Hybrid Alginate-Protein-Coated Graphene Oxide Microcapsules Enhance the Functionality of Erythropoietin Secreting C2C12 Myoblasts.

The beneficial effect of combining alginate hydrogel with graphene oxide (GO) on microencapsulated C2C12-myoblast viability has recently been described. However, the commercially available GO lacks homogeneity in size, this parameter being of high relevance for the cell fate in two-dimensional studies. In three-dimensional applications the capacity of this material for binding different kinds of proteins can result in the reduction of de novo released protein that can effectively reach the vicinity of the microcapsules. Undoubtedly, this could be an important hurdle in its clinical use when combined with alginate-PLL microcapsules. Here, we demonstrate that the homogenization of GO nanoparticles is not a mandatory preparation step in order to get the best of this material upon cell microencapsulation. In fact, when the superficial area of these particles is increased, higher amounts of the therapeutic protein erythropoietin (EPO) are adsorbed on their surface. On the other hand, we have been able to improve even more the favorable effects of this graphene derivative on microencapsulated cell viability by forming a protein biocorona. These proteins block the potential binding sites of EPO and, therefore, enhance the amount of therapeutic drug that is released. Finally, we prove that these hybrid alginate-protein-coated GO-microcapsules are functional in vivo.

Graphene oxide enhances alginate encapsulated cells viability and functionality while not affecting the foreign body response.

The combination of protein-coated graphene oxide (GO) and microencapsulation technology has moved a step forward in the challenge of improving long-term alginate encapsulated cell survival and sustainable therapeutic protein release, bringing closer its translation from bench to the clinic. Although this new approach in cell microencapsulation represents a great promise for long-term drug delivery, previous studies have been performed only with encapsulated murine C2C12 myoblasts genetically engineered to secrete murine erythropoietin (C2C12-EPO) within 160 µm diameter hybrid alginate protein-coated GO microcapsules implanted into syngeneic mice. Here, we show that encapsulated C2C12-EPO myoblasts survive longer and release more therapeutic protein by doubling the micron diameter of hybrid alginate-protein-coated GO microcapsules to 380 µm range. Encapsulated mesenchymal stem cells (MSC) genetically modified to secrete erythropoietin (D1-MSCs-EPO) within 380 µm-diameter hybrid alginate-protein-coated GO microcapsules confirmed this improvement in survival and sustained protein release in vitro. This improved behavior is reflected in the hematocrit increase of allogeneic mice implanted with both encapsulated cell types within 380 µm diameter hybrid alginate-protein-coated GO microcapsules, showing lower immune response with encapsulated MSCs. These results provide a new relevant step for the future clinical application of protein-coated GO on cell microencapsulation.

Cryopreservation of Human Mesenchymal Stem Cells in an Allogeneic Bioscaffold based on Platelet Rich Plasma and Synovial Fluid.

Transplantation of mesenchymal stem cells (MSCs) has emerged as an alternative strategy to treat knee osteoarthritis. In this context, MSCs derived from synovial fluid could provide higher chondrogenic and cartilage regeneration, presenting synovial fluid as an appropriate MSCs source. An allogeneic and biomimetic bioscaffold composed of Platelet Rich Plasma and synovial fluid that preserve and mimics the natural environment of MSCs isolated from knee has also been developed. We have optimized the cryopreservation of knee-isolated MSCs embedded within the aforementioned biomimetic scaffold, in order to create a reserve of young autologous embedded knee MSCs for future clinical applications. We have tested several cryoprotectant solutions combining dimethyl sulfoxide (DMSO), sucrose and human serum and quantifying the viability and functionality of the embedded MSCs after thawing. MSCs embedded in bioscaffolds cryopreserved with DMSO 10% or the combination of DMSO 10% and Sucrose 0,2 M displayed the best cell viabilities maintaining the multilineage differentiation potential of MSCs after thawing. In conclusion, embedded young MSCs within allogeneic biomimetic bioscaffold can be cryopreserved with the cryoprotectant solutions described in this work, allowing their future clinical use in patients with cartilage defects.

Advances in cell encapsulation technology and its application in drug delivery.

INTRODUCTION: Cell encapsulation technology has improved enormously since it was proposed 50 years ago. The advantages offered over other alternative systems, such as the prevention of repetitive drug administration, have triggered the use of this technology in multiple therapeutic applications. AREAS COVERED: In this article, improvements in cell encapsulation technology and strategies to overcome the drawbacks that prevent its use in the clinic have been summarized and discussed. Different studies and clinical trials that have been performed in several therapeutic applications have also been described. EXPERT OPINION: The authors believe that the future translation of this technology from bench to bedside requires the optimization of diverse aspects: i) biosafety, controlling and monitoring cell viability; ii) biocompatibility, reducing pericapsular fibrotic growth and hypoxia suffered by the graft; iii) control over drug delivery; iv) and the final scale up. On the other hand, an area that deserves more attention is the cryopreservation of encapsulated cells as this will facilitate the arrival of these biosystems to the clinic.

Cryopreservation of microencapsulated murine mesenchymal stem cells genetically engineered to secrete erythropoietin.

The ability to cryopreserve and store for long term the structure and function of therapeutic cells and tissues plays a pivotal role in clinical medicine. In fact, it is an essential pre-requisite for the commercial and clinical application of stem cells since preserves cells at low temperature and creates a reserve for future uses. This requisite may also affect the encapsulated stem cells. Several parameters should be considered on encapsulated cell cryopreservation such as the time and temperature during the cryopreservation process, or the cryoprotectant solutions used. In this study, we have compared the influence of penetrating and nonpenetrating cryoprotectants on the viability and functionality of encapsulated mesenchymal stem cells genetically modified to secrete erythropoeitin. Several cryoprotectant solutions combining DMSO, glycerol and trehalose at different concentrations were studied. Although almost no differences among the studied cryoprotectant solutions were observed on the differentiation potential of encapsulated mesenchymal stem cells, the penetrating cryoprotectant DMSO at a concentration of 10% displayed the best viability and erythropoietin secretion profile compared to the other cryoprotectant solutions. These results were confirmed after subcutaneous implantation of thawed encapsulated mesenchymal stem cells secreting erythropoeitin on Balb/c mice. The hematocrit levels of these animals increased to similar levels of those detected on animals transplanted with noncryopreserved encapsulated cells. Therefore, DMSO 10% represents the most suitable cryoprotectant solution among the solutions here studied, for encapsulated mesenchymal stem cells cryopreservation and its translation into the clinic. Similar studies should be performed for the encapsulation of other cell types before they can be translated into the clinic.