Vasculogenic Potential of MNC-QQ

Release tests for MNC-QQ were conducted to evaluate potency based on the ratio of cells and their vasculogenic potential. The presence of cell types essential for vascular and tissue regeneration in the MNC-QQ culture was confirmed using flow cytometry antibody-based detection of the following cell surface markers: CD34-PE, CD206-PE/Cy7, CD192(CCR2)-PerCP/Cy5.5, CD3-Alexa700, CD14-APC/Cy7, CD4-FITC, CD8-APC/Cy7, CD31-APC/Cy7, CD19-FITC, CD56-BV421, CD25-PerCP/Cy5.5, CD127-BV421 (BioLegend, San Diego, CA, USA), CD133-APC (Miltenyi Biotec, San Diego, CA, USA), and CXCR4-APC (BD Biosciences).^{17 (link),23 (link)}The vasculogenic potential of MNC-QQ was evaluated using a MethoCult SF H4236 colony formation assay (STEMCELL Technologies, Vancouver, BC, Canada).^{22 (link)} Cells (2 × 10⁵ per dish) were inoculated into a 35-mm Primaria culture dish and cultured for 14 days under routine conditions. Clusters comprising more than 100 cells were counted as primitive EPC CFU or definitive CFU based on the cell size and shape under phase-contrast microscopy (Nikon, Tokyo, Japan). PB collected less than 1 month before cell therapy was used to compare the baseline vasculogenic potential and cell surface markers between PBMNCs and MNC-QQ cells harvested on the treatment date.

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Tanaka R., Fujimura S., Kado M., Fukuta T., Arita K., Hirano-Ito R., Mita T., Watada H., Kato Y., Miyauchi K, & Mizuno H. (2022). Phase I/IIa Feasibility Trial of Autologous Quality- and Quantity-Cultured Peripheral Blood Mononuclear Cell Therapy for Non-Healing Extremity Ulcers. Stem Cells Translational Medicine, 11(2), 146-158.

Publication 2022

CD34-PE CD3-Alexa700 CD14-APC/Cy7 CD4-FITC CD8-APC/Cy7 CD31-APC/Cy7 CD19-FITC CD56-BV421 CD25-PerCP/Cy5.5 CD127-BV421 CD133-APC CXCR4-APC

Assay Cd25 Cell therapy Cells Cxcr4 Cy5 5 Dish Fitc Flow cytometry Phase contrast microscopy Regeneration Stemcell Surface antibody Tissue Vascular

Corresponding Organization : Juntendo University

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Variable analysis

independent variables

Ratio of cells in the MNC-QQ culture

dependent variables

Vasculogenic potential of MNC-QQ cells
Presence of cell types essential for vascular and tissue regeneration in the MNC-QQ culture

control variables

PBMNC (peripheral blood mononuclear cells) collected less than 1 month before cell therapy, used to compare the baseline vasculogenic potential and cell surface markers between PBMNCs and MNC-QQ cells harvested on the treatment date

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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