3.1. Hanging Drop Method

The hanging drop technique is a simple scaffold-free approach in which small droplets of the cell suspension are placed on a reversed upside-down culture lid [32]. This method involves the action of surface tension that anchors the drop of cell suspension as a hanging drop on the inverted lid [16]. Consequently, spheroids form as droplets owing to the gravity force that concentrates the cell suspension at the bottom tip [16]. Generally, the spheroids formed are tightly packed, rather than loose cell aggregates [12]. In addition to the simplicity and cost-effectiveness, this method has other advantages. This technique can form spheroids of consistent sizes and morphologies, which can be controlled by the volume of the drop or density of cell seeding suspension [32]. In addition, this method applies to a variety of cell lines and enables the creation of co-cultures with various cell types [16]. Studies reported that 3D spheroids produced by using the hanging drop technique have 100% reproducibility [12]. Nonetheless, the hanging drop protocol is one of the most time consuming and labor-intensive techniques in terms of maintaining spheroids and changing of spent culture medium, without disturbing the culture spheroid [10,12]. Another downside associated with this protocol is the inability to maintain long-term spheroids due to the generally limited volume of the seeding suspension that cannot provide sufficient nutrients [33]. Recently, studies have developed several tools such as commercial 384-well droplet suspension plates and bioassay plates to surpass some of these limitations and enhance the efficiency of spheroid formation [10,12,33].