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1.
PLoS One ; 15(7): e0235827, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32667933

RESUMO

Homogenization of the initial cell distribution is essential for effective cell development. However, there are few previous reports on efficient cell seeding methods, even though the initial cell distribution has a large effect on cell proliferation. Dense cell regions have an inverse impact on cell development, known as contact inhibition. In this study, we developed a method to homogenize the cell seeding density using secondary flow, or Ekman transportation, induced by orbital movement of the culture dish. We developed an orbital shaker device that can stir the medium in a 35-mm culture dish by shaking the dish along a circular orbit with 2 mm of eccentricity. The distribution of cells in the culture dish can be controlled by the rotational speed of the orbital shaker, enabling dispersion of the initial cell distribution. The experimental results indicated that the cell density became most homogeneous at 61 rpm. We further evaluated the cell proliferation after homogenization of the initial cell density at 61 rpm. The results revealed 36% higher proliferation for the stirred samples compared with the non-stirred control samples. The present findings indicate that homogenization of the initial cell density by Ekman transportation contributes to the achievement of higher cell proliferation.


Assuntos
Técnicas de Cultura de Células/instrumentação , Mioblastos/citologia , Animais , Contagem de Células , Técnicas de Cultura de Células/economia , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Desenho de Equipamento , Camundongos
2.
PLoS One ; 15(6): e0234441, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32525941

RESUMO

Human iPSC-derived hepatocytes hold great promise as a cell source for cell therapy and drug screening. However, the culture method for highly-quantified hepatocytes has not yet been established. Herein, we have developed an encapsulation and 3D cultivation method for iPSC-hepatocytes in core-shell hydrogel microfibers (a.k.a. cell fiber). In the fiber-shaped 3D microenvironment consisting of abundant extracellular matrix (ECM), the iPSC-hepatocytes exhibited many hepatic characteristics, including the albumin secretion, and the expression of the hepatic marker genes (ALB, HNF4α, ASGPR1, CYP2C19, and CYP3A4). Furthermore, we found that the fibers were mechanically stable and can be applicable to hepatocyte transplantation. Three days after transplantation of the microfibers into the abdominal cavity of immunodeficient mice, human albumin was detected in the peripheral blood of the transplanted mice. These results indicate that the iPSC-hepatocyte fibers are promising either as in vitro models for drug screening or as implantation grafts to treat liver failure.


Assuntos
Técnicas de Cultura de Células/métodos , Encapsulamento de Células/métodos , Hepatócitos/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Cavidade Abdominal/cirurgia , Alginatos/química , Animais , Técnicas de Cultura de Células/instrumentação , Diferenciação Celular , Encapsulamento de Células/instrumentação , Terapia Baseada em Transplante de Células e Tecidos/métodos , Perfilação da Expressão Gênica , Hepatócitos/transplante , Humanos , Hidrogéis/química , Falência Hepática/terapia , Masculino , Camundongos , Esferoides Celulares
3.
J Vis Exp ; (160)2020 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-32597844

RESUMO

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. While isolating myocytes from neonatal mouse hearts is relatively straightforward, myocytes from the adult murine heart are preferred. This is because compared to neonatal cells, adult myocytes more accurately recapitulate cell function as it occurs in the adult heart in vivo. However, it is technically difficult to isolate adult mouse cardiac myocytes in the necessary quantities and viability, which contributes to an experimental impasse. Furthermore, published procedures are specific for the isolation of either atrial or ventricular myocytes at the expense of atrial and ventricular non-myocyte cells. Described here is a detailed method for isolating both atrial and ventricular cardiac myocytes, along with atrial and ventricular non-myocytes, simultaneously from a single mouse heart. Also provided are the details for optimal cell-specific culturing methods, which enhance cell viability and function. This protocol aims not only to expedite the process of adult murine cardiac cell isolation, but also to increase the yield and viability of cells for investigations of atrial and ventricular cardiac cells.


Assuntos
Técnicas de Cultura de Células/métodos , Separação Celular/métodos , Átrios do Coração/citologia , Ventrículos do Coração/citologia , Miócitos Cardíacos/citologia , Envelhecimento , Animais , Técnicas de Cultura de Células/instrumentação , Sobrevivência Celular , Células Cultivadas , Camundongos
4.
J Vis Exp ; (159)2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32449725

RESUMO

Motor neurons (MNs) are highly polarized cells with very long axons. Axonal transport is a crucial mechanism for MN health, contributing to neuronal growth, development, and survival. We describe a detailed method for the use of microfluidic chambers (MFCs) for tracking axonal transport of fluorescently labeled organelles in MN axons. This method is rapid, relatively inexpensive, and allows for the monitoring of intracellular cues in space and time. We describe a step by step protocol for: 1) Fabrication of polydimethylsiloxane (PDMS) MFCs; 2) Plating of ventral spinal cord explants and MN dissociated culture in MFCs; 3) Labeling of mitochondria and acidic compartments followed by live confocal imagining; 4) Manual and semiautomated axonal transport analysis. Lastly, we demonstrate a difference in the transport of mitochondria and acidic compartments of HB9::GFP ventral spinal cord explant axons as a proof of the system validity. Altogether, this protocol provides an efficient tool for studying the axonal transport of various axonal components, as well as a simplified manual for MFC usage to help discover spatial experimental possibilities.


Assuntos
Transporte Axonal , Técnicas de Cultura de Células/instrumentação , Dispositivos Lab-On-A-Chip , Neurônios Motores/citologia , Organelas/metabolismo , Animais , Dimetilpolisiloxanos , Mitocôndrias/metabolismo , Medula Espinal/citologia
5.
PLoS One ; 15(5): e0232081, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32374763

RESUMO

The reproduction of reliable in vitro models of human skeletal muscle is made harder by the intrinsic 3D structural complexity of this tissue. Here we coupled engineered hydrogel with 3D structural cues and specific mechanical properties to derive human 3D muscle constructs ("myobundles") at the scale of single fibers, by using primary myoblasts or myoblasts derived from embryonic stem cells. To this aim, cell culture was performed in confined, laminin-coated micrometric channels obtained inside a 3D hydrogel characterized by the optimal stiffness for skeletal muscle myogenesis. Primary myoblasts cultured in our 3D culture system were able to undergo myotube differentiation and maturation, as demonstrated by the proper expression and localization of key components of the sarcomere and sarcolemma. Such approach allowed the generation of human myobundles of ~10 mm in length and ~120 µm in diameter, showing spontaneous contraction 7 days after cell seeding. Transcriptome analyses showed higher similarity between 3D myobundles and skeletal signature, compared to that found between 2D myotubes and skeletal muscle, mainly resulting from expression in 3D myobundles of categories of genes involved in skeletal muscle maturation, including extracellular matrix organization. Moreover, imaging analyses confirmed that structured 3D culture system was conducive to differentiation/maturation also when using myoblasts derived from embryonic stem cells. In conclusion, our structured 3D model is a promising tool for modelling human skeletal muscle in healthy and diseases conditions.


Assuntos
Técnicas de Cultura de Células , Fibras Musculares Esqueléticas/citologia , Músculo Esquelético/citologia , Engenharia Tecidual , Tecidos Suporte/química , Animais , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Células Cultivadas , Dimetilpolisiloxanos/química , Humanos , Hidrogéis/química , Teste de Materiais , Camundongos , Modelos Biológicos , Conformação Molecular , Desenvolvimento Muscular , Músculo Esquelético/fisiologia , Mioblastos/citologia , Mioblastos/fisiologia , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos , Engenharia Tecidual/instrumentação , Engenharia Tecidual/métodos
6.
Rev Sci Instrum ; 91(1): 014101, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-32012597

RESUMO

Gravity is the only constant stimulus during the evolution of life. To investigate the impact of the absence of gravity on living systems, their molecular and morphological status has to be studied under microgravity conditions. The experiment unit CellFix was developed in order to provide the possibility of exposure and chemical fixation of small biological systems, such as neurons, stem cells, small animals, yeast cultures, plants, etc., at dedicated time points during a sounding rocket flight. The current version of CellFix consists of two culture bags containing cell cultures in a temperature-controlled pressure vessel. The biosystems in the culture bags can be fixed by pumping the fixative [e.g., paraformaldehyde (PFA), methanol, RNAlater, or others] from a connected bag into the cell suspension. The mechatronic basis of the experiment unit is constructed from compartments of the shelf parts. Open source microcontroller systems (Arduino) or gear pumps, accumulators, etc., from the model making sector are affordable and reliable components to build up an experiment on an unmanned space mission such as a sounding rocket flight. Also, new technologies such as fused deposition modeling were used to construct structures and brackets, which were tested successfully in environmental tests and real space flights (MAPHEUS 7 and 8 sounding rocket missions). In combination with the possibility to handle the experiment as a late access insert in a standardized rocket compartment, CellFix provides a multiusable experiment unit for performing life science experiments in space.


Assuntos
Astrócitos , Técnicas de Cultura de Células , Análise de Célula Única , Ausência de Peso , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Camundongos , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos
7.
Methods Mol Biol ; 2102: 531-555, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31989575

RESUMO

Endoplasmic reticulum (ER) stress is one of the major mechanisms underlying the etiology of multiple diseases and drug-induced toxicity. Gaussia luciferase (Gluc) is a naturally secreted protein that has been used as a reporter for the secretory pathway of ER to enable efficient and real-time monitoring of the ER function. The Gluc assay has been widely used and optimized in various labs. In this chapter, we provide an example of the application of the Gluc assay by establishing a stable cell line expressing both Gluc and firefly luciferase (Fluc) to study ER stress in liver cells. We describe the detailed procedures used in our laboratory for Gluc- and Fluc-containing lentivirus production and titration, for establishing a HepG2-based stable cell line through lentivirus transduction and the validation process. In addition, we provide an example of using the established stable cell line to investigate ER stress.


Assuntos
Técnicas de Cultura de Células/métodos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Hepatócitos/metabolismo , Luciferases de Vaga-Lume/metabolismo , Luciferases/metabolismo , Animais , Técnicas de Cultura de Células/instrumentação , Copépodes , Retículo Endoplasmático/química , Retículo Endoplasmático/efeitos dos fármacos , Genes Reporter , Células Hep G2 , Hepatócitos/química , Humanos , Lentivirus/genética , Luciferases/química , Luciferases/genética , Luciferases de Vaga-Lume/química , Luciferases de Vaga-Lume/genética , Fluxo de Trabalho
8.
Nat Biomed Eng ; 4(4): 407-420, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31988458

RESUMO

Organ chips can recapitulate organ-level (patho)physiology, yet pharmacokinetic and pharmacodynamic analyses require multi-organ systems linked by vascular perfusion. Here, we describe an 'interrogator' that employs liquid-handling robotics, custom software and an integrated mobile microscope for the automated culture, perfusion, medium addition, fluidic linking, sample collection and in situ microscopy imaging of up to ten organ chips inside a standard tissue-culture incubator. The robotic interrogator maintained the viability and organ-specific functions of eight vascularized, two-channel organ chips (intestine, liver, kidney, heart, lung, skin, blood-brain barrier and brain) for 3 weeks in culture when intermittently fluidically coupled via a common blood substitute through their reservoirs of medium and endothelium-lined vascular channels. We used the robotic interrogator and a physiological multicompartmental reduced-order model of the experimental system to quantitatively predict the distribution of an inulin tracer perfused through the multi-organ human-body-on-chips. The automated culture system enables the imaging of cells in the organ chips and the repeated sampling of both the vascular and interstitial compartments without compromising fluidic coupling.


Assuntos
Técnicas de Cultura de Células/métodos , Dispositivos Lab-On-A-Chip , Microfluídica/métodos , Robótica/métodos , Barreira Hematoencefálica , Encéfalo , Calibragem , Técnicas de Cultura de Células/instrumentação , Desenho de Equipamento , Coração , Humanos , Intestinos , Rim , Fígado , Pulmão , Robótica/instrumentação , Pele
9.
PLoS One ; 15(1): e0227553, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31923210

RESUMO

INTRODUCTION: Articular cartilage (AC) is a viscoelastic tissue with a limited regenerative capability because of the lack of vasculature. Mechanical stimulation contributes to the homeostasis of functional AC since it promotes the delivery of nutrients, cytokines and growth factors between the distant chondrocytes. We hypothesized that biomechanical stimulation might enhance mobilization of endogenous mesenchymal stem/stromal cells (MSCs) from neighboring niches as the bone marrow. AIM: This study aimed to introduce a bioreactor for inducing mobilization of MSCs from one compartment to another above by mechanical stimulation in vitro. METHODS: A novel mechanical system for evaluating mobilization of cells in a 3D context in vitro is presented. The system consists of a compression bioreactor able to induce loading on hydrogel-based scaffolds, custom-made software for settings management and data recording, and image based biological evaluation. Intermittent load was applied under a periodic regime with frequency of 0.3 Hz and unload phases of 10 seconds each 180 cycles over 24 hours. The mechanical stimulation acted on an alginate scaffold and a cell reservoir containing MSCs below it. The dynamic compression exerted amplitude of 200 µm as 10% strain regarding the original height of the scaffold. RESULTS: The bioreactor was able to stimulate the scaffolds and the cells for 24.4 (±1.7) hours, exerting compression with vertical displacements of 185.8 (±17.8) µm and a force-amplitude of 1.87 (±1.37; min 0.31, max 4.42) N. Our results suggest that continuous mechanical stimulation hampered the viability of the cells located at the cell reservoir when comparing to intermittent mechanical stimulation (34.4 ± 2.0% vs. 66.8 ± 5.9%, respectively). Functionalizing alginate scaffolds with laminin-521 (LN521) seemed to enhance the mobilization of cells from 48 (±21) to 194 (±39) cells/mm3 after applying intermittent mechanical loading. CONCLUSION: The bioreactor presented here was able to provide mechanical stimulation that seemed to induce the mobilization of MSCs into LN521-alginate scaffolds under an intermittent loading regime.


Assuntos
Reatores Biológicos , Técnicas de Cultura de Células/métodos , Tecidos Suporte/química , Alginatos/química , Animais , Células da Medula Óssea/citologia , Técnicas de Cultura de Células/instrumentação , Sobrevivência Celular , Células Cultivadas , Força Compressiva , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Estresse Mecânico , Suínos
10.
Nat Biomed Eng ; 4(4): 437-445, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31611679

RESUMO

Implanted bioengineered livers have not exceeded three days of continuous perfusion. Here we show that decellularized whole porcine livers revascularized with human umbilical vein endothelial cells and implanted heterotopically into immunosuppressed pigs whose spleens had been removed can sustain perfusion for up to 15 days. We identified peak glucose consumption rate as a main predictor of the patency of the revascularized bioengineered livers (rBELs). Heterotopic implantation of rBELs into pigs in the absence of anticoagulation therapy led to sustained perfusion for three days, followed by a pronounced immune responses directed against the human endothelial cells. A 10 day steroid-based immunosuppression protocol and a splenectomy at the time of rBEL implantation reduced the immune responses and resulted in continuous perfusion of the rBELs for over two weeks. We also show that the human endothelial cells in the perfused rBELs colonize the liver sinusoids and express sinusoidal endothelial markers similar to those in normal liver tissue. Revascularized liver scaffolds that can maintain blood perfusion at physiological pressures might eventually help to overcome the chronic shortage of transplantable human livers.


Assuntos
Engenharia Biomédica/métodos , Transplante de Fígado/métodos , Perfusão/métodos , Transplante Heterotópico/métodos , Animais , Reatores Biológicos , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Células Endoteliais , Glucose , Humanos , Imunossupressão , Cinética , Fígado/imunologia , Perfusão/instrumentação , Baço , Suínos , Tecidos Suporte , Grau de Desobstrução Vascular
11.
J Cell Physiol ; 235(2): 1155-1164, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31250436

RESUMO

Blood transfusion or blood products, such as plasma, have a long history in improving health, but today, platelet-rich plasma (PRP) is used in various medical areas such as surgery, orthopedics, and rheumatology in many ways. Considering the high efficiency of tissue engineering in repairing bone defects, in this study, we investigated the combined effect of nanofibrous scaffolds in combination with PRP on the osteogenic differentiation potential of human induced pluripotent stem cells (iPSCs). Electrospinning was used for fabricating nanofibrous scaffolds by polyvinylidene fluoride/collagen (PVDF/col) with and without PRP. After scaffold characterization, the osteoinductivity of the fabricated scaffolds was studied by culturing human iPSCs under osteogenic medium. The results showed that PRP has a considerable positive effect on the biocompatibility of the PVDF/col nanofibrous scaffold when examined by protein adsorption, cell attachment, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. In addition, the results obtained from alkaline phosphatase activity and calcium content assays demonstrated that nanofibers have higher osteoinductivity while grown on PRP-incorporated PVDF/col nanofibers. These results were also confirmed while the osteogenic differentiation of the iPSCs was more investigated by evaluating the most important bone-related genes expression level. According to the results, it can be concluded that PVDF/col/PRP has much more osteoinductivity while compared with the PVDF/col, and it can be introduced as a promising bone bio-implant for use in bone tissue engineering applications.


Assuntos
Técnicas de Cultura de Células/instrumentação , Colágeno/química , Células-Tronco Pluripotentes Induzidas/fisiologia , Nanofibras , Plasma Rico em Plaquetas/química , Polivinil/química , Adesão Celular , Humanos , Microscopia de Força Atômica
12.
Mater Sci Eng C Mater Biol Appl ; 107: 110218, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31761204

RESUMO

In regenerative medicine, self-regulated tissue regeneration is perceived by Mesenchymal Stem Cells (MSCs) fate due to their tissue-specific differentiation, which is an emerging yet promising tool for therapeutics. MSCs with their innate nature like secretion of bioactive molecules, multilineage differentiation and proliferation supported tissue repair. MSCs interact with extracellular matrix (ECM) components like collagen, glycosaminoglycans (GAGs), proteoglycans and various proteins that are present in the form of nanofibers representing variable matrix elasticity along with topographies and bioactive cues. Synthetic nanofibers also showed to mimic native tissue microenvironment and supported regeneration owing to structural resemblance with ECM for anchorage-dependent cells. Different nanofibers generated using various polymer precursors and their resultant scaffolds, architectures, compositions etc. were studied for their influence on MSCs activities to improvise cell-cell and cell-material interactions. Electrospinning, popular nanotechnology for fiber formation based on electrohydrodynamic theory, is widely used for many applications due to its simplicity, efficacy and environmentally friendliness. Electrospun nanofibers were extensively investigated to understand the influence of material towards manipulating stem cells based on regenerative medicine. Subsequently, the influence of different solutions and process parameters were studied for nanofiber structure repeatability and emphasized on fiber properties such as diameter, mechanical properties, degradation rate, and porosity. Recent approaches towards scale-up for nanofiber production by electrospinning and other novel techniques are also presented briefly. The fate of MSCs, while seeded on nanofibers under external stimuli viz. electrical, mechanical, magnetic and electromagnetic field, is reviewed to find the niche for differentiation pathways. Further, several external stimuli presented as important factors motivating cellular differentiation in combination with specific conditions without the use of any chemical cues.


Assuntos
Células-Tronco Mesenquimais/citologia , Nanofibras/química , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Módulo de Elasticidade , Matriz Extracelular/química , Humanos , Células-Tronco Mesenquimais/metabolismo , Polímeros/química , Medicina Regenerativa , Engenharia Tecidual , Tecidos Suporte/química
13.
Mater Sci Eng C Mater Biol Appl ; 107: 110312, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31761174

RESUMO

The versatile properties of graphene-based materials are enabling various tissue regeneration, towards meeting an ever increasing demand for replacement tissues due to injury through trauma and disease. In particular, an innate ability for graphene to promote osteogenic differentiation of stem cells, combined with the potential to enhance the biological activity of cells through electrical stimulation (ES) using graphene, supports its use for osteoinduction or reconstruction. In this paper, we describe a miniaturized graphene-cellulose (G-C) scaffold-based device that incorporates electroactive G-C 'paper' within a polystyrene chamber for concomitant cell culture and ES. The G-C electrodes possessed lower impedance and higher charge injection capacity than gold (Au) electrodes, with high stability. By coupling ES with previously reported properties of the G-C scaffolds, we have advanced the platform for improved adipose derived stem cell (ADSC) support and osteogenic differentiation. We anticipate using the G-C scaffold-based ES device for in vitro modelling of osteogenic induction, bone tissue engineering and in vivo bone regeneration towards new therapeutic strategies for bone injury and disease. Furthermore, the device could reasonably be used for ES and culture of other cell types and engineering other tissues.


Assuntos
Tecido Adiposo/citologia , Estimulação Elétrica/instrumentação , Osteogênese/efeitos da radiação , Células-Tronco , Tecidos Suporte/química , Técnicas de Cultura de Células/instrumentação , Células Cultivadas , Celulose/química , Desenho de Equipamento , Grafite/química , Humanos , Células-Tronco/citologia , Células-Tronco/metabolismo , Células-Tronco/efeitos da radiação
14.
Mater Sci Eng C Mater Biol Appl ; 106: 110259, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31753381

RESUMO

Polymeric hydrogel-based 3D scaffolds are well-known structures, being used for cultivation and differentiation of stem cells. However, scalable systems that provide a native-like microenvironment with suitable biological and physical properties are still needed. Incorporation of nanomaterials into the polymeric systems is expected to influence the physical properties of the structure but also the stem cells fate. Here, alginate/gelatin hydrogel beads incorporated with mesoporous silica nanoparticles (MSNs) (average diameter 80.9 ±â€¯10 nm) and various surface chemistries were prepared. Human adipose-derived mesenchymal stem cells (hASCs) were subsequently encapsulated into the alginate/gelatin/silica hydrogels. Incorporation of amine- and carboxyl-functionalized MSNs (A-MSNs and C-MSNs) significantly enhances the stability of the hydrogel beads. In addition, the expression levels of Nanog and OCT4 imply that the incorporation of A-MSNs into the alginate/gelatin beads significantly improves the proliferation and the stemness of encapsulated hASCs. Importantly, our findings show that the presence of A-MSNs slightly suppresses in vivo inflammation. In contrast, the results of marker gene expression analyses indicate that cultivation of hASCs in alginate beads incorporated with C-MSNs (10% w/w) leads to a heterogeneously differentiated population of the cells, i.e., osteocytes, chondrocytes, and adipocytes, which is not appropriate for both cell culture and differentiation applications.


Assuntos
Técnicas de Cultura de Células/métodos , Hidrogéis/química , Nanopartículas/química , Dióxido de Silício/química , Tecido Adiposo/citologia , Alginatos/química , Animais , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Técnicas de Cultura de Células/instrumentação , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Gelatina/química , Humanos , Hidrogéis/farmacologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Proteína Homeobox Nanog/metabolismo , Fator 3 de Transcrição de Octâmero/metabolismo , Porosidade , Ratos , Ratos Wistar , Tecidos Suporte/química
15.
Mater Sci Eng C Mater Biol Appl ; 106: 110286, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31753397

RESUMO

Autologous chondrocyte implantation (ACI) is a promising approach to repair cartilage defects; however, the cartilage trauma-induced inflammatory environment compromises its clinical outcomes. Cell-derived decellularized extracellular matrix (DECM) has been used as a culture substrate for mesenchymal stem cells (MSCs) to improve the cell proliferation and lineage-specific differentiation. In this study, DECM deposited by synovium-derived MSCs was used as an in vitro expansion system for rabbit articular chondrocytes and the response of DECM-expanded chondrocytes to pro-inflammatory cytokines such as interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) was evaluated. Compared with those grown on tissue culture polystyrene (TCPS), the proliferation rate was significantly improved in DECM-expanded chondrocytes. TCPS- and DECM-expanded chondrocytes were isolated and induced to redifferentiation in a high-density pellet culture. DECM-expanded chondrocytes exerted a stronger resistance to 1 ng/mL of IL-1ß than TCPS-expanded cells, but the production of cartilage matrix in both groups was inhibited by 5 ng/mL of IL-1ß. When exposed to 1 or 5 ng/mL of TNF-α, DECM-expanded chondrocytes showed higher levels of cartilage matrix synthesis than TCPS-expanded cells. In addition, the gene expression of IL-1ß- or TNF-α-induced matrix degrading enzymes (MMP3, MMP9, MMP13, and ADAMTS5) was significantly lower in DECM-expanded chondrocytes than TCPS-expanded cells. Furthermore, we found that SIRT1 inhibition by nicotinamide completely counteracted the protective effect of DECM on chondrocytes in the presence of IL-1ß or TNF-α, indicating that the SIRT1 signaling pathway was involved in the DECM-mediated enhancement of anti-inflammatory properties of chondrocytes. Taken together, this work suggests that stem cell-derived DECM is a superior culture substrate for in vitro chondrocyte expansion by improving proliferation and enhancing the anti-inflammatory properties of chondrocytes. DECM-expanded chondrocytes with enhanced anti-inflammatory properties hold great potential in clinically ACI-based cartilage repair.


Assuntos
Condrócitos/metabolismo , Matriz Extracelular/metabolismo , Células-Tronco Mesenquimais/metabolismo , Sirtuína 1/metabolismo , Agrecanas/metabolismo , Animais , Cartilagem Articular/citologia , Técnicas de Cultura de Células/instrumentação , Técnicas de Cultura de Células/métodos , Proliferação de Células/efeitos dos fármacos , Condrócitos/citologia , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/farmacologia , Regulação para Baixo/efeitos dos fármacos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-1beta/farmacologia , Células-Tronco Mesenquimais/citologia , Coelhos , Fatores de Transcrição SOX9/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sirtuína 1/genética , Membrana Sinovial/citologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologia
16.
Int J Nanomedicine ; 14: 9295-9306, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31819431

RESUMO

Background: The advent of 3D printing technology allowed the realization of custom devices that can be used not only in the everyday life but also in the nanotechnology and biomedical fields. In nanotechnology, the use of bi-dimensional nanostructures based on carbon nanotubes, generally referred as buckypapers, have received considerable attention for their versatility and potential application in many biomedical fields. Unfortunately, buckypapers are extremely hydrophobic and cannot be used in aqueous media to culture cells. Methods: A polymeric device able to accommodate buckypapers and facilitate cell growth was fabricated by using 3D printing technology. We imparted hydrophilicity to buckypapers by coating them with polyamidoamine (PAMAM) dendrimers. Results: We found that by using novel techniques such as polymer coating the buckypaper hydrophilicity increased, whereas the use of 3D printing technology allowed us to obtain custom devices that have been used to culture cells on buckypapers for many days. We characterized in details the morphology of these structures and studied for the first time the kinetic of cell proliferation. We found that these scaffolds, if properly functionalized, are suitable materials to grow cells for long time and potentially employable in the biomedical field. Conclusion: Although these materials are cytotoxic under certain circumstances, we have found a suitable coating and specific experimental conditions that encourage using buckypapers as novel scaffolds for cell growth and for potential applications in tissue repair and regeneration.


Assuntos
Tecnologia Biomédica/métodos , Técnicas de Cultura de Células/instrumentação , Dendrímeros/química , Nanotubos de Carbono/química , Impressão Tridimensional/instrumentação , Linhagem Celular , Proliferação de Células , Humanos , MicroRNAs/metabolismo , Nanotubos de Carbono/ultraestrutura , Propriedades de Superfície
17.
PLoS One ; 14(11): e0224635, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31693673

RESUMO

Contamination control and removal are very important technical aspects of microbiological research. Bacterial contamination is very common in fungal cultures. Currently, the commonly used approach for inhibiting bacteria is antibiotic treatment; however, there are drawbacks to using antibiotics, including incomplete removal, limited antibacterial spectra, tendency toward recontamination, effects to fungal strains, and potential risks to the environment. Therefore, in the present work, we developed a new method for bacterial removal from fungi cultured on solid medium, the Cabin-Sequestering (CS) method, based on the different culture characteristics between fungi and bacteria. First, 3-5 mm round or square holes (the "cabin") are excavated on a solid medium plate. The fungal strain containing possible bacterial contamination is inoculated into the cabin. The cabin is then covered with a sterilized coverslip, followed by incubation at the appropriate temperature. After 7-10 days of culturing, fungal hyphae grow out along the edge of the coverslip; however, the contaminating bacteria cannot pass through the space formed between the medium and the coverslip and, thus, remain in the cabin. The newly grown fungal hyphae around the coverslip are re-inoculated into fresh culture plates, where they form bacteria-free fungal colonies. The CS method is easy handling, with a short experimental cycle and rare recontamination. When necessary, it can also be used in combination with antibiotics in bacterial removal operations.


Assuntos
Bactérias , Técnicas de Cultura de Células/métodos , Fungos , Técnicas Microbiológicas/métodos , Técnicas de Cultura de Células/instrumentação , Meios de Cultura , Estudos de Viabilidade , Hifas , Técnicas Microbiológicas/instrumentação
18.
Biomed Microdevices ; 21(4): 94, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31686216

RESUMO

Nowadays, cancer disease is continuously identified as the leading cause of mortality worldwide. Cancer chemotherapeutic agents have been continuously developing to achieve high curative effectiveness and low side effects. However, solid tumors present the properties of low drug penetration and resistance of quiescent cells. Radiation therapy is concurrently given in some cases; but it induces different levels of adverse effects. In the current work, uniform sized multicellular spheroids were raised by microwell arrays to mimic the architecture of solid tumors. Investigation of the response of the spheroids was conducted after the treatment of alternating electric field. The result showed that the electric field could induce early apoptosis by disturbing cell membrane. Moreover, combined treatment of electric field and anti-cancer drug was applied to the spheroids. The electric field synergistically enhanced the treatment efficacy because the anti-cancer drug could permeate through the disrupted cell membrane. Significant improvement of late apoptosis was shown by the combined treatment. Because the electric field treatment induces limited side effect to the patient, lower dosage of anti-cancer drug may be applied to the patients for achieving curative effectiveness.


Assuntos
Antineoplásicos/farmacologia , Técnicas de Cultura de Células/instrumentação , Eletricidade , Esferoides Celulares/efeitos dos fármacos , Análise Serial de Tecidos/instrumentação , Linhagem Celular Tumoral , Terapia Combinada , Relação Dose-Resposta a Droga , Doxorrubicina/farmacologia , Células HeLa , Humanos , Esferoides Celulares/patologia
19.
Nat Commun ; 10(1): 4824, 2019 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-31645567

RESUMO

Industrial applications of anchorage-dependent cells require large-scale cell culture with multifunctional monitoring of culture conditions and control of cell behaviour. Here, we introduce a large-scale, integrated, and smart cell-culture platform (LISCCP) that facilitates digital mass culture of anchorage-dependent cells. LISCCP is devised through large-scale integration of ultrathin sensors and stimulator arrays in multiple layers. LISCCP provides real-time, 3D, and multimodal monitoring and localized control of the cultured cells, which thereby allows minimizing operation labour and maximizing cell culture performance. Wireless integration of multiple LISCCPs across multiple incubators further amplifies the culture scale and enables digital monitoring and local control of numerous culture layers, making the large-scale culture more efficient. Thus, LISCCP can transform conventional labour-intensive and high-cost cell cultures into efficient digital mass cell cultures. This platform could be useful for industrial applications of cell cultures such as in vitro toxicity testing of drugs and cosmetics and clinical scale production of cells for cell therapy.


Assuntos
Técnicas de Cultura de Células/métodos , Dispositivos Lab-On-A-Chip , Animais , Engenharia Biomédica , Técnicas de Cultura de Células/instrumentação , Fibroblastos , Humanos , Células-Tronco Mesenquimais , Camundongos , Mioblastos , Miócitos Cardíacos , Tecnologia sem Fio
20.
Nat Commun ; 10(1): 4650, 2019 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-31604956

RESUMO

Gluconacetobacter xylinus (G. xylinus) metabolism is activated by oxygen, which makes the formation of an air-medium interface critical. Here we report solid matrix-assisted 3D printing (SMAP) of an incubation medium surface and the 3D fabrication of bacterial cellulose (BC) hydrogels by in situ biosynthesis of G. xylinus. A printing matrix of polytetrafluoroethylene (PTFE) microparticles and a hydrogel ink containing an incubation medium, bacteria, and cellulose nanofibers (CNFs) are used in the SMAP process. The hydrogel ink can be printed in the solid matrix with control over the topology and dimensional stability. Furthermore, bioactive bacteria produce BC hydrogels at the surface of the medium due to the permeability of oxygen through the PTFE microparticle layer. The flexibility of the design is verified by fabricating complex 3D structures that were not reported previously. The resulting tubular BC structures suggest future biomedical applications, such as artificial blood vessels and engineered vascular tissue scaffolding. The fabrication of a versatile free-form structure of BC has been challenged due to restricted oxygen supplies at the medium and the dimensional instability of hydrogel printing. SMAP is a solution to the problem of fabricating free-form biopolymer structures, providing both printability and design diversity.


Assuntos
Gluconacetobacter xylinus/fisiologia , Engenharia Tecidual/métodos , Técnicas de Cultura de Células/instrumentação , Celulose , Meios de Cultura , Gluconacetobacter xylinus/crescimento & desenvolvimento , Gluconacetobacter xylinus/metabolismo , Hidrogéis/química , Nanofibras , Oxigênio , Impressão Tridimensional , Tecidos Suporte/química
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