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Primary Cells Culture Tips and Tricks

Primary cells are isolated directly from living tissue and grown in vitro.  These cells haven’t multiplied excessively i.e., they have experienced very few passage numbers and thus, possess native characteristics of parent tissue. Primary cells shouldn’t be confused with cell lines as cell lines are cultured from immortalized cultures of primary cells.

Biomedical research is gaining a higher ground with the idea of using primary cells in clinical translational domains. Primary cells have found extensive applications in the field of 3D modeling, bioprinting, tissue engineering, cancer studies, cell therapy, etc. but when it comes to working with primary cells in the lab, researchers face various problems. This article contains a few cell culture tips and tricks that can help in mitigating these problems and make working with primary cells easier.

Cell Growth Requirements

Primary cells can grow on both suspension (cells that don’t need a solid surface to grow)as well as adherent (cells that need a solid surface to grow) cultures. As contamination chances of primary cell cultures are higher, it is vital that antibiotics are added to the growth medium.

For a longer life span of the cells, cell culture handling skills should be excellent; also, suitable culture conditions such as pH, growth factors, concentration, growth medium,  temperature, presence of nutrients, and glucose are essential. The practice of the aseptic technique is recommended.

Confluence of Cell Culture

Cellular confluence, in adherent cultures, denotes the surface area populated by attached cells in the culture vessel. For example, 100% cellular confluence means that cells cover the complete surface area of the vessel, while 50% confluence means that cells cover only half of the surface area. It is a significant and crucial parameter to track as different cells require different confluence endpoints i.e., points at which the cells are required to be sub-cultured.

Maintenance and cell Subculture

The maintenance of cells starts as soon as they are isolated and attached to the culture vessel surface. Attachment of cells takes about 12 hours from the point of seeding. When the desired confluence percent is achieved and the cells are actively multiplying, it is time to subculture. The best time to begin subculturing primary cell cultures is around 80-90% confluence as fully confluent cells may undergo differentiation and start exhibiting slower growth after subculturing.

Cryopreservation of Cells

Cryopreservation is the method by which the living cells are preserved, using low temperatures. This is generally done to increase primary cell shelf life. It is vital that cells are properly cryopreserved, in an effort to minimalize cell damage and death during experimental processes. In the case of primary cells, DMSO is generally used as a cryoprotectant along with FBS.

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