Morphological and Functional Analysis of Hepatocyte Spheroids Generated on Poly-HEMA-Treated Surfaces under the Influence of Fetal Calf Serum and Nonparenchymal Cells

Abstract

Poly (2-hydroxyethyl methacrylate) (HEMA) has been used as a clinical material, in the form of a soft hydrogel, for various surgical procedures, including endovascular surgery of liver. It is a clear liquid compound and, as a soft, flexible, water-absorbing material, has been used to make soft contact lenses from small, concave, spinning molds. Primary rat hepatocyte spheroids were created on a poly-HEMA-coated surface with the intention of inducing hepatic tissue formation and improving liver functions. We investigated spheroid formation of primary adult rat hepatocyte cells and characterized hepatic-specific functions under the special influence of fetal calf serum (FCS) and nonparencymal cells (NPC) up to six days in different culture systems (e.g., hepatocytes + FCS, hepatocytes - FCS, NPC + FCS, NPC - FCS, co-culture + FCS, co-culture - FCS) in both the spheroid model and sandwich model. Immunohistologically, we detected gap junctions, Ito cell/Kupffer cells, sinusoidal endothelial cells and an extracellular matrix in the spheroid model. FCS has no positive effect in the sandwich model, but has a negative effect in the spheroid model on albumin production, and no influence in urea production in either model. We found more cell viability in smaller diameter spheroids than larger ones by using the apoptosis test. Furthermore, there is no positive influence of the serum or NPC on spheroid formation, suggesting that it may only depend on the physical condition of the culture system. Since the sandwich culture has been considered a "gold standard" in vitro culture model, the hepatocyte spheroids generated on the poly-HEMA-coated surface were compared with those in the sandwich model. Major liver-specific functions, such as albumin secretion and urea synthesis, were evaluated in both the spheroid and sandwich model. The synthesis performance in the spheroid compared to the sandwich culture increases approximately by a factor of 1.5. Disintegration of plasma membranes in both models was measured by lactate dehydrogenase (LDH) release in both models. Additionally, diazepam was used as a substrate in drug metabolism studies to characterize the differences in the biotransformation potential with metabolite profiles in both models. It showed that the diazepam metabolism activities in the spheroid model is about 10-fold lower than the sandwich model. The poly-HEMA-based hepatocyte spheroid is a promising new platform towards hepatic tissue engineering leading to in vitro hepatic tissue formation.

Figure 1

Live/dead test of hepatic spheroid culture in poly-HEMA-treated surfaces. 1 A and B show the live/dead staining of hepatic spheroid culture in poly-HEMA-treated surfaces on Petri dishes on the fifth day of culture, Live Cell: green, dead cells: red (A) spheroid with 5% FCS, (B) without FCS (magnification 50×). Scale bar is 100 μm. Figure 1C,D show the frequency distribution of Spheroid diameter on fifth day of culture, hepatocytes (250,000 cells/mL) (C) with 5% FCS or (D) serum-free culture. The diameters of hepatocyte spheroids were measured using a Windows computer with computer-assisted image analyzer. The diameter of spheroids was calculated by converting the spheroid area into an equivalent circle diameter.

Figure 2

Spheroid formation in serum free culture. Spheroid in serum-free co-culture on day 0 (A); day 3 (B) and day 5 (C); (Magnification: 100×). Scale bar is 100μm. Average diameter of the spheroids in the period of five days in a culture serum-free culture of spheroid. (D) Monoculture from serum-free hepatocytes and a co-culture of hepatocytes (Hep) and NPC.

Figure 3

Immunological detection of hepatospecific marker in spheroid culture model (A and B) Extracellular matrix in the spheroid (Magnification 400×), (B) individual cells (magnification 400×). (C) Localization of the gap junctions in Spheroid after incubation with anti-Cx32; (Magnification 400×). (D) Distribution of hepatocytes in the Spheroid (magnification 400×). Scale bar is 100 μm.

Figure 4

Immunological detection of different liver cells in spheroid culture model (i) A and B: localization of Ito cells (A) and Kupffer cells (B) in the Spheroid (magnification 400×). (ii) A and B: distribution of the product ductal epithelial cells (A) and endothelial cells (B) in the spheroid; (Magnification 400×), (iii) A and B: Negative controls (A) anti-rabbit secondary (B) anti-mouse secondary; (Magnification 200×). Scale bar is 100 μm.

Figure 5

TUNEL staining test in spheroid culture. (i) A and B Vitality of cells in the spheroid (A) on day 4 and (B) on day 5 of culture; (Magnification 100×). (ii) A and B: (A) positive control, (B) Negative control of TUNEL staining (magnification 200×), (iii) A and B: TUNEL staining of 6-μm sections of spheroids (magnification 200×).

Figure 6

Speriod formation before the Percoll purification and after the Percoll purification. (i) A and B: live Tot staining of the cells in the spheroid; serum-free culture (A) before the Percoll purification and (B) after the Percoll purification (magnification 50×). (ii) A and B: Serum-free spheroid (A) spheroids before purification, (B) with spheroids Diameter <100 μm (C) spheroids with a diameter of 40–100 μm; (Magnification 100×). (iii) A and B: live/dead staining the spheroids after serum-free culture (magnification 100×). Scale bar is 100 μm. (A). Frequency distribution of spheroid diameter after serum-free culture (B).

Figure 7

LDH activity of the cells in the sandwich and spheroid culture. (A): LDH activity of the cells in the sandwich culture per 10 million from day 1 to day 5. (B): LDH activity of the cells in the spheroid culture per 10 million cells, day 1 to day 5. Each point represents the mean ± S.E.M. of five experiments during which LDH activities were measured in triplicate.

Figure 8

Albumin production in the sandwich and spheroid culture. (A) Total albumin production of the cells in serum-free and serum-containing mono-and co-cultures in the sandwich model; Hep + FCS, HEP – FCS, NPC + FCS, NPC – FCS, co-culture + FCS, co-culture – FCS. (B) Total albumin production of the cells in serum-free and serum-containing mono-and co-cultures in the spheroid model; Hep + FCS, HEP – FCS, co-culture + FCS, co-culture – FCS. Each point represents the mean ± S.E.M. of five experiments during which LDH activities were measured in triplicate.

Figure 9

Urea productions in the sandwich and spheroid culture. (A) Total urea production of the cells in serum-free and serum-containing mono-and co-cultures in the sandwich model; Hep + FCS, HEP – FCS, NPC + FCS, NPC – FCS, co-culture + FCS, co-culture – FCS. (B) Total urea production of the cells in serum-free and serum-containing mono-and co-cultures in the spheroid model; Hep + FCS, HEP – FCS, co-culture + FCS, co-culture – FCS. Each point represents the mean ± S.E.M. of five experiments during which LDH activities were measured in triplicate.

Figure 10

Diazepam metabolism in the sandwich and spheroid culture. (A), Percentage of diazepam metabolism in mono- and co-cultures in the sandwich model; Hep, co-culture and NPC (B), Percentage of diazepam metabolism in mono-and co-cultures in spheroid model; Hep, co-culture and NPC (C) Diazepam’s metabolite profile (percentage) in mono- and co-cultures in the sandwich model; Hep, co-culture and NPC, (D) Diazepam’s metabolite profile (percentage) inmono and co-cultures in the sandwich model; Hep, co-culture and NPC. Each point represents the mean ± S.E.M. of five experiments during which LDH activities were measured in triplicate.