University of California, Berkeley
At A Glance
Name: Ravi Chandra
Position: Graduate student, National Science Foundation pre-doctoral fellow, chemistry, University of California, Berkeley (laboratories of Carolyn Bertozzi and Matthew Francis)
Background: BS, chemistry and biology, Stanford University
One of the hottest areas in cell-based assays is cellular arrays, following on the heels of DNA and protein arrays. Many researchers, including those at the Translational Genomics Institute (see CBA News, 9/26/2005), University of California, Los Angeles (see CBA News, 5/23/2005), and the Whitehead Institute (see CBA News, 6/8/2004), are experimenting with ways to array cells for high-throughput or high-content screening, biosensing, or functional genomics applications.
There are even commercial entities interested in cellular arrays, such as Biolog and Molecular Cytomics. Of these entities, only Molecular Cytomics has developed a way to array individual live cells (see CBA News, 12/19/2005).
Now, researchers from the University of California, Berkeley, and Lawrence Berkeley National Laboratories have developed a method to array single live cells using DNA-based adhesion technology, and are exploring its use in a number of applications, including drug screening, biosensing, artificial tissue, and neural network design. The group's work was recently published in the January edition of Angewandte Chemie. Earlier this week, lead author Ravi Chandra took a few minutes to discuss the technology with CBA News.
How did this work get started?
It got started with the desire to be able to localize cells — particularly in devices — but if you think of any of the possible applications, it's the need to be able to precisely array cells in a manner that's not dependent on things like robotics or other lower-throughput methods. The technologies that are out there are really quite limited. There have been some fundamental studies of cell adhesion that have really been quite useful from a cell biology standpoint. But from a device standpoint, and engineering standpoint, and an application standpoint, the toolkit was pretty bare. We really wanted to start developing some tools that we could use in conjunction with other technologies.
The whole idea of DNA, for us, came from the fact that we know that, based on the (Carolyn) Bertozzi technology we can deliver these special functional groups to the surfaces of cells, and get very specific chemoselective reactions to occur there. The idea is that she's been able to exploit that reaction on the cell surface many times to specifically attach molecules of interest. We thought that if we have this thing at our disposal, can we then put something on the cell surface … Let's say we had a ligand and receptor, with the receptor being on the device. Could we engender specificity by doing this? We considered the fact that DNA, of course, is a natural recognition target with its sequence specificity. And we wondered whether it would be viable to use the DNA as a bar code of sorts to put on the surface of the cell, effectively tag it, and direct the cells to where we want them to go. That was the principal question, and the [Angewandte Chemie] paper, as a whole, really is focused on figuring out ways to deliver DNA appropriately to the surfaces of cells, to keep them there, and to really validate that they can be useful for something.
Do you have a particular interest in the drug-screening applications of this?
I would say that I probably have the largest interest in the drug-screening/biosensing applications. Those are both pretty much fruits from the same tree in the sense that if you can develop the technology to make a biosensor, you should be able to develop something to do drug screening. In either case you would like to have an array of cells — so maybe you have a 1,000-pixel array — where each pixel is a discrete population of cells. If you were to screen a pathogen over it and detect some sort of interaction, those cells laid down would have to be immune cells — T-cells or B-cells. If you're talking about drug screening, you'd put down whatever cells of interest you had. If you wanted to screen chemotherapy drugs, for example, the [National Cancer Institute] has [its] well-known 60-cell line screen, and you now have a miniature array to be able to test these variables quite quickly.
Then you can start thinking about the technology from a device standpoint, if you think about DNA microarray technology and other types of functionality people are starting to include in devices, we envision being able to dovetail our technology very nicely into that. If you have the ability to array DNA of any sequence in appropriate patterns and incorporate other functionalities into that device, if you now have the ability to incorporate cells, you have an even more powerful device.
You said that one of the potential applications of this technology might be sort of a mini-immune system. Can you elaborate?
That falls more into the biosensor realm. The immune system in the body, when a pathogen comes in, you have T-cells and B-cells that are specific for pathogens. They float around and if they find that a particular virus or bacteria has infected you, will recognize it by binding to it, and initiate a whole series of molecular-level signaling to get your immune system up and running. We envision a situation where, for example, we had an array that we could assemble with a number of different cells that recognize pathogens — for example, T-cells — and each had discrete pathogen specificity.
Let's say we were to flow anthrax over the chip, we could identify the pathogen based on which pixel elicited a response. We should note, of course, that the T-cell specific for anthrax should elicit a response, but it's possible that other pixels would elicit a more muted response, and potentially we could even begin to look at how the immune system recognizes pathogens. Is it more of a one-cell, one-pathogen deal? Or is it more of a molecular fingerprint? The advantages of using this technology to create a mini-immune system are (A), the immune system is entirely non-adherent; it's designed to float around and not stick to things, so that works well for us; and (B), if you have the ability to make a complex array, you can now start screening for pathogens. Even more interesting, beyond the biosensing, let's say we had something to which we'd like to generate an immune response, but have not been able to identify an immune cell that would react to it. For instance, if we had some antigens from cancer cell, we could try screening that against our little immune chip to see if any of those immune cells actually elicit a response. If they do, that could be very useful therapeutically, because you can then imagine taking those T-cells, giving them to a patient, and hopefully having the patient's immune system go after the cancer. At this point there is a certain degree of science fiction here, but we're driving toward a platform with which we can start asking these questions.
In the paper, you mentioned that the cells can stay bound and alive for long periods of time. Have you tried conducting long-term live cell assays of any sort?
We have, but at first our initial studies have been to develop a basic understanding of how the binding process works and what variables we need to tweak. We've looked at it for a number of days, and the best way to put it is that the same proportion of the cells survives using this method as compared with cells that would be cultured under identical conditions. That's not meant to be a circular statement; there are just no additional adverse reactions that cells have to this technology. Having said that, if the desire is to culture cells for a long period of time, then it really falls on the engineering side of things, in terms of being able to deliver nutrients appropriately, gas exchange, et cetera.
Additionally, the fact that you're using DNA gives you some flexibility in terms of being able to detach cells. If cells can self-assemble as readily as we hope, you might have the ability to be able to take them off once you're done and lay down some new cells without wasting your device — effectively, you're most expensive component of this whole thing.
Have you brainstormed procedures for being able to array the cells quickly?
We're really trying to get better procedures for making the designer arrays we're going to need to start testing some of these questions. Believe it or not, a lot of the gene chips that are out there are great, but in many cases, they would like to have arrays that have lots of different DNAs on them, very small feature sizes — these are things that are great for their applications. For our applications, we'd like to start a little bit simpler — change the feature sizes around, start to look at the dependence of how cells bind in terms of the area and concentration of DNA that's there. If everything is working like we expect it to, we don't anticipate needing to change the conditions in any way.
Obviously this could be used with array scanner technology, but what about sub-cellular type of imaging?
Of course my instinct would be to say yes. From our perspective, I don't think there would be a problem there. The question is whether a technology exists. If you had an array of live cells, often times for these types of confocal localization microscopy studies, you need to permeabilize the cells, fix them, et cetera. As long as this type of platform could work in that setup, I wouldn't expect that to be a problem. At first we'll probably be looking for a binary readout, to see if the pixel itself has a response, but I see no reason in principle that you couldn't look at this more closely.
What is the most immediate application area you'd like to explore next?
The next thing we'd like to do is show we can rapidly assemble complex arrays of cells. That would be to make kind of a simple microarray on which to throw populations of cells with different DNAs added, and keep cells assembled. At that point, there are a number of convenient assays we could run to validate the ability to use those types of platforms for either drug screening or biosensing.
Has your group applied for patents?
Not yet, but that's the next thing on our agenda. We'd like to get to the point where we have a viable application.
Is there an interest in commercializing this?
It's definitely something that would be valuable for us to do, mostly because some of these questions would be sort of trial-and-error, and would need to use the kind of researcher power that you don't often find in academia. At the same time, we'd like to at least package it up into a situation where we have the ability to make the model platform quickly, and from there you could effectively scale it up. It's at that stage where it would probably be best to hand it off to some commercial entity.