Alginate microcapsules for cell therapy : a study of functional properties of capsules made of native and enzymatically tailored alginates

Alginate-poly-L-lysine(PLL)-alginate capsules are the far most studied system for encapsulation of living cells. Immobilisation in ionically cross-linked alginate provides a simple and safe encapsulation procedure at physiological conditions. The properties of alginate, a linear natural polysaccharide consisting of mannuronic acid (M) and guluronic acid (G), is very much dependent on the composition of monomers in the chain. Enzymatic modification of the alginate by in-chain conversion of M to G by mannuronan C-5 epimerases enables us to tailor alginate to specific properties. In this study, an epimerase that converts M-blocks to blocks of alternating M and G was used. By this conversion, more flexible segments are formed, but the molecular weight and the number and length of the G-blocks that determine the cross-linking sequences of the alginate is not altered. The enzymatic modification increased the elasticity of diffusion set Caalginate gels. Increased syneresis was also obtained that could be seen as reduction in size of the Ca-alginate gel beads. The osmotic stability was increased after epimerisation and reduced permeability to immunoglobulin G (IgG) and tumour necrosis factor (TNF) was obtained.We have developed an electrostatic droplet generator that enables large-scale production of small alginate gel beads with a narrow size distribution. Encapsulated islets of Langerhans preserved their function in the new system. We observed that the smaller beads were more vulnerable to the encapsulation procedure, but stable alginate PLLalginate capsules of 200μm in diameter that were impermeable to IgG could be made by small changes in the encapsulation procedure. As Ca-alginate beads of epimerised alginate are initially smaller and more resistant to swelling than those made from the original alginate, the use of epimerised alginate reduces the problems of making small capsules.Visualisation of the capsules in the confocal laser scanning microscopy (CLSM) by fluorescent-labelled alginate and PLL, revealed an easy method to assess the distribution of polymers in intact capsules. It thus makes it easier to characterise the final capsules also by means of polymer distribution. This has previously been shown to vary with different encapsulation procedures and is important for the capsule properties.PLL was shown to be the major factor causing overgrowth of implanted empty capsules in mice as the fibrotic reaction depended on the PLL exposure. Further studies revealed that at low concentrations, PLL induced TNF production in human monocytes. At higher concentrations, PLL was toxic, causing necrosis of the exposed cells. Both the TNF inducing and the toxic effect of PLL was reduced by addition of soluble alginate, but the study shows that for producing biocompatible capsules, low amounts of PLL should be used to prevent inflammation. The effect of PLL was further verified, as better coating with compositionally homogeneous alginates with MG as repeating unit reduced the overgrowth on the implanted capsules. This strictly alternating coating alginate was produced by modifying mannuronan with a recombinant mannuronan C-5 epimerase. No differences in biocompatibility was seen on alginate gel beads of high-G and epimerised high-G alginates.Hence, increasing the flexible segments in the alginate chain by epimerisation leads to capsules of improved properties as it comes to size, stability, permeability and biocompatibility. The use of specific mannuronan C-5 epimerases thus provides a novel method for tailoring alginates to specific uses, such as immunoisolation of living cells.