Platelet Lysate for Mesenchymal Stromal Cell Culture in the Canine and Equine Species: Analogous but Not the Same

Platelet lysate (PL) is an attractive platelet-based therapeutic tool and has shown promise as xeno-free replacement for fetal bovine serum (FBS) in human and equine mesenchymal stromal cell (MSC) culture. Here, we established a scalable buffy-coat-based protocol for canine PL (cPL) production (n = 12). The cPL was tested in canine adipose MSC (n = 5) culture compared to FBS. For further comparison, equine adipose MSC (n = 5) were cultured with analogous equine PL (ePL) or FBS. During canine blood processing, platelet and transforming growth factor-β1 concentrations increased (p < 0.05 and p < 0.001), while white blood cell concentrations decreased (p < 0.05). However, while equine MSC showed good results when cultured with 10% ePL, canine MSC cultured with 2.5% or 10% cPL changed their morphology and showed decreased metabolic activity (p < 0.05). Apoptosis and necrosis in canine MSC were increased with 2.5% cPL (p < 0.05). Surprisingly, passage 5 canine MSC showed less genetic aberrations after culture with 10% cPL than with FBS. Our data reveal that using analogous canine and equine biologicals does not entail the same results. The buffy-coat-based cPL was not adequate for canine MSC culture, but may still be useful for therapeutic applications.

Chitosan Reinforced with Kenaf Nanocrystalline Cellulose as an Effective Carrier for the Delivery of Platelet Lysate in the Acceleration of Wound Healing

The clinical use of platelet lysate (PL) in the treatment of wounds is limited by its rapid degradation by proteases at the tissue site. This research aims to develop a chitosan (CS) and kenaf nanocrystalline cellulose (NCC) hydrogel composite, which intend to stabilize PL and control its release onto the wound site for prolonged action. NCC was synthesized from raw kenaf bast fibers and incorporated into the CS hydrogel. The physicochemical properties, in vitro cytocompatibility, cell proliferation, wound scratch assay, PL release, and CS stabilizing effect of the hydrogel composites were analyzed. The study of swelling ratio (>1000%) and moisture loss (60–90%) showed the excellent water retention capacity of the CS-NCC-PL hydrogels as compared with the commercial product. In vitro release PL study (flux = 0.165 mg/cm2/h) indicated that NCC act as a nanofiller and provided the sustained release of PL compared with the CS hydrogel alone. The CS also showed the protective effect of growth factor (GF) present in PL, thereby promoting fast wound healing via the formulation. The CS-NCC hydrogels also augmented fibroblast proliferation in vitro and enhanced wound closures over 72 h. This study provides a new insight on CS with renewable source kenaf NCC as a nanofiller as a potential autologous PL wound therapy.

Phospholipid Profiles for Phenotypic Characterization of Adipose-Derived Multipotent Mesenchymal Stromal Cells

Multipotent mesenchymal stromal cells (MSC) have emerged as therapeutic tools for a wide range of pathological conditions. Yet, the still existing deficits regarding MSC phenotype characterization and the resulting heterogeneity of MSC used in different preclinical and clinical studies hamper the translational success. In search for novel MSC characterization approaches to complement the traditional trilineage differentiation and immunophenotyping assays reliably across species and culture conditions, this study explored the applicability of lipid phenotyping for MSC characterization and discrimination. Human peripheral blood mononuclear cells (PBMC), human fibroblasts, and human and equine adipose-derived MSC were used to compare different mesodermal cell types and MSC from different species. For MSC, cells cultured in different conditions, including medium supplementation with either fetal bovine serum or platelet lysate as well as culture on collagen-coated dishes, were additionally investigated. After cell harvest, lipids were extracted by chloroform/methanol according to Bligh and Dyer. The lipid profiles were analysed by an untargeted approach using liquid chromatography coupled to mass spectrometry (LC-MS) with a reversed phase column and an ion trap mass spectrometer. In all samples, phospholipids and sphingomyelins were found, while other lipids were not detected with the current approach. The phospholipids included different species of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylserine (PS) in all cell types, whereas phosphatidylglycerol (PG) species were only present in MSC. MSC from both species showed a higher phospholipid species diversity than PBMC and fibroblasts. Few differences were found between MSC from different culture conditions, except that human MSC cultured with platelet lysate exhibited a unique phenotype in that they exclusively featured PE O-40:4, PG 38:6 and PG 40:6. In search for specific and inclusive candidate MSC lipid markers, we identified PE O-36:3 and PG 40:7 as potentially suitable markers across culture conditions, at which PE O-36:3 might even be used across species. On that basis, phospholipid phenotyping is a highly promising approach for MSC characterization, which might condone some heterogeneity within the MSC while still achieving a clear discrimination even from fibroblasts. Particularly the presence or absence of PG might emerge as a decisive criterion for future MSC characterization.