Micro-fragmented adipose tissue (MFAT) is a promising autologous therapy for knee osteoarthritis. To avoid repeated liposuction procedures for its clinical application, MFAT obtained from patients with knee osteoarthritis was stored at −80 °C in a tissue bank. This study describes the preparation, cryopreservation, thawing, and washing, as well as comprehensive analysis of cell populations in fresh and MFAT thawed after two years. Immunophenotyping of both fresh and thawed MFAT showed a significant presence of endothelial progenitors and pericytes in the stromal vascular fraction. Viability before (59.75%) and after freezing (55.73%) showed no significant difference. However, the average cell count per gram of MFAT was significantly reduced in thawed samples (3.00 × 10⁵) compared to fresh ones (5.64 × 10⁵), likely due to processing steps. Thawed MFAT samples showed increased CD73 expression on the CD31^highCD34^high subset of EP and SA-ASC, as well as increased expression of CD105 on EP, the CD31^lowCD34^low subset of EP, pericytes, and SA-ASC. Microbiological testing confirmed 100% sterility, and double washing efficiently removed DMSO, confirming sample safety. Histological analysis revealed healthy, uniformly shaped adipocytes with intact membranes. This approach allows accurate estimation of cell yield for intra-articular injection, ensuring delivery of the target cell number into the knee. Quality control analysis confirms that cryopreserved MFAT retains high cellular and structural integrity, supporting its safety and suitability for clinical application.