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

• US 20040067482A1
• Filed: 10/07/2003
• Published: 04/08/2004
• Est. Priority Date: 11/22/2000
• Status: Active Grant

• Current Assignee: On-Chip Cellomics Consortium
• Original Assignee: Japan Science And Technology Corpration
• Inventors: Yasuda, Kenji, Inoue, Ippei, Wakamoto, Yuichi, Kaneko, Kunihiko, Yomo, Tetsuya
• Granted Patent: US 7,092,154 B2
• Time in Patent Office: Days
• US Class Current: 435/4
• 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

Novel technical means 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, means for supplying a cell culture medium into the cell culture container, and microscopic optical means for enabling long-term observation of the cell within the cell culture region. The novel technical means 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, to spray a substance which interacts with cells, for example, a drug such as a signal substance onto only a particular cell in a cell group which is being cultured so that cells are cultured at a constant cell density, and observe a difference in variation between the particular cell and other calls. There is also provided novel means 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. (Amended) An apparatus for the microscopic observation of long-term culture of a single cell, which enables the isolation, long-term culture and observation of a particular cell of aim, comprising

a cell culture container comprising a cell culture region consisting of a hole with 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 created on a substrate, said cell culture region comprising a means for trapping and mowing one cell for isolating a particular cell, a semi-permeable membrane with a molecular weight of 10,000 of more and a pore size of 0.2 μ

m or smaller covering the cell culture region, and a culture medium exchange region on top of the semi-permeable membrane;

a means for supplying culture medium to the cell culture container; and

an optical microscopic means for long-term microscopic observation of a cell within the cell culture region.

3. The apparatus for the microscopic observation of long-term culture of a single cell of claim 1, wherein the cell culture container is made of an optically transparent material.

4. Th apparatus for the microscopic observation of long-term culture of a single cell of claim 1, wherein the semi-permeable membrane is fixed to the top surface of the substrate by avidin-biotin bonding.

7. (Amended) The apparatus for the microscopic observation of long-term culture of a single cell of claim, 1, wherein the cell culture region contains at least two holes created on the top surface of the substrate, of which at least two are connected to each other by passage created on the top surface of the substrate through which a cell can pass.

8. The apparatus for the microscopic observation of long-term culture of a single cell of claim 1, wherein the cell culture container further comprises a draining means, so that the culture medium supplied to the culture medium exchange region by the culture medium supplying means is exchanged with the used medium in the hole of the cell culture region through the semi-permeable membrane and the used medium is discharged by the draining means.

9. The apparatus for the microscopic observation of long-term culture of a single cell of claim 1, which further comprises a valve for discharging gas remaining in the container.

10. The apparatus for the microscopic observation of long-term culture of a single cell of claim 1, which further comprises a means for controlling the temperature of the culture medium.

12. (Amended) The apparatus for the microscopic observation of long-term culture of a single cell of claim 1, wherein the means for trapping and moving a cell is an optical tweezers.

13. (Amended) The apparatus for the microscopic observation of long-term culture of a single cell of claim 1, wherein ultrasonic wave is used as the means for trapping and moving a cell.

14. (Amended) The apparatus for the microscopic observation of long-term culture of a single cell of claim 1, wherein electric field is used as the means for trapping and moving a cell.

15. The apparatus for the microscopic observation of long-term culture of a single cell of claim 1, which further comprises a pipet for spraying reagents into the hole of the cell culture region and for collecting the reagent.

16. The apparatus for the microscopic observation of long-term culture of a single cell of claim 1, which further comprises a filter inserted into the optical path of the optical microscopic means to enable fluorescence observation of the cell.

17. The apparatus for the microscopic observation of long-term culture of a single cell of claim 1, which further comprises a means for acquiring image data, a means for recognizing the shape of a particular cell in the image data, and a means for controlling the position of the stage and the focal length of an objective lens to maintain the particular cell in the center of the visual field.

18. The apparatus for the microscopic observation of long-term culture of a single cell of claim 1, which further comprises a means for measuring the number of cells in the hole of the cell culture region.

19. The apparatus for the micrscopic observation of long-term culture of a single cell of claim 1 or 15, which further comprises a pipet that can by inserted into the hole of the cell culture region through the semi-permeable membrane for collecting a particular cell from the hole or for injecting reagents and filling materials to the hole.

20. A method for the microscopic observation of long-term culture of a single cell, which comprises the use of the apparatus of claims 1 to 19.

2. (Cancelled)

5. (Cancelled)

6. (Cancelled)

11. (Cancelled)