Introduction
3D bioprinting is defined as functional 3D structures or artificial tissue models of cells and biocompatible materials. This technology has changed the field of organizational engineering, making it possible to create complex pre-defined design structures while ensuring repeatability. Several 3D bioprinting based methods have been reported for engineering various tissues, such as cartilage, 2-bone, 3-bone, and skin regeneration. This technology has also been used to create new preclinical tumor models. In fact, as 2D cell culture models are increasingly questioned for their lack of predictability, 3D bioprinting has become an alternative approach to circumvent this issue by processing different cells and extracellular matrix derived molecules, modeling/displaying the tumor microenvironment (TME). Many tumor models based on 3D biological printing include lung cancer, 5 breast cancer and 6 glioblastoma. These models demonstrate advantages in design, with flexibility and repeatability compared to other strategies such as cell spheres and organoids. Ovarian cancer is a major public health issue, and 3D bioprinting is still being studied as a model for ovarian cancer. In this type of tumor, cancer associated fibroblasts (CAFs) exhibit close interactions with cancer cells and play a major role in cancer invasion and drug resistance. Therefore, CAFs are crucial for designing ovarian cancer models.
advantage
High content screening and transient transfection are helpful for characterizing and validating 3D bioprinting experiments. Transient gene expression is a powerful tool for increasing the multifunctionality of 3D bioprinting models. Multi wavelength analysis tools are necessary for fully utilizing the powerful functions of 3D bioprinting models to study the interactions of different cell types within the model. In this application description, 3D bioprinting is used to create ovarian cancer models containing cancer cells (SKOV3 cells) and CAF fibroblasts (MeWo cells). Transfect SKOV3 cells with jetOPTIMUS (Polyplus) GFP plasmid and stain MeWo cells with CellTracker and Orange CMRA Dye (ThermoFisher). Both cell types are included in gelatin alginate based hydrogels to obtain a cylindrical tumor like structure. The homogeneity of the model and the reproducibility of cell distribution within these tumors were evaluated using the ImageXpress Pico imaging system.
Results and Discussion
As shown in Figure 1, bioprinted structural imaging showed that SKOV3 cells expressing GFP and MeWo cells stained with fluorescence were uniformly distributed in a gelatin alginate matrix (Figures 1A and B). This is very important because the uniform distribution of homogeneous cells in bio ink is an important factor to consider in the 3D printing process. In addition, natural TME is known to contain multiple cell types with close interactions. Therefore, reproducing this point is crucial, while constructing in vitro tumor models to promote the interaction between cancer and stromal cells.
Multiple cell types should be combined in tumors to better simulate TME heterogeneity, and the number and proportion of each cell type need to be controlled to ensure reproducibility. ImageXpress Pico is particularly suitable for implementing such controls as it can quickly and easily capture multiple microscope images. The CellReporterXpress software allows quantification of multiple cells, even for thick samples, using the maximum projection of Z-stack. In this application description, the system was used to analyze our bioprinted tumor model, which showed good repeatability in terms of cell number and proportion. Among the 6 bioprinted structures, the average number of SKOV3 cells (FITC cell count) was 837 ± 200 (standard deviation ≤ 25%). For MeWo cells (cell count TRITC), the average number is 3264.50 ± 462 (StD ≤ 15%).

Figure 1 analyzes the 3D bioprinting structure of CMRA dye in SKOV3 cells transfected with GFP plasmid and MeWO cells stained with CellTracker Orange. A) FITC and TRITC images of 3D bioprinted structures in well B3 were collected using the ImageXpress Pico system (4X, Z-stack). B) Analyze the masks of FITC and TRITC cell counts created using CellReporterXpress software. C) The average number of cells in the FITC and TRITC channels of the sample (5 wells, stained and transfected) and negative control (3 wells, unstained and untransfected). D) The evaluation of transfection efficiency was performed using GFP plasmid and jetOPTIMUS Polyplus Transfection Transfection assay kit (FITC positive cell percentage/RITC positive cells).

Materials and Methods
Hydrogel preparation
For the preparation of hydrogel, the required mass of gelatin and sodium alginate powder were weighed, and then dissolved in Dulbecco modified Eagle Medium for 1 hour under ultraviolet light, mixed with Lowest Essential Medium (MEM), supplemented with 10% fetal bovine serum. The obtained solution was then kept overnight at 37 ℃ under magnetic stirring to complete homogenization. According to the manufacturer's instructions, SKOV3 cells were transfected with jetOPTIMUS (Polyplus) GFP plasmid for cell transfection and staining. In short, an appropriate amount of DNA is diluted into jetOPTIMUS buffer and jetOPTIMUS transfection reagent is added appropriately. The transfection solution was stored at room temperature for 10 minutes, then added to the cultured SKOV3 cells and cultured in T75 flasks at a fusion rate of 60-80%. Stain MeWO cells with CellTracker Orange CMRA dye (ThermoFisher) according to the manufacturer's instructions. In short, the dye is dissolved in DMSO to prepare a working solution, which is then diluted in MEM medium. Incubate the cells at 37 ℃ for 30 minutes. Then take out the reagent solution and replace it with fresh onecompleteCulture medium.

Figure 2 illustrates the method for bioprinting and imaging the structure of an ovarian cancer model.