Three-dimensional Cell Structure Research Articles

Preparation of a photocured GelMA hydrogel co-cultured with HOKs and HGFs for an artificial oral mucosal tissue model

Reversible or irreversible damage causes oral mucosal defects, the treatment of which requires biomaterials with good biomechanical properties. In this study, we prepared an artificial oral mucosa tissue model using a photocured methacrylate gelatin (GelMA) hydrogel co-cultured with human gingival fibroblasts (HGFs) and human oral keratinocytes (HOKs). GelMA was fabricated via a chemical reaction between gelatin and methacrylic anhydride (MA), and its structure was confirmed via1H NMR analysis. The culture of HOKs and HGFs was observed on the surface and in the bulk of the GelMA hydrogel, indicating that the hydrogel was biocompatible for cell adhesion and proliferation. In addition, immunofluorescent staining results indicated that HOKs form a three-dimensional cell structure while maintaining their intrinsic keratinocyte properties. Histological evaluations demonstrated that the HGF and HOK bi-layered GelMA hydrogel can be used to develop artificial oral mucosa tissue.

On-Chip Fabrication of Cell-Attached Microstructures using Photo-Cross-Linkable Biodegradable Hydrogel.

We developed a procedure for fabricating movable biological cell structures using biodegradable materials on a microfluidic chip. A photo-cross-linkable biodegradable hydrogel gelatin methacrylate (GelMA) was used to fabricate arbitrary microstructure shapes under a microscope using patterned ultraviolet light. The GelMA microstructures were movable inside the microfluidic channel after applying a hydrophobic coating material. The fabricated microstructures were self-assembled inside the microfluidic chip using our method of fluid forcing. The synthesis procedure of GelMA was optimized by changing the dialysis temperature, which kept the GelMA at a suitable pH for cell culture. RLC-18 rat liver cells (Riken BioResource Research Center, Tsukuba, Japan) were cultured inside the GelMA and on the GelMA microstructures to check cell growth. The cells were then stretched for 1 day in the cell culture and grew well on the GelMA microstructures. However, they did not grow well inside the GelMA microstructures. The GelMA microstructures were partially dissolved after 4 days of cell culture because of their biodegradability after the cells were placed on the microstructures. The results indicated that the proposed procedure used to fabricate cell structures using GelMA can be used as a building block to assemble three-dimensional tissue-like cell structures in vitro inside microfluidic devices.

Mathematical modelling of fluid flow and solute transport to define operating parameters for in vitro perfusion cell culture systems.

In recent years, there has been a move away from the use of static in vitro two-dimensional cell culture models for testing the chemical safety and efficacy of drugs. Such models are increasingly being replaced by more physiologically relevant cell culture systems featuring dynamic flow and/or three-dimensional structures of cells. While it is acknowledged that such systems provide a more realistic environment within which to test drugs, progress is being hindered by a lack of understanding of the physical and chemical environment that the cells are exposed to. Mathematical and computational modelling may be exploited in this regard to unravel the dependency of the cell response on spatio-temporal differences in chemical and mechanical cues, thereby assisting with the understanding and design of these systems. In this paper, we present a mathematical modelling framework that characterizes the fluid flow and solute transport in perfusion bioreactors featuring an inlet and an outlet. To demonstrate the utility of our model, we simulated the fluid dynamics and solute concentration profiles for a variety of different flow rates, inlet solute concentrations and cell types within a specific commercial bioreactor chamber. Our subsequent analysis has elucidated the basic relationship between inlet flow rate and cell surface flow speed, shear stress and solute concentrations, allowing us to derive simple but useful relationships that enable prediction of the behaviour of the system under a variety of experimental conditions, prior to experimentation. We describe how the model may used by experimentalists to define operating parameters for their particular perfusion cell culture systems and highlight some operating conditions that should be avoided. Finally, we critically comment on the limitations of mathematical and computational modelling in this field, and the challenges associated with the adoption of such methods.

A rapid and effective optical-clearing technique for deep tissue fluorescence imaging in trees

This non-invasive clearing technique allows for deep clear imaging of plant tissue and 3D structural analysis of leaf cells. Optical-clearing technology allows the reduction of light scattering and absorption in internal biological tissues. This technology allows microscopy techniques, such as confocal laser scanning microscopy, to obtain structural information deep within the internal tissue and also allows for three-dimensional (3D) tissue analysis. However, the transparent method in plants remains to be further elucidated. In this study, we first report on a non-invasive, rapid and effective clearing technique that increases the transmission of light through plants. Importantly, we found that the leaf terminal veins of four species had different characteristics based on fluorescence imaging observations. Moreover, through the combination of transparent technology and microscopic observation, we clearly observed the three-dimensional structure of plant leaf cells. Our simple and rapid clearing method is useful for fluorescence imaging the intact internal structure. Taken together, the results of this study will provide valuable information for further microscopic experimental investigations in trees.

In Situ Structural Analysis of Leptospira spp. by Electron Cryotomography.

Spirochetes such as Treponema, Borrelia, and Leptospira species can rotate their bodies to swim in liquid environments by rotating periplasmic flagella or endoflagella, which are present inside the cell. Electron cryotomography (ECT) is an imaging technique that directly provides three-dimensional (3D) structures of cells and molecular complexes in their cellular environment at nanometer resolution. Here, I present a general protocol of ECT that covers the sample preparation, data collection, tilt series alignment, and tomographic reconstruction for visualization of intact periplasmic flagella in Leptospira spp. This protocol is capable of determining protein structures at resolutions high enough to visualize their individual domains and secondary structures in their cellular environment.

Mechanical properties of the three-dimensional compression-twist cellular structure

The cellular structure can exhibit many special mechanical behaviors due to its variable cell shape. A three-dimensional compression-twist cell structure based on the rotation mechanism of two-dimensional chiral cell structure is developed, which has twist deformation under axial compression. The shape of three-dimensional compression-twist cell structure is determined through cell angle, cell length, and thickness ratio. Analytical expressions of effective Young’s modulus, Poisson’s ratio, and twist angle are derived by using beam theory, which have a good agreement with the finite element calculations and the deformation process of the cell is discussed. To work on the effect of geometric parameters of cell on the mechanical properties, a finite element analysis model of compression-twist cell structure is carried out, which shows the process of elastic and plastic deformation under compression. Effects of cell angle, cell length, and thickness ratio are fully discussed, which indicate that cell angle has obvious nonlinear effect on relative twist angle and could stiffen it. Finally, a compression-twist cell structure sample is made through three-dimensional printing, and an in-plane compressive experiment is carried out to prove analytical and finite element analysis results.

K-3 Scanning electron microscopy and scanning probe microscopy for visualizing the three-dimensional structure of cells and tissues

K-3 Scanning electron microscopy and scanning probe microscopy for visualizing the three-dimensional structure of cells and tissues

Defining and detecting links in chromosomes

Sophisticated methods for mapping chromatin contacts enable to generate data of the genome structure that provide deep insights into the formation of chromatin interactions within cell nuclei. Due to the recent progress in this field, three-dimensional genomic structures of individual haploid mouse embryonic stem cells have been determined. Here, we analyze these data (8 cells) and determine comprehensive landscape of entanglements between interphase chromosomes. We find a significant number of stable links formed by chromosome pairs. Some links are even conserved between cells. Moreover, examples of stable multiple links, with at least three chromosomes engaged, are also identified. Types of links and their location along chromosomes are determined based on computations of HOMFLY-PT polynomials and Gauss Linking Numbers. Furthermore, stability of links is studied between different models, cells, and based on relaxation simulations of the genomic structure in a simplified structure-based representation. Identified links suggest that small fraction of chromosomes are entangled not only locally. How topoisomerases engineer such configurations remains an open question. Furthermore, presented methods can be used as a quantitative assessment – descriptor – to distinguish the quality of modeled data.

An equivalent cylindrical shell model of vibration analysis based on simplified repeating unit cell for ring truss structure

This paper investigates the potential use of an equivalent cylindrical shell model to analyze the vibration of a ring truss structure for a large deployable circular mesh antenna clamped along one generatrix. The simplified method, which reduces the three-dimensional cell structure into a two-dimensional “flattened” cell structure of the ring truss, is presented. According to the drum-like structure of the antenna, the equivalent cylindrical shell model of the ring truss structure is established. Using the flattened cell structure, the equivalent membrane stiffness and bending stiffness are derived by using the equivalent process and the homogenization method. The differential governing equation of motion for the equivalent cylindrical shell clamped along one generatrix is established based on the Donnell thin shell theory. The theoretical results are verified by numerical simulations. The natural frequencies of the equivalent cylindrical shell are calculated with the non-normal boundary condition clamped along one generatrix. The natural frequencies are compared to ones obtained using a full-scale model by using the finite element method (FEM). The mode shapes of the equivalent cylindrical shell agree well with the ones of the ring truss structure for the full-scale finite element.

Three-dimensional culture of endometrial cells from domestic cats: A new in vitro platform for assessing plastic toxicity.

Plastic polymers can be combined with additives that modify physical properties and stability of the material. However, the biocompatibility of those additives is not well known. The objective of the study was to characterize the impact of zinc stearate–a common additive–through the development of a novel three-dimensional (3-D) in vitro platform with endometrial cells from domestic cats. Epithelial and stromal cells from adult uteri were isolated and cultured in medium supplemented with 3% Matrigel for two weeks in plastic tissue culture dishes that had been identified as polystyrene with and without zinc stearate by Raman, FTIR, and X-ray fluorescence spectroscopies. Three-dimensional cell structures that were obtained were measured and categorized by shape. Cell viability, proliferation, differentiation, organization, and apoptosis then were assessed by immuno-staining. Results indicated that zinc stearate did not affect 3-D endometrial cell structure morphology, viability, or cellular composition. This first study of a new in vitro platform will be useful for studies testing the influence of other additives, drugs, or exogenous hormones.

IL-33 Induces Murine Intestinal Goblet Cell Differentiation Indirectly via Innate Lymphoid Cell IL-13 Secretion.

Regulation of the intestinal mucus layer by goblet cells is important for preventing inflammation and controlling infection. IL-33, a cytokine upregulated in inflammatory bowel disease and helminth infection, induces intestinal goblet cells, but the mechanism remains unclear. Enteroids are three-dimensional structures of primary small intestinal epithelial cells that contain all differentiated intestinal epithelial cell types. We developed an enteroid-immune cell coculture model to determine the mechanism through which IL-33 affects intestinal goblet cell differentiation. We report that IL-33 does not directly induce goblet cell differentiation in murine enteroids; however, IL-13, a cytokine induced by IL-33, markedly induces goblet cells and gene expression consistent with goblet cell differentiation. When enteroids are cocultured with CD90+ mesenteric lymph node cells from IL-33-treated mice, IL-33 then induces IL-13 secretion by group 2 innate lymphoid cells and enteroid gene expression consistent with goblet cell differentiation. In cocultures, IL-33-induced Muc2 expression is dependent on enteroid Il4ra expression, demonstrating a requirement for IL-13 signaling in epithelial cells. In vivo, IL-33-induced intestinal goblet cell hyperplasia is dependent on IL-13. These studies demonstrate that IL-33 induces intestinal goblet cell differentiation not through direct action on epithelial cells but indirectly through IL-13 production by goup 2 innate lymphoid cells.

The Introduction of Thin Open-Cell Metal Foams and their Wider Engineering Applications

Metal foams, having a great of specific surface areas and three-dimensional open cell structures, can be used for electromagnetic shielding and thermal management in electronics, battery electrodes in new energy, catalyst carriers in chemical engineering and lightweight structure in aerospace. The key performance indicators of these open cell metal foams are how to effectively control their pore structures such as pore diameter distribution and porosity and how to make it thinner that can meet different requirements for the component preparation. Summarized about three manufacturing processes are usually used to build metal foams. The first is well-known as physical process. The second is chemical or electrochemical method. The third is a combined process between physical and chemical processes. No matter what kind of process is selected to manufacture open cell metal foam, the specific surface area directly related to the microstructure of the foam is an important parameter in a material selection and design for its application.This paper will introduce a special powder composite plus manufacture process that is developed by Jiangsu Green Materials Hi-Tech. Co. Ltd. The leading manufacture process is a new one that is combined by powder metallurgy, casting, deformation and physical-chemical synthesis. It is environmentally friendly and recyclable from raw material selection, manufacturing process to thermal-mechanical treatment. This paper will also focus on the introduction of the microstructure characterization of open-cell metal foams such as copper, nickel, iron, silver and their alloying foams that we manufactured and give some examples to demonstrate their potential applications in the field of new energy, such as being an electrode for lithium-ion battery, membranes for fuel cell and super-capacitor, in the field of electronic engineering such as thermal management and electromagnetic shielding, in the field of chemical engineering such as separation and catalysts. These examples show their lead roles of these open-cell metal foams and different applications by our developed process.

Three-dimensional genome structures of single diploid human cells.

Three-dimensional genome structures play a key role in gene regulation and cell functions. Characterization of genome structures necessitates single-cell measurements. This has been achieved for haploid cells but has remained a challenge for diploid cells. We developed a single-cell chromatin conformation capture method, termed Dip-C, that combines a transposon-based whole-genome amplification method to detect many chromatin contacts, called META (multiplex end-tagging amplification), and an algorithm to impute the two chromosome haplotypes linked by each contact. We reconstructed the genome structures of single diploid human cells from a lymphoblastoid cell line and from primary blood cells with high spatial resolution, locating specific single-nucleotide and copy number variations in the nucleus. The two alleles of imprinted loci and the two X chromosomes were structurally different. Cells of different types displayed statistically distinct genome structures. Such structural cell typing is crucial for understanding cell functions.

Cell shape affects nanoparticle uptake and toxicity: An overlooked factor at the nanobio interfaces

HypothesisIt is now being increasingly accepted that cells in their native tissue show different morphologies than those grown on a culture plate. Culturing cells on the conventional two-dimensional (2D) culture plates does not closely resemble the in vivo three-dimensional (3D) structure of cells which in turn seems to affect cellular function. This is one of the reasons, among many others, that nanoparticles uptake and toxicology data from 2D culture plates and in vivo environments are not correlated with one another. In this study, we offer a novel platform technology for producing more in vivo-like models of in vitro cell culture. ExperimentsThe normal fibroblast cells (HU02) were cultured on “pseudo-3D” substrates, made from cell imprinting approach. The respond of the cells to a model nanoparticle (gold nanorod) were compared in 2D and “pseudo-3D” cultures modes, by cytotoxicological assays. FindingsIt is illustrated here that the cells’ respond to the exact same type of nanoparticles is majorly dependant in their shape. The use of “pseudo-3D” substrates which could partially mimic the shape of cells in vivo is strongly proposed as a means of better predicting the efficacy of the 2D cell culture plates.

Three-dimensional analysis of micro- and nanostructure of biomaterials and cells by method of scanning probe nanotomography

Aim: to perform a three-dimensional analysis of micro- and nanosctucture and quantitative morphological parameters of alginate spherical microcarriers and porous regenerated silk macrocarriers modifi ed by microparticles of decellularized rat liver matrix and human hepatoma HepG2 cells adhered to micro- and macro carriers.Materials and methods. Three-dimensional porous matrices made from regenerated silk by salt leaching technique and alginate spherical microcarriers fabricated by encapsulation were vitalized by human hepatome HepG2 cells. Study of three-dimensional structure of cells and micro- and macro carriers was carried out at –120 °С by scanning probe cryonanotomography technique with use of experimental setup combining cryoultramicrotome and scanning probe microscope.Results. Three-dimensional nanotomographical reconstructions of HepG2 cells adhered to macropore wall of regenerated silk macrocarrier and to spherical alginate microcarrier are obtained. Morphological parameters (mean roughness, effective surface area and autocorrelation length) are determined for surfaces of macro and microcarriers and adhered cells. The determined mean roughness of alginate microcarrier surface is 76.4 ± 7.5 nm, while that of surface of macropore wall of regenerated silk macrocarrier is 133.8 ± 16.2 nm. At the same time mean roughness of cells adhered to micro- and macrocarriers are 118.5 ± 9.0 и 158.8 ± 21.6 nm correspondingly. Three-dimensional reconstructions of intracellular compartments with dimensions from 140 to 500 nm are also obtained.Conclusion. Obtained as a result of study quantitative morphology characteristics of surfaces of cell carriers and adhered cells show signifi cant degree of correlation of morphological parameters of cells and their carriers. Use of scanning probe cryonanotomography technique for three-dimensional analysis of structure and characteristics of biomaterials, cells and bio-artifi cial cellular systems enables to improve effi ciency of development of novel cell-engineered constructions with predicted morphological, physical, chemical and biological characteristics for tasks of tissue engineering and regenerative medicine.

What qualifies depression to be an aetiological risk factor for coronary artery disease?

It is now being increasingly accepted that cells in their native tissue show different morphologies than those grown on a culture plate. Culturing cells on the conventional two-dimensional (2D) culture plates does not closely resemble the in vivo three-dimensional (3D) structure of cells which in turn seems to affect cellular function. This is one of the reasons, among many others, that nanoparticles uptake and toxicology data from 2D culture plates and in vivo environments are not correlated with one another. In this study, we offer a novel platform technology for producing more in vivo-like models of in vitro cell culture.The normal fibroblast cells (HU02) were cultured on “pseudo-3D” substrates, made from cell imprinting approach. The respond of the cells to a model nanoparticle (gold nanorod) were compared in 2D and “pseudo-3D” cultures modes, by cytotoxicological assays.It is illustrated here that the cells’ respond to the exact same type of nanoparticles is majorly dependant in their shape. The use of “pseudo-3D” substrates which could partially mimic the shape of cells in vivo is strongly proposed as a means of better predicting the efficacy of the 2D cell culture plates.

2 牛骨膜細胞の3次元構造と骨再生過程の解析(第500回 大阪歯科学会例会)

2 牛骨膜細胞の3次元構造と骨再生過程の解析(第500回 大阪歯科学会例会)

Three-dimensional ultrastructural analysis of cells in the periodontal ligament using focused ion beam/scanning electron microscope tomography.

The accurate comprehension of normal tissue provides essential data to analyse abnormalities such as disease and regenerative processes. In addition, understanding the proper structure of the target tissue and its microenvironment may facilitate successful novel treatment strategies. Many studies have examined the nature and structure of periodontal ligaments (PDLs); however, the three-dimensional (3D) structure of cells in normal PDLs remains poorly understood. In this study, we used focused ion beam/scanning electron microscope tomography to investigate the whole 3D ultrastructure of PDL cells along with quantitatively analysing their structural properties and ascertaining their orientation to the direction of the collagen fibre. PDL cells were shown to be in contact with each other, forming a widespread mesh-like network between the cementum and the alveolar bone. The volume of the cells in the horizontal fibre area was significantly larger than in other areas, whereas the anisotropy of these cells was lower than in other areas. Furthermore, the orientation of cells to the PDL fibres was not parallel to the PDL fibres in each area. As similar evaluations are recognized as being challenging using conventional two-dimensional methods, these novel 3D findings may contribute necessary knowledge for the comprehensive understanding and analysis of PDLs.

Observation of the Oil Content of Fried Lotus (Nelumbo nuciferaGaertn.) Root Slices by Confocal Laser Scanning Microscopy Based on Three-Dimensional Model

Microstructures of fried lotus root slices were observed using confocal laser scanning microscopy (CLSM) in fluorescent modes. The three-dimensional (3D) cell structure and morphology of samples were analyzed, for the first time, using 3D reconstructed fluorescent images to determine the cell morphology, oil location and content. Results show that oil was mainly located in the intercellular spaces, broken cells and voids created by the cutting operation, as well as in the wrinkles and distortions caused by the water leakage after frying, cell detachment, starch swelling and rapid dehydration of the first cell layer. Besides, the image stacks obtained by CLSM were processed independently to determine the oil-volume measurements using objective threshold selection. The 3D image data were used for quantitative characterization of oil content by calculating the oil volume. A good correlation between the CLSM results and normal analysis is obtained with the correlation coefficients of 0.892 and 0.875 in calibration and predication, respectively. This study demonstrated that CLSM could determine the oil location and content in fried snacks. Practical Application One of the most important quality parameters for the oil uptake of vacuum fried (VF) and atmospheric fried (AF) lotus root slices were determined and compared under the same frying condition. Microstructures of fried root slices of lotus (Nelumbo nucifera Gaertn.) were observed using CLSM in fluorescent modes. The three-dimensional (3D) cell structure and morphology of samples were analyzed, for the first time, using 3D reconstructed fluorescent images to determine the cell morphology, oil location and content. This optimal way of fried and fired condition was selected by the analytical results of CLSM.

Flow control for cell growth by movement of magnetic particles utilizing electromagnetic force

Three-dimensional (3D) cell structures are required to fabricate artificial organ. Inkjet technology is applied for fabrication of 3D cell structures in order to fabricate artificial organ and inve...