Abstract
Study question
Is there a efficient establishing method of human embryonic stem cells directly from the human blastocysts independent of feeder cells?
Summary answer
We established a novel method of generating human embryonic stem cells directly from human blastocysts independent of feeder layer cells.
What is known already
Establishing embryonic stem cells lines mainly needed to coculture ICM clumps with feeder cells (like mouse or human fibroblasts) ,this brought in potential heterogeneous pollution.Although there had be some reports about generating human ESCs independent of feeder cells,but the efficiency was low and conditioned medium were unstable and also had the biological contamination.
Study design, size, duration
We used ten day5/6 donated human blastocysts from our reproductive center ,most of them were genetically diseased embryos with abnormal PGT diagnosis.After establishing ESCs procedure , all the cell lines were identified with pluripotency and differentiation potential tests.The success rate of system was calculated and compared with the conventional methods.
Participants/materials, setting, methods
In brief, ICM clumps were separated mechanically by using a micromanipulation system,and then transferred to a 30ul mTESR plus culture media drop pretreated with the geltrex (1:100 dilution) matrix and oxygen concentration was 5%. When cells attached and migrated,we also used laser to destroy the remaining trophoblast cells.About 10 days,the typical ES clone can be mechanically passaged and cells can be cultured in normal oxygen concentrations after passage 2. .
Main results and the role of chance
Using this method we had successfully established nine embryonic stem cell lines from donated human blastocysts ,the success rate was 90% (9/10). Each cell lines had passed the evaluation test of embryonic stem cell. When compared with the conventional feeder cells dependent method,our novol methods not only eliminated the pollution from heterogeneous cells,but also had higher success rate (90% vs 25%).
Limitations, reasons for caution
Due to the scarcity of donated human blastocysts, this experiment was a single-center experiment with small samples.
Wider implications of the findings: We speculated that the batch differences of culture dishes, matrix and culture medium might affect the establish efficiency , and how to carry out a high level of quality control work might be the key factor to keep the system stable.
Trial registration number
basic research
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