Systems, devices, and methods for cell-based therapies

Abstract

A method includes dissociating tissue into individual cells using at least one of heat, motion, or an enzyme. The method includes directing the individual cells into a mixing chamber. The method includes conveying a first volume of buffer fluid into the mixing chamber from a buffer storage such that the first volume of buffer fluid mixes with the individual cells. The method includes draining the first volume of buffer fluid into a waste via a microfilter, the microfilter configured to capture washed individual cells. The method includes conveying the washed individual cells into an illumination chamber, the illumination chamber containing at least one additive. The method includes illuminating the washed individual cells until a desired level of damage occurs to the individual cells.

Description

Atty. Dkt. PHTN-003 / 01WO 338747-2027SYSTEMS, DEVICES, AND METHODS FOR CELL-BASED THERAPIESCross-reference to Related Applications

[0001] The present application claims priority to and the benefit of U.S. Provisional Application No. 63 / 763,139, filed February 25, 2025, and titled “Systems, Devices, and Methods for Cell-Based Therapies,” and U.S. Provisional Application No. 63 / 789,840, filed April 16, 2025, and titled “Systems, Devices, and Methods for Cell-Based Therapies,” the disclosures of which are hereby incorporated by reference herein in their entirety.Technical Field

[0002] The present application relates generally to processing cells for cell-based therapies, specifically integrated systems, devices, and methods for inactivating or damaging cells for immunotherapy.Background

[0003] Immunotherapy has emerged as an approach in the treatment of various diseases, particularly cancers, by harnessing the body's own immune system to fight cancers. A critical component of this treatment strategy involves the isolation and modification of tumor cells before reinfusion into patients. However, the current methods for immunotherapy cell processing present several challenges that hinder efficiency, scalability, and accessibility.

[0004] Current methods often use specialized environments and equipment for producing such immunotherapies. Such equipment or environments can include tightly controlled, sterile environments to ensure the safety and efficacy of cell processing. These platforms typically use cleanrooms, advanced equipment, and highly trained personnel, which increases both the cost and complexity of the treatment. This reliance on specialized infrastructure limits the availability of immunotherapy to a small number of advanced medical facilities and makes it challenging to scale these treatments for broader use.

[0005] Another challenge arises from the desire to transfer cells between multiple devices during the process. Current cell therapies often involve the use of separate machines for different stages of the process. Each transfer introduces a risk of contamination, cell loss, and inconsistent processing conditions. Moreover, the manual intervention often performed during transfers adds operational complexity and lengthens the treatment timeline. These 305463503Atty. Dkt. PHTN-003 / 01WO 338747-2027inefficiencies hinder the overall workflow, reducing the speed and reliability of immunotherapy production.Summary

[0006] In some embodiments, a method includes: dissociating tissue into individual cells using at least one of heat, motion, or an enzyme; communicating the individual cells into a mixing chamber; conveying a first volume of a buffer fluid into the mixing chamber such that the first volume of the buffer fluid mixes with the individual cells and washes the individual cells; draining the first volume of the buffer fluid from the mixing chamber via a separator, the separator configured to capture the washed individual cells; conveying the washed individual cells into an illumination chamber, the illumination chamber containing at least one additive; and illuminating the washed individual cells with optical energy in the illumination chamber until a desired damage occurs to the washed individual cells.

[0007] In some embodiments, an apparatus includes: a fragmenter configured to fragment tissue in a plurality of tissue segments; a dissociation assembly configured to dissociate the plurality of tissue segments into individual cells using at least one of heat, motion, or an enzyme; a filter configured to selectively allow individual cells to pass through; a mixing chamber including a separator, the mixing chamber configured to: receive the individual cells through the filter, mix a first volume of a buffer with the individual cells to wash the individual cells, separate at least a portion of the washed individual cells from the first volume via the separator, and drain at least a portion of the first volume of the buffer; an illumination chamber containing at least one additive, the illumination chamber configured to receive at least a portion of the washed individual cells from the mixing chamber; and an illumination source configured to illuminate the portion of the washed individual cells in the illumination chamber with optical energy, the optical energy configured to deactivate the individual cells in the presence of the at least one additive.Brief Description of the Drawings

[0008] Reference should be made to the following detailed description which should be read in conjunction with the following figures, wherein like numerals represent like parts.305463503Atty. Dkt. PHTN-003 / 01WO 338747-2027

[0009] FIG. l is a schematic block diagram of a cellular processing system, according to an embodiment.

[0010] FIG. 2 is a schematic diagram of a cellular processing system, according to an embodiment.

[0011] FIGS. 3A-3C depict various views of a cellular processing system, according to an embodiment.

[0012] FIG. 4 is a schematic flow diagram of a method for processing cells, according to an embodiment.

[0013] FIG. 5 A depict a side view of a fragmenter, according to an embodiment.

[0014] FIG. 5B depicts a receiving portion of the fragmenter of FIG. 5 A.

[0015] FIGS. 5C-5D depict various configurations of a cutting portion of the fragmenter of FIG. 5A.

[0016] FIG. 5E depicts a bottom view of the cutting portion of the fragmenter of FIG. 5 A.Detailed Description

[0017] The present disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The examples described herein may be capable of other embodiments and of being practiced or being carried out in various ways. Also, it may be appreciated that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting as such may be understood by one of skill in the art. Throughout the present description, like reference characters may indicate like structure throughout the several views, and such structure need not be separately discussed. Furthermore, any particular feature(s) of a particular exemplary embodiment may be equally applied to any other exemplary embodiment s) of this specification as suitable. In other words, features between the various exemplary embodiments described herein are interchangeable, and not exclusive.

[0018] Some embodiments described herein relate to systems, devices, and methods for processing cells for immunotherapy. Cell processing can include genetic engineering of immune cells (e.g., T cells, B cells, macrophages, dendritic cell, tumor-infiltrating 305463503 ~Atty. Dkt. PHTN-003 / 01WO 338747-2027lymphocytes, etc.). Cell processing can include modifying tumor cells such that they are replication-incompetent (e.g., unable to replicate), for example, are damaged or inactivated. When the processed cells are reinjected into a patient, they may induce an immune response against tumor cells. In some embodiments, the tumor cells are isolated from a benign tumor. In some embodiments, the tumor cells, also referred to as cancer cells, are isolated from a malignant tumor. In some embodiments, to prepare tumor cells for immunotherapy, it may be desirable to dissociate (e.g., break apart, split apart, digest enzymatically etc.) a benign or malignant tumor into single cells. The tumor cells can be modified desirably. In some embodiments, modification to the tumor cells can include inducing damage to the cells’ DNA and / or RNA, thereby making them replication-incompetent, while leaving the cell membranes intact. When the processed tumor cells are injected into a patient, they may activate the patient’s immune system to destroy tumor and metastatic cells. In some embodiments, the systems, devices, and methods described herein may be implemented as a unitary device that increase the efficacy, efficiency, and reduce contamination associated with preparing cells for immunotherapy. In some embodiments, the replication incompetent tumor cells are injected into the same patient that they were originally extracted from.

[0019] Some embodiments described herein use one or more method to dissociate a tissue sample (e.g., tumor, human tumor, animal tumor, etc.) into individual cells. For example, dissociation can be biochemical (e.g., via an enzyme, heat, etc.) and / or physical (e.g., via motion, filter, mincing, grating, etc.). The separated individual cells can then be washed (e.g., via a buffer) by a system to allows for the washing fluid to be used and directed to a waste (e.g., waste chamber, waste collector, etc.) while allowing for capture of the individual cells. After washing, the individual cells can be exposed to an additive (e.g., such as riboflavin) and illuminated (e.g., exposed to energy) such that the cell is damaged to a desired degree. In some embodiments, the systems, devices, and methods described herein can be operated manually, automatically, or via a combination of automatic and manual operation. For example, certain processes can be manual, and certain processes can be automatic. In some embodiments, a valve system including a plurality of valves is used to control how fluids and / or cells are transferred.

[0020] Some embodiments described herein can be used in a clinical setting, a hospital setting, laboratory setting, and / or the like. The systems, devices, and methods described here are capable of being used outside of a clean room or a laboratory setting as the processes 305463503Atty. Dkt. PHTN-003 / 01WO 338747-2027described herein may be contained within a sealed housing that decreases or eliminates the likelihood of contamination. In some embodiments, the systems, devices, and methods described herein can be used for processing tumors or tumor cells removed from a patient with one or more types of cancers. The cancer may be a solid tumor or a liquid tumor. The tumor cells may be isolated or derived from a primary tumor, or a metastatic tumor.

[0021] In some embodiments, the tumor cells are autologous tumor cells. As used herein “autologous” refers to cells that were removed from or derived from the same patient to whom the processed cells are administered. In some embodiments, the tumor cells are allogeneic cells. As used herein, “allogeneic” refers to cells that were removed from or derived from a donor who is not the patient to whom the processed cells are administered. Non-limiting examples of cancers include breast cancer, lung cancer, liver cancer, bladder cancer, gynecological cancer, brain cancer, stomach cancer, prostate cancer, skin cancer, thyroid cancer, pancreatic cancer, colon cancer, or blood cancer. In some embodiments, the skin cancer is a melanoma. In some embodiments, the blood cancer is a leukemia, a lymphoma, or a myeloma. In some embodiments, the leukemia is Acute Lymphocytic Leukemia or Acute Myeloid Leukemia. In some embodiments, the lymphoma is Hodgkin's Lymphoma or Non-Hodgkins Lymphoma. In some embodiments, the myeloma is multiple myeloma. The cancer may be stage I, stage II, stage III, or stage IV. In some embodiments, the tumor cells are derived from an immortalized cancer cell line. As used herein, a “cancer cell line” refers to a transformed cell line derived from a cancer sample (e.g., HeLa cells).

[0022] Usually, a cancer cell line is capable of generating a tumor upon explant into an appropriate host. A cancer cell line usually retains, in vitro, properties in common with the cancer from which it is derived, including, e.g., loss of differentiation, loss of contact inhibition, and will undergo essentially unlimited cell divisions in vitro. Cancer cell lines may include cell lines which have been genetically modified, for example, to express a protein that allows the cells to be recognized better by antigen-presenting cells. In some embodiments, the tumor cells are cancer stem cells. In some embodiments, the cells are derived from a non-cancerous but abnormal growth, i.e., a benign tumor or growth. In some embodiments, the cancer vaccine includes, comprises, consists essentially of, or consists of white blood cells (e.g., tumor-associated macrophages), tumor-associated endothelial cells, tumor-associated fibroblasts, or any other cell type present in the tumor micro-environment. Examples of inactivating cancer cells, using such inactivated cells for immunotherapy for cancer treatment, and compositions 305463503Atty. Dkt. PHTN-003 / 01WO 338747-2027thereof are described in International Patent Publication No. WO 2019 / 183320, published September 26, 2019, and entitled “Cancer Vaccine Compositions and Methods of Use Thereof,” the entire disclosure of which is hereby incorporated herein by reference.

[0023] Embodiments of the systems, devices, and methods described herein may provide one or more benefits including, for example: (1) increasing efficiency by decreasing the number of manual processes and time to train personnel; (2) decreasing the likelihood of cross contamination by keeping processes together in one device; (3) decreasing the amount of space used for processing cells for immunotherapy by consolidating processes. Specifically, the systems, devices, and methods described herein are configured to reduce the challenges associated with current methods of personalized immunotherapy, which generally use a clean room laboratory environment and highly trained personnel to ensure sterility and manage complex bioprocesses; (4) increasing accessibility by allowing for the systems, devices, and methods described herein to be used in more locations than just specialized centers, for example, distributed manufacturing at local clinics and hospitals; (6) allow manufacturing in non clean room and / or non-sterile settings significantly reducing time and cost.

[0024] While the systems, devices, and methods described herein may be described specifically with reference to tumors, the systems, devices, and methods described herein, or portions thereof, can be used with any tissue where it may be desirable to separate tissue into individual cells and further process the individual cells. In some embodiments, the individual cells can refer to singular cells as well as micro-aggregates of cells that can include clumps of any number of cells. For example the micro-aggregates can include 2 cells, 3 cells, 4 cells, 5 cells, or 10 cells, inclusive of all ranges and values therebetween.

[0025] As described herein, “dissociate” (e.g., dissociation, etc.) can refer to splitting, breaking, decomposing, etc. of tissue into constituent cells. For example, dissociation can include physical dissociation, heat, chemical dissociation, filter, and / or the like.

[0026] FIG. 1 is a schematic block diagram of a cellular processing system 100, according to an embodiment. The cellular processing system 100 is configured to receive tissue T and to process the tissue T into individual inactive cells which can be used for immunotherapy. In some embodiments, the tissue T can be a tumor, a portion of a tumor, and / or the like. In some embodiments, the cellular processing system 100 can be implemented as a unitary system, a tabletop system, and / or the like. The cellular processing system 100 can be used in a laboratory, 305463503Atty. Dkt. PHTN-003 / 01WO 338747-2027in a clean room, a hospital, a clinic, and / or the like. In some embodiments, the cellular processing system 100 can be an automatic system or a semi-automatic system that is configured to be operable with minimal training from a user. The cellular processing system 100 includes a housing 101, a fragmenter 102 (optional), dissociation assembly 110, a first valve 150a (optional), a first filter 120 (optional), a second valve 150b (optional), a mixing chamber 130, a volume adjuster 132 (optional), a buffer reservoir 140, a third valve 150c (optional), a separator 160, a fourth valve 150d (optional), a waste 170, a fifth valve 150e (optional), and an illumination chamber 180 that may have an illumination system operatively coupled thereto.

[0027] The housing 101 can be a physical structure that is configured to cover at least a portion of the other components of the cellular processing system 100. The housing 101 can be configured to cover, protect, seal, and / or the like the components. For example, the housing 101 can be configured to enclose the components of the system 100 to shield them from environmental factors and contaminants such as dust, moisture, cross-contamination, and mechanical damage. In some embodiments, the housing 101 may include gaskets, seals, or similar structures to ensure that the system 100 remains impermeable to liquids or other contaminants. The housing 101 may be formed from various materials, including plastic, metal, or composites, and / or the like. Additionally, the housing 101 can be designed with openings and / or ports to allow for access to at least a portion of the components of the housing 101. In some embodiments, the housing 101 may be configured to maintain an internal volume thereof at a predetermined humidity, gas concentration, pressure, and / or temperature (e.g., a temperature of about 37.5 degrees Celsius, about 5% carbon dioxide, and / or about 95% relative humidity). In some embodiments, the housing 101 may be optional.

[0028] In some embodiments, the housing 101 can be implemented as and / or include a cartridge configured to house one or more of the components of the cellular processing system 100. For example, the cartridge can include a sterile path for the tissue T to be processed into inactive cells. The cartridge can be coupled to the remainder of the cellular processors system 100 for use. In some embodiments, the cartridge may be disposable. The cartridge can allow for the cellular processing system 100 to be used (e.g., with a new cartridge) without having to clean and / or sterilize one or more components. In some embodiments, at least some components of the system 100 described herein may be reusable. In some embodiments,305463503Atty. Dkt. PHTN-003 / 01WO 338747-2027components of the system described herein may be modular, for example, removable and replaceable with fresh components as needed.

[0029] The fragmenter 102 is configured to receive the tissue T and break apart the tissue T into smaller parts, for example, a plurality of tissue segments. In some embodiments, the tissue T can include tumor tissue harvested during surgery and / or a biopsy. In some embodiments, the fragmenter 102 can include a grate, a mesh (e.g., a mesh filter), a perforated structure, and / or the like that is configured to break (e.g., grate, mince, segment, etc.) the tissue T into smaller components, segments, or pieces. In some embodiments, the fragmenter 102 is formed of metal (e.g., stainless steel, titanium, etc.) or any other strong and rigid structure, that is resistant to deformation during operation, heavy use, or in contact with tough tissue. In some embodiments, the fragmenter 102 may be coated with a biocompatible or non-stick material (e.g., PTFE) to reduce friction and prevent tissue adhesion, ensuring smooth operation and easy cleaning. In some embodiments, the tissue T may be pressed into the fragmenter 102 via a piston, a syringe, and / or other force applying mechanisms. In some embodiments, the force applied by the piston or syringe can be manually controlled by the user or automatically regulated by a motorized mechanism (e.g., a rack and pinion, a hydraulic pressure system, a pneumatic pressure system, etc.).

[0030] In some embodiments in which the fragmenter includes a grate, the size of the grate of the fragmenter 102 (e.g., a mesh size) can vary depending on a desired size of resultant tissue T. In some embodiments, the fragmenter 102 is configured to mince tissue T into pieces that are between about 1 mm and about 5 mm in cross-sectional width (e.g., diameter). In some embodiments, the fragmenter 102 is configured to produce tissue fragments of at least about 1 mm, at least about 1.5 mm, at least about 2 mm, at least about 2.5 mm, at least about 3 mm, at least about 3.5 mm, at least about 4 mm, at least about 4.5 mm, or at least about 5 mm cross-sectional width. In some embodiments, the fragmenter 102 is configured to produce tissue fragments of no more than about 5 mm, no more than about 4.5 mm, no more than about 4 mm, no more than about 3.5 mm, no more than about 3 mm, no more than about 2.5 mm, no more than about 2 mm, or no more than about 1.5 mm cross-sectional width. Combinations of the above-referenced ranges are also possible (e.g., at least about 1 mm and no more than about 5 mm, at least about 2 mm and no more than about 3 mm, etc.), inclusive of all ranges and values therebetween. In some embodiments, the tissue T is minced into fragments of about 1 mm, about 2 mm, about 3 mm, about 4 mm, or about 5 mm in diameter. In some embodiments, the 305463503Atty. Dkt. PHTN-003 / 01WO 338747-2027fragmenter 102 can include a metal grate with shaped (e.g., square, circular, etc.) holes or pores with a width of between about 1 mm and about 5 mm, inclusive. In some embodiments, the fragmenter 102 is optional. For example, the dissociation assembly 110 can receive the tissue T directly. An example fragmenter 102 is further seen in and described in reference to FIGS.5A-5E.

[0031] The dissociation assembly 110 is configured to receive the tissue T directly and / or from the fragmenter 102, after the fragmenter 102 has at least partially broken down the tissue T into the plurality of tissue segments. For example, the tissue T, after fragmentation, can drop into the dissociation assembly 110 via gravity, or communicated fluidically from the fragmenter 102 to the dissociation assembly 110. The dissociation assembly 110 is configured to further break down the tissue T into individual cells via at least one of heat, motion, and / or an enzyme E. Examples of suitable enzymes E include, but are not limited to, hyaluronidase, collagenases, dispase, trypsin, papain, nucleases, proteases, and combinations thereof. The specific enzymes E and incubation times may be different depending on tumor and / or tissue type and tumor size.

[0032] In some embodiments, the dissociation assembly 110 can include a chamber filled with a fluid (e.g., saline, buffer, etc.) configured to receive the tissue T or the plurality of tissue segments. In some embodiments, the fluid can include one or more enzyme E configured to aid in breaking down the tissue T into individual cells. In some embodiments, the enzyme can be added via one or more blister pack configured to contain and selectively release the enzyme E when the blister pack is burst or broken. In some embodiments, the enzyme E can be added from an outside source to the dissociation assembly 110. In some embodiments, the dissociation assembly 110 can include one or more heat sources (e.g., heating element, etc.) configured to increase the temperature within the chamber. In some embodiments, the one or more heat sources can be configured to increase the temperature to a predetermined temperature. For example, the predetermined temperature can be a temperature associated with dissociation of the tissue T with no or minimal damage to the tissue T. As another example, the predetermined temperature can be associated with an activation temperature of the enzyme E. In some embodiments, the temperature of the tissue T may be measured by one or more temperature sensors. In some embodiments, the predetermined temperature can be between about 34 degrees C and about 40 degrees C, inclusive of all ranges and values therebetween.305463503Atty. Dkt. PHTN-003 / 01WO 338747-2027

[0033] In some embodiments, the dissociation assembly 110 can include one or more devices configured to induce a mechanical energy or mechanical disruption in the tissue T. For example, mechanical disruption can be induced by one or more of a blender, a stir-rod, and / or the like. In some embodiments, the mechanical disruption can be configured to further mix the tissue T with the enzyme E. In some embodiments, the dissociation assembly 110 can operate in phases (e.g., heating phase, mixing phase, etc.). In some embodiments, the dissociation assembly 110 can operate until a desired portion of the tissue T is broken down into individual cells. For example, the desired portion can be at least about 90% of the tissue T being broken down into individual cells. In some embodiments, the dissociation assembly 110 can operate for a predetermined amount of time (e.g., in a range of 1 minute to 1 hour, inclusive). After dissociation, the individual cells of the tissue T can be in a single cell suspension with the fluid and the enzyme E.

[0034] The first valve 150a is positioned downstream of the dissociation assembly 110. The first valve 150a is operable between a closed position and an open position. In the closed position, the first valve 150a inhibits or prevents the contents of the dissociation assembly 110 from exiting the dissociation assembly 110. In the open position, the first valve 150a allow the contents of the dissociation assembly 110 to flow downstream to the first filter 120 and / or towards the mixing chamber 130 (e.g., via the second valve 150b). In some embodiments, the first valve 150a may be a ball valve, a solenoid valve, a check valve, and / or the like. In some embodiments, the first valve 150a may be operated automatically. For example, the first valve 150a may be operated from the closed position to the open position in response to dissociation assembly 110 finishing dissociating the tissue T. In some embodiments, the first valve 150a may be optional.

[0035] The first filter 120 is configured to receive the at least partially dissociated tissue T from the dissociation assembly 110 (e.g., via the first valve 150a). The first filter 120 is a filter (e.g., mesh filter, a cellulose filter, a filter paper, etc.) configured to allow for the individual cells and the fluid to flow through while capturing particles (e.g., undissociated cells, non-cellular materials, cell aggregates or clumps, cell debris, etc.) greater than a predetermined size. In some embodiments, the predetermined size is at least about 1 mm, at least about 1.1 mm, at least about 1.2 mm, at least about 1.3 mm, at least about 1.4 mm, at least about 1.5 mm, at least about 2 mm, at least about 3 mm, at least about 4 mm, at least about 5 mm. In some embodiments, the first filter 120 is formed of metal. In some embodiments, the first filter 120 305463503Atty. Dkt. PHTN-003 / 01WO 338747-2027may be optional, for example, if the dissociation assembly 110 is configured to completely dissociate the tissue T into the individual cells.

[0036] In some embodiments, the first filter 120 may be disposed in a first filter chamber having an upstream portion configured to receive the individual cells from the dissociation assembly 110 (e.g., once the first valve 150 is in the open position, and a downstream portion located downstream of the first filter 120 configured to receive the filtered individual cells. The second valve 150b may positioned downstream of the first filter 120, for example, at an outlet of the downstream portion of the first filter chamber. The second valve 150b is operable between a closed position and an open position. In the closed position, the second valve 150b prevents the contents upstream from exiting. In the open position, the second valve 150b allow for single cell suspension to flow downstream to the mixing chamber 130. In some embodiments, the second valve 150b may be a ball valve, a solenoid valve, a check valve, and / or the like. In some embodiments, the second valve 150b may be operated automatically. For example, the second valve 150b may be operated from the closed position to the open position in response to dissociation assembly 110 finishing dissociating the tissue T, after a predetermined amount of time, after the liquid passes through the first filter 120, and / or the like. In some embodiments, the second valve 150b can be operated manually by a user. In some embodiments, the second valve 150b may be optional.

[0037] Referring generally to the mixing chamber 130, the volume adjuster 132, the buffer reservoir, the separator 160, the waste 170d, the third valve 150c, and / or the fourth valve 150d, may be configured to wash and capture the individual cells obtained from the tissue T in one or more washing cycles. The washing cycle can be configured to use clean, sterile fluid to wash enzyme, debris, other undesirable components, and / or the like from the individual cells and then capture the washed individual cells.

[0038] The mixing chamber 130 is configured to receive the individual cells after dissociation of the tissue T. The mixing chamber 130 is configured to provide a space for the individual cells to be mixed with and washed (e.g., of the enzyme, of the fluid, etc.) one or more time (e.g., washing cycles) with a suitable fluid (e.g., a buffer) while inhibiting the individual cells from escaping, for example, by capturing the individual cells. The mixing chamber 130, in some embodiments, is operatively coupled to a volume adjuster 132 and fluidically coupled to the buffer reservoir 140 (e.g., via the third valve 150c and the separator 160) and the waste 170 (e.g., via the fourth valve 150d and the separator 160). In some 305463503Atty. Dkt. PHTN-003 / 01WO 338747-2027embodiments, the mixing chamber can include a rotor-stator configuration, a stir bar, a blender, and / or the like configured to induce mixing in the mixing chamber. In some embodiments, mixing can be induced automatically via the washing process. In some embodiments, the third valve 150c and the fourth valve 150d may include on way valves (e.g., one way pinch valves, or duck bill valves). In such embodiments, the third valve 150c may be configured to allow fluid to only flow unidirectionally from the buffer reservoir 140 to the mixing chamber 130 (e.g., in response to a negative pressure in the mixing chamber 130), and the fourth valve 150d may be configured to allow used fluid to only flow unidirectionally from the mixing chamber 130 to the waste 170 (e.g., in response to a positive pressure in the mixing chamber 130).

[0039] The buffer reservoir 140 is configured to store a volume of fluid such as a buffer fluid (e.g., phosphate buffered saline (PBS), HEPES, etc.). In some embodiments, the volume of fluid may be associated with a desired number of wash cycles. For example, if N wash cycles are desired and each wash cycle uses X of the fluid, then the volume of fluid is configured to store, at least, N x X of the fluid. In some embodiments, the number of wash cycles is associated with how much of the enzyme is removed from the single cell suspension. In some embodiments, the buffer reservoir 140 may be configured to store the fluid at a predetermined temperature and can use at least one of a cooling and / or heating system to maintain the fluid at a desired temperature.

[0040] The buffer reservoir 140 is fluidically coupled to the mixing chamber 130 via the third valve 150c. In some embodiments, the third valve 150c is configured to allow for fluid to only flow from the buffer reservoir 140 to the mixing chamber 130 while inhibiting the fluid from flowing back into the buffer reservoir 140. This can allow for waste flow to not contaminate the fluid in the buffer reservoir 140. In some embodiments, the third valve 150c is operable between a closed position and an open position. In the closed position, the third valve 150c inhibits the contents of the buffer reservoir 140 from exiting the buffer reservoir and from fluid in the mixing chamber 130 from entering the buffer reservoir 140. In the open position, the third valve 150c allows for buffer to flow from the buffer reservoir 140 to the mixing chamber 130. In some embodiments, the third valve 150c may be a ball valve, a solenoid valve, a check valve, and / or the like. In some embodiments, the third valve 150c may be operated automatically. For example, the third valve 150c may be operated from the closed position to the open position to the beginning of a wash cycle, after a predetermined amount of305463503Atty. Dkt. PHTN-003 / 01WO 338747-2027time, and / or the like. In some embodiments, the third valve 150c can be operated manually by a user. In some embodiments, the third valve 150c may be optional.

[0041] The waste 170 is configured to store waste fluid that is a byproduct of the washing cycle. For example, the waste fluid can include fluid with enzyme removed or washed from the individual cells (e.g., individual cells suspended in the fluid). The waste 170 is configured to store at least as much fluid as is associated with the wash cycles. The waste 170 is fluidically coupled to the mixing chamber 130 via the fourth valve 150d. In some embodiments, the fourth valve 150d is configured to allow for fluid to only flow from the mixing chamber 130 to the waste 170 without allowing waste to flow back into the mixing chamber 130. This can allow for waste flow to not contaminate the fluid in the mixing chamber 130.

[0042] In some embodiments, the fourth valve 150d is operable between a closed position and an open position. In the closed position, the fourth valve 150d inhibits the contents of the mixing chamber 130 from exiting the mixing chamber 130 and the waste in the waste 170 from entering the mixing chamber 130. In the open position, the fourth valve 150d allows for waste from the mixing chamber 130 to enter the waste 170. In some embodiments, the fourth valve 150d may be a ball valve, a solenoid valve, a check valve, and / or the like. In some embodiments, the fourth valve 150d may be operated automatically. For example, the fourth valve 150d may be operated from the closed position to the open position during the end of a wash cycle, after a predetermined amount of time, and / or the like. In some embodiments, the fourth valve 150d can be operated manually by a user. In some embodiments, the fourth valve 150d may be optional. In some embodiments, the fourth valve 150d and the third valve 150c operate in an inverse. Specifically, when the fourth valve 150d is open, the third valve 150c is closed and when the fourth valve 150d is closed, the third valve 150c is open.

[0043] In some embodiments, the third valve 150c and the fourth valve 150d can be fluidly coupled to ajunction including an additional valve (e.g., functionally and / or structurally similar to any of the vales described herein). The additional valve is between the mixing chamber 130 and the junction. In some embodiments, such as when the third vale 150c and the fourth valve 150d are one way valves, the additional valve can be operated before and after the cycle to allow for flow out of the buffer reservoir 140 and flow into the waste 170, thus decreasing the number of valves operated during a cycle.305463503Atty. Dkt. PHTN-003 / 01WO 338747-2027

[0044] The separator 160 may be disposed in the mixing chamber 130 or between the mixing chamber 130 and the buffer reservoir 140, as well as between the mixing chamber 130 and the waste 170. In some embodiment, the separator 160 may include a second filter configured to inhibit flow of the individual cells from the mixing chamber 130 to the waste 170 and / or the buffer reservoir 140. In such embodiments, the separator 160 may be disposed between the mixing chamber 130 and the buffer reservoir 140 and between the mixing chamber 130 and the waste 170. The second filter may be configured to specifically filter the fluid when being drained into the waste 170. The second filter may be configured to allow for the enzymes and the fluid to pass through and continue to the waste 170, while capturing the individual cells. This allows for the washed individual cells to be separated from the enzyme and prepared for delivery to the illumination chamber 180.

[0045] In some embodiments, the separator 160 may include a centrifuge disposed in the mixing chamber. For example, the centrifuge may include a rotor, a rotary plate, a stir bar, or any other suitable mechanism configured to generate centrifugal forces in the mixing chamber 130 to facilitate washing of the individual cells as well as propel the cells under centrifugal force to a designated area of the mixing chamber 130, for example, as cell pellets. This may allow the cells to be separated from the fluid after washing to facilitate drainage of the fluid after washing from the mixing chamber 130 to the waste 170 such that a substantial portion of the cells remain in the mixing chamber 130 as the used fluid is communicated out of the mixing chamber 130 to the waste 170. In some embodiments, the separator 160 may include a combination of the second filter and the centrifuge, which may be disposed at any suitable location within the mixing chamber 130 or between the mixing chamber 130 and the buffer reservoir 140 and waste 170.

[0046] The volume adjuster 132 is configured to adjust (e.g., increase, decrease, etc.) the volume in the mixing chamber 130 as to either draw in fluid (e.g., buffer from the buffer reservoir 140) and / or push out fluid (e.g., into the waste 170). In some embodiments, the volume adjuster 132 may be a piston, a bellows, a diaphragm, a plunger, a pump (e.g., a positive pressure pump or vacuum pump) and / or any similar feature configured to adjust the volume of the mixing chamber 130. The volume adjuster 132 is operable between a minimum volume position, where the mixing chamber 130 is at a minimum, and a maximum volume position, where the volume of the mixing chamber 130 is at a maximum. In some embodiments, the volume adjuster 132 can be moved to any position between the minimum and the maximum 305463503Atty. Dkt. PHTN-003 / 01WO 338747-2027volume positions. The movement of the volume adjuster 132 can allow for fluid to be drawn out of the buffer reservoir 140 (e.g., when the third valve 150c is in the open configuration) and pushed into the waste 170 (e.g., when the fourth valve 150d is in the open configuration). The volume adjuster 132 may be operated manually (e.g., by a user) and / or automatically. For example, the volume adjuster 132 may be configured to operate after a predetermined amount of time, after a desired number of individual cells are disposed within the mixing chamber 130, during a wash cycle, and / or the like. In some embodiments, the volume adjuster 132 may include a plunger or piston configured to displace within the mixing chamber 130. In some embodiments, the volume adjuster 132 may be excluded, for example, in embodiments, where a continuous flow of the fluid is maintained through the mixing chamber 130 (e.g., in a flow through configuration).

[0047] During operation, the volume adjuster 132 may be moved to increase the volume in the mixing chamber 130 and thus draw the fluid out of the buffer reservoir 140 while the third valve 150c is open and the fourth valve 150d is closed. The volume adjuster 132 may be moved until a desired amount of fluid is drawn out of the buffer reservoir 140 into the mixing chamber 130. In some embodiments, the drawing of the fluid allows for the fluid to wash the individual cells in the mixing chamber 130 as the fluid and the individual cells extracted from tissue T mix automatically while the fluid is being drawn into the mixing chamber 130. In some embodiments, a stir rod or similar mixing feature can be used to mix the fluid and the tissue T to wash the tissue T. In some embodiments, in which the separator 160 includes a centrifuge, a speed of the centrifuge may be selectively adjusted to cause mixing of the individual cells with the fluid without separation from the fluid.

[0048] After the individual cells are washed (e.g., after a predetermined amount of time, etc.), the volume adjuster 132 may be moved to decrease the volume in the mixing chamber 130 and thus pushing the fluid and individual cells into the separator 160 (e.g., a second filter), which captures the individual cells while allowing the fluid and the enzyme to continue to the waste while the third valve 150c is closed and the fourth valve 150d is open, thus concluding one wash cycle. The wash cycles can repeat until the individual cells are cleaned desirably. Examples of a system including a second filter are shown in FIG. 2.

[0049] In some embodiments in which the separator 160 includes a centrifuge, after the cells have been washed for a desired time period or wash cycles, the centrifuge may be activated or a speed of the centrifuge adjusted to cause the cells to be separated under 305463503Atty. Dkt. PHTN-003 / 01WO 338747-2027centrifugal force from the fluid and be collected. The remaining fluid can then be expelled from the mixing chamber. Fresh fluid may be introduced into the mixing chamber 130, for example, to resuspend the cells therein (e.g., by adjusting a rotational speed of the centrifuge to facilitate remixing of the cells with the fluid). Examples of a system with a separator that includes a centrifuge is described in FIGS. 3A-3C.

[0050] In some embodiments, the mixing chamber 130 can use alternative methods of mixing the tissue T and the fluid and / or separating the enzyme from the tissue T such as blending, mixing, tangential flow filtration, density separation, electrostatic separation, any other suitable method, or any suitable combination thereof.

[0051] After the wash cycle(s) are completed, the individual cells are directed to the illumination chamber 180. In some embodiments, fluid is drawn through the separator 160 (e.g., a second filter) to separate the individual cells and aid in draining the mixing chamber 130 into the illumination chamber 180 via the fifth valve 150e. The fifth valve 150e is positioned downstream of the mixing chamber 130. The fifth valve 150e is operable between a closed position and an open position. In the closed position, the fifth valve 150e inhibits the contents upstream thereof from exiting. In the open position, the fifth valve 150e allow for the cells of the tissue T to flow downstream to the illumination chamber 180. In some embodiments, the fifth valve 150e may be a ball valve, a solenoid valve, a check valve, and / or the like. In some embodiments, the fifth valve 150e may be operated automatically or manually. For example, the fifth valve 150e may be operated from the closed position to the open position in response to a completion of the wash cycle(s), after a predetermined amount of time, and / or the like. In some embodiments, the fifth valve 150e can be operated manually by a user. In some embodiments, a filter functionally and / or structurally similar to the first filter 120 can be located upstream of the fifth valve 150e and after the mixing chamber 130. In some embodiments, the fifth valve 150e may be optional.

[0052] The illumination chamber 180 is configured to receive the tissue T that is dissociated into individual cells and washed (and a buffer fluid) for further processing. For example, the illumination chamber 180 may have an illumination source operatively coupled thereto and configured to deliver optical energy (e.g., light having a predetermined wavelength) to the individual cells and, in some embodiments, mix (e.g., stir, etc.) the fluid with the cells during illumination. In some embodiments, the illumination chamber 180 can include a stir rod, a linear agitator, a rotor, piston, and / or the like for mixing the fluid in the illumination chamber 305463503Atty. Dkt. PHTN-003 / 01WO 338747-2027180. In some embodiments, the illumination may occur via one or more illumination or light sources. For example, the illumination source can be configured to deliver ultraviolet (UV) light. In some embodiments, the light source can include light sources disposed in or around the illumination chamber 180. In some embodiments, the lights sources are light-emitting diodes (LEDs). In some embodiments, the illumination chamber 180 may be formed from a transparent material (e.g., glass, plexiglass, plastic, acrylic, etc.) or may have at least one sidewall that is transparent to allow the optical energy to be communicated into an internal volume thereof.

[0053] The illumination chamber 180 includes an additive A disposed in the internal volume thereof, and / or is configured to allow for an additive to be communicated thereto. In some embodiments, the additive A may be disposed in a frangible container within the illumination chamber 180 (e.g., disposed in a blister pack). The frangible container may be configured to selectively rupture to communicate the additive A into the internal volume of the illumination chamber 180 and mix with the individual cells therein. In some embodiments, the additive A can be added to the illumination chamber 180 from an external source. In some embodiments, the additive A is a photosensitizer, that are configured to attack nucleic acids. In some embodiments, the photosensitizer is riboflavin (e.g., vitamin B2; 7,8-dimethyl-10-ribityl isoalloxazine), which has been reported to attack nucleic acids. For example, riboflavin and its derivatives can inactivate microorganisms which may be contained in blood or blood products in the presence of optical energy such as UV light or may also be used to inactivate microorganisms.

[0054] In some embodiments, after the riboflavin is added, the fluid and the riboflavin can be mixed so that the riboflavin attaches to the nucleic acids in the cells. When the cells are exposed to UV light, the riboflavin has a sensitizing effect on the DNA of the tissue T and causes damage to the DNA but not proteins or other cell structures, thereby leaving the cells incapable of replicating while preserving surface proteins and their antigenicity. Specifically, the riboflavin absorbs the UV light and releases the energy into specific DNA bonds, fracturing the bonds and rendering the tissue T cells inactivated and incapable of replication. Inactivated cells with preserved surface proteins have a greater chance of provoking an immune response while ensuring safety for the patient. In some embodiments, the illumination chamber 180 can use alternative such as a peristaltic pump configured to recirculate flow, which can reduce the number of light sources desired for delivering energy to the cells of the tissue T.305463503Atty. Dkt. PHTN-003 / 01WO 338747-2027

[0055] After the cells are inactivated, the illumination chamber 180 can be accessed to remove the cells (e.g., the processed cells P) from the cellular processing system 100 and / or the illumination chamber 180 can be caused to deliver the processed cells P to a collection chamber. The processed cells P can then be harvested and used for immunotherapy. In some embodiments, the cells from the illumination chamber 180 can be further filtered using a filter structurally and / or functionally similar to the first filter 120 to filter out clumped cells.

[0056] FIG. 2 is a schematic diagram of a cellular processing system 200, according to an embodiment. In some embodiments, the cellular processing system 200 is functionally and / or structurally similar to the cellular processing system 100 of FIG. 1. The cellular processing system 200 is also configured to receive tissue (such as the tissue T) and process the tissue into inactive individual cells. The cellular processing system 200 includes a fragmenter (e.g., functionally and / or structurally similar to the fragmenter 102 of FIG. 1) including a pusher 202a and a grate 102b, a dissociation assembly 210 (e.g., functionally and / or structurally similar to the dissociation assembly 110 of FIG. 1), a stir rod 212, a first valve 250a (e.g., functionally and / or structurally similar to the first valve 150a of FIG. 1), a filter 220 (e.g., functionally and / or structurally similar to the filter 120 of FIG. 1), a second valve 250b (e.g., functionally and / or structurally similar to the second valve 150b of FIG. 1), a mixing chamber 230 (e.g., functionally and / or structurally similar to the mixing chamber 130 of FIG. 1), a volume adjuster 232 (e.g., functionally and / or structurally similar to the volume adjuster 132 of FIG. 1), a buffer reservoir 240 (e.g., functionally and / or structurally similar to the buffer reservoir 140 of FIG. 1), a third valve 250c (e.g., functionally and / or structurally similar to the third valve 150c of FIG. 1), waste 270 (e.g., functionally and / or structurally similar to the waste 170 of FIG. 1), a fourth valve 250d (e.g., functionally and / or structurally similar to the fourth valve 150d of FIG. 1), a microfilter 260, a fifth valve 250e (e.g., functionally and / or structurally similar to the fifth valve 150e of FIG. 1), and an illumination chamber 280 (e.g., functionally and / or structurally similar to the illumination chamber 180 of FIG. 1) including a stir rod 282 disposed therein, and a light source 284 operatively coupled thereto. In some embodiments, the components of the cellular processing system 200 may be disposed within a housing such as the housing 101 of FIG. 1.

[0057] The cellular processing system 200 is configured to receive tissue between the pusher 202a and the grate 202b. The pusher 202a is a piston, a plunger, or the like configured to press the tissue through the grate 202b. The grate 202b is configured to slice, mince, and / or 305463503Atty. Dkt. PHTN-003 / 01WO 338747-2027otherwise reduce the size of the tissue into a smaller size, for example, a plurality of tissue segments, as described in reference to the fragmenter 202 of FIG. 1. In some embodiments, the pusher 202a can be pushed manually and / or automatically (e.g., via a motor, drive, etc.) to push the tissue through the grate 202b.

[0058] After the tissue is pushed through the grate 202b, the plurality of tissue segments is communicated to (e.g., fall into) the dissociation assembly 210 which is configured to further dissociate the plurality of tissue segments into individual cells. The dissociation assembly 210 is filled with a fluid that can have enzymes E added before, during, and / or after the plurality of tissue segments are communicated into the dissociation assembly 210. In some embodiments, the enzyme E can be added from an outside source into the dissociation assembly 210. To further dissociate the tissue, the stir rod 212 is operated to agitate the enzymes E and the tissue and aid in dissociation. Agitation can continue for a predetermined amount of time and / or until a desired amount of the tissue is dissociated. After the agitation, the first valve 250a can be operated from a closed position to an open position, which can allow for the fluid, including the enzymes E and tissue, to flow through the filter 220, which is configured to capture tissue that did not dissociate or large aggregates or clumps of cells (e.g., tissue or cell aggregates larger than a threshold size).

[0059] The second valve 250b is then opened to allow for the filtered fluid to continue into the mixing chamber 230. The mixing chamber 230 includes the volume adjuster 232 implemented as a piston configured to alter the volume of the mixing chamber 230 and a microfilter 260 opposite the volume adjuster 232. The volume adjuster 232 is configured to move toward the microfilter 260 to decrease the volume within the mixing chamber 230 and away from the microfilter 260 to increase the volume within the mixing chamber 230. The operation of the volume adjuster 232 can be used for washing the cells of the tissue, for example, remove the enzyme from the cells. During a wash cycle, the volume adjuster 232 moves away from the microfilter 260 while the third valve 250c is open and the fourth valve 250d is closed so that fluid from the buffer reservoir 240 is drawn through the microfilter 260 into the mixing chamber 230. Once drawn into the mixing chamber 230, the fluid mixes with the cells and the enzyme to separate the enzyme from the cells. The third valve 250c and the fourth valve 250d are then closed and the volume adjuster 232 is then moved towards the microfilter 260 to push the fluid and the enzyme through the microfilter 260 into the waste 270305463503Atty. Dkt. PHTN-003 / 01WO 338747-2027while the microfilter 260 captures the washed tissue. The wash cycles can be repeated for a desired number of times.

[0060] After the final wash cycle, a final draw of fluid from the buffer reservoir 240 into the mixing chamber 230 allows for the tissue on the microfilter 260 to separate. The washed tissue and fluid are then delivered to the illumination chamber 280 by opening the fifth valve 250e. After the washed tissue and fluid are in the illumination chamber 280, the fifth valve 250e may close. In some embodiments, riboflavin (and / or another additive A) is added to the illumination chamber 280. In some embodiments, the riboflavin may already be present in the illumination chamber 280 when the fluid and washed tissue are conveyed into the illumination chamber 280. The stir rod 282 is configured to agitate the fluid, tissue, and riboflavin so that the tissue is exposed to the riboflavin. During and / or after agitation, the light sources, which surround the perimeter of the illumination chamber 280, are configured to deliver energy to the inactivate the cells (e.g., as described in more detail above). In some embodiments, the light sources are configured to generate UV light. In some embodiments, the light sources are LED lights. In some embodiments, the energy is delivered for a predetermined amount of time.

[0061] FIGS. 3 A-3C depict various views of a cellular processing system 300, according to an embodiment. Specifically, FIG. 3B depicts the cellular processing system 300 at crosssection A-A of FIG. 3 A, and FIG. 3C depicts the cellular processing system 300 at cross-section B-B of FIG. 3 A. The cellular processing system 300 can be substantially similar in structure and function to the cellular processing system 100 of FIG. 1 and / or the cellular processing system 200 of FIG. 2. In some embodiments, the cellular processing system 300 can be vertically arranged so that gravity can aid in the transfer of fluid, tissue, and / or the like. In some embodiments, the cellular processing system 300 may be configured to be inserted into a centrifuge during operation. The cellular processing system 300 includes a fragmenter (e.g., functionally and / or structurally similar to the fragmenter 102 FIG.l) including a syringe 302a and a grate 302b, a dissociation assembly 310 (e.g., functionally and / or structurally similar to the dissociation assembly 110 of FIG. 1 and / or the dissociation assembly 210 of FIG. 2) including an enzyme reservoir 314, a filter 320 (e.g., functionally and / or structurally similar to the first filter 120 of FIG. 1 and / or the filter 220 of FIG. 2), a buffer reservoir 340 (e.g., functionally and / or structurally similar to the buffer reservoir 140 of FIG. 1 and / or the buffer reservoir 240 of FIG. 2), an additive reservoir 386, a processing chamber 390 including a pellet 392, and waste 370. The cellular processing system 300 includes a first valve 350a, a second 305463503Atty. Dkt. PHTN-003 / 01WO 338747-2027valve 350b, a third valve 350c, a fourth valve 350d, a fifth valve 350e, and a sixth valve 350f, collectively referred to as “the valves 350”. In some embodiments, the valves are functionally and / or structurally similar to the valves described with reference to FIG. 1 and / or FIG. 2. In some embodiments, the cellular processing system 300 may be disposed within a housing such as the housing 101 of FIG. 1.

[0062] The dissociation assembly 310 is generally cylindrical and includes the enzyme reservoir 314 and the grate 302b disposed concentrically. The syringe 302a may be configured to store tissue, such as the tissue T, and insert the tissue into the dissociation assembly 310 through a grate 302b. For example, the tissue may be disposed in a volume of the syringe 302a and an outlet of the syringe 302a may be disposed adjacent to, in contact with, or in communication with the grate 302b. When a plunger 302c of the syringe 302a is depressed, the plunger 302c presses the tissue into and through the grate 302b causing the tissue to fragment (e.g., be crushed, cut, minced, chopped, broken down, etc.) into a plurality of tissue segments that pass through the grate 302b and enter the dissociation assembly 310. The first valve 350a can be operated to allow for an enzyme and / or a fluid to be conveyed into the dissociation assembly 310, thus allowing for the enzyme to aid in the further dissociation of the tissue. After enzymatic dissociation, the second valve 350b can be operated from a closed to an open configuration to allow for the tissue, fluid, and enzyme to pass through the filter 320, which is configured to filter out undissociated tissue.

[0063] The third valve 350c is operated from the closed to the open configuration to allow for the filtered tissue and fluid to be conveyed into the processing chamber 390. The processing chamber 390 may be functionally similar to both the mixing chambers described herein and the illumination chambers described herein. For example, the processing chamber 390 may be configured to wash the cells obtained by dissociating the tissue to remove the enzyme therefrom and then illuminate the tissue to inactivate the cells. In other words, the processing chamber 390 may be configured to perform washing, illumination, and / or separation of cells from the fluid before and / or after illumination.

[0064] The processing chamber 390 is in fluid communication with a buffer reservoir 340 via a fourth valve 350d and an additive reservoir 386 via a fifth valve 350e. To first wash the cells, the fourth valve 350d is activated to communicate buffer fluid into the processing chamber 390. During a centrifugal process, the pellet 392 forms, which includes a mass of cells that are separate from the fluid. After washing, the fluid can be drained via the sixth valve 350f 305463503Atty. Dkt. PHTN-003 / 01WO 338747-2027into the waste 370 without draining the pellet 392. In some embodiments, the sixth valve 350f may include a filter to prevent the cells from being directed into the waste. Once the washing fluid is drained, the fifth valve 350e can be operated into the open position to deliver an additive (e.g., riboflavin) into the processing chamber 390 for illumination. The cells can then be illuminated by one or more light sources disposed within or around the processing chamber 390 to allow for the cells to be inactivated and thus prepared for immunotherapy, as previously described herein.

[0065] FIG. 4 is a schematic flow diagram of a method 400 for processing cells, according to an embodiment. In some embodiments, the method 400 can be executed by a cellular processing system such as the cellular processing system 100 of FIG. 1, the cellular processing system 200 of FIG. 2, and / or the cellular processing system 300 of FIGS. 3A-3C. In some embodiments, the method 400 can be used for inactivating cells for immunotherapy. The method 400 can be used in a hospital, clinical, laboratory, and / or similar setting. In some embodiments, the method 400 may be executed automatically by a cellular processing system and / or can use manual intervention for one or more processes. In some embodiments, the method 400 can be repeated for different patients.

[0066] At 402, the method 400 includes dissociating tissue into individual cells using at least one of heat, motion, or an enzyme. In some embodiments, prior to dissociating the tissue using at least one of heat, motion, or an enzyme, the tissue can be minced, grated, cut, and / or the like to reduce the size of the tissue. In some embodiments, applying heat can include activating one or more heating elements to increase the temperature of a fluid (e.g., water) in which the tissue is disposed. In some embodiments, the heat can increase the reactiveness of an enzyme disposed within the fluid. In some embodiments, motion can include using a stir rod, blender, and / or the like to further dissociate the tissue. In some embodiments, dissociation occurs until the tissue is substantially broken down into the individual cells. In some embodiments, prior to 402, the tissue can be inserted into a cellular processing system. At 404, the method 400 includes filtering the dissociated tissue. For example, the dissociated tissue may be filtered using a first filter (e.g., the first filter 120, 220) to filter undissociated portions of the tissue, or large cell aggregates, as these may be difficult to fully inactivate relative to individual cells.

[0067] At 406, the method 400 includes directing the individual cells into a mixing chamber after filtering. In some embodiments, the directing the individual cells can include operating 305463503Atty. Dkt. PHTN-003 / 01WO 338747-2027one or more valves to allow for the individual cells in a fluid to flow into the mixing chamber. At 408, the method 400 includes conveying into the mixing chamber (e.g., from a buffer storage such as buffer reservoir 140), a first volume of a buffer fluid (e.g., any of the buffer fluids described herein). The first volume of buffer fluid mixes with and washes the individual cells. In some embodiments, the first volume of buffer fluid can be drawn into the mixing chamber via a volume adjuster configured to adjust the volume of the mixing chamber. In some embodiments, one or more valves can be operated to allow for the fluid to be directed into the mixing chamber. The first volume of the buffer fluid may wash the cells obtained from the dissociated tissue, for example, to remove enzymes that may have been used to dissociate the tissue from which the cells are obtained.

[0068] At 410, the method 400 includes communicating the first volume of the buffer fluid out of the mixing chamber through a separator (e.g., the separator 160 such as a microfilter) that is configured to capture the washed individual cells. In some embodiments, communicating or draining the first volume of the buffer fluid, similar to the conveying the first volume of the buffer of the buffer fluid into the mixing chamber, can include using the volume adjuster to push the fluid out of the mixing chamber into the waste via the separator. The separator may be configured to capture the individual cells while allowing the first volume of the buffer fluid including enzymes to be communicated out of the mixing chamber thus separating the cells from the first volume of the fluid and allowing the cells to be washed. In some embodiments, 408 and 410 can be repeated until the cells are washed to a desired degree (e.g., enough enzyme is washed off). In some embodiments, the separator may include a centrifuge configured to separate cells from the first volume of the buffer fluid via centrifugal force, as previously described herein.

[0069] At 412, the method 400 includes, optionally, conveying into the mixing chamber (e.g., from the buffer storage), a second volume of buffer fluid such that the second volume of buffer fluid mixes with the washed individual cells. In some embodiments, the second volume of buffer fluid is configured to remove the cells from the separator and be suspended into the second volume of the buffer fluid in the mixing chamber. At 414, the method 400 includes conveying the washed individual cells into an illumination chamber (e.g., the illumination chamber 180, 280) containing at least one additive (e.g., riboflavin). In some embodiments, conveying can include operating at least one valve to allow for the washed cells to be directed into the illumination chamber. In some embodiments, the additive can include riboflavin. In 305463503Atty. Dkt. PHTN-003 / 01WO 338747-2027some embodiments, after the cells and / or the additive is added, the mixture can be agitated so that the cells and the additive can come into contact.

[0070] At 416, the method 400 includes illuminating the washed individual cells until a desired damage occurs, for example, the individual cels are inactivated. In some embodiments, illuminating includes directing optical energy at the cells and / or the riboflavin so that the cells are inactivated, as previously described herein. In some embodiments, the optical energy is UV light. In some embodiments, at least one light source is used to illuminate the cells. In some embodiments, the light source can include LEDs. In some embodiments, the cells are illuminated until at least a desired portion of the cells are inactivated. After the cells are inactivated, the cells can be used for immunotherapy. For example, the cells can be injected back into a patient. In some embodiments, 416 can include collecting the processed cells.

[0071] FIG. 5A depicts a side view of a fragmenter 502 (e.g., functionally and / or structurally similar to the fragmenter 102 of FIG. 1), according to an embodiment. Similar to the other fragmenters described herein, the fragmenter 502 is configured to receive the tissue and break apart the tissue into smaller parts, for example, a plurality of tissue segments. The fragmenter 502 is configured to specifically cut the tissue into smaller parts. In some embodiments, cutting can allow for the fragmenter 502 to cut through tougher tissue. The fragmenter 502 includes a cutting portion 503 and a receiving portion 504. As seen in FIG. 5A, the cutting portion 503 and the receiving portion 504 are configured to engage together for operation.

[0072] The receiving portion 504 is configured to receive the tissue and the cutting portion 503. The receiving portion 504 includes engagement members 504a, a tissue support 504b, and an aperture 504c, as seen in FIGS. 5A-5B. In some embodiments, the receiving portion 504 is a unitary object. In some embodiments, the receiving portion 504 is formed of multiple components. The engagement members 504a are configured to engage associated portion of the receiving portion 504 so that the receiving portion 504 can be selectively coupled to the cutting portion 503. The engagement members 504a are sized so that during operation of the fragmenter 502, the engagement members 504a are unlikely to deform, bend, and / or the like. The engagement members 504a are configured to extend away (e.g., axially, radially, etc.) from a body of the receiving portion 504. In some embodiments, such as seen in FIG. 5B, there may be four engagement members 504a. In some embodiments, there may be any number of engagement members 504a. The tissue support 504b is configured to support the tissue during 305463503Atty. Dkt. PHTN-003 / 01WO 338747-2027cutting. The tissue support includes flexible supports that may be moved during cutting to allow for the tissue to be cut while being supported. The aperture 504c is configured to receive a distal end of the cutting portion 503. The shape of the aperture 504c corresponds to the shape of the cutting portion 503 to prevent rotation of the cutting portion 503 during operation. As seen in FIG. 5B, the aperture 504c is rectangular but can be any other shape.

[0073] The cutting portion 503 is configured to operate between a deployed (as seen in FIG.5C) and an undeployed configuration (as seen in the cutting portion 503’ of FIG. 5D) to allow for the tissue to be cut. As seen in FIGS. 5A and 5C-5E, the cutting portion 503 includes a plunger 503a, a handle 503b, coupling elements 503c, a sheath 503d, and a cutting element 503e. The coupling elements 503c are configured to engage the engagement members 504a such that the receiving portion 504 and the cutting portion 503 can be selectively coupled. In some embodiments, the engagement members 504a are configured to engage the coupling elements 503c via a rotational motion. The sheath 503d is configured to cover the blades when the cutting portion 503 is in the undeployed configuration and defines a plurality of slots 503f, as seen in FIG. 5E, through which the cutting elements 503e can extend. In some embodiments, the sheath may be configured to rotate relative to the handle 503b during coupling of the cutting portion 503 and the receiving portion 504. The cutting elements 503e extend through the slots 503f when the plunger 503a is pressed in relative to the handle 503b. In some embodiments, the cutting elements 503e can include one or more blades, scissors, and / or the like. The cutting elements 503e are configured to be pressured into the tissue support 504b repeatedly by the plunger 503a so that the tissue can be reduced to smaller pieces of the desirable size. In some embodiments, one or more openings may be defined at a distal end of the receiving portion 504, for example, a distal end of the tissue support 504b. The one or more openings may allow the smaller sized pieces of the tissue to be ejected from the receiving portion 504 of the fragmenter 502, for example, to be communicated to the dissociation assembly 110 or any other dissociation assembly described herein.

[0074] The present disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the preceding description or illustrated in the drawings. The present technology is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.305463503Atty. Dkt. PHTN-003 / 01WO 338747-2027

[0075] Thus, particular implementations of the invention have been described. Other implementations are within the scope of the following claims. In some cases, the actions recited in the claims can be performed in a different order and still achieve desirable results. In addition, the processes depicted in the accompanying figures do not necessarily require the particular order shown, or sequential order, to achieve desirable results. In certain implementations, multitasking and parallel processing may be advantageous.

[0076] It should be noted that the term “example” as used herein to describe various embodiments or arrangements is intended to indicate that such embodiments or arrangements are possible examples, representations, and / or illustrations of possible embodiments or arrangements (and such term is not intended to connote that such embodiments or arrangements are necessarily crucial, extraordinary, or superlative examples).

[0077] The use of “including”, “comprising”, or “having”, “containing”, “involving” and variations thereof herein, is meant to encompass the items listed thereafter as well as, optionally, additional items. In the description the same numerical references refer to similar elements.

[0078] It must be noted that, as used in this specification and the appended claims, the singular form “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0079] As used herein, the term “about” or “generally” or the like in the context of a given value or range (whether direct or indirect, e.g., “generally in line”, “generally aligned”, “generally parallel”, etc.) refers to a value or range that is within 20%, preferably within 10%, and more preferably within 5% of the given value or range.

[0080] As used herein, the term “and / or” is to be taken as specific disclosure of each of the two 10 specified features or components with or without the other. For example, “A and / or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

[0081] Modifications and improvements to the above-described implementations of the present technology may become apparent to those skilled in the art. The foregoing description is intended to be exemplary rather than limiting. The scope of the present technology is therefore intended to be limited solely by the scope of the appended claims.305463503Atty. Dkt. PHTN-003 / 01WO 338747-2027

[0082] In the context of the present specification, the words “first”, “second”, “third”, etc. have been used as adjectives only for the purpose of allowing for distinction between the nouns that they modify from one another, and not for the purpose of describing any particular relationship between those nouns. Thus, for example, it should be understood that the use of the terms “first unit” and “third unit” is not intended to imply any particular type, hierarchy or ranking (for example) of / between the units. Nor is their use (by itself) intended to imply that any “second unit” must necessarily exist in any given situation.

[0083] As utilized herein, the terms “substantially’ and similar terms are intended to have a broad meaning in harmony with the common and accepted usage by those of ordinary skill in the art to which the subject matter of this disclosure pertains. For example, the term “substantially flat” would mean that there may be de minimis amount of surface variations or undulations present due to manufacturing variations present on an otherwise flat surface. It should be understood by those of skill in the art who review this disclosure that these terms are intended to allow a description of certain features described and claimed without restricting the scope of these features to the precise arrangements and / or numerical ranges provided. Accordingly, these terms should be interpreted as indicating that insubstantial or inconsequential modifications or alterations of the subject matter described and claimed are considered to be within the scope of the inventions as recited in the appended claims.

[0084] The terms “coupled,” and the like as used herein mean the joining of two members directly or indirectly to one another. Such joining may be stationary (e.g., permanent) or moveable (e.g., removable, or releasable). Such joining may be achieved with the two members, or the two members and any additional intermediate members being integrally formed as a single unitary body with one another or with the two members or the two members and any additional intermediate members being attached to one another.

[0085] The arrangements described herein have been described with reference to drawings. The drawings illustrate certain details of specific arrangements that implement the systems, methods and programs described herein. However, describing the arrangements with drawings should not be construed as imposing on the disclosure any limitations that may be present in the drawings.305463503Atty. Dkt. PHTN-003 / 01WO 338747-2027

[0086] It should be understood that no claim element herein is to be construed under the provisions of 35 U.S.C. § 112(f), unless the element is expressly recited using the phrase “means for.”

[0087] It should be noted that although the diagrams herein may show a specific order and composition of method steps, it is understood that the order of these steps may differ from what is depicted. For example, two or more steps may be performed concurrently or with partial concurrence. Also, some method steps that are performed as discrete steps may be combined, steps being performed as a combined step may be separated into discrete steps, the sequence of certain processes may be reversed or otherwise varied, and the nature or number of discrete processes may be altered or varied. The order or sequence of any element or apparatus may be varied or substituted according to alternative arrangements. Accordingly, all such modifications are intended to be included within the scope of the present disclosure as defined in the appended claims. Such variations will depend on the machine-readable media and hardware systems chosen and on designer choice. It is understood that all such variations are within the scope of the disclosure. Likewise, software and web implementations of the present disclosure could be accomplished with standard programming techniques with rule based logic and other logic to accomplish the various database searching steps, correlation steps, comparison steps and the like.

[0088] It is important to note that the construction and arrangement of the various exemplary embodiments are illustrative only. Although only a few embodiments have been described in detail in this disclosure, those skilled in the art who review this disclosure will readily appreciate that many modifications are possible (e.g., variations in sizes, dimensions, structures, shapes and proportions of the various elements, values of parameters, mounting arrangements, use of materials, colors, orientations, etc.) without materially departing from the novel teachings and advantages of the subject matter described herein. Other substitutions, modifications, changes, and omissions may also be made in the design, operating conditions, and arrangement of the various exemplary embodiments without departing from the scope of the present invention.

[0089] While this specification contains many specific implementation details, these should not be construed as limitations on the scope of any inventions or of what may be claimed, but rather as descriptions of features specific to particular implementations of particular inventions. Certain features described in this specification in the context of separate implementations can 305463503Atty. Dkt. PHTN-003 / 01WO 338747-2027also be implemented in combination in a single implementation. Conversely, various features described in the context of a single implementation can also be implemented in multiple implementations separately or in any suitable subcombination. Moreover, although features may be described above as acting in certain combinations and even initially claimed as such, one or more features from a claimed combination can in some cases be excised from the combination, and the claimed combination may be directed to a subcombination or variation of a subcombination.305463503

Claims

Atty. Dkt. PHTN-003 / 01WO 338747-2027ClaimsWhat is claimed is:

1. A method, comprising:dissociating tissue into individual cells using at least one of heat, motion, or an enzyme;communicating the individual cells into a mixing chamber;conveying a first volume of a buffer fluid into the mixing chamber such that the first volume of the buffer fluid mixes with the individual cells and washes the individual cells; draining the first volume of the buffer fluid from the mixing chamber via a separator, the separator configured to capture the washed individual cells;conveying the washed individual cells into an illumination chamber, the illumination chamber containing at least one additive; andilluminating the washed individual cells with optical energy in the illumination chamber until a desired damage occurs to the washed individual cells.

2. The method of claim 1, further comprising:conveying one or more additional volumes of buffer fluid into the mixing chamber such that the one or more additional volumes of the buffer fluid mixes with the washed individual cells; andconveying a second volume of buffer fluid and the washed individual cells into the illumination chamber.

3. The method of claim 1, further comprising:filtering the dissociated tissue via a first filter, the first filter configured to filter cell aggregates while allowing the individual cells to pass through.

4. The method of claim 1, wherein the optical energy includes ultra-violet light.

5. The method of claim 1, wherein the additive includes riboflavin.

6. The method of claim 1, wherein the buffer fluid includes saline.305463503Atty. Dkt. PHTN-003 / 01WO 338747-20277. The method of claim 1, wherein the illumination chamber includes at least one mixing device configured to mix the washed individual cells during illumination.

8. The method of claim 1, wherein dissociating the tissue includes breaking the tissue into a plurality of tissue segments.

9. The method of claim 8, wherein the breaking the tissue into a plurality of tissue segments includes passing the tissue through a metal grate.

10. The method of claim 8, wherein the plurality of tissue segments have a cross-sectional width of no more than about 5 mm.

11. An apparatus, comprising:a fragmenter configured to fragment tissue in a plurality of tissue segments;a dissociation assembly configured to dissociate the plurality of tissue segments into individual cells using at least one of heat, motion, or an enzyme;a filter configured to selectively allow the individual cells to pass through;a mixing chamber including a separator, the mixing chamber configured to:receive the individual cells through the filter,mix a first volume of a buffer with the individual cells to wash the individual cells,separate at least a portion of the washed individual cells from the first volume via the separator, anddrain at least a portion of the first volume of the buffer;an illumination chamber containing at least one additive, the illumination chamber configured to receive at least a portion of the washed individual cells from the mixing chamber; andan illumination source configured to illuminate the portion of the washed individual cells in the illumination chamber with optical energy, the optical energy configured to inactivate the individual cells in presence of the at least one additive.

12. The apparatus of claim 11, wherein fragmenter includes at least one of a grate, a mesh filter, or a mincer.305463503Atty. Dkt. PHTN-003 / 01WO 338747-202713. The apparatus of claim 12, further comprising:a piston configured to press the tissue against the fragmenter to fragment the tissue into the plurality of tissue segments.

14. The apparatus of claim 11, wherein the filter is a first filter, and the separator includes a second filter, the second filter configured to inhibit the individual from being drained from the mixing chamber.

15. The apparatus of claim 11, wherein the separator includes a centrifuge.

16. The apparatus of claim 11, wherein the dissociation assembly includes a chamber filled with fluid configured to receive the plurality of tissue segments.

17. The apparatus of claim 16, wherein the fluid includes an enzyme configured to breakdown the plurality of tissue segments into individual cells.

18. The apparatus of claim 17, wherein the illumination chamber includes an additive disposed therein.

19. The apparatus of claim 18, wherein the additive includes a photosensitizer.

20. The apparatus of claim 18, wherein the additive is disposed in a frangible container within the illumination chamber, the frangible container configured to selectively rupture to communicate the additive into the internal volume of the illumination chamber.305463503

Images