This article introduces a simple approach to providing non-continuous gradient static strains on a concentric cell-laden hydrogel to regulate cell alignment for tissue engineering.
Artificial guidance for cellular alignment is a hot topic in the field of tissue engineering. Most of the previous research has investigated single strain-induced cellular alignment on a cell-laden hydrogel by using complex experimental processes and mass controlling systems, which are usually associated with contamination issues. Thus, in this article, we propose a simple approach to building a gradient static strain using a fluidic chip with a plastic PDMS cover and a UV transparent glass substrate for the stimulation of cellular behavior in a 3D hydrogel. Overloading photo-patternable cell prepolymer in the fluidic chamber can generate a convex curved PDMS membrane on the cover. After UV crosslinking, through a concentric circular micropattern under the curved PDMS membrane, and buffer washing, a microenvironment for investigating cell behaviors under a variety of gradient strains is self-established in a single fluidic chip, without external instruments. NIH3T3 cells were demonstrated after observing the change in the cellular alignment trend under geometry guidance, in cooperation with strain stimulation, which varied from 15 – 65% on hydrogels. After a 3-day incubation, the hydrogel geometry dominated the cell alignment under low compressive strain, where cells aligned along the hydrogel elongation direction under high compressive strain. Between these, the cells showed random alignment due to the dissipation of the radical guidance of hydrogel elongation and the geometry guidance of the patterned hydrogel.
Serving as a block material that mimics a native microenvironment, a hydrogel containing extracellular matrix (ECM) can re-build biomimetic tissue scaffolds to support cell growth. To possess the functions of a tissue, organized cell alignment is an essential requirement. Various 2D (i.e., cells cultured on a surface) and 3D (i.e., cells encapsulated in a hydrogel) cell alignments have been achieved by culturing or encapsulating cells in or on flexible substrates with micro-or nano-patterns1. 3D cell alignment in microarchitecture is more attractive, as the microenvironment is closer to the native tissue construct2,3,4. One common approach for 3D cell alignment is the geometric cue of hydrogel shape2,3. Because of the restricted space for cell proliferation in the short-axis direction, cells aim to align along the long-axis direction in a micro-patterned hydrogel. Another approach is to apply tensile stretch to the hydrogels to achieve cell alignment parallel to the stretch direction4,5.
Biophysical stimulation on ECM hydrogels, such as compressive strain or an electrical field, can regulate cell functions for proper tissue integration, proliferation, and differentiation1,2,3. Much research has been done to investigate cellular behavior by applying one strain condition at a time using multiple mechanical control units4,6,7,8,9. For example, the use of mechanical step motors squeezed or stretched on a 3D cell-encapsulated collagen hydrogel has been a common approach7,10. However, such controlling equipment requires extra space and faces the issue of contamination in the incubator7,9,11,12. In addition, the large instrument cannot give a precise control environment to provide high reproducibility13.
Considering that cell-laden hydrogels are usually employed on the micro-scale for biomedical applications, it is advantageous to combine MEMS techniques to generate a range of strain/stretch stimulation to simultaneously investigate cell behaviors in 3D biomimetic constructs in vitro2,14,15,16,17,18. For example, using gas pressure to deform the PDMS membrane in microfluidic chips can give rise to various strains, driving cell differentiation to different lineages9,16. However, there are many technical challenges, such as complicated chip fabrication processes in a clean room and the software control integration of motors, pumps, valves, and compressed gases.
In this work, we demonstrate a simple approach to obtain a self-sustaining gradient static-strain microfluidic chip by employing a concentric circular hydrogel pattern and a flexible PDMS membrane. Unlike most of the existing approaches, our platform is a portable and disposable miniature device that can be fabricated outside a yellow room and that possesses self-generating gradient strains on concentric cell-encapsulated hydrogels, without external mechanical equipment during the incubation. 3T3 fibroblast cell behaviors influenced by a combination of hydrogel shape and a variety of tensile stretch guidance cues were demonstrated during the observation of cell alignment within 3D ECM-mimetic environments in the gradient strain chip for 3 days.
1. GelMA Synthesis
2. 3-(Trimethoxysilyl)propyl Methacrylate (TMSPMA) Modification
3. Chip Fabrication
4. Static Gradient Strain on the Cell-laden Hydrogel
5. Cell Staining for Alignment Analysis
To compare the mechanical variations between each circular hydrogel in the completed gradient strain stimulation chip, we measured the line widths of each circular hydrogel in two of the same chips, with injection volumes of 0 µL (Figure 4a) and 40 µL (Figure 4b), respectively. The percent elongations at each circle were calculated by dividing the elongations in the 40 µL-injected chip by the line widths of the corresponding hydrogels in the 0 µL-injected chip (Figure 4c). The hydrogel is an incompressible material, so the vertical contracting strain will be equivalent to the lateral elongation strain. Therefore, the percent elongations in the chip with the 40-µL injection volume show 15 – 65% elongation, which can be converted to the compressive strain (see Supplementary File 1).
Fluorescent staining of the cell nuclei and F-actin with DAPI and phalloidin, respectively, was done to analyze the cellular alignment. The DAPI staining provided data on nuclear orientation, and the phalloidin staining was applied to assess cell spreading. Figure 5a-c shows the cell alignment in the gradient strain chip. In line 1, the 3T3 cells aligned along the radial direction. In hydrogel 7, the cells aligned randomly, and cells aligned along the circular direction in line 12. In accordance with the fluorescent staining images, a 90° shift from the angle of cell alignment of the 3T3 cells in line 1 (maximum hydrogel elongation in the radial direction) to the alignment angle of the cells with long-axis alignment in line 12 (lowest hydrogel elongation in the radial direction) was discovered.
In previous research2,9, cells in 200-µm line-patterned hydrogels aim to align along the long-axis direction of the hydrogel. However, in this study, we observed that the elongated stretch on the 200-µm hydrogels in the short-axis direction provided another factor to affect and dominate the cellular alignment by controlling the percentage of the strain on the hydrogel. For the 65% strain on line 1, the radical alignment proved that the elongation stretch of the hydrogel dominates the cell alignment. For the 15% strain on line 12, the circular alignment proved that the long-axis effect dominated the cell alignment. For the 40% strain on hydrogel 7, the cells aligned randomly due to the neutralization of the geometry guidance and strain effect.
Figure 1. PMMA Mother Mold for PDMS Sheet and Plug Fabrication. (a) The separated components of the PMMA mold, including a bottom plate, boundary frame, and flow channel. After assembly with double-sided tape, (b) the PMMA mother mold for the flow sheet is formed. (c) Another PMMA mold is assembled for the PDMS plug. The red number represents the depth. (unit: mm) Please click here to view a larger version of this figure.
Figure 2. Plastic Photomask Design for the Cell-laden Hydrogel Micropattern. There are two openings with triangle shapes (2 mm at the bottom line and 6.5 mm in height) connecting to the flow channels to supply fresh cell culture medium. (a) The plastic photomask is labeled with the dimension. The period of the concentric circle is 400 nm, and the duty cycle is 50%. The diameter of the center circle is 2 mm. (b) The photomask layout, without labels for laser printing on a plastic transparent film. Please click here to view a larger version of this figure.
Figure 3.Fabrication Processes of the Gradient Strain Cell-laden Hydrogel Along the Radial Direction in a PDMS Fluidic Chip. (a) A plastic photomask is aligned and stuck under the chip with a TMSPMA-coated fluidic channel. A micro-syringe with prepolymer cell solution is inserted into the inlet of the chip and used to inject about 50 µL to fill the flow channel. (b) The outlet of the flow channel is closed with the PDMS plug and an additional 40 µL of prepolymer cell solution is injected. The glass bottom is UV-patterned for 30 s to fabricate the concentric circular hydrogel in the flow chip. (c) The liquid pressure is released in the flow channel by unplugging the outlet and the un-crosslinking mixture is washed out with DPBS. (d) A chip with static gradient strain applies concentric cell-laden hydrogels that are ready for cell culturing. During the UV crosslinking process, (e) a non-continuous gradient height of hydrogel along the radius is formed. (f) After unplugging the outlet, the PDMS membrane becomes flat and applies gradient stress on the cell-encapsulated hydrogels. Please click here to view a larger version of this figure.
Figure 4.The Pure Hydrogel Elongation Gradient. The elongation line width and percentage of pure hydrogel without cell encapsulation in the gradient strain chip on day 3 with the (a) 0-µL (control group) and (b) 40-µL injection volumes. (c) The elongation percentage is calculated by dividing the value of the line width difference between 40 µL and 0 µL by the line width of 40 µL. Please click here to view a larger version of this figure.
Figure 5. Fluorescent Actin-nucleus Stain images of the 3T3 Cells Encapsulated in the Gradient Chips on Day 3. The cell alignment direction in (a-c) line 1, (d-f) line 7, and (g-i) line 12 reveal radial alignment, random alignment, and circular alignment, respectively. The green and blue colors show the actin and nucleus stains, respectively. The dotted white line represents the boundary of the hydrogel. Scale bar: 200 µm. Please click here to view a larger version of this figure.
Supplementary Figure 1. Calculation of the Dome-shaped PDMS Curvature. H(x): the convex PDMS curve; H0: the maximum height difference between the PDMS dome before and after deformation; r: the radius of the dome; V: the over-injection volume of the blue region, which causes the PDMS deformation as a dome. See Supplementary File 1 for details. Please click here to download this figure.
Supplementary File 1. Please click here to download this file.
In this paper, we report on a simple approach to compare cell alignment behavior after hydrogel shape guidance and tensile stretch. A flexible PDMS membrane creates a dome-shaped curvature for generating different heights of concentric circular hydrogels. After releasing the pressure, the PDMS membrane automatically applies force to the micro-patterned hydrogels to form gradient strain/elongation, with a maximum at the center and a minimum at the outer boundary. As the formation of the gradient strain is designed by the flexible PDMS membrane and the handling of the fluidic chip, there are several important parameters that should be attended to: (i) Precise control of the thickness of the PDMS membrane is crucial to adjust the gradient strain value. If the membrane is too thick, even the maximum injection volume of cell prepolymer will not be able to generate a proper convex curve in the PDMS membrane for crosslinking a gradient height of cell-laden hydrogels. In contrast, a too-thin PDMS membrane cannot apply sufficient force to the hydrogels. Please check that the weight of uncured PDMS in the PDMS cover mold is around 1.6-2.0 g per chip. (ii) Contamination prevention is very important during the crosslink handling of the cell-laden hydrogel in the fluidic chip. Before cell culturing in the incubator, thoroughly washing with sterilized PBS in the fluidic channel and using 75% ethanol to wipe the surface of the chip can help to avoid the contamination issue. (iii) The concentration of photoinitiator and the dosage of UV exposure should be careful controlled and in the range of ~0.1% – 2% (0.5% is recommended). Over-crosslinking the hydrogel and overdosing the UV irradiation will result in low cell viability. (iv) The line-width of the patterned hydrogel should not be too large. Otherwise, the nutrient replacement rate in the thick hydrogels will not be able to support cell proliferation. Usually, less than 300 µm is recommended. The spacing between two hydrogel circles can be varied, and a 50% duty cycle is recommended. (v) While washing or refilling the flow channel with solution, the formation of bubbles should be avoided. Gently pipetting the solution in the chip can help to remove bubbles.
The concept of gradient strain generated by PDMS-deformed curvature can be further upgraded to applying dynamic gradient strains and can be integrated with biochemical stimulation, which can benefit many studies on functional tissue regeneration. The simple fluidic injection module with a PDMS plug can be replaced by any advanced fluidic system for extended experimental control. The PMMA mold can also be replaced by a microfabricated SU-8 mold or a bulk-etched silicon mold.
This gradient strain chip with a circular cell-laden hydrogel can generate static compressive force on the 3D hydrogel without external mechanical or electrical machines. Therefore, it provides a fast screening platform for investigating cell behaviors in a series of strain conditions, without the risk of contamination issues caused by the operation of external machines. However, time-controlled strain stimulation is not achievable because the PDMS membrane generates strains until the degradation of the hydrogel.
The authors have nothing to disclose.
This project was supported by the Graduate Student Study Abroad Program (NSC-101-2917-I-007-010); the Biomedical Engineering Program (NSC-101-2221-E-007-032-MY3); and the Nanotechnology National Program (NSC-101-2120-M-007-001-); and the Ministry of Science and Technology (MOST-104-2221-E-007-072-MY3), National Science Council of the R.O.C., Taiwan. The authors would like to thank Prof. Ali Khademhosseini, Gulden Camci-Unal, Arghya Paul, and Ronglih Liao at Harvard Medical School for sharing the hydrogel and cell encapsulation technology.
1.5-mL black microcentrifuge tube | Argos Technologies | 03-391-161 | This one can be replaced with a neutral color of 1.5-mL tube covered with aluminun foil |
10X DPBS | Sigma-Aldrich | 56064C | |
Alexa Fluor 488 phalloidin | Invitrogen | A12379 | |
BSA | Sigma | A1595 | |
Calcein | Molecular Probe | C1430 | For labeling viable cells |
CCD | PCO. Imaging | Pixelfly qe | |
Cell membrane permeating solution | Sigma-Aldrich | X100 | 0.5% Triton X-100 for permeating cell membrane |
DAPI | Sigma-Aldrich | D8417 | Cell nucleus staining |
Dialysis membrane | Sigma-Aldrich | D9527 | Molecular weight cut-off = 14,000 |
DMEM | Gibco | 11995-065 | |
Double-side tape | 3M | 8003 | |
FBS | Hyclone | SH30071.03 | |
Gelatin | Sigma-Aldrich | G2500 | gel strength 300, type A, from porcine skin |
High frequency electronic corona generator | Electro-technic products | MODEL BD-20 | |
Methacrylic Anhydride | Sigma-Aldrich | 276685 | |
Micro syringe | Hamilton | 80501 | 50 μL |
Microscope | Olympus | IX71 | Include two filter sets: LF405/LP-B-000 and LF488/LP-C-000 from Semrock |
Oxygen plasma machine | Harrick plasma | PDC-001 | |
Paraformaldehyde | Sigma-Aldrich | P6148 | For fixing cell |
PDMS | DOW CORNING | Sylgard 184 | Mixture for PDMS chip cast-molding fabrication |
Pen-Strep | Gibco | 10378-016 | penicillin/streptomycin |
Photoinitiator | CIBA | Irgacure 2959 | |
Propidium iodide | Sigma-Aldrich | P4170 | For labeling dead cells |
Sterile Filtration cup | Millipore | SCGPT05RE | |
TMSPMA | Sigma-Aldrich | 440159 | For hydrogel immobilization |
Ultrasonicator | Delta | D150H | 150W, 43kHz |
UV light | DAIHAN | WUV-L10 | |
Freeze Dryer | FIRSTEK | 150311025 | |
NIH3T3(fibroblast) | Food Industry Research and Development Institute(FIRDI) | 08C0011 | |
MOXI Z Mini Automated Cell Counter | ORFLO | MXZ001 |