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Neurosphere Co-culture Assay
神经球共培养试验

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Abstract

The hippocampal niche is one of two areas in the brain where stem cells reside. In this neurogenic niche, stem cells can be found in close proximity to astrocytes and in contact with microvessels consisting of pericytes and endothelial cells. To study the regulatory interplay of this complex niche network in a simplified in vitro model, we established a co-culture system. We investigate the formation of neurosphere under different co-culture conditions by using primary niche cells. Here, we describe the isolation procedure for primary niche cells culture of astrocytes, endothelial cells and pericytes/smooth muscle cells from mouse brain. These niche cells are co-cultured (by hanging inserts) with fresh isolated stem and precursor cells from the adult hippocampus to study the influence of soluble factors. This method is used to investigate factors and cell types regulating stem cell behavior in a niche-like environment.

Keywords: Neuroscience(神经科学), Neurosphere(神经球), Dentate gyrus(齿状回), Neurogenic niche(神经性的利基)

Materials and Reagents

  1. Petri dishes (diameter 5.5 cm to 9 cm)
  2. Falcon tubes 50 ml and 15 ml
  3. Eppendorf tubes 2 ml (sterilized by autoclave)
  4. Pasteur pipettes (1 mm diameter)
  5. Brush (da Vinci, model: Nova synthetics series 1570, #3 )
  6. 12-well and 24-well tissue culture plates (growth-enhance treated, gamma-sterilised, free of pyrogens, free of DNA/RNA, DNase/RNase)
  7. Filter discs (Sartorius AG, catalog number: FT-3-205-055 )
  8. Falcon cell strainers 40 µm and 100 µm (Thermo Fisher Scientific, catalog number: 08-771-1 and 08-771-19 )
  9. Hanging insert for 24-well plates, 1 µm PET (Merck Millipore Corporation, catalog number: PIRP12R48 )
  10. 27G ¾ needle (Braun, StericanTM, catalog number: 4657705 )
  11. Mice at the age of six weeks
    Note: 6-8 mice for endothelial and pericyte isolation, at least 1 mouse for neurosphere co-culture assay
  12. Mice at the age of four weeks
    Note: 4 mice for astrocyte isolation are needed for one preparation.
  13. Distilled water
  14. Poly-D-lysine hydrobromide (PDL) (Sigma-Aldrich, catalog number: P7280 )
  15. Laminin (Sigma-Aldrich, Roche, catalog number: R11243217001 )
  16. 70% ethanol
  17. HBSS (Thermo Fisher Scientific, GibcoTM, catalog number: 14175 )
  18. Dextran from Leuconostoc mesenteroides average molecular weight 150,000 (Sigma-Aldrich, catalog number: D4876 )
  19. DMEM/F-12 without glutamine (store at 4 °C) (Thermo Fisher Scientific, GibcoTM, catalog number: 21331020 )
  20. DMEM/F-12 with L-glutamine and 15 mM HEPES (store at 4 °C) (Thermo Fisher Scientific, GibcoTM, catalog number: 11330032 )
  21. B27 (Thermo Fisher Scientific, GibcoTM, catalog number: 0080085SA )
  22. MCDB131 Media (Thermo Fisher Scientific, catalog number: 10372-019 )
  23. Glutamax (100x stock) (store at 4 °C) (Thermo Fisher Scientific, CTSTM, catalog number: A12860-01 )
  24. Insulin-transferrin-selenium (ITS) premix (store at -20 °C) (Thermo Fischer Scientific, CorningTM, catalog number: CB-40351 )
  25. Non-essential amino acids (100x stock) (store at 4 °C) (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11140-035 )
  26. Pen/Strep 100,000 U/ml (-20 °C) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140 )
  27. Heparin (store at 4 °C) (MP Biomedicals, catalog number: 0210193125 )
  28. Collagenase A (Sigma-Aldrich, Roche, catalog number: 10103578001 )
  29. Fetal bovine serum (FBS) (-20 °C) (Merck Millipore Corporation, Biochrom, catalog number: S0613 )
  30. Plasma derived platelet poor serum (Sigma-Aldrich, catalog number: P2918 )
  31. Human EGF (store aliquots at -20 °C) (PEPROTECH, catalog number: AF100-15 )
  32. Human FGF-basic (store aliquots at -20 °C) (PEPROTECH, catalog number: 100-18B )
  33. Human VEGF (store aliquots at -20 °C) (PEPROTECH, catalog number: 100-20 )
  34. Accutase (store at 4 °C) (GENTAUR, PAA Laboratories GmbH, catalog number: L11-007 )
  35. Neuronal tissue dissociation kit (P) (Miltenyl Biotec, catalog number. 130-092-628 )
  36. Trypsin/EDTA (store at -20 °C) (Merck Millipore Corporation, Biochrom, catalog number: L2153 )
  37. PBS with 2% FBS (see Recipes)
  38. 30% dextran solution (see Recipes)
  39. Endogrow media (see Recipes)
  40. Astrocyte media (see Recipes)
  41. Pericyte/smooth muscle cell media (see Recipes)
  42. Neurosphere media (see Recipes)

Equipment

  1. Dissection tools: scissors, small spatula, fine curved forceps and scalpel (see Figure 2B)
  2. 7 ml dounce tissue grinder (Capitol Scientific, Wheaton, catalog number: 357542 )
  3. Autoclave
  4. Vacuum pump
  5. Dounce homogenizer
  6. Dissection microscope (Olympus, model: SZ61 )
  7. Inverted microscope (Olympus, model: CKX41 )
  8. Scale plate insert into the eyepiece of the microscope (Olympus)
  9. Cooling centrifuge fitting 2 ml tubes (Eppendorf, model: 5430R )
  10. Swing bucket centrifuge for 15 ml tubes (Eppendorf, model: 5810R )
  11. Incubator at 37 °C with 5% CO2
  12. Tube rotator or orbital shaker

Procedure

Note: A general overview of the isolation procedure can be found in Figure 1.

  1. General Preparations
    1. At least two days prior to isolation of endothelial cells from tissue, prepare 4 poly-D-lysine (PDL)/laminin coated 12-well plates (with at least 3 wells coated per plate). To prepare wells add enough PDL (5 µg/ml in dH2O) to coat the surface and incubate overnight at room temperature. Remove the solution from the dish and wash the dish three times with dH2O. Allow to air dry. Add laminin (5 µg/ml in cold DMEM:F12) and incubate at 37 °C overnight. Remove the laminin and either use the plates immediately or store with the laminin at -20 °C until required.
    2. At least one day prior to astrocyte isolation prepare PDL coated plates (at least 3 wells coated per plate). Per well add enough PDL (5 µg/ml in dH2O) to coat the surface and incubate overnight at room temperature. Remove the solution from the dish and wash the dish three times with dH2O. Add dH2O and freeze at -20 °C or use directly.
    3. Prepare fire polished pipettes with "medium" and "small" bores by rotating glass Pasteur pipettes in a flame until the edges become rounded. We measured that our “small” bores are around 0.3-0.4 mm, whereas the “medium” pores are around 0.6-0.8 mm internal diameter. Autoclave pipettes to sterilize.

  2. Isolation of microvascular endothelial cells and pericyte/vascular smooth muscle cells(vSMC)
    Note: An overview of the isolation procedure can be found in Figure 1. A step-by-step picture series of the isolation procedure can be found in Figures 2 and 3.
    Two weeks prior to co-culture isolate endothelial cells and pericytes/vSMC from six to eight mice at the age of 6 to 8 weeks. The protocol for endothelial cell isolation was modified from Wu et al., 2003.
    1. Anesthetize 6-8 adult mice according to the appropriate institutional guidelines. Perform cervical dislocation.
    2. Spray the head with 70% ethanol to sterilize the area to minimize contamination through fur.
    3. With sharp scissors decapitate the mouse at the base of the brain stem.
    4. Cut the skin sagittal along the midline till an arbitrary point between the eyes. Expose the skull free of overlying skin and subcutaneous tissue (Figure 2C).
    5. Cut the skull between eyes by placing each blade of the scissors into the each eye cavity. Next, make two lateral cuts at the base of the skull, following a cut through the skull along the sagittal suture.
    6. Expose the brain by peeling back the skull with the scissors or forceps and remove the brain from the skull with a small spatula (Figure 2D).
    7. Rinse the brain with PBS and transfer to a Falcon tube with PBS/2% FBS, which is kept on ice.
    8. After collecting all brains transfer one brain to a petri dish with a filter disc inside filled up with PBS/2% FBS.
    9. With the curved forceps cut of the midbrain and the cerebellum (Figure 2F).
    10. Remove the meninges and any visible surface blood vessels with a fine paint brush, the big vessels will get trapped easily on the filter disc (Figure 2G-H).
    11. With the brush remove the white matter tracts of the corpus callosum (Figure 2I).
    12. Transfer the tissue now to a new petri dish and keep cortex tissue on ice till further processing. Continue to process the rest of the brains in the same way (Figure 2J).
    13. After processing all brain samples, chop the cortex tissue into small pieces with the scalpel. Homogenize the tissue in 2 ml PBS/2% FBS using a dounce homogenizer with a loose pestle by applying 6-8 strokes (Figures 2K-L and 3A).
    14. Mix the tissue homogenate (should be ~3 ml) with 3 ml of 30% dextran to get a final concentration of 15% (Figure 3B).
    15. Transfer the mixture into 2 ml Eppendorf tubes and centrifuge at 6,000 x g for 20 min at 4 °C (Figure 3C tube after centrifugation).
    16. Remove the thick supernatant carefully with a 1 ml pipette or by aspiration using a vacuum pump (Figure 3D-E). Take care to remove tissue homogenate properly from the walls. Resuspend the loose pellets in PBS with 2% FBS with a 1 ml pipette by rapidity pipetting up and down (Figure 3F).
    17. Place a 100 µm sterile nylon cell strainer inverse on top of a 50 ml tube. Filter the solution through the 100 µm filter and wash extensively with at least 10ml PBS containing 2% FBS. Label tube as “flow through 1” and keep on ice till further processing (Figure 3G).
    18. Take the 100 µm filter off and flip it into a new 50 ml tube labeled “pericytes/vSMC” (Figure 3H). Wash the filter extensively with at least 10 ml PBS containing 2% FBS (Figure 3I).
    19. Keep the big vessels in solution (pericytes/vSMC) on ice till centrifugation.
    20. Take flow through 1 and filter through a 40 µm sterile nylon cell strainer (again put it inverse on a 50 ml tube) (Figure 3J), wash with PBS containing 2% FBS to remove single cells and debris.
    21. The flow through can be discarded and the 40 µm nylon strainer should be flipped and inserted into a new 50 ml tube.
    22. Wash the nylon membrane extensively with at least 10 ml PBS containing 2% FBS to get all the microvessels back into solution, label the tube “endothelial cells”.
    23. Spin both tubes by centrifugation at 800 x g for 5 min at 4 °C.
    24. Prepare collagenase solution by dissolving 1 mg/ml collagenase A in respective growth medium endogrow and pericyte media without addition of growth factors. Dissolves best at 37 °C (see Recipes 3 and 4).
      Note: Volume of collagenase solution depends on pellet size we used 3-4 ml per pellet/ cell type.
    25. Dissolve the vessel pellet from step B23 in respective collagenase solution. Incubate the vessels in solution in a 15 ml tube for 4-5 h at 37 °C on tube rotator (Figure 3K-L). In between vigorously pipette up and down with a 1 ml pipette to break up cellular clumps. Take care that vessels do not gets attached to pipette tips. Once per hour with 5 times up and down is in most cases sufficient.
    26. Spin down the cells at 800 x g for 5 min, remove the supernatant and dissolve the pellets in respective growth medium with growth factors.
    27. Plate endothelial cells in one PDL/laminin coated well of a 12-well plate and pericytes/smooth muscle cells on two uncoated wells of a 24-well plate at 37 °C with 5% CO2 overnight.
    28. The next day transfer the supernatant to a new well of the same dish which was also coated with PDL/laminin (but still contains the laminin media till use) for endothelial cells or uncoated for pericyte/vSMCs. Add fresh growth medium with growth factors.
    29. Change medium every 3-4 days. Niche cells need several days until they begin to proliferate. Don’t split cells until transfer into co-culture hanging inserts. They should be grown at a high density in order to proliferate (Figure 4 shows the initial cell stages).

  3. Isolation of astrocytes
    Note: An overview of the isolation procedure can be found in Figure 1. The initial steps are the same as for neurosphere isolation, you can see in the video of Babu et al., 2011, how to dissect the hippocampus.
    1. Two weeks prior to co-culture isolate astrocytes from four 4-week old mice.
    2. Anesthetize adult mice according to the appropriate institutional guidelines. Perform cervical dislocation.
    3. Spray the head with 70% ethanol to sterilize the area to minimize contamination through fur.
    4. With sharp scissors decapitate the mouse at the base of the brain stem.
    5. Cut the skin sagittal along the midline till an arbitrary point between the eyes. Expose the skull free of overlying skin and subcutaneous tissue.
    6. Cut the skull between eyes by placing each blade of the scissors into the each eye cavity. Next, make two lateral cuts at the base of the skull, following a cut through the skull along the sagittal suture.
    7. Expose the brain by peeling back the skull with the scissors or forceps and remove the brain from the skull with a small spatula.
    8. Rinse the brain with PBS and transfer to a plastic dish containing PBS.
    9. Place petri dish containing the brain under a dissecting microscope at low magnification and position the brain on its ventral surface. Using fine curved forceps remove the olfactory bulbs while holding the brain in position by the cerebellum.
    10. Rotate the brain onto the dorsal aspect and using a scalpel make a coronal cut through the brain at the level of the optic chiasm.
    11. Place the caudal portion of the brain in the petri dish and cut along the longitudinal fissure using a scalpel.
    12. Under the dissection microscope remove the cerebellum and the diencephalon with forceps.
    13. Remove the hippocampus by sliding with a 27 G needle around the hippocampus formation. Using fine forceps, take of the hippocampus from the surrounding tissue and remove the attached myelin sheets (white matter), which can be found along the medial edge and at the backside of the hippocampus.
    14. Place the dissected hippocampus into an Eppendorf tube filled with HBSS on ice until all hippocampi are dissected.
    15. Prepare enzyme mix 1 from neuronal tissue dissociation kit by adding 50 µl of enzyme P and 1,900 µl of buffer X plus 2 µl of β-mercaptoethanol. Vortex and preheat the mixture at 37 °C for 10-15 min before use.
    16. Remove the HBSS carefully from the hippocampi and mince the tissue in a petri dish using a scalpel blade until no large pieces remain.
    17. Transfer the minced tissue into a 15 ml Falcon tube with a transfer pipette and add preheated enzyme mix 1.
    18. Incubate for 15 min at 37 °C with continuous shaking using a tube rotator.
    19. Prepare enzyme mix 2 from neuronal tissue dissociation kit by adding 40 µl of buffer Y and 20 µl of enzyme A.
    20. Add enzyme mix 2.
    21. Triturate 10 times slowly using a medium-sized fire-polished pipette.
    22. Incubate 10 min at 37 °C.
    23. Triturate 10 times slowly using a small-sized fire-polished pipette.
    24. Wash the cells by adding HBSS buffer to a final volume of 10 ml.
    25. Apply cells in solution to a 40 µm cell strainer, wash the strainer with additional 4 ml HBSS.
    26. Centrifuge the cells in the 15 ml tube at 300 x g for 5 min.
    27. Remove the supernatant and resuspend the pellet in the appropriate amount of astrocyte media (see Recipe 5) with 10 ng/ml EGF and 2% FBS.
    28. Seed onto one well of 12-well plate PDL coated plates and incubate at 37 °C with 5% CO2.
    29. After 36 h take off the medium and wash the remaining adherent cells thoroughly with pre-warmed PBS and add fresh media with 5 ng/ml EGF and 0.5% FBS.
    30. Change medium every 3 days, do not passage cells before use in co-culture experiment (see Figure 5 for representative images).

  4. Seeding of the niche co-culture cells into cell inserts
    1. Two days prior to co-culture dissociate the cells from the different preparations with accutase or trypsin.
      Note: We use accutase for astrocyte preparations and trypsin for endothelial and pericyte preparations.
    2. Seed ~5,000 cells per hanging insert in their respective growth medium for the different niche cells cultures (approx. 100 µl per insert, see Recipes 3-5 for media composition), no media is placed in the well below the hanging insert.
    3. 5-10 h prior to co-culture remove respective growth medium from hanging insert (by aspiration or with pipette) and replace by DMEM with B27 (see Recipe 6) without adding any growth factors.

  5. Isolating hippocampal precursor cells for neurosphere co-culture assay
    The isolation procedure for neural precursor cell is according to the protocol of Babu et al. (2011), Walker and Kempermann (2014) with light modifications.
    1. Anesthetize one adult 6 to 8-week-old mouse according to the appropriate institutional guidelines. Perform cervical dislocation.
    2. Spray the head with 70% ethanol to sterilize the area to minimize contamination through fur.
    3. With sharp scissors decapitate the mouse at the base of the brain stem.
    4. Cut the skin sagittal along the midline till an arbitrary point between the eyes. Expose the skull free of overlying skin and subcutaneous tissue.
    5. Cut the skull between eyes by placing each blade of the scissors into the each eye cavity. Next, make two lateral cuts at the base of the skull, following a cut through the skull along the sagittal suture.
    6. Expose the brain by peeling back the skull with the scissors or forceps and remove the brain from the skull with a small spatula.
    7. Rinse the brain with PBS and transfer to a plastic dish containing PBS.
    8. Place petri dish containing the brain under a dissecting microscope at low magnification and position the brain on its ventral surface. Using fine curved forceps remove the olfactory bulbs while holding the brain in position by the cerebellum.
    9. Rotate the brain onto the dorsal aspect and using a scalpel make a coronal cut through the brain at the level of the optic chiasm.
    10. For the microdisection of the dentate gyrus (for more details see Walker and Kempermann, 2014) place the caudal portion of the brain in the petri dish and cut along the longitudinal fissure using a scalpel.
    11. With forceps remove under the dissection microscope the cerebellum and the diencephalon.
    12. To remove the dentate gyrus, insert the tip of a 27 G needle and slide along the border between the dentate gyrus and Ammon’s horn. Using fine forceps, take of the dentate from the surrounding tissue.
    13. Place the dissected dentate gyrus in a petri dish with HBSS on ice.
    14. Prepare enzyme mix 1 from Neuronal tissue dissociation kit by adding 25 µl of enzyme P and 950 µl of buffer x plus 1 µl of β-mercaptoethanol. Vortex and preheat the mixture at 37 °C for 10-15 min before use.
    15. Remove the HBSS and mince the tissue using a scalpel blade until no larger pieces remain.
    16. Transfer the minced tissue from one mouse into a 15 ml Falcon tube and add preheated mix 1.
    17. Incubate for 15 min at 37 °C with continuous shaking using a tube rotator.
    18. Prepare enzyme mix 2 from Neuronal tissue dissociation kit by adding 20 µl of buffer Y and 10 µl of enzyme A.
    19. Add enzyme mix 2.
    20. Triturate 10 times slowly using a medium-sized fire-polished pipette.
    21. Incubate 10 min at 37 °C with continuous shaking using a tube rotator.
    22. Triturate 10 times slowly using a small-sized fire-polished pipette.
    23. Wash the cells by adding HBSS Buffer to a final volume of 10 ml.
    24. Apply cells in solution to a 40 µm cell strainer, wash the strainer with additional 4 ml HBSS.
    25. Centrifuge the cells in the 15 ml tube at 300 x g for 5 min.
    26. Remove the supernatant and resuspend the pellet in the appropriate amount of DMEM media (see Recipe 6), add 20 ng/ml EGF, 20 ng/ml bFGF and 2 µg/ml heparin.
      Note: Depending on how many co-culture conditions are going to be analyzed, titrate the volume of the medium so that enough but not too many cells are seeded per 24-well, otherwise spheres start to fuse. Per 24-well at least 500 µl medium needs to be used in order to have contact to the hanging cell insert.

  6. Hippocampal niche neurosphere co-culture
    1. Transfer 500 µl of neural cell suspension (from step E26) in DMEM media with growth factors to each well of the 24-well plate.
      Note: The number of wells seeded depends on the amount of co-culture conditions; in our experiments we seeded neurospheres from one animal into 12 wells of a 24-well plate (using 6 different co-cultures conditions). Do not forget one or two reference wells without niche cells in co-culture, where only stem and progenitor cells are seeded in but only an empty hanging insert with medium is placed on top. The growth of neurospheres in these reference wells can be taken as a baseline to evaluate the influence of the niche co-culture.
    2. Incubate for 30 min at 37 °C with 5% CO2.
    3. After cells from the isolation have settled down, add hanging inserts with the appropriate cells in DMEM/B27 without growth factors and incubate at 37 °C with 5% CO2 for 10 days. After 10 days take out the hanging inserts and count and measure the spheres using a 10x objective with a scale plate inside on an inverted microscope.
      Note: For easier analysis of spheres number draw gridlines on the surface of the 24-well plate or use a transparency with gridline (Figure 6). 
    4. For further analysis of differentiation, remove medium containing the spheres from the well and centrifuge at 300 x g for 5 min.
    5. Resuspend in DMEM medium with B27 but without growth factors and transfer to 24-well plate either with a glass bottom or containing coverslips (both need to be coated with laminin).
    6. After approximately 7 days at 37 °C with 5% CO2, the differentiation into neurons and astrocytes can be analyzed. Therefore fix cells with 4% paraformaldehyde (pre-warmed to 37 °C) and stain against Map2ab and GFAP (for detail see Walker and Kempermann, 2014).

Representative data



Figure 1. Isolation paradigm for neurosphere co-culture assay. Illustration outlines the primary cell isolation paradigm and the source of tissue, which can be used in this neurosphere co-culture assay. Pericytes/vascular smooth muscle cells (vSMCs) and endothelial cells can be isolated from the cortex, astrocytes were isolated from hippocampus and the dentate gyrus was used for isolation of neurospheres. Niche cells were plated into transwell hanging inserts, while isolated stem cells get seeded below.


Figure 2. Part 1-Isolation of endothelial cells and pericytes/vSMCs. Step-by-step procedure of the endothelial cell and pericyte/vSMC protocol. A. Sterile filtration of dextran. B. Set of instruments needed for the isolation. C. Sagittal cut through the skull and peel back of the skin and the skull. D. Remove the brain from the skull. E. Coronal cut through the brain with scalpel. F. Take the midbrain off with forceps. G. Remove visible surface blood vessels with a fine brush. H. No blood vessels are visible on the surface after brushing. I. Remove myelin sheets (white matter of corpus callosum) with the brush. J. Transfer the processed cortex to a new petri dish with PBS/FBS on ice. K. Cut the cortex with scalpel blades. L. Transfer the tissue to the douncer.


Figure 3. Part 2-Isolation of endothelial cells and pericytes/vSMCs. Second part of the step-by-step procedure of pericyte/vSMC and endothelial cell isolation. A. Homogenize tissue in the douncer with a loose pestle. B. Mix the tissue 1:1 with 30% dextran. C. After centrifugation a red vessel pellet is observable at the bottom and tissue homogenate floats on top. D. Aspirate carefully the tissue homogenate and the sucrose. E. The clean vessel pellet can be found at the bottom. F. Resuspend the vessel pellet in PBS/FBS. G. Filter vessels in solution though 100 µm nylon strainer, which is turned upside down. H. Take a new tube and flip on top to safely transfer the big vessels into it. I. Rinse the strainer till all vessels are moved back into solution. J. Transfer flow-through from previous step to 40 µm strainer. K. After centrifugation of big and small vessel fraction, a pellets can be found. These pellets get now resuspend in collagenase solution for digestion. L. Transfer the tubes with vessel preparations to rotary shaker in the incubator.


Figure 4. Endothelial cell and pericyte culture after vessel digestion. Representative images of endothelial cells and pericytes/vSMCs during the early time points of preparation. Images taken during the critical steps of endothelial cells (top panel) isolation and pericyte/vSMCs isolation (bottom panel). Scale bar, 10 µm


Figure 5. Astrocytes isolation. Representative Images of astrocytes during the early time points of preparation. Images taken during the critical steps of astrocyte isolation. Scale bar, 10 µm


Figure 6. Analysis of neurosphere coculture. Analysis of neurosphere co-culture assay by placing gridlines below. Left, gridlines underneath the 24-well plate. Middle, close-up view of one well. Right, view through the microscope with visible gridlines. Scale bar, 500 µm

Notes

In order to confirm enrichment and sustainability of the different primary niche cell preparations, always seed some cells onto PDL/laminin coated glass slides to evaluate the preparation by immunocytochemistry or take samples for RNA isolation (for details see original publication Ehret et al., 2015).

Recipes

  1. PBS with 2% FBS
    Add 1 ml sterile filtered FBS to 49 ml autoclaved PBS

  2. 30% dextran solution
    Dissolve 0.9 g dextran (avg. MW 150,000) in 3 ml HBSS
    Dissolve overnight on shaker, filter sterile with tip filters and store at 4 °C for maximal 3 days
  3. Endogrow media (for endothelial cell culture)
    MCDB131 media
    5% plasma derived platelet poor serum
    15 U/ml heparin
    ITS premix (5 μg/ml insulin, 5 μg/ml transferrin and 5 ng/ml selenium) (100x stock)
    100 U/ml pen/strep (100x stock)
    Just before use, add 10 ng/ml EGF, 10 ng/ml FGF-basic and 5 ng/ml VEGF
  4. Pericyte/vSMC media
    DMEM with glutamax (100x stock)
    10% FBS
    0.1 mM non-essential amino acids (100x stock)
    100 U/ml Pen/Strep (100x stock)
    Just before use, add 10 ng/ml VEGF
  5. Astrocyte media
    DMEM with glutamine and HEPES
    Glutamax (100x stock)
    B27 (50x stock)
    Growth factors (EGF and FBS) are added just before use, see steps C27-29 for the different concentrations
  6. Neurosphere media
    DMEM without glutamine
    B27 (50x stock)
    Glutamax (100x stock)
    Pen/Strep (100x stock)
    Growth factors (EGF and FGF 20 µg/ml each) are added just before use

Acknowledgments

This protocol was used in our previous published study (Ehret et al., 2015). This work was financed by basic institutional grants from the DZNE.

References

  1. 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.
  2. Ehret, F., Vogler, S. and Kempermann, G. (2015). A co-culture model of the hippocampal neurogenic niche reveals differential effects of astrocytes, endothelial cells and pericytes on proliferation and differentiation of adult murine precursor cells. Stem Cell Res 15(3): 514-521.
  3. 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.
  4. Wu, Z., Hofman, F. M. and Zlokovic, B. V. (2003). A simple method for isolation and characterization of mouse brain microvascular endothelial cells. J Neurosci Methods 130(1): 53-63.

简介

真菌形态发生需要细胞壁的修饰和可塑性,这意味着其组分(包括壳多糖和葡聚糖)的合成和重塑。因此几丁质酶和葡聚糖酶活性对于细胞壁生物发生和细胞分裂是至关重要的。几丁质酶活性的定量可能有助于鉴定可能负面影响一些丝状真菌如产生细胞内和分泌型几丁质酶的尖孢镰刀菌的生长和形态发生的结构缺陷。基于它们对壳多糖底物的酶作用将壳聚糖分解酶分类。内酯酶被定义为催化几丁质链中内部点的随机裂解的酶。外切球蛋白酶催化乙酰壳寡糖或N-乙酰葡糖胺从几丁质的非还原端逐步释放,因此分别称为壳聚糖酶和β-N-乙酰氨基葡糖苷酶。在这里,我们描述了一种简单的方法来轻易地纯化几丁质酶,以便比较不同的F的内切几丁质酶活性和外切几丁质酶活性。尖孢镰孢菌株。该方案可适用于任何真菌物种。...

关键字:神经科学, 神经球, 齿状回, 神经性的利基

材料和试剂

  1. 培养皿(直径5.5cm至9cm)
  2. Falcon管50ml和15ml
  3. Eppendorf管2ml(通过高压灭菌器灭菌)
  4. 巴斯德移液管(直径1mm)
  5. 刷(达芬奇,型号:Nova合成系列1570,#3)
  6. 12孔和24孔组织培养板(生长增强处理,γ-灭菌,不含热原,不含DNA/RNA,DNase/RNase)
  7. 滤盘(Sartorius AG,??目录号:FT-3-205-055)
  8. Falcon细胞过滤器40μm和100μm(Thermo Fisher Scientific,目录号:08-771-1和08-771-19)
  9. 用于24孔板,1μmPET(Merck Millipore Corporation,目录号:PIRP12R48)的悬挂插入件
  10. 27G针(Braun,Sterican TM ,目录号:4657705)
  11. 六周龄的小鼠
    注意:6-8只小鼠用于内皮和周细胞分离,至少1只小鼠用于神经球共培养测定
  12. 4周龄的小鼠
    注意:一次准备需要4只用于星形胶质细胞分离的小鼠。
  13. 蒸馏水
  14. 聚-D-赖氨酸氢溴酸盐(PDL)(Sigma-Aldrich,目录号:P7280)
  15. 层粘连蛋白(Sigma-Aldrich,Roche,目录号:R11243217001)
  16. 70%乙醇
  17. HBSS(Thermo Fisher Scientific,Gibco TM ,目录号:14175)
  18. 来自肠炎明串珠菌的葡聚糖平均分子量为150,000(Sigma-Aldrich,目录号:D4876)
  19. 没有谷氨酰胺的DMEM/F-12(储存在4℃)(Thermo Fisher Scientific,Gibco TM,目录号:21331020)
  20. 具有L-谷氨酰胺和15mM HEPES(储存在4℃)的DMEM/F-12(Thermo Fisher Scientific,Gibco TM ,目录号:11330032)
  21. B27(Thermo Fisher Scientific,Gibco TM ,目录号:0080085SA)
  22. MCDB131培养基(Thermo Fisher Scientific,目录号:10372-019)
  23. Glutamax(100×储液)(在4℃储存)(Thermo Fisher Scientific,CTS TM ,目录号:A12860-01)
  24. 胰蛋白转铁蛋白 - 硒(ITS)预混合物(-20℃保存)(Thermo Fischer Scientific,Corning TM,目录号:CB-40351)
  25. 非必需氨基酸(100×储液)(在4℃储存)(Thermo Fisher Scientific,Invitrogen TM,目录号:11140-035)
  26. Pen/Strep 100,000U/ml(-20℃)(Thermo Fisher Scientific,Gibco TM ,目录号:15140)
  27. 肝素(储存在4℃)(MP Biomedicals,目录号:0210193125)
  28. 胶原酶A(Sigma-Aldrich,Roche,目录号:10103578001)
  29. 胎牛血清(FBS)(-20℃)(Merck Millipore Corporation,Biochrom,目录号:S0613)
  30. 血浆来源的血小板血清(Sigma-Aldrich,目录号:P2918)
  31. 人EGF(在-20℃下储存等分试样)(PEPROTECH,目录号:AF100-15)
  32. 人类FGF碱性(在-20℃下储存等分试样)(PEPROTECH,目录号:100-18B)
  33. 人VEGF(储存在-20℃的等分试样)(PEPROTECH,目录号:100-20)
  34. Accutase(储存在4℃)(GENTAUR,PAA Laboratories GmbH,目录号:L11-007)
  35. 神经组织解离试剂盒(P)(Miltenyl Biotec,目录号130-092-628)
  36. 胰蛋白酶/EDTA(-20℃保存)(Merck Millipore Corporation,Biochrom,目录号:L2153)
  37. 含2%FBS的PBS(参见配方)
  38. 30%葡聚糖溶液(见配方)
  39. 内镜媒体(见配方)
  40. 星形胶质细胞(见食谱)
  41. 周细胞/平滑肌细胞培养基(见配方)
  42. 神经球媒体(见配方)

设备

  1. 解剖工具:剪刀,小刮刀,细弯曲镊子和手术刀(见图2B)
  2. 7ml dounce组织研磨机(Capitol Scientific,Wheaton,目录号:357542)
  3. 高压灭菌器
  4. 真空泵
  5. Dounce匀浆器
  6. 解剖显微镜(奥林巴斯,型号:SZ61)
  7. 倒置显微镜(Olympus,型号:CKX41)
  8. 刻度板插入显微镜(Olympus)的目镜
  9. 冷却离心机配备2ml管(Eppendorf,型号:5430R)
  10. 用于15ml管(Eppendorf,型号:5810R)的回转桶离心机
  11. 在37℃,5%CO 2/v/v的培养箱中
  12. 管旋转器或轨道振动器

程序

注意:有关隔离过程的一般概述,请参见图1。

  1. 一般准备
    1. 在从组织分离内皮细胞之前至少两天,制备4个聚-D-赖氨酸(PDL)/层粘连蛋白包被的12孔板(每个板包被至少3个孔)。为了制备孔,加入足够的PDL(在dH 2 O中5μg/ml)以包被表面并在室温下温育过夜。从盘中取出溶液,用dH 2 O洗涤该盘三次。让空气干燥。加入层粘连蛋白(5μg/ml,在冷DMEM:F12中),并在37℃下孵育过夜。去除层粘连蛋白,并立即使用板或与层粘连蛋白储存在-20°C,直到需要。
    2. 在星形胶质细胞分离之前至少一天制备PDL包被的平板(每个平板涂覆至少3个孔)。每孔加入足够的PDL(5μg/ml,在dH 2 O中)以包被表面,并在室温下温育过夜。从盘中取出溶液,用dH 2 O洗涤该盘三次。加入dH 2 O并在-20℃下冷冻或直接使用。
    3. 通过在火焰中旋转玻璃巴斯德移液管,准备具有"中"和"小"孔的火抛光移液管,直到边缘变圆。我们测量到我们的"小"孔大约为0.3-0.4mm,而"中"孔的内径大约为0.6-0.8mm。高压灭菌移液器消毒。

  2. 微血管内皮细胞和周细胞/血管平滑肌细胞(vSMC)的分离
    注意:隔离程序的概述可以在图1中找到。隔离程序的逐步图片系列可以在图2和3中找到。
    在共培养前两周,在6至8周龄时,从6只至8只小鼠分离内皮细胞和周细胞/vSMC。内皮细胞分离的方案从Wu等人2003年修改。
    1. 麻醉6-8成年小鼠根据适当的机构指南。执行颈椎脱位。
    2. 用70%乙醇喷洒头部以对该区域进行灭菌,以尽量减少毛皮的污染。
    3. 用锋利的剪刀将鼠标打断在脑干的基部。
    4. 沿着中线切开皮肤矢状,直到眼睛之间的任意点。暴露颅骨没有覆盖皮肤和皮下组织(图2C)。
    5. 通过将剪刀的每个刀片放入每个眼腔,在眼睛之间切开颅骨。接下来,在沿着矢状缝线穿过颅骨切开后,在颅骨的基部进行两个横向切割。
    6. 暴露大脑通过用剪刀或镊子回去的头骨,并用小刮刀从头骨去除大脑(图2D)。
    7. 用PBS冲洗大脑,转移到Falcon管用PBS/2%FBS,保存在冰上。
    8. 收集所有脑后,将一个脑转移到培养皿中,其中用填充有PBS/2%FBS的滤盘填充。
    9. 用中脑和小脑的弯钳钳(图2F)。
    10. 用细毛刷清除脑膜和任何可见表面血管,大血管将容易被捕获在滤盘上(图2G-H)。
    11. 用刷子去除胼the体的白质束(图2I)。
    12. 转移组织现在到一个新的培养皿,保持皮质组织在冰上,直到进一步处理。继续以相同的方式处理大脑的其余部分(图2J)。
    13. 处理所有脑样品后,用手术刀将皮质组织切成小块。使用带有松散杵的dounce匀浆器通过施加6-8次冲程使组织在2ml PBS/2%FBS中匀浆化(图2K-L和3A)。
    14. 混合组织匀浆(应该?3毫升)与3毫升30%葡聚糖,得到15%的最终浓度(图3B)。
    15. 将混合物转移到2ml Eppendorf管中,并在6℃下以4℃离心20分钟(图3C,离心后的管)。
    16. 用1ml移液管小心地除去厚的上清液或通过使用真空泵抽吸(图3D-E)。小心从壁上适当地去除组织匀浆。用1ml移液管通过快速吸移上下吸取,用含2%FBS的PBS重悬浮松散的沉淀(图3F)。
    17. 将100微米无菌尼龙细胞过滤器倒置在50ml管的顶部。将溶液过滤通过100μm过滤器,并用至少10ml含有2%FBS的PBS充分洗涤。标签管作为"流过1"并保持在冰上直到进一步处理(图3G)。
    18. 取100微米过滤器,将其翻转成标记为"周细胞/vSMC"的新的50ml管(图3H)。用至少10ml含有2%FBS的PBS充分洗涤过滤器(图3I)。
    19. 保持大容器在溶液(周细胞/vSMC)在冰上,直到离心
    20. 取流过1并过滤通过40μm无菌尼龙细胞过滤器(再次将其倒在50ml管上)(图3J),用含有2%FBS的PBS洗涤以除去单个细胞和碎片。
    21. 可以丢弃流出物,并且将40μm尼龙过滤器翻转并插入新的50ml管中。
    22. 用至少10ml含有2%FBS的PBS彻底洗涤尼龙膜,使所有微血管回到溶液中,标记管"内皮细胞"。
    23. 通过在4℃下以800×g离心5分钟来旋转两个管。
    24. 通过溶解1毫克/毫升胶原酶A在相应的生长培养基内生长和周细胞培养基中,而不添加生长因子制备胶原酶溶液。在37℃最好溶解(见配方3和4)。
      注意:胶原酶溶液的体积取决于我们使用的丸粒大小,每个丸/细胞类型3-4ml。
    25. 将来自步骤B23的容器沉淀溶解在各自的胶原酶溶液中。孵育在15毫升管中的溶液中的血管4-5小时,在37℃下管旋转器(图3K-L)。在用1ml移液管向上和向下用力吸取以破碎细胞团块之间。小心,容器不要连接到移液器吸头。每小时一次,5次上下,在大多数情况下是足够的。
    26. 在800×g下旋转细胞5分钟,除去上清液并将沉淀溶解在含有生长因子的相应生长培养基中。
    27. 将板内皮细胞在12孔板的一个PDL /层粘连蛋白包被的孔中,并在37℃,5%CO 2过夜,在24孔板的两个未涂布孔上形成周细胞/平滑肌细胞。
    28. 第二天将上清液转移到同样培养皿的新孔中,其也用PDL /层粘连蛋白(但是仍含有层粘连蛋白培养基,直到使用)包被内皮细胞或未包被于周细胞/vSMC。添加新鲜生长培养基与生长因子。
    29. 每3-4天更换培养基。小细胞需要几天,直到它们开始增殖。不要分裂细胞,直到转移到共培养悬挂插入。它们应当以高密度生长以增殖(图4显示初始细胞阶段)。

  3. 星形胶质细胞的分离
    注意:可以在图1中找到隔离程序的概述。初始步骤与神经球隔离的相同,您可以在Babu等人,2011年的视频中看到如何解剖海马。/em>
    1. 在共培养前两周从四只4周龄小鼠分离星形胶质细胞。
    2. 麻醉成年小鼠根据适当的机构指南。执行颈椎脱位。
    3. 用70%乙醇喷洒头部以对该区域进行灭菌,以尽量减少毛皮的污染。
    4. 用锋利的剪刀将鼠标打断在脑干的基部。
    5. 沿着中线切开皮肤矢状,直到眼睛之间的任意点。暴露颅骨没有覆盖皮肤和皮下组织。
    6. 通过将剪刀的每个刀片放入每个眼腔,在眼睛之间切开颅骨。接下来,在沿着矢状缝线穿过颅骨切开后,在颅骨的基部进行两个横向切割。
    7. 暴露大脑通过用剪刀或镊子回去的头骨,并用小刮刀从头骨去除大脑。
    8. 用PBS冲洗大脑,转移到含有PBS的塑料盘。
    9. 在低放大倍率下将含有脑的培养皿放置在解剖显微镜下,并将脑定位在其腹侧表面上。使用精细弯曲的钳子删除嗅球,同时通过小脑保持大脑的位置。
    10. 将大脑旋转到背部方面,并使用解剖刀在视交叉的水平做出冠状切口通过大脑。
    11. 将大脑的尾部置于培养皿中,使用手术刀沿纵向裂缝切开。
    12. 在解剖显微镜下,用镊子取出小脑和间脑。
    13. 通过用27 G针在海马结构周围滑动来移除海马。使用细镊子,从周围组织的海马,取出附加的髓磷脂床单(白质),可以沿着海马的内侧边缘和背面找到。
    14. 将解剖的海马放置在充满HBSS的冰上的Eppendorf管中,直到所有的海马解剖。
    15. 通过加入50μl酶P和1,900μl缓冲液X加2μlβ-巯基乙醇从神经元组织解离试剂盒制备酶混合物1。涡旋并在37°C预热混合物10-15分钟,然后使用。
    16. 从海马小心地取出HBSS,并使用手术刀刀片在培养皿中切碎组织,直到没有大块剩余。
    17. 使用转移移液管将切碎的组织转移到15ml Falcon管中,加入预热的酶混合物1.
    18. 在37℃下用管旋转器连续振荡孵育15分钟。
    19. 通过加入40μl缓冲液Y和20μl酶A从神经元组织解离试剂盒制备酶混合物2
    20. 添加酶混合物2.
    21. 使用中型火抛光移液管缓慢研磨10次。
    22. 在37℃孵育10分钟。
    23. 使用小型火抛光移液器缓慢研磨10次。
    24. 通过加入HBSS缓冲液至终体积为10ml来洗涤细胞。
    25. 将溶液中的细胞应用于40μm细胞过滤器,用额外的4ml HBSS洗涤过滤器。
    26. 将细胞在15ml管中以300×g离心5分钟
    27. 除去上清液,并用10ng/ml EGF和2%FBS在适当量的星形胶质细胞培养基(见配方5)中重悬沉淀。
    28. 种子在12孔板的PDL包被的板的一个孔上,并在37℃下用5%CO 2孵育。
    29. 36小时后取出培养基,用预热的PBS彻底洗涤剩余的贴壁细胞,加入含有5ng/ml EGF和0.5%FBS的新鲜培养基。
    30. 每3天更换培养基,在共培养实验中使用前不要传代细胞(见图5代表性图像)
  4. 将小生境共培养细胞接种到细胞插入物中
    1. 在共培养前两天用accutase或胰蛋白酶从不同的制剂中分离细胞 注意:我们使用accutase星形胶质细胞制剂和胰蛋白酶内皮和周细胞制备。
    2. 种子?5,000个细胞/悬挂插入物在它们各自的生长培养基中用于不同的细胞培养物(每个插入物约100μl,参见用于培养基组合物的配方3-5),没有培养基放置在悬挂插入物下面的孔中。
    3. 在共培养前5-10小时,从悬挂的插入物(通过抽吸或用移液管)中取出相应的生长培养基,并且用不含任何生长因子的B27(参见配方6)替换DMEM。
  5. 隔离海马前体细胞神经球共培养测定
    神经前体细胞的分离程序根据Babu等人的方案。 (2011),Walker和Kempermann(2014)轻微修改。
    1. 麻醉一个成人6至8周龄小鼠根据适当的机构指南。执行颈椎脱位。
    2. 用70%乙醇喷洒头部以对该区域进行灭菌,以尽量减少毛皮的污染。
    3. 用锋利的剪刀将鼠标打断在脑干的基部。
    4. 沿着中线切开皮肤矢状,直到眼睛之间的任意点。暴露颅骨没有覆盖皮肤和皮下组织。
    5. 通过将剪刀的每个刀片放入每个眼腔,在眼睛之间切开颅骨。接下来,在沿着矢状缝线穿过颅骨切开后,在颅骨的基部进行两个横向切割。
    6. 暴露大脑通过用剪刀或镊子回去的头骨,并用小刮刀从头骨去除大脑。
    7. 用PBS冲洗大脑,转移到含有PBS的塑料盘。
    8. 在低放大倍率下将含有脑的培养皿放置在解剖显微镜下,并将脑定位在其腹侧表面上。使用精细弯曲的钳子删除嗅球,同时通过小脑保持大脑的位置。
    9. 将大脑旋转到背部方面,并使用解剖刀在视交叉的水平做出冠状切口通过大脑。
    10. 对于齿状回的显微切片(更多细节参见Walker和Kempermann,2014)将脑的尾部置于培养皿中,并使用手术刀沿着纵向裂缝切割。
    11. 用钳子在解剖显微镜下取下小脑和间脑。
    12. 要去除齿状回,插入27 G针头的尖端,沿齿状回和Ammon号角之间的边界滑动。使用细镊子,从周围的组织取牙。
    13. 将解剖的齿状回置于带有HBSS的培养皿中的冰上。
    14. 通过加入25μl酶P和950μl缓冲液x加1μlβ-巯基乙醇从神经元组织解离试剂盒制备酶混合物1。涡旋并在37°C预热混合物10-15分钟,然后使用。
    15. 取出HBSS,并使用手术刀刀片切碎组织,直到没有更大的碎片剩下。
    16. 将切碎的组织从一只老鼠转移到15毫升Falcon管,并加入预热的混合物1
    17. 在37℃下用管旋转器连续振荡孵育15分钟。
    18. 通过加入20μl缓冲液Y和10μl酶A从神经元组织解离试剂盒制备酶混合物2
    19. 添加酶混合物2.
    20. 使用中型火抛光移液管缓慢研磨10次。
    21. 在37℃下用管旋转器连续振荡孵育10分钟。
    22. 使用小型火抛光移液器缓慢研磨10次。
    23. 通过加入HBSS缓冲液至终体积为10ml来洗涤细胞。
    24. 将溶液中的细胞应用于40μm细胞过滤器,用额外的4ml HBSS洗涤过滤器。
    25. 离心在15ml管中的细胞在300×g离心5分钟。
    26. 除去上清液并将沉淀重悬在适量的DMEM培养基中(参见配方6),加入20ng/ml EGF,20ng/ml bFGF和2μg/ml肝素。
      注意:根据将要分析多少共培养条件,滴定培养基的体积,使得每24孔接种足够但不太多的细胞,否则球体开始融合。每24孔需要使用至少500μl培养基以接触悬挂细胞插入物
  6. Hippocampal niche neurosphere co-cultural
    1. 转移500微升神经细胞悬液(从步骤E26)在含有生长因子的DMEM培养基中的24孔板的每个孔。
      注意:接种的孔数取决于共培养条件的数量;在我们的实验中,我们将来自一只动物的神经球接种到24孔板的12个孔中(使用6种不同的共培养条件)。不要忘记一个或两个参考井无共同培养的细胞,其中只有干细胞和祖细胞接种,但只有一个空悬挂插入物与培养基放置在顶部。这些参考孔中的神经球的生长可以作为基线,以评价利基共培养物的影响。
    2. 在37℃下用5%CO 2孵育30分钟
    3. 在来自分离的细胞沉降后,将具有合适细胞的悬挂插入物添加到没有生长因子的DMEM/B27中,并在37℃下用5%CO 2孵育10天。 10天后取出悬挂的插入物并在倒置显微镜上使用具有刻度板内部的10x物镜计数和测量球体。
      注意:为了更容易地分析24孔板表面上的球体数量绘制网格线,或使用具有网格线的透明度(图6)。
    4. 对于分化的进一步分析,从孔中移除含有球体的培养基,并在300×g离心5分钟。
    5. 重悬在含有B27但不含生长因子的DMEM培养基中,并转移到具有玻璃底部或含有盖玻片(都需要用层粘连蛋白包被)的24孔板中。
    6. 在37℃,5%CO 2下大约7天后,可以分析成神经元和星形胶质细胞。因此,用4%多聚甲醛(预热至37℃)和针对Map2ab和GFAP的染色来固定细胞(详见Walker和Kempermann,2014)。

代表数据



图1.神经球共培养测定的分离范例。图示概述了原代细胞分离模式和组织来源,其可以用于这种神经球共培养测定。外周细胞/血管平滑肌细胞(vSMC)和内皮细胞可以从皮层分离,星形胶质细胞从海马中分离,齿状回用于分离神经球。将细胞接种到transwell悬挂插入物中,而将分离的干细胞接种在下面

图2.第1部分 - 内皮细胞和周细胞/vSMC的分离。内皮细胞和周细胞/vSMC方案的逐步程序。 A.葡聚糖的无菌过滤。 B.隔离所需的一套仪器。 C.矢状切开通过头骨和剥皮的皮肤和头骨。 D.从头骨中除去大脑。 E.冠状动脉用手术刀切开大脑。 F.用镊子摘下中脑。 G.用细毛刷除去可见表面血管。 H.刷牙后在表面上看不见血管。 I.用刷子去除髓磷脂片(胼(体的白质)。 J.在冰上用PBS/FBS将处理过的皮质转移到新的培养皿中。 K.用手术刀切割皮质。 L.将组织转移到douncer。


图3.第2部分 - 内皮细胞和周细胞/vSMC的分离周细胞/vSMC和内皮细胞分离的逐步程序的第二部分。 A.用松散的杵均质化douncer中的组织。 B.将组织与30%葡聚糖混合1:1。离心后,可以在底部观察到红色血管沉淀,并且组织匀浆浮在顶部。 D.小心吸出组织匀浆和蔗糖。 E.可以在底部找到清洁的容器颗粒。 F.在PBS/FBS中重悬血管沉淀。 G.通过100微米的尼龙过滤器过滤容器在溶液中,上下颠倒。 H.取一个新的管,翻转在上面安全地转移大容器到它。 I.冲洗过滤器,直到所有容器都移回溶液中。 J.从上一步转移到40μm过滤器。在大小容器级分离心后,可以发现颗粒。这些颗粒现在重悬在胶原酶溶液中消化。 L.将容器准备的试管转移到培养箱中的旋转振荡器中

图4.血管消化后的内皮细胞和周细胞培养物。在早期制备时间点期间内皮细胞和周细胞/vSMC的代表性图像。在内皮细胞(上图)分离和周细胞/vSMCs分离的关键步骤期间拍摄的图像(下图)。比例尺,10μm


图5.星形胶质细胞分离。在早期制备时间点的星形胶质细胞的代表性图像。在星形胶质细胞分离的关键步骤期间拍摄的图象。比例尺,10μm


图6.神经球共培养分析。通过放置网格线进行神经球共培养分析。左,24孔板下的网格线。中间,一口井的特写镜头视图。右,通过显微镜用可见的网格线查看。比例尺,500μm

笔记

为了确认不同的原代细胞制备物的富集和可持续性,总是将一些细胞接种到PDL /层粘连蛋白包被的载玻片上以通过免疫细胞化学来评估制备,或取样品用于RNA分离(详见原始出版物Ehret等人。,2015)。

食谱

  1. 含2%FBS的PBS
    将1ml无菌过滤的FBS加入到49ml高压灭菌的PBS中
  2. 30%葡聚糖溶液 将0.9g葡聚糖(平均分子量150000)溶于3ml HBSS中
    在振荡器上溶解过夜,用尖端过滤器过滤灭菌并在4℃下储存最多3天
  3. 内皮细胞培养基(用于内皮细胞培养)
    MCDB131媒体
    5%血浆来源的血小板血清
    15 U/ml肝素 ITS预混物(5μg/ml胰岛素,5μg/ml转铁蛋白和5ng/ml硒)(100×储液)
    100U/ml青霉素/链霉素(100x原液) 使用前,加入10 ng/ml EGF,10 ng/ml FGF碱性和5 ng/ml VEGF
  4. Pericyte/vSMC媒体
    含glutamax的DMEM(100x储备液)
    10%FBS
    0.1mM非必需氨基酸(100x原液)
    100U/ml Pen/Strep(100×储备液) 在使用前,加入10 ng/ml VEGF
  5. 星形细胞媒介
    含谷氨酰胺和HEPES的DMEM
    Glutamax(100x股票)
    B27(50x股票)
    生长因子(EGF和FBS)在使用前加入,对于不同浓度,参见步骤C27-29
  6. 神经球媒体
    不含谷氨酰胺的DMEM
    B27(50x股票)
    Glutamax(100x股票)
    笔/Strep(100x股票)
    在即将使用之前加入生长因子(EGF和FGF各20μg/ml)

致谢

该方案用于我们先前发表的研究中(Ehret等人,2015)。这项工作由DZNE的基本机构赠款资助。

参考文献

  1. Babu,H.,Claasen,JH,Kannan,S.,Runker,AE,P??almer,T。和Kempermann,G(2011)。  用于从小鼠齿状回分离和富集单层培养神经前体细胞的方案。前脑神经瘤 5 :89.
  2. Ehret,F.,Vogler,S.和Kempermann,G.(2015)。 
  3. Walker,TL和Kempermann,G.(2014)。  一只小鼠,两种培养物:从单个小鼠的两个神经源区分离和培养成熟神经干细胞。(84):e51225。
  4. Wu,Z.,Hofman,FM和Zlokovic,BV(2003)。  一种用于分离和表征小鼠脑微血管内皮细胞的简单方法。 J Neurosci Methods 130(1):53-63。
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引用:Ehret, F., Vogler, S. and Kempermann, G. (2016). Neurosphere Co-culture Assay. Bio-protocol 6(15): e1883. DOI: 10.21769/BioProtoc.1883.
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