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cytocentric visionaries at p1Cytocentric Visionaries: Abhilasha Tiwari, PhD, Monash University

What do you look for when you want real answers? Real questions.

Dr. Abhilasha Tiwari is a SIEF-STEM+ Business Postdoctoral Researcher at The Ritchie Centre, Hudson Institute of Medical Research and Monash University. Her work with Graham Jenkin and Mark Kirkland focuses on optimal expansion of hematopoietic stem cells (HSC) from umbilical cord blood.

Here, we talk with Dr. Tiwari about her recent publication in Stem Cells and Development, “Impact of Oxygen Levels on Human Hematopoietic Stem and Progenitor Cell Expansion.” [1] This interview on how pericellular oxygen affects cord blood HSCs was edited for brevity.

AH: We see you as a Cytocentric Visionary because of your work in optimizing HSC culture conditions. What made you want to look at pericellular oxygen as a critical parameter for cord-derived HSC?

AT:About two years back, we were working on preparing a closed system HSC expansion kit and planning to use cell culture bags. Even though these bags are gas permeable, we wondered about the oxygen levels in the bags ie. if is it at 5% (similar to the environment of the incubator etc.)? So instead of going to the bags first, we started with a tissue culture plate, measuring oxygen in the media first and then we moved on to the cells. I think this was really important.

AH: Were you surprised by your findings?

AT: I wouldn’t say we were surprised because we were sort of expecting it, but it was good to see on a screen that the oxygen changed a lot. Because we are trying to comply with GMP processes to use these stem cells for clinical purposes, I think it was really crucial to check the actual oxygen available to the cultured cells. Even if 20% oxygen is shown on the incubator screen, it takes almost 4-19 hours to get to that oxygen level in the cell culture media.

AH: Because oxygen is so insoluble in medium?

AT: Yes, and before it gets to the right level, someone else opens the incubator door.

AH: It must cause variability that most people don’t even think about. Was the incubator open the same number of times this week as last week? Who knows?

AT: This was another reason we have to look at the cells and their oxygen levels, because we were getting inconsistent results. Reproducibility would totally depend on how many times people opened your incubator when you were running those experiments. It was hard to explain why this time we got better expansion and the last time we got less expansion. It’s so critical and we never thought about that point.

AH: Do you think that cell culture oxygen levels should be considered a major factor affecting in vitroreproducibility?

AT: Oh yes, definitely. Because we have seen that with this exponential rise in our knowledge in hypoxia and basic signaling pathways, it should be made very clear to every scientist cultivating cells that not only temperature, humidity, and CO2, but oxygen needs to be closely controlled and monitored as well. One of the reasons we started looking at pericellular oxygen levels was because we were not able to replicate our earlier results. So I think it is really critical to look at oxygen. It is going to affect the metabolic conditions for the cells. Every time we change the oxygen there is going to be stress on the cells that can affect reproducibility.

AH: What other struggles did you have getting your system established?

AT: One of the biggest technical challenges was that the oxygen monitor probe had an extremely fragile bulb at the bottom. We had to place it in the incubator in the tissue culture plates for seven days, the whole expansion time, while maintaining sterility. We drilled a hole in the tissue culture plate cover and put a protective sheet on the probe and ensured that the length of the sheet was so that when the probe was inserted into the tissue culture wells it was just sitting above the cell layer without hitting the bottom.

The other thing was that we did not want to open the doors for the whole expansion period. We had showed that once we open the door, like we normally do in a big lab with shared incubators, it takes 4 to 19 hours for the medium to equilibrate to the oxygen level that is indicated on the incubator screen. Even the slightest opening of the door would ruin the whole experiment. I had to get two different incubators for two different oxygen concentrations, making sure no one else touched them for the whole time.

AH: You suggest in your paper that pericellular oxygen levels should be reported in clinical validation studies. Do you think that this is something that should be reported as a release criterion for all in vitro expanded cells?

AT: Yeah, I completely agree with that. Oxygen plays a critical role when we are culturing any cell type. When we’re talking about stem cells, it’s even more crucial. It depends on several factors such as atmospheric oxygen, sample geometry, depth of the media, cell density, whether it’s static or dynamic, and the respiration rates of the cells. We and other researchers have shown that low oxygen has been reported to promote proliferation, and high atmospheric oxygen just promotes differentiation. The exposure to different oxygen levels critically affects the commitment level of the stem cells.

For scientific publications as well, I would say that oxygen concentrations along with other metabolic conditions to which the cells are exposed in vitro must be disclosed.

AH: What would you say to someone that said “I’d love to do that but there’s no way we would ever have the money to buy equipment for that kind of thing.”

AT: This is one of the reasons why there was this huge gap in these types of studies. We did some studies back in early 70s and 80s. Then people just took it for granted and they were not really worried about all of these conditions. One of the reasons for that might be the money thing. I don’t know why they don’t think of it as very important criteria because every time you have problems with reproducing your results I think this could be one of the culprits there.

AH: I was impressed by your citation of our forefathers in oxygen research. You cited Metzen[2] and Balin, these are big guys that have been lost in the stacks for many current researchers. What made you look them up?

AT: I must admit these classic ones came from my supervisor, Mark Kirkland. I was really surprised to see this huge gap in these types of basic cell culture studies. A lot of work was done on human cell line culture in the 1960s, 70s and 80s. And then other researchers just took it for granted and assumed that the cells in the culture plates would be exposed to the set environment of the incubator. However, diffusion of oxygen is influenced by a number of parameters like the height of the media, area of the containment and consumption by cells. So, it stands to reason why the oxygen levels of the incubator would not be the same as the plates or bags as it has to diffuse through air then media and then to the cells. And if the culture is static, it’s a bigger difference as we’ve shown in our study. There were a few more papers like one by Taylor et al. in 1978 in the Journal of Cell Physiology[3], where he discussed the density-dependent effects of oxygen on the growth of mammalian fibroblasts in cultures. Balin is obviously one of the forefathers of these studies. I liked one of his papers in Journal of Experimental Medicine in 1984[4] where he has shown the oxygen toxicity that is contributed to the cell density dependence of cellular growth.

AH; Anything else that you would like people to know about your work?

AT: Just pay attention to what you’re providing to your cells. If you want reproducibility, you have to monitor how many times someone opened your incubator, what growth factors, which companies, what’s their stability. If you want real answers, look for real questions.

AH: Thank you for your time and your insights. Dr. Tiwari. We will be watching for more exciting publications from you in the future.

If you would like to be featured in our Cytocentric Visionary Series, contact us. We would love to hear about your work.

1. Tiwari A, Wong CS, Nekkanti LP et al. Impact of Oxygen Levels on Human Hematopoietic Stem and Progenitor Cell Expansion. Stem Cells Dev doi:10.1089/scd.2016.0153 (2016).

2. Metzen E, Wolff M, Fandrey J, Jelkmann W. Pericellular PO2 and O2 consumption in monolayer cell cultures.Respiration physiology 100(2), 101-106 (1995).

3. Taylor WG, Camalier RF, Sanford KK. Densitydependent effects of oxygen on the growth of mammalian fibroblasts in culture.Journal of cellular physiology 95(1), 33-40 (1978).

4. Balin AK, Fisher AJ, Carter DM. Oxygen modulates growth of human cells at physiologic partial pressures.The Journal of experimental medicine 160(1), 152-166 (1984).

 


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About The Author

Alicia D Henn, PhD, MBA

Alicia D Henn, PhD, MBA

Chief Scientific Officer of BioSpherix, Ltd

 

 

Alicia Henn has been the Chief Scientific Officer of BioSpherix, Ltd since 2013. Previously, she was a researcher at the Center for Biodefense Immune Modeling in Rochester, NY. Alicia obtained her PhD in molecular pharmacology and cancer therapeutics from Roswell Park Cancer Institute in Buffalo, NY and her MBA from the Simon School at University of Rochester in Rochester, NY.

ahenn@biospherix.com

 

 

If you would like to be featured in our Cytocentric Visionary Seriescontact us. We would love to hear about your work.