WO2020176449A1 - Systèmes et procédés de séléction à haut débit. - Google Patents

Systèmes et procédés de séléction à haut débit. Download PDF

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Publication number
WO2020176449A1
WO2020176449A1 PCT/US2020/019607 US2020019607W WO2020176449A1 WO 2020176449 A1 WO2020176449 A1 WO 2020176449A1 US 2020019607 W US2020019607 W US 2020019607W WO 2020176449 A1 WO2020176449 A1 WO 2020176449A1
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cells
gel
droplets
liquid droplets
droplet
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PCT/US2020/019607
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English (en)
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David A. Weitz
Raoul Gedalja ROSENTHAL
Liangliang Qu
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President And Fellows Of Harvard College
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5005—Wall or coating material
    • A61K9/5021—Organic macromolecular compounds
    • A61K9/5036—Polysaccharides, e.g. gums, alginate; Cyclodextrin
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01F—MIXING, e.g. DISSOLVING, EMULSIFYING OR DISPERSING
    • B01F33/00—Other mixers; Mixing plants; Combinations of mixers
    • B01F33/30—Micromixers
    • B01F33/301—Micromixers using specific means for arranging the streams to be mixed, e.g. channel geometries or dispositions
    • B01F33/3011—Micromixers using specific means for arranging the streams to be mixed, e.g. channel geometries or dispositions using a sheathing stream of a fluid surrounding a central stream of a different fluid, e.g. for reducing the cross-section of the central stream or to produce droplets from the central stream
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
    • B01J13/0052—Preparation of gels
    • B01J13/0065—Preparation of gels containing an organic phase
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
    • B01J13/02—Making microcapsules or microballoons
    • B01J13/04—Making microcapsules or microballoons by physical processes, e.g. drying, spraying
    • B01J13/046—Making microcapsules or microballoons by physical processes, e.g. drying, spraying combined with gelification or coagulation
    • 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
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/16—Particles; Beads; Granular material; Encapsulation
    • 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
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0012—Cell encapsulation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/0074—Biological products
    • B01J2219/00743—Cells
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors

Definitions

  • the present invention generally relates to systems and methods for high throughput selection.
  • Directed evolution can increase the specificity or turnover of enzymes, adapt the temperature or pH optima, and even increase the expression yield of proteins.
  • the general approach for directed evolution experiments is (a) generating variation in the gene of interest that is to be evolved, (b) expressing the different variants, and (c) selecting the best variants for the specific application. The best variants are used as a starting point for the next cycle to create further improved variants.
  • improved selection for directed evolution are needed.
  • the present invention generally relates to systems and methods for high throughput selection.
  • the subject matter of the present invention involves, in some cases, interrelated products, alternative solutions to a particular problem, and/or a plurality of different uses of one or more systems and/or articles.
  • the present invention is generally directed to a method.
  • the method comprises encapsulating cells within liquid droplets, causing the liquid droplets containing the cells to form gel droplets, causing at least some of the cells within the gel droplets to degrade the gel, and separating the gel-degrading cells from the non-gel-degrading cells via a filter.
  • the method comprises encapsulating cells within liquid droplets, causing at least some of the cells to gel the liquid droplet to form a gel droplet, and separating the cells contained within gel droplets from cells contained within liquid droplets via a filter.
  • the present invention is generally directed to devices able to perform any of the methods described herein.
  • the present invention encompasses methods of making one or more of the embodiments described herein. In still another aspect, the present invention encompasses methods of using one or more of the embodiments described herein.
  • Fig. 1 is a schematic diagram illustrating droplet filtration, in one embodiment of the invention
  • Fig. 2 is a schematic diagram illustrating separation of cells using a filter, in another embodiment of the invention.
  • Fig. 3 is a schematic diagram of a microfluidic device in yet another embodiment of the invention.
  • Fig. 4 is the DNA sequence of a plasmid containing the subtilisin gene (SEQ ID NO:
  • the present invention generally relates to systems and methods for high throughput selection.
  • cells are contained within gel droplets, and the cells may interact with the gel droplet in some manner, e.g., to form the gel and/or to degrade the gel.
  • the interaction of cells with the gel droplet may result in some cells being contained within gel droplets and other cells not being contained within gel droplets, which may form the basis by which the cells are sorted.
  • Such cells can be relatively rapidly sorted, e.g., on the basis of size, for example, using filtration, centrifugation, or other similar techniques.
  • unlike sorting techniques which can only sort one entity, such as droplets, at a time, such techniques can allow for more than one entity (and in some cases, relatively large numbers of entities) to be simultaneously sorted, dramatically increasing throughput.
  • One aspect of the invention is thus generally directed to relatively high throughput selection of cells.
  • enzymes such as proteases that are expressed by cells can be sorted relatively quickly.
  • such techniques may be useful to simultaneously assay relatively large numbers of different variants of a protein, e.g., 10 7 , 10 s , 10 9 , or more different variants, without necessarily requiring separate assays for each possible genetic variant.
  • microfluidic droplets are used, which can easily and cheaply be parallelized with microfluidic techniques to increase the throughput as necessary.
  • Cells may be encapsulated within gelatin hydrogel droplets or microspheres, e.g., such that each droplet has, on average, 1 cell (or less).
  • proteases at least some of the encapsulated cells may secrete a protease that can degrade the gelatin hydrogel.
  • the encapsulated cell may be able to escape the gelatin droplet, and those cells can be separated from the cells that do not produce a protease (or produce inadequate amounts of proteases, or less active proteases, etc.), and which are accordingly still contained within hydrogels, for example, using filtration or other techniques.
  • the selection stringency can be controlled, for instance, by altering the gelatin concentration or reducing the incubation time, etc.
  • Such techniques may also be used in a variety of other contexts, not just for proteases or directed evolution experiments.
  • enzymes that degrade other hydrogel forming polymers may be may be screened, or cells that are able to form a hydrogel may be retained.
  • One non-limiting example of a hydrogel-forming reaction is the cross-linking of tyrosine residues in proteins with horseradish peroxidase and hydrogen peroxide. Since many enzymes can produce hydrogen peroxide, and/or can be coupled to hydrogen peroxide forming reactions, such enzymes can be screened by determining if cells containing such enzymes can produce a hydrogel when contained within a droplet.
  • a plurality of cells 10 are to be sorted. There may be at least a million cells, or more (e.g., at least 10 7 , at least 10 8 , at least 10 9 , etc.) to be sorted, for example, on the basis of protease production, enzymes that produce hydrogen peroxide, acid secretion, or the like.
  • the cells initially are encapsulated within droplets, such as microfluidic droplets, using techniques such as flow-focusing that are known to those of ordinary skill in the art. See, for example, U.S. Pat. Nos. 7,708,949, 8,337,778, 8,765,485, or Int. Pat.
  • the cells may be encapsulated within droplets 20 within a carrier fluid, for example, such that they are encapsulated at an average of 1 cell/droplet, or less (for example, such that most or all of the droplets contain either no cell or 1 cell).
  • the droplets may be converted into gel droplets 30.
  • the droplets may be produced containing a gel precursor, although in some cases, the gel precursor may be added to the droplet after the droplet has been formed, e.g., using picoinjection, merging the droplet with another droplet, or other techniques known by those of ordinary skill in the art. See, for example, Int. Pat. Apl. Pub. No. WO 2010/151776 incorporated herein by reference in its entirety.
  • a liquid droplet containing cells and gelatin may be produced at an elevated temperature, and the droplets subsequently cooled to cause the gelatin to gel, causing the droplet to form a gel droplet.
  • the cell may take part in the formation of a gel droplet.
  • a cell may indirectly or directly produce hydrogen peroxide, which can react with a peroxidase and proteins (for example, with tyrosine residues) to cause the droplet to form a gel (e.g., shown as shaded in Fig. 1).
  • the cell produces sufficient hydrogen peroxide, or compounds which can be coupled through enzymatic or non-enzymatic reactions to hydrogen peroxide formation, the droplet containing the cell forms a gel; otherwise, the droplet stays liquid, as is shown here.
  • One non-limiting example is glucose oxidase, which reacts glucose with oxygen to produce gluconic acid and hydrogen peroxide.
  • Such reactions can also be coupled to other reactions in certain cases, such as with a dehydrogenase, in which glucose can be prepared from other sources.
  • a variety of reactions, including enzymatic reactions can be coupled to gel formation in various embodiments.
  • some of the cells are contained within gel droplets, while others are not. Such cells may be contained within liquid droplets, and/or may be free-floating, e.g., in the carrier fluid. Such cells contained within gel droplets can then be sorted from cells not contained within gel droplets. In some cases, sorting can be applied to more than one cell or droplet at a time, which can allow for more rapid sorting than techniques that can only sort one droplet at a time.
  • sorting can be applied to more than one cell or droplet at a time, which can allow for more rapid sorting than techniques that can only sort one droplet at a time.
  • filtration is shown in Fig. 1.
  • a filter 40 having pores 45 having a size that allows free cells to pass, but does not allow cells contained within gels to pass can be used to efficiently separate the cells.
  • the pore size of the filter may be selected based on the types of cells being studied. Different levels of stringency can also be applied, for example, by using filters with different pore sizes.
  • the cells may be removed from the droplets, nucleic acids (coding for proteins such as enzymes) may be sequenced, amplified, etc., and/or other techniques applied to the some or all of the sorted droplets.
  • nucleic acids coding for proteins such as enzymes
  • amplified amplified
  • other techniques applied to the some or all of the sorted droplets.
  • suitable techniques for analyzing cells will be known to those of ordinary skill in the art.
  • certain aspects are generally directed to containing cells in droplets, such as microfluidic droplets.
  • a relatively large number of droplets may be created, e.g., at least about 10, at least about 30, at least about 50, at least about 100, at least about 300, at least about 500, at least about 1,000, at least about 3,000, at least about 5,000, at least about 10,000, at least about 30,000, at least about 50,000, at least about 100,000, at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 droplets, etc.
  • some or all of the droplets may contain cells, and in some cases, such that substantially each of the droplets contains 1 cell or no cell.
  • the droplets and the carrying fluid may be substantially immiscible, i.e., the droplets may be aqueous while the carrying fluid may be an oil, or vice versa.
  • oil merely refers to a fluid that is generally more hydrophobic, or not miscible or soluble in water, as is known in the art.
  • the oil may be a hydrocarbon in some embodiments, but in other embodiments, the oil may comprise other hydrophobic fluids.
  • a junction of channels may be used to create the droplets.
  • the junction may be, for instance, a T-junction, a Y-junction, a channel- within-a- channel junction (e.g., in a coaxial arrangement, or comprising an inner channel and an outer channel surrounding at least a portion of the inner channel), a cross (or“X”) junction, a flow- focusing junction, or any other suitable junction for creating droplets. See, for example, International Patent Application No.
  • PCT/US2004/010903 filed April 9, 2004, entitled “Formation and Control of Fluidic Species,” by Link, el al., published as WO 2004/091763 on October 28, 2004, or International Patent Application No. PCT/US2003/020542, filed June 30, 2003, entitled“Method and Apparatus for Fluid Dispersion,” by Stone, et al., published as WO 2004/002627 on January 8, 2004, each of which is incorporated herein by reference in its entirety.
  • Other techniques for creating droplets include, but are not limited to, bulk emulsification, ink-jet printing, acoustophoretic printing, or the like.
  • the junction may be configured and arranged to produce substantially monodisperse droplets.
  • the droplets may also be created on the fluidic device, and/or the droplets may be created separately then brought to the device.
  • the droplets may be of substantially the same shape and/or size (i.e.,“monodisperse”), or of different shapes and/or sizes, depending on the particular application.
  • the droplets may have a homogenous distribution of cross-sectional diameters, i.e., the droplets may have a distribution of diameters such that no more than about 5%, no more than about 2%, or no more than about 1% of the droplets have a diameter less than about 90% (or less than about 95%, or less than about 99%) and/or greater than about 110% (or greater than about 105%, or greater than about 101 ) of the overall average diameter of the plurality of droplets.
  • the average diameter of the droplets may be less than about 1 mm, less than about 500 micrometers, less than about 300 micrometers, less than about 200 micrometers, less than about 100 micrometers, less than about 75 micrometers, less than about 50 micrometers, less than about 30 micrometers, less than about 25 micrometers, less than about 20 micrometers, less than about 15 micrometers, less than about 10 micrometers, less than about 5 micrometers, less than about 3 micrometers, less than about 2 micrometers, less than about 1 micrometer, less than about 500 nm, less than about 300 nm, less than about 100 nm, or less than about 50 nm.
  • the average diameter of the droplets may also be at least about 30 nm, at least about 50 nm, at least about 100 nm, at least about 300 nm, at least about 500 nm, at least about 1 micrometer, at least about 2 micrometers, at least about 3 micrometers, at least about 5 micrometers, at least about 10 micrometers, at least about 15 micrometers, or at least about 20 micrometers in certain cases.
  • The“average diameter” of a population of droplets is the arithmetic average of the diameters of the droplets.
  • the cells contained within the droplets may be added during formation of the droplet, and/or added after formation of the droplet, for example, using techniques such as picoinjection, merging the droplet with another droplet, or the like. See, for example, Int.
  • the cells may arise from any suitable source, and may include one, or more than one, cell type.
  • the cells may be for example, from a specific population of cells, such as from a certain organ or tissue (e.g., cardiac cells, immune cells, muscle cells, cancer cells, etc.), cells from a specific individual or species (e.g., prokyaryotes, eukaryotes, human cells, mouse cells, bacteria, mammalian cells, etc.), cells from different organisms, cells from a naturally- occurring sample (e.g., pond water, soil, etc.), or the like. In some cases, the cells may be dissociated from tissue.
  • relatively large number of cells may be determined, sorted, etc., e.g., at least about 10, at least about 30, at least about 50, at least about 100, at least about 300, at least about 500, at least about 1,000, at least about 3,000, at least about 5,000, at least about 10,000, at least about 30,000, at least about 50,000, at least about 100,000, at least 10 6 , at least 10 7 , at least 10 8 , or at least 10 9 cells, etc.
  • the droplets are loaded such that, on the average, each droplet has 1 cell in it, or less.
  • the average loading rate may be less than about 1 cell/droplet, less than about 0.9 cells/droplet, less than about 0.8 cells/droplet, less than about 0.7 cells/droplet, less than about 0.6 cells/droplet, less than about 0.5 cells/droplet, less than about 0.4 cells/droplet, less than about 0.3 cells/droplet, less than about 0.2 cells/droplet, less than about 0.1 cells/droplet, less than about 0.05 cells/droplet, less than about 0.03 cells/droplet, less than about 0.02 cells/droplet, or less than about 0.01 cells/droplet.
  • lower cell loading rates may be chosen to minimize the probability that a droplet will be produced having two or more cells in it.
  • at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% of the droplets may contain either no cell or only one cell.
  • the droplets after encapsulation of the cells within droplets, the droplets may be caused to form gels.
  • the gel may be formed initially, and the cells assayed by their ability to degrade the gel. In other cases, the cells may be sorted by their ability to form a gel.
  • a gel may be formed after forming the droplet, and the cells assayed by their ability to degrade the gel.
  • a gel may be formed using a protein, and a protease or other enzyme may be assayed for its ability to degrade the gel.
  • the protease or other enzyme may be one that is excreted by the cell, and/or the cell may be lysed to release the protease into the gel.
  • the cells may be lysed via exposure to a lysing chemical or a cell lysis reagent (e.g., a surfactant such as Triton-X or SDS, an enzyme such as lysozyme, lysostaphin, zymolyase, cellulase, mutanolysin, glucanases, proteases, mannase, proteinase K, etc.), or a physical condition (e.g., ultrasound, ultraviolet light, mechanical agitation, etc.).
  • a lysing chemical e.g., the lysing chemical may be introduced into the droplet after formation of the droplet, e.g., through picoinjection, through fusion of the droplets with droplets containing the chemical or enzyme, etc.
  • hydrogels include, but are not limited to gelatin, collagen, agarose, or acrylamide-based gels, such as polyacrylamide, or poly-N-isopropylacrylamide.
  • acrylamide-based gels such as polyacrylamide, or poly-N-isopropylacrylamide.
  • Other non-limiting examples of gels can be seen in Int. Pat. Apl. Pub. No. WO 2008/109176, incorporated herein by reference.
  • an aqueous solution of a monomer or other gel precursor may be dispersed in a droplet, and then polymerized or reacted, e.g., to form a gel.
  • a hydrogel such as alginic acid that can be gelled by the addition of calcium ions.
  • a temperature change e.g., cooling
  • gelatin or agarose e.g., low-melt agarose gel or ultra low-melt agarose gel.
  • the gel may be chosen to be able to solidify upon exposure to relatively low temperatures, e.g., below about 60 °C, below about 50 °C, below about 40 °C, below about 35 °C, below 30 °C, below about 25 °C, below about 20 °C, below about 15 °C, below about 10 °C, below about 5 °C, etc.
  • relatively low temperatures e.g., below about 60 °C, below about 50 °C, below about 40 °C, below about 35 °C, below 30 °C, below about 25 °C, below about 20 °C, below about 15 °C, below about 10 °C, below about 5 °C, etc.
  • Such low-melt agarose gels may be readily obtained commercially.
  • gelation initiators (ammonium persulfate and TEMED for acrylamide, or Ca 2+ or other divalent ions such as Zn 2+ , Mg 2+ , etc. for alginate and other gels) can be added to a droplet, for example, by co-flow with the aqueous phase, by co-flow through the oil phase, or by coalescence of two different drops, e.g., as discussed in U.S. Patent
  • the droplet may be converted to a gel within a droplet without fusing the droplet to another droplet, or by injecting or otherwise adding an external species to the droplet after formation of the droplet. This may be advantageous, for example, in reducing errors that might be created by fusing two sets of droplets together.
  • a droplet may be formed containing a gel precursor, which may then be caused to form a gel (for example, by polymerization) upon an appropriate stimulus.
  • additional materials may also be present in the gels, e.g., before or after formation of the gels. For instance, some materials may be added to the droplets, before they are formed into gel, or the gels after formation may be exposed to the materials. In some cases, such materials may be able to react with agents released by the cells, e.g., to facilitate or inhibit degradation of the gels.
  • the cells may release, or caused to be released, one or more agents that can degrade the gel.
  • a plurality of cells may be contained within a plurality of droplets, and after the droplets form gels, the cells may be assayed for their ability to secrete proteins (e.g., proteases) that are able to degrade the gels.
  • the cells may secrete such agents, and/or the cells may be lysed to release the agents.
  • the agents can interact with the gel, e.g., to degrade the gel, via a variety of mechanisms, depending on the application.
  • certain types of proteases may cleave protein-based gels (such as gelatin, collagen, silk, peroxidase, transglutaminase polymerized proteins, etc.), agarases may cleave agarose, cellulases may cleave cellulose-derived gels, or pH changes (e.g., caused by secretion of acid or acidic compounds) may disrupt gel formation.
  • cells may be assayed for their ability to produced different types of protease, produce acid, or the like.
  • the cells may be incubated within the gels to allow such degradation to occur, e.g., at
  • the cell may actively take part in gel formation, which can be used to assay the cells, e.g., based on their ability to form the gel.
  • the cell may secrete one or more agents that can take part in gel formation.
  • a gel may be formed through the cross-linking of tyrosine residues in proteins with horseradish peroxidase and hydrogen peroxide. Cells may be assayed based on their ability to secrete a protein or other enzyme that produces hydrogen peroxide.
  • a suitable substrate e.g. protein or synthetic polymers containing phenols
  • a suitable substrate may react with hydrogen peroxide to form a hydrogel.
  • the reaction may, for example, be catalyzed using a peroxidase, such as horseradish peroxidase, which produces a peroxidase-mediated crosslinking reaction, which chemically crosslinks phenolic groups between different molecules to form a gel.
  • a peroxidase such as horseradish peroxidase
  • a peroxidase-mediated crosslinking reaction which chemically crosslinks phenolic groups between different molecules to form a gel.
  • cells may be assayed based on their ability to secrete proteins, enzymes, synthetic polymers, or the like, or the activity of such secreted compounds, based on how much gel forms within a droplet.
  • glucose oxidase reacts glucose with oxygen to produce gluconic acid and hydrogen peroxide.
  • sorbitol may be oxidized with NAD + or NADP + by an alcohol dehydrogenase (e.g., EC 1.1.1.2) to produce glucose, and the enzyme to be assayed may form NAD + or NADP + .
  • an enzyme efficient at producing NAD + or NADP + may provide the NAD + or NADP + for alcohol dehydrogenase to oxidize sorbitol to glucose, and a more efficient enzyme may result in more production of NAD + or NADP + , more production of glucose, more production of hydrogen peroxide, and consequently, better and/or faster gel formation.
  • gluconic acid delta-lactone may be reduced using NADH or NADPH (or another aldehyde) to produce glucose, for example, using a glucose
  • dehydrogenase such as glucose 1 -dehydrogenase or EC 1.1.1.47.
  • an enzyme efficient at forming NADH or NADPH may provide the substrate for glucose dehydrogenase in the form of NADH or NADPH, and a more efficient enzyme may result in more reduction of gluconic acid, more production of glucose, more production of hydrogen peroxide, and consequently, better and/or faster gel formation.
  • reactions such as these may be useful for assaying NADH/NAD + or NADPH/NADP + dependent enzymes, e.g., by causing the droplets to gel.
  • NADH-dependent enzymes include dehydrogenases (e.g., which may be NAD + /NADH or NADP + /NADPH dependent, oxidoreductases acting on the CH-OH group of donors with NAD + or NADP + as an acceptor (EC 1.1) (i.e., alcohol
  • oxidoreductases acting on the CH-Nfb group of donors with NAD + or NADP + as an acceptor (EC 1.4)
  • EC 1.4 e.g., amino acid oxidoreductases, monoamine oxidases, etc., acting on the CH-NH group of donors with NAD + or NADP + as an acceptor (EC 1.5), acting on NADH or NADPH with NAD + or NADP + as an acceptor (EC 1.6), acting on other nitrogenous compounds as donors, with oxidase NAD + or NADP + as an acceptor (EC 1.7), acting on a sulfur group of donors with NAD + or NADP + as an acceptor (EC 1.8), or the like.
  • Those of ordinary skill in the art will be familiar with such dehydrogenases and oxidoreductases.
  • any of the methods herein using proteins such as enzymes (e.g., proteases, dehydrogenases, etc.), such proteins may be endogenous within the cell, or transfected or transformed into the cell.
  • any of the enzymes described herein may be transfected into a suitable cell.
  • a gene such as a protease
  • the cells and droplets may accordingly be sorted or separated based on their size, for example, using filtration or other techniques. For instance, some cells may be contained within fully-gelled droplets, while other cells may be contained within liquid droplets or free of any droplets. While in some cases, such droplets can be sorted using sorting techniques which can only sort one droplet at a time, such as FACS or microfluidic single-cell sorters, in other cases, relatively large numbers of droplets can be simultaneously sorted using techniques that operate on larger numbers of cells, such as filtration or centrifugation. These may operate on the basis of size, or other suitable parameters.
  • the cells (which may or may not be in gel droplets) may be passed through a filter.
  • Larger or more rigid droplets e.g., gel droplets
  • smaller or more flexible droplets, or free cells may be able to pass through the filter and can be collected within the permeate.
  • a wide variety of filters may be obtained commercially, made from a variety of wide variety of materials (e.g., polymers such as polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), polyethersulfone (PES), ceramics, etc.), and may be selected to allow different levels of stringency or filtration, i.e., based on the percentage of cells that are desired in the filtrate versus the retentate.
  • materials e.g., polymers such as polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), polyethersulfone (PES), ceramics, etc.
  • the filter may have an average pore size of less than about 1 mm, less than about 500 micrometers, less than about 300 micrometers, less than about 200 micrometers, less than about 100 micrometers, less than about 75 micrometers, less than about 50 micrometers, less than about 30 micrometers, less than about 25 micrometers, less than about 20 micrometers, less than about 15 micrometers, less than about 10 micrometers, less than about 5 micrometers, less than about 3 micrometers, less than about 2 micrometers, less than about 1 micrometer, less than about 500 nm, less than about 300 nm, less than about 100 nm, or less than about 50 nm, and/or at least about 30 nm, at least about 50 nm, at least about 100 nm, at least about 300 nm, at least about 500 nm, at least about 1 micrometer, at least about 2 micrometers, at least about 3 micrometers, at least about 5 micrometers, at least about 10 micrometers, at least about
  • the filtration process may not necessarily be perfect, and some gel droplets may pass through into the filtrate and/or some non-gel droplets may stay within retentate, e.g., based on the pore sizes and droplets sizes used.
  • more than one filter may be used in some cases.
  • centrifugation may be used to sort droplets, e.g., based on their size. For example, after sorting, cells free of gel, and/or incomplete or degraded gel droplets, may be smaller and lighter than cells contained within gel droplets. Accordingly, by centrifuging such mixtures, the cells may be sorted and selectively removed.
  • a sample containing cells (which may or may not be contained within gels) is centrifuged to cause differential separation of the cells/gels to occur, e.g., at speeds of at least 100 g, at least 300 g, at least 500 g, at least 1,000 g, etc. In some cases, the speeds may be sufficient to cause differential separation to occur, e.g., without lysing large percentages of the cells.
  • a variety of materials and methods, according to certain aspects of the invention, can be used to form articles or components such as those described herein, e.g., channels such as microfluidic channels, chambers, etc.
  • various articles or components can be formed from solid materials, in which the channels can be formed via micromachining, film deposition processes such as spin coating and chemical vapor deposition, laser fabrication, photolithographic techniques, etching methods including wet chemical or plasma processes, and the like. See, for example, Scientific American, 248:44-55, 1983 (Angell, et al).
  • various structures or components of the articles described herein can be formed of a polymer, for example, an elastomeric polymer such as polydimethylsiloxane (“PDMS”), polytetrafluoroethylene (“PTFE” or Teflon ® ), or the like.
  • a microfluidic channel may be implemented by fabricating the fluidic system separately using PDMS or other soft lithography techniques (details of soft lithography techniques suitable for this embodiment are discussed in the references entitled“Soft Lithography,” by Younan Xia and George M. Whitesides, published in the Annual Review of Material Science, 1998, Vol. 28, pages 153-184, and“Soft
  • polymers include, but are not limited to, polyethylene terephthalate (PET), polyacrylate, polymethacrylate, polycarbonate, polystyrene, polyethylene, polypropylene, polyvinylchloride, cyclic olefin copolymer (COC), polytetrafluoroethylene, a fluorinated polymer, a silicone such as polydimethylsiloxane, polyvinylidene chloride, bis-benzocyclobutene (“BCB”), a polyimide, a fluorinated derivative of a polyimide, or the like. Combinations, copolymers, or blends involving polymers including those described above are also envisioned.
  • the device may also be formed from composite materials, for example, a composite of a polymer and a
  • various structures or components of the article are fabricated from polymeric and/or flexible and/or elastomeric materials, and can be conveniently formed of a hardenable fluid, facilitating fabrication via molding (e.g. replica molding, injection molding, cast molding, etc.).
  • the hardenable fluid can be essentially any fluid that can be induced to solidify, or that spontaneously solidifies, into a solid capable of containing and/or transporting fluids contemplated for use in and with the fluidic network.
  • the hardenable fluid comprises a polymeric liquid or a liquid polymeric precursor (i.e. a “prepolymer”).
  • Suitable polymeric liquids can include, for example, thermoplastic polymers, thermoset polymers, waxes, metals, or mixtures or composites thereof heated above their melting point.
  • a suitable polymeric liquid may include a solution of one or more polymers in a suitable solvent, which solution forms a solid polymeric material upon removal of the solvent, for example, by evaporation.
  • Such polymeric materials which can be solidified from, for example, a melt state or by solvent evaporation, are well known to those of ordinary skill in the art.
  • a variety of polymeric materials, many of which are elastomeric, are suitable, and are also suitable for forming molds or mold masters, for embodiments where one or both of the mold masters is composed of an elastomeric material.
  • a non-limiting list of examples of such polymers includes polymers of the general classes of silicone polymers, epoxy polymers, and acrylate polymers.
  • Epoxy polymers are characterized by the presence of a three-membered cyclic ether group commonly referred to as an epoxy group, 1,2- epoxide, or oxirane.
  • diglycidyl ethers of bisphenol A can be used, in addition to compounds based on aromatic amine, triazine, and cycloaliphatic backbones.
  • Another example includes the well-known Novolac polymers.
  • Non-limiting examples of silicone elastomers suitable for use according to the invention include those formed from precursors including the chlorosilanes such as methylchlorosilanes, ethylchlorosilanes,
  • Silicone polymers are used in certain embodiments, for example, the silicone elastomer polydimethylsiloxane.
  • Non-limiting examples of PDMS polymers include those sold under the trademark Sylgard by Dow Chemical Co., Midland, MI, and particularly Sylgard 182, Sylgard 184, and Sylgard 186.
  • Silicone polymers including PDMS have several beneficial properties simplifying fabrication of various structures of the invention. For instance, such materials are inexpensive, readily available, and can be solidified from a prepolymeric liquid via curing with heat.
  • PDMSs are typically curable by exposure of the prepolymeric liquid to temperatures of about, for example, about 65 °C to about 75 °C for exposure times of, for example, about an hour.
  • silicone polymers such as PDMS
  • PDMS polymethyl methacrylate copolymer
  • flexible (e.g., elastomeric) molds or masters can be advantageous in this regard.
  • One advantage of forming structures such as microfluidic structures or channels from silicone polymers, such as PDMS, is the ability of such polymers to be oxidized, for example by exposure to an oxygen-containing plasma such as an air plasma, so that the oxidized structures contain, at their surface, chemical groups capable of cross-linking to other oxidized silicone polymer surfaces or to the oxidized surfaces of a variety of other polymeric and non polymeric materials.
  • structures can be fabricated and then oxidized and essentially irreversibly sealed to other silicone polymer surfaces, or to the surfaces of other substrates reactive with the oxidized silicone polymer surfaces, without the need for separate adhesives or other sealing means.
  • oxidized silicone such as oxidized PDMS can also be sealed irreversibly to a range of oxidized materials other than itself including, for example, glass, silicon, silicon oxide, quartz, silicon nitride, polyethylene, polystyrene, glassy carbon, and epoxy polymers, which have been oxidized in a similar fashion to the PDMS surface (for example, via exposure to an oxygen-containing plasma).
  • Oxidation and sealing methods useful in the context of the present invention, as well as overall molding techniques, are described in the art, for example, in an article entitled“Rapid Prototyping of Micro fluidic Systems and Polydimethylsiloxane,” Anal. Chem., 70:474-480, 1998 (Duffy el al), incorporated herein by reference.
  • the design and/or fabrication of the article may be relatively simple, e.g., by using relatively well-known soft lithography and other techniques such as those described herein.
  • rapid and/or customized design of the article is possible, for example, in terms of geometry.
  • the article may be produced to be disposable, for example, in embodiments where the article is used with substances that are radioactive, toxic, poisonous, reactive, biohazardous, etc., and/or where the profile of the substance (e.g., the toxicology profile, the radioactivity profile, etc.) is unknown.
  • Another advantage to forming channels or other structures (or interior, fluid-contacting surfaces) from oxidized silicone polymers is that these surfaces can be much more hydrophilic than the surfaces of typical elastomeric polymers (where a hydrophilic interior surface is desired). Such hydrophilic channel surfaces can thus be more easily filled and wetted with aqueous solutions than can structures comprised of typical, unoxidized elastomeric polymers or other hydrophobic materials.
  • Fig. 2 illustrates schematically an experiment demonstrating a selection assay.
  • two bacterial populations one of which produced a lot of protease and one of which produced less protease, were mixed at a ratio of 1 : 1.
  • the gelatin droplets were filtered and plated onto a suitable growth medium.
  • the bacterial population producing increased protease was found to be highly enriched in the filtrate.
  • the mixture of cells was diluted in mineral medium (22 mM KH2PO4, 20 mM Na 2 HP0 , 18.7 mM NH 4 C1, 11 mM NaCl,l mM MgS0 , 0.1 mM CaS0 4 , Trace elements (per liter): 50 mg EDTA, 8.3 mg FeCb -6H20, 0.84 mg ZnCF. 130 micrograms CUCI2 2H2O, 100 micrograms C0Q2 6H2O, 100 micrograms H3BO3, 16 micrograms MnCk-6 H 2 0) containing 15% OptiPrepTM (Cat.
  • Dispersed phase #1 used 4% (w/w) gelatin from porcine skin (Cat. No. G2500, Sigma Aldrich, Darmstadt, Germany) in mineral medium at 37 °C.
  • the continuous phase was 3MTM NovecTM 7500 Engineered Fluid containing 2% (v/v) 008-FluoroSurfactant (RAN Biotechnologies, Beverly, MA 01915, USA).
  • a polydimethylsiloxane (PDMS) microfluidics drop-maker of about 35 micrometers thick see Fig. 3
  • cells were encapsulated by co-flowing dispersed phase #1 at a flowrate of 50-100 microliters/h and dispersed phase #2 at a flowrate of 150-300 microliters/h with the continuous phase at a flowrate of 1000-1500 microliters/h.
  • Disperse phase #1 was kept cold with ice during the encapsulation and disperse phase #2 was kept at around 37 °C to keep the gelatin liquid.
  • the encapsulated cells with gelatin were collected into a 1.5 ml EppendorfTM tube which was kept on ice during the collection.
  • the droplets were incubated at 37 °C for 2-4 h, followed by a transfer back on ice, and maintained for 0.5 to 1 hour to ensure the complete gelling of gelatin in the droplets.
  • the emulsion was disrupted with lH,lH,2H,2H-perfluoro-l-octanol (Cat. No.: 370533, Sigma Aldrich, Darmstadt, Germany) and the gelatin hydrogel microspheres were passed through a pluriStrainer Mini 10 micrometer cell strainer (pluriSelect Life Science, Leipzig, Germany).
  • the best protease- secreting cells pass through the filter because the gelatin which they were encapsulated in was digested while worse secreting cells are retained on the filter because they were entrapped in the gelatin hydrogel matrix.
  • the filtrate was plated on Luria-Bertani agar plates containing 6 microgram/ml chloramphenicol incubated overnight at 37 °C. After overnight growth, the best protease- secreting cells could be used for further analysis and validation.
  • a reference to“A and/or B”, when used in conjunction with open-ended language such as“comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.
  • “or” should be understood to have the same meaning as“and/or” as defined above.
  • “or” or“and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as“only one of’ or“exactly one of,” or, when used in the claims,“consisting of,” will refer to the inclusion of exactly one element of a number or list of elements.
  • the term“or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e.“one or the other but not both”) when preceded by terms of exclusivity, such as“either,”“one of,”“only one of,” or “exactly one of.”
  • the phrase“at least one,” in reference to a list of one or more elements should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements.
  • “at least one of A and B” can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another

Abstract

La présente invention concerne de manière générale des systèmes et des procédés de sélection à haut débit. Dans un aspect, des cellules sont contenues dans des gouttelettes de gel, et les cellules peuvent interagir avec la gouttelette de gel d'une certaine manière, par exemple, pour former le gel et/ou pour dégrader le gel. L'interaction de cellules avec la gouttelette de gel peut conduire à certaines cellules contenues dans des gouttelettes de gel et d'autres cellules qui ne sont pas contenues dans des gouttelettes de gel, qui peuvent former la base par laquelle les cellules sont triées. De telles cellules peuvent être triées relativement rapidement, par exemple, sur la base d'une taille, par exemple, à l'aide d'une filtration, d'une centrifugation ou d'autres techniques similaires. Dans certains cas, à la différence des techniques de tri qui ne peuvent trier qu'une entité, telle que des gouttelettes, à la fois, de telles techniques peuvent permettre à plus d'une entité (et dans certains cas, des nombres relativement importants d'entités) d'être triés simultanément, augmentant considérablement le débit.
PCT/US2020/019607 2019-02-26 2020-02-25 Systèmes et procédés de séléction à haut débit. WO2020176449A1 (fr)

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