RESUMO
With recent advances in biotechnology, mammalian cells are used in biopharmaceutical industries to produce valuable protein therapeutics and investigated as effective therapeutic agents to permanently degenerative diseases in cell based therapy. In these exciting and actively expanding fields, a reliable, efficient, and affordable platform to culture mammalian cells on a large scale is one of the most vital necessities. To produce and maintain a very large population of anchorage-dependent cells, a microcarrier-based stirred tank bioreactor is commonly used. In this approach, the cells are exposed to harmful hydrodynamic shear stress in the bioreactor and the mass transfer rates of nutrients and gases in the bioreactor are often kept below an optimal level to prevent cellular damages from the shear stress. In this paper, a hollow microcarrier (HMC) is presented as a novel solution to protect cells from shear stress in stirred bioreactors, while ensuring sufficient and uniform mass transfer rate of gases and nutrients. HMC is a hollow microsphere and cells are cultured on its inner surface to be protected, while openings on the HMC provide sufficient exchange of media inside the HMC. As a proof of concept, we demonstrated the expansion of fibroblasts, NIH/3T3 and the expansion and cardiac differentiation of human induced pluripotent stem cells, along with detailed numerical analysis. We believe that the developed HMC can be a practical solution to enable large-scale expansion of shear-sensitive anchorage-dependent cells in an industrial scale with stirred bioreactors.
Assuntos
Reatores Biológicos , Adesão Celular , Técnicas de Cultura de Células/métodos , Células Imobilizadas/fisiologia , Microesferas , Animais , Biotecnologia/métodos , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Camundongos , Células NIH 3T3/fisiologia , Tecnologia Farmacêutica/métodosRESUMO
BACKGROUND: Tissue injury triggers inflammatory responses that promote tissue fibrosis; however, the mechanisms that couple tissue injury, inflammation, and fibroblast activation are not known. Given that dying cells release proinflammatory "damage-associated molecular patterns" (DAMPs), we asked whether proteins released by necrotic myocardial cells (NMCs) were sufficient to activate fibroblasts in vitro by examining fibroblast activation after stimulation with proteins released by necrotic myocardial tissue, as well as in vivo by injecting proteins released by necrotic myocardial tissue into the hearts of mice and determining the extent of myocardial inflammation and fibrosis at 72 hours. METHODS AND RESULTS: The freeze-thaw technique was used to induce myocardial necrosis in freshly excised mouse hearts. Supernatants from NMCs contained multiple DAMPs, including high mobility group box-1 (HMGB1), galectin-3, S100ß, S100A8, S100A9, and interleukin-1α. NMCs provoked a significant increase in fibroblast proliferation, α-smooth muscle actin activation, and collagen 1A1 and 3A1 mRNA expression and significantly increased fibroblast motility in a cell-wounding assay in a Toll-like receptor 4 (TLR4)- and receptor for advanced glycation end products-dependent manner. NMC stimulation resulted in a significant 3- to 4-fold activation of Akt and Erk, whereas pretreatment with Akt (A6730) and Erk (U0126) inhibitors decreased NMC-induced fibroblast proliferation dose-dependently. The effects of NMCs on cell proliferation and collagen gene expression were mimicked by several recombinant DAMPs, including HMGB1 and galectin-3. Moreover, immunodepletion of HMGB1 in NMC supernatants abrogated NMC-induced cell proliferation. Finally, injection of NMC supernatants or recombinant HMGB1 into the heart provoked increased myocardial inflammation and fibrosis in wild-type mice but not in TLR4-deficient mice. CONCLUSIONS: These studies constitute the initial demonstration that DAMPs released by NMCs induce fibroblast activation in vitro, as well as myocardial inflammation and fibrosis in vivo, at least in part, through TLR4-dependent signaling.
Assuntos
Fibroblastos/fisiologia , Miocardite/fisiopatologia , Miocárdio/citologia , Actinas/metabolismo , Animais , Western Blotting , Proliferação de Células/fisiologia , Colágeno/metabolismo , Expressão Gênica/fisiologia , Técnicas In Vitro , Fígado/citologia , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Miocárdio/patologia , Células NIH 3T3/fisiologia , Necrose/fisiopatologiaRESUMO
Radiation induced fibrosis of the skin is a late toxicity that may result in loss of function due to reduced range of motion and pain. The current study sought to determine if oral delivery of quercetin mitigates radiation-induced cutaneous injury. Female C3H/HeN mice were fed control chow or quercetin-formulated chow (1% by weight). The right hind leg was exposed to 35 Gy of X rays and the mice were followed serially to assess acute toxicity and hind leg extension. Tissue samples were collected for assessment of soluble collagen and tissue cytokines. Human and murine fibroblasts were subjected to clonogenic assays to determine the effects of quercetin on radiation response. Contractility of fibroblasts was assessed with a collagen contraction assay in the presence or absence of quercetin and transforming growth factor-ß (TGF-ß). Western blotting of proteins involved in fibroblast contractility and TGF-ß signaling were performed. Quercetin treatment significantly reduced hind limb contracture, collagen accumulation and expression of TGF-ß in irradiated skin. Quercetin had no effect on the radioresponse of fibroblasts or murine tumors, but was capable of reducing the contractility of fibroblasts in response to TGF-ß, an effect that correlated with partial stabilization of phosphorylated cofilin. Quercetin is capable of mitigating radiation induced skin fibrosis and should be further explored as a therapy for radiation fibrosis.
Assuntos
Antioxidantes/uso terapêutico , Fitoterapia , Quercetina/uso terapêutico , Protetores contra Radiação/uso terapêutico , Radiodermite/prevenção & controle , Administração Oral , Animais , Antioxidantes/administração & dosagem , Antioxidantes/farmacocinética , Biotransformação , Carcinoma de Células Escamosas/radioterapia , Forma Celular , Colágeno/biossíntese , Colágeno/genética , Ensaio de Unidades Formadoras de Colônias , Avaliação Pré-Clínica de Medicamentos , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Fibroblastos/efeitos da radiação , Fibrose , Genes Reporter , Membro Posterior/efeitos da radiação , Humanos , Camundongos , Camundongos Endogâmicos C3H , Células NIH 3T3/efeitos dos fármacos , Células NIH 3T3/fisiologia , Células NIH 3T3/efeitos da radiação , Quercetina/administração & dosagem , Quercetina/farmacocinética , Protetores contra Radiação/administração & dosagem , Protetores contra Radiação/farmacocinética , Radiodermite/etiologia , Radioterapia/efeitos adversos , Distribuição Aleatória , Pele/efeitos dos fármacos , Pele/patologia , Pele/efeitos da radiação , Neoplasias Cutâneas/radioterapia , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/efeitos da radiação , Fator de Crescimento Transformador beta/biossíntese , Fator de Crescimento Transformador beta/genéticaRESUMO
Current fundamental investigations of human biology and the development of therapeutic drugs commonly rely on 2D monolayer cell culture systems. However, 2D cell culture systems do not accurately recapitulate the structure, function or physiology of living tissues, nor the highly complex and dynamic 3D environments in vivo. Microfluidic technology can provide microscale complex structures and well-controlled parameters to mimic the in vivo environment of cells. The combination of microfluidic technology with 3D cell culture offers great potential for in vivo-like tissue-based applications, such as the emerging organ-on-a-chip system. This article will review recent advances in the microfluidic technology for 3D cell culture and their biological applications.
Assuntos
Bioensaio/métodos , Técnicas de Cultura de Células/métodos , Microfluídica/métodos , Animais , Bioensaio/tendências , Técnicas de Cultura de Células/instrumentação , Linhagem Celular Tumoral/fisiologia , Proliferação de Células , Sobrevivência Celular , Hepatócitos/fisiologia , Humanos , Camundongos , Microfluídica/tendências , Miócitos Cardíacos/fisiologia , Células NIH 3T3/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Ratos , Células-Tronco/fisiologia , Engenharia Tecidual/métodos , Engenharia Tecidual/tendênciasRESUMO
Soluble eggshell membrane protein (SEP), isolated from natural eggshell membrane, was co-electrospun with biodegradable synthetic polymers poly(propylene carbonate) (PPC) and poly(lactic acid) (PLA) in various proportions from 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) solutions in order to prepare fibrous scaffolds having simultaneously good mechanical properties and biocompatibility. The fiber morphology was observed by field emission scanning electron microscopy, showing uniform fibers with diameter of 1.2-1.0 and 1.3-0.7 um for PPC/SEP and PLA/SEP blend fibers, respectively. Transmission electron microscopy observation shows that the blend fibers have domain-matrix phase morphology with fiber-like SEP domains in the PPC or PLA matrix, indicating the occurrence of phase separation, although interaction exists between PPC (or PLA) and SEP, as revealed by attenuated total reflectance Fourier transform infrared spectroscopy. The mechanical properties were evaluated by uniaxial tensile tests and showed that both the tensile strength and elongation at break increase with increasing incorporation of PPC (or PLA). The surface composition was investigated by X-ray photoelectron spectroscopy and SEP was found on the fiber surfaces, and as a result the surfaces of the fibrous scaffolds are superhydrophilic. NIH3T3 cell culture tests demonstrate that the PPC/SEP and PLA/SEP blend fibrous scaffolds have a much improved biocompatibility compared to pure PPC or PLA fibrous scaffolds.
Assuntos
Proteínas do Ovo/química , Ácido Láctico/química , Polímeros/química , Polipropilenos/química , Engenharia Tecidual/instrumentação , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Animais , Proteínas do Ovo/isolamento & purificação , Interações Hidrofóbicas e Hidrofílicas , Teste de Materiais , Camundongos , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Células NIH 3T3/citologia , Células NIH 3T3/fisiologia , Espectroscopia Fotoeletrônica , Poliésteres , Propanóis/química , Soluções , Espectroscopia de Infravermelho com Transformada de Fourier , Propriedades de Superfície , Resistência à TraçãoRESUMO
The spatial presentation of soluble growth factors, immobilized extracellular matrix molecules, as well as matrix rigidity, plays an important role in directed and guided cell migration. Synthetic hydrogel scaffolds offer the ability to systematically introduce gradients of these factors contributing to our understanding of how the 3D arrangement of biochemical and mechanical cues influence cell behavior. Using a novel photopolymerization technique, perfusion-based frontal photopolymerization (PBFP), we have engineered poly(ethylene glycol) diacrylate (PEGDA) hydrogel scaffolds with gradients of mechanical properties and immobilized biofunctionality. The controlled delivery of a buoyant photoinitiator, eosin Y, through a glass frit filter results in the formation and subsequent propagation of a polymer reaction front that is self-sustained and able to propagate through the monomeric mixture. Propagation of this front results in monomer depletion, leading to variations in cross-linking, as well as spatial gradients of elastic modulus and immobilized concentrations of the YRGDS cell adhesion ligand within PEGDA hydrogels. Furthermore, the magnitudes of the resulting gradients are controlled through alterations in polymerization conditions. Preliminary in vitro cell-culture studies demonstrate that the gradients generated stimulate directed 2D cell growth on the surface of PEGDA hydrogels. By day 14, fibroblast aggregates spread roughly twice as far in the direction parallel to the slope of the gradient as compared to the perpendicular direction. The presented technique has great potential in controlling gradients of mechanical properties and immobilized biofunctionality for directing and guiding 3D cell behavior within tissue-engineered scaffolds.
Assuntos
Hidrogéis/química , Hidrogéis/síntese química , Polietilenoglicóis/química , Polietilenoglicóis/síntese química , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Animais , Movimento Celular , Módulo de Elasticidade , Amarelo de Eosina-(YS)/química , Hidrogéis/efeitos da radiação , Lasers , Teste de Materiais , Camundongos , Células NIH 3T3/citologia , Células NIH 3T3/fisiologia , Processos Fotoquímicos , Polietilenoglicóis/efeitos da radiação , PolimerizaçãoRESUMO
Congenital diaphragmatic hernia (CDH) is a frequently occurring cause of neonatal respiratory distress and is associated with high mortality and long-term morbidity. Evidence from animal models suggests that CDH has its origins in the malformation of the pleuroperitoneal fold (PPF), a key structure in embryonic diaphragm formation. The aims of this study were to characterize the embryogenesis of the PPF in rats and humans, and to determine the potential mechanism that leads to abnormal PPF development in the nitrofen model of CDH. Analysis of rat embryos, and archived human embryo sections, allowed the timeframe of PPF formation to be determined for both species, thus delineating a critical period of diaphragm development in relation to CDH. Experiments on nitrofen-exposed NIH 3T3 cells in vitro led us to hypothesize that nitrofen might cause diaphragmatic hernia in vivo by two possible mechanisms: through decreased cell proliferation or by inducing apoptosis. Data from nitrofen-exposed rat embryos indicates that the primary mechanism of nitrofen teratogenesis in the PPF is through decreased cell proliferation. This study provides novel insight into the embryogenesis of the PPF in rats and humans, and it indicates that impaired cell proliferation might contribute to abnormal diaphragm development in the nitrofen model of CDH.
Assuntos
Anormalidades Induzidas por Medicamentos , Diafragma/anormalidades , Embrião de Mamíferos/anormalidades , Hérnia Diafragmática/embriologia , Praguicidas/toxicidade , Éteres Fenílicos/toxicidade , Animais , Apoptose/efeitos dos fármacos , Bromodesoxiuridina/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Diafragma/efeitos dos fármacos , Modelos Animais de Doenças , Embrião de Mamíferos/efeitos dos fármacos , Feminino , Hérnia Diafragmática/induzido quimicamente , Hérnias Diafragmáticas Congênitas , Humanos , Técnicas Imunoenzimáticas , Marcação In Situ das Extremidades Cortadas , Exposição Materna , Camundongos , Células NIH 3T3/efeitos dos fármacos , Células NIH 3T3/patologia , Células NIH 3T3/fisiologia , Peritônio/anormalidades , Peritônio/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
Most cells in our body form a single primary cilium when entering growth arrest. During the past decade, a number of studies have revealed a key role for primary cilia in coordinating a variety of signaling pathways that control important cellular and developmental processes. Consequently, significant effort has been directed toward the identification of genes involved in ciliary assembly and function. Many candidate ciliary genes and proteins have been identified using large-scale "omics" approaches, including proteomics, transcriptomics, and comparative genomics. Although such large-scale approaches can be extremely informative, additional validation of candidate ciliary genes using alternative "small-scale" approaches is often necessary. Here we describe a quantitative PCR-based method that can be used to screen groups of genes for those that are upregulated during growth arrest in cultured mouse NIH3T3 cells and those that might have cilia-related functions. We employed this method to specifically search for mouse kinesin-3 genes that are upregulated during growth arrest and identified three such genes (Kif13A, Kif13B, and Kif16A). In principle, however, the method can be extended to identify other genes or gene families that are upregulated during growth arrest.
Assuntos
Regulação da Expressão Gênica , Cinesinas/genética , Células NIH 3T3 , Reação em Cadeia da Polimerase/métodos , Isoformas de Proteínas/genética , Animais , Técnicas de Cultura de Células , Cinesinas/metabolismo , Camundongos , Células NIH 3T3/fisiologia , Reação em Cadeia da Polimerase/instrumentação , Isoformas de Proteínas/metabolismo , Regulação para CimaRESUMO
INTRODUCTION: In order to find a suitable carrier to deliver the product of gene transfection to improve the performance of bioengineered dermis, we used microencapsulation and gene transfection technology together for the first time and found that it was feasible. METHODS: We used a recombinant nerve growth factor (pcDNA3.1+/NGF) to modify NIH3T3 cells genetically. Control of NIH3T3-NGF cells were encapsulated within microspheres composed of alginate-poly-L-lysine-alginate and cultivated in-vitro. The concentration of NGF released from the microencapsulated NIH3T3-NGF cells was confirmed using ELISA assay. We co-cultivated microencapsulated NIH3T3-NGF cells, NIH3T3 cells (control) with human keratinocytes and fibroblasts, and tested the percentage of cycle of these cells. The alkaline hydrolysis method was used to analyse the content of hydroxyproline (Hyp). Immunohistochemistry method was used to calculate the transformation efficiency from fibroblasts to myofibroblasts. RESULTS: The concentration of NGF released from the microencapsulated NIH3T3-NGF cells lasted about six weeks in the supernatant of bioengineered dermis in-vitro. The proliferation of keratinocytes, as well as the concentration of Hyp in supernatant of fibroblasts, were promoted about three times. Transformation efficiency from fibroblasts to myofibroblasts was increased approximately two-fold because of the bio-effects of NGF. Two kinds of microencapsulations were seeded into collagen which contained human fibroblasts to form bioengineered skin. Microencapsulated NIH3T3-NGF cells formed a thicker dermis. The concentration of Hyp in the bioengineered skin which indicated the level of collagen synthesis was increased due to existing NGF. CONCLUSION: Microencapsulated NIH3T3-NGF cells can be used to enhance performance of bioengineered dermis and it also can be deduced that other cytokines can be used to treat local wound areas.
Assuntos
Núcleo Celular/genética , Composição de Medicamentos , Regulação da Expressão Gênica/genética , Células NIH 3T3/fisiologia , Fator de Crescimento Neural/genética , Receptores de Fator de Crescimento Neural/genética , Regeneração/genética , Fenômenos Fisiológicos da Pele , Engenharia Tecidual/métodos , Transfecção/métodos , Alginatos , Animais , Estudos de Viabilidade , Fibroblastos , Proteínas de Homeodomínio , Humanos , Camundongos , Polilisina/análogos & derivadosRESUMO
Based on several previous studies indicating that transfection of genomic DNA can stably alter the character of the cells that take up the exogenous DNA, we investigated antitumor immunity conferred by fusions of syngeneic dendritic cells (DCs) and allogeneic fibroblasts (NIH3T3) transfected with genomic DNA from B16 tumor cells. Fusion cells (FCs) composed of dendritic and genetically engineered NIH3T3 cells were prepared with polyethylene glycol, and fusion efficiency was 30.3%. Prior immunization with FCs prevented tumor formation upon challenge with B16 tumor cells. Efficacy was reduced when studies were performed in mice depleted of NK cells. Vaccination with FCs containing DCs and fibroblasts transfected with denatured DNA did not inhibit tumor growth. Cytotoxic T cell (CTL) activity of spleen cells from immunized mice against both Yac-1 and tumor cells was also stimulated by administration of FCs compared with the activity observed for cells obtained from naïve mice. These data demonstrate the therapeutic efficacy of fusion cell-based vaccine therapy using syngeneic DCs and allogeneic fibroblasts transfected with tumor-derived genomic DNA.
Assuntos
DNA de Neoplasias/genética , Células Dendríticas/fisiologia , Imunização , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Células NIH 3T3/fisiologia , Transfecção , Animais , Fusão Celular , Células Cultivadas , Imunoterapia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Polietilenoglicóis/metabolismo , Retroviridae/genética , Taxa de Sobrevida , Linfócitos T Citotóxicos/imunologiaRESUMO
OBJECTIVE: Endothelial progenitor cells (EPC) in one study group is not the same as EPC in other investigators, suggesting that EPC is not a single type of cell population. In this study, we tried to demonstrate the heterogeneity of EPC. METHODS AND RESULTS: We cultured total mononuclear cells from human peripheral blood to get two types of EPC sequentially from the same donors. We called them early EPC and late EPC. Early EPC with spindle shape showed peak growth at 2 to 3 weeks and died at 4 weeks, whereas late EPC with cobblestone shape appeared late at 2 to 3 weeks, showed exponential growth at 4 to 8 weeks, and lived up to 12 weeks. Late EPC was different from early EPC in the expression of VE-cadherin, Flt-1, KDR, and CD45. Late EPC produced more nitric oxide, incorporated more readily into human umbilical vein endothelial cells monolayer, and formed capillary tube better than early EPC. Early EPC secreted angiogenic cytokines (vascular endothelial growth factor, interleukin 8) more so than late EPC during culture in vitro. Both types of EPC showed comparable in vivo vasculogenic capacity. CONCLUSIONS: We found two types of EPC from a source of adult peripheral blood that might have different roles in neovasculogenesis based on the identified differences.
Assuntos
Endotélio Vascular/citologia , Neovascularização Fisiológica/fisiologia , Células-Tronco/química , Células-Tronco/fisiologia , Animais , Divisão Celular/fisiologia , Linhagem Celular , Sobrevivência Celular/fisiologia , Células Cultivadas , Meios de Cultivo Condicionados/química , Feminino , Artéria Femoral/fisiopatologia , Artéria Femoral/cirurgia , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Membro Posterior/fisiopatologia , Humanos , Interleucina-8/metabolismo , Isquemia/fisiopatologia , Camundongos , Camundongos Nus , Células NIH 3T3/fisiologia , Células-Tronco/metabolismo , Veias Umbilicais/citologia , Veias Umbilicais/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
OBJECTIVE: To investigate whether the soluble form of interleukin-1 (IL-1) receptor accessory protein (sIL-1RAcP), whose physiologic function remains to be established, can serve as a specific inhibitor of IL-1 signaling in vitro, and to evaluate its applicability in collagen-induced arthritis (CIA). METHODS: Soluble IL-1RAcP was cloned from murine liver complementary DNA and expressed by the use of either an adenoviral vector (AdRGD) for sIL-1RAcP or a stable-transfected NIH3T3 fibroblast cell line. The ability of affinity-purified sIL-1RAcP to inhibit IL-1 signaling was tested on NF-kappaB luciferase reporter fibroblasts and quantified by luminometer. To investigate therapeutic efficacy, sIL-1RAcP was both locally (knee joint) and systemically overexpressed in collagen-immunized male DBA/1 mice. Severity of arthritis was monitored visually, and the pathologic process in the joint was examined histologically. Serum was obtained from mice to quantify IL-6 and anti-bovine type II collagen (BCII) antibody levels. RESULTS: Incubation of the NF-kappaB reporter fibroblast with purified sIL-1RAcP protein showed a marked reduction of IL-1-induced, but not tumor necrosis factor-induced, NF-kappaB activation. This showed a novel role for sIL-1RAcP as a specific inhibitor of IL-1 signaling. Local transplantation of sIL-1RAcP-producing NIH3T3 fibroblasts into the knee before onset of CIA had little or no effect on general disease severity in these mice. Histologic evaluation of the knee joints receiving sIL-1RAcP cell transplantation showed a marked reduction in both joint inflammation and bone and cartilage erosion. Local treatment with sIL-1RAcP had no profound effect on serum levels of IL-6 and anti-BCII antibodies, which is indicative of the ongoing presence of arthritis in distal joints. In contrast to local treatment, systemic treatment with the AdRGD for sIL-1RAcP markedly ameliorated CIA in all joints. CONCLUSION: In this study we demonstrated that sIL-1RAcP is a biologically active and innovative inhibitor of IL-1, and treatment of mice with sIL-1RAcP had a profound prophylactic effect on collagen-induced arthritis.