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


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

July 22, 2015

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

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.


July 13, 2015

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!

April 22, 2015

Adipose- and Bone Marrow-Derived hMSCs: What's the Difference?


Human Mesenchymal Stem/Stromal Cells, or hMSC, are key components of future therapeutics, engineered tissues, and medical devices and are currently in use in over 400 clinical trials (1). Bone marrow-derived MSC (hBM-MSC) have historically been the most widely used hMSC, but hMSC can be isolated from many tissues of the body including fat, umbilical cord blood, dental pulp, Wharton’s jelly, and peripheral blood. In recent years, human Adipose (or fat) tissue-derived MSC (hAD-MSC) are increasingly used in studies due to adipose tissue having a higher frequency of MSC than bone marrow and the relative ease of collection (2). You can find more information on hMSC by following these links:

A common MSC misconception is that MSC isolated from different tissues are equivalent. hAD-MSC and hBM-MSC, and cells from other tissues, can meet the “traditional” ISCT criteria to identify a cell as an MSC (3,4): adherence to plastic, characteristic surface marker expression profiles (positive for CD73, CD90, CD105; negative for CD34, CD45), and trilineage differentiation to fat, bone, and cartilage.  However, there is widespread acceptance that hMSC achieve their biologic and therapeutic effects in vivo by secreting many bioactive molecules (referred to as the hMSC secretome) that moderate a variety of processes including angiogenesis, immunosuppression, and overall “tissue repair” (5). Despite being similar overall, hMSC isolated from adipose and bone marrow display some differences in functional capabilities (2,6). For example, hBM-MSC are more robust in bone and cartilage differentiation than hAD-MSC and hAD-MSC are more efficient at stimulating angiogenesis than hBM-MSC (2,6,7).

We have recently been applying our manufacturing protocols to adipose-derived hMSC (our newest product) and would like to share some of the similarities and differences in function between hBM-MSC and hAD-MSC that we have observed when these cells are cultured in our media systems with our protocols.  Both populations of hMSC have been manufactured using our GMP-compatible and scalable manufacturing processes, with standardized procedures and with rigorous quality control.  By reporting the differential functional characteristics of these hMSC populations, we assist our customers in making more informed choices on the cell type best-suited to their application(s).


Materials & Reagents:  Cell culture reagents, excluding RoosterBio materials, were purchased from Life Technologies, chemicals and reagents for kynurenine measurement were from Sigma, and cultureware was from Corning.  Two vials (1 million cells each) of hAD-MSC, representing two donors, were purchased from ZenBio, and used only for comparison. Other cell products used were RoosterBio hMSC products: Bone Marrow-derived MSC (hBM-MSC, part # MSC-001, MSC-003) and Adipose-derived MSC (hAD-MSC, part # MSC-020, MSC-021). Cells were cultured in RoosterBio High Performance Media (part # KT-001) or DMEM + 10% FBS

Methods: All methods for the analyses shown below are documented under RoosterBio’s Quality Control systems.  For more information, please contact us at info@roosterbio.com.  Detailed methods for priming hMSC can be found in a previous blog post here.


April 13, 2015

NIST Workshop Aims to Educate on Improving Confidence in Measurements Critical for Cell Therapy Products

We are always looking for ways to enable the commercialization of Cell-based Therapies and Technologies.  The ultimate success of the field is dependent on the convergence of several technology fields, and one that isn't given sufficient attention is the Measurement Sciences (or metrology).  The National Institute of Standards and Technology (NIST) has a Biosystems and Biomaterials Division that has several projects focused on their mission of "Building Confidence in Biological Measurements", and they have several people that are working to advance measurements in regenerative medicine.

The NIST BioSystems and Biomaterials Division has several projects related to stem cells and regenerative medicine.
On May 11&12, NIST will be holding a workshop focused on Measurement Assurance for Cell Therapy Products.  The Registration for the workshop is open and is limited to the first 100 registrants, and the Agenda (below) has speakers from Industry, the FDA, NIST, as well as several breakout workshops to focus on some of the analytics that are the low hanging fruit to bring standardization too.

The adage of "Measure Twice, Cut Once" only holds true if you have robust and precise methods and assays, and solid reference standards for which everyone can compare.  Our previous posts on Regenerative Medicine Standardization, and of course the great content on this at the Stem Cell Assays blog are good places for background reading for those interested.

February 6, 2015

An Open Letter to the Builders of the Cell-based BioEconomy

Dear Stem Cell Pioneer:

February marks the one year anniversary of RoosterBio shipping our first stem cell products to our valued customers, and I personally am very excited for the coming year ahead. We look forward to delivering even more high quality stem cells to people like you that are doing amazing things.

Looking forward into 2015 and beyond, I want to make sure we are staying true to our mission: to greatly increase the availability and accessibility of stem cell technology to researchers and product developers across the globe – and that we are committed to our vision of accelerating the pace of product development in the cellular therapy, bioprinting and tissue engineering markets.  I am hoping to focus our efforts to making sure that we are moving your discoveries and developments forward faster than anticipated.  This will not just be a win for our customers and RoosterBio, but for the entire Regenerative Medicine field.

In 2014 after launching our hMSCs in the unprecedented product format of 10 million cell vials to glowing reviews, we quickly implemented our Starter Kits and Working Cell Bank formats based on your feedback.  These new formats allow for accelerated testing, performance verification, and standardized small scale experimentation with reproducible outcomes.  We also initiated multiple collaborations with leaders in the tissue engineering and biofabrication fields – which has led to multiple conference posters, presentations, and soon to be submitted publications.

We also find ourselves at the precipice of a boom in biofabrication technologies, and we consider this the beginning of the Golden Age of Tissue Engineering.  I anticipate great progress will be made at an increasingly rapid pace.  Now that many of the tools required for bioprinting are becoming “democratized” (simpler, less expensive, more accessible) such as 3D BioPrinters, biomaterials, and primary cells – laboratories can get up and running in a matter of weeks with limited initial resources, something that would have taken months to years and extensive capital in the past.  We are at a special time, and the entire field will be accelerating forward at a rapid pace, making biofabrication truly an exponential medical technology.

2015 will truly be an exciting year for RoosterBio.  We will be participating in a Stem Cell Manufacturing Training Program, helping to organize several conferences on Cell Therapy BioProcessing and BioPrinting, exhibiting at multiple conferences, as well are contributing to initiatives such as the Georgia Tech Cell Manufacturing Consortium and the NIST Workshop on Strategies to Achieve Measurement Assurance for Cell Therapy Products.  The primary motivation behind these initiatives are to make sure that we are driving forward our vision and delivering on our mission.

None of this would be possible without the hard work and dedication of the entire RoosterBio team, 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 & Technology Officer
RoosterBio Inc.

January 27, 2015

The Rise of BioFabrication and BioPrinting in Tissue Engineering & Regenerative Medicine – notes from TERMIS 2014 Annual Meeting

RoosterBio participated in the annual Tissue Engineering and Regenerative Medicine International Society’s Annual Meeting of the Americas chapter (or TERMIS-AM for short) in Dec 2014.  You can find a lot of content on the meeting at the conference website where you can download the program for free, as well as read the published abstracts in the journal Tissue Engineering.  The final registration numbers for the 2014 TERMIS-AM conference in DC was 842 (about a 7% increase over last year’s conference).  There were 30 countries represented at the conference, with a total of 202 oral presentations and 338 poster presentations (Stats from Sarah Wilburn at the TERMIS head office).  We are looking forward to the 2015 TERMIS World Congress, which will be in Boston in early September, 2015.

There were two striking trends that were gleaned from the conference that I wanted to outline over a couple of blog posts.  First, there was a noticeable rise in the number (and quality) of the Biofabrication-related talks and posters (this blog post will focus on this).  The second trend to note was the rise in Product Development content at the 2014 meeting – and this will be the focus of a subsequent blog post.  Interestingly, the intersection of these two topics (manufacturing process technologies and product development) has traditionally been crucial for the successful commercialization of high tech products, including biopharmaceuticals (see recent HBR article by Pisano and Shih here).  

Our favorite booth (after the RoosterBio booth, or course) was BioBots', who were
showing off the beta version of the BioBot Rapid 3D Bio-Prototyper.

The Rise of BioFabrication and BioPrinting in Tissue Engineering
TERMIS has always been a great conference for academic Tissue Engineering technologies.  The major comment that I always heard from fellow industrialists was just how “academically” focused the conference was.  Meaning that the

January 17, 2015

Welcome to the Golden Age of BioPrinting, Tissue Engineering and BioFabrication

A"Golden Age" is defined as a period of time in a field where "great tasks are accomplished."  The ancient Greek philosopher Hesiod initially coined this phrase, and I think if he were alive today, he would agree with us that we are in a special time of technology convergence where innovations and advancements are progressing at an accelerating rate.  The fields of Tissue Engineering and Regenerative Medicine are benefiting from these rapid technology advancements.

We are now at the beginnings of the Golden Age of BioFabrication.  The last 20 years has seen steady progress in the Tissue Engineering field, but the cost and time it has taken to develop products based on these technologies has been prohibitive.  Thus, only the best funded labs have been able to perform this very expensive R&D.  Within the last year, products such as high volume stem cells (via RoosterBio) and low cost bioprinters (from our collaborators BioBots) have been coming to market and dramatically reduce the cost, the time, and the complexity to fabricate three dimensional biological structures that are the precursors to tomorrow's tissue engineered products.  By removing the technology and cost barriers and democratizing biofabrication technology, more labs can now afford to do the applied R&D, allowing more work to be accomplished faster, completely changing the equation of how labs function.  This is accelerating the development of this entire field.

 Walter Isaacson makes the point over and over in his new book The Innovators that collaboration between people and groups with complementary skill sets is essential to innovation and technology progress.  We, at RoosterBio, have always said that communication platforms (such as social networks, conferences, biohacker spacers, blogs, and journals) are also critical for those in a field to share knowledge and experiences - further progressing the thought convergence.  This February 9th and 10th in Boston is a focused conference on Tissue Engineering and BioPrinting that SelectBio is hosting.  The top researchers, thought leaders, and product developers in the field will be presenting cutting edge research, technology development, and commercialization strategies.  We hope to see you there.