Advances in Primary Cell Culture Driving Biomedical Research

“The fields of cell biology and cell culture have changed dramatically in the last decade,” says Jackie Damen, senior scientific advisor at STEMCELL Technologies. Although scientists used to rely on immortalized cell lines, new technology and better reagents now make growing and utilizing primary cells in the lab more feasible than ever before. And the intersection of primary cells with ever-evolving 3D culture models gives scientists the power to recreate a comprehensive microenvironment of normal biology ex vivo, with boundless implications.

Derived directly from the living tissue of donors (human or animal), primary cells are transferred, unmodified (aside from dissociation), to in vitro culture. These cells retain the major features of the original tissue, including both growth and senescence. Depending on the cell type, primary cells may replicate a handful of times, or not at all (as is the case with neurons). This makes working with primary cells a double edged-sword. Culturing primary cells makes for more relevant paradigms, better cell-based modeling of disease, and is making precision medicine a reality. But this is assuming you can figure out how to cost-effectively and efficiently integrate these potentially challenging cells into your cell culture practice, and design experiments around limited numbers.

The good, the bad, and the frustrating

Once upon a time, primary cells were notoriously difficult to culture, requiring lots of money, time, and resources. Even if the cells survived the extremely rigorous dissociation steps, contamination still loomed over the fragile cultures, threatening hours of work (not to mention the weeks or months of generating animals from which the tissue may have come). And then there are the issues of potential contamination by unwanted cells types, slow growth, and limited numbers on which to do actual experiments.

But today, much of the headache can be outsourced, with numerous companies selling ready-to-go primary cells complete with matching media and growth factors. Debra Sauvé, director, primary & cultured cells at STEMCELL, says, “To ensure that there is a reliable source of cells, researchers should work with vendors who are able to recall donors, who have access to a large network of donors, and who allow batches to be placed on hold during the testing process.” This can help ease the pressure of limited cell numbers that comes from working with primary cells. Sauvé notes that STEMCELL Technologies checks all of these boxes. The company offers a range of human primary cells, including peripheral blood mononuclear cells.

Kevin Grady, senior product line business manager at ATCC, says it’s a trade-off. The costs associated with culturing primary cells may be higher, but “having results based on a more in vivo-like model allows researchers to find answers in a more efficient manner.” He says that ATCC’s primary cells are thoroughly tested for purity and performance.

Primary cells are fast becoming the preferred tool for cellular and molecular biology research, providing high-quality contextual models for the study of normal cell physiology and signaling, disease, and therapeutic development (such as screening anticancer drug candidates). Patient-derived organoids (PDOs), for example, may represent the next frontier of precision medicine. While cancer lines and patient-derived xenograft (PDX) models (in which tumor from a patient is transplanted into immunocompromised mice) are prevalent preclinical models, PDOs offer the same drug response predictability as PDX models minus the expense, low-throughput nature, and potential ethical complication that may come with mice.

“Primary cells should be considered over immortalized cells for study if researchers want to understand physiological and developmental pathways, especially those that control disease,” says Damen. According to her, immortalized cell lines are just too far removed from the context of the original tissue and cannot replicate many of the same physiological activities.

Cell lines: Abundance at a cost

Traditionally a source of convenience as they are abundantly available and fairly easy to culture, cell lines can also be a source of liability. Cell lines mutate. Serial passaging causes both genotypic and phenotypic changes. They can mutate so much so that they become completely unrecognizable compared to the original donor cells. One research group found that adenovirus transformation of the popular HEK293 line produced cells that more closely resembled immature neurons than embryonic kidney cells.

Misidentification is another big issue. Consider the MDA-435 line, presumed to be a breast cancer cell line and used in thousands of publications. It turned out to be a melanoma cell line, instead.

In the early aughts researchers started to demand action be taken to curb the continued usage of the numerous misidentified and contaminated cell lines. These lines resulted is an astronomical waste of time and reagents, producing unreliable, irreproducible data. It is estimated that up to 20% of scientific publications using cultured cells have tainted data and the International Cell Line Authentication Committee currently lists 529 cell lines as cross-contaminated or misidentified.

Tips for Working with Primary Cells

• Primary cells are bridging the gap between the inherent shortcomings of cell lines and the difficulty, expense, and time required of animal models. When optimally utilized, primary cells offer the advantage of efficient biologically relevant results, that in the end, can save money on time and reagents.
• Commercially available primary cells are now available from a variety of vendors and come with fully optimized media and protocols making working with primary cells easier than ever before. Consider working with vendors who are able to recall donors or let you test or sample lots prior to starting a large study.
• Use caution when generating your own primary cells. Improper isolation technique can result in phenotypic inconsistencies, contamination, poor growth, the presence of unwanted cell types, or cell death. If you choose to isolate primary cells on your own, experts advise that you still purchase specialty media and incorporate commercial cells into experiments as a control.
• Use antibiotics during the isolation process. Antibiotic solutions should be made fresh or, if using prepared frozen solution, thawed and used just once before discarding. And use a mycoplasma detection kit, such as ATCC’s PCR-based Universal Mycoplasma Detection Kit (ATCC^® 30-1012K™).
• Never underestimate the impact of seemingly simple techniques. Primary cells should be kept in liquid nitrogen until just prior to thawing. Thawing should be quick. Once in culture, primary cells are temperature sensitive and so repeated warming and cooling should be avoided. Pay attention to confluency: 100% confluency will cause senescence.
• Complications from low cell numbers due to limited or no replication may be managed with newer cell imaging and western blotting techniques that require much less sample. However, hTERT and PSC-derived primary-like cells are valuable alternatives and remove the constraint of working with limited numbers.

And it’s no secret that cell lines are lacking when it comes to recapitulating numerous physiological aspects of the tissue from which they were derived. “Immortalized cells grown in 2D cultures are functionally one-dimensional,” says Damen. She says that the link between growth and maturation is dissociated in these lines, uncoupled from the normal processes of cellular differentiation.

This is not to say that immortalized cell lines are useless, only that greater care must be taken when using this model (such as adhering to validated lines) along with an understanding of the model’s limitations. “Immortalized continuous cell lines still have an important place in the research work flow,” adds Grady. The availability and limited variability of cell lines makes them good reagents for standardizing protocols and confirming results.

Sylvain Baron, research scientist at Cellecta, says that unlimited division and straightforward culture conditions make cell lines ideal when experiments require a large number of cells, such as genome-wide genetic screening. He warns that the finicky nature of primary cells and their limited numbers “drastically reduce the array of experiments that can be performed with them.”

Next-gen “primary” cell lines

“While it is true that it is difficult to get enough primary cells for analysis, there is no really good way to increase their numbers except to start with more tissue,” explains Amanda Fentiman, product manager at STEMCELL. But she says that newer imaging systems and immunofluorescence techniques may be able to replace more traditional types of analysis like western blot. “Single-cell based western blot, single-cell based sequencing, and microfluidic devices for cell-based assays require much less sample input compared to traditional analytic methods,” Fentiman explains.

But there are still times when an old-fashioned western blot is necessary. And there are times when limited numbers of primary cells just won’t cut it, despite their numerous advantages. The decision to use primary culture may ultimately depend on the field and the tissue needed—blood and skin is more readily available than brain. “For the most difficult to access cell types, hPSC [human pluripotent stem cell]-derived and hTERT [human Telomerase Reverse Transcriptase Protein] lines become most valuable,” says Erin Knock, senior scientist at STEMCELL.

While hTERT, PSC-, and iPSC (inducible pluripotent stem cell)-derived lines are not the true equivalent of primary cells, they are important alternatives that embody some of the best qualities of primary cells and cell lines. For example, forced expression of hTERT enables primary cells to maintain sufficient telomere length and avoid replicative senescence.

“Primary cells with the hTERT modification have the advantage of extended proliferation without chromosomal instability,” says Knock. hTERT cells are expandable, and can be frozen, banked, and thawed, to accommodate the timing of experiments, she explains. Similarly, she says that the true value of PSCs lies in their ability to differentiate into multiple cell types from one cell source. iPSCs lets scientists take easily accessible cells and turn them into cells that are not easily accessible, allowing for the probing of disease in a relevant cell type without the need for post-mortem or surgical samples.

Damen explains that research is significantly increasing in the areas of PSCs and organoids. “With the ability to produce induced PSCs, many new ‘primary’ cell lines are now being established to allow tremendous advancements in identifying important players and pathways controlling cellular function in health and disease.”