BACKGROUND: For clinical grade human embryonic stem cell (hESC) lines, a robust derivation system without any substances having animal origin would be required. We have gradually improved our hESC derivations. Human skin fibroblasts were used as feeder cells in derivation of all our 25 permanent fully characterized hESC lines. In the first four derivations, fetal calf serum was used as a supplement in the medium, thereafter, serum replacement medium was used. Immunosurgery generally used for isolation of the inner cell mass (ICM) still involves animal serum and complement. METHODS: We developed a practical mechanical isolation method for the ICM. Two flexible metal needles with sharpened tips, 0.125 mm in diameter, were used to open the zona pellucida and extract the ICM under a stereomicroscope. Immunohistochemical and karyotype characterization of the new hESC lines was carried out, and pluripotency was tested in vitro (immunocytochemistry and RT-PCR) and in vivo (teratoma growth). RESULTS: Five hESC lines were obtained from 19 supernumerary blastocysts collected in 2005-2006 (26%), whereas in similar conditions, we obtained 16 lines from 100 blastocysts (16%) using immunosurgery in 2003-2005. The new lines had a normal karyotype and tissues originating from the three embryonic germ cell layers were present. CONCLUSIONS: Mechanical isolation of the ICM proved to be an effective way to derive new hESC lines. The technique is fast, does not require any extra investment and the xeno-components of immunosurgery could be avoided.
BACKGROUND: For clinical grade human embryonic stem cell (hESC) lines, a robust derivation system without any substances having animal origin would be required. We have gradually improved our hESC derivations. Human skin fibroblasts were used as feeder cells in derivation of all our 25 permanent fully characterized hESC lines. In the first four derivations, fetal calf serum was used as a supplement in the medium, thereafter, serum replacement medium was used. Immunosurgery generally used for isolation of the inner cell mass (ICM) still involves animal serum and complement. METHODS: We developed a practical mechanical isolation method for the ICM. Two flexible metal needles with sharpened tips, 0.125 mm in diameter, were used to open the zona pellucida and extract the ICM under a stereomicroscope. Immunohistochemical and karyotype characterization of the new hESC lines was carried out, and pluripotency was tested in vitro (immunocytochemistry and RT-PCR) and in vivo (teratoma growth). RESULTS: Five hESC lines were obtained from 19 supernumerary blastocysts collected in 2005-2006 (26%), whereas in similar conditions, we obtained 16 lines from 100 blastocysts (16%) using immunosurgery in 2003-2005. The new lines had a normal karyotype and tissues originating from the three embryonic germ cell layers were present. CONCLUSIONS: Mechanical isolation of the ICM proved to be an effective way to derive new hESC lines. The technique is fast, does not require any extra investment and the xeno-components of immunosurgery could be avoided.
Authors: Thomas O'Leary; Björn Heindryckx; Sylvie Lierman; David van Bruggen; Jelle J Goeman; Mado Vandewoestyne; Dieter Deforce; Susana M Chuva de Sousa Lopes; Petra De Sutter Journal: Nat Biotechnol Date: 2012-02-26 Impact factor: 54.908
Authors: Frances Anne Brook; Sally A Cowley; Edward P Evans; Karen Turner; William S James; Helen J Mardon Journal: In Vitro Cell Dev Biol Anim Date: 2010-03-24 Impact factor: 2.416
Authors: Johan Stenberg; Maria Elovsson; Raimund Strehl; Eva Kilmare; Johan Hyllner; Anders Lindahl Journal: Cytotechnology Date: 2011-03-16 Impact factor: 2.058