Human duodenal cell culture in vesicles: A suitable model for examination of Ca2+ and pH-regulation

With the development of spharoid-like three-dimensional cell culture from h uman duodenum, there is a new cell modell available with the morphologie an d the functionality of the original tissue. Aim of our work was to study th e physiological properties of the spharoids and their congruence to the ori ginal tissue. As an example we tested the intracellular pH- and Ca2+ regula tion of the spheroide. Methods: From normal human biopsies spheroids were prepared according to th e method of Boxberger and Sessler. The spheroid was mounted in a special pe rfusion chamber on the microscope desk and perfused continuously. The pH wa s measured by ratio imaging of BCECF fluorescence and Ca-i(2+) with Fura2. Sodium free solution, amiloride and H2DIDS were used to specify the proton backregulation after intracellular acidification. The calciumregulation was determined by Verapamil, a calcium channel inhibitor. Results: a) pH-measurement: The intracellular pH of the spheroids was 7.31. The backregulation after rapid acidification was inhibited completely with Na+-withdrawal, which indicate a Na-dependent backregulation. The partial inhibition of the backregulation with amiloride and the total inhibiton wit h amiloride and H2DIDS indicate the involvement of a Na+-H+-exchanger and a Na+-HCO3--cotransporter. b) Ca2+-measurement: We measured a intracellular calcium concentration of 1 92 mu M in the spheroids. The backregulation after calcium withdrawal was i nhibited with Verapamil which indicate the participation of a calcium chann el. Conclusion: The physiological congruence of the spheroides with their nativ e tissue is evidenced by the measured ionregulation. This spheroides from h uman duodenal cells are very suitable for further investigations of the phy siological system as well as to enlighten various pathomechansims.