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1.
Int J Mol Sci ; 22(11)2021 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-34073710

RESUMO

Cortical circuit dysfunction is thought to be an underlying mechanism of schizophrenia (SZ) pathophysiology with normalization of aberrant circuit activity proposed as a biomarker for antipsychotic efficacy. Cannabidiol (CBD) shows potential as an adjunctive antipsychotic therapy; however, potential sex effects in these drug interactions remain unknown. In the present study, we sought to elucidate sex effects of CBD coadministration with the atypical antipsychotic iloperidone (ILO) on the activity of primary cortical neuron cultures derived from the rat methylazoxymethanol acetate (MAM) model used for the study of SZ. Spontaneous network activity measurements were obtained using a multielectrode array at baseline and following administration of CBD or ILO alone, or combined. At baseline, MAM male neurons displayed increased bursting activity whereas MAM female neurons exhibited no difference in bursting activity compared to sex-matched controls. CBD administered alone showed a rapid but transient increase in neuronal activity in the MAM networks, an effect more pronounced in females. Furthermore, ILO had an additive effect on CBD-induced elevations in activity in the MAM male neurons. In the MAM female neurons, CBD or ILO administration resulted in time-dependent elevations in neuronal activity, but the short-term CBD-induced increases in activity were lost when CBD and ILO were combined. Our findings indicate that CBD induces rapid increases in cortical neuronal activity, with sex-specific drug interactions upon ILO coadministration. This suggests that sex should be a consideration when implementing adjunct therapy for treatment of SZ.


Assuntos
Canabidiol/farmacologia , Isoxazóis/farmacologia , Neurônios/efeitos dos fármacos , Piperidinas/farmacologia , Esquizofrenia/tratamento farmacológico , Animais , Animais Recém-Nascidos , Antipsicóticos/farmacologia , Antipsicóticos/uso terapêutico , Canabidiol/uso terapêutico , Técnicas de Cultura de Células , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/fisiopatologia , Modelos Animais de Doenças , Feminino , Isoxazóis/uso terapêutico , Masculino , Neurônios/fisiologia , Piperidinas/uso terapêutico , Ratos , Ratos Sprague-Dawley , Esquizofrenia/fisiopatologia , Caracteres Sexuais
2.
Nat Commun ; 12(1): 3413, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099701

RESUMO

Bottom-up approaches using building blocks of modules to fabricate scaffolds for tissue engineering applications have enabled the fabrication of structurally complex and multifunctional materials allowing for physical and chemical flexibility to better mimic the native extracellular matrix. Here we report a vapor-phased fabrication process for constructing three-dimensional modulated scaffold materials via simple steps based on controlling mass transport of vapor sublimation and deposition. We demonstrate the fabrication of scaffolds comprised of multiple biomolecules and living cells with built-in boundaries separating the distinct compartments containing defined biological configurations and functions. We show that the fabricated scaffolds have mass production potential. We demonstrate overall >80% cell viability of encapsulated cells and that modulated scaffolds exhibit enhanced cell proliferation, osteogenesis, and neurogenesis, which can be assembled into various geometric configurations. We perform cell co-culture experiments to show independent osteogenesis and angiogenesis activities from separate compartments in one scaffold construct.


Assuntos
Materiais Biomiméticos/química , Vapor , Engenharia Tecidual/métodos , Tecidos Suporte/química , Animais , Técnicas de Cultura de Células , Linhagem Celular , Proliferação de Células , Técnicas de Cocultura , Matriz Extracelular , Humanos , Hidrogéis/química , Camundongos , Neovascularização Fisiológica , Neurogênese , Osteogênese , Ratos
3.
Int J Mol Sci ; 22(10)2021 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-34068404

RESUMO

Numerous cell-based therapeutics are currently being tested in clinical trials. Human platelet lysate (HPL) is a valuable alternative to fetal bovine serum as a cell culture medium supplement for a variety of different cell types. HPL as a raw material permits animal serum-free cell propagation with highly efficient stimulation of cell proliferation, enabling humanized manufacturing of cell therapeutics within a reasonable timeframe. Providers of HPL have to consider dedicated quality issues regarding identity, purity, potency, traceability and safety. Release criteria have to be defined, characterizing the suitability of HPL batches for the support of a specific cell culture. Fresh or expired platelet concentrates from healthy blood donors are the starting material for HPL preparation, according to regulatory requirements. Pooling of individual platelet lysate units into one HPL batch can balance donor variation with regard to essential platelet-derived growth factors and cytokines. The increasingly applied pathogen reduction technologies will further increase HPL safety. In this review article, aspects and regulatory requirements of whole blood donation and details of human platelet lysate manufacturing are presented. International guidelines for raw materials are discussed, and defined quality controls, as well as release criteria for safe and GMP-compliant HPL production, are summarized.


Assuntos
Plaquetas/citologia , Técnicas de Cultura de Células/normas , Diferenciação Celular , Animais , Proliferação de Células , Meios de Cultura , Humanos
4.
Nat Commun ; 12(1): 3192, 2021 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-34045434

RESUMO

Tissues achieve their complex spatial organization through an interplay between gene regulatory networks, cell-cell communication, and physical interactions mediated by mechanical forces. Current strategies to generate in-vitro tissues have largely failed to implement such active, dynamically coordinated mechanical manipulations, relying instead on extracellular matrices which respond to, rather than impose mechanical forces. Here, we develop devices that enable the actuation of organoids. We show that active mechanical forces increase growth and lead to enhanced patterning in an organoid model of the neural tube derived from single human pluripotent stem cells (hPSC). Using a combination of single-cell transcriptomics and immunohistochemistry, we demonstrate that organoid mechanoregulation due to actuation operates in a temporally restricted competence window, and that organoid response to stretch is mediated extracellularly by matrix stiffness and intracellularly by cytoskeleton contractility and planar cell polarity. Exerting active mechanical forces on organoids using the approaches developed here is widely applicable and should enable the generation of more reproducible, programmable organoid shape, identity and patterns, opening avenues for the use of these tools in regenerative medicine and disease modelling applications.


Assuntos
Tubo Neural/citologia , Organoides/fisiologia , Engenharia Tecidual/métodos , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Linhagem Celular , Matriz Extracelular/fisiologia , Humanos , Hidrogéis/química , Mecanotransdução Celular/fisiologia , Células-Tronco Pluripotentes , Polietilenoglicóis/química , RNA-Seq , Medicina Regenerativa/métodos , Análise de Célula Única , Engenharia Tecidual/instrumentação
5.
Int J Mol Sci ; 22(9)2021 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-33946876

RESUMO

The hair follicle dermal papilla is critical for hair generation and de novo regeneration. When cultured in vitro, dermal papilla cells from different species demonstrate two distinguishable growth patterns under the conventional culture condition: a self-aggregative three dimensional spheroidal (3D) cell pattern and a two dimensional (2D) monolayer cell pattern, correlating with different hair inducing properties. Whether the loss of self-aggregative behavior relates to species-specific differences or the improper culture condition remains unclear. Can the fixed 2D patterned dermal papilla cells recover the self-aggregative behavior and 3D pattern also remains undetected. Here, we successfully constructed the two growth patterns using sika deer (Cervus nippon) dermal papilla cells and proved it was the culture condition that determined the dermal papilla growth pattern. The two growth patterns could transit mutually as the culture condition was exchanged. The fixed 2D patterned sika deer dermal papilla cells could recover the self-aggregative behavior and transit back to 3D pattern, accompanied by the restoration of hair inducing capability when the culture condition was changed. In addition, the global gene expressions during the transition from 2D pattern to 3D pattern were compared to detect the potential regulating genes and pathways involved in the recovery of 3D pattern and hair inducing capability.


Assuntos
Cervos/anatomia & histologia , Folículo Piloso/citologia , Antígeno AC133/biossíntese , Antígeno AC133/genética , Fosfatase Alcalina/biossíntese , Fosfatase Alcalina/genética , Animais , Biomarcadores , Agregação Celular , Técnicas de Cultura de Células , Divisão Celular , Células Cultivadas , Cervos/genética , Regulação da Expressão Gênica , Ontologia Genética , Cabelo , Folículo Piloso/crescimento & desenvolvimento , Folículo Piloso/metabolismo , Mesoderma/citologia , Fatores de Transcrição SOXB1/biossíntese , Fatores de Transcrição SOXB1/genética , Especificidade da Espécie , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo , Transcriptoma , Versicanas/biossíntese , Versicanas/genética
6.
Mater Sci Eng C Mater Biol Appl ; 125: 112100, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33965110

RESUMO

Complex three-dimensional (3D) cell cultures are being increasingly implemented in biomedical research as they provide important insights into complex cancer biology, and cell-cell and cell-matrix interactions in the tumor microenvironment. However, most methods used today for 3D cell culture are limited by high cost, the need for specialized skills, low throughput and the use of unnatural culture environments. We report the development of a unique biomimetic hydrogel microwell array platform for the generation and stress-free isolation of cancer spheroids. The poly N-isopropylacrylamide-based hydrogel microwell array (PHMA) has thermoresponsive properties allowing for the attachment and growth of cell aggregates/ spheroids at 37 °C, and their easy isolation at room temperature (RT). The reversible phase transition of the microwell arrays at 35 °C was confirmed visually and by differential scanning calorimetry. Swelling/ shrinking studies and EVOS imaging established that the microwell arrays are hydrophilic and swollen at temperatures <35 °C, while they shrink and are hydrophobic at temperatures >35 °C. Spheroid development within the PHMA was optimized for seeding density, incubation time and cell viability. Spheroids of A549, HeLa and MG-63 cancer cell lines, and human lung fibroblast (HLF) cell line generated within the PHMAs had relatively spherical morphology with hypoxic cores. Finally, using MG-63 cell spheroids as representative models, a proof-of-concept drug response study using doxorubicin hydrochloride was conducted. Overall, we demonstrate that the PHMAs are an innovative alternative to currently used 3D cell culture techniques, for the high-throughput generation of cell spheroids for disease modeling and drug screening applications.


Assuntos
Hidrogéis , Neoplasias , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Sobrevivência Celular , Humanos , Esferoides Celulares , Microambiente Tumoral
7.
Mater Sci Eng C Mater Biol Appl ; 124: 112051, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33947545

RESUMO

Three-dimensional (3D) cell culture systems include bioengineered microenvironments that mimic the complexity of human tissues and organs in vitro. Robust biological models, like organoids and spheroids, rely on biomaterials to emulate the biochemical and biomechanical properties found in the extracellular matrix (ECM). Collagen (COL) is the main protein component of the ECM and has been used to generate fibrous matrices for 3D cell culture. Whilst neat COL gels are commonly blended with inert polymers to improve their poor mechanical properties, whether nanocellulose (NC) fibers interact or can develop some synergic bioactive effect to support organoid systems has never been demonstrated. Here, we investigate collagen-nanocellulose (COL-NC) hydrogels as a thermo-responsive matrix for the formation and growth of intestinal organoids. Cellulose nanofibres grafted with fibronectin-like adhesive sites form a porous network with type I collagen, presenting a sol-gel transition and viscoelastic profile similar to those of standard animal-based matrices. Crypts embedded in COL-NC form organoids with evidence of epithelial budding. Cell viability and metabolic activity are preserved as well as the expression of key cell markers. The stiffness of COL-NC hydrogels is shown to be a determinant element for the formation and development organoids. COL-NC hydrogels provide an affordable, performant thermo-responsive and sustainable matrix for organoid growth.


Assuntos
Hidrogéis , Organoides , Animais , Técnicas de Cultura de Células , Colágeno , Matriz Extracelular , Humanos
8.
Nat Commun ; 12(1): 2581, 2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-33972544

RESUMO

While the potential of patient-derived organoids (PDOs) to predict patients' responses to anti-cancer treatments has been well recognized, the lengthy time and the low efficiency in establishing PDOs hamper the implementation of PDO-based drug sensitivity tests in clinics. We first adapt a mechanical sample processing method to generate lung cancer organoids (LCOs) from surgically resected and biopsy tumor tissues. The LCOs recapitulate the histological and genetic features of the parental tumors and have the potential to expand indefinitely. By employing an integrated superhydrophobic microwell array chip (InSMAR-chip), we demonstrate hundreds of LCOs, a number that can be generated from most of the samples at passage 0, are sufficient to produce clinically meaningful drug responses within a week. The results prove our one-week drug tests are in good agreement with patient-derived xenografts, genetic mutations of tumors, and clinical outcomes. The LCO model coupled with the microwell device provides a technically feasible means for predicting patient-specific drug responses in clinical settings.


Assuntos
Adenocarcinoma/tratamento farmacológico , Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/tratamento farmacológico , Técnicas de Cultura de Células/métodos , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Neoplasias Pulmonares/tratamento farmacológico , Organoides/efeitos dos fármacos , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais/instrumentação , Gefitinibe/farmacologia , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Organoides/citologia , Organoides/patologia , Preparações Farmacêuticas , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Nat Commun ; 12(1): 3017, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-34021145

RESUMO

Extended pluripotent stem (EPS) cells have shown great applicative potentials in generating synthetic embryos, directed differentiation and disease modeling. However, the lack of a xeno-free culture condition has significantly limited their applications. Here, we report a chemically defined and xeno-free culture system for culturing and deriving human EPS cells in vitro. Xeno-free human EPS cells can be long-term and genetically stably maintained in vitro, as well as preserve their embryonic and extraembryonic developmental potentials. Furthermore, the xeno-free culturing system also permits efficient derivation of human EPS cells from human fibroblast through reprogramming. Our study could have broad utility in future applications of human EPS cells in biomedicine.


Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Pluripotentes/citologia , Animais , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Fibroblastos/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Fator 3 de Transcrição de Octâmero
10.
Nat Commun ; 12(1): 3155, 2021 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-34039977

RESUMO

Compact cardiomyocytes that make up the ventricular wall of the adult heart represent an important therapeutic target population for modeling and treating cardiovascular diseases. Here, we established a differentiation strategy that promotes the specification, proliferation and maturation of compact ventricular cardiomyocytes from human pluripotent stem cells (hPSCs). The cardiomyocytes generated under these conditions display the ability to use fatty acids as an energy source, a high mitochondrial mass, well-defined sarcomere structures and enhanced contraction force. These ventricular cells undergo metabolic changes indicative of those associated with heart failure when challenged in vitro with pathological stimuli and were found to generate grafts consisting of more mature cells than those derived from immature cardiomyocytes following transplantation into infarcted rat hearts. hPSC-derived atrial cardiomyocytes also responded to the maturation cues identified in this study, indicating that the approach is broadly applicable to different subtypes of the heart. Collectively, these findings highlight the power of recapitulating key aspects of embryonic and postnatal development for generating therapeutically relevant cell types from hPSCs.


Assuntos
Técnicas de Cultura de Células/métodos , Insuficiência Cardíaca/terapia , Infarto do Miocárdio/terapia , Miócitos Cardíacos/transplante , Células-Tronco Pluripotentes/fisiologia , Animais , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Modelos Animais de Doenças , Embrião de Mamíferos , Desenvolvimento Embrionário/fisiologia , Átrios do Coração/citologia , Átrios do Coração/embriologia , Insuficiência Cardíaca/patologia , Ventrículos do Coração/citologia , Ventrículos do Coração/embriologia , Ventrículos do Coração/patologia , Humanos , Infarto do Miocárdio/complicações , Infarto do Miocárdio/patologia , Miócitos Cardíacos/fisiologia , Ratos
11.
Exerc Immunol Rev ; 27: 24-41, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33965899

RESUMO

Numerous epidemiological studies have shown the existence of a relationship between exercise and reduced risk of different types of cancer. In vitro studies have identified a direct effect of exercise-conditioned human serum on cancer cell lines of the lung, breast, prostate, and colon. The aim of this systematic review with meta-analysis (SRM) was to estimate the magnitude of the effect that exercise-conditioned human serum produced on the viability of cancer cell cultures. The design followed the PRISMA guidelines and the TREND statement to assess the quality of information (QoI) in each study. Nine in vitro studies were included in the SRM, involving a total of nine cancer cell lines and serum from 244 individuals from different countries, including namely healthy sedentary individuals, at risk of prostate cancer individuals and cancer patients, with ages ranging from 18 to 73 years. The impact of exerciseconditioned human serum on the viability of cancer cell cultures was analysed by a variety of assays, using pre-exercise human serum for comparison purposes. Globally, cultures of cancer cell lines exposed to human serum conditioned by exercise of various intensities exhibited a reduced viability, when compared with control cultures, with an overall effect size of -1.126 (95% CI; -1.300 to -0.952; p < 0.001). When the analysis only included human serum conditioned by high intensity exercise, the effect became more pronounced (ES -1.350; -1.522 to -1.179 (95% CI); p < 0.001). These results are in line with the hypothesis that changes in human serum induced by exercise might play a role in the beneficial effects of physical activity in cancer prevention and management and that these effects depend on exercise intensity.


Assuntos
Meios de Cultivo Condicionados , Exercício Físico , Neoplasias/prevenção & controle , Adolescente , Adulto , Idoso , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias da Próstata , Adulto Jovem
12.
Int J Mol Sci ; 22(9)2021 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-33946403

RESUMO

Extracellular vesicles (EVs) are cell-derived nanostructures that mediate intercellular communication by delivering complex signals in normal tissues and cancer. The cellular coordination required for tumor development and maintenance is mediated, in part, through EV transport of molecular cargo to resident and distant cells. Most studies on EV-mediated signaling have been performed in two-dimensional (2D) monolayer cell cultures, largely because of their simplicity and high-throughput screening capacity. Three-dimensional (3D) cell cultures can be used to study cell-to-cell and cell-to-matrix interactions, enabling the study of EV-mediated cellular communication. 3D cultures may best model the role of EVs in formation of the tumor microenvironment (TME) and cancer cell-stromal interactions that sustain tumor growth. In this review, we discuss EV biology in 3D culture correlates of the TME. This includes EV communication between cell types of the TME, differences in EV biogenesis and signaling associated with differing scaffold choices and in scaffold-free 3D cultures and cultivation of the premetastatic niche. An understanding of EV biogenesis and signaling within a 3D TME will improve culture correlates of oncogenesis, enable molecular control of the TME and aid development of drug delivery tools based on EV-mediated signaling.


Assuntos
Técnicas de Cultura de Células/métodos , Vesículas Extracelulares/patologia , Neoplasias/patologia , Microambiente Tumoral , Animais , Humanos , Tecidos Suporte/química
13.
Cell Rep ; 35(7): 109133, 2021 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-33984267

RESUMO

Effective control of COVID-19 requires antivirals directed against SARS-CoV-2. We assessed 10 hepatitis C virus (HCV) protease-inhibitor drugs as potential SARS-CoV-2 antivirals. There is a striking structural similarity of the substrate binding clefts of SARS-CoV-2 main protease (Mpro) and HCV NS3/4A protease. Virtual docking experiments show that these HCV drugs can potentially bind into the Mpro substrate-binding cleft. We show that seven HCV drugs inhibit both SARS-CoV-2 Mpro protease activity and SARS-CoV-2 virus replication in Vero and/or human cells. However, their Mpro inhibiting activities did not correlate with their antiviral activities. This conundrum is resolved by demonstrating that four HCV protease inhibitor drugs, simeprevir, vaniprevir, paritaprevir, and grazoprevir inhibit the SARS CoV-2 papain-like protease (PLpro). HCV drugs that inhibit PLpro synergize with the viral polymerase inhibitor remdesivir to inhibit virus replication, increasing remdesivir's antiviral activity as much as 10-fold, while those that only inhibit Mpro do not synergize with remdesivir.


Assuntos
Antivirais/farmacologia , /antagonistas & inibidores , /enzimologia , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/farmacologia , Alanina/análogos & derivados , Alanina/farmacologia , Técnicas de Cultura de Células , Linhagem Celular , Reposicionamento de Medicamentos/métodos , Sinergismo Farmacológico , Hepacivirus/efeitos dos fármacos , Hepatite C/tratamento farmacológico , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Inibidores de Proteases/farmacologia , Replicação Viral/efeitos dos fármacos
14.
J Vis Exp ; (170)2021 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-33938879

RESUMO

Physiological electric fields (EF) play vital roles in cell migration, differentiation, division, and death. This paper describes a microfluidic cell culture system that was used for a long-term cell differentiation study using microscopy. The microfluidic system consists of the following major components: an optically transparent electrotactic chip, a transparent indium-tin-oxide (ITO) heater, a culture media-filling pump, an electrical power supply, a high-frequency power amplifier, an EF multiplexer, a programmable X-Y-Z motorized stage, and an inverted phase-contrast microscope equipped with a digital camera. The microfluidic system is beneficial in simplifying the overall experimental setup and, in turn, the reagent and sample consumption. This work involves the differentiation of neural stem and progenitor cells (NPCs) induced by direct current (DC) pulse stimulation. In the stem cell maintenance medium, the mouse NPCs (mNPCs) differentiated into neurons, astrocytes, and oligodendrocytes after the DC pulse stimulation. The results suggest that simple DC pulse treatment could control the fate of mNPCs and could be used to develop therapeutic strategies for nervous system disorders. The system can be used for cell culture in multiple channels, for long-term EF stimulation, for cell morphological observation, and for automatic time-lapse image acquisition. This microfluidic system not only shortens the required experimental time, but also increases the accuracy of control on the microenvironment.


Assuntos
Diferenciação Celular , Animais , Astrócitos/citologia , Técnicas de Cultura de Células , Eletricidade , Dispositivos Lab-On-A-Chip , Camundongos , Células-Tronco Neurais/citologia , Neurônios/citologia , Oligodendroglia/citologia
15.
Gene ; 788: 145662, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-33887373

RESUMO

INTRODUCTION: Culture conditions and differentiation cocktails may facilitate cell maturation and extracellular matrix (ECM) secretion and support the production of engineered fibroblastic tissues with applications in ligament regeneration. The objective of this study is to investigate the potential of two connective tissue-related ligands (i.e., BMP6 and GDF5) to mediate collagenous ECM synthesis and tissue maturation in vitro under normoxic and hypoxic conditions based on the hypothesis that BMP6 and GDF5 are components of normal paracrine signalling events that support connective tissue homeostasis. METHODS: Human adipose-derived MSCs were seeded on 3D-printed medical-grade polycaprolactone (PCL) scaffolds using a bioreactor and incubated in media containing GDF5 and/or BMP6 for 21 days in either normoxic (5% oxygen) or hypoxic (2% oxygen) conditions. Constructs were harvested on Day 3 and 21 for cell viability analysis by live/dead staining, structural analysis by scanning electron microscopy, mRNA levels by RTqPCR analysis, and in situ deposition of proteins by immunofluorescence microscopy. RESULTS: Pro-fibroblastic gene expression is enhanced by hypoxic culture conditions compared to normoxic conditions. Hypoxia renders cells more responsive to treatment with BMP6 as reflected by increased expression of ECM mRNA levels on Day 3 with sustained expression until Day 21. GDF5 was not particularly effective either in the absence or presence of BMP6. CONCLUSIONS: Fibroblastic differentiation of MSCs is selectively enhanced by BMP6 and not GDF5. Environmental factors (i.e., hypoxia) also influenced the responsiveness of cells to this morphogen.


Assuntos
Proteína Morfogenética Óssea 6/farmacologia , Técnicas de Cultura de Células/métodos , Fibroblastos/citologia , Fator 5 de Diferenciação de Crescimento/farmacologia , Células-Tronco Mesenquimais/citologia , Reatores Biológicos , Diferenciação Celular/efeitos dos fármacos , Hipóxia Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Fibroblastos/química , Fibroblastos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Células-Tronco Mesenquimais/química , Células-Tronco Mesenquimais/efeitos dos fármacos , Tecidos Suporte
16.
Mar Drugs ; 19(5)2021 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-33924764

RESUMO

Cancer cells grown in spheroid conditions interact with each other and the extracellular matrix, providing a better representation of the in vivo environment than two-dimensional cultures and are a more clinically relevant model. A discrete screening of genetically diverse marine samples in the spheroid assay led to the identification of a novel activity for the known compound furospinulosin 1. This compound shows activity against MDA-MB-231 triple negative breast cancer cells grown as spheroids and treated for 24 or 48 h. No cytotoxicity was seen in traditional two-dimensional adherent cultures treated for a longer time (72 h). A reverse phase protein array (RPPA) confirmed the limited activity of the compound in cells grown traditionally and revealed changes in protein expression when cells are grown as spheroids that are associated with better clinical prognosis. Analysis of the RPPA data through the Broad institute's connectivity map suggested the hypothesis that furospinulosin 1 functions as an MEK inhibitor. Analysis of the RPPA data through STRING supports the apoptosis observed. The selectivity exhibited by furospinulosin 1 for triple negative breast cancer cells only when grown as spheroids makes it an interesting compound with strong therapeutic potential that merits further study.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Sesterterpenos/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Biologia Computacional , Feminino , Humanos , Mapas de Interação de Proteínas , Proteoma , Proteômica , Transdução de Sinais , Esferoides Celulares , Fatores de Tempo , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia
18.
Sensors (Basel) ; 21(7)2021 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-33916287

RESUMO

The study of cell proliferation is of great importance for medical and biological research, as well as for industrial applications. To render the proliferation process accurately over time, real-time cell proliferation assay methods are required. This work presents a novel real-time and label-free approach for monitoring cell proliferation by continuously measuring changes in thermal properties that occur at the sensor interface during the process. The sensor consists of a single planar resistive structure deposited on a thin foil substrate, integrated at the bottom of a cell culture reservoir. During measurement, the structure is excited with square wave current pulses. Meanwhile, the temperature-induced voltage change measured over the structure is used to derive variations in the number of cells at the interface. This principle is demonstrated first by performing cell sedimentation measurements to quantify the presence of cells at the sensor interface in the absence of cell growth. Later, cell proliferation experiments were performed, whereby parameters such as the available nutrient content and the cell starting concentration were modified. Results from these experiments show that the thermal-based sensor is able to accurately measure variations in the number of cells at the interface. Moreover, the influence of the modified parameters could be observed in the obtained proliferation curves. These findings highlight the potential for the presented thermal method to be incorporated in a standardized well plate format for high-throughput monitoring of cell proliferation.


Assuntos
Técnicas de Cultura de Células , Proliferação de Células , Fenômenos Físicos
19.
J Biomed Nanotechnol ; 17(3): 399-406, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33875074

RESUMO

Pelvic organ prolapse (POP) has become one of the most common serious diseases affecting parous women. Weakening of pelvic ligaments plays an essential role in the pathophysiology of POP. Currently, synthetic materials are widely applied for pelvic reconstructive surgery. However, synthetic nondegradable meshes for POP therapy cannot meet the clinical requirements due to its poor biocompatibility. Herein, we fabricated electrospun core-shell nanofibers of poly(l-lactic acid)-hyaluronic acid (PLLA/HA). After that, we combined them with mouse bone marrow-derived mesenchymal stem cells (mBMSCs) to assess the cellular response and pelvic ligament tissue engineering in vitro. The cellular responses on the composite nanofibers showed that the core-shell structure nanofibers displayed with excellent biocompatibility and enhanced cellular activity without cytotoxicity. Moreover, compared with PLLA nanofibers seeded with mBMSCs, PLLA/HA nanofibers exhibited more cellular function, as revealed by the quantitative real-time polymerase chain reaction (RT-qPCR) for pelvic ligament-related gene markers including Col1a1, Col1a3 and Tnc. These features suggested that this novel core-shell nanofiber is promising in stem cell-based tissue engineering for pelvic reconstruction.


Assuntos
Nanofibras , Animais , Técnicas de Cultura de Células , Proliferação de Células , Ácido Hialurônico , Ácido Láctico , Ligamentos , Camundongos , Poliésteres , Engenharia Tecidual , Tecidos Suporte
20.
Int J Mol Sci ; 22(5)2021 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-33806404

RESUMO

A conditioned medium of a cell culture is widely used for various biological applications and frequently analyzed to characterize the functional proteins responsible for observed biological functions. However, a large number of abundant proteins in fetal bovine serum (FBS), usually included in the conditioned medium of a mammalian cell culture medium, hampers in-depth proteomic analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS). For a deep proteomic analysis of a conditioned medium by LC-MS/MS, we developed a simple albumin depletion approach coupled with data-independent acquisition (DIA)-mode LC-MS/MS for the conditioned medium of mammalian cells in this study. The results showed that this approach enabled the detection of more than 3700 cell-derived proteins in the cell culture supernatant containing FBS. We further demonstrated the potency of this approach by analyzing proteins in the conditioned media of HeLa cells with and without tumor necrosis factor (TNF) stimulation: >40 differentially accumulated proteins, including four cytokines, upon TNF stimulation were identified in the culture media, which were hardly detected by conventional proteome approaches in the literature.


Assuntos
Técnicas de Cultura de Células/métodos , Meios de Cultivo Condicionados/química , Proteoma/análise , Animais , Bovinos , Cromatografia Líquida , Células HeLa , Humanos , Proteômica/métodos , Soroalbumina Bovina/química , Soroalbumina Bovina/isolamento & purificação , Espectrometria de Massas em Tandem , Fator de Necrose Tumoral alfa/farmacologia
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