3.2.3. Electron Microscopic Features of HepG2 and HEK293 Spheroids

All published studies with HepG2 spheroids describe the "general appearance" of entire spheroids, noting the irregularity of shape and surface. Control of spheroids growth and analysis of their changes is carried out by means of light-optical observation of intravital characteristics as the perimeter length and square of spheroids, their roundness and compactness. Routine staining of paraffin sections mostly is used to show absence or presence of necrosis. Immunohistochemistry and fluorescence methods are increasingly introduced in recent years [11–15,45]. Published TEM data are scarce and usually present small portions of HepG2 cells [14,48]. Here we describe the ultrastructure of HepG2 spheroids with an emphasis on cell relationships and structure of spheroid outer surface and adjacent areas. We used a

semithin section of each spheroid to choose a location for the pyramid, so we knew exactly which part of the spheroid was being explored in the TEM.

**Figure 3.** Characteristics of spheroids in culture. Representative light microscopic images of HepG2 spheroids (**A**) and HEK293 spheroids (**B**), days 2–7 after seeding. Bars correspond to 200 μm. Growth curves of spheroids square: (**C**) HepG2, (**D**) HEK293. Representative images of 7-day HepG2 (**E**) and HEK293 (**F)** spheroids obtained with SEM.

SEM examination of the HepG2 spheroids revealed an uneven surface resembling a "lunar landscape" with some bulging cells and "craters" extending into the interior of spheroid (Figure 4A). "Craters" looked as openings between spheroid cells and were randomly located on spheroid surface. Surface of the cells was covered with many small outgrowths.

**Figure 4.** Ultrastructure of HepG2 spheroids. (**A**) Representative SEM image of spheroid surface. 1—bulging cells; arrows show openings on spheroid surface; (**B**–**D**) HepG2 spheroid periphery; (**E**) area at a distance of 50 μm from the surface; (**F**) bile capillary with microvilli. 1—nucleus; 2—cytoplasm; 3—lumen of cross-sectioned psedosinusoid; asterisks show external space; ovals show bile capillaries; yellow arrows show a space between hepatocytes (openings in SEM); black arrows show a space between lateral surfaces of neighboring cells; red arrows show microvilli; a frame shows desmosome between cells, and insert shows this desmosome at high magnification. (**B**–**F**) Ultrathin sections, TEM.

Analyzing the results obtained by light microscopy, SEM and TEM we found that the openings are expansions of the space between the lateral surfaces of neighboring cells, serving as an "entrance" into the spheroid (Figure 4B,C and Figure S1). This "entrance" continued in the form of a space expanded up to one μm between the lateral surfaces of cells, covered with cytoplasmic outgrowths of different lengths and shapes. It should be noted that these outgrowths were distinctly different from microvilli on the surface of bile capillaries (Figure 4C–F). The openings were not formed by all cells: the "entrance" between the lateral cell surfaces could be closed with desmosomes (Figure 4D). Observed patterns suggest that cell basal membranes form outer surface of HepG2 spheroids contacting with culture medium and receiving the nutrients and external influences. Presence of the "pores" on surface of HepG2 spheroids were noted in SEM and TEM earlier [48].

Hepatocytes are epithelial cells with unique type of polarization, which sets a direction of their morphology and function, determines formation of bile capillaries by the apical surface, and retrieval of metabolites and other substances from sinusoid blood by basolateral surfaces [11,49]. In the liver, sinusoids formed by endothelial cells provide blood flow that is cleared by hepatocytes. Inside the spheroids we observed empty spaces between HepG2 cells resembling the sinusoids up to 2–2.5 μm width (Figure 4E and Figure S2). This similarity allowed us to designate these spaces as "pseudosinusoids". It is unknown whether pseudosinusoids form a common network that permeates the spheroid; a special research is required to establish this.

Bile capillaries (bile canaliculi) formed by hepatocyte apical plasmalemma are bright morphological feature of hepatic tissue [11]. Bile capillaries are clearly visible in groups of HepG2 cells in monolayer (Figure 2A,B) and in ultrathin sections of HepG2 spheroids (Figure 4B,E,F and Figure S3). These structures are easily differentiated by the presence of microvilli on their surface, and could be found in various parts of spheroid, there are no visible ordering in their location. Analysis of the data obtained by TEM, SEM and light microscopy, clearly indicates that bile capillaries never come to the surface; they are "hidden" inside the spheroids. Correct identification of bile capillaries in HepG2 spheroids by TEM was reported in [14], and some published TEM studies of HEpG2 spheroids demonstrated pseudosinusoids instead bile capillaries [48,50].

Thus, the spheroids formed by HepG2 cells maintain typical for liver histology parameters: separation of the plasmalemma into apical and basolateral parts, formation of bile capillaries and pseudosinusoids. It is important that HepG2 spheroids face the environment with cell basal plasmalemma, which provides contact of hepatocytes with blood components in the liver. This feature of HepG2 spheroids should be taken into account when studying the effects of various preparations, including nanoparticles.

Examination of HEK293 spheroids in a SEM revealed a significantly flatter surface than those in HepG2 spheroids due to absence of "craters" (Figure 3E,F, 4A and 5A). Thin flat folds of plasmalemma indicating macropinocytosis were visible between bulging cell bodies. Small cytoplasm protrusions were present on cell surface in different amount, some cells had smooth surface (Figure 5A). Different appearance of cell surface could reflect different functional state of the cells in HEK293 spheroids.

**Figure 5.** Ultrastructure of HEK293 spheroids. (**A**) Representative SEM image of spheroid surface. 1—cell body; 2—cell surface with small microvilli; 3—flat cell surface; white arrows show flat folds. (**B**–**F**) Cells at the periphery of spheroid, ultrathin sections. 1—nucleus; 2—cytoplasm; asterisks show external space; circle shows a conglomerate of apical outgrowths; arrows show outgrowths protruding in external space; yellow arrows show narrow space between lateral cell surfaces. (**D1**) Outgrowth conglomerate at higher magnification, the insert shows desmosomes; (**D2**) this photo presents a structure similar to apical tight junction.

HEK293 cell were isolated from human embryo kidney and transformed with sheared DNA of adenovirus type 5 [42]. The obtained cell line was considered as epithelial cells. Presence of morphologically visible tight junctions in HEK293 monolayer were not reported [51,52], although expression of markers of epithelial tight junctions (zonula occludens) including ZO-1

and occludins was detected [53]. We did not observe tight junctions in monolayer HEK293 cells (Figure 2C,D).

Ultrathin sections of HEK293 spheroids showed groups of pyramidal cells (Figure 5C,E) connected to each other with narrow apical parts, which formed a conglomerate of interlaced cytoplasmic outgrowths bound by desmosomes and structures similar to tight junctions (Figure 5(D1,D2)). The conglomerates were observed throughout entire thickness of spheroids, not only at the periphery. On the ultrathin sections, these conglomerates looked like a disheveled skein of ribbons with ends sticking out in different directions. The center of conglomerate looked solid; there were no signs of a lumen formation, as in bile capillaries or glandular acini. We propose that apical cytoplasmic conglomerates organize HEK293 cells into groups, reflecting non-complete (without a lumen) formation of epithelial tube, determined by "columnar" polarization typical for of non-hepatic epithelia [39,41]. Thus, it is clear that cultivation of HEK293 cells in the 3D system induced formation of typical for epithelia structures (tight junctions and desmosomes) that were absent in the monolayer of these cells.

Surface of HEK293 spheroids was formed by basal plasmalemma of the cells, which was flat or covered with small protrusions. Some cells showed large outgrowths protruding in external space usually located near cell lateral borders (Figure 5B,E,F). It is obvious that formation of these structures is associated with the ability of HEK293 cells to macropinocytosis, which we noted on a monolayer culture. Similar structures were not observed in cells of HepG2 spheroid.

The lateral surfaces of spheroid cells were usually smooth and separated by narrow gaps led deep into the spheroid, widening of these gaps were observed in area of apical conglomerates (Figure 5B,C,E). Lateral plasmalemma of neighboring cells did not form interdigitations.

The results we obtained showed that cultivation of HepG2 and HEK293 cells in non-adhesive conditions in full media and without scaffold provide formation of spheroids; their morphological characteristics reflect structural polarization of epithelium in maternal organ. While HepG2 cells form bile capillaries and pseudosinusoids, HEK293 cells in these conditions are unable create complete structural units of epithelium with "columnar" polarization. Interestingly, the cells of both cultures were exposed to the culture medium by their basal plasmalemma, and the apical parts of the cells were inside spheroids.
