Protocol for Preparation and Cultivation of PromoCell^® Cryopreserved and Proliferating Cells

These products are available in the US, Canada, and select European countries.

• Protocol for Handling and Cultivating Cryopreserved Primary Cells
• Protocol for Handling and Cultivating Proliferating Primary Cells
• Primary Cell Subcultivation Protocol

Protocol for Handling and Cultivating Cryopreserved Primary Cells

Important Notes:

• Upon arrival, store the cryopreserved cells in liquid nitrogen, or seed them immediately. Storage at -80 °C is not sufficient for cell preservation and causes irreversible cell damage.
• Handle using aseptic technique and a laminar flow biosafety cabinet.

1. Prepare the medium
   Calculate the area of culture surface needed according to the plating density (see table below) and the lot-specific cell numbers stated on the certificate of analysis. Fill the appropriate volume of PromoCell^® Growth Medium (at least 9 mL per vial of cells) in cell culture vessels. Place the vessels in an incubator (37 °C, 5% CO₂) for 30 minutes.
   
   
2. Thaw the cells
   Remove the cryovial from the liquid nitrogen container and immediately place on dry ice - even for short transportation. Under a laminar flow cabinet, briefly twist the cap a quarter turn to relieve pressure, then re-tighten. Immerse the vial into a water bath (37 °C) just up to the screw cap for 2 minutes. Ensure that no water enters the thread of the screw cap.
   
   
3. Disinfect the vial and seed the cells
   Thoroughly rinse the cryovial with 70 % ethanol in a laminar flow cabinet. Then, aspirate the excess ethanol from the thread area of the screw cap. Open the vial and transfer the cells to a cell culture vessel containing the prewarmed medium from step 1.
   
   
4. Incubate the cells
   Place the vessel in an incubator (37 °C, 5% CO₂) for cell attachment. Replace the medium after 16–24 hours and every two to three days thereafter. The cells should be sub-cultured according to the subcultivation protocol (see below), once they have reached 70 – 90 % confluency.

Protocol for Handling and Cultivating Proliferating Primary Cells

PromoCell primary cells are available in a proliferating (non-cryopreserved) format.

Important notes:

• Start immediately after delivery.
• Use aseptic technique and work in a laminar flow biosafety cabinet.

1. Incubate the cells
   Unpack the culture vessel, do not open the cap, and immediately place it in an incubator (37 °C, 5% CO₂) for 3 hours to allow the cells to recover from transportation.
   
   
2. Replace the transport medium
   Carefully open the vessel, rinse the inner side of the lid with 70% ethanol, and let air dry. Aspirate the transport medium from the vessel. Add 10 mL of the appropriate PromoCell^® Cell Growth Medium.
   
   
3. Check and incubate the cells
   Check the cell density. Open the cap half a turn and place the vessel in an incubator (37 °C, 5% CO₂). Change the medium every two to three days. The cells should be subcultured according to the subcultivation protocol (see below), once they have reached 70 - 90% confluency.

Primary Cell Subcultivation Protocol

Important Notes:

• Use aseptic technique and a laminar flow biosafety cabinet.
• Due to their low-serum content or the absence of serum, PromoCell^® media are not suitable for trypsin neutralization (when subculturing the cells, for example). Instead, we recommend using the DetachKit (C-41200, C-41210, C-41220), which contains HEPES BSS, Trypsin/EDTA and Trypsin Neutralizing Solution.

1. Prepare the reagents and wash the cells
   Place the PromoCell^® DetachKit at room temperature for at least 30 minutes to adjust the temperature of the reagents. Carefully aspirate the medium from the culture vessel. Add 100 µL HEPES BSS Solution per cm² of vessel surface to wash the cells and agitate the vessel carefully for 15 seconds.
   
   
2. Detach the cells
   Note: We recommend detaching the cells at room temperature.
   
   Carefully aspirate the HEPES BSS from the culture vessel. Add 100 µL Trypsin/EDTA Solution per cm² of vessel surface. Close the vessel and examine the cells under a microscope. When the cells begin to detach, gently tap the side of the culture vessel to dislodge the remaining cells.
   
   
3. Neutralize the trypsin and harvest the cells
   Add 100 µL Trypsin Neutralization Solution per cm² of vessel surface and gently rock the vessel to mix. Carefully aspirate the cell suspension by pipette and transfer to a centrifugation tube. Spin down the cells for three minutes at 220 x g.
   
   
4. Incubate the cells
   Discard the supernatant from the cell pellet. Add 1 mL of the appropriate PromoCell^® Cell Growth Medium and resuspend the cells by gently pipetting up and down. Plate the cells according to the recommended seeding density in new cell culture vessels containing prewarmed Cell Growth Medium. Place the vessels in an incubator (37 °C, 5% CO₂) and change the media every two to three days.

*catalog numbers in this table are for cryopreserved cells and media in ready-to-use packaging