Biomedical Engineering Reference
In-Depth Information
Fig. 12 Amount of DNA
molecules of various genes,
as measured by qPCR assay.
The qPCR was carried out
after 1 day of culture in the
PMBV/PVA hydrogel or on
TCPS. See text for gene
descriptions
10
4
PMBV/ PVA hydrogel
Tissue culture polystyrene
10
3
10
2
10
1
10
0
B2m
Eef1g
Sdha
Tbp
Gene
7 Encapsulation of Stem Cells and Their Undifferentiated
Character
Mouse ES cells (129/SvEv, passage 11) were suspended in 5 wt% PMBV solution
dissolved in the ES cell culture medium. The PMBV solution containing ES cells
was mixed with 2.5 wt% PVA solutions. The mixture was gently shaken to form the
PMBV/PVA hydrogel. After confirmation of gelation, the PMBV/PVA hydrogel
containing ES cells was stored in an incubator. The cell morphology was observed
with a phase contrast microscope.
The ES cells were encapsulated in the PMBV/PVA hydrogel by the same
method used for the L929 cells. In general, ES cells form a cell aggregate called
an embryoid body in suspension culture. However, it was observed that the
encapsulated ES cells in the hydrogel did not form any embryoid body for 72 h.
Figure
13
shows the phase contrast microscope images of the encapsulated ES
cells in the PMBV/PVA hydrogel, and those cultured on the water-insoluble
phospholipid polymer, poly(MPC-
co
-BMA) (PMB30, MPC unit mole fraction
0.30). The ES cells were uniformly suspended in the PMBV/PVA hydrogel. On
the other hand, the ES cells cultured in suspension culture (cell adhesion was
completely inhibited on the PMB30 surface) were aggregated and formed an
embryoid body. It should be noted that the PMBV/PVA hydrogel did not affect
direct cell-cell interaction between the ES cells. This is important because
increased cell-cell interaction is a trigger for ES cell differentiation.
The PMBV/PVA hydrogel containing ES cells was dissociated by the addition
of 0.2 M
D
-fructose solution after 3 days. After dissociation of the PMBV/PVA
hydrogel, the recovered ES cells were cultured on gelatin-coated TCPS in the
culture medium as usual, and the differentiation characters of recovered ES cells
were estimated by alkaline phosphatase (ALP) staining (Fig.
14
).
ALP staining was performed to estimate the functionality of recovered ES cells,
and it was found that the ES cells with undifferentiated character were well stained
