BACKGROUND: Recently we reported about a stem cell factor (SCF)-dependent culture system for human mast cells (MC), isolated from intestinal mucosa. Here we present a method to obtain highly purified human intestinal MC. METHODS: MC were isolated from surgery specimens and purified by positive selection using the magnetic-activated cell sorting (MACStrade mark) system and subsequent culture of the MC in medium supplemented with SCF. RESULTS: In the presence of SCF, purified MC (50-85% purity after MACS) maintained in culture for up to 3 months. MC purity increased during culture and reached nearly 100%. During the first week of culture, MC numbers decreased, but after that time they started to proliferate. Cultured MC did not change their histamine content, phenotype or morphology. They were even more responsive towards IgE-dependent stimulation, which caused the release of high amounts of histamine, leukotrienes and cytokines such as TNF-alpha and IL-5. CONCLUSION: We show that mature human intestinal MC can be purified, maintained in culture, and triggered for proliferation in the presence of SCF. After culture, they are viable, release high amounts of mediators and cytokines upon stimulation, and thus are a valuable tool for further experiments on human mucosal MC.
BACKGROUND: Recently we reported about a stem cell factor (SCF)-dependent culture system for human mast cells (MC), isolated from intestinal mucosa. Here we present a method to obtain highly purified human intestinal MC. METHODS: MC were isolated from surgery specimens and purified by positive selection using the magnetic-activated cell sorting (MACStrade mark) system and subsequent culture of the MC in medium supplemented with SCF. RESULTS: In the presence of SCF, purified MC (50-85% purity after MACS) maintained in culture for up to 3 months. MC purity increased during culture and reached nearly 100%. During the first week of culture, MC numbers decreased, but after that time they started to proliferate. Cultured MC did not change their histamine content, phenotype or morphology. They were even more responsive towards IgE-dependent stimulation, which caused the release of high amounts of histamine, leukotrienes and cytokines such as TNF-alpha and IL-5. CONCLUSION: We show that mature human intestinal MC can be purified, maintained in culture, and triggered for proliferation in the presence of SCF. After culture, they are viable, release high amounts of mediators and cytokines upon stimulation, and thus are a valuable tool for further experiments on human mucosal MC.
Authors: Celalettin Ustun; Todd E DeFor; Fatma K Karadag; Hyun Don Yun; Sunita Nathan; Claudio G Brunstein; Bruce R Blazar; Daniel J Weisdorf; Shernan G Holtan; Khalid Amin Journal: Blood Adv Date: 2020-05-26