Optimization of Albumin Secretion and Metabolic Activity of Cytochrome P450 1A1 of Human Hepatoblastoma HepG2 Cells in Multicellular Spheroids by Controlling Spheroid Size.

Multicellular spheroids are useful as three-dimensional cell culture systems and for cell-based therapies. Their successful application requires an understanding of the consequences of spheroid size for cellular functions. In the present study, we prepared multicellular spheroids of different sizes using the human hepatoblastoma HepG2 cells, as hepatocytes are frequently used for in vitro drug screening and cell-based therapy. Precise polydimethylsiloxane-based microwells with widths of 360, 450, 560, and 770 µm were fabricated using a micromolding technique. Incubation of HepG2 cells in cell culture plates containing the microwells resulted in the formation of HepG2 spheroids with average diameters of 195, 320, 493, and 548 µm. The cell number per spheroid positively correlated with its diameter, and the viability of HepG2 cells was 94% or above for all samples. The smallest HepG2 spheroids showed the highest albumin secretion. On the other hand, the metabolic activity of 7-ethoxyresorufin, a fluorometric substrate for CYP1A1, increased with increasing spheroid size. These results indicate that controlling spheroid size is important when preparing HepG2 spheroids and that the size of HepG2 spheroids greatly influences the cellular function of HepG2 cells in the spheroids.

Using size-controlled multicellular spheroids of murine adenocarcinoma cells to efficiently establish pulmonary tumors in mice.

Previous studies demonstrated that multicellular spheroids developed using polydimethylsiloxane-based microwells exhibited superior functions, such as insulin secretion from pancreatic cells, over suspended cells. To successfully apply these spheroids, the effect of spheroid size on cellular functions must be determined. In this study, using murine adenocarcinoma colon26 cells, the authors examined whether such spheroids were useful for developing tumor-bearing animal models, which requires the efficient and stable engraftment of cancer cells at implanted sites and/or metastatic sites. The authors prepared microwells with widths of 360, 450, 560, and 770 µm through a micromolding technique, and obtained colon26 spheroids with average diameters of 169, 240, 272, and 341 µm, respectively. Small and medium spheroids were subsequently used. mRNA levels of integrin ß1, CD44, and fibronectin, molecules involved in cell adhesion, increased with increasing colon26 spheroid size. Approximately 1.5 × 104 colon26 cells in suspension or in spheroids were intravenously inoculated into BALB/c mice. At 21 days after inoculation, the lung weight of both colon26 spheroid groups, especially the group injected with small spheroids, was significantly higher than that of mice in the suspended colon26 cell group. These results indicate that controlling cancer cell spheroid size is crucial for tumor development in tumor-bearing mouse models.

Transplantation of insulin-secreting multicellular spheroids for the treatment of type 1 diabetes in mice.

The efficacy of cell-based therapy depends on the function and survival of transplanted cells, which have been suggested to be enhanced by spheroid formation. However, few attempts at spheroid generation from insulin-secreting cells, which may be used to treat type 1 diabetes, have been reported. We therefore developed spheroids from the mouse insulinoma cell line NIT-1 by using polydimethylsiloxane (PDMS)-based microwells with a coating of poly(N-isopropylacrylamide) (PNIPAAm). The prepared NIT-1 spheroids or dissociated NIT-1 cells were transplanted into the subrenal capsule in streptozotocin-induced diabetic mice. NIT-1 spheroids prepared using the PNIPAAm-coated PDMS-based microwells had a uniformly sized spherical structure with a diameter of 200-300µm. The PNIPAAm coating increased cell survival in the spheroids and the recovery of the spheroids from the microwells. In diabetic mice, the transplanted NIT-1 spheroids reduced blood glucose levels to normal values faster than dissociated NIT-1 cells did. Additionally, survival was higher among NIT-1 cells in spheroids than among dissociated NIT-1 cells 24h after transplantation. These results indicate that insulin-secreting NIT-1 spheroids prepared using PNIPAAm-coated PDMS-based microwells are more effective for the treatment of type 1 diabetes than dissociated cells in suspension.

Poly(N-isopropylacrylamide)-coated microwell arrays for construction and recovery of multicellular spheroids.

Microwell arrays that have many micro-sized cavities on the device have been employed to form multicellular spheroids. However, methods to efficiently harvest the constructed spheroids from the microwell arrays have not been thoroughly investigated. We evaluated the effects of poly(N-isopropylacrylamide) (PNIPAAm) for constructing and harvesting spheroids from microwell arrays. Microwell arrays were coated with ethanol containing 1%, 2.5%, 5%, or 10% PNIPAAm by a solvent-casting method and then dried. Spheroids formed using the coated microwell arrays were harvested. Highly efficient and rapid recovery of NIH3T3 mouse fibroblast spheroids were achieved for the 5% and 10% coated wells (93.2% ± 1.6% and 93.6% ± 1.1% at 60 s, respectively), whereas recovery was not efficient for 0%, 1%, and 2.5% coated wells (0.2% ± 0.2%, 1.1% ± 0.6%, and 7.8% ± 4.0% at 60 s, respectively). Because PNIPAAm is a thermoresponsive polymer that exhibits a lower critical solution temperature (LCST) of 32°C, we examined the effects of temperature on the recovery rate. The recovery rates at 4°C (below LCST) were equivalent to or higher than those at 37°C (above LCST) for all four cell types examined. Functional assessment suggests that the PNIPAAm microwell arrays are not toxic to the formed spheroids. The PNIPAAm microwell array developed in the present study will be useful for constructing and harvesting spheroids.