Name: preparation of 3d fibrin scaffolds for stem cell culture applications
Uploaded: 2012-03-02T12:00:00
Description: Watch this Scientific Journal Video about Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications at JoVE.com

Författarna vill tacka för NSERC Discovery bidrag 402462 &quot;vävnadsteknisk ställningar för att styra inducerade pluripotenta beteende stamceller&quot;.

Anteckningar innan du börjar protokollet: Fibrinogen är en blod härstammar protein och därför lämpligt säkerhetsutbildning måste slutföras innan hantering. Detta protokoll kräver 2 dagar att slutföra så tiden önskade stamcellerna kulturerna lämpligt att säkerställa att de är redo för sådd. I termer av att beräkna hur mycket fibrinogen till väga, tre 35 mm petriskålar av tris-buffrad saltlösning (TBS, pH 7,4) innehållande 110-130 mg av fibrinogen upplöst i 3 ml av TBS kommer att ...

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Detta protokoll beskrivs ovan tillhandahåller ett förfarande för att generera 3D fibrin ställningar för pluripotenta stamceller kultur, särskilt för mus embryonala och inducerade pluripotenta stamceller. Denna 3D biomaterialet baserade odlingssystem mer exakt efterliknar nisch stamcell återfinnes in vivo och som ett resultat av detta kan den användas för att screena biologiska signaler för att bestämma deras effekter på stamceller differentiering 6. Våra observationer har visat att dess...

Utrustning som behövs: Analysvåg pH-mätare Vävnadsodlingsinkubator (37 ° C, 5% CO2) Omrörningsplatta Spektrofotometer Steril vävnadsodling huv Tris-buffrad saltlösning (pH 7,4) (Behöver 4 L plus tillräckligt för att lösa upp fibrinogen) 50 mM kalciumkloridlösning Sterila koniska rör (15 eller 50 ml) 35 mm Petri-skålar Dialysrör (7000 MW cutoff) Dialys klipp 5,0 fim sprutfilter Individuellt förpackade sterilt 0,22 fim sprutfilter Spruta 24 brunnars vävnadskulturplattor <table border="1"><tbody><tr><td> Namnet på reagens </td><td> Företaget </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> Fibrinogen (humant) </td><td> Calbiochem </td><td> 341.578 </td><td></td></tr><tr><td> Trombin (humant) </td><td> Sigma </td><td> T7009 </td><td></td></tr></tbody></table>

preparation of 3d fibrin scaffolds for stem cell culture applications

Stamceller som finns i naturligt förekommande 3D ​​mikromiljöer in vivo, vilket ofta refereras till som stamceller nisch 1. Odling av stamceller inuti 3D biomaterial byggnadsställningar är ett sätt att exakt efterlikna dessa mikromiljöer, vilket ger en fördel jämfört med traditionella 2D kultur metoder med polystyren samt en metod för att vävnader tekniska ersättning 2. Medan 2D vävnadskultur polystyren har använts för de flesta av experiment cellodling kan 3D biomaterial ställningar närmare replikera mikromiljöer som finns in vivo genom att möjliggöra mer exakt fastställande av cell polaritet i miljön och som har biokemiska och mekaniska egenskaper som liknar mjuk vävnad. 3 En mängd olika naturligt framställda och syntetiska biomaterial byggnadsställningar har undersökts som 3D-miljöer för att stödja tillväxt stamceller. Även syntetiska ställningar kan syntetiseras ha en större rorange av mekaniska och kemiska egenskaper och har ofta större reproducerbarhet, är naturliga biomaterial ofta sammansatta av proteiner och polysackarider som finns i den extracellulära matrisen och som ett resultat innehåller bindningsställen för celladhesion och lätthet cellkultur. Fibrin ställningar, som produceras genom polymerisation av proteinet fibrinogen erhålles från plasma, har undersökts i stor utsträckning för en mängd olika tillämpningar vävnadsutvecklingssteg både in vitro och in vivo 4. Sådana ställningar kan ändras med hjälp av olika metoder för att integrera system med kontrollerad frisättning för att leverera terapeutiska faktorer 5. Tidigare arbete har visat att sådana ställningar kan användas för att framgångsrikt kultur embryonala stamceller, och denna stödstruktur-baserade odlingssystem kan användas för att screena effekterna av olika tillväxtfaktorer på differentiering av stamcellerna ympade inuti 6,7. Detta protokoll detaljer processen för polymerizing fibrin stödstrukturer från fibrinogen lösningar med den enzymatiska aktiviteten av trombin. Processen tar 2 dagar att slutföra, inklusive en övernattning dialys steg för fibrinogen lösningen att ta bort citrater som hämmar polymerisation. Dessa detaljerade metoder förlitar sig på fibrinogenkoncentrationer bestämts vara optimal för embryonal och inducerade pluripotenta stamceller kultur. Andra grupper har ytterligare undersökt fibrin ställningar för ett brett spektrum av celltyper och applikationer - visar mångsidigheten av denna strategi 8-12.

Watch this Scientific Journal Video about Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications at JoVE.com

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Detta arbete detaljer förberedelse av 3D fibrin ställningar för odling och differentiering plutipotent stamceller. Sådana ställningar kan användas för att screena effekterna av olika biologiska föreningar på stamceller beteende samt modifierats för att innehålla läkemedelsavgivningssystem.

Beredning av 3D Fibrin ställningar för Kultur Stem Cell Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

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Research

JoVE Journal

Bioengineering

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Självrapporterings Ställningar för 3-dimensionell cellodling

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.

Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.

Provberedning Strategier för masspektrometri avbildning av 3D cellodlingsmodeller

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with ...

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

3D Hydrogel Byggnadsställningar för Articular kondrocyt kultur och brosk Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

Fabrication of Mechanically Tunable and Bioactive Metal Scaffolds for Biomedical Applications

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness and low bioactivity of metals. In this study, we have developed a new method towards fabricating a new type of bioactive and mechanically reliable porous metal scaffolds-densified porous Ti scaffolds. The method consists of two fabrication processes, 1) the fabrication of porous Ti scaffolds by dynamic freeze casting, and 2) coating and densification of the porous scaffolds. The dynamic freeze casting method to fabricate porous Ti scaffolds allowed the densification of porous scaffolds by minimizing the chemical contamination and structural defects. The densification process is distinctive for three reasons. First, the densification process is simple, because it requires a control of only one parameter (degree of densification). Second, it is effective, as it achieves mechanical enhancement and sustainable release of biomolecules from porous scaffolds. Third, it has broad applications, as it is also applicable to the fabrication of functionally graded porous scaffolds by spatially varied strain during densification.

Bioactive and mechanically reliable metal scaffolds have been fabricated through a method which consists of two processes, dynamic freeze casting for the fabrication of porous Ti, and coating and densification of the Ti scaffolds. The densification process is simple, effective and applicable to the fabrication of functionally graded scaffolds.

Tillverkning av mekaniskt Avstämbara och Bioactive Metal Byggnadsställningar för biomedicinska tillämpningar

Biometal systems have been widely used for biomedical applications, in particular, as load-bearing materials. However, major challenges are high stiffness ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

Micropatterning och montering av 3D mikrokärl

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Ljus medierad Bildning och mönstring av Hydrogeler för cellodling Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Synthesis of Biocompatible Liquid Crystal Elastomer Foams as Cell Scaffolds for 3D Spatial Cell Cultures

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block co-polymers featuring cholesterol units as side-chain pendant groups, resulting in smectic-A (SmA) liquid crystal elastomers (LCEs). Foam-like scaffolds, prepared using metal templates, feature interconnected microchannels, making them suitable as 3D cell culture scaffolds. The combined properties of the regular structure of the metal foam and of the elastomer result in a 3D cell scaffold that promotes not only higher cell proliferation compared to conventional porous templated films, but also better management of mass transport (i.e., nutrients, gases, waste, etc.). The nature of the metal template allows for the easy manipulation of foam shapes (i.e., rolls or films) and for the preparation of scaffolds of different pore sizes for different cell studies while preserving the interconnected porous nature of the template. The etching process does not affect the chemistry of the elastomers, preserving their biocompatible and biodegradable nature. We show that these smectic LCEs, when grown for extensive time periods, enable the study of clinically relevant and complex tissue constructs while promoting the growth and proliferation of cells.

This study presents a methodology to prepare 3D, biodegradable, foam-like cell scaffolds based on biocompatible side-chain liquid crystal elastomers (LCEs). Confocal microscopy experiments show that foam-like LCEs allow for cell attachment, proliferation, and the spontaneous alignment of C2C12s myoblasts.

Syntes av biokompatibla Liquid Crystal Elastomer Skum som Cell Ställningar för 3D Spatial cellkulturer

Here, we present a step-by-step preparation of a 3D, biodegradable, foam-like cell scaffold. These scaffolds were prepared by cross-linking star block ...

Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low cell retention and poor survival within the target zone. However, during the in vitro construction process, a solution for maintaining stem cell bioactivity and increasing the cell amount within the cell sheet is urgently needed. Here, this protocol presents a method for constructing a multilayered cell sheet with favorable stem cell bioactivity and optimal operability. Decellularized porcine pericardium (DPP) is prepared by phospholipase A2 (PLA2) decellularization method as the cell sheet scaffold, and rat bone marrow mesenchymal stem cells (BMSCs) are isolated and expanded as the seeded cells. The temporary multilayered cell sheet structure is constructed by using RAD16-I peptide hydrogel. Finally, the cell sheet is cultured with a dynamic perfusion system to stabilize the three-dimensional (3D) structure, and the cell sheet could be obtained following a 48-hour culture in vitro. This protocol provides an efficient and feasible method for constructing a multilayered stem cell sheet, and the cell sheet could be developed as a favorable stem cell therapy product in the future.

This article provides an efficient and feasible method for constructing multilayered stem cell sheets with favorable stem cell property.

Byggandet av ett Multilayered mesenkymala stamceller blad med ett 3D dynamisk kultur System

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after chemotherapy or irradiation to treat bone marrow injury or in vitro culture. By mimicking the bone marrow microenvironment in the presence of abundant fatty acids, cholesterol, low concentration of cytokines, and hypoxia, human HSCs maintain quiescence in vitro. Here, a detailed protocol for maintaining functional HSCs in the quiescent state in vitro is described. This method will enable studies of the behavior of human HSCs under physiological conditions.

This protocol enables the maintenance of quiescent human hematopoietic stem cells in vitro by mimicking the bone marrow microenvironment using abundant fatty acids, cholesterol, lower concentrations of cytokines, and hypoxia.

En kulturmetod för att upprätthålla quiescenta mänskliga hematopoetiska stamceller

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...

Preparation and Characterization of Graphene-Based 3D Biohybrid Hydrogel Bioink for Peripheral Neuroengineering

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global problem that occurs in 1.5%-5% of emergency patients and may lead to significant job losses. Today, tissue engineering-based approaches, consisting of scaffolds, appropriate cell lines, and biosignals, have become more applicable with the development of three-dimensional (3D) bioprinting technologies. The combination of various hydrogel biomaterials with stem cells, exosomes, or bio-signaling molecules is frequently studied to overcome the existing problems in peripheral nerve regeneration. Accordingly, the production of injectable systems, such as hydrogels, or implantable conduit structures formed by various bioprinting methods has gained importance in peripheral neuro-engineering. Under normal conditions, stem cells are the regenerative cells of the body, and their number and functions do not decrease with time to protect their populations; these are not specialized cells but can differentiate upon appropriate stimulation in response to injury. The stem cell system is under the influence of its microenvironment, called the stem cell niche. In peripheral nerve injuries, especially in neurotmesis, this microenvironment cannot be fully rescued even after surgically binding severed nerve endings together. The composite biomaterials and combined cellular therapies approach increases the functionality and applicability of materials in terms of various properties such as biodegradability, biocompatibility, and processability. Accordingly, this study aims to demonstrate the preparation and use of graphene-based biohybrid hydrogel patterning and to examine the differentiation efficiency of stem cells into nerve cells, which can be an effective solution in nerve regeneration.

In this manuscript, we demonstrate the preparation of a biohybrid hydrogel bioink containing graphene for use in peripheral tissue engineering. Using this 3D biohybrid material, the neural differentiation protocol of stem cells is performed. This can be an important step in bringing similar biomaterials to the clinic.

Beredning och karakterisering av grafenbaserat 3D-biohybridhydrogelbiobläck för perifer neuroengineering

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global ...