Surface-modified magnetic human cells for scaffold-free tissue engineering

Abstract

We report the magnetically-facilitated scaffold-free assembly of lung tissue mimicking two-layered multicellular clusters. Polymer-stabilized magnetic nanoparticles were deposited on surfaces of viable human cells (A549 and skin fibroblasts), allowing the formation of two-layered porous tissue prototypes.

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Tissue engineering aims at the fabrication of human tissues and organs de novo.¹ In particular, for lung tissue engineering, the directed assembly of isolated human cells as building blocks is considered to be a promising approach.² Normally, the cells are spatially arranged on artificial scaffolds accommodating the cells and providing them with structural support.³ Magnetic nanoparticles (MNPs) are regarded as a promising tool to assemble the magnetically-labelled cells into larger tissue prototypes^4–7 and multicellular clusters and coatings.⁸ Magnetically-facilitated tissue engineering offers the scaffold-free arrangement of the cells allowing (i) avoiding the use of synthetic or natural scaffolds⁷ and (ii) fabricating of prototype tissues with elaborate morphologies.⁹ However, the scaffold-based tissue engineering approach can also benefit from the use of magnetically-labelled cells, especially when MNPs functionalized scaffolds are considered.¹⁰ In addition, magnetically-labelled tissues could be easily delivered or immobilised within the body guided by an external magnetic field⁹ or visualised using magnetic resonance imaging.⁶ Thus, the application of magnetic nanoparticles in human tissue engineering methodology is currently regarded as an advanced technique to drive tissue regeneration.

The conventional techniques for magnetic labelling of human cells are largely based on intracellular uptake of citrate¹⁰ or dextran¹² stabilised anionic MNPs, magnetic cationic liposomes (MCLs)^4,13 or poly-L-lysine-coated citrate-stabilized MNPs¹⁵via endocytosis. The procedure typically starts with the introduction of the dispersed MNPs or MCLs into the cell growth media. The cells are normally exposed to the media-dissolved nanoparticles for up to 72 hours.^11–15 During the incubation, the MNPs are spontaneously absorbed by the cells and randomly distributed within the cytoplasm. This approach has several drawbacks: (i) the nanoparticles concentrate in intracellular endosomes,⁵ which requires long incubation periods; (ii) the nanoparticles uptake (hence, the magnetisation rate) differs considerably in different cell cultures⁵ and depends strongly on surface chemistry of nanoparticles¹⁴ and (iii) apart from MNPs, lipid mixtures⁴ and serum proteins¹⁴ can be spontaneously delivered into the cells during the MNPs uptake. Although most of the recent studies report the low toxicity of MNPs used, the internalised nanoparticles can implement the yet unexplored long-term toxic effects seriously affecting the cellular functions.¹⁵ Obviously, an approach which would provide human cells with magnetic function without MNPs internalisation is required.

Earlier we proposed an alternative strategy for magnetic labelling of human cells, which is based on employing the cationic polyelectrolyte-stabilised MNPs for the direct one-step surface modification of human cell membranes.¹⁶ We demonstrated that the polyelectrolyte-stabilised MNPs assemble exclusively on the cellular membranes of the HeLa carcinoma cells. MNPs do not penetrate into the cytoplasm and do not affect the viability of the functionalised cells. Here we apply our technique for magnetic functionalisation of adenocarcinomic human alveolar epithelial cells (A549) and human skin fibroblasts (HSF) used here as the model cells for prototype lung tissue engineering. We demonstrate the effective and fast surface modification of these cells using (poly)allyl amine-coated MNPs (PAH-MNPs) (Fig. S1 in the ESI†) and the subsequent fabrication of lung-tissue mimicking multicellular clusters using the viable MNPs-coated A549 and HSF cells and the permanent magnet. Our technique is sketched in Scheme 1. Both A549 and HSF cells are coated with PAH-MNPs and then sequentially deposited onto the scaffold-free culture wells with a 3 mm NdFeB magnet. The first layer of the cells was assembled from the MNPs-coated HSF, while the second was formed by the MNPs-coated A549 cells, which after the removal of the magnet were able to form lung-tissue mimicking clusters. The experimental details are given in the ESI.†

Scheme 1 A sketch illustrating the lung-mimicking tissue fabrication employing magnetically-facilitated assembly of MNPs-functionalised HSF (first layer) and A549 (second layer) cells.

Here we employed for the first time the direct deposition of PAH-stabilised iron oxide magnetic nanoparticles to render the magnetic function on A549 and HSF cells. Due to electrostatic interactions, positively-charged PAH-MNPs (zeta-potential +27.5 mV in water; +26 mV in 0.15 M NaCl) self-assembled on negatively-charged cells (zeta-potential of A549 cells −25 mV; HSF −28 mV; measured in HEPES buffer), resulting in the formation of the uniform monolayer of MNPs on both A549 and HFS cells. The procedure is very simple and fast, involving the introduction of the sterile suspension of PAH-MNPs (0.05 mg mL⁻¹) in 0.15 M NaCl into the cell suspensions (10⁶ mL⁻¹) in Dulbecco phosphate buffered saline (DPBS). The cells were incubated for 3 min under gentle shaking and then separated from the unattached MNPs by centrifugation and washed twice with DPBS. Control samples were subjected to the same treatment with pure DPBS instead of MNPs. The optical microscopy images (Fig. S2 in the ESI†) demonstrate the characteristic brown hue of MNPs-coated suspended cells immediately after nanoparticle deposition when compared with non-coated cells. Ultrathin sections of magnetically-functionalised cells were examined using transmission electron microscopy (TEM). As shown in Fig. 1a and b, PAH-MNPs are assembled in the dense network of cellular microvilli, whereas no MNPs were detected inside the cytoplasm. These observations correspond well with our previous studies with HeLa cells.¹⁶ We suppose that the relatively large aggregates of polymer-coated MNPs (hydrodynamic diameter: 88 nm in water, 102 nm in 0.15 M NaCl) cannot be absorbed by the cells via endocytosis, which makes this approach more biocompatible than the conventional endocytosis-based MNPs-labelling techniques.^12,17

Fig. 1 Transmission electron microscopy images of MNPs-functionalised A549 (a) and HSF cells (b) demonstrating the deposition of MNPs (indicated by arrows) on the cellular membranes; fluorescence microscopy images of MNPs-coated A549 (c) and HSF (d) cells stained with ethidium bromide (red, dead cells) and acridin orange (green, live cells); optical microscopy images of MNPs-functionalised A549 (e) and HSF (f) cells colonising the substrates 24 hours after the introduction into the nutrient media (note the brown aggregates of MNPs on both types of cells).

After magnetic functionalisation, we investigated the viability and proliferation of the MNPs-coated cells. We employed the ethidium bromide/acridin orange viability stain and found that the cells were not affected by magnetic surface functionalisation. The percentage of viable MNPs-coated A549 and HSF cells (91.9% and 90.9%, respectively) (Fig. 1c and d) is almost equal to that in intact cells (A549 – 97.4%; HSF – 91.9%). Viability stain strongly suggests that the surface-deposited MNPs do not jeopardize the integrity of cellular membranes. Next, we found that the MNPs-coated cells proliferate normally if introduced into the nutrient media (Fig. 1e and f), reaching confluency as effectively as the intact cells (intact A549 – 85%, MNPs-coated A549 – 90%, intact HSF – 30%, MNPs-coated HSF – 30%), as shown in Fig. S3 in the ESI.† Importantly, we observed the circular brown aggregates of MNPs concentrated at central dome-like regions of the attached cells (Fig. 1e and f), which indicates that the cells actively relocate the MNPs coating after attaching to the substrates. We noticed that the MNPs-coated A549 and HSF cells proliferated somewhat faster than their intact counterparts; therefore we employed the xCELLigence analyser to investigate the adhesive properties and proliferation rate via the label-free monitoring of the impedance changes (expressed as cell index) in real time. Cell index is a dimensionless parameter derived as a relative change in the measured electrical impedance,¹⁸ indicating the adhesion and growth of the viable cells on the gold electrodes.¹⁹ As shown in Fig. 2a and b, both A549 and HSF cells demonstrate the higher cell index values after magnetic functionalisation when compared with intact cells. We attribute this to the increased adhesive properties of the MNPs-coated cells, due to the mechanical facilitation of adhesion of cell surfaces labelled with positively-charged MNPs onto polystyrene plates or gold xCELLigence electrodes. Importantly, deposited MNPs do not severely affect the surface charge of the coated cells (zeta-potential of the MNPs-coated A549 cells: −23 mV; HSF: −26 mV; measured in HEPES buffer). Moreover, we suppose that the MNPs-coated cells increase the division rate to remove the artificial nanoparticulate coatings from the cellular membranes. Additional experiments are required to elucidate the exact mechanisms of this phenomenon; however, at this point we suppose that the viable human cells are able to remove the surface-attached coatings, which is supported by the optical microscopy observations demonstrating the lack of MNPs in the distal regions of attaching and actively proliferating cells (Fig. 1e and f).

Fig. 2 Real time cell index monitoring demonstrates the increased adhesive properties and proliferation of MNPs-functionalised (green curves) A549 (a) and HSF (b) cells in comparison with intact cells (blue curves). Reference (media) signal is shown in red curves.

Finally, we employed the viable MNPs-coated A549 and HSF cells to fabricate two-layered scaffold-free lung tissue mimicking multicellular clusters. First, MNPs-coated HSF cells (0.3 × 10⁶ cells per well) were introduced in 6-well culture plates equipped with 3 mm NdFeB cylindrical magnets positioned under the wells (as shown in Scheme 1). The introduced MNPs-coated cells were incubated for 24 hours; then the loosely-attached cells were washed away with DPBS and the culture media was replaced. Next, we repeated the procedure with MNPs-coated A549 cells (0.5 × 10⁶ cells per well), which were assembled precisely above the magnetically-retained HSF cells. Then the culture plates were incubated for another 24 hours. Once the magnets were removed, the resulting multicellular clusters could be easily defoliated from the wells and studied using optical microscopy. The diameter of the round-shaped two-layered cluster was 3 mm. As one can see in Fig. 3a, the two-layered architecture of the clusters is supported by the proliferating viable cells. Brown aggregated MNPs can be clearly seen between the cells, which would allow, if necessary, magnetic manipulation with the clusters. Interestingly, the porous morphology of the artificial lung tissue-mimicking clusters to a certain extent repeats the morphology of human lung tissue (Fig. 3b). Remarkably, we noticed both relatively large mature and smaller emerging pores. This may suggest that the surface modification of human A549 and HSF cells with PAH-MNPs does not inhibit the functionality of the cells; therefore the magnetically-functionalised human cells reconstitute the original tissue. This approach is not limited to human epithelial cells and fibroblasts and can be applied to MNPs-mediated functionalisation of other types of cells, as we have shown earlier with HeLa cells.¹⁶ Future research will be focused on the application of cell surface engineering with MNPs in combination with other types of cells, including primary human cell lines.

Fig. 3 Optical microscopy images of thin sectioned paraffin-embedded hematoxylin–eosin stained magnetically-facilitated lung-mimicking multicellular clusters fabricated from MNPs-functionalised A549 and HSF cells (a) and human lung tissue (b). Note the alveoli-mimicking emerging and mature pores and brown MNPs aggregates.

Conclusions

To conclude, here we report for the first time the effective application of surface-functionalised magnetic human cells for the fabrication of lung-tissue mimicking multicellular clusters. Magnetically-facilitated assembly of MNPs-coated cells resulted in the formation of two-layered structures having the characteristic porous alveoli-mimicking morphologies. We believe that the approach described in this communication can be further extended to other types of cells/tissues.

Acknowledgements

This study was supported by RFBR 12-04-33290 (leading young scientists support) grant. The authors thank Dr R. A. Dzamukov (Republic of Tatarstan Clinical Infirmary) for real human lung tissue samples.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Experimental details and additional figures, as noted in the text. See DOI: 10.1039/c3bm60054h

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