quantitative measurement of epithelial barrier integrity and confluency of 2d bovine ileal enteroid-derived monolayer

3 Boyutlu Sığır İleal Enteroid Kültüründen 2 Boyutlu Monotabaka Oluşturulması

Animal models in research play a crucial role in enhancing our understanding of disease pathophysiology and the dynamics of the host immune response during infection and support the development of novel preventative and therapeutic strategies1,2,3,4. These models support research discovery and advancement in animals and are key to the progress of human health research. For decades, rodent models have underpinned advances in immune mechanisms and fundamental biology research for human diseases3,5,6,7. While rodent models are critical in screening and early development research, large animal models offer a more relevant comparison in researching human diseases in both early discovery and later development studies, including therapeutic efficacy and safety testing1,3,4,5. Livestock offers clear advantages compared to rodent models for more efficient translation for human applications for some diseases, including cryptosporidiosis, salmonellosis, tuberculosis, respiratory syncytial virus, and brucellosis1,7,8. Indeed, these diseases and others develop spontaneously in cattle, which share several analogous disease pathogenesis and immune processes to humans, and as an outbred population, cattle mimic the genetic and environmental heterogeneity influencing human immune responses5,8,9,10. The benefits of bovine models for infectious disease research can be maximized by first employing a sophisticated culture system and then implementing in vivo studies stepwise. The initial use of a highly complex bovine-derived culture system can considerably reduce the number of live animal studies while improving the chances of successful translational and applied research. Culture models should recapitulate the disease processes at an organ level for optimal predictive validity, retaining the native tissue microenvironment spatially and functionally.
The mucosal immune response is a multifaceted system comprised of a highly efficient barrier formed by gastrointestinal enterocytes and diverse populations of immune cells located below the mucosal surface11. This highly complex system is critical during infection in maintaining GI homeostasis and initiating immune defenses against enteric pathogens11. Communication between enterocytes and underlying innate immune cells initiates the development of protective immune responses against pathogenic microorganisms. As such, culture systems that are comparative in their level of complexity are necessary for an optimal investigation into host-enteric pathogen interactions and are highly effective in understanding enteric physiology and drug discovery and development12,13. Organoids are a robust culture system that resembles the architecture and function of the tissue of origin14,15. The multicellularity of these models permits investigation into the role of diverse cell populations and the cellular interactions involved in enteric health and disease12,14. However, human-derived organoid models in research are currently limited by the difficulty of obtaining a sufficient quantity and consistent quality of human intestinal epithelial cells and limited cell viability in culture. Immortalized cell lines can be used to obtain high yields of homologous cultures in these models consistently; however, transformed cells inherently lack the diversity and functional complexity of non-transformed epithelial cells16,17. The advantages of using cultures derived from bovine tissue as a model for investigating gastrointestinal diseases and physiology include the ease with which tissue samples can be consistently obtained from healthy donors, improved cell viability, and greater cellular diversity achievable only with non-immortalized tissue. Comparative tissue transcriptomics and characterization of intestinal organoids reveal similarities in conserved orthologous genes and cellular potentials between humans and cattle18. Therefore, a bovine organoid-derived culture system may be advantageous in investigating human intestinal diseases, with findings easily translatable to human medicine. 
The protocol described herein details an effective platform to evaluate host responses to enteric pathogens or compounds and intestinal physiology using a bovine enteroid-derived 2D primary cell culture system. Unlike 3D organoids, 2D culture systems generated on transwell inserts permit a dual culture of intestinal cells with immune or stromal cells, allowing study into the tissue-level dynamics. With applications in biomedical research, pharmaceutical development, and efficacy testing, this physiologically relevant model can benefit the health and advancement of both cattle and people alike.

Yazar Spotlight: Konak-patojen etkileşimlerini incelemek için gelişmiş enteroid model 

Translasyonel Biyomedikal Araştırma Uygulamaları için Sığır Primer Enteroid Türevli İki Boyutlu Tek Katmanlı Kültür Sisteminin Oluşturulması

Our lab focuses on examining host-pathogen interactions and the immune cell dynamics that occur following infection. By using a robust cell culture model, we hope to more closely mirror the complex interactions occurring in vivo at a tissue level. Some of the published literature describing intergeneration in cattle lacks the necessary details for the protocol to be easily reproducible, making its application by inexperienced researchers challenging.The benefit of our model is that we provide an extremely detailed protocol that can be used successfully by novice researchers. Creating Transwell monolayer enables co-culture with immune cells, revealing insights into tissue pathogen interaction while maintaining in vivo like organization. This versatile model is valuable for studying numerous cattle diseases with a complexity beyond single cell models.Our lab will employ this model to investigate the immune factors influencing susceptibility to cryptosporidium infection. It will facilitate comparison between adult cattle and calves while preserving tissue architecture in the immune cell microenvironment. The findings from this work have direct translational potential to human health.

Watch this Scientific Journal Video about Quantitative Measurement of Epithelial Barrier Integrity and Confluency of 2D Bovine Ileal Enteroid-Derived Monolayer at JoVE.com

Quantitative Measurement of Epithelial Barrier Integrity and Confluency of 2D Bovine Ileal Enteroid-Derived Monolayer  

2D Sığır İleal Enteroid Kaynaklı Tek Tabakanın Epitelyal Bariyer Bütünlüğü ve Birleşmesinin Kantitatif Ölçümü

Başlamak için, 2D sığır ileal enteroid tek tabakasını içeren Transwell kültür plakasını inkübatörden çıkarın. Plakanın biyogüvenlik kabininde oda sıcaklığında birkaç dakika dengelenmesine izin verin. STX2 elektrotlarının önceden koşullandırıldığından ve volt ohm metrenin üreticinin talimatlarına göre 1000 ohm'a kalibre edildiğinden emin olun.Probun uzun ucunu bazolateral bölmeye ve kısa ucunu Transwell epitel hücre kültürünün apikal bölmesine yerleştirin. Kurulum stabilize edildiğinde, hücrelerden yoksun kesici uç da dahil olmak üzere Transwell kesici uç başına üç trans-epitelyal elektrik direnci ölçümü yapın. Her bir kesici uç için ölçümlerin ortalamasını hesaplayın.Boş kuyucukların ortalama ölçümünü deneyselden çıkarın, ardından epitel bariyerinin direncini belirlemek için ekin yüzey alanı ile çarpın. Bir haftadan daha kısa bir kültürde birleşik 2D tek tabakalar gözlendi. TEER ölçümleri, 12. günde düşmeden önce yedi gün boyunca değerlerde istikrarlı bir artış gösterdi.Boyanmış 2D tek tabakanın konfokal mikroskobu, DAPI nükleer boyası, E-kaderin ve F-aktin boyamasının lokalizasyonunu gösterir. Hücreler, enteroendokrin hücreler, paneth hücreleri ve enterosit hücre soyları ile farklılaşmış gibi görünmektedir. Tek tabakanın apikal stimülasyonu, sitokin üretiminin artmasına neden oldu.

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Research

JoVE Journal

Immunology and Infection

Quantitative Measurement of Epithelial Barrier Integrity and Confluency of 2D Bovine Ileal Enteroid-Derived Monolayer

372 Görüntüleme.  Midwestern University. Başlamak için, 2D sığır ileal enteroid tek tabakasını içeren Transwell kültür plakasını inkübatörden çıkarın. Plakanın biyogüvenlik kabininde oda sıcaklığında birkaç dakika dengelenmesine izin verin. STX2 elektrotlarının önceden koşullandırıldığından ve volt ohm metrenin üreticinin talimatlarına göre 1000 ohm'a kalibre edildiğinden emin olun.Probun uzun ucunu bazolateral bölmeye ve kısa ucunu Transwell epitel hücre kültürünün apikal bölmesin...

Video: Quantitative Measurement of Epithelial Barrier Integrity and Confluency of 2D Bovine Ileal Enteroid-Derived Monolayer  

Generation of a Bovine Primary Enteroid-Derived Two-Dimensional Monolayer Culture System for Applications in Translational Biomedical Research

Organoid cell culture systems can recapitulate the complexity observed in tissues, making them useful in studying host-pathogen interactions, evaluating drug efficacy and toxicity, and tissue bioengineering. However, applying these models for the described reasons may be limited because of the three-dimensional (3D) nature of these models. For example, using 3D enteroid culture systems to study digestive diseases is challenging due to the inaccessibility of the intestinal lumen and its secreted substances. Indeed, stimulation of 3D organoids with pathogens requires either luminal microinjection, mechanical disruption of the 3D structure, or generation of apical-out enteroids. Moreover, these organoids cannot be co-cultured with immune and stromal cells, limiting in-depth mechanistic analysis into pathophysiological dynamics. To circumvent this, we optimized a bovine primary cell two-dimensional (2D) enteroid-derived monolayer culture system, allowing co-culture with other relevant cell types. Ileal crypts isolated from healthy adult cattle were cultured to generate 3D organoids that were cryopreserved for future use. A 2D monolayer was created using revived 3D enteroids that were passaged and disrupted to yield single cells, which were seeded on basement membrane extract-coated transwell cell culture inserts, thereby exposing their apical surface. The intestinal monolayer polarity, cellular differentiation, and barrier function were characterized using immunofluorescence microscopy and measuring transepithelial electrical resistance. Stimulation of the apical surface of the monolayer revealed the expected functionality of the monolayer, as demonstrated by cytokine secretion from both apical and basal compartments. The described 2D enteroid-derived monolayer model holds great promise in investigating host-pathogen interactions and intestinal physiology, drug development, and regenerative medicine.

Enteroids are emerging as a novel model for studying tissue physiology and pathophysiology, drug development, and regenerative medicine. Here, we describe a bovine primary cell 2D enteroid-derived culture system that permits co-culture with relevant tissue cell types. This model offers a translational advantage for gastrointestinal research modeling.

Organoid cell culture systems can recapitulate the complexity observed in tissues, making them useful in studying host-pathogen interactions, evaluating ...