Cells as Bioinks for 3D Bioprinting

Authored by Mayasari Lim, PhD, Regional Account Manager, West Coast, RoosterBio

Bioprinting overview

The field of 3D bioprinting has exploded in recent years largely due to the advances in additive manufacturing technology along with progress in material chemistry and tissue engineering techniques. The key ingredients that make up the complex bioprinted structures are comprised of hydrogel-based biomaterial/s often coined as ‘bioinks’ and a cellular component that serves as building blocks to create a 3D printed biological tissue. Clearly, the choice of the cell source, proteins and other biological ingredients depends largely on the desired final application. For the purpose of this blog, we will only focus on the desire to create bioprinted tissue for clinical translation.

Which cell should I use?

Cells for clinical use can be derived from the patient (autologous) or a donor (allogeneic). In many tissue engineering applications, stem cells are used due to their properties in self-renewal and differentiation. Adult stem cells, currently being the most clinically viable solution, include hematopoietic, mesenchymal, neural and epithelial. While hematopoietic stem cell transplantation is widespread, it has limited utility in tissue engineering applications due to its limited differentiation capabilities primarily toward blood lineages. Neural stem cells are most effective in neural regeneration but the limited source makes it very challenging to become clinically relevant. Mesenchymal stem/stromal cells (MSCs), on the other hand, has a significant advantage due to its tri-lineage differentiation ability and immunomodulatory functions thus it has been widely used in various therapeutic indications including brain trauma, graft-versus-host disease and cardiovascular disease. Moreover, MSCs have already demonstrated clinical safety in > 800 clinical trials treating over 30,000 patients to-date.

How many cells do I need?

In order to print a 3D tissue, we need a significant number of cells seeded at a relatively high density to achieve full tissue mimicry. Exactly how many cells would one require? Let us take a look at a simple example of the knee meniscus. The figure below (left) illustrates a 3D model of an adult meniscus with rough dimensions of 3.5 cm in diameter and 5.3 mm in height. If we were to print this structure at 30% infill, it would require a total volume of ~2.5 mL of bioink. Several studies in bioprinting cartilage tissues have reported that cells would need to be seeded at high densities, a minimum of 10-25 million cells/mL in order to form cartilage in vivo[1, 2]. Thus, in this print, we would require roughly 62.5 million MSCs for a single print. For an intervertebral disc with a diameter of 4 cm and height of 10 mm (Figure on the right), the total volume of bioink required to perform a print would be 4 mL thus the total number of cells required would be 100 million for a single print. These two examples serve to illustrate the number of cells required to bioprint a simple 3D tissue. For larger tissues or organs, one can imagine that you will need a significantly higher number of cells, in the orders to 10 billion or more, to achieve desirable cell distribution through the tissue.

Traditional methods of expanding MSCs in the lab using 2D tissue culture flasks will require significant amount of time (weeks) and labor to obtain enough cell numbers given that most commercially available MSC vials are sold in 0.5-1 million cells/vial. Fortunately, RoosterBio has developed a ready-to-print cell vial (RoosterRTP™) with 50 million cells in each vial that significantly reduces and/or eliminates the need for growing cells in the lab so that researchers in the field of tissue engineering and bioprinting can focus on their research rather than worry about optimizing their expansion protocols. At the development stage, this would significantly reduce the amount of time for researchers to conduct multiple experiments thus generating more data in a shorter time. To scale such a process for commercial production, it would be necessary to estimate the required manufacturing lot size as described in our previous blog to build an effective multi-year process development program. In the case of the knee meniscus, there are over 700,000 patients requiring meniscus surgery in the US alone. To supply even 20% of this requirement, one would require 140,000 bioprinted knee meniscus which would be equivalent to 8.75 trillion cells annually.

References:

1. Cohen et al., 2018. Tissue engineering the human auricle by auricular chondrocyte-mesenchymal stem cell co-implantation. PLoSOne13(10): e0202356. doi: 10.1371/journal.pone.0202356

2. Moller et al., 2017. In vivochondrogenesis in 3D bioprinted human cell-laden hydrogel constructs. Plast Reconstr Surg Glob Open. 5(2): e1227. Doi: 10.1097/GOX.000000000000127

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.

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! 

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:

Enabling a New Paradigm in hMSC Suspension Bioreactor Cultures

Introduction

RoosterBio is introducing a new product for highly efficient bioreactor expansion of human Mesenchymal Stem/Stromal Cells (hMSCs) that we are calling RoosterReplenish-MSC.  This innovative, first-in-class stem cell product is a concentrated bioreactor feed that replaces nutrients and growth factors that have been depleted during microcarrier expansion of hMSCs and replaces the need for a media exchange, enabling scalable and efficient fed-batch hMSC bioreactor expansion processes.  This is the first of several new products that we will launch enabling a cell therapy and tissue engineering bioprocess revolution that will be the foundation of a sustainable Regenerative Medicine Industry.

Media Designed for Scale-up

Human stem cells, characterized by their multi-lineage differentiation potential, tissue regenerative capacity, and high proliferation rates, are the most critical raw material in Regenerative Medicine today. Most cell-based therapies require between 50 million and >1 billion cells per patient application, necessitating efficient expansion (i.e. manufacturing) of starting cell sources.  Today, the most widely used cell expansion platforms for stem cell culture are planar technologies such as flasks and multi-layer cell factories (Rowley et. al), but it is generally accepted that lot sizes and COGS generated from these platforms are insufficient to meet the demand of a widely-used commercial product (Simaria et al).

Production technologies such as single use suspension bioreactors (used routinely in protein, monoclonal antibody and vaccine production) are proven to be robust, scalable manufacturing platforms. These platforms operate in a closed and controlled environment, which minimizes the risk of contamination, and are shown to reduce the time, expense, and carbon footprint required for cell processing. More importantly, it has been shown that such systems can yield lot sizes of hundreds of billions to (eventually) >1 trillion cells per manufacturing run, producing commercially-relevant lot sizes (Rowley et. al). 

MSC expansion in suspension bioreactors is typically done by growing cells on adherent substrates, such as microcarriers (Chen et al, Schnitzler et al, Szczypka et al).  Optimization of an efficient MSC bioreactor culture is central to maximizing yields and recovering healthy, functional cells at harvest. Another key attribute to efficient manufacturing processes is cost, and minimizing cost is crucial for building successful business models around MSC-based regenerative therapies.  Cell culture media is consistently the main cost driver of any stem cell production process, and it is critical to minimize media usage to keep production costs to a minimum (Rowley et al). Optimization of MSC-microcarrier cultures typically involves either full or partial media exchanges to manage nutrient supply and waste build-up (Goh et. al, Reichmann et al, Nienow et al, Santos et al, and Heathman et al), which is expensive and impractical at larger scales of >50L culture. This media exchange mentality is driven by the fact that commercially-available hMSC media formulations have been designed for flask-based culture processes and full media exchanges.  

Half media exchanges are the simplest to perform in small scale; however, when spent medium is only partially replaced with growth medium, the final concentration of nutrients and growth factors required for optimum cell proliferation are significantly reduced, resulting in lower cell proliferation rates. In addition, this procedure is time consuming, and the feasibility at larger scales decreases. Fed-batch culture, on the other hand, is more efficient in reducing processing time, mitigating contamination risk, and reducing costs associated with waste management such as time, labor, equipment and facility required to prepare and handle spent media. Hence, a new media design philosophy is required for suspension-based hMSC culture, and RoosterReplenish-MSC, coupled with RoosterBio’s High Perfromance Media kit, is the first media system designed specifically for hMSC bioreactor culture.

RoosterReplenish-MSC, a concentrated bioreactor feed, replaces nutrients and growth factors that have been depleted from RoosterBio’s High Performance Growth Media (KT-001) during extended culture. The nutrient boost provided by RoosterReplenish-MSC replaces the need for partial or full media exchanges when using our rich basal media, yielding a more streamlined culture process for hMSC expansion in bioreactors, and enabling efficiency in media utilization. 

In the next section, we will describe a series of studies performed with RoosterReplenish-MSC in microcarrier suspension culture.

Experimental Methods, Results & Discusssions

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!