Biology Reference
In-Depth Information
oxygen and required nutrients and releases bioactive cell secretions, but restricts pas-
sage of larger cytotoxic agents from the host immune defense system.
To date, all studies using encapsulated CP for CNS transplant studies have utilized
microcapsules formed using alginate. Alginate—one of the most frequently investigated
biomaterials for cell encapsulation—is a polysaccharide composed of guluronic (G) and
mannuronic (M) acid linked by - and -glycoside bonds. The ratio of these monomers
contributes directly to certain physical characteristics of the polysaccharide. Once cat-
ionically crosslinked, materials high in G are more brittle, because of a more networked
structure resulting from bonds, whereas those high in M, with more linear linkages,
exhibit decreased three-dimensional crosslinking and greater elasticity
[36,39,40]
.
Prior to cell encapsulation, alginate powder is typically reconstituted in a suitable
buffer, and a variety of purification techniques are employed to rid the solution of
proteins, endotoxins, and polyphenols. These techniques include solvent extraction,
sequential filtration, charcoal extraction, dialysis, and others. Contaminant removal is
essential to maintaining the optimal balance of hydrophilicity as well as to preventing
inflammation related to endotoxin. The purification process ultimately determines the
final physical and chemical characteristics of the encapsulated cell product, as fine
variations in copolymer ratio, molecular weight, and purity can all be controlled at
this step. Following purification and reconstitution of the alginate solution at a suit-
able pH, quality control analysis is carried out to maintain optimal operating specifi-
cations for encapsulation and subsequent
in vivo
longevity. The final purified alginate
can be characterized both as a raw material and as a formed capsule using the analyti-
cal techniques shown in
Table 4.2
.
For the xenogeneic transplantation paradigm, neonatal porcine CP (7-14 days of
age) is isolated from the lateral ventricles and dissociated using conventional col-
lagenase digestion procedures. The resulting cell clusters are groupings of epithelial
cells ranging from 50 to 200 m in diameter. Prior to encapsulation, viability, which
is typically greater than 95%, is confirmed by staining the cells with a vital dye. The
encapsulation process does not affect cell viability, so these cells can be maintained
in culture for months if needed or desired. The cultured CP clusters maintain the
typical genotypic and phenotypic characteristics of the native, undigested tissue.
Based on the knowledge that the CP epithelium is rich in tight junctions and lined
with microvilli, we have used immunocytochemical techniques (
zonnula occludens
;
ZO-1) to identify tight junctional complexes and the tubulin associated with the
cytoskeleton of the microvilli (
Figure 4.3
).
Following confirmation of cell viability and phenotype, CP cells are encapsulated
in alginate microcapsules by extruding a mixture of cells dispersed in 1.7% sodium
alginate through a droplet-generating apparatus into a bath of cations. This process
is typically performed at an encapsulation density of 10,000-50,000 clusters or
200,000-5 M cells/ml alginate. The cells, entrapped in the calcium-alginate gel, are
coated twice with poly L-ornithine (PLO), followed by an outer coat of alginate. The
central core of alginate is liquefied by chelation. The resulting microcapsules have a
diameter of between 500 and 750 m.
The alginate/PLO microcapsule appears to be very stable when implanted into
the brain relative to a commonly used transplant site such as the peritoneum. Using
