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 used cells are either autologous or allogenic, whereas the latter enables, especially by using of stem cell lines, a production of cell therapeutic or tissue engineered implants in stock. Therefore, the usually small initial cell number has to be increased. For that purpose bioreactors are demanded, which offer the controlled expansion of the hMSC under GMP-conform conditions. In this study, divided in 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 a comfortable automation of the inoculation, cultivation, and harvesting procedures. Furthermore the bioreactor owns a simple design which benefits the manufacturing as disposable. 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. It could be shown 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

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Cell-based Regeneration of Intervertebral Disc Defects: Review & Concepts

During the last century low back pain has been emerged as a widespread disease often caused by intervertebral disc degeneration (IDD). IDD in turn is a complex problem, in which a variety of causes play a crucial role. As IDD causes high costs, a corporate interest leads to a number of therapies developed. Today, these therapies focus on the restoration of the IVD function and not only on minimizing the pain caused by this disease. These approaches are often biological and aim to stimulate the regeneration of the intervertebral disc by injection of activator proteins, biomaterials, different cell types or complex cell-matrix-composites. Furthermore, the genetic engineering of disc cells and the in vitro tissue engineering offer a possibility for curing IDD. This article gives an overview on the concepts mentioned above

Use of encapsulated stem cells to overcome the bottleneck of cell availability for cell therapy approaches

Nowadays cell-based therapy is rarely in clinical practice because of the availability of appropriate cells. To apply cells therapeutically, they may not cause any immune response wherefore up to now mainly autologous cells were used. The amount of vital cells in patients is limited and under certain circumstances in highly degenerated tissues no vital cells are left. Moreover, the extraction of these cells is connected with additional surgery; also the expansion in vitro is difficult. Other approaches avoid these problems by using allo- or even xenogenic cells. These cells are more stable concerning their therapeutic behavior and can be produced in stock. To prevent an immune response caused by these cells, cell encapsulation (e.g. with alginate) can be performed. Certain studies showed that encapsulated allo- and xenogenic cells achieve promising results in treatment of several diseases. At this, stem cells, especially mesenchymal stem cells, are an interesting cell source for cell therapy approaches. This review deals on the one hand with cell therapy of encapsulated cells with focus on the use of stem cells and on the other hand with bioreactor systems for the expansion and differentiation of mesenchymal stem cells in reproducible and sufficient amounts for potential clinical use