SeedEZ 3D cell culture application notes - gel and drug embedding. Many inert polymers used as scaffolds for 3D cell cultures and colony formation are also used in drug delivery systems both in vitro and in vivo. Read this practical guide to learn how SeedEZ lets you merge these two worlds in order to integrate 3D cell cultures into standard drug delivery and testing applications.
By incorporating or adding drugs to SeedEZ, or in polymer matrices embedded in SeedEZ, dosage forms which release a drug over a period of time may be prepared in a desired shape and size. More importantly, all SeedEZ-based dosage forms may be tested in situ, with cells in a 3D cell culture. SeedEZ wicks most sol-state hydrogels, hydrogel precursors, semisolid media, excipient formulations, pharmaceuticals and test compounds. As a result, SeedEZ offers a novel 3D framework for (A) development of sustained release drug delivery systems that are simple to make and convenient to use in vitro; (B) localized or distributed drug delivery into 3D cell cultures using spot-a-culture and spot-a-drug approach, wick, dip or SeedEZ-stack method; (C) gradient formation and testing of drug combination strategies; (D) quality control testing and assurance; and (E) development of test platforms for quasi-steady drug release.
Notably, in most diffusion-driven drug delivery systems, a drug release rate declines in time. A degradable polymer matrix embedded in SeedEZ may enable quasi-steady drug release from a defined volume, defined by SeedEZ, when the matrix degradation rate is adjusted to compensate for this decline via increased drug permeability from the SeedEZ/polymer matrix system.
The application note covers use of common biomaterials, including extracellular matrix hydrogels (Collagen and Matrigel), gels from natural sources for spheroid cultures and controlled drug release (Agarose, Alginate, Methylcellulose, Gelatin), and synthetic materials such as Poloxamers (Pluronic - used for cell encapsulation, drug delivery and pharmaceutical formulations), and Carbomers used in ocular, transdermal, oral and nasal delivery systems.
SeedEZ 3D cell culture methods and protocols - cell seedingLena Biosciences
This document provides protocols for seeding various cell types into 3D cell culture substrates called SeedEZ. It describes seeding mixed cell cultures, adherent cells, and cells embedded in extracellular matrix or gel using different methods. These include seeding cells in a sol-state gel suspension, on top of an ECM barrier, or in a sandwich between ECM layers. The document also provides guidance on seeding feeder layers, stacking multiple SeedEZ substrates, and designing migration and invasion assays using SeedEZ.
SeedEZ 3D culture methods and protocols - total protein extractionLena Biosciences
SeedEZ 3D cell culture methods and protocols – total protein extraction and quantification. 3D cell culturing conditions influence protein extraction from cells for downstream analysis. Depending on extraction buffer used, an amount of protein from the extracellular matrix (in addition to cell protein) may be extracted. For these reasons, setup suitable controls. Here, it is shown how to extract total protein from cells cultured in 3D in the SeedEZ under different culturing conditions and how to apply a protein assay to quantify it. Protein assay results with 3D brain co-culture models comprising primary cortical neurons and primary-harvested and one-time passaged mixed glia (astrocytes and microglia) seeded into (a) uncoated SeedEZ substrate, (b) Poly-D-Lysine coated SeedEZ substrate, and (c) seeded in 7.5 mg/ml protein suspension into uncoated SeedEZ are shown.
SeedEZ 3D cell culture methods and protocols - tissue culture coatingLena Biosciences
SeedEZ 3D cell culture methods and protocols – tissue culture coating. 3D culturing conditions influence selection and application of coatings for anchorage-dependent cells. Depending on cell types and research objectives, SeedEZ may be coated or uncoated. If coated, SeedEZ may be coated with ligands which promote cell adhesion, or molecules which prevent cell adhesion to the SeedEZ substrate. The former provides a 3D network of cells adhered to and spread inside the SeedEZ. The latter provides an aggregate 3D cell culture model or 3D cell spheroids cultured suspended within the interior of the SeedEZ. Follow our user guidelines to learn which coatings are best suited for your application, whether spheroid cultures or 3D cell cultures of substrate adhered cells. Coating recommendations for diverse 3D cells culture models and detailed coating protocols for bone 3D cell culture models (cultured up to 8 weeks) are provided.
SeedEZ 3D culture methods and protocols - cell isolation techniquesLena Biosciences
This document provides protocols and guidelines for recovering cells that have been cultured in 3D using Lena Biosciences' SeedEZ cell culture system. It discusses general guidelines for recovering adherent cells coated on SeedEZ substrates or embedded in hydrogels. It provides details on commonly used reagents for recovery and factors to consider like coating composition and culturing time. The document includes two specific protocols for recovering cells coated on poly-D-lysine coated SeedEZ and embedded in an extracellular matrix gel in SeedEZ. It discusses expected results and recommendations for automation and increased throughput.
3D cell culture techniques for the tumor modelsDurgesh Jha
The document discusses 3D cell culture techniques for developing in vitro tumor models. It begins with an introduction to the advantages of 3D culture over 2D culture in mimicking the complex tumor microenvironment. Various 3D culture techniques are described, including spontaneous aggregation, liquid overlay, hanging drop method, and scaffold-based cultures. The mechanisms of spheroid formation and applications of 3D tumor models in cancer research are also summarized.
Three key points are summarized:
1. Three-dimensional cell cultures provide a more natural environment for cells compared to traditional 2D cultures, allowing cells to behave more like they do in vivo.
2. 3D cell culture technology is used for applications like tissue engineering, drug discovery, and analysis of cell biology. It involves engineering scaffolds and growth factors to direct cell differentiation.
3. Mathematical modeling is important for understanding the complex biological and physical factors influencing 3D cell cultures, but optimization of cultures remains an ongoing area of research due to the large number of tunable parameters.
Genes and Tissue Culture Technology Assignment (G6)Rohini Krishnan
The culture of cells in two dimensions does not reproduce the histological characteristics of a tissue for informative or useful study. Growing cells as three-dimensional (3D) models more analogous to their existence in vivo may be more clinically relevant.
3D tumor spheroid models for in vitro therapeutic screening: a systematic app...Arun kumar
This document describes a study that aimed to identify and validate a cytotoxicity test for large tumor spheroids. Several methods were used to culture 3D tumor spheroids from lung cancer cells. Spheroid size, shape, and heterogeneity were characterized using imaging software. Cell viability was assessed using different assays under drug and radiation treatment. The results showed that spheroid shape and volume influence cytotoxicity assay results. Careful selection of homogeneous, spherical spheroids is important for reliable data in drug screening tests using 3D tumor models.
SeedEZ 3D cell culture methods and protocols - cell seedingLena Biosciences
This document provides protocols for seeding various cell types into 3D cell culture substrates called SeedEZ. It describes seeding mixed cell cultures, adherent cells, and cells embedded in extracellular matrix or gel using different methods. These include seeding cells in a sol-state gel suspension, on top of an ECM barrier, or in a sandwich between ECM layers. The document also provides guidance on seeding feeder layers, stacking multiple SeedEZ substrates, and designing migration and invasion assays using SeedEZ.
SeedEZ 3D culture methods and protocols - total protein extractionLena Biosciences
SeedEZ 3D cell culture methods and protocols – total protein extraction and quantification. 3D cell culturing conditions influence protein extraction from cells for downstream analysis. Depending on extraction buffer used, an amount of protein from the extracellular matrix (in addition to cell protein) may be extracted. For these reasons, setup suitable controls. Here, it is shown how to extract total protein from cells cultured in 3D in the SeedEZ under different culturing conditions and how to apply a protein assay to quantify it. Protein assay results with 3D brain co-culture models comprising primary cortical neurons and primary-harvested and one-time passaged mixed glia (astrocytes and microglia) seeded into (a) uncoated SeedEZ substrate, (b) Poly-D-Lysine coated SeedEZ substrate, and (c) seeded in 7.5 mg/ml protein suspension into uncoated SeedEZ are shown.
SeedEZ 3D cell culture methods and protocols - tissue culture coatingLena Biosciences
SeedEZ 3D cell culture methods and protocols – tissue culture coating. 3D culturing conditions influence selection and application of coatings for anchorage-dependent cells. Depending on cell types and research objectives, SeedEZ may be coated or uncoated. If coated, SeedEZ may be coated with ligands which promote cell adhesion, or molecules which prevent cell adhesion to the SeedEZ substrate. The former provides a 3D network of cells adhered to and spread inside the SeedEZ. The latter provides an aggregate 3D cell culture model or 3D cell spheroids cultured suspended within the interior of the SeedEZ. Follow our user guidelines to learn which coatings are best suited for your application, whether spheroid cultures or 3D cell cultures of substrate adhered cells. Coating recommendations for diverse 3D cells culture models and detailed coating protocols for bone 3D cell culture models (cultured up to 8 weeks) are provided.
SeedEZ 3D culture methods and protocols - cell isolation techniquesLena Biosciences
This document provides protocols and guidelines for recovering cells that have been cultured in 3D using Lena Biosciences' SeedEZ cell culture system. It discusses general guidelines for recovering adherent cells coated on SeedEZ substrates or embedded in hydrogels. It provides details on commonly used reagents for recovery and factors to consider like coating composition and culturing time. The document includes two specific protocols for recovering cells coated on poly-D-lysine coated SeedEZ and embedded in an extracellular matrix gel in SeedEZ. It discusses expected results and recommendations for automation and increased throughput.
3D cell culture techniques for the tumor modelsDurgesh Jha
The document discusses 3D cell culture techniques for developing in vitro tumor models. It begins with an introduction to the advantages of 3D culture over 2D culture in mimicking the complex tumor microenvironment. Various 3D culture techniques are described, including spontaneous aggregation, liquid overlay, hanging drop method, and scaffold-based cultures. The mechanisms of spheroid formation and applications of 3D tumor models in cancer research are also summarized.
Three key points are summarized:
1. Three-dimensional cell cultures provide a more natural environment for cells compared to traditional 2D cultures, allowing cells to behave more like they do in vivo.
2. 3D cell culture technology is used for applications like tissue engineering, drug discovery, and analysis of cell biology. It involves engineering scaffolds and growth factors to direct cell differentiation.
3. Mathematical modeling is important for understanding the complex biological and physical factors influencing 3D cell cultures, but optimization of cultures remains an ongoing area of research due to the large number of tunable parameters.
Genes and Tissue Culture Technology Assignment (G6)Rohini Krishnan
The culture of cells in two dimensions does not reproduce the histological characteristics of a tissue for informative or useful study. Growing cells as three-dimensional (3D) models more analogous to their existence in vivo may be more clinically relevant.
3D tumor spheroid models for in vitro therapeutic screening: a systematic app...Arun kumar
This document describes a study that aimed to identify and validate a cytotoxicity test for large tumor spheroids. Several methods were used to culture 3D tumor spheroids from lung cancer cells. Spheroid size, shape, and heterogeneity were characterized using imaging software. Cell viability was assessed using different assays under drug and radiation treatment. The results showed that spheroid shape and volume influence cytotoxicity assay results. Careful selection of homogeneous, spherical spheroids is important for reliable data in drug screening tests using 3D tumor models.
Development of cancer therapeutics is often carried out in 2D cultures prior to testing on animal models. 3D in vitro models better mimic the in vivo tumor microenvironment and cell-cell interactions compared to 2D cultures. A recent study tested the efficacy of cancer drugs on ovarian cancer cells cultured in a 3D model. The study found that two experimental drugs had stronger dose-dependent effects on cell viability compared to a market competitor drug when tested on cells in 3D culture.
3D cell culture allows for more natural cell-to-cell attachments and communication through gap junctions compared to 2D culture. This results in greater tissue differentiation and resistance to chemotherapeutic drugs and radiation in 3D culture, whereas cells in 2D culture are more easily killed. A lab-on-a-chip integrates multiple laboratory functions onto a single microfluidic chip and offers advantages like lower reagent costs, portability, faster reactions, and lower fabrication costs compared to traditional labs.
This document discusses cell and tissue engineering, specifically focusing on how substrate stiffness influences cell fate and differentiation. It summarizes key studies that demonstrated mesenchymal stem cells differentiate into different cell types (osteogenic, myogenic, adipogenic) depending on the stiffness of the substrate they are cultured on. Similarly, embryonic stem cells were shown to express different genes associated with various differentiation pathways depending on whether they were cultured on stiff or soft surfaces. The document concludes that substrate stiffness is an important regulator of stem cell behavior that can guide their differentiation.
3D In Vitro Model for Drug Efficiency Testingjudoublen
This document discusses the potential advantages of using 3D in vitro models compared to traditional 2D models for drug testing. It notes that 3D cultures more closely mimic the in vivo microenvironment and cell morphology. This allows 3D cultures to better predict cellular responses to drugs and provide more accurate models of disease. The document outlines several applications of 3D cultures, such as studying tumor development, evaluating drug sensitivity, and developing organs-on-chips microfluidic devices that model human organ functions.
Building on the sell-out success of the launch event, SMi Group is delighted to announce the return of 3D Cell Culture, taking place on 21st and 22nd of February 2018, in London UK.
3D Cell Culture is rapidly growing with incredible potential for industrial application and a widespread reach that can be seen across many different fields, such as 3D bioprinting and microfluidics.
The 2nd annual conference will explore these overlapping areas and will combine pioneering breakthroughs with scientific research to strengthen your commercial success. Join us for exclusive insight into key topics such as disease models, organoids, organ-on-a-chip technologies, Ipsc advances and CRISPR technology. Notable speakers on the agenda for 2018 will include experts from Aurelia Bioscience, ReInnervate Ltd, Cell and Gene Therapy Catapult, University College London, Novartis Institutes for Biomedical Research, Kugelmeiers, GSK, AstraZeneca, Roche and more!
3 d biomatrix-white-paper-3d-cell-culture-101ratna azizah
This document provides an introduction to various 3D cell culture tools and techniques. It begins by explaining how 3D cell culture has evolved from being expensive and difficult to a wider array of options that better model the in vivo environment. Five main 3D culture methods are then described in detail: scaffold-free spheroid culture, scaffolds, gels, bioreactors, and microchips. Each method is explored in terms of materials, advantages, limitations, and example applications. Review articles are also cited for additional information on each technique.
Advancement in Cell culture Techniques 2000 onward Sarwar A.D
This document summarizes advances in animal cell culture techniques after 2000. It discusses several studies from 2000-2010 that established new techniques such as culturing hepatitis C virus in cell culture, developing temperature-responsive cell culture membranes, and deriving human embryonic stem cells from blastocysts. It also summarizes the development of 3D cell culture, microfluidic cell culture platforms, and generation of induced pluripotent stem cells and their use in disease modeling and transplantation therapies.
3D culture in phenotypic screening : advantages, process changes and new tech...HCS Pharma
It was a real pleasure to be in the « High-Content and Phenotypic Screening » meeting in Cambridge. We were invited by our partner Molecular Devices to give a talk during the "User meeting Molecular Devices" about our vision of 3D culture in phenotypic screening and the impact of new technologies. We also presented a poster about "Neurotoxicity assay on 2D and 3D culture using High Content Screening technology".
The potential of using 3D in vitro models for drug efficiency testing compare...Josiah Sim
Three key points:
1) 3D cell cultures provide a more physiologically relevant model than 2D cultures by mimicking the in vivo microenvironment and cell-cell interactions. However, 3D cultures are more complex and expensive.
2) Studies show 3D cultures better maintain tumor dormancy states and drug resistance patterns observed in patients. Ki-67 indexes indicate higher fractions of non-proliferating cells in 3D.
3) While 3D models are improving, they do not fully replicate the in vivo tumor microenvironment and are not yet standardized for high-throughput drug screening. Further development is still needed to address challenges like customizing the microenvironment and expanding models.
This document discusses biomaterials inspired by the extracellular matrix (ECM). It defines biomimicry, bioinspiration and bioderivation as three paradigms for learning from biological systems. The document then describes three main types of ECM mimicry in biomaterials: 1) mimicking ECM functions by using ECM components, either through direct copying or partial copying, 2) mimicking ECM architecture and topography through hierarchical microstructure and topographical features, and 3) mimicking ECM protein design and assembly. The goal is to create biomaterials that interact with cells in similar ways to the native ECM environment.
This document discusses methods for clinical testing, specifically 3D cell culture and organ-on-chip technologies. It notes that animal testing is time-consuming, costly, and often does not predict human outcomes. Organ-on-chip technologies use microfabrication and microfluidics to create microenvironments that better simulate human physiology and organs. This allows for testing of drugs and toxins using human cells in a way that may replace animal models. Examples discussed include a lung-on-a-chip to study pulmonary edema and a proposed "body on a chip" with 3D printed miniature organs to improve drug development and reduce costs.
3D In Vitro Models for Drug Efficiency TestingTiffany Ho
3D cell cultures more accurately model the in vivo microenvironment compared to traditional 2D cultures. 3D cultures form cell aggregates or spheroids, mimic tumor development, and allow for more effective drug testing compared to flat monolayers. Emerging technologies like organ-on-chip microfluidic devices and 3D printing have the potential to further advance 3D cell culture models by replicating the functions of human organs and embedding living cells in scaffolds.
3D BIOPRINTING USING ORGAN DERIVED SCAFFOLDS AND HUMAN PLURIPOTENT STEM CELLNivaasvignopathy
This document discusses 3D bioprinting of human organs using organ-derived extracellular matrix scaffolds and human pluripotent stem cells. The authors hypothesize that decellularized liver tissue can act as a suitable scaffold for stem cells to reorganize and potentially give rise to a fully functional liver organ when combined with 3D bioprinting techniques. The objectives are to decellularize liver tissue, recellularize it with stem cells, and 3D bioprint a liver graft. The methodology involves decellularizing a liver organ using perfusion, recellularizing the decellularized extracellular matrix with stem cells, and 3D bioprinting the stem cell-seeded scaffold
Tissue engineering uses scaffolds, cells, and signaling molecules to regenerate tissues and organs. Scaffolds provide a structure for cell attachment, growth, and tissue formation. Natural polymers like collagen and hyaluronic acid, and synthetic polymers like poly-lactic-co-glycolic acid are commonly used as scaffold materials. Scaffolds can be fabricated using various methods including freeze drying, electrospinning, 3D printing, and textile technologies to produce scaffolds with desirable properties like porosity and pore size for tissue growth. Scaffolds seeded with stem cells or tissue-specific cells aim to repair and regenerate tissues for applications in skin, bone, cartilage, and other organs.
1. Drug release scaffolds can be categorized as cell delivery scaffolds or drug delivery scaffolds, with common parameters including 3D architecture, porosity, composition, interfaces, degradability, and mimicking the ECM.
2. Specific parameters for drug release scaffolds include loading capacity, drug distribution, binding affinity, and stability.
3. Injectable hydrogel scaffolds show advantages over implantation scaffolds for drug release, including enabling sustained release upon swelling to control release behavior with minimal invasiveness.
Development of cancer therapeutics is often carried out in 2D cultures prior to testing on animal model. In comparison to 2D cultures, discuss the potential of using 3D in vitro models for drug efficiency testing.
Prof Ian Marison, Director, National Institute for Bio-processing Research & ...Investnet
This document discusses encapsulation techniques for non-parenteral drug and cell delivery. It presents Prof. Ian Marison's research at Dublin City University on using encapsulation for high cell density cultures and microcapsule characterization. Specific examples discussed include encapsulating the antibiotic geldanamycin and NSAIDs to allow their selective removal from environments and downstream purification. The research aims to develop novel encapsulation methods for bioprocessing applications such as increasing product yields from degradation environments.
Anticancer drug discovery using multicellular tumor spheroid modelsHasnat Tariq
Cancer, drug discovery, tumor spheroids, organoids, 3D tumor spheroids, 3D scaffold-based models, Scaffold-free models, 3D Scaffolds, Hanging drop, Low adhesion microplate, Magnetic levitation and bio printing, bioprinting, anticancer,, tumor models, Drug screening assays, flow cytometry, expansion microscopy.
This study encapsulated equine endothelial progenitor cells and mesenchymal stem cells alone and in co-culture within a PEG-fibrinogen hydrogel scaffold to assess neovascularization. Equine EPCs formed tubules within 1 day when encapsulated alone or with MSCs, and vascularization increased over 4 days. Tubules did not form with MSCs alone. The hydrogel scaffold supported long-term cell viability and vascular structure formation, demonstrating its potential for tissue engineering and wound healing applications in horses. Future work will quantify vessel formation to determine scaffold thickness and MSC effects.
The document discusses various ophthalmic drug delivery systems including eye drops, ointments, gels, and inserts. It describes common types of eye drops like solutions and suspensions. Factors that influence drug absorption in the eye are also covered such as drainage, permeability, and molecular weight. The anatomy of the eye is summarized including structures like the cornea, iris, lens, and retina. Different dosage forms for intraocular administration like irrigating solutions and intravitreal implants are also mentioned.
Development of cancer therapeutics is often carried out in 2D cultures prior to testing on animal models. 3D in vitro models better mimic the in vivo tumor microenvironment and cell-cell interactions compared to 2D cultures. A recent study tested the efficacy of cancer drugs on ovarian cancer cells cultured in a 3D model. The study found that two experimental drugs had stronger dose-dependent effects on cell viability compared to a market competitor drug when tested on cells in 3D culture.
3D cell culture allows for more natural cell-to-cell attachments and communication through gap junctions compared to 2D culture. This results in greater tissue differentiation and resistance to chemotherapeutic drugs and radiation in 3D culture, whereas cells in 2D culture are more easily killed. A lab-on-a-chip integrates multiple laboratory functions onto a single microfluidic chip and offers advantages like lower reagent costs, portability, faster reactions, and lower fabrication costs compared to traditional labs.
This document discusses cell and tissue engineering, specifically focusing on how substrate stiffness influences cell fate and differentiation. It summarizes key studies that demonstrated mesenchymal stem cells differentiate into different cell types (osteogenic, myogenic, adipogenic) depending on the stiffness of the substrate they are cultured on. Similarly, embryonic stem cells were shown to express different genes associated with various differentiation pathways depending on whether they were cultured on stiff or soft surfaces. The document concludes that substrate stiffness is an important regulator of stem cell behavior that can guide their differentiation.
3D In Vitro Model for Drug Efficiency Testingjudoublen
This document discusses the potential advantages of using 3D in vitro models compared to traditional 2D models for drug testing. It notes that 3D cultures more closely mimic the in vivo microenvironment and cell morphology. This allows 3D cultures to better predict cellular responses to drugs and provide more accurate models of disease. The document outlines several applications of 3D cultures, such as studying tumor development, evaluating drug sensitivity, and developing organs-on-chips microfluidic devices that model human organ functions.
Building on the sell-out success of the launch event, SMi Group is delighted to announce the return of 3D Cell Culture, taking place on 21st and 22nd of February 2018, in London UK.
3D Cell Culture is rapidly growing with incredible potential for industrial application and a widespread reach that can be seen across many different fields, such as 3D bioprinting and microfluidics.
The 2nd annual conference will explore these overlapping areas and will combine pioneering breakthroughs with scientific research to strengthen your commercial success. Join us for exclusive insight into key topics such as disease models, organoids, organ-on-a-chip technologies, Ipsc advances and CRISPR technology. Notable speakers on the agenda for 2018 will include experts from Aurelia Bioscience, ReInnervate Ltd, Cell and Gene Therapy Catapult, University College London, Novartis Institutes for Biomedical Research, Kugelmeiers, GSK, AstraZeneca, Roche and more!
3 d biomatrix-white-paper-3d-cell-culture-101ratna azizah
This document provides an introduction to various 3D cell culture tools and techniques. It begins by explaining how 3D cell culture has evolved from being expensive and difficult to a wider array of options that better model the in vivo environment. Five main 3D culture methods are then described in detail: scaffold-free spheroid culture, scaffolds, gels, bioreactors, and microchips. Each method is explored in terms of materials, advantages, limitations, and example applications. Review articles are also cited for additional information on each technique.
Advancement in Cell culture Techniques 2000 onward Sarwar A.D
This document summarizes advances in animal cell culture techniques after 2000. It discusses several studies from 2000-2010 that established new techniques such as culturing hepatitis C virus in cell culture, developing temperature-responsive cell culture membranes, and deriving human embryonic stem cells from blastocysts. It also summarizes the development of 3D cell culture, microfluidic cell culture platforms, and generation of induced pluripotent stem cells and their use in disease modeling and transplantation therapies.
3D culture in phenotypic screening : advantages, process changes and new tech...HCS Pharma
It was a real pleasure to be in the « High-Content and Phenotypic Screening » meeting in Cambridge. We were invited by our partner Molecular Devices to give a talk during the "User meeting Molecular Devices" about our vision of 3D culture in phenotypic screening and the impact of new technologies. We also presented a poster about "Neurotoxicity assay on 2D and 3D culture using High Content Screening technology".
The potential of using 3D in vitro models for drug efficiency testing compare...Josiah Sim
Three key points:
1) 3D cell cultures provide a more physiologically relevant model than 2D cultures by mimicking the in vivo microenvironment and cell-cell interactions. However, 3D cultures are more complex and expensive.
2) Studies show 3D cultures better maintain tumor dormancy states and drug resistance patterns observed in patients. Ki-67 indexes indicate higher fractions of non-proliferating cells in 3D.
3) While 3D models are improving, they do not fully replicate the in vivo tumor microenvironment and are not yet standardized for high-throughput drug screening. Further development is still needed to address challenges like customizing the microenvironment and expanding models.
This document discusses biomaterials inspired by the extracellular matrix (ECM). It defines biomimicry, bioinspiration and bioderivation as three paradigms for learning from biological systems. The document then describes three main types of ECM mimicry in biomaterials: 1) mimicking ECM functions by using ECM components, either through direct copying or partial copying, 2) mimicking ECM architecture and topography through hierarchical microstructure and topographical features, and 3) mimicking ECM protein design and assembly. The goal is to create biomaterials that interact with cells in similar ways to the native ECM environment.
This document discusses methods for clinical testing, specifically 3D cell culture and organ-on-chip technologies. It notes that animal testing is time-consuming, costly, and often does not predict human outcomes. Organ-on-chip technologies use microfabrication and microfluidics to create microenvironments that better simulate human physiology and organs. This allows for testing of drugs and toxins using human cells in a way that may replace animal models. Examples discussed include a lung-on-a-chip to study pulmonary edema and a proposed "body on a chip" with 3D printed miniature organs to improve drug development and reduce costs.
3D In Vitro Models for Drug Efficiency TestingTiffany Ho
3D cell cultures more accurately model the in vivo microenvironment compared to traditional 2D cultures. 3D cultures form cell aggregates or spheroids, mimic tumor development, and allow for more effective drug testing compared to flat monolayers. Emerging technologies like organ-on-chip microfluidic devices and 3D printing have the potential to further advance 3D cell culture models by replicating the functions of human organs and embedding living cells in scaffolds.
3D BIOPRINTING USING ORGAN DERIVED SCAFFOLDS AND HUMAN PLURIPOTENT STEM CELLNivaasvignopathy
This document discusses 3D bioprinting of human organs using organ-derived extracellular matrix scaffolds and human pluripotent stem cells. The authors hypothesize that decellularized liver tissue can act as a suitable scaffold for stem cells to reorganize and potentially give rise to a fully functional liver organ when combined with 3D bioprinting techniques. The objectives are to decellularize liver tissue, recellularize it with stem cells, and 3D bioprint a liver graft. The methodology involves decellularizing a liver organ using perfusion, recellularizing the decellularized extracellular matrix with stem cells, and 3D bioprinting the stem cell-seeded scaffold
Tissue engineering uses scaffolds, cells, and signaling molecules to regenerate tissues and organs. Scaffolds provide a structure for cell attachment, growth, and tissue formation. Natural polymers like collagen and hyaluronic acid, and synthetic polymers like poly-lactic-co-glycolic acid are commonly used as scaffold materials. Scaffolds can be fabricated using various methods including freeze drying, electrospinning, 3D printing, and textile technologies to produce scaffolds with desirable properties like porosity and pore size for tissue growth. Scaffolds seeded with stem cells or tissue-specific cells aim to repair and regenerate tissues for applications in skin, bone, cartilage, and other organs.
1. Drug release scaffolds can be categorized as cell delivery scaffolds or drug delivery scaffolds, with common parameters including 3D architecture, porosity, composition, interfaces, degradability, and mimicking the ECM.
2. Specific parameters for drug release scaffolds include loading capacity, drug distribution, binding affinity, and stability.
3. Injectable hydrogel scaffolds show advantages over implantation scaffolds for drug release, including enabling sustained release upon swelling to control release behavior with minimal invasiveness.
Development of cancer therapeutics is often carried out in 2D cultures prior to testing on animal model. In comparison to 2D cultures, discuss the potential of using 3D in vitro models for drug efficiency testing.
Prof Ian Marison, Director, National Institute for Bio-processing Research & ...Investnet
This document discusses encapsulation techniques for non-parenteral drug and cell delivery. It presents Prof. Ian Marison's research at Dublin City University on using encapsulation for high cell density cultures and microcapsule characterization. Specific examples discussed include encapsulating the antibiotic geldanamycin and NSAIDs to allow their selective removal from environments and downstream purification. The research aims to develop novel encapsulation methods for bioprocessing applications such as increasing product yields from degradation environments.
Anticancer drug discovery using multicellular tumor spheroid modelsHasnat Tariq
Cancer, drug discovery, tumor spheroids, organoids, 3D tumor spheroids, 3D scaffold-based models, Scaffold-free models, 3D Scaffolds, Hanging drop, Low adhesion microplate, Magnetic levitation and bio printing, bioprinting, anticancer,, tumor models, Drug screening assays, flow cytometry, expansion microscopy.
This study encapsulated equine endothelial progenitor cells and mesenchymal stem cells alone and in co-culture within a PEG-fibrinogen hydrogel scaffold to assess neovascularization. Equine EPCs formed tubules within 1 day when encapsulated alone or with MSCs, and vascularization increased over 4 days. Tubules did not form with MSCs alone. The hydrogel scaffold supported long-term cell viability and vascular structure formation, demonstrating its potential for tissue engineering and wound healing applications in horses. Future work will quantify vessel formation to determine scaffold thickness and MSC effects.
The document discusses various ophthalmic drug delivery systems including eye drops, ointments, gels, and inserts. It describes common types of eye drops like solutions and suspensions. Factors that influence drug absorption in the eye are also covered such as drainage, permeability, and molecular weight. The anatomy of the eye is summarized including structures like the cornea, iris, lens, and retina. Different dosage forms for intraocular administration like irrigating solutions and intravitreal implants are also mentioned.
Cell culture is the process of growing cells under controlled conditions outside of their natural environment. Key developments in cell culture technology include the use of antibiotics to prevent contamination, trypsin to detach adherent cells for subculturing, and chemically defined culture media. Cell culture is used in a variety of areas including basic research, toxicity testing, cancer research, virology, genetic engineering, and gene therapy. Successful cryopreservation of cell lines involves slow freezing and quick thawing to minimize ice crystal formation and damage to cells.
The document discusses mammalian cell culture and its applications. It provides details on the conditions needed for cell culture, including aseptic conditions, growth medium, temperature, pH, and gas exchange. It also discusses primary and continuous mammalian cell cultures. Primary cultures have a limited lifespan while continuous cell lines can divide indefinitely and are used for research and biotechnology applications.
The document discusses ocular drug delivery systems. It begins with an agenda that outlines the objectives and topics to be covered, including the anatomy and physiology of the eye, factors affecting intraocular bioavailability, and various approaches and classifications of ocular drug delivery systems. The document then provides details on the anatomy of the eye, mechanisms of ocular absorption, factors that influence drug availability in the eye, and different approaches to improve ocular bioavailability such as using viscosity enhancers, penetration enhancers, prodrugs, and mucoadhesives. It also describes various types of ocular delivery systems including solutions, suspensions, gels, ointments, inserts, and intraocular implants and injections.
The document discusses ocular drug delivery systems. It begins with an introduction to eye anatomy and factors affecting drug absorption in the eye. It then describes various ophthalmic formulations like solutions, suspensions, and ointments. It discusses advances in controlled release ocular systems including inserts, contact lenses, and nanoparticles to prolong drug release. Finally, it outlines new approaches in ocular drug delivery research focusing on combining technologies for targeted and sustained drug delivery to the eye.
This document discusses ocular drug delivery systems. It begins by introducing the need for ocular drug delivery and routes of administration to the eye. It then describes the anatomy and barriers of the eye. The document outlines various traditional and advanced ocular drug delivery systems including solutions, suspensions, ointments, inserts, and vesicular systems like liposomes and niosomes. It discusses factors influencing drug absorption in the eye and characteristics of ideal ocular drug delivery formulations. The trends in ocular drug delivery include controlled release systems like implants and iontophoresis.
This document discusses 3D cell culture systems and their application in drug discovery. It notes that 3D cell cultures better mimic the in vivo cellular environment compared to traditional 2D cultures. Cells in 3D cultures exhibit different gene expression, morphology, proliferation rates, and responses to drugs compared to 2D cultures. This makes 3D cultures more predictive of in vivo responses during drug testing. The document outlines different types of 3D culture systems, such as scaffold-based, scaffold-free, spheroids and organoids. It also discusses advantages of 3D cultures for applications in areas like developmental biology, disease modeling, regenerative medicine, and personalized drug testing.
Effect of Surface Engineering on Stem Cells.pdfaman15nanavaty
A thorough review covering the nascent domain of Surface Engineering on Stem Cells. This short review will cover basic details of Stem Cell Engineering and Cell Culture Surface Engineering.
Characterization of the adhesive interactions between cells and biomaterialsDr. Sitansu Sekhar Nanda
This document discusses characterization of adhesive interactions between cells and biomaterials. It begins by introducing how biomaterials are used to facilitate cell adhesion during tissue repair or replacement. It then discusses various adhesion receptors in native tissue like integrins, their classification and role in connecting the extracellular and intracellular environments. The document also covers optimization of cellular adhesion through biomaterial modification and different methods to measure cell adhesion like micromanipulation, centrifugation and applying hydrodynamic shear stress. It concludes that modifying biomaterials to mimic native adhesive interactions can help control downstream cell responses and have therapeutic applications.
Tissue engineering involves using scaffolds, cells, and biomolecules to create functional 3D tissues. It aims to develop biological substitutes to restore tissue function and repair damaged tissues, avoiding problems with organ transplants, mechanical devices, and surgery. A major goal is designing scaffolds that recreate the in vivo microenvironment through biophysical and biochemical signaling. Stem cells are a promising cell source for their ability to integrate into tissues and secrete growth factors. Signaling molecules can also be used to modulate cell behavior. Magnetic targeting of stem cells may help with the challenge of cell retention in tissue engineering applications like cardiac repair.
1. Three dimensional cell cultures are more clinically relevant for anti-cancer drug screening compared to traditional two dimensional cell cultures as they better mimic the tumor microenvironment.
2. In 3D cultures, cells can interact and organize in all three dimensions and form natural cell-cell attachments, barriers to drug diffusion, and gradients of soluble molecules similar to real tumors.
3. Studies show 3D cultures alter gene expression and protein production in cells to make them more resistant to drugs, providing more accurate predictions of drug responses than 2D cultures.
1. Three dimensional cell cultures are more clinically relevant for anti-cancer drug screening compared to traditional two dimensional cell cultures as they better mimic the in vivo tumor microenvironment.
2. In 3D cultures, cells can interact and organize in all three dimensions to form natural cell-cell attachments and cell-extracellular matrix interactions similar to real tumors.
3. 3D cultures have been shown to more accurately reproduce drug resistance mechanisms found in tumors and provide more predictive results for drug efficacy compared to 2D monolayer cultures.
1. Three dimensional cell cultures are more clinically relevant for anti-cancer drug screening compared to traditional two dimensional cell cultures as they better mimic the in vivo tumor microenvironment.
2. In 3D cultures, cells can interact and organize in all three dimensions to form natural cell-cell attachments and cell-extracellular matrix interactions similar to real tumors.
3. 3D cultures have been shown to more accurately reproduce drug resistance mechanisms found in tumors and provide more predictive results for drug efficacy compared to 2D monolayer cultures.
The document discusses different types of stem cells and how the elasticity of the extracellular matrix can influence stem cell differentiation. It finds that mesenchymal stem cells cultured on matrices of varying elasticity corresponding to brain, muscle, and bone tissues preferentially differentiate into neuron-like, myoblast-like, and osteoblast-like cells respectively. The stem cells appear to sense and respond to matrix stiffness through focal adhesions and actin-myosin contractility. Controlling the microenvironment may help direct stem cell differentiation for therapeutic applications.
From 3D cell culture to organs-on-chips.pdfgangligon
This document discusses the development of 3D cell culture and organ-on-chip models. It describes how microfabrication techniques have been applied to cell culture to better mimic the in vivo cellular microenvironment. Early work involved micropatterning cells to control their shape. More recent work involves organ-on-chips that integrate microfluidics to recreate tissue-tissue interfaces, chemical gradients, and mechanical cues found in living organs. These models allow more physiologically relevant study of human cells and disease modeling than traditional culture methods.
For decades, cell lines have played a critical role in scientific developments. In most cases, researchers just got data generated from cell lines. However, due to some weaknesses of cell lines, scientists become increasingly cautious about these generated results. But now the game has changed! Primary cells now are believed to be a more biologically relevant tool than cell lines for studying human and animal biology. And we design this primary cell culture guide aimed at showing new investigators the basic principles of primary cell and some practical culture skills.
Stem cells can be used as a raw material in tissue engineering to potentially replace curative medicine for healing illnesses. There are two main types of stem cells - embryonic stem cells which can be collected from embryos, and adult stem cells which can be obtained from limited tissues in the body. Stem cells have the ability to continuously divide and differentiate into various cell types and tissues. Researchers are working to apply stem cell therapy more broadly, but it raises some legal and ethical issues regarding embryo harvesting that need to be addressed. Tissue engineering using stem cells could revolutionize healthcare by restoring or enhancing tissue and organ function through growing tissues in vivo or in vitro for therapeutic or diagnostic applications.
Stem Cell, a raw material to be used in tissue engineering unit to have the solution against any of the ailments. Stem cell therapy may be used in treating any multi cellular organism (MCO).
Stem cell therapy may be the solution against most of ailments of multi cellular organism (MCO). It can be worked as a raw material for tissue engineering unit
This document discusses 3D cell culture techniques. It defines 3D cell culture as permitting biological cells to grow and interact in all three dimensions, mimicking their natural environment. This is an improvement over 2D cultures. The document outlines various 3D culture methods including scaffold-based techniques using polymeric scaffolds, biological scaffolds, or micropatterned surfaces. Non-scaffold methods include hanging drop plates, spheroid plates, and microfluidics. Bioreactors and gels are also discussed. The document notes applications in regenerative medicine, drug testing, and modeling tumors or tissues. Considerations in choosing a 3D culture method include the specific application and ability to recreate in vivo barriers or cancers.
This document discusses 3D cell culture techniques. It defines 3D cell culture as permitting biological cells to grow and interact in all three dimensions, mimicking their natural environment. This is an improvement over 2D cultures where cells grow unnaturally. The document describes various 3D culture methods including scaffold-based techniques using polymeric scaffolds or biological scaffolds, and non-scaffold methods like hanging drop plates, spheroid plates, microfluidics, and gels. It also discusses bioreactors and lists applications of 3D cultures.
Cytes Biotechnologies S.L. offers in its portfolio an assortment of various Human Endothelial Cells for research. These hepatic non-parenchymal liver cells are offered both fresh and cryopreserved to better meet the customer’s specific needs. All products are fully characterized by phenotypic markers, induction, metabolism, 3D, and transporters.
Cultured animal cells have many important applications. They can be used as (1) model systems to study basic cell biology and interactions between cells and pathogens, (2) for toxicity testing of new drugs and chemicals, and (3) in cancer research to study normal and cancerous cell differences. Animal cell culture is also used for virology research, manufacturing of vaccines and proteins, genetic counseling, genetic engineering of cells, and gene and drug screening and development. Proper growth media, aseptic techniques, cryopreservation, and applications in various fields make animal cell culture a valuable tool.
This document summarizes a study that examined the effects of culture environment on the mechanical and biochemical properties of cartilage tissue engineered using chondrocytes cultured in agarose gel constructs. The study found that total GAG synthesis was higher in constructs with 4% agarose gel concentration after 7 days of culture. Mechanical testing showed the peak load stress was lower in constructs as the agarose concentration increased. Analysis of the constructs indicated that the total GAG synthesis and mechanical strength of the engineered tissue can be influenced by the agarose concentration and culture time.
Similar to SeedEZ 3D cell culture application notes - gel and drug embedding (20)
Dr. Sean Tan, Head of Data Science, Changi Airport Group
Discover how Changi Airport Group (CAG) leverages graph technologies and generative AI to revolutionize their search capabilities. This session delves into the unique search needs of CAG’s diverse passengers and customers, showcasing how graph data structures enhance the accuracy and relevance of AI-generated search results, mitigating the risk of “hallucinations” and improving the overall customer journey.
Essentials of Automations: The Art of Triggers and Actions in FMESafe Software
In this second installment of our Essentials of Automations webinar series, we’ll explore the landscape of triggers and actions, guiding you through the nuances of authoring and adapting workspaces for seamless automations. Gain an understanding of the full spectrum of triggers and actions available in FME, empowering you to enhance your workspaces for efficient automation.
We’ll kick things off by showcasing the most commonly used event-based triggers, introducing you to various automation workflows like manual triggers, schedules, directory watchers, and more. Plus, see how these elements play out in real scenarios.
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UiPath Test Automation using UiPath Test Suite series, part 6DianaGray10
Welcome to UiPath Test Automation using UiPath Test Suite series part 6. In this session, we will cover Test Automation with generative AI and Open AI.
UiPath Test Automation with generative AI and Open AI webinar offers an in-depth exploration of leveraging cutting-edge technologies for test automation within the UiPath platform. Attendees will delve into the integration of generative AI, a test automation solution, with Open AI advanced natural language processing capabilities.
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1. Insights into integrating generative AI.
2. Understanding how this integration enhances test automation within the UiPath platform
3. Practical demonstrations
4. Exploration of real-world use cases illustrating the benefits of AI-driven test automation for UiPath
Topics covered:
What is generative AI
Test Automation with generative AI and Open AI.
UiPath integration with generative AI
Speaker:
Deepak Rai, Automation Practice Lead, Boundaryless Group and UiPath MVP
Alt. GDG Cloud Southlake #33: Boule & Rebala: Effective AppSec in SDLC using ...James Anderson
Effective Application Security in Software Delivery lifecycle using Deployment Firewall and DBOM
The modern software delivery process (or the CI/CD process) includes many tools, distributed teams, open-source code, and cloud platforms. Constant focus on speed to release software to market, along with the traditional slow and manual security checks has caused gaps in continuous security as an important piece in the software supply chain. Today organizations feel more susceptible to external and internal cyber threats due to the vast attack surface in their applications supply chain and the lack of end-to-end governance and risk management.
The software team must secure its software delivery process to avoid vulnerability and security breaches. This needs to be achieved with existing tool chains and without extensive rework of the delivery processes. This talk will present strategies and techniques for providing visibility into the true risk of the existing vulnerabilities, preventing the introduction of security issues in the software, resolving vulnerabilities in production environments quickly, and capturing the deployment bill of materials (DBOM).
Speakers:
Bob Boule
Robert Boule is a technology enthusiast with PASSION for technology and making things work along with a knack for helping others understand how things work. He comes with around 20 years of solution engineering experience in application security, software continuous delivery, and SaaS platforms. He is known for his dynamic presentations in CI/CD and application security integrated in software delivery lifecycle.
Gopinath Rebala
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Cosa hanno in comune un mattoncino Lego e la backdoor XZ?Speck&Tech
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Partecipate alla presentazione per immergervi in una storia di interoperabilità, standard e formati aperti, per poi discutere del ruolo importante che i contributori hanno in una comunità open source sostenibile.
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Presented by Vladimir Iglovikov:
- https://www.linkedin.com/in/iglovikov/
- https://x.com/viglovikov
- https://www.instagram.com/ternaus/
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Explore more about Albumentations and join the community at:
GitHub: https://github.com/albumentations-team/albumentations
Website: https://albumentations.ai/
LinkedIn: https://www.linkedin.com/company/100504475
Twitter: https://x.com/albumentations
Removing Uninteresting Bytes in Software FuzzingAftab Hussain
Imagine a world where software fuzzing, the process of mutating bytes in test seeds to uncover hidden and erroneous program behaviors, becomes faster and more effective. A lot depends on the initial seeds, which can significantly dictate the trajectory of a fuzzing campaign, particularly in terms of how long it takes to uncover interesting behaviour in your code. We introduce DIAR, a technique designed to speedup fuzzing campaigns by pinpointing and eliminating those uninteresting bytes in the seeds. Picture this: instead of wasting valuable resources on meaningless mutations in large, bloated seeds, DIAR removes the unnecessary bytes, streamlining the entire process.
In this work, we equipped AFL, a popular fuzzer, with DIAR and examined two critical Linux libraries -- Libxml's xmllint, a tool for parsing xml documents, and Binutil's readelf, an essential debugging and security analysis command-line tool used to display detailed information about ELF (Executable and Linkable Format). Our preliminary results show that AFL+DIAR does not only discover new paths more quickly but also achieves higher coverage overall. This work thus showcases how starting with lean and optimized seeds can lead to faster, more comprehensive fuzzing campaigns -- and DIAR helps you find such seeds.
- These are slides of the talk given at IEEE International Conference on Software Testing Verification and Validation Workshop, ICSTW 2022.
SeedEZ 3D cell culture application notes - gel and drug embedding
1. Lena Biosciences
Innovative 3D Culture Tools for Life Sciences
SeedEZ
TM
APPLICATION NOTES
GEL EMBEDDING INTO THE SeedEZ TM
FOR PHYSIOLOGICALLY CLOSER EXTRACELLULAR ENVIRONMENTS IN 3D CELL CULTURES,
INVASION, CHEMO-INVASION AND ANGIOGENSIS ASSAYS, AND DRUG RELEASE STUDIES
support@lenabio.com
www.lenabio.com
APRIL 2013| V1.6
3. Gel Embedding into the SeedEZTM
April 2013
TABLE OF CONTENTS
INTRODUCTION _____________________________________________________________________ 1
Why to seed cells in a sol-state gel? __________________________________________________________ 1
The role of extracellular matrix ______________________________________________________________ 2
Why to seed cells in a sol-state gel into the SeedEZ? ____________________________________________ 3
Why to embed a gel into the SeedEZ for cell motility assays, invasion and angiogenesis? _____________ 4
Why to embed gel and other reagents into the SeedEZ for in vitro controlled drug release studies? ____ 5
GUIDELINES AND RECOMMENDATIONS FOR SEEDING CELLS IN A GEL __________________________ 6
GELS USED IN 3D CELL CULTURE APPLICATIONS AND DRUG DELIVERY __________________________ 6
Gels comprising extracellular matrix constituents _________________________________________________ 7
Collagen I _______________________________________________________________________________________ 7
Matrigel ________________________________________________________________________________________ 7
Gels from natural sources for 3D cell cultures and controlled drug release _____________________________ 8
Gels of animal origin ______________________________________________________________________________ 8
Gelatin _______________________________________________________________________________________ 8
Gels of non-animal origin __________________________________________________________________________ 9
Agarose ______________________________________________________________________________________ 9
Alginate _____________________________________________________________________________________ 10
Methyl cellulose ______________________________________________________________________________ 11
Synthetic gels ____________________________________________________________________________ 12
Pluronic _______________________________________________________________________________________ 12
Carbomer______________________________________________________________________________________ 12
4. Lena Biosciences
Innovative 3D Culture Tools for Life Sciences
SeedEZTM Application Notes
www.lenabio.com
INTRODUCTION
When compared to a 2D cell culture comprising one layer of flat cells on a non-physiologically stiff and flat Petri dish, threedimensional (3D) cell culture provides tissue-like round cell morphology and tissue-soft cell environment with 3D cell-cell
and 3D cell-extracellular matrix interactions, signaling, extracellular availability of soluble factors, oxygen etc.
SeedEZ is a substrate which makes transition to physiologically closer 3D tissue models easy. It makes the handling of 3D cell
cultures as easy as handling a 2D culture so that you may continue with your protocols. It also makes 3D cell cultures
consistent, even in high throughput and even if your 3D tissue models include extracellular matrices.
Extracellular matrix (ECM) is an important component of cell environment in vivo. ECM is a tissue-soft 3D support structure
which holds cells together and to which cells adhere; a support structure which provides signaling molecules and diffusible
guidance cues which influence cell fate. Cells respond to mechanical and biochemical changes in the ECM through the
cross-talk between integrins and the actin cytoskeleton. In vitro, certain cell types are able to grow only if they are attached
to surfaces comprising extracellular matrix (ECM) or its constituents.
SeedEZ allows incorporation of extracellular matrix into 3D culture models. It allows to seed cells in an ECM suspension,
while providing lot-to-lot consistent cultures of defined dimensions in x, y, and z, and with reproducible cell and ECM gel
distribution in 3D. With simple seeding methods and with its ability to wick viscous cell culture reagents, SeedEZ facilitates
gel and cell handling to an extent that any user can generate consistent 3D gel cell cultures in minutes and in highthroughput. By allowing users to seed cells and extracellular matrix they choose, SeedEZ provides for the control of
extracellular environment in which cells can be driven to better approximate function of their in vivo counterparts; thus,
creating more robust, high-throughput and high-content assays.
Why to seed cells in a sol-state gel?
A. Most cells require cell-extracellular matrix interactions to maintain viability and function.
B. Not all cells secrete extracellular matrix (ECM) constituents they need and they are surrounded by in vivo.
C.
Many cells require specific ECM constituents to maintain their characteristic polarized organization, differentiated state
and specific gene expression.
D. Many extracellular matrix proteins have binding sites for both cell adhesion and growth factors.
E.
When cells are surrounded by an ECM and other cells in 3D, their morphology is closer to in vivo, and this influences
virtually all cell functions.
F.
An ECM gel mimicking biochemical and biomechanical cues present to cells in vivo is likely a better model of the
extracellular environment than is the extracellular environment in which half the cell surface is exposed to medium and
half-surface stuck to unphysiologically stiff plastic dish.
G. Soluble, diffusible guiding cues such as morphogen concentration gradients are difficult to establish, maintain and
control in a 2D cell culture pool of medium.
H. ECM gel provides for mass transport of soluble factors and gas intra-3D-culture which is physiologically closer than is
mass transport in a 2D culture in which half cell surface is bathed in medium and exposed to:
SeedEZTM Gel Embedding | 1
5. Lena Biosciences
Innovative 3D Culture Tools for Life Sciences
SeedEZTM Application Notes
www.lenabio.com
a.
Abnormally high concentration of soluble factors.
b.
Abnormally high oxygen tension.
c.
Abnormal variations in extracellular conditions each time medium is changed (cells in a gel are “shielded” better).
I.
The ECM gel stiffness and dimensionality have a profound effect on cell fate and function.
J.
Extracellular matrix gel is the substrate for cell migration and invasion.
K.
Cell movement and tissue remodeling are important for normal physiological processes and in pathology of diseases.
For these processes to happen, the ECM must be present and frequently degraded for cells to migrate or to deposit a
new matrix.
ECM PROVIDES FOR A MYRIAD OF IMPORTANT SIGNALS AND FUNCTIONS FOR RESIDENT CELLS, FUNCTIONS WHICH
FLAT-CELL-ON-A-PLASTIC SURFACE MAY NOT BE ABLE TO SUBSTITUTE BUT ROUND-CELL IN AN ECM IN A 3D CELL
CULTURE MIGHT BE ABLE TO. Recent studies have revealed the role of ECM in many pathological conditions, and ECM and
its constituents have emerged as an important drug target.
“..It seems possible for cancer to be induced to become quiescent or revert to a normal state, if provided with the correct set
of ECM signals, similarly to the normal embryogenesis.”
Kim SH, Turnbull J, Guimond S.
Extracellular matrix and cell signalling: the dynamic cooperation of integrin, proteoglycan and growth factor receptor.
J Endocrinol. 2011, 209(2):139-51. Review.
Kenny PA, Bissell MJ.
Tumor reversion: correction of malignant behavior by microenvironmental cues.
Int J Cancer. 2003, 107(5):688-95.
The role of extracellular matrix
In vivo, extracellular matrix (ECM) is the scaffolding which holds cells together and includes collagen, elastin, proteoglycans,
and glycoproteins such as fibronectin and laminin in varying composition depending on the tissue type. In vitro studies have
shown that cell-ECM adhesion is not merely to immobilize cells. Cell-ECM signaling and interactions bring about functional
changes including cell survival, growth, proliferation, metabolism, extracellular availability of soluble and diffusible factors,
migration, protein synthesis, and gene expression, all of which can be manipulated in vitro to mirror the in vivo conditions.
A large body of evidence suggests that cell adhesive molecules and biochemical cues in the extracellular environment
influence cell function. Cell adhesion is mediated by specific receptors on the cell surface (integrins) which interact with the
extracellular matrix constituents. Soluble and diffusible guidance cues in the ECM regulate cell fate. Biomechanical cues,
mechanosensing and mechanotransduction are increasingly cited as important factors of the extracellular environment
with profound effect on cytoskeletal organization and cell function. Cells sense stiffness, topography and dimensionality of
the extracellular environment through a process called mechanosensing which influences cell signaling, function and fate.
Cells respond to biomechanical properties of the environment through a process called mechanotransduction by altering
their organization, traction, protein expression, motility, etc. Clearly, the role of ECM on cell fate is profound.
SeedEZTM Gel Embedding | 2
6. Lena Biosciences
Innovative 3D Culture Tools for Life Sciences
SeedEZTM Application Notes
www.lenabio.com
In a physiologically closer tissue model, ECM needs to be present and cell-ECM cross-talk needs to be present. SeedEZ allows
effortless incorporation of the ECM into high-throughput cell-based assays.
References:
Fischer RS, Myers KA, Gardel ML, Waterman CM.
Stiffness-controlled three-dimensional extracellular matrices for high-resolution imaging of cell behavior.
Nat Protoc. 2012, 7(11):2056-66.
http://www.ncbi.nlm.nih.gov/pubmed/23099487
Wells RG.
The role of matrix stiffness in regulating cell behavior.
Hepatology. 2008, 47(4):1394-400.
http://www.ncbi.nlm.nih.gov/pubmed/18307210
Yeung T, Georges PC, Flanagan LA, Marg B, Ortiz M, Funaki M, Zahir N, Ming W, Weaver V, Janmey PA.
Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion.
Cell Motil Cytoskeleton. 2005, 60(1):24-34.
http://www.ncbi.nlm.nih.gov/pubmed/15573414
Yamada, K. M.
Extracellular Matrix.
Current Protocols in Cell Biology. 2009, 45:10.0.1–10.0.3.
http://onlinelibrary.wiley.com/doi/10.1002/0471143030.cb1000s45/pdf
Why to seed cells in a sol-state gel into the SeedEZ?
While seeding cells in a sol-state gel is a standard technique, it requires skill. Without automated equipment, 3D gel culture
shape is often inconsistent culture-to-culture and culture dimensions in x, y, and z are difficult to control at seeding. For
those gels which gelation is initiated with rise in temperature, cultures tend to spread out in routine transfer to incubator
for gelling. In essence, they flatten out even before they are gelled. In chemically cross-linked hydrogels, cells are seeded in
a precursor. Unfortunately, cells settle during the process of cross-linker addition to cultures in different wells. All this yields
questionable 3D cell cultures, with significant variations in x, y and z dimensions, and cell and gel distribution in x, y, and z
at seeding or following gelling.
Inconsistent culture dimensions and cell distribution across these dimensions at seeding yield culture-to-culture varying
supply of nutrients, removal of catabolic waste products, and intra-culture concentrations of trophic factors, autocrine and
paracrine signaling molecules cells secrete to regulate their environment, growth and many other functions. Some signaling
molecules degrade quickly, limiting the scope of their effectiveness to the immediate cell surroundings. Others affect only
nearby cells because they are taken up quickly, or because their transport is hindered by the extracellular matrix. In sum,
inconsistent cultures at plating yield inconsistent tissue analogs for life sciences research and drug testing. Variations in cell
function and secretion of drug metabolizing enzymes, as well as variations in intra-culture availability of test compounds
influence pharmacokinetic studies, resulting in less conclusive cell outcomes and problems with interpretation of results.
SeedEZ SOLVES THESE PROBLEMS AND TRANSFORMS 3D GEL CULTURES INTO CONSISTENT 3D CELL CULTURES
WHICH ARE EASY TO SEED AND EASY TO HANDLE IN HIGH-THROUGHPUT.
SeedEZTM Gel Embedding | 3
7. Lena Biosciences
Innovative 3D Culture Tools for Life Sciences
SeedEZTM Application Notes
www.lenabio.com
IF EMBEDDED IN THE SeedEZ, 3D GEL CULTURES CANNOT BE ACCIDENTALLY ASPIRATED OR DETACHED IN ROUTINE
MEDIA EXCHANGES. THIS ALLOWS REPRODUCIBLE STUDIES TO CONTINUE DAYS AND WEEKS AFTER CELL SEEDING.
With simple seeding methods and with its ability to wick viscous reagents, SeedEZ provides remarkable experimental
flexibility to embed cells, gels, and compounds of choice in order to, for example, model stiff extracellular matrices in aging
tissues with important ramifications to cell fate and potentially higher relevance of the in vitro compound screening results.
Additional advantage of SeedEZ is that it provides 3D cell support even when a gel degrades. When gel dissolves or thins
down due to enzymatic or hydrolytic degradation, cell digestion, or other factors, cells remain supported in 3D by the
SeedEZ. If your studies cannot be completed, are too short to answer specific questions, or simply difficult to repeat due to
inconsistent and time-dependent gel decay, SeedEZ can help. Gels which degrade too quickly may not allow cells to mature
and form functional networks for compound screening. On the other hand, gels which are difficult to remodel, may prevent
cells from invading or depositing their endogenous matrix constituents. In both cases, SeedEZ is the solution. When gel
degrades too fast, SeedEZ continues to support cells. If gel concentration is too high, SeedEZ may enable use of a lower
concentration gel which is still held in 3D by the SeedEZ.
Why to embed a gel into the SeedEZ for cell motility assays, invasion and angiogenesis?
A. In a truly 3D cell migration, representative of in vivo conditions, invasive cells are embedded in the extracellular
matrix from the assay start to its end.
Standard trans-membrane cell assays study cell invasion in an environment that is quite different from that in vivo. Cells are
plated in a monolayer at the liquid-ECM interface, and the trans-membrane is permissive only to uni-directional (vertical)
cell migration through the membrane pores.
The key difference between a thin layer of ECM on a membrane and the ECM in the SeedEZ is that the migration/invasion in
the SeedEZ is truly 3D; cells are embedded in 3D and migrate in 3D which is closer to in vivo conditions.
B. Physiologically closer extracellular environment with respect to ECM composition, gel stiffness, and the presence
of diffusible or substrate-bound cues, for physiologically closer assay results.
Emerging evidence elucidates role of physical resistance present to cells when migrating and invading in 3D, a resistance
which is often negligible in 2D but present in vivo. In 3D, ECM resistance forces cells to develop proteolytic and amoeboid
(non-proteolytic) strategies to either degrade the matrix to migrate (proteolytic migration) or to “deform and squeeze”
(amoeboid migration) through the 3D ECM.
SeedEZ allows embedding of most ECMs at high protein concentration to study cell invasion in 3D in a physiologically closer
setting with respect to biochemical and biomechanical cues present to cells taxing or invading in 3D. Putative modulators of
cell motility, invasion, angiogenic inhibitors etc., can all be tested using Spot-an-Agent and Spot-a-CultureTM approach. This
may be done in “side-by-side” testing in one SeedEZ substrate, or in a SeedEZ sandwich or stack.
C.
Cell-based assays with preserved cell morphology, heterogeneity of cell types, cell-cell and cell-ECM interactions
and signaling, and with extracellular cues having sufficient specificities to model pathologies in question may be
able to provide physiologically closer cell-based assay results.
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Normal and malignant cells can be distinguished morphologically in 3D environments. Solid tumors have pronounced
cellular heterogeneity on the histological, genetic, and gene expression level. Cells differ in size, morphology, motility,
angiogenic, proliferative, invasive potential, and drug resistance. Still, the majority of invasion assays still employ a single
cell type, and even for that cell type its size has to “match” the pore size on the trans-membrane to even conduct an assay.
SeedEZ allows to seed heterogeneous cell populations and sub-populations for physiologically closer tissue modeling and 3D
cell-based assay development.
D. Gel deteriorates too fast in angiogenesis/ transmigration assays.
SeedEZ solves this problem because it continues to support cells even when a gel degrades and allows embedding of high
protein gels. Substrates may also be stacked for consistent and reproducible longer term studies.
References:
Ehrbar M, Sala A, Lienemann P, Ranga A, Mosiewicz K, Bittermann A, Rizzi SC, Weber FE, Lutolf MP.
Elucidating the role of matrix stiffness in 3D cell migration and remodeling.
Biophys J. 2011, 100(2):284-93.
http://www.ncbi.nlm.nih.gov/pubmed/21244824
Brekhman V, Neufeld G.
A novel asymmetric 3D in-vitro assay for the study of tumor cell invasion.
BMC Cancer. 2009, 9:415.
http://www.ncbi.nlm.nih.gov/pubmed/19948022
Albini A, Benelli R, Noonan DM, Brigati C.
The "chemoinvasion assay": a tool to study tumor and endothelial cell invasion of basement membranes.
Int J Dev Biol. 2004, 48(5-6):563-71.
http://www.ncbi.nlm.nih.gov/pubmed/15349831
Bissell MJ, Rizki A, Mian IS.
Tissue architecture: the ultimate regulator of breast epithelial function.
Curr Opin Cell Biol. 2003, 15(6):753-62.
http://www.ncbi.nlm.nih.gov/pubmed/14644202
Marusyk A, Polyak K.
Tumor heterogeneity: causes and consequences.
Biochim Biophys Acta. 2010, 1805(1):105-17.
http://www.ncbi.nlm.nih.gov/pubmed/19931353
Why to embed gel and other reagents into the SeedEZ for in vitro controlled drug release studies?
Many inert polymers (gels) have been used as drug delivery systems both in vitro and in vivo. By incorporating drugs into
the SeedEZ or in biodegradable polymer matrices into the SeedEZ, the dosage forms which release a drug over a period of
time may be prepared in a desired shape and size without complex protocols and special equipment. More importantly, all
SeedEZ-based dosage forms can be tested in situ, with the cells in a 3D cell culture. Rigid, yet absorbent SeedEZ wicks
diverse sol-state gels, pharmaceuticals and test compounds. As a result, SeedEZ offers a novel 3D framework for:
A. Development of sustained release drug delivery systems that are simple and convenient to use in vitro.
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B. In situ testing of the above drug delivery systems with 3D cultures.
C.
Localized or distributed drug delivery into 3D cultures using Spot-a-CultureTM and Spot-a-DrugTM method in a single
substrate or in a SeedEZ-stack.
D. Development of test platforms for quasi-steady drug release.
In diffusion driven drug delivery systems, drug release rate often declines in time. A BIODEGRADABLE POLYMER
MATRIX EMBEDDED IN THE SeedEZ MAY ENABLE QUASI-STEADY DRUG RELEASE FROM A DEFINED VOLUME,
DEFINED BY THE SeedEZ, WHEN THE EMBEDDED MATRIX DEGRADATION RATE IS ADJUSTED TO COMPENSATE
FOR THIS DECLINE WITH AN INCREASED DRUG PERMEABILITY FROM THE SYSTEM.
GUIDELINES AND RECOMMENDATIONS FOR SEEDING CELLS IN A GEL
While the SeedEZ allows embedding of sol-state and even semi-sol-state gels from various origins and sources, for the most
consistent results embed gel in as much sol state as possible. This may depend on the nature of the gel; for example, if the
gel gels via physical or chemical cross-linking methods:
1.
For temperature-dependent hydrogels, and thermo-reversible hydrogels dispense at a suitable temperature when
the gel is in sol-state provided that that temperature is not detrimental to cells seeded in the gel.
2.
For chemically cross-linked hydrogels, dispense, wick or dip a gel precursor first, if possible, and then add crosslinking agent when precursor is embedded in the SeedEZ.
For example, for Alginate gels, you may embed cells in sodium alginate into the SeedEZ followed by addition of Calcium ions
in a buffer to crosslink the gel precursor at close to physiological conditions (or dip SeedEZ into Calcium containing buffer),
followed by 3D culture incubation in medium.
Pipette sol-state gel solutions carefully to avoid bubble formation. Use a positive displacement micropipette to dispense
viscous solutions, or if you find that losses in pipetting are significant using an ordinary micropipette. In some cases,
concentrated sol-state gels may be too viscous to dispense even with a positive displacement pipette. In this event, use dipin method to embed gel into the SeedEZ. Dip-in method works for many gels even when the gel viscosity is approximately
half the honey-like viscosity.
Most commonly, cells are seeded in a sol-state gel into the SeedEZ. This provides truly 3D cell distribution at seeding. If the
objective is to seed cells on top of a gel embedded in the SeedEZ, you may still do so; however, you may need to centrifuge
the plate to make cells ingress into the gel. To achieve desired culture conditions at seeding, you may also overlap or stack
the SeedEZ substrates, or you may add a layer of ECM between two SeedEZ substrates which already comprise the same or
a different gel.
GELS USED IN 3D CELL CULTURE APPLICATIONS AND DRUG DELIVERY
MOST GEL FORMING POLYMERS ARE SUSCEPTIBLE TO DEGRADATION BY REACTIVE OXYGEN SPECIES. THE USE OF
SeedEZ IS RECOMMENDED WITH MOST GELS USED IN 3D CELL CULTURE APPLICATIONS, ESPECIALLY IF CELLS DO NOT
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SECRETE THEIR ENDOGENEOUS EXTRACELLULAR MATRIX COMPONENTS THEY NEED TO MAINTAIN VIABILITY AND
FUNCTION.
GELS COMPRISING EXTRACELLULAR MATRIX CONSTITUENTS
COLLAGEN I
Collagen Type I is a fibrillar protein which provides
structural support to cells and facilitates cell attachment,
growth, differentiation and migration.
When used as a gelled matrix to support cells in 3D,
Collagen Type I derived from different sources may not gel
equally fast. For example, Bovine Collagen may gel slower
than a Rat Tail Collagen. In general, a faster gelling gel
allows cells to remain better distributed in 3D but the
working time may be short, while in slow-to-gel gels cells
tend to settle. The latter is of concern if you are seeding
cells in a sol-state gel (without the SeedEZ) with the
objective of obtaining 3D cell distribution.
SeedEZ accepts Collagen I as a sol-state gel (Fig. 1). With the
SeedEZ, you may find that cell distribution is more uniform
in 3D even with Bovine Collagen I. This is because SeedEZ
opposes cell settling, instantly wicks sol-Collagen, and
may be kept at 37 oC prior to cell seeding in Collagen
suspension to accelerate its gelation. When preparing
Collagen I for use with 3D cell cultures, follow
manufacturer protocols or other protocols you
normally use.
Among cells cultured in or on Collagen I are primary
colon carcinoma cells, mouse liver progenitor cells, rat
pancreatic islet cells, endothelial cells, hepatocytes,
muscle cells, breast carcinoma cells, osteoclasts, and
transfected cell lines including NIH3T3, PC12, and HEK293. The type of Collagen used, its concentration, cell
types, cell seeding method, and a culturing period
depend on application and research objectives. In
general, cells may be seeded distributed in Collagen in
the SeedEZ, seeded on top of the Collagen in the
SeedEZ, or seeded between two or more layers of
Collagen embedded in two or more SeedEZ substrates.
MATRIGEL
Fig. 1
Collagen Type I, gel embedded in the SeedEZ.
Without pre-wetting treatments, the SeedEZ instantly
wicks Bovine Collagen Type I, BD Biosciences Product
No. 354231, at a protein concentration of 2.9 mg/ml.
According to the supplier, under suitable conditions,
this product gelled in solutions containing as little as
0.5 mg/ml of protein.
Fig. 2
Matrigel gel (16 mg/ml) embedded in the SeedEZ.
Without pre-wetting treatments, the SeedEZ instantly wicks
Growth Factor Reduced Matrigel, diluted to 16 mg/ml in
HBSS (from the high protein concentration stock); BD
Biosciences Product No. 354263.
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Matrigel is a gelatinous protein mixture secreted by Engelbreth-Holm-Swarm mouse sarcoma cells (EHS matrix). This ECM
resembles extracellular environment in many tissues and is used by cell biologists as a gelled extracellular matrix for 3D
cultures. Main components of Matrigel are structural proteins (Laminin, Collagen IV and Entactin). Matrigel promotes and
maintains differentiated phenotypes in diverse cultures.
SeedEZ accepts Matrigel (Fig. 2) delivered as a sol-state gel up to 16 mg/ml protein concentration (the highest protein
concentration tested). According to manufacturer, Matrigel is a physiologically relevant surface for many applications and
cell types, including human embryonic and induced pluripotent stem cells, myogenic cells, mammary epithelial cells,
hepatocytes, rat brain microvessels, mammary acinar formation, and endothelial tube formation.
GELS FROM NATURAL SOURCES FOR 3D CELL CULTURES AND CONTROLLED DRUG RELEASE
Hydrocolloidal materials derived from natural sources are fully or partially soluble in water and used as gelling agents in 3D
cell culture applications. Commonly, they are either protein-based or polysaccharide-based biomaterials. An example of gel
derived from animal proteins is gelatin. Polysaccharide-based polymers represent a large class of biomaterials used in 3D
cell culture applications including agarose, alginate, carageenan, dextran, chitosan, cellulose derivatives etc.
THESE BIOMATERIALS ARE ALSO USED IN PREPARATION OF DRUG DELIVERY SYSTEMS (ALGINATES, GELATINS ETC.),
OR USED AS SUBSTRATES FOR CONTROLLED DRUG RELEASE (CHITOSAN, CELLULOSE DERIVATIVES, AGAROSE ETC.).
SeedEZ allows embedding of most sol-state gels while providing a convenient 3D framework for cell cultures studies, cellbased assay development, drug release studies and other applications.
GELS OF ANIMAL ORIGIN
GELATIN
A
Fig. 3
B
Type A gelatin embedded in the SeedEZ.
A. Without pre-wetting treatments, the SeedEZ instantly wicks porcine skin gelatin; a 2%
w/v solution delivered to the SeedEZ substrates at 37 oC. Type A indicates that the
gelatin was derived from acid cured tissue, Sigma-Aldrich Product No. G1890.
B. Room temperature gelled Gelatin; the same as delivered to the SeedEZ substrates in A.
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Gelatin is a mixture of water soluble peptides and proteins produced by partial hydrolysis of Collagen. It provides an
attachment and growth promoting substrate for the culture of many cell types. Gelatin is a thermo-reversible hydrogel
suitable for 3D cell cultures either alone or in combination with other molecules.
When an aqueous solution of gelatin at a concentration > 0.5% is cooled to 35-40°C it first increases in viscosity and then
gels. The gel stiffness depends on gelatin composition, method of manufacture, thermal history, concentration in solution,
pH and temperature. The optimal gel stiffness depends on cell type, application and the research objectives. In addition to
thermally-cooled or physically cross-linked gelatin gel, gelatin may be also cross-linked chemically:
Yung CW, Wu LQ, Tullman JA, Payne GF, Bentley WE, Barbari TA.
Transglutaminase crosslinked gelatin as a tissue engineering scaffold.
J Biomed Mater Res A. 2007, 83(4):1039-46.
Gelatin is biodegradable, thermally degradable, and susceptible to hydrolysis. SeedEZ accepts gelatin as a sol-state gel (Figs.
3-4) and helps with its application in long-term cell culture studies.
GELS OF NON-ANIMAL ORIGIN
AGAROSE
Agarose is a linear polysaccharide derived from agar, a hydrophilic colloid which forms thermo-reversible gels. In its gelled
state, agarose is used as matrix for 3D cell aggregates (spheroids) and 3D cell cultures of dissociated cells. Agarose melts at
higher than physiological temperatures and gels at temperatures close to 37°C. This makes it suitable for cell-based assay
development, molecular biology applications and drug release studies. The use of agarose as a matrix through which
chemoattractants diffuse provides a system to study cell motility using the “agarose drop” assay, the “agarose plug” assay,
and the “under agarose” assay.
A
B
INSTANTANEOUS WICKING
Fig. 5
Agarose gel embedded in the SeedEZ.
A. Without pre-wetting treatments, the SeedEZ instantly wicks SeaPrep Agarose, Lonza Rockland
Product. No. 50302; a 2.5% w/v solution delivered to the substrates at 60 oC.
B. Room temperature gelled agarose; the same as delivered to the SeedEZ substrates in A.
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Stiffness and porosity of agarose gels depend on agarose concentration in solution, while gelling and melting temperatures
may be adjusted by controlled introduction of hydroxyethyl groups. Agarose gels are frequently used as matrices for 3D cell
cultures at concentrations higher than about 2% w/v.
SeedEZ accepts sol-state agarose gel (Fig. 5). Most commonly, agarose is used to support 3D cell cultures of cartilage
phenotype. With covalently coupled Laminin, agarose gels were found to significantly enhance neurite extension from 3D
cultured embryonic day 9 chick dorsal root ganglia, and PC 12 cells.
ALGINATE
Alginates are hydrocolloids, water-soluble biopolymers produced by brown seaweeds. They are often supplied as sodium
salts which are soluble in water. In the presence of divalent cations, sodium alginates form cross-linked gels. Dissociated
cells and 3D cell spheroids are seeded in sodium alginate, followed by dipping or short-term incubation in a Calcium buffer
at close to physiological conditions. This procedure yields a cross-linked gel in which cells are embedded. Cells may be also
encapsulated in alginate beads or microcarriers using the same approach. Unlike ECM proteins, alginate is an inert material
lacking Arg-Gly-Asp peptide sequence which enables cell anchorage through integrins. Alginate is often used to culture cell
types that aggregate and form 3D cell spheroids. However, covalently bound peptides (integrin binding ligands) may be
necessary for the culture of anchorage dependent cells which do not aggregate to model cell-matrix interactions.
A
Fig. 6
B
Sodium alginate embedded in the SeedEZ.
A. Without pre-wetting treatments, the SeedEZ instantly wicks sodium alginate Keltone LVCR,
FMC Biopolymer; a 2% w/v solution delivered using a positive displacement pipettor.
B. Without pre-wetting treatments, the SeedEZ SC-C048 wicks sodium alginate Keltone HVCR,
FMC Biopolymer; a 2% w/v solution in less than 10 seconds.
SeedEZ wicks viscous sodium Alginate solutions 2% w/v (Fig. 6). The two sodium alginates tested were Keltone LVCR and
Keltone HVCR. Both products are used as drug-polymer matrices for extended release (polymer matrices for tablets used in
orally administered controlled drug delivery) and polymer matrices for 3D cell cultures or spheroids (for neural stem cell
growth, embryonic stem cell growth, or chondrocytes in alginate gels or beads). According to supplier, viscosity of a 2%
Keltone LVCR solution is 100-300 cPs. The viscosity of a 1.25% Keltone HVCR solution is 600-900 cPs; a 2% solution was used
for wicking into the SeedEZ. For reference, at room temperature, viscosity of water is 1 cP and that of honey about 10,000
cPs. Solutions were delivered to the SeedEZ substrates using a positive displacement pipette. For higher viscosity alginate
(Keltone HVCR), dip-in method was more appropriate.
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At a concentration higher than 1 % w/v, sodium alginates may be too viscous and difficult to dispense consistently cultureto-culture. This is inconvenient for 3D cell cultures where cell suspensions at seeding and following seeding may be less
homogeneous with the corresponding non-uniformity in 3D cell distribution. The SeedEZ solves this problem and wicks solstate 2% w/v viscous sodium alginate solutions by spot- and dip-in method (Fig. 6). As the SeedEZ holds cells in 3D by itself,
it also enables the use of lower concentration alginates for less constrained cell growth. Alginate gels degrade by methods
which reverse gelation. As the gel disintegrates due to gradual exchange of Calcium ions with Sodium, the use of SeedEZ is
recommended, particularly if low concentration gels were used.
METHYL CELLULOSE
Cellulose is the most abundant naturally occurring polymer of glucose. Cellulose-based hydrogels are formed by crosslinking aqueous solutions of cellulose ethers such as methylcellulose, hydroxypropyl methylcellulose, ethyl cellulose,
hydroxyethyl cellulose and sodium carboxymethylcellulose. Thermo-reversible gels are prepared from aqueous solutions of
methylcellulose and/or hydroxypropyl methylcellulose. While up to 10% methylcellulose solutions can be prepared using
low-viscosity products (< 10-50 cPs), the high-viscosity methylcellulose solutions are normally limited to 2-3%.
A
Fig. 7
B
Methylcellulose semi-solid media embedded in the SeedEZ.
A. Without pre-wetting treatments, the SeedEZ accepts a 2% w/v aqueous solution of methylcellulose,
Sigma Aldrich Product No. M0387 using a dip-in method.
B. The drops of methylcelluose semi-solid media as in A, shown in an almost vertically held Petri dish;
the solution viscosity was 1,500 cPs.
Relatively high gelling temperature of 2% methyl cellulose, approximately 48 oC, prevents its use in a gelled state for 3D cell
culture applications. While 10% methylcellulose solutions gel at approximately 30 oC, the solution viscosity is often too high
for routine use. For this reason, methyl cellulose semi-solid media rather than a gel is used for the culture of human cells,
clonal growth of cells, embryoid bodies, neurospheres etc, and further used in methylcellulose-based colony forming assays
and anchorage independence assays. In addition to cell culture applications, cellulose ethers are used as excipients in drug
formulations, or sustained release of other biomolecules. In solid tablets, cellulose ethers enable a swelling-driven release
of the drug in contact with physiological fluids.
Methylcellulose is biodegradable (although not by animal and human cells in culture as they do not synthesize cellulases).
The SeedEZ accepts methylcellulose semi-solid media as a 2% w/v aqueous solution (Fig. 7) at a viscosity of approximately
1500 cPs (Sigma Aldrich Product No. M0387).
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SYNTHETIC GELS
PLURONIC
Concentrated aqueous solutions of poloxamers form thermo-reversible gels which revert to a liquid when the temperature
is reduced. Poloxamers are triblock copolymers of a central hydrophobic chain of poly(propylene oxide) and two hydrophilic
chains of (poly(ethylene oxide), PEO-PPO-PEO. Pluronic F127 is the trade name for nontoxic Poloxamer 407. The advantage
of Pluronic F127 for 3D cell culture applications is that it forms a gel at physiological temperatures at concentrations higher
than approximately 20% w/v. It is frequently used for cell encapsulation, as a substrate in tissue engineering (e.g. cartilage),
as a component in drug delivery and pharmaceutical formulations, an additive to culture media in bioreactors, a surfactant
and reagent which facilitates solubilization of hydrophobic molecules in water etc.
A
Fig. 8
B
Pluronic F127 (PEO-PPO-PEO) gel embedded in the SeedEZ.
A. Without pre-wetting treatments, the SeedEZ instantly wicks a 25% w/v Pluronic F127 solution
in Neurobasal medium. The use of positive displacement pipette is recommended.
B. A sample of gelled Pluronic F127; the same as delivered to the SeedEZ substrates in A.
The making of aqueous poloxamer gels is simple and requires merely the addition of weighed amount of the poloxamer to
cold water with slow mixing to prevent foaming. The gelling temperature depends on the poloxamer concentration, pH,
and on the type and amount of additives, if anySeedEZ instantly wicks sol-state 25% w/v Pluronic F127 solution (Fig. 8). .
Pluronic is biodegradable.
CARBOMER
Carbomer is a generic name for synthetic high molecular weight polymers of acrylic acid. These polymers have widespread
use in pharmaceutical formulations for ocular, oral, transdermal and nasal drug delivery.
SeedEZ accepts Carbopol polymers by dip-in method (Fig. 9). Carbopol is the trade name for polymers of acrylic acid
crosslinked with polyalkenyl ethers or divinyl glycol manufactured by Lubrizol Corporation. Carbopol 971P NF is used as the
matrix ingredient for controlled release tablets and capsules, suspending agent for oral liquids, bioadhesive in drug delivery
systems, etc. Lightly crosslinked polymers, such as Carbopol® 971P NF polymer tend to be more efficient in controlling drug
release than highly crosslinked polymers such as Carbopol 974P NF polymer. Carbopol® 974P NF was introduced for use in
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oral and mucosal contact applications such as extended release tablets, oral liquids and bioadhesive formulations. It is a
highly crosslinked polymer and produces highly viscous gels with rheology similar to mayonnaise.
A
B
C
D
Fig. 9
Poly(acrylic acid) gel embedded in the SeedEZ.
A. Without pre-wetting treatments, the SeedEZ accepts Carbopol 971P NF, a 2% w/v solution in DI
water using a dip-in seeding method.
B. A sample of gelled Carbopol 971P NF; the same as delivered to the SeedEZ substrates in A.
C. Without pre-wetting treatments, the SeedEZ accepts Carbopol 974P NF, a 2% w/v solution in DI
water using a dip-in seeding method.
D. A sample of gelled Carbopol 974P NF; the same as delivered to the SeedEZ substrates in C.
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