The quality and quantity media-cultured mononuclear cell transplantation is safe and effective in ischemic hindlimb mouse model

OBJECTIVE: This study was conducted to investigate in vitro proangiogenic and anti-inflammatory phenotypes and functions and the in vivo efficacy and safety of quality and quantity (QQ) media-cultured mononuclear cells (MNCs) compared with standard cultured MNCs from the peripheral blood of patients with chronic limb-threatening ischemia (CLTI) with atherosclerotic risk factors. METHODS: Peripheral blood MNCs (PBMNCs) from patients with CLTI were cultured in QQ culture media or standard culture media. Phenotypic analysis of progenitor cells (CD34(+)CD133(+)), M2 macrophages (CD206(+)), and inactivated T regulatory cells (CD4(+)CD25(+)CD127(+)), colony-forming assay, and tube formation assay of QQ media-cultured MNCs (QQMNCs) and PBMNCs, were conducted. Intramuscular transplantation of QQMNCs or PBMNCs was performed in the ischemic hindlimb model. The clinical appearance of ischemic limbs was observed, and blood flow in ischemic limbs was measured using a laser Doppler perfusion imager. Outcomes were compared between the QQMNC and PBMNC groups. RESULTS: Twenty patients with CLTI were included. The mean percentages of CD34(+) cells, CD133(+) cells, CD34(+)CD133(+) progenitor cells, CD206(+) cells, colony-forming cells, and tube formation were significantly higher in the QQMNCs. The mean percentage of CD4(+)CD25(+)CD127(+) cells was significantly lower in QQMNC. The colony-forming unit count and Dil-acetylated low-density lipoprotein uptake were significantly greater in QQMNCs. The clinical appearance of post-QQMNC-injected limbs was less severe than the appearance of post-PBMNC-injected limbs. Limb perfusion was significantly better in the QQMNCs. CONCLUSIONS: Proangiogenic and anti-inflammatory phenotypes of MNCs cultured in QQ culture media were reproducible. Intramuscular QQMNC transplantation was safe and resulted in better reperfusion of ischemic hindlimbs compared with PBMNCs.