Hypoxic Cell Culture Technique

For hypoxic treatments, we applied the BIONIX-1 hypoxic culture kit (Sugiyamagen, Tokyo, Japan) as previously described [42 (link), 43 (link)]. This system consists of an AnaeroPack-Anaero 5% (oxygen absorber; Mitsubishi Gas Chemical, Tokyo, Japan), an OXY-1 oxygen monitor (JIKCO, Tokyo, Japan), an AnaeroPouch (Mitsubishi Gas Chemical), and plastic clips for sealing the pouch. Briefly, PCAs, the oxygen absorber, and the oxygen monitor were arranged in the pouch, and the left open side was sealed with a clip. The O₂ concentration in the pouch rapidly decreased after sealing; once a low O₂ concentration (approximately 0.1%) was reached, the pouch was sealed with another clip between the culture dish and the oxygen absorber to stop further oxygen absorption. Then, the pouch was maintained in an incubator at 37°C.