Spheroids and Organoids
The single-cell or multicellular spheroid model is based on the ability
to homotypic cell-cell adhesion, when its adhesion to plastic of the
culture flasks is prevented. In general, the methods to develop a
spheroid include hanging drop technique, in which cells are cultivated
suspended due to superficial tension; cultivation on non-adherent
surfaces, with the use of a scaffold or gel; and the magnetic levitation
method (MLM), in which cells are cultured with nanoparticles and kept in
culture with a magnetic field, allowing the formation of cell clusters
that are detached from dish bottom and manipulated with help of a
magnet. The size of the spheres varies depending on the number of cells
cultured and the cell type. In addition, differences in the ability to
establish cell-cell adhesions influence the formation of spheroids,
which may be looser, with an irregular surface, or firmer. The model
allows cultivation of single cell lineage as well as co-cultivation of
different cell types, and presents the capacity of cells to
self-organize spontaneously, deposit extracellular matrix and form
specific microenvironments (Haisler et al., 2013; Kelm and Fussenegger,
2004; Layer et al., 2002; Mueller-Klieser, 1997). In oncology research,
several trials have shown that while monolayer tumors were sensitive to
the action of several chemotherapeutic agents, the same cells, when
grown as spheroids, were resistant to them. On the other hand, some
drugs were effective only when the cells were in a 3D environment. The
central hypoxia and the diverse regions formed make the model especially
advantageous in the oncology area because of its resemblance to
non-vascularized tumor nodules. However, spheroids must be used with
discernment due to the possible development of central necrosis (Laschke
and Menger, 2017; Verjans et al., 2018).
Alternatively, organoids are artificial structures that represent
fragments of functional organs created for in vitro studies, capable of
exercising the primordial functions of the corresponding organ, and are
more complex in comparison to spheroids. They must exhibit
morphofunctional units of respective in vivo tissues (Simian and
Bissell, 2017). Normally, organoids are composed by different cell types
organized in a specific arrangement, and can be initiated from
spheroids, as well as with help of barriers, with a layered deposition
of different cell types. In the latter case, it is possible to control
the culture composition, and as the cells continue to proliferate after
the formation of the sphere, they present self-renewal and can survive
at long-term, as previously stated. The spontaneous arrangement of cells
to form the 3D structure increases the chances of presenting an
organotypic phenotype, thus representing more realistically the in vivo
tissue (Danielson et al., 2018). Furthermore, the possibility of using
different cell types in the same organoid allows heterotypic
intercellular contacts, providing additional advances in realistically
representing tissue functionality and differentiation (Marin and Pagani,
2018; Simian and Bissell, 2017). A summary of characteristics from
monolayer, spheroids and organoids are represented in Figure 2.
Getting started: Cell lineages for spheroid / organoid
generation
Spheroids and organoids can be derived basically from two originating
cell types: pluripotent stem cells (PSCs) or adult-tissue cells (ATCs).
Concerning PSCs, they can be either embryonic stem cells or induced PSCs
(iPSCs), de-differentiated from adult cells (mainly fibroblasts).
Because of the greater differentiation capacity, the resulting 3D model
presents diverse properties of the target-organ; they are highly
expandable; and can survive transplantation (Hohwieler et al., 2017;
Huang et al., 2015). On the other hand, 3D models derived from adult
tissue can be either adult stem cells (ASCs) or fully differentiated
cells, including immortalized cells and lineages commercially available.
In either case, the generation depends on a range of growth factors (Boj
et al., 2015), and are limited in regard to the possibility of
originating developmental intermediates, but the starting cells are of
easier access, such as small amounts of biopsy material.
A single ASC type can start an organoid, while usually a cell pool of
PSCs is required (Ootani et al., 2009). As a consequence, ASCs-derived
organoids develop with a spheroid phase, while organoids derived from
PSCs are usually more complex models, able to represent complex organs
such as brain parts (Monzel et al., 2016), kidney and lung (Lancaster et
al., 2013; Pasca, 2018), and they also might undergo a spheroid stage
prior to organoid completion. In the case of visceral models, the
complete process from PSCs to organoids usually include a definitive
endoderm induction (Clevers, 2016), through activin treatment usually; a
subsequent anterior foregut induction; and then organ-specific
progenitor spheroids (Dye et al., 2015).
The advantage of using PSCs to generate 3D cultures is that they can
differentiate into almost every cell lineage present in the living
organism, and associated with the correct differentiating and inducing
factors, a variety of spheroids and organoids can be achieved. Also, the
resulting culture are of great value in development and regenerative
medicine (Qu et al., 2014; Weidgang et al., 2013; Xu et al., 2015), in
addition to generating specific disease and patient conditions (Rezania
et al., 2014; Sampaziotis et al., 2015). On the downside, the generation
from PSCs can take an enormous amount of time because of the need of
step-wise differentiations before obtaining the final 3D model. In
addition, the model usually does not represent a mature tissue or organ.
For ATCs-derived models, the starting cells range from fetal cells (9+
weeks of development); stem cells isolated from bone marrow or adipose
tissue, such as mesenchymal cells; adult progenitors isolated from the
fully mature target-organ, whose cell fate is pre-determined; and fully
differentiated cells. In regard to mesenchymal stem cells / stromal
cells, which derive mesodermal-origin cells, they have been employed in
clinical trials involving cell engineering (Aldahmash et al., 2012;
Zhang et al., 2012), and because they secrete several important growth
factors that support vascularization, immune response and extracellular
matrix production, this results in a complex 3D culture. It has been
previously reported that the microenvironment in the core of
ASCs-derived spheroids can trigger more growth factor secretion, what in
turn helps nurture the culture (Potapova et al., 2007). In this case of
using differentiated cells, different cell types either isolated from
the tissue (usually obtained by biopsy) or commercially acquired
lineages are cultivated separately and later allocated in a
co-cultivation system. The selected cell lines, for the generation of
visceral models for instance, usually include epithelial and endothelial
cells, and fibroblasts. The co-culture cells, once placed onto a
scaffold such as Matrigel or collagen, tend to spontaneously agglomerate
and grow, forming a 3D structure containing co-cultures.