Valved, microwell cell-culture device and method

• US 8,673,625 B2
• Filed: 09/04/2012
• Issued: 03/18/2014
• Est. Priority Date: 01/04/2006
• Status: Active Grant

• Current Assignee: EMD Millipore Corporation (Merck & Co., Inc.)
• Original Assignee: EMD Millipore Corporation (Merck & Co., Inc.)
• Inventors: Hung, Paul Ju-Sung, Lee, Philip Janmin
• Primary Examiner(s): HOBBS, MICHAEL L
• Application Number:
  US13/602,328
• Publication Number:
  US 20130090268A1
• Time in Patent Office: 560 Days
• Field of Search: 435/287.1, 506/37, 264/81, 264/219, 264/227, 264/241, 156/228, 436/43, 436/180, 422/50, 422/81, 422/82, 422/502
• US Class Current: 435/287.1
• CPC Class Codes: B01L 2200/12 Specific details about manufacturing devices
• CPC Class Codes: B01L 2300/0681 Filter
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2300/0819 Microarrays; Biochips
• CPC Class Codes: B01L 2300/10 Means to control humidity and/or other gases
• CPC Class Codes: B01L 2300/123 Flexible; Elastomeric
• CPC Class Codes: B01L 2300/14 Means for pressure control
• CPC Class Codes: B01L 2400/0481 squeezing of channels or chambers
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 2400/0655 pinch valves
• CPC Class Codes: B01L 3/5025 for parallel transport of multiple samples
• 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 3/50273 characterised by the means or forces applied to move the fluids micropumps F04B19/006, of the membrane type F04B43/043
• CPC Class Codes: B01L 3/502738 characterised by integrated valves microvalves F16K99/0001
• 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: B29C 65/002 Joining methods not otherwise provided for
• CPC Class Codes: C12M 23/12 Well or multiwell plates C12M25/04 takes precedence
• CPC Class Codes: C12M 23/16 Microfluidic devices; Capillary tubes integrated microfluidic structures B01L3/5027; microreactors B01J19/0093
• CPC Class Codes: C12M 29/10 Perfusion
• CPC Class Codes: C12M 29/14 : Pressurized fluid
• CPC Class Codes: C12M 41/00 : Means for regulation, monitoring, measurement or control, e.g. flow regulation controlling or regulating chemical, physical or physicochemical processes B01J19/0006; heating or cooling apparatus for laboratory use B01L7/00; electro optical investigation of individual particles, flow cytometers G01N15/14; automatic analysis G01N35/00; controlling or regulating in general G06N
• CPC Class Codes: C12M 41/48 : Automatic or computerized control automatic analysis G01N35/00
• CPC Class Codes: C12Q 1/025 : for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics antimicrobial activity C12Q1/18
• CPC Class Codes: F16K 2099/0074 : using photolithography, e.g. etching
• CPC Class Codes: F16K 2099/0078 : using moulding or stamping
• CPC Class Codes: F16K 2099/0084 : Chemistry or biology, e.g. "lab-on-a-chip" technology
• CPC Class Codes: F16K 27/0236 : Diaphragm cut-off apparatus
• CPC Class Codes: F16K 99/0001 : Microvalves microdevices B81B1/00; manufacture or treatment of devices or systems in or on a substrate B81C1/00; microfluidic structures B01L3/5027; micropumps F04B19/006
• CPC Class Codes: F16K 99/0015 : Diaphragm or membrane valves
• CPC Class Codes: F16K 99/0059 : actuated by a pilot fluid
• CPC Class Codes: G01N 2035/00158 : Elements containing microarrays, i.e. "biochip"
• CPC Class Codes: G01N 33/5008 : for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• CPC Class Codes: G01N 33/54366 : Apparatus specially adapted for solid-phase testing
• CPC Class Codes: Y10T 137/2202 : By movable element

Abstract

A valved microfluidics device, microfluidics cell-culture device and system incorporating the devices are disclosed. The valved microfluidics device includes a substrate, a microchannel through which liquid can be moved from one station to another within the device, and a pneumatic microvalve adapted to be switched between open and closed states to control the flow of fluid through a microchannel. The microvalve is formed of three flexible membranes, one of which is responsive to pneumatic pressure applied to the valve and the other two of which deform to produce a more sealable channel cross-section. The cell culture device provides valving to allow controlled loading of cells into the individual well of the device, and exchange of cell-culture components in the wells.

Claims

1. A microarray culture system, comprising

(a) a substrate,

(b) formed in said substrate, a plurality of microfluidics passageways, each having (i) a well for receiving particles therein, (ii) an inlet channel segment, (iii) an outlet channel segment, (iv) a channel intersection segment having a first channel arm that communicates the inlet and outlet channel segments, and a second channel arm communicating the inlet channel segment with the interior of said well, where the first channel arm flows around a portion of the well, and (v) a porous barrier through which fluid, but not cells, in said well can perfuse from the well into said first arm,

(c) associated with each passageway, a first valve disposed in said first channel arm for controlling the flow of fluid from the inlet channel segment into the first channel arm, and a second valve disposed in said second channel arm for controlling the flow of fluid from the inlet channel segment into the passageway well,

(d) a microchannel well-distribution network adapted for supplying input fluid to each of a selected one or more of wells in the passageways, under the control of a plurality of valves associated with the network,

(e) first and second microchannel valve-supply networks for supplying fluid pressure to first and second valves in said passageways, respectively,

(f) a plurality of reservoirs, each in fluid communication with a channel in said well-distribution network or in said valve-supply network, and

(g) a controller for supplying pressurized fluid to selected ones of said reservoirs, thereby to supply fluid to a selected one or more of said microfluidic passageways and to selected ones of said valves.

2. The system of claim 1, which contains N passageways, wherein said microchannel distribution network has X separate valved channels, where X=2 log₂N.

3. The system of claim 1, wherein said first and second valve-supply networks are operable to supply fluid pressure simultaneously to all of said first and second valves, respectively.

4. The system of claim 1, which further includes a detector by which the presence or absence of cells in a passageway intersection can be determined, and a controller for sequentially activating said second and first valves for capturing a cell in such intersection in the associated well.

5. The system of claim 1, wherein said reservoirs include at least one cell reservoir for holding cells to be introduced into said passageways, and at least one reagent reservoir for holding a solution to be perfused through said passageways, and which further includes a sample-control network for controlling the flow of fluid in said cell and reagent reservoirs to said well-distribution network.

6. The system of claim 5, wherein said controller operates in one mode to (i) control the sample-control network to direct fluid in said cell reservoir into said well-distribution network, (ii) to control the well-distribution network to direct fluid from said cell reservoir to a selected passageway, and (iii) control said first and second valve-supply networks to open the second valve and close the first valve associated with the selected passageway.

7. The system of claim 5, wherein:

said controller operates in one mode to open said first valve, and close the second valve in each passageway, so that a cell-culture medium contained in a reagent reservoir flowing from a device inlet to a device outlet can exchange solute components with cell culture medium in each well across said porous barrier; and

said controller operates in one mode to close said first valve, and open the second valve of said passageways, so that a medium flowing from a device outlet to a device inlet will carry the cells in the device wells out of the device.

8. A microfluidics culture device, comprising

(a) a substrate,

(b) formed in said substrate, a microfluidics passageway having (i) a well for receiving particles therein, (ii) an inlet channel segment, (iii) an outlet channel segment, (iv) a channel intersection segment having a first channel arm that communicates the inlet and outlet channel segments, and a second channel arm communicating the inlet channel segment with the interior of said well, where the first channel arm flows around a portion of the well, and (v) a porous barrier through which fluid, but not cells, in said well can perfuse from the well into said first arm, and

(c) a valve disposed in said second channel arm, operable to control the flow of fluid from the inlet channel segment into said well, wherein a cell carried in a fluid moving through said inlet segment can be diverted into said well by opening said valve, and once diverted into said well, can be captured therein, with fluid flowing through said passageway, by closing said valve, and culture medium flowing through said first arm can exchange solute components with culture medium within said well by diffusion of such components across said porous barrier.

9. The device of claim 8, where said valve is a pneumatic valves having two or more flexible membranes forming wall portions in valved regions of said second channel arm.

10. The device of claim 9, wherein said two or more flexible membranes include a primary membrane and one or more secondary membranes that are each joined to said primary membrane at a common edge, and the valve further includes a chamber formed in said substrate, separated from said second channel arm by said primary membrane and adapted to receive a positive or negative fluid pressure, thus to deform said primary membrane, wherein deformation of said primary membrane causes said one or more secondary membranes to deform and the combined deformation of the primary and secondary membranes is effective to switch the condition of the valve between its open and closed states.

11. The device of claim 8, which further includes another valve disposed in said first channel arm, operable to control the flow of fluid from the inlet channel to the outlet channel.

12. The device of claim 11, wherein the first channel arm has a pair of branches that flow around opposite sides of the well, and said another valve includes a pair of valves controlling fluid flow through each branch.