Sasha Bakhru: Research Abstract

Towards the controllable and reproducible fabrication of microcapsules, a new microcapsule generator affording picoliter-scale control over capsule volume has been designed and assembled. By volumetric dispensing of polyelectrolyte solution into a reciprocating receiving bath, microcapsules are formed in a manner conducive to maintaining cell viability.

Using this microcapsule generator, microcapsules have been fabricated by complex coacervation of a positively charged methylated collagen (MC) and a synthetic, negatively-charged terpolymer (TP) of methyl methacrylate, methacrylate, and hydroxyethyl methacrylate. The MC/TP system provides substrate functionality through RGD cellular binding sites on MC. The synthetic TP component comprises hydrophobic, hydrophilic and charge carrying blocks, the relative amounts of which could be tuned to achieve desired capsule properties such as porosity/permeability and charge density.

Microcapsules ranging in size from tens of microns to several hundreds of microns in diameter have been generated with a high degree of reproducibility. This microcapsule system potentially offers control over physical (i.e. size, shape, permeability, moduli) and biochemical parameters relevant to cellular proliferation and differentiation. Stem cells are pluripotent, undifferentiated cells with the ability to both self renew and differentiate into other cell types. Their proliferation, differentiation, and self renewal are each effected by topographical and biochemical cues from the cellular niche in which the stem cells reside.

In recent years, neural stem cells (NSCs) in particular have demonstrated promise as the basis for treatment of neurodegenerative disorders such as Parkinson's and Huntingdon's diseases, for which the expansion of transplant ready NSCs is critical. Towards realizing efficient neural stem cell expansion in a manner amenable to cell harvesting, NSCs were cultured in MC/TP microcapsules of tunable physical and biochemical constitution. Diffusive ingress of FGF-2 has proven sufficient to support intracapsular NSC expansion and phenotype maintenance, and NR6R-3T3 cells (FGF-dependent mouse fibroblasts) were proliferative at substantially lower concentrations than on similar planar controls.

NSCs formed loosely associated clusters in microcapsules with a high Nestin+ fraction and no observable glial differentiation. These clusters were dissociated by mechanical shearing alone (without trypsin or EDTA treatment), an advantage relevant to harvesting of cells for clinical transplantation applications. This microcapsule-based culture system provides a platform to investigate the influence of topographical cues and biochemical signals on the self renewal and differentiation of NSCs.

Sasha Bakhru: Research Abstract

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