Multi-Scale Investigation of Human Renal Tissue in Three Dimensions.

Histopathology as a diagnostic mainstay for tissue evaluation is strictly a 2D technology. Combining and supplementing this technology with 3D imaging has been proposed as one future avenue towards refining comprehensive tissue analysis. To this end, we have developed a laboratory-based X-ray method allowing for the investigation of tissue samples in three dimensions with isotropic volume information. To assess the potential of our method for micro-morphology evaluation, we selected several kidney regions from three patients with cystic kidney disease, obstructive nephropathy and diabetic glomerulopathy. Tissue specimens were processed using our in-house-developed X-ray eosin stain and investigated with a commercial microCT and our in-house-built NanoCT. The microCT system provided overview scans with voxel sizes of [Formula: see text] and the NanoCT was employed for higher resolutions including voxel sizes from [Formula: see text] to 210 nm. We present a methodology allowing for a precise micro-morphologic investigation in three dimensions which is compatible with conventional histology. Advantages of our methodology are its versatility with respect to multi-scale investigations, being laboratory-based, allowing for non-destructive imaging and providing isotropic volume information. We believe, that after future developmental work this method might contribute to advanced multi-modal tissue diagnostics.

Revealing the Microscopic Structure of Human Renal Cell Carcinoma in Three Dimensions.

For fully characterizing renal cell carcinoma (RCC), information about the 3D tissue microstructure is essential. Histopathology, which represents the current diagnostic gold standard, is destructive and only provides 2D information. 3D X-ray histology endeavors to overcome these limitations by generating 3D data. In a laboratory environment, most techniques struggle with limited resolution and the weak X-ray attenuation contrast of soft tissue. We recently developed a laboratory-based method combining nanoscopic X-ray CT with a cytoplasm-specific X-ray stain. Here, we present the application of this method to human RCC biopsies. The NanoCT slices enable pathological characterization of crucial structures by reproducing tissue morphology with a similar detail level as corresponding histological light microscopy images. Beyond that, our data offer deeper insights into the 3D configuration of the tumor. By demonstrating the compatibility of the X-ray stain with standard pathological stains, we highlight the feasibility of integrating staining based NanoCT into the pathological routine.

A positive immunoselection method to isolate villous cytotrophoblast cells from first trimester and term placenta to high purity.

We developed a method for isolating highly pure villous cytotrophoblast cells from first trimester and term placenta that excludes extravillous trophoblast and syncytiotrophoblast fragments. The method is based on positive immunoselection using an antibody (mAb C76/18) reacting with hepatocyte growth factor activator inhibitor 1, HAI-1, a membrane antigen on villous cytotrophoblast. As a comparison, we also immunopurified cells using an antibody against CD105, present on syncytiotrophoblast and some extravillous trophoblast cells. The isolates were characterized by flow cytometry. HAI-1-positive cells from first trimester and term placentae were highly pure (>98 per cent cytokeratin 7-positive) mononuclear trophoblast cells. These isolations were contaminated with only very small percentages of vimentin and CD45-positive cells. HAI-1-positive trophoblast cells lacked CD105 and also HLA class I, a marker for extravillous trophoblast. In culture HAI-1-positive cells adhered, displayed an epithelial morphology, and survived for more than three days. In contrast, CD105-positive cell fractions from first trimester placenta were a heterogeneous mixture of mononuclear and multinuclear elements consisting of syncytiotrophoblast fragments, extravillous trophoblast cells, as well as around 5 per cent non-trophoblastic contaminants. In conclusion, the positive immunoselection method using antibody C76/18 yielded highly pure villous cytotrophoblast cells devoid of elements derived from syncytiotrophoblast or extravillous trophoblast.