Gmp scgm

Primary human CD34⁺ HSPCs were seeded at 1 × 10⁶ cells per milliliter in culture well plates in GMP SCGM (CellGenix, Freiburg, Germany) supplemented with 1 U/mL penicillin plus 100 μg/mL streptomycin (GIBCO, Thermo Fisher Scientific, Rochford, UK), 300 ng/mL hSCF, 300 ng/mL hFlt3-L, and 100ng/mL hTPO (either manufactured to cGMP from CellGenix or animal-free from PeproTech, London, UK), referred to as STF.⁴⁵ (link) For comparison of HSPC culture media, using the same supplements as described earlier for SCGM, cells were cultured in X-Vivo 15 (Lonza, Slough, UK) additionally supplemented with 1% human albumin (Zenalb 20, Bio Products Laboratory, Herts, UK), in StemSpan-ACF (STEMCELL Technologies, London, UK) additionally supplemented with 1% human albumin, or in HSC Brew GMP medium plus HSC Brew GMP supplement (Miltenyi Biotec, Surrey, UK) additionally supplemented with 2% human albumin.
For the GMP experiment, HD CD34⁺ HSPCs were isolated from a fresh mobilized leukapheresis (AllCells, Alameda, CA, USA) following standard immunomagnetic procedure (CliniMACS, Miltenyi Biotec). Cells were seeded at 1–1.5 × 10⁶ cells per milliliter in SCGM supplemented with 300 ng/mL hSCF, 300 ng/mL hFlt3-L, and 100 ng/mL hTPO (manufactured to cGMP from CellGenix). Cells were cultured in VueLife Culture Bags (CellGenix).

Cells were isolated using a human negative selection-based NK cell isolation kit (EasySep-19615, STEMCELL). Purified NK cells were then cultured in stem cell serum-free growth medium (CellGenix GMP SCGM, 20802-0500) supplemented with 10% heat-inactivated human AB plasma from healthy donors (SIGMA, male AB, H-4522, Israel, Jerusalem), 1% l-glutamine, 1% Pen-Strep, 1% sodium pyruvate, 1% MEM-Eagle, 1% HEPES 1M, and 300 IU/mL recombinant human IL-2 (PeproTech, 200-02-500UG, Cranbury, NJ, USA).

The mobilized peripheral blood was collected from four healthy donors, after informed consent and approval by the local ethics committee (Ethikkommission an der TUD, Ethic board no. EK201092004). Mobilization was achieved by subcutaneous injection of granulocyte colony-stimulating factor (7.5 µg/kg per day; Granocyte, Chugai Pharma) [14 (link)]. CD34⁺ HSPCs were isolated directly after leukapheresis by magnetic-activated cell sorting (MACS) (#130-046-702, Miltenyi Biotec, Bergisch Gladbach, Germany) technology based on CD34, as we described previously [14 (link), 20 (link), 37 (link)]. CD34⁺ HSPCs were cultured in serum-free HSPC medium (CellGenix® GMP SCGM, CellGenix GmbH, Freiburg, Germany) supplemented with early acting cytokines (50 ng/mL stem cell factor, (CellGenix), 50 ng/mL fms-related tyrosine kinase-3 ligand (PeproTech, Cranbury, NJ) and 15 ng/mL interleukin-3 (R&D Systems, MN)) at a density of 7.5 × 10⁴/cm² of surface area for 1–2 days on confluent MSCs in a humidified 5% CO₂ atmosphere at 37 °C. Afterward, they were collected and cultured on sub-confluent MSCs (see below).

Mobilized peripheral blood HSPCs were isolated from mobilized peripheral blood (IRB#329/10) using the CD34 MicroBead Kit UltraPure (Miltenyi Biotec, Bergisch-Gladbach, Germany, Cat# 130-100-453) according to the manufacturer’s instructions. Purified cells were resuspended in CryoStor CS10 (StemCell Technologies, Bothell, WA, USA, Cat# 07930) at 1 × 10⁶ cells/mL and stored in liquid nitrogen until usage. After thawing, and for 72 h prior to nucleofection, CD34+ cells were cultured in CellGenix GMP SCGM (CellGenix, Freiburg, Germany, Cat# 20802-0500) supplemented with recombinant human cytokines SCF (300 ng/mL), Flt3-L (300 ng/mL), IL-3 (60 ng/mL; all ImmunoTools, Friesoythe, Germany, Cat# 11343327, 11343307, 11340037), TPO (100 ng/mL; PeproTech, Hamburg, Germany, Cat# 300-18) as previously described [50 (link)]. After nucleofection (see section below), IL-3 was removed from the medium and HSPCs cultured at 37 °C with 5% CO₂ until harvested for downstream analyses [50 (link)].