ABSTRACT
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.
Subject(s)
Connective Tissue Cells , Stem Cells , Adipose Tissue , Cartilage , Glucose , HumansABSTRACT
Membrane proteins are responsible for conducting essential biological functions that are necessary for the survival of living organisms. In spite of their physiological importance, limited structural information is currently available as a result of challenges in applying biophysical techniques for studying these protein systems. Electron paramagnetic resonance (EPR) spectroscopy is a very powerful technique to study the structural and dynamic properties of membrane proteins. However, the application of EPR spectroscopy to membrane proteins in a native membrane-bound state is extremely challenging due to the complexity observed in inhomogeneity sample preparation and the dynamic motion of the spin label. Detergent micelles are very popular membrane mimetics for membrane proteins due to their smaller size and homogeneity, providing high-resolution structure analysis by solution NMR spectroscopy. However, it is important to test whether the protein structure in a micelle environment is the same as that of its membrane-bound state. Lipodisq nanoparticles or styrene-maleic acid copolymer-lipid nanoparticles (SMALPs) have been introduced as a potentially good membrane-mimetic system for structural studies of membrane proteins. Recently, we reported on the EPR characterization of the KCNE1 membrane protein having a single transmembrane incorporated into lipodisq nanoparticles. In this work, lipodisq nanoparticles were used as a membrane mimic system for probing the structural and dynamic properties of the more complicated membrane protein system human KCNQ1 voltage sensing domain (Q1-VSD) having four transmembrane helices using site-directed spin-labeling EPR spectroscopy. Characterization of spin-labeled Q1-VSD incorporated into lipodisq nanoparticles was carried out using CW-EPR spectral line shape analysis and pulsed EPR double-electron electron resonance (DEER) measurements. The CW-EPR spectra indicate an increase in spectral line broadening with the addition of the styrene-maleic acid (SMA) polymer which approaches close to the rigid limit providing a homogeneous stabilization of the protein-lipid complex. Similarly, EPR DEER measurements indicated a superior quality of distance measurement with an increase in the phase memory time (Tm) values upon incorporation of the sample into lipodisq nanoparticles when compared to proteoliposomes. These results are consistent with the solution NMR structural studies on the Q1-VSD. This study will be beneficial for researchers working on investigating the structural and dynamic properties of more complicated membrane protein systems using lipodisq nanoparticles.
