Establishment Of An In Vitro Culture System Of Human Gastroids

Background:Currently,studies of gastric pathophysiology have been limited due to the lack of primary cultures system of gastric epithelium.Organoids were three-dimensional structures which can be derived from many organs such as brain,prostate,breast tissues and kidney.These special structures provide a model of human organ development in a system remarkably similar to that in vivo.Gastroids were generated from gastric epithelium containing human or mouse pluripotent stem cells.Recently,the in vitro expansion of intestinal tissues has been well-established worldwide,however,the culture technologies of human Gastroids have not been specially reported in our country.The aim of the study was to build upon an in vitro culture system of human Gastroids which could benefit the basic study of gastric function and disease in the future.Methods:Gastric tissue samples were obtained from individuals undergoing endoscopic biopsies.Different combination of cellular factors were used to investigate the effects on gastroids growth in culture.Gastric samples of the gastric fundus and antrum were collected from healthy adults and patients previously diagnosed with low grade intraepithelial neoplasia(LIN),respectively.The microscopical morphology and Immunohistochemistry were applied to compare the difference between normal epithelium derived gastroids and LIN-derived ones.Genes with stable expression of GFP(green fluorescent protein)were integrated into gastroids by electroporation via a piggyBac vector.We also explored the transfection efficacy of gastroids under different electroporation conditions.Data were analyzed using GraphPad Prism 5(GraphPad Software 5.0,San Diego,CA,USA).P values<0.05 were considered as statistically significant(*p?0.05;**p ?0.01;**p?0.001).Results:In this study,we successfully established an in vitro culture system of human gastroids which can be stable cultured,passaged and frozen.We found significantly decreased growth efficacy of gastroids when lacking each of the following cellular factor:Wnt-3A,R-spondin-1,Noggin,EGF,Gastrin-1,Y-27632,while there is were no effects of gastroids growth by FGF-10,Nicotinamie and N-Acetylcysteine.Compared with normal epithelium-derived gastroids,LIN-derived gastroids grew more quickly under the same culture condition.There is no significant difference of Ki-67 positive rate between LIN-derived gastroids and LIN epithelium tissues(Fundus,8.95%vs 9.53%,p=0.153;Antrum,4.09%vs 4.67%,p=0.280)Genes with stable expression of GFP were successfully integrated into gastroids via a GFP-expressing piggyBac vector by electroporation.The transfection efficacy was significantly increased when using the BTXpress fluid or adding DMSO into culture medium before electroporation.Conclusions:This study built upon a novel culture system of gastroids which reflects the successful building of the in vitro gastrointestinal environment.We also completed the primary transfection protocol of Genes delivery into the gastroids,which is crucial to the further study of gene signal pathways in this new model.