Cryostor

At day 6 or 7 of culture, medium was removed from wells and 500-μl room temperature DPBS added. Organoid-containing Matrigel was resuspended in the DPBS and transferred to a centrifuge tube. The organoids were fragmented by gentle pipetting in the tube before centrifugation. The organoid fragment suspension was centrifuged at 50g for 3 min at 4 °C (bovine) or 300g for 10 min at 4 °C (porcine). Supernatant was removed and organoid pellets resuspended in CryoStor® (Stem Cell Technologies) using a volume of 1-ml CryoStor® per three wells organoids. Vials were frozen at – 80 °C for 24 h in a Nalgene® Mr Frosty freezing container (Thermo Fisher Scientific, Cheshire, UK) before being transferred to liquid nitrogen for long-term storage.
For resuscitation, vials were removed from liquid nitrogen and defrosted in a waterbath at 37 °C until the freezing medium became liquid but not warm. The solution in the vial was transferred to a 15-ml tube with 5-ml IntestiCult added. Suspensions were centrifuged at 50g for 3 min at 4 °C (bovine) or 300g for 10 min at 4 °C (porcine). Organoid pellets were then resuspended in Matrigel and covered with medium as per the passage process.

OT-I splenocytes frozen in CryoStor (STEMCELL technologies, Vancouver, Canada) and stored in liquid nitrogen were thawed in RPMI+ and labelled with 0.5 µM CellTrace™ Violet (CTV, Thermo Fisher Scientific) for 20 min in the dark at 37 °C and 5% CO₂. Staining was neutralized by adding 5 times the volume of medium and incubating for an additional 5 min. CTV⁺ OT-I splenocytes were subsequently plated in RPMI+ in round-bottomed 96-well plates at 200,000 cells per well. Treated or transfected CD103⁺ DCs were harvested and placed in co-culture at a 1:10 DC:T-cell ratio in a total volume of 200 µL. As a positive control, OT-I splenocytes were stimulated with 2 µg/mL SIINFEKL. After 2 days, 2 µM monensin and 5 µg/mL brefeldin A (BioLegend) were added to the co-cultures to stop cytokine secretion. After 16 h, cells were harvested and analyzed for polyfunctionality, defined as T cells displaying multiple functions on a single cell level by flow cytometry (first described in the context of HIV by Betts et al. [30 (link)]). Cells were categorized via Boolean gating containing 0–4 functions: IL-2, TNF-α, and/or IFN-γ production, with or without proliferation (measured as dilution of the CTV signal).

Within 2 h of whole blood collection, PBMCs were isolated from peripheral blood. T cells were enriched using RosetteSep™ Human T‐Cell Enrichment Cocktail (StemCell Technologies, Cat #15061) according to the manufacturer's protocol. Both PBMCs and T cells were collected via SepMate™ collection tubes (StemCell Technologies, Cat #85460), using Lymphoprep™ (StemCell Technologies, Cat #7861) density gradient, and washed twice. All isolated cells were frozen in CryoStor® (StemCell Technologies, Cat #7930) at a controlled rate using Mr. Frosty containers.