Human Bronchial Epithelial Cells (HBEpC)

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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.

• Avoid nutrient gradients. We recommend using a small; drop-like BME gel bead and high volumes of medium. Change the medium every day as we’ve observed that less frequent medium changes results in yellow culture medium indicative of a pH shift likely from an increase in cellular metabolism.
• For optimal cell distribution;  it is important to control the “gelling” or setting of the ECM gel. Therefore; the temperature is critical! BME is liquid in the cold (4°C) and solidifies at 37°C. The gelling must be done in a very short time; otherwise the cells in the BME would sink to the bottom.

• https://pubmed.ncbi.nlm.nih.gov/31871466/
• https://pubmed.ncbi.nlm.nih.gov/35169685/