胰岛细胞资源中心美国国立卫生研究院授予（05-003 RFA - RR），克里斯托弗基金会，Efroymson基金会，和泰乐基金会成立在芝加哥伊利诺伊大学以及支持。

1。胰岛的分离纯化<ol><li>要启动胰岛的纯化过程，加载的COBE袋，所有的管子钳，除了绿管，并设置速度到1500超到0。 </li><li>在引擎盖成立泵和梯度烧杯和收集管连接到绿管。 </li><li>现在可以开始加载聚蔗糖1.100：推“旋转”，然后添加110毫升聚蔗糖1.100前烧杯，并启动水泵，以负载的COBE袋。由于聚蔗糖加载，按超，停泵，松开泵的扬程，并设置超出速度100。 </li><li>当聚蔗糖达到烧杯中，所有空气管，泵头重新钳开除。设置到3000的转速，超出速度为0。 </li><li>现在，准备装载前的烧杯中倒入沉重的梯度梯度。稍微松开钳之间的两个烧杯，以消除任何空气。然后倒入后面的烧杯中的光线梯度。 </li><li>打开磁力搅拌器上，按“旋转”的COBE机上，取出钳管连接两个烧杯，直观地验证，重工业和轻工业的梯度混合。 </li><li>梯度一旦开始搅拌，打开离心机。等待一分钟，然后打开泵负载的混合梯度。 </li><li>要加载的组织让跑在前面的烧杯中低梯度，但没有足够低，让空气进入装载管，然后reclamp管连接烧杯和负载的组织。 </li><li>继续添加其他组织。然后洗管，洗涤液50毫升举行组织，并用它来洗前烧杯。 </li><li>作为最后的组织进入囊中，同时停泵，松开在泵头，和钳管袋以上。继续离心5分钟。 </li><li>虽然离心继续，带来了12个收集管罩，松开帽子。将收集管，黄管，并放置到无菌收集尖锥形管1。然后松开黄色管，夹绿管。 </li><li>当旋转结束时，慢慢提高到100 Superout。收集管1 150毫升。然后收集管2-12 30毫升（200-230毫升）。 </li><li>一旦收集完成后，按下“停止”的COBE。现在已经结束净化，胰岛，可评估和培养。 </li></ol> 2。采样和文化<ol><li>收集纯化胰岛分数后，取出的12个收集管中的每一个样品，并用双硫腙染色。染色层含有高纯度低纯度的胰岛，胰岛细胞与层。 </li><li>高和低纯度的组织和游泳池，每年收集。 </li><li>自旋和洗两次澡。第二次洗涤后，将每250毫升的量，最后洗媒体。 </li><li>为了评估汇总分数开始汇总分数和一个1 ml样品从低到两个1毫升样品。每1ml样品转移到一个圆锥形含9毫升洗涤液管。 </li><li>然后将1毫升每15毫升管计数盘。检查和计数细胞，在显微镜下。 </li><li>后计数细胞计算需要文化的胰岛，使用下列公式烧瓶号码：（总的EIN /十进制胰岛纯度）/（30,000元烧瓶的EIN）。例如，10万个胰岛，纯度90％，是4烧瓶培养。 </li><li>传输T175烧瓶和文化的小岛在37˚C和5％ 二氧化碳培养箱。 </li></ol>

<ol>
	<li>Shapiro, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+transplantation+in+seven+patients+with+type+1+diabetes+mellitus+using+a+glucocorticoid-free+immunosuppressive+regimen.">Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen.</a> N Engl J Med. 343, 230-238 (2000).</li><li>Nano, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+isolation+for+allotransplantation:+variables+associated+with+successful+islet+yield+and+graft+function.">Islet isolation for allotransplantation: variables associated with successful islet yield and graft function.</a> Diabetologia. 48, 906-912 (2005).</li><li>Barbaro, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+human+pancreatic+islet+purification+with+the+refined+UIC-UB+density+gradient.">Improved human pancreatic islet purification with the refined UIC-UB density gradient.</a> Transplantation. 84, 1200-1203 (2007).</li><li>Lakey, J. R., Rajotte, R. V., Warnock, G. L., Kneteman, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+pancreas+preservation+prior+to+islet+isolation.+Cold+ischemic+tolerance.">Human pancreas preservation prior to islet isolation. Cold ischemic tolerance.</a> Transplantation. 59, 689-694 (1995).</li><li>Fridell, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+histidine-tryptophan-ketoglutarate+solution+and+University+of+Wisconsin+solution+for+organ+preservation+in+clinical+pancreas+transplantation.">Comparison of histidine-tryptophan-ketoglutarate solution and University of Wisconsin solution for organ preservation in clinical pancreas transplantation.</a> Transplantation. 77, 1304-1306 (2004).</li><li>Potdar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+experience+using+histidine-tryptophan-ketoglutarate+solution+in+clinical+pancreas+transplantation.">Initial experience using histidine-tryptophan-ketoglutarate solution in clinical pancreas transplantation.</a> Clin Transplant. 18, 661-665 (2004).</li><li>Salehi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+islet+isolation+outcomes+from+pancreata+preserved+with+Histidine-Tryptophan+Ketoglutarate+versus+University+of+Wisconsin+solution.">Human islet isolation outcomes from pancreata preserved with Histidine-Tryptophan Ketoglutarate versus University of Wisconsin solution.</a> Transplantation. 82, 983-985 (2006).</li><li>Ricordi, C., Lacy, P. E., Scharp, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+islet+isolation+from+human+pancreas.">Automated islet isolation from human pancreas.</a> Diabetes. 38 Suppl 1, 140-142 (1989).</li><li>Ricordi, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+isolation+assessment+in+man+and+large+animals.">Islet isolation assessment in man and large animals.</a> Acta Diabetol Lat. 27, 185-195 (1990).</li></ol>

尽管在人胰岛分离技术方面取得了长足的进步，胰岛产量仍充满变数和不可预知的。净化消化胰腺组织移植成功的酶消化后的恢复足够岛大规模的关键。 UIC的净化方法的建议，因为优越的高纯度的人类胰岛复苏，通过使用这种方法来证明。此外，可装载高达50毫升的消化组织进行净化处理运行一个单一的COBE，从而最大限度地减少缺血相关的人类胰岛的损伤，单独使用的总时间缩短。

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Centrifuge</td>
<td>equipment</td>
<td>Beckman Coulter</td>
<td>424803</td>
<td>&nbsp;</td>
<tr>
<td>Cobe 2991 Cell Processor</td>
<td>equipment</td>
<td>Gambro. BCT</td>
<td>2556</td>
<td>islet purification</td>
</tr>
<tr>
<td>MZ6 Inverted Microscopy TLBD4.1</td>
<td>equipment</td>
<td>Leica Diagnostic Instrument Co</td>
<td>4103</td>
<td>&nbsp;</td>
<tr>
<td>Cell Culture Dish with 2 mm Grid</td>
<td>equipment</td>
<td>Nunc Brand Products</td>
<td>174926</td>
<td>islet counting</td>
</tr>
<tr>
<td>Digital Sight DS L1</td>
<td>equipment</td>
<td>Nikon</td>
<td>217267</td>
<td>&nbsp;</td>
<tr>
<td>COBE bag</td>
<td>disposable equipment</td>
<td>O.R. Solution</td>
<td>66707003</td>
<td>&nbsp;</td>
<tr>
<td>T175 culture flask</td>
<td>disposable equipment</td>
<td>Sarstedt</td>
<td>83.1812.500</td>
<td>&nbsp;</td>
<tr>
<td>University of Wisconcin (UW) solution</td>
<td>reagent</td>
<td>DURAMED</td>
<td>1000-46-06</td>
<td>&nbsp;</td>
<tr>
<td>Hank's Buffered Salt Solution (HBSS)</td>
<td>reagent</td>
<td>Mediatech Inc</td>
<td>99-597-CM</td>
<td>&nbsp;</td>
<tr>
<td>Ficoll 1.100</td>
<td>reagent</td>
<td>Bichrom AG</td>
<td>L6155</td>
<td>&nbsp;</td>
<tr>
<td>M199 media (wash solution)</td>
<td>reagent</td>
<td>Mediatech Inc</td>
<td>99-784-CM</td>
<td>&nbsp;</td>
<tr>
<td>Final wash/Culture Medium</td>
<td>reagent</td>
<td>Mediatech Inc</td>
<td>99-785-CV</td>
<td>&nbsp;</td>
<tr>
<td>Dithizone</td>
<td>reagent</td>
<td>Sigma </td>
<td>D5130</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

human pancreatic islet isolation: part ii: purification and culture of human islets

1型糖尿病的管理负担，既要对个人和社会，每年耗资超过100亿美元。尽管血糖监测和新的胰岛素制剂的广泛使用，许多人仍然发展毁灭性的继发性并发症。胰岛移植可以恢复接近正常的糖尿病患者血糖控制<sup&gt; 1</sup&gt;胰岛素强化治疗相关的严重的低血糖发作的危险，没有。胰岛移植成功的，重要的是提供足够的胰岛质量。但是，捐助者的特征，器官获取和保存的影响隔离的结果<sup&gt; 2</sup&gt;。在美国伊利诺伊大学芝加哥分校（UIC），我们开发了一种改进的净化梯度成功分离协议<sup&gt; 3</sup&gt;。该计划开始于2004年1月和2008年11月超过300隔离。 “胰腺被送往在冷保存液（UW液，威斯康星州或HTK的大学，组氨酸，色氨酸酮戊二酸）<sup&gt; 4-7</sup&gt;在UIC的细胞分离胰岛隔离的实验室。使用UIC的方法，这是最初所Ricordi描述的方法修改后的版本中分离出胰岛<em&gt;等</em&gt;<sup&gt; 8</sup&gt;。正如在第一部分所述：消化和胰腺组织收集，人体胰腺被修剪，空心，灌注，消化。组织收集和UW液中孵育至少30分钟后，被装在细胞分离机（COBE 2991，COBE，翠湖，二氧化碳）净化<sup&gt; 3</sup&gt;。经过净化，胰岛产量（表现为胰岛等值，IEQ），组织的体积，和纯度测定按标准方法<sup&gt; 9</sup&gt;。隔离胰岛培养媒体CMRL -​​ 1066（Mediatech，赫恩登，VA），辅以1.5％的人血白蛋白，0.1％的胰岛素转硒（简称ITS），1毫升，5毫升1M HEPES环丙沙星，和14.5毫升7.5％，在T175烧瓶钠碳酸氢盐在37 ° C过夜培养胰岛移植前或用于研究。

Watch this Scientific Journal Video about Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets at JoVE.com

Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets

实现高​​品质和适当数量的人类胰岛是胰岛移植成功的突出的先决条件之一。在这个视频中，我们描述一步一步人类胰岛隔离（第二部分：人类胰岛的纯化和文化）的程序，采用改良的自动方法。

人类胰岛的分离：第二部分：人类胰岛的分离纯化及文化

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of ...

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Research

JoVE Journal

Biology

15.4K 次观看.  University of Illinois, Chicago. 实现高​​品质和适当数量的人类胰岛是胰岛移植成功的突出的先决条件之一。在这个视频中，我们描述一步一步人类胰岛隔离（第二部分：人类胰岛的纯化和文化）的程序，采用改良的自动方法。

视频: Human Pancreatic Islet Isolation: Part II: Purification and Culture of Human Islets

Murine Pancreatic Islet Isolation

小鼠胰岛隔离

科研

生物学

Human Pancreatic Islet Isolation: Part I: Digestion and Collection of Pancreatic Tissue

Management of Type 1 diabetes is burdensome, both to the individual and society, costing over 100 billion dollars annually. Despite the widespread use of glucose monitoring and new insulin formulations, many individuals still develop devastating secondary complications. Pancreatic islet transplantation can restore near normal glucose control in diabetic patients 1, without the risk of serious hypoglycemic episodes that are associated with intensive insulin therapy. Providing sufficient islet mass is important for successful islet transplantation. However, donor characteristic, organ procurement and preservation affect the isolation outcome 2. At University of Illinois at Chicago (UIC) we have developed a successful isolation protocol with an improved purification gradient 3. The program started in January 2004, and more than 300 isolations were performed up to November 2008. The pancreata were sent in cold preservation solutions (UW, University of Wisconsin or HTK, Histidine-Tryptophan Ketoglutarate) 4-7 to the Cell Isolation Laboratory at UIC for islet isolation. Pancreatic islets were isolated using the UIC method, which is a modified version of the method originally described by Ricordi et al 8. Briefly, after cleaning the pancreas from the surrounding tissue, it was perfused with enzyme solution (Serva Collagenase + Neutral Protease or Sigma V enzyme). The distended pancreas was then transferred to the Ricordi digestion chamber, connected to a modified, closed circulation tubing system, and warmed up to 37&deg;C. During the digestion, the chamber was shaken gently. Samples were taken continuously to monitor the digestion progress. Once free islets were detected under the microscope, the digestion was stopped by flushing cold (4&deg;C) RPMI dilution solution (Mediatech, Herndon, VA) into the circulation system to dilute the enzyme. After being collected and washed in M199 media supplemented with human albumin, the tissue was sampled for pre-purification count and incubated with UW solution before purification. Purification process will be described in Part II: Purification and Culture of Human Islets.

Achieving high quality and appropriate quantity of human islets is one of the prominent prerequisites for successful islet transplantation. In this video, we describe step by step the procedures for human pancreatic islet isolation (part I: digestion and collection of pancreatic tissue) using a modified automated method.

人类胰岛的分离：第一部分：收集和胰腺组织消化

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny1. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24low/-)2. Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues3-5.
Recently, we and others identified CSCs from pancreatic adenocarcinoma based on
ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD1336-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic, but ALDH+ cells are relatively more invasive8. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human
xenografts9. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent,
a fluorescent substrate of ALDH10. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.

Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies.

人胰腺癌移植瘤的干细胞的分离

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

成人从人体皮肤的上皮干细胞的分离和培养

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The &#946;-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the &#946;-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on &#946;-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased &#946;-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression.

Assaying in vitro &#946;-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many&#160;downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining &#946;-cell function.

对小鼠胰腺胰岛分离和细胞内cAMP的测定方法

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

小鼠原代胰腺腺泡细胞的分离和培养

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

高效的生产和纯化重组小鼠Kindlin-3从昆虫细胞生物物理研究

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts migrate through the epidermis of the embryo where they subsequently colonize the developing hair follicles1,2. Neural crest cell migration is extensively studied in vitro but in vivo methods are still not well developed, especially in mammalian systems. One alternative is to use ex vivo organotypic culture3-6. Culture of mouse embryonic skin requires the maintenance of an air-liquid interface (ALI) across the surface of the tissue3,6. High resolution live-imaging of mouse embryonic skin has been hampered by the lack of a good method that not only maintains this ALI but also allows the culture to be inverted and therefore compatible with short working distance objective lenses and most confocal microscopes. This article describes recent improvements to a method that uses a gas permeable membrane to overcome these problems and allow high-resolution confocal imaging of embryonic skin in ex vivo culture6. By using a melanoblast specific Cre-recombinase expressing mouse line combined with the R26YFPR reporter line we are able to fluorescently label the melanoblast population within these skin cultures. The technique allows live-imaging of melanoblasts and observation of their behavior and interactions with the tissue in which they develop. Representative results are included to demonstrate the capability to live-image 6&#160;cultures in parallel.

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

We describe the dissection and ex vivo culture of mouse embryonic skin. The culture system maintains an air-liquid interface across the tissue surface and allows imaging on an inverted microscope. Melanoblasts, a component of the developing skin, are fluorescently labeled allowing their behavior to be observed using confocal microscopy.

小鼠胚胎皮肤和Live成像黑素细胞迁移的体外培养

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.

胰腺胰岛与小鼠分离的简单高效协议

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other ...

Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, epithelial progenitor cells can self-renew and generate differentiating progeny that exhibit cellular functions similar to those of their in vivo counterparts. Herein we describe a step-by-step protocol to isolate region-specific progenitors from human lung and generate 3D organoid cultures as an experimental and validation tool. We define proximal and distal regions of the lung with the goal of isolating region-specific progenitor cells. We utilized a combination of enzymatic and mechanical dissociation to isolate total cells from the lung and trachea. Specific progenitor cells were then fractionated from the proximal or distal origin cells using fluorescence associated cell sorting (FACS) based on cell type-specific surface markers, such as NGFR&#160;for sorting basal cells and HTII-280 for sorting alveolar type II cells. Isolated basal or alveolar type II progenitors were used to generate 3D organoid cultures. Both distal and proximal progenitors formed organoids with a colony forming efficiency of 9-13% in distal region and 7-10% in proximal region when plated 5000 cell/well on day 30. Distal organoids maintained HTII-280+ alveolar type II cells in culture whereas proximal organoids differentiated into ciliated and secretory cells by day 30. These 3D organoid cultures can be used as an experimental tool for studying the cell biology of lung epithelium and epithelial mesenchymal interactions, as well as for the development and validation of therapeutic strategies targeting epithelial dysfunction in a disease.

This article provides a detailed methodology for tissue dissociation and cellular fractionation approaches allowing enrichment of viable epithelial cells from proximal and distal regions of the human lung. Herein these approaches are applied for the functional analysis of lung epithelial progenitor cells through the use of 3D organoids culture models.

分离和富集人肺上皮祖细胞用于类器官培养

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, ...