This work was supported by the National Institutes of Health / National Institute of Mental Health (NIH/NIMH) grant #1R21MH085267.

Part 1: Preparing a master mold for multi-compartment neuron co-culture device fabrication
<ol>
<li>First step to fabricate the PMMA master is to make 3.5 mm deep compartments. One soma compartment and six axon/glia compartments are defined on a PMMA block using a micro-milling machine (MDX-40, Roland or any other CNC milling machine). PMMA master is then sonicated in isopropyl alcohol for 10 minutes to remove debris.</li>
<li>Arrays of 2.5 &mu;m high ridge structures are patterned on a 50.8 mm x 50.8 mm glass slide by a standard photolithography process, followed by wet etching of the glass. First, array of ridge structures are patterned on a cleaned glass substrate with positive photoresist S1818 using photolithography. S1818 is spin-coated on the substrate (4000 rpm), soft based at 110&deg;C for 5 min, and then exposed to UV light (84 mJ/cm2) using a mask aligner followed by developing of the photoresist in MF319 for 40 seconds. Next, the photoresist patterned glass substrate is immersed in buffered oxide etch (BOE) at room temperature for 6-7 minute to obtain a 2.5 &mu;m tall ridge structures. Finally, the photoresist layer is removed by acetone and isopropyl alcohol, followed by thorough rinsing in de-ionized (DI) water.</li>
<li>Glass substrate with micro-ridge structures is hot-embossed against the PMMA master to transfer its patterns. First, PMMA mold master and glass substrates are manually aligned and placed on a hot-press. Then, temperature is raised to 115 &deg;C. Once desired temperature is reached, 0.5 tons of weight is applied for 5 minutes. After 5 minutes, temperature is cooled down to 40 &deg;C and pressure is released. The PMMA master is then cut into a 50.8 mm x 50.8 mm size block.</li>
<li>PDMS pre-polymer (Sylgard 184) is mixed with curing agent at the ratio of 10: 1 by weight.</li>
<li>PMMA master is placed inside a cask and PDMS mixture is poured on top of the PMMA master to fabricate a PDMS master. It is then placed in a desiccator connected to a vacuum pump. PDMS poured on top of PMMA master is then degassed for 15 minutes to remove bubbles generated during the PDMS mixing process.</li>
<li>When all bubbles are removed, PDMS is put inside a leveled 85 &deg;C oven for one hour for polymerization.</li>
<li>Once PDMS is fully cured, PDMS master is peeled off from the PMMA master and placed inside a desiccator with 2-3 drops of trichlorosilane and vacuumed for 15 minutes to vapor coat the PDMS master surface. Trichlorosilane coating facilitates the release of the final PDMS neuron co-culture devices from the PDMS master. After coating, the PDMS master is briefly rinsed with isopropyl alcohol to remove excessive trichlorosilane, and dried with N2 gas.</li>
</ol>
Part 2: Neuron culture device replication
<ol>
<li>PDMS pre-polymer (Sylgard 184) is mixed with curing agent at the ratio of 10: 1 by weight.</li>
<li>PDMS mixture is poured on top of the PDMS master and degassed inside a desiccator connected to a vacuum pump for 15 minutes. It is important not to pour too much PDMS so that the 3.5 mm high PDMS structure on the master is not fully immersed.</li>
<li>When all bubbles are removed, PDMS is put inside a leveled 85 &deg;C oven for one hour for polymerization.</li>
<li>PDMS is taken out from the oven after one hour and left at room temperature to cool down. Then, PDMS neuron culture devices are gently peeled off from the PDMS master. Peeled off PDMS devices are wrapped with parafilm to protect them from dusts or debris.</li>
<li>PDMS devices, wrapped with parafilm, are cut into individual pieces using a drill press equipped with a blade.</li>
</ol>
Part 3: Device assembly
<ol>
<li>PDMS neuron culture device is placed inside the plasma cleaner and exposed to air plasma for 90 seconds to make the surface of the PDMS device hydrophilic. This plasma treatment will facilitate microchannels to be filled with culture media after assembly to poly-d-lysine coated substrate.</li>
<li>Devices treated with plasma are then immersed in 70 % ethanol for 30 minutes for sterilization and moved inside the sterilized bio-hood.</li>
<li>Sterilized devices are taken out from 70 % ethanol and dried with Millipore filtered N2 gas. Dried devices are placed on top of poly-d-lysine coated polystyrene 6-well culture plate and gentle pressure is applied to ensure the seal between the substrate and the device.</li>
<li>Culture media is then filled into the soma compartment, followed by filling the six axon/glia compartments. It is better to give 1-2 minutes pause before filling axon/glia compartments after adding culture media to the soma compartment to ensure that no bubbles are trapped within the microchannels.</li>
<li>Devices filled with culture media is incubated inside an 37 &deg;C incubator overnight to rinse any possible debris or toxic residue.</li>
</ol>
Part 4: Cell loading and neuron-oligodendrocyte co-culture
<ol>
<li>On the first day of the cell culture, primary forebrain neuron cells from embryonic day 16-18 rats are loaded into the soma compartment at an areal density of 500 cells/mm2. First, culture media inside both the soma compartment and axon/glia compartments are aspirated using pipette prior to cell loading. Then, 38500 cells, diluted in 40 &mu;l of culture media, are loaded to the soma compartment around the microchannel inlets. It is important not to touch the PDMS device while loading the cells, since it can break the seal between the device and the substrate.</li>
<li>Incubate the cell loaded devices for 30 minutes inside an 37 &deg;C incubator to enhance cell adhesion to the substrate. After incubation, all compartments are filled up with culture media.</li>
<li>Cells are cultured at 37 &deg;C in a humidified 5 % CO2 incubator and only half of culture media is changed out every 3-4 days.</li>
<li>After 14 days of neuron culture in vitro, oligodendrocytes are added to the axon/glia compartments for co-culture. Oligodendrocytes, dissected from the cerebral hemispheres of Sprague-Dawley rats at postnatal day 1-2, are added to each axon/glia compartment at an area density of 1000 cells/mm2. Prior to loading cells, approximately 60 % of culture media inside the compartments are aspirated with pipette. Caution must be made not to touch the bottom substrate, since it can damage the axonal network formed on the substrate. Then, 6700 oligodendrocyte cells, diluted in 5 &mu;l of culture media, are added to the axon/glia compartments. Cells are incubated for 1 hour and compartments are filled up with culture media.</li>
<li>Cells are cultured at 37 &deg;C in a humidified 5 % CO2 incubator and only half of culture media is changed out every 3-4 days for two more weeks.</li>
</ol>
Part 5: Representative Results:
When experiments are carried out properly, neuron cells loaded to the soma compartment locate close to microchannel inlets and axonal layer start to form inside the axon/glia compartments after 1 week of culture. Sign of neuron cell aggregation inside the soma compartment can be an indication that cells are not healthy. When loading oligodendrocytes after two weeks of neuron culture, axon/glia compartments should be covered with dense layer of axons and oligodendrocytes should be loaded on top of the axonal layer.
<img src="/files/ftp_upload/1399/1399fig1tmb.jpg" border="0" alt="figure 1" /> Figure 1. Schematic illustrations of the high-throughput microfluidic multi-compartment CNS neuron co-culture platform. Cross-sectional view showing isolation of axons from neuronal soma and dendrites as well as fluidic isolation between compartments with minute fluidic level difference. Please <a href="https://www.jove.com/files/ftp_upload/1399/1399fig1.jpg">click here</a> to see a larger version of figure 1.
<img src="/files/ftp_upload/1399/1399fig2tmb.jpg" border="0" alt="figure 2" /> Figure 2. Fabrication and assembly steps for the multi-compartment PDMS microfluidic co-culture device. Each device fits into one well of a conventional 6-well polystyrene cell culture plate. Please <a href="https://www.jove.com/files/ftp_upload/1399/1399fig2.jpg">click here</a> to see a larger version of figure 2.

<ol>
	<li>Sherman, D. L., Brophy, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+axon+ensheathment+and+myelin+growth.">Mechanisms of axon ensheathment and myelin growth.</a> Nature Reviews. 6, 683-690 (2005).</li><li>Xia, Y., Whitesides, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soft+Lithography.">Soft Lithography.</a> Angew. Chem. 37, 550-575 (1998).</li><li>Taylor, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+culture+platform+for+neuroscience+research.">Microfluidic culture platform for neuroscience research.</a> Nature Methods. 2, 599-605 (2005).</li><li>Li, J., Baud, O., Vartanian, T., Volpe, J. J., Rosenberg, P. A., A, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peroxynitrite+generated+by+inducible+nitric+oxide+synthase+and+NADPH+oxidase+mediates+microglial+toxicity+to+oligodendrocytes.">Peroxynitrite generated by inducible nitric oxide synthase and NADPH oxidase mediates microglial toxicity to oligodendrocytes.</a> Proceedings of the National Academy of Sciences. 102, 9936-9941 (2005).</li></ol>

The authors have nothing to disclose.

We have developed a multi-compartment co-culture platform for studying mammalian CNS axon-glia interaction. CNS axons were successfully isolated from neuronal cell bodies/dendrites, and oligodendrocytes were successfully co-cultured inside the device. Neuron density can be varied by applications, but too low or too high concentration can cause cells to die. Also, it is important to change only half of culture media so that the cells or axonal layer on the substrate are not damaged. We expect that this system will be a po...

a multi-compartment cns neuron-glia co-culture microfluidic platform

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to six independent experiments in parallel for higher-throughput. We developed a new fabrication method to create microfluidic devices having both micro and macro scale structures within the same device through a single soft-lithography process, enabling mass fabrication with good repeatability.
The multi-compartment microfluidic co-culture platform is composed of one soma compartment for neurons and six axon/glia compartments for oligodendrocytes (OLs). The soma compartment and axon/glia compartments are connected by arrays of axon-guiding microchannels that function as physical barriers to confine neuronal soma in the soma compartment, while allowing axons to grow into axon/glia compartments. OLs loaded into axon/glia compartments can interact only with axons but not with neuronal soma or dendrites, enabling localized axon-glia interaction studies. The microchannels also enabled fluidic isolation between compartments, allowing six independent experiments to be conducted on a single device for higher throughput.
Soft-lithography using poly(dimethylsiloxane) (PDMS) is a commonly used technique in biomedical microdevices. Reservoirs on these devices are commonly defined by manual punching. Although simple, poor alignment and time consuming nature of the process makes this process not suitable when large numbers of reservoirs have to be repeatedly created. The newly developed method did not require manual punching of reservoirs, overcoming such limitations. First, seven reservoirs (depth: 3.5 mm) were made on a poly(methyl methacrylate) (PMMA) block using a micro-milling machine. Then, arrays of ridge microstructures, fabricated on a glass substrate, were hot-embossed against the PMMA block to define microchannels that connect the soma and axon/glia compartments. This process resulted in macro-scale reservoirs (3.5 mm) and micro-scale channels (2.5 &mu;m) to coincide within a single PMMA master. A PDMS replica that served as a mold master was obtained using soft-lithography and the final PDMS device was replicated from this master.
Primary neurons from E16-18 rats were loaded to the soma compartment and cultured for two weeks. After one week of cell culture, axons crossed microchannels and formed axonal only network layer inside axon/glia compartments. Axons grew uniformly throughout six axon/glia compartments and OLs from P1-2 rats were added to axon/glia compartments at 14 days in vitro for co-culture.

Watch this Scientific Journal Video about A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform at JoVE.com

A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method.

We present a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research, capable of conducting up to ...

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11.9K Views.  Texas A&M University (TAMU). We developed a novel multi-compartment neuron co-culture microsystem platform for in vitro CNS axon-glia interaction research. The platform is capable of conducting up to six independent experiments in parallel and was fabricated using a newly developed macro/micro hybrid fabrication method. 

Video: A Multi-compartment CNS Neuron-glia Co-culture Microfluidic Platform

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

A Gradient-generating Microfluidic Device for Cell Biology

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an enabling experimental tool, because it can precisely manipulate fluid flows, enable high-throughput experiments, and generate stable soluble concentration gradients.  Compared to conventional gradient generators, poly(dimethylsiloxane) (PDMS)-based  microfluidic devices can generate stable concentration gradients of growth factors with well-defined profiles.  Here, we developed simple gradient-generating microfluidic devices with three separate inlets.  Three microchannels combined into one microchannel to generate concentration gradients.  The stability and shape of growth factor gradients were confirmed by fluorescein isothyiocyanate (FITC)-dextran with a molecular weight similar to epidermal growth factor (EGF).  Using this microfluidic device, we demonstrated that fibroblasts exposed to concentration gradients of EGF migrated toward higher concentrations.  The directional orientation of cell migration and motility of migrating cells were quantitatively assessed by cell tracking analysis.  Thus, this gradient-generating microfluidic device might be useful for studying and analyzing the behavior of migrating cells.

We describe a protocol for the microfabrication of the gradient-generating microfluidic device that can generate spatial and temporal gradients in well-defined microenvironment. In this approach, the gradient-generating microfluidic device can be used to study directed cell migration, embryogenesis, wound healing, and cancer metastasis.

 The fabrication and operation of a gradient-generating microfluidic device for studying cellular behavior is described.  A microfluidic platform is an ...

Cell Capture Using a Microfluidic Device

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

The Microfluidic Probe: Operation and Use for Localized Surface Processing

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. Microfluidics is commonly associated with closed microchannels which limit their use to samples that can be introduced, and cultured in the case of cells, within a confined volume. On the other hand, micropipetting system have been used to locally perfuse cells and surfaces, notably using push-pull setups where one pipette acts as source and the other one as sink, but the confinement of the flow is difficult in three dimensions. Furthermore, pipettes are fragile and difficult to position and hence are used in static configuration only. 
The microfluidic probe (MFP) circumvents the constraints imposed by the construction of closed microfluidic channels and instead of enclosing the sample into the microfluidic system, the microfluidic flow can be directly delivered onto the sample, and scanned across the sample, using the MFP. . The injection and aspiration openings are located within a few tens of micrometers of one another so that a microjet injected into the gap is confined by the hydrodynamic forces of the surrounding liquid and entirely aspirated back into the other opening. The microjet can be flushed across the substrate surface and provides a precise tool for localized deposition/delivery of reagents which can be used over large areas by scanning the probe across the surface. 
In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer.

In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer solution.

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. ...

Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions. Extracellular proteins, collagen I or von Willebrand factor are deposited within the microfluidic channel using active perfusion with a pneumatic pump. The matrix proteins are then washed with buffer and blocked to prepare the microfluidic channel for platelet interactions. Whole blood labeled with fluorescent dye is perfused through the channel at various flow rates in order to achieve platelet activation and aggregation. Inhibitors of platelet aggregation can be added prior to the flow cell experiment to generate IC50 dose response data.

The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions.

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion ...

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has been proposed that the microenvironment that the pathogen encounters in the commensal layer is important in determining the extent of colonization. Current culture methods for investigating pathogen colonization are not well suited for investigating this hypothesis as they do not enable co-culture of bacteria and epithelial cells in a manner that mimics the GI tract microenvironment. Here we describe a microfluidic co-culture model that enables independent culture of eukaryotic cells and bacteria, and testing the effect of the commensal microenvironment on pathogen colonization. The co-culture model is demonstrated by developing a commensal Escherichia coli biofilm among HeLa cells, followed by introduction of enterohemorrhagic E. coli (EHEC) into the commensal island, in a sequence that mimics the sequence of events in GI tract infection.

This protocol describes a microfluidic co-culture model for simultaneous and localized culture of epithelial cells and bacteria. This model can be used for investigating the role of different soluble molecular signals on pathogenesis as well as screen the effectiveness of putative probiotic bacterial strains.

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has ...

A Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable Concentration Gradients

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the concentration of specific chemoeffectors by comparing the current concentration to the concentration detected a few seconds earlier. This comparison determines the net direction of movement. Although multiple, competing gradients often coexist in nature, conventional approaches for investigating bacterial chemotaxis are suboptimal for quantifying migration in response to concentration gradients of attractants and repellents. Here, we describe the development of a microfluidic chemotaxis model for presenting precise and stable concentration gradients of chemoeffectors to bacteria and quantitatively investigating their response to the applied gradient. The device is versatile in that concentration gradients of any desired absolute concentration and gradient strength can be easily generated by diffusive mixing. The device is demonstrated using the response of Escherichia coli RP437 to gradients of amino acids and nickel ions.

This protocol describes the development of a microfluidic device for investigating bacterial chemotaxis in stable concentration gradients of chemoeffectors.

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the ...

Modified ES / OP9 Co-Culture Protocol Provides Enhanced Characterization of Hematopoietic Progeny

The in vitro differentiation of ES cells towards a hematopoietic cell fate is useful when studying cell populations that are difficult to access in vivo and for characterizing the earliest genes involved in hematopoiesis, without having to deal with embryonic lethalities. The ES/OP9 co-culture system was originally designed to produce hematopoietic progeny, without the over production of macrophages, as the OP9 stromal cell line is derived from the calvaria of osteopetrosis mutant mice that lack functional M-CSF. The in vitro ES/OP9 co-culture system can be used in order to recapitulate early hematopoietic development. When cultured on OP9 stromal cells, ES cells differentiate into Flk-1+ hemangioblasts, hematopoietic progenitors, and finally mature, terminally differentiated lineages. The standard ES/OP9 co-culture protocol entails the placement of ES cells onto a confluent layer of OP9 cells; as well as, periodic replating steps in order to remove old, contaminating OP9 cells. Furthermore, current protocols involve evaluating only the hematopoietic cells found in suspension and are not optimized for evaluation of ES-derived progeny at each day of differentiation. However, with replating steps and the harvesting of only suspension cells one potentially misses a large portion of ES-derived progeny and developing hematopoietic cells. This issue becomes important to address when trying to characterize hematopoietic defects associated with knockout ES lines. Here we describe a modified ES/mStrawberry OP9 co-culture, which allows for the elimination of contaminating OP9 cells from downstream assays. This method allows for the complete evaluation of all ES-derived progeny at all days of co-culture, resulting in a hematopoietic differentiation pattern, which more directly corresponds to the hematopoietic differentiation pattern observed within the embryo.

mStrawberry OP9 cells allow for complete evaluation of all ES-derived progeny from co-culture.

The in vitro differentiation of ES cells towards a hematopoietic cell fate is useful when studying cell populations that are difficult to access in vivo ...

Endothelial Cell Co-culture Mediates Maturation of Human Embryonic Stem Cell to Pancreatic Insulin Producing Cells in a Directed Differentiation Approach

Embryonic stem cells (ESC) have two main characteristics: they can be indefinitely propagated in vitro in an undifferentiated state and they are pluripotent, thus having the potential to differentiate into multiple lineages. Such properties make ESCs extremely attractive for cell based therapy and regenerative treatment applications 1. However for its full potential to be realized the cells have to be differentiated into mature and functional phenotypes, which is a daunting task. A promising approach in inducing cellular differentiation is to closely mimic the path of organogenesis in the in vitro setting. Pancreatic development is known to occur in specific stages 2, starting with endoderm, which can develop into several organs, including liver and pancreas. Endoderm induction can be achieved by modulation of the nodal pathway through addition of Activin A 3 in combination with several growth factors 4-7. Definitive endoderm cells then undergo pancreatic commitment by inhibition of sonic hedgehog inhibition, which can be achieved in vitro by addition of cyclopamine 8. Pancreatic maturation is mediated by several parallel events including inhibition of notch signaling; aggregation of pancreatic progenitors into 3-dimentional clusters; induction of vascularization; to name a few. By far the most successful in vitro maturation of ESC derived pancreatic progenitor cells have been achieved through inhibition of notch signaling by DAPT supplementation 9. Although successful, this results in low yield of the mature phenotype with reduced functionality. A less studied area is the effect of endothelial cell signaling in pancreatic maturation, which is increasingly being appreciated as an important contributing factor in in-vivo pancreatic islet maturation 10,11.
The current study explores such effect of endothelial cell signaling in maturation of human ESC derived pancreatic progenitor cells into insulin producing islet-like cells. We report a multi-stage directed differentiation protocol where the human ESCs are first induced towards endoderm by Activin A along with inhibition of PI3K pathway. Pancreatic specification of endoderm cells is achieved by inhibition of sonic hedgehog signaling by Cyclopamine along with retinoid induction by addition of Retinoic Acid. The final stage of maturation is induced by endothelial cell signaling achieved by a co-culture configuration. While several endothelial cells have been tested in the co-culture, herein we present our data with rat heart microvascular endothelial Cells (RHMVEC), primarily for the ease of analysis.

The current study describes a directed differentiation approach in inducing pancreatic differentiation of human embryonic stem cells. Of great significance is the finding that endothelial cell co-culture mediates maturation of human embryonic stem cell derived pancreatic progenitors into insulin expressing cells.