A mastectomy is a curative treatment for breast cancer. It causes breast and soft tissue deficits, resulting in a chest with poor vascularity. Autologous tissue breast reconstruction is commonly associated with donor site morbidity. Breast implants are another reconstruction alternative, but they are associated with infection, rupture, and the need for replacement. Autologous aspirated fat grafting has appeared as an ideal breast reconstruction method, but low graft viability and high resorption remain as the main shortcomings. We developed a novel method for fat-only grafts using cultured mature adipocytes (CMAs) mixed with their condition medium. Twenty-five mastectomy patients, aged 32–72 years, received a mixed grafting of CMAs, MCP1-containing condition medium, and fat grafts for total breast reconstruction. In follow-up periods of 24–75 months, MRI analysis showed full thickness fat-engraftment. The cell proliferation marker Ki67 was negative in post-transplant biopsy specimens from all patients. Aesthetic full breast morphology was achieved, patient satisfaction was evaluated 1 year and 3–6 years after surgery. All grafts were confirmed safe, demonstrating high reliability and long-term sustainability.