Choline chloride

Tadalafil (TDF) and lidocaine HCl (LDC) were kindly gifted by the Jordanian Pharmaceutical Company (JPM) Amman, Jordan. Choline chloride and malonic acid were purchased from Tokyo Chemical Industry Co., Ltd. (TCI), Tokyo, Japan.

The medium combinations were prepared with 31 commercially available compounds, in which choline chloride and pyridoxal hydrochloride were from Tokyo Chemical Industry, FBS was from Japan Bio Serum, and the remaining compounds were from Wako. The abundance of penicillin‒streptomycin and phenol red were maintained at 1% and 0.03 mM, respectively. The concentrations of FBS were changed in the range of 0.1–10%. The remaining 28 components were changed zero- to 10–100-fold of their concentrations in EMEM. In brief, four to five different concentrations were used for each component, and the changes were roughly on a logarithmic scale, because a broad range of concentration gradients benefited the ML-assisted optimization^{38 (link)}. The medium combinations were prepared by mixing the stock solutions of the chemical compounds, which were individually prepared in advance, with highly pure water (Direct-Q UV, Merck). The stock solutions were sterilized by sterile syringe filters (Merck) with hydrophilic PVDF membranes of 0.22 µm pore size, dispensed in a small volume and stored at −20 °C for future use. Note that all medium combinations were prepared immediately before use, and the stock solutions were used once. All medium combinations tested in the present study were summarized in Supplementary Data 1 and Supplementary Data 2 for the regular and time-saving modes, respectively.

Choline chloride (C0329:Tokyo Chemical Industry), trimethylamine (92262:Sigma-Aldrich), N8-acetylspermidine dihydrochloride (A3658: Sigma-Aldrich), cholic acid (032–03042: FUJIFILM Wako), and propionic acid (169–04723: FUJIFILM Wako) were purchased from the companies indicated.