A half-dozen Rice University bioengineering and nanoscale physics student interns working at Houston-based biotech startup Nano3D Biosciences (n3D) have wrapped up a yearlong project to aid the company in developing a high-throughput method for in vitro cytotoxicity assays that uses a free iPod app and analytical software for automated data collection and analysis.
“This literally collects about 100,000 data points during a 12-hour, overnight experiment,” says study co-author Shane Neeley in a Rice University release, a Rice bioengineering graduate student who has interned at n3D for nine months. “That’s all relevant publishable data that relate to the different for conducting high-throughput, in vitro cytotoxicity assays.
“This would not have been possible without the students,” says Glauco Souza, n3D’s president and chief scientific officer. “They helped develop the scientific protocols and hardware for this, and they wrote both the iPod app and the analytic software.”
The new assay method, which n3D has dubbed the “BiO Assay,” uses a free iPod app to collect time-lapse images of 3-D cell cultures that have been exposed to varying levels of a drug. Those images are then fed through an analytical program that measures each sample and creates time-lapse movies, graphs and charts of the drug’s cytotoxic profile.
A research paper about the new method was published this month in Nature’s open-access journal Scientific Reports. The paper, entitled “A high-throughput three-dimensional cell migration assay for toxicity screening with mobile device-based macroscopic image analysis (3000 doi:10.1038/srep03000),” is co-authored by David M. Timm, Jianbo Chen, David Sing, Jacob A. Gage of n3D, Rice graduate and n3D graduate student intern William L. Haisler, Shane K. Neeley, Robert M. Raphael — professor of bioengineering at Rice, Mehdi Dehghani and Kevin Rosenblatt — both of the University of Texas Health Science Center at Houston, T. C. Killian — professor and chair of physics and astronomy at RIce U., former Rice undergraduate and n3D’s senior research scientist Hubert Tseng, and Glauco R. Souza.
The paper’s preamble notes that: “Screening for toxicity plays an important role in the drug development pipeline, as it accounts for 20% of total failures of candidate compounds. Improvements in this process could significantly reduce the cost and time-to-market of new therapies. Common screens for drug toxicity use animal models that are similar in composition and structure to the human tissue they represent. However, these models are expensive, time-consuming, low-throughput, ethically challenging, vary widely in results between species, and predict human toxicity with varied success. In vitro assays have been used as early screens and cheaper alternatives to animal models, but they predominantly use two-dimensional (2D) environments that do not accurately replicate the human tissue they purport to represent. In particular, 2D models have different spatial gradients of soluble factor concentrations and substrate stiffnesses than those of native tissue, and they do not support the wide array of cell-cell and cell-matrix interactions that cells natively experience. As a result, biomedical research has moved towards the use of three-dimensional (3D) models, which can more accurately match the structure and biochemical environment of native tissue to predict in vivo toxicity.”
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The co-authors also observe that there is a growing demand for in vitro assays for toxicity screening in three-dimensional (3D) environments, and in this study, 3D cell culture using magnetic levitation was used to create an assay in which cells were patterned into 3D rings that close over time. The rate of closure was determined from time-lapse images taken with a mobile device and related to drug concentration. Rings of human embryonic kidney cells (HEK293) and tracheal smooth muscle cells (SMCs) were tested with ibuprofen and sodium dodecyl sulfate (SDS). Ring closure correlated with the viability and migration of cells in two dimensions (2D). Images taken using a mobile device were similar in analysis to images taken with a microscope. Ring closure may serve as a promising label-free and quantitative assay for high-throughput in vivo toxicity in 3D cultures.
They further note that: “One such method to construct 3D models is magnetic levitation magnetic levitation, cells are incubated with a magnetic nanoparticle assembly consisting of gold nanoparticles, poly-L-lysine, and magnetic iron oxide that non-specifically and electrostatically binds to cells. These nanoparticles are nontoxic and do not induce an inflammatory cytokine (IL-6, IL-8) response by cells. By binding to the nanoparticles, the cells become magnetic and can be manipulated with the external application of a magnetic field. In particular, when a magnetic field is applied above the culture plate, cells are levitated from the bottom surface, where they interact and aggregate with each other to form larger 3D cultures.”
Glauco Souza and Rice faculty members Tom Killian and Robert Raphael co-founded n3D in 2008 based on technology they created to grow 3-D cell cultures using magnetic levitation. The technology relies on inert, nontoxic magnetic nanoparticles that attach to living cells. Magnets can then be used to lift and suspend the cells as they grow and divide.
The research done by the Rice student team and Nano3D Biosciences is part of a growing trend to create better lab techniques for testing drug toxicity. At issue is the fact that the toxic side effects of many new drugs are discovered only during human clinical testing, which means tests on 2-D cell cultures and on lab animals failed to identify the toxicity risk in humans. Cells grown in 3-D cultures behave more like the body’s native tissues, and scientists have scrambled to find ways of using 3-D cultures to reduce the need for animal testing and to rule out toxic drug candidates earlier.
The Nano3D Biosciences (n3D) Bio-Assembler product brings the power of 3-dimensional cell culturing to experiments. The Bio-Assembler is a device for culturing cells in three-dimensions by magnetic levitation through combining nanoparticle based Nanoshuttle-PL with designed magnetic fields needed to levitate cells and provide the 3D cell growth environment.
Advantages the Bio-Assembler provides include:
• Faster set up times (minutes vs. hrs.)
• Easier preparation with 80% less likelihood of sample contamination
• Faster growth of cells (hrs. vs. weeks)
• No use of animal components
The Bio-Assembler uses nanoparticle-based Nanoshuttle-PL solution to deliver magnetic nanoparticles to cells. Magnetic drives then levitate cells to create the 3D cell growth environment. Using the Bio-Assembler is as easy as performing standard monolayer (2D) culture. Preparation time and cell manipulation requirements are equivalent to 2D culture.
Advantages of n3D’s 3-Dimensional Cell Culturing System include:
• Rapid formation of 3D structures and cell-cell interactions
• More accurate representation of in vivo tissue
• Compatibility with virtually any cell type including primary and stem cells
• Compatibility with any media type, standard culturing protocols, and diagnostic techniques.
Two simple additional steps differentiate the Bio-Assembler from 2D cell culturing:
1) Adding the Nanoshuttle to cells prior to levitation
2) Placing the magnetic drive on top of the cell culturing plate
“It’s been estimated that improving the accuracy of early cytotoxicity screenings by even 10 percent could save drug companies as much as $100 million per drug,” says study co-author Hubert Tseng, n3D’s senior research scientist In the Rice release. Dr. Tseng, who interned with the company prior to earning his Ph.D. in bioengineering in March, played an instrumental role in developing several of the company’s products, including the BiO Assay.
In the Rice release, Mr. Souza says the company developed the BiO Assay out of necessity; Interns in the lab were spending hour after hour snapping photos of individual cell cultures on the microscope. Each experiment involved exposing a hundreds of cell cultures to varying doses of a drug. The microscopic images revealed how much smaller the culture became over time, as the toxic drug slowly killed off the cells in the colony. Each culture was grown in its own tiny chamber on standard plates that each contained 96 chambers.
“Without looking in the microscope, just looking at the camera and clicking like a robot, it would take 20 minutes to take pictures of all 96 wells on one plate,” Souza notes. “To analyze that, all 96, with a ruler, took even longer.”
Study first authors David Timm and Jianbo Chen, professional masters students at Rice in nanoscale physics, had to repeat that tedious process over and over, as often as possible, on dozens of 96-well plates that were being used in multiple experiments.
“We decided there had to be a better way, so we began experimenting with using an iPod,” Souza notes. “It was promising, but none of the available apps worked very well, so we decided we needed to make our own. I called Apple and asked them to give me the name of a developer here in Houston. When they heard where I was, they said, ‘Don’t (hire a developer). Go to Rice University and get a couple of students instead. You’ll get a better app, and it will do exactly what you want.’”
Study co-author William Haisler had just graduated from Rice and joined n3D as a graduate student intern. Haisler was no stranger to n3D’s cell culturing technology because he and four other Rice seniors worked with the company as part of their senior capstone design project at Rice’s Oshman Engineering Design Kitchen. The team, Cells in 3-D, created a magnetic pen to help manipulate suspended cell cultures.
Haisler, who’d done some Java coding in high school and written several programs during his time at Rice, volunteered to create the iPod app. However, he had to learn a new programming language — Objective C — to write the iPod app.
“Apple provides a good amount of sample code, and it’s an object-oriented language like Java, so learning the language wasn’t too bad,” Haisler comments. “In terms of developing the app, the biggest challenge was using the tools to access the photo album and the camera. The troubleshooting was difficult because you don’t get full access because of how they use their security.”
Dr. Tseng says the team considered going with Android, an open platform that would have eliminated such issues, but they needed the standard hardware format that the iPod offered. Unlike Android handsets, every iPod is the same size and has the same camera location — two factors that were key for getting the repeatable, standardized results required for scientific experiments.
N3D’s technology grows 3-D cell cultures using magnetic levitation. The technology relies on inert, nontoxic magnetic nanoparticles that attach to living cells. Magnets can then be used lift and suspend the cell cultures as they grow.
But even with the standard camera location, the team found that slight shadows on the wells furthest from the camera were throwing off their measurements. Correcting the system’s optics fell to Chen, now a Ph.D. student in electrical and computer engineering. He researched various designs before choosing a compact Fresnel lens as being the easiest to use and the most cost-effective.
The problem of capturing images was solved; the new system could snap a photo every few seconds for days at a time. Even setting it for 15-minute intervals produced hundreds of pictures in short order, so the problem of analyzing photos became even more acute.
Chen wrote the first analytical software using MatLab, and Neeley modified that after joining the group in February. Neeley had several years experience writing code in Python, a versatile, high-level language, so he used that to convert Chen’s code and add new features. “Basically, the idea was to automate everything and process all the data uniformly,” Neeley says in the Rice release. “No matter what time points you use or what types of cells or the angles of the plates, you will still get nice figures that will look good at the end.”
Mr. Souza notes that the ease of use and low startup costs for using the system will make high-throughput screening feasible in places, including many classrooms, where it would have been unthinkable. “We’re hoping to get this into some high school classrooms here in Houston, and we’re working with one of Houston’s largest community colleges, Lone Star College, to see if it can be used there.
The research was supported by the National Science Foundation’s Small Business Innovation Research program and the Texas Emerging Technology Fund.
Nature.c0m Scientific Reports