Apparatus for microscopic observation of long-term culture of single cell

• US 7,092,154 B2
• Filed: 11/22/2001
• Issued: 08/15/2006
• Est. Priority Date: 11/22/2000
• Status: Expired due to Fees

• Current Assignee: On-Chip Cellomics Consortium
• Original Assignee: Japan Science And Technology Corpration (Japan Science And Technology Agency)
• Inventors: Inoue, Ippei, Wakamoto, Yuichi, Kaneko, Kunihiko, Yomo, Tetsuya, Yasuda, Kenji
• Primary Examiner(s): Dunn, Drew A.
• Assistant Examiner(s): Pritchett, Joshua L.
• Application Number:
  US10/432,396
• Publication Number:
  US 20040067482A1
• Time in Patent Office: 1,727 Days
• Field of Search: 359396-398, 356/244, 356/246
• US Class Current: 359/398
• CPC Class Codes: B01L 3/502 with fluid transport, e.g. in multi-compartment structures centrifugal-type cuvettes G01N21/07; analysis by separation into components G01N30/00; automatic analysers G01N35/00
• CPC Class Codes: C12M 25/00 Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
• CPC Class Codes: C12M 25/02 Membranes; Filters filters or filtration in general B01D24/00-B01D41/00
• CPC Class Codes: C12M 41/36 of biomass, e.g. colony counters or by turbidity measurements electrooptical investigation of individual particles G01N15/14, flow cytometers G01N15/1404
• CPC Class Codes: C12M 41/46 of cellular or enzymatic activity or functionality, e.g. cell viability
• CPC Class Codes: G01N 35/028 having reaction cells in the form of microtitration plates
• CPC Class Codes: G01N 35/1002 Reagent dispensers
• CPC Class Codes: G02B 21/34 Microscope slides, e.g. mounting specimens on microscope slides

Abstract

An apparatus is provided with a cell culture container having a cell culture region made of a hole formed on a substrate, a semi-permeable membrane covering a top plane of the cell culture region and a culture medium replacement part provided over the semi-permeable membrane, a mechanism for supplying a cell culture medium into the cell culture container, and a microscopic optical mechanism for enabling long-term observation of the cell within the cell culture region. This apparatus makes it possible to culture a cell group originating from a particular single cell, to perform culture and observation while identifying cells to be subjected to interaction during the process of culturing cells, and observe a difference in variation between the particular cell and other cells. There is also provided a mechanism which makes it possible to collect only a cell assuming a particular state and perform analysis or biochemical measurement of a gene of the cell, an expressed mRNA and the like.

Claims

1. An apparatus for enabling isolation, long-term culture and observation of a cell, said apparatus comprising:

a cell culture container having a culture medium exchange region;

a culture medium supplying device that supplies culture medium to said cell culture container;

a substrate coupled to said cell culture container;

a first cell culture region formed in said substrate, said first cell culture region comprising a first hole and being formed to hold one or more cells therein;

a second cell culture region formed in said substrate, said second cell culture region comprising a second hole and being formed to hold one or more cells therein;

a semi-permeable membrane arranged so as to cover said first cell culture region and said second cell culture region; and

a groove formed in said substrate,

wherein said groove connects said first cell culture region to said second cell culture region, and serves as a thin passage for a cell such that the cell can pass between said first cell culture region and said second cell culture region, and

wherein said culture medium exchange region is disposed above said semi-permeable membrane.

2. The apparatus according to claim 1, further comprising:

a first electrode disposed in a vicinity of said first cell culture region; and

a second electrode disposed in a vicinity of said second cell culture region;

wherein, when a charge is applied to said first electrode, a cell disposed within said second cell culture region moves from said second cell culture region to said first cell culture region via said groove that connects said first cell culture region to said second cell culture region.

3. A method for the microscopic observation of long-term culture of a single cell, which comprises the use of the apparatus of claim 2.

4. The apparatus according to claim 1,

wherein each of said first cell culture region and said second cell culture region have a diameter larger than or equal to 1 μ

m and smaller than or equal to 1 mm, and a depth less than or equal to 100 μ

m, and

wherein said semi-permeable membrane has a molecular weight of 10,000 or more and a pore size of 0.2 μ

m or smaller.

5. A method for the microscopic observation of long-term culture of a single cell, which comprises the use of the apparatus of claim 4.

6. The apparatus according to claim 1, wherein said cell culture container is made of an optically transparent material.

7. A method for the microscopic observation of long-term culture of a single cell, which comprises the use of the apparatus of claim 6.

8. The apparatus according to claim 1, wherein said semi-permeable membrane is fixed to a top surface of said substrate by avidin-biotin bonding.

9. A method for the microscopic observation of long-term culture of a single cell, which comprises the use of the apparatus of claim 8.

10. The apparatus according to claim 1, wherein said cell culture container further comprises a draining mechanism, so that the culture medium supplied to said culture medium exchange region from said culture medium supplying device is exchanged with used culture medium in said first and second cell culture regions through said semi-permeable membrane, wherein the used culture medium is discharged by said draining mechanism.

11. A method for the microscopic observation of long-term culture of a single cell, which comprises the use of the apparatus of claim 10.

12. The apparatus according to claim 1, further comprising a valve operable to discharge gas remaining in said cell culture container.

13. A method for the microscopic observation of long-term culture of a single cell, which comprises the use of the apparatus of claim 12.

14. The apparatus according to claim 1, further comprising a mechanism for controlling a temperature of the culture medium.

15. A method for the microscopic observation of long-term culture of a single cell, which comprises the use of the apparatus of claim 14.

16. The apparatus according to claim 1, wherein the cell can be trapped and moved between said first cell culture region and said second cell culture region by an optical tweezers.

17. A method for the microscopic observation of long-term culture of a single cell, which comprises the use of the apparatus of claim 16.

18. The apparatus according to claim 1, wherein the cell can be tapped and moved between said first cell culture region and said second cell culture region by an ultrasonic wave.

19. A method for the microscopic observation of long-term culture of a single cell, which comprises the use of the apparatus of claim 18.

20. The apparatus according to claim 1, wherein the cell can be trapped and moved between said first cell culture region and said second cell culture region by an electric field.

21. A method for the microscopic observation of long-term culture of a single cell, which comprises the use of the apparatus of claim 20.

22. The apparatus according to claim 1, wherein a pipet can be utilized for spraying reagents into said first cell culture region and said second cell culture region and for collecting the reagents.

23. A method for the microscopic observation of long-term culture of a single cell, which comprises the use of the apparatus of claim 22.

24. The apparatus according to claim 1, further comprising:

an optical microscope which enables long-term microscopic observation of a cell within said first cell culture region or said second cell culture region; and

a filter inserted into an optical path of said optical microscope so as to enable fluorescence observation of the cell.

25. A method for the microscopic observation of long-term culture of a single cell, which comprises the use of the apparatus of claim 24.

26. The apparatus according to claim 1, further comprising:

a means for acquiring image data;

a means for recognizing a shape of a particular cell in the image data; and

a means for controlling a position of a stage and a focal length of an objective lens so as to maintain the particular cell in a center of a visual field.

27. A method for the microscopic observation of long-term culture of a single cell, which comprises the use of the apparatus of claim 26.

28. The apparatus according to claim 1, further comprising a means for measuring the number of cells in said first and second cell culture regions.

29. A method for the microscopic observation of long-term culture of a single cell, which comprises the use of the apparatus of claim 28.

30. A method for the microscopic observation of long-term culture of a single cell, which comprises the use of the apparatus of claim 1.