A novel 3D cell culture system was developed and tested. The cell culture device consists of a microfluidic chamber on an optically absorbing substrate. Cells are suspended in a thermoresponsive hydrogel solution, and optical patterns are utilized to heat the solution, producing localized hydrogel formation around cells of interest. The hydrogel traps only the desired cells in place while also serving as a biocompatible scaffold for supporting the cultivation of cells in 3D. This is demonstrated with the trapping of MDCK II and HeLa cells. The light intensity from the optically induced hydrogel formation does not significantly affect cell viability.
Drug- and cell-based therapies are being explored for many medical conditions [
Cell-based drug assays can be improved by culturing cells
3D cultures have been developed where cells are seeded on porous biomaterials [
One remaining challenge for microfluidic culture systems is the culturing of specific individual cells. The isolation and culture of specific, individual, and potentially rare cells of interest, such as cancerous cells, can be used to test the effectiveness of drugs, which can lead to the development of treatments that are better tailored to their target. When isolating specific cells, it is desirable to simultaneously select the cells of interest and seed them into a 3D scaffold. This reduces unnecessary handling of the cells, saving time and money, while negating the question of how to recover the selected cells. Minimizing the manipulation required to arrive at the culturing stage also simplifies the equipment necessary to carry out the process.
Here, we demonstrate a microfluidic system that can isolate specific cells from a suspension and seed and culture them in 3D structures. This system creates 3D hydrogel structures for cell culture using projected light patterns. Specific cells can be captured for homogeneous or heterogeneous 3D culturing while simultaneously creating the scaffolds necessary to support cell growth in 3D. The 3D hydrogel scaffolds are formed using a thermosensitive polymer instead of photoinitated polymerization [
Our cell culture system uses a device with an optically absorbing substrate that converts the optical energy of the light patterns into heat (Figure
(a) Schematic representation of the 3D cell culture device. A microfluidic chamber is imprinted on polydimethylsiloxane (PDMS) using soft lithography and reversibly bonded to an optically absorbent substrate. Tubing is connected at the inlet and outlet ports. Fluid is introduced at the inlet and is drawn through the device by applying negative pressure at the outlet. (b) A prototype device is shown with fluidic tubing connected at the ports. In this design, the hexagonal chamber is approximately 9 mm × 5 mm; the PDMS piece and accompanying substrate are approximately 30 mm × 15 mm.
The thermoresponsive hydrogel used is composed of poly(
The optically generated heat in the culture device is sufficient to increase the temperature of the PNIPAAm solution above its LCST, thus inducing a sol-gel transition and forming a 3D hydrogel in the heated region. The dimensions of the hydrogel are determined by the heat distribution generated by the optical patterns, and the height of the microfluidic chamber. The height of the microfluidic chamber in these experiments was maintained at 50
A typical culturing procedure starts by mixing cells at a desired density with a PNIPAAm solution (Figure
Optically controlled 3D culturing process. (a) The cell sample in hydrogel solution is introduced to the device chamber. (b) Desired cells are trapped in PNIPAAm hydrogel (cylindrical volume) using localized optical heating. (c) Undesired cells that remain in solution are flushed from the device. (d) While maintaining the hydrogel above the LCST, the PDMS chamber is removed, and the device substrate with the patterned 3D hydrogel is placed into culture media to allow cell growth.
A thermoresponsive polymer solution that comprised 10% (w/v) PNIPAAm (Sigma-Aldrich, St. Louis, Mo, USA) dissolved in deionized (DI) water or phosphate-buffered saline (PBS) (ATCC, Manassas, Va, USA) was used in the experiments discussed in this paper.
Madin Darby Canine Kidney (MDCK II) epithelial cells and HeLa cells were cultivated in a standard 2D culture using Dulbecco’s Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin streptomycin. The cells were incubated at 37°C in a humidified 95% air, 5% CO2 environment. The cells were harvested using 0.25% Trypsin/0.53 mM EDTA and mixed into a solution of 10% (w/v) PNIPAAm in PBS for the experiments described here. Viability tests were performed using a LIVE/DEAD assay kit (Invitrogen, Carlsbad, Calif, USA) and fluorescent imaging.
The cell culture device is positioned on the stage of an upright microscope, on top of an indium tin oxide (ITO) thin-film heater. The ITO heater is used to warm the device above room temperature, but below the LCST, to facilitate the optically induced gelation. A CCD camera mounted on the microscope provides real-time visualization of the procedure. The thermoresponsive polymer solution containing the cell sample is introduced into the device.
Optical patterns are directed through a hole in the microscope stage to the device substrate. The light patterns are created by focusing the output of a commercial projector (Dell 2400MP) through a 10X objective lens, resulting in an average intensity of 4.07 W/cm2. An alternate light source that can be used to cause gelation is a 635-nm, 10-mW diode laser. Irradiating the absorbing substrate results in localized heating within the device.
The 3D culture device consists of a 50-
The temperature profile in the culture device was simulated using COMSOL Multiphysics (Figure
Simulated temperature (degrees Kelvin) in the culture device. In this model, two glass layers sandwich a 100-
In order to precisely select single, individual cells for culture, the hydrogel resolution needs to be optimized. While it is desirable to obtain the smallest hydrogel area necessary to fully encompass the cell, the area of hydrogel should also remain relatively constant over the short period of time between trapping and flushing steps. If the heated region of the substrate continues to spread from the point where the light pattern is aimed, the hydrogel region will continue to expand, potentially trapping additional, undesired cells in the immediate area. For this purpose, the various substrates were compared by measuring the area of the hydrogel while exposing the device to the smallest circular light pattern capable of producing gel within the first 30 seconds of exposure. First, the minimum optical pattern dimensions that produced gelling were determined for each substrate. The time point at which hydrogel initially appeared varied across the substrates. The area of the hydrogel was measured over an exposure time of 60 s to include the effects of continued hydrogel formation with time (Figure
Hydrogel area versus time for various optically absorbent substrates. The optically smallest pattern that effectively produced gel was determined for each substrate, using the computer projector light source. The graph shows the average hydrogel area measured across five trials and the standard deviation for each. A two-minute delay between subsequent trials allowed sufficient time for the device to return to room temperature.
The resolution of this technique can be improved by replacing the computer projector with a laser operating in the near-infrared, visible, or UV wavelength range. Hydrogel formation using the computer projector light source is limited by the intensity of the light from the lamp in the projector. The relatively low intensity requires larger illuminated areas to create sufficient heat to trigger the PNIPAAm gelation. Thus, the resolution of hydrogel formation was examined further using the a-Si substrate and a 635-nm, 10-mW diode laser as the optical source. The laser was focused down to a spot of approximately 108
Hydrogel area as a function of power, using a 635-nm diode laser as the optical source. The area of the laser spot was approximately 108
The capability of trapping specific objects in 3D hydrogels was demonstrated using polystyrene beads, which also served as model particles for cells. A group of 20-
Trapping of 20-
The optically controlled culturing system was also tested with mammalian cells. Single- and multiple-cell trapping as well as parallel trapping were demonstrated with MDCK II cells. The cells were added to PNIPAAm/PBS solution, resulting in a cell density of approximately 1 × 105 cells/mL and 10% (w/v) concentration of PNIPAAm. The MDCK II cells were selectively trapped using optically induced hydrogel formation (Figure
Gelation of hydrogels using optical control, demonstrating the trapping of MDCK II cells in 3D hydrogels. (a) Initial cell distribution. The two target cells are circled in the inset. Other cells will be flushed away. The inset scale bar is 100
Cell viability tests on MDCK II and HeLa cells were performed immediately after the trapping procedure using a fluorescent LIVE/DEAD assay. The cells were suspended in 10% (w/v) PNIPAAm in PBS. The cells were irradiated with a circular light pattern 200
Cell viability testing using LIVE/DEAD assay on trapped MDCK II cells. (a) Initial distribution of MDCK II cells after 50 minutes in an optically patterned hydrogel. The starting cell density was approximately 1 × 105 cells/mL in solution. The dashed circle encloses the area that was gelled. The circular objects that are visible are the cells. (b) Fluorescent image of the same cells from (a), demonstrating approximately 100% viability. Cells lying outside the gel were not taken into account. Live cells fluoresce green, while dead cells fluoresce red. Fluorescent cells appear as bright spots in these images.
Cell viability testing using LIVE/DEAD assay on trapped HeLa cells. (a) Initial distribution of HeLa cells after 45 minutes in an optically patterned hydrogel. The starting cell density was approximately 1 × 106 cells/mL in solution. (b-c) Fluorescent images of the same cells from (a), demonstrating approximately 81% viability. Live cells fluoresce green (b), while dead cells fluoresce red (c). Fluorescent cells appear as bright spots in these images.
In this work, a cell culture system was demonstrated with the ability to trap cells of interest in reversible 3D scaffolds composed of a thermoresponsive hydrogel, PNIPAAm. The culture system utilizes a simple microfluidic device that acts as a repository for the hydrogel solution containing the cell sample while the trapping procedure is performed. Optically generated heating is used to induce localized gelation of cells within the device, enabling the trapping of specific cells. Two possible methods of generating localized heating at the cellular level were shown: using a modified commercial projector as a source of light and using a diode laser. The projector offers a higher degree of flexibility for pattern generation, whereas the higher intensity of the laser provides a stronger heat source and can thus create gels with smaller areas. The intensity of either source is not at a level where it is expected to damage the irradiated cells. To test the trapping capabilities of the culture system, experiments involving microbeads as well as MDCK II and HeLa cells have been performed. With MDCK II cells, single- and multiple-cell trapping was achieved, using a single light pattern or multiple simultaneous patterns. Preliminary viability tests show that over 80% cells survive the trapping procedure. Future work includes performing further viability tests at later time points during the cell cultivation process.
The authors would like to thank Yujia Li and Dr. Hongwei Li for providing the MDCK II cells and Dr. Ava Dykes and Dr. Shiv Sharma for the HeLa cells. This project is supported by the National Science Foundation, Grant no. EEC09-26632. The project described was also supported by Grant no. U54RR022762 from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH), and its contents are solely the responsibility of the authors and do not necessarily represent the official view of NCRR or NIH.