Automated cell culture system and method

• US 10,202,568 B2
• Filed: 08/12/2014
• Issued: 02/12/2019
• Est. Priority Date: 08/12/2013
• Status: Active Grant

• Current Assignee: InvivoSciences Inc.
• Original Assignee: InvivoSciences Inc.
• Inventors: Conway, Michael, Gerger, Michael, Wakatsuki, Tetsuro
• Primary Examiner(s): Ariani, Kade
• Application Number:
  US14/910,641
• Publication Number:
  US 20160177244A1
• Time in Patent Office: 1,645 Days
• Field of Search: None
• CPC Class Codes: C12M 23/12 Well or multiwell plates C12M25/04 takes precedence
• CPC Class Codes: C12M 33/06 for multiple inoculation or multiple collection of samples
• CPC Class Codes: C12M 33/07 Dosage or metering devices therefore
• CPC Class Codes: C12M 41/12 of temperature controlling the temperature of chemical or physical processes B01J19/0013, heating or cooling apparatus for laboratory use B01L7/00
• 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 41/48 Automatic or computerized control automatic analysis G01N35/00

Abstract

An automated method for culturing stem cells using a robotic liquid handling system including a translatable bed and a movable multichannel pipette. The method includes the steps of: locating a first multi-well cell culture plate and a multi-trough plate on the bed; placing a suspension of stem cells in at least one trough of the multi-trough plate; using the multi-channel pipette, transferring a portion of the suspension of stem cells to each well of the first multi-well cell culture plate such that at least two of the wells of the first multi-well cell culture plate have different densities of stem cells; selecting a well of the first multi-well cell culture plate having a desired stem cell density; locating a second multi-well cell culture plate; and using the multi-channel pipette, transferring the cells of the selected well to a plurality of wells of the second multi-well cell culture plate.

Claims

1. An automated method for culturing stem cells using a robotic liquid handling system including a translatable bed and a movable multi-channel pipette, the method comprising the steps of:

placing a suspension of stem cells in a first trough of the multi-trough plate positioned on the bed;

placing a cell culture media in a second trough of the multiple-trough plate;

mixing the suspension of stem cells in the multi-trough plate by linearly moving the translatable bed back and forth;

plating stem cells in a first multi-well cell culture plate located on the bed such that at least two wells of the first multi-well cell culture plate have different densities of stem cells by

using the multi-channel pipette, transferring different volumes of the suspension of stem cells from the first trough to the at least two wells of the first multi-well cell culture plate, and

using the multi-channel pipette, transferring different volumes of the cell culture media from the second trough to the at least two wells of the first multi-well cell culture plate,

wherein the different volumes of the suspension of stem cells and the different volumes of cell culture media are defined to achieve the different densities of stem cells in the at least two wells of the first multi-well cell culture plate, and

wherein the back and forth linear motion of the translatable bed maintains the suspension of stem cells in the first trough at a uniform concentration during the plating;

selecting a well of the first multi-well cell culture plate having a desired density of stem cells after growth of cell colonies in the first multi-well cell culture plate;

using the multi-channel pipette, transferring the cells of the selected well of the first multi-well cell culture plate to a plurality of wells of a second multi-well cell culture plate, wherein the first multi-well cell culture plate is positioned on a heated, angled adapter located on the bed while the cells are transferred from the selected well of the first multi-well cell culture plate to the plurality of wells of the second multi-well cell culture plate;

using the multi-channel pipette, transferring a portion of the cell culture media to the plurality of wells of the second multi-well cell culture plate; and

feeding the stem cells in the plurality of wells of the second multi-well cell culture plate, by removing the cell culture media from the plurality of wells of the second multi-well cell culture plate and adding new cell culture media to the plurality of wells of the second multi-well cell culture plate.

2. The method of claim 1, wherein transferring the cells of the selected well to a plurality of wells of the second multi-well cell culture plate comprises detaching the stem cells from the first multi-well cell culture plate using one of an enzymatic method or a chemical method.

3. The method of claim 2, wherein transferring the cells of the selected well to a plurality of wells of the second multi-well cell culture plate further comprises guiding the multi-channel pipette such that a plurality of pipette tips of the multi-channel pipette are adjacent side walls of a plurality of the wells of the second multi-well cell culture plate.

4. The method of claim 3, wherein guiding the multi-channel pipette comprises guiding the multi-channel pipette with an accuracy of at least one micrometer.

5. The method of claim 1, wherein the heated, angled adapter is angled at an angle of approximately 10°

.

6. The method of claim 1, further comprising determining a density of stem cells in at least one well of the second multi-well cell culture plate and splitting the stem cells of the at least one well of the second multi-well cell culture plate if the density is greater than a predetermined level.

7. The method of claim 6, wherein determining a density of stem cells in at least one well of the second multi-well cell culture plate comprises comparing the stem cells in at least one well of the second multi-well cell culture plate with a plurality of images of cultured stem cells to determine a density.

8. The method of claim 1, wherein removing cell culture media from the plurality of wells of the second multi-well cell culture plate comprises guiding the multi-channel pipette such that a plurality of pipette tips of the multi-channel pipette are located at or near a lowest point of the plurality of the wells of the second multi-well cell culture plate.

9. The method of claim 1, wherein the multi-well plate is one of a 24-well plate, a 48-well plate, and a 96-well plate.

10. The method of claim 1, wherein the wells of the first and second multi-well cell culture plate are coated with extracellular matrix material.

11. The method of claim 1, wherein the stem cells are pluripotent stem cells.

12. The method of claim 11, further comprising testing the stem cells of the second multi-well cell culture plate for pluripotency.

13. The method of claim 1, wherein each of the wells of the second multi-well cell culture plate has a bottom surface area of between 0.3 and 0.4 square centimeters.

14. The method of claim 1, wherein a bottom of each of the wells of the second multi-well cell culture plate has a ratio of circumference to area of between 0.56 and 0.64.

15. The method of claim 1, further comprising adding at least one of a compound and a gene to at least one of the wells of the second multi-well cell culture plate to induce differentiation.

16. The method of claim 15, wherein the at least one of a compound and a gene induces differentiation of the stem cells into cardiac myocytes.

17. The method of claim 1, further comprising:

positioning the first multi-well cell culture plate on the heated, angled adapter; and

treating the stem cells in the selected well of the first multi-well cell culture plate with an enzyme to promote cell detachment from the surface of the selected well, wherein the heated, angled adapter is configured to maintain the first multi-well cell culture plate at an elevated temperature while stem cells in the selected well are treated with the enzyme.

18. The method of claim 1, wherein plating the stem cells in the first multi-well cell culture plate located on the bed further includes controllably dispensing different volumes of stem cells in wells of each column of the first multi-well cell culture plate to create a density gradient by

transferring a first volume of the suspension of stem cells from the first trough into at least one well in a first column of the first multi-well cell culture plate;

and

transferring a second volume of the suspension of stem cells from the first trough into at least one well in a second column of the first multi-well cell culture plate, wherein the first volume of the suspension of stem cells is different from the second volume of the suspension of stem cells.

19. The method of claim 18, wherein selecting the well of the first multi-well cell culture plate having the desired density of stem cells includes identifying a well of the first multi-well cell culture plate with a density of stem cells determined to promote differentiation of pluripotent stem cells into a particular cell or tissue type.