June 23, 2017

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.

June 15, 2017

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:

April 14, 2017

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:

January 3, 2017

Guest Blog Post: The Ambitious Future of Tissue Engineering

Bagrat Grigoryan and Jordan MillerPhysiologic 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.

June 16, 2016

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 10th 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! 

June 14, 2016

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! 

May 10, 2016

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.


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.


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.

April 4, 2016

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:

March 28, 2016

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:

February 23, 2016

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!


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.

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.

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