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).
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%.
|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.
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.
|Figure 6. Comparison of media consumption in different|
bioreactor culture processes demonstrates a significant
advantage to fed-batch culture over others.
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?
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