Editorial

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Open AccessEditorial

A Never-Ending Journey in Search for Novel Cell Biology Techniques

by Alexander E. Kalyuzhny

Cells 2022, 11(9), 1393; https://doi.org/10.3390/cells11091393 - 20 Apr 2022

Viewed by 1070

Abstract

Cell techniques undergo rapid advancement across different areas of biomedical research [...] Full article

(This article belongs to the Special Issue 10th Anniversary of Cells—Advances in Cell Techniques)

Research

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22 pages, 17322 KiB

Open AccessArticle

An Optimized Workflow for the Analysis of Metabolic Fluxes in Cancer Spheroids Using Seahorse Technology

by Gloria Campioni, Valentina Pasquale, Stefano Busti, Giacomo Ducci, Elena Sacco and Marco Vanoni

Cells 2022, 11(5), 866; https://doi.org/10.3390/cells11050866 - 02 Mar 2022

Cited by 10 | Viewed by 7935

Abstract

Three-dimensional cancer models, such as spheroids, are increasingly being used to study cancer metabolism because they can better recapitulate the molecular and physiological aspects of the tumor architecture than conventional monolayer cultures. Although Agilent Seahorse XFe96 (Agilent Technologies, Santa Clara, CA, United States) [...] Read more.

Three-dimensional cancer models, such as spheroids, are increasingly being used to study cancer metabolism because they can better recapitulate the molecular and physiological aspects of the tumor architecture than conventional monolayer cultures. Although Agilent Seahorse XFe96 (Agilent Technologies, Santa Clara, CA, United States) is a valuable technology for studying metabolic alterations occurring in cancer cells, its application to three-dimensional cultures is still poorly optimized. We present a reliable and reproducible workflow for the Seahorse metabolic analysis of three-dimensional cultures. An optimized protocol enables the formation of spheroids highly regular in shape and homogenous in size, reducing variability in metabolic parameters among the experimental replicates, both under basal and drug treatment conditions. High-resolution imaging allows the calculation of the number of viable cells in each spheroid, the normalization of metabolic parameters on a per-cell basis, and grouping of the spheroids as a function of their size. Multivariate statistical tests on metabolic parameters determined by the Mito Stress test on two breast cancer cell lines show that metabolic differences among the studied spheroids are mostly related to the cell line rather than to the size of the spheroid. The optimized workflow allows high-resolution metabolic characterization of three-dimensional cultures, their comparison with monolayer cultures, and may aid in the design and interpretation of (multi)drug protocols. Full article

(This article belongs to the Special Issue 10th Anniversary of Cells—Advances in Cell Techniques)

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15 pages, 2429 KiB

Open AccessArticle

Method for Isolation of Myxozoan Proliferative Stages from Fish at High Yield and Purity: An Essential Prerequisite for In Vitro, In Vivo and Genomics-Based Research Developments

by Ana Born-Torrijos, Anush Kosakyan, Sneha Patra, Joana Pimentel-Santos, Brian Panicucci, Justin Tze Ho Chan, Tomáš Korytář and Astrid S. Holzer

Cells 2022, 11(3), 377; https://doi.org/10.3390/cells11030377 - 23 Jan 2022

Cited by 5 | Viewed by 3612

Abstract

Myxozoans are a diverse group of microscopic cnidarian parasites and some representatives are associated with important diseases in fish, in both marine and freshwater aquaculture systems. Research on myxozoans has been largely hampered by the inability to isolate myxozoan parasites from their host [...] Read more.

Myxozoans are a diverse group of microscopic cnidarian parasites and some representatives are associated with important diseases in fish, in both marine and freshwater aquaculture systems. Research on myxozoans has been largely hampered by the inability to isolate myxozoan parasites from their host tissues. In this study, we developed and optimized a method to isolate the myxozoan proliferative stages of different size and cellularity from fish blood, using DEAE-cellulose ion exchange chromatography. We optimized several parameters and obtained 99–100% parasite purity, as well as high survival and infectivity. Using polyclonal pan-carp blood cell-specific antibodies, we further developed a rapid cytometric assay for quantification of the proliferative stages, not only in highly concentrated DEAE-C isolates but also in dilute conditions in full blood. Early developmental stages of myxozoans are key to parasite proliferation, establishment, and pathology in their hosts. The isolation of these stages not only opens new possibilities for in vivo and in vitro studies, but also for obtaining purified DNA and protein extracts for downstream analyses. Hence, we provide a long-desired tool that will advance the functional research into the mechanisms of host exploitation and immune stimulation/evasion in this group, which could contribute greatly to the development of therapeutic strategies against myxozoans. Full article

(This article belongs to the Special Issue 10th Anniversary of Cells—Advances in Cell Techniques)

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23 pages, 7118 KiB

Open AccessFeature PaperArticle

Large-Scale, Wavelet-Based Analysis of Lysosomal Trajectories and Co-Movements of Lysosomes with Nanoparticle Cargos

by Konstantin Polev, Diana V. Kolygina, Kristiana Kandere-Grzybowska and Bartosz A. Grzybowski

Cells 2022, 11(2), 270; https://doi.org/10.3390/cells11020270 - 13 Jan 2022

Cited by 4 | Viewed by 2568

Abstract

Lysosomes—that is, acidic organelles known for degradation/recycling—move through the cytoplasm alternating between bursts of active transport and short, diffusive motions or even pauses. While their mobility is essential for lysosomes’ fusogenic and non-fusogenic interactions with target organelles, their movements have not been characterized [...] Read more.

Lysosomes—that is, acidic organelles known for degradation/recycling—move through the cytoplasm alternating between bursts of active transport and short, diffusive motions or even pauses. While their mobility is essential for lysosomes’ fusogenic and non-fusogenic interactions with target organelles, their movements have not been characterized in adequate detail. Here, large-scale statistical analysis of lysosomal movement trajectories reveals that lysosome trajectories in all examined cell types—both cancer and noncancerous ones—are superdiffusive and characterized by heavy-tailed distributions of run and flight lengths. Consideration of Akaike weights for various potential models (lognormal, power law, truncated power law, stretched exponential, and exponential) indicates that the experimental data are best described by the lognormal distribution, which, in turn, can be related to one of the space-search strategies particularly effective when “thorough” search needs to balance search for rare target(s) (organelles). In addition, automated, wavelet-based analysis allows for co-tracking the motions of lysosomes and the cargos they carry—particularly the nanoparticle aggregates known to cause selective lysosome disruption in cancerous cells. The methods we describe here could help study nanoparticle assemblies, viruses, and other objects transported inside various vesicle types, as well as coordinated movements of organelles/particles in the cytoplasm. Custom-written code that includes integrated workflow for our analyses is made available for academic use. Full article

(This article belongs to the Special Issue 10th Anniversary of Cells—Advances in Cell Techniques)

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16 pages, 9076 KiB

Open AccessArticle

Protocols for Generating Surfaces and Measuring 3D Organelle Morphology Using Amira

by Edgar Garza-Lopez, Zer Vue, Prasanna Katti, Kit Neikirk, Michelle Biete, Jacob Lam, Heather K. Beasley, Andrea G. Marshall, Taylor A. Rodman, Trace A. Christensen, Jeffrey L. Salisbury, Larry Vang, Margaret Mungai, Salma AshShareef, Sandra A. Murray, Jianqiang Shao, Jennifer Streeter, Brian Glancy, Renata O. Pereira, E. Dale Abel and Antentor Hinton, Jr. Show full author list Hide full author list

Cells 2022, 11(1), 65; https://doi.org/10.3390/cells11010065 - 27 Dec 2021

Cited by 26 | Viewed by 6263 | Correction

Abstract

High-resolution 3D images of organelles are of paramount importance in cellular biology. Although light microscopy and transmission electron microscopy (TEM) have provided the standard for imaging cellular structures, they cannot provide 3D images. However, recent technological advances such as serial block-face scanning electron [...] Read more.

High-resolution 3D images of organelles are of paramount importance in cellular biology. Although light microscopy and transmission electron microscopy (TEM) have provided the standard for imaging cellular structures, they cannot provide 3D images. However, recent technological advances such as serial block-face scanning electron microscopy (SBF-SEM) and focused ion beam scanning electron microscopy (FIB-SEM) provide the tools to create 3D images for the ultrastructural analysis of organelles. Here, we describe a standardized protocol using the visualization software, Amira, to quantify organelle morphologies in 3D, thereby providing accurate and reproducible measurements of these cellular substructures. We demonstrate applications of SBF-SEM and Amira to quantify mitochondria and endoplasmic reticulum (ER) structures. Full article

(This article belongs to the Special Issue 10th Anniversary of Cells—Advances in Cell Techniques)

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14 pages, 3079 KiB

Open AccessArticle

Proliferation and Differentiation of Intestinal Caco-2 Cells Are Maintained in Culture with Human Platelet Lysate Instead of Fetal Calf Serum

by Dalanda Wanes, Hassan Y. Naim and Franziska Dengler

Cells 2021, 10(11), 3038; https://doi.org/10.3390/cells10113038 - 05 Nov 2021

Cited by 6 | Viewed by 3844

Abstract

Cell lines are widely used as in vitro model systems and substitute for animal experiments. The frequently used Caco-2 cell line is considered to reflect characteristics of differentiated intestinal epithelium. However, the need to culture the cells with fetal calf serum (FCS) induces [...] Read more.

Cell lines are widely used as in vitro model systems and substitute for animal experiments. The frequently used Caco-2 cell line is considered to reflect characteristics of differentiated intestinal epithelium. However, the need to culture the cells with fetal calf serum (FCS) induces a high variability, risk of contamination and is ethically disputed. We tested the culture of Caco-2 cells with human platelet lysate (PL) instead of FCS. We compared cell viability and differentiation by measuring ATP levels, gene and protein expression of specific markers in total cell extracts, brush border membrane vesicles (BBM) and lipid rafts (LR). Cell viability was slightly enhanced in cells grown with PL compared to FCS. The cells differentiated to an intestinal phenotype like the cells cultured in FCS, as indicated by the similar gene expression levels of hexose and protein transport proteins and the structural protein VILLIN. BBM showed a comparable distribution of the intestinal hydrolases, indicating a maintained cell membrane polarity. The distribution of the marker protein FLOTILLIN-2 in LR was also similar. We conclude that PL is an exquisite and suitable replacement for FCS in the culture of Caco-2 cells that can eliminate many disadvantages incurred due to the use of FCS. Full article

(This article belongs to the Special Issue 10th Anniversary of Cells—Advances in Cell Techniques)

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13 pages, 2482 KiB

Open AccessArticle

Comparison of Three CD3-Specific Separation Methods Leading to Labeled and Label-Free T Cells

by Ronald Weiss, Wilhelm Gerdes, Rommy Berthold, Ulrich Sack, Ulrike Koehl, Sunna Hauschildt and Anja Grahnert

Cells 2021, 10(11), 2824; https://doi.org/10.3390/cells10112824 - 21 Oct 2021

Cited by 4 | Viewed by 4082

Abstract

T cells are an essential part of the immune system. They determine the specificity of the immune response to foreign substances and, thus, help to protect the body from infections and cancer. Recently, T cells have gained much attention as promising tools in [...] Read more.

T cells are an essential part of the immune system. They determine the specificity of the immune response to foreign substances and, thus, help to protect the body from infections and cancer. Recently, T cells have gained much attention as promising tools in adoptive T cell transfer for cancer treatment. However, it is crucial not only for medical purposes but also for research to obtain T cells in large quantities, of high purity and functionality. To fulfill these criteria, efficient and robust isolation methods are needed. We used three different isolation methods to separate CD3-specific T cells from leukocyte concentrates (buffy coats) and Ficoll purified PBMCs. To catch the target cells, the Traceless Affinity Cell Selection (TACS^®) method, based on immune affinity chromatography, uses CD-specific low affinity Fab-fragments; while the classical Magnetic Activated Cell Sorting (MACS^®) method relies on magnetic beads coated with specific high affinity monoclonal antibodies. The REAlease^® system also works with magnetic beads but, in contrast to MACS^®, low-affinity antibody fragments are used. The target cells separated by TACS^® and REAlease^® are “label-free”, while cells isolated by MACS^® still carry the cell specific label. The time required to isolate T cells from buffy coat by TACS^® and MACS^® amounted to 90 min and 50 min, respectively, while it took 150 min to isolate T cells from PBMCs by TACS^® and 110 min by REAlease^®. All methods used are well suited to obtain T cells in large quantities of high viability (>92%) and purity (>98%). Only the median CD4:CD8 ratio of approximately 6.8 after REAlease^® separation differed greatly from the physiological conditions. MACS^® separation was found to induce proliferation and cytokine secretion. However, independent of the isolation methods used, stimulation of T cells by anti CD3/CD28 resulted in similar rates of proliferation and cytokine production, verifying the functional activity of the isolated cells. Full article

(This article belongs to the Special Issue 10th Anniversary of Cells—Advances in Cell Techniques)

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21 pages, 6571 KiB

Open AccessArticle

Weight Pulling: A Novel Mouse Model of Human Progressive Resistance Exercise

by Wenyuan G. Zhu, Jamie E. Hibbert, Kuan Hung Lin, Nathaniel D. Steinert, Jake L. Lemens, Kent W. Jorgenson, Sarah M. Newman, Dudley W. Lamming and Troy A. Hornberger

Cells 2021, 10(9), 2459; https://doi.org/10.3390/cells10092459 - 17 Sep 2021

Cited by 17 | Viewed by 6076

Abstract

This study describes a mouse model of progressive resistance exercise that utilizes a full-body/multi-joint exercise (weight pulling) along with a training protocol that mimics a traditional human paradigm (three training sessions per week, ~8–12 repetitions per set, 2 min of rest between sets, [...] Read more.

This study describes a mouse model of progressive resistance exercise that utilizes a full-body/multi-joint exercise (weight pulling) along with a training protocol that mimics a traditional human paradigm (three training sessions per week, ~8–12 repetitions per set, 2 min of rest between sets, approximately two maximal-intensity sets per session, last set taken to failure, and a progressive increase in loading that is based on the individual’s performance). We demonstrate that weight pulling can induce an increase in the mass of numerous muscles throughout the body. The relative increase in muscle mass is similar to what has been observed in human studies, and is associated with the same type of long-term adaptations that occur in humans (e.g., fiber hypertrophy, myonuclear accretion, and, in some instances, a fast-to-slow transition in Type II fiber composition). Moreover, we demonstrate that weight pulling can induce the same type of acute responses that are thought to drive these long-term adaptations (e.g., the activation of signaling through mTORC1 and the induction of protein synthesis at 1 h post-exercise). Collectively, the results of this study indicate that weight pulling can serve as a highly translatable mouse model of progressive resistance exercise. Full article

(This article belongs to the Special Issue 10th Anniversary of Cells—Advances in Cell Techniques)

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14 pages, 3341 KiB

Open AccessArticle

Chronic Alcohol Exposure of Cells Using Controlled Alcohol-Releasing Capillaries

by Wanil Kim, Hye-Seon Jeong, Sang-Chan Kim, Chang-Hyung Choi and Kyung-Ha Lee

Cells 2021, 10(5), 1120; https://doi.org/10.3390/cells10051120 - 06 May 2021

Cited by 4 | Viewed by 2125

Abstract

Alcohol is one of the main causes of liver diseases such as fatty liver, alcoholic hepatitis, and chronic hepatitis with liver fibrosis or cirrhosis. To reproduce the conditions of alcohol-induced liver diseases and to identify the disease-causing mechanisms at the cellular level, several [...] Read more.

Alcohol is one of the main causes of liver diseases such as fatty liver, alcoholic hepatitis, and chronic hepatitis with liver fibrosis or cirrhosis. To reproduce the conditions of alcohol-induced liver diseases and to identify the disease-causing mechanisms at the cellular level, several methods have been used to expose the cells to ethanol. As ethanol evaporates easily, it is difficult to mimic chronic alcohol exposure conditions at the cellular level. In this study, we developed a glass capillary system containing ethanol, which could steadily release ethanol from the polyethylene tubing and hydrogel portion at both sides of the capillary. The ethanol-containing capillary could release ethanol in the cell culture medium for up to 144 h, and the concentration of ethanol in the cell culture medium could be adjusted by controlling the number of capillaries. A long-term exposure to ethanol by the capillary system led to an increased toxicity of cells and altered the cellular physiologies, such as increasing the lipid accumulation and hepatic transaminase release in cells, as compared to the traditional direct ethanol addition method. Ethanol capillaries showed different gene expression patterns of lipid accumulation- or chronic alcoholism-related genes. Our results suggest that our ethanol-containing capillary system can be used as a valuable tool for studying the mechanism of chronic alcohol-mediated hepatic diseases at the cellular level. Full article

(This article belongs to the Special Issue 10th Anniversary of Cells—Advances in Cell Techniques)

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13 pages, 6998 KiB

Open AccessArticle

PicPreview and PicSummary: Two Timesaving Plugins for the Fluorescence Microscopist

by Gabrielle Vieyres

Cells 2021, 10(4), 846; https://doi.org/10.3390/cells10040846 - 08 Apr 2021

Cited by 1 | Viewed by 2068

Abstract

This article targets cell biologists who use fluorescence microscopy but lack automatic tools to summarize and manage their image datasets. When using microscopy to document a phenotype, multiple and random pictures are required to reflect the biological diversity of each imaged sample. Managing, [...] Read more.

This article targets cell biologists who use fluorescence microscopy but lack automatic tools to summarize and manage their image datasets. When using microscopy to document a phenotype, multiple and random pictures are required to reflect the biological diversity of each imaged sample. Managing, integrating and summarizing the acquired data can be a daunting task that becomes extremely time-consuming unless one automatizes it. Unfortunately, if many biologists use microscopy, only a few have automatized procedures to cope with the data generated. For the majority of microscope users, the two developed complementary ImageJ plugins, PicPreview and PicSummary, will allow, in a few clicks and in an instant, to obtain an overview of all pictures taken for each sample of an experiment and a summary with one user-defined representative picture per sample. The plugins and a video tutorial, as well as demonstration pictures, are available as supplementary data at the journal website. PicPreview and PicSummary should save precious time in managing microscopy datasets and in preparing figures for publications. Full article

(This article belongs to the Special Issue 10th Anniversary of Cells—Advances in Cell Techniques)

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Review

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11 pages, 1765 KiB

Open AccessReview

Machine Learning-Driven Multiobjective Optimization: An Opportunity of Microfluidic Platforms Applied in Cancer Research

by Yi Liu, Sijing Li and Yaling Liu

Cells 2022, 11(5), 905; https://doi.org/10.3390/cells11050905 - 05 Mar 2022

Cited by 9 | Viewed by 3191

Abstract

Cancer metastasis is one of the primary reasons for cancer-related fatalities. Despite the achievements of cancer research with microfluidic platforms, understanding the interplay of multiple factors when it comes to cancer cells is still a great challenge. Crosstalk and causality of different factors [...] Read more.

Cancer metastasis is one of the primary reasons for cancer-related fatalities. Despite the achievements of cancer research with microfluidic platforms, understanding the interplay of multiple factors when it comes to cancer cells is still a great challenge. Crosstalk and causality of different factors in pathogenesis are two important areas in need of further research. With the assistance of machine learning, microfluidic platforms can reach a higher level of detection and classification of cancer metastasis. This article reviews the development history of microfluidics used for cancer research and summarizes how the utilization of machine learning benefits cancer studies, particularly in biomarker detection, wherein causality analysis is useful. To optimize microfluidic platforms, researchers are encouraged to use causality analysis when detecting biomarkers, analyzing tumor microenvironments, choosing materials, and designing structures. Full article

(This article belongs to the Special Issue 10th Anniversary of Cells—Advances in Cell Techniques)

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3 pages, 4176 KiB

Open AccessCorrection

Correction: Garza-Lopez et al. Protocols for Generating Surfaces and Measuring 3D Organelle Morphology Using Amira. Cells 2022, 11, 65

by Edgar Garza-Lopez, Zer Vue, Prasanna Katti, Kit Neikirk, Michelle Biete, Jacob Lam, Heather K. Beasley, Andrea G. Marshall, Taylor A. Rodman, Trace A. Christensen, Jeffrey L. Salisbury, Larry Vang, Margaret Mungai, Salma AshShareef, Sandra A. Murray, Jianqiang Shao, Jennifer Streeter, Brian Glancy, Renata O. Pereira, E. Dale Abel and Antentor Hinton, Jr. Show full author list Hide full author list

Cells 2023, 12(10), 1356; https://doi.org/10.3390/cells12101356 - 10 May 2023

Cited by 4 | Viewed by 887

Abstract

In the original publication [...] Full article

(This article belongs to the Special Issue 10th Anniversary of Cells—Advances in Cell Techniques)

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18 pages, 3983 KiB

Open AccessProtocol

Isolation and Propagation of Human Corneal Stromal Keratocytes for Tissue Engineering and Cell Therapy

by Nur Zahirah binte M. Yusoff, Andri K. Riau, Gary H. F. Yam, Nuur Shahinda Humaira binte Halim and Jodhbir S. Mehta

Cells 2022, 11(1), 178; https://doi.org/10.3390/cells11010178 - 05 Jan 2022

Cited by 20 | Viewed by 2879

Abstract

The human corneal stroma contains corneal stromal keratocytes (CSKs) that synthesize and deposit collagens and keratan sulfate proteoglycans into the stromal matrix to maintain the corneal structural integrity and transparency. In adult corneas, CSKs are quiescent and arrested in the G0 phase of [...] Read more.

The human corneal stroma contains corneal stromal keratocytes (CSKs) that synthesize and deposit collagens and keratan sulfate proteoglycans into the stromal matrix to maintain the corneal structural integrity and transparency. In adult corneas, CSKs are quiescent and arrested in the G0 phase of the cell cycle. Following injury, some CSKs undergo apoptosis, whereas the surviving cells are activated to become stromal fibroblasts (SFs) and myofibroblasts (MyoFBs), as a natural mechanism of wound healing. The SFs and MyoFBs secrete abnormal extracellular matrix proteins, leading to corneal fibrosis and scar formation (corneal opacification). The issue is compounded by the fact that CSK transformation into SFs or MyoFBs is irreversible in vivo, which leads to chronic opacification. In this scenario, corneal transplantation is the only recourse. The application of cell therapy by replenishing CSKs, propagated in vitro, in the injured corneas has been demonstrated to be efficacious in resolving early-onset corneal opacification. However, expanding CSKs is challenging and has been the limiting factor for the application in corneal tissue engineering and cell therapy. The supplementation of serum in the culture medium promotes cell division but inevitably converts the CSKs into SFs. Similar to the in vivo conditions, the transformation is irreversible, even when the SF culture is switched to a serum-free medium. In the current article, we present a detailed protocol on the isolation and propagation of bona fide human CSKs and the morphological and genotypic differences from SFs. Full article

(This article belongs to the Special Issue 10th Anniversary of Cells—Advances in Cell Techniques)

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9 pages, 1977 KiB

Open AccessProtocol

Simple Method for Establishing Primary Leporidae Skin Fibroblast Cultures

by Fábio A. Abade dos Santos, C. L. Carvalho, Isabel Almeida, Teresa Fagulha, Fernanda Rammos, Sílvia C. Barros, Margarida Henriques, Tiago Luís and Margarida D. Duarte

Cells 2021, 10(8), 2100; https://doi.org/10.3390/cells10082100 - 16 Aug 2021

Cited by 5 | Viewed by 2589

Abstract

Commercial hare and rabbit immortalized cell lines are extremely limited regarding the many species within the lagomorpha order. To overcome this limitation, researchers and technicians must establish primary cell cultures derived from biopsies or embryos. Among all cell types, fibroblasts are plastic and [...] Read more.

Commercial hare and rabbit immortalized cell lines are extremely limited regarding the many species within the lagomorpha order. To overcome this limitation, researchers and technicians must establish primary cell cultures derived from biopsies or embryos. Among all cell types, fibroblasts are plastic and resilient cells, highly convenient for clinical and fundamental research but also for diagnosis, particularly for viral isolation. Here, we describe a fast and cheap method to produce primary fibroblast cell cultures from leporid species, using dispase II, a protease that allows dermal–epidermal separation, followed by a simple enzymatic digestion with trypsin. This method allows for the establishment of an in vitro cell culture system with an excellent viability yield and purity level higher than 85% and enables the maintenance and even immortalization of leporid fibroblastic cells derived from tissues already differentiated. Full article

(This article belongs to the Special Issue 10th Anniversary of Cells—Advances in Cell Techniques)

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