Microengineering double layer hydrogel structures towards the recapitulation of the hematopoietic stem cell niche

Adult stem cells, especially from bone marrow, have a tremendous therapeutic potential, and its application in regenerative medicine has been increasingly studied. It is well known that three-dimensional (3D) cell culture matrix promotes many biological relevant functions not observed in two-dimensional (2D) monolayer cell culture and therefore, engineering 3D cell culture platforms to mimic the native in vivo biological systems represents a realistic approach for studies of bone marrow stem cells. High-throughput microfluidics constitutes a promising technology to generate hydrogel three dimensional constructs, however, its application to host adult stem cells remains still a challenge.

Adult stem cells, especially from bone marrow, have a tremendous therapeutic potential, and its application in regenerative medicine has been increasingly studied [1,2]. It is well known that three-dimensional (3D) cell culture matrix promotes many biological relevant functions not observed in two-dimensional (2D) monolayer cell culture [3] and therefore, engineering 3D cell culture platforms to mimic the native in vivo biological systems represents a realistic approach for studies of bone marrow stem cells. High-throughput microfluidics constitutes a promising technology to generate hydrogel three dimensional constructs [4,5], however, its application to host adult stem cells remains still a challenge.
Approaches for 3D-organized cell culture have included scaffoldfree systems, which primarily consist of the spontaneous formation of cellular aggregates [6]. Stem cell maintenance in suspension or in spinner flask, a relatively simple method but typically leading to large heterogeneity in spheroid size [7], and hanging-drop techniques that provide some control over the spheroid size while lacking of aggregated generation control [8,9]. Dielectrophoresis has also been used to provide external guiding to introduce cells into multicellular aggregates forming some niche-like construction [10]. However, electrophoretic guiding does not provide a permanent template, thus neither cell stratification nor cell 3D-positional control are possible inside the aggregate. Other attempts for 3D cell culture have used scaffolds which should provide sufficient control on the spatial distribution of the different cellular contents in the single culture of co-culture spheroids [11]. Printing techniques for tissue microfabrication in flat supports [10] have also been attempted, however no standard methods for 3D scaffolding with adult stem cells have been reported.
Specifically, droplet-based microfluidics owns the unique ability to encapsulate cells into templated matrices in a controlled manner; however current application in 3D cell culture is mostly limited to co-culture in droplets [12,13]. Some studies focused on double encapsulation system for cell studies using laminar coflows [14]. Whereas this technology allows the fabrication of biopolymer capsules, it is not suitable for the generation of double layered alginate-alginate beads, presents limitations over the change of layer size and requires a complicated set-up. Other studies using coaxial capillaries reported the successful generation of multilayered hydrogel beads [15]. However, the system requires a complex set up and preparation, it is not reusable or highthroughput and it presents limitations on cell allocation area, as they can only be allocated in an outer layer.
Our route to achieve a 3D multi-cellular culture vehicle with double layered structure is the presented device where controlled generation of the structure geometry, composition and cell distribution can be achieved. In the presented droplet-based microdevice, hydrogel droplets are produced by hydrodynamic focusing techniques and gelled by the circulation of the droplet between two laminar flows, one containing the cross-linking agent. Droplet coating is achieved by synchronizing droplet production rates so that the gelled bead merges with the second layer droplet nearby its generation area, forming a double layered bead.
The presented device is designed for the use of alginate as the inner core scaffold and both Puramatrix (Becton Dickinson, USA) or alginate as the outer core scaffold. When using alginate as the outer core scaffold, gelation is achieved by internal gelation. Experiments using Puramatrix as the outer layer matrix used Dulbecco's Modified Eagle's medium (DMEM) powder as cross-linking agent at collection. This technology also permits rapid extraction of the cell constructs for immediate washing and culture, limiting the  This droplet-based niche mimicry strategy represents a promising technique able to generate 3D hydrogel scaffolds for the generation of multicellular stem cell constructs generation towards the recapitulation of the hematopoietic stem cell niche. Further studies would include long term stem cell culture and clinical applications opening a wide field of potential uses within uTAS for tissue engineering.
The experimental conditions used in our approach for a double hydrogel bead generation are described in Supporting Information (online). The presented device operational process is depicted in Fig. 1a. First, alginate droplets containing calcium carbonate nanopowder are generated by hydrodynamic focusing using gravity to drive the flow pressure. For cell assays, cells are re-suspended in 200 lL phosphate buffered saline (PBS) and premixed with the corresponding hydrogel previous to injection. The generated droplets travel through a meandering channel containing two laminar oil flows. Due to acetic acid being added to the second laminar oil flow continuous phase, calcium ions are released after droplet formation by the diffusion of the acetic acid across the oil interface. By separating drop formation and crosslinking in this system, we eliminate clogging issues and generate a stream of alginate microgel structures as the inner core. The platform is designed so that the second oil flow containing the cross-linking agent has a double function for both cross-linking and adjusting the droplet production rates/size of the generated droplet without affecting the gelation process.
The generated inner core droplets are then passed by a second inlet. The second inlet is a stream of second sample layer alginate droplets which are generated in a synchronous way. Hence, when the inner core droplet reaches the area of the second droplet production, the two droplets (one gelated and another ungelated) passively mix along the meander, generating a double layered hydrogel bead (Fig. S1 online).
The passive nature of the mixing principle underlying this construction process enhances the suitability of these resulting structures for accommodating adult hematopoietic stem cells. We demonstrated that both the gelation process and the double layer bead generation technique are promising for the encapsulation of living cells by using human Mesenchymal stem cells (hMSCs) and human Hematopoietic adult stem cells (hHSCs). This mild double layered bead generation technique allowed us to successfully encapsulate these cells using alginate as the inner core and both alginate and Puramatrix as an outer matrix.
The experimental procedure was performed as previously described. Premixed aqueous samples containing alginate (1.5 wt%) and Calcium carbonate nanopowder (1.0 wt%) were prepared as sample 1 and 2. Traces of Cresol red were added to the sample 1 in order to assess the gelation process. The second oil was prepared by adding glacial acetic acid to a pre-dyed Sudan red mineral oil (0.5%; v:v).
Experiments run by fine tuning sample 1, sample 2, oil 1 and oil 2 flow rates by adjusting their corresponding barrel heights, so that sample 1 droplets will be regularly produced and cross-linked along the meander (cresol red color changes from red to yellow) while sample 2 flows along its inlet but does not flow into the main channel. Then, the second sample flow has been adjusted to achieve a synchronized droplet production rate and therefore mixing along the meander. Most suitable flow rates for the given samples were adjusted to achieve the maximum rate of droplet mixing. Once these conditions were registered (initial height conditions h oil the cross-linking oil flow rate to assess its influence on the mixing efficiency and droplet size. Versatility of the designed platform was demonstrated by using different hydrogel types as matrix. Sample 1 was fixed to 1.5 wt% alginate whereas sample 2 was varied using alginate (1.5 wt%, 2.0 wt%, and 3.0 wt%) and Puramatrix (0.1%, 0.3% and 0.5% (v:v)).
For all sets of experiments, the inner hydrogel core was clearly embedded in the outer hydrogel droplet and the shape of the gelated droplet did not vary after the double layer generation. Droplet production rates of both sample 1 and sample 2 were measured as well as the percentage of successful droplet mixing as the percentage of inner core droplet produced that are coated by a second layer. Fig. 1d-i shows the results of these characterization studies.
For most of the tested materials, flow rate conditions could be set to achieve a double layer generation efficiency of 90%-100%. For experiments using Puramatrix as a sample of the outer layer, it was observed that maximum double layer bead generation efficiency was achieved when oil 1 and oil 2 were flowing equally distributed along the main meander. Increasing or decreasing oil 2 flow rate would result in a slight loss of synchronization and therefore mixing efficiency while still being able to generate 65%-100% double layered beads (for 0.3% and 0.5% Puramatrix, v:v).
The presented microfluidic platform also allows the variation of the dimensions of both the second layer and inner core on demand by just varying the second oil flow rates or the microfluidic device geometry. Some preliminary experiments using patent blue dye in sample 1 instead of cresol red in order to enhance the double layer imaging were performed. Two sets of experiments were run, varying the height of the oil 2 (Fig. 2a) and using three different geometries with different channel dimensions on the inner core generation area (Fig. 2b, Supporting Information). The initial conditions that using patent blue dye for imaging the inner core were set as h oil 1 = 61 cm, h oil 2 = 66 cm, h sample 1 = 34 cm, h sample 2 = 22 cm.
These experiments represented a proof of concept for the possibility of a quick and reliable size variation, leading to a wide range of possibilities of constructing on demand sized double layered beads.
From the operational point of view, it was concluded that while maintaining both oil flow rates equilibrated to co-flow equally along the main meander, heights of sample 1 and 2 can be finetuned to achieve a situation close to full synchronization leading to successful double layer hydrogel bead generation. However, it is important to note that if full synchronization of the flow rates is desired, second layer thickness cannot be varied.
The suitability of the presented method for double-layer 3D hydrogel generation for cell encapsulation was demonstrated by accommodating living hMSCs into both the inner and outer layer, and hHSCs into the outer layer. We choose these cell types as a recapitulation model for the hematopoietic stem cell niche.
Prior to re-suspension in PBS and mixture in premixed alginate emulsification, hMSCs were routinely cultured and trypsinized. We choose the cell density between 1.2 Â 10 7 and 1.7 Â 10 7 cells/mL when using hMSCs and 8.0 Â 10 6 cells/mL when using hHSCs. These concentrations represented a suitable amount of cells for control of visualization. Both double layered (Fig. 2c-h) and single (Fig. 2i) structures were generated by the described method.
The The experimental procedure compiled a collection batch double layered hydrogel beads generated using hMSCs in alginate for the Operational conditions of the device for double layered bead production containing: alginate inner core (1.5 wt%) and alginate outer core (1 wt%, 5 wt%, 2 wt%, 2.5 wt%). (g-i) Characterization for 1.5 wt% alginate inner core and 0.1%, 0.3% and 0.5% (v:v) Puramatrix outer core. Mixing efficiency is measured in percentage of mixed droplets (left y-axis). Sample 1 and sample 2 droplet production rates are also represented in droplet production rate (droplet/min) (right y-axis).
inner core with both empty alginate as the outer layer ( Fig. 2d and  e), or hMSCs in alginate as the outer layer (Fig. 2f). Additionally, a collection of hydrodynamically generated single layer beads containing hMSCs (Fig. 2i) was also performed.
Once post-encapsulation viability of hMSC was demonstrated, Hematopoietic adult stem cells were tested in order to assess the suitability of the presented technology as a method to model the hematopoietic stem cell niche. Hence, hMSCs were accommodated on the inner core (alginate 1.5 wt%) of the generated structure and hHSCs on the outer core (Puramatrix 0.3%, v:v).
In order to visualize the two types of cells, HLA-DR V450 was used as a marker for the hHSCs prior to the calcein staining ( Fig. 2g and h). As hHSC cell density was low for the given matrix volume, two different focus plane captions of the same structure were taken to visualize both types of cells. Both hMSCs and hHSCs were successfully encapsulated using this method.
Longer crosslinking times, i.e., extended exposure of the encapsulated cells to the acidic environment on the meander or at collection, or higher concentrations of acetic acid on the second oil would result in a lower cell viability (data not shown). However, the presented technique is able to minimize the acid exposure together with the low mechanical stresses that cells experiment to be embedded in this double layered construct, and shows high cell viability after encapsulation.
Furthermore, the cells were clearly embedded in the inner core and outer layer formed by the alginate and Puramatrix hydrogels. The shape of the spherical micro droplets remained unchanged after gelation and no leakage of cells after encapsulation was observed. Considering that cell viability could be increased by adjustment of the hydrogel's mechanical properties, this method could represent a technology not only for living cell encapsulation but also for long term culturing of cells in highly controlled microgel matrices in the near future.
In conclusion, we present a novel technology for the generation of double layered hydrogel beads with controlled size and composition suitable for adult stem cell encapsulation. The use of a double laminar oil flow where only one contains the cross-linking agent allows both the uniform gelation of the inner core and the continuous generation of a stream of cross-linked hydrogel beads without clogging. The second layer generation by passive mixing ensures the soft coating of the inner core without exposing the encapsulated cells to external forces that might reduce their viability. Furthermore, we demonstrate the suitability of the presented technology for encapsulation of stem cells by using hMSCs and hHSCs. We present a promising approach for modeling a hematopoietic stem cell niche comprising mesenchymal stem cells in the inner core and hematopoietic stem cells in the outer layer and evaluating their viability after encapsulation. This method for the artificial reconstruction of these niche components represents a promising approach to simulate in vivo situation of HSCs with the goal to study stem cell behavior in vitro under controlled conditions. This tailored coating of an inner hydrogel core could be extended to a third coating layer to reconstruct more complex cell arrangements and mimic better the complexity of the stem cell niche. Therefore, the presented device represents a valuable and mild technology to generate 3D multicellular stem cell structures towards gaining insights in understanding the regulation factors influencing the stemness maintenance of HSCs and more generally, in understanding stem cell behavior in 3D environments.