December 12, 2014

Priming of hMSCs to Improve Potency


By Iain Farrance, Priya Baraniak, and Jon Rowley. RoosterBio.

In this blog, we will present internal data and information on priming RoosterBio’s bone marrow derived human MSCs (hMSC) with pro-inflammatory molecules and the impact of these priming protocols on hMSC immunomodulatory function and angiogenic cytokine secretion.

INTRODUCTION:
Human Mesenchymal Stem/Stromal Cells, or hMSCs, are key components of future therapeutics, engineered tissues, and medical devices. There are currently over 400 clinical trials investigating hMSCs as therapies (1). The trials have produced some promising results, with hMSCs generally deemed safe, and in some cases effective (2). It is believed that these versatile cells achieve their biologic and therapeutic effects by secreting a plethora of biomolecules (referred to as the MSC secretome) that moderate a variety of processes including angiogenesis, immunosuppression, and overall “tissue repair” (3-6). As the secretome is one of the likely Mechanisms of Actions (MOA) of hMSC therapies, there is a significant amount of recent work on engineering hMSC preparations to enhance secreted factors by genetic modification, by culture strategies, or by engineering the hMSC microenvironment (5, 7-11). In addition, a recent ISCT paper (12) advances the concept of “priming” hMSCs by exposing the cells to pro-inflammatory cytokines prior to implantation.

Thus, priming of hMSCs can have two primary purposes:

  1. To assess human MSC preparations in vitro as recommended by the ISCT and the FDA (12, 13), and
  2. To enhance hMSC potency (survival, immunosuppression, homing) prior to implantation (8, 9, 14).

As part of our standard quality control (QC) testing, RoosterBio analyzes the immunosuppressive capability of our cell lots through priming with IFN-γ. The hMSC response (i.e. immunomodulatory potential) is reported as a measure of indoleamine 2,3-dioxygenase (IDO) activity, determined by measuring the amino acid kynurenine in the culture supernatant.  The IDO enzyme converts L-tryptophan to N-formylkynurenine (or kynurenine), an immunosuppressive molecule that acts as an inhibitor of immune cell proliferation - including T cells (12, 15, 16).  Testing every hMSC lot for inducible IDO activity provides a quality assurance that the cells we release have some level of functional potency as it relates to immunomodulation– which we consider a key quality attribute of hMSCs.

While researchers are beginning to implement testing of hMSC preparations for inducible IDO activity prior to implantation, few are looking at the impact of priming on other hMSC functions.  Here, we present information on priming of RoosterBio’s hMSCs with IFN-γ ± TNF-α across multiple lots and donors and the impact of such treatment on hMSC IDO activity and angiogenic cytokine secretion.  The goal of this blog post is to demonstrate that priming has impacts on several functional properties of hMSCs, and that researchers should consider priming regimens to (a) understand the potency of their specific cell products, especially in inflammatory environments, and (b) to potentially increase potency of these cell products upon therapeutic administration.

METHODS & EXPERIMENTAL DESIGN:

Materials & Reagents
Cell culture reagents were purchased from Life Technologies, chemicals and reagents for kynurenine measurement were from Sigma, and cultureware was from Corning.  Other products are: Bone Marrow-derived human MSCs (BM-hMSC, part # MSC-001, RoosterBio) and RoosterBio High Performance Media kit (part # KT-001).

Table 1: Experimental design.

hMSC Priming

November 6, 2014

Current Bottlenecks in MSC Research: MSC Misconceptions - Part II

 http://andreyev.com.au/wp-content/uploads/Misconceptions.jpg
We blogged recently about Mesenchymal Stem/Stromal Cell (MSC) Misconceptions that are holding the translational cell therapy field back, as identified by Donald Phinney and Luc Sensebé.  Since we have come to market with our own hMSC product lines, we have spoken with hundreds of MSC researchers and engineers, and we have compiled our own set of misconceptions that we think build off of Dr. Phinney’s and Dr. Sensebe’s initial concept.  This blog post is to share some of the market-based feedback that we have received.

To the list that was published in Cytotherapy, we would like to contribute the following list to the conversation:

1. Tracking MSC passage number is an accurate and reliable means of tracking cell age and standardizing experimental workflow
In many research laboratory environments, cellular age is most often tracked by the number of times a cell has been passaged; however, Passage Number is quite imprecise and not very acceptable as one gets into regulated environments such as translational clinical activities.  It is generally accepted that tracking the Population Doubling Level (PDL) or Cumulative Population Doublings (CPD) of primary cells is a best practice on understanding cellular age in vitro Since it is well documented that PDL impacts hMSC function (see here, here and here), in order to drive consistency into experiments, it has become a best practice to perform experiments or develop products with cells in a consistent range of population doublings where the cell function of interest is still robust.  Furthermore, regulatory agencies are beginning to require reporting of PDLs, or at least cell seeding and harvest densities, for primary cells intended for therapeutic use.  In an effort to drive adoption of PDL tracking and reporting, we’ve created a Best Practices Educational Powerpoint, and free, easy-to-use PDL calculator worksheet we’re happy to share with colleagues.  For your copy, just email us at info@roosterbio.com or subscribe to our blog!

2. Performing experiments with one MSC donor and/or lot is adequate for publication and moving forward with pre-clinical studies
Despite indications of clinical effectiveness of MSCs, there is repeated news of the failure of high-profile MSC trials to demonstrate efficacy in a number of therapeutic applications.  It has been suggested that the large amount of intra- and inter-donor variability in the MSC populations used in these trials may be responsible for their falling short of expectations despite highly encouraging in vitro and in vivo pre-clinical data.  Thus, to ensure the robust production of functional MSC products over a range of applications, experiments should be conducted and systems validated with MSCs from several donors It has been reported that best practices to qualify a manufacturing process should include “at least 3-5 donors”, and it is likely that proper Validation will require many more, and that donor selection may be required (i.e. not every donor will work in the manufacturing process). This is why we, at RoosterBio, believe in providing a number of donor MSC lots, ranging in age and sex, for use in our customer’s research and development experiments.

3. MSCs accelerate cancer…..MSCs can combat cancer

October 24, 2014

Current Bottlenecks in MSC Research: Widespread MSC Misconceptions



We blogged several months ago about bottlenecks in the bioprocessing of Mesenchymal Stem Cells that are impeding their clinical translation.  While the development of robust and scalable manufacturing methods, reduced cost of goods, and implementation of solid Quality Systems are all necessary for increased clinical use of MSCs, there are also current misconceptions surrounding MSCs, rooted in decades-old science, that are holding the field back.  I recently came across a paper from last year on this topic and decided to put it forth for discussion here – with a few targeted opinions from us scientists at RoosterBio.  It is my hope that you’ll provide your own opinions on the misconceptions detailed here, as well as your suggestions on other misconceptions you think could be holding the MSC field back.

The authors of the 2013 Cytotherapy Paper: Mesenchymal stromal cells: misconceptions and evolving concepts, Donald Phinney and Luc Sensebé, identify six major misconceptions that have persisted over the years, despite widely-accepted paradigm shifts on MSC nature and function.  Here, I will summarize four of these misconceptions and add our take to them.
Four of the misconceptions identified by Phinney and Sensebé:

1. MSCs isolated from different tissues are equivalent
Initially isolated from bone marrow in the 1950s, MSCs were then discovered in adipose tissue, and have since been found in a number of tissues including, but not limited to: Wharton’s jelly, umbilical cord blood, placenta, amnion, and dental pulp.  While MSCs from all these sources are somewhat similar in surface profile marker expression, phenotype, and gene expression profiles, their functionality, in terms of differentiation potential, immunomodulatory activity, and paracrine factor secretion, can vary widely depending on the tissue of origin.  ** Given that MSCs from a single tissue and donor are not equivalent (see below), it comes as no surprise that MSCs from different tissues vary in function! **

October 2, 2014

Rapid and Economic Generation of hMSC Spheroids for Macroscopic Tissue Biofabrication


Mesenchymal stem cells (MSCs) aggregated into three-dimensional (3D) cellular spheroids are a potent configuration for cell therapy and tissue engineering research and product development (5), and cellular spheroids are a preferred format for many bioprinting applications (10).  Cellular spheroids are essentially micro-tissues that can be manufactured as standardized “living materials” with certain controllable, measurable, and evolving material properties (10). Studies have shown that MSC aggregation into spheroids yield improved in vitro biological functionality over MSCs grown as 2D monolayer; likely due to the 3D tissue-like structure resembling the native configuration of cells in vivo with a microenvironment that allows for direct cell-cell signaling and cell-matrix interactions. MSC spheroids demonstrate enhanced cartilage, bone, and fat differentiation, as well as increased paracrine factor secretion over 2D MSC cultures (1-7). In vivo administration of hMSC spheroids has also showed enhanced therapeutic properties in pre-clinical models of myocardial infarction, bone and cartilage repair, and limb ischemia (3, 5, 8).  

Traditionally, aggregates were formed using suspension culture in spinners or shake flasks or in hanging drop cultures (5). The advancement of technologies has allowed one to quickly and easily generate large numbers of spheroids consistent in size and shape using forced aggregation in micro-wells (AggreWells, Stem Cell Technologies), or using liquid handling automation and 96 or 384 well hanging-droplet plates. There are also tools available that allow researchers to mold micro-tissues into interesting shapes such as rods, toroids, honeycombs, or whatever one can dream up (12-13).  While there are multiple methods for creating hMSC micro-tissues, the biggest challenge is reproducibly growing up sufficient hMSCs to create enough spheroids to start an experiment.  For example, if a researcher needs 10,000 spheroids with an average of 1000 cells per spheroid, then he/she will need at least 10 million cells to begin the experiment, which can take weeks to grow (see process flow diagrams below).  If he/she wishes to use 5000, or 10,000 cells per spheroid, then he/she will need 50 million or 100 million cells for his/her experiment.  This volume of cells has traditionally been very costly and time consuming to generate.

This Application Blog Post will provide a simple protocol to rapidly and economically generate tens of millions of high quality hMSCs so that researchers can minimize their time spent on routine cell culture and maximize their effort on performing hMSC spheroid-based experiments.


September 24, 2014

How Quickly are Cell-based Products Really Developing? Thoughts from the IBC Cell Therapy Bioprocessing Conference

Last week was the 4th Annual IBC Cell Therapy Bioprocessing Conference.  IBC (home of the BioProcess International conference) was the first conference organizer to dedicate a focused meeting on Cell Therapy Manufacturing Technologies 4 years ago.  Since the first conference in 2011, the growth in the field, and the conference, has been amazing.  The attendance has grown from less than 90 in year 1 to over 200 this year.  The content has also evolved heavily over the last 4 years, demonstrating a high level of sophistication and maturity in a field that seems “early stage” to those looking in from the outside.  The talks this year increased in the amount and quality of data presented. Topics included the impact of automation on the simplification, streamlining, and cost reduction of autologous therapies, the use of Quality by Design (QbD) in bioreactor scale-up and analytical development, advances in tissue engineering and biofabrication techniques, and even 2 year data on marketed products.   Phil Vanek, the General Manager of GE Healthcare’s Cell Therapy business, summed it up during his talk where he stated that: GE is interested in 1) big problems, 2) compelling clinical data, and 3) opportunities for “industrialization”, and “Cell Therapy/Regenerative Medicine has all three”.

Various cell manufacturing and processing devices seen throughout the exhibits at IBC's
4th Annual Cell Therapy BioProcessing Conference - No BioPrinters (yet!)
There are many signs that the Cell Therapy field is moving much faster than the protein therapeutics field before it and demonstrating rapid progress.  What we have here is a traditional case of  “standing on the shoulders of giants”, which has been paraphrased on Wikipedia as "discovering truth by building on previous discoveries”.  


September 10, 2014

Scale-up Production of hMSCs: Highlights from the BioProcess Summit Cell Therapy BioProduction Sessions – Post 2 of 2

From www.CellTherapyWonk.com
Fall is almost here, and that means it is Cell Therapy BioProcessing and Manufacturing conference season.  This year it started a few weeks earlier as conference organizer CHI put together a Cell Therapy BioProduction session as part of their Annual BioProcessing Summit in Boston from August 18-22.  In our last post (Scale-up Production – Post 1 of 2), I summarized some of the cool technologies that vendors had on display, as well as some of the poster highlights.  In this post, I want to highlight just a few talks that were focused on manufacturing, scale-up and Cost of Goods of allogeneic cell therapies.  There were several other great talks, but I just wanted to focus on these three due to topic and brevity.

Manufacturing, Cost of Goods, and Unprecedented Stem Cell Process Yields:
On the first day, we had a dynamic duo from Loughborough University give a pair of excellent talks.  Experienced Manufacturing Engineer David Williams gave a great talk on precision manufacturing of living products, highlighting the challenges of working with the inherent variability that comes with primary cell culture.  Dr Williams is the Director of the Center for Innovative Manufacturing in Regenerative Medicine that “works to equip the regenerative medicine industry with manufacturing tools, technologies and platforms by considering the ‘right therapy, right patient, right time’ supply chain from end to end.”  In his talk, he highlighted the need for solid quality characteristics so you know exactly what you are manufacturing – and can do it “again, and again, and again, and again….”.  He points out that, without knowing what characteristics are important for your product (the identity and functional potency of your cells), you: can’t manufacture to specification, can’t scale up, can’t implement new raw materials in your process, can’t transfer manufacturing to another facility, and can’t reduce COGS through process optimization.  Hearing David talk is always a reminder of how important the basics are.

September 5, 2014

Scale-up Production of hMSCs: Highlights from the BioProcess Summit Cell Therapy BioProduction Sessions – Post 1 of 2

Closed system 10 layer and media bags are 
now readilyavailable from several vendors.
Fall is almost here, and that means it is Cell Therapy BioProcessing and Manufacturing conference season.  This year, it started a few weeks earlier as conference organizer CHI put together a Cell Therapy BioProduction session as part of their Annual BioProcessing Summit in Boston from August 18-22.  It is always great to see new conferences including Cell Therapy content, as it shows the maturation of the field.  There is a now a “market” that the organizers believe is worth creating content for.

CHI was kind enough to invite RoosterBio to give the kick-off presentation, so I was able to get re-immersed on all that is new and improved in Cell Therapy Manufacturing Scale-up.  This blog post is meant to share a few of the interesting observations from the meeting and will focus on highlights from the vendor exhibits (i.e. product innovations) and the posters.  The next blog post will share some take-homes from the talks.  There were indeed some valuable and exciting new reports that I want to communicate. 

One key observation is that, while it is still my belief that most of the Allo-products are currently manufactured in 10-layer culture vessels (see above pic), most of the presentations focused on the next generation bioreactor-based processes.  There will continue to be a major shift over the next few years to more automated platforms such as these.

Products on Display for BioProduction of Therapeutic Cells

I always find it worth noting what products the vendors have on display, as they will be developing products based on requests from the market – so more new products displayed means "growing market", which turns into better tools available for everyone.  Interestingly, even at a general BioProcessing conference, there were several booths with Cell Therapy-focused products, and there were 3-5 posters (out of maybe 30) on the scale-up and processing of human MSCs (hMSCs). 

There were a few product innovations and focused product areas among the vendors that I want to highlight here (and we are not getting paid for this, I promise – no sponsorship at all).
Ready-to-Use Microcarriers from Pall


·         Pre-sterilized and ready-to-use microcarriers.  Both Pall/Solohill (see pic) and Corning now offer these products in bottles, but more importantly, in closed system bags ready to seed into a bioreactor system.  Microcarriers in the past had to be prepared and autoclaved by the end user, creating work and yet another set of variables to control when trying to implement this technology.  By providing pre-sterilized microcarriers that are QC’d for efficient cell attachment (I am assuming this QC step; I will try to confirm that), this takes one less process step out of the hands of the process development scientist and makes implementation that much simpler.  This is an important advancement in the field.

·         PBS Biotech reported hMSC densities (expanded in their bioreactors) consistently north of 1 million cells/mL, and up to 3 million cells/mL in a serum containing media.  These numbers are a good 10-fold greater than any number published or presented just 5 years ago, and the highest and most consistent I have seen presented to date.  It is unclear if the reason they achieved these targets was due to the microcarrier and media combination, their novel low shear bioreactor design, or just plain good bioprocess engineering - likely a combination of all three.  In any case, it is a significant achievement, and it demonstrates that it can be done.  Other vendors will now be trying to beat it, and cell therapy companies will be trying to implement it.

·         Vendors are beginning to focus, at least some, on the post-expansion/downstream processing (microcarrier removal, cell concentration) of the cells as well.  Millipore had a presentation and a poster that discussed microcarrier removal using single use filters and the concentration of hMSCs post-harvest using scalable tangential flow filtration (TFF) technology – both with good post-processing viability and recovery.  See poster summary below.  Downstream processing continues to be an under-appreciated aspect of the field and cannot be an afterthought to scale-up culture.  If you scale your expansion to several hundred liters before beginning to think about how you will process the massive cell volumes you have, it could set your program back over a year while these technologies are developed and integrated into the manufacturing process.  It is good to see these aspects of manufacturing get some focus here.

Poster Highlights: 

August 25, 2014

Best Practices in MSC R&D: Addressing Donor Variability within your Experimental System

Human MSCs are the single most used cell source for tissue engineering and regenerative medicine applications, and clinical trials involving hMSCs have outpaced all other cell types in recent years (see here and here).  However, despite indications of clinical effectiveness (see here and here), there is repeated news of the failure of high-profile MSC trials to demonstrate efficacy in a number of therapeutic applications (see here, here, here, here and here).  It has been suggested that the large amount of intra- and inter-donor variability in the MSC populations used in these trials may be responsible for their falling short of expectations despite highly encouraging in vitro and in vivo pre-clinical data.

A team led by Steve Bauer at the US FDA has reported that large variations in proliferation, morphology, differentiation capacity, and cell surface marker expression profiles exist within any population of MSCs and that these intra-population heterogeneities may arise as a result of long-term in vitro culture and the in vivo microenvironment (Free article available here.)  In addition, their work has demonstrated that there are inherent differences in MSCs from donors of similar age, and they have noted the “potential for other donor-related factors in MSC biological variability, which may play a role in their clinical usefulness or performance in various model systems.” Other research groups have also corroborated donor-related differences in MSC function, including in response to stimuli, such as challenge with inflammatory cytokines (see here and here).  A review article on developing cell therapy manufacturing processes reinforces that several donors should be tested prior to implementing; 1) changes in media composition (such as serum reduction/elimination or addition of growth supplements), 2) extensions of the product dose population doubling level (PDL), or 3) changes in lot size during scale-up.

July 7, 2014

Best Practices in MSC Culture: Tracking and Reporting Cellular Age Using Population Doubling Level (PDL) and not Passage Number


There has been much discussion in the literature and the blogosphere (here, here, and here) lately about keeping track of cellular age, and there is very good reason for this.  First, there are multiple regulatory guidelines that propose tracking the age of cells used in biologics manufacturing.  Secondly, it is well documented that cell phenotype and function can be compromised the older a cell is.  A few examples from the literature as this relates to human Mesenchymal Stem Cells (hMSCs) are:

  • Nikbin shows loss of adipogenic and osteogenic differentiation of hMSCs with increasing cumulative population doublings here
  • Lo Surdo and Bauer show here that while flow marker expression is stable, there is a decrease in proliferation rate and a loss of adipose differentiation in hMSCs from passages 3 to 7.
  • Le Blanc retrospectively proposes here that hMSCs from passages 1 or 2 are more therapeutically functional in GvHD than hMSCs from “later” passage 3 or 4 cells (the later passage cells were also cryopreserved).

In many research laboratory environments, cellular age is most often tracked by the number of times a cell has been passaged (such as in the  papers above, excluding Nikbin).  However, Passage Number is quite imprecise and not very acceptable as one gets into regulated environments such as translational clinical activities.  It is generally accepted that tracking the Population Doubling Level (PDL) or Cumulative Population Doublings (CPD) of primary cells is a best practice on understanding cellular age in vitro.  It is the goal of this blog post to help to explain how Passage Number and PDL are related, and how varying cell culture techniques can create a divergence in the reporting of Passage Number, versus PDL.  We also aim to provide guidance and tools to help labs adopt the best practice in tracking PDL of their cell cultures to help bring standardization to their own experimental protocols and the field.

Regulatory Guidelines Propose Tracking Population Doubling Levels

There are pharmaceutical regulatory guidelines such as the ICH Q5D (Titled “Derivation and Characterization of Cell Substrates Used for Production of Biotech/Biological Products” that state “For diploid cell lines possessing finite in vitro lifespan, accurate estimation of the number of population doublings during all stages of research, development, and manufacturing is important.” However, Bauer states in a

May 19, 2014

MSCs as the Workhorse of Regenerative Medicine - Part II - Orthopaedic Roundup

MSCs truly are the Workhorse of Regenerative Medicine, and their use in Orthopaedic applications is where clinical translation was initially imagined.  However, the robust signaling activity of MSCs has widened the range of clinical indications to include cardiac, vascular, and neurological regeneration, as well as immunological applications (eg. GVHD, Chron’s disease), and more recently, cancer therapies.  
It is thus not surprising that MSCs were a strong component of the annual meeting of the Orthopaedic Research Society in New Orleans, LA in March.  We were fortunate enough to attend this conference and see the vast amount of work being conducted in the orthopaedic tissue engineering arena.  Not surprising to us, there were many presentations on the use of MSCs for musculoskeletal tissue repair and regeneration.  Below is some of the MSC research that caught our eye.


ORS 2014 abstracts can be downloaded here.
Orthopaedics, Image from: http://healthcare.utah.edu/orthopaedics/images/body_skeleton.png.

Tissue Engineered Periosteum Approaches to Heal Bone Allograft Transplants 
  • Michael Hoffman, Benoit Lab, University of Rochester
  • Transplantation of decellularized bone allografts seeded with both undifferentiated MSCs and MSCs differentiated to the osteogenic lineage lead to modulation of VEGF production and an increase in BMP2 production that resulted in an increase in torsional biomechanical graft host stability and rate of endochondral ossification compared to allograft alone and allograft seeded with undifferentiated MSCs alone.
  • A related publication can be found here.

Anatomically Shaped, Vascularized Bone Grafts for Craniomaxillofacial Reconstruction
  • Joshua Temple, Grayson Lab, Johns Hopkins University
  • 3D printed porous scaffolds with correct anatomical features were successfully created to regenerate complex craniofacial deformities. Scaffolds containing adipose MSC aggregates formed extensive vascular networks as well as bone.  Implanted scaffolds demonstrated patent human vasculature and bone formation.
  • A related publication can be found here.

Anatomic Hypertrophic Cartilaginous Grafts For Whole Bone Tissue Engineering
  • Eamon Sheehy, Kelly Lab, Trinity College, Dublin
  • Alginate, chitosan and fibrin hydrogels seeded with MSCs were investigated for bone formation through endochondral ossification.  While all constructs underwent robust chondrogenesis in vitro, alginate supported the greatest degree of endochondral bone formation in vivo, with the establishment of a hematopoietic marrow component with evidence of blood vessel infiltration. Subsequently, anatomically correct, MSC-seeded alginate hydrogels were used as the osseous layer of an engineered phalanx construct, which successfully underwent spatially regulated endochondral ossification in vivo.
  • A related publication can be found here.

April 30, 2014

Is It Impolite to Ask an MSC Its Real Age?

Extreme cellular stress can trigger senescence, a mechanism protecting
against malignant cell transformation.
Adapted from: 
Kovacic J C et al. Circulation. 2011;123:1650-1660
We’ve mentioned several times on this blog how standardization of materials, equipment and processes is critical to driving reproducibility and robustness of living cell technologies.   As we continue to engage researchers in Industry and Academia in high-level scientific discussions, it has become apparent that we, as a community, need to adopt more standardized means of accounting for our cell culture practices and what that really means in terms of Mesenchymal Stem Cell (MSC) age and function.

Given MSCs definition as the plastic-adherent fraction of the bone marrow, and their need to be expanded ex vivo prior to therapeutic administration, age has often been associated with and reported as a function of passage level, or the number of times these cells have been plated onto and harvested from tissue culture plastic.  True MSC age, however, is actually related to when these cells will senesce (i.e. stop growing), and this is a function of how many times the cells have divided, or their population doubling level (PDL).

April 9, 2014

Regenerative Medicine Standards Development – an FDA Workshop and an Initiative in Need of Structure

As we stated in an earlier blog post, standardization of materials, equipment and processes will be critical to drive reproducibility and robustness of living cell technologies to the point where they can be widely used and considered “Democratized”.  Some day in the future, an advanced degree and several years of training will not be a pre-requisite for a person to use living cells for some type of particular task, be it for personal or professional applications.  Since standards are so important to RoosterBio’s long-term mission of Democratizing Cellular Technologies, I devoted my March 31st to attending an FDA-hosted public workshop titled Synergizing Efforts in Standards Development for Cellular Therapies and Regenerative Medicine.  The agenda for the day can be found here.

There has been a robust ongoing effort forged between several academic and industry groups (ISCT, ARM, TERMIS) and standards-related organizations (NIST, ASTM, ISO, AABB, ICCBBA, USP, FACT) to establish standards for cell therapy and regenerative medicine; enough that the FDA thought it wise to begin to try to organize and coordinate these activities.  Thus, the purpose of this workshop was to “bring together a broad range of stakeholders to discuss current and future standards development activities involving cellular therapies and regenerative medicine products”.  While there wasn’t a lot of “action” at this meeting, it was a very good mechanism for understanding the landscape of the various activities, who some of the key players are, and how to get actively involved in the dialogue.

March 14, 2014

Using MSCs to Beat Cancer: The Next Big MSC Application

Genetically-modified MSCs home to tumor cells and
accumulate at the tumor site. Image adapted from
http://dx.doi.org/10.1016/j.canlet.2011.02.012.
In the body, MSCs are known to home to sites of acute injury and inflammation and migrate to tumors in response to tumor secretion of growth factors, cytokines, and extracellular matrix (ECM) molecules. However, given their secretion of biomolecules that augment new blood vessel formation, increase inflammation, and degrade the ECM (lending to tumor metastasis), MSCs may promote rather than impede tumor growth and migration, and confounding results from a number of in vitro and in vivo studies have been published to date.  Furthermore, it has been suggested that the ability of MSCs to interact with malignant cells and cancer stem cells might preclude their safe therapeutic application, particularly in patients with dormant or undiagnosed cancers. Despite these concerns, MSCs can serve as an effective ‘Trojan Horse’ for the targeted delivery of anticancer genes, proteins and drugs to tumor cells.  Such targeted delivery can reduce the unsavory systemic side effects that often result from the use of anti-cancer agents, reducing patient morbidity and improving quality of life.  

MSCs can serve as an effective ‘Trojan Horse’ for the targeted delivery of anticancer genes, proteins and drugs to tumor cells.

Recently, a review was published focusing not only on the application of MSCs for the targeted delivery of anti-cancer agents to tumors, but also on the molecular mechanisms of MSC accumulation in tumors, a poorly understood mechanism.  For MSC-based anti-cancer therapies to be effective clinically, these mechanisms must be understood and successfully exploited.  The authors identified several methods to genetically-modify MSCs that resulted in tumor growth inhibition, metastasis suppression, and prolonged survival upon MSC injection in various tumor-laden animal models.  However, in addition to modification with anti-cancer agents, MSCs must be able to accumulate at the site of the tumor for effective cancer eradication.   The authors postulate that increasing the accumulation efficiency of MSCs at tumor sites can effectively target not only primary tumors but also metastatic lesions. 

March 10, 2014

Mesenchymal Stem Cells: The Workhorse of Regenerative Medicine

Prolific Stem Cell and Cell Therapy Blogger Alexey Bersenev (@cells_nnm on Twitter) has recently written a post on his Cell Trials blog titled "Trends in cell therapy clinical trials 2011 – 2013".  If you are interested in Stem Cells and Cell Therapy, it is highly likely that you already know Alexey.  He is one of the most passionate and dedicated technologists in the area.  If you don't know him,  you can follow his activities at the above links, as well as at his Stem Cell Assays blog and among several LinkedIn Discussion groups.  Alexey reported that his own research shows that Cell Therapy Clinical Trials that are listed in international databases have doubled from 2011 to 2013, going from of 161 to 324. Alexey evaluated the data by the split between industry and academia (three academic trials in 2013 for every industry-sponsored trial), by country, by region, as well as by cell type and clinical indication.  His analysis is very important because with his technical background and expertise (Alexey is an MD/PhD researcher who has been doing hands stem cell research or therapeutic cell manufacturing as his day job for several years), if anyone can correctly categorize a trial, it is Alexey.

When tweeting highlights of his Cell Therapy Clinical Trials blog post (above inset) an astute follower of his picked up on the fact that Mesenchymal Stem/Stromal Cell (MSC) trials are out-pacing the other cell types.  This fact is consistent with another often-cited recent manuscript printed in the journal Regenerative Medicine titled "The Global Landscape of Stem Cell Clinical Trials" (free download available here).  This manuscript also creates its own database and analyzes the various types of trials, with the conclusion that "most of the increase (in Cell Therapy Clinical Trials) since 2006 was due to trials using MSCs".

So WHY are MSC trials outpacing other cell sources?  There are several reasons for this.  We believe this is likely due mostly to:

February 26, 2014

Democratizing Living Cellular Technology

@JennWebb recently wrote an article for the O’Reilly Radar titled Democratizing Technology and the Road to Empowerment.  She starts out the article with a nice summary of what it means to Democratize Technology.  Jenn writes “Advancements in technology are making what once was relegated only to highly educated scientists, engineers and developers accessible to — and affordable for — the mainstream.“  Now, the blog she writes for is focused on the intersection of Hardware and Software (or the “physical and digital worlds” is how they phrase it), while we at RoosterBio are imagining a World where biotechnology, specifically living cellular technologies, are simplified and cost-reduced to the point that you don’t have to be a PhD researcher in a well-funded laboratory to perform your own experiments or build novel things out of living cells. The concept of biology paralleling the advances of IT are well laid out elsewhere.

Today, it is much easier to incorporate living cells into your research than it was 20 years ago.  This is evidenced by the proliferation of Cell Biology capabilities in Engineering departments all over the world as Biomedical Engineering has turned into a formalized academic discipline.  When I was doing undergraduate research at the University of Michigan in the early 1990’s, it took months and several collaboration attempts before we could get living cells onto the biomaterial constructs we were making at the time.  Today, it is more commonplace to find the tools to marry the Worlds of Cell Biology and Engineering in the same laboratory.  Despite this, the total number of labs with such capabilities and expertise is still very small.

 We believe that the steps required to fully Democratize Cellular Technologies will be to:

February 20, 2014

Current Bottlenecks in MSC Research

Mesenchymal Stem Cells (MSCs) are widely studied in academic circles and an attractive cell source for clinical applications. MSCs not only possess the ability to self-renew and differentiate to a number of mesenchymal lineages in vitro and in vivo [1,2], but these cells also secrete a cadre of potent trophic factors that contribute to tissue remodeling and modulate the host immune response, making them an attractive cellular biopharmaceutical for the treatment of a number of degenerative diseases and traumatic injuries [3]). However, there is a significant need to improve current methods to efficiently expand standardized, well-characterized MSCs in vitro to the cell numbers needed for widespread, off-the-shelf clinical use

Despite their immense therapeutic potential, MSCs are very rare, comprising only 0.001%-0.01% of the mononuclear cells in the bone marrow [1]. Since a typical adult bone marrow aspirate yields very few MSCs (roughly 1 out of every 10,000 cells) [4] prolonged in vitro expansion is typically necessary before clinical use. However, MSCs will often senesce (i.e. stop growing) in culture before adequate cell numbers for transplantation (on the order of a billion cells) can be obtained. In addition, prolonged in vitro culture of MSCs has been shown to diminish their multilineage potential and impair their immunosuppressive activity [5-8]. The aforementioned challenges associated with MSC culture currently limit their therapeutic potential, and a significant need remains for methods to efficiently expand multipotent MSCs ex vivo.  Therefore, extensive effort has been put into developing methods to expand MSCs while maintaining their differentiation potential and paracrine activity. Cell plating density, culture surfaces, and the addition of growth factor supplements have all been investigated. Of these variables, the use of growth factor and cytokine supplements has proven to effectively modulate MSC growth and self-renewal [9] while maintaining desirable cell characteristics.   

There are three major challenges that cell and tissue engineering technology efforts face today. These challenges are:

February 15, 2014

What are MSCs?

A lot of discussion on this blog will surround MSCs.  So, what is an MSC, why are they relevant, and what’s there to talk about?  A little intro:

The continuing debate on “What is an MSC”?
Adherent bone marrow-derived cells that differentiate down various tissue lineages have traditionally been called Mesenchymal Stem Cells, or MSCs.  There is great debate in academic circles whether this is an appropriate term, and alternative names such as Multipotent Stromal Cells, Marrow Stromal Cells, and even Multi-factor Secretory Cells have been proposed.  While there is debate on the technical name, there is agreement that the “MSC” acronym be maintained.

A Brief History of MSCs and their Therapeutic Use
MSC differentiation potential. Taken from www.sci-therapies.info.
In the 1950s, it was discovered that the adult bone marrow contained a rare, plastic-adherent stromal cell population, Mesenchymal Stromal Cells (MSCs)[1,2], that participate in maintaining the blood-forming, or hematopoietic, niche, and thus, they are implicated in organ homeostasis, wound healing, and aging[3].  Ex vivo, MSCs grow as long, spindle-shaped cells with prominent nuclei and are capable of forming single-cell colonies on tissue culture plastic[4,5].  MSCs have the potential for self-renewal and are multipotent, i.e. they have the ability to differentiate to cells from a number of mesenchymal lineages including fat, bone, cartilage, and muscle both in vitro and in vivo[4-7].  Thus, these cells are also referred to as Mesenchymal Stem Cells and Mulitpotent Stem/Stromal Cells. 

Since their initial discovery in bone marrow, MSCs have been found to reside in many tissues in the body, including fat, umbilical cord blood, dental pulp, and peripheral blood, to name a few.  Three major criteria have been established to define a cell as an MSC[8], including their plastic-adherence, their surface marker profile as measured by flow cytometry, and their ability to differentiate to osteoblasts (bone), adipocytes (fat), and chondrocytes (cartilage) under standard in vitro differentiation conditions.  However, these historical criteria have not kept up with how these cells are used in application.  Tri-lineage differentiation is relevant to less than 20% of all therapeutic applications.  Furthermore, flow marker expression has recently been seen as having little relevance to function[9-11]. 

February 12, 2014

Welcome to the RoosterBio Blog

Everyone at RoosterBio is extremely excited to be launching our company, shipping our first products, and interacting with tissue engineers, cell therapists, synthetic biologists, and our customers to learn about innovative research in these fields, share our knowledge, and contribute to the Cell and Tissue Engineering Revolution. The central theme behind RoosterBio, or what we call our “business hypothesis”, is that as living cellular technologies become more affordable, easier to access, and much simpler to incorporate into product development efforts– there will be a rapid acceleration in products coming to market that incorporate these technologies. We believe that we can help shape this new market by Democratizing Cell Technologies and making them abundant, affordable, and much simpler to translate into the clinic.

We have assembled a team at RoosterBio that have years of experience in stem cell R&D, cell therapeutic product and process development, and manufacturing operations, and with this blog we hope to share many of the topics that we are so enthusiastic about.  We gravitate toward technologies at the technical interfaces of fields such as 3D printing and tissue engineering; thus 3D bioprinting of tissues will be a big topic on this blog.  We will be sharing our thoughts on step-changes in technology on topics like stem cell therapies, engineered tissues and organs, biological robots, manufacturing technologies, and synthetic biology.  We will also engage in educational posts related to our expertise, and comment on broad themes that are facing the industries that we care about, such as establishing standards in the stem cell arena.  The developments in progressive fields always have profound effects on science and society, and we believe we are on the verge of the cell and tissue engineering revolution. 

This blog is targeted at the scientists, technologists, engineers, and doctors that are working passionately and feverishly to bring cell-based products to patients, as well as for general audiences that are interested in the driving forces behind cellular therapies, regenerative medicines, and tissue engineering technologies.  We hope you will come back to learn and engage in the conversation, and also be a part of the next technology revolution as Biology becomes Technology

We encourage everyone to leave comments, and to feel free to say what is on your mind. We look forward to the dialogue and helping to accelerate the Cell-based BioEconomy!


- The RoosterBio Team