Printer Friendly

Oxygen: too much of a good thing: Oxygen-controlled incubators can provide the low [O.sub.2] conditions necessary for stem cells derived from bone marrow and adipose tissue.

Stem cell research, whether using embryonic or somatic sources, promises to provide not only a better understanding of the fundamental principles of cell differentiation, but also a whole host of life-changing or even life-saving cures.


Although it is still a developing area, scientists are learning just as much about how to culture and use stem cells most effectively as they are about their unique potential. For example, optimizing culturing conditions is essential for maximizing the data obtained from any experiments, as well as the quality of daughter populations and subsequent differentiated cells.

Often overlooked yet important, certain stem cell populations naturally reside in locations with very low oxygen tensions, such as bone marrow and adipose tissue. This means that when culturing cells from such sources, in addition to monitoring [CO.sub.2], [O.sub.2] levels should be carefully controlled to the lower end of the physiological [O.sub.2] scale to best duplicate natural conditions. The most efficient way of controlling this is through the precise introduction of [N.sub.2] into the incubator culturing system.

The following looks at the measurable differences in stem cell maintenance and subsequent differentiation between a standard [CO.sub.2] incubator and one capable of controlling [O.sub.2] levels.

Oxygen's importance

Oxygen is one of the most essential elements to life on earth and forms about 21% of the "air" around us. As a result, laboratory cell culture is generally carried out without control over oxygen levels, and this does not always provide the correct [O.sub.2] tensions at the level of the cells. This is due to the proximity of the in vitro culture to the atmosphere as opposed to the in vivo situation where oxygen is transported around the body via the blood and delivered to different cells and tissues at a lower [O.sub.2] tension.

In addition, a growing number of research studies have shown that drastically reduced oxygen conditions are favored by a number of stem cell populations, especially those derived from bone marrow and adipose tissue. Traditionally, these lower [O.sub.2] levels have been referred to as hypoxia, but it is actually an in situ normoxia for the cells and forms part of a scale of [O.sub.2] tensions referred to as physiological [O.sub.2].

This range covers the in situ normoxic conditions for all cell types, from stem cells to fully differentiated and [O.sub.2] saturated cells. Therefore, to recreate this physiological [O.sub.2] range in an incubator, the chamber [O.sub.2] must be in the range of 2% to 10%, depending on the cell type.


We focused on the lower end of this spectrum and looked at the advantages of controlling incubator [O.sub.2] levels for stem cells. Low [O.sub.2] conditions were shown to improve the overall therapeutic function of bone-marrow-derived mesenchymal stem cells via a variety of biochemical effects.

Key factors such as cMet--the major receptor for hepatocyte growth factor (HGF)--were upregulated, and the Akt signaling pathway was activated, which is important in cell signaling and survival. Stem cells cultured under atmospheric and low [O.sub.2] conditions both increased revascularization following ischemic injury, but this occurred much quicker with cells conditioned in the latter environment.

Research also showed that the low [O.sub.2] incubator environment improved stem cell self-renewal without reducing their ability to differentiate. Such an environment was even proposed as one of the key factors in the maintenance of undifferentiated stem cells in vivo. This research demonstrated that preadipocytes remained undifferentiated in these conditions and noted the involvement of hypoxia-inducible factor-1 (HIF-1) and pref-1, key genes in the inhibition of differentiation.

In addition to HIF-1 stabilization, VEGF and other angiogenic factors were upregulated, which have previously been noted as important to stem cell survival and self-renewal.

Interestingly, research has suggested that the level of [O.sub.2] is important in determining the survival of stem cells and subsequent levels of progenitor cells. Work on bone-marrow-derived hematopoietic stem cells showed that cells in increasingly differentiated stages within the hematopoietic cell hierarchy reside in areas with increasing levels of [O.sub.2] saturation. Furthermore, the special low [O.sub.2] metabolic properties and concomitant self-renewal capabilities have been described as the "stem cell paradigm."

As a result, being able to accurately control [O.sub.2] levels during culturing is essential, not only for work on stem cells but also for subsequent generations of progenitor cells, and even those that have fully differentiated.

Evaluating [O.sub.2] control

Although stem cell research in low oxygen environments has been conducted for a number of years, most has been carried out in specialized low [O.sub.2] chambers. With such research increasing, modern multi-gas incubators now offer precise control of oxygen, as per the specialized chambers, and combine this with superior capacity and functionality.

The Thermo Scientific Heracell 240i [CO.sub.2] incubator features precise oxygen concentration maintenance (between 1% to 21%) using [N.sub.2] which is controlled via its interactive touchscreen navigator, iCAN. The incubator also has a built-in high-temperature decontamination system to ensure the chamber is aseptic, which essential for all cell culture processes.

The Heracell 240i was recently evaluated for culturing stem cells in a low [O.sub.2] environment (4% [+ or -] 0.2% [O.sub.2]) using human mesenchymal stem cells from adipose tissue and bone marrow in conjunction with specialized media developed to support stem cell culturing activities.

The data and general observation of the cells showed that the Heracell oxygen-controlled incubator successfully created a stable low [O.sub.2] environment suitable for advanced stem cell culture.

The advantages of hypoxic culture conditions became clear early on, as both cell types had greater viable cell counts (Figures A and B).



The HAMSCs began to senesce following passage 9.[dagger] This senescence is common with MSCs and became apparent sooner in the low [O.sub.2] condition, perhaps due to the faster growth of these cells in this condition.

Furthermore, the population doubling data showed a clear advantage for the hypoxic environment (Figures C and D).



When cultured for 18 days to differentiate into adipocytes, the HAMSCs achieved double the viable cell yield under hypoxic conditions compared to normoxic (Figure E).


Some differentiation was observed with the HAMSCs, as demonstrated by the colonies of red stained adipocytes (Figure F). This differentiation was much less than would normally be expected, even in the control condition. Our experience indicates that this is likely due to the age (passage 11) of the cultures. Little or no differentiation was observed in all of the adipose-derived MSC conditions, which again is likely due to the age of the cultures. The data also shows that growing cells in standard conditions and differentiating the cells under hypoxic conditions seems to provide no benefit.



Routine cell culture requires that cells are incubated at 37 C and 5% [CO.sub.2] A number of research studies though have shown that, when human stem cells are cultured at 37 C, 5% [CO.sub2] and [less than or equal to]4% [O.sub.2] (low [O.sub.2] conditions), the effect prolongs proliferate on/self-renewal and yields more cells when the cultures are allowed to differentiate.

In this experiment, the Thermo Scientific Heracell 240i [CO.sub.2] incubator with [O.sub.2] control simulated a low [O.sub.2] environment and was compared to incubation under atmospheric conditions in a standard [CO.sub.2] incubator. The results align with published results and demonstrate that this instrument can provide the correct environment for moving stem cell research forward.

Furthermore, the highly precise maintenance of the defined conditions and the flexibility of the incubator ensure that it is also suited for studying the hierarchy of cell lines, from stem ceils to fully differentiated cells.

[dagger] If cells in culture are left to multiply for too long, growth can he restricted and unwanted differentiation accelerated. To avoid this, cultures are split and supplied with fresh media on a regular basis (dependant on cell type and established protocols) in a process known as "passage". As a result, "passage"' is also a useful comparative measure of the age and potential status of cell cultures.


For more information, please contact Douglas Wernerspach, Global Product Manager, [CO.sub.2] Incubation, Thermo Fisher Scientific, at

by Douglas Wernerspach, Global Product Manager, Julie Morris, Stem Cell Scientist, and Mark Wight, R&D Manager, Thermo fisher Scientific
COPYRIGHT 2010 Advantage Business Media
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2010 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Title Annotation:Life Science & Biomedical Labs
Author:Wernerspach, Douglas
Publication:Laboratory Equipment
Date:May 1, 2010
Previous Article:Spectroscopy data software tailors itself to users.
Next Article:Magnetic beads improve multiplex assay automation compatibility.

Terms of use | Privacy policy | Copyright © 2019 Farlex, Inc. | Feedback | For webmasters