Rapid formation of functional muscle in vitro using fibrin gels

2005 ◽  
Vol 98 (2) ◽  
pp. 706-713 ◽  
Author(s):  
Yen-Chih Huang ◽  
Robert G. Dennis ◽  
Lisa Larkin ◽  
Keith Baar
Keyword(s):  
Physiological Function ◽  
Three Dimensional ◽  
Force Production ◽  
Force Transducer ◽  
Force Frequency ◽  
Platinum Electrodes ◽  
Fibrin Gel ◽  
Novel Method

The transition of a muscle cell from a differentiated myotube into an adult myofiber is largely unstudied. This is primarily due to the difficulty of isolating specific developmental stimuli in vivo and the inability to maintain viable myotubes in culture for sufficient lengths of time. To address these limitations, a novel method for rapidly generating three-dimensional engineered muscles using fibrin gel casting has been developed. Myoblasts were seeded and differentiated on top of a fibrin gel. Cell-mediated contraction of the gel around artificial anchors placed 12 mm apart culminates 10 days after plating in a tubular structure of small myotubes (10-μm diameter) surrounded by a fibrin gel matrix. These tissues can be connected to a force transducer and electrically stimulated between parallel platinum electrodes to monitor physiological function. Three weeks after plating, the three-dimensional engineered muscle generated a maximum twitch force of 329 ± 26.3 μN and a maximal tetanic force of 805.8 ± 55 μN. The engineered muscles demonstrated normal physiological function including length-tension and force-frequency relationships. Treatment with IGF-I resulted in a 50% increase in force production, demonstrating that these muscles responded to hormonal interventions. Although the force production was maximal at 3 wk, constructs can be maintained in culture for up to 6 wk with no intervention. We conclude that fibrin-based gels provide a novel method to engineer three-dimensional functional muscle tissue and that these tissues may be used to model the development of skeletal muscle in vitro.

Cultured slow vs. fast skeletal muscle cells differ in physiology and responsiveness to stimulation

2006 ◽  
Vol 291 (1) ◽  
pp. C11-C17 ◽  
Author(s):  
Yen-Chih Huang ◽  
Robert G. Dennis ◽  
Keith Baar
Keyword(s):  
Skeletal Muscle ◽  
Electrical Stimulation ◽  
Relaxation Times ◽  
Three Dimensional ◽  
Low Frequency ◽  
Force Production ◽  
Force Frequency ◽  
Protein Markers ◽  
Myogenic Cells

In vitro studies have used protein markers to distinguish between myogenic cells isolated from fast and slow skeletal muscles. The protein markers provide some support for the hypothesis that satellite cells from fast and slow muscles are different, but the data are equivocal. To test this hypothesis directly, three-dimensional skeletal muscle constructs were engineered from myogenic cells isolated from fast tibialis anterior (TA) and slow soleus (SOL) muscles of rats and functionality was tested. Time to peak twitch tension (TPT) and half relaxation time (RT1/2) were ∼30% slower in constructs from the SOL. The slower contraction and relaxation times for the SOL constructs resulted in left shift of the force-frequency curve compared with those from the TA. Western blot analysis showed a 60% greater quantity of fast myosin heavy chain in the TA constructs. 14 days of chronic low-frequency electrical stimulation resulted in a 15% slower TPT and a 14% slower RT1/2, but no change in absolute force production in the TA constructs. In SOL constructs, slow electrical stimulation resulted in an 80% increase in absolute force production with no change in TPT or RT1/2. The addition of cyclosporine A did not prevent the increase in force in SOL constructs after chronic low-frequency electrical stimulation, suggesting that calcineurin is not responsible for the increase in force. We conclude that myogenic cells associated with a slow muscle are imprinted to produce muscle that contracts and relaxes slowly and that calcineurin activity cannot explain the response to a slow pattern of electrical stimulation.

In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

1978 ◽  
Vol 36 (2) ◽  
pp. 434-435
Author(s):  
D. Reis ◽  
B. Vian ◽  
J. C. Roland
Keyword(s):  
Cell Wall ◽  
Self Assembly ◽  
Plant Cell Wall ◽  
Higher Plants ◽  
Three Dimensional ◽  
Cytochemical Study ◽  
Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

Epithelial Organotypic Cultures: A Viable Model to Address Mechanisms of Carcinogenesis by Epitheliotropic Viruses

2018 ◽  
Vol 18 (4) ◽  
pp. 246-255 ◽  
Author(s):  
Lara Termini ◽  
Enrique Boccardo
Keyword(s):  
Three Dimensional ◽  
Cell Types ◽  
Experimental Models ◽  
Biological Research ◽  
Specific Gene ◽  
Specific Cell ◽  
Organotypic Cultures ◽  
Skin Physiology

In vitro culture of primary or established cell lines is one of the leading techniques in many areas of basic biological research. The use of pure or highly enriched cultures of specific cell types obtained from different tissues and genetics backgrounds has greatly contributed to our current understanding of normal and pathological cellular processes. Cells in culture are easily propagated generating an almost endless source of material for experimentation. Besides, they can be manipulated to achieve gene silencing, gene overexpression and genome editing turning possible the dissection of specific gene functions and signaling pathways. However, monolayer and suspension cultures of cells do not reproduce the cell type diversity, cell-cell contacts, cell-matrix interactions and differentiation pathways typical of the three-dimensional environment of tissues and organs from where they were originated. Therefore, different experimental animal models have been developed and applied to address these and other complex issues in vivo. However, these systems are costly and time consuming. Most importantly the use of animals in scientific research poses moral and ethical concerns facing a steadily increasing opposition from different sectors of the society. Therefore, there is an urgent need for the development of alternative in vitro experimental models that accurately reproduce the events observed in vivo to reduce the use of animals. Organotypic cultures combine the flexibility of traditional culture systems with the possibility of culturing different cell types in a 3D environment that reproduces both the structure and the physiology of the parental organ. Here we present a summarized description of the use of epithelial organotypic for the study of skin physiology, human papillomavirus biology and associated tumorigenesis.

scholarly journals Functional Characterization of the mazEF Toxin-Antitoxin System in the Pathogenic Bacterium Agrobacterium tumefaciens

2021 ◽  
Vol 9 (5) ◽  
pp. 1107
Author(s):  
Wonho Choi ◽  
Yoshihiro Yamaguchi ◽  
Ji-Young Park ◽  
Sang-Hyun Park ◽  
Hyeok-Won Lee ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Physiological Function ◽  
Functional Characterization ◽  
Site Directed Mutagenesis ◽  
In Vitro Assays ◽  
Cell Growth Inhibition ◽  
The Right

Agrobacterium tumefaciens is a pathogen of various plants which transfers its own DNA (T-DNA) to the host plants. It is used for producing genetically modified plants with this ability. To control T-DNA transfer to the right place, toxin-antitoxin (TA) systems of A. tumefaciens were used to control the target site of transfer without any unintentional targeting. Here, we describe a toxin-antitoxin system, Atu0939 (mazE-at) and Atu0940 (mazF-at), in the chromosome of Agrobacterium tumefaciens. The toxin in the TA system has 33.3% identity and 45.5% similarity with MazF in Escherichia coli. The expression of MazF-at caused cell growth inhibition, while cells with MazF-at co-expressed with MazE-at grew normally. In vivo and in vitro assays revealed that MazF-at inhibited protein synthesis by decreasing the cellular mRNA stability. Moreover, the catalytic residue of MazF-at was determined to be the 24th glutamic acid using site-directed mutagenesis. From the results, we concluded that MazF-at is a type II toxin-antitoxin system and a ribosome-independent endoribonuclease. Here, we characterized a TA system in A. tumefaciens whose understanding might help to find its physiological function and to develop further applications.

scholarly journals The Revolutionary Roads to Study Cell–Cell Interactions in 3D In Vitro Pancreatic Cancer Models

Cancers ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 930
Author(s):  
Donatella Delle Cave ◽  
Riccardo Rizzo ◽  
Bruno Sainz ◽  
Giuseppe Gigli ◽  
Loretta L. del Mercato ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Therapeutic Response ◽  
Three Dimensional ◽  
Cellular Models ◽  
Common Cancer ◽  
Cancer Models ◽  
Anti Cancer ◽  
Tumor Environment

Pancreatic cancer, the fourth most common cancer worldwide, shows a highly unsuccessful therapeutic response. In the last 10 years, neither important advancements nor new therapeutic strategies have significantly impacted patient survival, highlighting the need to pursue new avenues for drug development discovery and design. Advanced cellular models, resembling as much as possible the original in vivo tumor environment, may be more successful in predicting the efficacy of future anti-cancer candidates in clinical trials. In this review, we discuss novel bioengineered platforms for anticancer drug discovery in pancreatic cancer, from traditional two-dimensional models to innovative three-dimensional ones.

Three-dimensional Growth of Renal Epithelial Cells in Vitro: A Tool in Toxicity Testing

1993 ◽  
Vol 21 (2) ◽  
pp. 191-195 ◽  
Author(s):  
Knut-Jan Andersen ◽  
Erik Ilsø Christensen ◽  
Hogne Vik
Keyword(s):  
Epithelial Cells ◽  
Three Dimensional ◽  
Toxicity Testing ◽  
Renal Tissue ◽  
Epithelial Cell Line ◽  
Multicellular Spheroids ◽  
Renal Epithelial Cell ◽  
Renal Epithelial Cells

The tissue culture of multicellular spheroids from the renal epithelial cell line LLC-PK1 (proximal tubule) is described. This represents a biological system of intermediate complexity between renal tissue in vivo and simple monolayer cultures. The multicellular structures, which show many similarities to kidney tubules in vivo, including a vectorial water transport, should prove useful for studying the potential nephrotoxicity of drugs and chemicals in vitro. In addition, the propagation of renal epithelial cells as multicellular spheroids in serum-free culture may provide information on the release of specific biological parameters, which may be suppressed or masked in serum-supplemented media.

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 Three-Dimensional Zirconia-Based Scaffolds for Load-Bearing Bone-Regeneration Applications: Prospects and Challenges

Materials ◽  
2021 ◽  
Vol 14 (12) ◽  
pp. 3207
Author(s):  
Kumaresan Sakthiabirami ◽  
Vaiyapuri Soundharrajan ◽  
Jin-Ho Kang ◽  
Yunzhi Peter Yang ◽  
Sang-Won Park
Keyword(s):  
Bone Regeneration ◽  
Biological Activities ◽  
Three Dimensional ◽  
In Vivo Studies ◽  
High Mechanical Strength ◽  
Real World Applications ◽  
3D Fabrication ◽  
Dental Applications

The design of zirconia-based scaffolds using conventional techniques for bone-regeneration applications has been studied extensively. Similar to dental applications, the use of three-dimensional (3D) zirconia-based ceramics for bone tissue engineering (BTE) has recently attracted considerable attention because of their high mechanical strength and biocompatibility. However, techniques to fabricate zirconia-based scaffolds for bone regeneration are in a stage of infancy. Hence, the biological activities of zirconia-based ceramics for bone-regeneration applications have not been fully investigated, in contrast to the well-established calcium phosphate-based ceramics for bone-regeneration applications. This paper outlines recent research developments and challenges concerning numerous three-dimensional (3D) zirconia-based scaffolds and reviews the associated fundamental fabrication techniques, key 3D fabrication developments and practical encounters to identify the optimal 3D fabrication technique for obtaining 3D zirconia-based scaffolds suitable for real-world applications. This review mainly summarized the articles that focused on in vitro and in vivo studies along with the fundamental mechanical characterizations on the 3D zirconia-based scaffolds.

scholarly journals BanLec-eGFP Chimera as a Tool for Evaluation of Lectin Binding to High-Mannose Glycans on Microorganisms

Biomolecules ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 180
Author(s):  
Zorana Lopandić ◽  
Luka Dragačević ◽  
Dragan Popović ◽  
Uros Andjelković ◽  
Rajna Minić ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Lectin Binding ◽  
Three Dimensional ◽  
Data Bank ◽  
Salmonella Typhi ◽  
Excitation Wavelength ◽  
Protein Data Bank Entry ◽  
High Mannose

Fluorescently labeled lectins are useful tools for in vivo and in vitro studies of the structure and function of tissues and various pathogens such as viruses, bacteria, and fungi. For the evaluation of high-mannose glycans present on various glycoproteins, a three-dimensional (3D) model of the chimera was designed from the crystal structures of recombinant banana lectin (BanLec, Protein Data Bank entry (PDB): 5EXG) and an enhanced green fluorescent protein (eGFP, PDB 4EUL) by applying molecular modeling and molecular mechanics and expressed in Escherichia coli. BanLec-eGFP, produced as a soluble cytosolic protein of about 42 kDa, revealed β-sheets (41%) as the predominant secondary structures, with the emission peak maximum detected at 509 nm (excitation wavelength 488 nm). More than 65% of the primary structure was confirmed by mass spectrometry. Competitive BanLec-eGFP binding to high mannose glycans of the influenza vaccine (Vaxigrip®) was shown in a fluorescence-linked lectin sorbent assay (FLLSA) with monosaccharides (mannose and glucose) and wild type BanLec and H84T BanLec mutant. BanLec-eGFP exhibited binding to mannose residues on different strains of Salmonella in flow cytometry, with especially pronounced binding to a Salmonella Typhi clinical isolate. BanLec-eGFP can be a useful tool for screening high-mannose glycosylation sites on different microorganisms.

scholarly journals Reversal of senescence-associated beta-galactosidase expression during in vitro three-dimensional tissue-engineering of human chondrocytes in a polymer scaffold

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shojiro Katoh ◽  
Atsuki Fujimaru ◽  
Masaru Iwasaki ◽  
Hiroshi Yoshioka ◽  
Rajappa Senthilkumar ◽  
...  
Keyword(s):  
Regenerative Medicine ◽  
Replicative Senescence ◽  
Cultured Cells ◽  
Three Dimensional ◽  
Human Chondrocytes ◽  
Cartilage Tissue ◽  
Optimal Method ◽  
Beta Galactosidase

AbstractRegenerative medicine applications require cells that are not inflicted with senescence after in vitro culture for an optimal in vivo outcome. Methods to overcome replicative senescence include genomic modifications which have their own disadvantages. We have evaluated a three-dimensional (3D) thermo-reversible gelation polymer (TGP) matrix environment for its capabilities to reverse cellular senescence. The expression of senescence-associated beta-galactosidase (SA-βgal) by human chondrocytes from osteoarthritis-affected cartilage tissue, grown in a conventional two-dimensional (2D) monolayer culture versus in 3D-TGP were compared. In 2D, the cells de-differentiated into fibroblasts, expressed higher SA-βgal and started degenerating at 25 days. SA-βgal levels decreased when the chondrocytes were transferred from the 2D to the 3D-TGP culture, with cells exhibiting a tissue-like growth until 42–45 days. Other senescence associated markers such as p16INK4a and p21 were also expressed only in 2D cultured cells but not in 3D-TGP tissue engineered cartilage. This is a first-of-its-kind report of a chemically synthesized and reproducible in vitro environment yielding an advantageous reversal of aging of human chondrocytes without any genomic modifications. The method is worth consideration as an optimal method for growing cells for regenerative medicine applications.

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