这项工作得到了国家自然科学基金（82000624）、陕西省自然科学基础研究计划（2022JQ-899和2021JM-268）、陕西省创新能力支持计划（2023KJXX-030）、陕西省重点研发计划大学联合项目-重点项目（2021GXLH-Z-047）、习安交通大学第一附属医院机构基金（2021HL-42和2021HL-21）的支持。

本研究经习交通大学动物实验伦理委员会批准，按照《实验动物护理与使用指南》开展。
1.脾脏采摘
<ol><li>使用体重250-280g的雄性Sprague Dawley大鼠。将大鼠安置在温度和湿度受控的房间中，并 随意为它们提供食物和水，手术前禁食除外。</li>
	<li>术前 1 小时皮下注射丁丙诺啡 （0.05 mL/kg） 作为镇痛剂。通过异氟烷吸入麻醉大鼠。在有机玻璃盒中使用1.5 L/min的流速的5%异氟烷进行诱导麻醉，并通过面罩以0.6-0.8 L/min的流速保持麻醉2%异氟烷。通过捏住脚趾来确认麻醉的深度。</li>
	<li>使用电动剃须刀刮除整个腹部的皮肤。将大鼠仰卧在手术台上。通过背静脉注射 2 mL 肝素化盐水（1,000 U 肝素）以达到全身抗凝。用聚维酮碘溶液对剃光的皮肤进行消毒，并用无菌布覆盖。</li>
	<li>用手术剪刀在腹部做一个十字形切口，用止血钳拉伸暴露腹腔，然后将肝脏朝向横膈膜翻转。将胃肠道外侧置于腹部右侧，并用湿纱布覆盖。</li>
	<li>小心地分离并切断脾胃韧带，露出脾门。 注意:脾脏呈淡红色细长结构，大小约为 3.0 cm x 0.6 cm x 0.6 cm，可在左腹部识别。</li>
	<li>沿脾门解剖，逐渐分离并暴露肝总动脉、胃十二指肠动脉和脾动脉。结扎并切断胃十二指肠动脉和肝总动脉，同时逐渐解离周围组织。</li>
	<li>将脾脏向右侧翻转，露出腹主动脉。轻轻进行钝性解剖，用棉签露出腹主动脉和腹腔干。在乳糜泻干的枝条上方和下方放置一条长约 3 厘米的 3-0 丝缝线，并在乳糜泻干的枝条上放置一条长约 10 厘米的 6-0 丝缝线。</li>
	<li>将腹主动脉结扎在腹腔主动脉的下方和上方，位于腹腔干的分支上方。在动脉分支处做一个小切口。轻轻提起 6-0 丝线缝合线，将 24 G 静脉导管沿腹腔干插入脾动脉，然后结扎并固定。</li>
	<li>使用注射泵以 4 mL/min 的速率输注肝素化的生理盐水 （25 U/mL），体积为 50 mL。同时，切断门静脉作为流出通道，让输液从脾脏流出。动物通过放血被安乐死。</li>
	<li>小心解剖脾脏周围的组织，避免对胰腺造成损伤，同时保留主要的附属血管。</li>
	<li>检查脾脏周围是否有渗漏，然后切除脾脏和胰腺，并用生理盐水冲洗。 注意:大鼠的脾脏和胰腺紧密相连，胰腺包裹着脾动脉。如果单独切除脾脏，结扎许多小血管可能会很困难。在这个过程中，脾脏和胰腺一起被切除。脱细胞后，脾脏和胰腺变得透明，微血管系统可见，有利于保存具有完整血管的脾脏。</li>
	<li>将脾脏转移到装有生理盐水的50mL离心管中，并将其储存在-80°C冰箱中。 注意:插入脾动脉的脾脏和静脉导管将一起冷冻保存，以便在灌注实验期间方便连接。</li>
</ol>2.脾脏脱细胞
<ol><li>在无菌容器中重复冻融循环 3 次以裂解脾细胞。 注意:冻融循环是一种用于支架脱细胞的物理方法。将脾组织置于-80°C冰箱中冷冻过夜，然后从低温环境中取出，置于室温或37°C水浴中解冻。这种冻融过程重复 3-6 次，会破坏细胞膜并导致细胞裂解。</li>
	<li>在洁净工作台内设置灌注系统，包括蠕动泵、2 L 储液罐、内径为 2.4 mm 的硅胶管和气泡收集器（图 1）。</li>
	<li>用去离子水（ddH2O）填充灌注系统，并保持其运行10分钟。</li>
	<li>小心地将收获的脾脏转移到 ddH2O 填充的容器中。</li>
	<li>将硅胶管连接到已插入脾动脉的静脉导管。</li>
	<li>以 2 mL/min 的速率开始用 ddH2O 灌注 30 分钟。</li>
	<li>继续以 4 mL/min 的速率用 ddH2O 灌注 30 分钟。</li>
	<li>用0.1%（w / v）SDS溶液灌注4小时。</li>
	<li>用1%（v / v）Triton X-100溶液灌注2小时。</li>
	<li>以 4 mL/min 的速率用 PBS 灌注 4 小时以洗涤 DSM。 注意: 使用蠕动泵单向灌注所有液体。</li>
	<li>灌注完成后，将胰腺和脾脏之间的血管分支结扎并横断，然后切除胰腺。将DSM储存在清洁密封的50mL离心管中，该离心管浸泡在含有10%青霉素 - 链霉素的PBS中，温度为-20°C，直至准备用于未来的实验。</li>
</ol>

用于肝组织工程的脱细胞大鼠脾基质制备

<ol>
	<li>Xu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=State+of+the+art+and+perspectives+in+liver+transplantation.">State of the art and perspectives in liver transplantation.</a> Hepatobiliary Pancreat Dis Int. 22 (1), 1-3 (2023).</li><li>Hautz, T., 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=Immune+cell+dynamics+deconvoluted+by+single-cell+RNA+sequencing+in+normothermic+machine+perfusion+of+the+liver.">Immune cell dynamics deconvoluted by single-cell RNA sequencing in normothermic machine perfusion of the liver.</a> Nat Commun. 14 (1), 2285 (2023).</li><li>Cardini, B., 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=Live+confocal+imaging+as+a+novel+tool+to+assess+liver+quality:+insights+from+a+murine+model.">Live confocal imaging as a novel tool to assess liver quality: insights from a murine model.</a> Transplantation. 104 (12), 2528-2537 (2020).</li><li>Ding, Y., 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=Mesenchymal+stem+cell-derived+exosomes:+a+promising+therapeutic+agent+for+the+treatment+of+liver+diseases.">Mesenchymal stem cell-derived exosomes: a promising therapeutic agent for the treatment of liver diseases.</a> Int J Mol Sci. 23 (18), 10972 (2022).</li><li>Yaghoubi, A., 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=Prednisolone+and+mesenchymal+stem+cell+preloading+protect+liver+cell+migration+and+mitigate+extracellular+matrix+modification+in+transplanted+decellularized+rat+liver.">Prednisolone and mesenchymal stem cell preloading protect liver cell migration and mitigate extracellular matrix modification in transplanted decellularized rat liver.</a> Stem Cell Res Ther. 13 (1), 36 (2022).</li><li>Uygun, B. 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=Organ+reengineering+through+development+of+a+transplantable+recellularized+liver+graft+using+decellularized+liver+matrix.">Organ reengineering through development of a transplantable recellularized liver graft using decellularized liver matrix.</a> Nat Med. 16 (7), 814-820 (2010).</li><li>Xiang, J., 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+effect+of+riboflavin/UVA+cross-linking+on+anti-degeneration+and+promoting+angiogenic+capability+of+decellularized+liver+matrix.">The effect of riboflavin/UVA cross-linking on anti-degeneration and promoting angiogenic capability of decellularized liver matrix.</a> J Biomed Mater Res A. 105 (10), 2662-2669 (2017).</li><li>Liu, P., 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=Implantation+strategy+of+tissue-engineered+liver+based+on+decellularized+spleen+matrix+in+rats.">Implantation strategy of tissue-engineered liver based on decellularized spleen matrix in rats.</a> J South Med Univ. 38 (6), 698-703 (2018).</li><li>Xiang, J., 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=Decellularized+spleen+matrix+for+reengineering+functional+hepatic-like+tissue+based+on+bone+marrow+mesenchymal+stem+cells.">Decellularized spleen matrix for reengineering functional hepatic-like tissue based on bone marrow mesenchymal stem cells.</a> Organogenesis. 12 (3), 128-142 (2016).</li><li>Gao, R., 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=Hepatocyte+culture+in+autologous+decellularized+spleen+matrix.">Hepatocyte culture in autologous decellularized spleen matrix.</a> Organogenesis. 11 (1), 16-29 (2015).</li><li>Liu, P., 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=Hemocompatibility+improvement+of+decellularized+spleen+matrix+for+constructing+transplantable+bioartificial+liver.">Hemocompatibility improvement of decellularized spleen matrix for constructing transplantable bioartificial liver.</a> Biomed Mater. 14 (2), 25003 (2019).</li><li>Somuncu, &. #. 2. 1. 4. ;. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularization+concept+in+regenerative+medicine.">Decellularization concept in regenerative medicine.</a> Adv Exp Med Biol. 1212, 71-85 (2020).</li><li>Neishabouri, A., Soltani, K. A., Daghigh, F., Kajbafzadeh, A. M., Majidi, Z. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularization+in+tissue+engineering+and+regenerative+medicine:+evaluation,+modification,+and+application+methods.">Decellularization in tissue engineering and regenerative medicine: evaluation, modification, and application methods.</a> Front Bioeng Biotech. 10, 805299 (2022).</li><li>Brown, M., Li, J., Moraes, C., Tabrizian, M., Li-Jessen, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decellularized+extracellular+matrix:+New+promising+and+challenging+biomaterials+for+regenerative+medicine.">Decellularized extracellular matrix: New promising and challenging biomaterials for regenerative medicine.</a> Biomaterials. 289, 121786 (2022).</li><li>Gui, L., Muto, A., Chan, S. A., Breuer, C. K., Niklason, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+decellularized+human+umbilical+arteries+as+small-diameter+vascular+grafts.">Development of decellularized human umbilical arteries as small-diameter vascular grafts.</a> Tissue Eng Pt A. 15 (9), 2665-2676 (2009).</li><li>Li, T., Javed, R., Ao, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenogeneic+decellularized+extracellular+matrix-based+biomaterials+For+peripheral+nerve+repair+and+regeneration.">Xenogeneic decellularized extracellular matrix-based biomaterials For peripheral nerve repair and regeneration.</a> Curr Neuropharmacol. 19 (12), 2152-2163 (2021).</li><li>Crapo, P. M., Gilbert, T. W., Badylak, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+overview+of+tissue+and+whole+organ+decellularization+processes.">An overview of tissue and whole organ decellularization processes.</a> Biomaterials. 32 (12), 3233-3243 (2011).</li></ol>

提交人未声明存在任何利益冲突。

BALs代表了治疗终末期肝病的有效方法，特别是在肝移植因目前供体器官短缺而受到阻碍的情况下6。制造 BAL 的一个有前途的选择是利用 DLM，它保留了天然肝脏的天然 ECM 和血管结构。然而，人类DLM的稀缺性以及与动物DLM相关的感染和免疫原性的潜在风险构成了重大限制。为了应对这一挑战，我们提出了一种新的策略，涉及采用脱细胞脾基质 （DSM） 作为BAL、<sup...

肝移植仍然是终末期肝病的唯一确定性治疗方法1,2,3。然而，供体器官的严重短缺和质量下降增加了对替代疗法的需求4.在再生医学领域，利用脱细胞肝基质 （DLM） 的生物人工肝 （BAL） 已成为有前途的解决方案5,6,7。DLM 保留了原始的肝脏结构，包括其错综复杂的微血管网络和 ECM 的组成部分，为创建可移植的 BAL 提供了支架，这可能会缓解肝脏疾病。
尽管前景广阔，但这项技术的采用仍面临挑战，特别是在寻找合适的DLM方面。 人源性DLMs供不应求，而来自动物源的DLMs则存在疾病传播和免疫排斥的风险。在一种创新的方法中，我们的研究探索了使用脱细胞脾基质 （DSM） 作为 BAL的基础 8,9,10,11。脾脏在各种医疗情况下更容易获得，例如门静脉高压、创伤性破裂、特发性血小板减少性紫癜和心源性死亡后的捐献。因此，出于研究目的，脾脏比肝脏更广泛地可用。接受过脾切除术的患者不会患有严重的疾病，这进一步证实了脾脏的可有可无性。脾脏的微环境，特别是细胞外基质和鼻窦，与肝脏的微环境相似。这使得脾脏成为肝细胞移植研究中细胞粘附和增殖的合适器官。基于这些发现，我们之前的研究表明，DSMs与DLMs具有相当的微观结构和成分，可以支持肝细胞的存活和功能，包括白蛋白和尿素的产生。此外，DSM已被证明可以增强骨髓间充质干细胞的肝脏分化，从而改善和一致的功能。
通过使用用肝素治疗的 DSM，我们设计了能够证明有效的短期抗凝和部分肝功能补偿的功能性 BAL11。因此，这种三维DSM对于肝组织工程和细胞疗法的发展具有重要的前景。在这项工作中，我们介绍了收获大鼠脾脏和制备 DSM 的详细方法，这些方法保留了 ECM 的微观结构和成分。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anesthesia Machine</td>
			<td>Harvard Apparatus</td>
			<td>tabletop</td>
			<td>animal anesthesia</td>
		</tr>
		<tr>
			<td>bubble trap</td>
			<td>Shandong Weigao Group Medical Polymer Co., Ltd.</td>
			<td>pore diameter: 5 &#956;m</td>
			<td>prevent air bubbles</td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>TIPR Pharmaceutical Responsible Co.,Ltd</td>
			<td></td>
			<td>an analgesic</td>
		</tr>
		<tr>
			<td>Hemostatic Forceps</td>
			<td>Shanghai Medical Instruments&#160; Co., Ltd</td>
			<td>J31020</td>
			<td>surgical tool</td>
		</tr>
		<tr>
			<td>Heparinized Saline</td>
			<td>SPH No.1 Biochemical &#38; Pharmaceutical Co., LTD&#160;</td>
			<td></td>
			<td>prevent the formation of thrombosis&#160;</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>RWD life Science Co.</td>
			<td></td>
			<td>anesthetic:for the induction and maintenanceof anesthesia</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin&#160;</td>
			<td>Beyotime Biotechnology Co., Ltd.</td>
			<td>C0222</td>
			<td>antibiotics in vitro to prevent microbial contamination</td>
		</tr>
		<tr>
			<td>Peristaltic Pump</td>
			<td>Baoding Longer Precision Pump Co., Ltd.</td>
			<td>BT100-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline</td>
			<td>Shanghai Titan Scientific Co., Ltd.</td>
			<td>4481228</td>
			<td>phosphoric acid buffer salt solution</td>
		</tr>
		<tr>
			<td>Silicone Tube</td>
			<td>Baoding Longer Precision Pump Co., Ltd.</td>
			<td>2.4&#215;0.8mm</td>
			<td></td>
		</tr>
		<tr>
			<td>Silk Suture</td>
			<td>Yangzhou Jinhuan Medical Instrument Factory</td>
			<td>6-0 and 3-0</td>
			<td>ligate blood vessels</td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulfate</td>
			<td>Shanghai Titan Scientific Co., Ltd.</td>
			<td>151-21-3</td>
			<td>ionic detergent, dissolves both cell and nuclear membranes</td>
		</tr>
		<tr>
			<td>Syringe Pump</td>
			<td>Shenzhen Mindray Bio-Medical Electronics Co., Ltd</td>
			<td>BeneFusion SP5</td>
			<td>intravenous infusion</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Shanghai Titan Scientific Co., Ltd.</td>
			<td>9002-93-1</td>
			<td>non-ionic detergent, disrupts lipid-lipid, lipid-protein, and DNA-protein interactions</td>
		</tr>
		<tr>
			<td>Venous Catheter</td>
			<td>B. Braun Company</td>
			<td>24G</td>
			<td>inserting the spleen artery</td>
		</tr>
	</tbody>
</table>

fabrication of decellularized spleen matrix derived from rats

肝移植是终末期肝病的主要治疗方法。然而，供体器官的短缺和质量不足需要开发替代疗法。利用脱细胞肝基质 （DLM） 的生物人工肝 （BAL） 已成为有前途的解决方案。然而，寻找合适的 DLM 仍然具有挑战性。已经探索使用脱细胞脾基质 （DSM） 作为 BAL 的基础，提供了一种现成的替代方案。在这项研究中，使用冻融循环和去细胞试剂灌注相结合的方式收获大鼠脾脏并进行脱细胞。该协议保留了 DSM 内细胞外基质 （ECM） 的微观结构和成分。完整的脱细胞过程大约需要 11 小时，从而在 DSM 内产生完整的 ECM。组织学分析证实了细胞成分的去除，同时保留了 ECM 的结构和组成。所提出的方案提供了一种获得 DSM 的综合方法，在肝组织工程和细胞治疗中提供了潜在的应用。这些发现有助于开发治疗终末期肝病的替代方法。

该方案利用重复冻融循环和与脱细胞试剂灌注的组合来使大鼠脾脏脱细胞。脾脏的完全脱细胞在大约11小时内实现（图2A）。在整个脱细胞过程中，脾脏的颜色逐渐从深红色转变为斑驳的浅红色，最终呈现白色半透明外观（图2B）。整体形态保持相对完整，具有可见的血管结构（图2B）。
苏木精-伊红染色证实?...

Watch this Scientific Journal Video about Utilization of Decellularized Spleen Matrix for Bioartificial Livers at JoVE.com

作者聚焦:脱细胞脾基质在生物人工肝脏中的应用

脱细胞脾基质（DSM）在肝组织工程领域具有广阔的应用前景。该方案概述了制备大鼠DSM的程序，其中包括收获大鼠脾脏，通过灌注使它们脱细胞，并评估所得的DSM以确认其特性。

来源于大鼠的脱细胞脾基质的制备

Liver transplantation is the primary treatment for end-stage liver disease. However, the shortage and inadequate quality of donor organs necessitate the ...

我们采用了一种新颖的策略，该策略涉及采用脱细胞的脾脏基质作为生物人工肝的替代支架。我们展示了收获大鼠脾脏和制备脱细胞脾基质的详细过程，该基质保留了细胞外基质的宏观结构和成分。我们的方案提供了一个现成的脾脏基质，作为稀缺供体肝脏的替代品。我们的方案提出了一种制备脱细胞脾脏基质的方法。该方法具有效率、稳定性和微创性，可保持脾脏固有的结构、组成和自然血管网络。它为细胞植入和三维动态培养提供了支架，从而为推进组织工程肝脏的研究奠定了基础。

fabrication-of-decellularized-spleen-matrix-derived-from-rats

Research

JoVE Journal

Bioengineering

Fabrication of Decellularized Spleen Matrix Derived from Rats

584 次观看.  The First Affiliated Hospital of Xi'an Jiaotong University. 我们采用了一种新颖的策略，该策略涉及采用脱细胞的脾脏基质作为生物人工肝的替代支架。我们展示了收获大鼠脾脏和制备脱细胞脾基质的详细过程，该基质保留了细胞外基质的宏观结构和成分。我们的方案提供了一个现成的脾脏基质，作为稀缺供体肝脏的替代品。我们的方案提出了一种制备脱细胞脾脏基质的方法。该方法具有效率、稳定性和微创性，可保持脾脏固?...

视频: 作者聚焦:脱细胞脾基质在生物人工肝脏中的应用

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and cell-matrix interactions. Due to this complexity, recapitulation of lung development in vitro to promote differentiation of stem cells to lung epithelial cells has been challenging. In this protocol, decellularized lung scaffolds are used to mimic the 3-dimensional environment of the lung and generate stem cell-derived airway epithelial cells. Mouse embryonic stem cell are first differentiated to the endoderm lineage using an embryoid body (EB) culture method with activin A. Endoderm cells are then seeded onto decellularized scaffolds and cultured at air-liquid interface for up to 21 days. This technique promotes differentiation of seeded cells to functional airway epithelial cells (ciliated cells, club cells, and basal cells) without additional growth factor supplementation. This culture setup is defined, serum-free, inexpensive, and reproducible. Although there is limited contamination from non-lung endoderm lineages in culture, this protocol only generates airway epithelial populations and does not give rise to alveolar epithelial cells. Airway epithelia generated with this protocol can be used to study cell-matrix interactions during lung organogenesis and for disease modeling or drug-discovery platforms of airway-related pathologies such as cystic fibrosis.

This protocol efficiently directs mouse embryonic stem cell-derived definitive endoderm to mature airway epithelial cells. This differentiation technique uses 3-dimensional decellularized lung scaffolds to direct lung lineage specification, in a defined, serum-free culture setting.

用脱细胞支架龙ESC衍生鼠气道上皮细胞的生成

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and ...

科研

生物工程

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

细胞外基质衍生的泡沫和微载体如组织特异性细胞培养和交付平台的制造

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

肾皮质细胞外基质衍生水凝胶的制备

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage research has focused on the development of regenerative scaffolds. This article describes the development of scaffolds that are completely derived from natural cartilage extracellular matrix, coming from an equine donor. Potential applications of the scaffolds include producing allografts for cartilage repair, serving as a scaffold for osteochondral tissue engineering, and providing in vitro models to study tissue formation. By decellularizing the tissue, the donor cells are removed, but many of the natural bioactive cues are thought to be retained. The main advantage of using such a natural scaffold in comparison to a synthetically produced scaffold is that no further functionalization of polymers is required to drive osteochondral tissue regeneration. The cartilage-derived matrix scaffolds can be used for bone and cartilage tissue regeneration in both in vivo and in vitro settings.

Decellularized cartilage-derived scaffolds can be used as a scaffold to guide cartilage repair and as a means to regenerate osteochondral tissue. This paper describes the decellularization process in detail and provides suggestions to use these scaffolds in in vitro settings.

脱碳软骨衍生基质支架的制备

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

成纤维细胞衍生的人类工程结缔组织用于筛查应用

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...

Synthesis of Decellularized Cartilage Extracellular Matrix Hydrogels

Decellularized cartilage extracellular matrix (DC-ECM) hydrogels are promising biomaterials for tissue engineering and regenerative medicine due to their biocompatibility and ability to mimic natural tissue properties. This protocol aims to produce DC-ECM hydrogels that closely mimic the native ECM of cartilage tissue. The protocol involves a combination of physical and chemical disruption and enzymatic digestion to remove the cellular material while preserving the structure and composition of the ECM. The DC-ECM is cross-linked using a chemical agent to form a stable and biologically active hydrogel. The DC-ECM hydrogel has excellent biological activity, spatial structure, and biological induction function, as well as low immunogenicity. These characteristics are beneficial in promoting cell adhesion, proliferation, differentiation, and migration and for creating a superior microenvironment for cell growth. This protocol provides a valuable resource for researchers and clinicians in the field of tissue engineering. Biomimetic hydrogels can potentially enhance the development of effective tissue engineering strategies for cartilage repair and regeneration.

This paper introduces a new method for the synthesis of decellularized cartilage extracellular matrix (DC-ECM) hydrogels. DC-ECM hydrogels have excellent biocompatibility and provide a superior microenvironment for cell growth. Therefore, they can be ideal cell scaffolds and biological delivery systems.

脱细胞软骨细胞外基质水凝胶的合成

Decellularized cartilage extracellular matrix (DC-ECM) hydrogels are promising biomaterials for tissue engineering and regenerative medicine due to their ...

牛肺 ECM 水凝胶脱细胞技术的比较研究

Development and Characterization of Decellularized Lung Extracellular Matrix Hydrogels

The use of extracellular matrix (ECM)-derived hydrogels in tissue engineering has become increasingly popular, as they can mimic cells&#39; natural environment in vitro. However, maintaining the native biochemical content of the ECM, achieving mechanical stability, and comprehending the impact of the decellularization process on the mechanical properties of the ECM hydrogels are challenging. Here, a pipeline for decellularization of bovine lung tissue using two different protocols, downstream characterization of the effectiveness of decellularization, fabrication of reconstituted decellularized lung ECM hydrogels and assessment of their mechanical and cytocompatibility properties were described. Decellularization of the bovine lung was pursued using a physical (freeze-thaw cycles) or chemical (detergent-based) method. Hematoxylin and Eosin staining was performed to validate the decellularization and retention of major ECM components. For the evaluation of residual collagen and sulfated glycosaminoglycan (sGAG) content within the decellularized samples, Sirius red and Alcian blue staining techniques were employed, respectively. Mechanical properties of the decellularized lung ECM hydrogels were characterized by oscillatory rheology. The results suggest that decellularized bovine lung hydrogels can provide a reliable organotypic<span style="font-size: 11pt; font-family: Calibri, sans-serif; color: black; background-image: initial; background-position: initial; background-size: initial; background-repeat: initial; background-attachment: initial; background-origin: initial; background-clip: initial;">&#160;alternative to commercial ECM products by retaining most native ECM components. Furthermore, these findings reveal that the decellularization method of choice significantly affects gelation kinetics as well as the stiffness and viscoelastic properties of resulting hydrogels.

作者聚焦：优化器官型水凝胶的牛肺脱细胞

The protocol elucidates two distinct decellularization methodologies applied to native bovine pulmonary tissues, providing a comprehensive account of their respective characterizations.

脱细胞肺细胞外基质水凝胶的开发与表征

The use of extracellular matrix (ECM)-derived hydrogels in tissue engineering has become increasingly popular, as they can mimic cells&#39; natural ...

分析细胞接种的脱细胞苹果衍生支架用于骨组织工程

Decellularized Apple-Derived Scaffolds for Bone Tissue Engineering In Vitro and In Vivo

Plant-derived&#160;cellulose biomaterials have been employed in various tissue engineering applications. In vivo studies have shown the remarkable biocompatibility of scaffolds made of cellulose derived from natural sources. Additionally, these scaffolds possess structural characteristics that are relevant for multiple tissues, and they promote the invasion and proliferation of mammalian cells. Recent research using decellularized apple hypanthium tissue has demonstrated the similarity of its pore size to that of trabecular bone as well as&#160;its ability to effectively support osteogenic differentiation. The present study further&#160;examined the potential of apple-derived cellulose scaffolds for bone tissue engineering (BTE) applications and evaluated their in vitro and in vivo mechanical properties. MC3T3-E1 preosteoblasts were seeded in apple-derived cellulose scaffolds that were then&#160;assessed for their osteogenic potential and mechanical properties. Alkaline phosphatase and alizarin red S staining confirmed osteogenic differentiation in scaffolds cultured in differentiation medium. Histological examination demonstrated widespread cell invasion and mineralization across the scaffolds. Scanning electron microscopy (SEM) revealed mineral aggregates on the surface of the scaffolds, and&#160;energy-dispersive spectroscopy (EDS)&#160;confirmed the presence of phosphate and calcium elements. However, despite a significant increase in the Young&#39;s modulus following cell differentiation, it remained lower than that of healthy bone tissue. In vivo studies showed cell infiltration and deposition of extracellular matrix within the decellularized apple-derived&#160;scaffolds after 8 weeks of implantation in rat calvaria. In addition, the force required to remove the scaffolds from the bone defect was similar to the previously reported fracture load of native calvarial bone. Overall, this study confirms that apple-derived cellulose is a promising candidate for BTE applications. However, the dissimilarity between its mechanical properties and those of healthy bone tissue may restrict its application to low load-bearing scenarios. Additional structural re-engineering and optimization may be necessary to enhance the mechanical properties of apple-derived cellulose scaffolds for load-bearing applications.

作者聚焦：深入了解 Apple-derived Cellulose 支架在骨组织工程中的应用 

In this study, we detail methods of decellularization, physical characterization, imaging, and in vivo implantation of plant-based biomaterials, as well as methods for cell seeding and differentiation in the scaffolds. The described methods allow the evaluation of plant-based biomaterials for bone tissue engineering applications.