Following the establishment of human malignant choroids plexus papilloma (HIBCPP) cell line in human cells (2005), we describe a more detailed protocol for establishing functional HIBCPP cell line. Generally, fibroblasts proliferate faster than tumor cells in primary culture; their presence is often essential for selecting functional epithelial cells in the culture process of establishing cell lines. Simply passing (subculturing) on the cells in the culture process will result in the selection of cells with a high proliferative potential. However, cells with a high proliferative potential often have no function. Considering these phenomena, fibroblasts present in the primary culture of tumor cells derived from the choroid plexus are separated and cultured in advance. Primary cultures of choroid plexus cells are cultured in a growth medium (GM) to which 10% of fibroblast-conditioned medium (F-CM) is added; F-CM tends to inhibit the proliferation of fibroblasts and promote the proliferation of epithelial cells. Next, to obtain slow-growing cells with function, we seeded a small number of cells (1 × 104 cells/10 cm dish) and cultured them in a laminin-coated petri dish with GM (DMEM/F12 supplemented 10% FBS) supplemented with 10% F-CM to form colonies. The smallest possible colony (a cell population with low proliferative potential) was subjected to colonial cloning and continued to be cultured in GM supplemented with 10% F-CM to establish a cell line. Subsequently, the cell lines were passaged only with GM in a noncoated petri dish. The established HIBCPP cells produced transthyretin (prealbumin), and immunostaining revealed presence of growth hormone (GH) receptors. GH increased transthyretin secretion by HIBCPP cells in a dose-dependent manner. Cells cultured under slightly hypoxic conditions (oxygen concentration, 15%) showed higher levels of secreted transthyretin than in those cultured under normoxic conditions. Furthermore, transthyretin increased the viability of neurons cultured under oxygen–glucose deprived conditions in a dose-dependent manner and decreased the number of terminal deoxynucleotidyl transferase dUTP nick-end labeling positive (apoptosis) cells. These results indicated that transthyretin produced by the choroid plexus epithelial cells is regulated by GH, and the secreted transthyretin has neuroprotective effects.