**5. Stages of organoid development**

Generally, the 3D culture of organoids was performed by using Matrigel derived from EHS (Engelbreth-Holam-Swarm) mouse sarcoma cells which were rich in adhesive proteins such as laminin, entactin, collagen, and proteoglycans. The Matrigel structural support, ECM signals along with the extracellular environment that supports the growth of cells [64]. In other techniques, Matrigel or fibroblasts were submerged in a medium to expose the upper layers of cells in the air. Later, the air-liquid interface will be utilized to culture the cells for better polarization and differentiation [65]. The organoid technology developed drastically after the successful generation of in-vitro organ generation from sponge cells [66], amphibians pronephros [67], chick embryos [68] followed by surface adhesion of cells using thermodynamic differentiation [69]. After the invention of pluripotent stem cells from mouse embryos, there was a significant impact on organoid development by utilizing stem cells [70].

The use of animal models has been considered in the initial days to predict the efficacy of drugs to tumor cells, later cell lines cultured in-vitro were used. But both these studies did not represent the actual model of tumor cells for which treatment is required [19]. A report by Radhakrishnan et al., 2017 [71] was used to analyze the immune target therapy for the treatment of cancer was carried out by utilizing the CANscript exvivo platform technology.

The various stages of organoid development were explained below [72] and the overview showed in **Figure 3**.


#### **Figure 3.**

*Flowchart of various stages of organoid development [10, 19].*


• 2000–2020: The development of the first organoid from pluripotent stem cells was performed in 2006 from mouse fibroblast, followed by the development of organoids from cortex tissue, adult stem cells, development of gastric organoids, development of retinal, kidney, pancreas, lungs, fallopian tubes and snake venom were performed successfully in subsequent years.
