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

파라핀 Embedding을 이용한 3D 장 점막 모델 구축

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.

저자 스포트라이트: 3D 인간 세포주 모델을 사용하여 화합물이 장 조직에 미치는 영향 조사 

면역 성분이 있는 기본 3차원(3D) 장 모델 시스템

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  

고정 장 등가물의 현미경 평가를 위한 기본 3D 재구성 장 점막 모델 준비

시작하려면 0.4 미크론 필터가 있는 인서트가 포함된 24웰 플레이트를 선택하십시오. 피펫을 사용하여 필터 인서트 아래와 위에 500마이크로리터의 HBSS를 추가합니다. 그런 다음 멀티웰 플레이트를 닫고 인큐베이터에 2시간 동안 넣습니다.배양 후 플레이트에서 HBSS를 조심스럽게 흡입합니다. 무세포 콜라겐 용액을 DMEM의 멸균 50밀리리터 튜브에 얼음 위에 준비합니다. 멤브레인 필터 위에 250마이크로리터의 콜라겐을 추가하고 접시 위에 뚜껑을 놓습니다.용액이 실온에서 중합되도록 합니다. 다음으로 인큐베이터에서 L-929 섬유아세포 배양을 제거합니다. 2 밀리리터의 예열된 트립신 EDTA 용액을 플라스크에 넣고 인큐베이터에 3-5분 동안 넣습니다.세포 현탁액을 멸균된 15mL 튜브로 옮기고 645G에서 5분 동안 원심분리합니다. 진공 펌프를 사용하여 상층액을 흡입하고 펠릿을 1밀리리터의 DMEM에 재현탁시킵니다. 20마이크로리터의 Trypan Blue 용액이 들어 있는 튜브에 20마이크로리터의 셀 현탁액을 추가합니다.그리고 세포 계수 챔버를 사용하여 세포 밀도를 평가합니다. 다음으로, 인큐베이터에서 U-937 단핵구 세포 배양을 제거합니다. 셀 현탁액을 원심분리하고 펠릿을 RPMI 1밀리리터로 재현탁시킵니다.세포가 균질하게 현탁되었는지 확인한 후 세포 계수 챔버를 사용하여 세포를 계수합니다. 다음에, 50, 000의 L-929 및 15의 DMEM의 50 마이크로리터에서 000의 U-937 세포를 각각 포함하는 세포 현탁액을 준비하십시오. 각 50 마이크로리터 셀 현탁액을 450 마이크로리터의 콜라겐에 첨가하고 잘 섞습니다.사전 코딩된 cell-free lamina propria에 500마이크로리터의 콜라겐 세포 용액을 오버레이합니다. 플레이트를 닫고 용액이 굳을 때까지 2시간 동안 인큐베이터에 넣습니다. 그런 다음 인큐베이터에서 Caco-2 세포 배양을 제거합니다.진공 펌프를 사용하여 배지를 흡인하고 10mL의 PBS로 세포를 헹굽니다. 5 밀리리터의 PBS 트립신 EDTA 용액을 넣고 인큐베이터에 5-8 분 동안 넣으십시오. 그런 다음 셀 현탁액을 원심분리하고 펠릿을 DMEM 1밀리리터에 재현탁시킵니다.세기 후에, DMEM의 50 마이크로리터에 있는 150, 000의 Caco-2 세포를 포함하는 세포 현탁액을 준비하십시오. 플레이트를 인큐베이터로 옮기기 전에 10분 동안 층류 후드에 놓습니다. 30분 후 재구성된 모델 위에 500마이크로리터의 DMEM을 추가하고 필터 아래에 500마이크로리터를 추가하고 플레이트를 인큐베이터로 되돌립니다.다음날 배지를 필터 위와 아래에 각각 500마이크로리터의 새 DMEM으로 조심스럽게 교체합니다.

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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 ...