Towards Stronger Microcapsules for Non-Autologous Somatic Gene Therapy
Contact Person: Jeremy M. Van Raamsdonk ([email protected])
Materials and Methods
Our lab primarily uses alginate-polylysine-alginate(APA) microcapsules. Alginate is a polysaccharide composed of mannuronic M)
and guluronic(G) acid units.
Microencapsulation Protocol:
Myoblasts are transfected
with a plasmid vector to create universal cell lines that secrete a therapeutic
protein(eg. factor IX). The myoblasts are harvested and mixed with a 1.5% or 2%
alginate solution at concentrations of about 2 million cells per ml. of
alginate. This cell-alginate mixture is extruded through a 27 gauge blunt ended
needle into calcium chloride with concentric air flow to induce droplet
formation. When the alginate droplet contacts the calcium chloride bath, the
calcium ions cross-link the alginate molecules inducing gel bead formation. The
resulting cell-alginate beads are washed with a series of solutions that
provide for capsule strength while maintaining cell viability. The polylysine PLL)
wash coats the capsule with a layer of polylysine
which gives strength to the microcapsule and determines the molecular weight
cutoff of the capsules(ie. pore size).An outer layer
of alginate is added after the polylysine coating for
biocompatability. The inner core can be dissolved
with sodium citrate to create hollow capsules. The microcapsules are generally
between 200 and 500 um in size.
Figure 5. On the left from top to bottom:
Structure of alginate composed of M and G units, the cross-linking of alginate
with calcium ions, chelation of calcium by 2 G units.
On the right is a diagrammatic overview of encapsulation process.
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