hUC-MSC Exhibit Robust Proliferation in 3D Bioreactor Systems

Authored by: Joseph Takacs, MS, Research Associate, RoosterBio and Katrina Adlerz, PhD, Scientist, RoosterBio

Scalable Manufacturing Solution Needed

Historically, pre-clinical studies and clinical trials have predominantly used hMSCs derived from bone marrow and adipose tissue sources. However, over the last 10 to 15 years, the number of publications and clinical trials in the regenerative medicine field using hMSCs derived from human umbilical cord (hUC-MSCs) as a raw material has significantly increased (1,2,3).

The commercial hUC-MSC therapies that will follow will require hundreds of millions to billions of cells per lot for effective patient dosing (4,5). Traditional 2-dimensional (2D) flask expansion platforms are not cost-effective for such large-scale expansion of cell-based therapeutics. However, 3-dimensional (3D), microcarrier-based, bioreactor systems offer a scalable manufacturing platform that can achieve the lot sizes needed. Here, we demonstrate that hUC-MSCs achieve high cell densities in a 3D bioreactor culture system and maintain their critical quality attributes (CQAs) after harvest.

hUC-MSC Expansion in Scalable Bioreactor Technologies

hUC-MSCs derived from three different donors were cultured in a xeno-free (XF), fed-batch suspension 100mL bioreactor system according to RoosterBio’s bioreactor process recommendations. hUC-MSCs from all three donors reached high cell densities of between 0.8 x 10⁶and 1.2 x 10⁶ cells/mL after five days of culture (Figure 1). Previous experience with bone marrow derived hMSCs suggests that bioreactor systems can be scaled to larger volumes such as 3, 15, and 50L systems with similar or even improved cell growth (6).

Figure 1: hUC-MSCs reach new expansion potential.

(A) hUC-MSCs were cultured for five days in a fed-batch bioreactor system with a RoosterReplenish-MSC-XF feed on Day 3. hUC-MSCs reached cell densities of at least 0.8 x 10⁶cells/mL by Day 5. (B) By Day 5, cell-microcarrier aggregation and sampled cell counts indicated that cultures were ready for harvest.

hUC-MSCs Robust Functional Capability

To ensure these cells maintained hMSC CQAs, hUC-MSCs expanded in 3D bioreactors were compared to hUC-MSCs expanded in 2D flasks in a panel of assays (7). After expansion in control flasks (2D) or bioreactors (3D), harvested cells were analyzed for: expansion over a subsequent passage, surface marker expression by flow cytometry, immunomodulatory properties, angiogenic cytokine secretion, and trilineage differentiation (Figure 2). While there was donor-to donor variability, hUC-MSCs that were expanded in 3D bioreactors performed comparably to hUC-MSCs that were expanded in 2D flasks. 

This study demonstrates that hUC-MSCs can be expanded in a 3D bioreactor system, reach high cell densities, and maintain their CQAs after harvest. Therefore, hUC-MSCs paired with a bioreactor platform is a system that can be scaled to yield the cell numbers required for product development and commercial therapeutics.

Figure 2: hUC-MSCs maintain critical quality attributes after 3D culture.

Freshly harvested cells were plated into functional assays to determine: (A) expansion capacity over a subsequent passage (B) surface marker expression by flow cytometry (representative donor shown) (C) immunomodulatory properties through the secretion of indoleamine-2,3-dioxygenase (D) angiogenic cytokine secretion and (E) trilineage differentiation through media induction (representative donor shown).

References

1. Davies JE, Walker JT, Keating A. (2017) Concise Review: Wharton's Jelly: The Rich, but Enigmatic, Source of Mesenchymal Stromal Cells. Stem Cells Transl Med.,6(7):1620-1630. https://www.ncbi.nlm.nih.gov/pubmed/28488282

2.Zhao J, Yu G, Cai M, Lei X, Yang Y, Wang Q, Zhai X (2018) Bibliometric analysis of global scientific activity on umbilical cord mesenchymal stem cells: a swiftly expanding and shifting focus. Stem Cell Research & Therapy,9(32). https://www.ncbi.nlm.nih.gov/pubmed/29415771

3.Farrance I (2019) Meeting the growing needs of the perinatal RegenMed Industry: the only Umbilical Cord hMSC (hUC-MSC) system designed for today’s translationally focused research and product development. RoosterBio Blog, 18 September 2019. http://roosterbio.blogspot.com/2019/09/meeting-growing-needs-of-perinatal.html

4. Lembong J, Rowley J (2018) Building Effective Multi-Year Process Development Programs: Evolution of Technology Platform Decisions Based on Lot Size. RoosterBio Blog, 15 December 2018. http://roosterbio.blogspot.com/2018/12/building-effective-multi-year-process.html

5. Olsen TR, Ng KS, Lock LT, Ahsan T, Rowley JA (2018) Peak MSC – are we there yet? Front. Med.,21 June 2018. https://www.ncbi.nlm.nih.gov/pubmed/29977893

6. Kirian RD, Wang D, Takacs J, Tsai A, Cruz K, Rosello F, Cox K, Hashimura Y, Lembong J, Rowley JA, Jung S (2019) Scaling A Xeno-Free Fed-Batch Microcarrier Suspension Bioreactor System From Development to Production Scale for Manufacturing XF hMSCs. Cytotherapy, 2019 May 1;21(5):S71-2.

7. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop D, & Horwitz E (2006) Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy, 8(4):315-317. http://www.ncbi.nlm.nih.gov/pubmed/16923606.

Meeting the growing needs of the perinatal RegenMed Industry: the only Umbilical Cord hMSC (hUC-MSC) system designed for today’s translationally focused research and product development

Authored by Iain Farrance, PhD, Technical Marketing Associate

Introduction

Human mesenchymal stromal cells (hMSC) are considered the "workhorse" of Regenerative Medicine (RegenMed).  hMSC are a critical starting material in a growing variety of established and emerging RegenMed products, including cellular therapies, cell-based gene therapies, hMSC-derived extracellular vesicles (EVs), and bioprinted engineered tissues (Olsen, 2018). Accordingly, there have been greater than 100 clinical trials initiated each year since 2011 using hMSC from various sources (database purchased from celltrials.org) across a host of indications and therapeutic strategies (clinical trials.gov).  hMSC have benefitted from having an excellent safety profile, and there have been nine (9) products approved globally over the last 10 years. The growth in use of hMSC in a variety of product types has created the opportunity to standardize the supply chain and provide economies of scale for a rapidly growing industry. RoosterBio was founded to industrialize and standardize the RegenMed supply chain and to radically simplify the incorporation of living cells into therapeutic product development. Our goal is to have the same impact on the RegenMed industry that Intel had on the computer industry.

Use of Human Umbilical Cord-derived MSCs (hUC-MSC) in research and clinical trials (CT) has grown rapidly over the last 10 to 15 years with quickest adoption in APAC (Figure 1, Davies, 2017; Zhao, 2018; Moll, 2019). hUC-MSC publications per year increased 19-fold increase from 2006 to 2016 (Zhao, 2018). CT with hUC-MSC have shown a similar growth pattern as publications. hUC-MSC are the second most used hMSC type in CT (Moll, 2019) and 178 CT using hUC-MSC were registered, are ongoing, or were completed between 2007 and 2017 (Couto, 2019). In fact, >30% of hMSC trials registered in 2019 use hUC-MSC as the cell source.  These drive the need for hUC-MSC to use in product development. Until now, IP surrounding hUC-MSC has been a primary roadblock to the widespread adoption of hUC-MSC. We have collaborated with leaders in Wharton’s Jelly/umbilical cord hMSC at Tissue RegenerationTherapeutics Inc. (TRT) and have brought to market a complete bioprocess cell and media system. RoosterBio’s hUC-MSC are available for licensing and are provided in scalable formulations and cGMP compatible processes that enable anyone to obtain hUC-MSC in numbers needed for incorporation into RegenMed product development.

Until now RoosterBio has paired our batch (2D) and fed-batch (3D bioreactor) bioprocess media systems with hMSC from two sources: adipose-derived (hAD-MSC) and bone marrow-derived (hBM-MSC and xeno-free (XF) hBM-MSC). RoosterBio’s launch of our XF hUC-MSC (RoosterVial™-hUC-MSC-XF) introduces the first umbilical cord-derived hMSC in the North American and worldwide market designed to meet the quality and volume needs of today’s translationally focused cell therapy product developers. For RoosterBio’s hMSC product lines see here.

RoosterBio’s RoosterVial-hUC-MSC-XF and RoosterNourish™-MSC cell and medium bioprocess system has several key advantages over the limited number of suppliers of perinatal hMSC. Being XF, and manufactured with RoosterBio’s existing cGMP compatible processes, our system is the only hUC-MSC commercially available with a clear line of sight to clinical translation.  Additionally, other suppliers (a) provide low cell number vials at a high price per M cells, (b) supply serum-based cells, or (c) require specialized, non-scalable culture vessels.  Finally, RoosterBio provides first in class characterization of hMSC key quality attributes (PDL, identity, expansion potential) and functional assays (cytokine secretion, trilineage differentiation, immunomodulation).