Optimized geometry of cell processing cartridges

Abstract

The present invention provides an AutoCell Platform (ACP), an advanced, fully automated, functionally closed system designed to revolutionize the manufacturing of genetically modified cell therapies. This innovative platform integrates automation, closed-loop processing, and novel technologies to address the inefficiencies and high costs of traditional methods. Key features include automated centrifugal seeding for enhanced genetic modification efficiency, microbubble-assisted cell selection for precise cell isolation, and a modular design for scalable amplification across clinical and research environments. By reducing production timelines from 30-40 days to less than 3 days and orders of magnitude in cost, the ACP enables instant manufacturing, decentralization, and broader accessibility. Advanced quality control measures and standardized design ensure compliance and consistent therapeutic outcomes. The ACP supports a variety of applications from CAR-T therapies to regenerative medicine, representing a transformative leap in global accessibility of precision medicine and life-saving cell therapies.

Description

Related applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 616,710, filed December 31, 2023, which is incorporated herein by reference in its entirety. Technical Field

[0002] This disclosure relates generally to cell therapy, and more particularly to an apparatus capable of separating target cells from non-target cells from blood or blood products and genetically modifying said target cells for clinical use in the fields of cell and gene therapy. Background Technology

[0003] Genetically engineered autologous cell therapies, utilizing a patient's own cells that have been recombined and expanded in vitro, represent a revolutionary approach to addressing complex diseases with high mortality and morbidity rates. These therapies use genetic modification to correct underlying defects, providing transformative health outcomes for conditions where conventional treatments have limited or no effect. Such gene-modified cell therapies, including those targeting hematopoietic cells (e.g., T cells, NK (natural killer) cells, and stem cells), have become a cornerstone of modern precision medicine, enabling targeted and durable interventions.

[0004] The field is rapidly evolving, driven by recent regulatory approvals and a robust development pipeline. Since 2017, multiple CAR-T cell therapies have received FDA approval for hematologic malignancies, and as of the end of 2019, over 1,000 additional gene-modified therapies were in clinical trials in the United States. In addition to CAR-T therapies, CD34+ hematopoietic stem cells (HSCs) have shown similar promise for hereditary diseases, including β-thalassemia and severe combined immunodeficiency (SCID), with two therapies approved in the European Union as of 2019 and over 31 pediatric trials underway in the United States. Market forecasts indicate a significant expansion of the global gene therapy market, projected to grow from $18 billion in 2023 to over $97 billion in 2033. This growth reflects the increasing potential and demand for gene-modified cell therapies.

[0005] CAR-T therapy, initially developed to combat blood cancers, is now being explored for a broader spectrum of conditions, significantly expanding the potential patient population. These therapies have shown remarkable promise in addressing a wide range of indications: Genetically modified immune cells, particularly CAR-T cells and NK cells, can precisely target and eliminate cancer cells in hematologic malignancies and solid tumors, providing a superior alternative to traditional treatments such as chemotherapy, radiotherapy, and stem cell transplantation for hematopoietic reconstitution, which are often accompanied by severe side effects and limited efficacy.

[0006] For hemoglobinopathies, available therapies include the use of genetically modified hematopoietic stem cells, which offer a potential cure for genetic disorders such as sickle cell disease and beta-thalassemia by addressing the fundamental defects in hemoglobin production. Gene-modified therapies are also used to treat autoimmune diseases by engineering immune cells to modulate or eliminate dysfunctional immune responses. These therapies offer new hope for chronic autoimmune diseases for which there are currently no definitive cures, such as lupus, rheumatoid arthritis, and multiple sclerosis.

[0007] Emerging research has shown that senescent cells play a role in aging and related diseases. Gene-modified cell therapies can selectively target and eliminate these cells, alleviating age-related symptoms, improving quality of life, and potentially extending healthy lifespan.

[0008] These advances mark a paradigm shift in medicine, highlighting the precision and adaptability of gene modification in addressing the root causes of complex diseases rather than merely controlling symptoms. However, the potential of gene-modified cell therapies is hampered by significant challenges in manufacturing, scalability, and accessibility.

[0009] Current challenges in manufacturing processes—despite their clinical promise, the production of gene-modified cell therapies is hampered by inefficiencies, high costs, and difficulty in scaling up. Reliance on outdated technologies and labor-intensive workflows exacerbates these problems, making it difficult to meet the growing demand for these therapies.

[0010] Outdated and complex technologies—conventional manufacturing relies on a variety of different, aging equipment and processes that are inefficient, prone to cell loss, and pose a risk of contamination, reportedly resulting in up to 92.1% T cell loss, requiring additional in vitro expansion steps to restore the cell population. These steps increase production time, variability, cell exhaustion, and contamination risks. Traditional magnetic bead-based systems permanently attach magnetic beads to cells, are inefficient, and lack the flexibility required for sequential sorting, limiting their practicality for producing specialized cell subpopulations such as memory T cells. Similarly, while flow cytometry can achieve high purity through multi-parameter sorting, its low throughput makes it unsuitable for clinical-scale production.

[0011] The lengthy and inefficient processing—the current CAR-T production vein-to-vein timeline is 30-40 days, with most of that time spent on in vitro expansion to compensate for cell loss. These extended timelines increase the risk of cell exhaustion and depletion of key subsets, such as memory T cells, which are crucial for sustained therapeutic efficacy. Centralized manufacturing further exacerbates the delays, as cells must be frozen and then transported between different facilities for different processing stages, which reduces cell viability, poses manufacturing challenges due to tumor cell lysis, and increases logistical complexity.

[0012] High costs and limited accessibility—the inefficiency of traditional approaches translates into extremely high costs, typically ranging from $350,000 to $500,000 per patient, with some gene therapies costing over $4 million per dose. These costs, coupled with the slow production process, limit access to these treatments for many patients and restrict their adoption within the healthcare system.

[0013] Limited capacity for clinical and research applications—existing production systems lack the capability to handle the demands of large-scale clinical applications or rapid iterations for research purposes. This limited capacity slows the development and testing of next-generation therapies, hindering innovation and the ability to serve a broader patient population.

[0014] As this trend intensifies over time—with gene-modified cell therapies being tailored to treat a wider range of diseases—such as solid tumors, autoimmune diseases, neuromuscular disorders, and age-related conditions—the shortcomings of current manufacturing methods will become increasingly apparent. This growing demand underscores the urgent need for scalable, efficient, and cost-effective production solutions.

[0015] Emerging approaches and other challenges – innovative methods are being developed to address these challenges. Alternatives such as automation, nonviral vectors, and stable cell line platforms for recombinant adeno-associated virus (rAAV) production show promise in improving scalability, consistency, and cost-effectiveness. However, these approaches are often in their early stages and face hurdles in regulatory approval and commercial scalability. For example, while stable rAAV production cell lines can streamline production timelines and improve reliability, they must overcome difficulties in balancing cell viability with productivity and stability, particularly in the presence of toxic reps / helper genes.

[0016] This invention This invention aims to address key challenges in current manufacturing processes for gene-modified cell therapies, providing a streamlined, reliable, and cost-effective platform for producing these transformative therapies. This invention introduces a transformative platform designed to overcome the limitations of conventional manufacturing processes. This platform, sometimes referred to herein as the AutoCell platform (ACP), integrates advanced automation, closed-loop processing, and innovative technologies designed to streamline workflows, reduce cell loss, and shorten production timelines, resulting in orders of magnitude lower costs. The main objectives of this invention are as follows: The first objective of this invention is to combine advanced automation with functionally closed system technology to achieve a highly efficient and controlled manufacturing process. By minimizing human intervention, this closed-loop design significantly reduces the risk of contamination and human error, improving the safety, consistency, and scalability of gene-modified cell therapy manufacturing. Traditional methods typically require 30-40 days to complete a batch due to labor-intensive steps such as washing, selection, activation, transduction, and formulation; this is reduced to less than 3 days using this platform. This eliminates the need for in vitro expansion to replace cells lost during manufacturing, increasing efficiency and reducing manufacturing costs to approximately one-tenth of conventional levels, making these therapies more affordable and readily available.

[0017] Another objective of this invention is to provide a platform for introducing an automated spinoculation process, a novel centrifugation-based method that combines centrifugation and inoculation (i.e., spinoculation) to significantly improve the uptake of genetic material by target cells. Conventional methods typically achieve only 25%-35% efficiency. The automated spinoculation of this invention can consistently achieve 70% efficiency, improving the quality and reliability of genetically modified cells. This technology is particularly critical for gene modification of target cells within a single container, ensuring the successful delivery of high levels of genetic material to target cells with minimal variability.

[0018] Another objective of this invention is to provide a scalable platform with a modular, standardized design that allows for standardized manufacturing in a variety of clinical and research settings. Unlike traditional custom systems tailored to specific facilities and therapies, this platform can be replicated and deployed in diverse locations, including smaller or remote medical centers. This adaptability ensures that patients can receive treatment directly at their treatment centers, reducing logistical challenges such as transportation, freezing, and storage that often compromise cell viability. By supporting both centralized and decentralized manufacturing models, this invention expands the accessibility of advanced therapies while maintaining a high level of consistency and quality.

[0019] Another objective of this invention is to provide precision in cell selection and modification, such as microvesicle-assisted cell selection (MBCSA) and aptamer-guided targeting, to achieve unprecedented accuracy in the isolation and activation of specific cell populations. These advanced methods, by focusing on key subpopulations to maximize therapeutic efficacy, enable the production of high-quality CAR-T cells, NK (natural killer) cells, and genetically modified stem cells. Unlike conventional techniques that typically result in high levels of cell loss and contamination, ACP's precise selection and modification process ensures optimal purity and functionality of the final therapeutic product.

[0020] Microbubble-assisted cell selection (MBCSA) is a key innovation within ACP, enabling sequential and highly specific target cell separation. By utilizing microbubbles that attach to target cells and provide buoyancy, the platform separates selected cells from the overall population, achieving multi-step enrichment without compromising cell viability. Then, after separation has occurred and non-target cells have been removed, buoyancy is eliminated by a moderate increase in pressure within the container, causing the microbubbles to burst and allowing the target cells to proceed to further processing steps. Similarly, aptamer-guided targeting employs molecularly bound agents with high specificity for specific cell surface markers, ensuring precise activation and retention of desired cell subpopulations.

[0021] The precision of ACP is particularly important for the development of CAR-T cell therapies, where the composition and quality of T cell subsets directly impact clinical outcomes. CAR-T products derived from carefully enriched subsets (such as CD8+ cytotoxic T cells and CD4+ helper T cells) have been known to exhibit superior anti-tumor efficacy. ACP has demonstrated its ability to successfully enrich CD8+ T cells from the general T cell population, highlighting its ability to enhance the therapeutic efficacy of genetically modified cell products.

[0022] Another object of the present invention is to provide a versatile and customizable platform that can accommodate a variety of cell types and therapies, including hematopoietic stem cells, T cells, and NK cells. The customizable approach allows the system to be adapted to the unique requirements of different therapies, from CAR-T therapy for cancer to regenerative therapies for age-related conditions. This versatility makes the platform a valuable tool across a range of gene-modification therapies.

[0023] Another object of the present invention is to provide a compact, automated platform that can be deployed in FDA-approved transplant centers or other localized healthcare facilities. This patient-centric setup reduces patient accessibility issues 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 advanced therapies for underserved populations and eliminates many of the inefficiencies inherent in centralized manufacturing models.

[0024] Another objective of this invention is to provide research and clinical support. The platform's automated and standardized processes allow researchers to focus on developing effective gene constructs, rather than labor-intensive manufacturing tasks. This simplification accelerates translational research, enabling scientists to rapidly iterate on therapeutic vector designs and advance innovative therapies to clinical trials more quickly. By supporting 592 cell biology research facilities across the United States, this invention fosters a more dynamic and productive research environment.

[0025] Another objective of this invention is to provide a platform incorporating robust and automated quality control measures, including automated pressure decay testing for filter integrity checks and a septum sterilization module for aseptic transfer operations, ensuring compliance with stringent FDA standards. These built-in features reduce the risks of contamination, batch failures, and regulatory delays. The platform's integrated data tracking and batch release record collection in real time, provided concurrently with the harvesting of genetically modified target cells, further simplifies regulatory submissions, accelerates the approval of new therapies, and ensures that each batch meets stringent quality standards.

[0026] Another objective of this invention is to address the long-standing challenges of high cost, inefficiency, and logistical constraints that have limited the application of gene-editing therapies. By reducing production costs and shortening timelines, this platform significantly enhances the scalability of these therapies, making them a viable option for a broader patient population. This shift expands market opportunities and positions the platform as a foundational technology for next-generation cell therapies.

[0027] Another objective of this invention is to provide innovative capabilities to support the development of therapies for diseases previously considered untreatable, including solid tumors, autoimmune diseases, and age-related degenerative diseases. Faster production timelines enable patients with aggressive diseases to receive timely treatment, including at initial diagnosis; while reduced costs make these therapies a sustainable option for chronic diseases. The platform's flexibility and precision open new therapeutic possibilities, paving the way for life-saving and life-extending treatments.

[0028] A further objective of this invention is to provide a platform that supports both centralized and decentralized manufacturing methods, offering flexibility to meet diverse clinical and logistical needs. Centralized facilities benefit from streamlined, high-throughput production, while decentralized setups enable faster “vein-to-vein” times, shortening the overall timeline from apheresis to reinfusion and improving patient outcomes. This dual capability transforms the logistical paradigm of cell therapy manufacturing, ensuring that therapies can be delivered when and where they are needed.

[0029] This invention represents a transformative approach to producing gene-modified cell therapies. By addressing the inefficiencies, high costs, and scalability challenges of conventional methods, this platform improves access to these life-saving treatments for a broader patient population. Its innovative design enhances research, accelerates regulatory approval, and enables the development of new treatment options, ultimately advancing the frontiers of precision medicine and expanding the global reach of gene-modified therapies. Summary of the Invention

[0030] The AutoCell platform (ACP) is a fully automated, functionally closed cell processing system designed to facilitate the sterile, FDA-compliant transfer of reagents, buffers, culture media, and gases through various processing cartridges. The platform employs advanced robotic control to coordinate the precise movement and interactions between the cartridges. This integration of robotic precision with a sterile, functionally closed environment makes ACP highly effective for manufacturing CAR T cells and other cell-based therapies. By minimizing contamination risk and optimizing automation, ACP ensures the production of high-quality cell products suitable for clinical applications.

[0031] The system employs a series of boxes, including a cell processing box (CPC), in which target cells are introduced, selected, enriched, genetically modified, washed, and concentrated.

[0032] The robotic control system ensures that each cartridge operates in a coordinated sequence, maintaining sterile conditions and eliminating the need for manual intervention. This system minimizes the risk of contamination while improving the accuracy and efficiency of reagent and fluid management. In one implementation, ACP's robotic mechanism precisely controls the positioning and manipulation of the support cartridges: Transfer syringe cartridge (TSC): This cartridge manages the precise transfer of reagents and fluids into the CPC, enabling accurate volumetric quantification for processes such as washing, activation, and transduction.

[0033] Reagent / Sample Cassette (RSC): The RSC contains multiple 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, carriers, and adapters. Additionally, the RSC contains designated sample vials, ready to receive cell samples for quality control (QC) analysis throughout the processing cycle.

[0034] Process Fluid Container (PFC): PFC stores various process fluids required for different stages of cell processing, such as buffers and culture media. These fluids are automatically transferred to the CPC as needed to support cell washing, sedimentation, and formulation.

[0035] The CPC incorporates an innovative internal shape for efficient CAR-T cell processing. The funnel-shaped internal chamber not only allows for efficient and uniform sedimentation but also enables targeted sedimentation, as reagents or carriers can be injected directly into the bottom of the sedimentation column, maximizing contact with the cell population and achieving microbubble integration. This shape also allows for better microbubble integration: due to the natural bubbling of microbubbles, the system is optimized to connect microbubbles to target cells in an efficient and controlled manner. Throughout the process, an automated balancing mechanism maintains perfect equilibrium, even during barrel and CPC movement. This balance ensures stable operation and prevents mechanical stress on the centrifuge system and target cells. The ACP retains more cells than traditional centralized manufacturing, where cells often experience loss due to freezing, transport, and multiple transfers. A central pneumatic system enables precise fluid movement and aseptic transfer, further ensuring cells remain uncontaminated.

[0036] After manufacturing is complete, ACP automatically compiles and publishes a QC batch record, which includes all tracking parameters and relevant data for that batch. This streamlined record enables rapid access and review, ensuring compliance with regulatory requirements while minimizing the time required for documentation. Pharmaceutical-grade cell therapy facilities, particularly those specializing in CAR T-cell production, typically employ more QC personnel than manufacturing personnel due to the traditionally exhaustive documentation review approach. ACP's integrated QC automation and exception release capabilities address this imbalance, allowing facilities to allocate resources more efficiently and reduce costs associated with cell therapy manufacturing. Attached Figure Description

[0037] To facilitate a clearer understanding of the various elements and embodiments illustrated herein, the accompanying drawings are not necessarily drawn to scale. Furthermore, to provide a clear view of the various embodiments of the invention, elements well-known and familiar in the art are not depicted; therefore, for the purposes of clarity and brevity, the drawings are general in form.

[0038] Figure 1 A perspective view of a cell processing device in a closed mode according to a preferred embodiment of the present invention is shown; Figure 2 A perspective view of the cell processing device in open mode is shown; Figure 3 It shows Figure 1 and 2 The depicted cross-sectional side view of the cell processing apparatus illustrates a centrifuge tank according to a preferred embodiment of the present invention; Figure 4 A preferred embodiment of the invention is shown, relative to Figure 1-3A perspective view of multiple boxes in the lower housing assembly of the depicted cell processing device; Figure 5 A perspective view of the bottle and multiple boxes is shown, wherein, according to a preferred embodiment of the invention, the depiction of the upper housing assembly and the lower housing assembly is removed; Figure 6 and Figure 7 Two operating modes of the centrifuge in the cell processing apparatus according to a preferred embodiment of the present invention are shown; Figure 8 A perspective view of the top surface of the CPC according to a preferred embodiment is depicted; Figure 9 This is a perspective view of the top surface of CPC according to an alternative implementation scheme; Figure 10A and 10B This is an exploded perspective view of three filters according to an embodiment of the present invention; Figure 11 A partial exploded view of an alternative embodiment of the centrifuge tank is shown; Figure 12 An exploded view of the harvest valve assembly is shown; Figure 13-15 An exploded view of a CPC according to an embodiment of the present invention is shown; Figure 16 and 17 This is a cross-sectional view of CPC; Figure 18 This is a cross-sectional side view of the CPC according to an alternative embodiment of the present invention; Figure 19 A plan view of a CPC according to an embodiment of the present invention is shown; Figure 20 A side view of a CPC according to an embodiment of the present invention is shown, wherein section lines DD, EE, and FF are respectively located at... Figure 26 , 27 As shown in more detail in 28; Figure 21-25 A cross-sectional view of a CPC according to an embodiment of the present invention is shown; Figure 26 It shows in Figure 20 A top view of a CPC filter according to an embodiment of the present invention, shown at section line DD in the figure; Figure 27 Depicting in Figure 20 A top view of the CPC main chamber according to an embodiment of the present invention is shown at section line EE in the figure; Figure 28 Depicting in Figure 20 The top view of the CPC rotary valve according to an embodiment of the present invention is shown at section line FF in the figure; Figure 29 A cross-sectional view of the CPC rotary valve is depicted, showing a section of the recovery pipe; Figure 30 This is the top perspective view of CPC; Figure 31 This is a top section view of CPC, showing section lines AA, BB, and CC, which define... Figure 32 , 33 and the views provided in 34; Figure 32 This is a side view of CPC, showing section lines D1-D1, E1-E1, and F1-F1; Figure 33 It comes from Figure 31 A cross-sectional view of section line BB; Figure 34 It comes from Figure 21 A cross-sectional view of section line CC; Figure 35 It comes from Figure 32 Cross-sectional view along section line D1-D1; Figure 36 It comes from Figure 32 Cross-sectional view of section line E1-E1; Figure 37 It comes from Figure 32 Cross-sectional view of section line F1-F1; Figure 38 A plan view of a CPC according to an embodiment of the present invention is shown; Figure 39 A cross-section of CPC is shown; Figure 40A A perspective view of a process fluid cartridge (PFC) according to a preferred embodiment of the present invention is shown; Figure 40B A plan view of the PFC is shown; Figure 40C A first cross-sectional view of the PFC is shown; Figure 42A A perspective view of a reagent / sample cartridge (RSC) according to a preferred embodiment of the present invention is shown; Figure 42B A plan view of the reagent / sample cartridge (RSC) is shown; Figures 43A-43C Various views of a transfer syringe cartridge (TSC) according to a preferred embodiment of the present invention are shown; Figures 44A-44B A cross-sectional side view of the interaction between the reagent syringe and the vial cartridge of a TSC according to a preferred embodiment of the present invention is shown. Figure 44C-44DA cross-sectional side view of the interaction between the sampling syringe of the TSC according to a preferred embodiment of the present invention and the processing cartridge and vial cartridge is shown. Figure 45A An actuator comprising a first clamping mechanism and a second clamping mechanism according to a preferred embodiment of the present invention is shown; Figure 45B A preferred embodiment of the invention is shown, in which the second clamping mechanism of the actuator is from Figure 45A Move the position shown downwards; Figure 45C The invention illustrates a preferred embodiment of the first clamping mechanism of a downwardly moving actuator and a finger of the first clamping mechanism that moves to a closed state. Figure 45D The invention illustrates a preferred embodiment in which the actuator moves downward to push the needle of the reagent syringe out of the bottom end of the TSC; Figure 45E The invention shows a preferred embodiment in which the second clamping mechanism moves upward while the first clamping mechanism remains stationary. Figure 45F According to a preferred embodiment of the invention, the actuator moves upward to retract the needle; Figure 45G This illustration shows a configuration in which the actuator pushes the reagent syringe downwards to engage the needle into another cartridge, according to a preferred embodiment of the invention. Figure 45H The invention illustrates a preferred embodiment in which the second clamping mechanism is moved downward to press the plunger downward, thereby discharging fluid into other cartridges; Figure 45I An actuator is shown that moves upward to retract the needle after the TSC, according to a preferred embodiment of the invention; Figure 45J-45K According to a preferred embodiment of the invention, the actuator is disengaging the first and second clamping mechanisms from the reagent syringe; Figure 46 A cross-sectional side view illustrating how a process fluid cartridge (PFC) according to a preferred embodiment of the present invention transfers fluid to a CPC is shown. Figures 47-48 This is a perspective view of an exemplary CAN bus wiring according to an alternative embodiment of the present invention; Figures 49A-49B This is a cross-sectional view of a centrifuge according to a preferred embodiment of the present invention, wherein the centrifuge drum is in its mixing mode; Figure 49C This is a cross-sectional view of a centrifuge in rotation according to a preferred embodiment of the present invention; Figure 49DThis is a cross-sectional view of a centrifuge in a stationary state according to a preferred embodiment of the present invention; Figure 50 Features of the heating mechanism of a centrifuge tank according to a preferred embodiment of the present invention are shown; Figure 51A and 51B The invention illustrates the use of microbeads to facilitate unique compression and release cycles for cell selection and concentration according to a preferred embodiment of the invention. Figure 52 This invention illustrates a preferred embodiment of which an optical encoder is used to optically measure valve rotation; Figures 53A-53B The four main rotational positions of the CPC valve are shown according to a preferred embodiment of the present invention; Figure 54 A cell processing cassette in active mode is shown according to a preferred embodiment of the present invention; Figure 55A-55J The invention illustrates a preferred embodiment in which the CPC valve rotates through a series of positions to facilitate the step-by-step processing involved in CAR T cell production; Figure 56 A plan view of a portion of the CPC is shown, with the highlighted component being part of the aseptic transfer process; Figure 57A This is a perspective view of a part of the ACP, showing the waste container; Figure 57B A carbon dioxide-rich container is shown as part of an aseptic transfer process; Figures 58A-58D The cell suspension when CPC is tilted is shown according to a preferred embodiment of the present invention; Figure 59A The cross-sectional line HH passing through CPC is shown according to a preferred embodiment of the invention; Figure 59B A cross section along HH is shown, illustrating the cell suspension fluid in the main chamber according to a preferred embodiment of the present invention; Figure 59C According to a preferred embodiment of the invention, fluid moves to other chambers; Figure 59D This illustration shows that, according to a preferred embodiment of the invention, fluid from the main chamber continues to push fluid upwards in the standpipe; Figure 60A A radar system for use with a CPC is shown according to a preferred embodiment of the present invention; Figure 60B An S-shaped channel according to a preferred embodiment of the invention is shown, which guides radar from a radar transmitter positioned above and centered on the CPC cover; Figure 60C A preferred embodiment of the present invention is shown for use in Figure 60A and 60B The rod of the radar system shown; and Figure 61A and 61B A perspective view of CPC is shown, which has... Figure 61A In the form of coiling and in Figure 61B The short section of the sealed tube is shown in a non-coiled form. Detailed Implementation

[0039] In the following discussion, various embodiments and applications of the invention are discussed, with reference to the accompanying drawings, which form a part of the invention, and specific embodiments in which the invention can be practiced are illustrated by way of example in the drawings. It should be understood that other embodiments can be utilized and changes can be made without departing from the scope of the invention.

[0040] Various inventive features are described below, each of which can be used independently of each other or in combination with other features. However, no single inventive feature may solve all or any of the problems discussed above. Furthermore, one or more of the problems discussed above may not be fully solved by any of the features described below.

[0041] As used herein, unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” include plural referents. Unless otherwise clearly stated, “and” and “or” are used interchangeably herein. As used herein, the term “about” means + / - 5% of the stated parameter. Unless the context clearly indicates otherwise, all embodiments of any aspect of the invention may be used in combination.

[0042] Unless the context explicitly requires it, throughout the specification and claims, the terms "comprising," "including," etc., should be interpreted in an inclusive sense rather than an exclusive or exhaustive sense; that is, they mean "including but not limited to." The use of singular or plural terms also includes both singular and plural forms, respectively. Furthermore, the terms "this article," "wherein," "wherein," "above," "below," and similar terms, when used in this application, should refer to the entire application and not any specific part thereof.

[0043] The description of embodiments of this disclosure is not intended to be exhaustive or to limit this disclosure to the precise form disclosed. While specific embodiments and examples of this disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of this disclosure, as will be recognized by those skilled in the art.

[0044] This application describes an AutoCell platform (ACP), an advanced, fully automated, functionally closed cell processing system specifically designed for the aseptic and FDA-compliant transfer of critical reagents, buffers, culture media, and gases via the CPC's cap. The platform utilizes robotic control to meticulously plan the precise movement and coordinated interaction between its integral components, including the transfer syringe cartridge (TSC), reagent / sample cartridge (RSC), and process fluid cartridge (PFC). By seamlessly integrating robotic precision with a sterile, functionally closed processing environment, ACP ensures high-quality, contamination-free production of CAR T-cell and other cell-based therapies. This innovative system not only mitigates the risk of contamination but also streamlines manufacturing workflows, enabling the efficient and consistent production of therapeutic-grade cell products for clinical applications.

[0045] ACP operates as a functionally closed system. A functionally closed system is a controlled environment that can operate without exposure to external contamination. All material transfers into and out of the system are performed aseptically or through sealed mechanisms designed to maintain the aseptic integrity of the internal processes. Asepticity is ensured throughout all operations by maintaining the aseptic state within the CPC through the use of aseptic connectors, aseptic transfer ports, and sealed paths. The internal environment is also completely isolated from the risk of external contamination. Finally, the system is designed to support material exchange without compromising asepticity, ensuring consistent and reliable functionality.

[0046] The various embodiments of the present invention described herein address at least the technical problem of autonomously applying multiple cell processing steps without the need for manual intervention by laboratory personnel. Specifically, a cell processing device configured to separate target cells from non-target cells and then modify the separated target cells is described. The ACP can be configured to operate on many different types of target cells, including but not limited to T cells, NK cells, and CD34+ HPSCs.

[0047] Now refer to the attached diagram, Figure 1-2 A perspective view of the cell processing device 100 is shown. Specifically, Figure 1A perspective view of a cell processing device 100 in a closed state is shown. The cell processing device 100 includes an upper housing assembly 102 hingedly attached to a lower housing assembly 104. The upper housing assembly 102 includes a first door 106 that allows one or more cartridges to be inserted into or removed from the upper housing assembly 102. The first door 106 can be configured to slide or swing between its open and closed states. The lower housing assembly 104 may include one or more controls for manipulating settings of the cell processing device 100. Specifically, the one or more controls may include an on / off button 108, a touch-sensitive electronic display 110, and an auxiliary control 112. In some embodiments, the auxiliary control 112 may be configured to initiate the shutdown or termination of operation of the cell processing device 100. The touch-sensitive electronic display 110 may also be configured to display multiple operating parameters associated with the cell processing device 100 and may be configured to allow an operator to change various operating parameters of the cell processing device 100. The lower housing assembly 104 further includes a second door 113 that allows one or more vials to be inserted into or removed from the lower housing assembly 104 without separating the upper housing assembly 102 from the lower housing assembly 104.

[0048] Figure 2 The upper housing assembly 102 is shown rotating away from the lower housing assembly 104 to allow one or more disposable cartridges to be removed from or inserted into the lower housing assembly 104. The lower housing assembly 104 further defines a centrifuge well 114 for accommodating the operation of a processing device in the form of a centrifuge 116. As depicted, the centrifuge 116 can be a horizontal or bucket centrifuge, including racks or centrifuge buckets 120 for holding sample containers. As depicted, the buckets 120 are rotatably coupled to opposite ends of a rotor yoke 122. During some operating modes of the centrifuge 116, the rotor yoke 122 rotates about an axis of rotation 124. In some embodiments, a fairing or aerodynamic cover may be employed around the rotating components to minimize unwanted air resistance during centrifugation, improve system operating efficiency, and optimize energy use during high-speed rotation. Further cross-sectional views are shown in... Figures 49A-49D As shown in the image.

[0049] It should be understood that in some embodiments, the processing device may include a greater number of buckets to accommodate additional containers (e.g., processing container 212 described below). For example, the rotor yoke 122 may include multiple buckets with additional buckets, which would allow the cell processing device to perform a greater number of parallel operations. In alternative embodiments, the platform may operate using two buckets and two counterweights, thereby effectively running two independent processes simultaneously. For example, if the platform is processing two leukocyte apheresis collections—one containing 150 mL and the other containing 200 mL—an additional 50 mL can be added to the smaller collection to make the volumes equal at 200 mL each. This approach eliminates the need for counterweights because the identical processes running in parallel in balanced buckets ensure system stability without the need for additional adjustments.

[0050] The lower housing assembly 104 further includes a vial holder recess 126 to receive a cartridge configured to hold a plurality of vials containing different reagents to assist in the operation of the cell processing device 100. In some embodiments, the cartridge received by the vial holder recess 126 may also include empty vials configured to receive material samples during operation of the cell processing device 100. Material samples can help confirm the correct operation of the cell processing device 100 and / or calibrate subsequent operation of the cell processing device 100. The upper housing assembly 102 includes through-holes 128 and 130 through which cartridges disposed within the upper housing assembly 102 extend to interact with cartridges disposed within the vial holder recess 126 and the cartridge 120. The lower housing assembly 104 also defines a bottle recess 127 configured to receive a bottle for collecting waste.

[0051] In some embodiments, the cell processing device 100 may include rubberized feet 101, which help dissipate any vibrations transmitted through the lower housing assembly 104 via the rotation of the centrifuge 116. In some embodiments, the motor may be a stepper motor. In other embodiments, a custom-designed centrifuge is used, wherein the space between the drums is substantially filled to minimize airflow and thus air resistance and noise as the centrifuge rotates. In this embodiment, the shape may be contoured to allow rotation of the drums and to largely fill the air space when the drums are nearly horizontal, i.e., when the centrifuge is at a high RPM.

[0052] Figure 3A cross-sectional side view of the cell processing device 100 is shown. The upper housing assembly 102 is shown to include a cartridge positioning assembly 132 that defines a syringe cartridge recess 134 and a fluid cartridge recess 136. The cartridge positioning assembly 132 is configured to exchange the positions of cartridges positioned within recesses 134 and 136 in a Lazy Susan turntable manner. In addition to repositioning the cartridges within recesses 134 and 136, the cartridge positioning assembly 132 may be further configured to influence the rotation of the cartridges within their respective recesses about their own axis of rotation (not shown) and to lower the cartridges at least partially into through-holes 128 or 130 to allow the cartridges located in recesses 134 and 136 to interact with the cartridges located in vial cartridge recesses 126 or barrels 120. In some embodiments, recesses 134 and 136 of the cartridge positioning assembly 132 may each include an aperture, thereby allowing the cartridges to be lowered through through-holes 128 or 130, respectively. It should be noted that in some embodiments, the cartridge positioning assembly 132 may be configured to carry more than two cartridges. For example, the third box may take the form of a test box to verify the correct operation of the box placed in the centrifuge tank 114. The test box allows the cell processing device 100 to verify the correct operation of various consumables (e.g., filters) during and / or after various cell processing operations.

[0053] Figure 4 This is a perspective view showing the relative positioning of the cartridge with respect to the lower housing assembly 104. The upper housing assembly 102 has been removed to more clearly show the position of the cartridge relative to the lower housing assembly 104. Specifically, RSC 202 is shown positioned within the vial recess 126 directly below PFC 204. Waste tube 208 is shown, configured to receive waste generated during operation of the cell processing device. Waste tube 208 may be attached to PFC 204 and / or TSC 210, each of which may include a waste channel allowing waste to be extracted through a corresponding cartridge. Figure 4 As depicted, only PFC204 is attached to waste tube 208. PFC204 stores various process fluids required for different stages of cell processing, such as buffers and culture media. These fluids are automatically transferred to the cell processing cartridge (CPC) 212 as needed to support cell washing, sedimentation, and formulation. Removal of waste from CPC212 is achieved by guiding pressurized air through pneumatic channels extending through PFC204 and subsequently into processing cartridge 212, which in turn forces the waste liquid out of CPC212.

[0054] The wastewater port and fluid waste disposal system control liquid removal while maintaining target cell integrity and minimizing cell loss during cell treatment. The wastewater port serves as a one-way outlet for fluid removal and is positioned at the center of the CPC cap. Its height and placement are configured to ensure efficient fluid removal while leaving a precise minimum volume of liquid—15 mL—at the bottom of the settling chamber when the CPC is upright and stationary. This design ensures that the target cells, safely settled and isolated at the bottom of the settling chamber, remain undisturbed during fluid extraction. The 15 mL volume left is consistent with the design intent of protecting the target cell population during treatment. Figure 1 The exact volume of residual fluid may vary slightly during multiple runs due to variations in the initial fluid volume or operating conditions. The fluid waste disposal tube is located in the center of the CPC and extends downward into the cone, reaching a fluid level of 15 mL when the CPC is upright and stationary.

[0055] The waste disposal tube serves as the primary discharge path for fluid removal, extending upwards through the hollow central tube of the docked PFC and into an expandable, sealed waste container. Positive air pressure is introduced into the CPC via a hydrophobic filter in the CPC cap, propelling fluid upwards through the waste disposal tube. During this process, the CPC valve rotates to one of four residence positions in the stator. This ensures that there is no other path for fluid to leave the CPC except through the centrally positioned waste disposal tube. The centered positioning of the tube also ensures uniform and controlled fluid movement, preventing turbulence or disturbance of cell deposits. The flow rate is regulated to avoid wicking a significant number of clinically significant cells—99.9% of the target cells remain undisturbed. This precision preserves the integrity of the isolated target cells while ensuring effective waste removal.

[0056] Figure 5 A perspective view of all the boxes in the system according to a preferred embodiment is shown, with particular emphasis on RSC202, PFC204, TSC210, and CPC212. Figure 5 The illustration also shows how the drum 120 is configured to rotate about the axis of rotation 218 during centrifuge 116 operation.

[0057] Figure 6-7 Two operating modes of centrifuge 116 are shown. Specifically, Figure 6 The first operating mode is shown, in which centrifuge 116 operates in centrifugation mode, and Figure 7 A second operating mode is shown, in which centrifuge 116 operates in mixing mode. Figure 6 In the first operating mode shown, the mixing motor associated with barrel 120 can be configured to further tilt barrel 120 so that the top surface of processing container 212 directly faces the axis of rotation 124. The angle depicted is a typical angle achieved when a force of approximately 50G is applied to the sample contained within processing container 212.

[0058] Figure 7 Centrifuge 116 is depicted operating in a second operating mode, and therefore, the bottom surface of barrel 120 is shown tilted towards the axis of rotation 124. The rotation angle and speed of barrel 120 can vary depending on the desired mixing motion of the sample material within processing chamber 212. Furthermore, when actively in mixing operating mode, barrel 120 will typically tilt towards and away from the axis of rotation 124. Typically, rotor yoke 122 will not rotate about the axis of rotation 124, and mixing will be limited to the rotation of barrel 120 about the axis of rotation 124. In some embodiments, rotor yoke 122 may be configured to rotate much slower in the second operating mode than in the first operating mode. For example, a rotational speed of approximately 10-20 RPM about the axis of rotation 124 can cooperate with rotation about the axis of rotation 124 to provide a more complex mixing pattern.

[0059] Figure 8 This is a perspective view of CPC212. Specifically, the processing cartridge typically includes a cover 402, an upper housing component 404, and a lower housing component 406. The cover 402 includes multiple ports for assisting in the introduction and extraction of sample material into and out of the processing cartridge 212.

[0060] The CPC port includes a fluid port 408 (FIT receiver 3) for receiving culture medium and buffer solution from PFC 204. Fluid port 408 may include a one-way valve, in the form of a check valve, which allows fluid to enter the processing chamber 212 only when fluid port 408 mates with a port on PFC 204. Cover 402 further includes pneumatic ports 410 and 412. Pneumatic outlet port 412 (FIT receiver 1) allows pressurized air to be discharged from processing chamber 212 to reduce or equalize the air pressure within processing chamber 212. Pneumatic inlet port 410 (FIT receiver 2) allows air to be introduced into processing chamber 212 to increase the main chamber 424 of processing chamber 212 (see [link to relevant documentation]). Figure 16 The air pressure within the container. Details of the air pressure control system will be described below. Pneumatic ports 410 and 412 may also include check valves, with the check valve in pneumatic outlet port 412 oriented in the opposite direction to the check valve in pneumatic inlet port 410. Alignment flaps 427 for aligning the processing box 212 within the container 120 are shown at the bottom.

[0061] Cover 402 further includes diaphragms 414 and 416 (see, for example, see...) Figure 24These diaphragms are configured to interact with and receive fluid supplied by the syringes of the TSC210, or to allow fluid to be collected by the extractor of the TSC210. ​​Specifically, the diaphragms include a harvesting diaphragm 414 and a dissolving O2 well diaphragm 416. All syringes within the TSC210 are sterilized before use, and each syringe is used for only one fluid transfer operation. In some embodiments, the syringes within the TSC210 can be sterilized simultaneously by irradiating the entire TSC while it is placed inside the TSC210. ​​The upward-facing surfaces or diaphragms can be aligned in a single plane to reduce the complexity of sterilizing them before engaging diaphragms 414 and 416 with the needles of the syringes. The cap 402 includes a waste port 418 through which waste liquid exits the treatment cartridge 212 when pressurized gas (e.g., air, nitrogen, or carbon dioxide) is introduced into the treatment cartridge 212 using the pneumatic inlet port 410 and all other outlets are restricted. The cover 402 further includes an inlet port 420, which facilitates the introduction of blood samples into the processing cartridge 212 before or after insertion of the cell processing device 100 into the processing cartridge 212. Finally, the cover 402 may include an emergency recovery tube 490 and a fluid level laser window 430 that detects the internal fluid level, preferably with an accuracy of 1 mm. Figure 29 The recovery tube 490, shown again, provides an alternative means of recovering fluid or material from the CPC during processing. The fluid level laser window 430 allows for fluid volume and weight sensing, enabling real-time monitoring of fluid level and weight to aid in precise volume management and quality control.

[0062] The following features describe this multi-port configuration in detail: CPC212 includes multiple ports located in the top section of cover 402, including fluid port 408 and pneumatic ports 410 and 412, for fluid and gas transfer. The internal openings of these ports are aligned in a straight line along the highest region inside CPC212. This straight alignment ensures that when CPC212 is tilted or rotated, the fluid level can rise to its maximum permissible angle along the conical wall of the main chamber 424 without reaching any port openings, thus preventing unwanted fluid from entering any port.

[0063] To facilitate fluid injection, removal, and gas exchange, CPC requires access points, which may not align with the inline configuration inside the CPC cap. To achieve this, the external ports are positioned radially at a precise distance from the cap's geometric center to ensure compatibility and docking with automated TSCs or PFCs. Generally, the external ports are positioned to allow TSC or PFC docking by precisely rotating and lowering either cartridge directly above and centered on the CPC cap to align its outlet port with the appropriate outlet port on the CPC cap.

[0064] Fluid pathways are constructed between the external ports and their respective in-line internal openings to guide each port to its designated linear location within the CPC. These pathways bridge the offset locations of the external ports with the uniform in-line configuration of the interior. This arrangement allows the system to access each port from the outside without disrupting the internal alignment that prevents fluid from contacting the port openings during cell processing movement.

[0065] This inline internal port configuration offers several functional advantages. First, by preventing fluid from reaching the ports, the CPC can be tilted to a wider range of angles, maximizing mixing and fluid dynamics without the risk of accidental fluid ingress. Second, it provides sterile access for the syringe mechanism. The offset external ports allow the syringe or other transfer mechanism to access the CPC without compromising the safety of the inline internal configuration. Third, the fluid pathway ensures that only designated fluids or gases can enter the CPC through a controlled pathway, thus maintaining sterility and preventing accidental mixing.

[0066] Figure 8 and Figure 9 A venting assembly 425 is also shown on the outer surface of the upper housing component 404. The venting assembly 425 includes vents configured to facilitate the venting of air from the staged chambers located within the lower housing component 406. This prevents undesirable pressure build-up caused by the addition of solution from the main chamber 424 into the staged chambers. Alternatively, the treatment cassette 212 may include vents leading to the area between the cover 402 and the filters 426 or 428 to prevent any contaminants traveling through the vents from entering the main chamber 424.

[0067] Figure 9A partially exploded view of an alternative embodiment of the treatment cartridge 212 is shown, with the cover 402 removed from the upper housing component 404 to reveal a plurality of filters arranged in the top region of the upper housing component 404. Specifically, the hydrophilic filter 422 is the largest of the filters. The hydrophilic filter 422 is positioned below the fluid port 408 and is configured to prevent any contaminants present in the fluid introduced through the fluid port 408 from entering and contaminating the main chamber 424 of the treatment cartridge 212 immediately below the hydrophilic filter 422. The large area and horizontal orientation of the hydrophilic filter 422 allow the fluid introduced into the treatment cartridge 212 through the fluid port 408 to be uniformly distributed across the hydrophilic filter 422 for passage through it. In some embodiments, the hydrophilic filter 422 may have a pore size of about 0.2 micrometers, which can be used to clean impurities in buffer or culture medium solutions introduced through the fluid port 408. The processing cartridge 212 further includes hydrophobic filters 426 and 428, which allow air and / or other gases to enter and exit the processing cartridge 212 using pneumatic ports 410 and 412. Although filters 422, 426, and 428 are shown positioned within the upper housing component 404 to indicate their position when the processing cartridge 212 is closed, these filters are secured to the inward-facing surface of the cover 402 to prevent the escape of contaminants located between the filters 422, 426, and 428 and the inward-facing surface of the cover 402. Alternatively, filters 422, 426, and 428 may be attached to and / or sealed to the outward-facing surface of the cover 402, in which case ports 408, 410, and 412 may be encapsulated with the respective filters 422, 426, and 428 to form an external filter assembly.

[0068] The cover 402 is also depicted as including multiple indentations. Specifically, a fluid level laser window 430 is positioned directly below a laser sensor located on the outward-facing surface of the cover 402 and configured to measure the fluid volume within the main chamber 424 of the processing cartridge 212. An indentation 432 is positioned directly above a pH probe 434 configured to monitor the pH value of sample material placed in the main chamber 424 of the processing cartridge 212. An indentation 436 is positioned directly above a dissolved oxygen sensor that helps confirm that the amount of dissolved oxygen in the main chamber of the processing cartridge 212 is maintained at a desired level during operation of the cell processing device 100. The cover 402 further includes a waste riser 438, which provides a path through which waste liquid within the CPC 212 can be discharged via a waste port 418.

[0069] Alignment fins 427 are distributed around the periphery of the upper housing component 404 and interact with grooves in the barrel 120 to radially align the processing cassette 212 with the barrel 120. This ensures proper alignment of the openings in the downward-facing surface of the processing cassette 212. The lower housing component 406 further includes discharge ports 429-1 and 429-2, which receive material removed from the staged chamber located within the lower housing component 406.

[0070] Figure 10A and Figure 10B Three filters 422, 426 and 428 are shown arranged in the top region of the upper housing component 404.

[0071] Figure 11 A partial exploded view of an alternative embodiment of barrel 120 is shown. Specifically, Figure 11 A magnetic valve actuator is shown. This actuator includes a motor 440 configured to rotate a shaft, the distal end of which is attached to a first gear 442. The first gear 442 is configured to mesh with teeth on a second gear 444, which is orthogonally oriented relative to the first gear 442, forming a configuration sometimes referred to as a miter gear. It should be noted that spur, helical, zero-degree, miter, or quasi-hyperboloid gear configurations are also possible. Therefore, rotation of the first gear 442 by the motor 440 causes rotation of the second gear 444. The second gear 444 includes multiple grooves for accommodating a magnet 446. The magnet 446 is typically a rare-earth magnet made of a material such as neodymium or samarium cobalt. After the magnet 446 is placed in the groove of the second gear 444, rotation of the second gear 444 generates a rotating magnetic field capable of rotating a valve positioned at the bottom of the CPC212. Other methods of rotating the valve can also be used.

[0072] Figure 12 An exploded view of a harvest valve assembly is shown, comprising a first valve section 454 and a second valve section 456. The first valve section 454 is configured to rotate about its longitudinal axis relative to the second valve section 456, which is configured to remain stationary. The first valve section 454 defines a first channel section 458 configured to guide a solution received in a tapered recess 460 disposed at a first end of the first valve section 454 into one of a plurality of channel sections defined by the second valve section 456. Although in Figure 12Only the second channel segment 462 is depicted, but it should be understood that the second valve segment 456 may include multiple channel segments, at least one of which is used to guide sample material into each of a plurality of staged chambers arranged around the lower periphery of the processing cartridge 212. The first valve segment 454 also includes a non-circular second end opposite to the first end, which is configured to engage a central opening in the magnet carrier 466. This central opening may have a shape and size that matches the shape and size of the non-circular second end 464 of the first valve segment 454. Although the second end 464 is depicted as generally square, an important aspect is that its geometry allows the central opening of the magnet carrier 466 to exert a force on the second end 464 sufficient to rotate the first valve segment 454 relative to the second valve segment 456.

[0073] The magnet carrier 466 is configured with a peripheral groove sized to accommodate a plurality of magnets 468 and formed of a non-magnetic material. In this context, non-magnetic materials refer to materials such as plastics or ceramics, as well as most magnetically neutral metals such as aluminum. The use of a non-magnetic material prevents interference with the magnetic coupling between magnets 468 and magnets 446. In this way, rotation of magnets 446 in the second gear 444 causes rotation of magnets 468, which then applies a force to the first valve section 454 sufficient to align the channel section 458 with the channel section defined by the second valve section 456 when sample material needs to be discharged from the main chamber 424 of the processing cartridge 212. Although magnets 468 are described as magnets, it should be understood that in some embodiments, the magnet carrier 466 may instead carry magnetically attractive elements formed of a ferrite material, which are still responsive to the moving magnetic field generated by the rotation of the magnets carried by the second gear 444. The magnet carrier 466 is situated in a circular opening defined by the bottom of the valve section carrier 470. The valve section carrier 470 is sized to also receive the second valve section 456 and includes a vertical wall 472 to prevent rotation of the second valve section 456. The harvest valve assembly also includes a non-magnetic cap 474, which prevents scratching of the interior of the barrel 120 during rotation of the magnetic carrier 466.

[0074] Figure 13-15 An exploded view of a centrifuge tank 120 according to a preferred embodiment is shown. Specifically, the tank 120 may be modified to include an improved second gear 444, which is coupled to a drive shaft 445 via a plurality of fasteners 450. Bearing elements 447-1 and 447-2 are positioned within a through-hole extending through the bottom wall of the tank 120 and assist rotation of the second gear 444 and the drive shaft 445 within the through-hole. Any liquid that accidentally escapes from the processing chamber can be prevented from entering the recess housing the motor 440 by an environmental seal 449 surrounding the base of the drive shaft 445. Figure 13 The illustration also shows how the distal end of the drive shaft 445 can be non-circular. Specifically, the distal end of the drive shaft 445 may have a D-shape or a crescent shape.

[0075] like Figure 16 and 17 As shown, the top of the CPC212 is configured with multiple inlet and outlet ports, each playing a crucial role in maintaining a sterile and controlled environment for CAR-T cell production. These ports allow for efficient, sterile transfer of various fluids, gases, and samples, supporting the entire cell processing cycle, from initial input to final harvest. Each inlet port is equipped with a sterile filter or needle septum, which is UV-sterilized before each use to ensure sterile entry or extraction of fluids, preventing contamination and maintaining the purity of the internal environment. This strategic arrangement of the inlet and outlet ports and the integrated filtration system enables the ACP to perform complex cell processing operations in a closed, sterile environment, supporting the production of high-purity CAR T cells with minimal risk of contamination. Figure 17 As shown, there is a short section 1114 of a sealed tube from a blood or white blood cell apheresis bag.

[0076] CPC212 includes dedicated inlet ports for gas inlet 1104, fluid inlet 1118, and reagent inlet 1116. The gas inlet extends through hydrophobic filters 426 and 428, which allow the introduction of sterile gases as needed to maintain cell culture conditions or control internal pressure at certain processing stages. Fluid port 1118 enables the addition of various fluids required for cell processing, such as wash solutions, culture media, or transduction agents, to the CPC. The fluid inlet extends through hydrophilic filter 422. Reagent inlet 1116 provides a pathway for the controlled introduction of specific reagents, including those required for cell activation or gene modification steps.

[0077] CPC212 includes a sealed tube truncated section 1114 from a blood or white blood cell apheresis bag. This tube... Figure 61A and 61B The images are shown again in both coiled and uncoiled forms. The patient's cells enter through this port for processing. A blood clot filter in the tube prevents blood clots from passing through. A bag containing a cell sample from the patient is sealed to this port by the tube.

[0078] The CPC212 is also characterized by its output ports: a CAR T cell harvesting output 1110, a sterile air access port 1106, a gas output 1102, a fluid output 1108, and a sample output 1112. Output port 1110 is dedicated to the final extraction of the resulting CAR T cell product after the cell processing step. The sterile air output 1102 facilitates the controlled release of gases to maintain internal pressure and prevent unwanted air buildup within the cartridge 212. Through gas output 1102, air is released via a hydrophobic filter 426, ensuring that any gas leaving the system is sterile. Gas output 1102 and fluid output 1108 allow for the safe removal of gases and fluids from the CPC 212 during washing, transduction, and other intermediate stages. The CAR T cell output port 1110 is dedicated to the final extraction of the CAR T cell product after the cell processing step. Sample output 1112 provides a pathway for collecting cell samples or aliquots at different stages, enabling quality control testing or process monitoring without disturbing the internal environment.

[0079] The CPC212 ports are equipped with specialized filters, such as 0.2 µm hydrophobic filters 426 and 428 and a 0.2 µm hydrophilic filter 422, to support sterility. The 0.2 µm hydrophobic filters 426 and 428 are used for various gas ports to ensure sterile air enters or exits, thus preventing contamination by external particles. The 0.2 µm hydrophilic filter 422, positioned below the coiled tube, allows sterile fluid flow and ensures sterile conditions are maintained during all fluid transfers.

[0080] The wider top section of the CPC houses multiple ports for fluid and gas transfer. These ports collectively allow for the aseptic introduction of fluids such as cell suspensions, reagents, or culture media, as well as the removal of waste liquids and the recovery of formulated volumes of genetically modified cells. The inclusion of gas exchange ports also provides for the aseptic introduction and removal of gases within the CPC, maintaining aseptic conditions and supporting processes that may require varying levels of oxygen or carbon dioxide.

[0081] The CPC's circular cap design achieves precise mating alignment using multiple alignment features 1100 to engage with other system components such as PFC and TSC. The CPC cap is characterized by multiple fluid transfer ports (septa) designed to enable precise, sterile fluid handling and improve overall cell processing efficiency.

[0082] Figure 18 A cross-sectional side view of an alternative embodiment of the present invention, with CPC212 arranged within barrel 120, is shown. Specifically, Figure 18In this embodiment, fastener 450 is shown to directly engage the second gear 444 with drive shaft 445, and environmental seal 449 is shown to prevent any fluid from the tank 120 from entering the motor 440 and interrupting its operation. The distal end of drive shaft 445 is shown protruding into a recess defined by first valve section 454.

[0083] Figure 19 A plan view of CPC212 according to a preferred embodiment of the present invention is shown.

[0084] Figure 20 The diagram shows a side view of CPC212, as well as section lines DD, EE, and FF, which are respectively located at... Figure 26 , 27 As shown in more detail in 28; Section line DD is an upward view of the filter and sensor configurations arranged directly below cover 402. Section line EE is a downward view of the main chamber 424 of CPC 212, and section line FF cuts through the staged chambers defined by the lower periphery of CPC 212.

[0085] Figure 21 A cross-sectional side view of the CPC212 is shown. The main chamber 424 can be configured to contain approximately 350 mL of fluid; however, it should be understood that the dimensions of the main chamber 424 may vary depending on the size of the blood sample being processed and the required level of dilution for that blood sample. Figure 21 Waste port 418 is shown, which includes a first check valve that allows fluid to flow out of the main chamber 424 and prevents fluid from entering the main chamber 424. Figure 21 The description also depicts how fluid port 408 includes a second check valve oriented in the opposite direction to the first check valve, which allows fluid to enter fluid port 408 but prevents fluid from leaving fluid port 408.

[0086] CPC212 is characterized by a conical, conical, or elliptical conical shape that gradually narrows from a wider diameter at the top to a narrower diameter at the bottom, wherein the diameter ratio is preferably between 4 and 5, and less than 4 in a less preferred embodiment. This design provides a range of functional advantages: the conical, conical, or elliptical internal geometry of the CPC plays a key role in achieving precise cell concentration, sedimentation, and mixing.

[0087] During centrifugation or sedimentation, the conical shape of the main chamber 424 naturally guides the precipitate—such as target cells, including hematopoietic stem cells and progenitor cells, T cells, or NK cells—to the narrow bottom section of CPC212, which is referred to as sedimentation column 431 (see [link to article]). Figure 18The sedimentation column is characterized by its substantially vertical cylindrical walls, which enhance measurement accuracy. This geometry concentrates the sediment (preferably cells) within a narrow area, improving the sensitivity of optical detection. In one embodiment, the sedimentation column 431 is designed with a flat, vertical surface wide enough to accommodate a light beam emitted from the optical transmitter to the sensor.

[0088] An optical detection system for cell monitoring in CPC212 utilizes multiple pairs of optical emitters / detectors positioned along the sedimentation column to measure changes in light transmittance caused by the presence, movement, and distribution of cells in the suspension. The system supports light across visible and invisible wavelengths (e.g., infrared or ultraviolet), currently using visible light. The system is adaptable to accommodate invisible wavelengths based on specific requirements such as improved sensitivity, reduced interference, or the optical properties of the sample or reagent. The detection system operates via a configurable sequencing protocol to optimize cell detection, sedimentation monitoring, and real-time feedback during cell handling operations. The sequencing algorithm includes the following features.

[0089] The plurality of optical emitter / sensor pairs are at least two, preferably three, and in some embodiments more than three, to detect optical signals passing through the settling column of the CPC main chamber. Although most of the CPC main chamber is funnel-shaped and has curved walls, the settling column walls are substantially vertical and parallel and include flat sections through which the emitter-sensor pairs operate, minimizing destructive reflections that would occur if those surfaces were curved. The flat sections of the settling column are on both the emitter and sensor sides. Preferably, the wavelength is adjustable white light to enable optimal detection of all cells, particularly leukocytes, during various phases of system operation.

[0090] The intensity of each emitter is configurable to optimize sensitivity and detection performance. The system can operate at absolute intensity (comparing the detected light intensity to a predefined threshold to identify the presence or movement of cells) or utilize relative intensity (monitoring changes across the entire array to determine sedimentation dynamics, cell stratification, and distribution), thus taking into account variations in hydrodynamics, optical path length, and cell density. The system can also utilize color variations in transmitted or received light as additional parameters to measure cell density or identify specific characteristics of cell suspensions. It is known that cells or particles in a suspension can scatter, absorb, or reflect light differently depending on their density, size, or type. The color (wavelength) of light passing through or being detected by the optical emitter / receiver pair can vary based on how the light interacts with the suspension. As cell density increases, the opacity of the fluid changes, but the spectrum (color) of the transmitted or scattered light may also shift. For example, densely packed cells may absorb more light within a specific wavelength range (e.g., red or blue), resulting in a measurable color shift in the detection signal. By analyzing both light intensity (opacity) and color changes, this system enhances sensitivity to small changes in cell density and potentially distinguishes cell types (e.g., erythrocytes, T cells, or NK cells) based on unique optical absorption characteristics. This provides a more robust and nuanced mechanism for detecting cell concentration or composition. In summary, the optical emitter can use multi-wavelength light sources (e.g., white light or specific RGB LEDs), and the receiver can detect intensity and wavelength shifts to measure color changes caused by variations in the suspension. The system's algorithm incorporates these readings into its cell counting or concentration calculations.

[0091] Multiple optical emitter-sensor pairs allow for real-time monitoring and control of cell stratification and density, crucial for precise cell recovery and distribution. For example, data from the optical emitter-sensor pairs can be used to control the centrifuge RPM, such as the centrifuge speed, which can be correlated with the cell stacking level. Once the cells are infused, they are allowed to settle to an empirically determined stacking level. This controlled centrifugation ensures that cells are optimally stacked to receive fluids from the reagent septum 1116 and the liquid transfer port. 1170 Delivery of carriers or reagents. When the valve is configured to allow this, it allows for the precise, direct introduction of reagents into the centrifuged, concentrated cell population located at the bottom of the sedimentation column.

[0092] In one example, if the bottommost sensor detects that cells are piled up too densely, this information can be sent to the platform, which then reduces the centrifuge's RPM. The transmitters can be activated sequentially or in parallel, depending on system requirements. For sequential activation, transmitters can be enabled and disabled in a controlled, top-to-bottom manner along the vertical array of transmitter / detector pairs. The delay between enabling sequential transmitters is configurable, allowing for precise adjustments to optimize detection sensitivity, resolution, or the dynamic characteristics of the cell suspension. Configurable delays can also be introduced before rerunning the sequence.

[0093] In operation, all cells eventually pass through the top emitter-sensor pair, and then, based on particle density, hydrodynamics, and centrifugal force, after a certain time, they will pass through the middle emitter-sensor pair, and finally the bottom emitter-sensor pair. In practice, the system can slow down or stop centrifugation after the cells have passed 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 liquid removal, which is preferably performed by applying positive pressure. The system is scalable to accommodate additional emitter / detector pairs, allowing for flexible adaptation to cartridges or similar devices of different sizes. The number of emitter / detector pairs can be proportional to the size of the sedimentation column used for cell detection and sedimentation. The system can also include an array of multiple emitter / detector pairs positioned at a 90-degree offset to allow detection from two orthogonal angles, thereby improving measurement accuracy and reliability.

[0094] In embodiments where the entire main chamber 424 or settling column is not constructed of a transparent or translucent material, at least one optically transparent window in the settling column 431 allows the sensor-transmitter pair 1062 to operate through it.

[0095] Real-time total cell concentration calculations are achieved by combining optical sensor data with the known volume of the cell suspension. The total volume of the suspension is determined by weight (via strain gauges) or vertical height measurement (using a laser or radar system). The narrow geometry of the sedimentation column significantly improves measurement sensitivity, facilitating the detection of subtle changes in light transmission associated with the presence of target cells, such as hematopoietic stem cells and progenitor cells, T cells, or NK cells.

[0096] To mitigate the limitation of optical sensors that only detect sediment within the sedimentation column, the system employs a rapid yet gentle mixing process immediately before measurement. This ensures that cells are uniformly distributed throughout the entire cell suspension volume. Once mixing is complete, the system tracks the elapsed time as the CPC rotates to a vertical, stationary position. This timing factor is crucial for the accurate algorithmic calculation of cell counting.

[0097] The system uses an empirically determined algorithm that takes into account one or more of the following to refine the cell count estimate, ensuring high accuracy even when settling begins shortly after mixing stops: the time elapsed since mixing stopped; light signal obstruction measured at each of the three optical sensor pairs; and the settling rate, which is substantially uniform throughout the settling column and the overall cell suspension above it.

[0098] The conical geometry of the main chamber 424, tapering from a wider top to a narrower bottom, ensures controlled movement of cells in the suspension during chamber tilting. This gravity- and shape-guided motion can generate uniform laminar mixing optimized for different solution volumes by adjusting the rotation rate, while minimizing shear stress on the cells. The conical geometry, tapering from a wider top to a narrower bottom, also ensures controlled movement of cells in the suspension as the chamber rotates about its axis. This gravity- and shape-guided motion can generate uniform laminar mixing optimized for different solution volumes by adjusting the rotation rate.

[0099] To optimize performance, the inner walls of the CPC are constructed using low-adhesion materials, such as biocompatible polymers with smooth, diamond-polished surfaces. This surface treatment minimizes the possibility of cell adhesion, ensuring that cells remain suspended in the fluid and can be effectively concentrated, isolated, or removed as needed. The low-adhesion materials significantly reduce cell loss due to adhesion, thereby increasing recovery rates and improving overall process efficiency. Through careful selection of anti-adhesion materials and surface treatments, the CPC maximizes the yield of live cells at every stage of the processing cycle, supporting consistent and high-quality results.

[0100] Effective and thorough mixing is crucial for cell manufacturing processes. The provided geometry enables CPC to perform essential steps such as cell washing, activation, gene modification, and harvesting in a closed, sterile, and automated environment. During these processes, cells must remain uniformly distributed to ensure reliable cell selection, activation, and transduction. Uniform mixing also guarantees that all cells interact equally with reagents and carriers, while maintaining a representative distribution for quality control sampling. Without this optimized mixing, cells will settle unevenly, leading to inconsistent recovery rates, reduced activation efficiency, and lower transduction rates.

[0101] The natural tendency of cells to settle and concentrate is overcome by a combination of conical or cone-shaped geometry, precise rotation, and optimized fluid motion. During centrifugation or sedimentation, the cone shape naturally guides the precipitate—such as target cells, including hematopoietic stem cells and progenitor cells, T cells, or NK cells—to a narrow bottom section called a sedimentation column.

[0102] By ensuring rapid and thorough mixing, the CPC is designed to prevent premature cell sedimentation and maximize exposure to reagents and genetic material, significantly improving overall treatment outcomes.

[0103] Figure 22 Alternative embodiments of the invention are depicted, showing the relative positions of the worm gear driven valve assemblies. The worm gear driven valve actuator includes a motor 440 positioned and electrically connected to a printed circuit board assembly 476. While details of the individual circuit components are not provided herein, it should be noted that those skilled in the art will understand how the components on the printed circuit board assembly 476 can be configured to provide power and control commands for the operation of the motor 440 when actuation of the valve assembly is required. Alternative Embodiments Figure 22 A second gear 444 is also depicted positioned within a recess defined by the downward-facing surface of the barrel 120, the recess being shaped to receive the second gear 444. A first valve section 454 is shown extending into an opening defined by the lower housing component 406 of the CPC212.

[0104] A hydrophilic filter 422 is shown directly below the fluid port 408 and is responsible for filtering out any impurities in the fluid introduced through the fluid port 408. Diaphragms 414 and 416 have a tapered geometry to guide the receiving syringe needle.

[0105] Figure 23 A cross-sectional side view of CPC212, taken along section line CC, according to a preferred embodiment of the invention, is shown.

[0106] like Figure 24 As shown, the center of gravity (CG) of barrel 120 varies based on the liquid level. Pivot axis 1092 mixes the cell suspension in CPC212. Figure 24The changes in CG are shown at 350 ml (point 1094) and 25 ml (point 1096). The vertical centering of the CG in both the centrifuge bucket and CPC212 is crucial for ensuring efficient operation of both the mixing and centrifugation processes. Mixing of the cell suspension occurs only when the centrifuge bucket is not rotating around the rotor. To minimize the torque required by the mixing motor, the pivot axis of the bucket / CPC assembly must be positioned extremely close to its CG. This ensures efficient mixing regardless of the cell suspension volume, whether it is as low as 15 mL or as high as 350 mL. By aligning the pivot axis close to the CG, the system achieves smooth, controlled oscillations with minimal energy consumption. As the bucket and CPC rotate around the centrifuge rotor, the bucket rotates upwards, tilting nearly 90°. This occurs because the geometric center of the mass is closely aligned with the geometric center of the counterweight, ensuring near-perfect balance. This alignment provides the most uniform sedimentation rate for the cells, which move consistently away from the axis of rotation under centrifugal force. If a perfect 90° rotation of the drum / CPC assembly is required during centrifugation, the mixing motor can be precisely adjusted due to the minimal torque requirements in this state. This fine control ensures optimized conditions for cell sedimentation and subsequent processing.

[0107] In one implementation, the system employs a relative centrifugal force (RCF) compensation mechanism, designed to regulate the sedimentation rate of cells in the cell suspension as they enter the sedimentation column during centrifugation. If the sedimentation rate deviates from an empirically determined optimal rate—whether too fast or too slow—the RCF is dynamically adjusted to achieve ideal cell stacking at the bottom of the sedimentation column. This mechanism is particularly critical during centrifugation seeding, when cells and carriers are co-mixed to ensure efficient and uniform interaction between cells and carriers.

[0108] like Figure 25 As shown, for small volumes such as 100 ml, the container 120 can rotate up to 135 degrees in either direction to achieve maximum mixing without liquid entering the fluid channel ports of the multiple filters. Therefore, the possible degree of tilt is inversely proportional to the amount of liquid in the main chamber. For example, for a volume of 115 ml, it may be able to tilt to approximately 120 degrees in either direction, or for 160 ml, it may only be able to rotate to 110 degrees in either direction. The CPC212 can hold a maximum liquid capacity of 350 ml and a minimum capacity of 25 ml. The motor enables the container 120 to rotate with a rotational accuracy of one-tenth of a degree. Therefore, it is clear that gravity is not the sole cause of the rotation of the container 120. The pivot point of the container 120 is designed to maximize the clearance of the container 120 during rotation.

[0109] Figure 26 It shows along Figure 20A cross-sectional view of the CPC212, viewed from inside the CPC looking upwards at the lower side of filters 422, 426, and 428. Filters 426 and 428 are preferably hydrophobic filters, allowing sterile gases to enter and exit; their structural integrity has been verified through automated pressure / attenuation testing. A hydrophilic filter 422, positioned below the coiled tube on the CPC cap, allows sterile fluid transfer into the CPC; this filter has also been verified through pressure / attenuation testing. Preferably, three filters with 0.2-micron pore sizes are integrated on the top of the CPC cap. These filters facilitate the sterile transfer of fluids into the CPC and the controlled exchange (entry and exit) of gases while maintaining a sterile environment. These filters ensure that all fluid and gas pathways remain uncontaminated during cell processing.

[0110] Figure 27 It shows along Figure 20 A view of the cross-section line EE, looking down at the main chamber 424 and waste riser 438, which provides a path through which waste liquid within the CPC can be discharged via waste port 418. Several fins are also shown, designed to improve heat transfer from the heater in barrel 120 to the fluid in CPC 212.

[0111] Figure 28 It shows along Figure 20 A view of the cross-section line FF shows the four rotational positions of the valve. The valve rotates to four positions, each creating a different fluid connection for a different process. Positions between these four positions do not create any fluid connection; in these positions, the CPC valve is referred to as being in a resident or closed state. When the valve is in position 1070, the fluid connection is open, allowing the CPC to receive reagents from the reagent septum. Position 1072 creates a fluid connection to isolate non-target cells. Position 1074 creates a fluid connection to the compartment for isolating secondary T cells or non-target cells, preferably entering a 30 mL volume-restricted compartment. Position 1076 creates a fluid connection from the CPC chamber to the harvest septum and ultimately to the harvest container. When the valve is configured to allow this, the reagent septum 1116 is connected via a conduit to valve position 1070, i.e., the liquid transfer port, which allows for precise, direct introduction of reagents into the centrifuged, concentrated cell population located at the bottom of the sedimentation column 431. This targeted delivery maximizes reagent efficiency and enhances cell processing outcomes.

[0112] The top of the CPC in the ACP is equipped with multiple inlet and outlet ports, each playing a crucial role in maintaining a sterile and controlled environment for CAR T cell manufacturing. These ports enable efficient and sterile transfer of fluids, gases, and samples through the cap, supporting the entire cell processing workflow—from the initial introduction of cells to the final harvest of genetically modified cells. Each inlet / outlet septum port integrates a UV mechanism that sterilizes the opposing septum during docking. At the bottom of the TSC, a septum is located for each syringe; this septum will be one of two opposing septa sterilized by the UV sterilization module during any fluid transfer. The opposing septa to be sterilized simultaneously will be located on the RSC container or the CPC cap. Specifically, the UV mechanism ensures that both the TSC septum and the CPC cap septum, or the RSC septum, are sterilized during reagent aspiration or sample deposition. Furthermore, a hydrophobic filter allows for sterile gas exchange, while a hydrophilic filter manages the sterile transfer of fluids. Both filter types undergo pressure / attenuation testing to confirm their structural integrity before and after processing clinical-grade genetically modified cell populations.

[0113] The aseptic transfer ports on the CPC cap preferably include a septum port and a filter port. The input and output septa are positioned on the CPC cap for precise alignment with the TSC syringe, enabling the aseptic transfer of reagents, samples, and harvested cells.

[0114] This strategic combination of port design, UV sterilization, and filtration systems ensures sterile entry and exit of gases and fluids, effectively preventing contamination and maintaining the purity of the CPC's internal environment. By maintaining these sterile conditions, the ACP can precisely and automatically perform complex cell processing tasks, ensuring the production of high-purity CAR T cells with minimal risk of contamination.

[0115] The CPC212 is designed with dedicated input and output ports to ensure sterile and efficient handling of gases, fluids, reagents, and samples throughout the cell processing cycle. Starting with the input ports, these can be categorized into four types: (1) Gas Inlet: Hydrophobic filters on the CPC cap allow sterile gases to enter without contaminating the internal environment. These filters also sterilely release displaced air as fluids are introduced into the CPC, maintaining cell culture conditions. (2) Fluid Inlet: Fluids required for cell processing, such as wash solutions, culture media, or transduction agents, pass through hydrophilic filters. These filters have a large surface area to accommodate the high fluid flow rates required for efficient processing. (3) Reagent Inlet: Dedicated input ports provide a controlled pathway for the introduction of specific reagents, such as those used for cell selection, activation, or gene modification. (4) Sealed Tube with Clot Filter: A tube that connects to a blood or leukocyte apheresis bag enables sterile docking and the introduction of patient cells. This tube includes a clot filter to remove unwanted blood clots before cells enter the CPC. After the transfer, the tube was sealed one inch from the CPC cap inlet and secured in a nearby plastic clip to ensure safety and avoid obstruction.

[0116] Now let's turn to the output ports, which can also be categorized into four types. (1) Gas venting: A hydrophobic filter ensures that the gas leaving the CPC is sterile and free of contaminants. (2) Harvested genetically modified cell output: A dedicated diaphragm allows for the sterile final harvesting of the genetically modified cell product after the processing steps are completed. (3) Waste discharge: This port is equipped with a one-way valve to safely discharge waste after the cells have been centrifuged and isolated at the bottom of the sedimentation column. Optical sensors confirm cell location before waste removal, and positive pressure pushes the waste into an expandable, sealed container at a controlled rate to prevent accidental removal of isolated cells. (4) Sample output: The diaphragm port allows the TSC to aseptically collect cell suspension samples at various stages of the process for quality control or monitoring without disturbing the sterile internal environment.

[0117] Figure 30 A top perspective view of CPC212 is shown. Diaphragms 414 and 416 are configured to interact with the injector of TSC210 and receive fluid supplied therefrom, or allow fluid to be collected by the extractor of TSC210. ​​Specifically, the diaphragms include a harvesting diaphragm 414 and a dissolving O2 well diaphragm 416.

[0118] Figure 31 The side line of sight AA, as well as the section lines BB and CC, are also shown, which define the sides respectively. Figure 32 , 33 The views provided in 34. Figures 33-34 It is a cross-sectional view, and Figure 32This is a side view with section lines D1-D1, E1-E1, and F1-F1, which are respectively located at... Figure 35 , 36 As described in 37.

[0119] Figure 38 The top of CPC212 is shown. The figure also shows the line of sight AA and the section lines BB, CC, and GG, which define the... Figure 20 , 21 And the views provided in 59B-59D. Line of sight AA in Figure 20 As shown, the cross-sectional line BB extends through the fluid port 408, the waste port 418, and the diaphragm 414, and... Figure 21 As shown in the image.

[0120] The pneumatic system within the ACP utilizes precise gas pressure control to perform several key functions throughout the cell processing workflow. First, it performs pressure decay testing to verify the integrity of the hydrophobic and hydrophilic filters within the CPC212, ensuring the sterility and reliability of fluid and gas transfer. Second, it uses pressurized gas to burst microbubbles, facilitating cell release during certain processing steps. Third, the system applies pressure to the top of the PFC, enabling fluid transfer to the CPC chamber through the disc filter outlet receiver. During this process, radar or laser sensors monitor the fluid volume entering the chamber to ensure accuracy. Fourth, the pneumatic system pressurizes the waste line to expel waste liquid from the CPC while preventing contamination. Once the liquid is removed, gas is applied to purge any residual liquid from the line. Finally, the pneumatic system propels the genetically modified cells into the harvest syringe, ensuring the precise and sterile transfer of the final cell product for collection. These multi-functional capabilities enhance the efficiency and reliability of the ACP in delivering high-quality cell therapies.

[0121] Figure 39 It is a cross-sectional view showing CPC212 undergoing a centrifugation operation, in which the target cell solution 1022 is pressed against the bottom of the main chamber 424, and then the first valve section 454 is rotated to align the first channel section 458 with the second channel section 482, as depicted.

[0122] The port allows for the aseptic passage of genetically modified and formulated target cell solutions. A capillary tube connects to a TSC syringe, which completes the transfer of the cell solution to the RSC final harvest container with minimal to no cell loss. This allows the target cell solution 1022 to be collected in the primary target cell compartment 480. Once the desired amount of target cell solution 1022 has been added to the primary target cell compartment 480, additional target cell solution 1022 is added to the secondary target cell compartment 488 by rotating the first valve segment 454 to align the first channel segment 458 with the channel segment associated with the secondary target cell compartment 488 (see [link to documentation]). Figure 22 ).

[0123] Once the primary and secondary fractionation chambers 480, 488 have received the target cell solution 1022, the centrifugation operation can be terminated. Before removing the processing cartridge 212 from the cell processing device 100 and drawing the final therapeutic dose or multiple doses, the cell processing device 100 may report any irregularities observed during the process, as evidenced by sensor readings monitored throughout the cell processing operation. The integrity of the filters 422, 426, and 428 of the processing cartridge 212 may also be checked, and any irregularities related to the filters may be reported. The integrity of the pre- and post-processing filters can be verified using a pressure decay method to check each filter individually. The pre- and post-processing filter integrity checks can be performed within the cell processing device 100 by a test cartridge positioned within the cartridge positioning assembly 132. This test cartridge can be configured to apply a known gas pressure to the inlet port of each filter (422, 426, or 428) while closing all outlet ports. Once pressurized, a sterile ventilation port (i.e., port 412 or equivalent) is opened, allowing gas to flow only through the filter being tested. The filter integrity is determined by measuring the pressure decay time in the inlet port volume against known limits. The filter integrity test results are included in the batch release report.

[0124] Figure 40A-40C Various views of PFC204 are shown. Figure 40AA perspective view of the PFC 204 and how it has an overall cylindrical geometry is shown. The upper surface of the PFC 204 includes multiple needle-free inlet ports 502 for adding solution into the PFC 204. Adding solution to the PFC 204 is typically performed before placing the PFC 204 into the cell processing device 100. While four needle-free inlet ports 502 are depicted and correspond to four pie-shaped chambers within the PFC 204, it should be understood that the PFC 204 may be divided into more or fewer pie-shaped chambers. Alternatively, the PFC 204 may include a single chamber containing only a single type of fluid, in which case the cartridge positioning assembly 132 may include multiple fluid cartridges, one for each desired liquid. Each chamber of the PFC 204 further includes a corresponding pneumatic port 504 configured to receive a fixed amount of compressed air to eject a precise amount of fluid from the corresponding chamber of the PFC 204.

[0125] Each pneumatic port 504 may include a filter to prevent accidental addition of contaminants to the PFC 204 when a specific chamber of the PFC 204 is pressurized. The PFC cap preferably contains four filters, corresponding to each of its four disc-shaped compartments. Each compartment can hold up to 500 mL of buffer solution or other fluid. The 0.2-micron filter allows aseptic airflow at a controlled pressure (1 to 5 psi), which modulates the flow rate of fluid through the docking port and the hydrophilic filter on the CPC cap.

[0126] In some implementations, the amount of fluid injected from PFC204 into treatment cassette 212 can also be controlled based on feedback from radar or laser level sensors. Figure 40A The upper end of the waste through-channel 506, extending along the longitudinal axis of PFC 204, is also shown, allowing waste sample material exiting the waste port 418 of processing cartridge 212 to continue upward into waste tube 208 and into a bottle for collection. The upper end of the pneumatic through-channel 508 is also shown. The pneumatic through-channel 508 allows air to be added to or removed from processing cartridge 212 by applying positive or negative pressure to the pneumatic through-channel 508 when it is coupled to the pneumatic port of processing cartridge 212. Any applied air pressure in each pie compartment is carefully managed to ensure precise fluid transfer into the CPC, supporting efficient and aseptic processing.

[0127] Figure 40B A top view of PFC204 is shown, illustrating how the pneumatic port 504 is positioned along the periphery of PFC204 and how the needleless inlet port 502 is arranged in the central region of PFC204. A waste through-channel 506 is located at the center of PFC204, and a pneumatic through-channel 508 is shown outside the needleless inlet port 502 and inside the pneumatic port 504. Figure 40B The section line FF is also shown, which corresponds to Figure 41 .

[0128] Figure 40C A bottom view of PFC204 is shown, including the other end of waste through channel 506. The opposite ends of waste through channel 506 are typically connected by flexible or rigid tubes extending along the longitudinal axis of PFC204. Figure 40C The other end of the pneumatic through-channel 508 is also shown, configured to connect pneumatic ports 410 and 412 on the processing housing 212. A fluid outlet port 510 is also shown, configured to connect fluid port 408 on the processing housing 212.

[0129] Figure 41 It shows the data from Figure 40B A side view of the cross-section of PFC204 with section line FF. Specifically, Figure 41 The diagram shows two filled fluid chambers of the PFC 204, as well as the internal components of the needleless inlet port 502, pneumatic port 504, waste through channel 506, and fluid outlet port 510. Specifically, the waste through channel 506 and fluid outlet port 510 include check valves to prevent contamination of the fluid stored within the PFC 204 when the PFC 204 is not in use. For example, the check valve of the waste through channel 506 is configured to compress a spring when pressurized sample material leaves the processing cartridge 212 and enters the waste through channel 506.

[0130] Figures 42A-42B Different views of RSC202 are shown. Figure 42A A perspective view of RSC202 is shown, and Figure 42B A top view of the RSC202 is shown. Figures 42A-42BThe diagram depicts a total of six reagent vials 602 and six pre-labeled sample vials 604; however, it should be understood that fewer or more vials are possible and considered within the scope of the invention. The RSC 202 contains up to six pre-filled reagent vials, which will be used throughout the cell processing, preferably each having a volumetric capacity of up to 20 mL. These vials may contain necessary components such as microbubbles, carriers, and adapters. Additionally, the RSC 202 contains designated sample vials, ready to receive cell samples for quality control analysis throughout the processing cycle. Sample vials 604 are positioned on a support 606 such that the upper surface of sample vials 604 is flush with the upper surface of reagent vials 602. Each vial can be slid into position on the RSC 202 by sliding the corresponding vial into a clip 608, which engages with a collar 610 to prevent lateral and vertical movement of the vial while mounted on the RSC 202. Each vial 602 and 604 includes a cap having a region 612 configured to be pierced by a needle to allow insertion or extraction of fluid from the vial. The system may include automated quality control in the form of integrated sensors to monitor syringe volume and flow rate to detect any deviations, triggering alarms and adjustments as needed.

[0131] The ACP is characterized by a UV sterilization module specifically designed to sterilize the port septa prior to each fluid transfer, ensuring that each needle transfer occurs under aseptic conditions. This is because the sterilization module (DM) uses strong UV radiation or light to sterilize the surfaces of both the TSC septa and the opposing RSC or CPC septa. UV light is focused on both opposing septa before each needle penetrates either septum. Only after both septa have been sterilized does the needle emerge from the bottom septum of the TSC and proceed downwards into the septum of the CPC cap or the RSC. The UV sterilization module is movable and will always be precisely positioned to ensure aseptic transfer involving the TSC and RSC or CPC. No needle will ever protrude beyond the TSC unless docked with the UV component and mechanically operated and under the automated control of the system software. Preferably, the syringe will penetrate the septum on the CPC cap and the septum on the sample vial 604, or the septum on the CPC cap and the septum on the reagent container. The diaphragm can preferably be found on the top of each sample vial 604 or reagent vial 602, on the cap of the CPC, and on the TSC itself. Key components include: UV-C Light Source: This module utilizes UV-C wavelength light or radiation (254–280 nm), which is the optimal wavelength for microbial inactivation, effectively eliminating bacteria, viruses, and other contaminants on the membrane surface. The UV light source is designed to provide full exposure to the membrane.

[0132] Reflective internal coating: The UV sterilization chamber utilizes a UV-reflective material that guides UV-C light, ensuring comprehensive and uniform exposure of the diaphragm from multiple angles. This design minimizes the risk of any shadows or missed areas on the diaphragm, achieving complete sterilization before any syringe needle punctures the diaphragm.

[0133] Automated detection and activation system: This module includes optical sensors that detect the approach of the syringe to the inlet port. Once detected, the control system automatically initiates a sterilization cycle, activating the UV radiation source for a precise duration. This automated activation eliminates the need for manual operation, ensuring that each diaphragm is sterilized precisely before use and reducing the likelihood of contaminants settling on the diaphragm after sterilization.

[0134] Timing and Control System: The UV module is programmed to deliver controlled, timed exposures (typically between 5 and 15 seconds) for effective sterilization without degrading the diaphragm material. This timing mechanism is integrated with ACP's central control system and calibrated to match treatment needs and UV exposure requirements, allowing for customized cycles based on usage needs and material specifications.

[0135] In use, the UV sterilization module follows a simplified process to sterilize the diaphragms as part of an automated fluid handling sequence: Syringe Detection: An optical sensor detects the syringe as it approaches the CPC inlet port, triggering a sterilization cycle. UV Exposure: The UV radiation source is activated to sterilize the surfaces of opposing diaphragms before the TSC syringe needle penetrates both diaphragms, continuing until transfer occurs and the TSC needle has been withdrawn over its diaphragm. Reflective internal materials ensure uniform UV exposure, effectively inactivating microbial contaminants on all exposed surfaces within the UV sterilization module. Completion and Shutdown: After the sterilization cycle, the UV radiation source is deactivated.

[0136] The UV sterilization module has been validated to achieve a preferred 6-log reduction in microbial load on the diaphragm surface, ensuring aseptic conditions during fluid transfer. The aseptic integrity of the TSC syringe needle, initially achieved through gamma irradiation of the packaging before use with ACP, remains uncompromised during the UV sterilization process, meeting all industry standards for aseptic handling environments. The module delivers approximately 750 mJ / cm³. 2 This high disinfection efficiency is achieved using UV energy, utilizing 170 mW of continuous direct current over 4.4 seconds. While direct current is currently the standard, future designs could incorporate pulsed electromagnetic energy to further improve efficiency.

[0137] Sample vials 604 are typically empty at the start of an operation performed by the cell processing device 100 and are gradually filled with samples collected from processing cartridge 212 throughout the operation. This allows laboratory technicians operating the device to examine the samples during the operation to verify that the operation is proceeding as intended. Reagent vials 602 typically contain fluids at the start of the operation, which are extracted by TSC 210 and transferred to processing cartridge 212 during the operation. Exemplary fluids contained in reagent vials 602 include microbubbles, antibody adapters, and disease vectors. In one embodiment, each reagent vial 602 carries up to 20 mL of fluid, and each sample vial 604 carries up to 5 mL of fluid.

[0138] Figures 43A-43C Various views of the TSC210 are shown. Figure 43A A perspective view of the TSC210 is shown, illustrating how it may include a plurality of reagent syringes 702 arranged in a first circular configuration and a plurality of sampling syringes 704 arranged in a second circular configuration within the first circular configuration. The number of reagent and sampling syringes 702 and 704 on the PFC204 will generally correspond to the number of reagent and sample vials 602 and 604 contained on the RSC202. Although Figures 43A-43B Only the plunger portion of each syringe 702 and 704 is visible, but it should be understood that each syringe 702 and 704 also includes at least a needle and a solution receiving container.

[0139] Figures 43B-43C The corresponding top and bottom views of the TSC210 are shown, a key component of the TSC designed to manage precise fluid transfer between other platform cassettes (preferably at least CPC and RSC). The TSC houses multiple independently controlled syringes that transfer fluids such as reagents, buffers, and cell suspensions with high precision, maintaining sterility and consistency throughout the various stages of cell processing. By integrating independent syringe controls, pressure regulation, and rotary docking mechanisms, the TSC provides a reliable and efficient means of managing fluids in closed-loop cell processing, ensuring consistency, safety, and scalability in cell therapy manufacturing and other biological applications.

[0140] Figure 43B The diagram illustrates how, in some embodiments, the syringe cartridge may include a waste through-channel 706 for removing waste sampling material from the CPC212. When the waste through-channel 706 is applied to the TSC210, it will typically also include a pneumatic through-channel for supplying the TSC210 with sufficient positive pressure to drive the waste sample material through the waste through-channel 706. Figure 43CThe diagram illustrates how each syringe 702 and 704 may have a corresponding port 708, with its needle extending through the diaphragm and through port 708 to add or draw fluid from the main chamber of the processing cartridge 212 or from a vial on the RSC 202. A first subset of ports 708 is arranged along the periphery of the PFC 204, corresponding to reagent syringe 702, and a second subset of ports 708 is arranged within the central portion of the PFC 204, corresponding to sampling syringe 704.

[0141] The TSC210 manages the precise transfer of reagents and fluids into the CPC212, enabling accurate volumetric quantification of processes such as washing, activation, and transduction.

[0142] The TSC210 is a key component of the ACP and is designed to manage precise fluid transfer between other platform cartridges, such as the CPC212 and RSC202. The TSC210 houses multiple independently controlled syringes that transfer fluids such as reagents, buffers, and cell suspensions with high precision, thus maintaining sterility and consistency throughout the various stages of cell processing.

[0143] The TSC210's structure may include a TSC housing, syringes, a rotary docking mechanism, fluid transfer channels and ports, a pressure control system, and feedback sensors. The housing is a structurally reinforced, enclosed enclosure that houses a series of syringes arranged in parallel, each located within a designated compartment. This housing provides sterile containment and is designed to prevent cross-contamination between fluids handled by different syringes. Each syringe within the TSC210 is configured to maintain a specific fluid volume, allowing for independent control of the fluid type, such as culture media, buffer solutions, or treated cell suspensions. The syringes are equipped with adjustable plungers for volumetric accuracy.

[0144] The rotating docking interface enables alignment with other platform cartridges. This mechanism allows the TSC210 to pivot between docking positions for each cartridge (specifically CPC212 and RSC202), ensuring precise fluid transfer between different stages of cell processing. The TSC210 integrates fluid transfer pathways and docking ports that align with corresponding ports on CPC212 and RSC202. These pathways are sterile and sealed with automatic check valves that open only when the TSC210 is docked with a specific cartridge, preventing contamination and ensuring fluid containment. Each syringe is equipped with a pressure control system capable of dispensing fluid over a pressure range of approximately 1.5 to 45 PSI. This control is crucial for managing various fluid types and viscosities, ensuring precise flow rates during dispensing into or from CPC212 into RSC202. The TSC210 includes feedback sensors positioned to monitor the fluid level within each syringe, providing real-time feedback on dispensing volume and fluid level. These sensors communicate with ACP's central control system to dynamically adjust fluid volume and pressure.

[0145] During stages requiring the transfer of fluids to or from the cell processing environment, the TSC210 is aligned with the CPC212. For example, during the washing and activation stages, the TSC210 dispenses reagents, buffers, and activation solutions into the CPC212 with precise volume and pressure to achieve optimal cell washing, separation, or activation. During cell modification stages, such as gene insertion or modification, the TSC210 transfers viral vectors or gene reagents into the CPC212, with controlled syringe actuation ensuring uniform distribution.

[0146] The TSC210 can also dock with the PFC204 to extract specific fluids required for cell processing. The PFC204 acts as a reservoir for various buffers, culture media, and reagents, which the TSC210 transfers to the CPC212 as needed. The TSC210 syringe extracts precise amounts of fluid from the PFC204 to inject into the CPC212 for specific processing steps, including cell isolation, incubation, or buffer exchange.

[0147] The TSC interacts with the RSC. Reagents are obtained from the RSC by the TSC210 and then transferred to the CPC212 via at least one syringe, wherein each large-volume syringe in the TSC210 selectively draws fluid from the RSC and injects it into the CPC212, and each small syringe in the TSC210 draws a cell suspension sample from the CPC and injects it into a sample vial in the septum of the RSC202. After the cell processing steps are completed, the TSC210 selectively draws genetically modified and formulated cell suspensions from the CPC and dispenses them into the harvest container of the RSC for final storage or analysis by aligning the TSC210 with the CPC212. The TSC210 transfers the final cell product, such as activated or genetically modified cells, from the CPC212 to the RSC202, ensuring minimal cell loss during aseptic containment and transfer. The TSC is aligned with the CPC, whereby the TSC selectively draws genetically modified cell suspensions from the CPC and dispenses them into the RSC.

[0148] Figure 44A A cross-sectional side view of the interaction between TSC210 and RSC202 is shown. Specifically, TSC210 is shown engaged with RSC202 such that syringe 702 is aligned with vial 602. Syringe 702 is held in position at the top of TSC210 by spring 802. UV sterilizer 800 is positioned between RSC202 and TSC210 and is configured to sterilize the outer surfaces of the diaphragms of TSC210 and RSC202 to prevent contamination of the needle of syringe 702. Although UV sterilizer 800 is shown not attached to another device, it should be understood that UV sterilizer 800 can be positioned between RSC202 and TSC210 by a swing arm capable of precisely positioning UV sterilizer 800. Since the top surfaces of vials 602 and 604 are flush with each other, the swing arm can maintain UV sterilizer 800 at a constant height and is only responsible for manipulating UV sterilizer 800 in the x and y directions. A close-up view of the UV sterilizer 800 shows its internal structure and how it includes channels that allow a needle 804 to extend through the UV sterilizer 800 to engage vial 602 and draw fluid from it. The UV sterilizer 800 operates by emitting UV light in two opposite directions. The UV sterilizer emits light upwards to sterilize the surface of the TSC 210 through which the needle 804 extends, and emits light downwards to sterilize the surface of the vial 602 through which the needle 804 extends. This is important because treatment of these vials before they are placed into the cell processing device 100 can cause contaminants to adhere to the exterior of the septa of each vial. Therefore, this sterilization procedure greatly reduces the likelihood of the needle 804 becoming contaminated when drawing fluid from the vial 602.

[0149] Figure 44BA configuration is depicted in which, after the exterior of the TSC210 and the top of the vial 602 are cleaned by a UV sterilizer 800, a needle extends through the UV sterilizer 800, pierces the fluid contained within the vial 602, and aspirates at least a portion of the fluid contained within the vial 602. A syringe 702 may be pressed against a spring 802 to engage the vial 602 as depicted. Downward movement of the syringe 702 may be achieved by an actuator of the cell processing device 100 positioned directly above the TSC210, which applies force to the upward-facing surface of the plunger 806 of the syringe 702, pressing the syringe 702 against the spring 802. In some embodiments, the TSC210 may include a locking mechanism that prevents the syringe 702 from moving backward upward once it reaches the fluid aspiration position within the tube 808, as... Figure 44B As shown. Once syringe 704 is locked in the fluid extraction position, the same actuator responsible for pushing the syringe downward can apply an upward force to the plunger 806 of syringe 702. This upward force can be applied to the plunger 806 by the actuator using a suction cup, vacuum tip, or mechanical gripper of the actuator until the solution receiving container 810 is filled to the desired level. Once the desired level is reached, the actuator can be withdrawn from TSC210, and the locking mechanism holding syringe 702 can be released. In some embodiments, the locking mechanism may include a damping element that prevents the syringe from being suddenly and violently bounced upward back to the starting position, instead allowing syringe 702 to be smoothly retracted into the tube 808 of TSC210. ​​Alternatively, each spring can be tuned such that its force output only results in a gradual upward movement of the corresponding syringe. Alternative actuator configurations operate in Figure 45A-45K As described in the accompanying text.

[0150] Figure 44C-44D The sampling syringe 704 is shown as being used to extract a sample from the processing cartridge 212 and to transfer the extracted sample to a sample vial 604 in the RSC 202. Figure 44C –44D also demonstrates how the UV sterilizer 800-1 is used when extracting sample material from the processing cassette 212, and how the UV sterilizer 800-2 is used when depositing the extracted sample material into the sample vial 604. It should be noted that the centrifuge tank 114 may be equipped with its own swing arm for positioning the UV sterilizer 800-1 between the processing cassette 212 and the TSC 210. Although the cell processing apparatus 100 is described as including multiple UV sterilizers 800, in some embodiments, the cell processing apparatus 100 includes only a single UV sterilizer 800, which is moved between the centrifuge tank 114 and the vial box recess 126 by the cassette positioning assembly 132.

[0151] Figure 45A-45KThe illustration shows the operation of actuator 850, which is configured to manipulate reagent syringe 702 disposed within TSC210. ​​Actuator 850 can be positioned at the upper end of recess 134 in cartridge positioning assembly 132, such as... Figure 3 As shown. Figure 45A The actuator 850 is shown to include a first clamping mechanism 852 and a second clamping mechanism 854. The second clamping mechanism 854 is carried within the first clamping mechanism 852 and is movable independently relative to the first clamping mechanism 852. The first and second clamping mechanisms 852 and 854 each include a finger shown in an open state.

[0152] Figure 45B This illustrates how the second clamping mechanism 854 of the actuator 850 is engaged with its... Figure 45A The previous position in the syringe moves downward and its fingers move to the closed state to secure the second clamping mechanism 854 to the first flange at the upper end of the plunger 806 of the reagent syringe 702. Figure 45C The first clamping mechanism 852 of the actuator 850 has been moved downward, and the fingers of the first clamping mechanism 852 have been moved to the closed state to clamp the flange at the upper end of the solution collection container 810 of the reagent syringe 702.

[0153] Figure 45D The actuator 850 is shown moving downward to push the needle 804 of the reagent injector 702 out of the bottom of the TSC 210. Although neither the RSC 202 nor the processing cartridge 212 for receiving the needle of the reagent injector 702 is shown, these are omitted only to focus on the movement of the actuator 850 and the reagent injector 702 relative to the TSC 210. Figure 45E The second clamping mechanism 854 is shown moving upward while the first clamping mechanism 852 remains stationary, so as to move the plunger 806 of the reagent syringe 702 upward, thereby drawing fluid into the solution collection container 810 of the reagent syringe 702.

[0154] Figure 45F The actuator 850 is shown moving upward to withdraw the needle 804 and the solution reagent syringe 702 back into the TSC210, allowing the cartridge positioning assembly 132 to move the TSC210. Figure 45G This illustrates how actuator 850 is configured to push reagent syringe 702 downwards so that needle 804 engages in another cartridge. Once needle 804 is engaged in the other cartridge, Figure 45H The second clamping mechanism 854 is shown to have moved downward to press down on the plunger 806, thereby discharging fluid into another cartridge. Figure 45I The actuator 850 is shown after it moves upward to retract the needle 804 into the TSC210. Figure 45J–45K illustrates actuator 850 disengaging from the first and second clamping mechanisms 852 and 854 of reagent syringe 702. The first and second clamping mechanisms 852 and 854 may disengage from reagent syringe 702 simultaneously or sequentially. It should be noted that, as depicted, actuator 850 is sized to work with the reagent syringe; however, it should be understood that the cell processing apparatus 100 may also include a second actuator of similar configuration, reduced in size to be compatible with sampling syringe 704.

[0155] This invention includes a method for analyzing cell suspensions during a cell processing workflow to provide accurate cell counting, population distribution, and purity assessment. This analysis is achieved by integrating a cell analysis module (CAM) within the cell processing system, which enables precise optical and biochemical cell characterization.

[0156] Analysis and Sampling Process: The process begins after the cells have been mixed to ensure homogeneity, and the TSC aspirates a precise volume of cell suspension. The precise volume of the sample (e.g., 5 µL, 10 µL, or a predetermined amount) is controlled by system programming or pre-selected by the operator. As previously mentioned, the TSC is equipped with rotational capability, allowing it to align with various functional modules within the system, including the CAM. Once the TSC has aspirated the sample, it rotates to align with the CAM, which can be integrated as a stand-alone analytical device or incorporated into one of the slots of a reagent / sample cartridge (RSC). The sample is then ejected through microfluidic channels within the CAM, enabling precise analysis.

[0157] Cell Analysis Module (CAM): The CAM is a dedicated analysis unit designed to process cell suspension samples for real-time assessment of cell characteristics. It includes the following components: Microfluidic channels for single-cell detection: The CAM includes microfluidic channels whose diameter is precisely designed to allow single cells to pass through. This ensures that cells flow through the channel one by one, allowing for high-resolution analysis. As each cell passes through the channel, an optical or impedance-based detection system measures key parameters, including the cell diameter, which can be used to estimate the volume of a single cell. The CAM records the rate at which cells pass through, enabling the system to calculate the cell concentration within the sample. By multiplying this concentration by the known sample volume, the total cell count in the suspension can be accurately determined.

[0158] Optical and Biochemical Detection of Surface Markers: CAM integrates advanced optical systems and reagents for the detection of specific cell surface markers. The system uses biochemical reagents capable of binding to target markers, such as CD3 (specific to T cells) and CD14 (specific to monocytes). This detection system assesses marker expression and provides quantitative breakdown of cell populations. For example, T cells are identified as CD3-positive and CD14-negative; monocytes are identified as CD14-positive and CD3-negative.

[0159] Quantitative and purity analysis of cell populations: Based on the detection of specific markers, CAM quantifies the different cell populations present in a sample. It calculates the number of T cells and monocytes, and determines the purity of T cells by dividing their count by the total cell count in the sample.

[0160] Integration with system control: CAM is fully integrated with the system's central control unit. This integration allows for real-time feedback of analytical results, which can be used to optimize downstream processes such as gene modification, washing, or formulation. Analytical data ensures that the cell suspension meets quality thresholds before proceeding to subsequent steps.

[0161] Portability and Design: CAM is designed to seamlessly integrate into the modular architecture of cell processing systems. It can be integrated within the RSC or included as a standalone module accessible via the TSC. The modular design facilitates replacement and maintenance while ensuring sterility and compliance with regulatory standards.

[0162] Advantages of CAM Integration: Integrating CAM into cell processing systems significantly improves the accuracy and efficiency of cell characterization. Microfluidic design ensures single-cell resolution for concentration measurements, while the use of optical and biochemical detection methods enables precise differentiation of cell populations. By providing real-time analytical results, CAM streamlines workflows and improves the reliability of therapeutic cell products, ensuring consistent and high-quality results.

[0163] Figure 46 A cross-sectional side view is shown illustrating how the PFC 204 can deposit solution into the main chamber 424 of the treatment cartridge 212. In particular, it should be noted that a UV sterilizer is not used in this step because the treatment cartridge 212 has a high-quality filter capable of removing any contaminants entering the fluid outlet port 510 of the PFC 204 and / or the fluid port 408 of the treatment cartridge 212. Figure 46A nozzle 902 attached to the pneumatic port 504 is also depicted, which supplies pressurized gas to the liquid chamber 904 of the PFC 204, causing the solution disposed within the liquid chamber 904 to exit through the fluid outlet port 510 and enter the treatment cassette 212 through the fluid port 408. In some embodiments, the pressure supplied by the nozzle 902 is tuned so that a filter disposed below the fluid port 408 can keep up with the rate of fluid reception.

[0164] This system features a robust communication and power delivery design to ensure reliable operation in the dynamic environment of a centrifuge, where noise and motion can pose challenges. Suitable communication methods may include a CAN bus system, a flexible robot cable, or an Ethernet connection. Figure 47 and 48 As shown, the system utilizes robotic flexibility and a CAN bus cable 443, positioned near the hinge of the centrifuge drum, providing sufficient flexibility to allow the drum to pivot up to 135° in each direction. Power cables supply energy to critical components such as the motor and heater, while also transmitting real-time data (such as the temperature of aluminum components) back to the control system. This cable is specifically designed to maintain effective data transmission even in environments with electromagnetic noise.

[0165] In one alternative implementation, the robot's flexible and CAN bus cable (see...) Figure 30 The drum is strategically positioned near the hinge of the centrifuge drum to facilitate movement. This arrangement allows the drum to tilt in either direction, with a preferred pivot range of up to 135°, but in some configurations it can extend to 165° or 180°, and in certain applications, a full 360° rotation is possible. Power cables supply energy to the motor and heater, while the CAN bus transmits real-time feedback (such as aluminum temperature, process sensor values, valve positions, and other operating parameters) to the control system. The CAN bus architecture ensures robust and noise-resistant communication, making it particularly suitable for the dynamic and electromagnetic noise environments of centrifuges.

[0166] Figures 49A-49D The diagram illustrates the automated balancing system (automatic balancer) used in ACP. Go to... Figure 49A and Figure 49B The image shows a cross-sectional view of a centrifuge with the drum in its mixing mode. Figure 49C It is a cross-sectional view of the centrifuge during rotation, and Figure 49D This is a cross-sectional view of the centrifuge when it is stationary.

[0167] The automated balancing system enhances the centrifuge's ability to maintain equilibrium amidst fluid level changes within the CPC212. This system achieves dynamic balancing through the use of specific linear actuators, each with a movable mass block, allowing for adjustable balancing of the centrifuge, centrifuge drum, and variable mass sample within the CPC during operation. In alternative implementations, additional actuators provide fine-tuning capabilities, particularly effective at higher RPMs where torque demands are greater. The CPC212 includes locking mechanisms or tabs for stability, ensuring the CPC is properly positioned and remains stable throughout the process. Each actuator incorporates a locking mechanism to prevent unwanted movement of the automated balancing system under load. This mechanism allows movement toward the centrifuge's center of rotation but restricts it to the opposite direction when engaged, thus stabilizing the system during high-speed operation.

[0168] A three-axis accelerometer, precisely aligned with the centrifuge's rotation axis, measures vibration and imbalance in real time. If vibration exceeds programmed limits, the system automatically cuts off the centrifuge motor's power drive and engages inductive braking to stop rotation. This accelerometer is part of an active imbalance compensation feedback loop, with multiple accelerometers on different excitation axes to provide enhanced signal accuracy. The accelerometer is synchronized with a rotary position encoder, enabling precise, rotation-related data collection at any speed. Fluid transfer into or out of the system, or within the system, can occur when the centrifuge is stationary or operating with various relative centrifugal forces, ensuring accuracy and minimizing turbulence. The balancing system employs a counterweight mechanism, where each complete rotation of the motor corresponds to a 2mm adjustment in the counterweight position. By correlating accelerometer data with position information from the encoder, the system achieves precise real-time indexing of vibration data with the motor's rotational position, enabling dynamic balancing, improved stability, and enhanced motion tracking along the X, Y, and Z axes. Furthermore, the system can transmit accelerometer data to any rotor, providing greater compatibility in some implementations.

[0169] An example of the use of an automated balancing system is as follows: The process begins after the centrifuge tank positions the CPC in an upright orientation. The PFC descends, docks with the CPC, and performs the necessary fluid transfer operations, including dispensing fresh fluid into the CPC or receiving waste liquid from the CPC for transfer to a waste container. After fluid transfer is complete, the system uses integrated sensors to determine the volume or weight of the cell suspension inside the CPC. This measurement provides crucial data for precise balancing of the system. The automatic balancer uses data from the sensors to reposition the movable mass block along a threaded rod. Linear actuators precisely adjust the position of the mass block to minimize vibration during centrifugation. This balancing step ensures stable operation by counteracting any imbalance caused by changes in the volume or weight of the fluid within the CPC. Centrifugation is only allowed to resume after the automatic balancing procedure is complete. Precise balancing prevents excessive vibration, reduces mechanical stress, and ensures consistent sedimentation rates and hydrodynamics during centrifugation.

[0170] This balancing system integrates seamlessly with the centrifuge tank for precise rotational control and active oscillation for in-situ mixing when the rotor is stationary. The system employs a motion-inducing mechanism that applies reciprocating motion to the container along a predefined path, ensuring effective mixing of the contents. This motion actuator supports non-linear motion modes designed to promote homogeneous mixing. Its velocity profile follows a sinusoidal trajectory, decelerating at the endpoints of the oscillating motion to minimize turbulence and ensure smooth handling of sensitive materials. The actuator also features adjustable speed settings, enabling optimized mixing at specific time intervals for thorough homogenization. Furthermore, precise motor position control allows for rotational mixing, which can generate a swirling effect in some applications to improve mixing efficiency. This versatile system supports multiple mixing techniques, including oscillation, tilting, and rotation, ensuring optimal content homogeneity.

[0171] like Figure 16 As shown, to ensure precise temperature control and monitoring, a temperature sensor 150 is integrated within the housing 212 and positioned near the liquid contents. The cell processing device 100 can be configured to increase or decrease heat to preferably maintain the solution 1022 at a temperature of approximately 37°C; however, in different embodiments, a temperature different from approximately 37°C may be desired.

[0172] The cell processing device 100 features improved temperature control for efficient heating of biomaterials without wasting excessive energy. In some embodiments, the container 120 may include a heating element capable of raising the temperature of the material contained within the processing chamber 212. In some embodiments, this design incorporates a thin, anodized aluminum heating element to heat only specific areas, supported by a low thermal conductivity material such as thermally stable cast nylon and air gaps to prevent unnecessary heat loss.

[0173] like Figure 50 As shown, this heating configuration is characterized by a main heating element, which is a thin anodized aluminum cylinder 1050, approximately 2 mm thick, positioned close to CPC212 where the biomaterial is most concentrated, thus ensuring targeted heating without unnecessary energy consumption. The aluminum is black-anodized to improve its thermal emissivity, which enhances heat transfer toward the contents of the container. This heating element covers only specific portions of CPC212, particularly the areas where the bio-liquid is primarily concentrated, ensuring targeted heating without unnecessary energy consumption. This heating element may be surrounded by a low thermal conductivity insulating barrier made of thermally stable cast nylon or a similar material, along with air gaps. The insulating properties of the cast nylon prevent heat loss to surrounding components, thus maintaining a focused heating area. An insulating cast nylon layer 1054, approximately 2 mm thick, circumferentially encapsulates the aluminum heating element to restrict heat outward flow. The aluminum tube with the heating element is wrapped by the insulating barrier. The heating element is adhered to the tube by pressure-sensitive adhesive (PSA) and surrounded by a polyester film shrink wrap. This mechanical belt is used to prevent heater stratification during centrifugation. To prevent heat loss, a black material is used, and certain thermal path links are severed using plastic and air gaps. In this configuration, the box design incorporates multiple ribs that are not otherwise used for structural purposes. These ribs facilitate heat conduction. This preferred configuration of the heating elements maintains the internal temperature even after heating is turned off. A 2 mm thick black anodized aluminum heating cylinder 1050 and a thin-film heating element 1052 maintain the temperature of the cell solution, which is crucial for cell viability and optimized processing conditions. A printed circuit board (PCB) 1060, fixed to the distal end of one of the sensor / emitter housings, is connected via a spring pin on a temperature sensor 150 (CPC212) to monitor temperature and ensure consistent thermal conditions. An additional air gap 1058 of approximately 6 mm is placed between the insulation layer 1054 and the outer barrel structure 1056. This air gap acts as a buffer to trap heat within the target area, further preventing heat transfer to unnecessary areas.

[0174] The purpose of this heating mechanism is to efficiently control the temperature of the biomaterial contained within the CPC while minimizing energy waste and preventing overheating of non-target areas. This is crucial for maintaining stable conditions at approximately 37°C, which is optimal for cell viability and processing without causing thermal stress or denaturation to the temperature-sensitive biomaterial, and simultaneously minimizing heat dissipation to the outside of the centrifuge tank and the centrifuge itself. Further benefits include reduced power consumption and extended component lifespan.

[0175] like Figure 50As shown, the barrel 120 includes three optical sensor / emitter pairs 1062 to track cell sedimentation and confirm correct valve rotation 1064. The barrel 120 is pivotally connected to a rotation axis 124 for mixing the cell suspension in CPC 212. The barrel further includes a valve rotation drive assembly 1066. Therefore, the barrel 120 of the cell processing device 100 includes a 2 mm thick black anodized aluminum heated cylinder 1050 for emissivity, a thin-film heating element 1052 having a thickness of approximately 1 / 2 mm, and an air gap.

[0176] In some alternative embodiments, the cell processing apparatus 100 may also include a cooling / heating unit capable of supplying cooling / heating air to the centrifuge tub 114. In some embodiments, heat is conducted into the tub via a conductive device (e.g., a thin black anodized aluminum tube around which heating elements are wound). To suppress or minimize heat transfer to the rest of the centrifuge tub, a ceramic shell and air gaps may be used. In some embodiments, the tub is multi-layered with materials of different thermal conductivity and insulation properties to specifically control heat transfer.

[0177] like Figure 51A As shown, T cells connect to microvesicle 1122 during barrel mixing. Figure 51B The diagram illustrates how a barrel rotates at 250 xg, separating buoyant T cells from sedimenting non-target cells. In this setup, microbubbles facilitate a unique compression and release cycle for cell selection and concentration. The main steps include: Centripetal force during rotation: As the centrifuge rotates, the centripetal force pushes non-target cells downward toward the narrower end of the funnel and eventually toward the sedimentation column 431. Here, microbubbles remain in close proximity to each other and to the target cells, thus preventing the target cells from settling.

[0178] Buoyancy rebound and separation: Cell buoyancy counteracts this downward force, causing cells to move upward within the funnel. As they rise, they encounter the gradually widening cross-section of the funnel, which gives them more space to disperse. This separation step reduces the density of cell clumps and allows cells to be distributed more evenly within the fluid.

[0179] This balance of forces—compression of the microbeads against the funnel wall and buoyancy separation as the cells rise—allows cells to be tightly packed and then gently separated, thus optimizing selection and distribution across the funnel. This design utilizes mechanical and physical properties to enhance the precision of cell handling and separation.

[0180] Figure 28 and Figure 53AThe four main rotational positions of the CPC valve are shown, corresponding to different fluid transfer points within the cartridge. The valve rotates to four positions, such as reagent septum 1070, non-target cells 1072, second-phase T cells 1074, and harvest septum 1076. The reagent septum position allows reagents to enter the cartridge, preferably for cell processing steps such as washing, activation, or transduction. This position allows reagents to enter the bottom of the cartridge's sedimentation column, where cells may or may not have been transferred by centrifugation in the CPC, potentially for cell processing steps such as washing, activation, or transduction. When in the non-target cells position, the valve is positioned to separate and dispose of or transfer non-target cells, ensuring that only the desired cell population remains in the cartridge. Here, it isolates non-target cells, ensuring that only the desired cell population remains in the CPC's main conical or cone-shaped cell processing chamber for subsequent processing steps. The harvest septum position is used to harvest the final cell product, allowing selected and processed cells (e.g., CAR-T cells) to be extracted from the cartridge. The second T cell location was designed to isolate a second subset of T cells, which may be used for sequential processing or further refinement of the cell population.

[0181] like Figure 53B As shown, in addition to these four main positions, the diagram also indicates four other rotational positions between the four positions listed above, each of which will prevent fluid transfer through the valve. These can act as “closed” positions, where the valve rotates to align with the section that blocks any fluid movement, ensuring containment and preventing accidental mixing or contamination.

[0182] A rotary valve positioning system can use up to three independent mechanisms to definitively verify that the valve is correctly positioned. These are a position sensor, an optical through-hole sensor, and an alignment sensor, as described below.

[0183] Position sensor on valve drive shaft: The position sensor is used in conjunction with a servo motor controller to move the valve to a commanded angular position. While any position sensor can be used in a preferred embodiment, in one embodiment, an optical encoder is used and reports the drive shaft position, as well as the backlash / clearance between the drive shaft and the valve rotor that may introduce uncertainties into the valve position. This encoder has an index position signal defining 0° rotation, which is calibrated using a fixture during manufacturing. For enhanced robustness, this is not the only position feedback used for the actual valve position. In one embodiment, the position sensor may have a 1:1 match with the valve drive gear position.

[0184] Optical through-hole sensors. For example... Figure 52As shown, alignment sensors are used for final valve position alignment and include a narrow slot in the valve rotor that allows light signals to be transmitted from an optical transmitter 1071a on one side to an optical detector 1071b on the other side (for this patent, the left and right positions of these sensors may not be important; however, if important, Figure 52 The numbering from left to right in the middle is the reverse of the actual position in the Trenchant barrel. This transmitter-detector provides visual confirmation through the holes in the valve stator, allowing the light signal to pass only if the valve is in the correct alignment position between the valve rotor and the stator of interest. In some instances, the correct alignment position is for opening or closing the port. In a preferred embodiment, the optical encoder guides the valve rotation within + / - 1.0°, such as... Figure 52 As described. The alignment sensor signal depends only on the relative positioning of the rotor and stator and is independent of changes in backlash or encoders or other position sensors in the drive components. Furthermore, additional sensors may include, but are not limited to, Hall effect sensors, additional optical encoders, inductive sensors, magnetic or optical sensors.

[0185] The system employs zone sensors to indicate which of the four (or more) port positions is active during valve operation. While this implementation preferably includes four Hall effect sensors, the system is not limited to this sensor type; alternative position sensing technologies, such as optical encoders, inductive sensors, magnetic sensors, or capacitive sensors, may also be used depending on application requirements.

[0186] In a preferred configuration, zone sensors interact with a single magnet located on a driveshaft gear to determine the approximate position of the valve relative to a port. Each zone sensor corresponds to an effective zone range centered on the ideal port position. The effective zone range can be between ±1° and ±20°. At any given time, only one zone sensor is active, indicating that the valve is approximately aligned with one of the ports. When the valve is between ports, all zone sensors are passive, ensuring the system recognizes that no port is currently engaged. This transitional state helps prevent misalignment or false reports. Zone sensors play a crucial role in valve positioning by confirming that the valve is approximately positioned with the desired port. This confirmation allows the system to subsequently employ an optical alignment sensor for final, precise positioning adjustments. This optical sensor ensures the valve achieves the required precise alignment, in some cases, to open or close a selected port, utilizing its ability to detect alignment peaks with high accuracy.

[0187] By combining the general positioning capability of a zone sensor with the precise alignment detection of an optical through-hole sensor, the system achieves robust and reliable valve positioning. This redundant sensing method enables seamless operation even under conditions where slight mechanical backlash or tolerances may introduce uncertainties.

[0188] The simplified usage model of the rotary valve system is as follows. The initial position assumes that the valve is in the "stayed / closed" position with no ports open.

[0189] Initial positioning to the nominal port position: The servo controller rotates the valve approximately 45° to the nominal angular position corresponding to port A. This movement relies on pre-calibrated position data. Next, the operation and accuracy of the encoder or other position sensors are verified by checking the zone sensors (e.g., Hall effect sensors or alternative techniques). At this stage, only the zone sensor for port A is active, confirming that the valve is within the correct general range. If no zone sensor is active, or more than one is active, the system reports a potential fault condition to the monitoring system controller for further diagnostics.

[0190] Precise positioning is achieved using optical alignment: The valve is rotated from its nominal position while monitoring an analog alignment sensor signal generated by an optical through-hole sensor. The system algorithm identifies a peak alignment signal (the maximum value of the analog signal), indicating precise alignment of the valve with port A. Valve rotation is stopped at this peak position. Optionally, a final positioning algorithm can be executed to ensure maximum accuracy, taking into account mechanical backlash, tolerances, or drift.

[0191] Verification and fault detection: At this point, the system performs a position cross-check to confirm alignment. Here, the encoder position should indicate the correct angular position of port A. The area sensor confirms the approximate position. The optical alignment sensor verifies precise alignment with the open port.

[0192] If all three detection mechanisms (encoder, zone sensor, and optical alignment sensor) are consistent, the valve is confirmed to be in the desired port open position. If a mismatch is detected between any of these position indicators, the system reports a fault condition to the monitoring system controller. The fault signal can trigger appropriate diagnostic, error handling, or corrective actions.

[0193] Figure 54 The CPC212 in active mode is shown. White blood cells are detected using LED white light frequency. Figure 55A-55J The diagram illustrates the CPC valve rotating through a series of positions to facilitate the stepwise processing of cells, reagents, and other components involved in CAR T cell production. Valve rotation is primarily in 45° increments, with a single 135° rotation in either direction. This allows for both fine and coarse adjustments to precisely position the valve for each task. Each position is associated with a specific action or step in the manufacturing workflow, allowing the device to separate or combine materials as needed for optimal processing.

[0194] This rotation sequence demonstrates the versatility and automation of the CPC, highlighting how the device systematically performs complex cell processing steps. By rotating valves to control access to different compartments, the system enables a closed, automated, and efficient process for CAR T cell manufacturing, where each step is finely tuned to maintain cell quality, safety, and efficacy. There are four residence positions (1, 3, 5, and 7 are residence positions), and four positions that allow cell movement.

[0195] Step 1: Filling, Buffer Addition and Washing - Position 1, shown Figure 55A The valve position allows buffer solution to be introduced into the chamber to wash cells in preparation for subsequent processing.

[0196] Step 2: Add connector - position 8, as shown Figure 55B This step allows for the addition of connectors, which may be necessary for cells to bind to microvesicles or other components.

[0197] Step 3: Mixing, removing, and adding buffer solution - Position 1, shown Figure 55C The combined steps allow for the mixing and removal of used or excess buffer within the cartridge, as well as the addition of fresh buffer, to maintain optimal conditions for cell health and viability. The buffer acts as a carrier to remove impurities and non-target components.

[0198] Step 4: Add microbubbles (MB) - Position 8, as shown Figure 55D In addition, microbubbles are added to assist in cell selection or separation. Microbubbles can be manipulated to help concentrate, separate, or isolate specific cell populations.

[0199] Step 5: Mix, remove buffer, pressurize, and centrifuge - Position 1, shown Figure 55E The valve position facilitates further mixing, buffer removal, pressurization, and centrifugation. This step may aid in cell isolation, concentration, or preparation for gene transfer.

[0200] Step 6: Isolate non-target cells - Location 2, shown Figure 55F In this step, non-target cells are isolated and removed to ensure that only the desired cell population (e.g., T cells) remains for subsequent treatment.

[0201] Step 7: Add culture medium, mix and control temperature - Position 1, shown Figure 55G In the middle, the valve introduces the culture medium to support cell growth, mixes the cells, and adjusts the temperature (heating to 37°C and cooling to 32°C) to create conditions favorable for cell viability and activation.

[0202] Step 8: Add carrier - position 8, shown Figure 55HIn CAR T-cell therapy, a key step involves introducing a gene transfer vector, whether viral or non-viral, to deliver the CAR gene into T cells, enabling the cells to target and eliminate cancer cells. The vector is introduced at the bottom of the cell suspension, allowing it to rise naturally through the small volume of T cells. Because both the vector and cells are in suspension, they begin to mix immediately upon contact, facilitating efficient gene transfer and uniform distribution throughout the cell population.

[0203] Step 9: Centrifugation, inoculation, washing, and formulation - Position 3, shown Figure 55I In this study, centrifugal inoculation was used to enhance gene transfer efficiency, followed by cell washing and preparation for final harvest. Centrifugal inoculation helps ensure close contact between the vector and cells, thereby improving transduction rates.

[0204] Position 10: Harvest - Position 4, shown Figure 55J The final step involves collecting CAR T cells and preparing them for therapeutic use.

[0205] Key insights into the rotating system include controlled sequential processing, customizable angle and rotation control, and closed-system integrity. In controlled sequential processing, the rotational position enables a precise, sequential workflow, minimizing cross-contamination and optimizing conditions for each process step. Furthermore, in customizable angle and rotation control, valve rotation is primarily in 45° increments with a 135° rotation. This allows for both fine and coarse adjustments to precisely position the valve for each task. Regarding closed-system integrity, the system design ensures sterility and containment. Moreover, this design is crucial for sterile cell handling, particularly when processing sensitive therapeutic cells.

[0206] ACP incorporates a rigorous Pressure Decay Filter Integrity Test (FIT) protocol to ensure the structural integrity and proper functionality of all filters within the CPC. For example... Figure 56 and Figure 57A As shown in -B, the FIT process was specifically used to validate aseptic transfer capability through a filter (e.g., a 0.2 µm hydrophilic input filter located in the CPC cap). This test was performed both before and after processing to confirm filter performance. Waste removal and fresh culture transfer were achieved individually via a PFC that rotates, descends, and docks with the CPC cap. Fresh culture enters the CPC through the input port, while waste exits through the waste outlet. The locations used for the FIT pressure decay test were indicated as 1080, 1082, and 1084, while the paths for waste removal and culture input were... Figure 57A The values ​​are shown as 1088 and 1090 respectively. Furthermore, Figure 57BThe CO2-rich container is highlighted, which facilitates the transfer of fresh culture medium into the CPC and the removal of waste culture medium at approximately 1.5 PSI. For filter integrity testing or microbubble degassing, a pressure of approximately 45.0 PSI is applied.

[0207] This process ensures that fluid and gas exchange with the CPC occurs only through the filter, eliminating bypasses that could compromise sterility. The FIT procedure plays a crucial role in maintaining a sterile environment throughout the CAR T-cell manufacturing workflow, minimizing the risk of contamination and ensuring the production of high-quality therapeutic products. Filter integrity is validated both before introducing patient cells and after the processing cycle is completed to ensure that no degradation or structural failure compromises the sterility of the CPC.

[0208] The automated pressure decay (FIT) process is performed independently for both hydrophobic and hydrophilic filters, which are key components of the ACP quality control system. Due to differences in surface area and decay rate thresholds, each filter is tested sequentially, ensuring accurate and individualized test parameters. This approach enhances sterility and reliability, ultimately improving the safety and efficacy of CAR T-cell products.

[0209] Test Procedure Overview - Pressure Decay Test (FIT) measures the pressure drop (ΔP) in the upstream volume connected to the filter over predefined time intervals. This procedure ensures that the filter's flow rate is within permissible limits, ruling out blockages or leaks. Hydrophobic and hydrophilic filters are tested individually. Due to their different surface areas, each filter requires a unique pressure decay parameter and lookup value to determine the acceptable decay time. The FIT protocol identifies potential problems, including low-flow failures (indicating potential blockages or contamination in the filter or associated ports or passages) and high-flow failures (indicating cracks, breaks, seal failures, or leaks in the filter housing, CPC structure, or test assembly). By sequentially testing each filter and employing precise decay thresholds tailored to specific filter areas, ACP ensures comprehensive quality control. This robust testing protocol not only minimizes the risk of contamination but also enhances the platform's ability to produce safe and effective CAR T-cell therapies.

[0210] Test procedure, example - (1) Pre-process FIT: Before introducing patient cells, gas flows through a sterile vent and the system is pressurized. (2) Attenuation measurement: Pressure attenuation test uses the formula to calculate the pressure drop over time: ΔP=DR×T×PaVupΔP=VupDR×T×Pa, where: ΔP is the pressure drop, DR is the diffusion rate, T is time, Pa is atmospheric pressure, and Vup is the upstream volume.

[0211] Step-by-step testing program Close the CPC valve to prevent flow. Closed outlet filter port Close the upstream volume (UV) valve Close the FIT valve Fit the FIT box to the hydrophobic inlet filter port. Open the UV valve to pressurize the upstream volume to the starting pressure. Start the decay timer & open the FIT valve Wait for the decay time and record the decay decrease value of ΔP. Close the PC FIT vent valve Close the FIT valve If the ΔP attenuation is within the minimum / maximum test limit or < the attenuation limit, then pass. Release all CPC stress to complete the test Pass / Fail Determination: A pass result is obtained if the pressure drop is within an acceptable range. A failure is caused by excessive or insufficient pressure drop, indicating an underlying filter problem.

[0212] The calculations for the representative test are shown below. For an initial upstream pressure of 45 psi, under these conditions, a pressure drop of 24.5 psi will result in a final upstream pressure of 20.5 psi. A failed low-flow filter will result in a higher final upstream pressure, while a failed high-flow filter will result in a lower final upstream pressure.

[0213] DR = 10,000 mL / min; Test duration = 1 minute; Vup = 100 mL ΔP = (10,000 mL / min) 1 minute 14.7 psi / 100 mL = 24.5 psi Post-process FIT: Following treatment, a repeat FIT ensures the filter remains intact throughout the cell processing and does not suffer any performance degradation. The results of this post-process test are automatically recorded in the batch record data, which helps to meet quality control and regulatory requirements.

[0214] In addition to performing filter integrity tests, the air pressure control system plays a crucial role in managing and maintaining precise fluid flow rates within the system during critical operations. It ensures controlled fluid distribution to the CPC, removal of waste liquid, transfer of formulated genetically modified target cells, and specific manipulations during selection and activation processes, such as microbubble bursting. By regulating air pressure across multiple operations, the system ensures accurate and efficient fluid transfer and removal. The system operates by selecting air pressure modes from at least three different modes to optimize fluid handling and system integrity.

[0215] The initial fixed pressure can be used for microbubble bursting and filter integrity testing. During the target cell selection and activation phase, a fixed air pressure is applied to cause the microbubbles attached to the target cells to burst. This eliminates the buoyancy imparted to the target cells, allowing them to settle effectively for subsequent processing. The same fixed pressure (or different pressures) can be used to test the structural integrity of both hydrophobic and hydrophilic filters on the CPC cap. This ensures the structural integrity of the filters, meaning they are leak-free and able to remain sterile throughout the process.

[0216] The second fixed pressure is associated with waste removal. Positive air pressure is applied to propel the waste from the CPC through a central waste tube into an expandable, sealed waste container. This controlled pressure ensures effective fluid removal without disturbing the isolated target cells at the bottom of the settling chamber.

[0217] A third fixed pressure setting is used for fluid transfer from the PFC to the CPC. When the PFC is docked with the CPC, air pressure is applied to the fluid compartment within the PFC to control the fluid transfer. This pressure ensures a regulated flow rate through a hydrophilic filter located on the CPC cap, enabling precise delivery of fluid into the CPC for processes such as washing, reagent addition, or volume adjustment. It should be further understood that these different fixed pressures can alternatively be supplied by a single electronically controlled regulator, which is programmed to adjust the pressure to meet the specific requirements of each process.

[0218] Figures 58A-58D The cell suspension 1122 is shown when CPC212 is tilted. As described above, CPC can be tilted to facilitate mixing. CPC preferably oscillates back and forth between 1° and 360°, and in some cases can rotate an unlimited number of times. In some embodiments, where a port or filter is found along the top cover of the CPC, the degree of tilt in either direction is inversely proportional to the fluid level in the CPC. For example, a 90° tilt in either direction is typical for a particular fluid level, but with reduced fluid, tilting beyond, for example, 135° is possible, and in this embodiment, a larger volume of fluid may only allow a tilt of less than 90° in either direction.

[0219] Figure 59A The cross-section line HH passing through CPC212 is shown, and this cross-section line is used for Figure 59B The cross-sectional image shown is -59D. Figure 59B In the figure, a cross section along HH shows the cell suspension fluid 1122 in the main chamber 424. Figure 59CThe illustration shows a portion of the fluid 1122 moving into a 30 mL auxiliary chamber 1124, from which a small riser 1126 extends upwards. In this embodiment, the riser has a volume of 0.2 mL. As the downward pressure from the weight of the fluid in the main chamber 424 fills the auxiliary chamber 1124 under centrifugation, this pressure pushes the fluid in the other chamber upwards along the riser 1126. Therefore, the small-aperture riser design ensures precise fluid volume control under centrifugation, addressing challenges related to fluid displacement, sterility, and measurement accuracy.

[0220] More specifically, the second-stage compartment, sealed with a cap, precisely holds 30 mL of fluid when full. During centrifugation, as fluid enters this compartment, the displaced air must be expelled. This is achieved through a fine-aperture tube extending upwards along the interior of the CPC and connecting to a port below a hydrophobic filter. The internal volume of this tube is less than 0.2 mL, ensuring precise air displacement while maintaining sterility by preventing communication with external air. When the CPC valve is rotated from its residence position to open the port to the 30 mL compartment, centrifugal force propels the cell suspension into this compartment. As fluid enters, the displaced air travels upwards along the fine-aperture tube and is transferred to the main chamber 424. Any pressure differential that may be caused by this fluid movement can be expelled through the hydrophobic filter. The fluid rises within the fine-aperture tube only to the same distance from the axis of rotation as the fluid level falling in the main conical or conical compartment, thus ensuring balance of centrifugal force. This dynamic prevents overfilling or underfilling of the compartment regardless of the duration of centrifugation.

[0221] The riser's main contributions are enhanced volumetric accuracy, measurement precision, and functional integration. The riser limits the variability of fluid measurements. Any change in fluid height within the main chamber is precisely mirrored in the riser, ensuring accurate volumetric control. The riser's small orifice volume (0.2 mL) ensures high measurement accuracy, with variability limited to ±0.1 mL. This precise design allows for reliable determination of fluid level without significantly impacting the overall system capacity. The design provides a simple, reliable, and highly accurate mechanism for determining fluid volume in secondary chambers during centrifugation. The riser system operates automatically without control mechanisms, making it cost-effective, minimally invasive, and without interfering with the overall functionality or capacity of the CPC.

[0222] This riser system is particularly advantageous for processes requiring high precision, such as cell separation and reagent handling, where minute changes in volume can significantly impact results. Its integration within the CPC enhances the closed-system design, minimizing the risk of fluid mismanagement and contamination. During operation, the process relies on empirically determined timeframes because radar or laser systems cannot monitor fluid levels during rotation. Precise fluid transfer of 30 mL volumes is consistently achieved by maintaining a centrifugal force of 50 G and keeping the valve open to the compartment input for a standardized duration (e.g., exceeding the minimum by 10+ seconds).

[0223] Figure 59D The diagram shows that fluid from the main chamber 424 continues to push the fluid in riser 1126 upwards until it is in equilibrium with the fluid in the main chamber 424. The fact that riser 1126 is so small (0.2 mL) means that there is very little uncertainty about the 30 mL volume in the other chamber, because such a small amount would need to move upwards along the riser to reach equilibrium with the liquid level in the main chamber 424, regardless of the liquid level.

[0224] The dynamic nature of the CPC weight, caused by the entry or exit of fluid during operation, requires precise monitoring to ensure stability and accuracy within the centrifuge tank. 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 the fluid level with high precision. Here, the radar system is positioned above the CPC cap, where it emits radar waves via a guide rod. The radar waves reflect off the fluid surface at a precise point where they contact the liquid. The radar system eliminates reflections from the plastic walls, focusing only on the fluid surface to obtain an accurate reading. Figure 60A and 60B This radar system 1130 is shown connected to cover 402. S-shaped channel 1128 guides radar 1130 from radar transmitter 1132 (not shown).

[0225] A radar transmitter / receiver 1130 (not shown) is configured to detect reflections from a fluid surface within a sensing range of 15 to 85 mm, wherein the detected fluid height is converted into a weight estimate for balancing purposes. The system software calculates the fluid level by subtracting the reflection data between the transmitter / sensor / rod interface and the fluid contact point along the straight portion of the rod extending vertically through the CPC cover.

[0226] In one exemplary case, this utilizes a 61 GHz radar transceiver (RFbeam V-LD1) combined with a polycarbonate rod that transmits radar waves. The radar measures fluid levels with exceptional accuracy, achieving a resolution better than 1 mm over a range of 0 mm to 63.47 mm, corresponding to fluid volumes from approximately 15.4 mL to 350 mL. This implementation ensures a resolution better than 1 mm; lower liquid levels require calibration to maintain this accuracy. Nonlinearity observed in the 0–3 mm measurement range due to rod-end reflections is corrected using a software filtering algorithm. The effect of the epoxy adhesive used to secure the rod to the cap is visible in the radar signal but is effectively filtered out through advanced signal processing. The radar signal dispersion is controlled within ±10°, ensuring that only reflections from the fluid surface are captured, eliminating interference from the containment walls. The rod's positioning is critical, avoiding contact with the containment except at designated anchor points to maintain measurement fidelity.

[0227] The radar system integrated into the platform achieves 99% energy reflection accuracy at the fluid contact point, ensuring precise fluid level detection. This facilitates real-time adjustments during critical processing cycles, including fluid input, centrifugation, and extraction, as the radar detects changes in fluid height within critical areas such as the neck and funnel regions. This data allows the system to calculate fluid volume with high precision, accurately estimate weight, and adjust operating parameters, such as rotational speed and relative centrifugal force (RCF), for optimal balance and performance. Measurement accuracy improves as fluid volume decreases, achieving ±4.5 g accuracy at a maximum fluid level of 350 mL, and improving to less than ±1 g at 100 mL. Even at the highest fluid levels, measurement accuracy remains well within the centrifuge's operating tolerances, which can accommodate imbalances of up to 50 g without compromising safety or performance.

[0228] Figure 60C A rod 1134 for radar 1130—the radar transmitter / sensor (not shown)—is shown located at the upper right end of rod 1128. The distance in the curved section up to the straight portion of the rod can be removed electronically. The straight length of rod 1134 enters through cover 402 and occupies exactly the vertical distance. Reflection brings back 99% of the energy. Rod 1134 extends downward into the liquid in the main chamber 424; the liquid does not move upward along the rod, yet radar 1130 can detect the liquid level. This is one means of detecting liquid level. Lasers, ultrasonic detectors, and strain gauges are other alternatives.

[0229] In addition to load cells and / or radar / laser systems, time-of-flight (ToF) monitoring systems can be used to detect fluid levels within a centrifuge (CPC). ToF systems use light pulses (e.g., infrared or laser) or ultrasound to measure the time required for a signal to travel to the fluid surface and return to the detector. Based on this time, the system calculates the distance to the fluid level. This enables non-contact, high-precision, real-time fluid level detection, ensuring accurate volumetric measurements and stable centrifuge performance.

[0230] In a preferred embodiment, the radar or other detection system engages only when the fluid level is changing or about to change. Alternatively, in some embodiments, the radar system is activated only when the CPC is in its in-situ position—i.e., vertical and stationary—thus ensuring consistent and interference-free measurements.

[0231] In use, ACP automates the entire CAR T-cell manufacturing process, starting with leukocyte apheresis and sequentially performing cell selection, activation, transduction, and final product purification. Key steps include washing, centrifugation, mixing, gene transduction, and multiple wash cycles to ensure a pure, viable, and effective cell product. While there may be additional or fewer steps, the following is an example of the platform process.

[0232] 1. Single-sample washing—Here, the initial single-sample leukocyte specimen is prepared by washing away unwanted materials while maximizing leukocyte purity and viability. The input is a single-sample leukocyte specimen containing red blood cells, white blood cells, platelets, and any other particulate matter. This single-sample leukocyte specimen is mixed with a buffer solution. The system removes unwanted components, such as excess antibodies, viral particles, and non-target debris, directing them to a waste container. Flow rate, temperature, and wash solution are strictly controlled to maintain leukocyte viability and ensure a high-purity sample.

[0233] 2. Selection and Activation – The goal of this step is to isolate target cells (e.g., hematopoietic stem cells, T cells, NK cells) using biotinylated aptamers or antibody-based adapters and prepare them for activation and genetic modification. This system can use specific markers, such as CD3+, CD4, CD8, and CD28, to identify and select target cells. Alternative implementations may use CD4 and CD8 markers alone, avoiding over-reliance on CD3. Biotinylated and non-biotinylated molecules play distinctly different roles in the selection and activation systems of cell processing machinery. Biotinylation is typically used to functionalize molecules such as aptamers or antibodies with biotinylated groups that can bind firmly to streptavidin-coated surfaces or streptavidin-linked microvesicles within the machinery. This approach allows biotinylated molecules to precisely attach to microvesicles, enhancing the selection and activation of target cells by allowing these functionalized microvesicles to bind to specific cell surface antigens via biotin-streptavidin interactions. Conversely, non-biotinylated molecules are used when reversible binding is required, or when applications demand aptamers and antibodies that do not form permanent bonds with the target. This combination allows the machine to balance stable binding with the flexibility to release or further process cells as needed during the selection and activation steps, thereby optimizing specificity and operational flexibility in cell handling.

[0234] 3. Connection mechanism: Biotinylated adapters are introduced to bind to target cell markers. Then, streptavidin-coated microvesicles are added and attached to the adapters to form a cell-microvesicle complex. This complex reduces the density of target cells, causing them to become buoyant in the cell suspension.

[0235] 4. Centrifugation: During centrifugation, the buoyant target cell-microbubble complex migrates away from the axis of rotation (upwards in the CPC), while non-target cells (due to their higher density than the fluid in the cell suspension) settle towards the bottom of the CPC's conical compartment. The conical or cylindrical shape of the CPC's central chamber increases in diameter closer to the axis of rotation, ensuring that the larger, buoyant target cell complex avoids contact with the descending non-target cells. Additional microbubbles can be added if needed to improve cell buoyancy and separation efficiency. Target cells with attached microbubbles are concentrated at the top of the CPC, while non-target cells are isolated in the sealable compartment at the bottom.

[0236] 5. Centrifugation and Transduction – In this embodiment, a viral vector is used to introduce the CAR gene into target cells with the aid of controlled centrifugation to optimize gene transfer efficiency. Specifically, the viral vector is introduced into CPCs to target selected and activated cells. The system employs centrifugation, where controlled centrifugation brings the viral particles into close proximity to the target cells, thereby improving transduction efficiency. This process may involve multiple steps, such as (1) centrifuging the target cells to improve vector penetration into the cell nucleus; (2) pivoting or rocking the CPCs back and forth to gently mix and redistribute the cells and vector, ensuring adequate interaction; and (3) recentrifugation and repeating as needed to improve gene transfer. The process is carefully timed and monitored to optimize transduction while minimizing cellular stress. The result is the production of genetically modified CAR T cells with high transduction efficiency.

[0237] 6. Washing and Harvesting – The purpose of this step is to purify and formulate the final cell suspension volume for genetically modified CAR T cells to meet therapeutic standards of purity, viability, and potency. The system performs multiple wash cycles to remove excess viral vectors, reagents, and particulate matter. Each wash significantly reduces contaminants. For example, 150 mL of wash fluid is introduced, thoroughly mixed, and positive pressure is applied to transfer contaminants to waste. In this case, a 90% contaminant reduction is achieved with each wash. Repeating this process three times achieves a 1000-fold reduction. After washing, the cells are concentrated to a convenient volume (e.g., 15 mL) and then adjusted to the required formulation volume for harvesting with buffer. Final quality control assessment confirms the purity, viability, and readiness of the CAR T cell product. The result is a purified, concentrated, and ready-to-use CAR T cell product suitable for therapeutic use.

[0238] MBCSA (Microvesicle Cell Selection and Activation) technology enables sequential cell selection, overcoming a key limitation in current CAR T cell production. Traditional methods, such as magnetic bead-based selection, have limitations because once T cells are selected with magnetic beads, they cannot undergo further magnetic-based separation. In contrast, MBCSA allows for the degassing of the initial selection reagent, enabling the sequential selection of different cell subpopulations using secondary reagents. This capability facilitates a more precise and customizable approach to cell composition, thereby enhancing control over T cell subpopulations in the final therapeutic product.

[0239] Using controlled pressure-based MBCSA—ACP employs innovative MBCSA technology, which utilizes microvesicles to selectively bind to and detach target cells (such as T cells or stem cells) based on surface markers. In this context, microvesicles are small, aerated bubbles with lipid, polymer, or protein shells, typically functionalized with specific molecules (such as aptamers or antibodies) to bind to target cells. This process is facilitated by surface-modified, phospholipid-shelled microvesicles that are stable, lyophilized, and easily reconfigurable for use. By utilizing pressure to selectively rupture microvesicles, ACP improves the efficiency and effectiveness of cell selection, providing high-purity, high-viability cell products that meet the stringent standards required for therapeutic applications.

[0240] In the context of this application, lyophilized microvesicles refer to microvesicles that have been freeze-dried to maintain their structure and functionality for long-term storage and subsequent use. Compared to their hydrated counterparts, lyophilized microvesicles are easier to store, transport, and integrate into automated systems, which may require specific storage conditions. In their lyophilized form, microvesicles are dehydrated under low temperature and vacuum conditions, removing moisture while maintaining their structural integrity and biological functional properties. Lyophilization stabilizes the microvesicles, making them suitable for extended storage without loss of efficacy. Before use, lyophilized microvesicles can be rehydrated (e.g., with saline solution) to restore their functional form for binding to target cells within a cell processing platform. Functional groups on the microvesicles, such as biotinylated aptamers or antibodies, are retained during the lyophilization process, ensuring specificity and binding efficiency. They then selectively and predictably bind to cell surface markers and facilitate separation, selection, or modification processes, such as centrifugal seeding or controlled cell processing cycles.

[0241] Microbubble Preparation and Targeting—Microbubbles are coated with streptavidin, enabling them to connect to biotinylated aptamers or antibodies that specifically bind to desired cell surface markers. This binding process causes target cells to become buoyant, allowing for easy separation based on their relative buoyancy within the CPC212. As they rise, they encounter the widened cross-section of the funnel, giving them more space to disperse. The balance of forces—compression of cells against the funnel wall and buoyancy separation as cells rise—allows cells to be tightly packed and then gently separated, optimizing selection and distribution within the funnel. This design leverages mechanical and physical properties to enhance the precision of cell handling and separation. This separation step reduces the density of cell clumps and allows for a more uniform distribution of cells within the fluid. To prepare the MBCSA reagent, lyophilized microbubbles are reconstituted with 6 cc of sterile saline and agitated for approximately 8 seconds to obtain a homogeneous suspension. Once reconstituted, the microbubbles remain stable in the fluid environment of the CPC212, enabling efficient cell targeting and separation.

[0242] Pressure-induced microbubble rupture—a unique feature of the MBCSA process—is the ability to disrupt the buoyancy of microbubbles and release bound cells by applying controlled air pressure. After target cells have been separated and bound to microbubbles, the internal pressure of the CPC is increased to an appropriate level, approximately 2.5 atmospheres in one example. This increase in pressure causes the phospholipid shells of the microbubbles to rupture, effectively “bursting” them and releasing the bound cells without causing cell damage. The rapid rupture of microbubbles allows for quick transitions between cell selection and subsequent processing steps, streamlining the overall cell handling workflow. The lipid shells of the microbubbles are later washed away. Controlled pressure rupture is a key aspect of MBCSA technology because it provides a gentle, non-destructive means of cell release, allowing them to proceed to subsequent processing stages, such as washing, activation, or formulation, while these cells remain viable and functional for therapeutic applications.

[0243] fit This system further integrates aptamers as high-affinity binders within the MBCSA system, targeting antigens such as CD3, CD8, CD28, and CD34. Compared to traditional antibody-based methods, aptamers offer a cost-effective and rapidly customizable alternative, while exhibiting superior stability and reduced degradation under optimized conditions. For example, aptamers can be selectively removed by introducing DNase to degrade DNA and release attached microvesicles—a flexibility unattainable with antibodies. Furthermore, antibodies can cause unintended physiological effects on cells through internalization, a concern that does not arise with aptamer-based methods. From an intellectual property perspective, aptamers offer significant advantages because they can be synthesized internally, avoiding reliance on commercially available antibodies.

[0244] Key findings highlight the effectiveness of the system, with aptamer-linked microbubbles achieving higher purity and recovery rates than competing technologies. For example, the platform consistently produces CD8+ cell-positive fractions with 95.1% purity and 89.1% recovery, outperforming conventional antibody- and magnetic bead-based methods that typically produce around 50% recovery.

[0245] ACPs combine aptamers—short, single-stranded oligonucleotides (DNA or RNA)—that fold into specific three-dimensional shapes, enabling them to selectively bind to target molecules with high affinity. Compared to antibodies, aptamers are more versatile and cost-effective in production and modification, making them suitable for a wide range of targets, including proteins, cells, and small molecules. Their customizable nature supports tailored applications in biotechnology, diagnostics, and therapeutics.

[0246] This platform ensures aptamer functionality through precise folding schemes and optimized buffer composition, maximizing binding efficiency while minimizing degradation. This design achieves results comparable to or even surpassing traditional antibody-based methods, 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.

[0247] Aptamers are used in conjunction with a microbubble system comprising a lipid shell and a gas core microbubble, which imparts buoyancy to target cells such as hematopoietic stem cells (HSCs), T cells, and NK cells. During low-speed centrifugation, target cells bound to the microbubbles float, effectively separating from non-target cells. This process is highly specific, with the aptamer binding to a unique surface antigen on the target cell. The use of aptamer-linked microbubbles enhances the precision of cell selection, enabling the automated separation of target and non-target cells during centrifugation. By binding to specific cell surface markers, aptamers facilitate the separation and concentration of desired cell populations, such as T cells, within the CPC. Furthermore, aptamers are designed to synergize with microbubbles and other separation aids, further improving the efficiency and specificity of the cell selection process.

[0248] The aptamers integrated into the platform are structurally optimized to mimic antibody binding potency, utilizing modifications such as dimerization and spacer enhancement. These adjustments maintain flexibility while ensuring high specificity for a variety of cellular markers. Furthermore, the aptamers exhibit reversible binding properties through tailored adapters, allowing for the release of bound cells post-selection. This reversibility enables further purification steps or reuse of treated cells, which is particularly advantageous for T-cell-based therapies requiring sequential selection and activation of multiple subsets.

[0249] Integrating aptamers into the system offers numerous benefits, including compatibility with automated, high-throughput closed systems that ensure sterility, precision, and reproducibility. The lower immunogenicity of aptamers compared to monoclonal antibodies (mAbs) enhances the platform's safety profile. Working synergistically with microbubbles, aptamers enable CPCs to efficiently isolate and process cells while maintaining adaptability for a wide range of therapeutic applications. This approach ensures the production of high-quality, safe cell therapy products suitable for clinical use.

[0250] The system envisions a configuration with the following adaptability design advantages: The development of bistranded aptamers—reversible aptamer adapters, such as bistranded aptamers—presents a promising avenue for enhancing flexibility in cell processing. These aptamers can be engineered to detach by introducing a complementary strand with a higher binding affinity to the aptamer. This reversible mechanism will allow for (1) the reuse of selected cells for additional processes or treatments, (2) further purification steps to achieve higher specificity and purity in target cell populations, and (3) expanding the utility of aptamer-based cell processing in a variety of applications, including research, manufacturing, and therapeutic fields.

[0251] Thermally pretreated aptamers undergo a controlled heating process to optimize their structural conformation, significantly enhancing their binding efficiency, stability, and application potential in cell processing workflows. By heating aptamers to specific temperatures (typically 70–95°C) and rapidly cooling them, they fold into an energy-stable and functional conformation. This prevents misfolding and aggregation, resulting in a homogeneous population of active aptamers. Thermally pretreated aptamers exhibit stronger and more specific binding to target antigens, ensuring high-purity cell selection and reducing non-specific interactions. Integrating thermally pretreated aptamers into the ACP workflow significantly enhances the precision and reliability of cell selection processes. Their adaptability and enhanced performance make them an ideal solution for applications requiring high specificity and efficiency.

[0252] Reversible aptamer design—aptamers offer the potential for reversibility, further expanding their versatility. For example, double-stranded aptamer hybrids can act as reversible linkers. As another example, single-stranded aptamers with an additional "tail" for connecting complementary strands can be used to facilitate detachment. By introducing disruptive strands with higher affinity for target antigens, aptamers and attached microvesicles can be efficiently removed from the cell surface.

[0253] SELEX and Aptamer Development: The SELEX (Spiritually Enriched Ligand Systematic Evolution) process provides access to a large library of aptamer candidates. Once funding is secured, the SELEX procedure will be performed to identify aptamers specific for the desired antigen. This process will generate multiple aptamer candidates, enabling the selection of the best-performing sequences for cell treatment. Heat-pretreated aptamers can also be selected and optimized during the SELEX process to ensure their stability and functionality under various physiological and treatment conditions.

[0254] The optimal condition range is as follows:

[0255] Secondary choice: In one implementation, an initial cell selection process is performed to isolate T cells, followed by subsequent selection steps to refine subsets, such as CD8+ or CD4+ T cells or memory T cells. This sequential selection can be performed using aptamers, where a first aptamer is digested and replaced with a second aptamer specific to the desired subset. Alternatively, a primary selection reagent, such as microvesicles functionalized with antibodies or aptamers targeting T cells, is used to isolate a broader target population. A secondary selection reagent, comprising microvesicles coated with antibodies or aptamers for selectively binding to CD8+ or CD4+ T cell subsets, is then employed for further purification and selection. Between selection steps, a degassed solution is used to remove the buoyancy of the microvesicles, ensuring effective removal and preventing contamination of the primary cell population. This method facilitates precise, sequential separation of target cell subsets for downstream applications.

[0256] Uses for leukapheresis The system's functionally closed design and advanced capabilities make it ideal for direct use in apheresis procedures, particularly by significantly reducing target cell loss during manufacturing. Its precision enables efficient production of genetically modified CAR T cells, even with smaller white blood cell counts typically found in whole blood samples rather than apheresis collections. Apheresis involves the selective separation and collection of white blood cells—granulocytes, lymphocytes, monocytes, and stem cells—from whole blood. The system's combination of closed-system sterility, controlled centrifugation, selective cell targeting, and automated quality control makes it highly suitable for apheresis applications. Its adaptability to handling smaller white blood cell counts and its ability to minimize target cell loss ensure efficient, high-yield recovery of white blood cells, supporting downstream processes such as CAR T cell manufacturing and immunotherapy development, as detailed below: Controlled centrifugation and sedimentation: This system optimizes the degree of target cell accumulation during centrifugation inoculation by dynamically adjusting the centrifugation speed. Cell detection sensors located in the sedimentation column monitor the target cells as they settle, ensuring precise control to improve accumulation and carrier delivery efficiency.

[0257] Aseptic Transfer and Contamination Prevention: The closed system design eliminates the risk of contamination by providing a sterile fluid transfer pathway. Pressure decay testing is performed before cell insertion to ensure the integrity of both the hydrophobic and hydrophilic filters on the CPC cap. These features maintain cell viability and meet the stringent aseptic standards required for leukapheresis and clinical applications.

[0258] Selective cell separation and enrichment: The system's aptamer and microvesicle technologies enable targeted selection and separation of leukocytes. Aptamers or antibodies designed to recognize specific leukocyte markers (e.g., CD45 or CD34) can be used to enrich leukocyte subsets. This targeted enrichment is particularly valuable in immunotherapy applications.

[0259] Cell sedimentation monitoring: An optical detection system utilizing three optical sensors provides real-time monitoring of target cell sedimentation. This ensures: optimal centrifugal concentration of target cells; precise cell stacking for enhanced vector exposure during gene modification; and improved overall transduction efficiency.

[0260] Uses for senescent cells The described device can be adapted to target, isolate, and remove senescent cells that accumulate with age and contribute to the progression of various age-related diseases. By specifically targeting these dysfunctional cells, the platform provides a novel approach to mitigate the adverse effects of cellular senescence on aging tissues.

[0261] This device utilizes aptamers specifically designed to bind to uniquely expressed biomarkers in senescent cells. These biomarkers include overexpressed surface proteins such as p16, p21, or SA-β-gal, which are characteristic of senescent cells. Using SELEX (evolution of exponentially enriched ligand systems), aptamers can be customized to selectively recognize these biomarkers, ensuring precise targeting within heterogeneous cell populations.

[0262] Aptamers conjugated to buoyancy microbubbles can selectively bind to and separate senescent cells. During low-speed centrifugation, microbubbles carrying bound senescent cells rise to the top of the funnel chamber. This buoyancy-based separation effectively separates senescent cells from healthy, non-senescent cells in the sample, enabling efficient enrichment and collection.

[0263] To enhance specificity and purity, reversible aptamer design can be used to allow for multiple rounds of selection. After initial separation, the aptamer can be disrupted using a complementary strand or a higher affinity reagent, releasing the bound senescent cells. This prepares the system for subsequent rounds of processing, refining the separation of specific senescent cell subtypes to achieve optimal purity and recovery.

[0264] This device helps to concentrate senescent cells within the funnel chamber, creating a high-density collection at the bottom. Once separated, these senescent cells can be extracted or subjected to depletion strategies. Their selective removal has the potential to revitalize surrounding tissues by mitigating age-related inflammation and harmful effects.

[0265] Isolated senescent cells can serve as a valuable resource for therapeutic research, enabling the development of cellular rejuvenation strategies. Alternatively, after removing senescent cells from patient samples, the system can reintroduce purified healthy cells into the patient. This approach will provide a tailored cellular composition that promotes tissue repair and extends the healthy lifespan of patients, offering a transformative solution to combat age-related diseases.

[0266] ACP integrates a comprehensive pre-operational diagnostic protocol to ensure the integrity and functionality of the CPC and related components before the introduction of any biomaterials. When the clinician inserts the cartridge into the machine, the system automatically performs a series of integrity and performance tests. If any fault is detected in the cartridge or its components, the machine will prevent cell fluid from being introduced, prompting the clinician to remove and discard the defective cartridge. This ensures that only fully functional cartridges are used in the cell processing workflow. The initial testing sequence that occurs before cells are loaded into the cartridge includes, but is not limited to: Filter integrity test: Perform a pressure decay test to confirm the structural integrity and functionality of all filters in the CPC.

[0267] UV Module Validation: Test the UV disinfection module to ensure it radiates at the correct intensity for effective disinfection.

[0268] Centrifuge diagnostics: Assess the operational readiness of the centrifuge system, including balance and motion parameters.

[0269] Hybrid actuator testing: Testing the functionality and precise motion control of a swing or hybrid mechanism.

[0270] Heater calibration: Verify the heating system to ensure it can maintain the specified temperature profile during cell treatment.

[0271] Once the diagnostic test is successfully completed, a green light signals to the operator that the system is ready for use. At this point, the operator can lift the lid, introduce the cells into the box, secure the tube, and insert the short segment into place for processing.

[0272] Following the initial run, the machine undergoes secondary validation of critical aseptic-related components, with a particular focus on retesting the filters and UV module. This post-operation testing ensures the integrity of the CPC and the aseptic nature of the process throughout the entire cell processing workflow. Other subsystems, such as the centrifuge, heater, and mixing mechanism, are not retested after the run.

[0273] Recovery rate and purity The disclosed system achieves superior recovery and purity in target cell selection, with a particular focus on CD8+ T cell enrichment and isolation. Efficiency is calculated as the fraction of live, genetically modified cells retained from the initial cell population. For example, conventional manufacturing typically starts with 2 billion T cells, with losses up to 92.1% occurring during processing over days or weeks. This leaves approximately 150 million activated T cells, which are then expanded to 1 billion within 8 days. However, another 50% of these cells may be lost during reinfusion into the patient. ACP minimizes these losses, shortens the timeline, and optimizes cell recovery for superior therapeutic outcomes.

[0274] The platform disclosed in this paper demonstrates near 90% recovery and 95% purity for CD8+ T cell selection through optimized aptamer functionality. This optimization involves controlled folding and binding conditions, including the use of buffer compositions such as 40 mM HEPES and 100 mM NaCl to effectively eliminate impurities and stabilize aptamer structures. The temperature protocol was also fine-tuned, incorporating a denaturation step at 95°C followed by structured cooling to promote aptamer refolding into the desired conformation. Importantly, non-functional or misfolded aptamers are presented as inactive without disrupting the process.

[0275] The platform also demonstrated excellent enrichment and depletion performance across various cell types. During enrichment, monocytes (MNCs) were recovered at 95%, while CD34+ hematopoietic stem cells (HSCs) were recovered at an impressive 99%. Non-target cells, including erythrocytes (RBCs), platelets (PLTs), and neutrophils (NEUs), were efficiently depleted to levels of 99%, 84%, and 67%, respectively, ensuring a highly purified final cell population.

[0276] For T cell isolation, this system produces a population of CD3+ T cells with a purity of at least 95% and a recovery rate exceeding 95%. The resulting cell product contains 90-95% activated T cells to ensure immediate therapeutic efficacy and includes a significant subset of memory T cells (20-30%) to enhance long-term in vivo persistence. When HSCs are included in the workflow, they are enriched to similar high levels of purity and recovery, supporting their use in applications such as stem cell transplantation.

[0277] This platform outperforms conventional platforms in both T-cell purity (99%) and recovery rate (90-95%), while alternative systems typically achieve only about 50% recovery and about 90% purity. Achieving superior purity is crucial, as contamination of patient samples with non-T cells or cancer cells can impair therapeutic efficacy. Notably, the entire process is completed within a single device at a single location, minimizing loss and contamination. This platform ensures less than 1% target cell loss during processing, with a further threshold of less than 2% or less than 5% depending on operating parameters, demonstrating unprecedented precision and efficiency in cell therapy manufacturing.

[0278] Comparative efficiency in key process stages Leukocyte collection and washing: ACP achieves 97.2% efficiency in leukocyte collection and 95.3% efficiency in the washing stage, compared to industry standards of 50% and 49.9%, respectively. This significant improvement stems from the platform's closed-loop automation and optimized fluid handling, minimizing cell loss and contamination risks.

[0279] Cell selection and activation: The microbubble cell selection and activation (MBCSA) process achieves an efficiency of 74.1% in cell selection and activation, compared to a standard efficiency of 39.4% for conventional magnetic bead-based or flow cytometry systems. The sequential selection capability of MBCSA ensures higher cell purity and viability, contributing to refined therapeutic products.

[0280] Transduction: During the transduction phase, ACP achieved an efficiency of 94.7%, significantly exceeding the industry standard of 80%. The controlled mixing and uniform cell distribution provided by the geometry of CPC allowed for effective cell exposure to the carrier, improving transduction efficiency while maintaining cell viability. Mixing was designed for uniform cell distribution, resulting in better cell washing and preventing shaking, bubbling, and air bubbles.

[0281] Harvesting, washing, and formulation: The final stages of the process, including harvesting, washing, and formulation, demonstrated significant efficiency improvements with ACP, achieving 65.0% efficiency, compared to the industry standard of 7.9%. This improvement is attributed to precise fluid handling, reduced cell loss, and minimized processing steps, which collectively enhance the integrity of the final cell product.

[0282] Cumulative Process Efficiency: ACP achieves a cumulative process efficiency of 65.0% across all stages of cell processing, compared to the current industry standard of 7.9%. This 823% efficiency improvement eliminates the need for extended ex vivo cell expansion, a costly and time-consuming step traditionally required to reach clinical doses in CAR T cell production. By maintaining high cell viability and functionality throughout the processing, ACP enables simplified manufacturing, reducing cost and time requirements.

[0283] The ACP described in this article offers several key functional advantages that enhance its practicality in automated cell processing applications. These include: Targeted cell selection: The use of aptamers enables selective binding and enrichment of target cells, significantly reducing contamination from non-target cells and improving the purity of downstream applications. This precision ensures high-quality therapeutic output.

[0284] Microbubble-assisted separation: When combined with microbubbles, aptamers provide a buoyancy-based cell separation mechanism. This method effectively separates target cells from non-target cells with minimal loss by using differential flotation during centrifugation.

[0285] Reversible binding and multifunctional control: Aptamers can be engineered with reversible adapters or hybridization structures, allowing for the controlled release of bound cells after separation. This reversibility supports additional purification steps or cell reuse, adding flexibility to the manufacturing process.

[0286] Compatibility with automated processing: The stability and customizability of aptamers make them highly compatible with automated, high-throughput cell processing platforms. This compatibility is essential for closed-system devices where sterility, precision, and reproducibility are critical.

[0287] Batch release / document record This platform can generate detailed batch release records for regulatory or other purposes. This capability supports personalized treatment, providing extensive data on each batch, similar to the extensive documentation requirements in large-scale pharmaceutical manufacturing. ACP continuously tracks and records various key parameters throughout the cell processing cycle, ensuring that each step adheres to defined protocols and maintains optimal cell viability and product quality.

[0288] The tracked QC data may include, but is not limited to: Identification: Protocol, patient, and cell origin identifiers ensure each batch meets the correct treatment requirements. Instrument and CPC identifiers verify the equipment and consumables used in each production cycle. Recording operator details maintains a complete accountability record, allowing for traceability and assessment of human involvement when necessary.

[0289] Cell culture parameters: Continuously monitor temperature, fluid composition (e.g., dissolved oxygen ([O2]) concentration), culture volume, and cell density to maintain ideal cell culture conditions. Record medium replacement time and volume to verify compliance with specified growth conditions and ensure cell viability and batch-to-batch consistency.

[0290] Physical process parameters: Track centrifugation time and applied force to confirm that cells are adequately separated or concentrated according to protocol specifications. Record mixing speed, angle, frequency, and duration to ensure uniform reagent distribution, effective cell washing, and optimal cell suspension. Monitor aspiration time and volume to maintain sterility and ensure precise fluid handling. QA sampling volume and time for consistency and to support reliable analytical testing during manufacturing. Perform pre- and post-operation filter integrity tests to verify that all filters maintain sterility, prevent contamination, and ensure product safety.

[0291] Reagent Information: Record reagent identification, receipt and expiration dates, addition time and volume to confirm the correct material was used within its stability window. Track the storage temperature of all reagents to prevent degradation and ensure that only viable materials are used in cell treatment.

[0292] Automated Error Reporting and Exception Handling: ACP incorporates an automated error reporting system that continuously monitors data for any deviations from preset limits. If any parameter deviates from the established threshold, an error condition is immediately flagged, and the system records detailed information about the deviation. This automated detection allows for real-time error identification and corrective actions when necessary.

[0293] Exception Release: The “Exception Release” model implemented within ACP is a transformative approach to batch release in cell therapy manufacturing. Traditionally, all batch records must be thoroughly reviewed before product release, a highly labor-intensive process prone to human error. In contrast, the Exception Release model requires only QC review of batches where deviations or anomalies are detected, allowing error-free batches to be automatically approved for release based on the system’s verified compliance with all critical parameters.

[0294] The fully automated nature of ACP reduces human intervention, significantly lowering the risk of contamination and operational variability. Compared to traditional methods, ACP accelerates the manufacturing process, enabling the production of clinical-dose CAR T cells in just 2.5 days without time-consuming in vitro expansion. This simplified approach ensures precise control over every stage of the process, optimizing flow rates, centrifugation forces, and timing to improve product reliability, purity, and cell viability. Multiple wash cycles integrated into the workflow further reduce contaminants, ensuring high-quality therapeutic products.

[0295] ACP's compact and automated design enhances accessibility, enabling direct deployment within FDA-approved transplant centers. This on-site collaborative capability supports research and clinical applications, allowing scientists to focus on gene-building development rather than labor-intensive manufacturing processes. Furthermore, ACP's robust quality control and aseptic processing steps ensure consistent and reproducible manufacturing results. By simplifying and accelerating cell therapy production, ACP has the potential to make life-saving treatments more accessible and affordable to a wider patient population.

[0296] In summary, the CPC is designed with a conical internal geometry, transitioning from a wide width in the top section to a narrow width in the bottom section. This geometry creates a gradient that concentrates and isolates the precipitate in the bottom section during centrifugation or sedimentation. The narrow bottom section is specifically configured to enhance the accuracy of target cell count measurements; changes in the light sensor reading correspond to the total target cell count in the suspension. The wide top section is at least four times the width of the narrow bottom section and includes multiple fluid transfer ports and gas exchange ports. These ports facilitate the entry and removal of sterile fluids, controlled gas exchange, and minimize foam formation during operation. Interface caps enable the sterile transfer of fluids, cells, reagents, or gases into and out of the CPC. The conical gradient further enhances buoyancy-based separation of target cells during processing.

[0297] The CPC allows for a tilting range of 1 to 360 degrees in any direction from a vertical position, facilitating the redistribution of deposits within the chamber. A narrow bottom section features an optically clear window aligned with the target cell detection sensor for precise monitoring. The conical inner wall is constructed of a low-adhesion material with a smooth, trimmed surface, preventing biofilm formation and ensuring efficient recovery of cells, including T cells, NK cells, or hematopoietic stem cells. The interface cap integrates multiple fluid and gas transfer ports. Internally, the port openings are aligned linearly along the inner surface of the interface cap to prevent fluid contact with the ports during tilting. Externally, these ports are offset from the internal alignment, providing pathways to maintain aseptic conditions during transfer operations.

[0298] The geometry of the CPC ensures precise sedimentation and optical monitoring. The diameter of the wide section of the chamber is at least 4.7 times that of the narrow section, and the conical walls facilitate the sedimentation process. A fluid waste disposal tube is positioned near the geometric center of the port cap and extends vertically when the CPC is upright. This tube effectively removes waste liquid while maintaining residual volume for cell integrity. A filter, including at least one hydrophilic and one hydrophobic filter, is integrated into the port to facilitate sterile gas and fluid exchange.

[0299] The CPC's internal chambers are designed to concentrate and isolate cells during centrifugation. The chambers narrow towards a bottom section with flat, parallel walls, improving optical monitoring and enabling selective fluid removal while preserving cells. Motion actuators induce controlled oscillatory motion, allowing the CPC to oscillate between 1 and 360 degrees. The upper section of the CPC features a wide surface area and multiple docking ports, ensuring proper alignment for sterile gas and fluid transfer. The bottom sedimentation column has a diameter no greater than one-quarter the diameter of the upper section, providing a controlled path for optical cell monitoring and fluid removal.

[0300] During cell processing, the CPC's ports enable gas exchange and fluid transfer without compromising sterility. The internal chamber geometry, low-adhesion surfaces, and optimized flow paths ensure efficient sedimentation, mixing, and fluid handling. Utilizing advanced optical sensors and algorithms, the CPC provides real-time monitoring of cell sedimentation rates and densities. These features, along with its closed-loop design, make the CPC ideal for high-precision, sterile cell processing applications.

Claims

1. A cell processing kit (CPC) comprising: a. A conical internal geometry, wherein the CPC tapers from a wide width in the top section to a narrow width in the bottom section, thereby forming a gradient that concentrates and isolates the precipitate in the bottom section during centrifugation or sedimentation; b. A narrow bottom section configured to enhance the accuracy of target cell count measurements, wherein the change measured by the light sensor within the bottom section corresponds to the total target cell count in the total volume of the cell suspension; c. A wide top section having a width at least four times that of the narrow width, including one or more fluid transfer ports and gas exchange ports, thereby allowing sterile fluid entry and removal and controlled gas exchange, which minimizes foam formation and supports aseptic operation within the CPC; d. An interface cap configured to facilitate the aseptic transfer of fluids, cells, reagents, or gases to and from the CPC; and e. The gradient is configured to enhance buoyancy-based target cell separation during centrifugation or sedimentation.

2. The CPC of claim 1, wherein the cartridge has a tiltable range of 1 to 360 degrees in any direction from a vertical position, and wherein the tilting promotes the redistribution of deposits within the CPC.

3. The CPC of claim 1, wherein the precipitate is a target cell, and wherein the narrow bottom section further comprises at least one optically transparent window aligned with a target cell detection sensor.

4. The CPC of claim 1, wherein the walls of the conical interior are made of a low-adhesion material with a smooth, trimmed surface, configured to prevent biofilm formation and ensure the recovery of cells including T cells, NK cells, or hematopoietic stem cells.

5. The CPC according to claim 1, further comprising: a. One or more fluid and / or gas transfer ports arranged along the interface cover of the CPC, each port comprising: i. An internal port opening aligned with a straight line bisecting the center of the internal surface of the interface cover, thereby allowing the fluid level within the CPC to rise to its maximum angle during tilting without contacting any of the port openings; and ii. An external access point for each port, wherein the external access point is offset from the inline configuration of the internal port opening, thereby enabling independent access to an external syringe or fluid transfer mechanism; and b. Connect each external access point to a fluid or gas passage through its corresponding inline internal port opening, which is configured to allow fluid or gas transfer through the port while maintaining the inline arrangement of the internal port openings within the CPC.

6. The CPC of claim 1, wherein the wide width is at least 4.7 times the narrow width.

7. The CPC according to claim 1, wherein the conical wall is a conical wall.

8. The CPC of claim 1, further comprising a fluid waste disposal tube arranged substantially close to the geometric center of the interface cover.

9. The CPC of claim 8, wherein the treatment tube is vertical when the CPC is vertical.

10. The CPC of claim 8, wherein the fluid waste disposal tube extends vertically downward from the interface cover to a depth less than the depth of the internal geometry of the CPC.

11. The CPC of claim 1, wherein at least two of the ports are filters.

12. The CPC of claim 11, wherein at least one of the filters is hydrophilic and at least one of the filters is hydrophobic.

13. A CPC for use in a cell processing platform, comprising: a. An internal chamber having a central axis and configured to concentrate and isolate cells by centrifugation, wherein the chamber narrows toward a bottom section that transitions from a conical wall to a flat opposing wall, thereby providing an improved optical path to monitor cell movement and allowing fluid to be washed away through a waste disposal tube during a washing cycle while retaining cells; b. A motion actuator configured to induce controlled oscillating motion of the CPC and facilitated by the geometry of the internal chamber, wherein the CPC is configured to oscillate reciprocally between 1 and 360 degrees in any direction; c. The upper section, which is arranged at the top of the funnel, is configured for gas exchange, fluid intake and filtration, wherein the surface area of ​​the upper section provides a 2D docking area with multiple ports, thereby allowing alignment with external docking mechanisms and ensuring proper engagement for gas and fluid transfer. and d. A sedimentation column defined by a wall section at the bottom of a funnel-shaped internal chamber that is substantially parallel to the axis of the funnel-shaped internal chamber, the wall section having a diameter not greater than 1 / 4 the diameter of the upper section, and configured to provide a predictable path for optical monitoring of cell sedimentation, thereby enabling the selective removal of non-cell-containing fluids while maintaining the cell count and purity within the chamber.

14. The disposable CPC of claim 13, wherein the upper section has a diameter of approximately 4.7 times the diameter of the settling column.

15. A method for controlling fluid movement within a CPC during tilting, comprising the steps of: a. Fluid and gas transfer ports are arranged in a straight line along the top inner surface of the CPC, wherein each inner port opening is configured to prevent fluid from entering during CPC tilting; b. Tilt the CPC at least 180 degrees from its vertical position in any direction and along a line perpendicular to the port, such that the fluid level inside the CPC rises along the inner wall without reaching any of the vertical internal port openings; c. Each internal port opening is connected to an external access point via a fluid or gas passage, wherein the external access point is offset from the inline configuration of the internal port opening to allow access to the fluid or gas transfer mechanism; as well as d. Maintaining fluid containment and sterility during the tilting process, wherein the inline configuration of the internal port openings ensures that the maximum permissible fluid level does not contact any port opening, thereby preventing contamination and fluid loss.

16. The method of claim 15, wherein the degree of tilt is adjusted based on the fluid volume, such that a higher volume results in a reduced tilt angle to maintain stability and sterile conditions.

17. A method for processing cells within a CPC, the method comprising: a. Introducing patient cells from a blood or leukocyte collection container into the CPC, the CPC comprising a funnel-shaped internal chamber that narrows toward a narrow bottom section, the narrow bottom section being cylindrical in shape and having substantially vertical and parallel walls; b. Cells are concentrated and settled into the narrow bottom section of the CPC via centrifugation, wherein the funnel-shaped internal chamber promotes cell concentration by guiding cells downward into the narrow bottom section; and c. The cells and fluids within the chamber are mixed by rotating the CPC, wherein the CPC is rotated 1 to 180 degrees in any direction to achieve effective mixing, thereby utilizing gravity and the geometry of the chamber to control the distribution of cells and fluids by a motion-inducing mechanism configured to apply reciprocating motion to the CPC.

18. The method of claim 17 for processing cells within a CPC, further comprising facilitating gas exchange and fluid transfer via a top section having a wide surface area, wherein the top section of the funnel includes a docking region having multiple ports, thereby enabling alignment with an external docking mechanism during the processing cycle for controlled transfer of sterile gases and fluids into and out of the CPC.

19. The method of processing cells within a CPC according to claim 18, wherein the narrow bottom section comprises a wall substantially parallel to the axis of the internal chamber.

20. The method for processing cells within a CPC according to claim 19, wherein: a. The substantially parallel wall segments provide an optical path for monitoring cell movement and enabling the selective removal of non-cell-containing fluids during washing cycles; and b. The optical path is integrated with automated sensors for real-time monitoring of cell density and sedimentation rate.

21. The method of claim 17 for processing cells within a CPC, further comprising adjusting the centrifugal force applied during sedimentation based on real-time feedback to ensure optimal cell packing density in the narrow bottom section.

22. The method of claim 17 for treating cells within a CPC, further comprising sequentially introducing reagents into the CPC during a periodic time in which the CPC is in a locked position.

23. The method for processing cells within a CPC according to claim 17, wherein the motion-inducing mechanism applies a sinusoidal motion that slows the rotation at each end.

24. A CPC comprising: a. A main chamber having a narrowing geometry that tapers from a wider diameter in an upper region to a narrower diameter in a lower region, wherein the narrower diameter includes flat sides; b. A plurality of optical emitter and receiver pairs arranged along the flat side portion of the narrower diameter, configured to measure the opacity of the cell suspension fluid within the chamber; c. The optical transmitter and receiver pair are adapted to: i. Detect changes in light transmission caused by the presence of cells within the fluid; ii. The measured opacity values ​​are compared with a lookup table to estimate the total cell count in the chamber; and iii. Due to the narrowed geometry, cell concentration can be predicted with enhanced sensitivity; d. A mixing system configured to uniformly distribute cells throughout the chamber prior to optical measurements; and e. An algorithm that takes into account the time elapsed since mixing to adjust cell count calculations based on sedimentation dynamics.

25. The CPC of claim 24, wherein the optical transmitter and receiver are arranged in a configuration that allows measurement of cell sedimentation in the lower region of the chamber.

26. The CPC of claim 24, further comprising a closed-loop mixing system that ensures that cells are uniformly distributed within the chamber prior to opacity measurement, thereby improving the accuracy of cell counting calculations.

27. The CPC of claim 24, wherein the algorithm is configured to interpret data from the optical sensor to calculate cell counts based on a known fluid volume and to optimize the measurement by correlating the degree of occlusion at the sensor with the time elapsed since mixing.

28. The CPC of claim 24, wherein the narrowed geometry promotes enhanced optical sensitivity by focusing light transmission through a smaller cross-sectional area, thereby enabling precise detection of cell concentration.

29. The CPC of claim 24, wherein the light transmission has a changing color, and the receiver detects the changing color data, and wherein the algorithm combines the changing color data to calculate the cell density within the fluid.

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