Perfecting Primary Cell Culture

Setting up and maintaining a successful primary cell culture can be a daunting task, even for experienced researchers. To shed light on best practices, common pitfalls, and expert troubleshooting strategies, we talked to James Sherley, M.D., Ph.D., a leading cellular biologist and CEO of Asymmetrex. Drawing from years of hands-on experience with primary cells, Dr. Sherley provided practical tips on everything from monitoring cell viability to optimizing culture conditions and preventing contamination.

Biocompare: What are the main steps involved in isolating primary cells from tissue samples for culture?

Dr. Sherley: For blood cells, it’s a straightforward sterile blood collection followed by removal of red blood cells by either gradient sedimentation (Ficoll) or selective lysis. The white blood cells, which contain hematopoietic stem cells (HSCs), are usually the cells of interest for study. The white blood cells can even be removed from red blood cells after centrifugation of blood (supplemented with additives to prevent clotting) as the “buffy coat” that settles on top of denser red blood cells.

For organ and solid tissue cells, the process begins with some form of sterile tissue mincing to provide better access for the entry of added enzymes that break down proteins like collagen and other extracellular matrix proteins that hold cells together. After this treatment, released cells are filtered through large-pore nylon mesh membranes to separate them from the digested tissue debris.

BC: How do you determine the optimal culture conditions for a specific type of primary cell?

Dr. Sherley: Back at the start of in vitro cell culture, it was by experimental trial and error. First finding a mixture of likely components that worked, and then reducing or removing components to get to a medium with the minimal requirements, known as the “minimal medium.” Animal blood serum, like the serum of fetal cows, is rich with cell growth factors, and is usually the growth factor supplement of choice. Many of the main growth factors in sera have been defined and can now be purchased as purified preparations.

If starting a first-time primary cell culture today, depending on the cell type of interest, two main information sources can be searched first to find effective media and growth factors: the scientific literature and the catalogs of commercial suppliers of cell culture media and growth factors. No sense recreating well-established wheels! When one or more media are identified, they are tried with the isolated primary cells of interest, and the ones that provide the desired high viability and active cell proliferation are used.

BC: How do you monitor and maintain the health and viability of primary cells during culture?

Dr. Sherley: Most cell culture scientists simply look at their cells regularly as a matter of course during their maintenance. Generally, when scientists are engaged in cell research, they are maintaining several cell stock cultures as cell sources for starting experiments and analyses. Since actively proliferating cells fill up the culture vessel and expend the nutrients in their medium, depending on the cell type, the cells must be given more space and fresh medium at least weekly and sometimes as often as every other day. This is achieved by taking a fraction of the cells in a grown cell culture and transferring them to a new culture vessel with fresh medium. This process is called “passaging” or “splitting.”

At each passage, before the cell transfer, investigators use inverted phase microscopes to qualitatively confirm that the cell number has increased during the preceding culture period. During this inspection, they also take note of the fraction of dead cells. Well-trained investigators can distinguish dead cells from live cells by the difference in their appearance in phase microscopy. In cultures of adherent cells, floating cells are often dead. In addition, cells undergoing division by the process of mitosis can also be detected by phase microscopy and their frequency roughly assessed. Their frequency in a culture, formally called the “mitotic index,” is proportional to the cell proliferation rate of the culture.

To get an exact and precise measurement of cell proliferation rate, direct cell counting is required. At each passage, a sample of cells is taken from cultures immediately after their seeding with transferred cells and counted directly, either manually with a special slide for counting cells with the phase microscope or with one of many commercially available electronic cell counters. The culture is sampled again before it is next passaged and counted. By comparing the beginning and ending cell counts, the proliferation rate during the culture period can be determined with accuracy and precision. A widely used parameter for cell culture proliferation rate is the number of times the cell population in the culture doubled during the culture period.

The running sum of the number of population doublings during the serial passage of cell cultures, called a cumulative population doubling (CPD) curve, was first described by Leonard Hayflick in the 1960s. In addition to being indicators of the health and proliferative capacity of cell cultures, CPD curves have been widely used for cancer research, aging research, toxicology, and drug development. Just recently, ASTM International established a new standard for this general procedure for quantifying the proliferation rate of serial cell cultures (ASTM F3716-25). Knowing how important this process is to supporting excellent science, I proposed the new standard and led the working group that drafted it (https://www.astm.org/news/tissue-cell-cultivation-covered-in-new-standard).

BC: What strategies do you use to prevent and detect contamination in primary cell cultures?

Dr. Sherley: As a precaution, the inclusion of antibiotics in medium, like penicillin and streptomycin, is a common practice. Of course, they do not prevent culture infections by common yeasts, molds, and insidious mycoplasma.

Good antimicrobial contamination practice begins with good training in sterile technique and cell culture hood use for all cell culture personnel. Humidified incubators and their shelves should be cleaned with mild bleach and 70% ethanol regularly, at least twice a year. Incubator water pans should be similarly cleaned and filled only with autoclaved water. There should be a quarantine incubator where new cell lines are maintained until they are tested to be mycoplasma-negative before being introduced into the main core of incubators.

Microbial contamination should always be considered when there is an unexplained change in the color of the phenol red pH indicator dye or an unexplained change in cell proliferation rate and cell health by microscopy inspection. Yeasts and molds are usually readily identified by routine phase microscopy, but bacterial contamination may require evaluations of suspect medium streaked on LB agar plates. Mycoplasma detection requires more sophisticated testing with fluorescent antibodies.

BC: When and how do you decide to subculture or passage primary cells, and what are the signs that a culture is approaching senescence?

Dr. Sherley: The best practice for maintaining primary cells is passaging them on a regular basis, not allowing them to achieve complete confluency and avoiding high cell dilutions for passaging (i.e., < 1/10 confluency splits). Allowing cultures to achieve confluency causes cell stress from nutrient and growth factor deprivation and increases selective pressure for the successful emergence of transformed cell variants. Some primary cells may require very low dilution passaging for maintenance with ½-confluency splits.

As culture senescence approaches, the proliferation rate of the culture decreases. Decreased proliferation rate leads to cultures taking longer to achieve the same degree of confluency as during earlier passages. Cells may also display changes in morphology, such as increased cytoplasm to nucleus ratio; the presence of more dead cells; and a decrease of the mitotic index, which can be indicative of a decreasing proliferation rate.

BC: Can you share any specific recommendations for addressing common primary cell culture challenges?

Dr. Sherley: Good luck subcloning primary cells! Without either an immortalizing agent or an agent that induces symmetric self-renewal by the tissue stem cells in the culture, it is quite difficult to subclone and propagate primary cells. The rare subclones that may be obtained will contain mutations that cause immortalization and/or conversion of tissue stem cells to stable symmetric self-renewal (which is often the essential mechanism of immortalization). Mutation of the p53 gene is the best-known example of this immortalization mechanism.

Dr. Sherley founded Asymmetrex to develop technologies to advance stem cell medicine. The company has patents covering differential stem cell quantification and tissue stem cell expansion. Its proprietary kinetic stem cell counting technology can be used to determine differential stem cell count of preparations for use in stem cell transplantation medicine and pre-clinical drug evaluations.

BC: How is Asymmetrex’s cell counting technology used to optimize primary cell culture?

Dr. Sherley: Today, Asymmetrex offers several free online calculators for standard cell counting and for differential tissue stem cell counting. Anyone can register for a free account on our website to use these calculators, which make it possible to get precise counts of population doublings, population doubling time, and cumulative population doublings for optimal measures of cell health—no more guesswork needed.

We also have a new instrument in development, with an anticipated commercial release next year. The Asymmetrex^® counter will automate the process of maintaining serial stock cell cultures with quantitative monitoring. It will implement application of the new ASTM standard for obtaining quantitative CPD data at any time for stock cell cultures, even when users are not in the lab.

The proliferation rate of stock cell cultures is critical for the success of the experiments and analyses they supply. Because of the inconvenience of performing population doubling measurements on multiple stock cultures routinely, investigators guesstimate it from qualitative microscopy inspections. They often get it wrong, especially with primary cell cultures that are continually evolving. This results in a lot of wasted time and money. With the new Asymmetrex counter, users will always know the proliferation rate of their cell cultures with accuracy and precision.