Abstract
We live in an age where new technologies, and organizations involved in the distribution of biological materials, such as cell culture lines, have eased accessibility to a variety of in vitro models, developed, and/or harvested from different sources. In translational and basic ophthalmology research, in vitro assays are an essential component to discovery and preclinical studies. It is, therefore, of utmost importance for vision researchers to be cognizant of the risks surrounding the use of newly developed cell culture models and how scientific integrity could be impacted when standard operating procedures are not followed for cell line validation and identification. Herein, we discuss authentication challenges we faced when we obtained a newly marketed human choroidal endothelial cell line for vision research, and outline our process of validating and characterizing primary human choroidal endothelial cell lines in the laboratory.
Misidentification and cross-contamination of cell lines were originally recognized by Rothfels et al. in 1958, and have since become well-documented phenomena in the basic sciences.
1–14 To date, the International Cell Line Authentication Committee has identified a growing list of 486 misidentified cell lines
15 that have, in turn, negatively impacted more than 32,000 published articles.
16 In a 2007 press release, the National Institutes of Health (NIH) responded to these concerns by adopting cell line authentication guidelines. Subsequently, in early 2016, all proposals submitted to the NIH were required to include a dedicated section reporting protocols utilized to “authenticate key biological resources,” and, of relevance here, to provide information on how the identity, purity, and fidelity of cell lines used in the grant will be verified. Other grant-awarding institutions have since followed suit, yet strict authentication practices across disciplines remain inconsistent.
The field of vision research is not exempt from the woes of misidentified cell lines.
17–20 The human eye is a complex organ structure where specialized niches of cells reside. The retinal pigment epithelium and choriocapillaris endothelium are two examples of cell types that are frequently isolated for the study of blind eye diseases of the posterior pole of the eye, such as age-related macular degeneration, proliferative vitreoretinopathy, and diabetic retinopathy, to name a few. The challenge of culturing these specialized cell types can be exacerbated by cell heterogeneity. To ensure culture purity, laboratories are expected to consider implementing additional isolation methods, such as fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS; Miltenyi Biotech Inc., Somerville, MA, USA), both of which may be cumbersome and costly. Another important factor affecting in vitro models is that most adult ocular cell types are non-proliferative in vivo, and cell sorting of ocular tissues, an expensive endeavor, does not guarantee the survival of the cells unless placed in a specific “proliferation-friendly” environment. Determining said “proper” environment in and of itself requires supplementary trial-and-error assays and further expenses. Critically, when the culture environment is unfavorable, cells can rapidly dedifferentiate and/or reach phases of senescence and/or apoptosis. To circumvent issues of cell senescence in vitro, researchers have turned to exploiting a variety of stem cell technologies,
21–24 immortalization techniques,
25–27 and cell line transformations.
28 These modern approaches have again proven to be costly and untimely, and have ultimately resulted in the emergence of biotechnology organizations who fill the void with the promise of providing “authenticated” primary cell lines, the acquisition of which often needs to be accompanied by purchasing proprietary reagents to propagate and culture at a high cost. Here, we present a narrative of our experience with a commercially obtained, purported human choroidal endothelial cell line and outline methods associated with authentication in support of the principles of rigor and reproducibility.
The Need for Authentic Human Primary Choroidal Cell Lines: We Won't Know if We Don't Test
Here, we provide one example of the imperative need to authenticate biological materials used in the laboratory regardless of the originating source. We found “human choroidal endothelial cells” obtained from a life sciences and biotechnology company failed to meet all five benchmarks used to authenticate the nature of the cells. They lacked typical choroidal endothelial morphology, did not express pan-endothelial or cell-specific markers, were functionally dissimilar to primary human choroidal cells, and lacked human DNA markers. Although the initial reaction to the commercial availability of a highly sought after cell line may be to operate on a sense of “trust” and begin utilizing the cells without further in-house characterization, these findings strongly reinforce the need for vision research laboratories to use robust standard operating procedures for authentication when receiving new cell lines regardless of origin. By creating a baseline, cell lines may be periodically monitored for genetic drift and cross contamination. When possible, comparison across culture models is preferable. In-house derived and characterized primary cell lines are particularly useful for confirming new cell culture models. Without such due diligence, laboratories will likely fall victim to the risks associated with the steady flow of new technological advances in cellular biology that are either created in-house or enter through the market.
The authors thank the donors and donor families for their generosity and BioSight for their assistance and timely procurement of donor eyes. Furthermore, the authors appreciate the assistances of Scott Langdon, Manager of the Duke DNA analysis facility; J. Michael Cook, Director of the Duke Flow Cytometry Core; and Joan Kalnitsky for support with flow cytometry.
Supported by the National Eye Institute grants R01 EY027802 (to GM), R01 EY028160 (to GM), P30 EY005722 (to the Duke Eye Center), and the Research to Prevent Blindness, Inc. (RPB) Core grant (to the Duke Eye Center).
Disclosure: J. Peavey, None; G. Malek, None