Optimization of microbeads for amplification of stem cells

The adoption of auto- or xeno- grafts is frequently used in surgery, nevertheless this approach still raises several concerns, amongst other in terms of risk of rejection, infection, availability, limited amount, weakening of tissue or even pain of the collection site. For all these reasons, regenerative medicine becomes more and more attractive, allowing to restore or replace tissues and organs, which have been damaged by diseases or trauma.
The daily application of this new clinical strategy imposes to solve several challenges. One of them is linked to the low number of stem cells (~40,000) which can typically be recovered from a human donor compared to the needs required in clinical therapy. Up to 500 million of stem cells are indeed needed to be injected to achieve efficient therapy. Such a high dose of stem cells is explained due to their rapid diffusion from the injection site and their low survival rate upon implantation. The adoption of biocompatible and biodegradable microcarriers to amplify stem cells in vitro should allow to answer these two main issues. Stem cells can be already pre-cultured on these microcarriers before proceeding to their local injection within the injury site. This approach combines the following main advantages by the formation of microcarrier-cells (Figure 1) :
i) a large surface-to-volume ratio promoting cell expansion,
ii) an easiness for parenteral administration without needs to detach MSCs, a main source for low survival rate,
iii) biocompatibility and biodegradability enhancing tissue reconstruction,
iv) to promote the specific targeting of stem cells within a specific tissue and to facilitate their integration.
The objective of this work relies on the optimization of biodegradable and biocompatible microcarriers tailored to promote the amplification of stem cells in vitro. To avoid any biocompatibility problems, these microparticles will be manufactured from FDA-approved degradable aliphatic polyesters. The degradation rate is also an important specification of our microcarriers in order to guarantee the lifetime of the final product while promoting tissue reconstruction after implantation.
Our microcarriers have been designed in two stages. The first step represents the manufacture of the raw microbeads using an emulsion/evaporation process. The size of the microcarriers has been easily adjusted by playing on the viscosity of the oil phase of the emulsion.
To promote cell adhesion and proliferation, microbeads have been functionalized by physical deposition of polyelectrolytes. The efficacy and the stability of different coatings have been demonstrated using fluorescent polyelectrolytes (Figure 2). In order to adjust the thickness, stability and surface density of this coating, different polymers have been assessed differing in terms of macromolecular characteristics, i.e. molecular weight, charge density and chain mobility. Indeed, all these surface features are well known to influence the cellular behavior, including their mobility, gene expression or their cytoskeleton reorganization. After sterilization, biocompatibility of these microbeads has been analyzed in vitro using cytotoxicity tests. The adhesion efficiency and viability of cells on the microcarrier surfaces have been quantified adopting the MTT assay. Cells have been also visualized using the Hoechst dye which allows to label the nucleus of the cells (Figure 3).
Cell detachment from these microcarriers has been also investigated as an alternative strategy.