CELL CULTURE ARRAY SYSTEM FOR AUTOMATED ASSAYS AND METHODS OF OPERATION AND MANUFACTURE THEREOF

• US 20090203126A1
• Filed: 01/05/2009
• Published: 08/13/2009
• Est. Priority Date: 01/03/2008
• Status: Active Grant

• Current Assignee: EMD Millipore Corporation (Merck & Co., Inc.)
• Original Assignee: CellASIC Co.
• Inventors: Lee, Philip J., Hung, Paul J.
• Granted Patent: US 9,376,658 B2
• Time in Patent Office: Days
• US Class Current: 435/325
• CPC Class Codes: B01L 2300/0829 Multi-well plates; Microtitration plates
• CPC Class Codes: B01L 2300/0848 Specific forms of parts of containers
• CPC Class Codes: B01L 2300/0861 Configuration of multiple channels and/or chambers in a single devices
• CPC Class Codes: B01L 2400/0457 passive flow or gravitation
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 3/5025 for parallel transport of multiple samples
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• 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 3/502761 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: 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 41/48 Automatic or computerized control automatic analysis G01N35/00
• CPC Class Codes: G01N 2035/00148 Test cards, e.g. Biomerieux or McDonnel multiwell test cards
• CPC Class Codes: G01N 35/00029 provided with flat sample substrates, e.g. slides G01N35/028 takes precedence
• CPC Class Codes: G01N 35/0099 comprising robots or similar manipulators robots per se B25J

Abstract

A number of novel improved microfluidic configurations and systems and methods of manufacture and operation. In one embodiment, three wells are used for independent cell culture systems in a cell culture array. In a second aspect, artificial sinusoids with artificial epithelial barriers are provided with just one (optionally shared or multiplexed) fluidic inlet and one (optionally shared or multiplexed) fluidic output, where the medium output also functions as a cellular input. A pneumatic cell loader combined with other components provides a fully automated cell culture system. Magnetic alignment of plate molds provides advantages and ease of molded manufacture.

Claims

1. A microfluidic cell culture system comprising:

a medium inlet reservoir fluidically connected to a plurality of microfluidic medium channels;

a plurality of cell culture areas each located at an end of a cell culture channel;

said cell culture channel having a cell inlet opening fluidically connected to a cell inlet reservoir for introducing cells to said plurality of cell culture areas;

a perfusion barrier between said cell culture areas and said plurality of microfluidic medium channels, said perfusion barrier containing a plurality of perfusion passages;

a media outlet reservoir fluidically connected to said cell culture channels;

such that fluid introduced into said medium inlet reservoir must pass through a part of said cell culture channels before reaching said media outlet reservoir.

3. The system of claim 1 further wherein said system is integrated into a standard well plate and further wherein each independent device occupies three well positions, a first well acting as a medium inlet, a second well acting as a medium outlet/cell inlet, and a middle well acting as a viewing/cell culture area.

4. The system of claim 1 further wherein said cell culture channels include a first portion near said cell inlet that contains solid barriers walls with no perfusion passages to prevent cell adhesion.

5. The system of claim 1 further wherein said cell culture channels include a second portion nearer to said cell inlet than said first portion, said second portion having a plurality of perfusion passages to facilitate media flow out of said cell culture region.

6. The system of claim 1 further wherein said standard well plate is handled by robotic equipment allowing for automated cell culture and analysis, said robotic equipment being in part equipment designed for use with standard well plates.

7. The system of claim 1 further wherein said standard well plate is configured to pneumatically couple with a pneumatic manifold, said manifold providing pneumatic pressure to drive cells into said cell culture areas.

8. The system of claim 1 further wherein:

said cell culture areas are separated into at least three blocks, each block containing at least two cell culture areas;

each block having an air channel adjacent thereto.

9. The system of claim 1 further wherein:

cells are introduced into said cell culture areas using pneumatic pressure, and thereafter, cells are maintained by perfusion of media using a passive gravity driven fluid flow.

10. The system of claim 1 further wherein:

a plurality of said perfusion passages are substantially narrower than a diameter of cells to be cultured.

11. The system of claim 1 further wherein:

a plurality of said perfusion passages are substantially narrower than about 6 microns.

12. The system of claim 1 further comprising:

a large fluidic opening directly connected to said cell culture channels to allow for easier cell flow.

13. The system of claim 1 further comprising:

a fluidic multiplexor connecting said media inlet reservoir to said microfluidic medium channels.

15. The system of claim 1 further wherein:

a plurality of said cell culture channels comprise a cell culture area with perfusion passages to said media channels and a non perfusion cell channel portion for better localizing cells introduced under pneumatic pressure and an outlet portion with perfusion passages near to an inlet of said cell culture channel.

16. The system of claim 1 further wherein:

said cell culture areas are elongated areas mimetic of a liver sinusoid;

said cell culture areas are loaded with cells from said cell inlet reservoir;

after cell loading and settling, media is loaded from said media inlet and removed from said cell inlet/medium outlet reservoir.

2. (canceled)

14. (canceled)

17. A microfluidic cell culture device comprising:

a medium inlet reservoir and a channel for introducing liquid media to a plurality of cell culture areas;

a cell inlet/medium outlet reservoir for introducing cells to said plurality of cell culture areas;

wherein said cell culture areas are elongated areas mimetic of a liver sinusoid;

wherein said cell culture areas are loaded with cells from said cell inlet/medium outlet reservoir;

wherein after cell loading and settling, media is loaded from said media inlet and received and/or removed from said cell inlet/medium outlet reservoir;

such that a portion of said media passes through cells in said culture area.

19. The device of claim 17 further wherein said device is integrated into a standard well plate and further wherein each independent device utilizes at least three well positions, a first well acting as a medium inlet, a second well acting as a medium outlet/cell inlet, and a middle well acting as a viewing/cell culture area.

24. The device of claim 17 further wherein:

said cell culture areas are separated into at least three blocks, each block containing at least two cell culture areas;

each block having an air channel adjacent thereto.

18. (canceled)

20-23. -23. (canceled)

25. A method of culturing cells comprising:

placing cells into a well of a standard well plate, said well fluidically connected to a plurality of microfluidic channels, said microfluidic channels connected to a plurality of cell culture areas;

placing media into a different well of said standard well plate, said well fluidically connected to a plurality of microfluidic perfusion passages, said microfluidic perfusion passages providing perfusion passages through said micro cell culture areas;

allowing said cells to culture for an appropriate time;

observing and/or assaying cells and or media in said standard well plate.

26. The method of claim 25 further comprising:

interfacing said standard well plate with a pneumatic manifold for providing air pressure to drive said cells into said cell culture areas, said pneumatic manifold providing sufficient pressure to form cell aggregates in said cell culture areas, wherein said pneumatic manifold interfaces with said well plate using a vacuum seal.

28. The method of claim 26 further comprising:

using a pipette to introduce and/or remove fluids from said reservoirs, said fluids passively perfusing through said cell culture areas as a result of gravity and/or differential fluid levels; and

or surface tensions.

30. The method of claim 25 further wherein said steps are performed by fully automated robotic equipment and further wherein said combination of pneumatic cell loading and passive perfusion allows simultaneous automatic culture of a large number of plates using one pneumatic manifold and one automated pipettor because plates do not need to be attached to any equipment during perfusion fluid flow.

27. (canceled)

29. (canceled)

31-87. -87. (canceled)