Abstract
Hollow alginate microfibers (od = 1.3 mm, id = 0.9 mm, th = 400 µm, L = 3.5 cm) comprised of 2% (w/v) medium molecular weight alginate cross-linked with 0.9 M CaCl2 were fabricated to model outward diffusion capture by 2D fluorescent microscopy. A two-fold comparison of diffusivity determination based on real-time diffusion of Fluorescein isothiocyanate molecular weight (FITC MW) markers was conducted using a proposed Fickian-based approach in conjunction with a previously established numerical model developed based on spectrophotometric data. Computed empirical/numerical (Dempiricial/Dnumerical) diffusivities characterized by small standard deviations for the 4-, 70- and 500-kDa markers expressed in m2/s are (1.06 × 10−9 ± 1.96 × 10−10)/(2.03 × 10−11), (5.89 × 10−11 ± 2.83 × 10−12)/(4.6 × 10−12) and (4.89 × 10−12 ± 3.94 × 10−13)/(1.27 × 10−12), respectively, with the discrimination between the computation techniques narrowing down as a function of MW. The use of the numerical approach is recommended for fluorescence-based measurements as the standard computational method for effective diffusivity determination until capture rates (minimum 12 fps for the 4-kDa marker) and the use of linear instead of polynomial interpolating functions to model temporal intensity gradients have been proven to minimize the extent of systematic errors associated with the proposed empirical method.
Highlights
Cross-linked hydrogel-based bio-membranes conferred with selective diffusivities and mechanical properties have been used since the inception of cell-based therapies and implantation [1], with the most common hydrogel biopolymer used being alginate [2,3,4,5]
De was estimated to be in units of m2/s 1.06 × 10−9, 5.89 × 10−11 and 4.89 × 10−12, for the 4-kDa, 70-kDa and 500-kDa molecular weight (MW) probes, respectively
While the membrane was permeable to the 4-kDa marker, transport of the 70-kDa and 500-kDa transport across the membrane were hindered over time proven by real-time fluorescence imaging
Summary
Cross-linked hydrogel-based bio-membranes conferred with selective diffusivities and mechanical properties have been used since the inception of cell-based therapies and implantation [1], with the most common hydrogel biopolymer used being alginate [2,3,4,5]. Other methods include spectrophotometry [17,18,19,20], size exclusion chromatography [8,21], mechanical release tests [22,23] and fluorescence microscopy [23,24,25]. Regardless of the diffusivity characterization method, the following are a sub-set of factors affecting cross-link homogeneity, size and density, gel isotropy and, membrane diffusivity [13,19,20]: (1) the β-D-mannuronic (M) to α-L-guluronic (G) ratio, the distribution of each monomer within the chain, and alginate molecular weight (MW); (2) the kinetics of gelation, external gelation (chelation) vs internal gelation. Spectrophotometry and fluorescence microscopy are non-destructive as compared to SPM methods requiring sample immobilization, morphology modifications and cross-sectioning, introducing artifacts
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