Innovative process engineering for semi-industrial culture of human mesenchymal stem cells – STEMCellREAcTOR

For this, the project is divided into five work tasks. The objective of the first task is to develop numerical simulation tools allowing Euler-Lagrange description of the solid-liquid flow. The eulerian spatial distribution of liquid velocity and turbulence within mini-bioreactors will be obtained through CFD-RANS simulations, experimentally validated. Further description of the turbulence will be conducted by LES simulations for the most promising conditions. Particle dynamics is studied numerically and experimentally establishing, for the microcarriers, their concentration gradients, their probability of collision and their paths, in order to record particulate hydrodynamic history. Specific work dedicated to the tailor-made design and synthesis of functionalized microcarriers and microcapsules will be conducted to meet the physiological needs of hMSCs by mimicking their natural organic niches and to consider, eventually, a significant increase of cell production performance. Based on these initial results, mixing and aeration configurations and materials used for cell adhesion will be retained. For these ones, hMSCs cultures will be conducted in mixed mini-bioreactors and analyzed by bioengineering and cell biology tools. The overall results will be analyzed to model the coupling between the cellular response (growth, stemness, differentiation) and the cellular microenvironment (surface adhesion, substrate concentrations and hydromechanical stresses). This hydro-biological model will be used as a basis to propose scale-up and operation rules for a larger scale process. Finally, the pilot-scale bioreactor will be designed by meeting not only the constraints imposed by the hydro-biological model but also various specifications including regulatory criteria, choice of material and (eco) integrated design for user-friendly applications, particularly in hospitals. This system will be validated numerically and experimentally by implementing hMSCs culture

Task 1
Establishing an experimental system for measuring the local concentration of microcarriers in a stirred tank by measuring the intensity of transmission light.
Numerical simulation of liquid-microcarrier flows in stirred tanks.
Task 2
Implementing microcarrier to heart PLA and dextran ring that will be probably the first completely biodegradable microcarrier. The importance of the covalent bond between the core and the ring has been demonstrated for nanoparticles in terms of their colloidal stability in the stem cells culture medium or by comparison with a powerful competitive surfactant. Finally, the very first photo-sensitive microcarrier (totally innovative in literature) have been developed.

Task 3. Culture of mesenchymal stem cells from bone marrow (BM-MSCs) was performed on the microcarriers based on PLA, synthesized previously in the context of the 2.1 subtask. Preliminary results showed that the cells had a low adhesion probably because of the hydrophilic and neutral character of the surface of microcarriers. Thus, tests on 2D support are currently underway to validate the formulation of surfaces. Also, a bibliographical work was carried out to determine the optimal composition of the culture medium for the expansion of mesenchymal stem cells from umbilical cord (UC-MSCs). A culture medium dedicated and responsive to the regulation of Good Manufacturing Practice has been identified and based on the use of clinical grade human platelet lysate. The first cultures of these cells in the bioreactor will be performed by using this specific culture medium as scheduled in autumn 2016

ARTICLES
Collignon, M. L., Delafosse, A., Calvo, S., Martin, C., Marc, A., Toye, D., & Olmos, E. (2016). Large-Eddy Simulations of microcarrier exposure to potentially damaging eddies inside mini-bioreactors. Biochemical Engineering Journal, 108, 30-43.
Martin, C., Olmos, É., Collignon, M. L., De Isla, N., Blanchard, F., Chevalot, I., ... & Guedon, E. (2016). Revisiting MSC expansion from critical quality attributes to critical culture process parameters. ProcessBiochemistry. In press..
Aljawish, A; Muniglia, L; Chevalot, I (2016) Growth of human mesenchymal stem cells (MSCs) on films of enzymatically modified chitosan Biotechnology Progress, 2016, 32 (2), 491-500.
Olmos, E., Loubiere, K., Martin, C., Delaplace, G., & Marc, A. (2015). Critical agitation for microcarrier suspension in orbital shaken bioreactors: Experimental study and dimensional analysis. Chemical Engineering Science, 122, 545-554.
Laurent, C.P.; Vaquette, C.; Martin, C.; Guedon, E.; Wu, X.; Delconte, A.; Dumas, D.; Hupont, S.; Isla, N.D.; Rahouadj, R.; Wang, X(2014). Towards a Tissue-Engineered Ligament: Design and Preliminary Evaluation of a Dedicated Multi-Chamber Tension-Torsion Bioreactor. Processes, 2, 167–179
Communications /diffusion
Martin C., Piccini A., Chevalot I., Olmos E., Guedon E., Marc A., (2015). Serum-free media for Mesenchymal Stem Cells expansion on microcarriers. 24th ESACT meeting «Cells, Culture, Patients, Products«, 31 mai-3 juin 2015, Barcelone. BMC Proceedings, 9 (Suppl 9): P70.
Olmos, E. (2016). Analyse dimensionnelle de la mise en suspension de particules en fioles agitées sur table orbitale. Journée thématique de la Société Française de Génie des Pro-
cédés, Paris, 8 mars 2016.
A.Roy, J. Babin, M. Leonard, A. Durand, J.-L. Six, «Microporteurs biodégradables pour la culture de cellules souches mésenchymateuses humaines en bioréacteur« ,14e Journée Scientifique du GFP section Grand-Est, Nancy, 16 juin 2016.

Human mesenchymal stem cells (hMSC) exhibit very promising applications in regenerative medicine but these cells show high specificities (limited growth capacity in time, cell differentiation capabilities related to biochemical and hydromechanical microenvironment). Thus, the large-scale use of these cells as products still presents a number of limitations including a reliable cell expansion process (quality and quantity of the cells produced). Noting that bioreactors available for stem cell culture are either directly transposed from the culture of continuous cell lines or only systems poorly or not "controlled", the STEMCREATOR project will build a rigorous scientific methodology combining the skills and know-how of French (LRGP, Nancy; LPCM, Nancy) and international (LGC, Liège, Belgium; IBC, Moscow, Russia) academic teams and two SMEs involved in the design of bioreactors (GPC, La Rochelle; Bio-Inox, Bergerac, France). This methodology will lead to the design and construction of an innovative ten liters bioreactor specifically dedicated to the culture of hMSC. For this purpose, the project is divided into four work tasks.
The objective of Task 1 (LGC, LRGP, GPC and Bio-Inox) is to develop numerical simulation tools allowing Euler-Lagrange description of the solid-liquid flow. The eulerian spatial distribution of liquid velocity and turbulence quantities within mini-bioreactors will be obtained through CFD-RANS simulations experimentally validated (PIV, PLIF, measurements of P/V). Further description of the turbulence will be conducted by LES simulations for the most promising conditions. Particle dynamics will be studied numerically (lagrangian tracking) and experimentally (3D trajectory), establishing, for the microcarriers, their concentration gradients, their probability of collision and their paths, in order to record particulate hydrodynamic history. Specific work dedicated to the tailor-made design and synthesis of functionalized microcarriers (Task 2.1, LCPM) and microcapsules (Task 2.2, IBC) will be carried out to meet the physiological needs of hMSC by mimicking their natural organic niches and to achieve a significant increase of cell production performance. Based on these initial results, mixing and aeration configurations and materials used for cell adhesion will be retained. For these ones, hMSC cultures will be carried out in stirred mini-bioreactors and analyzed by bioengineering (specific rates of growth, consumption and production) and cell biology (flow cytometry, RT-qPCR) tools (Task 3.1, LRGP, LCPM, IBC). The overall results will be integrated to model the coupling between the cellular microenvironment (surface adhesion, substrate concentrations and hydromechanical stresses) and the cellular response (growth, stemness, differentiation) (Task 3.2, LRGP, LGC). This hydro-biological model will be used as a basis to propose scale-up and operation rules for a larger scale process (Task 4.1, LRGP, LGC, LCPM, GPC, Bio-Inox, IBC). Finally, the pilot-scale bioreactor will be designed by fulfilling not only the constraints imposed by the hydro-biological model but also various specifications including regulatory criteria, choice of material and (eco) integrated design for user-friendly applications, particularly in hospitals (Task 4.2, GPC, Bio-Inox). This system will be validated numerically and experimentally by implementing hMSC cultures (Task 4.3, LRGP, GPC, Bio-Inox).