It supports cell–cell and cell–matrix interaction, cell attachment, and proliferation, resulting in rapid and reliable drug screening models. We have generated an ovarian cancer spheroid in interconnected porous scaffolds. The scaffold is fabricated using low-temperature synthesized graphene, cellulose acetate, and sodium alginate. Graphene nanosheets enhance cell proliferation and aggregation, which aids in the formation of cancer spheroids. The spheroids are assessed after day 7 and 14 for the generation of reactive oxygen species (ROS), expression of the hypoxia inducing factor (HIF-1⍺) and vascular endothelial growth factor (VEGF). Production of ROS was observed due to the aggregated tumor mass, and enhanced production of HIF-1⍺ and VEGF results from a lack of oxygen and nutrition. Furthermore, the efficacy of anticancer drug doxorubicin at varying concentrations is assessed on ovarian cancer spheroids by studying the expression of caspase-3/7 at day 7 and 14. The current findings imply that the graphene-cellulose-alginate (GCA) scaffold generates a reliable ovarian cancer spheroid model to test the efficacy of the anticancer drug. 3D cell culture can mimic tumor pathophysiology, which reflects cellular morphology and heterogeneity, strongly influencing gene expression, cell behavior, and intracellular signaling. However, only around 50% of patients benefit from the treatment with adverse side effects,3 consequently demanding an effective alternative. Developing new therapeutics is challenging, expensive, and time-consuming, as many drugs fail in clinical trials,4,5 due to the lack of in vitro models that mimic in vivo conditions. Therefore, a reliable primary drug screening model is required to investigate the effectiveness of novel therapies.In incidence and diagnosis, ovarian cancer is the eighth most common gynecological cancer worldwide.6,7Globally, researchers have been employing three-dimensional (3D) cell culture platforms for drug screening as they provide the advantage of cell–cell and cell–matrix interaction. It represents the physiological properties of the cell: cellular morphology and heterogeneity, which strongly influence gene expression, cell behavior, and intracellular signaling.8 The interconnectivity of pores and high surface area with macro-and microstructural features influence cell survival, signaling, growth, and motility. The scaffold's mechanical stiffness helps maintain the adhered tumor's phenotype and aggressiveness, influencing the intercellular organization and metastasis ability.9 Furthermore, the spheroid mimics the pathophysiology of the in vivo tumor and provides a microenvironment to influence its behavior.10Lately, porous scaffold-based methods to generate tumor spheroids have been used. Porous scaffolds are vital in cell adhesion, proliferation, and migration, as they allow cells to form aggregates and produce the extracellular matrix (ECM).et al. developed a thiol–acrylate hydrogel scaffold to generate breast cancer spheroids for high throughput drug screening and endocrine response monitoring.11 Obayemi et al. fabricated microporous paclitaxel loaded PLGA-PEG and PLGA-PCL scaffolds by a particulate leaching process to treat triple-negative breast cancer.12 Xu et al. demonstrated a chitosan-chondroitin sulphate 3D porous scaffold to promote epithelial to mesenchymal transition in prostate cancer13 Yang et al. fabricated an alginate-gelatin 3D scaffold to generate cancer spheroids and tumoroids.14 Le et al. demonstrated the effect of a chitosan–alginate porous scaffold on the proliferation and growth of MDA-MB-231 cells.15 Wu et al. developed a microfluidic-based vessel-assisted heterotypic tumor model for preclinical drug screening.16Khan 17–19 The hydrophilic nature of graphene permits interaction with extracellular matrix (ECM) components through hydrogen bonds and electrostatic and covalent interactions, enhancing cell adherence.20 Additionally, it is highly adherent to the phospholipid bilayer and polar head groups of the outermost layer of the cell membrane.21 Moreover, graphene is carbon-based and less toxic to cells and tissues and, thus, serves as an attractive biocompatible material for tissue engineering and developing regenerative medicine.22Herein, we report the generation of ovarian cancer spheroids in a graphene-cellulose acetate-sodium alginate (GCA) scaffold followed by assessment of drug efficacy. Graphene exhibits unique physicochemical properties such as small size and high surface area, aiding cell attachment and increasing cell growth.23 and differentiation24 that supports cell attachment. Chen et al. investigated the proliferation and differentiation of induced pluripotent stem cells (iPSCs) on graphene and graphene oxide-based surfaces.25 Pilato et al. fabricated a graphene oxide-based 3D porous scaffold for cardiac tissue engineering.26Recently, 3D graphene foams have been used as a scaffold for a neural stem cell culture27,28 Furthermore, cellulose acetate is biologically inert and forms nanofibers with surface porosity, promoting cell adhesion, cellular migration, infiltration, tissue ingrowth, and vascularization.29Sodium alginate is a naturally derived and biocompatible polymer widely used to culture different types of cells.30,31 The GCA scaffold is fabricated by varying graphene concentrations and assessed for spheroidal structure generation, cell adhesion, viability, and proliferation. Furthermore, the ovarian cancer spheroids generated in the porous GCA scaffold are subjected to doxorubicin. The spheroid is assessed for expression of early apoptotic cell death marker caspase-3/7.Analytical, morphological, and biological studies were used to characterize high-quality graphene synthesized at low temperature by a modified Hummer's method.32 The interconnectivity of the pores in the scaffold aids in the perfusion of nutrients and oxygen, replicating native tissue vasculature.33 3D scaffolds promote the generation of cancer spheroids through cellular aggregates from micrometers to millimeters and offer a better cancer drug screening model than 2D in vitro cell culture.34Polymer-based 3D scaffolds offer high water-holding capacity due to their porous structures. The flexibility and stiffness of the scaffold support cell–cell and cell–matrix interaction by secretion of the extracellular matrix. Furthermore, it permits the transfer of nutrients and the removal of waste products from tissues without disturbing the culture conditions.35 3D printing,36 and drop-casting.16 In the present work, cellulose acetate and sodium alginate are drop cast to form a cellulose-alginate (CA) scaffold. The drop-casting technique provides ease of fabrication, aiding in uniformity over the size and thickness of the scaffold.Different fabrication methods are used to fabricate the scaffolds, including electrospinning, freeze-drying,B is calculated as a free parameter with a value of 1.09.37,38The thickness of the fabricated CA scaffolds is 0.5 mm, and the scanning electron micrograph  shows anFurthermore, NIH 3T3 cells are grown on the scaffold, and their growth is assessed by confocal microscopy after day 3 and 7 refer to . An incrGraphene is synthesized at low temperature using a modified Hummer's method. Briefly, graphite is reduced to graphitic oxide, followed by a further reduction to reduced graphene oxide (rGO) to obtain high-quality graphene. The product at every synthesis step is characterized structurally and analytically using scanning electron microscopy (SEM), atomic force microscopy (AFM), Raman spectroscopy, Fourier transform infrared (FTIR) spectroscopy, ultraviolet-visible (UV-Vis) spectroscopy, X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS) techniques.Graphite, rGO, and graphene structures are shown in the SEM images . Overlap39,40AFM study of the surface topology is conducted to verify the SEM results. There is no evidence of separation between the sheets of carbon in the graphite image obtained using an AFM . rGO demPreliminary assessments of the precursors graphite, reduced graphene oxide, and graphene are performed using UV-Vis, FTIR, and XRD  are carried out to assess the chemical composition accurately and, to prove the quality of the synthesized graphene. The Raman peak for the 2D band for graphite is observed at 2687.2 cmrefer to . Graphengraphene . The for2 hybridized carbon. The binding energy of the C–OOR bond is observed at 288.9 eV, while the C–O and C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O bonds are seen from 286–287 eV. The deconvoluted peaks of graphite, reduced graphene oxide, and graphene show varying concentrations of these groups. Graphite shows predominantly sp2 hybridized C–C bonds and a shake-up peak at 289.02 eV. The residual oxygen content of rGO is seen to be reduced during the heating process to obtain graphene. Additionally, a sample containing many sp2 carbons will have an asymmetric tail in the direction of increased binding energy for the C1s spectrum.42 This is clear in the graphene spectrum, which attests to its excellent quality. A similar result is obtained by EDAX analysis (refer to Table S1−1 to 1000 μg mL−1) of graphene and rGO. The results  up to 7 days are shown in GCA scaffolds with various graphene concentrations are evaluated qualitatively for cell proliferation, adhesion, and aggregation. Confocal images of NIH 3T3 cells cultured on GCA scaffolds  cell line. A panel of the cell growth assay  demonstr44 Spheroids are treated with the caspase-3/7 green detection dye after 24 h to assess the expression of early apoptotic markers. Caspase-3/7 are expressed in ovarian cancer spheroids, along with inhibition of tumor mass and fragmentation of nuclei as can be seen in After validating the tumor spheroids and their characteristic properties, such as reactive oxygen species (ROS) generation and expression of HIF-1⍺ and VEGF, they are treated with doxorubicin at 0.1, 1, and 5 μM.in vivo physiological conditions could help better translate therapeutics to avoid drug failure in clinical trials. Consequently, 3D scaffolds have been widely used in investigating cancer therapeutics as they increase cell adhesion and proliferation.45 In this study, a cellulose acetate and sodium alginate porous scaffold is cast, characterized, and assessed for cell adherence and growth on NIH 3T3 cells. Even though the scaffold is porous with interconnected pores  using a modified Hummer's method.M images .30,31 AF quality .3030,31 The well-defined nanosheets and higher degree of freedom are responsible for the smooth peaks obtained from the FTIR spectrum  cellulose acetate sodium alginate (GCA) scaffolds show porosity and tortuosity of 0.91 and 1.21 respectively. The higher values, compared to the CA scaffold, indicate an increase in surface area and interconnection of pores. Furthermore, GCA scaffolds with 5 μg mL−1 graphene show increased cell growth with cellular aggregates for NIH 3T3 cells, indicating spheroidal formation , 1% calcium chloride (HiMedia), and 10% cellulose acetate with average molecular weight 100 000 (Acros Organics) in a ratio of 5 : 1 : 10 followed by drop casting 1 mL of the mixture on a cover slip. The scaffolds are allowed to polymerize overnight at room temperature.Scaffolds are fabricated by mixing 1% sodium alginate M.W. 749 g mol3 (HiMedia) are added to 90 mL H2SO4 (98%) (SDFCL) and stirred for 30 min in an ice bath. 10 g KMnO4  is added slowly and stirred at 50 °C for 2 h, then 200 mL deionised (DI) water and 12 mL H2O2 (Sigma Aldrich USA) are gradually added until the solution cools to room temperature. The solution is centrifuged at 5000 rpm for 60 min, and the supernatant is decanted. The product is washed with DI water and centrifuged repeatedly until the pH turns neutral (7.0). Thus, the graphitic oxide is dissolved in DI water and ultrasonicated for 1 h to yield graphene oxide. The suspension is centrifuged at 5000 rpm for 60 min, and the supernatant is decanted. Graphene oxide is mixed with DI water, followed by NaBH4 (LOBA CHEMIE) addition, and further reduced with 10% HCl (HiMedia). The reduced graphene oxide (rGO) is washed with DI water and purified by repeated centrifugation for 60 min at 5000 rpm, followed by lyophilization to obtain a powder. The rGO powder is heated until flashpoint (120 °C) to obtain pure graphene.2 g graphite (Sisco Research Laboratory (SRL) and 2 g NaNO−1). It is added to the CA scaffold to obtain the GCA scaffold.Graphene powder obtained by a modified Hummer's method at low temperature is dissolved in DI water to create different concentrations  EVO MA15, Zeiss (Germany). An atomic force microscope (AFM) from Nanonics, Israel, is used to analyze the surface of graphene. Raman spectra are obtained using a Renishaw Raman spectrometer, and an ESCA-3000 XPS is used to assess the purity of graphene. An X-ray diffractogram (XRD) is recorded to analyze the crystallinity, and Fourier transform infrared spectroscopy (FTIR) is used for bonding. UV-Vis spectroscopic data is assessed to deduce the interaction of graphite, rGO, and graphene with light.4 cells per well are seeded in 96 well plates and allowed to grow for 24 h, followed by treatment with 1 μg mL−1 to 1000 μg mL−1 graphene. After treatment, 2 mg mL−1 of 3--2,5-diphenyltetrazolium bromide (MTT) (Thermofisher Scientific) is added to each well and incubated at 37 °C for 4 h. 200 μL of DMSO is added and absorbance is recorded at 512 nm using a plate reader.Graphene is assessed for cytotoxicity by toxicity assay (MTT) using NIH 3T3 (mouse fibroblast cells). 1 × 106 NIH 3T3 cells are seeded on scaffolds and allowed to grow. On days 3 and 7, scaffolds are washed with phosphate buffered saline (PBS), fixed with 4% paraformaldehyde, and permeabilized with 0.5% Triton X-100 (Sigma Aldrich USA). Cell nuclei are stained with Hoechst 33342, and the cytoskeleton with rhodamine-phalloidin (Invitrogen USA) and visualized under a confocal microscope .NIH 3T3 cells  repository, Pune, India) are grown in Dulbecco's modified Eagle’s medium (DMEM), 10% fetal bovine serum (FBS), and 1% antibiotic antimycotic (Invitrogen USA). CA and GCA scaffolds are washed with 70% ethanol, followed by washing with double distilled water twice and autoclaved. Furthermore, 1 × 106 PA-1 cells are seeded on the scaffolds, and the growth of ovarian cancer spheroids is assessed. The ovarian cancer spheroids are observed for the generation of reactive oxygen species (ROS) and expression of the hypoxia-inducing factor (HIF-1⍺) and vascular endothelial growth factor (VEGF) on days 7 and 14. The formation of ROS is checked by labeling the spheroids with a CellROX green reagent (Invitrogen USA). Furthermore, the expression of HIF-1⍺ is checked by fixing the spheroids with 4% paraformaldehyde and permeabilized with 0.5% Triton-X-100 (Sigma Aldrich USA). The cells are blocked with 2% bovine serum albumin (BSA) , followed by treatment with HIF-1⍺ recombinant rabbit monoclonal primary antibody  in PBST , and labeled with donkey anti-rabbit IgG (H + L) highly cross-adsorbed secondary antibody, Alexa Fluor™ Plus (Invitrogen USA). Similarly, the expression of VEGF is checked by labeling VEGF monoclonal antibody (Invitrogen USA) and rabbit anti-mouse IgG (H + L) cross-adsorbed secondary antibody, Alexa Fluor™ 488 (Invitrogen USA).To generate ovarian cancer spheroids, PA-1 (ovarian teratocarcinoma) cells  repository, Pune) are maintained in minimum essential medium (MEM) with 10% FBS and 1% antibiotic antimycotic (Invitrogen USA). 1 × 1051 A control group is maintained under similar experimental conditions as compared to the test group; however, the control group is not treated with drugs. The expression of the early apoptotic marker is checked by staining the spheroids with a CellEvent™ caspase-3/7 green detection kit (Invitrogen USA) and observed under the confocal microscope.Spheroids of PA-1 grown on GCA scaffolds are tested for efficacy of the anticancer drug doxorubicin after day 7 and 14. The spheroids are treated with 0.1, 1, and 5 μM doxorubicin hydrochloride (European Pharmacopoeia Reference standard) for 24 h.in vivo tumor microenvironment by expressing ROS, HIF-1⍺, and VEGF. The ovarian cancer spheroids are subjected to different concentrations of doxorubicin and assessed after days 7 and 14. The drug treatment results show the expression of caspase-3/7, an early apoptotic marker, indicating the apoptotic nature of cell death. Thus, the GCA scaffold is an excellent substrate for cellular adhesion, proliferation, spheroid formation, and drug screening. Further developments can lead to the generation of cancer spheroids with tumor properties and serve as an alternative tool to rapid and high throughput drug screening platforms. Adding perfusion and microfluidics to the spheroid model will enable a close mimicry of in vivo conditions.This paper reports the fabrication of a GCA scaffold for the growth of ovarian cancer spheroids. A low-temperature modified Hummer's synthesis method yields high-quality and non-toxic graphene nanosheets. The GCA scaffold shows a porosity and tortuosity of 0.91 and 1.21, respectively, indicating its porous nature with interconnected pores. Due to a higher surface area, enhanced animal cell growth and adhesion are obtained using graphene nanosheets. Furthermore, graphene plays a crucial role in forming cellular aggregates. The spheroid formation closely resembles the tumor morphology and characteristic features of the Dhananjay Bodas conceptualized, designed, and supervised the work and contributed to data analysis. Pooja Suryavanshi fabricated and characterized the graphene-cellulose-alginate (GCA) scaffold, generated ovarian cancer spheroids, characterized the structures, and performed studies with the drug. Yohaan Kudtarkar synthesized graphene and characterized the product using morphological and analytical techniques. Mangesh Chaudhari was involved with the graphene characterization. The manuscript was written and revised with the contributions of all authors. All authors have approved the final version of the manuscript.The author declares no conflict of interest.NA-005-D3NA00420A-s001