Why Evercell Bio chose VIPS: a new workflow for hiPSC in cell therapy

Phil Manos Philip D. Manos, Founder and President of EverCell Bio, Natick, Massachusetts, USA
Our mission is to accelerate the implementation of human and disease-specific stem cell technologies for drug discovery and personalized medicine”

EverCell Bio is a single-source provider of customized services for the application of stem cell technology in cell therapy. EverCell offers human cell-modelling capabilities, such as donor cell line establishment, reprogramming, gene-editing and differentiation. When the decision was made to improve its iPSC workflows, EverCell chose Solentim’s Verified In-Situ Plate Seeding (VIPS™) solution for its single cell cloning capacity, image-based evidence of clonality and ‘double lock’ for regulatory submission. Philip Manos, President at EverCell, spoke to Emma Pickup, from Sciad Communications to explain why.

Evercell Bio is at the forefront of hiPSC technology. Can you explain why stem cell technology is such an important focus for your company?

“There is a significant need for stem cell therapies and more effective human cell models around the world and we feel that hiPSC (human-induced pluripotent stem cells) technology will play a critical role. I’ve been involved in the hiPSC field since its infancy and have experienced and observed many of the associated pain points and challenges. We feel that by improving access to the next generation of stem cell technologies we can lower the barrier to entry and increase the rate of adoption for stem cell therapies in personalized medicine and therapeutics. To achieve this, we’re very much focused on new and enabling technology for our service platform, such as the VIPS.”

As you look ahead to potential future uses of hiPSC in the cell therapy arena, what are the biggest challenges facing researchers today?

“Well, there are many! Two of the key challenges are the sensitivity of hiPSCs, meaning they require specialized media and matrices, and the poor survival/attachment of single hiPSCs without the aid of ROCK-inhibition. hiPSCs are generally required to be passaged as small clumps and cannot be cultured as single cells for extended culture. This creates barriers for in vitro strategies, such as gene editing, which require single cell manipulation. Moreover, hiPSCs are inherently variable, meaning ways of working with stem cells need to be as standardized as possible to secure meaningful data. However, current culture methods are almost entirely manual, leaving room for human error and presenting a significant technical challenge to standardize workflows.

With the addition of select small molecules it is possible to manipulate hiPSCs as single cells for a short period of time. However, in reality, much of our work with hiPSCs came down to manual, often tedious, methods and this opened up the need for automation. For example, in gene editing experiments, we could plate hiPSCs as single cells after delivery with a Rho kinase inhibitor, but i) we had to make the assumption that they were clonal and ii) we had to manually pick hundreds of clones for the downstream screening, overcoming major challenges with brute force and dedicated manual efforts. In order to scale-up this process, and to overcome these challenges, new workflow technology was needed.”

Implementing a new hiPSC workflow could have a big impact for EverCell Bio. Why did you choose Solentim’s VIPS instrument for your hiPSC workflow?

“While we are very selective in taking on projects involving instrumentation, I felt that with modern automation there would be real potential to significantly improve our hiPSC culture workflows. When we looked around the market, we found VIPS. The technology was convincingly robust with a number of validated applications already in existence, albeit in other cell types. Furthermore, Solentim’s focus on quality, standardization and data report generation aligns very well with EverCell’s overall strategy, mission and vision.

Knowing that Solentim is building appropriate reporting software within its instruments was really important for us. We build our lab processes around efficiency via quality which requires instrumentation that can accommodate the unique needs of hiPSCs. Standardization using an instrument such as the VIPS has a direct, positive impact on the quality of our biology and services. There may also be secondary benefits if we can harness the VIPS for specific assay readouts. Given the inherent variability within the hiPSC industry, any opportunity to standardize is valuable, particularly within a commercial setting, and especially when approaching more translational or therapeutic aims.

Solentim’s instruments are developed with evidencing clonality in mind. This will be key in building a regulatory structure for the future of stem-cell therapies, securing reliable growth and demonstrating clonal derivation. Starting with the end in mind, it is good to know that the hiPSC development we do at this early stage will be documented appropriately for any potential IND submission further down the line. As the interest in therapeutic hiPSCs grows, I see VIPS becoming the standard to manage effective growth and assurance.”

Having worked with Solentim on your hiPSC workflow, how did the VIPS iPSC compare to other cell seeding and imaging technologies?

“VIPS seemed unique and far ahead when it comes to single-cell validation and the accompanying clonal regulatory reports. In areas where technical capabilities can be compared against other imaging or cell seeding technologies respectively, the AI-driven automatic single-cell detection stands out from most systems that we have used. The VIPS is also easy to use and generates high quality, high confidence data.

I was also pleased with the ‘double lock assurance’ offered by Solentim, achieved by combining in-well single-cell verification imaging with outgrowth and clonal characterization imaging from day 0. This is documented in the Clonality Report, making it easy to review and share.”

You mentioned earlier that the sensitivity of hiPSC requires special matrices. How did Solentim’s MatriClone™ matrix enable automation of the hiPSC workflow?

“We were very pleased with the MatriClone. It was easy to work with and supported very healthy hiPSC cultures for expansion and sub-cloning. It is a defined reagent which is a benefit over the commonly used Matrigel. Matrigel is derived from mouse cancer cell lines which is non-defined and not xeno-free. This only adds to the inherent variability within many hiPSC culture systems and requires us to perform more rigorous lot reserve testing. Moreover, MatriClone can be added in the cell suspension when seeding using the VIPS, rather than requiring pre-coating of the plates.  This allows hiPSCs to be plated using the VIPS and also saves a lot of time in preparation. The combination of VIPS and MatriClone proved ideal for our hiPSC workflows.”

How much optimization was required by EverCell Bio for the technology to work optimally?

“Due to the sensitivity of hiPSCs, we had to adapt cell culture conditions to work optimally in the VIPS system. We also optimized balancing the cell concentration for seeding cells with the right droplet parameters. After optimizing just these two steps we saw significant improvements over our previous manual methods.”

EverCell Bio offers a wide range of stem cell services. Which areas do you imagine will benefit most significantly from the implementation of Solentim’s new workflow solution?

“Potentially quite a few, but we feel that our hiPSC gene-editing workflows will be the first to benefit from the VIPS. It is in this workflow where both single cell plating and manual culture/screening are required of hiPSCs, highlighting the two major challenges discussed earlier. We foresee the VIPS system aiding us in speed, quality and clonal confidence for gene editing-related projects.”

Do you have any additional data you can share on the following factors?

Yes, absolutely. These are the specific areas where we saw significant improvement:

Variable Data
Seeding efficiency When optimized, we were achieving around 70-75 % seeding efficiency
Outgrowth Range of 40%-60% successful clonal outgrowth from confirmed single cells
Evidence of clonality We were really impressed with the AI and ability to track single cells during seeding and outgrowth. Having the images to be able to visually confirm was critical.
Retention of pluripotency / karyotype All clones assessed from over four different hiPSC lines, some being disease affected, retained pluripotency and maintained healthy karyotypes throughout the VIPS subcloning process.
Sub-clone quality We could be confident of the sub-clones’ quality and traceability for downstream biology
MatriClone MatriClone allowed for increased cell outgrowth compared to Matrigel, cutting the time required for subcloning by almost 50%

Finally, would you recommend this technology to others working with hiPSCs and why?

“While we are currently validating other applications, which would broaden its reach, I’d absolutely recommend the VIPS now for any labs looking for true clonal studies or cell line development using hiPSCs. With both the seeding robustness and data reporting, this system simplifies the process and strengthens the quality of the biology for cell therapy development.”