Cellstart ctstm

Tissue fragments were placed in 115 cm² tissue-culture flasks with reclosable lids (TPP, Switzerland) in the presence of StemMACS-MSC expansion Media kit XF (Miltenyi Biotec GmbH, Germany) after coating with CELLstart^TM CTS^TM (Gibco/Thermo Fisher Scientific) or Synthemax^® II-SC (Corning) as adhesion substrates for “GMP grade I” and “GMP grade II” conditions, respectively (see Table 1). For the coating with CELLstart^TM CTS^TM, flasks were incubated with the reagent diluted 1:50 in DPBS for 2 h at 37°C. Regarding the coating with Synthemax^® II-SC, flasks were incubated with the reagent diluted 1:40 in sterile water for 2 h at RT.
At confluence, CPC were harvested using TrypLE^TM Select Enzyme (Gibco/Thermo Fisher Scientific), then seeded at 8–10 × 10⁴ cells/cm² and expanded in T flasks (75–150 cm²), HYPERFlask^® (1720 cm²) or HYPERStack^®-12 (6000 cm²) culture vessels (Corning). Thanks to their negatively charged, highly hydrophilic CellBIND^® surface (Corning), designed to facilitate cell attachment and spreading, HYPERFlasks and HYPERStacks did not require any coating.
For CPC MCB and PPCB generation (see below), the above indicated StemMACS-MSC expansion Media kit XF was substituted by its GMP counterpart, MSC-Brew GMP Medium (test lot kindly provided by Miltenyi Biotec GmbH).
CPC expanded in GMP grade conditions I and II are thereafter indicated as CPC-I and CPC-II, respectively.

On day 8, a CELLstart^TM CTS^TM (CTS; Gibco Thermo Fisher Scientific)-coated (for the transfection of H460) or CTS/Matrigel-coated (for the transfection of N87) 6-well plate was prepared. For the preparation of the CTS-coated plate, 1 ml of diluted CTS (1:50 in Dulbecco’s PBS, Gibco Thermo Fisher Scientific) was added per well according to the instructions, and for the preparation of the CTS/Matrigel-coated plate, 1 ml of diluted CTS and 1 ml of diluted Matrigel solution (mixture of one vial of Matrigel stock solution [Corning] and 25 ml of Pluriton^TM medium) were mixed. Thereafter, 1 ml of the mixture was added per well in a 6-well plate, and the plate was incubated first on a clean bench for 30 min and then overnight at 4 °C. To transfer the target cancer cells to the prepared CTS- or CTS/Matrigel-coated plates, the cancer cells on the feeder layer were detached and seeded in a CTS-coated (for H460) or CTS/Matrigel-coated (for N87) plate in conditioned-R-medium.