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The aim of this work was the combination of ratio imaging and light sheet fluorescence microscopy for examination of the redox state in tumor cell spheroids using the redox sensitive green fluorescent protein Grx1-roGFP2. This sensor mirrors the redox state of glutathione and is characterized by different excitation spectra in the reduced and oxidized state, whereas the emission spectra are identical. In the scope of this work add-on modules for an existing inverted microscope were developed. The SPIM (Single Plane Illumination Microscopy) module mounted to the fixed part of the positioning stage of the microscope allows for simultaneous light sheet illumination of the sample with two different wavelengths. Samples are located in rectangular glass capillaries placed in special capillary holders adapted to the movable part of the positioning stage. The capillaries can be attached to a fluidic system providing the sample with agents during the measurement. The module for ratio imaging is mounted to a side port of the microscope. Its main components are a field aperture, a beam splitter, an image transmitting fiber bundle and a time-gated camera synchronized with two pulsed laser diodes (shifted in time against each other) for excitation. It thus allows for simultaneous recording of two fluorescence images generated by different excitation wavelengths each on one side of the camera’s sensor although the fluorescence emission is in the same spectral range for both excitation wavelengths. Validation was performed by imaging changes of the redox state in tumor cell spheroids induced by hydrogen peroxide or staurosporine-induced apoptosis. Furthermore fluorescence lifetime imaging in single layers of cell spheroids was performed with light sheet illumination, and z-stacks of cell spheroids are presented to demonstrate the possibilities opened up by the SPIM module and corresponding sample holding.

Untreated focal articular cartilage defects may lead to secondary osteoarthritis. Symptomatic full-thickness defects in young patients that are located in the medial or lateral femoral condyle, trochlea, or patella and range in size from 3 cm2 to 10 cm2 as well as defects of the ventral talus are indications for autologous chondrocyte transplantation (ACT). Another indication for ACT are cartilage defects for which previous operative measures have failed. Osteoarthritis and kissing lesions are contraindications. Axial malalignment and knee joint instability need to be corrected. Second-generation ACT procedures are based on three-dimensional biomaterials in which the articular chondrocytes are seeded prior to implantation. Randomized trials demonstrate clinical, radiographic, and histologic findings that are similar to marrow-stimulating techniques such as microfracture. Long-term studies are needed to determine the effect of ACT on the development of osteoarthritis.

Therapy of advanced renal cell carcinoma remains difficult. New therapeutic schemes besides cytokine treatment should be evaluated. The following study analyzes the in vitro toxicity of treosulfan on spheroids of 8 primary cultures of renal cell carcinoma cells. these data were compared to the toxicity of vinblastine. All investigations were performed in regard to the P-glycoprotein (Pgp) expression of the cells, which is one of the main causes of multidrug resistance. Four Pgp positive and four Pgp negative spheroids were incubated with the drugs in increasing doses. Toxicity was measured using the MTT toxicity assay as well as trypan blue exclusion. Significantly higher toxicity of treosulfan compared to vinblastine could be demonstrated. In addition, the effects of treosulfan were not related to Pgp expression. These results are encouraging and a phase II study analyzing the efficacy of treosulfan in patients with advanced renal cell carcinoma has been initiated in our institution.

Calculations on the basis of the LQ-model have been focussed on the possible radiobiological equivalence between common continuous low dose rate irradiation (CLDR) and a superfractionated irradiation (PDR = pulsed dose rate) provided that the same total dose will be prescribed in the same overall time as with the low doserate. A clinically usable fractionation scheme for brachytherapy was recommended by Brenner and Hall and should replace the classical CLDR brachytherapy with line sources with an afterloading technique using a stepping source. The hypothes is that LDR equivalency can be achieved by superfractionation was tested by means of in vitro experiments on V79 cells in monolayer and spheroid cultures as well as on HeLa monolayers.Simulating the clinical situation in PDR brachytherapy, fractionation experiments were carried out in the dose rate gradient of afterloading sources. Different dose levels were produced with the same number of fractions in the same overall incubation time. The fractionation schedules which were to be compared with a CLDR reference curve were: 40 x 0.47 Gy, 20 x 0.94 Gy, 10 x 1.88 Gy, 5 x 3.76 Gy, 2 x 9.4 Gy given in a period of 20 h and 1 x 18.8 Gy as a "single dose" exposition. As measured by flow cytometry, the influence of the dose rate in the pulse on cell survival and on cell cycle distribution under superfractionation was examined on V79 cells.V79 spheroids as a model for a slowly growing tumor, reacted according to the radiobiological calculations, as a CLDR equivalency was achieved with increasing fractionation. Rapidly growing V79 monolayer cells showed an inverse fractionation effect. A superfractionated irradiation with pulses of 0.94 Gy/h respectively 0.47 Gy/0.5 h was significantly more effective than the CLDR irradiation. This inverse fractionation effect in log-phase V79 cells could be attributed to the accumulation of cycling cells in the radiosensitive G2/M phase (G2 block) during protected exposure which was drastically more pronounced for the pulsed scheme. HeLa cells were rather insensitive to changes of fractionation. Superfractionation as well as hypofractionation yielded CLDR equivalent survival curves.The fractionation scheme, derived from the PDR theory to achieve CLDR equivalent effects, is valid for many cell lines, however not for all. Proliferation and dose rate dependend cell cycle effects modify predictions derived from the sublethal damage recovery model and can influence acute irradiation effects significantly. Dose rate sensitivity and rapid proliferation favour cell cycle effects and substantiate, applied to the clinical situation, the possibility of a higher effectiveness of the pulsed irradiation on rapidly growing tumors.

The need to improve therapy regimes, determine prognosis, and study biological properties of tumors extracorporally led to development of different experimental systems. In order to approach the in vivo situation, specific properties of the tumors of origin should be retained by the cells in culture over relatively long periods. However, culture conditions may change expression of cellular antigens.Cryosections of a hypopharyngeal carcinoma were compared in this respect with different cultivation systems (2-dimensional monolayers [ML], 3-dimensional multicellular tumor spheroids [MTS] and substrate cultures on Gelita) in regard to expression of cytokeratins (CK) 1, 7, 10, 14, 18 and 19, vimentin, neurofilament (NF) kD200 and 68, ganglioside GD2, oncogene products (P53 mutant and wild), and membrane-associated antigens (HLA-ABC and -DR, epidermal growth factor receptor EGFR).Semiquantitative immunohistochemical methods revealed differences in expression of CK1, 14 and 19, GD2, and P53 mutant between these systems.Pronounced expression of markers in MTS compared to original biopsy and monolayer emphasizes the importance of cell-cell contact and 3-dimensionality or metabolic stress. However, weak marker expression within substrate cultures may reflect loose cell-cell contact observed. In these experiments, the antigenic configuration of MTS resembled the one of the original tumor more than the other culture systems: monolayer and growth on substrate. As vimentin and NF are not expressed by healthy epithelial cells of adults, occurrence of intratumoral vimentin and NF could point to derepression of early differentiation antigens.