Isolation, Culture, and Preservation of Primary Cells

Primary cells—cells isolated directly from tissue—are prized for their abilities to better mimic in vivo physiological metabolism and behavior than cell lines derived from tumors or immortalized cells. Yet at the same time they tend to be more finicky to grow. Primary cells are more sensitive and often have a limited capacity to proliferate in vitro, if they are even able to do so at all.

This article, largely based on a conversation with Sujoy Lahiri, who leads the primary cell portfolio in research and development at the American Type Culture Collection (ATCC), will provide some expert tips regarding the isolation, culture, and preservation of primary cells.

Single-cell suspensions

Primary cell cultures generally start with single-cell suspensions. (There are some exceptions, such as some organoid cultures derived from small tissue fragments, which are beyond the scope of this article.) Single cells are generally isolated from tissues digested with a protease—classically a collagenase because the majority of cells are bound in the tissue by collagen fiber. Different flavors of collagenase are used depending on the specific type of collagen fiber. The amount and time of digestion should be titrated and calibrated carefully to achieve a balance between maximal digestion and avoiding sacrificing too many cells in the process. Pay attention to the specific activity listed on the manufacturer’s website and the certificate of analysis. Do not try to achieve 100% digestion!

Digestion takes place in one of two ways, depending upon the type of tissue. In one case, the organ is first digested as a whole by first perfusing it with some clearing solution and then with digestion solution. This is typically the case for tissues such as liver or kidney. After that, the tissue is minced or manually dismembered to release the cells into a liquid suspension.

In the other scenario, for muscle or skin, for example, it is best to first mince or tease apart the organs or tissue. This will increase the accessible surface area and allow the collagenase broth to reach inside the vasculature and sinusoidal space as much as possible.

The mixture is then put through a series of purification steps to systematically separate out the dead cells, undigested tissue, and debris, and recover live cells as much as possible. This can be done using filters of sequentially smaller pore sizes, or sometimes with a mesh. For a whole minced organ, start by pouring the entire contents into a funnel containing a big mesh cloth.

For best practices, it’s always a good idea to see how others have been successful with your particular cell type. Consult the literature and manufacturers’ websites.

Recovery

Many cell types react adversely to being separated from other cells and extracellular matrix (ECM), by undergoing a programmed cell death process called anoikis. To stave off anoikis, sensitive cells are often incubated at 4°C, with shaking, for about an hour in a recovery medium containing supplements like serum, sugar, antioxidants, and anti-apoptotic agents like pan-caspase inhibitors.

Cells that can proliferate in vitro are generally allowed to do so before being cryopreserved. This has several advantages, including the ability to further purify the cells by selectively favorable culture conditions. They can at this point also be more easily sorted using specific antibodies. Culture also helps the cells to recover from the isolation procedure, and allows for expansion if greater numbers of cells are desired than have been harvested.

Primary cells that do not proliferate in vitro, such as hepatocytes, should be cryopreserved in aliquots immediately after recovery, leaving aside only what will be immediately used.

Extra caution needs to be taken when freezing down primary cells, and especially non-proliferating cells for which any losses in cell number incurred during the freezing process cannot be recovered. Cells should be frozen in a preservative such as DMSO or galactose, which helps prevent the buildup of ice crystals.

While immortalized cells are often frozen down in an isopropanol container such as Mr. Frosty at -80°C, that process is not ideal for primary cells, which are arguably more precious. Instead, it is highly recommended that a controlled rate freezer (CRF), which can rapidly drop the temperature with appropriate timing so as to minimize ice formation within the cells, be used. Most cryopreserved cells are best stored and transported in liquid nitrogen.

Culture and passaging

Culture of adherent cells typically takes place on a plastic surface, which may be treated with plasma to change the surface chemistry, for example, or coated with natural or synthetic ECM proteins, to allow the cells to adhere. The specifics are dependent on cell type being cultured and the goals of the culture.

Cells will generally behave differently, and may reach senescence sooner, when their culture nears confluence, so they should be passaged before that point. Non-confluent cells will aggregate less when released from the plate—usually by treating with a protease—making it easier to achieve a single-cell suspension and thus easier to get an accurate count.

Once cells are released, the protease should be diluted out and inactivated, usually with culture medium containing high concentrations of serum. Cells are centrifuged, resuspended in fresh medium, counted, and re-seeded at an appropriate density. Essentially the same steps, minus protease treatment, apply to passaging cultures of cells such as lymphocytes that grow in suspension.

Remember

The belief in the scientific community is that primary cells will recapitulate the molecular, metabolic, and physiological properties of the organs from which they were derived, better than continuous cell lines. They can be derived from healthy donors, patients, and disease model organisms. That makes them arguably better models for drug discovery, disease and toxicology research, and indeed most uses that cell lines are put to in a research lab.

But it is important to remember that, unlike immortalized cells they have a very finite lifespan and the line will senesce. Their properties may also change as they adapt to their new, plastic, environment. So for the greatest fidelity to the host, it’s best to use them with as little culturing as the research allows.