Hydrogel-based microfluidic device with multiplexed 3D in vitro cell culture

Abstract

Microfluidic devices that combine an extracellular matrix environment, cells, and physiologically relevant perfusion, are advantageous as cell culture platforms. We developed a hydrogel-based, microfluidic cell culture platform by loading polyethylene glycol (PEG) hydrogel-encapsulated U87 glioblastoma cells into membrane-capped wells in polydimethyl siloxane (PDMS). The multilayer microfluidic cell culture system combines previously reported design features in a configuration that loads and biomimetically perfuses a 2D array of cell culture chambers. One dimension of the array is fed by a microfluidic concentration gradient generator (MCGG) while the orthogonal dimension provides loading channels that fill rows of cell culture chambers in a separate layer. In contrast to typical tree-like MCGG mixers, a fractional serial dilution of 1, ½, ¼, and 0 of the initial solute concentration is achieved by tailoring the input microchannel widths. Hydrogels are efficiently and reproducibly loaded in all wells and cells are evenly distributed throughout the hydrogel, maintaining > 90% viability for up to 4 days. In a drug screening assay, diffusion of temozolomide and carmustine to hydrogel-encapsulated U87 cells from the perfusion solution is measured, and dose–response curves are generated, demonstrating utility as an in vitro mimic of the glioblastoma microenvironment.

Optimization of 3D-aggregated spheroid model (3D-ASM) for selecting high efficacy drugs

“…In the Scaffold method, cells are cultured in 3D by mixing various ECMs such as collagen 41,42 and Matrigel 43,44 with the cells. In this case, a special culture vessel such as a well plate for 3D cell culture [45][46][47] or a microfluidics chip 48,49 is required. However, the 3D cell culture method has not yet been standardized, and manual dispensing of the cell/ECM mixture into 96 and 384 well plates is very inefficient and labor-consuming, so it is difficult to implement 3D cultured cells reproducibly.…”

Fabrication of a Low-Cost Microfluidic Device for High-Throughput Drug Testing on Static and Dynamic Cancer Spheroid Culture Models

“…The reason for the significant difference before and after washing is that excess cells in the hydrogel surface were dropped into the microwell during the washing process, thus increasing the cell number. When cells were seeded at different velocities, the number of cells falling into the well rose with the increasing flow velocity [ 43 ]. This increase is similar to the results described by Liu et al for the quantification of cell trapping in microwells at various flow velocities.…”

“…This increase is similar to the results described by Liu et al for the quantification of cell trapping in microwells at various flow velocities. With flow rates of 0.03 mL/min and 0.05 mL/min, which Lui et al considered to be suitable for culturing dynamic medium 3D spheroids, the number of cells in the microwell was 63 cells and 92 cells, respectively [ 43 ]. However, when the velocity was increased to over 0.1 mL/min, the number of cells falling into the microwell decreased both before and after washing.…”

“…The dimensions of the spheroids measured on Day 3, Day 5 and Day 7 were 83.31 μm, 215.53 μm, and 367.24 μm, respectively. According to the study by Liu et al, HepG2 tumor spheroids in the 400 μm diameter microwell, which grew not to exceed the size of the microwell, had a diameter ranging from 330 μm to 370 μm [ 43 ]. The trend towards better growth of spheroids in dynamic environments was also shown in the study by Yun et al, where the culture of pancreatic islet spheroids in a dynamic environment led to an enhancement in the viability and function of islet spheroids [ 12 ].…”

“…Furthermore, the IC 50 value in the static environment was higher (about 8.02 mg/mL) than in the dynamic environment (about 4.46 mg/mL). The above results show that in the dynamic culture system, the cancer spheroids were more strongly affected than in the static system when treated with the drug [ 12 , 43 ].…”