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


Figure 1. hBM-MSC demonstrate rapid cell growth with the
addition of RoosterReplenish-MSC on day 3 of suspension
culture.Viability at harvest was 96%.
RoosterBio hBM-MSC (MSC-001) were thawed or passaged from T-flasks, and seeded onto SoloHill microcarriers (part # CS-215-040) in RoosterBio’s High Performance Media (KT-001) at 1.2 x 104 cells/ml (1667 cells/cm2 seeding onto uC). Cell-seeded microcarriers were inoculated into a PBS VerticalWheel bioreactor with a total media volume of 500mL. Cells were cultured for 3 days in 5% CO2 at 25 rpm agitation, and on day 3, one 10mL vial of RoosterReplenish-MSC was added to the culture to sustain hMSC proliferation and reach desired cell density (Figure 1).  hBM-MSC fed-batch cultures typically expand to 3-5 x 105 cells/mL within 6 days of culture depending on donor, seeding density and culture platform. Some aggregation of cells and microcarriers is typically observed on day 3 of culture and is an indication of healthy, proliferating hMSCs (Figure 2). (Note: With increased cell-microcarrier agglomeration, higher agitation rates (up to 35rpm) may be needed to keep microcarriers suspended and avoid sedimentation.)
 
Figure 2. Agglomeration of cell-laden microcarriers indicates presence of healthy, proliferating hMSCs.
Figure 3. Waste product and nutrient levels in bioreactor culture
over 6 days were within acceptable limits.
 Important parameters to monitor in a fed-batch process are the maintenance of sufficient nutrient levels in the growth media and the accumulation of hMSC waste products in culture. Here, we measured these levels, and our data indicated that lactate and ammonia levels remained below growth inhibitory levels (Schop et. al) until day 6 when they reach 2 g/L and 2.45 mM respectively (Figure 3 A-B). Glucose and glutamine levels were adequate for sustained cell growth (2.25 g/L and 0.93 mM respectively) in the fed-batch culture (Figure 3 C-D).

Cell expansion and waste and nutrient levels were also compared for hMSC microcarrier cultures between half media exchange and RoosterReplenish-MSC feed (Figure 4A) processes. While cells reached comparable final densities in both cases, hMSC doubling rate was higher in the RoosterReplenish-MSC culture (cells maintained their exponential growth rate to reach confluency within 5 days vs. 7 days for the half media exchange process). In addition, no differences in nutrient levels or waste product accumulation were noted between the two culture processes (Figure 4B).
Figure 4. RoosterReplenish-MSC and half media exchange resulted in similar hMSC growth profiles (A) and nutrient and waste product concentrations in suspension culture (B).

Figure 5. RoosterReplenish-MSC feed regimen was optimized
to attain maximum hMSC growth on microcarriers.
Identifying the optimum feeding time is also key to achieving an efficient hMSCs culture process. Addition of RoosterReplenish-MSC on different days (Figure 5) demonstrated an optimal time for nutrient replacement (day 3 with our process). It is important to note that cells should be maintained in the exponential growth phase and harvested while they in this highly proliferative state.  We found from initial studies that final cell harvest densities can be donor dependent, underscoring the need to establish and optimize processes over a range of donors.  Moreover, there are many variables in suspension culture that could be optimized, depending on application, including seeding density, time and amount of nutrient replenishment, time and method of harvest and microcarrier separation.  More importantly, demonstrating efficiency of the fed-batch process in >50L scale, utilizing the RoosterReplenish-MSC concentrated media feed, will be crucial for validating process consistency and scalability.


Figure 6. Comparison of media consumption in different
bioreactor culture processes demonstrates a significant
advantage to fed-batch culture over others.
This series of studies demonstrates that utilization of RoosterReplenish-MSC feed in a microcarrier-based hMSC bioreactor culture process achieves similar cell yield compared to a typical partial media exchanges process. A simple comparison of media utilized between a fed-batch, half media exchange and perfusion system at 0.1mL/min is summarized in Figure 6, showing a drastic reduction (thousands of liters) in media use in a fed-batch culture process. This reduction in media usage can translate to hundreds of thousands of dollars in savings for commercial-scale hMSC production.  Soon, RoosterBio will be publishing a Starter Protocol to help researchers get started with scalable bioreactor expansion of hMSCs. We anticipate researchers will improve upon the suggested processes to achieve an optimum process for each of their specific applications. 

We at RoosterBio hope to create a resource for researchers to share protocols and data from small studies, to eventually crowd-source an efficient, scalable protocol for bioreactor-based hMSC expansion where a cost effective, standard culture process for 3D suspension culture can be generated and widely-adopted, as in today’s 2D flask culture process.  Will you join us in such an endeavor?


References
  1. Meeting lot-size challenges of manufacturing adherent cells for therapy. J Rowley, E Abraham, A Campbell, H Brandwein, S Oh. BioProcess Int 10 (3), 7.
  2.  Increasing efficiency of human mesenchymal stromal cell culture by optimization of microcarrier concentration and design of medium feed. Allen Kuan-Liang Chen, , Yi Kong Chew, Hong Yu Tan, Shaul Reuveny, Steve Kah Weng Oh. Cytotherapy 17 (2), 163-173
  3. Scale-up of Human Mesenchymal Stem Cells on Microcarriers in Suspension in a Single-use Bioreactor. Aletta Schnitzler, Daniel Kehoe, Janice Simler, Anthony DiLeo, Andrew Ball. BioPharm International. 25(2).
  4. Growth, metabolism, and growth inhibitors of mesenchymal stem cells. Schop D1, Janssen FW, van Rijn LD, Fernandes H, Bloem RM, de Bruijn JD, van Dijkhuizen-Radersma R. Tissue Eng Part A. 2009 Aug;15(8):1877-86.
  5. Microcarrier Culture for Efficient Expansion and Osteogenic Differentiation of Human Fetal Mesenchymal Stem Cells. Tony Kwang-Poh Goh, Zhi-Yong Zhang, Allen Kuan-Liang Chen, Shaul Reuveny, Mahesh Choolani, Jerry Kok Yen Chan and Steve Kah-Weng Oh. Biores Open Access. 2013 Apr; 2(2):84-97.
  6. A microcarrier-based cultivation system for expansion of primary mesenchymal stem cells. Frauenschuh S, Reichmann E, Goetz PM, Sittinger M, Ringe J. Biotechnology Progress. 2007 Feb; 23(1):187-93.
  7. A potentially scalable method for the harvesting of hMSCs from microcarriers. Alvin W. Nienow, Qasim A. Rafiq, Karen Coopman, Christopher J. Hewitt. Biomedical Engineering. 2014 Apr; 85:79-88
  8.  Toward a Clinical-Grade Expansion of Mesenchymal Stem Cells from Human Sources: A Microcarrier-Based Culture System Under Xeno-Free Conditions. Francisco dos Santos, Pedro Z. Andrade, Manuel M. Abecasis, Jeffrey M. Gimble, Lucas G. Chase, Andrew M. Campbell, Shayne Boucher, Mohan C. Vemuri, Cl√°udia Lobato da Silva, and Joaquim M.S. Cabral. Tissue Eng Part C Methods. 2011 Dec; 17(12): 1201–1210.
  9. Expansion, harvest and cryopreservation of human mesenchymal stem cells in a serum-free microcarrier process. Thomas R. J. Heathman, Veronica A. M. Glyn, Andrew Picken, Qasim A. Rafiq, Karen Coopman, Alvin W. Nienow, Bo Kara and Christopher J. Hewitt. Biotechnology and Bioengineering. 2015 Aug; 112(8): 1696–1707.
  10.  Single-Use Bioreactors and Microcarrier. Mark Szczypka, David Splan, Heather Woolls and Harvey Brandwein. BioProcess International. Mar 2014.
  11. Allogeneic cell therapy bioprocess economics and optimization: Single-use cell expansion technologies. Ana S. Simaria, Sally Hassan, Hemanthram Varadaraju, Jon Rowley, Kim Warren2, Philip Vanek and Suzanne S. Farid. J. of Biotechnology & Bioengineering. Jan 2014. 111 (1): 69-83.
  12.  Developing Cell Therapy Biomanufacturing Processes. Chem. Eng. Progress. Rowley, JA. 2010. (SBE Stem Cell Engineering Supplement):50-55.





1 comment:

  1. There is no doubt that new scientific platform can boost the production. According to the displayed data,it really make a difference.

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