טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentGalia Yirme
SubjectControlled Bio-Processes for Proliferation and
Differentiation of Human Embryonic Stem Cells
DepartmentDepartment of Biotechnology
Supervisor Professor Emeritus Itskovitz Joseph
Full Thesis textFull thesis text - English Version


Abstract

Human embryonic stem cells (hESCs) have tremendous potential as a cell source for cell-based therapies. Realization of that potential will depend on our ability to understand and manipulate the factors that influence cell fate decisions and to develop scalable methods of cell production. By definition they are self-renewing cells. They also hold the capacity to produce every type of cell and tissue in the body. Differentiation of hESCs can be instigates through the formation of embryo-like aggregates in suspension, termed embryoid bodies (EB). Controlling cell aggregation and agglomeration during human EB formation has a profound effect on the extent of cell proliferation and differentiation. The main goal of this research was to develop a controlled dynamic bioprocess for the propagation of differentiated hESCs. We hypothesize that such propagation can be affected and manipulated by physicochemical and cellular parameters. Here we describe a robust scale-up technological platform for the formation, propagation and differentiation of hESC-derived embryoid bodies (EBs). The efficiency of the stirred process compared to static cultivation was analyzed. Seeding and propagation parameters were optimized to achieve efficient tissue culture scale up. Quantitative analyses of viable cells and EB yield revealed 6.7-fold enhancement in the generation of hESC-derived cells after ten days of cultivation in our system. Other metabolic indices, such as glucose and glutamine consumption, lactic acid production, lactate dehydrogenase (LDH) secretion and pH, pointed to efficient cell expansion in the stirred cultures. Maintenance of suitable hydrodynamic conditions during hESCs seeding and cultivation was found to be crucial for the EB formation and propagation. The duration of EB formation in static Petri dishes and EB cultivation in stirred systems resulted in high EB yield, round homogenous shape and fastest growth rate. The appearance of representative tissues derived from the three germ layers as well as primitive neuronal tube organization, blood vessel formation, and specific-endocrine secretion indicated that the initial developmental events were not altered in the stirred formed human EBs. Furthermore, well developed endothelial networks and contracting EBs with functional cardiac muscle cells, as well as well developed neural dendrites, were obtained after two weeks of cultivation. Linear scaling up of our 250ml Glass Boll Impeller spinner flask was established up to 250ml, the maximum volume available. Linear extrapolation can be done to 1L spinner flask supplied by the manufacture. Collectively, our study defines the technological platform for controlled large-scale generation of hESC-derived cells for clinical and industrial applications.