Efficient manufacturing of therapeutic mesenchymal stromal cells with the use of the Quantum Cell Expansion System.

BACKGROUND: The use of bone marrow-derived mesenchymal stromal cells (MSCs) as a cellular therapy for various diseases, such as graft-versus-host disease, diabetes, ischemic cardiomyopathy and Crohn's disease, has produced promising results in early-phase clinical trials. However, for widespread application and use in later phase studies, manufacture of these cells must be cost-effective, safe and reproducible. Current methods of manufacturing in flasks or cell factories are labor-intensive, involve a large number of open procedures and require prolonged culture times. METHODS: We evaluated the Quantum Cell Expansion System for the expansion of large numbers of MSCs from unprocessed bone marrow in a functionally closed system and compared the results with a flask-based method currently in clinical trials. RESULTS: After only two passages, we were able to expand a mean of 6.6 × 10(8) MSCs from 25 mL of bone marrow reproducibly. The mean expansion time was 21 days, and cells obtained were able to differentiate into all three lineages: chondrocytes, osteoblasts and adipocytes. The Quantum was able to generate the target cell number of 2.0 × 10(8) cells in an average of 9 fewer days and in half the number of passages required during flask-based expansion. We estimated that the Quantum would involve 133 open procedures versus 54,400 in flasks when manufacturing for a clinical trial. Quantum-expanded MSCs infused into an ischemic stroke rat model were therapeutically active. CONCLUSIONS: The Quantum is a novel method of generating high numbers of MSCs in less time and at lower passages when compared with flasks. In the Quantum, the risk of contamination is substantially reduced because of the substantial decrease in open procedures.

Scale-up of human embryonic stem cell culture using a hollow fibre bioreactor.

The commercialisation of human embryonic stem cell derived cell therapies for large patient populations is reliant on both minimising expensive and variable manual-handling methods whilst realising economies of scale. The Quantum Cell Expansion System, a hollow fibre bioreactor (Terumo BCT), was used in a pilot study to expand 60 million human embryonic stem cells to 708 million cells. Further improvements can be expected with optimisation of media flow rates throughout the run to better control the cellular microenvironment. High levels of pluripotency marker expression were maintained on the bioreactor, with 97.7 % of cells expressing SSEA-4 when harvested.

Characterization of clusters in rapid granular flows.

The clustering phenomenon within two-dimensional, rapid granular, simple shear flows is investigated. Two characterizations are developed and implemented for monodisperse systems, revealing physically meaningful insight. First, a new feature of the radial distribution function is identified for these dissipative granular systems, which is not present in molecular (nondissipative) systems. Namely, a long-scale minimum occurs at a distance representing the average distance between the center of a cluster and the center of a dilute region. Results indicate that center-to-center distances are least (i.e., clusters are most tightly packed) for systems of moderate particle concentrations and low restitution coefficients. In addition, concentration and temperature measurements of clustered and dilute regions are also obtained using a Gaussian filter that is based on this center-to-center distance and, thus, provides a means of appropriately defining local concentrations. These results confirm previous findings that cluster prevalence increases with decreasing dissipation and that clustered regions have lower temperatures than their dilute counterparts. Surprisingly, however, the results indicate that cluster prevalence, defined by normalized concentration differences between the two regions, decrease monotonically with an increase in overall particle concentration.

Clustering in rapid granular flows of binary and continuous particle size distributions.

The dynamic clustering phenomenon in two-dimensional simple shear flows has been investigated using molecular dynamic simulations of systems containing binary and continuous size distributions of equal-material-density particles. Particular attention has been paid to two questions: (1) Does the presence of size nonuniformities serve to enhance or attenuate the presence of clusters? (2) Do particles of a given size preferentially segregate within the clusters? With respect to the first question, the prominence of clustered regions increases with increasing deviation from the monodisperse limit in the case of both binary and continuous size distributions. With respect to the second question, the larger particles of both binary and continuous size distributions are consistently observed to segregate within the transient clustered regions. Further investigation of granular temperatures within the clustered and dilute regions reveals that this segregation is consistent with previously observed temperature-driven segregation in steady-state systems; large particles favor the lower-temperature (clustered) regions. Moreover, observation of clustering length scales suggests that large particles may favor the center of the clustered regions, where granular temperatures are expected to reach a minimum.