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Establishing embryonic mouse neural stem cell culture using the neurosphere assay.

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Establishing embryonic mouse neural stem cell culture using the neurosphere assay.
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  See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/49768503 Establishing Embryonic Mouse Neural StemCell Culture Using the Neurosphere Assay   Article   in  Journal of Visualized Experiments · January 2011 DOI: 10.3791/2457 · Source: PubMed CITATIONS 25 READS 246 5 authors , including:Hassan AzariShiraz University of Medical Sciences 82   PUBLICATIONS   455   CITATIONS   SEE PROFILE Sharareh Sharif ifarUniversity of Florida 11   PUBLICATIONS   108   CITATIONS   SEE PROFILE Saeed AnsariUniversity of Florida 37   PUBLICATIONS   281   CITATIONS   SEE PROFILE Brent ReynoldsMcKnight Brain Institute 183   PUBLICATIONS   16,561   CITATIONS   SEE PROFILE All content following this page was uploaded by Saeed Ansari on 06 January 2017. The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the srcinal documentand are linked to publications on ResearchGate, letting you access and read them immediately.  VideoArticle Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay Hassan Azari 1,2 , Sharareh Sharififar  2 , Maryam Rahman 2 , Saeed Ansari 2 , BrentA. Reynolds 21 Department ofAnatomical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran 2 Department of Neurosurgery, The University of FloridaCorrespondence to: Hassan Azari at hassan.azari@neurosurgery.ufl.eduURL: http://www.jove.com/index/Details.stp?ID=2457DOI: 10.3791/2457 Citation: Azari H., Sharififar S., Rahman M., Ansari S., Reynolds B.A. (2011). Establishing Embryonic Mouse Neural Stem Cell Culture Using the Neurosphere Assay. JoVE. 47.http://www.jove.com/index/Details.stp?ID=2457, doi: 10.3791/2457  Abstract In mammalians, stem cells acts as a source of undifferentiated cells to maintain cell genesis and renewal in different tissues and organs duringthe life span of the animal. They can potentially replace cells that are lost in the aging process or in the process of injury and disease. Theexistence of neural stem cells (NSCs) was first described by Reynolds and Weiss (1992) in the adult mammalian central nervous system (CNS)using a novel serum‐ free culture system, the neurosphere assay (NSA). Using this assay, it is also feasible to isolate and expand NSCs fromdifferent regions of the embryonic CNS. These  in vitro  expanded NSCs are multipotent and can give rise to the three major cell types of the CNS.While the NSA seems relatively simple to perform, attention to the procedures demonstrated here is required in order to achieve reliable andconsistent results. This video practically demonstrates NSA to generate and expand NSCs from embryonic day 14-mouse brain tissue andprovides technical details so one can achieve reproducible neurosphere cultures. The procedure includes harvesting E14 mouse embryos, brainmicrodissection to harvest the ganglionic eminences, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, andfinally plating cells in NSA culture.After 5-7 days in culture, the resulting primary neurospheres are passaged to further expand the number of theNSCs for future experiments. Protocol 1. Basic Set Up Before Proceeding to Dissection:2. Harvesting E14 Mouse Brain and Micro-dissection: Page 1 of 4Journal of Visualized Experiments www.jove.comCopyright © 2011 Journal of Visualized Experiments1. Appropriate volume of complete NSC medium is prepared by mixing NeuroCult NSC Basal Medium and NeuroCult NSC ProliferationSupplements at a 9:1 ratio, respectively.2. The medium is warmed up in a 37°C water bath.3. Cold HEPES-buffered minimum essential medium (HEM) with high concentration of antibiotics (10%) is prepared for dissection and washingpurpose.Alternatively, NSC basal medium with antibiotics supplementation may also be used for this purpose.4. 25-30 mL of cold HEM containing antibiotics is dispensed into sterile 50 mL tubes for collection of the embryos.5. Several 10cm plastic Petri dishes are needed to hold the embryos and brains during dissection and also to hold dissected tissue.6. The surgical tools, needed to remove the embryos (large scissors, small pointed scissors, large forceps, small curved forceps) or for embryonic brain dissection (small forceps, curved fine forceps, 45° angled fine forceps, and small scissors) are sterilized using glass beadsterilizer at 250°C or other available autoclave methods.7. Dissection microscope is wiped with 70% alcohol and set up inside a laminar flow or PC2 hood.1. A time mated pregnant mouse is anesthetized on day 14 th of gestation according to one's institutional approved animal protocol.2. Cervical dislocation is performed to make sure the animal does not suffer pain and distress.3. The anesthetized mouse is laid on its back on an absorbent tissue paper, and the abdomen is rinsed with 70% ethanol to sterilize the area.4. The skin over the abdomen is grasped using a large forceps, and then the skin and the underlying fascia is cut with large scissors to exposethe abdominal cavity and the uterine horns.5. The uteri are removed with small forceps and scissors and are transferred into a 50‐ ml conical tube-containing cold HEM.6. The uterine tissues are transferred to the hood and then rinsed once or twice with enough volume of fresh sterile cold HEM to removepossible contaminants like blood and hair.7. The uterine tissues are then transferred to a 10cm Petri dish containing cold HEM.8. The uterine horns are opened using small curved forceps and scissors and the embryos are transferred to a new 10cm dish, which containscold HEM.9. The heads of the embryos are separated at the cervical spinal cord level and transferred to another Petri dish containing cold HEM.10. The Petri dish is transferred under a dissecting microscope to remove the brain from the skull.11. The heads are held with a fine curved forceps from the caudal side so as the dorsal side of the heads are facing upwards. Usingmicro-scissors, first a horizontal cut is made above the eyes and then continued in the midline from the forehead towards the back of thehead. Make sure to cut through the skin and the skull and not to damage the underlying brain.12. The brain is removed from the skull by pushing the edges of the cut section in a backward motion using the curved forceps. This procedure iscontinued until all brains are removed from the skulls.13. The brain is held steady using the curved forceps so that the dorsal side is facing upwards and then using microscissors a cut is performedthrough the cortex of each hemisphere extending from the olfactory bulbs to the back of the hemisphere to expose the ganglionic eminences.14. Using the curved forceps in one hand and the 45° angled forceps in the other, the cut flaps of cortices are spread and the ganglioniceminences are dissected out.  3. Passaging and Expansion of Embryonic NSCs:4. Representative Results: In primary embryonic NSC culture, the majority of the cells will become hypertrophic and attach to the tissue culture dishes upon plating. Whilethe majority of cells will either die or differentiate, after 2-3 days, proliferative cells make small clusters of cells that will detach from the substrate(Figure 1). Formation of large spheroidal aggregates in the first 48-hour of culture should not be mistaken for primary spheres.Aggregateformation mainly depends on the amounts of debris and non-dissociated tissue clumps in the culture. True neurospheres are phase bright andbecome more spherical as size increases (Figure 2). Small microspikes appears on the outer surface of viable and healthy spheres (Figure 3). After 5-7 days, the spheres must be round but not compacted; and should measure between 150 and 200 μm in diameter. If neurospheres areallowed to grow too large (after 9-10 days in culture), they might form aggregates or become dark in color because of cell death at the center of the spheres (see video). Large neurospheres might eventually begin to differentiate  in situ  (attaching to the substrate and migrating toward theperiphery). It is also difficult to dissociate large neurospheres and subculture them. Figure 1.  Primary E14 NSC culture 3 days after plating.Arrows show the proliferating clusters of NSCs. Original magnification; 20xPage 2 of 4Journal of Visualized Experiments www.jove.comCopyright © 2011 Journal of Visualized Experiments15. The dissected tissue is placed in a sterile 10cm Petri dish, and this procedure is repeated until all the brains have been micro-dissected.16. Tissue pieces are collected using 1 mL of NSC medium in a 1 mL pipette tip and transferred to a 15 mL centrifuge tube.17. Tissue is then dissociated thoroughly, but gently by pressing the pipette tip to the bottom of the tube and pipetting the suspension up anddown. This way the clumps are broken up into single cells. Pipetting is performed 3-4 times so that a milky like suspension is achieved. Then,the suspension is allowed to settle for 1-2 minutes so as the non-dissociated clumps precipitates.18. Almost the entire cell suspension is transferred to the other tube and then another 1 mL of NSC medium is added to the remaining clumpsand dissociated to single cell as described.19. The content of the tubes are pooled and centrifuged for 5 minutes at room temperature, at 700rpm (110g).20. The supernatant is vacuumed off down to the actual pellet, and the cells are resuspended in 1 mL of complete NSC medium.21. The medium is gently pipetted up and down to have a homogeneous single cell suspension.22. 10 μL of the cell suspension is mixed with 90 μL of trypan blue to perform a cell count.23. Finally, the cells are plated at the density of 2x10 5 cells/ mL in complete NSC medium supplemented with 20ng/ mL epidermal growth factor (EGF). 5 mL medium is used for T25, 20 mL for T75 and 40 mL for T175 flasks.24. Neurospheres are formed in 5-7 days when incubated at 37° C in a humidified incubator with 5% CO 2 . In 6-7 days, the spheres shouldmeasure between 150-200 microns and will be ready to subculture (passage).1. When the neurospheres are ready for subculture (150-200 μm in diameter), the medium with suspended spheres is removed from the flasks,placed in an appropriate size sterile tissue culture tube, and centrifuged at 700 rpm (110 g) for 5 min at room temperature.2. The supernatant is discarded and the spheres are resuspended in 1 mL of %0.05 trypsin-EDTA.3. The cell suspension is then incubated in a 37°C water bath for 2-3 min, then an equal volume of soybean trypsin inhibitor is used to stop thetrypsin activity.4. The cell suspension is gently pipetted up and down to ensure that the trypsin has been completely inactivated.5. The cell suspension is centrifuged at 700 rpm (110g) for 5 min. Then, the supernatant is removed and the cells are resuspended in 1 mL of complete NSC medium.6. 10 μL of the cell suspension is mixed with 90 μL of trypan blue to perform a cell count.7. The cells are plated at a concentration of 5x10 4 cells/ mL in complete NSC medium supplemented with 20ng/ mL EGF in an appropriate sizetissue culture flask. 5 mL medium is used for T25, 20 mL for T75 and 40 mL for T175 flasks.8. Secondary neurospheres are formed in 5-7 days when incubated at 37° C in a humidified incubator with 5% CO 2 .  Figure 2.  Primary E14 NSC culture 7 days after plating. Original magnification; 10x Figure 3.  Passage one E14 neurospheres 5 days after plating. Note the micro-spikes (arrows) at the periphery of the spheres. OriginalMagnification; 20x Disclosures No conflicts of interest declared. Discussion The neurosphere assay is the method of choice for the isolation and expansion of neural stem cells  1-5 because of its simplicity andreproducibility. This assay is an invaluable tool for large-scale generation of undifferentiated CNS precursor cells, which could be used for both  invitro  and  in vivo  studies. It should be emphasized that neurospheres could be generated from both the bona fide neural stem cells and morerestricted progenitors. Therefore, calculating the neurosphere forming frequency simply overestimates the number of bona fide NSCs in any givenneural cell population  6 . To estimate the frequency of bona fide NSCs, it is strongly recommended to use the Neural Colony Forming Cell Assay(N-CFCA), which has been developed for this purpose 7 .To have a consistent high quality neurosphere culture of E14 NSCs, we recommend:  Acknowledgements This work was supported by funding from the Overstreet Foundation. References Page 3 of 4Journal of Visualized Experiments www.jove.comCopyright © 2011 Journal of Visualized Experiments1. To prepare needed items before starting to harvest the embryos.2. To keep the embryos in cold HEM or basal NSC medium throughout dissection.3. To perform dissection as quickly as possible (within 1 h), as tissue becomes soft and sticky over time and may be difficult to dissect.4. Not to let the sphere grow too much. Usually they should be sub-cultured every 5-7 days depending on the size of spheres.5. To trypsinize the spheres at the size of 150-200 μm for 2-3 minute. Leaving trypsin for more than 3 minutes causes damage to the cells andthe sphere forming efficiency will decrease and the cells will tend to attach the culture dishes and differentiate.1. Reynolds, B.A. & Weiss, S., Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.Science 255 (5052), 1707-1710 (1992).  Page 4 of 4Journal of Visualized Experiments www.jove.comCopyright © 2011 Journal of Visualized Experiments2. Morshead, C.M.  et al. , Neural stem cells in the adult mammalian forebrain: a relatively quiescent subpopulation of subependymal cells.Neuron 13 (5), 1071-1082 (1994).3. Craig, C.G.  et al. ,  In vivo  growth factor expansion of endogenous subependymal neural precursor cell populations in the adult mouse brain. JNeurosci 16 (8), 2649-2658 (1996).4. Golmohammadi, M.G.  et al. , Comparative analysis of the frequency and distribution of stem and progenitor cells in the adult mouse brain.Stem Cells 26 (4), 979-987 (2008).5. Azari H, Rahman M, Sharififar S, Reynolds BA (2010). Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay. J Vis Exp. http://www.jove.com/index/details.stp?id=2393, doi: 10.3791/23936. Reynolds, B.A. & Rietze, R.L., Neural stem cells and neurospheres--re-evaluating the relationship. Nat Methods 2 (5), 333-336 (2005).7. Louis, S.A.  et al. , Enumeration of neural stem and progenitor cells in the neural colony-forming cell assay. Stem Cells 26 (4), 988-996 (2008). View publication statsView publication stats
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