# Alginate-Chitosan Microencapsulated Cells for Improving CD34+
## 3 Results and Discussion
### Importance of Microenvironment for CD34\({}^{+}\) Progenitor Maintenance  
Hematopoiesis, including self-renewal and differentiation, is highly regulated by the bone marrow microenvironment. Autocrine/paracrine factors such as growth factors, cytokines, and chemokines are secreted by early and differentiated hematopoietic cells, may play important roles as chemo-attractants for other hematopoietic cells (accessory cells, facilitating cells, etc. ), stimulate the secretion of other regulatory molecules, modulate the expression of adhesion molecules on the cell surface (e.g., adhesion and homing), and/or influence the survival of the hematopoietic cells [25]. Figure 1: (**A**) Illustration of microencapsulation with a semi-permeable barrier. (**B**) Schematic and microscopic image of double-layered alginate-chitosan microcapsule. (**C**) Stability of alginate-chitosan microcapsules in culture media. (**D**) Microscopic images of cell-loaded microcapsule after DNA staining. Scale bars in (**B–D**) represent 100 and 200 μm, respectively. (**E**) Cytotoxicity of alginate-chitosan microcapsules for external CD34\({}^{+}\) cells. In a recent study, a micro-cavity platform was developed for isolating HSC to grow solely or in certain cell numbers thus that it can be used to investigate the impact of autocrine and paracrine signals in HSC culture [26]. The hypothesis was autocrine signals are predominantly involved in maintaining the quiescent state of HSC in single-cell niches [26]. However, it was implied that single-cell or multi-cell cultures were unable to preserve stemness state although various growth factors were introduced (stem cell factor, thrombopioetin, and FLT3L). The experimental results showed that decreasing in CD34\({}^{+}\) ratio is more likely to happen in lower cell density. The CD34\({}^{+}\) cell population was preserved by co-culturing with microencapsulated CD34\({}^{+}\) cells. Moreover, cell expansion increased significantly in the presence of microencapsulated CD34\({}^{+}\) cells (Figure S2). The number of loaded cells had a significant impact on the effectiveness of microencapsulated CD34\({}^{+}\) cells. The paracrine release from microencapsulated cells was hypothesized to play an important role in promoting the proliferation and maintenance of cultured CD34\({}^{+}\) cells. Figure 2: (**A**) Illustration of co-culture system of CD34\({}^{+}\) cell-loaded microcapsules (\(3\times 10^{4}\) cells/well) with bare CD34\({}^{+}\) cells from unrelated donors. (**B**) Viable cell concentrations of bare cells (lower well) on day 8. (**C**) Ratio of CD34\({}^{+}\) cell population (lower well) on day 8. Striped line in C indicates percentage of CD34\({}^{+}\) cell population on day 0. Asterisks (*) in (**B**,**C**) indicate \(p<0.05\). Additional experiments were conducted to address this hypothesis. The cells (Donor 6; lower well) were co-cultured with equivalent numbers of bare or encapsulated cells from an unrelated donor (Donor 5; upper well). On day 8, the cell viabilities of bare cells (lower well) were 94.4 \(\pm\) 3.2; 91.7 \(\pm\) 4.8 and 100 \(\pm\) 0.0% for monoculture, co-culture with bare cells, and co-culture with microencapsulated cells. It was noticed that co-cultures with encapsulated or bare cells from the unrelated donor could improve the cell expansion (Figure S3A). Moreover, co-cultures produced higher percentages of CD34\({}^{+}\) cells than of monoculture (Figure S3B). These results suggested that co-culture with encapsulated cells, isolated from an unrelated donor, have beneficial effects for CD34\({}^{+}\) cell expansion and progenitor maintenance. In the present study, thrombopoetin, which can promote cell proliferation and megakaryocytic differentiation [27], was added because platelet lysate alone could not provide enough growth factors for CD34\({}^{+}\) cell survival and proliferation in vitro (Figure S4). CD41 is expressed throughout megakaryocytic lineage and thus considered as a representative marker for megakaryotic differentiation [3; 28].