Correction to: Microtissue size and cell-cell communication modulate cell migration in arrayed 3D collagen gels.

The original version of this article unfortunately contained a mistake. One line indicating statistical significance was improperly placed in Fig. 5.

Microtissue size and cell-cell communication modulate cell migration in arrayed 3D collagen gels.

Cells communicate through the extracellular matrix (ECM) in many physiological and pathological processes. This is particularly important during cell migration, where cell communication can alter both the speed and the direction of migration. However, most cell culture systems operate with large volumes relative to cell numbers, creating low cell densities and diluting factors that mediate cell communication. Furthermore, they lack the ability to isolate single cells or small groups of cells. Droplet forming devices allow for an ability to embed single or small groups of cells into small volume segregated 3D environments, increasing the cell density to physiological levels. In this paper we show a microfluidic droplet device for fabricating 3D collagen-based microtissues to study breast cancer cell motility. MDA-MB-231 cells fail to spread and divide in small, thin chambers. Cell migration is also stunted as compared to thick 3D gels. However, larger chambers formed by a thicker devices promote cell spreading, cell division and faster migration. In the large devices, both cell-ECM and cell-cell interactions affect cell motility. Increasing collagen density decreases cell migration and increasing the number of cells per chamber increases cell migration speed. Furthermore, cells appear to sense both the ECM-chamber wall interface as well as other cells. Cells migrate towards the ECM-chamber interface if within roughly 150 µm, whereas cells further than 150 µm tend to move towards the center of the chamber. Finally, while cells do not show enhanced movement towards the center of mass of a cell cluster, their migration speed is more variable when further away from the cell cluster center of mass. These results show that microfluidic droplet devices can array 3D collagen gels and promote cell spreading, division and migration similar to what is seen in thick 3D collagen gels. Furthermore, they can provide a new avenue to study cell migration and cell-cell communication at physiologically relevant cell densities.

Contact guidance diversity in rotationally aligned collagen matrices.

Cancer cell metastasis is responsible for approximately 90% of deaths related to cancer. The migration of cancer cells away from the primary tumor and into healthy tissue is driven in part by contact guidance, or directed migration in response to aligned extracellular matrix. While contact guidance has been a focus of many studies, much of this research has explored environments that present 2D contact guidance structures. Contact guidance environments in 3D more closely resemble in vivo conditions and model cell-ECM interactions better than 2D environments. While most cells engage in directed migration on potent 2D contact guidance cues, there is diversity in response to contact guidance cues based on whether the cell migrates with a mesenchymal or amoeboid migration mode. In this paper, rotational alignment of collagen gels was used to study the differences in contact guidance between MDA-MB-231 (mesenchymal) and MTLn3 (amoeboid) cells. MDA-MB-231 cells migrate with high directional fidelity in aligned collagen gels, while MTLn3 cells show no directional migration. The collagen stiffness was increased through glycation, resulting in decreased MDA-MB-231 directionality in aligned collagen gels. Interestingly, partial inhibition of cell contractility dramatically decreased directionality in MDA-MB-231 cells. The directionality of MDA-MB-231 cells was most sensitive to ROCK inhibition, but unlike in 2D contact guidance environments, cell directionality and speed are more tightly coupled. Modulation of the contractile apparatus appears to more potently affect contact guidance than modulation of extracellular mechanical properties of the contact guidance cue. STATEMENT OF SIGNIFICANCE: Collagen fiber alignment in the tumor microenvironment directs migration, a process called contact guidance, enhancing the efficiency of cancer invasion and metastasis. 3D systems that assess contact guidance by locally orienting collagen fiber alignment are lacking. Furthermore, cell type differences and the role of extracellular matrix stiffness in tuning contact guidance fidelity are not well characterized. In this paper rotational alignment of collagen fibers is used as a 3D contact guidance cue to illuminate cell type differences and the role of extracellular matrix stiffness in guiding cell migration along aligned fibers of collagen. This local alignment offers a simple approach by which to couple collagen alignment with gradients in other directional cues in devices such as microfluidic chambers.

Direct expression of active human tissue inhibitors of metalloproteinases by periplasmic secretion in Escherichia coli.

BACKGROUND: As regulators of multifunctional metalloproteinases including MMP, ADAM and ADAMTS families, tissue inhibitors of metalloproteinases (TIMPs) play a pivotal role in extracellular matrix remodeling, which is involved in a wide variety of physiological processes. Since abnormal metalloproteinase activities are related to numerous diseases such as arthritis, cancer, atherosclerosis, and neurological disorders, TIMPs and their engineered mutants hold therapeutic potential and thus have been extensively studied. Traditional productions of functional TIMPs and their N-terminal inhibitory domains (N-TIMPs) rely on costly and time-consuming insect and mammalian cell systems, or tedious and inefficient refolding from denatured inclusion bodies. The later process is also associated with heterogeneous products and batch-to-batch variation. RESULTS: In this study, we developed a simple approach to directly produce high yields of active TIMPs in the periplasmic space of Escherichia coli without refolding. Facilitated by disulfide isomerase (DsbC) co-expression in protease-deficient strain BL21 (DE3), N-TIMP-1/-2 and TIMP-2 which contain multiple disulfide bonds were produced without unwanted truncations. 0.2-1.4 mg purified monomeric TIMPs were typically yielded per liter of culture media. Periplasmically produced TIMPs exhibited expected inhibition potencies towards MMP-1/2/7/14, and were functional in competitive ELISA to elucidate the binding epitopes of MMP specific antibodies. In addition, prepared N-TIMPs were fully active in a cellular context, i.e. regulating cancer cell morphology and migration in 2D and 3D bioassays. CONCLUSION: Periplasmic expression in E. coli is an excellent strategy to recombinantly produce active TIMPs and N-TIMPs.