Culture Macrophages

General protocol for the culture of Primary Macrophages

All cell culture procedures must be conducted in a bio-safety cabinet. Any and all media, supplements, and reagents must be sterilized by filtration through a 0.2 µm filter. Use aseptic technique to prevent microbial contamination.

Cryo-preserved cells must be stored in liquid nitrogen or seeded immediately upon arrival. Medium: Review the information provided on the Cell Biologics website about appropriate culture media (e.g. serum and other supplements). Use pre-warmed (37°C) cell culture media (30-50 ML) to recover cryo-preserved cells or when changing media or splitting cells.

Cell recovery from cryovial:

• Quickly thaw cells in cryo-vial by incubating them in a 37°C water bath for <1 min until there is just a small bit of ice left in the vial.
• Promptly remove the vial and wipe it down with 70% ethanol.
• Transfer cells from the vial to a sterile centrifuge tube. Add 10 ml of pre-warmed Cell Biologics Cell Culture Medium. Flush the vial with an additional 0.5-1.0 ml of medium to ensure complete transfer of cells to the centrifuge tube.
• Centrifuge cells at 100 g for 5 minutes.
• Aspirate the supernatant and resuspend the cell pellet in 5-7 ml of Cell Biologics’  Cell Culture Growth Medium.
• Add resuspended cells into a plate (tissue culture treated).

Recommended Cell Seeding:

• 0.7-0.8 million cells are seeded per well of a 12-well plate or 1-1.5 million macrophages are seeded per well of a 6-well plate.
• Place a plate in a humidified, 5%-CO₂ incubator at 37°C until experiments.
• Change fresh cell culture medium every 24-48 hours.
• Cells should be checked daily under a microscopy to verify appropriate cell morphology.

Primary cells differ from cell lines in many ways.  Here’s what you should know about primary cells:

Primary cells have a finite life span. They are exactly like the cells that you would find in your own body, so they do die after a certain period of time in culture. The amount of time primary cells survive in culture varies according to cell type.  

1.  Primary cells can be diverse. Cancer cell lines often come from a single patient – for example, HeLa cells came from one woman, Henrietta Lacks. Primary cells, on the other hand, can come from a variety of people. Having a variety of primary cell types from many different donors is especially useful when carrying out early drug testing— it’s important to ensure that the drug is effective for everyone. Before testing the drug in human patients, researchers can use primary cells from different donors to verify that the same effects are observed. 
2. Primary cells can change in culture.  For this reason, and because primary cells do not live forever, Lifeline Cell Technology strongly suggests carrying out experiments on primary cells using earlier passages.
3. Primary cells have not been modified in any way. Except for the enzymatic and/or physical dissociation required for extracting the cells from their tissue of origin, primary cells are not altered in any way. Immortalized cell lines, on the other hand, are often transformed either with cancer genes, viruses or other inducible modifications, which could alter the outcome of experiments.
4. Primary cells are finicky. Cell lines are hardy, mostly because they have to last a long time in culture, and be capable of surviving multiple rounds of cryopreservation and thawing. Primary cells, on the other hand, tend to be more difficult to maintain in culture.

Thank you!

CellBiologics