In Vitro Construction Of Three-dimensional Cell Culture Scaffolds And Its Application In Screening Of Chemotherapeutic Drugs

?Objective?Using natural silk fibroin(SF)and chitosan(CS)as the main raw material,two kinds of SF/CS three–dimensional cell culture scaffolds with different biological cross-linking agents(TPP,EDC)were prepared by vacuum freeze-drying.In order to screen out suitable three-dimensional culture scaffolds for tumor cell growth,the physicochemical properties and cell compatibility tests were examined.Using the scaffold selected above as the material,susceptibility tests were conducted to provide a new technology platform in vitro tumor chemotherapeutic screening.?Methods?Part I: 1.Mix the silk fibroin solution(2% to 3%)extracted from clean silkworm cocoons after degumming,drying,dissolving,filtering and dialyzing with the 3%chitosan solution at a ratio of 1:1.Add two different cross-linkers(EDC/NHS or TPP)to the above mixture to obtain two kinds of SF / CS three – dimensional cell culture scaffolds by vacuum freeze-drying.2.The pure silk fibroin and pure chitosan scaffolds were prepared by vacuum freeze-drying,namely SF group and CS group.3.Light microscopy,Scanning Electron Microscopy(SEM)and HE staining were used to observe the morphological characteristics of three dimensional scaffolds.4.The functional group composition of the scaffolds was analyzed by fourier transform attenuated total reflectance infrared spectroscopy(FTIR).5.The basic composition was analyzed using X-ray diffraction(XRD).6.The water absorption and swelling rate of the three-dimensional scaffold of the EDC group and the TPP group were measured.Part II: 1.SEM was used to observe the growth state of colorectal cancer cell CT26 in three-dimensional cell culture scaffolds of EDC and TPP groups.2.MTT assay was used to detect the proliferation of CT26 cells in two-dimensional culture(2D)and three-dimensional culture(EDC group,TPP group)environment.3.Establish xenograft tumor nude mice model to extract tumor tissue extract(TLTT)and use MTT assay to detect the effect of different concentrations of tumor tissue extract on the proliferation of CT26 cells.4.DAPI fluorescence staining was used to detect the apoptotic ability of CT26 cells in three-dimensional cell culture scaffolds(EDC group,TPP group)stimulated by tumor tissue extracts.Part III: Using six common chemotheraputic drugs(fluorouracil,oxaliplatin,paclitaxel,irinotecan,methotrexate and capecitabine)as research objects,five drug concentrations(0.01?M,0.1?M,1?M,10?M,100?M)were used to stimulate the Lovo cell line of colon cancer and the MDA-MB-231 cell line of breast cancer grown in 2D and EDC three-dimensional cell culture scaffolds(3D,3D+TLTT).CCK-8 assay was used to detect the sensitivity of tumor cells to different kinds of tumor chemotherapy drugs.?Results?Part I: 1.The pore size was more uniform in the EDC group than in the TPP group,the pure SF group,and the pure CS group,and the arrangement was relatively regular.Most of them were homogeneous stable void structures.2.ART-FTIR results showed that there were no new characteristic peaks in the cross-linked SF/CS three-dimensional cell culture scaffolds.In this process,no new chemical bonds were formed,but only simple physical binding.3.The results of XRD showed that the crystallinity of the SF/CS three-dimensional scaffolds cross-linked by EDC or TPP was different from that of pure silk fibroin protein,and it was characterized by the crystalline form of chitosan.4.The water absorption and swelling rate of the three-dimensional scaffolds EDC group were significantly higher than those of TPP group SF/CS three-dimensional scaffolds.Part II: 1.The results of SEM showed that the three-dimensional scaffold of EDCgroup was more conducive to the adhesion,growth and proliferation of CT26 cells than TPP group.2.The proliferation ability of CT26 cells in three-dimensional culture scaffolds(EDC group,TPP group)was significantly higher than that in two-dimensional culture environment(2D),and the proliferation ability of CT26 cells grown in the three-dimensional cell culture scaffold of EDC group was significantly higher than that of TPP group.3.Tumor tissue extracts can promote the growth of CT26 cells to a certain extent,and 10% tumor tissue extracts have the best proliferation promoting effect.4.The results of DAPI fluorescent staining showed that the apoptotic state of CT26 cells in the EDC group was better than that in the TPP group when stimulated by the same tumor tissue extract.Part III: Tumor cells in different culture environments have distinct sensitivities to chemotherapeutic drugs and tumor cells in a three-dimensional scaffold environment have high sensitivity to low concentrations of chemotherapeutic drugs.?Conclusions?EDC cross-linked SF/CS three-dimensional scaffold is more suitable for tumor cell growth than TPP cross-linked and the appropriate concentration of tumor tissue extract can promote tumor cell growth and proliferation.Comparing the results of chemosensitivity in the traditional two-dimensional cell culture environment and SF/CS three-dimensional cell culture scaffold,the sensitivity of tumor cells to low-dose chemotherapy drugs in three-dimensional scaffold was higher than that in traditional two-dimensional cell culture environment.