Tari Parmely, head of the Tissue Culture Core Laboratory at the Stowers Institute, talks to contributing editor Tanuja Koppal, PhD, about the challenges involved in working with many different types of cells, including embryonic and induced pluripotent stem cells. She discusses some operating protocols and informatics integration that have been put in place to make the cell culture process more efficient and reliable. Parmley discusses some operating protocols and informatics integration that have been put in place to make the cell culture process more efficient and reliable.
Q: Can you give me some information about your lab and describe some of the cell culture work you do?
A: I joined one of the research laboratories at the Stowers Institute in 2002 specifically to do cell culture. However, it soon came to a point where cell culture became integral to the research being done at several laboratories on campus, and it became apparent that a core lab was needed. I was one of the people who helped establish that core lab in 2008 and became its head. At the core lab, we culture a variety of cell types, such as insect cells, immortalized cell lines, mammalian embryonic stem cells, induced pluripotent stem cells, and more. We do not culture bacteria or viruses, but we do consult and help people working with viral cells.
Q: What are some challenges you face working with all these different kinds of cells at the same time?
A: We work with several different cell lines, and keeping those straight is a challenge. We have eight technicians in the lab, and many hands are involved in carrying a project from start to finish. The first issue that we had to address was recordkeeping. In 2009, along with our scientific operations team, we wrote a laboratory information management system (LIMS) module that was specific for our lab. Our LIMS is rather unique, as it was custom-written for us, and it is very detailed. The module has nine different tabs. There is one for cell lines, and it lists all the relevant information about the cell lines that have been deposited in our lab. Then we have other tabs, such as an order tab and a mycoplasma test tab. There is a cell bank tab for the cell lines that are frozen in the core lab so we can access all the related information at a moment’s notice just by going through the LIMS module. We have a cell log, so anytime the cell bank is touched, that information is recorded. There are computers at every hood, so the technician can access the LIMS module while working on the cells. If there are any special instructions for a specific cell line, we save that in the module. So even if we haven’t cultured those cell lines for a while, we can go back and see when and how those cells were cultured and see the trends of how they grew.
There is a calendar in the module, so every day we can look and go through the daily tasks. The calendar is color-coded, so we know at a glance what tasks are to be done. If you click on each one of those tasks, there are detailed instructions, so we know what [the] concentration [is], what media to use, and where to deliver the cells. With many different hands working on the cells, we have to make sure that the cells are handled the same way. We have an automated cell counter, so we know that each of our cells is handled and counted the same way each time and there is no variation.
Q: Are different cells kept in separate incubators? Have you run into problems with crosscontamination?
A: We do worry about cross-contamination, but we have never run into this problem. We never have more than one cell line in our hood at any given time, and that is the rule. We do have separate incubators for some cells, and a separate area with hoods for embryonic stem cell work. Insect incubators are separate, and that’s dictated by the temperature they need to be at. The other cell lines are placed together, but we flag the outside so we know the exact location where the cells are kept inside the incubator. The cells for transfections have their own incubator. We have a quarantine incubator for a new cell line or for those cell lines that are [being watched] for some reason.
Q: Have there been any major improvements in the equipment for cell culture?
A: We use incubators with SafeCell UV and InCu-saFe to reduce contamination issues. The automated cell counters have been very useful. There are automated cell-thawing instruments that maintain the cell viability and eliminate the risk of contamination, which can occur when you thaw cells in a water bath. For mouse embryonic stem cells, we have now switched to feeder-free cultures, which have saved us enormous amounts of time and money.
Q: Do you use a lot of lab automation for cell culture?
A: If you work with a limited number of cell lines repeatedly, then automation makes sense. When working with hundreds of cell lines, it doesn’t make sense to use automation, and there is nothing out there that is efficient for what we are doing. We just use multichannel pipettes for our work, nothing very glamorous. Instead, we prefer to invest in ergonomic equipment and furniture, such as pipettes that are lightweight or chairs with footrests, so technicians can be comfortable working in the cell culture hood for eight hours a day.
Q: Are you seeing any new trends in the cell culture projects that are coming to you?
A: We are working in collaboration with the molecular biology, the screening, and the lab animal services core labs on campus for using clustered regularly interspaced short palindromic repeats (CRISPR)-based gene editing in cells. We started working with organoid cultures a few months ago. We have grown 3-D cultures for intestinal cells and are currently troubleshooting how to freeze-thaw these cells efficiently. We are trying to standardize the protocols so we can move into other types of 3-D cultures. We are also working with some new methods and technologies for mouse embryonic stem cells and induced pluripotent stem cells. We are seeing more requests for cell authentication, and that’s becoming important as journals are now asking for it. These authentication reports are now fast and easy to obtain; there are no surprises, and researchers are happy with the types of information provided. Mycoplasma test kits are also becoming more reliable and easy to work with, and we test every cell line that comes into the lab.
Q: Can you discuss the types of cell culture training you provide as a core lab?
A: We offer a one-hour training course for any new student, postdoc, or anyone who has never done cell culture before. We teach them cell culture etiquette so they don’t leave the cell culture room messy for the next person. We show them how to culture both adherent and suspension cells and then have them culture [those] themselves. We teach them the basics of cell culture, such as how to passage a cell line, how to feed cells, what to look for, what [confluent means] and other concepts. We teach them how to count cells manually and [by] using automated counters. We provide them with a reagent list that we use. We can teach them how to harvest or transfect cells or how to work with mouse embryonic stem cells. People can also tell us ahead of time if they want training in a specialized technique. We hand them a standard operating protocol (SOP) that we have written out, and people have been very happy to have this service available on campus.
Tari Parmely is the head of the Tissue Culture Core Laboratory and of media prep, histology, and electron microscopy at the Stowers Institute. She joined the Stowers Institute as a research specialist in the Conaway laboratory in 2002 and took over the management of the Stowers Tissue Culture Core Laboratory in 2008. Working closely with Stowers investigators, postdoctoral researchers, and graduate students, Parmely assists with the development of new projects and ensures that all their tissue culture needs, including maintenance and large-scale expansion of cell lines, are met.