Edorta Santos

Novel advances in the design of three-dimensional bio-scaffolds to control cell fate: translation from 2D to 3D

Recreating the most critical aspects of the native extracellular matrix (ECM) is fundamental to understand and control the processes regulating cell fate and cell function. From the ill-defined complexity to the controlled simplicity, we discuss the different strategies that are being carried out by scientists worldwide to achieve the latest advances in the sophistication of three-dimensional (3D) scaffolds, stressing their impact on cell biology, tissue engineering and regenerative medicine. Synthetic and naturally derived polymers like polyethylene glycol, alginate, agarose, etc., together with micro- and nanofabrication techniques are allowing the creation of 3D models where biophysical and biochemical variables can be modified with high precision, orthogonality and even in real-time.

Application of cell encapsulation for controlled delivery of biological therapeutics.

Cell microencapsulation technology is likely to have an increasingly important role in new approaches rather than the classical and pioneering organ replacement. Apart from becoming a tool for protein and morphogen release and long-term drug delivery, it is becoming a new three-dimensional platform for stem cell research. Recent progress in the field has resulted in biodegradable scaffolds that are able to retain and release the cell content in different anatomical locations. Additional advances include the use biomimetic scaffolds that provide greater control over material-cell interactions and the development of more precise encapsulated cell-tracking systems. This review summarises the state of the art of cell microencapsulation and discusses the main directions and challenges of this field towards the controlled delivery of biological therapeutics.

Biomaterials in Cell Microencapsulation

The field of cell encapsulation is advancing rapidly. This cell-based technology permits the local and long-term delivery ofa desired therapeutic product reducing or even avoiding the need ofimmunosuppressant drugs. The choice of a suitable material preserving the viability and functionality of enclosed cells becomes fundamental if a therapeutic aim is intended. Alginate, which is by far the most frequently used biomaterial in the field of cell microencapsulation, has been demonstrated to be probably the best polymer for this purpose due to its biocompatibility, easy manipulation, gel forming capacity and in vivo performance.

Therapeutic cell encapsulation: Ten steps towards clinical translation

Since the conception of cell microencapsulation, many scientists bet on this biotechnology as they saw in it a promising alternative to protect transplanted cells from host immunoresponse. Some decades later, this initial enthusiasm is giving rise to a phase of certain conformism and lack of novel advances in the field. This perspective critically discusses current challenges needed to help this approach become a realistic clinical proposal. Alginate seems to be well established as the biomaterial of choice, but additional efforts are needed regarding current cross-linkers and coatings. Biofunctionalization of the matrices may provide the necessary biomimetic microenvironment to control cell behavior. Different alginate degradation rates would allow widening the applications of this biotechnology from drug delivery to cell delivery. In this sense, stem cells from stromal tissues could be the most suitable cell source due to their intrinsic hypoimmunogenicity, their immunomodulatory effects and their capacity to cell homing. The incorporation of suicide and reporter genes in the genome of enclosed cells may overcome some of the existing biosafety concerns. Administration and extraction by means of less invasive procedures also need to be developed to succeed in clinical translation. Finally, improving cost-effectiveness for the scale-up, together with establishing and fulfilling a series of strict regulatory aspects will be indispensable to make the final step to the clinic.

Improvement of the monitoring and biosafety of encapsulated cells using the SFGNESTGL triple reporter system.

Cell microencapsulation may represent a breakthrough to overcome problems associated with cell therapy. Advances in material biocompatibility and production protocols have put this field close to its clinical application. However, issues such as the possibility of tracking cell-containing microcapsules, monitoring cell viability, and discontinuation of the therapeutic activity when necessary, still remain unsolved. We demonstrate here simultaneous monitoring and pharmacological control of myoblasts-containing alginate microcapsules, injected in immunocompetent mice after transduction with the SFG(NES)TGL triple reporter retroviral vector, which contains green fluorescence protein (GFP), firefly luciferase and herpes simplex virus type 1 thymidine-kinase (HSV1-TK). Naked (as controls) or microencapsulated cells were subcutaneously injected in C57BL/6J mice and followed up by luminometry. Signal for naked cells disappeared 2 weeks after cell injection, whereas signal for microencapsulated cells remained strong for 8 months, thus demonstrating the presence of living cells. Treatment of mice with the thymidine-kinase substrate ganciclovir caused death of microencapsulated myoblasts, as seen by a drastic decay in the light emission and histological analysis. Hence, we conclude that incorporation of the SFG(NES)TGL vector into microencapsulated cells represents an accurate tool for controlling cell location and viability in a non-invasive way. Moreover, cell death can be induced by administration of ganciclovir, in case therapy needs to be interrupted. This system may represent a step forward in the control and biosafety of cell- and gene- therapy-based microencapsulation protocols.

Optimization of 100 μm alginate-poly-L-lysine-alginate capsules for intravitreous administration.

The field of cell microencapsulation is advancing rapidly. Particle size plays a critical role in terms of biocompatibility and limits decisively its applicability. Producing reduced size microcapsules involves broadening the possibilities to employ this technology in the treatment of many disorders. Nervous system diseases (NSD) represent a clear example of that. This work describes the feasibility of reducing the size of alginate-poly-L-lysine-alginate (APA) microcapsules up to 100 μm in a highly monodisperse way using the novel Flow Focusing technique. C(2)C(12) myoblasts genetically engineered to express the triple reporter gene thymidine kinase-green fluorescent protein-luciferase (TGL) and secrete vascular endothelial growth factor soluble receptor 2 (VEGFR2, also known as KDR) were encapsulated for further characterization. Resulting new particles were assayed in vitro to explore whether their functionality might be affected due to the physicochemical changes arising from such dramatic size reduction. Not only were negative effects at this level not noticed in terms of cell viability, cell proliferation and KDR secretion, but once again the suitability of APA microcapsules was also reinforced against other microcapsule designs. Furthermore, the fully viable and functional biosystems were successfully administered in the intravitreous space of rats, where the activity of encapsulated cells was monitoring over 3 weeks.

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.

Inactivation of encapsulated cells and their therapeutic effects by means of TGL triple-fusion reporter/biosafety gene.

The immobilization of cells within alginate-poly-l-lysine-alginate (APA) microcapsules has been demonstrated to be an effective technology design for long term delivery of therapeutic products. Despite promising advances, biosafety aspects still remain to be improved. Here, we describe a complete characterization of the strategy based on TGL triple-fusion reporter gene--which codifies for Herpes Simplex virus type 1 thymidine-kinase (HSV1-TK), green fluorescent protein (GFP) and Firefly Luciferase--(SFG(NES)TGL) to inactivate encapsulated cells and their therapeutic effects. Myoblasts genetically engineered to secrete erythropoietin (EPO) were retroviraly transduced with the SFG(NES)TGL plasmid to further characterize their ganciclovir (GCV)-mediated inactivation process. GCV sensitivity of encapsulated cells was 100-fold lower when compared to cells plated onto 2D surfaces. However, the number of cells per capsule and EPO secretion decayed to less than 15% at the same time that proliferation was arrested after 14 days of GCV treatment in vitro. In vivo, ten days of GCV treatment was enough to restore the increased hematocrit levels of mice implanted with encapsulated TGL-expressing and EPO-secreting cells. Altogether, these results show that TGL triple-fusion reporter gene may be a good starting point in the search of a suitable biosafety strategy to inactivate encapsulated cells and control their therapeutic effects.

Behaviour and ultrastructure of human bone marrow-derived mesenchymal stem cells immobilised in alginate-poly-l-lysine-alginate microcapsules

Abstract Context: Human bone marrow mesenchymal stem cells (hBM-MSCs) show a great promise for the treatment of a variety of diseases. Despite the previous trials to encapsulate hBM-MSCs in alginate-poly-l-lysine-alginate (APA) systems, the various changes that follow immobilisation have not been ascertained yet. Objective: Determine the various consequences derived from entrapment on cell behaviour, putting special emphasis on the ultrastructure. Methods: hBM-MSCs were immobilised in APA microcapsules to further characterise their viability, metabolic activity, proliferation, VEGF-secretability, and morphology. Results: The VEGF produced by monolayer hBM-MSCs increased significantly 1 d post-encapsulation, and was maintained for at least 4  weeks. TEM imaging of cells revealed well preserved ultrastructure indicating protein synthesis and high metabolic activity. Conclusion: Although APA microencapsulation did not support 100% of fully viable hBM-MSCs for long-term cultures, it was conceived to enhance both VEGF secretion and metabolic activity while not losing their stemness characteristics.

The synergistic effects of the RGD density and the microenvironment on the behavior of encapsulated cells: in vitro and in vivo direct comparative study.

The inclusion of the tripeptide RGD (Arg-Gly-Asp) in otherwise inert biomaterials employed for cell encapsulation has been observed to be an effective strategy to provide the immobilized cells with a more suitable microenvironment. However, some controversial results collected during the last years, especially in vivo, have questioned its effectiveness. Here, we have studied the behavior of C2 C12 myoblasts immobilized in alginate-poly-l-lysine-alginate microcapsules with different densities of RGD. The use of these microcapsules offer the advantage of avoiding native proteins influence permitting to establish direct comparisons between in vitro and in vivo assays. The results suggest that RGD-modified matrices provide higher dynamism, achieving therapeutically more active biosystems not only in vitro, but also in vivo. The highest functionality of the immobilized cells in vitro was obtained with the lowest RGD density. However, higher RGD densities were required in vivo to obtain the same effects observed in vitro. Altogether, these results suggest the lack of in vitro-in vivo correlation when cell behavior is evaluated within different RGD-tailored cell-loaded scaffolds.