Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Podsumowanie

Presented here is a method for the 3D bioprinting of gelatin methacryloyl.

Streszczenie

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed from mammal collagen. Thus, the arginine-glycine-aspartic acid (RGD) sequences and target motifs of matrix metalloproteinase (MMP) remain on the molecular chains, which help achieve cell attachment and degradation. Furthermore, formation properties of GelMA are versatile. The methacrylamide groups allow a material to become rapidly crosslinked under light irradiation in the presence of a photoinitiator. Therefore, it makes great sense to establish suitable methods for synthesizing three-dimensional (3D) structures with this promising material. However, its low viscosity restricts GelMA's printability. Presented here are methods to carry out 3D bioprinting of GelMA hydrogels, namely the fabrication of GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips. The resulting structures and biocompatibility of the materials as well as the printing methods are discussed. It is believed that this protocol may serve as a bridge between previously applied biomaterials and GelMA as well as contribute to the establishment of GelMA-based 3D architectures for biomedical applications.

Wprowadzenie

Hydrogels are thought to be a suitable material in the field of biofabrication^1,2,3,4. Among them, gelatin methacryloyl (GelMA) has become one of the most versatile biomaterials, initially proposed in 2000 by Van Den Bulcke et al.⁵. GelMA is synthesized by the direct reaction of gelatin with methacrylic anhydride (MA). The gelatin, which is hydrolyzed by the mammal collagen, is composed of target motifs of matrix metalloproteinase (MMP). Thus, in vitro three-dimensional (3D) tissue models established by GelMA can ideally mimic the interactions between cells and extracellular matrix (ECM) in vivo. Furthermore, arginine-glycine-aspartic acid (RGD) sequences, which are absent in some other hydrogels such as alginates, remain on the molecular chains of GelMA. This makes it possible to realize the attachment of encapsulated cells inside the hydrogel networks⁶. Additionally, the formation capability of GelMA is promising. The methacrylamide groups on the GelMA molecular chains react with the photoinitiator under mild reaction conditions and form covalent bonds upon exposure to light irradiation. Therefore, the printed structures can be rapidly crosslinked to maintain the designed shapes in a simple way.

Based on these properties, a series of fields utilize GelMA to carry out various applications, such as tissue engineering, basic cytology analysis, drug screening, and biosensing. Accordingly, various fabrication strategies have been also demonstrated^{7,8,9,10,11,12,13,14}. However, it is still challenging to carry out 3D bioprinting based on GelMA, which is due to its fundamental properties. GelMA is a temperature-sensitive material. During the printing process, the temperature of the printing atmosphere has to be strictly controlled in order to maintain the physical state of the bioink. Besides, the viscosity of GelMA is generally lower than other common hydrogels (i.e., alginate, chitosan, hyaluronic acid, etc.). However, other obstacles are faced when building 3D architectures with this material¹⁵.

This article summarizes several approaches for the 3D bioprinting of GelMA proposed by our lab and describes the printed samples (i.e., the synthesis of GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips). Each method has specialized functions and can be adopted in different situations with different requirements. GelMA microspheres are generated by an electroassisted module, which forms extra external electric force to shrink the droplet size. In terms of GelMA fibers, they are extruded by a coaxial bioprinting nozzle with the help of viscous sodium alginate. In addition, the establishment of complex 3D structures is achieved with a digital light processing (DLP) bioprinter. Finally, a twice crosslinking strategy is proposed to build GelMA-based microfluidic chips, combining GelMA hydrogel and traditional microfluidic chips. It is believed that this protocol is a significant summary of the GelMA bioprinting strategies used in our lab and may inspire other researchers in relative fields.

Protokół

1. Cell culturing

1. Prepare Dulbecco's modified Eagle medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin, used to culture human breast cancer cell (MDA-MB-231) lines and human umbilical vein endothelial cell (HUVEC) lines.
2. Prepare DMEM with L-glutamine (DMEM/F-12), supplemented with 10% FBS and 1% penicillin/streptomycin, used to culture bone marrow mesenchymal stem cell (BMSC) lines.
3. Set the culturing environment as 37 °C and 5% CO₂. Culture MDA-MB-231, HUVEC, and BMSC, and passage the cells in a 1:2 ratio when 90% confluence is reached.

2. Fabrication of GelMA microspheres

1. Print the fixture as Figure 1A with polylactic acid (PLA) on a fused deposition modeling (FDM) printer. Place two metal ring electrodes in the fixture.
2. Connect the two metal ring electrodes with ground and positive poles, respectively. Place the metal plate connected with the high voltage below the ring electrode and place a Petri dish with silicon oil on the metal plate as a droplet receiver.
3. Dissolve freeze-dried GelMA (5% w/v) and lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP, 0.5% w/v) in Dulbecco's phosphate-buffered saline (DPBS) as the bioink (10 mL). Filter the bioink through a 0.22 μm filter for sterility and heat it in a 37 °C water bath for 15 min.
4. Detach MDA-MB-231 cells with 3 mL of 0.25% trypsin-0.02% EDTA solution for 3 min at 37 °C. Centrifuge cells in a 15 mL centrifugal tube at 100 x g for 5 min to obtain a cell pellet.
5. Remove the supernatant. Mix the cell pellet with 1 mL of prepared bioink by slowly pipetting it to prevent the production of bubbles.
6. Place 1 mL of bioink (MDA-MB-231) into a 3 mL sterile syringe. Feed the bioink by the force of compressed air (~0.5 kPa). Put the syringe on the fixture.
   NOTE: The printing environment should be strictly controlled at a temperature of 30 °C and humidity of 50%.
7. Switch on the high voltage power and set the voltage as 0−4 kV. Simultaneously, turn on the 405 nm wavelength light to crosslink the GelMA droplets in 5 mL of silicon oil.
8. Pour the most part of the silicon oil away by decanting the Petri dish. Transfer the remained silicon oil and the GelMA microspheres into a 15 mL centrifugal tube using a spoon.
9. Add 5 mL of DPBS and shake the mixture uniformly. Centrifuge the tube at 100 x g for 5 min and remove the supernatant fluid.
10. Repeat step 2.9 3x.
11. Take out the GelMA microspheres with a spoon and culture them in DMEM in a Petri dish at 37 °C and 5% CO₂ for 3 days.
12. Discard the medium and wash the microspheres with DPBS. Fix with 2 mL of 4% paraformaldehyde (PFA) for 30 min at room temperature (RT).
13. Discard the PFA and wash the microspheres with DPBS. Permeabilize with 2 mL of 0.5% nonionic surfactant (i.e., Triton X-100) for 5 min at RT.
14. Discard the nonionic surfactant and wash the microspheres with DPBS. Stain them with 2 mL of tetramethylrhodamine (TRITC) phalloidin for 30 min in darkness at RT.
15. Discard the TRITC and wash the microspheres with DPBS. Stain them with 2 mL of 4–,6-diamidino-2-phenylindole (DAPI) for 10 min in darkness at RT.
16. Discard the DAPI and wash the microspheres with DPBS. Capture the morphology with a confocal fluorescence microscope.

3. Fabrication of GelMA fibers

1. Prepare a coaxial nozzle as shown in Figure 2A. Fix an inner nozzle (25 G, OD = 510 μm, ID = 250 μm) and outer nozzle (18 G, OD = 1200 μm, ID = 900 μm) with soldering. Connect a glass tube (length = 50 mm, inside diameter = 1.2 mm) to the end of the coaxial nozzle.
2. Dissolve sodium alginate (Na-Alg) powder that is sterilized under ultraviolet (UV) light for 30 min in deionized water at 2% (w/v).
3. Prepare a sterile bioink solution following step 2.3. Heat the GelMA bioink and Na-Alg solution in a 37 °C water bath for 15 min.
4. Detach BMSCs cells with 3 mL of 0.25% trypsin-0.02% EDTA solution for 3 min at 37 °C. Centrifuge cells in a 15 mL centrifugal tube at 100 x g for 5 min to obtain a cell pellet.
5. Remove the supernatant fluid. Mix the cell pellet with 2 mL of prepared GelMA bioink by slowly pipetting it to prevent the production of bubbles.
6. Place 2 mL of bioink (BMSCs) into a 10 mL syringe. Place 2 mL of Na-Alg solution into another syringe (10 mL). Feed them with two syringe pumps, respectively (here, bioink at 50 μm/min and Na-Alg solution at 350 μm/min).
   NOTE: The printing environment should be strictly controlled at a temperature of 30 °C and humidity of 50%.
7. Turn on the 405 nm wavelength light to irradiate the transparent tube to crosslink the GelMA fibers. Use a Petri dish with DPBS to receive the fibers.
8. Take out the GelMA fibers with a spoon from DPBS and culture them for 3 days in the prepared DMEM/F-12 in 37 °C and 5% CO₂.
9. Follow steps 2.12−2.16 to prepare the GelMA fibers for morphological observation with a confocal fluorescence microscope.

4. Fabrication of complex 3D GelMA structures

NOTE: Figure 3A shows the fabrication sketch of the complex 3D GelMA structures.

1. Wipe the DLP bioprinter (Figure 3E) with 75% alcohol and expose it to the UV irradiation for 30 min for sterility.
2. Dissolve the freeze-dried GelMA (10% w/v) and LAP (0.5% w/v) in DPBS. Add magenta edible pigment into the solution (3% v/v) to improve the printing accuracy.
3. Filter the solution through a 0.22 μm filter for sterility and heat it in a 37 °C water bath for 15 min.
4. Build the 3D models with computer-aided design (CAD) software. Import the model documents to the upper software (EFL) of the applied DLP bioprinter.
5. Add 10 mL of prepared bioink into the trough of the DLP bioprinter.
6. Set the printing parameters in the upper software as follows: light intensity = 12 mW/cm², irradiation duration = 30 s, and slice height = 100 μm. Start printing.
7. Remove the printed structure from the bioprinter and immerse it in DPBS in a Petri dish.
8. Detach the MDA-MB-231s cells with 3 mL of 0.25% trypsin-0.02% EDTA solution for 3 min at 37 °C. Centrifuge cells at 100 x g for 5 min in a 15 mL tube to obtain a cell pellet.
9. Remove the supernatant fluid and mix the cell pellet with 2 mL of DMEM.
10. Add 100 μL of cell suspension on the printed structures. Culture them for 3 days in the prepared DMEM at 37 °C and 5% CO₂.
11. Follow steps 2.12−2.16 to prepare the complex 3D structures for morphological observation with a confocal fluorescence microscope.

5. Fabrication of GelMA-based microfluidic chips

NOTE: Figure 4A shows the fabrication sketch of the GelMA-based microfluidic chip.

1. Dissolve the freeze-dried GelMA 10% (w/v) and LAP (0.5% w/v) in DPBS. Filter the GelMA solution through a 0.22 μm filter for sterility.
2. Sterilize the gelatin powder under UV light for 30 min and add it to the GelMA-LAP solution prepared in step 5.1 to a final concentration of gelatin of 5% (w/v). Heat the mixture in a 37 °C water bath for 15 min.
3. Design a group of molds (Figure 4B,C) with CAD software and manufacture them with photopolymer resin on a DLP printer.
4. Fill the molds fully with the prepared bioink.
5. Put the molds into a 4 °C refrigerator to crosslink the gelatin for 30 min.
6. Remove the molds and demold with a blade the partially (physically) crosslinked hydrogel sheets from the molds.
7. Combine the two demolded hydrogel sheets and bond them with the help of GelMA by irradiating at 405 nm for 1 min.
8. Detach the HUVECs cells with 3 mL of 0.25% trypsin-0.02% EDTA solution for 3 min at 37 °C. Centrifuge cells in a 15 mL centrifugal tube to obtain a cell pellet at 100 x g for 5 min.
9. Remove the supernatant fluid and mix the cell pellet with 2 mL of DMEM.
10. Fill the microchannel fully by injecting the cell suspension with a nozzle and syringe.
11. Flip the chip upside down every 15 min during the next 3 h to achieve uniform and complete cell seeding. Culture the chips in the Petri dish for 3 days in the prepared DMEM at 37 °C and 5% CO₂.
12. Follow steps 2.12−2.16 to prepare the microfluidic chips for morphological observation with a confocal fluorescence microscope.

Wyniki

During the fabrication of GelMA microspheres, the GelMA droplets were separated by the external electric field force. When the droplets fell into the receiving silicon oil, they remained standard spheroid shape without tails. This is because the GelMA droplets were in an aqueous phase, while the silicon oil was in an oil phase. The surface tension that formed between the two phases caused the GelMA droplets to maintain a standard spheroid shape. In terms of the cell-laden microspheres, cells experienced the high voltage ...

Dyskusje

This article describes several strategies to fabricate GelMA 3D structures, namely GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips. GelMA has promising biocompatibility and formation capability and is widely used in the field of biofabrication. Microsphere structures are suitable for controlled drug release, tissue culturing, and injection into organisms for further therapy^21,22,23<...

Materiały

Name	Company	Catalog Number	Comments
0.22 μm filter membrane	Millipore
2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI)	Yeasen Biological Technology Co., Ltd., Shanghai, China
3D bioprinter	SuZhou Intelligent Manufacturing Research Institute, SuZhou, China
405nm wavelength light	SuZhou Intelligent Manufacturing Research Institute, SuZhou, China
co-axial nozzle	SuZhou Intelligent Manufacturing Research Institute, SuZhou, China
confocal fluorescence microscope	OLYMPUS FV3000
digital light processing (DLP) bioprinter	SuZhou Intelligent Manufacturing Research Institute, SuZhou, China
DLP printer	SuZhou Intelligent Manufacturing Research Institute, SuZhou, China
Dulbecco's Phosphate Buffered Saline (DPBS)	Tangpu Biological Technology Co., Ltd., Hangzhou, China
Dulbecco's Modified Eagle Medium (DMEM)	Tangpu Biological Technology Co., Ltd., Hangzhou, China
Dulbecco's Modified Eagle Medium with L-glutamine (DMEM/F-12)	Tangpu Biological Technology Co., Ltd., Hangzhou, China
EFL Software	SuZhou Intelligent Manufacturing Research Institute, SuZhou, China
fetal bovine serum (FBS)	Tangpu Biological Technology Co., Ltd., Hangzhou, China
gelatin	Sigma-Aldrich, Shanghai, China
gelatin methacryloyl (GelMA)	SuZhou Intelligent Manufacturing Research Institute, SuZhou, China
high voltage power	SuZhou Intelligent Manufacturing Research Institute, SuZhou, China
lithium phenyl-2, 4, 6-trimethylbenzoylphosphinate (LAP)	SuZhou Intelligent Manufacturing Research Institute, SuZhou, China
paraformaldehyde	Tangpu Biological Technology Co., Ltd., Hangzhou, China
penicillin/streptomycin	Tangpu Biological Technology Co., Ltd., Hangzhou, China
sodium alginate (Na-Alg)	Sigma-Aldrich, Shanghai, China
TRITC phalloidin	Yeasen Biological Technology Co., Ltd., Shanghai, China
Triton X-100	Solarbio Co., Ltd., Shanghai, China