Calcium pre-conditioning substitution enhances viability and glucose sensitivity of pancreatic beta-cells encapsulated using polyelectrolyte multilayer coating method.

Type I diabetics are dependent on daily insulin injections. A therapy capable of immunoisolating pancreatic beta-cells and providing normoglycaemia is an alternative since it would avoid the late complications associated with insulin use. Here, 3D-concave agarose micro-wells were used to culture robust pancreatic MIN-6 cell spheroids within 24 hours that were shown to exhibit cell-cell contact and uniform size (201 ± 2 µm). A polyelectrolyte multilayer (PEM) approach using alginate and poly-l-lysine was employed to coat cell spheroids. In comparison to conventional PEM, use of a novel Ca2+ pre-coating step enhanced beta-cells viability (89 ± 6%) and metabolic activity since it reduced the toxic effect of the cationic polymer. Pre-coating was achieved by treating MIN-6 spheroids with calcium chloride, which enabled the adhesion of anionic polymer to the cells surface. Pre-coated cells coated with four bilayers of polymers were successfully immunoisolated from FITC-mouse antibody and pro-inflammatory cytokines. Novel PEM coated cells were shown to secret significantly (P < 0.05) different amounts of insulin in response to changes in glucose concentration (2 vs. 20 mM). This work presents a 3D culture model and novel PEM coating procedure that enhances viability, maintains functionality and immunoisolates beta-cells, which is a promising step towards an alternative therapy to insulin.

Alginate hydrogel has a negative impact on in vitro collagen 1 deposition by fibroblasts.

Hydrogels have been widely investigated as 3D culture substrates because of their reported structural similarity to the extracellular matrix (ECM). Limited ECM deposition, however, occurs within these materials, so the resulting "tissues" bear little resemblance to those found in the body. Here matrix deposition by fibroblasts encapsulated within a calcium alginate (Ca-alg) hydrogel was investigated. Although the cells transcribed mRNA for coll Iα over a period of 3 weeks, very little collagen protein deposition was observed within the gel by histology or immunohistochemistry (IHC). Although molecular diffusion demonstrated charge dependency, this did not prevent the flux of both positively and negative charged amino acids through the gel, suggesting that the absence of ECM could not be attributed to substrate limitation. The flux of protein, however, was charge-dependent as proteins with a net negative charge passed quickly through the Ca-alg into the medium. The minimal collagen deposition within the Ca-alg was attributed to a combination of rapid movement of negatively charged procollagen through the gel and steric hindrance of fibril formation.

Modification of alginate degradation properties using orthosilicic acid.

Biopolymers such as alginates have been widely researched for clinical use. Their clinical application, however, have been limited due to their unpredictable and often rapid degradation rates. Here we show that the degradation of an alginate hydrogel can be tailored through the addition of orthosilicic acid (OSA). On immersion in aqueous media a negligible quantity of orthosilicic acid was released from the gel matrix. The presence of the OSA within the gel was shown to significantly slow degradation of the alginate hydrogel when immersed in a potent calcium chelator (EDTA) when compared with the control group. Sample degradation was associated with a significant calcium release from the non-modified gel; however, the orthosilicic acid modified gel did not release detectable levels of calcium over the same period. This suggests that the orthosilicic acid inhibits degradation of the gel by forming an interaction with the calcium cross-links. A rapid reduction in the storage modulus G', was observed in alginate made without OSA, however, the G'exhibited by the orthosilicic acid modified alginate did not reduce significantly (p<0.05). Furthermore, although both the OSA and alginate exhibit negative charges in solution, it is likely that they form weak interactions, this hypothesis was proven by demonstrating the efficacy of OSA for binding the alginate hydrocolloid. The findings of this study are likely to have utility in applications where controlling gel degradation is desirable, such as in cell delivery or in the controlled release of molecules in the body.