The recent advancement of organoid technology has provided new opportunities for researchers to study the mechanism of the broad range of interactions existing within an organ in health and disease [1]. Although great improvement had been achieved in reproducing the in vitro and in vivo chemical and cellular microenvironment, there are still very few studies that have illustrated mechanistic explanations of how organoids can mimic the maturation, differentiation, and function of the different cell types found in vivo [1]. One of the novel directions is to boost the development of a more relevant, reproducible, and amenable organoid models by using cross-disciplinary of science [1].

In a review published by Cellular and Molecular Gastroenterology and Hepatology, Hautefort et al. provide a general introduction for improving the accessibility to the organoid models while highlight areas for cross-disciplinary collaboration with biomaterial, tissue engineering, genome engineering, and nanofabrication sciences as one of the novel directions to broaden the application of organoids.

Dolly the reporter from Lab-A-Porter extracted some of the highlights from the review article while we featured some of the organoid research reagents and tools that may support your next cross-disciplinary organoid research projects.

• General introduction
• Methods to improve organoid’s accessibility
• Current challenges
• Future: using cross-disciplinary of science
• Selected organoid research tools by LAB-A-PORTER

Why stem cell-based in vitro models are developed?

• Decoding the complex molecular and cellular interactions taking place in each organ, and how they malfunction in diseases, is instrumental to the progress of biomedical research and eventually to personalized medicine [1].

• Previously established in vitro models (cell lines, primary cell cultures) were either too simplified or not translatable to human beings and had limitations in recapitulating the different cell types and their interactions[1].

• Scientists have had to re-explore embryology and tissue development to devise and develop novel stem cell-based in vitro models that allow studying the mechanism of the vast range of interactions taking place within an organ in health and disease[1].

What criteria are considered when adapting organoid-based models to unexplored research fields?

• Diverse environmental triggers and instrumental in shaping the condition required for multicellular structures to grow in vitro[1].

• Mechanical shear forces from the fluid passing over cells or from pulling and pushing through muscle contraction also influence the accuracy of the model developed[1].

• It, therefore, is paramount for new and existing organoid model users to choose an organoid model based on many known factors, such as the source and types of cells to include, the level of simplification achievable, the availability of growth condition reagents, the scale of the planned experiments, and the different readouts applicable to that model[1].

• Despite the clear overlap in many existing protocols, there is no universal approach and many of the following factors will need to be considered separately and also in synergy for developing the appropriate model and answering specific biological questions[1].

How are cell proliferation and differentiation influenced by the surrounding extracellular matrix (ECM) and cells?

• In living tissues, mesenchymal and epithelial cells produce different components of the ECM, generating a gradient of signalling mediators important for tuning different pathways involved in tissue assembly, wound healing, and tissue regeneration[1].
  • • These include molecules such as integrins, laminin, collagen, fibronectin, entactin, and glycosaminoglycans[1].
    • These components or their concentrations are unique to the different organs or specific tissue regions [1].

• ECM-like products derived from living tissue promote cell adhesion with high efficiency and have become the by-default material scientists use for most organoid cultures[1].
  • • However, these products are very expensive and are derived from natural extracts, preventing researchers from labelling organoid experiments as animal-free[1].
    • More, each of these ECM products presents high batch-to-batch variability, especially in their protein content, causing reproducibility issues in organoid culture if not monitored[1].

• Some research groups have developed a wide assortment of basal cell-matrix protein-containing hydrogels, reproducing certain tissue-specific properties (different protein isoforms for different parts of tissue)[1].
• • • Initially, for organoid model experts these alternative ECMs, of more defined compositions, offer much-improved reproducibility and versatility than animal-derived matrixes to accommodate diverse organ-mimicking organoid cultures [1].
    • Some allow the ECM to evolve/degrade dynamically as the epithelial structure grows, some offer reduced stiffness, while others have tuneable biomolecular and biophysical properties[1].

• These technical advances enable optimizing the organoid cell size and differentiation, thus broadening the range of readout approaches that can be applied to organoids[1].

How do tissue topology, cell positioning, and mechanical forces impact cell differentiation and maturation?

• Among the factors influencing the development and homeostasis of an organ, organ 3D architecture increasingly is recognized as important[1].
  • • The 3D architecture encompasses the respective positioning and the distribution of the different cell types within the tissue[1].
    • Little is fully understood about what regulates the spatial resolution of what makes an organ a functional organ[1].
    • This highlights how useful it is to recapitulate at least part of this 3D landscape in an in vitro model to understand how it contributes to regulating cell functions[1].

• Attention to the tissue topology, cell positioning, and the shear forces applied to them, therefore, has gained importance as a valuable strategy in the development of more accurate organoid models[1].
  • • Successful strategies to co-culture different cell types have included aggregating cells on coated surfaces or, conversely, in rotating vessels to prevent their adherence to the vessel itself[1].
    • In parallel, using special scaffold coating or co-encapsulating the cells into defined ECM-mimicking hydrogels remains a preferred and more controllable approach[1].
    • These options allow for studying the different factors that influence cell survival in 3D cellular structures, including organoids[1].

• The stem cell niche maintenance and development are influenced strongly by the tissue topology (e.g., the curvature of the underlying tissue), the biomechanics (e.g., shear forces from smooth muscle contractions of the digestive tract), and the permanent circulation of luminal flow[1].
  • • In vitro control of these additional factors strongly impacts the degree of proliferation, polarization, and differentiation of the pluripotent stem cell–derived structures, and it is clear that simplification of such variables is inevitable in mechanistic studies[1].
    • Spatiotemporal control of the microcellular environment is important when studying the cell type–specific function homeostasis and the involved intercellular crosstalk[1].

What is the possible adaptation of novel hydrogels and scaffolds to organoids and other cell co-culture?

• Considering the high level of versatility of classic co-culture systems, similar strategies are being adapted to organoid culture systems. Cells interacting in vivo can be first cultured separately in vitro before being seeded together[1]. Several tools can be used for this purpose, for example, cell culture inserts or patterning scaffolds[1].

• Similarly, the pattern and layering of different cell types are of prime importance to better recapitulate cell-cell interactions, offering more control of the proliferation rate and differentiation state of the resulting organoid cells[1]. These microenvironmental signals will dictate how well the culture of organoids reflects the cell assembly and organization observed in the tissue of origin[1].

• Some technologies use magnetic nanoparticles and micromagnetic forces to help position different cell types, obtaining a more accurate cellular arrangement when studying their interactions[1]. Similarly, different materials such as synthetic polymers can be used as scaffolds to control the levels of homotypic or heterotypic cell interactions in in-vitro models[1]. The materials first are modified and necessitate conjugation with bioactive molecules to permit their interactions with living cells[1]. 

• Engineering biomaterials can involve, such as:
  • • Scaffolds with in vivo-mimicking curvature[1]
    • Perfusable systems for supplying nutrients and oxygens to complex 3D cell structures[1]
    • Defined hydrogels and relevant cell layering/positioning for mimicking in vivo intracellular crosstalks[1]
      

Technical limitations, however, still are restricting the possibility to conduct longitudinal studies and explore the full differentiation of these heterotypic and quite large cellular structures[1].

Classic ECM-embedded intestinal organoid cultures do not include a functional vascularization system[1]. The larger the 3D structure is, the more limited the oxygen supply becomes in the central part of the organoids, leading to hypoxia and exacerbated cell necrosis[1].

To date, one possible way to culture organoids for a prolonged length of time is their xenograft to a living animal model tissue[1]. Recently, an in vitro method was proposed using a patterned tubular matrix to grow organoids that self-arrange into an epithelial monolayer with crypt and villi regions. This system allows perfusion of primary cell monolayers and culture of them for several weeks without hypoxia-induced damages. In the future, additional cell types could be included in the hydrogel scaffold of such models to investigate, for example, epithelial/immune cell interactions[1].

What are the current challenges for organoid models?

• Despite the great advances made in reproducing in vitro the in vivo chemical and cellular microenvironment, very few studies have produced mechanistic explanations of how organoids can mimic the maturation, differentiation, and function of the different cell types found in vivo[1].

• Applying organoid culture protocols to samples that originated from diseased tissue to recapitulate a disease phenotype is much more challenging than for healthy tissue[1].

• Choosing a relevant model strictly depends on the exact scientific questions asked, hence, all different possible approaches should be considered while having that in mind, For example, is the studied disease monogenic or are there any genetic factors to control[1]?

• Different ways to improve the controllability and reproducibility of this technology should be pursued based on the specific scientific questions asked. Additional parameters will need to be added to control the cellular complexity, tissue geometry and cellular patterning and layering of the modelled tissue/organ[1].

What are examples of novel directions for organoid models?

• Currently, improved models are emerging from bridging stem cell research with biomaterial and bioengineering research fields in an attempt to replicate the cellular patterns, tissue curvature, heterotypic diversity, shear forces from fluid flux, and neighbouring cell movements[1].

• The next generation of organoid models is likely to contain most of the essential cell types present in an organ (e.g., nerves, stroma, immune cells) [1].

• They also will be developed following the concept of narrative engineering, that is, recapitulate the chronological changes (biochemical, mechanical, and physiological environment) as they would occur in vivo[1].

Once the various factors mentioned earlier become controllable, harmonized and standardized organoid-based models will be used by a larger part of the scientific community, providing the costs are reduced as well[1].

• Standardization of organoid expansion alongside a generation of stable organoid lines will form reliable tools for drug screening using high-throughput readouts (e.g., single-cell RNA sequencing [RNAseq], Assay for Transposase-Accessible Chromatin using sequencing, ATAC sequencing, bisulfite sequencing, spatially resolved RNAseq, proteomics, and bioimaging) [1].

• Recently, a multiplex single-cell analysis pipeline was developed on organoids co-cultured with fibroblast and leukocytes to establish posttranslational modification signalling networks that can baltered in diseases[1].

• For example, growing organoids from patient-derived stem cell aggregates in preformed U-shaped microcavities imprinted in the hydrogel achieves highly homogenous cultures, both in size and maturation level[1].

• In addition, in this high-throughput single organoid model, cells will be positioned on the same Z plane, thus facilitating the automated live bioimaging screening of various drugs for the development of personalized medicine[1].

• These recent advances are instrumental in the reproducibility of experiments among different research laboratories across the world[1].

Harmonizing these approaches at an international level will enable successful translational biomedical applications for global pharmaceutical and biomedical companies/hospitals[1].

• Structural and mechanical scaffolds mimicking the microenvironment of the epithelial cells now are being developed and will increase the capability of organoid cells to self-organize following layering or pattern that is important for those cells to fully mature and function as they would in vivo[1].

• It now is foreseeable to combine organoid models of different organs into assembloids to study further intracellular interactions between different body systems such as the lungs, heart, gut, and nervous system[1].

• The tissue engineering research field has been a great source of innovation for developing improved organoid models dedicated to basic or translational research[1].

• What microfluidics systems have enabled more recently was to recreate separated compartments, with the nutrient-containing medium side and the apical side of the epithelial barrier. These systems still are under improvement, but already can be exploited to recreate in vitro organ-specific features such as epithelium exposure to circulating fluid and flow-associated shear forces [1].

• In addition to the multiple platforms emerging for using organoid-based models, the increasing accessibility to gene editing technology (e.g. CRISPR-Cas 9) will bring forward more advanced regenerative and personalized medicine. It is now possible to confirm the genetic association of a mutation with a disease phenotype and to bring back functioning gene alleles, thus homeostatic functions in defective tissue [1].

• In parallel, assay for mats and readout technologies also have evolved and now have become applicable to organoid-based approaches (e.g. single-cell RNAseq, in situ RNAseq, and high-content live bioimaging), enabling high-resolution and longitudinal studies[1].

Such technologies definitely will complement the development of better disease organoid models, as well as the understanding of the different levels of interaction that regulate tissue homeostasis, fostering future therapeutic approaches in human and animal health[1].

For research use only (RUO). Not for human use. Not for therapeutic or diagnostic use. 

What is the future of organoid technology?

• In the past 10 years, stem cell-based research has made a huge leap forward, benefiting a myriad of other sectors, creating unforeseen collaborations between research fields such as biomaterials, microfluidics, high-throughput live bioimaging, mathematical modelling, data sciences, cellular biology, and multi-omics[1].

• Several biotechnology companies now offer already-made reagents/media to grow organoids, or alternative compounds to make growth medium from individual components, allowing the creation of diverse culture conditions for expansion, differentiations, or screening of organoids[1].

• Various microfluidic platforms already are available to grow cell monolaters from organoids and offer accessibility to both apical and basolateral sides of epithelial cell layers[1].

• The links currently developing between biology, biomedicine, biomaterials, and biophysics research with biotechnologies is a remarkable international initiative [1]. It will boost the development of more relevant, reproducible, and amenable tools to study intercellular interactions and their roles in health and disease[1].

• Future organoid-based models will become a goldmine resource for understanding the development and function of tissue at cell type-specific levels and in a patient-specific manner[1].

• With those models becoming more reliable, clinical trials of biologics pretested on organoids hopefully will be accelerated and tissue reconstruction will be elaborated with direct application in regenerative and personalized medicine[1].

Bioactive and cost-effective growth factors

CRISPR kits