Hypoxic conditions: the lowdown on the importance of hypoxia in stem cell culture

Hypoxia chambers vs cell culture incubators

When cells are grown outside of their source organism, they must be cultured under conditions that closely mimic their native in vivo environment. Thus, various parameters in cell culture must be rigorously optimized, including the culture pH, temperature, and media composition. One parameter that is being increasingly recognized as having a major influence on cell behavior and health is oxygen content. Indeed, many cell systems, such as stem cells, require hypoxic conditions – with low oxygen tension – for the maintenance of optimal behavior. However, standard cell culture incubation chambers, which allow for control over CO2, temperature, and humidity (typically maintained at 5%, 37°C, and 95%, respectively), do not take oxygen concentration into consideration and instead expose cells to ambient oxygen levels of around 21%1.

In contrast, hypoxia chambers are specifically designed to maintain strict control over both CO2 and oxygen content, and therefore allow for more physiologically relevant oxygen levels to be utilized during cell culture. Hypoxia chambers range in simplicity from cost-effective sealed food storage vacuum bags with a defined oxygen level2, to glass or plastic boxes with inlet and outlet ports for flushing the chamber with a gas mixture, to high-end commercial hoods. Using these chambers, oxygen can be maintained at hypoxic levels (for example, at or below 5%) to support the growth and maintain optimal characteristics of various cell types.

In the article below we will discuss the properties of stem cells, stem cell niches, and why hypoxia chambers can be highly beneficial when culturing stem cells.

 

Properties of stem cell cultures

Stem cells have the capacity to self-renew and differentiate into specified cell types. They have proven useful for the study of basic developmental processes and have provided new avenues for the treatment of diseases, with applications in tissue engineering, cell-based therapies, regenerative medicine, and disease models for drug discovery3,4. A variety of stem cell and progenitor cell types are cultured in vitro, including adult stem/progenitor cells, mesenchymal stem cells (MSCs)/mesenchymal progenitor cells (MPCs), embryonic stem cells (ES), and induced pluripotent stem cells (iPSCs).

Multipotent Cell Types

Adult stem cells are multipotent, therefore, they can differentiate into a limited number of cell lineages, are found in differentiated tissues, and exhibit limited self-renewal in culture. For example, epidermal stem cells, found in hair follicles and the basal layer of the epidermis, replenish the outer layers of the skin, mediate hair regeneration, and support wound healing5. MSCs, isolated from umbilical cord, cord blood, placenta, and amniotic fluid, also exhibit multipotency and limited self-renewal in culture6. These cells are characterized by adherence to plastic, surface expression of a defined set of markers (including CD73, CD90, and CD105), and the capacity to differentiate into adipocytes, chondrocytes, or osteocytes7.

Pluripotent cell types

Both ES cells and iPSCs are pluripotent, and therefore have the ability to differentiate into cells of all three primary embryonic germ layers: endoderm, mesoderm, and ectoderm. ES cells are derived from the inner cell mass of blastocysts. In contrast, iPSCs are derived from adult somatic cells that have been “reprogrammed” into a pluripotent state through the ectopic expression of a core set of transcription factors, such as Oct4, Sox2, Klf4, and C-Myc8.

 

Oxygenation in the stem cell niche

In vivo, stem cells exist in distinct 3D microenvironments known as stem cell niches. These regions were originally conceived of in a paper published in 1978, and their existence was later confirmed in the following decades through the study of worms, flies, and mammals9. Stem cell niches are unique compartments in tissues that include the neighboring cells, vasculature, extracellular matrix, and 3D environment9, providing a multitude of chemical and physical signals that dictate stem cells’ balance between quiescence, self-renewal, and differentiation10.

As with other in vivo tissues, stem cell niches are a hypoxic environment and exhibit oxygen levels that are notably lower than the ambient environment. Indeed, in most tissues, physiological normoxia ranges from <1-9% oxygen, and stem cell niches are no different9. For example, ES cells in intact blastocysts in monkeys are likely subject to oxygen levels as low as 1.5%10. Likewise, hematopoietic stem cells (HSCs) in their niche are exposed to an oxygen gradient ranging from 1-6%, depending on their proximity to blood vessels9.

 

The importance of hypoxia in stem cell culture

Precise control of oxygenation in the culture environment is highly beneficial for labs that strive to achieve a high yield of stem cells with optimal self-renewal and potency properties11,12,13. Several studies have shown that stem cells exhibit higher proliferation rates in culture under hypoxic conditions9,10. For example, human MSCs exhibited a 30-fold increase in proliferative capacity when grown in hypoxic conditions compared to ambient air14. However, it has been noted that ES cell proliferation can actually be suppressed at even lower oxygen levels (5% versus 1%), suggesting a range of oxygen-dependent behaviors in these cells15 and further highlighting the importance of maintaining strict control over oxygen content in the culture environment.

In addition, recent work has shown that HSCs exposed to ambient air during collection from bone marrow or cord blood exhibit a phenomenon called “Extra Physiologic Oxygen Shock/Stress,” which reduces the long-term quiescence and self-renewal capacity of the cells, as well as their transplantation efficiency into host animals16. This process was found to be mediated by activation of a pathway involving cyclophilin D, p53, and mitochondrial permeability transition pore (MPTP), leading to the production of reactive oxygen species16.

Stem cells also exhibit improved maintenance of stemness when cultured under physiologic oxygen levels. For example, while human ES cells will spontaneously differentiate when exposed to ambient oxygen, differentiation is suppressed when they are cultured under hypoxic (3-5% oxygen) conditions15. As another example, dental pulp stem cells (DPSCs) – which can be isolated from extracted teeth and have applications in tissue regeneration – grown under hypoxia (5% oxygen) have also been shown to exhibit higher expression of stem cell markers (CXCR4 and G-CSFR)17. Further, genes in the transcriptional pathway that regulates stemness, including Oct4 and Notch, are influenced by hypoxia-dependent signaling18.

----------------------

Hypoxia chambers can be useful tools to ensure that the oxygenation of cells is much closer to the in situ environment, however, gathering microscopic data from within a controlled environment can be a problem. The CytoSMART Lux2 mini live-cell imager fits inside hypoxia chambers and works via a remote control system. Gather data and operate the device without the need to take samples out of the hypoxia chamber.

----------------------

 

Assessing the quality of stem cell cultures

The use of hypoxia chambers allows researchers to carefully control the properties of stem cells, including their viability, yield, and potency, by providing them with a uniform, well-controlled oxygen environment. The quality of a stem cell culture can be assessed using both high-throughput brightfield microscopy (Table 1), as well as additional dye-, immunofluorescence-, flow cytometry-, and PCR-based assays. For example, iPSCs exhibit expression of several markers of self-renewal and potency, including Oct4, Nanog, and SSEA4, which can be assessed using flow cytometry19. For the purposes of this article, only methods compatible with brightfield microscopy will be discussed.

Table 1 | Methods for assessing the quality of a stem cell culture using high-throughput brightfield microscopy.

Quality parameterAssay

YeldCalculate the doubling time of the culture over multiple passages

ViabilityAssess cell death over time;
Use a dye exclusion assay (eg. trypan blue)

MorphologyMSCs: Small, spindle-shaped, fibroblast-like
IPSCS: High nucleus-to-cytoplasm ratio; prominent nucleoli;
uniform colonies with defined edges

HomogeneityUniform cell morphology within the culture

PotencyMSCs: Lineage-specific induction into adipocytes, osteocytes,or chondrocytes
IPSCs: Suspension culture to induce embryoid body formation/differentiation

 

Increasing stem cell yield and viability

First, for stem cells to be useful in regenerative medicine and tissue engineering, a sufficient number of cells must be isolated or obtained through subsequent passage. Thus, the cells must be viable and exhibit robust proliferation over multiple passages in culture. Cell viability can be assessed using morphology and dye exclusion assays, while proliferation can be examined by measuring the doubling time of the culture19.

Assessing cell morphology and homogeneity

Second, the morphology of the cells can be used as an indicator of stemness and culture health. In addition, the homogeneity of the culture, defined by the uniformity in cell morphology, can be assessed. The use of hypoxia chambers for stem cell culture can improve cell homogeneity by providing a uniform oxygen environment to the cells.

The morphology of MSCs, for example, is correlated with their capacity for self-renewal and differentiation potential, where smaller, spindle-shaped cells exhibit improved behavior14,17,20,21. Hypoxia can promote this morphology and therefore the viability, yield, and stemness of MSC cultures14. In contrast, MSC cultures with irregularly-sized, flattened, and enlarged cells likely represent cells with reduced self-renewal and differentiation potential.

Likewise, iPSCs should exhibit a high nucleus-to-cytoplasm ratio and prominent nucleoli, and will form uniformly-shaped colonies with dense centers and clearly defined borders22. iPSCs that are beginning to differentiate will form ill-defined colonies with darkened regions, and individual cells with endothelial- or fibroblast-like morphologies will break off from the colonies22.

Examining cell potency/differentiation capacity

Third, assays of stem cell functionality, and in particular the cells’ capacity for differentiation down distinct lineages, can also be used to determine the quality of a stem cell culture23. By culturing stem cells in a hypoxia chamber, researchers can carefully control the timing of these assays. Indeed, as indicated earlier, differentiation can be activated simply by exposing stem cells to elevated oxygen concentrations15.

The potency of MSCs can be assessed by exposing the cells to lineage-specific inducers and determining their ability to differentiate down osteogenic, adipogenic, and/or chondrogenic lineages24,25. MSCs that have been differentiated into adipocytes will enlarge, flatten, and develop prominent lipid droplets; in contrast, MSCs differentiated into osteocytes will flatten and take on an angular morphology7. Likewise, the potency of ES cells and iPSCs can be assessed using the embryoid body formation assay, where culturing the cells in suspension will drive the formation of three-dimensional aggregates that exhibit trilineage differentiation19.

 

The importance of maintaining a closed system in hypoxia culture

Ideally, cells that must be cultured under hypoxic conditions should remain in a hypoxia chamber at all times. The low oxygen environment in a cell culture dish is quickly lost when the culture plate is removed from its hypoxia chamber and exposed to ambient air; even opening the door of a hypoxia chamber for a brief period of time can affect culture oxygen levels26. According to Wenger et al., the medium in a standard 10 cm culture dish will require at least 38 minutes of incubation in a hypoxia chamber to drop from 20% to 2% oxygen, further highlighting the importance of reducing culture exposure to high oxygen environments26.

As cells respond to changes in oxygen levels within minutes16,26, the best course of action is to perform all procedures in a hypoxia chamber, including media changes, subculturing, and live-cell imaging. While the former two procedures may not be economically or practically feasible in all laboratories, remote microscopic analysis of stem cell cultures is. Live imaging can be performed using miniature microscopes that sit within the hypoxia chamber with the culture dishes, providing 24/7 remote imaging capabilities without disrupting the sensitive oxygen environment of the cells.

Learn more about the CytoSMART Lux2 mini live-cell imager.