Simple method of thawing cryo-stored samples preserves ultrastructural features in electron microscopy.

Preservation of ultrastructural features in biological samples for electron microscopy (EM) is a challenging task that is routinely accomplished through chemical fixation or high-pressure freezing coupled to automated freeze substitution (AFS) using specialized devices. However, samples from clinical (e.g. "biobanking" of bulk biopsies) and preclinical (e.g. whole mouse tissues) specimens are often not specifically prepared for ultrastructural analyses but simply immersed in liquid nitrogen before long-term cryo-storage. We demonstrate that ultrastructural features of such samples are insufficiently conserved using AFS and developed a simple, rapid, and effective method for thawing that does not require specific instrumentation. This procedure consists of dry ice-cooled pre-trimming of frozen tissue and aldehyde fixation for 3 h at 37 °C followed by standard embedding steps. Herein investigated tissues comprised human term placentae, clinical lung samples, as well as mouse tissues of different composition (brown adipose tissue, white adipose tissue, cardiac muscle, skeletal muscle, liver). For all these tissues, we compared electron micrographs prepared from cryo-stored material with our method to images derived from directly prepared fresh tissues with standard chemical fixation. Our protocol yielded highly conserved ultrastructural features and tissue-specific details, largely matching the quality of fresh tissue samples. Furthermore, morphometric analysis of lipid droplets and mitochondria in livers of fasted mice demonstrated that statistically valid quantifications can be derived from samples prepared with our method. Overall, we provide a simple and effective protocol for accurate ultrastructural and morphometric analyses of cryo-stored bulk tissue samples.

Testicular endothelial cells promote self-renewal of spermatogonial stem cells in rats†.

Spermatogonial stem cells (SSCs) are the basis of spermatogenesis in male due to their capability to multiply in numbers by self-renewal and subsequent meiotic processes. However, as SSCs are present in a very small proportion in the testis, in vitro proliferation of undifferentiated SSCs will facilitate the study of germ cell biology. In this study, we investigated the effectiveness of various cell lines as a feeder layer for rat SSCs. Germ cells enriched for SSCs were cultured on feeder layers including SIM mouse embryo-derived thioguanine and ouabain-resistant cells, C166 cells, and mouse and rat testicular endothelial cells (TECs) and their stem cell potential for generating donor-derived colonies and offspring was assessed by transplantation into recipient testes. Rat germ cells cultured on TECs showed increased mRNA and protein levels of undifferentiated spermatogonial markers. Rat SSCs derived from these germ cells underwent spermatogenesis and generated offspring when transplanted into recipients. Collectively, TECs can serve as an effective feeder layer that enhances the proliferative and self-renewal capacity of cultured rat SSCs while preserving their stemness properties.

Liver p53 is stabilized upon starvation and required for amino acid catabolism and gluconeogenesis.

The ability to adapt cellular metabolism to nutrient availability is critical for survival. The liver plays a central role in the adaptation to starvation by switching from glucose-consuming processes and lipid synthesis to providing energy substrates like glucose to the organism. Here we report a previously unrecognized role of the tumor suppressor p53 in the physiologic adaptation to food withdrawal. We found that starvation robustly increases p53 protein in mouse liver. This induction was posttranscriptional and mediated by a hepatocyte-autonomous and AMP-activated protein kinase-dependent mechanism. p53 stabilization was required for the adaptive expression of genes involved in amino acid catabolism. Indeed, acute deletion of p53 in livers of adult mice impaired hepatic glycogen storage and induced steatosis. Upon food withdrawal, p53-deleted mice became hypoglycemic and showed defects in the starvation-associated utilization of hepatic amino acids. In summary, we provide novel evidence for a p53-dependent integration of acute changes of cellular energy status and the metabolic adaptation to starvation. Because of its tumor suppressor function, p53 stabilization by starvation could have implications for both metabolic and oncological diseases of the liver.-Prokesch, A., Graef, F. A., Madl, T., Kahlhofer, J., Heidenreich, S., Schumann, A., Moyschewitz, E., Pristoynik, P., Blaschitz, A., Knauer, M., Muenzner, M., Bogner-Strauss, J. G., Dohr, G., Schulz, T. J., Schupp, M. Liver p53 is stabilized upon starvation and required for amino acid catabolism and gluconeogenesis.

Human mesenchymal progenitor cells derived from alveolar bone and human bone marrow stromal cells: a comparative study.

The aim of the present study was to evaluate the potential of intraoral harvested alveolar bone as an alternative source of multipotent mesenchymal stromal cells for future applications in oral and maxillofacial tissue engineering. Explant cultures were established from 20 alveolar bone samples harvested from the oblique line immediately before wisdom tooth removal. Morphology and proliferation characteristics of the in vitro expanded cells, referred to as human alveolar bone-derived cells (hABDCs), were studied using phase-contrast microscopy. Immunocytochemical analysis of their surface marker expression was conducted using monoclonal antibodies defining mesenchymal stromal cells. To evaluate their multilineage differentiation potential, hABDCs were induced to differentiate along the osteogenic, adipogenic, and chondrogenic lineage and compared to bone marrow mesenchymal stromal cells (hBMSCs) on mRNA and protein levels applying RT-PCR and cytochemical staining methods. hABDCs showed typical morphological characteristics comparable to those of hBMSCs such as being mononuclear, fibroblast-like, spindle-shaped, and plastic adherent. Immunophenotypically, cells were positive for CD105, CD90, and CD73 while negative for CD45, CD34, CD14, CD79α, and HLA-DR surface molecules, indicating an antigen expression pattern considered typical for multipotent mesenchymal stromal cells. As evidenced by RT-PCR and cytochemistry, hABDCs showed multilineage differentiation and similar chondrogenic and osteogenic differentiation potentials when compared to hBMSCs. Our findings demonstrate that human alveolar bone contains mesenchymal progenitor cells that can be isolated and expanded in vitro and are capable of trilineage differentiation, providing a reservoir of multipotent mesenchymal cells from an easily accessible tissue source.

Evaluation of graft cell viability-efficacy of piezoelectric versus manual bone scraper technique.

PURPOSE: The aim of the present study was to compare the influence of 2 different bone scrapers with respect to graft quality. MATERIALS AND METHODS: The study was conducted as a prospective, controlled experimental study of patients selected from the outpatient unit of the Department of Oral Surgery and Radiology (Dental Clinic, Medical University, Graz, Austria). Bone samples were obtained during routine lower third molar removal. Both a manual bone scraper (MS) and a piezoelectric device (PD) were used in directly adjacent regions in each case. As variables, the chip morphology, cell viability, and osteogenic differentiation were investigated. For statistical analysis, the Student t test and Fisher's exact test (P < .05) were applied. RESULTS: A total of 20 patients (12 women and 8 men, mean age 28.15 ± 5.8 years) were included in the study. A series of 40 bone samples was obtained during lower third molar removal. MS and PD enabled similar intraoral harvest of bone chips. In vitro outgrowth of adherent cells was found in 90% of the MS and 80% of the PD samples after 7 to 18 days, without statistical significance (P = .67). Similar cell viability of outgrowing cells in both groups was observed (94.7% ± 2.2% in the MS group and 94.1% ± 1.6% in the PD group). Reverse transcriptase-polymerase chain reaction analysis and the staining pattern verified osteopotent cells in both groups. CONCLUSIONS: Both manual and piezoelectric techniques are adequate harvesting technologies for limited intraoral augmentations. Our results did not show an advantage for the piezoelectric device.

Behaviour of human physeal chondro-progenitorcells in early growth plate injury response in vitro.

PURPOSE: The aim of this study was to investigate the proliferation and differentiation behaviour of a defined cell population gained from the human growth plate, namely, chondro-progenitorcells (CPCs), in the initial inflammatory phase of growth plate injury response in vitro. METHODS: Growth plate cells were sorted via FACS and differentiated along adipogenic and osteogenic lineage to confirm their progenitor features. To mimic the inflammatory phase of injury response at the growth plate they were treated with IL-1ß and exposed to cyclic mechanical loading. A BrdU assay was used to investigate CPC proliferation. CPC differentiation behaviour was analysed by RT-PCR. RESULTS: CPCs (CD45-, CD34-, CD73+, CD90+, and CD105+) showed a successful differentiation along adipogenic and osteogenic lineage. Under conditions simulating the inflammatory phase of injury response at the growth plate in vitro CPCs differentiated towards hypertrophy while chondrogenesis and ossification were inhibited. Proliferation was not significantly altered. CONCLUSION: This study showed that CPCs can be isolated from the human growth plate and expanded in vitro. In the first phase of injury response at the growth plate these cells differentiate towards hypertrophy. As longitudinal growth is obtained by chondrocyte proliferation and volume increase during hypertrophy this maturation might be the first step towards post-traumatic growth disorders such as unwanted premature ossification of the growth plate.

HLA class I expression in the human placenta.

Placental trophoblast cells of the semi-allogenic human conceptus invade deeply into maternal uterine tissue. From a classical immunoiogic point of view this invasion and the following growth and development of the fetus in the uterus have to be tolerated by a pregnant woman's immune system. Among the various possible protective mechanisms that may be involved, the unique expression pattern of HLA class I molecules seems to be relevant. Besides many other differences between placentation and organ transplantation, this extraordinary HLA class I expression on trophoblast explains why pregnancy should not be considered an immunologic paradox but rather a fascinating example of a very special challenge for the female immune system.

Combined molecular genetic and cytogenetic analysis from single cells after isothermal whole-genome amplification.

BACKGROUND: Analysis of chromosomal aberrations or single-gene disorders from rare fetal cells circulating in the blood of pregnant women requires verification of the cells' genomic identity. We have developed a method enabling multiple analyses at the single-cell level that combines verification of the genomic identity of microchimeric cells with molecular genetic and cytogenetic diagnosis. METHODS: We used a model system of peripheral blood mononuclear cells spiked with a colon adenocarcinoma cell line and immunofluorescence staining for cytokeratin in combination with DNA staining with the nuclear dye TO-PRO-3 in a preliminary study to define candidate microchimeric (tumor) cells in Cytospin preparations. After laser microdissection, we performed low-volume on-chip isothermal whole-genome amplification (iWGA) of single and pooled cells. RESULTS: DNA fingerprint analysis of iWGA aliquots permitted successful identification of all analyzed candidate microchimeric cell preparations (6 samples of pooled cells, 7 samples of single cells). Sequencing of 3 single-nucleotide polymorphisms was successful at the single-cell level for 20 of 32 allelic loci. Metaphase comparative genomic hybridization (mCGH) with iWGA products of single cells showed the gains and losses known to be present in the genomic DNA of the target cells. CONCLUSIONS: This method may be instrumental in cell-based noninvasive prenatal diagnosis. Furthermore, the possibility to perform mCGH with amplified DNA from single cells offers a perspective for the analysis of nonmicrochimeric rare cells exhibiting genomic alterations, such as circulating tumor cells.

Maternal platelets pass interstices of trophoblast columns and are not activated by HLA-G in early human pregnancy.

In early human gestation, maternal arterial blood flow into the intervillous space of the developing placenta is obstructed by invaded trophoblasts, which form cellular plugs in uterine spiral arteries. These trophoblast plugs have recently been described to be loosely cohesive with clear capillary-sized channels into the intervillous space by 7 weeks of gestation. Here, we analysed localisation of maternal platelets at the maternal-foetal interface of human first trimester pregnancy, and tested the hypothesis whether HLA-G, which is primarily expressed by extravillous trophoblasts, affects aggregation and adhesion of isolated platelets. Immunohistochemistry of first trimester placental sections localised maternal platelets in vessel-like channels and adjacent intercellular gaps of extravillous trophoblasts in distal parts of columns. Furthermore, this localisation was confirmed by transmission electron microscopy. Neither co-incubation of HLA-G overexpressing JAR cells with isolated platelets, nor incubation with cell-derived soluble HLA-G or recombinant HLA-G affected platelet adhesion and aggregation. Our study suggests that maternal platelets flow through vessel-like channels of distal trophoblast columns and spread into adjacent lateral intercellular gaps, where platelet-derived factors could contribute to trophoblast differentiation into the invasive phenotype.

Automatic retrieval of single microchimeric cells and verification of identity by on-chip multiplex PCR.

The analysis of rare cells is not an easy task. This is especially true when cells representing a fetal microchimerism are to be utilized for the purpose of non-invasive prenatal diagnosis because it is both imperative and difficult to avoid contaminating the minority of fetal cells with maternal ones. Under these conditions, even highly specific biochemical markers are not perfectly reliable. We have developed a method to verify the genomic identity of rare cells that combines automatic screening for enriched target cells (based on immunofluorescence labelling) with isolation of single candidate microchimeric cells (by laser microdissection and subsequent laser catapulting) and low-volume on-chip multiplex PCR for DNA fingerprint analysis. The power of the method was tested using samples containing mixed cells of related and non-related individuals. Single-cell DNA fingerprinting was successful in 74% of the cells analysed (55/74), with a PCR efficiency of 59.2% (860/1452) for heterozygous loci. The identification of cells by means of DNA profiling was achieved in 100% (12/12) of non-related cells in artificial mixtures and in 86% (37/43) of cells sharing a haploid set of chromosomes and was performed on cells enriched from blood and cells isolated from tissue. We suggest DNA profiling as a standard for the identification of microchimerism on a single-cell basis.

Human fetal placental endothelial cells have a mature arterial and a juvenile venous phenotype with adipogenic and osteogenic differentiation potential.

Growing interest in the sources of origin of blood vessel related diseases has led to an increasing knowledge about the heterogeneity and plasticity of endothelial cells lining arteries and veins. So far, most of these studies were performed on animal models. Here, we hypothesized that the plasticity of human fetal endothelial cells depends on their vascular bed of origin i.e. vein or artery and further that the differences between arterial and venous endothelial cells would extend to phenotype and genotype. We established a method for the isolation of fetal arterial and venous endothelial cells from the human placenta and studied the characteristics of both cell types. Human placental arterial endothelial cells (HPAEC) and human placental venous endothelial cells (HPVEC) express classical endothelial markers and differ in their phenotypic, genotypic, and functional characteristics: HPAEC are polygonal cells with a smooth surface growing in loose arrangements and forming monolayers with classical endothelial cobblestone morphology. They express artery-related genes (hey-2, connexin 40, depp) and more endothelial-associated genes than HPVEC. Functional testing demonstrated that vascular endothelial growth factors (VEGFs) induce a higher proliferative response on HPAEC, whereas placental growth factors (PlGFs) are only effective on HPVEC. HPVEC are spindle-shaped cells with numerous microvilli at their surface. They grow closely apposed to each other, form fibroblastoid swirling patterns at confluence and have shorter generation and population doubling times than HPAEC. HPVEC overexpress development-associated genes (gremlin, mesenchyme homeobox 2, stem cell protein DSC54) and show an enhanced differentiation potential into adipocytes and osteoblasts in contrast to HPAEC. These data provide collective evidence for a juvenile venous and a more mature arterial phenotype of human fetal endothelial cells. The high plasticity of the fetal venous endothelial cells may reflect their role as tissue-resident endothelial progenitors during embryonic development with a possible benefit for regenerative cell therapy.

Amnion-derived mesenchymal stem cells improve viability of endothelial cells exposed to shear stress in ePTFE grafts.

PURPOSE:: Blood vessel reconstruction is an increasing need of patients suffering from cardiovascular diseases. For the development of microvascular prostheses, efficient endothelialization is mandatory to prevent graft occlusion. Here, we assessed the impact of amnion-derived mesenchymal stem/stromal cells (hAMSC), known for their important angiogenic potential, on the integrity and stability of endothelial cells exposed to shear stress in vascular grafts. METHODS:: Human placental endothelial cells (hPEC) were cultured at the inner surface of an expanded polytetrafluoroethylene (ePTFE) graft positioned within a bioreactor and exposed to a minimal shear stress of 0.015 dyne/cm2 or a physiological shear stress of 0.92 dyne/cm2. hAMSC attached to the outer graft surface were able to interact with human placental endothelial cells by paracrine factors. RESULTS:: Microscopical analysis and evaluation of glucose/lactate metabolism evidenced successful cell seeding of the graft: hPEC formed a stable monolayer, hAMSC showed a continuous growth during 72 h incubation. hAMSC improved the viability of hPEC exposed to 0.015 dyne/cm2 as shown by a decreased lactate dehydrogenase release of 13% after 72 h compared to hPEC single culture. The viability-enhancing effect of hAMSC on hPEC was further improved by 13% under physiological shear stress. Angiogenesis array analysis revealed that hPEC exposed to physiological shear stress and hAMSC co-culture reduced the secretion of angiogenin, GRO, MCP-1, and TIMP-2. CONCLUSION:: hAMSC exerted best survival-enhancing effects on hPEC under exposure to physiological shear stress and modulated endothelial function by paracrine factors. Our data support further studies on the development of grafts functionalized with hAMSC-derived secretomes to enable fast clinical application.

Dendritic polyglycerol nanoparticles show charge dependent bio-distribution in early human placental explants and reduce hCG secretion.

A thorough understanding of nanoparticle bio-distribution at the feto-maternal interface will be a prerequisite for their diagnostic or therapeutic application in women of childbearing age and for teratologic risk assessment. Therefore, the tissue interaction of biocompatible dendritic polyglycerol nanoparticles (dPG-NPs) with first- trimester human placental explants were analyzed and compared to less sophisticated trophoblast-cell based models. First-trimester human placental explants, BeWo cells and primary trophoblast cells from human term placenta were exposed to fluorescence labeled, ∼5 nm dPG-NPs, with differently charged surfaces, at concentrations of 1 µM and 10 nM, for 6 and 24 h. Accumulation of dPGs was visualized by fluorescence microscopy. To assess the impact of dPG-NP on trophoblast integrity and endocrine function, LDH, and hCG releases were measured. A dose- and charge-dependent accumulation of dPG-NPs was observed at the early placental barrier and in cell lines, with positive dPG-NP-surface causing deposits even in the mesenchymal core of the placental villi. No signs of plasma membrane damage could be detected. After 24 h we observed a significant reduction of hCG secretion in placental explants, without significant changes in trophoblast apoptosis, at low concentrations of charged dPG-NPs. In conclusion, dPG-NP's surface charge substantially influences their bio-distribution at the feto-maternal interface, with positive charge facilitating trans-trophoblast passage, and in contrast to more artificial models, the first-trimester placental explant culture model reveals potentially hazardous influences of charged dPG-NPs on early placental physiology.

A novel sandwich ELISA for alpha1 domain based detection of soluble HLA-G heavy chains.

The detection of soluble human leukocyte antigen G (HLA-G) has been a technically demanding task for several years now and various enzyme linked immunosorbent assay (ELISA) formats have been designed. However, no ELISA test has been described so far which is able to detect all possible kinds of soluble HLA-G (sHLA-G) molecules that might occur in bio fluids. Here we describe a new ELISA approach able to recognize soluble alpha1 domain containing heavy chains of all HLA-G isoforms. The detection limit is shown to be at about 150 pg soluble recombinant HLA-G1 heavy chain per milliliters. Detectable HLA-G fragments are shown to occur in the supernatants of different HLA-G transfected cell lines and appear to be particularly abundant in supernatant of trophoblast derived choriocarcinoma cell lines. The novel ELISA employs the well characterized HLA-G mAbs 4H84 and MEM-G1 which ensure high HLA-G specificity. A negative control ELISA format, designed against non-existing analytes, has been established to reveal non-specific signal interference.

Heterogeneity of microvascular endothelial cells isolated from human term placenta and macrovascular umbilical vein endothelial cells.

The present study compares some phenotypic and physiologic characteristics of microvascular and macrovascular endothelial cells from within one human organ. To this end microvascular endothelial cells from human full-term placenta (PLEC) were isolated using a new method and compared with macrovascular human umbilical vein endothelial cells (HUVEC) and an SV40-transformed placental venous endothelial cell line (HPEC-A2). PLEC were isolated by enzymatic perfusion of small placental vessels, purified on a density gradient and cultured subsequently. Histological sections of the enzyme-treated vessels showed a selective removal of the endothelial lining in the perfused placental cotyledons. The endothelial identity of the cells was confirmed by staining with the endothelial markers anti-von Willebrand factor, Ulex europaeus lectin and anti-QBEND10. The cells internalized acetylated low-density lipoprotein and did not show immunoreactivity with markers for macrophages, smooth muscle cells and fibroblasts. The spindle-shaped PLEC grew in swirling patterns similar to that described for venous placental endothelial cells. However, scanning electron microscopic examination clearly showed that PLEC remained elongated at the confluent state, in contrast to the more polygonal phenotype of HPEC-A2 and HUVEC that were studied in parallel. The amount of vasoactive substances (endothelin-1,2, thromboxane, angiotensin II, prostacyclin) released into the culture medium and the proliferative response to cytokines was more similar to human dermal microvessels (MIEC) derived from non-fetal tissue than to HUVEC. Potent mitogens such as vascular endothelial growth factors (VEGF121, VEGF165) and basic fibroblast growth factor (FGF-2) induced proliferation of all endothelial cell types. Placental growth factors PIGF-1 and PIGF-2 effectively stimulated cell proliferation on PLEC (142 +/- 7% and 173 +/- 10%) and MIEC (160 +/- 20% and 143 +/- 28%) in contrast to HUVEC (9 +/- 8% and 15 +/- 20%) and HPEC-A2 (15 +/- 7% and 24 +/- 6%) after 48 h incubation time under serum-free conditions. These data support evidence for (1) the microvascular identity of the isolated PLEC described in this study, and (2) the phenotypic and physiologic heterogeneity of micro- and macrovascular endothelial cells within one human organ.

Detection and relocation of rare events. A comparative study using the laser scanning cytometer and the Metafer/RCDetect microscope scanning system.

We compared instrumental analysis of enriched cord blood nucleated red blood cells (CB-NRBC) out of in vitro contamination preparations of dilutions of minute volumes of male cord blood into peripheral blood from nonpregnant women. This was done using the laser scanning cytometer (LSC) and the Metafer/RCDetect microscope scanning system, both allowing for relocation of positive cells defined on the basis of fluorescence parameters. Both instruments were efficient in performing scanning and relocation; a difference in the recovery of CB-NRBC was not significant and can be explained by the method of preparation used.

Expression of indoleamine 2,3-dioxygenase in carcinoma of human endometrium and uterine cervix.

Expression of indoleamine 2,3-dioxygenase (IDO) in epithelium of the endometrium and the cervix is not restricted to normal but also present in carcinomatous tissue. The enzyme was found in the majority of cases studied, pioneer cells at the invasion front of the tumors being especially strongly reactive in immunohistology. In addition, also cells in the peritumoral infiltrate of the stroma expressed IDO. Taken together, these findings together with previous data on the immunosuppressive impact of tryptophan depletion suggest IDO-induced suppression of antitumoral immune response in both adenocarcinoma and squamous cell carcinoma of endometrium and cervix. On the other hand, IDO as also known to inhibit tumor cell proliferation by tryptophan depletion.

Exercise-induced norepinephrine decreases circulating hematopoietic stem and progenitor cell colony-forming capacity.

A recent study showed that ergometry increased circulating hematopoietic stem and progenitor cell (CPC) numbers, but reduced hematopoietic colony forming capacity/functionality under normoxia and normobaric hypoxia. Herein we investigated whether an exercise-induced elevated plasma free/bound norepinephrine (NE) concentration could be responsible for directly influencing CPC functionality. Venous blood was taken from ten healthy male subjects (25.3+/-4.4 yrs) before and 4 times after ergometry under normoxia and normobaric hypoxia (FiO2<0.15). The circulating hematopoietic stem and progenitor cell numbers were correlated with free/bound NE, free/bound epinephrine (EPI), cortisol (Co) and interleukin-6 (IL-6). Additionally, the influence of exercise-induced NE and blood lactate (La) on CPC functionality was analyzed in a randomly selected group of subjects (n = 6) in vitro under normoxia by secondary colony-forming unit granulocyte macrophage assays. Concentrations of free NE, EPI, Co and IL-6 were significantly increased post-exercise under normoxia/hypoxia. Ergometry-induced free NE concentrations found in vivo showed a significant impairment of CPC functionality in vitro under normoxia. Thus, ergometry-induced free NE was thought to trigger CPC mobilization 10 minutes post-exercise, but as previously shown impairs CPC proliferative capacity/functionality at the same time. The obtained results suggest that an ergometry-induced free NE concentration has a direct negative effect on CPC functionality. Cortisol may further influence CPC dynamics and functionality.

Extended ultrastructural characterization of chordoma cells: the link to new therapeutic options.

Chordomas are rare bone tumors, developed from the notochord and largely resistant to chemotherapy. A special feature of this tumor is the heterogeneity of its cells. By combining high pressure freezing (HPF) with electron tomography we were able to illustrate the connections within the cells, the cell-cell interface, and the mitochondria-associated endoplasmic reticulum membrane complex that appears to play a special role among the characteristics of chordoma. These lipid raft-like regions are responsible for lipid syntheses and for calcium signaling. Compared to other tumor cells, chordoma cells show a close connection of rough endoplasmic reticulum and mitochondria, which may influence the sphingolipid metabolism and calcium release. We quantified levels of ceramide and glycosylceramide species by the methyl tert-butyl ether extraction method and we assessed the intracellular calcium concentration with the ratiometric fluorescent dye Fura-2AM. Measurements of the changes in the intracellular calcium concentration revealed an increase in calcium due to the application of acetylcholine. With regard to lipid synthesis, glucosylceramide levels in the chordoma cell line were significantly higher than those in normal healthy cells. The accumulation of glycosylceramide in drug resistant cancer cells has been confirmed in many types of cancer and may also account for drug resistance in chordoma. This study aimed to provide a deep morphological description of chordoma cells, it demonstrated that HPF analysis is useful in elucidating detailed structural information. Furthermore we demonstrate how an accumulation of glycosylceramide in chordoma provides links to drug resistance and opens up the field for new research options.