Building Effective Multi-Year Process Development Programs: Estimating hMSC Lot Size Ranges for Clinical Manufacturing through Commercial Demand – it is all about the assumptions

Authored by Josephine Lembong, Ph.D., Scientist, Product & Process Development & Jon A. Rowley, Ph.D., Founder and Chief Product Officer, RoosterBio Inc.

Jon Rowley, RoosterBio’s Founder and Chief Product Officer, gave a talk at ISCT 2018 in May and had a set of slides about estimating lot size that had a lot of people scrambling to take notes. We thought we would share the content here at the RoosterBio blog with more discussion and make the content broadly available and open for discussion.  

The concept is consistent with all good strategic planning; 

1. Understand what the future looks like and work backwards,
2. Create a model using reasonable to conservative assumptions along the way to estimate a range of needs,
3. Use this model to lay out what the next several years will look like assuming successful achievement of intermediate milestones, and create multi-year programs that are right-sized, with the right technology platform, for the stage of product / clinical development of your company.

This is the type of consultation that we provide in our Process Design and Acceleration business unit at RoosterBio, which we offer to our customers that are incorporating RoosterBio hMSC cell banks and bioprocess media systems into their product and process development efforts. However, we love to share our knowledge with the broader RegenMed Industry and are offering it up here.

Notes to readers: this exercise has the base assumption that this is an Allogenic, Universal Donor manufacturing process [1] and is not applicable to Autologous or patient-specific products. The core logic would still hold when applied to autologous, but at a much smaller scale.

Lot Size Targets Dictate the Production Platform 

The goal of any process development program is to create a right-sized manufacturing process for your immediate business goals, but with future scalability in mind. Understanding the future lot size requirements will help strategically align manufacturing requirements today with commercial scalability while laying a platform foundation to minimize comparability risks.

First Start with the End in Mind – Engage with Marketing

Get with the business team within your company (usually Strategic Marketing and/or Commercial Operations) and request a numbers-driven discussion on which of the multiple indications that a process development program should be built for. There are usually several therapeutic indications of interest, but it is important to realize that a manufacturing process for an ocular indication that has a dose of 1 million cells/dose is very different than a Crohn's disease indication that has a dose size of greater than 1 billion cells/dose [2]. Each therapeutic indication from a company must be treated as a distinct product and target, with a distinct process. Just because the same cell type is used in both therapeutic products does not mean they share a manufacturing process. This is critical to bring clarity to each program.

“A common pitfall that many cell therapy companies fall into is that each therapeutic indication is not addressed as a unique product with a unique manufacturing process”

Once the team is aligned on the target indication (or set of prioritized target indications), then you need to understand what a reasonable peak commercial market demand would be – this is likely to be found in the business model projections that are built for investors and is a good place to start. If there are 500,000 patients a year in your target indication and the business is aiming to capture 20% of them (or treat 100,000 patients/year) at peak commercial demand, then you need a process that will scale with that over time, assuming both clinical and market success. It is a good idea to establish a range. We often like to perform this exercise with a low, middle and high assumptions (such as 30,000 patients as low, 100,000 as the target, and 250,000 patients treated/year if wildly successful). For this article, we will simply focus on the '100,000 patients treated assumption' for our calculations.

Dosing Assumptions: this is one of the hardest parts

Estimating dosing is sometimes the most difficult part. Does the patient need a single dose or multiple doses, and how many cells per dose? In many cases a dose escalation trial has not yet been performed, so some educated guesstimates are required. Taking a low/mid/high range of estimates also works here, and there is sufficient published work related to hMSCs in different indications that you can get close enough for these purposes [2,3]. For this example, we will assume a cardiac indication. There is a good amount of published work on hMSCs for cardiac indication and we can assume a conservative low dose of 25 million cells, a mid-range dose of 50 million, and a high dose of 100 million [4,5]. In this blog post we will perform all calculations with an assumption of 50 million cells per dose.

Calculating Yearly Product Needs at Peak Commercial Demand

• At peak demand, we aim to produce 100,000 doses per year, with a size of 50 million cells per dose.
• 100,000 doses per year ≈ 2,000 doses per week
• Weekly production is not recommended – it is simply unsustainable, so let’s assume one (1) lot production every two (2) weeks, or 25 lots total in a year (which is still a lot for any manufacturing site)
• Not all lots will be successful – assume a 10% scrap rate, thus 2-3 lots are lost per year, giving us 22-23 lots per year
• 100,000 doses per year / ~23 lots per year = 4,450 doses/lot 
• Assume 10% of lot goes to testing (~450 dose), so a final target lot size of 4,900 doses/lot will be needed to be manufactured at peak commercial demand

It is important to remember that it typically takes multiple years for a successful therapeutic to reach peak demand, so it is not necessary to go to market with a manufacturing process capable of meeting peak demand – especially for an early stage field like Regenerative Medicine. We will create estimates for peak demand, but then focus on building a reasonable “go-to-market” lot size and manufacturing process.

Calculating Total Cells to Manufacture that are Required to Fill into the Final Container

• We will assume 1 dose goes into one container.
• We assumed a mid-range dose size of 50 million (i.e. 50 million viable cells per vial, post-thaw).
• Cell Count is a strict QC parameter with a straight forward specification. It is important to point out that often there is a solid safety factor, or overfill, applied to this metric. You never want to fail a lot on cell count after you spent hundreds of thousands of dollars creating the units – so the risk averse will overfill vials until sampling and cell enumeration is an accurate, consistent, and precise method.
• Assume 15% (worst case) viability drop and 20% recovery loss during cryopreservation (these assumptions should be data driven from your process, and constantly trended and updated during manufacturing).
• Based on the above assumptions, to consistently achieve 50 million viable cells after thaw, it is required to target filling at 75 million viable cells into the final container:
• 75 million viable cells – 15 million (20% total cell loss on recovery) = 60 million viable cells 
• 60 million – 9 million (15% viability drop) = 51 million viable cell target specification
• NOTE: some programs will only overfill by 10-20%, but we find that with this range it is very difficult to routinely obtain successful cell counts in a manufacturing/QC environment, so we recommend 30-50% overfill for calculations.

Calculating Lot Size Requirements at Peak Commercial Demand

One unfortunate fact of cell manufacturing is that you need to manufacture more cells than you need to fill, since there will be losses associated with the post-harvest processing steps. Many biological manufacturing processes have losses in the 40%-60% range in this “downstream” processing.  For our process, we go through the following math:

• 75 million cells/dose × 4900 doses per manufacturing lot = 368 billion viable cells needed to target 4900 doses from every manufacturing lot.
• We assume 30% post-harvest cell loss during processing for this exercise – thus at least 525 billion cells need to be manufactured in order to have 368 billion cells during the fill unit operation.
• 525 billion viable cells – 157 billion (30% post-harvest cell loss) = 368 billion viable cells 
• 30% cell loss does seem significant, however every process development group will have a team working to optimize their process to minimize this number.  It is likely that recoveries from earlier processes will be much greater than 30%, and with optimization at large scale it is possible to improve.  For modeling, it is always worth using reasonable to conservative estimates.

In conclusion, we arrived at the following numbers:

• At peak commercial demand, 23 successful lots of 525 billion cells per lot need to be manufactured in order to meet the demand, assuming a mid-range dose for a cardiac indication aimed to treat 100,000 patients per year. 

This calculation does not take into account further downstream losses associated with stability, multiple distribution sites, safety stocks – which will just make the numbers worse. For this exercise, we will stop here. The 525 billion viable cells per lot is the number we were looking for, and it is this number that will drive the production platform decisions for streamlined hMSC clinical manufacturing.

For an early-stage RegenMed company, a go-to-market lot size of ~20% of the “peak commercial scale” is a reasonable target to plan for, so a ~100 billion cells/lot would be a good target number to develop and run your Phase III trial at.

We’d like to summarize this post with a few number recommendations:

Phase III/Go-to-Market: ~100B cells

Phase II (100-200 patients over 3-4 lots): 15-25B cells – this is an intermediate step up from Phase I and within a log of the Phase III/go-to-market process.

Phase I (small scale): 5-10B cells

The next stage is to lay out the technology platforms for the various manufacturing unit operations that are capable of processing these cell numbers. This will be the topic of our next blog post.

References:

1. Simaria et al., 2013. Allogeneic cell therapy bioprocess economics and optimization: Single‐use cell expansion technologies. Biotechnol Bioeng 111(1): 69-83. doi: 10.1002/bit.25008 
2. Olsen et al., 2018. Peak MSC—Are We There Yet? Front Med 5:178. doi: 10.3389/fmed.2018.00178 
3. Squillaro et al., 2016. Clinical Trials With Mesenchymal Stem Cells: An Update. Cell Transplant 25(5):829-848. doi: 10.3727/096368915X689622 
4. Majka et al., 2017. Concise Review: Mesenchymal Stem Cells in Cardiovascular Regeneration: Emerging Research Directions and Clinical Applications. Stem Cells Transl Med 6(10):1859-1867. doi: 10.1002/sctm.16-0484 
5. Golpanian et al., 2016. Concise Review: Review and Perspective of Cell Dosage and Routes of Administration From Preclinical and Clinical Studies of Stem Cell Therapy for Heart Disease. Stem Cells Transl Med 5(2):186-191. doi: 10.5966/sctm.2015-0101

Orthopaedic Research Spotlight: ORS 2017 Annual Meeting

A guest blog post by RoosterBio Travel Award winner, Poonam Sharma.

The annual Orthopaedic Research Society meeting was an energetic and collaborative conference attended by clinicians, industry professionals, and researchers. While the attendees brought diverse perspectives to this meeting, the varied presentation styles, such as short poster teasers, mid-length research talks, and longer, broader spotlight oral presentations helped bring the audience together in scientific discourse. With over 300 oral presentations and over 2200 poster presentations, the variety in presentation styles made ORS 2017 an engaging and dynamic conference to attend.

Knockdown of vimentin may affect chondrogenic
extracellular matrix deposition in high density pellets.
shVim-vimentinknockdown, shLacZ-control.

My research in Dr. Adam H. Hsieh’s Orthopaedic Mechanobiology Lab at the University of Maryland centers on the role of vimentin intermediate filaments in governing mesenchymal stem cell (MSC) properties and behavior, including cellular deformability, adhesion, and differentiation, specifically chondrogenesis. After knocking down the expression of vimentin intermediate filaments in human MSCs using RNA interference, we observe how a decrease in vimentin affects differentiation (abstract here). Preliminarily, we’ve found that a decrease in vimentin did not affect adipogenesis or osteogenesis, but may lead to a potential decrease in chondrogenic extracellular matrix deposition, but this needs further exploration. MSCs from RoosterBio have been singular in the progression of my graduate research. The fast growth and consistency of the high quality MSCs have taken the bottleneck of MSC growth out of the equation for my research. Further, using these MSCs and media has dramatically decreased the labor, time, and resources needed to obtain the cell numbers needed for conducting my experiments.

During this conference, I was able to have in-depth conversations about my research as well as exchange ideas and technical tips that will help strengthen my work. Attending ORS allowed me to both present my research through a poster presentation and network with both industry professionals and academic researchers. As I will soon be taking the next step in my career, these interactions helped me to start to home in on the types of opportunities that I would like to pursue and how to prepare myself to excel. Further, attending professional development seminars, such as one regarding the art of negotiation, helped me identify techniques for further developing soft skills.   

One of the most engaging sessions in this conference was a really fun debate about the related futures of regenerative medicine and orthopaedic implants – Will Regenerative Medicine Make Orthopaedic Implants Obsolete in Our Time? It was captivating to hear the discussion about two research and clinical areas that continue to intersect and diverge. Also, the keynote by Dr. Jennifer Doudna summarizing the CRISPR technology that she helped develop was a great overview and the brief discussion about the ethics of gene therapy was thought-provoking.

The research presentations and broader spotlight sessions gave me a great overview of the latest research in my interest areas of regenerative medicine, tissue engineering, cell therapies, and biomaterials. Many of the oral presentations I attended focused on bettering the design of tissue engineered scaffolds. Here are just a few of the research presentations that inspired me:

World Biomaterials Congress 2016 Meeting Round Up Part I: Guest Blog Post

A guest blog post by RoosterBio Travel Award winner, Gisele Calderon.

The World Biomaterials Congress (WBC) takes place every four years with an energy rivaling the Olympics. This Congress is the largest gathering of biomaterials-focused researchers with over 1,200 oral presentations and 2,400 poster presentations representing over 60 countries. I am incredibly grateful to have been given the opportunity to present my work to and learn from the World’s finest leaders of the field.

Our work in the Miller Lab at Rice University focuses on vascularizing engineered tissues to address the metabolic needs of these complex tissues via various techniques. I develop a system to monitor cellular morphogenesis toward a stable capillary plexus allowing biology to dictate the architectural hierarchy. The cell-cell interactions between the endothelial cells derived from an iPS source and human mesenchymal stem cells tend to enhance the stability of the putative capillaries we form. Our novel multicolor genetic reporter system is enabling a new class of longitudinal studies of tubulogenesis and their integration with 3D printed vasculature.

I was overjoyed to present my work to curious peer grad students, esteemed thought-leaders, and industry representatives. I was fortunately located near the coffee so my poster received a high amount of traffic! I particularly enjoyed how accessible all of my science idols were during the Congress. WBC ran an event where discussion and learning were the central mission.  

The Congress highlighted the field’s latest work and how critical using therapeutically relevant cell types (like RoosterBio’s hMSCs) is for successful tissue engineering strategies and forward progress. I especially enjoyed engaging with investigators sharing the following presentations:

3D Tissue Printing

•    Dr. Jennifer Lewis, Lewis lab, Wyss Institute, Harvard University
•    How can we directly print human tissue? Their approach utilizes top-down bioprinting elegantly recreating complex vascular geometries.
•      A recent publication features RoosterBio’s hMSCs!

3D Printing complex scaffolds using Freeform Reversible Embedding of Suspended Hydrogels (FRESH)

•    Dr. Adam Feinberg, Regenerative Biomaterials & Therapeutics Group, Carnegie Mellon University
•      They 3DP crazy complex structures in a gel within gel fashion for ubiquitous support in their soft constructs. A fun note - he was inspired by Salvador Dali’s painting where everything droops down without support!
•       Here’s their FRESH printing paper.

 Hydrogels with continuously variable stiffness defined by dual-color micro-stereolithography

•             Dr. Neils B Larsen, PolyCell group, Technical University of Denmark
•    This group constructs 3DP soft constructs using stereolithography techniques in order to incorporate microvessels in their hydrogels. They are able to achieve consistent channels under 200um with tunable elasticity dependent on wavelength and exposure tie of incident light.
•        More details can be found here.

In reality, there are details of many more presentations that I would love to share here, but I just couldn’t do justice to the high spirit of scientific rigor. Throughout the Congress, I was actively tweeting about all the excellent work. Follow me @g_caldero! And lastly, thank you, RoosterBio, for the awesome cells AND also the travel award support to attend this exciting Congress! 

ORS 2016 Annual Meeting Round-Up

A guest post by RoosterBio Travel Award winner, Katherine Hudson. 

Rocking some RoosterBio swag!

The Orthopaedic Research Society (ORS) Annual Meeting brings together clinicians, scientists, and engineers dedicated to addressing the current challenges facing orthopaedic research. With over 2,200 abstracts being presented, it can be a difficult landscape to navigate. Luckily, the organizers make it easy to connect with researchers with similar interests while still facilitating expanded horizons.

My research focuses on tissue engineering of the intervertebral disc (IVD), using mechanical and chemical cues to encourage Mesenchymal Stem Cell (MSC) differentiation and tissue maturation within my constructs. This subject spans the topics of stem cell biology, biomaterial development, and in vivo preclinical trials, making the ORS a perfect place to present my work. Attending the ORS meeting allowed me to accomplish many things including sharing my most recent work, networking with potential employers and collaborators, and learning about the latest scientific developments and techniques.

During the conference, my posters received plenty of traffic, which extended the impact of my findings. Both posters challenge traditional tissue engineering paradigms, and my aim was to make other tissue engineers aware of the potential benefits of culturing (and expanding) MSCs in hypoxia, and immunophenotyping cells before and after their use in 3D scaffolds (See my ORS abstracts here and here for details). Additionally, I was able to get valuable feedback on my research that will make my upcoming dissertation stronger.

The ORS encourages and facilitates networking with both clinicians and other scientists. While at the conference, I met with researchers from across the country, and even interviewed for postdoctoral positions, the next step after I finish my PhD work this May. Through these discussions and the presentation sessions organized by the ORS, I was exposed to the latest research in my current and proposed fields of study. This included the newest cell culture techniques, evaluation tools, and IVD biology.

Although I am biased towards tissue engineering and development, I feel that these topics were the highlight of the ORS meeting this year. The source of cells used in regenerative therapies, be they primary or stem cells, was a focus throughout the conference. Additionally, novel biomaterials and stimulation techniques to drive the behavior of cells was a focus. It is important that researchers understand the structure of orthopaedic tissues and their failure modes over multiple scales before we can truly develop successful repair and regeneration strategies. Appropriate cells types and materials facilitate these studies.

Some presentations that stood out to me:

At the Cutting-Edge of Regenerative Medicine: Bioengineering Human-Sized Bone

Fisher et al published the first attempt at engineering

a full-scale adult human femur head from hMSCs.  This

is the largest reported tissue to have been engineered

and took over 700 million hMSCs to fabricate.

John Fisher’s laboratory at the University of Maryland, College Park recently published what can be considered a significant advance for Tissue Engineering and Regenerative Medicine. Graduate student Bao Nguyen and her colleagues have engineered a bone construct that is 20 times larger than any reported previously, and the size of an adult human femur.

Why is this important? Critical size bone defects are a significant health problem (resulting in over $1 billion in annual healthcare costs incurred in the U.S.) and are currently treated with grafts, decellularized bone, or synthetic bone grafts, with sometimes unsucessful results. As such, modern medicine has been looking to tissue engineered bone grafts as future treatments for such defects. Human bone marrow-derived Mesenchymal Stem Cells (hBM-MSCs) are a promising cell source for such applications because they efficiently differentiate down the osteogenic path and also secrete paracrine factors that may aid survival and vascularization of engineered bone. Prior to this publication, engineered constructs have been relatively small due to cell and culture limits.  One major challenge has been growing hBM-MSCs, while maintaining their function, to sufficient numbers needed for an adult human-sized construct; a challenge adressed by RoosterBio.  In addition, nutrient and O₂ transfer are often insufficient to maintain cell viability and function throughout larger constructs, especially those of adult human dimension.

To address this cell culture limitation, the Fisher laboratory developed a Tubular Perfusion System (TPS) bioreactor where cells and scaffolds are cultured in a cylindrical chamber and subject to circular media flow. This system has high nutrient and O₂ transfer and efficient waste removal and has been previously used to produce smaller engineered bone and cartilage constructs (See here and here).

In the study detailed here, the authors had access to and combined, for the first time, advanced technologies required for biofabrication: 3D printing, the TPS bioreactor, and scalable production of hBM-MSCs. The goal of the study was to scale-up bone constructs to adult human size. A full size mold of the superior portion of a human femur (the largest bone in the human body) was 3D printed using information from an opensource database. The mold measured 23 cm long and 10 cm at its widest point with a volume of  200 cm³. The mold was filled with hBM-MSCs in alginate beads (3 mm beads, 100,000 cells per bead). The entire construct utlillized 7200 aliginate beads containing a total of 7.2 x 10⁸ cells (yes, that is 720 million cells!). The high volume hBM-MSC cell and media systems used were from RoosterBio, and technical support for the efficient production of large volumes of hBM-MSCs was provided by our company.

After 8 days of culture in the TPS, the construct was examined for cell viability and bone differentiation. High cell viability was seen in all parts of the construct, both on the outside and the inside (interior). In addition, hBM-MSCs committed to the osteogenic lineage throughout the construct, demonstrating efficiency of the TPS culture system. Both early (Alkaline Phosphotase, ALP) and late (Bone Morphogenic Protein-2, BMP-2) markers of osteogenesis were upregulated relative to day 0. Interestingly, ALP and BMP-2 expression was 25- to 30-fold higher in the construct shaft relative to other portions of the construct. The authors speculate that this is due to shear stress exerted on the parts of the construct closest to the inlet, which activates hBM-MSC signaling pathways, causing release of paracrine factors that stimulate osteogenesis of the “downstream” shaft portion. Taken together, these results demonstrate that the confluence of cutting-edge technologies such as 3D printing, TPS bioreactors, and best-in-class hBM-MSC manufacturing processes enable the engineering of adult human-sized tissue constructs.

While “…this first foray into full-scale bone engineering provides the foundation for future clinical applications of bioengineered bone grafts…” the authors point out some limitations to this study. The 8 day culture period was relatively short, given the weeks usually needed for high efficiency bone differentiation. Thus, extended time points and the fabrication of additional large constructs are needed to fully explore the capabilities of the TPS system. Further, alginate is a soft material, and its mechanical properties do not render it the best suited for bone differentiation.  In addition, hBM-MSCs within aliginate beads lack cell:cell contact, which may also limit their osteogenic differentiation.  To address these limitations of the current system, the Fisher group is developing a 3D printed shell made of an implantable rigid material better suited for the engineering of bone constructs.  Finally, the construct lacks a vascular network, which can be overcome by including endothelial cells (EC) in addition to hBM-MSCs or by incorporating micro-channels in the engineered constructs through a variety of methods (e.g. biomaterial fabrication and 3D printing). Despite the aforementioned limitations, the work presented is a significant advance towards clinical-sized tissue-engineered bone constructs for use in patients.

In an attempt to elicit discussion, I will mention other methods that harbor potential for use in such large-scale tissue engineering applications. For one, hBM-MSC aggregates could be used in place of cells in alginate beads. These 3D-MSC not only maintain cell:cell contact but also undergo osteogenic differentiation more efficiently than cells grown on tissue culture plastic, are resistant to hypoxia, and secrete angiogenic cytokines. Secondly, factors that stimulate bone differentiation of hBM-MSCs, and/or alter mechanical properties of the construct, could be incorporated into the polymer scaffolding, or could be introduced into 3D-MSC aggregates. Finally, once bio-inks are developed further, the bone construct could be patterned by 3D printing of cells (hBM-MSC, EC, 3D-MSC) and materials.  Now that human-sized constructs are possible in terms of cell numbers and O₂ and nutrient diffusion, the possibilities are virtually endless.

Finally, we sincerely thank Bao Nguyen and John Fisher for being early adopters of RoosterBio hBM-MSCs and joining us in accelerating Regenerative Medicine!

References:

Nguyen BB, Ko H, Moriarty RA, Etheridge JM, Fisher JP. Dynamic Bioreactor Culture of High Volume Engineered Bone Tissue. Tissue Engineering Part A. Volume 22, Numbers 3 and 4, 2016, ahead of print. doi:10.1089/ten.tea.2015.0395.  http://online.liebertpub.com/doi/abs/10.1089/ten.tea.2015.0395 

I’m sorry that this is paywalled!

Yeatts, A.B., and Fisher, J.P. Tubular perfusion system for the long-term dynamic culture of human mesenchymal stem cells. Tissue Eng Part C 17, 337, 2011.

http://online.liebertpub.com/doi/abs/10.1089/ten.tec.2011.0263

Yeatts, A.B., Choquette, D.T., and Fisher, J.P. Bioreactors to influence stem cell fate: augmentation of mesenchymal stem cell signaling pathways via dynamic culture systems. Biochim Biophys Acta 1830, 2470, 2013.

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3461086/

Ma, X et al. Deterministically patterned biomimetic human iPSC-derived hepatic model via rapid 3D bioprinting  PNAS, Early Edition doi: 10.1073/pnas.1524510113 http://www.pnas.org/content/early/2016/02/04/1524510113

Priming of hMSCs to Improve Potency

By Iain Farrance, Priya Baraniak, and Jon Rowley. RoosterBio.

In this blog, we will present internal data and information on priming RoosterBio’s bone marrow derived human MSCs (hMSC) with pro-inflammatory molecules and the impact of these priming protocols on hMSC immunomodulatory function and angiogenic cytokine secretion.

INTRODUCTION:

Human Mesenchymal Stem/Stromal Cells, or hMSCs, are key components of future therapeutics, engineered tissues, and medical devices. There are currently over 400 clinical trials investigating hMSCs as therapies (1). The trials have produced some promising results, with hMSCs generally deemed safe, and in some cases effective (2). It is believed that these versatile cells achieve their biologic and therapeutic effects by secreting a plethora of biomolecules (referred to as the MSC secretome) that moderate a variety of processes including angiogenesis, immunosuppression, and overall “tissue repair” (3-6). As the secretome is one of the likely Mechanisms of Actions (MOA) of hMSC therapies, there is a significant amount of recent work on engineering hMSC preparations to enhance secreted factors by genetic modification, by culture strategies, or by engineering the hMSC microenvironment (5, 7-11). In addition, a recent ISCT paper (12) advances the concept of “priming” hMSCs by exposing the cells to pro-inflammatory cytokines prior to implantation.

Thus, priming of hMSCs can have two primary purposes:

1. To assess human MSC preparations in vitro as recommended by the ISCT and the FDA (12, 13), and
2. To enhance hMSC potency (survival, immunosuppression, homing) prior to implantation (8, 9, 14).

As part of our standard quality control (QC) testing, RoosterBio analyzes the immunosuppressive capability of our cell lots through priming with IFN-γ. The hMSC response (i.e. immunomodulatory potential) is reported as a measure of indoleamine 2,3-dioxygenase (IDO) activity, determined by measuring the amino acid kynurenine in the culture supernatant.  The IDO enzyme converts L-tryptophan to N-formylkynurenine (or kynurenine), an immunosuppressive molecule that acts as an inhibitor of immune cell proliferation - including T cells (12, 15, 16).  Testing every hMSC lot for inducible IDO activity provides a quality assurance that the cells we release have some level of functional potency as it relates to immunomodulation– which we consider a key quality attribute of hMSCs.

While researchers are beginning to implement testing of hMSC preparations for inducible IDO activity prior to implantation, few are looking at the impact of priming on other hMSC functions.  Here, we present information on priming of RoosterBio’s hMSCs with IFN-γ ± TNF-α across multiple lots and donors and the impact of such treatment on hMSC IDO activity and angiogenic cytokine secretion.  The goal of this blog post is to demonstrate that priming has impacts on several functional properties of hMSCs, and that researchers should consider priming regimens to (a) understand the potency of their specific cell products, especially in inflammatory environments, and (b) to potentially increase potency of these cell products upon therapeutic administration.

METHODS & EXPERIMENTAL DESIGN:

Materials & Reagents

Cell culture reagents were purchased from Life Technologies, chemicals and reagents for kynurenine measurement were from Sigma, and cultureware was from Corning.  Other products are: Bone Marrow-derived human MSCs (BM-hMSC, part # MSC-001, RoosterBio) and RoosterBio High Performance Media kit (part # KT-001).

Table 1: Experimental design.

hMSC Priming

Current Bottlenecks in MSC Research: MSC Misconceptions - Part II

 http://andreyev.com.au/wp-content/uploads/Misconceptions.jpg

We blogged recently about Mesenchymal Stem/Stromal Cell (MSC) Misconceptions that are holding the translational cell therapy field back, as identified by Donald Phinney and Luc Sensebé.  Since we have come to market with our own hMSC product lines, we have spoken with hundreds of MSC researchers and engineers, and we have compiled our own set of misconceptions that we think build off of Dr. Phinney’s and Dr. Sensebe’s initial concept.  This blog post is to share some of the market-based feedback that we have received.

To the list that was published in Cytotherapy, we would like to contribute the following list to the conversation:

1. Tracking MSC passage number is an accurate and reliable means of tracking cell age and standardizing experimental workflow

In many research laboratory environments, cellular age is most often tracked by the number of times a cell has been passaged; however, Passage Number is quite imprecise and not very acceptable as one gets into regulated environments such as translational clinical activities.  It is generally accepted that tracking the Population Doubling Level (PDL) or Cumulative Population Doublings (CPD) of primary cells is a best practice on understanding cellular age in vitro.   Since it is well documented that PDL impacts hMSC function (see here, here and here), in order to drive consistency into experiments, it has become a best practice to perform experiments or develop products with cells in a consistent range of population doublings where the cell function of interest is still robust.  Furthermore, regulatory agencies are beginning to require reporting of PDLs, or at least cell seeding and harvest densities, for primary cells intended for therapeutic use.  In an effort to drive adoption of PDL tracking and reporting, we’ve created a Best Practices Educational Powerpoint, and free, easy-to-use PDL calculator worksheet we’re happy to share with colleagues.  For your copy, just email us at info@roosterbio.com or subscribe to our blog!

2. Performing experiments with one MSC donor and/or lot is adequate for publication and moving forward with pre-clinical studies

Despite indications of clinical effectiveness of MSCs, there is repeated news of the failure of high-profile MSC trials to demonstrate efficacy in a number of therapeutic applications.  It has been suggested that the large amount of intra- and inter-donor variability in the MSC populations used in these trials may be responsible for their falling short of expectations despite highly encouraging in vitro and in vivo pre-clinical data.  Thus, to ensure the robust production of functional MSC products over a range of applications, experiments should be conducted and systems validated with MSCs from several donors.  It has been reported that best practices to qualify a manufacturing process should include “at least 3-5 donors”, and it is likely that proper Validation will require many more, and that donor selection may be required (i.e. not every donor will work in the manufacturing process). This is why we, at RoosterBio, believe in providing a number of donor MSC lots, ranging in age and sex, for use in our customer’s research and development experiments.

3. MSCs accelerate cancer…..MSCs can combat cancer

Current Bottlenecks in MSC Research: Widespread MSC Misconceptions

We blogged several months ago about bottlenecks in the bioprocessing of Mesenchymal Stem Cells that are impeding their clinical translation.  While the development of robust and scalable manufacturing methods, reduced cost of goods, and implementation of solid Quality Systems are all necessary for increased clinical use of MSCs, there are also current misconceptions surrounding MSCs, rooted in decades-old science, that are holding the field back.  I recently came across a paper from last year on this topic and decided to put it forth for discussion here – with a few targeted opinions from us scientists at RoosterBio.  It is my hope that you’ll provide your own opinions on the misconceptions detailed here, as well as your suggestions on other misconceptions you think could be holding the MSC field back.

The authors of the 2013 Cytotherapy Paper: Mesenchymal stromal cells: misconceptions and evolving concepts, Donald Phinney and Luc Sensebé, identify six major misconceptions that have persisted over the years, despite widely-accepted paradigm shifts on MSC nature and function.  Here, I will summarize four of these misconceptions and add our take to them.

Four of the misconceptions identified by Phinney and Sensebé:

1. MSCs isolated from different tissues are equivalent

Initially isolated from bone marrow in the 1950s, MSCs were then discovered in adipose tissue, and have since been found in a number of tissues including, but not limited to: Wharton’s jelly, umbilical cord blood, placenta, amnion, and dental pulp.  While MSCs from all these sources are somewhat similar in surface profile marker expression, phenotype, and gene expression profiles, their functionality, in terms of differentiation potential, immunomodulatory activity, and paracrine factor secretion, can vary widely depending on the tissue of origin.  ** Given that MSCs from a single tissue and donor are not equivalent (see below), it comes as no surprise that MSCs from different tissues vary in function! **

Rapid and Economic Generation of hMSC Spheroids for Macroscopic Tissue Biofabrication

Mesenchymal stem cells (MSCs) aggregated into three-dimensional (3D) cellular spheroids are a potent configuration for cell therapy and tissue engineering research and product development (5), and cellular spheroids are a preferred format for many bioprinting applications (10).  Cellular spheroids are essentially micro-tissues that can be manufactured as standardized “living materials” with certain controllable, measurable, and evolving material properties (10). Studies have shown that MSC aggregation into spheroids yield improved in vitro biological functionality over MSCs grown as 2D monolayer; likely due to the 3D tissue-like structure resembling the native configuration of cells in vivo with a microenvironment that allows for direct cell-cell signaling and cell-matrix interactions. MSC spheroids demonstrate enhanced cartilage, bone, and fat differentiation, as well as increased paracrine factor secretion over 2D MSC cultures (1-7). In vivo administration of hMSC spheroids has also showed enhanced therapeutic properties in pre-clinical models of myocardial infarction, bone and cartilage repair, and limb ischemia (3, 5, 8).  

Traditionally, aggregates were formed using suspension culture in spinners or shake flasks or in hanging drop cultures (5). The advancement of technologies has allowed one to quickly and easily generate large numbers of spheroids consistent in size and shape using forced aggregation in micro-wells (AggreWells, Stem Cell Technologies), or using liquid handling automation and 96 or 384 well hanging-droplet plates. There are also tools available that allow researchers to mold micro-tissues into interesting shapes such as rods, toroids, honeycombs, or whatever one can dream up (12-13).  While there are multiple methods for creating hMSC micro-tissues, the biggest challenge is reproducibly growing up sufficient hMSCs to create enough spheroids to start an experiment.  For example, if a researcher needs 10,000 spheroids with an average of 1000 cells per spheroid, then he/she will need at least 10 million cells to begin the experiment, which can take weeks to grow (see process flow diagrams below).  If he/she wishes to use 5000, or 10,000 cells per spheroid, then he/she will need 50 million or 100 million cells for his/her experiment.  This volume of cells has traditionally been very costly and time consuming to generate.

This Application Blog Post will provide a simple protocol to rapidly and economically generate tens of millions of high quality hMSCs so that researchers can minimize their time spent on routine cell culture and maximize their effort on performing hMSC spheroid-based experiments.