Development of a model bladder extracellular matrix combining disulfide cross-linked hyaluronan with decellularized bladder tissue

[Image: see text] In this work we investigate the feasibility of modifying porcine-derived BAM to include HA with a view to developing a model, artificial extracellular matrix for the study of bladder cell-matrix interactions. HA-DPTH was incorporated into BAM disks and then cross-linked oxidatively to a disulfide containing hydrogel. Disks were seeded with bladder smooth muscle cells (BSMC) and UEC under three culture configurations and incubated for 3, 7, and 14 d. At each time point, matrix contraction was measured, and media supernatants assayed for cell-secreted gelatinase activity. To evaluate cell adherence and organization, triple immunofluorescent labeling of cell nuclei, actin cytoskeleton, and focal contacts was performed. HA-modified BAM exhibited a significant increase in matrix contraction and induced a higher level of cell-secreted gelatinase activity compared to unmodified BAM. Immunofluorescent labeling demonstrated that BSMCs remained adherent to both scaffold types over time. The distribution and organization of the cytoskeleton and focal contacts did not appear to be altered by the presence of HA. Interestingly, cellular infiltration into modified BAM was evident by 7 d and continued beyond 14 d, while BSMCs seeded onto unmodified BAM remained localized to the surface out to 14 d, with minimal infiltration evident only at day 28. These differences in cell infiltration support the gelatinase activity results. Increases in cell migration and matrix proteolysis in the presence of HA may be contributing factors toward BAM remodeling leading to increased matrix contraction with time. The model ECM developed in this work will be utilized for future studies aimed at elucidating the mechanisms controlling key remodeling events associated with bladder repair. Matrix contraction of cell-seeded BAM scaffolds.     

Mechanisms of reduced human vascular cell migration after photodynamic therapy

Restenosis results from intimal hyperplasia and constrictive remodeling following cardiovascular interventions. Photodynamic therapy (PDT) has been shown to inhibit intimal hyperplasia in vivo by preventing neointimal repopulation of the treated vessel. This study was undertaken in an attempt to further dissect the mechanisms by which PDT acts on secreted and extracellular matrix proteins to inhibit migration of cultured human vascular cells. PDT of three-dimensional collagen gels inhibited invasive human smooth muscle cell (SMC) migration, whereas cell-derived matrix metalloproteinase production remained unaltered. Additionally, PDT generated cross-links in the collagen gels, a result substantiated in an ex vivo model whereby PDT rendered the treated vessels resistant to pepsin digestion and inhibited invasive migration of SMC and fibroblasts. These data support the premise that by inducing matrix protein cross-links, rendering the vessel resistant to degradation, in vivo PDT inhibits repopulation of the vessel and therefore intimal hyperplasia.     

Fabrication of Stiffness Gradients of GelMA Hydrogels Using a 3D Printed Micromixer

Many properties in both healthy and pathological tissues are highly influenced by the mechanical properties of the extracellular matrix. Stiffness gradient hydrogels are frequently used for exploring these complex relationships in mechanobiology. In this study, the fabrication of a simple, cost‐efficient, and versatile system is reported for creation of stiffness gradients from photoactive hydrogels like gelatin‐methacryloyl (GelMA). The setup includes syringe pumps for gradient generation and a 3D printed microfluidic device for homogenous mixing of GelMA precursors with different crosslinker concentration. The stiffness gradient is investigated by using rheology. A co‐culture consisting of human adipose tissue‐derived mesenchymal stem cells (hAD‐MSCs) and human umbilical cord vein endothelial cells (HUVECs) is encapsulated in the gradient construct. It is possible to locate the stiffness ranges at which the studied cells displayed specific spreading morphology and migration rates. With the help of the described system, variable mechanical gradient constructs can be created and optimal 3D cell culture conditions can be experientially identified.     

Artocarpin‐enriched (Artocarpus altilis) Heartwood Extract Provides Protection Against UVB‐induced Mechanical Damage in Dermal Fibroblasts

This study aimed to evaluate the protective effect of artocarpin‐enriched (Artocarpus altilis) heartwood extract on the mechanical properties of UVB‐irradiated fibroblasts. Human skin fibroblasts were pretreated with 50 μg/mL^?1 extract and later irradiated with UVB (200 mJ/cm^?2). They were then cultured within three‐dimensional of free‐floating and tense collagen lattices. The pretreatment of fibroblasts with the extract prior to UVB radiation showed cells protection against UVB‐induced suppression of α‐SMA expression, fibroblast migration and contraction. These results reveal that the extract prevents mechanical damages induced by UVB irradiation in fibroblast‐embedded collagen lattices, and therefore, has a potential as a natural photo‐protectant.     

A novel 3D mammalian cell perfusion-culture system in microfluidic channels

Mammalian cells cultured on 2D surfaces in microfluidic channels are increasingly used in drug development and biological research applications. These systems would have more biological or clinical relevance if the cells exhibit 3D phenotypes similar to the cells in vivo. We have developed a microfluidic channel based system that allows cells to be perfusion-cultured in 3D by supporting them with adequate 3D cell-cell and cell-matrix interactions. The maximal cell-cell interaction was achieved by perfusion-seeding cells through an array of micropillars; and 3D cell-matrix interactions were achieved by a polyelectrolyte complex coacervation process to form a thin layer of matrix conforming to the 3D cell shapes. Carcinoma cell lines (HepG2, MCF7), primary differentiated (hepatocytes) and primary progenitor cells (bone marrow mesenchymal stem cells) were perfusion-cultured for 72 hours to 1 week in the microfluidic channel, which preserved their 3D cyto-architecture and cell-specific functions or differentiation competence. This transparent 3D microfluidic channel-based cell culture system also allows direct optical monitoring of cellular events for a wide range of applications.     

The Role of Mechanical Properties and Structure of Type I Collagen Hydrogels on Colorectal Cancer Cell Migration

Mechanical interactions between cells and their microenvironment play an important role in determining cell fate, which is particularly relevant in metastasis, a process where cells invade tissue matrices with different mechanical properties. In vitro, type I collagen hydrogels have been commonly used for modeling the microenvironment due to its ubiquity in the human body. In this work, the combined influence of the stiffness of these hydrogels and their ultrastructure on the migration patterns of HCT-116 and HT-29 spheroids are analyzed. For this, six different types of pure type I collagen hydrogels by changing the collagen concentration and the gelation temperature are prepared. The stiffness of each sample is measured and its ultrastructure is characterized. Cell migration studies are then performed by seeding the spheroids in three different spatial conditions. It is shown that changes in the aforementioned parameters lead to differences in the mechanical stiffness of the matrices as well as the ultrastructure. These differences, in turn, lead to distinct cell migration patterns of HCT-116 and HT-29 spheroids in either of the spatial conditions tested. Based on these results, it is concluded that the stiffness and the ultrastructural organization of the matrix can actively modulate cell migration behavior in colorectal cancer spheroids.     

Biostimulation of dermal fibroblast by sublethal Q-switched Nd:YAG 532 nm laser: collagen remodeling and pigmentation

The application of medical lasers in treating pigmented lesions has rapidly developed over the past decade. In both clinical and cosmetic application, melanin is targeted in pigmented areas and destroyed by the mechanism of selective photothermolysis. When laser radiation passes through superficial pigmented tissue, energy will be further reduced by dermal collagen scattering and absorption. Non-pigmented dermal fibroblasts will be exposed to co-incidental laser irradiation at lower energy levels. Biostimulation of dermal fibroblasts by low energy laser is reported in this paper. The Q-switched frequency doubled Nd:YAG 532nm laser used in clinical laser therapy was used in this study. Sublethal laser fluence was determined at 0.8J/cm(2) and used to stimulate normal human fibroblasts in monolayer culture. The results showed that there was no significant difference in collagen synthesis between the stimulated fibroblasts and controls. However, significant delay in collagen remodeling activity was demonstrated in the irradiated group by measuring fibroblast populated collagen lattice (FPCL) contraction. The stimulation of SCF, HGF and b-FGF gene expression was determined by RT-PCR analysis and demonstrated to vary between cases. Two out of six cell lineages that showed stronger responses to laser stimulation on SCF, HGF and b-FGF gene expressions were used to prepare conditioned media. The conditioned media from irradiated groups showed significant increase in SCF and b-FGF content and stimulated SK-mel-3 melanoma cells to synthesize more melanin in vitro. These results suggest that sublethal laser stimulation of fibroblasts may cause post-laser hyperpigmentation through production of melanogenic stimulatory cytokines. The degree of stimulation of SCF, HGF and b-FGF production varied between individual cell lineages, which may reflect the true variation of post-laser hyperpigmentation in clinical practice.     

Hydrogels with Dynamically Controllable Mechanics and Biochemistry for 3D Cell Culture Platforms

Many cell-matrix interaction studies have proved that dynamic changes in the extracellular matrix(ECM)are crucial to maintain cellular properties and behaviors.Thus,developing materials that can recapitulate the dynamic attributes of the ECM is highly desired for threedimensional(3 D)cell culture platforms.To this end,we sought to develop a hydrogel system that would enable dynamic and reversible turning of its mechanical and biochemical properties,thus facilitating the control of cell culture to imitate the natural ECM.Herein,a hydrogel with dynamic mechanics and a biochemistry based on an addition-fragmentation chain transfer(AFCT)reaction was constructed.Thiol-modified hyaluronic acid(HA)and allyl sulfide-modifiedε-poly-L-lysine(EPL)were synthesized to form hydrogels,which were non-swellable and biocompatible.The reversible modulus of the hydrogel was first achieved through the AFCT reaction;the modulus can also be regulated stepwise by changing the dose of UVA irradiation.Dynamic patterning of fluorescent markers in the hydrogel was also realized.Therefore,this dynamically controllable hydrogel has great potential as a 3 D cell culture platform for tissue engineering applications.     

Myofibroblasts in the infarct area: concepts and challenges

Myofibroblasts are differentiated fibroblasts that hold a key role in wound healing and remodeling following myocardial infarction (MI). A large repertoire of stimuli, such as mechanical stretch, growth factors, cytokines, and vasoactive peptides, induces myofibroblast differentiation. Myofibroblasts are responsible for the production and deposition of collagen, leading to the establishment of a dense extracellular matrix that strengthens the infarcted tissue and minimizes dilatation of the infarct area. In addition, cells contributing to fibrosis act on sites distal from the infarct area and promote collagen deposition in noninfarcted tissue, thus contributing to adverse remodeling and consequently to the development of congestive heart failure (CHF). Current drugs that are used to treat post-MI CHF do influence fibroblasts and myofibroblasts; however, their therapeutic efficacy is far from being regarded as ideal. Novel therapeutic agents targeting (myo)fibroblasts are being developed to successfully prevent the cardiac remodeling of sites remote from the infarct area and therefore hinder the establishment of CHF. The purpose of this review article is to discuss the basic concepts of the myofibroblasts' actions in cardiac wound healing processes, factors that influence them, currently available pharmacological agents, and future challenges in this area.     

Co-culture of tumor spheroids and monocytes in a collagen matrix-embedded microfluidic device to study the migration of breast cancer cells

A 3D co-culture microfluidic device was developed to study the effects of ECM stiffness and TAMs on tumor cells migration.     

A practical guide to microfluidic perfusion culture of adherent mammalian cells

Culturing cells at microscales allows control over microenvironmental cues, such as cell-cell and cell-matrix interactions; the potential to scale experiments; the use of small culture volumes; and the ability to integrate with microsystem technologies for on-chip experimentation. Microfluidic perfusion culture in particular allows controlled delivery and removal of soluble biochemical molecules in the extracellular microenvironment, and controlled application of mechanical forces exerted via fluid flow. There are many challenges to designing and operating a robust microfluidic perfusion culture system for routine culture of adherent mammalian cells. The current literature on microfluidic perfusion culture treats microfluidic design, device fabrication, cell culture, and micro-assays independently. Here we systematically present and discuss important design considerations in the context of the entire microfluidic perfusion culture system. These design considerations include the choice of materials, culture configurations, microfluidic network fabrication and micro-assays. We also present technical issues such as sterilization; seeding cells in both 2D and 3D configurations; and operating the system under optimized mass transport and shear stress conditions, free of air-bubbles. The integrative and systematic treatment of the microfluidic system design and fabrication, cell culture, and micro-assays provides novices with an effective starting point to build and operate a robust microfludic perfusion culture system for various applications.     

An axial periodic fibrillar arrangement of antigenic determinants for fibronectin and procollagen on ascorbate treated human fibroblasts

Fibronectin and collagens are major constituents of the cell matrix of fibroblasts. Fibronectin is a 220,000 dalton glycoprotein that mediates a variety of adhesive functions of cells examined in vitro. Fibronectin is secreted in a soluble form and interacts with collagen to form extracellular filaments. Fibronectin and procollagen type I were localized using the peroxidase anti-peroxidase method. Under standard culture conditions, fibronectin and procollagen were localized to non-periodic 10 nm extracellular fibrils, the cell membrane and plasma membrane vesicles. Ascorbate treatment of cells leads to a new larger fibril with a diameter of approximately 40 nm. Antibodies to fibronectin and procollagen I react to these native collagen fibrils with an axial periodicity of approximately 70 nm. Fibronectin is clearly associated with native collagen fibrils produced by ascorbate treated cells and there is an asymetric distribution or segregation of fibronectin on these collagen fibrils with a 70 nm axial repeat.     

Three-Dimensional Collagen Gel Networks for Neural Stem Cell-Based Neural Tissue Engineering

Stem and progenitor cells isolated from the embryonic rat cerebral cortex were immobilized by matrix entrapment in three-dimensional (3D) Type I collagen gels, and cultured in serum-free medium containing basic fibroblast growth factor. The cells trapped within the collagen networks actively proliferated and formed clone-like aggregates. Neurons were the first differentiated cells to appear within the aggregates, followed by generation of astrocytes and oligodendrocytes. In addition, necrotic cores were developed as the aggregate diameter increased and cell viability declined significantly after 3 weeks in culture. To overcome these problems, the cell-collagen constructs were transferred to Rotary Wall Vessel bioreactors for up to 10 weeks. In the rotary culture, the collagen gels compacted 3-4 folds and a long-term growth and differentiation of neural stem and progenitor cells was dynamically maintained. Remarkably, the cell-collagen constructs formed a complex two-layered structure that superficially emulated to a certain extent the cerebral cortex of the embryonic brain in architecture and functionality. The engineered 3D tissue-like constructs displaying characteristic properties of neuronal circuits may have potential use in tissue replacement therapy for injured brain and spinal cord.     

Phospholipid Polymer Hydrogel Matrices with Dually Immobilized Cytokines for Accelerating Secretion of the Extracellular Matrix by Encapsulated Cells

Construction of 3D tissues by various types of cells with specific characteristics is an important and fundamental technology in tissue reconstruction medicine and animal‐free diagnosis system. To do so, an excellent extracellular matrix (ECM) is needed for encapsulation of cells and maintaining cell activity. Spontaneously forming hydrogel matrix is used by complexation between two water‐soluble polymers, 2‐methacryloyloxyethyl phosphorylcholine polymer bearing phenylboronic acid groups and poly(vinyl alcohol). Two cytokines for cell proliferation are immobilized in the hydrogel matrix to control the activities of the encapsulated cells. The cytokine‐immobilized hydrogel matrix can encapsulate both L929 fibroblasts and normal human dermal fibroblasts under mild condition. The physical properties of the hydrogel matrix can follow the proliferation process of the encapsulated cells. The encapsulated cells secrete ECM in the polymer hydrogel networks upon 3D culturing for 7 days. Consequently, the tissue‐mimicking ECM hybrid hydrogels are fabricated successfully.     

Oxygen consumption rate of cells in 3D culture: the use of experiment and simulation to measure kinetic parameters and optimise culture conditions

Understanding the basal O(2) and nutrient requirements of cells is paramount when culturing cells in 3D tissue models. Any scaffold design will need to take such parameters into consideration, especially as the addition of cells introduces gradients of consumption of such molecules from the surface to the core of scaffolds. We have cultured two cell types in 3D native collagen type I scaffolds, and measured the O(2) tension at specific locations within the scaffold. By changing the density of cells, we have established O(2) consumption gradients within these scaffolds and using mathematical modeling have derived rates of consumption for O(2). For human dermal fibroblasts the average rate constant was 1.19 × 10(-17) mol cell(-1) s(-1), and for human bone marrow derived stromal cells the average rate constant was 7.91 × 10(-18) mol cell(-1) s(-1). These values are lower than previously published rates for similar cells cultured in 2D, but the values established in this current study are more representative of rates of consumption measured in vivo. These values will dictate 3D culture parameters, including maximum cell-seeding density and maximum size of the constructs, for long-term viability of tissue models.     

Enzyme‐Induced Matrix Softening Regulates Hepatocarcinoma Cancer Cell Phenotypes

The progression of cancer is often accompanied by changes in the mechanical properties of an extracellular matrix. However, limited efforts have been made to reproduce these biological events in vitro. To this end, this study demonstrates that matrix remodeling caused by matrix metalloproteinase (MMP)‐1 regulates phenotypic activities and modulates radiosensitivity of cancer cells exclusively in a 3D matrix. In this study, hepatocarcinoma cells are cultured in a collagen‐based gel tailored to present an elastic modulus of ≈4.0 kPa. The subsequent exposure of the gel to MMP‐1 decreases the elastic modulus from 4.0 to 0.5 kPa. In response to MMP‐1, liver cancer cells undergo active proliferation, downregulation of E‐cadherin, and the loss of detoxification capacity. The resulting spheroids are more sensitive to radiation than the spheroids cultured in the stiffer gel not exposed to MMP‐1. Overall, this study serves to better understand and control the effects of MMP‐induced matrix remodeling.     

Engineering Photocleavable Protein-decorated Hydrogels to Regulate Cell Adhesion and Migration

Cell adhesion and migration play essential roles in tissue development and maintenance, and abnormal cell migration is involved in life-threatening diseases, including vascular disease, tumor formation, and metastasis. The advances in hydrogel-based 3D cell culture development facilitated the investigation of cell motility behavior, including cell-cell and cell-matrix adhesion and cell migration in a microenvironment more related to in vivo situations. Establishing advanced methods for these in vitro studies is thus necessary. Photo-sensitive proteins show advantages in remote and non-invasive regulation of hydrogels' properties, and thus are of great potential in regulating 3D cultured cells' behavior. In the presented study, we engineered photocleavable protein(PhoCl)-decorated hydrogels to regulate cell adhesion and migration of MDA-MB-231. The integrin-binding motif RGD was fused to the PhoCl and was decorated on the hydrogel. After being exposed to light at 405 nm, the PhoCl was cleaved and the RGD motif was released, resulting in detachment of the binding cells. The regulatory effect of the light illumination showed a time-dependent and cell density-dependent manner. Furthermore, the elimination of RGD by patterned light exposure completely suspended the cell migration to the corresponding region, suggesting a controllable regulation of the cell migration direction.     

Three-dimensional microfiber devices that mimic physiological environments to probe cell mechanics and signaling

Many physiological systems are regulated by cells that alter their behavior in response to changes in their biochemical and mechanical environment. These cells experience this dynamic environment through an endogenous biomaterial matrix that transmits mechanical force and permits chemical exchange with the surrounding tissue. As a result, in vitro systems that mimic three-dimensional, in vivo cellular environments can enable experiments that reveal the nuanced interplay between biomechanics and physiology. Here we report the development of a minimal-profile, three-dimensional (MP3D) experimental microdevice that confines cells to a single focal plane, while allowing the precise application of mechanical displacement to cells and concomitant access to the cell membrane for perfusion with biochemical agonists. The MP3D device--an ordered microfiber scaffold erected on glass--provides a cellular environment that induces physiological cell morphologies. Small manipulations of the scaffold's microfibers allow attached cells to be mechanically probed. Due to the scaffold's minimal height profile, MP3D devices confine cells to a single focal plane, facilitating observation with conventional epifluorescent microscopy. When examining fibroblasts within MP3D devices, we observed robust cellular calcium responses to both a chemical stimulus as well as mechanical displacement of the cell membrane. The observed response differed significantly from previously reported, mechanically-induced calcium responses in the same cell type. Our findings demonstrate a key link between environment, cell morphology, mechanics, and intracellular signal transduction. We anticipate that this device will broadly impact research in fields including biomaterials, tissue engineering, and biophysics.     

Exploring peptide‐functionalized alginate scaffolds for engineering cardiac tissue from human embryonic stem cell‐derived cardiomyocytes in serum‐free medium

Engineering human cardiac tissue is a promising solution for myocardial repair of injured hearts and for drug screening. Herein, we examined the capability of chemically defined alginate scaffolds to promote cardiac tissue regeneration from human embryonic stem cell‐derived cardiomyocytes (hESC‐CMs) in serum‐free, chemically defined medium. The cells were single seeded or coseeded with human dermal fibroblasts (HFs) in macroporous scaffolds made from pristine alginate or alginate modified with arginine‐glycine‐aspartate (RGD) peptide and heparin‐binding peptide (HBP). Our results show that the addition of fibroblasts to the 3‐D culture is indispensable for the formation of functional cardiac tissues and that the presence of RGD/HBP attached to the alginate matrix further improves its functionality. The engineered tissue displayed the typical fiber morphology with massive striation. An increase in contraction amplitude and calcium transients with time, together with a decrease in excitation threshold, indicated advancement toward tissue maturation. Our results thus point to the importance of co‐cultivating fibroblasts with hESCs‐CMs in chemically defined peptide‐functionalized alginate scaffolds and culture medium for regenerating functional cardiac tissue in vitro.     

Microfluidic adhesion analysis of single glioma cells for evaluating the effect of drugs

Cancer metastasis is one of the most serious problems for tumor therapy, which is closely related to cell adhesion and deadhesion process. Better comprehension of cell adhesion ability will benefit drug research. Here, a biomimetic microfluidic enzyme digestion method was proposed to gently measure the influence of drugs on cell-matrix adhesion ability at the single cell level.The method can selectively digest the extracellular matrix(ECM) that linked to a single cell, and the trypsin concentration around the cell is relatively uniform and constant, thus the measured cell adhesion strength should be precise. Commercially available anti-cancer agents including 5-fluorouracil(5-FU), actinomycin D(Act D), temozolomide(TMZ) and allicin were evaluated, and the data showed only TMZ and allicin can inhibit cell adhesion significantly under our experiment conditions. The influence of TMZ became more and more obvious as the increase of duration and the effect became prominent only after 6 h adhesion process, which could provide a quick evaluation of whether the drugs are effective to cancer cell(compared with Calcein-AM/PI cell viability test). The adhesion strength of U87 cells decreased when the concentration of TMZ increased, and the effect of TMZ can be effectively inhibited by adding lactic acid to culture medium, which indicated acidic tumor microenvironment could promote drug resistance of tumor cells. Different from conventional evaluation methods which focus on the drugs' influence on cellular viability or metabolism, this work provides a new perspective to study the effect of drugs, which is helpful to enrich the drug evaluation system.