Replicating 3D printed structures into hydrogels Ho Nam Chan, Qian Tian, Yiwei Shu and Hongkai Wu*

Department of Chemistry, The Hong Kong University of Science and Technology, Hong Kong

ABSTRACT Fabricating hydrogel with arbitrary 3D geometries is challenging. Current additive manufacturing

technique developed for hydrogel suffers from low resolution and simple object architecture. Here, by addressing the challenges of mold removal and pores blocking in applying traditional lost-wax casting technique to replicate 3D printed structures into hydrogels, we demonstrate the first replication of 3D structure such as dodecahedron from 3D printed material into alginate and genipin-crosslinked gelatin. Furthermore, we show the fabrication of perfusable, complicated 3D microfluidic network in cell-seeded hydrogel by using the alginate replica as the sacrificial template. KEYWORDS: 3D printing, 3D replication, perfusable hydrogel, 3D culturing

INTRODUCTION

Fabricating hydrogel with arbitrary 3D geometries is challenging and important to tissue engineer.[1] Although 3D manufacturing techniques tailored for hydrogel are available, their performance including resolution, smallest printable feature size and printable structural complexity are subjected to and influenced by the gelation rate, rheological properties and crosslink mechanism of the hydrogel.[2,3] In contrast, conventional 3D printing systems, which do not have the constraints of using biocompatible processes and materials, naturally offer better printing resolution and higher structural design freedom than the bioprinting counterpart together with an order lower in equipment cost. Therefore, replicating conventional 3D printed master from the original materials into hydrogel would be an attractive alternative to enhance the resolution and structural complexity of 3D hydrogel construct.

Inspired by the lost-wax casting technique introduced a thousand years ago, here, we report a 3D replication method that replicates 3D printed masters into hydrogel and potentially into other 3D unprintable materials.

EXPERIMENTAL

Replication 3D printed structure into calcium alginate: The schematic representation is shown in Figure 1a. To prepare 1.625g of the plaster paste, 0.300 g of calcium sulphate hemihydrate was mixed with 0.700 g of calcium carbonate followed by mixing with 0.625 g of de-ionized water. The mixed paste was placed in a centrifugation tube and close packed with the 3D printed structure by centrifugation under 1400 r.c.f. for 30 s. The plaster paste was partially dried in room temperature for 2 h then heated in an 80oC oven overnight for complete setting. After taking the mold out of the tube, the mold was heated under 500oC in a muffle furnace for 2 h to remove the 3D printed template through combustion. After cooling to room temperature, remaining ashes inside the channel were blow out by a blowgun. Then, 0.01M CaCl2 solution was pipetted into the mold with a volume (mL) equal to 25% of the weight of the mold (g). Afterwards, 3% (w/v) sodium alginate solution was centrifuged into the mold under 1400 r.c.f. for 60 s. After cleaning the calcium alginate residue on the outer surface of the mold, the mold was placed in a 4% HCl solution for 1 h to retrieve the casted alginate. The alginate was further sonicated in a 2% HCl bath for 10 min to remove the calcium crystal stuck on the alginate surface. Finally, the alginate was washed with de-ionized water 3 times, and stored in de-ionized water to adjust the pH to 5.5.

Fabrication of cell-seeded agarose gel with perfusable microfluidic network: HepG2 cells were cultured with Dulbecco's Modified Eagle Medium (DMEM), penicillin and streptomycin, and 10% fetal bovine serum (FBS) in the petri dish. The cells were cultured for 4 days then harvested and suspended in fresh DMEM. The suspension was added to the warm agarose solution and mixed with other components to prepare the gel precursor with 3% agarose, 10 U/mL alginate lyase, 1X DMEM and 1 x 107 cells/mL HepG2. The schematic is shown in Figure 1b. After poured on the alginate replica with a trifurcating

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network structure and gelled by cooling, the cell-seeded agarose was incubated at 37ºC for 1.5 h to reverse the gelation of alginate and reduce its viscosity. Then, the head and tail of the incubated agarose was sliced to expose the inlet and outlet of the microfluidic network. Tubings were then inserted and connected to the microfluidic network. After removing the alginate solution by suction, DMEM (pre-equilibrated with 5.0% CO2 overnight in the cell incubator) was perfused into the network with a syringe pump at a flow rate of 2 µL/min for 3 days in a 5.0% CO2 cell incubator. To assess the cell viability in the perfused agarose, the agarose was sliced at the middle and stained with live/dead viability assay solution (2 µM calcein-AM-green and 4 µM ethidium homodimer-1). For the control, the agarose precursor was prepared with the same composition and cell density as the agarose used in the perfusion experiment. Instead, the precursor was poured in a plastic mold without the alginate template. After gelled, the agarose was incubated at 37 ºC for 1.5 h. It was then submerged in DMEM bath for 3 days inside the cell incubator. To determine the cell viability at the center of the agarose, it was sliced at the center and stained with live/dead viability assay solution. The cell viability was calculated at the edge and center of the gel.

RESULTS AND DISCUSSION

The 3D replication results are shown in Figure 1c. With this method, we can replicate different design of 3D structures from their original material into calcium alginate. The casted dodecahedron had the same 3D geometry as the original 3d printed master. The key steps in the replication process are: (1) incorporating CaCO3 as a major filler in the plaster mold (hence, the mold can be removed easily with HCl), (2) adding CaCl2 solution to fill the pores in the plaster mold so that the hydrogel precursor will not be wicked into the plaster mold by capillary force. We expect our replication technique can be used on most of the conventional 3D printers because combustion is a versatile method to remove the printed material. Moreover, with a slight modification in the casting procedure, other hydrogel such as genipin-crosslinked gelatin can also be casted (Figure 1c), which may found useful in fabricating cellular constructs.

Currently, it is difficult to bioprint cellular construct together with complicated 3D microfluidic network. However, incorporating microfluidic network in hydrogel is important in engineering large tissue as the network is required to ensure effective mass transport in the core of the cell construct. Alginate replica was used as the sacrificial template in fabricating microfluidic network in hydrogel. To demonstrate the capability of our casting technique to fabricate cell-seeded perfusable hydrogel, 3% (w/v) agarose solution that contained culture medium, alginate lyase and HepG2 cells (107 cells/mL) was prepared. In this experiment, a trifurcating network was used as the 3D model. Following the procedures described in the experimental section, a cell-seeded perfusable agarose gel was prepared. The initial viability of the cells after removing the alginate solution was determined as about 85%, indicating the alginate replica is biocompatible. Afterwards, tubings were inserted into the agarose and perfused with culture medium at a flow rate of 2 µL/min for 3 days in a cell incubator. The agarose was sliced as illustrated in Figure 1e and the embedded cells viability was determined as about 60% with live/dead staining (Figure 1g). For control experiment, another piece of HepG2 seeded agarose with the same cell density but not the microfluidic network was submerged in culture medium and incubated for 3 days. The agarose was then sliced at the center and stained with live/dead viability assay solution (Figure 1f). It was observed that the viability of cells seeded near the edge of the agarose was higher than those seeded at the center, which is expected because the nutrient from the culture medium cannot effectively diffuse into the center of the gel. From Figure 1h, we can observe that the viability of the cells seeded in the perfused agarose had the similar viability as the cells seeded at the edge of the control, meaning the perfusable microchannel network can support the cells viability inside the core of a large slab of hydrogel.

CONCLUSION

To conclude, we developed a 3D replication method to replicate 3D printed material into hydrogel including alginate and gelatin. With this strategy, we can fabricate 3D structure of hydrogel with high structural complexity and design freedom. Although the involvement of strong acid prohibited the

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technique to replicate cell-seeded hydrogel directly, the replica can be used as a sacrificial template to fabricate cell-seeded perfusable hydrogel. Finally, it should be noted that 3D printable materials are highly limited and the desired materials for a particular application are seldom 3D printable. 3D replication techniques would be essential in broadening the array of materials that can be constructed freely in three-dimension.

Figure 1: (a) Schematic representation of the strategy of the 3D replication technique. (b) Schematic representation of the fabrication of perfusable microfluidic network in cell seeded agarose by sacrificial molding. (c) The replication results of the model 3D structure – dodecahedron. (d) The microfluidic network generated by using the dodecahedron calcium alginate as a sacrificial template. The channel is loaded with 1% green fluorescent microbeads suspension for visualization. (e) Illustration of the trifurcating network used in the perfusion experiment. After 3 days of perfusion, the cell-seeded agarose was sliced as shown in the diagram. (f, g) The fluorescence microgram of the control experiment and the agarose slices after 3 days of perfusion. Green: live. Red: dead. (h) Histogram of the calculated cell viability in different experimental conditions.

ACKNOWLEDGEMENTS

Authors thank the financial support from HKRGC (#604712).

REFERENCES [1] A. Khademhosseini, R. Langer, J. Borenstein, J. P. Vacanti, Proc. Natl. Acad. Sci. U. S. A. 2006,

103, 2480–7. [2] S. V Murphy, A. Atala, Nat. Biotechnol. 2014, 32, 773–785. [3] A. Skardal, A. Atala, Ann. Biomed. Eng. 2014, 43, 730–746.

CONTACT * phone: +852-2358-7246; chhkwu@ust.hk

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