Optimising In Vitro Detection of CXCR4 in Human Dental Pulp Stromal Cells and Stromal Cells From the Apical Papilla

ABSTRACT Aim This study aims to comprehensively investigate the dynamic expression pattern of CXCR4 in human dental pulp stromal cells (hDPSCs) and stromal cells from the apical papilla (SCAPs) and assess the impact of different cell dissociation methods on its precise assessment. On this basis, a standardised CXCR4 detection and cell sorting strategy can be established to ensure the high purity and viability of the sorted population. Methodology hDPSCs and SCAPs were collected by the outgrowth method from human third molars from three independent healthy (ASA I) patients (< 18 years old). Cells were characterised as mesenchymal stromal cells (MSCs) and expanded to passage 3–6 for experiments. Three different dissociation methods (Enzymatic digestion, Non‐enzymatic digestion, Mechanical collection) were used to collect single‐cell suspensions from culture flasks. Afterwards, the cells were resuspended in culture medium and allowed to recover at 37°C/5% CO 2 for 0–4 h. Surface expression of CXCR4 on viable cells was assessed using flow cytometry. Cell metabolic activity and senescence after dissociation treatment were tested by XTT Assay and senescence staining. Cell migration capacity was evaluated by wound healing assay. The data were statistically analysed using p < 0.05 as a statistical significance reference. Results hDPSCs and SCAPs exhibited typical MSC profiles and comparable cell viability under the same dissociation methods and time points. hDPSCs showed higher CXCR4 expression than SCAPs. CXCR4 expression displayed time‐dependent fluctuations in both cell types and cell viability was significantly affected by dissociation method and recovery period. Enzymatic digestion resulted in higher CXCR4 expression after 2‐h recovery and preserved over 90% cell viability. In contrast, non‐enzymatic dissociation and mechanical scraping impaired cell metabolic activity, migration capacity, and induced premature senescence. Conclusion In in vitro expanded hDPSCs and SCAPs, the cell‐detachment method and recovery time can significantly affect the CXCR4 membrane detection and subsequent cell‐survival performance. To balance optimal cell viability and detection reliability, enzymatic cell dissociation followed by a 2‐h recovery time seems to be the best protocol for CXCR4 detection and subsequent downstream experiments within the 0–4 h time frame.