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Fibroblasts are the major producers of the extracellular matrix and regulate its organization. Aberrant signaling in diseases such as fibrosis and cancer can impact the deposition of the matrix proteins, which can in turn act as an adhesion scaffold and signaling reservoir promoting disease progression. To study the composition and organization of the extracellular matrix as well as its interactions with (tumor) cells, this protocol describes the generation and analysis of 3D fibroblast-derived matrices and the investigation of (tumor) cells seeded onto the 3D scaffolds by immunofluorescent imaging and cell adhesion, colony formation, migration, and invasion/transmigration assays.
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Adesão Celular , Movimento Celular , Matriz Extracelular , Fibroblastos , Transdução de Sinais , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Humanos , Linhagem Celular Tumoral , Técnicas de Cultura de Células/métodos , Neoplasias/metabolismo , Neoplasias/patologia , Comunicação Celular , Técnicas de Cultura de Células em Três Dimensões/métodos , Animais , Alicerces Teciduais/químicaRESUMO
Microphysiological and organ-on-chip platforms seek to address critical gaps in human disease models and drug development that underlie poor rates of clinical success for novel interventions. While the fabrication technology and model cells used to synthesize organs-on-chip have advanced considerably, most platforms rely on animal-derived or synthetic extracellular matrix as a cell substrate, limiting mimicry of human physiology and precluding use in modeling diseases in which matrix dynamics play a role in pathogenesis. Here, the development of human cell-derived matrix (hCDM) composite hydrogels for use in 3D microphysiologic models of the vasculature is reported. hCDM composite hydrogels are derived from human donor fibroblasts and maintain a complex milieu of basement membrane, proteoglycans, and nonfibrillar matrix components. The use of hCDM composite hydrogels as 2D and 3D cell culture substrates is demonstrated, and hCDM composite hydrogels are patterned to form engineered human microvessels. Interestingly, hCDM composite hydrogels are enriched in proteins associated with vascular morphogenesis as determined by mass spectrometry, and functional analysis demonstrates proangiogenic signatures in human endothelial cells cultured in these hydrogels. In conclusion, this study suggests that human donor-derived hCDM composite hydrogels could address technical gaps in human organs-on-chip development and serve as substrates to promote vascularization.
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Matriz Extracelular , Hidrogéis , Humanos , Hidrogéis/química , Matriz Extracelular/metabolismo , Matriz Extracelular/química , Dispositivos Lab-On-A-Chip , Engenharia Tecidual/métodos , Fibroblastos/metabolismo , Fibroblastos/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Neovascularização Fisiológica/efeitos dos fármacosRESUMO
Collagen, the most abundant structural protein in the human extracellular matrix (ECM), provides essential support for tissues and guides tissue development. Despite its widespread use in tissue engineering, there remains uncertainty regarding the optimal selection of collagen sources. Animal-derived sources pose challenges such as immunogenicity, while the recombinant system is hindered by diminished bioactivity. In this study, we hypothesized that human ECM-like collagen (hCol) could offer an alternative for tissue engineering. In this study, a facile platform was provided for generating hCol derived from mesenchymal stem cells with a hierarchical structure and biochemical properties resembling native collagen. Our results further demonstrated that hCol could facilitate basal biological behaviors of human adipose-derived stem cells, including viability, proliferation, migration and adipocyte-like phenotype. Additionally, it could promote cutaneous wound closure. Due to its high similarity to native collagen and good bioactivity, hCol holds promise as a prospective candidate for in vitro and in vivo applications in tissue engineering.
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Tissue-relevant O2 levels are considered as an important tool for the preconditioning of multipotent mesenchymal stromal cells (MSCs) for regenerative medicine needs. The present study investigated the quality and functions of the extracellular matrix (ECM) of MSCs under low O2 levels. Human adipose tissue-derived MSCs were continuously expanded under normoxia (20% O2, N) or "physiological" hypoxia (5% O2, Hyp). Decellularized ECM (dcECM) was prepared. The structure of the dcECM was analyzed using confocal laser and scanning electron microscopy. Collagen, dcECM-N, and dcECM-Hyp were recellularized with MSC-N and further cultured at normoxia. The efficacy of adhesion, spreading, growth, osteogenic potential, and paracrine activity of recellularized MSC-N were evaluated. At low O2, the dcECM showed an increased alignment of fibrillar structures and provided accelerated spreading of MSC-N, indicating increased dcECM-Hyp stiffness. We described O2-dependent "ECM-education" of MSC-N when cultured on dcECM-Hyp. This was manifested as attenuated spontaneous osteo-commitment, increased susceptibility to osteo-induction, and a shift in the paracrine profile. It has been suggested that the ECM after physiological hypoxia is able to ensure the maintenance of a low-commitment state of MSCs. DcECM, which preserves the competence of the natural microenvironment of cells and is capable of "educating" others, appears to be a prospective tool for guiding cell modifications for cell therapy and tissue engineering.
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Low back pain is the leading cause of disability worldwide, but current therapeutic interventions are palliative or surgical in nature. Loss of notochordal cells (NCs) and degradation of the healthy matrix in the nucleus pulposus (NP), the central tissue of intervertebral discs (IVDs), has been associated with onset of degenerative disc changes. Recently, we established a protocol for decellularization of notochordal cell derived matrix (NCM) and found that it can provide regenerative cues to nucleus pulposus cells of the IVD. Here, we combined the biologically regenerative properties of decellularized NCM with the mechanical tunability of a poly(ethylene glycol) hydrogel to additionally address biomechanics in the degenerate IVD. We further introduced a hydrolysable PEG-diurethane crosslinker for slow degradation of the gels in vivo. The resulting hydrogels were tunable over a broad range of stiffness's (0.2 to 4.5 kPa), matching that of NC-rich and -poor NP tissues, respectively. Gels formed within 30 min, giving ample time for handling, and remained shear-thinning post-polymerization. Gels also slowly released dNCM over 28 days as measured by GAG effusion. Viability of encapsulated bone marrow stromal cells after extrusion through a needle remained high. Although encapsulated NCs stayed viable over two weeks, their metabolic activity decreased, and their phenotype was lost in physiological medium conditions in vitro. Overall, the obtained gels hold promise for application in degenerated IVDs but require further tuning for combined use with NCs.
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Degeneração do Disco Intervertebral , Disco Intervertebral , Núcleo Pulposo , Humanos , Hidrogéis/farmacologia , Hidrogéis/metabolismo , Degeneração do Disco Intervertebral/terapia , Núcleo Pulposo/metabolismo , Células CultivadasRESUMO
Vascular Ehlers-Danlos Syndrome (vEDS) is a rare autosomal dominant disease caused by mutations in the COL3A1 gene, which renders patients susceptible to aneurysm and arterial dissection and rupture. To determine the role of COL3A1 variants in the biochemical and biophysical properties of human arterial ECM, we developed a method for synthesizing ECM directly from vEDS donor fibroblasts. We found that the protein content of the ECM generated from vEDS donor fibroblasts differed significantly from ECM from healthy donors, including upregulation of collagen subtypes and other proteins related to ECM structural integrity. We further found that ECM generated from a donor with a glycine substitution mutation was characterized by increased glycosaminoglycan content and unique viscoelastic mechanical properties, including increased time constant for stress relaxation, resulting in a decrease in migratory speed of human aortic endothelial cells when seeded on the ECM. Collectively, these results demonstrate that vEDS patient-derived fibroblasts harboring COL3A1 mutations synthesize ECM that differs in composition, structure, and mechanical properties from healthy donors. These results further suggest that ECM mechanical properties could serve as a prognostic indicator for patients with vEDS, and the insights provided by the approach demonstrate the broader utility of cell-derived ECM in disease modeling. STATEMENT OF SIGNIFICANCE: The role of collagen III ECM mechanics remains unclear, despite reported roles in diseases including fibrosis and cancer. Here, we generate fibrous, collagen-rich ECM from primary donor cells from patients with vascular Ehlers-Danlos syndrome (vEDS), a disease caused by mutations in the gene that encodes collagen III. We observe that ECM grown from vEDS patients is characterized by unique mechanical signatures, including altered viscoelastic properties. By quantifying the structural, biochemical, and mechanical properties of patient-derived ECM, we identify potential drug targets for vEDS, while defining a role for collagen III in ECM mechanics more broadly. Furthermore, the structure/function relationships of collagen III in ECM assembly and mechanics will inform the design of substrates for tissue engineering and regenerative medicine.
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Síndrome de Ehlers-Danlos Tipo IV , Síndrome de Ehlers-Danlos , Humanos , Células Endoteliais/metabolismo , Síndrome de Ehlers-Danlos/genética , Síndrome de Ehlers-Danlos/metabolismo , Mutação de Sentido Incorreto , Mutação/genética , Matriz Extracelular/metabolismo , Colágeno Tipo III/genética , Colágeno Tipo III/químicaRESUMO
Porcine notochordal cell-derived matrix (NCM) has anti-inflammatory and regenerative effects on degenerated intervertebral discs. For its clinical use, safety must be assured. The porcine DNA is concerning because of (1) the transmission of endogenous retroviruses and (2) the inflammatory potential of cell-free DNA. Here, we present a simple, detergent-free protocol: tissue lyophilization lyses cells, and matrix integrity is preserved by limiting swelling during decellularization. DNA is digested quickly by a high nuclease concentration, followed by a short washout. Ninety-four percent of DNA was removed, and there was no loss of glycosaminoglycans or collagen. Forty-three percent of the total proteins remained in the decellularized NCM (dNCM). dNCM stimulated as much GAG production as NCM in nucleus pulposus cells but lost some anti-inflammatory effects. Reconstituted pulverized dNCM yielded a soft, shear-thinning biomaterial with a swelling ratio of 350% that also acted as an injectable cell carrier (cell viability >70%). dNCM can therefore be used as the basis for future biomaterials aimed at disc regeneration on a biological level and may restore joint mechanics by creating swelling pressure within the intervertebral disc.
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Degeneração do Disco Intervertebral , Núcleo Pulposo , Animais , Anti-Inflamatórios/metabolismo , Materiais Biocompatíveis/farmacologia , DNA/metabolismo , Degeneração do Disco Intervertebral/metabolismo , Núcleo Pulposo/metabolismo , SuínosRESUMO
Directional cell locomotion requires symmetry breaking between the front and rear of the cell. In some cells, symmetry breaking manifests itself in a directional flow of actin from the front to the rear of the cell. Many cells, especially in physiological 3D matrices, do not show such coherent actin dynamics and present seemingly competing protrusion/retraction dynamics at their front and back. How symmetry breaking manifests itself for such cells is therefore elusive. We take inspiration from the scallop theorem proposed by Purcell for micro-swimmers in Newtonian fluids: self-propelled objects undergoing persistent motion at low Reynolds number must follow a cycle of shape changes that breaks temporal symmetry. We report similar observations for cells crawling in 3D. We quantified cell motion using a combination of 3D live cell imaging, visualization of the matrix displacement, and a minimal model with multipolar expansion. We show that our cells embedded in a 3D matrix form myosin-driven force dipoles at both sides of the nucleus, that locally and periodically pinch the matrix. The existence of a phase shift between the two dipoles is required for directed cell motion which manifests itself as cycles with finite area in the dipole-quadrupole diagram, a formal equivalence to the Purcell cycle. We confirm this mechanism by triggering local dipolar contractions with a laser. This leads to directed motion. Our study reveals that these cells control their motility by synchronizing dipolar forces distributed at front and back. This result opens new strategies to externally control cell motion as well as for the design of micro-crawlers.
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Actinas , Polaridade Celular , Actinas/metabolismo , Movimento Celular/fisiologia , Núcleo Celular/metabolismo , Polaridade Celular/fisiologia , Miosinas/metabolismoRESUMO
Due to its availability and minimal invasive harvesting human adipose tissue-derived extracellular matrix (dECM) is often used as a biomaterial in various tissue engineering and healthcare applications. Next to dECM, cell-derived ECM (cdECM) can be generated by and isolated from in vitro cultured cells. So far both types of ECM were investigated extensively toward their application as (bio)material in tissue engineering and healthcare. However, a systematic characterization and comparison of soft tissue dECM and cdECM is still missing. In this study, we characterized dECM from human adipose tissue, as well as cdECM from human adipose-derived stem cells, toward their molecular composition, structural characteristics, and biological purity. The dECM was found to exhibit higher levels of collagens and lower levels of sulfated glycosaminoglycans compared with cdECMs. Structural characteristics revealed an immature state of the fibrous part of cdECM samples. By the identified differences, we aim to support researchers in the selection of a suitable ECM-based biomaterial for their specific application and the interpretation of obtained results.
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Materiais Biocompatíveis , Matriz Extracelular , Matriz Extracelular Descelularizada , Matriz Extracelular/química , Humanos , Células-Tronco , Engenharia Tecidual/métodos , Alicerces Teciduais/químicaRESUMO
The extracellular matrix (ECM) is a non-cellular network of macromolecules, which provides cells and tissues with structural support and biomechanical feedback to regulate cellular function, tissue tension, and homeostasis. Even subtle changes to ECM abundance, architecture, and organization can affect downstream biological pathways, thereby influencing normal cell and tissue function and also driving disease conditions. For example, in cancer, the ECM is well known to provide both biophysical and biochemical cues that influence cancer initiation, progression, and metastasis, highlighting the need to better understand cell-ECM interactions in cancer and other ECM-enriched diseases. Initial cell-derived matrix (CDM) models were used as an in vitro system to mimic and assess the physiologically relevant three-dimensional (3D) cell-ECM interactions. Here, we describe an expansion to these initial CDM models generated by fibroblasts to assess the effect of genetic or pharmacological intervention on fibroblast-mediated matrix production and organization. Additionally, we highlight current methodologies to quantify changes in the ultrastructure and isotropy of the resulting ECM and also provide protocols for assessing cancer cell interaction with CDMs. Understanding the nature and influence of these complex and heterogeneous processes can offer insights into the biomechanical and biochemical mechanisms, which drive cancer development and metastasis, and how we can target them to improve cancer outcomes. This protocol was validated in: Sci Adv (2021), DOI: 10.1126/sciadv.abh0363.
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Scaffolds for tissue engineering aim to mimic the native extracellular matrix (ECM) that provides physical support and biochemical signals to modulate multiple cell behaviors. However, the majority of currently used biomaterials are oversimplified and therefore fail to provide a niche required for the stimulation of tissue regeneration. In the present study, 3D decellularized ECM (dECM) scaffolds derived from mesenchymal stem cell (MSC) spheroids and with intricate matrix composition are developed. Specifically, application of macromolecular crowding (MMC) to MSC spheroid cultures facilitate ECM assembly in a 3D configuration, resulting in the accumulation of ECM and associated bioactive components. Decellularized 3D dECM constructs produced under MMC are able to adequately preserve the microarchitecture of structural ECM components and are characterized by higher retention of growth factors. This results in a stronger proangiogenic bioactivity as compared to constructs produced under uncrowded conditions. These dECM scaffolds can be homogenously populated by endothelial cells, which direct the macroassembly of the structures into larger cell-carrying constructs. Application of empty scaffolds enhances intrinsic revascularization in vivo, indicating that the 3D dECM scaffolds represent optimal proangiogenic bioactive blocks for the construction of larger engineered tissue constructs.
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Células-Tronco Mesenquimais , Engenharia Tecidual , Células Endoteliais , Matriz Extracelular , Células-Tronco , Alicerces TeciduaisRESUMO
Tissue engineering and regenerative medicine approaches use biomaterials in combination with cells to regenerate lost functions of tissues and organs to prevent organ transplantation. However, most of the current strategies fail in mimicking the tissue's extracellular matrix properties. In order to mimic native tissue conditions, we developed cell-derived matrix (CDM) microtissues (MT). Our methodology uses poly-lactic acid (PLA) and Cultispher® S microcarriers' (MCs') as scaffold templates, which are seeded with rat bone marrow mesenchymal stem cells (rBM-MSCs). The scaffold template allows cells to generate an extracellular matrix, which is then extracted for downstream use. The newly formed CDM provides cells with a complex physical (MT architecture) and biochemical (deposited ECM proteins) environment, also showing spontaneous angiogenic potential. Our results suggest that MTs generated from the combination of these two MCs (mixed MTs) are excellent candidates for tissue vascularization. Overall, this study provides a methodology for in-house fabrication of microtissues with angiogenic potential for downstream use in various tissue regenerative strategies.
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Cardiac fibroblasts and myofibroblasts assemble and maintain extracellular matrix during normal development and following injury. Culture expansion of these cells yield a bioengineered matrix that could lead to intriguing therapeutic opportunities. For example, we reported that cultured rat cardiac fibroblasts form a matrix that can be used to delivery therapeutic stem cells. Furthermore, we reported that matrix derived from cultured human cardiac fibroblasts/myofibroblasts converted monocytes into macrophages that express interesting anti-inflammatory and pro-angiogenic properties. Expanding these matrix investigations require characterization of the source cells for quality control. In these efforts, we observed and herein report that Sushi Containing Domain 2 (SUSD2) is a novel and consistent marker for cultured human cardiac fibroblast and myofibroblasts.
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Matriz Extracelular/metabolismo , Glicoproteínas de Membrana/metabolismo , Miocárdio/metabolismo , Biomarcadores/metabolismo , Células Cultivadas , Matriz Extracelular/fisiologia , Feminino , Fibroblastos/metabolismo , Fibronectinas/metabolismo , Humanos , Masculino , Glicoproteínas de Membrana/genética , Miocárdio/citologia , Miofibroblastos/metabolismoRESUMO
A recent study has shown that demineralized cortical bone (DCB) did not improve the healing of tendon-bone interface. Considering that there is a gradient of mineral content in the tendon-bone interface, we designed a segmentally demineralized cortical bone (sDCB) scaffold with two different regions: undemineralized cortical bone section within the scaffold (sDCB-B) and complete demineralized cortical bone section within the scaffold (sDCB-D), to mimic the natural structure of the tendon-bone interface. Furthermore, the extracellular matrix (ECM) from tendon-derived stem cells (TDSCs) was used to modify the sDCB-D region of sDCB to construct a novel scaffold (sDCB-ECM) for enhancing the bioactivity of the sDCB-D. The surface topography, elemental distribution, histological structure, and surface elastic modulus of the scaffold were observed using scanning electron microscopy, energy-dispersive X-ray spectroscopy, Fourier transform infrared spectroscopy, histological staining and atomic force microscopy. Cell proliferation of bone marrow mesenchymal stem cells (BMSCs) and TDSCs cultured on scaffolds was evaluated using the Cell Counting kit-8, and cell viability was assessed by Live/Dead cell staining. Cell morphology was detected by fluorescent staining. The ability of the scaffolds to recruit stem cells was tested using transwell migration assay. The expression levels of bone-, cartilage- and tendon-related genes and proteins in stem cells were assessed by the polymerase chain reaction and western blotting. Our results demonstrated that there was a gradient of Ca and P elements in sDCB, and TDSC-derived ECM existed on the surface of the sDCB-D region of sDCB. The sDCB-ECM could promote stem cell proliferation and migration. Moreover, the sDCB-B region of sDCB-ECM could stimulate osteogenic and chondrogenic differentiation of BMSCs, and the sDCB-D-ECM region of sDCB-ECM could stimulate chondrogenic and tenogenic differentiation of TDSCs when compared to DCB. Our study indicated that sDCB-ECM might be a potential bioscaffold to enhance the tendon-bone interface regeneration.
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Epidermal Growth Factor Receptor (EGFR) is a known promoter of tumor progression and is overexpressed in lung cancers. Growth factor receptors (including EGFR) are known to interact with extracellular matrix (ECM) proteins, which regulate their activation and function. Fibulin-1 (FBLN1) is a major component of the ECM in lung tissue, and its levels are known to be downregulated in non-small cell lung cancers (NSCLC). To test the possible role FBLN1 isoforms could have in regulating EGFR signaling and function in lung cancer, we performed siRNA mediated knockdown of FBLN1C and FBLN1D in NSCLC Calu-1 cells. Their loss significantly increased basal (with serum) and EGF (Epidermal Growth Factor) mediated EGFR activation without affecting net EGFR levels. Overexpression of FBLN1C and FBLN1D also inhibits EGFR activation confirming their regulatory crosstalk. Loss of FBLN1C and FBLN1D promotes EGFR-dependent cell migration, inhibited upon Erlotinib treatment. Mechanistically, both FBLN1 isoforms interact with EGFR, their association not dependent on its activation. Notably, cell-derived matrix (CDM) enriched FBLN1 binds EGFR. Calu-1 cells plated on CDM derived from FBLN1C and FBLN1D knockdown cells show a significant increase in EGF mediated EGFR activation. This promotes cell adhesion and spreading with active EGFR enriched at membrane ruffles. Both adhesion and spreading on CDMs is significantly reduced by Erlotinib treatment. Together, these findings show FBLN1C/1D, as part of the ECM, can bind and regulate EGFR activation and function in NSCLC Calu-1 cells. They further highlight the role tumor ECM composition could have in influencing EGFR dependent lung cancers.
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Polymicrobial biofilms are major complications of various chronic infections. Therefore, in vitro biorelevant polymicrobial biofilm models are essential tools for medical studies. This study presents an in vitro model for dual species biofilm of Pseudomonas aeruginosa and Staphylococcus aureus developed on cell-derived matrices (CDMs), in order to simulate the microenvironment of in vivo biofilms. P. aeruginosa and S. aureus are two of the most frequent pathogens in polymicrobial biofilms of wound infections. Although they are commonly isolated from polymicrobial biofilms, their interaction is antagonistic; and there is severe battle between them for nutrients and space. We introduced a nutritious formulation supporting co-cultures of P. aeruginosa and S. aureus in order to study the interaction of these gram-positive and gram-negative bacterial species. Quantitative analyses demonstrated that the enrichment of tryptic soy broth (TSB) with NaCl and glucose facilitate dual-species biofilm formation of P. aeruginosa and S. aureus when it is mixed with fetal bovine serum (FBS). Furthermore, the dual species biofilm was incubated on CDMs. Characterization of the model by fluorescent and electron microscopy techniques revealed realistic features of chronic multi-species biofilms, including competitive distribution pattern of two bacterial species and small-colony variants (SCVs) morphology of S. aureus.
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Biofilmes/crescimento & desenvolvimento , Técnicas de Cocultura/métodos , Meios de Cultura , Pseudomonas aeruginosa/crescimento & desenvolvimento , Staphylococcus aureus/crescimento & desenvolvimento , Humanos , Infecção dos Ferimentos/microbiologiaRESUMO
Collagen is the main component of the extracellular matrix and it plays a key role in tumor progression. Commercial collagen solutions are derived from animals, such as rat-tail and bovine or porcine skin. Their cost is quite high and the product is stable only at low temperature, with the disadvantage of a short expiring date. Most importantly, lot-to-lot variability can occur and the reconstituted collagen gels differ significantly from native tissues in terms of both structure and stiffness. In this chapter, we describe a straightforward method to use native, collagen rich skin samples derived from by-products of the tanning industry. The protocol proposed preserves the microstructure of the ovine skin collagen network, offering structurally competent and more relevant model to investigate cell behavior in vitro. Other advantages of the proposed procedure consist in the cost-effectiveness of the process and an increased level of reproducibility. The decellularized ovine skin samples support the adhesion and growth of different cancer cell lines (pancreatic, breast and melanoma cells). The proposed decellularized skin scaffolds are meant as future low-cost competitors for conventional porous scaffold derived by biomaterials, since they offer a biomimetic environment for the cells.
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Técnicas de Cultura de Células/métodos , Colágeno/isolamento & purificação , Matriz Extracelular/química , Engenharia Tecidual/métodos , Animais , Técnicas de Cultura de Células/economia , Linhagem Celular Tumoral , Colágeno/química , Reprodutibilidade dos Testes , Ovinos , Pele/química , Pele/citologia , Engenharia Tecidual/economia , Alicerces Teciduais/economiaRESUMO
The composition and architecture of the extracellular matrix (ECM) and their dynamic alterations, play an important regulatory role on numerous cellular processes. Cells embedded in 3D scaffolds show phenotypes and morphodynamics reminiscent of the native scenario. This is in contrast to flat environments, where cells display artificial phenotypes. The structural and biomolecular properties of the ECM are critical in regulating cell behavior via mechanical, chemical and topological cues, which induce cytoskeleton rearrangement and gene expression. Indeed, distinct ECM architectures are encountered in the native stroma, which depend on tissue type and function. For instance, anisotropic geometries are associated with ECM degradation and remodeling during tumor progression, favoring tumor cell invasion. Overall, the development of innovative in vitro ECM models of the ECM that reproduce the structural and physicochemical properties of the native scenario is of upmost importance to investigate the mechanistic determinants of tumor dissemination. In this chapter, we describe an extremely versatile technique to engineer three-dimensional (3D) matrices with controlled architectures for the study of pathophysiological processes in vitro. To this aim, a confluent culture of "sacrificial" fibroblasts was seeded on top of microfabricated guiding templates to induce the 3D ECM growth with specific isotropic or anisotropic architectures. The resulting matrices, and cells seeded on them, recapitulated the structure, composition, phenotypes and morphodynamics typically found in the native scenario. Overall, this method paves the way for the development of in vitro ECMs for pathophysiological studies with potential clinical relevance.
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Matriz Extracelular/metabolismo , Engenharia Tecidual/métodos , Animais , Dimetilpolisiloxanos/química , Camundongos , Células NIH 3T3RESUMO
Cell migration is involved in key phenomena in biology, ranging from development to cancer. Fibroblasts move between organs in 3D polymeric networks. So far, motile cells were mainly tracked in vitro on Petri dishes or on coverslips, i.e., 2D flat surfaces, which made the extrapolation to 3D physiological environments difficult. We therefore prepared 3D Cell Derived Matrices (CDM) with specific characteristics with the goal of extracting the main readouts required to measure and characterize cell motion: cell specific matrix deformation through the tracking of fluorescent fibronectin within CDM, focal contacts as the cell anchor and acto-myosin cytoskeleton which applies cellular forces. We report our method for generating this assay of physiological-like gel with relevant readouts together with its potential impact in explaining cell motility in vivo.
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Movimento Celular , Técnicas Citológicas/métodos , Matriz Extracelular/metabolismo , Imageamento Tridimensional , Algoritmos , Animais , Fluorescência , Células HeLa , Humanos , Camundongos , Células NIH 3T3RESUMO
Extracellular matrix (ECM) provides both physical support and bioactive signals such as growth factors and cytokines to cells at their microenvironment or niche. Engineering the matrix niche becomes an important approach to study or manipulate cellular fate. This work presents an overview on the reconstitution of the ECM niche through a wide range of approaches ranging from coating culture dish with ECM molecules to decellularization of native tissues. In particular, we focused on reconstituting the complex ECM niche through cell-derived matrix (CDM) by reviewing the methodological approaches used in our group to derive ECM from mature cells such as chondrocytes and nucleus pulposus cells (NPCs), undifferentiated stem cells such as mesenchymal stem cells (MSCs), as well as MSCs undergoing chondrogenic and osteogenic differentiation, in 2D or 3D models. Specific attention has also been given to key factors that should be considered in various applications and challenges in relation to the CDM. Last but not the least, a few future perspectives and their significance have been proposed.