Evolution of research with in vitro models
Cell culturing has enabled diverse advances in cell biology (Achilli et al., 2012), including what we know especially on extracellular signals (Bissell et al., 2002), and started being used during the XX century, by researches such as Harrison and Carrel, who aimed at better understanding cell behavior and function (Breslin and O’Driscoll, 2013). In 1907, Harrison successfully cultivated nervous cells isolated from amphibious spinal cord (Harrison, 1907); in 1912, Carrel proved it was possible to cultivate cells for longer periods since maintained under aseptic conditions and given enough nutrient supply (Carrel and Ingebrigsten, 1912). Some decades later, in 1951, George Otto Gey stablished the first human cultured cell line, derived from cervix cancer collected from a patient named Henrietta Lacks, the HeLa cells, a lineage that is employed to this day for cancer research (Ambrose, 2017; Earle et al., 1951; Ehrmann and Gey, 1956; Rahbari et al., 2009; Scherer et al., 1953). Monolayer cell culturing relies on the cell adherence into a flat surface, and on the constant addition of culture medium as nutritious resource to cells; the plaque is maintained at a temperature of 37oC and with 5% CO2 flow, what resembles the human body conditions (Breslin and O’Driscoll, 2013).
In an in vivo situation, cells communicate to one another in addition to interacting with an extracellular matrix (ECM). They are in intimate contact to cells of the same type as well as other cell lineages, in a three-dimensional architecture, what constitutes the tissue or organ. Concerning drug testing, for example, the cell-cell connections in addition to cell-ECM interactions are mandatory in order to obtain a more valid replication of the in vivo context (Cushing and Anseth, 2007). Considering these aspects, although 2D cell cultures are still widely used for diverse researches, they present serious limitations. They can be improved using a transition to 3D culturing (Rimann and Graf-Hausner, 2012).
Tridimensional cultures are more sensitive to drugs due to the organization of surface receptors; also, cells are often in different stages, similar to in vivo conditions (Jensen and Teng, 2020). The petri dishes and the cellular monolayer are replaced by a gelatinous scaffold that resembles the ECM, and the culture, now unable to attach to the bottom of the plate, assembles in a resulting 3D morphology (Haisler et al., 2013). The resulting cultures allow co-cultivation of different cell lineages of the desired tissue or organ represent a more accurate replication of the in vivo condition, including morphology and functions, due to engineering and biology principles to construct the functional substitutes of the target-organ (Doryab et al., 2016). Because of the representation of histological and physiological key characteristics of the target-organ, 3D models can even be employed as research tools or even to replace damaged living tissue in human patients.
Despite the rapid boom observed in concern to organoid research, seen from the late 2000’s on, methods using 3D cultivation dates back to the XX century. The cultivation of cell aggregations instead of the regular monolayer display was first mentioned in the 50s by George Gey, the same researcher who isolated HeLa cells. In a paper from 1956, Ehrmann and Gey report they cultivated different human cell lineages using collagen isolated from rat tail as a substrate, and originated cell agglomerates with no interference from the scaffold (Ehrmann and Gey, 1956). Two decades later, the first 3D culture was described by James Rheinwald and Howard Green, who developed in vitro tridimensional tissues derived from human progenitor cells (Rheinwatd and Green, 1975). In the late 80’s and beginning of 90’s, in vitro cultures derived from neuroblastomas (Hachitanda and Tsuneyoshi, 1994) and lung tissue (Zimmermann, 1987) were developed and referred to as organoids. Researchers from Rheinwald’s and Green’s lab continued working on 3D cell cultures and expanded their know-how. In the 90’s, they successfully cultivated skin organoids from small amounts of primary cells of human donors, and the organoids were successfully used in the treatment of third-degree burn. Still in the 90’s, the 3D cornea cultures were used to treat blindness in over 100 patients (Lindberg et al., 1993; Pellegrini et al., 1997). A brief description of the evolution of cell cultures, from the first cultivation to the generation of spheroids and organoids are in Figure 1.
A grand part of these 3D cultures is called “organoids” because the cells in a 3D environment spontaneously organize themselves, forming complex histological structures, similar to those observed in the organs from which they are derived; for example, cells derived from mammary glands form structures similar to branched ducts or acini (Lee et al., 2007). The name “organoid” derives from the junction of the suffix “oid” (from Greek “eîdos”, similar) to the word “organ”, referring to a structure that resembles an organ; also, the name is also commonly taken as a small version of an organ, such as “mini-guts” (Sato et al., 2009) or “mini-tissues” (Almeqdadi et al., 2019).
Although there is a generalization of the use of the name “organoid” to refer to 3D cultures that presents multiple cell lineages in co-culture that are able to spatially organize and form clusters (Almeqdadi et al., 2019), there are some particularities among 3D models that must be taken into consideration. Both Sato and Clevers’ laboratories referred to their models as intestinal organoids (Barker et al., 2007; Sato et al., 2009), but the cultures developed consisted purely of epithelial cell lines. Thus, in 2012, the Intestinal Stem Cell Consortium established some nomenclature guidelines: the term ”organoid” refers to cultures containing multiple cell types, but must include epithelial- and mesenchymal-originated cells; epithelial-only 3D cultures must be referred to as ”spheroid” (Almeqdadi et al., 2019; Mustata et al., 2013). The epithelial-mesenchymal interactions have been shows to guarantee the stability of the 3D culture at long-term (at least one year) (Almeqdadi et al., 2019) and is one of the characteristics presented by organoids, while not all spheroids are viable for longer periods in culture.