Letter www.acsami.org
Microfluidic Generation of Porous Microcarriers for ThreeDimensional Cell Culture Jie Wang, Yao Cheng, Yunru Yu, Fanfan Fu, Zhuoyue Chen, Yuanjin Zhao,* and Zhongze Gu* State Key Laboratory of Bioelectronics, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China Laboratory of Environment and Biosafety, Research Institute of Southeast University in Suzhou, Suzhou 215123, China S Supporting Information *
ABSTRACT: Inspired by the microstructure of the stem cell niche, which is generally composed of adjacent cell protection layers and an extracellular matrix (ECM), we present novel microfluidic porous microcarriers for cell culture that consist of external− internal connected scaffold structures and biopolymer matrix fillers. The biomimetic scaffold structure of the porous microcarriers not only avoids the imposition of shear forces on the encapsulated cells but also provides a confined microenvironment for cell selfassembly, whereas the biopolymers in the porous cores of the microcarriers can act as an ECM microenvironment to promote the formation of multicellular spheroid aggregates for biomedical applications. KEYWORDS: microcarrier, cell culture, cell spheroid, microfluidics, niche
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In this paper, we present a simple microfluidic approach for generating porous microcarriers for cell culture, as outlined in Figure 1. Because of their advantages in controlling the
n recent decades, methods for culturing cells in threedimensional (3D) environments have received increasing attention in such fields as cell biology, drug discovery, and tissue engineering.1−4 Compared with conventional twodimensional (2D) cultures, the 3D approaches have shown great ability to mimic the complex cellular interactions and in vivo microenvironmental conditions that make the recovery of 3D structures and partial functions of the original tissue physiology possible.5−7 To resemble the cell’s natural surroundings more closely, numerous 3D biomaterial scaffolds that incorporate different biochemical, mechanical, or architectural cues have been developed.8−10 Among them, microcarriers, defined as a kind of support matrix for biomaterials, have emerged as novel biomimetic platforms because of their suitable chemical composition, surface topography, and degree of porosity.11 Moreover, these microcarriers combine the advantages of both adherent cultures and suspension cultures, which not only enable nutrient transport but also provide a high surface area for the large-scale cultivation of cells. Because of these features, many kinds of microcarriers have been fabricated using various methods for 3D cell culture.12−15 However, besides the disadvantage of polydispersity, most of the fabricated microcarriers have uniform structures and cells can only be cultured on their external surfaces in an analogously 3D manner. These cells suffer flow-induced shear stress that makes it difficult for them to spread and form real 3D multicellular systems, such as cell spheroids, which have a critical role in maintaining the viability and differentiated functions of cells in vitro. Therefore, the development of novel microcarriers to realize multicellular systems is desirable. © XXXX American Chemical Society
Figure 1. (a) Schematic diagram of the capillary microfluidic device used to generate the W/O/W double emulsion droplets; (b) schematic diagram of the fabrication process of the cell microcarriers and formation of spheroid aggregates.
structures of emulsions precisely, microfluidic techniques have emerged as a versatile method for generating monodisperse emulsion droplets with complex structures.16−20 These emulsion droplets have been used as templates to fabricate functional cell microcarriers with cells cultured on their surfaces or encapsulated in their inner structures.21−24 However, cells on the surfaces of the microfluidic microcarriers suffer the Received: October 30, 2015 Accepted: December 4, 2015
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DOI: 10.1021/acsami.5b10442 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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ACS Applied Materials & Interfaces disadvantages of shear forces while the cells encapsulated in the microcarriers usually face insufficient exchange of nutrients, oxygen, and waste during their culture. Herein, inspired by the microstructure of stem cell niches, which are generally composed of adjacent cell protection layers and an extracellular matrix (ECM),25−27 we propose the use of microfluidic microcarriers composed of external-internal connected scaffolds and biopolymer matrix fillers for cell culture. The biomimetic scaffold structure could not only offer the ability to avoid imposing shear forces on the cells, but also provide a confined microenvironment for cell self-assembly, whereas the presence of biopolymers in the porous cores of the microcarriers could act as an ECM microenvironment for promoting the formation of multicellular spheroid aggregates (outlined in Figure 1b). These features make the porous microcarriers described here ideal for 3D cell culture. In a typical experiment, water/oil/water (W/O/W) double emulsions with multiple encapsulated inner droplets were used as templates for the fabrication of the porous microcarriers. These monodisperse double emulsions were generated by a capillary microfluidic device, which was assembled by coaxially aligning three (inner, middle, and outer) cylindrical capillaries inside a square capillary. The inner phase was pumped through the inner capillary, while the middle phase flowed through the region between the inner round capillary and the middle capillary. The square capillary was used for better observation of the generation of the droplets, the pumping of the outer phase, and the rinsing channel of the microfluidic device. In this system, aqueous surfactant solution was used as the inner and outer phases, while ethoxylated trimethylolpropane triacrylate (ETPTA) was employed as the middle phase. When these fluids flowed through the corresponding capillaries, aqueous core droplets were generated at the end of the inner capillary and then encapsulated by a shell drop of the ETPTA at the end of the middle capillary (Figure 2). As the ETPTA used in this study was a kind of photopolymer (Figure S1a), the generated emulsion templates could be photopolymerized under ultraviolet (UV) illumination downstream from the fluidic channel, producing stable polymeric microcarriers with ETPTA resin shells and aqueous cores.
The overall sizes of the microcarriers and the numbers and the diameter of the encapsulated core droplets could be adjusted by using different orifice sizes and tuning the velocities of the three phases. To achieve microcarriers with suitable external-internal connected open structures for cell culture, we should fabricate the ETPTA double emulsions with dense packing of the aqueous core droplets. Thus, the ratio of the ETPTA flow rate to the inner aqueous flow rate was maintained much smaller than the ratio of the shell to core volume in the critical packing state (Figure S2). The shell phase then drained out of the films between the outer envelope and the overhanging surfaces of the core droplets. The fragile shell layers on the overhanging surfaces could easily be destroyed to achieve the open structure. In addition, it is known that the 3D cell spheroids with diameters greater than 200 μm may suffer from hypoxia because of the depletion of oxygen at the center.14,24 Thus, microcarriers with core diameters not exceeding 200 μm were fabricated to confine the cells’ growth space and to guarantee sufficient supply of oxygen and nutrients during cell culture. The microcarriers generated under these optimized conditions are shown in Figure 3. We observed that the aqueous cores were well encapsulated in the transparent ETPTA shells. The transparency of the shells was beneficial for observations during the cell culture, which was also considered as an essential element for cell microcarriers. Under the multiple encapsulations, the aqueous cores showed dense packing structures in the polymeric particles. The boundaries of these cores adjoined to the particle surfaces had ultrathin and fragile shell layers, which could easily be destroyed by washing with ethanol. Once the boundaries were ruptured, the internal aqueous phase encapsulated in the cores could leak out, leaving the particles with external−internal connected porous structures. The microstructures of the porous microcarriers were also characterized using a scanning electron microscope (SEM), as shown in Figure 4. We can confirm that the microcarriers had a uniform external-internal connected porous structure with large openings to the particle’s surface (Figure 4a−c). In addition, the microcarriers had small windows connecting adjacent pores (Figure 4d), which ensured the formation of open channels through the entire volume of the particle and facilitated nutrient transport. The sizes of the interconnected windows in the microcarriers could be tailored from about 20 to 70 μm for a 500 μm microcarrier by using microfluidic manipulation. This structure was ascribed to the dense packing of core droplets in the double emulsions and subsequent washing with ethanol. The Young’s modulus of the ETPTA scaffold was approximate 15.36 MPa according to the stress− strain curve in Figure S1b, which indicated their stability during the cell culture. The resultant porous microcarriers were employed to culture a human hepatocarcinoma cell line, HepG2 cells. This was carried out under a CO2 atmosphere at 37 °C followed by staining with calcein AM after 72 h. Figure S3 compares the culture results using a traditional multiwell (Figure S3a) and the microcarriers (Figure S3b). It is apparent that the HepG2 cells adhered to both the outer and inner surfaces of the microcarriers. In addition, in contrast to the cells on the bottom of the multiwell that mainly formed flat and fusiform morphologies, the cells adhering to the microcarriers mainly formed oval morphologies, which were more obviously threedimensional. Although some improvements were achieved toward 3D cell culture in comparison with the planar substrate
Figure 2. Microfluidic generation process of the double emulsion templates encapsulated with various numbers of cores; a to d were with 1 to 4 cores, respectively. The scale bar is 500 μm. B
DOI: 10.1021/acsami.5b10442 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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Figure 3. Transmission optical images of the polymeric microcarriers encapsulated with different numbers of cores; from a to f, the cores were 1 to 6, respectively. The scale bar is 500 μm.
are generally composed of adjacent cell protection layers and an ECM. For this purpose, the HepG2 cells were dispersed into a biopolymer ECM solution (Matrigel Basement Membrane Matrix) and the mixture was infiltrated into the porous spaces of the microcarriers. As the ECM solution could be transformed to a semihydrogel state above 10 °C, the dispersed cells were strongly locked in the porous cores of the microcarriers. In contrast to the traditional cell-encapsulated hydrogels, which are usually at high concentrations or have a high degree of cross-linking to maintain their structures and therefore limit the exchange of nutrients,22−24 our ECM hydrogel was in the scaffold of the porous microcarriers, which could maintain their structures even at low concentrations, just as in living tissues. The cells encapsulated in this ECM hydrogel without the porous carrier could aggregate at random and form multiple small aggregates with inhomogeneous sizes, as shown in Figure S3c. Only when they were cultured in the porous microcarriers, the formation and the sizes of these cell aggregations showed good controllability. The initial cell seeding profile and the subsequent 3D cell aggregation process in the porous microcarriers were investigated and recorded continuously for 8 days, as shown in Figure 5 and Figure S4. The transparency of the ECM and the ETPTA shell allowed the use of optical microscopy for this task. The cell aggregates were generally 2D and individual cells were easily distinguishable during the first 2 days after seeding. Subsequently, the cells began to coalesce, driven by cell−cell interactions and acquired spherical shapes after 3 days of culturing. After 7 days, the cell aggregates matured and cell
Figure 4. Scanning electron microscopy (SEM) images of the porous microcarriers: (a−c) external view of the microcarriers encapsulated with (a) two pores, (b) three pores, and (c) six pores; (d) crosssection image of the microcarrier in d. The scale bars are 200 μm.
culture, the porous microcarriers did not show obvious advantages over commercial microcarriers, which were not only more biocompatible but also provided a porous surfaces for cells culture. To exploit the distinct advantages of our porous microcarriers in 3D cell culture, we employed them with novel biomimetic microstructures copied from stem cell niches, which
Figure 5. (a−d) Transmission optical microscopy images of 3D cell aggregation in the porous microcarriers with three cores at (a) day 1, (b) day 3, (c) day 5 and (d) day 7. (e−h) Enlarged images from a−d, respectively. The scale bars are 250 μm. C
DOI: 10.1021/acsami.5b10442 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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which indicated strong ability to mimic the complex cellular interactions in in vivo microenvironmental conditions. Another attractive feature of our methods is that different kinds of hydrogel materials can be incorporated into the porous cores of the microcarriers for biomimetic 3D cell cultures, making these porous microcarriers ideal for evaluating biomaterials. To demonstrate this, we chosen alginate hydrogel and the above ECM hydrogel as representatives to investigate their impacts on the growth of HepG2 cells. HepG2 cells were homogeneously dispersed in these pregel solutions and infiltrated into the pores of the microcarriers. Because of the fast, diffusion-controlled ionic cross-linking, the cells were locked in Ca-alginate hydrogel when CaCl2 solution was added. For the evaluation, the different porous cell microcarriers encapsulated the same quantities of cells and were cultured under the same conditions. The cell viabilities on these microcarriers were investigated by using 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT assays, Figure 7),
spheroids were formed in most of the microcarriers (as confirmed in Figure S5). During this process, cells in most of the pores of the microcarriers could form one large cell spheroid directly. However, in some cases, they first formed multiple small spheroids in single pores, and these small spheroids fused together to form large cell spheroids during culture. This can be ascribed to the restriction forces of the biopolymer matrix. When the contractile forces of cells are greater than the restriction forces of the matrix, cells generally aggregate into clumps and proliferate to form large spheroids, whereas the cells just proliferate based on local cells and grow into small multispheroids. It should be noted that the HepG2 cells in our microcarriers could self-assemble 3D mature spheroids over a culture period of 7 days. This is fast for spheroid formation compared with most 3D cell culture methods.28−30 In addition, several large cell spheroids could be achieved in single microcarriers that had multiple pores (Figure 6). As these pores are interconnected via
Figure 7. MTT assays of the HepG2 cells cultured in microcarriers with different filled materials. Results are presented as mean value ± standard error. The number of replicates for each group was 6.
which is the most common method of quantitative study of cell viability of different biomaterials. The results indicated that the HepG2 cells could proliferate much more quickly in the Caalginate and ECM microcarriers than in the bare microcarriers. This indicated the advantages of the 3D matrix for cell proliferation. In addition, the cell proliferation in the ECM microcarriers showed better performance than in the Caalginate microcarriers, which is consistent with the properties of the biomaterials. These results demonstrate that the porous microcarrier is also a versatile platform for the evaluation of different kinds of biomaterials at the 3D level. In conclusion, inspired by the cell niche microstructure of protection layers and an ECM microenvironment, we have demonstrated a simple microfluidic approach for generating porous microcarriers that consist of an external-internal connected scaffold structure and biopolymer matrix fillers for cell culture. The biomimetic scaffold structure of the porous microcarriers not only offered the ability to avoid the imposition of shear forces on the encapsulated cells but also provided a confined microenvironment for the cell selfassembly, whereas the presence of biopolymers in the porous cores of the microcarriers could act as an ECM microenvironment for promoting the formation of multicellular spheroid aggregates. In addition, different kinds of hydrogel materials can be incorporated into the porous cores of the microcarriers to evaluate their biological functions in 3D cell culture. These
Figure 6. Confocal laser scanning microscopy 3D reconstruction images of the microcarriers with (a−c) two spheroids residing, (d−f) three spheroids residing, and (g−i) four spheroids residing. (a, d, g) HepG2 cells were stained green in the fluorescence microscopy images; (b, e, h) transmission optical microscopy images of the microcarriers; (c, f, i) Superimposed image of (c) a and b, (f) d and e, and (i) g and h, respectively. The scale bar is 200 μm.
windows, the microcarriers provided significant structures toward simulation of actual liver tissue, in which the functional lobule units are connected by a network of blood vessels. To confirm the viability of the encapsulated cells and their formed mature spheroids, we observedthe cell-cultured porous microcarriers by fluorescence and confocal microscopies at intervals of 5 μm in different Z-planes (Figure S6). We found that almost every pore of the microcarriers contained one large cell spheroid with high viability, and the cells in both the exterior and inner part of the spheroids were alive with bright fluorescence. Because of the elimination of the interfacial tension or other compressing forces, the HepG2 cells showed a less dense multicellular structure for the exchange of nutrients, D
DOI: 10.1021/acsami.5b10442 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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unique features of our porous microcarriers make them highly promising for biomimetic cell culture.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.5b10442. Experimental procedure details; Quantitative analysis of cell viability; Critical packing of droplet clusters and corresponding volume ratio of middle and inner phases; Fluorescence microscopy images, confocal laser scanning microscopy images and scanning electron microscopy images of HepG2 cells (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the National Science Foundation of China (Grants 21473029, 91227124, and 51522302), the NASF Foundation of China (Grant U1530260), the National Science Foundation of Jiangsu (Grant BK20140028), the Science and Technology Development Program of Suzhou (Grant ZXG2012021), the Research Fund for the Doctoral Program of Higher Education of China (20120092130006), the Program for Changjiang Scholars and Innovative Research Team in University (IRT1222), and the Program for New Century Excellent Talents in University.
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REFERENCES
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DOI: 10.1021/acsami.5b10442 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
