Annie Marc

Investigation of Growth Conditions for the Expansion of Porcine Mesenchymal Stem Cells on Microcarriers in Stirred Cultures

The extensive use of mesenchymal stem cells (MCS) in tissue engineering and cell therapy increases the necessity to improve their expansion. Among these, porcine MCS are valuable models for tissue engineering and are classically expanded in static T-flasks. In this work, different processes of stirred cultures were evaluated and compared. First, the effect of glucose, glutamine, antioxidant, and growth factors concentrations on porcine MSC expansion were analyzed in a suitable medium by performing kinetic studies. Results showed that a lower glucose concentration (5.5xa0mM) enabled to increase maximal cell concentration by 40xa0% compared with a higher one (25xa0mM), while addition of 2 to 6xa0mM of glutamine increased maximal cell concentration by more than 25xa0% compared with no glutamine supplementation. Moreover, supplementation with 1xa0μM thioctic acid increased maximal cell concentration by 40xa0% compared with no supplementation. Using this adapted medium, microcarriers cultures were performed and compared with T-flasks expansion. Porcine MSC were shown to be able to proliferate on the five types of microcarriers tested. Moreover, cultures on Cytodex 1, Cytopore 2, and Cultispher G exhibited a MSC growth rate more than 40xa0% higher compared with expansion in T-flasks, while MSC metabolism was similar.

Revisiting the determination of hydromechanical stresses encountered by microcarriers in stem cell culture bioreactors

Background Expansion of mesenchymal stem cells (MSC) is one of the key steps for their use in tissue engineering or cell therapies. Today, expansion processes are mainly based on the use of microcarriers to allow large interfacial adherence areas [1]. However, this culture technology is known to be practically limited to low agitation intensity and microcarrier concentrations due to possible cell damage arising from particle hydromechanical stress or collisions between microcarriers [2]. Unfortunately, the description of the relationship between bioreactor hydrodynamics, microcarrier suspension and occurrence of collisions was neither clearly established in the case of stem cell cultures, nor based on a local description of the bioreactor hydrodynamics heterogeneity. Thus, in the present study, it is proposed to use numerical simulations to describe not only the liquid phase but also the microcarrier dispersion and the occurrence of hydromechanical stress encountered by the microcarriers. Two kinds of hydromechanical stress can be distinguished: (i) fluid-solid interactions (fluid shear stress) arising from turbulent eddies and (ii) solid-solid interactions arising from collisions between microcarriers or between microcarriers and bioreactor walls [2].

Multicellular tumor spheroids in microcapsules as a novel 3D in vitro model in tumor biology

Background Advantages of microencapsulation as a 3D growth system are chemically and spatially defined 3D network of extracellular matrix components, cell-to-cell and cell-tomatrix interactions governing differentiation, proliferation and cell function in vivo. The study is aimed at i) optimization of techniques for preparing microcapsules; ii) generation of multicellular tumor spheroids (MTS) by culturing tumor cells in the microcapsules; iii) study of anticancer treatment effects for both photodynamic therapy (PDT) and anti-cancer drug screening. The model allows to estimate drug doses or parameters for PDT in vitro before carrying out preclinical tests, and thereby to reduce a number and costs of experiments with animals commonly used.

A dual near-infrared and dielectric spectroscopies strategy to monitor populations of Chinese hamster ovary cells in bioreactor.

Objectiveto develop a new strategy combining near-infrared (NIR) and dielectric spectroscopies for real-time monitoring and in-depth characterizing populations of Chinese hamster ovary cells throughout cultures performed in bioreactors.ResultsSpectral data processing was based on off-line analyses of the cells, including trypan blue exclusion method, and lactate dehydrogenase activity (LDH). Viable cell density showed a linear correlation with permittivity up to 6xa0×xa0106 cells ml−1, while a logarithmic correlation was found between non-lysed dead cell density and conductivity up to 107 cells ml−1. Additionally, partial least square technique was used to develop a calibration model of the supernatant LDH activity based on online NIR spectra with a RMSEC of 55 U l−1. Considering the LDH content of viable cells measured to be 110 U per 109 cells, the lysed dead cell density could be then estimated. These calibration models provided real-time prediction accuracy (R2xa0≥xa00.95) for the three types of cell populations.ConclusionThe high potential of a dual spectroscopy strategy to enhance the online bioprocesses characterization is demonstrated since it allows the simultaneous determination of viable, dead and lysed cell populations in real time.

Serum-free media for mesenchymal stem cells expansion on microcarriers

Background Expansion of mesenchymal stromal cells (MSC) is one of the key steps for their use in tissue engineering or cell therapies. To increase cells expansion yields, two milestones have to be achieved that will allow a wider MSC therapeutic use, namely an optimal serum-free medium and a process intensification via 3D suspension culture on microcarriers [1]. Indeed, one of the major obstacles to obtain a reliable manufacturing process is that most of MSC cultivation methods still rely on media being supplemented by a significant volume of fetal calf serum. While efforts have been made to develop serum-free media (SFM) for MSC expansion, they were systematically designed for planar plastic cultivation systems. The aim of this study was to compare usual serum alternatives on 2D and 3D cultures. However, these alternatives can be ill-defined, either when the medium formulation is proprietary or when it has been humanized with blood derivatives. Thus several supplements were also investigated to design a more defined serum-free formulation that could be used to expand stem cells on microcarriers.

Real-time monitoring of the physiological state of CHO cells in bioreactor by using a dual spectroscopic strategy

Background Bioprocesses of mammalian cell culture have become essential in pharmaceutical fields for the production of recombinant therapeutic proteins such as monoclonal antibodies or for tissue therapy. Following the FDA’s recommendation to apply Process Analytical Technology (PAT) approach as the mean to control production processes and ensure the quality of the end-product for patients, Near Infra-Red (NIR)and dielectric spectroscopic technologies have gained great attention over the past decade as online tools for cellular bioprocesses monitoring [1,2]. In such processes, cells are one of the most critical parameter, because their physiological state directly impacts the final product titer through their productivity as well as the product quality. Animal cells are typically classified into viable or dead entities via Trypan Blue exclusion technique, but lysed cells are not accounted for, although they could represent a significant proportion of the cell population in bioreactors. In addition to reducing productivity, cell lysis causes the release of intracellular proteases and glycosidases, which can degrade secreted recombinant protein of interest. Therefore, they should be considered for optimal control of processes. In this work, we propose the first online strategy combining NIR and dielectric spectroscopies for in-depth characterization of cellular growth and physiology of CHO (Chinese Hamster Ovary) cells cultivated in bioreactors.

Adhesion and colonization of mesenchymal stem cells on polylactide or PLCL fibers dedicated for tissue engineering

Background Tissue engineering covers a broad range of applications dedicated to the repair or the replacement of part or whole tissue such as blood vessels, bones, cartilages, ligaments, etc [1]. Practically, a bio substitute, made with cells cultivated on scaffold, is needed. Mesenchymal stem cells (MSC) are generally the most suitable cells for such application since they are self-renewable with a great potential for differentiation and immuno suppression [2]. However, materials used for bio functional scaffold synthesis have to meet several criteria, such as biocompatibility and biodegradability. Thus, the aim of the study was to screen several biopolymers differing in their composition for their capability to promote adhesion and growth of MSC.