title: Towards neuronal organoids: a method for long-term culturing of high-density hippocampal neurons.
abstract: One of the goals in neuroscience is to obtain tractable laboratory cultures that closely recapitulate in vivo systems while still providing ease of use in the lab. Because neurons can exist in the body over a lifetime, long-term culture systems are necessary so as to closely mimic the physiological conditions under laboratory culture conditions. Ideally, such a neuronal organoid culture would contain multiple cell types, be highly differentiated, and have a high density of interconnected cells. However, before these types of cultures can be created, certain problems associated with long-term neuronal culturing must be addressed. We sought to develop a new protocol which may further prolong the duration and integrity of E18 rat hippocampal cultures. We have developed a protocol that allows for culturing of E18 hippocampal neurons at high densities for more than 120 days. These cultured hippocampal neurons are (i) well differentiated with high numbers of synapses, (ii) anchored securely to their substrate, (iii) have high levels of functional connectivity, and (iv) form dense multi-layered cellular networks. We propose that our culture methodology is likely to be effective for multiple neuronal subtypes-particularly those that can be grown in Neurobasal/B27 media. This methodology presents new avenues for long-term functional studies in neurons.
authors: Department of Physiology and Membrane Biology, School of Medicine, University of California Davis, Davis, California, United States of America.
authors: George K
authors: GK
authors: Todd
authors: Casey A
authors: CA
authors: Boosalis
authors: Aaron A
authors: AA
authors: Burzycki
authors: Michael Q
authors: MQ
authors: Steinman
authors: Lynda D
authors: LD
authors: Hester
authors: Pete W
authors: PW
authors: Shuster
authors: Randen L
authors: RL
authors: Patterson
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002452:Cell Count; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D003470:Culture Media / Q000737:chemistry; D006624:Hippocampus / Q000166:cytology*; D042541:Intracellular Space / Q000378:metabolism; D009474:Neurons / Q000166:cytology*; D009940:Organoids / Q000166:cytology*; D051381:Rats; D013569:Synapses / Q000378:metabolism; D013997:Time Factors
chemical_list: D003470:Culture Media; D002118:Calcium
references: J Neurosci. 1998 Jul 15;18(14):5294-300
references: 9651212
references: J Neurosci. 2007 Jan 10;27(2):331-40
references: 17215394
references: Glia. 2004 Aug 15;47(3):226-32
references: 15252811
references: Proc Natl Acad Sci U S A. 2000 Jul 18;97(15):8196-7
references: 10899989
references: BMC Neurosci. 2011 May 10;12:38
references: 21569253
references: Anat Embryol (Berl). 1993 Jan;187(1):67-73
references: 8430901
references: Nat Neurosci. 2006 Jun;9(6):816-23
references: 16699507
references: Phys Biol. 2010 Oct 26;7(4):046004
references: 20978314
references: PLoS One. 2012;7(1):e29641
references: 22235318
references: J Histochem Cytochem. 2003 Jan;51(1):89-103
references: 12502758
references: Cell. 1999 Aug 20;98(4):487-99
references: 10481913
references: J Neurosci. 2002 Jan 1;22(1):183-92
references: 11756501
references: Mol Neurodegener. 2011 Dec 22;6:85
references: 22192775
references: J Comp Neurol. 1995 Mar 6;353(2):260-74
references: 7745135
references: FASEB J. 2003 Mar;17(3):341-8
references: 12631574
references: Trends Neurosci. 2003 Oct;26(10):531-5
references: 14522145
references: Brain Res. 1977 May 13;126(3):397-42
references: 861729
references: Neuron. 1997 Dec;19(6):1297-308
references: 9427252
references: Neuron. 2004 Feb 5;41(3):405-15
references: 14766179
references: Iran Biomed J. 2008 Apr;12(2):101-7
references: 18506216
references: J Neurosci. 1998 Oct 1;18(19):7709-16
references: 9742141
references: Science. 2001 Jan 26;291(5504):657-61
references: 11158678
references: Nat Rev Neurosci. 2001 Mar;2(3):185-93
references: 11256079
references: Eur J Neurosci. 1999 Feb;11(2):389-97
references: 10051739
references: Glia. 2012 Jul;60(7):1067-77
references: 22488940
references: Eur J Neurosci. 2011 Apr;33(8):1366-75
references: 21375603
references: J Biol Chem. 2000 Sep 15;275(37):28562-8
references: 10878007
references: Dokl Biol Sci. 2001 Sep-Oct;380:424-6
references: 12918393
references: Nat Methods. 2011 Dec 04;9(1):81-3
references: 22138822
references: J Neurosci. 2005 Sep 21;25(38):8680-5
references: 16177036
references: J Biol Chem. 1998 Nov 20;273(47):30855-8
references: 9812976
references: Trends Neurosci. 1999 May;22(5):208-15
references: 10322493
references: J Neurosci. 1999 Aug 15;19(16):6897-906
references: 10436047
references: Exp Gerontol. 2005 May;40(5):426-37
references: 15919595
references: Methods. 2004 Jun;33(2):95-103
references: 15121163
references: Anat Rec. 1963 Apr;145:573-91
references: 14012334