Application Of Tissue Engineering Techniques To Construct Tumor Models And Liver-related Models In Vitro

Background:In the fields of clinical and scientific research,in vitro models derived from tissues,organs or tumors are usually used to study mechanisms of diseases progression and metastasis,test and screen drugs,and develop new drugs.However,traditional 2D models differ greatly from the real in vivo environments,which may lead to misleading results.3D models can better simulate real environmental states of cells in vivo,as well as the characteristics of organs and cancers.They can be used for research into the functions of cells and organs,disease microenvironments,and the development and evaluation of drugs.Materials and methods:In the study of 3D bioprinting techniques to construct an immune microenvironment model of cholangiocarcinoma(CCA)and explore the function of tumor-associated stromal cells,we first cultured CCA cell line RBE,human umbilical vein endothelial cells HUVEC,fibroblasts CCC-HPF-1,and human mononuclear leukemia cells THP-1 in a 2D environment.The above-mentioned stromal cells were then activated into tumor-associated stromal cells as reported.THP-1 cells were activated into M2 macrophages using PMA and IL-4/IL-13.Considering that direct mixing of tumor cells and stromal cells in a 3D environment would lead to contact inhibition effects of two types of cells,we used a 3D bioprinter to construct a structure consisting of a hydrogel containing stromal cells(outer ring structure)enclosing a hydrogel containing CCA cells(inner gridlike structure).Subsequently,the survival status of tumor cells,cell proliferation ability,mRNA level and protein expression of related genes,anti-tumor drugs resistance and other indicators in different models were explored.Subsequently,the survival status of cells,cell proliferation ability,the expression of genes in mRNA and protein levels,the metabolism of antitumor drugs,and other indicators were investigated in different models.In the study of constructing liver function model with primary mouse hepatocytes(PMH)by acoustic holography(AH,acoustic tweezers),we modified the original method of separation of PMH,combined with self-made medium,and realized the long-term culture of PMH in vitro.Subsequently,we designed and constructed the instruments for AH by ourselves to construct the PMH AH model.Next,we verify the characteristics of PMH AH model,2D and 3D cultured(3DC)models were used as the control groups.In the experiment,hematoxylin and eosin staining was used to observe PMH spheroids in AH model.Cell survival ratio,cell proliferation status,expression activity of hepatocytesrelated genes,hepatocyte-related functions(glycogen storage capacity,indocyanine green uptake/release capacity,protein uptake,synthesis and secretion,etc.),and cytochrome P450 enzyme induced expression capacity,etc.were also detected in different models.Results:In this study,we successfully constructed a 3D bioprinted model to simulate the tumor immune microenvironment of CCA.The viability of RBE cells and tumorassociated stromal cells in each group remained above 90%throughout the entire experimental period.Our CCK8 assay demonstrated that,compared to cells cultured in 2D environments,cells in 3D bioprinted models exhibited a higher proliferation rate.Additionally,RBE cells co-cultured with tumor-associated fibroblasts(TAFs)or tumorassociated macrophages(TAMs)demonstrated a higher proliferative capacity compared to RBE cells cultured alone or co-cultured with tumor-associated endothelial cells(TECs).The results of qRT-PCR and immunofluorescence staining(IF)experiments showed that,the expression levels of genes related to proliferation,tumor stem cell heterogeneity,and drug resistance in the 3D bioprinted model were significantly higher than those in the 2D environment.In addition,the presence of TAF and TAM increased the malignancy of RBE,whereas TEC had no such effect on RBE.To evaluate the response of tumor cells to antitumor drugs in different models,3D bioprinted cells and 2D RBE were treated with different concentrations of gemcitabine,cisplatin,or 5-fluorouracil,respectively.Results showed that the presence of TAF and TAM increased the resistance of RBE to the above drugs.Finally,changes in epithelial-mesenchymal transition(EMT)in CCA were detected using qRT-PCR and IF.The 3D bioprinting environment significantly promoted the migration and invasion of RBE,increased the expression of Vimentin,N-cadherin and MMP9,and decreased the expression of E-cadherin.The expression of Wnt/β-catanin pathway genes(β-catenin,cyclin D1 and c-Myc)in cells in the 3D bioprinted environment was increased.They suggested that 3D bioprint may promote the expression of mesenchymal phenotype of tumor cells in a Wnt/β-catenin signaling-dependent manner.Also,we successfully constructed the PMH culture model using the AH device.PMH successfully proliferated and divided to form cell spheroids in the AH model.The proportion of living cells in the AH model was always above 96%throughout the experimental period,while that in 3DC model was 86%to 93%.The results of CCK-8 showed that,2D cells gradually apoptotic around day 5,and PMH in 3DC and AH models maintained stable proliferation all the time.We also checked the expression of hepatocyte function-related proteins by IF,compared with the cells in 3DC model and 2D,cells in AH model higher showed expression of albumin(ALB),α-1 antitrypsin(AAT,alAT),CYP2E1,CYP3A11,and MRP2.qRT-PCR results also showed that,the mRNA expression levels of genes related to hepatocytes’ function,such as ALB,AAT,TRF,CK18,Ki-67,FOXA2,HNF4A were higher in AH model than that in 3DC and 2D environments.In addition,the expression levels of the above genes in the AH model were closest to those of PMH just 24h after isolation from mice.PMH in AH model has function of ICG uptake and release,intracellular accumulation of glycogen,uptake of acetylated low-density lipoprotein(ace-LDL),urea synthesis and glucose metabolism.Besides,ALB,alAT,transferrin,and FOXA2 secreted by PMH in AH model were higher and had a longer secretion time,compared with the control groups.The experiments of CYP induction showed that,PMH in the AH model responded to some known inducers,such as acetaminophen(CYP1a2,2e1,and 3a11),3-methylcholanthrene(CYP1a1,1a2,and 1b1),rifampicin(CYP1a1,1a2,1b1 and 3a11),phenytoin(CYPla1 and 3a11),and phenobarbital(CYP1a2,2a4,2e1 and 3a11).The expression of CYP of PMH in AH model was higher than that in control groups.Conclusion:The immune microenvironment model based on 3D bioprinting is an effective method to study the effect of stromal cells on CCA cells.3D bioprinted tumor models can better mimic multifarious tumor types,and may become a powerful platform for preclinical research and drugs testing,as well as a suitable alternative to animal models.The model constructed by AH is a reliable solution for long-term culture of primary hepatocytes in vitro.Cells in this model can well maintain their original specific functions.This technology has great potential in many fields such as the research of cell metabolism,drugs testing,and liver replacement therapy in the future.