The authors acknowledge the Microscopy core facility at the Faculty of Health Sciences, Link&#246;ping University for providing access to advanced imaging systems; Karl-Eric Magnusson (Emeritus Scientist) at the Dept. of Clinical and Experimental Medicine, Link&#246;ping University for providing access to Imaris 3D/4D image processing software (Bitplane, Switzerland); and S. Braian for his help with the lung model cartoon. This work was supported by funds from the Swedish Research Council (Alternatives to animal research, 2012-1951) and Swedish Research Council (2012-3349) to M.L. and Swedish Foundation for Strategic Research to S.B. S.B. receive grants from the Karolinska Institutet, Swedish Research Council, the Swedish International Development Cooperation Agency (Sida) and the Swedish Civil Contingencies Agency (MSB), and the Swedish Heart and Lung Foundation (HLF). M.S. received grants from the Karolinska Institutet and Stockholm County Council.

<ol>
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The authors declare no competing financial interests.

The ability to recruit and form organized cell clusters at the site of infection is the hallmark of human TB 11. These dynamic structures known as tubercle granulomas primarily consist of immune cells (macrophages, monocytes, T-cells and B-cells) and multi-nucleated giant cells surrounding M. tuberculosis. The role of the granuloma has long been considered to wall off the infection, preventing local spread of bacteria. However, more recent studies show that granuloma formation is critical for early bacterial survival, growth and dissemination 12. A strategy of new studies is to identify molecules or pathways that could efficiently be targeted to inhibit the cellular migration in granuloma formation and/or TB dissemination. 
A caveat for novel studies on TB is the lack of models that recapitulate human TB. The most widely used experimental animals do not form true granuloma upon M. tuberculosis infection, and are therefore not appropriate choices for studies of TB 13-16. Non-human primates have the closest resemblance to human TB 17, but are not the preferred choice owing to high operational costs and ethical issues. Human TB is a complex immunological process and is difficult to model in vitro. Cell cultures of monolayers or co-cultures lack the 3D environment and tissue responses. Therefore, we have developed an innovative lung tissue model based on human primary immune cells and human lung-specific cell lines 8,9. The model displays characteristic features of human lung tissue, including epithelia with evenly integrated macrophages, formation of extracellular matrix, stratified epithelia and mucus secretion 9. 
The 3D human lung tissue model has several benefits over the in vitro single or co-cultures seeded on tissue culture plates or transwell inserts. First, the human lung-specific cells (fibroblasts and epithelial cells) are not commonly included in the in vitro single or co-cultures. Second, the immune cells and lung-specific cells are embedded in a 3D physiological context (collagen rich extra-cellular matrix products). The response of cells to a stimulus/infection and the migratory behaviour of cells, for instance formation of a granuloma, differ significantly between a 2D and 3D environment. Furthermore, the described method enables the 3D visualization and robust 3D quantitative analysis that provides pivotal information on spatial distribution and intricate cellular interactions. 
Experimental infection in the model tissue with M. tuberculosis resulted in clustering of macrophages at the site of infection, reminiscent of early TB granuloma (Figure 2 and 3). We have recently demonstrated that mutant strains defective in the ability to secrete the virulence factor ESAT-6 or Mycobacterium bovis BCG that lacks ESAT-6 did not induce the clustering of monocytes (no early granuloma), in contrast to the virulent M. tuberculosis 9. These data are consistent with the observations made from Mycobacterium marinum-infected zebrafish embryos, whose transparency allows for elegant live imaging of granuloma formation 12. As there is no gold-standard model for TB, we took advantage of the surgically resected tissue biopsies from TB patients for validation of the method 9. Our in vitro tissue model shares several characteristics with the lung and lymph node biopsies from TB patients, including the aggregation of macrophages in granuloma, the presence of both intra- and extracellular bacteria 18 and induction of necrosis 11. 
Although the described model has physiological relevance to human TB and has several advantages over other in vitro models, it has some limitations. For instance, out of more than 20 collagen proteins identified in humans, only type I is included to the model to mimic the extra-cellular matrix. However, type I collagen is a complex mixture of extra-cellular matrix products and is the most abundant collagen in the human body. Further, we have demonstrated the presence of collagen IV and several extra-cellular matrix proteins such as tropoelastin, vimentin and laminin, which are produced by the epithelial cells and fibroblasts in the tissue model, indicating the synthesis of new collagen 8. Presently, the lung tissue model only has monocytes and macrophages, besides lung-specific cells. It lacks neutrophils and lymphocytes that are also known to be present in the granuloma. Remarkably the model is not limited to the introduction of additional immune cells and is of interest to explore how they contribute to the complex cellular interactions in human TB. Implantation of primary alveolar macrophages, skin-specific cells and lung carcinoma cells has already been tested in the model. Since our objective was to use a model that closely resembles human TB, introduction of mouse cells have not been attempted.
In summary, the lung tissue model has implications for both basic mechanistic and applied studies. Potential applications of the lung model include the study of innate immunity, investigating mechanistic aspects of host defences such as phagosomal maturation, autophagy, production of cytokines, chemokines and anti-microbial peptides, and functional characterization of individual cell types. Strikingly, the in vitro tissue model allows manipulation of one or more cells types and provides a relevant tissue micro-environment, not only for studies on TB, but for a variety of infectious and non-infectious diseases that affect the lungs.

في البشر، والاستجابات للعدوى، والتهاب الأنسجة، والتوظيف الخلوية، وإعادة عرض الأنسجة وتنظيم توازن الأنسجة هي الأحداث المعقدة التي تنطوي على أنواع مختلفة من الخلايا. وبالتالي، فإن هذه العمليات أفضل درس في بيئة النسيج المحلية. سابقا، وقد كان هذا أساسا الممكن استخدام النماذج الحيوانية التجريبية. ومع ذلك، فإن حيوانات التجارب المستخدمة على نطاق واسع عقد العديد من حدود لأنها غالبا ما تستجيب لمسببات الأمراض بطريقة مختلفة من البشر وأيضا عرض مسارا مختلفا من مرض 1. A البشرية في المختبر نموذج أنسجة الرئة يحمل إمكانيات دراسة الاستجابات المناعية محددة في رئة الإنسان. عدوى السل البشري (TB) هو أساسا مرض يصيب الرئتين. المتفطرة السلية (M. السل)، العامل المسبب للمرض السل، تصل إلى الرئة عن طريق قطرات التي يتم نقلها إلى الفضاء السنخية، حيث اجتاحت البكتيريا عن طريق dendri الرئويةخلايا التشنج والضامة السنخية كجزء من الاستجابة المناعية الفطرية للعدوى 2،3. البلعمة من مسببات المرض يؤدي إلى تجزئة علة ضمن يبلوع ومثالي يؤدي إلى تحييد وقتل العامل الممرض من قبل بلعمية. ما يصل الى 50٪ من الأشخاص الذين تعرضوا لM. ويعتقد أن مرض السل لتكون قادرة على مسح العدوى عن طريق الاستجابة المناعية الفطرية 4. النتائج الأخرى للعدوى هي تطهير من قبل النظام المناعي التكيفي في مرحلة لاحقة، والعدوى كامنة أو في أسوأ الحالات المرض المزمن النشط 5. في السابق لم تكن هناك نماذج الأنسجة في المختبر للدراسات السل البشري. وكثيرا ما استخدمت الثقافات وحيدة الخلية الضامة الإنسان أو خلايا الدم الطرفية الأخرى 6،7. وعيب هذا النهج هو أنها لا يمكن أن تعكس ديناميكية أنواع مختلفة من الخلايا التي تعمل معا في نسيج الرئة تتعرض لM. مرض السل </eم&gt;. وبالتالي، هناك حاجة إلى نموذج في المختبر لتكون قادرة على إجراء دراسات الفنية والميكانيكية على السل. أنشئت في المختبر نموذج أنسجة الرئة الإنسان هو موضح هنا خلية القاعدة في الأصل من قبل مجموعتنا لإجراء دراسات عن وظائف الخلية الجذعية 8 لقد تكيفت هذه الطريقة لدراسة مرض السل. نموذج أنسجة الرئة البشرية المقدمة هنا يتكون من خلايا والخلايا الليفية 8 الظهارية الأنسجة محددة. هذه الخلايا يتم تربيتها في مصفوفة الكولاجين على رأس غشاء مسامي في إدراج transwell وهياكل تشبه شكل العادية أنسجة الرئة الإنسان (الشكل 1). عند تعرضها للهواء تبدأ الخلايا على إفراز المخاط في الجانب قمي 8. من خلال زرع الضامة الإنسان الأساسية المصابين M. السل للنموذج، لاحظنا كيف تهاجر الخلايا المناعية في الأنسجة وتشكل المراحل الأولى من حبيبية TB 9. وهذا هو أول البشري نموذج الأنسجة described لمكافحة السل وأنه يشكل أداة واعدة لدراسة الاستجابات المناعية الفطرية لمرض السل وغيرها من الأمراض في الرئة. حتى الآن، وقد استخدمنا فقط وحيدات الضامة كما الخلايا المناعية في النموذج ولكن يمكن زيادة مستوى التعقيد من خلال إدراج أنواع الخلايا الإضافية ذات الصلة. <img alt="الشكل 1" src="/files/ftp_upload/53084/53084fig1.jpg" /> الشكل 1. مخطط تخطيطي لنموذج أنسجة الرئة. (A) ويتكون هذا النموذج من خلايا الرئة البشرية خاصة الظهارية، M. السل -infected الضامة الابتدائية والمصنفة صبغة حمراء حيدات المسمى على الكولاجين جزءا لا يتجزأ من الخلايا الليفية التي أعدت حول مرشح transwell. التعرض للنموذج النسيج في الهواء يبدأ إنتاج البروتينات المصفوفة خارج الخلوي وإفراز المخاط والطبقية التي كتبها الظهارة. نموذج الأنسجة 3D بالتالي المتقدمة هو أداة مفيدة لدراسة M. عدوى السل في بيئة CLOيشبه كتبها sely رئة الإنسان. (B) الصور المجهرية التمثيلية للخطوات المختلفة في إعداد نموذج الأنسجة. (C) الهيكل الكامل للقسم الأنسجة نموذج الرئة. على نطاق و- 100 ميكرون <a href="https://www.jove.com/files/ftp_upload/53084/53084fig1large.jpg" target="_blank">الرجاء انقر هنا لعرض نسخة أكبر من هذا الرقم.</a>

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Cell culture inserts</td>
			<td>BD Falcon</td>
			<td>353092</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well culture plates</td>
			<td>BD Falcon</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>MRC-5 cells, lung fibroblasts</td>
			<td></td>
			<td></td>
			<td>ATCC#CCL-171</td>
		</tr>
		<tr>
			<td>16HBE cells, lung epithelial cells</td>
			<td></td>
			<td></td>
			<td>Gift from Dr. Dieter Gruenert, Mt. Zion Cancer Center, University of California, San Fransisco, USA&#160;</td>
		</tr>
		<tr>
			<td>5 x Dulbecco&#8217;s modified Eagle&#8217;s medium (5 x DMEM)</td>
			<td>Gibco</td>
			<td>12800-082</td>
			<td>Made from powder but add 5 times less water. Adjust pH to 7.3 and filter it using a 0.2 &#181;m filter.</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified Eagle&#8217;s medium with glucose (DMEM) 1x</td>
			<td>Gibco</td>
			<td>41965-039</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimum Essential Medium (MEM) 1x with Earle&#8217;s salts</td>
			<td>Sigma</td>
			<td>M4655</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-Essential Amino Acids Solution, 100x</td>
			<td>Life Technologies</td>
			<td>11140-035</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine 200 mM (100x)</td>
			<td>Gibco</td>
			<td>25030-024</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Life Technologies</td>
			<td>11360-039</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3 (71.2 mg/ml)</td>
			<td></td>
			<td></td>
			<td>Prepared in house</td>
		</tr>
		<tr>
			<td>Heat inactivated Fetal Bovine Serum (FBS)</td>
			<td>Gibco</td>
			<td>10270-106</td>
			<td>Heat inactivated for 30 min, 56 &#176;C</td>
		</tr>
		<tr>
			<td>Gentamicin (50 mg/ml)</td>
			<td>Gibco</td>
			<td>15750-060</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes buffer solution 1M</td>
			<td>Gibco</td>
			<td>15630-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin (Pen Strep)</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Lymphoprep</td>
			<td>Axis-Shield</td>
			<td>7801</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure 0.5 M EDTA</td>
			<td>Gibco</td>
			<td>15575</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Collagen PA treated (500 ml)</td>
			<td>Organogenesis</td>
			<td>200-055</td>
			<td></td>
		</tr>
		<tr>
			<td>Pure col purified Bovine Collagen solution (100 ml)</td>
			<td>Advanced biomatrix</td>
			<td>5005-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Extracellular matrix protein, Fibronectin (1 mg)</td>
			<td>BD</td>
			<td>354008</td>
			<td></td>
		</tr>
		<tr>
			<td>Primary human monocytes/macrophages</td>
			<td></td>
			<td></td>
			<td>Isolated from human whole blood or buffy coats.</td>
		</tr>
		<tr>
			<td>PKH26 Red fluorescent cell linker</td>
			<td>Sigma</td>
			<td>MINI26</td>
			<td></td>
		</tr>
		<tr>
			<td>Mycobacterium tuberculosis H37Rv expressing green fluorescent protein</td>
			<td></td>
			<td></td>
			<td>M. tuberculosis H37Rv wild type was transformed with the pFPV2 plasmid constitutively expressing GFP.</td>
		</tr>
		<tr>
			<td>Middlebrook 7H9 medium&#160;</td>
			<td>Difco</td>
			<td>271310</td>
			<td></td>
		</tr>
		<tr>
			<td>BBL Middlebrook ADC Enrichment</td>
			<td>BBL</td>
			<td>211887</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-80</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin B sulfate (20 &#181;g/ml)</td>
			<td>Sigma</td>
			<td>B5264</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolong Gold anti=-fade reagent with DAPI</td>
			<td>Invitrogen</td>
			<td>P36935</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin -EDTA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LSM700 Confocal microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>iMaris Scientific 3D/4D image processing software, version 7.6.8</td>
			<td>Bitplane AG</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a 3d human lung tissue model for functional studies on mycobacterium tuberculosis infection

السل (TB) لا يزال يحمل خطرا كبيرا على صحة الناس في جميع أنحاء العالم، وهناك حاجة لنماذج فعالة من حيث التكلفة ولكن يمكن الاعتماد عليها لتساعدنا على فهم آليات المرض وتقدم الاكتشافات من خيارات العلاج الجديدة. في المختبر الثقافات خلية من الطبقات الوحيدة أو زملاء الثقافات تفتقر إلى ثلاثية الأبعاد (3D) والبيئة والردود الأنسجة. وهنا، نحن تصف مبتكر في نموذج المختبر من أنسجة الرئة الإنسان، والتي تبشر بالخير لتكون أداة فعالة لدراسة الأحداث المعقدة التي تحدث أثناء الإصابة بمرض السل المتفطرة (M. السل). يتكون نموذج الأنسجة 3D من الخلايا والخلايا الليفية الظهارية الأنسجة محددة، والتي يتم تربيتها في مصفوفة الكولاجين على رأس غشاء مسامي. عند التعرض للهواء، والخلايا الظهارية تطبق وتفرز المخاط في الجانب قمي. عن طريق إدخال الضامة الأولية مع M. الإنسان بالعدوى السل إلى وضع الأنسجةلتر، ونحن قد أظهرت أن الخلايا المناعية تهاجر إلى الأنسجة المصابة وتشكل المراحل المبكرة من الورم الحبيبي السل. هذه الهياكل تلخيص سمة مميزة لمرض السل البشري، الحبيبي، والذي يختلف جذريا أو لم يلاحظ عادة في استخدامها على نطاق واسع النماذج الحيوانية التجريبية. هذه الطريقة ثقافة عضوي النمط يمكن التصور 3D والتحليل الكمي القوي الذي يوفر معلومات مهمة حول الميزات المكانية والزمانية لخلايا المضيف الممرض التفاعلات. معا، ويقدم نموذجا أنسجة الرئة والأنسجة البيئة الصغرى ذات الصلة من الناحية الفسيولوجية للدراسات على مرض السل. وبالتالي، فإن النموذج أنسجة الرئة آثار محتملة على كل الدراسات الميكانيكية والتطبيقية الأساسية. الأهم من ذلك أن النموذج يسمح إضافة أو التلاعب من أنواع الخلايا الفردية، والتي بالتالي يوسع استخدامه لنمذجة مجموعة متنوعة من الأمراض المعدية التي تؤثر على الرئتين.

نموذج الأنسجة 3D الرئة لمكافحة السل البشري يمكن استخدامها بشكل فعال لدراسة التفاعلات المضيف الممرض في M. عدوى السل. الخطوات الأساسية لهذا الأسلوب، وترد الصور المجهرية التمثيلية لمختلف الخطوات وبنية المجهرية العام للقسم الأنسجة في الشكل 1. نموذج لديها العديد من مكونات أنسجة الرئة الإنسان، بما في ذلك الخلايا الليفية الرئة، الخلايا الظهارية الشعب الهوائية وحيدات الأولية / الضامة جزءا لا يتجزأ من البيئة الأنسجة 3D. بالإضافة إلى دمج مكونات أنسجة الرئة الإنسان، ونموذج يشبه الظروف الفسيولوجية وهي الطبقية من ظهائر وإفراز المخاط. وقدم مثالا لاستخدام نموذج أنسجة الرئة في مراقبة عدوى السل في الشكل 2. لتصور M. السل -immune هجرة الخلايا والتفاعل، قدمنا ​​الضامة المصابة M. السل التي تعبر عن GFP(الأخضر) مع حيدات المسمى PKH26 المعزولة حديثا (الأحمر) في نموذج الأنسجة (الأزرق وملطخة دابي لنوى). في يوم 7 بالإضافة منصب M. السل خلايا -infected لنموذج الأنسجة، والفحص المجهري متحد البؤر يكشف تجميع حيدات حمراء في موقع الإصابة (الأخضر) (الشكل 2)، الذي يحاكي الآفات السمة المميزة لمرض السل البشري 9. سلسلة من الصور التمثيلية لل3D التصور من M. -infected السل يظهر نموذج الأنسجة وتقدير من مجموعات الخلايا في الشكل (3). والتصور 3D يعطي مرونة للمستخدم التفاعل وفحصها وتحديد العديد من الميزات في صورة 3D. ويمكن النظر إلى الترتيب المكاني من البكتيريا الخضراء ومجموعات الوحيدات الحمراء من وجهة نظر القمي، والتناوب الجانبي كما هو موضح في الشكل 3B، الذي يكشف عن تجميع وحيدات في موقع M. السل. العناقيد لم أوبخدم في الأنسجة غير المصابة (الشكل 3A). نحن كميا حجم وعدد من مجموعات الخلايا الوحيدات وجدت أن حجم (حجم) من مجموعات الخلايا مما يعزز (P &lt;0.001)، في حين انخفض عدد حيدات الفردية (p &lt;0.01) في M. السل إصابة الأنسجة بالمقارنة مع نماذج الأنسجة غير المصابة (الشكل 3C و 3D). بالتحقق من صحة هذه البيانات لدينا الحقائق السابقة من تشكيل الحبيبي في وقت مبكر من M. لاحظ عدوى السل في نماذج أنسجة الرئة تحليلها في 2D أقسام الأنسجة 9. وتشير البيانات المتوفرة لدينا أن هذا النموذج الأنسجة يوفر الموائل 3D الطبيعي للتحقيق الخلوي المضيف معقدة M. شبكة الاتصالات السل. وجدنا أيضا أن التصور 3D والتحليل الكمي وأدوات أفضل لدراسة الخصائص الموجودة في نموذج الأنسجة (الشكل 3). الكمي من كتلة الخلية (الورم الحبيبي على سبيل المثال) غالبا ما تمتد<html> [هس إلى عدة طبقات الخلايا ويمكن الحصول عليها تماما من قبل التحليل الكمي 3D. وعلاوة على ذلك، والتصور الدقيق من الميزات المكانية والزمانية لخلايا فردية أو البكتيريا الموجودة في نموذج يسمح الحية التصوير والهجرة وتتبع الدراسات في مختبر مخصص. <img alt="الرقم 2" src="/files/ftp_upload/53084/53084fig2.jpg" /> الشكل 2. الوحيدات في الكتلة نموذج الأنسجة المحيطة ضراوة M. السل. الصور مبائر التمثيلية للغير مصاب وM. ويرد السل نموذج الأنسجة المصابة. لوحات من الأخضر (M. tuberculosis- GFP) والأحمر (وحيدات المسمى PKH26) والأزرق (نوى دابي الملون) وقنوات اندمجت تظهر تجنيد حيدات في الأنسجة المصابة بالمقارنة مع الأنسجة غير المصابة. على نطاق و- 100 ميكرون.large.jpg &quot;على غرار =&quot; FONT-SIZE: 14px؛ خط الطول: 28px؛ &quot;الهدف =&quot; _ فارغة &quot;&gt; الرجاء انقر هنا لعرض نسخة أكبر من هذا الرقم. <img alt="الشكل (3)" src="/files/ftp_upload/53084/53084fig3.jpg" /> الرقم التصور 3. 3D والتحليل الكمي للنموذج الأنسجة توفير معلومات مفيدة. صور الممثل التصور 3D نموذج الأنسجة كامل (A) الأنسجة غير المصابة، (B) مصابة M. السل، من خلال باجتزاء البصرية باستخدام زايس LSM700 المجهر متحد البؤر والتحليل الكمي Imaris برامج معالجة الصور (الإصدار 7.6.8). تم الحصول على هذه الصور في 20X التكبير، 14 ض مداخن تغطي سمك الأنسجة 19.5 ميكرون مع 1.5 ميكرون الفاصل، مما يسمح التصور من قمية، أفقي التناوب، وعرض الرأسي والأفقي التناوب (A و B). (C) رابعةتحليل ntitative من مجموعات الخلايا الوحيدات تكشف محسن (P &lt;0.0001) حجم الكتل الحبيبي في وقت مبكر بعد M. عدوى السل بالمقارنة مع عدم وجود عدوى. (D) الكمي لعدد من وحيدات أظهر انخفاضا (p &lt;0.01) في الأنسجة المصابة بالمقارنة مع الأنسجة غير المصابة، مؤكدا المزيد من التجمعات في الأنسجة المصابة. الأخضر - M. -GFP السل، والأحمر - وحيدات PKH26 المسمى، الأزرق - نوى الخلايا، مقياس - 100 ميكرون <a href="https://www.jove.com/files/ftp_upload/53084/53084fig3large.jpg" target="_blank">الرجاء انقر هنا لعرض نسخة أكبر من هذا الرقم.</a>

Watch this Scientific Journal Video about A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection at JoVE.com

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Human tuberculosis infection is a complex process, which is difficult to model in vitro. Here we describe a novel 3D human lung tissue model that recapitulates the dynamics that occur during infection, including the migration of immune cells and early granuloma formation in a physiological environment.

نموذج الأنسجة 3D الرئة الإنسان للدراسات وظيفية على<em&gt; المتفطرة السلية</em&gt; العدوى

Tuberculosis (TB) still holds a major threat to the health of people worldwide, and there is a need for cost-efficient but reliable models to help us ...

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Immunology and Infection

A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

18.7K Views.  Linköping University. Human tuberculosis infection is a complex process, which is difficult to model in vitro. Here we describe a novel 3D human lung tissue model that recapitulates the dynamics that occur during infection, including the migration of immune cells and early granuloma formation in a physiological environment.

Video: A 3D Human Lung Tissue Model for Functional Studies on Mycobacterium tuberculosis Infection

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) drug-resistant strains threatens to derail current disease control efforts. Thus, there is an urgent need to develop drugs and vaccines that are more effective than those currently available. The genome of M. tuberculosis has been known for more than 10 years, yet there are important gaps in our knowledge of gene function and essentiality. Many studies have since used gene expression analysis at both the transcriptomic and proteomic levels to determine the effects of drugs, oxidants, and growth conditions on the global patterns of gene expression. Ultimately, the final response of these changes is reflected in the metabolic composition of the bacterium including a few thousand small molecular weight chemicals. Comparing the metabolic profiles of wild type and mutant strains, either untreated or treated with a particular drug, can effectively allow target identification and may lead to the development of novel inhibitors with anti-tubercular activity. Likewise, the effects of two or more conditions on the metabolome can also be assessed. Nuclear magnetic resonance (NMR) is a powerful technology that is used to identify and quantify metabolic intermediates. In this protocol, procedures for the preparation of M. tuberculosis cell extracts for NMR metabolomic analysis are described. Cell cultures are grown under appropriate conditions and required Biosafety Level 3 containment,1 harvested, and subjected to mechanical lysis while maintaining cold temperatures to maximize preservation of metabolites. Cell lysates are recovered, filtered sterilized, and stored at ultra-low temperatures. Aliquots from these cell extracts are plated on Middlebrook 7H9 agar for colony-forming units to verify absence of viable cells. Upon two months of incubation at 37 &deg;C, if no viable colonies are observed, samples are removed from the containment facility for downstream processing. Extracts are lyophilized, resuspended in deuterated buffer and injected in the NMR instrument, capturing spectroscopic data that is then subjected to statistical analysis. The procedures described can be applied for both one-dimensional (1D) 1H NMR and two-dimensional (2D) 1H-13C NMR analyses. This methodology provides more reliable small molecular weight metabolite identification and more reliable and sensitive quantitative analyses of cell extract metabolic compositions than chromatographic methods. Variations of the procedure described following the cell lysis step can also be adapted for parallel proteomic analysis.

Sample Preparation of Mycobacterium tuberculosis Extracts for Nuclear Magnetic Resonance Metabolomic Studies

The metabolomic profile of Mycobacterium tuberculosis is determined after growth in broth cultures. Conditions can be varied to test the effects of nutritional supplements, oxidants, and anti-tuberculosis agents on the metabolic profile of this microorganism. Procedure for extract preparation is applicable for both 1D 1H and 2D 1H-13C NMR analyses.

تحضير العينة من<em&gt; المتفطرة السلية</em&gt; خلاصات لدراسات النووية المغناطيسي الرنين Metabolomic

Mycobacterium tuberculosis is a major cause of mortality in human beings on a global scale. The emergence of both multi- (MDR) and extensively-(XDR) ...

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including antibiotics. Although stress tolerance of M. tuberculosis is one of the likely contributors to the 6-month long chemotherapy of tuberculosis 1, the molecular mechanisms underlying this characteristic phenotype of the pathogen remain unclear. Many microbial species have evolved to survive in stressful environments by self-assembling in highly organized, surface attached, and matrix encapsulated structures called biofilms 2-4. Growth in communities appears to be a preferred survival strategy of microbes, and is achieved through genetic components that regulate surface attachment, intercellular communications, and synthesis of extracellular polymeric substances (EPS) 5,6. The tolerance to environmental stress is likely facilitated by EPS, and perhaps by the physiological adaptation of individual bacilli to heterogeneous microenvironments within the complex architecture of biofilms 7.
In a series of recent papers we established that M. tuberculosis and Mycobacterium smegmatis have a strong propensity to grow in organized multicellular structures, called biofilms, which can tolerate more than 50 times the minimal inhibitory concentrations of the anti-tuberculosis drugs isoniazid and rifampicin 8-10. M. tuberculosis, however, intriguingly requires specific conditions to form mature biofilms, in particular 9:1 ratio of headspace: media as well as limited exchange of air with the atmosphere 9. Requirements of specialized environmental conditions could possibly be linked to the fact that M. tuberculosis is an obligate human pathogen and thus has adapted to tissue environments. In this publication we demonstrate methods for culturing M. tuberculosis biofilms in a bottle and a 12-well plate format, which is convenient for bacteriological as well as genetic studies. We have described the protocol for an attenuated strain of M. tuberculosis, mc27000, with deletion in the two loci, panCD and RD1, that are critical for in vivo growth of the pathogen 9. This strain can be safely used in a BSL-2 containment for understanding the basic biology of the tuberculosis pathogen thus avoiding the requirement of an expensive BSL-3 facility. The method can be extended, with appropriate modification in media, to grow biofilm of other culturable mycobacterial species.
Overall, a uniform protocol of culturing mycobacterial biofilms will help the investigators interested in studying the basic resilient characteristics of mycobacteria. In addition, a clear and concise method of growing mycobacterial biofilms will also help the clinical and pharmaceutical investigators to test the efficacy of a potential drug.

Growth of Mycobacterium tuberculosis Biofilms

Mycobacterium tuberculosis forms drug tolerant biofilms when cultured in certain conditions. Here we describe methods for culturing M. tuberculosis biofilms and determining the frequency of drug tolerant persisters. These protocols will be useful for further studies into the mechanisms of drug tolerance in M. tuberculosis.

نمو المتفطرة السلية الأغشية الحيوية

Mycobacterium tuberculosis, the etiologic agent of human tuberculosis, has an extraordinary ability to survive against environmental stresses including ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

عزل تدفق Cytometric من نوع خلايا الفئران الابتدائية السنخية II طلائي للدراسات الوظيفية والجزيئية

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

نموذج تجريبي لدراسة الملاريا والسل عدوى مرافقة على انتقال الطبيعية السل المتفطرة و المتصورة berghei</i

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource environments. An amplification microarray combines asymmetric PCR amplification, target size selection, target labeling, and microarray hybridization within a single solution and into a single microfluidic chamber. A batch processing method is demonstrated with a 9-plex asymmetric master mix and low-density gel element microarray for genotyping multi-drug resistant Mycobacterium tuberculosis (MDR-TB). The protocol described here can be completed in 6 hr and provide correct genotyping with at least 1,000 cell equivalents of genomic DNA. Incorporating on-chip wash steps is feasible, which will result in an entirely closed amplicon method and system. The extent of multiplexing with an amplification microarray is ultimately constrained by the number of primer pairs that can be combined into a single master mix and still achieve desired sensitivity and specificity performance metrics, rather than the number of probes that are immobilized on the array. Likewise, the total analysis time can be shortened or lengthened depending on the specific intended use, research question, and desired limits of detection. Nevertheless, the general approach significantly streamlines microarray workflow for the end user by reducing the number of manually intensive and time-consuming processing steps, and provides a simplified biochemical and microfluidic path for translating microarray-based diagnostics into routine clinical practice.

Demonstrating a Multi-drug Resistant Mycobacterium tuberculosis Amplification Microarray

An amplification microarray combines asymmetric PCR amplification and microarray hybridization into a single chamber, which significantly streamlines microarray workflow for the end user. Simplifying microarray workflow is a necessary first step for creating microarray-based diagnostics that can be routinely used in lower-resource environments.

مما يدل على متعدد المخدرات مقاومة<em&gt; السل المتفطرة</em&gt; التضخيم ميكروأري

Simplifying microarray workflow is a necessary first step for creating MDR-TB microarray-based diagnostics that can be routinely used in lower-resource ...

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most common microdissection technique used today. In this technique, a laser is used to focally melt a thermoplastic membrane that overlies a dehydrated tissue section1. The tissue section composite is then lifted and separated from the membrane. Although this technique can be used successfully for tissue examination, it is time consuming and expensive. Furthermore, the successful completion of procedures using this technique requires the use of a laser, thus limiting its use. A new more affordable and practical microdissection approach called mesodissection is a possible solution to the pitfalls of LCM. This technique employs the MESO-1/MeSectr system to mill the desired tissue from a slide mounted tissue sample while concurrently dispensing and aspirating fluid to recover the desired tissue sample into a consumable mill bit. Before the dissection process begins, the user aligns the formalin fixed paraffin embedded (FFPE) slide with a hematoxylin and eosin stained (H&#38;E) reference slide. Thereafter, the operator annotates the desired dissection area and proceeds to dissect the appropriate segment. The program generates an archived image of the dissection. The main advantage of mesodissection is the short duration needed to dissect a slide, taking an average of ten minutes from set up to sample generation in this experiment. Additionally, the system is significantly more cost effective and user friendly. A slight disadvantage is that it is not as precise as laser capture microscopy. In this article we demonstrate how mesodissection can be used to extract RNA from slides from FFPE granulomas caused by Mycobacterium tuberculosis (Mtb).

A Novel Microdissection Approach to Recovering Mycobacterium tuberculosis Specific Transcripts from Formalin Fixed Paraffin Embedded Lung Granulomas

Microdissection has been extensively employed for the examination of DNA, RNA, and protein within tissue. Laser capture microscopy (LCM) is the most commonly used method, but a new milling technique, mesodissection, is recently available. We demonstrate RNA extraction from mesodissected formalin fixed paraffin embedded tissue slides of Mycobacterium tuberculosis granulomas.

مقاربة الرواية تسليخ مجهري ليتعافى<em&gt; السل المتفطرة</em&gt; نصوص محددة من الفورمالين الثابتة جزءا لا يتجزأ من البارافين الرئة أورام حبيبية

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

نظام لفعالية والسمسة فحص مثبطات استهداف بين الخلايا<em&gt; المتفطرة السلية</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

وإنتاجية عالية الفحص المدعم لتقييم فعالية الدواء ضد بلعم Passaged<em&gt; المتفطرة السلية</em

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to ...

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

فيروس الإنفلونزا أ الفيروس دراسات في نموذج ماوس للإصابة

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically relevant methods are needed that evaluate the disease process and screen for potential antibiotic and vaccine treatments. Positron Emission Tomography/Computed Tomography (PET/CT) has been established as a valuable tool for studying a number of afflictions such as cancer, Alzheimer&#39;s disease, and inflammation/infection. Outlined here are a number of strategies that have been employed to evaluate PET/CT images in cynomolgus macaques that are infected intrabronchially with low doses of M. tuberculosis. Through evaluation of lesion size on CT and uptake of 18F-fluorodeoxyglucose (FDG) in lesions and lymph nodes in PET images, these described methods show that PET/CT imaging can predict future development of active versus latent disease and the propensity for reactivation from a latent state of infection. Additionally, by analyzing the overall level of lung inflammation, these methods determine antibiotic efficacy of drugs against M. tuberculosis in the most clinically relevant existing animal model. These image analysis methods are some of the most powerful tools in the arsenal against this disease as not only can they evaluate a number of characteristics of infection and drug treatment, but they are also directly translatable to a clinical setting for use in human studies.

Analysis of 18FDG PET/CT Imaging as a Tool for Studying Mycobacterium tuberculosis Infection and Treatment in Non-human Primates

Here, we present a protocol to describe the analysis of 18F-FDG PET/CT imaging in non-human primates that have been infected with M. tuberculosis to study disease process, drug treatment, and disease reactivation.

تحليل 18"فدج التصوير" PET/CT كأداة لدراسة بكتريا السل العدوى والعلاج في الإنسان عدم الرئيسات

Mycobacterium tuberculosis remains the number one infectious agent in the world today. With the emergence of antibiotic resistant strains, new clinically ...