Good Manufacturing Practice for Cell and Cell-Based Therapies: Facilities & Quality Control

Novel cell and cell-based therapies require stringent manufacturing, testing and oversight to ensure integrity, function, and above all else, patient safety upon administration.  Tissue-derived cellular products are considered to be manufactured products and are regulated as such.  Thus, you must ensure that your cell manufacturing process is aligned to current Good Manufacturing Practice requirements.  (Note the “current”.  This means that these are evolving requirements, so you must stay up-to-date!)  In the United States, human cells, tissue and cellular- and tissue-based products (HCT/Ps) are regulated by the Center for Biologics Evaluation and Research (CBER), a division of the U.S. Food and Drug Administration (FDA).

When manufacturing cell and cell-based products, a production facility under strict Quality Control must be used.  Ideally, this facility includes the cell manufacturing suites, the storage space for raw and finished product and any laboratory/testing areas.  Thus, (1) facility design, access and maintenance, (2) equipment purchase, installation and operational qualification (I/OQ), use and maintenance and (3) raw material specifications, purchase, use and storage must all be carefully controlled.  For cell and cell-based therapies, terminal sterilization of the final product is often not possible.  As such, quality by design (QbD) is highly important in cell therapy, with stringent testing conducted on the tissue Donor (our next blog post in this series will cover this topic) to preclude risk of contamination at the source.  In addition, facility and equipment standards and monitoring must be instituted to ensure aseptic processing of cell and cell-based products.  To this end, closed systems and single-use disposables should be used whenever possible to minimize risk of contamination.  Therefore, a cGMP manufacturing facility must include clean rooms which control for temperature, humidity, pressure and air particulates, preventing any contamination of manufactured product due to the environment, materials, human handlers and cross-contamination from other products manufactured in the same facility.  As such, there should be uni-directional flow of materials and people through these areas and personnel must follow proper gowning procedures.  Facilities and equipment requirements are defined under US FDA 21CFR§211 and 21CFR§1271.

As mentioned above, stringent Quality Control systems must be in place to qualify all reagents and processes and to institute Standard Operating Procedures (SOPs) to ensure quality and consistency in manufacturing processes and the end product. 

Facility and Quality Control considerations for cGMP cell manufacturing.

Demystifying Clinical Translation of Stem Cell-Based Therapies

As our company matures and we continue to execute to our mission of accelerating Regenerative Medicine, we find ourselves increasingly involved in conversations around scalable cGMP cell manufacturing, qualification of cell and cell-based therapies, and regulations around clinical translation of hMSCs and hMSC-based therapeutics.  While the US FDA and other global regulatory agencies provide guidances on the regulations surrounding clinical translation of cellular products, it is evident that there is still great uncertainty around rules, regulations, processes, timelines and costs associated with clinical translation.

How do you move from pre-clinical to clinical studies?

How must you manufacture your cells?

What data do you need to present to the FDA to move to clinical trials?

What sort of characterization assays are needed? 

What is the process for engaging the FDA and moving to clinical trials?

When do I need to do all of this?!

These are just a few of the questions that we commonly hear from our customers.  In an effort to facilitate our customers’ path to the clinic, we will be publishing here a series of blog posts on clinical translation of hMSCs.  These posts are meant to point you to the relevant guidances and points to consider surrounding clinical translation of hMSCs and are not meant to be the definitive authority on these matters.  We recommend that you engage your Quality and Regulatory teams (and/or consultants) early on in your Process Development efforts to ensure that you are considering all regulations and guidelines in developing a scalable, clinically- and commercially-relevant process and product.

What other questions do you have around clinical translation of hMSCs and hMSC-based therapies?

Check back in next week for our first blog post in this series.

Opportunities for BioManufacturing Sciences to Accelerate Upscaled Mesenchymal Stem Cell Manufacturing Technologies

Contributed by: Timothy R. Olsen, PhD - Sr. Scientist, Process and Product Development at RoosterBio Inc.

After the National Institute for Standards and Technology (NIST) identified upcoming challenges in the U.S. biopharmaceutical manufacturing landscape (see document here), the National Institute for Innovation in Manufacturing Biopharmaceuticals (NIIMBL) was formed to help solve these challenges through the formation of a public-private partnership between industry, government, academia, and non-profits. The goal of NIIMBL is to accelerate Biopharmaceutical manufacturing innovation, support the development of standards that enable more efficient and rapid manufacturing capabilities, and educate and train a world-leading Biopharmaceutical manufacturing workforce to maintain the United States’ global lead and competitiveness in this industry. NIIMBL will be leveraging a $70 million investment from NIST, with at least $129 million more in funds from private partners. One of the great aspects of this institute is that they are encouraging partnership between large companies and smaller or medium size companies (called Small to Medium Enterprises “SME”), which will be sure to bolster innovation and streamline commercialization and implementation of new technologies. Kelvin Lee, the NIIMBL Institute Director, did a fabulous job taking the lead on organizing the Consortium's first National Meeting, where members from the United States Congress, Directors from the Food and Drug Administration, and many executive level industry representatives were invited to speak about the importance of manufacturing sciences and the current challenges we are facing as an industry. I had the opportunity to represent RoosterBio as an SME at this inaugural NIIMBL National Meeting, and I gave a talk in the “Rapid Fire” SME Innovation Showcase, as well as presented some of our work on how we are radically shortening the development timelines of Biopharmaceuticals that include a stem cell-derived component.

Confluent hMSCs on Solohill microcarriers.
Image from RoosterBio Inc

Among the many relevant talks given throughout the day, one specifically caught my attention. In his talk titled “Key Process & Assay Challenges in Cell Therapy Development,” Greg Russotti (Vice President, Technical Operations at Celgene Cellular Therapeutics) laid out challenges in the upscaled manufacturing of human mesenchymal stem cells (hMSCs). To meet the pressing need for economical manufacturing of hMSCs at clinically- and commercially- relevant scales, researchers have turned to single-use bioreactor systems that have successfully been used to manufacture other biomolecules, such as monocolonal antibodies which make up the lion’s share of blockbuster pharmaceuticals. However, unlike small molecule or large molecule production, cell therapy products are living, breathing cells, which presents unique bioprocessing constraints and challenges. Greg noted that there are technologies based on monoclonal production that can expand hMSCs in large quantities, like using 3D microcarrier-based bioreactor systems, but there are still many manufacturing innovations required before these manufacturing platforms can support a commercial cell therapy product.  

The technology gaps that he specifically mentioned for upscaled hMSC manufacturing were downstream processing technologies, specifically the unit operations related to:

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:

Guest Blog Post: The Ambitious Future of Tissue Engineering

Bagrat Grigoryan and Jordan Miller, Physiologic Systems Engineering & Advanced Materials Lab at Rice University.

Over the last several decades, various advances in tissue engineering have allowed for the not so distant possibility of replacing, repairing, or regenerating injured tissues¹. Significant progress has been made in understanding cellular biology as well as pathophysiology and healthy states of tissues. Additionally, a suite of diverse biofabrication technologies and biomaterials has been conceived, enabling fabrication of complex 3D tissues with greater physiological relevance compared to the traditional 2D context that cells are studied in². However, the field of tissue engineering still has unresolved questions involving choice of fabrication technique, biomaterial, cellular niche, or even cell type when designing a synthetic tissue.

While different fabrication techniques and biomaterials have been explored in fabricating tissues in vitro, the use of stem cells in engineered tissues is ubiquitous. Not surprisingly so, as biologists continually demonstrate novel ways of directing different lineage commitment of stem cells and further unlocking their vast regenerative potential³. Indeed, stem cell banks have emerged to cryogenically store a patient’s own cells as the therapeutic potential of stem cells is being positively demonstrated in multiple clinical trials⁴. With over 450 mesenchymal stem cell (MSC)-based trials alone currently ongoing or completed, the regenerative and immunoregulatory properties of MSCs are constantly being exploited to improve the quality of human life⁵.

Due to the immense therapeutic potential of MSCs, there is a need to rapidly and reproducibly grow a vast amount of MSCs for clinical and research purposes. Although MSCs were identified and isolated from bone marrow more than 40 years ago, we still have not fully mapped their biological characteristics³.

At the Cutting Edge of 3D Bioprinting: WBC 2016 Round Up Part II

A guest blog post by RoosterBio Travel Award winner, Ian Kinstlinger.

Presenting on Open-source Selective Laser 

Sintering at WBC2016!

Since 1980, the international community of biomaterials scientists and engineers has convened every four years to discuss the cutting edge of biomaterials research. This year’s 10^th World Biomaterials Congress (WBC) brought us to lovely Montreal, Canada for a stimulating week of workshops, talks, posters, and social activities. I was honored to present my work from the Miller Lab at Rice University in both a podium talk and a poster session.

Our lab is broadly interested in developing strategies to construct vascular networks within engineered tissues. In my research, I have developed a platform technology which uses 3D printed carbohydrates as templates around which cells and biomaterials can be assembled. Dissolving the sugar away gives you an engineered tissue with perfusable channels; we believe that these constructs will be useful for understanding the mass transport requirements and emergent properties of engineered living tissue.

An overview of one method our lab has introduced to create 

embedded vascular networks in biomaterials.

I used my poster to spread the word about our lab’s Open-source Selective Laser Sintering technology and my podium talk to describe how we’ve adapted this system to perform laser-based 3D printing of carbohydrate materials. I was thrilled to have a large audience for my talk, followed by several insightful questions. My poster also received a steady stream of visitors, many of whom are involved in the open-source hardware community and were eager to talk about hardware hacking for biomaterials. That work was actually published earlier this year – and RoosterBio hMSCs were absolutely central. Their high quality and robust differentiation response made characterizing biocompatibility of materials quite straightforward.

A couple of key presentations stood out at WBC 2016:

Nano- and Micro-fabricated Hydrogels for Regenerative Engineering

• Dr. Ali Khademhosseini, Khademhosseini Lab, Harvard University
•  Dr. Khademhosseini gave an illuminating keynote on the many angles from which his lab is using bioprinting technologies to fabricate functional biological structures. He is also emerging as a leader in the field of integrated organ-on-chip drug screening platforms.

Injection of Dual-Crosslinking Hydrogels to Limit Infarct Induced Left Ventricular Remodeling

• Dr. Jason Burdick, Polymeric Biomaterials Laboratory, University of Pennsylvania
•  The Burdick lab has developed an innovative class of supramolecular biomaterials specifically targeted for 3D printing applications. The gels are shear-thinning due to their non-covalent crosslinks, and thus are amenable to extrusion printing. These materials are also useful as injectables for reducing left ventricular remodeling after heart attack.

Photoreversible patterning of hydrogel biomaterials with site-specifically-modified proteins

• Dr. Cole DeForest,  DeForest Research Group, University of Washington
•  Much like our lab is interested in patterning biomaterial architecture via 3D printing, the DeForest group is patterning functional proteins into materials through some very clever photochemistries. Their techniques give them spatiotemporal control over the incorporation of various full proteins into synthetic hydrogels.

It was tremendously exciting to see so many investigators working on 3D printing of biomaterials. I counted at least seven sessions devoted to the topic and was also impressed by the low-cost printers and inks now hitting the market, including RoosterBio’s new ready-to-print hMSC products. The diverse hardware and materials that have been introduced in the past few years are already transforming the field! It will be very interesting to see in the coming years whether these new techniques give way to novel insights into cell and tissue function in vitro, as many groups are currently promising.

It is also not yet clear whether the same groups who are mastering the materials and fabrication technology have the resources and expertise to analyze complex biological phenomena in their printed structures. A greater level of collaboration between biologists and materials/fabrication engineers may be necessary in the future to make progress in this area. I am going to end with shameless plug for my recent review article in Lab on a Chip which discusses 3D printing approaches for fabricating vascular networks and addresses the need for increased communication between biologists and materials scientists.

WBC 2016 was an incredible conference in which I got to present my work, learn about key advances in biomaterials, meet leaders in the field, and explore Montreal. Thanks so much to RoosterBio for providing the highest quality hMSCs and for their support of my work through a travel grant! 

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! 

Comparability of hMSC Economic and Quality Attributes after Expansion in Bovine Serum Containing vs Xeno-Free Bioprocessing Media Formulations

Human Mesenchymal Stem/Stromal Cells (hMSCs), from bone marrow or other tissues, are poised to have the most significant impact on Regenerative Medicine compared to any other single cell type.  This is due to their ability to be utilized across multiple therapeutic indications due to the wide ranging functional nature of the cells (1-3).  hMSCs are not only capable of differentiating into tissue-specific cell types, but also have angiogenic, immunomodulatory, anti-inflammatory and anti-bacterial abilities (4).  hMSCs are true Tissue Repair Cells – setting the stage for all phases of wound healing and tissue repair: promoting new blood vessel growth, reducing inflammation to aid healing, secreting several mitogenic factors important for tissue building and stimulating tissue-specific stem cells. 

However, hMSCs have traditionally been challenging to source in significant volumes and at sufficient quality levels, hindering the advancement of the science into medical products.  At RoosterBio, we focus on transitioning hMSCs from a scarce into an abundant resource, and we achieve this by borrowing best practices from the Manufacturing Sciences and applying them towards the grand challenge of producing billions of hMSCs, with critical quality and functional parameters in place, and at costs and volumes that enable the rapid and wide-spread adoption of hMSC technology into clinical practice.

RoosterBio came to market 2 years ago with hMSC cell and media systems that include a highly efficient hMSC bioprocess expansion media  that simply and consistently produces greater than 100x expansion of cells with 8-10 days of culture. Our cell and media system was designed for a “batch” culture process (no media exchange required between passages), removing labor-intensive and costly media exchanges, and enabling rapid expansion with little in process intervention (thus fewer risks for contamination).  While the cell and media system has now been used in several translational and high impact publications (5-8), the expansion medium does utilize low levels of high quality bovine serum to maximize the performance and robustness of the overall system. 

In recent years, the field has been shifting towards xeno-free (XF) cell and media systems to remove any remaining safety issues related to xeno-sourced animal components (9-13). Furthermore, our customers have been requesting XF expansion options. We have listened to our customers and spent the last year developing and optimizing a fully XF media formulation based on our innovative bioprocess media platform.  The goals of this media were to remove all xeno-sourced raw materials from the formulation, while maintaining all hMSC functional properties, as well as the economic and production efficiency of our initial bovine serum containing (BSC) media formulation.  We are now ready to commercially launch our XF media to advance the industry, and this blog post will outline the initial work we have performed to evaluate the comparability of expansion, cost and functional properties of hMSCs expanded in the new XF media compared to our flagship BSC media.

Table 1. Media formulations and nomenclature.

For the purpose of this blog post, we will be comparing RoosterBio hMSC products expanded in either our initial bovine serum containing High Performance Media, or our new xeno-free High Performance Media XF formulation.

METHODS

Cell expansion. RoosterBio hBM-MSC were expanded in BSC Media and XF Media. Frozen cells were thawed and plated in triplicate at 3,000 cells/cm² in T-75 flasks and cultured for 4 days. At 4 days, cells were harvested with TrypLE (Gibco) and cell number and viability were determined on a Nucleocounter. These cells were used for the analyses below or plated again for further expansion.

Cell surface marker expression. To determine if the cells grown in XF Media were capable of expressing MSC markers, hBM-MSC expanded in both BSC and XF Media were plated and incubated in DMEM/10% FBS for 5 days prior to flow cytometry.

Immunomodulatory function. Induction of indoleamine 2,3-dioxygenase (IDO) activity by exposure of hMSCs to the pro-inflamatory cytokine IFN-γ is central to the immunosuppressive function of hMSCs (14,15). See here for a blog post on this topic. hBM-MSCs were expanded in BSC and XF Media (Donors 1 and 2) or XF Media alone (Donor 3), harvested and plated in High Performance Basal medium (SU-005) with 2% FBS at 40,000 cells/cm². After 18-22 hr of incubation, cells were treated with IFN-γ (10 ng/ml) for 24hr±1hr. The cell supernatant was collected, and the kynurenine concentration was measured using a spectrophotometric assay and normalized to number of cells and days of incubation to obtain the amount of IDO secreted (expressed as pg kynurenine secreted per cell per day).  

Angiogenic cytokine secretion. hBM-MSCs were expanded in BSC or XF Media, harvested and plated in High Performance Basal Medium with 2% FBS at 40,000 cells/cm². After 24hr±1hr culture supernatant was collected and assayed for FGF, HGF, IL-8, TIMP-1, TIMP-2 and VEGF concentration using a MultiPlex ELISA (Quansys). Cytokine concentration was normalized to number of cells and days of incubation to obtain cytokine secretion rates.

Trilineage differentiation. hBM-MSCs were expanded in BSC or XF Media, harvested and plated in High Performance Basal Medium with 2% FBS at 5,000-10,000 cells/cm²  for adipogenesis and osteogenesis or formed into 100,000 cell micromasses for chondrogenesis. On day 1, cells were switched to differentiation or control media (LifeTech StemPro Differentiation Kits) and cultured per kit protocols for 10-21 days. Differentiation was detected by Oil Red O (adipogenesis), Alizarin Red (osteogenesis), or Toluidine Blue (chondrogenesis) stains.

COMPARATIVE ANALYSES

Cell expansion. A key characteristic of RoosterBio hMSC cell and media systems is rapid cell expansion with a guaranteed 10-fold expansion within 7 days. In engineering our XF Media system, we aimed to preserve this hMSC expansion profile.  hBM-MSCs display rapid and comparable growth in both our BSC Media and the new XF Media formulations, with similar doubling times and expansion rates.  We see the typical variability across donors, but all donors are harvested at greater than 30,000 cells/cm², after plating at 3,000 cells/cm², within 5 days (Figure 1). hBM-MSC growth over 2 passages yields greater than 1 billion cells using both our BSC and XF Media (and 10M cell product vials) in less than 2 weeks (Figure 2), leading to tremendous economic benefits (described below).

Figure 1. hBM-MSCs expand efficiently in XF Media. All cell lots expanded at least 10-fold (>30,000 cells/cm2) in XF and BSC Media (data shown for 2 donor lots). Data are mean of 3 replicates +/- SD.

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:

Towards a Cell Therapy Manufacturing Technology Roadmap -- Resources

RoosterBio was founded to accelerate the development of the Regenerative Medicine field by utilizing advancements in Manufacturing Sciences.  As such, we have long been involved in laying the groundwork for a Technology Roadmap for sustainable Cell Therapy Manufacturing.  

Recently, RoosterBio was part of the first-ever Cell Manufacturing Consortium aiming to position the United States as a leading developer of Cell Manufacturing Technologies and the Chief Authority on Cell Manufacturing Standards Worldwide.  The goal of this Consortium was to establish a collaborative public-private partnership that engages industry, academia, regulators, and other stakeholders in removing barriers to the advancement of the cell-manufacturing industry, thereby bringing new therapies and diagnostics to the healthcare market.  As a result, the Consortium members have formulated an extensive Cell Therapy Manufacturing Technology Roadmap to the year 2025.  This document is in final review and should be available shortly.  In the meantime, we've compiled some resources (to which we will continue to add and seek your input as well) on Cell Therapy Manufacturing for those interested.

Relevant Publications:

• Developing Cell Therapy Biomanufacturing Processes
• Cell Therapy Bioprocessing
• Meeting Lot-Size Challenges of Manufacturing Adherent Cells for Therapy
• Developing assays to address identity, potency, purity and safety: cell characterization in cell therapy process development.

The Use of Animal Serum in the Clinical Translation of hMSCs

Human mesenchymal stem or stromal cells (hMSCs) are an integral part of cell-based therapeutics, with over 400 clinical trials recently completed or in progress using hMSCs. As more research teams transition their stem cell-based regenerative technologies to the clinic, the use of serum in the cell production process has been, and will continue to be, a necessary evil that must be managed.  Luckily, pharmaceutical regulatory agencies, driven by the biologics industry over the last 30 years, have established guidances and guidelines that have helped to demystify and clarify some critical aspects of dealing with animal components. As it is important to have an understanding of how to manage serum during the clinical translation of hMSCs, we have focused this blog post on this specific topic

There are many researchers in the MSC community who firmly believe that the FDA simply does not allow hMSCs into clinical trials if the cells have been cultured in media supplemented with animal serum. This is currently not the case, and in fact, Mendicino et al. from the Center for Biologics Evaluation and Research at the FDA reviewed all MSC regulatory filings and found that over 80% of all regulatory submissions described the use of fetal bovine serum (FBS) during the hMSC manufacturing process [1]. Several other analyses of hMSC-based clinical trials in recent years have similarly shown that at least 65-75% of trials utilize FBS [2,3,4]. Regardless, a push to remove serum from the manufacturing process still continues due to regulatory, production and supply chain concerns.  Each of these areas is detailed below.

(Note: when we refer to "clinical-grade" products below, this is not an official regulatory classification, it is meant to generally refer to materials that are destined for use in clinical testing of cell therapies.)

Regulatory Compliance      

The main regulatory concerns associated with the use of xenogeneic serum include the risk of contamination with non-human pathogens and inducing an unwanted immune response. To account for such serious consequences, the FDA has put several requirements in place for the production process of both clinical-grade FBS and hMSCs:

• FBS: Clinical-grade FBS must be derived from cattle herds grown in countries that are USDA approved for import, with well-monitored animal health status [2]. The FBS should be processed under current good manufacturing practice (cGMP) standards that set minimum requirements for the facilities, materials and protocols used [2]. Every batch or lot of FBS must be traceable back to its country, slaughterhouse and herd of origin. Finally, all lots must be tested for adventitious agents (viral contamination), sterility (bacterial and fungal elements), endotoxin levels, mycoplasma content and other constituents [2,5]. While regulatory agencies address safety, it is up to the cell manufacturer to establish metrics around performance, as FBS has traditionally been both a major cost driver and a source of process variablity.
• Clinical-grade hMSCs: Clinical-grade hMSCs must be manufactured under cGMP standards, and this topic is covered extensively in the literature.  As it pertains to serum use, each lot of serum used during the cell production process must be documented [5], and the final cell product must meet specific standards of identity, potency, purity and safety. Purity standards include freedom from unwanted contaminants (such as other cell types, endotoxins, residual proteins and animal serum) [6]. The FDA Code of Regulations for Biologics provides a guideline for vaccines that animal serum levels must be under 1 ppm in the final product formulation when serum is used in any part of the process [US FDA. 21 CFR 610.15 ].  While there is no direct guidance for cellular therapies, the 1ppm residual level has been used as a target in some cell therapy manufacturing processes [6] and is a good place to start when developing process specifications.

These checks and balances have allowed clinical trials using FBS-cultured hMSCs to be conducted safely. A meta-analysis by Lalu et al. showed that there was no evidence of infection or toxicity in any subjects involved in clinical trials using FBS-cultured hMSCs [7]. Several other clinical trials have described the use of FBS in cellular therapeutics and biologics without any adverse side effects [8-12]. That said, it is best practice to develop sufficient cell washing protocols after cell harvest, and before formulation, to remove process impurities and get serum protein levels down to acceptable levels [13].

For FDA resources on this topic, see:

Cryopreserved hMSCs maintain comparable in vitro functional activity compared to fresh hMSCs

INTRODUCTION:

Human mesenchymal stem cells (hMSC) are currently in use in over 400 clinical trials and are critical components of tomorrow’s cell-based products and devices (1, 2, 3). Secretion of biomolecules by hMSC influences many biological processes and is thought to be central to the mechanism of action. Since widespread clinical use of hMSC and cell-based therapies with positive economic outcomes will be facilitated by frozen storage, cryopreserved hMSC must maintain high levels of biological function upon thaw.  Additionally, while hMSC have an excellent clinical track record in terms of safety, efficacy data has been difficult to come by, suggesting that more standardized cell formats are needed.  This too could be addressed by effective means of cryopreservation, allowing off-the-shelf hMSC products to be widely used in Regenerative Medicine, Tissue Engineering and for 3D BioPrinting of cells and tissues.

To date, there have been conflicting results on the impact of cryopreservation on hMSC function.  The Galipeau lab showed that cryopreserved MSC have impaired immunosuppressive function in response to the pro-inflammatory cytokine, IFN-γ (lower IDO response, and decreased T-cell suppression) relative to proliferating cells (5, 6). The LeBlanc group similarly found that cryopreserved hMSC have reduced responsiveness to IFN-γ, decreased production of anti-inflammatory mediators, and impaired blood regulatory properties (7). In contrast, other studies support the use of cryopreserved hMSC.  The Mueller lab showed that cryopreservation of hMSC did not change the cells’ immunomodulatory activity, viability, or differentiation (8). The Weiss group also performed in vivo tests of thawed hMSC and found that “in an immunocompetent mouse model of allergic airways inflammation … thawed MSCs are as effective as fresh MSCs.” (9)  The difference in results is likely due to differences in the cryopreservation formulations, controlled rate freezing protocols, and how the cells are thawed and handled prior to implantation.

To address the critical issue of cryopreservation in our hMSC systems, we compared the biological activity of RoosterBio hMSCs from 2 donors either (a) with cells straight out of cryopreservation (THAW) or (b) with cells that had been in culture for at least 5 days (FRESH), while controlling for PDL.  Based on the literature, we established a conservative  hypothesis for this study that cryopreserved hBM-MSC would exhibit diminished immunosuppression and altered angiogenic cytokine secretion compared to proliferating hBM-MSC in response to challenge by inflammatory cytokines. We tested this hypothesis with RoosterBio’s hBM-MSC, produced with GMP-compatible and scalable manufacturing processes, by comparing the immunomodulatory activity and angiogenic cytokine secretion of proliferating (FRESH) to cryopreserved and thawed (THAW) hBM-MSC.  By presenting the results of this study, we hope to provide additional data points for the industry on the use of cryopreserved, off-the-shelf hMSCs for Regenerative Medicine, Tissue Engineering and 3D BioPrinting.

METHODS AND EXPERIMENTAL DESIGN:

See you at MSC 2015?

MSC 2015 is quickly approaching next month and we at RoosterBio are getting ready.  This conference is arguably the single most important conference related to MSCs, and Cleveland is the considered by many to be the birthplace of the current paradigm of MSCs used in therapeutic contexts.  We will be sending most of our company, and we do look forward to seeing everyone there. Not only is this conference full of great sessions and talks, but the networking at this bi-yearly MSC conference is always top notch and yet another reason to attend.

The faculty and sessions at MSC 2015 are hyper-relevant to today’s more important topics, and the sessions are organized by several key themes.  Day 1 of the conference will be kicked off with a Keynote from Arnold Caplan , the godfather of MSCs (and yes, if you Google “MSC Godfather” you get Arnold Caplan), who is always entertaining and insightful to where MSC technology is going. The sessions look to be focused on Clinical Trial updates by the likes of Athersys, Katerina LeBlanc, Dan Weiss and Jacques Galipeau, among others. 

Day 2 of the conference gets kicked off with a keynote from Frank Barry from The National University of Ireland at Galway, and he will be speaking on MSC Translation.  My favorite topic, MSC BioManufacturing, will be covered that morning, and we all know that MSC technology cannot be translated into humans without consistent, robust and cost effective manufacturing processes that are capable of maintaining the quality parameters and functions of these critical cells.  Sessions on MSCs in applications like cardiology and organ transplantation will follow, and the day will end with the session I am most excited about – Next Generation MSCs.  Jan Nolta and Mike West will highlight this “not to miss” session.

The final day of the conference will have a keynote from Stanton Gerson, followed by many new and impactful applications including MSCs in Cancer and Sepsis.  The last two sessions are on potentially the most impactful translational areas of MSCs (as it pertains with shear numbers of patients treated), which are the use in Sports Medicine and Veterinary Sciences.  I will bet that Bob Harman at Vet Stem has treated more patients with MSCs than any other clinic or company in the World – and I plan on asking him what that number is at the conference, so look for it in our Twitter feed.

It does look like the dedicated organizing team at Case has done a great job at organizing yet another stellar event, and we look forward to seeing you there.  Be sure to stop by our Booth and posters and say hello!