SENSITIVITY OF VARIOUS METHODS FOR ASSESSING THE CYTOTOXICITY OF MEDICAL DEVICES

Highlights

• There is no universal method for assessing the cytotoxicity of a material. A valid result can be obtained using several fundamentally different research methods. It is also necessary to take into account the features of the tested material.

Resume

Background. Despite the prevalence and frequency of use, the issues of standardization of methods for determining the cytotoxicity of materials for medical devices remain unresolved. The wide variability of approaches to assessing cytotoxicity in vitro requires the researcher to carefully plan the test to according with characteristics of the object being studied.

Aim. To conduct a comparative analysis of the sensitivity of different cell cultures and methods for assessing cytotoxicity materials for creating cardiovascular prostheses using preserved xenopericardium as an example.

Methods. The experiment was carried out on three cell cultures: fibroblasts, HUVEC, Ea.hy926, with the specific culture medium for each case. We used xenopericardium samples stored in a paraben solution as an object with a cytotoxic effect. We analyzed reaction of cells in direct and indirect contact with xenopericardium samples, as well as in the presence of its extract. An MTT test was carried out, cell growth dynamics were studied using the xCelligence cell analyzer and cell proliferative activity was assessed using a commercial Click-IT kit.

Results. According to the results of the MTT test, in all cases the presence of xenopericardium extract in the culture medium led to a pronounced decrease in cell viability and population density. According to the assessment of growth dynamics, in groups with pericardial samples, complete cell death was noted. The addition of preserved xenopericardium extract did not have a noticeable effect on the change in the cellular growth index of the fibroblast culture, however, in the presence of xenopericardium samples, there was a pronounced decrease in it. In HUVEC culture, the addition of both the extract and xenopericardium samples caused the death of a significant portion of the cells in a short time. In the Ea.hy926 culture, a decrease in the rate of proliferation was observed, but throughout the experiment the positive dynamics of cell culture growth continued. In the presence of the extract, the absence of proliferating cells in the fibroblast culture and a decrease in their number in the Ea.hy926 culture were noted.

Conclusion. Experimental conditions, test culture and evaluation methods must be selected individually based on the characteristics of the material being tested. To obtain reliable results, it is necessary to use several fundamentally different research methods to more fully characterize the effect of cytotoxic agents on cell culture.

1. GOST ISO 10993-5-2011. Medical products. Assessment of the biological effect of medical products. Part 5. Cytotoxicity studies: in vitro methods. - Introduction. 2013.01.01 – Moscow: Standard inform, 2014. – 10 p. – (System of standards for information, librarianship and publishing).

2. Gruber S., Nickel A. Toxic or not toxic? The specifications of the standard ISO 10993-5 are not explicit enough to yield comparable results in the cytotoxicity assessment of an identical medical device. Front. Med. Technol. 2023;5:1195529. https://doi.org/10.3389/fmedt.2023.1195529.

3. Bellucci D., Salvatori R., Anesi A., Chiarini L., Cannillo V. SBF assays, direct and indirect cell culture tests to evaluate the biological performance of bioglasses and bioglass-based composites: Three paradigmatic cases. Materials Science and Engineering: C. 2019;96:757-764. https://doi.org/10.1016/j.msec.2018.12.006.

4. Braun K., Stürzel C.M., Biskupek J., Kaiser U., Kirchhoff F., Lindén M. Comparison of different cytotoxicity assays for in vitro evaluation of mesoporous silica nanoparticles. Toxicology in Vitro. 2018;52:214-221. https://doi.org/10.1016/j.tiv.2018.06.019.

5. Diemer F., Stark H., Helfgen E.H., Enkling N., Probstmeier R., Winter J., Kraus D. In vitro cytotoxicity of different dental resin-cements on human cell lines. J Mater Sci Mater Med. 2021;32(1):4. https://doi.org/10.1007/s10856-020-06471-w.

6. Wang Y., Ma B., Yin A., Zhang B., Luo R., Pan J., Wang Y. Polycaprolactone vascular graft with epigallocatechin gallate embedded sandwiched layer-by-layer functionalization for enhanced antithrombogenicity and anti-inflammation. J Control Release. 2020;320:226-238. https://doi.org/10.1016/j.jconrel.2020.01.043.

7. Zhou J., Wang M., Wei T., Bai L., Zhao J., Wang K., Feng Y. Endothelial cell-mediated gene delivery for in situ accelerated endothelialization of a vascular graft. ACS Appl Mater Interfaces. 2021;13(14):16097-16105. https://doi.org/10.1021/acsami.1c01869.

8. Kabirian F., Brouki Milan P., Zamanian A., Heying R., Mozafari M.. Nitric oxide-releasing vascular grafts: A therapeutic strategy to promote angiogenic activity and endothelium regeneration. Acta Biomater. 2019;92:82-91. https://doi.org/10.1016/j.actbio.2019.05.002.

9. Lee S.J., Kim M.E., Nah H., Seok J.M., Jeong M.H., Park K., Kwon I.K., Lee J.S., Park S.A. Vascular endothelial growth factor immobilized on mussel-inspired three-dimensional bilayered scaffold for artificial vascular graft application: In vitro and in vivo evaluations. J Colloid Interface Sci. 2019;537:333-344. https://doi.org/10.1016/j.jcis.2018.11.039.

10. Daum R., Visser D., Wild C., Kutuzova L., Schneider M., Lorenz G., et al. Fibronectin adsorption on electrospun synthetic vascular grafts attracts endothelial progenitor cells and promotes endothelialization in dynamic in vitro culture. Cells. 2020;9(3):778. https://doi.org/10.3390/cells9030778.

11. Guan G., Yu C., Xing M., Wu Y., Hu X., Wang H., Wang L. Hydrogel small-diameter vascular graft reinforced with a braided fiber strut with improved mechanical properties. Polymers. 2019;11:810. https://doi.org/10.3390/polym11050810.

12. Jirofti N., Mohebbi-Kalhori D., Samimi A., Hadjizadeh A., Kazemzadeh G.H. Small-diameter vascular graft using co-electrospun composite PCL/PU nanofibers. Biomed Mater. 2018;13(5):055014. https://doi.org/10.1088/1748-605X/aad4b5.

13. Fiqrianti I.A., Widiyanti P., Manaf M.A., Savira C.Y., Cahyani N.R., Bella F.R. Poly-L-lactic Acid (PLLA)-chitosan-collagen electrospun tube for vascular graft Application. J Funct Biomater.2018;9(2):32. https://doi.org/10.3390/jfb9020032.

14. Rosellini E., Barbani N., Lazzeri L., Cascone M.G. Biomimetic and bioactive small diameter tubular scaffolds for vascular tissue engineering. Biomimetics (Basel). 2022;7(4):199. https://doi.org/10.3390/biomimetics7040199.

15. Jaffe E.A., Nachman R.L., Becker C.G., Minick C.R. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. Clin Invest. 1973; 52: 2745–2756. https://doi.org/10.1172/JCI107470.

16. Ghasemi M., Turnbull T., Sebastian S., Kempson I. The MTT Assay: utility, limitations, pitfalls, and interpretation in bulk and single-cell analysis. Int. J. Mol. Sci. 2021;22:12827. https://doi.org/10.3390/ijms222312827.

17. Velikanova E.A., Matveeva V.G., Khanova M.Yu., Antonova L.V. Effects of shear stress on the properties of colonyforming endothelial cells in comparison with coronary artery endothelial cells. Complex Issues of Cardiovascular Diseases. 2022;11(4):90-97. (In Russ.) https://doi.org/10.17802/2306-1278-2022-11-4-90-97.

18. Li W., Zhou J., Xu Y. Study of the in vitro cytotoxicity testing of medical devices. Biomed Rep. 2015;3(5):617-620. https://doi.org/10.3892/br.2015.481

19.