Cell encapsulation: technical and clinical advances.

Treating many chronic diseases will require a tight, minute-to-minute regulation of therapeutic molecules that is currently not achievable with most pharmaceutical therapies. For these diseases, implantable living cellular systems may be able to provide unlimited drug delivery, enabling seamless matching of treatment duration with disease longevity. Cell encapsulation is an advanced technology that achieves this goal and represents a viable therapeutic option. The advanced state of the field has allowed researchers to inch forward into therapeutic domains previously untouchable because of the myriad disparate fields that intersect biomaterials and cells. Here, we discuss the next generation of clinical trials and potential approaches, 'smart' and responsive encapsulation systems, sophisticated and multifunctional devices, and novel imaging tools, together with the future challenges in the field.

Evaluation of different RGD ligand densities in the development of cell-based drug delivery systems.

BACKGROUND: The inclusion of the tripeptide Arg-Gly-Asp (RGD) 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. PURPOSE: The objective of this study was to determine the impact of different RGD densities on the behavior of baby hamster kidney (BHK) fibroblasts able to secrete vascular endothelial growth factor (VEGF) encapsulated in alginate microcapsules. METHODS: Alginate was modified by varying the concentration of RGD peptides in the coupling reaction. After obtaining four different types of alginate, cells were encapsulated in alginate-poly-L-lysine-alginate (APA) microcapsules. RESULTS AND DISCUSSION: The results obtained after viability, cell proliferation and VEGF secretion assays showed that the inclusion of RGD in alginate enhances the functionality of immobilized cells, obtaining the highest values with the intermediate RGD density. CONCLUSION: These results put in evidence that alginate modification influences the behavior of immobilized cells but even more that the employed density of the tripeptide is of crucial importance, obtaining in some cases even excessive activity of the encapsulated cells.

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.

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

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.

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.

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.

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.

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.

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.