Differences In Cell Behavior Research Articles

Effects of platelet-rich fibrin on human endometrial stromal cells behavior in comparison to platelet-rich plasma.

Intrauterine transfusion of platelet-rich plasma (PRP) has become a new treatment for thin endometrium (TE) in recent years, but its low efficacy due to rapid release of growth factors limits its clinical use. Platelet-rich fibrin (PRF) starts the coagulation cascade reaction immediately after the blood comes into contact with the test tube. The natural coagulation process results in stable platelet activation and the slow release of growth factors. In our study, primary human endometrial stromal cells (hESCs) were extracted from endometrial tissue. PRP and PRF were prepared from the patient cubital vein blood. Stromal cells were cultured in conditioned medium supplemented with PRP and PRF. Differences in cell behavior were observed by cell proliferation test and cell migration test. The relative expression levels of apoptotic Bax and antiapoptotic Bcl-2 genes were measured by qRT-PCR. The release of growth factors from PRP and PRF was detected by ELISA. We found that both PRP and PRF inhibited apoptosis of hESCs, which favored cell proliferation and migration. In addition, PRF releases growth factors for a longer period of time compared to PRP. PRF offer a more sustained therapeutic effect compared to PRP, which provides a new idea for endometrial regeneration and repair.

Osteogenic differentiation of human mesenchymal stem cells on electroactive substrates

This study investigates the effect of electroactivity and electrical charge distribution on the biological response of human bone marrow stem cells (hBMSCs) cultured in monolayer on flat poly(vinylidene fluoride), PVDF, substrates. Differences in cell behaviour, including proliferation, expression of multipotency markers CD90, CD105 and CD73, and expression of genes characteristic of different mesenchymal lineages, were observed both during expansion in basal medium before reaching confluence and in confluent cultures in osteogenic induction medium. The crystallisation of PVDF in the electrically neutral α-phase or in the electroactive phase β, both unpoled and poled, has been found to have an important influence on the biological response. In addition, the presence of a permanent positive or negative surface electrical charge distribution in phase β substrates has also shown a significant effect on cell behaviour.

Design and characterisation of a cell exposure system with high magnetic field homogeneity: RILZ coils.

In vitro studies requiring controlled exposure to low-frequency electromagnetic fields employ exposure systems with different geometries and configurations, the Helmholtz configuration being one of the most widely used. This configuration has limitations in the homogeneity of the spatial distribution of the magnetic field intensity values. We present the design, manufacturing, and characterisation of a new coil system, called RILZ configuration, which improves the distribution of magnetic field intensity values in the three dimensions of space for three different heights in comparison with the traditional circular coils in Helmholtz configuration. In addition, a comparative study of the cellular response in CT2A cultures exposed to a magnetic field of 50 Hz and 100µT for 48hrs is performed with both exposure systems. The results of the study show reduced values of deviation from the central value of magnetic field intensity using the RILZ coil system. These differences are statistically significant compared to the Helmholtz configuration for the three Cartesian directions: x (p < 0.01), y (p < 0.01), z (p < 0.01). In addition, the intensity values for three different heights are statistically significantly correlated using the RILZ coil system (p < 0.01). The differences in cell behaviour are also statistically significant between the two systems (p < 0.01) and may be directly related to the differences found in the distribution of intensity values between the two systems. This study highlights the importance of the homogeneity of the magnetic field intensity generated by the exposure systems used and offers an effective solution to control the magnetic field exposure parameters in vitro assays.

Spatial control of self-organizing vascular networks with programmable aptamer-tethered growth factor photopatterning.

Given the dynamic nature of engineered vascular networks within biofabricated tissue analogues, it is instrumental to have control over the constantly evolving biochemical cues within synthetic matrices throughout tissue remodeling. Incorporation of pro-angiogenic vascular endothelial growth factor (VEGF165) specific aptamers into cell-instructive polymer networks is shown to be pivotal for spatiotemporally controlling the local bioactivity of VEGF that selectively elicit specific cell responses. To harness this effect and quantitatively unravel its spatial resolution, herein, bicomponent micropatterns consisting of VEGF165 specific aptamer-functionalized gelatin methacryloyl (GelMA) (aptamer regions) overlaid with pristine GelMA regions using visible-light photoinitiators (Ru/SPS) were fabricated via two-step photopatterning approach. For the 3D co-culture study, human umbilical vein-derived endothelial cells and mesenchymal stromal cells were used as model cell types. Bicomponent micropatterns with spatially defined spacings (300/500/800​μm) displayed high aptamer retention, aptamer-fluorescent complementary sequence (CSF) molecular recognition and VEGF sequestration localized within patterned aptamer regions. Stiffness gradient at the interface of aptamer and GelMA regions was observed with high modulus in aptamer region followed by low stiffness GelMA regions. Leveraging aptamer-tethered VEGF's dynamic affinity interactions with CS that upon hybridization facilitates triggered VEGF release, co-culture studies revealed unique characteristics of aptamer-tethered VEGF to form spatially defined luminal vascular networks covered with filopodia-like structures in vitro (spatial control) and highlights their ability to control network properties including orientation over time using CS as an external trigger (temporal control). Moreover, the comparison of single and double exposed regions within micropatterns revealed differences in cell behavior among both regions. Specifically, the localized aptamer-tethered VEGF within single exposed aptamer regions exhibited higher cellular alignment within the micropatterns till d5 of culture. Taken together, this study highlights the potential of photopatterned aptamer-tethered VEGF to spatiotemporally regulate vascular morphogenesis as a tool for controlling vascular remodeling in situ.

Application of sequential cyclic compression on cancer cells in a flexible microdevice.

Mechanical forces shape physiological structure and function within cell and tissue microenvironments, during which cells strive to restore their shape or develop an adaptive mechanism to maintain cell integrity depending on strength and type of the mechanical loading. While some cells are shown to experience permanent plastic deformation after a repetitive mechanical tensile loading and unloading, the impact of such repetitive compression on deformation of cells is yet to be understood. As such, the ability to apply cyclic compression is crucial for any experimental setup aimed at the study of mechanical compression taking place in cell and tissue microenvironments. Here, we demonstrate such cyclic compression using a microfluidic compression platform on live cell actin in SKOV-3 ovarian cancer cells. Live imaging of the actin cytoskeleton dynamics of the compressed cells was performed for varying pressures applied sequentially in ascending order during cell compression. Additionally, recovery of the compressed cells was investigated by capturing actin cytoskeleton and nuclei profiles of the cells at zero time and 24 h-recovery after compression in end point assays. This was performed for a range of mild pressures within the physiological range. Results showed that the phenotypical response of compressed cells during recovery after compression with 20.8 kPa differed observably from that for 15.6 kPa. This demonstrated the ability of the platform to aid in the capture of differences in cell behaviour as a result of being compressed at various pressures in physiologically relevant manner. Differences observed between compressed cells fixed at zero time or after 24 h-recovery suggest that SKOV-3 cells exhibit deformations at the time of the compression, a proposed mechanism cells use to prevent mechanical damage. Thus, biomechanical responses of SKOV-3 ovarian cancer cells to sequential cyclic compression and during recovery after compression could be revealed in a flexible microdevice. As demonstrated in this work, the observation of morphological, cytoskeletal and nuclear differences in compressed and non-compressed cells, with controlled micro-scale mechanical cell compression and recovery and using live-cell imaging, fluorescent tagging and end point assays, can give insights into the mechanics of cancer cells.

Novel microsphere-packing synthesis, microstructure, formation mechanism and in vitro biocompatibility of porous gelatin/hydroxyapatite microsphere scaffolds

In this study, three types of porous gelatin/HAp composite-based scaffolds were prepared and their microstructure, mechanical property, and in vitro biocompatibility were evaluated. FD-gelatin/HAp scaffolds were prepared via a conventional freeze-drying (FD) method, while DCPD-bound gelatin/HAp (DCPD-gelatin/HAp) microsphere scaffolds and HAp-bound gelatin/HAp (HAp-gelatin/HAp) microsphere scaffolds were prepared via the combined method of emulsion technique and novel microsphere-packing method. It was found that the microstructure and mechanical property of porous gelatin/HAp scaffolds were highly dependent on the preparation method and type of scaffolds. FD-gelatin/HAp scaffolds had few pores with a comparatively flat surface and had a compressive strength was 24 ± 1 MPa. DCPD-gelatin/HAp microsphere scaffolds had a porous structure with the average pore size of 121 ± 12 μm and had the compressive strength of 50 ± 3 MPa. HAp-gelatin/HAp microsphere scaffolds also had a porous structure with the average pore size of 190 ± 26 μm and had the compressive strength of 17 ± 4 MPa. In vitro biocompatibility was performed by culturing the osteoblast-like MC3T3-E1 cells with all types of scaffolds. The results showed that all types of scaffolds were biocompatible to support cell attachment and proliferation. However, a significant difference in cell behavior was clearly observed. Most cells were grown on the surface of FD-gelatin/HAp scaffolds due to the lack of the porous structure, while those were grown on the surface and inside the pore of both DCPD-gelatin/HAp microsphere scaffolds and HAp-gelatin/HAp microsphere scaffolds due to the presence of the porous structure. Thus, the porous gelatin/HAp microsphere-based scaffolds have a potential as novel bone tissue regenerative materials.

Preparation of Absorbable Suture Biomaterial Formed by Glycolide/Lactide and Its Application in the Congenital Thumb Deformity Surgery

Conducting polymers (CPs) can regulate cell behavior and promote the regeneration of damaged tissues, which are widely applied in tissue engineering. In this study, poly(3-hexylthiophene) (PHTP) was first obtained by the catalyst + transfer poly-condensation method, and then, it was mixed with poly(lactide-co-glycolide) (PLGA) to obtain a composite material with superconductivity (PHTP/PLGA). Besides, ferric chloride (FeCl3) was selected as the dopant, and the doping time was changed, so as to obtain 5 PHTP/PLGA materials with different conductivity (10-7 S/cm, 10-6 S/cm, 10-5 S/cm, 10-4 S/cm, and 10-3 S/cm). While PHTP/PLGA was physically characterized, the cell proliferation ability and blood compatibility of this series of materials were further analyzed, and the materials were made into medical sutures for the treatment of congenital thumb deformities. In the experiment, the surface morphology, hydrophilicity, and hydrophobicity of materials with different conductivity would not affect the difference in cell behavior. Using mouse embryonic osteoblasts (MC3T3-E1) as the research objects, increasing PHTP/PLGA conductivity could promote MC3T3-E1 alkaline phosphatase activity cell mineralization, and collagen type I expression. PHTP/PLGA series materials had a low hemolysis rate and were not easily affected by platelet adhesion and activation, meaning that they would not cause coagulation. The material was applied in the direct suture method of hammer deformity, and the results showed that the patient’s deformed thumb had a knot reaction and local skin edge necrosis. After dressing change, it gradually improved. Moreover, the excellent and good rate of deformed thumb based on this material was higher than that of wire extraction method and Kirschner wire fixation method (P &lt; 0.05).

Cell proliferation and migration explain pore bridging dynamics in 3D printed scaffolds of different pore size

Tissue growth in bioscaffolds is influenced significantly by pore geometry, but how this geometric dependence emerges from dynamic cellular processes such as cell proliferation and cell migration remains poorly understood. Here we investigate the influence of pore size on the time required to bridge pores in thin 3D-printed scaffolds. Experimentally, new tissue infills the pores continually from their perimeter under strong curvature control, which leads the tissue front to round off with time. Despite the varied shapes assumed by the tissue during this evolution, we find that time to bridge a pore simply increases linearly with the overall pore size. To disentangle the biological influence of cell behaviour and the mechanistic influence of geometry in this experimental observation, we propose a simple reaction–diffusion model of tissue growth based on Porous-Fisher invasion of cells into the pores. First, this model provides a good qualitative representation of the evolution of the tissue; new tissue in the model grows at an effective rate that depends on the local curvature of the tissue substrate. Second, the model suggests that a linear dependence of bridging time with pore size arises due to geometric reasons alone, not to differences in cell behaviours across pores of different sizes. Our analysis suggests that tissue growth dynamics in these experimental constructs is dominated by mechanistic crowding effects that influence collective cell proliferation and migration processes, and that can be predicted by simple reaction–diffusion models of cells that have robust, consistent behaviours.

Stygamoeba cauta n. sp. (Amoebozoa, Discosea) – a new brackish-water species from Nivå Bay (Baltic Sea, The Sound)

Several evolutionary lineages of Amoebozoa are characterized by unusual morphological and ultrastructural features that impede resolving of their position in the phylogenetic tree. Among them is the genus Stygamoeba, not yet reliably placed on the phylogenetic tree even by a phylogenomic analysis. Only two species of Stygamoeba are known at present, and molecular data exists on one species only. Here, we present a description of the mesohaline species Stygamoeba cauta n. sp. isolated from the bottom sediments of Nivå Bay (Baltic Sea, The Sound). This stick-like, flattened amoeba morphologically resembles the previously described species Stygamoeba regulataSmirnov, 1996. However, the molecular analysis based on the 18S rRNA gene sequences and differences in cell behavior and pattern of locomotion provide strong support for establishing a new species.

A Growth Factor-Free Co-Culture System of Osteoblasts and Peripheral Blood Mononuclear Cells for the Evaluation of the Osteogenesis Potential of Melt-Electrowritten Polycaprolactone Scaffolds.

Scaffolds made of biodegradable biomaterials are widely used to guide bone regeneration. Commonly, in vitro assessment of scaffolds’ osteogenesis potential has been performed predominantly in monoculture settings. Hence, this study evaluated the potential of an unstimulated, growth factor-free co-culture system comprised of osteoblasts (OB) and peripheral blood mononuclear cells (PBMC) over monoculture of OB as an in vitro platform for screening of bone regeneration potential of scaffolds. Particularly, this study focuses on the osteogenic differentiation and mineralized matrix formation aspects of cells. The study was performed using scaffolds fabricated by means of a melt electrowriting (MEW) technique made of medical-grade polycaprolactone (PCL), with or without a surface coating of calcium phosphate (CaP). Qualitative results, i.e., cell morphology by fluorescence imaging and matrix mineralization by von Kossa staining, indicated the differences in cell behaviours in response to scaffolds’ biomaterial. However, no obvious differences were noted between OB and OB+PBMC groups. Hence, quantitative investigation, i.e., alkaline phosphatase (ALP), tartrate-resistant acid phosphatase (TRAP) activities, and gene expression were quantitatively evaluated by reverse transcription-polymerase chain reaction (RT-qPCR), were evaluated only of PCL/CaP scaffolds cultured with OB+PBMC, while PCL/CaP scaffolds cultured with OB or PBMC acted as a control. Although this study showed no differences in terms of osteogenic differentiation and ECM mineralization, preliminary qualitative results indicate an obvious difference in the cell/non-mineralized ECM density between scaffolds cultured with OB or OB+PBMC that could be worth further investigation. Collectively, the unstimulated, growth factor-free co-culture (OB+PBMC) system presented in this study could be beneficial for the pre-screening of scaffolds’ in vitro bone regeneration potential prior to validation in vivo.

Assessing the Minimum Time-Period of Normoxic Preincubation for Stable Adipose Stromal Cell-Derived Vascular Networks.

Pre-vascularization of tissue engineered grafts is a promising strategy to facilitate their improved viability following in vivo implantation. In this process, endothelial cells (ECs) form capillary-like networks that can anastomose with host vasculature. Adipose-derived stromal cells (ASCs) are a commonly used cell population for tissue engineering and contain a subpopulation of ECs capable of assembling into robust vascular networks and anastomosing with the host. However, their initial vascular assembly is significantly impaired in hypoxic conditions (2% O2). In this study, we explored the minimum period of normoxic (20% O2) pre-treatment required to enable the formation of stable vascular networks. ASC-derived vascular structures were allowed to preassemble in fibrin hydrogels in normoxia for 0, 2, 4, or 6days and then transplanted into hypoxic environments for 6days. Total vascular length, pericyte coverage, cell proliferation, apoptosis rates, and ECM production was assessed. Vascular assembly increased with time over the 6days of culture. We found that 4days was the minimum period of time required for stable vascular assembly. We compared the major differences in cell behavior and network structure at Days 2 and 4. Neither proliferation nor apoptosis differed, however, the Day 4 time-point was associated with a significant increase in pericyte coverage (46.1±2.6%) compared to Day 2 (24.3±5.3%). These data suggest oxygen tension may be a mediator of EC-pericyte interactions during vascular assembly. Pre-vascularization strategies should incorporate a normoxic period of to enable successful vascular formation and development.

Tissue Engineering Laboratory Models of the Small Intestine.

In recent years, three-dimensional (3D) cell culture models of the small intestine have gained much attention. These models support cell proliferation, migration, and differentiation and encourage tissue organization which is not possible in two-dimensional (2D) culture systems. Furthermore, the use of a wide variety of cell culture scaffolds and support substrates has revealed considerable differences in cell behavior and tissue organization. These systems have been used in combination with intestinal stem cells, organoid units, or human colonic adenocarcinoma cell lines such as Caco-2 and HT29-MTX to generate a number of in vitro and in vivo models of the intestine. In this study, we review the current 2D and 3D tissue engineering models of the intestine to determine the most effective sources of intestinal cells and current research on support scaffolds capable of inducing the morphological architecture and function of the intestinal mucosa.

DIPG-30. DIFFERENTIAL HYPOXIC RESPONSE IN HUMAN DIPG CELL LINES

BACKGROUND: Diffuse Intrinsic Pontine Glioma (DIPG) are rare and aggressive childhood tumors, with an abysmal prognosis and limited experimental models available for study. A recent report showed that DIPG are hypoperfused compared to surrounding brain tissue, suggesting that the tumor cells are exposed to a hypoxic microenvironment. This stimulus may induce widespread transcriptional changes through activation of Hypoxia-inducible Factors (HIF), which have been associated with invasion, metastasis, angiogenesis, and resistance to radiation therapy and chemotherapy. Therefore, in vitro models using DIPG cells cultured in conditions of normal oxygen tension may not be representative of in vivo tumors because of artificial differences in HIF activation. METHODS: To test cellular responses to hypoxia, human DIPG cell lines with H3.1 K27M and H3.3 K27M mutations were treated with the hypoxia-mimetic compound, cobalt (II) chloride, for 24 hours. Simulated hypoxic responses were assayed using Western blot analysis, proliferation assays, and extracellular flux analysis. RESULTS: The H3.3 K27M cell line increased expression of HIF1a with cobalt (II) chloride treatment and correlated with an increase in glycolytic rates and increased expression of proteins involved in glucose transport and glycolysis. The H3.1 K27M cell line increased expression of HIF2a and not HIF1a after cobalt (II) chloride treatment, correlating with an increase in some glycolysis-associated proteins but no change in glycolytic rate. CONCLUSION: These data suggest that the effects of a hypoxic microenvironment should be considered in experimental models using cultured DIPG cell lines to understand differences in cell behavior, patient prognosis, and predicted treatment responses. Activation of specific HIF isoforms may underlie differences in hypoxic responses among cell lines.

Effects of concave and convex substrate curvature on cell mechanics and the cytoskeleton

In order to understand how cells respond to concave and convex subcellular surface structures, colloidal crystal array and honeycomb-structured surfaces composed of highly ordered hexagonal units with completely inverse curvature were fabricated via facile self-assembly and breath figure approaches, respectively. The influence of hexagonal surface curvature on cell fate was subsequently investigated. Cells underwent more extensive spreading on the convex colloidal crystal array surface, while adhesive forces were higher on the concave honeycomb surface. The behaviors of cells on the different surfaces were investigated by comparing cell morphology, cellular adhesive force and cytoskeleton structure. The results revealed comprehensive differences in cell behavior between those on concave honeycomb surfaces and convex colloidal crystal arrays.

One size does not fit all: developing a cell-specific niche for in vitro study of cell behavior

For more than 100years, cells and tissues have been studied in vitro using glass and plastic surfaces. Over the last 10–20years, a great body of research has shown that cells are acutely sensitive to their local environment (extracellular matrix, ECM) which contains both chemical and physical cues that influence cell behavior. These observations suggest that modern cell culture systems, using tissue culture polystyrene (TCP) surfaces, may fail to reproduce authentic cell behavior in vitro, resulting in “artificial outcomes.” In the current study, we use bone marrow (BM)- and adipose (AD)-derived stromal cells to prepare BM-ECM and AD-ECM, which are decellularized after synthesis by the cells, to mimic the cellular niche for each of these tissues. Each ECM was characterized for its ability to affect BM- and AD-mesenchymal stem cell (MSC) proliferation, as well as proliferation of three cancer cell lines (HeLa, MCF-7, and MDA-MB-231), modulate cell spreading, and direct differentiation relative to standard TCP surfaces. We found that both ECMs promoted the proliferation of MSCs, but that this effect was enhanced when the tissue-origin of the cells matched that of the ECM (i.e. BM-ECM promoted the proliferation of BM-MSCs over AD-MSCs, and vice versa). Moreover, BM- and AD-ECM were shown to preferentially direct MSC differentiation towards either osteogenic or adipogenic lineage, respectively, suggesting that the effects of the ECM were tissue-specific. Further, each ECM influenced cell morphology (i.e. circularity), irrespective of the origin of the MSCs, lending more support to the idea that effects were tissue specific. Interestingly, unlike MSCs, these ECMs did not promote the proliferation of the cancer cells. In an effort to further understand how these three culture substrates influence cell behavior, we evaluated the chemical (protein composition) and physical properties (architecture and mechanical) of the two ECMs. While many structural proteins (e.g. collagen and fibronectin) were found at equivalent levels in both BM- and AD-ECM, the architecture (i.e. fiber orientation; surface roughness) and physical properties (storage modulus, surface energy) of each were unique. These results, demonstrating differences in cell behavior when cultured on the three different substrates (BM- and AD-ECM and TCP) with differences in chemical and physical properties, provide evidence that the two ECMs may recapitulate specific elements of the native stem cell niche for bone marrow and adipose tissues. More broadly, it could be argued that ECMs, elaborated by cells ex vivo, serve as an ideal starting point for developing tissue-specific culture environments. In contrast to TCP, which relies on the “one size fits all” paradigm, native tissue-specific ECM may be a more rational model to approach engineering 3D tissue-specific culture systems to replicate the in vivo niche. We suggest that this approach will provide more meaningful information for basic research studies of cell behavior as well as cell-based therapeutics.

Surface Chemical Gradient Affects the Differentiation of Human Adipose-Derived Stem Cells via ERK1/2 Signaling Pathway.

To understand the role of surface chemistry on cell behavior and the associated molecular mechanisms, we developed and utilized a surface chemical gradient of amine functional groups by carefully adjusting the gas composition of 1,7-octadiene (OD) and allylamine (AA) of the plasma phase above a moving substrate. The chemical gradient surface used in the present work shows an increasing N/C ratio and wettability from the OD side toward the AA side with no change in surface topography. Under standard culture conditions (with serum), human adipose-derived stem cells (hASCs) adhesion and spreading area increased toward the AA side of the gradient. However, there were no differences in cell behavior in the absence of serum. These results, supported by the trends in proteins adsorption on the gradient surface, demonstrated that surface chemistry affects the response of hASCs through cell-adhesive serum proteins, rather than interacting directly with the cells. The expression of p-ERK and the osteogenic differentiation increased toward the AA side of the gradient, while adipogenic differentiation decreased in the same direction; however, when the activation of ERK1/2 was blocked by PD98059, the levels of osteogenic or adipogenic differentiation on different regions of the chemical gradient were the same. This indicates that ERK1/2 may be an important downstream signaling pathway of surface chemistry directed stem cell fate.

Automated, contour-based tracking and analysis of cell behaviour over long time scales in environments of varying complexity and cell density.

Understanding single and collective cell motility in model environments is foundational to many current research efforts in biology and bioengineering. To elucidate subtle differences in cell behaviour despite cell-to-cell variability, we introduce an algorithm for tracking large numbers of cells for long time periods and present a set of physics-based metrics that quantify differences in cell trajectories. Our algorithm, termed automated contour-based tracking for in vitro environments (ACTIVE), was designed for adherent cell populations subject to nuclear staining or transfection. ACTIVE is distinct from existing tracking software because it accommodates both variability in image intensity and multi-cell interactions, such as divisions and occlusions. When applied to low-contrast images from live-cell experiments, ACTIVE reduced error in analysing cell occlusion events by as much as 43% compared with a benchmark-tracking program while simultaneously tracking cell divisions and resulting daughter-daughter cell relationships. The large dataset generated by ACTIVE allowed us to develop metrics that capture subtle differences between cell trajectories on different substrates. We present cell motility data for thousands of cells studied at varying densities on shape-memory-polymer-based nanotopographies and identify several quantitative differences, including an unanticipated difference between two 'control' substrates. We expect that ACTIVE will be immediately useful to researchers who require accurate, long-time-scale motility data for many cells.

Integration of microfluidic chip with biomimetic hydrogel for 3D controlling and monitoring of cell alignment and migration

A biomimetic hydrogel was integrated into microfluidic chips to monitor glioma cell alignment and migration. The extracellular matrix-based biomimetic hydrogel was remodeled by matrix metalloprotease (MMP) secreted by glioma cells and the hydrogel could thus be used to assess cellular behavior. Both static and dynamic cell growth conditions (flow rate of 0.1 mL/h) were used. Cell culture medium with and without vascular endothelial growth factor (VEGF), insensitive VEGF and tissue inhibitor of metalloproteinases (TIMP) were employed to monitor cell behavior. A concentration gradient formed in the hydrogel resulted in differences in cell behavior. Glioma cell viability in the microchannel was 75-85%. Cells in the VEGF-loaded microchannels spread extensively, degrading the MMP-sensitive hydrogel, and achieved cell sizes almost fivefold larger than seen in the control medium. Our integrated system can be used as a model for the study of cellular behavior in a controlled microenvironment generated by fluidic conditions in a biomimetic matrix.

Expression patterns of Oct4, Cdx2, Tead4, and Yap1 proteins during blastocyst formation in embryos of the marsupial, Monodelphis domestica Wagner

The marsupial blastocyst forms in an entirely different manner from its eutherian counterpart, involving cell-zona rather than cell-cell adhesion during the 8- to-16-cell transition. While the eutherian blastocyst consists of a spherical trophoblast completely enveloping a pluripotent inner cell mass, or pluriblast, the marsupial blastocyst forms initially as a bowl-shaped monolayer of cells lining the zona pellucida at the embryonic pole (ep). This monolayer contains a small patch of centrally positioned pluriblast cells edged with trophoblast cells that later coalesce at the abembryonic pole. Using immunocytochemistry, we examined the localization of the proteins Oct4, Cdx2, Tead4, Sox2, and Yap1 in opossum embryos to determine if their temporal expression pattern differed from that in the mouse, given the important differences in cell behavior preceding blastocyst formation in these mammals. Our results indicate that these proteins are expressed in similar temporal patterns despite the topological differences between mouse and opossum cleavage-stage embryos and blastocysts. That the Hippo-pathway protein Yap1 localized specifically around the approximately 128-cell stage to opossum trophoblast nuclei but remained in the cytoplasm of pluriblast cells suggests that this transcriptional regulator participates in allocating cells to the trophoblast lineage, as it does in mouse. Interestingly, in both mouse and opossum embryos, expression of the pluripotency marker Oct4 persisted after Cdx2, which signals trophoblast specification, began to be expressed in trophoblast cells. This and the observation that Cdx2 is present in opossum embryos well before blastomere-zona adhesion even occurs suggests that the proteins studied may have other roles in early mammalian embryonic development.

Ultrastructural Patterns of the Activated Cell Death Programs in the Human Brain

The authors analyzed by transmission electron microscopy (TEM) neurosurgical samples obtained from patients with cerebral tumors, neurotrauma, cerebral ischemia, Moyamoya disease, encephalitis, etc. Their observations concern a variety of dying cell types by different programmed death pathways, including apoptosis, paraptosis, autophagy, autoschizis, programmed necrosis, as well as combined and coexisting forms. This ample work pointed out not only the role of TEM in cell death diagnosis, but the biological differences in cell behavior and beneficial or detrimental effects of suicides for homeostasis, survival, or normal functioning of a tissue, like the integrated vascular tissue and brain parenchyma.