Background

Tears are a vital component of the ocular surface, serving as a first line of defense against pathogens and extreme changes in the environment [1, 2] One of the main functions of tears is to act as lubricant, keeping the eye moist while also protecting it from physical damage [2]. Tears also help to maintain conjunctival and corneal health by providing nutrition, regulating optical properties, and boosting tissue cooling. The unique composition of tears also protects the ocular surface against various bacterial, fungal, and viral infections. The tear film is comprised of three layers: the innermost mucous layer, the middle aqueous part, and the outermost lipid layer. The biological composition of metabolites, electrolytes, mucins, lipids, water, proteins, salts, and organic molecules that are present in tears can be used to distinguish healthy and diseased states [1]. Several studies have shown how tears can be used as an indicator of ocular health and pathology, for example in diabetic retinopathy [3], dry eye disease [4], glaucoma, Graves’ ophthalmopathy, and in some ocular tumors [5, 6].

Given that tears are readily available and can be collected via non-invasive procedures, they have recently been recognized as more valuable tools in the identification of ocular health and in the diagnosis of diseased states when compared to other biological fluids. Therefore, studies are being dedicated towards characterizing the composition of tears and understanding how changes in this composition relate to pathology. In several ocular disorders, tears have been proposed to promote inflammation, and the precise secretome of the tears directly relates to the ocular microenvironment [6]. However, characterizing how tears affect ocular health and more specifically how tears affect corneal epithelial cells remains poorly understood. Herein, we describe procedures that can be easily implemented to study how healthy vs. diseased tears affect the corneal epithelial cells in vitro. This protocol is based on our recently published paper, which focuses on how the tear proteins of Persistent Epithelial Defect (PED) patients have the potential to alter the inflammatory, fibrotic, and corneal epithelial barrier profile of other healthy cornea cells [7]. Taken together, we can infer that patient-derived tears can be a valuable source of factors to create a disease-specific in vitro model to study the key features of the pathology. This system can also be used to study the patient-specific clinicopathological features.

There are standard protocols describing the methods for collecting tears, extracting the protein, and using it for biochemical characterization, proteomic analysis, and inflammatory cytokines analysis. Recently, tears have been explored as a non-invasive method to identify disease-specific markers. Recent reports highlight the presence of exosomes in keratoconus tears, hypothesized as a communication vehicle [8]. The in vitro model used for studying disease pathology, such as in dry eye, allergy, or inflammatory models, is based on using a single growth factor/cytokine, which does not represent the actual pathology, as the disease state is governed by multiple factors such as cytokines, growth factors, and metabolites. The novel aspect of the described protocol is the use of patient-derived factors in the form of tears, which is the true representation of the diseased state (including growth factors, cytokines, and metabolites). Co-culturing patient-specific and control tears with relevant cells (corneal epithelial cells in this case) will provide an in vitro model that truly depicts the diseased state, whereas the healthy individual will be taken as a control. To the best of our knowledge, the ease of collecting tears and using it as a supplement in cell culture has never been explored previously, and this is the first study describing the detailed methodology for establishing a tear-based in vitro model of ocular diseases.

This protocol highlights the effect of patient-derived tears on corneal epithelial cells’ morphology and gene expression pattern. The procedure to collect tears can be done using either the Schirmer strip or the plastic capillary tube method. Sampling with Schirmer strip is relatively easy, rapid, reliable, and free of risk, whereas obtaining tears from capillary tube requires a degree of practice and experience; this procedure is also interrupted by blinking and the investigator needs to hold the tube for the duration of the sampling process. Hence, the protocol developed using the Schirmer’s test strip is easy to carry out, reproducible, and can be used to further understand ocular disease progression. Proteins are isolated post tear collection using the Schirmer’s test strip from both healthy and diseased individuals. Isolated proteins are then used to treat human corneal limbal epithelial (HCLE) cells at a specified concentration for a defined period (48 h in our study) until visual changes are observed. Next, morphological changes in inflammatory or fibrotic gene expression are recorded and studied. Matrix metalloproteinases (MMPs) are iron- and calcium-dependent enzymes that degrade the corneal collagen and the extracellular matrix. The level of MMP9 activity in the tear fluids has been regarded as an indicator of ocular inflammation and damage in dry eye disease, vernal keratoconjunctivitis, and glaucoma. Thus, MMP9 activity of the tears of PED patients has been studied via gelatin zymography and is represented in Figure 1. This novel assay uses tear fluid as a medium for testing and analyzing biomarkers, allowing for a more cost-effective and non-invasive diagnosis. The technique is also beneficial for tracking disease progression, as it can be used to detect changes in biomarkers over time. Furthermore, this method presents a convenient and time-saving method for healthcare professionals to monitor the health of their patients. Tears can be used for in vitro drug testing as a more effective and efficient means to test for the efficacy and safety of drugs. This model also mimics the in vivo conditions and is thus useful in drug testing.