The debate over using primary cells versus cell lines continues to challenge researchers. Primary cells are believed to be more biologically relevant tools than cell lines for studying human and animal biology. With the emergence of newer technologies such as 3D culture, the use of primary cells are becoming increasingly prevalent to achieve improved results.


For decades, cell lines, with their ability to proliferate virtually indefinitely, have been used as a cost-effective tool in basic research. However, some cell lines may not be the cell type they have historically been reported to be. For example, a review about cell line quality summarized that 18–36% of cell lines are misidentified or cross-contaminated (Hughes P et al., 2007).

A recent study just showed the impact of misidentified cell lines on published scientific papers. The authors found out that approximately 32,755 articles report on research with misidentified cells which in turn are cited by an estimated half a million other papers. "As a result of mislabelled samples, cross-contaminations, or inadequate protocols, some research papers report results for lung cancer cells that turn out to be liver carcinoma, or human cell lines that turn out to be rat [..]. In some cases, these errors may only marginally affect results; in others they render results meaningless" (Halffman et al. 2017).

Serial passaging of cell lines over time can cause genotypic and phenotypic variation. Bioinformatic analysis of proteomic phenotypes revealed that the Hepa1–6 cell lines were deficient in mitochondria, reflecting rearrangement of metabolic pathways when compared to primary hepatocytes (Pan et al., 2009).


Therefore, researchers should be careful about only relying on data generated with cell lines. For example, NIH highlights authentication of cultured cell lines as critical for grant applications. And the current recommendation from the scientific community is to use key control experiments with primary cells to qualify the findings seen in cell lines.  


Primary Cells in Research

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Primary Cells vs. Cell Lines

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Cell Culture Procedures are Conducted with Two Types of Cells:

Primary Cells Cells isolated directly from human or animal tissue using enzymatic or mechanical methods. Once isolated, they are placed in an artificial environment in plastic or glass containers supported with specialized medium containing essential nutrients and growth factors to support proliferation. Primary cells could be of two types – adherent or suspension. Adherent cells require attachment for growth and are said to be anchorage-dependent cells. The adherent cells are usually derived from tissues of organs. Suspension cells do not require attachment for growth and are said to be anchorage-independent cells. All suspension cells are isolated from blood system. Although primary cells usually have a limited lifespan, they offer a huge number of advantages compared to cell lines.



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Human Primary Cells

Animal Primary Cells 

Cell LinesCells that have been continually passaged over a long period of time and have acquired homogenous genotypic and phenotypic characteristics. Cell lines can be finite or continuous. An immortalized or continuous cell line has acquired the ability to proliferate indefinitely, either through genetic mutations or artificial modifications. A finite cell line has been sub-cultured for 20-80 passages after which they senesce. Cell lines are preferably used for convenience as they are easy to handle and widely published. However, they are less preferred as a biologically relevant option, since they have lost the true characteristics of the original tissue from which they were isolated.


Characteristics: Primary Cells Immortalized Cell Lines
Lifespan: Limited, resembles tissue characteristics Infinite, loses tissue characteristics
Closer to an in vivo model: Yes, isolated directly from the tissue No, clonally selected over time
Reduces animal testing costs: Yes, used in advanced cell culture models to refine experiments Limited ability to develop biologically relevant complex in vitro models
Mutations/Modifications: Low High
Authentication required before use: No, if bought commercially Yes, mandated by many government institutes and scientific journals
Availability of donor characteristics: Yes No


Primary Cells and Cell Lines Show Variability in
Drug Dose Response   

Primary Cells and Cell Lines Show Variability in Drug Dose Response


Figure 1. Primary cells and cell lines show variability in drug dose, so data acquired through cell lines cannot easily be replicated in an in vivo model. The three cell types were dosed with Camptothecin for 48h and their cytotoxic response assessed using the ViaLight™ Assay.The ViaLight™ Assay measures the number of viable cells left after drug challenge, measuring the ATP donated to the assay reaction by viable cells. In this assay the data points are the mean of 40 replicates (8 replicates per plate; 5 plates). We have a marked difference in EC50 dose response almost 10 fold. The HEK 293 cell line required only 68nM Camptothecin to kill 50% of the cells. In the case of the primary cells (HRE = human renal epithelial cells, HRCE = human renal cortical epithelial cells) this dose was over 500nM a strikingly different response.



Figure 2. Some pathways can only be studied in primary cells, as there is no cell line to represent that pathway. Cytolytic T lymphocytes (CTLs) recognize foreign cells and lyse them via the “immunological synapse”. At the immunological synapse CTLs secrete cytolytic granules containing Perforin. hMUNC13-4 is involved in an activation step preceding vesicle membrane fusion in cytolytic granule secretion. Nucleofection™of a plasmid expressing hMUNC13-4/RFP fusion shows only partial co-localization of hMUNC13-4 (red, H) and Perforin (green, I) in isolated CTLs (overlay J; blue: nuclei stained by DAPI). Upon target cell contact (M) hMUNC13-4 polarizes and extensively co-localizes with Perforincontaining cytolytic granules at the immunological synapse (L). Reprinted from: Feldmann J et al. (2003) Cell 115(4):461-73; with permission of Elsevier.



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Some Pathways Can Only Be Studied in Primary Cells

Some Pathways Can Only Be Studied in Primary Cells