Spheroid-Based In Vivo Angiogenesis Model

The spheroid-based In Vivo Angiogenesis Model allows studying the efficacy of pro- and anti-angiogenic compounds in a living organism.

This mouse model for studying angiogenesis is based on spheroids from human endothelial cells (HUVECs), which are implanted subcutaneously into mice embedded in an extracellular matrix containing angiogenic growth factors. Subsequently, the formation of a human vasculature and the effect of the pro-or anti-angiogenic treatment can be monitored in vivo and ex vivo.

  • Spheroid-based engineering of a human vasculature in mice
  • Live monitoring of vessel formation via luciferase-expressing human endothelial cells
  • Histology-based readouts available for in-depth analysis of vessel characteristics

Human vessel formation

In 2008, we first described the spheroid-based in vivo angiogenesis assay in Nature Methods. In the assay spheroids of human endothelial cells are implanted into mice for the development of a human vasculature and efficacy testing of anti- or pro-angiogenic drug candidates.

Additional Information

Standard Study Layout

Primary human endothelial cells (HUVEC) get transduced for expression of luciferase and spheroids are formed overnight (see Cellular Angiogenesis Assay for more details). The spheroids are harvested and mixed in a matrigel/fibrin matrix, which is subcutaneously injected into immunocompromised mice. The matrix mixture contains pro-angiogenic factors to stimulate vessel formation. The treatment may be started directly after implantation (preventive study) or after a mature human vasculature has been established (interventive study). The growing human vasculature is monitored by bioluminescence imaging during therapy once weekly. At the endpoint, the matrix plug is removed, and the luciferase signal is analyzed ex vivo as a readout for human vascular formation.

Additional readouts:

  1. Implantation of spheroids from untransfected HUVECs into the other flank
  2. Counting of human blood vessels by human CD34 staining
  3. Determination of relative pericyte coverage and/or perfusion rate of human vessels
Luciferase-based readout

Luciferase-based readout

Study example. Sunitinib and PTK787were tested in the In Vivo Angiogenesis Assay using luciferase-expressing HUVEC cells. Bioluminescence imaging of the mice was performed during therapy. After spheroid implantation, the mice of both groups showed similar luciferase signals which were strongly reduced in the Sunitinib treated group after 20 days of treatment (left). At necropsy, the matrix plugs were removed from the mice, and luciferase activity was measured ex vivo which showed a comparable result as the in vivo imaging (right).

Histology-based readout

Histology-based readout

Histology-based readouts. At necropsy, the matrix plugs are removed from the mice, and human vessels are stained with an anti-human CD34 antibody. Co-staining with anti-SMA (smooth muscle actin) is performed for visualization of recruited murine mural cells. Optional: perfusion of the vessels can be visualized by injection of a dye.


The in vivo facility of Reaction Biology is located in Freiburg, Germany.

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Quality Assurance

Animal work

  • Routine health monitoring of sentinel animals (according to FELASA guidelines)
  • Standardized operation procedures are in place of every step and every model

Study support

  • Studies generally start (tumor implantation) 3 to 5 weeks after receipt of the order
  • Suggestions for study layout to get statistically relevant results
  • Weekly study update and personal contact with the study supervisor
  • Reports are written by medical writers on Ph.D. level

Ethical principles

  • Our animal work is conducted according to the 3R (Replacement, Reduction, and Refinement)
  • We are working closely with our animal care committee to ensure timely adaptions of our animal care licenses to custom-tailor our client’s project to ensure the most meaningful study outcome
Animal Welfare

Reaction Biology uses laboratory animals to help our customers to understand the fundamental mechanisms behind malignancies and to discover therapeutics to prevent and treat cancer. Data obtained from animal models is critical to predicting the clinical outcome for an oncology drug candidate in development.

Animal welfare is of the utmost importance to us. Animal-based research is highly regulated to ensure ethical and responsible treatment. The mice in our facility are specifically bred for research purposes, and they are cared for to the very high standards.

We are working under GV-SOLAS and ISO9001 regulations in regard to standards of animal welfare and code of practice.

We employ three veterinarians and appropriately trained staff to ensure animal welfare is maintained at the highest standards. Regulation officers inspect our unit regularly.