Martin Danner is acknowledged for his assistance in preparing samples and establishment of cell culture procedures. The authors would like to thank Biesterfeld Spezialchemie GmbH (Hamburg, Germany), especially Robert Radsziwill for continuous support and discussions. The research leading to these results has received funding from the European Research Council under the European Union&#39;s Seventh Framework Programme (FP/2007-2013) /ERC Grant Agreement n. 340929.

<ol>
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The authors have nothing to disclose.

The design of composite structures enables the simple adjustment of material properties, such as Young&#39;s modulus or the thickness of the samples. The Young&#39;s modulus of PDMS can be effectively changed in a wide range by either altering the mixing ratio between the two components or manufacturing of blends using a different silicone elastomer30,31. The described methods are not limited to the PDMS&#39;s used in the current investigation, but especially the adhesive performance depends strongly on the specific type used. A critical step within this protocol is the manufacturing process of the composite films (Figure 1). It was shown that thickness of the films affects significantly the adhesion behavior of the films on different substrates, including skin (Figure 5 and Figure 6). In addition to the film thickness, time and temperature during the curing process affects the material properties32. Therefore, parameters as the thickness of the polymeric layers have to be carefully adapted and verified.
Analysis of the adhesive properties of the thin films was performed with normal force adhesion measurements using two glass substrates with different surface roughness up to Ra = 0.338 &#181;m (Figure 3). In general, roughness impacts significantly the adhesion of surfaces, especially of elastic materials33,34. The roughness of glass can be easily varied by grinding with sandpaper of different asperity sizes, therefore allowing the fabrication of substrates exhibiting higher roughness values21. In addition, other materials, for example epoxy resin can be used for the production of substrates15,35. This might be an important modification strategy of the presented protocol. For example, if substrates exhibiting different surface free energies are needed or specific topographies are required. Here, pull-off stress and work of separation of the thin films manufactured of PDMS and SSA were analyzed with a custom-built setup (macroscopic adhesion measurement device (MAD, Figure 4)).36 Optical alignment of substrate and indenter is a critical step for the analysis of the measurement results. Therefore, adjustment of the tilt angle needs to be performed with the goniometer, as precise as possible. This can be achieved with sufficient precision by manually bringing the substrate into contact with the film surface until a horizontal contact is achieved.
In the current protocol the hold time was kept constant at one second (Figure 5 and Figure 7). Especially for the investigation of the adhesive performance of an elastic film to a rough substrate surface, an extension of the hold time provides additional information. For example, an increase in pull-off stress with increasing hold time has been reported8. In addition to the measurements performed in the current protocol, other methods, for example peel tests could be performed, allowing a more comprehensive investigation of adhesion performance37.
The adhesive properties of composite films exhibiting different film thicknesses of the soft skin adhesive were determined (Figure 7). Our results are in line with published data, showing that a decrease of film thickness lead to an increase of the pull-off stress as the confinement, i.e., the ratio between substrate diameter and film thickness, increases38,39. Based on these results and the data depicted in Figure 7, we conclude that composite films with a total thickness of approx. 100 &#181;m (a thickness of the SSA layer of approx. 60 &#181;m applied to a PDMS film with a thickness of approx. 40 &#181;m) exhibit favorable adhesion properties on rough surfaces.
Next, experiments related to the biological characterization have been performed on pristine composite films and plasma treated composite films (Figure 8). Plasma treatment of silicon elastomers is an often applied, versatile technique to increase the hydrophilic properties of surfaces and promoting cellular attachment and cellular spreading40,41. Silicones are well known for their low toxicity and high biostability but may contain residual monomers or catalysts which might influence physiological processes, leading also to cytotoxicity42,43. In the conducted experiments we have observed less than 5% cytotoxicity using LDH release as an indicator and a Trypan Blue exclusion assay. In the presented protocol, the entire cellular population, including cellular aggregates detached form the surface has been analyzed for proliferation analysis (Figure 9B). A modification of the protocol could produce more differentiated results. For each sample, the supernatant containing detached cellular aggregates could be transferred to a separate reaction tube and not combined with the cells enzymatically removed from the polymer surface. This would allow the exact assessment of cells attached to the surface and eventually reveal a more detailed determination of the influence of the polymers on the cellular adhesion process. In addition to the immunocytochemical methods presented here, cells might be harvested for investigation with immunoblot methods, allowing a detailed quantitative assessment of protein expression.
In summary, we have established manufacturing conditions for the production of thin elastomeric composite films for applications in advanced cell culture research. Additionally, these thin films possess high adaptability to skin roughness, enabling sophisticated design of skin adhesives.

In this protocol, detailed methods for manufacturing of thin elastomer films are presented. The widely available doctor blade technique has been used for the production of thin composite films. The manufacturing technique has been performed on polyethylenterephtalate (PET) foils, enabling subsequent production of these films in large scale. Emphasis of this protocol is the assessment of reproducibility, precise manufacturing of the different layers of the composite films and determination of the biological and adhesion properties of the final composite patch. The silicone elastomer poly-(dimethylsiloxane) (PDMS) is extensively used in biomedical technology, including production of skin adhesives, microfluidics applications and additional research fields1,2,3,4. Recently, another subclass of PDMS, so called Soft Skin Adhesives (SSAs) have been introduced, particular for gentle skin bonding and de-bonding.
Silicone SSAs are vinyl functionalized elastomers, differing from analogous polymers by the absence of reinforcing silica5. Similar to other PDMS, SSA&#39;s Young&#39;s modulus can be adapted in a wide range by modulating cross-linker concentration or curing time6,7,8. This change in Young&#39;s modulus of silicone elastomers affects the adhesive properties of the material significantly and has also profound consequences on prokaryotic and eukaryotic cells cultured on the surface9,10,11. On the cellular biological level, it was shown, that eukaryotic cells respond on the signal transduction level to a modulation of the matrix elasticity or thickness of the surface9,10,12,13,14. Therefore, a broad interest in cell culture applications of polymers with tunable mechanical properties exist. Importantly, the intrinsically low surface energy of silicone based elastomers does not provide optimal conditions for cell culture of eukaryotic cells. Oxygen plasma treatment is a widely used technique to increase PDMS low surface energy temporarily, leading to an enhancement of its pull-off strength, decreased surface adsorption of molecules, while in parallel promoting attachment, spreading and proliferation of eukaryotic cells15,16,17,18.
In addition to the materials properties, the surface topography significantly affects cellular adhesion and the adhesive interaction between two materials19,20,21,22. Surface roughness has several effects on the contact formation between two surfaces: reduction of the contact area, high stored elastic energy surrounding asperities as well as influence on the crack propagation can alter the adhesive strength23,24. Adhesion of self-adhesive films to human skin is an emerging application field, e.g., wound dressings, fixation of ECG electrodes or other wearable electronic devices25,26,27,28. To measure the adhesive performance of self-adhesives in relation to surface topography, glass substrates with varying degrees of roughness can be used in normal adhesion measurements8,21. Here, two glass substrates have been selected to investigate the adhesive properties of the polymer films. First, composite films with a PDMS backing layer in a mixing ratio of 10 to 1 weight parts covered by PDMS with different mixing ratio were characterized. In a second step a adhesive SSA layer was prepared with equal weight amounts of both components and with varying film thickness on top of a supporting PDMS film.

<table>
	<tbody>
		<tr>
			<td>2-Propanol, 97%</td>
			<td>Stockmeier Chemie</td>
			<td>1000452610000</td>
			<td>Isopropanol</td>
		</tr>
		<tr>
			<td>Abrasive diamnod hand pad</td>
			<td>Bohle</td>
			<td>MO 5007522</td>
			<td>Grit: 220</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Capricorn Scientific</td>
			<td>ACC-1B</td>
			<td></td>
		</tr>
		<tr>
			<td>Albumin Fraktion V</td>
			<td>Roth</td>
			<td>0163.2</td>
			<td>BSA</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Phalloidin</td>
			<td>ThermoFischer Scientific</td>
			<td>A12379</td>
			<td>highly toxic</td>
		</tr>
		<tr>
			<td>Aquamount</td>
			<td>Polysciences</td>
			<td>18606-20</td>
			<td>water soluble mounting medium</td>
		</tr>
		<tr>
			<td>CytoTox-ONE Homogeneous Membrane Integrity Assay</td>
			<td>Promega</td>
			<td>G7890</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS, without Ca2+, Mg2+</td>
			<td>ThermoFischer Scientific</td>
			<td>14190094</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum gold</td>
			<td>GE Health Care Life Science</td>
			<td>A15-151</td>
			<td>FBS</td>
		</tr>
		<tr>
			<td>Goniometer OCA35</td>
			<td>Dataphysics</td>
			<td></td>
			<td>for the determination of the static water contact angle</td>
		</tr>
		<tr>
			<td>Hoechst Dye 33342</td>
			<td>Sigma-Aldrich</td>
			<td>B1155-100MG</td>
			<td>bisBenzimide H 33342 trihydrochloride, highly toxic</td>
		</tr>
		<tr>
			<td>Microscope Axiovert 25</td>
			<td>Zeiss</td>
			<td></td>
			<td>Microscope used for cell culture documentation</td>
		</tr>
		<tr>
			<td>Microscope Eclipse LV100ND</td>
			<td>Nikon</td>
			<td></td>
			<td>Microscope used for film thickness determination</td>
		</tr>
		<tr>
			<td>Paraformaldehyde, aqueous solution 16%</td>
			<td>Electron Microscopy Sciences</td>
			<td>RT 15710</td>
			<td>electron microscopy grade</td>
		</tr>
		<tr>
			<td>penicillin und streptomycin solution</td>
			<td>Sigma-Aldrich</td>
			<td>P4333-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenom XL Scanning Electron Microscope (SEM)</td>
			<td>Phenom</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-(vinyl alcohol) 4-88, MW 31000</td>
			<td>Sigma-Aldrich</td>
			<td>81381-1KG</td>
			<td>Mowiol 4-88</td>
		</tr>
		<tr>
			<td>Poly-dimethyl siloxanes, Sylgard 184</td>
			<td>Dow Corning</td>
			<td>(400)000108351397</td>
			<td>PDMS</td>
		</tr>
		<tr>
			<td>RPMI 1640 basal medium</td>
			<td>ThermoFischer Scientific</td>
			<td>21875034</td>
			<td></td>
		</tr>
		<tr>
			<td>soft skin adhesive (SSA)</td>
			<td>Dow Corning</td>
			<td>(400)000108251792</td>
			<td>MG 7-9800 Soft Skin Adhesive (SSA)</td>
		</tr>
		<tr>
			<td>speed mixer DAC 600.2 VAC-P</td>
			<td>Hauschild</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>stylus profilomter</td>
			<td>Zeiss</td>
			<td></td>
			<td>Model: SURFCOM 1500SD3</td>
		</tr>
		<tr>
			<td>Tecan Infinite M200 pro</td>
			<td>Tecan</td>
			<td></td>
			<td>fluorescence plate reader</td>
		</tr>
		<tr>
			<td>Triton X 100</td>
			<td>Calbiochem</td>
			<td>648466</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue solution</td>
			<td>Sigma-Aldrich</td>
			<td>T8154-100ML</td>
			<td>highly toxic</td>
		</tr>
		<tr>
			<td>Trypsin/EDTA solution</td>
			<td>PAN-Biotech</td>
			<td>P10-023500</td>
			<td>0.05% Trypsin, 0.02% EDTA in PBS</td>
		</tr>
		<tr>
			<td>UV glue</td>
			<td>Bohle</td>
			<td>BO MV76002</td>
			<td>medium viscosity</td>
		</tr>
	</tbody>
</table>

thin film composite silicon elastomers for cell culture and skin applications: manufacturing and characterization

In this protocol, we present methods to fabricate thin elastomer composite films for advanced cell culture applications and for the development of skin adhesives. Two different poly-(dimethyl siloxanes) (PDMS and soft skin adhesive (SSA)), have been used for in depth investigation of biological effects and adhesive characteristics. The composite films consist of a flexible backing layer and an adhesive top coating. Both layers have been manufactured by doctor blade application technique. In the present investigation, the adhesive behavior of the composite films has been investigated as a function of the layer thickness or a variation of the Young&#39;s modulus of the top layer. The Young&#39;s modulus of PDMS has been changed by varying the base to crosslinker mixing ratio. In addition, the thickness of SSA films has been varied from approx. 16 &#181;m to approx. 320 &#181;m. Scanning electron microscopy (SEM) and optical microscopy have been used for thickness measurements. The adhesive properties of elastomer films depend strongly on the film thickness, the Young&#39;s modulus of the polymers and surface characteristics. Therefore, normal adhesion of these films on glass substrates exhibiting smooth and rough surfaces has been investigated. Pull-off stress and work of separation are dependent on the mixing ratio of silicone elastomers.
Additionally, the thickness of the soft skin adhesive placed on top of a supportive backing layer has been varied in order to produce patches for skin applications. Cytotoxicity, proliferation and cellular adhesion of L929 murine fibroblasts on PDMS films (mixing ratio 10:1) and SSA films (mixing ratio 50:50) have been conducted. We have shown here, for the first time, the side by side comparison of thin composite films manufactured of both polymers and present the investigation of their biological- and adhesive properties.

Watch this Scientific Journal Video about Thin Film Composite Silicon Elastomers for Cell Culture and Skin Applications: Manufacturing and Characterization at JoVE.com

Thin Film Composite Silicon Elastomers for Cell Culture and Skin Applications: Manufacturing and Characterization

A protocol for the manufacturing process of polymeric thin film composite structures possessing either different Young&#39;s moduli or thicknesses is presented. Films are produced for advanced cell culture studies or as skin adhesives.

In this protocol, we present methods to fabricate thin elastomer composite films for advanced cell culture applications and for the development of skin ...

thin-film-composite-silicon-elastomers-for-cell-culture-skin

Research

JoVE Journal

Developmental Biology

10.5K Views.  INM - Leibniz Institute for New Materials. A protocol for the manufacturing process of polymeric thin film composite structures possessing either different Young's moduli or thicknesses is presented. Films are produced for advanced cell culture studies or as skin adhesives.

Video: Thin Film Composite Silicon Elastomers for Cell Culture and Skin Applications: Manufacturing and Characterization

Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS expression system1 in zebrafish larvae, and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells. The KalTA4-ERT2/UAS system is both inducible and reversible which allows us to induce cell transformation with precise temporal/spatial resolution in vivo. This provides us with a unique opportunity to live image how individual preneoplastic cells interact with host tissues as soon as they emerge, then follow their progression as well as regression. Recent studies in zebrafish larvae have shown a trophic function of innate immunity in the earliest stages of tumorigenesis2,3. Our inducible system would allow us to live image the onset of cellular transformation and the subsequent host response, which may lead to important insights on the underlying mechanisms for the regulation of oncogenic trophic inflammatory responses. We also discuss how one might adapt our protocol to achieve temporal and spatial control of ectopic gene expression in any tissue of interest.

Studying the earliest events of preneoplastic cell progression and innate immune cell interaction is pivotal to understand and treat cancer. Here we describe a method to conditionally induce epithelial cell transformations and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells in zebrafish larvae.

Here we describe a method to conditionally induce epithelial cell transformation by the use of the 4-Hydroxytamoxifen (4-OHT) inducible KalTA4-ERT2/UAS ...

Culture and Co-Culture of Mouse Ovaries and Ovarian Follicles

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together within a basement membrane. A finite pool of follicles is laid down during embryonic development, when oocytes in meiotic arrest form a close association with flattened granulosa cells, forming primordial follicles. By or shortly after birth, mammalian ovaries contain their lifetime&#8217;s supply of primordial follicles, from which point onwards there is a steady release of follicles into the growing follicular pool.
The ovary is particularly amenable to development in vitro, with follicles growing in a highly physiological manner in culture. This work describes the culture of whole neonatal ovaries containing primordial follicles, and the culture of individual ovarian follicles, a method which can support the development of follicles from an immature through to the preovulatory stage, after which their oocytes are able to undergo fertilization in vitro. The work outlined here uses culture systems to determine how the ovary is affected by exposure to external compounds. We also describe a co-culture system, which allows investigation of the interactions that occur between growing follicles and the non-growing pool of primordial follicles.

This protocol describes the primary culture/co-culture of mouse ovarian tissue, using ovaries from neonatal mice and individual ovarian follicles from prepubertal mice. The culture techniques support development in a highly physiological manner, allowing investigation of the effect of extrinsic agents on the ovary, and of interactions between ovarian follicles.

The mammalian ovary is composed of ovarian follicles, each follicle consisting of a single oocyte surrounded by somatic granulosa cells, enclosed together ...

Transfection, Selection, and Colony-picking of Human Induced Pluripotent Stem Cells TALEN-targeted with a GFP Gene into the AAVS1 Safe Harbor

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral vector mediated random integration. This protocol describes a universal and efficient transgene targeted addition platform in human iPSCs based on utilization of validated open-source TALENs and a gene-trap-like donor to deliver transgenes into a safe harbor locus. Importantly, effective gene editing is rate-limited by the delivery efficiency of gene editing vectors. Therefore, this protocol first focuses on preparation of iPSCs for transfection to achieve high nuclear delivery efficiency. When iPSCs are dissociated into single cells using a gentle-cell dissociation reagent and transfected using an optimized program, >50% cells can be induced to take up the large gene editing vectors. Because the AAVS1 locus is located in the intron of an active gene (PPP1R12C), a splicing acceptor (SA)-linked puromycin resistant gene (PAC) was used to select targeted iPSCs while excluding random integration-only and untransfected cells. This strategy greatly increases the chance of obtaining targeted clones, and can be used in other active gene targeting experiments as well. Two weeks after puromycin selection at the dose adjusted for the specific iPSC line, clones are ready to be picked by manual dissection of large, isolated colonies into smaller pieces that are transferred to fresh medium in a smaller well for further expansion and genetic and functional screening. One can follow this protocol to readily obtain multiple GFP reporter iPSC lines that are useful for in vivo and in vitro imaging and cell isolation.

TALEN-mediated gene editing at the safe harbor AAVS1 locus enables high-efficiency transgene addition in human iPSCs. This protocol describes the procedures for preparing iPSCs for TALEN and donor vector delivery, transfecting iPSCs, and selecting and isolating iPSC clones to achieve targeted integration of a GFP gene to generate reporter lines.

Targeted transgene addition can provide persistent gene expression while circumventing the gene silencing and insertional mutagenesis caused by viral ...

3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse this scaffold in a highly coordinated temporal and spatial order while sequentially passing obligatory check points, i.e., T cell lineage commitment, followed by T cell receptor repertoire generation and selection prior to their export into the periphery. The two major resident cell types forming this scaffold are cortical (cTECs) and medullary thymic epithelial cells (mTECs). A key feature of mTECs is the so-called promiscuous expression of numerous tissue-restricted antigens. These tissue-restricted antigens are presented to immature thymocytes directly or indirectly by mTECs or thymic dendritic cells, respectively resulting in self-tolerance.
Suitable in vitro models emulating the developmental pathways and functions of cTECs and mTECs are currently lacking. This lack of adequate experimental models has for instance hampered the analysis of promiscuous gene expression, which is still poorly understood at the cellular and molecular level. We adapted a 3D organotypic co-culture model to culture ex vivo isolated mTECs. This model was originally devised to cultivate keratinocytes in such a way as to generate a skin equivalent in vitro. The 3D model preserved key functional features of mTEC biology: (i) proliferation and terminal differentiation of CD80lo, Aire-negative into CD80hi, Aire-positive mTECs, (ii) responsiveness to RANKL, and (iii) sustained expression of FoxN1, Aire and tissue-restricted genes in CD80hi mTECs.

Studying medullary thymic epithelial cells in vitro has been largely unsuccessful, as current 2D culture systems do not mimic the in vivo scenario. The 3D culture system described herein - a modified skin organotypic culture model - has proven superior in recapitulating mTEC proliferation, differentiation and maintenance of promiscuous gene expression.

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse ...

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head and heart progenitor cell development with pharyngeal arches (also known as branchial arches) ex vivo. Using this method, we have recently described that the second pharyngeal arch contains self-renewing heart progenitors and serves as a microenvironment for expansion of the progenitors during mouse heart development. The progenitor cells remain undifferentiated and expansive inside the arch, but quickly become functional cardiomyocytes as they migrate out of the arch. We also reported that first pharyngeal arch contains muscle progenitors giving rise to myotubes after leaving the arch. Here, we demonstrate the procedure for the dissection and ex vivo culture of first and second pharyngeal arches from developing mouse embryos. The method enables one to study head and heart progenitor/muscle development, including cardiomyocyte and myotube formation in detail ex vivo.

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

Here, we present a protocol to culture pharyngeal arches to study the biology of heart and muscle progenitor cells and their microenvironment.

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head ...

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy.

Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Epigenetic Conversion as a Safe and Simple Method to Obtain Insulin-secreting Cells from Adult Skin Fibroblasts

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such cells are not readily available, they can be obtained using different approaches that include, among the many, reprogramming and trans-differentiation, with advantages and limitations that are specific of the different techniques. Here a new strategy for the conversion of an adult mature fibroblast into an insulin-secreting cell, arbitrarily designated as epigenetic converted cells (EpiCC), is described. The method has been developed, based on the increasing understanding of the mechanisms controlling epigenetic regulation of cell fate and differentiation. In particular, the first step uses an epigenetic modifier, namely 5-aza-cytidine, to drive adult cells into a "highly permissive" state. It then takes advantage of this brief and reversible window of epigenetic plasticity, to re-address cells toward a different lineage. The approach is designated "epigenetic cell conversion". It is a simple and robust way to obtain an efficient, controlled and stable cellular inter-lineage switch. Since the protocol does not involve the use of any gene transfection, it is free of viral vectors and does not involve a stable pluripotent state, it is highly promising for translational medicine applications.

Here, a new method that allows the conversion of adult skin fibroblasts into insulin-secreting cells is presented. This technique is based on epigenetic conversion, does not involve the use of retroviral vectors nor the acquisition of a stable pluripotent state. It is therefore highly promising for translational medicine applications.

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...