Isolation of Neural Stem Progenitor Cells from the Spinal Cord Periventricular Region of an Adult Rat

Abstract

Source: Mothe, A., et al., Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord. J. Vis. Exp. (2015).

This video presents a method for isolating neural stem/progenitor cells from the periventricular tissue of the spinal cord in adult rats. The technique includes incubating the tissue fragments in a solution containing proteolytic enzymes to disrupt the extracellular matrix and release the cells. Subsequently, the released cells are plated in a medium containing specific growth factors that enhance neural stem/progenitor proliferation and promote aggregation into three-dimensional neurospheres.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Preparation of Dissection Buffers and Culture Media

1. For the isolation of rat spinal cord, prepare 100 ml dissection buffer (1x PBS + 0.6% glucose + 2% penicillin-streptomycin) and refrigerate.
2. Prepare 100 ml serum-free medium (SFM) and warm at 37 °C. To prepare SFM, add 2 mM L-glutamine, 100 μg/ml penicillin-streptomycin, 2% B27, and 10% hormone mix to Neurobasal-A medium. To prepare hormone mix, make a 1:1 DMEM/F12 medium containing 0.6% glucose, 3 mM NaHCO₃

Results

Figure 1. Laminectomy of rat spinal cord and dissection of periventricular region. (A) At the exposed caudal end of the spinal cord, rongeurs are inserted extradurally into the lateral aspect of the spinal canal. (B) Small c.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1x PBS	Life Technologies	#10010023	Dissection buffer
1x HBSS	Life Technologies	#14175095	Dissection buffer
D-glucose	Sigma	# G-6152	Prepare 30% glucose stock solution for dissection buffer and hormone mix
Penicillin-Streptomycin	Life Technologies	#15140-148	Dissection buffer and culture medium
Neurobasal-A	Life Technologies	#10888-022	Culture medium
L-glutamine, 200 mM	Life Technologies	#25030-081	Culture medium
B27	Life Technologies	#12587010	Culture medium
DMEM	Life Technologies	#11885084	Hormone mix
F12	Life Technologies	#21700-075	Hormone mix
NaHCO3	Sigma	# S-5761	Prepare 7.5% NaHCO3 stock solution for hormone mix
HEPES	Sigma	#H9136	Prepare 1M HEPES stock solution for hormone mix
Insulin	Sigma	#I-5500	Hormone mix
Apo-transferrin	Sigma	#T-2252	Hormone mix
Putrescine	Sigma	# P7505	Hormone mix
Selenium	Sigma	#S-9133	Hormone mix
Progesterone	Sigma	#P-6149	Hormone mix
EGF, mouse	Sigma	#E4127	Prepare 100 μg/ml stocks in B27 and aliquot; EFH medium
bFGF, human recombinant	Sigma	#F0291	Prepare 100 μg/ml stocks in B27 and aliquot; EFH medium
Heparin, 10,000 U	Sigma	#H3149	Prepare 27.3 mg/ml stocks in hormone mix and aliquot; EFH medium
Papain dissociation kit	Worthington Biochemicals	#LK003150	Contains EBSS, papain, DNase, ovomucoid protease inhibitor with BSA
Sodium Pentobarbital	Bimeda – MTC Animal Health Inc	DIN 00141704
Tissue Forceps: Addisons	Fine Science Tools	#11006-12	Serrated standard tip; micro-tip also available
Fine Forceps: Dumont #4	Fine Science Tools	#11241-30
Microscissors	Fine Science Tools	#15024-10	Round-handled Vannas
Rongeurs	Bausch & Lomb	N1430
10 mm Petri dishes	Nunc	1501
T25 Culture flasks	Nunc	156367
6 well plates	Nunc	CA73520-906