1. Test Set-up <ol><li> 4 ° C (yaklaşık 2 saat) olarak buz üzerinde Matrigel küçük bir alikotu (&lt;500 ul) çözünmesi. </li><li> Jel tarifi (aşağıdaki tabloda örnek sayılarını görmek) hazırlayın: 10x PBS (toplam hacim 1x), 1N sodyum hidroksit (ekledi kollajen 0.023 hacimleri eşit veya kollajen üreticinin önerilerine uygun olarak), Matrigel ve kollajen tip I 1 mg / ml ve 1.3 mg / ml, sırasıyla (diğer matris formülasyonlarının hücre tipi ve deneye bağlı olarak kullanılabilir) son konsantrasyonları. </li></ol> Örnek jel tarifi <table border="1"><tbody><tr><td> Bileşenleri </td><td> Stok Konsantrasyon </td><td> Nihai konsantrasyon </td><td> Hacmi ekle </td></tr><tr><td> 10X PBS </td><td> 10X </td><td> 1X </td><td> 0.090 ml </td></tr><tr><td> Steril su </td><td></td><td></td><td> 0.346 ml </td></tr><tr><td> 1N NaOH </td><td></td><td></td><td> 0.008 ml </td></tr><tr><td> Matrigel </td><td> 9,90 mg / ml </td><td> 1 mg / ml </td><td> 0.101 ml </td></tr><tr><td> Sıçan kuyruk tip I kollajen </td><td> 3,66 mg / ml </td><td> 1.3 mg / ml </td><td> 0.355 ml </td></tr></tbody></table><ol start="3"><li> 4 ° C&#39;de 1 saat inkübe yukarıdaki gibi ve nihai çözelti, aynı sırayla buz üzerinde jel bileşenlerinin karıştırın Deneyimlerimize göre, kollajen daha düzgün bir jelleşme hücre ekim sonuçları öncesinde 1 saat inkübasyon. Her zaman buz üzerinde Matrigel ve kollajen tutmak ve jelleşme önlemek için mümkün olduğunca hızlı biçimde çalışmaya emin olun. </li><li> Steril forseps kullanarak 12-iyi plakasına Sıra 12 mm çapında 8 mikron gözenek hücre kültürü ekler. </li><li> 5 x 10 6 hücre / ml (toplam hacim jeli çözeltisi% 10 olmalıdır) azından serumsuz ortam içinde hücreleri ve sayma Pastör pipetiyle. </li><li> Hücre su 100 ul eklejel solüsyonu 900 ul (son hücre konsantrasyonu 5 x 10 5 hücre / ml) spension ve yukarı ve aşağı yavaşça pipet ile iyice karıştırın. </li><li> Jeli polimerize kadar 30 dakika boyunca 37 ° C&#39;de,% 5 CO2 inkübatöründe için her bir ekleme ve transfer etmek Nihai karışımın 150 ul ekleyin. </li><li> Serumsuz ortam kullanılması, </li></ol><ul><li> STATİK durum için takın altından jel ve 1200 ul üstüne 100 ul ekleyin. Iyi ekleme ve dış içindeki sıvı seviyeleri jel ve hiçbir interstisyel akışı üzerinde minimal basınç farkı ile sonuçlanan, yaklaşık eşit olmalıdır. </li><li> AKIŞ durum için jel üzerinde ekleme ve 650 ul altında 100 ul ekleyin. Onlar bu noktalarda zarını geçerek göç hücreleri önleyecek şekilde, insert altında hava kabarcığı olmamasına dikkat ediniz. Bu birimler tarafından üretilen basınç farkı yaklaşık 1.3 cm H2O (ya da 1 mm Hg) &#39;dir. </li></ul><ol start="9"><li> 37 koyun plaka° C, 24 saat süreyle% 5 CO2 inkübatöründe. </li></ol> 2. Hücre Boyama ve Sayma <ol><li> 500 ul PBS 1X başına kuyuya yeni bir 24-kuyulu plakalı Add; Bu uçlar yıkamak için kullanılacaktır. </li><li> Akış transwells üst kısmı içinde kalan orta kaldırmak ve hacim belirler. Başlangıçta, 650 ul (bu aşağıya bakın, akış hızı hesaplamaları için kullanılacak) eklenen toplam hacmin dışında kalan hacmi çıkarılarak toplam Yıkanan hacmini hesaplayın. Uçlar gelen jel kaldırmak ve non-istila hücreleri çıkarmak için membran üst yüzeyi temizlemek için pamuklu kullanabilir. Ekler yıkamak için 15 sn 1X PBS içeren 24 sıra plaka içine ekler yerleştirin. </li><li> PBS kaldırmak ve nakledildi hücreleri düzeltmek için oda sıcaklığında 30 dakika boyunca her ekleme ve inkübe altında 4% paraformaldehid (PFA) ile 500 ul ilave edin. </li><li> PFA çıkarın ve kalan sabitleştirici kaldırmak için 500 ul 1X PBS ile bir kez yıkayın. </li><li> 500 ul o eklehücrelerinin geçirgenliği, oda sıcaklığında 10 dakika süreyle inkübe uçlar ve altından f% 0.5 Triton X-100 çözeltisi. </li><li> Aşağı membran yüzün alt yerleştirmek için dikkatli olmak, 2 mikrogram / 1X PBS solüsyonu ml DAPI 100 ul içine bir jilet ve yer kullanarak ucun zarları Kes (hücreler nakledildi yüz aşağı). </li><li> Oda sıcaklığında 30 dakika boyunca 150 rpm&#39;de bir çalkalayıcı içinde alüminyum folyo ve yerinde sargı plakası. </li><li> Çalkalayıcı üzerinde 500 ul 1X PBS ücretsiz DAPI kaldırmak için (her 10 dakika için 3 defa tekrar) membran yıkayın. </li><li> Yukarı bakacak şekilde nakledildi hücreleri ile cam lamlara Yeri membranlar, montaj çözümü ve kapak kayma ekleyin. </li></ol> 3. Veri Analizi <ol><li> Her membran 5 rastgele seçilmiş yerleri (uzak kenarlarından kalmak ve 10X veya 20X objektif lens kullanın) DAPI lekeli çekirdekleri sayın. </li><li> Ortalama hücre sayısı hesaplayın ve aşağıdaki formül kullanılarak yüzde işgali edinin: Yüzde işgali =% 100 x (ortalama hücre sayısı x membran yüzey alanı) / (hücre numaralı seribaşı mikroskop görüntüsü x sayısının yüzey alanı) Yüzde işgali, bağımsız deneyler arasında karşılaştırma için izin vermek için bazı kontrol koşulu (genellikle statik koşul) normalize edilebilir. </li><li> Inkübasyon süresi (örneğin, 24 saat) tarafından akış durumda toplam Yıkanan hacmi bölmek: ortalama debisi hesaplayın. </li><li> Ortalama akış hızı hesaplayınız: jel / membran (bu durumda, 60 mm 2) kesit alanı tarafından ortalama akış hızı böler. </li></ol> 4. Temsilcisi Sonuçlar Interstisyel akışı altında tümör hücre invazyonu ölçmek için, MDA-MB-435S metastatik melanom hücreleri kullanarak, bizim 3 boyutlu akış işgali test yapmış. Bu hücreler daha önce interstisyel sıvı akışı 13-14 yanıt olarak işgal etmek için gösterilmiştir. Hücrelerinde 1.3 mg / ml sıçan tai oluşan bir matris içine gömülül tendonu kollajen tip I ve 5 x 10 5 hücre / ml &#39;lik nihai bir konsantrasyon hücre 1 mg / ml Matrigel bazal membran matris. İki farklı koşulları karşılaştırıldı: (1) ortalama interstisyel akım = 0,1 mikron s -1 ve (2) statik koşul = ölçülebilir akış hızı (Şekil 1). 24 saat sonra, membran gözenekler yoluyla istila hücreleri hücre sayımı kolaylaştırmak için DAPI ile boyanmıştır. Şekil 2 istila hücreleri temsili bir görüntüsü gösterilmektedir. Aydınlık altında, membran sadece gözenekler görülebilir. Floresans kullanarak, boyalı DAPI-çekirdekler ve hücre sayımı Phalloidin lekeli F-aktin yapılar için kullanılmıştır hücre gövdesi (isteğe bağlı) canlandırmak için kullanıldı. Eşit değişkenlerle varsayarak bir Student t-testi kullanılarak, bu interstisyel akımı anlamlı (Şekil 3) (p = 0.003), statik koşullar üzerinde 2.3 kat MDA-MB-435S hücre invazyonu artırır gösterdi. Bu corroboraBu hücre hattı 13-14 kullanarak benzer bulgular (ama farklı matris koşulları ve bu nedenle akım hızları ile) da görülüyor. <img alt="Şekil 1" src="/files/ftp_upload/4159/4159fig1.jpg" /> Şekil 1. 3-D interstisyel sıvı akışı işgali tahlil şematik. İlk uygun konsantrasyonları ve hacimleri kullanılarak jel solüsyon hazırlanır. Daha sonra jel çözüm hücre eklemek ve hücre kültürü ekler transfer. Son olarak her bir durum için ortam uygun hacim kazandırmak ve inkübe edin. İnterstisyel sıvı akışının bir sıvı basıncı baş tarafından tahrik edilir. <img alt="Şekil 2" src="/files/ftp_upload/4159/4159fig2.jpg" /> Şekil 2,. Membran üzerindeki MDA-MB-435S hücreleri nakledildi. Parlak bir alan altında membran A) Resim;; işgal hücreleri istila hücrelerinin sayımı kolaylaştırmak için DAPI ve Alexa Fluor ile interstisyel sıvı akışı işgali tahlil ve lekeli 488-konjuge Phalloidin sonra tespit edildi B) DAPI stained çekirdeklerin (mavi); C) Alexa Fluor 488-Phalloidin lekeli F-aktin (yeşil). Ölçek çubuğu 50 um temsil eder. <img alt="Şekil 3" src="/files/ftp_upload/4159/4159fig3.jpg" /> Şekil 3. Akışı altında MDA-MB-435S hücreleri istilası yükseltildi. 24 saat sonra Hücre işgali belirgin interstisyel akışı (p = 0.003) ile güçlendirilmiştir. Sonuçlar ortalama statik durumu normalize ve değerleri 6 hücre kültürü ekler ortalama ± SEM temsil edilir.

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Çıkar çatışması ilan etti.

Burada bir hücre kültürü içinde bir uç 3-D matriks içine gömülü hücreleri kullanılarak, tümör hücresi istilası, interstisyel akımının etkisiyle hesaplamak için bir yöntem de tarif etmiştik. Bu ve benzeri yöntemler hücre tipleri 13-15, 17, çeşitli üzerine interstisyel akış etkisini araştırmak için kullanılmıştır. Bizim yaklaşım kısmen türünü kullanarak tümörün matris mikro taklit Ben epitel doku ve çevresindeki stromada 21-22 bazal membran bulunan protei...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> Kollajen (Rat Kuyruk) </td><td> BD </td><td> 354236 </td><td> Steril tutun </td></tr><tr><td> Millicell hücre kültürü insert </td><td> Millipore </td><td> PI8P01250 </td><td> 8 mikron gözenek çapı, polikarbonat membran </td></tr><tr><td> Matrigel </td><td> BD </td><td> 354234 </td><td> Steril tutun </td></tr><tr><td> PBS </td><td> Sigma Aldrich </td><td> 100M-8202 </td><td> Adımları yıkama için jel solüsyonu, 1x hazırlanması için 10x </td></tr><tr><td> Sodyum hidroksit, 1.0N Çözüm </td><td> Sigma Aldrich </td><td> S2770 </td><td> Steril tutun </td></tr><tr><td> DMEM 1X </td><td> CellGro </td><td> 10-013-CV </td><td> Steril tutun </td></tr><tr><td> Sığır fetüs serumu </td><td> Atlanta Biologicals </td><td> 511150 </td><td> Steril tutun </td></tr><tr><td> Penisilin Streptomisin </td><td> CellGro </td><td> 30002CI </td><td> Steril tutun </td></tr><tr><td> Triton X-100 </td><td> Sigma Aldrich </td><td> X100-500 ml </td><td> PBS içinde% 0.5 </td></tr><tr><td> Paraformaldehyde </td><td> Fisher Scientific </td><td> 04042-500 </td><td> PBS içinde% 4 </td></tr><tr><td> Deiyonize Su </td><td></td><td></td><td> Steril tutun </td></tr><tr><td> 4 &#39;,6-diaminido-2-fenilindol (DAPI) </td><td> MP Biomedicals </td><td> 0215757401 </td><td> 1 mg / ml stok çözeltisi </td></tr><tr><td> Montaj Çözüm </td><td> Thermo Scientific </td><td> TA-030-FM </td><td></td></tr><tr><td> Tripsin-EDTA </td><td> CellGro </td><td> 25-052-CI </td><td> Steril tutun </td></tr></tbody> &lt;/ Table&gt;

three-dimensional cell culture model for measuring the effects of interstitial fluid flow on tumor cell invasion

En katı tümörlerin büyümesini ve ilerlemesini kanser hücreleri ve stroma ilişkili tümör mikro 1 in sinyalleşme tepkilerini başlangıç ​​dönüşümü bağlıdır. Daha önce, tümör mikro araştırma öncelikle tümör-stroma etkileşim 1-2 odaklanmıştır. Bununla birlikte, tümör mikro de etkiler iyi anlaşılamamıştır kalır biofiziksel kuvvetleri, çeşitli içerir. Bu güçler gen ekspresyonu değişiklikleri, hücre bölünmesi, farklılaşma ve işgali 3 yol tümör büyüme biyomekanik sonuçları. Matrix yoğunluğu 4, sertliği 5-6 ve 6-7 yapısı, interstisyel sıvı basıncı 8, ve interstisyel sıvı akış 8 Tüm kanser gelişimi süresince değiştirilir. Özellikle interstisyel sıvı akışını, normal dokularda 8-10 göre tümörlerin daha yüksektir. Tahmini interstisyel sıvı flooo hızları ölçülür ve tümör büyüklüğü ve farklılaşmasını 9, 11 bağlı olarak, 0,1-3 um s -1 aralığında olduğu bulunmuştur. Bu tümör-uyarımlı anjiyojenaz ve artan vasküler permeabilite 12 kaynaklanır yükseltilmiş interstisyel sıvının basıncı kaynaklanmaktadır. Interstisyel sıvı akışı kanser hücreleri 13-14, vasküler düz kas fibroblast ve hücreler 15 istilası artırmak için gösterilmiştir. Bu işgali 3-D 16 hücre etrafında oluşturulmuş veya matriks metalloproteinaz (MMP) ifadesi 15, kemokin sekresyon ve hücre adezyon molekül 17 arttı otolog kemotaktik gradyanlar bağlı olabilir. Ancak, hücreler sıvı akış seziyorum hangi mekanizma tam olarak anlaşılamamıştır. Tümör hücresi davranışı değiştiren ek olarak, interstisyel sıvı akışı tümör mikro diğer hücrelerin aktivitesini modüle. Bu tümör terfili myofibr içine fibroblastlann (a) sürüş farklılaşma ile ilişkilioblastlar 18, (b) antijenleri ve lenf düğümleri 19, ve (c) modüle lenfatik endotel hücre morfojenezini 20 diğer faktörlere çözünür taşıma. Burada sunulan tekniği Vitro hücreleri üzerinde interstisyel sıvı akışı getirir ve invazyonu (Şekil 1) üzerindeki etkilerini rakamlarla. Bu yöntem, stromal ve kanser hücresi işgali 13-15, 17 sıvı akışı etkilerini ölçmek için birçok çalışma yayınlanmıştır. Matriks kompozisyonu, hücre tipi ve hücre konsantrasyonunu değiştirerek, bu yöntem bu işgali, farklılaşma, proliferasyon ve gen ekspresyonu gibi hücresel süreçleri interstisyel akımının etkilerini incelemek amacıyla, diğer hastalıklar ve fizyolojik sistemler uygulanabilir.

Watch this Scientific Journal Video about Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion at JoVE.com

Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

İnterstisyel sıvı akışı solid tümörler yüksek olan ve tümör hücre invazyonu modüle olabilir. Burada bir matris içinde gömülü hücrelere interstisyel sıvı akışı uygulayıp sonra hücre işgali üzerine etkilerini ölçmek için bir tekniği açıklar. Bu teknik, kolayca diğer sistemlerle çalışmaya adapte edilebilir.

Tümör Hücre Invasion üzerine İnterstisyel Akışkan Etkilerinin Ölçülmesi için üç boyutlu Hücre Kültürü Modeli

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling ...

three-dimensional-cell-culture-model-for-measuring-effects

Research

JoVE Journal

Bioengineering

16.4K Görüntüleme.  Drexel University. İnterstisyel sıvı akışı solid tümörler yüksek olan ve tümör hücre invazyonu modüle olabilir. Burada bir matris içinde gömülü hücrelere interstisyel sıvı akışı uygulayıp sonra hücre işgali üzerine etkilerini ölçmek için bir tekniği açıklar. Bu teknik, kolayca diğer sistemlerle çalışmaya adapte edilebilir.

Video: Three-dimensional Cell Culture Model for Measuring the Effects of Interstitial Fluid Flow on Tumor Cell Invasion

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

Yumurtalıklar ve fallop borularında Üç Boyutlu Organ Kültür Aljinatta Hidrojeller

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

Biyomühendislik

Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in&#160;vitro driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e. a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.

Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Three-dimensional flow separation induced by a wall-mounted model polyp and its impact on the wall pressure loading are examined using particle image velocimetry, skin friction line visualization, and wall pressure measurements.

Model Vocal Fold Polip'in Neden Olduğu Üç Boyutlu Akış Ayrımının Araştırılması

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. ...

Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and invasion. Rat tail type I collagen and/or matrix derived from Engelbreth-Holm-Swarm mouse sarcoma cells have been traditionally employed as the substrates to model the matrix or stromal microenvironment into which mesenchymal cells (usually fibroblasts) are populated. Although experiments using such matrices are very informative, it can be argued that due to an overriding presence of a single protein (such as in type I Collagen) or a high content of basement membrane components and growth factors (such as in matrix derived from mouse sarcoma cells), these substrates do not best reflect the contribution to matrix composition made by the stromal cells themselves. To study native matrices produced by primary dermal fibroblasts isolated from patients with a tumor prone, genetic blistering disorder (recessive dystrophic epidermolysis bullosa), we have adapted an existing native matrix protocol to study tumor cell invasion. Fibroblasts are induced to produce their own matrix over a prolonged period in culture. This native matrix is then detached from the culture dish and epithelial cells are seeded onto it before the entire coculture is raised to the air-liquid interface. Cellular differentiation and/or invasion can then be assessed over time. This technique provides the ability to assess epithelial-mesenchymal cell interactions in a 3D setting without the need for a synthetic or foreign matrix with the only disadvantage being the prolonged period of time required to produce the native matrix. Here we describe the application of this technique to assess the ability of a single molecule expressed by fibroblasts, type VII collagen, to inhibit tumor cell invasion.

Tissue engineered fibroblast-derived native matrix is an emerging tool to generate a stromal substrate which supports epithelial cell proliferation and differentiation. Here a protocol applying this methodology to assess the impact of different stromal cell types on tumor cell biology is presented.

Kanser Hücre Invasion Uygulama ile Tümör Stromal mikroçevresinin Doku Mühendisliği

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

Bir Üç boyutlu Kollajen Matrix içindeki Hücre Göç Analizi

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 &#181;m diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

Genetik olarak tadil edilmiş hücre transplantasyonu için Micropatterned kültür plakalarında Sferoid hücrelere karşı gene

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

Ters Kolloidal Kristal Poli (etilen glikol) İskele imalatı: Karaciğer Doku Mühendisliği için Üç boyutlu Hücre Kültürü Platformu

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.

İçinde üç boyutlu Vitro hücre göç karakterize ortamlar yara

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on ...

Evaluation of Keratinocyte Proliferation on Two- and Three-dimensional Type I Collagen Substrates

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. Both forms can be prepared with the same type I collagen. In general, the non-fibrous form promotes cell adhesion and proliferation. The fibril form (gels) provides more physiological conditions in many types of cells; therefore, gel culture is useful for examining physiological behaviors of cells, such as drug efficacy.
Researchers can select the appropriate form according to the purpose of its use. For example, in the case of keratinocytes, on-gel culture has been used as a wound healing model. FEPE1L-8, a keratinocyte cell line cultured on the non-fibrous form of type I collagen, promote cell adhesion. Notably, keratinocyte proliferation is slower on the fibril form than the non-fibrous form. Protocols for the preparation of type I collagen for cell culture are simple and have wide applications depending on the experimental needs.

Here, we present a protocol for the preparation of two different forms of culture substrates utilizing type I collagen. Depending on how collagen is handled, collagen molecules either maintain two-dimensional, non-fibrous form or reassemble into three-dimensional, fibril form. Cell proliferation on type I collagen is drastically affected by fibril formation.

Keratinosit yayılması üzerine iki ve üç boyutlu değerlendirilmesi tip ı kollajen yüzeylerde

Type I collagen, useful as a substrate for cell culture, exists in two forms: the two-dimensional, non-fibrous form and three-dimensional, fibril form. ...

Using Multilayered Hydrogel Bioink in Three-Dimensional Bioprinting for Homogeneous Cell Distribution

During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by shear stress and improve the survival of the encapsulated cells. However, rapid gravity-driven cell sedimentation in the reservoir could lead to an inhomogeneous cell distribution in bioprinted structures and therefore hinder the application of liquid-like bioinks. Here, we developed a novel multilayered modified strategy for liquid-like bioinks (e.g., gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells. Multiple liquid interfaces were manipulated in the multilayered bioink to provide interfacial retention. Consequently, the cell sedimentation action going across adjacent layers in the multilayered system was retarded in the bioink reservoir. It was found that the interfacial retention was much higher than the sedimental pull of cells, demonstrating a critical role of the interfacial retention in preventing cell sedimentation and promoting a more homogeneous dispersion of cells in the multilayered bioink.

Here, we developed a novel multilayered modified strategy for liquid-like bioinks (gelatin methacryloyl with low viscosity) to prevent the sedimentation of encapsulated cells.

Homojen Hücre Dağılımı için Üç Boyutlu Biyobaskıda Çok Katmanlı Hidrojel Bioink'in Kullanılması

During the extrusion-based three-dimensional bioprinting process, liquid-like bioinks with low viscosity can protect cells from membrane damage induced by ...

Primary Clarification of CHO Harvested Cell Culture Fluid using an Acoustic Separator

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested cell culture fluid. While traditional methods like centrifugation or filtration are widely implemented for cell removal, the equipment for these processes have large footprints and operation can involve contamination risks and filter fouling. Additionally, traditional methods may not be ideal for continuous bioprocessing schemes for primary clarification. Thus, an alternate application using acoustic (sound) waves was investigated to continuously separate cells from the cell culture fluid. Presented in this study is a detailed protocol for using a bench-scale acoustic wave separator (AWS) for the primary separation of culture fluid containing a monoclonal IgG1 antibody from a CHO cell bioreactor harvest. Representative data are presented from the AWS and demonstrate how to achieve effective cell clarification and product recovery. Finally, potential applications for AWS in continuous bioprocessing are discussed. Overall, this study provides a practical and general protocol for the implementation of AWS in primary clarification for CHO cell cultures and further describes its application potential in continuous bioprocessing.

Presented here is a protocol for the primary clarification of CHO cell culture using an acoustic separator. This protocol can be used for the primary clarification of shake flask cultures or bioreactor harvests and has the potential application for continuous clarification of the cell bleed material during perfusion bioreactor operations.

Akustik Ayırıcı Kullanarak CHO Hasat Edilen Hücre Kültürü Sıvısının Birincil Netleştirilmesi

Primary clarification is an essential step in a biomanufacturing process for the initial removal of cells from therapeutic products within the harvested ...