טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentShai Meretzki
SubjectStationary Packed Bed Bioreactor for Propagation of Human
Haemopoietic Stem Cells
DepartmentDepartment of Biotechnology
Supervisor Assistant Professor Merchav Shoshana


Abstract

The haemopoietic system in mammals is composed of a heterogeneous population of cells that range in function from mature cells with limited proliferative potential to Pluripotent Haemopoietic Stem Cells (PHSC) with extensive proliferative, differentiative and self-renewal capacities. The PHSC are exclusively required for haemopoietic reconstitution following transplantation and serve as a primary target for gene therapy. In spite of the key role of PHSC in maintaining the haemopoietic system, current strategies aimed at ex vivo maintenance or expansion of transplantable PHSC, have so far been met with limited success.

     

 It is widely accepted that PHSC are intimately associated in vivo with discrete niches within the marrow, which provide molecular signals that mediate their differentiation and self-renewal, via short-range interactions and the secretion of stromal cell associated soluble factors. In this work we developed a novel three dimensional (3D) stationary phase bioreactor which closely mimics the bone marrow microenvironment and which is capable of supporting the growth and prolonged maintenance of marrow stromal cells. The latter are seeded on porrosive carriers made of a non woven fabric matrix of polyester, packed in a glass column, thereby enabling the propagation of large cell numbers in a relatively small volume.


The three dimensional (3D) stromal cell cultures were superior to stromal cell monolayers (2D) in supporting the maintenance/expansion of human cord blood     (CB) CD34+38-CXCR4+ cells representing NOD/SCID repopulating cells. Furthermore, media conditioned by stromal cells within the bioreactor (3D SCM) was better than stromal cell monolayer SCM, in supporting the expansion of CB CD34+38-CXCR4+ cells. These findings demonstrate that our 3D bioreactor provides a suitable system for ex vivo expansion of human HSC via superior stromal-stem cell contact and stromal cell production of soluble stem cell self renewal regulators.


The establishment of 3D culture system for stem cell expansion is highly essential for potential expansion of undifferentiated stem cells for transplantation and genetic modification and may facilitate the detection of novel cytokines inducing the expansion of undifferentiated stem cells.