Electrospun fibers for in vitro biological studies

Electrospun nanofibers structure have attracted much interests from researchers in biology and medical field as its resemblance to natural extracellular matrix (ECM) may encourage cell response that is closer to in vivo situation. This will enable more accurate understanding of cell behavior to facilitate medical development. In mid 1990s to early 2000s, much studies were focused on the suitability of electrospun membrane as a tissue scaffold. Later studies have shown that the 3D environment provided by the electrospun nanofibers were able to better replicate cell behavior in natural extracellular matrix (ECM) [Wolun-Cholewa et al 2013]. Now, there are commercially available, electrospun nanofibers coated cell culture plates and coverslips for research into cell behavior. The use of electrospun nanofibers for in vitro cell studies have progressed beyond just coating on culture plates and coverslips. Electrospun scaffold are been used in other ways for in vitro biological studies in particular, as model for organ and tissue response.

Cells growing on the scaffold visualized using light microscopy after 48 hours of incubation. Original magnification x200 (A). Scanning electron microscopy image of HeLa cells on the nanostructured grid fibers after 48 hours of incubation. Original magnification x100 (B). [Wolun-Cholewa et al PLoS ONE 2013; 8: e72936. This work is licensed under a Creative Commons Attribution 4.0 International.]

Electrospun fibers on chip allow real time monitoring of cell response to the environment. Liu et al (2016) was able to create hepatocyte spheroid formation on integrated poly-DL-lactide (PLA) patterned electrospun fibers with a polydimethylsiloxane (PDMS) microfluidic chip. With a dynamic culture system, there was hepatocyte polarity which supported biliary excretion and maintained high levels of albumin and urea secretion over 15 days. A flow rate of 10 µL/min was found to be optimum in maintaining hepatocyte viability and significantly stronger biliary excretory function. At this parameters, they found that hepatocyte was most sensitive to 120 µg/ml Ag nanoparticles with 50% cell mortality after 24 hrs exposure to the nanoparticles. Cells cultured to day 7 and day 15 also showed similar mortality after 24 hrs exposure. The same setup has also been used for metabolism tests on tolbutamide and testosterone by hepatocytes [Liu et al 2017].

The glomerular filtration barrier of kidney functions as a biological sieve and is comprised of layers of two specialised cells, glomerular endothelial cells (GEnC) and podocytes, separated by a basement membrane. To create a model of the glomerulus, Slater et al (2011) electrospin a membrane layer made of collagen type I/polycaprolactine (PCL) on a supporting micro photo-electroformed (micro-PEF) nickel mesh. To mimic the cell layers of glomerular filtration barrier, GEnC and podocytes were co-cultured on opposite sides of the collagen/PCL/mesh composite. Their result demonstrated GEnC was able to grow in a uniform monolayer whilst podocytes were less densely packed. Highly porous and thin electrospun membrane enables the close contact of the cells and crosstalk between the cocultures.

The blood brain barrier (BBB) is a selectively permeable membrane that plays a critical role in maintaining brain homeostasis preventing entry of undesirable substances and organisms from reaching the brain. Having a physiologically relevant _{in vitro} BBB model is crucial to the development of drug therapy meant for the brain. Electrospun membrane is highly porous and known to promote various cells adhesion and proliferations. This has prompted several researchers to examine its potential use as an _{in vitro} BBB model. Pensabene et al (2016) developed electrospun poly (ε-caprolactone) (PCL) and polyethylene glycol (PEG) copolymer membranes to support the proliferation of human-derived endothelial cells, pericytes and astrocytes. They found that collagen coated electrospun PCL/PEG was able to promote a confluent and tight endothelium confirmed by transendothelial electrical resistance measurements (TEER). Bischel et al (2016) used electrospun gelatin to support a co-culture of astrocytes and endothelial cells (EC). Compared to standard PET inserts, electrospun gelatin inserts were found to have improved TEER, decreased permeability, and permitted a smaller separation between co-cultured cells at 21 days.

In cancer research, it is useful to establish a reliable cancer cell or tumor model for testing treatment response. Since electrospun scaffold is able to mimic ECM, cancer cell cultured on electrospun scaffold may response to treatment in a manner closer to clinical outcome. The effect of anti-cancer drugs seem to be reduced when cancer cells are cultured on electrospun scaffold compared to culture plates. Using gastric cancer cell line (MKN28), Kim et al (2009) showed lower inhibition of the cancer cells cultured on electrospun poly(3-hydroxybutyrate-co-3-hydroxyvalerate)/collagen nanofibers when anti-cancer drugs, namely, 5-FU, oxaliplatin and cisplatin are added compared to 2D cell culture plate cultures at 24 hours. However, paclitaxel and irinotecan are able to exhibit comparable cell inhibition to 2D culture. Breast cancer stem cells which are self-renewal and chemo-resistance are known to stay in a dormant state for many years as single cells and are resistant to therapies targeting proliferating cells. These cells have been described as having low rates of cell division, exhibit resistance to primary chemotherapy and radiation and expressing CD44+ and CD24-. Isolated MDA-MB-231 stem cells did not show significant difference in growth when cultured on fibrous scaffold and TCP. Comparison of a heterogeneous population of breast cancer cells on electrospun polycaprolactone fibers and tissue culture polystyrene (TCP) showed that aggressive breast cancer cells (MDA-MB-231) adopt a dormant phenotype with reduced growth on fibrous scaffold but the cells cultured on TCP maintains a high growth [Guiro et al 2015]. This gives electrospun scaffold the potential for enrichment of cancer stem cells for in vitro models.

In the research of fibrosis where tissue scarring is caused by activated myofibroblast (MF), it has been shown that conventional 2D hydrogel alone may not be able to elicit appropriate myofibroblast differentiation [Matera et al 2020]. The addition of fibers to form a multi-component hydrogel matrix is better able to imitate the 3D fibrous structure of interstitial tissue regions. Matera et al (2020) used a functionalized a biocompatible and protein-resistant polysaccharide, dextran, with pendant vinyl sulfone groups amenable to peptide conjugation (DexVS) for electrospinning into fibers and constructing a hydrogel. Electrospun DexVS fibers were added into the DeVS hydrogel precursors prior to gelation. MF differentiation as measured by α-smooth muscle actin (α-SMA) was found to be absent in transforming growth factor-β1 (TGF-β1) supplemented conditions that lacked fibrous architecture and other markers suggested that higher fiber density drives fibrotic phenotype and gene expression in the absence of a stiff hydrogel environment. Tests using primary human dermal fibroblasts and mammary fibroblasts showed that higher fiber density promoted proliferation in dermal fibroblast while mammary fibroblasts underwent MF differentiation. Higher fiber density also prompted greater hydrogel contraction compared to hydrogels with no or low fiber density. Greater collagen production was also found in hydrogels with higher fiber density. This study showed a clear influence of fiber density in hydrogel on MF differentiation and phenotype in 3D environment.

In most environments, microorganisms do not exist as a monoculture. Instead, a community of microorganisms would form a biofilm on surfaces. On human bodies, such biofilm may cause infections that are resistant to the host immune system and even to antibiotics. To enhance the understanding of biofilms in human body systems, researchers are trying to develop biofilms in vitro. Biagini et al (2022) tested the adhesion of human gut microbiota microorganisms on electrospun substrates to develop a 3D in vitro model of the human gut microbiota. Gelatin and polycaprolactone (PCL) was selected for this purpose with gelatin being a natural, hydrophilic material and PCL, a synthetic and hydrophobic material. Mucin, a protein produced by epithelial tissues, was coated on half the samples of electrospun gelatin and PCL scaffolds. Interestingly, on the 24 h and 48 h time points, electrospun uncoated PCL scaffold and mucin-coated PCL scaffolds performed better than gelatin scaffolds. However, at 72 h and 7 days, uncoated gelatin scaffolds outperform all other scaffolds. Between gelatin and PCL, gelatin could be digested by microorganisms and their degradation over time may have helped to support their proliferation. PCL is more resistant to degradation and its degraded products may not support proliferation of the microorganisms.

Electrospun scaffold may also be used to create whole organ model. Replicating skin in vitro will allow testing for various medical and cosmetic treatment to determine its toxicity to skin cells and to examine their response. The distribution of cells in the skin is such that the inner layer comprises mainly of fibroblast while the upper layer closer to the skin surface is mainly populated by keratinocytes. With this organization in mind, Yang et al (2009) cultured fibroblast and keratinocytes on separate electrospun PCL/collagen membranes. These membranes were constructed by alternating electrospinning of fibers directly into a cell culture media and seeding of cells. The layers were built up such that the fibroblast layers were at the bottom and the keratinocyte layers above. After culturing for 3 days, the individual layers were found to be tightly bounded together. This method allows for rapid formation of the 3D scaffold with cell distributions that mimics native skin.