Angiogenesis within Stem Cell–Seeded Silk Scaffolds Cultured on the Chorioallantoic Membrane and Visualized by 3D Imaging

Anna Woloszyk1, Thimios A. Mitsiadis1

1 Orofacial Development and Regeneration, Institute of Oral Biology, University of Zurich, Zurich
Publication Name:  Current Protocols in Stem Cell Biology
Unit Number:  Unit 1F.19
DOI:  10.1002/cpsc.27
Online Posting Date:  May, 2017
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Abstract

The long‐term survival and successful integration of implants for tissue replacement and regeneration highly depends upon the fast ingrowth of blood vessels from the surrounding tissues. Before selecting potential biomaterials for clinical applications, they must be thoroughly tested with proper analytical tools. This unit provides a protocol for studying the potential of cell‐seeded scaffolds to attract vessels that will form vascular networks within biomaterials. It includes seeding of stem cells into silk fibroin scaffolds, angiogenesis assay on the chorioallantoic membrane (CAM) of fertilized chicken eggs, a procedure for perfusion with MicroFil, and finally microcomputed tomography (µCT) scanning. This technique can help screen potential biomaterial implants, thereby reducing the amount of animals needed for pre‐clinical in vivo studies. © 2017 by John Wiley & Sons, Inc.

Keywords: microcomputed tomography (µCT); tissue engineering; scaffold biomaterial; stem cells; vasculature

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Culture of Cryopreserved Human Dental Pulp Stem Cells
  • Basic Protocol 2: Seeding Human Dental Pulp Stem Cells Onto Silk Fibroin Scaffolds
  • Basic Protocol 3: Preparation of Fertilized Chicken Eggs for CAM Assay and Placing Cell‐Seeded Samples Onto the CAM
  • Basic Protocol 4: Perfusion of the Developing Vasculature
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Culture of Cryopreserved Human Dental Pulp Stem Cells

  Materials
  • Human dental pulp stem cells (cryopreserved hDPSCs)
  • Growth medium (see recipe), 4ºC
  • 37ºC water bath
  • 15‐ml centrifuge tubes (Falcon)
  • Centrifuge
  • 75‐cm2 cell culture flask
  • 37ºC, 5% CO 2 humidified incubator

Basic Protocol 2: Seeding Human Dental Pulp Stem Cells Onto Silk Fibroin Scaffolds

  Materials
  • Growth medium (see recipe)
  • hDPSCs (see protocol 1)
  • Trypsin
  • Phosphate buffered saline (PBS)
  • 37ºC water bath
  • 70‐µm strainer
  • Centrifuge
  • Neubauer chamber (or other cell counting device)
  • Microscope
  • Sterile silk fibroin scaffold (5‐mm diameter, 3‐mm height, pore‐size of 200 to 300 µm, 90% porosity)
  • Sterile filter paper
  • 24‐ and 96‐well plates
  • Sterile tweezers
  • 37ºC, 5% CO 2 humidified incubator
  • 50‐ml centrifuge tubes (Falcon)

Basic Protocol 3: Preparation of Fertilized Chicken Eggs for CAM Assay and Placing Cell‐Seeded Samples Onto the CAM

  Materials
  • Fertilized Lohman white Lohman Selected Leghorn (LSL) chicken eggs
  • 70% ethanol
  • Cell‐seeded scaffolds (see protocol 2)
  • 38ºC egg incubator (e.g., Bruja 3000, Brutmaschinen‐Janeschitz)
  • 60‐mm petri dishes
  • Autoclave tape
  • 5‐ml syringes
  • 21‐G × 1 ½‐in. (40 mm × 0.8–mm) needles
  • Sterile pointy scissors
  • Clear tape (e.g., Scotch tape)
  • 500‐ml glass beaker
  • Pencil
  • 37ºC, 0% CO 2 humidified incubator
  • Silicone rings (taken from sterile cryovials; e.g., Sigma‐Aldrich, cat. no. CLS430488‐500EA)

Basic Protocol 4: Perfusion of the Developing Vasculature

  Materials
  • Chicken embryos (see protocol 3)
  • MicroFil set (MV‐diluent, MV‐curing agent, yellow silicone rubber injection compound; Flow Tech)
  • Superglue
  • PBS
  • 4% paraformaldehyde
  • Small containers (e.g., Sterilin 7‐mL Polystyrene Bijou Containers, Thermo Fisher Scientific)
  • 5‐ml syringes
  • Three‐way valves (Discofix C; B. Braun Melsungen AG)
  • 10‐cm tube
  • 30‐G × 1‐in. (25 × 0.3–mm) needles
  • 24‐well plates
  • Stereo microscope
  • Sterile, blunt‐end tweezers
  • Microcomputed tomography scanner
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Figures

Videos

Literature Cited

 
  Anderson‐Berry, A., O'Brien, E. A., Bleyl, S. B., Lawson, A., Gundersen, N., Ryssman, D., & Albertine, K. H. (2005). Vasculogenesis drives pulmonary vascular growth in the developing chick embryo. Developmental Dynamics, 233, 145–153. doi:10.1002/dvdy.20296. doi: 10.1002/dvdy.20296
  Carmeliet, P., & Jain, R. K. (2011). Molecular mechanisms and clinical applications of angiogenesis. Nature, 473, 298–307. doi:10.1038/nature10144
  Garcia‐Sanz, A., Rodnguez‐Barbero, A., Bentley, M. D., Ritman, E. L., & Romero, J. C. 1998. Three‐dimensional microcomputed tomography of renal vasculature in rats. Hypertension, 31(part 2), 440–444. doi:10.1161/01.HYP.31.1.440
  Gronthos, S., Mankani, M., Brahim, J., Gehron Robey, P., & Shi, S. (2000). Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proceedings of the National Academy of Sciences, 97(25), 13625–13630. doi:10.1073/pnas.240309797
  Stoppato, M., Stevens, H. Y., Carletti, E., Migliaresi, C., Motta, A., & Guldberg, R. E. (2013). Effects of silk fibroin fiber incorporation on mechanical properties, endothelial cell colonization and vascularization of PDLLA scaffolds. Biomaterials, 34(19). Elsevier Ltd., 4573–4581. doi:10.1016/j.biomaterials.2013.02.009
  Tirino, V., Paino, F., d'Aquino, R., Desiderio, V., De Rosa, A., & Papaccio, G. (2011). Methods for the identification, characterization and banking of human DPSCs: Current strategies and perspectives. Stem Cell Reviews, 7(3), 608–615. doi:10.1007/s12015‐011‐9235‐9
  Upputuri, P. K., Sivasubramanian, K., Mark, C. S. K., & Pramanik, M. (2015). Recent developments in vascular imaging techniques in tissue engineering and regenerative medicine. BioMed Research International, 2015, 1–9. doi:10.1155/2015/783983
  Woloszyk, A., Liccardo, D., & Mitsiadis, T. A. (2016). Three‐dimensional imaging of the developing vasculature within stem cell‐seeded scaffolds cultured in ovo. Frontiers in Physiology, 7, 1–5. doi:10.3389/fphys.2016.00146
  Xuan, J. W., Bygrave, M., Jiang, H., Valiyeva, F., Dunmore‐Buyze, J., Holdsworth, D. W., & Lacefield, J. C. (2007). Functional neoangiogenesis imaging of genetically engineered mouse prostate cancer using three‐dimensional power Doppler ultrasound. Cancer Research, 67(6), 2830–2839. doi:10.1158/0008‐5472.CAN‐06‐3944
Key Reference
  Woloszyk, A., Liccardo, D., & Mitsiadis, T. A. (2016). Three‐dimensional imaging of the developing vasculature within stem cell‐seeded scaffolds cultured in ovo. Frontiers in Physiology, 7, 1–5. doi: 10.3389/fphys.2016.00146
  The presented method was first published in the referenced paper. However, in Current Protocols the procedure is described in more detail and with more figures giving step‐by‐step instructions.
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