Cell selective chitosan microparticles as injectable cell carriers for tissue regeneration
Introduction
The success of many cell therapies is highly dependent on the development of techniques for isolation and selection of cells that guarantee high yield and purity. One of the limitations in stem cell isolation is the restricted quantity that can be isolated from a tissue and the typical heterogeneity of the cell population. Obtain a pure cell population is particularly challenging when the target cells are rare in the total amount of cells in the sample. Methods for cell separation based on a difference in physiochemical properties, such as density and size have been reported, but the purity of the obtained population is generally low [1], [2]. Thus the need for alternative methods to select subsets of cells with increased yield decreasing cell manipulation and expansion time is a reality. Moreover, the development of systems suitable for an efficient and effective cell isolation/separation that can act as a cell expansion platforms may result in a separation method that are much more specific for the population of interest [3]. An efficient system for adhesion-based cell separation lies in the specificity of an immobilized biomolecule to the target cell population. Fluorescence-activated cell sorting (FACS) effectively provides high efficiency in cell sorting, but this technique has associated high cell manipulation and requires a demanding operational training [4], [5]. Magnetic activated cell sorting is another separation technique based in surface markers, where cells bind to antibody labeled magnetic particles [6], [7]. Antibody-coated microchips have also been used to successfully detect and isolate rare circulating tumor cells from peripheral blood [8], [9]. All of these techniques have been in use for years and have shown some degree of success. Nevertheless, none of the described techniques provide support for cell expansion or proliferation. In an effort to overcome some of these limitations Nguyen et al. have recently reported the use of multilayered magnetic microparticles as a novel strategy to isolate, expand and detach endothelial progenitor cells [10]. Still, these particles are not suitable for the implementation of an injectable system to form a biodegradable construct in situ. Thus efforts are necessary to develop implantable supports for specific cell populations [11]. To overcome these issues while decreasing cell manipulation and time consumption, we report a new system for cell separation and expansion that may ultimately be injected to form a scaffold in situ for tissue regeneration purposes.
Chitosan is well known as a biodegradable and biocompatible material [12], [13], [14]. In addition, the amino and hydroxyl chemical groups along chitosan chains enable this polysaccharide to form stable covalent bonds with many molecules of interest [14], [15], [16]. In our previous work, we demonstrated that chitosan films grafted with antibodies were able to promote selective cell attachment and growth [15]. Additionally to cell isolation and expansion, we envisage the development of a system for directly deliver cells in vivo, decreasing cell manipulation. The goal of this work was to explore the use of functional chitosan microparticles, as a strategy for cell separation and expansion that may be used as an injectable system to form tissue constructs at the lesion site for tissue regeneration purposes.
Chitosan microparticles were firstly functionalized with biotin. Such modification allows engineering the surface of microparticles with a variety of biotinylated biomolecules via streptavidin (SaV), increasing its versatility and yield due to the multiple binding sites for biotinylated molecules. We tested the immobilization of biotinylated antibodies to target endothelial cells and human adipose stem cells (ASCs). Biotinylated antibody anti-CD31 was used to target human umbilical vein endothelial cells (HUVECs) cells while biotinylated antibody anti-CD90 was used to capture ASCs.
Section snippets
Preparation of chitosan microparticles
Medical grade chitosan (150–300 kDa and a deacetylation degree of 95%) (Heppe Medical Chitosan GmbH, Germany) was dissolved in a 2% v/v aqueous acetic acid (VWR, UK) solution to a final concentration of 2% w/v. Subsequently, the chitosan solution was passed through an aerodynamically-assisted jetting equipment (Nisco Encapsulation Units VAR J30, Switzerland) at a speed of 1 ml/min. The injected air led the chitosan solution to break up into a spray at the outlet of the nozzle. The generated
Fabrication of microparticles
Chitosan-based microparticles were generated by forming small droplets using a coaxial air-flow that were hardened in a NaOH solution. The size and morphology of the chitosan microparticles was determined using optical microscopy. The obtained particles exhibited rounded shape and the size ranged from 80 to 140 μm (Fig. 2A). The average diameter of the microparticles was 115.8 ± 10.61 μm (Fig. 2B) and they exhibited a rough surface (Fig. 2C and D).
Bioconjugated microparticles
The chitosan particles were modified with
Discussion
In this study the goal was to develop polymeric microparticles that along with cell selection/isolation allow cell expansion i.e., that may work as selective microcarriers to expand a target cell type (Fig. 1). Additionally we hypothesize that the developed bioconjugated microparticles may be used as an injectable system for in situ formation of small tissue constructs for regeneration purposes.
Cell microcarriers are typically submillimeter-sized polymeric particles that provide sites for
Conclusions
Here was demonstrated the ability of biofunctionalized particles to select specific cell types from mixed cell populations and to promote cell expansion, by using hASCs and HUVECs as examples. The versatility of this method allows the combination of the biotin-SaV conjugated microparticles with any biotinylated molecule as antibodies, growth factors or peptides of interest. It was shown that biodegradable and biocompatible particles functionalized with antibodies presented selective affinity to
Acknowledgments
This work was supported by European Research Council grant agreement ERC-2012-ADG 20120216-321266 for project ComplexiTE and by the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreement n° REGPOT-CT2012-316331-POLARIS. The authors acknowledge the FCT for the fellowship SFRH/BD/61390/2009 (C.A.C.) for the financial support. We are grateful to Hospital da Prelada for the lipoaspirates donations.
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