RESUMO
Three-dimensional cell spheroids show promise for the reconstruction of native tissues. Herein, we report a sophisticated, uniform, and highly reproducible spheroid culture system for tissue reconstruction. A mesh-integrated culture system was designed to precisely control the uniformity and reproducibility of spheroid formation. Furthermore, we synthesized hexanoyl glycol chitosan, a material with ultralow cell adhesion properties, to further improve spheroid formation efficiency and biological function. Our results demonstrate improved biological function in various types of cells and ability to generate spheroids with complex structures composed of multiple cell types. In conclusion, our spheroid culture system offers a highly effective and widely applicable approach to generating customized spheroids with desired structural and biological features for a variety of biomedical applications.
Assuntos
Técnicas de Cultura de Células , Quitosana , Medicina Regenerativa , Esferoides Celulares , Esferoides Celulares/citologia , Quitosana/química , Humanos , Técnicas de Cultura de Células/métodos , Engenharia Tecidual/métodos , AnimaisRESUMO
Organoids significantly advanced our comprehension of organ development, function, and disease modeling. This Perspective underscores the potential of heart-kidney-connected organoids in understanding the intricate relationship between these vital organs, notably the cardiorenal syndrome, where dysfunction in one organ can negatively impact the other. Conventional models fall short in replicating this complexity, necessitating an integrated approach. By co-culturing heart and kidney organoids, combined with microfluidic and 3D bioprinting technologies, a more accurate representation of in vivo conditions can be achieved. Such interconnected systems could revolutionize our grasp of multi-organ diseases, drive drug discovery by evaluating therapeutic agents on both organs simultaneously, and reduce the need for animal models. In essence, heart-kidney-connected organoids present a promising avenue to delve deeper into the pathophysiology underlying cardiorenal disorders, bridging existing knowledge gaps, and advancing biomedical research.
RESUMO
Cell therapy using chondrocytes has shown promise for cartilage regeneration, but maintaining functional characteristics during in vitro culture and ensuring survival after transplantation are challenges. Three-dimensional (3D) cell culture methods, such as spheroid culture, and hydrogels can improve cell survival and functionality. In this study, a new method of culturing spheroids using hyaluronic acid (HA) microparticles was developed. The spheroids mixed with HA microparticles effectively maintained the functional characteristics of chondrocytes during in vitro culture, resulting in improved cell survival and successful cartilage formation in vivo following transplantation. This new method has the potential to improve cell therapy production for cartilage regeneration.
Assuntos
Cartilagem Articular , Ácido Hialurônico , Ácido Hialurônico/farmacologia , Engenharia Tecidual/métodos , Cartilagem , Condrócitos , Regeneração , Hidrogéis/farmacologiaRESUMO
Despite recent progress in medical and endovascular therapy, the prognosis for patients with critical limb ischemia (CLI) remains poor. In response, various stem cells and growth factors have been assessed for use in therapeutic neovascularization and limb salvage in CLI patients. However, the clinical outcomes of cell-based therapeutic angiogenesis have not provided the promised benefits, reinforcing the need for novel cell-based therapeutic angiogenic strategies to cure untreatable CLI. In the present study, we investigated genetically engineered mesenchymal stem cells (MSCs) derived from human bone marrow that continuously secrete stromal-derived factor-1α (SDF1α-eMSCs) and demonstrated that intramuscular injection of SDF1α-eMSCs can provide long-term paracrine effects in limb ischemia and effectively contribute to vascular regeneration as well as skeletal muscle repair through increased phosphorylation of ERK and Akt within the SDF1α/CXCR4 axis. These results provide compelling evidence that genetically engineered MSCs with SDF-1α can be an effective strategy for successful limb salvage in limb ischemia.
Assuntos
Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Animais , Humanos , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Quimiocina CXCL12/farmacologia , Membro Posterior/irrigação sanguínea , Isquemia/terapia , Isquemia/metabolismo , Células-Tronco Mesenquimais/metabolismo , Músculo Esquelético/metabolismo , Neovascularização FisiológicaRESUMO
3D spheroids, which have the potential to bridge the gap between 2D cell culture and native tissue, are used as tissue models in many applications, particularly in cancer, stem cell, and pharmaceutical research. A considerable amount of effort has focused on the development of more relevant physiological models. However, spheroids still have limitations in that they cannot replicate the components and structure of the ECM in the natural environment. In this study, we proposed new concept of scaffold-based techniques for the generation of spheroids. Spheroids were successfully generated by single cell or small number of aggregated cells between HA particles. The size of each spheroid was uniform, a necrotic core didn't form, and the system showed high viability. The expression levels of the proteins and genes required to maintain cell-specific functions increased. Thus, our system provides more physiologically relevant models and could be applied to regenerative medicine or drug screening.
Assuntos
Neoplasias , Esferoides Celulares , Biomimética , Técnicas de Cultura de Células/métodos , Humanos , Células-TroncoRESUMO
Cell culture technology has evolved into three-dimensional (3D) artificial tissue models for better reproduction of human native tissues. However, there are some unresolved limitations that arise due to the adhesive properties of cells. In this study, we developed a hexanoyl glycol chitosan (HGC) as a non-cell adhesive polymer for scaffold-based and -free 3D culture. The uniform cell distribution in a porous scaffold was well maintained during the long culutre period on the HGC-coated substrate by preventing ectopic adhesion and migration of cells on the substrate. In addition, when culturing many spheroids in one dish, supplementation of the culture medium with HGC prevented the aggregation of spheroids and maintained the shape and size of spheroids for a long culture duration. Collectively, the use of HGC in 3D culture systems is expected to contribute greatly to creating excellent regenerative therapeutics and screening models of bioproducts.
RESUMO
BACKGROUND: Tissue engineering approaches to treat damaged bone include various tissue transplants such as autologous, allogeneic, and xenografts. Artificial materials have been widely introduced to meet the demand for graft materials, but insufficiency in supply is still not resolved. In this study, human adipose tissue, easily obtained from the human body, was harvested, and the tissue was decellularized to fabricate a decellularized human adipose tissue matrix (DM) as an alternative graft material. METHODS: Human adipose tissue was obtained via liposuction. The obtained fresh adipose tissue sample was cut into pieces then put into decellularization solution (1% antibiotic-antimycotic solution and 1% phenylmethanesulphonyl fluoride). Lipids were further removed via treatment in isopropanol. The sample was then subjected to another enzymatic digestion and lipid removal processes. The obtained decellularized adipose tissue matrix was lyophilized to form a graft material in disc shape. RESULTS: Decellularization was confirmed by nuclear staining methods and detection of RNA and DNA via PCR. Bone morphogenetic protein 2 (BMP2)-loaded DM showed the ability to form new bone tissue when implanted in subcutaneous tissue. In recovery of a mouse calvarial defect model, BMP2-loaded DM exhibited similar levels of bone tissue regeneration efficiency compared with a well-defined commercial product, BMP2-loaded CollaCote®. CONCLUSION: The DM developed in this study is expected to address the problem of insufficient supply of graft materials and contribute to the treatment of bone defects of critical size as an alternative bone graft material with preserved extracellular matrix components.
Assuntos
Proteína Morfogenética Óssea 2 , Alicerces Teciduais , 2-Propanol/metabolismo , Tecido Adiposo , Animais , Antibacterianos , Proteína Morfogenética Óssea 2/metabolismo , Regeneração Óssea , DNA/metabolismo , Matriz Extracelular/metabolismo , Fluoretos/metabolismo , Humanos , Lipídeos , Camundongos , RNA/metabolismoRESUMO
Direct lineage conversion holds great promise in the regenerative medicine field for restoring damaged tissues using functionally engineered counterparts. However, current methods of direct lineage conversion, even those using virus-mediated transgenic expression of tumorigenic factors, are extremely inefficient (~25%). Thus, advanced methodologies capable of revolutionizing efficiency and addressing safety concerns are key to clinical translation of these technologies. Here, we propose an extracellular vesicle (EV)-guided, nonviral, direct lineage conversion strategy to enhance transdifferentiation of fibroblasts to induced cardiomyocyte-like cells (iCMs). The resulting iCMs have typical cardiac Ca2+ transients and electrophysiological features and exhibit global gene expression profiles similar to those of cardiomyocytes. This is the first demonstration of the use of EVs derived from embryonic stem cells undergoing cardiac differentiation as biomimetic tools to induce cardiac reprogramming with extremely high efficiency (>60%), establishing a general, more readily accessible platform for generating a variety of specialized somatic cells through direct lineage conversion.
RESUMO
Wheatgrass and barley grass are freshly sprouted leaves of wheat and barley seeds and are rich sources of phytochemicals. This study was conducted to investigate the effects of drought stress on the biochemical compounds and antioxidant activities of barley grass and wheatgrass extracts. The grass was cultivated in an organic soil growing medium with different levels of drought stress (a control with 100% water holding capacity (WHC), mild drought stress with 75% WHC, moderate drought stress with 50% WHC, and severe drought stress with 25% WHC) in a growth chamber by controlling temperature (20/15 °C, day/night), light (12/12 h, light/dark; intensity 150 µmol m-2 s-1 with quantum dot light-emitting diodes), and relative humidity (60%) for 7 days. The drought stress showed increased levels of biochemical compounds, especially phenolics, flavonoids, and vitamin C, in both barley grass and wheatgrass extracts. The wheatgrass extracts showed 1.38-1.67 times higher phenolics, flavonoids, and vitamin C contents than the barley grass extracts did. The antioxidant (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), 2,2-diphenyl-1-picrylhydrazyl radical-scavenging activity, and nitrite-scavenging activity) and antioxidant enzymes (guaiacol peroxidase, catalase, and glutathione reductase) were the highest under severe drought stress in both barley grass and wheatgrass extracts; and the wheatgrass extracts showed 1.20-5.70 times higher antioxidant enzyme activities than the barley grass extracts did. Proper drought-stress treatment of barley grass and wheatgrass may be a convenient and efficient method to increase biochemical compounds and antioxidants in our diet to exploit the related health benefits. © 2021 Society of Chemical Industry.
Assuntos
Antioxidantes , Hordeum , Antioxidantes/química , Ácido Ascórbico , Secas , Hordeum/química , Água/químicaRESUMO
Three-dimensional (3D) culture systems that provide a more physiologically similar environment than conventional two-dimensional (2D) cultures have been extensively developed. Previously we have provided a facile method for the formation of 3D spheroids using non-adhesive N-hexanoyl glycol chitosan (HGC) hydrogel-coated dishes, but with limitations such as low gel stability and weak mechanical properties. In this study, chemically crosslinked hydrogels were prepared by photocrosslinking of methacrylated HGCs (M-HGCs), and their spheroid-forming abilities were evaluated for long-term 3D cell cultures. The M-HGC hydrogels demonstrated not only enhanced gel stability, but also good spheroid-forming abilities. Furthermore, the M-HGC-coated dishes were effective in generating spheroids of larger size and higher cell density depending on the crosslinking density of the M-HGCs. These results indicate that our hydrogel-coated dish system could be widely applied as an effective technique to produce cell spheroids with customized sizes and densities that are essential for tissue engineering and drug screening.
Assuntos
Quitosana/química , Fibroblastos/fisiologia , Técnicas de Cultura de Células , Células Cultivadas , Quitosana/análogos & derivados , Quitosana/efeitos da radiação , Humanos , Hidrogéis , Processos Fotoquímicos , Esferoides Celulares , Propriedades de Superfície , Temperatura , Raios UltravioletaRESUMO
The current 2D culture model systems developed for drug screening are not sufficient to reflect the characteristics of in vivo solid tumors. Therefore, more effective in vitro tumor model systems must be developed for translational studies on therapeutic drug screening and testing. Herein, we report a new ultra-low adhesion (ULA) hydrogel for generating 3D cancer cell spheroids as tumor models in vitro. N-octanoyl glycol chitosan (OGC) was synthesized and coated onto the surface of a typical cell culture dish. Cell spheroids were effectively formed on the OGC-coated surface, and phenotypes of the tumor cells were well maintained during culture. More importantly, U373-MG cells cultured on OGC-coated plates were more resistant to doxorubicin than cells cultured on typical plates. Our OGC-based ULA system may offer a convenient method for 3D cell culture to provide enhanced performance in cancer research, drug screening and toxicology.
Assuntos
1-Octanol/química , Neoplasias Encefálicas/tratamento farmacológico , Quitosana/química , Glioblastoma/tratamento farmacológico , Esferoides Celulares/citologia , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Avaliação Pré-Clínica de Medicamentos , Humanos , Hidrogéis , Esferoides Celulares/química , Esferoides Celulares/efeitos dos fármacosRESUMO
Biofunctional polymers have been widely used to enhance the proliferation and functionality of stem cells. Here, we report the development of a new biofunctional polymer, octanoyl glycol chitosan (OGC), and demonstrate its effects on the cell cycle and stem cell function using tonsil-derived mesenchymal stem cells (TMSCs). OGC treatment (100 µg/mL) significantly increased the proliferation of TMSCs, which could be attributed to cyclin D1 up-regulation in the G1 phase of the cell cycle. Additionally, OGC enhanced the ability of TMSCs to differentiate into adipocytes, chondrocytes, and osteoblasts. Taken together, this new biofunctional polymer, OGC, can promote stemness and osteogenesis, as well as induce stem cell proliferation by enhancing the intracellular metabolic rate and regulating the cell cycle. Thus, in the future, OGC could be a potential therapeutic additive for improving stem cell function.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Quitosana/farmacologia , Células-Tronco Mesenquimais/metabolismo , Tonsila Palatina/citologia , Ciclo Celular/efeitos dos fármacos , Células Cultivadas , Quitosana/química , Ciclina D1/metabolismo , Humanos , Osteogênese/efeitos dos fármacos , Consumo de Oxigênio , Tonsila Palatina/metabolismo , Polímeros/química , Polímeros/farmacologia , Engenharia Tecidual/métodos , Cicatrização/efeitos dos fármacosRESUMO
The process of evaluating the efficacy and toxicity of drugs is important in the production of new drugs to treat diseases. Testing in humans is the most accurate method, but there are technical and ethical limitations. To overcome these limitations, various models have been developed in which responses to various external stimuli can be observed to help guide future trials. In particular, three-dimensional (3D) cell culture has a great advantage in simulating the physical and biological functions of tissues in the human body. This article reviews the biomaterials currently used to improve cellular functions in 3D culture and the contributions of 3D culture to cancer research, stem cell culture and drug and toxicity screening.
Assuntos
Antineoplásicos/farmacologia , Materiais Biocompatíveis/química , Pesquisa Biomédica , Técnicas de Cultura de Células/métodos , Desenvolvimento de Medicamentos , Neoplasias/tratamento farmacológico , Células-Tronco/efeitos dos fármacos , Animais , Humanos , Neoplasias/patologia , Células-Tronco/citologiaRESUMO
Human embryonic stem cells-derived endothelial progenitor cells (hEPCs) were utilized as cell therapeutics for the treatment of ischemic diseases. However, in vivo tracking of hEPCs for predicting their therapeutic efficacy is very difficult. Herein, we developed bioorthogonal labeling strategy of hEPCs that could non-invasively track them after transplantation in hind limb ischemia models. First, hEPCs were treated with tetraacylated N-azidomannosamine (Ac4ManNAz) for generating unnatural azide groups on the hEPCs surface. Second, near-infrared fluorescence (NIRF) dye, Cy5, conjugated dibenzocylooctyne (DBCO-Cy5) was chemically conjugated to the azide groups on the hEPC surface via copper-free click chemistry, resulting Cy5-hEPCs. The bioorthogonally labeled Cy5-hEPCs showed strong NIRF signal without cytotoxicity and functional perturbation in tubular formation, oxygen consumption and paracrine effect of hEPCs in vitro. In hind limb ischemia models, the distribution and migration of transplanted Cy5-hEPCs were successfully monitored via fluorescence molecular tomography (FMT) for 28 days. Notably, blood reperfusion and therapeutic neovascularization effects were significantly correlated with the initial transplantation forms of Cy5-hEPCs such as 'condensed round shape' and 'spread shape' in the ischemic lesion. The condensed transplanted Cy5-hEPCs substantially increased the therapeutic efficacy of hind limb ischemia, compared to that of spread Cy5-hEPCs. Therefore, our new stem cell labeling strategy can be used to predict therapeutic efficacy in hind limb ischemia and it can be applied a potential application in developing cell therapeutics for regenerative medicine.
Assuntos
Células Progenitoras Endoteliais , Animais , Química Click , Modelos Animais de Doenças , Membro Posterior , Humanos , Isquemia/diagnóstico por imagem , Isquemia/terapia , Neovascularização Fisiológica , Células-Tronco , TomografiaRESUMO
Thermogels that undergo temperature-dependent sol-gel transition have recently attracted attention as a promising biomaterial for injectable tissue engineering. However, conventional thermogels usually suffer from poor physical properties and low cell binding affinity, limiting their practical applications. Here, a simple approach for developing a new thermogel with enhanced physical properties and cell binding affinity is proposed. This thermogel (AcHA/HGC) was obtained by simple blending of a new class of polysaccharide-based thermogel, N-hexanoyl glycol chitosan (HGC), with a polysaccharide possessing good cell binding affinity, acetylated hyaluronic acid (AcHA). Gelation of AcHA/HGC was initially triggered by the thermosensitive response of HGC and gradually intensified by additional physical crosslinking mechanisms between HGC and AcHA, resulting in thermo-irreversible gelation. Compared to the thermos-reversible HGC hydrogel, the thermo-irreversible AcHA/HGC hydrogel exhibited enhanced physical stability, mechanical properties, cell binding affinity, and tissue compatibility. These results suggest that our thermo-irreversible hydrogel is a promising biomaterial for injectable tissue engineering.
Assuntos
Materiais Biocompatíveis , Quitosana , Ácido Hialurônico , Hidrogéis , Engenharia Tecidual , Animais , Materiais Biocompatíveis/síntese química , Materiais Biocompatíveis/química , Materiais Biocompatíveis/uso terapêutico , Células Cultivadas , Quitosana/química , Quitosana/uso terapêutico , Condrócitos , Ácido Hialurônico/química , Ácido Hialurônico/uso terapêutico , Hidrogéis/síntese química , Hidrogéis/química , Hidrogéis/uso terapêutico , Masculino , Camundongos , Camundongos Endogâmicos ICRRESUMO
BACKGROUND: We investigated whether electrical stimulation via indium tin oxide (ITO) could enhance the in vitro culture of neonatal rat ventricular myocytes (NRVMs), which are important in vitro models for studying the mechanisms underlying many aspects of cardiology. METHODS: Cardiomyocytes were obtained from 1-day-old neonatal rat heart ventricles. To evaluate function of NRVMs cultured on ITO with electrical stimulation, the cell viability, change of cell morphology, immunochemistry using cardiac-specific antibodies, and gene expression were tested. RESULTS: Defined sarcomeric structure, cell enlargement, and increased distribution of NRVMs appeared in the presence of electrical stimulation. These characteristics were absent in NRVMs cultured under standard culture conditions. In addition, the expression levels of cardiomyocyte-specific and ion channel markers were higher in NRVMs seeded on ITO-coated dishes than in the control group at 14 days after seeding. ITO-coated dishes could effectively provide electrical cues to support the in vitro culture of NRVMs. CONCLUSIONS: These results provide supporting evidence that electrical stimulation via ITO can be effectively used to maintain culture and enhance function of cardiomyocytes in vitro.
RESUMO
Various nanopatterning techniques have been developed to improve cell proliferation and differentiation efficiency. As we previously reported, nanopillars and pores are able to sustain human pluripotent stem cells and differentiate pancreatic cells. From this, the nanoscale patterns would be effective environment for the co-culturing of epithelial and mesenchymal cell types. Interestingly, the nanopatterning selectively reduced the proliferative rate of mesenchymal cells while increasing the expression of adhesion protein in epithelial type cells. Additionally, co-cultured cells on the nanopatterning were not negatively affected in terms of cell function metabolic ability or cell survival. This is in contrast to conventional co-culturing methods such as ultraviolet or chemical treatments. The nanopatterning appears to be an effective environment for mesenchymal co-cultures with typically low proliferative rates cells such as astrocytes, neurons, melanocytes, and fibroblasts without using potentially damaging treatments.
Assuntos
Técnicas de Cocultura/instrumentação , Células Epiteliais , Células-Tronco Mesenquimais , Nanoestruturas , Animais , Adesão Celular , Proliferação de Células , Sobrevivência Celular , Células Epiteliais/citologia , Células Epiteliais/fisiologia , Fibroblastos/citologia , Fibroblastos/fisiologia , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Camundongos , Propriedades de SuperfícieRESUMO
Cell labeling technologies are required to monitor the fate of transplanted cells in vivo and to select target cells for the observation of certain changes in vitro. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been transplanted for the treatment of heart injuries or used in vitro for preclinical cardiac safety assessments. Cardiomyocyte (CM) labeling has been used in these processes to facilitate target cell monitoring. However, the functional effect of the labeling agent on hiPSC-CMs has not been studied. Therefore, we investigated the effects of labeling agents on CM cellular functions. 3'-Dioctadecyloxacarbocyanine perchlorate (DiO), quantum dots (QDs), and a DNA plasmid expressing EGFP using Lipo2K were used to label hiPSC-CMs. We conclude that the hiPSC-CM labeling with DiO and QDs does not induce arrhythmogenic effects but rather improves the mRNA expression of cardiac ion channels and Ca2ï¼ influx by L-type Ca2ï¼ channels. Thus, DiO and QD labeling agents may be useful tools to monitor transplanted CMs, and further in vivo influences of the labeling agents should be investigated in the future.
RESUMO
Discovering and developing ideal cell culture methods is important for cell biology, drug development, and cell therapy. Recent studies have explored and demonstrated the use of nanoscale structures and patterns that influence cell behavior, such as 3D scaffolds. In this study, we analyzed the effects of nanopatterned-surface dishes using chondrocytes as model cells. Chondrocytes grown on nanopatterned dishes exhibited rounded shapes. Interestingly, chondrocytes have a lower COL10 mRNA level when cultured using nanopatterned dishes. The nanopatterned dishes induced G0-/G1-phase cell cycle arrest and reduced the rate of proliferation. Our results suggest that nanoscale structures can directly control cellular behaviors and can be used for chondrocyte cell culture without causing chondrocytes to lose their functions. These results help to elucidate cellular responses and behaviors in native-like environments, and this information can be used to improve human health.