Cell-based delivery of glucagon-like peptide-1 using encapsulated mesenchymal stem cells.

Glucagon-like peptide-1 (GLP-1) CellBeads are cell-based implants for the sustained local delivery of bioactive factors. They consist of GLP-1 secreting mesenchymal stem cells encapsulated in a spherically shaped immuno-isolating alginate matrix. A highly standardized and reproducible encapsulation method is described for the manufacturing of homogeneous CellBeads. Viability and sustained secretion was shown for the recombinant GLP-1 and the cell endogenous bioactive factors like vascular endothelial growth factor, neurotrophin 3 (NT-3) and glial cell line-derived neurotrophic factor. Manufacturing and quality control is performed in compliance with good manufacturing practice and fulfils all regulatory requirements for human clinical use. GLP-1 CellBeads combine the neuro- and cardioprotective properties of both GLP-1 and mesenchymal stem cells. First promising results were obtained from preclinical studies and an ongoing safety trial in humans but further studies have to prove the overall potential of CellBead technology in cell-based regenerative medicine.

Evaluation of biocompatibility and anti-glioma efficacy of doxorubicin and irinotecan drug-eluting bead suspensions in alginate.

INTRODUCTION: Chemotherapeutic drug-eluting beads (DEBs) are microspheres that are in clinical use for intraarterial chemoembolisation of liver cancer. Here we report on the biocompatibility and anti-tumour efficacy of DEBs after intratumoral application in a rat BT4Ca glioma model. METHODS AND RESULTS: Doxorubicin and irinotecan-eluting DEBs were suspended in a Ca(2+)-free aqueous alginate solution that provides a sol-gel transition when injected into the Ca(2+) rich brain tissue. In this way the DEBs are immobilised at the implantation site. Forced elution studies in vitro using a USP-4 flow-through apparatus demonstrated that the alginate excipient helped to reduce the burst effect and rate the elution from the beads. From the in vivo evaluation, doxorubicin DEBs demonstrated a significant local toxicity, while irinotecan-loaded DEBs showed good local tissue compatibility. Doxorubicin at higher concentrations and irinotecan-loaded DEBs were found to decrease tumour volume, increase survival time and decrease the Ki67 proliferation index of the tumour. Doxorubicin was shown by fluorescent microscopy to diffuse into the peritumoral tissue, but also penetrates along white matter tracts, to more distant areas. DISCUSSION: We conclude that the alginate suspension of irinotecan DEBs can be considered safe and effective in a clinical setting.

Alginate encapsulated human mesenchymal stem cells suppress syngeneic glioma growth in the immunocompetent rat.

Human mesenchymal stem cells (MSC) are promising candidates for cell therapy of neurological diseases. However, co-transplantation of MSC with tumour cell lines has been reported to promote tumour growth. In this study, we co-transplant glioma cells together with alginate-encapsulated MSC. Immunocompetent BD-IX rats were inoculated with syngeneic BT4Ca glioma cells. Encapsulated unmodified MSC, endostatin producing (endoMSC) or cell-free alginate capsules were stereotactically implanted into the tumour bed. After 12 days, tumour volumes were significantly diminished in the MSC-treated group. The decrease in tumour volume found with endoMSC was statistically not significant, despite significantly reduced tumour vascularization. We conclude that, under syngeneic conditions in the immunocompetent animal, (1) the intracranial, orthotopic co-transplantation of MSC with glioma cells leads to a suppression in tumour growth and (2) the tumour can escape the antiangiogenic treatment with endostatin. Our finding may facilitate the clinical translation of encapsulated cell therapy.

Therapeutic concentrations of glucagon-like peptide-1 in cerebrospinal fluid following cell-based delivery into the cerebral ventricles of cats.

BACKGROUND: Neuropeptides may have considerable potential in the treatment of acute and chronic neurological diseases. Encapsulated genetically engineered cells have been suggested as a means for sustained local delivery of such peptides to the brain. In our experiments, we studied human mesenchymal stem cells which were transfected to produce glucagon-like peptide-1 (GLP-1). METHODS: Cells were packed in a water-permeable mesh bag containing 400 polymeric microcapsules, each containing 3000 cells. The mesh bags were either transplanted into the subdural space, into the brain parenchyma or into the cerebral ventricles of the cat brain. Mesh bags were explanted after two weeks, and cell viability, as well as GLP-1 concentration in the cerebrospinal fluid (CSF), was measured. RESULTS: Viability of cells did not significantly differ between the three implantation sites. However, CSF concentration of GLP-1 was significantly elevated only after ventricular transplantation with a maximum concentration of 73 pM (binding constant = 70 pM). CONCLUSIONS: This study showed that ventricular cell-based delivery of soluble factors has the capability to achieve concentrations in the CSF which may become pharmacologically active. Despite the controversy about the pharmacokinetic limitations of ventricular drug delivery, there might be a niche in this for encapsulated cell biodelivery of soluble, highly biologically-effective neuropeptides of low molecular weight like GLP-1.

Encapsulated native and glucagon-like peptide-1 transfected human mesenchymal stem cells in a transgenic mouse model of Alzheimer's disease.

Encapsulated human mesenchymal stem cells(MSC) are studied in a double transgenic mouse model of Alzheimer's disease (AD) after intraventricular implantation at 3 months of age. Abeta 40/42 deposition, and glial (GFAP) and microglial (CD11b) immunoreactivity were investigated 2 months after transplantation of either native MSC or MSC transfected with glucagon-like peptide-1 (GLP-1). CD11b immunostaining in the frontal lobes was significantly decreased in the GLP-1 MSC group compared to the untreated controls. Also, the plaque associated GFAP immunoreactivity was only observed in one of four animals in the GLP-1 MSC group. Abeta 40 whole brain ELISA was decreased in the MSC group: 86.06±5.2 pg/ml (untreated control) vs. 78.67±11.2 pg/ml (GLP-1 MSC group) vs.70.9±11.1 pg/ml (MSC group, p<0.05). Intraventricular transplantation of native and GLP-1 transfected MSC has been shown effective. Decreased amyloid deposition or suppression of glial and microglial responses were observed. However, encapsulation of MSC may alter their biological activity.

Expansion of human mesenchymal stem cells in a fixed-bed bioreactor system based on non-porous glass carrier - Part B: Modeling and scale-up of the system.

Human mesenchymal stem cells (hMSC) are a promising cell source for the manufacturing of cell therapy or tissue-engineered implants. In part A of this publication a fixed-bed bioreactor system based on non-porous borosilicate glass spheres and procedures for the automated expansion of hMSC with high yield and vitality was introduced. Part B of this study deals with the modeling of the process in order to transfer the bioreactor system from the laboratory to the production scale. Relevant model parameters were obtained by fitting them to the experimental data of hMSC-TERT cultivations in scales up to 300 cm3. Scale-up calculations were carried out exemplarily for a target cell number of twenty billion cells.

Expansion of human mesenchymal stem cells in a fixed-bed bioreactor system based on non-porous glass carrier--part A: inoculation, cultivation, and cell harvest procedures.

Human mesenchymal stem cells (hMSC) are a promising cell source for several applications of regenerative medicine. The cells employed are either autologous or allogenic; by using stem cell lines in particular, allogenic cells enable the production of therapeutic cell implants or tissue engineered implants in stock. For these purposes, the generally small initial cell number has to be increased; this requires the use of bioreactors, which offer controlled expansion of the hMSC under GMP-conform conditions. In this study, divided into part A and B, a fixed bed bioreactor system based on non-porous borosilicate glass spheres for the expansion of hMSC, demonstrated with the model cell line hMSC-TERT, is introduced. The system offers convenient automation of the inoculation, cultivation, and harvesting procedures. Furthermore, the bioreactor has a simple design which favors its manufacturing as a disposable unit. Part A is focused on the inoculation, cultivation, and harvesting procedures. Cultivations were performed in lab scales up to a bed volume of 300 cm³. The study showed that the fixed bed system, based on 2-mm borosilicate glass spheres, as well as the inoculation, cultivation, and harvesting procedures are suitable for the expansion of hMSC with high yield and vitality.

Expansion and Harvesting of hMSC-TERT.

The expansion of human mesenchymal stem cells as suspension culture by means of spinner flasks and microcarriers, compared to the cultivation in tissue culture flasks, offers the advantage of reducing the requirements of large incubator capacities as well as reducing the handling effort during cultivation and harvesting. Nonporous microcarriers are preferable when the cells need to be kept in viable condition for further applications like tissue engineering or cell therapy. In this study, the qualification of Biosilon, Cytodex 1, Cytodex 3, RapidCell and P102-L for expansion of hMSC-TERT with an associated harvesting process using either trypsin, accutase, collagenase or a trypsin-accutase mixture was investigated. A subsequent adipogenic differentiation of harvested hMSC-TERT was performed in order to observe possible negative effects on their (adipogenic) differentiation potential as a result of the cultivation and harvesting method. The cultivated cells showed an average growth rate of 0.52 d(-1). The cells cultivated on Biosilon, RapidCell and P102-L were harvested succesfully achieving high cell yield and vitalities near 100%. This was not the case for cells on Cytodex 1 and Cytodex 3. The trypsin-accutase mix was most effective. After spinner expansion and harvesting the cells were successfully differentiated to adipocytes.

Cultivation and Differentiation of Encapsulated hMSC-TERT in a Disposable Small-Scale Syringe-Like Fixed Bed Reactor.

The use of commercially available plastic syringes is introduced as disposable small-scale fixed bed bioreactors for the cultivation of implantable therapeutic cell systems on the basis of an alginate-encapsulated human mesenchymal stem cell line. The system introduced is fitted with a noninvasive oxygen sensor for the continuous monitoring of the cultivation process. Fixed bed bioreactors offer advantages in comparison to other systems due to their ease of automation and online monitoring capability during the cultivation process. These benefits combined with the advantage of single-use make the fixed bed reactor an interesting option for GMP processes. The cultivation of the encapsulated cells in the fixed bed bioreactor system offered vitalities and adipogenic differentiation similar to well-mixed suspension cultures.