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

Abstract

A method of establishing a cell line from the inner cell mass of a blastocyst, comprising: isolating a blastocyst having a zona pellucida, a trophectoderm, and an inner cell mass; creating an aperture in the blastocyst by laser ablation; isolating cells from the inner cell mass from the blastocyst through the aperture; and culturing the cells to establish a cell line, wherein the cells are cultured under feeder free conditions.

1 - 20 . (canceled)  
     
     
         21 . A method of establishing a cell line from the inner cell mass of a human blastocyst, comprising: 
 (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, wherein the cells are cultured under feeder free conditions.    
     
     
         22 . The method of  claim 21 , wherein the aperture is through the zona pellucida.  
     
     
         23 . The method of  claim 21 , wherein the aperture is through the zona pellucida and the trophectoderm.  
     
     
         24 . The method of  claim 21 , wherein the laser ablation is achieved using a non contact diode laser.  
     
     
         25 . The method of  claim 24 , wherein the non contact diode laser is a continuous 1.48 μm diode laser.  
     
     
         26 . The method of  claim 21 , wherein cells from the inner cell mass are isolated by aspiration.  
     
     
         27 . The method of  claim 21 , wherein isolating cells from the inner cell mass is carried out in the absence of animal generated antibodies and sera.  
     
     
         28 . The method of  claim 21 , 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.  
     
     
         29 . The method of  claim 21 , wherein the feeder free conditions comprise an extracellular matrix.  
     
     
         30 . The method of  claim 29 , wherein the feeder free conditions further comprise conditioned medium.  
     
     
         31 . The method of  claim 21 , wherein the isolated blastocyst is the product of in vitro fertilization.  
     
     
         32 . A method of establishing a human embryonic stem cell line, comprising: 
 (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; and    (d) culturing the cells under feeder free conditions to obtain an isolated human embryonic stem cell line.    
     
     
         33 . The method of  claim 32 , wherein the aperture is through the zona pellucida and the trophectoderm.  
     
     
         34 . The method of  claim 32 , wherein the laser ablation is achieved using a non contact diode laser.  
     
     
         35 . The method of  claim 32 , wherein the cells of the inner cell mass are cultured to produce inner cell mass-derived cell masses.  
     
     
         36 . The method of  claim 35 , wherein the inner cell mass-derived cell masses are dissociated and re-plated on an extracellular matrix.  
     
     
         37 . The method of  claim 32 , wherein cells of the inner cell mass are isolated by aspiration.  
     
     
         38 . The method of  claim 32 , wherein the human embryonic stem cell line is established in the absence of animal generated antibodies and sera.  
     
     
         39 . The method of  claim 32 , wherein the feeder free condition comprises plating the cells of the inner cell mass on an extracellular matrix.  
     
     
         40 . A method of establishing a human embryonic stem cell line, comprising: 
 (a) isolating cells of an inner cell mass from a blastocyst by creating an aperture in the blastocyst by laser ablation and removing cells of the inner cell mass from the blastocyst through the aperture;    (b) culturing the cells of the inner cell mass to produce inner cell mass derived masses; and    (c) culturing the inner cell mass derived masses under feeder free conditions to produce an isolated human embryonic stem cell line.    
     
     
         41 . The method of  claim 40 , wherein the feeder free conditions comprise an extracellular matrix.  
     
     
         42 . The method of  claim 40 , 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.