Array-based microenvironment for cell culturing, cell monitoring and drug-target validation

• US 6,653,124 B1
• Filed: 11/10/2000
• Issued: 11/25/2003
• Est. Priority Date: 11/10/2000
• Status: Expired due to Fees

• Current Assignee: Middle Atlantic Products Incorporated (Legrand SA)
• Original Assignee: Cytoplex Biosciences Incorporated
• Inventors: Freeman, Alex R.
• Primary Examiner(s): Redding, David A.
• Application Number:
  US09/710,700
• Time in Patent Office: 1,110 Days
• Field of Search: 435/297.5, 435/286.5, 435/287.1, 435/287.2, 435/288.4, 435/288.5, 435/288.7, 435/297.1, 435/305.2
• US Class Current: 435/297.1
• CPC Class Codes: B01L 2200/10 Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• 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 2300/0877 Flow chambers
• CPC Class Codes: B01L 2300/0887 Laminated structure
• CPC Class Codes: B01L 2400/0475 specific mechanical means and fluid pressure
• CPC Class Codes: B01L 2400/0487 fluid pressure, pneumatics
• CPC Class Codes: B01L 2400/0638 membrane valves, flap valves
• 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 3/50273 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: B33Y 80/00 Products made by additive manufacturing
• CPC Class Codes: C08L 2201/12 Shape memory
• CPC Class Codes: C12M 23/16 Microfluidic devices; Capillary tubes integrated microfluidic structures B01L3/5027; microreactors B01J19/0093
• CPC Class Codes: C12M 23/58 Reaction vessels connected in series or in parallel combinations of bioreactors with other apparatus, C12M43/00
• CPC Class Codes: F16K 2099/0069 Bistable microvalves
• CPC Class Codes: F16K 2099/0074 using photolithography, e.g. etching
• CPC Class Codes: F16K 2099/0076 using electrical discharge machining [EDM], milling or drilling
• 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 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 99/0013 : Rotary valves
• CPC Class Codes: F16K 99/0015 : Diaphragm or membrane valves
• CPC Class Codes: F16K 99/0034 : Operating means specially adapted for microvalves
• CPC Class Codes: F16K 99/0036 : operated by temperature variations
• CPC Class Codes: F16K 99/0038 : using shape memory alloys
• CPC Class Codes: F16K 99/0042 : Electric operating means therefor
• CPC Class Codes: F16K 99/0046 : using magnets
• CPC Class Codes: F16K 99/0048 : using piezoelectric means
• CPC Class Codes: F16K 99/0051 : using electrostatic means
• CPC Class Codes: F16K 99/0055 : actuated by fluids

Abstract

An apparatus and method for cell culture in microchambers that are formed on or adjacent to a substrate. The microchambers are connected by one or more fluid conduits, valves and reagent reservoirs, is disclosed.

Claims

1. An apparatus, comprising:

one or more microchambers;

wherein the one or more of the microchambers are adapted to contain cells;

one or more fluid conduits in fluid communication with the microchamber;

one or more valves adapted to regulate the flow of a fluid to the one or more microchambers;

one or more reagent reservoirs in fluid communication with the one or more fluid conduits, wherein the valve regulates the flow of fluids between the one or more microchambers and the one or more reagent reservoirs adapted to exchange nutrients, oxygen and remove excretions; and

the one or more microchambers adapted to contain cells is substantially planar.

2. The apparatus of claim 1, further comprising at least one pump in fluid communication with the fluid conduit.

3. The apparatus of claim 2, further comprising a valve controller adapted to control the at least one pump.

4. The apparatus of claim 2, further comprising a pump controller.

5. The apparatus of claim 1, further comprising one or more sensors in fluid communication with the one or more microchambers, the one or more fluid conduits or the one or more reagent reservoirs.

6. The apparatus of claim 1, integrated on a substrate.

7. The apparatus of claim 6, wherein the substrate is integral with at least one of a following element from a group consisting of:

the fluid conduit;

the microchamber; and

the reagent reservoir.

8. The apparatus of claim 1, further comprising an array of arrays coupled to the substrate.

9. The apparatus of claim 1, further comprising an array associated with the one or more microchambers.

10. The apparatus of claim 1, wherein the one or more microchambers form a part of an array.

11. The apparatus of claim 1, further comprising encapsulation of the one or more microchambers, one or more fluid conduits and one or more fluid reservoirs.

12. The apparatus of claim 1, wherein the one or more microchambers comprise a cell adherent surface.

13. The apparatus of claim 1, wherein the microchambers form a 4-, 6-, 12-, 48-, 96- or 384-microchamber array.

14. The apparatus of claim 1, further comprising a diode array in fluid communication with the microchamber or the reagent reservoir.

15. The apparatus of claim 1, further comprising a micromirror array adjacent to and facing the microchamber or the reagent reservoir.

16. The apparatus of claim 15, wherein the microchamber further comprises a chemically treated substrate integral with the microchamber suitable for culturing cells wherein the micromirror array directs a light source onto the chemically treated substrate to produce a patterned array of photochemically treated substrate in the microchamber.

17. The apparatus of claim 16, wherein the light source is integral to the apparatus.

18. The apparatus of claim 16, wherein the light source comprises a laser.

19. The apparatus of claim 16, wherein the light source comprises ultraviolet light.

20. The apparatus of claim 16, wherein the substrate is treated with an organosilane.

21. The apparatus of claim 20, wherein the organosilane further comprises a biologically functional group.

22. The apparatus of claim 21, wherein the biologically functional group comprises a free amino moiety.

23. The apparatus of claim 22, wherein lectin is reacted with the free amino moiety to provide a cell adherent substrate.

24. The apparatus of claim 16, wherein a microprocessor controls the micromirror direction of the light source onto the substrate.

25. The apparatus of claim 16, wherein the micromirrors are positioned to direct light into the microchamber to regulate the temperature of the microchamber.

26. The apparatus of claim 16, wherein the patterned array is produced in a covered microwell.

27. A method for producing a patterned.array of cultured cells in the cell culture apparatus of claim 16, the method comprising the steps of:

treating the substrate with one or more photosensitive chemicals;

directing light of a selected wavelength at the micromirrors whereby the micromirrors re-direct the light onto the treated substrate in an array pattern to produce a substrate having photochemically treated portions wherein the photochemically treated portions of the substrate form a patterned array of photochemically treated substrate; and

adhering cells to the substrate whereby the cells adhere to the photochemically treated portions of the substrate to produce a patterned array of cultured cells.

28. The method of claim 27, wherein the light source comprises a laser.

29. The method of claim 27, wherein the light source comprises ultraviolet light.

30. The method of claim 27, wherein the substrate is treated with an organosilane.

31. The method of claim 30, wherein the organosilane further comprises a biologically functionaligroup.

32. The method of claim 31, wherein the biologically functional group comprises a free amino moiety.

33. The method of claim 32, wherein lectin is reacted with the free amino moiety to provide a cell adherent substrate.

34. The method of claim 27, wherein a computer controls the micromirror direction of the light source onto the substrate.

35. The method of claim 27, further comprising a laser in fluid communication with the microchamber or the reagent reservoir.

36. The method of claim 27, further comprising a light amplifier in fluid communication with the microchamber or the reagent reservoir.

37. The method of claim 27, further comprising a mirror in fluid communication with the microchamber or the reagent reservoir.

38. An apparatus, comprising:

one or more microchambers;

wherein the one or more of the microchambers are adapted to contain cells;

one or more fluid conduits in fluid communication with the microchamber;

one or more valves adapted to regulate the flow of a fluid to the one or more microchambers;

one or more reagent reservoirs in fluid communication with the one or more fluid conduits, wherein the valve regulates the flow of fluids between the one or more microchambers and the one or more reagent reservoirs adapted to exchange nutrients, oxygen and remove excretions;

the one or more microchambers adapted to contain cells is substantially planar; and

one or more micromirrors arranged to direct light into the microchambers.

39. A cell culturing system, comprising:

one or more nano-scale microchambers for housing cultured cells;

one or more of nano-scale fluid inlets and outlets in fluid communication with the microchambers;

one or more controls to regulate the inlet and the outlet of fluids between the inlets and the outlets and the microchambers; and

one or more controls in communication with the microchambers for detecting biological parameters of the cultured cells.

40. An apparatus, comprising:

one or more microchambers;

wherein the one or more of the microchambers are adapted to contain cells;

one or more fluid conduits in fluid communication with the microchamber;

one or more valves adapted to regulate the flow of a fluid to the one or more microchambers;

one or more reagent reservoirs in fluid communication with the one or more fluid conduits, wherein the valve regulates the flow of fluids between the one or more microchambers and the one or more reagent reservoirs adapted to exchange nutrients, oxygen and remove excretions; and

the one or more microchambers adapted to contain cells is substantially planar for detecting biological parameters of the cultured cells.

41. A cell culturing system comprising:

a chamber having a bottom substrate for growing cultured cells, the chamber further comprising through-holes for controlled porosity;

a nanoporous semipermeable membrane attached to the substrate; and

a top head on the bottom substrate, the head being adapted to provide space for cells to grow on the substrate and to sandwich the membrane between the substrate and the head,

wherein fluid exchange occurs across the membrane through the through-holes.