Home › Analytical Chem › Tissue Engineering of a Human 3D in vitro Tumor Test System
Analytical Chem JoVE (Open Access) Citable · DOI

Tissue Engineering of a Human 3D in vitro Tumor Test System

DOI: 10.3791/50460-v
What you'll learn
  • ✓ Decellularize biological vascularized scaffolds for tissue engineering applications.
  • ✓ Isolate and culture primary human endothelial cells and fibroblasts.
  • ✓ Engineer 3D tumor tissue models under static and dynamic conditions.
  • ✓ Characterize tissue constructs using immunohistological analysis.
Protocol

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Difficulty
advanced
Total time
~7–10 days (decellularization 2–3 days, cell isolation 1–2 days, culture optimization 4–5 days)
Model organism
Primary human cells (dermal microvascular endothelial cells, fibroblasts); tumor cell line
Biosafety
BSL-2

Steps

1
Decellularize biological vascularized scaffold

Remove cellular material from tissue scaffolds using appropriate decellularization protocols to preserve extracellular matrix and vascular architecture. Resulting scaffolds serve as a foundation for recellularization.

▶ 02:16
2
Isolate primary human dermal microvascular endothelial cells

Extract and culture human dermal microvascular endothelial cells and fibroblasts from primary tissue sources. Prepare cell populations for seeding onto decellularized scaffolds.

▶ 03:52
3
Establish 3D tumor model under static culture

Seed primary cells and tumor cell lines onto decellularized scaffolds and maintain culture under static conditions without fluid flow. Monitor tissue maturation and cell integration over culture period.

▶ 05:50
4
Culture 3D tumor model in bioreactor with flow

Perfuse recellularized scaffolds in a flow bioreactor under dynamic conditions to enhance nutrient transport and simulate in vivo hemodynamic environment. Maintain culture parameters and harvest tissue at defined timepoints.

▶ 07:36
5
Characterize 3D tumor tissue by immunohistology

Process engineered tissue constructs for histological analysis and perform immunohistochemical staining to assess cell phenotype, organization, and marker expression within the 3D model.

▶ 08:45
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