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.

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.

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!



References:

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

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

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

February 12, 2016

Dear Stem Cell Pioneers:

     I am as excited as ever about Cell Therapy and Regenerative Medicine, and the role that RoosterBio is playing in advancing these Industries.  Each February marks the anniversary of when we shipped our first stem cell products – 2 years now on the market!  We have shipped our products to 13 countries worldwide, across 5 different continents, and are continuing to expand our reach in the stem cell marketplace.  As the CEO of RoosterBio, I like to take time to reflect during these milestones on where we have been, where we currently are, and where we are going.  This is important to make sure that we are still being true to the mission of the company, the vision that we hold for the industry, and to course correct if necessary.  This keeps us on our trajectory of helping to build a sustainable, lasting business that is helping you, our customers, do amazing things with living cells. 

     Looking forward into 2016 and beyond, our mission is still intact: to greatly increase the availability and accessibility of stem cell technologies to researchers and product developers across the globe.  We remain firmly committed to our vision of accelerating the pace of product development and clinical translation in cellular therapy, bioprinting and tissue engineering.  We have made great strides in 2015 towards these goals, and 2016 brings great opportunity as we continue our journey.

     In 2015, we built upon our hMSC technology platform to launch products focused on two key areas critical to the future of Regenerative Medicine: bioprinting and stem cell manufacturing sciences.  We launched several unique First-In-Class products that greatly simplify R&D in these critical stem cell sub-disciplines, lowering barriers and accelerating discovery and eventual commercial translation. These unprecedented products include:
  • RTP (Ready-to-Print) cells, the first stem cell reagent
  • RoosterReplenish, the first hMSC suspension bioreactor media feed
  • Bioreactor kits and accompanying protocols to simplify and streamline scalable suspension bioreactor expansion of MSCs
  • Bioprinting kits and accompanying protocols for simplified mixing and printing of living cellular bioinks
     Our customers have also been very busy working on their own innovations, and the last year has brought multiple abstracts presented at international conferences (including the ORS, ISCT and ISSCR Annual Meetings and the TERMIS World Congress), the first publications using our cells for bioreactor expansion and engineering massive tissue constructs, as well as numerous grant proposals that have been submitted using our products as key reagents. 

     The government’s interest in Therapeutic Cell Manufacturing Sciences has also expanded, and it is accepted now that the US must invest in these technologies and build our own infrastructure and talent base in this critical field (see here and here).  We had the pleasure of participating on the executive committee of a NIST-funded Cell Manufacturing Consortium with many academic and industry leaders in the field of Therapeutic Cell Manufacturing. The output of this is a Technology Roadmap for Scalable Cell Therapy Manufacturing that will drive funding areas in this field for years to come (link to be added as soon as the Roadmap is finalized and published).

     None of this would be possible without the hard work and dedication of the entire RoosterBio team (and we have already added to the team in 2016!), as well as the support that we are getting from you, our valued customers.  Please continue to join us on our journey as we accelerate the development of the cell-based BioEconomy.



All the Best from Frederick, Maryland. 

Jon A Rowley

Chief Executive and Technology Officer

RoosterBio, Inc.

October 8, 2015

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:

August 12, 2015

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