WO2017004676A1 - Encapsulation de cellules uniques sur demande à l'aide d'un champ acoustique localisé - Google Patents

Encapsulation de cellules uniques sur demande à l'aide d'un champ acoustique localisé Download PDF

Info

Publication number
WO2017004676A1
WO2017004676A1 PCT/AU2016/050590 AU2016050590W WO2017004676A1 WO 2017004676 A1 WO2017004676 A1 WO 2017004676A1 AU 2016050590 W AU2016050590 W AU 2016050590W WO 2017004676 A1 WO2017004676 A1 WO 2017004676A1
Authority
WO
WIPO (PCT)
Prior art keywords
cell
interface
microfluidic channel
carrier liquid
single cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/AU2016/050590
Other languages
English (en)
Inventor
David John Collins
Tuncay ALAN
Adrian NEILD
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Monash University
Original Assignee
Monash University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from AU2015902713A external-priority patent/AU2015902713A0/en
Application filed by Monash University filed Critical Monash University
Publication of WO2017004676A1 publication Critical patent/WO2017004676A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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

  • the present invention is directed to a device, system and method for single cell encapsulation using a localised acoustic field.
  • Microfluidics is a research area which is fuelled by its ability to perform unique experiments. By reducing the size of a fluidic system it becomes possible to control the fluid environment, cell location and flow regimes highly accurately.
  • One developing role of microfluidics is for encapsulated single cell analysis, where a single cell is fully contained in an aqueous droplet that is suspended in a mutually immiscible second fluid phase.
  • the fluid interface acts as a physical barrier, eliminating undesired diffusion gradients, (2) undesired cell-cell interactions and cell-environment reactions, with the benefit of also (3) all but eliminating cell-cell or cell-wall adherence so that (4) mass quantities of encapsulated cells can be sorted, stored and handled independently. Because of these benefits, single cell analysis can be performed with significantly greater power, especially with regard to a cell's influence on its neighbouring
  • the cell concentration must be so low that the vast majority of vesicles contain no cells at all. This, however, limits the encapsulated cell production rate, and necessitates a downstream process to sort droplets containing cells from those that do not.
  • inertial focussing to increase the seeding ratio.
  • inertial focussing to increase the seeding ratio.
  • some work has been undertaken to combine the ability to order particles using inertial focussing with passive droplet generation.
  • the concept is that an ordered line of cells arrives at the droplet generating region in sequence with the generation of droplets themselves, resulting in a single-cell emulsion that is mostly (but not completely) composed of single cells in droplets.
  • Some droplets may not contain any cells and some droplets may contain multiple cells. This combination of inertial focussing and droplet generation has the potential to overcome the barrier imposed by the poisson distribution.
  • a microfluidic device for isolating a single cell on-demand, the device including: a substrate; a first microfluidic channel provided on the substrate adapted to have a solution containing cells flow within; a second microfluidic channel provided on the substrate adapted to have a carrier liquid flow within; an interface zone having an interface where the solution containing cells flowing in the first microfluidic channel and the carrier liquid flowing in the second microfluidic channel meet; and an acoustic signal source; wherein: the solution containing cells and the carrier liquid are immiscible; and application of an acoustic signal from the acoustic signal source when the single cell passes into the interface zone causes deformation of the interface and thereby directs the single cell through the interface into the carrier liquid, such that the single cell is encapsulated in a droplet of the solution the cell was originally flowing within.
  • the device preferably further includes an interrogation area upstream of the interface zone for detecting the cell prior to encapsulation.
  • the interface zone may further include in the first microfluidic channel any one or more of: (a) obstacles; (b) electrically generated forces; or (c) acoustically generated forces, to encourage or direct the cell to the interface and/or to hold the single cell at the interface zone.
  • An acoustic mismatch may occur at the interface and the movement of the cell into the carrier liquid occurs because the interface is displaced.
  • the substrate is preferably a piezoelectric substrate with patterned electrodes for generating surface acoustic waves.
  • the carrier liquid is preferably a form of oil and the solution containing cells is preferably water, cell nutrient, biological fluid or buffer solution, such as phosphate buffer solution or phosphate buffer saline (PBS).
  • PBS phosphate buffer saline
  • a method of isolating a single cell on-demand using a device having: a substrate; a first microfluidic channel adapted to have a solution containing cells flow within; a second microfluidic channel adapted to have a carrier liquid flow within; an interface zone having an interface where the solution containing cells flowing in the first microfluidic channel and the carrier liquid flowing in the second
  • microfluidic channel meet; and an acoustic signal source, the method including: introducing into the first microfluidic channel a solution including cells; introducing into the second microfluidic channel a carrier liquid, wherein the solution including cells and the carrier liquid are immiscible fluids; and applying an acoustic signal from the acoustic signal source which produces a force at the interface zone, causing deformation of the interface and thereby directing the single cell through the interface into the carrier liquid in the second microfluidic channel, such that the single cell is encapsulated in a droplet of the solution the cell was originally flowing within.
  • the acoustic signal used in the method or device described above is a surface acoustic wave signal.
  • the signal may be a travelling surface acoustic wave signal or a standing surface acoustic wave signal.
  • the method may further include the step of detecting the cell upstream of the interface zone prior to encapsulation.
  • an acoustic mismatch may occurs at the interface and the movement of the cell preferably occurs because the interface is displaced.
  • the method may further include the step of holding the single cell at the interface zone through the use of any one or more of: (a) obstacles; (b) electrically generated forces; or (c) acoustically generated forces, in the interface zone of the first microfluidic channel.
  • the method further includes the step of directing one encapsulated cell into the second microfluidic channel and directing another encapsulated cell into a third microfluidic channel.
  • the substrate used is preferably a piezoelectric substrate with patterned electrodes for generating surface acoustic waves.
  • Figures 1 (a) to 1 (e) show the steps of a method according to an embodiment of the present invention. These figures also show a cross-sectional schematic of a device according to another embodiment of the present invention.
  • Figures 2(a) and 2(b) show a cross-sectional schematic of a device according to yet another embodiment of the present invention.
  • Figure 1 (a) shows a part of a device or system for encapsulating single cells using surface acoustic waves (SAW).
  • SAW surface acoustic waves
  • the device of the present invention produces droplets to encapsulate cells on-demand, that is, a single droplet is formed when required, and in this case as a result of a pulsed actuation of the acoustic source. As shown in
  • the device 1 includes at least two channels, a cell solution channel 3 through which a solution 6 containing cells flows, and a carrier liquid channel 4, through which a carrier liquid 7 flows.
  • the device 1 also includes an acoustic signal source 2 for generating an acoustic signal.
  • the acoustic signal source is a surface acoustic wave (SAW) device for generating SAWs.
  • the device 1 also has a cell solution-carrier liquid interface 5. This interface 5 occurs at an area where there is a gap in a wall which is common to each channel 3, 4 , such that an interface 5 forms when the cell solution fluid 6 in one channel 3 meets the carrier liquid 7 in the other channel 4.
  • the channels are arranged on a substrate (not shown).
  • the channels may be made using a material which is bonded to the substrate.
  • the channel material is preferably polydimethylsiloxane (PDMS) but may be any other suitable polymer or other material.
  • the channels may be made in the material which is then bonded to the substrate.
  • the channels may be formed by a combination of the material and the substrate.
  • the material may form the side walls and top of the channel and the substrate forms the base of the channel.
  • the channels may be etched into the substrate.
  • the substrate may be a piezoelectric substrate with patterned electrodes for generating acoustic signals.
  • the acoustic signal source may be formed as part of the substrate or is provided on the substrate.
  • Figures 1 (a) to 1 (e) show the different stages in a method for producing encapsulated single cells using the device 1 .
  • Figure 1 (a) shows cells 8 in the cell solution 6 flowing through the cell solution channel 3.
  • the figure also shows single cells 9 that have been encapsulated in droplets of the cell solution 6 flowing along the carrier liquid 7 in the carrier liquid channel 4.
  • FIG. 1 (b) the box 33 around cell 8 has been used to illustrate the interrogation area 33. It will be appreciated that in the embodiment shown the interrogation area does not have physical barriers such as a box or square, however, other embodiments may have physical obstacles in or which form the interrogation area.
  • An individual cell 8 is detected as it flows through the channel 3 when it approaches or is within the interrogation area 33 as shown in Figure 1 (b). The individual cell may be detected by any conventional method including an electrical, optical or acoustic method.
  • a focussed SAW 22 is applied (as shown in Figure 1 (d)) which forces the cell 8 toward the interface 5.
  • Applying the SAW also deforms the interface 5 as shown in Figure 1 (d).
  • the single cell 8 is encapsulated by a small amount of cell solution 66 (see Figure 1 (e)).
  • the encapsulated single cell 9 is forced into the carrier liquid channel 4 and flows within the carrier liquid 7 along the channel 4 down stream for further processing, sorting or other diagnostics.
  • a burst of SAWs 22 pushes the cell to the interface 5 of the cell solution 6 and the carrier liquid 7, for example, a water-oil interface (that is, the solution 6 containing the cells is water, and the carrier liquid 7 is oil).
  • the solution containing the cells could also be cell nutrient, biological fluid or buffer solution, such as phosphate buffer solution or phosphate buffer saline (PBS).
  • the SAW 22 pushes the cell 8 towards the interface 5
  • the SAW 22 also deforms the interface 5 so that it extends into the carrier liquid 7 phase (See Figure 1 (d)). If the SAW 22 is of sufficiently high amplitude, pressure conditions on either side of the distended interface 5 will result in droplet pinch-off, where the droplet of cell solution 66, for example water, contains the now encapsulated cell 9.
  • the required energy which results in pinch-off may depend on a number of factors including: the frequency of excitation of the ultrasonic surface acoustic wave; the size of electrodes in the SAW generating device; the viscosity of the carrier liquid and the viscosity of the cell solution; the length of the electrical pulse applied to the electrodes; the size of the cell to be encapsulated; the flow rates of each of the carrier liquid and the cell solution; and the surface tension at the interface of the carrier liquid and cell solution.
  • the cell can be encouraged, directed or brought to the interface zone through various means, including obstacles, electrically generated forces or acoustically generated forces.
  • the cells 8 travel in the channel 3 with the cell solution 6. As the cells near the interface 5, the device 1 encourages and forces the cells 8 to move towards the interface 5 of the cell solution 6 and the carrier liquid 7. Whilst similar to the previous embodiment described, in this embodiment, the cells are then held at the interface zone 55. This may be achieved by using obstacles in the channel or using electrically or acoustically generated forces (of various methods). As shown in Figure 2(b), once held at the interface, a burst of SAWs are applied from the SAW generating device 2. The SAW generating device 2 may have curved electrodes as shown in Figure 2.
  • the SAWs push the cell 8 into the interface 5 and as a result cause part of the cell solution in which the cell is flowing to be pinched off from the rest of the cell solution 6. This results in a droplet 66 containing a cell 9 being pushed and directed into the carrier liquid 7.
  • the pulse of acoustic waves pushes the cell (with a small amount of solution 66) into the carrier liquid (or secondary) stream 7, for example, an oil stream, such that a droplet 66 is formed which encapsulates the cell 9.
  • the present invention allows a cell to be encapsulated on-demand as the cell passes a fixed point.
  • the present invention detects the cell as it approaches the acoustic wave signal source and times the droplet formation by the acoustic signal around the timing or flow of the cell, rather than the other way around as taught by the prior art.
  • the prior art tries to order the cells so that they arrive at an interface in time with a natural droplet formation rate, effectively controlling the cells to arrive at the interface of the cell solution and carrier liquid at the same periodicity as the natural production of a droplet.
  • the present invention waits for a cell to arrive in a liquid stream, which does not necessarily arrive at a uniform spacing or periodicity.
  • the present invention then forms a droplet around the cell while it travels.
  • the present invention advantageously uses actively-controlled acoustic forces to control droplet formation, as and when it is needed, rather than controlling the cells to coincide with droplets that are naturally formed.
  • This device and method of the present invention has application in drug delivery and dosage control as well as for the analysis and diagnostics of cells in isolation allowing interrogation of physical properties without the influence of other cell, for example using flow cytometry.
  • the device and method of the present invention could also be used for drug testing, for example, the droplets containing the single cell could be merged with droplets containing a drug solution and a response detected.
  • the device may contain droplets of different drug dosages to ascertain the response.
  • This device and method could be used to identify knowledge of the cell behaviour could be used to establish deviations from the standard response indicative of disease.
  • SAWs are optimal for this acoustic microfluidics application due to their planar nature, and SAWs are easily integrated directly into microfluidic systems with efficient energy transfer from the substrate to the fluid.
  • the active nature of the system of the present invention using an acoustic field improves cell seeding and substantially increases the cell seeding ratio. Further, the present invention controls when a droplet is formed, rather than controlling when the cell arrives to align with the produced droplet, as many prior art devices and methods do.
  • the system of the present invention is able to be integrated onto a microchip unlike many prior art systems.
  • the present invention significantly improves the efficiency of encapsulating single cells with no instances of multiple cells contained in a droplet, or no cells contained in a droplet.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Sustainable Development (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

La présente invention concerne un dispositif microfluidique et un procédé permettant d'isoler une cellule unique sur demande, le dispositif comprenant : un substrat; un premier canal microfluidique (3) prévu sur le substrat adapté pour avoir une solution contenant des cellules s'écoulant à l'intérieur; un second canal microfluidique (4) prévu sur le substrat adapté pour avoir un liquide porteur s'écoulant à l'intérieur; une zone d'interface (55) ayant une interface (5) où la solution contenant des cellules s'écoulant dans le premier canal microfluidique et le liquide porteur s'écoulant dans le second canal microfluidique se rejoignent; et une source de signal acoustique (2); la solution contenant des cellules et le liquide porteur étant non miscibles; et l'application d'un signal acoustique à partir de la source de signaux acoustiques lorsque la cellule unique passe dans la zone d'interface provoque la déformation de l'interface et, de ce fait, dirige la cellule unique par l'intermédiaire de l'interface dans le liquide porteur, de telle sorte que la cellule unique soit encapsulée dans une gouttelette de la solution à l'intérieur de laquelle la cellule s'écoulait initialement.
PCT/AU2016/050590 2015-07-09 2016-07-07 Encapsulation de cellules uniques sur demande à l'aide d'un champ acoustique localisé Ceased WO2017004676A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AU2015902713A AU2015902713A0 (en) 2015-07-09 On demand single cell encapsulation using localised acoustic field
AU2015902713 2015-07-09

Publications (1)

Publication Number Publication Date
WO2017004676A1 true WO2017004676A1 (fr) 2017-01-12

Family

ID=57684740

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/AU2016/050590 Ceased WO2017004676A1 (fr) 2015-07-09 2016-07-07 Encapsulation de cellules uniques sur demande à l'aide d'un champ acoustique localisé

Country Status (1)

Country Link
WO (1) WO2017004676A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020155231A1 (en) * 2000-09-25 2002-10-24 Ellson Richard N. Use of immiscible fluids in droplet ejection through application of focused acoustic energy
US20120236299A1 (en) * 2011-02-11 2012-09-20 The Regents Of The University Of California High-speed on demand droplet generation and single cell encapsulation driven by induced cavitation
US20130213488A1 (en) * 2010-08-23 2013-08-22 President And Fellows Of Harvard College Acoustic waves in microfluidics
WO2014066624A1 (fr) * 2012-10-26 2014-05-01 President And Fellows Of Harvard College Systèmes et procédés de production et de manipulation de gouttelettes à l'aide d'ondes acoustiques

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020155231A1 (en) * 2000-09-25 2002-10-24 Ellson Richard N. Use of immiscible fluids in droplet ejection through application of focused acoustic energy
US20130213488A1 (en) * 2010-08-23 2013-08-22 President And Fellows Of Harvard College Acoustic waves in microfluidics
US20120236299A1 (en) * 2011-02-11 2012-09-20 The Regents Of The University Of California High-speed on demand droplet generation and single cell encapsulation driven by induced cavitation
WO2014066624A1 (fr) * 2012-10-26 2014-05-01 President And Fellows Of Harvard College Systèmes et procédés de production et de manipulation de gouttelettes à l'aide d'ondes acoustiques

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
COLLINS, D. J. ET AL.: "Surface acoustic waves for on-demand production of picoliter droplets and particle encapsulation", LAB ON A CHIP, vol. 13, no. 16, 2013, pages 3225 - 3231, XP055332059 *
LAGUS, T. P. ET AL.: "A review of the theory, methods and recent applications of high- throughput single- cell droplet microfluidics", JOURNAL OF PHYSICS D: APPLIED PHYSICS, vol. 46, no. 11, 2013, pages 114005 - 114025, XP020242379 *

Similar Documents

Publication Publication Date Title
Kersaudy-Kerhoas et al. Recent advances in microparticle continuous separation
Xi et al. Active droplet sorting in microfluidics: a review
Collins et al. The Poisson distribution and beyond: methods for microfluidic droplet production and single cell encapsulation
US10780413B2 (en) High-speed on demand microfluidic droplet generation and manipulation
CN108432132B (zh) 微流体颗粒操纵
Collins et al. Highly localized acoustic streaming and size-selective submicrometer particle concentration using high frequency microscale focused acoustic fields
Destgeer et al. Recent advances in microfluidic actuation and micro-object manipulation via surface acoustic waves
Pit et al. Droplet manipulations in two phase flow microfluidics
Kim et al. Nanowire-integrated microfluidic devices for facile and reagent-free mechanical cell lysis
Feng et al. Advances in micro-droplets coalescence using microfluidics
TW200536601A (en) Micorfluidic treatment method and device
Fergola et al. Droplet generation and manipulation in microfluidics: a comprehensive overview of passive and active strategies
Bussiere et al. High-throughput triggered merging of surfactant-stabilized droplet pairs using traveling surface acoustic waves
CN110918139B (zh) 微流控芯片、含有该微流控芯片的装置及样本浓缩的方法
CN108212236A (zh) 一种实现液滴对/气泡对同步运动并融合的微流控芯片
Li et al. Monodisperse water-in-oil-in-water emulsions generation for synthesising alginate hydrogel microspheres via locally hydrophobic modification to PMMA microchannels
CN105013544B (zh) 一种基于亲水纤维丝诱导的微液滴融合方法
Xu et al. Fusion and sorting of two parallel trains of droplets using a railroad-like channel network and guiding tracks
Xu et al. Droplet coalescence in microfluidic systems
CN110918141B (zh) 微流控芯片及含有该微流控芯片的装置，以及用于制备微乳化液滴的应用
KR102354673B1 (ko) 관성을 기반으로 한 세포 내 전달 미세 유체 플랫폼
CN108607376B (zh) 一种基于振荡流的液滴融合方法及器件
WO2017004676A1 (fr) Encapsulation de cellules uniques sur demande à l'aide d'un champ acoustique localisé
CN111215159B (zh) 微流控芯片及基于该芯片融合样本的方法
CN114867556A (zh) 将材料输送到细胞内的基于液滴变形方法和用于其的芯片

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16820572

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 16820572

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 16820572

Country of ref document: EP

Kind code of ref document: A1