A Novel Model For Organotypic Human Salivary Gland Slice Culture

OBJECTIVE:It is a challenge to maintain the salivary cells alive in vitro with the three-dimensional (3D) morphology. The purpose of this paper is to establish a novel organotypic slice culture model for human salivary gland tissue and maintain its physiological functions and expression of protein and gene on ex vivo platform. It can maintain the intercellular environment of salivary glands and diversity of cells. Deeper research of salivary gland can be accomplished by the completion of slice culture model.METHODS:Six human salivary glands were sliced to 35 to 50μm thickness pieces and cultured. The specimens were all from the patients from 34 to 75 years old. Cell viability, proliferation and physiological function as well as the gene expression of salivary gland tissue were tested.The morphology was detected by the Live/dead, proliferation and apoptosis staining and all the pictures were observed by confocal microscope. Amylase activity and the calcium detection helped us to assay the physiological function.Immunofluorescence (IF) and polymerase chain reaction (PCR) was detected to demonstrate the protein and the gene expression. The proteins of Na+-K+-2 Cl—cotransporter (NKCC1), aquaporin 5 (AQP5), cytokeratin 5 (CK5) and alpha smooth muscle actin (a-SMA) was measure by IF staining. And NKCC1、 AQP5、CK5 and E-cadherin were the gene detected by q PCR. The GAPDH gene was used as a reference gene to analyze the human gene quantitatively.There were four different time points during the whole procedure (day0,3,7 and 14 respectively).RESULTES:Tissue slices could be viable and maintain the proliferation capacity. Briefly,～70% and ～40% cells alive in day0 and day 14 respectively, and the proliferation rate rage from 6% to 17% during the culture period. Interestingly, an increase tendency was observed from day3 to day7 compared with the proliferation rate in day0, which demonstrated the salivary cells kept the ability of proliferation for more than two weeks. Amylase activity proved that the cells still have physiological functions post 14 days culture in vitro. The positive results from the calcium detection also represented the function remained. Protein synthesis and gene expression could be also observed in culture through the IF staining and q PCR. Acinar cells, ductal cells and myoepithelial cells were all be detected, which means all of them were alive with their polarity in this 3D slice culture system.CONCLUSIONS:Slice culture system can be used as a model to study the human salivary gland tissues. This model allows sliced tissue to be viable and maintain important physiological functions of salivary gland for 7 to 14 days. It could be used to study the mechanisms of the salivary gland dysfunction, which would be the chance for us to find out an effective therapies in the next study.