In to see the recognition of the importance of

In the last ten
years, there has been a dramatic surge in the number of publications where
single or groups of cells are grown in substrata that have elements of basement
membrane leading to the formation of tissue-like structures referred to as
organoids. However, this field of research began many decades ago, when the
pioneers of cell culture began to ask questions we still ask today: How does
organogenesis occur? How do signals integrate to make such vastly different
tissues and organs given that the sequence of the genome in our trillions of
cells is identical? Here, we summarize how work over the past century generated
the conceptual framework that has allowed us to make progress in the understanding
of tissue-specific morphogenetic programs. The development of cell culture
systems that provide accurate and physiologically relevant models are proving
to be key in establishing appropriate platforms for the development of new
therapeutic strategies.

How do we define
organoids and 3D cultures?

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That functional differentiation is
dependent on 3D architecture has become accepted recently. Many papers over the
last 50 years have shown that cells cultured in 2D are not representative of
the in vivo situation. Structurally, 2D cultures do not provide the conditions
for the organization and cellular relationships observed in vivo. Moreover,
cell signaling networks are altered in 2D versus 3D, and this probably explains
why drug screening outcomes many times do not reproduce the in vivo setting (Wang et al., 1998; Weaver et al., 2002). It
is encouraging to see the recognition of the importance of 3D cultures to model
signaling, differentiation, and drug development. Many of the studies use
elegant images and sophisticated animations that are a delight to see and hear
about and clearly show the similarity between organoids and the tissues and
organs from which the cells were derived in vivo. We applaud the excitement and
cheer the general enthusiasm that the work has deservedly generated. What is
most exciting is that the combined effort is finally a critical mass and as a
result has caught the attention of many new scientists who are emphasizing the
importance of 3D culture by pointing out the relevance and the significance of
this work to clinical research. However, the term “organoid” is being treated,
or has come to imply, that this is a completely new field, separate from what
several scientists from as early as the turn of the previous century have been
engaged in for years, essentially in isolation, introducing the term 3D cultures,
beginning the field of microenvironment, and pointing out the significance of
tissue architecture.

The first use of the words
“three-dimensional culture models,” we believe, started with the assays
developed by Barcellos-Hoff et al. (1989) and Petersen et al. (1992),
although floating collagen gels were described in the 1970s and were certainly
3D (see Fig. 2). Before 2005, the
word organoid was an extension of 3D cultures. Typically, it referred to small
tissue fragments taken from organs, mostly epithelial tissues, separated from
stroma by mechanical and enzymatic digestion and grown in different types of 3D
gels to produce an organ-like structure. As an example, see Simian et al. (2001), in
which rodent mammary fragments were grown in collagen gels to produce a
branching structure resembling branching in the mammary gland of virgin mice,
or Fata et al. (2007), in
which rodent mammary fragments were grown in laminin-rich gels giving rise to
alveogenesis. However, in the last decade, the meaning of “organoid” has lost
precision and has come to cover a series of cell culture techniques that are
not necessarily a single technique. Below are examples of definitions of
organoids taken from some recent papers in appropriate journals for the field.
We come across the following definitions:

(1) “Various subfields use these terms either
interchangeably or distinctly; for example, in the field of mammary gland
biology, the term organoids refers to primary explants of epithelial ducts into
3D extracellular matrix (ECM) gels. Conversely, in studies of intestinal
biology, organoids can refer to clonal derivatives of primary epithelial stem
cells that are grown without mesenchyme or can refer to epithelial–mesenchymal
co-cultures that are derived from embryonic stem cells or induced pluripotent
stem cells” (Shamir and Ewald, 2014).

(2) “Thus, we would like to define an
organoid as containing several cell
types that develop from stem cells or organ progenitors and self-organize through cell
sorting and spatially restricted lineage commitment, similar to the process in
vivo” (Lancaster and Knoblich, 2014).

(3) “An organoid is now defined as a 3D
structure grown from stem cells and consisting
of organ-specific cell types that self-organizes through cell sorting and spatially restricted lineage commitment…”
(Clevers, 2016).

(4) “Here we define an organoid as an
in vitro 3D cellular cluster derived exclusively from primary tissue, embryonic
stem cells, or induced pluripotent stem cells, capable of self-renewal and
self-organization, and exhibiting similar organ functionality as the tissue of
origin” (Fatehullah et al., 2016).

“The character and organization of
tissues are determined by the spatial arrangement, the mutual relations, and
the typical groupings of cells, which, together with the intercellular
material, combine into developmental and functional patterns. To the structure
and integrity of these cellular patterns are related the course of the
prospective development…” Moscona and Moscona, 1952

To our minds, the first definition,
provided by Shamir and Ewald (2014) is
the most inclusive and accurate, given that it includes the different
definitions the word “organoid” has been associated with. It avoids the
specific restriction imposed by other definitions and includes organoids
generated from induced or embryonic stem cells. Researchers indeed are able to
generate organoids in laminin-rich gels from single cells of normal tissues or
malignant tumors, or even cell lines, without necessarily starting from cells
that express stem cell markers (Weaver and Bissell, 1999).
This is especially relevant given that we still do not understand whether a
stem cell can be defined independently of its niche (Schofield, 1978; Mesa et al., 2015). Niches
are specialized microenvironments located within each tissue where stem cells
reside. Niches exert a key influence over stem cell function and are defined as
the sum of the cell–cell, cell–ECM, and cell–soluble factor interactions, in
the context of the physical and geometric constraints that a cell may
experience at a given time (Kaplan et al., 2007). The
ability of the niche to determine the functional spectrum of stem cell
activities leads to the hypothesis that stem cell niche microenvironments are
critical in the definition of stem cell functions (Kaplan et al., 2007; LaBarge et al., 2007).

It should be acknowledged that the
development of the culture conditions that were established by scientists
working on organoids (as originally defined) has contributed to the significant
advances reported in the stem cell field in the last 10 years. Independently of
the methods used to generate the organoids and keep them in culture, these
advances represent outstanding model systems to study human development and
disease. For many organs, such as the brain, mouse and human development are
not the same (Lancaster et al., 2013).
Moreover, induced pluripotent stem cells derived from skin fibroblasts as well
as 3D cultures of normal and diseased human organs offer models for human
diseases that are not easy to study in animal models (Lancaster et al., 2013).
Additionally, developing screening platforms based on human organoids may
provide a more cost-effective and precise preclinical setting for drug
discovery in the long term.

Interestingly, the word organoid
initially had a different meaning from all of the above. In the 1950s and
1960s, papers referring to organoids often centered on intracellular structures
(organelles), with titles such as “Quantitative cine analysis of cell organoid
activity” (Pomerat et al., 1954) or
“Nuclear and cytoplasmic organoids in the living cell” (Duryee and Doherty, 1954).
The word organoid was used also for tumors (Gordienko, 1964) or
abnormal cellular growths (Wolter, 1967). Many papers
described cases of “Organoid Nevus,” a malformation of the skin, most commonly
in the scalp (Pinkus, 1976). Other
researchers seemed to use organoids for cellular clusters that maintained the
structural characteristics of the tissue of origin. For instance, Schneider et al. (1963),
in a paper titled “Some unusual observations of organoid tissues and blood
elements in monolayer cultures,” observed organoids as 1-mm nodules attached to
the flask or floating after mechanical and enzymatic digestion of mammalian
tissues. From 1980 on, however, papers referring to 3D cultures included the
use of the word organoids.

The aforementioned use and misuse of
the word organoids appears to have contributed to significant divergence
regarding when the organoid field began to develop. In a recent review, Clevers (2016)states that
there was an initial increase in organoid research in the 1965 to 1985 period (Fig. 1, organoids, red
squares), showing an astounding 563 papers in 1980. This number and the fact
that the graph shows a sudden drop in 1985 surprised us and caught our
attention. A close look at the papers referenced for this period shows that the
PubMed search picked up many papers that included the word “organ,” but not
necessarily “organoids.” A different search using “organoid” followed by the
“text word” tag shows papers actually using the word “organoid” (Fig. 1, blue circles).
This search, in a way, also overestimates the number of papers about organoids
because the results encompass research on organoids as defined before 1980,
which included small structures within the cells’ cytoplasm. From 1980,
researchers began to use collagen and laminin-rich matrices to culture cells
and organoids in 3D, and thus 3D included organoids as discussed above. The
start of the dramatic increase in the number of published papers on organoids
was in fact around 2011, with 150 papers published in 2016 up to the time this
article was written. For comparison, we also show the search that referred to
“3D cell culture” (Fig. 1, green triangles).
In reality, as we have argued, the two terms are one and the same until such
time that those scientists who are active in this field would subdivide and
define the different types of 3D/organoids practiced in different laboratories.
(Indeed, M.J. Bissell and H. Clever discussed this nomenclature problem in a
meeting entitled “Organoids” organized by the European Molecular Biology
Organization in October of 2016. The aim is to bring clarity in nomenclature
and keep the focus on showing the overriding importance of context and
architecture in how tissues and organs are formed and maintained.) Organoid
cultures as models for the study of development and disease could not have occurred
without the advances in what is now referred to as 3D cell cultures. Even
though the first papers that were essentially doing 3D cultures/organoids
started in the 1960s, the number of publications began to increase steadily
from 2003 on, with a total of 640 publications up to the time this article was
written in 2016. What the data in Fig. 2 show is that
it took about half a century for the recognition that this way of thinking is
not simply utilization of a technique but that form and function are
fundamentally intertwined and that once the organism is formed, essentially,
phenotype is dominant over genotype.


            Recent technical advances in the stem cell field have
enabled the in vitro generation of complex structures resembling whole organs
termed organoids. Most of these approaches employ three-dimensional (3D)
culture systems that allow stem cell-derived or tissue progenitor cells to
self-organize into 3D structures. These systems evolved, methodologically and
conceptually, from classical reaggregation experiments, showing that
dissociated cells from embryonic organs can reaggregate and re-create the
original organ architecture. Since organoids can be grown from human stem cells
and from patient-derived induced pluripotent stem cells, they create
significant prospects for modelling development and diseases, for toxicology
and drug discovery studies, and in the field of regenerative medicine. Here, we
outline historical advances in the field and describe some of the major recent
developments in 3D animal organoid formation. Finally, we underline current
limitations and highlight examples of how organoid technology can be applied in
biomedical research. (XINARIS et al., 2015.)