Stemmacs msc expansion media kit xf

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.

MSCs were obtained from iliac crest aspirates from a human male post-mortem organ donor (protocol agreed by the French Agency of Biomedicine), and were isolated as previously described [17 (link), 40 (link)]. This cell population was expanded by culture in StemMACS™ MSC Expansion Media Kit XF (Miltenyi Biotec, Paris, France) in a humidified incubator at 37 °C, under an atmosphere containing 5% CO₂, until 70% confluence. All experiments were performed with cells between passages 4 and 5.
The human U87MG GB cell line was obtained from the ATCC (LGC Promochem, Molsheim, France). Cells were maintained in Dulbecco’s modified eagle medium-high glucose medium (DMEM-HG, Lonza, Verviers, Belgium) containing 10% fetal bovine serum (FBS) (Fisher Scientific, Illkirch, France) and 1% antibiotics (Sigma-Aldrich, St. Quentin Fallavier, France), under an atmosphere containing 5% CO₂ (37 °C), in a humidified incubator, until they reached 80% confluence.
Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza. Cells were cultured according to the supplier’s instructions, in endothelial cell growth medium-2 (EGM-2) in a humidified chamber at 37 °C, under an atmosphere containing 5% CO₂.
SFN was purchased from LC Laboratories (Woburn, USA). The stock solution was prepared in DMSO (Sigma-Aldrich), at a concentration of 100 mM. Aliquots were stored at −20 °C.

Umbilical cords were collected from Hue Central Hospital with the approval of the Research Ethics Committee of Hue Central Hospital and transported to the Stem cells Laboratory, Department of Biology, College of Sciences, Hue University in 0.9% normal saline containing 100 U/mL penicillin and 100 mg/mL streptomycin at 4 °C with written consents of mothers and families.
Initially, blood vessels were removed in saline, and the umbilical cords were sliced into 1–2 cm² sections. These fractions were rinsed with PBS and subsequently suspended in StemMACS™ MSC Expansion Media Kit XF (Miltenyi Biotec, Germany). Incubation was performed at 37 °C in a humidified atmosphere containing 5% CO₂, and the medium was replaced every three days. The morphology of UC-MSCs was followed with Olympus CKX31SF inverted microscope (Olympus Corporation, Tokyo, Japan).
When the cell population's confluence reached around 80%, the cells were trypsinized, counted, and re-seeded into culture dishes at a density of approximately 1000 cells/cm².