Study Of 3D Bioprinted Glioma Model On Enrichment Of Glioma Stem Cells,Tumor Vascularization And Drug Resistance

Part I Enrichment of glioma stem cell-like cells on 3D porous scaffoldscomposed of different extracellular matrixBackground and objective:Cancer stem cells(CSCs),being tumor-initiating with self-renewal capacity and heterogeneity,are mostlikely the cause of tumor resistance,reoccurrence and metastasis.To further investigate the role of CSCsin tumor biology,there is a need to develop an effective culture system to grow,maintain and enrichCSCs.Three-dimensional(3D)cell culture model has been widely used in tumor research and drugscreening.At present,a variety of natural and synthetic scaffolds has been used to construct 3D porousscaffolds for the study of CSCs enrichment.However,themost effective composition that enriches CSCs is still unclear.In this study,glioma cell line was cultured with 3D porous chitosan(CS)scaffolds orchitosan-hyaluronic acid(CS-HA)scaffolds to explore the possibility of glioma stem cells(GSCs)-like cellsenrichment,to study the morphology,gene expression,and in vivo tumorigenicity of 3D scaffolds cells,and to compare results to 2D controls.Methods:1.CS and CS-HA solution were prepared respectively.After freeze-drying,3D porous CS and CS-HA scaffolds were obtained,which could be sterilized by gamma ray irradiation.2.CCK-8 reagent was used to analyze the cytotoxicity of scaffolds,and the porosity and swelling of the scaffold were determined by impregnation method.3.U87 glioma cells were inoculated on CS and CS-HA scaffolds respectively.The activity and proliferation of glioma cells on CS and CS-HA scaffolds were assessed by fluorescent living/dead reagents and Alma blue reagents.The morphological structure of glioma cells was observed by hematoxylin and eosin staining and scanning electron microscopy.4.Immunofluorescence staining was used to analyze the expression of CD 133 and Nestin in U87 cells under different culture conditions.The expression of CD44,HIF-la,Snail,CD133 and Nestin in U87 cells under different culture conditions was analyzed by qRT-PCR.Flow cytometry was used to analyze the expression of CD133+in U87 cells under three-dimensional scaffolds and two-dimensional culture conditions.5.Tumor cells were harvested from 2-D culture and 3-D porous scaffolds and inoculated subcutaneously in nude mice to observe the occurrence and volume changes of tumors.After 6 weeks of inoculation,specimens of transplanted tumors were collected for HE staining,CD31 and Ki67 immunohistochemical detection.Results:1.The average diameter of CS and CS-HA scaffolds obtained after freeze-drying is 13±0.8mm,and the thickness is2±0.5mm.2.Both scaffolds have good porosity after freeze-drying.The porosity of CS scaffolds is 90±3%,and the pore size is 98.37±5.57μm.The pore size of CS scaffolds is irregular.The porosity of CS-HA scaffolds is 86±2%,and the pore size is 94.13±3.67μm.Both CS and CS-HA scaffolds have no cytotoxicity and are suitable for cell growth.Both scaffolds have good water absorption and the swelling capacity of CS-HA scaffolds is higher than that of CS scaffolds.3.U87 cells have good cell viability on CS and CS-HA scaffolds.Compared with the traditional 2D culture,the proliferation of U87 cells on 3D porous scaffolds is more stable.U87 cells in 3D scaffolds gradually formed multicellular clusters and aggregated into tumor cell spheres,while the tumor cells on 2D dishes grew in epithelioid form.4.CD133 and Nestin mRNA contents in CS and CS-HA scaffolds were higher than those in CS-HA scaffolds on the 10th day of culture.The relative expression of CD 133 and Nestin in CS-HA scaffolds was higher than that in CS-HA scaffolds.In addition,the expression of epithelial-mesenchymal transformation(EMT)related genes CD44,HIF-1α,and Snail in CS-HA scaffold was higher than that in CS scaffold and the proportion of GSC-like cells in CS-HA scaffold was higher than that in 2D monolayer and CS scaffold culture.5.The tumorigenic rates of tumor cells cultured by CS-HA and CS scaffolds were 100%and 80%,respectively,which were significantly higher than 40%of 2D,and the tumor volume formed by 3D cultured cells was significantly larger than that of 2D cultured cells.Conclusion:Compared with 2D culture,both CS and CS-HA scaffolds can mimic the microenvironment of glioma.3D porous scaffolds promote cell-cell and cell-extracellular matrix interactions.Particularly,binding to HA induces activation of downstream signaling pathways.CS and CS-HA scaffold culture not only promoted the formation of tumor cell spheres,but also compared with 2D monolayer culture,both scaffolds have the ability to enrich GSC-like cells,especially CS-HA scaffold culture.Part Ⅱ 3D bioprinted glioma cell-laden hydrogelscaffolds enriching glioma stem cellsBackground and objective:Glioma stem cells(GSC)are considered to be the source of glioma recurrence,progression,and resistance to chemoradiotherapy.GSC accounts for less than 1%of the total number of glioma cells.Establishing an ideal in vitro model for culture and enrichment of GSC will help to further study the biological behavior of GSC.In the second part,gelatin/alginate/fibrinogen hydrogel scaffolds containing glioma cell line U118 were used to enrich GSC like cells by 3D bioprinting technology.To evaluate the activity,morphology,proliferation and expression of GSC markers and EMT-related genes in 3D bioprinted scaffolds and compare them with 2D.Finally,the sensitivity and tumorigenicity of tumor cells to temozolomian under different culture conditions were analyzed.Methods:1.The printing materials with 1%sodium alginate,10%gelatin and 1%fibrinogen were prepared by mixing the cell suspension with 4%sodium alginate,20%gelatin and 4%fibrinogen in volume ratio of 1:1:2:1.2.A grid hydrogel scaffold with a width of 15 x 15mm and a thickness of lmmwas printed by a bioprinter.(LivPrintTM).3.The activity and proliferation of cells in hydrogel scaffolds were evaluated by fluorescent living/dead reagents and Alma blue reagents.The morphological structure of cells in hydrogel scaffolds and 2D culture was observed by hematoxylin and eosin staining,phalloidin stainingand scanning electron microscopy.4.Immunofluorescence staining was used to analyze the expression of glioma stem cell markers CD133 and Nestin in U118 cells under 3D and 2D culture conditions,the gene expression of Twistl,HIF-la,Snail,VEGF,CD 133 and Nestin in U118 cells under different culture conditions was analyzed by qRT-PCR.Flow cytometry was performed to analyze the proportion of CD133+ cells in U118 cells under 3D hydrogel scaffolds and 2D culture conditions.5.Temozolomide solution with concentration of 0,100,200,400,800,1600 ug/mL was added to cells cultured under two different conditions for 48 hours,cell viability was detected by Alma blue reagent.6.Tumor cells were harvested from 2-D culture and 3-D hydrogel scaffolds and inoculated subcutaneously in nude mice to observe the occurrence and volume changes of the tumors.After 6 weeks of inoculation,the transplanted tumors were collected for HE staining,and immunofluorescence detection of CD31 and vWF.Results:1.3D bioprinted hydrogel scaffold with interworking structure facilitates the exchange of nutrients and metabolic waste,and hydrogel scaffold maintains a good morphological structure even after 15 days of culture.2.The cell survival rate immediately after printing was 89.06±3.58%,and it was 84.30±2.67%after culture for 15 days.U118 cells grew in a single layer in traditional 2D culture,while cell gradually aggregate to form tumor spheres in 3D printed hydrogel scaffold.Cells proliferated rapidly fromday 1 to day 5 in 2D culture,and proliferation became slower after 5 days.After 10 days,the cell proliferation decreased rapidly,while cell proliferation in 3D bioprinted hydrogel scaffold gradually increased,and tended to be stable after 15 days.3.The mRNA contents of CD133,Nestin and epithelial-mesenchymal transformation(EMT)related genesHIF-1α,Snail,Twist and VEGF in 3D hydrogel scaffold were higher than those in 2D culture,and flow cytometry results indicated that the proportion of CD133+cells in 3D was higher than that in 2D culture.4.Cells cultured in 3D have stronger drug resistance than those cultured in 2D.5.Compared with 2D culture,U118 cells cultured in 3D hydrogel scaffolds have higher tumorigenic rate,larger tumor volume and stronger angiogenesis.Conclusion:Compared with conventional 2D culture,3D bioprinted tumor model can better mimic tumor microenvironment in vivo.The bioprinted glioma cell-laden hydrogel scaffold can enrich glioma stem cell-like cells,and cultured glioma cells have stronger drug resistance and tumorigenicity in vivo.Part Ⅲ Bioprinting of glioma stem cells improves their endotheliogenic potentialBackground and objective:Glioblastoma(GBM)is a highly malignant primary brain tumor with highdegree of vascularization.Studies have shown that glioma stem cells(GSC)are involved in the angiogenesis of tumors.However,most of GSCsused for in vivo and in vitro GBM angiogenesis studies were suspensioncultured in serum-free medium.The secretedproducts of suspension cultured GSCs,such as vascular endothelialgrowth factor(VEGF),diffuse in medium,which is not beneficial to improve theparacrine and autocrine function of cells.Furthermore,suspensionculture cannot well mimic the 3D microenvironment of GSCs.In this study,glioma stem cell GSC23-laden hydrogel scaffolds wereprinted by a 3D bioprinter.Viability,proliferation and morphology of3D bioprinted GSC23 were analyzed and compared with those of traditional suspension cultured cells.Furthermore,the level of vascular endothelial growth factor A(VEGFA)secreted by GSC23 and the expressionof tumor angiogenesis-related genes were evaluated.Finally,we investigated the in vitro vascularization capability of GSC23 underdifferent culture conditions.Methods:1.The printing materials with 1%sodium alginate,10%gelatin and 1%fibrinogen were prepared by mixing the cell suspension with 4%sodium alginate,20%gelatin and 4%fibrinogen in volume ratio of 1:1:2:1.2.A circular grid hydrogel scaffold with a diameter of 15 mm and a thickness of 1 mm was printed bya bioprinter(LivPrintTM).3.The activity and proliferation of cells in 3D scaffolds were evaluated by fluorescent living/dead reagents and Alma blue reagents.The morphological structure of cells in 3D scaffolds and suspension culture was observed by hematoxylin and eosin staining,transmission electron microscopeand scanning electron microscopy.4.The secretion of VEGFA by GSC23 was detected by ELISA kit in 3D hydrogel and suspension culture.The gene expression of CD31,VEGFR2 and CD 133 in GSC23 under different culture conditions was analyzed by qRT-PCR.Flow cytometry was performed to analyze the proportion of CD 133+cells in GSC23 cells under 3D hydrogel scaffolds and suspension culture conditions.5.GSC23 from suspension culture and 3D hydrogel scaffolds were seeded on Matrigel-coated well plates,and tubular structure formed by GSC23 was observed under microscope.Results:1.3D bioprinted hydrogel scaffold with porous channelsfacilitates the exchange of nutrients and metabolic waste,and hydrogel scaffold maintains a good morphological structure even after 15 days of culture.2.Cell survival rate immediately after printing was 86.27±2.41%,and after 15 days of culture was 89.39±1.86%.GSC23 cells grew gradually from cluster to sheet in traditional suspension culture,while GSC23 cells in 3D printed hydrogel scaffold gradually accumulated to form tumor cell spheres,Cell proliferation in suspension culture increased significantly from day 1 to day 5,and decreased rapidly after day 5.However,cell proliferation in 3D scaffolds gradually increased,reaching a peak on day 10,and then stabilized.3.VEGFA content in suspension culture gradually increased within the first 7 days and then decreased slowly.However,VEGFA in 3D bioprinted hydrogel scaffolds gradually increased and stabilized after 9 days of culture.4.The mRNA expressions of CD31,VEGFR2 and CD 133 in 3D hydrogel scaffolds were higher than those in suspension culture,and flow cytometry results indicated that the proportion of CD133+phenotype cells was higher than that in suspension culture.5.Compared with suspension culture,3D cultured GSC23 cells formed a more defined tubular structure with more meshes.6.The microvilli of suspension cultured GSC23 were not developed,and microvilli of GSC23 cultured in 3D bioprinted scaffold was long and numerous.In addition,GSC23 in 3D hydrogel scaffold has abundant mitochondria and rough endoplasmic reticulum.Conclusion:The microenvironment provided by 3D bioprinted scaffolds can not only promote cell growth and proliferation,but also affect cell morphology and biological behavior.Compared with traditional suspension culture,the stemness of GSC,the expression of tumor angiogenesis related genes and the ability of in vitro vascularization were improved.Part Ⅳ Coaxial extrusion bioprinted shell-core hydrogel microfibers mimic gliomamicroenvironment and enhance the drug resistance of cancer cellsBackground and objective:Although chemotherapy can further kill the residual tumor cellsnafter glioblastoma(GBM)surgery,the drug resistance of tumor cells causes recurrence of GBM.Achieving a desired tumor model fordrug evaluation remains a challenge,because of the inability of thecurrent models to mimic in vivo tumor microenvironment.Tumor microenvironment consists of cancer cells,cancer stemcells and mesenchymal cells,among which cancer stem cells play a keyrole in tumorigenesis,progression,recurrence and drug resistance.Cancer stem cells interact with tumor microenvironment to promote thedevelopment of tumor.Up to this date,glioma models constructedwith bioprinting platform either contain only glioma cells or gliomastem cells(GSCs).Constructing a model consisting of gliomacells and GSCs is beneficial for mimicking glioma microenvironmentand studying of biological behavior of glioma cells,as well as drugresistance.In this study,shell-GSC23/core-U 118(G/U)hydrogel microfiberswere fabricated by coaxial extrusion bioprinting.Here,cell viability and morphology of shell-corehydrogel microfibers were analyzed.Furthermore,the expression ofdrug resistance-related genes of U118 cultured in G/U microfiber wasevaluated and compared to that of U microfiber cultured cells.Moreover,the in vitro sensitivity of U118 cells cultured in differentconditions to chemotherapeutic drug temozolomide(TMZ)was investigated.Finally,we explored the mechanism of increased drug resistancein U118 cells derived from G/U hydrogel microfibers.Methods:1.Glioma stem cells GSC23 and 4%sodium alginate solution were mixed to obtain the shell-material needed for coaxial printing.Glioma cells U118 were suspended in medium to obtain the core-material needed for printing.The shell-GSC23/core-U118 and shell/core-U118 hydrogel microfibers were printed with a customized two-coaxial printing device.2.The viability and proliferative ability of printed cells were evaluated by living/dead reagents and Alma blue reagents.The morphological structure of cells in hydrogel microfibers was observed by hematoxylin and eosin(HE)staining and scanning electron microscopy.3.The expression of MMP9,MMP2,MGMT and VEGFR2 in U118 cells under different culture conditions was analyzed by qRT-PCR.The expression of MMP9 and MGMT related to drug resistance was detected by Western blot.4.Temozolomide solution with concentration of 0,100,200,400,800,1600 ug/mL was added to cells cultured under two different conditions for 48 hours,cell viability was detected by Alma blue reagent.5.DNA methylation rate of U118 cells cultured under different conditions was evaluated by bisulfite sequencing PCR(BSP).Results:1.The inner and outer diameters of shell-core hydrogel microfibers constructed by coaxial extrusion bioprinting are407.36±11.42μm and 867.53±12.28μm,respectively.2.Cell viability was 93.72±2.51%after 2 h of coaxial printing.After 15 days of culture,cells in shell-core hydrogel microfibers maintained a high survival rate of 90.63±1.54%.HE staining showed that GSC23 cells were encapsulated in the shell,and the core was filled with U118 cells.SEM further revealed that the sodium alginate shell enveloped and supported U118 cells to form tumor cell spheres.Cells proliferated stably in hydrogel microfibersduring culture.3.Compared with shell/core-U118 hydrogel microfibers,the expression of MMP9,MMP2,MGMT and VEGFR2 in U118 cells in shell-GSC23/core-U 118 hydrogel microfibers was significantly increased,and the expression of MMP9 and MGMT related to drug resistance was also significantly increased.4.With the increase of TMZ concentration,cell viability decreased gradually.Compared with cells cultured in shell/core-U118 hydrogel microfibers,cells cultured in shell-GSC23/core-U118 hydrogel microfibers showed stronger viability,which indicated that U118 cells cultured in shell-GSC23/core-U118 hydrogel microfibers had stronger drug resistance.5.The methylation rate of MGMT gene in shell-GSC23/core-U118 hydrogel microfibers cultured U118 cells was 18.95%,while the methylation rate of MGMT in shell/core-U118 hydrogel microfibers was 40.26%.The higher methylation degree of MGMT in tumor cells,the lower drug resistance of tumors.Conclusion:Shell-GSC23/core-U118 hydrogel microfiber showed greatpotential in mimicking glioma microenvironment that can be potentiallyused to evaluate the drug resistance of cancer cells.Comparedwith shell/core-U118 hydrogel microfibers,expression of core-U118genes associated with tumor invasion and drug resistance was significantlyincreased in shell-GSC23/core-U118 microfibers.More importantly,U118 cells derived from shell-GSC23/core-U118 microfibershad greater chemoresistance in vitro.