A Novel Suspended Hydrogel Membrane Platform for Cell Culture

2015 ◽  
Vol 6 (2) ◽  
Author(s):  
Yong X. Chen ◽  
Shihao Yang ◽  
Jiahan Yan ◽  
Ming-Han Hsieh ◽  
Lingyan Weng ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Biology ◽  
3D Cell Culture ◽  
Cell Encapsulation ◽  
Cell Types ◽  
Ease Of Use ◽  
In Vitro Assays ◽  
Hydrogel Membrane

Current cell-culture is largely performed on synthetic two-dimensional (2D) petri dishes or permeable supports such as Boyden chambers, mostly because of their ease of use and established protocols. It is generally accepted that modern cell biology research requires new physiologically relevant three-dimensional (3D) cell culture platform to mimic in vivo cell responses. To that end, we report the design and development of a suspended hydrogel membrane (ShyM) platform using gelatin methacrylate (GelMA) hydrogel. ShyM thickness (0.25–1 mm) and mechanical properties (10–70 kPa) can be varied by controlling the size of the supporting grid and concentration of GelMA prepolymer, respectively. GelMA ShyMs, with dual media exposure, were found to be compatible with both the cell-seeding and the cell-encapsulation approach as tested using murine 10T1/2 cells and demonstrated higher cellular spreading and proliferation as compared to flat GelMA unsuspended control. The utility of ShyM was also demonstrated using a case-study of invasion of cancer cells. ShyMs, similar to Boyden chambers, are compatible with standard well-plates designs and can be printed using commonly available 3D printers. In the future, ShyM can be potentially extended to variety of photosensitive hydrogels and cell types, to develop new in vitro assays to investigate complex cell–cell and cell–extracellular matrix (ECM) interactions.

Ratiometric Nanoviscometers: Applications for Measuring Cellular Physical Properties in 3D Cultures

2020 ◽  
Vol 25 (3) ◽  
pp. 234-246
Author(s):  
Charles McRae White ◽  
Mark A. Haidekker ◽  
William S. Kisaalita
Keyword(s):  
Cell Culture ◽  
Cell Cultures ◽  
Cell Biology ◽  
Low Cost ◽  
3D Cell Culture ◽  
Biomechanical Properties ◽  
Practical Applications ◽  
3D Cell Cultures

New insights into the biomechanical properties of cells are revealing the importance of these properties and how they relate to underlying molecular, architectural, and behavioral changes associated with cell state and disease processes. However, the current understanding of how these in vitro biomechanical properties are associated with in vivo processes has been developed based on the traditional monolayer (two-dimensional [2D]) cell culture, which traditionally has not translated well to the three-dimensional (3D) cell culture and in vivo function. Many gold standard methods and tools used to observe the biomechanical properties of 2D cell cultures cannot be used with 3D cell cultures. Fluorescent molecules can respond to external factors almost instantaneously and require relatively low-cost instrumentation. In this review, we provide the background on fluorescent molecular rotors, which are attractive tools due to the relationship of their emission quantum yield with environmental microviscosity. We make the case for their use in both 2D and 3D cell cultures and speculate on their fundamental and practical applications in cell biology.

scholarly journals Simplified Bioprinting-Based 3D Cell Culture Infection Models for Virus Detection

Viruses ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 1298
Author(s):  
Robert Koban ◽  
Tobias Lam ◽  
Franziska Schwarz ◽  
Lutz Kloke ◽  
Silvio Bürge ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cell Cultures ◽  
Virus Detection ◽  
3D Cell Culture ◽  
Cell Types ◽  
Promising Alternative ◽  
Host Interactions ◽  
Infection Models

Studies of virus–host interactions in vitro may be hindered by biological characteristics of conventional monolayer cell cultures that differ from in vivo infection. Three-dimensional (3D) cell cultures show more in vivo-like characteristics and may represent a promising alternative for characterisation of infections. In this study, we established easy-to-handle cell culture platforms based on bioprinted 3D matrices for virus detection and characterisation. Different cell types were cultivated on these matrices and characterised for tissue-like growth characteristics regarding cell morphology and polarisation. Cells developed an in vivo-like morphology and long-term cultivation was possible on the matrices. Cell cultures were infected with viruses which differed in host range, tissue tropism, cytopathogenicity, and genomic organisation and virus morphology. Infections were characterised on molecular and imaging level. The transparent matrix substance allowed easy optical monitoring of cells and infection even via live-cell microscopy. In conclusion, we established an enhanced, standardised, easy-to-handle bioprinted 3D-cell culture system. The infection models are suitable for sensitive monitoring and characterisation of virus–host interactions and replication of different viruses under physiologically relevant conditions. Individual cell culture models can further be combined to a multicellular array. This generates a potent diagnostic tool for propagation and characterisation of viruses from diagnostic samples.

scholarly journals Mammary gland 3D cell culture systems in farm animals

2021 ◽  
Vol 52 (1) ◽  
Author(s):  
Laurence Finot ◽  
Eric Chanat ◽  
Frederic Dessauge
Keyword(s):  
Cell Culture ◽  
Mammary Gland ◽  
Bacterial Infections ◽  
Three Dimensional ◽  
3D Cell Culture ◽  
Farm Animals ◽  
Culture Systems ◽  
Cell Culture Systems

AbstractIn vivo study of tissue or organ biology in mammals is very complex and progress is slowed by poor accessibility of samples and ethical concerns. Fortunately, however, advances in stem cell identification and culture have made it possible to derive in vitro 3D “tissues” called organoids, these three-dimensional structures partly or fully mimicking the in vivo functioning of organs. The mammary gland produces milk, the source of nutrition for newborn mammals. Milk is synthesized and secreted by the differentiated polarized mammary epithelial cells of the gland. Reconstructing in vitro a mammary-like structure mimicking the functional tissue represents a major challenge in mammary gland biology, especially for farm animals for which specific agronomic questions arise. This would greatly facilitate the study of mammary gland development, milk secretion processes and pathological effects of viral or bacterial infections at the cellular level, all with the objective of improving milk production at the animal level. With this aim, various 3D cell culture models have been developed such as mammospheres and, more recently, efforts to develop organoids in vitro have been considerable. Researchers are now starting to draw inspiration from other fields, such as bioengineering, to generate organoids that would be more physiologically relevant. In this chapter, we will discuss 3D cell culture systems as organoids and their relevance for agronomic research.

scholarly journals Self-organization and culture of Mesenchymal Stem Cell spheroids in acoustic levitation

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Nathan Jeger-Madiot ◽  
Lousineh Arakelian ◽  
Niclas Setterblad ◽  
Patrick Bruneval ◽  
Mauricio Hoyos ◽  
...  
Keyword(s):  
Cell Culture ◽  
3D Cell Culture ◽  
Culture Conditions ◽  
Acoustic Levitation ◽  
Cell Sheets ◽  
Cell Therapies ◽  
Cellular Models ◽  
Cell Spheroids

AbstractIn recent years, 3D cell culture models such as spheroid or organoid technologies have known important developments. Many studies have shown that 3D cultures exhibit better biomimetic properties compared to 2D cultures. These properties are important for in-vitro modeling systems, as well as for in-vivo cell therapies and tissue engineering approaches. A reliable use of 3D cellular models still requires standardized protocols with well-controlled and reproducible parameters. To address this challenge, a robust and scaffold-free approach is proposed, which relies on multi-trap acoustic levitation. This technology is successfully applied to Mesenchymal Stem Cells (MSCs) maintained in acoustic levitation over a 24-h period. During the culture, MSCs spontaneously self-organized from cell sheets to cell spheroids with a characteristic time of about 10 h. Each acoustofluidic chip could contain up to 30 spheroids in acoustic levitation and four chips could be ran in parallel, leading to the production of 120 spheroids per experiment. Various biological characterizations showed that the cells inside the spheroids were viable, maintained the expression of their cell surface markers and had a higher differentiation capacity compared to standard 2D culture conditions. These results open the path to long-time cell culture in acoustic levitation of cell sheets or spheroids for any type of cells.

scholarly journals Robust and Scalable Angiogenesis Assay of Perfused 3D Human iPSC-Derived Endothelium for Anti-Angiogenic Drug Screening

2020 ◽  
Vol 21 (13) ◽  
pp. 4804
Author(s):  
Vincent van Duinen ◽  
Wendy Stam ◽  
Eva Mulder ◽  
Farbod Famili ◽  
Arie Reijerkerk ◽  
...  
Keyword(s):  
Drug Screening ◽  
Cell Formation ◽  
Cell Types ◽  
Culture Conditions ◽  
In Vitro Assays ◽  
Vegf Receptor ◽  
Screening Assay ◽  
Angiogenesis Assay

To advance pre-clinical vascular drug research, in vitro assays are needed that closely mimic the process of angiogenesis in vivo. Such assays should combine physiological relevant culture conditions with robustness and scalability to enable drug screening. We developed a perfused 3D angiogenesis assay that includes endothelial cells (ECs) from induced pluripotent stem cells (iPSC) and assessed its performance and suitability for anti-angiogenic drug screening. Angiogenic sprouting was compared with primary ECs and showed that the microvessels from iPSC-EC exhibit similar sprouting behavior, including tip cell formation, directional sprouting and lumen formation. Inhibition with sunitinib, a clinically used vascular endothelial growth factor (VEGF) receptor type 2 inhibitor, and 3-(3-pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO), a transient glycolysis inhibitor, both significantly reduced the sprouting of both iPSC-ECs and primary ECs, supporting that both cell types show VEGF gradient-driven angiogenic sprouting. The assay performance was quantified for sunitinib, yielding a minimal signal window of 11 and Z-factor of at least 0.75, both meeting the criteria to be used as screening assay. In conclusion, we have developed a robust and scalable assay that includes physiological relevant culture conditions and is amenable to screening of anti-angiogenic compounds.

scholarly journals Lipodendriplexes mediated enhanced gene delivery: a cellular to pre-clinical investigation

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Imran Tariq ◽  
Muhammad Yasir Ali ◽  
Muhammad Farhan Sohail ◽  
Muhammad Umair Amin ◽  
Sajid Ali ◽  
...  
Keyword(s):  
Cell Culture ◽  
Gene Delivery ◽  
Cell Viability ◽  
3D Cell Culture ◽  
Serum Biochemistry ◽  
Cellular Protein ◽  
Ros Generation ◽  
Gfp Expression

AbstractClinical success of effective gene therapy is mainly hampered by the insufficiency of safe and efficient internalization of a transgene to the targeted cellular site. Therefore, the development of a safe and efficient nanocarrier system is one of the fundamental challenges to transfer the therapeutic genes to the diseased cells. Polyamidoamine (PAMAM) dendrimer has been used as an efficient non-viral gene vector (dendriplexes) but the toxicity and unusual biodistribution induced by the terminal amino groups (–NH2) limit its in vivo applications. Hence, a state of the art lipid modification with PAMAM based gene carrier (lipodendriplexes) was planned to investigate theirs in vitro (2D and 3D cell culture) and in vivo behaviour. In vitro pDNA transfection, lactate dehydrogenase (LDH) release, reactive oxygen species (ROS) generation, cellular protein contents, live/dead staining and apoptosis were studied in 2D cell culture of HEK-293 cells while GFP transfection, 3D cell viability and live/dead staining of spheroids were performed in its 3D cell culture. Acute toxicity studies including organ to body index ratio, hematological parameters, serum biochemistry, histopathological profiles and in vivo transgene expression were assessed in female BALB/c mice. The results suggested that, in comparison to dendriplexes the lipodendriplexes exhibited significant improvement of pDNA transfection (p < 0.001) with lower LDH release (p < 0.01) and ROS generation (p < 0.05). A substantially higher cellular protein content (p < 0.01) and cell viability were also observed in 2D culture. A strong GFP expression with an improved cell viability profile (p < 0.05) was indicated in lipodendriplexes treated 3D spheroids. In vivo archives showed the superiority of lipid-modified nanocarrier system, depicted a significant increase in green fluorescent protein (GFP) expression in the lungs (p < 0.01), heart (p < 0.001), liver (p < 0.001) and kidneys (p < 0.001) with improved serum biochemistry and hematological profile as compared to unmodified dendriplexes. No tissue necrosis was evident in the animal groups treated with lipid-shielded molecules. Therefore, a non-covalent conjugation of lipids with PAMAM based carrier system could be considered as a promising approach for an efficient and biocompatible gene delivery system.

scholarly journals Tailoring 3D cell culture templates with common hydrogels

2018 ◽  
Author(s):  
Aurélien Pasturel ◽  
Pierre-Olivier Strale ◽  
Vincent Studer
Keyword(s):  
Cell Culture ◽  
3D Cell Culture ◽  
In Vitro Models ◽  
Manufacturing Method ◽  
Biologically Relevant ◽  
Physiological Conditions ◽  
Subtractive Manufacturing ◽  
User Friendly

3D cell culture aims at reconciliating the simplicity of in vitro models with the human like properties encountered in vivo. Soft permeable hydrogels have emerged as user-friendly materials to grow cells in more physiological conditions. With the intent on turning these homogeneous substrates into biomimetic templates, we introduce a generic solution compatible with the most biologically relevant and often frail materials. Here we take control of the chemical environment driving generic radical reactions to craft common gels with patterned light. In a simple microreactor, we harness the well-known inhibition of radicals by oxygen to enable topographical photopolymerization. Strikingly, by sustaining an oxygen rich environment, we can also induce hydrogel photo-scission which turns out to be a powerful and generic subtractive manufacturing method. We finally introduce a flexible patterned functionalization protocol based on available photo-linkers. Using these common tools on the most popular hydrogels, we tailored soft templates where cells grow or self-organize into standardized structures. The platform we describe has the potential to set a standard in future 3D cell culture experiments.

scholarly journals A reliable and affordable 3D tumor spheroid model for natural product drug discovery: A case study of curcumin

2019 ◽  
Vol 2 (1) ◽  
Author(s):  
Loh Teng Hern Tan ◽  
Liang Ee Low ◽  
Siah Ying Tang ◽  
Wei Hsum Yap ◽  
Lay Hong Chuah ◽  
...  
Keyword(s):  
Cell Culture ◽  
Drug Discovery ◽  
Three Dimensional ◽  
3D Cell Culture ◽  
Automated Image Analysis ◽  
Tumor Spheroids ◽  
Culture Methods ◽  
In Vivo Models

Three-dimensional cell culture methods revolutionize the field of anticancer drug discovery, forming an important link-bridge between conventional in vitro and in vivo models and conferring significant clinical and biological relevant data. The current work presents an affordable yet reproducible method of generating homogenous 3D tumor spheroids. Also, a new open source software is adapted to perform an automated image analysis of 3D tumor spheroids and subsequently generate a list of morphological parameters of which could be utilized to determine the response of these spheroids toward treatments. Our data showed that this work could serve as a reliable 3D cell culture platform for preclinical cytotoxicity testing of natural products prior to the expensive and time-consuming animal models

scholarly journals Kidney-based in vitro models for drug-induced toxicity testing

2019 ◽  
Vol 93 (12) ◽  
pp. 3397-3418 ◽  
Author(s):  
João Faria ◽  
Sabbir Ahmed ◽  
Karin G. F. Gerritsen ◽  
Silvia M. Mihaila ◽  
Rosalinde Masereeuw
Keyword(s):  
Adverse Effects ◽  
Drug Disposition ◽  
Drug Effects ◽  
Toxicity Testing ◽  
Cell Types ◽  
In Vitro Assays ◽  
Vascular Effects ◽  
Drug Induced

Abstract The kidney is frequently involved in adverse effects caused by exposure to foreign compounds, including drugs. An early prediction of those effects is crucial for allowing novel, safe drugs entering the market. Yet, in current pharmacotherapy, drug-induced nephrotoxicity accounts for up to 25% of the reported serious adverse effects, of which one-third is attributed to antimicrobials use. Adverse drug effects can be due to direct toxicity, for instance as a result of kidney-specific determinants, or indirectly by, e.g., vascular effects or crystals deposition. Currently used in vitro assays do not adequately predict in vivo observed effects, predominantly due to an inadequate preservation of the organs’ microenvironment in the models applied. The kidney is highly complex, composed of a filter unit and a tubular segment, together containing over 20 different cell types. The tubular epithelium is highly polarized, and the maintenance of this polarity is critical for optimal functioning and response to environmental signals. Cell polarity is dependent on communication between cells, which includes paracrine and autocrine signals, as well as biomechanic and chemotactic processes. These processes all influence kidney cell proliferation, migration, and differentiation. For drug disposition studies, this microenvironment is essential for prediction of toxic responses. This review provides an overview of drug-induced injuries to the kidney, details on relevant and translational biomarkers, and advances in 3D cultures of human renal cells, including organoids and kidney-on-a-chip platforms.

Developmental complexity of early mammalian pluripotent cell populations in vivo and in vitro

1998 ◽  
Vol 10 (8) ◽  
pp. 535 ◽  
Author(s):  
T. A. Pelton ◽  
M. D. Bettess ◽  
J. Lake ◽  
J. Rathjen ◽  
P. D. Rathjen
Keyword(s):  
Cell Biology ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Cell Types ◽  
Experimental Manipulation ◽  
Pluripotent Cell ◽  
Pluripotent Cells ◽  
Cellular Origin

Early mammalian embryogenesis is characterised by the coordinated proliferation, differentiation, migration and apoptosis of a pluripotent cell pool that is able to give rise to extraembryonic lineages and all the cell types of the embryo proper. These cells retain pluripotent differentiation capability, defined in this paper as the ability to form all cell types of the embryo and adult, until differentiation into the three embryonic germ layers at gastrulation. Our understanding of pluripotent cell biology and molecular regulation has been hampered by the difficulties associated with experimental manipulation of these cells in vivo. However, a more detailed understanding of pluripotent cell behaviour is emerging from the application of molecular technologies to early mouse embryogenesis. The construction of mouse mutants by gene targeting, mapping of gene expression in vivo, and modelling of cell decisions in vitro are providing insight into the cellular origin, identity and action of key developmental regulators, and the nature of pluripotent cells themselves. In this review we discuss the properties of early embryonic pluripotent cells in vitro and in vivo, focusing on progression from inner cell mass (ICM) cells in the blastocyst to the onset of gastrulation.

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