# Alginate-Chitosan Microencapsulated Cells for Improving CD34+
## 1 Introduction  
Hematopoietic stem cells (HSCs) are multipotent cells capable of generating all blood components, including erythrocytes, leucocytes, and thrombocytes [1]. HSCs represent only a fraction of the cell population in umbilical cord blood, bone marrow, or peripheral blood. To isolate the HSCs from heterogenous populations, specific surface markers such as HLA-DR, CD38, CD117 (c-kit), CD45, CD133, and/or CD34 have been used [2]. One of the prominent surface markers that is widely used to isolate hematopoietic progenitorfrom heterogenous populations is CD34 [3,4]. CD34\({}^{+}\) cells that have enhanced progenitor activity represent a small proportion of the population [4]. Previous studies have reported that transplantation of CD34\({}^{+}\) cells successfully established multi-lineage hematopoietic engraftment [5] and improved neurobehavior of animals with stroke [6,7], brain injury [8], and cerebral palsy [9].  
In clinical cases, HSCs transplantation is required for various applications, including repeated chemo- or radiotherapy for cancer patients, leukemia, lymphoma, or blood and bone marrow disorders [10]. Although the efficacy has been proven and protocol has been established, providing HLA-matched HSCs is challenging due to the diversity of HLA types and because the cells hardly propagate ex vivo.  
In this study, we propose microencapsulation of non- or low-matched CD34\({}^{+}\) cells for protection against immune rejection and facilitating paracrine excretion. Microencapsulated CD34\({}^{+}\) cells can be utilized to deliver paracrine factors of unrelated CD34\({}^{+}\) cells for ex vivo expansion with a bioreactor system. For a long-term target, the microencapsulation system can be further developed to deliver paracrine factors, which provide signals to resident HSCs for regulating the homeostasis of HSCs in their niche. Two main activities that should be performed by HSCs to maintain homeostasis are cell proliferation and stemness preservation.  
The novelty of this study lies in the design of a 3-dimensional (3D) scaffold for encapsulating CD34\({}^{+}\) cells and the idea for co-cultures between CD34\({}^{+}\) cells from different donors. To our knowledge, published works on 3D HSC scaffolds were generally used for ex vivo HSC culture and expansion systems and as a model for interaction between HSCs and their microenvironment [11,12]. Alginate-based capsules [13,14], 3D polyethylene terephthalate-based nanofiber [15], and collagen gel [16] have been studied as mesenchymal stem cells (MSCs) loaded scaffolds [17] and HSC-MSC micro-aggregates [16]. In these studies, biocompatibility, durability, and transplant feasibility have not always been the main considerations for designing the systems. We initially developed collagen-based scaffolds for encapsulating CD34\({}^{+}\) cells [18]. Cell viability was not affected by the collagen-based encapsulation, but it is likely that collagen promotes cell differentiation as indicated by decreasing CD34\({}^{+}\) cell population [18]. In the next step, alginate-based microcapsules were constructed [19]. In this paper, chitosan, a biocompatible polymer [20], was cross-linked with glutaraldehyde [21] and used as an outer layer to further improve alginate microcapsule stability [22]. The impact of co-culture between microencapsulated CD34\({}^{+}\) cells and bare CD34\({}^{+}\) cells from different donors was investigated. Paracrine effects by the microencapsulated CD34\({}^{+}\) cells towards the bare CD34\({}^{+}\) cells in the lower well were analyzed for cell viability and progenitor maintenance represented by the CD34 marker.