Research highlights

Directed cell migration using micropatterns

Cell migration is critical for the development of multicellular organisms, as well as in (patho)physiological processes, such as wound healing, immune response and carcinogenesis. Although several studies have been conducted to characterize the effect of simple chemical and topographical patterns on cell-substrate interactions,¹ the influence of shape and dimensions of micropatterns on cell migration is still not fully understood.

Recently Mofrad and colleagues² have explored the effect of spatial cues (shape and dimensions of micropatterns) on the locational and migratory behavior of adherent cells. Using a microengraved substrate, Yoon et al.² demonstrated passive control over the locomotion of adherent cells. The authors also performed a quantitative analysis of the migratory behavior of undisturbed adherent cells and were able to manipulate the speed of cell migration.

The platform was generated from a transparent, biodegradable, UV-curable resin (ORMOCOMP) by printing the microstructures with a PDMS mold. The topography of the platform was designed to give rise to several migratory paths, all of which would lead the cells to a single target site (Rome). These paths were formed by a series of microscale ridges and troughs that were aligned in three distinct zones, in parallel and diverging configurations (Fig. 1), and were comparable in dimensions to cells (3 μm deep and 3–75 μm wide). The researchers observed the attachment and locomotion of NIH-3T3 fibroblasts after seeding. Analysis of the imaged cells indicated that the cells chose to reside inside the troughs rather than on the ridges, and preferred wider features (larger than a single cell) compared to narrower ones. Thus, the localization of adherent cells was mainly influenced by the width of the physical spatial cues. Furthermore, the microscale features on the Rome platform were oriented at various angles to each other to help guide the cells towards such collection sites. More specifically, cells predominantly moved away from points of convergence, which is in line with the observation that the cells preferred wide structures to narrow ones. Also, the average speed of cell motion was inversely proportional to the structure width; the narrower the feature, the faster cells moved away.

Fig. 1 The micropatterned Rome platform consists of three distinct zones with parallel and angled troughs and ridges of varying widths. This gradient of spatial cues is used to passively direct cell migration. Details of the cell structure integral in locomotion are also shown. Figure adapted and reprinted with permission from the Royal Society of Chemistry from Yoon et al.²

This study offers observation of cell locomotion in 2D, and it is unclear to what extent it can be extrapolated to physiologically relevant 3D systems. However, it is conceivable that this information could be used for applications, such as engineering smart wound dressings with patterned surfaces, such that fibroblasts—a key cell type in wound healing—would migrate towards sites of injury in a controlled fashion. Similarly, drug-delivery and other implantable devices could be surface patterned to either limit or enhance cell migration to a particular site, depending on the application. In this case, understanding the locomotion behavior of different cell types (e.g. healthy and carcinogenic) can also be utilized in development of novel therapeutic strategies.

‘True Blood’ hydrogels

The shortage of blood supply coupled with other challenges, such as the short shelf-life of donor blood, the need for proper matching and infection risks, have made the quest for blood alternatives a necessity. Synthetic oxygen carriers have been proposed as a viable alternative to blood, both as a means of supplementation and possibly even replacement of blood transfusions altogether. Hemoglobin (Hb) based oxygen carriers were amongst the first to be utilized, however, free Hb is rapidly cleared from circulation. In addition, it is severely toxic and has several adverse side effects. Previous approaches (conjugation, crosslinking or polymerizing Hb molecules) have led to altered oxygen affinity and poor bioactivity. Similarly, methods of encapsulating Hb have resulted in rigid particles with insufficient circulation times that promote Hb autooxidation.³

DeSimone and colleagues have recently challenged conventional wisdom and proposed that synthetic microparticles would not be quickly cleared from the circulation due to mechanical filtration. This led them to further expand on their previous work⁴ and to report the development of extremely deformable red blood cell (RBC)-like hydrogel microparticles loaded with bovine hemoglobin (Hb) that was meant to potentiate oxygen transport (Chen et al.⁵).

These microparticles matched the size, shape and deformability of RBCs when tested inside a novel microfluidic device (Fig. 2). The group was able to demonstrate homogenous distribution and a controlled variation in the amount of Hb conjugates throughout the microparticles, known to affect the loading ratio of Hb. Volumes as high as 5 times the amount of Hb to polymer (by weight) could be incorporated into these hydrogel microparticles, while having little or no effect on particle stability and shape. Interestingly, although a slight decrease in particle diameter was observed after Hb conjugation, the particles remained deformable and intact even after high shear rates (1000 per second for 10 min). Maintaining a RBC-like elasticity is particularly important given that increased particle flexibility has been shown to enable their passage through constricted pores and correlated with longer in vivo circulation times. In addition the inactive autooxidated metHemoglobin (metHb) in these RBC-mimics could be maintained at low levels.

Fig. 2 (A) A scheme for the conjugation chemistry of Hb to RBC-mimicking microparticles. (B) An Illustration of the microfluidic device. Figure reprinted with permission from Chen et al.⁵

To evaluate the deformability of these RBC-mimicking hydrogel microparticles the group utilized a novel microfluidic setup. This microfluidic setup mimicked the naturally occurring vascular constrictions by containing 3 μm pores inside a series of channels of the same height. The small channel dimensions inhibited vertical passage of microparticles, forcing them to deform in order to pass through the pores. Therefore, this design allowed for evaluating particle deformations, while maintaining constant fluid flow and therefore low pressure inside the device.

This approach represents a significant step forward in the applications of oxygen carriers toward artificial blood constructs, tackling major obstacles in functional Hb conjugation and microparticle deformability. However, given that reducing agents preventing Hb to metHb conversion might only work in storage fluids and not post injection, some challenges still remain. To overcome this the authors proposed additional functionalization of microparticles with low amounts of reactive oxygen species (ROS) scavenger enzymes that could regulate metHb levels and prolong Hb stability, but such ideas remain to be tested. In addition, immunogenicity concerns linger with the use of non-human Hb and need to be addressed in detail. But it is clear that the merger of microscale technologies in fabricating and testing RBC-mimetic particles may provide solutions to long-standing challenges in the field of artificial blood.

Tumor cell intravasation

Tumor cells have the ability to enter the circulatory system and mix with blood by intravasation, a process that is critical in cancer metastasis due to the possibility of forming secondary tumors.⁶ While contact or communication between cells (e.g. via secretion of cytokines and growth factors) is thought to be regulating intravasation, the exact mechanism of this rate-limiting step in metastasis remains poorly understood. Due to the absence of physiologically relevant in vitro models and the difficulties in investigating cellular interactions in vivo, the intravasation mechanism remains unclear. One means to overcome this challenge would be to investigate tumor–endothelial cell interactions with the use of microfluidic platforms and real time visualization techniques.⁷

Kamm and colleagues have recently developed a novel microfluidic chip to study dynamic tumor–endothelial cell interactions in 3D,⁸ allowing for measurements of endothelial permeability and real-time observation of cytokine mediated tumor cell intravasation. The microfluidic device consisted of two parallel channels connected with a 3D biomimetic hydrogel. One channel was seeded with tumor cells and the other with endothelial cells, such that a confluent monolayer of endothelial cells was formed on the ECM hydrogel (Fig. 3). This structure was shown to be superior to conventional transwell plates due to its ability to facilitate cellular signalling during the 3D intravasation process. In addition, the microfluidic device was capable of precisely controlling the micro-environment of the cells, such as gradients of growth factors. Another unique feature of the microfluidic platform was the ability to accurately measure the permeability of the endothelial cell layer.

Fig. 3 The experimental set-up for monitoring interactions between tumor and endothelial cells. (A) The microfluidic device with the endothelial channel (green), tumor channel (red) and 3D ECM (dark gray) between the two channels. (B) Migration of fibrosarcoma cells toward endothelial cells. (C) Immunostaining for endothelial cells. (D) Invasion of endothelium by tumor cells. Figure adapted and reprinted with permission of the National Academy of Sciences from Zervantonakis et al.⁸

Experimental results showed that the presence of macrophages increased the intravasation rate of breast tumor cells in the microfluidic platform. In this case, tumor cells migrated through the 3D biomimetic hydrogel and intravasated into the flow channel passing through the endothelial cell layer. Similarly, upon endothelial layer stimulation with TNF-α, a higher number of tumor cells migrated through the 3D hydrogel beyond the endothelial surface, pointing to an increased permeability of the endothelium. As a result, it was suggested that a damaged endothelium contributes to tumor cell intravasation.

To further probe the dynamics of the tumor migration process, human fibrosarcoma cells were tested in both normal and impaired endothelial barrier conditions stimulated by TNF-α. The results indicated that an impaired endothelium increased the rate of tumor cell invasion, which is in agreement with previous findings.⁹ The influence of paracrine signaling from macrophages and TNF-α stimulation on tumor cell migration was also tested. When TNF-α was blocked, the amount of intravasation decreased significantly.

In short, an in vitro microfluidic assay was developed to monitor interactions between tumor cells and endothelial cells during the intravasation process. The microfluidic device was identified as an effective tool for direct observation of tumor cells in real-time.

In the future, high-resolution dynamic imaging strategies could potentially be used to monitor cellular interactions for the diagnosis of various pathological events. These could include inflammation, cellular damage, tissue injury and apoptosis, as well as wound healing. The integration of quantitative real-time imaging platforms to microfluidic systems is also critical for toxin screening and drug discovery, which may lead to new research avenues in biomedical research.

References

1. (a) J. Y. Lim and H. J. Donahue, Tissue Eng., 2007, 13, 1879–1891; (b) M. N. Yousaf, B. T. Houseman and M. Mrksich, Proc. Natl. Acad. Sci. U. S. A., 2001, 98, 5992–5996.
2. S. H. Yoon, Y. K. Kim, E. D. Han, Y. H. Seo, B. H. Kim and M. R. K. Mofrad, Lab Chip, 2012, 12, 2391–2402.
3. R. M. Winslow, Adv. Drug Delivery Rev., 2000, 40, 131–142.
4. T. J. Merkel, S. W. Jones, K. P. Herlihy, F. R. Kersey, A. R. Shields, M. Napier, J. C. Luft, H. Wu, W. C. Zamboni, A. Z. Wang, J. E. Bear and J. M. DeSimone, Proc. Natl. Acad. Sci. U. S. A., 2011, 108, 586–591.
5. K. Chen, T. J. Merkel, A. Pandya, M. E. Napier, J. C. Luft, W. Daniel, S. Sheiko and J. M. DeSimone, Biomacromolecules, 2012, 13, 2748–2759.
6. (a) A. F. Chambers, A. C. Groom and I. C. MacDonald, Nat. Rev. Cancer, 2002, 2, 563–572; (b) P. S. Steeg, Nat. Med., 2006, 12, 895–904.
7. (a) V. V. Abhyankar, M. W. Toepke, C. L. Cortesio, M. A. Lokuta, A. Huttenlocher and D. J. Beebe, Lab Chip, 2008, 8, 1507–1515; (b) J. W. Song, S. P. Cavnar, A. C. Walker, K. E. Luker, M. Gupta, Y. C. Tung, G. D. Luker and S. Takayama, Plos One, 2009, 4.
8. I. K. Zervantonakis, S. K. Hughes-Alford, J. L. Charest, J. S. Condeelis, F. B. Gertler and R. D. Kamm, Proc. Natl. Acad. Sci. U. S. A., 2012, 109, 13515–13520.
9. B. D. Robinson, G. L. Sica, Y. F. Liu, T. E. Rohan, F. B. Gertler, J. S. Condeelis and J. G. Jones, Clin. Cancer Res., 2009, 15, 2433–2441.

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