Technical Tips For Primary Cell Culture
When working with primary cells, it is important to remember that they are not cell lines and cannot be treated the same. Because iXCells Biotechnologies has extensively worked with primary cells, we are very familiar with the common problems researchers encounter when culturing them. Here are five common mistakes that are made with primary cells and how to correct these mistakes.
Mistake #1: Thawing a vial of primary cells in a water bath for an extended period of time
Correction #1: Primary cells are very sensitive to the thawing process so it is important that the vial be placed in a 37oC water bath, held and rotated gently until the contents are just thawed. Then remove the vial from the water bath promptly and transfer in into a sterile hood. Make sure your culture vessel is ready before thawing so the cells can immediately be seeded and placed in the incubator.
Mistake #2: Allowing primary cells to become too confluent
Correction #2: Primary cells can become senescent when grown to 100% confluent. Remember that primary cells are not 100% pure, so it is important to minimize growth of contaminating cells. We recommend subculturing primary cells when they are 90-95% confluent.
Mistake #3: Over-trypsinization when passaging primary cells
Correction #3: When passaging cells, use low concentrations of trypsin and monitor cells closely under the microscope. Also, remember to completely neutralize the cells after trypsinization because any active trypsin will damage the cells.
Mistake #4: Primary cells can easily be refrozen
Correction #4: Normally we do not recommend refreezing primary cells as this can promote a senescent phenotype and/or cause functional changes. Primary cells are extremely sensitive and refreezing may result in cell death or damage.
Mistake #5: Primary cells can proliferate indefinitely
Correction #5: Unlike cell lines, primary cells have a limited expansion capacity. We recommend using primary cells as early as possible for experiments to prevent genetic drift. In addition, if you are working with a difficult cell type you should monitor cell morphology closely for contaminating cells as they can take over the culture over time.