Construction of Floating Cell Spheroid Culture System Using Hydrogel Membrane as Culture Scaffold for Expression of Liver-specific Function

概要

1. Title of the Thesis/Dissertation :
Construction of Floating Cell Spheroid Culture System Using Hydrogel Membrane as Culture Scaffold for Expression of Liver-specific Function

2. Summary (1,000 - 1,200 words in English)

1. Introduction
Drug discovery takes a long time and high cost with low success rate in clinical trials. According to a recent report, efficacy or safety issues occupied more than 80 % of causes of failure in Phase II and Phase III clinical trials. Therefore, evaluation of the efficacy and the safety of drug candidates with high precision before clinical trials is very important to decrease both enormous period and expense of drug development. In the preclinical trials, animal experiments are carried out for the evaluations. However, they have considerable problems such as difference of drug response between species, and protection of animal welfare. Therefore, alternatives to them are required strongly. Nowadays, bioassay using human derived cells, which is called cell-based assay, is gaining increased attention due to recent active research on stem cells and development of cell culture techniques. Cell-based assay is expected to meet the species-specific elements that cannot be covered with animal experiments.

Development of cell-based assay using hepatocytes is significant for evaluation of metabolism and hepatotoxicity of drug candidates. In most cases of incubating hepatocytes, monolayer cell culture systems on rigid substrate composed of polystyrene (PS) have been utilized conventionally. However, it has been indicated that the cells do not express their own liver-specific physiological functions in such culture systems because the culture environments are far from those in human living body. In order to improve these situations, I proposed a novel cell spheroid culture system using flexible hydrogel membrane floating in the culture medium as cell culture scaffold. This cell culture system is expected to have three advantages: flexibility of cell culture scaffold, adequate supply of oxygen and nutrition to cells through semi-permeable hydrogel membrane, and enhancement of three-dimensional cell-cell interaction. Due to them, the culture system is expected to provide suitable cell culture environment and enhance liver-specific function of cells compared to the conventional one. In this study, I examined the preparation of a floating hydrogel membrane with patterned cell adhesion to construct the objective culture system. Then, I evaluated liver-specific function of HepG2 cells, which is one of human hepatoma cell lines, incubated in the system to confirm the effect of the constructed culture system for cells.

2. Construction of monolayer culture on semi 3-D hydrogel microstructure fabricated through photolithography
Firstly, I developed the method for fabricating semi 3-D microstructures composed of flexible hydrogel membrane through photolithography. Cross-linked bilayer was prepared, and by removing sacrificial layer composed of poly (acrylic acid) (PAAc), semi 3-D hydrogel microstructures composed of hydroxypropyl cellulose (HPC) was obtained. Both areas of cross-linking and detachment of the membrane from the substrate can be controlled by varying irradiation patterns. Therefore, various microstructures whose shape is controlled arbitrarily can be fabricated through photolithography. Next, the method for providing hydrogel membrane with cell adhesive property was examined. As a result, coating of poly (styrene-co-maleic anhydride) on hydrogel membrane enabled several kinds of cells such as HeLa, MDCK, and HepG2 cells to adhere and proliferate on the whole microstructure. Thus, monolayer cell culture system on semi 3-D hydrogel microstructure was constructed.

3. Construction of spheroid culture on floating hydrogel membrane
In the method described above, only monolayer cell culture system can be constructed, and it takes a long time to prepare the sufficient microstructures to incubate a lot of cells. Therefore, I examined a simple method for preparing floating hydrogel membrane on the whole culture surface of a dish, and the method for providing the membrane with patterned cell adhesion to construct cell spheroid culture.

After flushing the bilayer composed of cross-linked HPC and PAAc with water, cross-linked HPC layer lifted off of the PS substrate, and swelled in water in the only area where PAAc had been coated. The results showed that both stable and flexible membrane could be prepared by adjusting the cross-linking density of cross-linked HPC and the thickness of PAAc. Next, cell adhesive area on the hydrogel membrane was specified by using photo- responsive poly [(methyl methacrylaye)-co-(7-(4-trifluoromethyl) coumarin methacrylamide)] (PCMM). HepG2 cells adhered only in the irradiated area where PCMM was cross-linked through photodimerization of coumarin. Cross-linking of PCMM did not inhibit hydrogel from lifting off. As a result, spheroid culture system composed of HepG2 cells on the floating membrane was constructed, and the size and the spacing of spheroids could be controlled arbitrarily by varying irradiation pattern.

4. Evaluation of liver-specific function of HepG2 cells by spheroid culture on floating hydrogel membrane
In order to clarify the advantage of the constructed floating spheroid culture system compared to conventional one, respiratory metabolism of cells and gene expression of liver-specific drug metabolizing enzyme were evaluated. The culture systems were made move like seesaw in order to induce convection of culture medium and promote oxygen and nutrition supply to HepG2 cells more than conventional stagnant culture. Compared to the cell culture immobilized on the substrate, the cell culture on the floating membrane decreased the lactate production against glucose consumption of cells, and increased CYP1A2, which is one of the drug-metabolizing enzymes expressed in human liver in both spheroid and monolayer culture. These results suggest that liver-specific function of HepG2 cells was enhanced due to the activation of aerobic respiration through the improvement of oxygen supply on the floating membrane. Moreover, the result obtained for spheroid culture suggests that the enhancement of three- dimensional cell-cell interaction improved CYP1A2 activity. According to above results, it was demonstrated that the combination of incubation on floating hydrogel membrane scaffold and spheroid culture enhanced liver-specific function of HepG2 cells.

5. Conclusion
I established a method to prepare a flexible floating hydrogel membrane with patterned cell adhesion based on photolithography. By using this method, floating spheroid culture composed of HepG2 cells was constructed successfully. Compared to the conventional immobilized monolayer culture, liver-specific function of HepG2 cells was improved through the activation of respiratory metabolism on floating membrane and enhancement of three- dimensional cell-cell interaction in spheroid culture. These findings and established elemental technology to construct a novel cell culture system are expected to contribute to development of alternatives to animal experiments.