Patent Application
20180015471
The present invention, in some embodiments thereof, relates broadly to methods and apparatus for single-cell isolation (such as for processing or analysis) and, more particularly to techniques for isolating single cells using microfluidic technology.
Microfluidics is a multidisciplinary field intersecting engineering, physics, chemistry, biochemistry, nanotechnology, and biotechnology, with practical applications to the design of systems in which low volumes of fluids are processed to achieve multiplexing, automation, and high-throughput screening. Advances in microfluidics technology are revolutionizing molecular biology procedures for enzymatic analysis (e.g., glucose and lactate assays), DNA analysis (e.g., polymerase chain reaction and high-throughput sequencing), and proteomics. The basic idea of microfluidic biochips is to integrate assay operations such as detection, as well as sample pre-treatment and sample preparation on one chip.
There is increased evidence that phenotypic and genotypic heterogeneity in cell populations widely exists. The key information from individual rare cells may be masked by bulk cell analysis. Single-cell analysis, especially sequencing of DNA and RNA, has therefore become significantly important for clonal mutation, tumor evolution, embryonic development, and immunological intervention.
The initial and key step for such downstream single-cell genetic analysis is to effectively isolate live single cells of interest from heterogeneous cell populations into submicroliter medium volume, followed by PCR (polymerase chain reaction) analysis. (PCR is a technology in molecular biology used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.)
Besides laser capture microdissection primarily used to isolate single cells from formalin-fixed paraffin-embedded tissue, there are currently four main approaches for single-cell isolation from cell suspensions.
In the original Microfluidic Aliquot Chip (MA-Chip), 120 channels connect directly to one center inlet well in a radial pattern, resulting in a space of less than 40 μm between two neighboring channels around the center inlet well. Although the 40 μm of fabrication resolution can be well achieved by photolithography to make PDMS MA-Chips, it is challenging to fabricate such MA-Chips with plastic materials, such as polystyrene (PS), polypropylene (PP), polycarbonate (PC), and polymethyl methacrylate (PMMA), by using injection molding or laser cutting. Therefore, an alternative design of MA-Chip with the increased space of two neighboring channels is required for the mass fabrication of plastic MA-Chips.
The branched MA-Chip Type 1 (bMA-Chip T1) in the present invention is designed to increase the space between two neighboring channels around the center inlet well while maintaining uniform liquid distribution from the center inlet well to the outlet wells. The original MA-Chip contains one segment, in contrast, the bMA-Chip T1 contains multiple segments, allowing the channel number around the center inlet well to decrease from 120 to 24 and even 12. These improvements reduce channel density and provide an extra space around the center inlet well. As a result, the space between neighboring channels increases from less than 40 μm to more than 400 μm. The improved design in bMA-Chip T1 can meet the requirement for the mass fabrication of plastic MA-Chips.
An objective of the present invention is to provide a technique for simple, rapid, and versatile single-cell isolation using microfluidic technology. In this invention, a single cell is isolated by aliquoting from a suspension of a large number of cells, independently of cell size, shape, and motility. The original microfluidic aliquot chip provides such functions, however it consists of a plurality of straight channels in the chip, resulting in the densely positioned channels. The branched Microfluidic Aliquot Chip—Type 2 (bMA Chip-T2) and the branched Microfluidic Aliquot Chip—Type 3 (bMA Chip-T3) in the present invention are designed as an improvement to the original MA-chip due to the multiple branched channels. This offers the advantage of reduced clogging, strengthened sealing, and enhanced isolation through uniform distribution of flow resistance into the branched channels.
The original design and fabrication methods of the original MA-Chip are based on photolithography followed by soft PDMS casting on the mold. While the photolithography based manufacturing process ensures high resolution of the microstructures in MA-Chip, the daily manufacturing output is limited. The roughly estimated production cycle time for a MA-Chip is 1 hr. This low production rate results in high production costs. To reduce both production costs and final market price, the MA-Chip must be redesigned so that it is appropriate for standard mass production strategy such as the injection molding process. The new design is suitable for a high output production process such as injection molding to greatly increase the production rate.
The injection mold design of the present MA-Chip allows mass production by the injection molding process. With standard operation, the estimated production cycle time for a single device is 10 s, which is 360 times higher than the original rate. Compared to the original design, the new design also improves MA-Chip function, operation, and compatibilities. The device is assembled and packaged for ready to use application. It reduces additional operations such as placing the MA-Chip on a flat sterile surface and ensuring sealing of the flow channel.
The original MA-Chip has a unique design of radial channels connecting a center inlet to surrounding outlet wells and provides the capability to isolate and identify rare single cells in a mixed population with a simple pipetting operation. The vital design and fabrication element is the smooth connection of micrometer scale channels with millimeter scale wells. In the original manufacturing scheme, the micrometer size channels (30-80 μm) are fabricated by soft lithography followed by PDMS molding, and the millimeter scale wells (1.5-2 mm) are created by mechanical punch press. Thus, this manual operation demands a significant amount of time and labor. To improve throughput of the hole punch process, a multiplexed hole punch device is designed.
A multiplex hole punch strategy for the rapid fabrication of MA-Chip is designed to meet the requirement of mass production while maintaining the original MA-Chip manufacturing format. Current operation requires the holes to be punched manually by trained individuals. In one MA-Chip, there are 96-120 holes and alignment is required in each hole punch process. The quality of outcome and labor time in this process highly depends on the operator's skill and experience. The multiplex hole punch is designed to increase throughput of the hole punch process while maintaining the original design format of the MA-Chip.
Photolithography is suitable for fabricating high quality PDMS channels but difficult for making holes. In contrast, laser cutting or injection molding can easily achieve mass production of plastic holes but difficult to make high quality channels. The two methods can be combined to achieve rapid fabrication of MA-Chips. The present invention includes a basic strategy for the rapid fabrication of MA-Chip to meet the requirement of mass production. The operation is to align and combine two patterned layers.
In the original MA-Chip, the outlet wells are primarily located in the edge of the device with a radial pattern. However, the majority of the MA-Chip is occupied by radial channels, resulting in wasted space and difficulty in further increasing the number of outlet wells to meet the requirement of high-throughput assay, such as a device containing hundreds to thousands of wells. Therefore, a new design of the MA-Chip is required. The present invention has a rectangular MA-Chip (rMA-Chip) that has the potential to integrate hundreds to thousands of outlet wells in the size of a standard 96-well plate.
U.S. Pat. No. 6,632,656 (2003 Oct. 14; Thomas et al.), incorporated by reference herein, discloses apparatus and methods for performing cell growth and cell based assays in a liquid medium. The apparatus comprises a base plate supporting a plurality of micro-channel elements, each micro-channel element comprising a cell growth chamber, an inlet channel for supplying liquid sample thereto and an outlet channel for removal of liquid sample therefrom, a cover plate positioned over the base plate to define the chambers and connecting channels, the cover plate being supplied with holes to provide access to the channels. Means are incorporated in the cell growth chambers, for cell attachment and cell growth. More particularly, as shown and described therein:
Referring to FIG. 1b, the apparatus comprises a rotatable disc (18) microfabricated to provide a sample introduction port located towards the centre of the disc and connected to an annular sample reservoir (9) which in turn is connected to a plurality of radially dispersed micro-channel assay elements (6) each of said micro-channel elements comprising a cell growth chamber, a sample inlet channel and an outlet channel for removal of liquid therefrom and a cover plate positioned onto said disc so as to define closed chambers and connecting channels. Each micro-channel element is connected at one end to the central sample reservoir (9) and at the opposing end to a common waste channel (10).
Each of the radially-dispersed micro-channel elements (6) of the microfabricated apparatus (shown in FIG. 1a) comprises a sample inlet channel (1) connected at its left hand-end end to the reservoir (9), a cell growth chamber (2) for performing cell growth and connected through a channel (4) to an assay chamber (3) and an outlet channel (5) connected at its right-hand end to the waste channel (10).
Suitably the disc (18) is of a one- or two-piece moulded construction and is formed of an optionally transparent plastic or polymeric material by means of separate mouldings which are assembled together to provide a closed structure with openings at defined positions to allow loading of the device with liquids and removal of waste liquids. In the simplest form, the device is produced as two complementary parts, one or each carrying moulded structures which, when affixed together, form a series of interconnected micro-channel elements within the body of a solid disc. Alternatively the micro-channel elements may be formed by micro-machining methods in which the micro-channels and chambers forming the micro-channel elements are micro-machined into the surface of a disc, and a cover plate, for example a plastic film, is adhered to the surface so as to enclose the channels and chambers.
The scale of the device will to a certain extent be dictated by its use, that is the device will be of a size which is compatible with use with eukaryotic cells. This will impose a lower limit on any channel designed to allow movement of cells and will determine the size of cell containment or growth areas according to the number of cells present in each assay. An average mammalian cell growing as an adherent culture has an area of −300 μm2; non-adherent cells and non-attached adherent cells have a spherical diameter of −10 μm. Consequently channels for movement of cells within the device are likely to have dimensions of the order of 20-30 μm or greater. Sizes of cell holding areas will depend on the number of cells required to carry out an assay (the number being determined both by sensitivity and statistical requirements). It is envisaged that a typical assay would require a minimum of 500-1000 cells which for adherent cells would require structures of 150,000-300,000 μm2, i.e. circular ‘wells’ of 400-600 μm diameter.
The configuration of the micro-channels . . . is preferably chosen to allow simultaneous seeding of the cell growth chamber by application of a suspension of cells in a fluid medium to the sample reservoir by means of the sample inlet port, followed by rotation of the disc (18) by suitable means at a speed sufficient to cause movement of the cell suspension outward towards the periphery of the disc by centrifugal force. The movement of liquid distributes the cell suspension along each of the inlet micro-channels (1, 8) towards the cell growth chambers (2, 7). The rotation speed of the disc is chosen provide sufficient centrifugal force to allow liquid to flow to fill the cell growth chamber (2, 7), but with insufficient force for liquid to enter the restricted channel (4, 16) of smaller diameter on the opposing side of the cell growth chamber.
In a variant, a microfluidic aliquot (MA) chip for performing single-cell isolation, comprises a chip having a radius, a center, a top surface, a bottom surface, an outer edge and a thickness; a single center inlet well disposed substantially at the center of the chip, extending into and accessible from the top surface of the chip; a plurality of side outlet wells disposed in an annular outer portion of the chip, extending into and accessible from the top surface of the chip; and a plurality of channels having multiple segments that extend into the bottom surface of the chip and extending from the center inlet well to the side outlet wells in fluid communication with the center inlet well and the side outlet wells, maintaining uniform distribution of liquid from the center inlet well to the side outlet wells.
In another variant, the chip is an MA chip type 1 (bMA Chip T1), that comprises a first segment that is an inner layer, a last segment is an outer layer, and any segment between the first segment and the last segment is a middle layer; a liquid and a plurality of cells, which are uniformly distributed into the side outlet wells through the multiple segments; wherein the first segment is divided from the center inlet well in a radial pattern; and wherein each segment after the first segment is divided from a prior segment in a radial pattern.
In a further variant, wherein the chip is an MA chip type 2 (bMA Chip T2), comprising a sheet of flexible or semi-rigid material selected from a group consisting of polydimethylsiloxane (PDMS), PMMA (poly(methyl methacrylate)), PS (polystyrene), and PC (polycarbonate); a center inlet well in a form of a round hole located at the geometric center; a cap having a hole, disposed on a top of the center inlet well, and comprising a lip or shoulder extending from a bottom surface to the center inlet well; wherein the sheet has a top surface and a bottom surface, that correspond to the top and bottom surfaces of the bMA Chip T2, respectively; wherein the side outlet wells are arranged into a set of branched channels; wherein the channels extend along four cardinal directions; and wherein μm-scale markings are disposed on an inside of the side outlet wells and mm-scale markings are disposed on an outside of a first segment of a branched channel along four cardinal directions.
In yet another variant, characterized by at least one of the sheet has a thickness of approximately 1 mm; the chip has a shape of a square with a geometric center and a side of approximately 6 cm; the center inlet well has a diameter of approximately 3 mm and a volume of 4 μl; the side outlet wells have a diameter of approximately 1.5 mm and a volume of 1 μl; the branched channels have a width of approximately 50 μm; and the chip is sized and shaped to fit within a Petri dish of approximately 8.5 cm or 10 cm.
In another variant, wherein the chip is an MA chip type 3 (bMA Chip T3), comprising a sheet of flexible or semi-rigid material selected from a group consisting of polydimethylsiloxane (PDMS), PMMA (poly(methyl methacrylate)), PS (polystyrene), and PC (polycarbonate); a center inlet well in a form of a round hole located at the geometric center; a cap having a hole, disposed on a top of the center inlet well, and comprising a lip or shoulder extending from a bottom surface to the center inlet well; wherein the side outlet wells are arranged into a set of branched channels; wherein the sheet has a top surface and a bottom surface, that correspond to the top and bottom surfaces of the bMA Chip T3, respectively; and wherein mm-scale markings are disposed on an outside of the side outlet wells.
In a further variant, characterized by at least one of the sheet has a thickness of approximately 1 mm; the chip has a shape of a disk with a geometric center and a diameter of approximately 8 cm; the center inlet well has a diameter of approximately 3 mm and a volume of 4 μl; the side outlet wells have a diameter of approximately 1.5 mm and a volume of 1 μl; the branched channels have a width of approximately 50 μm; and the chip is sized and shaped to fit within a Petri dish of approximately 8.5 cm or 10 cm.
In yet another variant, wherein the chip is made of two patterned layers, comprising a top layer made of plastic materials such as PS, PP, PMMA, and PC, and contains a through hole array and a central through hole; a bottom layer made of PDMS and contains radial channels and a well array; and wherein when the two patterned layers are assembled and bonded, the through hole array and the open well array overlap.
In a further variant, characterized by at least one of the through hole array is 1-2 mm in diameter; the central through hole is 2-4 mm in diameter; the radial channels are 0.03-0.1 mm in width and 2-4 mm in length; and the well array is 0.5-1.5 mm in diameter.
In another variant, wherein the chip is a rectangular MA chip (rMA Chip), comprising a top layer; a bottom layer comprising a well array and a channel array; wells; wherein the top layer is a membrane; wherein the top layer is gas permeable and liquid impermeable; wherein one side of the top layer is adhesive and a second side is not; wherein an adhesive side of the top layer reversibly adheres to the bottom layer; wherein the bottom layer can be made by PS (polystyrene) PC (polycarbonate), PP (polypropylene), PMMA (poly (methyl methacrylate)), COC (cyclic olefin copolymer) and polydimethylsiloxane (PDMS); and wherein each well is labeled by an alphabetic letter.
In a further variant, wherein the channel array contains long and wide channels, and short and narrow channels, wherein wells are symmetrically distributed on both sides of the long and wide channels; and wherein liquids first go into the long and wide channels, then into the short and narrow channels, and finally into the outlet wells.
In yet another variant, characterized by at least one of the chip is rectangular in shape; the chip is 127.8±5 mm long, 85.5±3 mm wide, and 1-10 mm in height; the membrane is 0.03-0.3 mm thick; the well array is rectangular in shape; and the wells are 0.5-5 mm in diameter and 1-10 mm in height.
In another variant, the chip is produced from an injection mold design. The injection mold design comprises an insert and a base. The injection mold design comprises a thermoplastic having a radius, patterned bottom surface, a top surface, center hole (inlet), edge wells, a patterned sink plateau surface, and a flattened edge. The injection mold design comprises a channel sealing mechanism between the insert and the base by a rivet, wherein the flattened edge is located between the insert and the base. Each well is assigned an alphabetic letter and the patterned sink plateau surface matches the insert exactly.
In another variant, a multiplex hole punch comprises a metal alloy substrate having a radius, a center, a top surface, tapered holes, a bottom surface, an outer edge and a thickness. An aluminum block has a radius, a center, a top surface, through holes, a bottom sink plateau, an outer edge and a thickness; wherein a shape of the bottom sink plateau with a flattened edge matches a reverse mold of the MA Chip; wherein the flattened edge aligns the reverse mold to the top surface; wherein a dimension of the top surface is a mirror image of a dimension of the bottom surface; wherein a position of pins is matched with a position of the wells; and wherein hollow metal alloy punch pins are secured on the metal alloy substrate.
In a further variant, wherein a reverse mold PDMS block comprises a radius with a flattened edge, a center, a top surface, an outer edge and a thickness; wherein a patterned surface of the reverse mold PDMS block matches the channel and well design of the MA Chip; and wherein the radial shape with a flattened edge matches the bottom sink plateau in the top and bottom surfaces.
The following Reference Numbers are used in this document:
Referring generally to
Section 2: Branched Microfluidic Aliquot Chip Type 2 (bMA-Chip T2)
In a variant, referring to
The side wells serve as outlets, and are in the form of round holes extending completely through the bMA Chip-T2. The side wells may be other than in the form of round holes, such as oval, triangle, square, rectangle, rhombus, trapezoid, and pentagon. In the main, hereinafter, the side wells which are round holes will be described. The side wells have a diameter of approximately 1.5 mm. The side wells each have a volume of 1 μl. The side wells are distributed along four cardinal directions at sides of the bMA Chip-T2. The side wells are accessible to a user from the top surface of the bMA Chip-T2, for retrieving isolated cells from the bMA Chip-T2.
In another variant, the 16 side wells are arranged into a set of branched channels, corresponding to the total 64 side wells arranged into four sets of branched channels. All channels have a width of approximately 50 μm. Each set of branched channels consists of four segments that the channels are evenly divided and are connected to 16 side wells. The total number of branch channels in each set from 1st segment to 4th segment is 2, 4, 8, and 16, respectively. Relatively small μm-scale markings are disposed inboard of the side wells for identifying the side wells under microscopic observation, and relatively large mm-scale markings are disposed outboard of 1st segment along four cardinal directions.
Section 3: Branched Microfluidic Aliquot Chip Type 3 (bMA-Chip T3)
In a further variant, referring to
The bMA Chip-T3 has a single center well, connected by a plurality of branched channels extending radially from the center well to a plurality of side wells. The channels are in fluid communication with the center well and the side wells. The center well serves as an inlet, and is in the form of a round hole extending completely through the bMA Chip-T3. The center well has a diameter of approximately 3 mm. The center well has a volume of 4 μl. The center well is disposed at the geometric center of the bMA Chip-T3. The center well is accessible to a user from the top surface of the bMA Chip-T3, for loading a cell suspension into the bMA Chip-T3. The side wells serve as outlets, and are in the form of round holes extending completely through the bMA Chip-T3. The side wells may be other than in the form of round holes, such as oval, triangle, square, rectangle, rhombus, trapezoid, and pentagon. In the main, hereinafter, the side wells which are round holes will be described.
The side wells have a diameter of approximately 1.5 mm. The side wells each have a volume of 1 μl. The side wells are distributed around an outer annular portion of the bMA Chip-T3 with the branched channels. The side wells are accessible to a user from the top surface of the bMA Chip-T3, for retrieving isolated cells from the bMA Chip-T3.
The four side wells are arranged into a set of branched channels, corresponding to the total 64 side wells arranged into 16 sets of branched channels. All channels have a width of approximately 50 μm. Each set of branched channels consists of two segments that the channels are evenly divided and are connected to 4 side wells. The total number of branch channels of 1st segment and 2nd segment is 32 and 64, respectively.
Relatively large mm-scale markings are disposed outboard of the side wells for identifying the side wells under naked-eye observation. The markings may be other than numbers or letters, such as 1D and 2D barcodes for identifying the side wells by using an imaging software. For the linear 1D barcodes, the information is stored in the relationship of the widths of the bars (spaces) to each other. For the stacked 2D barcodes, several stacked linear barcodes are used to encode the information. Compared to stacked barcodes the information of the matrix 2D barcodes is not stored by using different bar (space) widths. Instead the position of black or white dots is relevant.
In another variant, referring to
In another variant, referring to
In a further variant, an engineered aluminum block having a radius, a center, a top surface, through holes, a bottom sink plateau, an outer edge and a thickness. The enclosure hold MA-Chip fabricated with soft PDMS material in a place to prevent distortion during multiplex hole punch process. The through holes guide punch pins to the exact position of MA-Chip well. The shape of the bottom sink plateau with a flatten edge matches reverse mold of MA-Chip. The flattened edge is used to align the reverse mold to the top enclosure. The dimension of top and bottom enclosure is mirror image tween.
In another variant, reverse mold PDMS block having a radius with flattened edge, a center, a top surface, an outer edge and a thickness. The patterned surface of the reverse mold PDMS block matches with the channel and well design of MA-Chip for ensuring the alignment between MA-Chip holes and channels. The radial shape with a flattened edge matches the sink plateau in top and bottom enclosure. The flattened edge is used to automatically align the reverse mold to the enclosure.
In a further variant, referring to
In a further variant, referring to
In another variant, the rMA-Chip is rectangular in shape and 127.8±5 mm in length, 85.5±3 mm in width, and 1-10 mm in height. The rMA-Chip has two layers: the top layer and the bottom layer. The top layer is a membrane with 0.03-0.3 mm in thickness. The top layer is gas permeable and liquid impermeable. The top layer is biocompatible and no harmful to cells. The top layer is transparent. The top layer is flexible. One side of the top layer is adhesive and the other side in not adhesive. The adhesive side of the top layer can adhere reversibly to the bottom layer. The top layer can be easily peeled off from the bottom layer by fingers. The bottom layer is hard and half-hard materials. The bottom layer can be made by PS (polystyrene) PC (polycarbonate), PP (polypropylene), PMMA (poly (methyl methacrylate)), COC (cyclic olefin copolymer) and polydimethylsiloxane (PDMS) by using injection molding, laser cutting, or photolithography. The bottom layer is transparent. The bottom layer contains a well array and a channel array. The well array is rectangular. Wells are usually round. Wells are 0.5-5 mm in diameter and 1-10 mm in height. Wells are used as outlets. The number of wells in one rMA-Chip can be 32, 64 (32×2), 96 (32×4), 384 (96×4), or 1536 (384×4). Wells can be other shapes such as rectangle, triangle, and oval. Wells are uniformly distributed in all the area of rMA-Chip. Each well is labeled by micro-scale digital & letter designed for microscopic observation and macro-scale digital & letter designed for naked-eye observation. The top of well array is connected by the channel array. The channel array contains long & wide channels and short & narrow channels.
In another variant, wells are symmetrically distributed on both sides of long & wide channels (20-120 mm in length, 0.2-1 mm in width, and 0.01-0.5 mm in depth). Short & narrow channels (0.5-2 mm in length, 0.01-0.2 mm in width, and 0.01-0.5 mm in depth) are used to connect wells with long & wide channels. Both well array and channel array are covered and sealed by gas permeable and liquid impermeable membrane. There is only one inlet well to load liquid into channels and outlet wells. There is one cylindrical PDMS cap on the top of the inlet well for liquid loading. Liquid loading can be manually achieved by pipette and syringes. After liquid loading, the cap can be removed from the inlet well. Liquids firstly go into the long & wide channels, then short & narrow channels, and finally outlet wells. The air within channels and outlet wells are pushed to the outside of the top layer because it is gas permeable. The injected liquid will be kept within the channels and outlet wells because the top layer is liquid impermeable. After liquid loading, the top layer can be easily peeled off from the top of the bottom layer by fingers. After removing the top layer, a well array containing 0.2 μl-30 μl liquid is obtained. After removing the top layer, wells are open. rMA-Chip can be used in liquid distribution and single-cell isolation. Liquids and cells within outlet wells can be easily retrieved and transferred by commonly used pipette.
The present application is a continuation of U.S. application Ser. No. 15/006,634 filed Jan. 26, 2016, which is hereby incorporated herein by reference in its respective entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 15006634 | Jan 2016 | US |
| Child | 15701401 | US |
