Biomedical Engineering Reference
In-Depth Information
and a dilution factor at each passage (1:2-1:4). All cultured cells were also trans-
planted periodically, and SSC numbers were measured by counting the number of
colonies in recipient testes 2 months later. As the figure shows, each parameter
increases linearly over time, when cluster and colony numbers are expressed in
log 10 values. This observation indicates that cluster-forming cells are capable of
long-term self-renewal and proliferate continuously in vitro while sustaining
spermatogenic regeneration activity. Importantly, proliferation kinetics of clusters
parallel with those of colonies determined by transplantation, demonstrating that
cluster numbers directly correlate with SSC numbers. Using the data shown in
Fig. 6.1b , therefore, it is calculated that both clusters and colonies increase in
number with a near-identical population-doubling time: 5.5 days with clusters and
5.6 days with colonies (Yeh et al. 2007 ).
This strong correlation addresses the above two questions. First, the parallelism
between the two proliferation kinetics assures that the cluster number faithfully
reflects the number of functional SSCs, even though the regeneration of spermato-
genesis is not demonstrated. Second, on this basis, the strong linearity of cluster
proliferation kinetics that is consistent over an extended culture period demonstrates
that relative SSC activity can be quantified by cluster counts at any single culture
period, even though the period is only 6-7 days. Therefore, although the regenera-
tion and long-term maintenance of complete spermatogenesis cannot be recapitu-
lated in vitro , the CFA assay detects SSC activity in a semi-quantitative manner in a
short time, based strictly on its correlation to the transplantation assay.
The faithfulness of the CFA assay to the transplantation assay can also be demon-
strated under an experimental condition (Yeh et al. 2007 ). For example, we measured
the number of SSCs surviving exposure to a cytotoxic hypotonic solution using both
the CFA and transplantation assays, and obtained near identical results with both
techniques (Fig. 6.1c ). We have used the CFA assay in combination with the trans-
plantation assay under various experimental situations, and data thus far obtained in
our laboratory have been supportive of the correlation between the two methods.
6.2
Advantages of the CFA Assay
The CFA assay has several advantages over spermatogonial transplantation (Yeh
et al. 2007 ). First, it generates results in 1 week. Although the transplantation assay
is the unequivocal SSC detection method, its time-consuming nature makes it dif-
ficult to conduct a given experimental program in a stepwise manner; it often forces
us to take a second step without the data of a first step in hand. Hence, the CFA
assay facilitates research progress by markedly shortening the time until data acqui-
sition, while allowing for semi-quantitative detection of SSC activity. Second, the culture
technique is simple and enables an analysis of a wide range of experimental condi-
tions at once. In contrast, the transplantation assay requires microinjection into
individual testes, which limits the number of conditions to be examined at a time.
Third, while the transplantation assay requires genetic compatibility between donor
Search WWH ::




Custom Search