Cells

Research

Jump to: Review

14 pages, 17095 KiB

Open AccessArticle

Cell Dome as an Evaluation Platform for Organized HepG2 Cells

by Ryotaro Kazama, Satoshi Fujita and Shinji Sakai

Cells 2023, 12(1), 69; https://doi.org/10.3390/cells12010069 - 23 Dec 2022

Cited by 2 | Viewed by 2495

Abstract

Human-hepatoblastoma-derived cell line, HepG2, has been widely used in liver and liver cancer studies. HepG2 spheroids produced in a three-dimensional (3D) culture system provide a better biological model than cells cultured in a two-dimensional (2D) culture system. Since cells at the center of [...] Read more.

Human-hepatoblastoma-derived cell line, HepG2, has been widely used in liver and liver cancer studies. HepG2 spheroids produced in a three-dimensional (3D) culture system provide a better biological model than cells cultured in a two-dimensional (2D) culture system. Since cells at the center of spheroids exhibit specific behaviors attributed to hypoxic conditions, a 3D cell culture system that allows the observation of such cells using conventional optical or fluorescence microscopes would be useful. In this study, HepG2 cells were cultured in “Cell Dome”, a micro-dome in which cells are enclosed in a cavity consisting of a hemispherical hydrogel shell. HepG2 cells formed hemispherical cell aggregates which filled the cavity of Cell Domes on 18 days of culture and the cells could continue to be cultured for 29 days. The cells at the center of hemispherical cell aggregates were observed using a fluorescence microscope. The cells grew in Cell Domes for 18 days exhibited higher Pi-class Glutathione S-Transferase enzymatic activity, hypoxia inducible factor-1α gene expression, and higher tolerance to mitomycin C than those cultured in 2D on tissue culture dishes (* p < 0.05). These results indicate that the center of the glass adhesive surface of hemispherical cell aggregates which is expected to have the similar environment as the center of the spheroids can be directly observed through glass plates. In conclusion, Cell Dome would be useful as an evaluation platform for organized HepG2 cells. Full article

(This article belongs to the Special Issue Cell and Tissue Engineering for Functional Analysis)

► Show Figures

Figure 1

25 pages, 4671 KiB

Open AccessArticle

Liposome-Mediated Gene Transfer in Differentiated HepaRG™ Cells: Expression of Liver Specific Functions and Application to the Cytochrome P450 2D6 Expression

by Manuel Vlach, Hugo Coppens-Exandier, Agnès Jamin, Mathieu Berchel, Julien Scaviner, Christophe Chesné, Tristan Montier, Paul-Alain Jaffrès, Anne Corlu and Pascal Loyer

Cells 2022, 11(23), 3904; https://doi.org/10.3390/cells11233904 - 2 Dec 2022

Cited by 5 | Viewed by 2731

Abstract

The goal of this study was to establish a procedure for gene delivery mediated by cationic liposomes in quiescent differentiated HepaRG™ human hepatoma cells. We first identified several cationic lipids promoting efficient gene transfer with low toxicity in actively dividing HepG2, HuH7, BC2 [...] Read more.

The goal of this study was to establish a procedure for gene delivery mediated by cationic liposomes in quiescent differentiated HepaRG™ human hepatoma cells. We first identified several cationic lipids promoting efficient gene transfer with low toxicity in actively dividing HepG2, HuH7, BC2 and progenitor HepaRG™ human hepatoma cells. The lipophosphoramidate Syn1-based nanovector, which allowed the highest transfection efficiencies of progenitor HepaRG™ cells, was next used to transfect differentiated HepaRG™ cells. Lipofection of these cells using Syn1-based liposome was poorly efficient most likely because the differentiated HepaRG™ cells are highly quiescent. Thus, we engineered the differentiated HepaRG™ Mitogenic medium supplement (ADD1001) that triggered robust proliferation of differentiated cells. Importantly, we characterized the phenotypical changes occurring during proliferation of differentiated HepaRG™ cells and demonstrated that mitogenic stimulation induced a partial and transient decrease in the expression levels of some liver specific functions followed by a fast recovery of the full differentiation status upon removal of the mitogens. Taking advantage of the proliferation of HepaRG™ cells, we defined lipofection conditions using Syn1-based liposomes allowing transient expression of the cytochrome P450 2D6, a phase I enzyme poorly expressed in HepaRG cells, which opens new means for drug metabolism studies in HepaRG™ cells. Full article

(This article belongs to the Special Issue Cell and Tissue Engineering for Functional Analysis)

► Show Figures

Figure 1

11 pages, 1644 KiB

Open AccessArticle

Alignment of Skeletal Muscle Cells Facilitates Acetylcholine Receptor Clustering and Neuromuscular Junction Formation with Co-Cultured Human iPSC-Derived Motor Neurons

by Kazunori Shimizu, Haruo Kassai, Yuhei Kamei, Kazuki Yamamoto, Takunori Nagashima, Tadayoshi Maekawa, Hirokazu Akiyama and Hiroyuki Honda

Cells 2022, 11(23), 3760; https://doi.org/10.3390/cells11233760 - 24 Nov 2022

Cited by 4 | Viewed by 2488

Abstract

In vitro neuromuscular junction (NMJ) models are powerful tools for studying neuromuscular disorders. Although linearly patterned culture surfaces have been reported to be useful for the formation of in vitro NMJ models using mouse motor neuron (MNs) and skeletal muscle (SkM) myotubes, it [...] Read more.

In vitro neuromuscular junction (NMJ) models are powerful tools for studying neuromuscular disorders. Although linearly patterned culture surfaces have been reported to be useful for the formation of in vitro NMJ models using mouse motor neuron (MNs) and skeletal muscle (SkM) myotubes, it is unclear how the linearly patterned culture surface increases acetylcholine receptor (AChR) clustering, one of the steps in the process of NMJ formation, and whether this increases the in vitro NMJ formation efficiency of co-cultured human MNs and SkM myotubes. In this study, we investigated the effects of a linearly patterned culture surface on AChR clustering in myotubes and examined the possible mechanism of the increase in AChR clustering using gene expression analysis, as well as the effects of the patterned surface on the efficiency of NMJ formation between co-cultured human SkM myotubes and human iPSC-derived MNs. Our results suggest that better differentiation of myotubes on the patterned surface, compared to the flat surface, induced gene expression of integrin α7 and AChR ε-subunit, thereby increasing AChR clustering. Furthermore, we found that the number of NMJs between human SkM cells and MNs increased upon co-culture on the linearly patterned surface, suggesting the usefulness of the patterned surface for creating in vitro human NMJ models. Full article

(This article belongs to the Special Issue Cell and Tissue Engineering for Functional Analysis)

► Show Figures

Figure 1

17 pages, 2393 KiB

Open AccessArticle

Construction of a Versatile, Programmable RNA-Binding Protein Using Designer PPR Proteins and Its Application for Splicing Control in Mammalian Cells

by Yusuke Yagi, Takamasa Teramoto, Shuji Kaieda, Takayoshi Imai, Tadamasa Sasaki, Maiko Yagi, Nana Maekawa and Takahiro Nakamura

Cells 2022, 11(22), 3529; https://doi.org/10.3390/cells11223529 - 8 Nov 2022

Cited by 6 | Viewed by 3265

Abstract

RNAs play many essential roles in gene expression and are involved in various human diseases. Although genome editing technologies have been established, the engineering of sequence-specific RNA-binding proteins that manipulate particular cellular RNA molecules is immature, in contrast to nucleotide-based RNA manipulation technology, [...] Read more.

RNAs play many essential roles in gene expression and are involved in various human diseases. Although genome editing technologies have been established, the engineering of sequence-specific RNA-binding proteins that manipulate particular cellular RNA molecules is immature, in contrast to nucleotide-based RNA manipulation technology, such as siRNA- and RNA-targeting CRISPR/Cas. Here, we demonstrate a versatile RNA manipulation technology using pentatricopeptide-repeat (PPR)-motif-containing proteins. First, we developed a rapid construction and evaluation method for PPR-based designer sequence-specific RNA-binding proteins. This system has enabled the steady construction of dozens of functional designer PPR proteins targeting long 18 nt RNA, which targets a single specific RNA in the mammalian transcriptome. Furthermore, the cellular functionality of the designer PPR proteins was first demonstrated by the control of alternative splicing of either a reporter gene or an endogenous CHK1 mRNA. Our results present a versatile protein-based RNA manipulation technology using PPR proteins that facilitates the understanding of unknown RNA functions and the creation of gene circuits and has potential for use in future therapeutics. Full article

(This article belongs to the Special Issue Cell and Tissue Engineering for Functional Analysis)

► Show Figures

Figure 1

23 pages, 7634 KiB

Open AccessArticle

Development of Human Pituitary Neuroendocrine Tumor Organoids to Facilitate Effective Targeted Treatments of Cushing’s Disease

by Jayati Chakrabarti, Ritu Pandey, Jared M. Churko, Jennifer Eschbacher, Saptarshi Mallick, Yuliang Chen, Beth Hermes, Palash Mallick, Ben N. Stansfield, Kelvin W. Pond, Curtis A. Thorne, Kevin C. J. Yuen, Andrew S. Little and Yana Zavros

Cells 2022, 11(21), 3344; https://doi.org/10.3390/cells11213344 - 23 Oct 2022

Cited by 7 | Viewed by 4565

Abstract

(1) Background: Cushing’s disease (CD) is a serious endocrine disorder caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary neuroendocrine tumor (PitNET) that stimulates the adrenal glands to overproduce cortisol. Chronic exposure to excess cortisol has detrimental effects on health, including increased stroke rates, diabetes, [...] Read more.

(1) Background: Cushing’s disease (CD) is a serious endocrine disorder caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary neuroendocrine tumor (PitNET) that stimulates the adrenal glands to overproduce cortisol. Chronic exposure to excess cortisol has detrimental effects on health, including increased stroke rates, diabetes, obesity, cognitive impairment, anxiety, depression, and death. The first-line treatment for CD is pituitary surgery. Current surgical remission rates reported in only 56% of patients depending on several criteria. The lack of specificity, poor tolerability, and low efficacy of the subsequent second-line medical therapies make CD a medical therapeutic challenge. One major limitation that hinders the development of specific medical therapies is the lack of relevant human model systems that recapitulate the cellular composition of PitNET microenvironment. (2) Methods: human pituitary tumor tissue was harvested during transsphenoidal surgery from CD patients to generate organoids (hPITOs). (3) Results: hPITOs generated from corticotroph, lactotroph, gonadotroph, and somatotroph tumors exhibited morphological diversity among the organoid lines between individual patients and amongst subtypes. The similarity in cell lineages between the organoid line and the patient’s tumor was validated by comparing the neuropathology report to the expression pattern of PitNET specific markers, using spectral flow cytometry and exome sequencing. A high-throughput drug screen demonstrated patient-specific drug responses of hPITOs amongst each tumor subtype. Generation of induced pluripotent stem cells (iPSCs) from a CD patient carrying germline mutation CDH23 exhibited dysregulated cell lineage commitment. (4) Conclusions: The human pituitary neuroendocrine tumor organoids represent a novel approach in how we model complex pathologies in CD patients, which will enable effective personalized medicine for these patients. Full article

(This article belongs to the Special Issue Cell and Tissue Engineering for Functional Analysis)

► Show Figures

Figure 1

16 pages, 3121 KiB

Open AccessArticle

HepG2-Based Designer Cells with Heat-Inducible Enhanced Liver Functions

by Hiroyuki Kitano, Yoshinori Kawabe and Masamichi Kamihira

Cells 2022, 11(7), 1194; https://doi.org/10.3390/cells11071194 - 1 Apr 2022

Cited by 4 | Viewed by 3622

Abstract

Functional human hepatocytes have been a pivotal tool in pharmacological studies such as those investigating drug metabolism and hepatotoxicity. However, primary human hepatocytes are difficult to obtain in large quantities and may cause ethical problems, necessitating the development of a new cell source [...] Read more.

Functional human hepatocytes have been a pivotal tool in pharmacological studies such as those investigating drug metabolism and hepatotoxicity. However, primary human hepatocytes are difficult to obtain in large quantities and may cause ethical problems, necessitating the development of a new cell source to replace human primary hepatocytes. We previously developed genetically modified murine hepatoma cell lines with inducible enhanced liver functions, in which eight liver-enriched transcription factor (LETF) genes were introduced into hepatoma cells as inducible transgene expression cassettes. Here, we establish a human hepatoma cell line with heat-inducible liver functions using HepG2 cells. The genetically modified hepatoma cells, designated HepG2/8F_HS, actively proliferated under normal culture conditions and, therefore, can be easily prepared in large quantities. When the expression of LETFs was induced by heat treatment at 43 °C for 30 min, cells ceased proliferation and demonstrated enhanced liver functions. Furthermore, three-dimensional spheroid cultures of HepG2/8F_HS cells showed a further increase in liver functions upon heat treatment. Comprehensive transcriptome analysis using DNA microarrays revealed that HepG2/8F_HS cells had enhanced overall expression of many liver function-related genes following heat treatment. HepG2/8F_HS cells could be useful as a new cell source for pharmacological studies and for constructing bioartificial liver systems. Full article

(This article belongs to the Special Issue Cell and Tissue Engineering for Functional Analysis)

► Show Figures

Figure 1

13 pages, 3815 KiB

Open AccessArticle

Contractile Activity of Myotubes Derived from Human Induced Pluripotent Stem Cells: A Model of Duchenne Muscular Dystrophy

by Kantaro Yoshioka, Akira Ito, Masanobu Horie, Kazushi Ikeda, Sho Kataoka, Keiichiro Sato, Taichi Yoshigai, Hidetoshi Sakurai, Akitsu Hotta, Yoshinori Kawabe and Masamichi Kamihira

Cells 2021, 10(10), 2556; https://doi.org/10.3390/cells10102556 - 27 Sep 2021

Cited by 5 | Viewed by 3106

Abstract

Duchenne muscular dystrophy (DMD) is a genetic disorder that results from deficiency of the dystrophin protein. In recent years, DMD pathological models have been created using induced pluripotent stem (iPS) cells derived from DMD patients. In addition, gene therapy using CRISPR-Cas9 technology to [...] Read more.

Duchenne muscular dystrophy (DMD) is a genetic disorder that results from deficiency of the dystrophin protein. In recent years, DMD pathological models have been created using induced pluripotent stem (iPS) cells derived from DMD patients. In addition, gene therapy using CRISPR-Cas9 technology to repair the dystrophin gene has been proposed as a new treatment method for DMD. However, it is not known whether the contractile function of myotubes derived from gene-repaired iPS cells can be restored. We therefore investigated the maturation of myotubes in electrical pulse stimulation culture and examined the effect of gene repair by observing the contractile behaviour of myotubes. The contraction activity of myotubes derived from dystrophin-gene repaired iPS cells was improved by electrical pulse stimulation culture. The iPS cell method used in this study for evaluating muscle contractile activity is a useful technique for analysing the mechanism of hereditary muscular disease pathogenesis and for evaluating the efficacy of new drugs and gene therapy. Full article

(This article belongs to the Special Issue Cell and Tissue Engineering for Functional Analysis)

► Show Figures

Figure 1

Review

Jump to: Research

23 pages, 1166 KiB

Open AccessReview

Breakthroughs and Applications of Organ-on-a-Chip Technology

by Mufeeda C. Koyilot, Priyadarshini Natarajan, Clayton R. Hunt, Sonish Sivarajkumar, Romy Roy, Shreeram Joglekar, Shruti Pandita, Carl W. Tong, Shamsudheen Marakkar, Lakshminarayanan Subramanian, Shalini S. Yadav, Anoop V. Cherian, Tej K. Pandita, Khader Shameer and Kamlesh K. Yadav

Cells 2022, 11(11), 1828; https://doi.org/10.3390/cells11111828 - 2 Jun 2022

Cited by 39 | Viewed by 11278

Abstract

Organ-on-a-chip (OOAC) is an emerging technology based on microfluid platforms and in vitro cell culture that has a promising future in the healthcare industry. The numerous advantages of OOAC over conventional systems make it highly popular. The chip is an innovative combination of [...] Read more.

Organ-on-a-chip (OOAC) is an emerging technology based on microfluid platforms and in vitro cell culture that has a promising future in the healthcare industry. The numerous advantages of OOAC over conventional systems make it highly popular. The chip is an innovative combination of novel technologies, including lab-on-a-chip, microfluidics, biomaterials, and tissue engineering. This paper begins by analyzing the need for the development of OOAC followed by a brief introduction to the technology. Later sections discuss and review the various types of OOACs and the fabrication materials used. The implementation of artificial intelligence in the system makes it more advanced, thereby helping to provide a more accurate diagnosis as well as convenient data management. We introduce selected OOAC projects, including applications to organ/disease modelling, pharmacology, personalized medicine, and dentistry. Finally, we point out certain challenges that need to be surmounted in order to further develop and upgrade the current systems. Full article

(This article belongs to the Special Issue Cell and Tissue Engineering for Functional Analysis)

► Show Figures

Graphical abstract

Journal Menu

Journal Browser

Cell and Tissue Engineering for Functional Analysis

Share This Special Issue

Special Issue Editor

Special Issue Information

Keywords

Benefits of Publishing in a Special Issue

Related Special Issue

Published Papers (8 papers)

Research

Review

Further Information

Guidelines

MDPI Initiatives

Follow MDPI