Sixty years after its arrival, one molecule still dominates cell cryopreservation: DMSO. It’s unmatched in its ability to get cells through the freeze–thaw cycle alive, and its clinical track record is extensive. Yet beneath the surface of every viable vial lurk toxicity risks and a legacy of side effects that have regulators and innovators hungry for change.

Why hasn’t DMSO been dethroned—and what’s finally threatening its reign?

Joining David Brühlmann is Steve Oh, whose 22 years at Singapore’s A*STAR produced 43 patents, breakthroughs in stem cell microcarrier technologies, and hard-won expertise on the toughest bottlenecks in bioprocessing. Steve Oh isn’t just theorizing about better cryoprotectants—he’s lived the old problems and is now advising startup trailblazers trying to solve them for good.

Key Topics Discussed

• Steve Oh’s career reflects major industry shifts, from early antibody production challenges to advanced stem cell bioprocessing.
• His transition into entrepreneurship highlighted the gap between scientific innovation and market readiness in cell therapy.
• Cryopreservation remains a critical bottleneck across bioprocessing, with broad applications beyond cell therapy.
• DMSO persists as the standard cryoprotectant due to reliability, despite known toxicity and regulatory concerns.
• DMSO negatively impacts cellular function, affecting potency, metabolism, and therapeutic performance.
• Molecular-level effects of DMSO include altered gene expression and DNA methylation, with uncertain long-term consequences.
• New cryopreservation approaches, including antifreeze protein mimics and alternative formulations, aim to improve safety and efficacy.

Episode Highlights

• How antifreeze protein-inspired peptide chemistry reduces ice crystal size and protects cells during freezing and thawing [00:23]
• T cell and MSC performance data comparing XT Thrive to DMSO and CryoStor CS10, including a 2.5-fold increase in cell yield on microcarriers post-thaw [02:23]
• Why ice crystal formation causes more damage during thawing than freezing, and how XT Thrive addresses this [05:32]
• Elimination of the post-thaw wash step and what that means for contamination risk and manufacturing simplicity [07:00]
• Hold time extended to 24 hours and storage performance across 4°C, -80°C, and -196°C [08:01]
• Applicability beyond single cells: organoids, islets, and potential implications for organ preservation [09:50]
• How to transition from DMSO to XT Thrive: GMP grade, Drug Master File, same concentration, no protocol overhaul required [10:49]
• Broader cell therapy challenges: differentiation time, cell population consistency, and cost of goods [12:03]

In Their Words

DMSO has been the gold standard because of its unique chemical properties and extensive clinical track record, making it difficult to fully replace. But that's a 60-year-old product, and a lot of things have changed since then. So some of the properties are as follows: physicochemical efficiency. It has rapid membrane permeability, and it enters the cells and equilibrates with the intracellular environment faster than other penetrating agents like glycerol. Water displacement: it forms hydrogen bonds with water about 30% stronger than between water molecules themselves, thereby preventing the formation of intracellular ice crystals.

DMSO in Cell Therapy: Why Viability Scores Hide the Real Toxicity - Part 1

David Brühlmann [00:00:42]:
For 60 years, one molecule has defined cell cryopreservation—effective enough to become the universal standard, yet problematic enough that the FDA attempted to ban it twice. My guest today, Steve Oh, spent 22 years at Singapore's A*STAR, invented stem cell microcarrier technologies, CRISPR activation, and a lot more. He holds 43 patents and has seen this problem from every angle. Steve brings hard-won wisdom to one of bioprocessing’s most persistent challenges. Why has DMSO survived this long, and what finally threatens its reign? Let's find out!

Welcome, Steve. It's good to have you on today.

Steve Oh [00:02:50]:
Thank you, David, for the invitation.

David Brühlmann [00:02:52]:
Steve, share something that you believe about bioprocess development that has made the most impact.

Steve Oh [00:02:59]:
Okay, love to. I think there are 3 that I can think of right now. The development of the AMBR system for small-scale volume optimization that has made a lot of impact in the biologics industry. The development of high-intensity perfusion cultures that has reduced the volume and the footprint of bioreactors for generating high titers of proteins, recombinant antibodies. And I think one that's coming in the future is the use of digital twins to reduce the number of experiments and then focus on the key experiments. I believe you're going to be talking about that in the future, right?

David Brühlmann [00:03:38]:
Yes, absolutely. And we have already covered this topic—digital twins and hybrid modeling—a few times on previous episodes. So if you're interested in that, Smart Biotech Scientists, go back and listen to these episodes. It's a huge pleasure to have you back on, Steve. For those listeners who have not had a chance to listen to our interview we did, I think a couple of years back, take us back to the beginning. What sparked your passion for biotech and cell therapy, and what pivotal moments during your long career shaped your path to becoming a leader in stem cell bioprocessing solutions?

Steve Oh [00:04:17]:
I think back in the '90s, when the production of antibodies was challenging, I had the opportunity to complete my PhD and get into learning about the use of bioreactors for manufacturing cells, which at that time was a big challenge. People were doing bacterial fermentation but not animal cells. So that was the first pivotal moment that gave me the training and the opportunity to get into this field.

And then around 2001, post the discovery of pluripotent embryonic stem cells, again that was a challenge—how to produce stem cells at scale in bioreactors. We built a small team to look at growing these cells, which were very dependent on support cells—feeder cells—and Matrigel, all kind of undefined conditions. And finally, we found a way to switch them to grow on microcarriers in bioreactors. So those were the two big opportunities I had to make an impact in bioprocessing and come up with new solutions to scale.

David Brühlmann [00:05:22]:
And what were some pivotal moments you experienced during your long years at A*STAR that finally led you to also take a leap and pursue entrepreneurial endeavors?

Steve Oh [00:05:32]:
After the discovery of microcarriers for manufacturing stem cells, I did try my hand at forming a spin-off company, but the challenge was raising cash to build a CDMO-type business model and then finding a CEO. So my entrepreneurial skills were not that strong, and we didn't succeed. I'm glad to see that there are companies out there now that are able to make that impact. Still, no products made from bioreactors yet, but people are trying.

David Brühlmann [00:06:03]:
Wow, that's fantastic. You hold a lot of patents—exactly, if I'm not mistaken, 43 patents across microcarrier technologies, serum-free media, and CRISPR activation. So you could have focused on many different areas. What drew you to cryopreservation as one of your key advisory roles? And tell us also what made you believe that this was an area ripe for innovation.

Steve Oh [00:06:31]:
It was in collaboration with a company called X-Therma back in 2015, I believe—or maybe a bit later—where I saw that they had this amazing solution that could transport whole hearts across the country over 3 days. And we had seen there were challenges with DMSO for cryopreservation of pluripotent stem cells and the first challenging differentiated cell type—neural stem cells. So I approached the CEO, Xiaoxi, and looked at potentially testing out that solution for stem cell applications. And I realized that in the final cell therapy, you wouldn't be giving fresh cells—you'd have to freeze them down and use the thawed cells for patient injection.

So we needed something that would be robust and give potent cell properties as close to fresh cells as possible. And really, nobody at that time was looking at the replacement of DMSO, the standard solution. So that's why I felt that there was a blue ocean opportunity to collaborate with them. And then once I left A*STAR about 4 years ago, I sought the opportunity to be a scientific advisor. That's why I'm working with X-Therma on educating the field in alternatives to DMSO.

David Brühlmann [00:07:48]:
Let's start with the fundamentals, Steve, because cryopreservation—or shall I say the preservation of cells—is a big, big topic, not only in cell therapy, but I'd say in various areas of biologics. Help our listeners understand what are the main challenges scientists face when preserving cells, freezing cells, and why has DMSO remained the gold standard despite its limitations for many decades?

Steve Oh [00:08:17]:
So DMSO has been the gold standard because of its unique chemical properties and extensive clinical track record, making it difficult to fully replace. But that's a 60-year-old product, and a lot of things have changed since then. So some of the properties are as follows: physicochemical efficiency. It has rapid membrane permeability, and it enters the cells and equilibrates with the intracellular environment faster than other penetrating agents like glycerol.

Water displacement: it forms hydrogen bonds with water that are about 30% stronger than between water molecules themselves, thereby preventing the formation of intracellular ice crystals. It's versatile. In the pre-cell therapy days, hematopoietic stem cells (HSCs) were mostly the cell type used in cryopreservation—blood products, some immune cells, and mesenchymal stromal cells (MSCs).

And the HSCs have been used in clinical and regulatory environments—so lots of stem cell transplants. Its side effects are documented and manageable within existing medical protocols, such as maximum daily dose limits, typically 1 gram per kilogram of patient weight. There are standardized protocols and workflows built around the 5–10% DMSO formulation. Transitioning to alternatives requires extensive and costly validation to ensure that therapeutic efficacy is not affected.

Then the practical and economic advantages include low cost, high stability, long shelf life at low temperatures, and wide availability in high-purity grades—such as United States Pharmacopeia (USP) grade for clinical use. And it's easy to use in controlled-rate freezers at about −1°C per minute, so fairly simple to implement. So that's the reason why it has been in use for 60 years.

David Brühlmann [00:10:14]:
What I'm hearing here, Steve, is that DMSO has a lot of advantages, and that's the reason why it has been used for so long. Isn't it interesting that the FDA has tried to ban DMSO twice? So what are the specific issues with the toxicity of DMSO, and what are some other aspects that make its use problematic?

Steve Oh [00:10:39]:
One example was back in 1965—there was a ban on human testing because of safety alarms regarding lens (eye) toxicity in animals, and then a sudden death of an Irish woman after topical use. So these early concerns established regulatory wariness that persists today.

Now, for modern cell therapies, there are new issues that we've never had—the challenges of the variety of cell types now being used. This includes cellular dysfunction and loss of potency. For T cells, at concentrations as low as 0.25–1%, DMSO inhibits CD4-positive T cell activation, proliferation, and cytokine production such as IL-2, IL-4, and IL-17A. It downregulates genes in early signaling and T-cell receptor pathways.

Then for NK cells, DMSO is associated with altered expression of natural killer cell markers and diminished effector functions. For stem cells, DMSO is known as a differentiation inducer, so it can downregulate pluripotency factors like OCT4 and NANOG at concentrations as low as 0.125%, potentially biasing their therapeutic identity before they reach the patient.

In terms of physical and structural damage, DMSO induces pore formation in cell membranes and can disrupt the cytoskeleton by dehydrating lipids and interacting with proteins. In terms of mitochondrial and metabolic stress, it can compromise mitochondrial respiration, induce oxidative stress, and trigger apoptosis through caspase-9 and caspase-3 activation.

And then in patients, DMSO toxicity significantly increases dose-dependent cell damage and adverse reactions. Clinical risks include cardiovascular issues, severe nausea, neurological symptoms, and allergic reactions, which necessitate rapid removal or lower concentrations.

David Brühlmann [00:12:42]:
Let me reframe this because you're making an excellent point here, Steve. I have worked in biologics for most of my career, and DMSO is used to freeze cell banks. And pretty much the only parameter many teams were interested in is viability when you thaw your cells.

But now what I'm hearing is that in cell therapy, we are playing a completely different ball game. Because we're not just cultivating cells in a bioreactor—we are using these cells to treat patients. So we have to look much more carefully at what's going on at the genetic level, at the transcriptomics level, and at the metabolic level. So we have a plethora of aspects to watch out for.

Steve Oh [00:13:28]:
Correct. Because the cells are the functional entities, just having viable cells is not sufficient. You have to have the cells be able to perform the function that they were intended for, as they would when fresh.

David Brühlmann [00:13:40]:
Can we look into the cells a bit? What's happening when you're using DMSO and you're freezing and then thawing the cells? What exactly happens at the microscopic level? What are these changes that affect the genetic stability, the transcriptomics, or the metabolism of these cells?

Steve Oh [00:14:01]:
Some of the toxicity issues in cryopreservation are that when cells are exposed to DMSO at temperatures above 4°C during thawing, it can disrupt the cellular membrane, cause mitochondrial damage, and lead to the production of reactive oxygen species. So this is cumulative, and it can start to create all those metabolic damages that I mentioned earlier.

On top of that, exposure to DMSO can lead to undesirable phenotypic changes in stem cells due to alterations in DNA methylation and histone-modifying enzymes that can open up the DNA for priming towards differentiation or close it down such that they can't differentiate.

Then there is the time- and dose-dependent risk. Toxicity is directly related to concentration and exposure time. Typically, DMSO is used in the 5–10% range, but even up to 40% DMSO has been used, and this completely destroys hematopoietic stem cell viability. And then, as I mentioned earlier, when residual DMSO is infused into patients, you can have nausea, vomiting, cardiovascular events like bradycardia and arrhythmia, neurological symptoms like seizures and dizziness, and allergic reactions.

And finally, in terms of manipulating the DMSO itself, because of these toxicity issues, it needs to be washed away to remove residuals and reduce the overall volume to decrease the dose. This can lead to potential opportunities for contamination of the product by having to do this additional step.

David Brühlmann [00:15:38]:
To what extent can these detrimental effects happen even though the viability looks okay?

Steve Oh [00:15:46]:
So even at low concentrations post-wash, the types of disruptions that can happen are as follows: Alterations in DNA methylation: DMSO disrupts the balance between the “writers” and “erasers” of DNA methylation, leading to widespread genomic instability.

• On one hand, hypermethylation—DMSO can upregulate methyltransferases like DNMT3A or DNMT3B, leading to global hypermethylation in cell types such as fibroblasts or embryoid bodies. This reduces transcriptional activity by silencing genes.

• Then the opposite can also happen—hypomethylation. DMSO can induce active demethylation by promoting TET family enzymes such as TET1 and TET2, which convert 5-methylcytosine to 5-hydroxymethylcytosine.

The major risk in preserving stem cells and related products is that we don’t fully understand how these methylation changes will affect the long-term behavior of the cell product.

David Brühlmann [00:16:48]:
Given what we've discussed so far, Steve, what are the options we have at our disposal as scientists? Because we could use DMSO, but there are some detrimental effects you just mentioned, and we have to be aware of that. And there are now some alternative products. So what are these products scientists listening can use?

Steve Oh [00:17:10]:
So there are a couple of approaches that have been taken. Companies have looked at trying to reformulate alternatives to DMSO using other small molecules like glycerol and additional cryoprotective agents. These are complex formulations that have to be optimized for each cell type.

Then there are companies like X-Therma, which have come up with antifreeze peptide mimics—essentially single-molecule solutions that aim to do the same thing as DMSO but with processing benefits and without the significant toxicity seen with DMSO. So those are the two approaches: either complex formulations or a completely new entity to replace DMSO.

David Brühlmann [00:17:52]:
And looking at X-Therma, I saw that you took inspiration from antifreeze proteins found in Arctic fish. How did this come about? Explain how these specifically protect the cells from freezing damage.

Steve Oh [00:18:06]:
So these antifreeze proteins improve cryopreservation by binding to ice crystals, inhibiting their growth and recrystallization, and reducing damage to cells at sub-zero temperatures. They were derived from cold-adapted organisms and create a thermal hysteresis gap that lowers the freezing point without affecting the melting point, thereby protecting biological samples and enhancing survival rates.

Essentially, how it works is that they cluster around water molecules and prevent the rapid growth of crystals. Because of these much smaller crystals, they don’t damage the cellular structure either when frozen or during thawing. They can return to the liquid state more smoothly than large crystals would, even without traditional cryoprotectants.

David Brühlmann [00:18:55]:
We've just unpacked why DMSO has persisted for six decades despite its well-documented limitations—and why that persistence has real consequences for your cells, your patients, and your manufacturing process.

In part 2, Steve Oh takes us into the solutions—the science behind next-generation cryoprotectants, the data that's turning heads, and what a DMSO-free workflow actually looks like in practice.
If this episode added value, please leave a review on Apple Podcasts or your preferred platform. This enables more scientists like you to discover the show. Thank you for tuning in today—see you next time.

For additional bioprocessing tips, visit us at www.smartbiotechscientist.com. Stay tuned for more inspiring biotech insights in the next episode. Until then, let’s continue to smarten up biotech.

Disclaimer: This transcript was generated with the assistance of artificial intelligence. While efforts have been made to ensure accuracy, it may contain errors, omissions, or misinterpretations. The text has been lightly edited and optimized for readability and flow. Please do not rely on it as a verbatim record.

Next Step

Book a free consultation to help you get started on any questions you may have about bioprocess development: https://bruehlmann-consulting.com/call

About Steve Oh

Steve Oh is a biotechnology leader with over 35 years of international experience spanning academia and industry. He spent 22 years at A*STAR in Singapore as an Institute Professor at the Bioprocessing Technology Institute, where he pioneered innovations in stem cell bioprocessing, including microcarrier systems, serum-free media, and gene activation technologies. 

He holds 43 patents, has published over 150 scientific papers, and has led more than $34 million in research funding. Today, Steve serves as a scientific advisor to multiple global biotech companies, supporting advances in gene therapy, cryopreservation, and cell manufacturing.

Connect with Steve Oh on LinkedIn.

Further Listening

If you’re interested in this topic, check out these episodes, where we explore how Minnesota’s frozen forests inspired a new wave of biotech innovation, transforming how life-saving cells are frozen, stored, and shipped.

Episodes 161 - 162: How to Achieve 85%+ Cell Recovery Without DMSO's Toxic Side Effects with Jeffrey Allen

This is Steve’s second appearance on the podcast. You can also catch his earlier conversation with David, where they explored the challenges and opportunities of cell and gene therapy.

Episodes 11 - 12: From Lab to Patient: Steve Oh’s Guide to Mastering Cell Therapy Process Development.

He is also a biotech technology innovation coach, technology transfer leader, and host of the Smart Biotech Scientist podcast—the go-to podcast for biotech scientists who want to master biopharma CMC development and biomanufacturing.  

Hear It From The Horse’s Mouth

Want to listen to the full interview? Go to Smart Biotech Scientist Podcast. 

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