Automated system for maintenance and differentiation of pluripotent stem cells

Patent metadata

Field	Value
Publication number	US-11149245-B2
Application number	US-201715478585-A
Country	US
Kind code	B2
Filing date	Apr 4, 2017
Priority date	Apr 4, 2017
Publication date	Oct 19, 2021
Grant date	Oct 19, 2021

Abstract

The present invention relates to the automated maintenance of cells particularly reprogrammed somatic cells into iPSCs to enable the large-scale culture and passaging of human pluripotent stem cells (PSCs) that can be adapted to a Freedom EVO®.

First claim

The claims defining the invention are as follows:

1. An automated instrument for sterile cell culture, said instrument comprising:
a platform for receiving and moving a cell culture plate from a housing;
a manipulator arm for moving the culture plate between the housing and the platform and placing the culture plate on the platform;
a receptacle containing a sterile solution to be dispensed controllably and sterilely to the culture plate;
a disposable tip carrier carrying sterile disposable tips having a capacity of 5 mL for dispensing the sterile solution from the receptacle to the culture plate and removing solution from the culture plate;
a liquid handling system including a robotic arm which interacts with the disposable tip carrier to fetch one or more disposable tips, and a syringe pump which is controlled to aliquot a first predetermined volume of the sterile solution from the receptacle into the one or more disposable tips, and to dispense the first predetermined volume of the sterile solution to and remove a second predetermined volume of solution from the culture plate such that the solution is sterilely dispensed from the disposable tips without dispersion outside the sterile cell culture while preventing contamination from the syringe pump, and wherein the syringe pump has at least 5 mL capacity and is controlled to remove the second predetermined volume of solution from the culture plate without causing additional air displacement in the disposable tip; and
a controlling system configured to control the operation of each of the manipulator arm, the platform and the liquid handling system such that the liquid handling system controllably and sterilely dispenses and removes individual predetermined volumes of solution to and from the culture plate before the manipulator arm optionally returns the culture plate to the housing, thereby achieving automated maintenance, within the instrument, of cells in the cell culture.

2. An automated instrument according to claim 1 wherein the housing is an incubator for housing a culture plate comprising a cell culture well containing the cells or a hotel to hold the culture plate for later use.

3. An automated instrument according to claim 1 wherein said housing is an incubator or a hotel to which the manipulator arm has access to obtain or return the culture plate.

4. An automated instrument according to claim 1 wherein the receptacle contains a solution selected from a culture medium, PBS, cell releasing solution for dislodging cells or a coating for cell culture plates.

5. An automated instrument according to claim 1 wherein the platform is moveable.

6. An automated instrument according to claim 5 wherein the platform is moveable by tilting, shaking, rotating or any combination thereof.

7. An automated instrument according to claim 6 wherein the controlling system is programmed to tilt the platform at a predetermined angle for a predetermined number of times sufficient to spread the solution over the cells in culture.

8. An automated instrument according to claim 7 wherein the controlling system is programmed to tilt the platform for approximately 4 times at ±40° at each addition of solution.

9. An automated instrument according to claim 1 wherein the platform, one or more receptacles containing solution and disposable tip carrier are housed in a biosafety cabinet of the instrument.

10. An automated instrument according to claim 9 wherein the biosafety cabinet further includes the liquid handling system and the manipulator arm.

11. An automated instrument according to claim 1 wherein the disposable tips are filtered disposable tips.

12. An automated instrument according to claim 1 where the second predetermined volume is less than the first predetermined volume such that the liquid handling system removes a volume less than a volume present in the culture plate prior to delivering a volume of solution to the culture plate suitable to maintain the cells in cell culture.

13. An automated instrument according to claim 1 for sterile passaging of cells, wherein said sterile solution is a cell releasing solution.

14. An automated instrument for sterile passaging of cells according to claim 13 , said system further including:
a dispenser for controllably and sterilely dispensing a cell releasing solution to the cells in the culture plate.

15. An automated instrument for sterile passaging of cells according to claim 13 wherein the controlling system is configured to cause the liquid handling system to collect the cells from the culture plate for passaging to another culture plate and wherein a separate disposable tip is assigned for each culture plate for collection of cells and passage of cells.

16. An automated instrument for sterile passaging of cells according to claim 13 wherein the controlling system is configured to cause the liquid handling system to dispense the cell releasing solution to the culture plate for incubation with the cells for a predetermined time that is sufficient to detach the cells.

17. An automated instrument for sterile passaging of cells according to claim 13 wherein the controlling system is programmed to tilt the platform at a predetermined angle for a predetermined number of times sufficient to spread the cell releasing solution over the cell culture.

18. An automated instrument for sterile passaging of cells according to claim 13 wherein the controlling system is programmed to tilt the platform for approximately 20 times at ±10° to rinse the cells.

19. An automated instrument for sterile passaging of cells according to claim 13 wherein the controlling system is programmed to tilt the platform for approximately 20 times at ±10° with the cell releasing solution exposed to the cells.

20. An automated instrument for sterile passaging of cells according to claim 13 wherein the controlling system is programmed to tilt the platform for approximately 4 times at ±40° to change medium.

21. An automated instrument for sterile passaging of cells according to claim 13 wherein the controlling system is programmed to tilt the platform to shake for approximately 100 times ±5°; approximately 20 times ±40°, and then approximately 1000 times ±0.5° to detach the cells.

22. An automated instrument for sterile passaging of cells according to claim 13 wherein the controlling system is programmed to tilt the platform at an approximately 52° angle to aspirate and collect the cells following detachment from the culture plate for further passaging.

23. An automated instrument for sterile passaging of cells according to claim 13 wherein a split ratio for each culture plate for passaging cells is determined from a density of released cells following detachment from the culture plate.

24. An automated instrument for sterile passaging of cells according to claim 23 wherein the split ratio is in the range of approximately 1:1 to 1:6.

25. An automated instrument according to claim 1 for sterile differentiation of cells, wherein the sterile solution comprises a differentiation factor.

26. An automated instrument for sterile differentiation of cells, said system comprising an automated instrument according to claim 25 and further comprising:
a differentiation unit for sterilely dispensing a differentiation factor to the cells in the culture plate to induce differentiation of the cells and wherein the liquid handling system further interacts

Assignees

• Tecan Trading Ag

Inventors

• Pebay Alice
• Hewitt Alex
• Crombie Duncan
• Liang Helena
• Wong Raymond
• Daniszewski Maciej
• Zalivani Marco

Classifications

• C12M37/00

  Means for sterilizing, maintaining sterile conditions or avoiding chemical or biological contamination (C12M23/38 takes precedence; filtration in general and filters per se B01D24/00-B01D41/00; autoclaves B01J3/04; treatment of microorganisms with electrical or wave energy C12N13/00) · CPC title

• C12M33/04

  by injection or suction, e.g. using pipettes, syringes, needles (pipettes in general B01L3/02) · CPC title

• C12M41/14

  Incubators; Climatic chambers (per se B01L1/00) · CPC title

• C12M47/02

  Separating microorganisms from the culture medium; Concentration of biomass (separating microorganisms from their culture media C12N1/02) · CPC title

• C12M41/48 Primary

  Automatic or computerized control (automatic analysis G01N35/00) · CPC title