Optimization For Culture Conditions And Cocultivation Of Umbilical Cord Blood Derived Stem Cells

Hematopoietic stem cells (HSCs) have been used for gene therapy, tumor depollution and hematopoiesis reconstitution. But the shortage of HSCs greatly limits their widespread clinical applications. Though a great deal of research work about ex-vivo expansion of HSCs has been done, expansion results varied in a considerably wide range under different culture conditions because of the complicated relationship between them. Till now, there is no report about optimization for HSCs ex-vivo expansion. Hence, it is important to build up the quantitative relationship between culture conditions and expansion results for HSCs through a suitable mathematic method for optimizing the HSCs ex-vivo expansion culture conditions and solving shortage of HSCs in clinical applications undoubtedly.An evaluating and predictive model for the ex-vivo expansion of HSCs was firstly built up in this study with artifical neural network (ANN) technology. 341 groups of data were summarized from literatures, in which 124, 86 and 90 data were employed to train the network and 17, 14 and 10 data were applied to predict respectively. Expansion folds of nuclear cells (NCs), CD34⁺ cells and Colony-forming units (CFU-Cs) were chosen as evaluation objectives and inoculated density, cytokines, cell resources, serum, stromal cells, culture time and bioreactor types were chosen as network inputs. The calculated results show that for the training of network, the interval accuracy of the expansion folds for the different cells is 85.5%, 86.1% and 86.7% respectively. While for the prediction of network, the interval accuracy can be up to 82.4%, 71.4% and 70.0% respectively. Therefore this nonlinear modeling makes it possible to describe quantitatively the effects of the culture conditions on the HSCs expansion and to predict the optimal culture conditions for higher ex-vivo expansion of HSCs.Besides the evaluation and prediction for the existing literature data, more optimizations for culture condition and predictions for the HSC expansion were carried out with the founded ANN model. The quantification of all the 7 factors influencing the ex-vivo expansion of HSCs was defined and the case combination of the values taken by these influencing factors could result in 18480 groups of possible culture conditions. Only quite few predictive results of these conditions have been verified with experiments, most of them were still not. In order to use the optimized culture conditions with ANN model safely and reliably, the extrapolated predictive reliability of the network need to be confirmed with more verification experiments. Therefore, 6 groups of representative culture conditions were selected as verification experiments from all these 18480 cases. Since alginate chitosan (AC) beads would be applied in the encapsulation of stromal cells to coculture HSCs in most verification experiments, the optimized preparation parameters for AC beads were firstly determined by fractional factorial experiments, which were 1% (wt%) alginate, 3% (wt%) CaCl₂, 1% (wt%) chitosan, 8 minutes for both the first and the second step reaction. Based on this, the optimized operation conditions for stromal cells (Rabbit mesenchymal stem cells, Rabbit-MSCs) encapsulated in AC beads to support the ex-vivo expansion of UCB-HSCs were also determined by fractional factorial tests, which were 2×l0⁵cells·mL^-1 inoculate density for Rabbit-MSCs in alginate, 3:1 for the weight ratio of high and low molecular weight of chitosan and 5:1 for cell inoculate density ratio of UCB-MNCs and Rabbit-MSCs. Thus the optimized protocol was determined to encapsulate stromal cells in AC beads for the next verification experiments and subsequent coculture of UCB-HSCs and UCB-MSCs in bioreactors.The results of 6 groups of verification experiments showed that there were 4, 5 and 6 experimental results in agreement with the predictive results for the ex-vivo expansion of CD34⁺ cells, NCs and CFU-Cs respectively, which meaned that the predictive accuracy of this ANN was 67.7%, 83.3% and 100%. Therefore the ANN model was reliable and suitable.UCB-MSC is another type of stem cells in UCB, which can not only support the expansion of HSCs in vitro as stromal cells but also alleviate complications and accelerate recovery of hematopoiesis during hematopoietic stem cell transplantation. However it proved challenging to culture MSCs from UCB with a low probability of 50%～60% even after optimized study. In this work, in order to improve the probability of separating and obtaining UCB-MSCs, three fractional factorial experiments were designed and performed to investigate the main influencing factors for the primary culture, the key cytokines and the dose of the suitable cytokines used. The cultured UCB-MSC-like cells were characterized by immunophenotypic and multi-lineage induced differentiation analysis. The experimental results showed that the probability of culture UCB-MSCs could be improved from 50%～60% to 90% with adding 18ng·ml^-1 IL-3 and 5ng·ml^-1 GM-CSF based on high cell inoculated density. Moreover, the UCB-MSC-like cells expressed MSCs-related surface markers of CD13, CD29, CD44 and CD105, but not hematopoietic cells-related surface markers of CD34, CD45 and HLA-DR. Meanwhile, these cells could differentiate into osteoblasts, chondrocytes and adipocytes similarly to MSCs derived from bone marrow. Therefore, it is possible to provide enough UCB-MSCs for clinical application with this opitimized culture method.Lastly, the feasibility of coculture autologous UCB-HSCs and UCB-MSCs was investigated because of the great importance of clinical application. Spinner flasks and rotating wall vessel bioreactor (RWVB) together with glass coated styrene copolymer (GCSC) microcarriers were applied in the study. IMDM medium without serum but supported with the combination of cytokines, including SCF 15ng·mL^-1, FL 5ng·mL^-1, TPO 6ng·mL^-1, IL-3 15ng·mL^-1, G-CSF 1ng·mL^-1 and GM-CSF 5ng·mL^-1, was adopted. And allogeneic adipose derived stem cells encapsulated in AC beads were used for the coculture with UCB-HSCs and UCB-MSCs. Meanwhile diluted-feeding protocol was applied during the culture process. The results indicated that the expansions of total cell number, CFU-Cs, CD34⁺CD45⁺CD105^-(HSCs) cells and CD34^-CD45^-CD105⁺ (MSCs) cells in RWVB were 3.7+0.3-fold, 5.1 + 1.2-fold, 5.2+0.4-fold and 13.9+1.2-fold respectively in RWVB, better than those in spinner flasks. After coculture, UCB-HSCs and UCB-MSCs can be easily separated by gravity sedimentation since UCB-MSCs are adhered on microcarriers. At the same time, the fibroblast-like cells appeared on the surface of GCSC microcarriers can be induced and differentiate into osteoblasts, chondrocytes and adipocytes and expressed MSCs-related surface markers of CD13, CD44, CD73 and CD105, while not hematpopietic cells-related surface markers of CD34, CD45 and HLA-DR, which is similarly to MSCs derived from bone marrow. In conclusion we have developed a feasible culture system to cocultivate UCB-HSCs and UCB-MSCs by adding cytokines and stromal cells together with GCSC microcarriers in RWVB.