1. `
3D Engineered bio-degradable scaffolds to study cell
microenvironment
Diana H. Pham, Amina Rahimi, Mariëtte Wessels, Julia Binger, and Carla V. Finkielstein
Integrated Cellular Responses Laboratory-Department of Biological Sciences-Virginia Tech
Abstract
The 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 Results
The 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 C
Figure 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).
B
C
D
E
F G
H
J
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