Nonadherent Surface Research Articles

“A lactose-modified chitosan accelerates chondrogenic differentiation in mesenchymal stem cells spheroids”

Spheroids derived from human mesenchymal stem cells (hMSCs) are of limited use for cartilage regeneration, as the viability of the cells progressively decreases during the period required for chondrogenic differentiation (21 days). In this work, spheroids based on hMSCs and a lactose-modified chitosan (CTL) were formed by seeding cells onto an air-dried coating of CTL. The polymer coating can inhibit cell adhesion and it is simultaneously incorporated into spheroid structure. CTL-spheroids were characterized from a morphological and biological perspective, and their properties were compared with those of spheroids obtained by seeding the cells onto a non-adherent surface (agar gel). Compared to the latter, smaller and more viable spheroids form in the presence of CTL as early as 4 days of culture. At this time point, analysis of stem cells differentiation in spheroids showed a remarkable increase in collagen type-2 (COL2A1) gene expression (~700-fold compared to day 0), whereas only a 2-fold increase was observed in the control spheroids at day 21. These results were confirmed by histological and transmission electron microscopy (TEM) analyses, which showed that in CTL-spheroids an early deposition of collagen with a banding structure already occurred at day 7. Overall, these results support the use of CTL-spheroids as a novel system for cartilage regeneration, characterized by increased cell viability and differentiation capacity within a short time-frame. This will pave the way for approaches aimed at increasing the success rate of procedures and reducing the time required for tissue regeneration.

Research on the Corrosion Resistance of an Epoxy Resin-Based Self-Healing Propylene Glycol-Loaded Ethyl Cellulose Microcapsule Coating

In this work, ethyl cellulose was used as a wall material, propanetriol as a core material, polyvinyl alcohol as a stabilizer and gelatin as an emulsifier. Self-healing microcapsules with a slow-release effect were prepared using the solvent evaporation method. Various analytical techniques, such as 3D confocal microscopy (LCSM), optical microscopy (OM), scanning electron microscopy (SEM), infrared spectroscopy (FT-IR), energy dispersive spectroscopy (EDS), thermal weight loss analysis (TGA), laser particle size tester and electrochemical impedance polarization, are utilized. The morphology, distribution, particle size, corrosion resistance and self-healing ability of the prepared microcapsules and resin-based coatings were characterized and analyzed. The results show that the cross-sectional core–shell structure is clearly seen in the LCSM, showing a smooth, hollow, spherical shape. OM and laser particle size testers have shown that the size of the microcapsules decreases over time. Also, in OM, the microcapsules are uniformly distributed in the emulsion with a smooth and non-adherent surface. In SEM, the microcapsule particle size is about 150 μm, the shell wall thickness is about 18 μm, and the hollow structure of ruptured microcapsules is obvious. FT-IR and TGA confirmed the successful encapsulation of the formulated microcapsules. The results show that when the core-wall mass ratio is 1.2:1 and the amount of microcapsule is 10% of the coating amount, the prepared microcapsule has high thermal stability and certain wear resistance. By electrochemical and immersion experiments, it was found that a 3.5 wt % NaCl solution has the best impedance, the lowest corrosion current density, and good adhesion and tensile toughness. The results showed that glycerol was successfully released from the broken microcapsules and self-healed, forming an anticorrosive coating with excellent corrosion resistance and self-healing ability.

Nanofabrication Technologies to Control Cell and Tissue Function in Three-Dimension.

In the 2000s, advances in cellular micropatterning using microfabrication contributed to the development of cell-based biosensors for the functional evaluation of newly synthesized drugs, resulting in a revolutionary evolution in drug screening. To this end, it is essential to utilize cell patterning to control the morphology of adherent cells and to understand contact and paracrine-mediated interactions between heterogeneous cells. This suggests that the regulation of the cellular environment by means of microfabricated synthetic surfaces is not only a valuable endeavor for basic research in biology and histology, but is also highly useful to engineer artificial cell scaffolds for tissue regeneration. This review particularly focuses on surface engineering techniques for the cellular micropatterning of three-dimensional (3D) spheroids. To establish cell microarrays, composed of a cell adhesive region surrounded by a cell non-adherent surface, it is quite important to control a protein-repellent surface in the micro-scale. Thus, this review is focused on the surface chemistries of the biologically inspired micropatterning of two-dimensional non-fouling characters. As cells are formed into spheroids, their survival, functions, and engraftment in the transplanted site are significantly improved compared to single-cell transplantation. To improve the therapeutic effect of cell spheroids even further, various biomaterials (e.g., fibers and hydrogels) have been developed for spheroid engineering. These biomaterials not only can control the overall spheroid formation (e.g., size, shape, aggregation speed, and degree of compaction), but also can regulate cell-to-cell and cell-to-matrix interactions in spheroids. These important approaches to cell engineering result in their applications to tissue regeneration, where the cell-biomaterial composite is injected into diseased area. This approach allows the operating surgeon to implant the cell and polymer combinations with minimum invasiveness. The polymers utilized in hydrogels are structurally similar to components of the extracellular matrix in vivo, and are considered biocompatible. This review will provide an overview of the critical design to make hydrogels when used as cell scaffolds for tissue engineering. In addition, the new strategy of injectable hydrogel will be discussed as future directions.

Effect of Mir-4270 Inhibitor and Mimic on Viability and Stemness in Gastric Cancer Stem-Like Cells Derived from MKN-45 Cell Line.

MicroRNAs (miRNAs) are significant regulatory factors in stem cell proliferation, and change in miRNA expression influences the cancer stem cell viability and gene expression. Herein, we evaluated the effect of the hsa-miR-4270 inhibitor and its mimic on the expression of stem cell markers in gastric cancer (GC) stem-like cells. GC stem-like cells were isolated from the MKN-45 cell line by a non-adherent surface system. The cells were confirmed by differentiation assays using dexamethasone and insulin as adipogenesis-inducing agents and also Staurosporine as a neural-inducing agent. Isolated GC stem-like cells were treated with different concentrations (0, 15, 20, 25, 30, 40, 50, and 60 nM) of hsa-miR-4270 inhibitor and its mimic. The quantity of cell viability was determined by trypan blue method. Transcription of the stem cell marker genes, including CD44, OCT3/4, SOX2, Nanog, and KLF4, was evaluated by real-time RT-PCR. The results showed that GC stem-like cells were differentiated into both adipose cells using dexamethasone and insulin and neural cells by Staurosporine. Treatment of GC stem-like cells with hsa-miR-4270 inhibitor decreased cell viability and downregulated OCT3/4, CD44, and Nanog to 86%, 79%, and 91% respectively. Also, SOX2 and KLF4 were overexpressed to 8.1- and 1.94-folds, respectively. However, hsa-miR-4270 mimic had opposite effects on the cell viability and gene expression of the stem cell markers. The effect of hsa-miR-4270 inhibitor and its mimic on the expression of the stem cell markers in GCSCs indicated that hsa-miR-4270 stimulates the stemness property of GCSCs, likely through stimulating the development of gastric stem cells.

The Effect of Pantoprazole on Tumor Growth and Apoptosis in Gastric Cancer Stem-Like Cells

Background: Gastric cancer is a daunting global problem with unsatisfactory treatment. Due to the key role of Gastric Cancer Stem-like Cells (GCSCs) in all stages of gastric cancer and the failure of contemporary anticancer therapies, many research efforts are focusing on these treatmentresistant cells. Pantoprazole, as recently considered antitumor agent with well-documented effects on tumorigenesis inhibition, has seldom been investigated in GCSCs in previous studies. We aimed to study the influence of pantoprazole on cell proliferation, apoptosis, and the transcription of genes involved in the cell proliferation and apoptosis pathways. Materials and Methods: Herein, we isolated GCSCs from MKN-45 cell line, on a non-adherent surface and then evaluated the effect of pantoprazole on cell growth and apoptosis of GCSCs by means of MTT, DNA laddering and quantitative real-time RT-PCR techniques. Results: Our findings showed that treated cells with pantoprazole decreased cell proliferation and induced apoptosis. PCNA (Proliferating Cell Nuclear Antigen) and antiapoptotic Bcl2 genes were downregulated and Bax and CASP3 proapoptotic genes, as well as tumor suppressor p53 gene, were overexpressed. Conclusion: Our results revealed that pantoprazole induced apoptosis and declined tumor growth and support the idea that pantoprazole played as a promising breakthrough in gastric cancer therapy.

Development of plant-based biopolymer coatings for 3D cell culture: boron-silica-enriched quince seed mucilage nanocomposites.

Spheroid formation with spontaneous aggregation has captured interest in most cell culture studies due to its easy set-up and more reliable results. However, the economic and technical costs of the advanced systems and commercial ultra-low adhesive platforms have pushed researchers into pursuing alternatives. Nowadays, polymeric coatings, including poly-hydroxyethyl methacrylate and agar/agarose, are the commonly used polymers for non-adhesive plate fabrication, yet the costs and working solvent or heat-dependent preparation procedures maintain the need for the development of novel biomaterials. Here, we propose a greener and more economical approach for producing non-adherent surfaces and spheroid formation. For this, a plant waste-based biopolymer from quince fruit (Cydonia oblonga Miller, from Rosaceae family) seeds and boron-silica precursors were introduced. The unique water-holding capacity of quince seed mucilage (Q) was enriched with silanol and borate groups to form bioactive and hydrophilic nanocomposite overlays for spheroid studies. Moreover, 3D gel plates from the nanocomposite material were fabricated and tested in vitro as a proof-of-concept. The surface properties of coatings and the biochemical and mechanical properties of the nanocomposite materials were evaluated in-depth with techniques, and extra hydrophilic coatings were obtained. Three different cell lines were cultured on these nanocomposite surfaces, and spheroid formation with increased cellular viability was recorded on day 3 with a >200 μm spheroid size. Overall, Q-based nanocomposites are believed to be a fantastic alternative for non-adherent surface fabrication due to their low-cost, easy operation, and intrinsic hydration layer forming capacity with biocompatible nature in vitro.

Multiplexed Viability Assays for High-Throughput Screening of Spheroids of Multiple Sizes.

High-throughput (HT) drug screening is in high demand for successful drug discovery and personalized medicine. Spheroids act as a promising preclinical model for HT drug screening, which may decrease drug failures in clinical trials. Numerous spheroid-forming technological platforms are currently under development, which include synchronous, jumbo-sized, hanging drop, rotary, and nonadherent surface spheroid growth. Initial cell seeding concentration and time of culture play a vital role for spheroids to mimic the extracellular microenvironment of natural tissue, especially for HT preclinical evaluation. Hence microfluidic platforms become a potential technology to provide a confined space for the oxygen and nutrient gradients within the tissues while controlling the cell count and spheroid size in an HT manner. We describe here a microfluidic platform capable of generating spheroids of multiple sizes in a controlled manner with a predefined cell concentration for HT drug screening. Ovarian cancer spheroids grown on this microfluidic platform were evaluated for viability using a confocal microscope and flow cytometer. In addition, screening of the HT chemotherapeutic drug carboplatin was carried out on-chip to evaluate the impact of spheroid size on drug toxicity. This chapter summarizes a detailed protocol on microfluidic platform fabrication for spheroid growth, on-chip multi-sized spheroid analysis, and chemotherapeutic drug screening.

Discriminating bladder cancer cells through rheological mechanomarkers at cell and spheroid levels

The stiffening or softening of cancers observed in nanoindentation experiments has been recognized as a marker of cancer-related changes. In bladder cancers, continuous stretching/destretching is observed due to its functionality, indicating that shear forces dominate the mechanical response of these cells. Thus, nanoindentation and microrheological measurements conducted in parallel allow for a fully reliable mechanomarker of cancer progression. Here, bladder cancer cell lines, i.e., non-malignant cell cancer of the ureter (HCV29), bladder carcinoma (HT1376), and transitional cell carcinoma (T24), were studied. Nanoindentation and microrheological experiments were conducted on individual cells, cell monolayers, and spheroids that were formed using non-adherent surface plates. The results show that nanoindentation experiments can only differentiate between non-malignant HCV29 (stiffer) and cancerous HT1376 and T24 (softer) cells. Applying microrheology recognizes the type of grade 3 bladder cancers (carcinoma HT1376 or transitional cell carcinoma T24 cells). We showed that actin filaments are a vital element defining the rheological properties of spheroids. Differences in mechanical properties of cell monolayers could be associated with thick actin bundles and intercellular connections, with some extracellular matrix (ECM) contributing to the stiffening of such monolayers. Our findings demonstrate that a complete image of how cancer cells respond to mechanical stress (compressive and shear forces) can only be obtained after microrheological measurements using the transition frequency separating elastic and viscous regimes as a non-labeled biomarker of bladder cancer progression.

Non-coating method for non-adherent cell culture using high molecular weight dextran sulfate and bovine serum albumin

Non-adherent cell culture surface has been widely used for producing cell spheroids and cell aggregates. The purpose of this study was to formulate a new method for non-adherent cell culture without coating or surface-modification that has been needed. We found that high-molecular-weight dextran sulfate (DS) and bovine serum albumin (BSA) synergistically prevented cell adhesion in media supplemented with no or low serum. This method worked on tissue culture-treated polystyrene surfaces as well as on commercially available low-attachment- and untreated polystyrene surfaces. Further investigation revealed that BSA may mediate the adsorption of DS to the surface. In addition, as the adsorption of fluorescently labeled fibronectin was inhibited by BSA alone, it appears that protein adsorption and cell adhesion do not always correlate. Finally, we demonstrated the successful formation of HepG2 spheroids and cardiomyocyte aggregates using this method. In conclusion, cell adhesion can be effectively suppressed by simply adding DS and BSA to the culture medium without coating or surface modification, and it may be useful for generating cell spheroids and aggregates.

The Culture Dish Surface Influences the Phenotype and Cytokine Production of Human Monocyte-Derived Dendritic Cells.

Monocyte-derived dendritic cells (moDC) are an important scientific and clinical source of functional dendritic cells (DC). However, the optimization of the generation process has to date mainly been limited to the variation of soluble factors. In this study, we investigated the impact of the cell culture dish surface on phenotype and cytokine profile. We compared a standard cell culture dish to a non-adherent culture dish for two immunogenic maturation conditions, two tolerogenic conditions, and an unstimulated control. Phenotype, cytokine profile and T cell stimulatory capacity were determined after a 3-day culture. Light microscopy revealed an increase in homotypic cluster formation correlated with the use of non-adherent surfaces, which could be reduced by using blocking antibodies against CD18. All surface markers analyzed showed moderate to strong differences depending on the culture dish surface, including significantly decreased expression of key maturation markers such as CD80, CD86, and CCR7 as well as PD-L1 on cells stimulated with the Jonuleit cytokine cocktail cultured on a non-adherent surface. Significant differences in the secretion of many cytokines were observed, especially for cells stimulated with LPS, with over 10-fold decreased secretion of IL-10, IL12-p40, and TNF-α from the cells cultured on the non-adherent surface. All immunogenic moDC populations showed similar capacity to induce antigen-specific T cells. These results provide evidence that the DC phenotype depends on the surface used during moDC generation. This has important implications for the optimization of DC-based immunotherapy development and underlines that the local surrounding can interfere with the final DC population beyond the soluble factors.

Bone Sialoprotein Shows Enhanced Expression in Early, High-Proliferation Stages of Three-Dimensional Spheroid Cell Cultures of Breast Cancer Cell Line MDA-MB-231.

Normally, bone sialoprotein (BSP) is an important contributor to bone micro-calcification. However, it is also highly expressed in bone-metastatic malignancies, including prostate, lung, and breast cancer. In these disorders, BSP correlates with poor prognosis. Its expression in triple-negative breast cancer cells is enhanced by the transcription factor RUNX2, and both, BSP and RUNX2 are under control of IGF-1 and TGFβ1. Knockdown of BSP or its inactivation by specific antibodies were found to reduce the metastatic potential of MDA-MB-231 triple-negative breast cancer cells in xenografts. While the role of BSP in bone metastasis was studied using such in vivo models, valid in vitro test systems to investigate BSP biology have been lacking since this protein is expressed at very low levels in classical 2D cell cultures and the frequently used breast cancer cell line MDA-MB-231 is difficult to grow in 3D. Here, we have developed a long-term 3D spheroid culture model using MDA-MB-231 cells in a sandwich approach using cell embedding between a non-adherent surface and basement membrane extracts. This allowed consistent growth of spheroids for more than 21 days. Also, co-culturing of MDA-MB-231 with CCD-1137Sk fibroblasts yielded stably growing spheroids, suggesting the importance of extracellular matrix (ECM) in this process. In addition, we have set up a novel and simple open source analysis tool to characterize protein expression in 2D cultures and spheroids by immunofluorescence. Using this approach in combination with Western blot analysis, the expression profile of BSP was analyzed. BSP was enriched at the rims of spheroids, both in mono- and co-cultures and its abundance in general correlated with that of TGFβ1 under different conditions, including spheroid maturation, cytostatic treatment, and fibroblast co-culture. Conversely, correlation of IGF-1 and BSP was limited to mono-culture time course profiles. In conclusion, we present novel tools to study the regulation of gene expression in combination with cell proliferation and apoptosis in a long-term 3D model of breast cancer and find dynamic abundance profiles of the metastasis-relevant protein BSP and its regulators.

Characteristics of multicellular tumor spheroids formed by pancreatic cells expressing different adhesion molecules

AimsMulticellular tumor spheroids (MCTS) produced by different methods vary in forms, sizes, and properties. The aim of this work was to characterize MCTS formed by six pancreatic cell lines on a non-adherent surface. Materials and methodsHuman pancreatic cells were grown in 2D and 3D conditions and compared for the expression of E- and desmosomal cadherins (PCR, confocal microscopy), growth, cell cycling, apoptosis (flow cytometry), and a response to antitumor drugs doxorubicin and gemcitabine (MTT-assay). Key findingsThree types of MCTS were identified: BxPC-3, T3M4 formed small number of large and dense spheroids representing type I MCTS; COLO-357 and AsPC-1 generated type II multiple and loose MCTS of different sizes while MiaPaCa-2 and PANC-1 represented type III cultures which grew almost as floating monolayer films. Formation of type I MCTS depended on the simultaneous expression of DSG3 and several DSC proteins; II MCTS expressed solely DSG2-DSC2 but not DSG3, while type III cells either did not express E-cadherin or a pair of DSG and DSC proteins. Cells in type I MCTS but not in types II and III ones quickly became quiescent which correlated with a decrease in the proliferation, increased apoptosis, and a higher resistance to antitumor drugs doxorubicin and gemcitabine. SignificanceTaken collectively, pancreatic cells significantly vary in the expression of desmosomal cadherins, resulting in the formation of MCTS with different characteristics. The sensitivity of MCTS to various drugs depends on the type of cells and the method of spheroid preparation used.

Non-Invasive Quantification of the Growth of Cancer Cell Colonies by a Portable Optical Coherence Tomography.

Investigation of tumor development is essential in cancer research. In the laboratory, living cell culture is a standard bio-technology for studying cellular response under tested conditions to predict in vivo cellular response. In particular, the colony formation assay has become a standard experiment for characterizing the tumor development in vitro. However, quantification of the growth of cell colonies under a microscope is difficult because they are suspended in a three-dimensional environment. Thus, optical coherence tomography (OCT) imaging was develop in this study to monitor the growth of cell colonies. Cancer cell line of Huh 7 was used and the cells were applied on a layer of agarose hydrogel, i.e., a non-adherent surface. Then, cell colonies were gradually formed on the surface. The OCT technique was used to scan the cell colonies every day to obtain quantitative data for describing their growth. The results revealed the average volume increased with time due to the formation of cell colonies day-by-day. Additionally, the distribution of cell colony volume was analyzed to show the detailed information of the growth of the cell colonies. In summary, the OCT provides a non-invasive quantification technique for monitoring the growth of the cell colonies. From the OCT images, objective and precise information is obtained for higher prediction of the in vivo tumor development.

Characterization of breast cancer stem cells isolated from 4T1 cell line cultured in vitro

Breast cancer is the most common cancer in women of which the main causes of death are its relapse and drug resistance. Recently, researchers clarified a small population of cancer cells holding the abilities of self-renewal, unlimited propagation responsible for tumor progression, metastasis, therapy resistance, and tumor recurrence. These cells are termed as cancer sterm cells (CSCs), possessing characteristics of stem cells. Thus, cancer stem cells have become to be a potential target for cancerous drug discovery. They can be used in a new research model for establishing novel therapy as well as sreening new compounds that target CSCs. In our studies, we isolated cancer stem cells from 4T1 cell line using serum-free medium and non-adherent culture surface. Those CSCs clones were formed after 10 culturing days from 1 initial cell. The clones could initiate subtumorsphere better than 4T1 cells themselves. There were 3 selected clones named as Đ3G5, Đ3C3 and Đ4E7 which had mammosphere formation efficiency over 30% (31.67%, 36.33%, and 35.33%, respectively). These clones showed the significant withstanding against ellipticine and/or doxorobicin stronger than that of 4T1 cells (P<0.05). The IC50 values (inhibition of cell growth at 50%) of elliticine was 0.89 ± 0.07 μg/mL and of doxorobicin was 0.58 ± 0.04 μg/mL toward 4T1 cell growth while those values were 2 - 3 folds higher in our isolated cancer stem cell clones (P<0.05). Those clones also exhibited strong expression of several typical genes of stem cells such as nanog and Sox2.

PlGF knockdown induced apoptosis through Wnt signaling pathway in gastric cancer stem cells.

Despite the fact that much research has focused on gastric cancer, it is still a worldwide concern, because of the difficulties with factors such as signaling pathway crosstalk and gastric cancer stem cell (GCSC). Placental growth factor (PlGF) is one of these factors, and its tumorigenicity potential still remains a question. As a result, we have investigated the effect of PlGF knockdown on apoptosis and genes involved in the Wnt signaling pathway, and apoptosis in cancer stem cells derived from AGS an MKN-45 gastric cancer cell lines. We isolated GCSCs from MKN-45 and AGS cell lines on a nonadherent surface. Then the cell viability, the real-time reverse transcription-polymerase chain reaction data of the genes involved in the Wnt signaling pathway, and apoptosis were evaluated. Furthermore, DNA laddering was used to show the apoptotic effect and DNA fragmentation caused by the PlGF knockdown. Our investigation revealed that the PlGF knockdown with PlGF-specific small interfering RNA at 40 pmol for GCSCs derived from MKN-45 and AGS at 24 hours can significantly affect the cell viability, the Wnt signaling pathway, and the apoptosis-related genes expression. In conclusion, we showed the PlGF knockdown may induce apoptosis via the Wnt signaling pathway in GCSCs.

A Combination of Two Receptor Tyrosine Kinase Inhibitors, Canertinib and PHA665752 Compromises Ovarian Cancer Cell Growth in 3D Cell Models

IntroductionAdvanced ovarian cancer is often a fatal disease as chemotherapeutic drugs have limited effectiveness. Better targeted therapy is needed to improve the survival and quality of life for these women. Receptor tyrosine kinases including EGFR, Her-2 and c-Met are associated with a poor prognosis in ovarian cancer. Therefore, the co-activation of these receptors may be crucial for growth promoting activity. In this study, we explored the effect of combining two small molecule inhibitors that target the EGFR/Her-2 and c-Met receptor tyrosine kinases in two ovarian cancer cell lines. The aim of this study was to investigate the combined inhibition activity of a dual EGFR/Her-2 inhibitor (canertinib) and a c-Met inhibitor (PHA665752) in ovarian cancer cell lines in 3D cell aggregates.MethodsOVCAR-5 and SKOV-3 ovarian cancer cell lines were cultured on a non-adherent surface to produce 3D cell clusters and aggregates. Cells were exposed to canertinib and PHA665752, both individually and in combination, for 48 h. The effect on growth, metabolism and the expression/phosphorylation of selective signaling proteins associated with EGFR, Her-2 and c-Met were investigated.ResultsThe single drug treatments significantly decreased cell growth and altered the expression of signaling proteins in OVCAR-5 and SKOV-3 cell lines. The combination treatment showed greater reduction of cell numbers for both cell lines. Total expression and phosphorylation of signaling proteins were further reduced in the combination drug treatments, compared to the single inhibitor treatments.ConclusionOur findings suggest that the concurrent targeting of more than one receptor tyrosine kinase may be useful in developing more effective targeted drug regimens for patients, who have EGFR, Her-2 and c-Met positive ovarian cancer cells.

The monolayer–spheres–monolayer cycle: Ultrastructural changes in stem cells

Stem cell sphere formation is applied as a preconditioning treatment prior to transplantation. Here, stem cells (SC) isolated from human subepicardial adipose tissue were analyzed at different stages of the monolayer–spheres–monolayer cycle by transmission electron microscopy. Spheres obtained on a non-adherent surface or through hanging drops gave similar results. At the first two or three passages (stage 1), isolated SC displayed an embryonal cell-like ultrastructure. With increased passage number (stage 2), SC became larger and more electron-dense with a multilobed nucleus, well-developed rough endoplasmic reticulum (RER), well-developed Golgi apparatus, and numerous vacuoles. At 2 h after sphere induction (stage 3), SC gathered into clusters and formed desmosome-like intercellular contacts. Their nuclei possessed a large loose fibrillo-granular nucleolus, the cytoplasm was tightly packed with disintegrated cisternae of RER, and Golgi apparatus was not identified. Twenty-four hours afterward (stage 4), SC in well-formed spheres exhibited a large dense nucleolus and poorly developed Golgi apparatus and RER. One day after sphere dissociation (stage 5), SC looked like embryonal cells and were morphologically similar to the cells of the first stage except for the presence of a large nucleolus and several Golgi complexes. At 48 h after sphere dissociation (stage 6), SC became electron-dense and resembled SC of the second stage, bearing irregular nuclei and cytoplasm that was rich in RER. We interpret these results as SC senescence with increasing passage number after isolation from the tissue or 1 day after sphere dissociation and rejuvenation of the SC just after sphere dissociation. Further research is needed to determine the genetic, biochemical, and physiological parameters of the SC corresponding to morphologically defined distinct stages in order to provide high quality cellular material for cell therapy.

Microgravity as a means to incorporate HepG2 aggregates in polysaccharide-protein hybrid scaffold.

Tissue culture under microgravity provides a venue which promotes cell-cell association while avoiding the detrimental effects of high shear stress. Hepatocytes cultured on carriers or entrapped within matrices under simulated microgravity conditions showed improved cell function and proliferation. In the present study, a new approach was adopted where a non-cell adherent scaffold was incorporated with hepatospheroids (HepG2) under microgravity. Gum arabic (GA) was cross-linked with gelatin (GA-Gel) and collagen (GA-Col) to prepare non-cell adherent scaffolds. Microgravity experiments with GA-Gel and GA-Col indicated that GA-Col is a better substrate compared to GA-Gel. Microgravity experiments of GA-Col scaffolds with HepG2 cells confirmed that the non-adherent surface with porous architecture can incorporate hepatocyte spheroids and maintain liver specific functions. Albumin and urea synthesis of hepatocytes was sustained up to 6days under microgravity conditions in the presence of GA-Col scaffold. This new approach of using non-cell adherent matrix and microgravity environment for developing biological substitutes will be beneficial in tissue engineering, bioartificial liver devices and in vitro safety assessment of drugs.

Cancer stem cell marker-expressing cell-rich spheroid fabrication from PANC-1 cells using alginate microcapsules with spherical cavities templated by gelatin microparticles.

Cancer stem-like cells (CSCs) are rare subpopulations of cancer cells. The development of three-dimensional tissues abundant in CSCs is important to both the understanding and establishment of novel therapeutics targeting them. Here, we describe the fabrication of multicellular tumor spheroids (MTSs) abundant in CSCs by employing alginate microcapsules with spherical cavities templated by cell-enclosing gelatin microparticles. Encapsulated human pancreatic cancer cell line PANC-1 cells grew for 14 days until they filled the cavities. The percentage of cells expressing reported CSC markers CD24, CD44, and epithelial-specific antigen (ESA), increased during this growth period. The percentage at 24 days of incubation, 22%, was 1.6 times higher than that of MTSs formed on a nonadherent surface in the same period of incubation. The MTSs in microcapsules could be cryopreserved in liquid nitrogen using a conventional method. No significant difference in the content of CSC marker-expressing cells was detected at 3 days of incubation when thawed after cryopreservation for 2 weeks, compared with cells incubated without prior cryopreservation.

Abstract P2-09-15: Effect of [4-valine 5-glutamine] desmopressin, a novel vasopressin analog, on human breast cancer cell growth in two-dimensional (2D) and three-dimensional (3D) cultures

Abstract Background: Desmopressin (dDAVP), a synthetic nonapeptide derivative of arginine vasopressin, is a safe hemostatic compound that acts as a selective agonist for the vasopressin V2 membrane receptor (V2R). This receptor is expressed in endothelium and also in some tumor cells. V2R stimulation is associated with increased levels of cyclic AMP and cyclic AMP-dependent protein kinase activation. We previously reported that dDAVP can inhibit progression of residual metastatic cells in preclinical models. Among other mechanisms, the compound induces an agonist effect on V2R present in tumor cells. This nonapeptide was substituted in positions 4 and 5 searching for compounds with improved biological activity. Such positions belong to the conformational peptide loop which has a key role in ligand-receptor interaction. After an extensive in vitro and in vivo characterization, novel peptide compound [4-valine 5-glutamine] dDAVP ([V4Q5]dDAVP) exerted a remarkable and improved antitumor effect in comparison to parental peptide dDAVP. [V4Q5]dDAVP inhibited tumor growth in both syngeneic F3II and xenogeneic MDA-MB-231 breast cancer models, modulating tumor aggressiveness and vasculature. In this study, to further understand direct cytostatic action of this novel vasopressin analog on tumor cells, we assessed the effect of [V4Q5]dDAVP on clonogenic and three dimensional growth of V2R-expressing human breast carcinoma cell lines. Methods and results: We used MCF7 and triple negative MDA-MB-231 human breast cancer cell lines to conduct the experiments. Vasopressin V2 membrane receptor expression in tumor cell lines was assessed by RT-PCR and immunofluorescence. We first examined peptide ability to modify in vitro low density cell growth by the clonogenic growth assay. Incubation with [V4Q5]dDAVP (100-1500 nM) during one week significantly reduced the number of tumor cell colonies showing a concentration dependant manner and an IC50 value of 1135 nM. To assess the effect of novel analog on three dimensional growth of cancer cells we used two different assays. The first one evaluates the ability of basement membrane extract (BME)-embedded single cells to proliferate and form spheroids. The second assay evaluates growth of single fully established spheroids (Approx. 300 mm diameter) transplanted individually on a non-adherent surface. [V4Q5]dDAVP, at a concentration of 1000 nM, was able to significantly reduce the number of BME-embedded spheroids formed from single cells after 14 days of treatment. In a similar manner, incubation of established and isolated spheroids with [V4Q5]dDAVP using the same concentration for 9 days, caused a decrease in multicellular spheroids final diameter. Cellular metabolic activity analysis by the MTS assay showed no significant differences between treated and control groups. Conclusions: We report for the first time the inhibitory effect of [V4Q5]dDAVP on colony and spheroid formation and growth of human breast carcinoma cells. Our results using three dimensional models suggest that V2R stimulation in cancer cells may affect early stages of microtumor establishment. Further analysis of involved signaling pathways in treated 3D cultures is needed. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-09-15.