Biomedical Engineering Reference
In-Depth Information
a suspension of gelatin in aqueous solution to more than around 40°C. However, this
hydrogel can remelt and shows a gel-to-sol transition if subsequently heated at around 40°C
[70]. This means the impossibility of using unmodified gelatin as a material for cell-enclosing
capsules for transplantation
in vivo
and culture under normal mammalian cell culturing
temperatures. Thus we used the gelatin conjugate with agarose for supporting cellular growth
in microcapsules. As with the agarose and alginate-gelatin microcapsules, we could prepare
cell-enclosing microcapsules via the droplet breakup process using liquid paraffin and the
subsequent thermal gelation process [37]. As we expected, the adherent cells enclosed in the
agarose-gelatin conjugate microcapsules showed a higher degree of proliferation than those in
the unmodified agarose microcapsules (Figure 9).
Enzymatically-crosslinked Alginate Microcapsules
As a novel method for gelation of droplets obtained via the breakup process in ambient
co-flowing liquid paraffin, we developed a method using enzymatic polymerization [39].
Enzymatic polymerization
in vitro
has been studied for decades because specific enzyme
catalysis provides a novel synthetic route for functional and useful polymeric materials
[71,72]. We used peroxidase for obtaining microcapsules. Peroxidases function as
oxidoreductases that catalyze the oxidation of donors using H
2
O
2
resulting in polyphenols
linked at the aromatic ring by a C-C and C-O coupling of phenols. The alginate incorporated
hydroxyl phenol moieties into about 3% of the original carboxyl groups (Alg-Ph) and was
used as the material of microcapsules. We synthesized this alginate derivative [73] based on
the report by Kurisawa
et al.
that a hyaluronic acid conjugate with tyramine forms a hydrogel
via a peroxidase-catalyzed oxidative reaction [74]. Because more than 90% of the original
carboxyl groups remain, the alginate derivative is gellable via not only the peroxidase
catalyzed reaction, but also via the conventional gelation process coming into contact with
multivalent cations [73]. One of the problems at the start point of this study was how to
supply the H
2
O
2
necessary for the enzymatic reaction. One option was to extrude the mixture
of Alg-Ph, peroxidase, and H
2
O
2
into the ambient co-flowing stream of liquid paraffin.
However, this is not practical for continuous production of cell-enclosing microcapsules
because of the formation of crosslinks between the Alg-Ph molecules over time in the syringe
before being extruded from the needle. Next, we attempted to dissolve H
2
O
2
in liquid
paraffin. Although liquid paraffin is a 'water-immiscible liquid', it can dissolve a small
quantity of water. The liquid paraffin containing H
2
O
2
is obtained by vigorously stirring an
aqueous H
2
O
2
solution with liquid paraffin followed by a centrifugation step to separate the
liquid paraffin and non-dissolved aqueous H
2
O
2
solution. We added 5 mL of aqueous H
2
O
2
solution (31 wt%) into 1000 mL liquid paraffin. Cell-suspending 1.5 w/v% Alg-Ph solution
containing 1.6 units/mL of horse radish peroxidase (HRP) was extruded into a co-flowing
stream of liquid paraffin containing dissolved H
2
O
2
(Figure 10a). The liquid paraffin
suspension with partially gelated Alg-Ph microcapsules was collected in a plastic tube. After
10 min of standing to allow for further progress of the enzymatic crosslinking reaction
(Figure 10b), Alg-Ph microcapsules were collected via centrifugation. The resultant
microcapsules had high sphericity (Figure 11). This high sphericity means that the sufficient
degree of enzymatic gelation necessary for fixing the final shape of the gel did not occur
instantaneously.
