Special review series on 3D organotypic culture models: Introduction and historical perspective

Patrick J. Hayden , John W. Harbell

Three dimensional (3D) organ-like (organotypic) culture models are a rapidly advancing area of in vitro biological science. In contrast to monolayer cell culture methods which were developed to achieve proliferation of animal cells in the beginning of in vitro biology, the 3D culture methods are designed to promote cellular differentiation, and to achieve in vivo-like 3D structure and organotypic functions. 3D organotypic models may be composed of a single cell type, such as in simple reconstructed human epidermal, corneal or airway epithelial models.  More advanced co-culture models that consist of multiple cell types may include stromal components and/or other types of functional cells such as fibroblasts, pericytes, melanocytes or immune cells. This project was conceived under the auspices of the Society for In Vitro Biology to draw from the experiences of individual authors who are experts in the development and application of specific 3D organotypic constructs. Each author has been asked to prepare a review that focuses on a specific tissue type in 3D organotypic culture. Our introductory paper is intended to provide a brief overview and historical perspective of some of the major achievements of the pioneers in early tissue culture who set the stage for the science as we know it today. Early leaders appreciated the limitations of the monolayer systems for cellular differentiation. These limitations were particularly evident in the efforts to produce functionally differentiated parenchymal cells of human liver, mammary, skin and other organs. Based on work with many types of cells, it became clear that submerged monolayer culture conditions were missing essential influences that are required for functional differentiation. 

Patrick J. Hayden, John W. Harbell. Special review series on 3D organotypic culture models: introduction and historical perspective. In Vitro Cellular Developmental Biology – Animal, 57:95-103, 2021. 

In vitro reconstructed 3D corneal tissue models for ocular toxicology and ophthalmic drug development

Yulia Kaluzhny & Mitchell Klausner 

The goal of replacing the Draize rabbit eye irritation test has spurred the development of numerous 2-dimensional (2D) monolayer culture systems and 3-dimensional (3D) corneal tissue equivalent models.  These alternatives have been used extensively to test a plethora of cosmetic, personal care, household product, chemical, and pharmaceutical products to satisfy regulatory requirement for determining eye irritation. In addition, more complex 3D corneal equivalent models have been developed to study the permeation of ophthalmic pharmaceuticals, wound healing, drug bioavailability, and ocular diseases.  This paper reviews in vitro reconstructed 3D corneal tissue models and their utilization in ocular toxicology as well as their application in pharmacology and ophthalmic research. 

Yulia Kaluzhny and Mitchell Klausner.  In vitro reconstructed 3D corneal tissue models for ocular toxicology and ophthalmic drug development.In Vitro Cellular & Developmental Biology – Animal, 57:207-237, 2021.     

In Vitro Three Dimensional Organotypic Culture Models of the Human Mucosa

From left to right: Mitchell Klausner, Yuki Handa, and Seiya Aizawa

Three dimensional (3D), organotypic tissue models offer insight into tissue specific phenomena which monolayer cultures cannot provide.  In addition to recapitulating the 3D structure of the tissue, the barrier properties, reactions to xenobiotic materials, and the underlying biochemistry of these models more closely approximate those of native tissues.  Also, many of these models are cultured using primary human cells.  This paper reviews commercially available models of the human gingival and buccal mucosa.  Utilization of these tissue models to investigate irritancy caused by oral care products, the effects of cigarette smoke, the mechanisms of oral mucositis and oral candidiasis, transbuccal drug delivery rates, infection by HIV-1, the effects of commensal and pathogenic bacteria, and the absorption of nanoparticles are reviewed.   QC testing in the US and Japan demonstrate the suitability of these models for worldwide shipment.  As presented in the paper, the models are used in a broad variety of applications and often offer advantages versus animal models in terms of reproducibility, avoiding species extrapolation, and the ethical concerns related to animal experimentation.   

Mitchell Klausner, Yuki Handa, Seiya Aizawa. In vitro three-dimensional organotypic culture models of the oral mucosa.In Vitro Cellular & Developmental Biology – Animal, 57:148-159, 2021. 

Human air-liquid-interface organotypic airway tissue models derived from primary tracheobronchial epithelial cells-overview and perspectives

Top row: Xuefei Cao, Jayme P. Coyle, Rui Xiong, Yiying Wang; Bottom row: Robert H. Heflich, Baiping Ren, William M. Gwinn, Patrick Hayden, Liying Rojanasakul

The lung is the key interface between the external environment and the systemic circulation. Although there are elaborate and effective mechanisms protecting the lung and maintaining its homeostasis, the respiratory system remains highly susceptible to respiratory pathogens, environmental pollutants, tobacco smoke, and allergens. Given the impact of respiratory exposure on human health and diseases, there has been a longstanding interest in the development of reliable and predictive in vitro model systems for respiratory toxicology research. Submerged monolayer cultures of human lung origin have been used for decades; however, the cells in these cultures lack the differentiated phenotypes of the in vivo airway epithelium and do not grow under the native air interface as occurs in vivo. To overcome these limitations, alternative airway models have been developed. Among them is the well-differentiated air-liquid-interface (ALI) airway model that possesses multiple types of epithelial cells as well as an air interface that can be exposed directly to airborne substances. This article provides a comprehensive overview of the lower large airway ALI tissue model. The history of its development, its structure and functions, methods for conducting ALI exposures, and key applications for chemical toxicity testing, pulmonary drug evaluation, and host-pathogen interactions are reviewed. The strengths and limitations of the current technology, status of using these in vitro systems as a supplement or replacement for animal models, and recommendations for future directions also are discussed.

Xuefei Cao, Jayme P. Coyle, Rui Xiong, Yiying Wang, Robert H. Heflich, Baiping Ren, William M. Gwinn, Patrick Hayden, and Liying Rojanasakul. Human air-liquid-interface organotypic airway tissue models derived from primary tracheobronchial epithelial cells-overview and perspectives. In Vitro Cellular & Developmental Biology – Animal,57:104-132, 2021.

Expression of storage lipid biosynthesis transcription factors and enzymes in Jatropha curcas L. cell suspension cultures and seeds

Members of Biotechnology laboratory at Universidad de Antioquia, Colombia, including the authors. First row from left to right: Laura Michell Carmona (first author), Dr. Aura Urrea, Dr. Natalia Pabón (Evo-Devo in Plants), Dr. Lucia Atehortua (Director). Second row from left to right: Ana Maria Henao. Tatiana Osorio, Liliana Monsalve, Juan Felipe Tamayo. Third row from left to right: Catalina Botero, Sandra Macias, Maria Isabel Quintero, Anngy Amaya. Fourth row from left to right: Erika Obando, Dr. Adriana Gallego, Liuda Sepulveda, Monica Arias.  

The oleaginous plant Jatropha curcas has been proposed as a promising source for biodiesel production, using its seeds or in vitro production in cell cultures. In this paper, we showed a new approach to understand the regulation of storage lipids. We compared gene expression between endosperm cells in planta and endosperm-derived cell suspension cultures (EDCCs). We found a unique expression of some transcription factors that could participate in regulating different processes, among them LEC1, FUS3, ABI3, and WRI1. They could play a pivotal role in regulating the early stages of seed development and control the accumulation of storage compounds during the maturation of J. curcas seeds. Conversely, these genes displayed a lower genetic expression in cell suspensions and less content of total lipids, except for WRI1, which had comparable expression levels in the two systems. The remarkable differences between both types of cells allowed us to establish the important role of those genes in regulating storage lipids biosynthesis. Our results provide valuable information to understand the regulation processes carried out during seed development. Moreover, plant suspension cultures have proven to be an invaluable system to carry out studies at biochemical and molecular levels for lipids and other metabolites and future biotechnological applications. In this study, we test the Jatropha cell suspensions as an oil-producing platform in vitro and provide evidence of changes in response to the carbon-nitrogen ratio. We lay the foundation for future studies on carbon flow towards the biosynthesis of both pathways, starches, and oils in this oleaginous plant. The main areas of the Biotechnology laboratory include biodiversity, biotechnology of plants, microalgae, and fungi. Currently, our lab is working on several projects, like the establishment of embryogenic suspension cultures of Theobroma cacao as a model to study the gene expression of transcription factors involved in the acquisition of embryogenic potential and the optimization of the storage compounds accumulation on somatic embryos to improve plant conversion rates.

Laura Carmona-Rojas, Aura Urrea-Trujillo, Daniel Gil-Arrendondo, Lucia Atehortúa-Garcés, Natalia Pabón-Mora. Expression of storage lipid biosynthesis transcription factors and enzymes in Jatropha curcas L. cell suspension cultures and seeds. In Vitro Cellular & Developmental Biology – Plant, 57, 164-177, 2021.

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