Microfluidic Aliquoting For Single-Cell Isolation

Abstract

According to the invention, generally, a microfluidic aliquoting (MA) chip, adapted to fit in a Petri dish, has a center well (inlet) connected by branched channels to a plurality of side wells (outlets). The chip comes in various types, including a bMA Chip T1, bMA Chip T2, bMA Chip T3, and an rMA Chip. The branched channel improvement provides for a greater distance between neighboring channels and a decreased density near the center well. Design improvements including an injection mold design for an insert and a base and a multiplex hole punch allow for rapid fabrication of the MA chip.

Claims

1. A microfluidic aliquot (MA) chip for performing single-cell isolation, comprising: 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, configured to maintain uniform distribution of a liquid from the center inlet well to the side outlet wells.

2. The MA chip of claim 1, wherein the multiple segments comprise: a plurality of first segments that form an inner layer, a plurality of last segments that form an outer layer, and a plurality of segments between the first segments and the last segments form a middle layer; wherein the side outlet wells and the multiple segments are configured to uniformly distribute liquid and a plurality of cells when the liquid and cells are placed in the MA chip; wherein the first segment is connected to 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.

3. The MA chip of claim 2, wherein a plurality of segments in the middle layer form a curved portion generally in the shape of a bend, whereby one of the first segments is joined to the bend of a segment in the middle layer.

4. The MA chip of claim 1, wherein the multiple segments comprise: a plurality of first segments that extend from the center inlet well to a plurality of second segments; wherein an end of a first segment branches at a 90 degree angle into two opposite directions that each lead into second segments; and wherein subsequent branches of segments branch at 90 degree angles.

5. The MA chip of claim 4, wherein the chip comprises: 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); wherein the sheet has a top surface and a bottom surface, that correspond to the top and bottom surfaces of the chip, respectively.

6. The MA chip of claim 4, wherein m-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.

7. The MA chip of claim 4, wherein mm-scale markings are disposed on an outside of the side outlet wells.

8. The MA chip of claim 4, wherein the chip comprises: a center inlet well having a diameter of approximately 3 mm and a volume of 4 l; a plurality of side outlet wells having a diameter of approximately 1.5 mm and a volume of 1 l; a plurality of branched channels having a width of approximately 50 m; wherein the chip is sized and shaped to fit within a Petri dish of approximately 8.5 cm; and wherein the chip is sized and shaped to fit within a Petri dish of approximately 10 cm.

9. The MA chip of claim 1, wherein the chip comprises: a top layer having a center inlet well and a first plurality of channels; a bottom layer having a well array and a second plurality of channels; and wherein the second plurality of channels on the bottom layer are shorter and wider than the first plurality of channels on the top layer.

10. The MA chip of claim 1, wherein the chip comprises: a top layer having a center inlet well and a well array; and a bottom layer having an alignment mark array and a plurality of channels that extend outwards radially from a center of the bottom layer.

11. The MA chip of claim 12, wherein the chip comprises: a well array having a diameter of 1-2 mm; a center inlet well having a diameter of 2-4 mm; a plurality of radial channels having a width of 0.03-0.1 mm and a length of 2-4 mm; and an alignment mark array having a diameter of 0.5-1.5 mm.

12. The MA chip of claim 1, wherein the chip comprises: a top layer having an adhesive side; a bottom layer having a well array and a channel array; and wherein the adhesive side of the top layer adheres to the bottom layer.

13. The MA chip of claim 14, wherein the chip comprises a plurality of wells, each well labeled with an alphabetic letter.

14. The MA chip of claim 14, wherein the channel array comprises: a first plurality of channels; a second plurality of channels; wherein the second plurality of channels is shorter and narrower than the first plurality of channels; wherein wells are symmetrically distributed on both sides of the first plurality of channels; and wherein the first plurality of channels are configured to distribute liquid into the second plurality of channels, and the second plurality of channels is configured to distribute liquid into the outlet wells.

15. The MA chip of claim 14, wherein the chip comprises: a membrane on the top layer having a thickness of 0.03-0.3 mm; a plurality of wells having a diameter of 0.5-5 mm and a height of 1-10 mm; and wherein the chip has a length of 127.85 mm, a width of 85.53 mm, and a height of 1-10 mm.

16. The MA chip of claim 1, wherein the center inlet well is configured to receive and distribute liquid through the multiple segments and into the wells.

17. A method for making an MA chip, comprising: forming a pair of metal injection molds using a Computer Numerical Control (CNC) machine tool; inserting a first metal injection mold into an injection molding machine; injecting thermoplastic into the first metal injection mold to produce an insert; inserting a second metal injection mold into the injection molding machine; inserting thermoplastic into the second metal injection mold to produce a base; and inserting a plurality of rivets on the base into a plurality of rivet through holes on the insert, thereby sealing the insert into the base.

18. A method for creating holes in an MA chip, comprising: inserting an MA chip between a top and a bottom of an enclosure; aligning the MA chip directly above a mold in the enclosure; inserting a plurality of pins attached to a bottom of a pin head into a plurality of through holes on the top of the enclosure; and pressing a top of the pin head so that the MA chip is pushed into the mold.

19. A method for mass production of MA chips, comprising: conducting photolithography to produce a silicon mold; injecting Polydimethylsiloxane (PDMS) into the silicon mold; heating the silicon mold containing the PDMS to produce a first layer of an MA chip; using a laser to cut a well array into a plastic material to produce a second layer of an MA chip; and placing the first layer on a top of the second layer aligned directly below the first layer.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0040] FIG. 1A is a bMA Chip Type 1.

[0041] FIG. 1B is a magnified portion of the MA chip in FIG. 1A.

[0042] FIG. 1C is a magnified portion of an MA chip with four segments.

[0043] FIG. 2A is a magnified portion of a first segment of an MA chip.

[0044] FIG. 2B is a magnified portion of a second segment of an MA chip.

[0045] FIG. 2C is a magnified portion of a third segment of an MA chip.

[0046] FIG. 2D is a magnified portion of a second segment of an MA chip.

[0047] FIG. 3A is a diagram of a bMA Chip Type 2.

[0048] FIG. 3B is a magnified portion of a bMA Chip Type 2.

[0049] FIG. 3C is a bMA Chip Type 3.

[0050] FIG. 3D is a magnified portion of a bMA Chip Type 3.

[0051] FIG. 4A is an MA chip insert.

[0052] FIG. 4B is an MA chip base.

[0053] FIG. 5 is a multiplex hole punch.

[0054] FIG. 5A is an MA Chip top and bottom enclosure assembly.

[0055] FIG. 5B is an MA Chip with microchannel array.

[0056] FIG. 5C is a reverse mold of an MA chip.

[0057] FIG. 5D is an assembled enclosure of an MA chip without the top.

[0058] FIG. 5E is a complete assembled enclosure of an MA Chip.

[0059] FIG. 6A is an MA chip assembly with long and narrow channels on a top layer.

[0060] FIG. 6B is an MA chip assembly with short and wide channels on a bottom layer.

[0061] FIG. 6C is an MA chip assembly with the top and bottom layer combined.

[0062] FIG. 6D is an MA chip assembly with short and wide channels on a top layer.

[0063] FIG. 6E is an MA chip assembly with long and narrow channels on a bottom layer.

[0064] FIG. 6F is an MA chip assembly with the top and bottom layer combined.

[0065] FIG. 7 is a side view diagram of the rMA chip.

[0066] FIG. 8 is a top view diagram of the rMA chip.

[0067] FIG. 9A is an rMA chip before loading liquid into the chip.

[0068] FIG. 9B is an rMA chip after loading liquid into the chip.

[0069] FIG. 9C is an rMA chip after removing the tape.

[0070] FIG. 10A is a design of an rMA chip.

[0071] FIG. 10B is a magnified portion of an rMA chip.

[0072] FIG. 10C is a prototype of a PMMA rMA chip.

DETAILED DESCRIPTION

[0073] The following Reference Numbers are used in this document: [0074] 100 Microfluidic Aliquoting (MA) chip [0075] 110 Center well (inlet) [0076] 112 Plurality of channels [0077] 114 Plurality of side wells (outlets) [0078] 114a First set of side wells (first segment) [0079] 114b Second set of side wells (second segment) [0080] 114c Third set of side wells (third segment) [0081] 114d Fourth set of side wells (fourth segment) [0082] 116 Width between first segments [0083] 118 Width between second segments [0084] 120 Cap [0085] 122 Width between third segments [0086] 124 Width between fourth segments [0087] 126 Top surface of MA chip [0088] 128 Identifying micrometer-scale number [0089] 130 MA Chip Base [0090] 132 Liquid reservoir wells [0091] 134 Joining rivet [0092] 136 Engraved identification number [0093] 138 Flattened edge [0094] 140 Multiplex hole punch pin head [0095] 142 Top enclosure of multiplex hole punch [0096] 144 Reverse mold of MA chip [0097] 146 Bottom enclosure of multiplex hole punch [0098] 148 MA chip with microchannel array [0099] 150 Bottom surface of MA chip [0100] 152 Impermeable membrane [0101] 154 Well filled with liquid [0102] 156 rMA Chip [0103] 158 rMA Chip with liquid [0104] 160 384 well rMA Chip
Section 1: Branched Microfluidic Aliquot Chip Type 1 (bMA-Chip T1)

[0105] Referring generally to FIGS. 1A-2D, in a variant, the bMA-Chip Type 1 contains multiple segments (branched channels) to connect the common inlet well with the 96 outlet wells. The bMA-Chip T1 can contain three segments: the 1st segment (inside layer), the 2nd segment (middle layer), and the 3rd segment (outside layer). The channel number of each segment, from the inside layer to the outside layer, is 24, 48, and 96, respectively. The bMA-Chip T1 can also contain four segments: the 1st segment (inside layer), the 2nd and 3rd segments (middle layers), and the 4th segment (outside layer). The channel number of each segment, from the inside layer to the outside layer, is 12, 24, 48, and 96, respectively. The bMA-Chip T1 can also contain other segments, such as 2, 5, and 6. The 1st segment is divided from the center inlet well with radial pattern. The next segment, such as the 2nd segment, is divided from the previous segment, such as the 1st segment, with radial pattern. The liquid and cells can be uniformly distributed into 96 outlet wells through the multiple segments of bMA-Chip T1. The improved design with decreased channel density around the inlet well reduces machining requirement, allowing plastic bMA-Chip T1 to be mass produced by injection molding or laser cutting. Besides the newly designed and validated branched structures, other features, such as size and application, of bMA-Chip T1 is similar to the original MA-Chip.

Section 2: Branched Microfluidic Aliquot Chip Type 2 (bMA-Chip T2)

[0106] In a variant, referring to FIGS. 3A and 3B, the bMA Chip-T2 comprises a thin sheet of flexible or semi-rigid material such as, polydimethylsiloxane (PDMS), PS (polystyrene) PC (polycarbonate), and PMMA (poly (methyl methacrylate)) with a thickness of approximately lmm. The bMA Chip-T2 has the overall form of a square, having a geometric center and a side of approximately 6 cm. The bMA Chip-T2 is sized and shaped to fin in a Petri dish having a diameter of approximately 8.5 or 10 cm. The sheet forming the bMA Chip-T2 has a top surface and a bottom surface, corresponding to the top and bottom surfaces of the overall bMA Chip-T2, respectively. The bMA Chip-T2 has a single center well, connected by a plurality of branched channels extending along four cardinal directions 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-T2. The center well has a diameter of approximately 3 mm. The center well has a volume of 4 l. The center well is disposed at the geometric center of the bMA Chip-T2. The center well is accessible to a user from the top surface of the bMA Chip-T2, for loading a cell suspension into the bMA Chip-T2.

[0107] 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 l. 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.

[0108] 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 m. 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 m-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)

[0109] In a further variant, referring to FIGS. 3C and 3D, the bMA Chip-T3 comprises a thin sheet of flexible or semi-rigid material such as, polydimethylsiloxane (PDMS), PS (polystyrene) PC (polycarbonate), and PMMA (poly (methyl methacrylate)) with a thickness of approximately 1 mm. The bMA Chip-T3 has the overall form of a disk, having a geometric center and a diameter of approximately 8 cm. The bMA Chip-T3 is sized and shaped to fit in a Petri dish having a diameter of approximately 8.5 or 10 cm. The sheet forming the bMA Chip-T3 has a top surface and a bottom surface, corresponding to the top and bottom surfaces of the overall bMA Chip-T3, respectively.

[0110] 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 l. 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.

[0111] The side wells have a diameter of approximately 1.5 mm. The side wells each have a volume of 1 l. 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.

[0112] 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 m. 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.

[0113] 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.

Section 4: Injection Mold Design

[0114] In another variant, referring to FIGS. 4A and 4B, a thermoplastic having a radius, patterned bottom surface, a top surface, center hole (inlet), and edge wells, and a flattened edge. The branched channel design provides large spacing (>0.4 mm) between channels. The large space allows to add a channel sealing mechanism between the insert and base by rivet. The flattened edge is added to ensure the alignment between the MA-Chip insert and base. A thermoplastic having a radius, patterned bottom surface, a patterned sink plateau surface, and edge wells. The 24 edge wells are designed for carrying buffers, culture mediums, or cell suspension. Each well can contain 30 to 400 l volume of liquid. The aliquot cells can be transferred to the edge wells for long term culture and cell expansion. The wells can serve as a medium reservoir during on-chip tissue culture to prevent culture medium evaporation. Each well is assigned with an alphabet as a well identification method. The well identification alphabets are positively engraved on the top. Patterned bottom surface prevents the viewing area from scratch when it lays down by providing small gap between the bottom surface and the rough surface. A patterned sink plateau surface exactly matches to the MA-Chip insert by the flattened edge for perfect alignment. The identification numbers for aliquot wells are engraved on the base allowing large spacing between MA-Chip channels. The large spacing between MA-Chip channels provide better manufacturability.

Section 5: Multiplex Hole Punch

[0115] In another variant, referring to FIGS. 5, and 5A-5E, a multiplex design of hole punch pin head, comprising a metal alloy substrate having a radius, a center, a top surface, tapered holes, a bottom surface, an outer edge and a thickness. The position of pins is matched with the position of MA-Chip wells to punch the MA-Chip holes at the same time. This design reduces hole punch process time. Hollow metal alloy punch pins are secured on the rigid metal alloy substrate such as stainless steel or brass. The metal substrate holds the pins by tapered hole, which allows to replace the pin at the end of life time.

[0116] 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.

[0117] 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.

Section 6: Method of Mass-Producing an MA Chip

[0118] In a further variant, referring to FIGS. 6A-6F, the assembled MA-Chip is made of two patterned layers. The top layer is made of PDMS by photolithography and contains long and narrow channels (2-4 cm in length and 0.03-0.1 mm in width) and a central through hole (2-4 mm in diameter). The bottom layer is made of plastic materials by laser cutting or injection molding, such as PS, PP, PMMA, and PC, and contains a well array (1-2 mm in diameter) and associated short and wide channels (0.5-2.5 mm in length and 0.3-0.5 mm in width). When the two patterned layers are assembled and bonded, the long and narrow channels and short and wide channels can be well overlapped for uniform liquid distribution. A microliter of liquid, typically between 100 L and 200 L, can be injected into the central hole by a pipette and uniformly dispensed into 100 open wells. Besides the new design and assembling method, other features, such as size and application, of this kind of MA-Chip is similar to the original MA-Chip.

Section 7: Rectangular MA Chip

[0119] In a further variant, referring to FIGS. 7-10C, a rectangular MA-Chip (rMA-Chip) that has the potential to integrate hundreds to thousands of outlet wells in the size of standard 96-well plate. In this design, all outlet wells are patterned in a rectangular array but not radial pattern. The top of the well array is connected by the channel array and both of them are sealed by gas permeable and liquid impermeable membrane. Liquids are loaded by commonly used pipette or syringes and will firstly flow into the channel array and then well array by continuously pushing the air to the outside. After liquid loading, the cover membrane is removed and the well array with liquid inside is obtained. After removing the gas permeable and liquid impermeable membrane, the wells are open to the air and the liquids associated single cells can be confirmed by microscope and then retrieved by commonly used pipette.

[0120] In another variant, the rMA-Chip is rectangular in shape and 127.85 mm in length, 85.53 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 (322), 96 (324), 384 (964), or 1536 (3844). 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.

[0121] 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 l-30 l 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.