Introduction: Subcutaneous (SC) implantation of encapsulated cells producing complex biologics (e.g. antibodies) holds promise for long-term, affordable, and accessible treatment. Cells implanted in encapsulation devices at high density in order to reduce device footprint are exposed to low oxygen (O₂) levels that potentially affect their viability and protein production; e.g., antibody production from hybridomas is influenced by ambient oxygen concentrations. Here, we assess the effect of oxygen levels predicted at the SC implantation site on ADC26.2 cells, an epithelial cell line genetically modified to produce monoclonal human antibodies (mAb) capable of neutralizing HIV. Our global motivation is to address logistical challenges in the treatment of diseases endemic to medically underserved countries. A living, implantable cell therapy capable of sustained mAb production might offer a transformative solution.
Methods: ADC26.2 cells obtained from Rice University were cultured as monolayers in T75 flasks and exposed to controlled oxygen concentrations (1%, 5%, and 21%) with 5% CO2 in the gas phase using a specialized incubator (BioSpherix). O2 levels were monitored throughout the study. At each time point (days 1 and 3 of regulated O₂ exposure), media samples were collected to measure IgG concentrations via ELISA (Abcam) and metabolic markers (glucose consumption rate – GCR and lactate production rate - LPR) via a YSI biochemistry analyzer. Additional assays included viability by membrane integrity staining (acridine orange/propidium iodide) and quantification by nuclei counts and DNA. At least n=3 replicates were obtained for each O2 concentration at each time point. Data are presented as the mean ± standard error mean (SEM).
Results: ADC26.2 cells retained cell viability under all oxygen conditions [Tables 1 and 2]; however, 1% O2 inhibited cell growth by 25% at day 3 compared to the normoxic control [Table 2]. Importantly, after 3 days of 1% and 5% O2 exposure, ADC26.2 cells continued to produce human IgG without substantial impairment compared to that observed with 21% O2 [Tables 1 and 2]. Metabolic measurements showed increased LPR and GCR in 1% O₂ compared to the 21% control, consistent with their known ability to shift to anaerobic glycolysis under oxygen limiting conditions. In all cases, GCR and LPR, when normalized per cell [Table 2], declined with culture time relative to the rates measured on day 1 [Table 1]. 
Conclusion: This first report on the effects of O2 concentration upon ADC26.2 cells demonstrates the extraordinary resilience of the cell line under physiologically relevant O2 conditions at the SC transplant site and adds evidence of their promise as implantable antibody factories for HIV vaccines. These initial insights will be critical to future work optimizing the design of implantable encapsulated cell factories. Further investigations will serve to evaluate long-term functionality and in vivo performance.