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Isolation, Culture and Differentiation of Adult Hippocampal Precursor Cells
成年海马前体细胞的分离,培养和分化   

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Abstract

There are two neurogenic niches in the adult mammalian brain: the subventricular zone of the lateral ventricle and the subgranular zone of the hippocampal dentate gyrus. Cells from these areas can be isolated and maintained in vitro, using two different culture systems to assess their potential regarding proliferation and differentiation in a reductionist model. While the neurosphere assay is primarily performed to directly study the proliferative and differentiation potential of cells in individual brains, the monolayer culture allows single cell analysis in a rather homogeneous cell population. Here, we describe the isolation, culturing methods and differentiation of neural precursor cells in both systems.

Keywords: Neuroscience(神经科学), Precursor cell(前体细胞), Neurosphere(神经球), Adherent monolayer(粘附单层), Differentiation(分化), Subventricular zone(室下区), Dentate gyrus(齿状回), Adult mouse(成年老鼠)

Background

In the mammalian brain, adult neural stem cells reside in two main neurogenic niches, the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG) and the lateral ventricle of the subventricular zone (SVZ), that allow the generation of new neurons in the adult brain. Neural precursor cells from the neurogenic niches can be isolated and cultured in vitro to model cellular processes, especially proliferation and differentiation. Two standard culture systems, the adherent monolayer culture (Palmer et al., 1995; Ray et al., 1995) and the neurosphere assay (Reynolds and Weiss, 1992 and 1996), both introduced in the 1990s, represent valuable tools to study neural progenitor cell biology in vitro.

Depending on the research question, each system has advantages and disadvantages that should be considered carefully before choosing one or the other culture method. In adherent monolayer cultures cells grow rather isolated and form more homogeneous cultures. Monolayers allow the direct investigation and monitoring of neural precursor cells at the single cell level. Characteristics like morphology, proliferation and differentiation under controlled conditions, can easily be analysed and visualised. However, compared to neurosphere cultures, cells cultured as monolayer represent a more reductionist model as the cells grow with fewer cell-to-cell contacts that are usually present in the niche.

Neurosphere cultures are free-floating aggregate cultures that are easy to obtain from adult tissue. Primary neurospheres are more heterogeneous and presumably represent a more niche-like environment. Neurospheres can be used to model the interaction of different cell types and allow relative comparisons of precursor cell number and potential, but does not allow absolute conclusions about stem cell numbers in vivo. Also, the sphere-forming capacity is not identical to ‘stemness’.

This protocol describes the detailed workflow of the generation and analysis of adult neural precursor cultures as neurospheres and monolayers from both neurogenic regions, the SVZ and the DG. The protocol represents an optimized version of our previously published protocols that have been successfully applied to many research projects within our group and by other groups (Babu et al., 2011; Walker and Kempermann, 2014; Ehret et al., 2016; Hörster et al., 2017).

Materials and Reagents

  1. Animals
    Mice: C57BL/6J (8 weeks old)
    Note: We recommend three to four mice for establishing a monolayer cell culture. For neurosphere assay experiments we recommend one mouse per 96-well plate.

  2. General materials and reagents
    1. Centrifugation tubes 15 ml and 50 ml
    2. Reaction tubes 1.5 ml
    3. Parafilm
    4. 70% ethanol
    5. Double distilled water (ddH2O)
    6. 1x phosphate-buffered saline (PBS)
    7. DMEM/F-12 without glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 21331020 )
    8. 4% paraformaldehyde (PFA) in 0.1 M phosphate buffer pH 7.4
      1. PFA (Merck, catalog number: 1040051000 )
      2. Sodium dihydrogen phosphate (Merck, catalog number: 1063421000 )
      3. Disodium phosphate dihydrate (Acros Organics, catalog number: 343810025 )
      4. Sodium hydroxide (NaOH) (Carl Roth, catalog number: 6771 )
    9. Growth media (see Recipes)
      1. Neurobasal® medium (Thermo Fisher Scientific, GibcoTM, catalog number: 21103049 )
      2. B-27® supplement (50x) (Thermo Fisher Scientific, GibcoTM, catalog number: 17504044 )
      3. Pen/Strep 100,000 U/ml (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
      4. GlutaMAXTM supplement (100x stock) (Thermo Fisher Scientific, GibcoTM, catalog number: 35050061 )

  3. Fire polished pipettes
    1. Glass Pasteur pipette (1 mm diameter)

  4. Coating
    1. Poly-D-Lysine hydrobromide (PDL) (Sigma-Aldrich, catalog number: P7280 )
    2. Laminin (Roche Diagnostics, catalog number: 11243217001 )

  5. Brain dissection
    Petri dishes (10 cm diameter)

  6. SVZ tissue dissociation
    1. Petri dishes (6 cm diameter)
    2. Scalpel (#10) (Fisher Scientific, catalog number: 11995756)
      Manufacturer: B. Braun Melsungen, catalog number: 5518059 .
    3. Falcon® 40 µm cell strainer (Corning, Falcon®, catalog number: 352340 )
    4. 0.05% trypsin-EDTA (Thermo Fisher Scientific, GibcoTM, catalog number: 25300054 )
    5. Trypsin inhibitor containing DNAse I (see Recipes)
      1. Trypsin inhibitor (Sigma-Aldrich, catalog number: T6522 )
      2. DNase I (Roche Diagnostics, catalog number: 10104159001 )

  7. DG tissue dissociation
    1. Petri dishes (6 cm diameter)
    2. Falcon® 40 µm cell strainer (Corning, Falcon®, catalog number: 352340 )
    3. Neural tissue dissociation kit (P) (Miltenyi Biotech, catalog number: 130-092-628 )
    4. Beta-mercaptoethanol (Sigma-Aldrich, catalog number: M7522 )
      Note: This product has been discontinued.
    5. Hank’s buffered salt solution (HBSS) (Thermo Fisher Scientific, GibcoTM, catalog number: 14175 )

  8. Monolayer culture
    1. Tissue culture flasks (T25 and T75)
    2. 24-well tissue culture plates (growth-enhance treated, gamma-sterilized, free of pyrogens, free of RNA/DNA, DNase, RNase)
    3. Coverglass slips 12 mm (Fisher Scientific, catalog number: 12-545-82 )
      Note: This product has been discontinued.
    4. Heparin (MP Biomedicals, catalog number: 0210193125 )
    5. Human EGF (PeproTech, catalog number: AF-100-15 )
    6. Human FGF2 (PeproTech, catalog number: 100-18B )
    7. Accutase solution (Sigma-Aldrich, catalog number: A6964 )
    8. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418 )
    9. Normal donkey serum (Jackson ImmunoResearch Laboratories, catalog number: 017-000-121 )
    10. Trypan blue solution, 0.4% (Thermo Fisher Scientific, GibcoTM, catalog number: 15250061 )
    11. BrdU (Sigma-Aldrich, catalog number: B5002 )
    12. Freezing mix (see Recipes)
    13. Antibody solution (see Recipes)

  9. Neurosphere assay
    1. Petri dishes (10 cm diameter)
    2. 96-well tissue culture plates (growth-enhance treated, gamma-sterilized, free of pyrogens, free of RNA/DNA, DNase, RNase)
    3. 24-well tissue culture plates (growth-enhance treated, gamma-sterilized, free of pyrogens, free of RNA/DNA, DNase, RNase)
    4. Coverglass slips 12 mm (Fisher Scientific, catalog number: 12-545-82 )
    5. Heparin (MP Biomedicals, catalog number: 0210193125 )
    6. Human EGF (Peprotech, catalog number: AF-100-15 )
    7. Human FGF2 (PeproTech, catalog number: 100-18B )
    8. Blocking solution (see Recipes)

  10. Staining reagents
    1. Microscope slides SuperFrost® (VWR, Thermo Scientific, catalog number: 631-0706 )
    2. Triton® X-100 (Carl Roth, catalog number: 3051 )
    3. 1 N HCl (from 37% stock solution) (Sigma-Aldrich, catalog number: 435570 )
      Note: This product has been discontinued.
    4. 0.9% NaCl
    5. Normal donkey serum (Jackson ImmunoResearch Laboratories, catalog number: 017-000-121 )
    6. Hoechst 33342 (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 62249 )
    7. Primary antibodies (see Table 1)
    8. Aqua-Poly/Mount (Polysciences, catalog number: 18606 )
    9. Borate buffer (see Recipes)
      1. Boric acid (Carl Roth, catalog number: 6943.1 )
      2. Sodium hydroxide (NaOH) (Carl Roth, catalog number: 6771 )

        Table 1. Primary antibodies for immunocytochemistry
        Antibody
        Host Clone Isotype
        Company
        Catalog number
        β-III-tubulin (β-tubulin)
        Mouse
        5G8
        IgG1
        Promega
        G7121
        5’-bromo-2’-deoxyuridine (BrdU)
        Rat
        BU1/75 (ICR1)
        IgG2a
        Bio-Rad Laboratories
        OBT0030
        Glial fibrillary acidic protein (GFAP)
        Rabbit
        polyclonal
        -
        Agilent Technologies
        Z0334
        Map2ab
        Mouse
        AP-20
        IgG1
        Sigma-Aldrich
        M1406
        Nestin
        Mouse
        25/NESTIN
        IgG1, κ
        BD
        611658
        Oligodendrocyte marker 4 (O4)
        Mouse
        O4
        IgM
        R&D Systems
        MAB1326
        Sox2
        Rabbit
        polyclonal
        -
        Merck
        AB5603

Equipment

  1. Bunsen burner
  2. Autoclave
  3. Dissection tools
    1. Scissors
    2. Small spatula (Fine Science Tools, catalog number: 10093-13 )
    3. Curved forceps (Fine Science Tools, model: Dumont #7, catalog number: 11271-30 )
    4. Angled forceps (Fine Science Tools, model: Dumont #5-45, catalog number: 11253-25 )
    5. 27 G ¾ needle (B. Braun Melsungen, catalog number: 4657705-02 )
  4. Vacuum pump
  5. Stereo microscope (Olympus, model: SZ61 )
  6. Inverted microscope (Olympus, model: CKX42 )
  7. Centrifuge with swing bucket rotor for 15 ml and 50 ml centrifuge tubes (Eppendorf, model: 5430 R )
  8. Incubator at 37 °C with 5% CO2
  9. Sterile laminar flow hood
  10. Hemocytometer (Neugebauer improved)
  11. Fluorescence microscope (ZEISS, model: Axio Imager.M2 )
  12. Freezing containers, Mr. FrostyTM (Thermo Fisher Scientific, Thermo ScientificTM, model: Mr. FrostyTM, catalog number: 5100-0001 )
  13. 37 °C water bath
  14. -80 °C freezer
  15. Pipettes
  16. Multistepper pipette (and tips)

Procedure

  1. General preparations
    Coating of cell culture vessels
    Monolayer cultures as well as the differentiation of neurospheres require poly-D-lysine (PDL)/Laminin coated surfaces for attachment.
    1. Add the appropriate amount of PDL (5 µg/ml in ddH2O) and assure that the surface or the coverslips are fully covered (for volumes see Table 2).
    2. Incubate for at least 4 h or overnight at room temperature.
    3. Remove the solution and wash the vessels three times with ddH2O.
    4. Let them dry properly until no residual water remains.
    5. Add Laminin (5 µg/ml in cold DMEM/F-12) and incubate for at least 4 h or overnight at 37 °C.
    6. Either use them directly or store them at -20 °C until required.

    Fire polished pipettes
    During the isolation process, fire-polished pipettes with either small or medium size bores are required.
    1. Rotate the glass Pasteur pipettes for about 3 sec over a hot blue flame of a Bunsen burner until the edges become rounded. Small Pasteur pipettes show an internal diameter of 0.3-0.4 mm while the medium size bore of the pipettes measures approximately 0.6-0.8 mm.
    2. Pipettes should be autoclaved prior to use.

  2. Brain dissection
    Note: Monolayer cultures require coated 24-well plates, which should be prepared at least two days prior to isolation.
    The protocol for neural precursor cell isolation is based on the protocols of Babu et al. (2011), Walker and Kempermann (2014) (which also includes a video of the dissection procedure) and Ehret et al. (2016). Neurosphere assays can be performed using one mouse per neurosphere experiment (per 96-well plate). Monolayer cultures can also be generated using a single mouse, however we recommend pooling 3-4 brains.
    1. Anesthetize 6-8 weeks old mice according to the appropriate institutional guidelines. Perform cervical dislocation.
    2. Spray the head with 70% ethanol to sterilize the area and to reduce the amount of fur adhering to the dissection tools and tissue.
    3. Decapitate the animal at the base of the brain stem using sharp scissors.
    4. Cut the skin sagittally along the midline approximately until a point between the eyes. Expose the skull.
    5. Place one blade of a small pair of scissors into each eye cavity to perform a coronal cut of the skull between the eyes. Afterwards, make two lateral cuts at the base of the skull, followed by a longitudinal cut along the sagittal suture.
    6. Expose the brain by peeling back the skull with a pair of forceps.
    7. Remove the brain from the skull by using a small spatula and place it into cold PBS.
    8. Transfer the brain into a 10 cm plastic Petri dish containing PBS (see Figure 1B).
    9. Place the Petri dish with the brains under a stereo microscope at low magnification and position the brain on its ventral surface. Remove the olfactory bulbs using fine angled forceps while holding the brain in position by the cerebellum.

    SVZ dissection
    1. Rotate the brain onto the dorsal aspect and make a coronal cut through the brain at the area of the optic chiasm using a scalpel (see Figure 1C).
    2. For microdissection of the SVZ (for more details see Azari et al. (2010) and Walker and Kempermann, 2014), have the rostral portion of the brain exposing the lateral ventricles of the cut coronal surface. Increase the magnification, remove and discard the septum using fine curved forceps.
    3. Dissect the SVZ by placing the tip of one blade of a pair of fine curved forceps in the lateral corner of the lateral ventricle immediately under the corpus callosum and the other roughly 1 mm into the tissue immediately adjacent to the ventricle. Press down the forceps towards the base of the dish and the ventral aspect of the ventricle to remove a small triangular piece of tissue (see Figure 1E). Place the dissected tissue into a small Petri dish on ice.

    DG dissection
    1. For microdissection of the DG (for more details see Hagihara et al. (2009) and Walker and Kempermann, 2014), place the caudal portion of the brain into the Petri dish and use a scalpel to cut along the longitudinal fissure (see Figure 1D).
    2. Carefully remove the cerebellum and the diencephalon using fine angled forceps.
    3. Refocus the microscope to visualize the borders around the DG. To remove the DG, use a 27 G ¾ needle and carefully slide along the border between the DG and Ammons’s horn.
    4. Free the DG from the surrounding tissue using fine angled forceps (see Figure 1F). Place the dissected tissue into a small Petri dish in 10 µl PBS on ice.

  3. SVZ tissue dissociation
    1. Preheat 1 ml of 0.05% trypsin-EDTA in a 15 ml centrifuge tube in a water bath at 37 °C for 10-15 min.
    2. Mince the dissected SVZ tissue with a scalpel in a 6 cm Petri dish for about 1 min (see Figure 1G).
    3. Transfer tissue pieces into the warm trypsin-EDTA and incubate for 7 min at 37 °C. Gently mix by inverting the tube occasionally.
    4. Stop the enzyme reaction by adding 1 ml of trypsin inhibitor containing DNaseI (see Recipes) and mix gently by flicking the tube.
    5. Centrifuge for 5 min at 300 x g.
    6. Gently resuspend the pellet in 1 ml of growth medium (see Recipes) by carefully pipetting up and down 7 to 10 times using a P1000 pipette (see Figure 1H).
      Note: To increased cell death, it is important to triturate the tissue very gently.
    7. Add up to 5 ml of growth medium and pass the cell suspension through a 40 µm cell strainer into a 50 ml centrifuge tube.
    8. Centrifuge for 5 min at 300 x g.
    9. Resuspend the cell suspension in 500 µl of growth medium.

  4. DG tissue dissociation
    Note: The average yield of cells that can be obtained from one animal varies depending on the age of the animal as well as on the strain. However, to provide a rough estimate, we usually gain about 100,000-200,000 cells/ml from one 8-week old C57BL/6J animal.
    To obtain a single-cell suspension from the isolated DG tissue, the Neural Tissue Dissociation Kit (P) containing papain is applied as follows:
    1. To prepare Enzyme Mix 1 add 1,900 µl Buffer X and 50 µl Enzyme P (both supplied in the kit) to a 15 ml centrifuge tube. To increase the stability of the enzymes and viability of the cells add beta-mercaptoethanol to Buffer X to a final concentration of 0.067 mM.
    2. Preheat Enzyme Mix 1 in a water bath at 37 °C for 10-15 min.
    3. Mince the dissected DG tissue with a scalpel in a 6 cm Petri dish for about 1 min (see Figure 1G).
    4. Transfer the tissue pieces into 1,950 µl of preheated Enzyme Mix 1.
    5. Incubate for 15 min at 37 °C and gently mix by inverting the tube every 3-5 min.
    6. Prepare Enzyme Mix 2 by adding 20 µl of Buffer Y to 10 µl of Enzyme A (both supplied in the kit).
    7. Add Enzyme Mix 2 to the tissue-enzyme mix.
    8. Dissociate the tissue mechanically by gently pipetting up and down 10 x with a fire-polished Pasteur pipette with the medium bore.
      Note: To avoid increased cell death, it is important to triturate the tissue very gently.
    9. Incubate for 10 min at 37 °C and gently mix by inverting the tube every 3-5 min.
    10. Further dissociate the tissue mechanically using a small bore, fire-polished Pasteur pipette by gently pipetting up and down 10 x (see Figure 1I).
    11. To wash the cell suspension, add Hank’s buffered salt solution up to 10 ml.
    12. Centrifuge at 300 x g for 5 min.
    13. Resuspend the pellet in 500 µl of growth medium and apply to 40 µm cell strainer placed in a 50 ml centrifuge tube.


      Figure 1. Isolation of neural precursor cells from the SVZ and the DG. A. Required tools; B. Isolated brain; C. Coronal cut through the brain at the area of the optic chiasm using a scalpel; D. Cut along the longitudinal fissure; E. Dissected SVZ tissue; F. Dissected DG; G. Mincing of the dissected tissue using a scalpel; H. Resuspended SVZ tissue; I. DG tissue after enzymatic incubation.

  5. Monolayer culture
    Monolayer cultures are established from primary cells, which are then cultured as adherent cells over several passages under proliferation conditions (Figure 2A). This leads to a rather homogeneous culture that can be used to perform single cell analyses.


    Figure 2. Neural precursor cells from the adult mouse brain can be cultured as adherent monolayer cultures (A) or as neurospheres (B). Scale bars = 50 µm.

    Culturing
    1. Add 20 ng/ml FGF2, 20 ng/ml EGF and 2 µg/ml Heparin to the single cell suspension in growth medium obtained from the tissue dissociation.
    2. Seed the cell suspension into a coated well of a 24-well plate.
    3. 24 h after seeding, remove the growth medium and exchange it with fresh growth medium with growth factors (20 ng/ml FGF2, 20 ng/ml EGF and 2 µg/ml Heparin).
      Note: This step can be skipped if the cells don’t seem to be sufficiently attached.
    4. 24 h later, wash the cells with prewarmed PBS and add fresh growth medium with growth factors (20 ng/ml FGF2, 20 ng/ml EGF and 2 µg/ml Heparin).
    5. Every other day, exchange 50% of the old growth medium with fresh growth medium containing 100% of the growth factors to counteract the growth factor consumption in the residual medium (e.g., for a T25 flask containing 5 ml medium, remove 2.5 ml and add 2.5 ml fresh medium containing 40 ng/ml EGF and FGF2 and 4 µg/ml Heparin).
    6. Once cells reach 70-80% confluency, remove the medium and wash once with PBS.
      Note: This can take up to two weeks.
    7. Add 150 µl of Accutase and incubate at 37 °C for 3 min.
    8. Tap the plate strongly onto its surface as well as from the side to agitate the attached cells.
    9. Check under the microscope if the cells are detached. If not, prolong incubation time (max 10 min).
    10. Add 2 ml of growth medium to the cell suspension and spin at 300 x g for 3 min to pellet the cells.
    11. Remove the supernatant and resuspend the cells in 1 ml of growth medium.
    12. Take out 10 µl of cell suspension and mix it with 10 µl of trypan blue.
    13. Count the cells with a hemocytometer.
    14. Seed the cells in a density of min. 1 x 104 c/cm2 in growth medium with growth factors (20 ng/ml FGF2 and 20 ng/ml EGF; no more Heparin) into a new vessel.

    Further passaging (passage only once cells have reached 70-80% confluency)
    1. Remove the medium and wash once with PBS.
    2. Add Accutase and incubate at 37 °C for 3 min.
    3. Tap the plate strongly onto its surface as well as from the side to agitate the attached cells.
    4. Check under the microscope if the cells are detached. If not, prolong incubation time (max 10 min).
    5. Collect the cells in 4.5 ml of growth medium (volume for a T25 flask) and spin at 300 x g for 3 min to pellet the cells.
    6. Add 10 µl of trypan blue to 10 µl of cell suspension and count cells with a hemocytometer.
    7. Seed the cells in a density of min. 1 x 104 c/cm2 (live cells) in growth medium containing growth factors (20 ng/ml FGF2 and 20 ng/ml EGF) into a new vessel.

    Freezing
    1. Prepare freezing mix (see Recipes) and store at 4 °C.
    2. Remove growth medium from flask/plate and wash with 5 ml of PBS.
    3. Add Accutase to the flask/plate and incubate for 3 min at 37 °C, check if all the cells are detached. If not, prolong incubation time (max 10 min).
    4. Collect cells in 4.5 ml of growth medium (volume for a T25 flask) and spin at 300 x g for 3 min to pellet the cells.
    5. Remove the supernatant.
    6. Resuspend cells in 1 ml of growth medium.
    7. Add 10 µl of trypan blue to 10 µl of cell suspension.
    8. Count the cells and adjust the cell density to 2 x 106 c/ml (live cells) with growth medium.
    9. Add 0.5 ml of freezing mix to each freezing vial.
    10. Add 0.5 ml of cell suspension to each freezing vial, mix and put into a freezing container.
    11. Place the freezing container directly in the -80 °C freezer.
    12. After 12 h, vials can be transferred into liquid nitrogen.

    Cell seeding and analysis of cell characteristics under proliferation conditions (Figure 3)
    Note: To analyse cell characteristics under proliferation conditions, cells can either be fixed directly 48 h after seeding or labeled with BrdU.


    Figure 3. Monolayer cultures under proliferation conditions. A. Monolayer cultures under proliferation conditions express cellular markers for progenitor cells of the nervous system such as Nestin (green) and Sox2 (magenta). B. Progenitor cells can be labeled to assess their proliferation capacity by examining the fraction of cells undergoing S-phase (BrdU label, green) relative to the overall number of cells (Hoechst 33342, blue). Scale bars = 50 µm.

    1. Thaw PDL/Laminin-coated plates (24-well plates with coverslips) for about 15 min at 37 °C.
    2. Remove the Laminin from the 24-well plates.
    3. Plate 20,000 cells/well onto coated coverslips in 24-well plate wells under proliferation conditions (add 20 ng/ml EGF and 20 ng/ml FGF2 to the medium)
    4. Incubate for 48 h.
    5. Add BrdU (final concentration 10 µM) to each well and incubate for 2 h at 37 °C.
      Note: Skip this step if no BrdU labeling is required and continue with step 6.
    6. Remove the medium, add 4% PFA in 0.1 M phosphate buffer to each well and incubate for 20 min at room temperature.
    7. Wash twice with PBS and add 1 ml of fresh PBS into the wells.
    8. Store at 4 °C until staining is performed. To avoid evaporation, seal the plate with Parafilm.

    Cell seeding and differentiation (Figure 4A)
    1. Thaw PDL/Laminin-coated plates (24-well plates with coverslips) for about 15 min at 37 °C.
    2. Remove the Laminin from the 24-well plates and wash the plate with PBS.
    3. Plate 20,000 cells/well onto coated coverslips in 24-well plate wells under proliferation conditions (add 20 ng/ml EGF and 20 ng/ml FGF2 to the medium)
    4. Incubate for 48 h.
    5. Remove growth medium and add fresh growth medium containing 5 ng/ml FGF2.
    6. After another 48 h, remove growth medium and add fresh growth medium without growth factors.
    7. Let the cells differentiate for 3-5 days.

    Fixation
    1. Remove the growth medium.
    2. Add 300 µl of 4% PFA in 0.1 M phosphate buffer to each well and incubate for 20 min at room temperature.
    3. Wash twice with PBS and add 1 ml of fresh PBS into the wells.
    4. Store at 4 °C until staining is performed. To avoid evaporation, seal the plate with Parafilm.

    Staining
    Note: Steps 1-4 are required for BrdU stainings only, for other stainings start directly with step 5.
    1. Wash 2 x with 0.9% NaCl.
    2. Incubate for 30 min with 1 N HCl at 37 °C.
    3. Wash 1 x with Borate buffer (see Recipes).
    4. Rinse 3 x 10 min with PBS.
    5. Permeabilize with 0.1% Triton X-100 in PBS for 10 min.
    6. Block with blocking solution at room temperature for 1 h.
    7. Incubate with primary antibodies (see Table 1) in antibody solution for 2 h at room temperature otherwise overnight at 4 °C (300 µl/well).
    8. Wash 3 x with PBS.
    9. Incubate with appropriate secondary antibodies diluted in antibody solution (see Recipes) for 1 h at room temperature (300 µl/well) in the dark.
    10. Wash 10 min with PBS.
    11. Incubate with Hoechst 33342 (1:4,000 in PBS) for 10 min at room temperature in the dark.
    12. Wash 1 x with PBS.
    13. Dip-wash in ddH2O.
    14. Dry coverslip by gently tapping on tissue paper and mount with fluorescence mounting medium.
    15. Dry overnight in darkness.
    16. Store at 4 °C.
    17. Image using a fluorescence microscope.

  6. Neurosphere assay
    Neurosphere cultures are easy to generate and allow straightforward data analysis with regards to precursor cell numbers and potential in brain tissue derived from individual mice (Figure 2B).
    Culture
    Note: Neurospheres are cultured in growth medium that additionally contains 2 µg/ml Heparin.
    1. Dilute the single cell suspension obtained from one mouse in 20 ml of neurosphere growth medium containing 20 ng/ml FGF2, 20 ng/ml EGF and 2 µg/ml Heparin.
    2. Plate 200 µl per well across a 96-well plate using a 10 ml multistepper pipette.
    3. Incubate SVZ-derived neurosphere cultures for 7 days and DG-derived neurosphere cultures for 12 days at 37 °C.
      Note: Extended incubation times can lead to overgrowth and may result in spontaneous attachment, differentiation or cell death in the core of the neurospheres.
    4. Count and size neurospheres using an inverted light microscope.

    Differentiation (Figures 4B and 4C)
    Note: For one experiment, we recommend seeding at least 4 coverslips with randomly sized neurospheres per 96-well plate.
    1. Thaw PDL/Laminin-coated plates (24-well plates with coverslips) for about 15 min at 37 °C.
    2. Remove the Laminin from the 24-well plates and wash the plate with PBS.
    3. Add 1 ml of growth medium to each well.
    4. Collect the medium containing the neurospheres from all wells of the 96-well plate into a 10 cm Petri dish.
    5. Using a stereo microscope collect the neurospheres from the Petri dish with a P100 pipette (final volume of 75 µl) and transfer them onto the coverslips in the 24-well plates. Per coverslip we recommend to seed about 15 neurospheres.
    6. Differentiate the neurospheres for 7 days in the incubator at 37 °C. The cells will spread out over the coverslip and become adherent.


      Figure 4. Monolayer cultures as well as neurospheres can be differentiated. A. Differentiated cells of a monolayer culture with GFAP-positive astrocytes (green) and Map2ab-positive neurons (magenta). B. Differentiated neurosphere culture with β-III-tubulin-positive neurons (magenta) and GFAP-positive astrocytes (green). C. Differentiated neurosphere culture with O4-positive Oligodendrocytes (red). Scale bars = 50 µm.

    Fixation
    1. Remove the medium and wash 2 x with PBS to remove dead cells and debris.
    2. Add 300 µl of 4% PFA in 0.1 M phosphate buffer to each well and incubate for 20 min at room temperature.
    3. Remove PFA and wash twice with PBS.
    4. Store in 1 ml PBS at 4 °C. To avoid evaporation, seal the plate with Parafilm.

    Staining
    Note: For staining with the recommended O4 antibody, do not add Triton to the blocking and antibody solutions and use an appropriate IgM secondary antibody.
    1. Wash the coverslips containing the differentiated neurospheres 2 x with PBS.
    2. Incubate the coverslips in blocking solution (see Recipes) containing 0.2% Triton X-100 for 30 min at room temperature.
    3. Incubate with primary antibodies against the neuronal and astrocytic markers or the oligodendrocyte marker O4 (see Table 1) in antibody solution containing 0.2% Triton X-100 for 1 h at room temperature (300 µl/well).
    4. Wash 4 x with PBS.
    5. Incubate for 30 min with appropriate secondary antibodies in antibody solution containing 0.2% Triton X-100 at room temperature in the dark (300 µl/well).
    6. Wash 2 x with PBS.
    7. Incubate with Hoechst 33342 (1:4,000 in PBS) for 10 min at room temperature in the dark.
    8. Wash 2 x with PBS.
    9. Dip-wash in ddH2O.
    10. Mount the coverslips onto microscope slides with fluorescence mounting medium and air dry in the dark overnight.
    11. Store at 4 °C.
    12. Image using a fluorescence microscope.

  7. Cell culture volumes and densities (Table 2)

    Table 2. Cell culture volumes and densities

Data analysis

  1. In the neurosphere assay, the size and number of neurospheres can be determined and used as an indirect measure of the proliferation potential of precursor cells, for example under the influence of different external stimuli or treatments, or between differing genetic backgrounds. The neurospheres further allow the analysis of the differentiation potential of the sphere-forming cells. This can be quantified as the percentage of Map2ab/β-III-tubulin and GFAP positive cells of the total number of cells determined by Hoechst 3342 (DAPI) staining. The neurosphere assay does not allow conclusions about absolute stem cell numbers.
  2. In addition, to biological replications, we advise to perform neurosphere assay experiments at least five times as technical replicates since there might be some day to day differences and we always strongly advise to perform control and treatment experiments at the same time to minimize the possibility of a batch effect.
  3. Data analysis is performed using a Student’s t-test or an ANOVA in combination with the appropriate post-hoc test, depending on the specific experimental setup.
  4. Monolayer culture-based experiments allow the assessment of characteristics on a single cell level. BrdU data serve as an indirect measure of the proliferation potential of the cells by evaluating the number of cells that went through S-Phase while BrdU was present. For the differentiation experiments the number of Map2ab/β-III-tubulin positive cells and GFAP positive cells are analyzed as the percentage of the total number of cells determined by Hochest 33342 (DAPI) staining. We advise to perform both stainings simultaneously on the same coverslip for comparable results. For one differentiation experiment, we analyze four coverslips and take at least five images of each coverslip on random positions to count the cells.
  5. Note that freezing/thawing cycles and passaging events may influence the cells and the resulting data. It is therefore crucial to perform experiments in the same passage and with cells that have been treated similarly, to obtain reproducible data. We recommend to perform at least five technical replicates. Data analysis is performed using a Student’s t-test or an ANOVA in combination with the appropriate post-hoc test, depending on the experimental setup.

Recipes

  1. Trypsin inhibitor containing DNase I
    0.125 mg/ml trypsin inhibitor
    0.01 mg/ml DNase I
    in DMEM/F-12 without glutamine
  2. Growth medium
    Neural basal medium
    0.5% B-27® supplement (50x)
    0.25% Pen/Strep 100,000 U/ml
    0.25% GlutaMAXTM (100x stock)
  3. Freezing medium
    Growth medium
    20% DMSO
  4. Borate buffer
    Boric acid
    ddH2O
    10 N NaOH to adjust pH to 8.5
  5. Antibody solution
    1x PBS
    3% normal donkey serum
  6. Blocking solution
    1x PBS
    10% normal donkey serum

Acknowledgments

The authors declare no competing financial interests. This work was partly funded by the Deutsche Forschungsgemeinschaft SFB655 and the Bundesministerium für Bildung und Forschung. Images were acquired and processed using equipment of the Imaging Platform at the DZNE Dresden. We thank Dr. Fanny Ehret for her helpful comments on the manuscript. The protocols described here represent a variation and further development of protocols described in Babu et al. (2011), Walker and Kempermann (2014) and Ehret et al. (2016).

References

  1. Azari, H., Rahman, M., Sharififar, S. and Reynolds, B. A. (2010). Isolation and expansion of the adult mouse neural stem cells using the neurosphere assay. J Vis Exp (45).
  2. Babu, H., Claasen, J. H., Kannan, S., Runker, A. E., Palmer, T. and Kempermann, G. (2011). A protocol for isolation and enriched monolayer cultivation of neural precursor cells from mouse dentate gyrus. Front Neurosci 5: 89.
  3. Ehret, F., Vogler, S. and Kempermann, G. (2016). Neurosphere co-culture assay. Bio Protoc e1883.
  4. Hagihara, H., Toyama, K., Yamasaki, N. and Miyakawa, T. (2009). Dissection of hippocampal dentate gyrus from adult mouse. J Vis Exp (33).
  5. Hörster, H., Garthe, A., Walker, T. L., Ichwan, M., Steiner, B., Khan, M. A., Lie, D. C., Nicola, Z., Ramirez-Rodriguez, G. and Kempermann, G. (2017). p27kip1 is required for functionally relevant adult hippocampal neurogenesis in mice. Stem Cells 35(3):787-799.
  6. Palmer, T. D., Ray, J. and Gage, F. H. (1995). FGF-2 responsive neuronal progenitors reside in proliferative and quiescent regions of the adult rodent brain. Mol Cell Neurosci (5):474-86.
  7. Ray, J., Raymond, H. K. and Gage, F. H. (1995). Generation and culturing of precursor cells and neuroblasts from embryonic and adult central nervous system. Methods Enzymol 254:20-37.
  8. Reynolds, B. A. and Weiss, S. (1992). Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 255(5052):1707-10.
  9. Reynolds, B. A. and Weiss, S. (1996). Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic CNS precursor is a stem cell. Dev Biol 175(1):1-13.
  10. Walker, T. L. and Kempermann, G. (2014). One mouse, two cultures: isolation and culture of adult neural stem cells from the two neurogenic zones of individual mice. J Vis Exp (84): e51225.

简介

成年哺乳动物脑中有两个神经生态位:侧脑室下脑室区和海马齿状回颗粒下区。 来自这些区域的细胞可以在体外分离和维持,使用两种不同的培养系统评估它们在还原模型中的增殖和分化的潜力。 虽然神经球测定主要是为了直接研究个体脑中细胞的增殖和分化潜能,单层培养允许在相当均匀的细胞群中进行单细胞分析。 在这里,我们描述了两个系统中的神经前体细胞的分离,培养方法和分化。

【背景】在哺乳动物脑中,成人神经干细胞存在于两个主要神经生态位中,即海马齿状回(DG)的下颗粒区(SGZ)和室下区(SVZ)的侧脑室,其允许新生神经元成人的大脑。来自神经生态位的神经前体细胞可以在体外分离和培养以模拟细胞过程,尤其是增殖和分化。两种标准培养系统,贴壁单层培养(Palmer等人,1995; Ray等人,1995)和神经球测定(Reynolds和Weiss,1992和1996 )在20世纪90年代被引入,代表了在体外研究神经祖细胞生物学的有价值的工具。

根据研究问题,每个系统都有其优点和缺点,在选择其中一种或另一种培养方法之前应该仔细考虑。在贴壁单层培养中,细胞生长相当孤立,形成更均匀的培养物。单层允许直接调查和监测单细胞水平的神经前体细胞。受控条件下的形态,增殖和分化等特征可以很容易地分析和可视化。然而,与神经球培养物相比,以单层培养的细胞代表更复杂的模型,因为细胞通常以更少的通常存在于细胞壁中的细胞 - 细胞接触来生长。

神经球培养物是易于从成人组织中获得的自由漂浮的聚集培养物。主要的神经球是更多的异构,可能代表了一个更利基的环境。神经球可以用来模拟不同细胞类型的相互作用,并允许前体细胞数量和潜力的相对比较,但是不能对体内干细胞数量做出绝对的结论。此外,球形成能力与“干性”不相同。

该协议描述了成人神经前体培养物的生成和分析的详细工作流程,其为来自神经原性区域SVZ和DG的神经球和单层细胞。该协议代表了我们以前发表的协议的优化版本,已经成功地应用于我们小组和其他小组的许多研究项目(Babu et al。,2011; Walker和Kempermann,2014; Ehret 等,2016;Hörster等,,2017)。

关键字:神经科学, 前体细胞, 神经球, 粘附单层, 分化, 室下区, 齿状回, 成年老鼠

材料和试剂

  1. 动物
    小鼠:C57BL / 6J(8周龄)
    注:我们建议三到四只小鼠建立单层细胞培养。对于神经球分析实验,我们推荐每个96孔板使用一只小鼠。

  2. 一般材料和试剂
    1. 离心管15毫升和50毫升
    2. 反应管1.5毫升
    3. Parafilm
    4. 70%乙醇
    5. 双蒸水(ddH2O)
    6. 1x磷酸盐缓冲盐水(PBS)
    7. 不含谷氨酰胺的DMEM / F-12(Thermo Fisher Scientific,Gibco TM,目录号:21331020)
    8. 4%多聚甲醛(PFA)在0.1M磷酸盐缓冲液pH7.4中
      1. PFA(Merck,目录号:1040051000)
      2. 磷酸二氢钠(Merck,目录号:1063421000)
      3. 磷酸氢二钠二水合物(Acros Organics,目录号:343810025)
      4. 氢氧化钠(NaOH)(Carl Roth,目录号:6771)
    9. 生长介质(见食谱)
      1. Neurobasal培养基(Thermo Fisher Scientific,Gibco TM,目录号:21103049)
      2. B-27补充物(50x)(Thermo Fisher Scientific,Gibco TM,目录号:17504044)
      3. Pen / Strep 100,000U / ml(Thermo Fisher Scientific,Gibco TM,产品目录号:15140122)
      4. GlutaMAX TM补充物(100×储备液)(Thermo Fisher Scientific,Gibco TM,目录号:35050061)

  3. 火抛光移液器
    1. 玻璃巴斯德吸管(1毫米直径)

  4. 涂层
    1. 聚-D-赖氨酸氢溴酸盐(PDL)(Sigma-Aldrich,目录号:P7280)
    2. 层粘连蛋白(Roche Diagnostics,目录号:11243217001)

  5. 大脑解剖
    培养皿(直径10厘米)

  6. SVZ组织解离
    1. 培养皿(直径6厘米)
    2. 手术刀(#10)(Fisher Scientific,目录号:11995756)
      制造商:B.BraunMelsungen,目录号:5518059。
    3. Falcon40μm细胞过滤器(Corning,Falcon ,产品目录号:352340)
    4. 0.05%胰蛋白酶-EDTA(Thermo Fisher Scientific,Gibco TM,目录号:25300054)。
    5. 含有DNAse I的胰蛋白酶抑制剂(见食谱)
      1. 胰蛋白酶抑制剂(Sigma-Aldrich,目录号:T6522)
      2. DNA酶I(Roche Diagnostics,目录号:10104159001)

  7. DG组织解离
    1. 培养皿(直径6厘米)
    2. Falcon40μm细胞过滤器(Corning,Falcon ,产品目录号:352340)
    3. 神经组织解离试剂盒(P)(Miltenyi Biotech,目录号:130-092-628)
    4. β-巯基乙醇(Sigma-Aldrich,目录号:M7522)
      注:此产品已停产。
    5. Hank's缓冲盐溶液(HBSS)(Thermo Fisher Scientific,Gibco TM,目录号:14175)

  8. 单层文化
    1. 组织培养瓶(T25和T75)
    2. 24孔组织培养板(生长增强处理,γ消毒,不含热原,不含RNA / DNA,DNA酶,核糖核酸酶)
    3. 盖玻片滑动12毫米(Fisher Scientific,目录号:12-545-82)
      注:此产品已停产。
    4. 肝素(MP Biomedicals,目录号:0210193125)
    5. 人类EGF(PeproTech,产品目录号:AF-100-15)
    6. 人类FGF2(PeproTech,目录号:100-18B)
    7. Accutase溶液(Sigma-Aldrich,目录号:A6964)
    8. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D8418)
    9. 正常的驴血清(Jackson ImmunoResearch Laboratories,目录号:017-000-121)
    10. 台盼蓝溶液,0.4%(Thermo Fisher Scientific,Gibco TM,产品目录号:15250061)
    11. BrdU(Sigma-Aldrich,目录号:B5002)
    12. 冷冻混合(见食谱)
    13. 抗体溶液(见食谱)

  9. 神经球测定
    1. 培养皿(直径10厘米)
    2. 96孔组织培养板(生长增强处理,γ消毒,不含热原,不含RNA / DNA,DNA酶,核糖核酸酶)
    3. 24孔组织培养板(生长增强处理,γ消毒,不含热原,不含RNA / DNA,DNA酶,核糖核酸酶)
    4. 盖玻片滑动12毫米(Fisher Scientific,目录号:12-545-82)
    5. 肝素(MP Biomedicals,目录号:0210193125)
    6. 人EGF(Peprotech,目录号:AF-100-15)
    7. 人类FGF2(PeproTech,目录号:100-18B)
    8. 阻止解决方案(见食谱)

  10. 染色剂
    1. 显微镜载玻片SuperFrost®(VWR,Thermo Scientific,目录号:631-0706)
    2. Triton X-100(Carl Roth,目录号:3051)
    3. 1N HCl(来自37%储备液)(Sigma-Aldrich,目录号:435570)
      注:此产品已停产。
    4. 0.9%NaCl
    5. 正常的驴血清(Jackson ImmunoResearch Laboratories,目录号:017-000-121)
    6. Hoechst 33342(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:62249)
    7. 一抗(见表1)
    8. Aqua-Poly / Mount(Polysciences,目录号:18606)
    9. 硼酸盐缓冲液(见食谱)
      1. 硼酸(Carl Roth,目录号:6943.1)
      2. 氢氧化钠(NaOH)(Carl Roth,目录号:6771)

        表1.用于免疫细胞化学的一级抗体
        Antibody
        Host Clone Isotype
        Company
        Catalog number
        β-III-tubulin (β-tubulin)
        Mouse
        5G8
        IgG1
        Promega
        G7121
        5’-bromo-2’-deoxyuridine (BrdU)
        Rat
        BU1/75 (ICR1)
        IgG2a
        Bio-Rad Laboratories
        OBT0030
        Glial fibrillary acidic protein (GFAP)
        Rabbit
        polyclonal
        -
        Agilent Technologies
        Z0334
        Map2ab
        Mouse
        AP-20
        IgG1
        Sigma-Aldrich
        M1406
        Nestin
        Mouse
        25/NESTIN
        IgG1, κ
        BD
        611658
        Oligodendrocyte marker 4 (O4)
        Mouse
        O4
        IgM
        R&D Systems
        MAB1326
        Sox2
        Rabbit
        polyclonal
        -
        Merck
        AB5603

设备

  1. 本生燃烧器
  2. 高压灭菌器
  3. 解剖工具
    1. 剪刀
    2. 小铲子(精细科学工具,目录号:10093-13)
    3. 弧形镊子(Fine Science Tools,型号:Dumont#7,产品目录号:11271-30)
    4. 有角度的镊子(Fine Science Tools,型号:Dumont#5-45,目录号:11253-25)
    5. (B.BraunMelsungen,目录号:4657705-02)
  4. 真空泵
  5. 立体显微镜(奥林巴斯,型号:SZ61)
  6. 倒置显微镜(奥林巴斯,型号:CKX42)
  7. 用15毫升和50毫升离心管(Eppendorf,型号:5430 R)的摆动斗转子离心。
  8. 37℃,5%CO 2
    培养箱
  9. 无菌层流罩
  10. 血细胞计数器(Neugebauer改进)
  11. 荧光显微镜(ZEISS,型号:Axio Imager.M2)
  12. 冷冻容器,Frosty TM先生(Thermo Fisher Scientific,Thermo Scientific TM,型号:Frosty TM先生,产品目录号:5100-0001 )
  13. 37°C水浴
  14. -80°C冷冻机
  15. 移液器
  16. 多级移液器(和提示)

程序

  1. 一般准备
    细胞培养皿的涂层
    单层培养物以及神经球的分化需要聚-D-赖氨酸(PDL)/层粘连蛋白涂覆的表面进行附着。
    1. 加入适量的PDL(ddH2O中5μg/ ml),并确保表面或盖玻片完全覆盖(体积见表2)。

    2. 在室温下孵育至少4小时或过夜
    3. 去除溶液并用ddH 2 O清洗容器三次。
    4. 让它们适当干燥,直到没有残留的水。
    5. 加入层粘连蛋白(5毫克/毫升冷DMEM / F-12)孵育至少4小时或37°C过夜。
    6. 直接使用它们或将其储存在-20°C直到需要。

    火抛光移液器
    在隔离过程中,需要具有小或中等大小的孔的火焰抛光移液器。
    1. 在本生灯的热蓝色火焰上旋转玻璃巴斯德吸液管约3秒,直到边缘变圆。小巴斯德移液管显示内径为0.3-0.4毫米,而中等大小的移液管孔大小为0.6-0.8毫米。
    2. 移液器应在使用前进行高压灭菌。

  2. 大脑解剖
    注意:单层培养需要涂层的24孔板,应至少在分离前两天准备好。
    神经前体细胞分离的方案基于Babu等人的方案。 (2011),Walker和Kempermann(2014)(其中还包括解剖程序视频)以及Ehret et al。(2016)。可以使用一个小鼠/神经球实验(每个96孔板)进行神经球测定。单层培养也可以使用一只小鼠产生,但是我们建议将3-4个脑组合起来。
    1. 麻醉6-8周龄的老鼠根据适当的制度指导。执行颈椎脱臼。
    2. 用70%乙醇喷头消毒,并减少毛皮附着在解剖工具和组织上。

    3. 使用锋利的剪刀将脑干基部的动物斩首
    4. 沿着中线纵向切开皮肤,直到眼睛之间的一点。暴露头骨。
    5. 将一把小小的剪刀插入每个眼腔中,以在眼睛之间进行颅骨的冠状切割。之后,在颅底做两个侧切,然后沿着矢状缝进行纵切。

    6. 用一把镊子将颅骨剥去,露出大脑

    7. 使用小铲子从颅骨中取出大脑,并将其放入冷PBS中。
    8. 将大脑转移到含有PBS的10cm塑料培养皿中(见图1B)。
    9. 将培养皿放在低倍镜下的立体显微镜下,将大脑置于腹面。用小角钳取出嗅球,小脑保持大脑正常。

    SVZ解剖
    1. 将大脑旋转到背侧,使用手术刀在视交叉区域进行冠状切割(见图1C)。
    2. 对于SVZ的显微切割(更多细节请参阅Azari等人(2010)和Walker和Kempermann,2014),使脑部的头侧部分暴露切割的冠状面的侧脑室。增加放大倍率,使用细弯钳除去和去除隔膜。
    3. 通过将一对细弯钳中的一个刀片的尖端放置在紧靠胼os体的侧脑室的侧角并且另一个大致1mm进入紧邻心室的组织中来解剖SVZ。将钳子向下压向盘基部和心室的腹侧以去除小的三角形组织(参见图1E)。将解剖的组织放入冰上的小培养皿中。

    DG解剖
    1. 对于DG的显微切割(更多细节参见Hagihara等人(2009)和Walker和Kempermann,2014),将大脑的尾部置于培养皿中并使用手术刀切割纵向裂缝(见图1D)。

    2. 用小角钳小心地去除小脑和间脑
    3. 重新调整显微镜的焦距以显示DG周围的边界。要卸下DG,请使用27 G¾针,并小心地沿着DG和Ammons喇叭之间的边界滑动。
    4. 使用细角钳将周围组织中的DG释放(见图1F)。将解剖的组织放入10μlPBS冰上的小培养皿中。

  3. SVZ组织解离
    1. 在15ml离心管中,在37℃的水浴中预热1ml的0.05%胰蛋白酶-EDTA 10-15分钟。

    2. 用6厘米培养皿中的解剖刀切碎SVZ组织约1分钟(见图1G)。
    3. 将组织碎片转移到温热的胰蛋白酶-EDTA中并在37℃孵育7分钟。
      轻轻颠倒管道轻轻混合
    4. 通过加入1毫升含有DNaseI的胰蛋白酶抑制剂(参见食谱)停止酶反应,并轻轻地搅拌混合。

    5. 在300×g离心5分钟
    6. 使用P1000移液器小心吸取7-10次,轻轻地将沉淀重悬于1ml生长培养基中(见食谱)(见图1H)。
      注意:为了增加细胞死亡,非常温和地研磨组织非常重要。
    7. 加入多达5毫升的生长培养基,并通过一个40微米的细胞过滤器细胞悬液进入一个50毫升的离心管。

    8. 在300×g离心5分钟
    9. 重悬细胞悬液在500μL的生长培养基。

  4. DG组织解离
    注意:可以从一只动物获得的细胞的平均产量取决于动物的年龄以及应变。但是,为了提供一个粗略的估计,我们通常从一只8周龄的C57BL / 6J动物中获得约100,000-200,000个细胞/ ml。
    为了从分离的DG组织获得单细胞悬浮液,含有木瓜蛋白酶的神经组织解离试剂盒(P)如下应用:
    1. 为了制备酶混合物1,将1,900μl缓冲液X和50μl酶P(均在试剂盒中提供)加入到15ml离心管中。为了增加酶的稳定性和细胞活力,向缓冲液X中加入β-巯基乙醇至终浓度为0.067mM。
    2. 预热酶混合物1在37℃的水浴中10-15分钟。

    3. 用解剖刀在6厘米培养皿中解剖DG组织约1分钟(见图1G)。
    4. 将组织块转移到1,950μl预热的酶混合物1中。

    5. 在37°C孵育15分钟,每3-5分钟轻轻搅拌一次
    6. 准备酶混合2通过添加20μL的缓冲液Y到10μL的酶A(都在试剂盒中提供)。
    7. 将酶混合物2添加到组织酶混合物中。
    8. 用火焰抛光的巴斯德吸管用中孔轻轻吹打上下吸取组织,机械分离组织。
      注意:为避免细胞死亡增加,非常温和地研磨组织非常重要。

    9. 在37°C孵育10分钟,每3-5分钟轻轻搅拌一次
    10. 进一步使用小口径火焰抛光巴斯德吸管通过上下轻轻移液10次(见图1I),机械地分离组织。
    11. 要洗细胞悬液,添加汉克的缓冲盐溶液达10毫升。

    12. 在300×g离心5分钟
    13. 用500μl生长培养基重悬沉淀,适用于置于50ml离心管中的40μm细胞过滤器。


      图1.从SVZ和DG分离神经前体细胞A.需要的工具; B.孤立的大脑; C.使用手术刀在视交叉区域切除大脑的冠状动脉; D.切开纵向裂隙; E.解剖SVZ组织; F.解剖DG; G.用解剖刀切碎组织; H.重悬SVZ组织; I.酶促孵育后的DG组织。

  5. 单层文化
    从原代细胞建立单层培养物,然后在增殖条件下将其作为贴壁细胞培养数代(图2A)。这导致相当均匀的文化,可用于执行单细胞分析。


    图2.来自成年小鼠脑的神经前体细胞可以作为贴壁单层培养物(A)或作为神经球(B)培养。比例尺= 50微米。

    培养

    1. 添加20ng / ml FGF2,20ng / ml EGF和2μg/ ml肝素至从组织解离获得的生长培养基中的单细胞悬浮液中。
    2. 将细胞悬浮液接种到24孔板的涂层孔中。
    3. 接种后24小时,取出生长培养基,并用含有生长因子(20ng / ml FGF2,20ng / ml EGF和2μg/ ml肝素)的新鲜生长培养基交换。
      注意:如果单元格似乎没有充分连接,则可以跳过此步骤。
    4. 24小时后,用预热的PBS洗涤细胞并添加具有生长因子(20ng / ml FGF2,20ng / ml EGF和2μg/ ml肝素)的新鲜生长培养基。
    5. 每隔一天,用含有100%生长因子的新鲜生长培养基交换50%旧生长培养基以抵消残余培养基中的生长因子消耗(例如,对于含有5ml的T25烧瓶培养基中,取出2.5ml,加入含有40ng / ml EGF和FGF2和4μg/ ml肝素的2.5ml新鲜培养基)。
    6. 一旦细胞达到70-80%汇合,取出介质,并用PBS洗一次。
      注意:这可能需要两周时间。
    7. 加入150μl的Accutase,37°C孵育3分钟。


    8. 强力地将钢板轻轻敲击表面以及侧面,以便搅动连接的电池。
    9. 检查显微镜下如果细胞分离。如果没有,延长孵化时间(最多10分钟)。
    10. 向细胞悬浮液中加入2ml生长培养基,并在300×g下旋转3分钟以沉淀细胞。
    11. 取出上清液,并在1毫升生长培养基中重悬细胞。
    12. 取出10微升的细胞悬液,并与10微升的台盼蓝混合。
    13. 用血细胞计数器计数细胞。
    14. 以最小密度种植细胞。在具有生长因子(20ng / ml FGF2和20ng / ml EGF;不再含有肝素)的生长培养基中培养1×10 4个/ cm 2。

    进一步传代(一旦细胞达到70-80%汇合,通过)
    1. 取出培养基,用PBS清洗一次。
    2. 加入Accutase并在37°C孵育3分钟。


    3. 强力地将钢板轻轻敲击表面以及侧面,以便搅动连接的电池。
    4. 检查显微镜下如果细胞分离。如果没有,延长孵化时间(最多10分钟)。
    5. 将细胞收集在4.5ml生长培养基中(体积为T25烧瓶),并在300xg下旋转3分钟以沉淀细胞。
    6. 加10μl台盼蓝至10μl细胞悬液,用血细胞计数器计数细胞。
    7. 以最小密度种植细胞。 (20ng / ml FGF2和20ng / mlEGF)的生长培养基中培养1×10 4 c / cm 2(活细胞)。

    冷冻
    1. 准备冷冻混合物(见食谱),并在4°C储存。
    2. 从烧瓶/平板上移除生长培养基,并用5毫升PBS洗。
    3. 加入Accutase到培养瓶/培养板中,在37°C孵育3分钟,检查是否所有细胞都脱落。如果没有,延长孵化时间(最多10分钟)。
    4. 将细胞收集在4.5ml生长培养基中(体积为T25烧瓶)并在300xg下旋转3分钟以沉淀细胞。
    5. 去除上清液。
    6. 重悬细胞在1毫升的生长介质。
    7. 加入10μl台盼蓝到10μl细胞悬液。
    8. 计数细胞并用生长培养基调整细胞密度至2×10 6 c / ml(活细胞)。

    9. 每个冷冻小瓶加入0.5毫升的冷冻混合物
    10. 每个冷冻小瓶加入0.5毫升的细胞悬液,混合,并放入冷冻容器。
    11. 将冷冻容器直接放在-80°C冷冻箱中。
    12. 12小时后,可将小瓶转移到液氮中。

    在增殖条件下进行细胞接种和细胞特征分析(图3)
    注意:为了分析增殖条件下的细胞特征,细胞可以在接种后48小时直接固定或用BrdU标记。


    图3.在增殖条件下的单层培养物A.在增殖条件下的单层培养物表达神经系统的祖细胞例如巢蛋白(绿色)和Sox2(洋红色)的细胞标记物。 B.可以通过检查相对于细胞总数(Hoechst 33342,蓝色)的经历S-期的细胞部分(BrdU标记,绿色)来标记祖细胞以评估其增殖能力。比例尺= 50微米。


    1. 解冻PDL /层粘连蛋白包被板(带盖玻片的24孔板)在37℃约15分钟。

    2. 从24孔板中取出层粘连蛋白
    3. 在增殖条件下(在培养基中添加20ng / ml EGF和20ng / ml FGF2),将20,000个细胞/孔平板涂布在24孔板孔中的涂布的盖玻片上。
    4. 孵育48小时。
    5. 将BrdU(终浓度10μM)加入到每个孔中,并在37℃孵育2小时。
      注意:如果不需要BrdU标签,则跳过此步骤并继续执行步骤6.
    6. 去除培养基,在0.1M磷酸盐缓冲液中加入4%PFA到每个孔中并在室温下孵育20分钟。
    7. 用PBS清洗两次,加入1毫升新鲜的PBS到井中。
    8. 在4°C储存,直到进行染色。为了避免蒸发,用Parafilm密封板。

    细胞接种和分化(图4A)

    1. 解冻PDL /层粘连蛋白包被板(带盖玻片的24孔板)在37℃约15分钟。
    2. 从24孔板中取出层粘连蛋白,用PBS洗板。
    3. 在增殖条件下(在培养基中添加20ng / ml EGF和20ng / ml FGF2),将20,000个细胞/孔平板涂布在24孔板孔中的涂布的盖玻片上。
    4. 孵化48小时。
    5. 去除生长培养基,并添加含有5ng / ml FGF2的新鲜生长培养基。
    6. 再过48小时后,取出生长培养基,加入不含生长因子的新鲜生长培养基。
    7. 让细胞分化3-5天。

    的 固定
    1. 删除生长介质。

    2. 在每孔中加入300μl4%PFA的0.1M磷酸盐缓冲液并在室温下孵育20分钟
    3. 用PBS清洗两次,加入1毫升新鲜的PBS到井中。
    4. 在4°C储存,直到进行染色。为了避免蒸发,用Parafilm密封板。

    染色
    注意:步骤1-4只需要BrdU染色,其他染色直接从步骤5开始。
    1. 用0.9%NaCl洗2次。

    2. 用1N HCl在37℃孵育30分钟
    3. 用硼酸盐缓冲液清洗1次(见食谱)。
    4. 用PBS冲洗3次10分钟。
    5. 用0.1%Triton X-100在PBS中透化10分钟。

    6. 室温封闭溶液1小时
    7. 在室温下用抗体溶液孵育1小时(参见表1)2小时,否则在4℃孵育过夜(300μl/孔)。
    8. 用PBS洗3次。
    9. 在室温下(300μl/孔)在黑暗中用稀释在抗体溶液中的适当的二抗(参见食谱)孵育1小时。
    10. 用PBS清洗10分钟。
    11. 孵育Hoechst 33342(1:4,000的PBS)10分钟在室温在黑暗中。
    12. 用PBS洗1次。
    13. 在ddH 2 O中浸洗。
    14. 通过轻轻拍打薄纸干燥盖玻片,并使用荧光固定介质进行安装。
    15. 在黑暗中过夜。
    16. 在4°C储存。
    17. 使用荧光显微镜的图像。

  6. 神经球分析
    神经球培养很容易产生,并允许直接的数据分析方面的前体细胞数量和潜力的脑组织从个别小鼠(图2B)。
    文化
    注意:神经球在另外含有2μg/ ml肝素的生长培养基中培养
    1. 稀释从20毫升含20ng / ml FGF2,20ng / ml EGF和2μg/ ml肝素的神经球生长培养基中获得的单细胞悬浮液。

    2. 使用10 ml多步骤移液器,在96孔板上每孔平板200μl
    3. 培养7天SVZ衍生的神经球培养物和DG衍生的神经球培养物12天在37℃。
      注意:延长孵育时间可能导致过度生长,并可能导致神经球核心中的自发附着,分化或细胞死亡。
    4. 使用倒置光学显微镜计数和大小的神经球。

    微分(图4B和4C)
    注意:对于一个实验,我们建议每个96孔板至少播种4个随机大小的神经球的盖玻片。

    1. 解冻PDL /层粘连蛋白包被板(带盖玻片的24孔板)在37℃约15分钟。
    2. 从24孔板中取出层粘连蛋白,用PBS洗板。

    3. 加入1毫升的生长培养基
    4. 将含有神经球的培养基从96孔板的所有孔中收集到10厘米培养皿中。
    5. 使用立体显微镜用P100移液管(终体积75μl)从培养皿中收集神经球,并将它们转移到24孔板的盖玻片上。每盖玻片,我们建议种子约15个神经球。
    6. 在37°C的培养箱中分化神经球7天。这些细胞将在盖玻片上扩散并变得粘附。


      图4.单层培养物和神经球可以分化A.单层培养的分化细胞与GFAP阳性星形胶质细胞(绿色)和Map2ab阳性神经元(洋红色)。 B.用β-III-微管蛋白阳性神经元(品红)和GFAP阳性星形胶质细胞(绿色)分化的神经球培养物。 C.用O4阳性少突胶质细胞分化的神经球培养物(红色)。比例尺= 50微米。

    的 固定
    1. 取出培养基,用PBS洗2次,去除死细胞和碎片。

    2. 在每孔中加入300μl4%PFA的0.1M磷酸盐缓冲液并在室温下孵育20分钟
    3. 去除PFA,用PBS洗两次。
    4. 在4°C储存在1毫升PBS。为了避免蒸发,用Parafilm密封板。

    染色
    注意:使用推荐的O4抗体进行染色时,请勿将Triton加入封闭液和抗体溶液中,并使用合适的IgM二抗。

    1. 用含有2倍分化的神经球的盖玻片清洗

    2. 在室温下孵育含有0.2%Triton X-100封闭液(参见食谱)30分钟的盖玻片。
    3. 在含有0.2%Triton X-100的抗体溶液中,在室温下(300μl/孔)与针对神经元和星形细胞标记物或少突神经胶质细胞标记物O4(参见表1)的一级抗体孵育1小时。
    4. 用PBS洗4次。
    5. 在室温下避光孵育30分钟,加入含有0.2%Triton X-100的抗体溶液(300μl/孔)。
    6. 用PBS洗2次。
    7. 孵育Hoechst 33342(1:4,000的PBS)10分钟在室温在黑暗中。
    8. 用PBS洗2次。
    9. 在ddH 2 O中浸洗。
    10. 将盖玻片安装在带有荧光固定介质的显微镜载玻片上,在黑暗中风干一夜。
    11. 在4°C储存。
    12. 使用荧光显微镜的图像。

  7. 细胞培养体积和密度(表2)

    表2.细胞培养体积和密度

数据分析

  1. 在神经球测定中,神经球的大小和数量可以被确定,并用作前体细胞增殖潜力的间接测量,例如在不同的外部刺激或处理的影响下,或在不同的遗传背景之间。神经球进一步允许分析球形细胞的分化潜能。这可以通过Hoechst 3342(DAPI)染色确定的细胞总数中Map2ab /β-III-微管蛋白和GFAP阳性细胞的百分比来量化。神经球测定不能得出关于绝对干细胞数量的结论。
  2. 此外,对于生物学复制,我们建议进行神经球测定实验至少5次作为技术重复,因为可能会有一些日常的差异,我们总是强烈建议在同一时间进行控制和治疗实验,以尽量减少一个批处理效果。
  3. 根据具体的实验设置,数据分析使用学生的检验或方差分析结合适当的事后检验进行。
  4. 基于单层培养的实验可以评估单细胞水平上的特征。 BrdU数据通过评估在BrdU存在的情况下通过S-相的细胞的数量作为间接测量细胞增殖潜力的量度。对于分化实验,分析Map2ab /β-III-微管蛋白阳性细胞和GFAP阳性细胞的数量,作为通过Hochest33342(DAPI)染色测定的细胞总数的百分比。我们建议在相同的盖玻片上同时进行两种染色以获得可比较的结果。对于一个分化实验,我们分析四个盖玻片,并采取随机位置上的每个盖玻片至少五个图像来计数细胞。
  5. 请注意,冻融循环和传代事件可能会影响细胞和结果数据。因此,在同一段落中进行实验以及对经过类似处理的细胞进行实验,以获得可再现的数据至关重要。我们建议执行至少五个技术重复。数据分析是根据学生的实验设置,使用Student's test或者ANOVA结合适当的post-hoc test进行的。

食谱

  1. 含有脱氧核糖核酸酶I的胰蛋白酶抑制剂
    0.125毫克/毫升胰蛋白酶抑制剂
    0.01毫克/毫升DNase I
    在没有谷氨酰胺的DMEM / F-12中
  2. 生长介质
    神经基础培养基
    0.5%B-27补充剂(50x)
    0.25%Pen / Strep 100,000 U / ml
    0.25%GlutaMAX TM(100x股票)
  3. 冻结介质
    生长介质
    20%DMSO
  4. 硼酸盐缓冲液
    硼酸
    ddH 2
    10N氢氧化钠调节pH至8.5
  5. 抗体解决方案
    1x PBS
    3%正常驴血清
  6. 阻止解决方案
    1x PBS
    10%正常驴血清

致谢

作者宣称没有竞争的经济利益。这项工作是由德国联邦银行SFB655和德国联邦理工学院联合资助的。图像采集和处理使用DZNE德累斯顿成像平台的设备。我们感谢Fanny Ehret博士对稿件的有益评论。这里描述的协议代表了Babu等人(2011),Walker和Kempermann(2014)和Ehret等人描述的协议的变化和进一步发展。 (2016)。

参考

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  2. Babu,H.,Claasen,J.H.,Kannan,S.,Runker,A.E。,Palmer,T。和Kempermann,G。(2011)。 从小鼠齿状回分离和富集单层培养神经前体细胞的方案
    前面的Neurosci 5:89
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引用:Bernas, S. N., Leiter, O., Walker, T. L. and Kempermann, G. (2017). Isolation, Culture and Differentiation of Adult Hippocampal Precursor Cells. Bio-protocol 7(21): e2603. DOI: 10.21769/BioProtoc.2603.
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