caa a22 4500 44529213X CHVBK 20180317142531.0 cr unu---uuuuu 170323e20100401xx s 000 0 eng 10.1007/s11626-010-9294-2 doi (NATIONALLICENCE)springer-10.1007/s11626-010-9294-2 Generation of Sheffield (Shef) human embryonic stem cell lines using a microdrop culture system [Elektronische Daten] [Behrouz Aflatoonian, Ludmila Ruban, Shamsul Shamsuddin, Duncan Baker, Peter Andrews, Harry Moore] The conventional method for the derivation of human embryonic stem cells (hESCs) involves inner cell mass (ICM) co-culture with a feeder layer of inactivated mouse or human embryonic fibroblasts in an in vitro fertilisation culture dish. Growth factors potentially involved in primary derivation of hESCs may be lost or diluted in such a system. We established a microdrop method which maintained feeder cells and efficiently generated hESCs. Embryos were donated for stem cell research after fully informed patient consent. A feeder cell layer was made by incubating inactivated mouse embryonic fibroblasts (MEFs) feeder cells in a 50μl drop of medium (DMEM/10% foetal calf serum) under mineral oil in a small tissue culture dish. MEFs formed a confluent layer and medium was replaced with human embryonic stem medium supplemented with 10% Plasmanate (Bayer) and incubated overnight. Cryopreserved embryos were thawed and cultured until the blastocyst stage and the zona pellucida removed with pronase (2mg/ml; Calbiochem). A zona-free intact blastocyst was placed in the feeder microdrop and monitored for ES derivation with medium changed every 2-3d. Proliferating hESCs were passaged into other feeder drops and standard feeder preparation by manual dissection until a stable cell line was established. Six hESC lines (Shef 3-8) were derived. From a total of 46 blastocysts (early to expanded), five hESC lines were generated (Shef 3-7). Shef 3-6 were generated on MEFs from 25 blastocysts. Shef7 was generated on human foetal gonadal embryonic fibroblasts from a further 21 blastocysts. From our experience, microdrop technique is more efficient than conventional method for derivation of hESCs and it is much easier to monitor early hESC derivation. The microdrop method lends itself to good manufacturing practice derivation of hESCs. The Society for In Vitro Biology, 2010 Blastocyst nationallicence Derivation nationallicence Differentiation nationallicence Inner cell mass nationallicence Microdrop system nationallicence Open system nationallicence Human embryonic stem cells (hESCs) nationallicence Aflatoonian Behrouz Centre for Stem Cell Biology, Sheffield University, S10 2TN, Sheffield, UK aut Ruban Ludmila Centre for Stem Cell Biology, Sheffield University, S10 2TN, Sheffield, UK aut Shamsuddin Shamsul Centre for Stem Cell Biology, Sheffield University, S10 2TN, Sheffield, UK aut Baker Duncan Centre for Stem Cell Biology, Sheffield University, S10 2TN, Sheffield, UK aut Andrews Peter Centre for Stem Cell Biology, Sheffield University, S10 2TN, Sheffield, UK aut Moore Harry Centre for Stem Cell Biology, Sheffield University, S10 2TN, Sheffield, UK aut In Vitro Cellular & Developmental Biology - Animal Springer-Verlag 46/3-4(2010-04-01), 236-241 1071-2690 46:3-4<236 2010 46 11626 https://doi.org/10.1007/s11626-010-9294-2 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/s11626-010-9294-2 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Aflatoonian Behrouz Centre for Stem Cell Biology, Sheffield University, S10 2TN, Sheffield, UK aut NATIONALLICENCE 700 1- Ruban Ludmila Centre for Stem Cell Biology, Sheffield University, S10 2TN, Sheffield, UK aut NATIONALLICENCE 700 1- Shamsuddin Shamsul Centre for Stem Cell Biology, Sheffield University, S10 2TN, Sheffield, UK aut NATIONALLICENCE 700 1- Baker Duncan Centre for Stem Cell Biology, Sheffield University, S10 2TN, Sheffield, UK aut NATIONALLICENCE 700 1- Andrews Peter Centre for Stem Cell Biology, Sheffield University, S10 2TN, Sheffield, UK aut NATIONALLICENCE 700 1- Moore Harry Centre for Stem Cell Biology, Sheffield University, S10 2TN, Sheffield, UK aut NATIONALLICENCE 773 0- In Vitro Cellular & Developmental Biology - Animal Springer-Verlag 46/3-4(2010-04-01), 236-241 1071-2690 46:3-4<236 2010 46 11626 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer