Recent Advances in Electrospinning Technique to Mimic Natural Tissue

By Benedette Cuffari, M.Sc.Mar 30 2017

Researchers at the Lund University in Sweden have recently developed an artificial three-dimensional (3D) electrically active human neuronal network¹. Albin Jakobsson and Maximilian Ottosson’s team grew the neural progenitor cells of the brain on non-toxic, biodegradable and biocompatible low density poly-ε-caprolactone (PCL) fiber scaffolds which were synthesized by utilizing an improved electrospinning technique^2,3.

Electrospinning is an electrostatic fiber fabrication technique where nanoscale fibers measuring from 2 nanometers (nm) to several micrometers (mm) are produced from the application of a strong electric field on top of a solution or melt composed of both natural and synthetic polymers².

This relatively robust and simple technique technique has attracted researchers due to its potential applications in tissue engineering, biosensors, filtration, wound dressing, drug delivery and enzyme immobilization².

The nanofibrous spaghetti-like matrix produced by electrospinning technique more closely resembles the extracellular matrix (ECM) components present within a human body, as compared to traditional cell culture methods which often employ a flat laboratory glass dish¹.

The spun fibers produced by this technique offer various advantages including a high surface area to volume ratio, an adjustable porosity and the ability to customize the nanofiber composition to obtain various properties and functions³.

The 3D modeling of the neuronal network holds great promise in cell-based neuroscience research to further the understanding of the development and function of central nervous system (CNS), along with its corresponding diseases. The interactions between neural cells and the ECM play a key role in the proper development and functionality of the CNS³. The ECM is particularly important for the maintenance of homeostasis by regulating key cellular functions during development such as migration, differentiation and synapse formation³.

In brain cells cultured on a two dimensional (2D) flat laboratory dish using traditional laboratory techniques, the nerve cells grow on top of the supporting sheath of glial cells to produce abnormal cell-cell contacts and network formation³. This arrangement of both nerve and glial cells as different layers is not identical to the mixed arrangement of these cells that is present in actual brain tissue³.

In order to address this problem, researchers have tried to develop a 3D neuronal model in which the cells mimic the real brain tissue. Earlier studies have revealed that 3D neuronal cultures, which are based on cell-aggregate cultures, as well as hydrogels, have shown improved cell survival, varying differentiation patterns, longer neuronite outgrowth and formation of higher density network as compared to the traditional 2D cultures.

Based on these previous studies, The Lund research team employed an improved electrospinning method that utilizes a type of polymer that has been approved for medical purposes to produce 3D ECM-mimicking scaffolds. The advantage of using such a polymer could potentially facilitate neuronal growth in a similar to that which takes place in the human brain³.

While standard electrospinning methods usually yield dense and tightly packed fiber meshes that can result in poor cell infiltration and integration, the improved electrospinning method employed by this group yielded a highly porous and low density fiber scaffold with maintained interconnecting pores³.

Albin Jakobsson’s team designed and tested a novel collector consisting of a concave array of metal needles mounted on a non-conductive base in order to overcome the layer-on-layer effect of the compact fiber that is typically produced by a flat and static collector.

The uncompressed low-density PCL fiber scaffolds generated were evaluated for their ability to facilitate the growth of human neural progenitor cells (HNPC). Several parameters of the neural cells cultured onto 3D PCL scaffolds³ including cell survival, infiltration, phenotypic differentiation and form function were assessed and compared to the control, traditional 2D cultures of the neural progenitor cells³.

Scanning electron microscope (SEM) images confirmed that the 3D PCL fibers produced by the new and improved electrospinning technique utilizing a metal probe collector appeared to be uncompressed, which differed greatly from the compressed fibers that are typically generated following the use of standard electrospinning technique within a standard flat collector³.

Confocal microscopy images of HNPC cultured for 20 days in 3D cultures showed amalgamation of glial and neuronal cells, whereas the 2D cultures showed a layer of neuronal cells on top of supporting glial cells³. Furthermore, confocal images of HNPC cultured in 2D and 3D substrates, where the β -Tubulin cytoskeleton is stained with Texas red, demonstrated that cell infiltration and neurite extension in z-direction only occurred within the 3D substrates³.

Phenotypic analysis using immunohistochemistry (IHC) and western blot analysis also revealed that HNPC cultured on 2D substrates showed increased expression of nestin, which revealed that many cells were still in an immature development stage, which was comparable to the HNPC cultured on 3D substrates, which expressed much smaller amounts of nestin³.

The data obtained from this study determined that the uncompressed low-density nanofiber 3D scaffolds are able to adequately support the maturation of HNPC and other functional neuronal networks. This research suggests that the new electrospinning technique can open-up new opportunities for the development of cell cultures to mimic the natural environment of these cells, therefore allowing for the effective testing of potential drug candidates.

References

1. "Cells Grow More Naturally in 'spaghetti'." Phys.org. 27 Mar. 2017. Web. https://phys.org/news/2017-03-cells-naturally-spaghetti.html.
2. Bhardwaj, Nandana, and Subhas C. Kundu. "Electrospinning: A Fascinating Fiber Fabrication Technique." Biotechnology Advances 28.3 (2010): 325-47. Web.
3. Albin Jakobsson et al. Three-dimensional functional human neuronal networks in uncompressed low-density electrospun fiber scaffolds, Nanomedicine: Nanotechnology, Biology and Medicine (2017).
4. Image Credit: Shutterstock.com/pixelparticle

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