A scalable and xeno-free bioreactor system for biomanufacturing of hUC-MSCs

Background & Aim Mesenchymal stem/stromal cells (MSCs) are a prominent cell type in cellular therapies with over 900 registered clinical trials. A critical bottleneck in using MSCs, however, is generating the large number of cells required for both clinical trials and commercial therapies. Many commercial therapies will require 100's of millions to billions of cells. While 2D cell culture may be able to generate sufficient cells for development and early clinical trials, later clinical trials and commercial therapies will require 3D bioreactors in a scalable manufacturing platform. Thus for consistency in the therapeutic product from the development stage through clinical trials and commercial therapies, it is necessary that the state of the cells be comparable across the transition from 2D to 3D culture and then maintained with scale-up in 3D. Here, we establish a scalable 3D manufacturing process using MSCs isolated from human umbilical cord tissue (hUC-MSCs) to generate cells at a consistent PDL that maintain comparable critical quality attributes (CQAs) to 2D flask culture. Methods, Results & Conclusion hUC-MSCs were isolated from the perivascular region of human umbilical cords. Using a xeno-free culture system (medium, reagents and materials), cells were cryopreserved to create a Working Cell Bank (WCB). WCB vials were expanded either in 2D cell stacks or on microcarriers in a small-scale (0.1L) or larger scale (15L) bioreactor. Cells were then assessed for CQAs: final PDL, typical MSC surface marker expression (positive for CD90 and CD166, negative for CD34 and CD45), trilineage differentiation potential (osteogenesis, adipogenesis, and chondrogenesis), and functional properties including IDO secretion and angiogenic cytokine secretion (VEGF, IL-8, bFGF, HGF, TIMP1 and TIMP2). hUC-MSCs were successfully expanded in 2D flasks, a 0.1L bioreactor, and a 15L bioreactor. Cells maintained all the tested CQAs across both 2D and 3D manufacturing platforms as well as with increases in scale in 3D. Therefore, this study establishes a scalable xeno-free manufacturing paradigm to reproducibly generate populations of hUC-MSCs that have consistent properties. Repeatedly generating cell populations with similar attributes is critical for well-designed studies during the development of cellular therapy approaches and as these approaches move towards clinical trials and commercial therapies. Mesenchymal stem/stromal cells (MSCs) are a prominent cell type in cellular therapies with over 900 registered clinical trials. A critical bottleneck in using MSCs, however, is generating the large number of cells required for both clinical trials and commercial therapies. Many commercial therapies will require 100's of millions to billions of cells. While 2D cell culture may be able to generate sufficient cells for development and early clinical trials, later clinical trials and commercial therapies will require 3D bioreactors in a scalable manufacturing platform. Thus for consistency in the therapeutic product from the development stage through clinical trials and commercial therapies, it is necessary that the state of the cells be comparable across the transition from 2D to 3D culture and then maintained with scale-up in 3D. Here, we establish a scalable 3D manufacturing process using MSCs isolated from human umbilical cord tissue (hUC-MSCs) to generate cells at a consistent PDL that maintain comparable critical quality attributes (CQAs) to 2D flask culture. hUC-MSCs were isolated from the perivascular region of human umbilical cords. Using a xeno-free culture system (medium, reagents and materials), cells were cryopreserved to create a Working Cell Bank (WCB). WCB vials were expanded either in 2D cell stacks or on microcarriers in a small-scale (0.1L) or larger scale (15L) bioreactor. Cells were then assessed for CQAs: final PDL, typical MSC surface marker expression (positive for CD90 and CD166, negative for CD34 and CD45), trilineage differentiation potential (osteogenesis, adipogenesis, and chondrogenesis), and functional properties including IDO secretion and angiogenic cytokine secretion (VEGF, IL-8, bFGF, HGF, TIMP1 and TIMP2).