Isolation of Inner Cell Mass for the Establishment of Human Embryonic Stem Cell (hESC) Lines

• US 20070048864A2
• Filed: 08/23/2002
• Published: 03/01/2007
• Est. Priority Date: 08/23/2001
• Status: Active Grant

• Current Assignee: Reliance Life Sciences Pvt. Ltd.
• Original Assignee: Reliance Life Sciences Pvt. Ltd.
• Inventors: TOTEY, SATISH, SAXENA, SHAILAJA, PARIKH, FIRUZA
• Granted Patent: US 7,294,508 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: A61B 17/435 for embryo or ova transplantation
• CPC Class Codes: A61B 18/20 using laser
• 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

Abstract

A method for isolating an inner cell mass comprising the steps of immobilizing a blastocyst stage embryo having a zona pellucida, trophectoderm, and inner cell mass, creating an aperture in the blastocyst stage embryo by laser ablation, and removing the inner cell mass from the blastocyst stage embryo through the aperture. The aperture is through the zona pellucida and the trophectoderm. The laser ablation is acheived using a non-contact diode laser. The inner cell mass removed from the blastocyst stage embryo is used to establish human Embryonic Stem Cell lines.

Claims

1-10. -10. (canceled)

11. A method for establishing a cell line from the inner cell mass of a human blastocyst, comprising the steps of:

(a) isolating a blastocyst having a zona pellucida, a trophectoderm, and an inner cell mass;

(b) creating an aperture in the blastocyst by laser ablation;

(c) isolating cells from the inner cell mass from the blastocyst through the aperture; and

(d) culturing the cells to establish a cell line.

12. The method of claim 11, wherein the aperture is through the zona pellucida.

13. The method of claim 11, wherein the aperture is through the zona pellucida and the trophectoderm.

14. The method of claim 11, wherein the laser ablation is achieved using a non-contact diode laser.

15. The method of claim 14, wherein the non-contact diode laser is a continuous 1.48 μ

m diode laser.

16. The method of claim 11, wherein cells from the inner cell mass are isolated by aspiration using an aspiration pipette introduced through the aperture.

17. The method of claim 11, wherein isolating cells from the inner cell mass is carried out in the absence of animal generated antibodies and sera.

21. The method of claim 11, wherein the isolated blastocyst of step (a) is placed in conventional Embryo biopsy medium.

22. The method of claim 21, wherein the Embryo biopsy medium is Ca++/Mg++ free.

23. The method of claim 11, further comprising a micromanipulator system comprising a microscope with a heating stage, a holding pipette, an aspiration pipette, and an air syringe, wherein the isolated blastocyst of step (a) is placed on the heating stage, the micromanipulator system is adjusted so that the blastocyst is at the center of the microscope field, and the blastocyst is secured with the holding pipette by suction through the air syringe, so that the inner cell mass is opposite the holding pipette.

24. The method of claim 12, wherein the aperture is generated by laser ablation using a 1.48 μ

diode laser.

25. The method of claim 13, wherein the aperture is generated by laser ablation using a 1.48 μ

m diode laser, and is adjacent to the inner cell mass.

26. The method of claim 16, wherein the cells of the inner cell mass are washed one or more times in embryonic stem cell media, and placed on a feeder layer in the presence of embryonic stem cell media.

27. The method of claim 26, where in the feeder layer is of murine or human origin.

28. The method of claim 26, where in the feeder layer is an embryonic fibroblast feeder layer.

29. The method of claim 11, wherein the isolated cells of the inner cell mass are cultured under feeder free conditions.

30. The method of claim 29, wherein the feeder free conditions comprise an extracellular matrix.

31. The method of claim 30, wherein the feeder free conditions further comprise conditioned medium.

32. The method of claim 11, wherein the isolated blastocyst is the product of in vitro fertilization.

33. The method of claim 11, wherein the isolated blastocyst is the product of intracytoplasmic sperm injection.

18. A method for establishing a human embryonic stem cell line comprising the steps of:

(a) isolating cells of an inner cell mass from a blastocyst stage embryo by creating an aperture in the blastocyst stage embryo by laser ablation, and removing cells of the inner cell mass from the blastocyst stage embryo through the aperture;

(b) culturing the cells of the inner cell mass in the presence of an embryonic stem cell medium and an inactivated feeder layer to produce inner cell mass derived masses; and

(c) culturing the inner cell mass derived masses to produce an isolated human embryonic stem cell line.

19. The method of claim 18, wherein the embryonic stem cell medium comprises:

(a) Dulbecco'"'"'s modified Eagle'"'"'s medium with high glucose content in an amount from about 70% to about 90%, and without sodium pyruvate;

(b) fetal bovine serum in an amount from 10% to 30% of the volume of the embryonic stem cell medium;

(c) beta-mercaptoethanol;

(d) non-essential amino acids;

(e) L-glutamine; and

(f) basic fibroblast growth factor.

20. The method of claim 18, further comprising mechanically dissociating the inner cell mass derived masses of step (b) and re-plating the mechanically dissociated cells of the inner cell mass derived masses on a feeder layer.

34. The method of claim 18, where in the feeder layer is of murine or human origin.

35. The method of claim 18, wherein the feeder layer is an embryonic fibroblast feeder layer.

36. The method of claim 20, where in the feeder layer is of murine or human origin.

37. The method of claim 20, wherein the feeder layer is an embryonic fibroblast feeder layer.

38. A method of establishing a human embryonic stem cell line, comprising the steps of:

(a) isolating a human blastocyst comprising an inner cell mass;

(b) creating an aperture in the blastocyst by laser ablation;

(c) isolating cells from the inner cell mass of the blastocyst through the aperture;

(d) culturing the cells to obtain an isolated human embryonic stem cell line.

39. The method of claim 38, wherein the aperture is through the zona pellucida and the trophectoderm.

40. The method of claim 38, wherein the laser ablation is achieved using a non-contact diode laser.

41. The method of claim 38, wherein cells of the inner cell mass are isolated by aspiration through the aperture.

42. The method of claim 38, wherein the human embryonic stem cell line is established in the absence of animal generated antibodies and sera.

43. The method of claim 38, wherein step (c) further comprises plating the cells of the inner cell mass on a feeder layer, wherein inner cell mass-derived cell masses are formed.

44. The method of claim 43, wherein the feeder layer is an embryonic fibroblast feeder layer.

45. The method of claim 43, where in the feeder layer is of murine or human origin.

46. The method of claim 43, wherein the inner cell mass-derived cell masses are dissociated and replated on a feeder layer.

47. The method of claim 38, wherein step (c) further comprises plating the cells of the inner cell mass in a feeder free condition, wherein inner cell mass-derived cell masses are formed.

48. The method of claim 47, wherein the feeder free condition comprises plating the cells of the inner cell mass on an extracellular matrix.

49. The method of claim 48, wherein the cells are cultured in the presence of conditioned medium.

50. The method of claim 47, wherein the inner cell mass-derived cell masses are dissociated and replated on an extracellular matrix.

51. The method of claim 50, wherein the cells are cultured in the presence of conditioned medium.

52. A method for establishing a human embryonic stem cell line comprising the steps of:

(a) isolating cells of an inner cell mass from a blastocyst stage embryo by creating an aperture in the blastocyst stage embryo by laser ablation, and removing cells of the inner cell mass from the blastocyst stage embryo through the aperture;

(b) culturing the cells of the inner cell mass under feeder free conditions to produce inner cell mass derived masses; and

(c) culturing the inner cell mass derived masses to produce an isolated human embryonic stem cell line.

53. The method of claim 52, wherein the feeder free conditions comprise an extracellular matrix.

54. The method of claim 53, wherein the feeder free conditions further comprise conditioned medium.

55. The method of claim 52, further comprising mechanically dissociating the inner cell mass derived masses of step (b) and re-plating the mechanically dissociated cells of the inner cell mass derived masses under feeder free conditions.

56. The method of claim 55, wherein the feeder free conditions comprise an extracellular matrix.

57. The method of claim 56, wherein the feeder free conditions further comprise conditioned medium.