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Setting up a Stem Cell Culture Lab: Dos and Don'ts

Philip Schwartz, PhD, supervisor and senior scientist and director of the National Human Neural Stem Cell Resource at the Children’s Hospital of Orange County Research Institute, talks to Contributing Editor Tanuja Koppal, PhD, about the subtle particulars and expertise needed to design and maintain a laboratory dedicated to culturing stem cells.

by Tanuja Koppal, PhD
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Philip Schwartz, PhD, supervisor and senior scientist and director of the National Human Neural Stem Cell Resource at the Children’s Hospital of Orange County Research Institute, talks to Contributing Editor Tanuja Koppal, PhD, about the subtle particulars and expertise needed to design and maintain a laboratory dedicated to culturing stem cells. He also talks about the common mistakes made and the need for adequate personnel training, and portends what the future holds for this promising field.

Q: What does your lab work on?

A: We are a research lab with about a dozen people working mostly with human stem cells, primarily neuronal stem cells. It’s a comprehensive setup involving six different aspects. One involves the R&D work for methods development on how to grow stem cells, which we then publish in scientific journals and methods publications. We are also a training lab where we teach people how to do what we do. Our training is meant for groups of students from the California state universities and for our collaborators who get cells from us. We are also a resource for different kinds of stem cells, including human stem cells that are associated with specific diseases. This is made possible under the aegis of the National Human Neural Stem Cell Resource (NHSCR). We also have ongoing research projects using stem cells to understand and treat disease. In order to understand diseases, we create stem cells from skin biopsies taken from patients and use them to study various disorders. For treating diseases we are looking at transplantation using mouse models of disease. We are using stem cells in an effort to come up with a cure for certain types of pediatric brain diseases. Finally, we provide stem cells as a service by isolating fibroblasts from skin biopsies, characterizing them, and turning them into induced pluripotent stem (iPS) cells, which are then differentiated into neural stem cells and sent to people who need these cells to study various diseases.

Q: How does a stem cell laboratory differ from a regular cell culture lab?

A: For the most part the differences are not obvious, but there are important differences. There are lots of approvals and much oversight is needed for stem cell work. For instance, approvals from the Stem Cell Research Oversight Committee (SCRO), the Institutional Review Board (IRB), the Biosafety Committee Review (IBC), and the Institutional Animal Committee (IACUC) are typically needed for this work. The cell culture process is largely the same, but things are different from the very beginning and definitely more steps are involved. In order to change the skin fibroblasts into neural stem cells, we use either non-integrating viruses or viruses that integrate into the DNA and express transcription factors that lead to changes in the phenotype of the cell. The iPS cells are grown as little colonies on a feeder layer of mouse fibroblasts, and those colonies have to be manually picked out for further passaging. The iPS cells are then differentiated into neural stem cells and the cell phenotype and methodologies change once again. So although the lab equipment is the same, there are differences in the details. These details are important, which is why we offer a training course so people gain the expertise to do this work efficiently and reproducibly.

Q: Can the processes in a stem cell lab be automated?

A: In our laboratory there is no automation at all, and there are a couple of reasons for that. One is that culturing stem cells is still an art form to a certain extent, and passaging these little colonies of iPS cells requires an eye. But that’s changing and the methods are getting better and becoming more amenable to automation. The second reason is that we are constantly improving what we are doing. So there is no point automating something that might very well change next week. However, people are definitely looking into more automation. There is a big initiative in California to provide 3,000 stem cell lines over three years. (More details are available at http://www.cirm. This is a tall order and is going to require robotics and automation.

Q: Are there any special considerations around lab design for this work?

A: The primary consideration when designing any cell culture lab is to have the atmosphere set in such a way that you don’t contaminate your cells or the people in the hallways. We have a separate virus transfection room, which has restricted access since we use recombinant DNA techniques. Our lentiviral rooms have negative pressure to keep the virus inside the room and the cell culture rooms have positive pressure to keep the various contaminants out of the room. So there are ventilation requirements put in place to protect the cells and the lab personnel at all times. If you are culturing different types of cells it’s best to work with each of them in separate rooms to avoid cross-contamination of cell lines, which is a fairly common problem.

Q: What practices have you put in place to minimize contamination?

A: Right now we have four separate cell culture rooms, and in about six months we will have two more. Each room has its own biosafety cabinet, incubator, centrifuge, refrigerator, and other equipment to keep the cell lines separate. The protocols for testing contamination are also very stringent, and we do some testing ourselves and outsource the rest. I am a firm believer in not reinventing the wheel and not doing things that are easily, commercially available. For instance, one of the tests that need to be regularly done with iPS cells is karyotyping, since these cells are known to develop karyotypic abnormalities in culture. So we routinely send our cells for karyotyping. The testing may be expensive, but in the long term it’s cheaper because the tests are well validated and used for all types of clinical purposes. We do the same for phenotypic testing for our animal work. If there are good services available we make use of them.

Q: Have you put any contingency plans in place in case of a disaster?

A: No, we don’t have any contingency plans in place. Some investigators have suggested housing the cells in two different locations, which essentially means in a different state, in case of a natural disaster like an earthquake. In order to do that you have to set up contracts with labs and doing so involves expenditure that sometimes becomes hard to justify. It sounds like a great idea, but we don’t have anything set up for a major catastrophe. However, in case of power outages we are on an emergency power supply and all the critical equipment such as freezers and incubators get powered in seven seconds. We also have a contingency in terms of manpower, since a cell culture lab operates 24/7. The staff shares responsibility for feeding and maintaining the cells and our employees come in during the week, weekends, and holidays so there is constant coverage of the lab.

Q: What is your advice to lab managers who work with stem cells?

A: My advice would be to get good training so you can visualize how to modify your setup to meet the lab’s needs. The most critical aspect is to know what you are doing. Stem cells are very finicky and trying to keep them from differentiating is not that easy. It requires an attention to detail that far surpasses anything in a cell culture laboratory. Unless you have good training, you will spend a lot of time with failed cultures because of technical issues.

Q: What are some of the common mistakes that people make when using stem cells?

A: The most common mistake is that people think they know what they are doing and do not follow the detailed protocols that are given to them. This is common because in some ways cell culture is an art form and the people who have been in it for a while have their own ways and superstitions, and have a mind-set on how things should be done. However, an advanced cell culture technique, such as working with stem cells, requires a real attention to detail. Hence, people should never change the protocol before they learn how to do it the original way that was given to them.

Q: What are some of the big changes you see coming?

A: The automation that we discussed earlier is definitely happening and that will make stem cell work more manageable and easier to scale up. Clinical trials using stem cells will also become commonplace in the coming years.

Key Considerations for Operating a Successful Cell Culture Laboratory
  • Skilled Personnel (training, knowledge, resources)
  • Established Standard Operating Protocols (quarantine incoming cells, culture cells, long- and shortterm storage)
  • Laboratory Planning and Design (budget, space, scope, mechanical, engineering considerations)
  • Proper Equipment (for tissue culture, microscopy, biochemistry, molecular biology)
  • Quality Control (reliable techniques, validated reagents, checklists)
  • Documentation and Approvals