Adipose tissue as a source of growth factors to promote wound healing: a human study of skin graft donor sites.

OBJECTIVE: In the microenvironment of wound sites, naturally occurring growth factors are crucial for cell migration, opsonisation, chemotaxis, differentiation and angiogenesis. Exogenous growth factors, such as platelet-rich plasma (PRP) and adipose tissue, also improve healing. METHOD: In the present within-subject study, we described the effects of PRP and adipose tissue extract (ATE) on skin graft donor site wound healing in patients requiring split-thickness skin grafts. Each patient, having at least two donor sites, received both control (no growth factor) and experimental (PRP or ATE) treatments. Wounds were evaluated on days 5, 7, 10, 15, 30 and 60. Digital photography and spectral images were used to analyse haemoglobin and melanin content, and re-epithelialisation area. Pain was assessed by visual analogue scale. Scar characteristics were scored on days 30 and 60. Biomaterial samples were analysed for growth factor and protein content. RESULTS: The study included 24 patients (18 male and six female; mean age: 59.1 years). PRP was topically applied to wounds in 11 patients (13 donor sites) and ATE in 13 patients (15 sites). ATE-treated donor sites exhibited significantly accelerated wound re-epithelialisation on days 5 and 7 compared with control sites (p=0.003 and 0.04, respectively). PRP accelerated healing on day 7 compared with control sites (p=0.001). Additionally, the application of ATE improved scar quality on days 30 and 60 (p=0.0005 and 0.02, respectively). Pain scores did not differ significantly between treatments. CONCLUSION: In this study, both growth factor sources stimulated wound healing. ATE is an alternative source of growth factors that promote early wound healing and improve scar quality.

Development of novel human in vitro vascularized adipose tissue model with functional macrophages.

Inflammation has been proven significant factor in development of type 2 diabetes. So far, most of the adipose tissue related research has been performed in animals, mainly rodent models. The relevance of translation of animal results to humans is questionable. However, in vitro model with relevant human cell source, such as human adipose tissue stromal cells (hASC), can be developed and should be utilized for human adipose tissue research. We developed in vitro models of human adipose tissue utilizing hASC, endothelial cells and monocytes/macrophages. By isolating endothelial cells and macrophages from same adipose tissue as hASC, we were able to provide method for constructing personalized models of adipose tissue. With these models, we studied the effect of macrophages on adipogenesis and protein secretion, with and without vasculature. The models were analyzed for immunocytochemical markers, cell number, triglyceride accumulation and protein secretion. We found that lipid accumulation was greater in adipocytes in the presence of macrophages. Interferon gamma increased this difference between adipocyte culture and Adipocyte-Macrophage co-culture. Protein secretion was affected more by macrophages when vasculature was not present compared to the mild effect when vasculature was present. The vascularized adipose model with macrophages is valuable tool for human adipose tissue research, especially for the personalized medicine approaches; for choosing the right treatments and for studying rare medical conditions.

Presence of vasculature results in faster insulin response in adipocytes in vascularized adipose tissue model.

Besides being an energy storage, adipose tissue is an endocrine organ closely associated with vascular system. Human relevant in vitro models are needed to study adipose tissue and related diseases. Vasculature plays a central role in the development and inhibition of adipose tissue related diseases. Here, adipocyte culture was established from hASC (human adipose stromal cells), and a vascularized adipose tissue model was established from hASC and HUVEC (human umbilical cord vein endothelial cell) co-culture, utilizing the same differentiation procedure. Using these models together allowed analysis of the effect of vascularization on adipocytes. Adipocyte culture and Vascularized adipose tissue model were characterized on gene (adipocyte and vasculature-related), protein (von Willebrand factor, CollagenIV, CD140b and CD144, secretion of leptin, adiponectin and FABP4) and functional (triglyceride accumulation, glucose uptake and lipolysis) levels. Additionally, vascularized adipose tissue model was exposed to chemicals with known effects on adipogenesis and angiogenesis (rosiglitazone, chlorpyrifos, prochloraz, mancozeb, butylparaben, 15-deoxy-δ12,14-prostaglandin j2, bisphenol a, bis-(2-ethylhexyl) phthalate, tributyltin chloride) to compare their effects to the literature. The in vitro vascularized adipose tissue model showed presence of functional adipocytes and extensive vascular network. Adipocytes and the vasculature showed relevant gene and protein markers. Insulin induced glucose uptake, inhibited lipolysis and influenced vasculature-related genes. The results showed that vasculature led to faster insulin response in lipolysis inhibition and modulated responses to chemicals. This novel thoroughly characterized vascularized adipose tissue model is a promising new tool for studying adipose tissue as well as effect of chemicals on adipogenesis and angiogenesis in adipose tissue.

Adipose tissue extract shows potential for wound healing: in vitro proliferation and migration of cell types contributing to wound healing in the presence of adipose tissue preparation and platelet rich plasma.

Growth factors are the key elements in wound healing signaling for cell migration, differentiation and proliferation. Platelet-rich plasma (PRP), one of the most studied sources of growth factors, has demonstrated to promote wound healing in vitro and in vivo. Adipose tissue is an alternative source of growth factors. Through a simple lipoaspirate method, adipose derived growth factor-rich preparation (adipose tissue extract; ATE) can be obtained. The authors set out to compare the effects of these two growth factor sources in cell proliferation and migration (scratch) assays of keratinocyte, fibroblast, endothelial and adipose derived stem cells. Growth factors involved in wound healing were measured: keratinocyte growth factor, epidermal growth factor, insulin-like growth factor, interleukin 6, platelet-derived growth factor beta, tumor necrosis factor alfa, transforming growth factor beta and vascular endothelial growth factor. PRP showed higher growth factor concentrations, except for keratinocyte growth factor, that was present in adipose tissue in greater quantities. This was reflected in vitro, where ATE significantly induced proliferation of keratinocytes at day 6 (p < 0.001), compared to plasma and control. Similarly, ATE-treated fibroblast and adipose stem cell cultures showed accelerated migration in scratch assays. Moreover, both sources showed accelerated keratinocyte migration. Adipose tissue preparation has an inductive effect in wound healing by proliferation and migration of cells involved in wound closure. Adipose tissue preparation appears to offer the distinct advantage of containing the adequate quantities of growth factors that induce cell activation, proliferation and migration, particularly in the early phase of wound healing.

Development of Versatile Human In Vitro Vascularized Adipose Tissue Model with Serum-Free Angiogenesis and Natural Adipogenesis Induction.

Many adipose tissue-related diseases, such as obesity and type 2 diabetes, are worldwide epidemics. For studying these diseases, relevant human cell models are needed. In this study, we developed a vascularized adipose tissue model where human adipose stromal cells and human umbilical cord vein endothelial cells were cocultured with natural adipogenic and defined serum-free angiogenic media for 14 days. Several different protocols were compared to each other. The protocols varied in cell numbers and plating sequences. Lipid accumulation was studied with AdipoRed reagent, relative cell number with WST-1 reagent, gene expression of glut4, leptin, aP2, adiponectin, PPARγ and PPARγ2 with RT-qPCR. Secretion of adiponectin, leptin and aP2 was analysed with ELISA. The immunostained vascular network was imaged with Cell-IQ and area quantified using ImageJ. In this study, both angiogenesis and adipogenesis were successfully induced. Protocols produced strong lipid accumulation, good vascular network formation and induced adipocyte-specific protein secretion and expression of studied adipocyte genes. Results showed that cell numbers and cell plating sequences are important factors when aiming at in vitro standardized tissue model. Presence of mature vasculature appeared leads to faster the maturation of adipocytes judged by the lipid accumulation and gene expression results. The developed vascularized adipose tissue model is simple to use, easily modifiable to suit various applications and as such, a promising new tool for adipose tissue research when, for example, studying the effect of different cell types on adipose tissue function or for mechanistic studies.

Human BJ Fibroblasts is an Alternative to Mouse BALB/c 3T3 Cells in In Vitro Neutral Red Uptake Assay.

The OECD GD 129 BALB/c 3T3 neutral red uptake (NRU) assay is a standardized test method for estimating starting dose for an acute oral systemic toxicity test in rodents. Mouse BALB/c 3T3 fibroblasts are the most commonly used cells in the NRU assay. We have previously transferred and validated BALB/c 3T3 NRU assay in our GLP laboratory. Subsequently, in order to obtain more human-relevant cytotoxicity data, we performed an intralaboratory validation using human BJ fibroblasts in the NRU assay instead of mouse BALB/c 3T3 fibroblasts. Here, we present comparative cytotoxicity data of 26 different test chemicals (pharmaceuticals, industrial chemicals, pesticides and food additives) produced with both BALB/c 3T3 NRU and BJ NRU assays.

Maturation of human pluripotent stem cell derived cardiomyocytes is improved in cardiovascular construct.

In order to translate preclinical data into the clinical studies, relevant in vitro models with structure and key functional properties similar to native human tissue should be used. In vitro cardiac models with vascular structures mimic the highly vascularized myocardium and provide interactions between endothelial cells, stromal cells and cardiomyocytes. Currently, human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been shown to present immature morphology and fetal-like electrophysiological properties that may limit their use as physiological test platform. The aim of this study was to develop multicellular in vitro cardiovascular construct modeling human heart tissue. In the cardiovascular construct, hPSC-CMs were cultured with a vascular-like network formed by human foreskin fibroblasts and human umbilical vein endothelial cells that served as a platform in the construct. Cardiomyocyte orientation, maturation, electrophysiological properties and drug responses of the cardiovascular construct were characterized and compared to CM monoculture. hPSC-CMs in cardiovascular construct showed elongated morphology and aligned with the vascular-like network. Electrophysiological properties and calcium metabolism of hPSC-CMs as well as response to E-4031 and adrenaline demonstrated normal physiological behavior. Increased expression of cardiac structural proteins and ion channels in cardiovascular construct compared to CM monoculture were detected. In conclusion, vascular-like network supports the structural and functional maturation of hPSC-CMs. Our results suggest that cardiovascular construct presents more mature in vitro cardiac model compared to CM monoculture and could therefore serve as an advanced test system for cardiac safety and efficacy assessment as well as a model system for biomedical research.

Strong inhibitory effect of pre-eclampsia serum on angiogenesis detected in vitro by human cell-based angiogenesis tests.

OBJECTIVE: To explore in vitro angiogenic properties of maternal and umbilical cord blood sera from women with symptomatic pre-eclampsia in comparison with sera from women with normotensive pregnancies. STUDY DESIGN: Maternal and umbilical blood serum samples were collected from eleven primiparous women with pre-eclampsia and ten healthy gestational-age-matched primiparous controls. The samples were tested for tubule formation in two different types of in vitro angiogenesis tests. The first test (fibroblast-HUVEC) showed effects on angiogenesis and the second test (hASC-HUVEC), in addition to angiogenesis, also showed effects on vasculogenesis. The pro-angiogenic and inhibitory properties of the samples were microscopically quantified after immunostaining tubular structures, using markers for von Willebrand factor (vWf) and collagen IV. RESULTS: Serum samples from pre-eclamptic women inhibited tubule formation in both models, while those from normal pregnancy didn't. Umbilical blood samples were inhibitory both after pre-eclampsia and normal pregnancy. In the fibroblast-HUVEC model the inhibition was stronger after preeclampsia pregnancy, and the difference between groups was statistically significant. In the pre-eclampsia group a correlation between the inhibitory effect of umbilical blood and birth weight adjusted to gestational age was found. No clear correlation between sera from pregnant women and corresponding umbilical sera was found. CONCLUSION: The strong inhibitory effect of maternal serum samples on tubule formation reflects the anti-angiogenic state that is present in pre-eclampsia.

Cytokine-Rich Adipose Tissue Extract Production from Water-Assisted Lipoaspirate: Methodology for Clinical Use.

Proper functioning wound healing strategies are sparse. Adequate vascular formation to the injured area, as well as replacement of the volume loss, is fundamental in soft tissue repair. Tissue engineering strategies have been proposed for the treatment of these injury sites. Novel cell-free substance, human adipose tissue extract (ATE), has been previously shown to induce in vitro angiogenesis and adipogenesis and in vivo soft tissue formation. This study reports the translation of ATE preparation from laboratory to the operating room (OR). ATE samples for this study were derived from adipose tissue obtained with the water-jet assisted liposuction technique from 27 healthy patients. The variables studied included incubation time (15, 30, and 45 min), temperature (room temperature vs. 37°C), and filter type to determine the optimal method yielding the most consistent total protein content, as well as consistent and high expression of adipose-derived growth factors and cytokines, including: vascular endothelial growth factor, basic fibroblast growth factor, interleukin-6, adiponectin, leptin, and insulin-like growth factor. Following the optimization, samples were produced in the OR and tested for their sterility. No significant differences were observed when comparing extract incubation time points or incubation temperature. Nonetheless, when studying the different filter types used, a syringe filter with PES membrane with larger filter area showed significantly higher protein concentration (p ≤ 0.018). When studying the different growth factor concentrations, ELISA results showed less variation in cytokine concentrations in the OR samples with the optimized protocol. All of the OR samples were tested sterile. The devised protocol is an easy and reproducible OR-ready method for ATE generation. As an attractive source of growth factors, ATE is a promising alternative in the vast field of tissue engineering. Its clinical applications include volume replacement as a complement to fillers and improvement of the permanence of fat grafts and wound healing, among other bioactive functions.

The effects of culture conditions on the functionality of efficiently obtained mesenchymal stromal cells from human cord blood.

BACKGROUND AIMS: Cord blood (CB) is an attractive source of mesenchymal stromal cells (MSCs) because of its abundant availability and ease of collection. However, the success rate of generating CB-MSCs is low. In this study, our aim was to demonstrate the efficiency of our previously described method to obtain MSCs from CB and further characterize them and to study the effects of different culture conditions on MSCs. METHODS: CB-MSC cultures were established in low oxygen (3%) conditions on fibronectin in 10% fetal bovine serum containing culture medium supplemented with combinations of growth factors. Cells were characterized for their adipogenic, osteogenic and chondrogenic differentiation capacity; phenotype; and HOX gene expression profile. The functionality of the cells cultured in different media was tested in vitro with angiogenesis and T-cell proliferation assays. RESULTS: We demonstrate 87% efficacy in generating MSCs from CB. The established cells had typical MSC characteristics with reduced adipogenic differentiation potential and a unique HOX gene fingerprint. Growth factor-rich medium and a 3% oxygen condition enhanced cell proliferation; however, the growth factor-rich medium had a negative effect on the expression of CD90. Dexamethasone-containing medium improved the capacity of the cells to suppress T-cell proliferation, whereas the cells grown without dexamethasone were more able to support angiogenesis. CONCLUSIONS: Our results demonstrate that the composition of expansion medium is critical for the functionality of MSCs and should always be appropriately defined for each purpose.

Morphological Differentiation Towards Neuronal Phenotype of SH-SY5Y Neuroblastoma Cells by Estradiol, Retinoic Acid and Cholesterol.

Human SH-SY5Y neuroblastoma cells maintain their potential for differentiation and regression in culture conditions. The induction of differentiation could serve as a strategy to inhibit cell proliferation and tumor growth. Previous studies have shown that differentiation of SH-SY5Y cells can be induced by all-trans-retinoic-acid (RA) and cholesterol (CHOL). However, signaling pathways that lead to terminal differentiation of SH-SY5Y cells are still largely unknown. The goal of this study was to examine in the RA and CHOL treated SH-SY5Y cells the additive impacts of estradiol (E2) and brain-derived neurotrophic factor (BDNF) on cell morphology, cell population growth, synaptic vesicle recycling and presence of neurofilaments. The above features indicate a higher level of neuronal differentiation. Our data show that treatment for 10 days in vitro (DIV) with RA alone or when combined with E2 (RE) or CHOL (RC), but not when combined with BDNF (RB), significantly (p < 0.01) inhibited the cell population growth. Synaptic vesicle recycling, induced by high-K(+) depolarization, was significantly increased in all treatments where RA was included (RE, RC, RB, RCB), and when all agents were added together (RCBE). Specifically, our results show for the first time that E2 treatment can alone increase synaptic vesicle recycling in SH-SY5Y cells. This work contributes to the understanding of the ways to improve suppression of neuroblastoma cells' population growth by inducing maturation and differentiation.

Human vascular model with defined stimulation medium - a characterization study.

The formation of blood vessels is a vital process in embryonic development and in normal physiology. Current vascular modelling is mainly based on animal biology leading to species-to-species variation when extrapolating the results to humans. Although there are a few human cell based vascular models available these assays are insufficiently characterized in terms of culture conditions and developmental stage of vascular structures. Therefore, well characterized vascular models with human relevance are needed for basic research, embryotoxicity testing, development of therapeutic strategies and for tissue engineering. We have previously shown that the in vitro vascular model based on co-culture of human adipose stromal cells (hASC) and human umbilical vein endothelial cells (HUVEC) is able to induce an extensive vascular-like network with high reproducibility. In this work we developed a defined serum-free vascular stimulation medium (VSM) and performed further characterization in terms of cell identity, maturation and structure to obtain a thoroughly characterized in vitro vascular model to replace or reduce corresponding animal experiments. The results showed that the novel vascular stimulation medium induced intact and evenly distributed vascular-like network with morphology of mature vessels. Electron microscopic analysis assured the three-dimensional microstructure of the network containing lumen. Additionally, elevated expressions of the main human angiogenesis-related genes were detected. In conclusion, with the new defined medium the vascular model can be utilized as a characterized test system for chemical testing as well as in creating vascularized tissue models.

Novel in vitro cardiovascular constructs composed of vascular-like networks and cardiomyocytes.

The interaction between different cardiac cells has shown to be important for critical biological properties including cell survival, proliferation, differentiation and function. The improvement of culture conditions with different cell types and to study their effects on cardiomyocyte viability and functionality is essential. For practical applications including general toxicity testing, drug development and tissue engineering it is important to study whether co-cultures have additional advantages over cardiomyocyte monoculture. Two multicellular in vitro cardiovascular constructs devoid of added biomaterial were developed in this study. In the first construct, neonatal rat cardiomyocytes (CM) were seeded on vascular-like network formed by human umbilical vein endothelial cells (HUVEC) and human adipose stromal cells (hASC). In the second construct, CMs were seeded on vascular-like network formed by HUVECs and human foreskin fibroblasts. The ability of these two vascular-like networks to support the viability and functionality of CMs was analyzed. Different culture media compositions were evaluated to support the development of optimal cardiovascular construct. Our results demonstrate that both vascular-like networks markedly improved CM viability and functionality. In the constructs, co-localization of CMs and vascular-like networks was seen. Multicellular constructs also allowed synchronized contractility of CMs. Serum-free medium supplemented with vascular endothelial growth factor and basic fibroblast growth factor was found to provide the most optimal conditions for cardiovascular construct as an entity. In conclusion, when combining a vascular-like network with CMs, the viability and functionality of CMs was markedly improved. The results suggest that the cardiovascular constructs developed provide a promising new tool for the assessment of toxicological and safety pharmacological effects of compounds in vitro.

Bioactive acellular implant induces angiogenesis and adipogenesis and sustained soft tissue restoration in vivo.

Soft tissue defects resulting from trauma, tumor resection, or congenital causes provide a challenging problem to reconstructive surgery and tissue engineering. Current therapeutic procedures lack the ability to induce rapid formation of neovascularization. Therefore, to date, no adequate application for the reconstruction of soft tissue defects is available. We have previously shown that bioactive factors extracted from adipose tissue (adipose tissue extract [ATE]) induce both adipogenesis and angiogenesis in vitro. These bioactive factors were incorporated into hyaluronan (HA) hydrogel, and the ATE-HA implant-induced angiogenesis and adipogenesis were studied. The developed implant was shown to gradually release the bioactive factors, and the presence of the implant in human adipose stem cell culture was able to induce adipogenic differentiation as evaluated by Oil-red-O staining. In animal experiments, the implants were placed under dorsal subcutis of rodents. Either rat- (rATE, allograft) or human- (hATE, xenograft) derived ATE was incorporated into implants. Local inflammation reactions, angiogenesis, and adipogenesis were followed from 1 week to 40 weeks. Angiogenesis was assessed by microvessel density analysis; adipogenesis was assessed by automated image analysis, and immunological effects by immunostaining and counting inflammatory cells. The key requirements for soft tissue replacement--host compatibility, bioactivity, and sustainability--were all achieved with the novel ATE-HA implant. This acellular implant induced microvessel induction early after implantation and adipose tissue deposition from 12 weeks onward as well as subcutaneous tissue volume increase. The ATE-HA implant was replaced by mature adipose tissue with capillaries, nerve bundles, and healthy connective tissue without local inflammation or capsule formation. The large fat pads remained in tissue until the end of the follow-up time, for 9 months. No adverse effects were detected at the site of implantation, and according to irritating ranking, the ATE-implant was considered to have excellent biocompatibility. The results demonstrate that an acellular HA hydrogel implant induces significant increase in adipogenesis and angiogenesis in vivo compared to the plain HA implant, and ATE has excellent potential for use in tissue engineering for sustained reconstruction of soft tissue defects.

Adipose stromal cell tubule network model provides a versatile tool for vascular research and tissue engineering.

The current limitation in designing three-dimensional tissue models is the lack of adequate vascularization with mature and stable vessels. Adipose tissue is known to secrete several angiogenic factors, and human adipose stromal cells (hASC) are known to promote vessel growth, maturation and stabilization. In this study, hASC were induced to angiogenesis with growth factor-enriched medium either in monoculture or in coculture with human umbilical vein endothelial cells (HUVEC) and analyzed for vascular, pericytic and smooth muscle cell markers. hASC and HUVEC cocultures showed an accelerated proliferation rate and the cells self-assembled, independent of the cell passage number, into multilayered three-dimensional tubular networks. The networks of hASC and HUVEC expressed endothelial markers, a complete basement membrane and vessel-supporting cells with contractile properties. A hASC and green fluorescence protein-HUVEC-infection model revealed that cocultures consisted of a mosaic of von Willebrand factor-positive cells derived from both cell populations - hASC and HUVEC. hASC monoculture had passage- and donor-dependent ability to form tubular networks, with half of the cultures presenting tubule structures and basement membrane formation. Pericytic and smooth muscle cell markers were expressed in hASC monoculture even when tubules were absent. By combining the potential properties of hASC and features from the present angiogenesis assays, we generated a natural-like, xeno-free, prevascular-like network in vitro model with excellent reproducibility and minimal limitations in technical performance. This tubular network model is an excellent tool for studying cell interactions during vascular development, for chemical and drug testing and for developing natural-like, multilayered, vascularized, scaffold-free tissue models.

Human adipose tissue extract induces angiogenesis and adipogenesis in vitro.

The induction of adequate vascularization, a major challenge in tissue engineering, has been tried with numerous methods but with unsatisfactory results. Adipose tissue, an active endocrine organ with dense vasculature, secretes a wide number of angiogenic and adipogenic factors and seems an attractive source for these bioactive factors. We produced a novel cell-free extract from mature human adipose tissue (adipose tissue extract [ATE]) and analyzed the ability of this extract to induce angiogenesis and adipogenesis in vitro and studied the cytokine and growth factor composition of ATE with ELISA and cytokine array. We demonstrate that ATE, when added as cell culture supplement, effectively induced triglyceride accumulation in human adipose stem cells at concentrations from 200 µg/mL upward in less than a week and caused elevated levels of adipocyte differentiation markers (proliferator-activated receptor gamma and acyl-CoA-binding protein) when treated with at least 350 µg/mL of ATE. ATE induced angiogenesis from 450 µg/mL upward after a week in vitro. ATE contained numerous angiogenic and adipogenic factors, for example, vascular endothelial growth factor, basic fibroblast growth factor, interleukin-6, adiponectin, angiogenin, leptin, and insulin-like growth factor-I, as well as lower levels of a wide variety of other cytokines. We here present a novel cell-free angiogenesis- and adipogenesis-inducing agent that is cell-free and easy to produce, and its effect is dose dependent and its composition can be easily modified. Therefore, ATE is a promising novel agent to be used for angiogenesis induction to overcome the challenge of vascularization and for adipogenesis induction in a wide variety of tissue engineering applications in vitro and in vivo. ATE is also efficient for reproduction and modeling of natural adipogenesis in vitro for, for example, obesity and diabetes studies.

Intra-Laboratory Pre-Validation of a Human Cell Based in vitro Angiogenesis Assay for Testing Angiogenesis Modulators.

The developed standardized human cell based in vitro angiogenesis assay was intra-laboratory pre-validated to verify that the method is reliable and relevant for routine testing of modulators of angiogenesis, e.g., pharmaceuticals and industrial chemicals. This assay is based on the earlier published method but it was improved and shown to be more sensitive and rapid than the previous assay. The performance of the assay was assessed by using six reference chemicals, which are widely used pharmaceuticals that inhibit angiogenesis: acetyl salicylic acid, erlotinib, 2-methoxyestradiol, levamisole, thalidomide, and anti-vascular endothelial growth factor. In the intra-laboratory pre-validation, the sensitivity of the assay (upper and lower limits of detection and linearity of response in tubule formation), batch to batch variation in tubule formation between different Master cell bank batches, and precision as well as the reliability of the assay (reproducibility and repeatability) were tested. The pre-set acceptance criteria for the intra-laboratory pre-validation study were met. The relevance of the assay in man was investigated by comparing the effects of reference chemicals and their concentrations to the published human data. The comparison showed a good concordance, which indicates that this human cell based angiogenesis model predicts well the effects in man and has the potential to be used to supplement and/or replace of animal tests.

Cholesterol supports the retinoic acid-induced synaptic vesicle formation in differentiating human SH-SY5Y neuroblastoma cells.

Synaptic vesicle formation, vesicle activation and exo/endocytosis in the pre-synaptic area are central steps in neuronal communication. The formation and localization of synaptic vesicles in human SH-SY5Y neuroblastoma cells, differentiated with 12-o-tetradecanoyl-phorbol-13-acetate, dibutyryl cyclic AMP, all-trans-retinoic acid (RA) and cholesterol, was studied by fluorescence microscopy and immunocytochemical methods. RA alone or together with cholesterol, produced significant neurite extension and formation of cell-to-cell contacts. Synaptic vesicle formation was followed by anti-synaptophysin (SypI) and AM1-43 staining. SypI was only weakly detected, mainly in cell somata, before 7 days in vitro, after which it was found in neurites. Depolarization of the differentiated cells with high potassium solution increased the number of fluorescent puncta, as well as SypI and AM1-43 co-localization. In addition to increase in the number of synaptic vesicles, RA and cholesterol also increased the number and distribution of lysosome-associated membrane protein 2 labeled lysosomes. RA-induced Golgi apparatus fragmentation was partly avoided by co-treatment with cholesterol. The SH-SY5Y neuroblastoma cell line, differentiated by RA and cholesterol and with good viability in culture, is a valuable tool for basic studies of neuronal metabolism, specifically as a model for dopaminergic neurons.

Quantification of vesicles in differentiating human SH-SY5Y neuroblastoma cells by automated image analysis.

A new automated image analysis method for quantification of fluorescent dots is presented. This method facilitates counting the number of fluorescent puncta in specific locations of individual cells and also enables estimation of the number of cells by detecting the labeled nuclei. The method is here used for counting the AM1-43 labeled fluorescent puncta in human SH-SY5Y neuroblastoma cells induced to differentiate with all-trans retinoic acid (RA), and further stimulated with high potassium (K+) containing solution. The automated quantification results correlate well with the results obtained manually through visual inspection. The manual method has the disadvantage of being slow, labor-intensive, and subjective, and the results may not be reproducible even in the intra-observer case. The automated method, however, has the advantage of allowing fast quantification with explicitly defined methods, with no user intervention. This ensures objectivity of the quantification. In addition to the number of fluorescent dots, further development of the method allows its use for quantification of several other parameters, such as intensity, size, and shape of the puncta, that are difficult to quantify manually.