Biomedical Engineering Reference
In-Depth Information
6.3.3
Does the Number of Cells Placed in Culture
Affect Assay Outcomes?
It is conceivable that the CFA assay can be affected by the number or density of
cluster-forming cells placed in culture. If such cells are densely cultured, multiple
cells located nearby may form one cluster, or multiple clusters may merge as they
grow, causing an underestimation of cluster numbers. We addressed this issue in
two ways (Yeh et al.
2007
). The first was to examine the correlation between cluster
numbers and testis cells that were seeded after SSC enrichment, ranging from 5 to
2.5 × 10
4
cells/cm
2
(i.e., limiting dilution of SSC-enriched testis cells). We found
that these two parameters (both in a log scale) linearly correlated. Next, we cultured
a 1:1 mixture of two populations of SSC-enriched testis cells, which can be distin-
guished from each other by the presence or absence of a transgene, and examined
the proportion of clusters comprised of cells from both populations; clusters with
both cell types were judged to have arisen from multiple cluster-forming cells
placed nearby or merging of multiple clusters. The results showed that such clusters
represented less than 10% of all clusters when the density of total SSC-enriched
cells seeded was 0.5 × 10
4
cells/cm
2
or lower, while 14% of total clusters were made
of the two cell types when the cell density was 2.5 × 10
4
cells/cm
2
; 1.25 × 10
4
cells/cm
2
gave approximately 10% of such clusters. Total cluster numbers were ~160, ~343,
and ~520 clusters/cm
2
, at 0.5, 1.25, and 2.5 cells seeded/cm
2
.
The strong linear correlation between the cell density and cluster number, as
well as the observation that clusters composed of the cells with two different
origins are a minority in general, indicate that an underestimation of SSC quantifi-
cation by the CFA assay may not be a significant concern. Perhaps, this is because
cluster-forming cells represent a rare cell population. Nevertheless, the cell-
density- dependent increase in the proportion of multi-origin clusters implies that
the possibility exists that a higher cell density affects the fidelity of assay outcomes.
Based on the above results, the density of testis cells seeded at ~1 × 10
4
SSC-
enriched cells/cm
2
or ~350 clusters/cm
2
, with which multi-clonal clusters arise at a
frequency of 10% or less, generate the most reliable assay condition.
6.3.4
Can the CFA Assay Distinguish SSC Proliferation
and Maintenance?
To address this question, we want to introduce a hypothetical experimental situa-
tion. Here, the aim is to evaluate the effect of a growth factor (Factor A) on SSC
activity
in vitro
in the presence of GDNF, GFRA1, and FGF2 (Fig.
6.2
,
“experimental culture”). Test cells were harvested from an established cluster cul-
ture, which had been maintained for experimental use. This culture was then split
into to the hypothetical cultures with or without Factor A, and cluster numbers were
measured after 6 days
in vitro
. The culture with Factor A produced 200 clusters,
whereas the control group generated 100 clusters.
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