Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)

Published: June 01, 2011

doi:

Lenard Conradi², Christiane Pahrmann, Stephanie Schmidt, Tobias Deuse³, Arne Hansen, Alexandra Eder, Hermann Reichenspurner, Robert C. Robbins, Thomas Eschenhagen, Sonja Schrepfer³

¹Transplant and Stem Cell Immunobiology Lab (TSI) and CVRC, University Hospital Hamburg,University Heart Center Hamburg, ²Department of Experimental and Clinical Pharmacology and Toxicology,University Heart Center Hamburg, ³CT Surgery,Stanford University School of Medicine

Summary

This video demonstrates the use of in vivo bioluminescence imaging to study immune responses after implantation of Engineered Heart Tissue (EHT) in rats.

Abstract

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or bioartificial scaffold materials, entrapment of cardiac myocytes in hydrogels such as fibrin or collagen and stacking of myocyte monolayers ¹. These concepts aim at restoration of compromised cardiac function (e.g. after myocardial infarction) or as experimental models (e.g. predictive toxicology and substance screening or disease modelling). Precise monitoring of cell survival after implantation of engineered heart tissue (EHT) has now become possible using in-vivo bioluminescence imaging (BLI) techniques ². Here we describe the generation of fibrin-based EHT from a transgenic rat strain with ubiquitous expression of firefly luciferase (ROSA/luciferase-LEW Tg; ³). Implantation is performed into the greater omentum of different rat strains to assess immune responses of the recipient organism following EHT implantation. Comparison of results generated by BLI and the Enzyme Linked Immuno Spot Technique (ELISPOT) confirm the usability of BLI for the assessment of immune responses.

Protocol

1. EHT Generation and Culture Conditions Manufacture of fibrin-based EHT was performed as described elsewhere 4. In brief, to generate fibrin-based EHT for implantation purposes, a master mix was prepared containing cells isolated from neonatal ROSA/luciferase-LEW transgenic rat hearts, bovine fibrinogen and Matrigel. Agarose casting molds were prepared using Teflon spacers placed in 6-well culture dishes. After solidification of agarose, spacers were removed and silicone post racks …

Discussion

Before clinical implementation of EHT technology for cardiac repair, fundamental questions such as immunogenicity of both cellular and matrix components or graft survival after implantation need to be assessed in experimental models. In-vivo bioluminescence imaging represents a useful new technique for monitoring cell survival after transplantation. We successfully generated fibrin-based EHT containing luciferase positive (luc+) heart cells by adaptation of an established protocol ⁴. These EHT were im…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Sonja Schrepfer, Lenard Conradi and Arne Hansen received funding from the Deutsche Forschungsgemeinschaft (DFG; SCHR992/3-1, SCHR992/4-1, CO 858/1-1, HA 3423/3-1). The work was also supported by grants from the European Union to Thomas Eschenhagen (EUGeneHeart and Angioscaff).

Materials

LEW-Tg(Gt(ROSA)26Sor-luc)11Jmsk rats (NBPR rat number 0299, http://www.anim.med.kyoto-u.ac.jp/NBR/)
Bovine fibrinogen (stock solution: 200 mg/ml plus aprotinin 5 μg/ml fibrinogen in NaCl 0.9%, Sigma F4753)
Matrigel (BD Bioscience 356235)
DMEM (Biochrome F0415)
Horse serum (Gibco 26050)
Penicillin/streptomycin (Gibco 15140)
Insulin (Sigma-Aldrich I9278)
Aprotinin (Sigma Aldrich A1153)
d-Luciferin (Biosynth L-8220)
Enzyme-linked immunosorbent spot (ELISpot) assay plates (Hassa Laborbedarf, S2EM004M99)

References

Eschenhagen, T., Zimmermann, W. H. Engineering myocardial tissue. Circ Res. 97, 1220-1231 (2005).
van der Bogt, K. E., Schrepfer, S., Yu, J. Comparison of transplantation of adipose tissue- and bone marrow-derived mesenchymal stem cells in the infarcted heart. Transplantation. 87, 642-652 (2009).
Hakamata, Y., Murakami, T., Kobayashi, E. “Firefly rats” as an organ/cellular source for long-term in vivo bioluminescent imaging. Transplantation. 81, 1179-1184 (2006).
Hansen, A., Eder, A. Development of a Drug Screening Platform Based on Engineered Heart Tissue. Circ Res. , (2010).

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Cite This Article

Conradi, L., Pahrmann, C., Schmidt, S., Deuse, T., Hansen, A., Eder, A., Reichenspurner, H., Robbins, R. C., Eschenhagen, T., Schrepfer, S. Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT). J. Vis. Exp. (52), e2605, doi:10.3791/2605 (2011).