Microencapsulated Cell Cytokine Release Evaluation

Microencapsulated cells were gradually deprived of fetal calf serum (10% on the day of encapsulation, 5% from day 1 to day 4, and 0.75% on day 4) to evaluate their ability to release cytokines and growth factors. Non-encapsulated cells were used as controls. On day 4, cells were stimulated with a culture medium containing 0.75% FCS, supplemented with 20 ​ng/mL of TNF-α (Miltenyi Biotec, Germany) and 20 ​ng/mL of IFN-γ (Miltenyi Biotec, Germany). Non-stimulated cells were cultured in a culture medium containing 0.75% of FCS. After 72 ​h (day 7 after encapsulation), the release of soluble factors into the supernatant was evaluated. Prostaglandin E₂ (PGE₂) concentration was determined using an ELISA kit (Cayman Chemical, USA), following the manufacturer's instructions. Human Growth Factor (HGF) and Transforming Growth Factor-beta (TGF-β) concentrations were also determined using an ELISA kit (DuoSet®, R&D Systems, Canada). Indoleamine 2,3-dioxygenase (IDO) enzymatic activity was measured through tryptophan-to-kynurenine conversion with a photometric determination of kynurenine concentration in the supernatant described before [34 (link)]. The experiments were performed in triplicate for each condition, with cells from four donors (Donors A-D, see Table S2). All results were normalized by the number of cells per sample.
Another experiment was performed using OA synovial fluids from 6 patients to stimulate the cells for 72 ​h. Encapsulated cells were gradually deprived of calf serum for 4 days and then stimulated with a culture medium containing 0.75% FCS supplemented with 10% v/v of synovial fluids harvested from OA patients (Patients A-F, see Table 1). After 72 ​h, PGE₂ concentration and IDO activity were evaluated in the supernatant. The experiments were performed once for each condition, with cells from one donor (Donor A). All results were normalized by the number of cells per sample.
In another experiment, non-encapsulated and microencapsulated cells were mock injected through a 26G needle, gradually deprived of calf serum for 4 days, then stimulated with culture medium containing 0.75% FCS, supplemented with 20 ​ng/mL of TNF-α and 20 ​ng/mL of IFN-γ or supplemented with 10% v/v of synovial fluids harvested from 3 another OA patients (Patients G-I, see Table 1). Non-stimulated cells were cultured in a culture medium containing 0.75% of FCS. After 72 ​h, PGE₂ concentration and IDO activity were evaluated in the supernatant. As a control, a study of the secretory function of cells in 2D monolayer culture was performed simultaneously. 2D experiments were performed using the same number of cells as 3D experiments, with 20 ​000 ​cells seeded in a 24-well plate. Cells were used at the same passage and were donor-matched to 3D experiments. The experiments were performed in triplicate for each condition, with hASCs from four donors (Donors B-E, see Table S2). All results were normalized by the number of cells per sample.