| Haematopoietic stem/progenitor cells (HSPC) are transplanted for patients with malignant and nonmalignant conditions who lost the haematogenous faction. However, haematopoietic stem/progenitor cells (HSPC) from all source is far from sufficient. Alginate-Poly-lysine- Alginate(APA) encapsulation has been developed in our Lab in the form of scalable, controlled culture systems to engineer various types of tissues. Wang et al had noted APA liquefied miroencapsulation system which was able to support mESC maintenance for extended periods without the addition of cytokines or MEF. Encapsulation not only directly controls the key culture parameters through variations in the encapsulation process, but also facilitates rapid cell recovery for doun-stream analysis and application. APA encapsulation has been investigated for use as scalable, controlled culture systems to engineer various types of tissues. In this paper, we employed APA microencapsulation as a 3-D close scaffold for the expansion of HSPCs by mimicking the bone marrow microenvironment. We chose mononuclear cells (MNCs) as starting culture cells for APA culture system to avoid the costly and time-consuming CD34+ cells isolated procedure. To observe whether or not APA capsulation system can support ex vivo expansion of CD34+ cells, we assessed the number of CD34+ cells by Fluorescence Activated Cell Sorter(FACS). To further confirm intrinsic ex vivo expansion capacity of capsule system, we performed Clonal Formation Unit (CFU) culture in different days.Results: 1.The data indicated that 4×106 cells/mol alginate (about 150-200 cells per capsule) was the optical cell density, which could makethe best proliferation profile among all the different cell densities tested; 2. Encapsulated cell growth profile and metabolic activity of the enclosed cells were retained during 18 days of culture, these results showed that UCB-derived MNCs were quiescent initially and in low metabolic activity, then increased gradually and maximal activity occurred after approximately 15 days of capsulated culture; 3. The higher total cell number, CD34+ cell and CFU production were observed in APA microcapsule compared to plating culture.Discussion: In vivo, the marrow microenvironment plays a critical role in the induction of proliferation and differentiation of hematopoietic stem/progenitor cells (HSC/HPC). The intricate structure of the microenvironment is depended cell-cell and cell-matrix interactions are facilitated by its 3-D conformation and high cell density. We developed umbilical cord blood (UCB)cell culture in APA capsule as a new approach to simulate an in vivo microenvironment and simultaneous expand HSPC in this study. Our results showed that expression of marker remained over 15 days of culture in vitro, and the APA-MNC system being a new system for the expansion of hematopoietic stem cells. Our 3-D capsule culture system supported significant progenitor production from CB cells, and the possible mechanism is as follows. (1)Microcapsules provide a relatively close, not open, 3-D culture system resembling to the physical topography of bone marrow microenvironment. (2) The nature polysaccharides, alginate matrix, provide a fine surface for stromal cells gained and HSPCs expansion. (3) The amount of protein around cells was higher than that in 2-D culture system. And the capsule membrane or alginate could hold back some proteins and ECM from diffusion. Confined environment of APA microcapsules makes the supporting cells of MNCs adhere to the inner surface of capsule and develop into stromal cells. The cells will excrete ECM and cytokines to maintain hematopoietic stem/progenitor cells proliferation. (4) The alginate entrapment may either promote cell-cell contact or decrease intercellular spacing.Conclusion: These findings implied that the integration of the intrinsic characteristics of MNC and their microenvironment determines their fate. In summary, we investigated APA microcapsule as a novel 3-D system toconstruct a hematopoietic microenvironment. UCB MNCs cultured in APA capsule yielded higher total cell, CD34+ cell and CFU production compared to 2-D Petri dish. The establishment of APA-MNC system provides a new system for the expansion of hematopoietic stem cells.1...
|
PDF Full Text Request