Martin Oberringer

The liquid overlay technique is the key to formation of co-culture spheroids consisting of primary osteoblasts, fibroblasts and endothelial cells.

BACKGROUND AIMS The 3-dimensional (3-D) culture of various cell types reflects the in vivo situation more precisely than 2-dimensional (2-D) cell culture techniques. Spheroids as 3-D cell constructs have been used in tumor research for a long time. They have also been used to study angiogenic mechanisms, which are essential for the success of many tissue-engineering approaches. Several methods of forming spheroids are known, but there is a lack of systematic studies evaluating the performance of these techniques. METHODS We evaluated the performance of the hanging drop technique, carboxymethyl cellulose technique and liquid overlay technique to form both mono- and co-culture spheroids consisting of primary osteoblasts, fibroblasts and endothelial cells. The performance of the three techniques was evaluated in terms of rate of yield and reproducibility. The size of the generated spheroids was determined systematically. RESULTS The liquid overlay technique was the most suitable for generating spheroids reproducibly. The rate of yield for this technique was between 60% and 100% for monoculture spheroids and 100% for co-culture spheroids. The size of the spheroids could be adjusted easily and precisely by varying the number of seeded cells organized in one spheroid. The formation of co-culture spheroids consisting of three different cell types was possible. CONCLUSIONS Our results show that the most suitable technique for forming spheroids can vary from the chosen cell type, especially if primary cells are used. Co-culture spheroids consisting of three different cell types will be used to study angiogenic phenomena in further studies.

A new in vitro wound model based on the co-culture of human dermal microvascular endothelial cells and human dermal fibroblasts.

Background information. Different in vitro models, based on co‐culturing techniques, can be used to investigate the behaviour of cell types, which are relevant for human wound and soft‐tissue healing. Currently, no model exists to describe the behaviour of fibroblasts and microvascular endothelial cells under wound‐specific conditions. In order to develop a suitable in vitro model, we characterized co‐cultures comprising NHDFs (normal human dermal fibroblasts) and HDMECs (human dermal microvascular endothelial cells). The CCSWMA (co‐culture scratch wound migration assay) developed was supported by direct visualization techniques in order to investigate a broad spectrum of cellular parameters, such as migration and proliferation activity, the differentiation of NHDFs into MFs (myofibroblasts) and the expression of endothelin‐1 and ED‐A‐fibronectin (extra domain A fibronectin). The cellular response to hypoxia treatment, as one of the crucial conditions in wound healing, was monitored.

Reduced myofibroblast differentiation on femtosecond laser treated 316LS stainless steel

In-stent restenosis is a common complication after stent surgery which leads to a dangerous wall narrowing of a blood vessel. Laser assisted patterning is one of the effective methods to modify the stent surface to control cell-surface interactions which play a major role in the restenosis. In this current study, 316 LS stainless steel substrates are structured by focusing a femtosecond laser beam down to a spot size of 50 μm. By altering the laser induced spot density three distinct surfaces (low density (LD), medium density (MD) and high density (HD)) were prepared. While such surfaces are composed of primary microstructures, due to fast melting and re-solidification by ultra-short laser pulses, nanofeatures are also observed as secondary structures. Following a detailed surface characterization (chemical and physical properties of the surface), we used a well-established co-culture assay of human microvascular endothelial cells and human fibroblasts to check the cell compatibility of the prepared surfaces. The surfaces were analyzed in terms of cell adherence, proliferation, cell morphology and the differentiation of the fibroblast into the myofibroblast, which is a process indicating a general fibrotic shift within a certain tissue. It is observed that myofibroblast proliferation decreases significantly on laser treated samples in comparison to non-treated ones. On the other hand endothelial cell proliferation is not affected by the surface topography which is composed of micro- and nanostructures. Such surfaces may be used to modify stent surfaces for prevention or at least reduction of restenosis.

Analysis of cellular composition of co-culture spheroids.

3D spheroids and in particular co-culture spheroids reflect the natural organization of cells in tissues much better than 2D cell cultures as indicated by differences in cellular phyisology. However, most methods to analyze cells were established for 2D cultures and cannot easily be applied to spheroids. This study has aimed to demonstrate the possibility of quantification of the cellular composition of co-culture spheroids without previous dissociation into single cells. Prior to the generation of the spheroids, human endothelial cells, osteoblasts and fibroblasts were stained with fluoresent dyes for living cells. Co-culture spheroids of defined stoichiometric compositions were generated by the liquid overlay technique, cultivated for one, three or six days, respectively, and afterwards snap-frozen in liquid nitrogen. Cryo-sections of co-culture spheroids were analyzed by fluorescence microscopy and a newly established semi-automatic measuring routine. In order to compare the results, spheroids of one group were dissociated and the cellular composition was quantified by FACS-analysis. Staining efficiencies were higher than 95% as quantified in preliminary experiments with 2D cultures. Depending on the staining procedure, variations from uniform to punctate signals were detected. The size of all co-culture spheroids decreased over time and snap-freezing did not lead to shrinkage of the spheroids. We were able to detect organizational patterns of different cell types within the spheroids. It was possible to determine the cellular composition by quantitative microscopic analyses of cryo-sections as it could be confirmed by flow cytometric analyses. Depending on the experimental requirements, a combination of both methods might lead to valuable synergy.

Two- and three-dimensional co-culture models of soft tissue healing: pericyte-endothelial cell interaction

The demographic change in western countries towards an older population is being shadowed by an increased appearance of chronic diseases influencing soft tissue healing in a negative manner. Although various promising therapeutic approaches are available for treating chronic wounds, no in vitro model exists that successfully allows the analysis of interacting cells and of the effect of therapeutic drugs within a wound. Granulation tissue assures wound stability, neo-angiogenesis and revascularization finally leading to functional soft tissue repair. As one of the first steps in developing a model for human granulation tissue, we examined microvascular endothelial cells and pericytes in conventional 2D and in 3D spheroid co-cultures. We determined which parameters could be used in a standardized manner and whether the cultures were responsive to hypoxia and to erythropoietin supplementation. The read-out parameters of cell migration, cell density, rate of apoptotic cells, spatial cell distribution in the spheroid and spheroid volume were shown to be excellent analytic measures. In addition, quantification of hypoxia-related genes identified a total of 13 genes that were up-regulated in spheroids after hypoxia. As these parameters delivered reliable results in the present approach and as the general morphological distribution of pericytes and endothelial cells within the spheroid occurred in a typical manner, we believe that this basic in vitro model will serve for the future study of diverse aspects of soft tissue healing.

Expansion and differentiation of human primary osteoblasts in two- and three-dimensional culture.

Abstract Despite the regenerative capability of bone, treatment of large defects often requires bone grafts. The challenge for bone grafting is to establish rapid and sufficient vascularization. Three-dimensional (3D) multicellular spheroids consisting of the relevant cell types can be used as “mini tissues” to study the complexity of angiogenesis. We investigated two-dimensional (2D) expansion, differentiation and characterization of primary osteoblasts as steps toward the establishment of 3D multicellular spheroids. Supplementation of cell culture medium with vitamin D3 induces the osteocalcin expression of osteoblasts. An increased osteocalcin concentration of 10.8 ± 0.58 ng/ml could be measured after 19 days in supplemented medium. Vitamin D3 has no influence on the expression of alkaline phosphatase or the deposition of calcium. Expression of these additional osteogenic markers requires addition of a cocktail of osteogenic factors that, conversely, have no influence on the expression of osteocalcin. Supplementation of the cell culture medium with both vitamin D3 and a cocktail of osteogenic factors is recommended to produce an osteoblast phenotype that secretes osteocalcin, expresses alkaline phosphatase and deposits calcium. In such a supplemented medium, a mean osteocalcin concentration of 11.63 ± 4.85 ng/ml was secreted by the osteoblasts. Distinguishing osteoblasts and fibroblasts remains a challenge. Neither differentiated nor undifferentiated osteoblasts can be distinguished from fibroblasts by the expression of CD90, ED-A-fibronectin or α-smooth muscle actin; however, these cell types exhibit clear differences in their growth characteristics. Osteoblasts can be arranged as 3D spheroids by coating the bottom of the cell culture device with agarose. The cellular composition of 3D multicellular spheroids can be evaluated quantitatively using vital fluorescence labeling techniques. Spheroids are a promising tool for studying angiogenic and osteogenic phenomena in vivo and in vitro.

Reoxygenation Attenuates the Adhesion of Neutrophils to Microvascular Endothelial Cells

In humans, the pathophysiological inflammation response subsequent to hypoxia and reoxygenation often leads to systemic inflammation and multiorgan failure. We applied a newly developed static interaction model using human polymorphonuclear neutrophils and microvasular endothelial cells to clarify the role of hypoxia and hypoxia/reoxygenation in vitro. Human dermal microvasular endothelial cell cultures (n = 7) were exposed to hypoxia and different reoxygenation periods and the adherence rate of neutrophils to the endothelial cells as well as to the protein matrix on the culture slide surface were determined by quantitative microscopy. Hypoxia clearly triggered neutrophil adhesion to human dermal microvasular endothelial cells whereas additional reoxygenation significantly decreased neutrophil adhesion. These in vitro findings suggest that systemic inflammation caused by increased neutrophil adherence to the microvascular endothelium is already initiated by hypoxia rather than by subsequent reoxygenation.

Dissociation of mono- and co-culture spheroids into single cells for subsequent flow cytometric analysis

BACKGROUND Spheroids are considered to reflect the natural organization of cells better than 2D cell cultures, but their analysis by flow cytometry requires dissociation into single cells. METHODS We established protocols for dissociation of mono- and co-culture spheroids consisting of human fibroblasts and human endothelial cells. Cell recovery rate and viability after dissociation were evaluated with hemocytometer and by flow cytometry. The diameter of cells and the amount of cell aggregates were quantified by Casy®-technology and the cellular composition was analyzed by flow cytometry. RESULTS Optimal dissociation conditions with low cell aggregation were determined by size, cultivation time and cellular composition of the spheroids. Smaller spheroids (10,000 cells) could be dissociated with Accutase®, whereas larger spheroids (50,000 cells) required more stringent dissociation conditions. The size of the cells decreased with increasing cultivation time. Cell recovery rate was dependent upon cellular composition and spheroid size. The highest cell recovery rate was found for co-culture spheroids. The highest cell viability was detected for dissociated fibroblast spheroids. A quantitative analysis of the cellular composition of dissociated co-culture spheroids was possible. DISCUSSION Spheroids can be successfully dissociated into singular cells for subsequent flow cytometric analysis. Dissociation conditions as well as cell recovery rate and cell viability depend on size, cultivation time and cellular composition of the spheroids. The observed decrease in cell size in spheroids over time might be responsible for the well-known time-dependent decrease in spheroid size.

The role of adipose-derived stem cells in a self-organizing 3D model with regard to human soft tissue healing

The clinical phenomenon of inadequate soft tissue healing still remains an important issue. The occurrence of chronic wounds is correlated to the life span, which is still increasing in western countries. Tissue engineering products containing adipose-derived stem cells are discussed as a promising therapeutic approach. Several studies confirmed the value of these cells for soft tissue healing improvement, suggesting a paracrine as well as a direct effect on vessel repair and angiogenesis. In an attempt to figure out specific effects of adipose-derived stem cells on dermal microvascular endothelial cells with respect to the different phases of soft tissue healing, we designed a 3D in vitro model on the basis of spheroids. Basic parameters like spheroid volume, cell numbers, and rate of apoptotic cells were determined in dependence on culture time, on different oxygen conditions and using mono- as well as co-cultures of both cell types. Furthermore we focused on gene expression and protein levels of interleukin-6, interleukin-8, monocyte chemoattractant protein-1, and vascular endothelial growth factor, which are discussed against the background of therapies for chronic wounds. The visualization of α-smooth muscle actin allowed the estimation of the function of adipose-derived stem cells as stabilizer for dermal microvascular endothelial cells. The results of the present 3D model underscore a paracrine effect of adipose-derived stem cells on microvessel repair during early hypoxic conditions, whereas a stabilizing effect occurs during a later phase of soft tissue healing, simultaneously to reoxygenation.