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Compared to cell therapy, where cells are injected into a defect region, the treatment of heart infarction with cells seeded in a vascularized scaffold bears advantages, such as an immediate nutrient supply or a controllable and persistent localization of cells. For this purpose, decellularized native tissues are a preferable choice as they provide an in vivo-like microenvironment. However, the quality of such scaffolds strongly depends on the decellularization process. Therefore, two protocols based on sodium dodecyl sulfate or sodium deoxycholate were tailored and optimized for the decellularization of a porcine heart. The obtained scaffolds were tested for their applicability to generate vascularized cardiac patches. Decellularization with sodium dodecyl sulfate was found to be more suitable and resulted in scaffolds with a low amount of DNA, a highly preserved extracellular matrix composition, and structure shown by GAG quantification and immunohistochemistry. After seeding human endothelial cells into the vasculature, a coagulation assay demonstrated the functionality of the endothelial cells to minimize the clotting of blood. Human-induced pluripotent-stem-cell-derived cardiomyocytes in co-culture with fibroblasts and mesenchymal stem cells transferred the scaffold into a vascularized cardiac patch spontaneously contracting with a frequency of 25.61 ± 5.99 beats/min for over 16 weeks. The customized decellularization protocol based on sodium dodecyl sulfate renders a step towards a preclinical evaluation of the scaffolds.
Diabetes mellitus is an incurable, metabolic disease, which is associated with severe long-term complications. The in vitro generation of pancreatic β-cells from human induced pluripotent stem cells (hiPSCs) represent a promising strategy for a curative therapy of diabetes mellitus. However, current differentiation strategies largely fail to produce functional β-cells in vitro and require an additional in vivo transplantation to achieve terminal maturation. Previous studies demonstrated a beneficial effect of the extracellular matrix (ECM) on the survival and sustained function of adult, isolated islets of Langerhans. This raises the question whether organ-specific cell-ECM interactions might represent the missing link driving the final stage of β-cell development. In order to address this issue, this study investigated the impact of the pancreas ECM on in vitro β-cell differentiation and its use for the establishment of a pancreatic endocrine organ model.
To this purpose, a pancreas-specific ECM scaffolds (PanMa) was derived from porcine pancreata using whole organ decellularization with Sodium Deoxycholate. In a first step, the generated PanMa was thoroughly characterized using (immuno-) histological stainings, scanning electron microscopy and DNA quantification as well as perfusion and recellularization experiments with endothelial cells. Based on these data, a scoring system (PancScore) for a standardized PanMa generation was developed. Next, the generated PanMa was tested for the presence of tissue-specific ECM features. Therefore, the biophysical and physico-structural characteristics, such as rigidity, porosity and hygroscopy were analyzed using rheological measurements, particle diffusion analyses as well as a water evaporation assay and compared to the properties of ECM scaffolds derived from porcine small intestine (SISser) and lung (LungMa) to examine organ-specific scaffold cues. Following the thorough scaffold characterization, the impact of the PanMa on pluripotency and early development of hiPSC was studied. To this purpose, gene and protein expression of hiPSCs during maintenance culture and spontaneous differentiation on the PanMa were assessed. In a next step, the impact of the PanMa on the pancreatic endocrine differentiation of hiPSCs was tested. Therefore, the PanMa was used as a liquid media supplement or as a solid scaffold during the directed differentiation of hiPSC towards either pancreatic hormone-expressing cells (Rezania et al. 2012; Rezania et al. 2014) or maturing β-cells (Rezania et al. 2014). The impact of the PanMa on the generated cells was examined by gene expression analysis, immunohistochemical staining of important stage markers, as well as glucose stimulated insulin secretion assays. In a last part of this study, the potential of the PanMa for the prolonged culture of hiPSC derived endocrine cells for the establishment of an in vitro organ model of the endocrine pancreas was examined. Therefore, a PanMa-derived hydrogel was generated and used for the encapsulation and culture of hiPSC-derived hormone-expressing cells (HECs). The influence of the PanMa-hydrogel culture was analyzed on gene, protein and functional level by gene expression analysis, immunohistochemical stainings and glucose stimulated insulin secretion.
Whole organ decellularization resulted in the generation of an acellular PanMa scaffold, with low amounts of residual DNA and a preserved ECM micro- and ultrastructure, including important ECM components, such as collagen I, III and IV. Furthermore, the PanMa maintained an intact vessel system and was verified as cytocompatible as demonstrated by the successful recellularization of the arterial system with human endothelial cells. In comparison to SISser and LungMa, the PanMa was characterized as a relative soft, hygroscopic scaffold with a collagen-fiber based structure. Furthermore, the findings indicate that the ECM-specific properties have a relevant effect on the stem cell character and early multi-lineage decisions of hiPSCs. In this regard, maintenance of hiPSCs on the PanMa resulted in a slightly changed expression of pluripotency genes (OCT4, SOX2 and NANOG) and a weak immunohistochemical signal for NANOG protein, indicating a PanMa-dependent impact on hiPSC pluripotency. Strikingly, this presumption was corroborated by the finding that culture on the PanMa promoted an endodermal development of hiPSCs during spontaneous differentiation. In line with that, pancreatic differentiation of hiPSC on both the PanMa and SISser resulted in a significant decrease of glucagon and somatostatin gene expression as well as an unaltered insulin expression, suggesting an ECM-driven suppression of the development of non β-cell endocrine cells. However, this change did not result in an improved glucose stimulated insulin secretion of the generated HECs. Moreover, use of the PanMa as a hydrogel allowed prolonged culture of these cells in a defined culture system. HECs were viable after 21 days of culture, however already showed an altered islet morphology as well as a slightly decreased glucose stimulated insulin secretion.
Altogether, this study demonstrates a relevant biological effect of tissue specific ECM cues on the in vitro differentiation of hiPSCs. More specifically, the data indicate an involvement of the ECM in the endocrine commitment of hiPSC-derived pancreatic cells during directed differentiation highlighting the ECM as an important regulator of pancreatic development. Collectively, these findings emphasize the relevance of the ECM for the fabrication of functional hiPSC-derived cell types and suggest a much stronger consideration of organ specific ECM cues for tissue engineering approaches as well as clinical translation in regenerative medicine.
Despite advances in cartilage repair strategies, treatment of focal chondral lesions remains an important challenge to prevent osteoarthritis. Articular cartilage is organized into several layers and lack of zonal organization of current grafts is held responsible for insufficient biomechanical and biochemical quality of repair-tissue. The aim was to develop a zonal approach for cartilage regeneration to determine whether the outcome can be improved compared to a non-zonal strategy. Hydrogel-filled polycaprolactone (PCL)-constructs with a chondrocyte-seeded upper-layer deemed to induce hyaline cartilage and a mesenchymal stromal cell (MSC)-containing bottom-layer deemed to induce calcified cartilage were compared to chondrocyte-based non-zonal grafts in a minipig model. Grafts showed comparable hardness at implantation and did not cause visible signs of inflammation. After 6 months, X-ray microtomography (µCT)-analysis revealed significant bone-loss in both treatment groups compared to empty controls. PCL-enforcement and some hydrogel-remnants were retained in all defects, but most implants were pressed into the subchondral bone. Despite important heterogeneities, both treatments reached a significantly lower modified O’Driscoll-score compared to empty controls. Thus, PCL may have induced bone-erosion during joint loading and misplacement of grafts in vivo precluding adequate permanent orientation of zones compared to surrounding native cartilage.
Tissue-engineered skin equivalents mimic key aspects of the human skin, and can thus be employed as wound coverage for large skin defects or as in vitro test systems as an alternative to animal models. However, current skin equivalents lack a functional vasculature limiting clinical and research applications. This study demonstrates the generation of a vascularized skin equivalent with a perfused vascular network by combining a biological vascularized scaffold (BioVaSc) based on a decellularized segment of a porcine jejunum and a tailored bioreactor system. Briefly, the BioVaSc was seeded with human fibroblasts, keratinocytes, and human microvascular endothelial cells. After 14 days at the air-liquid interface, hematoxylin & eosin and immunohistological staining revealed a specific histological architecture representative of the human dermis and epidermis including a papillary-like architecture at the dermal-epidermal-junction. The formation of the skin barrier was measured non-destructively using impedance spectroscopy. Additionally, endothelial cells lined the walls of the formed vessels that could be perfused with a physiological volume flow. Due to the presence of a complex in-vivo-like vasculature, the here shown skin equivalent has the potential for skin grafting and represents a sophisticated in vitro model for dermatological research.
Advanced Therapy Medicinal Products (ATMP) provide promising treatment options particularly for unmet clinical needs, such as progressive and chronic diseases where currently no satisfying treatment exists. Especially from the ATMP subclass of Tissue Engineered Products (TEPs), only a few have yet been translated from an academic setting to clinic and beyond. A reason for low numbers of TEPs in current clinical trials and one main key hurdle for TEPs is the cost and labor-intensive manufacturing process. Manual production steps require experienced personnel, are challenging to standardize and to scale up. Automated manufacturing has the potential to overcome these challenges, toward an increasing cost-effectiveness. One major obstacle for automation is the control and risk prevention of cross contaminations, especially when handling parallel production lines of different patient material. These critical steps necessitate validated effective and efficient cleaning procedures in an automated system. In this perspective, possible technologies, concepts and solutions to existing ATMP manufacturing hurdles are discussed on the example of a late clinical phase II trial TEP. In compliance to Good Manufacturing Practice (GMP) guidelines, we propose a dual arm robot based isolator approach. Our novel concept enables complete process automation for adherent cell culture, and the translation of all manual process steps with standard laboratory equipment. Moreover, we discuss novel solutions for automated cleaning, without the need for human intervention. Consequently, our automation concept offers the unique chance to scale up production while becoming more cost-effective, which will ultimately increase TEP availability to a broader number of patients.
This thesis concerned the design and examination of a scaffold for tissue engineering applications. The template for the presented scaffold came from nature itself: the intercellular space in tissues that provides structure and support to the cells of the respective tissue, known as extracellular matrix (ECM). Fibres are a predominant characteristic feature of ECM, providing adhesion sites for cell-matrix interactions. In this dissertation a fibrous mesh was generated using the electrospinning technique to mimic the fibrous structure of the ECM. Two base polymers were explored: a biodegradable polyester, poly(D,L-lactide-co-glycolide); and a functional PEG-based star polymer, NCO-sP(EO-stat-PO). This topic was described in three major parts: the first part was materials based, concerning the chemical design and characterisation of the polymer scaffolds; the focus was then shifted to the cellular response to this fibrous scaffold; and finally the in vivo performance of the material was preliminarily assessed. The first steps towards an electrospun mesh started with adjusting the spinning parameters for the generation of homogeneous fibres. As reported in Chapter 3 a suitable setup configuration was on the one hand comprised of a spinning solution that consisted of 28.5 w/v% PLGA RG 504 and 6 w/v% NCO-sP(EO-stat-PO) in 450 µL acetone, 50 µL DMSO and 10 µL of an aqueous trifluoroacetic acid solution. On the other hand an ideal spinning behaviour was achieved at process parameters such as a flow rate of 0.5 mL/h, spinneret to collector distance of 12-16 cm and a voltage of 13 kV. The NCO-sP(EO-stat-PO) containing fibres proved to be highly hydrophilic as the functional additive was present on the fibre surface. Furthermore, the fibres featured a bulk degradation pattern as a consequence of the proportion of PLGA. Besides the morphologic similarity to ECM fibres, the functionality of the electrospun fibres is also decisive for a successful ECM mimicry. In Chapter 4, the passive as well as active functionality of the fibres was investigated. The fibres were required to be protein repellent to prevent an unspecific cell adhesion. This was proven as even 6.5 % sP(EO-stat-PO) in the PLGA fibres reduced any unspecific protein adsorption of bovine serum albumin and foetal calf serum to less than 1 %. However, avidin based proteins attached to the fibres. This adhesion process was avoided by an additional fibre surface treatment with glycidol. The active functionalisation of NCO-sP(EO-stat-PO)/PLGA fibres was investigated with two fluorescent dyes and biocytin. A threefold, chemically orthogonal, fibre modification was achieved with these dyes. The chapters about the chemical and mechanical properties laid the basis for the in vitro chapters where a specific fibre functionalisation with peptides was conducted to analyse the cell adhesion and biochemical expressions. Beginning with fibroblasts in Chapter 5 the focus was on the specific cell adhesion on the electrospun fibres. While NCO-sP(EO-stat-PO)/PLGA fibres without peptides did not allow any adhesion of fibroblasts, a fibre modification with GRGDS (an adhesion mediating peptide sequence) induced the adhesion and spreading of human dermal fibroblasts on the fibrous scaffolds. The control sequence GRGES that has no adhesion mediating qualities did not lead to any cell adhesion as observed on fibres without modifications. While the experiments of Chapter 5 were a proof-of-concept, in Chapter 6 a possible application in cartilage tissue engineering was examined. Therefore, primary human chondrocytes were seeded on fibrous scaffolds with various peptide sequences. Though the chondrocytes exhibited high viability on all scaffolds, an active interaction of cells and fibres was only found for the decorin derived sequence CGKLER. Live-cell-imaging revealed both cell attachment and migration within CGKLER-modified meshes. As chondrocytes undergo a de-differentiation towards a fibroblast-like phenotype, the chondrogenic re-differentiation on these scaffolds was investigated in a long term cell culture experiment of 28 days. Therefore, the glycosaminoglycan production was analysed as well as the mRNA expression of genes coding for collagen I and II, aggrecan and proteoglycan 4. In general only low amounts of the chondrogenic markers were measured, suggesting no chondrogenic differentiation. For conclusive evidence follow-up experiments are required that support or reject the findings. The success of an implant for tissue engineering relies not only on the response of the targeted cell type but also on the immune reaction caused by leukocytes. Hence, Chapter 7 dealt with primary human macrophages and their behaviour and phenotype on two-dimensional (2D) surfaces compared to three-dimensional (3D) fibrous substrates. It was found that the general non-adhesiveness of NCO-sP(EO-stat-PO) surfaces and fibres does not apply to macrophages. The cells aligned along the fibres on surfaces or resided in the pores of the meshes. On flat surfaces without 3D structure the macrophages showed a retarded adhesion kinetic accompanied with a high migratory activity indicating their search for a topographical feature to adhere to. Moreover, a detailed investigation of cell surface markers and chemokine signalling revealed that macrophages on 2D surfaces exhibited surface markers indicating a healing phenotype while the chemokine release suggested a pro-inflammatory phenotype. Interestingly, the opposite situation was found on 3D fibrous substrates with pro-inflammatory surface markers and pro-angiogenic cytokine release. As the immune response largely depends on cellular communication, it was concluded that the NCO-sP(EO-stat-PO)/PLGA fibres induce an adequate immune response with promising prospects to be used in a scaffold for tissue engineering. The final chapter of this thesis reports on a first in vivo study conducted with the presented electrospun fibres. Here, the fibres were combined with a polypropylene mesh for the treatment of diaphragmatic hernias in a rabbit model. Two scaffold series were described that differed in the overall surface morphology: while the fibres of Series A were incorporated into a thick gel of NCO-sP(EO-stat-PO), the scaffolds of Series B featured only a thin hydrogel layer so that the overall fibrous structure could be retained. After four months in vivo the treated defects of the diaphragm were significantly smaller and filled mainly with scar tissue. Thick granulomas occurred on scaffolds of Series A while the implants of Series B did not induce any granuloma formation. As a consequence of the generally positive outcome of this study, the constructs were enhanced with a drug release system in a follow-up project. The incorporated drug was the MMP-inhibitor Ilomastat which is intended to reduce the formation of scar tissue. In conclusion, the simple and straight forward fabrication, the threefold functionalisation possibility and general versatile applicability makes the meshes of NCO-sP(EO-stat-PO)/PLGA fibres a promising candidate to be applied in tissue engineering scaffolds in the future.
Elastic fibers are essential for the proper function of organs including cardiovascular tissues such as heart valves and blood vessels. Although (tropo)elastin production in a tissue-engineered construct has previously been described, the assembly to functional elastic fibers in vitro using human cells has been highly challenging. In the present study, we seeded primary isolated human vascular smooth muscle cells (VSMCs) onto 3D electrospun scaffolds and exposed them to defined laminar shear stress using a customized bioreactor system. Increased elastin expression followed by elastin deposition onto the electrospun scaffolds, as well as on newly formed fibers, was observed after six days. Most interestingly, we identified the successful deposition of elastogenesis-associated proteins, including fibrillin-1 and -2, fibulin-4 and -5, fibronectin, elastin microfibril interface located protein 1 (EMILIN-1) and lysyl oxidase (LOX) within our engineered constructs. Ultrastructural analyses revealed a developing extracellular matrix (ECM) similar to native human fetal tissue, which is composed of collagens, microfibrils and elastin. To conclude, the combination of a novel dynamic flow bioreactor and an electrospun hybrid polymer scaffold allowed the production and assembly of an elastic fiber-containing ECM.
In order to mimic the extracellular matrix for tissue engineering, recent research approaches often involve 3D printing or electrospinning of fibres to scaffolds as cell carrier material. Within this thesis, a micron fibre printing process, called melt electrospinning writing (MEW), combining both additive manufacturing and electrospinning, has been investigated and improved. Thus, a unique device was developed for accurate process control and manufacturing of high quality constructs. Thereby, different studies could be conducted in order to understand the electrohydrodynamic printing behaviour of different medically relevant thermoplastics as well as to characterise the influence of MEW on the resulting scaffold performance.
For reproducible scaffold printing, a commonly occurring processing instability was investigated and defined as pulsing, or in extreme cases as long beading. Here, processing analysis could be performed with the aim to overcome those instabilities and prevent the resulting manufacturing issues. Two different biocompatible polymers were utilised for this study: poly(ε-caprolactone) (PCL) as the only material available for MEW until then and poly(2-ethyl-2-oxazoline) for the first time. A hypothesis including the dependency of pulsing regarding involved mass flows regulated by the feeding pressure and the electrical field strength could be presented. Further, a guide via fibre diameter quantification was established to assess and accomplish high quality printing of scaffolds for subsequent research tasks.
By following a combined approach including small sized spinnerets, small flow rates and high field strengths, PCL fibres with submicron-sized fibre diameters (fØ = 817 ± 165 nm) were deposited to defined scaffolds. The resulting material characteristics could be investigated regarding molecular orientation and morphological aspects. Thereby, an alignment and isotropic crystallinity was observed that can be attributed to the distinct acceleration of the solidifying jet in the electrical field and by the collector uptake. Resulting submicron fibres formed accurate but mechanically sensitive structures requiring further preparation for a suitable use in cell biology. To overcome this handling issue, a coating procedure, by using hydrophilic and cross-linkable star-shaped molecules for preparing fibre adhesive but cell repellent collector surfaces, was used.
Printing PCL fibre patterns below the critical translation speed (CTS) revealed the opportunity to manufacture sinusoidal shaped fibres analogously to those observed using purely viscous fluids falling on a moving belt. No significant influence of the high voltage field during MEW processing could be observed on the buckling phenomenon. A study on the sinusoidal geometry revealed increasing peak-to-peak values and decreasing wavelengths as a function of decreasing collector speeds sc between CTS > sc ≥ 2/3 CTS independent of feeding pressures. Resulting scaffolds printed at 100 %, 90 %, 80 % and 70 % of CTS exhibited significantly different tensile properties, foremost regarding Young’s moduli (E = 42 ± 7 MPa to 173 ± 22 MPa at 1 – 3 % strain). As known from literature, a changed morphology and mechanical environment can impact cell performance substantially leading to a new opportunity of tailoring TE scaffolds.
Further, poly(L-lactide-co-ε-caprolactone-co-acryloyl carbonate) as well as poly(ε-caprolactone-co-acryloyl carbonate) (PCLAC) copolymers could be used for MEW printing. Those exhibit the opportunity for UV-initiated radical cross-linking in a post-processing step leading to significantly increased mechanical characteristics. Here, single fibres of the polymer composed of 90 mol.% CL and 10 mol.% AC showed a considerable maximum tensile strength of σmax = 53 ± 16 MPa. Furthermore, sinusoidal meanders made of PCLAC yielded a specific tensile stress-strain characteristic mimicking the qualitative behaviour of tendons or ligaments. Cell viability by L929 murine fibroblasts and live/dead staining with human mesenchymal stem cells revealed a promising biomaterial behaviour pointing out MEW printed PCLAC scaffolds as promising choice for medical repair of load-bearing soft tissue.
Indeed, one apparent drawback, the small throughput similar to other AM methods, may still prevent MEW’s industrial application yet. However, ongoing research focusses on enlargement of manufacturing speed with the clear perspective of relevant improvement. Thereby, the utilisation of large spinneret sizes may enable printing of high volume rates, while downsizing the resulting fibre diameter via electrical field and mechanical stretching by the collector uptake. Using this approach, limitations of FDM by small nozzle sizes could be overcome. Thinking visionary, such printing devices could be placed in hospitals for patient-specific printing-on-demand therapies one day. Taking the evolved high deposition precision combined with the unique small fibre diameter sizes into account, technical processing of high performance membranes, filters or functional surface finishes also stands to reason.
In this study, well-defined, 3D arrays of air-suspended melt electrowritten fibers are made from medical grade poly(ɛ-caprolactone) (PCL). Low processing temperatures, lower voltages, lower ambient temperature, increased collector distance, and high collector speeds all aid to direct-write suspended fibers that can span gaps of several millimeters between support structures. Such processing parameters are quantitatively determined using a “wedge-design” melt electrowritten test frame to identify the conditions that increase the suspension probability of long-distance fibers. All the measured parameters impact the probability that a fiber is suspended over multimillimeter distances. The height of the suspended fibers can be controlled by a concurrently fabricated fiber wall and the 3D suspended PCL fiber arrays investigated with early post-natal mouse dorsal root ganglion explants. The resulting Schwann cell and neurite outgrowth extends substantial distances by 21 d, following the orientation of the suspended fibers and the supporting walls, often generating circular whorls of high density Schwann cells between the suspended fibers. This research provides a design perspective and the fundamental parametric basis for suspending individual melt electrowritten fibers into a form that facilitates cell culture.
Als Therapie des Diabetes mellitus Typ II stellt die Xenotransplantation von porzinen Langerhans-Inseln in mikroverkapselter Form eine attraktive Alternative zu den täglichen Insulininjektionen dar. Kultivierung und Funktionsdiagnostik der isolierten porzinen Langerhans-Inseln sind technisch anspruchsvoll und bieten noch immer Potential für Verbesserungen. Werden die Zellen in Zellkulturflaschen über mehrere Tage kultiviert, sinkt die Vitalität unter ein akzeptables Niveau. Bei der Beurteilung der Vitalität und Funktion der Inseln gehen wertvolle Zellen für eine spätere Transplantation verloren. Dies schränkt eine ausgiebige Diagnostik vor der Transplantation ein. Ziel der Arbeit ist es, eine Möglichkeit zur Verbesserung der Kulturbedingungen zu finden und exakte Ergebnisse bei der Funktions- und Vitalitätsdiagnostik ohne Verlust von Zellen zu erreichen. Vorversuche konnten beweisen, dass eine Verbesserung der Vitalität von Langerhans-Inseln in Kultur an der Methode der Kultivierung in Zellkulturflaschen scheitert. Stattdessen wurde das Prinzip der Perfusionskultur in spezialisierten Behältnissen für die systematische Verbesserung der Kulturbedingungen und zur genaueren Diagnostik mittels Mikroskopie und Funktionsdiagnostik gewählt. Mit einem solchen System ist sowohl Kultivierung als auch Funktions- und Vitalitätsdiagnostik in einem Behälter möglich. Beim Prinzip der Perfusionskultur befindet sich das Medium stets in Bewegung um die Zellen und Gewebe und sorgt so für einen kontinuierlichen Zustrom exakt definierten Mediums und permanenten Abtransport der Stoffwechselprodukte. Im Rahmen dieser Arbeit wurde für die Anforderungen im eigenen Labor ein maßgeschneidertes System mit mehreren Versionen von Behältnissen für Perfusionskulturen entwickelt, deren jeweils neuere Version auf den Erfahrungen mit den Vorversionen aufbaut. In die Entwicklung fließen ebenso umfangreiche theoretische Überlegungen ein, sowie systematische Tests zu den physikalischen Eigenschaften der Behältnisse. Die zuletzt entwickelte ist die Version V6SMTE, die in der Arbeit „Würzburger Kammer“ genannt wird. Dieser Behälter ist aus Edelstahl, mit einem Deckglas zur makro- und mikroskopischen Begutachtung, Zu- und Abläufen und einem Anschluss zum Entfernen von Gasblasen. Im Inneren befindet sich ein Einsatz, der eine stufenlose Regulierung des Volumens um die Zellen ermöglicht, so dass für Kultur und Funktionsprüfung bzw. Mikroskopie jeweils optimale Bedingungen erreicht werden. Weiterhin kann über einen Temperatursensor die Temperatur im Inneren des Behälters gemessen und über Heizelemente an der Außenwand computergesteuert reguliert werden. Die Zellen und Gewebe können auf unterschiedlichen Trägermaterialien eingesetzt werden. Während der Kultur kann ein Deckel geöffnet und die Zellen manipuliert werden. Das System ist unabhängig vom Brutschrank, ist sterilisierbar und wieder verwendbar. Kultiviert wurde endokrines Gewebe (isolierte Langerhans-Inseln, humanes Nebenschilddrüsengewebe). Dieses wurde zur Funktionsprüfung mit verschiedenen Mediatoren stimuliert, das Medium fraktioniert aufgefangen und sein Hormongehalt mit ELISA oder RIA bestimmt. Die Zellen wurden nativ und mit Fluoreszenzfarbstoffen (FDA, PI) gefärbt mit bis zu 400facher Vergrößerung unter dem Auflichtmikroskop beurteilt. Im Zuge der Auswertung der anfallenden Proben auf ihren Insulingehalt wurde für diese Arbeit ein Insulin-ELISA entwickelt, der bei vergleichbarer Genauigkeit deutlich günstiger ist, als der bisher verwendete kommerzielle ELISA. Mit der Würzburger Kammer kultivierte Langerhans-Inseln zeigten eine vergleichbare Vitalität im Vergleich zur Zellkulturflasche, die mit der Würzburger Kammer gewonnenen Perifusionskurven sind in hohem Maß reproduzierbar, Zusammenhänge von Höhe der Glukoseexposition und Kultivierungsdauer mit der Insulinausschüttungskurve konnten eindrucksvoll beschrieben werden. Erstmals wurde auch im eigenen Labor die aus der Literatur bekannte paradoxe Insulinausschüttung beschrieben. Beispielhaft für andere endokrine Gewebe wurde humanes Nebenschilddrüsengewebe erfolgreich in der Würzburger Kammer kultiviert und Vitalitäts- und Funktionsdiagnostik unterzogen. Das Kultursystem ermöglicht die Kultivierung und eine komplette Analyse von Funktion, Vitalität und Morphologie von endokrinen Zellen. Es kann somit in idealer Weise zur Verbesserung der Kulturbedingungen und zur Beurteilung von endokrinen Zellen vor der Transplantation herangezogen werden.