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theses

A leading cause of disease and death for all age groups are syndromes characterized by widespread uncontrollable inflammation. Severe trauma induces pro-inflammatory responses, increasing the risk of complications. Mesenchymal Stem Cells (MSCs) have the remarkable ability of altering the immune response by emitting soluble anti-inflammatory factors, which rebalance the immune system and thereby may prevent additional damage and promote recovery. Furthermore, alginate encapsulation of MSCs has proven to be an effective long-term delivery method. Encapsulation of MSCs involves a mixture of alginate and cells, which are cross-linked into microspheres with divalent cations. We hypothesized that optimization and scale-up of encapsulation of cells in alginate microspheres will provide a commercially viable solution for delivery of cell therapy. To analyze trends and characteristics of MSC clinical trials, a novel database was constructed using data derived from Clinical.Trial.gov, including data on disease targets, sources of cells, doses being delivered and routes of administration. Among the hundreds of clinical trials using free MSCs that were analyzed, several critical uncontrollable parameters may contribute to the relatively poor success rates. This includes rapid clearance in various organs, and uncertainty of numbers of surviving cells over time in target locations after transplantation. Encapsulation of MSCs in may overcome many of these challenges by prolonging MSC survival and localizing the cells in desired sites. Alginate encapsulation involves extrusion of a mixture of cells suspended in alginate into microdroplets that crosslinked into microspheres after falling into a bath with divalent cations. We compared two modes of encapsulation, fluidic control and pressure control, holding these parameters fixed. Pressure control is advantageous over fluidic control by reducing transient and residual flow, and eliminating dead volume. Our results with pressure control show that as inner needle diameter increases flow rates increase by a power of 4. Shortening the needle increases the flow rate linearly. Thus shorter and wider needles have higher flow rates, which yield larger capsules and increased yields of encapsulated cells after a given run time using the pressure control mode. The wide dynamic range with pressure control is advantageous over the more limited fluidic control by varying needle diameters. Measuring the number of cells per capsule is technically challenging and often not reported. The most common method for quantifying the number of live/dead cells per alginate microcapsule is imaging stained encapsulated cells using a confocal microscope. While confocal microscopy is advantageous in imaging all planes throughout a capsule, they have some disadvantages including high cost of imaging, long image acquisition time and technical limitation of measuring the number of cells per capsule in large and high density capsules. To overcome these problems, we have developed an alginate depolymerization method to visualize the contents of individual microcapsules in 2-D spreads. A key advantage of this method is that the capsules are distributed over a much larger area nearly eliminating cell overlap. This simplifies problems with confocal imaging by physically collapsing cells into a single plane rather than imaging all planes throughout the capsule and then computationally collapsing them into a single plane. In addition, compared to confocal microscopy, measuring the viability using a epifluorescent microscope is less expensive, faster, and diminishes the cell overlapping issue because the cells are dispersed in a single plane.

ETD_8861

M.S.

Includes bibliographical references

by Maciej Kabat

doi:10.7282/T3K35Z37

ETD graduate

The author owns the copyright to this work.