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.