Name: microdissection of primary renal tissue segments and incorporation with novel scaffold-free construct technology
Uploaded: 2018-03-27T13:30:00.000+00:00
Duration: 9 min
Description: Watch this Scientific Journal Video about Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology at JoVE.com

NIH 机构博士后培训补助金, NIH-HL-007260

所有的动物外科手术程序都是在南卡罗来纳州医科大学的机构动物护理和使用委员会 (IACUC) 批准的, 然后再进行任何动物手术或任何动物组织的使用。
1. 小鼠肾切除术
<ol><li>不要用手术面罩和蓬松帽来减少污染的风险。在手术区的设置中保持不孕。<ol>
<li>将非 fenestrated 的手术窗帘放在手术台上。</li>
		<li>在无菌窗帘上打开一包蒸压仪器。肾切除术所需的器械包括3小止血剂、牙精钳、无牙精钳、直虹膜剪刀。</li>
		<li>将另一种非 fenestrated 的手术悬垂放在手术台的中心。</li>
		<li>用钙和镁制备5毫升的 Dulbecco 磷酸盐缓冲盐水 (DPBS), 在15毫升无菌圆锥管中补充1% 青霉素/链霉素。将此解决方案放在操作表旁边的冰上。</li>
	</ol>
</li>
	<li>从动物住房综合体获得 8-12 周大的 C57BL/6 鼠 (男性或女性)。</li>
	<li>将鼠标放在麻醉感应腔内, 并诱导3.5% 吸入异氟醚。在鼠标上放置一个鼻锥, 使用2% 异氟醚进行维护麻醉。<ol>
<li>通过评估踏板反射 (牢固的脚趾捏), 在整个手术过程中周期性地监测足够的麻醉深度。</li>
	</ol>
</li>
	<li>使用脱毛膏去除腹侧躯干的所有毛发。用蒸馏水冲洗这个区域, 以确保所有的毛发都已从腹部取出。</li>
	<li>在顶部非 fenestrated 悬垂下, 将鼠标转移到仰卧位的无菌手术台上。切出一个 2 x 2 厘米2开窗, 在刚刚覆盖鼠标腹部的褶皱。</li>
	<li>应用聚维酮碘后, 70% 乙醇的腹部使用无菌纱布或棉花球。</li>
	<li>使用牙和虹膜剪刀精钳, 使 3-4 厘米垂直腹部皮肤切口平行的中线, 0.5 厘米到左中线 注意:如果试图删除右肾, 这将是一个切口0.5 厘米右侧的中线。<ol>
<li>用细钳无牙抓腹膜, 用虹膜剪刀切割进入腹腔。将止血剂应用于皮肤和腹膜的上、下侧边缘 , 以横向放置张力 , 增露。</li>
		<li>将肠道内容微妙地移到腹部右侧, 以暴露左肾。轻轻地把牵引放在肾脏上, 把它从腹部抬高。</li>
		<li>在左肾的脐处放置一个止血。用虹膜剪刀对输尿管和肾血管进行横断面。</li>
		<li>在冰上放置左肾1% 青霉素/链霉素 DPBS 溶液。</li>
		<li>将含有肾脏的管转移到无菌组织培养罩中。将肾脏从15毫升锥形管转移到60毫米培养皿 (图 1)。用5毫升 DPBS 与钙和镁冲洗两次。保持肾脏悬浮在5毫升 DPBS 在60毫米菜。<ol>
<li>准备一个额外的60毫米培养皿与5毫升 DPBS 将用于在显微解剖协议。</li>
		</ol>
</li>
		<li>根据批准的 IACUC 动物协议, 弄死通过开胸和放血后的肾脏的采购。</li>
	</ol>
</li>
</ol>2. 小鼠肾脏显微切割分离肾段
<ol><li>继续使用无菌技术的这一部分的协议, 包括无菌手套, 手术面罩, 蓬松, 以减少污染风险。<ol>
<li>将无菌窗帘放在立体声显微镜平台上, 以及在显微镜的左侧和右侧的区域有一个无菌的领域为仪器。将塑料粘合剂片放在显微镜聚焦旋钮上, 喷雾70% 乙醇。打开双鹅颈灯照明舞台。</li>
		<li>开放式消毒器械 (无牙精钳, 手术刀柄, #15 手术刀刀片, 两个直止血剂, 两个30½口径空心孔针) 到无菌窗帘。现在将 #15 刀片放到手术刀手柄上, 并将一个止血连接到每个针适配器/轮毂, 以创建用于以后使用的针式解剖仪器, 如图 1所示。</li>
	</ol>
</li>
	<li>移动60毫米培养皿, 一个包含肾脏在 DPBS 和其他仅 DPBS, 从组织培养敞篷到显微镜区域。<ol>
<li>将60毫米培养皿置于目标下, 取出盖子露出肾脏。将含有 DPBS 的盘子留在不育的田地旁, 供日后使用。</li>
		<li>移动的菜, 以便只有前或后表面的肾脏是在视图 (即, 大平脸的肾脏, 与另一张脸接触底部的菜)。缩放到 3.2 4X 的过程的这一部分。使用非显性手, 使用无牙的细钳刺穿和针在肾脏的下和上两极附近的盘子。</li>
		<li>保持不占支配地位的手固定肾脏。用主导的手, 使用 #15 刀片从暴露的表面去除半透明纤维囊层。从肾脏外露表面刮出最浅的0.5 毫米, 除去胶囊的其余部分。</li>
		<li>使用 #15 刀片从 decapsulated 区域解剖组织的倒置金字塔, 大小约为2毫米3 。检索60毫米的菜只含有 DPBS, 并将组织金字塔放到干净的盘子里。丢弃肾脏的其余部分。</li>
		<li>在解剖的其余部分减少 1.5-2.0X 的放大倍数。使用两个止血针仪器作为切割仪器, 切片的组织片段成逐渐小的片断, 直到组织段小于或等于针尖的直径。肾脏组织2毫米3的大小将产生超过50段, 一旦完全解剖。</li>
		<li>将60毫米的菜与肾段移至组织培养罩。用1000µL 吸管去除 DPBS。添加5毫升 DMEM, 辅以10% 胎牛血清 (血清) 和1% 青霉素/链霉素。</li>
		<li>培养3天在37°c 在孵化器或植入细胞结构立即。</li>
	</ol>
</li>
</ol>3. 肾段细胞结构制作
<ol><li>培养正常的人真皮成纤维细胞 (NHDF) 和人脂肪微血管内皮细胞 (HAMEC) 几个星期, 以获得 7.2 x 105成纤维细胞和 1.8 x 105内皮细胞每个结构所需的。在组织培养罩中, 将成纤维细胞的适当比例 (4:1) 放入琼脂糖模中的棒状井中, 形成无支架的前血管内皮-成纤维细胞结构 (规格), 如前文所述 Czajka et 19。</li>
	<li>获得灭菌止血, 30½ G 针, 和无菌铲与平端。<ol>
<li>从含有肾脏片段的60毫米盘子中取出大部分培养基, 小心不要吸入并取出肾部分。</li>
		<li>配备手术放大镜, 以可视化的片段植入。</li>
		<li>在60毫米的盘子底部轻轻地刮擦刮刀的末端, 以获得 10-15 段, 沿铲片的尖端均匀分布。把刮刀的尖端尽可能靠近规格的种子的唇口。</li>
		<li>另一方面, 使用 hemostat-30½的 G 针工具, 将每个部分从铲头移动到井的边缘。用针尖轻轻地将每个段向下移动到井中, 直到在先前播种的细胞悬浮液中略微淹没。</li>
		<li>培养肾段-规格在 2:1: 1 FGM-2/EGM-2/DMEM 培养基的比例 (2.5 毫升体积总数)。每24小时更改文化媒体3天。从模具中取出 72 h 的构造, 然后进行修复或闪速冻结处理。</li>
	</ol>
</li>
</ol>

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作者没有什么可透露的。

用于设计活体肾脏组织结构的方法在所使用的细胞类型和生物材料方面差别很大, 在许多情况下, 在文献7中已经过时或没有很好的特征。虽然许多人使用干细胞方法或综述独立的肾脏结构的个别成分, 但从细胞悬浮中人工再造26多个不同分化细胞类型的整个器官的前景是压倒性地考虑21。这很可能是其他人在组织工程应用中使用肾组织片段的方法。如上所述, 以前...

慢性肾脏疾病 (CKD) 是当前全球主要的公共卫生挑战之一1。在美国, CKD 患病率超过总人口的 14%, 超过60万的美国人患有最严重的疾病, 终末期肾病 (ESRD)2。目前的治疗方案可供那些 ESRD 包括透析和肾移植。虽然每年约有2.5万名患者接受肾移植, 但每年有大量患者被增加, 导致等待救生器官和接受移植的人之间的巨大差距3。除了对长寿和生活质量造成严重的负面影响外, 透析还带来了惊人的经济负担。在 2014年, 医疗保险支付的索赔总额超过300亿美元的 ESRD 患者2。由于器官供应有限, 需要透析的患者没有明显的下降趋势, 因此, 旨在确定透析和移植的替代解决方案的研究工作一直是重要的。即使是相对较短的延迟, 透析的需要增加了病人的质量调整生活年限和生产力的大量, 而推迟透析相关费用4,5,6。
在组织工程和再生医学实验室中, ESRD 的功能性组织丧失的解决方案目前正在研究中, 从脚手架为基础的 organoid 制造到整个器官工程都有广泛多样的方法。瓣膜细胞植入的器官结构7,8,9,10,11。综述复杂的肾脏结构从边缘或被放弃的肾脏仅部份地被调查了。事实上, 为移植而采购的肾脏的近20% 因各种原因被丢弃,12,13。这些假定的移植物的功能性肾脏组织可以被利用并纳入一个或多个组织工程结构。先前的研究表明, 与这些被丢弃的器官合作的可行性, 利用肾脏为组织工程目的的额外细胞矩阵14,15。然而, 很少有使用健康肾脏的主要 nephronal 组织为组织工程目的16,17,18。
Kim et以前描述的一种方法是将肾脏 "段" 从健康的大鼠肾脏中分离出来, 然后在羟基酸 (PGA) 支架上播种, 用于构造制造16。然而, 关于精确解剖方法的信息很少, 从精细切碎和过滤的结合中得到了部分。我们描述了这个协议的修改, 这同样产生了离散的肾段与完整的 nephronal 体系结构, 而是依靠显微切割技术。切除是对活体成年小鼠进行的, 然后将肾脏转移到切除肾囊的解剖显微镜, 并进一步解剖组织。小孔 30½ G 针作为切割仪器, 也作为辅助解剖辅助, 因为针直径等于肾段的靶直径。这种孤立的, 在这种情况下, 小鼠, 肾部分保持在培养的生存能力, 并纳入无支架内皮细胞成纤维细胞结构19。这些构造以前被用来设计其他器官, 包括生物人工胰腺20。

<table><tbody><tr><td>Non-fenestrated Sterile Field</td><td>Busse Hospital Disposables</td><td>696</td><td></td></tr><tr><td>Fenestrated Sterile Field</td><td>Busse Hospital Disposables</td><td>697</td><td></td></tr><tr><td>Halsted Mosquito Forceps 5 Curved</td><td>Miltex</td><td>Mil-7-4</td><td>&quot;Hemostat&quot; in manuscript</td></tr><tr><td>Extra Fine Graefe Forceps, Curved with teeth</td><td>Fine Science Tools</td><td>11155-10</td><td>Fine forceps with teeth</td></tr><tr><td>Extra Fine Graefe Forceps, Serrated (without teeth)</td><td>Fine Science Tools</td><td>11152-10</td><td>Fine forceps without teeth</td></tr><tr><td>Fine Scissors - Tungsten Carbide</td><td>Fine Science Tools</td><td>14568-09</td><td>Iris Scissors</td></tr><tr><td>Betadine Surgical Scrub with Pump, Povidone-iodine 7.5%</td><td>Purdue Products L.P.</td><td>67618-151-17</td><td></td></tr><tr><td>Sterile Cotton Gauze Pad (4&quot; x 4&quot;)</td><td>Fisher Healthcare</td><td>22-415-469</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Solution</td><td>Corning</td><td>21-030-CV</td><td></td></tr><tr><td>Penicillin/Streptomycin Solution, 100X</td><td>Corning</td><td>30-002-Cl</td><td></td></tr><tr><td>Isoflurane, USP</td><td>Manufacturer: Piramal, Distributor: McKesson</td><td>2254845</td><td></td></tr><tr><td>Nair Hair Remover</td><td>Nair</td><td>22600-23307</td><td>Hair Removal Cream in text</td></tr><tr><td>200 Proof Ethanol</td><td>Decon Laboratories</td><td>2705</td><td>Diluted to 70% Ethanol Solution</td></tr><tr><td>BioLite 60mm Tissue Culture Dish</td><td>Themo-Scientific</td><td>130181</td><td></td></tr><tr><td>Press&#039;n Seal</td><td>Glad</td><td>12587-70441</td><td>Applied to Stereoscope</td></tr><tr><td>SZX16 Stereo Microscope</td><td>Olympus</td><td>SZX16</td><td></td></tr><tr><td>Fiber Optic Illuminator</td><td>Cole Parmer</td><td>41720-20</td><td></td></tr><tr><td>Self-Supporting Dual-Light Pipe, 23&quot; L Gooseneck</td><td>Cole Parmer</td><td>EW-41720-60</td><td></td></tr><tr><td>Scalpel Handle #3</td><td>Miltex</td><td>Mil-4-7</td><td></td></tr><tr><td>Sterile Rib-Back Carbon Steel Blade, Blade Size 15</td><td>Bard-Parker</td><td>371115</td><td></td></tr><tr><td>31 1/2 Gauge Needle</td><td>ThermoFisher Scientific</td><td>14-826F</td><td>Becton Dickinson 305106</td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium</td><td>Corning</td><td>10-017-CV</td><td></td></tr><tr><td>Fetal Select 100% Bovine Serum</td><td>Atlas Biologicals</td><td>FS-0500-AD</td><td></td></tr><tr><td>Normal Human Dermal Fibroblasts</td><td>Lonza</td><td>CC-2511</td><td></td></tr><tr><td>Human Adipose Microvascular Endothelial Cells</td><td>Sciencell Research Laboratories</td><td>7200</td><td></td></tr><tr><td>Surgical Loupes (2.5x)</td><td>Orascoptic</td><td>(N/A) Custom Order</td><td></td></tr><tr><td>FGM-2 (Fibroblast Basal Medium with FGM-2 SingleQuots Added)</td><td>Lonza</td><td>CC-3131, CC-4126</td><td></td></tr><tr><td>EGM-2 (Endothelial Basal Medium with EGM-2 SingleQuots Added)</td><td>Lonza</td><td>CC-3156, CC-4176</td><td></td></tr><tr><td>Live/Dead Viability/Cytotoxicity Kit for Mammalian Cells</td><td>ThermoFisher Scientific</td><td>L3224</td><td></td></tr><tr><td>Anti-Cytokeratin-18 Antibody</td><td>Abcam</td><td>ab668</td><td></td></tr><tr><td>Goat anti-Mouse IgG, Alexa Fluor 633</td><td>ThermoFisher Scientific</td><td>A-21052</td><td></td></tr><tr><td>Goat anti-Rabbit IgG, Alexa Fluor 546</td><td>ThermoFisher Scientific</td><td>A-11010</td><td></td></tr><tr><td>Anti-Von Willebrand Factor Antibody</td><td>Abcam</td><td>ab6994</td><td></td></tr><tr><td>Albumin, Fluorescein isothiocyanate Conjugate</td><td>Sigma Aldrich</td><td>A9771-50MG</td><td></td></tr><tr><td>Hoescht 33342</td><td>BD Pharmingen</td><td>561908</td><td></td></tr><tr><td>Background Buster</td><td>Innovex Biosciences</td><td>NB306</td><td></td></tr></tbody></table>

microdissection of primary renal tissue segments and incorporation with novel scaffold-free construct technology

肾脏移植现在是治疗终末期肾脏疾病的主流疗法。然而, 约有9.6万人在等待名单上, 只有1/4 的患者实现移植, 迫切需要为那些器官衰竭者提供替代品。为了减少透析带来的有害后果, 以及它所招致的整体医疗费用, 正在积极调查寻找移植器官的替代办法。植入性组织工程的肾细胞结构是一种替代失去的肾脏功能的可行方法。这里, 第一次被描述, 是小鼠肾脏的显微解剖为分离生活皮质髓质肾脏片断。这些片段能够迅速纳入无支架的内皮纤维细胞结构, 这可能使与宿主血管的快速连接一旦植入。成年小鼠肾脏是从活体捐献者那里获得的, 其次是立体镜显微解剖, 得到 200-300 µm 直径的肾段。多肾结构是用从一肾中收获的原发性肾段制作而成。该方法证明了一种可以从器官中抢救功能性肾组织的程序, 否则将被丢弃。

所描述的协议产生大约50个肾脏片段每锥体2毫米3段肾脏组织。已处理和成像的肾段有不同比例的管状和肾小球成分 (请参见图 2)。完整的片段进行了化验, 以确定每24小时的不同片段的生存时间三天。绿色荧光 calcein 是存在的细胞内酯酶活性, 指示的活细胞。红荧光溴 homodimer-1 被认为是由于等离子膜完整性的丧失。虽然这些片段是完整的, 三?...

Watch this Scientific Journal Video about Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology at JoVE.com

Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology

组织工程的肾脏结构提供了解决的器官短缺和有害影响的透析。在这里, 我们描述了一个小鼠肾脏的显微解剖的协议, 以隔离皮质髓质段。这些片段植入无支架细胞结构, 形成肾 organoids。

原发性肾组织段的显微解剖与新型无支架结构技术的结合

Kidney transplantation is now a mainstream therapy for end-stage renal disease. However, with approximately 96,000 people on the waiting list and only ...

microdissection-primary-renal-tissue-segments-incorporation-with

Research

JoVE Journal

Bioengineering

7.4K 次观看.  Medical University of South Carolina. 组织工程的肾脏结构提供了解决的器官短缺和有害影响的透析。在这里, 我们描述了一个小鼠肾脏的显微解剖的协议, 以隔离皮质髓质段。这些片段植入无支架细胞结构, 形成肾 organoids。

视频: Microdissection of Primary Renal Tissue Segments and Incorporation with Novel Scaffold-free Construct Technology

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped human cell culture facilities. These methods include isolation of cells in single cell suspension and several laborious and time-consuming events before transplantation back to the patient. Previous studies suggest that the body itself could be used as a bioreactor for cell expansion and regeneration of tissue in order to minimize ex vivo manipulations of tissues and cells before transplanting to the patient. The aim of this study was to demonstrate a method for tissue harvesting, isolation of continuous epithelium, mincing of the epithelium into small pieces and incorporating them into a three-layered biomaterial. The three-layered biomaterial then served as a delivery vehicle, to allow surgical handling, exchange of nutrition across the transplant, and a controlled degradation. The biomaterial consisted of two outer layers of collagen and a core of a mechanically stable and slowly degradable polymer. The minced epithelium was incorporated into one of the collagen layers before transplantation. By mincing the epithelial tissue into small pieces, the pieces could be spread and thereby the propagation of cells was stimulated. After the initial take of the transplants, cell expansion and reorganization would take place and extracellular matrix mature to allow ingrowth of capillaries and nerves and further maturation of the extracellular matrix. The technique minimizes ex vivo manipulations and allow cell harvesting, preparation of autograft, and transplantation to the patient as a simple one-stage intervention. In the future, tissue expansion could be initiated around a 3D mold inside the body itself, according to the specific needs of the patient. Additionally, the technique could be performed in an ordinary surgical setting without the need for sophisticated cell culturing facilities.

Tissue engineering often includes in vitro expansion in order to create autografts for tissue regeneration. In this study a method for tissue expansion, regeneration, and reconstruction in vivo was developed in order to minimize the processing of cells and biological materials outside the body.

在压缩的胶原组织糜:含细胞Biotransplant单上演重建修复

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

科研

生物工程

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 &#176;C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 &#181;M resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

上皮细胞复育和肾组织的发展准备啮齿动物细胞外基质的支架

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper composition, targeted modulus, and well-defined architectural features. Biomaterials that recapitulate the intrinsic architecture of in vivo tissue are vital for studying diseases as well as to facilitate the regeneration of lost and malformed soft tissue. A novel biofabrication technique was developed which combines state of the art imaging, three-dimensional (3D) printing, and selective enzymatic activity to create a new generation of biomaterials for research and clinical application. The developed material, Bovine Serum Albumin rubber, is reaction injected into a mold that upholds specific geometrical features. This sacrificial material allows the adequate transfer of architectural features to a natural scaffold material. The prototype consists of a 3D collagen scaffold with 4 and 3 mm channels that represent a branched architecture. This paper emphasizes the use of this biofabrication technique for the generation of natural constructs. This protocol utilizes a computer-aided software (CAD) to manufacture a solid mold which will be reaction injected with BSA rubber followed by the enzymatic digestion of the rubber, leaving its architectural features within the scaffold material.

An innovative biofabrication technique was developed to engineer three-dimensional constructs that resemble the architectural features, components, and mechanical properties of in vivo tissue. This technique features a newly developed sacrificial material, BSA rubber, which transfers detailed spatial features, reproducing the in vivo architectures of a wide variety of tissues.

三维仿生技术:新型Biorubber创建自定义微观和宏观尺度架构的胶原凝胶

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper ...

Optimization of Renal Organoid and Organotypic Culture for Vascularization, Extended Development, and Improved Microscopy Imaging

Embryonic kidney organotypic cultures, and especially pluripotent stem cell-derived kidney organoids, are excellent tools for following developmental processes and modelling kidney disease. However, the models are limited by a lack of vascularization and functionality. To address this, an improved protocol for the method of xenografting cells and tissues to the chorioallantoic membrane (CAM) of an avian embryo to gain vascularization and restoration of blood flow was developed. The grafts are overlaid with custom-made minireservoirs that fix the samples to the CAM and supply them with culture medium that protects the grafts from drying. The improved culture method allows xenografts to grow for up to 9 days. The manuscript also describes how to provide optimal conditions for long-term confocal imaging of renal organoids and organotypic cultures using the previously published Fixed Z-Direction (FiZD) method. This method gently compresses an embryonic organ or organoid between a glass coverslip and membrane in a large amount of medium and provides excellent conditions for imaging for up to 12 days. Together, these methods allow vascularization and blood flow to renal organoids and organotypic kidney cultures with improved confocal imaging. The methods described here are highly beneficial for studying fundamental and applied functions of kidneys ex vivo. Both methods are applicable to various types of tissues and organoids.

This work describes two methods for studying organ development, an improved xenotransplantation setup on chorioallantoic membrane (CAM) from avian embryos that allows for vascularization of cultured embryonic organs and organoids and a novel fixed z-direction organ culture method with modified experimental conditions that allows for high-resolution time-lapse confocal imaging.

血管化、扩展发育和改进显微镜成像的肾器官和器官培养的优化

Embryonic kidney organotypic cultures, and especially pluripotent stem cell-derived kidney organoids, are excellent tools for following developmental ...

发育生物学

<meta charset="utf8"></head><body>使用微生理系统进行肾癌和健康组织共培养

Microfluidic Co-culture of Renal Healthy and Tumor Epithelium to Model Kidney Cancer Progression

Microphysiological systems (MPS) have enabled the introduction of more complex and relevant physiological elements into in vitro models, recreating intricate morphological features in three-dimensional environments with dynamic interactions lacking in conventional models. We implemented a renal cell carcinoma (RCC) co-culture model to recreate the cross-talk between healthy and malignant renal tissue. 
This model is based on the referenced multi-organ platform and consists of co-culturing a reconstructed renal proximal tubule with RCC spheroids. Custom-designed 3D-printed chambers were used to culture human renal epithelial proximal tubule cells (RPTEC) and facilitate their self-assembly into a renal tubule contained in a collagen type I matrix. Caki-1 RCC cells were embedded in an agar collagen matrix, subsequently forming cancer spheroids. Both collagen and agar/collagen gels were optimized to maintain their integrity during cyclic perfusion and withstand shear stress during a minimum culture period of 7 days. 
The gels also enable adequate nutrient supply and cell secretions. Moreover, the agar/collagen gels limit the overproliferation of RCC cells, ensuring relatively homogeneous spheroid size. The MPS chip microfluidic circuits comprise two independent culture chambers with the size of a standard 96-microplate well. The renal tubule and RCC gels populate separate chambers and share the same culture media, which is recirculated approximately twice per minute. Under these conditions, we observed upregulation of immune factor expression and secretion in the renal tubules (interleukin-8 and tumor necrosis factor-alpha). The renal tubules also shift their metabolic activity towards glycolysis under the influence of RCC. This novel approach demonstrates that a co-culture-based MPS can amplify the complexity of RCC in vitro and be employed to study the impact of cancer on non-tumor cells.

<meta charset="utf8"></head><body>肾脏健康和肿瘤上皮细胞的微流控共培养以模拟肾癌进展

Here, we present protocols that detail instructions for implementing 3D cell cultures using collagen and collagen-agarose matrixes in a microphysiological system. These protocols support renal proximal tubule and renal cell carcinoma spheroids co-culture, simulating in vivo conditions and enabling advanced investigation of kidney cancer cell interactions.

肾脏健康和肿瘤上皮细胞的微流控共培养以模拟肾癌进展

Microphysiological systems (MPS) have enabled the introduction of more complex and relevant physiological elements into in vitro models, recreating ...

癌症研究

Isogenic Kidney Glomerulus Chip Engineered from Human Induced Pluripotent Stem Cells

Chronic kidney disease (CKD) affects 15% of the U.S. adult population, but the establishment of targeted therapies has been limited by the lack of functional models that can accurately predict human biological responses and nephrotoxicity. Advancements in kidney precision medicine could help overcome these limitations. However, previously established in vitro models of the human kidney glomerulus-the primary site for blood filtration and a key target of many diseases and drug toxicities-typically employ heterogeneous cell populations with limited functional characteristics and unmatched genetic backgrounds. These characteristics significantly limit their application for patient-specific disease modeling and therapeutic discovery. 
This paper presents a protocol that integrates human induced pluripotent stem (iPS) cell-derived glomerular epithelium (podocytes) and vascular endothelium from a single patient to engineer an isogenic and vascularized microfluidic kidney glomerulus chip. The resulting glomerulus chip is comprised of stem cell-derived endothelial and epithelial cell layers that express lineage-specific markers, produce basement membrane proteins, and form a tissue-tissue interface resembling the kidney's glomerular filtration barrier. The engineered glomerulus chip selectively filters molecules and recapitulates drug-induced kidney injury. The ability to reconstitute the structure and function of the kidney glomerulus using isogenic cell types creates the opportunity to model kidney disease with patient specificity and advance the utility of organs-on-chips for kidney precision medicine and related applications.

Presented here is a protocol to engineer a personalized organ-on-a-chip system that recapitulates the structure and function of the kidney glomerular filtration barrier by integrating genetically matched epithelial and vascular endothelial cells differentiated from human induced pluripotent stem cells. This bioengineered system can advance kidney precision medicine and related applications.

由人诱导多能干细胞改造的同基因肾小球芯片

Chronic kidney disease (CKD) affects 15% of the U.S. adult population, but the establishment of targeted therapies has been limited by the lack of ...

An Efficient Sieving Method to Isolate Intact Glomeruli from Adult Rat Kidney

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by proteinuria which can eventually lead to chronic and end-stage renal disease. The glomerulus is a complex apparatus responsible for the filtration of plasma from the body. In disease, structural integrity is lost and allows for the abnormal leakage of plasma contents into the urine. A method to isolate and examine glomeruli in culture is critical for the study of these diseases. In this protocol, an efficient method of retrieving intact glomeruli from adult rat kidneys while conserving structural and morphological characteristics is described. This process is capable of generating high yields of glomeruli per kidney with minimal contamination from other nephron segments. With these glomeruli, injury conditions can be mimicked by incubating them with a variety of chemical toxins, including protamine sulfate, which causes foot process effacement and proteinuria in animal models. Degree of injury can be assessed using transmission electron microscopy, immunofluorescence staining, and western blotting. Nephrin and Wilms Tumor 1 (WT1) levels can also be assessed from these cultures. Due to the ease and flexibility of this protocol, the isolated glomeruli can be utilized as described or in a way that best suits the needs of the researcher to help better study glomerular health and structure in diseased states.

The main focus of this protocol is to efficiently isolate viable primary glomeruli cultures with minimal contaminants for use in a variety of downstream applications. The isolated glomeruli retain structural relationships between component cell types and can be cultured ex vivo for a short time.

一种从成年大鼠肾脏中分离完整型肾小球的有效筛分方法

Preservation of glomerular structure and function is pivotal in the prevention of glomerulonephritis, a category of kidney disease characterized by ...

医学

使用胶原支架进行猪单阶段尿流改道手术

Surgical Model for Single-Staged Tissue-Engineered Urothelial Tubes in Minipigs

Reconstructive surgeries are often challenged by a lack of grafting tissue. In the treatment of urogenital malformations, the conventional solution has been harvesting gastrointestinal tissue for non-orthotopic reconstruction due to its abundance to reestablish normal function in the patient. The clinical outcomes after rearranging native tissues within the body are often associated with significant morbidity; thus, tissue engineering holds specific potential within this field of surgery. Despite substantial advances, tissue-engineered scaffolds have not yet been established as a valid surgical treatment alternative, mainly due to the costly and complex requirements of materials, production, and implantation. In this protocol, we present a simple and accessible collagen-based tubular scaffold embedded with autologous organ-specific tissue particles, designed as a conduit for urinary diversion. The scaffold is constructed during the primary surgical procedure, comprises commonly available surgical materials, and requires conventional surgical skills. Secondly, the protocol describes an animal model designed to evaluate the short-term in vivo outcomes post-implantation, with the possibility of additional variations to the procedure. This publication aims to demonstrate the procedure step-by-step, with special attention to the use of autologous tissue and a tubular form.

作者聚焦:改善先天性畸形和后天性疾病重建手术结果的创新方法

Tissue-engineered implants for reconstructive surgery rarely progress beyond preclinical trials due to laborious ex vivo culturing, which includes complex and expensive scaffold components. Here, we present a single-staged procedure designed for urinary diversion with an accessible collagen-based tubular scaffold containing autologous micrografts.

小型猪单期组织工程尿路上皮管手术模型

Reconstructive surgeries are often challenged by a lack of grafting tissue. In the treatment of urogenital malformations, the conventional solution has ...

原发性肾组织段的显微解剖与新型无支架结构技术的结合

摘要

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此视频中的章节

在压缩的胶原组织糜:含细胞Biotransplant单上演重建修复

上皮细胞复育和肾组织的发展准备啮齿动物细胞外基质的支架

三维仿生技术:新型Biorubber创建自定义微观和宏观尺度架构的胶原凝胶

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