Name: Preparation of a Basic 3D Reconstructed Intestinal Mucosa Model for Microscopic Evaluation of Fixed Intestinal Equivalents
Uploaded: 2023-09-01T13:00:00
Description: Watch this Scientific Journal Video about Preparation of a Basic 3D Reconstructed Intestinal Mucosa Model for Microscopic Evaluation of Fixed Intestinal Equivalents at JoVE.com

preparation of a basic 3d reconstructed intestinal mucosa model for microscopic evaluation of fixed intestinal equivalents

Parafin Gömülü 3 Boyutlu Bağırsak Mukoza Modelinin Yapımı

The intestinal epithelial barrier, a one-cell-thick internal lining containing different types of epithelial cells, constitutes the first physical defensive barrier or interface between the outside and the internal milieu of the body1,2. Columnar-type enterocytes constitute the most abundant type of epithelial cells. These are&#160;responsible for maintaining epithelial barrier integrity through interactions between several barrier components, including tight junctions (TJs), playing a significant role in barrier tightening1,3. The TJ structure consists of intracellular plaque proteins, such as zonula occludens (ZO) and cingulin, cooperating with transmembrane proteins, including occludins, claudins, and junctional adhesion molecules (JAMs) that form zipper-like structure tightly linking the neighboring cells3,4. The transmembrane proteins regulate the passive paracellular diffusion of small compounds and exclude toxic large molecules.
Potentially toxic food compounds and food contaminants stimulate inflammatory cytokine production that disrupts the epithelial permeability, activating immune cells and causing chronic intestinal tissue inflammation5,6,7. In contrast, various anti-oxidant and anti-inflammatory phytochemicals have been reported to reduce inflammatory cytokine expression and enhance intestinal TJ barrier integrity through the restoration of TJ protein expression and assembly4,6,8. Hence, the regulation of epithelial barrier integrity by both beneficial and harmful compounds has seen an increase in the use of both in vivo and in vitro models aimed at mimicking the intestinal barrier for pharmaceutical screening and toxicity studies. This is particularly relevant given the increasing interest in understanding the pathophysiology of intestinal bowel diseases (IBD), necrotizing enterocolitis, and cancer, which can be simulated in experimental models8,9,10.
There has been a demand for the development of cell-based in vitro models in order to achieve the objective of the &#8220;3Rs&#8221; in animal testing. These include replacement alternatives to the use of animals, reduction in the number of animals used, and refinement in adopting methods that alleviate distress11,12,13. Moreover, the underlying molecular, cellular, and physiological mechanisms between human and murine models (rodents being the most widely used species) are distinctive, leading to controversy regarding the efficacy of the murine models as predictors in human responses12,13. Numerous advantages of in vitro human cell-line models include target-restricted experimentation, direct observation, and continuous analysis13.
Single-cell-type monolayers in two-dimensional (2D) cultures have served as powerful models. However, these cannot precisely reproduce the physiological complexity of human tissues8,13,14. As a result, 3D&#160;culture systems are being developed with ever-increasing improvements to recapitulate the physiological complexity of both healthy and diseased intestinal tissues as next-generation risk assessment toolboxes13,14. These models include 3D Transwell scaffolds with diverse cell lines, organoid cultures, and microfluidic devices (intestine-on-chip) using both cell lines and organoids (derived from both healthy and diseased tissues)8,13,14.&#160;
The 3D &#8220;healthy tissue&#8221; intestinal equivalent protocol presented in the present study was based on striking a balance between physiological complexity and experimental simplicity13. The model is representative of a 3D Transwell scaffold, comprised of three cell lines (enterocytes [the gold-standard colon adenocarcinoma Caco-2 line] with a supporting immune component [U937 monocytes and L929 fibroblasts]), constituting a standardized and easily repeatable system applicable for the preliminary screening of dietary molecules of interest on intestinal epithelial barrier integrity and immune response. The protocol includes paraffin embedding for light microscopic evaluation of epithelial barrier integrity using fixed intestinal equivalents. The advantage of the present approach is that numerous sections of the embedded tissues can be made to stain for multiple parameters from a single experiment.

Yazar Spotlight: 3D İnsan Hücre Hattı Modellerini Kullanarak Bileşiklerin Bağırsak Dokusu Üzerindeki Etkilerinin Araştırılması 

Bağışıklık Bileşenli Temel Üç Boyutlu (3D) Bağırsak Model Sistemi

The scope of the work is to use these in vitro three-dimensional human cell line models to investigate the fact of beneficial as well as helpful compounds on intestinal tissue. The models can be used to further research understanding and also as a basic drug model. Using the plant-derived compounds, spermidine and eugenol, both separately and together in supplement form, we measure beneficial increases in autophagy and decreases in inflammation using these models.Then we will also able to show the potentially harmful effect of gluten extracted from certain wheat varieties on intestinal tissues. We are currently using economically priced, standardized, and easily repeatable in vitro system that better reflect the physiology of the human intestine as an alternative to animal models. Since we also use commercial human cell line rather than plant-derived cells, the responses from the study are more applicable to the general population.The protocol is based entirely on the use of different lines. These renders the model most standardized and reproducible. Furthermore, the simultaneous cultivation of different cell lines, enterocytes, fibroblasts, and immune system cells, allows for cellular responses that are closer to what happens in the patient.

Watch this Scientific Journal Video about Preparation of a Basic 3D Reconstructed Intestinal Mucosa Model for Microscopic Evaluation of Fixed Intestinal Equivalents at JoVE.com

Preparation of a Basic 3D Reconstructed Intestinal Mucosa Model for Microscopic Evaluation of Fixed Intestinal Equivalents  

Sabit İntestinal Eşdeğerlerin Mikroskobik Değerlendirilmesi için Temel 3D Rekonstrükte İntestinal Mukoza Modelinin Hazırlanması

Başlamak için, 0,4 mikron filtreli ekler içeren 24 oyuklu bir plaka seçin. Bir pipet kullanarak, filtre eklerinin altına ve üstüne 500 mikrolitre HBSS ekleyin. Ardından çok kuyulu plakayı kapatın ve iki saat boyunca bir inkübatöre yerleştirin.İnkübasyondan sonra, HBSS'yi plakadan dikkatlice aspire edin. Buz üzerinde DMEM'de steril 50 mililitrelik bir tüpte hücresiz bir kollajen çözeltisi hazırlayın. Membran filtrenin üzerine 250 mikrolitre kolajen ekleyin ve kapağı plakaya yerleştirin.Çözeltinin oda sıcaklığında polimerize olmasına izin verin. Daha sonra, L-929 fibroblast hücre kültürünü inkübatörden çıkarın. Şişeye iki mililitre önceden ısıtılmış tripsin EDTA çözeltisi ekleyin ve üç ila beş dakika boyunca bir inkübatöre koyun.Hücre süspansiyonunu steril 15 mililitrelik bir tüpe aktarın ve beş dakika boyunca 645 G'de santrifüjleyin. Bir vakum pompası kullanarak, süpernatanı aspire edin ve peleti bir mililitre DMEM'de yeniden süspanse edin. 20 mikrolitre Tripan Blue çözeltisi içeren bir tüpe 20 mikrolitre hücre süspansiyonu ekleyin.Ve hücre yoğunluğunu değerlendirmek için bir hücre sayma odası kullanın. Ardından, U-937 monosit hücre kültürünü inkübatörden çıkarın. Hücre süspansiyonunu santrifüjleyin ve peleti bir mililitre RPMI'de yeniden süspanse edin.Hücrelerin homojen bir şekilde süspanse edildiğinden emin olduktan sonra, bir hücre sayma odası kullanarak bunları sayın. Daha sonra, 50 mikrolitre DMEM'de sırasıyla 50.000 L-929 ve 15.000 U-937 hücresi içeren hücre süspansiyonları hazırlayın. Her 50 mikrolitrelik hücre süspansiyonunu 450 mikrolitre kolajene ekleyin ve iyice karıştırın.Önceden kodlanmış hücresiz lamina propria'yı 500 mikrolitre kollajen hücre solüsyonu ile kaplayın. Plakayı kapatın ve çözeltinin sertleşmesini sağlamak için iki saat boyunca bir inkübatöre yerleştirin. Ardından, Caco-2 hücre kültürünü inkübatörden çıkarın.Bir vakum pompası kullanarak, ortamı aspire edin ve hücreleri 10 mililitre PBS ile durulayın. Beş mililitre PBS tripsin EDTA çözeltisi ekleyin ve beş ila sekiz dakika boyunca bir inkübatöre yerleştirin. Daha sonra hücre süspansiyonunu santrifüjleyin ve peleti bir mililitre DMEM'de yeniden süspanse edin.Saydıktan sonra, 50 mikrolitre DMEM'de 150.000 Caco-2 hücresi içeren bir hücre süspansiyonu hazırlayın. Plakayı inkübatöre aktarmadan önce 10 dakika boyunca laminer akış başlığına yerleştirin. 30 dakika sonra, yeniden yapılandırılmış modelin üzerine 500 mikrolitre DMEM ve filtrenin altına 500 mikrolitre ekleyin ve plakayı inkübatöre geri koyun.Ertesi gün, ortamı sırasıyla filtrenin üstünde ve altında 500 mikrolitre taze DMEM ile dikkatlice değiştirin.

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JoVE Journal

Biology

Preparation of a Basic 3D Reconstructed Intestinal Mucosa Model for Microscopic Evaluation of Fixed Intestinal Equivalents

Basic Three-Dimensional (3D) Intestinal Model System with an Immune Component

There has been an increase in the use of in vivo and in vitro intestinal models to study the pathophysiology of inflammatory intestinal diseases, for the ...