Category: Cell Therapy

Demand for human mesenchymal stem cells (hMSCs) is increasing dramatically as numerous cell therapy, tissue engineering, and related products advance through clinical trials. Greater efficiency and productivity in GMP manufacturing will be essential to produce sufficient quantities of hMSCs at an acceptable cost for patients and payers.

Critical Raw Material

Human mesenchymal stem cells (hMSCs) are a critical raw material for many regenerative medicine products including cell therapies, engineered tissues like bones and organs, and, crucially, cell-derived products, such as extracellular vesicles and growth factors. This mammalian cell type is also instrumental in ongoing research to 3D-model diseases ex vivo or to reconstitute skeletal muscle tissue for so-called “clean” or cruelty-free meat. 

A few cell therapy products have received regulatory approval around the world, and others are rapidly advancing through late-stage clinical trials. Bioprinted bones and organs and meat grown from animal muscle precursor cells derived from MSCs will require more research and technology advances to be realized, but the progress made to date suggests that multi-application success is likely to be achieved in the next decade or so.

Focusing on therapeutic applications, hMSCs have been identified for the potential treatment of many different diseases. Bone and cartilage and cardiovascular diseases and diabetes account for the largest percentage of clinical trials, but others receiving attention include Crohn’s, Alzheimer’s, hematological, lung, liver, kidney and host-graft diseases, spinal cord injury, multiple sclerosis, cancer, and many more.

A Few Growing Pains

Like other technologies, hMSC development has followed what is known as the Gartner hype cycle, in which an “innovation trigger” leads to a “peak of inflated expectations” followed by a “trough of disillusionment” and then a “slope of enlightenment” leading to a “plateau of productivity.”  The hMSC hype cycle can be seen in Figure 1.¹

Results of early studies with hMSCs led to significant interest and investment in the first decade of the 2000s. Nevertheless, owing to variable manufacturing practices, differences in isolation techniques, an underappreciation of the impact of culture methods on final product quality, a lack of understanding of hMSC mechanisms of action, and poor potency assays, there have been initial failures and limited adoption. Yet in recent years, manufacturing advances, greater knowledge gained from basic and applied research, and confirmation of therapeutic properties and MSCs’ mechanisms of action led to renewed interest and the implementation of many clinical trials. 

A Technology Akin to the Microchip

Given their widespread therapeutic potential for treating many indications and their usage in a variety of applications, hMSCs can be considered the “microchips” of tomorrow’s regenerative medicine products. Initially used as a biological tool to understand cellular mechanisms, hMSCs today have become fundamental components of regenerative medicine products and are needed in large quantities at pharmaceutical grade (Figure 2). On the basis of a review of the literature and clinical trial data, it has been estimated that the average academic non-clinical or preclinical study consumes just 43 million hMSCs, while a typical cell therapy clinical trial requires 60 billion hMSCs.² Today, there are greater than 10 trillion MSCs manufactured globally per year to support these trials.  

There is interest in accelerating the development of hMSC-based products from the U.S. Food and Drug Administration (FDA). With approvals for these therapies and engineered tissues already granted outside of the United States, a strong signal of therapeutic efficacy reported in early-stage trials, and growing momentum toward later-stage clinical trials, the potential for growth of hMSCs is tremendous. Indeed, their use in therapeutic applications is increasing exponentially in a manner similar to that observed for computing power.³

Estimating the Future Demand for hMSCs

In order to estimate the future demand for hMSCs, Olsen, et al.² considered the potential for success of different hMSC-based products and the quantity of cells required for each (Figure 3).

For cell-therapy products, given that there were 46 phase II/III and phase III clinical trials using hMSCs as of November 2017 and that the FDA estimates that 25–30% of phase III clinical drugs will receive approval,⁴ it can be conservatively assumed that 10 MSC-based cell therapy products will be on the U.S. market by 2030.  Further assuming there will be two “low-dose” products with 2 million cells/dose, five “medium-dose” products with 100 million cells/dose, two “high-dose” products with 500 million cells/dose, and one “monster-dose” product with 1.5 billion cells/dose and 100,000 patients per each if the 10 indications, 300 trillion hMSCs would be required per year to satisfy this demand. Of course, if any of these indications involved large patient populations, such as diabetes or stroke, this estimate could increase by 10-fold or more.²

Estimates for engineered tissues and organs were based on research involving the biofabrication of half an adult femur, which requires 720 million hMSCs,⁵ the number of patients requiring replacement bones per year and data regarding organ transplants. Assuming that 185,000 upper and lower limb amputations are performed per year in the United States,⁶ with each full bone requiring approximately 1.5 billion hMSCs, 278 trillion cells would be needed per year to fulfill the demand for the all the patients requiring replacement bones.

While creating a functional organ via bioprinting remains a future target, it will eventually be achieved and will address a critical need for the more than 100,000 people in the United States alone waiting to receive organ transplants.⁷ Using the liver as an example, stem cells are known to be a precursor to the non-parenchymal cells (endothelial cells, Kupffer cells, and stellate cells) that make up 40% of the liver⁸ and play a functional role in liver maintenance and regeneration.^9,10 In addition, only half of a human liver is required to support life and is suitable for transplant,¹¹ and acute liver failure patients can be supported with 5–10% liver mass or a bioartificial liver composed of 10 billion cells.^12,13 Assuming conservatively that 25% of the non-parenchymal cells in a tissue-engineered liver will be hMSCs, 1 billion hMSCs would be required per liver, and thus bioprinting 6,427 livers, which was the number of people at the end of 2016 waiting for a liver,⁶ would require 6.427 trillion hMSCs.

With liver transplants accounting for approximately 12% of all organ transplants⁷ — and using the same assumptions of 40% nonparenchymal cells in solid organs generated using 25% hMSCs — an additional 10-fold multiplication factor would be needed to satisfy all organ manufacturing and provide for off-the-shelf organs, or a total of 64 trillion cells.

Extracellular vesicles (EVs), such as exosomes or microvesicles collected via hMSCs, have been shown to be quite potent^14–16 and under some conditions can elicit similar responses to whole hMSCs.¹⁷  Along with gene-modified or edited cells and therapies that leverage synthetic biology, EVs can be referred to as “MSC 2.0” products.² Given their history of clinical use without significant adverse events in controlled and monitored studies, hMSCs are a relevant cell source valued for their potential in accelerating translation of EV therapies. Limited data are available on the quantity of hMSCs necessary to provide a therapeutic dose of hMSC-EVs, although an equivalent or greater amount of hMSCs for treatment appears reasonable. The same demand as for hMSC cell therapy products — ultimately, 300 trillion hMSCs per year — is therefore assumed over the next 20 years. This estimate is once again conservative, because it does not include the hMSCs required to generate other cell-derived materials, such as cytokines and cell lysates, both of which have shown therapeutic promise.^18–20

Cytokines and growth factors derived from hMSCs as biological ingredients in so-called “cosmeceuticals” or bioprinted human skin models for animal-free testing of cosmetics and personal care products are both being explored by companies such as L’Oreal and Johnson and Johnson.²¹ Cruelty-free, “clean meat” produced from non-human MSCs, if it were to address the predicted total meat consumption, would require approximately 1.25 ×10²² cells for bioreactor inoculation.²²

Peak hMSC Demand Predicted in 2040

The staggering growth in demand for hMSCs is similar to that experienced for monoclonal antibodies (mAbs) over the first two decades of their development. As a result, it can be expected — as was observed with mAbs — that a variety of approved products will be on the market over the next 20 years as the industry moves toward peak MSC demand and consumption. Indeed, more than 50 products are predicted to be on the market by 2040, with approximately four products added each year — a trajectory similar to that taken by mAbs.²

“Cosmeceutical” products could have success initially outside of the United States, owing to the interpretation that cell products (and not the cells themselves) are the active product. Similar clinical products containing MSC-derived, “secretome”-based materials will also eventually be common (Figure 4). With FDA approvals following successful phase III trials, applications of topical MSC-derived products could find potential for discretionary, “off-label” applications in dermatologic and/or cosmetic uses by prescription. Several cell therapies based on hMSCs should be on the market by 2025, and tissue-engineered products will receive approvals by 2030. By 2040, biofabricated organs will be in clinical trials. A summary of predictions is presented in Table 1.

Success Predicated on Manufacturing Advances

Realizing this full potential will require manufacturers to develop new technologies and processes that deliver high-quality hMSCs in massive volumes—and at radically lower costs. To become a truly abundant critical therapeutic material that enables innovation at the highest level, novel manufacturing solutions will be required to support commercial manufacture of hMSC products.  

Particular technical speed bumps have challenged the industry to determine a workable trypsin- and agitation-free release step from the microcarriers used to support the MSCs in order to minimize cell damage. Quenching of the proteolytic enzymes is often achieved with 10% fetal bovine serum, an animal-based product that introduces significant contamination risk. Furthermore, to isolate the suspended cells in an efficient and automated way, one must rapidly concentrate the cells while maintaining cell viability and functionality, a step necessitating filtration of the cells at large scale.² 

Yet, much recent progress has been made, such as the development of new, gentler dissociation reagents and animal-free materials, now manufactured according to Good Manufacturing Practice (GMP) requirements. Completely dissolvable microcarriers are also being explored, which would eliminate the need for cell/microcarrier separation and streamline downstream processing.²³

Driving Down Cost

Currently, there is a reasonable supply of high-quality hMSCs produced using robust manufacturing processes — prerequisites for clinical trials — but they come at a high cost. Scalable platform technologies are needed to economically fulfill clinically and commercially relevant lot sizes.² An intermediate step for reducing time to the clinic will be the production of hMSCs with proven manufacturability and clinical-grade quality.

Despite the manufacturing challenges that still exist today, companies like RoosterBio^® are increasing access to commercially-relevant hMSC cells through supply chain industrialization. RoosterBio has developed standardized cell bank product forms, coupled with scalable manufacturing, that includes fit-for-purpose cGMP-compatible cells and media systems supported by an FDA Master File.

Such solutions enable production of hMSC products in large-scale 3D bioreactors for streamlined clinical translation and reduced time to GMP product manufacturing. Overall, access to standardized, fully tested “off-the-shelf” cell banks as manufacturing starting materials for direct use in streamlined manufacturing processes provides product developers with reduced development and go-to-market timelines and, ultimately, reduced costs.²⁴

As manufacturing breakthroughs enable larger-scale production to increase the availability of hMSCs and drive down their cost due to efficiencies of scale, further innovation will follow (Figure 5).

References

1. Lembong, Josephine, Jon Carson and Jon Rowley. “The Stabilization of hMSCs as a Technology.” RoosterBio^® White Paper. 2020.
2. Olsen, Timothy R., Kelvin S. Ng, Lye T. Lock, Tabassum Ahsan and Jon A. Rowley. “Peak MSC—Are We There Yet?” Frontiers in Medicine. 5:178 (2018).
3. Maris B. “Medicine’s transistor moment: 8 emerging technologies that could revolutionize the life sciences.” Medium (2015) Available online at: https://www.speakingtree.in/article/medicine-s-transistor-moment 
4. The Drug Development Process. (2015) Available online at: http://www.fda.gov/forpatients/approvals/drugs/default.htm
5. Nguyen, BNB, Ko H, Moriarty RA, Etheridge JM, Fisher JP. “Dynamic bioreactor culture of high volume engineered bone tissue.” Tissue Eng. Part A. 22:263–71 (2016).
6. Ziegler-Graham, K, MacKenzie EJ, Ephraim PL, Travison TG, & Brookmeyer R. “Estimating the Prevalence of Limb Loss in the United States: 2005 to 2050.” Phys. Med. Rehabil. 89:422–9 (2017).
7. Organ Donation Statistics. U.S. Department of Health and Human Services. 2017. Available online at: https://www.organdonor.gov/statistics-stories/html
8. Godoy P, Hewitt NJ, Albrecht U, Andersen ME, Ansari N, Bhattacharya S, et al. “Recent advances in 2D and 3D in vitrosystems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME.” Arch Toxicol. 87:1315–530 (2013).
9. Chan C, Berthiaume F, Nath BD, Tilles AW, Toner M, Yarmush ML. “Hepatic tissue engineering for adjunct and temporary liver support: critical technologies.” Liver Transplant. 10:1331–42 (2004).
10. Wang B, Zhao L, Fish M, Logan CY, & Nusse R. “Self-renewing diploid Axin2(+) cells fuel homeostatic renewal of the liver.” Nature. 524:180–5 (2015).
11. Clavien PA, Petrowsky H, DeOliveira ML, & Graf R. “Strategies for safer liver surgery and partial liver transplantation.” Engl. J. Med. 356:1545–59 (2007).
12. Bianconi E, Piovesan A, Facchin F, Beraudi A, Casadei R, Frabetti F, Vitale L, Pelleri MC, Tassani S, Piva F, et al. “An estimation of the number of cells in the human body. Hum. Biol. 40:463–71 (2013).
13. Chan C, Berthiaume F, Nath BD, Tilles AW, Toner M, & Yarmush ML. Hepatic tissue engineering for adjunct and temporary liver support: critical technologies. Liver Transplant. 10:1331–42 (2004).
14. Von Bahr L, Batsis I, Moll G, Hägg M, Szakos A, Sundberg B, et al. “Analysis of tissues following mesenchymal stromal cell therapy in humans indicates limited long-term engraftment and no ectopic tissue formation.” Stem Cells. 30:1575–8 (2012).
15. Gnecchi M, Zhang Z, Ni A, & Dzau VJ. “Paracrine mechanisms in adult stem cell signaling and therapy.” Res. 103:1204–19 (2008).
16. Ng KS, Kuncewicz TM, & Karp JM. “Beyond Hit-and-Run: Stem Cells Leave a Lasting Memory.” Cell Metab. 22:541–3 (2015). doi:
17. Rani S, Ryan AE, Griffin MD, & Ritter T. “Mesenchymal stem cell-derived extracellular vesicles: toward cell-free therapeutic applications.” Mol Ther. 23:812–23 (2015).
18. Liang X, Ding Y, Zhang Y, Tse HF, & Lian Q. “Paracrine mechanisms of mesenchymal stem cell-based therapy: current status and perspectives.” Cell Transplant. 23:1–32 (2013).
19. Parekkadan B, Van Poll D, Suganuma K, Carter EA, Berthiaume F, Tilles AW, et al. “Mesenchymal stem cell-derived molecules reverse fulminant hepatic failure.” PLoS ONE. 2:e941 (2007).
20. Albersen M, Fandel TM, Lin G, Wang G, Banie L, Lin CS, et al. “Injections of adipose tissue-derived stem cells and stem cell lysate improve recovery of erectile function in a rat model of cavernous nerve injury.” Sex. Med. 7:3331–40 (2010).
21. Wood L. Global Cosmeceuticals Market Worth USD 61 Billion by 2020 – Analysis, Technologies & Forecasts Report 2016-2020 – Key Vendors: Avon, Bayer, Johnson & Johnson – Research and Markets. 2016.
22. van der Weele C & Tramper J. “Cultured meat: Every village its own factory?” Trends Biotechnol. 32:294–6 (2014).
23. Olsen TR, Lock LT, & Rowley JA. “Scaling up: how manufacturing sciences will dictate the future of cell therapy.” Med.11:S15–8 (2016).
24. Rowley, J.A. and S.A. Montgomery “The Need for Adherent Cell Manufacturing: Production Platform and Media Strategies Drive Cell Production Economics.” BioProcess International 16:34-49 (2018).

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*This article draws heavily (including the republished figures) from the white paper “Peak MSC — Are We There Yet?” by Timothy R. Olsen, Kelvin S. Ng, Lye T. Lock, Tabassum Ahsan, and Jon A. Rowley published in 2018 in Frontiers in Medicine.

Organoids, tiny yet powerful replicas of organs derived from stem cells, are revolutionizing diagnostics, drug discovery, and precision medicine. Researchers are making progress in creating more complex organoids that closely mimic the structure and function of real organs. As these advancements continue, the potential for using organoids in regenerative medicine to repair or replace damaged tissues becomes increasingly promising. Despite the exciting progress, manufacturing challenges still stand in the way of organoid-based therapies becoming widespread. The Celligent^™ platform from Cell X Technologies offers an innovative, automated, and customizable solution for producing high-quality, GMP-compliant 3D cell cultures, including organoids, paving the way for future breakthroughs.

Understanding Organoids: A Journey in Biopharma

Organoids, despite their roots stretching back to the 1970s, have only recently captured the biopharma spotlight. In the past decade, the conversation around organoids has ebbed and flowed, and now, they’re here again — and for good reason. These tiny, lab-grown organ replicas are revolutionizing therapeutics, yet they remain under-discussed. It’s high time we bring their versatility and potential to the forefront.

Think of organoids as miniature, meticulously crafted models of human organs. They mimic the structure and function of their full-sized counterparts, offering a unique window into human biology. Unlike the simpler spheroids—3D cell aggregates lacking the intricate architecture of specific organs—organoids are complex, containing multiple cell types that self-organize into structures that intend to mimic real organs. This distinction, though often blurred, is crucial.

Derived from various sources like induced pluripotent stem cells or tissue-based stem cells,¹ organoids can be tailored to model both healthy and diseased tissues, including infectious diseases, genetic disorders, and cancers. This versatility extends to their origin: they can be created from allogeneic stem cells to represent a general model or from autologous cells to reflect a patient’s unique biology.

The breadth of organoids’ applications is astounding. They’ve been generated from brain, lung, intestinal, stomach, kidney, liver, cardiac, skeletal, bone, retinal tissues, and even various tumors.^2,3 Each organoid serves as a powerful tool, offering insights that could lead to groundbreaking treatments and personalized medicine.

Let’s explore some the compelling uses of organoids the research and development of biopharmaceuticals.

Applications for Organoids Across the Industry

Regenerative Medicine

The buzz around organoids in regenerative medicine is growing. Researchers have long pursued the dream of building human organs in a dish to create accurate models of human diseases and replace worn-out or diseased organs. In regenerative medicine, the potential of organoids to repair and replace damaged tissues is markedly valuable. Imagine replacing diseased or damaged tissues with precisely engineered, functional tissues derived from organoids. This isn’t just a future possibility; it’s a rapidly approaching reality. For instance, liver and pancreatic organoids have shown great potential for restoring function in patients with organ failure. Studies have demonstrated that liver organoids can aid in liver regeneration and repair by mimicking the organ’s natural architecture and function.⁴ Similarly, pancreatic organoids have been explored for their ability to generate insulin-producing cells, offering a potential therapeutic approach for diabetes treatment.⁵ An additional example, intestinal organoids have demonstrated much promise for treating gastrointestinal diseases.⁶ These advancements highlighting the potential of organoids to develop into complex tissues open up new avenues for autologous and allogeneic cell therapies, promising to restore normal function to affected areas and change countless lives for the better. See Case Study below.

Drug Discovery and Development

In the field of drug discovery, organoids are truly transformative. These miniature organs could provide a more accurate model of human responses than traditional animal models, transforming how we identify drug targets and test potential therapies. By offering insights into efficacy and safety, organoids streamline the drug discovery process, potentially slashing costs and reducing time-to-market. Organoids from tissues like liver, kidney, and brain are pivotal for high-throughput screening, helping us select the most promising drug candidates and identify critical biomarkers. Their ability to mimic human organs surpasses traditional 2D cultures and animal models, giving us a front-row seat to disease progression and drug toxicity. The integration of engineering and computational sciences has only enhanced this technology, making organoids more scalable for widespread drug development applications. Moreover, for drug discovery and development, the promise of organoids as preclinical alternatives is immense. They can reduce costs and improve the relevance of preclinical data. Traditional preclinical models, such as mice, often fail to capture the complexities of human biology, leading to failed clinical trials due to lack of relevance and, subsequently, wasted resources. Organoids, on the other hand, can provide a better representation of human physiology, offering a path to more successful preclinical research. The FDA’s push for non-animal and in silico testing, highlighted in the Modernization Act 2.0, underscores the significance of this shift. Organoids are poised to play a critical role in this transformation, bridging the gap between preclinical promise and clinical success. 

Process Development

When it comes to cell therapy process development, even the simpler relatives of organoids, spheroids, are game-changers. The biopharmaceutical industry increasingly relies on 3D scale-down models, which are essential for process development, optimization, and validation. Given the complexity of biological products, their manufacturing processes must be thoroughly understood, characterized, and controlled. Typically, as development progresses, the process is refined and tested through benchtop bioreactors, pilot-scale bioreactors, and ultimately commercial-scale production. Scale-down models play a crucial role throughout this journey, enabling process specialists to acquire process knowledge and translate it into optimal operating conditions in a cost-effective and timely manner. For instance, these models are applied across a wide range of process development activities, such as cell line selection and high-throughput growth medium optimization. Multivariable experimental studies conducted in scale-down models allow for extensive testing of process parameters, which would be impractical or too costly to perform at commercial scale. paving the way for smoother, faster development cycles.

Personalized Medicine

Creating organoids from a patient’s cells allows us to study that individual’s unique condition in unprecedented detail, predicting how they will respond to various treatments. This ability is particularly important in rare diseases and when patient variability plays a huge role in the therapeutic decision, such as in cancer treatments. In rare diseases, research is significantly hindered by the small number of individuals available for participation in studies. Additionally, many rare diseases lack suitable animal models, making it difficult for scientists to conduct early testing of new treatments. One such example is the use of organoids to develop therapeutics for Timothy syndrome, a rare disease where 80% of children with this syndrome will die within 2.5 years after birth. Researchers developed an antisense oligonucleotide (ASO) therapeutic and successfully demonstrated correction of function in organoids derived from individuals with Timothy syndrome. Additionally, human-derived organoids were transplanted into newborn rat brains, where they integrated well, and ASO treatment reduced abnormal function.⁷ This study not only highlights a potential treatment for Timothy syndrome but also offers a promising approach for other rare genetic disorders. In cancer treatment, the ability to recapitulate the heterogeneity of a patient-specific tumor could potentially be the difference between recurrence and remission. Since cancer cells change over time, patient-specific organoids can be analyzed for responses to combinatorial treatments at various stages of disease progression. Indeed, research has shown that the timing and duration of drug exposure can significantly affect results, and since tumor organoids can be grown indefinitely, we can experiment with different drug doses and schedules in the lab until the best match for a specific patient.  In addition, patient-derived tumor organoids can also be used to select individual patients for novel targeted therapies.⁸ Altogether, these functional data help tailor therapies to individual needs, ensuring patients receive the most effective treatments for their specific conditions.

Overcoming Production Challenges: A Personal Journey into Organoids

The path to producing organoids is fraught with challenges. It demands immense time and effort, where each step often relies on the meticulous hands of skilled operators. This manual nature, while rooted in expertise, inevitably opens the door to errors, inconsistencies, and soaring production costs. The heart of the problem lies in the delicate art of culturing stem cells — the very foundation of organoids.⁹ 

Each organoid’s creation is a unique endeavor, where its specific type and function will be shaped by many factors. Factors such as the choice of starting cells, matrix material (whether biologically derived like laminins or synthetic like hydrogels), growth factors, cytokines, proteins, and physical cues like shaking or stirring, all play pivotal roles. The complexity of these variables means that producing high-quality organoids is no small feat.

To relevantly mimic the behavior and function of real organs, organoids need the right type of interactions with the right types of cells in the right places. Achieving this requires more than just careful planning — it demands the organoid produce differentiated cells, secrete crucial signaling compounds, and respond to external stimuli accurately. Yet, even under the same conditions, we often end up with a wide variety of shapes, sizes, and cell compositions. This intricate dance of biology often takes months, if not years, of painstaking experimentation and optimization.

Another hurdle is the characterization of these organoids. With such variability, aligning on standardized criteria for what makes a “good” organoid and defining and achieving critical quality attributes (CQAs) can feel like navigating a maze in the dark. This variability underscores the importance of optimizing and standardizing the workflow to minimize inconsistencies between batches.

To address these challenges, researchers are turning to innovative techniques. For instance, micropatterning in 2D cultures can create a more reproducible starting condition, which then evolves into the desired 3D structures. Controlling matrix properties and applying physical forces like stretching can direct specific tissue morphogenesis. 

As with other processes where automation provides the key to consistency and reproducibility to achieve the desired results, organoids can benefit from automated, reliable production processes. Imagine a robotic cell-culture platform where one technician oversees the simultaneous production of multiple cell lines. Imagine, furthermore, a scenario where different cell types are consistently placed in exact coordinates, ensuring the relevant spatial interactions. Furthermore, picture organoids in development undergoing automatic inspection, allowing only those that meet stringent criteria to progress, ultimately resulting in the most homogenous population of organoids possible. This would significantly accelerate the quality control process, ensuring consistency and reliability at an unprecedented level, all while boosting production rates.

Embracing Automation for Organoid Production

At Cell X Technologies, we’re passionate about tackling the hurdles in the cell therapy field, knowing that our efforts can profoundly impact the therapeutic industry. Consistent organoid production is one of those hurdles. Our solution? The Celligent™ robotic platform, designed to take the labor-intensive, error-prone steps of generating organoids and transform them into a seamless, automated process. By simplifying and standardizing the generation of organoids, Celligent ensures consistency and precision that manual methods struggle to achieve, especially at scale and under GMP conditions.

The beauty of the Celligent platform lies in its ability to automate critical tasks. Imagine having a system that handles imaging and tracking cells, changing cell culture media, selecting colonies of induced pluripotent stem cells (iPSCs), and eliminating unwanted cells. These tasks are often highly variable and operator dependent, poorly documented, and error-prone when done manually. These become effortlessly consistent, reliable, and rigorously documented when automated using the Celligent platform.

Celligent addresses two key challenges in organoid generation:

1. Consistency: By automating the preparation of initial starting materials (iPSCs) and the differentiation process (when multiple cell types are involved), Celligent ensures a uniform and high-quality foundation for organoid development.
2. Optimization: The platform tracks and fine-tunes the organoid generation process to produce organoids with most relevant attributes for specific applications, making them more effective and reliable.

Operating within a cGMP-compliant Biospherix Xvivo system, the Celligent platform maintains aseptic conditions during operations and uses disposable components, further reducing the risk of contamination. Advanced algorithms within Celligent integrate external data with real-time information generated during cell culture, identifying CQAs and crucial process parameters. Every action is documented in a GMP-compliant database, ensuring traceability.

At Cell X Technologies, we’re more than just automating processes; we’re dedicated to thoughtfully bridging gaps in cell therapy to drive down costs and improve patient access. We’ve envisioned a new era in organoid production—one where consistency, efficiency, and innovation lead to real breakthroughs in biopharma.

Case Study: Revolutionizing Clinical-Grade iPSCs with the Celligent Platform¹⁰

In an inspiring real-world application, the Celligent precision robotic platform generated high-quality, clinical-grade patient iPSCs that successfully formed organoids rich with transplantable retinal progenitor and photoreceptor precursor cells. 

The journey began with dermal fibroblasts isolated from skin biopsies of patients suffering from inherited retinal degenerative blindness. These cells were reprogrammed and, the subsequent clones of the reprogrammed iPSCs were “picked” using automated methods and  expanded. In today’s version, the Celligent platform automates the entire culture process — growth, picking, and clonal expansion of the iPSCs. Clonal populations were compared and selected based on proliferation, morphology, and differentiation attributes. Selected clones were used to generate high-quality neural organoids rich in photoreceptor cells with quality equal to traditional manual methods. 

After 10 passages, the iPSC lines underwent rigorous karyotyping and scorecard analysis to confirm their genetic integrity and potency. The subsequent retinal differentiation process yielded organoids that, by days 120 and 160, expressed key photoreceptor markers and displayed morphology and gene expression profiles akin to those generated via manual methods.

This case study illustrates the power of the Celligent platform to transform a very manual process into a series to automated, linked protocols. By automating complex and labor-intensive processes, the production of high-quality, consistent organoids suitable for clinical applications can be achieved. This streamlines workflows and  opens up new possibilities in regenerative medicine, providing hope and tangible progress in the fight against blindness and other debilitating conditions.

Cell X Technologies: Our Mission

At Cell X, our mission is to customize our manufacturing solutions to perfectly align with our customers’ unique processes and goals. We collaborate closely to design Celligent protocols tailored to customer’s specific needs. This personalized approach ensures that the operations of the Celligent system we create is suited to the unique processes and targeted iPSC- (or other stem cell) based products, including organoids.

We understand that every process is unique. As such, we have designed the Celligent platform to be programmable by a bench scientist and to adapt to the input of subject matter experts by refining cell selection and weeding algorithms as the process moves forward. We understand that time is the greatest limiting factor, and by automating cell and organoid processing steps, scientists are freed up to move to other priorities. 

We are putting a unique focus on creating documented, transferrable processes that are appropriate for GMP production. We understand that a process successful with a research-only cell line might not translate seamlessly to a GMP line. The key is to use the power of automation to establish GMP-ready cell lines earlier in the process, alleviating the necessity to have re-define process parameters when time is most critical.

By using the Celligent platform throughout the process development phase of cell therapy or organoid  development, we provide a traceable, trackable, and mineable data structure that is amenable to developing artificial intelligence approaches.

Looking to the future, Cell X is dedicated to continuously innovating and refining our solutions, remaining committed to supporting our customers in achieving their goals, and advancing the field of regenerative medicine.

References

1. Zhao, Z., Chen, X., Dowbaj, A.M. et al. “Organoids.” Nat Rev Methods Primers. 2: 94 (2022). 
2. Zieba, Jennifer. “What Are Organoids and How Are They Made?” The Scientist. 11 Aug. 2022. 
3. Chakraborty, Debomita. “An Introduction to Organoids, Organoid Creation, Culture and Applications.” Technology Networks. 17 Jan. 2023. 
4. Lam, D.T.U.H., Y.Y. Dan, Y.S. Chan, et al. “Emerging liver organoid platforms and technologies.” Cell Regen. 10: 27 (2021). 
5. Jin , Chen, Lu Jin , Wang Shu-Na , AND Miao Chao-Yu. “Application and challenge of pancreatic organoids in therapeutic research.” Frontiers in Pharmacology. 15 (2024). 
6. Sato, T., and H. Clevers. “Growing self-organizing mini-guts from a single intestinal stem cell: mechanism and applications.” Science. 340: 1190–1194 (2013).  
7. Chen X, et al. “Antisense oligonucleotide therapeutic approach for Timothy syndrome.” Nature. 628: 818–825 (2024). 
8. Weeber, Fleur, Salo N. Ooft, Krijn K. Dijkstra, and Emile E. Voest. “Tumor Organoids as a Pre-clinical Cancer Model for Drug Discovery.” Cell Chemical Biology. 9: 1092–1100 (2017). 
9. Weisinger, Karen. “Intelligent Cell Processing Enables Consistent, Reproducible, and Scalable GMP-Compliant Processes.” Pharma’s Almanac. 30 May 2024.
10. Bohrer, LR, et al. “Automating iPSC generation to enable autologous photoreceptor cell replacement therapy.” J. Transl. Med. 21: 161 (2023).

Cell Therapies: A Promising Horizon

The landscape of the biopharmaceutical industry has undergone transformative shifts over time, from a focus on small molecule drugs to the introduction of biologics and more recently the emergence of cell therapies.

Within the space of cell therapy, two distinct categories have emerged: autologous therapies, which utilize a patient’s own cells to treat individual cases, and allogeneic therapies, which draw from donors to potentially treat multiple patients. While cell therapy is often promoted as a common therapeutic modality, it’s crucial to acknowledge the truth; this field is still in its infancy.  Consider that FDA approval of the first autologous cell therapy, Kymriah® (tisagenlecleucel), a chimeric antigen receptor T cell (CAR-T) therapy, occurred just seven years ago (2017), in stark contrast to the six-decade history of chemical cancer treatments.

Since then, the landscape has rapidly evolved, with six additional autologous CAR-T therapies approved, primarily for hematological cancers. Interestingly, the most recent of these approvals, Amtagvi™ (Lifileucel), marks the first solid tumor cell therapy; a significant milestone underscoring the vast potential that lies ahead.

On the allogeneic side, there are currently no FDA-approved therapies. Encouragingly, clinical advancements, such as Vertex’s pioneering clinical trials for a fully allogeneic therapy for the treatment of type 1 diabetes and Cyanta Therapeutics’ entry into a phase II clinical trial of CYP-001 for the treatment of GvHD, as well as its CYP-004, an allogeneic, iPSC-derived mesenchymal stem cell product for osteoarthritis, are signaling a new era for allogeneic cell therapies.

We stand on the brink of an extraordinary frontier in medicine, where autologous and allogeneic therapies join the ranks of other therapeutic modalities, and where the prospect of curing diseases by restoring cellular components within the body, rather than merely alleviating symptoms, is within our grasp.

Overcoming Challenges and Charting a Path Forward

Navigating the promising yet intricate world of cell therapies, there are numerous challenges that stem from fact that the field is still in its infancy.

Manufacturing living therapies is a layered and complex process that demands meticulous attention from cell isolation to cultivation and often stretches over months to yield a single patient dose. The quest for quality in these therapies, promising as they are in their potential to cure diseases, necessitates robust processes and cutting-edge technologies to ensure consistency and compliance.

There are many process challenges encompassing variability in the product, prolonged process development times, scalability hurdles, contamination risks, cell licensing complexities, and evolving regulatory landscapes. Much of these challenges trace back to the notoriously labor-intensive manual work in the manufacturing processes, particularly evident in allogeneic induced pluripotent stem cell (iPSC)-derived therapies. Daily tasks like imaging, cell culture maintenance, passaging, and offline assays are performed manually by multiple operators, introducing an astonishing amount of variability.

These hurdles manifest in the pain points of cell therapies, including extended process development timelines, scalability issues, ambiguous quality metrics, regulatory bottlenecks, contamination, and substantial wastage, culminating in exorbitant costs per volume of product. Consequently, the lofty price tags attached to these therapeutics hinder patient accessibility and dampen investor enthusiasm for venturing into what is perceived as a high-risk domain.

Yet, in the face of these challenges, we must begin to formulate solutions. Experts may offer divergent strategies, but some common foundational threads emerge. One such common thread lies in starting with an acceptable cell population. Whether it involves sourcing T cells for autologous CAR-T therapies or procuring acceptably documented clinical-grade iPSC lines for allogeneic therapies, the importance of this initial step cannot be overstated. However, this journey is fraught with hurdles, from the scarcity of patient-derived T cells due to patients being in later stages of severe diseases, unable to readily donate large volumes of blood, in addition to being subject to time-sensitive cycles of immunosuppressants, to the labyrinth of licensing issues surrounding iPSC lines.

Another common thread that emerges as imperative is ensuring process robustness and leveraging cutting-edge technologies for characterization data. In a landscape where each bioprocess is unique, tailored characterization is essential, especially in the realm of allogeneic therapies. There’s no one-size-fits-all solution; each step demands meticulous attention and time-efficient approaches. Addressing these intricacies isn’t just essential — it’s pivotal to unlock the full potential of cell therapeutics, ensuring their reliability and reproducibility in the pursuit of transformative solutions.

Sidebar: How Cell X Came to Be

In the heart of Cell X lies a remarkable tale of innovation, born from an unexpected encounter over lunch at a Cleveland restaurant. Dr. George Muschler, from the Cleveland Clinic, voiced his need for a robotic system to handle precise and repeatable patient bone marrow culturing. Overhearing from a nearby table, an executive from Parker Hannifin, an industrial automation company, seized the moment and proposed a collaboration to tackle this challenge.

From this serendipitous meeting sprang Cell X Technologies, dedicated to the joint development of its foundational data and automation system. Fast forward to 2019, three patents were granted, and a pivotal Small Business Innovation Research grant was secured, laying the groundwork for Cell X Technologies’ establishment as a spinout. Amid the turbulence of the COVID-19 pandemic, the technology continued to evolve.

In 2021, Cell X underwent a transition in leadership, amplifying its focus on business aspects. Notably, strategic partnerships and funding rounds propelled its growth trajectory, validating its technology and addressing critical industry challenges.

Introducing the Cell X Technologies Solution: Balancing Innovation with Reliability

The realization of Cell X’s concept combines advanced engineering, proprietary algorithms, and cutting-edge biology. Using iterative optimization, Cell X integrated robotic capabilities with automated state-of-the-art, rapid, large-field-of-view imaging capabilities. This integration facilitated precision automation, enabling the selective removal of unwanted cells and the picking and transfer of desired colonies with known clump sizes. Each step was meticulously documented with high-quality images and time-stamped records, ensuring transparency and accountability throughout the process. The Celligent™ system was born.

At the core of Celligent’s success lies its algorithm-driven cell-selection process, which emulates human decision-making with unparalleled accuracy. The primary focus was not merely on technological innovation for its own sake but on value innovation. While scientists have adeptly managed cell culture for decades, the aim was not to reinvent the wheel but to bridge gaps that elevate existing practices to new heights.

The Celligent™ system demonstrates exceptional adaptability, allowing for the addition or removal of media, execution of cell maintenance tasks, and transfer of cells using various methods and combinations, all while ensuring delicate cell handling. Its user-friendly software eliminates the need for extensive programming expertise, simplifying the protocol-building process that would typically involve multiple operators in multiple hoods. Furthermore, the system excels in data collection, ensuring compliance with Title 21 of the Code of Federal Regulations (21CFR) while automatically generating, storing, and organizing data for convenient access and analysis.

As Cell X technologies pioneers the future of automation in cell processing, we continue to add to our proprietary cloud-based database called Cx Knowledge. This innovative platform allows customers to access the Cell X Technologies data sets used to train a multitude of algorithms. By leveraging these datasets, customers can significantly expedite the learning process for their own systems, fostering greater efficiency and effectiveness in their operations.

Additionally, CX Knowledge forms the basis of our ongoing developments in AI; combing knowledge graphs and models to assess data sets and ultimately develop critical quality attributes for cell therapies.

Addressing Current Bottlenecks in Cell Therapy with Celligent™

As we dive into the world of cell therapies, it’s clear that we’re on the brink of something incredible. There’s so much potential waiting to be unlocked, and automation is key to making it happen. That’s where CelligentTM comes in. It’s not just a solution; it’s a game-changing design to tackle the toughest challenges we face in the industry.

Regulatory

It’s important to recognize that, just as biotech and pharma companies are pushing the boundaries of therapeutic innovation, regulatory bodies are evolving their requirements to safeguard the introduction of new medicines. As technology progresses, so must our regulatory framework to keep pace with these advancements. Anticipating future regulatory demands is a daunting task for any company, as the regulatory environment of tomorrow may differ significantly from today’s standards Yet, foresight is essential for success. Celligent™ is designed to address this challenge head-on by prioritizing comprehensive documentation. Every action, no matter how big or small, is recorded in real time. This automated documentation serves as a valuable asset, providing a detailed record of the entire process. With Celligent™, companies can rest assured that they have a robust repository of information, enabling them to retrieve essential information and navigate future regulatory requirements whenever needed.

Licensing

Navigating the minefield of licensing clinical-grade iPSC lines can be daunting, especially for emerging companies with limited resources. The upfront costs, recurring fees, royalties, and potential freedom-to-operate constraints create formidable barriers to entry. Compounding this challenge is the scarcity of reputable iPSC-producing cell lines to choose from. In a race against time, many startups opt for research-use-only (RUO) grade lines to kickstart their R&D initiatives, intending to upgrade to GMP-grade lines later, once the company is adequately funded. However, this often proves to be a costly gamble. iPSC lines vary significantly in their kinetics and differentiation potential, leading to compatibility issues with the proposed manufacturing processes. The result: more resources are expended on process adjustments than would have been necessary with clinical-grade iPSC lines from the outset. It’s a catch-22! A common approach adopted by companies is to qualify their RUO line, thus avoiding the necessity of ensuring compatibility with another line. While this is a viable strategy, it comes with inherent risks. If previously undetected genetic abnormalities surface during the qualification process, or if any adverse events linked directly to the iPSC line occur during clinical trials conducted by other groups utilizing the same line and strategy, there is a possibility that the cell line will become unusable. This scenario not only results in wasted resources but also poses potential setbacks in research and development efforts.

With Celligent™, users have the power to generate their very own clinical-grade iPSC line from the convenience of their workspace. Whether opting to kickstart the journey with this strategy or already possessing an RUO line, Celligent™ enables efficient screening of numerous clones that seamlessly integrate into the manufacturing process rather than altering the processes to accommodate available GMP lines. What’s more, the Celligent™ platform is fully customizable, ensuring that the automation aligns perfectly with unique process requirements. With Cell X Technologies, the path to licensing clinical-grade iPSC lines becomes not just feasible, but transformative, offering a streamlined approach that puts control back in users’ hands.

Variability

Variability stands as a primary challenge in cell therapy, often compromising consistency and predictability. Unlike other therapeutic modalities, the inherent nature of living cells introduces a level of unpredictability that can be challenging to mitigate. Much of this variability stems from the intrinsic properties of the cells themselves, coupled with our limited ability to exert control over their behavior. While this topic warrants a deeper dive, for now, let’s acknowledge its pervasive influence.

Adding to the complexity is the variability introduced by human operators. In the labor-intensive landscape of cell therapy, individual approaches and subtle adjustments can inadvertently amplify variability. Despite best intentions, practices diverge, and undocumented steps further compound the issue. The cumulative effect of these nuances is a variability multiplier. The reality is, in most instances, we may not realize or fully understand how even the smallest differences among operators influence the overall variability of the product.

The solution to this conundrum echoes throughout the industry: automation. While Celligent™ cannot erase the inherent variability of living cells, it can automate all the operator steps and provide a consistent and reliable process. From scheduled media changes to real-time adjustments based on culture parameters, our algorithms ensure precision and comprehensive documentation. This not only aids in minimizing variability but also allows our scientists to dedicate their time to tasks where automation falls short –– creative and continued innovation.

Data and Analytics

In the landscape of cell therapy, navigating the intricacies of data and analytics presents a significant challenge. The quest for precise quality metrics within processes often feels like searching for a needle in a haystack. The difficulty lies not only in monitoring the right variables but also in deciphering correlations between cell behavior and molecular metrics.  This bottleneck stems largely from the initial step, which hinges on collecting comprehensive process characterization data. This crucial foundation for knowledge relies heavily on manual practices, which, as we’ve previously highlighted, introduces variability.

Furthermore, the integration of diverse data sets and analytical pipelines aimed at extracting meaningful and predictive metrics has yet to fully come to fruition. It’s a recurring scenario where companies, after investing months and years in processing, only later realize that they’ve been utilizing incorrect metrics. This revelation often coincides with ambitious scaling plans, and the absence of meaningful quality attributes sets them back significantly, in terms of both time and finances. Sadly, this setback all too frequently leads to companies shuttering their operations. The loss of potentially groundbreaking therapeutic innovations underscores the critical need for consistent process characterization data and comprehensive analysis.

Herein lies a critical juncture: the need for a paradigm shift in analytical methodologies to keep pace with the rapid evolution of this field. Celligent™ merges cutting-edge imaging technology with sophisticated data analytics. By harnessing high-quality, rapid whole-well imaging capabilities, Celligent™ offers temporal image-based tracking of iPSC clones and colonies throughout the process. What sets it apart is its ability to seamlessly integrate offline molecular measurements with image data, creating a comprehensive knowledge base in Cx Knowledge. This holistic approach captures every aspect of the process, from reagent additions to cell movements, while reducing variability and increasing the reliability of the data. Furthermore, Celligent™ leverages proprietary algorithms to extract predictive critical quality and process parameters, empowering practitioners with actionable insights. Its automated statistical testing capabilities expedite the validation process, surpassing the reproducibility of human operators. Implementing such technology promises to drive advancements across the entire industry.

Contamination

Cell culture contamination is an ever-present source of anxiety in therapeutic development and manufacturing, especially in the cell therapy world, where therapeutics can take months to manufacture. The ramifications of contamination extend beyond financial losses, reaching into the lives of patients reliant on these treatments. The imperative to monitor and prevent contamination remains paramount, yet current solutions often entail the construction, rental, or outsourcing of cleanroom facilities — a costly endeavor both in terms of finances and logistical challenges.

The Celligent™ platform offers a fully customizable and configurable solution that can be easily adapted from BSC to phase-appropriate enclosures to cleanrooms within the company’s premises.

Speed to Market

Ensuring speedy access to groundbreaking therapies is a top priority from both patient and investor perspectives. Patients eagerly await the arrival of innovative treatments, hoping for faster relief and better outcomes. Meanwhile, investors seek timely returns on their investments, which can significantly impact the trajectory of funding and development. However, the reality of creating cell therapies involves a process with numerous steps, each essential and non-negotiable. Despite these inherent challenges, there are avenues to accelerate progress. By streamlining intermediary steps, enhancing throughput, and expediting regulatory processes, we can expedite the journey from concept to market.

The Celligent™ platform emerges as a pivotal solution, facilitating consistent data collection, rapid extraction of quality metrics, and accelerated customized assay development. This capability results in error reduction and consequent time savings between successive steps.

Celligent’s automation capabilities enable continuous operation, boosting efficiency round the clock. Finally, with Celligent™ and Cx Knowledge, automated reporting and critical quality attributes enhance regulatory submissions, minimizing risks and increasing approval probabilities.

Building a Cross-Functional Team of Experts

Creating an automated imaging system like Celligent requires a collaborative effort from a multidisciplinary team encompassing engineering, biology, software, and various specialized domains.  As a company established during the onset of the COVID-19 pandemic, Cell X embraced a virtual operating model at its inception and has now moved to establishing core capabilities across three sites.

We have established an engineering and software center in Pittsburgh, made possible by investment in Cell X from Innovation Works and its Robotics Accelerator program. Through this program, we gained access to mechanical and electrical prototyping labs, as well as engineering expertise from CMU and the Pittsburgh robotic and AI ecosystem.

Our original laboratory, located at the Cleveland Clinic’s Lerner Center for Regenerative Medicine, continues to operate as a Cell X testing and development center, as well as a use case center across application areas.

We’re excited to announce our plans for a Boston-based applications lab, slated to open its doors by mid-2024. This facility offers dedicated space for application proof-of-concept studies, as well as customer projects and customer support. With GMP capabilities and a qualified cleanroom facility, it will serve as a hub for showcasing our technology to potential customers, facilitating external equipment integration prototyping, and hosting user networking groups for collaborative information sharing. 

References

1. “Sector Snapshot: Building a Next-Gen Workforce).” Alliance for Regenerative Medicine. Aug. 2023.
2. “Ongoing Clinical Trials by Phase and Therapeutic Approach.” Alliance for Regenerative Medicine. 2023.
3. “Investment Data – 2023 Q2.” Alliance for Regenerative Medicine. Oct. 2023.
4. Tobolowsky, Mark A. and Richard A. Lewis. “What Regenerative Medicine Manufacturers Have Been Waiting For.” Outsourced Pharma. 9 Nov. 2023.