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Freeze-drying of Platelet-rich plasma and its effect on wound healing: An in vivo pilot case study. Himanshu Bansal1,2*, Alnkrita Bansal1,2, Irfan Khan2, Anupama Bansal1, Shahnawaz Hussein Khan3, Jerry Leon4, Mustafa al Maini5, Matias Fernandez Viña6 1Mother Cell Spinal Injury and Stem Cell Research, Anupam Hospital, Rudrapur, Uttarakhand, India 2Revita Life Sciences, Rudrapur, Uttarakhand, India 3Department of regenerative medicine, Lovinium Biotech, Asturias, Spain 4PMR Advance Health Institute Mayaguez, Puerto Rico, USA 5Mafraq Hospital, Sheikh Shakhbout Medical City, Abu Dhabi, United Arab Emirates 6Consultant, Stem Cell Therapy, Argentina *Corresponding author Dr. Himanshu Bansal, MD, Consultant Regenerative Medicine, Revita Lifesciences, Rudrapur, India-263153 Phone: 09634501234 Email: hbansal@drhbf.org Abstract Background: Platelet-rich plasma (PRP) is widely used in regenerative medicine because of its high content of growth factors and cytokines that promote tissue repair. However, fresh PRP is limited by the rapid loss of bioactivity, compositional variability, and storage challenges. Freeze-dried PRP (FD-PRP) is a promising alternative with potential for long-term storage and standardized therapeutic efficacy. Objective: This study aimed to evaluate the biological activity, growth factor preservation, and wound healing efficacy of FD-PRP compared with fresh PRP using in vitro and in vivo assays. Methods: PRP from 20 healthy donors was standardized to 5 billion platelets per 4 mL and freeze-dried using proprietary stabilizers. Platelet morphology, aggregation, and growth factor concentrations (PDGF, TGF-β1, VEGF, EGF, bFGF, and IGF-1) were assessed before and after lyophilization. The stability of the samples was monitored over 26 weeks. In vitro wound healing was assessed in human mesenchymal stem cells (MSCs) using scratch assays. A pilot in vivo study evaluated the efficacy of FD-PRP in accelerating wound closure in 10 patients over three weeks. Results: FD-PRP retained 82.05% of platelets post-lyophilization, with preserved morphology and aggregation. Growth factor levels were maintained or increased in FD-PRP, notably TGF-β1 (2.1-fold) and IGF-1 (3.04-fold) levels. Stability testing demonstrated >90% retention of platelets and growth factors over 26 weeks. In vitro, FD-PRP accelerated wound closure (85% at 72 hours) compared to fresh PRP (70%) and saline (30%). In vivo, FD-PRP-treated wounds showed a mean size reduction of 56.17 % %by day 12, which was significantly higher than that in non-treated regions (23.5%). Conclusion: FD-PRP preserves platelet functionality and key growth factors, demonstrates excellent storage stability, and enhances wound healing both in vitro and in vivo. These findings support FD-PRP as a practical and effective alternative to fresh PRP for clinical applications that require standardized, off-the-shelf regenerative therapies. Keywords: Platelet-rich plasma, Freeze-drying, Lyophilization, Wound healing, Growth factors, Regenerative medicine.