Apparatus and methods for three-dimensional tissue measurements based on controlled media flow

• US 9,494,577 B2
• Filed: 11/13/2013
• Issued: 11/15/2016
• Est. Priority Date: 11/13/2012
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Seahorse Biosciences
• Inventors: McGarr, Paul, Neilson, Andy C., Teich, Jay S.
• Primary Examiner(s): BROWN, MELANIE YU
• Application Number:
  US14/079,022
• Publication Number:
  US 20140170671A1
• Time in Patent Office: 1,098 Days
• US Class Current: 1/1
• CPC Class Codes: B01L 2200/026 Fluid interfacing between devices or objects, e.g. connectors, inlet details
• CPC Class Codes: B01L 2300/046 Function or devices integrated in the closure
• CPC Class Codes: B01L 2300/0829 Multi-well plates; Microtitration plates
• CPC Class Codes: B01L 2400/0478 pistons
• CPC Class Codes: B01L 3/5025 for parallel transport of multiple samples
• CPC Class Codes: B01L 3/50853 with covers or lids
• CPC Class Codes: C12M 23/12 Well or multiwell plates C12M25/04 takes precedence
• CPC Class Codes: C12M 25/04 in combination with well or multiwell plates, i.e. culture inserts
• CPC Class Codes: C12M 27/00 Means for mixing, agitating or circulating fluids in the vessel by introduction of gas C12M29/06, C12M29/14, mixing in general or mixers per se B01F; mixing in apparatus for chemical or physical processes B01J
• CPC Class Codes: C12M 29/10 Perfusion
• 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 41/32 of substances in solution
• CPC Class Codes: G01N 21/6452 Individual samples arranged in a regular 2D-array, e.g. multiwell plates
• CPC Class Codes: G01N 33/5091 for testing the pathological state of an organism

Abstract

An apparatus including a plurality of wells for conducting analysis of three-dimensional cell samples (e.g., tissue samples) and methods for experimenting with a three-dimensional sample. A removable insert for use with the apparatus enables plunger-driven perfusion of the three-dimensional sample.

Claims

1. An apparatus for conducting analysis of a three-dimensional cell sample, the apparatus comprising:

a plurality of wells for holding respective samples and sample media, at least one of the wells comprising

a sample nesting site disposed in a bottom surface of the well,

disposed thereabove a bore dimensioned to interfit with a plunger which moves vertically down within and relative to the bore and within the sample media disposed in the well, and

a media channel defined by the well, a removable well insert, or both the well and the insert, in fluid communication with the sample nesting site, which permits flow of the sample media therethrough impelled by the plunger and permits exposure of the sample to flowing media as the plunger moves.

2. The apparatus of claim 1 wherein the plunger moves relative to the nesting site to induce perfusion of media about the sample.

3. The apparatus of claim 1 wherein said media channel comprises a fluid path which returns media perfused about the sample back to media disposed in the well.

4. The apparatus of claim 1 wherein at least said bore is defined by the removable well insert.

5. The apparatus of claim 1 wherein the well comprises a sump in fluid communication with the media channel.

6. The apparatus of claim 1 wherein said sample nesting site is disposed within a sump for collection of media and in fluid communication with said media channel.

7. The apparatus of claim 1 wherein said media channel comprises a fluid path defining a closed loop beneath the surface of media in the wells to permit media perfusive flow about said sample on both upward and downward movement of a plunger within said bore.

8. The apparatus of claim 1 further comprising a sensor for detecting the concentration of a solute in media disposed about said sample.

9. The apparatus of claim 1 comprising a plurality of plungers adapted for reciprocating movement within the bores of respective wells.

10. The apparatus of claim 1 wherein the plurality of wells defines a multiwell plate comprising 24 or 96 wells.

11. The apparatus of claim 1 further comprising a source of oxygen, carbon dioxide, and/or a biologically inert gas in fluid communication with media in a well or a headspace above the surface of media in the wells for controlling the composition of gas in the headspace or in the media.

12. The apparatus of claim 1 further comprising a source of a solution of a biologically active substance in fluid communication with media in wells for exposing a sample to said substance.

13. The apparatus of claim 1 further comprising a three-dimensional cell growth scaffold disposed on said sample nesting site.

14. The apparatus of claim 1 further comprising a check valve disposed in said media channel to inhibit media perfusion from the channel back to the sample.

15. An apparatus for conducting analysis of three-dimensional cell samples, the apparatus comprising:

a well for holding a sample and sample media comprising

a sample nesting site disposed in a bottom surface of the well,

disposed above said nesting site, a bore dimensioned to interfit with a plunger which moves vertically down within the bore and within media disposed in said well,

a media channel in fluid communication with said sample nesting site which permits media perfusion about said sample, wherein the media channel is defined by the well, a removable well insert, or both the well and the insert, and

a plunger adapted for reciprocal movement in said bore thereby to impel media perfusion about said sample,

wherein the media channel permits flow of the sample media disposed in the well therethrough impelled by the plunger and permits exposure of the sample to flowing media as the plunger moves.

16. The apparatus of claim 15 further comprising a sensor for detecting the concentration of a dissolved media component in media about the sample nesting site.

17. A method of impelling media flow about a three-dimensional cell culture sample, so as to maintain viability of the sample and exercise control over its microenvironment, the method comprising the steps of:

providing a well including a sample nesting site disposed in a bottom surface of the well, a bore dimensioned to interfit with a plunger disposed above the sample nesting site, a media channel in fluid communication with said sample nesting site, wherein the media channel is defined by the well, a removable well insert, or both the well and the insert, and a plunger adapted for reciprocal movement in said bore;

placing a sample on the sample nesting site in medium within the well, and

moving said plunger within the bore to impel media flow about the sample and through said channel thereby to perfuse the sample with media,

wherein the media channel permits flow of the sample media disposed in the well therethrough impelled by the plunger and permits exposure of the sample to flowing media as the plunger moves.

18. The method of claim 17 comprising the additional step of adding a gas to the media or to headspace in the well above the media to modify the microenvironment about the sample by altering dissolved gas composition.

19. The method of claim 17 comprising the additional step of adding a solution of a biologically active substance to the media to modify the microenvironment about the sample by exposing the sample to the biologically active substance.

20. The method of claim 17 comprising the additional step of measuring the concentration of one or more solutes in media about said sample.

21. The method of claim 17 comprising the additional step of making a plurality of measurements separated in time of the concentration of one or more solutes in media about said sample.

22. The method of claim 17 comprising the additional step of adding at least one of a metered amount of one or more gases and one or more solutes to media in said well thereby to set the microenvironment in the medium about said sample to a predetermined point.

23. The method of claim 17 wherein the sample is a tumor sample, the method comprising the additional step of adding a metered amount of one or more gases and/or one or more solutes to media in said well thereby to set the microenvironment in the medium about said sample to a predetermined point simulating the microenvironment of the tumor sample in vivo.

24. The method of claim 17 comprising multiplexing said method by the steps of:

providing a plurality of said wells including sample nesting sites, bores dimensioned to interfit with plungers disposed above the sample nesting sites, media channels in fluid communication with said sample nesting site, and plungers adapted for reciprocal movement in said bore;

placing a sample on the sample nesting sites in media within the plurality of wells,

moving the plurality of said plungers within the bores to impel media flow about the samples and through said channels thereby to perfuse the samples with media, and

measuring one or more times the concentration of one or more solutes in media about said samples in the plurality of said wells.

25. The method of claim 17 comprising the additional step of placing one or a plurality of beads bearing immobilized binders for biomolecules of interest secreted from or absorbed by the sample in the media and using the beads to detect the presence or concentration of said biomolecules in the microenvironment of the sample.