Este trabajo fue financiado por el NIH (U54HD41857 y PL1EB008542, un procesador Core P30 Biomateriales en el Consorcio Oncofertility conceder Hoja de Ruta).

<ol>
	<li>Cortvrindt, R., Smitz, J., VanSteirteghem, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-vitro+maturation,+fertilization+and+embryo+development+of+immature+oocytes+from+early+preantral+follicles+from+prepuberal+mice+in+a+simplified+culture+system.">In-vitro maturation, fertilization and embryo development of immature oocytes from early preantral follicles from prepuberal mice in a simplified culture system.</a> Human Reproduction. 11, 2656-2666 (1996).</li><li>Smitz, J., Cortvrindt, R., Hu, Y. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+growth+factor+combined+with+recombinant+human+chorionic+gonadotrophin+improves+meiotic+progression+in+mouse+follicle-enclosed+oocyte+culture.">Epidermal growth factor combined with recombinant human chorionic gonadotrophin improves meiotic progression in mouse follicle-enclosed oocyte culture.</a> Human Reproduction. 13, 664-669 (1998).</li><li>Xu, M., Banc, A., Woodruff, T. K., Shea, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secondary+Follicle+Growth+and+Oocyte+Maturation+by+Culture+in+Alginate+Hydrogel+Following+Cryopreservation+of+the+Ovary+or+Individual+Follicles.">Secondary Follicle Growth and Oocyte Maturation by Culture in Alginate Hydrogel Following Cryopreservation of the Ovary or Individual Follicles.</a> Biotechnology and Bioengineering. 103, 378-386 (2009).</li><li>Smitz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+achievements+and+future+research+directions+in+ovarian+tissue+culture,+in+vitro+follicle+development+and+transplantation:+implications+for+fertility+preservation.">Current achievements and future research directions in ovarian tissue culture, in vitro follicle development and transplantation: implications for fertility preservation.</a> Human Reproduction Update. 16, 395-414 (2010).</li><li>Shikanov, A., Xu, M., Woodruff, T. K., Shea, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interpenetrating+fibrin-alginate+matrices+for+in+vitro+ovarian+follicle+development.">Interpenetrating fibrin-alginate matrices for in vitro ovarian follicle development.</a> Biomaterials. 30, 5476-5485 (2009).</li><li>West, E. R., Xu, M., Woodruff, T. K., Shea, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+properties+of+alginate+hydrogels+and+their+effects+on+in+vitro+follicle+development.">Physical properties of alginate hydrogels and their effects on in vitro follicle development.</a> Biomaterials. 28, 4439-4448 (2007).</li><li>Kreeger, P. K., Fernandes, N. N., Woodruff, T. K., Shea, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+mouse+follicle+development+by+follicle-stimulating+hormone+in+a+three-dimensional+in+vitro+culture+system+is+dependent+on+follicle+stage+and+dose.">Regulation of mouse follicle development by follicle-stimulating hormone in a three-dimensional in vitro culture system is dependent on follicle stage and dose.</a> Biology of Reproduction. 73, 942-950 (2005).</li><li>Pangas, S. A., Saudye, H., Shea, L. D., Woodruff, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+approach+for+the+three-dimensional+culture+of+granulosa+cell-oocyte+complexes.">Novel approach for the three-dimensional culture of granulosa cell-oocyte complexes.</a> Tissue Engineering. 9, 1013-1021 (2003).</li><li>Xu, M., West, E., Shea, L. D., Woodruff, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+stage-specific+permissive+in+vitro+culture+environment+for+follicle+growth+and+oocyte+development.">Identification of a stage-specific permissive in vitro culture environment for follicle growth and oocyte development.</a> Biology of Reproduction. 75, 916-923 (2006).</li><li>Xu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encapsulated+Three-Dimensional+Culture+Supports+Development+of+Nonhuman+Primate+Secondary+Follicles.">Encapsulated Three-Dimensional Culture Supports Development of Nonhuman Primate Secondary Follicles.</a> Biology of Reproduction. 81, 587-594 (2009).</li><li>West, E. R., Shea, L. D., Woodruff, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+the+follicle+micro+environment.">Engineering the follicle micro environment.</a> Seminars in Reproductive Medicine. 25, 287-299 (2007).</li><li>Xu, M., Kreeger, P. K., Shea, L. D., Woodruff, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-engineered+follicles+produce+live,+fertile+offspring.">Tissue-engineered follicles produce live, fertile offspring.</a> Tissue Engineering. 12, 2739-2746 (2006).</li><li>Ebisch, I. M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+the+role+of+the+plasminogen+activator+system+and+vascular+endothelial+growth+factor+in+subfertility.">Review of the role of the plasminogen activator system and vascular endothelial growth factor in subfertility.</a> Fertility and Sterility. 90, 2340-2350 (2008).</li></ol>

El folículo ovárico presenta un método de encapsulación en FA-IPN permite cultivo de folículos en un entorno 3D in vitro. A FA-IPN es un proceso dinámico, de células sensibles a la matriz en la que las propiedades mecánicas iniciales están determinadas por la combinación de ambos fibrina y alginato. Durante el cultivo, el folículo encapsulado activa proteasas que degradan el único componente de la IPN, la fibrina, que se traduce en una rigidez gel disminuyendo gradualmente que es aportado exclusivam...

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		<th>Comment</th>
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<td>Fetuin</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich, St. Louis, MO</td>
<td>F3385</td>
<td>&nbsp;</td>
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<td>FBS</td>
<td>&nbsp;</td>
<td>Invitrogen, Gibco</td>
<td>10082-139</td>
<td>&nbsp;</td>
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<td>Aprotinin</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>10236624001</td>
<td>&nbsp;</td>
<tr>
<td>CaCl2</td>
<td>&nbsp;</td>
<td>Wako</td>
<td>039-00475</td>
<td>40 mM</td>
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<td>EGF</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A412</td>
<td>&nbsp;</td>
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<td>rFSH</td>
<td>&nbsp;</td>
<td>A.F. Parlow, National Hormone and Peptide Program, National Institute of Diabetes and Digestive and Kidney Diseases</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>hCG</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>CG-5</td>
<td>&nbsp;</td>
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<td>Hyaluronidase</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A1603</td>
<td>&nbsp;</td>
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<td>ITS</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>I1884-1VL</td>
<td>&nbsp;</td>
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<td>L-15</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>11415</td>
<td>&nbsp;</td>
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<td>&alpha;MEM+Gluta MAX</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>32561</td>
<td>&nbsp;</td>
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<td>Pen-Strep</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>30-002-CI</td>
<td>&nbsp;</td>
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<td>TBS</td>
<td>&nbsp;</td>
<td>Pierce</td>
<td>28379</td>
<td>&nbsp;</td>
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<td>Tisseel Fibrin kit</td>
<td>&nbsp;</td>
<td>Baxter</td>
<td>921030</td>
<td>&nbsp;</td>
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<td>Sodium Alginate</td>
<td>&nbsp;</td>
<td>FMC BioPolymers</td>
<td>LF200DL</td>
<td>Mw 418kDa</td>
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a method for ovarian follicle encapsulation and culture in a proteolytically degradable 3 dimensional system

El folículo ovárico es la unidad funcional del ovario que segrega las hormonas sexuales y apoya la maduración de ovocitos. En las técnicas in vitro de folículos proporcionar una herramienta para el desarrollo del folículo modelo para investigar la biología básica, y además se está desarrollando como una técnica para preservar la fertilidad en el clínica 1-4. Nuestro sistema de cultivo in vitro cuenta con hidrogeles con el fin de imitar el ambiente de ovario nativos mediante el mantenimiento de la arquitectura folicular en 3D, interacciones célula-célula y la señalización paracrina que el desarrollo del folículo directa 5. Anteriormente, los folículos se cultivaron con éxito en alginato, un derivado de algas inertes polisacárido que se somete a la gelificación de los iones de calcio 6-8. Hidrogeles de alginato forma a una concentración de 0,25% w / v fueron las más permisivas de la cultura del folículo, y mantiene la más alta competencia en el desarrollo 9. Hidrogeles de alginato no son degradables, por lo tanto un aumento en los resultados de folículos de diámetro en una fuerza de compresión en el folículo que pueden afectar el crecimiento del folículo 10. Hemos desarrollado posteriormente un sistema de cultivo basado en una red de fibrina, alginato de interpenetración (FA-IPN), en el que se gelifican una mezcla de fibrina y alginato de forma simultánea. Esta combinación proporciona un entorno dinámico mecánico debido a que ambos componentes contribuyen a la rigidez de la matriz inicial, sin embargo, las proteasas secretado por el folículo creciente degradación de fibrina en la matriz de alginato dejando sólo para brindar apoyo. Con el IPN, el contenido de alginato se puede reducir por debajo del 0,25%, lo que no es posible con alginato solo 5. Por lo tanto, cuando el folículo se expande, se experimentará una fuerza de compresión reducida debido a la reducida contenido de sólidos. En este documento, se describe un método de encapsulación y un sistema de cultivo in vitro de los folículos ováricos en un FA-IPN. El entorno mecánico dinámico simula el entorno del ovario natural en el que residen los pequeños folículos en la corteza rígida y pasar a una médula más permisiva, ya que aumentan de tamaño 11. El componente degradable puede ser particularmente importante para la traducción clínica con el fin de apoyar el mayor de 10 en 6 veces el aumento en el volumen que los folículos humanos normalmente pasan en vivo.

Watch this Scientific Journal Video about A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System at JoVE.com

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

Un nuevo método para la encapsulación de los folículos ováricos en 3D de fibrina-alginato red interpenetrante se describe. Este sistema combina un soporte estructural a la degradación proteolítica de apoyar el desarrollo de folículos inmaduros para producir ovocitos maduros. Este método puede ser aplicado a los agregados de cultivo celular para mantener contactos célula-célula, sin limitar la expansión.

Un método para la encapsulación de los folículos ováricos y la Cultura en un proteolíticamente degradables tres dimensiones del sistema

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

a-method-for-ovarian-follicle-encapsulation-culture-proteolytically

Research

JoVE Journal

Bioengineering

24.2K vistas.  Northwestern University. Un nuevo método para la encapsulación de los folículos ováricos en 3D de fibrina-alginato red interpenetrante se describe. Este sistema combina un soporte estructural a la degradación proteolítica de apoyar el desarrollo de folículos inmaduros para producir ovocitos maduros. Este método puede ser aplicado a los agregados de cultivo celular para mantener contactos célula-célula, sin limitar la expansión.

Video: A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

El colágeno organotípicos I del ensayo: Una plataforma maleable para evaluar el comportamiento celular en un contexto de 3 dimensiones

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

Investigación

Bioingeniería

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

La creación de dos dimensiones con dibujos sustratos para el confinamiento de proteínas y células

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

Cultivo de Células progenitoras neurales en un hidrogel de péptidos 3-dimensional de auto-montaje

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

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

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

Los andamios de autoinforme para el 3-Dimensional Cultivo Celular

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

A Novel Method for Localizing Reporter Fluorescent Beads Near the Cell Culture Surface for Traction Force Microscopy

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When the substrate is coated with an extracellular matrix protein, cells adhere to the gel and apply forces, causing the gel to deform. The deformation depends on the cell traction and the elastic properties of the gel. If the deformation field of the surface is known, surface traction can be calculated using elasticity theory. Gel deformation is commonly measured by embedding fluorescent marker beads uniformly into the gel. The probes displace as the gel deforms. The probes near the surface of the gel are tracked. The displacements reported by these probes are considered as surface displacements. Their depths from the surface are ignored. This assumption introduces error in traction force evaluations. For precise measurement of cell forces, it is critical for the location of the beads to be known. We have developed a technique that utilizes simple chemistry to confine fluorescent marker beads, 0.1 and 1 &#181;m in diameter, in PA gels, within 1.6 &#956;m of the surface. We coat a coverslip with poly-D-lysine (PDL) and fluorescent beads. PA gel solution is then sandwiched between the coverslip and an adherent surface. The fluorescent beads transfer to the gel solution during curing. After polymerization, the PA gel contains fluorescent beads on a plane close to the gel surface.

Traditional techniques for fabricating polyacrylamide (PA) gels containing fluorescent probes involve sandwiching a gel between an adherent surface and a glass slide. Here, we show that coating this slide with poly-D-lysine (PDL) and fluorescent probes localizes the probes to within 1.6 &#181;m from the gel surface.

Un nuevo método para la localización de cuentas fluorescentes Reporter cerca de la superficie de la célula de la cultura para Microscopía de Fuerza de Tracción

PA gels have long been used as a platform to study cell traction forces due to ease of fabrication and the ability to tune their elastic properties. When ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

Generación de una tridimensional grosor de la piel completa y automatizada Equivalente Heridas

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

A Method for Determination and Simulation of Permeability and Diffusion in a 3D Tissue Model in a Membrane Insert System for Multi-well Plates

In vitro cultivated skin models have become increasingly relevant for pharmaceutical and cosmetic applications, and are also used in drug development as well as substance testing. These models are mostly cultivated in membrane-insert systems, their permeability toward different substances being an essential factor. Typically, applied methods for determination of these parameters usually require large sample sizes (e.g., Franz diffusion cell) or laborious equipment (e.g., fluorescence recovery after photobleaching (FRAP)). This study presents a method for determining permeability coefficients directly in membrane-insert systems with diameter sizes of 4.26 mm and 12.2 mm (cultivation area). The method was validated with agarose and collagen gels as well as a collagen cell model representing skin models. The permeation processes of substances with different molecular sizes and permeation through different cell models (consisting of collagen gel, fibroblast, and HaCaT) were accurately described.
Moreover, to support the above experimental method, a simulation was established. The simulation fits the experimental data well for substances with small molecular size, up to 14 x 10-10 m Stokes radius (4,000 MW), and is therefore a promising tool to describe the system. Furthermore, the simulation can considerably reduce experimental efforts and is robust enough to be extended or adapted to more complex setups.

A method for determination of permeability in a membrane insert system for multi-well plates and in silico parameter optimization for the calculation of diffusion coefficients using simulation are presented.

Un método para la determinación y simulación de permeabilidad y difusión en un modelo de tejido 3D en una membrana Introduzca el sistema de placas de varios pocillos

In vitro cultivated skin models have become increasingly relevant for pharmaceutical and cosmetic applications, and are also used in drug development as ...

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.

Recombinant protein-engineered hydrogels are advantageous for 3D cell culture as they allow for complete tunability of the polymer backbone and therefore, the cell microenvironment. Here, we describe the process of recombinant elastin-like protein purification and its application in 3D hydrogel cell encapsulation.

Producción de la proteína elastina como hidrogeles para encapsulación y el Immunostaining de células en 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue ...

Hyaluronic-Acid Based Hydrogels for 3-Dimensional Culture of Patient-Derived Glioblastoma Cells

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular matrix (ECM) of the unique brain environment, such as hyaluronic acid (HA), facilitates GBM progression and invasion. However, most in vitro culture models include GBM cells outside of the context of an ECM. Murine xenografts of GBM cells are used commonly as well. However, in vivo models make it difficult to isolate the contributions of individual features of the complex tumor microenvironment to tumor behavior. Here, we describe an HA hydrogel-based, three-dimensional (3D) culture platform that allows researchers to independently alter HA concentration and stiffness. High molecular weight HA and polyethylene glycol (PEG) comprise hydrogels, which are crosslinked via Michael-type addition in the presence of live cells. 3D hydrogel cultures of patient-derived GBM cells exhibit viability and proliferation rates as good as, or better than, when cultured as standard gliomaspheres. The hydrogel system also enables incorporation of ECM-mimetic peptides to isolate effects of specific cell-ECM interactions. Hydrogels are optically transparent so that live cells can be imaged in 3D culture. Finally, HA hydrogel cultures are compatible with standard techniques for molecular and cellular analyses, including PCR, Western blotting and cryosectioning followed by immunofluorescence staining.

Here, we present a protocol for three-dimensional culture of patient-derived glioblastoma cells within orthogonally tunable biomaterials designed to mimic the brain matrix. This approach provides an in vitro, experimental platform that maintains many characteristics of in vivo glioblastoma cells typically lost in culture.

Ácido hialurónico basado en hidrogeles de 3 dimensiones cultura de células de Glioblastoma derivados del paciente

Glioblastoma (GBM) is the most common, yet most lethal, central nervous system cancer. In recent years, many studies have focused on how the extracellular ...

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

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

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

Construcción de una hoja de varias capas células madre mesenquimales con un sistema de cultivo dinámico 3D

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 ...