Culture And Observation Of Human Lacrimal Adenoid Cystic Carcinoma Cells And Isolation Of Cancer Stem-like Cells

Objective1. To obtain human adenoid cystic carcinoma cells of lacrimal gland(LACC),and lay the foundation for the establishment of an lacrimal adenoid cystic carcinoma cell line.2. To understand the morphological characteristics,including growth characteristics, cell morphology,cell markers and electron microscope,to provide evidence for the in-depth understanding of the lacrimal adenoid cystic carcinoma cells.3. To explore cancer stem-like cells exist in lacrimal adenoid cystic carcinoma cells, and lay theoretical foundation for cancer stem cells research.Methods1. Part1:Tumor tissue was obtained from the surgical material of a patient in our hospital.We gain primary cells via tissue culture techniques. Mixed cells were removed through several ways.Observe cell morphological characteristics by phase contrast microscope,transmission electron microscope(TEM) and scanning electron microscope(SEM).Cyto-immunochemical staining was applied to detect the expression of vimentin,desmin,S-100,cytokeratin(pan) and CD34.Their expressions were also detected in the tumor tissue except CD34. Make cell growth curve and culculate cell doubling time.2. Part2:Use surum free medium to,isolate and culture the cancer stem-like cell spheres. Similar standard fetal bovine serum was used to induce the suspension cells differentiation conversely.Results1. The outgrowth of cells was observed by day5after seeding,and then cells generated slowly.The first passage proceeded by day32,and the classical epithelioid cell colonies was observed by day69after inoculation.Purified cells of human lacrimal adenoid cystic carcinoma were gained after the removal of mixed cells through sevral ways,which have been successfully subcultured for more than100passages.2. The LACC cells appeared to be typically epithelioid cells,they showed contact inhibition as the density high enough.SEM and TEM showed LACC cells were malignant tumor cells poorly differentiated.The LACC cells showned positive reaction with vimentin、cytokeratin (pan) as well as S-100,but negative reaction with desmin and CD34,which were consistent with the tumor tissue. The cell growth curve turned like a sigmoid one,and the cell doubling time was37.1h.3. Cancer stem-like cells were abtained by serum free medium culturing, suspension cell spheres grew like balls or grapes24h later.Cell spheres grew bigger gradually.4. Suspension cell spheres appeared adherent growth in serum supplied medium,as similar as LACC cells.Conclusions1. Purified LACC cells were obtained for the first time,which lay the foundation for the establishment of an lacrimal adenoid cystic carcinoma cell line,and provide seed cells for further research.2. Cancer stem-like cells exist in LACC cells,which can be isolated by serum free medium,with characteristics of renewal,continuous proliferation and reverse differentiation.