By Gail M. Seigel, Ph.D.

University at Buffalo

It’s the holiday season! But let’s take a break from the spiced cider for a moment to mull over strategies to create cell lines. As I sit here waxing nostalgic about the R28 and E1A-NR.3 retinal cell lines from my own lab, I’d like to share a few tips about what goes into the making of a new and successful cell line.

Where do I begin? The most important consideration in making a new cell line is the initial choice of cell type and tissue of origin. Ask yourself what you hope to accomplish with the cells. Can these cells provide you with important information that cannot be gleaned from intact tissues? If the answer is yes, it will be essential to know as much as possible about your cells of interest and their functions. You need to be aware of cell surface markers, particularly any markers unique to the cell type, as well as the potential growth rate in cell culture. All of these factors will play a role in the establishment and characterization of your new cell line.

Enrichment strategies. If your cells of interest reside within heterogeneous tissue, it makes sense to dissociate the tissue and use a unique cell surface marker to enrich for your cells of interest prior to immortalizing them. In my experience, immunomagnetic separation is a gentle way to promote continued cell growth after the sorting process. There are helpful kits for immunomagnetic enrichment of specific cell types, or you can mix-and-match your cell surface antibody of choice with a do-it-yourself kit. Live cell sorting instruments can also be used, but I find they can be harsh on the cells in the process, leading to decreased viability. Regardless of the enrichment method used, you still may not attain 100% cell purity. However, you will have a highly enriched cell population that you can further purify at a later stage, if necessary.

Immortalization. At this point, you may wonder how to keep the cells proliferating once they have been sorted and plated in a dish. If they are cancer cells, they can sometimes grow indefinitely without further manipulation, due to loss of growth control checkpoints inherent in malignancies. But if they are primary cells from normal tissues, you will most likely need to introduce an immortalizing gene (with accompanying drug resistance marker) to promote continued cell growth and allow for selection based on drug resistance. There are a few cases of spontaneous immortalization of primary cells, but it’s difficult to rely on such rare events. Instead, there are growth-promoting genes that can be used to immortalize cells. Retroviral vectors and transfection of your gene of choice, with or without temperature-sensitive promoters, can be paired with your cell type for a custom fit. After the immortalizing gene is introduced to the cells, I generally wait a few days to allow for recovery time and additional cell growth.

Selection and Characterization. Once your cells express the immortalizing gene, they will proliferate at a faster rate. A few days after plating, you can begin to select for your cells of interest based on coexpression of drug resistance genes (e.g., G418 or Puromycin). Two weeks of treatment with a selection drug will kill residual cells that do not express the drug resistance gene, leaving you with cells that express both the drug resistance gene and immortalizing gene. At this point, you are ready to characterize your new cells by genetic, immunocytochemical and functional assays to compare with the primary cells of origin. Sometimes, the process of immortalization may cause the new cell line to lose some functions or behave more like a precursor cell type. If this is the case, it should be documented. These changes are not necessarily a detriment to the success of the cell line and its applications. On the plus side, precursor cells can be used for studies on differentiating conditions, tissue organization and regeneration.

Authentication. At some point during characterization, it is important to have the cells authenticated by an outside source to determine a baseline genetic profile. There are commercial labs that will analyze cell lines for less than $200 per sample. In return, you get a report that confirms the species of origin, as well as a listing of isoenzymes that impart a unique genetic profile to your cell line. This information will be used as a future point of reference for yourself and others. Periodic genetic authentication is not only essential to verify that the cell line has remained free of cross-contamination with other cell types, but is now a requirement for most high-impact journals and NIH grant proposals that require an authentication plan for biological reagents.

Maintenance. Once you have your new cell line, proper maintenance is key. Make sure to freeze down your cell line at early passages in liquid nitrogen. You’ll need these archival cells in case of contamination, incubator failure or unwanted biological changes that may occur at high passage number. Strict containment procedures will help ensure that your new cell line is not cross-contaminated with other cell lines present in your lab. In my lab, we make sure that there is only one cell line at a time in the laminar flow hood. We use separate bottles of medium, buffer and trypsin for each cell type. We also clean the hood surface with ethanol and wash hands/change gloves between handling cell lines to maintain cell line integrity. With careful attention to detail, your new cell lines should remain healthy and robust for years to come.

Spread the holiday cheer! Scrooges will say that immortalized cell lines will never take the place of primary cells or the intact organism. And they would be right. But a cell line can save a lot of time and effort by providing a large yield of cells that can be used for screening new drugs and treatment conditions, with subsequent validation requiring fewer primary cells and intact organisms. Once your new cells have been characterized and described in a publication, be sure to drop a line to Kerafast to become a provider and share them with the world. Good luck and all the best for 2017!