In vitro human cell-based models: What can they do and what are their limitations?

It is said that all models are wrong, but some are useful. In vitro human cell-based models are a prime example of this maxim. We asked researchers: when is your model system useful? How can it be made more useful? What are its limitations?

Probing the killing potency of tumor-infiltrating lymphocytes on microarrayed colorectal cancer tumoroids.

Immunotherapy has emerged as a new standard of care for certain cancer patients with specific cellular and molecular makeups. However, there is still an unmet need for ex vivo models able to readily assess the effectiveness of immunotherapeutic treatments in a high-throughput and patient-specific manner. To address this issue, we have developed a microarrayed system of patient-derived tumoroids with recreated immune microenvironments that are optimized for the high-content evaluation of tumor-infiltrating lymphocyte functionality.

Patient-derived mini-colons enable long-term modeling of tumor-microenvironment complexity.

Existing organoid models fall short of fully capturing the complexity of cancer because they lack sufficient multicellular diversity, tissue-level organization, biological durability and experimental flexibility. Thus, many multifactorial cancer processes, especially those involving the tumor microenvironment, are difficult to study ex vivo. To overcome these limitations, we herein implemented tissue-engineering and microfabrication technologies to develop topobiologically complex, patient-specific cancer avatars.

Nanoplasmonic Single-Tumoroid Microarray for Real-Time Secretion Analysis.

Organoid tumor models have emerged as a powerful tool in the fields of biology and medicine as such 3D structures grown from tumor cells recapitulate better tumor characteristics, making these tumoroids unique for personalized cancer research. Assessment of their functional behavior, particularly protein secretion, is of significant importance to provide comprehensive insights. Here, a label-free spectroscopic imaging platform is presented with advanced integrated optofluidic nanoplasmonic biosensor that enables real-time secretion analysis from single tumoroids.

Bioengineered human colon organoids with in vivo-like cellular complexity and function.

Organoids and organs-on-a-chip have emerged as powerful tools for modeling human gut physiology and disease in vitro. Although physiologically relevant, these systems often lack the environmental milieu, spatial organization, cell type diversity, and maturity necessary for mimicking human intestinal mucosa. To instead generate models closely resembling in vivo tissue, we herein integrated organoid and organ-on-a-chip technology to develop an advanced human organoid model, called "mini-colons.

Spatiotemporally resolved colorectal oncogenesis in mini-colons ex vivo.

Three-dimensional organoid culture technologies have revolutionized cancer research by allowing for more realistic and scalable reproductions of both tumour and microenvironmental structures. This has enabled better modelling of low-complexity cancer cell behaviours that occur over relatively short periods of time. However, available organoid systems do not capture the intricate evolutionary process of cancer development in terms of tissue architecture, cell diversity, homeostasis and lifespan.

Open-source microscope add-on for structured illumination microscopy.

Super-resolution techniques expand the abilities of researchers who have the knowledge and resources to either build or purchase a system. This excludes the part of the research community without these capabilities. Here we introduce the openSIM add-on to upgrade existing optical microscopes to Structured Illumination super-resolution Microscopes (SIM).

Bioengineered Tubular Biliary Organoids.

Liver organoids have emerged as promising in vitro models for toxicology, drug discovery, and disease modeling. However, conventional 3D epithelial organoid culture systems suffer from significant drawbacks, including limited culture duration, a nonphysiological 3D cystic anatomy with an inaccessible apical surface, and lack of in vivo-like cellular organization. To address these limitations, herein a hydrogel-based organoid-on-a-chip model for the development functional tubular biliary organoids is reported.

Embryo-uterine interaction coordinates mouse embryogenesis during implantation.

Embryo implantation into the uterus marks a key transition in mammalian development. In mice, implantation is mediated by the trophoblast and is accompanied by a morphological transition from the blastocyst to the egg cylinder. However, the roles of trophoblast-uterine interactions in embryo morphogenesis during implantation are poorly understood due to inaccessibility in utero and the remaining challenges to recapitulate it ex vivo from the blastocyst.

Multimodal characterization of murine gastruloid development.

Gastruloids are 3D structures generated from pluripotent stem cells recapitulating fundamental principles of embryonic pattern formation. Using single-cell genomic analysis, we provide a resource mapping cell states and types during gastruloid development and compare them with the in vivo embryo. We developed a high-throughput handling and imaging pipeline to spatially monitor symmetry breaking during gastruloid development and report an early spatial variability in pluripotency determining a binary response to Wnt activation.

Erratum: An automated do-it-yourself system for dynamic stem cell and organoid culture in standard multi-well plates.

[This corrects the article DOI: 10.1016/j.crmeth.

Self-organizing in vitro mouse neural tube organoids mimic embryonic development.

The embryonic neural tube is the origin of the entire adult nervous system, and disturbances in its development cause life-threatening birth defects. However, the study of mammalian neural tube development is limited by the lack of physiologically realistic three-dimensional (3D) in vitro models. Here, we report a self-organizing 3D neural tube organoid model derived from single mouse embryonic stem cells that exhibits an in vivo-like tissue architecture, cell type composition and anterior-posterior (AP) patterning.

Organoids as tools for fundamental discovery and translation-a Keystone Symposia report.

Complex three-dimensional in vitro organ-like models, or organoids, offer a unique biological tool with distinct advantages over two-dimensional cell culture systems, which can be too simplistic, and animal models, which can be too complex and may fail to recapitulate human physiology and pathology. Significant progress has been made in driving stem cells to differentiate into different organoid types, though several challenges remain. For example, many organoid models suffer from high heterogeneity, and it can be difficult to fully incorporate the complexity of in vivo tissue and organ development to faithfully reproduce human biology.

c-MAF coordinates enterocyte zonation and nutrient uptake transcriptional programs.

Small intestinal villi are structural and functional units present in higher vertebrates and uniquely adapted to nutrient absorption. Villus enterocytes are organized in transcriptional "zones" dedicated to specialized tasks such as absorption of specific nutrients. We report that the transcription factor c-MAF is expressed in differentiated lower and mid-villus enterocytes and is a target of BMP signaling.

Gastruloids as in vitro models of embryonic blood development with spatial and temporal resolution.

Gastruloids are three-dimensional embryonic organoids that reproduce key features of early mammalian development in vitro with unique scalability, accessibility, and spatiotemporal similarity to real embryos. Recently, we adapted the gastruloid culture conditions to promote cardiovascular development. In this work, we extended these conditions to capture features of embryonic blood development through a combination of immunophenotyping, detailed transcriptomics analysis, and identification of blood stem/progenitor cell potency.

An automated do-it-yourself system for dynamic stem cell and organoid culture in standard multi-well plates.

We present a low-cost, do-it-yourself system for complex mammalian cell culture under dynamically changing medium formulations by integrating conventional multi-well tissue culture plates with simple microfluidic control and system automation. We demonstrate the generation of complex concentration profiles, enabling the investigation of sophisticated input-response relations. We further apply our automated cell-culturing platform to the dynamic stimulation of two widely employed stem-cell-based models for early mammalian development: the conversion of naive mouse embryonic stem cells into epiblast-like cells and mouse 3D gastruloids.

Microarrayed human bone marrow organoids for modeling blood stem cell dynamics.

In many leukemia patients, a poor prognosis is attributed either to the development of chemotherapy resistance by leukemic stem cells (LSCs) or to the inefficient engraftment of transplanted hematopoietic stem/progenitor cells (HSPCs) into the bone marrow (BM). Here, we build a 3D model system of bone marrow organoids (BMOs) that recapitulate several structural and cellular components of native BM. These organoids are formed in a high-throughput manner from the aggregation of endothelial and mesenchymal cells within hydrogel microwells.

An engineered multicellular stem cell niche for the 3D derivation of human myogenic progenitors from iPSCs.

Fate decisions in the embryo are controlled by a plethora of microenvironmental interactions in a three-dimensional niche. To investigate whether aspects of this microenvironmental complexity can be engineered to direct myogenic human-induced pluripotent stem cell (hiPSC) differentiation, we here screened murine cell types present in the developmental or adult stem cell niche in heterotypic suspension embryoids. We identified embryonic endothelial cells and fibroblasts as highly permissive for myogenic specification of hiPSCs.

Dissecting mechanisms of chamber-specific cardiac differentiation and its perturbation following retinoic acid exposure.

The specification of distinct cardiac lineages occurs before chamber formation and acquisition of bona fide atrial or ventricular identity. However, the mechanisms underlying these early specification events remain poorly understood. Here, we performed single cell analysis at the murine cardiac crescent, primitive heart tube and heart tube stages to uncover the transcriptional mechanisms underlying formation of atrial and ventricular cells.