A novel method for purification of inner cell mass and trophectoderm cells from blastocysts using magnetic activated cell sorting.
OBJECTIVE
To develop a simple method to purify blastomeres of inner cell mass (ICM) and trophectoderm (TE) lineage using magnetic activated cell sorting.


METHODS
Prospective laboratory study.


METHODS
Embryology research laboratory.


METHODS
None.


METHODS
Trophectoderm cells of zona-free blastocysts were labeled with concanavalin A conjugated to FITC, and every nucleus in the blastocyst was labeled with Hoechst 33342. The labeled blastocyst was disaggregated to single cells by trypsin treatment followed by pipetting using a finely drawn, flame-polished micropipet. Disaggregated blastomeres were incubated with anti-FITC antibody conjugated to magnetic microbeads and subjected to magnetic cell sorting to separate cells into FITC-positive and -negative fractions.


METHODS
Purity and gene expression.


RESULTS
In the FITC-positive fraction, an average of 91.2% of cells was dual-labeled with FITC and Hoechst, whereas only 7.8% of FITC negative fractions were labeled with FITC. Expression of CDX2, a trophectoderm marker, was significantly higher in the FITC-positive fraction, whereas expression of NANOG, an inner cell mass marker, was significantly higher in the FITC-negative fraction.


CONCLUSIONS
Highly purified trophectoderm cells or inner cell mass cells can be collected using magnetic activated cell sorting. This method can be useful for understanding differentiation and function of cell lineages in the blastocyst.
Department of Animal Sciences and DH Barron Reproductive and Perinatal Biology Research Program, University of Florida, Gainesville, Florida 32601, USA.
Manabu
M
Ozawa
Peter J
PJ
Hansen
D000818:Animals; D015415:Biomarkers / Q000032:analysis / Q000378:metabolism; D053624:Blastocyst Inner Cell Mass / Q000166:cytology* / Q000378:metabolism; D001757:Blastomeres / Q000166:cytology / Q000378:metabolism / Q000502:physiology; D002417:Cattle; D019070:Cell Lineage / Q000235:genetics; D002478:Cells, Cultured; D004475:Ectoderm / Q000166:cytology* / Q000378:metabolism; D004526:Efficiency; D046149:Embryo Culture Techniques; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism; D018189:Immunomagnetic Separation / Q000379:methods*; D014327:Trophoblasts / Q000166:cytology* / Q000378:metabolism / Q000502:physiology
D015415:Biomarkers