Isolation of inner cell mass for the establishment of human embryonic stem cell (hESC) lines

• US 20030104616A1
• Filed: 08/23/2002
• Published: 06/05/2003
• Est. Priority Date: 08/23/2001
• Status: Active Grant

• Current Assignee: Reliance Life Sciences Pvt. Ltd.
• Original Assignee: Reliance Life Sciences Pvt. Ltd.
• Inventors: Parikh, Firuza Rajesh, Saxena, Shailaja Anupam, Totey, Satish Mahadoerao
• 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. A method of isolating inner cell mass (ICM) for establishing human Embryonic Stem Cell (hESC) lines using a non-contact diode laser technique comprising the steps of:

(i) placing the blastocyst in a 35 mm petridish in a 50 mico liter conventional Embryo biopsy medium covered with mineral oil;

(ii) setting up the micromanipulator system consisting of the microscope with the heating stage, tool holders to hold the holding pipette, aspiration pipette and air syringes;

(iii) placing the petridish of (i) above, containing the blastocyst on the heating stage of (ii) above and adjusting the blastocyst at the center of the field;

(iv) securing the blastocyst with holding pipette at 9 O'"'"'clock position by suction through the air syringe in such a way that the inner cell mass (ICM) which is to be isolated is at 3 O'"'"'clock position;

2. A method of claim 1, further comprising of dissolving zona pellucida, by laser ablation of zona pellucida using 3-5 pulse of 1.48 micro diode laser to create a hole in glycoprotein matrix present in zona pellucida.

3. A method of claim 1, further comprising of removing the trophectoderm cells adjacent to inner cell mass (ICM) by ablation of trophectoderm cells using 1.48 micro diode laser by cell lysis.

4. A method of claim 1, further comprising of aspiration of inner cell mass (ICM), comprising of the following steps:

(i) bringing the aspirating pipette close to blastocyst through the hole in the zona pellucida and trophectoderm cells close to inner cell mass (ICM);

(ii) aspirating the inner cell mass (ICM) into the aspiration pipette by gentle suction through air syringe of claim 4(i) above;

(iii) the aspirated inner cell mass (ICM) of claim 4(ii) above is slowly released into a culture droplet.

5. A method of claim 1, further comprising of culturing of Embryonic Stem Cells comprising of the following steps:

(i) washing the isolated inner cell mass (ICM) several times in micro drops of Embryonic Stem Cell media;

(ii) placing the inner cell mass (ICM) on the plate, containing feeder layer in the presence of a media, for the establishment of Embryonic Stem Cells;

6. A method of claim 1, wherein Embryo biopsy medium is Ca++/Mg++ free

7. A method of claim 5, wherein the media used is Embryonic Stem Cell media.

8. A method of claim 5, wherein the plate used is 0.5% gelatin coated plate.

9. A method of claim 5, wherein the Embryonic Stem Cell media is a combination of:

(i) Dulbecco'"'"'s Modified Eagle'"'"'s Medium (DMEM) without Sodium pyruvate with high glucose content (70-90%);

(ii) Fetal Bovine Serum (10-30%)

(iii) Beta-Mercapto Ethanol (0.1 mM)

(iv) Non-Essential Amino Acids (1%)

(v) L-Glutamine 2 mM

(vi) Basic Fibroblast Growth Factor (4 ng/ml)

10. A method of claim 5, wherein feeder layer is murine or human origin

11. 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.

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 acheived 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 the inner cell mass is removed by aspiration using an aspiration pipette introduced through the aperture.

17. The method of claim 11, wherein the method is carried out in the absence of animal generated antibodies and sera.

18. A method for establishing human Embryonic Stem Cell lines comprising the steps of:

isolating an inner cell mass from a blastocyst stage embryo by 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;

culturing the inner cell mass in the presence of an embryonic stem cell medium and a mouse inactivated embryonic fibroblast feeder layer to produce inner cell mass derived masses.

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

Dulbecco'"'"'s modified Eagle'"'"'s medium in an amount from about 70% to about 90% of the embryonic stem cell medium, the Dulbecco'"'"'s modified Eagle'"'"'s medium being without sodium pyrubate with high glucose content;

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

beta-mercaptoethanol in an amount of about 0.1 micro-mole based on the total moles of the embryonic stem cell medium;

non-essential amino acids in an amount of about 1% of the volume of the embryonic stem cell medium;

L-glutamine in an amount of about 2 micro-moles based on the total moles of the embryonic stem cell medium; and

basic fibroblast growth factor in an amount of about 4 nanograms per milliliter of the embryonic stem cell medium.

20. The method of claim 18 further comprising the steps of removing the inner cell mass derived masses, mechanically dissociating the inner cell mass derived masses, and re-plating the mechanically dissociated inner cell mass derived masses on fresh feeder cells.