这项工作是由ANR（计划相思神经科学），FRC（联合会倒拉RECHERCHE河畔乐Cerveau）支持补助，巴黎市（诞生计划）的，INSERM和LaPitié妇女救济院医院（临床转化研究合同），以NR，从Neuropôle德RECHERCHE Francilien（NERF）以ED，从UNIVERSITE皮埃尔与玛丽·居里UPMC（方案融合），并从研究所杜Cerveau等è拉Moelle epiniere（巴黎），生长激素

注：这里所描述的协议遵循法国的“法国精品行业Consultatif国家科特迪瓦Ethique”的指导方针，并宣布由INSERM一个征集活动。 1.手术切除癫痫人体组织<ol><li>患者<ol><li>取得患者难治性癫痫患脑组织。手术前获得书面知情同意书。 </li><li>对于所有操作的癫痫患者进行了广泛的手术前的后处理，包括神经系统检查，神经心理学评估，表面脑电图和神经影像（MRI和有时18 FDG-PET扫描的; 图1A），接着切除的组织的外科手术后的组织学检查（ 图1B） 。注：手术中指出，当不同的探索本地化类似的发作起始区，当其去除的好处超过了手术风险。 </li></ol></li><li>手术<ol><li>麦ntain抗癫痫药物预操作性。与吸入七氟醚（6％递送率和100％的吸入的O 2分），静脉内舒芬太尼（0.3微克/千克）和antracurium（0.5毫克/千克），接着用静脉内舒芬太尼维修感应：在全身麻醉下进行手术（0.5微克/千克/小时）和吸入七氟醚（1，MAC）。 </li><li>使用神经导航系统，以允许在受损皮层（ 图1C）的精确定位。后的皮肤切口，执行中的骨随后用高洒钻头开颅钻孔内。轻轻地提升骨瓣，然后打开用剪刀（ 图1D）硬脑膜。 </li><li>使用术中超声，以便在手术显微镜下识别的异常皮层，和显微器械和超声波抽吸。 </li><li>凝结和根据脑沟限制切断的PIO-蛛网膜平面。使用软膜下剥离至Release来自各地的受损区域的软皮质。 </li><li>切正常皮质下白质完成一件“整块”切除术，不遗余力，尽可能的血管。避免创伤性操作的皮质。 </li><li>切成1厘米厚的片出了切除的组织的沿一个方向正交的软膜表面，包括沟底部及过渡皮质。 </li></ol></li></ol> 2.人工脑脊液（学联）的切片准备，孵化和录音<ol><li>学联的切片准备<ol><li>制备1升的蔗糖为基础的ACSF 7,8，含有（以mM计）：250蔗糖，3的KCl，25的NaHCO 3，10 D-葡萄糖，1的CaCl 2，10的MgCl 2;溶解这些盐在去离子水和充氧与卡波金的溶液至少10分钟。 </li><li>放〜700毫升蔗糖的ACSF中，在-80℃下进行50-60分钟（或-150℃下进行15-20分钟）。不断的休息根据氧合冰的解决方案。注：ACSF中为片制剂可贮存于4℃;在这种情况下，加在实验的CaCl 2和MgCl 2的那一天，将其冷却之前。 </li></ol></li><li>学联的切片孵化和录音<ol><li>准备至少3L的记录的ACSF 7,8的，它含有（以mM计）：124氯化钠，氯化钾3，26的NaHCO 3，10 D-葡萄糖，1.6的CaCl 2，1.3 MgCl 2的;溶解这些盐在去离子水和充氧与卡波金的溶液。在添加氯化镁 ，保留一些学联在0 镁离子学联进行录音。注：孵化/记录的ACSF可贮存于4℃;在这种情况下，添加的CaCl 2和MgCl 2的实验的日子。 </li></ol></li></ol> 3.设备切片孵化和录音<ol><li>接口室孵化片<ol><li>采用脑片室维持生活片体外&lt;/ em&gt;的接口模式。注：该腔室是由一低部，其中包含了加热和传感元件和一个陶瓷鼓泡器，并填充有蒸馏水，和上部，其中，所述片上休息晶状体组织中的平坦区域的片材中的腔室的中心（ 图2A - B）中 。 </li><li>使用两个单独的细的PTFE管中，通过该preoxygenated ACSF进入腔室。注：将管螺旋中被加热的水并到达腔室的上部;然后将溶液排出通过毛细作用带镜头薄纸，该输送溶液进入出口孔装置。 </li><li>连接接口室的聚四氟乙烯管，以蠕动泵，以允许与preoxygenated介质的切片的连续灌注。 </li><li>由通过陶瓷扩散器与卡波金（95％O 2和5％CO 2）的充氧维持高氧张力。注：此温热和湿润的混合气体达到片在通过适当的孔腔的上部。 </li><li>通过将腔室与温度控制器，通过一个特殊的双端连接线，具有连接器，用于所述腔室的加热元件和一个外部温度传感器的一端保持在所述腔室的上部的温度。在附近放置切片传感器在整个温育（ 图2C）检查温度。 </li></ol></li><li>多电极阵列记录<ol><li>使用平面多电极阵列120的氮化钛电极（直径30微米）绝缘用氮化硅和内部参比电极，布置在一个12×12的矩阵与200μm的中心到中心的间距。注意：用不同的电极直径和间距的其它配置是可能的。特别是，在MEA芯片可以加宽来样较大的人体组织的切片。 </li><li>获得的电信号在10kHz使用MEA2100-120系统（前和音响滤波器放大器，通过专用的软件（MC_Rack）集成的数据采集和模拟 - 数字转换器，16位的数据的分辨率，输入电压范围和带宽，通过软件可调）。注：MEA2100探头被赋予一个金属板，通过磁持有人可加热灌注元素固定的，在整个录制过程持续灌注切片。 </li><li>持组织向下在MEA由小自制铂锚，这确保了片和电极之间有良好的电耦合。 </li></ol></li></ol> 4.编写界面商会和工具解剖和切片<ol><li>接口室<ol><li>装满蒸馏水的界面室的下段。确保水的水平完全沉浸在加热元件和为2至4mm的腔室的下部和上部部分之间的连接处下方。在加热开关前，每天都要检查这个层面上，为了避免加热元件的损害。 </li><li>该腔室连接到一个卡波金源，赋予了二次流量调节器对气体压力进行微调。 </li><li>切镜头纸的片材，以适合的堰板腔的平坦区域，并且其定位接近输入解管，让来自输入线路的任何气泡逃逸到达片之前。 </li><li>切两片滞留棉线的，只要片透镜的组织，并将它们沿腔的平坦区域的主面的位置。注意：这有助于略微提高腔室中的灌注溶液的水平。 </li><li>设置蠕动泵的流速为1毫升/分钟，并启动泵。 </li><li>启动水的氧合作用在腔室的底部部分。 </li><li>将温度控制器的温度在37℃，并打开它。盖上一片封口膜的界面室的上部，并等待至少30分钟的Temperat .. [温度URE增加和稳定。 </li></ol></li><li>工具解剖和切片<ol><li>准备解剖套件包括两个细镊子，铲子，一个小勺子和一个刀片;两个玻璃培养皿中，两个电刷以及氰基丙烯酸酯胶。 </li><li>设置的vibratome的参数：厚度400微米;频率，70 - 90赫兹;振幅，0.9 - 1毫米，速度：0.1毫米/秒。 </li></ol></li></ol> 5.人力皮质切片制备<ol><li>取下-80℃冷冻蔗糖为基础的学联，混匀，以获得一种搪塑的。与卡波含氧它，把〜250毫升玻璃瓶，并保持它在杜瓦瓶中装满冰块，而要去医院的手术室，以获得组织。在此期间，保持静止，在-20℃下。 注：在手术中，神经外科医生解剖皮层组织的一小块。立即放入冰冷的蔗糖学联样品转移到实验室。 </li><li>保持所需要的从医院转移到实验室到最多30分钟的时间。在实验室中，以蔗糖为基础的含氧学联出​​-20℃的冰箱中，混合它有行贿，填补了培养皿和的vibratome缓冲盘，并同时启动与卡波充氧。小心取出一块人体组织从瓶中，用勺子，把它的皮氏培养皿（ 图2D）。 </li><li>使用镊子，小心地取出血栓，血管及脑膜，避免切割过程中的阻力。除去脑膜，包括在软质，从组织块的侧面开始，注意不要损伤大脑皮质表面。用刀片，切割组织，以获得平坦的表面，这将在对标本的vibratome板胶合的一侧。注：脑膜，包括在软质，除去来自组织块的侧面开始，注意不要损伤大脑皮质表面。 </li><li>如果TI块ssue大于MEA的腔室，在样品板（ 图2E）胶合之前切下一块的相应尺寸。 </li><li>胶组织，以获得横向皮质切片，放入缓冲盘切400微米厚的薄片，在低速（0.1毫米/秒）（ 图2F）。 </li><li>镜头切与组织切片维度件;用刮铲和刷子，传送片上，这些透镜的组织和开始记录（ 图2G - I）的前孵育它们在接口室中于37℃下至少1小时。连续地存储在相同的条件下的切片，直到使用。注：对镜头纸放置片是为了便于片底侧的两个灌注和从接口室切片的记录前除去的重要步骤。 <ol><li>在传送片的镜头纸，管理起来非常仔细，避免与触摸皮质层区域刷。 </li><li>制备切片一次一个，并立即他们每个传送到接口室，以确保适当的氧气供应和温度尽可能快地切片切除组织的块。 </li></ol></li></ol> 6.准备MEA设置为录音<ol><li>堵塞灌注系统以蠕动泵与MEA系统到计算机。含氧录音学联。将放大器内部的空MEA芯片。 </li><li>设置温度控制器，用于加热套管元件达到37℃，在MEA室并开始灌注在5-6毫升/分钟。注意：通常是必要的，设置温度3-4℃，比所述一个需要的话，根据房间温度和流率。 <ol><li>检查在MEA室内的温度用细温度计，将其放置在尽可能接近该中心，而不触及电极面积。 </li></ol></li><li>开始录制仿真软件e和检查所有的MEA的电极连接好，并且有因灌注无噪音。 </li><li>开始MEA_Monitor检查摄像机表的功能。 </li></ol> 7.转移切片从界面室以MEA芯片记录<ol><li>倒一些温热的玻璃培养皿记录的ACSF;用钳子，采取从接口室切片，通过镜头的组织抓住它，并把它放在培养皿。仔细分离从组织切片。如果在接口室中的溶液的水平期间的温育时间是经过精心调节，限幅将分离从镜头纸浸没它在溶液中;否则，用一个小刷子，方便拆卸。 </li><li>填充的MEA芯片与一些ACSF和用抹刀，切片转移到它。用塑料巴斯德吸管，轻轻地取下的解决方案，使切片粘附在电极区域，并与铂ANCH保持它在的地方或。加入1 ml录音学联，将MEA芯片进入放大器，并在5-6毫升/分钟开始灌注（ 图2J - L）。 </li><li>检查在MEA记录区域中的切片位置使用MEA_Monitor，如果需要调整它，以便从所述皮质层来记录并拍照。开始录制。 </li></ol>

<ol>
	<li>Huberfeld, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamatergic+pre-ictal+discharges+emerge+at+the+transition+to+seizure+in+human+epilepsy.">Glutamatergic pre-ictal discharges emerge at the transition to seizure in human epilepsy.</a> Nature Neuroscience. 14 (5), 627-634 (2011).</li><li>Kwan, P., Brodie, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+identification+of+refractory+epilepsy.">Early identification of refractory epilepsy.</a> New England Journal of Medicine. 342 (5), 314-319 (2000).</li><li>Van Pelt, J., Corner, M. A., Wolters, P. S., Rutten, W. L. C., Ramakers, G. 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=Longterm+stability+and+developmental+changes+in+spontaneous+network+burst+firing+patterns+in+dissociated+rat+cerebral+cortex+cell+cultures+on+multielectrode+arrays.">Longterm stability and developmental changes in spontaneous network burst firing patterns in dissociated rat cerebral cortex cell cultures on multielectrode arrays.</a> Neuroscience Letters. 361 (1-3), 86-89 (2004).</li><li>Sun, J. -. J., Luhmann, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatio-temporal+dynamics+of+oscillatory+network+activity+in+the+neonatal+mouse+cerebral+cortex:+Network+oscillations+in+neonatal+cerebral+cortex.">Spatio-temporal dynamics of oscillatory network activity in the neonatal mouse cerebral cortex: Network oscillations in neonatal cerebral cortex.</a> European Journal of Neuroscience. 26 (7), 1995-2004 (2007).</li><li>Dossi, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+Regeneration+of+the+ex-vivo+Reconstructed+Mesocorticolimbic+Dopaminergic+System.">Functional Regeneration of the ex-vivo Reconstructed Mesocorticolimbic Dopaminergic System.</a> Cerebral cortex. 23 (12), 2905-2922 (1991).</li><li>Darbon, P., Scicluna, L., Tscherter, A., Streit, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+controlling+bursting+activity+induced+by+disinhibition+in+spinal+cord+networks.">Mechanisms controlling bursting activity induced by disinhibition in spinal cord networks.</a> The European journal of neuroscience. 15 (4), 671-683 (2002).</li><li>Cohen, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+Origin+of+Interictal+Activity+in+Human+Temporal+Lobe+Epilepsy+in+Vitro.">On the Origin of Interictal Activity in Human Temporal Lobe Epilepsy in Vitro.</a> Science. 298 (5597), 1418-1421 (1126).</li><li>Huberfeld, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perturbed+chloride+homeostasis+and+GABAergic+signaling+in+human+temporal+lobe+epilepsy.">Perturbed chloride homeostasis and GABAergic signaling in human temporal lobe epilepsy.</a> The Journal of neuroscience. 27 (37), 9866-9873 (2007).</li><li>Maier, N., Morris, G., Johenning, F. W., Schmitz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Approach+for+Reliably+Investigating+Hippocampal+Sharp+Wave-Ripples+In+Vitro.">An Approach for Reliably Investigating Hippocampal Sharp Wave-Ripples In Vitro.</a> PLoS ONE. 4 (9), (2009).</li><li>H&#225;jos, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintaining+network+activity+in+submerged+hippocampal+slices:+importance+of+oxygen+supply.">Maintaining network activity in submerged hippocampal slices: importance of oxygen supply.</a> European Journal of Neuroscience. 29 (2), 319-327 (2009).</li><li>Wittner, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epileptic+human+hippocampal+cornu+ammonis+2+region+generates+spontaneous+interictal-like+activity+in+vitro.">The epileptic human hippocampal cornu ammonis 2 region generates spontaneous interictal-like activity in vitro.</a> Brain. 132 (11), 3032-3046 (2009).</li><li>Dzhala, V., Valeeva, G., Glykys, J., Khazipov, R., Staley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Traumatic+alterations+in+GABA+signaling+disrupt+hippocampal+network+activity+in+the+developing+brain.">Traumatic alterations in GABA signaling disrupt hippocampal network activity in the developing brain.</a> The Journal of neuroscience. 32 (12), 4017-4031 (2012).</li><li>Menendezdela de la Prida, L., Huberfeld, G., Cohen, I., Miles, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Threshold+Behavior+in+the+Initiation+of+Hippocampal+Population+Bursts.">Threshold Behavior in the Initiation of Hippocampal Population Bursts.</a> Neuron. 49 (1), 131-142 (2006).</li><li>Avoli, M., Drapeau, C., Louvel, J., Pumain, R., Olivier, A., Villemure, J. -. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epileptiform+activity+induced+by+low+extracellular+magnesium+in+the+human+cortex+maintained+in+vitro.">Epileptiform activity induced by low extracellular magnesium in the human cortex maintained in vitro.</a> Annals of neurology. 30 (4), 589-596 (1991).</li><li>Heinemann, U., Dreier, J., Leschinger, A., Stabel, J., Draguhn, A., Zhang, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+anticonvulsant+drugs+on+hippocampal+neurons.">Effects of anticonvulsant drugs on hippocampal neurons.</a> Hippocampus. 4 (3), 291-296 (1994).</li><li>Li Zhang, C., Dreier, J. P., Heinemann, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paroxysmal+epileptiform+discharges+in+temporal+lobe+slices+after+prolonged+exposure+to+low+magnesium+are+resistant+to+clinically+used+anticonvulsants.">Paroxysmal epileptiform discharges in temporal lobe slices after prolonged exposure to low magnesium are resistant to clinically used anticonvulsants.</a> Epilepsy research. 20 (2), 105-111 (1995).</li><li>Buzs&#225;ki, G., Anastassiou, C. A., Koch, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+of+extracellular+fields+and+currents+-+EEG,+ECoG,+LFP+and+spikes.">The origin of extracellular fields and currents - EEG, ECoG, LFP and spikes.</a> Nature Reviews Neuroscience. 13 (6), 407-420 (2012).</li></ol>

提供了用于制作视频和开放存取出版的赞助多声道系统。

Pharmacoresistant癫痫病是一种罕见的疾病，它可以在体外培养人体组织进行研究。这使得研究人类癫痫的皮层，这显示只有部分转载在动物模型中特定的缺陷。这里介绍的方法可以准备和记录人术后组织离体保存与细胞活力和网络，使他们自发地产生癫痫活动。护比记录在体内类似的活动是非常重要的研究病理活动发生的机制。此外，这种方法允许探索人体组织和​​避免疾病的非完美的动物模型。然而，研究人体组织需要神经外科医生和实验室实验之间的同步。组织运输需要特定小心不要被创伤。另外，检体的数量和组织是有限的。最后，获得适当的控制的组织是迈ñ关注。有了这个准备，术后组织自发产生发作样放电正常学联7,8。发作样的事件，也可以在改性，proconvul​​sive ACSF中引起使癫痫发作起始和转换的机制，从发作状态癫痫发作可以调查。 人体组织可保持存活长达10小时的接口条件。片被认为是可行的，当观察到多单位活动或场电位自发并且当这样的活动是通过细胞外钾的增加和/或镁的减少增加的兴奋诱发。虽然变异性活动发生，可能反映了病理和皮质区的差异，我们只能在健康的切片呈现自发性发作探讨了组织癫痫的特点及诱发发作期放电。为了保存组织活力和活性，一个界面室用于存储吨他切片在36-37℃下恢复录制与MEA系统之前。事实上，几个小组已经清楚地表明相对于标准烧杯存储和温度对网络活动的保护的重要性接口基于存储系统的优点，如自发尖波涟漪或胆碱诱导的振荡9,10。片接口存储先前已用于记录从人类海马和subicular片1,11癫痫活动。与当前的MEA技术，从切片在界面条件的恢复期后，网络活动，在37℃下记录，在浸没的条件下，在高流动速率（5-6毫升/分钟）存在下，与MEA系统。 MEA芯片的直径减小（1.8厘米），其限定一个小容积室（&lt;1.5毫升），用增加的流量一起，提高了氧气供应的片，它已被证明是一个重要的因素为自发的和帕的rmacologically引起的网络活动，9,10。另外，循环的ACSF的减少量的方便的药理试验。 在切片过程是但一个创伤的组织12。两个神经元体系结构和氯化物稳态出现在组织表面（50微米）被扰动。记录MEA芯片，这样的组织大多是表面上无熔深大，可能引起创伤领域的活动的起源。但是，我们的数据表明，细胞外场电位检测到的局部被记录在最MEA的电极和以前的工作表明，它们被集成在从记录站点13中的 100至200微米的距离，表明记录在我们的制剂发作不太可能是受创伤的地区生产。此外，与钨电极使深层渗透进行的研究中，记录在人体组织中的癫痫样活动是相似对那些癫痫患者1,7,8观察。 离体组织的记录的另一个限制是不同脑区之间的连接的干扰，从而限制了动态neuromodulations。这也许可以解释为什么在这些组织中不发作状事件自发记录，但是需要通过离子操纵或药理学刺激增强的兴奋性来触发。因此，在该协议中，癫痫样事件是通过结合于细胞外K + 3至6毫米的变化，减少了外部的Mg 2+为1.3毫米的Mg 2+ -free ACSF中，为了提高组织的兴奋性诱导并去除镁离子依赖的 NMDA受体块。事实上，先前已经表明，癫痫样活动诱导使用的Mg 2+学联类似于记录在体内 14的脑电图癫痫人类新皮层和海马。此外，它已被证明在颞叶切片获得的癫痫样放电产生耐药性临床使用的抗惊厥药物长时间暴露于镁2+的ACSF 15,16之后，从而提供了一种模型， 在体外研究pharmacoresistant癫痫样事件。 多边环境协定允许两个记录，场电位和多单元的活动，包括在神经元动作电位体外的。因此，多边环境协定的较脑电图，探讨其在体内的神经元合奏的同步活动产生的场电位，但不给接入单个神经元的行为17一个强大的电工具。虽然最近的微电极能够记录在体内多单位的活动，包括在神经元动作电位，它们是侵入性的，所以它们的使用大多限于研究目的在颅内录音。特别是，记录的MEA代表所选择的技术研究癫痫的事件的时空格局，控制癫痫发作和传播的经典著作和新的抗癫痫药物的作用机制。值得注意的是，解开的细胞类型和癫痫放电的信令基础，穗排序技术和药理学试验应与MEA的技术进行组合。虽然多边环境协定可以给访问各个尖峰，他们不提供有关突触和生物物理特性的信息。在将来，其它技术应该被耦合到MEA的记录，以便更好样品细胞行为，网络活动和突触信号。例如，荧光成像解开神经元或神经胶质细胞的行为，以及离子动力学特性，可以用的MEA的录音进行组合。进一步事后组织学分析还可以揭示类型的细胞，蛋白质或受体的特异性改变，使得癫痫活动的位置可以用的具体的重排相关联神经结构。所述的MEA系统还可以嵌入在一个膜片钳设置以与人口活动相关联的单个细胞或电导。在未来，光遗传学工具也可以使用，条件是人类的切片可以培养在长期，作为执行用于器官切片，所以特定细胞类型的转染或感染可以被执行。

癫痫是一种慢性疾病，其中癫痫发作，这是间歇性的图案放电持续几秒钟到几十秒钟的临床表现相关的脑电图（EEG）记录，中断的发作状态，特点是神经元同步放电持续数十毫秒的存在并呼吁发作事件1。它会影响约1％的世界人口，虽然癫痫发作被控制在广大的患者，癫痫患者大约有三分之一不显示抗癫痫药2充分反应。在这种情况下，所谓的pharmacoresistant癫痫，其机制仍有待清楚地识别时，手术切除确定为癫痫发作区大脑的特定部位的仍然是唯一的替代治疗的患者给予积极的成果。因此，从手术切除的标本得到opportunity研究发作间放电和癫痫发作的产生和传播的上可行的人类中枢神经组织离体的机制，以及pharmacoresistance。 多电极阵列（MEAs）中，包括在空间上分布的微电极的布置，允许电生理学活性的同时刺激和记录来自多个站点的组织的，从而提供了一个极好的方法来研究自发和诱发活性的时空图案。该技术中，首先施加到监测神经元细胞培养物的活性3的发育变化，然后适于急性和器官型脑和脊髓切片4 - 6，目前被认为有价值的电生理工具。 目前的协议描述了如何从手术切除的组织准备的人脑片和多边环境协定可靠的前六记录VO发作，并从这些片癫痫样活动。这种技术因此提供了一种方法，以解决潜在的癫痫活动引发，繁殖和抗癫痫药物同时在蜂窝网络和水平的影响的基本机制。所有用于获取，编制，维护和记录人类切片的过程进行了详细的描述。

<table border="0" cellpadding="0" cellspacing="0" width="826">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Name of Material/ Equipment</td>
			<td style="width:201px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:279px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Brain Slice Chamber-2: Interface</td>
			<td style="width:201px;">AutoMate Scientific, Inc.</td>
			<td style="width:119px;">S-BSC2</td>
			<td style="width:279px;">It requires separate temperature controller</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">2-channel Temperature Controller</td>
			<td style="width:201px;">Multichannel Systems, Germany</td>
			<td style="width:119px;">TC02</td>
			<td style="width:279px;">It allows to plug in and use 2 interface chambers.&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:227px;">Special cable for connection of TC01 to S-BSC2 with external reference PT100</td>
			<td style="width:201px;">Multichannel Systems, Germany</td>
			<td style="width:119px;">CA3</td>
			<td style="width:279px;">The reference PT100 is placed near the slices&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">6pin plug-connector with PT100</td>
			<td style="width:201px;">Multichannel Systems, Germany</td>
			<td style="width:119px;">TS-PT100</td>
			<td style="width:279px;">External reference for CA3 cable</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:227px;">MEA workstation for recording data from 120-electrode MEAs</td>
			<td style="width:201px;">Multichannel Systems, Germany</td>
			<td style="width:119px;">MEA2100-120</td>
			<td style="width:279px;">It includes MEA2100-120 headstage and MEA2100 interface board. www.multichannelsystems.com</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:227px;">Microelectrode array for MEA2100-120</td>
			<td style="width:201px;">Multichannel Systems, Germany</td>
			<td style="width:119px;">120MEA200/30</td>
			<td style="width:279px;">Electrode spacing: 200 &#181;m; electrode diameter: 30 &#181;m; glass ring: 6 mm high. Different configurations (spacing, diameter, ring) possible.&#160;</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:227px;">Video Microscope Table</td>
			<td style="width:201px;">Multichannel Systems, Germany</td>
			<td style="width:119px;">MEA-VMT-1</td>
			<td style="width:279px;">Table with a video microscope underneath to image the electrode field of the MEAs in an amplifier placed on top of the table and transfer the image to a computer.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:227px;">Perfusion cannula</td>
			<td style="width:201px;">Multichannel Systems, Germany</td>
			<td style="width:119px;">PH01</td>
			<td style="width:279px;">Heatable perfusion cannula with temperature sensor; temperature can be programmed with TC02 controller</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">MC_Rack</td>
			<td style="width:201px;">Multichannel Systems, Germany</td>
			<td></td>
			<td style="width:279px;">Software for data acquisition and recordings</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:227px;">Magnetic Perfusion Holder</td>
			<td style="width:201px;">Multichannel Systems, Germany</td>
			<td style="width:119px;">MPH</td>
			<td style="width:279px;">Magnetic perfusion holder for PH01 element to fix the perfusion cannula and connect the perfusion system to the amplifier&#39;s ground</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Neuroexplorer</td>
			<td style="width:201px;">Nex Technologies</td>
			<td></td>
			<td style="width:279px;">Software for data analysis; info@neuroexplorer.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Peristaltic pump (drive unit)</td>
			<td style="width:201px;">Gilson</td>
			<td style="width:119px;">F155001</td>
			<td style="width:279px;">0,01 to 48 rp</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Peristaltic pump (pump head)</td>
			<td style="width:201px;">Gilson</td>
			<td style="width:119px;">F117800</td>
			<td style="width:279px;">R2 two channel</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Ultrasonic aspirator</td>
			<td style="width:201px;">Integra Life sciences, USA</td>
			<td style="width:119px;">Cusa Excel +</td>
			<td style="width:279px;">It allows blunt subpial dissection of the cortex</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Neuronavigation</td>
			<td style="width:201px;">Isis Solutions, France</td>
			<td style="width:119px;">Surgiscope</td>
			<td style="width:279px;">It allows real time identification of the brain structure on the preoperative MRI</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Vibratome HM 650 V</td>
			<td style="width:201px;">Microm</td>
			<td></td>
			<td style="width:279px;">Block slicing into 400 &#956;m thick slices</td>
		</tr>
	</tbody>
</table>

multi-electrode array recordings of human epileptic postoperative cortical tissue

癫痫，影响约1％的人口，包括一组特征为周期性发生癫痫发作的神经性疾病，这扰乱正常脑功能的影响。尽管与现有的抗癫痫药物靶向神经功能的治疗，对癫痫患者有三分之一是pharmacoresistant。在这种情况下，手术切除的脑区产生惊厥仍然是唯一的替代治疗。研究人类癫痫组织中作出了贡献，在过去10年时间理解新的致痫机制。的确，这些组织产生自发性发作癫痫放电以及其中可以记录与经典电生理技术，药理学诱导的发作的事件。值得注意的是，多电极阵列（多边环境协定），这是微加工设备中嵌入的空间排列的微电极阵列，提供了独特的机会，同时刺激并记录字段POTEntials，以及从组织的不同区域的多个神经元的动作电位。因此，多边环境协定的录音提供了一个很好的方法来研究自发性发作的时空格局和诱发癫痫样的事件和潜在的癫痫发作和传播的机制。在这里，我们描述了如何从手术切除的组织准备的人脑片和多边环境协定发作和发作般的事件在体外进行录制。

在灌注与正常记录的ACSF的组织（如前面所述）-7,8-人皮质切片的活动的记录开始。在这种状态下，当组织在手术过程中被完好地保存并稍后从医院和切片制备组织转移过程中，人们可以观察到自发活动，在小发作样的活动，由小簇的MEA的电极检测出的形式。发作样放电包括在一个场电位，通常是双相的，包括一个初始急剧负偏转，达到几十微伏，随后较长相对波持续几十毫秒。快的多单元活动通常嵌入在字段电位主要是在初始部分。发作间样活性的代表性实例示于图3Aii，其中记录了一个MEA的电极迹线被示出。 记录从癫痫发作体外人体皮质切片，必须灌注它们与一个“癫痫”的ACSF，其中的Mg 2+不存在和K +升高到6毫米，以提高组织的兴奋性1（K + 3至6毫米的增加独自不足以诱发癫痫发作;数据未示出）。一旦有0 镁离子6毫米的K +学联灌注开始，脑片的活性逐渐增加，首检出现在15〜20分钟（15.4±1.7分钟，N = 10，5例; 图3艾 ）。我们诱发发作样活性，如图3中所示，在患者进行测试的75％，并在所有的记录片的66.7％。 癫痫活动从MEA芯片的相邻电极被记录，并且场的潜在事件的发生动力学的比较研究允许发生和传播他们的网站。一位代表扣押的嗡嗡声一片记录一个大脑皮层与MEA系统示于图3B（记录由单个的MEA电极发作的代表性跟踪被显示在更高的时间分辨率， 如图3 AIII）。注意，癫痫发作是从MEA芯片的几乎所有的电极记录的，从而表明它能够传播到一个大的皮层区域。此外，在寻找从每个电极记录的单一的痕迹，所以能够观察到的发作横跨组织的逐渐传播。事实上，它开始先在皮质区覆盖电极阵列的右半部分，并且它逐渐扩散到覆盖左半侧区域（。即 ，比较从电极L6和B6的迹线; 图3B）。 <img alt="图1" fo:content-width="3in" src="/files/ftp_upload/51870/51870fig1highres.jpg" width="300" /> 图1：皮质发育不良的。ð其手术去除泰勒焦型皮质发育不良（白色箭头），嵌在左额叶脑沟可视化的术前MRI（A），后来被组织学检查（B）MAP激酶的标签确认;比例尺：200微米）示出与下白质的不完全迁移皮质dyslamination，异常的神经元和在轨道上的神经元。在手术中，在发育不良是根据与一个神经导航系统（C）的 MRI数据本地化。可视化手术（D）中含不典型增生的脑回的。 <img alt="图2" fo:content-width="4in" src="/files/ftp_upload/51870/51870fig2highres.jpg" width="400" /> 图2：设备和过程对人类大脑皮质切片制备的界面室用来维持人体皮质切片的离体 （A）的;切片休息晶状体组织中的平坦区域的片材在所述腔室（B）中 ，其中的温度传感器（C，左），通过一个双连接到温度控制器（C，向右，向上）的上半部分端线（C，右，下），置于保持37℃，整个切片的培养时间。一旦得到，将组织的切除块的培养皿中充满冰冷蔗糖基ACSF（D）和去除血凝块，血管和脑膜，以方便切割。如果组织块大于MEA室，隔离片的适当的尺寸，用刀片（E）和把它粘在所述的vibratome样品板（F）中 。切400微米厚的薄片，用抹刀（G）的帮助下转移他们的镜头纸（H）的作品，并培养他们在接口室（I）。之前的记录，圣雷莫已经透镜组织浸没在培养皿中充满人工脑脊液的切片，并用刮铲（J）传送到ACSF中填充的MEA芯片。除去溶液，让切片附着在MEA（K），并保持在适当的位置使用铂锚。放置在放大器（L）的 MEA中，开始灌注和记录。 <img alt="图3" fo:content-width="3in" src="/files/ftp_upload/51870/51870fig3highres.jpg" width="300" /> 图3：在人脑片痫性发作的MEA记录（A）记录由MEA的电极人类大脑皮层切片活动的代表痕迹显示在面板上我。在正常的ACSF的存在下​​，能够观察到间期状自发活性（小灰色矩形，放大面板ⅱ）;然后，当与0的Mg 2+ 6毫米的K +的ACSF灌流开始后，第一seizu重新出现了〜15分钟的延迟之后，并随后在2-3分钟间隔的其他事件。第三小组展示检获的大灰色矩形面板中的我和缩放比例。蓝色迹线示出了活动的变焦，用蓝线和箭头所示。面板IV）显示过滤，在250赫兹面板三所示的数据）高通，揭示多单位活动时放大（蓝色线）。（二）扣押在0存在的Mg 2+ 6毫米ķ代表录音+学联所有MEA的电极。每平方表示12×12的MEA阵列的一个电极和显示了一个80秒的时间窗口;虚线表示皮质表面的相对向的MEA阵列的位置。

Watch this Scientific Journal Video about Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue at JoVE.com

Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue

在这里，我们将介绍如何执行人类癫痫的皮层组织的多电极阵列记录。发作间期和发作性事件的癫痫组织切除，切片准备，多电极阵列记录都证明了细节。

人类癫痫术后皮层组织的多电极阵列录音

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which ...

multi-electrode-array-recordings-human-epileptic-postoperative

Research

JoVE Journal

Medicine

20.4K 次观看.  CNRS UMR 7241, INSERM U1050, Collège de France. 在这里，我们将介绍如何执行人类癫痫的皮层组织的多电极阵列记录。发作间期和发作性事件的癫痫组织切除，切片准备，多电极阵列记录都证明了细节。 

视频: Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue

Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

Progress in prostate cancer research is severely limited by the availability of human-derived and hormone-na&iuml;ve model systems, which limit our ability to understand genetic and molecular events underlying prostate disease initiation. Toward developing better model systems for studying human prostate carcinogenesis, we and others have taken advantage of the unique pro-prostatic inductive potential of embryonic rodent prostate stroma, termed urogenital sinus mesenchyme (UGSM). When recombined with certain pluripotent cell populations such as embryonic stem cells, UGSM induces the formation of normal human prostate epithelia in a testosterone-dependent manner. Such a human model system can be used to investigate and experimentally test the ability of candidate prostate cancer susceptibility genes at an accelerated pace compared to typical rodent transgenic studies. Since Human embryonic stem cells (hESCs) can be genetically modified in culture using inducible gene expression or siRNA knock-down vectors prior to tissue recombination, such a model facilitates testing the functional consequences of genes, or combinations of genes, which are thought to promote or prevent carcinogenesis.
The technique of isolating pure populations of UGSM cells, however, is challenging and learning often requires someone with previous expertise to personally teach. Moreover, inoculation of cell mixtures under the renal capsule of an immunocompromised host can be technically challenging. Here we outline and illustrate proper isolation of UGSM from rodent embryos and renal capsule implantation of tissue mixtures to form human prostate epithelium. Such an approach, at its current stage, requires in vivo xenografting of embryonic stem cells; future applications could potentially include in vitro gland formation or the use of induced pluripotent stem cell populations (iPSCs).

To unravel the earliest molecular mechanisms underlying prostate cancer initiation, novel and innovative human model systems and approaches are desperately needed. The potential of pre-prostatic urogenital sinus mesenchyme (UGSM) to induce pluripotent stem cell populations to form human prostate epithelium is a powerful experimental tool in prostate research.

人类前列腺上皮细胞形成的使用鼠类泌尿生殖窦间质细胞和人类干细胞的组织重组

Progress in prostate cancer research is severely  limited by the availability of human-derived and hormone-na&iuml;ve model systems,  which limit our ...

科研

医学

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular weight metabolites in tissues of the treated host to achieve long-term survival. The semipermeable membrane allows engrafted encapsulated cells to avoid rejection by the immune system. The encapsulation procedure was designed to enable a controlled release of bioactive compounds, such as insulin, other hormones, and cytokines. Here we describe a method for encapsulation of catabolic cells, which consume lipids for heat production and energy dissipation (thermogenesis) in the intra-abdominal adipose tissue of obese mice. Encapsulation of thermogenic catabolic cells may be potentially applicable to the prevention and treatment of obesity and type 2 diabetes. Another potential application of catabolic cells may include detoxification from alcohols or other toxic metabolites and environmental pollutants.

Here, we present a protocol for encapsulation of catabolic cells, which consume lipids for heat production in intra-abdominal adipose tissue and increase energy dissipation in obese mice.

封装产热前脂肪细胞移植到脂肪组织仓库

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular ...

Working with Human Tissues for Translational Cancer Research

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human specimens, precious samples must be handled with the greatest care in order to ensure integrity of organs, tissues, and cells to the highest degree. Unfortunately, tissue processing by definition requires extraction of tissues from the host, a change which can cause great cellular stress and have major repercussions on subsequent analyses. These stresses could result in the specimen being no longer representative of the site from which it was retrieved. Therefore, a strict protocol must be adhered to while processing these specimens to ensure representativeness. The desired assay(s) must also be taken into consideration in order to ensure that an optimal technique is used for sample processing. Outlined here is a protocol for tissue retrieval, processing and various analyses which may be performed on processed tissue in order to maximize downstream production from limited tissue samples.

Translational cancer research is dependent on extraction of human tissues. Much work has gone into optimizing extraction methods for ex vivo analysis. Here, we describe tissue processing methods allowing for maximal data output from limited samples.

与人体组织转化为癌症研究工作

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human ...

3D Ultrasound Imaging: Fast and Cost-effective Morphometry of Musculoskeletal Tissue

The developmental goal of 3D ultrasound imaging (3DUS) is to engineer a modality to perform 3D morphological ultrasound analysis of human muscles. 3DUS images are constructed from calibrated freehand 2D B-mode ultrasound images, which are positioned into a voxel array. Ultrasound (US) imaging allows quantification of muscle size, fascicle length, and angle of pennation. These morphological variables are important determinants of muscle force and length range of force exertion. The presented protocol describes an approach to determine volume and fascicle length of m.&#160;vastus lateralis and m.&#160;gastrocnemius medialis. 3DUS facilitates standardization using 3D anatomical references. This approach provides a fast and cost-effective approach for quantifying 3D morphology in skeletal muscles. In healthcare and sports, information on the morphometry of muscles is very valuable in diagnostics and/or follow-up evaluations after treatment or training.

3D ultrasound imaging (3DUS) allows fast and cost-effective morphometry of musculoskeletal tissues. We present a protocol to measure muscle volume and fascicle length using 3DUS.

3D 超声成像: 快速、经济高效的肌骨骼组织形态分析

The developmental goal of 3D ultrasound imaging (3DUS) is to engineer a modality to perform 3D morphological ultrasound analysis of human muscles. 3DUS ...

Assessment of Human Adipose Tissue Microvascular Function Using Videomicroscopy

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. It is hypothesized that pro-atherogenic mediators released from fat tissues particularly in association with central/visceral adiposity may promote pathogenic vascular changes locally and systemically, and the notion that cardiovascular disease may be the consequence of adipose tissue dysfunction continues to evolve. Here, we describe a unique method of videomicroscopy that involves analysis of vasodilator and vasoconstrictor responses of intact small human arterioles removed from the adipose depot of living human subjects. Videomicroscopy is used to examine functional properties of isolated microvessels in response to pharmacological or physiological stimuli using a pressured system that mimics in vivo conditions. The technique is a useful approach to gain understanding of the pathophysiology and molecular mechanisms that contribute to vascular dysfunction locally within the adipose tissue milieu. Moreover, abnormalities in the adipose tissue microvasculature have also been linked with systemic diseases. We applied this technique to examine depot-specific vascular responses in obese subjects. We assessed endothelium-dependent vasodilation to both increased flow and acetylcholine in adipose arterioles (50 - 350 &#181;m internal diameter, 2 - 3 mm in length) isolated from two different adipose depots during bariatric surgery from the same individual. We demonstrated that arterioles from visceral fat exhibit impaired endothelium-dependent vasodilation compared to vessels isolated from the subcutaneous depot. The findings suggest that the visceral microenvironment is associated with vascular endothelial dysfunction which may be relevant to clinical observation linking increased visceral adiposity to systemic disease mechanisms. The videomicroscopy technique can be used to examine vascular phenotypes from different fat depots as well as compare findings across individuals with different degrees of obesity and metabolic dysfunction. The method can also be used to examine vascular responses longitudinally in response to clinical interventions.

Videomicroscopy systems are used to examine functional properties of isolated adipose tissue arterioles in response to physiological and pharmacological stimuli. This technique can be used to examine microvascular phenotypes in different adipose tissue domains in obese humans.

应用 Videomicroscopy 评价人脂肪组织微血管功能

While obesity is closely linked to the development of metabolic and cardiovascular disease, little is known about mechanisms that govern these processes. ...

A Mouse Model of Orthopedic Surgery to Study Postoperative Cognitive Dysfunction and Tissue Regeneration

Surgery is commonly used to improve and maintain quality of life. Unfortunately, in vulnerable patients such as the elderly, complications may occur and significantly diminish the outcome. Indeed, after routine orthopedic surgery to repair a fracture, as many as 50% of elderly patients suffer from neurologic complications like delirium. Also, the capacity to heal and regenerate tissue after surgery decreases with age, and can impact the quality of fracture repair and even osseous integration of implants. Thus, a better understanding of mechanisms that drive these age-dependent changes could provide strategic targets to minimize risk for such complications and optimize outcomes. Here, we introduce a clinically relevant mouse model of tibial fracture. The postoperative changes in these mice mimic some of the cognitive impairments commonly observed after routine orthopedic surgery in humans. Briefly, an incision is performed in the right hind limb under strictly aseptic conditions. Muscles are disassociated, and a 0.38-mm stainless steel pin is inserted into the upper crest of the tibia, inside the intramedullary canal. Osteotomy is then performed, and the wound is stapled. We have used this model to investigate the effects of surgical trauma on postoperative neuroinflammation and behavioral changes. By applying this fracture model in combination with parabiosis, a surgical model in which 2 mice are anastomosed, we have studied cells and secreted factors that systemically rejuvenate organ function and tissue regeneration after injury. By following our step-by-step protocol, these models can be reproduced with high fidelity, and can be adapted to interrogate many biologic pathways that are altered by surgical trauma.

This protocol describes a mouse model of orthopedic surgery that has been used to study mechanisms of postoperative neuroinflammation and behavioral changes, and when combined with parabiosis, to study tissue regeneration during aging.

骨科手术的小鼠模型研究术后认知功能障碍与组织再生

Surgery is commonly used to improve and maintain quality of life. Unfortunately, in vulnerable patients such as the elderly, complications may occur and ...

Generation of Human 3D Lung Tissue Cultures (3D-LTCs) for Disease Modeling

Translation of novel discoveries to human disease is limited by the availability of human tissue-based models of disease. Precision-cut lung slices (PCLS) used as 3D lung tissue cultures (3D-LTCs) represent an elegant and biologically highly relevant 3D cell culture model, which highly resemble in situ tissue due to their complexity, biomechanics and molecular composition. Tissue slicing is widely applied in various animal models. 3D-LTCs derived from human PCLS can be used to analyze responses to novel drugs, which might further help to better understand the mechanisms and functional effects of drugs in human tissue. The preparation of PCLS from surgically resected lung tissue samples of patients, who experienced lung lobectomy, increases the accessibility of diseased and peritumoral tissue. Here, we describe a detailed protocol for the generation of human PCLS from surgically resected soft-elastic patient lung tissue. Agarose was introduced into the bronchoalveolar space of the resectates, thus preserving lung structure and increasing the tissue&#39;s stiffness, which is crucial for subsequent slicing. 500 &#181;m thick slices were prepared from the tissue block with a vibratome. Biopsy punches taken from PCLS ensure comparable tissue sample sizes and further increase the amount of tissue samples. The generated lung tissue cultures can be applied in a variety of studies in human lung biology, including the pathophysiology and mechanisms of different diseases, such as fibrotic processes at its best at (sub-)cellular levels. The highest benefit of the 3D-LTC ex vivo model is its close representation of the in situ human lung in respect of 3D tissue architecture, cell type diversity and lung anatomy as well as the potential for assessment of tissue from individual patients, which is relevant to further develop novel strategies for precision medicine.

Generation of Human 3D Lung Tissue Cultures 3D-LTCs for Disease Modeling

Here, we present a protocol for the preparation of agarose-filled human precision-cut lung slices from resected patient tissue that are suitable for generating 3D lung tissue cultures to model human lung diseases in biological and biomedical studies.

用于疾病建模的人三维肺组织培养 (3D-LTCs) 的生成

Translation of novel discoveries to human disease is limited by the availability of human tissue-based models of disease. Precision-cut lung slices (PCLS) ...

Nanoscopic Imaging of Human Tissue Sections via Physical and Isotropic Expansion

In modern pathology, optical microscopy plays an important role in disease diagnosis by revealing microscopic structures of clinical specimens. However, the fundamental physical diffraction limit prevents interrogation of nanoscale anatomy and subtle pathological changes when using conventional optical imaging approaches. Here, we describe a simple and inexpensive protocol, called expansion pathology (ExPath), for nanoscale optical imaging of common types of clinical primary tissue specimens, including both fixed-frozen or formalin-fixed paraffin embedded (FFPE) tissue sections. This method circumvents the optical diffraction limit by chemically transforming the tissue samples into tissue-hydrogel hybrid and physically expanding them isotropically across multiple scales in pure water. Due to expansion, previously unresolvable molecules are separated and thus can be observed using a conventional optical microscope.

Nanoscale imaging of clinical tissue samples can improve understanding of disease pathogenesis. Expansion pathology (ExPath) is a version of expansion microscopy (ExM), modified for compatibility with standard clinical tissue samples, to explore the nanoscale configuration of biomolecules using conventional diffraction limited microscopes.

通过物理和各向异性扩张对人体组织部分的纳米成像

In modern pathology, optical microscopy plays an important role in disease diagnosis by revealing microscopic structures of clinical specimens. However, ...

A Postoperative Evaluation Guideline for Computer-Assisted Reconstruction of the Mandible

Valid comparisons of postoperative accuracy results in computer-assisted reconstruction of the mandible are difficult due to heterogeneity in imaging modalities, mandibular defect classification, and evaluation methodologies between studies. This guideline uses a step-by-step approach guiding the process of imaging, classification of mandibular defects and volume assessment of three-dimensional (3D) models, after which a legitimized quantitative accuracy evaluation method can be performed between the postoperative clinical situation and the preoperative virtual plan. The condyles and the vertical and horizontal corners of the mandible are used as bony landmarks to define virtual lines in the computer-assisted surgery (CAS) software. Between these lines the axial, coronal, and both sagittal mandibular angles are calculated on both pre- and postoperative 3D models of the (neo)mandible and subsequently the deviations are calculated. By superimposing the postoperative 3D model to the preoperative virtually planned 3D model, which is fixed to the XYZ axis, the deviation between pre- and postoperative virtually planned dental implant positions can be calculated. This protocol continues and specifies an earlier publication of this evaluation guideline.

Here, we propose a practical, feasible and reproducible evaluation guideline for computer-assisted reconstruction of the mandible in order to create uniformity between studies regarding postoperative accuracy evaluation. This protocol continues and specifies an earlier publication of this evaluation guideline.

可读机辅助重建术后评估指南

Valid comparisons of postoperative accuracy results in computer-assisted reconstruction of the mandible are difficult due to heterogeneity in imaging ...

Preparation of Human Myocardial Tissue for Long-Term Cultivation

Cardiomyocyte cultivation has seen a vast number of developments, ranging from two-dimensional (2D) cell cultivation to iPSC derived organoids. In 2019, an ex vivo way to cultivate myocardial slices obtained from human heart samples was demonstrated, while approaching in vivo condition of myocardial contraction. These samples originate mostly from heart transplantations or left-ventricular assist device placements. Using a vibratome and a specially developed cultivation system, 300 &#181;m thick slices are placed between a fixed and a spring wire, allowing for stable and reproducible cultivation for several weeks. During cultivation, the slices are continuously stimulated according to individual settings. Contractions can be displayed and recorded in real-time, and pharmacological agents can be readily applied. User-defined stimulation protocols can be scheduled and performed to assess vital contraction parameters like post-pause-potentiation, stimulation threshold, force-frequency relation, and refractory period. Furthermore, the system enables a variable pre- and afterload setting for a more physiological cultivation.
Here, we present a step-by-step guide on how to generate a successful long-term cultivation of human left ventricular myocardial slices, using a commercial biomimetic cultivation solution.

We present a protocol for ex vivo cultivation of human ventricular myocardial tissue. It allows for detailed analysis of contraction force and kinetics, as well as the application of pre- and afterload to mimic the in vivo physiological environment more closely.

长期培养用人心肌组织的制备

Cardiomyocyte cultivation has seen a vast number of developments, ranging from two-dimensional (2D) cell cultivation to iPSC derived organoids. In 2019, ...