scholarly journals Gelatin-Coated Microfluidic Channels for 3D Microtissue Formation: On-Chip Production and Characterization

Micromachines ◽  
2019 ◽  
Vol 10 (4) ◽  
pp. 265 ◽  
Author(s):  
Gabriele Pitingolo ◽  
Antoine Riaud ◽  
Claudio Nastruzzi ◽  
Valerie Taly
Keyword(s):  
Cell Culture ◽  
Type I Collagen ◽  
3D Cell Culture ◽  
Gelatin Film ◽  
New Drugs ◽  
In Situ Monitoring ◽  
Microfluidic Channels ◽  
Type I ◽  
Gelatin Coating

Traditional two-dimensional (2D) cell culture models are limited in their ability to reproduce human structures and functions. On the contrary, three-dimensional (3D) microtissues have the potential to permit the development of new cell-based assays as advanced in vitro models to test new drugs. Here, we report the use of a dehydrated gelatin film to promote tumor cells aggregation and 3D microtissue formation. The simple and stable gelatin coating represents an alternative to conventional and expensive materials like type I collagen, hyaluronic acid, or matrigel. The gelatin coating is biocompatible with several culture formats including microfluidic chips, as well as standard micro-well plates. It also enables long-term 3D cell culture and in situ monitoring of live/dead assays.

scholarly journals SAT0293 EXOSOMES DERIVED FROM PLASMA OF SYSTEMIC SCLEROSIS (SSC) PATIENTS AND FROM SSC CULTURED FIBROBLASTS CONTAIN PRO-FIBROTIC MIRNA SIGNATURES AND COULD INDUCE MYOFIBROBLAST DIFFERENTIATION IN VITRO

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1091.2-1092
Author(s):  
C. Corallo ◽  
M. Cutolo ◽  
S. Soldano ◽  
E. Selvi ◽  
F. Bellisai ◽  
...  
Keyword(s):  
Cell Culture ◽  
Systemic Sclerosis ◽  
Real Time Pcr ◽  
Type I Collagen ◽  
Type I ◽  
Myofibroblast Differentiation ◽  
Plasma Samples ◽  
Fibroblast Cultures ◽  
Control Plasma

Background:Exosomes generated great resonance in the last few years due to their important roles in different biological pathways and diseases, including systemic sclerosis (SSc) (1). They are lipid-like nanovesicles containing biomarkers, such as proteins, lipids, macromolecules and nucleic acids, including microRNA (miRNA) (2). Exosomes are implicated in intercellular communication by fusing and releasing their cargo into the target cells (3).Objectives:In the present study, we evaluated the potential of exosomes deriving from plasma of SSc patients or generating from cultured SSc fibroblasts to drive the fibrotic signaling in the disease.Methods:Exosomes were isolated from plasma of n=10 SSc patients and from n=10 control subjects. Exosomes were also purified from cell culture supernatants of SSc fibroblasts and of control fibroblasts. Exosome size and concentration were assessed by Nanosight Particle Tracking Analysis (NTA) and by transmission electron microscopy (TEM). The content of anti-fibrotic (let-7a, 146a, 200a, 223a) and pro-fibrotic (150, 155) miRNAs was assessed in all the plasma-derived and cell culture-derived exosome populations by semiquantitative real time PCR. Finally, isolated exosomes were used to stimulate control dermal fibroblasts in culture. Gene expressions (COL1A1, ACTA2 and TAGLN) were assessed by quantitative real time PCR (qRT-PCR) and protein levels (type-I-collagen, α-SMA and SM22) by immunofluorescence (IF).Results:Exosomes isolated from SSc plasma samples showed higher concentration (3.3x1010±1.1x1010particles/mL) compared to those isolated from control plasma ones (1.5x1010±0.4x1010particles/mL) (p<0.01). The exosome size did not differ between SSc and control plasma samples and ranged from 50nm to 150nm. Similar results were obtained with exosomes generated from fibroblast cultures: the concentration was higher in SSc fibroblasts (1.1x1010±0.2x1010particles/mL) than in control ones (0.4x1010±0.1x1010particles/mL) (p<0.05) with no significant differences in size distribution. The content of all anti-fibrotic (let-7a, 146a, 200a, 223a) miRNAs was decreased in exosomes coming from both SSc plasma samples and from SSc fibroblasts with respect to control plasma samples (p<0.05) and to control fibroblasts (p<0.05). On the contrary, the pro-fibrotic (150, 155) miRNAs were significantly upregulated in exosomes deriving from SSc plasma samples and from SSc fibroblasts, with respect to control plasma samples (p<0.05) and to control fibroblasts (p<0.05). Finally, only exosomes coming from SSc plasma samples or SSc fibroblast cultures were able to induce pro-fibrotic gene (COL1A1, ACTA2 and TAGLN) and protein (type-I-collagen, α-SMA and SM22) expression in control fibroblasts. No pro-fibrotic induction was seen in presence of exosomes isolated from control plasma samples or control fibroblast cultures.Conclusion:This study demonstrates that plasma from SSc patients contains higher concentration of exosomes compared to plasma from control subjects and SSc-derived exosomes contain specific pro-fibrotic miRNA signatures that can induce myofibroblast differentiationin vitro. These results suggest that exosomes could be fibrotic drivers towards non-affected areasin vivo, and they might represent novel targets for precision medicine treatments in SSc.References:[1]Zhu T, Wang Y, Jin H, Li L. The role of exosome in autoimmune connective tissue disease. Ann Med. 2019 Mar;51(2):101-108.[2]Wermuth PJ, Piera-Velazquez S, Rosenbloom J, et al. Existing and novel biomarkers for precision medicine in systemic sclerosis. Nat Rev Rheumatol. 2018 Jul;14(7):421-432.[3]Colletti M, Galardi A, De Santis M, et al. Exosomes in Systemic Sclerosis: Messengers Between Immune, Vascular and Fibrotic Components? Int J Mol Sci. 2019 Sep 4;20(18). pii: E4337.Disclosure of Interests:Claudio Corallo: None declared, Maurizio Cutolo Grant/research support from: Bristol-Myers Squibb, Actelion, Celgene, Consultant of: Bristol-Myers Squibb, Speakers bureau: Sigma-Alpha, Stefano Soldano: None declared, Enrico Selvi: None declared, Francesca Bellisai: None declared, Nicola Giordano: None declared

scholarly journals 3D cell culture using a clinostat reproduces microgravity-induced skin changes

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Dong Hyun Choi ◽  
Byoungjun Jeon ◽  
Min Hyuk Lim ◽  
Dong Hun Lee ◽  
Sang-Kyu Ye ◽  
...  
Keyword(s):  
Type I Collagen ◽  
Skin Irritation ◽  
3D Culture ◽  
3D Cell Culture ◽  
Tissue Growth ◽  
Microgravity Environment ◽  
Type I ◽  
Physiological Changes ◽  
3D Culture System

AbstractExposure to microgravity affects human physiology in various ways, and astronauts frequently report skin-related problems. Skin rash and irritation are frequent complaints during space missions, and skin thinning has also been reported after returning to Earth. However, spaceflight missions for studying the physiological changes in microgravity are impractical. Thus, we used a previously developed 3D clinostat to simulate a microgravity environment and investigate whether physiological changes of the skin can be reproduced in a 3D in vitro setting. Our results showed that under time-averaged simulated microgravity (taSMG), the thickness of the endothelial cell arrangement increased by up to 59.75%, indicating skin irritation due to vasodilation, and that the diameter of keratinocytes and fibroblast co-cultured spheroids decreased by 6.66%, representing skin thinning. The α1 chain of type I collagen was upregulated, while the connective tissue growth factor was downregulated under taSMG. Cytokeratin-10 expression was significantly increased in the taSMG environment. The clinostat-based 3D culture system can reproduce physiological changes in the skin similar to those under microgravity, providing insight for understanding the effects of microgravity on human health before space exploration.

Collagen fibrillogenesis in vitro: interaction of types I and V collagen

1986 ◽  
Vol 44 ◽  
pp. 210-211
Author(s):  
Arthur J. Wasserman ◽  
Kathy C. Kloos ◽  
David E. Birk
Keyword(s):  
Type I Collagen ◽  
Corneal Stroma ◽  
Minor Component ◽  
Homogeneous Distribution ◽  
Type I ◽  
Type V Collagen ◽  
Fibril Morphology ◽  
Type V ◽  
In Vitro Interaction

Type I collagen is the predominant collagen in the cornea with type V collagen being a quantitatively minor component. However, the content of type V collagen (10-20%) in the cornea is high when compared to other tissues containing predominantly type I collagen. The corneal stroma has a homogeneous distribution of these two collagens, however, immunochemical localization of type V collagen requires the disruption of type I collagen structure. This indicates that these collagens may be arranged as heterpolymeric fibrils. This arrangement may be responsible for the control of fibril diameter necessary for corneal transparency. The purpose of this work is to study the in vitro assembly of collagen type V and to determine whether the interactions of these collagens influence fibril morphology.

scholarly journals Restoration of Osteogenesis by CRISPR/Cas9 Genome Editing of the Mutated COL1A1 Gene in Osteogenesis Imperfecta

2021 ◽  
Vol 10 (14) ◽  
pp. 3141
Author(s):  
Hyerin Jung ◽  
Yeri Alice Rim ◽  
Narae Park ◽  
Yoojun Nam ◽  
Ji Hyeon Ju
Keyword(s):  
Osteogenesis Imperfecta ◽  
Type I Collagen ◽  
Disease Modeling ◽  
Bone Fragility ◽  
Osteogenic Potential ◽  
Type I ◽  
Gene Correction ◽  
Col1a1 Gene ◽  
Collagen Formation

Osteogenesis imperfecta (OI) is a genetic disease characterized by bone fragility and repeated fractures. The bone fragility associated with OI is caused by a defect in collagen formation due to mutation of COL1A1 or COL1A2. Current strategies for treating OI are not curative. In this study, we generated induced pluripotent stem cells (iPSCs) from OI patient-derived blood cells harboring a mutation in the COL1A1 gene. Osteoblast (OB) differentiated from OI-iPSCs showed abnormally decreased levels of type I collagen and osteogenic differentiation ability. Gene correction of the COL1A1 gene using CRISPR/Cas9 recovered the decreased type I collagen expression in OBs differentiated from OI-iPSCs. The osteogenic potential of OI-iPSCs was also recovered by the gene correction. This study suggests a new possibility of treatment and in vitro disease modeling using patient-derived iPSCs and gene editing with CRISPR/Cas9.

scholarly journals Applications of Biomaterials in 3D Cell Culture and Contributions of 3D Cell Culture to Drug Development and Basic Biomedical Research

2021 ◽  
Vol 22 (5) ◽  
pp. 2491
Author(s):  
Yujin Park ◽  
Kang Moo Huh ◽  
Sun-Woong Kang
Keyword(s):  
Cell Culture ◽  
Three Dimensional ◽  
3D Culture ◽  
3D Cell Culture ◽  
Accurate Method ◽  
New Drugs ◽  
Toxicity Screening ◽  
Cellular Functions ◽  
Toxicity Of Drugs ◽  
External Stimuli

The process of evaluating the efficacy and toxicity of drugs is important in the production of new drugs to treat diseases. Testing in humans is the most accurate method, but there are technical and ethical limitations. To overcome these limitations, various models have been developed in which responses to various external stimuli can be observed to help guide future trials. In particular, three-dimensional (3D) cell culture has a great advantage in simulating the physical and biological functions of tissues in the human body. This article reviews the biomaterials currently used to improve cellular functions in 3D culture and the contributions of 3D culture to cancer research, stem cell culture and drug and toxicity screening.

scholarly journals In Vitro Human Joint Models Combining Advanced 3D Cell Culture and Cutting-Edge 3D Bioprinting Technologies

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 596
Author(s):  
Christian Jorgensen ◽  
Matthieu Simon
Keyword(s):  
New Technology ◽  
3D Cell Culture ◽  
External Pressure ◽  
New Drugs ◽  
Cellular Interactions ◽  
Joint Models ◽  
Physical Stimulation ◽  
Precise Control ◽  
Diarthrodial Joint

Joint-on-a-chip is a new technology able to replicate the joint functions into microscale systems close to pathophysiological conditions. Recent advances in 3D printing techniques allow the precise control of the architecture of the cellular compartments (including chondrocytes, stromal cells, osteocytes and synoviocytes). These tools integrate fluid circulation, the delivery of growth factors, physical stimulation including oxygen level, external pressure, and mobility. All of these structures must be able to mimic the specific functions of the diarthrodial joint: mobility, biomechanical aspects and cellular interactions. All the elements must be grouped together in space and reorganized in a manner close to the joint organ. This will allow the study of rheumatic disease physiopathology, the development of biomarkers and the screening of new drugs.

scholarly journals Effects of chitosan-collagen dressing on wound healing in vitro and in vivo assays

2021 ◽  
Vol 19 ◽  
pp. 228080002198969
Author(s):  
Min-Xia Zhang ◽  
Wan-Yi Zhao ◽  
Qing-Qing Fang ◽  
Xiao-Feng Wang ◽  
Chun-Ye Chen ◽  
...  
Keyword(s):  
Wound Healing ◽  
Wound Dressing ◽  
Type I Collagen ◽  
Inhibition Zone ◽  
Negative Control ◽  
Type I ◽  
Nih3t3 Cells ◽  
Post Operation

The present study was designed to fabricate a new chitosan-collagen sponge (CCS) for potential wound dressing applications. CCS was fabricated by a 3.0% chitosan mixture with a 1.0% type I collagen (7:3(w/w)) through freeze-drying. Then the dressing was prepared to evaluate its properties through a series of tests. The new-made dressing demonstrated its safety toward NIH3T3 cells. Furthermore, the CCS showed the significant surround inhibition zone than empty controls inoculated by E. coli and S. aureus. Moreover, the moisture rates of CCS were increased more rapidly than the collagen and blank sponge groups. The results revealed that the CCS had the characteristics of nontoxicity, biocompatibility, good antibacterial activity, and water retention. We used a full-thickness excisional wound healing model to evaluate the in vivo efficacy of the new dressing. The results showed remarkable healing at 14th day post-operation compared with injuries treated with collagen only as a negative control in addition to chitosan only. Our results suggest that the chitosan-collagen wound dressing were identified as a new promising candidate for further wound application.

scholarly journals Relative rates of biosynthesis of collagen type I, type V and type VI in calf cornea

1991 ◽  
Vol 274 (2) ◽  
pp. 615-617 ◽  
Author(s):  
P Kern ◽  
M Menasche ◽  
L Robert
Keyword(s):  
Type I Collagen ◽  
Collagen Type ◽  
Corneal Stroma ◽  
Collagen Type I ◽  
Type I ◽  
Type Vi Collagen ◽  
Sds Page ◽  
Proline Incorporation ◽  
Type V

The biosynthesis of type I, type V and type VI collagens was studied by incubation of calf corneas in vitro with [3H]proline as a marker. Pepsin-solubilized collagen types were isolated by salt fractionation and quantified by SDS/PAGE. Expressed as proportions of the total hydroxyproline solubilized, corneal stroma comprised 75% type I, 8% type V and 17% type VI collagen. The rates of [3H]proline incorporation, linear up to 24 h for each collagen type, were highest for type VI collagen and lowest for type I collagen. From pulse-chase experiments, the calculated apparent half-lives for types I, V and VI collagens were 36 h, 10 h and 6 h respectively.

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.

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