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3D Engineered bio-degradable scaffolds to study cell microenvironmentDiana H. Pham, Amina Rahimi, Mariëtte Wessels, Julia Binger, and Carla V. FinkielsteinIntegrated Cellular Responses Laboratory-Department of Biological Sciences-Virginia Tech

AbstractThe increased stiffness of the extracellular matrix (ECM) is believed to facilitate malignant cell migration likely due to clustering integrins and an increase in Rho activity. Since the rigid substratas of 2D cultures intensify this issue, studies move towards using compliant 3D ECM cultures. The aim of this study is to include a vascular system in a 3D-scaffold model to test the effects of ECM stiffness on the phenotype of malignant mammary epithelial cells. The effect of matrix stiffness on cell morphology is therefore examined in a system that more closely represents the physiological environment that cells experience in vivo. The procedure for creating the 3D ECM involves, essentially, printing a 3D carbohydrate structure that is used as a temporary structure. The carbohydrate structure is coated with poly(d-lactide-co-glycolide) (PDLGA) to facilitate controlled diffusion and support endothelial cells. When this structure is put into a mixture of collagen and cell media, the carbohydrate lattice in the structure dissolves, leaving the PDLGA as channels for flow of nutrients and other necessary substances while encasing it in a collagen ECM. The concentration of collagen can then be varied within the ECM to resemble different ECM stiffness. This study provides a way to examine how the ECM stiffness affects the morphology and malignancy of cells with a vascular system to further understand how mammary cells can undergo tumor formation and metastasis.

Background

tissue engineering in creating 3D tissue models is tissue scaffolding. This method uses a polymer structure as a temporary biocompatible surface that allows the attachment and proliferation of cells while the cells develop their own ECM. In this way the scaffold acts as a mediator for establishing a structural, mechanical, and biochemical environment similar to one in the human body.

Objectives

Methods• Rapid Prototyping with Carbohydrates• 3D Embedded Assay• 3D On-Top Assay• Cell Fixation with Immuno-staining and DNA Staining

Biomaterial ResultsThe 3D On-Top and 3D Embedded assays with MDA-MB-231 cells and collagen extracted from rat tails (Figure 5A) were done as a prototype for how the mammary cells would culture in the 3D system. Although there was concern that the 1:1 dilution of collagen with media to make 4 mg/mL of collagen would cause the cells to be poorly suspended in a 3D Embedded Assay, that was not the case for this collagen concentration.

Discussion

References• Huang S and Ingber DE. Cell tension, matrix mechanics, and cancer

development. Cancer Cell, [September 2005]; 8(3):175-176. http://dx.doi.org/10.1016/j.ccr.2005.08.009.

• Lee GY, Kenny PA, Lee EH and Bissell MJ. Three-dimensional culture models of normal and malignant breast epithelial cells. Nat Methods. [2007], 4: 359–365

• Miller JS, Stevens KR, Yang MT, Baker BM, Nguyen DH, et al. (2012) Rapid casting of patterned vascular networks for perfusable engineered three-dimensional tissues. Nat Mater 11: 768–774.

• Paszek MJ, Weaver VM. The tension mounts: Mechanics meets morphogenesis and malignancy. J Mammary Gland Biol Neoplasia. 2004;9:325–42. 

• Paszek MJ, Zahir N, Johnson KR, Lakins JN, Rozenberg GI, et al. Tensional homeostasis and the malignant phenotype. Cancer Cell. 2005;8:241–54.

ECM stiffness triggers integrins, which are transmembrane mechanotraducing receptors, to promote focal adhesion and fuel the Rho/ROCK pathway, leading to cell contractility that heighten ECM stiffness. As an interconnected pathway with the EGFR/Erk signaling cascade, the increase of cell contractility may in turn cause the EGFR/Erk signaling to maintain the malignant phenotype of mammary epithelial cells.

Figure 1. A mechanical autocrine loop regarding ECM stiffness and potential cell malignancy (From Huang and Ingber 2005).

The importance of using 3D models to replicate the human physiological environment has been well established. One of the most prominent methods of

• Building 3D-scaffold mammary tissue model with vascular system

• Produce carbohydrate structure with rapid prototyping

• Coat carbohydrate structure with PGLA polymer

• Dissolve carbohydrate structure while encasing it in ECM material with mammary endothelial cells leaving vascular channels

Carbohydrate structures are fabricated using rapid prototyping with an adapted MendelMax 3D printer (Figure 3A) and Gcode. Custom Gcode programs have been created to print certain standard carbohydrate structures. For more complicated figures, a ‘slicing software’ converts 3D drawings in STL files (Figure 3B) into horizontal layers (Figure 3C), calculates the path for the extruder to print, and converts this path into Gcode. Although the ‘slicing software’ provides rapid conversion of a 3D

B CFigure 3. A, The adapted MendelMax 3D printer with printer interface. B, Imported STL file of 3D model for scaffold. C, ‘Sliced’ 3D model.

models into Gcode, it currently does not calculate the extruder’s path efficiently. his method of rapid prototyping succeeds in printing simple and complex structures, including curved structures (Figure ). However, this method currently cannot print structures where the bottom layers do not directly support the upper layers.The carbohydrate solution, a mixture of various polysaccharides with water, is optimal after being heated slowly to 140°C in an aluminum beaker for approximately 2 hours. After printing at roughly 110-120°C and solidifying, the carbohydrate structure is ready to be coated in polymer.After PLGA is prepared in chloroform, it is found that the carbohydrate structure should be immersed in the PLGA solution for roughly 5 minutes and dried for at least 15 minutes. The use of dye confirmed that the carbohydrate lattice dissolves when structures are gelled in a collagen-media mixture, leaving the PDLGA as channels in a collagen ECM matrix.

Figure 5. A, 3D Embedded Assay. B, MDA-MB-231 cells with DAPI from a 3D On-Top Assay with a scale. C, MDA-MB-231 cells with images with DAPI, GFP, Tranmission, and a combination of all three from a 3D On-Top Assay.

Figure 6. Carbohydrate structures. A and B, Top and side view of simple scaffold designed with cylinder ends for inlet and outlet access. C, Curved Scaffold. D and E, Top and side views of Sphere Structures. F and G, Top and side view of square tree-like structure. H, Geometric patterned structure. I, Structure modeling a tree. J, Image of authentic tumor vasculature.

This study shows progress in the increased ability to model tissue. The increased complexity of the vasculatures that can be modeled, the determination of standard procedures for preparing the carbohydrate solution and coating the structure with PLGA, and the successful 3D assays show the promise that MDA-MB-231 cells can be successfully cultured on those scaffolds. When multiple cell types are seeded on the vasculature and flow established, the system can then test the effects of ECM stiffness on cell morphology with particular attention to integrin clustering, Rho activity, and the EGFR/Erk signaling pathways. Current work is done to create stable inlet and outlet flow (Figure 6A and B).The ability of the printing system to spheres and bridges can also be expanded to model tumor vasculature. Figure 6D and E particularly show promise in fabricating tumor vasculature where branches inside a sphere can resemble arteries and veins within a tumor. Additionally, Figure 6I shows the extensive branching that can be possibly printed to closely model actual tumor vasculature (Figure 6J).

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Figure 4. Images of the procedure for extracting and preparing collagen for use.

• Flow media, vascular endothelial cells, and other necessary biochemicals or biomaterials in the PLGA channels

• Test effects of ECM stiffness on the phenotype of malignant mammary epithelial cells• Vary collagen concentration in culture to resemble different ECM stiffness• Use staining and fluorescence to view cell morphology, particularly acini

formation and integrin clustering

Figure 2. Diagram modeling procedure for tissue scaffolding