RESUMO
Among all of the materials used in tissue engineering in order to develop bioequivalents, collagen shows to be the most promising due to its superb biocompatibility and biodegradability, thus becoming one of the most widely used materials for scaffold production. However, current imaging techniques of the cells within collagen scaffolds have several limitations, which lead to an urgent need for novel methods of visualization. In this work, we have obtained groups of collagen scaffolds and selected the contrasting agents in order to study pores and patterns of cell growth in a non-disruptive manner via X-ray computed microtomography (micro-CT). After the comparison of multiple contrast agents, a 3% aqueous phosphotungstic acid solution in distilled water was identified as the most effective amongst the media, requiring 24 h of incubation. The differences in intensity values between collagen fibers, pores, and masses of cells allow for the accurate segmentation needed for further analysis. Moreover, the presented protocol allows visualization of porous collagen scaffolds under aqueous conditions, which is crucial for the multimodal study of the native structure of samples.
Assuntos
Colágeno , Alicerces Teciduais , Microtomografia por Raio-X , Alicerces Teciduais/química , Microtomografia por Raio-X/métodos , Colágeno/química , Colágeno/metabolismo , Engenharia Tecidual/métodos , Animais , Água/química , Porosidade , Técnicas de Cultura de Células em Três Dimensões/métodos , HumanosRESUMO
An antifouling peptide hydrogel-based electrochemical biosensor was developed for real-time monitoring of hydrogen peroxide (H2O2) and nitric oxide (NO) released by 3D cultured breast cancer cells upon drug stimulation. Platinum nanoparticles (Pt NPs) were electrodeposited on titanium mesh (Pt NPs/TM) to enhance sensitivity and shown to possess excellent electrocatalytic ability toward H2O2 and NO. The composite hydrogel formed by co-assembling of N-fluorenylmethoxycarbonyl diphenylalanine (Fmoc-FF) and a fluorine methoxycarbonyl group-functionalized Lys-(Fmoc)-Asp was coated on Pt NPs/TM electrode surface to provide cellular scaffolding. Their favorable biocompatibility promoted cell adhesion and growth, while good hydrophilicity endowed the sensor with greatly enhanced antifouling capability in complex cell culture environments. The biosensor successfully determined H2O2 and NO secretion from both non-metastatic and metastatic breast cancer cells in real time. Our results demonstrated robust associations between reactive oxygen species (ROS) and reactive nitrogen species (RNS) production and cell malignancy, with the main difference in oxidative stress between the two subtypes of cells being NO release, particularly emphasizing RNS's critical leading in driving cancer metastasis and invasion progression. This sensor holds great potential for cell-release research under the in vivo-like microenvironment and could reveal RNS as an attractive therapeutic target for treating breast cancer.
Assuntos
Técnicas Biossensoriais , Neoplasias da Mama , Técnicas Eletroquímicas , Hidrogéis , Peróxido de Hidrogênio , Óxido Nítrico , Platina , Humanos , Técnicas Biossensoriais/métodos , Peróxido de Hidrogênio/química , Hidrogéis/química , Neoplasias da Mama/patologia , Óxido Nítrico/metabolismo , Óxido Nítrico/análise , Técnicas Eletroquímicas/métodos , Técnicas Eletroquímicas/instrumentação , Platina/química , Nanopartículas Metálicas/química , Feminino , Peptídeos/química , Peptídeos/farmacologia , Linhagem Celular Tumoral , Titânio/química , Células MCF-7 , Técnicas de Cultura de Células em Três Dimensões/métodosRESUMO
Skeletal muscle is a postmitotic tissue composed of contractile myofibers that are oriented and connected to different layers of connective tissue. Nevertheless, adult muscle fibers retain the capacity to regenerate in response to damage, activating the classical muscle stem cell compartment, namely, satellite cells (SCs), which are mitotically quiescent cells until required for growth or repair and are localized between the basal lamina and sarcolemma of myofibers. The transition of SCs from the quiescent state toward activation, commitment, and differentiation involves the genetic and epigenetic adaptation to novel biological functions, entailing dynamic changes in the protein expression profile. Interestingly, some of the activities and signaling regulating proliferation, commitment, differentiation, and survival/apoptosis of satellite cells have been also partially recapitulated in vitro, taking advantage of robust markers, reliable techniques, and reproducible protocols. Over the years, different techniques of muscular cell culture have been designed including primary cultures from embryonic or postnatal muscle, myogenic cell line, and three-dimensional (3D) skeletal muscle construct. Typical two-dimensional (2D) muscle cell culture cannot fully recapitulate the complexity of living muscle tissues, restricting their usefulness for physiological studies. The development of functional 3D culture models represents a valid alternative to overcome the limitations of already available in vitro model, increasing our understanding of the roles played by the various cell types and how they interact. In this chapter, the development of bidimensional and three-dimensional cell cultures have been described, improving the technical aspect of satellite cell isolation, the best culture-based conditions for muscle cell growth and differentiation, and the procedures required to develop a three-dimensional skeletal muscle construct.
Assuntos
Técnicas de Cultura de Células , Músculo Esquelético , Células Satélites de Músculo Esquelético , Animais , Células Satélites de Músculo Esquelético/citologia , Células Satélites de Músculo Esquelético/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Músculo Esquelético/crescimento & desenvolvimento , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Camundongos , Técnicas de Cultura de Células em Três Dimensões/métodos , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Desenvolvimento Muscular , Proliferação de Células , Células CultivadasRESUMO
The progression of cancer cell migration, invasion and subsequent metastasis is the main cause of mortality in cancer patients. Through creating more accurate cancer models, we can achieve more precise results, which will lead to a better understanding of the invasion process. This holds promise for more effective prevention and treatment strategies. Although numerous 2D and 3D cell culture systems have been developed, they poorly reflect the in vivo situation and many questions have remained unanswered. This work describes a novel dynamic 3D cell culture system aimed at advancing our comprehension of cancer cell migration. With the newly designed cultivation chamber, 3D tumor spheroids were cultivated within a collagen I matrix in the presence of fluid flow to study the migration of cancer cells from spheroids in the matrix. Using light sheet microscopy and histology, we demonstrated that the morphology of spheroids is influenced by dynamic culture and that, in contrast to static culture, spheroids in dynamic culture are characterized by the absence of a large necrotic core. Additionally, this influence extends to an increase in the size of migration area, coupled with an increase in expression of some genes related to epithelial-mesenchymal transition (EMT). The results here highlight the importance of dynamic culture in cancer research. Although the dynamic 3D cell culture system in this study was used to investigate migration of one cell type into a matrix, it has the potential to be further developed and used for more complex models consisting of different cell types or to analyze other steps of metastasis development such as transendothelial migration or extravasation.
Assuntos
Técnicas de Cultura de Células em Três Dimensões , Movimento Celular , Neoplasias do Colo , Transição Epitelial-Mesenquimal , Esferoides Celulares , Humanos , Neoplasias do Colo/patologia , Neoplasias do Colo/metabolismo , Esferoides Celulares/patologia , Técnicas de Cultura de Células em Três Dimensões/métodos , Técnicas de Cultura de Células/métodos , Linhagem Celular TumoralRESUMO
The preterm neonatal airway epithelium is constantly exposed to environmental stressors. One of these stressors in neonates with lung disease includes oxygen (O2) tension higher than the ambient atmosphere - termed hyperoxia (>21% O2). The effect of hyperoxia on the airway depends on various factors, including the developmental stage of the airway, the degree of hyperoxia, and the duration of exposure, with variable exposures potentially leading to unique phenotypes. While there has been extensive research on the effect of hyperoxia on neonatal lung alveolarization and airway hyperreactivity, little is known about the short and long-term underlying effect of hyperoxia on human neonatal airway epithelial cells. A major reason for this is the scarcity of an effective in vitro model to study human neonatal airway epithelial development and function. Here, we describe a method for isolating and expanding human neonatal tracheal airway epithelial cells (nTAECs) utilizing human neonatal tracheal aspirates and culturing these cells in air-liquid interface (ALI) culture. We demonstrate that nTAECs form a mature polarized cell-monolayer in ALI culture and undergo mucociliary differentiation. We also present a method for moderate hyperoxia exposure of the cell monolayer in ALI culture using a specialized incubator. Additionally, we describe an assay to measure cellular oxidative stress following hyperoxia exposure in ALI culture using fluorescent quantification, which confirms that moderate hyperoxia exposure induces cellular oxidative stress but does not cause significant cell membrane damage or apoptosis. This model can potentially be used to simulate clinically relevant hyperoxia exposure encountered by neonatal airways in the Neonatal Intensive Care Unit (NICU) and used to study the short and long-lasting effects of O2 on neonatal airway epithelial programming. Studies using this model could be utilized to explore ways to mitigate early-life oxidative injury to developing airways, which is implicated in the development of long-term airway diseases in former premature infants.
Assuntos
Células Epiteliais , Hiperóxia , Humanos , Recém-Nascido , Hiperóxia/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/citologia , Traqueia/citologia , Traqueia/metabolismo , Técnicas de Cultura de Células em Três Dimensões/métodos , Mucosa Respiratória/citologia , Mucosa Respiratória/metabolismo , Técnicas de Cultura de Células/métodosRESUMO
The cardiac perivascular niche is a cellular microenvironment of a blood vessel. The principles of niche regulation are still poorly understood. We studied the effect of TGFß1 on cells forming the cardiac perivascular niche using 3D cell culture (cardiospheres). Cardiospheres contained progenitor (c-Kit), endothelial (CD31), and mural (αSMA) cells, basement membrane proteins (laminin) and extracellular matrix proteins (collagen I, fibronectin). TGFß1 treatment decreased the length of CD31+ microvasculature, VE cadherin protein level, and proportion of NG2+ cells, and increased proportion of αSMA+ cells and transgelin/SM22α protein level. We supposed that this effect is related to the stabilizing function of TGFß1 on vascular cells: decreased endothelial cell proliferation, as shown for HUVEC, and activation of mural cell differentiation.
Assuntos
Diferenciação Celular , Proliferação de Células , Fator de Crescimento Transformador beta1 , Fator de Crescimento Transformador beta1/farmacologia , Fator de Crescimento Transformador beta1/metabolismo , Diferenciação Celular/efeitos dos fármacos , Humanos , Proliferação de Células/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Animais , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Caderinas/metabolismo , Laminina/metabolismo , Laminina/farmacologia , Proteínas Musculares/metabolismo , Células Cultivadas , Células Endoteliais/metabolismo , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/citologia , Fibronectinas/metabolismo , Fibronectinas/farmacologia , Antígenos CD/metabolismo , Miocárdio/metabolismo , Miocárdio/citologia , Nicho de Células-Tronco/efeitos dos fármacos , Nicho de Células-Tronco/fisiologia , Colágeno Tipo I/metabolismo , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/citologia , Técnicas de Cultura de Células em Três Dimensões/métodosRESUMO
Transformed lung organoids have extensive applications in lung cancer modeling and drug screening. Traditional two-dimensional (2D) cultures fail to propagate a large subpopulation of murine primary tumors in vitro. However, three-dimensional (3D) air-liquid interface (ALI) cultures, which are employed to grow normal lung organoids, can be used to efficiently culture cancerous lung tumor cells. Here, we detail a procedure for cultivating genetically modified lung organoids in 3D-ALI cultures. This protocol contains two parts. The first part describes how to transduce lung epithelial cells, which are either freshly sorted from lungs or from actively growing murine organoids, with virus in order to modify gene expression. The target lung cells are incubated with virus for 1-2 h for transduction. Then, the transduced cells are thoroughly washed and mixed with stromal support cells and Matrigel and are loaded into transwell inserts for culture and validated for genetic modifications through downstream assays. The second part describes how to isolate tumor cells growing orthotopically in genetically engineered mouse models to produce organoid cell lines that can be used for ex vivo drug discovery assays. For this protocol, tumors are isolated from lungs of mice, finely chopped and washed. Then, tumor chunks are mixed with Matrigel for 3D-ALI culture. Finally, organoids budding from tumor chunks are trypsinized and passaged to establish an organoid line. Together these two protocols provide a promising platform to study the genesis, progression, and treatment of lung cancer.
Assuntos
Neoplasias Pulmonares , Pulmão , Organoides , Organoides/citologia , Animais , Camundongos , Pulmão/citologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Técnicas de Cultura de Células em Três Dimensões/métodos , Humanos , Técnicas de Cultura de Células/métodos , Células Epiteliais/citologia , Transdução Genética/métodosRESUMO
Cell patterning for 3D culture has increased our understanding of how cells interact among themselves and with their environment during tissue morphogenesis. Building cell communities from the bottom up with size and compositional control is invaluable for studies of morphological transitions. Here, we detail Photolithographic DNA-programmed Assembly of Cells (pDPAC). pDPAC uses a photoactive polyacrylamide gel substrate to capture single-stranded DNA on a 2D surface in large-scale, highly resolved patterns using the photomask technology. Cells are then functionalized with a complementary DNA strand, enabling cells to be temporarily adhered to distinct locations only where their complementary strand is patterned. These temporary 2D patterns can be transferred to extracellular matrix hydrogels for 3D culture of cells in biomimetic microenvironments. Use of a polyacrylamide substrate has advantages, including a simpler photolithography workflow, lower non-specific cell adhesion, and lower stiction to ECM hydrogels during release of patterned hydrogels. The protocol is equally applicable to large (cm)-scale patterns and repetitive arrays of smaller-scale cell interaction or migration experiments.
Assuntos
Hidrogéis , Engenharia Tecidual , Hidrogéis/química , Humanos , Engenharia Tecidual/métodos , Resinas Acrílicas/química , Adesão Celular , Matriz Extracelular/metabolismo , Matriz Extracelular/química , Técnicas de Cultura de Células/métodos , Animais , Técnicas de Cultura de Células em Três Dimensões/métodosRESUMO
In vitro biofilm models have allowed researchers to investigate the role biofilms play in the pathogenesis, virulence, and antimicrobial drug susceptibility of a wide range of bacterial pathogens. Rotary cell culture systems create three-dimensional cellular structures, primarily applied to eukaryotic organoids, that better capture characteristics of the cells in vivo. Here, we describe how to apply a low-shear, detergent-free rotary cell culture system to generate biofilms of Mycobacterium bovis BCG. The three-dimensional biofilm model forms mycobacterial cell aggregates in suspension as surface-detached biomass, without severe nutrient starvation or environmental stress, that can be harvested for downstream experiments. Mycobacterium bovis BCG derived from cell clusters display antimicrobial drug tolerance, presence of an extracellular matrix, and evidence of cell wall remodeling, all features of biofilm-associated bacteria that may be relevant to the treatment of tuberculosis.
Assuntos
Biofilmes , Mycobacterium bovis , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Mycobacterium bovis/crescimento & desenvolvimento , Mycobacterium bovis/efeitos dos fármacos , Mycobacterium bovis/fisiologia , Técnicas de Cultura de Células/métodos , Técnicas de Cultura de Células em Três Dimensões/métodosRESUMO
Plasma cells (PCs) in bone marrow (BM) play an important role in both protective and pathogenic humoral immune responses, e.g. in various malignant and non-malignant diseases such as multiple myeloma, primary and secondary immunodeficiencies and autoimmune diseases. Dedicated microenvironmental niches in the BM provide PCs with biomechanical and soluble factors that support their long-term survival. There is a high need for appropriate and robust model systems to better understand PCs biology, to develop new therapeutic strategies for PCs-related diseases and perform targeted preclinical studies with high predictive value. Most preclinical data have been derived fromin vivostudies in mice, asin vitrostudies of human PCs are limited due to restricted survival and functionality in conventional 2D cultures that do not reflect the unique niche architecture of the BM. We have developed a microphysiological, dynamic 3D BM culture system (BM-MPS) based on human primary tissue (femoral biopsies), mechanically supported by a hydrogel scaffold casing. While a bioinert agarose casing did not support PCs survival, a photo-crosslinked collagen-hyaluronic acid (Col-HA) hydrogel preserved the native BM niche architecture and allowed PCs survivalin vitrofor up to 2 weeks. Further, the Col-HA hydrogel was permissive to lymphocyte migration into the microphysiological system´s circulation. Long-term PCs survival was related to the stable presence in the culture of soluble factors, as APRIL, BAFF, and IL-6. Increasing immunoglobulins concentrations in the medium confirm their functionality over culture time. To the best of our knowledge, this study is the first report of successful long-term maintenance of primary-derived non-malignant PCsin vitro. Our innovative model system is suitable for in-depthin vitrostudies of human PCs regulation and exploration of targeted therapeutic approaches such as CAR-T cell therapy or biologics.
Assuntos
Hidrogéis , Plasmócitos , Humanos , Plasmócitos/citologia , Plasmócitos/metabolismo , Hidrogéis/química , Sobrevivência Celular/efeitos dos fármacos , Ácido Hialurônico/química , Ácido Hialurônico/farmacologia , Células da Medula Óssea/citologia , Colágeno/química , Medula Óssea/metabolismo , Células Cultivadas , Técnicas de Cultura de Células em Três Dimensões , Modelos Biológicos , Alicerces Teciduais/química , Sefarose/químicaRESUMO
Recently, the need to develop a robust three-dimensional (3D) cell culture system that serves as a valuable in vitro tumor model has been emphasized. This system should closely mimic the tumor growth behaviors observed in vivo and replicate the key elements and characteristics of human tumors for the effective discovery and development of anti-tumor therapeutics. Therefore, in this study, we developed an effective 3D in vitro model of human prostate cancer (PC) using a marine collagen-based biomimetic 3D scaffold. The model displayed distinctive molecular profiles and cellular properties compared with those of the 2D PC cell culture. This was evidenced by (1) increased cell proliferation, migration, invasion, colony formation, and chemoresistance; (2) upregulated expression of crucial multidrug-resistance- and cancer-stemness-related genes; (3) heightened expression of key molecules associated with malignant progressions, such as epithelial-mesenchymal transition transcription factors, Notch, matrix metalloproteinases, and pluripotency biomarkers; (4) robust enrichment of prostate cancer stem cells (CSCs); and (5) enhanced expression of integrins. These results suggest that our 3D in vitro PC model has the potential to serve as a research platform for studying PC and prostate CSC biology, as well as for screening novel therapies targeting PC and prostate CSCs.
Assuntos
Antineoplásicos , Proliferação de Células , Colágeno , Células-Tronco Neoplásicas , Neoplasias da Próstata , Humanos , Masculino , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Linhagem Celular Tumoral , Células-Tronco Neoplásicas/efeitos dos fármacos , Técnicas de Cultura de Células em Três Dimensões/métodos , Animais , Movimento Celular/efeitos dos fármacos , Alicerces Teciduais , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Organismos Aquáticos , Descoberta de Drogas/métodosRESUMO
Three-dimensional (3D) cell culture models capable of emulating the biological functions of natural tissues are pivotal in tissue engineering and regenerative medicine. Despite progress, the fabrication ofin vitroheterocellular models that mimic the intricate structures of natural tissues remains a significant challenge. In this study, we introduce a novel, scaffold-free approach leveraging the inertial focusing effect in rotating hanging droplets for the reliable production of heterocellular spheroids with controllable core-shell structures. Our method offers precise control over the core-shell spheroid's size and geometry by adjusting the cell suspension density and droplet morphology. We successfully applied this technique to create hair follicle organoids, integrating dermal papilla cells within the core and epidermal cells in the shell, thereby achieving markedly enhanced hair inducibility compared to mixed-structure models. Furthermore, we have developed melanoma tumor spheroids that accurately mimic the dynamic interactions between tumor and stromal cells, showing increased invasion capabilities and altered expressions of cellular adhesion molecules and proteolytic enzymes. These findings underscore the critical role of cellular spatial organization in replicating tissue functionalityin vitro. Our method represents a significant advancement towards generating heterocellular spheroids with well-defined architectures, offering broad implications for biological research and applications in tissue engineering.
Assuntos
Técnicas de Cultura de Células em Três Dimensões , Esferoides Celulares , Esferoides Celulares/citologia , Técnicas de Cultura de Células em Três Dimensões/métodos , Humanos , Engenharia Tecidual/métodos , Organoides/citologia , Folículo Piloso/citologia , Animais , Linhagem Celular Tumoral , Alicerces Teciduais/química , Técnicas de Cultura de Células/métodos , Técnicas de Cultura de Células/instrumentaçãoRESUMO
The tumor microenvironment (TME) orchestrates cellular and extracellular matrix (ECM) interactions, playing a key role in tumorigenesis, tumor growth, and metastization. Investigating the interplay between stromal-epithelial cells within the TME is paramount for understanding cancer mechanisms but demands reliable biological models. 3D-models have emerged as powerful in vitro tools, but many fall short in replicating cell-cell/cell-matrix interactions. This study introduces a novel hybrid 3D-model of the breast TME, combining epithelial cells, cancer-associated fibroblasts (CAFs), and their ECM. To build the stromal compartment, porous 3D-printed alginate scaffolds were seeded with CAFs, which proliferated and produced ECM. The pores were infused with oxidized peptide-modified alginate hydrogel laden with MCF10A cells, forming the parenchymal compartment. The hybrid system supported epithelial morphogenesis into acini surrounded by fibroblasts and ECM, and could be readily solubilized to recover cells, their matrix, and sequestered soluble factors. Proteome profiling of the retrieved ECM showed upregulation of proteins associated with matrix assembly/remodeling, epithelial-to-mesenchymal transition (EMT), and cancer. The TME-like microenvironment induced a partial EMT in MCF10A cells, generating a hybrid population with epithelial and mesenchymal features, characteristic of aggressive phenotypes. Our model provided new insights into epithelial-stromal interactions within the TME, offering a valuable tool for cancer research in a physiologically-relevant 3D setting.
Assuntos
Alginatos , Neoplasias da Mama , Células Epiteliais , Matriz Extracelular , Microambiente Tumoral , Humanos , Alginatos/química , Neoplasias da Mama/patologia , Neoplasias da Mama/metabolismo , Feminino , Matriz Extracelular/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Células Estromais/metabolismo , Células Estromais/efeitos dos fármacos , Fibroblastos Associados a Câncer/patologia , Fibroblastos Associados a Câncer/metabolismo , Hidrogéis/química , Impressão Tridimensional , Alicerces Teciduais/química , Técnicas de Cultura de Células em Três Dimensões/métodos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacosRESUMO
Cell co-culture technology aims to study the communication mechanism between cells and to better reveal the interactions and regulatory mechanisms involved in processes such as cell growth, differentiation, apoptosis, and other cellular activities. This is achieved by simulating the complex organismic environment. Such studies are of great significance for understanding the physiological and pathological processes of multicellular organisms. As an emerging cell cultivation technology, 3D cell co-culture technology, based on microfluidic chips, can efficiently, rapidly, and accurately achieve cell co-culture. This is accomplished by leveraging the unique microchannel structures and flow characteristics of microfluidic chips. The technology can simulate the native microenvironment of cell growth, providing a new technical platform for studying intercellular communication. It has been widely used in the research of oncology, immunology, neuroscience, and other fields. In this review, we summarize and provide insights into the design of cell co-culture systems on microfluidic chips, the detection methods employed in co-culture systems, and the applications of these models.
Assuntos
Técnicas de Cocultura , Humanos , Técnicas de Cultura de Células em Três Dimensões , Microfluídica , Dispositivos Lab-On-A-Chip , Animais , Técnicas Analíticas MicrofluídicasRESUMO
Previously, we reported successful cellular expansion of a murine colorectal carcinoma cell line (CT-26) using a three-dimensional (3D) engineered extracellular matrix (EECM) fibrillar scaffold structure. CCL-247 were grown over a limited time period of 8 days on 3D EECM or tissue culture polystyrene (TCPS). Cells were then assayed for growth, electroporation efficiency and Vigil manufacturing release criteria. Using EECM scaffolds, we report an expansion of CCL-247 (HCT116), a colorectal carcinoma cell line, from a starting concentration of 2.45 × 105 cells to 1.9 × 106 cells per scaffold. Following expansion, 3D EECM-derived cells were assessed based on clinical release criteria of the Vigil manufacturing process utilized for Phase IIb trial operation with the FDA. 3D EECM-derived cells passed all Vigil manufacturing release criteria including cytokine expression. Here, we demonstrate successful Vigil product manufacture achieving the specifications necessary for the clinical trial product release of Vigil treatment. Our results confirm that 3D EECM can be utilized for the expansion of human cancer cell CCL-247, justifying further clinical development involving human tissue sample manufacturing including core needle biopsy and minimal ascites samples.
Assuntos
Matriz Extracelular , Imunoterapia , Alicerces Teciduais , Humanos , Alicerces Teciduais/química , Imunoterapia/métodos , Engenharia Tecidual/métodos , Células HCT116 , Neoplasias Colorretais/patologia , Animais , Camundongos , Proliferação de Células , Linhagem Celular Tumoral , Técnicas de Cultura de Células em Três Dimensões/métodosRESUMO
The demand for macroporous hydrogel scaffolds with interconnected porous and open-pore structures is crucial for advancing research and development in cell culture and tissue regeneration. Existing techniques for creating 3D porous materials and controlling their porosity are currently constrained. This study introduces a novel approach for producing highly interconnected aspartic acid-gelatin macroporous hydrogels (MHs) with precisely defined open pore structures using a one-step emulsification polymerization method with surface-modified silica nanoparticles as Pickering stabilizers. Macroporous hydrogels offer adjustable pore size and pore throat size within the ranges of 50 to 130 µm and 15 to 27 µm, respectively, achieved through variations in oil-in-water ratio and solid content. The pore wall thickness of the macroporous hydrogel can be as thin as 3.37 µm and as thick as 6.7 µm. In addition, the storage modulus of the macroporous hydrogels can be as high as 7250 Pa, and it maintains an intact rate of more than 92% after being soaked in PBS for 60 days, which is also good performance for use as a biomedical scaffold material. These hydrogels supported the proliferation of human dental pulp stem cells (hDPSCs) over a 30 day incubation period, stretching the cell morphology and demonstrating excellent biocompatibility and cell adhesion. The combination of these desirable attributes makes them highly promising for applications in stem cell culture and tissue regeneration, underscoring their potential significance in advancing these fields.
Assuntos
Quitosana , Emulsões , Gelatina , Hidrogéis , Alicerces Teciduais , Gelatina/química , Hidrogéis/química , Humanos , Porosidade , Quitosana/química , Alicerces Teciduais/química , Emulsões/química , Células-Tronco/citologia , Aminoácidos/química , Proliferação de Células/efeitos dos fármacos , Polpa Dentária/citologia , Técnicas de Cultura de Células em Três Dimensões/métodos , Células Cultivadas , Técnicas de Cultura de Células/métodos , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Engenharia Tecidual/métodosRESUMO
Introduction: Within adipose tissue (AT), different macrophage subsets have been described, which played pivotal and specific roles in upholding tissue homeostasis under both physiological and pathological conditions. Nonetheless, studying resident macrophages in-vitro poses challenges, as the isolation process and the culture for extended periods can alter their inherent properties. Methods: Stroma-vascular cells isolated from murine subcutaneous AT were seeded on ultra-low adherent plates in the presence of macrophage colony-stimulating factor. After 4 days of culture, the cells spontaneously aggregate to form spheroids. A week later, macrophages begin to spread out of the spheroid and adhere to the culture plate. Results: This innovative three-dimensional (3D) culture method enables the generation of functional mature macrophages that present distinct genic and phenotypic characteristics compared to bone marrow-derived macrophages. They also show specific metabolic activity and polarization in response to stimulation, but similar phagocytic capacity. Additionally, based on single-cell analysis, AT-macrophages generated in 3D culture mirror the phenotypic and functional traits of in-vivo AT resident macrophages. Discussion: Our study describes a 3D in-vitro system for generating and culturing functional AT-resident macrophages, without the need for cell sorting. This system thus stands as a valuable resource for exploring the differentiation and function of AT-macrophages in vitro in diverse physiological and pathological contexts.
Assuntos
Tecido Adiposo , Técnicas de Cultura de Células em Três Dimensões , Diferenciação Celular , Macrófagos , Animais , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Tecido Adiposo/citologia , Técnicas de Cultura de Células em Três Dimensões/métodos , Células Cultivadas , Fagocitose , Camundongos Endogâmicos C57BL , Esferoides Celulares/citologia , Técnicas de Cultura de Células/métodos , FenótipoRESUMO
Three-dimensional culture models of the brain enable the study of neuroinfection in the context of a complex interconnected cell matrix. Depending on the differentiation status of the neural cells, two models exist: 3D spheroids also called neurospheres and cerebral organoids. Here, we describe the preparation of 3D spheroids and cerebral organoids and give an outlook on their usage to study Rift Valley fever virus and other neurotropic viruses.
Assuntos
Organoides , Esferoides Celulares , Organoides/virologia , Organoides/citologia , Esferoides Celulares/virologia , Humanos , Animais , Vírus de RNA/fisiologia , Encéfalo/virologia , Encéfalo/citologia , Infecções por Vírus de RNA/virologia , Técnicas de Cultura de Células/métodos , Técnicas de Cultura de Células em Três Dimensões/métodosRESUMO
Three-dimensional (3D) cell culture creates a more physiologically relevant environment for enhanced drug screening capabilities using microcarriers. An automated 3D system that integrates robotic manipulators, liquid handling systems, sensors, and environment control systems has the capacity to handle multiple samples in parallel, perform repetitive tasks, and provide real-time monitoring and analysis. This chapter describes a potential 3D cell culture drug screening model by combining renal proximal tubule cells as a representative normal cell line with cancer cell lines. This combination is subjected to drug screening to evaluate the drug's efficacy in suppressing cancer cells while minimizing impact on normal cells with the added benefit of having the ability to separate the two cell types by magnetic isolation for high content screens including mass spectrometry-based proteomics. This study presents advancements in 3D cell culture techniques, emphasizing the importance of automation and the potential of microcarriers in drug screening and disease modeling.
Assuntos
Técnicas de Cultura de Células em Três Dimensões , Humanos , Técnicas de Cultura de Células em Três Dimensões/métodos , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos/métodos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Técnicas de Cultura de Células/métodos , Antineoplásicos/farmacologia , Automação , Automação Laboratorial/métodos , Neoplasias/patologia , Neoplasias/tratamento farmacológicoRESUMO
Addressing current challenges in solid tumor research requires advanced in vitro three-dimensional (3D) cellular models that replicate the inherently 3D architecture and microenvironment of tumor tissue, including the extracellular matrix (ECM). However, tumor cells exert mechanical forces that can disrupt the physical integrity of the matrix in long-term 3D culture. Therefore, it is necessary to find the optimal balance between cellular forces and the preservation of matrix integrity. This work proposes using polydopamine (PDA) coating for 3D microfluidic cultures of pancreatic cancer cells to overcome matrix adhesion challenges to sustain representative tumor 3D cultures. Using PDA's distinctive adhesion and biocompatibility, our model uses type I collagen hydrogels seeded with different pancreatic cancer cell lines, prompting distinct levels of matrix deformation and contraction. Optimizing the PDA coating enhances the adhesion and stability of collagen hydrogels within microfluidic devices, achieving a balance between the disruptive forces of tumor cells on matrix integrity and the maintenance of long-term 3D cultures. The findings reveal how this tension appears to be a critical determinant in spheroid morphology and growth dynamics. Stable and prolonged 3D culture platforms are crucial for understanding solid tumor cell behavior, dynamics, and responses within a controlled microenvironment. This advancement ultimately offers a powerful tool for drug screening, personalized medicine, and wider cancer therapeutics strategies.