Tridimensional Culture Research Articles

A three-dimensional cell culture approach to investigate cytotoxic effects and production of inflammatory mediators by epoxy resin-based and calcium silicate-based endodontic sealer.

The aim of the present study was to assess the cytocompatibility of epoxy resin-based AH Plus Jet (Dentsply De Trey, Konstanz, Germany), Sealer Plus (MK Life, Porto Alegre, Brazil), calcium silicate-based Bio-C Sealer (Angelus, Londrina, PR, Brazil), Sealer Plus BC (MK Life) and AH Plus BC (Dentsply) through a tridimensional (3D) culture model of human osteoblast-like cells. Spheroids of MG-63 cells were produced and exposed to fresh root canal sealers extracts by 24h, and the cytotoxicity was assessed by the Lactate Dehydrogenase assay (LDH). The distribution of dead cells within the microtissue was assessed by fluorescence microscopy, and morphological effects were investigated by histological analysis. The secreted inflammatory mediators were detected in cell supernatants through flow luminometry (XMap Luminex). Cells incubated with AH Plus Jet, AH Plus BC, Sealer Plus BC and Bio-C Sealer extracts showed high rates of cell viability, while the Sealer Plus induced a significant reduction of cell viability, causing reduction on the spheroid structure. Sealer Plus and Seaker Plus BC caused alterations on 3D microtissue morphology. The AH Plus BC extract was associated with the downregulation of secretion of pro-inflammatory cytokines IL-5, IL-7, IP-10 and RANTES. The new AH Plus BC calcium silicate-based endodontic sealer did not reduce cell viability in vitro, while led to the downregulation of pro-inflammatory cytokines. Choosing the appropriate endodontic sealer is a crucial step. AH Plus BC demonstrated high cell viability and downregulation of pro-inflammatory cytokines, appearing reliable for clinical use, while Sealer Plus presented lower cytocompatibility.

Property of Hemorrhoid Tissue Mesenchymal Stem Cell Tow-Dimensional and Tridimensional Cultures for the Treatment of Anal Incontinence

Abstract Background Autologous mesenchymal stem cells (MSC) from different origins, including adipose tissue, have been proposed for the treatment of anal incontinence with encouraging preclinical and clinical outcomes. Cell transplantation as tridimensional structures instead of individual cell suspension seems to increase cell viability and implantation. We previously developed a protocol for MSC isolation directly from haemorrhoidal tissue (he-MSC) in human and demonstrated that the isolated cells fulfil MSC criteria. Aims Evaluate the characteristics and secretome of he-MSC in both two-dimensional or tridimensional cultures. Methods Informed consent was obtained from all patients. Tissue samples were procured from haemorrhoidectomy specimens or liposuction waste product and processed and characterized according to previously reported methods. He-MSC were cultured as spheroids using the “hanging-drop technique” or agarose pits. Immunofluorescence was performed to assess the expression of mesenchymal proteins and cell viability. Conditioned media of tow-dimensional cultures or spheroids in suspension were generated using low serum culture medium and analysed with semi-quantitative cytokine arrays (120 cytokines) and ELISA assays. Results Cultured cells demonstrated expression of vimentin and good viability, even as spheroids (2.6±2% cell death). Cytokine profile of he-MSC secretome was similar to that of adipose tissue-derived MSC (AT-MSC), with shared core cytokines (FGF-9, OPG, CCL2, CCL11, CCL13, IGFBP-4, IGFBP-6). Concentration of HGF measured by ELISA was higher in he-MSC conditioned medium than in AT- MSC conditioned medium (19613±3528 versus 179±84 pg/ml p<0.001) whereas VEGF concentration followed an opposite trend (7627±947pg/ml versus 181±74 pg/ml p<0.001). Production of mesenchymal proteins and growth factors was not affected by the spheroid configuration of cells. Conclusion We demonstrated that he-MSC exhibit a similar secretome profile compared to AT-MSC. Generation of spheroids did not compromise cell viability or their ability to produce structural proteins and growth factors, validating he-MSC spheroids use for further in vivo studies prior to clinical validation.

Advances in the knowledge and therapeutics of schizophrenia, major depression disorder, and bipolar disorder from human brain organoid research.

Tridimensional cultures of human induced pluripotent cells (iPSCs) experimentally directed to neural differentiation, termed "brain organoids" are now employed as an in vitro assay that recapitulates early developmental stages of nervous tissue differentiation. Technical progress in culture methodology enabled the generation of regionally specialized organoids with structural and neurochemical characters of distinct encephalic regions. The technical process of organoid elaboration is undergoing progressively implementation, but current robustness of the assay has attracted the attention of psychiatric research to substitute/complement animal experimentation for analyzing the pathophysiology of psychiatric disorders. Numerous morphological, structural, molecular and functional insights of psychiatric disorders have been uncovered by comparing brain organoids made with iPSCs obtained from control healthy subjects and psychiatric patients. Brain organoids were also employed for analyzing the response to conventional treatments, to search for new drugs, and to anticipate the therapeutic response of individual patients in a personalized manner. In this review, we gather data obtained by studying cerebral organoids made from iPSCs of patients of the three most frequent serious psychiatric disorders: schizophrenia, major depression disorder, and bipolar disorder. Among the data obtained in these studies, we emphasize: (i) that the origin of these pathologies takes place in the stages of embryonic development; (ii) the existence of shared molecular pathogenic aspects among patients of the three distinct disorders; (iii) the occurrence of molecular differences between patients bearing the same disorder, and (iv) that functional alterations can be activated or aggravated by environmental signals in patients bearing genetic risk for these disorders.

Modeling the Differentiation of Embryonic Limb Chondroprogenitors by Cell Death and Cell Senescence in High Density Micromass Cultures and Their Regulation by FGF Signaling

Considering the importance of programmed cell death in the formation of the skeleton during embryonic development, the aim of the present study was to analyze whether regulated cell degeneration also accompanies the differentiation of embryonic limb skeletal progenitors in high-density tridimensional cultures (micromass cultures). Our results show that the formation of primary cartilage nodules in the micromass culture assay involves a patterned process of cell death and cell senescence, complementary to the pattern of chondrogenesis. As occurs in vivo, the degenerative events were preceded by DNA damage detectable by γH2AX immunolabeling and proceeded via apoptosis and cell senescence. Combined treatments of the cultures with growth factors active during limb skeletogenesis, including FGF, BMP, and WNT revealed that FGF signaling modulates the response of progenitors to signaling pathways implicated in cell death. Transcriptional changes induced by FGF treatments suggested that this function is mediated by the positive regulation of the genetic machinery responsible for apoptosis and cell senescence together with hypomethylation of the Sox9 gene promoter. We propose that FGF signaling exerts a primordial function in the embryonic limb conferring chondroprogenitors with their biological properties.

Stretchable mesh microelectronics for the biointegration and stimulation of human neural organoids

Advances in tridimensional (3D) culture approaches have led to the generation of organoids that recapitulate cellular and physiological features of domains of the human nervous system. Although microelectrodes have been developed for long-term electrophysiological interfaces with neural tissue, studies of long-term interfaces between microelectrodes and free-floating organoids remain limited. In this study, we report a stretchable, soft mesh electrode system that establishes an intimate in vitro electrical interface with human neurons in 3D organoids. Our mesh is constructed with poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) based electrically conductive hydrogel electrode arrays and elastomeric poly(styrene-ethylene-butylene-styrene) (SEBS) as the substrate and encapsulation materials. This mesh electrode can maintain a stable electrochemical impedance in buffer solution under 50% compressive and 50% tensile strain. We have successfully cultured pluripotent stem cell-derived human cortical organoids (hCO) on this polymeric mesh for more than 3 months and demonstrated that organoids readily integrate with the mesh. Using simultaneous stimulation and calcium imaging, we show that electrical stimulation through the mesh can elicit intensity-dependent calcium signals comparable to stimulation from a bipolar stereotrode. This platform may serve as a tool for monitoring and modulating the electrical activity of in vitro models of neuropsychiatric diseases.

Biophysical and Biomechanical Effect of Low Intensity US Treatments on Pancreatic Adenocarcinoma 3D Cultures

Current developments in medical technology have focused on therapeutic treatments that selectively and effectively address specific pathological areas, minimizing side effects on healthy tissues. In this regard, many procedures have been developed to provide non-invasive therapy, for example therapeutic ultrasound (US). In the medical field, in particular in cancer research, it has been observed how ultrasounds can cause cell death and inhibit cell proliferation of cancer cells, while preserving healthy ones with almost negligible side effects. Various studies have shown that low intensity pulse ultrasound (LIPUS) and low intensity continuous ultrasound (LICUS) regulate the proliferation, cell differentiation and cavitation phenomena. Nowadays, there are poorly known aspects of low intensity US treatment, in terms of biophysical and biomechanical effects on target cells. The aim of this study is to set up an innovative apparatus for US treatment of pancreatic ductal adenocarcinoma (PDAC) cells, monitoring parameters such as acoustic intensity, acoustic pressure, stimulation frequency and treatment protocol. To this purpose, we have developed a custom-made set up for the US stimulation at 1.2 and 3 MHz of tridimensional (3D) cultures of PDAC cells (PANC-1, Mia Paca-2 and BxPc3 cells). Images of the 3D cultures were acquired, and the Calcein/PI assay was applied to detect US-induced cell death. Overall, the setup we have presented paves the way to an innovative protocol for tumor treatment. The system can be used either alone or in combination with small molecules or recombinant antibodies in order to propose a novel combined therapeutic approach.

In Vitro Biological Performance of Alginate Hydrogel Capsules for Stem Cell Delivery.

Encapsulation of biological components in hydrogels is a well described method for controlled drug delivery of proteins, tissue engineering and intestinal colonization with beneficial bacteria. Given the potential of tissue engineering in clinical practice, this study aimed to evaluate the feasibility of encapsulation of adipose tissue-derived mesenchymal stem cells (MSCs) of mules in sodium alginate. We evaluated capsule morphology and cell viability, immunophenotype and release after encapsulation. Circular and irregular pores were observed on the hydrogel surface, in which MSCs were present and alive. Capsules demonstrated good capacity of absorption of liquid and cell viability was consistently high through the time points, indicating proper nutrient diffusion. Flow cytometry showed stability of stem cell surface markers, whereas immunohistochemistry revealed the expression of CD44 and absence of MHC-II through 7 days of culture. Stem cell encapsulation in sodium alginate hydrogel is a feasible technique that does not compromise cell viability and preserves their undifferentiated status, becoming a relevant option to further studies of tridimensional culture systems and in vivo bioactive agents delivery.

Simulated microgravity promotes the formation of tridimensional cultures and stimulates pluripotency and a glycolytic metabolism in human hepatic and biliary tree stem/progenitor cells

Many pivotal biological cell processes are affected by gravity. The aim of our study was to evaluate biological and functional effects, differentiation potential and exo-metabolome profile of simulated microgravity (SMG) on human hepatic cell line (HepG2) and human biliary tree stem/progenitor cells (hBTSCs). Both hBTSCs and HepG2 were cultured in a weightless and protected environment SGM produced by the Rotary Cell Culture System (Synthecon) and control condition in normal gravity (NG). Self-replication and differentiation toward mature cells were determined by culturing hBTSCs in Kubota’s Medium (KM) and in hormonally defined medium (HDM) tailored for hepatocyte differentiation. The effects on the expression and cell exo-metabolome profiles of SMG versus NG cultures were analyzed. SMG promotes tridimensional (3D) cultures of hBTSCs and HepG2. Significative increase of stemness gene expression (p < 0.05) has been observed in hBTSCs cultured in SMG when compared to NG condition. At the same time, the expression of hepatocyte lineage markers in hBTSCs differentiated by HDM was significantly lower (p < 0.05) in SMG compared to NG, demonstrating an impaired capability of hBTSCs to differentiate in vitro toward mature hepatocytes when cultured in SMG condition. Furthermore, in HepG2 cells the SMG caused a lower (p < 0.05 vs controls) transcription of CYP3A4, a marker of late-stage (i.e. Zone 3) hepatocytes. Exo-metabolome NMR-analysis showed that both cell cultures consumed a higher amount of glucose and lower glutamate in SMG respect to NG (p < 0.05). Moreover, hBTSCs media cultures resulted richer of released fermentation (lactate, acetate) and ketogenesis products (B-hydroxybutyrate) in SGM (p < 0.05) than NG. While, HepG2 cells showed higher consumption of amino acids and release of ketoacids (3-Methyl-2-oxovalerate, 2-oxo-4-methyl-valerate) and formiate with respect to normogravity condition (p < 0.05). Based on our results, SMG could be helpful for developing hBTSCs-derived liver devices. In conclusion, SMG favored the formation of hBTSCs and HepG2 3D cultures and the maintenance of stemness contrasting cell differentiation; these effects being associated with stimulation of glycolytic metabolism. Interestingly, the impact of SMG on stem cell biology should be taken into consideration for workers involved in space medicine programs.

Characterization of a novel decellularized bone marrow scaffold as an inductive environment for hematopoietic stem cells.

Due to the increasing demand for a bone marrow study model, we developed a natural scaffold from decellularized bovine bone marrow (DeBM). The obtained bioscaffold was analyzed after the decellularization process; histological staining, scanning and transmission electron microscopy confirmed the preservation of its native 3D-architecture; including blood vessels and cell niches as well as the integrity of important components of the extracellular matrix; Collagen III, IV and fibronectin. In addition to biochemical composition, physical properties of the bone marrow were also conserved. We evaluated the suitability of this bio-scaffold as a tridimensional culture platform. Seeding experiments with umbilical cord-derived hematopoietic stem cells and human bone marrow stromal cell line HS5 demonstrated that this scaffold is capable of supporting hematopoietic and stromal cell adhesion and proliferation without the need of exogenous factors. DeBM provided an inductive environment for the repopulation of the bone marrow inducing the expression of SDF-1, HGF and SCF by seeded stromal cells. The presence of these potent hematopoietic chemoattractants would be crucial for ex vivo long-term culture of HSCs, and for recreating the natural microenvironment of the bone marrow for bioengineering applications. We conclude that the decellularization process succeeded in preserving the 3D structure and mechanical properties of the bone marrow. The resulting scaffold is suitable for cell culture, representing an advantageous bone marrow experimental model, and potentially an effective platform for CD34+ HSC expansion and differentiation for clinical applications.

Mouse placental scaffolds: a three-dimensional environment model for recellularization

The rich extracellular matrix (ECM) and availability make placenta eligible as alternative biomaterial source. Herein we produced placental mouse scaffolds by decellularization, and structure, composition, and cytocompatibility were evaluated to be considered as a biomaterial. We obtained a cell-free scaffold containing 9.42 ± 5.2 ng dsDNA per mg of ECM, presenting well-preserved structure and composition. Proteoglycans were widespread throughout ECM without cell nuclei and cell remnants. Collagen I, weak in native placenta, clearly appears in the scaffold after recellularization, opposite distribution was observed for collagen III. Fibronectin was well-observed in placental scaffolds whereas laminin and collagen IV were strong expressed. Placental scaffolds recellularization potential was confirmed after mouse embryonic fibroblasts 3D dynamic culture, resulting in massive scaffold repopulation with cell–cell interactions, cell-matrix adhesion, and maintenance of natural morphology. Our small size scaffolds provide a useful tool for tissue engineering to produce grafts and organ fragments, as well as for cellular biology purposes for tridimensional culture substrate.

MYPT1 is targeted by miR-145 inhibiting viability, migration and invasion in 2D and 3D HeLa cultures.

The miR-143/145 cluster is down-regulated in cervical tumor cells suggesting a role in tumorigenesis including cytoskeleton remodeling, a key event for tumor progression. The aim of the present work was to determine the role of miR-143/145 in the modulation of the myosin regulator phospho-myosin light chain (pMLC). HeLa monolayer and tridimensional cultures were transfected with miR-143 or miR-145 mimics inhibiting cell viability, proliferation, migration and invasion, mainly through miR-145. MiR-145 transfection increased pMLC levels by targeting the MYPT1 subunit of the regulatory myosin phosphatase. MYPT1 knockdown by siRNAs reproduced miR-145 effects suggesting miR-145 as a tumor suppressor through MYPT1 targeting, leading to a subsequent increase of pMLC levels with implications for cervical cell viability, migration and invasion.

High-throughput 3-dimensional culture of epithelial ovarian cancer cells as preclinical model of disease

BackgroundRecent reports have identified distinct genomic patterns in ovarian carcinoma, including proliferative and mesenchymal-like groups, with worse outcome. The exact mechanisms driving the onset and progression of these tumors are still poorly understood. Additionally, researchers are concerned about the correct subtype stratification of the available cell line models, and the exploration of alternatives to monolayer culture. Identification of biomarkers to stratify cell lines, characterization of important processes as epithelial-mesenchymal transition (EMT), and the use of three-dimensional (3D) cultures as alternative models could be useful for cell line classification.Methods and ResultsIn this work, we present a descriptive analysis of 16 commonly used ovarian cancer cell lines. We have studied their morphology in 2- and 3D culture, and their response to cisplatin, observing in the majority of them an increased resistance in 3D. We have also performed an immunohistochemical analysis for proliferation marker Ki-67, and EMT related markers to establish phenotypes. Epithelial cells tend to show higher proliferative rates, and mesenchymal cells show an increase in EMT related markers, especially when cultured in 3D conditions.ConclusionsWe have stated the complex heterogeneity of ovarian cancer models, resembling primary tumors, agreeing with the argument that the cell line model for in vitro experiments must be carefully chosen. Our results also support that tridimensional culture could be a very helpful alternative in ovarian cancer research. Regarding EMT, a very important process for the development of this disease, some related biomarkers might be further characterized for their role in this disease development.

Characterization of Gastrospheres Using 3D Coculture System.

To understand the molecular mechanisms involved in gastric disorders and regeneration, we need an in vitro tridimensional (3D) culture model, which can mimic the in vivo gastric microenvironment. A 3D coculture system named gastrosphere is proposed herein, composed of primary human gastric epithelial and stromal cells. The primary cultures were obtained from endoscopic gastric biopsies, and after mechanical and enzymatic dispersion, epithelial (HGE3) and stromal (HGS12) cells were expanded. After extensive immunocytochemical characterization, cells were seeded onto 96-well round bottom plates previously covered with 1% agarose. Cells were cultured in KM-F12 culture medium with 10% fetal bovine serum (FBS), antibiotics, and antimycotics, in humidified air at 37°C and atmosphere containing 5% CO2 for 72h or until spheres formation. Then gastrospheres were carefully transferred to a rotary cell culture system (RCCS-4), and maintained for 07, 14, 21, and 28days. Gastrospheres were morphologically characterized by immunocytochemistry [cytokeratins (CK), vimentin, α-smooth muscle actin (α-SMA), laminin (LN), fibronectin (FN), and type IV collagen (CIV), proliferating cell nuclear antigen (PCNA)], and electron microscopy. In gastrospheres, the cytokeratin-positive epithelial cells were found in the outer layer, while vimentin-positive stromal cells were localized in the center of the gastrospheres. PCNA+ cells were mainly seen at the peripheral and in the intermediary region while nestin+ cells were also depicted in the latter zone. Scanning electron microscopy revealed groups of cohesive gastric cells at the periphery, while transmission electron microscopy demonstrated some differentiated mucous-like or zymogenic-like cells in the periphery and stromal structures located at the center of the 3D structures. Extracellular matrix was deposed between cells. Our data suggest that in vitro gastrospheres recapitulate the in vivo gastric microenvironment.

In Vitro Modeling of Tumor-Immune System Interaction.

Immunotherapy has emerged during the past two decades as an innovative and successful form of cancer treatment. However, frequently, mechanisms of actions are still unclear, predictive markers are insufficiently characterized, and preclinical assays for innovative treatments are poorly reliable. In this context, the analysis of tumor/immune system interaction plays key roles, but may be unreliably mirrored by in vivo experimental models and standard bidimensional culture systems. Tridimensional cultures of tumor cells have been developed to bridge the gap between in vitro and in vivo systems. Interestingly, defined aspects of the interaction of cells from adaptive and innate immune systems and tumor cells may also be mirrored by 3D cultures. Here we review in vitro models of cancer/immune cell interaction and we propose that updated technologies might help develop innovative treatments, identify biologicals of potential clinical relevance, and select patients eligible for immunotherapy treatments.

Ex vivo Culture of Adult Mouse Antral Glands.

The tri-dimensional culture, initially described by Sato et al. (2009) in order to isolate and characterize epithelial stem cells of the adult small intestine, has been subsequently adapted to many different organs. One of the first examples was the isolation and culture of antral stem cells by Barker et al. (2010), who efficiently generated organoids that recapitulate the mature pyloric epithelium in vitro. This ex vivo approach is suitable and promising to study gastric function in homeostasis as well as in disease. We have adapted Barker's protocol to compare homeostatic and regenerating tissues and here, we meticulously describe, step by step, the isolation and culture of antral glands as well as the isolation of single cells from antral glands that might be useful for culture after cell sorting as an example (Fernandez Vallone et al., 2016 ).

Ex vivo Culture of Fetal Mouse Gastric Epithelial Progenitors.

Isolation and tridimensional culture of murine fetal progenitors from the digestive tract represents a new approach to study the nature and the biological characteristics of these epithelial cells that are present before the onset of the cytodifferentiation process during development. In 2013, Mustata et al. described the isolation of intestinal fetal progenitors growing as spheroids in the ex vivo culture system initially implemented by Sato et al. (2009) to grow adult intestinal stem cells. Noteworthy, fetal-derived spheroids have high self-renewal capacity making easy their indefinite maintenance in culture. Here, we report an adapted protocol for isolation and ex vivo culture and maintenance of fetal epithelial progenitors from distal pre-glandular stomach growing as gastric spheroids (Fernandez Vallone et al., 2016 ).

IKKα regulates the stratification and differentiation of the epidermis: implications for skin cancer development.

IKKα plays a mandatory role in keratinocyte differentiation and exerts an important task in non-melanoma skin cancer development. However, it is not fully understood how IKKα exerts these functions. To analyze in detail the role of IKKα in epidermal stratification and differentiation, we have generated tridimensional (3D) cultures of human HaCaT keratinocytes and fibroblasts in fibrin gels, obtaining human skin equivalents that comprise an epidermal and a dermal compartments that resembles both the structure and differentiation of normal human skin. We have found that IKKα expression must be strictly regulated in epidermis, as alterations in its levels lead to histological defects and promote the development of malignant features. Specifically, we have found that the augmented expression of IKKα results in increased proliferation and clonogenicity of human keratinocytes, and leads to an accelerated and altered differentiation, augmented ability of invasive growth, induction of the expression of oncogenic proteins (Podoplanin, Snail, Cyclin D1) and increased extracellular matrix proteolytic activity. All these characteristics make keratinocytes overexpressing IKKα to be at a higher risk of developing skin cancer. Comparison of genetic profile obtained by analysis of microarrays of RNA of skin equivalents from both genotypes supports the above described findings.

Cell death in human articular chondrocyte: a morpho-functional study in micromass model.

Chondrocyte death and loss of extracellular matrix are the central features in articular cartilage degeneration during osteoarthritis pathogenesis. Cartilage diseases and, in particular, osteoarthritis are widely correlated to apoptosis but, chondrocytes undergoing apoptosis "in vivo" more often display peculiar features that correspond to a distinct process of programmed cell death termed "chondroptosis". Programmed cell death of primary human chondrocyte has been here investigated in micromasses, a tridimensional culture model, that represents a convenient means for studying chondrocyte biology. Cell death has been induced by different physical or chemical apoptotic agents, such as UVB radiation, hyperthermia and staurosporine delivered at both 1 and 3 weeks maturation. Conventional electron microscopy was used to analyse morphological changes. Occurrence of DNA fragmentation and caspase involvement were also investigated. At Transmission Electron Microscopy, control cells appear rounding or slightly elongated with plurilobated nucleus and diffusely dispersed chromatin. Typically UVB radiation and staurosporine induce chromatin apoptotic features, while hyperthermia triggers the "chondroptotic" phenotype. A weak TUNEL positivity appears in control, correlated to the well known cell death patterns occurring along cartilage differentiation. UVB radiation produces a strong positivity, mostly localized at the micromass periphery. After hyperthermia a higher number of fluorescent nuclei appears, in particular at 3 weeks. Staurosporine evidences a diffuse, but reduced, positivity. Therefore, DNA fragmentation is a common pattern in dying chondrocytes, both in apoptotic and "chondroptotic" cells. Moreover, all triggers induce caspase pathway activation, even if to a different extent, suggesting a fundamental role of apoptotic features, in chondrocyte cell death.

Label-free analysis of prostate acini-like 3D structures by lensfree imaging

We present a lensfree imaging method to analyze polarity in RWPE1 prostate epithelial cells that form polarized acini with lumen under standard tridimensional (3D) culture conditions. The first event in epithelial carcinogenesis is loss of polarity, followed by uncontrolled proliferation leading to metastasis. We demonstrate that it is possible to use optical signatures to discriminate 3D objects with distinct polarities in a large field of view. The three metrics we present here are designed as image processing tools to discriminate acini from spheroids without any 3D reconstruction. To demonstrate that our lensfree imaging platform may be used to study the 3D organization of epithelial cells, we analyzed and quantified the modulation of dynamic processes, e.g., the polarity of acini and the merging of polarized structures, upon transforming growth factor beta-1 (TGF beta-1) addition to the culture media. Hence, coupling lensfree microscopy with 3D cell culture provides an innovative tool to study epithelial tissue morphogenesis in a large field of view and to elucidate the regulation of growth, morphogenesis and differentiation in normal and cancerous human prostate cells. Moreover, such biosensor would be a powerful tool to follow cancer progression and to evaluate anti-cancer drugs.

Abstract 226: Trastuzumab effect on cell death and proliferation of HER2+ mammary adenocarcinoma cells using a tridimensional culture

Abstract HER2 is overexpressed in 20-25% invasive breast tumors, and associated with low free disease survival. Trastuzumab (Tz), monoclonal antibody anti HER2, is used as immunotherapy to treat HER2+ tumors; however more than half of them are resistant or acquire resistance during treatment. Cellular spheroids are a model of cell growth in 3D that mimic the structure of in vivo avascular tumors. We have previously demonstrated that tumor spheroids (TS) present different subpopulations, with a gradient of proliferative, quiescente and apoptotic cells from the outer rim towards the hypoxic core. The aim of this study was to analyze the biological characteristics of the different cell populations that constitute the TS and their response to Tz. TS of HER2+ human mammary tumor cells (cell line BT474) were cultured as cell suspensions on agar (hanging drop method) in 48-well plates (1 spheroid / well). Cell cycle at days 7, 14 and 21 of TS growth was analyzed by flow cytometry. A significant increase only in the G2/M phase (6.6%, 10.3% and 13.3% respectively) was detected. TS were incubated with Tz at a concentration of 50ug/ml; a commercial IgG (50ug/mL) was used as negative control. The effect on cell growth kinetics was evaluated with photographs taken at different times, observing a reduction of the spheroids volume until they reached a decrease by 71% at day 20 (p&amp;lt;0.01 vs controls). Tz induced an increase in the% of cells arrested in G1 (80.8% vs 64.7% control IgG, p&amp;lt;0.001) and a decrease in the proliferative subpopulation (7.4% vs 11.4%, p&amp;lt;0.05). A decrease in the% of apoptotic cells was also detected (Annexin V/IP). When we analyzed cell viability at different time points, we observed that Tz elicited changes in live to dead cells ratio. A reversion of the inhibitory effect of Tz on TS growth was observed after antibody withdrawal. Spheroids were fixed and included in paraffin to analyze the expression of HER2, pHER2, clived caspase 3, HIF-1a, p27, p21 and cyclinD1 by confocal microscopy. HER2 expression was not modulated by Tz; however, we observed lower expression of its activated receptor (pHER2). HIF-1a was expressed only in cells around the central core while clived caspase 3 was expressed in the central core. After Tz incubation, both markers were no longer detected. We can conclude that Tz exerted a potent antiproliferative and direct cytotoxic effect on BT474 tumor cells growing in 3D; these activities might be due to the partial inhibition of HER2 activation. Tz inhibited the formation of the hypoxic core and therefore the apoptotic subpopulation, generating thereby a smaller TS composed only of remaining Tz-resistant living cells. We propose that this model could be useful to study the mechanisms involved in the effect and on the resistance to Tz. Citation Format: Cristina E. Rodríguez, Sara Reidel, Luciana Moverer, Lina Marino, Elisa Bal de Kier Joffé, Maria A. Jasnis, Gabriel L. Fiszman. Trastuzumab effect on cell death and proliferation of HER2+ mammary adenocarcinoma cells using a tridimensional culture. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 226. doi:10.1158/1538-7445.AM2013-226