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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

MA Markus Auler
LP Lena Pitzler
EP Ernst Pöschl
ZZ Zhigang Zhou
BB Bent Brachvogel

发布: 2017年04月20日第7卷第8期 DOI: 10.21769/BioProtoc.2247 浏览次数: 18104

评审: Joseph C. ChenAnonymous reviewer(s)

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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) search Perivascular cells (血管周围细胞) search Pericytes (周细胞) search Angiogenesis (血管生成) search Co-culture (共培养) search Collagen gel (胶原蛋白凝胶) search

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

English
中文翻译

文章信息

版权信息

© 2017 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

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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分类

干细胞 > 成体干细胞 > 内皮干细胞/祖细胞

细胞生物学 > 细胞分离和培养 > 细胞分化

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