Establishment of Long-Term Primary Cortical Neuronal Cultures From Neonatal Opossum Monodelphis domestica

Abstract

Primary dissociated neuronal cultures have become a standard model for studying central nervous system (CNS) development. Such cultures are predominantly prepared from the hippocampus or cortex of rodents (mice and rats), while other mammals are less used. Here, we describe the establishment and extensive characterization of the primary dissociated neuronal cultures derived from the cortex of the gray South American short-tailed opossums, Monodelphis domestica. Opossums are unique in their ability to fully regenerate their CNS after an injury during their early postnatal development. Thus, we used cortex of postnatal day (P) 3–5 opossum to establish long-surviving and nearly pure neuronal cultures, as well as mixed cultures composed of radial glia cells (RGCs) in which their neurogenic and gliogenic potential was confirmed. Both types of cultures can survive for more than 1 month in vitro. We also prepared neuronal cultures from the P16–18 opossum cortex, which were composed of astrocytes and microglia, in addition to neurons. The long-surviving opossum primary dissociated neuronal cultures represent a novel mammalian in vitro platform particularly useful to study CNS development and regeneration.

Label-Free Long-Term Methods for Live Cell Imaging of Neurons: New Opportunities

“…Spinal cord primary cultures [ 28 , 29 , 30 , 31 ] are less frequently used, but recently, more effective protocols with extensive cell characterization have become available [ 32 , 33 , 34 , 35 ]. Among mammalian species, rodents (mice and rats) are used predominantly, but primary cultures from other mammals such as opossums [ 36 , 37 ], pigs [ 38 ], sheep [ 39 ] and monkeys [ 40 ] have also been established. Inter-species variabilities can, for many aspects including adult neurogenesis, be a cause of misinterpretation in translation of the obtained knowledge to humans [ 2 , 12 , 41 ], thereby necessitating the development of a wider source of mammalian CNS cells.…”

Epigenetic clock and methylation studies in marsupials: opossums, Tasmanian devils, kangaroos, and wallabies

“…This is especially the case as gray short-tailed opossums, although marsupials, lack a pouch. Researchers have taken advantage of these traits to study development of the lung [ 7 ], neural crest [ 8 ], brain [ 9 – 11 ], limb [ 12 , 13 ], reproductive [ 14 , 15 ], and other systems. Because of their early developmental state at birth, newborn opossums are also capable of totally regenerating their spinal cord even after it has been completely severed, and therefore provide an ideal model for spinal cord regeneration [ 9 , 16 – 18 ].…”

“…Researchers have taken advantage of these traits to study development of the lung [ 7 ], neural crest [ 8 ], brain [ 9 – 11 ], limb [ 12 , 13 ], reproductive [ 14 , 15 ], and other systems. Because of their early developmental state at birth, newborn opossums are also capable of totally regenerating their spinal cord even after it has been completely severed, and therefore provide an ideal model for spinal cord regeneration [ 9 , 16 – 18 ]. Opossums are also one of the only mammals besides humans that develop UV-induced melanoma, making them a good model for melanoma research [ 19 – 21 ].…”

Epigenetic clock and methylation studies in gray short-tailed opossums

“…This is especially the case as gray short-tailed opossums, although marsupials, lack a pouch. Researchers have taken advantage of these traits to study development of the lung [7], neural crest [8], brain [9][10][11], limb [12,13], reproductive [14,15], and other systems. Because of their early developmental state at birth, newborn opossums are also capable of totally regenerating their spinal cord even after it has been completely severed, and therefore provide an ideal model for spinal cord regeneration [9,[16][17][18].…”

“…Researchers have taken advantage of these traits to study development of the lung [7], neural crest [8], brain [9][10][11], limb [12,13], reproductive [14,15], and other systems. Because of their early developmental state at birth, newborn opossums are also capable of totally regenerating their spinal cord even after it has been completely severed, and therefore provide an ideal model for spinal cord regeneration [9,[16][17][18]. Opossums are also one of the only mammals besides humans that develop UV-induced melanoma, making them a good model for melanoma research [19][20][21].…”