Necrotic regions are absent in fiber-shaped cell aggregates, approximately 100 μm in diameter

Abstract

Microscopic, fiber-shaped cell aggregates, have been used as building blocks for fabricating macroscopic three-dimensional tissue architectures, in the field of tissue engineering. In this study, we examined the occurrence of necrotic regions in the most widely used, fiber-shaped cell aggregates, approximately 100 μm in diameter. Alginate hydrogel hollow microfibers were used as templates for the cell aggregates. We demonstrated negligible necrotic region formation occurred in the cell aggregates formed in the hollow microfibers. Furthermore, we improved on previously-reported methods for preparing the hollow microfibers to avoid common microfiber tangling during the fiber preparation process.

Keywords::

• hydrogel
• microfiber
• necrosis
• regenerative medicine
• tissue engineering

Introduction

Microscopic cell aggregates, formed during in vitro cell culture, have been used for tissue engineering, regenerative medicine and drug screening. Cells in the cell aggregates appear to mimic the morphology and physiology of those in native tissues, compared to conventional monolayer cell culture systems (Sutherland 1988, Koide et al. 1989). Cell aggregates with varying shapes, including spherical (spheroid), fiber-shaped and ring-shaped, have been previously generated (Lin and Chang 2008, Masuda et al. 2012, Onoe et al. 2013). It is important during preparation of cell aggregates to avoid the generation of central necrotic regions, which results in decreased functions of the constructs (Ijima et al. 1998). Necrotic regions result from an insufficient supply of oxygen and nutrients to the cells. During in vitro cell culture, delivery of oxygen and nutrients to cells in cell aggregates, is based solely on diffusion from the surrounding environment (cell culture media). Therefore, to avoid generation of necrotic cells, reduction and control of the cell aggregate size is essential. In the case of spheroids, the maximum diameter, to avoid formation of necrotic cells, is reported to be less than 150–200 μm (Lin and Chang 2008).

Recently, fiber-shaped cell aggregates have been utilized as building blocks for fabricating macroscopic three-dimensional (3D) tissue architecture, in the field of tissue engineering (Matsunaga et al. 2011). The cell aggregates were prepared using flexible alginate hydrogel hollow microfibers (Sugiura et al. 2008, Sakai et al. 2009, 2013, Raof et al. 2011). In the previous reports, cells were first encapsulated in the hollow core of the fibers and proliferated to form fiber-shaped cell aggregates. It appears obvious, that the maximum diameter for the cell aggregates to avoid formation of necrotic regions, is smaller than that (150–200 μm as described above) of spheroids, because their surface area per unit volume is smaller. Therefore, fiber-shaped cell aggregates, approximately 100 μm in diameter, have been most widely prepared in previous reports (Sugiura et al. 2008, Raof et al. 2011, Onoe et al. 2013, Sakai et al. 2013). However, to our knowledge, no reports demonstrated that there were no necrotic regions in the cell aggregates.

In this study, our primary aim was to investigate occurrence of necrotic regions in fiber-shaped cell aggregates, approximately 100 μm in diameter. Our findings will impact on the advance of research when applying cell aggregates for tissue engineering, regenerative medicine and drug screening. In addition to our primary aim, we wanted to improve on previous methods for preparing alginate hydrogel hollow microfibers, to avoid common microfiber tangling during the fiber preparation process, which would hinder application of the microfibers for tissue engineering and regenerative medicine (see Results and discussion for details).

Materials and methods

Materials and cells

Sodium alginate (Na-Alg), with a molecular weight of 70,000 (61% guluronic acid residues, Kimica I-1G), was kindly donated by Kimica Co. (Tokyo, Japan). Poly(vinyl alcohol) (PVA, degree of saponification: ≥ 96%, degree of polymerization: 400–600) was purchased from Wako Pure Chemical Industries (Osaka, Japan). A human hepatoblastoma cell line (HepG2, RCB1648) was obtained from Riken Cell Bank (Tsukuba, Japan) and was maintained in Dulbecco's Modified Eagle's Medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin.

Preparation of microfibers

A coaxial double cylinder was constructed from two stainless steel needles cut at 90° to their axial direction (Figure 1). Tip inner (id) and outer (od) diameters of the inner cylinder were 470 and 730 μm, respectively. Three outer cylinders with different id at their tips (240, 610 and 910 μm) were employed. The double cylinder was set at the bottom of a bath containing CaCl₂ solution. Aqueous solutions of 10% (w/v) PVA (core solution) and 1% (w/v) Na-Alg solutions (sheath solution) were extruded simultaneously into CaCl₂ solution from the inner and outer cylinders, respectively, using syringe pumps. The sheath solution gelled through uptake of Ca²⁺ in the CaCl₂ solution (the core solution did not form a hydrogel with Ca²⁺). The resultant alginate hydrogel hollow microfibers were collected by a roller. The microfibers were then incubated in a large volume of 100 mM CaCl₂ solution for 24 h to induce further gelation. Mean microfiber id and od were determined from measurements of approximately 100 randomly selected samples, using an optical microscope (DM IL LED, Leica, Tokyo, Japan). PVA was used to increase viscosity of the core solution, which was necessary to enable homogeneous distribution of cells in the solution during fiber preparation process.

Figure 1. Schematic depiction of calcium alginate hydrogel hollow microfiber formation.

HepG2 cell-enclosed microfibers were prepared using 10% (w/v) PVA in calcium-free Krebs-Ringer HEPES-buffered solution (CF-KRH, pH 7.4) suspending the cells (1.0 × 10⁶ cells/ml), 1% (w/v) sodium alginate in CF-KRH and 100 mM CaCl₂/10 mM HEPES solution (pH 7.4) under otherwise identical conditions. The microfibers were incubated in DMEM containing 10% (v/v) FBS at 37°C and 5% CO₂ to proliferate the cells in the hollow core. The microfibers containing cell aggregates were embedded in paraffin, sliced and stained with hematoxylin and eosin. Thickness of living cell layers in the cell aggregates was evaluated by measuring the thickness in more than 30 randomly selected parts in cross-sections of the cell aggregates. The ratio of necrotic area in cell aggregates ( = necrotic area in cross-sections of cell aggregates × 100/cross-sectional area of the cell aggregates) was also calculated.

Statistical analysis

Data are presented as mean ± standard deviation. Statistical differences between two groups were analyzed using two-tailed Student's unpaired t-test.

Results and discussion

Some researchers fabricated flexible, alginate hydrogel hollow microfibers, and applied them to preparing fiber-shaped cell aggregates (Sugiura et al. 2008, Raof et al. 2011, Onoe et al. 2013, Sakai et al. 2013). We also previously developed methods to produce cell-enclosed alginate hydrogel microfibers in both solid and hollow structures, using coaxial double and triple cylinders, respectively (Takei et al. 2006, 2007a, 2007b, Sakai et al. 2008, Takei et al. 2010). A common issue of our and other researcher's methods for fabricating the cell-enclosed microfibers is unavoidable tangle of the fibers immersed in aqueous solutions due to their flexibility and length (> 1 m). Tangle leads to failure of high-order assembly (folding, bundling and weaving) of the cell aggregates formed in the microfibers, required for creating 3D tissue architectures (Onoe et al. 2013). In this study therefore, we first modified on the previous methods to prevent tangle. In our present system, microfibers formed in a bath containing CaCl₂ solution were pulled and continuously reeled by a roller (Figure 1). This procedure was able to prevent tangle during the fiber preparation process.

The primary purpose of this study was to examine whether necrotic regions occurred in fiber-shaped cell aggregates, approximately 100 μm in diameter. To prepare these structures, precise control of the alginate hydrogel hollow microfiber size is essential. Here, we investigated the effect of preparation parameters on microfiber size, because there were no reports in which the size was controlled using an identical system to our present system. We first determined microfiber size by varying the tip id of the outer cylinders and flow rates of the core and sheath solutions at the tip (structure of the double cylinder ensured that the flow rates of the two solutions were identical at the tip). This was achieved by ensuring that core volume ratio ( = flow volume of core solution/total flow volume of core and sheath solutions) and pulling rate of the microfibers ( = rotation speed of the roller) were fixed at 0.1 and 28.8 cm/s, respectively. At a tip id of 240 μm and flow rate of 11.8 cm/s, the minimum microfiber od was 107 ± 10 μm (Figure 2a). The microfiber od increased with increasing flow rate. Tip id also influenced microfiber od. The od increased with increased tip id, and the maximum value was 565 ± 43 μm, with a tip id of 910 μm and flow rate of 21.8 cm/s.

Figure 2. Effects of outer cylinder tip id [910 μm (circles), 610 μm (triangles) and 240 μm (squares)] and flow rates of core and sheath solutions at the tip on od (a) and i/o ratio (b) of the microfibers.

Figure 2b shows i/o ratio ( = mean id/mean od of microfibers) for each condition shown in Figure 2a. Theoretically, the i/o ratio depends only on the core volume ratio. Actually, the i/o ratio hardly changed at fixed core volume ratio of 0.1, showing precision of our system. We subsequently examined the effect of core volume ratio on microfiber id and od in more detail under the condition that flow rates of core and sheath solutions and pulling rate of microfibers were fixed at 17.9 and 28.8 cm/s, respectively. Core volume ratio hardly influenced microfiber od when the outer cylinder tip id was fixed. Conversely, microfiber id varied by changing core volume ratio (Figure 3) and an id of approximately 100 μm, adequate to prepare fiber-shaped cell aggregates of approximately 100 μm in diameter, was achieved.

Figure 3. Effect of core volume ratio on microfiber od (closed symbols) and id (open symbols). Two outer cylinders with different tip id (910 μm: circles, 240 μm: triangles) were used.

Finally, we attempted to prepare fiber-shaped cell aggregates, approximately 100 μm in diameter, using thin microfibers, 96 ± 11 μm in id prepared at a core volume ratio of 0.10 and outer cylinder tip id of 240 μm (see Figure 3). For the control group, cells were encapsulated in thick microfibers, 351 ± 25 μm in id prepared at a core volume ratio of 0.20 and outer cylinder tip id of 910 μm. HepG2 cells were used as a model cell. As shown in Figure 4, HepG2 cells encapsulated in thin microfibers, proliferated and eventually formed fiber-shaped cell aggregates with identical diameter to microfiber id. Cells in the thick microfibers also formed cell aggregates. Hematoxylin and eosin-stained sections of the cell aggregates formed in thick microfibers showed existence of necrotic regions in the center of the aggregates (Figure 5a). The ratio of necrotic area and thickness of living cell layers in the thick cell aggregates were 28 ± 9% and 73 ± 22 μm, respectively. On the other hand, no necrotic regions were observed in the cell aggregates formed in the thin microfibers (Figure 5b, ratio of necrotic area = 0%, p < 0.001 vs. thick cell aggregates). These results show that the most widely used fiber-shaped cell aggregates, approximately 100 μm in diameter, are useful tools for tissue engineering, regenerative medicine and drug screening, because of the absence of necrotic regions.

Figure 4. HepG2 cell-enclosed thick (a, c) and thin (b, d) microfibers at 0 (a, b) and 7 (c, d) day(s) of culture. Arrows show encapsulated cells. Bars represent 500 μm.

Figure 5. Hematoxylin and eosin-stained sections of the cell aggregates formed in thick (a) and thin (b) microfibers. The area surrounded by dotted-line in (a) is the necrotic region. Bars represent 200 μm.

Conclusions

In this study, we improved on previous methods for preparing alginate hydrogel hollow microfibers to prevent tangle of the microfibers. Furthermore, we demonstrated that necrotic regions are absent in the most widely used, fiber-shaped cell aggregates, approximately 100 μm in diameter.

Declaration of interest

The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.

This work was supported by a Grant-in-Aid for Young Scientists (B), 25820383, 2013, from Japan Society for the Promotion of Science (JSPS).