Liver Injury Model and SHED-Heps Therapy

A CCl₄ solution in olive oil (1.0 mL/kg body weight; CCl₄ to olive oil = 1:4 volume/volume; Wako Pure Chemicals, Osaka, Japan) was intraperitoneally injected into C57BL/6J mice twice a week for 8 weeks, as described previously [12 (link), 19 (link)]. Age-matched C57BL/6J mice infused with olive oil (Wako Pure Chemicals) were used as controls. The generated SHED-Heps were washed with sterilized with phosphate-buffered saline and kept in cold phosphate-buffered saline (PBS) on ice and quickly infused into the recipient spleen within 10 min after the preparation to maintain the initially prepared donor cell viability. Four-week-CCl₄-treated C57BL/6J mice were intrasplenically infused SHED-Heps (1 × 10⁶/10 g body weight in 100 μL of PBS). Age-matched 4-week-CCl₄-treated C57BL/6J mice were infused with PBS (100 μL) as experimental controls. All mice did not receive any immunosuppressants throughout the experiment.