Modelling oxygen levels in the lab
Our lives are tied to the air that we breathe. Without it, our cells will die.
Even so, some experiments require an intricate control of oxygen levels, even to hypoxia. Cells can encounter low oxygen conditions during infection and inflammation. Some cell types are also conditioned to low oxygen levels, such as skeletal cells where the oxygen fraction varies between 2% and 10%. Scientists have striven to recreate these conditions in the lab. While hypoxic chambers have been indispensable for advancing studies about hypoxia, they remain prohibitively expensive and take up substantial lab space.
That’s where Insimili comes in. After beginning as a spinoff from the University of Bologna, the company seamlessly combines chemistry and biology to recreate the oxygen levels in our bodies. Although they started out studying cancer, they have since expanded to mimicking oxygen levels for many other cell types. Their efforts led to Enrico Grassilli receiving this year’s SLAS Ignite Award for their array of products that control oxygen levels in the lab.
Now, you get a chance to hear from Enrico’s expertise in oxygen control. Read on to hear more about his products and check them out on their website! Â
The Interview
What are oxygen levels like in our bodies?
PN: Let’s start by talking about the environment that you’re trying to mimic. What are oxygen levels like in our bodies?
EG: Oxygen is an essential nutrient for all life. Within the powerhouse of the cell — the mitochondria — our bodies perform respiration to produce energy with oxygen. However, cells don’t take up oxygen at the same rate. The kinds of tissues, the distance from blood vessels, and the levels of oxygen available all affect how much oxygens cells take up to operate. For instance, oxygen levels at the inner side of the bone are low because fewer blood vessels are connected to it.
Oxygen levels also play a big part in how stem cells develop. The external oxygen levels we expose them to determines whether stem cells will differentiate as it’s designed to. Some types of stem cells proliferate and maintain its multipotent state so long as hypoxic conditions are maintained. Low oxygen levels can also stimulate cytokine production and encourage the formation of new blood vessels. So, as you can see, our oxygen levels are diverse, and we must mimic them properly to better understand how our cells work.
PN: That’s not to mention how oxygen levels in our bodies change when we experience disease.
EG: Exactly. Much of my specialty lies in cancer, and those tumors are something else. As these cells grow uncontrollably, they consume a lot of glucose, oxygen, and other metabolites. If they don’t have a continuous source of energy, they will suffocate and starve themselves. That’s why they try to pull in and make as many blood vessels as they can. In the process, they create hugely hypoxic environments. Although that sounds beneficial for suffocating the cells, cancer cells can resist therapy under these conditions.
How do we make recreating oxygen levels in the lab easier?
PN: Ensuring the correct oxygen levels in the lab ensures good research. How do scientists measure oxygen levels right now? What are the biggest shortcomings with measuring oxygen levels this way?
EG: Hypoxic hoods or incubators are the most common ways we have to control oxygen levels in the lab. If you’ve worked with a hypoxic chamber, you’ll know the process of removing oxygen from the chamber or adding other gases such as nitrogen to model hypoxic conditions. You’ll see microbiologists doing this to grow anaerobic bacteria. The same idea goes for scientists who want to expose human cell cultures to low oxygen levels.
However, when you work with a hypoxic chamber, you’re confined within the incubator. You can only grow your cells within these large machines, and you’ll have to spend considerable time preparing to enter the chamber. Moreover, the moment you take your cultures out, you’ll lose the hypoxic state you worked so hard to achieve. Then, your cells may experience oxygen shock. If they don’t die, they will change their phenotypes so much that they’ll become useless for your experiments. The lack of convenience and the risk of losing your hypoxic conditions makes it nearly impossible for labs and companies to scale up their workflows for higher-volume applications.
PN: This is where your technology comes in, I presume. Tell us how your products minimize the need for large apparatuses like hypoxic chambers while maintaining hypoxic conditions.
EG: Our team has a lot of knowledge in chemistry, material science, and biomaterials. With the tools on hand, we could measure oxygen concentrations in cell cultures. The data was so accurate that we could also calculate a cell’s respiration rate in each well we tested. With our know-how, we created a coating that we could apply directly to the wells. This coating contains proteins that mediate a chemical reaction that consumes glucose and releases glucuronic acid. In doing so, the media will have an oxygen gradient where the areas closest to the coating will have the lower oxygen concentrations. This concentration then increases the farther away you are from the bottom of the well.
PN: It sounds like you can create lots of unique configurations with your coating.
GE: Yes, we can. The coating patterns give us a lot of flexibility and potential for customization. For one, we can create multiple gradients across different wells. This setup gives researchers multiple layers of control when creating conditions of varying oxygen concentrations. The coolest part about our platform is that you can manipulate oxygen levels at both a 2D and a 3D space (Figure 1). You can imagine it as a semi-sphere where the oxygen levels are removed to certain extents within the semi-sphere. We can create these semi-spheres in 96-well and 384-well formats, which creates so many avenues of future research.
How can we integrate automation when modelling oxygen levels in the lab?
PN: That’s quite a creative design you’ve come up with! With automation rapidly becoming a major theme in the life sciences, how do you make this reaction reproducible and easy to automate?
GE: We go through many control experiments to ensure that we have exactly the correct oxygen concentrations within the wells. This helps us create specific configurations, like the Full Oxygen Gradient and Uniform Oxygen kits that can create oxygen gradients within a well or create environments with fixed oxygen concentrations respectively.
Various research groups have already worked with our plates. We’ve seen lots of interest from researchers specializing in stem cells and T cells and we’ve already helped them better understand how stem cells differentiate and how oxygen levels in our bodies affect our immune responses,
PN: To end things off, Congratulations on winning the SLAS Ignite Award! How do you feel winning the award, and how will it help Insimili achieve its mission to ease the creation of hypoxic conditions in the lab?
GE: Winning the SLAS Ignite Award is great. I was honoured to be the recipient because the other companies who presented were also amazing and had some quite outstanding products and teams behind them. I see this award as recognition that we’re working on something that is relevant and interesting for the research and lab automation community. SLAS has also given us a robust network of researchers with whom we can now discuss to make our vision a reality. All in all, we are still an early-stage company. This award will help us access a wide pool of clients and investors so we can gather funds and scale our solution further.
