Cytocentric Blog

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Cytocentric Visionaries: Hal Broxmeyer, Indiana University

How the Shock of Room Air Oxygen Reduces Stem Cell Yields

Alicia Henn, PhD MBA, Chief Scientific Officer, BioSpherix

Dr. Hal Broxmeyer is a Distinguished Professor, Professor of Microbiology/Immunology and Co-Leader of the Program on Hematopoiesis, Malignant Hematology, and Immunology at Indiana University Simon Cancer Center. He has chalked up well over 1,000 scientific publications and his work has been seminal to the field of hematopoietic stem cell transplantation. His group’s 2015 paper in Cell and the subsequent review article are landmark papers that demonstrate how room air oxygen exposure during cell isolation inflicts damage on freshly isolated stem cells.

 

Here, we talk with Dr. Broxmeyer about his work. This interview was conducted by email and edited for length.

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Why the Lasker Awards are So Important This Year;

It’s All About the Oxygen

The Lasker Awards were won this year by a trio of incredibly important figures in physiologic oxygen research; William Kaelin, Peter Ratcliffe, and Gregg Semenza.

 

What did they do?

These three researchers, along with others, have traced the molecular mechanisms that connect changes in oxygen levels to cellular responses.

 Gregg Semenza, along with his post-doc, Guang Wang, first reported the structure of a protein that was regulated by oxygen levels [1]. They called it Hypoxia-inducible factor 1 (HIF-1). It had two subunits, alpha and beta. The beta subunit was expressed all the time, but the alpha subunit changed in levels when oxygen levels changed.

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Why Should We Care About In Vitro Pericellular Oxygen?

Alicia D. Henn, CSO BioSpherix

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 oxygen 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

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How to Clean a Closed Environment for Cells: Resisting the Misting

In a closed system like a barrier isolator, there are few routes for disinfectants to leave and no way for contaminants to float in.

In an open laboratory, you can spray surfaces liberally with disinfectant and it all evaporates into the room air. Keeping surfaces wet with disinfectant for maximum contact time is the major concern.

It is a big change, moving from an open lab, which exists in a constant state of high microbial contamination risks, into the closed Xvivo system, a very low risk environment. A risk-based balance has to be struck between much lower risk of contamination and the higher risk of disinfectant fume build-up.

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Cytocentric Visionaries: Jan Jensen, Trailhead BioSystems 

Part Three: Full-time Oxygen Control and Aseptic Conditions are Critical for Automated Protocol Optimization

Alicia Henn, Chief Scientific Officer, BioSpherix

In Part Two, we discussed Trailhead’s unbiased system of cell signaling optimization for differentiation. Today, Dr. Jan Jensen. CSO of Trailhead BioSystems discusses full-time optimization of cell culture conditions with us.

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