, Chapter 11 Pineal Cells Dissociation and Culture: Isolated Pinealocytes Isolated Astrocytes, and Co-culture Solange Castro Afeche, Diego de Piazza Pimentel, Luı ´s Felipe Ferro, and Jose ´ Cipolla-Neto Abstract Mammalian pineal glands are composed mostly of pinealocytes, which are the melatonin secretory cells, and also importantly of glial cells in special astrocytes. With the aim of studying the interactions between pinealocytes and astrocytes, the methodologies for obtaining and maintaining isolated pinealocytes and astrocytes in culture were standardized, in addition to the co-culture of both cell types. Some works of our group were published on the interactions between isolated astrocytes and pinealocytes from the pineal gland of Wistar rats, considering the modulatory role of glutamate and angiotensin on the synthesis of melatonin. In this chapter, the methodologies for obtaining and maintaining astrocytes and pinealocytes culture as well as co-culture of these two cell types will be presented. Key words Pinealocytes, Astrocytes, Pinealocytes-astrocytes co-culture, Pineal cells dissociation, Papain 1 Introduction The parenchymal cells of the mammalian pineal gland are the hormone-producing pinealocytes and the interstitial cells [1]. In addition, perivascular phagocytes are present and these share anti- genic properties with microglial and antigen-presenting cells. In certain species, the pineal gland also contains neurons and/or neuron-like peptidergic cells. The peptidergic cells might influence the pinealocyte by a paracrine secretion of peptides. With the purpose of studying the possible paracrine interac- tions between the cells like pinealocytes and astrocytes in the rat pineal gland, we standardized the methodology of cell separation allowing experimental work with one or two types of cells both in contact and physically isolated but sharing the culture medium. Several articles have been published on the use of pinealocytes cultured alone or co-cultured with astrocytes [2–5], and the Ralf Jockers and Erika Cecon (eds.), Melatonin: Methods and Protocols, Methods in Molecular Biology, vol. 2550, https://doi.org/10.1007/978-1-0716-2593-4_11, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022 85