Silica bioreplication preserves three-dimensional spheroid structures of human pluripotent stem cells and HepG2 cells

Abstract

Three-dimensional (3D) cell cultures produce more in vivo-like multicellular structures such as spheroids that cannot be obtained in two-dimensional (2D) cell cultures. Thus, they are increasingly employed as models for cancer and drug research, as well as tissue engineering. It has proven challenging to stabilize spheroid architectures for detailed morphological examination. Here we overcome this issue using a silica bioreplication (SBR) process employed on spheroids formed from human pluripotent stem cells (hPSCs) and hepatocellular carcinoma HepG2 cells cultured in the nanofibrillar cellulose (NFC) hydrogel. The cells in the spheroids are more round and tightly interacting with each other than those in 2D cultures, and they develop microvilli-like structures on the cell membranes as seen in 2D cultures. Furthermore, SBR preserves extracellular matrix-like materials and cellular proteins. These findings provide the first evidence of intact hPSC spheroid architectures and similar fine structures to 2D-cultured cells, providing a pathway to enable our understanding of morphogenesis in 3D cultures.

Figure 1. The morphology of hiPSCs iPS(IMR90)-4, hESCs WA07, and human hepatocellular carcinoma HepG2 cells cultured in 3D hydrogels and the pluripotency of WA07 cells.

(a) WA07 and iPS(IMR90)-4 cell spheroids in the NFC hydrogel (NFC) but not in the ExtraCel™ hydrogel (EC). HepG2 cell spheroids in both NFC and EC hydrogels. Images are representative of eight biological samples from NFC hydrogels and three biological samples from EC hydrogels. (b,c) Immunostaining of the pluripotency marker OCT4 in WA07 cells cultured in standard 2D culture system (b) and in the NFC hydrogel for 7 days (5 μm paraffin section) (c). (d) Flow cytometry analysis of the pluripotency marker SSEA-4 in WA07 cells after being cultured in 2D and in 3D NFC hydrogel for 7 days. Scale bars = 100 μm.

Figure 2. Morphology of human pluripotent stem cells and hepatocellular carcinoma cells with and without silica bioreplication.

(a) SEM images of HepG2, iPS(IMR90)-4, and WA07 cell spheroids show deformation of 3D spheroids. (b) SEM images of HepG2, iPS(IMR90)-4, and WA07 cell spheroids after silica bioreplication show well-preserved spheroid architecture and tight cell-cell contact in the NFC hydrogel-based 3D cultures. Images are representative of eight biological samples.

Figure 3. Scanning electron microscopy of silicified cells.

(a) Microvilli-like structures on the surface of HepG2, iPS(IMR90)-4, and WA07 cells in 2D culture and in 3D NFC hydrogel culture. (b) Extracellular matrix-like material on a HepG2 cell spheroid in the NFC hydrogel for 8 days. (c) Silica-replicas of WA07 spheroids (5 days in the NFC hydrogel) after calcination. Images are representative of eight biological samples.

Figure 4. Detection of cellular proteins in desilicified cells.

(a) Staining of filamentous actin (F-actin) and multidrug resistance-associated protein 2 (MRP2) in HepG2 spheroids after 8 day-culture in the NFC hydrogel. Immunostaining of the pluripotency marker OCT4 in WA07 cells after 5 day-culture in the NFC hydrogel. (b) Detection of F-actin, MRP2, and OCT4 after desilicification of spheroid-silica composites. Scale bars = 100 μm. Images are representative of eight biological samples.