Insights in Behavior of Variably Formulated Alginate-Based Microcapsules for Cell Transplantation

Abstract

Alginate-based microencapsulation of live cells may offer the opportunity to treat chronic and degenerative disorders. So far, a thorough assessment of physical-chemical behavior of alginate-based microbeads remains cloudy. A disputed issue is which divalent cation to choose for a high performing alginate gelling process. Having selected, in our system, high mannuronic (M) enriched alginates, we studied different gelling cations and their combinations to determine their eventual influence on physical-chemical properties of the final microcapsules preparation, in vitro and in vivo. We have shown that used of ultrapure alginate allows for high biocompatibility of the formed microcapsules, regardless of gelation agents, while use of different gelling cations is associated with corresponding variable effects on the capsules' basic architecture, as originally reported in this work. However, only the final application which the capsules are destined to will ultimately guide the selection of the ideal, specific gelling divalent cations, since in principle there are no capsules that are better than others.

Figure 1

Microcapsules' integrity and morphological evaluation. (a) Microcapsules 24 hours after gelification with 100 mM CaCl₂, 50 mM BaCl₂·2H₂O, 50 mM CaCl₂, and 25 mM BaCl₂·2H₂O, or 100 mM SrCl₂·6H₂O in 0.9% NaCl. All types of microcapsules show a spherical and smooth shape and are homogeneous in diameters' size for each type of counterion used. (b) Microcapsules retrieved from Nod/Scid mice after 40 days from transplantation retained their physical-chemical parameters, with no change in terms of morphology. (c) Microcapsules retrieved from CD1 mice after 40 days from transplantation and 6 days with LPS: they appear undamaged and free from any inflammatory response with no macrophage growth outside the membrane.

Figure 2

In vitro evaluation of microcapsules. (a) The microcapsules gelled with indicated conterions 7 months after gelification and maintained in saline at +4°C maintain a spherical and smooth shape. (b) Distribution of diameters' size over the time for each microcapsule's type at +4°C. All microcapsules show the aptitude to decrement their diameter both over time (P < 0.0001) and between ions (P < 0.0001). (c) The diameter sizes show similar differences over time (P < 0.001) and between ions (P < 0.0001) in both left (+4°C) and middle (+37°C) panels; the temperature is irrelevant on diameters' decrement for microcapsules made with various gelling solutions, as shown in the right panel in which, considering all the ions together, the within-subjects effect (time) is still significant (P < 0.0001), whilst the between-subjects effect (temperature) is not significant (P = 0.122). (d) Microcapsules left O/N in gelling solution swell when saline solution was changed as indicated in figure both over time (P < 0.0001) and between ions (P < 0.0001). (e) The illustrated graph summarizes at once, using the same capsules, some of the experiments carried out throughout this report. The initial part of the graph shows capsules maintained in saline for 7 months. The second part shows the experiment elsewhere named “fake saline changes,” while the last part coincides with the swelling test performed by changing saline at fixed times. Finally, the capsules have been left to rest in saline, like the beginning of the experiment. Looking at the graph, during the “fake saline changes” the capsules' diameters did not vary compared to the values seen after 7 months of incubation. Two actual changes of saline were performed; all capsules' types tended to increase their diameter. The capsules then were incubated for 1 hour at 4°C, overnight, or for 10 days with no saline changes. From the graph, it appears that the capsules tend to stabilize their diameters which eventually tend to decrease again.

Figure 3

Evaluation of mannuronic (M): guluronic (G) acidic residues ratio.