Embryonic motoneuron-skeletal muscle co-culture in a defined system.
This paper describes a significant biotechnological advancement by creating a minimalist serum-free defined system to co-culture rat mammalian nerve and muscle cells in order to form functional neuromuscular junctions. To date, all the known in vitro nerve and muscle co-culture models use serum containing media; and while functional neuromuscular junctions (NMJ) are described, they failed to detail or quantify the minimum factors needed to recreate the NMJ in vitro. In this work, we demonstrate the development of a defined motoneuron and muscle co-culture system resulting in the formation of NMJs including: 1) a new culture technique, 2) a novel serum-free medium formulation and 3) a synthetic self-assembled monolayer (SAM) substrate N-1 [3-(trimethoxysilyl) propyl] diethylenetriamine (DETA). We characterized the culture by morphology, immunocytochemistry, electrophysiology and videography. This model system provides a better understanding of the minimal growth factor and substrate interactions necessary for NMJ formation and provides a basic system that can be utilized for nerve-muscle tissue engineering, regenerative medicine and development of limb prosthetics.
Das
M
M
NanoScience Technology Center, 12424 Research Parkway, Suite 400, University of Central Florida, Orlando, FL 32826, USA.
Rumsey
J W
JW
Gregory
C A
CA
Bhargava
N
N
Kang
J-F
JF
Molnar
P
P
Riedel
L
L
Guo
X
X
Hickman
J J
JJ
D000818:Animals; D002038:Bungarotoxins / Q000378:metabolism; D002478:Cells, Cultured; D018920:Coculture Techniques; D017077:Culture Media, Conditioned / Q000378:metabolism*; D016895:Culture Media, Serum-Free / Q000378:metabolism; D004622:Embryo, Mammalian; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D008954:Models, Biological; D009046:Motor Neurons / Q000502:physiology*; D032446:Myoblasts / Q000502:physiology*; D018995:Myosin Heavy Chains / Q000378:metabolism; D016900:Neurofilament Proteins / Q000378:metabolism; D009469:Neuromuscular Junction / Q000187:drug effects / Q000502:physiology*; D018408:Patch-Clamp Techniques / Q000379:methods; D011073:Polyamines / Q000494:pharmacology; D051381:Rats; D016708:Synaptophysin / Q000378:metabolism
D002038:Bungarotoxins; D017077:Culture Media, Conditioned; D016895:Culture Media, Serum-Free; D016900:Neurofilament Proteins; D011073:Polyamines; D016708:Synaptophysin; C050934:neurofilament protein 150; C005391:diethylenetriamine; D018995:Myosin Heavy Chains