3D scaffolds prepared from platelet rich plasma versus conventional plastic surface for culturing adipose-derived canine mesenchymal stem cells and stromal vascular fraction cells

3D scaffolds prepared from Platelet Rich Plasma versus conventional plastic surface for culturing adipose-derived canine Mesenchymal Stromal Cells and Stromal Vascular Fraction cells. Suelzu C., Conti V., Basini G., Ramoni R. and Grolli S. Dipartimento di Scienze Mediche Veterinarie, Università di Parma OBJECTIVE: Mesenchymal Stem Cells (MSCs) are considered a valuable tool for regenerative medicine applications. Stromal vascular fraction (SVF), a heterogeneous population of cells derived from adipose tissue, represents a valid source of MSCs. We evaluated the ability of canine MSCs and adipose-derived SVF cells to be amplified in vitro by exploiting, in addition to the canonical plastic adhesion, a three-dimensional matrix obtained by gelling Platelet Rich Plasma (PRP) or Platelet Poor Plasma (PPP). The aim was to evaluate the possibility of obtaining autologous preparations capable of supporting MSCs and SVF cells growth for the "point of care" application in the veterinary clinic. MATERIALS AND METHODS: 1. MSCs growth on plastic surface vs 3D fibrin matrix. Total cell number, cell doubling time and cell doubling number of MSCs grown on plastic surface were compared to MSCs cells grown inside a 3D-fibrin matrix prepared by mixing PPP or PPP (50%v/v), DMEM, thrombin (10% v/v) and calcium gluconate (100mg/ml, 10%v/v). 2. SVF growth on plastic surface vs 3D fibrin matrix. SVF cells prepared by collagenase type-I digestion were cultured either on conventional culture dishes or within a 3D fibrin matrix. Total cell number, cell doubling time and cell doubling number were determined for both culture conditions until P3. 3.Phenotypic characterization by RT-PCR of MSCs and SVF cells. Gene expression was compared between MSCs and SVF grown in different culture conditions, i.e. plastic surface versus 3D fibrin matrix. A set of typical MSCs markers and gene involved in their biological properties were evaluated. RESULTS: Canine MSCs grow within 3D fibrin-based matrices, demonstrating a shorter doubling time and a higher duplication rate when compared to cells grown on the plastic surface: the number of cells obtained is about seven-fold higher in the 3D environment after 144 hours of culture (p<0.01). Moreover, the cells included herein can be frozen and sub-cultured. In our experimental setup, the use of PRP instead of PPP in the preparation of the matrix does not modify cell replication rate. RT-PCR characterisation of cells cultured within the 3D matrix confirms the expression framework of MSCs markers. Furthermore, 3D environment improves SVF cells replication rate, producing at the first culture passage, a mean 1.5-fold increase in cell number. CONCLUSION: 3D matrices prepared with autologous PRP or PPP are suitable for canine MSCs and SVF cells cultures. Cells grow faster than in standard 2D culture on the plastic surface, while they maintain their panel of gene expression. Furthermore, they can be sub-cultured and frozen for future applications. These results could contribute to set-up more effective MSCs-based therapies, with advantages regarding time shortening for the production of adequate amounts of cells to be applied in the clinical practice.