Functionalization, preparation and use of cell-laden gelatin methacryloyl–based hydrogels as modular tissue culture platforms

Journal name:
Nature Protocols
Volume:
11,
Pages:
727–746
Year published:
DOI:
doi:10.1038/nprot.2016.037
Published online

Abstract

Progress in advancing a system-level understanding of the complexity of human tissue development and regeneration is hampered by a lack of biological model systems that recapitulate key aspects of these processes in a physiological context. Hence, growing demand by cell biologists for organ-specific extracellular mimics has led to the development of a plethora of 3D cell culture assays based on natural and synthetic matrices. We developed a physiological microenvironment of semisynthetic origin, called gelatin methacryloyl (GelMA)-based hydrogels, which combine the biocompatibility of natural matrices with the reproducibility, stability and modularity of synthetic biomaterials. We describe here a step-by-step protocol for the preparation of the GelMA polymer, which takes 1–2 weeks to complete, and which can be used to prepare hydrogel-based 3D cell culture models for cancer and stem cell research, as well as for tissue engineering applications. We also describe quality control and validation procedures, including how to assess the degree of GelMA functionalization and mechanical properties, to ensure reproducibility in experimental and animal studies.

At a glance

Figures

  1. Overview of the GelMA-based hydrogel preparation protocol.
    Figure 1: Overview of the GelMA-based hydrogel preparation protocol.

    GelMA is dissolved in PBS at 37 °C and mixed with the photo-initiator to obtain the precursor solution. Cells, and optionally hyaluronic acid methacrylate (HAMA; indicated by a dashed line), are added to the precursor solution before casting it into a custom-made Teflon mold. The casting mold is then covered with glass slides and transferred into a UV cross-linker to enable the formation of the hydrogel by photo-polymerization. By using a custom-made cutting guide, the hydrogel stripes thus obtained are cut into constructs of equal size (4 mm × 4 mm × 2 mm), and can be used for biological assays and as a cell delivery vehicle for animal studies.

  2. GelMA-based hydrogel preparation and 3D cell culture.
    Figure 2: GelMA-based hydrogel preparation and 3D cell culture.

    The custom-made and sterilized Teflon mold is filled with cell-laden precursor solution under sterile conditions and covered with glass slides. After UV-initiated cross-linking, a custom-made cutting guide is required to cut the hydrogel strip into equal squares (4 mm × 4 mm × 2 mm), which are then transferred into culture medium to enable cell growth and spheroid formation. Bright-field microscopy indicates the appearance of spheroids after 14 d of 3D culture of ovarian cancer OV-MZ-6 cells, which can be visualized by confocal microscopy and maximal projections depicting cell nuclei (DAPI, blue) and F-actin (rhodamine 415–conjugated phalloidin, red).

  3. Application of GelMA-based hydrogels as cell delivery vehicles for an intraperitoneal animal model.
    Figure 3: Application of GelMA-based hydrogels as cell delivery vehicles for an intraperitoneal animal model.

    (a) Ovarian cancer OV-MZ-6 cells were transfected with a luciferase vector23 and encapsulated within GelMA-based hydrogels (5% (wt/vol) polymer concentration). After 2 weeks of 3D cell culture, bioluminescence signals (3.15 × 105 ± 9.32 × 104 photons/s/cm2/steradian; n = 12) of cell-laden GelMA-based hydrogels were confirmed before intraperitoneal implantation into NOD-SCID mice. (b) Bioluminescence imaging confirmed tumor growth 8 weeks after implantation (1.80 × 106 ± 1.70 × 106 photons/s/cm2/steradian; n = 6), and ex vivo bioluminescence imaging of the peritoneal organs indicated tumor spread. Human-derived tumor load was immunohistochemically detected by positive nuclear mitotic apparatus protein 1 (NuMA; Epitomics, cat. no. s2825, 1:100 dilution) staining, as shown by representative images from two different mice per group. (c) Four weeks after implantation, mice were treated with intraperitoneal paclitaxel injections (10 mg/kg, twice per week) over 4 weeks, leading to a decreased tumor load, as detected by bioluminescence imaging (6.59 × 105 ± 1.01 × 106 photons/s/cm2/steradian; n = 6). The effect of paclitaxel was also confirmed via ex vivo bioluminescent imaging, with only minor tumor spread within the peritoneum. Smaller tumors were formed upon paclitaxel treatment, as indicated by NuMA staining. The average radiance indicated a paclitaxel response of 63% (n = 6). All animal experiments conformed to Queensland University of Technology animal ethics approval.

  4. Application of GelMA-based hydrogels in a breast cancer bone colonization animal model.
    Figure 4: Application of GelMA-based hydrogels in a breast cancer bone colonization animal model.

    (a) Metastatic breast cancer MDA-MB-231BO (MDA-BO) and MDA-MB-231 (MDA) cells and nontumorigenic epithelial MCF10A cells were encapsulated within GelMA-based hydrogels (5% (wt/vol) polymer concentration). After 1 week of 3D cell culture, cell viability was assessed by confocal microscopy by live/dead staining (FDA, green; PI, red). After 2 weeks of 3D cell culture, cell proliferation was measured with an AlamarBlue assay (mean ± s.e.m.; n = 5). (b) Melt-electrospun scaffolds seeded with primary human osteoblastic cells were cultured over 8 weeks in vitro before subcutaneous implantation together with recombinant human bone morphogenetic protein 7 into NOD-SCID mice. After 8 weeks of ectopic bone formation in the humanized tissue-engineered bone, cell-laden hydrogels were implanted in close proximity to mimic invasion of the humanized bone by human breast cancer cells. Development of breast tumors in contact with the engineered bone was observed macroscopically (explant images) and microscopically (H&E staining) for both metastatic groups, but not for the control group. Human tumor and bone cells were detected immunohistochemically by positive NuMA (Epitomics, cat. no. s2825, 1:100 dilution) staining, as shown by representative images from each group. All animal experiments conformed to Queensland University of Technology animal ethics approval. AT, adipose tissue; BM, bone marrow; BV, blood vessel; CT, connective tissue; hTEBC, humanized tissue–engineered bone construct; NB, new bone; T, tumor.

  5. Formation of a humanized vascular network in GelMA-based hydrogels.
    Figure 5: Formation of a humanized vascular network in GelMA-based hydrogels.

    (a) Human ECFCs and MSCs were cocultured within GelMA-based hydrogels (5% (wt/vol) polymer concentration; DoF: 50%) at a 1:1 ratio, as indicated by confocal microscopy images of DsRed-ECFCs (left) and CMFDA-MSCs (right) after 6 d of coculture. (b) DsRed-ECFCs were imaged in the complete hydrogel construct by confocal microscopy, and a 2D projection (x-y plane) was collected along the z axis (left). A 3D reconstruction with a cross-section covering a thickness of 400 μm in the direction of the white arrowhead was acquired (right). (c) MSCs differentiated into perivascular cells surrounding the ECFC capillaries (left). A confocal microscopy image shows the spatial distribution of the DsRed-ECFC-lined capillaries surrounded by alpha smooth muscle actin–expressing (αSMA; Abcam, cat. no. ab9465, clone EA-53,1:200 dilution; anti-mouse Alexa Fluor 488, Life Technologies, cat. no. A-11001, 1:1,000 dilution) MSCs (right, top). The zoom-in view depicts details of a capillary and a cross-section image taken in the direction of the yellow arrowhead (right, bottom). (d) Confocal microscopy image showing that lumens were lined exclusively by DsRed-ECFC and surrounded by αSMA-expressing MSCs (yellow arrowhead); adapted and used with permission from Chen et al.40. Functional human vascular network generated in photo-cross-linkable gelatin methacrylate hydrogels, Adv. Funct. Mat. 22, 2027–2039 (2012), copyright Wiley-VCH Verlag GmbH & Co. KGaA.

  6. Microfluidic channels coated with GelMA-based hydrogels for use in a cardiomyocyte culture model.
    Figure 6: Microfluidic channels coated with GelMA-based hydrogels for use in a cardiomyocyte culture model.

    (a) Representation of the coating procedure, with cross-sections of a single microfluidic channel perpendicular to the direction of the flow (top) and channel cross-sections along the direction of the flow (bottom). GelMA pre-polymer (5% (wt/vol) polymer concentration; DoF: ~80%) is perfused through the microfluidic channel and exposed to UV-initiated cross-linking. Non-cross-linked prepolymer is then washed away with PBS. The channel can be subsequently seeded with cells and perfused with culture medium. PDMS, polydimethylsiloxane. (b) Confocal microscopy images of cardiomyocytes stained for F-actin (rhodamine 415–conjugated phalloidin, red) and cell nuclei (DAPI, blue) show an elongated morphology and alignment with the GelMA-coated microchannel on day 6 after seeding. (c) Confocal microscopy images show immunostaining of cardiomyocyte markers: troponin I (red, top; Abcam, cat. no. ab10231, clone 4C2, 1:200 dilution; anti-mouse Alexa Fluor 594, Life Technologies, cat. no. A-11005, 1:1,000 dilution), α-SMA (green; Abcam, cat. no. ab9465, clone EA-53, 1:200 dilution; anti-mouse Alexa Fluor 488, Life Technologies, cat. no. A-11001, 1:1,000 dilution) and connexin-43 (red, bottom; Abcam, cat. no. ab11370, 1:200 dilution; anti-rabbit Alexa Fluor 594, Life Technologies, cat. no. A-11012, 1:1,000 dilution) inside the GelMA-coated microchannel, with cell nuclei counterstaining (DAPI, blue); adapted with permission from Annabi et al.44. Scale bars, 50 μm.

  7. Effect of Teflon casting mold depth on mechanical and swelling properties of GelMA-based hydrogels.
    Figure 7: Effect of Teflon casting mold depth on mechanical and swelling properties of GelMA-based hydrogels.

    GelMA was dissolved to a final concentration of 10% (wt/vol) in PBS and photo-cross-linked in the presence of 0.05% (wt/vol) Irgacure 2959 by 15 min exposure to 365 nm light at an intensity of 2.6 mW/cm2 in a CL-1000 cross-linker (UVP). (a) Compressive moduli of cell-free GelMA-based hydrogels prepared in Teflon molds with either 1 or 2 mm depth were determined after incubation in PBS at 37 °C overnight (mean ± s.e.m.; n = 6), and they differ significantly (P < 0.001). (b) Effective hydrogel swelling was determined by weighing GelMA-based hydrogels immediately after cross-linking and again after swelling in PBS at 37 °C overnight and changes significantly (P < 0.001) with the casting mold depth. The difference in wet weights, before and after swelling, was expressed as a percentage (mean ± s.e.m.; n = 6).

  8. Hydrogel casting mould.
    Supplementary Fig. 1: Hydrogel casting mould.

    Technical drawing and dimensions for the custom-made Teflon casting mould. This mould produces hydrogel strips of 50 mm x 4 mm x 2 mm (length x width x height), which can be cut into smaller units using a cutting guide. All dimensions are in mm.

  9. Hydrogel cutting guide.
    Supplementary Fig. 2: Hydrogel cutting guide.

    Technical drawing and dimensions for the custom-made Teflon cutting guide. This guide can be used to cut hydrogel strips obtained after polymerization in the Teflon casting mould into smaller units of 4 mm x 4 mm x 2 mm (length x width x height). All dimensions are in mm.

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Author information

  1. These authors contributed equally to this work.

    • Daniela Loessner &
    • Christoph Meinert

Affiliations

  1. Queensland University of Technology (QUT), Brisbane, Queensland, Australia.

    • Daniela Loessner,
    • Christoph Meinert,
    • Elke Kaemmerer,
    • Laure C Martine,
    • Peter A Levett,
    • Travis J Klein,
    • Ferry P W Melchels &
    • Dietmar W Hutmacher
  2. Division of Biomedical Engineering, Department of Medicine, Biomaterials Innovation Research Center, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA.

    • Kan Yue &
    • Ali Khademhosseini
  3. Division of Health Sciences and Technology, Harvard-Massachusetts Institute of Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Kan Yue &
    • Ali Khademhosseini
  4. Department of Orthopaedics, University Medical Center Utrecht, Utrecht, the Netherlands.

    • Ferry P W Melchels
  5. Institute of Biological Chemistry, Biophysics and Bioengineering, School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh, UK.

    • Ferry P W Melchels
  6. Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts, USA.

    • Ali Khademhosseini
  7. Department of Bioindustrial Technologies, College of Animal Bioscience and Technology, Konkuk University, Hwayang-dong, Gwangjin-gu, Seoul, Republic of Korea.

    • Ali Khademhosseini
  8. Department of Physics, King Abdulaziz University, Jeddah, Saudi Arabia.

    • Ali Khademhosseini
  9. Australian Prostate Cancer Research Centre-Queensland, Translational Research Institute, Queensland University of Technology, Brisbane, Queensland, Australia.

    • Dietmar W Hutmacher
  10. George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, Georgia, USA.

    • Dietmar W Hutmacher
  11. Institute for Advanced Study, Technische Universität München, Munich, Germany.

    • Dietmar W Hutmacher

Contributions

D.L., C.M., E.K., A.K. and D.W.H. conceived and designed the experiments. D.L., C.M. and E.K. performed the experiments and analyzed the data. D.L., C.M. and D.W.H. wrote the manuscript; E.K. and K.Y. partially wrote the manuscript. L.C.M. performed the breast cancer bone colonization animal study. C.M., P.A.L. and T.J.K. established the cartilage tissue engineering. F.P.W.M. established the GelMA polymer production and physico-chemical characterization. A.K. and D.W.H. supervised this project. All authors read and critiqued the manuscript extensively.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: Hydrogel casting mould. (113 KB)

    Technical drawing and dimensions for the custom-made Teflon casting mould. This mould produces hydrogel strips of 50 mm x 4 mm x 2 mm (length x width x height), which can be cut into smaller units using a cutting guide. All dimensions are in mm.

  2. Supplementary Figure 2: Hydrogel cutting guide. (93 KB)

    Technical drawing and dimensions for the custom-made Teflon cutting guide. This guide can be used to cut hydrogel strips obtained after polymerization in the Teflon casting mould into smaller units of 4 mm x 4 mm x 2 mm (length x width x height). All dimensions are in mm.

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  1. Supplementary Text and Figures (324 KB)

    Supplementary Figures 1 and 2

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