Monolayer Tissue Research Articles

"I make efforts, people make comments": Prof. H. Zanyin Gaw-pioneering the world, the trailblazer and founder of China's virology research.

"I make efforts, people make comments": Prof. H. Zanyin Gaw-pioneering the world, the trailblazer and founder of China's virology research.

Use of Mesothelial Cells and Biological Matrices for Tissue Engineering of Simple Epithelium Surrogates.

Tissue-engineering technologies have progressed rapidly through last decades resulting in the manufacture of quite complex bioartificial tissues with potential use for human organ and tissue regeneration. The manufacture of avascular monolayered tissues such as simple squamous epithelia was initiated a few decades ago and is attracting increasing interest. Their relative morphostructural simplicity makes of their biomimetization a goal, which is currently accessible. The mesothelium is a simple squamous epithelium in nature and is the monolayered tissue lining the walls of large celomic cavities (peritoneal, pericardial, and pleural) and internal organs housed inside. Interestingly, mesothelial cells can be harvested in clinically relevant numbers from several anatomical sources and not less important, they also display high transdifferentiation capacities and are low immunogenic characteristics, which endow these cells with therapeutic interest. Their combination with a suitable scaffold (biocompatible, degradable, and non-immunogenic) may allow the manufacture of tailored serosal membranes biomimetics with potential spanning a wide range of therapeutic applications, principally for the regeneration of simple squamous-like epithelia such as the visceral and parietal mesothelium vascular endothelium and corneal endothelium among others. Herein, we review recent research progresses in mesothelial cells biology and their clinical sources. We make a particular emphasis on reviewing the different types of biological scaffolds suitable for the manufacture of serosal mesothelial membranes biomimetics. Finally, we also review progresses made in mesothelial cells-based therapeutic applications and propose some possible future directions.

Mechanical model of geometric cell and topological algorithm for cell dynamics from single-cell to formation of monolayered tissues with pattern.

Geometric and mechanical properties of individual cells and interactions among neighboring cells are the basis of formation of tissue patterns. Understanding the complex interplay of cells is essential for gaining insight into embryogenesis, tissue development, and other emerging behavior. Here we describe a cell model and an efficient geometric algorithm for studying the dynamic process of tissue formation in 2D (e.g. epithelial tissues). Our approach improves upon previous methods by incorporating properties of individual cells as well as detailed description of the dynamic growth process, with all topological changes accounted for. Cell size, shape, and division plane orientation are modeled realistically. In addition, cell birth, cell growth, cell shrinkage, cell death, cell division, cell collision, and cell rearrangements are now fully accounted for. Different models of cell-cell interactions, such as lateral inhibition during the process of growth, can be studied in detail. Cellular pattern formation for monolayered tissues from arbitrary initial conditions, including that of a single cell, can also be studied in detail. Computational efficiency is achieved through the employment of a special data structure that ensures access to neighboring cells in constant time, without additional space requirement. We have successfully generated tissues consisting of more than 20,000 cells starting from 2 cells within 1 hour. We show that our model can be used to study embryogenesis, tissue fusion, and cell apoptosis. We give detailed study of the classical developmental process of bristle formation on the epidermis of D. melanogaster and the fundamental problem of homeostatic size control in epithelial tissues. Simulation results reveal significant roles of solubility of secreted factors in both the bristle formation and the homeostatic control of tissue size. Our method can be used to study broad problems in monolayered tissue formation. Our software is publicly available.

Regenerating Retinal Pigment Epithelial Cells to Cure Blindness: A Road Towards Personalized Artificial Tissue.

Retinal pigment epithelium (RPE) is a polarized monolayer tissue that functions to support the health and integrity of retinal photoreceptors (PRs). RPE atrophy has been linked to pathogenesis of age-related macular degeneration (AMD), a leading cause of blindness in elderly in the USA. RPE atrophy in AMD leads to the PR cell death and vision loss. It is thought that replacing diseased RPE with healthy RPE tissue can prevent PR cell death. Retinal surgical innovations have provided proof-of-principle data that autologous RPE tissue can replace diseased macular RPE and provide visual rescue in AMD patients. Current efforts are focused on developing an in vitro tissue using natural and synthetic scaffolds to generate a polarized functional RPE monolayer. In the future, these tissue-engineering approaches combined with pluripotent stem cell technology will lead to the development of personalized and "off-the-shelf" cell therapies for AMD patients. This review summarizes the historical development and ongoing efforts in surgical and in vitro tissue engineering techniques to develop a three-dimensional therapeutic native RPE tissue substitute.

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Fetal vs adult mesenchymal stem cells achieve greater gene expression, but less osteoinduction.

To investigate adenoviral transduction in mesenchymal stem cells (MSCs) and effects on stemness in vitro and function as a cell therapy in vivo. Bone marrow-derived adult and fetal MSC were isolated from an equine source and expanded in monolayer tissue culture. Polyethylenimine (PEI)-mediated transfection of pcDNA3-eGFP or adenoviral transduction of green fluorescent protein (GFP) was evaluated in fetal MSCs. Adenoviral-mediated transduction was chosen for subsequent experiments. All experiments were carried out at least in triplicate unless otherwise noted. Outcome assessment was obtained by flow cytometry or immunohystochemistry and included transduction efficiency, cell viability, stemness (i.e., cell proliferation, osteogenic and chondrogenic cell differentiation), and quantification of GFP expression. Fetal and adult MSCs were then transduced with an adenoviral vector containing the gene for the bone morphogenic protein 2 (BMP2). In vitro BMP2 expression was assessed by enzyme linked immunosorbent assay. In addition, MSC-mediated gene delivery of BMP2 was evaluated in vivo in an osteoinduction nude mouse quadriceps model. New bone formation was evaluated by microradiography and histology. PEI provided greater transfection and viability in fetal MSCs than other commercial chemical reagents. Adenoviral transduction efficiency was superior to PEI-mediated transfection of GFP in fetal MSCs (81.3% ± 1.3% vs 35.0% ± 1.6%, P < 0.05) and was similar in adult MSCs (78.1% ± 1.9%). Adenoviral transduction provided significantly greater expression of GFP in fetal than adult MSCs (7.4 ± 0.1 vs 4.4 ± 0.3 millions of mean fluorescence intensity units, P < 0.01) as well as significantly greater in vitro BMP2 expression (0.16 pg/cell-day vs 0.10 pg/cell-day, P < 0.01). Fraction of fetal MSC GFP positive cells decreased significantly faster than adult MSCs (1.15% ± 0.05% vs 11.4% ± 2.1% GFP positive at 2 wk post-transduction, P < 0.05). Cell proliferation and osteogenic differentiation in vitro were not affected by Ad transduction in both fetal and adult MSCs, but fetal MSCs had reduced chondrogenic differentiation in vitro when compared to adult (P < 0.01). Chondrogenic differentiation was also significantly reduced in Ad-GFP transduced cells (P < 0.05). Ad-BMP2 transduced adult MSCs induced new bone formation in more thighs than Ad-BMP2 transduced fetal MSCs (83% vs 17% of the six treated thighs per group, P < 0.05) and resulted in increased femur midshaft diameter due to greater extent of periosteal new bone (1.57 ± 0.35 mm vs 1.27 ± 0.08 mm, P < 0.05). Fetal MSCs may be genetically manipulated ex vivo with adenoviral vectors. Nonetheless, the abbreviated expression of the exogenous gene may limit their applications in vivo.

Tumor cell-targeted delivery of nanoconjugated oligonucleotides in composite spheroids.

Standard tissue culture has often been a poor model for predicting the efficacy of anti-cancer agents including oligonucleotides. In contrast to the simplicity of monolayer tissue cultures, a tumor mass includes tightly packed tumor cells, tortuous blood vessels, high levels of extracellular matrix, and stromal cells that support the tumor. These complexities pose a challenge for delivering therapeutic agents throughout the tumor, with many drugs limited to cells proximal to the vasculature. Multicellular tumor spheroids are superior to traditional monolayer cell culture for the assessment of cancer drug delivery, since they possess many of the characteristics of metastatic tumor foci. However, homogeneous spheroids comprised solely of tumor cells do not account for some of the key aspects of metastatic tumors, particularly the interaction with host cells such as fibroblasts. Further, homogeneous culture does not allow for the assessment of targeted delivery to tumor versus host cells. Here we have evaluated delivery of targeted and untargeted oligonucleotide nanoconjugates and of oligonucleotide polyplexes in both homogeneous and composite tumor spheroids. We find that inclusion of fibroblasts in the spheroids reduces delivery efficacy of the polyplexes. In contrast, targeted multivalent RGD-oligonucleotide nanoconjugates were able to effectively discriminate between melanoma cells and fibroblasts, thus providing tumor-selective uptake and pharmacological effects.

Technical Aspects of 3-Dimentional Culture of Mammalian Cells

Three dimensional-tissue culture systems of human solid tumor cells has been implemented for effective chemotherapeutic screening and to avoid the disadvantages of the over simplified monolayer tissue culture system. The influence of tumor microenvironment to tumor therapeutics and screening of new anticancer drugs has been found to significant. The complex tumor microenvironment is poorly represented in the simple monolayer tissue culture system. The 3D culture models closely imitate several in vivo conditions of solid tumors in terms of geometry and expression of microenvironment. . However, several technical obstacles might discourage the use of this culture model. In the current mini-review, we are presenting the exact protocol for culturing mammalian cell as three dimensional tissue culture systems (multicellular spheroids and multicellular layers) and how to utilize these models for anti-cancer assessment.

Glucan HBP-A increase type II collagen expression of chondrocytes in vitro and tissue engineered cartilage in vivo

Although chondroprotective activities have been documented for polysaccharides, the potential target of different polysaccharide may differ. The study was aimed to explore the effect of glucan HBP-A in chondrocyte monolayer culture and chondrocytes-alginate hydrogel constructs in vivo, especially on the expression of type II collagen. Chondrocytes isolated from rabbit articular cartilage were cultured and verified by immunocytochemical staining of type II collagen. Chondrocyte viability was assessed after being treated with HBP-A in different concentrations. Morphological status of chondrocytes-alginate hydrogel constructs in vitro was observed by scanning electron microscope (SEM). The constructs were treated with HBP-A and then injected to nude mice subcutaneously. Six weeks after transplantation, the specimens were observed through transmission electron microscopy (TEM). The mRNA expressions of disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTs-5), aggrecan and type II collagen in both monolayer culture and constructs were determined by real time polymerase chain reaction (PCR). The expression of type II collagen and matrix metalloproteinases-3 (MMP-3) in chondrocyte monolayer culture was also tested through Western blot and enzyme linked immunosorbent assay (ELISA), respectively. MMP-3 secretion and ADAMTs-5 mRNA expression in vitro were inhibited by HBP-A at 0.3 mg/mL concentration. In morphological study, there were significant appearance of collagen in those constructs treated by HBP-A. Accordingly, in both chondrocyte monolayer culture and chondrocytes-alginate hydrogel constructs, the expression of type II collagen was increased significantly in HBP-A group when compared with control group (P<0.001). The study documented that the potential pharmacological target of glucan HBP-A in chondrocytes monolayer culture and tissue engineered cartilage in vivo may be concerned with the inhibition of catabolic enzymes MMP-3, ADAMTs-5, and increasing of type II collagen expression.

Intercellular Adhesion, Cytoskeletal Organization and Signaling in a Substrate-Free Cell Monolayer

Connections between cell-cell junctions and cytoskeleton profoundly influence cell shape, motility and gene expression. However, cell-cell interactions are easily masked by cell-substrate interactions thus remains poorly characterized. A cell monolayer can be considered the simplest multicellular tissue structure, yet they fulfill critical roles in human physiology and tissue engineering. A systematic characterization of intercellular adhesion, cytoskeletal organization and signaling in a substrate-free cell monolayer is crucial for unlocking the potential for cell-sheet tissue engineering. To test our hypothesis that intercellular junctions and cytoskeleton components likely play significant roles in establishing cell sheet cohesion, we have developed a unique partial-lifting technique that allows generation and investigation of the biological and mechanical properties of substrate-freehuman keratinocytecell monolayer.Results demonstrated that through alterations of spatial organization and expression of their cytoskeleton and specialized intercellular adhesions, substrate-free cells create a monolayer tissue that has a much stronger mechanical strength than their substrate-adhering controls. To determine the contribution of intercellular junctions and cytoskeleton to cell sheet mechanics, we altered the junctional protein plakoglobin by siRNA interference, altered actin-myosin interactions by ROCK kinase inhibitor and actin disruption agent. Knockdown of plakoglobin significantly diminished its participation in intercellular junctions and resulted in weakened cell sheet strength. Similarly, actin disruption and actin-myosin contraction inhibition also diminished cell sheet strength. Next, we demonstrated that external mechanical stimuli further boosted the sheet's mechanical strength via increased junctional localizations and elevated expression levels, suggesting possible involvement of plakoglobin-related Wnt/β-catenin signaling and actin-related RhoA/ROCK signaling.Given the near-ubiquity of plakoglobin and actin in multicellular organisms, these findings could have significant implications for understanding the fundamental mechanisms that regulating cell adhesion and biomechanics, and improving the mechanical properties of tissue engineering constructs.

Force communication in multicellular tissues addressed by laser nanosurgery

Cell contractility is a prominent mechanism driving multicellular tissue development and remodeling. Forces originated by the actomyosin cytoskeleton not only act within the cell body but can also propagate many layers away from the contraction source and grant tissues the ability to organize collectively and to achieve robust remodeling through development. Tissue tension is being thoroughly investigated in model organisms and increasing evidence is revealing the major role played by the communication, dynamics and propagation of cell-to-cell physical forces in multicellular remodeling. Recently, pulsed-laser-based surgery has fostered in vivo experimental studies to investigate intracellular and supracellular forces in action. The technique offers a unique method to perturb mechanical equilibrium in a subpopulation of cells or in a single cell, while the overall tissue remains intact. In particular, improved ablation precision with short laser pulses and the combination of this technique with biophysical models now allow an in-depth understanding of the role of cellular mechanics in tissue morphogenesis. We first characterize laser ablation modes available to perform intracellular, cellular, or multi-cellular ablation via the example of the model monolayer tissue of the amnioserosa of Drosophila by relating subnanosecond laser pulse energy to ablation efficiency and the probability of cavitation bubble formation. We then review recent laser nanosurgery experiments that have been performed in cultured cells and that tackle actomyosin mechanics and provide molecular insights into force-sensing mechanisms. We finally review studies showing the central role of laser ablation in revealing the nature and orientation of forces involved in intracellular contractility and force mechanosensing in tissue development, e.g., axis elongation, branching morphogenesis, or tissue invagination. We discuss the perspectives offered by the technique in force-based cell-cell communication and mechanosensing pathways.

Abstract 1333: Organ specific biomatrix scaffolds for ex vivo characterization of colorectal cancer metastases

Abstract Colorectal cancer is the second leading cause of cancer-related deaths in the United States with more than 140,000 new cases being diagnosed this year. However, morbidity and mortality is not generally caused by the primary tumor, but by metastases to other organs. There are few models in which to study colorectal cancer metastasis, either in two-dimensional (2D) monolayer tissue culture or in animal xenografts. Both of these models have limitations. Standard 2D tissue culture, though inexpensive and accessible, poorly mimics the metastatic process and xenograft models are costly and technically challenging. Two of the most common sites of colorectal metastasis are the liver and lungs, however, there are few ex vivo models in which to study metastases to these sites. Advances in materials science have led to the development of ex vivo tissue specific models of extracellular matrices (ECM). We have utilized a novel material, biomatrix scaffolds, to engineer a colorectal cancer metastasis ex vivo model. We hypothesized that through the use of liver and lung specific biomatrix, (1) we can better characterize the colorectal cancer metastatic process by mimicking the organ specific microenvironment and (2) more accurately determine the response to therapeutic agents to treat metastases. In this study, we characterized biomatrix derived from rat liver and lung as a growth substrate for human colon cancer cell lines. We used the human colon cancer derived cell lines: HT29, SW480, CaCo2 and Lovo. Chemotherapeutic response to oxaliplatin, irinotecan and 5-fluorouracil for these cell lines grown on biomatrix was examined. We also used primary cultures of colorectal cancer metastases derived from patient samples. The biomatrix scaffolds were generated through decellularization of rat liver or lungs. We preformed mass spectrometry and determined the ECM composition and the associated growth factors found in lysates of liver and lung remain bound to the biomatrix in concentrations similar to those found in vivo. Colorectal cancer cell lines demonstrated different growth rates and morphologies when grown on biomatrix, resulting in organized, 3-dimenasional growth of non-hollow spheroids several hundred microns in diameter. We demonstrated differential responses to chemotherapeutics and observed differential gene expression via microarray analysis when colorectal cancer cell lines were grown on biomatrix compared to plastic, collagen, or matrigel. We also demonstrated increased tumor cell colony formation in primary cultures of patient derived colorectal metastases on biomatrix compared to plastic or collagen. In conclusion, we have developed a decellularized tissue material, biomatrix, that can provide an ex vivo model of colorectal cancer metastasis to aid in therapeutic drug development and genetic characterization of metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1333. doi:1538-7445.AM2012-1333

Cytotoxicity testing of burn wound dressings, ointments and creams: A method using polycarbonate cell culture inserts on a cell culture system

We have developed a method to test the cytotoxicity of wound dressings, ointments, creams and gels used in our Burn Centre, by placing them on a permeable Nunc™ Polycarbonate cell culture insert, incubated with a monolayer of cells (HaCaTs and primary human keratinocytes). Methods We performed two different methods to determine the relative toxicity to cells. (1) Photo visualisation: The dressings or compounds were positioned on the insert's membrane which was placed onto the monolayer tissue culture plate. After 24 h the surviving adherent cells were stained with Toluidine Blue and photos of the plates were taken. The acellular area of non-adherent dead cells which had been washed off with buffer was measured as a percentage of the total area of the plate. (2) Cell count of surviving cells: After 24 h incubation with the test material, the remaining cells were detached with trypsin, spun down and counted in a Haemocytometer with Trypan Blue, which differentiates between live and dead cells. Results Seventeen products were tested. The least cytotoxic products were Melolite™, White soft Paraffin™ and Chlorsig1% Ointment. Some cytotoxicity was shown with Jelonet™, Mepitel ®, PolyMem ®, DuoDerm ® and Xeroform™. The most cytotoxic products included those which contained silver or Chlorhexidine and Paraffin Cream™ a moisturizer which contains the preservative Chlorocresol. Conclusion This in vitro cell culture insert method allows testing of agents without direct cell contact. It is easy and quick to perform, and should help the clinician to determine the relative cytotoxicity of various dressings and the optimal dressing for each individual wound.

Evaluation of Plasma Modified Polycaprolactone Honeycomb Scaffolds by Human Mesenchymal Stem Cells Cultured in Vitamin D Differentiation Medium

AbstractPolycaprolactone (PCL) scaffolds with a honeycomb (hc) microstructure have been prepared, modified at surface level by plasma processes and evaluated with human mesenchymal stem cells (hMSCs). Thin scaffolds were fabricated by self‐assembly of PCL in the presence of an amphiphilic copolymer under controlled humidity. The surface of the so formed PCLhc scaffolds was modified by Ar and ArO2 plasma discharges and by plasma deposition of allylamine (ALA). Such modification induces composition changes and microstructure modifications as determined by Fourier transformed infrared spectroscopy and scanning electron microscopy. As a consequence of these treatments, the hydrophobic character of PCLhc scaffolds is rectified as derived from water contact angle measurements. The hMSC cytocompatibility of these scaffolds was initially assayed under proliferation conditions evaluating surface–cell interactions by fluorescence and confocal microscopy. Most relevant behaviour was observed for hMSCs cultured onto ALA modified scaffolds in view of the cytoskeleton polarization. These modified surfaces were further explored for differentiation of hMSCs in the presence of vitamin D, giving rise to secreting vesicles and increased extension of the cytoskeleton. These results are promising for the development of cell monolayer tissue regenerating platforms.magnified image

Evaluation of Production Parameters with the Vaccinia Virus Expression System Using Microcarrier Attached HeLa Cells

Parameters that affect production of the recombinant reporter protein, EGFP, in the T7 promoter based VOTE vaccinia virus-HeLa cell expression system were examined. Length of infection phase, inducer concentration, and timing of its addition relative to infection were evaluated in 6-well plate monolayer cultures. One hour infection with 1.0 mM IPTG added at the time of infection provided a robust process. For larger scale experiments, anchorage-dependent HeLa cells were grown on 5 g/L Cytodex 3 microcarriers. The change to this dynamic culture environment, with cell-covered microcarriers suspended in culture medium in spinner flasks, suggested a re-examination of the multiplicity of infection (MOI) for this culture type that indicated a need for an increase in the number of virus particles per cell to 5.0, higher than that needed for complete infection in monolayer tissue flask culture. Additionally, dissolved oxygen level and temperature during the protein production phase were evaluated for their effect on EGFP expression in microcarrier spinner flask culture. Both increased dissolved oxygen, based on surface area to volume (SA/V) adjustments, and decreased temperature from 37 to 31 degrees C showed increases in EGFP production over the course of the production phase. The level of production achieved with this system reached approximately 17 microg EGFP/10(6) infected cells.

Co‐determination of ATP and proteins in Triton X 100 non‐ionic detergent‐opened monolayer cultured cells

Human monolayer cells (HEp-2 and Hep G2) were cultured in 96-well plates. A modified Triton X 100 nonionic detergent extraction method was used for releasing intracellular ATP and protein in one step. The detergent technique was compared to perchloric acid (PCA) extraction. ATP was determined by the firefly bioluminescence method and ATP values were referred to cell protein (ATP:protein ratio). There was no significant difference in ATP data between detergent and PCA treatments. The ATP:protein ratio seems to be a sensitive tool for characterizing the metabolic activity of monolayer tissue culture cells. The protein-mobilizing capability of Triton X 100 depends on the type of cell culture used. Our modified extraction gives reliable ATP:protein values with one simple extraction step.

Patterned biofunctional designs of thermoresponsive surfaces for spatiotemporally controlled cell adhesion, growth, and thermally induced detachment

In the present study, we report advanced patterned biofunctionalization of thermoresponsive surfaces for achievement of spatiotemporally controlled cell adhesion, growth, and thermally induced detachment. These patterned biofunctional thermoresponsive surfaces were prepared using dual surface modification techniques: electron beam-induced surface patterning of carboxyl-functional thermoresponsive polymers with appropriate metal masks and following site-selective biofunctionalization with biomolecules, the cell adhesive peptide (RGDS) and/or the cell growth factor (insulin; INS). Patterned co-immobilization of RGDS–INS onto thermoresponsive surfaces dominated site-selective cell adhesion and growth along with patterned biofunctional domains in the serum-free culture. Spatiotemporal detachment of sparsely adherent and confluent cells from these patterned biofunctional thermoresponsive surfaces were both achieved only by reducing temperature. Furthermore, RGDS–INS-patterned thermoresponsive surfaces also successfully demonstrated the selective fabrication and recovery of either contiguous monolayer or mesh-like designed monolayer tissue constructs on the identical surfaces. Thus, patterned biofunctional designs would be utilized for the creation and harvest of biomimetic-designed vascular networks having sufficient biofunctional activities in facilitated cell sheet engineering and regenerative medicine.

Guidance of engineered tissue collagen orientation by large-scale scaffold microstructures

The tensile strength and stiffness of load-bearing soft tissues are dominated by their collagen fiber orientation. While microgrooved substrates have demonstrated a capacity to orient cells and collagen in monolayer tissue culture, tissue engineering (TE) scaffolds are structurally distinct in that they consist of a three-dimensional (3-D) open pore network. It is thus unclear how the geometry of these open pores might influence cell and collagen orientation. In the current study we developed an in vitro model system for quantifying the capacity of large scale (∼200 μm), geometrically well-defined open pores to guide cell and collagen orientation in engineered tissues. Non-degradable scaffolds exhibiting a grid of 200 μm wide rectangular pores (1:1, 2:1, 5:1, and 10:1 aspect ratios) were fabricated from a transparent epoxy resin via high-resolution stereolithography. The scaffolds ( n = 6 per aspect ratio) were surface modified to support cell adhesion by covalently grafting GRGDS peptides, sterilized, and seeded with neonatal rat skin fibroblasts. Following 4 weeks of static incubation, the resultant collagen orientation was assessed quantitatively by small angle light scattering (SALS), and cell orientation was evaluated by laser confocal and scanning electron microscopy. Cells adhered to the struts of the pores and proceeded to span the pores in a generally circumferential pattern. While the cell and collagen orientations within 1:1 aspect ratio pores were effectively random, higher aspect ratio rectangular pores exhibited a significant capacity to guide global cell and collagen orientation. Preferential alignment parallel to the long strut axis and decreased spatial variability were observed to occur with increasing pore aspect ratio. Intra-pore variability depended in part on the spatial uniformity of cell attachment around the perimeter of each pore achieved during seeding. Evaluation of diamond-shaped pores [Sacks, M.S. et al., 1997. J. Biomech. Eng. 119(1), 124–127] suggests that they are less sensitive to initial conditions of cell attachment than rectangular pores, and thus more effective in guiding engineered tissue cell and collagen orientation.

Microscopical heat stress investigations under application of quantum dots

Heat stress responses are analyzed in cancer cells by applying different microscopy techniques for targeting various fluorescently labeled or native structures. Thermotreatments are performed at 40, 45, 50, and 56 degrees C, respectively, for 30 min each, while controls were kept at 37 degrees C. Actin cytoskeletons labeled with Alexa Fluor 488-conjugated phalloidin are imaged by wide-field fluorescence microscopy (WFFM). Structural plasma membrane stabilities are labeled with fluorescent quantum dots and analyzed by laser scanning microscopy (LSM). High-resolution atomic force microscopy (AFM) and scanning electron microscopy (SEM) are used to study morphological features and surface structures. Fluorescence images reveal F-actin to be a comparatively thermolabile cell component showing distinctive alteration after heat treatment at 40 degrees C. Destabilization of actin cytoskeletons proceed with increasing stress temperatures. Active reorganization of plasma membranes coincidental to heat-induced shrinkage and rounding of cell shapes, and loosening of monolayered tissue are observed after treatment at 45 or 50 degrees C. Active stress response is inhibited by stress at 56 degrees C, because actin cytoskeletons as well as plasma membranes are destroyed, resulting in necrotic cell phenotypes. Comparing data measured with the same microscopic technique and comparing the different datasets with each other reveal that heat stress response in MX1 cells results from the overlap of different heat-induced subcellular defects.

The establishment and characterization of an immortal cell line with a stable chondrocytic phenotype

A cell line was developed from the transplantable Swarm rat chondrosarcoma (RCS) and has been maintained in continuous monolayer tissue culture for a number of years. This long term-cultured (LTC) cell line exhibits the morphological and biochemical characteristics of chondrocytes and resembles the RCS tumor by electron and light microscopy. The cell line differs from the original tumor cells in that about 90% of the sulfated macromolecules are retained in the LTC extracellular matrix as compared to 30% by primary cultures of cells from the RCS tumor. An interesting and useful feature of this cell line is that it contains clonal populations of cells which differ in the quality and quantity of matrix produced. Two such clones serve to illustrate the diversity of cell types within the LTC cell line. One termed Rex accumulates an intensely staining matrix around it, while the other, Ng, accumulates a matrix, that remains virtually unstained. The chondrocytic nature and ease of cloning make these cells ideal for biochemical analysis of the chondrocyte and its extracellular matrix.