Name: construction of a multilayered mesenchymal stem cell sheet with a 3d dynamic culture system
Uploaded: 2018-10-20T15:00:00
Description: Watch this Scientific Journal Video about Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System at JoVE.com

This work was supported by the National Natural Science Foundation of China (grant number 31771064); the Science and Technology Planning Project of Guangdong Province (grant numbers 2013B010404030, 2014A010105029, and 2016A020214012); the Science and Technology Planning Project of Guangzhou (grant number 201607010063); and the Undergraduate Innovation and Entrepreneurship Training Program (grant number 201610559028); the National Science Foundation for Young Scientists of China (grant number 31800819).

All stem cell and animal experiment procedures were conducted according to the ethical guidelines of the National Guide for the Care and Use of Laboratory Animals and approved by the Jinan University Animal Care and Use Committee (Guangzhou, China).
1. Preparation of the DPP Scaffold with the PLA2 Decellularization Method14
Note: See Figure 1A for a schematic of the PLA2 decellularization meth...

<ol>
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The present protocol reports an efficient method for constructing a multilayered MSC sheet. This cell sheet exhibits optimal mechanical strength, high cell seeding density, and favorable stem cell bioactivity. Using BMSCs as an example, the 3D cell structure is quickly constructed with RAD16-I peptide hydrogel. After being cultured in the dynamic perfusion system, the multilayered BMSC sheet is successfully obtained and the BMSCs maintain a high expression of stem cell markers.
Constructing th...

Stem cell therapy has been reported as an effective treatment for many diseases; however, low cell retention and poor survival within the target zone remain critical issues following traditional stem cell injection. To solve this problem, tissue engineering scientists developed the cell sheet technique. A monolayered cell sheet with intact extracellular matrix was firstly prepared by using the temperature-response culture dish1, and its follow-up studies reported the significant improvements of stem cell retention and survival within the infarcted area2,3. Among the methods, constructing the multilayered cell sheet has been reported as an effective strategy for improving the cell survival and the cell sheet therapeutic effect3,4. Since then, scientists have worked on developing different cell sheet construction methods in order to increase the cell amount, stem cell property, and mechanical property of the cell sheets. So far, certain types of cell sheet have been constructed and studied in the treatment of myocardial infarction5, cartilage injury6, and skin wound7.
The bioactivity of stem cells before transplantation showed an emerging influence on injured tissue regeneration, and different cell sheet construction strategies have different effects on the stem cells. On one hand, confluent cell sheets only consisted of high-density stem cells, and natural extracellular matrices could be acquired by stacking monolayered cell sheets8 or by using magnetic tissue engineering techniques9. On the other hand, researchers developed different scaffolds to provide adequate mechanical strength and support cell growth10,11,12, which allowed a low stem cell seeding density to ensure the nutrition supply. However, despite these approaches, the low efficient nutrition supply within the multilayered cell sheet structure remains a major concern during the in vitro construction. Therefore, an efficient and feasible cell sheet construction system is urgently required.
This protocol describes the steps to prepare a multilayeredmesenchymal stem cell (MSC) cell sheet. In this construction system, the cell sheet mechanical strength is provided by a DPP. Based on this scaffold, the 3D cell structure can be quickly constructed with RAD16-I peptide hydrogel, and a dynamic perfusion system is used to culture the multilayered cell sheet, in order to stabilize the 3D cell sheet structure and provide sufficient nutrition supply for the cells. Using this system, a multilayered BMSC sheet was successfully prepared and exhibited an optimal therapeutic effect on the rat myocardial infarction model13.

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			<td height="21" style="height:21px;">MINUSHEET</td>
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			<td height="21" style="height:21px;">MINUSHEET 0002</td>
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			<td>500 mL glass bottle&#160;</td>
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			<td height="21" style="height:21px;">MINUSHEET 1301</td>
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			<td>perfusion culture container&#160;</td>
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construction of a multilayered mesenchymal stem cell sheet with a 3d dynamic culture system

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

The schematic of the multilayered stem cell sheet construction is shown in Figure 1. Preparing the cell sheet scaffold by the PLA2 decellularization method is the first step. Based on the scaffold, a temporary 3D cell structure is constructed by mixing the stem cells with the RAD16-1 peptide hydrogel. In order to obtain a multilayered cell sheet with favorable stem cell bioactivity and optimal mechanical strength, the cell sheet is cultured in a dy...

Watch this Scientific Journal Video about Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System at JoVE.com

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

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

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

Stem cell therapy has been widely reported as a promising treatment for many diseases. And the cell sheet techniques have been developed in order to improve the cell's retention and the survival within the targets, too. During cell sheets construction, the insufficient nutrition supply remain a key problems for the loss of stem cell's function, and is an unsatisfactory stem cell property would finally effect the therapeutic effects in vivo.Our purpose to prepare available multilayered stem cell sheet product, our team developed the cellularized polesized, pericardium and cell sheet scaffold. Based on the scaffold the multilayer cells starter would be quickly constructed with nonapeptide Hydrogen and a dynamics perfusion system is used to ensure the stem cell's nutrition supply in vitro. Take out a dried DBP scaffold and put it in the center of the black base.Put a white tension ring on the DBP scaffold, and affix it in the tissue carrier. Ensure the DBP scaffold is totally fixed in the tissue carrier. Add 100 microliters culture mediums on the DBP scaffold.Put the scaffold into the 37 degree Celsius incubator, and allow it to soak for 15 minutes. Take the cells out of the incubator, remove the culture mediums from the culture dish, gently wash the cells with two milliliters of warm PBS. Remove all PBS from the culture dish, and make sure no liquid remains.Add two milliliters to 1.25%trypsin solution to the dish, and incubate the cells for three minutes. Stop the trypsin effect by adding two milliliters culture medium. Gently wash the cells from the dish.Transfer the cell suspensions in a new 15 milliliter centrifugal tube. Centrifugate the cells at 220 times gravity for five minutes, and obtain cell sediments. Remove the supernatant, and wipe out the cell sediment.Re suspend the cells with three milliliter 10%sucrose solution. Aspirate 10 microliters cell suspension, and the content cell number reads a hemocytometer, extract three million cells and transfer in your new 1.5 milliliter centrifugal tube. Centrifugate the cells at 220 times square root for five minutes, completely remove the supernatants and obtain the cells sediment.Prepare 20 microliters 10%sucrose solution. Gently re-suspend the cells and obtain a uniform suspension. Add 20 microliter peptide hydro gel at the top of suspension gently mix the peptide hydro gel, and the cells suspension with the T, take out the DBP scaffold, leaving the tissue carrier, and then gently aspirate the calculi median with a T.Aspirate the mixture the mixture and evenly add on to the DBP scaffold.Add one milliliter calciumedetate to the bottom of the tissue carrier. Gently add four milliliter cultural medium in the culture dish and then emerge the cell sheet. Put the cells in the incubator for two hours let it culture.The dynamic perfusion system include a perfusion culture container the gas exchanging recrement and a glass bottle. Assembles are dynamic perfusion systems as the figure shown. Add two hydro milliliters culture medians in the glass bottle.Insert the cell sheet into the chamber of the culture container, add three milliliters culture medians in the container, put the dynamic perfusion systems in the incubators, and start to pump. Open the culture container in the bipositive recumbent. Take out the cell sheet from the culture container and put it in culture dish.Use one forceps to immobilize the tissue carrier and use another two forceps to separate the white tension ring from the black base. Finally, obtain the multilayered cell sheet. DBP scaffold is transparent.The SEM resells shows that colleges captured and the components are completely reserved. The cell sheets can be obtained and held by forceps for short preservation. These cell sheet could be transferred to 1.5 milliliter cylindriform tube.As a model for instance starting with cell showings of BMFC'S highly positive for the stem cells marker CD 9O and the CD 29. After cell construction the BMFC'S leaving the cell sheet remains high expansion levels of CD 90 and the CD 29. Constructing the multilayered cell structure is the critical step of the protocol.The pan time requires four hydro gel when the PH value changes from SA to neutral, therefore, it is important to wash the cell with sucrose solution to remove the surface ions. Also, the volume ratio of cell suspension and panthihydrogel is not good friend for constructing the temporary multilayer structure. After the formation of 3D stem cell structure, you will see the dynamic perfusion system is important for stabilizing the multilayered structure as well as maintaining the stem cell viabilities.The dynamic infiltration of the culture median could facilitate stem cells to proliferate and extrapolate cell context within the multilayered structure also the dynamic culture thermometers should be adjusted according to different stem cell types.

construction-multilayered-mesenchymal-stem-cell-sheet-with-3d-dynamic

Research

JoVE Journal

Bioengineering

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

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

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, direct visualization of individual viral complexes in their host cellular environment with cryoET is challenging2, due to the infrequent and dynamic nature of viral entry, particularly in the case of HIV-1. While time-lapse live-cell imaging has yielded a great deal of information about many aspects of the life cycle of HIV-13-7, the resolution afforded by live-cell microscopy is limited (~ 200 nm). Our work was aimed at developing a correlation method that permits direct visualization of early events of HIV-1 infection by combining live-cell fluorescent light microscopy, cryo-fluorescent microscopy, and cryoET. In this manner, live-cell and cryo-fluorescent signals can be used to accurately guide the sampling in cryoET. Furthermore, structural information obtained from cryoET can be complemented with the dynamic functional data gained through live-cell imaging of fluorescent labeled target.
In this video article, we provide detailed methods and protocols for structural investigation of HIV-1 and host-cell interactions using 3D correlative high-speed live-cell imaging and high-resolution cryoET structural analysis. HeLa cells infected with HIV-1 particles were characterized first by confocal live-cell microscopy, and the region containing the same viral particle was then analyzed by cryo-electron tomography for 3D structural details. The correlation between two sets of imaging data, optical imaging and electron imaging, was achieved using a home-built cryo-fluorescence light microscopy stage. The approach detailed here will be valuable, not only for study of virus-host cell interactions, but also for broader applications in cell biology, such as cell signaling, membrane receptor trafficking, and many other dynamic cellular processes.

We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, ...

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that replicate the biofidelity of three-dimensional native human myocardium, while also enabling a controlled level of biological complexity, and allowing non-destructive longitudinal monitoring of tissue contractile function. Initially, human engineered cardiac tissues (hECT) were created using the entire cell population obtained from directed differentiation of human pluripotent stem cells, which typically yielded less than 50% cardiomyocytes. However, to create reliable predictive models of human myocardium, and to elucidate mechanisms of heterocellular interaction, it is essential to accurately control the biological composition in engineered tissues. 
To address this limitation, we utilize live cell sorting for the cardiac surface marker SIRP&#945; and the fibroblast marker CD90 to create tissues containing a 3:1 ratio of these cell types, respectively, that are then mixed together and added to a collagen-based matrix solution. Resulting hECTs are, thus, completely defined in both their cellular and extracellular matrix composition.
Here we describe the construction of defined hECTs as a model system to understand mechanisms of cell-cell interactions in cell therapies, using an example of human bone marrow-derived mesenchymal stem cells (hMSC) that are currently being used in human clinical trials. The defined tissue composition is imperative to understand how the hMSCs may be interacting with the endogenous cardiac cell types to enhance tissue function. A bioreactor system is also described that simultaneously cultures six hECTs in parallel, permitting more efficient use of the cells after sorting.

This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

Micropatterning and Assembly of 3D Microvessels

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

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

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

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Exploring the Potential of Mesenchymal Stem Cell Sheet on The Development of Hepatocellular Carcinoma In Vivo

An in vivo animal model that mimics human cancer could have various applications that deliver significant clinical information. The currently used techniques for the development of in vivo cancer models have considerable limitations. Therefore, in this study, we aim to implement cell sheet technology to develop an in vivo cancer model. Hepatocellular carcinoma (HCC) is successfully developed in nude rats using cell sheets created from HCC cell line cells. The cancer cell sheets are generated through intracellular adhesion and the formation of a stratified structure, controlled by the extracellular matrix. This allows for the HCC sheet transplantation into the liver and the creation of a tumor-bearing animal model within a month. In addition, the role of mesenchymal stem cells (MSC) in the development of this cancer model is investigated. In addition to the HCC cell line sheet, another two cell sheets are created: a sheet of HCC cells and bone marrow MSCs (BMMSCs) and a sheet of HCC cells and umbilical cord MSCs (UCMSCs). Sheets that have a combination of both HCC cells and MSCs are also capable of producing a tumor-bearing animal. However, the addition of MSCs reduces the size of the formed tumor, and this adverse effect on tumor development varies depending on the used MSCs' source. This indicates that a cell sheet made of certain MSC subtypes could be utilized in tumor management and control.

Exploring the Potential of Mesenchymal Stem Cell Sheet on The Development of Hepatocellular Carcinoma In Vivo

Here, we present a protocol to develop an in vivo cancer model using cell sheet technology. Such a model could be very useful for the evaluation of anticancer therapeutics.

An in vivo animal model that mimics human cancer could have various applications that deliver significant clinical information. The currently used ...

A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth plate cartilage architecture and results in growth modulation of long bones of children. To determine the role of compressive stress in bone growth, we created a microfluidic device actuated by pneumatic pressure, to dynamically (or statically) compress growth plate chondrocytes embedded in alginate hydrogel cylinders. In this article, we describe detailed methods for fabricating and characterizing this device. The advantages of our protocol are: 1) Five different magnitudes of compressive stress can be generated on five technical replicates in a single platform, 2) It is easy to visualize cell morphology via a conventional light microscope, 3) Cells can be rapidly isolated from the device after compression to facilitate downstream assays, and 4) The platform can be applied to study mechanobiology of any cell type that can grow in hydrogels.

This article provides detailed methods for fabricating and characterizing a pneumatically actuating microfluidic device for chondrocyte compression.

Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...