Hermann Dertinger

Microstructured scaffolds for liver tissue cultures of high cell density: Morphological and biochemical characterization of tissue aggregates

Very high cell densities and optimal vascularization characterize among others organs and tissues in vivo. In order to study organ‐specific functions in vitro or to make use of them in medical devices/treatments in the future, this natural architecture should be rebuilt. An important aspect in this context is the appropriate ratio of medium to cell volume being so far not optimally reestablished in most of the currently available in vitro systems. To improve such culture conditions, we constructed a microstructure to culture hepatocytes and (without any addition of extracellular matrix material) characterized liver tissue in the form of evenly sized aggregates. The liver‐specific differentiation status of such aggregates was monitored by their ability to perform CYP450 dependent xenobiotic metabolism along with the measurement of albumin secretion. Freshly isolated adult rat hepatocytes show an initial loss of total CYP450 content and of associated activities (mixed function oxidases). However, in the aggregate system, this level did not decrease further but remained stable or even increased throughout the culture period of 10–13 days. The CYP450 dependent metabolism of the hepatocytes is able to respond to classic inducing agents. The described culture efficiently supports liver‐specific functions of adult rat hepatocytes and seems to be suited not only for use in an extracorporeal liver device but also for the formation of evenly sized small aggregates to be of use in transplantation of differentiated liver tissue. Moreover, after design variations, the microstructure can be applied for functional analysis of metabolically active hepatocytes as well as for toxicological and pharmacological validation.

A Comparative Study of Post-irradiation Growth Kinetics of Spheroids and Monolayers

Post-irradiation growth kinetics of gamma-irradiated spheroid and monolayer cells in exponential growth phase was investigated by means of dose-response curves based on cell counts after specified time intervals following irradiation. A mathematical model of cell-growth after irradiation was fitted to these curves. The model parameters (related to division delay and growth of non-surviving cellsy obtained from this analysis consistently indicate increasing resistance to sub-lethal damage of cells cultured as multicellular spheroids under conditions of increasing three-dimensional contact. In contrast, no indication of an increased radiation-resistance was found with cells cultured on a substratum under a variety of conditions.

Energy Requirements for Damaging DNA Molecules

SummaryStrand break formation and biological inactivation of infectious DNA of bacteriophage ΦX 174 exposed to vacuum-ultra-violet radiation of 4·9 to 21·2 eV quantum energy is investigated. At 21·2 eV as many as 84 per cent of the DNA molecules are inactivated by breaks whereas breaks do not contribute to inactivation at 4·9 eV. The quantum yield of break formation increases from 1·7 × 10−5 (4·9 eV) to 0·55 (21·2 eV) and shows a dependence on energy similar to that of electron emission (due to ionization) above 8 eV. The mechanisms leading to break formation and inactivation are discussed taking the absorption spectrum of DNA in the vacuum-ultra-violet region into consideration.

A computer-controlled E.P.R. analysis of free radical formation in dry thymine and derivatives after electron irradiation.

SummaryRadical formation and transformation in electron irradiated dry thymine, thymidine, thymidine-5′-monophosphate, and DNA have been studied with 35 GHz electron paramagnetic resonance. Dry samples were irradiated at 77°K and the E.P.R. spectra taken at 77 and 300°K under anaerobic conditions. The spectra of the cation and the anion radical of thymine were identified. It was shown that the cation easily undergoes deprotonization. Conversely, the anion radical is transformed into the 5,6-dihydro-5-thymyl radical by proton capture. These processes were found to be significant for all the compounds investigated.

The DNA Content of Some Mammalian Cells Measured by Flow Cytometry and its Influence on Radiation Sensitivity

The DNA content of nine mammalian cell lines was determined by flow cytometry. Using radiobiological data from this and other laboratories a correlation between DNA mass and 1/D0 for X-rays, alpha-particles, and heavy ions could be established when the quantities were plotted on a log-log scale. The slopes of the regression lines amounted to 0.65 (X-rays), 0.64 (alpha-particles) and 0.74 (heavy ions). A similar correlation was found between DNA content and mean inactivation dose. The rather uniform slopes close to 2/3 suggest that radiosensitivity may depend on the surface area of the sensitive target, (cell nucleus) indicating a possible non-uniform distribution of radiosensitive sites within the nucleus.

Negative pion irradiation of mammalian cells. 1. Survival characteristics of monolayers and spheroids of Chinese hamster lung cells.

Monolayers and spheroids of Chinese hamster cells (V79) were subjected to negative pion irradiation under aerobic conditions. R.b.e. values in the pion peak of 1-8 and 1-5 were obtained for monolayers and spheroids, respectively, whereas the r.b.e. for the plateau was found to be slightly higher than 1. In addition, it was observed that the higher resistance of the V79 spheroid cells than the monolayers to gamma-irradiation is not diminished in the pion peak, suggesting that the underlying phenomenon of intercellular communication influences cell survival even after high-LET irradiation.

Kinetic response of an in vitro “tumour-model” (V 79 spheroids) to 42°C hyperthermia

Abstract The proliferative changes in multicullular spheroids of Chinese hamster lung cells were investigated under conditions of hyperthermia which lead in combination with radiation treatment to a TER (thermal enhancement ratio) of 1·63 By fractionated trypsinization single cell suspensions from successive shells of spheroids were prepared. Cells from the outer region (cycling cells) were found to be partially synchronized by heat and killed in S-phase whereas cells from the center of spheroids (non-cycling cells) became actively proliferating after heat treatment.

Mechanisms of Radical Formation in Irradiated Purine Bases and Derivatives: An E.P.R. Study Using Computer Technique

SummaryThe formation and transformation of radicals induced in dry guanine and adenine and their deoxyribonucleosides and nucleotides by electron irradiation have been studied with Q-band E.P.R. at 77 and 300°K. Two basic mechanisms have been derived: (i) decay of the purine ion radicals with release of atomic hydrogen and (ii) reaction of the hydrogen atoms with the base moieties to yield the adducts at the positions C2 and C8, respectively.

Formation of hydroxyl radicals in thymine solution by excited hydrogen or argon from a gas discharge.

SummaryThe effects of hydrogen gas excited in a radiofrequency discharge on oxygen-free solutions of thymine have been followed chemically. Two main reaction products have been identified: 5,6-dihydrothymine and 5,6-thymine glycol. The latter product was found to be the only significant species if discharge-excited argon was pumped through the solutions instead of hydrogen. Although dihydrothymine clearly is the reaction product of atomic hydrogen generated in the hydrogen discharge, the glycol requires the formation of OH radicals in the solution. A mechanism is proposed by which this species is produced through (super) excitation and decay of water molecules by energy transfer from the excited gas.

Changes of Intercellular Communication Induced by Alternating Electric Fields Correlate with Changes of cAMP

The role of the cellular membrane as the main site for the interaction with low frequency electric fields has been confirmed by experimental evidence over the last years. Signal transduction processes, mediated by second messengers such as cyclic AMP or Ca, have been shown to be influenced by the interaction of the field with components of the cell membrane e.g. receptor molecules or ion channels1–3.