Ex Vivo Expansion Of Hematopoietic Stem Cells And Progenitors In Rotating Wall Vessel Bioreactor

Ex vivo expanded hematopoietic stem cells (HSCs) and progenitors have a wide range of potential applications in therapy, including 'graft engineering' in stem cell transplantation, gone and immunotherapy and the production of mature blood cells for transfusion. Human umbilical cord blood (UCB) is an attractive source of cells for transplantation therapy and regenerative medicine. However, the low number of HSCs and progenitors obtainable from a single donor significantly restricts its widespread application as a viable source of transplantable hematopoietic cells in adults. It is thcrefore necessary to develop ex vivo culture systems to scale up the expansion of UCB HSCs and progenitors. Conventional static culture systems, such as T-flasks, have several inherent limitations, which are not suitable to be used in the expansion of HSCs and progenitors at a large scale. Hence there is an urgent need for developing bioreactors for HSCs expansion, which overcomes the limitation of mass transport and keeps culture parameters constant.This research used rotating wall vessel (RWV) bioreactor with appropriate culture protocols, such as suspension culture without stromal cells and microencapsulated coculture, to expand human HSCs arid progenitors derived from UCB at a large-scale. And a mathematic model on the static and RWV microencapsulated coculture was also developed to simulate the mass transfer in those coculture systems.Firstly, expansion of UCB mononuclear cells (MNCs) alone was carried out in a RWV bioreactor and tissue culture flasks (T-flasks) in serum-containing medium supplemented with relatively low doses of purified recombinant human cytokines (5.33ng/ml IL-3, 16ng/ml SCF, 3.33ng/ml G-CSF, 2.13ng/ml GM-CSF, 7.47ng/ml FL and 7.47ng/ml TPO) for 8 days. The cell density, pH and osmolality of the culture medium in the two culture systems were measured every 24 hours. Flow cytometric assay for CD34~+ cells was carried out at Oh, 144h and 197h and methylcellulose colony assays were performed at Oh, 72h, 144h and 197h. The pH and osmolality of the medium in the two culture systems were maintained in the proper ranges for HSCs and progenitors culture. The RWV bioreactor, combined with a cell-dilution feeding protocol, was efficient to expand UCB M-NCs. At the end of 200h culture, the total cell number was multipled by 435.5±87.6 times, and CD34~+ cells 32.7±15.6 times, and colony-forming units of granulocyte-macrophage (CFU-GM) 21.7±4.9 times. While in T-flasks, however, total cells density changed mildly, CD34~+ cells and CFU-GM decreased in number. The results of section demonstrate that the RWV bioreactor can provide a better environment for UCB MNCs expansion, enhance the contact between HSCs and accessory cells and make the utilization of cytokines more effective than T-flasks.Since HSCs can be better expanded in the present of stromal cells, especially of early progenitors, a reasonable coculture protocol is very important and required for HSCs expansion with the support of stromal cells. The method of direct contact coculture was used in most research, which is however, not qualified for clinical application. Microencapsulation technology may provide a technical solution, which is the most commonly applied procedure for immunoisolation. In this research, the appropriate alginate bead (Alg bead) was determined to encapsulate stromal cells after strength test. And then stromal cells, rabbit rabbit bone marrow (BM) mesenchymal stem cells (MSCs) were encapsulated in these Alg beads (2.1mm diameter) for 7 days culture. Laser confocal microscope confirmed that MSCs in Alg beads grew well after culture, which indicated that microencapsulated stromal cells could be used in our next coculture experiment with hematopoietic cells.Then the static expansion of UCB MNCs was investigated in a novel coculmre system by means of encapsulation of rabbit BM MSCs in Alg beads with 2.1mm diameter. Three kinds of media were applied and the experiments lasted for 7 days. It was found that the encapsulated MSCs illustrated remarkable effects on UCB MNCs expansion regardless whether serum was present in culture media or not. At the end of 168h coculture, the total nucleated cell number was multipled by 154±2.85 times, and CD34~+ cells 5.33±0.32 times and colony-forming units in culture (CFU-Cs) 5.6±1.21 times respectively in the serum-free media supplemented with conventional dose of cytokines (SCF 50ng/ml, FL 50ng/ml, TPO 50ng/ml and IL-3 25ng/ml), which was very similar to the results of containing 20% serum media. While in the control, i.e. MNC expansion without encapsulated MSCs, however, total nucleated cells density changed mildly, CD34~+ cells and CFU-Cs showed little effective expansion. It can be concluded from this part of the experiment that the encapsulated stromal cells can support the expansion of UCB MNCs effectively under the static experimental condition.In addition, it was found that the coculture in perfusion and fixed bed bioreactors could not support the HSC expansion at a large-scale, which resulted from the less effective interaction between HSCs and stromal cells since they had little chance to contact each other with short distance and long period in these bioreactors. Therefore. it is necessary to develop a novel dynamic coculture system to realize the efficient communication of HSCs and stromal cells and easy separation of them after HSCs expansion. RWV bioreactor plus microencapsulation may provide the answer. Therefore, the expansion of UCB MNCs was also carried out with the support of alginate-chitosan-alginate (ACA) microcapsules containing rabbit BM MSCs in a RWV bioreactor and culture plates in serum-free medium supplemented with conventional doses of purified recombinant human cytokines (SCF 50ng/ml, FL 50ng/ml, TPO 50ng/ml and IL-3 25ng/ml) for 7 days. The pH and osmolality of the medium in the two coculture systems were still maintained in the proper ranges for HSCs and progenitors. The RWV bioreactor, combined with a cell-dilution feeding protocol, was efficient to expand UCB MNCs at a large-scale. At the end of 168h culture, the total nucleated cell number was multipled by about 107-fold, and CD34~+ cells 26-fold, and CFU-Cs 19-fold. While in culture plates, however, total nucleated Cell number was multipled by 10-fold and CD34~+ cells and CFU-Cs numbers changed mildly. So it is demonstrated that the expansion of HSCs and progenitors can be achieved at a large-scale with the support of microencapsulated stromal cells in the RWV bioreactor.Finally, since lots of factors affected the culture outcome of the microencapsulated coculture system, such as diameters of Alg beads, stromal cell density inside beads and the flow rate of medium in RWV bioreacter, a mathematic model was then developed to evaluate the mass transfer in static and dynamic microencapsulated coculture under different culture parameters. This model well reflected the glucose, lactic acid and dissolved oxygen bulk concentration in culture medium and concentration distribution inside or outside Alg beads. Therefore, it can be further applied to the optimization of microencapsulated coculture.