Bu çalışma, Ulusal Sağlık Enstitüleri (CA148828), Michigan Gastrointestinal Peptide Merkezi Üniversitesi ve Michigan Üniversitesi Kapsamlı Kanser Merkezi Jeffrey A. Colby Kolon Kanseri Araştırma ve Tom Liu Memorial fonlarından YMS için hibe tarafından desteklenmiştir.

1. Kolon Tümör İzolasyon ve Hücre Ayrışma <ol><li> Bağırsak tümörleri her sürekli veya tedaviye bağlı kolon kanseri modeli izole edilebilir. Farenin CO2 ile ötenazi edilmelidir. Kolon daha sonra soğuk fosfat-tamponlu salin (PBS) ile yıkanmış, toplanmış ve boylamasına açılmıştır. Bir stereomikroskopta tümör içeren bölgelerin belirlenmesi, bir makas ile incelemek ve soğuk PBS ile yıkayın. </li><li> EDTA şelasyon tampon tümörleri içeren bağırsak parçaları (2 mM EDTA, 5.6 mmol / L Na 2 HPO 4, 8.0 mmol / L KH 2 PO 4, 96.2 mmol / L NaCl, 1.6 mmol / L KCl, 43.4 mmol / L sakaroz, inkübe Buz üzerinde 60 dakika süre ile 54.9 mg / L D-sorbitol, 0.5 mmol / L damıtılmış su içinde DL-ditiyotretol). </li><li> Tümör hücreleri mezenşimin takılı kalır ise şelasyon sonra, normal bağırsak epitel hücrelerinin çoğu, ayrılacaktır. Normal epitel hücreleri içeren şelasyon tampon kapalı aspireve geriye kalan tümör yıkayın 5 ml soğuk şelasyon tamponu ile bir kez daha parçalar. </li><li> Şelasyon tampon kapalı aspire, 5 ml soğuk 1x PBS ile tümör parçaları yıkayın. </li><li> 1x PBS aspire, sindirim tamponu olarak tümör parçası (% 2.5 fetal sığır serumu, penisilin 1 ünite / ml streptomisin içinde 1 ug / ml ve 2.5 ng / ml amfoterisin B, 200 U / ml tip IV kollajenaz, inkübasyon 37 ° C&#39;de 2 saat boyunca 125 ug / Dulbecco Modified Eagle Medium mi tip II dispaz) ° C &#39; </li><li> Tümör parçası 1 dakika normal yerçekimi altında yerleşmek, ve 15 ml şahin tüp süpernatant toplamak için izin verin. Pelet 3 dakika boyunca 200 x g&#39;de santrifüj edilir ve 3 dakika boyunca 200 x g&#39;de santrifüj ile 5 ml PBS ile bir kez yıkayın ile tek bir hücre süspansiyonu tümör süpernatan. </li></ol> 2. Bağırsak Tümör Kültür <ol><li> Hemasitometre kullanarak izole tek tümör hücreleri saymak, 500 ul PBS ile tümör hücre pelletini tekrar. </li><li> Yüzde Pelet tümör hücreleri3 dakika boyunca 200 x g&#39;de rifuging ve oyuk başına 50 ul Matrigel başına 15.000 hücre, 24-yuvalı plakalar içinde buz ve plaka üzerinde 5 mg / ml Matrigel bunları tekrar süspansiyon haline getirin. </li><li> Matrigel 37 ° C&#39;de 15 dakika boyunca polimerize ve 500 de bazal / ml kültür ortamı (penisilin 1 ünite / ml streptomisin içinde 1 ug / ml ve 2.5 ng / ml amfoterisin B, 10 mmol / L Hepes katalım , 2mm Glutamax, 1x N2 ek, 1x B27 takviyesi, Gelişmiş Dulbecco&#39;nun Modifiye Kartal Medium/F12 1 mmol / L N-asetilsistein) 50 ng / ml fare EGF içeren. </li></ol> 3. Kurulmuş Organoids bakımı <ol><li> Haftada bir EGF 2 gün ve geçiş organoids 01:05 her içeren bazal kültür ortamı değiştirin. </li><li> Pasajı, taze bazal kültür ortamı ile kültür ortamı değiştirin. Mekanik kesilmiş ipuçları ile bir P1000 pipet kullanarak organoids ve Matrigel bozabilir ve 15 ml şahin tüp içine aktarın. Ayrıca mekanik ayrışma bir yangın cilalı Pasteur boru kullanılarak elde edilirtte. </li><li> 2 dakika boyunca 200 xg&#39;de bazal kültür ortamı ve santrifüj 5 ml ayrışmış organoids yıkayın. </li><li> Süpernatantı atın, Matrigel ile pelet tekrar süspansiyon ve yukarıda açıklandığı gibi kültür ortamı ekleyin. </li></ol> 4. Depolama ve Kuruluş Organoids Kurtarma <ol><li> Uzun süreli depolama için, en az 2 yıl boyunca kararlı sıvı N 2 organoids dondurma. Donma organoids için ipuçları kesilmiş ve 15 ml şahin tüp içine transferi ile bir P1000 pipet kullanarak bozabilir. </li><li> 2 dakika boyunca 200 xg&#39;de bazal kültür ortamı ve santrifüj 5 ml ayrışmış organoids yıkayın. </li><li> Süpernatant atılır, Dulbecco Modifiye Edilmiş Eagle Ortamı (DMEM)% 20 fetal sığır serumu (FBS) ve% 10 dimetil sülfoksit (DMSO) içeren pelletini. </li><li> 1.5 ml cryotubes içine transfer hücreleri, daha sonra ° C derin dondurucu bir soğutma hızı elde etmek için -80 bir Nalgene Bay Frosty dondurma konteyner ve mağaza tüpleri koymak-1 ° C / dak. Gece boyunca inkübasyondan sonra, sıvı N2 tüpler içine aktarın. </li><li> Geri almak için, dondurulmuş organoids hızla 37 ° yukarıda açıklanan koşullarla Matrigel ve kültür tekrar süspansiyon C su banyosunda, aşağı doğru döndürün, bazal kültür ortamı ile yıkayın. Tekrar çözülür </li></ol> 5. RNA Ekstraksiyon, Protein Ekstraksiyon ve İmmünohistokimya <ol><li> RNA ekstraksiyonu: geçişi için yukarıda tarif edildiği gibi hücreler toplanır. RNA, üretici talimatlarına göre PicoPure TM RNA İzolasyon Kiti ile izole edilmiştir. </li><li> Protein Ekstraksiyonu: Yukarıda tarif edilen ve (100 ul radyoimmüno deney tamponu içinde lize RIPA olarak Hücre topakları toplanır, 50 mmol / L Tris-HCl, pH 7.5, 150 mmol / L NaCl, 2 mmol / L EDTA,% 1 NP-40, 0.1 1x proteaz inhibitörü ile% SDS). </li><li> İmmünohistokimya: hücre kültür ortamı kapalı aspire, Cryo-kalıp içine kesme ipuçları ile P1000 pipet ile tümör organoids transferi ve tis hemen dondurmakkuru buz ile dondurma orta (OCT) dava. Dondurulmuş Bölüm 7 mikron kesilir ve daha önce 26 açıklandığı gibi boyandı. </li></ol>

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Biz ifşa hiçbir şey yok.

Bu protokolde tarif edilen deneysel prosedürler farelerdeki primer fare kolon tümörlerinde, izolasyonu ve kültürü için izin verir. Protokol Dr Clevers grup 1,24,27 tarafından yapılan ufuklar iş uyarlanmıştır. Biz tümör organoids daha iyi verim almak için sindirim süresi ve kollajenaz konsantrasyonu optimize edilmiş. Kritik adımlar tümör tek hücre içine hücre sindirim, Matrigel tabanda ve seçici kültürünü içerir. Tümör hücre sindirim için, sırayla kolon tümörlerinde etkin a...

Bağırsak epitel hücreleri çoğalmaya başlar ve omurgalı vücut 2,3 diğer tüm dokular geride bıraktı, olağanüstü bir hızla ters çevirin. Bağırsak kök hücreler (ISC) ve transit yalıtım hücreleri de dahil olmak üzere bölünen hücreleri ya salgı (kadeh, Paneth ve enteroendokrin) hücreleri veya enterositler 3 farklılaşırlar. ISC mahzenin tabanında yer alır. Paneth hücrelerinin kript altına aşağı hareket ve diğer soy villus 3,4 için yukarı göç ise, uzun ömürlü. Burada hücreler Mikrobiyota dahil olmak üzere mide içeriğine maruz kalan ve bir anoikis bağlı apoptotik mekanizma yoluyla villus ipuçları dökülür. Kolon villus ve Paneth hücrelerinin yoksun olsa da, dengesini korumak için mekanizma 4 benzer. Wnt sinyal yolu bağırsak çoğalması ve ISC bakım 4 çok önemli bir rol oynadığı edilmiştir. Inci silinmesiE transkripsiyon faktörü TCF4, Wnt sinyal bir alt efektör, bağırsak kök hücre ve doku 5 sonraki arıza kaybına yol açar. Benzer şekilde, Wnt inhibitör DKK1 transgenik ifade epitel proliferasyonu azaltır ve salgı hücre soyları 6 tüketir. Tersine, Wnt agonist R-spondin-1 aşırı bağırsak crypt hücrelerinin 7 güçlü ve hızla yayılması neden olur. Bağırsak dengesini için Wnt sinyal önemi göz önüne alındığında, Wnt yolu mutasyonlar sık kolon kanseri 8 gözlenmektedir. Kolon kanseri Amerika Birleşik Devletleri 9 kanserden ölüm üçüncü önde gelen nedenidir. Kırmızı et ve alkol ile aşırı diyet fiziksel aktivite azalır ve kalıtsal ve somatik mutasyonlar kolon kanseri 10,11 risk faktörleri olarak kabul edilir. Adenomatöz polipozis koli (Süm) gen, önemli bir Wnt sinyal faktör, pa bir çoğunda mutasyona uğramışailesel, sporadik ve kolit ile ilişkili kolon kanseri 12,13 ile hastaları. Axin2 ve β-katenin dahil olmak üzere Wnt sinyal yolu ile ilgili diğer faktörlerin mutasyonlar da kolon kanseri 14,15 gözlenmektedir. Ancak, kolon kanseri için kesin mekanizması ve etkin tedavi hala eksiktir. Kolon kanseri için moleküler mekanizmaların soruşturma kolaylaştırmak için, agresif hücre tipi için iyi huylu kanser ilerleme farklı aşamalarında temsil eden insan kolon kanseri hücre hatları 16-18 kurulmuştur. Farklı metastatik özelliklere sahip Fare kolon karsinoma hücre çizgileri 19,20 mevcuttur. Yakından in vivo durumda taklit eder ve daha fazla fizyolojik olarak ilgili verileri 21 oluşturmak için Bununla birlikte, birincil hücreler veya organoid kültürleri dönüştürülmüş hücre çizgileri tercih edilir. En kolon kanseri hücre çizgileri elde edilen sonik, plakasına bağlı olarak tek tabakalı ya da hücre süspansiyonları olarak büyürapikal-bazolateral yönelim ve hücreler arasındaki sıkı bağlantıları g. Farklılaşmış hücrelerin villus ipuçları ulaşmak ve 22 döken olarak Ayrıca, in vivo normal ve tümör bağırsak epitel hücrelerinde apoptoz olarak adlandırılan anoikis kendiliğinden bir formu tabi. Bu özellikler, hücre hatlarında özetlemek zordur, fakat kolon kanseri 23 gelişim sürecinde önemlidir. Bu özellikler, primer organoids tutulur. Buna ek olarak, tümör organoid in vivo çalışmalar, kültürler ile karşılaştırıldığında genlerin tümör otonom işlevleri değerlendirmek için etkili bir sistem sağlar. Bağırsak in vivo genetik manipülasyon özellikle bağırsak-özel sürücüler kullanılarak transgenik ve / veya nakavt fareler yaratarak bir zaman alan bir süreçtir. Ancak, tümör organoids kolayca viral aracılı genetik manipülasyon için uygun ve bu nedenle hassas moleküler mekanizmaları değerlendirmek için harika bir araçtır. Primer intestinal tümör organoid kültür have uygun bir ve güçlü bir teknik olduğu gösterilmiştir. Birincil bağırsak hücre kültürü tek bir yetişkin Lgr5 + kök hücre 24 in vitro crypt-villus yapısı ile fonksiyonel bağırsak organoids kurabilir. Bu organoids nakledilen ve rejenerasyon 25 için hasarlı kolon doku içine engrafted olabilir. Kültür koşulları daha da adaptasyon fare kolon ve insan ince bağırsak ve 1 uygun kolondan benzer epitel organoids yapmıştı. Bazal kültür ortamı ve EGF primer fare kolon tümör organoids 1 büyüme için yeterli iken, birinci, normal kolon epitel kültür, bazal kültür ortamı için hem de EGF, Noggin, R-spondin ve Wnt3a büyüme faktörleri dahil olmak üzere, çok önemlidir. Burada ayrıntılı bir protokol, izole kültür ve kolon tümör organoids oluşturmak için açıklar.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Matrigel Basement Membrane Matrix</td>
			<td>BD Biosciences</td>
			<td>356234</td>
			<td>5 mg/ml</td>
		</tr>
		<tr>
			<td>Collagenase Type IV</td>
			<td>Worthington</td>
			<td>LS004188</td>
			<td>375 U/mg</td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Gibco</td>
			<td>17105-041</td>
			<td>1.8 U/mg</td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>Invitrogen</td>
			<td>12634010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Epidermal Growth Factor (EGF), Murine, Natural</td>
			<td>Invitrogen</td>
			<td>53003-018</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>N2 Supplement</td>
			<td>Invitrogen</td>
			<td>17502-048</td>
			<td>100 x</td>
		</tr>
		<tr>
			<td>B27 Supplement</td>
			<td>Invitrogen</td>
			<td>17504-044</td>
			<td>50 x</td>
		</tr>
		<tr>
			<td>Glutamax-I</td>
			<td>Gibco</td>
			<td>35050-079</td>
			<td>100x</td>
		</tr>
		<tr>
			<td>N-Acetylcysteine</td>
			<td>Sigma</td>
			<td>A9165-5G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dulbecco's Modified Eagle Medium</td>
			<td>Invitrogen</td>
			<td>11965-092</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PicoPureTM RNA Isolation Kit</td>
			<td>Invitrogen</td>
			<td>KIT0204</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

in vitro organoid culture of primary mouse colon tumors

Çeşitli insan ve fare kolon kanseri hücre hatları kurulmuştur, bu tür birden fazla hücre katmanları, bazal-apikal kutup, ayırt etmek yeteneği ve anoikis gibi kolon tümörlerinin fizyolojik bütünlüğü kolon kanseri edilen hücre hatlarında korunmaz. Mevcut çalışmada, Sato T et al. 1, kolon tümörlerinde önemli fizyolojik özelliklerini korur uyarlanmıştır kültür birincil fare kolon tümör organoids için bir yöntemi gösterir. Bu yöntem, tümör hücrelerinin normal göre besin kısıtlayıcı koşullar üzerinde büyümeyi sürdürmek ilkesine göre fare kolon tümör doku toplanması, komşu normal kolon epitel ayrışma, tek bir hücre içine Kolon tümör hücreleri sindirim, Matrigel içine Kolon tümör hücreleri gömme ve seçici kültür oluşmaktadır epitel hücreleri. Genetik olarak değiştirilmiş fareler izole ise primer tümör organoids tümör özerk fonksiyon değerlendirmek için çok kullanışlı bir sistem sağlamakBelirli genlerin iyon. Ayrıca, tümör organoids gen teslim meditasyon virüsünün genetik manipülasyon için uygun olan, kolon kanser oluşumunda yer alan bu nedenle sinyal yolları da yoğun aşırı veya demonte incelenmiştir olabilir. Primer tümör organoids kültür kolon tümörogenez için mekanizmalar ve tedavi yöntemleri incelemek için bir fizyolojik ilgili ve uygulanabilir bir yol sağlar.

/ Mouse Şekil 1&#39;de gösterilmiştir üç aylık Apc dk bir kolon tümör organoid oluşumu zaman ders. 0. günde, tek hücre kaplama (Şekil 1A) takiben birkaç saat gözlenebilir. 1. günde, refrakter çekirdekleri ile kolon tümör epitel hücreleri gözlenebilir atlattı. 3. gün de, hücrelerin boyutunu iki katına çıktı. 6. günde, organoid büyüklüğü on kat daha fazla genişletilmiş ve orta apoptoz işaretleri göstermiştir. Gün 14, or...

Watch this Scientific Journal Video about In vitro Organoid Culture of Primary Mouse Colon Tumors at JoVE.com

In vitro Organoid Culture of Primary Mouse Colon Tumors

Primer fare kolon tümörü organoid kurmak için basit bir yöntem açıklanmıştır. Bu yöntem kolon tümör hücreleri hayatta kalmak ve epitel normal bir kolon yok ise, sınırlı büyüme faktörleri içeren ortamlarda organoids içine büyümeye bu özelliği kullanır.

Birincil Fare Colon Tümörler in vitro organoid Kültür

Several  human and murine colon cancer cell lines have been established, physiologic  integrity of colon tumors such as multiple cell layers, basal-apical ...

in-vitro-organoid-culture-of-primary-mouse-colon-tumors

Research

JoVE Journal

Medicine

In vitro Organoid Culture of Primary Mouse Colon Tumors

34.9K Görüntüleme.  University of Michigan. Primer fare kolon tümörü organoid kurmak için basit bir yöntem açıklanmıştır. Bu yöntem kolon tümör hücreleri hayatta kalmak ve epitel normal bir kolon yok ise, sınırlı büyüme faktörleri içeren ortamlarda organoids içine büyümeye bu özelliği kullanır.

Video: In vitro Organoid Culture of Primary Mouse Colon Tumors

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

Bağışıklık Eksik Fare Kuruluş ve İnsan Retinoblastom Tümörler yayılımı

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

Tıp

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts in this protocol have been successfully used in the derivation of induced pluripotent stem cells to model disease1. Early passages of these fibroblasts can also be used for cell-based functional assays to study specific disease pathways, mechanisms2 and subsequent drug screening approaches. The advantage of the presented protocol over enzymatic procedures are 1) the reproducibility of the technique from small amounts of tissue derived from older patients, e.g. patients affected with Parkinson's disease, 2) the technically simple approach over more challenging methodologies using enzymatic treatments, and 3) the time consideration: this protocol takes 15-20 min and can be performed immediately after biopsy arrival. Enzymatic treatments can take up to 4 hr and have the problems of overdigestion, reduction of cell viability and subsequent attachment of cells when not handled properly. This protocol describes the dissection and preparation of a 4-mm human skin biopsy for derivation of a fibroblast culture and has a very high success rate which is important when dealing with patient-derived tissue samples. In this culture, keratinocytes migrate out of the biopsy tissue within the first week after preparation. Fibroblasts appear 7-10 days after the first outgrowth of keratinocytes. DMEM high glucose media supplemented with 20% FBS favors the growth of fibroblasts over keratinocytes and fibroblasts will overgrow the keratinocytes. After 2 passages keratinocytes have been diluted out resulting in relatively homogenous fibroblast cultures which expresses the fibroblast marker SERPINH1 (HSP-47). Using this approach, 15-20 million fibroblasts can be derived in 4-8 weeks for cell banking. The skin dissection takes about 15-20 min, cells are then monitored once a day under the microscope, and media is changed every 2-3 days after attachment and outgrowth of cells.

The fibroblast explant culture protocol from human skin punch biopsies is a technically robust and simple way to derive skin cells within 4-8 weeks for banking of about 15-20 million cells at a low passage number.

İlköğretim İnsan Fibroblastlar türetilmesi için Cilt Punch Biyopsi Eksplantın Kültür

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts ...

Primary Culture of Human Vestibular Schwannomas

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in either sporadic or familial (neurofibromatosis type 2, NF2) forms, both associated with inactivating defects in the NF2 tumor suppressor gene. Treatment for VSs is generally surgical resection or radiosurgery, however the morbidity of such procedures has driven investigations into less invasive treatments. Historically, lack of access to fresh tissue specimens and the fact that schwannoma cells are not immortalized have significantly hampered the use of primary cultures for investigation of schwannoma tumorigenesis. To overcome the limited supply of primary cultures, the immortalized HEI193 VS cell line was generated by transduction with HPV E6 and E7 oncogenes. This oncogenic transduction introduced significant molecular and phenotypic alterations to the cells, which limit their use as a model for human schwannoma tumors. We therefore illustrate a simplified, reproducible protocol for culture of primary human VS cells. This easily mastered technique allows for molecular and cellular investigations that more accurately recapitulate the complexity of VS disease.

Vestibular Schwannomas (VSs) are non-malignant tumors of Schwann cell (SC) origin, associated with mutations in the NF2 tumor suppressor gene. We report a reproducible, efficient protocol for primary human VS cell culture that allows for molecular and cellular experimental manipulation and analysis and recapitulates the heterogeneous nature of human disease.

İnsan Vestibüler Schwannomalar İlköğretim Kültürü

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in ...

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both direct and rapid analysis of therapeutic sensitivity and resistance. However, recent evidence suggests that therapeutic response is not exclusive to the inherent molecular composition of cancer cells but rather is greatly influenced by the tumor cell microenvironment, a feature that cannot be recapitulated by traditional culturing methods. Even implementation of tumor xenografts, though providing a wealth of information on drug delivery/efficacy, cannot capture the tumor cell/microenvironment crosstalk (i.e., soluble factors) that occurs within human tumors and greatly impacts tumor response. To this extent, we have developed an ex vivo (fresh tissue sectioning) technique which allows for the direct assessment of treatment response for preclinical and clinical therapeutics development. This technique maintains tissue integrity and cellular architecture within the tumor cell/microenvironment context throughout treatment response providing a more precise means to assess drug efficacy.

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

Established cancer cell lines and xenografts have been the mainstay of cancer research for the past several decades. However, recent evidence suggests that therapeutic response is greatly influenced by the tumor cell microenvironment. Therefore, we have developed an ex vivo analysis of primary tumor specimens for drug development purposes.

İlköğretim Tümörleri ve / veya klinik öncesi için İlişkili Metastazlarının ve Tedavi Klinik Geliştirme Ex Vivo Tedavisi Tepki

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both ...

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The approach allows unbiased identification and functional characterization of individual colorectal afferents. Extracellular recordings of propagated action potentials (APs) that originate from one or a few afferent (i.e., single-fiber) receptive fields (RFs) in the colorectum are made from teased nerve fiber fascicles. The colorectum is removed with either the pelvic (PN) or lumbar splanchnic (LSN) nerve attached and opened longitudinally. The tissue is placed in a recording chamber, pinned flat and perfused with oxygenated Krebs solution. Focal electrical stimulation is used to locate the colorectal afferent endings, which are further tested by three distinct mechanical stimuli (blunt probing, mucosal stroking and circumferential stretch) to functionally categorize the afferents into five mechanosensitive classes. Endings responding to none of these mechanical stimuli are categorized as mechanically-insensitive afferents (MIAs). Both mechanosensitive and MIAs can be assessed for sensitization (i.e., enhanced response, reduced threshold, and/or acquisition of mechanosensitivity) by localized exposure of RFs to chemicals (e.g., inflammatory soup (IS), capsaicin, adenosine triphosphate (ATP)). We describe the equipment and colorectum&#8211;nerve recording preparation, harvest of colorectum with attached PN or LSN, identification of RFs in the colorectum, single-fiber recording from nerve fascicles, and localized application of chemicals to the RF. In addition, challenges of the preparation and application of standardized mechanical stimulation are also discussed.

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates a protocol for conducting single-fiber electrophysiological recordings on an in vitro mouse colorectum-nerve preparation.

Fare Kolorektal Afferent Endings in vitro Fonksiyonel Karakterizasyonu içinde

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The ...

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA (Locked Nucleic Acid) probe. The probe was recognized by anti-DIG alkaline phosphatase antibodies and later developed using alkaline phosphatase substrate producing fluorescence signals. Here we utilized miRNA in situ hybridization (MISH) technique to analyze expression of miR-489 in tissues from breast cancer patients. This technique can detect the localization of miRNA of interest in individual tissue samples. This technique can be used to compare the expression of desired miRNA in tumor tissue with that in adjacent normal tissue and to identify the specific structures responsible for expressing this miRNA. This technique can be very useful in answering certain clinical questions, such as role of specific miRNA in the development of cancer. Our results indicate that mammary epithelial cells express significantly higher levels of miR-489 than adjacent tumor cells.

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

Here we present a protocol to detect miRNA expression in breast cancer patient samples using miRNA in situ hybridization.

miRNA kullanarak Meme Kanseri Dokularındaki miRNA İfade Klinikopatolojik Analizi<em&gt; In Situ</em&gt; Melezleme

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA ...

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

Köpek arter ve venlerin Primer Endotel Hücreleri İzolasyon ve Kültür

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

Forskolin kaynaklı bağırsak Organoids şişme: Bir<em&gt; İn Vitro</em&gt; Deney Kistik Fibrozis Hastalarında İlaç Tepki değerlendirilmesi için

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Muscle Imbalances: Testing and Training Functional Eccentric Hamstring Strength in Athletic Populations

Many hamstring injuries that occur during physical activity occur while the muscles are lengthening, during eccentric hamstring muscle actions. Opposite of these eccentric hamstring actions are concentric quadriceps actions, where the larger and likely stronger quadriceps straighten the knee. Therefore, to stabilize the lower limbs during movement, the hamstrings must eccentrically combat against the strong knee-straightening torque of the quadriceps. As such, eccentric hamstring strength expressed relative to concentric quadricep strength is commonly referred to as the &#34;functional ratio&#34; as most movements in sports require simultaneous concentric knee extension and eccentric knee flexion. To increase the strength, resiliency, and functional performance of the hamstrings, it is necessary to test and train the hamstrings at different eccentric speeds. The main purpose of this work is to provide instructions for measuring and interpreting eccentric hamstring strength. Techniques for measuring the functional ratio using isokinetic dynamometry are provided and sample data will be compared. Additionally, we briefly describe how to address hamstring strength deficiencies or unilateral strength differences using exercises that specifically focus on increasing eccentric hamstring strength.

The hamstrings are a group of muscles that are sometimes problematic for athletes, resulting in soft tissue injury in the lower limbs. To prevent such injuries, functional training of the hamstrings requires intensive eccentric contractions. Additionally, hamstring function should be tested in relation to quadricep function at different contraction speeds.

Kas dengesizlikleri: Test ve fonksiyonel eksantrik Hamstring gücü atletik nüfus eğitim

Many hamstring injuries that occur during physical activity occur while the muscles are lengthening, during eccentric hamstring muscle actions. Opposite ...

On-Site Sampling and Extraction of Brain Tumors for Metabolomics and Lipidomics Analysis

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still in need. In recent years, mass spectrometry has become a method of choice for untargeted profiling of discriminatory compound as potential biomarkers of a disease. Biofluids are generally considered as preferable matrices given their accessibility and easier sample processing while direct tissue profiling provides more selective information about a given cancer. Preparation of tissues for the analysis via traditional methods is much more complex and time-consuming, and, therefore, not suitable for fast on-site analysis. The current work presents a protocol combining sample preparation and extraction of small molecules on-site, immediately after tumor resection. The sampling device, which is of the size of an acupuncture needle, can be inserted directly into the tissue and then transported to the nearby laboratory for instrumental analysis. The results of metabolomics and lipidomics analyses demonstrate the capability of the approach for the establishment of phenotypes of tumors related to the histological origin of the tumor, malignancy, and genetic mutations, as well as for the selection of discriminating compounds or potential biomarkers. The non-destructive nature of the technique permits subsequent performance of routinely used tests e.g., histological tests, on the same samples used for SPME analysis, thus enabling attainment of more comprehensive information to support personalized diagnostics.

The manuscript presents on-site sampling of human brain tumors with solid phase microextraction followed by their biochemical profiling towards the discovery of biomarkers.

Metabolomik ve Lipidomik Analizler için Beyin Tümörlerinin Yerinde Örnekleme sivesi ve ekstraksiyonu

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still ...