Cell Capturing using Electrospun Membrane

Figure 1. Hela cells on glass coverslip showing preferential proliferation and adhesion on nanofibers instead of exposed smooth surface.

A surface that allows rapid cell adhesion is useful for bedside cell seeding onto scaffold prior to implant or for isolation of cells for diagnostic purpose. A study using osteoblast showed more than 30% cell attachment on the surface of poly-L-lactic acid nanofiber surface compared to about 10% on tissue culture plate (TCP) [Ngiam 2010] at 30 min. Electrospun nanofibrous membrane is known to encourage early cell attachment on its surface. An electrospun membrane may be functionalized to improve cell adhesion rate or made selective towards specific cell type.

An electrospinning setup by Bioinicia [Video of FLUIDNATEK LE-50].

Isolation and in vitro cell expansion is a commonly suggested technique of introducing scaffold with cells. However, this requires two visits to the hospital and a lengthy wait for the cell to multiple to the desired quantity. An alternative is use a scaffold that is able to isolate stem cell from stem-cell rich blood source and implant the stem-cell enriched scaffold. The whole process of blood drawing, isolation and implantation can be completed within one hospital visit. Studies have shown that 25% of hematopoietic stem cell (HSC) was captured on collagen and PLGA/collagen blend in 30 min as compared to only about 2% on TCP[Ma et al 2008]. Significantly higher percentage of MSC was also found to adhere on electrospun membrane at 1 hour (50 to 71%) compared to smooth film (28%) of the same material [Finne-Wistrand et al 2008]. From table 1, it is apparent that a fibrous surface encourages faster cell adhesion compared to smooth surface. Apart from surface roughness, fibers with biomolecules incorporated would further encourage cell adhesion on the substrate [Chan et al 2009].

Table 1. Comparison of cell adhesion/capture on different substrates.

Reference	Cell type	Substrate	Duration	Percentage cell capture/adhesion
Ngiam 2010	Osteoblast	Poly-L-lactic nanofiber	30 min	30%
		TCP		10%
Ma et al 2008	Hematopoietic stem cell	PLGA/collagen blend (1:1),	30 min	23%
		TCP		2%
		PLGA		8%
		Collagen		26%
Finne-Wistrand et al 2008	MSC	Electrospun poly[(L-lactide)-co-(1,5-dioxepan-2-one)]	60 min	50 to 71%
		Film poly[(L-lactide)-co-(1,5-dioxepan-2-one)]		28%
Chan et al 2009	Human MSC	Tissue culture plastic bottom	30 min	3.2%
		Naked glass coverslip		3.3%
		Gelatin coated coverslip		5.3%
		Collagen-coated coverslip		3.6%
		P(LLA-CL) nanofiber, 480 nm		9.7%
		Air-plasma P(LLA-CL) nanofiber		27%
		Collagen-coated P(LLA-CL) nanofiber		40%
		Collagen microfiber, dia. 1140 nm		43%
		Collagen nanofiber, dia. 390 nm		45.1%
		CD49a antibody conjugated collagen nanofiber		50%
		CD29 antibody conjugated collagen nanofiber		55%

In a novel experiment, mineralized 3D nanofibrous scaffold with yarn microstructure was used to capture nucleated cells from bone marrow. After 20 min, 80% of the nucleated cells were captured on the mineralized 3D scaffold with 76% of the cells expressing MSC markers (CD44) [Ngiam 2010]. It is also possible to use thin nanofibrous membrane or cut nanofibrous membrane to capture the cells prior to meshing the membrane and cells to form a 3D nanofibrous scaffold where structural integrity is not required. Another way of forming 3D scaffold is to stack layers of fiber membrane or rolling the membrane to form a concentric layered cylindrical rod.

The ability to capture and isolate cells may also be used for the capturing of circulating tumor cells. These cancer cells are typically found in small quantities in the blood and the ability to capture it will aid diagnosis. Cao et al (2014) used collagen-blended poly(d,l-lactide-co-glycolide) nanofibers substrate for capturing of K562 leukemia cells. Adhesion of K562 leukemia cells was found to 70% better than tissue culture polystyrene surface with a shorter time. Zhang et al (2012) used electrospun TiO₂ functionalized with anti-EpCAM/EpCAM to capture circulating tumor cells from colorectal and gastric cancer patients. Similarly, Liu et al (2014) used MnO₂ grafted with anti-EpCAM to capture breast cancer cell line (MCF-7). The nanofibers placed in a microchannel were able to capture 90% of the cells while very few were captured on antibody-grafted glass substrate. Their study also showed that anti-EpCAM grafting contributes to capturing EpCAM-positive cancer cell while EpCAM-negative cancer cell are captured in much lower frequency. Oxalic acid was able to release the captured cancer cell by dissolving the nanofibers with sufficient surviving cells for re-culturing. Zhao et al (2014) used layer by layer technique to functionalize cellulose acetate using a bilayer of poly(diallyldimethylammonium chloride) (PDADMAC) and polyacrylic acid (PAA) through electrostatic interactions. G5.NH₂ dendrimer pre-modified with folic acid (FA)and florescein isothiocyanate (FI) was covalently conjugated to the bilayer-assembled nanofibers. With the folic acid, the membrane was able to capture significantly more KB cells (a human epithelial carcinoma cell line) overexpressing FA receptors at 83% after 1 hour compared to the control membrane without surface modifications (38%). This is also significantly more than L929 cells (a mouse fibroblast cell line) and KB cells with low level FA expression at 59% and 66% respectively captured by the surface modified membrane.

Rapid cell adhesion on electrospun fibers may encourage early drug uptakeby the cells. Chu et al (2023) tested the cellular uptake efficiency of released drugs by the nanofibers and nanoparticles using cultured mouse macrophages. At 0.5 h, the cell uptake from the nanofibers was 2.7 times higher than pure nanoparticles although there was no significant difference between the two at 4h. For celecoxib PLGA nanoparticles (Cel-NPs), the cumulative release of celecoxib was 44% within the first 3 hours. Subsequently, a cumulative release of 82% was recorded within 72 h. In contrast, electrospun PVA loaded with Cel-NPs (Cel-NPs-NFs) had a cumulative release of 68% at 168 h which is much slower than Cel-NPs. While drug-loaded nanoparticles showed rapid drug release, the lack of adhesion may delay the drug uptake process by the cells. In contrast, the faster adhesion of the cells on the nanofiber membrane may have allowed earlier absorption of the drugs.

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