Interaction of human Schwann cells with an artificial axon captured at high definition
Antonio Merolli, MD FBSE; Yong Mao, PhD; Viaceslav Manichev, MSc; Torgny Gustafsson, PhD; Leonard C Feldman, PhD; Joachim Kohn, PhD FBSE; Rutgers - The State University of New Jersey, Piscataway, NJ
INTRODUCTION: Understanding of myelination-remyelination process is essential to guide tissue engineering for nerve regeneration. In vitro models currently used are limited to cell population studies and had difficulty dissecting individual contributors to the process. In a limited number of reports, engineered fibers have been used to evaluate myelination in the absence of axons in vitro; however, by far, Oligodendrocytes but not Schwann cells have been used. We established a novel model in the absence of axons or neuronal factors to address the contribution of biophysical properties of axonal structure to the myelination process by human Schwann cells (HSCs).
MATERIALS AND METHODS: Our model uses a single carbon fiber (CF), suspended in culture media, to provide an elongated structure of defined diameter with 360-degree of surface available for HSCs cells to wrap around. Poly-Capro-lactone 3D printed scaffolds (n=76) supported the CF in a 24-wells-plate. HSCs (ScienCell Research Laboratories, Carlsbad, CA) were cultured. Live cell tracker (C2925, Thermofisher) was used to image cells day-by-day and for time-lapse studies. After fixation, scaffolds were incubated with anti-MAG (ab89780 AbCam) and anti-MBP (ab124439 AbCam) antibodies and imaged by fluorescence, confocal, scanning electron and Helium-Ion microscopy (without any coating).
RESULTS: We observed cell attachment, elongation and enwrapment around a fiber over a period of 2 weeks. The single wire construct allowed us to track and image each cell with different modalities. After 2-4 hours of incubation, elogantion begins and cytoplasmic prolongations start to spiral round the CF. Expression of myelin basic protein (MBP) was clearly detected; it was associated with the expansion of the cell membrane in a vast surface of the CF.
CONCLUSIONS: This model enabled us to spatially and temporally capture the interaction of human Schwann cells with an artificial axon. The possibility to apply multimodal analysis on each individual cell has never been reported in the past. Imaging by Helium-Ion microscopy is also a new modality and it allowed us to associate morphology at high resolution to functional the features previously identified by fluorescent markers (figure 1). The model is suited for the analysis of natural/artificial molecules and external physical factors (e.g. pulsed electrical current) as possible regulators of myelination by HSCs.
Figure 1. Human Schwann cells culture on a suspended Carbon Fiber of 6.7 micron in diameter: Helium-Ion microscopy at 2200X (HIM); Fluorescence microscopy at 400X (FLUO); combined picture (COMB). (blue=DNA; red=MBP; green=live tracker)
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