Semi-Synthetic Click-Gelatin Hydrogels as Tunable Platforms for 3D Cancer Cell Culture

Abstract

Basement membrane extracts (BME) derived from Engelbreth–Holm–Swarm (EHS) mouse sarcomas such as Matrigel® remain the gold standard extracellular matrix (ECM) for three-dimensional (3D) cell culture in cancer research. Yet, BMEs suffer from substantial batch-to-batch variation, ill-defined composition, and lack the ability for physichochemical manipulation. Here, we developed a novel 3D cell culture system based on thiolated gelatin (Gel-SH), an inexpensive and highly controlled raw material capable of forming hydrogels with a high level of biophysical control and cell-instructive bioactivity. We demonstrate the successful thiolation of gelatin raw materials to enable rapid covalent crosslinking upon mixing with a synthetic poly(ethylene glycol) (PEG)-based crosslinker. The mechanical properties of the resulting gelatin-based hydrogels were readily tuned by varying precursor material concentrations, with Young’s moduli ranging from ~2.5 to 5.8 kPa. All hydrogels of varying stiffnesses supported the viability and proliferation of MDA-MB-231 and MCF-7 breast cancer cell lines for 14 and 21 days of cell culture, respectively. Additionally, the gelatin-based hydrogels supported the growth, viability, and osteogenic differentiation of patient-derived preosteoblasts over 28 days of culture. Collectively, our data demonstrate that gelatin-based biomaterials provide an inexpensive and tunable 3D cell culture platform that may overcome the limitations of traditional BMEs.

GelMA, Click-Chemistry Gelatin and Bioprinted Polyethylene Glycol-Based Hydrogels as 3D Ex Vivo Drug Testing Platforms for Patient-Derived Breast Cancer Organoids

“…In this study, we assessed the ability of various hydrogel-based biomaterials to support the 3D organoid formation of patient-derived breast cancer (BCa) cells derived from patient tissue following tumor resection, surplus to pathology needs. The tumor pieces were successfully reduced to individual cells, according to established protocols [ 40 ]. Relying on tissue mincing, enzymatic digestion and mechanical agitation, cells were then cultured and expanded in 2D ( Figure 1 A) using a suitable media validated for human BCa-derived organoids [ 41 ].…”

“…Patients consented use of their tissues following admission for breast cancer surgery at the breast cancer unit either at the Princess Alexandra Hospital or Mater Hospital, Brisbane, Australia. The study was performed in accordance with the principles of the Declaration of Helsinki and within ethical and institutional guidelines (ethics number HREC/2020/QRBW/61294 and HREC/17/MHS/50, approved 28 July 2017), and in accordance with the standards and protocols of the Centre for Personalised Analysis of Cancers (CPAC, Brisbane, Australia) and the Mater-Queensland University of Technology (QUT) Breast Cancer Research Biobank [ 40 ]. Four primary patient-derived breast cancer tumor materials were used in this study ( Table 1 ); three directly from primary breast cancer (BCa) patients undergoing surgery and one from a mouse xenograft.…”

“…Y-27632 (5 µM) was purchased from Abmole, Australia and Hydrocortisone (1 µg/mL) from Sigma, Macquarie Park, NSW, Australia. After one to two hours of digestion, the samples were filtered through pre-wet reversible 100 µm and 37 µm strainers (STEMCELL Technologies, Tullamarine, VIC, Australia) into a new 50 mL centrifuge tube to remove large insoluble material [ 40 ]. While undigested material was kept and cultured with ORM in T75 flasks (Corning ® , Mulgrave, VIC, Australia), the cell eluates obtained from the 37 µm strainers were diluted in Dulbecco’s Phosphate Buffered Saline (DPBS, Thermofisher) and used to assess cell viability upon digestion using Trypan Blue (Thermofisher) and automatic cell counter (Bio-Rad-TC20 TM , South Granville, NSW, Australia).…”

Mechanics of gelatin-based hydrogels during finite strain tension, compression and shear

“…Neat Ge hydrogels – 30 mL of a gelatin aqueous solution (10% w/v) was obtained by dissolving 3 g of Ge powder in water for 30 min at 45°C ( Portier et al, 2017 ). This concentration was chosen as an intermediate value based on previous studies dealing with gelatin-based hydrogels, reporting Ge concentrations within the range 2% w/v up to 20% w/v ( Nichol et al, 2010 ; Bode et al, 2011 ; Farris et al, 2011 ; Pan et al, 2014 ; Poursamar et al, 2016 ; Goodarzi et al, 2019 ; Hipwood et al, 2022 ). Ultrapure water (18.2 MΩ) was used to minimize the non-uniform physical bonding network caused by unbalanced ionic charge distribution ( Xing et al, 2014 ).…”

Organoids: The current status and biomedical applications

“…Therefore, significant efforts have been devoted to developing artificially synthesized ECM or hydrogels for organoid culture, which are chemically defined with clear components, and their chemical and physical features can be adjusted on the basis of different requirements. [326][327][328][329][330][331] The advantages of these novel hydrogels have been demonstrated. A nanofibrillar hydrogel proposed by Prince et al 332 could support the initiation and growth of breast tumoroids and protect the PDOs from murine contamination.…”