Table 2.

The available cell lines and primary cells, culturing methods, induction, and end point of in vitro DED experiment.

Name of Cells Line	Culture	Induction Methods	Starting of Treatment	End of Experiment	References
Wong Kilbourne derivative of Chang conjunctival epithelial cell line (WKD; clone 1–5c-4)	Dulbecco’s minimum essential medium supplemented with 10% fetal bovine serum, 1% glutamine, 50 UI/mL penicillin, and 50 UI/mL streptomycin	Cells were grown for 24 h. Then, benzalkonium chloride was dissolved in phosphate-buffered saline (PBS). Different concentrations of BAC (10−2%, 10−3%, were analyzed.	–	15 min of treatment or 15 min of treatment followed by 24 h of cell recovery in complete medium	Brasnu et al. [47] 2008
	Dulbecco minimum essential medium supplemented with 10% fetal bovine serum (FBS), 1% glutamine (200 mM stock solution), and 1% penicillin (10,000 units/mL) and streptomycin (10,000 μg/mL)	Cells were grown for 24 h using hyperosmotic media 500 mOsM, achieved by adding 90 mM sodium chloride or media containing benzalkonium chloride at 10–4%, 3.10–4%, or 5.10–4%.	–	Cell was analyzed at 24 h and 48 h	Clouzeau et al. [48] 2012
	Dulbecco’s minimum essential medium (DMEM) supplemented with 10% fetal bovine serum, 1% glutamine, 0.1% ampicillin, and 2% kanamycin	A 15 min BAC 0.001% treatment and after 15 min culture media was removed, and normal culture medium was added and allowed for 24 h	Candidate drug was mixed 1 h before BAC treatment	After 24 h	Debbasch et al. [49] 2001, Debbasch et al. [50] 2001
	Eagle’s minimal essential medium supplemented with 5% fetal calf serum, 2 mM L-glutamine, 50 mg/mL streptomycin, and 50 IU/mL penicillin	0.0001% (1 μg/mL). Cells were treated for 10 min. After this time, the BAC-containing medium was removed, cells were rinsed twice with culture medium, and normal cell culture conditions were restored.		Examined before treatment and 3, 24, 48, and 72 h later	De Saint Jean et al. [51] 1999
IOBA-NHC cells	DMEM/F12 supplemented with 1 μg/mL bovine pancreas insulin, 2 ng/mL mouse epidermal growth factor, 0.1 μg/mL cholera toxin, 5 μg/mL hydrocortisone, 10% fetal bovine serum (FBS), 50 UI/mL penicillin, and 50 UI/mL streptomycin	Cells were grown for 24 h. Benzalkonium chloride was dissolved in PBS. Different concentrations of BAC (10–2%, 10–3%, were analyzed.	–	Two incubation times were applied to the cells: 15 min of treatment and 15 min of treatment followed by 24 h of cell recovery in complete medium	Diebold et al. [52] 2003; Brasnu et al. [47] 2008
	Dulbecco’s Modified Eagle Medium (DMEM)/HAM’s F12 (1:1) supplemented with 10% fetal calf serum (FCS, Biochrom AG, Berlin, Germany) in a humidified incubator containing 5% CO2 at 37 °C	For stimulation, cells (1 × 106) were seeded in Petri dishes and cultured until confluence was reached. Cells were washed PBS and changed to serum-free medium for 3 h. Afterward, cells were either treated with recombinant proinflammatory cytokine interleukin (IL)-1β (10 ng/mL) or tumor necrosis factor (TNF) α (10 ng/mL) for 6 h, 12 h, 24 h, or 48 h	–	6 h, 12 h, 24 h, or 48 h each	Schiicht et al. [53] 2018
Human corneal epithelial cells (HCECs), a human transformed SV40 immortalized corneal epithelial cell line	Were cultured in Dulbecco’s modified Eagle’s medium/F12 with 10% fetal bovine serum and 10 ng/mL human epidermal growth factor and the medium replaced every other day.	Cells were grown for 24 h. Then, they were treated with a different osmolarity, ranging from 312 to 550 mOsm/L, which was achieved by adding 0, 70, 90, or 120 mM sodium chloride (NaCl) with or without candidate drugs.	Candidate drugs were added 2 h before adding NaCl.	Samples were after 24 h treatment	Li et al. [54] 2020
	Were cultured in Dulbecco’s modified Eagle medium (DMEM)/HAM’s F12 supplemented with 10% fetal bovine serum, 50 U/mL penicillin, 50 μg/mL streptomycin mixture, 1% insulin-transferrin–selenium mixture, and 10 ng/mL human epidermal growth factor	Hyperosmotic stress (500 mOsm) was achieved by adding 90 mM sodium chloride (NaCl, Sigma-Aldrich) to isosmotic medium (310 mOsm).	Candidate drugs were added 2 h before adding NaCl.	Supernatants of conditioned medium were collected at 24 h after stimulation	Ma et al. [55] 2021
Human conjunctival cell line HCC	Cells were cultured according to the manufacturer’s instruction in RPMI medium supplemented with 100 IU/mL penicillin, 100 mg/mL streptomycin, and 10% heat-inactivated FBS	Hyperosmotic media (528 mOsM)	Candidate drugs were added 2 h before adding NaCl.	24 h after treatment	Park et al. [56] 2019
Primary Culture
Primary HCECs (human corneal epithelial cells) were cultured from donors within 72 h after death	Supplemented hormonal epidermal medium (SHEM) containing 5% FBS	The addition of 44, 69, and 94 mM of sodium chloride (NaCl) can achieve hyperosmolarity (400, 450, and 500 mOsM) from the isosmolar (312 mOsM) medium.	-	The HCECs co-incubated for 12 h, 24 h, or 48 h were used for immunostaining	Liu et al. [57] 2020
Primary culture of rabbit corneal epithelial cells (CECs) or Primary rabbit LG acinar cells (LGACs)	After isolation, cultured with DMEM/F12 (Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12) with 1% antibiotic–antimycotic solution	After 24 h culture, DED-like symptom was induced by the addition of IL-1β (10 ng/mL) with the medium.	Dexamethasone (10 μM) was used combined with IL-1β for treatment	Culture was maintained for 1 week and analyzed	Lu et al. [58] 2017