Evaluation of cleaning methods for change-over after the processing of cell products to avoid cross-contamination risk

Abstract

Introduction: Cell-processing facilities face the risk of environmental bacteria contaminating biosafety cabinets during processing, and manual handling of autologous cell products can result in contamination. We propose a risk- and evidence-based cleaning method for cross-contamination, emphasizing proteins and DNA.

Methods: The transition and residual risks of the culture medium were assessed by measuring both wet and dried media using fluorescence intensity. Residual proteins and DNA in dried culture medium containing HT-1080 cells were analyzed following ultraviolet (UV) irradiation, wiping, and disinfectant treatment.

Results: Wet conditions showed a higher transition to distilled water (DW), whereas dry conditions led to higher residual amounts on SUS304 plates. Various cleaning methods for residual culture medium were examined, including benzalkonium chloride with a corrosion inhibitor (BKC + I) and DW wiping, which demonstrated significantly lower residual protein and DNA compared to other methods. Furthermore, these cleaning methods were tested for residual medium containing cells, with BKC + I and DW wiping resulting in an undetectable number of cells. However, in some instances, proteins and DNA remained.

Conclusions: The study compared cleaning methods for proteins and DNA in cell products, revealing their advantages and disadvantages. Peracetic acid (PAA) proved effective for nucleic acids but not proteins, while UV irradiation was ineffective against both proteins and DNA. Wiping emerged as the most effective method, even though traceability remained challenging. However, wiping with ETH was not effective as it caused protein immobilization. Understanding the characteristics of these cleaning methods is crucial for developing effective contamination control strategies.

Keywords: Biosafety cabinet; Cell-product processing; Changeover; Contamination control strategy; Cross-contamination.

Fig. 1

Risk of cross-contamination during the processing of autologous cell products. Despite advances, cell processing is mostly performed manually, which can lead to inadvertent contamination of biosafety cabinet surfaces. Failure to properly address this contamination during production line switches can result in cross-contamination.

Fig. 2

Risk of transition and cross-contamination within biosafety cabinets. (A) Experimental setup depicting wet and dry conditions of culture medium on SUS304 plates. (B) Quantification of medium transition to DW. Medium amounts were assessed based on phenol red fluorescence intensity (Ex: 485 nm, Em: 535 nm). Data are presented as Median with IQR. ∗P < 0.05. P values were calculated using the Mann–Whitney test.

Fig. 3

Risk of residuals and cross-contamination within biosafety cabinets. (A) Experimental setup illustrating wet and dry conditions of culture medium on SUS304 plates after wiping with 2 mL spraying of 70% ETH. (B) Residual amounts of medium on SUS304 plates. Medium quantities were determined based on phenol red fluorescence intensity (Ex: 485 nm, Em: 535 nm). Data are presented as Median with IQR. ∗P < 0.05. P values were calculated using the Mann–Whitney test.

Fig. 4

Cleaning methods for biosafety cabinet and risk of residual culture medium. (A) Experimental setup for cleaning methods. (B) Air-dried 200 μL MEMα with 10% fetal bovine serum (FBS) on SUS304 plates after each cleaning method, collected by 200 μL DW into 1.5 mL tubes. (C) Measurement of residual protein amount (μg) (n = 12). ∗P < 0.05. P values were calculated using the Kruskal–Wallis test with Steel-Dwass's multiple comparison test. (D) Measurement of residual DNA amount (ng) (n = 12). ∗P < 0.05. P values were calculated using the Kruskal–Wallis test with Steel-Dwass's multiple comparison test.

Fig. 5

Cleaning methods for biosafety cabinets and risk of residual culture medium containing cells. (A) Experimental setup for cleaning methods. Air-dried 200 μL MEMα with 10% FBS containing 2 × 10⁴ HT-1080 cells on SUS304 plates after each cleaning method, collected by 200 μL DW into 1.5 mL tubes. (B) Cell counts performed after each treatment; live cells are stained green by AO and dead cells are stained red by PI. Wet represents the count before treatment and is the value used to adjust for cell count. (C) Number of cells were detected by live/dead assay. (D) Measurement of residual protein amount (μg) (n = 12). ∗P < 0.05. P values were calculated using the Kruskal–Wallis test with Steel–Dwass's multiple comparison test. (E) Measurement of residual DNA amount (ng) (n = 12). ∗P < 0.05. P values were calculated using the Kruskal–Wallis test with Steel–Dwass's multiple comparison test.