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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<li>Ogata, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Purified+human+synovium+mesenchymal+stem+cells+as+a+good+resource+for+cartilage+regeneration%5BTitle%5D)+AND+%22PLOS+One%22%5BJournal%5D)">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/pubmed?term=((Minimal+criteria+for+defining+multipotent+mesenchymal+stromal+cells%3A+the+International+Society+for+Cellular+Therapy+position+statement%5BTitle%5D)+AND+%22Cytotherapy%22%5BJournal%5D)">Minimal criteria for defining multipotent mesenchymal stromal cells: the International Society for Cellular Therapy position statement.</a> Cytotherapy. 8 (4), 315-317 (2006).</li>
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<li>Cooke, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structured+three-dimensional+co-culture+of+mesenchymal+stem+cells+with+chondrocytes+promotes+chondrogenic+differentiation+without+hypertrophy%5BTitle%5D)+AND+%22Osteoarthritis+and+Cartilage%22%5BJournal%5D)">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/pubmed?term=((Human+articular+chondrocytes+secrete+parathyroid+hormone-related+protein+and+inhibit+hypertrophy+of+mesenchymal+stem+cells+in+coculture+during+chondrogenesis%5BTitle%5D)+AND+%22Arthritis+and+Rheumatism%22%5BJournal%5D)">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>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>MesenGro</td><td>StemRD</td><td>MGro-500 1703</td><td>Warm in 37 &amp;deg;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 Ca&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt;</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&amp;beta;1</td><td>Peprotech</td><td>100-21</td><td>10 ng/mL, Component of MSCs chondrogenic differentiation</td></tr><tr><td>&amp;alpha;-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>&lt;strong&gt;Equipment&lt;/strong&gt;</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 &amp;mu;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 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?...

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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 ...

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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

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells (ASCs)

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix (ECM) remodeling. In vivo, ECM-rich environment consisting of fibrillar collagens provides a structural support to adipose tissues during the progression and regression of obesity. Physiological ECM remodeling mediated by matrix metalloproteinases (MMPs) plays a major role in regulating adipose tissue size and function1,2. The loss of physiological collagenolytic ECM remodeling may lead to excessive collagen accumulation (tissue fibrosis), macrophage infiltration, and ultimately, a loss of metabolic homeostasis including insulin resistance3,4. When a phenotypic change of the adipose tissue is observed in gene-targeted mouse models, isolating primary ASCs from fat depots for in vitro studies is an effective approach to define the role of the specific gene in regulating the function of ASCs. In the following, we define an immunomagnetic separation of Sca1high ASCs.

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells ASCs

We present the techniques required to isolate the stromal vascular fraction (SVF) from mouse inguinal (subcutaneous) and perigonadal (visceral) adipose tissue depots to assess their gene expression and collagenolytic activity. This method includes the enrichment of Sca1high adipose-derived stem cells (ASCs) using immunomagnetic cell separation.

脂肪库专用SCA1的免疫磁性分离<sup&gt;高</sup&gt;脂肪干细胞（ASCs）

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix ...

科研

发育生物学

Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular cartilage and subchondral bone in recent years led to increased attention to restoration of the entire osteochondral unit. In comparison to chondral lesions the regeneration of osteochondral defects is much more complex and a far greater surgical and therapeutic challenge. The damaged tissue does not only include the superficial cartilage layer but also the subchondral bone. For deep, osteochondral damage, as it occurs for example with osteochondrosis dissecans, the full thickness of the defect needs to be replaced to restore the joint surface 1. Eligible therapeutic procedures have to consider these two different tissues with their different intrinsic healing potential 2. In the last decades, several surgical treatment options have emerged and have already been clinically established 3-6.
Autologous or allogeneic osteochondral transplants consist of articular cartilage and subchondral bone and allow the replacement of the entire osteochondral unit. The defects are filled with cylindrical osteochondral grafts that aim to provide a congruent hyaline cartilage covered surface 3,7,8. Disadvantages are the limited amount of available grafts, donor site morbidity (for autologous transplants) and the incongruence of the surface; thereby the application of this method is especially limited for large defects.
New approaches in the field of tissue engineering opened up promising possibilities for regenerative osteochondral therapy. The implantation of autologous chondrocytes marked the first cell based biological approach for the treatment of full-thickness cartilage lesions and is now worldwide established with good clinical results even 10 to 20 years after implantation 9,10. However, to date, this technique is not suitable for the treatment of all types of lesions such as deep defects involving the subchondral bone 11.
The sandwich-technique combines bone grafting with current approaches in Tissue Engineering 5,6. This combination seems to be able to overcome the limitations seen in osteochondral grafts alone. After autologous bone grafting to the subchondral defect area, a membrane seeded with autologous chondrocytes is sutured above and facilitates to match the topology of the graft with the injured site. Of course, the previous bone reconstruction needs additional surgical time and often even an additional surgery. Moreover, to date, long-term data is missing 12.
Tissue Engineering without additional bone grafting aims to restore the complex structure and properties of native articular cartilage by chondrogenic and osteogenic potential of the transplanted cells. However, again, it is usually only the cartilage tissue that is more or less regenerated. Additional osteochondral damage needs a specific further treatment. In order to achieve a regeneration of the multilayered structure of osteochondral defects, three-dimensional tissue engineered products seeded with autologous/allogeneic cells might provide a good regeneration capacity 11.
Beside autologous chondrocytes, mesenchymal stem cells (MSC) seem to be an attractive alternative for the development of a full-thickness cartilage tissue. In numerous preclinical in vitro and in vivo studies, mesenchymal stem cells have displayed excellent tissue regeneration potential 13,14. The important advantage of mesenchymal stem cells especially for the treatment of osteochondral defects is that they have the capacity to differentiate in osteocytes as well as chondrocytes. Therefore, they potentially allow a multilayered regeneration of the defect.
In recent years, several scaffolds with osteochondral regenerative potential have therefore been developed and evaluated with promising preliminary results 1,15-18. Furthermore, fibrin glue as a cell carrier became one of the preferred techniques in experimental cartilage repair and has already successfully been used in several animal studies 19-21 and even first human trials 22.
The following protocol will demonstrate an experimental technique for isolating mesenchymal stem cells from a rabbit's bone marrow, for subsequent proliferation in cell culture and for preparing a standardized in vitro-model for fibrin-cell-clots. Finally, a technique for the implantation of pre-established fibrin-cell-clots into artificial osteochondral defects of the rabbit's knee joint will be described.

An experimental technique for the treatment of osteochondral defects in the rabbit's knee joint is described. The implantation of allogeneic mesenchymal stem cells into osteochondral defects provides a promising development in the field of tissue engineering. The preparation of fibrin-cell-clots in vitro offers a standardized method for implantation.

异基因骨髓间充质干细胞植入兔子的膝关节软骨缺损治疗纤维蛋白凝块

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular ...

医学

Isolation and Enrichment of Rat Mesenchymal Stem Cells (MSCs) and Separation of Single-colony Derived MSCs

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and can be easily separated from the hematopoietic stem cells (HSCs) due to their plastic adherence. This protocol describes how to isolate MSCs from rat femurs and tibias. The isolated cells were further enriched against two MSCs surface markers CD54 and CD90 by magnetic cell sorting. Expression of surface markers CD54 and CD90 were then confirmed by flow cytometry analysis. HSC marker CD45 was also included to check if the sorted MSCs were depleted of HSCs. MSCs are naturally quite heterogeneous. There are subpopulations of cells that have different shapes, proliferation and differentiation abilities. These subpopulations all express the known MSCs markers and no unique marker has yet been identified for the different subpopulations. Therefore, an alternative approach to separate out the different subpopulations is using cloning cylinders to separate out single-colony derived cells. The cells derived from the single-colonies can then be cultured and evaluated separately.

Isolation and Enrichment of Rat Mesenchymal Stem Cells MSCs and Separation of Single-colony Derived MSCs

Rat MSCs were isolated from femurs and tibias and then enriched by magnetic cell sorting. Sorted cells were confirmed for the expression of surface markers by flow cytometry. These cells were also cultured at clonal density to form single colonies and then these colonies were separated by cloning cylinders.

分离和富集大鼠间质干细胞（MSCS）及衍生的干细胞分离单细胞集落

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and ...

生物学

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach recently explored for the development of autologous replacements involves the differentiation of stem cells into chondrocytes. To initiate this chondrogenesis, a degree of compaction of the stem cells is required; hence, we demonstrated the feasibility of magnetically condensing cells, both within thick scaffolds and scaffold-free, using miniaturized magnetic field sources as cell attractors. This magnetic approach was also used to guide aggregate fusion and to build scaffold-free, organized, three-dimensional (3D) tissues several millimeters in size. In addition to having an enhanced size, the tissue formed by magnetic-driven fusion presented a significant increase in the expression of collagen II, and a similar trend was observed for aggrecan expression. As the native cartilage was subjected to forces that influenced its 3D structure, dynamic maturation was also performed. A bioreactor that provides mechanical stimuli was used to culture the magnetically seeded scaffolds over a 21-day period. Bioreactor maturation largely improved chondrogenesis into the cellularized scaffolds; the extracellular matrix obtained under these conditions was rich in collagen II and aggrecan. This work outlines the innovative potential of magnetic condensation of labeled stem cells and dynamic maturation in a bioreactor for improved chondrogenic differentiation, both scaffold-free and within polysaccharide scaffolds.

Chondrogenesis from stem cells requires fine tuning the culture conditions. Here, we present a magnetic approach for condensing cells, an essential step to initiate chondrogenesis. In addition, we show that dynamic maturation in a bioreactor applies mechanical stimulation to the cellular constructs and enhances cartilaginous extracellular matrix production.

3D磁干细胞聚集和生物反应器为成熟软骨再生

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach ...

生物工程

Isolation and Cellular Phenotyping of Mesenchymal Stem Cells Derived from Synovial Fluid and Bone Marrow of Minipigs

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, synovial-fluid-derived MSCs were reported to have multi-lineage differentiation potential and immunomodulatory features, which indicates that these cells can be used for tissue engineering and systemic treatments. This study presents a protocol for simple and non-invasive isolation of MSCs derived from the bone marrow and synovial fluid of minipigs to analyze cell surface markers for cell phenotyping and in vitro culturing. Using sexually mature six-month-old minipigs, bone marrow was extracted from the iliac crest bone using a bone marrow extractor, and the synovial fluid was aspirated from the femorotibial joint. Procedures for the collection of samples from both sources were non-invasive. The protocols for effective isolation of MSCs from harvested cell sources and for creating in vitro culture conditions to expand stable MSCs from minipigs and the application of systemic autologous treatments are provided. For cell phenotyping, the cell surface markers of both cells were analyzed using flow cytometry. In the results, the MSCs were isolated from the synovial fluid of the minipigs and showed that synovial-fluid-derived MSCs have a similar morphology and cell phenotype to bone-marrow-derived MSCs. Therefore, non-invasively obtained synovial fluid is a valuable source of MSCs.

A protocol establishing mesenchymal stem cells (MSCs) isolated from the synovial fluid and bone marrow of minipigs and non-invasively collected using syringe aspiration is presented. Cellular phenotyping was performed using flow cytometry after cell isolation and in vitro cultivation of bone marrow and synovial fluids derived from MSCs.

从小型猪关节液和骨髓间充质干细胞的分离和细胞表型

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, ...

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides the use of bone marrow and umbilical cord MSCs for exogenous cell therapy, there is also increasing interest in the repair and regenerative potential of resident tissue MSCs. Moreover, they likely have a role in normal organ development, and have been attributed roles in disease, particularly those with a fibrotic nature. The main hurdle for the study of these resident tissue MSCs is the lack of a clear marker for the isolation and identification of these cells. The isolation technique described here applies multiple characteristics of lung resident MSCs (L-MSCs). Upon sacrifice of the rats, lungs are removed and rinsed multiple times to remove blood. Following mechanical dissociation by scalpel, the lungs are digested for 2-3 hr using a mix of collagenase type I, neutral protease and DNase type I. The obtained single cell suspension is subsequently washed and layered over density gradient medium (density 1.073 g/ml). After centrifugation, cells from the interphase are washed and plated in culture-treated flasks. Cells are cultured for 4-7 days in physiological 5% O2, 5% CO2 conditions. To deplete fibroblasts (CD146-) and to ensure a population of only L-MSCs (CD146+), positive selection for CD146+ cells is performed through magnetic bead selection. In summary, this procedure reliably produces a population of primary L-MSCs for further in vitro study and manipulation. Because of the nature of the protocol, it can easily be translated to other experimental animal models.

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

This protocol describes an isolation technique for obtaining primary lung resident mesenchymal stromal cells from rats, through the use of enzymatic digestion, density gradient separation, plastic adherence and CD146+ magnetic bead selection.

CD146的分离<sup&gt; +</sup&gt;从大鼠肺组织驻地肺间质基质细胞

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides ...

从血管壁分离和磁性分离小鼠 CD34+ 干细胞

Immunomagnetic Isolation of the Vascular Wall-Resident CD34+ Stem Cells from Mice

Resident CD34+ vascular wall-resident stem and progenitor cells (VW-SCs) are increasingly recognized for their crucial role in regulating vascular injury and repair. Establishing a stable and efficient method to culture functional murine CD34+ VW-SCs is essential for further investigating the mechanisms involved in the proliferation, migration, and differentiation of these cells under various physiological and pathological conditions. The described method combines magnetic bead screening and flow cytometry to purify primary cultured resident CD34+ VW-SCs. The purified cells are then functionally identified through immunofluorescence staining and Ca2+ imaging. Briefly, vascular cells from the adventitia of the murine aorta and mesenteric artery are obtained through tissue block attachment, followed by subculturing until reaching a cell count of at least 1 &#215; 107. Subsequently, CD34+ VW-SCs are purified using magnetic bead sorting and flow cytometry. Identification of CD34+ VW-SCs involves cellular immunofluorescence staining, while functional multipotency is determined by exposing cells to a specific culture medium for oriented differentiation. Moreover, functional internal Ca2+ release and external Ca2+ entry is assessed using a commercially available imaging workstation in Fura-2/AM-loaded cells exposed to ATP, caffeine, or thapsigargin (TG). This method offers a stable and efficient technique for isolating, culturing, and identifying vascular wall-resident CD34+ stem cells, providing an opportunity for in vitro studies on the regulatory mechanisms of VW-SCs and the screening of targeted drugs.

作者聚焦:使用磁珠筛选偶联流式细胞术增强血管壁驻留小鼠 CD34+ 干细胞的分离和表征

This study has established a stable and efficient method for the isolation, culture, and functional determination of vascular wall-resident CD34+ stem cells (CD34+ VW-SCs). This easy-to-follow and time-effective isolation method can be utilized by other investigators to study the potential mechanisms involved in cardiovascular diseases.

从小鼠中免疫磁性分离血管壁驻留的 CD34+ 干细胞

Resident CD34+ vascular wall-resident stem and progenitor cells (VW-SCs) are increasingly recognized for their crucial role in regulating vascular injury ...

Isolation and Characterization of Adult Cardiac Fibroblasts and Myofibroblasts

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that mitigate fibrosis. However, fibroblast research has been hindered due to the lack of universally acceptable fibroblast markers to identify quiescent as well as activated fibroblasts. Fibroblasts are a heterogenous cell population, making them difficult to isolate and characterize. The presented protocol describes three different methods to enrich fibroblasts and myofibroblasts from uninjured and injured mouse hearts. Using a standard and reliable protocol to isolate fibroblasts will enable the study of their roles in homeostasis as well as fibrosis modulation.

Obtaining a pure population of fibroblasts is crucial to studying their role in wound repair and fibrosis. Described here is a detailed method to isolate fibroblasts and myofibroblasts from uninjured and injured mouse hearts followed by characterization of their purity and functionality by immunofluorescence, RTPCR, fluorescence-assisted cell sorting, and collagen gel contraction.

成人心脏纤维细胞和肌纤维细胞的分离与表征

Cardiac fibrosis in response to injury is a physiological response to wound healing. Efforts have been made to study and target fibroblast subtypes that ...

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

摘要

探索更多视频

脂肪库专用SCA1的免疫磁性分离

异基因骨髓间充质干细胞植入兔子的膝关节软骨缺损治疗纤维蛋白凝块

分离和富集大鼠间质干细胞（MSCS）及衍生的干细胞分离单细胞集落

3D磁干细胞聚集和生物反应器为成熟软骨再生

从小型猪关节液和骨髓间充质干细胞的分离和细胞表型

CD146的分离

从小鼠中免疫磁性分离血管壁驻留的 CD34⁺ 干细胞

从小鼠中免疫磁性分离血管壁驻留的 CD34⁺ 干细胞

从小鼠中免疫磁性分离血管壁驻留的 CD34⁺ 干细胞

成人心脏纤维细胞和肌纤维细胞的分离与表征