Bu araştırma Kanser Araştırma Ulusal Sağlık Enstitüleri İntramural Araştırma Programı, Ulusal Kanser Enstitüsü, Merkezi tarafından desteklenmiştir.

Not: Aşağıdaki protokol NUR-PIT kullanarak belirli hücreleri ortadan kaldırmak için gereken adımları açıklar. Kontroller ve NUR-PIT ve hücre canlılığı ile ilgili diğer ayrıntıları başka bir yerde 18 bulunabilir. Monoklonal antikorlara IR700 1. Konjugasyon (mAb) <ol><li> 2-5 mg / 0.1 M Na 2 HPO 4 (pH 8.6) çözeltisi içinde ml&#39;de ilgi mAb hazırlayın. </li><li> Mikrosantrifüj tüpü içinde 0.1 M Na 2 HPO 4 çözeltisi (pH 8.6) içinde 10 mM IR700 30.8 nmol olan mAb 6.8 nmol karıştırın ve alüminyum folyo ile kaplı 1 saat için oda sıcaklığında inkübe edilir. </li><li> iki kez 15 ml PBS ile PD-10 kolonu (Malzeme / Ekipman bkz: İçindekiler) yıkayın. Adım 1.2 numuneyi yükleyin. </li><li> Üreticinin talimatlarına uygun olarak, PBS ile akıtılarak PD-10 kolonu ile bir karışım saflaştınlır. Not: Burada, elüsyon IR700 band rengi boyunca oldu. 300 ul örnek, PBS elüsyon fraksiyonu 4.4 ml, tipik olarak 2,5 ila ml&#39;dir. </li><li> belirlemekBir spektrofotometre 9 595 nm&#39;de emilimin ölçülmesiyle, Coomassie boyama ile protein konsantrasyonu. Her mAb molekülüne 9 konjuge florofor moleküllerinin sayısını onaylamak için 689 nm&#39;de absorpsiyon ile IR700 konsantrasyonunu belirleyin. Not: bir spektrofotometre ile 1 MAB&#39;deki IR700 moleküllerinin optimal konjügasyon sayısını belirlemek için önemlidir. Genel olarak, 1 mAb molekül içinde 3 çevresinde IR700 molekülleri, in vitro ve in vivo çalışmaları en uygunudur. HPLC ve SDS-PAGE yöntemleri mAb ile IR700 bağlı olup olmadıklarını teyit etmek için kullanılabilir. </li><li> protein konsantrasyonu belirlendikten sonra 4 ° C&#39;de saklayın. </li></ol> Karışık 3D Hücre Kültürü 2. Hazırlık (spheroid Karışık) <ol><li> damla plakaları asılı levha rezervuar kısmına steril su (yaklaşık 1 ml) uygulayın. </li><li> A431-luc-GFP hücreleri ve 3T3-RFP hücrelerini - çeşitli ilgi hücre tiplerinin oranları hazırlayın -Kültür ortamı, 50 ul içerisinde süspansiyon haline toplam 5000 hücreleri ihtiva eden her bir örnek ile. Not: kültür ortamı daki hücre türlerinin oranı, hücre tipine bağlı olarak vb 100: 100, 10: 100, 25: 100, 50: 1 hazırlayın. </li><li> 37 ° C ve% 5 karbon dioksit nemlendirilmiş bir inkübatör içinde 96 gözenekli asılı bırakma plakasında 5-7 gün karışımı inkübe edin. 2 günde kültür ortamı değiştirin. Not: hücreleri içeren damlalar olarak, 3D tam küremsi yapmak hafifçe plaka işlemek için 3D şekiller oluşturan önce kolayca düşmek. </li><li> 40X büyütme - 10X bir ters aydınlık mikroskop kullanılarak küremsi morfolojisi ve boyutunu dikkate alınmalıdır. Not: Bu hücre tipleri ve asılı damla büyüklüğüne bağlı olmasına rağmen, sfero çapı de damla plaka asılı 96&#39;da kuluçkalamadan 7 gün sonra yaklaşık 400-600 um olduğundan emin olun. </li></ol> Karışık 3D Hücre Kültürü için İn Vitro NUR-PIT 3. <ol><li> ha ortamı değiştirmek10 ug / ml antikor-photoabsorber konjügatı (APC) ihtiva eden ortama açılan levhalar donanımının değiştirilmesi, ve 37 ° C&#39;de ve% 5 karbon dioksit nemlendirilmiş bir inkübatörde 6 saat inkübe edilir. </li><li> 6 saat kuluçkalamadan sonra, taze kültür ortamı (fenol kırmızısı Free) ile iki kez sfero yıkayın. ucu kesilmiş hafifçe steril bir 200 ul pipet kullanarak 100 ul taze fenol kırmızısı ücretsiz kültür ortamı ile bir cam tabanlı 50 mm çanak sfero aktarın. Her yemeğin bir sfero yerleştirin. </li><li> morfoloji değişikliği tespit etmek için ters aydınlık mikroskop ile sfero dikkate alınmalıdır. Aşağıdaki filtre ayarları ile floresan mikroskop kullanmak optik gazetecilere (örneğin, GFP ve RFP) gözlemlemek için: GFP - 469 nm uyarma filtresi ve 525 nm emisyon filtresi; RFP - 559 nm uyarma filtresi ve 630 nm emisyon filtresi. </li><li> ışınlanması için cam tabanlı tabak üzerinde ışık yayan diyot (LED) koyun. Not: küremsi NIR maruz kalabilirmikroskop veya laminer kaputu ya yanar. <ol><li> Bir optik güç ölçer 9 NIR ışık güç yoğunluğunu ölçün. Bu ölçüme göre, 2 J / cm2 de 670 nm 710 dalga boylarında ışık yayan ışık yayan diyotlar (LED&#39;ler) üzerinden NIR ışık saçmak. Not: LED ışık yaklaşık 5 inç maksimum derinliği nüfuz edebilir. NIR PIT sitotoksik etkileri bağımsız olarak güç yoğunluğu ve maruz kalma 23 süresi, sadece belirli bir enerji bağlıdır. </li></ol></li><li> Işık verilmesinden sonra taze kültür ortamı 50 ul yeni Asılı damla plakasına sfero aktarmak ve 37 ° C&#39;de ve% 5 karbon dioksit, bir nemlendirilmiş inkübatörde 1 gün süreyle inkübe edilir. </li><li> Yavaşça kesilmiş ucu ile steril 200 ul pipet kullanarak taze kültür ortamı (fenol kırmızısı ücretsiz), 100 ul bir cam tabanlı çanak sfero aktarın. NUR-PIT kullanarak fi sonra bir floresan mikroskop 1 gün sfero gözlemlemekltresi ayarları adım 3.3 tarif. <ol><li> Veya bir floresan mikroskobu (Şekil 2B) 14,18,22 kullanılarak sitoplazmik GFP floresans kaybı ile 2 ug / ml&#39;lik bir son konsantrasyonda ortamına propidyum iyodür eklenerek ölü hücreleri algılar. </li></ol></li><li> Hedef hücreler tamamen ortadan değilse tekrarlayın 3,1-3,6 adımları. </li></ol>

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Yazarlar ifşa hiçbir şey yok.

Bu NIR PIT kullanarak hedef olmayan hücrelere zarar vermeden karışık bir 3D hücre kültüründen spesifik hücre eliminasyonu için bir yöntem ortaya koymaktadır. Şimdiye kadar, hiçbir pratik hücre eleme yöntemi doku sağlandıktan sonra veya transplantasyon sonrasında bulunmamaktadır. Böylece, NIR-PIT bunu gerçekleştirmek için gelecek vadeden bir yöntemdir. APC &#39;ler mAb kendisi benzer farmakokinetiği göstermektedir, çünkü bu teknik, aynı zamanda, in vivo 18,22 yararlanılabilir. hedef hücre tipi, çeşitli APC ile uyarlanabilir. 21 - anti-EGFR, anti-PSMA, anti-mezotelin, ve anti-CEA dahil olmak üzere çeşitli antikorlar ya da antikor fragmanları, daha önce APC &#39;15 olarak kullanıldı. Buna ek olarak, NIR radyasyon bölgesini değiştirme tedavi bölgeyi en aza indirebilirsiniz. Burada, hedef hücreler üzerinde lusiferaz aktivitesi miktarının ve floresan ile spesifik hücreler tamamen ortadan kaldırılması doğrulandı. NUR-PIT böylece kritik bir nokta t, bir ışık tabanlı bir tedavidirışık enerjisi ve ters-kare yasa (adım 3.4) &#39;e göre, bu nedenle hücre nekrozu azalma beri NIR ışık kaynağı ve hedef arasındaki o mesafeyi. 3D doğrusu, plaka tedavi edilmelidir ve mümkün olduğunca nazikçe kuluçkaya sfero yapmak. damla içeren hücreleri kolayca tahrip ya da 3D şekiller oluşturan önce biraz şok düşmek vardır. NUR-PIT birçok benzersiz avantajlara sahiptir. İlk olarak, NIR-PIT ZPT nedeniyle hücreler ve minimal hedef dışı birikimini hedef bağlayıcı oldukça spesifik yol IR700 hidrofil özellikleri, ebeveyn olmayan konjuge antikor benzer intravenöz farmakokinetik göstermektedir. Bu nedenle, daha doku naklinden sonra, NIR PIT NIR için ilgili bölgenin maruz bırakılarak, ardından APC&#39;nin intravenöz enjeksiyon ile hedef hücrelerin tedavi edilebilir. İkinci olarak, NIR ışık UV veya görünür ışıktan daha dokuya daha derin nüfuz edebilir, bu nedenle, NIR-PIT yüzey değil, aynı zamanda enti sadece tedavi edilebileceğiyüzeyde NUR maruz nakledilen doku kalınlığı yeniden. Son olarak, NIR PIT sürekli sınırlama olmaksızın hedef hücreleri yok etmek için de kullanılabilir. Hedef hücreler, hücre yüzeyinde 18 hedef moleküllerin yeterli kopyalarını ifade bu yöntem yararlıdır. Ancak, bu tekniğin bir kaç sınırlamalar vardır. Bu büyük bir molekül olduğu için İlk olarak, APC geçirgenliği sınırlıdır. Bu sorunun üstesinden gelmek için, NIR PIT tedavisi ya da antikor fragmanları, 18,19,22 yararlanılabilir tekrarladı. Başka bir sınırlama ışık nüfuz olduğunu. NIR ışık düzgün in vitro kültürü, böyle bir hedef bölgeye yakın ışık kaynağını taşımak için fiberoptik ile sindirim endoskopi, bronkoskopi, ya da doğrudan torakoskopi olarak uyarlanmış yaklaşımları gerektirir in vivo tedaviye çeviri nüfuz edebilir rağmen. NIR PIT daha adaptasyonların lokal ve spec hem de bu yöntemin kullanımı için izin verir26 - Böyle bağışıklık azaltılması veya tümör mikroçevrelerde 24 olarak birçok alanda, IFIC hücre ortadan kaldırılması.

Diğer hücrelere zarar vermeden özel hücrelerin ortadan kaldırılması özellikle kurulmuş doku olarak, son derece zordur ve doku mühendisliği alanında bir hücre eleme yöntemi için acil bir ihtiyaç vardır. 3 - Günümüzde rejeneratif tıp alanında, embriyonik kök hücreleri (ES), pluripotent kök hücrelerini (PSC), ya da uyarılmış pluripotent kök hücre (iPS) kullanarak doku kültürleri umut verici malzemeler 1 bulunmaktadır. Bu doku rejenerasyonu umut verici olmasına rağmen, istenmeyen hücreleri ile kirlenme önemli bir husustur. Ayrıca, transplantasyon 4,5 sonra tümorijenisite &#39;nin güvenlik endişesi var. Birçok çalışmalar bu konular üzerinde duruldu olmasına rağmen özellikle rejeneratif tıp 6, belirli hücreleri ortadan kaldırmak için - 8, hiçbir pratik bir yöntem geliştirilmiştir. Yakın Kızılötesi photoimmunotherapy (NIR PIT) bir antikor-photoabsorber conjugat göre bir tedavi yöntemie (APC). Bir APC hücreye spesifik monoklonal antikor (mAb) ve photoabsorber, IR700 oluşur. IR700 bir hidrofilik silika ftalosiyanin türevi ve tek başına 9 ile fototoksisite indüklemez. IR700 kovalent lisin molekülü yan zinciri üzerinde amid artıkları ile antikora konjuge edilir. APC hücre membran hedef molekülüne bağlanması ve daha sonra 690 nm &#39;de, NIR ışığa maruz kaldıktan sonra yaklaşık anında hücre nekrozunu indükler. 14 - NUR-ışık, hücre ölümüne 9 yol açan hücresel membran rüptürü maruz kalma sırasında. 21 - NIR PIT anti-EGFR, anti-HER2, anti-PSMA, anti-CD25, anti-mezotelin, anti-GPC3 ve anti-CEA 15 de dahil olmak üzere birden fazla antikor ya da antikor fragmanları, etkili olduğu kanıtlanmıştır. Bu nedenle, NIR PIT hedef moleküller, çok çeşitli karşı kullanılabilir. Ayrıca, NIR-PIT NIR ligh kısıtlayarak belirli bölgelerde seçici tedavi sağlayan bir iyi-kontrollü tedavit ışınlama 18,22. Burada, karışık 3D kültürlerden gelen NIR PIT kullanarak belirli hücre eleme bir yöntem mevcut.

<table border="0" cellpadding="0" cellspacing="0" width="1083">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:424px;">IRDye 700DX Ester Infrared Dye</td>
			<td style="width:364px;">LI-COR Bioscience (Lincoln, NE, USA)</td>
			<td style="width:191px;">929-70011</td>
			<td style="width:104px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2HPO4</td>
			<td>SIGMA-ALDRICH (St. Louis, MO, USA)</td>
			<td>S9763</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sephadex G25 column (PD-10)&#160;</td>
			<td>GE Healthcare (Piscataway, NJ, USA)</td>
			<td>17-0851-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Coomassie (bradford) Plus protein assay</td>
			<td>Thermo Fisher Scientific Inc (Waltham, MA, USA)</td>
			<td>PI-23200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Perfecta3D 96-Well hanging Drop Plates</td>
			<td>3D Biomatrix Inc (Ann Arbor, MI, USA)</td>
			<td>HDP1096-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Optical power meter</td>
			<td>Thorlabs (Newton, NJ, USA)</td>
			<td>PM100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LED: L690-66-60</td>
			<td>Marubeni America Co. (Santa Clara, CA, USA)</td>
			<td>L690-66-60</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vectibix (panitumumab)</td>
			<td>Amgen (Thousand Oaks, CA, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35mm glass bottom dish, dish size 35mm, well size 10mm</td>
			<td>Cellvis (Mountain View, CA, USA)</td>
			<td>D35-10-0-N</td>
			<td></td>
		</tr>
	</tbody>
</table>

selective cell elimination from mixed 3d culture using a near infrared photoimmunotherapy technique

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem cell (iPS) emerged as a new method in regenerative medicine. Although this tissue regeneration is promising, contamination with unwanted cells during tissue cultures is a major concern. Moreover, there is a safety concern regarding tumorigenicity after transplantation. Therefore, there is an urgent need for eliminating specific cells without damaging other cells that need to be protected, especially in established tissue. Here, we present a method for specific cell elimination from a mixed 3D cell culture in vitro with near infrared photoimmunotherapy (NIR-PIT) without damaging non-targeted cells. This technique enables the elimination of specific cells from mixed cell cultures or tissues.

optik olarak EGFR aşın NIR PIT, A431 hücre hattı, etkin olabilmesi için, genetik olarak GFP ve lusiferaz (A431-Luc-GFP) ifade etmek için modifiye edilmiştir. NIR PIT olmayan bir hedef olarak Balb / 3T3 hücre çizgisi optik RFP (3T3-RFP) ifade etmek için modifiye edilmiştir. APC panitumumab-IR700 (pan-IR700) sentezlendi. Hücreleri (A431-luc-GFP ve 3T3-RFP) çeşitli oranlarda oluşmuştur Karışık sferoidler, bu protokol (Şekil 1) &#39;e göre imal edildi. Tekrarlanan NIR PIT pan-IR700 (Şekil 2A&#39;da rejim bakınız) ile inkübasyon gerçekleştirilmiştir. Bu karma 3D kültüründen hedef hücre eliminasyonu elde ve floresan ve lusiferaz aktiviteleri (Şekil 2B, C) ​​ile izlendi. Diğer yandan, hedef olmayan hücreler büyümeye devam etmiştir. <img alt="Şekil 1" src="/files/ftp_upload/53633/53633fig1.jpg" /> <strong&gt; karma küremsi çeşitli oranlarda Şekil 1. Temsilcisi görüntüler. karma sferoidlerde Temsilcisi görüntüleri A431-luc-GFP hücreleri ve 7. günde 3T3-RFP hücrelerin çeşitli oranları gösterilmiştir. Ölçek çubuğu = 200 mikron. Bu rakam 16 ile modifiye edilmiştir. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53633/53633fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <img alt="şekil 2" src="/files/ftp_upload/53633/53633fig2.jpg" /> 3D cep küresellerde Şekil 2. Hedef hücre eliminasyon (A431-luc-GFP ve 3T3-RFP hücrelerinin karışık sferoidler). (A) NIR PIT (2 J / cm2) rejimi gösterilir. NUR-PIT Tekrarlanan (B) tamamen karışık 3D sfero olarak, hedef olmayan hücrelere (3T3-RFP) hiçbir zarar ile hedef hücreleri (A431-luc-GFP) ortadan kaldırmıştır. Çubuk = 200 um. Lusiferaz aktivitelerinin (C) Niceleme (RLU oranı)hedef hücrelerde (n = her grupta 10 sferoidler) arasında gösterdi tamamen ortadan kaldırılması. Bu rakam, 18 modifiye edilmiştir. Veri ± sem aracı olarak ifade edilir <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53633/53633fig2large.jpg" target="_blank">, bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a>

Watch this Scientific Journal Video about Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique at JoVE.com

Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique

Eliminating specific cells without damaging other cells is extremely difficult, especially in established tissue, yet there is an urgent need for a cell elimination method in the tissue engineering field. Here, we present a method for specific cell elimination from a mixed 3D cell culture using near infrared photoimmunotherapy (NIR-PIT).

Bir Yakın Kızılötesi Photoimmunotherapy Tekniği Karışık 3D Kültür seçici hücre Eliminasyon

Recent developments in tissue engineering offer innovative solutions for many diseases. For example, tissue engineering using induced pluripotent stem ...

selective-cell-elimination-from-mixed-3d-culture-using-near-infrared

Nanyang Technological University

Research

JoVE Journal

Bioengineering

7.8K Görüntüleme.  National Cancer Institute. Eliminating specific cells without damaging other cells is extremely difficult, especially in established tissue, yet there is an urgent need for a cell elimination method in the tissue engineering field. Here, we present a method for specific cell elimination from a mixed 3D cell culture using near infrared photoimmunotherapy (NIR-PIT). 

Video: Selective Cell Elimination from Mixed 3D Culture Using a Near Infrared Photoimmunotherapy Technique

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1or in biomaterial scaffolds2. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

3D parçacıklarının Üretimi için Basit Asma Bırak Hücre Kültürü Protokolü

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

Biyomühendislik

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

İnsan Hücre Kültürü Döner Hücre Kültür Sistemleri: Bir Model Olarak İnsan trofoblast Hücreleri

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

3D Fibrin hazırlanması Kök Hücre Kültürü Uygulamaları için iskeleleri

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

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.

Çekme Kuvveti Mikroskopi Hücre Kültürü Yüzey civarında yerelleştirme raportör flöresan Beads için Yeni Bir Yöntem

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 ...

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous number of imaging techniques currently available and the progress in instrumentation, there is still a need for highly sensitive probes that are suitable for in vivo imaging. One typical problem of available preclinical fluorescent probes is their rapid clearance in vivo, which reduces their imaging sensitivity. To circumvent rapid clearance, increase number of dye molecules at the target site, and thereby reduce background autofluorescence, encapsulation of the near-infrared fluorescent dye, DY-676-COOH in liposomes and verification of its potential for in vivo imaging of inflammation was done. DY-676 is known for its ability to self-quench at high concentrations. We first determined the concentration suitable for self-quenching, and then encapsulated this quenching concentration into the aqueous interior of PEGylated liposomes. To substantiate the quenching and activation potential of the liposomes we use a harsh freezing method which leads to damage of liposomal membranes without affecting the encapsulated dye. The liposomes characterized by a high level of fluorescence quenching were termed Lip-Q. We show by experiments with different cell lines that uptake of Lip-Q is predominantly by phagocytosis which in turn enabled the characterization of its potential as a tool for in vivo imaging of inflammation in mice models. Furthermore, we use a zymosan-induced edema model in mice to substantiate the potential of Lip-Q in optical imaging of inflammation in vivo. Considering possible uptake due to inflammation-induced enhanced permeability and retention (EPR) effect, an always-on liposome formulation with low, non-quenched concentration of DY-676-COOH (termed Lip-dQ) and the free DY-676-COOH were compared with Lip-Q in animal trials.

Fluorescence-quenching of a Liposomal-encapsulated Near-infrared Fluorophore as a Tool for In Vivo Optical Imaging

The use of fluorophores for in vivo imaging can be greatly limited by opsonization, rapid clearance, low detection sensitivity and cytotoxic effects on the host. Encapsulation of fluorophores in liposomes by film hydration and extrusion leads to fluorescence quenching and protection which enables in vivo imaging with high detection sensitivity.

Aracı Olarak bir lipozomal-kapsüllü Yakın-Kızılötesi floroforun Floresan-söndürme<em&gt; İn Vivo</em&gt; Optik Görüntüleme

Optical imaging offers a wide range of diagnostic modalities and has attracted a lot of interest as a tool for biomedical imaging. Despite the enormous ...

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol adhesive through a metal stencil, the overall amount of adhesive used in assembling paper microfluidic devices can be significantly reduced. We show on a simple 4-layer planar paper microfluidic device that the optimal adhesive application technique and device construction style depends heavily on desired performance characteristics. By moderately increasing the overall area of a device, it is possible to dramatically decrease the wicking time and increase device success rates while also reducing the amount of adhesive required to keep the device together. Such adhesive application also causes the adhesive to form semi-permanent bonds instead of permanent bonds between paper layers, enabling single-use devices to be non-destructively disassembled after use. Nonplanar 3D origami devices also benefit from the semi-permanent bonds during folding, as it reduces the likelihood that unrelated faces may accidently stick together. Like planar devices, nonplanar structures see reduced wicking times with patterned adhesive application vs uniformly applied adhesive.

We demonstrate the use of patterned aerosol adhesives to construct 3D paper microfluidic devices. This method of adhesive application forms semi-permanent bonds between layers, enabling single-use devices to be non-destructively disassembled after use and to ease folding complex nonplanar structures.

3D Kağıt mikroakışkan Cihazlar Construct Yapışkan Desenlendirme kullanma

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

Micropatterning ve 3D mikrodamarlar Meclisi

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

3D Hücre Kültürü için Pulmoner Ekstrasellüler Matrix Ayarlanabilir Hidrojellerinin

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioprinting kıkırdak ve cilt doku analogları bir roman pasif birimi teknik Bioink Precellularization için karıştırma kullanmak

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease modeling. Self-assembled tissues offer advantages over scaffold-based tissue engineering, such as enhanced matrix deposition, strength, and function. However, there are few available methods for fabricating 3D tissues without seeding cells on or within a supporting scaffold. Previously, we developed a system for fabricating self-assembled tissue rings by seeding cells into non-adhesive agarose wells. A polydimethylsiloxane (PDMS) negative was first cast in a machined polycarbonate mold, and then agarose was gelled in the PDMS negative to create ring-shaped cell seeding wells. However, the versatility of this approach was limited by the resolution of the tools available for machining the polycarbonate mold. Here, we demonstrate that 3D-printed plastic can be used as an alternative to machined polycarbonate for fabricating PDMS negatives. The 3D-printed mold and revised mold design is simpler to use, inexpensive to produce, and requires significantly less agarose and PDMS per cell seeding well. We have demonstrated that the resulting agarose wells can be used to create self-assembled tissue rings with customized diameters from a variety of different cell types. Rings can then be used for mechanical, functional, and histological analysis, or for fabricating larger and more complex tubular tissues.

This protocol describes a platform for fabricating self-assembled tissue rings in variable sizes using a customized 3D-printed plastic mold. PDMS negatives are cured in the 3D-printed mold; then agarose is cast in the cured PDMS negatives. Cells are seeded into the resulting agarose wells where they aggregate into tissue rings.

Hücre tohum için özel özel Wells ve kendinden montajlı 3D baskı kalıpları kullanarak doku yüzük imalatı

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease ...