עבודה זו מומנה על ידי NIH (U54HD41857 ו PL1EB008542, P30 Biomaterials Core במסגרת קונסורציום Oncofertility מפת הדרכים מענק).

<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>

אין ניגודי אינטרסים הכריז.

זקיק הציגו השחלות אנקפסולציה השיטה FA-IPN מאפשר תרבות זקיק בסביבת 3D במבחנה. FA-IPN הוא דינאמי, תאים תגובה מטריצה ​​שבו תכונות מכניות הראשוני נקבעים על ידי שילוב של הפיברין והן אלגינט. במהלך התרבות, זקיק במארז מפעיל פרוטאזות כי לבזות רק מרכיב אחד של IPN, את הפיברין, שתוצ...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Fetuin</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich, St. Louis, MO</td>
<td>F3385</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Invitrogen, Gibco</td>
<td>10082-139</td>
<td>&nbsp;</td>
<tr>
<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>
</tr>
<tr>
<td>EGF</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A412</td>
<td>&nbsp;</td>
<tr>
<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>
<tr>
<td>Hyaluronidase</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A1603</td>
<td>&nbsp;</td>
<tr>
<td>ITS</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>I1884-1VL</td>
<td>&nbsp;</td>
<tr>
<td>L-15</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>11415</td>
<td>&nbsp;</td>
<tr>
<td>&alpha;MEM+Gluta MAX</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>32561</td>
<td>&nbsp;</td>
<tr>
<td>Pen-Strep</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>30-002-CI</td>
<td>&nbsp;</td>
<tr>
<td>TBS</td>
<td>&nbsp;</td>
<td>Pierce</td>
<td>28379</td>
<td>&nbsp;</td>
<tr>
<td>Tisseel Fibrin kit</td>
<td>&nbsp;</td>
<td>Baxter</td>
<td>921030</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Alginate</td>
<td>&nbsp;</td>
<td>FMC BioPolymers</td>
<td>LF200DL</td>
<td>Mw 418kDa</td>
</tr>		</tbody>
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a method for ovarian follicle encapsulation and culture in a proteolytically degradable 3 dimensional system

זקיק בשחלה היא היחידה תפקודית של השחלה כי מפריש הורמוני המין ותומך הבשלת הביצית. בטכניקות זקיק במבחנה לספק כלי להתפתחות זקיק מודל כדי לחקור את הביולוגיה הבסיסית, והם עוד יותר מפותח כטכניקה לשימור פוריות מרפאה 1-4. המערכת שלנו במבחנה תרבות מעסיקה הידרוג על מנת לחקות את הסביבה השחלות יליד ידי שמירה על ארכיטקטורת הזקיקים 3D, תאים תאים אינטראקציות איתות אוטוקריני כי התפתחות זקיק ישיר 5. בעבר, היו זקיקים תרבותי בהצלחה אלגינט, פוליסכריד אינרטי אצות נגזר כי עובר gelation עם יוני סידן 6-8. הידרוג אלגינט נוצר בריכוז של 0.25% w / v היו המתירני ביותר לתרבות זקיק, ושימר את יכולת ההתפתחות הגבוהה ביותר 9. הידרוג אלגינט לא מתכלה, ובכך להגדיל את קוטר תוצאות זקיק בכוח דחיסה על זקיק אשר יכול להשפיע על הצמיחה זקיק 10. לאחר מכן אנו פיתחה מערכת המבוססת על תרבות רשת הפיברין-אלגינט interpenetrating (FA-IPN), שבה תערובת של הפיברין וכן בג&#39;ל אלגינט הם בעת ובעונה אחת. שילוב זה מספק סביבת מכני דינמי כי שני הרכיבים לתרום קשיחות מטריצה ​​בתחילה, אולם פרוטאזות מופרש על ידי הזקיק גדל לבזות הפיברין במטריצה ​​עוזב אלגינט רק כדי לספק תמיכה. עם מהמכון נמסר, התוכן אלגינט יכול להיות מופחת מתחת 0.25%, דבר שאינו אפשרי עם אלגינט לבד 5. לכן, כפי הזקיק מתרחב, זה יהיה ניסיון כוח דחיסה מופחת עקב תוכן מוצקים מופחת. בזאת, אנו מתארים שיטה אנקפסולציה ומערכת במבחנה תרבות זקיקי השחלה בתוך FA-IPN. סביבת מכני דינמי מחקה את הסביבה הטבעית שבה השחלות זקיקים קטנים מתגוררים בקליפת נוקשה ולעבור לשד מתירני יותר כפי שהם גידול בגודל 11. רכיב מתכלה עשוי להיות קריטי במיוחד עבור תרגום קליניים כדי לתמוך יותר מ 10 6 פי הגידול בהיקף כי זקיקי אדם בדרך כלל עוברים in 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

שיטה חדשה אנקפסולציה השחלות זקיק ברשת 3D הפיברין-אלגינט interpenetrating מתואר. מערכת זו משלבת תמיכה מבנית עם השפלה פרוטאוליטי כדי לתמוך בפיתוח של זקיקים בשלים לייצר ביציות בוגרות. שיטה זו עשויה להיות מיושם אגרגטים תא התרבות לשמור תאים תאים מגעים ללא הגבלת ההתרחבות.

שיטה Encapsulation השחלות זקיק ותרבות מערכת 3 מתכלה Proteolytically מימדי

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

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Research

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Bioengineering

24.2K Views.  Northwestern University. שיטה חדשה אנקפסולציה השחלות זקיק ברשת 3D הפיברין-אלגינט interpenetrating מתואר. מערכת זו משלבת תמיכה מבנית עם השפלה פרוטאוליטי כדי לתמוך בפיתוח של זקיקים בשלים לייצר ביציות בוגרות. שיטה זו עשויה להיות מיושם אגרגטים תא התרבות לשמור תאים תאים מגעים ללא הגבלת ההתרחבות.

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.

קולגן אני Assay Organotypic: פלטפורמה חשילה לבחינת התנהגות תא בהקשר 3-Dimensional

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

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.

יצירת דו מימדי מצעים Patterned עבור החזקת חלבון ו-Cell

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.

טיפוח האדם ובתאים עצבית ב Hydrogel עצמית להרכבת 3 מימדי פפטיד

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.

פיגומים דיווח עצמי לתרבית תאים 3-Dimensional

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

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.

דור של עור בעובי מלא תלת ממדי Equivalent ואוטומטי פציעה

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.

שיטה של נחישות וסימולציות של חדירות ובהפצה במודל תלת-ממד רקמת קרום הוספת מערכת עבור לוחות רב טוב

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.

הייצור של חלבון כמו אלסטין Hydrogels כימוס, Immunostaining של תאים ב- 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.

חומצה היאלורונית מבוסס Hydrogels לתרבות תלת-ממדי של החולה-Derived גליובלסטומה תאים

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

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

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

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

שלושה תלת-ממד של מטריקס-Adipocyte מודל תרבות המודל ללימוד תא מטריקס-מטבולית הסלולר

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

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after chemotherapy or irradiation to treat bone marrow injury or in vitro culture. By mimicking the bone marrow microenvironment in the presence of abundant fatty acids, cholesterol, low concentration of cytokines, and hypoxia, human HSCs maintain quiescence in vitro. Here, a detailed protocol for maintaining functional HSCs in the quiescent state in vitro is described. This method will enable studies of the behavior of human HSCs under physiological conditions.

This protocol enables the maintenance of quiescent human hematopoietic stem cells in vitro by mimicking the bone marrow microenvironment using abundant fatty acids, cholesterol, lower concentrations of cytokines, and hypoxia.

שיטת תרבות לשמירה על תאי גזע ההמטוטופיאטיים האנושיים

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...