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

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.

Inhibition of glucose use improves structural recovery of injured Achilles tendon in mice

“…The findings are consistent with previous observations. High glucose medium supports high production of sulfated proteoglycans and GAGs in mesenchymal stem/progenitor cells and chondrocytes, 28,29 while the cultured fibroblasts reduced GAG production under the low glucose media 30 . In addition, inhibition of glucose use by 2DG inhibits sulfate proteoglycan accumulation in injured tendon derived progenitors in culture 19 .…”

“…High concentrations of glucose increase proteoglycan synthesis in mesenchymal stem/progenitor cells, 28,29 while a shortage of glucose reduces glycosaminoglycan synthesis in fibroblast cultures. 30 We tested whether inhibition of glucose use affects proteoglycan accumulation in injured tendons.…”

Assessment of Clinical, Tissue, and Cell-Level Metrics Identify Four Biologically Distinct Knee Osteoarthritis Patient Phenotypes

“…Cells from each tissue source were plated in Lab-tek TM chambers, at a plating density of 24,000 cells/cm 2 , and cultured at 37°C, 80% to 90% humidity, 20% O 2 and 5% CO 2 in chondrogenic media for 6 days for colony-forming unit (CFU) assay. 16 , 17 , 22 - 24 On Day 6, cells were fixed and stained with bisbenzimide (nuclei stain) and imaged for automated CFU analysis using Colonyze TM image analysis software using previously established protocols. 16 , 17 , 22 - 24 …”

“… 16 , 17 , 22 - 24 On Day 6, cells were fixed and stained with bisbenzimide (nuclei stain) and imaged for automated CFU analysis using Colonyze TM image analysis software using previously established protocols. 16 , 17 , 22 - 24 …”

From regeneration to osteoarthritis in the knee joint: The role shift of cartilage-derived progenitor cells

“…In cartilage, chondrocytes are more sensitive to physioxia (5% O 2 ) than CPCs (Anderson et al, 2018), but further physioxia (2% O 2 ) benefit CPC chondrogenesis (Anderson et al, 2016). High glucose (25 mM) inhibited CPC colony-forming efficiency (CFE), but it increased GAG expression (Mantripragada et al, 2021), which is a key component of hyaline cartilage, so that we may choose different culture condition based on our purposes. Low-density and low-glucose condition in vitro was proved to enhance CPC proliferation, achieving CPC amplification for large knee cartilage defect (6-13 cm 2 ) repair in humans (Jiang et al, 2016).…”