| With the increasing prevalence and mortality of malignant tumors,the pathogenesis and physiological characteristics of tumors is of essence to be understood while offering guidelines and knowledge to develop new effective tumor diagnosis and treatment technology for high cure rate.A three-dimensional(3D)cell culture method endows the in vitro cell model with a higher correlation with clinical cases and biology.3D tumor spheroid shows higher similarity to native tissues in terms of 3D structure,interactions between cell and microenvironment,metabolism,invasion,geno/phenotype than 2D cultured cells,and have therefore gained increasing attention in biology and biomedical research.Currently,3D tumor spheroid has been used for enrichment of tumor/cancer stem cells and preclinical evaluation of anti-tumor drugs.3D cell scaffolds,benefited from their flexible biochemical and biophysical characteristics,have been developed and explored in tissue engineering and oncobiology applications to closely mimic the critical features of the in vivo extracellular matrix(ECM)environment.Nevertheless,either the complexity of wet chemical reaction and residual organic solvent,or expensive machinery greatly hinders the applications of scaffolds in 3D tumor spheroid,especially in the large-scale applications in general biology laboratory with restricted resources.Due to its many attractive properties such as biocompatibility,three-dimensional fiber network,ease of chemical and physical modifications,cost efficiency,eco-friendliness and ease of large scale manufacturing,paper has been used as a new material in many biomedical applications.For instance,paper has been used as a substrate for cell calture.Nevertheless,the effect of physical and chemical properties of paper on cell behaviors have never been inverstigated,which hinders the development of paper-based cell assay for tumor biological studies.In this thesis,by studying the influence of physical and chemical properties of paper-based materials on cell behaviors,the potential of paper as the base material for 3D tumor spheroid formation was explored at the first time.More importantly,a series of 3D tumor spheroid platforms were constructed.The main research contents are as follows:1.Investigating the effect of physical and chemical properties of paper on cell behaviorsThe physical and chemical properties of biomaterials closely affect the biological response of cells,including cell adhesion,survival,cycle process and phenotype.Paper have been emerged as a new base matrial in microfluidic analytical devices and cell culture,but the effect of physical and chemical properties of paper on cell biohaviors has not been investigated.Take Whatman(?)114 filter paper and Chinese rice paper as two model,the properties of paper substat on cell adhesion and morphological characteristics were studied.The results show that there are significant differences in fiber substructure,hydrophobicity,element composition and valence between filter paper and rice paper.The obtained results suggested that the excellent hydrophilicity of the cellulose fibers retarded the hydrophobic interaction-mediated cell anchoring on the cellulose fibers,while the limited space/ niche between fibers promoted the aggregation of cells.While the adherent cells spread on smooth and comparatively less hydrophilic surface of rice paper exhibiting trigonal morphologies.This study indicates that paper show dual functions in cell culture,which can not only provide a three-dimensional network growth framework,but also influence cell behavior by means of physical and chemical properties,providing a theoretical basis for the design of ecnomical 3D tumor platform based on paper.2.Spontaneous formation of tumor spheroid on a hydrophilic filter paper for cancer stem cell enrichmentCancer stem cells(CSCs)play a key role in tumor invasion,metastasis and recurrence.However,isolation,enrichment and cultivation of these crucial and infrequent groups of tumor cells for in vitro analyses is a hard job.Our studies have found that 3D tumor spheroid can be spontaneously formated on a hydrophilic filter paper.3D tumor spheroids may prove to be an effective tool for isolation and enrichment of CSCs.Herein,we aimed to characterize the stemlike properties of prostate cancer cell lines cultured on filter paper.The results showed that the filter paper-grown cells had elevated expression of putative CSC markers(ALDH1 A1,OCT4,SOX2 and NANOG),indicating increased stemness of the cancer cells.Moreover,increased resistance of the chemotherapeutic drug doxorubicin was observed on the formed CSC spheroids compared to regular culture.The results of flow cytometry showed growing cells on filter paper for 96 h leading to an increase in fraction of ALDH1A1+,CD44+ and CD133+ in DU 145 and LNCap cells.This study demonstrated that hydrophilic filter papers could provide an effective and low-cost platform for inducing and enrichment of CSCs,which may facilitate more advanced investigations in cancer biology or screening of anti-tumor drugs.3.Novel core-shell silk fibroin @ rice paper fibrous scaffold for 3D tumor spheroids formationAlthough electrospinning is one of the most used approaches to fabricate silk fibroin fibrous scaffold,there are still many disadvantages,such as low yield,residual organic solvents,limited extensibility in fiber and lack of spatial control over pore size.Our hypothesis is that silk fibroin funcitonalzied paper sacffold can have the biocompatibility of silk fibroin and 3D fibros structure of paper.To this aim,a core-shell silk fibroin @ rice paper(SF@RP)fibrous scaffold was fabricated by a mild one-step dip coating method.The study indicated that by tunning the second-order protein conformation of silk fibroin on the surface of SF @ RP scaffold,the cell behavior on silk fibroin @ rice paper can be controlled.Tumor spheroids can be formed on SF @ RP composite scaffolds with the dominant conformation of α-Helical /random strutures.While cells adherent and spread on SF @ RP composite scaffolds with the dominant conformation of β-sheet strutures.The cell growth behavior of different tumor cell lines on SF@RP substrate have been studied.The physiological properties of hepatcarcinoma Hep G2,such as toxin metabolizing,protein secretion and drug resistance were inverstigated.The results show that tumor spheroids spontaneous formed on the SF @ RP composite scaffolds have higher level of CYP 3A4,CYP 1A1,albumin gene expression and drug durancy than Hep G2 monolayer cells.In summery,dip-coating method prepared SF@RP scaffolds is a novel biocompatible substrate for cell culture,especially for tumor cell spheroid formation.4.Ready-to-use microporose gelatin @ paper-like hybdrid scaffold for cancer stem cell enrichmentThe interaction of cells with micro-nano scale topography has proven to be a pivotal signal source in controlling cell behaviors or functions in 2D culture model.To investigate the impact of microstructures in a 3D scaffold on cell’s physiological behavior,gelatin was used to modify lens paper and polypropylene nonwoven,two paper-like substrate.The aperture-controlled porous gelatin was fabricated on paper-like substrate by grafting gelatin and subsequent lyophilizaiton.The gelain@paper-like sheet with different pore size were obtained by tunning the concentration of gelatin and pre-cooling temperature.The cell adhesion and growth assay were conducted with the as prepared gelain@paper-like sheet.The results showed that when the pore size was smaller than the cell size,the cells tended to aggregate to form 3D spheroid.A higher cell migration ability was witnessed from the cells grown on gelain@paper-like sheet.The q PCR and western-blot results showed that the cells in spheroids formed on gelain@paperlike sheet had an increased CSC – related biomarker expression than the cells cultured on TCP,which suggests that the gelain@paper-like scaffold can be used to fast generating of tumor spheroids and enrichment of CSC.More importantly,the gelatin @ paper-like scaffold can still induce cells aggregate to form 3D tumor spheroid after 90 days storage at 4 ℃,highlighting its ready-to-use potential in 3D cell culture. |
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