טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentIsakov Elina
SubjectCultivation of Human Embryonic Stem Cells in Microcapsules
DepartmentDepartment of Biotechnology
Supervisor Professor Emeritus Joseph Itskovitz
Full Thesis text - in Hebrew Full thesis text - Hebrew Version


Abstract

Embryonic stem cells (ESCs) have unique features that include unlimited growth capacity, expression of specific markers, and an ability to differentiate into any cell type of the adult body. Due to their unique capacity to regenerate functional tissue for the lifetime of an organism, human ESCs (hESCs) are an attractive "raw materials" for multiple biotechnological applications. Differentiation of hESCs can be achieved through formation of embryo-like aggregates in suspension, termed embryoid bodies (EBs). Controlling cell aggregation and agglomeration during human EBs formation has a profound effect on the extent of cell proliferation and differentiation. One of the methods to control cell-cell interactions in scalable culture is by mass encapsulation in size-specified hydrogel capsules. Cell encapsulation aims to entrap viable cells within the confines of semi-permeable membranes. The membranes are expected to be permeable for transport of molecules essential for cell survival, but not to allow transport of molecules larger than a desired critical size. Aims of the study: to develop manual and automatic microencapsulation systems for undifferentiated hESCs, compare their ability to differentiate in the microcapsules versus non-encapsulated cells and characterize the expression pattern of cell adhesion molecule E-cadherin in human EBs grown without capsules in suspension. Agarose and alginate capsules of 300-400 μm diameter were designed to encapsulate hESCs. Encapsulated cells were grown for 2 weeks in static culture. We can assume from the study that manual agarose encapsulation system was less efficient than automated microencapsulation system using alginate, in which hESCs viability was higher. The metabolic rate of encapsulated hEBs in alginate microcapsules was slower than of non-encapsulated hEBs. It is possible that static incubation of encapsulated hESCs may be crucial for the decrease of cell viability by preventing adequate transport of essential nutrients into the capsules. Furthermore, encapsulated hESCs did not form cellular aggregates and remained in their current formation in small clumps, which may have impeded their survival given the lack of cell-to-cell interactions. Our results demonstrated that E-cadherin was expressed equally not only in undifferentiated hESCs but also in most parts of the hEB. Encapsulation did not prevent hESCs from differentiating, as indicated by the up-regulation of the three lineage markers (βIII-tubulin, α-fetoprotein and α-cardiac actin). Differentiation pattern of encapsulated cells differed from that of non-encapsulated ones and should be further investigated. Due to the multilineage nature of the differentiating hESCs in the microcapsules, it would seem appropriate to differentiate hESCs prior to encapsulation.