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Cell Biology JoVE (Open Access) Citable · DOI

Bioengineering of Humanized Bone Marrow Microenvironments in Mouse and Their Visualization by Live Imaging

DOI: 10.3791/55914-v
What you'll learn
  • ✓ Prepare collagen-based scaffolds for humanized bone marrow implantation
  • ✓ Implant and explant bioengineered scaffolds in mice
  • ✓ Image humanized bone marrow niches using 2-photon microscopy
  • ✓ Interpret vascularization and osteogenesis in chimeric tissues
Protocol

Biopharma Insights A method to create and live-image different humanized bone-marrow niches in mice is presented. Based on the supportive niche created by human mesenchymal cells, the addition of human endothelial cells induces the formation of human vessels, while the addition of rhBMP-2 induces the formation of human-mouse chimeric mature bone tissue.

Difficulty
advanced
Total time
~4–6 weeks (scaffold fabrication ~1 week, implantation surgery ~1 hour/mouse, imaging and analysis ~2–4 weeks)
Model organism
Mouse (immunocompromised strain implied for human cell engraftment)
Biosafety
BSL-1

Steps

1
Prepare collagen-based scaffolds

Fabricate and crosslink collagen scaffolds as the structural foundation for humanized bone marrow niche engineering. This step establishes the extracellular matrix framework.

▶ 00:54
2
Implant scaffolds and harvest explants

Surgically implant bioengineered scaffolds seeded with human mesenchymal cells, endothelial cells, and/or rhBMP-2 into mice, then retrieve explanted tissue at designated timepoints.

▶ 02:40
3
Image scaffolds using 2-photon microscopy

Acquire high-resolution intravital or ex vivo 2-photon microscopy images of explanted scaffolds to visualize humanized vasculature, bone formation, and cellular organization.

▶ 04:43
4
Analyze scaffold explants and tissues

Quantify and characterize representative imaging and histological data from explanted scaffolds, including human vessel formation and human-mouse chimeric bone maturation.

▶ 07:44
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