טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentGoldshmid Revital
SubjectA 3D-Fibrinogen Based Microgel Suspension Culture System for
hMSC Bioprocessing in Bioreactors
DepartmentDepartment of Biotechnology
Supervisor Professor Dror Seliktar


Abstract

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The emerging approach to tissue therapy requires replacement of damaged cells with healthy multipotent stem cells, having the potential to differentiate into several mature cell types. The use of mesenchymal stem cells (MSCs) is a promising tool for regenerative stem cell therapy due to their ability to self-renew and their ability to differentiate into varied tissues. In order to direct cell fate, a 3D hydrogel microenvironment model can be applied. The interplay between the cells, the biomechanical attributes and the dynamic relationship between the physical and biochemical signals allows us to direct hMSC proliferation and differentiation and provides insights which will advance stem-cell-based clinical approaches for tissue regeneration.

In the present study, we integrated biological-synthetic polymer matrix and bioreactors for the development of a unique suspension cell culture system for 3D stem cell expansion. A fibrinogen based Polyethylene Glycol (PEG) hydrogel and fibrinogen based Pluronic F-127 were used to form a 3D niche for hMSC bioprocessing.  We focused on developing a methodology for the encapsulation and cultivation of hMSCs in hydrogel biomaterials to be utilized as a routine, efficient, and scalable solution for hMSC bioprocessing.

Our 3D-PF and FF-127 micro-engineering model offers the versatility required to undertake the arduous task of developing 3D bioprocessing technologies for stem cells which affect the self-renewal, proliferation and morphology of MSCs using various blood derivatives and variable mechanical stiffnesses of 3D hydrogel micro-carriers for optimized MSCs expansion and differentiation.

Herein, we present a method for efficient propagation of hMSCs. We demonstrated a gradient expression of multipotency, proliferation and differentiation markers guided by variable mechanical stiffnesses, converging towards a specific pattern of mechanical control on MSCs fate. In addition, we determined that using Von Willebrand Factor, (a human blood derivate), elevates MSC proliferation, while fibronectin affects hMSC morphology. In addition to 3D-PF, we utilized thermos-responsive micro-carriers (FF-127), and demonstrated that the hMSCs’ viability, proliferation and cell recovery yields were shown to be higher than photo-chemically cross-linked over physical crosslinking micro-carriers made from a similar material.

As for the differentiation of hMSCs, the results suggest that modification of storage modulus (G') of the 3D-PF hydrogel enables direction of cell fate. Chondrogenesis of hMSCs has been shown to increase in correlation with an increase in the storage modulus (G'). Neurogenesis, on the other hand, requires a soft hydrogel environment and the myogenic potential of hMSCs was shown to increase in the mid-stiffed milieu. In addition, we improved cell recovery after encapsulation using physical encapsulation method with the FF-127.

This study has established a microgel suspension culture system for hMSCs bioprocessing using a direct correlation between hydrogel attributes, including: stiffness, adhesiveness, and addetives manipulating the hMSC fate in 3D. Moreover, it illustrates the significance of meticulous formulation and a modular approach when designing biosynthetic materials that could ultimately be optimized for hMSCs bioprocessing applications.