Human CD34+ Progenitor Cells (hCD34+-CB)

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Short protocol:

• Trypsinize the cells as usual
• Centrifuge and resuspend in suitable cold freezing medium at a density of 1-4 x 10⁶ cells/ml
• Cool down the cells slowly to -80°C (approx. -1°C per min). We recommend to use "Mr. Frosty" from Nalge or "CoolCell" from Biocision; which both provide gradual and controlled cooling rates when placed in a -80°C freezer overnight.
• Transfer the vials into liquid nitrogen for long-term storage

• Mesenchymal Stem Cells (C-12974/C-12971/C-12977) need Fibronectin-coating when grown in PromoCell MSC Growth Medium XF (C-28019) and when differentiated in MSC Neurogenic (C-28015); Adipogenic (C-28016); or Osteogenic (C-28013) Differentiation Media.
• Human monocyte-derived macrophages (C-12914/C-12916/C-12915/C-12917) must be seeded into Fibronectin-coated culture vessels in combination with PromoCell's M1- and M2-Generation Media XF (C-28055; C-28056).
• For efficient induction of osteoblast mineralization with PromoCell's Osteoblast Mineralization Medium (C-27020); the TC plates should be pre-coated with collagen type I.

• Liquid phase storage provides a consistent temperature of -196°C; a longer holding time and a greater vial capacity but involves the risk of contamination issues.
• Storage in the gas phase is very safe with respect to contaminations but the holding time of the cells is shorter and the vial capacity is reduced.

• Thaw the cells (C-12921) for 2 min in a 37°C waterbath. Dilute in 9 ml of complete HPC Expansion Medium XF (+ Cytokine Mix E) and count the cells
• Spin down for 10 min at 240xg; aspirate the supernatant; resuspend the pellet at 20;000 cells/ml HPC Expansion Medium XF
• Plate in an appropriate suspension culture vessel and incubate the culture for 2-3 days at 37°C and 5% CO₂
• Then double the media volume by adding fresh complete medium; e.g.; 4 ml  suspension culture + 4 ml fresh medium (= 8 ml)
• Incubate the cells for an additional 10-12 days by performing a partial medium change every 2-3 days Example partial medium change: For a culture volume of 8 ml; spin down the cells; aspirate and discard 4 ml of the supernatant; resuspend the cells and add 12 ml of fresh complete medium (= 16 ml).