3.2.2. Spheroids of HepG2 and HEK293 Cells

Previously published studies showed changes in the shape and compactization of HepG2 spheroids during their development; early, middle and late stages of spheroid growth were identified, the duration of which varied in different works and mainly depended on the dose of cell seeding. Common recommendation was to work with HepG2 spheroids at middle stage (beginning from days 4–5 after cell seeding) which is characterized by active function of spheroid cells and absence of cell destruction [10,14,45]. Amount of published studies using HEK293 spheroids is incomparably less than studies with HepG2 spheroids; however, authors noted a very fast compactization of HEK293 cells on a non-adhesive surface and regular spherical shape of the spheroids [46,47].

To examine interaction of AuNPs, AuPEI-NPs and AuBSA-NPs with HepG2 and HEK293 cells in 3D-culture we used corresponding spheroids cultured for 7 days with daily monitoring and photographing. In the first three days after cell seeding HepG2 spheroids looked loose and had an irregular shape, while HEK293 spheroids became spherical within a day. The spheroids showed differences in shape and rate of growth (Figure 3A–D). HepG2 spheroids always had somewhat irregular shape and surface with various recesses and ledges, as it is clearly seen on SEM image (Figure 3E). In contrast, HEK293 spheroids had a shape very close to spherical and relatively smooth surface (Figure 3F). We did not set out to study the growth features of HepG2 and HEK293 spheroids, since they are sufficiently described in literature, and we present here brief information on the growth and "appearance" of spheroids only to confirm the adequacy of our 3D models with published data.

**Figure 2.** Cell cultures in monolayer: (**A**,**B**)—HepG2, (**C**,**D**)—HEK293. Inserts: (**A**) cisternae of endoplasmic reticulum; (**B**) tight junction and desmosome between cells at apical pole. 1—"bile capillaries", arrows show microvilli; 2—nucleus; 3—lipid droplet; asterisks show mitochondria; arrowheads show basolateral membranes; dotted arrow shows area of "simple" contact of two cells; oval shows site of macropinocytosis; tick arrows show cell surface with many outgrowths.
