WO2023277943A1 - Réseaux d'obstacles coniques pour le traitement de particules - Google Patents

Réseaux d'obstacles coniques pour le traitement de particules Download PDF

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Publication number
WO2023277943A1
WO2023277943A1 PCT/US2021/063468 US2021063468W WO2023277943A1 WO 2023277943 A1 WO2023277943 A1 WO 2023277943A1 US 2021063468 W US2021063468 W US 2021063468W WO 2023277943 A1 WO2023277943 A1 WO 2023277943A1
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degrees
separation device
particle separation
obstacles
cells
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PCT/US2021/063468
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English (en)
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Alison Skelley
Khushroo Gandhi
Anthony Ward
Yasna BEHMARDI
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Gpb Scientific, Inc.
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Publication of WO2023277943A1 publication Critical patent/WO2023277943A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502753—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
    • 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
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0647—Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0652—Sorting or classification of particles or molecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/08—Regulating or influencing the flow resistance
    • B01L2400/084—Passive control of flow resistance
    • B01L2400/086—Passive control of flow resistance using baffles or other fixed flow obstructions
    • 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

Definitions

  • Microfluidic deterministic lateral displacement (DLD) arrays offer a size-based separation process that is rapid, gentle and versatile.
  • DLD microfluidic deterministic lateral displacement
  • these arrays utilize carefully designed array geometries to achieve size- based separation. Imperfections or poor design in array geometry can lead to undesirable effects, including the deposit of biological debris during the operation of the arrays, lower throughput and poor separations due to mixing within the array.
  • the development of better device geometries that are more tolerant to manufacturing practices are of considerable interest.
  • DLD arrays utilizing tapered obstacles to provide size-based separations of particles of a predetermined size from a mixture containing particles of a plurality of sizes.
  • FIG. 1 illustrates tapered obstacles having various shapes for use in DLD arrays.
  • FIG. 2A illustrates the effect of tapered obstacles on the gap between adjacent obstacles.
  • FIG. 2B illustrates the effect of tapered obstacles on the gap between adjacent obstacles of three different aspect ratios.
  • FIG. 3 illustrates symmetric and asymmetric obstacle placements for example obstacle shapes.
  • FIG. 4 illustrates the effects of utilizing tapered obstacles in an asymmetric layout DLD array on gap spacing.
  • FIG. 5 illustrates a comparison of mixing effects between two tapered-obstacle DLD-array geometries.
  • FIG. 6 illustrates the effects of tapered-obstacle geometry on product contamination in a blood separation application.
  • FIG. 7 illustrates the effect of critical size range on white blood cell recover in a blood separation application of tapered-obstacle DLD arrays.
  • FIG. 8 illustrates a scanning electron micrograph of a DLD array with tapered hexagonal obstacles.
  • FIG. 9 illustrates the manufacturing advantages of parts with angled walls during injection molding.
  • FIG. 10 illustrates scanning electron micrographs of DLD arrays with tapered obstacles made by etching.
  • FIG. 11 illustrates the effect of input pressure on white blood cell recovery and throughput in a blood separation application using tapered-obstacle DLD arrays.
  • the present disclosure relates to size based microfluidic separations, and especially with the use of DLD in preparing cells that are of therapeutic value.
  • the disclosure herein describes the manufacturing/manufacturability and use of microfluidic devices and the use of DLD for carrying out separations involving biological materials.
  • Apheresis As used herein this term refers to a procedure in which blood from a patient or donor is separated into its components, e.g., white blood cells, platelets and red blood cells.
  • An "apheresis sample” is the product that is the end result of this procedure. More specific terms are “plateletpheresis” (referring to the separation of platelets) and “leukapheresis” (referring to the separation of leukocytes).
  • the term “separation” refers to the obtaining of a product that is enriched in a particular component compared to whole blood or other starting material and does not mean that absolute purity has been attained.
  • CAR T cells The term “CAR” is an acronym for "chimeric antigen receptor.” A “CAR T cell” is therefore a T cell that has been genetically engineered to express a chimeric receptor.
  • CAR T cell therapy This term refers to any procedure in which a disease or condition is treated with CAR T cells.
  • Diseases that may be treated include hematological and solid tumor cancers, autoimmune diseases and infectious diseases.
  • obstacle array and “DLD array” are used synonymously herein and describe an ordered array of obstacles that are disposed in a flow channel through which a cell or particle-bearing fluid can be passed.
  • An obstacle array comprises a plurality of obstacles arranged in a column (along the path of fluid flow). Gaps are formed between the obstacles (along the path of the fluid flow) that allows the passage of cells or other particles. Such arrays or columns can be arranged into one or more repeating rows (perpendicular to the path of fluid flow).
  • particles may include human or biological cells.
  • particles include nonbiological materials such as beads, spheres, or other discrete objects.
  • a “channel” or “lane” refers to a fluidic pathway comprising a plurality of obstacles that are arranged into a discrete separation unit. Such channels may be bounded on either side by walls such that discrete lanes are separated. Channels may run in parallel from one or more common inputs to one or more common outputs. Channels may be fluidly connected in series.
  • Deterministic Lateral Displacement As used herein, the term “Deterministic Lateral Displacement” or “DLD” refers to a process in which particles are deflected on a path through a microfluidic obstacle array deterministically, based on their size. This process can be used to separate cells, which is generally the context in which it is discussed herein. However, it is important to recognize that DLD can also be used to concentrate cells and for buffer exchange. Processes are generally described herein in terms of continuous flow (DC conditions; i.e., bulk fluid flow in only a single direction). However, DLD can also work under oscillatory flow (AC conditions; i.e., bulk fluid flow alternating between two directions).
  • DC conditions continuous flow
  • AC conditions oscillatory flow
  • Critical size The "critical size,” “critical diameter” or “predetermined size” of particles passing through an obstacle array describes the size limit of particles that are able to follow the bulk fluid flow. Particles larger than the critical size can be diverted from the flow path of the bulk fluid while particles having sizes lower than the critical size (or predetermined size) will not be displaced.
  • the "critical size,” “critical diameter” or “predetermined size” may be associated with size-based discrimination and behavior of particles passing through an obstacle array. For example, at above a critical size, certain particles may no longer follow the bulk fluid path. At below the critical size, certain particles will follow the bulk fluid path.
  • a critical size range may refer to an optimal or preferred range for which target particles of a certain size can be separated from contaminants of another size in a sample, while maintaining at least a predefined flow throughput rate along a flow channel comprising the obstacle array.
  • Fluid flow The terms "fluid flow” and “bulk fluid flow” as used herein in connection with DLD refer to the macroscopic movement of fluid in a general direction across an obstacle array. These terms do not take into account the temporary displacements of fluid streams for fluid to move around an obstacle in order for the fluid to continue to move in the general direction.
  • Tilt angle e In an obstacle array device, the tilt angle is the angle between the direction of bulk fluid flow and the direction defined by alignment of rows of sequential obstacles in the array. Tilt angle can be specified in degrees, radian, or as a fractional unit indicating the frequency of a repeated row position (i.e. a 1/60 tilt angle refers to an arrangement where each sequential row is shifted relative to the first row such that at the 60 th shift iteration results in a row with an identical arrangement to the first row).
  • Wall angle Q The wall angle is the angle formed between a given wall of an obstacle and the surface normal of a bottom surface of the obstacle, where the bottom surface is defined as the surface positioned opposite to the surface with the smallest surface area on the volume of the obstacle (e.g. see FIG. 1) .
  • Tapered obstacle is an obstacle having at least one wall wherein at least one wall angle (Q) is not equal to zero degrees.
  • Tapered profile as described herein refers to a side profile or cross-section profile of a tapered obstacle.
  • the “array direction” is a direction defined by the alignment of rows of sequential obstacles in the array.
  • a particle is "deflected” in an obstacle array if, upon passing through a gap and encountering a downstream obstacle, the particle's overall trajectory follows the array direction of the obstacle array (i.e., travels at the tilt angle e relative to bulk fluid flow).
  • a particle is not deflected if its overall trajectory follows the direction of bulk fluid flow under those circumstances.
  • sample generally refers to any sample containing or suspected of containing a nucleic acid molecule or cells.
  • a sample can be a biological sample containing one or more nucleic acid molecules or cells.
  • the biological sample can be obtained (e.g., extracted or isolated) from or include blood (e.g., whole blood), plasma, serum, urine, saliva, mucosal excretions, sputum, stool and tears.
  • the sample may contain blood, a blood product (such as a leukapheresis or apheresis product) also containing an anti-coagulant (e.g., EDTA, EGTA, heparin, citrate, ACD-A, or a thrombin inhibitor).
  • the biological sample can be a fluid or tissue sample (e.g., skin sample).
  • the sample is obtained from a cell-free bodily fluid, such as whole blood.
  • the sample can include circulating tumor cells.
  • the sample is an environmental sample (e.g., soil, waste, ambient air and etc.), industrial sample (e.g., samples from any industrial processes), and food samples (e.g., dairy products, vegetable products, and meat products).
  • the sample may be processed prior to loading into the microfluidic device.
  • the sample may suitably be an apheresis product or a leukapheresis product (e.g., leukopak).
  • Target cells are the cells that various procedures described herein require or are designed to purify, collect, engineer etc. What the specific cells are will depend on the context in which the term is used. For example, if the objective of a procedure is to isolate a particular kind of stem cell, that cell would be the target cell of the procedure.
  • Isolate or purify Unless otherwise indicated, these terms, as used herein, are synonymous and refer to the enrichment of a desired product relative to unwanted material. The terms do not necessarily mean that the product is completely isolated or completely pure. For example, if a starting sample had a target cell that constituted 2% of the cells in a sample, and a procedure was performed that resulted in a composition in which the target cell was 60% of the cells present, the procedure would have succeeded in isolating or purifying the target cell.
  • the present disclosure is directed to microfluidic devices in which size-based purifications are performed by passing a biological sample through an array of obstacles in a microfluidic channel.
  • Size-based separations in a DLD array require that mixing of a raw sample undergoing separation and a buffer used to recover a target particle does not occur as the two coflowing streams traverse the DLD array.
  • Manufacturing tolerances and the use of certain manufacturing practices for construction of a DLD array can result in deviations from the ideal array geometry, particularly with respect to the obstacle shape and arrangement, which are critical to the functioning of DLD array -based separations.
  • the microfluidic devices will be used to separate target particles or target cells having a size larger than the critical size of the device from contaminants with sizes smaller than the critical size.
  • a sample containing the target cells or particles When a sample containing the target cells or particles is applied to a device through a sample inlet and fluidically passed through the channel, the target cells or target particles will flow to one or more product outlets where a product enriched in target cells or target particles is obtained.
  • enriched as used in this context means that the ratio of target cells or particles to contaminants is higher in the product than in the sample.
  • Contaminants with a size smaller than the critical size will flow predominantly to one more waste outlets where they may be either collected or discarded.
  • a microfluidic device may be used with a critical size larger than the target cells or particles but smaller than the contaminants.
  • Combinations of two or more obstacle arrays with different critical sizes, either on a single device or on multiple devices, may also be used in separations.
  • a device may have channels with a first array of obstacles that has a critical size larger than T cells but smaller than granulocytes and monocytes and a second array with a critical size smaller than T cells but larger than platelets and red blood cells.
  • Processing of a blood sample on such a device allows for the collection of a product in which T cells have been separated from granulocytes, monocytes, platelets and red blood cells.
  • the order of the obstacle arrays should not be of major importance to the result, i.e., an array with a smaller critical size could come before or after an array with a larger critical size. Also arrays with different critical sizes can be on separate devices that cells pass through.
  • the sample may be obtained from an individual or a patient, especially a patient with cancer, an autoimmune disease or an infectious disease.
  • the sample is blood or is derived from blood (e.g ., an apheresis or leukapheresis sample), and the target cells are dendritic cells, leukocytes (especially T cells), stem cells, B-cells, NK-cells, monocytes or progenitor cells.
  • the contaminants in these instances will typically include red blood cells and/or platelets.
  • the purification should result in a product enriched in target cells and in which at least 80% (preferably 90% and more preferably 95%) of the platelets and/or red blood cells from the sample have been removed.
  • microfluidic cartridges i.e. devices, chips, cassettes, plates, microfluidic devices, cartridges, DLD devices, etc.
  • methods for purifying particles or cells which may comprise chimeric antigen receptor (CAR) T and NK cells.
  • the microfluidic cartridges i.e. devices, chips, cassettes, plates, microfluidic devices, cartridges, DLD devices, etc.
  • the use of the described cartridges may allow for production of more highly effective CAR T or NK cells by providing a purer T orNK cell product for downstream genetic engineering and CAR T or NK cell production.
  • a more effective CAR T or NK cell may be produced by removing platelets that other methods for producing CAR T or NK cells cannot accomplish.
  • a method for producing chimeric antigen receptor (CAR) T or NK cells may comprise obtaining sample comprising T or NK cells and separating the T or NK cells from contaminants.
  • Contaminants may comprise platelets, or other contaminants described herein. Separating contaminants may comprise applying the sample to the one or more sample inlets of any of the cartridges or devices described herein., flowing the sample to the outlets of the cartridge, obtaining a product enriched in T or NK cells from the product outlet, and genetically engineering the T cells in the enriched product to product chimeric antigen receptors on the surface of the T NK cells.
  • the sample of the method may include an apheresis product or a leukapheresis product.
  • the genetically engineering of the method may comprise genetic engineering methods as described herein.
  • the method may further comprise expanding the
  • CAR T cell therapeutics that can be engineered according to the device and methods herein include axicabtagene ciloleucel, tisagenlecleucel, and brexucabtagene autoleucel.
  • the target particles or target cells of the method may comprise stem cells, thrombocytes, synoviocytes, fibroblasts, beta cells, liver cells, megakaryocytes, pancreatic cells, DE3 lysogenized cell, yeast cells, plant cells, algae cells, monocytes, T cells, B cells, regulatory T cells, macrophages, dendritic cells, granulocytes, innate lymphoid cells, natural killer cells, leukocytes, peripheral blood mononuclear cells, CD3+ cells, neurons, platelets, cancer cells, muscle cells, or epithelial cells.
  • the method may comprise enriching target particles or target cells to produce enriched target cells comprising stem cells, thrombocytes, synoviocytes, fibroblasts, beta cells, liver cells, megakaryocytes, pancreatic cells, DE3 lysogenized cell, yeast cells, plant cells, algae cells, monocytes, T cells, B cells, regulatory T cells, macrophages, dendritic cells, granulocytes, innate lymphoid cells, natural killer cells, leukocytes, peripheral blood mononuclear cells, CD3+ cells, neurons, platelets, cancer cells, muscle cells, or epithelial cells.
  • stem cells thrombocytes, synoviocytes, fibroblasts, beta cells, liver cells, megakaryocytes, pancreatic cells, DE3 lysogenized cell, yeast cells, plant cells, algae cells, monocytes, T cells, B cells, regulatory T cells, macrophages, dendritic cells, granulocytes, innate lymphoid cells, natural killer cells, le
  • the contaminants of the method may comprise stem cells, thrombocytes, synoviocytes, fibroblasts, beta cells, liver cells, megakaryocytes, pancreatic cells, DE3 lysogenized cell, yeast cells, plant cells, algae cells, monocytes, T cells, B cells, regulatory T cells, macrophages, dendritic cells, granulocytes, innate lymphoid cells, natural killer cells, leukocytes, peripheral blood mononuclear cells, CD3+ cells, neurons, platelets, cancer cells, muscle cells, or epithelial cells.
  • the target cells may be peripheral blood mononuclear cells and the contaminants may be platelets.
  • the target cells may be CD3+ cells and the contaminants may be platelets.
  • the method may result in the removal of more than 90% of the platelets.
  • the method may result in the removal of about 50 % of the platelets to about 99 % of the platelets.
  • the method may result in the removal of about 50 % of the platelets to about 75 % of the platelets, about 50 % of the platelets to about 80 % of the platelets, about 50 % of the platelets to about 90 % of the platelets, about 50 % of the platelets to about 95 % of the platelets, about 50 % of the platelets to about 99 % of the platelets, about 75 % of the platelets to about 80
  • the method may result in the removal of about 50 % of the platelets, about 75 % of the platelets, about 80 % of the platelets, about 90 % of the platelets, about 95 % of the platelets, or about 99 % of the platelets.
  • the method may result in the removal of at least about 50 % of the platelets, about 75 % of the platelets, about 80 % of the platelets, about 90 % of the platelets, or about 95 % of the platelets.
  • the method may result in the removal of at most about 75 % of the platelets, about 80 % of the platelets, about 90 % of the platelets, about 95 % of the platelets, or about 99 % of the platelets.
  • the method may comprise modifying the enriched target cells.
  • the method may comprise genetically engineering the enriched target cells to obtain genetically engineered target cells.
  • Genetically engineering includes transfecting or transducing the target cells with a recombinant nucleic acid.
  • Methods of genetic engineering may include the use of TALENs, Zinc
  • the method may also include expanding the enriched target cells or genetically engineered cells by culturing them in vitro.
  • the present invention relates to DLD array geometries, that are compatible with existing manufacturing processes (for example, injection molding), and remain functional regardless of the manufacturing process used to construct them.
  • the devices herein include DLD arrays which incorporate obstacles with tapered profiles.
  • Tapered obstacles can include the following.
  • FIG. 1 illustrates examples of obstacles of three different shapes (diamond, hexagon, and triangle) in a tapered configuration (left) and a non-tapered configuration (right).
  • the wall taper on the tapered obstacles can be described in terms of wall angle (Q). In some embodiments, the wall angle is between 0.1 and 6 degrees.
  • the wall angle may be about 0.1 degrees to about 6 degrees. In some embodiments, the wall angle may be about 0.1 degrees to about 0.3 degrees, about 0.1 degrees to about 0.6 degrees, about 0.1 degrees to about 0.9 degrees, about 0.1 degrees to about 1 degree, about 0.1 degrees to about 1.5 degrees, about 0.1 degrees to about 2 degrees, about 0.1 degrees to about 2.5 degrees, about 0.1 degrees to about 3 degrees, about 0.1 degrees to about 3.5 degrees, about 0.1 degrees to about 4 degrees, about 0.1 degrees to about 6 degrees, about 0.3 degrees to about 0.6 degrees, about 0.3 degrees to about 0.9 degrees, about 0.3 degrees to about 1 degree, about 0.3 degrees to about 1.5 degrees, about 0.3 degrees to about 2 degrees, about 0.3 degrees to about 2.5 degrees, about 0.3 degrees to about 3 degrees, about 0.3 degrees to about 3.5 degrees, about 0.3 degrees to about 4 degrees, about 0.3 degrees to about 6 degrees, about 0.6 degrees to about 0.9 degrees, about 0.6 degrees to about 1 degree, about 0.1 degrees to about 1.5 degrees, about 0.3 degrees to about
  • the wall angle may be about 0.1 degrees, about 0.3 degrees, about 0.6 degrees, about 0.9 degrees, about 1 degree, about 1.5 degrees, about 2 degrees, about 2.5 degrees, about 3 degrees, about 3.5 degrees, about 4 degrees, or about 6 degrees.
  • the wall angle may be at least about 0.1 degrees, about 0.3 degrees, about 0.6 degrees, about 0.9 degrees, about 1 degree, about 1.5 degrees, about 2 degrees, about 2.5 degrees, about 3 degrees, about 3.5 degrees, or about 4 degrees. In some embodiments, the wall angle may be at most about 0.3 degrees, about 0.6 degrees, about 0.9 degrees, about 1 degree, about 1.5 degrees, about 2 degrees, about 2.5 degrees, about 3 degrees, about 3.5 degrees, about
  • the wall angle may be about 0.3 degrees to about 1.4 degrees. In some embodiments, the wall angle may be about 0.3 degrees to about 0.4 degrees, about 0.3 degrees to about 0.5 degrees, about 0.3 degrees to about 0.6 degrees, about 0.3 degrees to about 0.7 degrees, about 0.3 degrees to about 0.8 degrees, about 0.3 degrees to about 0.9 degrees, about 0.3 degrees to about 1 degree, about 0.3 degrees to about 1.1 degrees, about 0.3 degrees to about 1.2 degrees, about 0.3 degrees to about 1.3 degrees, about 0.3 degrees to about 1.4 degrees, about 0.4 degrees to about 0.5 degrees, about 0.4 degrees to about 0.6 degrees, about 0.4 degrees to about 0.7 degrees, about 0.4 degrees to about 0.8 degrees, about 0.4 degrees to about 0.9 degrees, about 0.4 degrees to about 1 degree, about 0.4 degrees to about 1.1 degrees, about 0.4 degrees to about 1.2 degrees, about 0.4 degrees to about 1.3 degrees, about 0.4 degrees to about 1.4 degrees, about 0.4 degrees to about 0.5 degrees, about 0.4 degrees to about 0.6 degrees, about
  • the wall angle may be about 0.3 degrees, about 0.4 degrees, about 0.5 degrees, about 0.6 degrees, about 0.7 degrees, about 0.8 degrees, about 0.9 degrees, about 1 degree, about 1.1 degrees, about 1.2 degrees, about 1.3 degrees, or about 1.4 degrees. In some embodiments, the wall angle may be at least about 0.3 degrees, about 0.4 degrees, about 0.5 degrees, about 0.6 degrees, about 0.7 degrees, about 0.8 degrees, about 0.9 degrees, about 1 degree, about 1.1 degrees, about 1.2 degrees, or about 1.3 degrees.
  • the wall angle may be at most about 0.4 degrees, about 0.5 degrees, about 0.6 degrees, about 0.7 degrees, about 0.8 degrees, about 0.9 degrees, about 1 degree, about 1.1 degrees, about 1.2 degrees, about 1.3 degrees, or about 1.4 degrees.
  • the wall angle may be about 1.5 degrees to about 2.6 degrees. In some embodiments, the wall angle may be about 1.5 degrees to about 1.6 degrees, about 1.5 degrees to about 1.7 degrees, about 1.5 degrees to about 1.8 degrees, about 1.5 degrees to about 1.9 degrees, about 1.5 degrees to about 2 degrees, about 1.5 degrees to about 2.1 degrees, about
  • the wall angle may be about 1.5 degrees, about 1.6 degrees, about 1.7 degrees, about 1.8 degrees, about 1.9 degrees, about 2 degrees, about 2.1 degrees, about 2.2 degrees, about 2.3 degrees, about 2.4 degrees, about 2.5 degrees, or about 2.6 degrees.
  • the wall angle may be at least about 1.5 degrees, about 1.6 degrees, about
  • the wall angle may be at most about 1.6 degrees, about 1.7 degrees, about 1.8 degrees, about 1.9 degrees, about 2 degrees, about 2.1 degrees, about 2.2 degrees, about 2.3 degrees, about 2.4 degrees, about 2.5 degrees, or about 2.6 degrees.
  • the wall angle may be about 2.7 degrees to about 3.5 degrees. In some embodiments, the wall angle may be about 2.7 degrees to about 2.8 degrees, about 2.7 degrees to about 2.9 degrees, about 2.7 degrees to about 3 degrees, about 2.7 degrees to about 3.1 degrees, about 2.7 degrees to about 3.2 degrees, about 2.7 degrees to about 3.3 degrees, about 2.7 degrees to about 3.4 degrees, about 2.7 degrees to about 3.5 degrees, about 2.8 degrees to about 2.9 degrees, about 2.8 degrees to about 3 degrees, about 2.8 degrees to about 3.1 degrees, about 2.8 degrees to about 3.2 degrees, about 2.8 degrees to about 3.3 degrees, about
  • the wall angle may be about 2.7 degrees, about 2.8 degrees, about 2.9 degrees, about 3 degrees, about 3.1 degrees, about 3.2 degrees, about 3.3 degrees, about 3.4 degrees, or about 3.5 degrees. In some embodiments, the wall angle may be at least about 2.7 degrees, about 2.8 degrees, about 2.9 degrees, about 3 degrees, about 3.1 degrees, about 3.2 degrees, about 3.3 degrees, or about 3.4 degrees. In some embodiments, the wall angle may be at most about 2.8 degrees, about 2.9 degrees, about 3 degrees, about 3.1 degrees, about 3.2 degrees, about 3.3 degrees, about 3.4 degrees, or about 3.5 degrees.
  • Gmin and Gmax are also useful in calculation of the maximum wall angle a DLD device can utilize, while remaining functional for separations. Examples of these calculations for obstacle heights of 50 pm and 100 pm are also described in FIG. 2A.
  • the height of the obstacles may be about 20 pm to about 200 pm. In some embodiments, the height of the obstacles may be about 20 pm to about 25 pm, about 20 pm to about 30 pm, about 20 pm to about 35 pm, about 20 pm to about 40 pm, about 20 pm to about 50 pm, about 20 pm to about 75 pm, about 20 pm to about 100 pm, about 20 pm to about 125 pm, about 20 pm to about 150 pm, about 20 pm to about 175 pm, about 20 pm to about 200 pm, about 25 pm to about 30 pm, about 25 pm to about 35 pm, about 25 pm to about 40 pm, about 25 pm to about 50 pm, about 25 pm to about 75 pm, about 25 pm to about 100 pm, about 25 mih to about 125 mih, about 25 mih to about 150 mih, about 25 mih to about 175 mih, about 25 mih to about 200 mih, about 30 mih to about 35 mih, about 30 mih to about 40 mih, about 30 mih to about 50 mih, about 30 mih to about 75 mih,
  • the height of the obstacles may be about 20 mih, about 25 mih, about 30 mih, about 35 mih, about 40 mih, about 50 mm, about 75 mih, about 100 mih, about 125 mm, about 150 mih, about 175 mm, or about 200 mih. In some embodiments, the height of the obstacles may be at least about 20 mih, about 25 mih, about 30 mih, about 35 mih, about 40 mih, about 50 mih, about 75 mih, about 100 gm, about 125 gm, about 150 gm, or about 175 gm.
  • the height of the obstacles may be at most about 25 gm, about 30 gm, about 35 gm, about 40 gm, about 50 gm, about 75 gm, about 100 gm, about 125 gm, about 150 gm, about 175 gm, or about 200 gm.
  • FIG. 2B shows two adjacent obstacles having tapered walls from both top and cross-sectional views in three different aspect ratios example aspect ratios. Any of these aspect ratios could be made and utilized to separate particles, if the wall angle limits calculated according to the equations of FIG. 2A are not exceeded.
  • the height of obstacles is in the range from 20 pm to 200 pm.
  • the aspect ratio is between 0.5:1 and 5:1.
  • DLD arrays having tapered obstacles may further incorporate either symmetric or asymmetric arrangements of obstacles.
  • asymmetric arrangements are preferred because they reduce the shear forces experienced by separated particles relative to symmetric array layouts.
  • Lengthening obstacle gaps perpendicular to the direction of fluid flow and decreasing the length of gaps parallel to fluid flow allows cells of a given size to be processed more rapidly. This can be achieved with a range of obstacle shapes, with the most preferred obstacles being diamond or hexagonally shaped.
  • Hexagonally shaped obstacles may be preferred because they provide the same processing advantages as diamonds but can result in a device that is easier to manufacture and more resistant to biofouling.
  • Using asymmetric gaps can improve throughput and allow devices to operate more efficiently.
  • Polygonal-shaped, elongated obstacles that have vertices pointing toward one another in parallel gaps but where the vertices are offset from one another (as opposed to being directly opposite one another) are preferred. This design reduces flow through the parallel gaps (also called minor flux) by making the gap longer, not narrower.
  • vertices in perpendicular gaps preferably are directly opposite from one another.
  • a primary characteristic of the devices disclosed herein is the presence of obstacle arrays in which perpendicular gaps and parallel gaps are asymmetric, i.e., they are not the same size.
  • FIG. 3 Examples of both symmetric and asymmetric obstacle placements are shown in FIG. 3. Arrangements are described in terms of the gaps between obstacles and the tilt angle e, which describes the offset between adjacent rows of obstacles. In some embodiments the tilt angle is between 0.1 degree and 6 degrees. In the left arrangement, a symmetric configuration is depicted, meaning that the gap is uniform between all obstacles (i.e. the same spacing is used for placement of obstacles in both the vertical and horizontal directions of the diagram).
  • FIG. 3 further provides examples of obstacle shape which are symmetric or asymmetric with respect to the axis of flow.
  • the left and center arrangements both demonstrate symmetric obstacles, since the symmetry axis of the diamond-shaped obstacles is aligned with the axis of fluid flow.
  • the right arrangement provides an additional example of an asymmetric obstacle layout (i.e. Gap 1 is not equal to Gap 2), which further demonstrates an asymmetric obstacle geometry. While the shape of the triangles in the particular example of the right arrangement is symmetric in of itself, the axis of symmetry is out of phase with the axis of flow, making the obstacle geometry asymmetric.
  • FIG. 4 depicts two example arrays of tapered obstacles in asymmetric layouts, tapered diamond-shaped obstacles (top) and hexagonal tapered obstacles (bottom).
  • the tapered walls of the obstacles in both cases result in the gaps between adjacent posts having gaps that increase in size going from the bottom of the array to the top, where for Gap 1 the minimum gap is Gi b and the maximum gap is Gi t. Similarly, for Gap 2 the minimum gap is G2 b and the maximum gap is G2 t.
  • Gap 1 is in the range of 5-150 pm and Gap 2 is in the range of 5-150 pm.
  • Gap 1 and Gap 2 may each independently be about 5 pm to about
  • Gap 1 and Gap 2 may each independently be about 5 pm to about 10 pm, about 5 pm to about 15 pm, about 5 pm to about 20 pm, about 5 pm to about 25 pm, about 5 pm to about 30 pm, about 5 pm to about 35 pm, about 5 pm to about 40 pm, about
  • 15 pm to about 30 pm about 15 pm to about 35 pm, about 15 pm to about 40 pm, about 15 pm to about 50 pm, about 15 pm to about 100 pm, about 15 pm to about 125 pm, about 15 pm to about 150 pm, about 20 pm to about 25 pm, about 20 pm to about 30 pm, about 20 pm to about
  • Gap 1 and Gap 2 may each independently be about 5 pm, about 10 pm, about 15 pm, about 20 pm, about 25 pm, about 30 pm, about 35 pm, about 40 pm, about 50 pm, about 100 pm, about
  • Gap 1 and Gap 2 may each independently be at least about 5 mih, about 10 mih, about 15 mih, about 20 mih, about 25 mih, about 30 mih, about
  • Gap 2 may each independently be at most about 10 pm, about 15 pm, about 20 pm, about
  • Gap 1 and Gap 2 may each independently be about 7 pm to about 18 pm. In some embodiments, Gap 1 and Gap 2 may each independently be about 7 pm to about
  • 8 pm about 7 pm to about 9 pm, about 7 pm to about 10 pm, about 7 pm to about 11 pm, about 7 pm to about 12 pm, about 7 pm to about 13 pm, about 7 pm to about 14 pm, about 7 pm to about 15 pm, about 7 pm to about 16 pm, about 7 pm to about 17 pm, about 7 pm to about 18 pm, about 8 pm to about 9 pm, about 8 pm to about 10 pm, about 8 pm to about 11 pm, about 8 pm to about 12 pm, about 8 pm to about 13 pm, about 8 pm to about 14 pm, about 8 pm to about 15 pm, about 8 pm to about 16 pm, about 8 pm to about 17 pm, about 8 pm to about 18 pm, about 9 pm to about 10 pm, about 9 pm to about 11 pm, about 9 pm to about 12 pm, about
  • 10 pm to about 18 pm about 11 pm to about 12 pm, about 11 pm to about 13 pm, about 11 pm to about 14 pm, about 11 pm to about 15 pm, about 11 pm to about 16 pm, about 11 pm to about 17 pm, about 11 pm to about 18 pm, about 12 pm to about 13 pm, about 12 pm to about 14 pm, about 12 pm to about 15 pm, about 12 pm to about 16 pm, about 12 pm to about 17 pm, about 12 pm to about 18 pm, about 13 pm to about 14 pm, about 13 pm to about 15 pm, about 13 pm to about 16 pm, about 13 pm to about 17 pm, about 13 pm to about 18 pm, about 14 pm to about 15 pm, about 14 pm to about 16 pm, about 14 pm to about 17 pm, about 14 pm to about 18 pm, about 15 pm to about 16 pm, about 15 pm to about 17 pm, about 15 pm to about 18 mih, about 16 mih to about 17 mih, about 16 mih to about 18 mih, or about 17 mih to about 18 mih.
  • Gap 1 and Gap 2 may each independently be about 7 pm, about 8 pm, about 9 pm, about 10 pm, about 11 pm, about 12 pm, about 13 pm, about 14 pm, about 15 pm, about 16 pm, about 17 pm, or about 18 pm. In some embodiments, Gap 1 and Gap 2 may each independently be at least about 7 pm, about 8 pm, about 9 pm, about 10 pm, about 11 pm, about 12 pm, about 13 pm, about 14 pm, about 15 pm, about 16 pm, or about 17 pm. In some embodiments, Gap 1 and Gap 2 may each independently be at most about 8 pm, about 9 pm, about 10 pm, about 11 pm, about 12 pm, about 13 pm, about 14 pm, about 15 pm, about 16 pm, about 17 pm, or about 18 pm.
  • tapered obstacle shapes can be employed in the DLD arrays described herein.
  • Tapered obstacles with symmetric cross- sectional shapes with respect to the axis of flow exhibit reduced or no mixing, which can lead to proper separation of particles.
  • Two DLD array devices with tapered obstacles are depicted in FIG. 5. The device on the right incorporates diamond-shaped tapered obstacles in an asymmetric arrangement similar to the one shown in FIG. 3, center arrangement.
  • the device on the left incorporates tapered triangle obstacles in an asymmetric layout, where the triangles are rotated such that they are asymmetric with respect to the axis of flow, as shown in FIG. 3, right arrangement.
  • food dye is added to the sample stream (502, outer flow), but not to the buffer stream (504, inner flow) in both devices.
  • the two streams remain laminar, parallel and coflowing as they travel down the device and exit without mixing.
  • the two streams are mixed by three-dimensional currents as they traverse the DLD array, resulting in a visible blurring of the boundary between the two flows (506).
  • tapered obstacles arrays can be as separation devices which employ flows that are nonlaminar.
  • FIG. 6 demonstrates the contamination effects of mixing, by applying DLD array separations to isolation of white blood cells from blood, removing the smaller red blood cell and platelet contaminants.
  • the x-axis of the graph shows the effective D c value for a given DLD array, while the y-axis shows level of platelet contamination in the product fraction for DLD arrays with geometries that correspond to each of the three example layouts detailed in FIG. 3.
  • the data are further divided by the degree to which the obstacles are tapered. Markedly, the asymmetric shaped triangles with Q > 0.5 produce the worst contamination, due to the mixing effects demonstrated for this geometry in FIG. 5.
  • the critical size of a given DLD array is set by the geometry of the gap spacings between the obstacles. For straight-walled obstacles, a single critical size value governs the separation process. Particles above that size are deflected into the product fraction flow stream, while particles below that size are carried by the sample flow to the waste outlet.
  • the gap size varies from the bottom of the array to the top of the array (see FIG. 2A and FIG. 4). This results in variability in the effective critical size which depends on the height at which the particles encounter the obstacles. If the critical size is encountered by the particles is too large, poor recoveries result since fewer particles will be deflected to the product fraction outlet. If the critical size encountered is too low, contamination will result since smaller particles will also be deflected to the product fraction outlet. Careful consideration of the separation application is needed to design an array using tapered obstacles that has an appropriate effective critical size range.
  • FIG. 7 shows white blood cell recoveries as a function of critical size, where error bars on the x-axis represent the range of possible effective critical sizes available in a particular tapered-DLD array. All arrays shown on the graph utilize diamond shape posts, which are symmetric with respect to the axis of flow.
  • a further parameter, which is an important consideration for all separations, is throughput.
  • the 40 x 20 pm hexagon design resulted from a need to process a more complex sample (apheresis vs. blood) and manufacturing considerations around obstacle/gap aspect ratios (post height to post width, or post height to gap).
  • This design was optimized to be manufacturable in plastic, using high-volume manufacturing, particularly by injection molding a plastic design off a rigid tool. This process requires surfaces to be angled to facilitate release of the part from the tool. The angle also reduces wear by prevents the plastic part dragging on the tool (see FIG. 9).
  • the initial design was a diamond array (20 x 20 pm diamonds with 16-17 um gaps).
  • the asymmetric arrays were designed (larger gaps perpendicular to flow, smaller gaps parallel to flow). This gave the same net D c , but a bigger gap, which was more resistant to biofouling, and also produced less resistance to flow, allowing operation at higher flow rate. Smaller gaps are more difficult to manufacture, so DLD arrays were designed to have a minimum gap size of 12-14 um, to facilitate manufacturing. In order to maintain the asymmetric geometry (and the processing benefits) a design was needed where instead of pinching the small gap to set the minor flux, the pathway is lengthened at a reasonable gap size. This results in the same net minor flux, which is critical to determine the D c.
  • Theoretical wall angle limitations were validated by construction of tapered obstacle devices with a variety of wall angles in silicon through a DRIE etch process. Scanning electron micrographs of these devices are shown in FIG. 10. Separation performance of the tapered devices was compared to straight-walled devices for blood sample separations in terms of recovery and depletion (i.e. lack of contamination) to determine the effects of tapered devices having a range of D c values from lid to floor.
  • the tolerable limits for a device with a 50 pm obstacle height were calculated to be 2-3 degrees of wall angle, which results in a maximum of about 0.5 pm to 1 pm Dc shift (AD C ) from the top of the array to the bottom of the array.
  • the posts are hexagonal with dimensions of 20 x 40 pm, sharp comer radii, heights of 50 pm, gap values at the top of the device (16-17 um), and gaps at the bottom of the device (12-14 um) driven by a 2-3 degree wall angle, where gaps are the minor flux gaps.
  • a wall angle (Q) of 2.3 degrees.
  • a 13 pm gap yields a D c of 3.7 pm in a straight-walled device, while a 17 pm gap yields a D c of 4.7 pm, meaning that a 50 pm device with a 2.3 degree wall angle and a G min of 13 pm will have an effective D c in the range of 3.7 pm to 4.7 pm.
  • WBC white blood cell
  • FIG. 11 A blood separation was performed, and white blood cell (WBC) recoveries were examined as a function of pressure in using three different array geometries incorporating tapered hexagonal obstacles with dimensions of 40 pm length x 20 pm width x 50 pm height, as depicted in FIG. 11. Pressures of 10, 15, and 20 psi were tested for each array. The three leftmost bars of the left graph show that high recoveries are maintained at all pressures for the array where the tilt angle is 1/60, and Gap 1 and Gap 2 are 21 and 15 pm, respectively, such that the D e value of the array is 4.1 pm. This particular array is a preferred configuration due to its versatility. The rightmost plot shows a measurement of throughput of this array at 10 and 20 psi, which resulted in processing rates of 1.8 and 3.4 mL/hr, where operating at higher pressures leads to faster throughput.
  • WBC white blood cell

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Abstract

L'invention concerne un dispositif de séparation de particules comprenant un réseau d'obstacles espacés le long d'un canal d'écoulement, chaque obstacle dans le réseau d'obstacles comprenant un profil effilé, une différence de tailles d'espace entre des obstacles adjacents le long de profils effilés opposés étant commandée pour obtenir une plage de taille critique qui permet au réseau d'obstacles de séparer des particules cibles d'une taille prédéfinie à partir de contaminants d'une taille différente dans un échantillon lorsque l'échantillon est transporté le long du canal d'écoulement.
PCT/US2021/063468 2021-07-01 2021-12-15 Réseaux d'obstacles coniques pour le traitement de particules WO2023277943A1 (fr)

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Citations (5)

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US8518705B2 (en) * 1999-08-13 2013-08-27 Pathogenetix, Inc. Methods and apparatuses for stretching polymers
US8906322B2 (en) * 2002-10-23 2014-12-09 The Trustees Of Princeton University Method for continuous particle separation using obstacle arrays asymmetrically aligned to fields
US20160363523A1 (en) * 2008-07-10 2016-12-15 Steven H. Reichenbach Method and apparatus for sorting particles using asymmetrical particle shifting
US20170209864A1 (en) * 2013-03-15 2017-07-27 Gpb Scientific, Llc Methods and systems for processing particles
US10324011B2 (en) * 2013-03-15 2019-06-18 The Trustees Of Princeton University Methods and devices for high throughput purification

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8518705B2 (en) * 1999-08-13 2013-08-27 Pathogenetix, Inc. Methods and apparatuses for stretching polymers
US8906322B2 (en) * 2002-10-23 2014-12-09 The Trustees Of Princeton University Method for continuous particle separation using obstacle arrays asymmetrically aligned to fields
US20160363523A1 (en) * 2008-07-10 2016-12-15 Steven H. Reichenbach Method and apparatus for sorting particles using asymmetrical particle shifting
US20170209864A1 (en) * 2013-03-15 2017-07-27 Gpb Scientific, Llc Methods and systems for processing particles
US10324011B2 (en) * 2013-03-15 2019-06-18 The Trustees Of Princeton University Methods and devices for high throughput purification

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