这项研究得到了以下赠款的资助: 中国自然科学基金 (81572198 号;81772394号);深圳大学高等医学学科建设基金 (2016031638 号);中国广东省医学研究基金会 (no。A2016314);和深圳科技项目 (no。JCYJ20170306092215436;大声 笑JCYJ20170412150609690;大声 笑JCYJ20170413161800287;大声 笑SGLH20161209105517753;大声 笑JCYJ20160301111338144)。

所有动物实验都是按照区域伦理委员会的指导方针进行的, 所有动物程序都是由深圳市第二人民医院机构动物护理和使用委员会批准的。
1. 分离和培养 rbSF-MSCs
<ol><li>
准备为动物做法
	<ol>
<li>制备 skeletally 成熟的新西兰雌性白兔, 用于收集 rbSF-MSCs. 在麻醉和冲洗术手术前一天对兔子进行一次临床检查。 注: 体格检查应包括体重 (2.0-2.5 公斤)、性别 (女性)、体温、呼吸率和心率。临床健康家兔的参数间隔为呼吸率为 30-50 分钟, 心率为 220-280 分钟, 体温为 38-39 摄氏度。</li>
		<li>在麻醉前将动物快速6小时。</li>
	</ol>
</li>
	<li>
动物麻醉的准备和做法
	<ol>
<li>用笼子约束兔子 (见材料表), 然后将3% 戊巴比妥钠注射到边缘耳静脉中, 剂量为1毫升/千克, 用于全身麻醉。</li>
		<li>把兔子放在一个舒适的背卧位置。</li>
		<li>监测麻醉期间家兔的呼吸速率、心率和体温。</li>
		<li>在其眼睛上使用眼科软膏, 以防止麻醉时的干燥。</li>
		<li>评估 Guedel 分类14后的麻醉深度。</li>
	</ol>
</li>
	<li>
MSCs 分离培养的制备
	<ol>
<li>培养 rbSF-MSCs, 准备500毫升的商业培养基 (见材料表) 补充10% 商业补充 (见材料表), 10% 胎牛血清 (血清) 和1% 青霉素-链霉素。</li>
		<li>在37摄氏度的水浴中孵化培养基。</li>
		<li>准备500毫升磷酸盐缓冲盐水 (PBS), 用于细胞分离和细胞洗涤。</li>
		<li>准备100毫升的等渗盐水溶液治疗膝关节腔冲洗术程序。</li>
	</ol>
</li>
	<li>
兔膝关节关节滑膜ﬂ uid 的采集
	<ol>
<li>选择一个面积约 5 x 5 厘米的大小在膝盖周围和剃须从这个地区的兔毛使用安全电动剃须刀。</li>
		<li>交替使用聚维酮碘溶液和75% 乙醇消毒程序现场3x。然后, 在该区域彻底干燥后应用无菌窗帘。</li>
		<li>注射 1-2 毫升的等渗盐水溶液, 使用无菌皮下注射注射器 (2 毫升), 进入膝关节腔从外侧关节空间, 移动膝盖 3-4x, 然后吸出所有的滑液在室温下。</li>
		<li>过滤滑膜ﬂuid 通过40µm 尼龙细胞过滤器去除任何碎片在4小时内。</li>
	</ol>
</li>
	<li>
rbSF-MSCs 的培养
	<ol>
<li>在室温下收集50毫升离心机管和离心机的过滤ﬂuid, 1500 转每分钟10分钟。</li>
		<li>离心后丢弃上清液, 用 PBS 清洗颗粒, 用完全培养基并用重悬颗粒, 然后将培养基在100毫米的盘子中。</li>
		<li>在含有 5% CO2的湿润气氛中孵化37摄氏度的菜肴。</li>
		<li>48小时后, 用新鲜培养基补充菜肴, 去除任何非黏附细胞。每星期更换两次培养基2周作为通道 0 (P0)。 注: 应该有大约 1 x 104黏附细胞。活细胞/毫升在 SF 大约 2 x 102/毫升。</li>
		<li>经过14天的初步电镀, 许多殖民地应该已经形成的文化菜肴。选择直径大于2毫米的菌落。通过在盘子底部追踪它们的圆周来标记所选菌落所在的位置。</li>
		<li>使用细胞刮刀丢弃菌落 < 2 毫米宽。用克隆的圆柱体, 用大约5µL 0.25% 胰蛋白酶消化选定的菌落, 并将菌落转移到新的盘子里, 作为通道 1 (P1)。</li>
	</ol>
</li>
	<li>
术后动物护理
	<ol>
<li>遵循标准的机构操作程序进行术后监测和从麻醉中恢复。</li>
		<li>每10分钟监测一只兔子的生命体征, 直到它恢复知觉为止。最后, 将有意识的动物转移到笼子里。在完全恢复之前, 不要将已经进行过手术的兔子退回到其他动物的公司。</li>
		<li>手术后, 用0.1% 聚维酮碘消毒现场, 每天2x 天3天。</li>
	</ol>
</li>
</ol>2. rbSF-MSCs 和原代培养的 CD90-positive 磁活化细胞分选 (mac)
<ol><li>
样品准备
	<ol>
<li>当细胞达到大约80% 融合, 吸入培养基, 然后添加 1-2 毫升0.25% 胰蛋白酶-EDTA 到每道菜。</li>
		<li>将盘子孵化 2-3 分钟, 允许细胞脱离。</li>
		<li>一旦细胞分离, 添加同等数量的培养基以使胰蛋白酶失效。</li>
		<li>通过40µm 细胞过滤器通过细胞悬浮液, 收集15毫升管中的滤液, 然后在室温下将细胞在 600 x g 处旋转10分钟。</li>
		<li>并用重悬细胞颗粒在 mac 运行缓冲 (PBS, pH 值 7.2, 0.5% 牛血清白蛋白, 2 毫米 EDTA), 然后计数的细胞数。</li>
	</ol>
</li>
	<li>
磁性标签 注意: 将 rbSF-MSCs 与包含列、支架和分隔符的磁性激活单元格分拣套件进行排序 (见材料表)。<ol>
<li>使用 hemocytometer 确定单元格编号。</li>
		<li>离心机细胞悬浮在 300 x g 10 分钟在4°c。完全吸入上清液。</li>
		<li>每 107个单元格中添加80µL 的泥沙缓冲区。</li>
		<li>对于 107的总细胞, 添加20µL 的微珠与单克隆抗兔 CD90 抗体结合。</li>
		<li>将磁性珠子和细胞均匀地混合在试管中, 然后在黑暗中孵化4摄氏度, 15 分钟。</li>
		<li>将每 107个细胞的1毫升缓冲液添加到管中, 然后将其离心在 300 x g 处以10摄氏度为4分钟, 以洗涤细胞。离心后要丢弃上清液。</li>
		<li>并用重悬每 107细胞的500µL 缓冲颗粒。</li>
	</ol>
</li>
	<li>
磁性分离
	<ol>
<li>将柱面与柱翼放在磁选机的磁场中。</li>
		<li>用每 107个单元格中500µL 的缓冲器冲洗磁选机 (MS) 柱。</li>
		<li>将单个单元格悬浮体转移到该列中。让阴性细胞通过磁场来丢弃这些未标记的细胞。</li>
		<li>用每 107个单元格的500µL, 将 1-2x 列洗净, 然后丢弃该流。</li>
		<li>将该列转移到离心管 (15 毫升)。</li>
		<li>将每 107个单元格的缓冲区的1毫升添加到该列, 然后立即将柱塞推入该列, 以冲出磁性标记的单元格。</li>
		<li>重复上述步骤与第二个 MS 专栏, 以提高 CD90+细胞纯度, 这可以丰富的洗脱分数。</li>
		<li>离心细胞悬浮在 300 x g 10 分钟, 吸入上清, 并与培养基并用重悬。</li>
	</ol>
</li>
	<li>
CD90+ rbSF-MSCs 的培养
	<ol>
<li>在磁性活化细胞分类后, 在100毫米的菜肴中接种细胞。</li>
		<li>孵化的菜肴在37°c 在一个湿润细胞孵化器与 5% CO2。</li>
		<li>当主要培养的 80-90% 汇合达到, 在大约 7-10 天, 消化黏附的细胞与0.25% 胰蛋白酶-EDTA 并且通过他们在1:2 稀释使段落 2 (P2)。</li>
		<li>使用相同的方法将细胞传递到 3 (P3), 可用于体外测定。 注: 亚培养纯化后, 可获得约 1 x 107 rbSF MSCs。</li>
	</ol>
</li>
</ol>3. rbSF-MSCs 的鉴定
<ol><li>
流式细胞仪 rbSF 的表面标记确认
	<ol>
<li>当 MSCs 在 P3 80-90% 汇合时, 用 PBS 冲洗细胞, 并用1毫升0.25% 胰蛋白酶-EDTA 进行治疗。然后, 在37摄氏度处孵育 MSCs 2-3 分钟, 直到细胞分离。</li>
		<li>用10毫升的 PBS 收割细胞, 将它们转移成锥形管 (15 毫升), 在室温下将其离心在 300 x g 上5分钟。</li>
		<li>丢弃上清液。并用重悬500µL 中的细胞颗粒, 并将其转化为1.5 毫升管。</li>
		<li>孵化1:100 稀释 FITC 共轭和 PE 共轭抗体 (同种, FITC-CD34/PE-CD45, FITC-CD105/PE-CD44) 为1小时在黑暗中4°c。</li>
		<li>将此混合物在室温下以 300 x g 为5分钟, 每两次离心一次, 用 300 x g 离心法将电池清洗一次。丢弃上清液。</li>
		<li>并用重悬500µL 中的细胞, 将细胞悬浮液转移到圆底管 (5 毫升) 中, 并用流式细胞仪进行分析。 注: 在装有两个荧光通道的细胞仪上获取数据: 533/30 和585/40。对于每个同种荧光, 使用同种控制来调整适当的激光电压, 并设置获得荧光直方图所需的最小强度, 以显示峰值的左和右边缘。为统计分析收集至少1万个事件。</li>
	</ol>
</li>
	<li>
rbSF-MSCs 的 Multidifferentiation 注: 使用 P3 rbSF 干细胞体外multidifferentiation 测定。<ol>
<li>
成骨分化
		<ol>
<li>制备成骨诱导培养基: DMEM 基本 (1x), 含有50毫米的 l-抗坏血酸 acid-2-phosphate,10 毫米的α甘油和100毫微米的地塞米松。</li>
			<li>种子细胞在 103细胞/cm2在6良好的组织培养板和培养他们在成骨诱导培养基。</li>
			<li>每3天更换一次感应培养基3周。</li>
			<li>在区分完成后, 在室温下固定4% 甲醛30分钟的细胞, 用1% 茜素红染成5分钟的细胞, 然后用 PBS15清洗3x。</li>
		</ol>
</li>
		<li>
脂肪分化
		<ol>
<li>制备脂肪诱导介质: DMEM 基本 (1x), 由100毫米消炎痛、10毫克/毫升重组人胰岛素、1毫米地塞米松和0.5 毫米3异丁基-1-甲基黄嘌呤组成。</li>
			<li>在脂肪诱导培养基中治疗 P3 rbSF-MSCs 3 周。</li>
			<li>3周后, 用油红 O 染色中性脂质泡, 确认脂肪分化。在室温下将4% 甲醛溶液中的细胞固定30分钟, 然后用 PBS16冲洗3x。</li>
		</ol>
</li>
		<li>
软骨分化
		<ol>
<li>制备软骨诱导介质: DMEM 基本 (1x), 由 TGFβ1 10 ng/毫升、1% 补充剂、0.35 毫米 l-脯氨酸、100 nM 地塞米松、50毫米 l-抗坏血酸 acid-2-phosphate 和1毫米丙酮酸钠组成。</li>
			<li>使用颗粒培养软骨诱导。离心机 5×105 P3 rbSF-MSCs, 1500 rpm 10 分钟, 在聚丙烯管 (15 毫升) 形成颗粒。</li>
			<li>用软骨诱导培养基培养3周的颗粒。</li>
			<li>每3天更换一次培养基3周。</li>
			<li>3周诱导后, 将细胞颗粒嵌入石蜡切片, 将其切片成4毫米切片, 在室温17时用0.1% 甲苯胺蓝染色30分钟。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
定量实时聚合酶链反应 (qRT PCR) 对总 RNA 的提取和分析
	<ol>
<li>用商业 RNA 萃取试剂盒分离出3周后细胞的总 RNA (见材料表)。<ol>
<li>去除培养基中的培养基, 然后加入1毫升的隔离试剂。</li>
			<li>通过重复吹打溶解细胞, 直到溶液是均匀的。在室温下孵化3分钟。</li>
			<li>将细胞裂解液转化为1.5 毫升离心管。</li>
			<li>加100µL 氯仿, 用手摇动 15x, 室温下将混合物孵化3分钟。</li>
			<li>离心管在 1.2万 x g 15 分钟在4摄氏度。将上清液转移到一个新的1.5 毫升离心管。</li>
			<li>添加500µL 异丙醇, 在室温下沉淀 RNA 10 分钟。</li>
			<li>离心机在 1.2万 x g 为 10 minat 4 °c。RNA 颗粒应该在管子的底部可见。</li>
			<li>取出上清, 然后添加500µL 75% 乙醇。简要旋转管几秒钟, 以洗涤 RNA 颗粒。离心机它在 7500 x g 为5分钟在4°c。</li>
			<li>除去残留的乙醇。</li>
			<li>将 RNA 颗粒溶解在10µL 的 DEPC 水中, 将管子放在冰上。</li>
		</ol>
</li>
		<li>用 DNA 合成试剂盒将总 RNA 反转转录成 cDNA (见材料表)。 注: 在冰上执行以下所有步骤。<ol>
<li>准备反向转录大师组合。</li>
			<li>添加25µL DNase 处理的 rna 到每个管与1µg 的 rna。</li>
			<li>使用 PCR 热循环仪在以下温度下孵化混合物:26 °c 10 分钟 (允许随机 hexamers 退火), 42 °c 45 分钟 (反转转录) 和75°c 10 分钟 (使逆转录酶失效)。</li>
			<li>立即通过定量的实时 PCR 分析结果 cDNA, 或将其存储在-20 摄氏度。</li>
		</ol>
</li>
		<li>采用定量实时 PCR 系统进行基因表达分析。<ol>
<li>使用 qRT pcr 的主组合 (见材料表) 进行 pcr。GAPDH、Runx2、Agg 和 PPARγ的 PCR 引物列于表 1。</li>
			<li>使用 2−ΔΔCT方法计算基因表达。</li>
			<li>使用标准统计软件进行统计分析。使用独立的样本 t 检验来比较组的方法。</li>
		</ol>
</li>
	</ol>
</li>
</ol>

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F., 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=Characterization+of+mesenchymal+stem+cells+derived+from+the+equine+synovial+fluid+and+membrane.">Characterization of mesenchymal stem cells derived from the equine synovial fluid and membrane.</a> BioMed Central Veterinary Research. 11 (1), 281 (2015).</li><li>Yoshimura, H., 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=Comparison+of+rat+mesenchymal+stem+cells+derived+from+bone+marrow,+synovium,+periosteum,+adipose+tissue,+and+muscle.">Comparison of rat mesenchymal stem cells derived from bone marrow, synovium, periosteum, adipose tissue, and muscle.</a> Cell and Tissue Research. 327 (3), 449-462 (2007).</li><li>Wu, C. C., 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=Intra-articular+injection+of+platelet-rich+fibrin+releasates+in+combination+with+bone+marrow-derived+mesenchymal+stem+cells+in+the+treatment+of+articular+cartilage+defects:+an+in+vivo+study+in+rabbits.">Intra-articular injection of platelet-rich fibrin releasates in combination with bone marrow-derived mesenchymal stem cells in the treatment of articular cartilage defects: an in vivo study in rabbits.</a> Journal of Biomedical Materials Research Part B: Applied Biomaterials. 105 (6), 1536-1543 (2017).</li><li>John, T., Lerche, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anesthesia and Analgesia for Veterinary Technicians. , (2011).</li><li>Koyama, 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=Pluripotency+of+mesenchymal+cells+derived+from+synovial+fluid+in+patients+with+temporomandibular+joint+disorder.">Pluripotency of mesenchymal cells derived from synovial fluid in patients with temporomandibular joint disorder.</a> Life Sciences. 89 (19-20), 741-747 (2011).</li><li>Kim, Y. 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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=Synovial+fluid+mesenchymal+stem+cells+in+health+and+early+osteoarthritis:+detection+and+functional+evaluation+at+the+single+cell+level.">Synovial fluid mesenchymal stem cells in health and early osteoarthritis: detection and functional evaluation at the single cell level.</a> Arthritis &amp; Rheumatism. 58 (6), 1731-1740 (2008).</li><li>Makker, K., Agarwal, A., Sharma, R. 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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=Synovial+fluid+progenitors+expressing+CD90++from+normal+but+not+osteoarthritic+joints+undergo+chondrogenic+differentiation+without+micro-mass+culture.">Synovial fluid progenitors expressing CD90+ from normal but not osteoarthritic joints undergo chondrogenic differentiation without micro-mass culture.</a> PLOS One. 7 (8), e43616 (2012).</li><li>Ogata, 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=Purified+human+synovium+mesenchymal+stem+cells+as+a+good+resource+for+cartilage+regeneration.">Purified human synovium mesenchymal stem cells as a good resource for cartilage regeneration.</a> PLOS One. 10 (6), e0129096 (2015).</li><li>Dominici, M., 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=Minimal+criteria+for+defining+multipotent+mesenchymal+stromal+cells:+the+International+Society+for+Cellular+Therapy+position+statement.">Minimal criteria for defining multipotent mesenchymal stromal cells: the International Society for Cellular Therapy position statement.</a> Cytotherapy. 8 (4), 315-317 (2006).</li><li>Gauci, S. 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=Modulating+chondrocyte+hypertrophy+in+growth+plate+and+osteoarthritic+cartilage.">Modulating chondrocyte hypertrophy in growth plate and osteoarthritic cartilage.</a> Journal of Musculoskeletal &amp; Neuronal Interact. 8 (4), 308 (2008).</li><li>Cooke, M. 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=Structured+three-dimensional+co-culture+of+mesenchymal+stem+cells+with+chondrocytes+promotes+chondrogenic+differentiation+without+hypertrophy.">Structured three-dimensional co-culture of mesenchymal stem cells with chondrocytes promotes chondrogenic differentiation without hypertrophy.</a> Osteoarthritis and Cartilage. 19 (10), 1210-1218 (2011).</li><li>Fischer, J., Dickhut, A., Rickert, M., Richter, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+articular+chondrocytes+secrete+parathyroid+hormone-related+protein+and+inhibit+hypertrophy+of+mesenchymal+stem+cells+in+coculture+during+chondrogenesis.">Human articular chondrocytes secrete parathyroid hormone-related protein and inhibit hypertrophy of mesenchymal stem cells in coculture during chondrogenesis.</a> Arthritis and Rheumatism. 62 (9), 2696-2706 (2010).</li></ol>

作者声明他们没有竞争的财政利益。

在滑膜液中 MSCs 的存在为细胞治疗提供了一种替代方法。以往的研究表明, 损伤部位在滑液中含有较高数量的间充质干细胞, 这可能与损伤后5期呈正相关。在滑膜液中的 MSCs 可能有利于组织, 以加强在伤害18,19的自发愈合。在文献中很少涉及 sf 间充质干细胞的临床应用, 主要是因为关节内的 sf-mscs 的机制仍未确定20。琼...

骨髓间充质干细胞被认为是再生医学的宝贵来源, 尤其是软骨损伤。骨髓间充质干细胞, 包括软骨细胞、成骨细胞、脂肪细胞、骨骼肌细胞和内脏基质干细胞, 由于其高扩张率和多谱系分化潜能1, 广泛扩大了干细胞移植的面积。MSCs 可从骨骼肌、滑膜、骨髓和脂肪组织中分离出2、3、4。研究结果还 conﬁrmed 了在滑膜液中的 MSCs 的存在, 先前的调查已经确定了滑膜液源性 mscs (SF-mscs) 作为关节再生5、6的有望候选者。
然而, 人体标本的研究和前期实验受到许多伦理问题的影响。相反, 兔子一直是并且继续是最常用的动物种类表明, 骨髓间充质干细胞移植可以修复软骨损伤。近年来, 越来越多的研究人员研究了兔间充质干细胞 (rbMSCs)的体外和体内作用, 因为这些细胞在细胞生物学和组织生理学上与人的 MSCs 相似。同样, rbMSCs 能够附着在塑料表面, 显示纺锤体成纤维细胞形态, 如在人类 MSCs 中。此外, 家兔间充质样品简单易获得7。此外, 最关键的一点是, rbMSCs 明确的表面标记, 如 CD44, CD90, 和 CD105, 并认为多血统分化潜力保持, 这是符合标准的鉴定的 MSC 人口作为由国际社会为细胞疗法定义8,9。特别是, 滑膜液生发能够在 TGF-β1诱导下的非肥厚软骨, 从而使其适合表型关节软骨再生10,11的细胞源, 12。
然而, 与其他组织 (包括脐带、脂肪组织、外周血、骨髓) 的分离有很大的不同。目前, 采用流式细胞术和磁珠分选方法对 SF MSCs 进行纯化和分类是最常用的方法, 尽管流式细胞术法需要特定的环境和高成本的仪器13。
本文介绍了一种简便、微创的新西兰白兔滑液标本采集方法。在该过程中, rbSF-MSCs 在体外稳定扩张, 然后以 CD90 正磁珠为基础进行分离。最后, 该协议显示了如何从收获的细胞来源获得高纯度和生存能力的 MSCs。
在该协议中, 分离的 rbSF-MSCs 的特点是基于它们的形态学, 特定标记的表达, 和干细胞的干细胞。基于流式细胞术的免疫分型揭示了 CD44 和 CD105 的显著阳性表达, 而 CD45 和 CD34 的表达呈阴性。最后, 对 rbSF 骨髓间充质干细胞的体外测定表明了这些细胞的成骨、脂肪和软骨分化。

<table>
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MesenGro</td>
			<td>StemRD</td>
			<td>MGro-500 1703</td>
			<td>Warm in 37 &#176;C water bath before use</td>
		</tr>
		<tr>
			<td>MesenGro Supplement</td>
			<td>StemRD</td>
			<td>MGro-500 M1512</td>
			<td>Component of MSCs culture medium</td>
		</tr>
		<tr>
			<td>DMEM basic</td>
			<td>Gibco Inc.</td>
			<td>C11995500BT</td>
			<td>MSCs differentiation medium</td>
		</tr>
		<tr>
			<td>Isotonic saline solution</td>
			<td>Litai, China</td>
			<td>5217080305</td>
			<td>Cavity arthrocentesis procedure reagent</td>
		</tr>
		<tr>
			<td>Phosphate-Buffered Saline (PBS)</td>
			<td>HyClone Inc.</td>
			<td>SH30256.01B</td>
			<td>PBS, free of Ca2+/Mg2+</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gibco Inc.</td>
			<td>10099-141</td>
			<td>Component of MSCs culture medium</td>
		</tr>
		<tr>
			<td>Povidone iodine solution</td>
			<td>Guangdong, China</td>
			<td>150605</td>
			<td>Sterilization agent</td>
		</tr>
		<tr>
			<td>75% ethanol</td>
			<td>Lircon, china</td>
			<td>170917</td>
			<td>Sterilization agent</td>
		</tr>
		<tr>
			<td>0.25% Trypsin/EDTA</td>
			<td>Gibco Inc.</td>
			<td>25200-056</td>
			<td>Cell dissociation reagent</td>
		</tr>
		<tr>
			<td>1% Penicillin-Streptomycin</td>
			<td>Gibco Inc.</td>
			<td>15140-122</td>
			<td>Component of MSCs medium</td>
		</tr>
		<tr>
			<td>MACS Running Buffer</td>
			<td>MiltenyiBiotec</td>
			<td>5160112089</td>
			<td>Containing phosphate-buffered saline (PBS), 0.5% bovine serum albumin(BSA), and 2 mMEDTA</td>
		</tr>
		<tr>
			<td>CD90 antibody conjugated MicroBeads</td>
			<td>MiltenyiBiotec</td>
			<td>5160801456</td>
			<td>For magnetic activated cell sorting</td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td>P2256</td>
			<td>Component of MSCs chondrogenic differentiation</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma-Aldrich</td>
			<td>D1756</td>
			<td>Component of MSCs osteogenic differentiation</td>
		</tr>
		<tr>
			<td>ITS</td>
			<td>BD</td>
			<td>354352</td>
			<td>1%, Component of MSCs chondrogenic differentiation</td>
		</tr>
		<tr>
			<td>L-proline</td>
			<td>Sigma-Aldrich</td>
			<td>P5607</td>
			<td>0.35 mM, Component of MSCs chondrogenic differentiation</td>
		</tr>
		<tr>
			<td>L-ascorbic acid-2-phosphate</td>
			<td>Sigma-Aldrich</td>
			<td>A8960</td>
			<td>50 mM, Component of MSCs chondrogenic differentiation</td>
		</tr>
		<tr>
			<td>3-isobutyl-1-methylxanthine</td>
			<td>Sigma-Aldrich</td>
			<td>I5879</td>
			<td>0.5 mM, Component of adipogenic differentiation</td>
		</tr>
		<tr>
			<td>Indomethacin</td>
			<td>Sigma-Aldrich</td>
			<td>I7378</td>
			<td>100 mM, Component of adipogenic differentiation</td>
		</tr>
		<tr>
			<td>TGF&#946;1</td>
			<td>Peprotech</td>
			<td>100-21</td>
			<td>10 ng/mL, Component of MSCs chondrogenic differentiation</td>
		</tr>
		<tr>
			<td>&#945;-glycerophsphate</td>
			<td>Sigma-Aldrich</td>
			<td>G6751</td>
			<td>Component of MSCs osteogenic differentiation</td>
		</tr>
		<tr>
			<td>CD34 Polyclonal Antibody, FITC Conjugated</td>
			<td>Bioss</td>
			<td>bs-0646R-FITC</td>
			<td>Hematopoietic stem cells marker</td>
		</tr>
		<tr>
			<td>Mouse antirabbit CD44</td>
			<td>Bio-Rad</td>
			<td>MCA806GA</td>
			<td>Thy-1 membrane glycoprotein (MSCs marker)</td>
		</tr>
		<tr>
			<td>CD45 (Monoclonal Antibody)</td>
			<td>Bio-Rad</td>
			<td>MCA808GA</td>
			<td>Hematopoietic stem cells marker</td>
		</tr>
		<tr>
			<td>CD105 antibody</td>
			<td>Genetex</td>
			<td>GTX11415</td>
			<td>MSCs marker</td>
		</tr>
		<tr>
			<td>Isopropyl alcohol</td>
			<td>Sigma-Aldrich</td>
			<td>I9030</td>
			<td>Precipitates RNA extraction organic phases</td>
		</tr>
		<tr>
			<td>Trichloromethane</td>
			<td>Wenge, China</td>
			<td>61553</td>
			<td>Extract total RNA</td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>Invitrogen</td>
			<td>15596-018</td>
			<td>Isolate total RNA</td>
		</tr>
		<tr>
			<td>SYBR green master mix</td>
			<td>Takara Bio, Japan</td>
			<td>RR420A</td>
			<td>PCR test</td>
		</tr>
		<tr>
			<td>cDNA synthesis kit</td>
			<td>Takara Bio, Japan</td>
			<td>RR047A</td>
			<td>Reverse-transcribed to complementary DNA</td>
		</tr>
		<tr>
			<td>Alizarin Red</td>
			<td>Sigma-Aldrich</td>
			<td>A5533</td>
			<td>Staining of calcium compounds</td>
		</tr>
		<tr>
			<td>Toluidine Blue</td>
			<td>Sigma-Aldrich</td>
			<td>89640</td>
			<td>Staining of cartilaginous tissue</td>
		</tr>
		<tr>
			<td>Oil Red O solution</td>
			<td>Sigma-Aldrich</td>
			<td>O1391L</td>
			<td>Lipid vacuole staining</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MiniMACS Separator</td>
			<td>MiltenyiBiotec</td>
			<td>130-042-102</td>
			<td>For magnetic activated cell sorting</td>
		</tr>
		<tr>
			<td>MultiStand</td>
			<td>MiltenyiBiotec</td>
			<td>130-042-303</td>
			<td>For magnetic activated cell sorting</td>
		</tr>
		<tr>
			<td>MS Columns</td>
			<td>MiltenyiBiotec</td>
			<td>130-042-201</td>
			<td>For magnetic activated cell sorting</td>
		</tr>
		<tr>
			<td>Cell Strainer</td>
			<td>FALCON Inc.</td>
			<td>352340</td>
			<td>40 &#956;m nylon</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>ISOLAB Inc.</td>
			<td>075.03.001</td>
			<td>Cell counting</td>
		</tr>
		<tr>
			<td>Falcon 100 mm&#160; dish</td>
			<td>Corning</td>
			<td>353003</td>
			<td>Cell culture dish</td>
		</tr>
		<tr>
			<td>Microcentrifuge tube</td>
			<td>Axygen</td>
			<td>MCT-150-C</td>
			<td>RNA Extraction and PCR</td>
		</tr>
		<tr>
			<td>Centrifuge Tubes</td>
			<td>Sigma-Aldrich</td>
			<td>91050</td>
			<td>Gamma-sterilized</td>
		</tr>
		<tr>
			<td>High-speed centrifuge</td>
			<td>Eppendorf</td>
			<td>5804R</td>
			<td>Centrifuge cells</td>
		</tr>
		<tr>
			<td>Carbon dioxide cell incubator</td>
			<td>Thermo scientific</td>
			<td>3111</td>
			<td>Cell culture</td>
		</tr>
		<tr>
			<td>Real-Time PCR Instrument</td>
			<td>Life Tech</td>
			<td>QuantStudio</td>
			<td>Real-Time quantitative polymerase chain reaction</td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>BD Biosciences</td>
			<td>342975</td>
			<td>Cell analyzer</td>
		</tr>
		<tr>
			<td>Pipettor</td>
			<td>Eppendorf</td>
			<td>O25456F</td>
			<td>Transfer the liquid</td>
		</tr>
		<tr>
			<td>Cloning cylinder</td>
			<td>Sigma-Aldrich</td>
			<td>C3983-50EA</td>
			<td>Isolate and pick individual cell colonies</td>
		</tr>
		<tr>
			<td>Sterile hypodermic syringe</td>
			<td>Double-Dove, China</td>
			<td>131010</td>
			<td>Arthrocentesis procedure</td>
		</tr>
		<tr>
			<td>Rabbit cage</td>
			<td>Zhike, China</td>
			<td>ZC-TGD</td>
			<td>Restrain the rabbit</td>
		</tr>
	</tbody>
</table>

magnetic-activated cell sorting strategies to isolate and purify synovial fluid-derived mesenchymal stem cells from a rabbit model

间充质干细胞 (MSCs) 是细胞治疗的主要细胞来源。关节腔滑液中的 MSCs 可能用于软骨组织工程。滑膜液中的骨髓间充质干细胞被认为是促进关节再生的候选者, 其潜在的治疗效果已成为晚期的一个重要研究课题。从新西兰白兔的膝腔中的 SF 干细胞可以作为一种优化的转化模型来评估人体再生医学。通过 CD90-based 磁活化细胞分选 (mac) 技术, 该协议成功地从该兔模型中获得了兔 SF-mscs (rbSF), 并通过诱导它们分化为进一步充分展示了这些细胞的 MSC 表型。成骨细胞, 脂肪细胞和软骨。因此, 这种方法可以应用于细胞生物学研究和组织工程中, 使用简单的设备和程序。

rbSF-MSCs 的分离、纯化和培养: 根据 MSC 表面标记 CD90 的表达式, 该协议使用 mac 隔离 rbSF MSCs。图 1显示了 rbSF-MSCs 的分离、纯化和表征以及体外培养协议的工艺流程图。
磁性活化细胞分选 (mac) 与 CD90 后的细胞形态学: 首先, 对于 mac, immunolabel MSCs 与 CD90 磁珠。离心后, 并?...

Watch this Scientific Journal Video about Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model at JoVE.com

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

从兔模型中分离纯化滑液源性间充质干细胞的磁活化细胞分选策略

本文提出了一种简单而经济的协议, 直接隔离和纯化新西兰白兔滑液间充质干细胞。

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

magnetic-activated-cell-sorting-strategies-to-isolate-purify-synovial

Research

JoVE Journal

Biology

11.1K 次观看.  Guangzhou Medical University. 本文提出了一种简单而经济的协议, 直接隔离和纯化新西兰白兔滑液间充质干细胞。

视频: Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

从小鼠胚胎干细胞的造血干细胞的推导

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

科研

生物学

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

人类胚胎干细胞的推导Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

隔离和动物血清自由膨胀的人脐血源性间质基质细胞（MSCs），内皮细胞集落形成祖细胞（ECFCs）

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting (LIMACS): a Novel Method to Analyze Protein-lipid Interaction

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification procedures. As an alternative, lipids can be coated on flat surfaces as used for lipid ELISA and Plasmon resonance spectroscopy. However, surface coating lipids do not form microdomain structures, which may be important for the lipid binding properties. Further, these methods do not allow for the purification of larger amounts of proteins binding to their target lipids.
 To overcome these limitations of testing lipid protein interaction and to purify lipid binding proteins we developed a novel method termed lipid vesicle-mediated affinity chromatography using magnetic-activated cell sorting (LIMACS). In this method, lipid vesicles are prepared with the target lipid and phosphatidylserine as the anchor lipid for Annexin V MACS. Phosphatidylserine is a ubiquitous cell membrane phospholipid that shows high affinity to the protein Annexin V. Using magnetic beads conjugated to Annexin V the phosphatidylserine-containing lipid vesicles will bind to the magnetic beads. When the lipid vesicles are incubated with a cell lysate the protein binding to the target lipid will also be bound to the beads and can be co-purified using MACS. This method can also be used to test if recombinant proteins reconstitute a protein complex binding to the target lipid. 
 We have used this method to show the interaction of atypical PKC (aPKC) with the sphingolipid ceramide and to co-purify prostate apoptosis response 4 (PAR-4), a protein binding to ceramide-associated aPKC. We have also used this method for the reconstitution of a ceramide-associated complex of recombinant aPKC with the cell polarity-related proteins Par6 and Cdc42. Since lipid vesicles can be prepared with a variety of sphingo- or phospholipids, LIMACS offers a versatile test for lipid-protein interaction in a lipid environment that resembles closely that of the cell membrane. Additional lipid protein complexes can be identified using proteomics analysis of lipid binding protein co-purified with the lipid vesicles.

Lipid Vesicle-mediated Affinity Chromatography using Magnetic Activated Cell Sorting LIMACS: a Novel Method to Analyze Protein-lipid Interaction

To test the interaction of a protein with its target lipid we used MACS and Annexin V-conjugated magnetic beads and lipid vesicles synthesized from the target lipid and Annexin V-binding phosphatidylserine. Proteins bound to the target lipid are co-purified and analyzed after elution from the beads.

脂质囊泡介导的亲和层析法，使用磁性细胞分选（LIMACS）:一种新的方法来分析蛋白质 - 脂质相互作用

The analysis of lipid protein interaction is difficult because lipids are embedded in cell membranes and therefore, inaccessible to most purification ...

Isolation &#38; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor formation. Taken together these studies suggest that resident lung MSC play a role during pulmonary tissue homeostasis, injury and repair during diseases such as pulmonary fibrosis (PF) and arterial hypertension (PAH). Here we describe a technology to define a population of resident lung MSC. The definition of this population in vivo pulmonary tissue using a define set of markers facilitates the repeated isolation of a well-characterized stem cell population by flow cytometry and the study of a specific cell type and function.

Isolation & Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

In this article we demonstrate the isolation of murine resident lung mesenchymal stem cells (lung MSC), their expansion, characterization and analysis of immunomodulatory properties.

对赫斯特的分离与表征低 CD45负小鼠肺间质干细胞

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

单个肌纤维和成人骨骼肌卫星细胞的分离和培养

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

肾毒素显微注射斑马鱼到模型急性肾损伤

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Fluorescence-Activated Cell Sorting for the Isolation of Scleractinian Cell Populations

Coral reefs are under threat due to anthropogenic stressors. The biological response of coral to these stressors may occur at a cellular level, but the mechanisms are not well understood. To investigate coral response to stressors, we need tools for analyzing cellular responses. In particular, we need tools that facilitate the application of functional assays to better understand how cell populations are reacting to stress. In the current study, we use fluorescence-activated cell sorting (FACS) to isolate and separate different cell populations in stony corals. This protocol includes: (1) the separation of coral tissues from the skeleton, (2) creation of a single cell suspension, (3) labeling the coral cells using various markers for flow cytometry, and (4) gating and cell sorting strategies. This method will enable researchers to work on corals at the cellular level for analysis, functional assays, and gene expression studies of different cell populations.

Corals create biodiverse ecosystems important for both humans and marine organisms. However, we still do not understand the full potential and function of many coral cells. Here, we present a protocol developed for the isolation, labeling, and separation of stony coral cell populations.

荧光激活细胞分拣，用于分离瘦杆菌细胞群

Coral reefs are under threat due to anthropogenic stressors. The biological response of coral to these stressors may occur at a cellular level, but the ...

Large-Scale Preparation of Synovial Fluid Mesenchymal Stem Cell-Derived Exosomes by 3D Bioreactor Culture

Exosomes secreted by mesenchymal stem cells (MSCs) have been suggested as promising candidates for cartilage injuries and osteoarthritis treatment. Exosomes for clinical application require large-scale production. To this aim, human synovial fluid MSCs (hSF-MSCs) were grown on microcarrier beads, and then cultured in a dynamic three-dimension (3D) culture system. Through utilizing 3D dynamic culture, this protocol successfully obtained large-scale exosomes from SF-MSC culture supernatants. Exosomes were harvested by ultracentrifugation and verified by a transmission electron microscope, nanoparticle transmission assay, and western blotting. Also, the microbiological safety of exosomes was detected. Results of exosome detection suggest that this approach can produce a large number of good manufacturing practices (GMP)-grade exosomes. These exosomes could be utilized in exosome biology research and clinical osteoarthritis treatment.

Here, we present a protocol to produce a large number of GMP-grade exosomes from synovial fluid mesenchymal stem cells using a 3D bioreactor.

3D生物反应器培养大规模制备滑液间充质干细胞来源外泌体

Exosomes secreted by mesenchymal stem cells (MSCs) have been suggested as promising candidates for cartilage injuries and osteoarthritis treatment. ...