METHODS AND APPARATUS FOR CELL CULTURE ARRAY

• US 20090023608A1
• Filed: 07/06/2006
• Published: 01/22/2009
• Est. Priority Date: 07/07/2005
• Status: Active Grant

• Current Assignee: Regents of the University of California (University of California)
• Original Assignee: Regents of the University of California (University of California)
• Inventors: Lee, Philip J., Hung, Paul J., Lee, Luke P.
• Granted Patent: US 9,260,688 B2
• Time in Patent Office: Days
• US Class Current: 506/32
• CPC Class Codes: B01L 2200/027 for microfluidic devices
• CPC Class Codes: B01L 2200/0647 Handling flowable solids, e.g. microscopic beads, cells, particles
• CPC Class Codes: B01L 2200/0689 Sealing
• CPC Class Codes: B01L 2300/0627 Sensor or part of a sensor is integrated
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 2400/086 using baffles or other fixed flow obstructions
• CPC Class Codes: B01L 2400/088 by specific surface properties
• CPC Class Codes: B01L 3/502707 characterised by the manufacture of the container or its components by shaping or joining plastic parts B29C59/00, B29C65/00; by laminating B32B37/00; manufacture of microstructural devices in general B81C
• CPC Class Codes: B01L 3/502715 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
• CPC Class Codes: B01L 3/502746 characterised by the means for controlling flow resistance, e.g. flow controllers, baffles B01L3/502738 takes precedence
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: B81B 2201/05 Microfluidics
• CPC Class Codes: B81B 2201/06 Bio-MEMS
• CPC Class Codes: B81C 2201/019 Bonding or gluing multiple substrate layers
• CPC Class Codes: C12M 21/08 for producing artificial tissue or for ex-vivo cultivation of tissue prostheses A61F2/00, grafts A61L27/00
• CPC Class Codes: C12M 23/16 Microfluidic devices; Capillary tubes integrated microfluidic structures B01L3/5027; microreactors B01J19/0093
• CPC Class Codes: C12M 23/34 Internal compartments or partitions
• CPC Class Codes: C12M 29/06 Nozzles; Sprayers; Spargers; Diffusers per se B01F23/231, B01J19/26
• CPC Class Codes: C12M 29/10 Perfusion
• CPC Class Codes: C12M 41/46 : of cellular or enzymatic activity or functionality, e.g. cell viability
• CPC Class Codes: C12N 5/0602 : Vertebrate cells

Abstract

Method and systems that provide improved handling and/or culturing and/or assaying of cells, chemically active beads, or similar materials in microfluidic systems and microfluidic culture arrays.

Claims

1. A method of fabricating a microculture microfluidic array comprising:

patterning small fluidic structures in a first layer of a mold material on a substrate;

patterning large fluidic structures in a second layer of a mold material adjacent to said first layer;

introducing a settable material into the mold and curing to form a main body of said array;

removing said array from said mold;

wherein there is a high fluidic resistance ratio between said small structures and said large structures.

3. The method of claim 1 further wherein said method comprises one or more of:

patterning a polymer mold on a silicon wafer followed by replication with a soft elastomer or thermoplastics;

electroplating metals on a silicon wafer followed by replication with a soft elastomer or thermoplastics;

etching on a silicon wafer followed by replication with a soft elastomer or a thermoplastic.

4. The method of claim 1 further wherein:

said patterning small fluidic structures comprises;

spinning a first thin layer on said substrate;

defining a micro passage structure in said thin layer;

said patterning larger fluidic structures comprises;

spinning a second thicker layer on said substrate;

defining a plurality of culture microchambers and/or larger fluidic channels in said thicker layer;

thereby to produce a 2-level mold.

8. The method of claim 1 further comprising:

providing a mold defining higher fluidic resistance passages and lower fluidic resistance areas;

injecting a settable plastic and/or polymer into said mold to form a molded microfluidic structure;

separating said mold from said molded structure; and

attaching said molded structure to a cover piece to thereby form a microfluidic device with one or more microfluidic passages and/or channels and/or areas.

9. The method of claim 8 further wherein;

said high fluidic resistance passages have a fluidic flow resistance ratio greater than about 10 to said lower fluidic resistance areas.

said moldable material comprising a material selected from the group consisting of;

Polydimethylsiloxane (PDMS);

Polymethylmethacrylate (PMMA);

Polycarbonate (PC); and

Polystyrene (PS)

said mold is constructed from a material selected from the group consisting of;

silicon;

a Polymer;

a metal;

a metal alloy.

and further comprising;

providing a thin adhesive on said cover piece, said thin adhesive enabling said attaching;

said providing comprises spin coating a thin adhesive on said cover piece, said thin adhesive having a substantially uniform thickness;

said cover piece comprises a plastic material, thereby providing an all plastic microfluidic structure.

subsequently bonding a molded device onto a cover material, said cover material comprising a material selected from the group consisting of;

glass;

polymer;

silicon.

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19. A microfluidic culture system capable of providing an environment for biologic growth and/or analysis and/or manipulation comprising:

a plurality of culture units, at least some of said culture units comprising;

a culture area;

a loading inlet, allowing loading of cells or other culture objects;

a large fluidic cross-section medium/reagent channel, said channel providing a desired flow of a culture medium and/or reagent and/or providing access to culture products; and

a micro fluidic passage structure, said structure separating said culture area and said medium/reagent channel while providing fluidic connection between said culture area and said medium/reagent channel without disturbing cells or other culture objects.

20. The system according to claim 19 further wherein:

said micro fluidic passage structure comprises a plurality of micro passages, each approximately between 0.25 to 10 μ

m wide.

21. The system according to claim 20 further wherein:

said micro passages are arranged in a grid-like structure, wherein said passage structure comprises a plurality of intersecting micro passages.

22. The system according to claim 19 further wherein:

said micro fluidic passage structure provides fluidic passages much smaller than the size of cells handled and effectively prevents cells from being flushed away or from migrating outside the culture area.

23. The system according to claim 20 further wherein:

said passages are each approximately between 1 to 5 μ

m high and connect to at least one large fluidic flow area that is between 20 and 200 μ

m high.

28. The system according to claim 19 further comprising:

at least four ports for fluidic access to a microchamber, two designated left and right and two designated top and bottom;

wherein said left and right ports are designed to provide continuous perfusion of the medium to the microchamber for sustaining growth;

wherein top and bottom ports are used to load and/or remove a cell or cells, beads, or other objects for culture.

29. The system according to claim 28 further comprising:

an inner microchamber wall substantially surrounding a microchamber with a largest opening near to said inlet port and containing one or more diffusion/perfusion fluidic passages;

said inner microchamber wall substantially blocking passage of culture objects between said inlet port and said outlet port while allowing diffusion/perfusion fluid flow;

said inner microchamber wall defining an outer channel around said wall, said outer channel having low resistance fluidic flow connection with an outlet port; and

an outer wall having a plurality of micro inlet passages therein separating said outer channel from said higher volume perfusion channels.

36. The system according to claim 19 further wherein said microchambers are positioned between two wells in a well plate thereby providing two fluidic access points to said microchambers.

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37. A microfluidic microchamber device comprising:

a microchamber having an inlet port and substantially surrounded by a chamber wall, said chamber wall comprising a diffusion micro structure;

a medium flow channel substantially surrounding at least one-half of said chamber wall; and

an inlet channel substantially aligned with said inlet port.

38. The system according to claim 37 further wherein:

said diffusion micro structure has a substantially higher fluidic resistance than other channels;

wherein said diffusion micro structures function as a passive barrier for sample immobilization.

39. A microfluidic culture system including a plurality of devices according to claim 37 providing an environment for biologic growth and manipulation and further comprising:

a set of one or more micro-sized units, at least one comprising;

a microchamber and/or a microchannel;

a single large volume inlet port;

a flow around channel proximate to said microchamber and connected thereto by one or more micro diffusion structures;

such that medium or reagents in said flow around channel can enter said microchamber and waste can exit said microchamber into said flow around channel by diffusion and/or other mass transfer through said micro diffusion structures.

40. A method of handling cells comprising:

introducing cells into one or more micro-sized micro culture units, at least one unit comprising;

a microchamber and/or a microchannel;

at least one large volume port;

a flow around channel proximate to said microchamber and connected thereto principally by a plurality of one or more micro diffusion structures;

such that medium and/or reagents in said flow around channel can enter said microchamber and waste can exit said microchamber into said flow around channel by diffusion and/or mass transfer through said micro diffusion structures;

wherein said microchamber and said micro diffusion structures have a high fluidic resistance ratio.

42. The method of claim 40 further wherein said micro diffusion structures comprise a plurality of micro passages that are arranged as part of a grid, said grid comprised of a plurality of intersecting passages that are substantially smaller in cross section than said flow around channel or said microchamber.

44. The method of claim 40 further comprising analyzing multiple parameters of one or more cells by additional steps comprising:

fluidically preparing said cells;

fluidically transporting said cells through one or more microfluidic channels to one or more micro culture areas;

culturing said cells through continuous perfusion of a medium through said micro culture areas;

fluidically addressing said micro culture areas with one or more compounds; and

acquiring data regarding cellular responses in said micro culture areas chambers.

45. The method of claim 44 wherein said acquiring comprises one or more steps selected form the group consisting of:

continuously capturing images of said cells in said micro culture areas

continuously measuring the perfusate in said microfluidic channels

electrically detecting cellular responses in said microchambers.

58. The method of claim 44 further comprising:

culturing cells in solid aggregates to provide functional artificial tissues and/or organs comprising;

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