Why Should We Care About In Vitro Pericellular Oxygen?

An important new publication in Stem Cells and Development by Tiwari et al in Australia has brought a critically important in vitro oxygen diffusion issue to the forefront[1]. In a murine model, they explore optimum tissue oxygenation levels for best in vitro expansion of HSC for transplant.

The Findings

They report:

– It took between 4 and 19 hours for human umbilical cord blood CD34⁺ cells pericellular oxygen to reach steady-state at ~4% O₂ (in the incubator set to 5%) O₂ and at about 18% O₂ (incubator set to 20%)

– A gradual decrease in HSC pericellular oxygen levels in 24-well plates in static culture over 7 days with the incubator set at 5% or 20% O₂

– Orbital shaking, “dynamic” incubation, of the plate at 5% O₂ maintained pericellular oxygen levels at 4% longer even as cell numbers increased to numbers 70% higher than without shaking

– Cells incubated at 20% O₂ also saw increased cell expansion with shaking

– HSC incubated with shaking at 5% O₂ expanded the most as assessed by colony forming assay and flow cytometry, producing multiple hematopoietic cell types

– The bone marrow and spleen of immunodeficient mice were engrafted better by HSC (a mix of cell types including progenitors) incubated at 5% O₂ with shaking

The Significance: Pericellular Oxygen

The importance of this study cannot be overstated in that it reported on the need to define the best pericellular oxygen levels for the in vitro expansion. All oxygen that the cells get has to be provided by the environment, but so many of us set the atmospheric oxygen level without considering what the cells actually experience sitting on the bottom of a tissue culture plate or flask. The many different levels of gas exchange needed for cells to get oxygen in static culture are covered in a previous post here.

“Those who don’t know history are doomed to repeat it.” – Edmund Burke

It has been a long time since optimal in vitro oxygen levels were first explored. The authors of the Stem Cells and Development paper recall for us Balin’s work in the 1970’s on the importance of in vitro oxygen[2] as well as Metzen’s 1995 equation for the pericellular oxygen level as balanced between the oxygen provided by the incubator and the oxygen consumed by the cells[3].

Tiwari et al didn’t repeat the work of the early in vitro oxygen giants, but extended their work on importance of oxygen control in a thoroughly modern and relevant context. Hematopoietic stem cells are among the most medically and financially valuable cell type in culture right now and they are extraordinarily sensitive to oxygen changes.

The Broxmeyer group published a landmark paper in Cell in 2014 in which they showed that exposure of umbilical cord HSC to room air oxygen levels can dramatically reduce the ability of the cells to repopulate animals [4]. Getting the in vitro oxygen right for these cells has the potential to extend the capacity of the system so that it can provide lifesaving treatments to more patients that need them.

Oxygen Levels, Even in a Static Culture, are Never “Static”

This is truly cytocentric work in that the authors considered all the factors that go into pericellular oxygen fluctuation and the fact that with live cells, the system can undergo change over time. They looked at cultures over 7 days of culture, considering oxygen consumption as well as the settings of the incubation chamber. They also introduced agitation of the culture as a variable.

Agitation of a cell culture, through bubbling, stirring or shaking, is a major factor in gas exchange that has been studied thoroughly in industrial cell culture settings. However, it is often overlooked in academic laboratories that are working to define the biology behind critically important physiologic and pathophysiologic states.

We will be discussing this paper on in vitro tissue oxygenation further in coming posts.

Questions? Comments? Contact us at the Cytocentric Blog. We would love to hear your thoughts.

1. Tiwari, A., et al., Impact of Oxygen Levels on Human Hematopoietic Stem and Progenitor Cell Expansion.Stem Cells Dev, 2016.

  2. Balin, A.K., et al., The effect of oxygen tension on the growth and metabolism of WI-38 cells. J Cell Physiol, 1976. 89(2): p. 235-49.

  3. Metzen, E., et al., Pericellular PO2 and O2 consumption in monolayer cell cultures. Respir Physiol, 1995. 100(2): p. 101-6.

  4. Mantel, C.R., et al., Enhancing Hematopoietic Stem Cell Transplantation Efficacy by Mitigating Oxygen Shock. Cell, 2015. 161(7): p. 1553-65.