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1. (WO2019002456) METHODS FOR IDENTIFYING AGENTS WHICH INDUCE (RE) DIFFERENTIATION IN UN- OR DEDIFFERENTIATED SOLID TUMOR CELLS
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Claims

1. Method for the identification of compounds inducing (re-)differentiation in non- or dedifferentiated cells, comprising :

a) provision of a cell culture sample consisting of de-/ or undifferentiated tumour cells,

b) bringing the compound of interest into contact with the cell culture sample, c) following the determination of the relative concentration of a first marker lactate in contrast to untreated cells, and

d) following the determination of the relative concentration of a second marker neutral lipids in contrast to untreated cells,

wherein steps c) and d) may be performed in reverse order if necessary

2. Method according to claim 1, characterised by lipid-free or lipid-reduced cultivation of the cell culture sample

3. Method according to claim 1 or 2, characterised by cultivating the cell culture sample under aerobic conditions

4. Method according to one or more of claims 1 to 3, characterised by the invented process which further includes the following steps:

e) Determination of the amount of viable adherent cells via quantification of absorbance after centrifugation, and/or

f) Determination of complete induction of apoptosis in the cells among a

complete signal reduction to background level

5. Method according to claim 4, for utilisation as viability test and/or for utilisation as test for apoptosis.

6. Method according to one or more of claims 1 to 5, characterised by cultivating the cell culture sample in the presence of insulin.

7. Method according to one or more of claims 1 to 6, characterised by cultivating (tumour) cell culture sample either negative for/ or low expressing insulin receptor isotype B or possessing a higher expression ratio of insulin receptor isotype A/B than differentiated cells in the presence vs. absence of insulin.

8. Method according to one or more of claims 1 to 7, characterised by a change in one or both previously described marker(s) in the presence vs. absence of insulin towards the direction of an anabolic change in the cell 's metabolism enabling the analysis of functional insulin receptor subtype B expression and ratio of subtype A to B towards a relative higher subtype B expression, respectively.

9. Method according to one or more of claims 1 to 8, characterised by cultivating a breast cell culture sample negative for or poorly expressing prolactin receptor in the presence vs. absence of prolactin.

10. Method according to one or more of claims 1 to 9, characterised by a change in one or both previously described marker(s) in the presence vs. absence of prolactin towards the direction of an anabolic change in the cell 's metabolism enabling the analysis of functional prolactin receptor expression.

11. Method according to one or more of claims 1 to 10, characterised by the provision of the cell culture sample in a microtiter plate.

12. Method according to one or more of claims 1 to 11, characterised by a gas permeable foil sealing the microtiter plate.

13. Method according to one or more of claims 1 to 12, characterised by quantification of lactate concentration in bicarbonate buffered cell culture medium incubated in an appropriate CCte-atmosphere by measuring the pH-dependent change of optical phenol red absorption.

14. Method according to one or more of claims 1 to 13, characterised by single/multiple media and/or washing buffer removal from the cell culture sample via centrifugation prior to addition of new media for further culturing or measurement of marker in step Id).

15. Method according to one or more of claims 1 to 14, characterised by performing single/multiple changes in media and/or washing buffer via partial device (A) and (B).

16. Method according to one or more of the claims 1 to 15, characterised by the combined utilisation of the markers lactate and neutrals lipids for the identification of compounds inducing (re-)differentiation in un- or dedifferentiated cells, especially tumour cells.

17. Method according to one or more of claims 1 to 16, characterised by quantification of lactate concentration in bicarbonate buffered cell culture medium incubated in an appropriate constant CCte-atmosphere by measuring the pH-dependent change of optical phenol red absorption and by quantification of neutral lipids using an appropriate neutral lipid staining (fluorescent) dye (e.g. BODIPY® 493/503).

18. Use of the markers lactate and neutrals lipids for the identification of compounds inducing (re-)differentiation in un- or dedifferentiated cells, especially tumour cells, wherein the markers are used combined.

19. Vessel for the removal of liquids from cells via centrifugation (partial device (A)) characterized by comprising :

- 4 side walls (ai, a2, bi, b2), where the two opposing side walls have the same length (la, lb), so that a rectangular shape is obtained,

- a flat bottom (c) being connected in such a way with each of the side walls over all of the connecting area in a liquid proof way,

- each of the side walls having a protrusion (da, db) directed towards the inside of the vessel,

- 2 of the 4 side walls being opposite to each other having recesses (ei, e2) positioned in the middle of the lengths la of the side wall on the upper surface of the respective side wall.

20. Microtiter plate (partial device (B)) for culturing cells enabling addition of liquid via centrifugation, said plate comprising a surface (1) and wells (2), said wells being tapered towards the bottom (3) where an opening, especially a circular opening, is present.

21. Microtiter plate according to claim 20 wherein the diameter of the

opening is proportional to the surface tension of a liquid inside each of the wells and adjusted in such way that liquid can escape only under influence of forces stronger than the gravitational force.