Spherical Cell Aggregates Research Articles

Development of 3-Dimensional Prevascularized Scaffold-Free Contractile Cardiac Patch for Heart Regeneration

Purpose The aim of our study is to develop completely scaffold-free, viable, contractile cardiac tissue which can be grafted to the damaged native heart. Methods and Materials Our technology is based on the fundamental characteristics of self-assembling nature of cells. We called these self-assembled spherical cell aggregates as “spheroids”. We created contractile cardiac spheroids by plating a mixture of 800 cells of rat neonate ventricular cardiomyocytes (RNVCM), 100 cells of human normal dermal fibroblasts (HNDFB), and 100 cells of human coronary micro-artery endothelial cells (HCMEC) in ultra-low-attachment plate. After 24 hr cultivation, prevascularized contractile cardiac spheroids were fabricated. Next, 10000 spheroids were fused into patch like construct with a maximum diameter of 1cm with 200μm in thickness. We evaluated morphological characteristics, and electro- and mechanical function. We also grafted the cardiac patch onto the heart of F344 nude rats, and performed histological study 3 weeks after transplantation. Results We confirmed synchronous beating of the cardiac patch electro-physiologically, and mechanically (video-motion analysis). Expression of connexin 43 (a component of gap junction) and micro network of endothelial cell in the construct were demonstrated. Histological study performed 3 weeks after transplantation showed that the grafts were viable with functioning micro-vascular structure inside the graft. [ figure 1 ] Conclusions We consider that applying our scaffold-free 3-Dimensinal tissue engineering technology to cardiac regeneration therapy is feasible. We expect that this technology will become a promising tool to treat end-stage heart failure.

Mechanical Pressure Arrests the Growth of Tumor Spheroids

Often tumors have to push their surroundings in order to grow. Thus, during their development, tumors must be able to both exert and sustain mechanical stresses. We study quantitatively the effect of an applied mechanical stress on the long-term growth of a spherical cell aggregate. Our results indicate the possibility to modulate tumor growth depending on the applied pressure. Moreover, we observe that a stress between 500 and 5000 Pa drastically reduces growth by inhibition of cell proliferation mainly in the core of the spheroid, while it sligthly affect the division at the perifery.FIGURE: Three cryosections of a spheroid grown for 2 days without stress, with a stress of 1 kPa or with a stress of 5 kPa. Dividing cells are stained incyan (Antibody against Ki67).View Large Image | View Hi-Res Image | Download PowerPoint Slide

Protein markers of cancer-associated fibroblasts and tumor-initiating cells reveal subpopulations in freshly isolated ovarian cancer ascites

BackgroundIn ovarian cancer, massive intraperitoneal dissemination is due to exfoliated tumor cells in ascites. Tumor-initiating cells (TICs or cancer stem cells) and cells showing epithelial-mesenchymal-transition (EMT) are particularly implicated. Spontaneous spherical cell aggregates are sometimes observed, but although similar to those formed by TICs in vitro, their significance is unclear.MethodsCells freshly isolated from malignant ascites were separated into sphere samples (S-type samples, n=9) and monolayer-forming single-cell suspensions (M-type, n=18). Using western blot, these were then compared for expression of protein markers of EMT, TIC, and of cancer-associated fibroblasts (CAFs).ResultsS-type cells differed significantly from M-type by expressing high levels of E-cadherin and no or little vimentin, integrin-β3 or stem cell transcription factor Oct-4A. By contrast, M-type samples were enriched for CD44, Oct-4A and for CAF markers. Independently of M- and S-type, there was a strong correlation between TIC markers Nanog and EpCAM. The CAF marker α-SMA correlated with clinical stage IV. This is the first report on CAF markers in malignant ascites and on SUMOylation of Oct-4A in ovarian cancer.ConclusionsIn addition to demonstrating potentially high levels of TICs in ascites, the results suggest that the S-type population is the less tumorigenic one. Nanoghigh/EpCAMhigh samples represent a TIC subset which may be either M- or S-type, and which is separate from the CD44high/Oct-4Ahigh subset observed only in M-type samples. This demonstrates a heterogeneity in TIC populations in vivo which has practical implications for TIC isolation based on cell sorting. The biological heterogeneity will need to be addressed in future therapeutical strategies.

Isotropic stress reduces cell proliferation in tumor spheroids

In most instances, tumors have to push their surroundings in order to grow. Thus, during their development, tumors must be able to both exert and sustain mechanical stresses. Using a novel experimental procedure, we study quantitatively the effect of an applied mechanical stress on the long-term growth of a spherical cell aggregate. Our results indicate the possibility to modulate tumor growth depending on the applied pressure. Moreover, we demonstrate quantitatively that the cells located in the core of the spheroid display a different response to stress than those in the periphery. We compare the results to a simple numerical model developed for describing the role of mechanics in cancer progression.

Kinetic Monte Carlo and cellular particle dynamics simulations of multicellular systems

Computer modeling of multicellular systems has been a valuable tool for interpreting and guiding in vitro experiments relevant to embryonic morphogenesis, tumor growth, angiogenesis and, lately, structure formation following the printing of cell aggregates as bioink particles. Here we formulate two computer simulation methods: (1) a kinetic Monte Carlo (KMC) and (2) a cellular particle dynamics (CPD) method, which are capable of describing and predicting the shape evolution in time of three-dimensional multicellular systems during their biomechanical relaxation. Our work is motivated by the need of developing quantitative methods for optimizing postprinting structure formation in bioprinting-assisted tissue engineering. The KMC and CPD model parameters are determined and calibrated by using an original computational-theoretical-experimental framework applied to the fusion of two spherical cell aggregates. The two methods are used to predict the (1) formation of a toroidal structure through fusion of spherical aggregates and (2) cell sorting within an aggregate formed by two types of cells with different adhesivities.

Physical Passaging of Embryoid Bodies Generated from Human Pluripotent Stem Cells

Spherical three-dimensional cell aggregates called embryoid bodies (EBs), have been widely used in in vitro differentiation protocols for human pluripotent stem cells including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Recent studies highlight the new devices and techniques for hEB formation and expansion, but are not involved in the passaging or subculture process. Here, we provide evidence that a simple periodic passaging markedly improved hEB culture condition and thus allowed the size-controlled, mass production of human embryoid bodies (hEBs) derived from both hESCs and hiPSCs. hEBs maintained in prolonged suspension culture without passaging (>2 weeks) showed a progressive decrease in the cell growth and proliferation and increase in the apoptosis compared to 7-day-old hEBs. However, when serially passaged in suspension, hEB cell populations were significantly increased in number while maintaining the normal rates of cell proliferation and apoptosis and the differentiation potential. Uniform-sized hEBs produced by manual passaging using a 1∶4 split ratio have been successfully maintained for over 20 continuous passages. The passaging culture method of hEBs, which is simple, readily expandable, and reproducible, could be a powerful tool for improving a robust and scalable in vitro differentiation system of human pluripotent stem cells.

Human Cardiospheres Are a Source of Stem Cells with Cardiomyogenic Potential

Several laboratories have shown that collections of adult cardiac stem cells can be grown directly from myocardial tissue.(1–7) Expanded cells are multipotent and clonogenic.(3–5) Spherical aggregates of cells grown from heart biopsies, termed cardiospheres, self-assemble in suspension culture and are enriched in stemness.(1, 6, 7) Andersen and colleagues, in work on neonatal murine heart tissue, used novel culture methods as a basis to question the cardiogenic potential of cardiosphere-cultured stem cells.(8) We contend that their sweeping conclusions are not applicable to studies using established methods (1, 2, 6, 7). To confirm the cardiogenic nature of human cardiosphere-derived cells (CDCs) in vivo, we evaluated the ability of CDCs to engraft and form new cardiomyocytes after transplantation into infarcted SCID mice. Immunostaining for human nuclear antigen (1:50, Chemicon MAB1281) and lentivirally-mediated GFP or beta-galactosidase labelling were used to track cells after injection. While the majority of CDCs could be found throughout the infarct (57±3% of the total engrafted) and the immediate border zone (30±5%), stable engraftment also existed in the remote myocardium (13±3%). Figure 1A and 1B show GFP-labelled CDCs within the border and infarct zones. Counterstaining for cardiac troponin I (cTnI; Chemicon MAB3150) demonstrates that many CDCs have differentiated into cardiomyocytes. As shown in Figure 1, portions of the mouse heart were reconstituted by human CDCs (Figure 1A), while CDC-derived cardiomyocytes in the dense central infarct zone remained small with little cTnI expression in their cytoplasm (Figure 1B). Human nuclear antigen expression also demonstrates differentiated cardiomyocytes, derived from transplanted human CDCs, within the border zone (Figure 1C). These human-derived cells express Cx43, suggesting, but not proving, functional integration within the infarcted tissue (Figure 1D). Such functional integration has, however, been rigorously documented in vitro with myocyte-CDC co-culture.(7) Further, beta-galactosidase-labelled CDCs expressing markers of endothelial (vWF, Figure 1E) and smooth muscle (αSMA; Figure 1F) lineages highlight the in vivo multilineage potential of CDCs. Figure 1 Cardiogenic differentiation of transplanted cells cultured from humans biopsies using the cardiosphere method. Human CDCs were injected into SCID mice at the time of myocardial infarction. (A): Example images of the infarct border zone demonstrating engraftment ... These results challenge the conclusions of Andersen et al. by demonstrating in vivo cardiomyogenesis of transplanted cells cultured using established cardiosphere methods. Our lab and others have shown that CDCs also decrease infarct size and improve myocardial function(1, 7), with the different sub-populations within CDCs acting synergistically to improve myocardial function.(9) Although cardiac differentiation of transplanted CDCs does occur consistently, long-term persistence of transplanted CDCs is relatively low (10), with important contributions of paracrine effects (both tissue preservation and recruitment of endogenous regeneration) to the functional benefit of CDC transplantation.(11). In this light, Andersen and colleagues(8) may have overstated the relevance of their findings, considering that their work made use of several unprecedented experimental procedures with novel experimental findings that occurred exclusively when neonatal rat heart was used as a tissue source. These investigators stored the myocardial tissue prior to processing in a novel buffer, potentially damaging the very cells of interest; performed prolonged enzymatic digestion to collect cells from explant culture, possibly favoring the survival of contaminating cells; and finally, failed to characterize their cells in vivo.(12) If the authors seek to convincingly critique the cardiosphere approach, they would be well-advised to reproduce established methods. As such, their work is unrelated to adult cardiosphere culture and, as performed, provides no justification for the indictment of the clinical utility of cardiospheres or CDCs, which are already being tested in human subjects. (13)

Cellular Particle Dynamics Simulation Of Bioprinted 3d Tissue Constructs

Previous studies have shown that under certain conditions living tissues and multicellular aggregates behave as highly visco-elastic liquids. Tissue liquidity, brought about by cellular adhesion and motility, forms the basis of the newly developed bioprinting technology, which is used to design and build 3D tissue constructs by employing computer-controlled layer-by-layer deposition of bioink (submillimeter size cell aggregate) droplets onto biopaper (biocompatible gel). In order to describe and predict the self-assembly process of bioprinted multicellular constructs we have developed a computer simulation method referred to as cellular particle dynamics (CPD). In CPD cells are modeled as an ensemble of cellular particles (CPs) that interact via short range contact interactions, characterized by an attractive (adhesive interaction) and a repulsive (excluded volume interaction) component. The time evolution of the spatial conformation of the multicellular system is determined directly by recording the trajectories of all CPs through integration of their equations of motion. The cellular level CP model parameters are related to the experimentally measurable tissue level biophysical quantities (e.g., surface tension, viscosity and shear modulus) by comparing the results from selected benchmark experiments (e.g., compression and fusion of spherical cell aggregates) with those from the corresponding CPD simulations. Here we apply the CPD method to describe and predict in silico the post-bioprinting time evolution of the formation of tubular multicellular structures (which resemble primitive blood vessels). Our CPD simulations take substantially less time and effort than the corresponding experiments and, most importantly, provide results in good agreement with the experimental ones.Work supported by the National Science Foundation [FIBR-0526854]. Computer time provided by the University of Missouri Bioinformatics Consortium.

Towards In Silico Bioprinting

Bioprinting is a computer-controlled procedure for building three-dimensional tissue constructs via layer-by-layer delivery of cells and supportive hydrogels. To describe the post-printing self-assembly of multicellular structures, we performed computer simulations that incorporate a basic principle of developmental biology, the differential adhesion hypothesis (DAH). DAH states that cell motility combined with differences in the adhesive properties of different cell types yields tissue conformations with the largest number of strong bonds between cells. Metropolis Monte Carlo (MMC) simulations based on DAH predicted the emergence of long-lived structures (cell sheets, toroidal and tubular constructs) in accord with experiments. The MMC method, however, does not describe time evolution. We propose a kinetic Monte Carlo (KMC) approach, where transition rates are associated with possible rearrangements of cells. The system is represented on a lattice, with sites occupied either by cells or by volume elements of cell culture medium. We associate rates to swapping cells with nearest neighbors of different types (cells or medium). The new approach was tested against experiments on cell sorting within an aggregate composed of two cell types. In quantitative studies, we determined the time evolution of the interfacial area between two fusing spherical cell aggregates experimentally, analytically and by KMC simulations. In the analytic approach, we used continuum hydrodynamics to describe the coalescence of two identical, highly viscous liquid droplets, and obtained good agreement with experiments on smooth muscle cell aggregates. Apart from the early stages of fusion, the KMC method predicted a fusion pattern similar to the experimental one. Comparison with measurements allowed relating KMC transition rates to experimental time scales. Our results indicate that the KMC method can give an accurate account of the time evolution of complex cellular structures, thus it may be a useful tool for tissue engineering applications. Work supported by NSF-056854.

Unrestricted somatic stem cells from human umbilical cord blood grow in serum-free medium as spheres

BackgroundHuman umbilical cord blood-derived unrestricted somatic stem cells (USSCs), which are capable of multilineage differentiation, are currently under investigation for a number of therapeutic applications. A major obstacle to their clinical use is the fact that in vitro expansion is still dependent upon fetal calf serum, which could be a source of pathogens. In this study, we investigate the capacity of three different stem cell culture media to support USSCs in serum-free conditions; HEScGRO™, PSM and USSC growth mediumACF. Our findings demonstrate that USSCs do not grow in HEScGRO™ or PSM, but we were able to isolate, proliferate and maintain multipotency of three USSC lines in USSC growth mediumACF.ResultsFor the first one to three passages, cells grown in USSC growth mediumACF proliferate and maintain their morphology, but with continued passaging the cells form spherical cell aggregates. Upon dissociation of spheres, cells continue to grow in suspension and form new spheres. Dissociated cells can also revert to monolayer growth when cultured on extracellular matrix support (fibronectin or gelatin), or in medium containing fetal calf serum. Analysis of markers associated with pluripotency (Oct4 and Sox2) and differentiation (FoxA2, Brachyury, Goosecoid, Nestin, Pax6, Gata6 and Cytokeratin 8) confirms that cells in the spheres maintain their gene expression profile. The cells in the spheres also retain the ability to differentiate in vitro to form cells representative of the three germline layers after five passages.ConclusionsThese data suggest that USSC growth mediumACF maintains USSCs in an undifferentiated state and supports growth in suspension. This is the first demonstration that USSCs can grow in a serum- and animal component-free medium and that USSCs can form spheres.

Interactions of coronary artery smooth muscle cells with 3D porous polyurethane scaffolds

One strategy in vascular tissue engineering is the design of hybrid vascular substitutes where vascular cells infiltrate biostable porous scaffolds that provides favorable environment for guided cell repopulation and acts as a mechanically supporting layer after the tissue regeneration process. The aim of the present work was to study the interaction of human coronary artery smooth muscle cells (HCASMC) with 3D porous polyurethane scaffolds. We therefore fabricated porous and highly interconnected 3D polyurethane scaffolds that can promote HCASMC attachment, proliferation, and migration. SEM and microCT studies of the fabricated scaffolds showed that the current scaffolds had highly open and interconnected pore structures, with an average porosity of 84%. HCASMC interaction on polyurethane films revealed that cells adhere and express specific marker proteins (vinculin and h-caldesmon). This expression was further enhanced by coating the polyurethane with Matrigel. On uncoated 3D scaffolds, dense spherical aggregates of cells were often encountered with little adhesion of individual cells alongside the struts of the scaffold, independent of the porogens used. In contrast, when cultured on Matrigel-coated scaffolds, cell numbers quickly increased after 14 days and spread along the entire scaffold. At the upper scaffold surface, elongated cells were seen adhering to one another and also to the scaffold surface. These cells were elongated, aligned in parallel and contained abundant F-actin bundles suggesting a differentiated contractile phenotype. Deep into the scaffold, cells were encountered that formed actin-rich lamellipodial extensions spreading along the strut and lacked stress fibers, suggesting active cell migration along the substrate.

Bioadhesive hydrogel microenvironments to modulate epithelial morphogenesis

Epithelial cells polarize and differentiate into organotypic cell aggregates in response to cell–cell and cell–matrix interactions. For example, Madin–Darby Canine Kidney (MDCK) cells form spherical cell aggregates (cysts) with distinct apical and basolateral polarity when cultured three dimensionally (embedded) in type I collagen gels. To investigate the effects of individual extracellular factors on epithelial morphogenesis, we engineered fast degrading protease-responsive polyethylene glycol (PEG) hydrogels functionalized with controlled densities of various bioligands (RGD peptide, laminin-1 (LN)) to allow 3D culturing of MDCK cells, cyst expansion, and morphogenesis/polarization. Cysts formed after 15 days of culture in these hydrogels were analyzed with multiphoton fluorescence microscopy for markers of apical and basolateral membrane domains. Epithelial cysts formed in bioadhesive ligand-functionalized PEG gels exhibited a higher frequency of central lumen and interior apical pole formation as well as basolateral polarization compared to those of unmodified PEG hydrogels. These results demonstrate that incorporation of specific bioadhesive motifs into synthetic hydrogels provides 3D culture environments that support epithelial morphogenesis. These microenvironments provide a flexible and controlled system for systematic investigations into normal and pathologic morphogenic behaviours as well as synthetic environments for promoting tissue morphogenesis for regenerative medicine applications.

Nestin-positive spheres derived from canine bone marrow stromal cells generate cells with early neuronal and glial phenotypic characteristics

Bone marrow stromal cells (BMSCs) isolated from humans and rodents have been shown to generate neural cells under specific culture conditions and after transplantation in the central nervous system. The apparent plasticity of BMSCs has therefore been a target of intensive research in attempt to develop a novel therapy for neurological diseases. Canines sustain neurological disorders (e.g., traumatic spinal cord injury) that closely mirror pathology of those in humans. Therefore, we evaluated neural differentiation properties of canine BMSCs to provide insights into basic characterization of these cells for future neurotransplantation trials in canine patients with neurological disorders. We demonstrate that canine BMSCs form spherical cellular aggregates on anti-adhesive culture substrate in serum-free culture media, which morphologically and phenotypically resemble spherical aggregates of neural progenitor cells, so-called neurospheres. Upon dissociation and subculture on adhesive substrate, canine BMSCs express neuronal (ss capital SHA, Cyrillic-tubulin) and glial (GFAP, A2B5, and CNPase) markers. Formation of spherical aggregates appears to be a critical preceding process for some of the glial marker expression (CNPase and A2B5). However, expression of more mature neuronal (MAP2) and glial (MBP) markers could not be induced with the protocol used in this study. We suggest that induction of canine BMSCs into cells with neural progenitor cell characteristics is possible and that these cells may have the potential for future cellular therapy for neurological disorders.

Efficient formation of uniform-sized embryoid bodies using a compartmentalized microchannel device

The formation of spherical aggregates of cells called embryoid bodies (EBs) is an indispensable step in many protocols in which embryonic stem (ES) cells are differentiated to other cell types. Appropriate morphology and embryo size are critical for the sequential developmental stages of naturally conceived embryos. Likewise, regulating the size of EBs and the timing of their formation is crucial for controlling the differentiation of ES cells within the EB. Existing methods of formation of EBs, however, are tedious or provide heterogeneously-sized EBs. Here we describe a microfluidic system for straightforward synchronized formation of uniform-sized EBs, the size of which can be controlled by changing the cross-sectional size of microchannels in the microfluidic device. The device consists of two microchannels separated by a semi-porous polycarbonate membrane treated to be resistant to cell adhesion. ES cells introduced into the upper channel self-aggregate to form uniformly-sized EBs. The semi-porous membrane also allows subsequent treatment of the non-attached EBs with different reagents from the lower channel without the need for wash out because of the compartmentalization afforded by the membrane. This method provides a simple yet robust means to control the formation of EBs and the subsequent differentiation of ES cells in a format compatible for ES cell processing on a chip.

Cultivation of Recombinant Chinese hamster ovary cells grown as suspended aggregates in stirred vessels

Recombinant Chinese hamster ovary (rCHO) cells capable of producing a prourokinase mutant (mPro-uk) grown as suspended aggregates in stirred vessels were described and characterized. The addition of chitosan to a mixture of DMEM and Ham's F12 (D-MEM/F-12) medium promoted cell aggregation and spheroid formation efficiently. Multicellular aggregates formed immediately after the rCHO cells were inoculated into the chitosan-added medium, and the mean diameter of the cell aggregates reflecting the aggregate size increased with culture time, shifting from 65 to 163 mum after 2 and 9 d of culture in spinner flasks. No significant difference in the metabolism performance of the rCHO cells was observed between suspended aggregates and anchored monolayers. However, the cells cultured as suspended aggregates showed a marked decrease in growth rate as evaluated from specific growth rate (mu). Replacing D-MEM/F-12 medium with CD 293 medium caused compact spherical cell aggregates to dissociate into small irregular aggregates and single cells without apparent effects on cell performance in subcultures. The perfusion culture of the rCHO cells grown as suspended aggregates in a 2-l stirred tank bioreactor for 15 d resulted in a maximum viable cell density of 5.6 x 10(6) cells ml(-1) and an mPro-uk concentration of about 2.6 x 10(3) IU ml(-1), and cell viability was remained at roughly 90% during the entire run.

De Novo Morphogenesis of Seminiferous Tubules From Dissociated Immature Rat Testicular Cells in Xenografts

Testicular development is initiated with the differentiation of Sertoli cells in the embryonic gonad. The aggregation of Sertoli cells is crucial for the generation of testicular cords and thus for the first sign of male gonadal development. To date, functional testicular tissue has not yet been generated in vitro. The objective of this study was to explore the de novo morphogenesis of testicular tissue from isolated postnatal rat testicular cells using a combination of in vitro culture and ectopic xenografting. Immature rat testicular cells were cultured in either a 2-dimensional (laminin-coated coverglass) or a 3-dimensional (extracellular matrix gel) culture system. Whereas testicular cells cultured on laminin showed a slow morphogenetic cascade resulting in cord formation after about 10 days of culture, cells cultured on extracellular matrix gel assembled to a network of cordlike structures within several hours after plating and formed spherical cell aggregates at day 3. Further progression of the morphogenetic cascade was not obtained in either the 2- or the 3-dimensional culture system. In contrast, structures resembling immature testicular tissue were obtained after xenografting of extracellular matrix gel-enclosed spherical testicular cell aggregates. The grafts were vascularized and contained elongated seminiferous tubules. Histologic analysis revealed the presence of a basement membrane, a histologically normal interstitium containing putative Leydig cells, the establishment of tubule lumen, and the integration of few putative spermatogonia into the seminiferous epithelium. We conclude that immature rat testicular cells carry the full potential to generate all somatic components of a testis in xenografts, thus opening fascinating pathways to study testicular organogenesis.

Suspended aggregates as an immobilization mode for high-density perfusion culture of HEK 293 cells in a stirred tank bioreactor

Cells of the human embryonic kidney cell line (HEK 293) grown in repeated suspension and perfusion systems were characterized and described. Cell aggregates that formed immediately after the HEK 293 cells were inoculated in stirred vessels in serum-containing Dulbecco's modified Eagle's medium (D-MEM)/F-12 medium. The mean diameter of the cell aggregates reflecting the aggregate size increased with culture time, shifting from 63 to 239 mum after 1 and 8 days of culture in spinner flasks, respectively. No significant differences in cell performance were observed between HEK 293 cell populations grown as suspended aggregates and those grown as anchored monolayers. Replacing the D-MEM/F-12 with CD 293 medium caused the compact spherical cell aggregates to dissociate into single cells and small irregular aggregates without any apparent effect on cell performance. Moreover, the spherical cell aggregates could reform from individual cells and small aggregates when exposed to the serum-containing D-MEM/F-12 dominant medium. Perfusion culture of HEK 293 cells grown as suspended aggregates in a 7.5-l stirred tank bioreactor for 17 days resulted in a maximum viable cell density of 1.2 x 10(7) cells ml(-1). These results demonstrate the feasibility and proof-of-concept for using aggregates as an immobilization system in large-scale stirred bioreactors because a small-scale culture can be used as easily as the inoculum for larger bioreactors.

Cell aggregates as self‐assembling bioink

Understanding the principles of biological self-organization is indispensable for developing efficient strategies to build living tissues and organs. Morphogenesis, the sequence of natural pattern-forming processes through which organs acquire their final shape, is under strict genetic control. However, genes do not create spatial structures: physical mechanisms do. Here we show how the self-assembly capacity of cells and tissues can be exploited to construct living structures of prescribed shape. The central tenet of our approach is that tissues composed of motile and adhesive cells mimic the behavior of liquids. To biologically validate this idea, we first show that the fusion of embryonic cushion tissue during heart morphogenesis proceeds similarly in vitro and in vivo and both qualitatively and quantitatively resembles the coalescence of liquid drops. Next, we demonstrate that, once given the right initial cues, that is directed, spherical cell aggregates mixed with appropriate hydrogel behave as self-assembling “bio-ink” particles. Finally, we show that these bio-ink particles can be dispensed by special “bio-printers.” We print cellular toroids, tubes and in particular “beating” sheets of cardiomyocytes.

The Neurofibromatosis Type 1 Gene Product Neurofibromin Enhances Cell Motility by Regulating Actin Filament Dynamics via the Rho-ROCK-LIMK2-Cofilin Pathway

Neurofibromin is a neurofibromatosis type 1 (NF1) tumor suppressor gene product with a domain that acts as a GTPase-activating protein and functions, in part, as a negative regulator of Ras. Loss of neurofibromin expression in NF1 patients is associated with elevated Ras activity and increased cell proliferation, predisposing to a variety of tumors of the peripheral and central nervous systems. We show here, using the small interfering RNA (siRNA) technique, that neurofibromin dynamically regulates actin cytoskeletal reorganization, followed by enhanced cell motility and gross cell aggregation in Matrigel matrix. NF1 siRNA induces characteristic morphological changes, such as excessive actin stress fiber formation, with elevated negative phosphorylation levels of cofilin, which regulates actin cytoskeletal reorganization by depolymerizing and severing actin filaments. We found that the elevated phosphorylation of cofilin in neurofibromin-depleted cells is promoted by activation of a Rho-ROCK-LIMK2 pathway, which requires Ras activation but is not transduced through three major Ras-mediated downstream pathways via Raf, phosphatidylinositol 3-kinase, and RalGEF. In addition, the exogenous expression of the NF1-GTPase-activating protein-related domain suppressed the NF1 siRNA-induced phenotypes. Neurofibromin was demonstrated to play a significant role in the machinery regulating cell proliferation and in actin cytoskeletal reorganization, which affects cell motility and adhesion. These findings may explain, in part, the mechanism of multiple neurofibroma formation in NF1 patients.

A Round-bottom 96-well Polystyrene Plate Coated with 2-methacryloyloxyethyl Phosphorylcholine as an Effective Tool for Embryoid Body Formation

In this study, we proposed a culture method for forming embryoid bodies (EBs) from mouse embryonic stem (ES) cells using a round-bottom 96-well polystyrene plate coated with 2-methacryloyloxyethyl phosphorylcholine (MPC plate). MPC is a phospholipid biocompatible polymer and prevents cells from adhering to the culture surface. The ES cells were seeded at 1000 cells per well in the MPC plate with 200 mul of medium. After 5 days of static incubation, a spherical cell aggregate termed EB was formed in a well. The size (diameter) of resulting EB was approximately 550 mum and it contained approx. 22,000 cells. It seems that the non-adhesiveness and the roundness of the well are important factors to form a good EB. Transferring the EBs to the attached differentiation culture, the EBs spread out and flattened, and the beating cells (cardiomyocytes) were effectively generated in the outgrowth of EBs. The round-bottom 96-well polystyrene plate coated with MPC is an effective tool for EB formation.