scholarly journals Zebrafish skeletal muscle cell cultures: Monolayer to three-dimensional tissue engineered collagen constructs

2020 ◽  
Author(s):  
K.K Vishnolia ◽  
N.R.W Martin ◽  
D.J Player ◽  
E Spikings ◽  
M.P Lewis
Keyword(s):  
Skeletal Muscle ◽  
Mrna Expression ◽  
Expression Profiles ◽  
Three Dimensional ◽  
3D Culture ◽  
Morphological Characteristics ◽  
Hypertrophic Growth ◽  
3D Cultures

AbstractZebrafish (Danio rerio) are a commonly used model organism to study human muscular myopathies and dystrophies. To date, much of the work has been conducted in vivo due to limitations surrounding the consistent isolation and culture of zebrafish muscle progenitor cells (MPCs) in vitro and the lack of physiologically relevant models.Here we report a robust, repeatable, and cost-effective protocol for the isolation and culture of zebrafish MPCs in conventional monolayer (2D) and have successfully transferred these cells to 3D culture in collagen based three-dimensional (3D) tissue-engineered constructs. Zebrafish MPC’s cultured in 2D were consistently reported to be Desmin positive reflecting their muscle specificity, with those demonstrating Desmin positivity in the 3D cultures. In addition, mRNA expression of muscle markers specific for proliferation, differentiation and maturation measured from both monolayer and 3D cultures at appropriate developmental stages were found consistent with previously published from other species in vitro and in vivo muscle data.Collagen constructs seeded with zebrafish MPC’s were initially characterised for optimal seeding density, followed by macroscopic characterisation (three-fold contraction) of the matrix. Direct comparison between the morphological characteristics (proportion of cells) and gene expression profiles of cells cultured in collagen constructs revealed higher maturation and differentiation compared to monolayer cultures. In this regard, cells embedded in 3D collagen constructs revealed higher fusion index, Desmin positivity, hypertrophic growth, myotube maturity and myogenic mRNA expression when compared to in monolayer.In conclusion, these methods and models developed herein will facilitate in vitro experiments, which would complement in vivo zebrafish studies used to investigate the basic developmental, myopathies and dystrophies in skeletal muscle cells.

scholarly journals Recapitulating tumour microenvironment in chitosan–gelatin three-dimensional scaffolds: an improved in vitro tumour model

2012 ◽  
Vol 9 (77) ◽  
pp. 3288-3302 ◽  
Author(s):  
Neha Arya ◽  
Viren Sardana ◽  
Meera Saxena ◽  
Annapoorni Rangarajan ◽  
Dhirendra S. Katti
Keyword(s):  
Cancer Progression ◽  
Expression Profiles ◽  
Three Dimensional ◽  
Gene Expression Profiles ◽  
Petri Dish ◽  
Tumour Microenvironment ◽  
Two Dimensional ◽  
Tumour Model

Owing to the reduced co-relationship between conventional flat Petri dish culture (two-dimensional) and the tumour microenvironment, there has been a shift towards three-dimensional culture systems that show an improved analogy to the same. In this work, an extracellular matrix (ECM)-mimicking three-dimensional scaffold based on chitosan and gelatin was fabricated and explored for its potential as a tumour model for lung cancer. It was demonstrated that the chitosan–gelatin (CG) scaffolds supported the formation of tumoroids that were similar to tumours grown in vivo for factors involved in tumour-cell–ECM interaction, invasion and metastasis, and response to anti-cancer drugs. On the other hand, the two-dimensional Petri dish surfaces did not demonstrate gene-expression profiles similar to tumours grown in vivo . Further, the three-dimensional CG scaffolds supported the formation of tumoroids, using other types of cancer cells such as breast, cervix and bone, indicating a possible wider potential for in vitro tumoroid generation. Overall, the results demonstrated that CG scaffolds can be an improved in vitro tool to study cancer progression and drug screening for solid tumours.

scholarly journals Selection of theIn VitroCulture Media Influences mRNA Expression of Hedgehog Genes,Il-6,and Important Genes regarding Reactive Oxygen Species in Single Murine Preimplantation Embryos

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
N. Pfeifer ◽  
D. M. Baston-Büst ◽  
J. Hirchenhain ◽  
U. Friebe-Hoffmann ◽  
D. T. Rein ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Mrna Expression ◽  
Culture Media ◽  
Expression Profiles ◽  
Reactive Oxygen ◽  
Female Reproductive Tract ◽  
Preimplantation Embryos ◽  
Oxygen Species

Background. The aim of this paper was to determine the influence of differentin vitroculture media on mRNA expression of Hedgehog genes,il-6,and important genes regarding reactive oxygen species in single mouse embryos.Methods. Reverse transcription of single embryos either culturedin vitrofrom day 0.5 until 3.5 (COOK’s Cleavage medium or Vitrolife’s G-1 PLUS medium) orin vivountil day 3.5post coitum. PCR was carried out forβ-actinfollowed by nested-PCR forshh, ihh, il-6, nox, gpx4, gpx1,andprdx2.Results. The number of murine blastocysts cultured in COOK medium which expressedil-6, gpx4, gpx1,andprdx2mRNA differed significantly compared to thein vivogroup. Except fornox, the mRNA profile of the Vitrolife media group embryos varied significantly from thein vivoones regarding the number of blastocysts expressing the mRNA ofshh, ihh, il-6, gpx4, gpx1andprdx2.Conclusions. The present study shows that differentin vitroculture media lead to different mRNA expression profiles during early development. Even the newly developedin vitroculture media are not able to mimic the female reproductive tract. The question of long-term consequences for children due to assisted reproduction techniques needs to be addressed in larger studies.

scholarly journals Three-Dimensional Spheroids as In Vitro Preclinical Models for Cancer Research

Pharmaceutics ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 1186
Author(s):  
Bárbara Pinto ◽  
Ana C. Henriques ◽  
Patrícia M. A. Silva ◽  
Hassan Bousbaa
Keyword(s):  
Cancer Research ◽  
Low Cost ◽  
Three Dimensional ◽  
3D Culture ◽  
Comprehensive Overview ◽  
Culture Techniques ◽  
Drug Candidates ◽  
In Vivo Testing

Most cancer biologists still rely on conventional two-dimensional (2D) monolayer culture techniques to test in vitro anti-tumor drugs prior to in vivo testing. However, the vast majority of promising preclinical drugs have no or weak efficacy in real patients with tumors, thereby delaying the discovery of successful therapeutics. This is because 2D culture lacks cell–cell contacts and natural tumor microenvironment, important in tumor signaling and drug response, thereby resulting in a reduced malignant phenotype compared to the real tumor. In this sense, three-dimensional (3D) cultures of cancer cells that better recapitulate in vivo cell environments emerged as scientifically accurate and low cost cancer models for preclinical screening and testing of new drug candidates before moving to expensive and time-consuming animal models. Here, we provide a comprehensive overview of 3D tumor systems and highlight the strategies for spheroid construction and evaluation tools of targeted therapies, focusing on their applicability in cancer research. Examples of the applicability of 3D culture for the evaluation of the therapeutic efficacy of nanomedicines are discussed.

scholarly journals VIP17/MAL expression modulates epithelial cyst formation and ciliogenesis

2012 ◽  
Vol 303 (8) ◽  
pp. C862-C871 ◽  
Author(s):  
Vinita Takiar ◽  
Kavita Mistry ◽  
Monica Carmosino ◽  
Nicole Schaeren-Wiemers ◽  
Michael J. Caplan
Keyword(s):  
Epithelial Cells ◽  
Primary Cilium ◽  
Three Dimensional ◽  
3D Culture ◽  
Cyst Formation ◽  
Unknown Etiology ◽  
Development In Vitro ◽  
Renal Cystic Diseases

The polarized organization of epithelial cells is required for vectorial solute transport and may be altered in renal cystic diseases. Vesicle integral protein of 17 kDa (VIP17/MAL) is involved in apical vesicle transport. VIP17/MAL overexpression in vivo results in renal cystogenesis of unknown etiology. Renal cystogenesis can occur as a consequence of defects of the primary cilium. To explore the role of VIP17/MAL in renal cystogenesis and ciliogenesis, we examined the polarization and ciliary morphology of wild-type and VIP17/MAL overexpressing Madin-Darby canine kidney renal epithelial cells grown in two-dimensional (2D) and three-dimensional (3D) cyst culture. VIP17/MAL is apically localized when expressed in cells maintained in 2D and 3D culture. VIP17/MAL overexpressing cells produce more multilumen cysts compared with controls. While the distributions of basolateral markers are not affected, VIP17/MAL expression results in aberrant sorting of the apical marker gp135 to the primary cilium. VIP17/MAL overexpression is also associated with shortened or absent cilia. Immunofluorescence analysis performed on kidney sections from VIP17/MAL transgenic mice also demonstrates fewer and shortened cilia within dilated lumens ( P < 0.01). These studies demonstrate that VIP17/MAL overexpression results in abnormal cilium and cyst development, in vitro and in vivo, suggesting that VIP17/MAL overexpressing mice may develop cysts secondary to a ciliary defect.

scholarly journals Transmigration of Trypanosoma cruzi trypomastigotes through 3D cultures resembling a physiological environment

2019 ◽  
Author(s):  
Matías Exequiel Rodríguez ◽  
Mariana Rizzi ◽  
Lucas D. Caeiro ◽  
Yamil E. Masip ◽  
Alina Perrone ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Three Dimensional ◽  
3D Cultures ◽  
Monolayer Cultures ◽  
Host Parasite Interactions ◽  
Virulent Strains ◽  
Host Parasite ◽  
3D Spheroids

AbstractChaga’ disease, caused by the kinetoplastid parasite Trypanosoma cruzi, presents a variety of chronic clinical manifestations whose determinants are still unknown but probably influenced by the host-parasite interplay established during the first stages of the infection, when bloodstream circulating trypomastigotes disseminate to different organs and tissues. After leaving the blood, trypomastigotes must migrate through tissues to invade cells and establish a chronic infection. How this process occurs remains unexplored. Three-dimensional (3D) cultures are physiologically relevant because mimic the microarchitecture of tissues and provide an environment similar to the encountered in natural infections. In this work, we combined the 3D culture technology with host-pathogen interaction, by studying transmigration of trypomastigotes into 3D spheroids. T. cruzi strains with similar infection dynamics in 2D monolayer cultures but with different in vivo behavior (CL Brener, virulent; SylvioX10 no virulent) presented different infection rates in spheroids (CL Brener ∼40%, SylvioX10 <10%). Confocal microscopy images evidenced that trypomastigotes from CL Brener and other highly virulent strains presented a great ability to transmigrate inside 3D spheroids: as soon as 4 hours post infection parasites were found at 50 µm in depth inside the spheroids. CL Brener trypomastigotes were evenly distributed and systematically observed in the space between cells, suggesting a paracellular route of transmigration to deepen into the spheroids. On the other hand, poor virulent strains presented a weak migratory capacity and remained in the external layers of spheroids (<10µm) with a patch-like distribution pattern. The invasiveness -understood as the ability to transmigrate deep into spheroids- was not a transferable feature between strains, neither by soluble or secreted factors nor by co-cultivation of trypomastigotes from invasive and non-invasive strains. We also studied the transmigration of recent T. cruzi isolates from children that were born congenitally infected, which showed a high migrant phenotype while an isolate form an infected mother (that never transmitted the infection to any of her 3 children) was significantly less migratory. Altogether, our results demonstrate that in a 3D microenvironment each strain presents a characteristic migration pattern and distribution of parasites in the spheroids that can be associated to their in vivo behavior. Certainly, the findings presented here could not have been studied with traditional 2D monolayer cultures.Author SummaryTrypanosoma cruzi is the protozoan parasite that causes Chaga’ disease, also known as American trypanosomiasis. Experimental models of the infection evidence that different strains of the parasite present different virulence in the host, which cannot be always reproduced in 2D monolayer cultures. Three dimensional (3D) cultures can be useful models to study complex host-parasite interactions because they mimic in vitro the microarchitecture of tissues and provide an environment similar to the encountered in natural infections. In particular, spheroids are small 3D aggregates of cells that interact with each other and with the extracellular matrix that they secrete resembling the original microenvironment both functionally and structurally. Spheroids have rarely been employed to explore infectious diseases and host-parasite interactions. In this work we studied how bloodstream trypomastigotes transmigrate through 3D spheroids mimicking the picture encountered by parasites in tissues soon after leaving circulation. We showed that the behavior of T. cruzi trypomastigotes in 3D cultures reflects their in vivo virulence: virulent strains transmigrate deeply into spheroids while non-virulent strains remain in the external layers of spheroids. Besides, this work demonstrates the usefulness of 3D cultures as an accurate in vitro model for the study of host-pathogen interactions that could not be addressed with conventional monolayer cultures.

scholarly journals Role of cellulose family in fibril organization of collagen for forming 3D cancer spheroids: In vitro and in silico approach

Bioimpacts ◽  
2020 ◽  
Vol 11 (2) ◽  
pp. 111-117
Author(s):  
Elaheh Dalir Abdolahinia ◽  
Behzad Jafari ◽  
Sepideh Parvizpour ◽  
Jaleh Barar ◽  
Samad Nadri ◽  
...  
Keyword(s):  
In Silico ◽  
Carboxymethyl Cellulose ◽  
Three Dimensional ◽  
3D Culture ◽  
Cell Aggregation ◽  
Cellulose Derivatives ◽  
Solid Media ◽  
Semi Solid

Introduction: Cell aggregation of three-dimensional (3D) culture systems (the so-called spheroids) are designed as in vitro platform to represent more accurately the in vivo environment for drug discovery by using semi-solid media. The uniform multicellular tumor spheroids can be generated based on the interaction of cells with extracellular matrix (ECM) macromolecules such as collagen and integrin. This study aimed to investigate the possible interactions between the cellulose family and collagen using both in vitro and in silico approaches. Methods: The 3D microtissue of JIMT-1 cells was generated using hanging drop method to study the effects of charge and viscosity of the medium containing cellulose family. To determine the mode of interaction between cellulose derivatives (CDs) and collagen-integrin, docking analysis and molecular simulation were further performed using open source web servers and chemical simulations (GROMACS), respectively. Results: The results confirmed that the addition of CDs into the 3D medium can promote the formation of solid spheroids, where methylcellulose (MC) yielded uniform spheroids compared to carboxymethyl cellulose (CMC). Moreover, the computational analysis showed that MC interacted with both integrin and collagen, while sodium carboxymethyl cellulose (NaCMC) only interacted with collagen residues. The stated different behaviors in the 3D culture formation and collagen interaction were found in the physicochemical properties of CDs. Conclusion: Based on in vitro and in silico findings, MC is suggested as an important ECM-mimicking entity that can support the semi-solid medium and promote the formation of the uniform spheroid in the 3D culture.

scholarly journals Sex steroids influence organizational but not functional decidualization of feline endometrial cells in a 3D culture system†

2019 ◽  
Vol 101 (5) ◽  
pp. 906-915 ◽  
Author(s):  
Kathryn Wilsterman ◽  
Xinmiao Bao ◽  
Allegra D Estrada ◽  
Pierre Comizzoli ◽  
George E Bentley
Keyword(s):  
Sex Steroids ◽  
Culture System ◽  
Three Dimensional ◽  
3D Culture ◽  
In Vitro Models ◽  
Sex Steroid ◽  
Endometrial Cells ◽  
Organizational Patterns

Abstract Successful implantation requires complex signaling between the uterine endometrium and the blastocyst. Prior to the blastocyst reaching the uterus, the endometrium is remodeled by sex steroids and other signals to render the endometrium receptive. In vitro models have facilitated major advances in our understanding of endometrium preparation and endometrial–blastocyst communication in mice and humans, but these systems have not been widely adapted for use in other models which might generate a deeper understanding of these processes. The objective of our study was to use a recently developed, three-dimensional culture system to identify specific roles of female sex steroids in remodeling the organization and function of feline endometrial cells. We treated endometrial cells with physiologically relevant concentrations of estradiol and progesterone, either in isolation or in combination, for 1 week. We then examined size and density of three-dimensional structures, and quantified expression of candidate genes known to vary in response to sex steroid treatments and that have functional relevance to the decidualization process. Combined sex steroid treatments recapitulated organizational patterns seen in vivo; however, sex steroid manipulations did not induce expected changes to expression of decidualization-related genes. Our results demonstrate that sex steroids may not be sufficient for complete decidualization and preparation of the feline endometrium, thereby highlighting key areas of opportunity for further study and suggesting some unique functions of felid uterine tissues.

Application of Three-dimensional (3D) Tumor Cell Culture Systems and Mechanism of Drug Resistance

2019 ◽  
Vol 25 (34) ◽  
pp. 3599-3607 ◽  
Author(s):  
Adeeb Shehzad ◽  
Vijaya Ravinayagam ◽  
Hamad AlRumaih ◽  
Meneerah Aljafary ◽  
Dana Almohazey ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cancer Cells ◽  
Anticancer Drugs ◽  
Three Dimensional ◽  
3D Culture ◽  
3D Cell Culture ◽  
Cancer Therapeutics ◽  
In Vivo Model

: The in-vitro experimental model for the development of cancer therapeutics has always been challenging. Recently, the scientific revolution has improved cell culturing techniques by applying three dimensional (3D) culture system, which provides a similar physiologically relevant in-vivo model for studying various diseases including cancer. In particular, cancer cells exhibiting in-vivo behavior in a model of 3D cell culture is a more accurate cell culture model to test the effectiveness of anticancer drugs or characterization of cancer cells in comparison with two dimensional (2D) monolayer. This study underpins various factors that cause resistance to anticancer drugs in forms of spheroids in 3D in-vitro cell culture and also outlines key challenges and possible solutions for the future development of these systems.

Organoid: Current Implications and Pharmaceutical Applications in Liver Diseases

2020 ◽  
Vol 13 ◽  
Author(s):  
Mengqi Zhu ◽  
Yuting Huang ◽  
Saiyan Bian ◽  
Qianqian Song ◽  
Jie Zhang ◽  
...  
Keyword(s):  
Structural Characteristics ◽  
Three Dimensional ◽  
3D Culture ◽  
Disease Models ◽  
In Vivo Models ◽  
Construction Methods ◽  
Full Picture ◽  
Liver Organoids

Background: Understanding organogenesis, disorders, and repairing processes particularly important for understanding disease occurrence and developing treatment approaches. At present, liver-related studies are mainly conducted using in vivo models and cell lines, making it difficult to generalize the full picture of the structural characteristics and functions of human organs. Organoid is a three-dimensional (3D) culture system in vitro, which holds the promise to establish various disease models and conduct in-depth research by generating organ-like tissues in a dish. Recent advances of human liver organoids have provided us a deeper understanding of this complex organ. Conclusion: In this review, we provide a systematic overview of the construction methods of organoids, focusing on their applications in the hepatic organogenesis and various liver disease models, as well as the limitations of current models. The development of organoid models is proving to be crucial in future liver research.

Evaluation of co-cultured spermatogonial stem cells encapsulated in alginate hydrogel with Sertoli cells and their transplantation into azoospermic mice

Zygote ◽  
2021 ◽  
pp. 1-8
Author(s):  
Mohammad Veisi ◽  
Kamran Mansouri ◽  
Vahideh Assadollahi ◽  
Cyrus Jalili ◽  
Afshin Pirnia ◽  
...  
Keyword(s):  
Sertoli Cells ◽  
Periodic Acid ◽  
Three Dimensional ◽  
3D Culture ◽  
Alginate Hydrogel ◽  
Periodic Acid Schiff ◽  
Expression Levels ◽  
Pas Staining

Summary An in vitro spermatogonial stem cell (SSC) culture can serve as an effective technique to study spermatogenesis and treatment for male infertility. In this research, we compared the effect of a three-dimensional alginate hydrogel with Sertoli cells in a 3D culture and co-cultured Sertoli cells. After harvest of SSCs from neonatal mice testes, the SSCs were divided into two groups: SSCs on a 3D alginate hydrogel with Sertoli cells and a co-culture of SSCs with Sertoli cells for 1 month. The samples were evaluated by quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays and bromodeoxyuridine (BrdU) tracing, haematoxylin and eosin (H&E) and periodic acid–Schiff (PAS) staining after transplantation into an azoospermic testis mouse. The 3D group showed rapid cell proliferation and numerous colonies compared with the co-culture group. Molecular assessment showed significantly increased integrin alpha-6, integrin beta-1, Nanog, Plzf, Thy-1, Oct4 and Bcl2 expression levels in the 3D group and decreased expression levels of P53, Fas, and Bax. BrdU tracing, and H&E and PAS staining results indicated that the hydrogel alginate improved spermatogenesis after transplantation in vivo. This finding suggested that cultivation of SSCs on alginate hydrogel with Sertoli cells in a 3D culture can lead to efficient proliferation and maintenance of SSC stemness and enhance the efficiency of SSC transplantation.

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