gms | German Medical Science

71. Jahrestagung der Deutschen Gesellschaft für Neurochirurgie (DGNC)
9. Joint Meeting mit der Japanischen Gesellschaft für Neurochirurgie

Deutsche Gesellschaft für Neurochirurgie (DGNC) e. V.

21.06. - 24.06.2020

Enriched human endothelial cells isolated from peripheral nerves – a potential tool for research and the further development of artificial nerve grafts

Angereicherte humane Endothelzellen isoliert aus peripheren Nerven – ein potentielles Werkzeug in der Forschung sowie in der Entwicklung von zellularisierten künstlichen Nerventransplantaten

Meeting Abstract

  • presenting/speaker Patrick Dömer - Carl von Ossietzky Universität Oldenburg, Abteilung für Neurowissenschaften, Oldenburg, Deutschland
  • Christian Heinen - Evangelisches Krankenhaus Oldenburg, Abteilung für Neurochirurgie, Oldenburg, Deutschland
  • Bettina Kewitz - Carl von Ossietzky Universität Oldenburg, Abteilung für Neurowissenschaften, Oldenburg, Deutschland
  • Johannes Woitzik - Evangelisches Krankenhaus Oldenburg, Abteilung für Neurochirurgie, Oldenburg, Deutschland
  • Ulrike Janssen-Bienhold - Carl von Ossietzky Universität Oldenburg, Abteilung für Neurowissenschaften, Oldenburg, Deutschland; Carl von Ossietzky Universität Oldenburg, Forschungszentrum für Neurosensorik, Oldenburg, Deutschland
  • Thomas Kretschmer - Klinikum Klagenfurt am Wörthersee, Abteilung für Neurochirurgie, Klagenfurt, Austria

Deutsche Gesellschaft für Neurochirurgie. 71. Jahrestagung der Deutschen Gesellschaft für Neurochirurgie (DGNC), 9. Joint Meeting mit der Japanischen Gesellschaft für Neurochirurgie. sine loco [digital], 21.-24.06.2020. Düsseldorf: German Medical Science GMS Publishing House; 2020. DocV031

doi: 10.3205/20dgnc035, urn:nbn:de:0183-20dgnc0350

Veröffentlicht: 26. Juni 2020

© 2020 Dömer et al.
Dieser Artikel ist ein Open-Access-Artikel und steht unter den Lizenzbedingungen der Creative Commons Attribution 4.0 License (Namensnennung). Lizenz-Angaben siehe http://creativecommons.org/licenses/by/4.0/.


Gliederung

Text

Objective: Over the last years, endothelial cells (ECs) have come into scientific focus in peripheral nerve research, as they were shown to be a major constituent of the blood-nerve-barrier (BNB) and provide axonal guidance in peripheral nerve regeneration. However, mainly animal models and commercially available primary human umbilical-vein endothelial cells (HUVECS) were used in research, since previous protocols for the isolation of human nerve derived ECs were labour-intensive and required substantial experience in density centrifugation. Additionally, studies have shown different physiological properties and protein expression profiles for human nerve ECs compared to commercially available HUVECS and animal derived ECs. Thus, we established an easy-to-follow, time-efficient and inexpensive EC isolation protocol for human sural nerves, facilitating immunomagnetic cell sorting by means of CD31 coated Dynabeads.

Methods: For the EC isolation, sural nerve tissue derived from autologous nerve transplantations was enzymatically digested to gain a single cell solution. In a second step, a highly specific anti-CD31 antibody, which selectively binds to ECs, was conjugated to streptavidin coated superparamagnetic Dynabeads (ø 1 µm, Invitrogen, Carlsbad, CA, USA). Following incubation of the nerve cell solution with the CD31-Dynabeads, bead-bound ECs were retained magnetically, while the supernatant containing the other cell-types was discarded.

Results: The ECs reached a purity of > 95% and were seeded in gelatin coated cell culture plastic, where they started to proliferate within 24 hours. When ECs reached confluency, splitting of cells was performed using trypsin-EDTA, which resulted in a removal of Dynabeads from the ECs probably due to CD31 epitope cleavage by trypsin. The ECs could be subcultured for up to 6 passages until the proliferation rate declined.

Conclusion: This easy-to-follow isolation protocol simplifies the access to human primary nerve-derived ECs in basic research and allows the use of these ECs in clinically relevant applications. One area of application would be the experimental in-vitrovascularization of artificial peripheral nerve grafts with autologous patient derived ECs, extracted for instance from neuroma biopsies. Another application could be the introduction of patient derived ECs into the suture site to improve the axonal crossing of the injury-site, since ECs were shown to mediate axonal guidance.