Primary cells mimic the tissue of origin as they are directly isolated from the tissue and processed for culturing under optimized media conditions. Therefore, primary cell culture provides excellent model systems for studying normal cell physiology and biochemistry. There are a lot of articles on primary cells vs cell lines to understand which one of them enhances research efficacy but in the case of primary cells (take for example, drug studies), the research results are closer to the effects of drugs on normal body physiology. Primary cell culture researchers work with different types of primary cells based on tissue-specific, species-specific, and disease-specific categories as per their research requirements. But to understand primary cell culture better, here are five things to know about primary cells.
* Growth Requirements
Primary cells can be grown in suspension or adherent cultures. Some primary cell examples cells like peripheral blood cells naturally grow in suspension, without surface attachment. These primary cells grow to a higher density than the possible limit of adherent conditions. For the types of primary cells that are anchorage-dependent, these adherent cells require a surface to grow in vitro properly. Adherent primary cells are mostly cultured in a plastic vessel, but they can also be cultured on a micro-carrier (coated with extracellular matrix proteins to increase adhesion and provide growth and differentiation signals. The cell culture media for primary cells is composed of a basal medium with growth factors and cytokine supplements. Primary cells are grown and maintained at typically 37 °C, 5% CO2 (for mammalian cells) in a cell incubator. The culture conditions widely vary depending up on the types of primary cells. Primary cell growth media can vary in pH, glucose concentration, growth factors, and nutrient composition, depending up on the primary cell types.
During the establishment of primary cell cultures, it is essential to include antibiotics (may include a mixture of gentamicin, penicillin, streptomycin and amphotericin B) in the growth medium for limiting contamination from the host tissue. However, the long-term use of antibiotics is not recommended, as some reagents may be cytotoxic or cause problems during secretome analysis.
Retaining the viability of primary cells after isolation is crucial as they undergo senescence and stop dividing after a certain number of passages. For long-term viability of the primary cells, excellent primary cell culture handling skills along with aseptic culture techniques and appropriate culture conditions are essential.
* Cellular confluence
Primary cell confluence generally refers to the percentage of the cell culture flask or dish that the cells inhabit. A 100% primary cell confluence means that the surface area of the culture vessel is completely covered by cells, whereas 50% primary cell confluence means half of the surface is covered. Primary cells are never grown to 100% confluency as the chances of senescence increases and leads to increased cell loss. The limit of confluence is a major factor that determines subculturing period and cell health after subculturing.
* Maintenance and Subculture
The primary cell culture maintenance phase begins after the attachment of the isolated cells to the dish or flask surface. Usually, cell attachment takes about 12-24 hours after initiation of the primary culture. When cells reach a desired confluence and are actively proliferating, subculturing process is the next step. Never let the cells reach a 100% confluency as post-confluent cells may undergo differentiation and exhibit slower proliferation.
Adherent primary cells grow in monolayers and needs sub-culturing at regular intervals with appropriate culture medium for maintaining exponential growth. Sub-cultivation involves the breakage of both inter-cellular and intra-cellular bonds using proteolytic enzymes like trypsin/EDTA. After cell-attachment dissociation and single-cell suspension, the primary cells are counted and diluted to appropriate concentration before transferring into fresh culture vessels for growing.
* Cell counting
Hemocytometers are generally used for primary cell count and cell viability determination using the exclusion dye Trypan Blue. For information on cell counting, check out https://www.sigmaaldrich.com/technical-documents/protocols/biology/cell-quantification.html . In case of primary cells, although the method for counting cells is simple and considered a universal gold standard, it is difficult to achieve consistency among lab researchers as it becomes subjective based on handling of the subculturing process.
* Cryopreservation and recovery
In case of primary cells, cryopreservation is achieved using a cryoprotectant, such as DMSO or glycerol. Generally, cryopreservation can be achieved in 80% complete growth medium supplemented with 10% FBS and 10% DMSO. The freezing has to be slow, at a rate of -1°C per minute, to minimize ice crystal formation within the cells. Finally, the sample needs to be stored in the vapor phase of liquid nitrogen (-196°C). Precaution should be taken to avoid centrifuging the primary cells right after thawing as they are extremely sensitive to damage during the recovery phase.
For more information on primary cell culture, contact Kosheeka at info@kosheeka.com with your primary cells and stem cells inquiries.
