3D Culture And Application Of Patient-derived Glioma Cells In Vitro

Glioma is the most common primary malignant tumor in the human central nervous system with a poor prognosis and the median survival time is generally only 12-15 months.At present,one of the main reasons for the failure of glioma treatment is the resistance of tumor cells to chemotherapy drugs.The disadvantage of the tumor model was that it was difficult to provide a tumor microenvironment in vitro,which has led to inconsistent drug results in vitro and in vivo.Recently,cell culture is gradually development from "monolayer" to "threedimensional".Patient-derived three-dimensional(3D)culture,also known as organoids,is a new model in vitro cancer that can simulate the tumor environment in vivo and provide guidance for clinical medication in the future.In this study,20 cases of fresh glioma tumor tissues were obtained including astrocytoma and glioblastoma.Single cells isolated from glioma tissue were directly sorted in a Matrigel-based 3D system without any sorting.It was found that 3D cultures from different patients showed different morphological characteristics,which may be related to the clinical characteristics of the patients.Our data revealed that glioblastoma organoids had irregular edges and more protrusions,while astrocytoma organoids displayed relatively round shape.To further explore the similarity between organoids and the original patient tumor,Hematoxylin and eosin staining(H&E)and immunohistochemical staining(IHC)were performed.H&E staining revealed morphological similarities between patient derived organoids(PDOs)and the patient glioma tissue from which they were originally derived,retaining the nuclear abnormality of the parental tissue.IHC staining revealed that the molecular expression of the parental tissue was maintained in the 3D culture.The CD133 expression was detected before and after cell culture,and it was found that the expression of CD133 was extremely low in freshly isolated tumor cells,but the expression was increased in 3D culture.Quantitative PCR showed that the mRNA expression levels of stem cell markers CD133,SRY-box 2(SOX2),and oligodendrocyte transcription factor 2(OLIG2)was greatly increased in organoids.To explore the effect of 3D culture of tumor cells on the drug.Drug test was performed gefitinib for primary cells by two different culture methods of,monolayer planar and 3D culture(Matrigel).The results indicated that 3D cultures had relatively poor drug sensitivity compared with monolayer culture,which may have caused the cells have a certain degree of drug resistance due to the increase of cell stem expression.In summary,in this study,a Matrigel-based three-dimensional culture system was established for glioma cells.Patient-derived organoids can maintain the morphology and molecular characteristics of the parent tissue.It was found that three-dimensional culture can up-regulate glioma cell stemness and increased cell resistance to drugs.The three-dimensional culture is expected to provide a better platform for the development of glioma drugs and the study of tumor microenvironment-related mechanisms.