Name: the ex vivo culture and pattern recognition receptor stimulation of mouse intestinal organoids
Uploaded: 2016-05-18T15:00:00.000+00:00
Duration: 9 min 9 s
Description: Watch this Scientific Journal Video about The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids at JoVE.com

The authors would like to thank Dr. Sheryl Coutermarsh-Ott, Dylan McDaniel and Bettina Heid for technical discussions. The authors thank Dr. Nanda Nanthakumar for providing the Caco-2 cells. The authors also thank The Multicultural Academic Opportunities Program (MAOP) at Virginia Tech. This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Award K01DK092355 (to I.C.A.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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The authors declare that they have no competing financial interests.

腸オルガノイドの文化やメンテナンスは、適切な組織培養技術を用いて任意の個人によって習得することが可能な手順です。そこより、従来の単層で増殖する細胞と比較した場合、微妙な継代しているが、これらの微妙な違いを克服することは困難ではありません。この方法の重要なステップは、最適な播種のために十分に高い密度にオルガノイドを成長させることができるという伴います?...

収穫する能力と文化の一次オルガノイドは、小腸、大腸、膵臓、肝臓および脳のために説明し、組織の生物学1-5のためのより多くの生理学的に代表的な現象の理解に密接エキサイティングな進歩ですされています。小腸オルガノイドの文化とメンテナンスを記述する最初の方法は、佐藤らによって報告された。ハンスClevers 1の研究室の外に。一次腸上皮細胞のこの方法、収穫培養前には、上皮細胞の成長を維持する上で制限され、無効であることが判明しました。方法は、最終的に混在し、一次線維芽細胞6の成長につながるようなコラゲナーゼおよびディスパーゼなどの酵素、とのインキュベーションを介した組織の解離が含まれていました。これらの条件は、時間は、上皮細胞の培養を維持するに制限されることになります。上皮細胞が起因してアポトーシスを入力しますようのない上皮細胞ニッチに最小限のは、形成するであろう適切な成長因子又は接触の完全性の損失の欠如は、anokis 7と呼ばれます。 3D-オルガノイド培養システムの出現は、持続的な文化1に腸の細胞型のスペクトルを含む培養一次腸細胞への方法を提供してきました。これらの上皮オルガノイドは、それらがいくつかの分化した細胞で構成されていることである細胞株上の利点を有し、より良いそれらはインビボ 8 に由来する器官を模倣します。最終的に「皿の中のミニ腸を成長させる」ためのプロセスは、様々な刺激下での腸上皮の応答を評価するための貴重なツールであることが証明されています。これらの分子パターンは、ホストと微生物9の両方からの多様な応答を調節することができるように微生物病原体関連分子パターン（のPAMPs）との一次腸細胞の相互作用を調査することは、免疫学の分野に関連します。研究者らは今、マウスオルガノイドとこれらの相互作用を探るが、彼らすることができますだけでなく、ウェル2のようなヒトから培養することができます。この技術は飛躍的にオーダーメイド医療を変える可能性を持っており、この技術は、近い将来に可能になることを進歩について推測したくなります。 この方法の全体的な目標は、種々の刺激で文化、拡張のためのプロトコル、および腸オルガノイドの治療を提供することにあります。このような刺激は、最終的なワクチン、細菌のPAMP、生きた病原体、胃腸（GI）癌治療の範囲とすることができます。マウスの腸オルガノイドの単離および培養は、佐藤らから適応されています。元のメソッドから若干のずれがありますが、このプロトコルを以下のとき、オルガノイド培養される最終製品がまだ達成されています。この方法は、セルnに基づいて分析を行う際に考慮されなければならない不均一なセル構造を操作するとき、適切な正規化のための適切な手法を説明に焦点を当てていますアンバー。

<table><tbody><tr><td>Fetal Bovine Serum (FBS)</td><td>Atlanta Biologicals</td><td>S11050</td><td>(Section 1,3,6) Or equivalent brand</td></tr><tr><td>Sorvall Legend XTR Centrifuge</td><td>Thermo</td><td></td><td>(Section 1,3)</td></tr><tr><td>DMEM</td><td>GE Healthcare</td><td>Sh30243.01</td><td>(Section 1,6) For Caco-2 and HEK293 Rspondin1 cells</td></tr><tr><td>HEK293T-Rspondin1 secreting cell line</td><td></td><td></td><td>(Section 1) Described and modified from Kim, K.A. et al. Lentiviral particles contained RSPO1(NM_138683) ORF cDNA cloned into a pReceiver-Lv105 backbone custom ordered and purchased from GeneCopoeia.&amp;nbsp;</td></tr><tr><td>50 ml conical tube</td><td>Falcon</td><td>352070</td><td>(Section 1) Or equivalent brand</td></tr><tr><td>T-175 Flask</td><td>Corning</td><td>431079</td><td>(Section 1) Or equivalent brand&amp;nbsp;</td></tr><tr><td>Protein Matrix</td><td>Corning</td><td>356231</td><td>(Section 2,3,4,5,6) Matrigel Growth Factor Reduced&amp;nbsp;</td></tr><tr><td>HyClone Dulbecco&#039;s (DPBS)</td><td>GE Healthcare</td><td>SH30264.01</td><td>(Section 2,3)</td></tr><tr><td>DMEM/F12&amp;nbsp;</td><td>Life Technologies</td><td>12634-010</td><td>(Section 2,3) Advanced DMEM/F12</td></tr><tr><td>Corning 24 Well TC Plates</td><td>Corning</td><td>3524</td><td>(Section 2)</td></tr><tr><td>N2 Supplement 100x&amp;nbsp;</td><td>Life Technologies</td><td>17502-048</td><td>(Section 2)</td></tr><tr><td>B27 without vitamin A 50x&amp;nbsp;</td><td>Life Technologies</td><td>12587-010&amp;nbsp;</td><td>(Section 2)</td></tr><tr><td>Trizol</td><td>Life Technologies</td><td>15596-026</td><td>(Section 2)</td></tr><tr><td>Glutamine Supplement (Glutamax)</td><td>Life Technologies</td><td>35050-061</td><td>(Section 2) Can Combine with Advanced DMEM/ F12</td></tr><tr><td>HEPES (1 M)</td><td>Life Technologies</td><td>15630-080</td><td>(Section 2) Can Combine with Advanced DMEM/ F12&amp;nbsp;</td></tr><tr><td>10ml Serological Pipet</td><td>Falcon</td><td>357551</td><td>(Section 2) Or equivalent brand</td></tr><tr><td>Murine Noggin</td><td>Peprotech</td><td>250-38</td><td>(Section 2) Stock = 100 mg/ml</td></tr><tr><td>N-Acetyl-L-cysteine</td><td>Sigma-Aldrich</td><td>A9165</td><td>(Section 2) Stock = 1M</td></tr><tr><td>Recombinant Mouse EGF</td><td>Biolegend</td><td>585608</td><td>(Section 2) Stock = 500 mg/ml</td></tr><tr><td>Rocker Variable</td><td>Bioexpres</td><td></td><td>(Section 3)</td></tr><tr><td>dissecting scissors</td><td></td><td></td><td>(Section 3)</td></tr><tr><td>forceps</td><td></td><td></td><td>(Section 3)</td></tr><tr><td>glass slides</td><td></td><td></td><td>(Section 3)</td></tr><tr><td>dissecting tweezers</td><td></td><td></td><td>(Section 3)</td></tr><tr><td>25 ml Serological Pipet</td><td>Falcon</td><td></td><td>(Section 3)</td></tr><tr><td>EDTA&amp;nbsp;</td><td>Sigma-Aldrich</td><td>SLBB9821</td><td>(Section 3) 0.5M or alternative TC grade EDTA</td></tr><tr><td>Sterile Petri Dish 100mm x 15mm</td><td>Fisher</td><td>FB0875712</td><td>(Section 3) Or equal sized TC dish</td></tr><tr><td>1ml Syringe</td><td>Becton Dickinson</td><td>309659</td><td>(Section 4)</td></tr><tr><td>Precision Glide Needle</td><td>Becton Dickinson</td><td>305120</td><td>(Section 4) 23G x 1 1/4 (0.6mm x 30mm)</td></tr><tr><td>Flagellin from Bacillus subtilis</td><td>Invivogen</td><td>tlrl-bsfla&amp;nbsp;</td><td>(Section 5,6)</td></tr><tr><td>Listeria monocytogenes</td><td>ATCC</td><td>19115</td><td>(Section 5,6)&amp;nbsp;(Murray&amp;nbsp;et&amp;nbsp;al.)&amp;nbsp;</td></tr><tr><td>Hemocytometer</td><td>Sigma-Aldrich</td><td>Z359629-1EA</td><td>(Section 5,6)Or equivalent brand</td></tr><tr><td>BBL Brain Heart Infusion Agar</td><td>Becton Dickinson</td><td>211065</td><td>(Section 5)</td></tr><tr><td>Bacto Brain Heart Infusion</td><td>Becton Dickinson</td><td>237500</td><td>(Section 5)</td></tr><tr><td>Caco-2</td><td>ATCC</td><td>HTB-37</td><td>(Section 6)</td></tr><tr><td>Trypsin&amp;nbsp;</td><td>gibco</td><td>25200056</td><td>(section 6)</td></tr><tr><td>Methanol</td><td>Fisher</td><td>A412-4</td><td>(Section 6)</td></tr><tr><td>SpectraMax M5</td><td>Molecuar Devices</td><td></td><td>(Section 6)</td></tr><tr><td>96 Well Assay Plate</td><td>Corning</td><td>3603</td><td>(Section 6) Black Plate, Clear Bottom TC treated</td></tr><tr><td>Nuclear Staining Dye</td><td>Life Technologies</td><td>H1399</td><td>(section 6) Hoechst 33342</td></tr><tr><td>T-75 Flask</td><td>Corning</td><td>430641</td><td>(Section 6) Or equivalent brand</td></tr><tr><td>15 ml conical tube</td><td>Falcon</td><td>352096</td><td>(Section1,3) Or equivalent brand</td></tr><tr><td>1.7 ml polypropylene tube</td><td>Bioexpress</td><td>C-3262-1</td><td>Or equivalent brand</td></tr><tr><td>Quick-RNA MiniPrep</td><td>Zymo Research</td><td>R1054</td><td>Or equivalent brand</td></tr><tr><td>TNF-alpha&amp;nbsp;</td><td>Applied Biosystems</td><td>Mm 00443260_g1</td><td>Taqman gene expression assay kit</td></tr><tr><td>IL-6&amp;nbsp;</td><td>Applied Biosystems</td><td>(Mm 00446190_m1</td><td>Taqman gene expression assay kit</td></tr><tr><td>IL-1beta</td><td>Applied Biosystems</td><td>Mm 00434228_m1</td><td>Taqman gene expression assay kit</td></tr><tr><td>IL-18</td><td>Applied Biosystems</td><td>Mm 00434225_m1</td><td>Taqman gene expression assay kit</td></tr><tr><td>18s</td><td>Applied Biosystems</td><td>Hs 99999901_s1</td><td>Taqman gene expression assay kit</td></tr><tr><td>7500 Fast Real Time PCR System</td><td>Applied Biosystems</td><td></td><td></td></tr><tr><td>Nexus gradient Mastercycler</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>TaqMan Fast Universal PCR Master Mix</td><td>Life Technologies</td><td>4352042</td><td></td></tr><tr><td>High Capacity cDNA Reverse Transcription Kit</td><td>Life Technologies/Applied Biosystems</td><td>4368814</td><td></td></tr><tr><td>Fast Optical 96-Well Reaction Plate, 0.1 mL</td><td>Life Technologies/Applied Biosystems</td><td>4346907</td><td></td></tr><tr><td>Recombinant Mouse R-Spondin 1 Protein</td><td>R&amp;amp;D Systems</td><td>3474-RS-050</td><td>500 ng/ml</td></tr><tr><td>chloroform</td><td>Sigma-Aldrich</td><td>C7559</td><td></td></tr></tbody></table>

the ex vivo culture and pattern recognition receptor stimulation of mouse intestinal organoids

主要腸オルガノイドを大幅粘膜免疫学の分野に影響を与える可能性があり、貴重なモデル系です。しかし、オルガノイド成長特性の複雑さは、研究者のための重要な注意事項を運びます。具体的には、各個々のオルガノイドの成長パターンは非常に可変であり、培養中の上皮細胞の異種集団を作成します。このような警告と、一般的な組織培養の実践は、単にによる細胞構造の複雑さオルガノイドシステムに適用することはできません。いくつかの正規化技術が適用されない限り、カウントし、このような細胞株として個々に分離された細胞のための一般的な細胞数、のみに基づいてメッキオルガノイドのための信頼できる方法はありません。総タンパク質含量に対して正規化が常駐タンパク質マトリックスによる複合体形成されています。分泌されたコンを評価する際に、セル数、形状、および細胞型の点でこれらの特性を考慮しなければなりませんオルガノイド塊からテント。このプロトコルは、文化に簡単な手順の概要を説明し、微生物病原体や病原体関連分子パターン（のPAMPs）と小腸オルガノイドを治療するために生成されています。それはまた、タンパク質の分析は、そのようなチャレンジ後に行われているときに適用されるべき正規化技術を強調する。

腸のオルガノイドを育成するには、このプロトコルを、以下の場合には、特徴的な球体状のオルガノイドは、収穫後に存在するであろう。 R-spondin1馴化培地の添加は、毎日オルガノイドの成長と出芽を開始します。オルガノイドの成長は、 図 1Aに示されている- F、および1日目に、腸オルガノイドの代表、2、4、5、6と14日目?...

Watch this Scientific Journal Video about The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids at JoVE.com

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

ここでは、収穫維持、および病原体関連分子パターン（のPAMPs）とリステリア菌でマウス小腸オルガノイドを治療するためのプロトコルが記載されているだけでなく、遺伝子発現やタンパク質のための適切な正規化技術を重視。

ザ<em&gt; ex vivoで</emマウス腸オルガノイドの&gt;文化とパターン認識受容体刺激

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the ...

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Research

JoVE Journal

Immunology and Infection

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

13.5K ビュー.  Virginia Maryland College of Veterinary Medicine, Virginia Tech. ここでは、収穫維持、および病原体関連分子パターン（のPAMPs）とリステリア菌でマウス小腸オルガノイドを治療するためのプロトコルが記載されているだけでなく、遺伝子発現やタンパク質のための適切な正規化技術を重視。 

ビデオ: The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

In vitro Organoid Culture of Primary Mouse Colon Tumors

Several human and murine colon cancer cell lines have been established, physiologic integrity of colon tumors such as multiple cell layers, basal-apical polarity, ability to differentiate, and anoikis are not maintained in colon cancer derived cell lines. The present study demonstrates a method for culturing primary mouse colon tumor organoids adapted from Sato T et al. 1, which retains important physiologic features of colon tumors. This method consists of mouse colon tumor tissue collection, adjacent normal colon epithelium dissociation, colon tumor cells digestion into single cells, embedding colon tumor cells into matrigel, and selective culture based on the principle that tumor cells maintain growth on limiting nutrient conditions compared to normal epithelial cells.
The primary tumor organoids if isolated from genetically modified mice provide a very useful system to assess tumor autonomous function of specific genes. Moreover, the tumor organoids are amenable to genetic manipulation by virus meditated gene delivery; therefore signaling pathways involved in the colon tumorigenesis could also be extensively investigated by overexpression or knockdown. Primary tumor organoids culture provides a physiologic relevant and feasible means to study the mechanisms and therapeutic modalities for colon tumorigenesis.

In vitro Organoid Culture of Primary Mouse Colon Tumors

A simple method to establish primary murine colon tumor organoid is described. This method utilizes the feature that colon tumor cells survive and grow into organoids in media containing limited growth factors, whereas normal colon epithelial do not.

プライマリマウス大腸腫瘍のin vitroオルガノイド培養で

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

医学

Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo

The intestinal epithelium regenerates every 5-7 days, and is controlled by the intestinal epithelial stem cell (IESC) population located at the bottom of the crypt region. IESCs include active stem cells, which self-renew and differentiate into various epithelial cell types, and quiescent stem cells, which serve as the reserve stem cells in the case of injury. Regeneration of the intestinal epithelium is controlled by the self-renewing and differentiating capabilities of these active IESCs. In addition, the balance of the crypt stem cell population and maintenance of the stem cell niche are essential for intestinal regeneration. Organoid culture is an important and attractive approach to studying proteins, signaling molecules, and environmental cues that regulate stem cell survival and functions. This model is less expensive, less time-consuming, and more manipulatable than animal models. Organoids also mimic the tissue microenvironment, providing in vivo relevance. The present protocol describes the isolation of colonic crypts, embedding these isolated crypt cells into a three-dimensional gel matrix system and culturing crypt cells to form colonic organoids capable of self-organization, proliferation, self-renewal, and differentiation. This model allows one to manipulate the environment-knocking out specific proteins such as claudin-7, activating/deactivating signaling pathways, etc.-to study how these effects influence the functioning of colonic stem cells. Specifically, the role of tight junction protein claudin-7 in colonic stem cell function was examined. Claudin-7 is vital for maintaining intestinal homeostasis and barrier function and integrity. Knockout of claudin-7 in mice induces an inflammatory bowel disease-like phenotype exhibiting intestinal inflammation, epithelial hyperplasia, weight loss, mucosal ulcerations, epithelial cell sloughing, and adenomas. Previously, it was reported that claudin-7 is required for intestinal epithelial stem cell functions in the small intestine. In this protocol, a colonic organoid culture system is established to study the role of claudin-7 in the large intestine.

Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo

The present protocol describes establishing a murine colonic organoid system to study the activity and functioning of colonic stem cells in a claudin-7 knockout model.

腸幹細胞機能をex vivoで研究するためのマウス結腸陰窩の三次元培養

The intestinal epithelium regenerates every 5-7 days, and is controlled by the intestinal epithelial stem cell (IESC) population located at the bottom of ...

がん研究

The Organoid Reconstitution Assay (ORA) for the Functional Analysis of Intestinal Stem and Niche Cells

The intestinal epithelium is characterized by an extremely rapid turnover rate. In mammals, the entire epithelial lining is renewed within 4 - 5 days. Adult intestinal stem cells reside at the bottom of the crypts of Lieberk&#252;hn, are earmarked by expression of the Lgr5 gene, and preserve homeostasis through their characteristic high proliferative rate1. Throughout the small intestine, Lgr5+ stem cells are intermingled with specialized secretory cells called Paneth cells. Paneth cells secrete antibacterial compounds (i.e., lysozyme and cryptdins/defensins) and exert a controlling role on the intestinal flora. More recently, a novel function has been discovered for Paneth cells, namely their capacity to provide niche support to Lgr5+ stem cells through several key ligands as Wnt3, EGF, and Dll12.
When isolated ex vivo and cultured in the presence of specific growth factors and extracellular matrix components, whole intestinal crypts give rise to long-lived and self-renewing 3D structures called organoids that highly resemble the crypt-villus epithelial architecture of the adult small intestine3. Organoid cultures, when established from whole crypts, allow the study of self-renewal and differentiation of the intestinal stem cell niche, though without addressing the contribution of its individual components, namely the Lgr5+ and Paneth cells.
Here, we describe a novel approach to the organoid assay that takes advantage of the ability of Paneth and Lgr5+ cells to associate and form organoids when co-cultured. This approach, here referred to as &#34;organoid reconstitution assay&#34; (ORA), allows the genetic and biochemical modification of Paneth or Lgr5+ stem cells, followed by reconstitution into organoids. As such, it allows the functional analysis of the two main components of the intestinal stem cell niche.

The Organoid Reconstitution Assay ORA for the Functional Analysis of Intestinal Stem and Niche Cells

Intestinal organoid cultures are established from whole crypts and do not allow the analysis of self-renewal and differentiation in a cell-specific fashion. This protocol describes reconstitution of sorted stem (Lgr5+) and niche (Paneth) cells, which give rise to organoids while enabling their prior biochemical and genetic modification and functional analysis.

腸幹とニッチ細胞機能解析における再構成アッセイ (ORA)

The intestinal epithelium is characterized by an extremely rapid turnover rate. In mammals, the entire epithelial lining is renewed within 4 - 5 days. ...

生物工学

The Ex Vivo Colon Organ Culture and Its Use in Antimicrobial Host Defense Studies

The intestine displays an architecture of repetitive crypt structures consisting of different types of epithelial cells, lamina propia containing immune cells, and stroma. All of these heterogeneous cells contribute to intestinal homeostasis and participate in antimicrobial host defense. Therefore, identifying a surrogate model for studying immune response and antimicrobial activity of the intestine in an in vitro setting is extremely challenging. In vitro studies using immortalized intestinal epithelial cell lines or even primary crypt organoid culture do not represent the exact physiology of normal intestine and its microenvironment. Here, we discuss a method of culturing mouse colon tissue in a culture dish and how this ex vivo organ culture system can be implemented in studies related to antimicrobial host defense responses. In representative experiments, we showed that colons in organ culture express antimicrobial peptides in response to exogenous IL-1&#946; and IL-18. Further, the antimicrobial effector molecules produced by the colon tissues in the organ culture efficiently kill Escherichia coli in vitro. This approach, therefore, can be utilized to dissect the role of pathogen- and danger-associated molecular patterns and their cellular receptors in regulating intestinal innate immune responses and antimicrobial host defense responses.

The Ex Vivo Colon Organ Culture and Its Use in Antimicrobial Host Defense Studies

The ex vivo organ culture allows investigation of biological processes in the context of the intact tissue architecture. Here, we introduce a method of ex vivo culture of the mouse colon, which can be used to study innate immunity and antimicrobial host defense in the intestine.

ザ<em&gt; ex vivoで</em&gt;コロン器官培養および抗菌生体防御研究におけるその使用

The intestine displays an architecture of repetitive crypt structures consisting of different types of epithelial cells, lamina propia containing immune ...

免疫・感染学

Co-Culture of Murine Small Intestine Epithelial Organoids with Innate Lymphoid Cells

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate balance of mucosal homeostasis. These 3D, multi-cellular systems offer a reductionist model for addressing multi-factorial diseases and resolving technical difficulties that arise when studying rare cell types such as tissue-resident innate lymphoid cells (ILCs). This article describes a murine system that combines small intestine organoids and small intestine lamina propria derived helper-like type-1 ILCs (ILC1s), which can be readily extended to other ILC or immune populations. ILCs are a tissue-resident population that is particularly enriched in the mucosa, where they promote homeostasis and rapidly respond to damage or infection. Organoid co-cultures with ILCs have already begun shedding light on new epithelial-immune signaling modules in the gut, revealing how different ILC subsets impact intestinal epithelial barrier integrity and regeneration. This protocol will enable further investigations into reciprocal interactions between epithelial and immune cells, which hold the potential to provide new insights into the mechanisms of mucosal homeostasis and inflammation.

This protocol offers detailed instructions for establishing murine small intestine organoids, isolating type-1 innate lymphoid cells from the murine small intestine lamina propria, and establishing 3-dimensional (3D) co-cultures between both cell types to study bi-directional interactions between intestinal epithelial cells and type-1 innate lymphoid cells.

マウス小腸上皮オルガノイドと先天性リンパ球細胞との共培養

Complex co-cultures of organoids with immune cells provide a versatile tool for interrogating the bi-directional interactions that underpin the delicate ...

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding primary 3D epithelial structures in vitro. Both the architecture and physiological properties of these &#39;mini-guts&#39;, also called organoids, closely resemble their in vivo counterparts. This makes them an attractive model system for the small intestinal epithelium. Using retroviral transduction, functional genetics can now be performed by conditional gene overexpression or knockdown. This video demonstrates the procedure of organoid culture, the generation of retroviruses, and the retroviral transduction of organoids to assist phenotypic analysis of the small intestinal epithelium in vitro. This novel organotypic model system in combination with retroviral mediated gene expression provides a valuable tool for rapid analysis of gene function in vitro without the need of costly and time-consuming generation for transgenic animals.

This protocol explains primary Lgr5-positve organoid culture and the subsequent performance of retroviral transduction. This enables Cre-inducible overexpression or knockdown of the delivered transgene and allows functional studies to be carried out in the novel in vitro organotypic model system.

初代腸オルガノイド培養におけるレトロウイルス感染のビデオプロトコル

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding ...

生物学

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine1. Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought1. The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology and three-dimensional reconstruction software, the ability to visualize intact, developing organs is rapidly improving. Two-photon excitation allows less damaging penetration deeper into tissues with high resolution. Two-photon imaging and 3D reconstruction of the whole fetal mouse intestines in Walton et al., 2012 helped to define the pattern of villus outgrowth2. Here we describe a whole organ culture system that allows ex vivo development of villi and extensions of that culture system to allow the intestines to be three-dimensionally imaged during their development.

Mouse Fetal Whole Intestine Culture System for Ex Vivo Manipulation of Signaling Pathways and Three-dimensional Live Imaging of Villus Development

Improved imaging technology is allowing three-dimensional imaging of organs during development. Here we describe a whole organ culture system that allows live imaging of the developing villi in the fetal mouse intestine.

マウス胎児腸全体培養システム<em&gt;生体外</em&gt;シグナル伝達経路の操作と絨毛開発の3次元ライブイメージング

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over ...

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.

In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry.

レンチウイルス形質導入と腸オルガノイドの下流分析のためのプロトコル

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth ...

Culture Methods to Study Apical-Specific Interactions using Intestinal Organoid Models

The lining of the gut epithelium is made up of a simple layer of specialized epithelial cells that expose their apical side to the lumen and respond to external cues. Recent optimization of in vitro culture conditions allows for the re-creation of the intestinal stem cell niche and the development of advanced 3-dimensional (3D) culture systems that recapitulate the cell composition and the organization of the epithelium. Intestinal organoids embedded in an extracellular matrix (ECM) can be maintained for long-term and self-organize to generate a well-defined, polarized epithelium that encompasses an internal lumen and an external exposed basal side. This restrictive nature of the intestinal organoids presents challenges in accessing the apical surface of the epithelium in vitro and limits the investigation of biological mechanisms such as nutrient uptake and host-microbiota/host-pathogen interactions. Here, we describe two methods that facilitate access to the apical side of the organoid epithelium and support the differentiation of specific intestinal cell types. First, we show how ECM removal induces an inversion of the epithelial cell polarity and allows for the generation of apical-out 3D organoids. Second, we describe how to generate 2-dimensional (2D) monolayers from single cell suspensions derived from intestinal organoids, comprised of&#160;mature and differentiated cell types. These techniques provide novel tools to study apical-specific interactions of the epithelium with external cues in vitro and promote the use of organoids as a platform to facilitate precision medicine.

Here we present two protocols that allow the modeling of intestinal apical-specific interactions. Organoid-derived intestinal monolayers and Air-Liquid Interface (ALI) cultures facilitate the generation of well-differentiated epithelia accessible from both luminal and basolateral sides, whereas polarity-inverted intestinal organoids expose their apical side and are amenable to high throughput assays.

腸オルガノイドモデルを用いたアプリカル特異的相互作用を研究する培養法

The lining of the gut epithelium is made up of a simple layer of specialized epithelial cells that expose their apical side to the lumen and respond to ...

マウス腸陰窩および単一幹細胞からのオルガノイドの3D培養

The 3D Culturing of Organoids from Murine Intestinal Crypts and a Single Stem Cell for Organoid Research

At present, organoid culture represents an important tool for in vitro studies of different biological aspects and diseases in different organs. Murine small intestinal crypts can form organoids that mimic the intestinal epithelium when cultured in a 3D extracellular matrix. The organoids are composed of all cell types that fulfill various intestinal homeostatic functions. These include Paneth cells, enteroendocrine cells, enterocytes, goblet cells, and tuft cells. Well-characterized molecules are added into the culture medium to enrich the intestinal stem cells (ISCs) labeled with leucine-rich repeats containing G protein-coupled receptor 5 and are used to drive differentiation down specific lineages; these molecules include epidermal growth factor, Noggin (a bone morphogenetic protein), and R-spondin 1. Additionally, a protocol to generate organoids from a single erythropoietin-producing hepatocellular receptor B2 (EphB2)-positive ISC is also detailed. In this methods article, techniques to isolate small intestinal crypts and a single ISC from tissues and ensure the efficient establishment of organoids are described.

著者スポットライト:オルガノイド研究のためのマウス腸管陰窩からのオルガノイドと単一幹細胞の3D培養  

We describe a protocol to isolate murine small intestinal crypts and culture intestinal 3D organoids from the crypts. Additionally, we describe a method to generate organoids from a single intestinal stem cell in the absence of a sub-epithelial cellular niche.

オルガノイド研究のためのマウス腸陰窩および単一幹細胞からのオルガノイドの3D培養

At present, organoid culture represents an important tool for in vitro studies of different biological aspects and diseases in different organs. Murine ...

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