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Development of Cell Culture Technology: Molecular Aspects and Beyond

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A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Toxicology".

Deadline for manuscript submissions: closed (30 September 2021) | Viewed by 22669

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Special Issue Editor

Dr. Sun-Woong Kang

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Guest Editor

Research Group for Biomimetic Advanced Technology, Korea Institute of Toxicology, Yusong, Republic of Korea
Interests: stem cell; tissue engineering; 3D cell culture; alternative toxicology; organoid

Special Issue Information

Dear Colleagues,

There are a number of cell culture systems that are already available. These systems are being used in a variety of research applications such as cancer research, drug and toxicity screening, stem cell research, and regenerative medicines. Therefore, cell culture systems have become very important and useful in toxicology and medicine.

This Special Issue, “Development of Cell Culture Technology: Molecular Aspects and Beyond”, of the International Journal of Molecular Sciences, will comprise a selection of research papers and reviews contributing to the evaluation of specialized cell cultures in toxicology and medicine.

Dr. Sun-Woong Kang
Guest Editor

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. International Journal of Molecular Sciences is an international peer-reviewed open access semimonthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. There is an Article Processing Charge (APC) for publication in this open access journal. For details about the APC please see here. Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

3D cell culture
Molecular toxicology
Cell therapy
Alternative toxicology test
Organoids
Stem cell
Tissue engineering

Published Papers (6 papers)

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Research

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20 pages, 2998 KiB

Open AccessArticle

Determination of Intra- and Extracellular Metabolic Adaptations of 3D Cell Cultures upon Challenges in Real-Time by NMR

by Christian Urzì, Damian Hertig, Christoph Meyer, Sally Maddah, Jean-Marc Nuoffer and Peter Vermathen

Int. J. Mol. Sci. 2022, 23(12), 6555; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms23126555 - 12 Jun 2022

Cited by 1 | Viewed by 1779

Abstract

NMR flow devices provide longitudinal real-time quantitative metabolome characterisation of living cells. However, discrimination of intra- and extracellular contributions to the spectra represents a major challenge in metabolomic NMR studies. The present NMR study demonstrates the possibility to quantitatively measure both metabolic intracellular fingerprints and extracellular footprints on human control fibroblasts by using a commercially available flow tube system with a standard 5 mm NMR probe. We performed a comprehensive 3D cell culture system characterisation. Diffusion NMR was employed for intra- and extracellular metabolites separation. In addition, complementary extracellular footprints were determined. The implemented perfused NMR bioreactor system allowed the determination of 35 metabolites and intra- and extracellular separation of 19 metabolites based on diffusion rate differences. We show the reliability and sensitivity of NMR diffusion measurements to detect metabolite concentration changes in both intra- and extracellular compartments during perfusion with different selective culture media, and upon complex I inhibition with rotenone. We also demonstrate the sensitivity of extracellular footprints to determine metabolic variations at different flow rates. The current method is of potential use for the metabolomic characterisation of defect fibroblasts and for improving physiological comprehension. Full article

(This article belongs to the Special Issue Development of Cell Culture Technology: Molecular Aspects and Beyond)

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13 pages, 5055 KiB

Open AccessArticle

Differences in the Central Energy Metabolism of Cancer Cells between Conventional 2D and Novel 3D Culture Systems

by Ryo Ikari, Ken-ichi Mukaisho, Susumu Kageyama, Masayuki Nagasawa, Shigehisa Kubota, Takahisa Nakayama, Shoko Murakami, Naoko Taniura, Hiroyuki Tanaka, Ryoji P. Kushima and Akihiro Kawauchi

Int. J. Mol. Sci. 2021, 22(4), 1805; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms22041805 - 11 Feb 2021

Cited by 17 | Viewed by 2889

Abstract

The conventional two-dimensional (2D) culture is available as an in vitro experimental model. However, the culture system reportedly does not recapitulate the in vivo cancer microenvironment. We recently developed a tissueoid cell culture system using Cellbed, which resembles the loose connective tissue in living organisms. The present study performed 2D and three-dimensional (3D) culture using prostate and bladder cancer cell lines and a comprehensive metabolome analysis. Compared to 3D, the 2D culture had significantly lower levels of most metabolites. The 3D culture system did not impair mitochondrial function in the cancer cells and produce energy through the mitochondria simultaneously with aerobic glycolysis. Conversely, ATP production, biomass (nucleotides, amino acids, lipids and NADPH) synthesis and redox balance maintenance were conducted in 3D culture. In contrast, in 2D culture, biomass production was delayed due to the suppression of metabolic activity. The 3D metabolome analysis using the tissueoid cell culture system capable of in vivo cancer cell culture yielded results consistent with previously reported cancer metabolism theories. This system is expected to be an essential experimental tool in a wide range of cancer research fields, especially in preclinical stages while transitioning from in vitro to in vivo. Full article

(This article belongs to the Special Issue Development of Cell Culture Technology: Molecular Aspects and Beyond)

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23 pages, 9050 KiB

Open AccessArticle

Co-Culturing of Endothelial and Cancer Cells in a Nanofibrous Scaffold-Based Two-Layer System

by Ye-Seul Oh, Min-Ho Choi, Jung-In Shin, Perry Ayn Mayson A. Maza and Jong-Young Kwak

Int. J. Mol. Sci. 2020, 21(11), 4128; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21114128 - 10 Jun 2020

Cited by 10 | Viewed by 3417

Abstract

Angiogenesis is critical for local tumor growth. This study aimed to develop a three-dimensional two-layer co-culture system to investigate effects of cancer cells on the growth of endothelial cells (ECs). Poly(ε-caprolactone) (PCL) nanofibrous membranes were generated via electrospinning of PCL in chloroform (C-PCL-M) and chloroform and dimethylformamide (C/DMF-PCL-M). We assembled a two-layer co-culture system using C-PCL-M and C/DMF-PCL-M for EC growth in the upper layer with co-cultured cancer cells in the lower layer. In the absence of vascular endothelial growth factor (VEGF), growth of bEND.3 ECs decreased on C/DMF-PCL-M but not on C-PCL-M with time. Growth of bEND.3 cells on C/DMF-PCL-M was enhanced through co-culturing of CT26 cancer cells and enhanced growth of bEND.3 cells was abrogated with anti-VEGF antibodies and sorafenib. However, EA.hy926 ECs displayed steady growth and proliferation on C/DMF-PCL-M, and their growth was not further increased through co-culturing of cancer cells. Moreover, chemical hypoxia in CT26 cancer cells upon treatment with CoCl₂ enhanced the growth of co-cultured bEND.3 cells in the two-layer system. Thus, EC growth on the nanofibrous scaffold is dependent on the types of ECs and composition of nanofibers and this co-culture system can be used to analyze EC growth induced by cancer cells. Full article

(This article belongs to the Special Issue Development of Cell Culture Technology: Molecular Aspects and Beyond)

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18 pages, 6173 KiB

Open AccessArticle

Use of 3D Human Liver Organoids to Predict Drug-Induced Phospholipidosis

by Ji-Young Lee, Hyo-Jeong Han, Sang-Joon Lee, Eun-Ho Cho, Han-Byul Lee, Ju-Hyung Seok, Hee Seon Lim and Woo-Chan Son

Int. J. Mol. Sci. 2020, 21(8), 2982; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21082982 - 23 Apr 2020

Cited by 16 | Viewed by 4603

Abstract

Drug-induced phospholipidosis (PL) is a storage disorder caused by the formation of phospholipid-drug complexes in lysosomes. Because of the diversity of PL between species, human cell-based assays have been used to predict drug-induced PL in humans. We established three-dimensional (3D) human liver organoids as described previously and investigated their liver characteristics through multiple analyses. Drug-induced PL was initiated in these organoids and in monolayer HepG2 cultures, and cellular changes were systemically examined. Organoids that underwent differentiation showed characteristics of hepatocytes rather than HepG2 cells. The organoids also survived under PL-inducing drug conditions for 48 h and maintained a more stable albumin secretion level than the HepG2 cells. More cytoplasmic vacuoles were observed in organoids and HepG2 cells treated with more potent PL-induced drugs, but to a greater extent in organoids than in HepG2 cells. Lysosome-associated membrane protein 2, a marker of lysosome membranes, showed a stronger immunohistochemical signal in the organoids. PL-distinctive lamellar bodies were observed only in amiodarone-treated organoids by transmission electron microscopy. Human liver organoids are thus more sensitive to drug-induced PL and less affected by cytotoxicity than HepG2 cells. Since PL is a chronic condition, these results indicate that organoids better reflect metabolite-mediated hepatotoxicity in vivo and could be a valuable system for evaluating the phospholipidogenic effects of different compounds during drug development. Full article

(This article belongs to the Special Issue Development of Cell Culture Technology: Molecular Aspects and Beyond)

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12 pages, 11978 KiB

Open AccessArticle

Existence of Circulating Mitochondria in Human and Animal Peripheral Blood

by Xiang Song, Wei Hu, Haibo Yu, Honglan Wang, Yelu Zhao, Robert Korngold and Yong Zhao

Int. J. Mol. Sci. 2020, 21(6), 2122; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms21062122 - 19 Mar 2020

Cited by 32 | Viewed by 5485

Abstract

Mitochondria are usually located in the cytoplasm of cells where they generate adenosine triphosphate (ATP) to empower cellular functions. However, we found circulating mitochondria in human and animal blood. Electron microscopy confirmed the presence of mitochondria in adult human blood plasma. Flow cytometry analyses demonstrated that circulating mitochondria from the plasma of human cord blood and adult peripheral blood displayed the immune tolerance-associated membrane molecules such as CD270 and PD-L1 (programmed cell death-ligand 1). Similar data were obtained from fetal bovine serum (FBS) and horse serum of different vendors. Mitochondria remained detectable even after 56 °C heat inactivation. A real-time PCR array revealed purified mitochondria from animal sera expressed several genes that contribute to human T- and B-cell activation. Transwell experiments confirmed the migration capability of mitochondria through their expression of the chemokine receptor CXCR4 in responses to its ligand stromal-derived factor-1α (SDF-1α). Functional analysis established that human plasma mitochondria stimulated the proliferation of anti-CD3/CD28 bead-activated PBMC, up-regulated the percentage of activated CD4⁺ T and CD8⁺ T cells, and reduced the production of inflammatory cytokines. These findings suggested that the existence of circulating mitochondria in blood may function as a novel mediator for cell-cell communications and maintenance of homeostasis. Plasma-related products should be cautiously utilized in cell cultures due to the mitochondrial contamination. Full article

(This article belongs to the Special Issue Development of Cell Culture Technology: Molecular Aspects and Beyond)

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Review

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28 pages, 11488 KiB

Open AccessReview

The Communication between Ocular Surface and Nasal Epithelia in 3D Cell Culture Technology for Translational Research: A Narrative Review

by Malik Aydin, Jana Dietrich, Joana Witt, Maximiliane S. C. Finkbeiner, Jonas J.-H. Park, Stefan Wirth, Christine E. Engeland, Friedrich Paulsen and Anja Ehrhardt

Int. J. Mol. Sci. 2021, 22(23), 12994; https://0-doi-org.brum.beds.ac.uk/10.3390/ijms222312994 - 30 Nov 2021

Cited by 1 | Viewed by 3591

Abstract

There is a lack of knowledge regarding the connection between the ocular and nasal epithelia. This narrative review focuses on conjunctival, corneal, ultrastructural corneal stroma, and nasal epithelia as well as an introduction into their interconnections. We describe in detail the morphology and physiology of the ocular surface, the nasolacrimal ducts, and the nasal cavity. This knowledge provides a basis for functional studies and the development of relevant cell culture models that can be used to investigate the pathogenesis of diseases related to these complex structures. Moreover, we also provide a state-of-the-art overview regarding the development of 3D culture models, which allow for addressing research questions in models resembling the in vivo situation. In particular, we give an overview of the current developments of corneal 3D and organoid models, as well as 3D cell culture models of epithelia with goblet cells (conjunctiva and nasal cavity). The benefits and shortcomings of these cell culture models are discussed. As examples for pathogens related to ocular and nasal epithelia, we discuss infections caused by adenovirus and measles virus. In addition to pathogens, also external triggers such as allergens can cause rhinoconjunctivitis. These diseases exemplify the interconnections between the ocular surface and nasal epithelia in a molecular and clinical context. With a final translational section on optical coherence tomography (OCT), we provide an overview about the applicability of this technique in basic research and clinical ophthalmology. The techniques presented herein will be instrumental in further elucidating the functional interrelations and crosstalk between ocular and nasal epithelia. Full article

(This article belongs to the Special Issue Development of Cell Culture Technology: Molecular Aspects and Beyond)

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