Continuous microfluidic encapsulation of single mesenchymal stem cells using alginate microgels as injectable fillers for bone regeneration

Abstract

The encapsulation of cells in microscale hydrogels can provide a mimic of a three-dimensional (3D) microenvironment to support cell viability and functions and to protect cells from the environmental stress, which have been widely used in tissue regeneration and cell therapies. Here, a microfluidics-based approach is developed for continuous encapsulation of mesenchymal stem cells (MSCs) at the single-cell level using alginate microgels. This microfluidic technique integrated on-chip encapsulation, gelation, and de-emulsification into a one-step fabrication process, which enables scalable cell encapsulation while retaining the viability and functionality of loaded cells. Remarkably, we observed MSCs encapsulated in Ca-alginate microgels at the single-cell level showed significantly enhanced osteogenesis and accelerated mineralization of the microgels which occurred only after 7 days of induction. Furthermore, MSCs laden in alginate microgels displayed significantly enhanced bone formation compared to MSCs mixed with microgels and acellular microgels in a rat tibial ablation model. To conclude, the current microfluidic technique represents a significant step toward continuous single cell encapsulation, fabrication, and purification. These microgels can boost bone regeneration by providing a controlled osteogenic microenvironment for encapsulated MSCs and facilitate stem cell therapy in the treatment of bone defects in a minimally invasive delivery way.

Statement of Significance

The biological functions and therapeutic activities of single cells laden in microgels for tissue engineering remains less investigated. Here, we reported a microfluidic-based method for continuous encapsulation of single MSCs with high viability and functionality by integrating on-chip encapsulation, gelation, and de-emulsification into a one-step fabrication process. More importantly, MSCs encapsulated in alginate microgels at the single-cell level showed significantly enhanced osteogenesis, remarkably accelerated mineralization in vitro and bone formation capacity in vivo. Therefore, this single-cell encapsulation technique can facilitate stem cell therapy for bone regeneration and be potentially used in a variety of tissue engineering applications.

Introduction

The gold standard for bone regeneration in the clinic still relies on the transplantation of autologous bone, which intrinsically contains all elements for successful bone regeneration, i.e. extracellular matrix (ECM), cells, and growth factors from bone tissue [1]. The strategy of tissue engineering and regenerative medicine basically aims to replicate the structural and compositional features of the natural bone tissues by combining biomaterials with multipotent stem cells and relevant signaling biomolecules [2]. Specifically, biomaterials play a pivotal role in the success of tissue engineering, which not only function as scaffolds to provide mechanical and structural support, but also to manipulate cell fate including cell attachment, proliferation, differentiation, and eventually tissue-specific functionality [3], [4], [5]. However, conventional monolithic scaffolds to load stem cells are far from the successful application of bone reconstruction in the clinic. This can be attributed to i) poor efficiency in nutrient/waste exchange in large scaffolds which typically lead to the necrosis of engineered tissues [6,7], ii) failure in replicating the biochemical and biophysical signals of natural ECM [8], and iii) requiring surgical incisions for conventional monolithic scaffolds to treat irregular-shaped defects [9]. Therefore, the concept of modular tissue engineering, in which microscale tissue-engineered modules as building blocks to bottom-up assemble biomimetic tissues, have attracted increasing attention due to its potential to address challenges in the conventional tissue engineering strategy [10].

Microgels are miniaturized hydrogels with a typical size of 10-1000 µm in diameter, which are composed of a hydrophilic polymer network dispersed in water [11]. They are of great interest for biomedical applications due to their biocompatibility, biodegradability, and bioactivity resembling the natural ECM [12,13]. The microscopic size renders microgels enhanced sensitivity to surrounding environmental stimuli, higher diffusion rate and distance, availability for mass transportation, and injectability via a minimally invasive surgery [14]. Microgels loaded with living cells are ideal candidates as building blocks to be assembled into large-size biomimetic tissues, or as delivery vesicles towards controlled cell delivery [15], [16], [17]. To this end, microfabrication techniques for high-throughput cell encapsulation in microgels without compromising cell viability and biological functions are imperatively needed.

Several microfabrication techniques have been developed to produce cell-laden microgels, including batch emulsion [18,19], microfluidic emulsion [12,20], electrohydrodynamic spraying [21,22] and lithography [23], [24], [25]. Among them, a droplet-based microfluidic technique has been recently proposed, which allows precise control over immiscible multiphase flows, and provides continuous and rapid production of monodisperse microgels with controllable size and morphology [11,26]. However, major hurdles that restrict this technique for cell encapsulation in microgels are the hazardous effects from the production process on cell viability such as shearing force during extrusion, long term exposure to the cytotoxic oil phase, surfactants and crosslinking reagents [27,28]. Especially, previous microfluidic droplet-based approaches to produce cell-laden microgels normally involve procedures including droplet formation, gelation on chip, and de-emulsification to transfer microgels from oil into aqueous phase by cyclic steps of centrifugation/re-dispersion [29]. These time- and labor-consuming steps invariably result in poor cell viability and low retrieval efficiency [30,31], which weakens their potential of high-throughput cell encapsulation for widespread biomedical applications.

The biocompatibility of microgels plays a pivotal role in expanding their applications by facilitating biological functions of encapsulated cells, either as deliver vesicles for regenerative medicine [14] or as a platform to study single-cell behavior [32]. Specifically, microgel should provide a physiological-relevant niche to accommodate cells and regulate their functions by replicating the biochemical signals from ECM (e.g. cytokines and growth factors) and biophysical signals (e.g. matrix topography and stiffness) [33,34]. Alginate is a naturally-derived anionic polymer containing D-mannuronate (M) and L-guluronate (G) residues, which can be crosslinked by divalent cations such as Ca²⁺ ions to form hydrogel [35]. Alginate has been proved numerous applications in the biomedical field due to its favorable properties including biocompatibility, facile gelation process, and ease of chemical functionalization [36,37]. Particularly, alginate has been widely used for microencapsulation of cells or bioactive components for controlled delivery or regenerative medicine [38]. These studies typically employed alginate hydrogel beads with the size ranging from hundreds of microns to several millimeters to support cell proliferation and functionality [39,40]. Previous studies have shown alginate beads containing multiple MSCs capable of inducing osteogenesis and even leading to ectopic bone formation in a rat subcutaneous model [41]. In addition, the bursa omentalis implantation of alginate beads encapsulating allogeneic islets has been applied for the treatment of type I diabetes, which can provide long-term protection to the implanted cells from rapid clearance by the host immune system [42]. Despite these favorable properties, these works relied on the encapsulation of multiple cells within macroscopic hydrogels, which may not assure quantifiable access of cells to nutrients and oxygen but also compromise the injectability due to the large size [12,43].

Microgels with a smaller size below a hundred microns to encapsulate single cells might boost the development of cell encapsulation for widespread biomedical applications due to the unprecedented advantages including the feasibility of direct intravenous delivery, enhanced mass exchange and ease of single cell monitor and analysis [32]. Previous studies demonstrated single MSCs encapsulated in alginate microgels can proliferate [30] and differentiated upon paracrine signals from neighboring cells [44]. Moreover, intravenous injection of singly encapsulated MSCs in alginate microgels effectively protected allogeneic cells from being rapidly cleared by the host immune system and significantly prolonged the viability and therapeutic effects of the transplanted cells [45]. Although these researches have facilitated applications of single cell-laden microgels, there has been little work to investigate the influence of microgel encapsulation at the single-cell level on the biological functions of encapsulated cells, and the therapeutic activities of single cell-laden microgels for tissue regeneration remains less explored.

In this work, we presented a microfluidics-based method for continuous encapsulation of single mesenchymal stem cells in monodisperse alginate microgels. We developed a microfluidic device that integrated drop preparation, on-chip gelation, and de-emulsification to produce stem cell-laden microgels in a biocompatible and high-throughput manner. The biocompatibility of developed alginate microgels, the osteogenic differentiation and mineralization of encapsulated MSCs in alginate microgels at the single-cell level were assessed. The feasibility and therapeutic effect of using those single stem cell-laden microgels as injectable fillers for bone regeneration were also evaluated using a rat tibial marrow ablation model.