Mesenchymal stromal cells (MSCs) are one of the most frequently used cell types in regenerative medicine and cell therapy. Generating sufficient cell numbers for MSC-based therapies is constrained by: 1) their low abundance in tissues of origin, which imposes the need for significant ex vivo cell amplification, 2) donor-specific characteristics including MSC frequency/quality that decline with disease state and increasing age, 3) cellular senescence, which is promoted by extensive cell expansion and results in decreased therapeutic functionality. The final yield of a manufacturing process is therefore primarily determined by the applied isolation procedure and its efficiency in isolating therapeutically active cells from donor tissue. To date, MSCs are predominantly isolated using media supplemented with either serum or its derivatives, which pose safety and consistency issues. To overcome those limitations while enabling robust MSC production with constant high yield and quality, we developed a chemically defined biomimetic surface coating, called isoMATRIX, that facilitates the isolation of significantly higher numbers of MSCs in xeno-/serum-free and chemically defined conditions. The isolated cells display a smaller cell size and higher proliferation rate than those derived from a serum-containing isolation procedure and a strong immunomodulatory capacity. In sum, the isoMATRIX promotes enhanced xeno-, serum-free, or chemically defined isolation of human MSCs and supports consistent and reliable cell performance for improved stem cell-based therapies.