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1.
Adv Exp Med Biol ; 1237: 49-60, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31376140

RESUMO

Cell proliferation, apoptosis and differentiation are essential processes from the early phases of embryogenesis to adult tissue formation and maintenance. These mechanisms also play a key role in embryonic stem cells (ESCs) that are able to proliferate maintaining pluripotency and, at the same time, to give rise to all populations belonging to the three germ layers, in response to specific stimuli. ESCs are, therefore, considered a well-established in vitro model to study the complexity of these processes. In this perspective, we previously generated parthenogenetic embryonic stem cells (ParthESC), that showed many features and regulatory pathways common to bi-parental ESCs. However, we observed that mono-parental cells demonstrate a high ability to form outgrowths and generate 3D spheroid colonies, which are distinctive signs of high-plasticity. Furthermore, preliminary evidence obtained by WTA, revealed the presence of several differentially expressed genes belonging to the Rho and Hippo signaling pathways. In the present study, we compare bi-parental ESCs and ParthESC and analyze by Real-Time PCR the differentially expressed genes. We demonstrate up-regulation of the Rho signaling pathway and an increased expression of YAP and TAZ in ParthESC. We also show that YAP remains in a dephosphorylated form. This allows its nuclear translocation and its direct binding to TEADs and SMADs, that are up-regulated in ParthESC. Altogether, these complex regulatory interactions result in overexpression of pluripotency related genes, in a global DNA hypomethylation and a histone-dependent chromatin high permissive state that may account for ParthESC high potency, possibly related to their exclusive maternal origin.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Células-Tronco Embrionárias/citologia , Partenogênese , Transdução de Sinais , Esferoides Celulares/citologia , Transativadores/metabolismo , Fatores de Transcrição/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Humanos
2.
Cancer Sci ; 111(2): 467-476, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31845453

RESUMO

Collective invasion of cancer cells is the key process of circulating tumor cell (CTC) cluster formation, and greatly contributes to metastasis. Cancer stem-like cells (CSC) have a distinct advantage of motility for metastatic dissemination. To verify the role of CSC in the collective invasion, we performed 3D assays to investigate the collective invasion from cancer cell spheroids. The results demonstrated that CSC can significantly promote both collective and single-cell invasion. Further study showed that CSC prefer to move outside and lead the collective invasion. More interestingly, approximately 60% of the leader CSC in collective invasion co-expressed both epithelial and mesenchymal genes, while only 4% co-expressed in single invasive CSC, indicating that CSC with hybrid epithelial/mesenchymal phenotype play a key role in cancer cell collective invasion.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Invasividade Neoplásica/patologia , Células-Tronco Neoplásicas/patologia , Esferoides Celulares/patologia , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Transição Epitelial-Mesenquimal , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Camundongos , Metástase Neoplásica , Transplante de Neoplasias , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/metabolismo , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo
3.
Int J Mol Sci ; 20(19)2019 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-31590325

RESUMO

Various tissue engineering techniques have been created in research spanning two centuries, resulting in new opportunities for growing cells in culture and the creation of 3-D tissue-like constructs. These techniques are classified as scaffold-based and scaffold-free techniques. Cell sheet, as a scaffold-free technique, has attracted research interest in the context of drug discovery and tissue repair, because it provides more predictive data for in vivo testing. It is one of the most promising techniques and has the potential to treat degenerative tissues such as heart, kidneys, and liver. In this paper, we argue the advantages of cell sheets as a scaffold-free approach, compared to other techniques, including scaffold-based and scaffold-free techniques such as the classic systemic injection of cell suspension.


Assuntos
Bioimpressão/métodos , Esferoides Celulares/citologia , Engenharia Tecidual/métodos , Animais , Modelos Animais de Doenças , Humanos , Tecidos Suporte/efeitos adversos
4.
Nature ; 574(7776): 112-116, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31554966

RESUMO

Organogenesis is a complex and interconnected process that is orchestrated by multiple boundary tissue interactions1-7. However, it remains unclear how individual, neighbouring components coordinate to establish an integral multi-organ structure. Here we report the continuous patterning and dynamic morphogenesis of hepatic, biliary and pancreatic structures, invaginating from a three-dimensional culture of human pluripotent stem cells. The boundary interactions between anterior and posterior gut spheroids differentiated from human pluripotent stem cells enables retinoic acid-dependent emergence of hepato-biliary-pancreatic organ domains specified at the foregut-midgut boundary organoids in the absence of extrinsic factors. Whereas transplant-derived tissues are dominated by midgut derivatives, long-term-cultured microdissected hepato-biliary-pancreatic organoids develop into segregated multi-organ anlages, which then recapitulate early morphogenetic events including the invagination and branching of three different and interconnected organ structures, reminiscent of tissues derived from mouse explanted foregut-midgut culture. Mis-segregation of multi-organ domains caused by a genetic mutation in HES1 abolishes the biliary specification potential in culture, as seen in vivo8,9. In sum, we demonstrate that the experimental multi-organ integrated model can be established by the juxtapositioning of foregut and midgut tissues, and potentially serves as a tractable, manipulatable and easily accessible model for the study of complex human endoderm organogenesis.


Assuntos
Sistema Biliar/embriologia , Intestinos/embriologia , Fígado/embriologia , Modelos Biológicos , Morfogênese , Pâncreas/embriologia , Animais , Sistema Biliar/citologia , Biomarcadores/análise , Biomarcadores/metabolismo , Padronização Corporal , Endoderma/citologia , Endoderma/embriologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Intestinos/citologia , Fígado/citologia , Masculino , Camundongos , Organoides/citologia , Organoides/embriologia , Pâncreas/citologia , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo , Esferoides Celulares/transplante , Fatores de Transcrição HES-1/análise , Fatores de Transcrição HES-1/metabolismo
5.
Oncol Rep ; 42(5): 1878-1892, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31545459

RESUMO

3D spheroids are built by heterogeneous cell types in different proliferative and metabolic states and are enriched in cancer stem cells. The main aim of the study was to investigate the usefulness of a novel metastatic renal cell carcinoma (RCC) 3D spheroid culture for in vitro cancer stem cell physiology research and drug toxicity screening. RCC cell lines, Caki­1 (skin metastasis derived) and ACHN (pleural effusion derived), were efficiently cultured in growth­factor/serum deprived, defined, StemXvivo and Nutristem medium on laminin­coated or poly­D­lysine­coated plates. In optimal 3D culture conditions, ACHN cells (StemXVivo/poly­D­lysine) formed small spheroids with remaining adherent cells of an epithelial phenotype, while Caki­1 cells (StemXVivo/laminin) formed large dark spheroids with significantly reduced cell viability in the center. In the 3D structures, expression levels of genes encoding stem transcription factors (OCT4, SOX2, NES) and RCC stem cell markers (CD105, CD133) were deregulated in comparison to these expression levels in traditional 2D culture. Sunitinib, epirubicin and doxycycline were more toxic to cells cultured in monolayers than for cells in 3D spheroids. High numbers of cells arrested in the G0/G1 phase of the cell cycle were found in spheroids under sunitinib treatment. We showed that metastatic RCC 3D spheroids supported with ECM are a useful model to determine the cancer cell growth characteristics that are not found in adherent 2D cultures. Due to the more complex architecture, spheroids may mimic in vivo micrometastases and may be more appropriate to investigate novel drug candidate responses, including the direct effects of tyrosine kinase inhibitor activity against RCC cells.


Assuntos
Biomarcadores Tumorais/genética , Carcinoma de Células Renais/genética , Técnicas de Cultura de Células/métodos , Meios de Cultura Livres de Soro/química , Resistencia a Medicamentos Antineoplásicos , Neoplasias Renais/genética , Células-Tronco Neoplásicas/química , Biomimética , Carcinoma de Células Renais/dietoterapia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Doxiciclina/farmacologia , Epirubicina/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Renais/tratamento farmacológico , Laminina/farmacologia , Metástase Neoplásica , Células-Tronco Neoplásicas/efeitos dos fármacos , Fenótipo , Esferoides Celulares/química , Esferoides Celulares/citologia , Esferoides Celulares/efeitos dos fármacos , Sunitinibe/farmacologia
6.
BMC Cancer ; 19(1): 864, 2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-31470802

RESUMO

BACKGROUND: Bronchial carcinoids are neuroendocrine tumors that present as typical (TC) and atypical (AC) variants, the latter being more aggressive, invasive and metastatic. Studies of tumor initiating cell (TIC) biology in bronchial carcinoids has been hindered by the lack of appropriate in-vitro and xenograft models representing the bronchial carcinoid phenotype and behavior. METHODS: Bronchial carcinoid cell lines (H727, TC and H720, AC) were cultured in serum-free growth factor supplemented medium to form 3D spheroids and serially passaged up to the 3rd generation permitting expansion of the TIC population as verified by expression of stemness markers, clonogenicity in-vitro and tumorigenicity in both subcutaneous and orthotopic (lung) models. Acetazolamide (AZ), sulforaphane (SFN) and the AZ + SFN combination were evaluated for targeting TIC in bronchial carcinoids. RESULTS: Data demonstrate that bronchial carcinoid cell line 3rd generation spheroid cells show increased drug resistance, clonogenicity, and tumorigenic potential compared with the parental cells, suggesting selection and expansion of a TIC fraction. Gene expression and immunolabeling studies demonstrated that the TIC expressed stemness factors Oct-4, Sox-2 and Nanog. In a lung orthotopic model bronchial carcinoid, cell line derived spheroids, and patient tumor derived 3rd generation spheroids when supported by a stroma, showed robust tumor formation. SFN and especially the AZ + SFN combination were effective in inhibiting tumor cell growth, spheroid formation and in reducing tumor formation in immunocompromised mice. CONCLUSIONS: Human bronchial carcinoid tumor cells serially passaged as spheroids contain a higher fraction of TIC exhibiting a stemness phenotype. This TIC population can be effectively targeted by the combination of AZ + SFN. Our work portends clinical relevance and supports the therapeutic use of the novel AZ+ SFN combination that may target the TIC population of bronchial carcinoids.


Assuntos
Acetazolamida/administração & dosagem , Anticarcinógenos/administração & dosagem , Neoplasias Brônquicas/tratamento farmacológico , Tumor Carcinoide/tratamento farmacológico , Isotiocianatos/administração & dosagem , Células-Tronco Neoplásicas/efeitos dos fármacos , Acetazolamida/farmacologia , Animais , Anticarcinógenos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Brônquicas/genética , Neoplasias Brônquicas/metabolismo , Tumor Carcinoide/genética , Tumor Carcinoide/metabolismo , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Isotiocianatos/farmacologia , Camundongos , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Esferoides Celulares/citologia , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Oncol Rep ; 42(5): 2029-2038, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31432145

RESUMO

In vitro culture of patient­derived tumor cells offers many advantages in the development of novel therapies for colorectal cancer. Although various culture systems have been developed, the long­term expansion of patient­derived tumor cells remains challenging. The present results suggested that tumor cells isolated from colorectal cancer patient­derived xenografts can be efficiently immortalized in conditioned medium from irradiated feeder cells containing Y­27632, a rho­associated coiled­coil containing protein kinase (ROCK) inhibitor. Patient­derived tumor cells proliferated rapidly, reaching 90­95% confluence in ~6 days. Short tandem repeat analysis suggested that these tumor tissues and cultured cells presented 13 identical short tandem repeat loci, including Amelogenin, Penta E, Penta D, D2S1338 and D19S433. Their epithelial phenotype was confirmed by staining for epithelial cell adhesion molecule and cytokeratin 20, whereas vimentin was used as a mesenchymal marker. When cells were transferred to 3D cultures, they continued to proliferate, forming well­defined tumor spheroids. Expression levels of human telomerase reverse transcriptase and C­Myc mRNA were increased in cultured cells. Finally, immortalized cells were used for the screening of 65 anticancer drugs approved by the Food and Drug Administration, allowing the identification of gene­drug associations. In the present study, primary culture models of colorectal cancer were efficiently established using a ROCK inhibitor and feeder cells, and this approach could be used for personalized treatment strategies for patients with colorectal cancer.


Assuntos
Amidas/farmacologia , Neoplasias Colorretais/patologia , Células Alimentadoras/citologia , Cultura Primária de Células/métodos , Piridinas/farmacologia , Células Tumorais Cultivadas/citologia , Idoso , Animais , Proliferação de Células , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Meios de Cultivo Condicionados/química , Feminino , Humanos , Masculino , Camundongos , Repetições de Microssatélites , Pessoa de Meia-Idade , Transplante de Neoplasias , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo , Células Tumorais Cultivadas/efeitos dos fármacos
8.
Nat Commun ; 10(1): 3838, 2019 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-31444335

RESUMO

Developing biomimetic nanoparticles without loss of the integrity of proteins remains a major challenge in cancer chemotherapy. Here, we develop a biocompatible tumor-cell-exocytosed exosome-biomimetic porous silicon nanoparticles (PSiNPs) as drug carrier for targeted cancer chemotherapy. Exosome-sheathed doxorubicin-loaded PSiNPs (DOX@E-PSiNPs), generated by exocytosis of the endocytosed DOX-loaded PSiNPs from tumor cells, exhibit enhanced tumor accumulation, extravasation from blood vessels and penetration into deep tumor parenchyma following intravenous administration. In addition, DOX@E-PSiNPs, regardless of their origin, possess significant cellular uptake and cytotoxicity in both bulk cancer cells and cancer stem cells (CSCs). These properties endow DOX@E-PSiNPs with great in vivo enrichment in total tumor cells and side population cells with features of CSCs, resulting in anticancer activity and CSCs reduction in subcutaneous, orthotopic and metastatic tumor models. These results provide a proof-of-concept for the use of exosome-biomimetic nanoparticles exocytosed from tumor cells as a promising drug carrier for efficient cancer chemotherapy.


Assuntos
Doxorrubicina/administração & dosagem , Portadores de Fármacos/química , Composição de Medicamentos/métodos , Exossomos/química , Neoplasias/tratamento farmacológico , Animais , Linhagem Celular Tumoral/citologia , Linhagem Celular Tumoral/metabolismo , Linhagem Celular Tumoral/transplante , Modelos Animais de Doenças , Exocitose , Feminino , Humanos , Masculino , Camundongos , Nanopartículas/química , Neoplasias/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Porosidade , Estudo de Prova de Conceito , Silício/química , Esferoides Celulares/citologia , Esferoides Celulares/metabolismo , Esferoides Celulares/transplante , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Ultrason Sonochem ; 58: 104615, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31450294

RESUMO

In vivo assays of graphene and its derivatives are big challenges in biological evaluations because they require simultaneous long-term stability in aqueous dispersion and controllable systemic toxicity. Bifunctional graphene nanosheets which have key function in biomedical area are expected to address this challenge. Here, novel bifunctional graphene nanosheets were successfully synthesized in the presence of Herceptin, a natural antibody, using a facile ultrasonic-assisted method. Graphite layers were successfully exfoliated which resulted excellent stability of separated layers in herceptin solution. In aqueous solution, graphene concentration was effectively controlled by varying the herceptin content and sonication time. Furthermore, the toxicity of graphene was tested in both 2D and 3D spheroid cultures. The results showed that graphene toxicity were considerably reduced in spheroid culture compared to the 2D culture data. Moreover, the toxicity behavior of graphene was dependent on the exposed concentration of graphene that the mortality rate was significantly decreased when the concentration of graphene was below 1 µg/mL. This bifunctional graphene which possessed long-term stability in aqueous solutions and induced slight toxicity offers a promising nanostructure in tumor-targeted drug delivery, regenerative medicine and tissue engineering. This proof-of-concept study demonstrates the feasibility of ultrasonic assisted method in one-step synthesis of bifunctional nanomaterials and biostructures for clinical applications.


Assuntos
Grafite/química , Grafite/toxicidade , Esferoides Celulares/efeitos dos fármacos , Trastuzumab/química , Ondas Ultrassônicas , Linhagem Celular Tumoral , Técnicas de Química Sintética , Estabilidade de Medicamentos , Humanos , Esferoides Celulares/citologia
10.
Mol Biol Rep ; 46(5): 5103-5112, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31290055

RESUMO

The therapeutic application of recombinant proteins is limited due to their inherent structural complexity. Additionally, screening of therapeutic potential of protein products requires an appropriate testing platform to achieve biological relevance. Fabrication of three dimensional cultures bridges the gap between in vitro based monolayer cultures and clinical applications. In this perspective, glioblastoma U-87 MG and breast cancer MCF7 spheroids were generated to assess the therapeutic prospect of recombinant PTEN protein. PTEN bound to silver nanoclusters was encapsulated within PEG coating, which resulted in fabrication of spherical nanocarriers named as PTEN-nanocomposites. Internalization of PTEN-nanocomposites in the spheroids was confirmed by confocal microscopy. Upon uptake, PTEN-nanocomposites led to modulation of cyclins and apoptosis gene regulators culminating in cell cycle arrest and reduced cell viability as confirmed by calcein-AM/PI dual staining and alamar blue assay. Further, combination of tamoxifen and PTEN-nanocomposites on U-87 MG spheroids resulted in two-fold reduction of drug dosage. The study revealed that the monolayer culture results translated to the 3D culture as well, however higher dose of the recombinant PTEN was required for the spheroid system. The anti-proliferative role of PTEN-nanocomposites in a complex 3D environment augments its biological implication and paves the way for recombinant PTEN based therapeutic applications.


Assuntos
PTEN Fosfo-Hidrolase/farmacologia , Polietilenoglicóis/química , Esferoides Celulares/citologia , Tamoxifeno/farmacologia , Técnicas de Cultura de Células , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Células MCF-7 , Nanopartículas Metálicas , Microscopia Confocal , Nanocompostos , PTEN Fosfo-Hidrolase/química , PTEN Fosfo-Hidrolase/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo
11.
Biofabrication ; 11(4): 045013, 2019 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-31290409

RESUMO

Multicellular aggregated tissues have grown critically important in benchtop biomedical research, both as stand-alone spheroids and when assembled into larger bioengineered constructs. However, typical systems for aggregate formation are limited in their capacity to reliably handle such cultures at various experimental stages in a broadly accessible, consistent, and scalable manner. In this work, we develop a broadly versatile all-in-one biofabrication strategy to form uniform, spherical, multicellular aggregates that can be maintained at precisely defined positions for analysis or transfer into a larger tissue. The 3D-printed MicroPocket Culture (MPoC) system consists of an array of simple geometry-based valves in a polyacrylamide hydrogel, and is able to produce hundreds of uniformly-sized aggregates in standard tissue culture well plates, using simple tools that are readily available in all standard biological wet-labs. The model breast cancer aggregates formed in these experiments are retained in defined positions on chip during all liquid handling steps required to stimulate, label, and image the experiment, enabling high-throughput studies on this culture model. Furthermore, MPoCs enable robust formation of aggregates in cell types that do not conventionally form such structures. Finally, we demonstrate that this single platform can also be used to generate complex 3D tissues from the precisely-positioned aggregate building blocks. To highlight the unique and broad versatility of this technique, we develop a simple 3D invasion assay and show that cancer cells preferentially migrate towards nearby model tumors; demonstrating the importance of spatial precision when engineering 3D tissues. Together, this platform presents a broadly accessible and uniquely capable system with which to develop advanced aggregate-based models for tissue engineering, fundamental research, and applied drug discovery.


Assuntos
Hidrogéis/química , Microtecnologia/instrumentação , Engenharia Tecidual/métodos , Agregação Celular , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Tamanho Celular , Matriz Extracelular/metabolismo , Humanos , Esferoides Celulares/citologia
12.
Comput Math Methods Med ; 2019: 7853586, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31236128

RESUMO

A long-standing problem in tissue engineering is the biofabrication of perfusable tissue constructs that can be readily connected to the patient's vasculature. It was partially solved by three-dimensional (3D) printing of sacrificial material (e.g., hydrogel) strands: upon incorporation in another cell-laden hydrogel, the strands were removed, leaving behind perfusable channels. Their complexity, however, did not match that of the native vasculature. Here, we propose to use multicellular spheroids as a sacrificial material and investigate their potential benefits in the context of 3D bioprinting of cell aggregates and/or cell-laden hydrogels. Our study is based on computer simulations of postprinting cellular rearrangements. The computational model of the biological system is built on a cubic lattice, whereas its evolution is simulated using the Metropolis Monte Carlo algorithm. The simulations describe structural changes in three types of tissue constructs: a tube made of a single cell type, a tube made of two cell types, and a cell-laden hydrogel slab that incorporates a branching tube. In all three constructs, the lumen is obtained after the elimination of the sacrificial cell population. Our study suggests that sacrificial cell spheroids (sacrospheres) enable one to print tissue constructs outfitted with a finer and more complex network of channels than the ones obtained so far. Moreover, cellular interactions might give rise to a tissue microarchitecture that lies beyond the bioprinter's resolution. Although more expensive than inert materials, sacrificial cells have the potential to bring further progress towards the biofabrication of fully vascularized tissue substitutes.


Assuntos
Bioimpressão/métodos , Hidrogéis/química , Impressão Tridimensional , Esferoides Celulares/citologia , Engenharia Tecidual/métodos , Células 3T3 , Algoritmos , Animais , Carcinoma Pulmonar de Lewis/metabolismo , Simulação por Computador , Humanos , Nanopartículas Metálicas/química , Camundongos , Método de Monte Carlo , Perfusão , Silício/química , Tecidos Suporte
13.
Biofabrication ; 11(4): 045011, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31247598

RESUMO

Neurological disorders affect millions of Americans and this number is expected to rise with the aging population. Development of drugs to treat these disorders may be facilitated by improved in vitro models that faithfully reproduce salient features of the relevant brain regions. Current 3D culture methods face challenges with reliably reproducing microarchitectural features of brain morphology such as cortical or hippocampal layers. In this work, polydimethylsiloxane (PDMS) mini-wells were used to create low aspect ratio, adherent 3D constructs where neurons self-assemble into layers. Layer self-assembly was determined to depend on the size of the PDMS mini-well. Layer formation occurred in cultures composed of primary rat cortical neurons or human induced pluripotent stem cell-derived neurons and astrocytes and was robust and reproducible. Layered 3D constructs were found to have spontaneous neural activity characterized by long bursts similar to activity in the developing cortex. The responses of layered 3D cultures to drugs were more similar to in vivo data than those of 2D cultures. 3D constructs created with this method may be thus suitable as in vitro models for drug discovery for neurological disorders.


Assuntos
Técnicas de Cultura de Células/métodos , Neurônios/citologia , Animais , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Ácido Cinurênico/farmacologia , Neurônios/efeitos dos fármacos , Ratos Sprague-Dawley , Esferoides Celulares/citologia , Esferoides Celulares/efeitos dos fármacos , Tetrodotoxina/farmacologia
14.
Int J Dev Biol ; 63(6-7): 271-280, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31250910

RESUMO

Human pluripotent stem cells (hPSCs), such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), are very attractive cell sources for the treatment of diabetes mellitus, because numerous cells can be obtained using their infinite proliferation potential to overcome the paucity of donor islets. Advances in differentiation protocols make it possible to generate glucose responsive hPSC-beta cells, which can ameliorate hyperglycemia in diabetic mice. These protocols have mainly been based on an adherent culture system. However, in clinical applications, suspension culture methods are more suitable for large-scale culture. There are reports that suspension culture and spheroid formation promote differentiation in various cell types, including hPSCs, but, to our knowledge, there are no reports comparing gene expression patterns between suspension and adherent cultured human iPSCs (hiPSCs) during definitive endoderm (DE) differentiation. In this study, we chose several stage marker genes, not only for DE but also for posterior epiblast and primitive streak, and we examined their time course expression in suspension and adherent cultures by quantitative PT-PCR (qPCR), western blot, flow cytometry and immunocytochemistry. Our results demonstrate that expressions of these marker genes are faster and more strongly induced in suspension culture than in adherent culture during the DE differentiation process, indicating that suspension culture favors DE differentiation.


Assuntos
Adesão Celular , Diferenciação Celular , Células-Tronco Embrionárias/citologia , Endoderma/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Esferoides Celulares/citologia , Técnicas de Cultura de Células , Células Cultivadas , Células-Tronco Embrionárias/metabolismo , Endoderma/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Esferoides Celulares/metabolismo
15.
Exp Eye Res ; 185: 107699, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31202832

RESUMO

The loss of retinal ganglion cells (RGCs) is one of the common pathological features associated with optic nerve diseases leading to blindness. The aims of our study were to compare the neuroprotection of two forms of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) on RGCs and axon regeneration after optic nerve crush (ONC) in vivo, and to investigate the molecular mechanism. The effects of intravitreally transplanted hUCB-MSCs cultured in two-dimensional (2D-MSCs) and spheroids (3D-MSCs) were assessed by the survival of RGCs, regenerating axons, and flash visual evoked potentials (fVEP); the level of signal factors secreted by transplanted MSCs in vitreous and the marker protein levels of JAK/STAT3, PI3K/Akt/mTOR and MAPK/ERK pathways were detected using Bead-Based analysis and Western blot, respectively. We found that RGCs began to lose at day 3 after ONC, rapidly decreased at day 7, and flattened at day 14. The neuroprotection of transplanted 2D-MSCs was much stronger than that of 3D-MSCs. The transplanted 2D-MSCs could survive at least 2 weeks without differentiation and keep the characters of MSCs, which secreted multiple tropic factors and accompanied by activation of JAK/STAT3 and MAPK/ERK signaling pathways, top three most abundant factors: stem cell growth factor- ß (SCGF-ß), hepatocyte growth factor (HGF), and monocyte chemoattractant protein-1 (MCP-1). These results indicate that intravitreal injection of 2D-MSCs is a promising therapeutic strategy for retinal pathological diseases characterized by the loss of RGCs and open the door for the application of SCGF-ß, HGF, and MCP-1 in the treatment of optic nerve diseases.


Assuntos
Axônios/fisiologia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/fisiologia , Regeneração Nervosa/fisiologia , Traumatismos do Nervo Óptico/metabolismo , Células Ganglionares da Retina/fisiologia , Animais , Biomarcadores/metabolismo , Western Blotting , Sobrevivência Celular/fisiologia , Células Cultivadas , Potenciais Evocados Visuais/fisiologia , Sangue Fetal/citologia , Humanos , Injeções Intravítreas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estimulação Luminosa , Esferoides Celulares/citologia
16.
In Vitro Cell Dev Biol Anim ; 55(6): 462-471, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31111346

RESUMO

To produce insulin-producing cells (IPCs) from bone marrow mesenchymal stem cells (BM-MSCs) using a simple and cost effective method. During the initial 7 days of three-dimensional (3D) culture, BM-MSCs were cultured on 1% agar or agarose to form multicellular spheroids. Spheroids and spheroid-derived single cells (SS and SSC, respectively) were cultured in the absence of any proteinaceous growth factor in a simple specific medium for a further 7 d. The insulin content of the differentiated cells was evaluated at the mRNA and protein levels. Furthermore, the expression of pancreatic beta cells-related genes other than INS as well as the in vitro responses of IPCs to different glucose concentrations were investigated. Cellular clusters generated on agar and SS conditions (agar+SS-IPCs) stained better with beta cell specific stains and were more reactive to serum-containing insulin reactive antibodies compared with agarose-SS-IPCs. Gene expression analysis revealed that in comparison to agarose + SS-IPCs, agar+SS-IPCs expressed significantly higher levels of INS-1, INS-2, PDX-1, NKX6.1, and XBP-1. Of interest, agar+SS-IPCs expressed 2215.3 ± 120.8-fold more INS-1 gene compared to BM-MSCs. The expression of ß-cell associated genes was also higher in agar+SS-IPCs compared to the agar+SSC-IPCs. Moreover, the expression of INS-1 gene was significantly higher in agar+SS-IPCs compared with agar+SSC-IPCs after culture in media with high concentration of glucose. Compared to the most expensive and time-consuming protocols, 3D culture of MSCs on agar followed by 2D culture of cellular clusters in a minimally supplemented high glucose media produced highly potent IPCs which may pay the way to the treatment of diabetic patients.


Assuntos
Insulina/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Técnicas de Cultura de Tecidos/métodos , Ágar , Animais , Células da Medula Óssea/citologia , Diferenciação Celular , Regulação da Expressão Gênica , Humanos , Células Secretoras de Insulina/fisiologia , Masculino , Ratos Sprague-Dawley , Esferoides Celulares/citologia , Cordão Umbilical/citologia
17.
BMC Cancer ; 19(1): 402, 2019 Apr 29.
Artigo em Inglês | MEDLINE | ID: mdl-31035967

RESUMO

BACKGROUND: Different 3D-cell culture approaches with varying degrees of complexity have been developed to serve as melanoma models for drug testing or mechanistic studies. While these 3D-culture initiatives are already often superior to classical 2D approaches, they are either composed of only melanoma cells or they are so complex that the behavior of individual cell types is hard to understand, and often they are difficult to establish and expensive. METHODS: This study used low-attachment based generation of spheroids composed of up to three cell types. Characterization of cells and spheroids involved cryosectioning, immunofluorescence, FACS, and quantitative analyses. Statistical evaluation used one-way ANOVA with post-hoc Tukey test or Student's t-test. RESULTS: The tri-culture model allowed to track cellular behavior in a cell-type specific manner and recapitulated different characteristics of early melanoma stages. Cells arranged into a collagen-IV rich fibroblast core, a ring of keratinocytes, and groups of highly proliferating melanoma cells on the outside. Regularly, some melanoma cells were also found to invade the fibroblast core. In the absence of melanoma cells, the keratinocyte ring stratified into central basal-like and peripheral, more differentiated cells. Conversely, keratinocyte differentiation was clearly reduced upon addition of melanoma cells. Treatment with the cytostatic drug, docetaxel, restored keratinocyte differentiation and induced apoptosis of external melanoma cells. Remaining intact external melanoma cells showed a significantly increased amount of ABCB5-immunoreactivity. CONCLUSIONS: In the present work, a novel, simple spheroid-based melanoma tri-culture model composed of fibroblasts, keratinocytes, and melanoma cells was described. This model mimicked features observed in early melanoma stages, including loss of keratinocyte differentiation, melanoma cell invasion, and drug-induced increase of ABCB5 expression in external melanoma cells.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Técnicas de Cultura de Células/métodos , Técnicas de Cocultura/métodos , Esferoides Celulares/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Docetaxel/farmacologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Queratinócitos/citologia , Queratinócitos/metabolismo , Melanoma/metabolismo , Melanoma/patologia , Esferoides Celulares/citologia
18.
Math Biosci Eng ; 16(4): 2795-2810, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-31137238

RESUMO

This work investigates the mechano-biological features of cells cultured in monolayers in response to different osmotic conditions. In-vitro experiments have been performed to quantify the long-term effects of prolonged osmotic stresses on the morphology and proliferation capacity of glioblastoma cells. The experimental results highlight that both hypotonic and hypertonic conditions affect the proliferative rate of glioblastoma cells on different cell cycle phases. Moreover, glioblastoma cells in hypertonic conditions display a flattened and elongated shape. The latter effect is explained using a nonlinear elastic model for the single cell. Due to a crossover between the free energy contributions related to the cytosol and the cytoskeletal fibers, a critical osmotic stress determines a morphological transition from a uniformly compressed to an elongated shape.


Assuntos
Neoplasias Encefálicas/fisiopatologia , Glioblastoma/fisiopatologia , Pressão Osmótica , Astrócitos/metabolismo , Divisão Celular , Linhagem Celular Tumoral , Proliferação de Células , Simulação por Computador , Citoesqueleto/metabolismo , Dextranos/química , Elasticidade , Humanos , Microscopia , Modelos Biológicos , Osmose , Pressão , Esferoides Celulares/citologia , Estresse Fisiológico , Resultado do Tratamento
19.
BMC Cancer ; 19(1): 502, 2019 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-31138163

RESUMO

BACKGROUND: Every biological experiment requires a choice of throughput balanced against physiological relevance. Most primary drug screens neglect critical parameters such as microenvironmental conditions, cell-cell heterogeneity, and specific readouts of cell fate for the sake of throughput. METHODS: Here we describe a methodology to quantify proliferation and viability of single cells in 3D culture conditions by leveraging automated microscopy and image analysis to facilitate reliable and high-throughput measurements. We detail experimental conditions that can be adjusted to increase either throughput or robustness of the assay, and we provide a stand alone image analysis program for users who wish to implement this 3D drug screening assay in high throughput. RESULTS: We demonstrate this approach by evaluating a combination of RAF and MEK inhibitors on melanoma cells, showing that cells cultured in 3D collagen-based matrices are more sensitive than cells grown in 2D culture, and that cell proliferation is much more sensitive than cell viability. We also find that cells grown in 3D cultured spheroids exhibit equivalent sensitivity to single cells grown in 3D collagen, suggesting that for the case of melanoma, a 3D single cell model may be equally effective for drug identification as 3D spheroids models. The single cell resolution of this approach enables stratification of heterogeneous populations of cells into differentially responsive subtypes upon drug treatment, which we demonstrate by determining the effect of RAK/MEK inhibition on melanoma cells co-cultured with fibroblasts. Furthermore, we show that spheroids grown from single cells exhibit dramatic heterogeneity to drug response, suggesting that heritable drug resistance can arise stochastically in single cells but be retained by subsequent generations. CONCLUSION: In summary, image-based analysis renders cell fate detection robust, sensitive, and high-throughput, enabling cell fate evaluation of single cells in more complex microenvironmental conditions.


Assuntos
Fibroblastos/citologia , Processamento de Imagem Assistida por Computador/métodos , Melanoma/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Esferoides Celulares/citologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Técnicas de Cocultura , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Fibroblastos/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Melanoma/tratamento farmacológico , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Análise de Célula Única , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Microambiente Tumoral , Quinases raf/antagonistas & inibidores
20.
Biofabrication ; 11(3): 035025, 2019 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-31096204

RESUMO

Recently, stromal cell spheroids have been actively studied for use in tissue regeneration. In this study, we report a method for the fabrication of size-controllable stromal cell spheroids in different sizes from micro-scaled cell sheets (µCS) using thermosensitive hydrogels and investigated their effects on stromal cell function. Mesenchymal stromal cells isolated from different tissues such as human turbinate tissue, bone marrow, and adipose tissue were adhered selectively to each micro-pattern (squares with widths of 100 and 400 µm) on the surface of the hydrogel and formed µCS. The diameters of the spheroids were modulated by the size of the patterns (45 ± 5 and 129 ± 4 µm in diameter for the 100 and 400 µm micro-patterns, respectively) and the seeding density (129 ± 4, 149 ± 6, and 163 ± 6 µm for 5.0, 10.0, and 15.0 × 104 cells cm-2, respectively, on 400 µm micro-pattern). In addition, the spheroids were successfully fabricated regardless of stromal cell origin, and the diameter of the spheroids was also affected by cell spreading area on a cell culture dish. Stemness markers were highly expressed in the spheroids regardless of the spheroid size. Furthermore, an increase in E-cadherin and decrease in N-cadherin gene expression showed the stable formation of spheroids of different sizes. Gene expression levels of hypoxia inducible factors and secretion of vascular endothelial growth factor were increased (13.2 ± 1.4, 325 ± 83.4 and 534.3 ± 121.5 pg ng-1 DNA in a monolayer, and 100 and 400 µm micro-patterned spheroids, respectively) proportional to the diameters of the spheroids. The size of spheroids were maintained even after injection, cryopreservation and 7 d of suspension culture with high viability (∼90%). In conclusion, this novel technique to fabricate spheroids with controlled size could be widely applied in various applications that require a controlled size in regenerative medicine.


Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Mesenquimais/citologia , Microtecnologia/métodos , Esferoides Celulares/citologia , Indutores da Angiogênese/metabolismo , Adesão Celular/efeitos dos fármacos , Contagem de Células , Tamanho Celular , Criopreservação , Dimetilpolisiloxanos/química , Humanos , Hidrogéis/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Esferoides Celulares/efeitos dos fármacos , Células Estromais/citologia , Células Estromais/efeitos dos fármacos
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