Ensuring sterility is a key quality requirement in the manufacturing of cell-based medicinal products. This study evaluated the capability of the BACTEC automated microbial detection system to identify viable microorganisms in adipose-derived stem cell-based products suspended in Ringer's Lactate. The validation process comprised four stages: i) sterility testing of uninoculated media to assess reagent quality; ii) growth promotion test to confirm medium performance; iii) suitability testing for compatibility with the sample matrix; iv) confirmation using pharmacopeial methods. All inocula met the European Pharmacopoeia recommendations. Validation was defined as the demonstration that the BACTEC system provides results equivalent to the pharmacopeial sterility test in terms of sensitivity, reliability, and detection time. Evaluated parameters included accuracy, repeatability, and detection limit, following European Pharmacopoeia requirements for alternative microbiological methods. The system successfully detected microbial contamination for all reference strains with a detection limit of 5 CFU and repeatability > 95 %. All microorganisms showed growth within 24-72 h, well inside the 7-day acceptance criterion. Quantitative comparison with the pharmacopeial method demonstrated full concordance (Cohen's κ = 1.0) and a strong correlation of detection times (r = 0.92, p < 0.001). These results confirm that the BACTEC system is a validated and reliable alternative to the pharmacopeial sterility test, providing faster detection without loss of analytical accuracy. The validation supports its applicability for sterility assurance of cell-based materials and integration into quality management during the manufacture of advanced therapy medicinal products.

Background: Adipose-derived mesenchymal stem/stromal cells (ADSCs) are gaining recognition in regenerative medicine for their potential for adipogenic, osteogenic, and chondrogenic differentiation, as well as their immunomodulatory properties. However, ADSC-based therapies focus either on differentiation for tissue replacement or on counteracting unrestrained inflammation to prevent tissue destruction and initiate regeneration. Here, we aim to examine the immunomodulatory potential of osteogenically differentiated ADSCs by analyzing their proteomic profile. Methods: Using LC-MS/MS, we generated the proteomic profiles of differentiated and undifferentiated ADSCs and compared them with the Reactome database. Transcriptomic analysis was also performed and compared with the proteomic profile. Results: Comparison of the proteomic (499 up-regulated; 355 down-regulated) and transcriptomic (212 up-regulated; 232 down-regulated) profiles showed 60.1% concordance—both proteins and transcripts showed the same trend. Significantly upregulated proteins in differentiating ADSCs (−log₁₀ p > 5 and >10) were grouped into four categories: propensity for osteogenic differentiation; immunomodulation/immune/inflammatory response; cell senescence; and cell cycle regulation. Among those proteins, thirteen were reported to play roles in processes such as immunomodulation, inflammatory signaling, or transplant rejection. Conclusions: We observed that differentiating ADSCs might still exert immunomodulatory effects, which could be used in the treatment of, e.g., bone defects.

The short-term stability of cell-based advanced therapy medicinal products (ATMPs) is a critical factor in their clinical handling and applicability. This study evaluates a multiparametric stability assessment approach using a GMP-manufactured ADSC-based ATMP as a model product. It consists of an ADSC suspension in Ringer’s lactate contained in polypropylene syringes. The in-use stability of the ADSC-based ATMP was evaluated over 36 h of hypothermic storage (2-8°C). Ten GMP-manufactured batches derived from a single qualified donor were analyzed immediately after preparation (T0) and after storage (T36) using validated methods for viability, sterility, mycoplasma status, endotoxin level assessment, and macroscopic appearance evaluation. Additionally, complementary quality tests using flow cytometry were applied. Cell integrity was characterized by Annexin V-FITC/7-AAD staining, whereas FSC/SSC analysis, doublet discrimination, and qualitative re-adhesion testing were used to investigate cell aggregation and post-storage recovery capacity. All tested parameters remained within predefined acceptance criteria at both time points. Mean cell viability decreased from 95.1 ± 1.8% to 88.2 ± 2.8% but remained above the acceptance threshold (≥70%) in all batches. Sterility, absence of mycoplasma, endotoxin levels, and suspension appearance met specifications. Flow cytometry demonstrated preserved membrane integrity, a small proportion of early apoptotic cells, and no changes in cell-size distribution or aggregation. These findings indicate that the investigated ADSC suspension maintains viability, structural integrity, and microbiological quality during short-term hypothermic storage under clinically relevant conditions. The study highlights the utility of complementary flow cytometry-based methods for stability assessment of fresh ATMPs.

Stromal vascular fraction (SVF) isolated from adipose tissue is an important source of starting material for adipose tissue-derived advanced therapy medicinal products (ATMPs), but the influence of donor-related and harvesting-related factors on SVF isolation outcomes remains unclear. In this study, SVF was isolated from adipose tissue samples obtained from 100 adult donors undergoing elective liposuction using a standardized enzymatic digestion protocol. Donor age, body mass index (BMI), waist-to-hip ratio (WHR), and harvesting site were evaluated in relation to SVF yield, isolation efficiency, and cell viability using correlation and non-parametric statistical analyses. SVF was successfully isolated from all samples. Mean SVF yield was 16.7 ± 9.68 × 10⁶ cells per 100 mL of adipose tissue, and mean viability was 80.9 ± 9.97%. No significant associations were observed between donor age and SVF yield, isolation efficiency, or viability. Similarly, BMI and WHR were not significantly associated with SVF isolation outcomes, although BMI showed a weak positive trend with isolation efficiency (ρ = 0.181, p = 0.086). Harvesting site had no significant effect on isolation outcomes despite numerically higher yield and viability in thigh-derived samples. Initial SVF viability was not associated with subsequent ADSC yield or viability after culture. Overall, demographic, anthropometric, and harvesting-site characteristics showed limited influence on quantitative SVF isolation outcomes. These findings support the robustness of standardized SVF isolation procedures and indicate that adipose tissue represents a reliable source of starting material for adipose tissue-derived ATMP manufacturing.