WO2021097456A1

WO2021097456A1 - Guided Microfluidic Flow for Cell Capture, Indexing, and Directed Release

Info

Publication number: WO2021097456A1
Application number: PCT/US2020/060796
Authority: WO
Inventors: Nathaniel Kegan LIU; Kristina Waiyen CHAN; Lydia L. Sohn
Original assignee: The Regents Of The University Of California
Priority date: 2019-11-17
Filing date: 2020-11-16
Publication date: 2021-05-20

Abstract

A guided microfluidic flow device comprising a flow-path comprising an inlet, a cell track, streamline guide structures, and an outlet configured for cell or microparticle capture, indexing, and/or directed release.

Description

Guided Microfluidic Flow for Cell Capture, Indexing, and Directed Release

This invention was made with government support under Grant Numbers CA190843 and EB024989 awarded by the National Institutes of Health. The government has certain rights in the invention.

[001] Introduction

[002] As single-cell testing has become increasingly common in biomedical research, the ability to identify and handle specific cells of interest is necessary to link experimental data to the corresponding cell from which it was obtained. Though there are several popular methods of marking and manipulating specific cells, such as fluorescence-activated cell sorting (FACS) and direct manual handling via micro-pipettes, these techniques have chemically or physically invasive effects which can compromise cell viability or cell function.

[003] The disclosed technology was originally developed to link data from a single-cell mechanical-phenotyping platform and a single-cell RNA-transcriptome sequencing platform. This allows for the information corresponding from each experimental setup to be coupled with the exact cell from which it came, to search for trends in gene expression and cellular mechanical properties. As the mechanical phenotyping setup utilized continuous flow of cell samples while the RNA sequencing device used sequential routing of cells, a subsystem capable of capturing cells between these experimental platforms while preserving each cell’s order and identity was needed. Such a system should be minimally invasive, both chemically and physically, to preserve the integrity of the data types to be obtained from each cell, and should be scalable in both size and quantity to accommodate a broad range of biomicrofluidic applications. The disclosed devices and systems provide these features and functionalities.

[004] Summary of the Invention

[005] The invention provides guided microfluidic devices, systems and methods for cell capture, indexing, and/or directed release

[006] In an aspect the invention provides a guided microfluidic flow device or system comprising a flowpath comprising an inlet, a cell track, streamline guide (fluidic resistance tuning) structures, and an outlet, essentially as disclosed, configured for cell or microparticle capture, indexing, and/or directed release (Fig. 1).

[007] In embodiments: [008] the device or system comprises railing features and divot capture mechanisms as described (Fig. 1);

[009] the device or system comprises or is bonded to a substrate which can be functionalized with one or more biochemical compounds such as antibodies or polynucleotides, or a bioactive agent, such as a bioactive ligand, cell, receptor, dmg, candidate agent, etc. (Fig. 2);

[010] the device or system comprises or is bonded to a substrate comprising incorporated patterned electrodes, integrated circuits and/or MEMS actuators (Fig. 2);

[Oil] the device or system comprises multi-height reliefs, wherein narrower full-height features serve as tracks for cells or other experimental particles or subjects to flow, shorter regions permit flow of carrier solution but are not tall enough for cells or particles or subjects of interest to flow, and isolated full-height features provide flow-modulating (tuning) cavities to strategically reduce fluidic resistance and produce desired flow profiles (Figs. 1, 2);

[012] the device or system or substrate comprises a surface, such as a glass or quartz surface, preferably functionalized for attaching probes, such as with aldehyde groups, or a plastic, such as cyclic olefin copolymer, polymethylmethacrylate (PMMA), polydimethylsiloxane (PDMS), or thermal plastic, or a composite of a glass or plastic surface layer bonded to a glass or plastic substrate (Fig. 2);

[013] the device or system is operably connected to a microfluidic pump and/or downstream fluid analyzer; and/or

[014] the device or system is operably connected to a plurality or array of such devices and configured for multiplex operation and/or operative for a combination of ligands, cells, etc.

[015] In an aspect the invention provides a method of using a disclosed device or system, comprising flowing the medium through the channel, and capturing, isolating and/or indexing the cells, particles, or subjects of interest In an aspect the invention provides a method of making a disclosed device or system such as comprising photolithography applied to successive layers of spin-coated resist to achieve the multiple feature heights (Fig. 1).

[016] The invention encompasses all combination of the particular embodiments recited herein, as if each combination had been laboriously recited.

[017] Brief Description of the Drawings

[018] Fig. 1 : 3D rendering of the dual-height mold used to cast the device. To achieve multiple feature heights, photolithography is applied to successive layers of spin-coated photoresist. The features are developed all at once to reveal the completed, multi-height mold. Feature sizes and ratios can be scaled to the application. An example device useful for circulating tumor cell (CTC) assays has feature heights of 20 mhi and secondary feature heights of 5 mhi. The inset cross-section view depicts the two feature heights.

[019] Fig. 2: 3D rendering of device features transferred into PDMS. The device can be bonded to a variety of substrates depending on the application, such as a plain silicon wafer or glass slide. Substrates can be functionalized with biochemical compounds such as antibodies or DNA strands, and further microfabrication techniques can be used to incorporate patterned electrodes, integrated circuits, or MEMS actuators. The inset depicts cell capture sites and streamline guide features constituting the region in which guided microfluidic flow for cell capture occurs.

[020] Fig. 3 : Flow simulation results depicting the flow-modulating effects of the streamline guide features. Carrier flow is modulated such that individual cells are carried along the cell track until they encounter an available semicircular divot capture site. A single cell will be immobilized against the semicircular divot due to continuous force from the flow of the carrier solution. The trajectory and capture of a single cell (represented as a spherical particle) is included in the simulation.

[021] Fig. 4: Simulation results demonstrating the effect of increasing streamline guide feature size. As the size of the streamline guide feature is increased, flow of the carrier solution is modulated through the cell capture sites and applies a hydrodynamic force that directs cells towards the single-cell capture sites. When the streamline guide feature is sufficiently large, the force is sufficient to cause single cells to become gently retained against the first vacant semicircular divot capture site (rightmost image).

[022] Fig. 5 : Still images from high-speed camera (500 fps) demonstrating guided microfluidic flow resulting in single-cell capture. Left panel: A cell approaches a vacant site and is captured. Right panel: A subsequent cell is carried past the now-occupied site. The device shown was optimized for 25 pm cells, and the cells captured in these images are this size.

[023] Fig. 6: Side-by-side visualizations of single-cell capture (left) and subsequent release (right). The guided microfluidic flow platform can be used to capture flows using flow driven by a single inlet pressure. After cells are captured in order and indexed, reversing the direction of actuation pressure can be used as a facile means of releasing the cells in the order of capture.

[024] Description of Particular Embodiments of the Invention

[025] Unless contraindicated or noted otherwise, in these descriptions and throughout this specification, the terms “a” and “an” mean one or more, the term “or” means and/or. The examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein, including citations therein, are hereby incorporated by reference in their entirety for all purposes.

[026] Disclosed are multilayer microfluidic devices designed and configured to actively categorize, sort, parse, capture, index, and/or pointedly release cells or other microscale objects of interest on a single platform. The device is generally configured for applicability to a multitude of cell processing platforms and generalized integration and/or connectability to such systems. The system permits minimal chemical and physical invasiveness, making it practical for preliminary, intermediary, or terminal analysis and sample transportation.

[027] The disclosed devices are less chemically and physically invasive to cells or particles as compared to existing methods. The disclosed devices and systems do not require any chemical modification of a cell or even the introduction of a surface marker, unlike methods such as fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS). They also do not require physical grasping or repeated deformation of cells, unlike manipulation via micro-pipette. As the technology can be directly integrated into experimental process flows it can circumvent the need for sample transport between devices, saving time and effort. Additionally the use of parallelizable fabrication process enables multiplexing for scalable throughput.

[028] The fabrication method is generally based on conventional photolithography methods for producing microchannels (see, e.g. A. Mata, S. Roy. Fabrication of multi-layer SU-8 microstructures. Journal of Micromechanics and Microengineering. 2006; D. Di Carlo, L. Lee. Dynamic Single Culture Array. Lab on a Chip. 2006), but differs in that the patterning steps are repeated to achieve multi-height features. Hence the device comprises individually standing features of different step heights without compromising the integrity or continuity of surrounding features.

[029] The readily-scalable nature of the fabrication process enables the fabrication of features tuned for cells of any size. Hence, the technology can be applied to specifically and reliably guide, separate, and isolate cells without compromising cellular integrity, the continuous flow of the experiment, or the order of the incoming cell samples and their associated data. The device’s fluidic railing features focus and confine the motion of cells to designated tracks within the channels while permitting fluid flow across the entirety of the cross-sectional area; see, e.g. Figs 1-6. [030] As the cells move through, travel along and/or are contained by the railing features, the device accommodates changes in the carrier solution such as replacement of cell media, flushing and rinsing with buffered solution, or introduction of other fluids.

[031] In one embodiment the mechanism of cell capture and control comprises divot features along the designated track for cell flow (Fig. 1); however this does not preclude the integration of other cell-interfacing features into the railing structures. The divots are preferably oriented against the directed laminar flow of the channel to intercept and retain one cell each. The release mechanism implemented to retrieve the sequestered cells is tailored to the specific application; some options include etched holes on the substrate for direct extraction, pneumatic valves to enable negative pressure extraction, or simply backflow to guide cells out of the device. Cell capture and containment may be further augmented by streamline guide features, which are strategically-placed regions or areas of empty flow volume used to direct streamlines through modulation of fluidic resistance. Such regions provide a larger open volume and encourage the fluid to flow towards and through those spaces (Fig. 3, 4).

[032] The disclosed devices and systems are preferably chemical label-free and physically gentle upon target cells, thus minimizing any detrimental effects to cell experiments. Preferred embodiments retain the identity of captured cells, which allows for the same cells to be routed through different experimental setups and for the resulting data to be linked back to the correct cell specimen. One application is to correlate a cell’s mechanical deformability to its gene expression, with practical applications in clinical diagnosis and treatment pathways, such as cancer. Another application is for targeted cell manipulation for drug screening or single-cell sequencing. Furthermore, with feature scaling this technology can be applied to retain, for example, larger white blood cells from a whole blood sample for subsequent experimental use; can be used to capture cells without restricting the flow of the carrier fluid; and can be used to efficiently change the fluid surrounding retained cells.

[033] Example: EXPERIMENTAL/FABRICATION

[034] Our platform was made using soft lithography. A negative mold consisting of two overlaid SU-8 photoresist layers was lithographically patterned onto a silicon wafer. The first SU-8 layer (SU-8 3005, Kayaku Advanced Materials), was spin-coated at 3000 rpm to achieve a thickness of 5 pm and the second layer of SU-8 (SU-8 3025, Kayaku Advanced Materials) was spin coated at 4000 rpm to achieve a thickness of 20 pm. The two layers were exposed using individual photomasks and developed simultaneously to produce a dual-height mold with a maximal thickness of 25 pm and a secondary thickness of 5 pm. PDMS (SYLGARD™ 184 Silicone Elastomer Kit, Dow Chemical) was cast and cured upon the mold, resulting in a negative-imprint PDMS device that was subsequently bonded to a glass substrate. Capture experiments were performed using MCF-7 breast epithelial cells and 25 pm reference microspheres (Sigma- Aldrich). COMSOL Multiphysics 5.3a was used to model the microfluidic flow and simulate the capture and release of single cells.

[035] Example: TESTING

[036] Testing of the guided microfluidic cell capture and release platform was conducted using both live cells (MCF-7 breast epithelial cell line) and reference microspheres (25 pm). Carrier solution was formed by suspending cells or microspheres in IX PBS buffer solution at a concentration of 10M/mL. A pneumatic microfluidic pressure controller (Elveflow OBI) was used to drive carrier solution containing either cells or microspheres at 20 kPa into the fabricated devices. Cell capture was observed using a high-speed camera at 500 fps (FASTEC IL-5).

Single cells or microspheres were observed to become captured in semicircular capture sites in the order of arrival (Fig. 5). To release captured cells or microspheres, a 20 kPa backflow pressure was applied and single-cell trap sites were vacated in the order of arrival.

Claims

CLAIMS:

1. A guided microfluidic flow device comprising a flowpath comprising an inlet, a cell track, streamline guide structures, and an outlet, configured for cell or microparticle capture, indexing, and directed release.

2. The device of claim 1 comprising multi-height reliefs, wherein narrower full-height features serve as tracks for the cells or microparticles to flow, while isolated full-height features provide flow-modulating cavities to strategically reduce fluidic resistance and produce desired flow profiles, wherein the shorter features are not tall enough for the cells or microparticles to flow, but permit flow of carrier solution (Fig. 1).

3. The device of claim 1 comprising railing features and a divot capture sites.

4. The device of claim 2 comprising railing features and a divot capture cites.

5. The device of claim 1, 2, 3 or 4 comprising or bonded to a substrate functionalized with one or more biochemical compounds such as antibodies or polynucleotides, or a bioactive agent, such as a bioactive ligand, cell, receptor, drug, candidate agent, etc. (Fig. 2)

6. The device claim 1, 2, 3 or 4 comprising or bonded to a substrate comprising incorporated patterned electrodes, integrated circuits and/or MEMS actuators (Fig. 2).

7. The device of claim 1, 2, 3 or 4 comprising a surface or a substrate surface, such as comprising glass or quartz, functionalized for attaching cell or particle capture probes, such as with aldehyde groups, or a plastic, such as cyclic olefin copolymer, polymethylmethacrylate (PMMA), polydimethylsiloxane (PDMS), or thermal plastic, or a composite of a glass or plastic surface layer bonded to a glass or plastic substrate (Fig. 2).

8. The device of claim 1, 2, 3 or 4, comprising a medium, wherein the medium is flowing through the flowpath.

9. The device of claim 1, 2, 3 or 4, operably connected to a microfluidic pump and/or downstream fluid analyzer.

10. The device of claim 1, 2, 3 or 4, operably connected to a plurality or array of such devices and configured for multiplex operation and/or operative for a combination of ligands, cells, etc.

11. A method of using the device of claim 1, 2, 3 or 4, comprising flowing the medium through the channel, and capturing, isolating, and/or indexing of the cells or microparticles.

12. A method of making the device of claim 1, 2, 3 or 4, comprising employing photolithography applied to successive layers of spin-coated resist to achieve the multiple feature heights.