Streptomycin

Human T lymphoblasts (CCRF-CEM) and human embryonic kidney (HEK293) cells were purchased from DSMZ (German collection of microorganisms and cell cultures, Braunschweig, Germany). CCRF-CEM and HEK293 cells were grown in 75-cm² culture flasks (Sarstedt, Nuemberecht, Germany) and maintained in culture at 37 °C in 95% humidity, 20% O₂ and 5% CO₂. RPMI-1640 and DMEM culture medium supplemented with 10% fetal calf serum, 100,000 U/L penicillin and 100 μg/L streptomycin (Biochrome, Berlin, Germany) were used to grow CCRF-CEM and HEK293 cells, respectively. Cell confluency was regularly checked, and the medium was changed accordingly.

The isolation of MSC followed a standardized protocol based on Ficoll (Ficoll Paque™ Plus, density 1.078 g/mL, GE Healthcare, Freiburg, Germany) density gradient centrifugation as reported previously [20 (link)]. 10 ×  10⁶ mononucleated cells (MNC) of each probe were cultivated in T75 tissue flasks in low-glucose DMEM (Gibco, Life Technologies, Darmstadt, Germany) culture media containing 10% (v/v) fetal calf serum (FCS; Biochrome, Berlin, Germany), 100 U/mL penicillin, 0.1 mg/mL streptomycin, 2 mM-glutamax, and 1 mM sodium pyruvate (all from Sigma-Aldrich).
Following the International Society for Cellular Therapy’s (ISCT) minimal criteria to define mesenchymal stromal cells (MSCs), we choose plastic adherence, as well as appropriate surface marker expression and trilineage differentiation for MSC characterization [21 (link)–25 (link)].

Intestinal epithelial organoids were cultured from 4- to 8-week-old Gpx4^+/−IEC and littermate WT mice using IntestiCult Organoid Growth Medium (Stemcell Technologies, 06005) and a protocol adapted from manufacturer’s instructions as initially described^{58 (link)}. Briefly, small intestines were flushed with ice cold PBS, minced to pieces of approximately 2–3 mm in size and washed up to five times with 10 ml ice cold PBS. Samples were transferred to 2 mM EDTA/PBS and incubated at 4 °C on a rocking platform for 30 min. After sedimentation supernatant was removed and crypts eluted in 10 ml PBS by shaking vigorously and passing crypts through a 70 µm cell strainer to obtain Fraction 1. This process was repeated three times to obtain Fractions 2–4. Fractions were analyzed under a light microscope and the optimal fraction was chosen to obtain crypts for organoid culture by centrifugation at 290g for 5 min at 4 °C. Crypts (N = 500) per well were seeded in 50 µl Matrigel (BD, 356231) on a pre-warmed 24-well plate and allowed to solidify for 10 min at 37 °C, after which 500 µl IntestiCult Growth Medium supplemented with 100U/ml penicillin and 100 µg/ml streptomycin (Biochrome, 0257F) was added. Medium was exchanged three times per week and organoids passaged with a split ratio of 1:6 every 7–14 days (Supplementary Fig. 8A-G).