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Mimicking Angiogenesis in vitro: Three-dimensional Co-culture of Vascular Endothelial Cells and Perivascular Cells in Collagen Type I Gels
体外模拟血管发生:血管内皮细胞和血管周围细胞在I型胶原蛋白凝胶中的三维共培养   

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Stem Cells
May 2016

Abstract

Angiogenesis defines the process of formation of new vascular structures form existing blood vessels, involved during development, repair processes like wound healing but also linked to pathological changes. During angiogenic processes, endothelial cells build a vascular network and recruit perivascular cells to form mature, stable vessels. Endothelial cells and perivascular cells secret and assemble a vascular basement membrane and interact via close cell-cell contacts. To mimic these processes in vitro we have developed a versatile three-dimensional culture system where perivascular cells (PVC) are co-cultured with human umbilical cord vascular endothelial cells (HUVEC) in a collagen type I gel. This co-culture system can be used to determine biochemical and cellular processes during neoangiogenic events with a wide range of analyses options.

Keywords: Endothelial cells (内皮细胞), Perivascular cells (血管周围细胞), Pericytes (周细胞), Angiogenesis (血管生成), Co-culture (共培养), Collagen gel (胶原蛋白凝胶)

Background

The coordinated interaction between endothelial and perivascular cells is essential to form a stable vascular network according to the local needs within a given tissue. Multiple molecular components contribute to the interactions but are still poorly understood. Various growth factors are needed to attract endothelial cells to sites of low oxygen concentrations and build new vessels that are then covered by perivascular cells. Both cell types interact to secrete a specialized extracellular matrix and stabilize the newly formed vessels. In the past multiple assays have been established to analyze vascular cell interaction and vessel-like network formation on two-dimensional matrigel substrates, but those are limited in providing information about initial steps of endothelial-perivascular cell interaction and vascular basement membrane formation in a three-dimensional microenvironment. In addition, well characterized perivascular cell suitable for culture experiments were missing.

We have previously isolated cells with perivascular characteristics, as they express pericyte-specific markers, produce and secrete extracellular matrix proteins and stimulate angiogenic processes in vivo (Brachvogel et al., 2005 and 2007). These cells were used to establish a co-culture system with human umbilical vein endothelial cells and study critical steps in neoangiogenesis upon interaction of the two cell types in a three-dimensional microenvironment (Pitzler et al., 2016; Zhou et al., 2016).

The co-cultures showed a superior activity to promote the formation endothelial tube formation and stabilization by perivascular cell recruitment and basement membrane formation. The culture system allows to monitor migration and interaction of vascular cells by time-lapse microscopy and to study the deposition of basement membrane protein by immunofluorescence analysis. To quantify and isolate endothelial and perivascular cells from co-cultures immunomagnetic or flow cytometry sorting approaches can be used and cell type-specific global changes in mRNA and miRNA expression can be analyzed by subjecting isolated RNA from the separated cell populations to microarray analysis or RNA sequencing. Moreover, the effects of anti- and pro-angiogenic substances on endothelial-perivascular cell interaction can be analyzed in vitro. Hence, the three-dimensional culture protocol allows to study cellular, biochemical and transcriptional events during neoangiogenesis and to characterize the pro- and anti-angiogenetic effects of molecules on endothelial perivascular cell interaction ex vivo.

Materials and Reagents

  1. 15 ml tubes (Greiner Bio One International, catalog number: 188271 )
  2. 100 mm TC-treated cell culture dish (Corning, Falcon®, catalog number: 353003 )
  3. 1.5 ml tubes (SARSTEDT, catalog number: 72.690.001 )
  4. 24-well-plate (Corning, Costar®, catalog number: 3524 )
  5. T75-flask (VWR, catalog number: 734-0050 )
  6. 48-well-plate (Corning, Costar®, catalog number: 3548 )
  7. 10 ml syringe (BD, catalog number: 309604 )
  8. Sterile syringe filter with a 0.22 µm pore size (EMD Millipore, catalog number: SLGP033RB )
  9. Disposable HSW FINE-JECT® needles (Henke Sass Wolf, catalog number: 4710004012 )
  10. Pipettes Gilson (10 µl, 20 µl, 100 µl, 1,000 µl, 5 ml)
  11. Macro pipet tips (VWR, catalog number: 89368-994 )
  12. Microscope cover glasses: squares (Fisher Scientific, catalog number: S175222 )
  13. Adhesion slides, Menzel Gläser, SuperFrost® Plus (VWR, catalog number: 631-9483 )
  14. HUVEC (human umbilical cord vascular endothelial cell) (Lonza, catalog number: CC-2519 or self-isolation of HUVECs according to Baudin et al., 2007)
  15. Isolated perivascular cells/pericytes (PVC) (see Note 1)
  16. AnxA5-LacZ mice (Anxa5tm1Epo) (Brachvogel et al., 2003)
  17. Collagenase type II (Worthington Biochemical, catalog number: LS004176 )
  18. DNase I recombinant (Roche Diagnostics, catalog number: 04536282001 )
  19. Fluorescein-di-(β-D-galactopyranoside) (FDG) (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: F1179 )
  20. Propidium iodide (PI) (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: P3566 )
  21. Dulbecco’s modified Eagle medium (DMEM) (Thermo Fisher Scientific, GibcoTM, catalog number: 31966021 )
  22. Fetal calf serum (FCS) (Biochrom, catalog number: S 0115 )
  23. Penicillin-streptomycin (P/S) (10,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  24. Gelatin from porcine skin (Sigma-Aldrich, catalog number: G2500 )
  25. Sodium bicarbonate (NaHCO3) (EMD Millipore, catalog number: 1.6329.1000 )
  26. Rat tail type I collagen solution (Corning, catalog number: 354236 )
  27. Sodium hydroxide (NaOH) (VWR, catalog number: 28244.295 )
  28. Phosphate-buffered saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 18912014 )
  29. Trypsin/EDTA (Biochrom, catalog number: L 2153 )
  30. Endothelial growth medium 2 (EGM2) (PromoCell, catalog number: C-22111 )
  31. Recombinant VEGF-A/Vascular endothelial growth factor (Biomol, catalog number: 94900 )
  32. Recombinant PDGF/Platelet-derived growth factor-BB (Biomol, catalog number: 94968 )
  33. Ascorbic acid phosphate (Sigma-Aldrich, catalog number: A8960-5G )
  34. Methanol (VWR, catalog number: 20903.461 )
  35. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: D8418-100ML )
  36. Tween-20 (Sigma-Aldrich, catalog number: P1379-500ML )
  37. Bovine serum albumin (BSA) (SERVA Electrophoresis, catalog number: 11930.03 )
  38. Anti-human CD31 (BD, BD Biosciences, catalog number: 555444 )
  39. Anti-NG2-proteoglycan (EMD Millipore, catalog number: AB5320 )
  40. Donkey-anti-rat Ig, Cy2 conjugated (Jackson ImmunoResearch, catalog number: 712-225-150 )
  41. Donkey, anti-rabbit Ig, Cy2 conjugated (Jackson ImmunoResearch, catalog number: 711-225-152 )
  42. Donkey, anti-rabbit Ig, Cy5 conjugated (Jackson ImmunoResearch, catalog number: 711-175-152 )
  43. Donkey, anti-goat Ig, Cy2 conjugated (Jackson ImmunoResearch, catalog number: 705-225-147 )
  44. Mounting solution
  45. CFSE (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: C1157 )
  46. SNARF-1 (Thermo Fisher Scientific, Molecular ProbesTM, catalog number: S22801 )
  47. MCDB-131 medium (Thermo Fisher Scientific, GibcoTM, catalog number: 10372019 )
  48. Basic fibroblast growth factor (bFGF) (ReliaTech, catalog number: 300-003 )
  49. Epidermal growth factor (EGF) (Biomol, catalog number: 97052 )
  50. Insulin solution human (Sigma-Aldrich, catalog number: I9278 )
  51. Heparin
  52. Hydrocortisone
  53. 10x DMEM, 10x Dulbecco’s modified Eagle’s medium (Sigma-Aldrich, catalog number: D2429-100ML )
  54. Sodium pyruvate (Thermo Fisher Scientific, GibcoTM, catalog number: 11360070 )
  55. Trizma® base (Sigma-Aldrich, catalog number: T1503 )
  56. Sodium chloride (NaCl) (EMD Millipore, catalog number: 1064041000 )
  57. Autoclaved distilled H2O
  58. Proliferation medium (see Recipes)
  59. PVC culture medium (see Recipes)
  60. HUVEC culture medium (see Recipes)
  61. 2x medium-mix (see Recipes)
  62. Collagen solution (see Recipes)
  63. Tris-buffered saline (TBS) (see Recipes)

Equipment

  1. Centrifuge (Eppendorf, model: 5415 R )
  2. Hemocytometer (LO-Laboroptik, model: Neubauer-Improved )
  3. CO2 cell incubator (Heraeus)
  4. Centrifuge Labofuge (Heraeus Sepatech, model: Labofuge GL )
  5. Autoclave
  6. SteREO Lumar.V12 (Zeiss, model: SteREO Lumar.V12 )
  7. Axiovert 40 CFL (Zeiss, model: Axiovert 40 CFL )
  8. Axioplan 2 (Zeiss, model: Axioplan 2 )
  9. Water bath (Köttermann)
  10. Vortex mixer (Scientific Industries)
  11. Inverted microscope Eclipse TE2000-U (Nikon Instruments, model: Eclipse TE2000-U )
  12. Fluorescence-activated cell sorting (MoFlow, Cytomation)
  13. Biosafety cabinet LaminAir HB 2448 (Heraeus, model: LaminAir HB 2448 )

Software

  1. ImageJ (Fiji)

Procedure

  1. Original protocol for pericyte isolation from meninges using AnxA5-LacZ mice
    1. Prepare approximately 200 ml of 5% FCS-PBS and cool down 100 ml of 5% FCS-PBS on ice.
    2. Carefully isolate the brain from 4-6 months old AnxA5-LacZ mice. Dissect the meninges under a stereomicroscope and transfer them into a 15 ml tube with 5% FCS-PBS on ice. Collect several meninges and then centrifuge the sample for five min at 400 x g at 4 °C.
    3. Aspirate supernatant and dissolve pellet in 1.5-3.0 ml of 0.4% collagenase type II. Incubate sample for 60 min at 37 °C slightly shaking. Subsequently, centrifuge digested tissue for five min at 400 x g at room temperature.
    4. Aspirate supernatant and resuspend in 1.5-3.0 ml of 2% trypsin/100 U DNAse I. Then, incubate digested tissue for 30 min at 37 °C slightly shaking. Next, centrifuge for five minutes at 400 x g at room temperature.
    5. Resuspend cell pellet in 5% FCS-PBS and filter cell suspension through a 100 μm cell strainer. After that, centrifuge cell suspension for 5 min at 400 x g at room temperature.
    6. Aspirate supernatant and resuspend cell pellet in 5% FCS-PBS. Determine cell number by using a hemocytometer, then dissolve 1 x 106 cells in 20 µl of 5% FCS-PBS and distribute 20 µl of the cell suspension into new 1.5 ml tubes. Add 20 µl of 2 mM fluorescein-di-(β-D-galactopyranoside) (FDG) to 20 µl cell suspension and incubate mix at 37 °C for 75 sec. Add 500 µl of ice-cold 5% FCS-PBS to the cells and incubate on ice in the dark for 3 h. Use a non-stained sample as control for cell sorting.
    7. Centrifuge cell suspension for 5 min at 400 x g at RT and resuspend cell pellet in 500 µl of 5% FCS-PBS. To exclude dead cells, add propidium iodide (final concentration 1 µg/ml)
    8. Sort stained cells by fluorescence-activated cell sorting (MoFlow, Cytomation) and collect propidium iodide negative and FDG positive cells. Next, plate 5 x 104 of PI negative/FDG positive cells per well in proliferation medium (see Recipes) on gelatin-coated 24-well-plate and store cells in a 5% CO2 humidity incubator at 37 °C. Change medium every second day and expanse isolated cells.
    9. After initial expansion, the cells are cultured in DMEM with 10% FCS and 1% P/S. Cells should be further characterized by flow cytometry. Cells are positive for Sca-1, CD140b and α-SMA and negative for PECAM, CD45, CD34, F4/F80 and c-kit. Isolated Anxa5-LacZ(+) PVC from mouse meninges retain their perivascular cell phenotype and can be cultured without undergoing senescence for multiple passages (Brachvogel et al., 2007).
      Note: An established and well characterized PVC cell line with superior angiogenic activity can be send to individual researcher upon request to perform coculture experiments with endothelial cells (Zhou et al., 2016). This eliminates the need to isolate the cells from mouse brain meninges.

  2. Co-culture of PVCs and HUVECs in collagen type I gels
    1. Dissolve 0.1 g of gelatin in 100 g of autoclaved distilled water (final concentration 0.1%) and autoclave the solution at 121 °C for 30 min. Then, coat 100 mm TC-treated cell culture dish with gelatin by adding 5 ml of 0.1% gelatin solution to the dish and distribute gelatin solution until the whole 100 mm TC-treated cell culture dish surface is fully covered. After at less two hours’ incubation at 37 °C, the residual gelatin solution is aspirated. Subsequently, dry dish at room temperature before use.
    2. HUVECs are cultured in EGM2 culture medium (see Recipes) on gelatin-coated plates in a 5% CO2 humidity incubator at 37 °C. Cells are cultured until subconfluent (~80%) and further passaged (1:3 dilution). HUVEC are only used in early passages (passage 3-6). PVCs are cultured in DMEM with 10% FCS and 1% P/S (see Recipes) on T75-flasks or on 10 cm dishes and expand to about 80% confluency (see Note 2 and Figure 1).


      Figure 1. Cultured perivascular cells (PVC) and human umbilical cord vein endothelial cells (HUVEC) with different cell densities. Representative phase contrast images of 80% and 100% confluency (cell density) cultured PVCs and HUVECs. The cell density plays an important role for this co-culture experiment and can affect the tube formation. Bars = 100 µm.

    3. Prepare 10 ml of 2x medium-mix (see Recipes). Therefore, dissolve 0.38 g of NaHCO3 in 10 ml of autoclaved distilled water (concentration 0.45 M) and after dissolving sterile filter the solution. Then, mix 2 ml of 10x DMEM, 200 µl of 100x sodium pyruvate and 1 ml of prepared 0.45 M NaHCO3 (final concentration 0.045 M) in 6.8 ml (final volume 10 ml) of autoclaved distilled H2O. Solution should be cool on ice until use.
    4. For 4 ml of collagen solution (see Recipes), mix 2 ml of 2x medium-mix, 800 µl FCS (final concentration 20%), 963 µl of 8.3 mg/ml rat tail type I collagen (see Note 3) (final concentration 2 mg/ml) and 23 µl of autoclaved distilled H2O on ice. Finally, neutralize collagen type I gel solution with 214 µl of 0.1 N NaOH on ice and adjust pH of solution to 7.4. Store the prepared collagen solution on ice until usage.
    5. Wash sub-confluent, cultured HUVECs and PVCs with 10 ml of PBS and detach cells with 1-2 ml trypsin/EDTA for 5 min at 37 °C. Add 9 ml of EGM2 culture medium to stop reaction and collect cells in a 15 ml tube.
    6. Centrifuge cells for 5 min at 400 x g at room temperature (RT). Aspirate supernatant and resuspend cell pellets in 5 ml of EGM2 culture medium. Add 10 µl of each cell suspension into a hemocytometer to determine cell concentration.
    7. For monoculture, transfer 6.25 x 105 HUVECs or 1.25 x 105 PVCs (see Note 4) in EGM2 medium in a new 15 ml tube. Mix 6.25 x 105 HUVECs and 1.25 x 105 PVCs for co-culture in 2 ml EGM2 medium in a new 15 ml tube (Figure 2). Alternatively, different endothelial cells or perivascular cells can be used, but the relative ratio of cells need to adapted and tested. Technical duplicates or triplicates are highly recommended. Centrifuge cell suspensions at 400 x g for 5 min at RT.


      Figure 2.  Workflow of the three-dimensional co-culture assay

    8. Carefully remove all medium and resuspend cell pellets in 1 ml of collagen type I gel solution. Add 200 µl into each well of a 48-well plate and incubate for 60 min at 37 °C until solutions turn into gel. To increase homogeneity of gels, collagen type I gel solution with cells should be properly mixed and gel solution be uniformly disturbed in the well. Try to prevent uneven distribution of the solution by adding the gel solution directly in the center of the well and distribute by moving the 48-well plate carefully. To improve gelling, 48-well plates should be prewarmed in the incubator at 37 °C
    9. Check after 60 min whether a homogenous gel has formed. Otherwise incubate additional 30 min. After gelling, carefully add 400 µl EGM2 culture medium supplemented with 10 ng/ml VEGF, 10 ng/ml PDGF and 250 μg/ml ascorbic acid phosphate on top of each gel. Other growth factors or drugs may be added at this stage for different experimental conditions.
    10. Incubate the gels for 3-6 days (see Note 6) in a 5% CO2 humidity incubator and take images (Figure 3) or videos (3 images/h) every 24 h. We used 10 or 20x objectives to take images of networks (Zeiss SteREO Lumar.V12 or Zeiss Axiovert 40 CFL). Higher magnification can be used but the thickness of the gel can hinder the acquisition of images. EGM2 culture medium with supplements should be replaced every 48 h.


      Figure 3. Tube formation studies of mono and co-culture HUVECs in a three-dimensional collagen type I gel. After 96 h, HUVECs form in present of perivascular cells a robust tube network in a collagen type I gel, whereas monocultured HUVECs few tubes. Representative phase contrast images were taken with 20x objective. Bars = 100 µm.

  3. Immunohistochemical detection of extracellular matrix and cellular proteins in 3D cultures
    1. Wash gels twice with 600 µl of PBS (see Note 6).
    2. Fix gels with 400 µl of 80% methanol/20% DMSO (Dent’s Fixative) for 30 min at RT.
    3. Rehydrate gels with 600 µl of 50% methanol/PBS for 1 h at RT. Remove solution and add 600 µl of 20% methanol/PBS. Next, wash gel in 600 µl of PBS-T (PBS, 0.1% Tween-20) for 1 h at RT.
    4. Aspirate solution and add 600 µl of 10% FCS, 5% BSA in PBS for 2-4 h at RT to block non-specific antibody-binding.
    5. Carefully remove blocking solution and incubate gels with 150-300 µl of primary antibody diluted in blocking solution (10% FCS, 5% BSA in PBS), overnight at 4 °C lightly shaking on a rocking plate. Antibody dilutions should be tested empirically before.
    6. Wash gels with 600 µl of TBS-T (0.1% Tween in TBS) for an hour. Repeat washing steps six times.
    7. Incubate samples with 150-300 µl of fluorescent chromophore-conjugated, secondary antibodies in blocking solution for 2-16 h in the dark at RT.
    8. Wash gels with 600 µl of TBS-T (0.1% Tween in TBS) for an hour. Then, repeat washing step six times.
    9. Carefully remove gels from the wall of the well using a 27 G needle. Use a 5 ml macro pipet tips to transfer gels on slides. Add 50 µl of mounting solution on top of the gels and carefully mount samples with a glass coverslip.
    10. Images are taken with Zeiss Axioplan 2 by using 5x and 63x objectives and adequate filter sets to get overview and close-up images, respectively. To visualize HUVECs in three-dimensional cultures gels can be immunostained with CD31 (Figures 4A and 4B) and PVCs can be immunostained with NG2-PG (Figure 4B) or α-SMA specific antibodies. The overview images of CD31+ HUVECs are used for tube formation analysis.


      Figure 4. Endothelial tube formation of human umbilical cord vein endothelial cells (HUVEC) was induced upon co-coculturing with PVC. A. HUVECs are cultured either alone (monoculture) or in co-culture with PVC in a three-dimensional collagen I gel. After 6 days in culture, the three-dimensional collagen I gels were stained with human CD31 as described in Procedure C. Scale bars = 500 µM. B. Immunostainings of co-cultured HUVECs and PVCs using CD31 and NG2-proteoglycan (NG2-PG) specific antibodies. Individual or as merged images with nuclear staining (DAPI) are shown. Scale bars = 100 µm.

  4. Cell labelling with fluorescent dyes (CFSE, SNARF-1) for live cell tracking
    1. Dissolve CFSE or SNARF-1 in DMSO to a final concentration of 5 mM at RT in the dark. After using fluorescent dyes, solution can be aliquoted and stored at -20 °C. For subsequent use of fluorescent dyes, thaw CFSE and SNARF-1 dye at RT in the dark. Freshly prepare 100 ml of 3% FCS-PBS solution. Store 25 ml at RT and cool the remaining 75 ml on ice for at least 30 min.
    2. Detach HUVECs and PVCs as described (see step A3) and collect cells in 15 ml tubes. Determine cell number using a hemocytometer, then centrifuge cells for 5 min at 400 x g at RT.
    3. Resuspend cell pellets in an appropriate amount of 3% FCS-PBS to a final concentration of 1 x 107 cells per 1 ml of 3% FCS-PBS. Do not use less than 50 µl of 3% FCS-PBS solution.
    4. Prior to use, prepare 0.5 mM solutions of CFSE and SNARF-1 using PBS. Then, further dilute CFSE or SNARF-1 solutions (0.5 mM) using cell suspensions to a final concentration of 0.01 mM. Typically, 2 µl of 0.5 mM CFSE are added to 100 µl cell suspension. All following steps are performed in the dark avoiding light exposure.
    5. Mix HUVECs and PVCs for 10 sec at maximum speed on a vortex mixer. Then, incubate cell suspensions for 10 min in a water bath, pre-heated to 37 °C, without shaking.
    6. Add 5 ml of ice cold 3% FCS-PBS to stop the reaction and centrifuge cells for 5 min at 400 x g at 4 °C. Aspirate supernatant and resuspend pellets in 5 ml of ice cold 3% FCS-PBS. Centrifuge cells for 5 min at 400 x g at 4 °C. After centrifugation, aspirate supernatant and resuspend pellet again in 5 ml of ice cold 3% FCS-PBS. Centrifuge cell suspensions for additional 5 min at 400 x g at 4 °C.
    7. Resuspend cells in an appropriate amount of EGM2 culture and proceed with step A4. Reduce light exposure during the subsequent steps and images (Figure 5) should be taken on an inverted microscope Eclipse TE2000-U (Nikon) or SteREO Lumar.V12 (Zeiss) over time. Here, a 20x objective was used for visualization. For each image, a phase contrast and fluorescence images in adequate channels (e.g., FITC-channel for CFSE labelled cells) were taken. SNARF-1 is normally used for detecting initial tube formation (a few hours after gelling). Later SNARF-1 labelling is difficult to detect. This system can be used to track the recruitment of perivascular cells to the endothelial cells during initial angiogenesis steps.


      Figure 5. Co-culture of human umbilical cord vein endothelial cells (HUVEC) and CFSE labelled perivascular cells (PVC) to track perivascular cells in three-dimensional condition. PVCs were labelled with CFSE as described in Procedure D and subsequently co-cocultured with HUVECs in a three-dimensional collagen type I gel. After 72 h in culture, phase contrast image and fluorescence images were taken with a 20x objective. The brightness and contrast of the CFSE fluorescent and merged image were adjusted for visualization. Scale bars = 100 µm. 

Data analysis

  1. The program ImageJ (Fiji) was used for image analysis.
  2. Open image file of interest (File → Open → Select image).
  3. Set scale. Choose ‘Arrow Tool’ in the toolbar and draw an arrow along the scale bar in the image. Next, click Analyze → Set Scale to open a new window, in which the known distance and unit of the length should be adjusted based on the distance and unit lengths acquired from the scale bar arrow. After, click ‘OK’.
  4. Go to toolbar and double click on ‘Arrow Tool’ to open a window. Choose a color for the arrow and click ‘keep after adding to overlay’. Next, click ‘OK’.
  5. Choose ‘Arrow Tool’ and draw an arrow along the tubes. Click Analyze → Measure (or STRG+M) to open a ‘Results’ window, which contains the length of this arrow. To add the arrow to the image, click Image → Overlay → Add Selection (or STRG+B).
  6. Draw an arrow along the next tube and measure the lengths as described in step D5.
  7. Once all the tubes have been measured, save the image with arrows (File → Save as → TIFF).
  8. Transfer the data from the ‘Results’ window into an Excel spreadsheet and determine the average of all measured tube lengths. It is recommended to acquire measurements from two or three, different images.

Notes

  1. Different endothelial cells (commercial providers) or perivascular cells isolated by various isolation procedures may be used for co-culture. In any case, cell numbers may have to be optimized for individual experiments.
  2. 100% confluent cultures should not be used for this assay, as it affects tube formation.
  3. Concentration of rat tail type I collagen can vary in different batches, volume have to be adjusted.
  4. Cell numbers that have not be adjusted accordingly can affect results.
  5. Include scale bar in each image for quantification.
  6. Carefully remove supernatant from gel without touching. Pipetting, rather than aspiration, is recommended for this step.

Recipes

  1. Proliferation medium
    MCDB-131
    5% FCS
    10 ng/ml epidermal growth factor (EGF)
    2 ng/ml basic fibroblast growth factor (bFGF)
    5 µg/ml insulin
    5 ng /ml platelet-derived growth factor-BB (PDGF-BB)
  2. PVC culture medium (DMEM, 10% FCS, 1% P/S)
    445 ml DMEM
    50 ml FCS
    5 ml P/S
    Pre-warm medium before use at 37 °C
  3. HUVEC culture medium (EGM2 with supplements)
    500 ml basal medium
    0.02 ml/ml FCS
    5 ng/ml epidermal growth factor (EGF)
    10 ng/ml basic fibroblast growth factor (bFGF)
    20 ng/ml insulin-like growth factor (IGF)
    0.5 ng/ml vascular endothelial growth factor (VEGF)
    1 µg/ml ascorbic acid
    22.5 µg/ml heparin
    0.2 µg/ml hydrocortisone
    Note: Basal medium should be ordered with supplement pack which includes all above listed supplements in the right concentration. Only small aliquots should be prewarmed at 37 °C.
  4. 2x medium-mix (final volume 10 ml)
    2 ml of 10x DMEM
    0.2 ml of 100x sodium pyruvate
    1 ml of 0.45 M NaHCO3
    6.8 ml of autoclaved distilled H2O
  5. Collagen solution (final volume 4 ml)
    2 ml of 2x medium-mix
    800 µl of FCS (final concentration 20%)
    963 µl of 8.3 mg/ml rat tail type I collagen solution
    214 µl of 0.1 N NaOH
    23 µl of autoclaved distilled H2O
    Prepare solution on ice and keep it on ice until usage
  6. Tris-buffered saline (TBS), pH 7.4
    150 mM NaCl
    50 mM Tris-HCl

Acknowledgments

The three-dimensional co-culture assay was used in two recent publications (Pitzler et al., 2016; Zhou et al., 2016). The work is supported by Deutsche Forschungsgemeinschaft (DFG 2304/5-3, 2304/7-1, 2304/9-1).

References

  1. Baudin, B., Bruneel, A., Bosselut, N. and Vaubourdolle, M. (2007). A protocol for isolation and culture of human umbilical vein endothelial cells. Nat Protoc 2(3): 481-485.
  2. Brachvogel, B., Dikschas, J., Moch, H., Welzel, H., von der Mark, K., Hofmann, C. and Poschl, E. (2003). Annexin A5 is not essential for skeletal development. Mol Cell Biol 23(8): 2907-2913.
  3. Brachvogel, B., Moch, H., Pausch, F., Schlotzer-Schrehardt, U., Hofmann, C., Hallmann, R., von der Mark, K., Winkler, T. And Poschl, E. (2005). Perivascular cells expressing annexin A5 define a novel mesenchymal stem cell-like population with the capacity to differentiate into multiple mesenchymal lineages. Dev 132(11): 2657-2668.
  4. Brachvogel, B., Pausch, F., Farlie, P., Gaipl, U., Etich, J., Zhou, Z., Cameron, T., von der Mark, K., Bateman, J. F. and Poschl, E. (2007). Isolated Anxa5+/Sca-1+ perivascular cells from mouse meningeal vasculature retain their perivascular phenotype in vitro and in vivo. Exp Cell Res 313(12): 2730-2743.
  5. Pitzler, L., Auler, M., Probst, K., Frie, C., Bergmeier, V., Holzer, T., Belluoccio, D., van den Bergen, J., Etich, J., Ehlen, H., Zhou, Z., Bielke, W., Poschl, E., Paulsson, M. and Brachvogel, B. (2016). miR-126-3p promotes matrix-dependent perivascular cell attachment, migration and intercellular interaction. Stem Cells 34(5): 1297-1309.
  6. Zhou, Z., Pausch, F., Schlotzer-Schrehardt, U., Brachvogel, B. and Poschl, E. (2016). Erratum to: Induction of initial steps of angiogenic differentiation and maturation of endothelial cells by pericytes in vitro and the role of collagen IV. Histochem Cell Biol 145(5): 527-529.

简介

血管发生定义了形成现有血管的新血管结构的形成过程,涉及发育过程中的修复过程,如伤口愈合,还与病理变化有关。 在血管生成过程中,内皮细胞建立血管网络并招募血管周围细胞以形成成熟稳定的血管。 内皮细胞和血管周围细胞秘密并组装血管基底膜,并通过细胞间接触进行相互作用。 为了体外模拟这些过程,我们开发了一种通用的三维培养系统,其中血管周围细胞(PVC)与胶原I型凝胶中的人脐带血管内皮细胞(HUVEC)共培养。 这种共培养系统可用于通过广泛的分析选项来确定新生血管生成事件期间的生物化学和细胞过程。
【背景】内皮细胞和血管周围细胞之间的协调相互作用对于根据给定组织内的局部需要形成稳定的血管网是非常重要的。多个分子组分有助于相互作用,但仍然很少了解。需要各种生长因子来吸引内皮细胞到低氧浓度的位点,并建立新的血管,然后被血管周围细胞覆盖。两种细胞类型相互作用以分泌特定的细胞外基质并稳定新形成的血管。在过去已经建立了多个测定法来分析二维基质胶底物上的血管细胞相互作用和血管样网络形成,但是这些测定在三维细胞内提供关于血管内血管周围细胞相互作用和血管基底膜形成的初始步骤的信息是有限的维度微环境。此外,缺乏适合于培养实验的良好表征的血管周围细胞。
我们以前分离出具有血管周围特征的细胞,因为它们表达周细胞特异性标记,产生和分泌细胞外基质蛋白并在体内刺激血管生成过程(Brachvogel等,2005和2007)。这些细胞用于与人脐静脉内皮细胞建立共培养系统,并研究三维微环境中两种细胞类型相互作用后新生血管发生的关键步骤(Pitzler等,2016; Zhou et al。,2016 )。
共培养物通过血管周围细胞募集和基底膜形成显示出优异的活性以促进内皮细管形成和稳定化。培养系统允许通过时间延迟显微镜监测血管细胞的迁移和相互作用,并通过免疫荧光分析研究基底膜蛋白的沉积。为了从共同培养物定量和分离内皮细胞和血管周围细胞,可以使用免疫磁性或流式细胞术分选方法,通过使来自分离的细胞群体的分离的RNA进行微阵列分析或RNA来分析mRNA和miRNA表达中的细胞类型特异性全局变化测序。此外,可以在体外分析抗血管生成物质和促血管生成物质对血管内血管周围细胞相互作用的影响。因此,三维培养方案允许研究新生血管生成过程中的细胞,生物化学和转录事件,并且表征分子对离体内皮血管周围细胞相互作用的促和抗血管生成作用。

关键字:内皮细胞, 血管周围细胞, 周细胞, 血管生成, 共培养, 胶原蛋白凝胶

材料和试剂

  1. (Greiner Bio One International,目录号:188271)
  2. 100毫米TC处理的细胞培养皿(Corning,Falcon ®,目录号:353003)
  3. 1.5毫升管(SARSTEDT,目录号:72.690.001)
  4. 24孔板(Corning,Costar ®,目录号:3524)
  5. T75烧瓶(VWR,目录号:734-0050)
  6. 48孔板(Corning,Costar ®,目录号:3548)
  7. 10ml注射器(BD,目录号:309604)
  8. 具有0.22μm孔径的无菌注射器过滤器(EMD Millipore,目录号:SLGP033RB)
  9. 一次性HSW FINE-JECT ®针(Henke Sass Wolf,目录号:4710004012)
  10. 移液器Gilson(10μl,20μl,100μl,1,000μl,5 ml)
  11. 微量吸头(VWR,目录号:89368-994)
  12. 显微镜盖眼镜:方格(Fisher Scientific,目录号:S175222)
  13. 粘合片,MenzelGläser,SuperFrost ® Plus(VWR,目录号:631-9483)
  14. HUVEC(人脐带血管内皮细胞)(Lonza,目录号:CC-2519或根据Baudin等人,2007)HUVEC的自我分离
  15. 隔离血管周围细胞/周细胞(PVC)(见注1)
  16. AnxA5-LacZ小鼠(Anxa5 tm1Epo )(Brachvogel等人,2003)
  17. 胶原酶II型(Worthington Biochemical,目录号:LS004176)
  18. DNase I重组(Roche Diagnostics,目录号:04536282001)
  19. 荧光素二(β-D-吡喃半乳糖苷)(FDG)(Thermo Fisher Scientific,Molecular Probes TM,目录号:F1179)
  20. 碘化丙啶(PI)(Thermo Fisher Scientific,Molecular Probes TM,目录号:P3566)
  21. Dulbecco改性Eagle培养基(DMEM)(Thermo Fisher Scientific,Gibco TM,目录号:31966021)
  22. 胎牛血清(FCS)(Biochrom,目录号:S 0115)
  23. 青霉素 - 链霉素(P/S)(10,000U/ml)(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  24. 来自猪皮的明胶(Sigma-Aldrich,目录号:G2500)
  25. 碳酸氢钠(NaHCO 3)(EMD Millipore,目录号:1.6329.1000)
  26. 鼠尾型I型胶原溶液(Corning,目录号:354236)
  27. 氢氧化钠(NaOH)(VWR,目录号:28244.295)
  28. 磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,Gibco TM,目录号:18912014)
  29. 胰蛋白酶/EDTA(Biochrom,目录号:L 2153)
  30. 内皮生长培养基2(EGM2)(PromoCell,目录号:C-22111)
  31. 重组VEGF-A /血管内皮生长因子(Biomol,目录号:94900)
  32. 重组PDGF /血小板衍生生长因子-BB(Biomol,目录号:94968)
  33. 抗坏血酸磷酸盐(Sigma-Aldrich,目录号:A8960-5G)
  34. 甲醇(VWR,目录号:20903.461)
  35. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D8418-100ML)
  36. 吐温-20(Sigma-Aldrich,目录号:P1379-500ML)
  37. 牛血清白蛋白(BSA)(SERVA Electrophoresis,目录号:11930.03)
  38. 抗人CD31(BD,BD Biosciences,目录号:555444)
  39. 抗NG2-蛋白聚糖(EMD Millipore,目录号:AB5320)
  40. 驴抗大鼠Ig,Cy2缀合(Jackson ImmunoResearch,目录号:712-225-150)
  41. 驴,抗兔Ig,Cy2缀合(Jackson ImmunoResearch,目录号:711-225-152)
  42. 驴,抗兔Ig,Cy5缀合(Jackson ImmunoResearch,目录号:711-175-152)
  43. 驴,抗山羊Ig,Cy2缀合(Jackson ImmunoResearch,目录号:705-225-147)
  44. 安装解决方案
  45. CFSE(Thermo Fisher Scientific,Molecular Probes TM,目录号:C1157)
  46. SNARF-1(Thermo Fisher Scientific,Molecular Probes TM,目录号:S22801)
  47. MCDB-131培养基(Thermo Fisher Scientific,Gibco TM,目录号:10372019)
  48. 碱性成纤维细胞生长因子(bFGF)(ReliaTech,目录号:300-003)
  49. 表皮生长因子(EGF)(Biomol,目录号:97052)
  50. 胰岛素溶液人(Sigma-Aldrich,目录号:I9278)
  51. 肝素
  52. 氢化可的松
  53. 10x DMEM,10x Dulbecco改良Eagle's培养基(Sigma-Aldrich,目录号:D2429-100ML)
  54. 丙酮酸钠(Thermo Fisher Scientific,Gibco TM,目录号:11360070)
  55. Trizma ®基质(Sigma-Aldrich,目录号:T1503)
  56. 氯化钠(NaCl)(EMD Millipore,目录号:1064041000)
  57. 高压灭菌蒸馏H 2 O O
  58. 增殖介质(见食谱)
  59. PVC培养基(参见食谱)
  60. HUVEC培养基(见食谱)
  61. 2x中混合(见配方)
  62. 胶原蛋白溶液(参见食谱)
  63. Tris缓冲盐水(TBS)(见食谱)

设备

  1. 离心机(Eppendorf,型号:5415 R)
  2. 血细胞计数器(LO-Laboroptik,型号:Neubauer-Improved)
  3. CO 2细胞培养箱(Heraeus)
  4. 离心机Labofuge(Heraeus Sepatech,型号:Labofuge GL)
  5. 高压灭菌器
  6. SteREO Lumar.V12(Zeiss,型号:SteREO Lumar.V12)
  7. Axiovert 40 CFL(Zeiss,型号:Axiovert 40 CFL)
  8. Axioplan 2(Zeiss,型号:Axioplan 2)
  9. 水浴(Köttermann)
  10. 涡旋搅拌机(科学工业)
  11. 倒置显微镜Eclipse TE2000-U(Nikon Instruments,型号:Eclipse TE2000-U)
  12. 荧光激活细胞分选(MoFlow,Cytomation)
  13. 生物安全柜LaminAir HB 2448(Heraeus,型号:LaminAir HB 2448)

软件

  1. ImageJ(斐济)

程序

  1. 使用AnxA5-LacZ小鼠从脑膜分离的周细胞的原始方案
    1. 准备约200毫升的5%FCS-PBS,并在冰上冷却100ml的5%FCS-PBS。
    2. 从4-6个月大的AnxA5-LacZ小鼠中仔细分离大脑。在立体显微镜下解剖脑膜,并将其转移到冰上用5%FCS-PBS的15ml管中。收集几个脑膜,然后在4℃以400×g离心样品5分钟。
    3. 吸出上清液并溶解在1.5-3.0ml的0.4%胶原酶II型中。在37℃下孵育样品60分钟轻微摇动。随后,在室温下以400×g离心5分钟消化组织。
    4. 吸出上清并重新悬浮于1.5-3.0ml的2%胰蛋白酶/100U DNA酶I.然后,37℃下将消化的组织温育30分钟,轻微摇动。接下来,在室温下以400×g离心5分钟。
    5. 将细胞沉淀重悬于5%FCS-PBS中,并通过100μm细胞过滤器过滤细胞悬液。之后,在室温下以400×g离心细胞悬浮5分钟
    6. 吸出上清液并将细胞沉淀重悬于5%FCS-PBS中。通过使用血细胞计数器确定细胞数,然后在20μl5%FCS-PBS中溶解1×10 6个细胞,并将20μl细胞悬浮液分配到新的1.5ml管中。向20μl细胞悬浮液中加入20μl的2mM荧光素二(β-D-吡喃半乳糖苷)(FDG),并在37℃下孵育混合物75秒。向细胞中加入500μl冰冷的5%FCS-PBS,并在黑暗中在冰上孵育3小时。使用未染色的样品作为细胞分选的对照
    7. 在室温下以400×g离心细胞悬浮液5分钟,并将细胞沉淀重新悬浮于500μl的5%FCS-PBS中。为了排除死细胞,加入碘化丙啶(终浓度1μg/ml)
    8. 通过荧光激活细胞分选(MoFlow,Cytomation)对染色细胞进行分选,并收集碘化丙啶阴性和FDG阳性细胞。接下来,在明胶涂覆的24孔板上,将细胞在5%CO 2中的细胞培养物(见Recipes)中,每孔5×10 4个PI阴性/FDG阳性细胞, 2℃培养箱中37℃。每隔一天更换培养基,并扩大分离的细胞。
    9. 初始扩增后,细胞在10%FCS和1%P/S的DMEM中培养。细胞应通过流式细胞术进一步表征。细胞对Sca-1,CD140b和α-SMA呈阳性,对于PECAM,CD45,CD34,F4/F80和c-kit为阴性。来自小鼠脑膜的分离的Anxa5-LacZ(+)PVC保留其血管周围细胞表型,并且可以在不经历衰老的情况下培养以进行多次传代(Brachvogel等人,2007)。
      注意:可以根据要求与个体研究者一起建立具有优异血管生成活性的成熟且良好表征的PVC细胞系,以与内皮细胞进行共培养实验(Zhou等人 。,2016)。这消除了将细胞与小鼠脑脑膜隔离的需要。

  2. 胶原蛋白凝胶中PVC和HUVEC的共培养
    1. 将0.1g明胶溶解在100g高压灭菌的蒸馏水(终浓度为0.1%)中,并将溶液在121℃下高压灭菌30分钟。然后,通过向培养皿中加入5ml 0.1%明胶溶液,用明胶涂覆100mm TC处理的细胞培养皿,并分配明胶溶液,直到整个100mm TC处理的细胞培养皿表面被完全覆盖。在37℃下孵育少于2小时后,抽吸残留的明胶溶液。随后,在室温下使用干盘。
    2. 将HUVEC在37℃的5%CO 2湿度培养箱中的明胶包被的平板上在EGM2培养基(参见食谱)中培养。将细胞培养直至亚汇合(〜80%)并进一步传代(1:3稀释)。 HUVEC只用于早期通道(3-6段)。将PVC在T75烧瓶或10cm培养皿上用10%FCS和1%P/S(参见食谱)的DMEM培养,并扩增至约80%汇合(见注2和图1)。


      图1.培养的血管周围细胞(PVC)和具有不同细胞密度的人脐静脉内皮细胞(HUVEC)。代表性的80%和100%融合(细胞密度)培养的PVC和HUVEC的相图。细胞密度在这种共培养实验中起重要作用,可影响管形成。酒吧= 100微米。

    3. 准备10毫升2x中等混合物(见食谱)。因此,将0.38g NaHCO 3溶解在10ml高压釜蒸馏水(浓度0.45M)中,并将溶液无菌过滤后溶解。然后,在6.8ml(最终体积为10ml)的高压蒸馏蒸馏水中混合2ml 10x DMEM,200μl100x丙酮酸钠和1ml制备的0.45M NaHCO 3(终浓度为0.045M)H 2 O。解决方案应该在冰上冷却直到使用。
    4. 对于4ml胶原溶液(参见食谱),混合2ml 2x中混合物,800μlFCS(终浓度20%),963μl8.3mg/ml大鼠尾型I型胶原(见注3)(终浓度2毫克/毫升)和23微升高压灭菌蒸馏的H 2 O在冰上。最后,用214μl0.1N NaOH中和I型胶原溶液,并将溶液pH调节至7.4。将制备的胶原蛋白溶液储存在冰上直至使用。
    5. 用10ml PBS洗涤亚汇合培养的HUVEC和PVCs,并在37℃下用1-2ml胰蛋白酶/EDTA分离细胞5分钟。加入9ml EGM2培养基以停止反应,并将细胞收集在15ml管中
    6. 在室温(RT)下以400×g离心细胞5分钟。吸出上清并将细胞沉淀重悬于5ml EGM2培养基中。加入10μl每个细胞悬液至血细胞计数器中,以确定细胞浓度
    7. 对于单次培养,在新的15ml管中,在EGM2培养基中转移6.25×10 5个HUVEC或1.25×10 5个(见注4)。混合6.25×10 5个HUVEC和1.25×10 5个PVC,用于在新的15ml管中的2ml EGM2培养基中共培养(图2)。或者,可以使用不同的内皮细胞或血管周围细胞,但细胞的相对比例需要适应和测试。强烈推荐技术重复或一式三份。在室温下以400×g离心细胞悬浮液5分钟。


      图2.三维共培养测定的工作流程

    8. 仔细清除所有培养基,并将细胞沉淀重悬于1ml胶原I型凝胶溶液中。在48孔板的每个孔中加入200μl,并在37℃下孵育60分钟,直到溶液变成凝胶。为了增加凝胶的均匀性,胶原蛋白I型凝胶溶液与细胞应适当混合,凝胶溶液在孔中均匀地受到干扰。尝试通过将凝胶溶液直接添加到孔的中心并通过小心移动48孔板来分布来防止溶液的不均匀分布。为了改善凝胶化,48孔板应在37℃的温室中预热
    9. 60分钟后检查是否形成均匀的凝胶。否则孵育30分钟。胶凝后,仔细添加400μl补充有10 ng/ml VEGF,10 ng/ml PDGF和250μg/ml抗坏血酸磷酸盐的EGM2培养基,每个凝胶顶部。在这个阶段可能会为不同的实验条件添加其他生长因子或药物。
    10. 在5%CO 2/2湿度培养箱中孵育3〜6天的凝胶(见附注6),每24小时拍摄图像(图3)或视频(3张图像/h)。我们使用10或20x目标拍摄网络图像(Zeiss SteREO Lumar.V12或Zeiss Axiovert 40 CFL)。可以使用更高的放大倍数,但凝胶的厚度可能阻碍图像的采集。每48小时应更换含有补充剂的EGM2培养基。


      图3.在三维胶原I型凝胶中单次和共培养HUVECs的管形成研究。96小时后,HUVEC在血管周围细胞中形成一种在I型胶原中稳定的管网络凝胶,而单培养HUVEC几个管。以20倍的目标采集代表性的相位图像。酒吧= 100微米。

  3. 免疫组织化学检测细胞外基质和细胞蛋白在三维文化中的应用
    1. 用600μlPBS洗涤凝胶两次(见附注6)
    2. 用400μl80%甲醇/20%DMSO(Dent固定剂)固定凝胶30分钟。
    3. 用600μl50%甲醇/PBS在室温下再溶解凝胶1小时。取出溶液,加入600μl20%甲醇/PBS。接下来,在室温下将600μlPBS-T(PBS,0.1%吐温-20)洗涤凝胶1小时。
    4. 吸入溶液,加入600μl10%FCS,5%BSA的PBS溶液2-4小时,以阻止非特异性抗体结合。
    5. 取出小心的封闭溶液,并用稀释在封闭溶液(10%FCS,PBS中5%BSA)的150-300μl初级抗体孵育凝胶,在4℃下摇摆过夜摇匀。抗生素稀释液应先经验检验。
    6. 用600μlTBS-T(0.1%吐温在TBS中)洗涤凝胶一小时。重复洗涤步骤六次。
    7. 孵育样品与150-300μl萤光发色团缀合的二抗在封闭溶液中在黑暗中在室温下2-16小时。
    8. 用600μlTBS-T(0.1%吐温在TBS中)洗涤凝胶一小时。然后重复洗涤步骤六次。
    9. 使用27 G针小心地从孔壁上除去凝胶。使用5毫升的微量吸管技巧将凝胶转移到载玻片上。在凝胶顶部加入50μl安装溶液,并用玻璃盖玻片小心地安装样品。
    10. 图像采用Zeiss Axioplan 2采用5x和63x目标和足够的滤镜组来分别获得概览和特写图像。为了在三维培养物中可视化HUVEC,可以用CD31免疫染色(图4A和4B),并且可以用NG2-PG(图4B)或α-SMA特异性抗体对PVC进行免疫染色。 CD31 + HUVEC的概述图像用于管形成分析。


      图4.与PVC共同培养,诱导人脐带静脉内皮细胞(HUVEC)的内皮管形成。 A.将HUVEC单独培养(单培养)或与PVC共培养在三维胶原蛋白I凝胶中。培养6天后,如步骤C所述,用人CD31染色三维胶原蛋白I凝胶。比例尺=500μM。 B.使用CD31和NG2蛋白聚糖(NG2-PG)特异性抗体对共培养HUVEC和PVC的免疫染色。显示了具有核染色(DAPI)的个体或合并图像。比例尺=100μm
  4. 用荧光染料(CFSE,SNARF-1)进行活细胞跟踪的细胞标记
    1. 在室温下将CFSE或SNARF-1在DMSO中溶解至终浓度为5mM。使用荧光染料后,溶液可以等分并储存在-20°C。为了随后使用荧光染料,在室温下在黑暗中融化CFSE和SNARF-1染料。新鲜制备100ml 3%FCS-PBS溶液。在室温下储存25毫升,并在冰上冷却剩余的75毫升至少30分钟
    2. 如所述分离HUVECs和PVC(参见步骤A3),并在15 ml管中收集细胞。使用血细胞计数器确定细胞数,然后在室温下以400×g离心细胞5分钟。
    3. 将细胞沉淀物重悬于适量的3%FCS-PBS中至终浓度为每1ml 3%FCS-PBS 1×10 7个细胞。不要使用少于50μl的3%FCS-PBS溶液。
    4. 在使用前,使用PBS制备0.5mM的CFSE和SNARF-1溶液。然后,使用细胞悬浮液进一步稀释CFSE或SNARF-1溶液(0.5mM)至终浓度为0.01mM。通常,将2μl0.5mM CFSE加入到100μl细胞悬浮液中。所有以下步骤均在黑暗中避免曝光。
    5. 在涡旋混合器上以最大速度将HUVEC和PVC混合10秒。然后,将细胞悬浮液在水浴中孵育10分钟,预热至37℃,不摇动
    6. 加入5ml冰冷的3%FCS-PBS以停止反应,并在4℃以400×g离心细胞5分钟。吸出上清液并将沉淀重悬于5ml冰冷的3%FCS-PBS中。在4℃下以400×g离心细胞5分钟。离心后,吸出上清并再次沉淀在5ml冰冷的3%FCS-PBS中。离心细胞悬浮液在4℃下以400×g的速度再静置5分钟
    7. 将细胞重悬在适量的EGM2培养物中,并进行步骤A4。在后续步骤和图像中减少曝光(图5)应在倒置的显微镜Eclipse TE2000-U(Nikon)或SteREO Lumar.V12(Zeiss)上进行。在这里,一个20x的目标被用于可视化。对于每个图像,拍摄相关通道(例如,用于CFSE标记细胞的Fitc通道)的相位对比度和荧光图像。 SNARF-1通常用于检测初始管形成(胶凝后几小时)。后来的SNARF-1标签难以检测。该系统可用于在初始血管生成步骤期间跟踪血管内细胞向内皮细胞的募集

      图5.人脐带静脉内皮细胞(HUVEC)和CFSE标记的血管周围细胞(PVC)的共培养,以三维条件跟踪血管周围细胞。如方法所述,将PVC用CFSE标记并随后与三维胶原I型凝胶共同培养HUVEC。培养72小时后,用20倍物镜拍摄相差图像和荧光图像。调整CFSE荧光和合并图像的亮度和对比度,以进行可视化。比例尺=100μm。 

数据分析

  1. ImageJ(斐济)用于图像分析。
  2. 打开感兴趣的图像文件(文件→打开→选择图像)。
  3. 设置刻度。在工具栏中选择"箭头工具",并沿图像中的比例尺绘制箭头。接下来,单击分析→设置缩放以打开一个新窗口,其中根据从比例尺箭头获得的距离和单位长度来调整长度的已知距离和单位。之后,点击"确定"。
  4. 转到工具栏,双击"箭头工具"打开一个窗口。选择箭头的颜色,然后点击"添加到覆盖后保留"。接下来,点击"确定"
  5. 选择"箭头工具",沿着管子绘制箭头。单击分析→测量(或STRG + M)打开一个"结果"窗口,其中包含此箭头的长度。要向图像添加箭头,请单击图像→覆盖→添加选择(或STRG + B)。
  6. 沿着下一个管绘制一个箭头,并按照步骤D5中所述测量长度。
  7. 一旦测量了所有的管子,用箭头保存图像(文件→另存为→TIFF)。
  8. 将数据从"结果"窗口传输到Excel电子表格中,并确定所有测量管长度的平均值。建议从两个或三个不同的图像采集测量。

笔记

  1. 通过各种分离程序分离的不同内皮细胞(商业供体)或血管周围细胞可用于共培养。在任何情况下,可能必须对单个实验优化单元格编号。
  2. 100%汇合培养物不应用于该测定,因为它影响管形成
  3. 大鼠尾巴I型胶原的浓度可以在不同批次中变化,体积必须调整。
  4. 没有相应调整的单元格编号可能会影响结果。
  5. 在每个图像中包括比例尺以进行定量。
  6. 小心地从凝胶中除去上清液而不接触。推荐这个步骤,而不是吸气。

食谱

  1. 增殖培养基
    MCDB-131
    5%FCS
    10ng/ml表皮生长因子(EGF)
    2ng/ml碱性成纤维细胞生长因子(bFGF)
    5μg/ml胰岛素 5ng/ml血小板衍生生长因子-BB(PDGF-BB)
  2. PVC培养基(DMEM,10%FCS,1%P/S)
    445毫升DMEM
    50 ml FCS
    5 ml P/S
    在37°C使用前的预热培养基
  3. HUVEC培养基(EGM2与补品)
    500毫升基础培养基
    0.02 ml/ml FCS
    5ng/ml表皮生长因子(EGF)
    10 ng/ml碱性成纤维细胞生长因子(bFGF)
    20ng/ml胰岛素样生长因子(IGF)
    0.5 ng/ml血管内皮生长因子(VEGF)
    1μg/ml抗坏血酸
    22.5μg/ml肝素 0.2μg/ml氢化可的松
    注意:基础培养基应订购补充包,其中包括所有上述补品,其浓度正确。只有小的等分试样应该在37°C预热。
  4. 2x中混(最终体积10 ml)
    2 ml 10x DMEM
    0.2ml 100x丙酮酸钠
    1ml 0.45M NaHCO 3
    6.8毫升高压灭菌的蒸馏H 2 O 2/
  5. 胶原溶液(终体积4毫升)
    2 ml 2x medium-mix
    800μlFCS(终浓度20%)
    963μl8.3 mg/ml大鼠尾型I型胶原溶液
    214μl0.1 N NaOH 23升高压灭菌的蒸馏H 2 O 2/
    在冰上准备解决方案,并保持在冰上直到使用
  6. Tris缓冲盐水(TBS),pH 7.4
    150 mM NaCl
    50 mM Tris-HCl

致谢

在最近的两篇出版物(Pitzler等人,2016; Zhou等人,2016)中使用了三维共培养测定法。这项工作得到德意志民主共和国(DFG 2304/5-3,2304/7-1,2304/9-1)的支持。

参考文献

  1. Baudin,B.,Bruneel,A.,Bosselut,N.和Vaubourdolle,M。(2007)。用于人脐静脉内皮细胞分离和培养的方案 Nat Protoc 2(3):481-485。
  2. Brachvogel,B.,Dikschas,J.,Moch,H.,Welzel,H.,von der Mark,K.,Hofmann,C.and Poschl,E。(2003)。 Annexin A5对于骨骼发育不是必需的。 Mol Cell Biol 23(8):2907-2913。
  3. Brachvogel,B.,Moch,H.,Pausch,F.,Schlotzer-Schrehardt,U.,Hofmann,C.,Hallmann,R.,von der Mark,K.,Winkler,T.and Poschl, )。表达膜联蛋白A5的血管周围细胞定义了一种小说间充质干细胞样群体,具有分化为多个间充质谱系的能力。 132(11):2657-2668。
  4. Brachvogel,B.,Pausch,F.,Farlie,P.,Gaipl,U.,Etich,J.,Zhou,Z.,Cameron,T.,von der Mark,K.,Bateman,JF和Poschl, (2007)。孤立的Anxa5 + 和体内。 > Exp Cell Res 313(12):2730-2743。
  5. Pitzler,L.,Auler,M.,Probst,K.,Frie,C.,Bergmeier,V.,Holzer,T.,Belluoccio,D.,van den Bergen,J.,Etich,J.,Ehlen,H 。,Zhou,Z.,Bielke,W.,Poschl,E.,Paulsson,M.and Brachvogel,B.(2016)。  miR-126-3p促进基质依赖性血管周围细胞附着,迁移和细胞间相互作用。干细胞 34(5):1297-1309。
  6. Zhou,Z.,Pausch,F.,Schlotzer-Schrehardt,U.,Brachvogel,B.and Poschl,E。(2016)。< a class ="ke-insertfile"href ="http: ncbi.nlm.nih.gov/pubmed/27038636"target ="_ blank">错误:诱导体外细胞血管生成分化和内皮细胞成熟的初始步骤和胶原蛋白IV的作用。 Histochem Cell Biol 145(5):527-529。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Auler, M., Pitzler, L., Pöschl, E., Zhou, Z. and Brachvogel, B. (2017). Mimicking Angiogenesis in vitro: Three-dimensional Co-culture of Vascular Endothelial Cells and Perivascular Cells in Collagen Type I Gels. Bio-protocol 7(8): e2247. DOI: 10.21769/BioProtoc.2247.
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