Name: isolation and transplantation of different aged murine thymic grafts.
Uploaded: 2015-05-13T14:00:00.000+00:00
Duration: 5 min 47 s
Description: Watch this Scientific Journal Video about Isolation and Transplantation of Different Aged Murine Thymic Grafts. at JoVE.com

This work was supported by funding received from the National Institutes of Health (1R01AI083269).

鼠研究批准了北卡罗来纳教堂山大学的机构动物护理和使用委员会（IACUC）和所有的动物护理是按照IACUC的指导方针。 1.准备新生儿和成人Thymi的<ol><li>准备所有安乐死供体小鼠之前，试剂和设备。 </li><li>消毒手术器械通过高压灭菌或其他合适的方法。所有的外科手术必须在层流罩保持无菌条件和避免污染进行。组装需要从供体小鼠提取thymi的工具。 </li><li>填充60mm培养皿用无菌的1×PBS（pH7.4）中，并放置在冰上的罩内。这将被用于简单地存储thymi从供体小鼠移植前切除。 </li><li>按道德准则，通过CO新生儿安乐死供体小鼠斩首和成年供体小鼠2窒息颈椎脱位。 </li><li>当r胸腺emoval:莱鼠标在背卧位在无菌吸水纸巾喷洒之前做一个切口70％的乙醇。 </li><li>通过皮肤制备中线切口暴露腹腔和胸腔。折叠的皮肤在胸部和前肢以显示胸腔。 </li><li>使通过隔膜和肋骨两侧切口，显露上纵隔和前胸腔。胸腺应该是可见的两个白色的凸起立即上方和相邻的心脏。 </li><li>除了捉弄周围用细镊子胸腺结缔组织，使某些不破坏胶囊。而憋尿用钳子胸腔，使用第二对镊子通过定位弯钳器官下方，垂直提拉提取胸腺的两叶。这可以使用解剖范围为从新生小鼠提取thymi来完成。 </li><li>放置在胸腺60毫米双含有SH在冰上并分离胸腺裂片无菌的1×PBS（pH7.4）中，通过所述连通峡部切割。从胸腺裂片使某些不损坏胶囊除去任何碎片，并切断胸腺成用于移植部分的适当数量。 </li><li>不要操纵新生小鼠获得的thymi。 注:用于最多两个受体小鼠（每个收件人1叶）中。 </li><li>移植从成年小鼠中得到的thymi分为最多4-6受体小鼠。使用一对镊子，小心地抓住一名成人胸腺叶， 如图1，切到胸腺用手术剪刀三个相等的部分。 </li><li>重复步骤1.3-1.4为每个供体小鼠。为了限制thymi暴露的时间，准备1施主胸腺的时间。 </li></ol> 2.植入胸腺肾囊下<ol><li>装配在表1中列出的预先消毒设备，适当的无菌技术应的过程，以防止暴露移植受者对污染的工具或试剂的过程中被利用。 </li><li>移植前，权衡和标记每个收件人鼠标。 </li><li>设置解剖显微镜和麻醉电路在层流罩。 </li><li>使用电动剃须刀，剃接受者小鼠的左侧，并确保没有头发仍然围绕用于使切口的区域。 </li><li>打开异氟烷蒸发器和麻醉使用的1-2％之间的剂量鼠标。通过检查以下脚趾捏缺乏反射开始手术前确定适当的麻醉。 </li><li>之后，鼠标正常麻醉，适用兽医药膏鼠​​标以防止干燥和准备鼠标移植如下的眼睛: </li><li>定位在解剖显微镜下鼠标在右侧卧位，使剃侧朝上。 Starti纳克在手术区的中心，使用一次性移液管的70％乙醇，随后碘伏（优碘）分配在一个圆周运动。重复乙醇/优碘治疗3次之前做一个切口。 </li><li>使用解剖剪刀做一个1-2厘米的切口侧翼肾脏正上方。 </li><li>使用镊子中等掌握邻近肾脏结缔组织和轻轻抬起肾使得其位于上盖的肌肉。为了保持肾脏暴露和地方在肌肉组织，插入介质镊子一只手臂肾下，要特别注意不要破坏肾髂骨。 </li><li>为了防止组织脱水，在每个步骤中，直至肾和肾囊操纵完成灌溉用无菌的1×PBS（pH7.4）中的肾。 </li><li>用细镊子夹住并提起肾脏最远端的肾上腺它从肾脏分离的边缘附近的胶囊。 </li><li>使用18号针，使一个切口足够大以插入准备好的片的供体胸腺（ 图2A）。使一定保持囊切口尽可能小，以防止移植物的错位随着时间的推移。 </li><li>而从肾脏保持胶囊拉远，使用第二对细镊子的插入胶囊下方的移植物和胸腺从囊切口尽可能（ 图2B）推远向前。 </li><li>从肾脏下方取出钳，并通过切口轻轻返回肾脏回原位。 </li><li>缝合腹膜壁和应用一旦关闭优碘关闭肌肉。缝合完成后，应用伤口夹关闭真皮并应用优碘到周边地区。 </li><li>返回鼠标已使用热灯加热笼子。 </li><li>监测每个术后鼠标，直到它恢复全部的意识和行动，也不要将鼠塔吨有没有恢复麻醉下与其他动物的笼子。 </li><li>根据需要治疗的小鼠手术后的疼痛管理药物。提供小鼠手术后止痛药如对乙酰氨基酚在饮用水以1.6毫克/毫升的浓度。 </li><li>重复步骤2.3-2.9为每个收件人鼠标。 </li><li>确保小鼠恢复正常活动中1小时后的手术。监视手术后的重量，运动技能，以及各小鼠的饮用和摄食活动，以确定所产生的移植过程的结果的并发症。 注:手术后体重减轻，相比手术前的重量，以及改变流动性或没有吃或喝可以指示并发症。 </li><li>监视外周血T细胞重建的出血的小鼠经尾缺口随后根据制造商的说明书使用Lympholyte细胞分离介质的淋巴细胞的分离。实现T细胞characteriza灰通过染色的淋巴细胞与特异于T细胞标记CD3，CD4，和CD8，以及一个活死鉴别荧光标记的抗体，例如荧光激活琥珀酰亚胺基酯8。 如图3分析可以通过选通上单峰，活细胞，和CD3阳性细胞来完成。 </li></ol>

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The authors do not have any competing financial interests to disclose.

Events regulating thymic selection are poorly understood. Recent studies have demonstrated ontogenic changes in the efficiency of selection. Coupling this approach with different transgenic thymus donor and recipient mice will further facilitate studies to identify the events and parameters that regulate thymic selection. Notably, thymic transplantation is used for the treatment and management of T cell and thymic disorders seen for instance in athymic infants lacking functional T cells and in patients with DiGeorge synd...

胸腺是在T细胞发育的关键事件发生1器官。驻地胸腺细胞，在T细胞受体（TCR）α和β基因的重排，进行一系列与在胸腺2的皮质和髓质区lymphostromal和抗原呈递细胞（APC）的相互作用。胸腺阳性选择由皮质胸腺上皮细胞（TEC）介导，以产生胸腺细胞识别的抗原肽在宿主主要组织相容性复合体（MHC）I类和II类分子2-3的上下文中。随后胸腺阴性选择嗣继承吹扫自身反应的胸腺细胞，通过与髓TEC或树突细胞（DC）从自身蛋白质结合的MHC I类和II类分子3推导出本发明的肽的相互作用来驱动。这些处理的最终结果是建立成熟的CD4 +和CD8 + T细胞能够对广谱响应一池外来抗原的同时表现出最小的反应性的自身蛋白质4。 胸腺选择事件的效率是由许多因素，包括胸腺成熟，髓和皮质TEC，胸腺DC亚群组成的频率，和胸腺前体3的源的影响。值得注意的是，异常胸腺选择可导致自身免疫性5或免疫缺陷的病状，从受损的阴性或阳性选择，它们分别出现。的分子事件调节胸腺选择，然而，却知之甚少。 体外方法，如reaggregate胸腺器官培养（RTOC）6，已被证明是用于分析与胸腺选择相关联的基本事件是有用的，但不能完全概括的正在进行的动态在体内的活动。这样一来，这个胸腺移植为基础的方式成立，以便更好地研究胸腺细胞评选活动在体内 7 </s向上&gt;。 本协议描述移栽从新生和成年供体小鼠thymi到免疫缺陷SCID受体小鼠。这种技术允许调节阳性和阴性胸腺选择，以及各种T细胞亚群的个体发育过程中胸腺输出机制的研究。最近，这种方法已被用于证明胸腺选择的效率出生的小鼠，导致自身反应性T细胞的增加发展后早期的限制，并降低T细胞剧目特异于外来抗原7。

<table><tbody><tr><td>Ethanol&amp;nbsp;</td><td>Decon Laboratories</td><td>2705HC</td><td></td></tr><tr><td>PBS</td><td>GIBCO</td><td>14190-144</td><td>pH 7.4</td></tr><tr><td>Betadine Solution</td><td>Purdue Products</td><td>67618-150-17</td><td></td></tr><tr><td>18 gauge needle</td><td>BD</td><td>305195</td><td></td></tr><tr><td>Sutures (1.0 metric)</td><td>Ethicon</td><td>J493G</td><td>18&quot; (45cm)</td></tr><tr><td>AUTOCLIP Wound Clips (9mm)</td><td>Clay Adams&amp;reg; Brand</td><td>427631</td><td></td></tr><tr><td>Transfer pipette</td><td>Fisher Scientific</td><td>13-711-20</td><td>Sterile, disposable</td></tr><tr><td>Mouse anti-CD3 Ab</td><td>eBioscience</td><td>11-0031-85</td><td>Clone: 1452C11</td></tr><tr><td>Mouse anti-CD4 Ab</td><td>eBioscience</td><td>48-0042-82</td><td>Clone: RM4-5</td></tr><tr><td>Mouse anti-CD8 Ab</td><td>eBioscience</td><td>25-0081-82</td><td>Clone: 53-6.7</td></tr><tr><td>Lancet</td><td>Medipoint</td><td>Goldenrod 4mm</td><td></td></tr><tr><td>Pacific Orange Succinimidyl Ester, Triethylammonium Salt</td><td>Invitrogen</td><td>P30253</td><td></td></tr><tr><td>96-well round botton polypropylene plates</td><td>Corning</td><td>3365</td><td></td></tr><tr><td>1.2 ml polypropylene cluster tubes</td><td>Corning</td><td>4401</td><td></td></tr><tr><td>5 ml polypropylene round-bottom tubes</td><td>BD</td><td>352002</td><td></td></tr><tr><td>40uM Nylon Cell Strainer</td><td>Falcon</td><td>352340</td><td></td></tr><tr><td>16% Paraformaldehyde Solution, EM Grade</td><td>Electron Microscopy Services</td><td>15710</td><td>Hazardous</td></tr><tr><td>Puralube Optical Ointment</td><td>Fisher Scientific</td><td>NC0138063</td><td></td></tr><tr><td>Lympholyte</td><td>Cedarlane</td><td>CL5030</td><td></td></tr><tr><td>60 mm cell culture dish</td><td>Corning</td><td>430196</td><td>60 mm x 15 mm, Sterile</td></tr></tbody></table>

isolation and transplantation of different aged murine thymic grafts.

调节胸腺选择的有效性的机制仍然不明确。这里介绍的方法允许在体内特异于自我和外来抗原的T细胞的发展和选择的分析。该方法需要来自各种老年小鼠免疫缺陷SCID收件人胸腺移植物的植入。在一段相对较短的时间的收件人完全重构与来自植入胸腺移植物衍生的T细胞。只有播种的胸腺细胞在胸腺在隔离的时间进行选择，并发展成为成熟的T细胞。这样，改变在移植的T细胞的年龄依赖性胸腺事件的函数的性质和特异性可以进行评估。虽然技术专长需要成功胸腺移植，这种方法提供了以研究在体内的广泛的病理是由于异常胸腺功能和/或homeostas或结果的一个独特的战略是。

这个过程的成功依赖于最小的手术创伤，以及肾囊下的移植物的准确定位。胸腺移植物应该被切割，以确保适当大小胸腺部分用于随后的移植， 如图1。按照图1中的示意图，从新生或成年小鼠thymi可用于成功包膜下移植具有一致和可重复的T细胞植入的结果。如所提到的，肾囊下的移植物的适当的定位是在保持长期存活的移植物，功能，和血管重要。正如在图2...

Watch this Scientific Journal Video about Isolation and Transplantation of Different Aged Murine Thymic Grafts. at JoVE.com

Isolation and Transplantation of Different Aged Murine Thymic Grafts.

This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo.

隔离和不同的老年小鼠胸腺移植的移植。

The mechanisms that regulate the efficacy of thymic selection remain ill-defined. The method presented here allows in vivo analyses of the development and ...

isolation-and-transplantation-of-different-aged-murine-thymic-grafts

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

13.7K 次观看.  University of North Carolina at Chapel Hill. This report provides a detailed description of transplanting murine thymi from different aged donor mice under the kidney capsule of immunodeficient mouse recipients. The goal of this approach is to model T cell development and thymic selection events in vivo. 

视频: Isolation and Transplantation of Different Aged Murine Thymic Grafts.

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T cell replacement in lymphopenic patients. We studied thymic settling progenitors from murine embryonic day 13 and 18 thymi by two complementary in vitro and in vivo techniques, both based on the &#8220;hanging drop&#8221; method. This method allowed colonizing irradiated fetal thymic lobes with E13 and/or E18 thymic progenitors distinguished by CD45 allotypic markers and thus following their progeny. Colonization with mixed populations allows analyzing cell autonomous differences in biologic properties of the progenitors while colonization with either population removes possible competitive selective pressures. The colonized thymic lobes can also be grafted in immunodeficient male recipient mice allowing the analysis of the mature T cell progeny in vivo, such as population dynamics of the peripheral immune system and colonization of different tissues and organs. Fetal thymic organ cultures revealed that E13 progenitors developed rapidly into all mature CD3+ cells and gave rise to the canonical &#947;&#948; T cell subset, known as dendritic epithelial T cells. In comparison, E18 progenitors have a delayed differentiation and were unable to generate dendritic epithelial T cells. The monitoring of peripheral blood of thymus-grafted CD3-/- mice further showed that E18 thymic settling progenitors generate, with time, larger numbers of mature T cells than their E13 counterparts, a feature that could not be appreciated in the short term fetal thymic organ cultures.

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny.

胸腺表征祖定居在小鼠胚胎使用<em&gt;在体内</em&gt;和<em&gt;在体外</em&gt;测定

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

科研

发育生物学

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

活体成像的鼠标使用2 - 光子显微镜的胸腺

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

免疫与感染

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological deficiencies. GVHD is caused by mature alloreactive T cells present in the bone marrow graft that are infused into the recipient and cause damage to host organs. However, in mice, T cells must be added to the bone marrow inoculum to cause GVHD. Although extensive work has been done to characterize T cell responses post transplant, bioluminescent imaging technology is a non-invasive method to monitor T cell trafficking patterns in vivo. 
Following lethal irradiation, recipient mice are transplanted with bone marrow cells and splenocytes from donor mice. T cell subsets from L2G85.B6 (transgenic mice that constitutively express luciferase) are included in the transplant. By only transplanting certain T cell subsets, one is able to track specific T cell subsets in vivo, and based on their location, develop hypotheses regarding the role of specific T cell subsets in promoting GVHD at various time points. At predetermined intervals post transplant, recipient mice are imaged using a Xenogen IVIS CCD camera. Light intensity can be quantified using Living Image software to generate a pseudo-color image based on photon intensity (red = high intensity, violet = low intensity). 
Between 4-7 days post transplant, recipient mice begin to show clinical signs of GVHD. Cooke et al.1 developed a scoring system to quantitate disease progression based on the recipient mice fur texture, skin integrity, activity, weight loss, and posture. Mice are scored daily, and euthanized when they become moribund. Recipient mice generally become moribund 20-30 days post transplant. 
Murine models are valuable tools for studying the immunology of GVHD. Selectively transplanting particular T cell subsets allows for careful identification of the roles each subset plays. Non-invasively tracking T cell responses in vivo adds another layer of value to murine GVHD models.

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Murine bone marrow transplantation is a widely used technique to study immunological mechanisms governing graft-versus-host disease in humans. The ability to monitor T cell trafficking patterns in vivo allows for detailed analysis of the development and perpetuation of T cell responses during graft-versus-host disease.

移植物抗宿主病的诱导和<em在体内</em&gt; T细胞监测中的应用MHC匹配的小鼠模型

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

尾部皮肤来研究异基因CD4 + T细胞应答的小鼠移植

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

分离，鉴定和小鼠胸腺上皮细胞的分离纯化

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Murine Full-thickness Skin Transplantation

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited application to humans, skin transplantation in mice has been widely employed for transplantation research. The procedure is easy to learn and perform, and it does not require delicate microsurgical techniques nor extensive training. Moreover, graft rejection in this model occurs in a very reproducible immunological reaction and is easily monitored by direct inspection and palpation. In addition, secondary skin transplantation with donor-matched or third-party skin grafts can be performed on more complex transplant models as an alternative and uncomplicated method to assess donor-specific tolerance. The complications are low and are in general limited to anesthesia overdose or respiratory distress after the procedure. Graft failure, on the other hand, occurs commonly as a result of poor preparation of the graft, incorrect positioning in the graft bed, or inappropriate placement of the bandage. In this article, we present a protocol for full-thickness skin transplantation in mice and describe the important steps necessary for a successful procedure.

Murine full-thickness skin transplantation is a well-established model to study rejection in an alloimmune setting. Here, we provide a tutorial of each step involved in performing a BALB/c--&#62;C57BL/6 full-thickness skin transplant.

鼠全层皮肤移植

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited ...

A Novel Microsurgical Model for Heterotopic, En Bloc Chest Wall, Thymus, and Heart Transplantation in Mice

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression must be pursued. Employing vascularized bone marrow transplantation and co-transplantation of the thymus have shown promise in this regard in various animal models.1-11 Vascularized bone marrow transplantation allows for the uninterrupted transfer of donor bone marrow cells within the preserved donor microenvironment, and the incorporation of thymus tissue with vascularized bone marrow transplantation has shown to increase T-cell chimerism ultimately playing a supportive role in the induction of immune regulation. The combination of solid organ and vascularized composite allotransplantation can uniquely combine these strategies in the form of a novel transplant model. Murine models serve as an excellent paradigm to explore the mechanisms of acute and chronic rejection, chimerism, and tolerance induction, thus providing the foundation to propagate superior allograft survival strategies for larger animal models and future clinical application. Herein, we developed a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique. The experience in syngeneic and allogeneic transplant settings is described for future broader immunological investigations via an instructional manuscript and video supplement.

To study combined solid organ and vascularized composite allotransplantation, we describe a novel heterotopic en bloc chest wall, thymus, and heart transplant model in mice using a cervical non-suture cuff technique.

一种新的显微模型异位，整块胸壁，胸腺，和心脏移植小鼠

Exploration of novel strategies in organ transplantation to prolong allograft survival and minimizing the need for long-term maintenance immunosuppression ...

医学

Renal Subcapsular Transplantation of 2'-Deoxyguanosine-Treated Murine Embryonic Thymus in Nude Mice

The thymus is an important central immune organ, which plays an essential role in the development and differentiation of T cells. Thymus transplantation is an important method for investigating thymic epithelial cell function and T cells maturation in vivo. Here we will describe the experimental methods used within our laboratory to transplant 2&#8217;-deoxyguanosine (to deplete donor&#8217;s lymphocytes) treated embryonic thymus into the renal capsule of an athymic nude mouse. This method is both simple and efficient and does not require special skills or devices. The results obtained via this simple method showed that transplanted thymus can effectively support the recipient&#8217;s T cells production. Additionally, several key points with regards to the protocol will be further elucidated.

We provide a simple and efficient method to transplant 2&#8217;-deoxyguanosine treated E18.5 thymus into the renal capsule of a nude mouse. This method should aide in the study of both thymic epithelial cells function and T cells maturation.

2'-脱氧核苷在裸体小鼠中治疗鼠胚胎胸腺的肾下囊移植

The thymus is an important central immune organ, which plays an essential role in the development and differentiation of T cells. Thymus transplantation ...

优化从胸腺和骨髓中分离基质细胞

Stromal Cell Isolation From Hematopoietic Organs

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens through which to study previously unresolved heterogeneity at steady state, as well as changes in cell type representation induced by extrinsic stresses or during aging. Here, we present step-wise protocols for the isolation of high-quality stromal cell populations from murine and human thymus, as well as murine bone and bone marrow. Cells isolated through these protocols are suitable for generating high-quality single-cell multiomics datasets. The impacts of sample digestion, hematopoietic lineage depletion, FACS analysis/sorting, and how these factors influence compatibility with single-cell sequencing are discussed here. With examples of FACS profiles indicating successful and inefficient dissociation and downstream stromal cell yields in post-sequencing analysis, recognizable pointers for users are provided. Considering the specific requirements of stromal cells is crucial for acquiring high-quality and reproducible results that can advance knowledge in the field.

作者聚焦：使用基质细胞分离进行单细胞测序推进造血研究

Here we present protocols that enable isolation of stromal cells from murine bone, bone marrow, thymus and human thymic tissue compatible with single-cell multiomics.

从造血器官中分离基质细胞

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens ...

隔离和不同的老年小鼠胸腺移植的移植。

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此视频中的章节

胸腺表征祖定居在小鼠胚胎使用

活体成像的鼠标使用2 - 光子显微镜的胸腺

移植物抗宿主病的诱导和

尾部皮肤来研究异基因CD4 + T细胞应答的小鼠移植

分离，鉴定和小鼠胸腺上皮细胞的分离纯化

鼠全层皮肤移植

一种新的显微模型异位，整块胸壁，胸腺，和心脏移植小鼠

2'-脱氧核苷在裸体小鼠中治疗鼠胚胎胸腺的肾下囊移植

从造血器官中分离基质细胞

从造血器官中分离基质细胞