Three‐dimensional cryogel matrix for spheroid formation and anti‐cancer drug screening

Abstract

Spheroid-based systems have been developed as alternatives to two-dimensional (2D) monolayer cultures for understanding 3D cell behavior and conducting in vitro drug screening tests. However, spheroids are easily disrupted while handling and do not mimic the presence of extracellular matrix (ECM) components. To address that, we have developed a cost-effective, polyethylene glycol diacrylate (PEGDA), and gelatin methacryloyl (GELMA) based semi-synthetic cryogel matrix system, which can be used to grow spheroids and conduct studies while providing architectural support and mimicking in vivo ECM components. These matrices are macroporous and support formation of tumor-like spheroids of breast tumor epithelial (MCF-7) cells in the absence of additional growth factors otherwise required for spheroid formation. Difference in morphology of cells as a function of matrix composition and increase in size and number of spheroids as a function of time was observed. Spheroids grown in cryogel matrices showed more drug resistance than their 2D counterparts, which can partially be explained by the epithelial to mesenchymal transition (EMT) observed in spheroids. We believe that spheroids formed through these PEGDA-GELMA cryogel matrices better represent in vivo pathological conditions and can help develop costeffective in vitro assays for screening new pharmacological drug candidates and performing cell mechanistic studies. K E Y W O R D S 3D cell culture, breast cancer cells, cryogels or scaffolds, polyethylene glycol diacrylate, gelatin methacryloyl, spheroids PEG (molecular weight 4,000 Mw), anhydrous dichloromethane (DCM), anhydrous magnesium sulfate (MgSO 4 ), potassium carbonate (K 2 CO 3 ), diethyl ether, and toluene were purchased from Merck (India). Acryloyl chloride and methacrylic anhydride were purchased from Alfa Aesar. 2-Hydroxy-4 0 -(2-hydroxyethoxy)-2-methylpropiophenone and Paclitaxel were purchased from Sigma Aldrich, triethylamine (TEA), tetramethylethylenediamine (TEMED), and dialysis tubing (12 kDa MWCO) were purchased from HiMedia lab (India) and ammonium persulfate (APS) was purchased from Sisco Research Laboratories (SRL) India. Gelatin was a gift from Nitta Gelatin, India and Calcein-AM, DAPI, Phalloidin, Propidium Iodide, and trizol were purchased from Invitrogen. Dulbecco's Phosphate Buffer Saline (PBS), Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum (FBS), and the antibiotic-antimycotic solution were purchased from HiMedia, India. EMT antibody sampler kit and Ki-67 antibody were purchased from Cell Signaling Technology and Brilliant II SYBR Green qPCR Master Mix and cDNA synthesis kit were purchased from Agilent Technologies and growth factor reduced Matrigel was purchased from BD Corning. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and bovine serum albumin (BSA) was purchased from Himedia Lab (India). | Cryogel preparationPEGDA and GELMA were synthesized and characterized as reported earlier in our previous work (Shrimali et al., 2018). Briefly, PEGDA was synthe...

Design and Assessment of Biodegradable Macroporous Cryogels as Advanced Tissue Engineering and Drug Carrying Materials

“…Significant degradation of PEGDA hydrogels occurred over 12 weeks In Vivo as a result of hydrolysis of the PEGDA end groups esters. PEG cryogels were synthesized by radical polymerization of PEGDA using APS/TEMED [ 128 , 129 , 130 ] or via UV radiation [ 112 , 131 ]. The morphology and swelling of the PEG cryogels were controlled by the freezing temperature and initiator concentration [ 58 ] and depended on the PEGDA concentration [ 131 ].…”

Mimicking tumor hypoxia and tumor-immune interactions employing three-dimensional in vitro models

“…To address these issues, 3D cell culture models are a reliable alternative, providing experimentally accessible human models to study the biological processes of cancer. Several 3D culture platforms such as spheroids, organoids, hydrogels, 3D scaffolds, 3D bio-printing, and microfluidics have attempted the recreation of certain aspects of tumor microenvironments present in tissues including the brain [30][31][32][33][34][35][36], breast [37][38][39][40][41][42][43], ovarian [44][45][46][47][48][49][50][51], bone [52][53][54][55][56][57][58], liver [59][60][61][62][63][64][65], lung [66][67][68][69][70][71][72], colon [73][74][75][76]…”

Semi‐interpenetrating polymer network cryogels based on poly(ethylene glycol) diacrylate and collagen as potential off‐the‐shelf platforms for cancer cell research

“…Along with their use as scaffolds for tissue regeneration, 18,[20][21][22] cryogels are also being increasingly explored to develop 3D functional tumor models in vitro. [23][24][25][26] Since cancer cell growth is modulated by the stiffness of the surrounding matrix, with cancerous tissues being generally stiffer than non-malignant tissues, 23,27 cryogels may indeed be useful to mimic the strength and the elasticity of tumors, thus allowing to analyze the biomechanical cues involved in cell-matrix interactions and pathogenesis.…”

“…), which suffer from large batch-to-batch variability and need particular handling care, thus making experimental reproducibility harder to achieve. In this scenario, PEG-based hydrogels 32,[37][38][39][40][41][42][43] and cryogels 23,24,26 appear as powerful alternatives to conventional organotypic culture models for the production of reproducible tumor tissue equivalents.…”

“…Firstly, we verified the feasibility of synthesizing the semi-IPN cryogels by means of fast ultraviolet (UV) irradiation. While the synthesis of PEGDA cryogels is commonly obtained through the use of the ammonium persulfate/tetramethylethylenediamine (APS/TEMED) redox initiating system, 23,25,26 we recently reported the faster synthesis of PEGDA cryogels by means of UV exposure, showing that the PEGDA concentration has a significant effect on the cryogel pore size and stiffness. 44 Here, we further tested whether the addition of low amounts of collagen (up to 1% w/v) to a fixed PEGDA concentration (10% w/v) could allow the formation of tunable semi-IPN cryogels.…”