JPWO2019241885A5

JPWO2019241885A5 -

Info

Publication number: JPWO2019241885A5
Application number: JP2020571616A
Authority: JP
Publication date: 2023-08-18
Anticipated expiration: 2039-06-19

Description

システム１は、液体ディスペンサモジュール３００をさらに備える。液体ディスペンサモジュール３００は、ピペットモジュール２００から離間され得る。明確にするために、離間することは、これらがシステム１の別個の要素であり、場合によってはシステム１の異なる場所にあることを意味する。液体ディスペンサモジュール３００は、溶液を細胞培養容器にバルクで分注するために使用されるが、ピペットモジュール２００は、細胞培養容器から流体の一部又は全部を引き出すために使用される。液体ディスペンサモジュール３００の例示的な使用例は、細胞の懸濁液を播種する前又は後のいずれかで、細胞培養容器又は娘細胞培養容器に細胞の培養物又は分化培地を分注すること、細胞培養容器内の細胞の培養物の自動継代プロトコル中の細胞培養容器への洗浄バッファ又は細胞剥離溶液を分注すること、又は細胞培養マトリックス溶液（例えば可溶化細胞外マトリックス）を分注することを含み得る。
System 1 further comprises a liquid dispenser module 300 . Liquid dispenser module 300 can be spaced apart from pipette module 200 . For clarity, spaced apart means that they are separate elements of system 1, possibly at different locations in system 1. FIG. The liquid dispenser module 300 is used to bulk dispense solutions into cell culture vessels, while the pipette module 200 is used to withdraw some or all of the fluid from the cell culture vessels. Exemplary uses of the liquid dispenser module 300 include dispensing cell culture or differentiation medium into cell culture vessels or daughter cell culture vessels either before or after seeding a suspension of cells; Dispensing washing buffers or cell detachment solutions into cell culture vessels during automated passaging protocols of cultures of cells in cell culture vessels, or dispensing cell culture matrix solutions (e.g., solubilized extracellular matrix) can include

Claims

An automated system for adaptively passaging an adherent culture of cells in a cell culture medium in a cell culture vessel, said automated system comprising:
an imaging module for capturing one or more images of the adherent culture of cells at a first time point and at one or more subsequent time points;
a pipette module having the one or more pipettes for drawing fluid from the cell culture vessel through a pipette tip mateable with the end of one or more pipettes;
A liquid dispenser module spaced from the pipette module and in fluid communication with a plurality of solution reservoirs , each of the plurality of solution reservoirs each containing at least a cell culture medium and a cell culture matrix solution, each solution reservoir comprising: is dispensed in bulk into said cell culture vessel via said liquid dispenser module, said liquid dispenser module comprising a plurality of conduits, each conduit in fluid communication with a separate one of said plurality of solution reservoirs a dispenser module having
a handling module having a pair of opposable arms for gripping and transporting the cell culture vessel or lid or daughter cell culture vessel or lid within the automated system;
at least one processor in communication with the imaging module, the pipetting module, the liquid dispenser, and the handling module, the processor comprising:
receiving the one or more images from the imaging module;
converting each of the one or more images from a background to a contrasting representation of the adherent culture of the cells;
calculating one or more properties of the adherent culture of cells based on the transformed one or more images received from the imaging module;
configured to run an automated passaging protocol to passage the adherent culture of cells, wherein the protocol includes the calculated one or more characteristics of the adherent culture of cells;
a) a learned threshold level for the one or more characteristics; or b) an input threshold level for the one or more characteristics. An automated system comprising a processor, including coordinating the operation of

The one or more properties are
a) a measurement of the confluency of an adherent culture of said cells;
b) a measure of differentiation of cells or colonies of adherent cultures of said cells;
c) a measurement of the change from said first time point in a) or b) to said one or more subsequent time points, or
4. The automated system of claim 1, comprising : d) the measurement of a) with respect to b).

wherein the learned threshold level or the input threshold level is
i. 30-90% with respect to the confluency of the cells or colonies in case a),
ii. 3. The method of claim 2, wherein in case b) 0-30% differentiation compared to control cultures in a maintenance protocol or 50%-100% differentiation compared to control cultures in a differentiation protocol. automatic system.

at said first time point or at said one or more subsequent time points, said one or more characteristics calculated for said adherent culture of said cells in said cell culture vessel; is different from the one or more properties calculated for adherent cultures of cells of , wherein the one or more properties become more consistent during subsequent passages 4. Automated system according to any one of Clauses 3.

5. The automated system of any one of claims 1-4, wherein the imaging module further comprises a stage module for supporting the cell culture vessel.

6. The automated system of claim 5, wherein the stage module is movable in a first plane along a first axis, a second axis, or both.

7. The automated system of claim 5 or claim 6, wherein the stage module or subcomponent thereof pivots vertically about a third axis defined by ends of the stage module or subcomponent thereof .

8. The automated system of any one of claims 5-7 , further comprising an enclosure for containing at least the imaging module, the pipette module, the liquid dispenser module, the stage module and the handling module.

9. The automated system of claim 8 , wherein said enclosure is sterile.

i) a refrigeration module for storing one or more of said plurality of solution reservoirs; and ii) a waste reservoir, wherein said plurality of solution reservoirs, said refrigeration module and said waste reservoir are connected to said enclosure. 10. An automated system according to claim 8 or claim 9 , external to the .

11. The automated system of claim 10 , wherein the waste reservoir is in fluid communication with a waste trough within the enclosure.

12. The automated system of claim 11 , wherein said waste trough is coated with a hydrophobic coating.

further comprising one or more sensors, the one or more sensors comprising:
a) detecting the mass of a load on the imaging module, on the stage module, or in the plurality of solution reservoirs;
13. The automated system of any one of claims 10-12 , configured to: b) trigger an alert if the mass of the load is different than expected.

14. Any one of claims 8 to 13 , further comprising a closed conveyor module connecting said enclosure and an incubator module for transporting said cell culture vessels from said incubator module near said handling module. automatic system.

15. The automated system of any one of claims 1-14 , further comprising a graphical user interface.

16. The automated system of claim 15 , wherein said graphical user interface displays a fluid level report for each of said plurality of solution reservoirs.

An automated method for adaptively passaging adherent cultures of cells comprising :
providing an adherent culture of said cells in a cell culture medium within a cell culture vessel;
capturing, by an imaging module, one or more images of the adherent culture of cells at a first time point and one or more subsequent time points;
converting the one or more images from a background to a contrasting representation of the adherent culture of the cells;
calculating, by at least one processor communicatively coupled to the imaging module, one or more properties of the adherent culture of cells based on the transformed one or more images ; The one or more characteristics include a measure of confluency of the adherent culture of the cells or a measure of cell or colony differentiation of the adherent culture of the cells.
the calculated one or more properties of the adherent culture of the cells;
a) learned threshold levels for the one or more characteristics; or b) predetermined threshold levels for the one or more characteristics. An automated method, comprising: performing a replacement protocol;

The one or more properties are
a) from said first time point to said one or more subsequent time points of a measurement of confluency of said adherent culture of cells or of a measurement of cell or colony differentiation of said adherent culture of cells; a measure of change, or
b) a measure of the confluency measure of an adherent culture of said cells relative to a measure of cell or colony differentiation of said adherent culture of cells;
18. The automated method of claim 17 , comprising :

wherein the learned threshold level or the input threshold level is
i. 30-90% in terms of cell or colony confluency,
ii. 18. The automated method of claim 17, wherein 0-30% differentiation compared to control cultures for maintenance protocols, or 50%-100% differentiation compared to control cultures for differentiation protocols.

at said first time point or at said one or more subsequent time points, said one or more characteristics calculated for said adherent culture of said cells in said cell culture vessel; is different from the one or more properties calculated for adherent cultures of cells of , wherein the one or more properties become more consistent during subsequent passages 20. An automated method according to any one of clauses 19 .

21. The automated method of any one of claims 17-20 , wherein providing the adherent culture of cells comprises transporting the adherent culture of cells from an incubator.

further comprising placing the cell culture vessel on a stage module, the stage module being movable in a first plane along a first axis, a second axis, or both; 22. The automated method of any one of claims 17-21 , wherein a stage module pivots vertically about a third axis defined by an end of said stage module or subcomponent thereof.

23. The method of claims 17 to 22, further comprising obtaining a cell suspension from said adherent culture of cells and seeding a daughter cell culture vessel with some or all of said cell suspension. An automated method according to any one of paragraphs.

Prior to seeding the daughter cell culture vessel, the cell suspension is passed through a first pipette tip to convert the adherent culture of cells to a single cell suspension or the caliber of the first pipette tip. 24. The automated method of claim 23 , dissociating into a plurality of clamps having an average diameter not exceeding.

4. The method of claim 1, wherein obtaining the cell suspension comprises aspirating cell culture medium from the cell culture vessel and contacting the adherent culture of cells in the cell culture vessel with a detachment solution. 25. An automated method according to claim 23 or claim 24.

26. The automated method of claim 25 , wherein the stripping solution selectively strips either the first population of differentiated cells or the second population of undifferentiated cells from the wall of the cell culture vessel.

27. The automated method of any one of claims 17-26 , wherein said adherent culture of cells are pluripotent stem cells.

28. The automated method of claim 27 , wherein said pluripotent stem cells are human pluripotent stem cells.

29. The automated method of claim 27 or claim 28 , wherein said pluripotent stem cells are passaged as clumps.