*3.1.2.2. Scaffold-free organoid cultures*

**Figure 2.** (a) A schematic of the hanging drop plate and (b) Schematic of spheroid formation techniques for hanging

dish containing media to maintain a humid atmosphere. Suspended cells then come together and form 3D spheroids at the apex of the droplet of media [72, 73]. This method has many advantages such as cost effectiveness, controlled spheroid size, and various cell types can be co-cultured and produced into spheroids [74, 75]. Moreover, it has been reported that 3D cell culture generated with hanging drop method have 100% reproducibility [69]. Due to limited volume of droplets generated with this technique, it is difficult to maintain spheroids and change the medium. Presently, there are many commercial devices for hanging drop culture

The use of low adhesion plates helps to promote self-aggregation of cells into spheroids [76]. Low adhesion plates have been developed as the commercial product of the liquid overlay technique, which is a low cost highly reproducible culture method that easily promotes 3D aggregates or spheroids [77]. Low adhesion plates are spheroid microplates with round, V-shaped bottoms and very low attachment surfaces to generate self-aggregation and spheroid formation. Plates are designed with hydrophilic or hydrophobic coating, which reduces cell from attaching to the surface. The main advantage of low adhesion plates is the potential to produce one spheroid per well making it appropriate for medium-throughput screening,

drop spheroids.

(**Figure 2**).

28 Cell Culture

Organoids are *in vitro* derived 3D cell aggregates that are capable of self-renewal, self-organization, and exhibit organ functionality [83]. Organoids are produced either from stem cells or primary tissues by providing suitable physical (support for cell attachment and survival) and biochemical (modulate signaling pathways) cues [84]. Organoids are classified into tissue organoids and stem cell organoids, based on how the organ buds are created [85]. Distinctive examples of tissue organoids culture are intestine, prostate, mammary and salivary glands. Stem cell organoids are created from either embryonic stem cells or primary stem cells (neonatal tissue) or induced pluripotent cells. Presently, different *in vitro* organoids have been set to simulate numerous tissues such as functional organoids for pancreas [86], liver [85], intestine [87], kidney [88], lung [89], retina [90], stomach [91] and thyroid [92]. Organoids mimic some of the structure and function of real organs [83]. Several approaches have been used to obtain organoids. The first approach is to culture cells as a monolayer on an ECM coated surface; organoids are then produced after the cells differentiate. The second is a mechanically supported cell culture to provide further differentiation of primary tissues. The third approach is to produce embryoid bodies through hang drop culture or on the low adhesion plates [93]. The main disadvantages of organoids are the lack of vasculature, lack of key cell types found *in vivo* and some organoids only replicate early stages of organ development [83].
