WO2020123655A1 - Cell isolation for use in automated bioreactors - Google Patents
Cell isolation for use in automated bioreactors Download PDFInfo
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- WO2020123655A1 WO2020123655A1 PCT/US2019/065731 US2019065731W WO2020123655A1 WO 2020123655 A1 WO2020123655 A1 WO 2020123655A1 US 2019065731 W US2019065731 W US 2019065731W WO 2020123655 A1 WO2020123655 A1 WO 2020123655A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
- C12M47/04—Cell isolation or sorting
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M21/00—Bioreactors or fermenters specially adapted for specific uses
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/42—Integrated assemblies, e.g. cassettes or cartridges
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/44—Multiple separable units; Modules
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
- C12M33/14—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus with filters, sieves or membranes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
- C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/48—Automatic or computerized control
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
Definitions
- the present disclosure provides cassettes for use in automated cell engineering systems that include cell separation filters for capturing a target cell population for automated processing.
- the disclosure also provides methods of separating a target cell population, as well as automated cell engineering systems that can utilize the cassettes and carry out the methods.
- a cassette for use in an automated cell engineering system comprising a cellular sample input, a cell separation filter fluidly connected to the cellular sample input, a cell culture chamber fluidly connected to the cell separation filter, and a cellular sample output fluidly connected to the cell culture chamber.
- the cassette does not include a centrifuge following the cell separation filter.
- a cassette for use in an automated cell engineering system comprising a cellular sample input, a cell separation filter fluidly connected to the cellular sample input, the cell separation filter including a matrix which captures immune cells, a cell culture chamber for carrying out activation, transduction and/or expansion of the immune cells having a chamber volume that is configured to house the immune cells, a back flush system fluidly connected to the cell separation filter, and a cellular sample output fluidly connected to the cell culture chamber.
- the cassette does not include a centrifuge following the cell separation filter.
- a method of preparing a target cell population for automated processing comprising introducing a cellular sample containing the target cell population into a cassette of an automated cell engineering system, passing the cellular sample through a cell separation filter, capturing the target cell population from the cellular sample onto a matrix of the cell separation filter, back flushing the cell separation filter, and transferring the target cell population from the cell separation filter, so that the target cell population can undergo automated processing.
- an automated cell engineering system comprising an enclosable housing, a cassette contained within the enclosable housing, the cassette comprising a cellular sample input, a cell separation filter fluidly connected to the cellular sample input, a cell culture chamber fluidly connected to the cell separation filter, and a cellular sample output fluidly connected to the cell culture chamber, wherein the cassette does not include a centrifuge following the cell separation filter, and a user interface for receiving input from a user.
- FIG. 1 shows various steps that can be performed with a cassette of an automated cell engineering system, as described in embodiments hereof.
- FIG. 2A shows an exemplary cassette in accordance with embodiments hereof.
- FIGS. 2B and 2C show exemplary cell separation filters in accordance with embodiments hereof.
- FIGS. 3A and 3B show images of an automated cell engineering system in accordance with embodiments hereof.
- FIG. 4 shows a lab space containing exemplary cell engineering systems as described in embodiments herein.
- FIG. 5 shows a flowpath for cell separation and isolation in a cassette of an automated cell engineering system as described in embodiments herein.
- FIG. 6A shows comparison of Donor 1 cell viability (%) post leukocyte isolation via whole blood cell isolation Ficoll and cell separation filtration methods.
- FIG. 6B shows comparison of Donor 1 total cell yield post whole blood Ficoll and cell separation filtration processing.
- FIG. 7A shows total cell yield over 11 days of culture post whole blood processing via Ficoll and filtration methods.
- FIG. 7B shows average culture viability (%) of duplicate T-25 flask culture post whole blood processing via Ficoll and filtration.
- FIG. 8A shows comparison of Donor 2 cell viability (%) post leukocyte isolation via whole blood cell isolation Ficoll and filtration methods.
- FIG. 8B shows comparison of Donor 2 total cell yield post whole blood Ficoll and filtration processing.
- FIG. 9A shows comparison of Leukopak Donor cell viability (%) post leukocyte isolation via Ficoll and filtration methods.
- FIG. 9B shows comparison of Leukopak Donor total cell yield post whole blood Ficoll and filtration processing.
- FIG. 10 shows gating strategy for FACS analysis.
- FIG. 1 1 shows percentage of CD3+CD4+ and CD3+CD8+ T-cells from Donor
- FIG. 12 shows percentage of CD3+CD4+ and CD3+CD8+ T-cells from Donor
- FIG. 13 shows percentage of CD3+CD4+ and CD3+CD8+ T-cells from filtered and Ficoll isolated leukopak collection samples.
- FIG. 1 shows an exemplary cassette 102, in which various processes can be carried out in an enclosed, automated system that allows for production of various cellular samples and populations. Such processes can include activating, transducing, expanding, concentrating, and collecting/harvesting steps
- the cassettes and methods are suitably utilized and carried out in a fully enclosed automated cell engineering system 300 (see FIGS. 3A, 3B), suitably having instructions thereon for performing steps such as, activating, transducing, expanding, concentrating, and harvesting.
- a fully enclosed automated cell engineering system 300 see FIGS. 3A, 3B
- FIGS. 3A, 3B suitably having instructions thereon for performing steps such as, activating, transducing, expanding, concentrating, and harvesting.
- Cell engineering systems for automated production of, for example genetically modified immune cells, including CAR T cells are described in U.S. Patent Application No. 16/1 19,618, filed August 31 , 2018 (the disclosure of which is incorporated by reference herein in its entirety), and are also called automated cell engineering system, COCOON, or COCOON system herein.
- a user can provide a automated cell engineering system pre-filled with a cell culture and reagents (e.g., an activation reagent, a vector, cell culture media, nutrients, selection reagent, and the like) and parameters for the cell production (e.g., starting number of cells, type of media, type of activation reagent, type of vector, number of cells or doses to be produced, and the like), the automated cell engineering system is able to carry out the various automated methods, including methods of producing genetically modified immune cell cultures, including CAR T cells, without further input from the user.
- the fully enclosed automated cell engineering system minimizes contamination of the cell cultures by reducing exposure of the cell culture to non-sterile environments.
- the fully enclosed automated cell engineering system minimizes contamination of the cell cultures by reducing user handling of the cells.
- the automated cell engineering systems 300 suitably include a cassette 102.
- a cassette for use in an automated cell engineering system.
- a“cassette” refers to a largely self-contained, removable and replaceable element of a automated cell engineering system that includes one or more chambers for carrying out the various elements of the methods described herein, and suitably also includes one or more of a cell media, an activation reagent, a wash media, etc.
- FIG. 2A shows an exemplary cassette 102 for use in an automated cell engineering system.
- cassette 102 includes a cellular sample input 202.
- Cellular sample input 202 is shown in FIG. 2A as a vial or chamber in which a cellular sample can be placed prior to introduction or loading into cassette 102.
- cellular sample input 202 can simply be a sterile-locking tubing (for example a luer lock tubing connection or the like) to which a syringe or a cell-containing bag, such as a blood bag, can be connected.
- Cassette 102 further includes a cell separation filter 204, located within the cassette, and fluidly connected to cellular sample input 202.
- fluidly connected means that one or more components of a system, including cassette 102, are connected via a suitable element that allows for fluids (including gasses and liquids) to pass between the components without leaking or losing volume.
- Exemplary fluid connections include various tubing, channels and connections known in the art, such as silicone or rubber tubing, luer lock connections, etc. It should be understood that components that are fluidly connected can also include additional elements between each of the components, while still maintaining a fluid connection. That is, fluidly connected components can include additional elements, such that a fluid passing between the components can also pass through these additional elements, but is not required to do so.
- Cassette 102 suitably further includes a cell culture chamber 206 fluidly connected to the cell separation filter. Examples of the characteristics and uses of cell culture chamber 206 are described herein.
- cassette 102 further includes one or more fluidics pathways connected to the cell culture chamber (see inside cassette 102 in FIG. 2A). Also included in cassette 102 is a cellular sample output 208 fluidly connected to cell culture chamber. As described herein, cellular sample output 208 is utilized to harvest the cells following the various automated procedures for either further processing, storage, or potential use in a patient. Examples of fluidics pathways include various tubing, channels, capillaries, microfluidics elements, etc., that provide nutrients, solutions, etc., to the elements of the cassette, as described herein.
- cassette 102 explicitly excludes a centrifuge following cell separation filter 204. “Following the cell separation filter” includes embodiments where a centrifuge is not included downstream of the cell separation filter, or downstream of the back flush from the cell separation filter. It has been determined that through the use of the various cell separation filters and methods described herein, additional cellular separation via centrifugation procedures and the use of a centrifuge is not required. In embodiments, however, a further filtration system, such as a column filtration, tangential flow filtration, and/or magnetic filtration system, can be utilized.
- a further filtration system such as a column filtration, tangential flow filtration, and/or magnetic filtration system
- cell separation filter 204 includes a matrix which captures a cell population, suitably target cells.
- Suitable matrix materials include various porous media that has been treated with a gas plasma.
- the porous media can be a natural or synthetic fiber or woven material, or a sintered powder material.
- Exemplary matrix materials include those disclosed in, for example, U.S. Patent Nos. 4,701 ,267, 4,936,998, 4,880,548, 4,923,620, 4,925,572, and 5,679,264, the disclosures of each of which are incorporated by reference herein in their entireties.
- target cell population refers to a desired sub-set of cells that is to be separated from a larger cell population, including from debris or other contaminants, such that the remaining target cell population is largely free of other cell types.
- target cell populations include immune cells, cancer cells, etc.
- Exemplary cell separation filters suitably include a matrix that allows for the capture of immune cells, that is the matrix retains immune cells on or within the matrix.
- “immune cells” includes basophils, eosinophils, neutrophils, leukocytes, etc., and include cells such as mast cells, dendritic cells, naturally killer cells, B cell, T cells, etc.
- the cassettes and cell separation filters are suitably used to separate immune cells from a cellular sample, including a whole blood cell sample or a leukophoresis sample (sample in which white blood cells are separated from whole blood).
- FIGS. 2B and 2C show exemplary cell separation filters for use in the cassettes and methods described herein.
- FIG. 2B shows a leukocyte filter for salvaged blood (Flaemonetics, Braintree, MA) and
- FIG. 2C a syringe filter (PALL ARCODISC ® , PALL Laboratory, Port Washington, NY).
- cassette 102 suitably includes a waste collection chamber 510 (contained within cassette 102 in FIG. 2A), following cell separation filter 204 and fluidly connected to the separation filter.
- An exemplary location for waste collection chamber 510 within the flowpath of a cassette is shown in FIG 5.
- Waste collection chamber 510 is suitably positioned following, or downstream (i.e. , fluidly connected after the cell separation filter) so that waste that passes through cell separation filter can be held for either further processing or disposal. Waste the can be collected suitably includes undesired cells, either whole or lysed, as well as blood components, as well as potential contaminants within a cellular sample that is being filtered.
- Waste chamber 510 can be in the form of a solid chamber or a bag within cassette 102, or can be a bag or chamber external to the cassette, but connected via a fluidic path, such as tubing and a sampling port.
- cassette 102 includes a cell wash system 512 that is suitably contained within cassette 102 (i.e. , within the structure shown in FIG. 2A), and fluidly connected to separation filter 204. As shown in FIG. 5, cell wash system 512 can be connected to one of the various input ports of cassette 102, to allow for a direct fluid path to separation filter 204.
- cell wash system 512 is a container or bag contained within cassette that suitably includes a cell wash media.
- the cell wash media is suitably used to clean the target cell population and the separation filter and remove any undesired waste cells or contamination from the target cell population, prior to transferring the target cell population from the cell separation filter to another portion of the cassette.
- Cell wash system 512 can also be included outside of cassette 102. In further embodiments, cell wash system 512 can be used to wash cells held in a target cell population holding chamber.
- cassette 102 includes a back flush system 514 (not visible in FIG. 2 as it is suitably located inside cassette 102), but shown in FIG. 5 as an element of the flowpath for the cassette.
- back flush system 514 is suitably a container or bag contained within the cassette and can be connected to one or of the various input ports of cassette 102, to allow a direct fluid path to separation filter 204.
- Back flush system 514 can also be included external to the cassette.
- Back flush system 514 is suitably fluidly connected to separation filter 204 in such a way that a back flush media contained within the back flush system can be introduced into or onto cell separation filter 204 in a reverse manner to transfer cells captured by the separation filter from the filter to another section of the cassette, including a holding chamber or a cell culture chamber, as described herein.
- Cassette 102 can also further optionally include a target cell population holding chamber 516 (not visible in FIG. 2 as it is located inside cassette 102) located between the cell separation filter and the cell culture chamber.
- FIG. 5 shows an exemplary location of target cell population holding chamber 516 in the flowpath for the cassette.
- Target cell population holding chamber 516 is suitably a reservoir or suitable chamber located within the cassette into which a target cell population that has been captured on the separation filter 204, and then back flushed via back flush system 514 to transfer the captured cells to target cell population holding chamber 516.
- the fluidics pathways which can include various tubing elements, suitably provide recirculation, removal of waste and homogenous gas exchange and distribution of nutrients to various parts of the cassette, including the cell culture chamber without disturbing cells within the cell culture chamber.
- Cassette 102 also further includes one or more pumps 520 and related tubing, including peristaltic pumps, for driving fluid through the cassette, as described herein, as well as one or more valves 522, for controlling the flow through the various fluidic pathways ( see FIG. 5 for exemplary locations within flowpath).
- cell culture chamber 206 is flat and non-flexible chamber (i.e. , made of a substantially non-flexible material such as a plastic) that does not readily bend or flex.
- the use of a non-flexible chamber allows the cells to be maintained in a substantially undisturbed state.
- cell culture chamber 206 is oriented so as to allow the immune cell culture to spread across the bottom of the cell culture chamber.
- cell culture chamber 206 is suitably maintained in a position that is parallel with the floor or table, maintaining the cell culture in an undisturbed state, allowing the cell culture to spread across a large area of the bottom of the cell culture chamber.
- the overall thickness of cell culture chamber 206 is low, on the order of about 0.5 cm to about 5 cm.
- the cell culture chamber has a volume of between about 0.50 ml and about 300 ml, more suitably between about 50 ml and about 200 ml, or the cell culture chamber has a volume of about 180 ml.
- the use of a low chamber height (less than 5 cm, suitably less than 4 cm, less than 3 cm, or less then 2 cm) allows for effective media and gas exchange in close proximity to the cells. Ports are configured to allow mixing via recirculation of the fluid without disturbing the cells. Larger height static vessels can produce concentration gradients, causing the area near the cells to be limited in oxygen and fresh nutrients. Through controlled flow dynamics, media exchanges can be performed without cell disturbance. Media can be removed from the additional chambers (no cells present) without risk of cell loss.
- cell culture chamber 206 is a bag or hard chamber.
- the cassette is pre-filled with one or more of a cell culture, a culture media, a cell wash media, a back flush media, an activation reagent, and/or a vector, including any combination of these.
- these various elements can be added later via suitable injection ports, etc.
- the back flush media suitably contains an anticoagulant, such as ethylenediaminetetraacetic acid (EDTA), to reduce clumping of the target cell population that is transferred from the separation filter.
- EDTA ethylenediaminetetraacetic acid
- the cassettes suitably further include one or more of a pH sensor 524, a glucose sensor (not shown), an oxygen sensor 526, a carbon dioxide sensor (not shown), a lactic acid sensor/monitor (not shown), and/or an optical density sensor (not shown). See FIG. 5 for exemplary positions within the flowpath.
- the cassettes can also include one or more sampling ports and/or injection ports. Examples of such sampling ports 220 and injection ports (222) are illustrated in FIG. 2A, and exemplary locations in the flowpath shown in FIG. 5, and can include an access port for connecting the cartridge to an external device, such as an electroporation unit or an additional media source.
- FIG. 2A also shows the location of the cellular sample input 202, reagent warming bag 224 which can be used to warm cell media, etc., and secondary chamber 230.
- cassette 102 suitably includes a low temperature chamber, which can include a refrigeration area 226 suitably for storage of a cell culture media, as well as a high temperature chamber, suitably for carrying out activation, transduction, transfection and/or expansion of a cell culture.
- the high temperature chamber is separated from the low temperature chamber by a thermal barrier.
- low temperature chamber refers to a chamber, suitably maintained below room temperature, and more suitably from about 4°C to about 8°C, for maintenance of cell media, etc., at a refrigerated temperature.
- the low temperature chamber can include a bag or other holder for media, including about 1 L, about 2L, about 3L, about 4L, or about 5L of fluid. Additional media bags or other fluid sources can be connected externally to the cassette, and connected to the cassette via an access port.
- high temperature chamber refers to chamber, suitably maintained above room temperature, and more suitably maintained at a temperature to allow for cell proliferation and growth, i.e. , between about 35-40°C, and more suitably about 37°C.
- high temperature chamber suitably includes cell culture chamber 206 (also called proliferation chamber or cell proliferation chamber throughout).
- FIGA. 3A-3B show the COCOON automated cell engineering system 300 with cassette 102 positioned inside (cover of automated cell engineering system opened in FIG. 3B). Also shown is an exemplary user interface, which can include a bar code reader, and the ability to receive using inputs by touch pad or other similar device.
- the automated cell engineering systems and cassettes described herein suitably have three relevant volumes, the cell culture chamber volume, the working volume, and the total volume.
- the working volume used in the cassette ranges from 180 mL to 460 ml_ based on the process step, and can be increased up to about 500 mL, about 600 mL, about 700 mL, about 800 mL, about 900 mL or about 1 L.
- the cassette can readily achieve 4*10 9 cells - 10*10 9 cells.
- the cell concentration during the process varies from 0.3*10 6 cells/ml to approximately 10*10 6 cells/ml.
- the cells are located in the cell culture chamber, but media is continuously recirculated through additional chambers (e.g., crossflow reservoir and satellite volume) to increase the working volume, as described herein.
- Fluidics pathways may be made from a gas- permeable material such as, e.g., silicone.
- the automated cell engineering system recirculates oxygen throughout the substantially non-yielding chamber during the cell production methods.
- the oxygen level of a cell culture in the automated cell engineering system is higher than the oxygen level of a cell culture in a flexible, gas-permeable bag. Higher oxygen levels may be important in the cell culture expansion step, as increased oxygen levels may support increased cell growth and proliferation.
- the methods and cartridges described herein are utilized the COCOON platform (Octane Biotech (Kingston, ON)), which integrates multiple unit operations in a single turnkey platform.
- Multiple cell protocols are provided with very specific cell processing objectives.
- the methods described utilize the concept of application-specific/sponsor- specific disposable cassettes that combine multiple unit operations - all focused on the core requirements of the final cell therapy product.
- Multiple automated cell engineering systems 300 can be integrated together into a large, multi-unit operation for production of large volumes of cells or multiple different cellular samples for individual patients ( see FIG. 4).
- cassette 102 for use in an automated cell engineering system 300.
- the cassette includes cellular sample input 202, cell separation filter 204 fluidly connected to the cellular sample input, the cell separation filter including a matrix which captures immune cells.
- Cassette 102 further includes cell culture chamber 206 for carrying out activation, transduction, transfection and/or expansion of the immune cells having a chamber volume that is configured to house the immune cells.
- Cassette 102 also suitably further includes back flush system 514 fluidly connected to the separation filter, and cellular sample output 208 fluidly connected to the cell culture chamber for harvesting the cells.
- the cassette does not include a centrifuge following the cell separation filter (or before the cell separation filter).
- the cassette can further include cell wash system 512 fluidly connected to the separation filter.
- the cassette can further include one or more fluidics pathways connected to the cell culture chamber, wherein the fluidics pathways provide recirculation, removal of waste and homogenous gas exchange and distribution of nutrients to the cell culture chamber suitably without disturbing immune cells within the cell culture chamber.
- the fluidic pathways comprise a silicon-based tubing component that allows oxygenation through the tubing component.
- the cassette also further includes waste collection chamber 510, suitably following separation filter 204.
- the cassette can include immune cell holding chamber 516, suitably located between the cell separation filter and the cell culture chamber.
- cell culture chamber 206 is flat and non- flexible chamber, having a low chamber height.
- the cassette is pre-filled with culture media, cell wash media, and back flush media, as described herein.
- a method of preparing a target cell population for automated processing As described herein, the methods suitably allow for the introduction of a sample of cells, including a whole blood sample, and then separating out a desired or target cell population from this cell sample for further processing, suitably further automated processing in a automated cell engineering systems, such as those described herein.
- a cellular sample that contains a target cell population is introduced into cassette 102 of automated cell engineering system 300.
- exemplary cellular samples include blood samples (including whole blood), tissue samples, bodily fluid samples, etc.
- a cellular sample is suitably introduced at cellular sample input 202.
- a cellular sample can be introduced for example, from a syringe, container, vial, blood bag, etc.
- the cellular sample passes through control valve (522) V3, and through the fluidic pathways (labeled generically as 540), while being driven by pump 520.
- cell separation filter 204 suitably includes a matrix for capturing the a desired cell population, including a target cell population from the cellular sample.
- a back flushing occurs, during which cell separation filter 204 is back flushed, suitably from back flush system 512.
- a back flush media is contained in back flush system 512, passed through valve V4, and driven via pump 520 through valve V12 and valve V1 , to back flush the cell separation filter.
- This back flushing transfers the target cell population that was captured on the matrix of the cell separation filter, so that the target cell population can be removed from the filter and undergo further processing, including further automated processing.
- the back flushing occurs using a back flush media containing an anticoagulant, so as to limit the coagulation of the target cell population as the cells undergo further automated processing procedures.
- the target cell population that is removed from the matrix of the cell separation filter can be transferred to a target cell population holding chamber 516, for example by passing through valve V1 1 .
- the target cell population that is removed from the matrix of the cell separation filter can be transferred to a transduction system (not shown), a transfection system (i.e. , a non-viral method), suitably through a sample port (e.g., R5 or R6), following passing through valves V1 1 and V9.
- a transduction system not shown
- a transfection system i.e. , a non-viral method
- a sample port e.g., R5 or R6
- Exemplary transduction systems are known in the art and exemplary transfection systems include electroporation systems, etc., and can be included within cassette 102 or can be external to cassette 102.
- the target cell population that is removed from the matrix of the cell separation filter can be transferred to cell culture chamber 206, for example, by passing through valve V1 1 and then valves V5 or V6.
- these various elements following the cell separation filter allow for the target cell population to undergo further automated processing, including transduction, transfection, growth, expansion, etc.
- the methods can further include washing the captured target cell population on the cell separation filter prior to the back flushing.
- cell wash system 512 which can be a bag contained within cassette 102 and include a cell wash media, can pass the cell wash media, via pump 520, through valve V4 and V1 1 to wash the captured target cell population on cell separation filter 204.
- the target cell population remains on the matrix of the cell separation filter, while additional unwanted waste is passed from the cell separation filter into waste collection chamber 510, via valves V1 and V13.
- unwanted waste from cellular sample can also pass through the cell separation filter and into waste collection chamber 510 via valves V1 and V13.
- additional embodiments allow for the further filtration of a cellular sample by re-passing the waste from the cellular sample back through the cell separation filter, for example by passing through valves V1 , V12 and V1 1 , to complete another filtration cycle.
- Cell washing can also occur via cell wash system 512 by transferring a cell wash media to target cell holding chamber 516, and wash the cells that are being held in the chamber prior to further processing.
- passing the cellular sample through cell separation filter 204 suitable occurs via gravity filtration. That is, no pumping mechanism is used to drive the cellular sample through the cell separation filter.
- pump 520 can be used to generate a positive or negative pressure on the cellular sample, so as to drive the sample through the cell separation filter.
- a syringe or other mechanism can also be used to provide additional positive or negative pressure if desired, to pass the cellular sample through the cell separation filter.
- the target cell population is suitably collected. This collection can occur via sample output 208, or via one of the various sample ports 220.
- the cassettes and methods described herein suitably exclude a centrifuge, and the use of centrifugation.
- the methods exclude centrifugation following the transferring the target cell population from the cell separation filter, whether that transfer occur directly following capture via the cell separation filter or via a back flush from the cell separation filter. It has been determined that by excluding centrifugation, a target cell population can be separated from a cellular sample via simple filtration, without the need for harsh centrifugation conditions. This includes removing a target cell population from a sample of whole blood.
- a magnetic separation process can be utilized to further eliminate and separate undesired cells and debris from a target cell population.
- a magnetic bead or other structure to which a biomolecule (e.g., antibody, antibody fragment, etc.) has been bound, can interact with a target cell.
- Various magnetic separation methods including the use of filters, columns, flow tubes or channels with magnetic fields, etc., can then be used to separate the target cell population from undesired cells, debris, etc., that may be in a cellular sample.
- a target cell population can flow through a tube or other structure and exposed to a magnetic filed, whereby the target cell population is retained or held-up by the magnetic field, allowing undesired cells and debris to pass through the tube.
- the magnetic field can then be turned off, allowing the target cell population to pass onto a further retention chamber or other area(s) of the cassette for further automated processing.
- the flowpath in FIG. 5 also shows the connection between cell culture chamber 206, and a satellite volume 550, which can be provide additional storage capabilities for the cassette, or to increase the overall volume of the automated processes.
- a satellite volume 550 which can be provide additional storage capabilities for the cassette, or to increase the overall volume of the automated processes.
- various sensors e.g., pH sensor 524, dissolved oxygen sensor 526
- sampling/sample ports and various valves including bypass check valves 552
- one or more fluidic pathways 540 suitably comprising a silicone-based tubing component, connecting the components.
- a silicone-based tubing component allows oxygenation through the tubing component to facilitate gas transfer and optimal oxygenation for the cell culture.
- hydrophobic filters 554 or hydrophilic filters 556 are also show in FIG.
- automated cell engineering system 300 suitably includes an enclosable housing 302, and cassette 102, contained within the enclosable housing.
- “enclosable housing” refers to a structure than can be opened and closed, and within which cassette 102 as described herein, can be placed and integrated with various components such as fluid supply lines, gas supply lines, power, cooling connections, heating connections, etc.
- enclosable housing can be opened (FIG. 3B) to allow insertion of the cassette, and closed (FIG. 3A) to maintain a closed, sealed environment to allow the various automated processes described herein to take place utilizing the cassette.
- cassette 102 suitably includes cellular sample input 206, cell separation filter 204 fluidly connected to the cellular sample input, cell culture chamber 206 fluidly connected to the cell separation filter, and cellular sample output 208 fluidly connected to the cell culture chamber.
- the cassette (as well as the automated cell engineering system) does not include a centrifuge following the cell separation filter, or suitably in any configuration.
- automated cell engineering system 300 also further includes a user interface 304 for receiving input from a user.
- User interface 304 can be a touch pad, tablet, keyboard, computer terminal, or other suitable interface, that allows a user to input desired controls and criteria to the automated cell engineering system to control the automated processes and flowpath.
- the user interface is coupled to a computer control system to provide instructions to the automated cell engineering system, and to control the overall activities of the automated cell engineering system.
- Such instructions can include when to open and close various valves, when to provide media or cell populations, when to increase or decrease a temperature, etc.
- the cell separation filter includes a matrix which captures a target cell population.
- the matrix captures immune cells.
- the cassette in the automated cell engineering systems further comprises a waste collection chamber following the separation filter.
- a cell wash system fluidly connected to the separation filter can also be included, as described herein.
- a back flush system fluidly connected to the separation filter can also be included, as well as optionally a target cell population holding chamber located between the cell separation filter and the cell culture chamber.
- the cassettes of the automated cell engineering systems further include one or more fluidics pathways, wherein the fluidics pathways provide recirculation, removal of waste and homogenous gas exchange and distribution of nutrients to the cell culture chamber without disturbing cells within the cell culture chamber.
- the cell culture chamber is flat and non-flexible chamber, having a low chamber height.
- the cassette is pre filled with culture media, cell wash media, and back flush media (suitably including an anticoagulant).
- the cassette of the automated cell engineering system can further include one or more of a pH sensor, a glucose sensor, an oxygen sensor, a carbon dioxide sensor, and/or an optical density sensor, and in suitable embodiments, one or more sampling ports.
- the automated cell engineering systems described herein comprise a plurality of chambers, and wherein each of steps of the various method described herein are performed in a different chamber of the plurality of chambers of the automated cell engineering system, each of the activation reagent, the vector, and cell culture medium are contained in a different chamber of the plurality of the chambers prior to starting the method, and wherein at least one of the plurality of chambers is maintained at a temperature for growing cells (e.g., at about 37°C) and at least one of the plurality of chambers is maintained at a refrigerated temperature (e.g., at about 4-8°C).
- a temperature for growing cells e.g., at about 37°C
- a refrigerated temperature e.g., at about 4-8°C
- the automated cell engineering systems described herein are monitored with a temperature sensor, a pH sensor, a glucose sensor, an oxygen sensor, a carbon dioxide sensor, and/or an optical density sensor.
- the automated cell engineering system includes one or more of a temperature sensor, a pH sensor, a glucose sensor, an oxygen sensor, a carbon dioxide sensor, and/or an optical density sensor.
- the automated cell engineering system is configured to adjust the temperature, pH, glucose, oxygen level, carbon dioxide level, and/or optical density of the cell culture, based on the pre-defined culture size.
- the automated cell engineering system will automatically increase the oxygen level of the cell culture by, e.g., introducing oxygenated cell culture media, by replacing the cell culture media with oxygenated cell culture media, or by flowing the cell culture media through an oxygenation component (i.e. , a silicone tubing).
- an oxygenation component i.e. , a silicone tubing
- the automated cell engineering system detects that the current temperature of the cell culture is too high and that the cells are growing too rapidly (e.g., possible overcrowding of the cells may lead to undesirable characteristics)
- the automated cell engineering system will automatically decrease the temperature of the cell culture to maintain a steady growth rate (or exponential growth rate, as desired) of the cells.
- the automated cell engineering system automatically adjusts the schedule of cell feeding (i.e., providing fresh media and/or nutrients to the cell culture) based on the cell growth rate and/or cell count, or other monitored factors, such as pH, oxygen, glucose, etc.
- the automated cell engineering system may be configured to store media (and other reagents, such as wash solutions, etc.) in a low-temperature chamber (e.g., 4°C or -20°C), and to warm the media in a room temperature chamber or a high-temperature chamber (e.g., 25°C or 37°C, respectively) before introducing the warmed media to the cell culture.
- a low-temperature chamber e.g., 4°C or -20°C
- a high-temperature chamber e.g. 25°C or 37°C, respectively
- Embodiment 1 is a cassette for use in an automated cell engineering system, comprising a cellular sample input, a cell separation filter fluidly connected to the cellular sample input, a cell culture chamber fluidly connected to the cell separation filter, and a cellular sample output fluidly connected to the cell culture chamber, wherein the cassette does not include a centrifuge following the cell separation filter.
- Embodiment 2 includes the cassette of embodiment 1 , wherein the cell separation filter includes a matrix which captures a cell population.
- Embodiment 3 includes the cassette of embodiment 1 , wherein the matrix captures target cells.
- Embodiment 4 includes the cassette of embodiments 1 -3, further comprising a waste collection chamber following the cell separation filter.
- Embodiment 5 includes the cassette of embodiments 1 -4, further comprising a cell wash system fluidly connected to the cell separation filter.
- Embodiment 6 includes the cassette of embodiments 1 -5, further comprising a back flush system fluidly connected to the cell separation filter, and optionally a target cell population holding chamber located between the cell separation filter and the cell culture chamber.
- Embodiment 7 includes the cassette of embodiments 1 -6, further comprising one or more fluidics pathways, wherein the fluidics pathways provide recirculation, removal of waste and homogenous gas exchange and distribution of nutrients to the cell culture chamber without disturbing cells within the cell culture chamber.
- Embodiment 8 includes the cassette of embodiments 1 -7, wherein the cell culture chamber is a flat and non-flexible chamber, having a low chamber height.
- Embodiment 9 includes the cassette of embodiments 1 -8, wherein the cassette is pre-filled with culture media, cell wash media, and back flush media.
- Embodiment 10 includes the cassette of embodiment 9, wherein the back flush media contains an anticoagulant.
- Embodiment 1 1 includes the cassette of embodiments 1 -10, further comprising one or more of a pH sensor, a glucose sensor, an oxygen sensor, a carbon dioxide sensor, and/or an optical density sensor.
- Embodiment 12 includes the cassette of embodiments 1 -1 1 , further comprising one or more sampling ports.
- Embodiment 13 is a cassette for use in an automated cell engineering system, comprising a cellular sample input, a cell separation filter fluidly connected to the cellular sample input, the cell separation filter including a matrix which captures immune cells, a cell culture chamber for carrying out activation, transduction and/or expansion of the immune cells having a chamber volume that is configured to house the immune cells, a back flush system fluidly connected to the cell separation filter, and a cellular sample output fluidly connected to the cell culture chamber, wherein the cassette does not include a centrifuge following the cell separation filter.
- Embodiment 14 includes the cassette of embodiment 13, further comprising a cell wash system fluidly connected to the cell separation filter.
- Embodiment 15 includes the cassette of embodiments 13-14, further comprising one or more fluidics pathways connected to the cell culture chamber, wherein the fluidics pathways provide recirculation, removal of waste and homogenous gas exchange and distribution of nutrients to the cell culture chamber without disturbing immune cells within the cell culture chamber.
- Embodiment 16 includes the cassette of embodiments 13-15, further comprising a waste collection chamber following the cell separation filter.
- Embodiment 17 includes the cassette of embodiments 13-16, further comprising an immune cell holding chamber located between the cell separation filter and the cell culture chamber.
- Embodiment 18 includes the cassette of embodiments 13-17, wherein the cell culture chamber is flat and non-flexible chamber, having a low chamber height.
- Embodiment 19 includes the cassette of embodiments 13-18, wherein the cassette is pre-filled with culture media, cell wash media, and back flush media.
- Embodiment 20 includes the cassette of embodiments 13-19, wherein one or more of the fluidic pathways comprise a silicon-based tubing component that allows oxygenation through the tubing component.
- Embodiment 21 is a method of preparing a target cell population for automated processing, the method comprising introducing a cellular sample containing the target cell population into a cassette of an automated cell engineering system, passing the cellular sample through a cell separation filter, capturing the target cell population from the cellular sample onto a matrix of the cell separation filter, back flushing the cell separation filter; and transferring the target cell population from the cell separation filter, so that the target cell population can undergo automated processing.
- Embodiment 22 includes the method of embodiment 21 , wherein the transferring comprises transferring the target cell population to a target cell population holding chamber, a transduction system, a system for transfection, or a cell culture chamber, so that the target cell population can undergo automated processing.
- Embodiment 23 includes the method of embodiment 22, wherein the transduction system is an electroporation system.
- Embodiment 24 includes the method of embodiments 21 -23, further comprising washing the captured target cell population on the cell separation filter prior to the back flushing.
- Embodiment 25 includes the method of embodiments 21 -24, further comprising passing unwanted waste from the cellular sample through the cell separation filter and into a waste collection chamber.
- Embodiment 26 includes the method of embodiments 21 -25, wherein the passing the cellular sample through the cell separation filter occurs via gravity filtration.
- Embodiment 27 includes the method of embodiments 21 -26, wherein the method excludes centrifugation following the transferring the target cell population from the cell separation filter.
- Embodiment 28 includes the method of embodiments 21 -26, further comprising collecting the target cell population from the cassette following the automated processing.
- Embodiment 29 is an automated cell engineering system, comprising an enclosable housing, a cassette contained within the enclosable housing, the cassette comprising a cellular sample input, a cell separation filter fluidly connected to the cellular sample input, a cell culture chamber fluidly connected to the cell separation filter, and a cellular sample output fluidly connected to the cell culture chamber, wherein the cassette does not include a centrifuge following the cell separation filter, and a user interface for receiving input from a user.
- Embodiment 30 includes the automated cell engineering system of embodiment 29, wherein the cell separation filter of the cassette includes a matrix which captures a cell population.
- Embodiment 31 includes the automated cell engineering system of embodiment 30, wherein the matrix captures target cells.
- Embodiment 32 includes the automated cell engineering system of embodiments 29-31 , wherein the cassette further comprises a waste collection chamber following the cell separation filter.
- Embodiment 33 includes the automated cell engineering system of embodiments 29-32, wherein the cassette further comprises a cell wash system fluidly connected to the cell separation filter.
- Embodiment 34 includes the automated cell engineering system of embodiments 29-33, wherein the cassette further comprises a back flush system fluidly connected to the cell separation filter, and optionally a target cell population holding chamber located between the cell separation filter and the cell culture chamber.
- Embodiment 35 includes the automated cell engineering system of embodiments 29-34, wherein the cassette further comprises one or more fluidics pathways, wherein the fluidics pathways provide recirculation, removal of waste and homogenous gas exchange and distribution of nutrients to the cell culture chamber without disturbing cells within the cell culture chamber.
- Embodiment 36 includes the automated cell engineering system of embodiments 29-35, wherein the cell culture chamber of the cassette is flat and non- flexible chamber, having a low chamber height.
- Embodiment 37 includes the automated cell engineering system of embodiments 29-35, wherein the cell culture chamber of the cassette is a bag or hard chamber.
- Embodiment 38 includes the automated cell engineering system of embodiments 29-37, wherein the cassette is pre-filled with culture media, cell wash media, and back flush media.
- Embodiment 39 includes the automated cell engineering system of embodiment 38, wherein the back flush media contains an anticoagulant.
- Embodiment 40 includes the automated cell engineering system of embodiments 29-39, wherein the cassette further comprises one or more of a pH sensor, a glucose sensor, an oxygen sensor, a carbon dioxide sensor, and/or an optical density sensor.
- Embodiment 41 includes the automated cell engineering system of embodiments 29-40, wherein the cassette further comprises one or more sampling ports.
- Embodiment 42 includes the automated cell engineering system of embodiments 29-41 , further comprising a computer control system, wherein the user interface is coupled to the computer control system to provide instructions to the automated cell engineering system.
- the Octane CocoonTM system is a closed, automated, end-to-end cell engineering system for the manufacture of cell therapy products.
- the CocoonTM is comprised of three main components: the base instrument, software, and customizable disposable cassette.
- the system is capable of automated cell isolation, expansion, concentration, and buffer exchange for both upstream and downstream cell culture processes; however, it does not have centrifugation functionality.
- Isolation of target cell populations by adherence can be applied to most adherent cell types including mesenchymal stem cells (MSCs), dendritic cells, and monocytes.
- MSCs mesenchymal stem cells
- dendritic cells dendritic cells
- monocytes monocytes.
- human bone marrow MSCs can be isolated by adherence in the CocoonTM cassette proliferation chamber. 1 - 2 days post-inoculation of the bone marrow tissue, contaminating red blood cells (RBCs) and other suspension cells are drained to waste, leaving behind adherent cell types in the CocoonTM cassette proliferation chamber. Media exchanges occurred every 2 - 3 days with media designed to promote MSC expansion.
- RBCs red blood cells
- T-cells in the CocoonTM cassette requires a purified population of either T-cells or peripheral blood mononuclear cells (PBMCs), typically from a whole blood collection from a donor.
- PBMCs peripheral blood mononuclear cells
- WBC White Blood Cell
- RS-1 Haemonetics Leukocyte Filter for Salvaged Blood
- the Acrodisc WBC Syringe Filters can process up to 12mL of donor whole blood or leukopheresis sample, while the Leukocyte Filter (Haemonetics) can process up to 450mL of donor whole blood or leukopheresis sample.
- human T-cells can be isolated from whole blood or Leukopak donor samples for CAR-T and other cell therapy products within the CocoonTM system.
- the proposed process flow path described herein allows end users to introduce donor whole blood or leukopheresis samples sterilely and directly into the CocoonTM system.
- the whole blood filter can be integrated within the CocoonTM disposable cassette fluidic pathway to separate leukocytes from the mixed cell population and further expand them in the CocoonTM proliferation chamber.
- the final therapeutic product can then be automatically harvested and used fresh or cryopreserved, as required.
- the initial donor sample was then divided into 2 collections: the first for processing via Ficoll density gradient and the second via a cell separation filter. For density gradient isolation, half of the initial donor sample was processed using standard procedures for the manufacturing of human PBMCs. Specifically, the donor sample was diluted 1 : 1 in an equal volume of 2mM EDTA/1X DPBS (Lonza). The diluted sample was then carefully layered in 30mL fractions onto 15mL of Ficoll Plaque Plus density gradient solution (GE Healthcare) for a total volume of up to 45mL per 50mL conical tube. The tubes were then centrifuge at 400 x g for 40min at room temperature.
- Ficoll Plaque Plus density gradient solution GE Healthcare
- the top layer of plasma was removed to approximately 10mL above the buffy coat layer of the tube which contained the PBMCs.
- the PBMCs were collected and washed in 2mM EDTA/1X DPBS at three times the collection volume. The collected cells were then counted in duplicate using the Nucleocounter NC-200 (Chemometec), analyzed via flow cytometry (FACS) analysis, and cryopreserved.
- the WBC filter was carefully removed from the syringe housing, a clean 150ml_ blood collection bag (WalkMed) attached to the inlet side of the filter, and a media bag filled with 10mL of PBS (Lonza) was attached to the outlet of the WBC filter. The filter was then back flushed with the PBS and collected in the 150ml_ blood collection bag (WalkMed). The collected cell suspension was then washed in 2mM EDTA/1X DPBS at three times the collection volume. Cells were then counted in duplicate using the Nucleocounter NC-200 (Chemometec) and samples cryopreserved for flow cytometry (FACS) analysis.
- FACS flow cytometry
- Donor 1 148mL of whole blood from a single donor was divided into 2 fractions. One 74mL fraction was diluted 1 : 1 in 0.2mM EDTA/1X DPBS (total 148mL diluted whole blood) and the second 74mL fraction was left undiluted. Both undiluted and diluted fractions were then split into two additional fractions at 2 x 74mL of diluted whole blood and 2 x 37mL of undiluted whole blood to use in both Ficoll separation gradient processing and Pall Acrodisc WBC cell separation filtration processing. The use of undiluted whole blood for Ficoll separation is not a standard laboratory practice and was only included in this evaluation to better understand process limitations.
- Pall Acrodisc WBC filtration sample volumes were 3mL undiluted, 6mL diluted and undiluted, 12mL diluted and undiluted, and 24mL diluted.
- Donor 2 279m L of whole blood from a second donor was divided by leaving 133ml_ of whole blood undiluted and diluting the second 145ml_ whole blood fraction 1 : 1 in 0.2mM EDTA/1X DPBS for a total volume of 290ml_ diluted whole blood. 54ml_ of the diluted whole blood was processed in triplicate by Ficoll. There were no undiluted samples processed via Ficoll for this donor. The remaining diluted and undiluted whole blood fractions were processed via Pall Acrodisc WBC filtration in 6ml_ and 12ml_ volumes.
- Donor 1 127ml_ of leukopheresis product from a single donor was divided into 28ml_ for unwashed sample filtration and the remaining 99ml_ washed by diluting the 99ml_ in 400ml_ 5mM EDTA-FIBSS, centrifuging, discarding the supernatant, and resuspending the cell pellet in 200ml_ of 5mM EDTA-FIBSS. 180ml_ of this washed sample was utilized for Ficoll separation density gradient and 20ml_ for 6ml_ and 12ml_ Pall Acrodisc WBC Filtration processing.
- total processing time for 6mL - 24mL of whole blood samples ranged between 5 minutes to 20 minutes depending on volume processed and whole blood dilution (Table 1 ).
- 6mL of undiluted whole blood required 10 minutes ⁇ 2 minutes to pass through the filter and 19 minutes ⁇ 2 minutes total processing time (filtration, two washes, and back flush collection).
- Whole Blood Donor 1 Two Donor 1 whole blood samples were omitted from data analysis shown in Table 3, but are shown in FIGS. 6A and 6B. Of the remaining samples, an average of 1x10 6 viable cells per mL of processed whole blood was collected via the Pall Acrodisc WBC filter compared to 0.9 x 10 6 cells per mL of whole blood processed via Ficoll density gradient separation. 27.3% less viable cells per mL of whole blood was obtained from undiluted Pall Acrodisc WBC filter samples compared to diluted WBC filtered samples. 8% more viable cells per mL of processed whole blood was obtained from undiluted Ficoll samples when compared to diluted Ficoll samples.
- FIGS. 6A and 6B show the difference in cell yield and viability.
- Leukopak Donor 1 On average, 32.9 x 10 6 viable cells per mL of Leukopheresis product processed was obtained via Ficoll separation gradient methods at 98.1 % ⁇ 0.6% viability. In comparison, 8.02 x 10 6 viable cells per mL of processed washed (1 :1 diluted) leukopheresis product and 4.36 x 10 6 viable cells of unwashed product was obtained via filtration methods at 95.8% ⁇ 0.3% viability and 98.4% ⁇ 0.6% viability, respectively (Table 5, FIG. 9A, and FIG. 9B).
- Whole Blood Donor 2 The collected fraction from the undiluted 6ml_ whole blood samples presented the lowest percentage of CD3+CD4+ T-cells (23.6% and 22%) and CD3+CD8+ T-cells (8%, and 7.5%) when compared to all other conditions with approximated 30% - 38% CD3+CD4+ T-cells and 9% - 12% CD3+CD8+ T-cells (FIG. 12). Flowever, all samples showed a 3: 1 ratio of CD3+CD4+ cells to CD3+CD8+ cells. Differences in CD4+ to CD8+ ratios between the two whole blood donors is likely a result of donor to donor variability
- Leukopak Donor Sample On average, cell fractions from whole blood processed via Ficoll isolation methods contained 40% ⁇ 2% CD3+CD4+ T-cells and 22.8% ⁇ 3% CD3+CD8+ T-cells (FIG. 13). This was approximately 15% more CD3+CD4+ T-cells and 5% more CD3+CD8+ T-cells collected than the unwashed filtered Leukopak samples. Ficoll isolation also yielded 21 % more CD3+CD4+ T-cells and 13% more CD3+CD8+ T-cells than washed (1 : 1 diluted) Pall Acrodisc WBC filtered Leukopak samples.
- the Pall Acrodisc WBC filter makes the capture and expansion of T-cells without centrifugation possible. Larger filters with increased whole blood and leukopheresis product process capabilities are also useful. In particular, the Whole Blood Filtration using Leukocyte Filter for Salvaged Blood by Haemonetics.
- CocoonTM in-line leukocyte isolation from whole blood can be carried out when using specialized filters with leukocyte capture media/matrixes. Suitable Pall or Haemonetics custom filters can be produced for use in the CocoonTM system.
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US11149244B2 (en) | 2018-04-04 | 2021-10-19 | Southwest Research Institute | Three-dimensional bioreactor for T-cell activation and expansion for immunotherapy |
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Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5459069A (en) * | 1989-06-15 | 1995-10-17 | The Regents Of The University Of Michigan | Device for maintaining and growing human stem and/or hematopoietics cells |
US5688687A (en) * | 1995-06-07 | 1997-11-18 | Aastrom Biosciences, Inc. | Bioreactor for mammalian cell growth and maintenance |
US5989913A (en) * | 1998-07-02 | 1999-11-23 | Charles Daniel Anderson | Culture vessel for growing or culturing cells, cellular aggregates, tissues and organoids and methods for using the same |
US20070202536A1 (en) * | 2001-10-11 | 2007-08-30 | Yamanishi Douglas T | Methods and compositions for separating rare cells from fluid samples |
US20160251628A1 (en) * | 2013-10-30 | 2016-09-01 | Exmoor Pharma Concepts Ltd | Apparatus And Method For Filtration Of A Suspension |
US20170096627A1 (en) * | 2004-05-26 | 2017-04-06 | Octane Biotech, Inc. | Advanced tissue engineering system |
WO2017109071A1 (en) * | 2015-12-22 | 2017-06-29 | Ge Healthcare Bio-Sciences Corp. | Improvements in and relating to cell harvesting apparatus |
Family Cites Families (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA2159005C (en) * | 1993-03-23 | 2007-06-19 | Yves Claude Nicolau | Method and apparatus for encapsulation of biologically-active substances in cells |
US20020146817A1 (en) * | 2000-10-02 | 2002-10-10 | Cannon Thomas F. | Automated bioculture and bioculture experiments system |
EP1495108A2 (en) * | 2002-04-08 | 2005-01-12 | Millenium Biologix Inc. | Automated tissue engineering system |
US20130143195A1 (en) * | 2011-12-05 | 2013-06-06 | Pall Corporation | Leukocyte purification |
EP3227003A1 (en) * | 2014-12-03 | 2017-10-11 | Bend Research, Inc. | Disposable cell removal system |
GB201617713D0 (en) * | 2016-10-19 | 2016-11-30 | Q-Linea Ab | Method for recovering microbial cells |
EP3645719B1 (en) * | 2017-06-30 | 2022-03-09 | Inscripta, Inc. | Automated cell processing methods, modules, instruments, and systems |
-
2019
- 2019-12-11 JP JP2021533208A patent/JP2022511935A/en active Pending
- 2019-12-11 WO PCT/US2019/065731 patent/WO2020123655A1/en unknown
- 2019-12-11 CA CA3122288A patent/CA3122288A1/en active Pending
- 2019-12-11 SG SG11202106030QA patent/SG11202106030QA/en unknown
- 2019-12-11 EP EP19894452.2A patent/EP3894540A4/en active Pending
- 2019-12-11 CN CN201980089612.3A patent/CN113330106A/en active Pending
- 2019-12-11 KR KR1020217020939A patent/KR20210102928A/en unknown
- 2019-12-11 US US16/710,952 patent/US20200181562A1/en not_active Abandoned
-
2021
- 2021-06-08 IL IL283819A patent/IL283819A/en unknown
-
2022
- 2022-04-28 US US17/732,041 patent/US20220267712A1/en not_active Abandoned
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5459069A (en) * | 1989-06-15 | 1995-10-17 | The Regents Of The University Of Michigan | Device for maintaining and growing human stem and/or hematopoietics cells |
US5688687A (en) * | 1995-06-07 | 1997-11-18 | Aastrom Biosciences, Inc. | Bioreactor for mammalian cell growth and maintenance |
US5989913A (en) * | 1998-07-02 | 1999-11-23 | Charles Daniel Anderson | Culture vessel for growing or culturing cells, cellular aggregates, tissues and organoids and methods for using the same |
US20070202536A1 (en) * | 2001-10-11 | 2007-08-30 | Yamanishi Douglas T | Methods and compositions for separating rare cells from fluid samples |
US20170096627A1 (en) * | 2004-05-26 | 2017-04-06 | Octane Biotech, Inc. | Advanced tissue engineering system |
US20160251628A1 (en) * | 2013-10-30 | 2016-09-01 | Exmoor Pharma Concepts Ltd | Apparatus And Method For Filtration Of A Suspension |
WO2017109071A1 (en) * | 2015-12-22 | 2017-06-29 | Ge Healthcare Bio-Sciences Corp. | Improvements in and relating to cell harvesting apparatus |
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