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.

In Vitro Vascular Network Modified to Function as Culture Platform and Angiogenic Induction Potential Test for Cancer Cells.

Drug treatments have been designed to inhibit tumor angiogenesis in hope of stopping tumor growth. However, not all tumor types respond to this type of treatment. A screening method which identifies angiogenesis inducing cancer types would help predict the efficacy of angiogenesis-inhibiting drugs for the patients. Our goal is to develop (1) a cell assay to assess the angiogenic induction potential of patient-derived tumor cells, and (2) a protocol for culturing cancer cells on a vascular platform. We optimized the media composition and seeding density of cells (hASC, HUVEC, and cancer cells) to 48-, 96-, and even 384-well plate sizes to allow vascular formation and cancer cell proliferation and subsequent analysis with high throughput. The angiogenic induction potential of patient-derived cancer cells was investigated by quantifying the formation of tubular structures and the drug response of cancer cells grown on a vascular platform was evaluated using gene expression and cell viability (WST-1) assay. Immunocytochemistry was performed with von Willebrand factor, collagen IV, CD44, cytokeratin 19 and ALDH1A1. The angiogenic induction potential test was shown to be responsive to the induction of angiogenesis by cancer cells. The responses of cancer cells were different when grown on a vascular platform or on plastic, seen in gene expression level and viability results. These two protocols are promising novel tools for aiding the selection of efficient cancer drugs for personalized medicine and as an alternative cancer cell culture platform.

Effects of nanofibrillated cellulose hydrogels on adipose tissue extract and hepatocellular carcinoma cell spheroids in freeze-drying.

The aim of this study was to evaluate the effects of two nanofibrillated cellulose (NFC) hydrogels on two human derivatives during freeze-drying. Native NFC hydrogel is a suitable platform to culture 3D cell spheroids and a hydrogel processed further, called anionic NFC (ANFC) hydrogel, is an excellent platform for controlled release of proteins. Moreover, it has been shown to be compatible with freeze-drying when correct lyoprotectants are implemented. Freeze-drying is a method, where substance is first frozen, and then vacuum dried trough sublimation of water in order to achieve dry matter without the loss of the original three-dimensional structures. The first chosen human derivative was adipose tissue extract (ATE) which is a cell-free growth factor-rich preparation capable of promoting growth of regenerative cells. The release of growth factors from the freeze-dried mixture of ATE and ANFC was compared to that of non-freeze-dried control mixtures. The release profiles remained at the same level after freeze-drying. The second derivative was hepatocellular carcinoma (HepG2) cell spheroids which were evaluated before and after freeze-drying. The 3D structure of the HepG2 cell spheroids was preserved and the spheroids retained 18% of their metabolic activity after rehydration. However, the freeze-dried and rehydrated HepG2 cell spheroids did not proliferate and the cell membrane was damaged by fusion and formation of crystals.

International STakeholder NETwork (ISTNET): creating a developmental neurotoxicity (DNT) testing road map for regulatory purposes.

A major problem in developmental neurotoxicity (DNT) risk assessment is the lack of toxicological hazard information for most compounds. Therefore, new approaches are being considered to provide adequate experimental data that allow regulatory decisions. This process requires a matching of regulatory needs on the one hand and the opportunities provided by new test systems and methods on the other hand. Alignment of academically and industrially driven assay development with regulatory needs in the field of DNT is a core mission of the International STakeholder NETwork (ISTNET) in DNT testing. The first meeting of ISTNET was held in Zurich on 23-24 January 2014 in order to explore the concept of adverse outcome pathway (AOP) to practical DNT testing. AOPs were considered promising tools to promote test systems development according to regulatory needs. Moreover, the AOP concept was identified as an important guiding principle to assemble predictive integrated testing strategies (ITSs) for DNT. The recommendations on a road map towards AOP-based DNT testing is considered a stepwise approach, operating initially with incomplete AOPs for compound grouping, and focussing on key events of neurodevelopment. Next steps to be considered in follow-up activities are the use of case studies to further apply the AOP concept in regulatory DNT testing, making use of AOP intersections (common key events) for economic development of screening assays, and addressing the transition from qualitative descriptions to quantitative network modelling.

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.

Decellularized In Vitro Capillaries for Studies of Metastatic Tendency and Selection of Treatment.

Vascularization plays an important role in the microenvironment of the tumor. Therefore, it should be a key element to be considered in the development of in vitro cancer assays. In this study, we decellularized in vitro capillaries to remove genetic material and optimized the medium used to increase the robustness and versatility of applications. The growth pattern and drug responses of cancer cell lines and patient-derived primary cells were studied on decellularized capillaries. Interestingly, two distinct growth patterns were seen when cancer cells were grown on decellularized capillaries: "network" and "cluster". Network formation correlated with the metastatic properties of the cells and cluster formation was observed in non-metastatic cells. Drug responses of patient-derived cells correlated better with clinical findings when cells were cultured on decellularized capillaries compared with those cultured on plastic. Decellularized capillaries provide a novel method for cancer cell culture applications. It bridges the gap between complex 3D culture methods and traditional 2D culture methods by providing the ease and robustness of 2D culture as well as an in vivo-like microenvironment and scaffolding for 3D cultures.

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.

Erratum: Multilabel segmentation of cancer cell culture on vascular structures with deep neural networks (Erratum).

[This corrects the article DOI: 10.1117/1.JMI.7.2.024001.].

Multilabel segmentation of cancer cell culture on vascular structures with deep neural networks.

New increasingly complex in vitro cancer cell models are being developed. These new models seem to represent the cell behavior in vivo more accurately and have better physiological relevance than prior models. An efficient testing method for selecting the most optimal drug treatment does not exist to date. One proposed solution to the problem involves isolation of cancer cells from the patients' cancer tissue, after which they are exposed to potential drugs alone or in combinations to find the most optimal medication. To achieve this goal, methods that can efficiently quantify and analyze changes in tested cell are needed. Our study aimed to detect and segment cells and structures from cancer cell cultures grown on vascular structures in phase-contrast microscope images using U-Net neural networks to enable future drug efficacy assessments. We cultivated prostate carcinoma cell lines PC3 and LNCaP on the top of a matrix containing vascular structures. The cells were imaged with a Cell-IQ phase-contrast microscope. Automatic analysis of microscope images could assess the efficacy of tested drugs. The dataset included 36 RGB images and ground-truth segmentations with mutually not exclusive classes. The used method could distinguish vascular structures, cells, spheroids, and cell matter around spheroids in the test images. Some invasive spikes were also detected, but the method could not distinguish the invasive cells in the test images.

Effects of simvastatin, acetylsalicylic acid, and rosiglitazone on proliferation of normal and cancerous prostate epithelial cells at therapeutic concentrations.

BACKGROUND: Non-steroidal anti-inflammatory drugs and cholesterol-lowering statins have been reported to inhibit prostate cancer cell growth suggesting their chemopreventive potential within the prostate. However, the effect has been demonstrated only with advanced prostate cancer cell lines and with drug concentrations above the clinical therapeutic range. In this study we compared the effect of therapeutic concentrations of acetylsalicylic acid, simvastatin and rosiglitazone on the growth of a set of prostatic primary cultures and various prostate epithelial cell lines. METHODS: Two primary epithelial cell lines isolated from surgical resecates of normal prostate tissue (P96E, P97E), a primary cell line isolated from untreated prostate carcinoma (ESTO1), two transformed prostate epithelial cell lines (PWR1-E, RWPE-1) and advanced cancer cell lines LNCaP and VCaP were used in the study. Cells were treated for seven days with therapeutic concentrations of acetylsalisylic acid, simvastatin, rosiglitazone or their combination. Cellular growth rate was measured by crystal violet staining method. RESULTS: Acetylsalicylic acid (0.5 mM) and simvastatin (10 nM) inhibited the growth of prostate epithelial cells of normal and primary cancer origin, whereas advanced cancer cell lines were resistant to the effect. Rosiglitazone at the therapeutic level of 1 microM did not reduce the growth of any cell type studied. CONCLUSIONS: Our results demonstrate that acetylsalicylic acid and simvastatin inhibit prostate epithelial cell growth at clinically relevant doses. This should be acknowledged when designing possible prostate cancer chemopreventive trials.

Concomitant exposure of ovarian cancer cells to docetaxel, CPT-11 or SN-38 and adenovirus-mediated p53 gene therapy.

Owing to its central role in multiple cellular functions, p53 is an attractive candidate for gene replacement therapy. We studied the role of adenovirus-mediated p53 gene (p53Ad) therapy on sensitivity of two ovarian cancer cell lines, OVCAR-3 (p53(mut)) and SK-OV-3 (p53(wt)), to docetaxel, CPT-11 and SN-38 exposures. Expressions of Bcl-XL, Bcl-XS, p53, Gadd45, c-fos, p21(waf1/cip1), Bax, Bcl-2 and Mcl-1 were measured after concomitant p53Ad and drug exposures. In SK-OV-3 cells containing a normal p53 gene, p53Ad alone or concomitantly with docetaxel, CPT-11 or SN-38 exposures did not have an effect on cell growth, cell cycle distribution or induction of apoptosis. In OVCAR-3 cells, p53 gene therapy inhibited the cell growth and sensitized cells to CPT-11/SN-38, but not to docetaxel. Growth inhibition and sensitization were results of G2M cell cycle arrest and increased apoptosis. In SK-OV-3 cells, but not in OVCAR-3 cells, CPT-11/SN-38 exposures alone increased p21(waf1/cip1) expression. The p53Ad therapy induced strong p21(waf1/cip1) expression in both cell lines. In addition, the expression of Bax and expression ratios Bax/Bcl-2 and Bax/Bcl-XL increased in p53Ad-infected OVCAR-3 cells, but not in SK-OV-3 cells. These expression ratios were further increased in p53Ad+CPT-11/SN-38-exposed OVCAR-3 samples. These results support the combination of p53 gene therapy with topoisomerase I inhibitors SN-38/CPT-11 when tumour cells contain mutated p53. When p53 status is normal, p53 gene therapy is not effective alone or concomitantly with CPT-11/SN-38. Increased expression ratios of Bax/Bcl-2 and Bax/Bcl-XL might serve as positive markers for effective p53 gene therapy and concomitant topoisomerase I inhibitor therapy.

Inhibition of P-glycoprotein-mediated docetaxel efflux sensitizes ovarian cancer cells to concomitant docetaxel and SN-38 exposure.

The first-line treatment of ovarian cancer is based on cytoreductive surgery and the use of anticancer drugs. The main disadvantage in the usage of anticancer drugs is the wide capacity of cancer cells to acquire a resistance to chemotherapeutic agents and therefore new treatment strategies have to be developed and tested. In this study, the responses of seven ovarian carcinoma cell lines to docetaxel and a camptothecin derivative, SN-38, were evaluated. We further studied the expression of P-glycoprotein (P-gp), the best described mechanism of drug resistance, in these cells and the effect of treatment with a specific P-gp inhibitor (PGP-4008). Simultaneous treatment with docetaxel and SN-38 (docetaxel+SN-38) had an antagonistic growth effect that was not dependent on the administration schedule. Both drugs alone or in combination induced G2M cell cycle arrest. Docetaxel was a more potent inducer of apoptosis than SN-38, but simultaneous treatment with docetaxel+SN-38 decreased the proportion of apoptotic cells to the same level observed after exposure to SN-38 alone. SN-38 increased P-gp expression in all cell lines. PGP-4008 enhanced docetaxel-mediated growth inhibition and apoptosis, but it did not have an effect when used simultaneously with SN-38. When cells were treated with docetaxel, SN-38, and PGP-4008 simultaneously, the growth was inhibited more efficiently and the proportion of apoptotic cells was higher than that without PGP-4008. Thus, treatment of ovarian cancer cells with docetaxel+SN-38 may have antagonistic effects. The simultaneous administration of a P-gp inhibitor may prevent docetaxel efflux, thereby sensitizing cells to docetaxel and other chemotherapeutic agents.

Gene expression changes during the development of estrogen-independent and antiestrogen-resistant growth in breast cancer cell culture models.

We have established estrogen-independent and antiestrogen-resistant cell lines from hormone-dependent MCF-7 breast cancer cells by long-term culture in the absence of estrogen, or in the presence of antiestrogen toremifene, respectively. By using a cDNA microarray we compared gene expression profiles among estrogen-independent, antiestrogen-resistant and long-term estrogen-treated MCF-7 cells. We also determined how the expression of the differentially expressed genes has developed during the long-term culture of the cell lines. Of the screened 1176 cancer-related genes, FOSL1, TIMP1, L1CAM, GDF15, and MYBL2 were found to be differentially expressed between the cell lines. A change in FOSL1 and TIMP1 expression could be attributed to the development of antiestrogen resistance, whereas induced L1CAM expression was implicated in the development of estrogen-independent growth of the cells. Estrogen regulated genes GDF15 and L1CAM became regulated by toremifene in the later passage number of toremifene-resistant cells, which might be an indication of the developed estrogen-agonistic activity of toremifene in these cells. Our findings suggest a pattern where the hormone-responsive cancer cells, which survive E2 deprivation and/or antiestrogen treatment, first acquire necessary changes in gene expression for transition to maximal growth in the new hormonal environment. Then, after prolonged treatment with antiestrogen, the antiestrogen-resistant cells may eventually generate an E2-agonistic response to antiestrogen, probably acquiring additional growth advantage.

Changes in protein tyrosine phosphatase type IVA member 1 and zinc finger protein 36 C3H type-like 1 expression demonstrate altered estrogen and progestin effect in medroxyprogesterone acetate-resistant and estrogen-independent breast cancer cell models.

Estrogen stimulates proliferation in hormone-responsive breast cancer cells. Progestins inhibit the estrogen-mediated growth in these cells and are used in the treatment of mammary carcinomas. We applied cDNA microarray and real-time RT-PCR methods to reveal 17beta-estradiol- and medroxyprogesterone acetate (MPA)-regulated genes in MCF-7 breast cancer cells. We identified six genes, two of which were novel MPA and/or 17beta-estradiol-regulated genes: protein tyrosine phosphatase type IVA, member 1 (PTP4A1) and zinc finger protein 36, C3H type-like 1 (ZFP36L1). PTP4A1 expression was upregulated by 17beta-estradiol and this was opposed by MPA treatment of the cells. ZFP36L1 proved to be a direct target of MPA. Since MPA has also been shown to bind to glucocorticoid- and androgen receptors, we studied the regulation of the genes with progesterone, synthetic progestin R5020, dexamethasone and dihydrotestosterone. We also assessed the expression and hormonal regulation of PTP4A1 and ZFP36L1 mRNA in MCF-7-derived MPA-resistant and estrogen-independent sublines. The regulation of PTP4A1 expression upon 17beta-estradiol and combined 17beta-estradiol and MPA treatment was completely reversed in the estrogen-independent and MPA-resistant sublines, respectively. This study suggests an important role for PTP4A1 in cell growth, and shows that MPA may potentiate the effect of 17beta-estradiol in the MPA-resistant breast cancer cells.

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.

Characterisation of human dental stem cells and buccal mucosa fibroblasts.

Human craniofacial stem cells are recently discovered sources of putative mesenchymal stem cells that hold great promise for autogenic or allogenic cell therapy and tissue engineering. Prior to employing these cells in clinical applications, they must be thoroughly investigated and characterized. In this study, the surface marker expression was investigated on dental pulp stem cells (DPSCs), dental follicle cells (DFCs), periodontal ligament stem cells (PDLSCs), and buccal mucosa fibroblasts (BMFs) utilising surface markers for flow cytometry. The osteogenic potential was also examined by bone-associated markers alkaline phosphatase, Runx2, collagen type I, osteocalcin, and osteopontin. The results from our study demonstrate that the dental cell sources exhibit comparable surface marker and bone-associated marker profiles parallel to those of other mesenchymal stem cell sources, yet distinct from the buccal mucosa fibroblasts. Our data support evidence towards clinical applicability of dental stem cells in hard tissue regeneration.

Effects of tamoxifen and raloxifene on normal human endometrial cells in an organotypic in vitro model.

The selective estrogen receptor modulator tamoxifen is widely used in breast cancer therapy though its use is associated with an elevated risk of endometrial carcinoma. An organotypic culture model was employed here to examine the effects of tamoxifen and raloxifene, a related compound with no known adverse uterine effects, on epithelial cells of the premenopausal human endometrium. Changes in the expression levels of the proliferation marker Ki67, and estrogen and progesterone receptors were evaluated. No change in the Ki67 index compared to untreated controls was detected in cultures exposed to tamoxifen or tamoxifen+estradiol. In response to tamoxifen, the level of progesterone receptor-expressing organoids was shown to vary markedly between individual samples, whereas no change in estrogen receptor expression could be demonstrated. A significant decrease in Ki67 expression was observed in raloxifene-exposed cultures. Raloxifene or raloxifene+estradiol had no effect on progesterone receptor expression. The expression of estrogen receptor was markedly inhibited in response to raloxifene or raloxifene+estradiol in all but two samples displaying an intense estrogen receptor labelling. The present observations add to current clinical data on the respective estrogen receptor agonist and antagonist activities of tamoxifen and raloxifene on the human uterus by providing novel insights into the interindividual variation in cellular responses. Our organotypic model may have uses as an alternative to animal experimentation in preclinical screening of the endometrial effects of selective estrogen receptor modulators and may serve as a tool in personalized medicine by identifying patients with an increased risk of developing endometrial pathologies.

Progestins regulate genes that can elicit both proliferative and antiproliferative effects in breast cancer cells.

Sex steroid hormone progesterone is known to have profound effects on the growth and differentiation of the normal mammary gland and malignant breast epithelial cells. In vitro progesterone and synthetic progesterone-like compounds (progestins) inhibit breast cancer cell growth. Medroxyprogesterone acetate (MPA) is a synthetic hormone widely used in the adjuvant treatment of advanced breast cancer, hormone replacement therapy and in oral contraceptives. It is a paradoxical hormone, since it inhibits breast cancer cell proliferation, but has also been implicated in increased breast cancer risk. To better understand the molecular mechanism by which cell proliferation and differentiation are regulated by progesterone and MPA in human breast cancer, we utilized cDNA microarray and quantitative real-time RT-PCR methods to identify their target genes. This study describes novel progestin/progesterone target genes in breast cancer cells and, notably, novel target genes that elucidate the underlying molecular mechanism of the dual role progestins play in the breast. A cDNA microarray containing 3000 genes showed notable regulation in 30 and 27 genes by MPA and progesterone, respectively. Only 6 out of the 30 genes regulated by MPA are down-regulated, but no progesterone down-regulation was observed. Overlapping in gene regulation by progesterone and MPA occurred, but the majority of genes regulated by these hormones were distinct. Given that progestins both stimulate and inhibit cancer cell growth, we report our findings on novel progestin and progesterone targets, which could explain the paradoxical actions of progestins in the breast.

Comparison of an automated pattern analysis machine vision time-lapse system with traditional endpoint measurements in the analysis of cell growth and cytotoxicity.

Machine vision is an application of computer vision. It both collects visual information and interprets the images. Although the machine obviously does not 'see' in the same sense that humans do, it is possible to acquire visual information and to create programmes to identify relevant image features in an effective and consistent manner. Machine vision is widely applied in industrial automation, but here we describe how we have used it to monitor and interpret data from cell cultures. The machine vision system used (Cell-IQ) consisted of an inbuilt atmosphere-controlled incubator, where cell culture plates were placed during the test. Artificial intelligence (AI) software, which uses machine vision technology, took care of the follow-up analysis of cellular morphological changes. Basic endpoint and staining methods to evaluate the condition of the cells, were conducted in parallel to the machine vision analysis. The results showed that the automated system for pattern analysis of morphological changes yielded comparable results to those obtained by conventional methods. The inbuilt software analysis offers a promising way of evaluating cell growth and various cell phases. The continuous follow-up and label-free analysis, as well as the possibility of measuring multiple parameters simultaneously from the same cell population, were major advantages of this system, as compared to conventional endpoint measurement methodology.

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.