Name: generation of genetically modified organotypic skin cultures using devitalized human dermis
Uploaded: 2015-12-14T14:00:00.000+00:00
Duration: 9 min 16 s
Description: Watch this Scientific Journal Video about Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis at JoVE.com

Questo lavoro è stato supportedby l&#39;American Cancer Society Research Scholars Grant (RSG-12-148-01-DDC) e il Premio Biologia CIRM di base (RB4-05779).

Il protocollo pelle umana è stata effettuata in conformità con le linee guida della University of California, Ricerca Comitato Etico di San Diego. La pelle umana può essere ottenuto da campioni chirurgici scartati o comprato da banche della pelle (pelle banca è elencato nella tabella Materiali / Equipment). La posizione in cui la pelle è realizzata o età del donatore non è critico per l&#39;esperimento finché le proteine ​​membrana basale (collagene / laminina) nel derma non sono degradati. 1. Preparazione di devitalizzate derma umano <ol><li> Alla ricezione di pelle umana, posizionare il tessuto nella cappa coltura tissutale per scongelare (~ 3-5 min). A seconda delle dimensioni della pelle, posizionare il tessuto scongelato in un monouso tubo da 50 ml o 125 ml sterile bottiglia. La dimensione tipica della pelle da parte della banca pelle è di 0,75 mq. </li><li> Riempire il serbatoio pieno al 75% con 4x la penicillina e streptomicina (penna / strep) in PBS 1x. Agitare energicamente per5 min e smaltire il liquido. Ripetere questa operazione altre 3 volte. </li><li> Dopo l&#39;ultimo lavaggio, trasferire il tessuto in una nuova provetta / bottiglia e aggiungere 4x fresco pen / strep / PBS per riempire 75% del contenitore. Incubare questa miscela in un incubatore di coltura tissutale a 37 ° C per 2 settimane per consentire la separazione del derma dell&#39;epidermide. </li><li> In condizioni sterili, utilizzare una pinza a staccarsi l&#39;epidermide dal derma. Il derma è completamente bianco, mentre l&#39;epidermide avranno colorazione a seconda della razza del donatore (Figura 1A). Eliminare l&#39;epidermide secondo il protocollo dell&#39;istituzione per affrontare con i tessuti umani. </li><li> Posizionare il derma in un tubo o bottiglia e lavarlo in 4x penna / strep diluito in PBS 1x con vigorosi tremanti (5 min lavaggi per 4 volte). Dopo l&#39;ultimo lavaggio, trasferire il derma in una nuova provetta / bottiglia in 4x penna / strep diluito in PBS 1x e conservare a 4 ° C fino al momento dell&#39;uso. Derma possono essere conservati a 4 ° C per un massimo di un anno. </li></ol&gt; 2. modificare geneticamente cheratinociti umani primari NOTA: utilizzare le cellule phoenix amphotropic cresciute nei media DMEM completo [DMEM + 10% siero bovino fetale (FBS) + pen / strep] per la produzione di virus per infettare cheratinociti umani primari. Geni imballaggio virali che codificano proteine ​​come gag-pol e busta sono stabilmente integrati nel genoma della cellula Phoenix che consente la produzione di virus quando trasfettate con un vettore retrovirale. Phoenix cellule possono essere transfettate con alta efficienza che consente un&#39;elevata produzione titolo virale. Virus prodotto da questa linea di confezionamento può infettare una grande varietà di cellule di mammiferi compreso umana. <ol><li> 1 ° giorno: Il giorno prima trasfezione, le cellule fenice seme in un 6-pozzetti ad una densità di 800.000 cellule per pozzetto nei media DMEM completo. </li><li> Giorno 2: Il giorno della transfezione, utilizzare 3 mg di vettori retrovirali per pozzetto. Aliquotare 3 mg di vettore retrovirale in una provetta da 1,5 ml. Utilizzare un mix di 100 μ; l DMEM più 6 ml di reagente di trasfezione per ogni 3 mg di vettore. Mescolare 6 ml di reagente di trasfezione con 100 microlitri DMEM in una provetta da 1,5 ml. (I vettori retrovirali che vengono utilizzati sono elencati nella tabella attrezzature / materiali). <ol><li> Incubare per 5 minuti e aggiungere il composto di 106 microlitri nel tubo pre-aliquotati contenente 3 mg di vettore retrovirale. Mescolare e incubare a temperatura ambiente per 30 min. Aggiungere questa intera miscela gocce a ciascun pozzetto di cellule Phoenix. </li></ol></li><li> 3 ° giorno: Il giorno dopo la trasfezione, rimuovere il supporto dalle cellule phoenix e aggiungere 2 ml di fresco supporto completo DMEM. Lo stesso giorno, seminare cheratinociti umani primari in 6 pozzetti ad una densità di 75.000 cellule per pozzetto. Mantenere i cheratinociti in un mezzo di siero cheratinociti gratuito (KCSFM) con antibiotici (penna / strep). NOTA: cheratinociti umani primari sono stati acquistati. Le cellule possono essere acquistati da una varietà di fornitori (elencati nella tabella Materiali / Equipment). </li><li> 4 ° giorno: Harconferire al supporto dalle cellule trasfettate fenice, che ora contiene particelle virali. Far passare il materiale attraverso un filtro da 0,45 micron con una siringa (per rimuovere tutte le cellule fenice contaminanti) e posizionare 2 ml sulle cheratinociti (placcato a 75.000 cellule / pozzetto del giorno precedente: vedere il punto 2.3). Aggiungere hexadimethrine bromuro (5 mg / ml) per i media per aiutare a mediare il processo di infezione. Titoli virali sono ~ 1 x 10 7 TU / ml. <ol><li> Spin piastre da 6 pozzetti in una centrifuga a 200 xg per 1 ora a RT. Dopo lo spin, rimuovere i supporti contenenti virus e lavare le cellule una volta con PBS 1x prima di aggiungere KCSFM. </li></ol></li><li> 5 ° giorno: infettare la stessa partita di cheratinociti di nuovo come al punto 2.4. NOTA: Selezionare i cheratinociti usando un farmaco, se vi è un marcatore sul vettore retrovirale ed espandere per l&#39;uso in analisi a valle o applicazioni come colture organotipiche. </li></ol> 3. Configurazione Culture pelle organotipiche <ol><li> Costruire ilcassetta organotipica da materiali comuni che si trovano in laboratorio o le cassette può essere personalizzato attraverso un negozio di fabbricazione di metallo (Figura 2A - F). Per rendere queste cassette da una piastra di coltura tissutale 3.5 cm, utilizzare un bisturi per rimuovere un 1,0 cm x 1,0 cm quadrato dal centro del coperchio di un piatto 3.5 cm (Figura 2A-B). Fate questo in condizioni di sterilità. <ol><li> Attaccare pioli quadrati sul fondo della cassetta (coperchio di 3.5 cm piatto) utilizzando smalto trasparente (Figura 2C). Lasciare 5 minuti per lo smalto si asciughi e capovolgere la cassetta sopra in modo che poggi sui pioli quadrati (Figura 2D). Posizionare la cassetta in un piatto 6 centimetri. NOTA: Piazza pioli possono essere acquistati da una varietà di arti e botteghe artigiane. </li></ol></li><li> Preparare terreno di crescita dei cheratinociti (KGM) composto da 66% DMEM, il 22% F12 Ham, 100 unità / ml di penicillina, 100 mg / ml di streptomicina, il 10% FBS, 2,67 mg / ml di adenina, 40 ng / ml idrocortisone, 10 ng tossina colerica ml /, 4,94 mg / ml di insulina, 5 mg / ml transferrina, 1.36 ng / ml triiodo-L-tironina, 10 ng / ml EGF, e 10 ug / ml cloridrato ciprofloxacina. Filtrare il mezzo attraverso un filtro e conservare 0,22 micron a 4 ° C fino al momento dell&#39;uso. </li><li> Prendere il derma dalla 4x pen / strep + soluzione PBS e lavare 2 volte in KGM e incubare in KGM a 37 ° C per due giorni prima dell&#39;uso. </li><li> Tagliare il derma con un bisturi a 1,5 cm x 1,5 cm pezzi di dimensioni e quindi posizionare sul vassoio organotipica. Posizionare il derma con la parte superiore del derma rivolte verso l&#39;alto. La parte superiore del derma sarà il lato che viene a contatto con i cheratinociti (Figura 1B). </li><li> Posizionare il derma pretagliati sul foro quadrato della cassetta organotipiche con il lato superiore del derma rivolti verso l&#39;alto (Figura 3A). </li><li> Capovolgere l&#39;intera cassetta organotipica contenente oltre il derma con pinze (Figura 3B). Scongelare la soluzione matrice extracellulare sul ghiaccio. Usare un ago e la siringa a tirar fuori 100 ml di soluzione di matrice extracellulare per cassetta. Aggiungere 5 gocce al fondo del derma (lato lucido) e agitare leggermente per assicurare una distribuzione derma (Figura 3B). </li><li> Lasciare 5 minuti per la soluzione matrice extracellulare commerciale a solidificare completamente (Figura 3C). Dopo la solidificazione, utilizzare pinze per capovolgere il cassetto sopra. Aggiungere 4 ml di KGM al piatto 6 cm. </li><li> Posizionare 0,5-1 milioni di cheratinociti geneticamente modificati sulle cassette organotipiche contenenti il derma (Figura 3D). <ol><li> Trypsinize cheratinociti mettendo 4 ml di 0,05% tripsina su piastre di 10 cm per 5 minuti e spegnere con 4 ml di mezzi DMEM completo. </li><li> Contare i cheratinociti utilizzando un emocitometro e poi spin down a 200 xg per 5 min. Risospendere 0.5-1 milioni cellule (a seconda del numero di cellule disponibili) in 90 μl di KGM e posizionare tutte le cellule sulle derma. NOTA: È importante posizionare lo stesso numero di celle sul derma nello stesso esperimento per garantire che eventuali differenze rilevate spessore dell&#39;epidermide rigenerata non è dovuta a differenze nel numero iniziale di cellule. Posizionamento di meno di 0,5 milioni di cellule sul derma può portare a poveri stratificazione e la differenziazione. </li></ol></li><li> Modificare i media KGM sulle culture organotipiche ogni altro giorno. Tessuto raccolto 1-14 giorni dopo il posizionamento dei cheratinociti sulle derma. Stratificazione e la differenziazione completa dell&#39;epidermide avviene di solito dopo giorno 5. </li></ol>

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We have nothing to disclose.

La manipolazione genetica in pelle umana colture organotipiche offrono molti vantaggi a comunemente studiato 2D cellule in coltura e modelli di mouse. Culture 2D mancano i tre cellula-cellula e cellula-matrice extracellulare interazioni dimensionali si trovano in tessuti e organi intatti. Recenti studi hanno anche trovato enormi differenze tra le colture di cellule tumorali della pelle 2D e 3D con le cellule in coltura 3D che mostra molto di più somiglianze di espressione genica per la pelle umana primaria tumori ...

L&#39;epidermide umana è un epitelio stratificato che si collega al derma sottostante attraverso una matrice extracellulare noto come l&#39;epidermide zone.The membrana basale non serve solo come una barriera impermeabile per evitare la perdita di umidità, ma anche come una prima linea di difesa per proteggere la corpo dalle sostanze estranee e tossiche 1. Lo strato basale, che è lo strato più profondo dell&#39;epidermide, contiene le cellule staminali epidermiche e progenitrici che danno luogo alla progenie differenziata che formano il resto dell&#39;epidermide 2. Come le cellule progenitrici epidermiche differenziano migrano verso l&#39;alto per formare il primo strato di cellule differenziate note come strato spinoso 3. Nello strato spinoso, cellule attivano l&#39;espressione di cheratine 1 e 10, che quindi fornisce la forza di resistere allo stress fisico per gli strati differenziati dell&#39;epidermide. Come le cellule strato spinoso ulteriormente differenziano, si muovono verso l&#39;alto nell&#39;epidermide a perm strato granulare che è caratterizzata dalla formazione di keratohyalin e lamellari granuli così come proteine ​​strutturali che vengono assemblati sotto la membrana plasmatica. Poiché le cellule procedere nel processo di differenziazione, le proteine ​​sotto la membrana plasmatica sono trasversali collegati tra loro, mentre i granuli vengono estrusi lamellari dalle cellule per formare una barriera lipidica ricco chiamato strato corneo 4. Malattie che coinvolgono alterazioni nella crescita epidermico e impatto differenziazione ~ 20% della popolazione 5. Così, la comprensione dei meccanismi di questo processo è di grande importanza. Dal momento che la manifestazione di molte di queste malattie è subordinato cellula-cellula o cellula-matrice contatto, colture organotipiche dove l&#39;epidermide umana è ricostituito in un ambiente 3D sono stati creati 6-10. Questi metodi tipicamente comportano l&#39;uso di cheratinociti primari o trasformati seminate su matrice extracellulare come devitalizzati umanaderma, Matrigel o collagene. Per comprendere gene meccanismi di regolamentazione che sono importanti nella crescita e differenziazione epidermica, cheratinociti possono essere geneticamente manipolati tramite vettori retrovirali per atterramento o iperespressione di geni nella cultura 2D e poi ricostituito in 3D. Questi metodi sono stati ampiamente utilizzati per caratterizzare i geni coinvolti nella epidermica staminali e cellule progenitrici auto-rinnovamento e la differenziazione, nonché la progressione di neoplasia 11-21. Qui, è previsto un protocollo approfondito su come modificare l&#39;espressione genica in colture organotipiche epidermiche attraverso l&#39;uso di retrovirus.

<table><tbody><tr><td>Human skin</td><td>New York Firefighters Skin Bank</td><td>&lt;a href=&quot;http://www.cornellsurgery.org/pro/services/burn-surgery/skin-bank.html&quot;&gt;http://www.cornellsurgery.org/pro/services/burn-surgery/skin-bank.html&lt;/a&gt;</td><td></td></tr><tr><td>PEN/STREP</td><td>GIBCO</td><td>15140-122</td><td></td></tr><tr><td>amphotropic phoenix cell lines</td><td>ATCC</td><td>CRL-3213</td><td></td></tr><tr><td>FUGENE 6 transfection reagent</td><td>Promega</td><td>E2691</td><td></td></tr><tr><td>Keratinocyte Media (KCSFM)</td><td>Life Technologies</td><td>17005042</td><td></td></tr><tr><td>DMEM</td><td>GIBCO</td><td>11995</td><td></td></tr><tr><td>Ham&#039;s F12</td><td>Cambrex</td><td>12-615F</td><td></td></tr><tr><td>FBS</td><td>GIBCO</td><td>10437-028</td><td></td></tr><tr><td>Adenine</td><td>Sigma</td><td>A-9795</td><td></td></tr><tr><td>Cholera Toxin</td><td>Sigma</td><td>&amp;nbsp;C-8052</td><td></td></tr><tr><td>Hydrocortisone</td><td>Calbiochem</td><td>3896</td><td></td></tr><tr><td>Insulin</td><td>Sigma</td><td>I-1882</td><td></td></tr><tr><td>EGF</td><td>Invitrogen</td><td>13247-051</td><td></td></tr><tr><td>Transferrin&amp;nbsp;</td><td>Sigma</td><td>T-0665</td><td></td></tr><tr><td>Ciprofloxacin Hydrochloride</td><td>Serologicals</td><td>89-001-1</td><td></td></tr><tr><td>cautery</td><td>Bovie Medical Corporation</td><td>AA01</td><td></td></tr><tr><td>Matrigel</td><td>Corning</td><td>354234</td><td></td></tr><tr><td>Keratin 1 antibody</td><td>Biolegend</td><td>PRB-149P</td><td></td></tr><tr><td>square pegs</td><td>Arts and crafts stores</td><td></td><td></td></tr><tr><td>human neonatal keratinocytes</td><td>ATCC</td><td>PCS-200-010</td><td></td></tr><tr><td>human neonatal keratinocytes</td><td>Cell Applications</td><td>102K-05n</td><td></td></tr><tr><td>MSCV retroviral vector</td><td>Clontech</td><td>634401</td><td></td></tr><tr><td>LZRS retroviral vector</td><td>Addgene</td><td></td><td></td></tr><tr><td>pSuper.Retro.Puro Retroviral vector</td><td>Oligoengine</td><td>VEC-PRT-0002&amp;nbsp;</td><td></td></tr><tr><td>hexadimethrine bromide&amp;nbsp;</td><td>Sigma</td><td>H9268-5G</td><td></td></tr></tbody></table>

generation of genetically modified organotypic skin cultures using devitalized human dermis

Colture organotipiche permettono la ricostituzione di un ambiente 3D critico per interazioni di contatto cellula-cellula e cellula-matrice che imita la funzione e la fisiologia dei loro omologhi dei tessuti in vivo. Questo è esemplificato da culture pelle organotipiche che riassume fedelmente la differenziazione epidermica e il programma di stratificazione. Cheratinociti epidermici primari umani sono geneticamente manipolabile attraverso retrovirus in cui i geni possono essere facilmente overexpressed o abbattuti. Questi cheratinociti geneticamente modificati possono poi essere utilizzati per rigenerare epidermide umana in colture organotipiche della pelle che forniscono un modello potente per studiare percorsi genetici di crescita epidermico impattante, la differenziazione e la progressione della malattia. I protocolli qui presentati descrivono metodi per preparare derma umano devitalizzati nonché manipolare geneticamente cheratinociti umani primari per generare culture organotipiche pelle. Pelle umana rigenerata può essere utilizzato in downstrapplicazioni EAM come il profilo di espressione genica, immunostaining, e immunoprecipitazioni cromatina seguiti da sequenziamento ad alta produttività. Così, la generazione di queste culture organotipiche pelle geneticamente modificati consentirà la determinazione di geni cruciali per mantenere l&#39;omeostasi della pelle.

Il primo passo nella generazione di pelle umana organotipica è rimuovere l&#39;epidermide dal derma. I due settimana incubazione della pelle a 37 ° C in 4x pen / strep / PBS dovrebbe consentire la separazione del derma dall&#39;epidermide (Figura 1A). Se la separazione dell&#39;epidermide e del derma è difficile posizionare il tessuto a 37 ° C a 4x penna / strep / PBS per un&#39;altra settimana e poi provare di nuovo peeling con pinze. Una delle chiavi per rigenerare il ...

Watch this Scientific Journal Video about Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis at JoVE.com

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

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.

Generazione di culture geneticamente modificate pelle organotipiche Uso devitalizzate derma umano

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

generation-genetically-modified-organotypic-skin-cultures-using

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

11.2K Views.  UCSD Stem Cell Program, University of California, San Diego. 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. 

Video: Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

A 3D Organotypic Melanoma Spheroid Skin Model

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased metastatic potential. Recently, mono-chemotherapies continue to improve by melanoma specific combination therapies with targeted kinase inhibitors. Still, metastatic melanoma remains a life-threatening disease because tumors exhibit primary resistance or develop resistance to novel therapies, thereby regaining tumorigenic capacity. In order to improve the therapeutic success of malignant melanoma, the determination of molecular mechanisms conferring resistance against conventional treatment approaches is necessary; however, it requires innovative cellular in vitro models. Here, we introduce an in vitro three-dimensional (3D) organotypic melanoma spheroid model that can portray the in vivo architecture of malignant melanoma and may warrant new insights into intra-tumoral as well as tumor-host interactions. The model incorporates defined numbers of mature and differentiated melanoma spheroids in a 3D human full skin reconstruction model consisting of primary skin cells. The cellular composition and differentiation status of the embedded melanoma spheroids is similar to the one of cutaneous melanoma metastasis in vivo. Using this organotypic melanoma spheroid model as a drug screening platform may support the identification of responders to selected combination therapies, while sparing the unnecessary treatment burden for non-responders, thereby increasing the benefit of therapeutic interventions.

Here, we present a protocol to generate a 3D organotypic melanoma spheroid skin model that recapitulates both the architecture and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention.

Un modello di pelle sferoide 3D Organotypic Melanoma

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased ...

Ricerca

Ricerca sul cancro

Generation and Culturing of Primary Human Keratinocytes from Adult Skin

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. In addition, keratinocytes play an essential role in the initiation, maintenance, and regulation of epidermal immune responses by being part of the innate immune system responding to antigenic stimuli in a fast, nonspecific manner. Here, we describe a protocol for isolation of primary human keratinocytes from adult skin, and demonstrate that these cells respond to calcium-induced terminal differentiation, as measured by an increased expression of the differentiation marker involucrin. In addition, we show that the isolated keratinocytes are responsive to IL-1&#946;-induced activation of intracellular signaling pathways as measured by the activation of the p38 MAPK pathway. Taken together, we describe a method for isolation and culturing of primary human keratinocytes from adult skin. Because the keratinocytes are the predominant cell type in the epidermis, this method is useful to study molecular mechanisms in cutaneous biology in vitro.

The human skin acts as a first line of defense against the external environment. We present a method for isolating primary human keratinocytes from adult skin. These isolated keratinocytes are useful in numerous experimental setups, and are a highly suitable model for studying molecular mechanisms in cutaneous biology in vitro.

Generazione e la coltura dei Keratinocytes umani primari da pelle adulta

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. ...

Biologia dello sviluppo

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.

Generazione di una tridimensionale a tutto spessore della pelle Equivalent e Automated Ferimento

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

Bioingegneria

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

Generazione di pelle 3D Organoid dal cordone emoderivati indotta da cellule staminali pluripotenti indotte

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern tissue engineering. A technical bottleneck is the deposition of collagen in vitro, as it is notoriously slow, resulting in sub-optimal formation of connective tissue and subsequent tissue cohesion, particularly in skin models. Here, we describe a method which involves the addition of differentially-sized sucrose co-polymers to skin cultures to generate macromolecular crowding (MMC), which results in a dramatic enhancement of collagen deposition. Particularly, dermal fibroblasts deposited a significant amount of collagen I/IV/VII and fibronectin under MMC in comparison to controls.
The protocol also describes a method to decellularize crowded cell layers, exposing significant amounts of extracellular matrix (ECM) which were retained on the culture surface as evidenced by immunocytochemistry. Total matrix mass and distribution pattern was studied using interference reflection microscopy. Interestingly, fibroblasts, keratinocytes and co-cultures produced cell-derived matrices (CDM) of varying composition and morphology. CDM could be used as &#34;bio-scaffolds&#34; for secondary cell seeding, where the current use of coatings or scaffolds, typically from xenogenic animal sources, can be avoided, thus moving towards more clinically relevant applications.
In addition, this protocol describes the application of MMC during the submerged phase of a 3D-organotypic skin co-culture model which was sufficient to enhance ECM deposition in the dermo-epidermal junction (DEJ), in particular, collagen VII, the major component of anchoring fibrils. Electron microscopy confirmed the presence of anchoring fibrils in cultures developed with MMC, as compared to controls. This is significant as anchoring fibrils tether the dermis to the epidermis, hence, having a pre-formed mature DEJ may benefit skin graft recipients in terms of graft stability and overall wound healing. Furthermore, culture time was condensed from 5 weeks to 3 weeks to obtain a mature construct, when using MMC, reducing costs.

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

We present a protocol to obtain cell-derived matrices rich in extracellular matrix proteins, using macromolecular crowders (MMC). In addition, we present a protocol which incorporates MMC in 3D organotypic skin co-culture generation, which reduces culture time while maintaining maturity of construct.

Migliorare 2D e 3D della pelle<em&gt; In Vitro</em&gt; modelli con Macromolecole affollamento

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

Una revisione completa dei protocolli di coltura di espianti di pelle embrionale aviaria

Using Avian Skin Explants to Study Tissue Patterning and Organogenesis

The developing avian skin during embryogenesis is a unique model that can provide valuable insights into tissue patterning. Here three variations on skin explant cultures to examine different aspects of skin development are described. First, ex vivo organ cultures and manipulations offer researchers opportunities to observe and study the development of feather buds directly. Skin explant culture can grow for 7 days enabling direct analysis of cellular behavior and 4D imaging at intervals during this growth period. This also allows for physical and molecular manipulations of culture conditions to visualize tissue response. For example, growth factor-coated beads can be applied locally to induce changes in feather patterning in a limited area. Alternatively, viral transduction can be delivered globally in the culture media to up or downregulate gene expression. Second, the skin recombination protocol allows researchers to investigate tissue interactions between the epidermis and mesenchyme that are derived from different skin regions, different life stages, or different species. This affords an opportunity to test the time window in which the epithelium is competent to respond to signals and its ability to form different skin appendages in response to signals from different mesenchymal sources. Third, skin reconstitution using dissociated dermal cells overlaid with intact epithelium resets skin development and enables the study of the initial processes of periodic patterning. This approach also enhances our ability to manipulate gene expression among the dissociated cells before creating the reconstituted skin explant. This paper provides the three culture protocols and exemplary experiments to demonstrate their utility.

Autore in primo piano: Approfondimento sulle attuali metodologie sperimentali di espianti di pelle aviaria

Here we describe protocols for three types of avian embryonic skin explant cultures that can be used to examine tissue interactions, 4D imaging timelapse movie (3D plus time), global or local perturbation of molecular function, and systems biology characterization.

Utilizzo di espianti di pelle aviaria per studiare il patterning e l'organogenesi dei tessuti

The developing avian skin during embryogenesis is a unique model that can provide valuable insights into tissue patterning. Here three variations on skin ...

Cultivating a Three-dimensional Reconstructed Human Epidermis at a Large Scale

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process and the characterization of the model is described. Neonatal primary keratinocytes are grown submerged on permeable polycarbonate inserts and lifted to the air-liquid interface three days after seeding. After fourteen days of stimulation with defined growth factors and ascorbic acid in high calcium culture medium, the model is fully differentiated. Histological analysis revealed a completely stratified epidermis, mimicking the morphology of native human skin. To characterize the model and its barrier functions, protein levels and localization specific for early-stage keratinocyte differentiation (i.e., keratin 10), late-stage differentiation (i.e., involucrin, loricrin, and filaggrin) and tissue adhesion (i.e., desmoglein 1), were assessed by immunofluorescence. The tissue barrier integrity was further evaluated by measuring transepithelial electrical resistance. Reconstructed human epidermis was responsive to proinflammatory stimuli (i.e., lipopolysaccharide and tumor necrosis factor alpha), leading to increased cytokine release (i.e., interleukin 1 alpha and interleukin 8). This protocol represents a straightforward and reproducible in vitro method to cultivate reconstructed human epidermis as a tool to assess environmental effects and a broad range of skin-related studies.

This protocol describes a straightforward method to cultivate three-dimensional reconstituted human epidermis in a reproducible and robust manner. Additionally, it characterizes the structure-function relationship of the epidermal barrier model. The biological responses of the reconstituted human epidermis upon proinflammatory stimuli are also presented.

Coltivare un'epidermide umana ricostruita tridimensionale su larga scala

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process ...

Generation of Self-assembled Vascularized Human Skin Equivalents

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to study skin development, wound healing, and grafting techniques. Many HSEs continue to lack vasculature and are additionally analyzed through post-culture histological sectioning which limits volumetric assessment of the structure. Presented here is a straightforward protocol utilizing accessible materials to generate vascularized human skin equivalents (VHSE); further described are volumetric imaging and quantification techniques of these constructs. Briefly, VHSEs are constructed in 12 well culture inserts in which dermal and epidermal cells are seeded into rat tail collagen type I gel. The dermal compartment is made up of fibroblast and endothelial cells dispersed throughout collagen gel. The epidermal compartment is made up of keratinocytes (skin epithelial cells) that differentiate at the air-liquid interface. Importantly, these methods are customizable based on needs of the researcher, with results demonstrating VHSE generation with two different fibroblast cell types: human dermal fibroblasts (hDF) and human lung fibroblasts (IMR90s). VHSEs were developed, imaged through confocal microscopy, and volumetrically analyzed using computational software at 4- and 8-week timepoints. An optimized process to fix, stain, image, and clear VHSEs for volumetric examination is described. This comprehensive model, imaging, and analysis techniques are readily customizable to the specific research needs of individual labs with or without prior HSE experience.

The goal of this protocol is to describe the generation and volumetric analysis of vascularized human skin equivalents using accessible and simple techniques for long term culture. To the extent possible, the rationale for steps is described to allow researchers the ability to customize based on their research needs.

Generazione di equivalenti di pelle umana vascolarizzata auto-assemblati

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to ...

Generazione di culture geneticamente modificate pelle organotipiche Uso devitalizzate derma umano

Riepilogo

Esplora Altri Video

Capitoli in questo video

Un modello di pelle sferoide 3D Organotypic Melanoma

Generazione e la coltura dei Keratinocytes umani primari da pelle adulta

Generazione di una tridimensionale a tutto spessore della pelle Equivalent e Automated Ferimento

Generazione di pelle 3D Organoid dal cordone emoderivati indotta da cellule staminali pluripotenti indotte

Migliorare 2D e 3D della pelle

Utilizzo di espianti di pelle aviaria per studiare il patterning e l'organogenesi dei tessuti

Utilizzo di espianti di pelle aviaria per studiare il patterning e l'organogenesi dei tessuti

Utilizzo di espianti di pelle aviaria per studiare il patterning e l'organogenesi dei tessuti

Coltivare un'epidermide umana ricostruita tridimensionale su larga scala

Generazione di equivalenti di pelle umana vascolarizzata auto-assemblati