Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture

Summary

We describe the fabrication of micropatterned hydrogel sheets using a simple process, which can be assembled and manipulated in a freestanding form. Using these modular hydrogel sheets, a simple macro-scaled 3D cell culture system can be generated with a controlled cellular microenvironment.

Abstract

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue geometry. The resulting 3D hydrogel-based cellular constructs have been introduced as an alternative to animal experiments for advanced biological studies, pharmacological assays and organ transplant applications. Although hydrogel-based particles and fibers can be easily fabricated, it is difficult to manipulate them for tissue reconstruction. In this video, we describe a fabrication method for micropatterned alginate hydrogel sheets, together with their assembly to form a macro-scale 3D cell culture system with a controlled cellular microenvironment. Using a mist form of the calcium gelling agent, thin hydrogel sheets are easily generated with a thickness in the range of 100 - 200 µm, and with precise micropatterns. Cells can then be cultured with the geometric guidance of the hydrogel sheets in freestanding conditions. Furthermore, the hydrogel sheets can be readily manipulated using a micropipette with an end-cut tip, and can be assembled into multi-layered structures by stacking them using a patterned polydimethylsiloxane (PDMS) frame. These modular hydrogel sheets, which can be fabricated using a facile process, have potential applications of in vitro drug assays and biological studies, including functional studies of micro- and macrostructure and tissue reconstruction.

Introduction

Hydrogels are particularly promising biomaterials, and are expected to be important in basic biology, pharmacological assays and medicine.¹ Biofabrication of hydrogel-based cellular constructs has been suggested to reduce the use of animal experiments,^2,3 replace transplantable tissues,⁴ and improve cell-based assays.^5,6 Water-containing (hydro-) viscoelastic materials (gels) allow a large number of cells to be encapsulated and maintained in a scaffold structure to control the 3D cellular microenvironment. In combination with the guidance of microfluidic or micropatterning technologies, the geometry of the hydrogel constructs can be precisely controlled at the cellular scale. To date, a variety of shapes of hydrogels, including particles,^7-9 fibers,^10-12 and sheets,^13-15 have been used as building units in bottom-up approaches to the fabrication of macro-scale multi-cellular architectures.

Both hydrogel-based particles and fibers have been readily and rapidly fabricated for applications as micro-scale cellular environments, with fluidic controls using microfluidic devices. However, as the basic units of engineered tissues, it would be complicated to rearrange them and to enlarge their volume as macro-scale constructs.¹⁶ It is more difficult to achieve macro-scaled constructs than to produce micron-sized basic modules. Sheet-like units of hydrogel-based constructs can be used to increase the volume of scaffolds via a simple assembly process. Consequentially, stacked layers of hydrogel sheets provide not only a volumetric increase but also a geometric extension in a 3D space.

We have previously reported a method of fabricating micropatterned hydrogel sheets,^13-15 together with their assembly into multi-layered cellular architectures. The technique enables complex micropatterning and modular design of cellular constructs via a stacking process of multi-layered structures. Through the fabrication of stacked modular hydrogel sheets, which are micropatterned, a 3D cell culture system with a controlled macro-scale cellular microenvironment can be realized. This video protocol describes a simple yet powerful fabrication method that can be used to construct modular hydrogel sheets, based on the human liver carcinoma cell line (HepG2). We demonstrate herein simple manipulation of these patterned modular hydrogel sheets, and their assembly into a multi-layered structure.

Protocol

   1. Preparation of the Micropatterned Molds and Hydrogels

1. Produce the desired micro-scale patterns using SU-8 photoresist on the surface of a silicon wafer via a standard two-step photolithography technique^15,17 for casting PDMS molds. The example shown uses a liver lobule-like mesh pattern (Figure 1).
2. Weigh out PDMS and a curing agent solution with a ratio of 1:5 (i.e., 12.5 g of PDMS and 2.5 g of curing agent).
3. Mix the 15 g of the solution thoroughly, degas the bubbles in a vacuum chamber, and then spread the mixed solution onto a micropatterned surface of the silicon wafer evenly within a foil casting dish.
4. Place the silicon wafer onto a 65 °C heated plate for 90 min on a flat surface to cure the PDMS.
5. Remove the cured PDMS from the casting dish and the silicon wafer.
6. Cut the edges of the PDMS and place it onto a 100-mm-diameter petri dish with the micropatterned side up.
7. Wash the micropatterned cured PDMS on the petri dish using 70% ethanol and distilled water for primary sterilization. Then, dry them completely for 10 min in an oven at 65 °C.
8. Dissolve and mix O/N 3 g of a powdered nonionic surfactant in 100 ml of distilled water, creating a 3% (w/v) coating solution.
9. Place the micropatterned PDMS molds in a plasma cleaner and clean them for 1 min (85 W, 0.73 mbar) to create a hydrophilic surface, to facilitate the addition of aqueous liquids. Then, coat the surface of the PDMS with the 100 ml surfactant solution for at least 3 hr (orO/N) using a laboratory rocker.
10. Wash the surfactant solution from the PDMS molds and dry them completely in an oven at 65 °C for 10 min. Then, sterilize each micropatterned mold by exposure to ultraviolet (UV) radiation over 30 min.

2. Prepare the Cell Suspension in a Hydrogel Precursor

1. To prepare hydrogel precursor, dissolve 0.1 g of sodium alginate powder in 10 ml of phosphate buffered saline (PBS), creating a 1% (w/v) alginate precursor. To dissolve the powder completely, incubate and mix them O/N.
2. Filter the solution through a 0.22 µm filter connected to a 1 ml syringe.
3. Culture the HepG2 cells in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin on a conventional tissue culture dish until 70% - 80% confluence in a 5% CO₂ humidified incubator at 37 °C.
4. Wash the cells once using PBS, and then trypsinize them by adding 1 ml of 0.05% trypsin-EDTA for 4 min in a 5% CO₂ humidified incubator at 37 °C. Centrifuge the cells harvested from the culture dish at 250 x g for 3 min and resuspend using 1 ml of PBS following removal of the supernatant.
5. Count the number of single distributed cells in PBS using an automated cell counter.
6. Following centrifugation at 250 x g for 3 min and PBS removal, add 1 ml of the 1% (w/v) of sodium alginate solution to the remaining cell pellet and mix them gently using pipetting. The final seeding density of the cells should be 5 x 10⁶ - 10⁷ cells/ml. Incubate the cell/hydrogel suspension in a 5% CO₂ humidified incubator at 37 °C.

3. Loading and Cross-linking of Cell/Hydrogel Suspension

1. Dissolve 1.47 g of calcium chloride dehydrate in 100 ml of ddH₂O to produce a cross-linking reagent (i.e., 100 mM CaCl₂·2H₂O in ddH₂O).
2. Rinse out the interior of a humidifier with ultrasonic transducer using 70% ethanol, and fill it with 200 ml of cross-linking reagent. The humidifier is 110 mm wide, 300 mm long and 170 mm deep (i.e., 110 mm x 300 mm x 170 mm (W x H x D)).
3. Place the micropatterned PDMS molds in a plasma cleaner and clean them for 1 min at 85 W to create a hydrophilic surface.
4. Steadily load 7.2 µl of the well-mixed cell/hydrogel suspension at the edge of the micropattern in the mold. The example shown uses a liver lobule-like mesh pattern (Figure 1). The volume of the suspension depends on the topographic pattern.
5. To achieve gelation of the cell/hydrogel suspension, turn on the humidifier and verify that the humidifier produces a mist of the cross-linking reagent. Spray the cross-linking reagent onto the hydrogel precursor for 5 min, covering the topographic surface of the PDMS molds within a range of 5 cm.
   Note: Distances longer and shorter than 5 cm could cause incomplete and uneven gelation, respectively. Ensure that the humidifier has a spraying rate of 250 ml/hr, producing 20 ml of mist of the cross-linking reagent in 5 min.
6. Following the cross-linking process, turn off the humidifier and fill the PDMS molds with PBS.

4. Handling of Single Modular Hydrogel Sheets

1. Detach each hardened hydrogel sheet from the micropatterned molds via pipetting PBS gently around the hydrogel sheet using a 200 µl pipette tip.
2. Pick up each floating hydrogel sheet using an end-cut 1,000 µl pipette tip end. Each liver lobule-like mesh hydrogel sheet has dimensions of 8 mm x 8.7 mm, and be 100 - 200 µm thick.
3. Transfer a single layer of the hydrogel sheet into 1 ml of DMEM in a 12-well plate, and culture the cells in vitro in a floating manner using the hydrogel construct as a unit component in the 12-well plate over a week in a 5% CO₂ humidified incubator at 37 °C. Exchange the culture medium every other day.

5. Assembly of Multi-layered Hydrogel Sheets

1. Repeat steps 1.1 to 1.5 to produce a PDMS frame with dimensions of 18 mm x 18 mm x 4 mm (W x H x D), and which contains 170-µm-high pillar structures at the lower surface. Use 42 g of the mixture of PDMS and curing agent, with the same ratio as in step 1.2. Place a specialized polycarbonate mold on the silicon wafer for the PDMS frame to create an interior frame with dimensions of 8 mm x 9 mm x 2 mm (W x H x D).
2. Sterilize the PDMS frame and 180-µm-pore nylon filter papers submerged in distilled water and tweezers in an autoclave for 15 min at 121 °C.
3. Place the sterilized PDMS frame onto a quarter of a piece of nylon filter paper (with a diameter of 5 cm) in a 60-mm diameter petri dish.
4. Transfer a modular hydrogel sheet into the interior of the PDMS frame using an end-cut 1,000 µl pipette tip. The hydrogel sheets used for assembly should be cultured for at least a day after they were fabricated.
5. Align the edge of each modular hydrogel sheet with the PDMS frame using an empty 200 µl pipette tip.
6. Repeat steps 5.4 and 5.5 using modular hydrogel sheets to form a stack of 4 - 6 layers.
7. Remove the culture medium by flowing it out through the pillar structures at the bottom of the PDMS frame. Then add 2 µl of alginate solution (2% w/v) at a corner of the multi-layer construct.
8. Spray a mist of the cross-linking reagent for 30 sec onto the multi-layer construct to attach the edges of each layer with those of another. Use 2 ml of mist of the cross-linking reagent (at a spraying rate of 250 ml/hr).
9. Rinse the multi-layered construct gently with 400 µl DMEM and remove the PDMS frame using tweezers.
10. Detach the multi-layered construct from the filter paper by gently wiping with a cell lifter following the addition of 4 ml of DMEM.
11. Transfer the multi-layered construct to a 6-well plate containing 3 ml of DMEM using filter paper, and culture the cells in vitro in a floating manner, with the hydrogel construct as a multi-scale cellular scaffold in the 6-well plate over a week in a 5% CO₂ humidified incubator at 37 °C. Exchange the culture medium every other day.

Results

We have described the fabrication and manipulation of freestanding cellular hydrogel sheets. As shown in Figure 1, we fabricated micropatterned PDMS molds, and cell-containing hydrogel was loaded onto the hydrophilic surface of these molds and cross-linked using a humidifier to generate an aerosolized mist of gelling agent. Following release from the molds, HepG2 cells were cultured in freestanding hydrogel sheets with various patterns (Figure 2). Thus, t...

Discussion

This protocol provides a simple method of fabricating modular hydrogel sheets, and assembling them to form 3D cellular scaffolds.

To construct clear-cut patterned alginate structures in a short time, we should identify a cross-linking process that can create sufficiently rigid structures to maintain the complex micropatterns from the mold, as well as maintain cell viability and metabolism. We have developed a cross-linking process, including a sol–gel transition, to spray a cross-linking...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Sylgard 184 Silicone Elastomer Kit	Dow Corning Corporation	000000000001064291
Pluronic F-127	Sigma-Aldrich	P2443	Powdered nonionic surfactant
Alginic acid sodium salt, low viscosity	Alfa Aesar	B25266
Calcium chloride dihydrate	Sigma-Aldrich	C7902
Ultrasonic humidifier	MediHeim	MH-2800	Modified equipment, Maximum sprayed rate: 250 ml/hr
Nylon net filter hydrofilic, 180 μm	EMD Millipore	NY8H04700
Polycarbonate mold			Customized mold for fabrication of a PDMS frame pattern