A new perfusion mode of culture for WJ-MSCs expansion in a stirred and online monitored bioreactor.

As a clinical dose requires a minimum of 106 cells per kilogram of patients, it is, therefore, crucial to develop a scalable method of production of Wharton Jelly mesenchymal stem cells (WJ-MSCs) with maintained inner characteristics. Scalable expansion of WJ-MSCs on microcarriers usually found in cell culture, involves specific cell detachment using trypsin and could have harmful effects on cells. In this study, the performance of batch, fed-batch, and perfused-continuous mode of culture were compared. The batch and fed-batch modes resulted in expansion factors of 5 and 43, respectively. The perfused-continuous mode strategy consisted of the implementation of a settling tube inside the bioreactor. The diameter of the tube was calculated to maintain microcarriers colonized by cells in the bioreactor whereas empty microcarriers (responsible for potentially damaging collisions) were removed, using a continuous flow rate based on MSCs physiological requirements. Thanks to this strategy, a maximal number of 800 million cells was obtained in a 1.5 L bioreactor in 10 days. Lastly, online dielectric spectroscopy was implemented in the bioreactor and indicated that cell growth could be monitored during the culture.

Quality by design to define critical process parameters for mesenchymal stem cell expansion.

Stem cell-based therapeutic products could be the key to treat the deadliest current pathologies, ranging from neuro-degenerative to respiratory diseases. However, in order to bring these innovative therapeutics to a commercialization stage, reproducible manufacturing of high quality cell products is required. Although advances in cell culture techniques have led to more robust production processes and dramatically accelerated the development of early-phase clinical studies, challenges remain before regulatory approval, particularly to define and implement science-based quality standards (essential pre-requisites for national health agencies). In this regard, using new methodologies, such as Quality By Design (QBD), to build the production process around drug quality, could significantly reduce the chance of product rejection. This review-based work aims to perform a QBD approach to Mesenchymal Stem Cell (MSC) manufacturing in standard two-dimensional flasks, using published studies which have determined the impact of individual process parameters on defined Critical Quality Attributes (CQA). Along with this bibliographic analysis, parameter criticality was determined during the two main manufacturing stages (cell extraction and cell amplification) along with an overall classification in view of identifying the Critical Process Parameters (CPP). The analysis was performed in view of an improved standardization between research teams, and should contribute to reduce the gap towards compliant Good Manufacturing Practice (cGMP) manufacturing.

Impact of the type of microcarrier and agitation modes on the expansion performances of mesenchymal stem cells derived from umbilical cord.

The present study proposed to compare the impact of agitation mode (static, orbital, and mechanical) on the culture of mesenchymal stem cells extracted from the Wharton's jelly of umbilical cords (WJ-MSC), in a clinical grade culture medium, using human platelet lysate and different xeno-free microcarriers. Attachment, expansion, and detachment performances were characterized by a new dedicated tool of microscopic image posttreatment, allowing an in situ cell counting without detachment step. Results showed that performances in static mode were not necessarily representative of those obtained in dynamic mode. Moreover, impacts on nutrient consumptions and metabolite productions were identified, such as a higher glutamine consumption when Cytodex-1 microcarriers were used. The detachment strategy used was relatively efficient for Star-Plus, Plastic-Plus, and Hillex II, but not sufficient for Cytodex-1. Despite Cytodex-1 presented promising attachment and expansion performances, Star-Plus and Plastic-Plus showed a better compromise, respectively, for the orbital and the mechanical agitation modes.