Pfhm 2 protein free hybridoma medium

NMuMG and BALB/3T3 clone A31 (“A31”) cell lines were purchased from ATCC. Cell lines were authenticated by STR profiling (ATCC), mycoplasma negative, used at low passage, and examined for correct morphology. The 8A7H5, H35-17.2, and GK1.5 hybridomas were grown in PFHM-II Protein-Free Hybridoma Medium (Thermofisher) (Dialynas et al., 1983 (link); Golstein et al., 1982 (link); Swimm et al., 2010 (link)). mAb was produced in CELLine bioreactor flasks (Corning). All other cells were kept in Dulbecco’s Minimal Eagle Media supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin.

A swelling test to investigate real‐world swelling under culture conditions was performed on transwell filters, as previously described.^[9] Briefly, hydrogel solutions were prepared (Table S1, Supporting Information) and 500 µL of this solution was added onto the transwells with a 0.4 µm pore size (Corning, 12‐well culture plate) without organoids and left to cross‐link for 1 h. STEMdiff APEL2 medium (1% (v/v) PFHM‐II protein‐free hybridoma medium (Thermo Fisher Scientific) and 1% (v/v) antibiotic/antimycotic (Thermo Fisher Scientific) was added below the transwells with hydrogels and incubated up to 98 h (37 °C). Transwells with hydrogels were weighed at 1, 2, 4, 6, 24, 48, 72, and 96 h. The hydrogel swelling ratio in % (S_r) over time was calculated by: Sr=wx−wowo, in which w₀ = initial hydrogel weight and w_x = hydrogel weight at the x time point (Figures S3d and S8d, Supporting Information). GraphPad 8.2.0 software was used for statistical analyses using two‐way ANOVA or unpaired t‐test.

The 8A7H5 hybridoma (rat IgG2b, κ) was previously generated by immunization of rats with MuPyV VP1 virus-like particles (Swimm et al., 2010 (link)). NMuMG, BALB/3T3 clone A31 ‘A31’, and mIMCD-3 cells were purchased from ATCC. Mouse embryonic fibroblasts (MEFs) were isolated from day 13 C57BL/6 embryos. Hybridomas 8A7H5 and H35-17.2 (anti-CD8β) (Pierres et al., 1982 (link)) were maintained in PFHM-II Protein-Free Hybridoma Medium (ThermoFisher) at 37°C in 5% CO₂. mAb was generated by growing the hybridomas in CELLine disposable bioreactor flasks (Corning). All other cells were maintained in Dulbecco’s Minimal Eagle Media supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin (DMEM) at 37°C in 5% CO₂. The sex of NMuMG cells is female, the sex of mIMCD-3 and A31 cells is not reported. Cell lines were mycoplasma negative, authenticated by STR profiling (ATCC), examined for correct cell morphology, and used at low passage number.