Preparation of cell culture to be irradiated with SPICE for studies on the process of proliferation and differentiation

Single Particle Irradiation System to Cells (SPICE) is an equipment to irradiate 3.4 MeV accelerated proton microbeam to the individual single cell target. This report describes the establishment of protocol for the irradiation of cell culture to investigate the process of its proliferation and differentiation. Cells used were undifferentiated neural stem cells originally derived from mouse ES (embryonic stem) cells of SV6 strain. They were cultured in the ordinary dish and seeded in SPICE dish to form colony. The SPICE dish was designed to have the bottom of an etched Mylar polyester film (No. 100, Mylar Indust., Inc.) of 2.5 mc-m thickness because the beam was vertical by which cells were to be irradiated from their culture bottom, the range of the proton ion was only 183 mc-m in water, and the beam scattering had to be minimized. Etching was performed by treatment of the film with NaOH. Cells were fixed by methanol and stained for their nuclei with iodide at 24 or 90 hr culture to get laser-excited fluorescent image for microscopy. The conditions established were: etching with 2M-NaOH, at 50 deg. C, for 30 min-1 hr, where colony formation was observable after 90 hr. Under the conditions, it was thought possible to apply the classical technique of radiation biology to SPICE experiments. (T.T.)