Influence of Glucose Concentration on Colony-Forming Efficiency and Biological Performance of Primary Human Tissue-Derived Progenitor Cells.

OBJECTIVE: Glucose concentrations used in current cell culture methods are a significant departure from physiological glucose levels. The study focuses on comparing the effects of glucose concentrations on primary human progenitors (connective tissue progenitors [CTPs]) used for cartilage repair. DESIGN: Cartilage- (Outerbridge grade 1, 2, 3; superficial and deep zone cartilage), infrapatellar fatpad-, synovium-, and periosteum-derived cells were obtained from 63 patients undergoing total knee arthroplasty and cultured simultaneously in fresh chondrogenic media containing 25 mM glucose (HGL) or 5 mM glucose (NGL) for pairwise comparison. Automated ASTM-based quantitative image analysis was used to determine colony-forming efficiency (CFE), effective proliferation rates (EPR), and sulfated-proteoglycan (GAG-ECM) staining of the CTPs across tissue sources. RESULTS: HGL resulted in increased cell cultures with CFE = 0 compared with NGL in all tissue sources (P = 0.049). The CFE in NGL was higher than HGL for superficial cartilage (P < 0.001), and contrary for synovium-derived CTPs (P = 0.046) when CFE > 0. EPR of the CTPs did not differ between the media in the 6-day assay time period (P = 0.082). The GAG-ECM area of the CTPs and their progeny was increased in presence of HGL (P = 0.027). CONCLUSION: Glucose concentration is critical to progenitor's physiology and should be taken into account in the setting of protocols for clinical or in vitro cell expansion strategies.

Native-Osteoarthritic Joint Resident Stem and Progenitor Cells for Cartilage Cell-Based Therapies: A Quantitative Comparison With Respect to Concentration and Biological Performance.

BACKGROUND: Cell-based therapy for cartilage repair is a promising approach and is becoming an established technique. Yet, there is no consensus on the optimal cell source. PURPOSE: To provide a donor-matched quantitative comparison of the connective tissue progenitors (CTPs) derived from cartilage (Outerbridge grade 1-3 [G1-2-3]), bone marrow aspirate concentrate (BMC), infrapatellar fat pad (IPFP), synovium, and periosteum with respect to (1) cell concentration ([Cell], cells/mL), (2) CTP prevalence (PCTP, colonies per million cells), and (3) biological performance based on in vitro proliferation potential (cells per colony) colony density, and differentiation potential (expression of negatively charged extracellular matrix: glycosaminoglycan-rich extra cellular matrix [GAG-ECM]). STUDY DESIGN: Descriptive laboratory study. METHODS: Tissues were obtained from 10 patients undergoing total knee arthroplasty (mean age, 59 years; women, n = 6). Automated quantitative colony-forming unit analysis was used to compare [Cell], PCTP, and CTP biological performance across tissue sources. RESULTS: [Cell] was highest in grade 3 cartilage (P = .002) and BMC (P = .001). Median PCTP was highest in IPFP (P = .001), synovium (P = .003), and G1-2 cartilage (P = .02). Proliferation was highest in synovium-derived CTPs (P < .001). Median colony density was highest in G1-2-3 (P < .001). Median GAG-ECM was highest in G1-2-3 (P < .001). Within each patient, CTPs derived from all tissues were highly heterogeneous in biological performance as determined by cells per colony, density, and GAG-ECM. CONCLUSION: Tissue sources differ in [Cell], PCTP, and biological attributes. The data presented in this study suggest that cartilage (G1-2-3) is the preferred tissue source for cartilage repair based on PCTP and GAG-ECM, followed by synovium, IPFP, BMC, and periosteum. However, due to the heterogeneous mixture of CTPs within each tissue source, there exists a subset of CTPs with biological performance similar to G1-2-3 cartilage, particularly in synovium and IPFP. Performance-based clonal selection and expansion of preferred CTPs and their progeny will potentially lead to improved cell population with predictive future. CLINICAL RELEVANCE: Optimal tissue regeneration strategies will require informed decisions regarding which of the available tissue sources to use. Optimizing cell sourcing in any tissue may require separation of CTPs with preferred attributes from those with less desirable attributes. The heterogeneity manifest in the early stage of colony formation represents an opportunity for performance-based clone selection for clinical cell processing and manufacturing.