Name: organotypic cultures of adult human cortex as an ex vivo model for human stem cell transplantation and validation
Uploaded: 2022-12-09T13:20:00
Description: Watch this Scientific Journal Video about Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation at JoVE.com

这项工作得到了瑞典研究委员会，瑞典脑基金会，瑞典中风基金会，斯科讷地区，Thorsten and Elsa Segerfalk基金会以及瑞典政府战略研究领域倡议（StemTherapy）的资助。

该协议遵循瑞典隆德区域伦理委员会批准的指南（道德许可证号 2021-07006-01）。从接受颞叶癫痫择期手术的患者获得健康的新皮质组织。获得所有患者的知情同意。
注意：无论其大小如何，所有获得的组织都经过处理。然而，小于 1-1.5 mm3 的组织在技术上具有挑战性，难以处理和用振动切片机切片。
1. 组织收集、维护、切割和电镀
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<ol>
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获得足够高质量的hACtx切片是该协议中最关键的一步。 皮质组织是从接受切除手术的癫痫患者获得的24。切除组织的质量以及切除和培养之间的组织暴露时间至关重要;组织从手术室转移到实验室并切割的速度越快，器官型培养就越理想。理想情况下，应在收集后的最初几个小时内切割组织并将其转移到细胞培养实验室。在此过程中组织的氧合也提高了切片的质量。?...

神经退行性疾病，如帕金森病、阿尔茨海默病或缺血性中风，是一组具有神经元功能障碍或死亡共同特征的疾病。它们在受影响的大脑区域和神经元群体方面是异质的。不幸的是，由于缺乏模仿人脑中发生的事情的动物模型，这些疾病的治疗方法很少或疗效有限1，2。干细胞疗法是大脑再生最有前途的策略之一3。近年来，从不同来源的干细胞中产生神经元祖细胞已经有了很大的发展4，5。最近的出版物表明，人类诱导多能干（iPS）细胞衍生的长期自我更新神经上皮样干（lt-NES）细胞，遵循皮质分化方案，并在缺血性中风影响躯体感觉皮层的大鼠模型中进行皮质内移植后，产生成熟的皮质神经元。此外，移植物衍生的神经元接收来自宿主神经元的传入和传出突触连接，显示它们整合到大鼠神经元网络中6，7。移植物来源的轴突被髓鞘化，发现在大鼠大脑的不同区域，包括梗死周围区域、胼胝体和对侧躯体感觉皮层。最重要的是，iPS细胞衍生的移植逆转了中风动物的运动缺陷7。
即使动物模型有助于研究移植存活，神经元整合以及移植细胞对运动和认知功能的影响，该系统中也缺少有关人类细胞（移植物 - 宿主）之间相互作用的信息8，9。出于这个原因，这里描述了一种长期人脑器官型培养与人iPS细胞来源神经元祖细胞离体移植的组合方法。从神经外科切除中获得的人脑器官型培养物是与大脑的生理相关的3D模型，使研究人员能够增加对人类中枢神经系统回路的理解以及测试人类脑部疾病治疗的最准确方法。然而，在这种情况下还没有进行足够的研究，在大多数情况下，已经使用了人类海马脑器官型培养物10，11。大脑皮层受到几种神经退行性疾病的影响，例如缺血性中风12 或阿尔茨海默病13，因此拥有一个人类皮质 3D 系统非常重要，该系统使我们能够扩展我们的知识并测试和验证不同的治疗策略。过去几年的几项研究使用成人皮质（hACtx）组织的培养物来模拟人类脑部疾病14，15，16，17，18，19;然而，在干细胞治疗方面可获得的信息有限。两项研究已经证明了此处描述的系统的可行性。2018年，用不同转录因子编程并移植到hACtx组织中的人类胚胎干细胞被证明会产生成熟的皮质神经元，这些神经元可以整合到成人皮质网络中20。2020 年，将 lt-NES 细胞移植到人类器官型系统中揭示了它们分化成具有功能神经元电生理特性的成熟、层特异性皮质神经元的能力。移植神经元与成人脑切片中的人类皮质神经元建立了传入和传出突触接触，狂犬病病毒逆行单突触追踪、全细胞膜片钳记录和免疫电子显微镜证实了这一点21。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Tissue Cutting and electrophysiology</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine 5&#39;-triphosphate magnesium salt</td>
			<td>Sigma</td>
			<td>A9187</td>
			<td></td>
		</tr>
		<tr>
			<td>Bath temperature controller&#160;</td>
			<td>Luigs &#38; Neumann</td>
			<td>TC0511354</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride dihydrate</td>
			<td>Merck</td>
			<td>102382</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbogen gas</td>
			<td>Air Liquide</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Cooler</td>
			<td>Julaba FL 300</td>
			<td>9661012.03</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>Double Patch-Clamp amplifier</td>
			<td>HEKA electronic</td>
			<td>EPC10</td>
			<td></td>
		</tr>
		<tr>
			<td>Guanosine 5&#39;-Triphosphate disodium salt</td>
			<td>Millipore</td>
			<td>371701</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>AppliChem</td>
			<td>A1069</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Chloride hexahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>M2670</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate heptahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>230391</td>
			<td></td>
		</tr>
		<tr>
			<td>Patchmaster</td>
			<td>HEKA electronic</td>
			<td>Patchmaster 2x91</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Puller</td>
			<td>Sutter</td>
			<td>P-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic Petri dish</td>
			<td>Any suitable</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Merck</td>
			<td>104936</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium D-gluconate</td>
			<td>ThermoFisher</td>
			<td>B25135</td>
			<td></td>
		</tr>
		<tr>
			<td>Rubber teat + glass pipette</td>
			<td>Any suitable</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dihydrogen phosphate monohydrate</td>
			<td>Merck</td>
			<td>106346</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S7903</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue adhesive: Acryl super glue</td>
			<td>Loctite</td>
			<td>2062278</td>
			<td></td>
		</tr>
		<tr>
			<td>Upright microscope</td>
			<td>Olympus</td>
			<td>BX51WI&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome&#160;</td>
			<td>Leica</td>
			<td>VT1200 S</td>
			<td></td>
		</tr>
		<tr>
			<td>RINSING SOLUTION</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7021</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS (without Ca, Mg, or PhenolRed)</td>
			<td>ThermoFisher Scientific</td>
			<td>14175095</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>AppliChem</td>
			<td>A1069</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>ThermoFisher Scientific</td>
			<td>15-140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>MANTAINANCE AND CULTURE OF HUMAN NEOCORTICAL TISSUE</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>ThermoFisher Scientific</td>
			<td>140675</td>
			<td></td>
		</tr>
		<tr>
			<td>Alvetex scaffold 6 well insert</td>
			<td>Reinnervate Ltd</td>
			<td>AVP004-96</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Supplement (50x)</td>
			<td>ThermoFisher Scientific</td>
			<td>17504001</td>
			<td></td>
		</tr>
		<tr>
			<td>BrainPhys without Phenol Red</td>
			<td>StemCell technologies</td>
			<td>#05791</td>
			<td>Referenced as neuronal medium in the text</td>
		</tr>
		<tr>
			<td>Filter units 250 mL or 500 mL</td>
			<td>Corning Sigma</td>
			<td>CLS431096/97</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Any suitable</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin (50 mg/mL)</td>
			<td>ThermoFisher Scientific</td>
			<td>15750037</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax Supplement (100x)</td>
			<td>ThermoFisher Scientific</td>
			<td>35050061</td>
			<td>Referenced as L-glutamine in the text</td>
		</tr>
		<tr>
			<td>Rubber teat + Glass pipette</td>
			<td>Any suitable</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GENERATION OF lt-NES cells</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol 50 mM</td>
			<td>ThermoFisher Scientific</td>
			<td>31350010</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal Free Recombinant EGF</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Suplemment (50x)</td>
			<td>Thermo Fisher Scientific</td>
			<td>17504001</td>
			<td></td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>Peprotech</td>
			<td>AF-100-18B</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Albumin Fraction V (7.5% solution)</td>
			<td>ThermoFisher Scientific</td>
			<td>15260037</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyclopamine, V. calcifornicum</td>
			<td>Calbiochem</td>
			<td># 239803</td>
			<td></td>
		</tr>
		<tr>
			<td>D (+) Glucose solution (45%)</td>
			<td>Sigma</td>
			<td>G8769</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma Aldrich</td>
			<td>D2438-10mL</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>ThermoFisher Scientific</td>
			<td>11320074</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffer Saline (DPBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>14190-144</td>
			<td>Without calcium and magnesium</td>
		</tr>
		<tr>
			<td>Laminin Mouse Protein, Natural</td>
			<td>Thermo Fisher Scientific</td>
			<td>23017015</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Non-essential aminoacids solutions (100x)</td>
			<td>ThermoFisher Scientific</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>N-2 Supplement (100 x)</td>
			<td>ThermoFisher Scientific</td>
			<td>17502001</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Ornithine</td>
			<td>Merk</td>
			<td>P3655</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human BMP-4 Protein</td>
			<td>R&#38;D Systems</td>
			<td>314-BP-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human Wnt-3a Protein</td>
			<td>R&#38;D Systems</td>
			<td>5036-WN</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate (100 mM)</td>
			<td>ThermoFisher Scientific</td>
			<td>11360070</td>
			<td></td>
		</tr>
		<tr>
			<td>Soybean Trypsin Inhibitor, powder</td>
			<td>Thermo Fisher Scientific</td>
			<td>17075029</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile deionized water</td>
			<td>MilliQ</td>
			<td></td>
			<td>MilliQ filter system</td>
		</tr>
		<tr>
			<td>Trypsin EDTA (0.25%)</td>
			<td>Sigma</td>
			<td>T4049-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>EQUIPMENT FOR CELL CULTURE&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adjustable volume pipettes 10, 100, 200, 1000 &#181;L</td>
			<td>Eppendorf</td>
			<td>Various</td>
			<td></td>
		</tr>
		<tr>
			<td>Basement membrane matrix ESC-qualified (Matrigel)</td>
			<td>Corning</td>
			<td>CLS354277-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Hettich Centrifugen</td>
			<td>Rotina 420R</td>
			<td>5% CO2, 37 &#176;C</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>ThermoForma Steri-Cult CO2</td>
			<td>HEPA Class100</td>
			<td></td>
		</tr>
		<tr>
			<td>Stem cell cutting tool 0.190-0.210 mm</td>
			<td>Vitrolife</td>
			<td>14601</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile tubes</td>
			<td>Sarstedt</td>
			<td>Various</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Disposable Glass Pasteur Pipettes 150 mm</td>
			<td>VWR</td>
			<td>612-1701</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile pipette tips 0.1-1000 &#160;&#181;L</td>
			<td>Biotix VWR</td>
			<td>Various</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Serological Pipettes 5, 10, 25, 50 mL</td>
			<td>Costar</td>
			<td>Various</td>
			<td></td>
		</tr>
		<tr>
			<td>T25 flasks Nunc</td>
			<td>ThermoFisher Scientific</td>
			<td>156367</td>
			<td></td>
		</tr>
		<tr>
			<td>IMMUNOHISTOCHEMISTRY</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>488-conjugated AffinityPure Donkey anti-mouse IgG</td>
			<td>Jackson ImmunoReserach</td>
			<td>715-545-151</td>
			<td></td>
		</tr>
		<tr>
			<td>488-conjugated AffinityPure Donkey anti-rabbit IgG</td>
			<td>Jackson ImmunoReserach</td>
			<td>711-545-152</td>
			<td></td>
		</tr>
		<tr>
			<td>488-conjugated AffinityPure Donkey anti-chicken IgG</td>
			<td>Jackson ImmunoReserach</td>
			<td>703-545-155</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa fluor 647-conjugated Streptavidin</td>
			<td>Jackson ImmunoReserach</td>
			<td>016-600-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Jackson ImmunoReserach</td>
			<td>001-000-162</td>
			<td></td>
		</tr>
		<tr>
			<td>Chicken anti-GFP</td>
			<td>Merk Millipore</td>
			<td>AB16901</td>
			<td></td>
		</tr>
		<tr>
			<td>Chicken anti-MAP2&#160;</td>
			<td>Abcam</td>
			<td>ab5392</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy3-conjugated AffinityPure Donkey anti-chicken IgG</td>
			<td>Jackson ImmunoReserach</td>
			<td>703-165-155</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy3-conjugated AffinityPure Donkey anti-goat IgG</td>
			<td>Jackson ImmunoReserach</td>
			<td>705-165-147</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy3-conjugated AffinityPure Donkey anti-mouse IgG</td>
			<td>Jackson ImmunoReserach</td>
			<td>715-165-151</td>
			<td></td>
		</tr>
		<tr>
			<td>Diazabicyclooctane (DABCO)</td>
			<td>Sigma Aldrich</td>
			<td>D27802</td>
			<td>Mounting media</td>
		</tr>
		<tr>
			<td>Goat anti-AIF1 (C-terminal)&#160;</td>
			<td>Biorad</td>
			<td>AHP2024</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Molecular Probes</td>
			<td></td>
			<td>Nuclear staining</td>
		</tr>
		<tr>
			<td>Mouse anti-MBP&#160;</td>
			<td>BioLegend</td>
			<td>808402</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-SC123&#160;</td>
			<td>Stem Cells Inc</td>
			<td>AB-123-U-050</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Donkey Serum</td>
			<td>Merk Millipore</td>
			<td>S30-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Paint brush</td>
			<td>Any suitable</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Sigma Aldrich</td>
			<td>150127</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phospate Buffer Saline, KPBS (1x)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; Distilled water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; Potassium dihydrogen Phospate (KH2PO4)</td>
			<td>Merk Millipore</td>
			<td>104873</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; Potassium phospate dibasic (K2HPO4)</td>
			<td>Sigma Aldrich</td>
			<td>P3786</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160; Sodium chloride (NaCl)</td>
			<td>Sigma Aldrich</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-NeuN&#160;</td>
			<td>Abcam</td>
			<td>ab104225</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-Olig2&#160;</td>
			<td>Abcam</td>
			<td>ab109186</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-TMEM119&#160;</td>
			<td>Abcam</td>
			<td>ab185333</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Sigma Aldrich</td>
			<td>S2002-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium citrate</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160;&#160; Distilled water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160;&#160; Tri-Sodium Citrate</td>
			<td>Sigma Aldrich</td>
			<td>S1804-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;&#160;&#160;&#160;&#160;&#160; Tween-20</td>
			<td>Sigma Aldrich</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>ThermoFisher Scientific</td>
			<td>327371000&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>EQUIPMENT FOR IMMUNOHISTOCHEMISTRY</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeiss</td>
			<td>LSM 780</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Slides 76 mm x 26 mm</td>
			<td>VWR</td>
			<td>630-1985</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Coverslips 24 mm x 60 mm</td>
			<td>Marienfeld</td>
			<td>107242</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Software</td>
			<td>Zeiss</td>
			<td>ZEN Black edition</td>
			<td></td>
		</tr>
		<tr>
			<td>Rubber teat + Glass pipette</td>
			<td>Any suitable</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

organotypic cultures of adult human cortex as an ex vivo model for human stem cell transplantation and validation

神经退行性疾病在症状和细胞影响方面是常见且异质的，由于缺乏完全模仿人类疾病的适当动物模型以及死后人类脑组织的可用性差，他们的研究变得复杂。成人神经组织培养为研究神经系统疾病的不同方面提供了可能性。分子，细胞和生化机制可以很容易地在这个系统中解决，以及测试和验证药物或不同的治疗方法，如基于细胞的疗法。该方法结合了从接受切除手术的癫痫患者获得的成人皮层的长期器官型培养物，以及诱导多能干细胞来源的皮质祖细胞的 体外 皮质移植。该方法将允许研究细胞存活，神经元分化，突触输入和输出的形成以及移植到完整的成人皮质组织后人类来源细胞的电生理特性。这种方法是开发3D人类疾病建模平台之前的重要一步，该平台将使基础研究更接近基于干细胞的治疗的临床转化，用于患有不同神经系统疾病的患者，并允许开发用于重建受损神经回路的新工具。

按照所描述的方案，如上所述，收集和处理来自颞叶癫痫患者的hACtx组织。在培养24小时后固定几片以研究宿主组织的起点。对不同神经细胞群的分析，如神经元（表达NeuN和Map2，图1A），少突胶质细胞（Olig2和MBP，图1B）和星形胶质细胞（人类特异性GFAP，也称为STEM123，图1C）显示组织的最佳保存。 
下一步是研...

Watch this Scientific Journal Video about Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation at JoVE.com

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

该协议描述了成人皮层的长期器官型培养与诱导多能干细胞衍生皮质祖细胞的 离体 皮质内移植相结合，这提供了一种新方法来进一步测试基于干细胞的治疗人类神经退行性疾病。

成人皮层的器官型培养作为人类干细胞移植和验证的 离体 模型

Neurodegenerative disorders are common and heterogeneous in terms of their symptoms and cellular affectation, making their study complicated due to the ...

迄今为止，人类成人皮层的器官型培养物是测试受损皮层干细胞疗法的唯一系统，可以研究人类移植细胞和人类宿主细胞之间的相互作用。由于啮齿动物和人类细胞之间的差异，在动物模型中测试的疗法在转化为临床环境时通常会失败。在这里，我们可以在人与人的环境中研究移植细胞、存活、分化和功能。这种方法将帮助我们了解神经回路在人脑中的运作方式，并将刺激这些知识转化为临床环境，以改善脑损伤后的功能恢复。演示该程序的将是来自我实验室的博士生Raquel Martinez-Curiel和硕士生Costanza Aretio-Medina。首先，在通风罩下的细胞培养实验室中使用镊子将培养插入物放入六孔板中。在插入物底部加入五毫升人类成人皮质培养基，直到它接触膜。避免形成气泡，并在插入物顶部添加两毫升培养基。在将组织切片转移到插入物之前，在培养箱中以37摄氏度和5%二氧化碳平衡至少两个小时。将手术室中患者的组织收集到装有冷冻、起泡和压碎的切割溶液的容器中。立即将冰上的封闭容器转移到实验室的切割区域。检查组织，并考虑皮质层的方向，找到最佳表面将其粘合到振动切片机的切割阶段。如果需要，用手术刀切割不平坦的表面，将组织放在载物台上，以获得最佳切片方向。然后，用纸巾粘合剂将纸巾粘在载物台上。将其放入切片室中，并立即用冷的气泡切割溶液填充。在整个切割过程中继续冒泡。现在根据手稿中描述的组织到刀片的方向切割冠状切片或矢状切片。将切片放入室温下带有鼓泡切割溶液的收集室中。切割完所有组织后，将切片转移到带有室温冲洗溶液的无菌培养皿中，以从切片中去除多余的蔗糖并将其运送到细胞培养实验室。接下来，将组织切片分别放在湿插入物和浸没的插入物上。24 小时后，更换培养基以去除切割过程中残留的蔗糖或其他残留物质。两周后，使用不含庆大霉素的成人皮质培养基。从细胞培养瓶中取出培养基。在分化的第七天，通过加入 500 微升胰蛋白酶分离皮质引发的 GFP-lt-NES 细胞，并在室温下孵育 5 至 10 分钟。接下来，加入等体积的胰蛋白酶抑制剂，然后加入五毫升DDM培养基。重悬细胞，轻轻地将细胞悬液转移到15毫升管中，并以300G离心5分钟，同时将含有人体组织的板从培养箱转移到罩上，并从插入物顶部取出两毫升培养基。在离心结束时，除去上清液，将细胞重悬于冷，纯的基底膜基质中，并将养老金转移到较小的管中。将细胞悬液收集到连接到橡胶奶嘴的冷玻璃毛细管中进行抽吸。通过在各个部位刺伤半干组织切片，将细胞悬液注射为微小滴剂。让凝胶在 37 摄氏度下凝固 30 分钟。然后，将板从培养箱转移回罩，并小心地将两毫升人成人皮质培养基添加到插入物的顶部以完全浸没组织。急性人类成人皮质组织显示神经元、少突胶质细胞和星形胶质细胞的存在，显示出组织的最佳保存。细胞培养两周后，组织显示神经元标志物NeuN和Map2的表达。全细胞膜片钳记录显示，神经元具有持续的静息膜电位和膜输入阻力，与急性制剂的神经元相当。这些细胞的活性略低于新鲜组织，尽管大多数细胞能够发射至少一个（如果不是多个）动作电位。急性皮质组织显示小胶质细胞标志物Iba1和Tmem119的表达。培养两周后，小胶质细胞的分支减少，并且比急性组织更活化。离体移植4周后，移植的GFP-lt-NES细胞在整个器官型培养过程中表现出延伸的神经突，具有广泛而复杂的树化。几乎没有任何移植细胞在保存不良的组织中存活。在整个人体切片中广泛观察到死细胞上的碎片和抗体的非特异性标记。在成功移植的情况下，细胞变得功能活跃，成熟的神经元显示出重复的，通常是自发的动作电位。快速向内钠、缓慢向外钾流和一定程度的突触活性表明移植物在移植后四周与宿主组织的功能整合。人体组织离开手术室后处理得越快，系统的可行性和实验程序的成功就越高。

Research

JoVE Journal

Neuroscience

Organotypic Cultures of Adult Human Cortex as an Ex vivo Model for Human Stem Cell Transplantation and Validation

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

嗅觉感官神经元的慢病毒介导的遗传操作和可视化在体内</em

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the ...

科研

神经科学

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

神经元形态的研究方法：<em&gt;体外</em&gt; RNAi技术在胚胎小鼠大脑皮质的电穿孔

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

Ex vivo Culturing of Whole, Developing Drosophila Brains

Ex vivo Culturing of Whole, Developing Drosophila Brains

前全体体内培养，发展的果蝇大脑

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for ...

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

体外实时成像在小鼠神经上皮单细胞分裂

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of ...

Mouse Hindbrain Ex Vivo Culture to Study Facial Branchiomotor Neuron Migration

Mouse Hindbrain Ex Vivo Culture to Study Facial Branchiomotor Neuron Migration

鼠标后脑<em&gt;前体内</em&gt;企业文化来研究面部Branchiomotor神经元迁移

Embryonic neurons are born in the ventricular zone of the brain, but subsequently migrate to new destinations to reach appropriate targets. Deciphering ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

从人多能神经嵴前体细胞的无饲养推导干细胞

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

器官切片文化研究少突胶质细胞动力学和髓鞘

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

人视网膜色素上皮细胞向大鼠眼巩膜视网膜移植精制方案的研制

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

器官海马切片培养模型研究肿瘤细胞的神经保护和侵袭性

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

利用干细胞衍生脑 Organoids体外培养人脑组织 Zika 病毒感染的模拟研究

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions ...

成人皮层的器官型培养作为人类干细胞移植和验证的 离体 模型

成人皮层的器官型培养作为人类干细胞移植和验证的离体模型