Three-dimensional cell culture for the study of nasal polyps

Abstract Objectives: Three-dimensional (3D) cell cultures have many applications such as stem cell biology research, new drug discovery, cancer, and Chronic Rhinosinusitis with Nasal Polyps (CRSwNP). This disease is characterized by a significant impact on quality of life and productivity. The diversity of factors that act in the progression of CRSwNP point to the creation of a cell culture model that allows the integration of different cell types with extracellular matrix. This work aimed to create a cell culture model in 3 dimensions (spheroids) for the study of Nasal Polyposis. Methods: Nasal polyp tissue from patients diagnosed with CRSwNP was mechanically dissociated using tweezers and a scalpel and the solution containing cells and small aggregates of nasal polyps was transferred to a Petri dish containing 5 mL of culture medium at the concentration of 106 cells/mL. Results: The spheroids were cultivated for 20 days, fixed and analyzed using confocal microscopy. In a 3D culture environment, the spheroids were formed both by clustering cells and from small tissue fragments. In the cultures analyzed, the ciliary beat was present from the dissociation of the cells up to 20 days in culture. Conclusion: Our findings also point to these characteristics showing the environment generated in our study, the cells remained differentiated for a longer time and with ciliary beating. Thus, this work shows that nasal polyp-derived cells can be maintained in a 3D environment, enabling better strategies for understanding CRSwNP in situations similar to those found in vivo. Level of evidence: Laboratory studies.

3D spheroid cell cultures and their role in bone regeneration: a systematic review / Cultivos de esferoides 3D y su papel en la regeneración ósea: una revisión sistemática

Abstract Recently, the 3D spheroid cell culture application has been extensively used in the treatment of bone defects. A wide variety of methodologies have been used, which has made the comparison of results complex. Therefore, this systematic review has two aims: (i) to perform an analysis focused on the role of 3D spheroid cell culture in bone regeneration strategies; and (ii) address the main challenges in clinical application. A search of the following keywords "3D cell culture", "spheroid", and "bone regeneration" was carried out in the PubMed, Scopus, and ScienceDirect databases and limited to the years 2010-2020. Studies were included if their primary objective was the behavior of cell aggregates to formed spheroids structures by different 3D cell culture techniques focused on the regeneration of bone tissue. To address the risk of bias for in vitro studies, the United States national toxicology program tool was applied, and descriptive statistics of the data were performed, with the SPSS V.22 program. A total of 16 studies were included, which met the established criteria corresponding to in vitro and in vitro/in vivo studies; most of these studies used stem cells for the 3D cell spheroids. The most often methods used for the 3D formation were low adherence surface and rotational methods, moreover, mesenchymal stem cells were the cell line most frequently used because of their regenerative potential in the field of bone tissue engineering. Although the advances in research on the potential use of 3D spheroids in bone regeneration have made great strides, the constant innovation in cell spheroid formation methodologies means that clinical application remains in the future as strategy for 3D tissue bioprinting.

Resumen Recientemente, la aplicación del cultivo 3D de esferoides se ha utilizado ampliamente en el tratamiento de defectos óseos. La variedad de metodologías para lograr los cultivos 3D de esferoides ha hecho compleja la comparación de resultados. Por tanto, esta revisión sistemática tiene dos objetivos: (i) realizar un análisis centrado en el papel de los cultivos 3D de esferoides en las estrategias de regeneración ósea; y (ii) abordar los principales desafíos en la aplicación clínica. Se realizó una búsqueda de las siguientes palabras clave "cultivo celular 3D", "esferoide" y "regeneración ósea" en las bases de datos PubMed, Scopus y ScienceDirect y se limitó a los años 2010-2020. Se incluyeron los estudios si su principal objetivo era el comportamiento de agregados celulares para generar las estructuras esferoidales desarrollados por diferentes técnicas de cultivo celular 3D enfocadas a la regeneración del tejido óseo. Para abordar el riesgo de sesgo de los estudios in vitro, se aplicó la herramienta del programa nacional de toxicología de Estados Unidos y se realizaron estadísticas descriptivas de los datos, con el programa SPSS V.22. Se incluyeron un total de 16 estudios, que cumplieron con los criterios establecidos correspondientes a estudios in vitro e in vitro/in vivo; la mayoría de estos estudios utilizaron células troncales para generar los esferoides celulares 3D. Los métodos más utilizados para la formación de los esferoides 3D fueron la superficie de baja adherencia y los métodos de rotación, asimismo, la línea celular de células troncales mesenquimales fueron las más utilizadas debido a su gran potencial regenerativo en el campo de la ingeniería de tejidos óseos. Aunque los avances en la investigación sobre el uso potencial de los cultivos celulares de esferoides 3D en la regeneración ósea han logrado grandes avances, la constante innovación en las metodologías de la generación de esferoides 3D deja claro que la aplicación clínica de estos permanecerá en el futuro como estrategia en la bioimpresión tisular.

Development of a novel high throughput brain-on-chip with 3D structure and its application in evaluation of pesticide-induced-neurotoxicity / 生物工程学报

We designed and fabricated a novel high throughput brain-on-chip with three dimensional structure with the aim to simulate the in vivo three-dimensional growth environment for brain tissues. The chip consists of a porous filter and 3D brain cell particles, and is loaded into a conventional 96-well plate for use. The filter and the particle molds were fabricated by using computer modeling, 3D printing of positive mold and agarose-PDMS double reversal mold. The 3D cell particles were made by pouring and solidifying a suspension of mouse embryonic brain cells with sodium alginate into a cell particle mold, and then cutting the resulting hydrogel into pieces. The loaded brain-on-chip was used to determine the neurotoxicity of pesticides. The cell particles were exposed to 0, 10, 30, 50, 100 and 200 µmol/L of chlorpyrifos or imidacloprid, separated conveniently from the medium by removing the porous filter after cultivation. Subsequently, cell proliferation, acetylcholinesterase activity and lactate dehydrogenase release were determined for toxicity evaluation. The embryonic brain cells were able to grow and proliferate normally in the hydrogel particles loaded into the filter in a 96-well plate. Pesticide neurotoxicity test showed that both chlorpyrifos and imidacloprid presented dose-dependent inhibition on cell growth and proliferation. Moreover, the pesticides showed inhibition on acetylcholinesterase activity and increase release of lactate dehydrogenase. However, the effect of imidacloprid was significantly weaker than that of chlorpyrifos. In conclusion, a novel brain-on-chip was developed in this study, which can be used to efficiently assess the drug neurotoxicity, pharmacodynamics, and disease mechanism by combining with a microtiterplate reader.

Research progress in 3D cell culture model of ovarian cancer tumor microenvironment / 实用肿瘤学杂志

Ovarian cancer(OC)is an aggressive and fatal cancer. A growing number of studies have shown that the tumor mi-croenvironment(TME)is involved in the promotion and development of ovarian cancer,immunosuppression and inflammatory response through various mechanisms. TME includes tumor blood vessels and lymphatic vessels,as well as cancer cells,mesenchymal cells,im-mune cells,and extracellular matrix(ECM). Based on recent literature reports,this paper reviews the commonly used three-dimen-sional(3D)cell culture model of ovarian tumor microenvironment,and summarizes many 3D models that do not contain primitive stro-mal cells,aiming to find new approaches for ovarian cancer treatment.

Three-dimensional Culture of Human Airway Epithelium in Matrigel for Evaluation of Human Rhinovirus C and Bocavirus Infections / 生物医学与环境科学（英文）

OBJECTIVE@#Newly identified human rhinovirus C (HRV-C) and human bocavirus (HBoV) cannot propagate in vitro in traditional cell culture models; thus obtaining knowledge about these viruses and developing related vaccines are difficult. Therefore, it is necessary to develop a novel platform for the propagation of these types of viruses.@*METHODS@#A platform for culturing human airway epithelia in a three-dimensional (3D) pattern using Matrigel as scaffold was developed. The features of 3D culture were identified by immunochemical staining and transmission electron microscopy. Nucleic acid levels of HRV-C and HBoV in 3D cells at designated time points were quantitated by real-time polymerase chain reaction (PCR). Levels of cytokines, whose secretion was induced by the viruses, were measured by ELISA.@*RESULTS@#Properties of bronchial-like tissues, such as the expression of biomarkers CK5, ZO-1, and PCK, and the development of cilium-like protuberances indicative of the human respiration tract, were observed in 3D-cultured human airway epithelial (HAE) cultures, but not in monolayer-cultured cells. Nucleic acid levels of HRV-C and HBoV and levels of virus-induced cytokines were also measured using the 3D culture system.@*CONCLUSION@#Our data provide a preliminary indication that the 3D culture model of primary epithelia using a Matrigel scaffold in vitro can be used to propagate HRV-C and HBoV.

Polypeptide Thermogels as Three-Dimensional Scaffolds for Cells

BACKGROUND: Thermogel is an aqueous solution that exhibits a sol-to-gel transition as the temperature increases. Stem cells, growth factors, and differentiating factors can be incorporated in situ in the matrix during the sol-to-gel transition, leading to the formation of a three-dimensional (3D) cell-culture scaffold. METHODS: The uses of thermogelling polypeptides, such as collagen, Matrigel™, elastin-like polypeptides, and synthetic polypeptides, as 3D scaffolds of cells, are summarized in this paper. RESULTS: The timely supply of growth factors to the cells, cell survival, and metabolite removal is to be insured in the cell culture matrix. Various growth factors were incorporated in the matrix during the sol-to-gel transition of the thermogelling polypeptide aqueous solutions, and preferential differentiation of the incorporated stem cells into specific target cells were investigated. In addition, modulus of the matrix was controlled by post-crosslinking reactions of thermogels or employing composite systems. Chemical functional groups as well as biological factors were selected appropriately for targeted differentiation of the incorporated stem cells. CONCLUSION: In addition to all the advantages of thermogels including mild conditions for cell-incorporation and controlled supplies of the growth factors, polypeptide thermogels provide neutral pH environments to the cells during the degradation of the gel. Polypeptide thermogels as an injectable scaffold can be a promising system for their eventual in vivo applications in stem cell therapy.

Advances on the study of limbal stem cells culture in vitro / 国际眼科杂志(Guoji Yanke Zazhi)

@#LSCs(limbal stem cells)are one of the adult unipotent stem cells located in the Vogt palisade area of the limbal cornea. It can supplement the repair of corneal epithelium by self renewal and play an important role in maintaining normal corneal epithelial integrity, corneal transparency and maintaining normal vision. The lack of corneal limbus stem cells caused by various reasons will lead to corneal turbid, ocular surface vascularization, and final blindness. The main methord to treat related diseases is to cultivate corneal limbal stem cells <i>in vitro</i> and retransplantation. How to effectively expand the limbal stem cells <i>in vitro</i> is the key to the success of the treatment. The location, acquisition methods and expansion methods of limbal stem cells were reviewed.

3D tissue engineering, an emerging technique for pharmaceutical research

Tissue engineering and the tissue engineering model have shown promise in improving methods of drug delivery, drug action, and drug discovery in pharmaceutical research for the attenuation of the central nervous system inflammatory response. Such inflammation contributes to the lack of regenerative ability of neural cells, as well as the temporary and permanent loss of function associated with neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, and traumatic brain injury. This review is focused specifically on the recent advances in the tissue engineering model made by altering scaffold biophysical and biochemical properties for use in the treatment of neurodegenerative diseases. A portion of this article will also be spent on the review of recent progress made in extracellular matrix decellularization as a new and innovative scaffold for disease treatment.

Application of three-dimensional cell culture in the study of Alzheimer's disease / 中国比较医学杂志

Cells in two-dimensional(2D)cultures gradually lose their original traits as they are passaged in vitro. Existing animal models are expensive and animal experiments require too much work to conduct large-scale experiments. Three-dimensional(3D)cell culture models have attracted increasing attention as they can circumvent the limitations of these previous two models. In 2014, it was reported that the incidence of Alzheimer's disease(AD)in people over 65 years old was 5.14%. In China,the number of AD patients reached 8 million in 2016,but the pathogenesis of the disease is not yet clear. Study of AD is a 'hot' yet complicated issue. This article will briefly introduce 2D and 3D cell cultures and the application of 3D cell culture to AD research in recent years.

Matrix stiffness regulates cell uptake of nanoparticles in 2D and 3D cultures of breast cancer cells / 药学学报

Fluorescent polystyrene nanospheres (PS) were used to explore the impact of substrate stiffness on cell uptake of nanoparticles in the breast cancer cells. Polyacrylamide (PAA) gels with varying stiffness were prepared by photopolymerization, and type I rat tail collagen was covalently conjugated on the surface of PAA gels to facilitate cell adhesion. Type I rat tail collagen was also used to fabricate collagen gels for 3D cell culture. Cells of human breast cancer cell line MCF-7 were incubated in the 2D culture on PAA gels and 3D culture within collagen gels. Next, nanospheres of 20 nm and 50 nm polystyrene were applied to MCF-7 cells in the 2D or 3D cultures. Cell morphology and uptake efficiency were observed with confocal microscopy. Our study demonstrates that substrate stiffness differentially regulated the cell morphology as well as the cell uptake behavior of polystyrene nanospheres in MCF-7 cells under 2D or 3D culture conditions.

The study on Notch1 pathway-related tubular formation of glioma cell lines / 中国神经精神疾病杂志

Objective To explore the mechanism of the formation of glioma cells derived vessels (GCDV).Methods The tubular formation assay was performed on 3D cell cultures of U87,U251,U373,SF295,T98G,SKMG-4 and C6 glioma cell lines.The expression of Notch1 、Dll4 were examined by western blot analysis.Result The mean number of vasculogenic channels of cell lines C6,U373,U87,T98G,SF295,U251 and SKMG-4 per area (100×) were 25.2±5.0,36.4±3.2,19.0±2.2,12.6±2.4,4±2.2,0.2±0.4 and 0,respectively.The relative protein densities of Notchl in U87,U251,T98G,SF295,SKMG-4,C6 and U373 were 0.34,0.21,0.79,0.04,0.28,1.75 and 1.19,which were significantly related with the tubular formation ability (P＜0.05).However,the expression of Dll4 was not associated with tubular formation ability (P＞0.05).Conclusion Notch1 may play a key role in tubular formation of glioma cells while the role of Dll4 in the process needs further study.

Cytotoxicity Profile of Endodontic Sealers Provided by 3D Cell Culture Experimental Model

Abstract The aim of the present study was to evaluate the cytotoxic effects of five endodontic sealers (AH Plus, Endomethasone N, EndoSequence BC, MTA Fillapex and Pulp Canal Sealer EWT) using a three-dimensional (3D) cell culture model. A conventional bi-dimensional (2D) cell culture model was used as reference technique for comparison. Balb/c 3T3 fibroblasts were cultured in conventional bi-dimensional cell culture and in rat-tail collagen type I three-dimensional cell culture models. Then, both cell cultures were incubated with elutes of freshly mixed endodontic sealers for 24 h. Cell viability was measured by the methyl-thiazol-diphenyltetrazolium assay (MTT). Data were statistically analyzed using ANOVA and the Tukey test at a significance level of p<0.05. All tested sealers exhibited cytotoxic effects; however, cytotoxic effect was culture model- and sealer-dependent. Sealers showed higher cytotoxicity in 2D than in 3D cell culture model (p<0.05). In both conditions, EndoSequence BC showed the lowest cytotoxicity (p<0.05). MTA Fillapex was much more cytotoxic than the other tested endodontic sealers (p<0.05), with the exception of AH Plus in the 2D cell culture model (p>0.05). Endomethasone N and Pulp Canal Sealer EWT showed lower cytotoxic effects than AH Plus in 2D cell culture model (p<0.05); however no statistical differences was observed among these sealers in 3D cell culture model. It may be concluded that cytotoxicity was higher in 2D cell culture compared to 3D cell culture. EndoSequence BC sealer exhibited the highest cytocompatibility and MTA Fillapex the lowest cytocompatibility.

Resumo O objetivo do presente estudo foi avaliar os efeitos citotóxicos de cinco cimentos endodônticos (AH Plus, Endomethasone N, EndoSequence BC, MTA Fillapex e Pulp Canal Sealer EWT) utilizando um modelo de cultura celular tridimensional (3D). Utilizou-se um modelo convencional de cultura de células bidimensionais (2D) como técnica de referência para comparação. Os fibroblastos Balb/c 3T3 foram cultivados em culturas de células bidimensionais convencionais e em modelos de cultura de células tridimensionais de colagéno de cauda de rato do tipo I. Em seguida, ambas as culturas de células foram incubadas com eluções dos cimentos endodônticos recém manipulados, durante 24 h. A viabilidade celular foi medida pelo ensaio de MTT. Os dados foram analisados estatisticamente utilizando ANOVA e o teste de Tukey com nível de significância de p<0,05. Todos os cimentos testados exibiram efeitos citotóxicos. Contudo, o efeito citotóxico foi dependente do modelo de cultura e do cimento testado. Os cimentos apresentaram maior citotoxicidade no modelo 2D do que no modelo 3D (p<0,05). Em ambas as condições, a EndoSequence BC apresentou a menor citotoxicidade (p<0,05). MTA Fillapex foi mais citotóxico do que os outros cimentos endodônticos testados (p<0,05), com exceção do AH Plus no modelo de cultura de células 2D (p>0,05). Endomethasone N e EWT mostraram efeitos citotóxicos mais baixos do que AH Plus no modelo de cultura de células 2D (p<0,05); entretanto, não houve diferenças estatísticas entre esses cimentos no modelo de cultura de células 3D. Pode concluir-se que a citotoxicidade foi maior na cultura de células 2D em comparação com a cultura de células 3D. EndoSequence BC selante exibiu a maior citocompatibilidade e MTA Fillapex a menor citocompatibilidade.

3D-printing microfludic device for breast cancer cell culture and pharmacological research / 中国药学杂志

OBJECTIVE: To develop a microfluidic mammalian cell culture device with human breast cancer cells co-cultured with extracellular matrix (ECM), reconstitute the 3-dimension human body microenvironment and preliminarily conduct drug screening on the breast cancer chip. METHODS: 3D Printing technology was utilized to build a two-layer microchip with cell culture reservoirs. The breast cancer cells (MCF7) were cultured in the matrix gel structure which mimiced the 3-dimension human body microenvironment. The cell toxicity of paclitaxel (PTX) on MCF7 cells was preliminarily observed using this microfluidic chip. With the use of AO/EB immunofluorescent staining and laser confocal microscopy, the cell death ratio induced by PTX was determined and compared with that determined by 2-dimension drug screening methods. RESULTS: MCF7 cells cultured on the chip successfully formed a 3D cavity structure in the matrix after 6-day dynamic incubation. The flow rate was 10 μL · h-1. After 24 h dynamic culture, the culture medium was changed to culture solution containing 0.02 μmol · mL-1 PTX, and the incubation was continued for 24 h. Obvious cell death was detected under laser confocal microscopy. CONCLUSION: The microfluidic chip developed in this study can successfully culture breast cancer cells in 3-dimension structure and perform drug screening, which lays a foundation for actualization of "human-on-a-chip".

Isolation and identification of lung cancer stem-like cells based on a 3D cell culture system / 中国肿瘤临床

Objective:To highlight the developmental process of 3D cell culture technology system, which is more suitable for isolating and identifying lung cancer stem-like cells than 2D cell culture technology system, and to explore the application of 3D cell cultures in the evaluation of proliferation, apoptosis, invasion, and drug resistance of lung cancer. Methods:Cells (104/well) from the human lung adenocarcinoma cell lines A549 and RPMI 1640 were cultured in complete medium containing 10%fetal bovine serum. Cell suspension was cultured in a BME basal medium. A growth curve was drawn after 7 d of culture. The stem-like cell was identified through a mammosphere culture, drug resistance and invasion assay, and flow cytometry. Data of A549 cells cultured in 3D and 2D tra-ditional cell culture technologies were compared. Results: Cells from the 3D cell culture had higher tumor formation rates [(20.75 ± 0.85) d vs. (60.25 ± 1.49) d, P<0.01)] and tumor sphere formation (28.50%± 1.17%vs. 8.67%± 0.80%, P<0.01) than those from the 2D cell culture. Moreover, cells from 3D cell culture were more invasive and resistant to therapy (58.17%± 2.19%vs. 41.70%±5.81%in 48 h, P<0.01;33.27%±5.76%vs. 27.30%±4.25%in 72 h, P<0.01). Phenotype experimental results demonstrated that the CD44 and CD326 cells were double-positive, whereas the CD24 cell was negative. Conclusion:The proportion of stem-like cells in A549 cell line after 3D cell culture significantly increased compared with 2D cell culture. The 3D cell culture can promote the proliferation of lung cancer stem cells.