Engineering tissues with a perfusable vessel-like network using endothelialized alginate hydrogel fiber and spheroid-enclosing microcapsules

Development of the technique for constructing an internal perfusable vascular network is a challenging issue in fabrication of dense three-dimensional tissues in vitro. Here, we report a method for realizing it. We assembled small tissue (about 200 μm in diameter)-enclosing hydrogel microcapsules and a single hydrogel fiber, both covered with human vascular endothelial cells in a collagen gel. The microcapsules and fiber were made from alginate and gelatin derivatives, and had cell adhesive surfaces. The endothelial cells on the hydrogel constructs sprouted and spontaneously formed a network connecting the hydrogel constructs with each other in the collagen gel. Perfusable vascular network-like structure formation after degrading the alginate-based hydrogel constructs by alginate lyase was confirmed by introducing solution containing tracer particles of about 3 μm in diameter into the lumen templated by the alginate hydrogel fiber. The introduced solution flowed into the spontaneously formed capillary branches and passed around the individual spherical tissues.


Introduction
The purpose of tissue engineering is the creation of three-dimensional (3D) tissue constructs that will benefit human health [1,2]. To date, the tissue constructs of quite thin [3,4,5,6] and/or those composed of the cells with a low oxygen demand [7,8,9,10] have been successfully fabricated. The fabrication of dense tissues faces problems in developing methods for constructing perfusable vascular-like network inside them [11]. Among varieties of reported approaches for solving the problem, a feasible approach is based on the combination of following two approaches: 1) Development of tubular structure (several hundred micrometer to several millimeter in diameter) large enough to flow medium directly from medium reservoirs in hydrogels using appropriate templates [12,13,14,15,16,17], and 2) spontaneous formation of capillary (less than dozens micrometers in diameter) network in hydrogels by the original ability of vascular endothelial cells [13,14,15,17,18,19,20]. It is expected that the network structure can be developed by combining the two approaches like a trunk and branches of a tree. In the preceding reports [14,15,17,18,20], the capillary branches formed randomly despite they were induced for transporting oxygen and nutrient to parenchymal cells. For more effective oxygenation and nutrition of the parenchymal cells, the capillary branches should be induced closer to the parenchymal cells.
The objective of the present study was to develop a method for fabricating a perfusable vascular network-like structure having a tubular construct and capillary branches close to spherical tissues of parenchymal cells. Our methodology for this objective is summarized in Fig. 1. First, we prepare hydrogel fiber and small-tissue enclosing microcapsules,[ 5 _ T D $ D I F F ] both covered with vascular endothelial cells based on previously reported methods [21,22]. The hydrogel constructs [ 6 _ T D $ D I F F ] are prepared from alginate [23] and gelatin derivatives [24].
Then, they [ 7 _ T D $ D I F F ] are assembled in a collagen gel (Step 1). After inducing sprouting and migration of the endothelial cells on the hydrogel constructs into the collagen gel resulting in the formation of capillary network between the hydrogel constructs in medium containing angiogenic factors (Step 2), the hydrogel templates, hydrogel fiber and microcapsules, are degraded using alginate lyase under a mild condition for cells (Step 3). As a result of the processes, we can obtain a perfusable vascular network-like structure having a perfusable tubular construct and capillary branches formed close to spherical tissues of parenchymal cells in the collagen gel.

Article No~e00067
Considering the potential application of the vascularized 3D tissue constructs, a variety of cells are possible candidates of the parenchymal and vascular cells.
In this study, human liver carcinoma cell line HepG2 cells and human umbilical vein endothelial cells (HUVEC) were used as model cells for revealing the feasibility of our approach.

Empty microbead and tissue-enclosed microcapsule preparation
Empty microbeads and cell-enclosing microcapsules were prepared using an axisymmetric, flow-focusing droplet generation device designed in our laboratory [21]. Briefly, empty microbeads were obtained by extruding phosphate-buffered saline (PBS) containing 5% (w/v) gelatin-Ph and 100 units/mL HRP at 4.5 mL/h from a 26-G stainless steel needle into co-flowing immiscible liquid paraffin containing 4% (w/w) lecithin and H 2 O 2 . The emulsion system was centrifuged to collect the microbeads after mixing with PBS. To prepare tissue-enclosed microcapsules, first, cell-enclosing AP-Ph microbeads were prepared. A PBS solution containing 10% (w/v) AP-Ph, 100 units/mL HRP, and 4.0 × 10 7 HepG2 cells/mL into co-flowing liquid paraffin containing lecithin and H 2 O 2 , and then processed as described for preparing empty microbeads. The diameter of the cell- template exposing in the medium containing FBS [25]. The viability of enclosed HepG2 cells right after the encapsulation was determined using trypan blue assay for the cells collected by degrading the microcapsules using alginate lyase. The growth profile of enclosed HepG2 cells was estimated based on the mitochondrial activity per microcapsule measured by a colorimetric method using a cell-counting kit-8 (Dojindo, Kumamoto, Japan) as previously described [21].

Hydrogel fiber preparation
Hydrogel fibers were prepared based on a reported method using a double co-axial cylinder system designed in our laboratory [15,16,22].

Covering with an endothelial cell layer
Empty microbeads, tissue-[ 1 3 _ T D $ D I F F ] enclosing microcapsules or hydrogel fiber were respectively soaked in medium containing 8.0 × 10 5 GFP-HUVECs/mL on non-adherent dishes for 3 h. For allowing homogeneous attachment of the GFP-HUVECs on the surfaces of the hydrogel constructs, the dishes were shaken slightly every 30 min. Then, the unattached cells were removed using a 100 μm cell strainer (BD Falcon, Durham, NC, USA).

Assembly of spherical vehicles and hydrogel fibers
Porcine skin collagen type I (Nitta Gelatin Inc., Osaka, Japan), 5 × DMEM, and neutralization solution were mixed on ice to produce a final collagen concentration of 2.1 mg/mL at pH 7.4. The empty microbeads or tissueenclosed microcapsules with GFP-HUVECs were suspended in the collagen solution and poured onto a gelled thin collagen sheet formed in a well of 5 mm (length) × 5 mm (width) × 0.5 mm (depth). The hydrogel fiber covered with GFP-HUVECs was laid in the center of the well. After incubation for 15 min, the collagen gel was formed while containing the hydrogel fiber and microbeads or microcapsules. Then, additional collagen solution was poured onto the collagen gel to form a thin collagen gel layer on top. Subsequently, the resultant specimen was incubated in medium containing angiogenic factors at 37°C in 5% CO 2 and humidified air. The behaviors of GFP-HUVECs were observed under a fluorescence microscope (BIOREVE BZ-9000, Keyence, Osaka, Japan) and confocal-laser scanning microscope (CLSM; FluoView1000, Olympus, Tokyo, Japan).

Article No~e00067
The nuclei of HUVECs were stained with Hoechst 33342 (Dojindo, Kumamoto, Japan) for cell counting. Surface areas of the microbeads were calculated based on their diameter. The grow profile of HUVECs from the surface of microbeads were demonstrated by comparing the numbers of nuclei per unit of surface area of the microbeads in a same volume of the 3D assembly before and after 1 day of culture.

Immunohistochemical staining and hematoxylin and eosin staining
Immunohistochemical staining was performed on deparaffinized and rehydrated sections of 4-μm-thick. Endogenous peroxidase activity was quenched by incubation for 10 min in 3% H 2 O 2 . Antigen retrieval was performed by incubation for 10 min at 98°C in 10 mM sodium citrate (pH 6.0). The sections were blocked in 5% goat serum (Sigma) for 1 h. Then, the sections were

Fluorescent particle perfusion
The specimen for this experiment was fabricated by assembling the tissueenclosing microcapsules covered with GFP-HUVECs in a collagen gel at 50% (vol.) with a hydrogel fiber covered with GFP-HUVECs in a mold of 5 mm × 5 mm × 0.5 mm (height; Fig. 2). After the endothelial cell network formed among the microcapsules and the hydrogel fibers at 1 day of culture in medium containing angiogenic factors, hydrogel fiber and membrane of microcapsules were removed by using 0.025 mg/mL alginate lyase. Then, the tissue specimen was cultured for an additional day. Fluorescent microparticles of 3 μm in diameter (Polysciences, Warrington, PA, USA) were suspended in PBS and perfused into the cavity of a tubular tissue of endothelial cells formed after degrading the hydrogel fiber crossing the tissue constructs by introducing the solution from the inlet in the polydimethylsiloxane (PDMS) device (Fig. 2). The surface level of the solution was set to be 5 mm higher than the level of the cavity of the tubular tissue. The solution was drawn by gravity and kept flowing through the tubular cavity for several minutes.  (Fig. 3A). Some endothelial cells from the adjacent microbeads were attached with each other at 3 days of culture (Fig. 3B). A neo-formed network of endothelial cells connected the majority of the microbeads at 5 days of culture (Fig. 3C). In contrast, poor cell migration was observed at 5 days of culture in medium without angiogenic factors (Fig. 3D). [13]. An interesting structure of the neo-formed, vessel-like network was revealed by [ 1 7 _ T D $ D I F F ] imagine with a confocal-laser scanning microscope (CLSM; Fig. 4).

Similar phenomena caused by angiogenic factors have been reported in previous studies
An additional endothelial cell layer appeared around the microbeads in the ambient collagen gel. In fact, this cell layer and the cell layer on the surface of the microbeads were the two sides of an integral tubule of endothelial cells (Fig. 4, arrows). The majority of empty microbeads were surrounded by these new tubules. The tubules connected with each other to create a vessel-like network.
[ ( F i g . _ 2 ) T D $ F I G ] In this study, HUVECs were used as a model of endothelial cells for confirming the feasibility of our approach of making a perfusable vessel-like network in engineered tissues. Considering the large heterogeneity of endothelial cells from different tissues and organs [29,30,31], it should be better to use the endothelial cells suitable for individual purposes to make the tissue constructs with specific functions.

Vessel-like network formation among tissue-enclosing microcapsules
Next, we assembled spherical tissue-enclosing microcapsules and a hydrogel fiber,[ 2 0 _ T D $ D I F F ] both obtained from Alg-Ph and gelatin-Ph and covered with GFP-HUVECs, in a collagen gel. To make it easy to observe the vascular-like network, the density of the tissue-enclosing microcapsules was set to be 50% (vol.) of total volume. Before the assembly, the spherical tissues in the microcapsules were obtained by incubating the enclosed HepG2 cells (> 95% viability) for 6 days (Fig. 1B). The measurement based on the cellular mitochondrial activity per microcapsule indicated the enclosed HepG2 cells

Perfusability study
Finally, we investigated the possibility of perfusion of solution in the spontaneously formed network of endothelial cells through the introduction of solution to the tubular cavity obtained by degrading the hydrogel fiber. A solution containing fluorescent microparticles of 3 μm in diameter was poured into the inlet well of the PDMS device for the introduction (Fig. 2). As shown in Fig. 7A  were essential for the formation of the perfusable vessel-like network between the spherical tissues. It may be thought that the degradation of the microcapsules by alginate lyase induced the cavity in which the solution flowed.
However, it was denied by the result for the specimen obtained by assembling gelatin microbeads (non-degradable by alginate lyase) and the alginate-based hydrogel fiber both covered with GFP-HUVECs. The solution containing the fluorescent microparticles traveled around the individual gelatin microbeads when we introduced the solution into the tubular cavity obtained by degrading the hydrogel fiber using alginate lyase (Fig. 8).
In the current method, the hydrogel fiber covered with endothelial cells was used as a template for preparing the endothelialized tubular cavity into which solution was introduced from outer environment. Another reported method to prepare an endothelialized tubular cavity is an extraction of a gold rod covered with endothelial cells from collagen gel after transferring the cells to an ambient collagen gel [12]. An advantage of using the hydrogel fiber is the possibility of preparing curved tubular cavity due to the flexibility of the hydrogel fiber as shown in Fig. 7. It enables to increase the volume occupied by the single tubular cavity in fabricated tissue constructs with a single inlets and outlet. This feature allows fabrication of larger tissue constructs with a lower number of inlets and outlets, which is desirable for vascular anastomosis in transplantation of the fabricated tissue constructs. Considering the practical applications of tissue fabrication in transplantation, the tubular cavity templated by the hydrogel fiber of 200 μm in diameter is very small. The diameter of hydrogel fibers can be easily increased during the preparation process as reported previously [15,16,22]. Because of the feasibility demonstrated in the current study, our next objective will be fabrication of larger tissue constructs by increasing the number of hydrogel fibers and/or the diameter of the fiber to facilitate medium flow throughout the tissues.

Conclusions
In this study, we developed a method to fabricate a perfusable vessel-like network having a tubular construct and capillary branches in a dense tissue construct. We assembled microcapsules enclosing spherical tissues of HepG2 cells and a hydrogel fiber, both of which were covered with endothelial cells in the collagen gel. These endothelial cells spontaneously formed capillary branches, sprouted into the collagen gel and connected as a vessel-like network. Therefore, the individual spherical tissues requiring oxygenation and nutrition could be surrounded with capillary branches. A liquid carrier could be delivered and flowed through the network by introducing the solution into the tubular tissue obtained by degrading the hydrogel fiber template using alginate lyase.
Considering the present results, we anticipate that the proposed method would greatly contribute to development of dense tissues in vitro.

Declarations
Author contribution statement