Isolation and Expansion of Adult Canine Hippocampal Neural Precursors

Summary

The canine brain is a valuable model in which to study adult neurogenesis. Presented here are protocols for isolating and expanding adult canine hippocampal neural precursor cells from primary brain tissue.

Abstract

The rate of neurogenesis within the adult hippocampus has been shown to vary across mammalian species. The canine hippocampus, demonstrating a structural intermediacy between the rodent and human hippocampi, is therefore a valuable model in which to study adult neurogenesis. In vitro culture assays are an essential component of characterizing neurogenesis and adult neural precursor cells, allowing for precise control over the cellular environment. To date however, culture protocols for canine cells remain under-represented in the literature. Detailed here are systematic protocols for the isolation and culture of hippocampal neural precursor cells from the adult canine brain. We demonstrate the expansion of canine neural precursor cells as floating neurospheres and as an adherent monolayer culture, producing stable cell lines that are able to differentiation into mature neural cell types in vitro. Adult canine neural precursors are an underused resource that may provide a more faithful analogue for the study of human neural precursors and the cellular mechanisms of adult neurogenesis.

Introduction

Regional variations in the rate of neurogenesis have been observed along the dorsoventral axis of the rodent hippocampus^1,2. Furthermore, the rates of hippocampal neurogenesis also show distinct inter-species variation, with precursor cell turnover in the subgranular zone shown to be significantly lower in adult humans than in rodents^3-5. Inter-species differences in hippocampal structural anatomy may be relevant here, as it has been postulated that neural stem cell distribution along the murine ventricular neuraxis may be influenced by cephalic flexures during embryological development⁶. To date, the rodent brain remains the most popu....

Protocol

In accordance with New South Wales, Australia law, post mortem brain tissue was acquired from adult dogs euthanized for reasons unrelated to the study.

1. Preparation of Culture Medium

1. Prepare a 0.1% gelatin solution by adding 0.1 g of gelatin powder to 100 ml of distilled water and agitating at 37 °C until dissolved. Sterilize the solution by UV irradiation for 15 min. This media can then be stored at 4 °C for up to 1 month.
2. Prepare a 3:1 ratio of Dulbecco's Modified Eagle Medium (DMEM) and nutrient mixture F12 by adding 167 ml of F-12 to 500 ml of DMEM (4.5 g/L D-glucose). Add 6.7 ml of Penicillin/Streptomycin solu....

Results

Through the use of in vitro neural precursor assays, neurogenesis and neural precursor cell populations were characterized and compared across the dorsoventral axis of the adult canine hippocampus. Neural precursor cells derived from isolated hippocampal tissue formed floating neurospheres within 14 days of isolation, reaching a diameter of 100 µm by 28 days of culture. Neurospheres derived from dorsal and ventral isolates showed no difference in mean size, and following enzymati.......

Discussion

The protocols described here are optimized to maintain favorable culture conditions for maximizing cell viability. The speed and care taken during extraction, isolation and expansion is of critical importance. A critical step for establishing adherent monolayer expansion is the effective dissociation of the primary neurospheres. Following passage, insufficiently dissociated neurospheres may generate secondary floating neurospheres. During media change, these neurospheres may be removed, dissociated and reseeded for adher.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1,000 μl filtered pipette tip	Axygen	TF1000
150 mm Petri dish	BD Biosciences	351058
15 ml centrifuge tubes	Greiner Bio One	188271
200 μl filtered pipette tip	Axygen	TF200
24 well culture plate	Greiner Bio One	662160
35 mm tissue culture dish	BD Biosciences	353001
40 µm cell strainer	BD Biosciences	352340
6 well culture plate	BD Biosciences	351146
B-27 Supplement (50x) serum free	Life Technologies	17504044
Basic fibroblast growth factor (bFGF)	Life Technologies	13256029
Brain derived neurotrophic factor (BDNF)	Millipore	GF029
Collagen solution	Stem Cell Technologies	04902	Also available in the Neurocult NCFC Assay Kit from Stem Cell Technologies. Cat: 05740
DMEM (4.5 g/L, D-glucose) 500 ml	Life Technologies	11960044
DPBS	Life Technologies	14190250
Epidermal growth factor (EGF)	BD Biosciences	354001
F-12 nutrient mixture (Ham) (1x) Liquid	Life Technologies	31765035
Fetal bovine serum (FBS)	Life Technologies	16141079
Gelatin from Porcine Skin Type A	Sigma-Aldrich	G1890
L-alanyl-L-glutamine dipeptide (GlutaMAX)	Life Technologies	35050061
Heparin sodium salt from (porcine)	Sigma-Aldrich	H314950KU
Laminin (mouse)	Life Technologies	23017015
NCFC serum free medium (NeuroCult)	Stem Cell Technologies	5720	Also available in the Neurocult NCFC Assay Kit from Stem Cell Technologies. Cat: 05740
Proliferation NS-A (NeuroCult)	Stem Cell Technologies	05773	Also available in the Neurocult NCFC Assay Kit Cat: 05740, and NS-A Prolieration Kit (Rat) Cat: 05771  from Stem Cell Technologies.
NSC basal medium (Rat; NeuroCult)	Stem Cell Technologies	5770	Also available in the Neurocult NS-A Prolieration Kit (Rat) from Stem Cell Technologies. Cat: 05771
Penicillin/Streptomycin (5,000 U/ml)	Life Technologies	15070063
Povidone-iodine	Munipharma	Betadine
Trypan blue (0.4%)	Life Technologies	15250061
Trypsin EDTA	Life Technologies	25200056
Class II biological safety cabinet	ThermoFisher Scientific	Safe 2020 1.2
Brain knife (disposable)	Macroknife
Cell culture incubator	ThermoFisher Scientific	HERAcell 150i
Centrifuge	Hettich	Universal 320R
Dumont #5 Forceps	Dumont
Easypet Electric pipette	Eppindorf
Hemocytometer	Boeco	Bright-Line Improved Neubauer
Manual pipettes	Eppindorf research
Oscillating bone saw
Scalpel blades (No.21)	Paramount
Scissors	Delta
Water bath	Grant	JB Aqua 18 plus