Photo-curing of GelMA A bioink is more preferable than chemical curing for creating 3D models of breast cancer tumor growth.
Authors: Filippova Svetlana Yu , Kit Oleg I. , Sitkovskaya Anastasia O. , Mezhevova Irina V. , Gnennaya Nadezhda V. , Shamova Tatiana V. , Timofeeva Sofia V. , Novikova Inna A. , Lapteva Tatiana O. , Vashchenko Larisa N. , Kechedzhieva Emma E. , Dashkova Irina R. , Ausheva Tatiana V. , Andreiko Elena А. , Pozdnyakova Viktoria V. 
Published: June 2022

e15067

Background: Biogels based on natural components of the extracellular matrix are traditionally used to obtain 3D models of breast cancer (BC) tumor growth. However, such biogels are difficult to use in 3D bioprinting due to their poor shape retention and suboptimal curing conditions. Artificial biogels based on gelatin methacrylate and/or alginate have a good printability and can include natural components of the extracellular matrix to improve interaction with breast cancer cells. Nevertheless, for successful reconstruction of the tumor microenvironment, not only the composition but also the microstructure of the resulting models is important. The aim of the study was to investigate the effect of gelatin methacrylate and alginate composed bioink curing method on the microstructure of the resulting 3D construct and the morphology of breast cancer cells enclosed in it. Methods: BT20 breast cancer cells were mixed with GelMA A bioink (Cellink, USA) at a concentration of 106 cells/ml. The construct was printed on a BIO X bioprinter (Cellink, USA) at an ink temperature of 26°C, a printing table temperature of 10°C, a pressure of 8 kPa, a print speed of 10 mm/s, and a needle diameter of 22G. Next, the printed constructs were cured chemically by immersion in a 100 mM CaCl2 solution for 1 min or photo-cured by irradiating with light at a wavelength of 405 nm for 20 seconds from a distance of 5 cm. After washing, the constructs with encapsulated cells were incubated in DMEM medium (Gibco, USA) supplemented with 10% FBS (HyClone, USA) for two weeks, after which they were fixed in 10% formalin and embedded in paraffin blocks. The sections were H&E stained and photographed at a magnification of 400X, then the area and roundness of the pores were determined using the ImageJ software. Results: During photo-curing of the printed construct, the pore area on the section averaged 1.5±1.07 µm2 (M±SD, n = 500), which is significantly less than with chemical curing (4.5±0.9 µm2, n = 500) (α = 0.05, df = 998). At the same time, the pores had an irregular shape (roundness 2.5±0.6, n = 500), which indicates their communication, in contrast to the chemically cured construct, the pores of which were almost perfectly round (roundness 1.2±0.2 M±SD, n = 500). BT20 culture cells encapsulated in bioink had an elongated process shape, as if squeezing through the fine-mesh structure of the light-cured construct, while in the chemically cured construct they had a rounded shape, not going beyond the boundaries of the pores. Conclusions: When creating 3D tumor growth models of breast cancer using bioinks based on gelatin methacrylate and alginate, photocuring is preferable, as it allows creating a spongy microstructure of communicating pores. Such a structure supports cell migration and helps maintain cell morphology close to that observed in vivo.