132 research outputs found
Simple and High Yielding Method for Preparing Tissue Specific Extracellular Matrix Coatings for Cell Culture
Background: The native extracellular matrix (ECM) consists of a highly complex, tissue-specific network of proteins and polysaccharides, which help regulate many cellular functions. Despite the complex nature of the ECM, in vitro cell-based studies traditionally assess cell behavior on single ECM component substrates, which do not adequately mimic the in vivo extracellular milieu. Methodology/Principal Findings: We present a simple approach for developing naturally derived ECM coatings for cell culture that provide important tissue-specific cues unlike traditional cell culture coatings, thereby enabling the maturation of committed C2C12 skeletal myoblast progenitors and human embryonic stem cells differentiated into cardiomyocytes. Here we show that natural muscle-specific coatings can (i) be derived from decellularized, solubilized adult porcine muscle, (ii) contain a complex mixture of ECM components including polysaccharides, (iii) adsorb onto tissue culture plastic and (iv) promote cell maturation of committed muscle progenitor and stem cells. Conclusions: This versatile method can create tissue-specific ECM coatings, which offer a promising platform for cell cultur
Extracellular Matrix Aggregates from Differentiating Embryoid Bodies as a Scaffold to Support ESC Proliferation and Differentiation
Embryonic stem cells (ESCs) have emerged as potential cell sources for tissue engineering and regeneration owing to its virtually unlimited replicative capacity and the potential to differentiate into a variety of cell types. Current differentiation strategies primarily involve various growth factor/inducer/repressor concoctions with less emphasis on the substrate. Developing biomaterials to promote stem cell proliferation and differentiation could aid in the realization of this goal. Extracellular matrix (ECM) components are important physiological regulators, and can provide cues to direct ESC expansion and differentiation. ECM undergoes constant remodeling with surrounding cells to accommodate specific developmental event. In this study, using ESC derived aggregates called embryoid bodies (EB) as a model, we characterized the biological nature of ECM in EB after exposure to different treatments: spontaneously differentiated and retinoic acid treated (denoted as SPT and RA, respectively). Next, we extracted this treatment-specific ECM by detergent decellularization methods (Triton X-100, DOC and SDS are compared). The resulting EB ECM scaffolds were seeded with undifferentiated ESCs using a novel cell seeding strategy, and the behavior of ESCs was studied. Our results showed that the optimized protocol efficiently removes cells while retaining crucial ECM and biochemical components. Decellularized ECM from SPT EB gave rise to a more favorable microenvironment for promoting ESC attachment, proliferation, and early differentiation, compared to native EB and decellularized ECM from RA EB. These findings suggest that various treatment conditions allow the formulation of unique ESC-ECM derived scaffolds to enhance ESC bioactivities, including proliferation and differentiation for tissue regeneration applications. © 2013 Goh et al
Extracellular matrix hydrogels from decellularized tissues: structure and function
Extracellular matrix (ECM) bioscaffolds prepared from decellularized tissues have been used to facilitate constructive and functional tissue remodeling in a variety of clinical applications. The discovery that these ECM materials could be solubilized and subsequently manipulated to form hydrogels expanded their potential in vitro and in vivo utility; i.e. as culture substrates comparable to collagen or Matrigel, and as injectable materials that fill irregularly-shaped defects. The mechanisms by which ECM hydrogels direct cell behavior and influence remodeling outcomes are only partially understood, but likely include structural and biological signals retained from the native source tissue. The present review describes the utility, formation, and physical and biological characterization of ECM hydrogels. Two examples of clinical application are presented to demonstrate in vivo utility of ECM hydrogels in different organ systems. Finally, new research directions and clinical translation of ECM hydrogels are discusse
The impact of detergents on the tissue decellularization process: a ToF-SIMS study
Biologic scaffolds are derived from mammalian tissues, which must be decellularized to remove cellular antigens that would otherwise incite an adverse immune response. Although widely used clinically, the optimum balance between cell removal and the disruption of matrix architecture and surface ligand landscape remains a considerable challenge. Here we describe the use of time of flight secondary ion mass spectroscopy (ToF-SIMS) to provide sensitive, molecular specific, localized analysis of detergent decellularized biologic scaffolds. We detected residual detergent fragments, specifically from Triton X-100, sodium deoxycholate and sodium dodecyl sulphate (SDS) in decellularized scaffolds; increased SDS concentrations from 0.1% to 1.0% increased both the intensity of SDS fragments and adverse cell outcomes. We also identified cellular remnants, by detecting phosphate and phosphocholine ions in PAA and CHAPS decellularized scaffolds. The present study demonstrates ToF-SIMS is not only a powerful tool for characterization of biologic scaffold surface molecular functionality, but also enables sensitive assessment of decellularization efficacy
Extracellular matrix-derived hydrogels for dental stem cell delivery
Decellularized mammalian extracellular matrices (ECM) have been widely accepted as an ideal substrate for repair and remodelling of numerous tissues in clinical and pre-clinical studies. Recent studies have demonstrated the ability of ECM scaffolds derived from site-specific homologous tissues to direct cell differentiation. The present study investigated the suitability of hydrogels derived from different source tissues: bone, spinal cord and dentine, as suitable carriers to deliver human apical papilla derived mesenchymal stem cells (SCAP) for spinal cord regeneration. Bone, spinal cord, and dentine ECM hydrogels exhibited distinct structural, mechanical, and biological characteristics. All three hydrogels supported SCAP viability and proliferation. However, only spinal cord and bone derived hydrogels promoted the expression of neural lineage markers. The specific environment of ECM scaffolds significantly affected the differentiation of SCAP to a neural lineage, with stronger responses observed with spinal cord ECM hydrogels, suggesting that site-specific tissues are more likely to facilitate optimal stem cell behavior for constructive spinal cord regeneration
In vitro models of medulloblastoma: choosing the right tool for the job
The recently-defined four molecular subgroups of medulloblastoma have required updating of our understanding of in vitro models to include molecular classification and risk stratification features from clinical practice. This review seeks to build a more comprehensive picture of the in vitro systems available for modelling medulloblastoma.
The subtype classification and molecular characterisation for over 40 medulloblastoma cell-lines has been compiled, making it possible to identify the strengths and weaknesses in current model systems. Less than half (18/44) of established medulloblastoma cell-lines have been subgrouped. The majority of the subgrouped cell-lines (11/18) are Group 3 with MYC-amplification. SHH cell-lines are the next most common (4/18), half of which exhibit TP53 mutation. WNT and Group 4 subgroups, accounting for 50% of patients, remain underrepresented with 1 and 2 cell-lines respectively.
In vitro modelling relies not only on incorporating appropriate tumour cells, but also on using systems with the relevant tissue architecture and phenotype as well as normal tissues. Novel ways of improving the clinical relevance of in vitro models are reviewed, focusing on 3D cell culture, extracellular matrix, co-cultures with normal cells and organotypic slices. This paper champions the establishment of a collaborative online-database and linked cell-bank to catalyse preclinical medulloblastoma research
Decellularized Matrix from Tumorigenic Human Mesenchymal Stem Cells Promotes Neovascularization with Galectin-1 Dependent Endothelial Interaction
BACKGROUND: Acquisition of a blood supply is fundamental for extensive tumor growth. We recently described vascular heterogeneity in tumours derived from cell clones of a human mesenchymal stem cell (hMSC) strain (hMSC-TERT20) immortalized by retroviral vector mediated human telomerase (hTERT) gene expression. Histological analysis showed that cells of the most vascularized tumorigenic clone, -BD11 had a pericyte-like alpha smooth muscle actin (ASMA+) and CD146+ positive phenotype. Upon serum withdrawal in culture, -BD11 cells formed cord-like structures mimicking capillary morphogenesis. In contrast, cells of the poorly tumorigenic clone, -BC8 did not stain for ASMA, tumours were less vascularized and serum withdrawal in culture led to cell death. By exploring the heterogeneity in hMSC-TERT20 clones we aimed to understand molecular mechanisms by which mesenchymal stem cells may promote neovascularization. METHODOLOGY/PRINCIPAL FINDINGS: Quantitative qRT-PCR analysis revealed similar mRNA levels for genes encoding the angiogenic cytokines VEGF and Angiopoietin-1 in both clones. However, clone-BD11 produced a denser extracellular matrix that supported stable ex vivo capillary morphogenesis of human endothelial cells and promoted in vivo neovascularization. Proteomic characterization of the -BD11 decellularized matrix identified 50 extracellular angiogenic proteins, including galectin-1. siRNA knock down of galectin-1 expression abrogated the ex vivo interaction between decellularized -BD11 matrix and endothelial cells. More stable shRNA knock down of galectin-1 expression did not prevent -BD11 tumorigenesis, but greatly reduced endothelial migration into -BD11 cell xenografts. CONCLUSIONS: Decellularized hMSC matrix had significant angiogenic potential with at least 50 angiogenic cell surface and extracellular proteins, implicated in attracting endothelial cells, their adhesion and activation to form tubular structures. hMSC -BD11 surface galectin-1 expression was required to bring about matrix-endothelial interactions and for xenografted hMSC -BD11 cells to optimally recruit host vasculature
Decellularized biomaterials for cell culture and repair after ischemic injury
Ischemic disease, which involves tissue death and dysfunction due to vessel blockage, is one of the largest causes of morbidity and mortality across the world. Ischemia that targets the brain can lead to a stroke, which causes functional impairment. Blockage of the coronary artery can lead to a myocardial infarction (MI) which can eventually lead to heart failure. Ischemia of the vessels in the skeletal muscle causes peripheral artery disease, which can lead to tissue damage that may necessitate amputation of the affected limb. The severity of the downstream effects of ischemia indicates the need for some sort of treatment to repair the tissue after ischemic attack. Yet, there are few clinical treatments available for patients, creating a need for novel therapies for treating this disease. The use of biomaterials in tissue engineering strategies have recently been studied to alleviate these conditions, however this approach has been met with limited success as many of these therapies require an invasive surgery for delivery to the affected site. Injectable biomaterials offer the advantage of minimally invasive delivery to improve patient outcomes, which would be attractive to reduce patient recovery time. The materials that have been studied often do not mimic the microenvironment of the tissue that it is trying to repair. This is similar to how cells are often cultured on a substrate that do not mimic the in vivo environment, which may be important for assessing cellular function. Thus, the objective of this dissertation was to generate biomaterials derived from decellularized tissue from the brain, skeletal muscle, and cardiac tissue, and test whether they could be used as cell culture platforms that would provide biomimetic substrates and be used as scaffolds for tissue engineering. In this work, I have developed a method to decellularize each tissue leaving behind only the extracellular matrix. The matrix material was then characterized using gel electrophoresis, mass spectrometry, glycosaminoglycan quantification and DNA quantification, indicating that the cellular remnants have been removed, but that the biocomplexity has been retained. These tissue specific biomaterials were tested as a cell culture coating platform in vitro and as a potential therapy for ischemia in vivo. The material was enzymatically digested and used as a cell culture coating and compared to conventionally used substrates. It was found that progenitor cells cultured on the tissue-matched coatings display a more mature morphology on the decellularized extracellular matrix (ECM) coatings. For instance, skeletal muscle progenitors differentiate into larger, thicker myotubes, cardiomyocytes derived from human embryonic stem cells localize their intracellular junctions into a more mature organization, and neurons from induced pluripotent stem cells display a clear axon and increased dendritic branching. The maturation of these cell types on the coatings demonstrate a more in vivo like phenotype which could be useful for studying cellular behavior and to translate in vitro findings into an in vivo setting. This material was able to self-assemble upon injection in vivo, forming a nanofibrous and porous scaffold that could be used as an injectable biomaterial for ischemic repair. Additionally, in vitro assays measuring proliferation and migration show that some of the matrix materials act as a chemoattractant and as a mitogenic agent on cells in culture. The skeletal muscle matrix has been used in a rat hindlimb ischemia model and compared to a collagen scaffold. It was shown that the skeletal muscle matrix stimulates increased neovascularization, which is important for bringing blood flow to an ischemic region, as well as recruits endogenous muscle progenitor cells into the scaffold. The cardiac matrix is able to gel in situ upon injection and has been explored by others in our lab. The brain matrix was also able to self-assemble and form a gel after subcutaneous injection into a mouse, demonstrating proof-of-concept for its use as a tissue engineering scaffold. To investigate whether the material might be derived from an allogeneic source instead of from porcine origin, the decellularization process was also performed on human cardiac tissue. It was found that additional steps were needed to fully decellularize the material and render it into a usable form. However, this could provide a potentially allogeneic source for this material. This work demonstrates that decellularized extracellular matrices derived from various tissues provide a biomimetic platform for cell culture that increases maturation of progenitor and stem cells cultured upon the surface. The maturation of these cells could be important for understanding and regulating cellular processes. The same material can be used as an injectable scaffold that could be delivered through minimally invasive means to treat ischemic damage in the brain, heart and skeletal muscle. When applied in a hindlimb ischemia model, the skeletal muscle matrix is able to increase neovascularization, recruit more muscle progenitor cells, and recruit more proliferating muscle cells when compared to a collagen control. This work shows that decellularized matrices hold great potential for both in vitro and in vivo application
Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering
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