COMPLEX IN-VITRO THREE-DIMENSIONAL TUMOR ARCHITECTURE WITH MULTIPLE CELL TYPES

Abstract Organoids are three dimensional structures consisting of multiple cell types that recapitulate the cellular architecture and functionality of native organs. Over the last decade, the advent of organoid research has opened up many avenues for basic and translational studies. Following suit of other disciplines, research groups working in the field of male reproductive biology have started establishing and characterizing testicular organoids. The three-dimensional architectural and functional similarities of organoids to their tissue of origin facilitate study of complex cell interactions, tissue development and establishment of representative, scalable models for drug and toxicity screening. In this review, we discuss the current state of testicular organoid research, their advantages over conventional monolayer culture and their potential applications in the field of reproductive biology and toxicology.

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Generation of rat lungs by blastocyst complementation in Fgfr2b-deficient mouse model

10.1101/2022.01.05.475149 ◽

2022 ◽

Author(s):

Shunsuke Yuri ◽

Yuki Murase ◽

Aayako Isotani

Keyword(s):

Mouse Model ◽

Three Dimensional ◽

Cell Types ◽

Lung Epithelial Cells ◽

Developmental Timing ◽

Deficient Mouse ◽

Blastocyst Complementation ◽

Multiple Cell ◽

Species Specific

Regenerative medicine is a tool to compensate for the shortage of lungs for transplantation, but it remains difficult to construct a lung in vitro due to the complex three-dimensional structures and multiple cell types required. A blastocyst complementation method using interspecies chimeric animals has been attracting attention as a way to create complex organs in animals, but successful lung formation has not yet been achieved. Here, we applied a reverse-blastocyst complementation method to clarify the conditions required to form lungs in an Fgfr2b-deficient mouse model. We then successfully formed a rat-derived lung in the mouse model without generating a mouse line by applying a tetraploid-based organ-complementation method. Importantly, rat lung epithelial cells retained their developmental timing even in the mouse body. This result provides useful insights regarding the need to overcome the barrier of species-specific developmental timing in order to generate functional lungs in interspecies chimeras.

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Epithelial Organotypic Cultures: A Viable Model to Address Mechanisms of Carcinogenesis by Epitheliotropic Viruses

Current Topics in Medicinal Chemistry ◽

10.2174/1568026618666180410125850 ◽

2018 ◽

Vol 18 (4) ◽

pp. 246-255 ◽

Cited By ~ 1

Author(s):

Lara Termini ◽

Enrique Boccardo

Keyword(s):

Three Dimensional ◽

Cell Types ◽

Experimental Models ◽

Biological Research ◽

Specific Gene ◽

Specific Cell ◽

Organotypic Cultures ◽

Skin Physiology

In vitro culture of primary or established cell lines is one of the leading techniques in many areas of basic biological research. The use of pure or highly enriched cultures of specific cell types obtained from different tissues and genetics backgrounds has greatly contributed to our current understanding of normal and pathological cellular processes. Cells in culture are easily propagated generating an almost endless source of material for experimentation. Besides, they can be manipulated to achieve gene silencing, gene overexpression and genome editing turning possible the dissection of specific gene functions and signaling pathways. However, monolayer and suspension cultures of cells do not reproduce the cell type diversity, cell-cell contacts, cell-matrix interactions and differentiation pathways typical of the three-dimensional environment of tissues and organs from where they were originated. Therefore, different experimental animal models have been developed and applied to address these and other complex issues in vivo. However, these systems are costly and time consuming. Most importantly the use of animals in scientific research poses moral and ethical concerns facing a steadily increasing opposition from different sectors of the society. Therefore, there is an urgent need for the development of alternative in vitro experimental models that accurately reproduce the events observed in vivo to reduce the use of animals. Organotypic cultures combine the flexibility of traditional culture systems with the possibility of culturing different cell types in a 3D environment that reproduces both the structure and the physiology of the parental organ. Here we present a summarized description of the use of epithelial organotypic for the study of skin physiology, human papillomavirus biology and associated tumorigenesis.

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MyD88 Is Not Required for Muscle Injury-Induced Endochondral Heterotopic Ossification in a Mouse Model of Fibrodysplasia Ossificans Progressiva

Biomedicines ◽

10.3390/biomedicines9060630 ◽

2021 ◽

Vol 9 (6) ◽

pp. 630

Author(s):

Huili Lyu ◽

Cody M. Elkins ◽

Jessica L. Pierce ◽

C. Henrique Serezani ◽

Daniel S. Perrien

Keyword(s):

Heterotopic Ossification ◽

Muscle Injury ◽

Cell Types ◽

Fibrodysplasia Ossificans Progressiva ◽

Inflammatory Signaling ◽

Conditional Deletion ◽

Pathway Crosstalk ◽

Multiple Cell

Excess inflammation and canonical BMP receptor (BMPR) signaling are coinciding hallmarks of the early stages of injury-induced endochondral heterotopic ossification (EHO), especially in the rare genetic disease fibrodysplasia ossificans progressiva (FOP). Multiple inflammatory signaling pathways can synergistically enhance BMP-induced Smad1/5/8 activity in multiple cell types, suggesting the importance of pathway crosstalk in EHO and FOP. Toll-like receptors (TLRs) and IL-1 receptors mediate many of the earliest injury-induced inflammatory signals largely via MyD88-dependent pathways. Thus, the hypothesis that MyD88-dependent signaling is required for EHO was tested in vitro and in vivo using global or Pdgfrα-conditional deletion of MyD88 in FOP mice. As expected, IL-1β or LPS synergistically increased Activin A (ActA)-induced phosphorylation of Smad 1/5 in fibroadipoprogenitors (FAPs) expressing Alk2R206H. However, conditional deletion of MyD88 in Pdgfrα-positive cells of FOP mice did not significantly alter the amount of muscle injury-induced EHO. Even more surprisingly, injury-induced EHO was not significantly affected by global deletion of MyD88. These studies demonstrate that MyD88-dependent signaling is dispensable for injury-induced EHO in FOP mice.

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Human iPSC-Based Modeling of Central Nerve System Disorders for Drug Discovery

International Journal of Molecular Sciences ◽

10.3390/ijms22031203 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1203

Author(s):

Lu Qian ◽

Julia TCW

Keyword(s):

Drug Discovery ◽

Disease Modeling ◽

Three Dimensional ◽

Structural Complexity ◽

Induced Pluripotent Stem Cell ◽

Cell Types ◽

Central Nerve System ◽

Human Ipscs ◽

Nerve System

A high-throughput drug screen identifies potentially promising therapeutics for clinical trials. However, limitations that persist in current disease modeling with limited physiological relevancy of human patients skew drug responses, hamper translation of clinical efficacy, and contribute to high clinical attritions. The emergence of induced pluripotent stem cell (iPSC) technology revolutionizes the paradigm of drug discovery. In particular, iPSC-based three-dimensional (3D) tissue engineering that appears as a promising vehicle of in vitro disease modeling provides more sophisticated tissue architectures and micro-environmental cues than a traditional two-dimensional (2D) culture. Here we discuss 3D based organoids/spheroids that construct the advanced modeling with evolved structural complexity, which propels drug discovery by exhibiting more human specific and diverse pathologies that are not perceived in 2D or animal models. We will then focus on various central nerve system (CNS) disease modeling using human iPSCs, leading to uncovering disease pathogenesis that guides the development of therapeutic strategies. Finally, we will address new opportunities of iPSC-assisted drug discovery with multi-disciplinary approaches from bioengineering to Omics technology. Despite technological challenges, iPSC-derived cytoarchitectures through interactions of diverse cell types mimic patients’ CNS and serve as a platform for therapeutic development and personalized precision medicine.

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Spheroid confrontation assay: A simple method to monitor the three-dimensional migration of different cell types in vitro

Annals of Anatomy - Anatomischer Anzeiger ◽

10.1016/j.aanat.2010.12.005 ◽

2011 ◽

Vol 193 (3) ◽

pp. 181-184 ◽

Cited By ~ 22

Author(s):

Kirsten Hattermann ◽

Janka Held-Feindt ◽

Rolf Mentlein

Keyword(s):

Three Dimensional ◽

Cell Types ◽

Simple Method ◽

Different Cell Types ◽

Confrontation Assay

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High-precision 3D inkjet technology for live cell bioprinting

International Journal of Bioprinting ◽

10.18063/ijb.v5i2.208 ◽

2019 ◽

Vol 5 (2) ◽

pp. 27 ◽

Cited By ~ 3

Author(s):

Daisuke Takagi ◽

Waka Lin ◽

Takahiko Matsumoto ◽

Hidekazu Yaginuma ◽

Natsuko Hemmi ◽

...

Keyword(s):

Three Dimensional ◽

Cell Types ◽

Cell Number ◽

Live Cell ◽

Drop On Demand ◽

Long Time ◽

Spatial Positioning ◽

Different Cell Types ◽

Membrane Vibrations

In recent years, bioprinting has emerged as a promising technology for the construction of three-dimensional (3D) tissues to be used in regenerative medicine or in vitro screening applications. In the present study, we present the development of an inkjet-based bioprinting system to arrange multiple cells and materials precisely into structurally organized constructs. A novel inkjet printhead has been specially designed for live cell ejection. Droplet formation is powered by piezoelectric membrane vibrations coupled with mixing movements to prevent cell sedimentation at the nozzle. Stable drop-on-demand dispensing and cell viability were validated over an adequately long time to allow the fabrication of 3D tissues. Reliable control of cell number and spatial positioning was demonstrated using two separate suspensions with different cell types printed sequentially. Finally, a process for constructing stratified Mille-Feuille-like 3D structures is proposed by alternately superimposing cell suspensions and hydrogel layers with a controlled vertical resolution. The results show that inkjet technology is effective for both two-dimensional patterning and 3D multilayering and has the potential to facilitate the achievement of live cell bioprinting with an unprecedented level of precision.

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Hydrogel-Coated Textile Scaffolds as Three-Dimensional Growth Support for Human Umbilical Vein Endothelial Cells (HUVECs): Possibilities as Coculture System in Liver Tissue Engineering

Cell Transplantation ◽

10.3727/000000002783985837 ◽

2002 ◽

Vol 11 (4) ◽

pp. 369-377 ◽

Cited By ~ 24

Author(s):

Makarand V. Risbud ◽

Erdal Karamuk ◽

René Moser ◽

Joerg Mayer

Keyword(s):

Endothelial Cells ◽

Umbilical Vein ◽

Three Dimensional ◽

Mesh Size ◽

Cell Types ◽

Cell Number ◽

Human Umbilical Vein ◽

Hydrogel Matrix ◽

Umbilical Vein Endothelial Cells

Three-dimensional (3-D) scaffolds offer an exciting possibility to develop cocultures of various cell types. Here we report chitosan–collagen hydrogel-coated fabric scaffolds with defined mesh size and fiber diameter for 3-D culture of human umbilical vein endothelial cells (HUVECs). These scaffolds did not require pre-coating with fibronectin and they supported proper HUVEC attachment and growth. Scaffolds preserved endothelial cell-specific cobblestone morphology and cells were growing in compartments defined by the textile mesh. HUVECs on the scaffold maintained the property of contact inhibition and did not exhibit overgrowth until the end of in vitro culture (day 6). MTT assay showed that cells had preserved mitochondrial functionality. It was also noted that cell number on the chitosan-coated scaffold was lower than that of collagen-coated scaffolds. Calcein AM and ethidium homodimer (EtD-1) dual staining demonstrated presence of viable and metabolically active cells, indicating growth supportive properties of the scaffolds. Actin labeling revealed absence of actin stress fibers and uniform distribution of F-actin in the cells, indicating their proper attachment to the scaffold matrix. Confocal microscopic studies showed that HUVECs growing on the scaffold had preserved functionality as seen by expression of von Willebrand (vW) factor. Observations also revealed that functional HUVECs were growing at various depths in the hydrogel matrix, thus demonstrating the potential of these scaffolds to support 3-D growth of cells. We foresee the application of this scaffold system in the design of liver bioreactors wherein hepatocytes could be cocultured in parallel with endothelial cells to enhance and preserve liver-specific functions.

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TMOD-21. A NOVEL IN-VITRO METHOD TO MODEL MACROPHAGES IN GLIOBLASTOMA

Neuro-Oncology ◽

10.1093/neuonc/noab196.882 ◽

2021 ◽

Vol 23 (Supplement_6) ◽

pp. vi220-vi220

Author(s):

Hasan Alrefai ◽

Andee Beierle ◽

Lauren Nassour ◽

Nicholas Eustace ◽

Zeel Patel ◽

...

Keyword(s):

Cell Types ◽

In Vitro Models ◽

Tumor Initiating Cells ◽

Serum Free ◽

Brain Tumor Initiating Cells ◽

Multiple Cell ◽

Anti Inflammatory ◽

Free Media ◽

Inflammatory Macrophages

Abstract BACKGROUND The GBM tumor microenvironment (TME) is comprised of a plethora of cancerous and non-cancerous cells that contribute to GBM growth, invasion, and chemoresistance. In-vitro models of GBM typically fail to incorporate multiple cell types. Others have addressed this problem by employing 3D bioprinting to incorporate astrocytes and macrophages in an extracellular matrix; however, they used serum-containing media and classically polarized anti-inflammatory macrophages. Serum has been shown to cause GBM brain-tumor initiating cells to lose their stem-like properties, highlighting the importance of excluding it from these models. Additionally, tumor-associated macrophages (TAMs) do not adhere to the traditional M2 phenotype. METHODS THP-1 monocytes and normal human astrocytes (NHAs) were transitioned into serum-free HL-1 and neurobasal-based media, respectively. Monocytes were stimulated towards a macrophage-like state with PMA and polarized by co-culturing them with GBM patient-derived xenograft(PDX) lines, using a transwell insert. CD206 expression was used to validate polarization and a cytokine array was used to characterize the cells. RESULTS There was no difference in proliferation rates at 72 hours for THP-1 monocytes grown in serum-free HL-1 media compared to serum-containing RPMI 1640 (p > 0.95). Macrophages polarized via transwell inserts expressed the lymphocyte chemoattractant protein, CCL2, whereas resting(M0), pro-inflammatory(M1), and anti-inflammatory(M2) macrophages did not. Additionally, these macrophages expressed more CXCL1 and IL-1ß relative to M1 macrophages. We have also demonstrated a method to maintain a tri-culture model of GBM PDX cells, NHAs, and TAMs in a serum-free media that supports the growth/maintenance of all cell types. CONCLUSIONS We have demonstrated a novel method by which we can polarize macrophages towards a tumor-supportive phenotype that differs in cytokine expression from traditionally polarized macrophages. This higher-fidelity method of modeling TAMs in GBM can aid in the development of targeted therapeutics that may one day enter the clinic in hopes of improving outcomes in GBM.

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Tough Double-Network Hydrogels as Scaffolds for Tissue Engineering

Technological Advancements in Biomedicine for Healthcare Applications - Advances in Bioinformatics and Biomedical Engineering ◽

10.4018/978-1-4666-2196-1.ch023 ◽

2012 ◽

pp. 213-222

Author(s):

Jing Jing Yang ◽

Jian Fang Liu ◽

Takayuki Kurokawa ◽

Nobuto Kitamura ◽

Kazunori Yasuda ◽

...

Keyword(s):

Tissue Engineering ◽

Three Dimensional ◽

Cartilage Regeneration ◽

Cell Types ◽

Embryoid Bodies ◽

Living Tissue ◽

Double Network ◽

Dimensional Network

Hydrogels are used as scaffolds for tissue engineering in vitro & in vivo because their three-dimensional network structure and viscoelasticity are similar to those of the macromolecular-based extracellular matrix (ECM) in living tissue. Especially, the synthetic hydrogels with controllable and reproducible properties were used as scaffolds to study the behaviors of cells in vitro and implanted test in vivo. In this review, two different structurally designed hydrogels, single-network (SN) hydrogels and double-network (DN) hydrogels, were used as scaffolds. The behavior of two cell types, anchorage-dependent cells and anchorage-independent cells, and the differentiation behaviors of embryoid bodies (EBs) were investigated on these hydrogels. Furthermore, the behavior of chondrocytes on DN hydrogels in vitro and the spontaneous cartilage regeneration induced by DN hydrogels in vivo was examined.

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