Heiko Prof. Dr. Zimmermann

A Novel Class of Amitogenic Alginate Microcapsules for Long-Term Immunoisolated Transplantation

Abstract: In the light of results of clinical trials with immunoisolated human parathyroid tissue Ba2+‐alginate capsules were developed that meet the requirements for long‐term immunoisolated transplantation of (allogeneic and xenogeneic) cells and tissue fragments. Biocompatibility of the capsules was achieved by subjecting high‐M alginate extracted from freshly collected brown algae to a simple purification protocol that removes quantitatively mitogenic and cytotoxic impurities without degradation of the alginate polymers. The final ultra‐high‐viscosity, clinical‐grade (UHV/CG) product did not evoke any (significant) foreign body reaction in BB rats or in baboons. Similarly, the very sensitive pERK assay did not reveal any mitogenic impurities. Encapsulated cells also exhibited excellent secretory properties under in vitro conditions. Despite biocompatible material, pericapsular fibrosis is also induced by imperfect capsule surfaces that can favor cell attachment and migration under the release of material traces. This material can interact with free end monomers of the alginate polymers under formation of mitogenic advanced glycation products. Smooth surfaces, and thus topographical biocompatibility of the capsules (visualized by atomic force microscopy), can be generated by appropriate crosslinking of the UHV/CG‐alginate with Ba2+ and simultaneous suppression of capsule swelling by incorporation of proteins and/or perfluorocarbons (i.e., medically approved compounds with high oxygen capacity). Perfluorocarbon‐loaded alginate capsules allow long‐term non‐invasive monitoring of the location and the oxygen supply of the transplants by using 19F‐MRI. Transplantation studies in rats demonstrated that these capsules were functional over a period of more than two years.

Topography of cell traces studied by atomic force microscopy

Abstract Migrating adherent cells release material onto artificial substrates like glass and silicon while moving. Traces of mouse fibroblasts (L929) have been visualised by atomic force microscopy (AFM). “Non-contact” mode AFM in a liquid environment can extract topographic information from these traces. This dynamic mode allows the study of these soft structures without damage or compression. The AFM images show crossing and branching networks (with specific angles of branching), structured patches, nodular elements, linear elements with irregular height and other features. Fourier analysis of segment spacing in the strands is presented. These spatial features of fibroblast traces are strong indications that actin linked to structural proteins is involved in the formation of cell traces. We also give methods for trace preparation and undistorted imaging and discuss further perspectives.

Trace formation during locomotion of L929 mouse fibroblasts continuously recorded by interference reflection microscopy (IRM)

The recently reported formation of highly ordered traces by migrating cells has been studied on L929 fibroblasts in time lapse experiments by means of interference reflection microscopy (IRM) as well as by conventional microscopy. Formation of pronounced traces on glass substrates correlates to migration after cell division, and the trace arrangement on the substrate depends on migration velocity: slow migration results in a highly branched, broad, and relatively short trace, while fast migration yields a slim and long trace with few branches. IRM-irradiation caused cessation of locomotion and trace formation and accelerated degradation of existing traces. Traces consist of cord-like cytoplasmic strands, which contain F-actin filaments and they seem to be enveloped by a membrane. It is supposed that cell traces are homologous to filopodia. Traces arise mainly from non-retracted filopodia at the rear margin of the migrating cell. The branches within the traces are the result of the repeated stretching out of a backwardly directed lamellipodium. They arise from the formation of new filopodia that emerge at the actin ribs of the lamellipodium.

Towards a Full Automation of the ELISpot Assay for Safe and Parallelized Immunomonitoring

The ELISpot assay, as a sensitive and specific method, enables the detection of cytokines for immunological purposes and in vaccine development. Here we describe the successful transfer of the manual procedure to a commercially available automated liquid handling platform, based on the work described in Neubauer et al. (Cytotechnology 69:57-73, 2017). Different kinds of technical issues (dead volume reduction, instrumental handling limitations, liquid class improvement) have been solved and biological effects (reagents concentration, selectivity tests, dispensing way, etc.) have been controlled during the implementation process. At the end a maximum of 6% mean delta difference and a lower mean dispersion than the manual assay were reached as well as a turnaround time of four to six times higher than the manual process.

P16-33. Standardized serum-free cryomedia have minimum cytotoxicity and maintain antigen-specific T-cell response

Background The ability to analyze cryopreserved PBMC for antigen specific T-cells immunity is needed in evaluating response to immune based therapies. Comprehensive studies have demonstrated that the quality of frozen PBMC is critical and maintaining cell viability and functionality by using appropriate cryopreservation techniques is a key to the successful outcome of assays using PBMC. Different cryomedia additives impact the cell viability. The most common additive is FCS although it has been recognized that lots used for this purpose have to be chosen based on careful testing to avoid using serum that may lead to nonspecific stimulation of T-cells. In addition, the most widely used cryopreservation procedure for cells is based on adding DMSO. However, the amount of DMSO added must be reduced significantly due to its toxic impact on cells at room temperature. Therefore, we have developed novel freezing approaches aiming at the use of cryoprotectants having minimum cytotoxicity and maintaining T-cell functionality.

Development and Application of New Storage-Technologies for Effective and Secure Cryobanking

HI virus collections are essential instruments for the development of new vaccines, because endless virus variations consistently raise new, unpredictable problems. In studies performed with HIV patients, blood samples are usually analyzed in laboratories located hundreds of kilometers away from sample origin, because of complicated conditions in the field. Thus, essential aspects for transporting blood samples are efficient cryopreservation without loss of viability or functionality, as well as a safe sample identification. Therefore, the Global HIV Vaccine Research Cryorepository (GHRC) was funded by the Bill&Melinda Gates Foundation. Goal of GHRC is to establish the first large-scale centralized biobank for lowtemperature storage of HIV-1 related specimens, providing standardized access to these important samples. Fraunhofer IBMT, member of GHRC designed a novel cryosubstrate, which is securely attached to an electronic memory chip in combination with a conventional barcode and a low temperature RFID-tag to prevent mismatches [1]. The recovery of peripheral blood mononuclear cells (PBMCs) after cryopreservation in the new cryosubstrates showed, especially for small sample volumes (0.1 ml), an increased viability in comparison to the preservation in standard cryovials. For optimization of cryopreservation procedures, an improvement of sample handling and transport conditions is also important. Therefore, the effects of temperature changes on PBMC viability and lymphocyte lethality were investigated.