# Alginate-Chitosan Microencapsulated Cells for Improving CD34+
## 3 Results and Discussion
### Importance of Microenvironment for CD34\({}^{+}\) Progenitor Maintenance
It was found that bare and encapsulated cells have opposite effects on CD41 expression of co-cultured cells (Figure S5). Co-culture with bare cells from different donors suppressed CD41 expression of cultured cells (lower well). On the contrary, when the equivalent cells were encapsulated with alginate-chitosan, it could promote CD41 expression of cultured cells (lower well). To our knowledge, this is the first time such a paracrine effect is reported. There are two possible mechanisms: (1) direct effect in which chitosan and/or alginate interact with thrombopoetin and subsequently promote megakaryocytic differentiation of external cells or (2) indirect effect in which chitosan and/or alginate interact with thrombopoetin or encapsulated cells and subsequently cause the entrapped cells to release paracrine factors that promote megakaryocytic differentiation of external cells. Further studies are needed to understand the detailed mechanism. Encapsulation for transplantation was initially developed to improve allogeneic pancreas islet transplantation for diabetic patients [29]. Semi-permeable membrane provides mechanical protection and immune rejection for transplanted islets while nutrients and cellular metabolites freely diffuse through the membrane [30]. Several clinical trials have been performed, and the follow-up evaluation showed a safe and promising result of alginate-based microencapsulated islets for diabetic patients [30]. Inspired by these findings, we developed CD34\({}^{+}\) cells-loaded microcapsule to demonstrate paracrine factor delivery. The microcapsule possessed no toxicity for external cells. The 3D capsules could restrict cell growth of mesenchymal stem cells (MSCs) [31], myoblast and fibroblast cells [32], which was postulated due to limited nutrients/oxygen exchange. Limited cell growth is beneficial to prevent overpopulation inside the capsule as long as the cells are still capable of releasing paracrine factors. In addition, microencapsulation can be an advantage in facilitating scaled-up co-culture systems for HSC or progenitor expansion with bioreactors (Figure S6). HSC homeostasis maintenance required paracrine factors secreted by niche cells to supply or feed the right signal for HSCs [33]. A study reported encapsulation of several cell types, including human amniotic-MSCs, umbilical cord-MSCs, and fibroblasts, to maintain CD34\({}^{+}\) cell expansion in vitro [31]. Their finding indicated that encapsulated MSCs or fibroblasts do not benefit CD34\({}^{+}\) cells proliferation [31]. Interestingly, improved cell proliferation of CD34\({}^{+}\) cells by co-culturing with osteosarcoma cell line has been reported [34]. Instead of employing other cells, our study used CD34\({}^{+}\) cells collected from unrelated donors as the source of paracrine factors required for hematopoietic progenitor maintenance. This system can be utilized to deliver paracrine factors for ex vivo HSC or progenitor expansion with bioreactor and further developed to support initial engraftment of allogeneic HSC transplantation.