Micropallet arrays for the capture, isolation and culture of circulating tumor cells from whole blood of mice engrafted with primary human pancreatic adenocarcinoma

Abstract

Circulating tumor cells (CTCs) are important biomarkers of cancer progression and metastatic potential. The rarity of CTCs in peripheral blood has driven the development of technologies to isolate these tumor cells with high specificity; however, there are limited techniques available for isolating target CTCs following enumeration. A strategy is described to capture and isolate viable tumor cells from whole blood using an array of releasable microstructures termed micropallets. Specific capture of nucleated cells or cells expressing epithelial cell adhesion molecules (EpCAM) was achieved by functionalizing micropallet surfaces with either fibronectin, Matrigel or anti-EpCAM antibody. Surface grafting of poly(acrylic acid) followed by covalent binding of protein A/G enabled efficient capture of EpCAM antibody on the micropallet surface. MCF-7 cells, a human breast adenocarcinoma, were retained on the array surface with 90 ± 8% efficiency when using an anti-EpCAM-coated array. To demonstrate the efficiency of tumor cell retention on micropallet arrays in the presence of blood, MCF-7 cells were mixed into whole blood and added to small arrays (71 mm2) coated with fibronectin, Matrigel or anti-EpCAM. These approaches achieved MCF-7 cell capture from ≤10 μL of whole blood with efficiencies greater than 85%. Furthermore, MCF-7 cells intermixed with 1 mL blood and loaded onto large arrays (7171 mm2) were captured with high efficiencies (≥97%), could be isolated from the array by a laser-based approach and were demonstrated to yield a high rate of colony formation (≥85%) after removal from the array. Clinical utility of this technology was shown through the capture, isolation and successful culture of CTCs from the blood of mice engrafted with primary human pancreatic tumors. Direct capture and isolation of living tumor cells from blood followed by analysis or culture will be a valuable tool for cancer cell characterization.

Fabrication of 3D Cellular Tissue Utilizing MEMS Technologies

“…Positively charged hydrophilic molecules such as poly-D-lysine [18][19][20] and poly-L-lysine also promote cellular adhesion. Antibody to specific cell adhesion molecules such as anti-EpCAM captures target cells [35]. Surface roughness, nano-sized topography and hydrophilicity are also important for cell adhesion.…”

“…The layer is often designed to be enduring enough for handling by externals forces, such as fluidic forces [12], and micromanipulators [15]. Typical materials selected for the core layers are glass [12], parylene [13][14][15][16], polystyrene [22,[25][26][27], photoresist such as SU-8 [18-23, 50, 58] and 1002F [22,24,27,28,30,34,35], 1009F epoxy resin [22], poly(lactic-co-glycolic acid) (PLGA) [32], PDMS [52], poly(ethylene glycol) (PEG) hydrogel [54], iron oxide nanoparticles [24,26], and metals such as copper (Cu), nickel (Ni) [49,55], and gold (Au) [49,50,55].…”

Biosensors for Cell Analysis

“…A number of dynamic substrates employing electrical potential (79)(80)(81)(82)(83)(84), chemical composition (85-87), or light irradiation (88)(89)(90) have been introduced to retrieve cells. Although these are promising, electrochemical detachment strategies are somewhat challenging to adapt to retrieve specific, single cells.…”

“…This strategy would require a high-density array of micropatterned electrodes that could be addressed sequentially to change their surface properties and release cells. In this regard, the Allbritton lab (88,89) developed micropallet arrays that can be used to culture cells. The adhesion of individual micropallets to the substrate can be disrupted by laser light, which can be used to dislodge a pallet with immobilized cells.…”

Highly efficient cellular cloning using Ferro-core Micropallet Arrays

“…Shadpour et al . reported the ability to identify colonies derived from murine ES cells by gross morphology using mechanical roughening of the surface, that was required for the incorporation of a gelatin substrate 16,17 and the coating of the micropallet array with a monoclonal antibody recognizing a cell surface molecule 18 , a functionalization of the micropallet array that has been used to capture rare cells from heterogeneous mixtures 10 . We described the ability to effectively coat the micropallet array with a variety of extracellular matrix components that could be tailored to the desired adherent cell type 19 , including methodologies to limit bridging of the extracellular matrix coating between micropallets.…”