185antiserum followed by separate exposure to complement (Solter and Knowles
1975 ). However, immunosurgery uses substances, like antihuman antiserum and
guinea pig complement, that are animal based and may carry pathogens and/or mol-
ecules that can contaminate the ICM and thus the ES cells derived from them
(Turetsky et al. 2008 ). There is also the risk of damaging ICM cells, thus decreasing
the chances of deriving new ES cell lines from these cells (Turksen 2012). Therefore,
to avoid immunosurgery, alternative isolation methods, mainly mechanical or
chemical dissolution of the TE layer, were developed, but still problems remained.
Firstly, mechanical dissection is a crude method relying heavily on the operator’s
technical skills. Secondly, the use of acid tyrode for chemical dissolution of the TE
layer can lead to acidifi cation of the medium resulting in damage to the cell of the
ICM (Turetsky et al. 2008 ). Thus, a better approach is to use a non-contact laser,
both for dissection of the ICM and assisted hatching, facilitating isolation without
the use of animal products or causing damage to cells (Turetsky et al. 2008 ). In addi-
tion, a large fraction of human ES cell lines have also been established from whole
plated embryos on feeder cells; in some cases good-quality embryos are used, but
mostly poor-quality embryos lacking a clear ICM are used. However, this method
can lead to the development of TE stem cell lines as TE cells proliferate much faster
than the cells of ICM and can suppress their growth (Turksen 2012). The best
method to isolate ICM from the blastocyst, in order to generate ES cell lines, has not
yet been consolidated as shown in Fig. 9.3. Although, over the years, new methods
have been developed, all methods continue to be used. The method used for isola-
tion depends on the availability of laser and micromanipulation equipment along
with the morphology and quality of the embryo.
Fig. 9.3 Number of ES cell lines derived over the years, based on the method employed for
extraction
9 Human Embryonic Stem Cells and Associated Clinical Concerns