Cells can be uniquely sensitive to their environment, and controlling that environment is crucial to efforts to use cells as protein factories or to study how they mutate and grow into tumors. However, in some cases, the cellular environment simply cannot be mimicked outside of tissue. Now researchers at the University of Illinois in Urbana hope that, using a three-dimensional optical trap, they have developed a technique that will allow them to build artificial cell structures similar to tissue, but that can be more easily controlled. “A 3-D array gives us the capability to arbitrarily position cells in the X, Y and Z directions as they are in living tissue,” explained Gregory Timp of the university’s Beckman Institute. He and his colleagues have developed a system that positions individual cells into a three-dimensional structure and holds them in place with a polymer scaffold. Using a specially designed system that couples time-multiplexed optical traps with a rapid-setting hydrogel, researchers created three overlapping 3 x 3 arrays (red, green and blue) of Pseudomona aeruginosa embedded in hydrogel. The arrays are shifted by 4 μm in the X and Y directions and 3 μm on the Z-axis. Images courtesy of Gregory Timp. The process has two phases. First, a time-multiplexed array of optical tweezers holds individual cells in position as they float freely in polymer solution. Once the cells are positioned, the researchers expose the solution to 10 seconds of UV light, which fixes it into a hydrogel that holds the cells in place and allows nutrients to enter and waste to exit. The time-multiplexed optical trap is the key component in the process. They created it using a combination of acousto-optical deflectors and a spatial light modulator. The deflectors move the beam in the X-Y direction, which enables placement of cells in a single plane. The ability of the deflectors to operate at high repetition rates allows use of a single beam to create the planar array. The beam stays in a single trapping location only long enough to fix a cell in position — at least 10 ms between trapping locations. When the beam moves off the trapping location, a cell caught in the trap will begin to disperse, so the researchers must carefully choose the “dark time” based on how quickly cells diffuse, how large the array is, how fast the beam is scanned and how long it stays on a position. To create additional layers of cells, relay lenses project the planar array onto a spatial light modulator. The spatial light modulator alters the wavefront of the laser beam. When the beam is focused by the microscope objective lens, the intensity distribution in the sample plane is the Fourier transform of the wavefront generated by the modulator. Using an iterative algorithm, they can determine how to use the modulator to distort the wavefront and create the desired array of traps. The modulator functions effectively as a Fresnel lens, allowing replication of the array at several focal planes. The system consists of a Zeiss inverted microscope and two lasers to create the optical traps. The scientists used a 20-W Coherent argon-ion laser at 514 nm and a Spectra-Physics CW Ti:sapphire laser that was tunable from 850 to 900 nm. The acousto-optical deflector was made by AA-Optoelectronics, and the spatial light modulator came from Hamamatsu. Timp said that they chose the two wavelengths to compare their trapping ability and their effect on the cells. “We used the 514-nm light for some of the trapping experiments and 900 nm for others. It is widely believed that there is less photodamage at 900 nm. However, 514-nm light provides a sharper focus and a stronger trap at lower power. We thought it might be advantageous to try 514-nm light, and we surmised that time-sharing the light might mitigate photodamage.” They did not observe adverse effects on cell viability at 514 nm for the conditions they used. They used polyethyleneglycoldiacrylate to make the hydrogel scaffold because it not only polymerizes in less than 3 seconds, but also is pliable and permeable and has been demonstrated to be biocompatible. The researchers populated the optical traps with cells in one of two ways. “The simplest method is to allow a cell that is undergoing random motion in the aqueous medium to come within the capture range of the optical trap,” Timp explained. The second method uses a separately controlled optical trap as a “shepherd beam” to move cells into the array traps. In the future, Timp added, they hope to develop an image-processing algorithm that will be able to find cells in a solution and use multiple shepherd beams to rapidly populate an array. To polymerize the hydrogel, the researchers used a 100-W mercury lamp filtered to a 360-nm beam of roughly 600 mm in diameter. They also tested the possibility that the UV light used to polymerize the hydrogel might kill or damage the cells and found that UV exposure needed to exceed 20 seconds to affect cell proliferation. One application for the new technique is to study cellular signaling and the methods by which bacterial pathogens infect cells, here demonstrated by a ring of 16 P. aeruginosa around a Swiss 3T3 fibroblast. They used the new system to create what they believe is the first example of a permanent, living cell array of this level of complexity. They formed an array containing 441 Pseudomonas aeruginosa bacteria, a common human pathogen that typically infects people who are immunocompromised. The array had a spatial period of about 1.5 mm and a separation between the bacteria of about 350 nm. To demonstrate the ability of the system to build an array that could be used to study cellular signaling and microenvironments, they constructed one containing a single fibroblast surrounded by 16 P. aeruginosa. The optical system allowed the bacteria to be packed very tightly together. In this image, P. aeruginosa have a center-to-center average distance of 1.52 μm with an average space between them of 354 nm. Timp said that the system offers several benefits and capabilities. Because it can be used to trap microspheres in addition to cells, it enables scientists to shape chemical gradients in 3-D. “Since cells communicate using chemical gradients, this facility allows us to test models for development, cancer, etcetera,” he said. It also gives researchers the ability to build tissue-like structures. “To examine cell interactions in an in-vivo-like environment, we need a way to mimic this organization. With the ability to create heterotypic 3-D living cell arrays, we can make tissue elements to study cellular interactions,” he added. Timp said that they plan to begin using the system to study intercellular communications, cell development and differentiation, immune function, cancer development and neuronal communications. They also plan to study the stochastic mechanisms of gene expression in bacteria. “To accomplish these two tasks, we plan to pursue arrays of optical traps containing many more elements — more than 1000 — and develop hydrogels compatible with these functions,” he said.