METHODS: Ten healthy subjects (aged 19-44 years) received 3 consecutive daily doses of filgrastim followed by an apheresis harvest of mononuclear cells on a fourth day. In a clean room, the apheresis product was prepared for cryopreservation and processed into 4 mL aliquots. Sterility and qualification testing were performed pre-processing and post-processing at multiple time points out to 2 years. Eight samples were shipped internationally to validate cell transport potential. One sample from all participants was cultured to test proliferative potential with colony forming unit (CFU) assay. Five samples, from 5 participants were tested for differentiation potential, including chondrogenic, adipogenic, osteogenic, endoderm, and ectoderm assays.

RESULTS: Fresh aliquots contained an average of 532.9 ± 166. × 106 total viable cells/4 mL vial and 2.1 ± 1.0 × 106 CD34+ cells/4 mL vial. After processing for cryopreservation, the average cell count decreased to 331.3 ± 79. × 106 total viable cells /4 mL vial and 1.5 ± 0.7 × 106 CD34+ cells/4 mL vial CD34+ cells. Preprocessing viability averaged 99% and postprocessing 88%. Viability remained constant after cryopreservation at all subsequent time points. All sterility testing was negative. All samples showed proliferative potential, with average CFU count 301.4 ± 63.9. All samples were pluripotent.

CONCLUSIONS: Peripheral blood stem cells are pluripotent and can be safely harvested/stored with filgrastim, apheresis, clean-room processing, and cryopreservation. These cells can be stored for 2 years and shipped without loss of viability.

METHODS: Osteoarthritis was induced at the right knee of sheep by complete resection of ACL and medial meniscus. Stem cells from sheep were induced to chondrogenic lineage. Test sheep received 5 mls single doses of 2 × 107 autologous PKH26-labelled ADSCs or BMSCs, while controls received basal medium. Functional recovery of the knees was evaluated via electromyography.

RESULTS: Induced ADSCs had 625, 255, 393, 908, 409, 157 and 1062 folds increases of collagen I, collagen II, aggrecan, SOX9, cartilage oligomeric protein, chondroadherin and fibromodullin compare to uninduced cells, while BMSCs had 702, 657, 321, 276, 337, 233 and 1163 respectively; p = .001. Immunocytochemistry was positive for these chondrogenic markers. 12 months post-treatment, controls scored 4 in most regions using ICRS, while the treated had 8; P = .001. Regenerated cartilages were positive to PKH26 and demonstrated the presence of condensing cartilages on haematoxylin and eosin; and Safranin O. OA degenerations caused significant amplitude shift from right to left hind limb. After treatments, controls persisted with significant decreases; while treated samples regained balance.

Methods: The OACs were expanded from passage 0 (P0) to P3, and cells in each passage were analyzed for gross morphology, growth rate, RNA expression and immunochemistry (IHC). The harvested OACs were assigned into two groups: low (1×10[7] cells/ml) and high (3×10[7] cells/ml) cell density. Three-dimensional (3D) constructs for each group were created using polymerised fibrin and cultured for 7, 14 and 21 days in vitro using chondrocyte growth medium. OAC constructs were analyzed with gross assessments and microscopic evaluation using standard histology, IHC and immunofluorescence staining, in addition to gene expression and biochemical analyses to evaluate tissue development.

Results: Constructs with a high seeding density of 3×10[7] cells/ml were associated with better quality cartilage-like tissue than those seeded with 1×10[7] cells/ml based on overall tissue formation, cell association and extracellular matrix distribution. The chondrogenic properties of the constructs were further confirmed by the expression of genes encoding aggrecan core protein and collagen type II.