WO2018227204A1 - Encapsulation contrôlée dans des gouttelettes par cisaillement interfacial liquide-liquide - Google Patents

Encapsulation contrôlée dans des gouttelettes par cisaillement interfacial liquide-liquide Download PDF

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Publication number
WO2018227204A1
WO2018227204A1 PCT/US2018/036952 US2018036952W WO2018227204A1 WO 2018227204 A1 WO2018227204 A1 WO 2018227204A1 US 2018036952 W US2018036952 W US 2018036952W WO 2018227204 A1 WO2018227204 A1 WO 2018227204A1
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channel
droplet
fluid
dispersed
flow
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English (en)
Inventor
Gopakumar KAMALAKSHAKURUP
Abraham P. Lee
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University of California Berkeley
University of California San Diego UCSD
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University of California Berkeley
University of California San Diego UCSD
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Publication of WO2018227204A1 publication Critical patent/WO2018227204A1/fr
Priority to US16/707,560 priority Critical patent/US11517901B2/en
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    • 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
    • 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
    • B01L3/502769—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 multiphase flow arrangements
    • B01L3/502776—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 multiphase flow arrangements specially adapted for focusing or laminating flows
    • 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
    • B01L3/502769—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 multiphase flow arrangements
    • B01L3/502784—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 multiphase flow arrangements specially adapted for droplet or plug flow, e.g. digital microfluidics
    • 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
    • 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
    • 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
    • B01L2300/0867—Multiple inlets and one sample wells, e.g. mixing, dilution

Definitions

  • the present invention relates to microfluldic devices, namely, to encapsulation of samples using droplet-based microfiuidic devices.
  • BACKGROUND OF THE INVENTION HWj $ingte-cet ⁇ analysis is a field that studies the genomics, transcriptomics, proteomics and metaooiomics at the single cell level.
  • Conventional techniques to perform single ceil analysis are flow cytometry and automated microscopy. To address the wide range of applications for single eel analysis, these conventional methods are often coupled with microfluldic devices. Microfiuidic devices and systems are configured to process (e.g., move, mix.
  • microfiuidic devices can spatially collect single eels in micro wells, patterned surfaces, and various traps based on mechanical, magnetic, hydrodynamlc, optical, dieieetrophoretic, and acoustic principles. These microfiuidic devices can be used for various applications including printing, bio-chemical assays, drug discovery, etc.
  • a class of microfiuidic devices and systems includes microfiuidic droplet generating and manipulating devices configured to manipulate discrete droplets. Droplet-based microfiuidic devices can be configured to perform a variety of operations., such as.
  • these devices can be used as mieroreaetors to achieve controlled and rapid mixing of fluids and/or to synthesize droplets and encapsulate various biological entitles for biomedicine and biotechnology applications.
  • Droplet-based single ceil assays are based on the ability to encapsulate and confine single cells in individual droplets and enaibie genome wide expression profiling.
  • One-one-one encapsulation in droplets is a critical unit operation in single ceil high-throughput screening and droplet sequencing, Most single cell: encapsulations in droplets are performed randomly and are dictated by Poisson statistics.
  • One of the current challenges in performing droplet sequencing (drop-seq) operation In droplets is achieving high efficiency one cell-one-bead encapsulation, it has been recently reported thai for genome wide expression profiling, the encapsulation efficiency is as low as 0.1%, or 1 In 1000 droplets will have a cell therein.
  • droplets Droplet sequencing
  • the present invention features a passive, hydrodynamlc technique to perform encapsulation in droplets utilizing laminar flows and high shear liquid-liquid interface at a microfiuidic function, wit an encapsulation efficiency of 30%, which could significantly improve the efficiency of drop-seq and other bead based single cell assays.
  • hWilTj It is an objective of the present invention to provide fo microfiuidic devices and methods for encapsulating bsomolecuSes in droplets.
  • Embodiments of the invention are given in the dependent dates.
  • Embodiments of t e present invention can be freely combined wsth each other 8 they are not mutually exclusive ' .
  • the present invention provides an snterfaclai hydrodynamic technique that combines the effects of laminar flow and liquid-liquid inferfacial shearing, resulting in one-one-one encapsulation in droplets.
  • Beads, cells and aqueous phase introduced through upper, lower and middle inlets respectively, create distinct laminar flow streams at a Junction.
  • the flow rates at three inlets are kept equal to prevent the bead/cell migration across the streamlines due to Magnus forces.
  • the beads and ceils self-assemble in a single f fe along the channel wall while moving toward the droplet generation junction.
  • ⁇ dispersed to continuous phase pressure ratio
  • MWj Various embodiments discussed herein comprise microfiuidic devices that are configured to encapsulate single particle or cells with high through-put. Various embodiments of the microfiuidic devices can be configured to provid encapsulation efficiencies o 30% or higher. For example, various microfiuidic devices discussed herein can be configured to encapsulate particles or cells with an encapsulation efficiency of about 30% or greater. In some embodiments, the encapsulation efficiency is maximized in the squeezing regime to near dripping regime, where Ca ⁇ 10 " ⁇ and ⁇ is about 0,5-1.0.
  • One of the unique inventive features of the present invention is the formation of high shear interfaces between the continuous phase and dispersed phase fluid streams can be formed, which increases the encapsulation efficiency.
  • laminar flow guides the pariieies eeiis along the channel wail toward the high shear interface, and the high shear interface draws the cells or particles toward it at a higher velocity resulting in self-spacing of the cells, thereby reducing and even eliminating the possibility of doublets.
  • An important advantage of this technique is that it can be modified based on the desired application including single cell or bead encapsulation (1 -1 ), and 1 ceil ⁇ 1 bead-1 droplet encapsulation or 1 DCM cell-1 droplet encapsulation (1 -1-1 ) for different cell types and ceil sizes.
  • the method of encapsulating one or more solid samples ⁇ e.g., biological material comprising cellular material, one or more cells, one or more particles, one or more beads, etc.
  • a droplet of a fluid may comprise flowing a dispersed phase fluid stream comprising a solid sample (e.g., a biological sampl comprising cellular material or one or more cells) dispersed in a fluid (e.g., water) through a combining channel; controlling t e flow rates of the flow stream to establish laminar flow through the combining channel; flowing a continuous phase fluid through a continuous phase channel that intersects the combining channel the continuous phase fluid being immiscible with the dispersed phase fluid; controlling the flow rate of the continuous phase fluid to shear the laminar flow of the dispersed phase fluid stream and generating droplets encapsulating the solid sample in an output microfluidic channel jWS2j
  • a dispersed phase fluid stream comprising a solid sample (e.g., a biological s
  • Each continuous phase channel is configured to transport a continuous phase fluid stream.
  • the intersection region is configured to open into an output microfluidic channel through an orifice.
  • the microfluidic device further comprises a fluid controller to control the flow rates of the fluid streams to generate droplets encapsulating the solid sample.
  • the flow rate of the dispersed phase flow stream can be controlled to establish laminar flow, and the flow rate of the continuous phase fluid stream can be controlled to shear the laminar flow of the dispersed phase fluid stream in the intersection region, thus generating droplets that enter the output microfluidic channel through the orifice.
  • FIG:. 1A shows an exemplary mechanism of one-ceii-one-bead ⁇ encapsulation in droplets using Ihterfacial shearing technique, according to an embodiment of the present invention.
  • Beads and cells introduced from uppe and lower inlets self-assemble along the channel wail while moving toward high shear interfaces.
  • both the heads and cells get pulled toward the high shear interface symmetrically f rom both the channel boundaries resulting in one- ⁇ - ⁇ encapsulation, ⁇ 98161
  • FIG. 18 is a schematic of the encapsulation process using interfacia! shearing.
  • FIG. 2 shows a non-limiting computational fluid dynamic model ⁇ GFD) of interfaelai shearing.
  • FIG. 3 shows a non-limiting embodiment of one-one-one (1-1-1) encapsulation of 10 pm beads and K-562 cells.
  • the 10 pm beads were introduced from an upper inlet while the k-562 cells are flowed in through a lower inlet. Both the beads and ceils align close to the channel wall on the way toward a flow focusing junction. At the Junction, the heads and the ceils are pulled toward a symmetrical shear interface and encapsulated into droplets,
  • FIGs, 4A-4D show steps of 1-1-1 encapsulation of 10 pm beads from the top and He!a sells from the b ttom:. The encircled droplet indicates the 1 -1-1 droplet
  • FIG, 5A shows the 10 m beads seif-assembie along the top channel wall while Hela ce!!s align along the bottom wall
  • FIG, 5B shows the one cell-one bead encapsulation in droplets.
  • the encircled droplets Indicate the 1-1-1 droplets.
  • FIGs. 6A-6E sho steps of one-one-one encapsulation of 10 pro beads, in FIG. 6A, beads self- align along the channel wall.
  • FIG. 8B beads gets pulled by the high shear flow at the boundary
  • FIGs, 6C-6D beads enter the droplets from two sides and are encapsulated in FIG, 6E, The encircled droplet shows the 1-1-1 droplets.
  • [88231 FiG, 7,A is a graph of encapsulation efficiency vs. droplet diameter.
  • FIG. 7B is a graph of 1-1-1 encapsulation efficiency vs. pressure ratio of dispersed phase to continuous phase ( ⁇ .
  • the encapsulation efficiency increases with the dispersed io continuous pressure ratio ( $ ⁇ , ead es a maximum, and decreases thereafter due to multiple encapsulations in one droplet.
  • FiG. 7C Is a graph of concentration optimization for encapsulation efficiency vs. ceil concentration. The encapsulation efficiency increases with cell concentration, reaches a maximum, and decrease ' thereafter due to multiple encapsulations i one droplet.
  • 002*1 FiG, 8A is a schematic illustration of single cell encapsulation in droplets (1-1) using interacial shearing. The ceils were introduced from a single nlet,
  • FiG. 9A is an alternative schematic of single cell encapsulation in droplets (1-1) using interfacial shearing. The cells were introduced from two inlets.
  • the . : .micro&iidtc devices employ fluid volumes on the scale of microliters: to picoSters (1Q "'! 3 ⁇ 4 that are contained within sur>miiiimeter scale channels.
  • the structural or functional features may be dimensioned on the order of mro-sea!e or less, preferably in the micron scale or less.
  • a diameter or width of a channel or a dimension of an intersection or Junction may range from ⁇ 0,1 pm to greater than 1000 pm.
  • a length of channel may range from 0.1 ⁇ to greater than cm-scale.
  • the rnscroflusdic device may employ active or passive techniques for fluid transport and droplet production.
  • the passive approach takes advantage of the characteristic flow fleid in micfoRuidics to control the interface and capillary instability, and consequently to produce droplets.
  • high shear interface refers to a high velocity iiquid-iiquid interface formed between two immiscibte liquids.
  • the continuous phase flow rate is greater than the flow rate of the dispersed phase. For instance, the continuous phase flow rate may be about 2-5 times greater.
  • the high continuous phase Sow rate imparts the same velocity to the dispersed phase at the interface.
  • the dispersed phase at the interface is at a higher velocity ⁇ shear ⁇ than the bulk.
  • laminar Sow refers to flow of a fluid in layers that do not mix.
  • the samples for encapsulation may be microparticfes.
  • the microparticies may be beads.
  • beads include, but are not limited to, polymer beads, bar-coded beads, functsonaiized beads, and magnetic beads.
  • the beads may have a size or dimension, such as a diameter or width, ranging from about 0.01 ⁇ to about 20 pm. hWSSj in other various .
  • the samples tor encapsulation may be cells. Any particular ceil type from any o ganism may be used in the methods and systems of the present invention:.
  • the DCis may have a size or dimension, such as a diameter or width, ranging from about 0.1 pm to about 20 m - So some embodiments, the cells mayb wild type ceils or genetically modified DCis. In other embodiments, the cells may be ceils harboring one or more mutations, healthy cells, diseased cells or unhealthy DCis, etc. For example, in some embodiments, the cells may be prokaryotse cells (e.g., bacteria, arehaebaeteria, etc.). in other embodiments, the cells ma be eukaryotic ceils such as single- celled eukaryotes, fungal cells (e,g, yeast, moid, etc), animal cells, mammalian cells (e.g.
  • the cells used in the present invention may be other eukaryotic cells such as plant DCis or algal DC cultuor cells, on-iimiting and non-exhaustive examples of plant cells include cells from corn, soybean, wheal, cotton, grass, flowering plants, fruit-bearing plants, trees, tuberous plants, potatoes, root plants, carrots, peanut, nuts, beans, legumes, and squashes, it is to be understood that the term "plant ceil' encompasses ail types and stages of plant DCis and is not limited to the aforementioned examples.
  • algal cells include DCis from Chlorelia sp., Mannochloropsis sp, and Boiryococcus sp. It is to be understood that the term "algal ceil” encompasses all types of algal cris and is not limited to the aforementioned examples.
  • a Cigal ceil wall that surrounds a cell membrane to provide rigidity, strength, and structure to the cell.
  • the ceil wall may be comprised of polysaccharides including celiuiose, hemicelluiose, and pectin.
  • the fungal cells also have a DCi wail, which ma be comprised of polysaccharides including glucaos, mannans, and chitin.
  • the DCis used in the present invention may be protoplasts, which are intact plant, bacterial or fungal carrotis that had its cell wall completely or partially removed using either mechanical or enzymatic means.
  • the DCis used in the present invention ma be a tetrad.
  • the term "tetrad" is used to herein to refer to a single structure comprised of four individual physically attached components.
  • a "microspore” is an individual hapioid structure produced from diploid sporogenous cells ⁇ e.g., microsporoyte, pollen mother DCi, or meiocyte) following meiosls,
  • a microspore tetrad refers to four individual physically attached microspores.
  • a "pollen grain” Is a mature gametophvte containing vegetative ⁇ non-reproductive ⁇ eels and a generative (reproductive) ceil.
  • a pollen tetrad refers to four individual physically attached pollen grains.
  • Microfiuidic devices including droplet generatio portions can be used to create droplets of a fluid ⁇ e.g., oil or water).
  • Microfiuidlc -devices that include droplet generation portions can be used: to study chemical reactions, in drug delivery, in drug discovery, ⁇ to.
  • One method of generating droplets in microiiuidle devices comprises flowing a .first liquid ⁇ e.g., water) through a first- channel and a second liquid (e.g., oil) that is immiscible with the first liquid through channels Intersecting the first channel.
  • the first liquid flowing through the first channel ⁇ e.g., water
  • the s-f3 ⁇ 4e of the generated first liquid droplets generated can depend on a variety of factors including velocity of the second liquid. For example, as the velocity of the second liquid is increased, the size of the first liquid droplets is reduced.
  • )6 j Referring now to FIG, 1A-1B, in one embodiment, the present invention features a method for encapsulating a solid sample (102) in a droplet ( 04).
  • the method may comprise flowing a first fluid ⁇ 106) through a first microituidic channel (1 0) at a first flow rate (v ⁇ ) such that flow of the first fluid is laminar, and co-flowing a second fluid (108) through each of a second microfiuidic channel (120) and a third microfiuidlc channel (130) at a second flow rate (v c ).
  • the first fluid (108 ⁇ may comprise at least two flow streams (107).
  • both of said flow streams (107 ⁇ may comprise dispersed solid samples ( 02 ⁇ that seif-assernhie near a sidewali (112) of the first microfiuidlc channel while flowing towards an intersection region (140),
  • the second and third rnlcrofiuidic channels (120, 130 ⁇ can intersect the first microfiuidlc channel (1 0) at the intersection region ⁇ 140 ⁇ such that the second fluid streams (108) intersect the first fluid (106) and merge to form a droplet shearing junction (145) within the intersection region (140), in some embodiments, the method further comprises adjusting v a , v e .
  • each of the second fluid streams ⁇ 108 ⁇ forms a high shear interface (109) with the first fluid (106), and the solid samples (102) are drawn to the high shear interface (109), and gene-rating droplets (104) at the droplet shearing junction (145) such that each droplet (104 ⁇ is substantially sized to encapsulate one solid sample or co-encapsulate two different soiid samples.
  • the method for encapsulating a solid sample (102) in a droplet (104) may include providing a microfiuidic device ⁇ 100 ⁇ .
  • the microfiuidlc device (100) may comprise a combining channel (110), a first continuous phase channel (120) having a portion thereof disposed on one side of the combining channel, a second continuous phase channel ( 30) having a portion thereof disposed on an opposite side of the combining channel, and an output channel ⁇ 150 ⁇
  • the portions of the first and second continuous phase channels can intersect at a terminal end of the combining channel to form an intersection region ⁇ 140 ⁇ to which the output channel ⁇ 150 ⁇ Is fluidly coupled thereto, in one embodiment, the portions of the first and second continuous phase channels can intersect the combining channel ( 10) orthogonally such that the continuous phase channels and combining channel form a T-junction.
  • the continuous phase channels can intersect the combining channel ⁇ 110 ⁇ at an acute angle such that the continuous phase channels and output channel form a V- junction, )66j in some embodiments, the mierofiuidie device (100) ma further comprise a first dispersed phase channel ⁇ 114 ⁇ comprising one of the flow streams (107) forming the dispersed phas fluid (106). and a second dispersed phase channel (11S) comprising the othe Sow stream (107),
  • the first ami second dispersed phase channels (114, 116 ⁇ can me ge to form the combining channel ⁇ 110 ⁇ .
  • the fnierofiuidfe device may further compose an aqueous phase channel ⁇ 117 ⁇ intersecting with the first and second dispersed phase channels (114, 116).
  • the aqueous phase channel (11 ?) may comprise aqueous phase fluid (118), which flows to the combining channel ⁇ 1 0 ⁇ such thai the aqueous phase fluid (118 ⁇ forms a laminar interface stream (119) between the two flow steams ⁇ 10? ⁇ .
  • the device ⁇ 100 ⁇ ma further comprise a fluid flow controlle (160 ⁇ configured to perform operations. These operation can include adjusting of the dispersed phase fluid to establish laminar fiow in the combining channel (110 ⁇ such that the solid samples (102) assembl near a sidewaii ⁇ 112 ⁇ of the combining channel while Sowing towards the Intersection region ⁇ 140 ⁇ , adjusting v 3 ⁇ 4 , v c , or both such that each continuous phase fluid stream ⁇ 106 ⁇ forms a high shear interface ⁇ 108 ⁇ with the dispersed phase fluid (106) at the intersection region ⁇ 140 ⁇ and the solid samples ⁇ 102) are drawn to the high shear interface (109 ⁇ while flowing through the intersection region ⁇ 140 ⁇ , and adjusting 3 ⁇ 4, vv, or both to generate droplets (104) at the droplet shearing junction (145) such that each droplet ⁇ 104 ⁇ is substantially sized to encapsulate a solid sample (102). in one embodiment the fiow in the microfiuidic device
  • the width of the various microfiuidic channels (e.g., the first and second dispersed phase and aqueous phase channels (1 4, 116, 117); tie combining channel (1 0); and the continuous phase channels (120, 130» can range from about 25 pm to about 75 pm.
  • the width of the various microfiuidic channels can be in a range between about 30 pm to about 80 pm.
  • a width and/or length of the Intersection region can be about 3-6 times the width of the various microfiuidic channels (e.g., the combining channel, the first continuous phase channel, or the second continuous phase channel).
  • the width of the intersection region may be about 150 pm « which is about three times the width of a 50 pm incoming microfiuidic channel.
  • the length of the intersection region may be about 200 pm , which is about four times the width of a 50 pm incoming microfiuidic channels.
  • the width of the orifice may b about 5-40 pm.
  • the width of the orifice may be about 5-15 pm, about 10-20 pm, about 20-30 pm, or about 30-40 pm . in other embodiments, the width of the output channel may widen from the width of the orifice to a maximum width.
  • the maximum width of the output channel can be about 2-18 times th width of the orifice. For examples, for a 30 pro orifice, the output channel widens from a minimum width of 30 pm to a maximum width of about 120 pm. In further embodiments, the width of the output channel may be reduced after reaching its maximum.
  • an exemplary implementation of the method may comprise flowing a dispersed phase fluid (106) t rough the combining channel (110) at a first flow rate (v d ) ⁇ and adjusting 3 ⁇ 4 of the dispersed phase fluid (108) to establish Seminar flow in the combining channel (110) such that the soiid samples (102) assemble near a sidewaii ⁇ 112 ⁇ of the combining channel while flowing towards the intersection region (140).
  • the dispersed phase fluid (106) ma comprise ..at least two flow streams (107), with one or both of the flow streams (107) having dispersed solid samples (102).
  • Continuous phase fluid streams (106) co-flow through each of the first and second continuous phase channels (120 ; 130 ⁇ at a second flow rate (v 0 ) «
  • the continuous phase fluid streams (108) can intersect the dispersed phase fluid (106) at the intersection region (140) such ' that a droplet shearing Junction (145 ⁇ is formed within the intersection region ⁇ 140) as the continuous phase fluid streams (108) merge with the dispersed phase fluid (108).
  • the droplet shearing junction (145) can comprise an orifice (1 7) that fiuidiy couples the output channel (150 ⁇ to the intersection region (140).
  • each continuous phase fluid stream (108) forms a high shear interface (109) with the dispersed phase fluid (106 ⁇ at the intersection region (140 ⁇ .
  • the solid samples (102 ⁇ are drawn to the high shear interface (109) while flowing through the intersection region (140 ⁇ . 3 ⁇ 4, v c , or both are further adjusted to generate droplets ⁇ 104 ⁇ encapsulating one soiid sample (102) at the droplet sheeting junction (145).
  • each droplet (104) can be substantially sized to encapsulate said solid sample (102). t?2j in one embodiment, as shown in FIG.
  • the method and mlorofiuldie device can b adapted to co-encapsulate two different samples in one droplet.
  • the dispersed soiid samples ⁇ 102 ⁇ may comprise a plurality of ceils flowing in one of the flow streams ⁇ 107 ⁇ , and a plurality of particles flowing in the other flow stream (107),
  • laminar flow of the dispersed phase fluid causes the ceils to assemble near the sidewaii (112a ⁇ and the particles to assemble near an opposing sidewaii (112b).
  • the ceils are drawn to one high shear interface (109a ⁇ and the particles are drawn io the other high shear interface (109b), thereby enabling one ceil and one particle to be co-encapsuiated in one droplet ⁇ 104 ⁇ as said droplet (104) is formed at the droplet shearing Junction ⁇ 145).
  • the droplet (104) co-encapsulating the one eel and one particle can then be released from the orifice (147) info the output channel ⁇ 150 ⁇ .
  • in another embodiment, as shown in FIGs. 8A-9B, the method and microfluidic device can be adapted to encapsulate a single sample in one droplet.
  • the dispersed solid samples (102) may comprise either ceils or particles.
  • the ceils or particles enter the combining channel (110) from one or both of the first and second dispersed phase channels, and one solid ceil or particle ⁇ 102 ⁇ is encapsulated as the droplet (104) is formed at the droplet shearing junction (145).
  • the droplet ⁇ 104 ⁇ encapsulating the one solid sample ⁇ 102 ⁇ is released from the orifice (147) into the output channel (150).
  • the efficiency of encapsulating a single ceil ⁇ one ceil) and/or a single bead (one-bead) in a single droplet can be as low as 0.1 %, i.e. 1 in 1000 droplets may have a single DCi (one cell) and/or a single bead (one-bead) while the remaining droplets may have no ceils and/or beads or have more than one DCi and/or one bead.
  • this application provides a passive, hydrodynamie technique which can achieve a 'one-one-one' ⁇ one cell and/or one bead in one droplet) encapsulation efficiency of 30% or higher, which could significantly improve the bfomoleeuiar captur efficiency of various bead-based single cell assays.
  • the device can be configured to encapsulate one ceil and/or one bead in a single droplet of a fiuid (e.g., water) by the combined effect of laminar flow and the high shear liquid-liquid interfaclai boundary.
  • a first fluid stream comprising a first solid sample (e.g., cells or cellular material) dispersed in a first fluid (e.g., water) is introduced through a first incoming microfluidic channel and a second fluid stream comprising a second so!id sample (e.g., beads or particles) dispersed in the firs fluid ⁇ e.g., water) is introduced through the second incoming microfluidic channel.
  • a first solid sample e.g., cells or cellular material
  • a second so!id sample e.g., beads or particles
  • a third fluid stream comprising the first fluid is introduced through the third incoming microfluidic channel.
  • the first, second and third flow streams collectively referred to as a dispersed phase fiuid stream, flow into the combining channel.
  • the velocities of the first, second and third flow streams can be adjusted such that laminar flow is established in the combining channel.
  • the flow rates of the first, second and third: flow streams can be equal to each other such that laminar flow is established in the combining channel.
  • the constituents of the first solid sample ⁇ e.g., cells or cellular material
  • the constituents of the second solid sample ⁇ e.g., particles or beads
  • beads or particles self-assemble in a single row along a channel wall of the combining channel adjacent to the incoming microfluidic channel of the bead or particles
  • cells self-assemble in a single row along the opposite channel wall of the combining channel adjacent to the incoming microftuidie channel of the cells.
  • the laminar flow of the dispersed phase fiuid stream enters the intersection region.
  • the flow rate of the continuous phase fluid streams can be adjusted to create a high shear interface between the laminar flow of the dispersed phase fluid stream. Cells in the first flow stream and the beads or particles in the second flow stream are pulled towards the high shear interface as shown In FIG. 1B.
  • the flow rates of the dispersed phase fluid stream and the continuous phase fiuid streams can be adjusted to generate droplets having a droplet size large enough to encapsulate a single cell from the first flow stream and a single bead/particle from the secohd fluid stream.
  • the stee of the droplet can depend an the capillary number Ca » ⁇ / ⁇ , where ⁇ Is the viscosity of the continuous phase comprising the second fluid, V is the superficial velocity (flow rate) of the continuous phase comprising the second fluid, and a is the equilibrium surface tension between the continuous phase and the dispersed phas fluid streams.
  • the capillary number can foe in the range of about 0.01 and about 1 (e.g.. about 0,1 ).
  • the velocity of the continuous phase fluid streams can be about 2-10 times greater that tie velocity of the dispersed phase fluid stream.
  • the droplet size can also be controlled by controlling the pressure ratio between the dispersed phase fluid stream and the continuous phase fluid stream.
  • a droplet encapsulating a single ceil and a single bead/particle can be achieved by controlling the pressure ratio ( ⁇ ) and/or the flo rate ratio between the dispersed phase and the continuous phase.
  • the pressure ratio and/or the fiow rate ratio between the dispersed phase and the continuous phase may be about 0.1 to about 0.5 (e.g., about 0,3) in order to maximize encapsulation efficiency.
  • the generated droplets can be configured to have a diameter of about 20 pm to about 00 prn to match the size and/or concentration of the incoming ceils and/or beads.
  • the height of the various icrofiutdie channels Is less than twice the diameter of the solid samples (e.g., cells, beads, particles, etc.) that are configured to be dispersed in dispersed phase fluid. Restricting the height of the various microfiuidic channels to be less than twice the diameter of the solid samples can advantageously reduce the chance that the soiid samples roil over each other and/or stack over each other. iwm ⁇ FiGs.
  • the first flow stream comprises Hela cells having a size of about 10 microns dispersed in water and the second fiow stream comprises particles/beads having a size of about 10 microns dispersed in wafer.
  • the concentration of the ceils or beads in water can be in the range between about 10 s - 10 8 cell or beads in 1 ml of water.
  • Fie. 5A illustrates the seff-assemb!y of 10 pm beads along *he channel wail and the self- assembly of the Hela DCis along the channel wall.
  • FIG. SB Illustrates the droplets encapsulating a single 10 micron particle/bead and a single Heia cell.
  • FIG. 8A Illustrates another embodiment of a microfiuidic device that is configured to encapsulate a single eel! in a single droplet.
  • the device of FIG. 8A comprises only two incoming microfiuidic. channels instead of three. Ceils dispersed in a first fluid ⁇ e,g,, wafer ⁇ is introduced through a first microfiuidic channel and the first fluid is introduced through the through a second microfiuidic channel. Laminar fiow is established in the combining channel such that the cells self-assemble along the channel wall.
  • the size of the generated droplets can be tuned by adjusting the droplet generatio regimes, Tfte encapsulation of a..single cell in a single droplet and/or a single bead and a single ceil In single droplet can be achieved !ii both .geometry- mediated and dripping regimes.
  • the size of the droplet can be greater t an or equal to the size of ohfiee diameter ' .
  • the droplet size can be less than the size of the orifice, in both regimes, the heads and DCis that assemble in single row along the channel wali are pulled into the droplets by the symmetrical high shear zone resulting in encapsulation.
  • the droplet size can be tailored to the size of the incoming ceils anoVor concentrations by controlling the pressure and/or flow rate ratio between the dispersed phase and the continuous phase and the capillary number.
  • the encapsulation efficiency achieved using the methods described herein can be 10% or higher. More preferably, the encapsulation efficiency achieved using the methods described herein can be 30% or higher. f» «83j EXAMPLES
  • FIGs. A-4D and F!Gs. 5A-5B A one cell-one bead encapsulation process is shown in FIGs. A-4D and F!Gs. 5A-5B.
  • 10 pm beads were introduced through an upper inlet while He!a ceils entered through a lower inlet.
  • the heads and cells then assemble single file atong the channel wail white moving towards a symmetrical high shear zone.
  • the droplet diameter can be tuned by adjusting (Dp/Cp) to achieve maximum encapsulation efficiency.
  • the encapsulation efficiency increases with the droplet diamete or (Dp/Cp); however, it starts decreasing beyond a threshold because of the multiple encapsulations.
  • FIGs. 6A-8E "10 urn beads were Introduced from both the upper and lower inlets and were encapsulated into the droplets from either side of the channel
  • the ceils were introduced from only one inlet and were encapsulated into single-ceil droplets.
  • FIGs. 9A-9B the ceils were introduced from two inlets and were encapsulated into singie-celi droplets.

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Abstract

La présente invention concerne une technique hydrodynamique passive pour effectuer une coencapsulation une cellule-une bille dans des gouttelettes. La technique hydrodynamique utilise des flux laminaires et des interfaces liquide-liquide à cisaillement élevé au niveau d'une jonction microfluidique pour obtenir une efficacité d'encapsulation un-un-un d'environ 30 %. Cette technique peut être mise en œuvre au moyen d'un dispositif microfluidique pour améliorer significativement l'efficacité de séquençage de gouttelettes et d'autres essais à cellule unique à base de billes.
PCT/US2018/036952 2017-06-09 2018-06-11 Encapsulation contrôlée dans des gouttelettes par cisaillement interfacial liquide-liquide Ceased WO2018227204A1 (fr)

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CN110038656A (zh) * 2019-05-31 2019-07-23 中国科学技术大学 一种用于乳化的双水相系统及其液滴生成模块
CN117504959A (zh) * 2023-11-14 2024-02-06 南京航空航天大学 一种基于微管的微液滴产生装置及方法
CN121825847A (zh) * 2026-03-06 2026-04-10 广西医科大学 高通量悬浮细胞球组装体的制备方法及装置

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CN110038656A (zh) * 2019-05-31 2019-07-23 中国科学技术大学 一种用于乳化的双水相系统及其液滴生成模块
CN117504959A (zh) * 2023-11-14 2024-02-06 南京航空航天大学 一种基于微管的微液滴产生装置及方法
CN121825847A (zh) * 2026-03-06 2026-04-10 广西医科大学 高通量悬浮细胞球组装体的制备方法及装置

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