Ralph Garippa, Ph.D., independent consultant and former head of cell-based high-throughput screening (HTS) and microscopic imaging-based high-content screening (HCS) at Hoffmann-La Roche, discusses with Tanuja Koppal, Ph.D., contributing editor at Lab Manager, the recent trends in cell culture that include the increasing use of stem cells in compound screening, the integration of labelfree assays along with conventional assays, and the use of innovative de-cellularized scaffolding and synthetic materials for cell growth.
Q: How do you see cell-based screening evolving with the use of different types of cells, besides just clonal cell lines?
A: The cells that we’ve used in the past, such as CHO, COS, and HEK cells, are still highly relevant. At one point, we started using cells that weren’t traditionally used in cell-based screening, such as U2OS, an osteosarcoma line that is a beautiful cell line for running FLIPR-based calcium flux assays. These cells grow very well, they’re very flat, and they’re so “imageable” in terms of fluorescent microscopy. You’re going to see more and more of that: finding a cell that fits the application. So the clonal lines are continually in use, and they will always be in use with better cell lines coming through. We do have problems with primary cells. They don’t last very long and you can’t propagate them for many generations. But they’re more or less relevant, particularly soon after they come out of the human or animal body. What is really exciting is the prospect of using induced pluripotent stem (iPS) cells from rodents, higher primates, or humans to either recapitulate the disease state or to stress them using chemical or mechanical means. Being able to use cells that were directly brought through a particular lineage (the endoderm, ectoderm, or mesoderm) and then brought into the specific cell or tissue type that we’re interested in—be it cardiac, hepatocyte, lung or another—will be very useful.
Q: So are we still largely dealing with clonal lines, and when and how do you see that changing?
A: It’s going to be a slow change, and the reason I say that is because the clonal lines are so useful and there’s such a huge legacy out there in repositories, libraries, and freezers filled with stable clones. But more and more you’re seeing people transfecting embryonic stem cells and iPS cells, and they’re getting better at that. The one little blip on the screen, and it’s not any surprise with a brand-new technology, is the reports that have come out over the past year about the number of irregularities that we’re seeing in iPS cells that are both genetic and epigenetic. I think there’s a little more caution now that should have been there from the beginning, and scientists are trying to learn more about the cell background, the degree of variability, and how often we can expect changes to occur. In our hands, prior to passage 35, it was rare to find genetic abnormalities. The aberrations start to appear with a higher frequency, around passage 50. So one has to frequently karyotype or use in situ hybridization to give an early indicator of genetic abnormalities. We’re also seeing companies popping up—CROs—that are able to provide epigenetic characteristics of cells. So as the field begins to spontaneously self-assemble and all these support services and more-detailed analyses of the iPS cell background fit into place, then I think you’ll see the second wave of iPS come. And once that happens, the percentages will start to change a little bit in terms of using pluripotent-derived cells in screening.
Q: What, in your mind, is the biggest advantage that stem cells, iPS cells particularly, offer when compared to clonal cells?
A: Well, like clonal cells, iPS cells are immortal, and if you handle them correctly, they’re immortal in the undifferentiated state. That’s a great advantage. By and large, they have fewer chromosomal abnormalities than a clonal cell line. However, the real attraction is that they can come from people who manifest abnormalities in later decades of life. So for late-onset diseases such as Huntington’s or Parkinson’s or even type 2 diabetes, we can take the cells from a skin punch or other source and bring them back to iPS, or even trans-differentiate them into cells of interest that would be relevant in the disease state. The bottom line is: these are human cells, immortal in the undifferentiated state, that can be brought forward again and again into the differentiated cell type that we’re interested in. We already know that the people from whom these were derived manifested pathologies that we’re interested in. So how can you possibly find a better context than that to screen your new treatments and your new drug modalities?
Q: Are there any good resources, journals, websites, or books that you have found particularly useful when learning about standard protocols and best practices for working with stem cells?
A: The International Society for Stem Cell Research (ISSCR) website, [www.isscr.org], I think, is a great starting point with lots of good links and accurate information. Second, I would recommend attending conferences. It could be a small Keystone conference or a very large conference like ISSCR, but I can’t possibly overstate how useful they are to help you get up to speed quickly.
Q: Besides the cells, there is also a lot of activity and innovation in cell reagents and the scaffolding on which cells grow. Is there anything there that has caught your attention?
A: With the advent of stem cells and the cells being so difficult to culture in the early days, people took a multipronged approach. They started looking at all types of variables, such as the cell media, the co-culture conditions, and the oxygenation, and one of the variables they looked at was attachment substrates. With the development of synthetic polymers, these cells were found to react differently on different surfaces. We’re now starting to see reports in regenerative medicine where people can use these de-cellularized tissues or synthetic scaffolds and repopulate them with stem cells. Repopulating scaffolds autologously with a person’s stem cells that are differentiated into the organ or the piece of the organ that’s been traumatized will announce real replacement therapy. So the promise of regenerative medicine is just starting. As you can imagine, there’s just an enormous range of choices in the scaffolding materials that are available in terms of the starting material—the porosity, the hydrophobicity, the hydrophilicity, and such. Whether it’s natural materials that are de-cellularized down to the support structures or whether it’s synthetic, varying just the substrate while keeping all other variables fixed is going to produce some very interesting results that will further the field.
Q: How do you go about choosing the right substrate for your cells? Is it all done by trial and error?
A: It’s all empirical design and trial and error. There’s no way to predict via an algorithm because this field is so new and we don’t have a database of reported results to go with. But the good news is that we already have some successes out there, so we know that the proof of concept is true. The cells won’t react well to all biomaterials or synthetic materials, but there’s a certain subset of materials that they do react to very well. You could imagine putting pluripotent cells on a material that would help drive differentiation through a specific pathway toward a certain cell fate, just as we would do by adding a certain cocktail of growth factors and extrinsic factors to cells. So it’s an area that I think up until now has been lightly explored, but I see a crest of a wave on the near horizon. I don’t think that for most lab managers there’s a necessity to explore new biomaterials or synthetic materials right now, but they should be aware of it. So if one sees a better attachment substrate or well coating, they would be able to explore and exploit it. If investigators are doing more sophisticated work in three dimensions or with multiplicity of cells in the well, that creates a scenario where you might want to go beyond the standard run-of-the-mill attachment surface and look for something that’s, perhaps, a little more cutting-edge.
Q: What advantages do these cell substrates offer?
A: There are always spatial constraints when growing cells—the amount of separation between cells, the ability to connect with each other, and the amount of perfusion that affects the amount of nutrients and oxygenation that reach the cells. This happens in our own bodies, and it’s very difficult to produce a synthetic layer of cells that’s thicker than 300 microns. The farthest cell in our smallest capillaries is not farther than 150 microns away from the nearest blood source. So if you have a slab of synthetic tissue that’s 300 microns thick, you would expect that at the center—which is at 150 microns—those cells would still receive enough nutrients and oxygenation. Once the slab of tissue becomes thicker, it’s much more difficult. And that’s part of the reason why people such as Dr. Anthony Atala at Wake Forest in Winston-Salem, N.C. are doing great pioneering work in creating hollow organs. But it’s a much more difficult road to create solid organs because you can’t get the perfusate down to the deepest cells. If materials that allow cells to grow also had some degree of porosity that allowed the perfusate to get into the deepest cells, you’d create a scenario where you could possibly break that 300-micron barrier and start to work with engineered cells in thicker slices.
Q: How do you see the field of label-free technologies shaping up?
A: Label-free technologies are being used to measure cells using photons or electrons penetrating the cells, in a noninvasive manner, without changing the biology. When we put together the known responses from labeled cells with those obtained from label-free systems, we are able to match and get a comprehensive and believable picture of what these measurements are providing about cell physiology. I don’t see label-free technologies replacing all the experiments that are out there for reporter systems and biochemical assays, for imaging and HCS. But this is the newest tool in a drug screener’s arsenal for looking at complex issues such as functional selectivity and receptor-based signaling. Now you have the ability to gain knowledge about pathways that we were unaware of because we could not get a readout on multiple pathways simultaneously or because there were events that we were not measuring, such as protein translocation, the strength of attachment of cells to the surface, and cell spreading. We have worked with both impedance and photon-based label-free technologies, and they are both good. It’s just a matter of letting the field evolve and see how it can be enabling.