Jesús Ciriza

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.

Assessment of the Behavior of Mesenchymal Stem Cells Immobilized in Biomimetic Alginate Microcapsules.

The combination of mesenchymal stem cells (MSCs) and biomimetic matrices for cell-based therapies has led to enormous advances, including the field of cell microencapsulation technology. In the present work, we have evaluated the potential of genetically modified MSCs from mice bone marrow, D1-MSCs, immobilized in alginate microcapsules with different RGD (Arg-Gly-Asp) densities. Results demonstrated that the microcapsules represent a suitable platform for D1-MSC encapsulation since cell immobilization into alginate matrices does not affect their main characteristics. The in vitro study showed a higher activity of D1-MSCs when they are immobilized in RGD-modified alginate microcapsules, obtaining the highest therapeutic factor secretion with low and intermediate densities of the bioactive molecule. In addition, the inclusion of RGD increased the differentiation potential of immobilized cells upon specific induction. However, subcutaneous implantation did not induce differentiation of D1-MSCs toward any lineage remaining at an undifferentiated state in vivo.

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.

Graphene oxide increases the viability of C2C12 myoblasts microencapsulated in alginate.

Cell microencapsulation represents a great promise for long-term drug delivery, but still several challenges need to be overcome before its translation into the clinic, such as the long term cell survival inside the capsules. On this regard, graphene oxide has shown to promote proliferation of different cell types either in two or three dimensions. Therefore, we planned to combine graphene oxide with the cell microencapsulation technology. We first studied the effect of this material on the stability of the capsules and next we analyzed the biocompatibility of this chemical compound with erythropoietin secreting C2C12 myoblasts within the microcapsule matrix. We produced 160 μm-diameter alginate microcapsules with increasing concentrations of graphene oxide and did not find modifications on the physicochemical parameters of traditional alginate microcapsules. Moreover, we observed that the viability of encapsulated cells within alginate microcapsules containing specific graphene oxide concentrations was enhanced. These results provide a relevant step for the future clinical application of graphene oxide on cell microencapsulation.

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.

Characterization of an encapsulated insulin secreting human pancreatic beta cell line in a modular microfluidic device

Abstract Type I diabetes mellitus is characterised by the destruction of the insulin producing beta cells within the pancreas by the immune system. After the success of Edmonton protocol, islet transplantation has shown to be a promising therapy, but with the Achilles´ heel of the need of using immunosuppressive drugs. Currently, cell encapsulation technology represents a real alternative to protect transplanted islets from the host´s immune attack. Although preliminary in vitro studies with encapsulated cells have been traditionally performed under static conditions in terms of viability and efficiency, these static cultures do not represent a close approach to in vivo environments. We have developed and characterised different alginate-poly-l-lysine-alginate (APA) microcapsules loaded with the insulin producing 1.1B4 cell line. Static in vitro studies confirmed a constant insulin secretion and a boost of the secretion when the medium was enriched with glucose. Nevertheless, these results were not completely reproduced in a dynamic system by APA liquefied microcapsules containing 1.1B4 cells. The dynamic culture setting created by a microfluidic device, allowed the determination of the glucose response in APA liquefied microcapsules, showing that dynamic conditions can mimic better physiological in vivo environments.

Current advanced therapy cell-based medicinal products for type-1-diabetes treatment

&NA; In the XXI century diabetes mellitus has become one of the main threats to human health with higher incidence in regions such as Europe and North America. Type 1 diabetes mellitus (T1DM) occurs as a consequence of the immune‐mediated destruction of insulin producing &bgr;‐cells located in the endocrine part of the pancreas, the islets of Langerhans. The administration of exogenous insulin through daily injections is the most prominent treatment for T1DM but its administration is frequently associated to failure in glucose metabolism control, finally leading to hyperglycemia episodes. Other approaches have been developed in the past decades, such as whole pancreas and islet allotransplantation, but they are restricted to patients who exhibit frequent episodes of hypoglycemia or renal failure because the lack of donors and islet survival. Moreover, patients transplanted with either whole pancreas or islets require of immune suppression to avoid the rejection of the transplant. Currently, advanced therapy medicinal products (ATMP), such as implantable devices, have been developed in order to reduce immune rejection response while increasing cell survival. To overcome these issues, ATMPs must promote vascularization, guaranteeing the nutritional contribution, while providing O2 until vasculature can surround the device. Moreover, it should help in the immune‐protection to avoid acute and chronic rejection. The transplanted cells or islets should be embedded within biomaterials with tunable properties like injectability, stiffness and porosity mimicking natural ECM structural characteristics. And finally, an infinitive cell source that solves the donor scarcity should be found such as insulin producing cells derived from mesenchymal stem cells (MSCs), embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Several companies have registered their ATMPs and future studies envision new prototypes. In this review, we will discuss the mechanisms and etiology of diabetes, comparing the clinical trials in the last decades in order to define the main characteristics for future ATMPs. Graphical abstract Figure. No caption available.

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.

Tunable injectable alginate-based hydrogel for cell therapy in Type 1 Diabetes Mellitus

Islet transplantation has the potential of reestablishing naturally-regulated insulin production in Type 1 diabetic patients. Nevertheless, this procedure is limited due to the low islet survival after transplantation and the lifelong immunosuppression to avoid rejection. Islet embedding within a biocompatible matrix provides mechanical protection and a physical barrier against the immune system thus, increasing islet survival. Alginate is the preferred biomaterial used for embedding insulin-producing cells because of its biocompatibility, low toxicity and ease of gelation. However, alginate gelation is poorly controlled, affecting its physicochemical properties as an injectable biomaterial. Including different concentrations of the phosphate salt Na2HPO4 in alginate hydrogels, we can modulate their gelation time, tuning their physicochemical properties like stiffness and porosity while maintaining an appropriate injectability. Moreover, these hydrogels showed good biocompatibility when embedding a rat insulinoma cell line, especially at low Na2HPO4 concentrations, indicating that these hydrogels have potential as injectable biomaterials for Type 1 Diabetes Mellitus treatment.

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

Abstract 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.