COMBINED SCALABLE IN VITRO DIFFERENTIATION SYSTEM FOR HUMAN BLASTOCYST-DERIVED STEM (HBS) CELLS OR CELLS DERIVED FROM HBS CELLS FOR DIRECT ASSAY APPLICATION IN MULTIWELL PLATES

• US 20100143913A1
• Filed: 03/17/2008
• Published: 06/10/2010
• Est. Priority Date: 03/16/2007
• Status: Abandoned Application

• Current Assignee: Cellartis AB
• Original Assignee: Cellartis AB
• Inventors: Adler, Sara, Strehl, Raimund
• Application Number:
  US12/531,527
• Publication Number:
  US 20100143913A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 2503/04 Screening or testing on artificial tissues
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 5/0606 Pluripotent embryonic cells, e.g. embryonic stem cells [ES] embryonic germ cells C12N5/0611, induced pluripotent stem cells C12N5/0696
• CPC Class Codes: C12N 5/0657 Cardiomyocytes; Heart cells
• CPC Class Codes: G01N 33/5014 for testing toxicity
• CPC Class Codes: G01N 33/5073 Stem cells

Abstract

A combined scalable in vitro differentiation and assay system based on human blastocyst-derived stem (hBS) cells or cells derived from hBS cells is provided. This system makes it possible to merge both the differentiation and the assay parts of the system into one. The advantage of the combined assay system is that the hBS cells or the cells derived from hBS cells in the differentiation system are directly applicable for assays, in a large variety of assay units, such as different multiwell plates. The system offers a major improvement compared to the prior art, since the cells are differentiated in the same format as when further being subject to analysis. The starting cell material can be homogenously distributed across a variety of different plates, for use and can be cultured when attached, when semi-attached or when in suspension.

Claims

1. A combined differentiation and assay method for hBS cells or cells derived from hBS cells wherein said cells are differentiated in the same format as they are further being subject to analysis in, the method comprising the steps:

(i) dissociation of the cells into single cells, clusters or a mixture thereof;

(ii) seeding of the dissociated cells into one or more wells of one or more multi-well format plates;

(iii) application of a centrifugal force to the seeded cells for enhanced coalescence;

(iv) incubation to differentiate the cells and without re-plating in steps (ii)-(iv) and assaying the cells without replating.

2. A combined differentiation and assay method according to claim 1, wherein the hBS-derived cells are any cells between undifferentiated hBS cells and fully differentiated cells.

3. A combined differentiation and assay method according to claim 2, wherein the hBS-derived cells are a mixture of cell types comprising all three germ layers.

4. A combined differentiation and assay method according to claim 2, wherein the cells derived from hBS cells are progenitor cells or fully differentiated cells of the endodermal lineage.

5. A combined differentiation and assay method according to claim 3, wherein the hBS-derived cells are hepatoblast-like cells.

6. A combined differentiation and assay method according to claim 2, wherein the hBS-derived cells are progenitor cells or fully differentiated cells of the ectodermal lineage.

7. A combined differentiation and assay method according to claim 6, wherein the hBS-derived cells are neural progenitor cells or glial cells.

8. A combined differentiation and assay method according to claim 2, wherein the hBS-derived cells are progenitor cells or fully differentiated cells of the mesodermal lineage.

9. A combined differentiation and assay method according to claim 8, wherein the hBS-derived cells are mesenchymal-like cells or multipotent cardiac progenitor cells.

10. A combined differentiation and assay method according to claim 1, wherein the hBS-derived cells are a mixed population of more than one cell type.

11. A combined differentiation and assay method according to claim 1, wherein the hBS-derived cells are an essentially pure population.

12. A combined differentiation and assay method according to claim 1, wherein the hBS cells after differentiation are any cells between undifferentiated hBS cells and fully differentiated cells.

13. A combined differentiation and assay method according to claim 12, wherein the hBS cells after differentiation are a mixture of cell types comprising all three germ layers simultaneously.

14. A combined differentiation and assay method according to claim 12, wherein the hBS cells after differentiation are progenitor cells or fully differentiated cells of the endodermal lineage.

15. A combined differentiation and assay method according to claim 14, wherein the hBS cells after differentiation are hepatoblast-like cells.

16. A combined differentiation and assay method according to claim 12, wherein the hBS cells after differentiation are progenitor cells or fully differentiated cells of the ectodermal lineage.

17. A combined differentiation and assay method according to claim 16, wherein the hBS cells after differentiation are neural progenitor cells or glial cells.

18. A combined differentiation and assay method according to claim 12, wherein the hBS cells after differentiation are progenitor cells or fully differentiated cells of the mesodermal lineage.

19. A combined differentiation and assay method according to claim 18, wherein the hBS cells after differentiation are mesenchymal-like cells or multipotent cardiac progenitor cells.

20. A combined differentiation and assay method according to claim 1, wherein the hBS cells after differentiation are a mixed population of more than one cell type.

21. A combined differentiation and assay method according to claim 1, wherein the hBS cells after differentiation are an essentially pure population.

22. A combined differentiation and assay method according to claim 1, wherein the differentiation and assay use take place without any transfer of the cells between different plates.

23. A combined differentiation and assay method according to claim 1, wherein the differentiation and assay analysis take place in the same multi-well plate.

24. A combined differentiation and assay method according to claim 1, wherein the cell dissociation agent in item (i) allows dissociation into single cells or to a cluster size of between 1 and 20 cells.

25. A combined differentiation and assay method according to claim 1, wherein the cell dissociation agent in step (i) allows dissociation to cluster sizes of between 20 and 500 cells.

26. A combined differentiation and assay method according to claim 1, wherein the cell dissociation agent in step (i) is a mechanical tool, allowing dissociation of hBS cells and cells derived from hBS cells into clusters.

27. A combined differentiation and assay method according to claim 1, wherein the multi-well format plates in step (ii) are plates with between 12 and 1536 wells.

28. A combined differentiation and assay method according to claim 1, wherein the multi-well format plates in step (ii) are conical-bottom or round-bottom bottom plates to allow enhanced coalescence to microtissue.

29. A combined differentiation and assay method according to claim 1, wherein the multi-well format plates in step (ii) are flat-bottom plates to allow enhanced coalescence to cell monolayers.

30. A combined differentiation and assay method according to claim 1, further comprising one or more cell strainers for the even distribution of the hBS cells or hBS-derived cells between individual wells of the multi-well format plates.

31. A combined differentiation and assay method according to claim 1, wherein the centrifugal force in step (iii) is applied by use of a centrifuge and rotor compatible with the multi-well format plate in step (ii).

32. A combined differentiation and assay method according to claim 1, where one or more dissociation agents are utilized in step (i).

33. A combined differentiation and assay method according to claim 1, where one or more culture media for incubation of the cells contains a serum component, selected from a group including FBS, serum replacement, human serum, albumin, insulin, and transferin, at a concentration of 0.5-50%.

34. A combined differentiation and assay method according to claim 1, where one or more multi-well format plates are selected from a group of shapes as illustrated in FIG. 1.

35. A combined differentiation and assay method according to claim 1, where a centrifugal force for enhanced coalescence of the cells of 5-10 000 G is utilized in step (iii)

36. A combined differentiation and assay method according to claim 1, wherein the cells are cultured and/or incubated for 1 hour-90 days.

37. A combined differentiation and assay method according to claim 1, where the cell number at seeding ranges from 500-500 000 cells.

38. A combined differentiation and assay method according to claim 1, comprising:

i) dissociating the cells with one or more dissociation agents;

ii) seeding of the cells into one or more individual wells in one or more multi-well format plates at a density of 1000-1000 000 cells per well;

iii) applying a centrifugal force of 100-1000 G for enhanced coalescence of the cells;

iv) incubation of the cells in one or more culture media containing a serum component, at a concentration of such as 5-30%, to differentiate cells into one or more of the germ layers for 1-30 days;

and assaying the cells without replating.

39. The combined differentiation and assay method according to claim 1, where a survival promoting substance is added during step (i) and/or step (ii).

40. The combined differentiation and assay method according to claim 1, wherein the cells are strained in step (ii).

41. The combined differentiation and assay method according to claim 1 for producing cells repeating the earliest embryo development in vitro

42. The combined differentiation and assay method according to claim 1 for producing cells to test embryo toxicity

43. The combined differentiation and assay method according to claim 1, for producing cells to test teratogenicity.

44. The combined differentiation and assay method according to claim 1, for use in drug discovery and/or for safety assessment and toxicity studies.

45. The combined differentiation and assay method according to claim 1, for preparing microtissues and monolayers from the very same cell suspension of hBS cells or cells derived from hBS cells for the performing of a comparative analysis.

46. The combined differentiation and assay method according to claim 1, for performing developmental toxicity assays and differentiation assays.

47. The combined differentiation and assay method according to claim 1, for preparing cells for detecting or measuring cell differentiation.

48. The combined differentiation and assay method according to claim 47, wherein the differentiation is measured by measuring gene or protein levels.

49. A method according to claim 1, for running an assay based on immuno-cytochemistry.

50. A method according to claim 1, for running an assay based on a genetically engineered hBS cell line carrying reporters for selected marker genes.

51. A method according to claim 1, for running an assay based on quantitative PCR.

52. A method according to claim 1, for running an assay measuring the levels of a substance or compounds in the cell culture medium.

53. A method according to claim 1, for running an assay to detect human toxicity.

54. A method according to claim 1, for running an assay to detect or measure cytotoxicity.

55. A kit for carrying out the method according to claim 1, comprising:

one or more dissociation agents, selected from Trypsin/EDTA, Collagenase IV and Dispase.

56. The kit according to claim 55, further comprising a rotor for plate centrifugation.

57. The kit according to claim 55, further comprising additional tools for in vitro assay performance.

58. The kit according to claim 57, wherein the additional tools are reagents for PCR.

59. The kit according to claim 57, wherein the additional tools are immunochemistry reagents for the detection or quantification of pluripotency.

60. The kit according to claim 57, wherein the additional tools are immunochemistry reagents for the detection or quantification of germ layer specific differentiation.

61. The kit according to claim 1, further comprising positive control substances, negative control substances, or additional cell types for use as control populations.