Culture phases, cytotoxicity and protein expressions of agarose hydrogel induced Sp2/0, A549, MCF-7 cell line 3D cultures

Abstract

Advancements in cell cultures are occurring at a rapid pace, an important direction is culturing cells in 3D conditions. We demonstrate the usefulness of agarose hydrogels in obtaining 3 dimensional aggregates of three cell lines, A549, MCF-7 and Sp2/0. The differences in culture phases, susceptibility to cisplatin-induced cytotoxicity are studied. Also, the 3D aggregates of the three cell lines were reverted into 2D cultures and the protein profile differences among the 2D, 3D and revert cultures were studied. The analysis of protein profile differences using UniProt data base further augment the usefulness of agarose hydrogels for obtaining 3D cell cultures.

Keywords: 3D aggregates; Agarose hydrogels; Cell culture phases; Cytotoxicity; Protein expressions.

Fig. 1

The morphological features of the three cell lines a s 2D, 3D and 3DRs showed unique characteristics for each of the cell line. While A549 exhibited a compact 3D structure with medium amount of extracellular matrix holding the single large aggregate together, the MCF-7 showed distinct spheroid structures. The 3D cultures of the suspension cell line Sp2/0 showed loosely formed aggregate with large amount of jelly-like extra cellular matrix. While the 3DRs of A-549 and Sp2/0 showed faster movement of cells from the aggregates, the MCF-7 took a longer time for the cells to migrate out of the spheroids to form a monolayer

Fig. 2

The human breast cancer cell line MCF-7 formed distinct spheroids, whose formation progress could be well observed and documented. The progress of the cultures towards the formation of the spheroids is presented as A1-A4 in the panel A. The B1 to B3 panels show the presence and the role of globular extracellular matrix entities towards the formation of spheroids. Arrows in B1 show the initial formation of globular structure, where the cells are surrounded with a spherical shaped extracellular matrix. Arrows in B2 show the cells dividing inside the globular structure leading to the formation of spheroids called as acinar structures. B3 shows the formation of multiacinar structures which is formed by attachment of several acinar structures. The Panel B shows the progressive formation of the acinar structures from the initial well with formed extracellular matrix. The individual cells appear to be trapped into the extracellular matrix with a definite spherical structure, divide and form an aggregate of cells that is spherical. Many such individual aggregates form the multiacinar structures. In the panel B, the acellular spherical extracellular matrix is presented at the extreme left, the progressive development of the acinar structures are present through the panel, ending with the multiacinar structures presented at the extreme right of the panel B

Fig. 3

The 3D cultures of the three cell lines showed a marked difference in the cell viability over time. The 3D cultures showed better viability and tended to last longer when compared to their 2D counterparts. Also, the panels 1,2 and 3 show that the 3D cultures of the three cell lines were less susceptible to the cisplatin induced cytotoxicity when compared to their 2D counterparts. Also, the differences were much pronounced for the suspension cell line Sp2/0 (3) than the monolayers. Among the monolayers, the A549 cell line showed more differences in higher concentration of the drug when compared to the MCF-7 cell line

Fig. 4

SDS-PAGE protein analysis of the whole protein extracts from the 2D, 3D and the 3DRs showed differences in the protein profiles. The bands that appear in higher intensity are marked with arrows pointing upwards and those bands that are comparatively of less intensity are marked with arrows pointing downwards. Specific molecular weight regions that show marked differences are presented below each of the three gel images. These differences can be useful for several applications such as for cancer biomarker discovery, Also the induced differences highlight the usefulness of simple agarose hydrogels as useful matrices for 3D cell cultures owing to their ease of use