In vitro cell culture has been a powerful model for in vivo biological processes. However, the majority of studies have been conducted using 2-D culture systems in which cells grow as flat monolayers. Two recent symposia—CHI’s “Bioprocessing Summit” and the “BioMethods Boston” conference—discussed a raft of studies on cellular behavior in 3-D platforms, and how this model may offer a more realistic view of how cells behave in their natural environments.
“Deregulated cell movement is a hallmark of pathological events, including pulmonary disease and cancer metastasis,” said Shuichi Takayama, Ph.D., professor of biomedical engineering at the University of Michigan. To investigate this phenomenon, Dr. Takayama and his team developed a high-throughput platform for cell-migration studies.
Traditionally, cell migration could be measured by the wound-healing assay, in which a cell monolayer is scraped with a tool. This leaves a vacant area, which in subsequent days is repaired as cells move to fill in the empty zone.
This approach is not entirely satisfactory, as there is a need for specialized tools to scrape the cell monolayer in high-throughput settings, and the scratching tool may disrupt both cells and the underlying substrate. Moreover, since the cell removal may be initiated by hand, the “wound” size (or width) may be variable, yielding inconsistent results in duplicate assays.
To meet these challenges, Dr. Takayama and his colleagues took advantage of an aqueous two-phase system consisting of polyethylene glycol (PEG) and dextran (DEX) as the phase-forming polymers.
A submicroliter droplet of the dextran phase is spotted and dried in a conventional microwell. Addition of the polyethylene glycol to the well results in the rehydration of the dextran spot to form an immiscible droplet.
The PEG-DEX biphasic system generates an interfacial tension, which prevents cells from adhering to the part of the substrate covered by the rehydrating drop. The result is a consistent, repeatable, clearly defined circular area from which the cells are cleared away, Dr. Takayama claimed.
He and his team have investigated another model for cell culture evaluation: spheroid formation. Using custom-designed 384-array plates, a pipette is used to introduce the cells into holes where the droplets are suspended. The pipette tip is first inserted through the access hole to the bottom surface of the plate, and the cell suspension is dispensed.
The cell suspension is attracted to the hydrophilic plate surface and a hanging drop is quickly formed and confined within the plateau. Within hours, individual cells will begin to aggregate and form into a single spheroid. The hanging drops can be fed and manipulated on a periodic basis. Dr. Takayama is developing this project in collaboration with 3D Biomatrix.
Breast Cancer Tumorigenesis
“Increased stiffness is a hallmark of solid tumors, due to the altered physiochemical properties of the extracellular matrix,” stated Claudia Fischbach-Teschl, Ph.D., assistant professor of biomedical engineering at Cornell University. “This is thought to be largely due to crosslinking of collagen, which occurs with greater incidence than in normal breast tissue.”
Other elements may participate in this process including fibronectin, whose stretching leads to unfolding and increased stiffness of the molecules. Furthermore, these modifications of the fibronectin matrix may contribute to the enhanced rigidity of mammary tumors, which is associated with tumorigenesis and metastasis.
Dr. Fischbach-Teschl, in collaboration with Delphine Gourdon, Ph.D., a researcher in the department of material science and engineering at Cornell University, have evaluated paracrine signaling between breast cancer cells and adipose progenitor cells in order to assess whether such signaling might promote tumor progression via changes in fibronectin matrix assembly.
They constructed in vitro and in vivo model systems combining adipose cells with a human breast cancer cell line. In order to measure, quantitatively, the changes in the fibronectin molecule, they employed FRET analysis.
Results demonstrated that the paracrine signaling between the tumor cells and the adipose cells contributes to fibronectin matrix stiffening in tumors. Furthermore, they showed that this stiffening is leading to phenotypic changes of adipose stem cells that lead to enhanced tumor progression.
“Our observations suggest that these changes promote tumor vascularization and growth by upregulating the pro-angiogenic capability of both the adipose stem cells and the endothelial cells,” Dr. Fischbach-Teschl explained. “In summary, these studies suggest that adipose stem cells stimulate tumor vascularization in a stiffness-dependent manner and represent a promising target for improved anti-angiogenic therapies.”