. UV sanitizer 800 works by emitting UV light in two opposingdirections. UV sanitizer emits light upward to sanitize a surface of TSC 210 through whichneedle 804 extends and emits light downward to sanitize a surface of vial 602 through whichneedle 804 extends. This is important as handling of these cassettes prior to placement in cellprocessing device 100 can result in contaminates adhering to an exterior of the septa of variouscassettes. Consequently, this sanitization procedure greatly reduces the likelihood of needle804 being contaminated while withdrawing fluid from vial 602.

[0231] FIG. 44B depicts a configuration where after UV sanitizer 800 cleans the exterior ofTSC 210 and top of vial 602, the needle extends through UV sanitizer 800, plunges into fluidcontained within vial 602 and withdraws at least a portion of the fluid contained within vial602. Syringe 702 may be pressed against spring 802 in order to engage vial 602 as depicted.Movement of syringe 702 downward may be accomplished by an actuator of cell processingdevice 100 positioned directly above TSC 210 that compresses syringe 702 against spring 802by asserting a force on an upward facing surface of a plunger 806 of syringe 702. In someembodiments, TSC 210 can include a locking mechanism that prevents syringe 702 fromtravelling back upward once syringe 702 reaches a fluid withdrawal position within tube 808,as shown in FIG. 44B. Once syringe 704 is locked into the fluid withdrawal position that sameactuator responsible for pushing syringe downward can assert an upward force on plunger 806of syringe 702. The upward force may be applied to plunger 806 by the actuator using a suctioncup, a vacuum head or a mechanical grip of the actuator until solution receptacle 810 is filledto a desired level. Once the desired level is reached the actuator may be withdrawn from TSC210 and the locking mechanism holding syringe 702 may be released. In some embodiments,the locking mechanism can include a dampening element that prevents syringe from beingabruptly slammed upward into a starting position and instead allows syringe 702 to be gentlyretracted into tube 808 of TSC 210. Alternatively, each spring may be tuned so that its forceoutput only results in a gradual upward movement of a respective syringe. Operation of analternative actuator configuration is described in the text accompanying FIGS. 45A – 45K.

[0232] FIGS. 44C – 44D show how sampling syringe 704 may be used to withdraw a samplefrom processing cassette 212 and transfer the retrieved sample into a sample vial 604 in RSC202. FIGS. 44C – 44D also demonstrate how UV sanitizer 800-1 is used when retrievingsample material from processing cassette 212 and how UV sanitizer 800-2 is used whendepositing the retrieved sample material into sample vial 604. It should be noted that centrifugewell 114 may be equipped with its own swing arm for positioning UV sanitizer 800-1 betweenprocessing cassette 212 and TSC 210. While cell processing device 100 is described asincluding multiple UV sanitizers 800, in some embodiments cell processing device 100includes only a single UV sanitizer 800 that is moved between centrifuge well 114 and vialcassette recess 126 by cassette positioning assembly 132.

[0233] FIGS. 45A – 45K illustrate operation of an actuator 850 configured to manipulatereagent syringes 702 disposed within TSC 210. Actuator 850 may be positioned at an upperend of recess 134 of cassette positioning assembly 132 as shown in FIG. 3. FIG. 45A showshow actuator 850 includes a first gripping mechanism 852 and a second gripping mechanism854. Second gripping mechanism 854 is carried within first gripping mechanism 852 andcapable of moving independently relative to first gripping mechanism 852. First and secondgripping mechanisms 852 and 854 each include fingers that are shown in an open state.

[0234] FIG. 45B shows how second gripping mechanism 854 of actuator 850 movesdownward from its previous position in FIG. 45A and its fingers move into a closed state tosecure second gripping mechanism 854 to a first lip at an upward end of a plunger 806 ofreagent syringe 702. FIG. 45C shows first gripping mechanism 852 of actuator 850 havingmoved downward and fingers of first gripping mechanism 852 having moved to a closed stateto grip a lip at an upward end of a solution receptacle 810 of reagent syringe 702.

[0235] FIG. 45D shows movement of actuator 850 downward to push a needle 804 of reagentsyringe 702 out of a bottom end of TSC 210. While neither RSC 202 nor processing cassette212 are shown receiving the needle of reagent syringe 702, these are omitted only to focus onthe movement of actuator 850 and reagent syringe 702 relative to TSC 210. FIG. 45E showsmovement of second gripping mechanism 854 upward while first gripping mechanism 852remains stationary in order to move plunger 806 of reagent syringe 702 upward to draw fluidinto solution receptacle 810 of reagent syringe 702.

[0236] FIG. 45F shows movement of actuator 850 upward to draw needle 804 and the solutionreagent syringe 702 back into TSC 210 to allow for movement of TSC 210 by cassettepositioning assembly 132. FIG. 45G shows how actuator 850 is configured to push reagentsyringe 702 back downward to engage needle 804 into another cassette. Once needle 804 isengaged within the other cassette, FIG. 45H shows second gripping mechanism 854 havingmoved downward to press plunger 806 downward to expel fluids into the other cassette. FIG.451 shows actuator 850 after moving upward to retract needle 804 into TSC 210. FIGS. 45J45K show actuator 850 disengaging first and second gripping mechanisms 852 and 854 fromreagent syringe 702. First and second gripping mechanism 852 and 854 can disengage fromreagent syringe 702 either concurrently or sequentially. It should be noted that actuator 850, asdepicted, is sized to work with reagent syringes but it should be appreciated that cell processingdevice 100 can also include a second similarly configured actuator scaled down in size forcompatibility with sampling syringes 704.

[0237] The present invention includes a method for analyzing cell suspensions during the cellprocessing workflow to provide accurate cell counts, population distributions, and purityassessments. This analysis is achieved by integrating a Cell Analytics Module (CAM) within thecell processing system, capable of precise optical and biochemical cell characterization.

[0238] Analytical Sampling Process: The process begins with the TSC drawing a precisevolume of cell suspension after the cells have been mixed to ensure uniformity. The precisevolume of the sample (e.g., 5 µL, 10 µL, or a predetermined amount) is controlledprogrammatically by the system or chosen in advance by the operator. As described previously,the TSC is equipped with rotational capabilities, allowing it to align with various functionalmodules within the system, including the CAM. Once the TSC has drawn the sample, it rotates toalign with the CAM, which may be integrated as a standalone analytical device or incorporatedinto one of the slots of the Reagent / Sample Cassette (RSC). The sample is then expelled througha microfluidic channel within the CAM, enabling precise analysis.

[0239] Cell Analytics Module (CAM): The CAM is a dedicated analytical unit designed toprocess cell suspension samples for real-time evaluation of cell characteristics. It comprisesthe following components:

[0240] Microfluidic Channel for Single-Cell Detection: The CAM includes a microfluidicchannel with a diameter precisely engineered to allow single-cell passage. This ensures thatcells flow individually through the channel, allowing high-resolution analysis. As each cell passesthrough the channel, an optical or impedance-based detection system measures keyparameters, including cell diameter, which can be used to estimate the volume of individualcells. The CAM records the rate of cell passage, enabling the system to calculate theconcentration of cells within the sample. By multiplying this concentration with the knownsample volume, the total cell count in the suspension can be accurately determined.

[0241] Optical and Biochemical Detection of Surface Markers: The CAM integrates advancedoptical systems and reagents for detecting specific cell surface markers. The system usesbiochemical reagents capable of binding to target markers, such as CD3 (specific to T-cells) andCD14 (specific to monocytes). The detection system evaluates marker expression and providesa quantitative breakdown of cell populations. For instance: A T-cell is identified as being positivefor CD3 and negative for CD14; A monocyte is identified as being positive for CD14 and negativefor CD3.

[0242] Quantification of Cell Populations and Purity Analysis: Based on the detection ofspecific markers, the CAM quantifies the distinct populations of cells present in the sample. Itcalculates the number of T-cells and monocytes and determines the purity of T-cells by dividingtheir count by the total cell count in the sample.

[0243] Integration with System Control: The CAM is fully integrated with the system's centralcontrol unit. This integration allows real-time feedback of the analytical results, which can beused to optimize downstream processes, such as gene modification, washing, or formulation.The analytical data ensures that cell suspensions meet quality thresholds before proceeding tosubsequent steps.

[0244] Portability and Design: The CAM is designed to fit seamlessly into the modulararchitecture of the cell processing system. It may be integrated within the RSC or included as aseparate module accessible via the TSC. The modular design allows for easy replacement andmaintenance while ensuring sterility and compliance with regulatory standards.

[0245] Advantages of the CAM Integration: The incorporation of the CAM into the cellprocessing system significantly enhances the accuracy and efficiency of cell characterization.The microfluidic design ensures single-cell resolution for concentration measurement, while theuse of optical and biochemical detection methods enables precise differentiation of cellpopulations. By providing real-time analytical results, the CAM streamlines workflow andimproves the reliability of therapeutic cell products, ensuring consistent and high-qualityoutcomes.

[0246] FIG. 46 shows a cross-sectional side view illustrating how PFC 204 can depositsolution into a main chamber 424 of processing cassette 212. In particular, it should be notedthat UV sanitizer is not used in this step since processing cassette 212 has a high-quality filtercapable of filtering out any contaminates that made their way on to fluid exit port 510 of PFC204 and / or fluid port 408 of processing cassette 212. FIG. 46 also depicts a nozzle 902 attachedto pneumatic port 504 that is responsible for supplying pressurized gas into a liquid chamber904 of PFC 204 to cause solution disposed within liquid chamber 904 to exit out of fluid exitport 510 and enter processing cassette 212 through fluid port 408. In some embodiments, anamount of pressure supplied by nozzle 902 is tuned so that the filter disposed beneath fluid port408 is able to keep up with a rate at which fluid is received.

[0247] The system features a robust communication and power transfer design to ensurereliable operation within the centrifuge's dynamic environment, where noise and motion cancreate challenges. Suitable communication methods may include CAN-bus systems, roboticflex cables, or Ethernet connections. As illustrated in FIGS. 47 and 48, the system utilizesrobotic flex and CAN-bus cabling 443 positioned near the centrifuge bucket's hinge, providingsufficient flexibility to allow the bucket to pivot up to 135^{\circ} in each direction. The power cablesupplies energy to critical components, such as the motor and heater, while also transmittingreal-time data such as the temperature of the aluminum components-back to the controlsystem. This cabling is specifically designed to maintain effective data transmission even inelectromagnetically noisy environments.

[0248] In one alternative embodiment, robotic flex and CAN-bus cabling (see FIG. 30) isstrategically positioned near the hinge of the centrifuge bucket to facilitate movement. Thissetup enables the bucket to tilt in either direction, with a preferred pivot range of up to 135^{\circ}but capable of extending to 165^{\circ} or 180^{\circ} in some configurations, and full 360^{c} rotation incertain use cases. The power cable provides energy to the motor and heater while the CAN-busdelivers real-time feedback, such as the aluminum temperature, process sensor values, valveposition and other operating parameters to the control system. The CAN-bus architectureensures robust and noise-resistant communication, making it particularly suited for thedynamic and electromagnetically noisy environment of the centrifuge.

[0249] FIGS. 49A-49D illustrate an automated balancing system (auto balancer), utilized inthe ACP. Turning to FIG. 49A and FIG. 49B, a cross-sectional view of a centrifuge whereinthe bucket is in its mixing mode is shown. FIG. 49C is a cross-sectional view of a centrifugewhen spinning, and FIG. 49D is a cross-sectional view of a centrifuge when stationary.

[0250] The automated balancing system enhances the centrifuge to remain in a balanced stateas fluid levels change within the CPC 212. The automated balancing system achieves dynamicbalancing by employing certain linear actuators, each with a moveable mass that allowsadjustable balancing of variable-mass samples within the centrifuge, centrifuge bucket andCPC during operation. The use of additional actuators in alternative embodiments may providefine-tuning capability, which is particularly effective at higher RPMs where torquerequirements are greater. The CPC 212 includes a locking mechanism or tab to achieve stabilityand to ensure that the CPC is properly seated and remains stable throughout the process. Eachactuator incorporates a locking mechanism to prevent unwanted movement of the automatedbalancing system when under load. This mechanism permits movement towards the center ofrotation of the centrifuge but restricts it in the opposite direction when engaged therebystabilizing the system during high-speed operation.

[0251] A 3-axis accelerometer, precisely aligned with the rotation axis of the centrifuge,measures vibrations and out-of-balance conditions in real time. If vibrations exceedprogrammed limits, the system automatically shuts off the electrical drive to the centrifugemotor and engages inductive braking to halt rotation. This accelerometer is part of a feedbackloop that actively compensates for imbalances, with multiple accelerometers on differentexcitation axes providing enhanced signal accuracy. The accelerometer synchronizes with arotation position encoder, enabling precise, rotation-referenced data collection at any speed.Fluid transfers into, out of, or within the system may occur when the centrifuge is stationary orwhen operating at various relative centrifugal forces, ensuring precision and minimizingturbulence. The balancing system employs a counterweight mechanism, where each full motorrotation corresponds to a 2mm adjustment in the counterweight's position. By correlatingaccelerometer data with positional information from the encoder, the system achieves precisereal-time indexing of vibration data with the motor's rotational position, allowing for dynamicbalancing, improved stability, and enhanced motion tracking along the X, Y, and Z axes.Additionally, the system can transmit accelerometer data to any rotor, providing increasedcompatibility in certain embodiments.

[0252] One use case example of the automated balancing system is as follows: The processbegins once the centrifuge bucket positions the CPC in an upright orientation. The PFCdescends, docks with the CPC, and performs the required fluid transfer operations, includingdispensing fresh fluids into the CPC, or receiving waste fluids from the CPC for transfer to thewaste container. After fluid transfer is complete, the system determines the volume or weightof the cell suspension inside the CPC using integrated sensors. This measurement providescritical data to enable accurate balancing of the system. The auto balancer utilizes the data fromthe sensors to reposition the moveable mass along a threaded rod. The linear actuator adjuststhe mass location precisely to minimize vibration during centrifugation. This balancing stepensures stable operation by counteracting any imbalance caused by variations in fluid volumeor weight within the CPC. Only after the automatic balancing procedure is completed does thesystem allow centrifugation to resume. The precise balancing prevents excessive vibration,reduces mechanical stress, and ensures consistent sedimentation rates and fluid dynamicsduring centrifugation.

[0253] The balancing system is seamlessly integrated with the centrifuge bucket to enableprecise rotational control and an active swing function for in-place mixing when the rotor isstationary. This system employs a motion-inducing mechanism that imparts reciprocalmovement to the container along a predefined path, ensuring effective mixing of the contents.The motion actuator supports non-linear movement patterns designed to promote uniformmixing. Its velocity profile follows a sinusoidal trajectory, decelerating at the endpoints of therocking motion to minimize turbulence and ensure gentle handling of sensitive materials. Theactuator also features adjustable speed settings, enabling optimized mixing at specific intervalsto achieve thorough homogenization. Furthermore, precise motor position control allows forrotational mixing, which can create a swirling effect to enhance mixing efficiency in certainapplications. This versatile system supports a variety of mixing techniques, including rocking,tilting, and rotation, ensuring optimal content uniformity.

[0254] As shown in FIG. 16, in order to ensure precise temperature control and monitoring,temperature sensor 150 is integrated within the cartridge 212 and positioned near the liquidcontents. The cell processing device 100 may be configured to add or subtract heat to keepsolution 1022 preferably approximately at a temperature of about 37 C, however, in differentembodiments temperatures other than approximately 37 C may be desired.

[0255] The cell processing device 100 has an improved temperature control for efficientlyheating the biological materials without excessive energy waste. In some embodiments, bucket120 may include a heating element capable of increasing a temperature of the materialcontained within processing cassette 212. In some embodiments this design incorporates a thin,anodized aluminum heating element to heat only specific areas, supported by low-conductivitymaterials like heat stabilized cast nylon and air gaps to prevent unnecessary heat loss.

[0256] As shown in FIG. 50, the heating configuration features a primary heating elementwhich is a thin, anodized aluminum cylinder 1050 approximately 2 millimeters thick,positioned close to the CPC 212 CPC where the biological materials are most concentrated,thus ensuring targeted heating without unnecessary energy expenditure. The aluminum is blackanodized to improve its thermal emissivity which enhances heat transfer towards the cartridgecontents. This heating element only covers a specific portion of the CPC 212, particularly theareas where the biological liquids are mostly concentrated, thus ensuring targeted heatingwithout unnecessary energy expenditure. The heating element may be surrounded by a lowconductivity insulative barrier made of heat-stabilized cast nylon or like materials and air gaps.The insulating properties of cast-nylon prevent heat from dispersing into the surroundingcomponents, thus maintaining a focused heating zone. The insulative cast-nylon layer 1054 isabout 2 millimeters thicks and circumferentially encapsulates the aluminum heating elementto restrict heat flow outward. An aluminum tube with the heating element is wrapped by theinsulative barrier. The heating element is adhered to the tube with pressure sensitive adhesive(PSA) and around that goes a mylar shrink belt. This mechanical belt is utilized to preventdelamination of the heater during centrifugation. In order to prevent heat dissipation, a blackmaterial is employed, and certain thermal path links are shut off utilizing plastic and an air gap.In this configuration, the cartridge design, incorporates a plurality of ribs which are nototherwise for structural purposes. The ribs facilitate heat conduction. This preferredconfiguration of the heating element retains the temperature inside even after the heat is turnedoff. The 2 mm thick, black-anodized aluminum warming cylinder 1050 and a film heatingelement 1052 maintain the temperature of cell solutions which are critical for cell viability andoptimized processing conditions. A printed circuit board (PCB) 1060 affixed to the distal endof one of the sensor / emitter housings connects via spring pins on temperature sensor 150 ofCPC 212 to monitor the temperature to ensure consistent thermal conditions. An additional airgap 1058 of approximately 6 mm is placed between the insulative layer 1054 and the outerbucket structure 1056. This air gap acts as a buffer to trap heat within the target region, furtherpreventing thermal transfer to non-essential areas.

[0257] The purpose of the heating mechanism is to efficiently control the temperature of thebiological material contained within the processing CPC while minimizing energy waste andpreventing excessive heating of non-target areas. It is crucial for maintaining stable conditionsat around 37^{\circ}C, optimal for cell viability and processing, without causing thermal stress ordenaturation of temperature-sensitive biological materials, and while minimizing heatdispersion to the exterior of the centrifuge bucket and the centrifuge itself. Further benefits arereduction of power consumption and the prolonging of component life.

[0258] As shown in FIG. 50, bucket 120 includes three optical sensor / emitter pairs 1062 totrack cell sedimentation and to confirm correct valve rotation 1064. The bucket 120 is pivotallyconnected to the axis of rotation 124 for mixing cell suspensions in the CPC 212. The bucketfurther includes a valve rotation drive assembly 1066. Thus, the bucket 120 of the cellprocessing device 100 includes the black-anodized aluminum warming cylinder 1050 of 2 mmthickness for emissivity, the film heating element 1052 having approximately 1 / 2 mm thicknessand air gaps.

[0259] In some alternative embodiments, cell processing device 100 can also include arefrigeration / heating unit capable of supplying chilled / heated air into centrifuge well 114. Insome embodiments, the heat is conducted into the cassette via a conduction apparatus such asa thin black anodized aluminum tube around which is wrapped a heating element. To inhibitor minimize heat from transferring out to the remainder of the centrifuge bucket, a ceramicshell and air gap may be used. In some embodiments, the bucket is multilayered with materialsof varying heat conductivity and insulation to specifically control the heat transfer.

[0260] As shown in FIG. 51A, T-cells link to microbubbles 1122 during bucketmixing. FIG. 51B shows the bucket spun up to 250 x g with the buoyant T-cells separate fromsedimenting non-target cells. In the setup, the microbubbles facilitate a unique compressionand release cycle for cell selection and concentration. The main steps include:

[0261] Centripetal Force during Spinning: As the centrifuge spins, centripetal force pushesnon-target cells down towards the funnel's narrower end and ultimately to the sedimentationcolumn 431. Here, the microbubbles maintain close proximity to each other and to the targetcells thereby keeping the target cells from Sedimenting.

[0262] Buoyant Rebound and Separation: Cell buoyancy works against this downwardforce, causing cells to move upward in the funnel. As they rise, they encounter a wideningcross-section of the funnel, which gives them more space to disperse. This separation stepreduces the density of cell clusters and allows for a more even distribution of cells within thefluid.

[0263] This balance of forces-compression by microbeads against the funnel walls andbuoyant separation as cells rise allows the cells to be tightly packed and then gently separatedthereby optimizing selection and distribution across the funnel. This design leverages bothmechanical and physical properties to enhance precision in cell processing and isolation.

[0264] FIGS. 28 and 53A, show four primary rotational positions for CPC valves thatcorrespond to different fluid transfer points within the cassette. The valve rotates to fourpositions such as reagent septa 1070, non-target cells 1072, 2^{nd} fraction T-cells 1074 andharvest septa 1076. Reagent Septa position allows reagents to enter the cassette, preferably forcell processing steps like washing, activation, or transduction. This position allows reagents toenter the bottom of sedimentation column of the cassette where the cells may, or may not, havebeen transferred by centrifugation of the CPC, likely for cell processing steps like washing,activation, or transduction. When in the Non-Target Cells location, the valve is positioned toseparate and dispose of or transfer out non-target cells, ensuring that only the desired cellpopulation remains in the cassette. Here, it sequesters non-target cells, ensuring that only thedesired cell population remains in the main tapered or conical cell processing chamber of theCPC for subsequent processing steps. Harvest Septa position is used for harvesting the finalcell product, allowing the selected and processed cells (e.g., CAR-T cells) to be extracted fromthe cassette. Second Fraction T-Cells position is designed for the isolation of a second fractionof T-cells, possibly for sequential processing or further refinement of the cell population.

[0265] As shown in FIG. 53B, In addition to these four primary positions, the diagram alsoindicates four other rotary positions, located in between the four locations listed above, eachof which will prevent fluid transfer through the valve. These may act as "closed" positionswhere the valve rotation aligns with segments that block any fluid movement, ensuringcontainment and preventing unintended mixing or contamination.

[0266] The rotary valve positioning system can use up to three separate mechanisms toexplicitly verify the valve is positioned correctly. These are a position sensor, an optical pass-through sensor, and an alignment sensor, as described below.

[0267] A position sensor on the valve drive shaft: The position sensor is used in conjunctionwith the servo motor controller to move the valve to a commanded angular position. Althoughin a preferred embodiment, any position sensor may be used, in one embodiment and opticalencoder is used and reports the drive shaft position and the existing backlash / slop between thedrive shaft and the valve rotor which may introduce uncertainty in the valve position. Theencoder has an index position signal which defines 0^{\circ} rotation which is calibrated duringmanufacturing using a fixture. For increased robustness, this is not the only position feedbackof actual valve position used. In one embodiment the position sensor may have a 1:1 matchwith valve drive gear position.

[0268] An optical pass-through sensor. As shown in FIG. 52, the alignment sensor is usedfor final valve position alignment, and includes a narrow slot in the valve rotor, which permitspassage of an optical signal from an optical sender 1071a on one side to an optical detector1071b on the other side (For the patent the position left-to-right of these sensors is likely notimportant, however, if it is important the numbers are reversed left-to-right in Figure 52 fromthe actual position in the Trenchant bucket.). This emitter-detector pair provides line of sightconfirmation through apertures in the valve stator, allowing the optical signal to pass throughonly when the valve is in the correct position of interest alignment between valve rotor andstator. In some instances, the correct alignment position is to open or close a port. In a preferredembodiment, the optical encoder directs valve rotation within + / -1.0^{\circ} as depicted by FIG. 52.The alignment sensor signal depends solely on the relative positioning of the rotor and statorand is independent of backlash in drive components or variations in encoder or other positionsensors. Furthermore, additional sensors may include but are not limited to Hall effect sensors,additional optical encoders, inductive sensors, magnetic or optical sensors.

[0269] The system employs zone sensors to indicate which of the four (or more) port locationsis active during valve operation. While the implementation preferably includes four Hall effectsensors, the system is not limited to this sensor type; alternative position-sensing technologies,such as optical encoders, inductive sensors, magnetic sensor, or capacitive sensors may also beutilized depending on application requirements.

[0270] In the preferred configuration, the zone sensors interact with a single magnet locatedon the drive shaft gear to determine the general position of the valve relative to the ports. Eachzone sensor corresponds to an active zone range, which is centered around an ideal portposition. The active zone range may be between \pm1^{\circ} to \pm20^{\circ}. At any given time, only one zonesensor is active, signaling the valve's general alignment with one of the ports. When the valveis between ports, all zone sensors are inactive, ensuring the system recognizes that no port iscurrently engaged. This transitional state helps prevent misalignment or erroneous reporting.The zone sensors serve a critical role in valve positioning by confirming the valve is in thegeneral location of the desired port. This confirmation enables the system to subsequentlyemploy the optical alignment sensor for final, precise positioning. The optical sensor ensuresthe valve achieves the exact alignment needed, in some cases this alignment to open or closethe selected port, leveraging its ability to detect alignment peaks with high accuracy.

[0271] By combining the general positioning capabilities of the zone sensors with the precisealignment detection of the optical pass-through sensor, the system achieves robust and reliablevalve positioning. This redundant sensing approach allows for seamless operation even underconditions where minor mechanical backlash or tolerances might otherwise introduceuncertainty.

[0272] A simplified usage model for the rotary valve system is as follows. Startingposition assumes the valve is in a “parked / closed", no port open, position.

[0273] Initial Positioning to Nominal Port Location: A servo controller rotates thevalve approximately 45^{\circ} to the nominal angular position corresponding to Port A. Thismovement relies on pre-calibrated positional data. Next, the operation and accuracy of theencoder or other position sensors is verified by checking the zone sensors (e.g., Hall effectsensors or alternative technologies). At this stage, only the zone sensor for Port A is active,confirming the valve is in the correct general range. If no zone sensor is active, or more thanone is active, the system will report a potential fault condition to the supervisory systemcontroller for further diagnostics.

[0274] Precise Positioning Using Optical Alignment: The valve is rotated from thenominal position while monitoring the analog alignment sensor signal generated by the opticalpass-through sensor. The system algorithm identifies the peak alignment signal (analog signalmaximum), indicating precise alignment of the valve with Port A. Valve rotation is stopped atthis peak position. Optionally, a final positioning algorithm may be executed to ensuremaximum precision, accounting for mechanical backlash, tolerances, or drift.

[0275] Validation and Fault Detection: At this point, the system performs a positionalcross-check to confirm alignment. Here, encoder position should indicate the correct angularposition for Port A. A zone sensor confirms the general location. An optical alignment sensorvalidates precise alignment with the open port.

[0276] If all three detection mechanisms (encoder, zone sensor, and optical alignmentsensor) agree, the valve is confirmed to be in the desired port-open position. If a mismatch isdetected between any of these positional indicators, the system reports a fault condition to thesupervisory system controller. The fault signal can trigger appropriate diagnostics, errorhandling, or corrective action.

[0277] FIG. 54 shows the CPC 212 in the active mode. LED white light frequencies detect thewhite blood cells. FIGS. 55A-55J show the CPC valve rotating through a series of positionsto facilitate the step-by-step processing of cells, reagents, and other components involved inCAR T-cell production. The valve rotation is mostly at 45^{\circ} increments, with one 135^{\circ} rotationin either direction. This allows for both fine and coarse adjustments to position the valveprecisely for each task. Each position correlates with a specific action or step in themanufacturing workflow, allowing the device to isolate or combine materials as needed foroptimal processing.

[0278] This rotation sequence demonstrates the CPC's versatility and automation, highlightinghow the device can systematically carry out complex cell processing steps. By rotating thevalve to control access to different compartments, the system enables a closed, automated, andhighly efficient process for CAR T-cell manufacturing, where each step is fine-tuned tomaintain cell quality, safety, and efficacy. There are four parked positions (1 3 5 and 7 areparked positioned) and there are four positions that allow cells to move.

[0279] Step 1: Filling, Buffer Addition, and Washing – Position 1, shown in FIG. 55A. Thevalve position allows for the introduction of buffer solution into the cassette, preparing the cellsby washing them in preparation for further processing.

[0280] Step 2: Adding Linkers – Position 8, shown in FIG. 55B. This step enables theaddition of linkers, which may be required for cell binding with microbubbles or othercomponents.

[0281] Step 3: Mixing, Removing, and Adding Buffer – Position 1, shown in FIG. 55C. Acombination step that allows mixing within the cassette, removal of spent or excess buffer, andaddition of fresh buffer to maintain optimal conditions for cell health and activity. Buffer actsas a carrier to remove impurities and non-target components.

[0282] Step 4: Adding microbubbles (MB) – Position 8, shown in FIG. 55D. Microbubblesare added to assist in cell selection or separation. The Microbubbles may be manipulated tohelp concentrate, isolate or sequester specific cell populations.

[0283] Step 5: Mixing, Removing Buffer, Pressurizing, and Centrifuging – Position 1,shown in FIG. 55E. The valve position facilitates further mixing, buffer removal,pressurization, and centrifugation. This step likely aids in cell separation, concentration, orpreparation for gene transfer.

[0284] Step 6: Sequestering Non-Target Cells – Position 2, shown in FIG. 55F. This stepisolates and removes non-target cells, ensuring that only the desired cell population (e.g., T-cells) remains for subsequent processing.

[0285] Step 7: Adding Culture Media, Mixing, and Temperature Control – Position 1,shown in FIG. 55G. The valve introduces culture media to support cell growth, performsmixing, and regulates temperature (warming to 37^{\circ}C and cooling to 32^{\circ}C) to create favorableconditions for cell viability and activation.

[0286] Step 8: Adding Vector – Position 8, shown in FIG. 55H. A gene transfer vector, eitherviral or non-viral, is introduced to deliver the CAR gene into the T-cells, a critical step in theCAR T-cell therapy process, which enables the cells to target and eliminate cancer cells. Thevector is introduced at the bottom of the cell suspension, allowing it to naturally rise throughthe small volume of T-cells. As both the vector and cells are in suspension, they begin to miximmediately upon contact, facilitating efficient gene transfer and uniform distributionthroughout the cell population.

[0287] Step 9: Spinoculation, Washing, and Formulation – Position 3, shown in FIG. 551.Spinoculation is used to enhance the gene transfer efficiency, followed by washing andformulating the cells for final harvest. Spinoculation helps bring the vector into close contactwith cells, improving transduction rates.

[0288] Position 10: Harvest – Position 4, shown in FIG. 55J. The final step, where the CART-cells are collected and prepared for therapeutic use.

[0289] Key insights into the rotation system include controlled sequential processing,customizable angle and rotation control and closed-system integrity. In controlled sequentialprocessing, the rotation positions enable a precise, sequential workflow thereby minimizingcross-contamination and optimizing the conditions for each process step. Further, incustomizable angle and rotation control, the valve rotation is mostly at 45^{c} increments, withone 135^{\circ} rotation. This allows for both fine and coarse adjustments to position the valveprecisely for each task. In Closed-System Integrity, the system design ensures sterility andcontainment. Also, this design is critical for aseptic cell processing, particularly when workingwith sensitive therapeutic cells.

[0290] The ACP incorporates a rigorous pressure decay filter integrity testing (FIT) protocolto ensure the structural integrity and proper functionality of all filters within the CPC. As shownin FIGS. 56 and 57A-B, the FIT process is specifically used to validate aseptic transfercapabilities through filters, such as the 0.2~\mu m hydrophilic input filter located in the CPC lid.This testing is performed both before and after processing to confirm filter performance.Separately, waste removal and fresh media transfer are achieved via the PFC, which rotates,descends, and docks with the CPC lid. Fresh media enters the CPC through the input port,while waste exits through the waste outlet. The locations for FIT pressure decay tests areindicated as 1080, 1082, and 1084, while the pathways for waste removal and media input areshown as 1088 and 1090, respectively, in FIG. 57A. Additionally, FIG. 57B highlights acarbon dioxide-enriched container that facilitates the movement of fresh media into the CPCand waste media out at approximately 1.5 PSI. For filter integrity tests or degassingmicrobubbles, pressures of approximately 45.0 PSI are applied.

[0291] This process guarantees that fluid and gas exchanges with the CPC's interior occurexclusively through the filters, eliminating bypasses that could compromise sterility. The FITprocedure plays a pivotal role in maintaining an aseptic environment throughout the CAR T-cell manufacturing workflow, minimizing contamination risks and safeguarding the productionof high-quality therapeutic products. Filter integrity is verified both prior to introducing patientcells and after completing the processing cycle, ensuring that no degradation or structuralfailures have compromised the sterility of the CPC

[0292] The automated pressure decay FIT process is independently conducted for hydrophobicand hydrophilic filters, which are critical components of the ACP's quality control system.Each filter is tested sequentially due to variations in surface area and decay rate thresholds,ensuring precise and individualized testing parameters. This protocol enhances sterility andreliability, ultimately improving the safety and efficacy of the CAR T-cell product.

[0293] Testing Process, Generally - The pressure decay test measures the pressure drop (\Delta P)in an upstream volume connected to the filter over a predefined time interval. The processensures confirms that the filter's flow rates are within permissible limits, ruling out obstructionor leakage. Hydrophobic and hydrophilic filters are tested separately. Due to differences insurface area, each filter requires unique pressure decay parameters and lookup values todetermine acceptable decay times. The FIT protocol identifies potential issues, including lowflow failures (indicating possible blockages or contamination in the filter or associated ports orpassageways) and high flow failures (suggesting cracks, ruptures, seal failure, or leaks in thefilter housing, CPC structure, or testing assembly). By testing each filter sequentially andemploying precise decay thresholds tailored to the specific filter area, the ACP ensurescomprehensive quality control. This robust testing protocol not only minimizes contaminationrisks but also reinforces the platform's ability to produce safe and effective CAR T-celltherapies.

[0294] Testing Process, Exemplary – (1) Pre-Process FIT: Before introducing patient cells,gas flows through sterile vents and the system is pressurized. (2) Decay Measurement: Thepressure decay test calculates the drop in pressure over time using the formula:\Delta P=DR\times T\times PaVup\Delta P=VupDR\times T\times Pa, where: AP is the pressure drop, DR is the diffusion rate,T is the time, Pa is atmospheric pressure, and Vup is the upstream volume.

[0295] Step-by-Step Testing Procedure

[0296] Close CPC Valve to prevent flow

[0297] Block Outlet Filter Port

[0298] Close Upstream Volume (UV) Valve

[0299] Close FIT Valve

[0300] Mate FIT Cassette to Hydrophobic Inlet Filter Port

[0301] Open UV Valve to Pressurize Upstream Volume to a starting pressure

[0302] Start Decay Timer & Open FIT Valve

[0303] Wait Decay Time and Record AP-decay Drop

[0304] Close PC FIT vent Valve

[0305] Close FIT Valve

[0306] PASS if AP-decay within min / max test limits or < Decay limit

[0307] Release all CPC pressure to complete test

[0308] Pass / Fail Determination: A Pass result is obtained if the pressure drops falls withinan acceptable range. Failure results from either excessive or insufficient pressure decay,indicating potential filter issues.

[0309] Calculation for a representative test is shown below. For a starting upstream pressureof 45 psi, with these conditions, a 24.5 psi pressure drop would result in a final upstreampressure of 20.5 psi. A failing low flow filter would result in a higher final upstream pressureand a failing high flow filter would result in a lower final upstream pressure.DR=10,000~mL / minute; Test Duration = 1 minute; Vup=100~mL\Delta P=(10,000~mL / minute*1~minute*14.7~psi) / 100~mL=24.5~psi

[0310] Post-Process FIT: After processing, a repeat FIT ensures that the filters remainedintact throughout cell processing and did not suffer any performance degradation. The resultsof this post-process test are automatically logged in the batch record data, facilitatingcompliance with quality control and regulatory requirements.

[0311] In addition to running the filter integrity tests, the air pressure control system plays acritical role in managing and maintaining precise fluid flow rates within the system during keyoperations. It ensures controlled fluid dispensing into the CPC, removal of waste fluids, transferof formulated gene-modified target cells, and specific manipulations during the selection andactivation process, such as microbubble implosion. By regulating air pressure across multipleoperations, the system ensures accurate and efficient fluid transfers and removals. The systemoperates by selecting air pressure in at least three distinct modes to optimize fluid handling andsystem integrity.

[0312] A first fixed pressure may be used for microbubble implosion and filter integrityTesting. A fixed air pressure is applied to collapse the microbubbles attached to target cellsduring the selection and activation stage. This removes the buoyancy imparted to the targetcells, allowing them to settle efficiently for subsequent processing. The same fixed pressure(or a different pressure) may be used to test the structural integrity of both hydrophobic andhydrophilic filters on the CPC lid. This ensures the structural integrity of the filters that theyare leak-free and able to maintain sterility throughout the process.

[0313] A second fixed pressure is associated with waste fluid removal. Positive air pressure isapplied to propel waste fluids from the CPC through the central fluid waste tube and into anexpandable, sealed waste container. This controlled pressure ensures efficient fluid removalwithout disturbing sequestered target cells at the bottom of the sedimentation chamber.

[0314] A third fixed pressure setting is utilized for fluid transfer from the PFC to the CPC.When the PFC is docked with the CPC, air pressure is applied to the fluid compartments withinthe PFC to control the transfer of fluids. This pressure ensures a regulated flow rate throughthe hydrophilic filter located on the CPC lid, enabling accurate delivery of fluids into the CPCfor processes such as washing, reagent addition, or volume adjustments. It is further understoodthat these distinct fixed pressures could alternatively be supplied by a single electricallycontrolled regulator, which programmatically adjusts the pressure to meet the specificrequirements of each process.

[0315] FIGS. 58A – 58D show the cell suspension 1122 when the CPC 212 is being tilted.The As described above, the CPC may be tilted to facilitate mixing. The CPC is preferablyreciprocally oscillated between 1^{\circ} and 360^{\circ}, and in some instances may be rotated an unlimitednumber of times. In some embodiments where ports or filters are found along the top lid of theCPC, the degree of tilt in either direction is inversely correlated to the level of fluid in the CPC.For instance, a 90^{\circ} of tilt in either direction is typical for some a certain fluid level, but withreduced fluid, tilting beyond to for instance 135^{\circ} may be possible, while in this embodiment agreater amount of fluid may only allow for some amount of tilting less than 90^{\circ} in eitherdirection.

[0316] FIG. 59A a shows cutline H-H through the CPC 212 that is used for the cross-sectionimages shown in FIGS 59B – 59D. In FIG. 59B, the cross section along H-H shows a cellsuspension fluid 1122 in the main chamber 424. FIG. 59C shows some of the fluid 1122moving into the 30mL ancillary chamber 1124, extending up from which is a small standpipe1126. In this embodiment the standpipe is 0.2 mL in volume. As the downward pressure fromthe weight of the fluid in the main chamber 424 fills the ancillary chamber 1124 undercentrifugation, the pressure urges the fluid in the other chamber up the standpipe 1126. Thus,the small-bore standpipe design ensures precise fluid volume control under centrifugation,addressing challenges related to fluid displacement, sterility, and measurement accuracy.

[0317] In more detail, the second fraction compartment, capped with a lid, holds exactly 30mL of fluid when full. During centrifugation, as fluid enters the compartment, the displaced airmust exit. This is achieved through a thin bore tube that extends up the CPC's interior andconnects to a port below a hydrophobic filter. The tubing's internal volume is less than 0.2 mL,ensuring precise air displacement while maintaining sterility by preventing communicationwith external air. When the CPC valve rotates from its park position to open the port leadingto the 30 mL compartment, centrifugal force propels the cell suspension into the compartment.As fluid enters, displaced air travels up the thin bore tubing and is transferred into the mainchamber 424. Any pressure differential that may be caused by this fluid movement can exitthrough the hydrophobic filter. The fluid rises within the thin bore tubing only to the samedistance from the axis of rotation as the descending fluid level in the main conical or taperedcompartment, ensuring equilibrium of centrifugal forces. Regardless of the centrifugationduration, this dynamic prevents fluid from overfilling or underfilling the compartment.

[0318] Key contributions of the standpipe are enhanced volume precision, measurementaccuracy, and functional integration. The standpipe limits variability in fluid measurements.Any change in fluid height within the main chamber is mirrored exactly in the standpipe,ensuring precise volume control. The standpipe's small-bore volume (0.2 mL) ensures highmeasurement accuracy, with variability limited to \pm0.1 mL. This precise design enables reliabledetermination of fluid levels without significantly affecting the overall capacity of the system.The design provides a simple, reliable, and highly accurate mechanism for determining thefluid volume in the secondary chamber during centrifugation. The standpipe system operatesautomatically, requiring no control mechanisms and is therefore cost-effective, minimallyintrusive, and does not interfere with the overall functionality or capacity of the CPC.

[0319] This standpipe system is particularly beneficial for processes requiring high precision,such as cell separation and reagent handling, where minor variations in volume cansignificantly impact outcomes. Its integration within the CPC reinforces the closed-systemdesign, minimizing fluid mismanagement and contamination risks. During operation, sinceradar or laser systems cannot monitor fluid levels while spinning, the process relies onempirically determined timing. By maintaining 50G centrifugal force and leaving the valveopen to the compartment input for a standardized duration (e.g., 10+ seconds beyond theminimum), precise fluid transfer of the 30 mL volume is consistently achieved.

[0320] FIG. 59D shows the fluid from the main chamber 424 that continues to urge the fluidupwards in the standpipe 1126 until it equalizes with the fluid in the main chamber 424. Thefact that this standpipe 1126 is so small (0.2 mL) means very little uncertainty as to the 30mLvolume of the other chamber, since such a small amount will need to move up the standpointin order to equalize with the liquid level of the main chamber 424, regardless of the level.

[0321] The dynamic nature of the CPC's weight, caused by fluid entering or exiting duringoperation, requires precise monitoring to ensure stability and accuracy within the centrifugebucket. This is achieved through integrated weight sensors or fluid level detection systems,such as radar, laser, weight, or time of flight monitoring systems. In a preferred embodiment,radar technology is used to measure fluid levels with high accuracy. Here, a radar system ispositioned above the CPC lid, where it emits radar waves through a guide rod. The radar wavesreflect off the fluid surface at the precise point where the liquid contacts the rod. The radarsystem eliminates reflections from plastic walls, focusing only on the fluid surface for accuratereadings. FIGS. 60A and 60B show this radar system 1130 connected to the cover 402. An Sshaped channel 1128 directs radar 1130 from a radar emitter 1132, not shown.

[0322] The radar emitter / receiver 1130 (not shown) are configured to detect reflections fromthe fluid surface within a sensing range of 15 to 85 mm, wherein the detected fluid height isconverted to a weight estimate for balancing purposes. The system software calculates the fluidlevel by subtracting reflection data between the emitter / sensor / rod interface and the fluidcontact point along the straight portion of the rod that extends vertically through the CPC lid.

[0323] In one exemplary case, this utilizes a 61 GHz radar transceiver (RFbeam V-LD1)coupled with a polycarbonate rod that transmits radar waves. The radar measures fluid levelswith exceptional accuracy, achieving better than 1 mm resolution across a range of 0 mm to63.47 mm, corresponding to fluid volumes from approximately 15.4 mL to 350 mL. Thisembodiment ensures a resolution finer than 1 mm, with calibration required for low liquidlevels to maintain this precision. Nonlinearities observed in the 0-3 mm measurement rangedue to end-of-rod reflections are corrected via software filtering algorithms. The impact ofepoxy glue used to secure the rod to the lid is visible in the radar signal but is effectively filteredout through advanced signal processing. The radar's signal dispersion is controlled to \pm10^{c},ensuring that only reflections from the fluid surface are captured, excluding interference fromthe containment walls. The rod's positioning is critical, avoiding contact with the containmentexcept at designated fixation points to maintain measurement fidelity.

[0324] The radar system incorporated into the platform achieves a 99% energy reflectionaccuracy at the fluid contact point, ensuring precise fluid level detection. Real-time adjustmentsare facilitated during critical processing cycles, including fluid input, centrifugation, andextraction, as the radar detects changes in fluid height within key regions, such as the neck andfunnel areas. This data allows the system to calculate fluid volumes with high precision,estimate weights accurately, and adjust operational parameters like rotational speeds andRelative Centrifugal Force (RCF) for optimal balance and performance. Measurementaccuracy improves as fluid volume decreases, with a precision of \pm4.5 grams at the maximumfluid level of 350 mL, improving to less than \pm1 gram at 100 mL. Even at the highest fluidlevel, the measurement precision is well within the centrifuge's operational tolerance, whichaccommodates up to 50 grams of imbalance without compromising safety or performance.

[0325] FIG. 60C shows a rod 1134 for use with the radar 1130 – the radar emitter / sensor (notshown) it as top right end of the rod 1128. The distance in the curved section up until thestraight portion of the rod can be electronically removed. The straight length of the rod 1134comes in through the cover 402 and just occupies a vertical distance. The reflection brings back99% of the energy. The rod 1134 extends down into the liquid in main chamber 424, liquiddoes not move up the rod, however, the radar 1130 can detect the liquid level. This is a meansfor detecting the liquid level. Laser, ultrasonic detector, strain gauge are other alternatives.

[0326] In addition to load cells and / or radar / laser systems, a time-of-flight (ToF) monitoringsystem may be utilized to detect fluid levels within the CPC. A ToF system uses light pulses(e.g., infrared or laser) or ultrasonic waves to measure the time it takes for the signal to travelto the fluid surface and back to the detector. Based on this time, the system calculates thedistance to the fluid level. This enables non-contact, high-precision detection of real-time fluidlevels, ensuring accurate volume measurement and stable centrifuge performance.

[0327] In a preferred embodiment, the radar or other detection system engages only when fluidlevels are changing or about to change. Alternatively, in some embodiments, the radar systemactivates only when the CPC is in its home position-vertically upright and motionless-ensuring consistent and interference-free measurements.

[0328] In use, the ACP automates the entire CAR-T cell manufacturing process, starting withleukapheresis and progressing through cell selection, activation, transduction, and final productpurification. Key steps include washing, centrifugation, mixing, gene transduction, andmultiple wash cycles to ensure a pure, viable, and potent cell product. Although there may beadditional or fewer steps, the following is one examples of the platform process.

[0329] 1. Apheresis Wash – Here, the initial leukapheresis sample is prepared by washingaway unwanted materials while maximizing white blood cell purity and viability. Input is aleukapheresis sample containing red blood cells, white blood cells, platelets, and any otherparticulate matter. The leukapheresis sample is mixed with a buffer solution. The systemremoves unwanted components, such as excess antibodies, viral particles, and non-targetdebris, directing them to the waste container. Flow rates, temperature, and wash solutions aretightly controlled to preserve white blood cell viability and ensure a high-purity sample.

[0330] 2. Selection and Activation – The objective in this step is to isolate target cells (e.g.,hematopoietic stem cells, T-cells, NK cells) using biotinylated aptamer or antibody-basedlinkers and prepare them for activation and genetic modification. The system may use specificmarkers, such as CD3+, CD4, CD8, and CD28, to identify and select target cells. Alternativeembodiments may use CD4 and CD8 markers alone, avoiding over-reliance on CD3.Biotinylated and non-biotinylated molecules play distinct roles in the cell processing machine'sselection and activation system. Biotinylation is commonly used to functionalize molecules,such as aptamers or antibodies, with biotin groups that can bind strongly to streptavidin-coatedsurfaces or to streptavidin-linked microbubbles in the machine. This approach enables preciseattachment of biotinylated molecules to microbubbles, enhancing target cell selection andactivation by allowing these functionalized microbubbles to bind to specific cell surfaceantigens via biotin-streptavidin interactions. Conversely, non-biotinylated molecules are usedwhen reversible binding is needed or when the application requires aptamers and antibodiesthat do not form permanent bonds with their targets. This combination allows the machine tobalance stable binding during selection and activation steps with the flexibility to release orfurther process cells when needed, optimizing both specificity and operational flexibility incell processing.

[0331] 3. Linking Mechanism: Biotinylated linkers are introduced to bind to the target cellmarkers. Streptavidin-coated microbubbles are then added and attach to the linkers, forming acell-microbubble complex that decreases the density of target cells, causing them to becomebuoyant in the fluid of the cell suspension.

[0332] 4. Centrifugation: During centrifugation, the buoyant target cell-microbubblecomplexes migrate away from the axis of rotation (upward in the CPC), while non-target cells,being denser than the fluid of the cell suspension, sediment toward the bottom of the CPC'stapered compartment. The tapered or conical shape of the central chamber of the CPC increasesin diameter as it is closer to the axis of rotation and ensures that the larger, buoyant target cellcomplexes avoid contact with descending non-target cells. Additional microbubbles may beadded if necessary to improve cell buoyancy and separation efficiency. Target cells withattached microbubbles are concentrated at the top of the CPC, while non-target cells aresequestered in a sealable compartment at the bottom

[0333] 5. Spinoculation and Transduction – In this embodiment, a CAR gene is introducedto target cells using viral vectors assisted by controlled centrifugation to optimize gene transferefficiency. In detail, viral vectors are introduced to the CPC, targeting the selected and activatedcells. The system employs spinoculation, where controlled centrifugation brings viral particlesinto close proximity with the target cells, enhancing transduction efficiency. The process mayinvolve multiple steps, such as (1) centrifuging the target cells to improve vector penetrationto the nucleus; (2) reciprocally pivot or rock the CPC back and forth to gently mixand redistribute cells and vectors, ensuring thorough interaction; and (3) re-centrifuge andrepeat as necessary to improve gene transfer. The process is carefully timed and monitored tooptimize transduction while minimizing cell stress. The outcome is gene-modified CAR-T cellsgenerated with high transduction efficiency.

[0334] 6. Wash and Harvest – The objective with this step is to purify and formulate a finalcell suspension volume for the gene-modified CAR-T cells to meet therapeutic standards forpurity, viability, and potency. The system performs multiple washing cycles to remove excessviral vectors, reagents, and particulate matter. Each wash significantly reduces contaminants.For example, introducing 150 mL of wash fluid, mixing thoroughly, and applying positivepressure transfers the contaminants to waste. In this case each wash achieves a 90% reductionin contaminants. Repeating the process three times achieves a 1000-fold reduction. Afterwashing, the cells are concentrated to a convenient volume (e.g., 15 mL) and then adjustedwith buffer up to the required formulation volume for harvest. Final quality control assessmentsconfirm the purity, viability, and readiness of the CAR-T cell product. The outcome is apurified, concentrated, and ready-to-use CAR-T cell product suitable for therapeutic use.

[0335] The MBCSA (microbubble Cell Selection and Activation) technology enablessequential cell selection, addressing a critical limitation in current CAR-T production.Traditional methods, such as magnetic bead-based selection, are limited because once T cellsare selected with magnetic beads, they cannot undergo further magnetic-based separations. Incontrast, the MBCSA allows for de-gassing of the initial selection reagent, making it possibleto sequentially select different cell sub-populations using secondary reagents. This capabilityfacilitates a more precise and customizable approach to cell composition, allowing forenhanced control over T cell subsets in the final therapeutic product.

[0336] MBCSA Using Controlled Pressure - The ACP employs an innovative MBCSAtechnology that uses microbubbles to selectively bind and isolate target cells, such as T cellsor stem cells, based on their surface markers. The microbubbles are in this instance small gas-filled bubbles with a lipid, polymer, or protein shell, often functionalized with specificmolecules like aptamers or antibodies to bind to target cells. This process is facilitated bysurface-modified, phospholipid-shell microbubbles that are stable, lyophilized, and easilyreconstituted for use. By leveraging pressure to selectively collapse the microbubbles, the ACPenhances both the efficiency and effectiveness of cell selection, providing a high-purity, high-viability cell product that meets the rigorous standards required for therapeutic applications.

[0337] In the context of this application, lyophilized microbubbles refer to microbubbles thathave been freeze-dried to preserve their structure and functionality for long-term storage andlater use. Lyophilized microbubbles are more convenient to store, transport, and integrate intoautomated systems compared to their hydrated counterparts, which may require specificstorage conditions. In their lyophilized form, the microbubbles are dehydrated under lowtemperature and vacuum conditions, removing moisture while retaining their structuralintegrity and biofunctional properties. Lyophilization stabilizes microbubbles, making themsuitable for extended storage without loss of efficacy. Before use, the lyophilized microbubblescan be rehydrated (e.g., with a saline solution) to restore their functional form for binding totarget cells within the cell processing platform. The functional groups on the microbubbles,such as biotinylated aptamers or antibodies, are preserved during the lyophilization process,ensuring specificity and binding efficiency. They then bind selectively and predictably to cellsurface markers and facilitate separation, selection, or modification processes likespinoculation or controlled cell processing cycles.

[0338] Preparation and Targeting of Microbubbles - The microbubbles are coated withstreptavidin, allowing them to link to biotinylated aptamers or antibodies that specifically bindto desired cell surface markers. This binding process causes the target cells to become buoyant,enabling easy separation based on their relative buoyancy within the CPC 212. As they rise,they encounter a widening cross-section of the funnel, which gives them more space todisperse. The balance of forces-compression of cells against the funnel walls and buoyantseparation as cells rise allows the cells to be tightly packed and then gently separated,optimizing selection and distribution across the funnel. This design leverages both mechanicaland physical properties to enhance precision in cell processing and isolation This separationstep reduces the density of cell clusters and allows for a more even distribution of cells withinthe fluid. To prepare the MBCSA reagent, lyophilized microbubbles are reconstituted with 6cc of sterile saline and shaken for approximately 8 seconds to achieve a uniform suspension.Once reconstituted, the microbubbles retain stability within the fluid environment of the CPC212, enabling efficient cell targeting and isolation.

[0339] Pressure-Induced Microbubble Collapse - A unique feature of the MBCSA processis the ability to disrupt the buoyancy of the microbubbles, releasing the bound cells by applyingcontrolled air pressure. After the target cells have been isolated and bound to the microbubbles,the CPC's internal pressure is raised to an appropriate pressure, in one example approximately2.5 atmospheres. This increase in pressure collapses the phospholipid shells of themicrobubbles, effectively "popping" them and releasing the bound cells without causingcellular damage. The rapid collapse of microbubbles enables swift transition between cellselection and subsequent processing steps, streamlining the overall cell processing workflowThe lipid shell of the microbubble is washed out later. The controlled pressure collapse is acritical aspect of the MBCSA technology, as it provides a gentle, non-destructive means ofreleasing the cells, allowing them to proceed to subsequent processing stages, such as washing,activation, or formulation of cells which remain viable and functional for therapeuticapplications

[0340] Aptamers

[0341] This system further integrates aptamers as high-affinity binding agents within theMBCSA system, targeting antigens such as CD3, CD8, CD28, and CD34. Compared totraditional antibody-based methods, aptamers offer cost-effective and rapidly customizablealternatives while demonstrating superior stability and reduced degradation under optimizedconditions. For instance, aptamers can be selectively removed by introducing DNase to degradethe DNA and release the attached microbubbles, a flexibility not possible with antibodies.Additionally, antibodies can cause unintended physiological effects on cells due tointernalization, a concern that does not arise with aptamer-based approaches. From anintellectual property perspective, aptamers offer a significant advantage as they can besynthesized in-house, avoiding reliance on commercially available antibodies.

[0342] Key findings highlight the system's effectiveness, with aptamer-linked microbubblesachieving higher purity and recovery rates than competing technologies. For example, theplatform consistently produces a positive fraction of CD8+ cells with 95.1% purity and 89.1%recovery, outperforming traditional antibody and bead-based methods that typically yieldapproximately 50% recovery.

[0343] The ACP incorporates aptamers-short, single-stranded oligonucleotides (DNA orRNA) that fold into specific three-dimensional shapes, allowing them to selectively bind totarget molecules with high affinity. Compared to antibodies, aptamers are more versatile andcost-efficient to produce and modify, making them suitable for a wide range of targets,including proteins, cells, and small molecules. Their customizable nature supports tailoredapplications in biotechnology, diagnostics, and therapeutic processes.

[0344] The platform ensures aptamer functionality by employing precise folding protocols andoptimized buffer compositions to maximize binding efficiency while minimizing degradation.This design achieves outcomes comparable to, or even surpassing, traditional antibody-basedmethods, providing a robust, scalable, and predictable solution for cell therapy manufacturing.The integration of aptamers guarantees high-quality cell products with minimal contamination,ensuring safe and effective therapeutic applications.

[0345] Aptamers are utilized alongside a microbubble system comprising lipid-shell, gas-coremicrobubbles, which confer buoyancy to target cells such as hematopoietic stem cells (HSCs),T-cells, and NK cells. During low-speed centrifugation, target cells bound to microbubblesfloat, effectively separating from non-target cells. This process is highly specific, withaptamers binding to unique surface antigens on target cells. The use of aptamer-linkedmicrobubbles enhances precision in cell selection, enabling automated separation of target andnon-target cells during centrifugation. By binding to specific cell surface markers, aptamersfacilitate the isolation and concentration of desired cell populations, such as T-cells, within theCPC. Additionally, aptamers are engineered to work synergistically with microbubbles andother separation aids, further improving the efficiency and specificity of cell selectionprocesses.

[0346] The aptamers integrated into the platform are structurally optimized to mimic thebinding efficacy of antibodies, utilizing modifications such as dimerization and spacerenhancements. These adjustments maintain flexibility while ensuring high specificity forvarious cell markers. Moreover, aptamers exhibit reversible binding properties through tailoredlinkers, allowing the release of bound cells post-selection. This reversibility enables furtherpurification steps or reuse of processed cells, particularly advantageous for T-cell-basedtherapies where sequential selection and activation of multiple subpopulations are required.

[0347] Integrating aptamers into the system delivers numerous benefits, includingcompatibility with automated, high throughput closed systems that ensure sterility, precision,and reproducibility. Aptamers' low immunogenicity compared to monoclonal antibodies(mAbs) enhances the platform's safety profile. In conjunction with microbubbles, aptamersenable the CPC to isolate and process cells efficiently while remaining adaptable for a widerange of therapeutic applications. This approach ensures the production of high-quality, safecell therapy products tailored for clinical use.

[0348] The system contemplates use with the following advantages for aptamer designs:

[0349] Dual-stranded aptamers - The development of reversible aptamer linkers, such asdual-stranded aptamers, presents a promising avenue for enhanced flexibility in cellprocessing. These aptamers can be designed to detach by introducing a complementary strandwith a higher binding affinity to the aptamer. This reversible mechanism would allow for (1)reuse of selected cells for additional processes or treatments, (2) Further purification steps toachieve higher specificity and purity in target cell populations, and (3) Expanded utility ofaptamer-based cell processing across diverse applications, including research, manufacturing,and therapeutic contexts.

[0350] Thermally pre-treated aptamers -Thermally pre-treated aptamers undergo acontrolled heating process to optimize their structural conformation, significantly enhancingtheir binding efficiency, stability, and application potential in cell processing workflows. Byheating aptamers to a specific temperature (typically 70-95^{\circ}C) and rapidly cooling them, theyfold into energetically stable and functional conformations. This prevents misfolding andaggregation, resulting in a homogenous population of active aptamers. Thermally treatedaptamers exhibit stronger and more specific binding to target antigens, ensuring high-puritycell selection and reducing nonspecific interactions. Incorporating thermally pre-treatedaptamers into the ACP's workflow would significantly enhance the precision and reliability ofcell selection processes. Their adaptability and enhanced performance make them an idealsolution for applications demanding high specificity and efficiency.

[0351] Reversible Aptamer Design - Aptamers offer the potential for reversibility, furtherexpanding their versatility. For example, a dual-stranded aptamer hybrid could act as areversible linker. As another example, a single-stranded aptamer with an additional "tail" forattachment to a complementary strand could be used to facilitate detachment. By introducinga disrupting strand with higher affinity for the target antigen, the aptamer and attachedmicrobubble could be effectively removed from the cell surface.

[0352] SELEX and Aptamer Development: The SELEX (Systematic Evolution of Ligandsby Exponential Enrichment) process offers access to a vast library of aptamer candidates. Oncefunding is secured, a SELEX procedure will be conducted to identify aptamers specific to thedesired antigens. This process will generate multiple aptamer candidates, enabling the selectionof the best-performing sequences for use in cell processing. Thermally pre-treated aptamerscan also be selected and optimized during the SELEX process to ensure they remain stable andfunctional under varying physiological and processing conditions.

[0353] The optimal range of conditions is as follows:ParameterOptimal ConditionsAptamer DesignAddition of Spacer-18 at 5^{,} end between biotin and basesto act as a hinge and enable easier access for MBAptamer Folding Buffer40mM HEPES, 100mM NaCl, 5mM MgCl_{2}.Aptamer Folding Temp. Profile10 min. 95^{\circ}C, 15 min ice, 30 min 37^{\circ}C, 45 min RTAptamer Folding Conc.10 nMAptamer Working Conc.0.5 nMAptamer: Cell Ratio0.075 nmol per 10\times10^{6^{\prime}} T cellsExperimental Buffer• DPBS, 1% HSA. (in short, DPBS and protein)• Addition of MgCl_{2} to experimental buffer wastested to maximize aptamer stability but did not improveperformance• This is the main buffer used during the processAptamer Incubation Duration30 minutesAptamer Incubation Temp.RT. 4^{\circ}C was tested to try and improve purity but hadlower purity and recovery than RTAptamer Mixing ProfileMix manually every 5 minutesMicrobubble: Cell Ratio450~\mu L per 10\times10^{6} T cellsMicrobubble Incubation Duration1 minuteMicrobubble Mixing ProfileManually Mix after addition and again at 30 secondsCentrifugation Profile250 x g for 5 minutesPositive Fraction WashImproves purity but reduction of recovery. Has not beenused in recent studies since purity is now acceptablewithout it

[0354] Secondary Selection:

[0355] In one embodiment, an initial cell selection process is conducted to isolate T cells,followed by a subsequent selection step to refine a subpopulation, such as CD8+ or CD4+ Tcells or memory T cells. This sequential selection can be performed using aptamers, where thefirst aptamer is digested and replaced with a second aptamer specific to the desiredsubpopulation. Alternatively, a primary selection reagent, such as microbubbles functionalizedwith specific antibodies or aptamers targeting T cells, is used to isolate the broader targetpopulation. A secondary selection reagent, comprising microbubbles coated with antibodies oraptamers for selective binding to CD8+ or CD4+T cell subpopulations, is then employed tofurther refine the selection. Between selection steps, a degassing solution is utilized to removethe buoyancy of the microbubbles, ensuring efficient removal and preventing contamination ofthe primary cell population. This method facilitates precise, sequential isolation of target cellsubsets for downstream applications.

[0356] Use for Leukapheresis

[0357] This system's functionally closed design and advanced capabilities make it ideal fordirect use in leukapheresis procedures, particularly by dramatically reducing target cell lossesduring manufacturing. Its precision enables efficient gene-modified CAR-T cell production,even with smaller white cell counts typically found in whole blood samples rather thanleukapheresis collections. Leukapheresis involves the selective separation and collection ofwhite blood cells-granulocytes, lymphocytes, monocytes, and stem cells-from whole blood.The system's combination of closed-system sterility, controlled centrifugation, selective celltargeting, and automated quality control makes it highly suitable for leukapheresis applications.Its adaptability to handle smaller white blood cell counts and its ability to minimize target celllosses ensure efficient, high-yield recovery of leukocytes, supporting downstream processessuch as CAR-T cell manufacturing and immunotherapy development, as detailed below:

[0358] Controlled Centrifugation and Sedimentation: The system optimizes the degree oftarget cell packing during spinoculation by dynamically adjusting centrifugation speeds. Celldetection sensors located in the sedimentation column monitor target cells as they sediment,ensuring precise control for improved packing and vector delivery efficiency.

[0359] Aseptic Transfer and Contamination Prevention: The closed-system designeliminates contamination risks by providing sterile fluid transfer pathways. Pressure decaytesting ensures the integrity of both hydrophobic and hydrophilic filters on the CPC lid beforecells are inserted. These features maintain cell viability and meet strict sterility standardsrequired for leukapheresis and clinical applications.

[0360] Selective Cell Isolation and Enrichment: The system's aptamer and microbubbletechnology enable selective targeting and isolation of leukocytes. Aptamers or antibodiesdesigned to recognize specific white blood cell markers (e.g., CD45 or CD34) can be appliedto enrich leukocyte subpopulations. This targeted enrichment is particularly valuable inimmunotherapy applications.

[0361] Cell Sedimentation Monitoring: The optical detection system, utilizing three opticalsensors, provides real-time monitoring of target cell sedimentation. This ensures: Optimalcentrifugal concentration of target cells, accurate packing of cells for enhanced vector exposureduring gene modification processes, improved overall transduction efficiency.

[0362] Use with Senescent Cells

[0363] The described device can be adapted to target, isolate, and clear senescent cells, whichaccumulate with age and contribute to the progression of various age-related diseases. Byspecifically targeting these dysfunctional cells, the platform offers a novel approach tomitigating the adverse effects of cellular senescence on aging tissues.

[0364] The device may leverage aptamers specifically designed to bind to markers uniquelyexpressed by senescent cells. These markers include overexpressed surface proteins such asp16, p21, or SA-\beta-gal, which are characteristic of senescent cells. Using SELEX (SystematicEvolution of Ligands by Exponential Enrichment), custom aptamers can be tailored toselectively recognize these markers, ensuring precise targeting within a heterogeneous cellpopulation.

[0365] Aptamers conjugated to buoyant microbubbles enable the selective binding andisolation of senescent cells. During low-speed centrifugation, microbubbles carrying boundsenescent cells rise to the top of the funnel chamber. This buoyancy-based separationeffectively isolates senescent cells from healthy, non-senescent cells in the sample, allowingfor efficient enrichment and collection.

[0366] For enhanced specificity and purity, reversible aptamer designs may be used to allowmultiple rounds of selection. After initial isolation, aptamers can be disrupted usingcomplementary strands or higher-affinity reagents, releasing the bound senescent cells. Thisprepares the system for subsequent rounds of processing, refining the isolation of specificsenescent cell subtypes for optimal purity and recovery.

[0367] The device facilitates the concentration of senescent cells within the funnel chamber,creating a high-density collection at the bottom. Once isolated, these senescent cells can beextracted or subjected to depletion strategies. Their selective removal has the potential torejuvenate surrounding tissues by alleviating the inflammatory and detrimental effectsassociated with senescence.

[0368] The isolated senescent cells may serve as valuable resources for therapeutic research,enabling the development of cellular rejuvenation strategies. Alternatively, after clearingsenescent cells from a patient's sample, the system could reintroduce purified healthy cells intothe patient. This approach would provide a tailored cellular composition that promotes tissuerepair and extends the patient's healthy lifespan, offering a transformative solution forcombating age-related disease.

[0369] The ACP incorporates a comprehensive pre-operation diagnostic protocol to ensure theintegrity and functionality of the CPC and associated components before any biologicalmaterials are introduced. When the clinician inserts the cartridge into the machine, the systemautomatically conducts a series of integrity and performance tests. If any failure is detected inthe cartridge or its components, the machine prevents cell fluids from being introduced,prompting the clinician to remove and discard the defective cartridge. This ensures that onlyfully functional cartridges are used in the cell processing workflow. The initial testingsequence, which occurs before cells are loaded into the cassette, includes but is not limited to:

[0370] Filter Integrity Testing: Pressure decay tests are performed to confirm thestructural integrity and functionality of all filters in the CPC.

[0371] UV Module Verification: The UV disinfection module is tested to ensure itradiates at the correct intensity for effective disinfection.

[0372] Centrifuge Diagnostics: The centrifuge system is assessed for operationalreadiness, including balance and motion parameters.

[0373] Mixing Actuator Testing: The rocking or mixing mechanism is tested forfunctionality and precise motion control.

[0374] Heater Calibration: The heating system is verified to ensure it can maintain thespecified temperature profile during cell processing.

[0375] Upon successful completion of the diagnostic tests, a green light signals the operatorthat the system is ready for use. At this point, the operator may lift the lid, introduce the cellsinto the cartridge, secure the tubing, and snap the stub into place for processing.

[0376] Following the processing run, the machine conducts a secondary verification of criticalsterility-related components, specifically re-testing the filters and the UV module. This post-operation testing ensures that the integrity of the CPC and the sterility of the process weremaintained throughout the cell processing workflow. Other subsystems, such as the centrifuge,heater, and mixing mechanisms, are not re-tested post-run.

[0377] Recovery and Purity

[0378] The disclosed system achieves exceptional recovery and purity rates in target cellselection, with a particular focus on CD8+ T-cell enrichment and isolation. Efficiency iscalculated as the fraction of viable gene-modified cells retained from the initial cell population.For example, conventional manufacturing often begins with 2 billion T-cells, with up to 92.1%lost during processing over days or weeks. This leaves around 150 million activated T-cells,which are then expanded to 1 billion over 8 days. However, an additional 50% of these cellsmay be lost during reinfusion into the patient. The ACP minimizes these losses, compressingtimelines and optimizing cell recovery for superior treatment outcomes

[0379] The platform disclosed herein demonstrates near 90% recovery and 95% purity forCD8+ T-cell selection, enabled by optimized aptamer functionality. This optimization involvescontrolled folding and binding conditions, including the use of buffer compositions such as 40mM HEPES and 100 mM NaCl to effectively eliminate impurities and stabilize aptamerstructures. Temperature protocols are also fine-tuned, incorporating a denaturation step at 95^{\circ}Cfollowed by structured cooling to facilitate aptamer refolding into ideal conformations.Importantly, non-functional or misfolded aptamers are rendered inactive and do not disrupt theprocess.

[0380] The platform also excels in enrichment and depletion performance across various celltypes. Mononuclear cell (MNC) recovery during enrichment achieves a rate of 95%, whileCD34+ hematopoietic stem cells (HSCs) are recovered at an impressive 99%. Non-target cells,including red blood cells (RBCs), platelets (PLTs), and neutrophils (NEUs), are effectivelydepleted to levels of 99%, 84%, and 67%, respectively, ensuring a highly purified final cellpopulation.

[0381] For T-cell isolation, the system produces CD3+ T-cell populations with a purity of atleast 95% and recovery rates exceeding 95%. The resulting cell product contains 90-95%activated T cells to ensure immediate therapeutic efficacy and includes a substantial subset ofmemory T cells (20-30%) to enhance long-term in vivo durability. When HSCs are includedin the workflow, they are enriched to similarly high purity and recovery levels, supporting theiruse in applications such as stem cell transplantation.

[0382] The present platform outperformed conventional platforms in both T-cell purity (99%)and recovery rates (90-95%), whereas alternative systems typically achieve only 50% recoverywith approximately 90% purity. The emphasis on achieving exceptional purity is crucial, ascontamination with non-T-cells or cancer cells in patient samples can compromise treatmentefficacy. Notably, the entire process is completed within a single device at a single location,minimizing loss and contamination. The platform ensures that less than 1% of target cells arelost during processing, with further thresholds of less than 2% or less than 5% depending onoperational parameters, demonstrating unparalleled precision and efficiency in cell therapymanufacturing.

[0383] Comparative Efficiency Across Key Process Stages

[0384] Leukapheresis and Washing: The ACP achieves a 97.2% efficiency in leukapheresisand 95.3% in the washing stage, compared to industry standards of 50% and 49.9%,respectively. This significant improvement results from the platform's closed-loop automationand optimized fluid handling, which minimize cell loss and contamination risk.

[0385] Cell Selection and Activation: The Microbubble Cell Selection and Activation(MBCSA) process enables a 74.1% efficiency in cell selection and activation, compared to astandard efficiency of 39.4% in traditional magnetic bead-based or flow cytometry systems.The sequential selection capability of MBCSA ensures higher cell purity and viability,contributing to a refined therapeutic product.

[0386] Transduction: During the transduction stage, the ACP achieves a 94.7% efficiency,greatly surpassing the industry standard of 80%. The controlled mixing and even celldistribution provided by the CPC's geometry enable efficient vector exposure to cells,improving transduction rates while preserving cell viability. The mixing is to distribute thecells evenly, so the cells are better washed, avoiding sloshing, frothing and bubbles.

[0387] Harvest, Wash, and Formulation: The final stage of the process, including harvest,washing, and formulation, demonstrates a substantial efficiency improvement with the ACP,achieving a 65.0% efficiency compared to the industry standard of 7.9%. This improvement isdue to precise fluid handling, reduced cell loss, and minimized handling steps, whichcollectively enhance the integrity of the final cell product.

[0388] Cumulative Process Efficiency: The cumulative process efficiency of the ACP acrossall stages of cell processing is 65.0%, compared to the current industry cumulative standard of7.9%. This 823% increase in efficiency removes the need for extended ex vivo cell expansion,a costly and time-consuming step traditionally required to reach clinical doses in CAR-T cellproduction. By maintaining high viability and functionality of cells throughout processing, theACP supports streamlined manufacturing with reduced cost and time requirements.

[0389] The ACP described herein provides several key functional advantages that enhance itsutility in automated cell processing applications. These include:

[0390] Targeted Cell Selection: The use of aptamers enables selective binding andenrichment of target cells, significantly reducing contamination from non-target cells andimproving purity for downstream applications. This precision ensures high-quality therapeuticoutputs.

[0391] Microbubble-Assisted Separation: When combined with microbubbles, aptamersprovide a buoyancy-based cell separation mechanism. This method isolates target cells viadifferential flotation during centrifugation, effectively segregating them from non-target cellswith minimal loss.

[0392] Reversible Binding and Versatile Control: Aptamers can be engineered withreversible linkers or hybrid structures, allowing for the controlled release of bound cellsfollowing separation. This reversibility supports additional purification steps or reuse of cells,adding flexibility to the manufacturing process.

[0393] Compatibility with Automated Processing: The stability and customizable nature ofaptamers make them highly compatible with automated, high-throughput cell processingplatforms. This compatibility is essential for closed-system devices where sterility, precision,and repeatability are critical.

[0394] Lot Release / Documentation

[0395] The platform may produce detailed lot release records for regulatory or other purposes.This capability supports personalized treatments, providing extensive data on each batch,similar to the extensive documentation requirements in large pharmaceutical manufacturing.The ACP continuously tracks and records a variety of critical parameters throughout the cellprocessing cycle, ensuring that each step adheres to the defined protocol and maintains optimalcell viability and product quality.

[0396] The tracked QC data may include, but is not limited to:

[0397] Identities: Protocol, patient, and cell source identifiers ensure each batch correspondsto the correct therapeutic requirements. Instrument and CPC identifiers verify the equipmentand consumables used for each production cycle. Operator details recorded to maintain a fullaccountability record, allowing traceability and assessment of human involvement wherenecessary.

[0398] Cell Culture Parameters: Temperature, fluid composition such as dissolved oxygen([O_{2}]) concentration, culture volume, and cell density are continuously monitored to maintainideal cell culture conditions. Medium change times and volumes are recorded to verifycompliance with specified growth conditions, ensuring cell viability and consistency acrossbatches.

[0399] Physical Process Parameters: Centrifugation time-courses and applied forces aretracked to confirm that cells are adequately separated or concentrated according to protocolspecifications. Mixing speeds, angles, frequencies, and durations are recorded to ensureuniform reagent distribution, efficient cell washing, and optimal cell suspension. Aspirationtimes and volumes are monitored to maintain sterile conditions and ensure precise fluidhandling. QA sampling volumes and times for consistency, and for supporting reliableanalytical testing across the manufacturing process. Pre- and post-processing filter integritytests are conducted to verify that all filters maintain aseptic conditions, preventingcontamination and ensuring product safety.

[0400] Reagent Information: Reagent identities, receipt and expiration dates, addition times,and volumes are documented to confirm the use of correct materials within their stabilitywindows. Storage temperatures for all reagents are tracked to prevent degradation, ensuringthat only viable materials are used in cell processing.

[0401] Automated Error Reporting and Release by Exception: The ACP incorporates anautomated error reporting system that continuously monitors the data for any deviations frompreset limits. If any parameter deviates from the established threshold, an error condition isimmediately flagged, and the system records the details of the deviation. This automateddetection allows for real-time error identification and corrective actions if necessary.

[0402] Release by Exception: The "release by exception" model implemented within the ACPis a transformative approach to batch release in cell therapy manufacturing. Traditionally, allbatch records must be exhaustively reviewed before product release, a process that is highlylabor-intensive and prone to human error. In contrast, the release by exception model onlyrequires QC review for batches where deviations or anomalies are detected, allowing batcheswithout errors to be automatically approved for release based on the system's verifiedcompliance with all critical parameters.

[0403] The fully automated nature of the ACP reduces manual intervention, significantlylowering contamination risks and operational variability. Compared to traditional methods, theACP accelerates the manufacturing process, enabling the production of a clinical dose of CART-cells in just 2.5 days without the need for time-intensive ex-vivo expansion. This streamlinedapproach ensures precise control over every stage of the process, optimizing flow rates,centrifugation forces, and timing to enhance product reliability, purity, and cell viability.Multiple washing cycles integrated into the workflow further reduce contaminants, ensuring ahigh-quality therapeutic product.

[0404] The compact and automated design of the ACP enhances accessibility, enablingdeployment directly within FDA-licensed transplant centers. This co-location capabilitysupports both research and clinical applications, allowing scientists to concentrate on geneconstruct development rather than labor-intensive manufacturing processes. Additionally, theACP's robust quality control and aseptic processing steps ensure consistent and reproduciblemanufacturing outcomes. By simplifying and accelerating cell therapy production, the ACPhas the potential to make life-saving treatments more accessible and affordable to a broaderpatient population.

[0405] In summary, the invention presents a functionally closed cell processing platform thatminimizes contamination and automates critical cell processing steps. The platformincorporates multiple specialized cassettes to perform various operations, including a RSC forreagent storage and cell suspension delivery, a PFC for managing waste fluids and buffers, aCPC designed to house cell suspensions and buoyant microbubbles for cell selection, and aTSC to retrieve processed cell suspensions from the CPC and transfer them to the RSC. Thesystem is equipped with a centrifuge mechanism capable of applying centrifugal forces to theCPC, and a pneumatic control system that performs functions such as expelling waste fluids,disrupting buoyant microbubbles to prepare cells for further processing, testing the integrity offilters, and expelling gene-modified cells.

[0406] The platform integrates hydrophobic and hydrophilic filters in the CPC lid, and itscentrifuge mechanism can operate in distinct modes for cell concentration and mixing, withautomatic transitions based on process requirements. UV sanitizers disinfect fluid pathwaysand septa interfaces, ensuring aseptic conditions during fluid transfers via the TSC. Some gene-modified cells can be sequestered, with provisions for selectively handling a second fractionof these cells. The CPC can rotate about two orthogonal axes to enhance fluid mixing andprevent sedimentation during processing.

[0407] The platform's housing accommodates the RSC, PFC, and TSC, and includes acentrifuge capable of multi-axis rotation of the CPC. A pneumatic control system ensuresefficient fluid transfer through filters and valves, while a waste management system integratedwith the PFC handles waste disposal. An automatic balancing system maintains stability andminimizes vibrations during centrifugation by adjusting counterweights and rotationalparameters. A sensor array, comprising optical and thermal sensors, monitors environmentalconditions within the CPC, enabling real-time adjustments during centrifugation. The platformfurther supports a microbubble cell selection system, utilizing functionalized buoyantmicrobubbles to selectively bind target cells for isolation.

[0408] The TSC includes multiple syringes with needles, plungers, and fluid receptacles, allaligned via a docking and disinfection module for aseptic insertion into the CPC lid. Thedisinfection module employs UV radiation to maintain sterility, while an automated plungeractuation system enables precise fluid handling without manual intervention.

[0409] The system incorporates a control module to monitor and manage CPC operations,including cell movement, temperature, rotation speed, and fluid volumes. The control systemalso disinfects CPC septa surfaces using UV radiation and tracks fluid composition and volumethrough radar, laser, or strain gauge sensors. Additional features include optical transmittersand receivers aligned across the CPC sedimentation column to monitor cell presence anddensity, with configurations for rotational optical data collection from multiple perspectives.

[0410] The invention also encompasses a method for analyzing cell suspensions, utilizing theTSC to extract samples for analysis in a Cell Analytics Module (CAM). The CAM measurescell concentrations via optical or impedance-based mechanisms, identifies cell surface markers,and determines the purity of T-cell populations based on marker expressions. The systemprovides real-time analytical feedback to monitor and control cell processing quality fordownstream applications.

[0411] This functionally closed platform offers an innovative, automated solution for cellprocessing with exceptional precision, sterility, and efficiency. It combines advanced cassette-based systems, automated controls, and real-time monitoring to meet the rigorous demands oftherapeutic and research applications in gene therapy, immunotherapy, and cell manufacturing.

Claims

CLAIMS1. A functionally closed cell processing platform, configured to minimize contaminationand automate cell processing steps the platform comprising:a. a reagent sample cassette (RSC) for reagent storage and delivery, and for cellsuspension sample receival and delivery;b. a process fluids cassette (PFC) for fluid waste and buffer management;c. a cell processing cassette (CPC) configured to house said cell suspension,linkers and buoyant microbubbles bound to said cells; andd. a transfer syringe cassette (TSC) for retrieving said cell suspension samplefrom the CPC and transferring it to the RSC;e. a centrifuge mechanism operable to apply centrifugal force to the CPC;f. a pneumatic control system, using pressurized gas configured to:i. expel waste fluids from the CPC via a fluid waste disposal tube;ii. selectively disrupt the buoyant microbubbles remove their buoyancyfrom attached cells for subsequent processing steps;iii. perform pressure decay tests on the hydrophobic and hydrophilic filtersto confirm their structural integrity; andiv. expel gene-modified target cells to the TSC.

2. The device for processing biological cells according to claim 1, further comprisinghydrophobic and hydrophilic filters within the lid of the CPC.

3. The device for processing biological cells according to claim 1 wherein the centrifugemechanism is operable in a centrifugation mode for cell concentration and a mixingmode for homogeneous distribution of cells and reagents, with automatic transitionsbetween modes based on process requirements.

4. The device for processing biological cells according to claim 1 further comprising UVsanitizers positioned to disinfect fluid pathways and septa interfaces, ensuring asepticconditions during fluid transfer by the TSC.

5. The device for processing biological cells according to claim 1 wherein at least someof the gene-modified target cells are sequestered.

6. The device for processing biological cells according to claim 5 wherein a secondfraction of the gene-modified target cells are sequestered.

7. The device for processing biological cells according to claim 1 wherein the rotation ofthe CPC is about two orthogonal axes, wherein the rotation of the CPC about twoorthogonal axes enhances fluid mixing and prevents sedimentation during cellprocessing.

8. A device for a functionally closed cell processing platform, the device comprising:a. a housing configured to contain multiple cassettes, including:i. a PFC;ii. a TSC; andiii. a RSC;b. a centrifuge, comprising at least bucket into which is nested a CPC, thecentrifuge operable to perform multi-axis rotation of the CPC;c. a pneumatic control system, configured to transfer fluids through at least onehydrophilic filter, at least one hydrophobic filter, and one-way valves via gaspressurized channels; andd. a waste management system, integrated with the PFC and configured to expelwaste fluids.

9. The cell processing platform according to claim 8, wherein the centrifuge mechanismfurther comprises an auto balancing system that adjusts counterweights and rotationalparameters to maintain stability and reduce vibration prior to and during multi-axiscentrifugation.

10. The cell processing platform according to claim 8, further comprising a sensor array,including optical, thermal, disposed within the bucket, configured to monitor andadjust environmental conditions within the CPC during centrifugation.

11. The cell processing platform according to claim 8, further comprising a microbubblecell selection system, wherein buoyant microbubbles, functionalized with antibody oraptamer linkers, selectively bind target cells, enabling isolation of these cells fromnon-target cells during centrifugation.

12. A TSC for use in a functionally closed cell processing platform, the TSC comprising:a. a plurality of syringes, each syringe including a needle, a plunger, and a fluidreceptacle for housing biological materials or reagents, and a septa for eachsyringe;b. a docking and disinfection module configured to align and engage eachsyringe with corresponding septa on another cassette;c. an automated plunger actuation system configured to control fluid transfer bymoving the syringe plunger in a precise sequence, handling; andd. at least one septum aligned with at least one additional septum, and adisinfection module to sanitize the exterior surfaces of each opposing septa.

13. The TSC for use in a functionally closed cell processing platform according to claim12, wherein the docking interface enables precise and controlled insertion of thesyringe needle into a target septa on the lid of the CPC.

14. The TSC for use in a functionally closed cell processing platform according to claim13, wherein the disinfection module ensures aseptic conditions are maintained as thesyringe is inserted.

15. The TSC for use in a functionally closed cell processing platform according to claim12, wherein the docking and disinfection module utilizes UV radiation.

16. The TSC for use in a functionally closed cell processing platform according to claim12, wherein the automated plunger actuation system allows for the intake anddispensing of fluids without manual assistance.

17. A control system for managing and monitoring cell processing within a CPC,comprising:a. a centrifuge capable of multi-mode operation, configured to align the CPCwith the TSC during fluid handling and operate independently during otherprocesses;b. a sensor array, configured to provide real-time tracking of:i. cell movement;ii. CPC temperature;iii. rotation speed during centrifugation;iv. volumes when the CPC is vertical and not under centrifugation andaligned for docking with additional cassettes;c. a control module in communication with said sensor array and configured toenable adjustments to centrifuge speed; fluid flow rates; and reagentdispensing intervals and flow rates; andd. a sanitization mechanism, comprising a UV radiation system positioned todisinfect the surface of septa on the CPC, and the exterior bottom surface ofthe septa for each syringe of the TSC.

18. The control system of claim 17, wherein the sensor array further measures fluidcomposition within the CPC.

19. The control system of claim 17, further comprising at least one sensor positionedwithin to monitor fluid volumes within the CPC when the CPC is not undercentrifugation.

20. The control system of claim 19 wherein said at least one sensor is a radar sensor.

21. The control system of claim 19 wherein said at least one sensor is a laser sensor.

22. The control system of claim 19 wherein said at least one sensor is a strain gauge.

23. The control system of claim 17 further comprising pneumatic control valvesconfigured to regulate waste fluid movement out of the CPC while maintainingaseptic conditions.

24. The control system of claim 17 further comprising:a. optical transmitters and receivers aligned across a sedimentation column in theCPC, where the transmitter emits light through flat sections on the exterior andinterior of the sedimentation column;b. the receiver is positioned approximately 180 degrees from the transmitter todetect the reduction of received light caused by the presence of cells passingbetween the flat sections, thereby generating a signal indicative of thepresence of cells within the sedimentation column.

25. The control system of claim 24 wherein the optical detection system is configured torotate about the sedimentation column at a configurable rotational velocity, acquiringtransmitted optical data at a set of rotational angles over a configurable period of time.

26. The control system of claim 24 wherein the receiver is positioned at a correspondingvertical location adjacent to the transmitter to detect the relative optical reflectiveintensity caused by the presence and density of cells within the column, therebygenerating a signal that corresponds to the cell presence and density within thesedimentation column of the cassette.

27. The control system of claim 26 wherein the optical detection system is configured torotate about the sedimentation column at a configurable rotational velocity, acquiringtransmitted optical data at a set of rotational angles over a configurable period of time.

28. The control system of claim 17, further comprising an optical detection systemcomprising two sets of at least one transmitter and receiver pair oppositely positionedacross the sedimentation column, wherein:a. each transmitter emits light through flat sections on the exterior and interior ofthe sedimentation column;b. each receiver is positioned approximately 180 degrees from its pairedtransmitter to detect the reduction of received light caused by the presence ofcells passing between the flat sections, thereby generating signals indicative ofthe presence of cells within the sedimentation column; andc. the second set of transmitter and receiver pairs is arranged at a 90-degreeoffset from the first set, providing cell presence data from two distinct angularperspectives.

29. A method for analyzing cell suspension within a cell processing system, comprising:a. utilizing a Transfer Syringe Cassette (TSC) to draw a predetermined volumeof cell suspension after the mixing process within the cell processing system;b. rotating the TSC to align with a Cell Analytics Module (CAM) integratedwithin the system or positioned as part of a Reagent / Sample Cassette (RSC),wherein the CAM is equipped to analyze the cell sample;c. expelling the sampled cell suspension from the TSC into the CAM foranalysis, wherein the CAM includes:i. an optical or impedance-based detection mechanism configured tomeasure cell concentration by detecting individual cells as they passthrough a microfluidic channel, wherein the total cell count iscalculated by multiplying the measured concentration by the knownsample volume;ii. reagents for identifying cell surface markers specific to different celltypes, such that T-cells are distinguished by the presence of CD3 andabsence of CD14; and monocytes are distinguished by the presence ofCD14 and absence of CD3;iii. quantifying separate populations of cells based on marker expression,wherein the CAM determines the number of T-cells and monocytes inthe sample, and calculates the purity of T-cells within the total cellsuspension based on the identified cell populations; andd. providing real-time feedback of the analytical results to the system, enablingprecise monitoring and control of cell suspension quality for downstreamprocesses.

Images