To this day, the evaluation of drugs and other treatments for use in humans is done using animal testing as the gold standard. No other method can give us a similar level of confidence in the efficacy and safety of a drug. However, animal testing is expensive, takes a long time, is ethically questionable and should therefore be avoided as much as possible.
Fortunately, methods that offer us good alternatives to animal testing have been expanding steadily in recent years. This offers us more and more tools that can be utilized up to the very last stages of development, while avoiding animal testing as much as possible. One such approach is cell-based assays, in which the effects of specific stimuli such as chemicals, pathogens or environmental factors are assessed in vitro by exposing cell cultures to them.
Cell-based tests have several advantages. For example, they are usually performed in a shorter time and therefore incur lower costs compared to animal testing. In addition, numerous cell types are available from healthy and pathologic tissues, allowing studies of disease treatment and prevention with a specific focus on one cell line. These targeted studies may allow researchers to discover the role of specific cells in more complex processes in the body. In addition, cell-based assays have long been used and there is extensive experience with them in the scientific community.
Cell culture is traditionally performed in Petri dishes. The cells grow side by side on a synthetic substrate, sometimes referred to as monolayer or two-dimensional (2D) cell culture. A drawback of this method is that these conditions do not accurately reflect the natural cell environment. To address this shortcoming, modern approaches attempt to mimic conditions in the body as closely as possible to increase the validity of cell culture results.
These alternatives include organ-on-a-chip systems, tissue bioprinting and three-dimensional (3D) cell culture – each of which is a whole field in itself. Here, we focus on 3D cell culture, which allows for a system that more closely resembles the environment of the cells in our bodies to achieve better results in our experiments.
Bronchial epithelial cells (BEC) are the cells that form the barrier between the small airways and the deeper tissues of the lungs. They act as a protective layer, producing mucus and creating movement to move particles and pathogens into the upper airways where we can more easily get rid of them, for example by coughing. When bronchial epithelial cell function is compromised, it can lead to serious health problems – even death in severe cases. Diseases of the lungs usually affect these cells in a variety of ways, making them perfect models for studying lung disease. Therefore, these cells have been a popular object of study in 2D cultures.
Functionality and phenotype of BEC can also be further enhanced by culturing them at the air-liquid interface with 3D cell culture tools such as membrane inserts [1] or magnetic levitation [2,3].
In recent years the Covid-19 pandemic has captivated the entire world. Since the outbreak began, numerous scientific publications have aimed to study the virus and develop treatments for infection. 3D cell culture of BECs is a promising model to study covid infections and has been used in several cases [4,5]. BECs can act as host cells for SARS-CoV-2 because they express the ACE2 receptor and TMPRSS2 protein, both of which are necessary for the virus to enter and infect a cell. Since SARS-CoV-2 is known to cause problems primarily in the upper and lower respiratory tract, it is not surprising that cells from these regions are ideally suited to study the virus in more detail. Using 3D cell culture, it is also possible to study the effects of SARS-CoV-2 infection on other organ tissues such as the intestines, kidneys, liver, and brain [6-9].
It is no coincidence that many of the more recent publications use 3D cell culture methods. Since the early 2000s, the number of publications using 3D cell culture has skyrocketed [10]. Scientists today prefer to work with 3D models because more valid results lead to more relevant research findings. In addition, researchers can re-evaluate the results of previous experiments conducted with 2D cell cultures, examining how 3D culture may affect the results. After all, if your competitors are working with 3D models, you don't want to be left with outdated methods.
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[1] THINCERT® cell culture plates - https://shop.gbo.com/en/row/products/bioscience/cell-culture-products/thincert-cell-culture-inserts/
[2] Tseng H, Daquinag AC, Souza GR, Kolonin MG. Three-Dimensional Magnetic Levitation Culture System Simulating White Adipose Tissue. Methods Mol Biol. 2018;1773:147-154. doi: 10.1007/978-1-4939-7799-4_12. PMID: 29687387.
[3] Tseng H, Gage JA, Raphael RM, Moore RH, Killian TC, Grande-Allen KJ, Souza GR. Assembly of a three-dimensional multitype bronchiole coculture model using magnetic levitation. Tissue Eng Part C Methods. 2013 Sep;19(9):665-75. doi: 10.1089/ten.TEC.2012.0157. Epub 2013 Feb 25. PMID: 23301612.
[4] Harb A, Fakhreddine M, Zaraket H, Saleh FA. Three-Dimensional Cell Culture Models to Study Respiratory Virus Infections Including COVID-19. Biomimetics (Basel). 2021 Dec 25;7(1):3. doi: 10.3390/biomimetics7010003. PMID: 35076456; PMCID: PMC8788432.
[5] de Dios-Figueroa GT, Aguilera-Marquez JDR, Camacho-Villegas TA, Lugo-Fabres PH. 3D Cell Culture Models in COVID-19 Times: A Review of 3D Technologies to Understand and Accelerate Therapeutic Drug Discovery. Biomedicines. 2021 May 26;9(6):602. doi: 10.3390/biomedicines9060602. PMID: 34073231; PMCID: PMC8226796.
[6] Lamers MM, Beumer J, van der Vaart J, Knoops K, Puschhof J, Breugem TI, Ravelli RBG, Paul van Schayck J, Mykytyn AZ, Duimel HQ, van Donselaar E, Riesebosch S, Kuijpers HJH, Schipper D, van de Wetering WJ, de Graaf M, Koopmans M, Cuppen E, Peters PJ, Haagmans BL, Clevers H. SARS-CoV-2 productively infects human gut enterocytes. Science. 2020 Jul 3;369(6499):50-54. doi: 10.1126/science.abc1669. Epub 2020 May 1. PMID: 32358202; PMCID: PMC7199907.
[7] Mahalingam R, Dharmalingam P, Santhanam A, Kotla S, Davuluri G, Karmouty-Quintana H, Ashrith G, Thandavarayan RA. Single-cell RNA sequencing analysis of SARS-CoV-2 entry receptors in human organoids. J Cell Physiol. 2021 Apr;236(4):2950-2958. doi: 10.1002/jcp.30054. Epub 2020 Sep 17. PMID: 32944935; PMCID: PMC7537521.
[8] Monteil V, Kwon H, Prado P, Hagelkrüys A, Wimmer RA, Stahl M, Leopoldi A, Garreta E, Hurtado Del Pozo C, Prosper F, Romero JP, Wirnsberger G, Zhang H, Slutsky AS, Conder R, Montserrat N, Mirazimi A, Penninger JM. Inhibition of SARS-CoV-2 Infections in Engineered Human Tissues Using Clinical-Grade Soluble Human ACE2. Cell. 2020 May 14;181(4):905-913.e7. doi: 10.1016/j.cell.2020.04.004. Epub 2020 Apr 24. PMID: 32333836; PMCID: PMC7181998.
[9] Tang H, Abouleila Y, Si L, Ortega-Prieto AM, Mummery CL, Ingber DE, Mashaghi A. Human Organs-on-Chips for Virology. Trends Microbiol. 2020 Nov;28(11):934-946. doi: 10.1016/j.tim.2020.06.005. Epub 2020 Jul 13. PMID: 32674988; PMCID: PMC7357975.
[10] Jensen C, Teng Y. Is It Time to Start Transitioning From 2D to 3D Cell Culture? Front Mol Biosci. 2020 Mar 6;7:33. doi: 10.3389/fmolb.2020.00033. PMID: 32211418; PMCID: PMC7067892.