Name: isolation, culture, and differentiation of bone marrow stromal cells and osteoclast progenitors from mice
Uploaded: 2018-01-06T14:00:00.000+00:00
Duration: 8 min 7 s
Description: Watch this Scientific Journal Video about Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice at JoVE.com

这项工作得到了国家卫生研究院 (R01 AR061164-01A1) 的支持。

所有实验程序均由缅因州医学中心研究所的机构动物保育和使用委员会批准。
1. 收集管制备
<ol><li>用10% 胎牛血清和1% 的青霉素/链霉素补充最低必需培养基α (骨髓) 来制作培养基。</li>
	<li>在1.5 毫升离心管中放置100µL 骨髓培养基。3骨通常适合在一个单一的管, 所以准备相应。</li>
	<li>削减200µL 吸管的两端足够的技巧, 使他们适合在离心管。插入管内的提示, 确保盖子可以关闭, 然后把管子放在冰上。 注: 另外, 0.75 毫升离心管与两端切割可以插入到1.5 毫升离心管。</li>
</ol>2. 小鼠骨的收获
<ol><li>安乐的动物使用 CO2其次是颈椎脱位, 并喷洒70% 乙醇。 注意: 确保使用相同性别和最好年龄的动物。我们经常使用7岁到8周的动物。我们建议每组至少3只动物聚集细胞, 以获得更好的代表性和可再生的细胞数量。</li>
	<li>
将安乐死的老鼠放回解剖板上。用镊子在腹部创建一个皮帐篷。用无菌解剖剪刀做一个小切口 (大约1厘米)。剥去皮肤对四肢和脚从切口为了暴露下腹部和腿。
	<ol>
<li>在踝关节下面切掉脚。沿髋部的髂嵴切开, 将股骨头与髋分开。当老鼠仍然在它的背部, 做一个切口在中间髋分离两个髂骨。</li>
	</ol>
</li>
	<li>使用无绒抹布, 小心地从股骨、胫骨和髂骨中取出肌肉。 注意: 尽可能多地从骨骼中切割肌腱和肌肉, 从而使清洁变得更快, 更容易使用无绒抹布 (例如, kimwipes)。在操纵骨骼时要小心, 因为它们容易破裂, 而且它们的脆弱性可能随着某些基因型而增加。</li>
	<li>一旦所有的样品都被解剖, 把它们放在 PBS 的冰上, 并移动到一个无菌的文化遮光罩的其余步骤隔离。 注意: 尽可能多的工作菌会减少文化中污染的可能性。</li>
	<li>在骨头的近端和远端都做小切口 (大约1到2毫米), 然后把它们放在离心管内的切片尖端。 注: 确保切割足够的数量, 使骨髓可以离心出来;然而, 必须注意避免切割太多, 因为细胞的数量往往根据骨髓空间的接近而变化。</li>
	<li>十五年代在室温下离心 1万 x g。确保所有的骨髓都在1.5 毫升管的底部被冲洗和颗粒化, 而骨骼则在插入物内 (200 µL 吸管尖端或0.75 毫升离心管)。一旦所有的骨髓收集, 删除与骨头从收集管插入。 注: 如果所有的骨髓都被冲洗, 骨头就会呈白色。然而, 如果骨髓仍然存在于某些骨骼中, 请尝试再次切割末端和离心。</li>
</ol>3. 细胞悬浮
<ol><li>使用25克针, 拉动细胞颗粒向上和向下慢慢地打破了团簇, 并结合所有的样本到一个单一的15毫升锥形管。每500µL 增加10毫升的骨髓培养基。</li>
	<li>放置一个70µm 过滤器顶部的一个50毫升锥管, 并通过这个过滤器通过细胞悬浮, 以消除可能的骨碎片。</li>
</ol>4. 骨髓细胞混合种群的电镀和培养 (总 bmc)
注: 细胞培养在37° c 的孵化器与 5% CO2。
<ol><li>在区域性媒体中计算单元格数, 并将其放在 1.0 x 106单元格/cm2中。允许 72 h 混合区域性以供单元格附加。 注意: 我们建议在培养基中稀释细胞悬浮 20x, 再在台盼蓝中稀释, 然后再用例来计数细胞。对于7周龄的雄性 C57Bl6/J 小鼠, 我们通常会产生大约 50 x 106细胞的细胞数, 但年龄、性别和应变会极大地影响细胞数量。 注: 细胞混合种群应在培养井的底部附着。</li>
	<li>72小时后, 取出介质并将其替换为新介质。每隔一天继续更换媒体并检查70-100。当细胞达到 80-100% 70-100, 它们就可以分化。 注: 由于这种文化与骨髓基质细胞和干细胞混合, 它可以直接用于 osteoblastogenesis、成以及破。</li>
</ol>5. 骨髓基质干细胞的分裂群体的电镀和培养
注: 细胞培养在37° c 的孵化器与 5% CO2。
<ol><li>将一只老鼠从股骨、胫骨和髂骨中收集的所有细胞放入一个10厘米的培养皿中 (55 厘米2的培养区)。 注意: 当共用 2-3 C57BL/6J 小鼠时, 我们通常会在150厘米2烧瓶中将这种 "总" 的骨髓填充。</li>
	<li>经过 48 h 混合文化, 删除文化媒体 (包含换药干细胞)。如果要进行破的实验, 将换药细胞的这一种群放在一旁 (见7节), 如果没有, 就抽吸并丢弃这些细胞。</li>
	<li>小心地用 PBS 清洗盘子/烧瓶, 以免干扰附着的细胞。</li>
	<li>通过加入0.25% 的胰蛋白酶, 在37° c 下孵育, 将细胞介质加入细胞悬浮液中, 以去除胰蛋白酶, 并将其提升1-3 分钟。此时, 骨髓基质细胞, 现在已经解除, 可以计算 (如先前在步骤 4.1) 和电镀为未来的实验。</li>
	<li>计算电镀所需的细胞数。离心机所需的细胞悬浮体积在 1000 x g 为5分钟的室温。 注意: 细胞通常是基于 end-point 实验的电镀。例如, 如果将蛋白质/RNA 分离, 我们通常会在更高的密度 (〜 1.0-2.0 x 106细胞/井在 6-井板) 板块。</li>
	<li>去除介质, 重所需的培养基中的细胞颗粒。根据实验结果, 将细胞在培养基中, 并允许它们附着几天。当骨髓基质细胞到达 70-100, 进行分化。</li>
</ol>6. 骨髓基质干细胞的分化
<ol><li>
骨
	<ol>
<li>在 pbs 中加入800µL 1 米β甘油磷酸酯和 pbs 中的200µL 0.5 m 抗坏血酸, 骨髓培养基的99毫升, 使成骨细胞分化培养基 (参见步骤1.1 为骨髓文化媒介的构成)。<ol>
<li>一旦骨髓基质细胞的培养 (从步骤4.3 的总 bmc 或从步骤5.6 的黏附的骨髓基质) 是汇合的, 通过把培养基转化成成骨分化培养基, 将它们分化成成成成成的</li>
		</ol>
</li>
		<li>每隔一天更换一次差异化介质。细胞开始产生成矿的白色结节。用肉眼观察它们的底部的井眼;此过程可能在几天到3周内发生。<ol>
<li>为了评估 osteoblastogenesis, 执行碱性磷酸酶染色和/或冯科萨染色的水井, 如8节所述。 注意: 为了避免细胞单层的破坏, 通过倾斜板的角度来仔细地改变介质。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
脂肪
	<ol>
<li>通过混合90毫升 Dulbecco 修饰鹰培养基 (DMEM) 高糖, 10 毫升胎牛血清, 1 毫升青霉素/链霉素, 100 µL 20 毫米罗格列酮和100毫米胰岛素µL, 使脂肪细胞基质培养基。如果需要, 在分化实验期间, 将此介质存储在4° c 的周内。</li>
		<li>通过添加200µL 62.5 毫米 3-异丁基-1-嘌呤 (IBMX) 和25毫米地塞米松µL 1 毫升脂肪细胞基质培养基, 使脂肪细胞诱导培养基。</li>
		<li>一旦骨髓基质细胞的培养 (从步骤4.3 的总 bmc 或从步骤5.6 的黏附的骨髓基质) 是汇合的, 改变媒介对脂肪细胞诱导介质。考虑到这一天0的分化。</li>
		<li>48小时后, 取出培养基, 用脂肪细胞诱导培养基刷新培养基。</li>
		<li>4天, 切换到脂肪细胞基质介质。 注: 脂滴可以在2或4天初开始出现。</li>
		<li>在7天, 观察细胞积累了最大的脂滴和显示一个表型类似于成熟的脂肪。不要在这一点上文化, 因为它可能导致细胞死亡/支队。</li>
	</ol>
</li>
</ol>7. 干细胞分化为破骨细胞
<ol><li>将 NFKB 配体 (RANKL) 和15µL 25 µg/毫升的巨噬细胞集落刺激因子 (mCSF) 的50µL 25 µg/毫升的受体激活剂添加到25毫升的骨髓培养基中, 使破骨的分化培养基。</li>
	<li>一旦总 bmc (从步骤 4.3) 或换药细胞 (从步骤 5.2) 连接到井, 文化媒体可以转换成破骨分化介质。每隔一天补充分化介质。</li>
	<li>每天在显微镜下以10X 的倍率观察破骨细胞的培养。观察破骨细胞的融合和形成核, 通常在5-7 天的分化。</li>
</ol>8. 染色
<ol><li>
骨 注: 对于碱性磷酸酶 (ap) 染色, 我们经常使用 AP 染色试剂盒 (参见材料表) 根据指南。<ol>
<li>按照试剂盒制造商的指导方针进行 AP 染色。<ol>
<li>用 PBS 吸吸培养基和冲洗细胞。在 PBS 中用4% 甲醛 (粉煤灰) 固定细胞, 室温下12分钟。</li>
			<li>同时, 在 ap 染色试剂盒中加入500µL 亚硝酸钠至500µL FRV 碱性溶液, 并轻轻搅拌;让我们站2分钟添加22.5 毫升 dH2O 和500µL 萘酚为铋碱性。轻轻地混合。 注意: 粉煤灰是有毒的, 必须小心处理。</li>
			<li>一旦细胞固定, 冲洗与 PBS 和添加 AP 解决方案, 每井 (1 至2毫升)。用铝箔盖上, 并让污渍在室温下保护30分钟。吸气溶液, 用蒸馏水冲洗, 让盘子晾干。</li>
		</ol>
</li>
		<li>利用摄像机获取染色菌落的图像, 以观察碱性磷酸酶阳性细胞。</li>
		<li>交替执行冯科萨而不是碱性磷酸酶染色。为此, 用蒸馏水彻底冲洗水井, 因为任何剩余的 PBS 都会与硝酸银沉淀成.
		<ol>
<li>在室温下, 在 PBS 中用4% 的粉煤灰固定细胞12分钟。然后, 添加足够的5% 硝酸银溶液覆盖井, 并暴露在室温下1小时强光。</li>
		</ol>
</li>
		<li>除去硝酸银溶液, 用蒸馏水冲洗每一个井。在细胞中加入5% 的硫代硫酸钠溶液, 并让3分钟。</li>
		<li>抽出硫代硫酸钠, 用蒸馏水冲洗。让盘子晾干, 用肉眼观察深棕色/黑色的矿床。</li>
	</ol>
</li>
	<li>
脂肪
	<ol>
<li>为油红色 O (破产) 染色, 固定在室温下使用10% 中性缓冲福尔马林1小时的细胞。</li>
		<li>同时, 通过在10毫升的异丙醇中溶解0.035 克, 使该公司的库存解决方案。这是足够的6井板。使用一张滤纸 (孔大小11µm) 混合并传递解决方案。</li>
		<li>用6毫升蒸馏水稀释9毫升的破产解决方案, 使破产处理署的工作解决办法。把溶液放在摇杆上, 直到准备好使用。</li>
		<li>在蒸馏水中制备60% 异丙醇 (使其足够覆盖每一个井)。</li>
		<li>一旦细胞修复后, 取出定影液, 用蒸馏水冲洗一次。用60% 异丙醇溶液代替水1分钟。 注: 在向盘中添加液体时要缓慢;脂肪细胞很容易脱落。</li>
		<li>取出异丙醇, 并添加足够的破产解决方案, 以覆盖所有的细胞。室温下在低速摇杆上孵育15分钟。用蒸馏水冲洗两次。 注意: 在这一点上, 在显微镜下可以直观地看到由破产人染色的脂滴。</li>
		<li>为量化破产人, destain 在每口井加1毫升100% 异丙醇, 并缓慢摇动10分钟。</li>
		<li>同时, 准备一个稀释系列, 根据以下8标准, 使用破产解决方案 (2100 µg/毫升) 和异丙醇作为稀释剂来创建标准曲线: 500、350、300、250、150、100、75和0µg/毫升。</li>
		<li>装载200µL 标准和脱色样品在 triplicates 在板材和读吸光度在490毫微米。<ol>
<li>根据标准曲线确定每个样本的破产浓度。 注意: 我们建议将浓度的变化归入细胞数。细胞数可以通过结晶紫染色或 DNA 数量来量化。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
破 注: 当准备好时, 我们建议使用诱捕染色试剂盒 (请参见材料表) 来染色破骨细胞的抗酒石酸酸性磷酸酶 (trap)。<ol>
<li>对于陷阱染色, 在室温下固定细胞30分钟, 2.5% 戊二醛在 PBS。同时, 将50µL 快速石碱溶液、50µL 亚硝酸钠、4.55 毫升蒸馏水、50µL Napthol 为氢、200µL 醋酸酯溶液和100µL 酒石酸溶液混合, 使疏水阀解决方案。</li>
		<li>在37° c 时, 在1小时内加入诱捕液, 将固定剂抽出, 用 PBS 冲洗两次细胞。</li>
		<li>用蒸馏水冲洗, 然后在显微镜下计数破骨细胞 (放大倍数 10X)。陷阱+单元格显示为紫色, 具有3或更多的核。</li>
	</ol>
</li>
</ol>

<ol>
	<li>Friedenstein, A. J., Latzinik, N. W., Grosheva, A. G., Gorskaya, U. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Marrow+microenvironment+transfer+by+heterotopic+transplantation+of+freshly+isolated+and+cultured+cells+in+porous+sponges.">Marrow microenvironment transfer by heterotopic transplantation of freshly isolated and cultured cells in porous sponges.</a> Exp Hematol. 10 (2), 217-227 (1982).</li><li>Chen, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+growth+and+differentiation+of+osteoprogenitors+in+mouse+bone+marrow+stromal+cell+cultures+by+increased+donor+age+and+glucocorticoid+treatment.">Inhibition of growth and differentiation of osteoprogenitors in mouse bone marrow stromal cell cultures by increased donor age and glucocorticoid treatment.</a> Bone. 35 (1), 83-95 (2004).</li><li>Kokabu, S., 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=BMP3+suppresses+osteoblast+differentiation+of+bone+marrow+stromal+cells+via+interaction+with+Acvr2b.">BMP3 suppresses osteoblast differentiation of bone marrow stromal cells via interaction with Acvr2b.</a> Mol Endocrinol. 26 (1), 87-94 (2012).</li><li>Ghali, O., 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=Dexamethasone+in+osteogenic+medium+strongly+induces+adipocyte+differentiation+of+mouse+bone+marrow+stromal+cells+and+increases+osteoblast+differentiation.">Dexamethasone in osteogenic medium strongly induces adipocyte differentiation of mouse bone marrow stromal cells and increases osteoblast differentiation.</a> BMC Cell Biol. 16, 9 (2015).</li><li>Kretlow, J. D., 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=Donor+age+and+cell+passage+affects+differentiation+potential+of+murine+bone+marrow-derived+stem+cells.">Donor age and cell passage affects differentiation potential of murine bone marrow-derived stem cells.</a> BMC Cell Biol. 9, 60 (2008).</li><li>DeMambro, V. 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=Igfbp2+Deletion+in+Ovariectomized+Mice+Enhances+Energy+Expenditure+but+Accelerates+Bone+Loss.">Igfbp2 Deletion in Ovariectomized Mice Enhances Energy Expenditure but Accelerates Bone Loss.</a> Endocrinology. 156 (11), 4129-4140 (2015).</li><li>Maridas, D. 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=IGFBP-4+regulates+adult+skeletal+growth+in+a+sex-specific+manner.">IGFBP-4 regulates adult skeletal growth in a sex-specific manner.</a> J Endocrinol. 233 (1), 131-144 (2017).</li><li>Nadri, S., 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=An+efficient+method+for+isolation+of+murine+bone+marrow+mesenchymal+stem+cells.">An efficient method for isolation of murine bone marrow mesenchymal stem cells.</a> Int J Dev Biol. 51 (8), 723-729 (2007).</li><li>Sreejit, P., Dilip, K. B., Verma, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+mesenchymal+stem+cell+lines+from+murine+bone+marrow.">Generation of mesenchymal stem cell lines from murine bone marrow.</a> Cell Tissue Res. 350 (1), 55-68 (2012).</li><li>Abdallah, B. 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=CD34+defines+an+osteoprogenitor+cell+population+in+mouse+bone+marrow+stromal+cells.">CD34 defines an osteoprogenitor cell population in mouse bone marrow stromal cells.</a> Stem Cell Res. 15 (3), 449-458 (2015).</li><li>Akiyama, K., 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+bone+marrow+derived+mesenchymal+stem+cells+in+suspension.">Characterization of bone marrow derived mesenchymal stem cells in suspension.</a> Stem Cell Res Ther. 3 (5), 40 (2012).</li><li>Lin, C. S., Ning, H., Lin, G., Lue, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+CD34+truly+a+negative+marker+for+mesenchymal+stromal+cells?.">Is CD34 truly a negative marker for mesenchymal stromal cells?.</a> Cytotherapy. 14 (10), 1159-1163 (2012).</li></ol>

作者透露, 他们没有金融利益冲突。

本文介绍了两种骨髓基质细胞的培养方法及其优点和局限性。从骨髓中分离出来的细胞是一个相对轻松的过程。然而, 由于骨髓腔的细胞环境的多样性, 获得细胞间充质干细胞或破祖先的代表可能具有挑战性。
培养完整的骨髓的内容提供了一个密切的表示,在体内微环境。然而, 分离细胞从不同的小组小鼠 (基因型, 治疗, 年龄,等) 可能导致一个非常不同数量的骨髓?...

小鼠骨髓间充质干细胞在二十世纪八十年代早期的1中被发现, 通常被作为一个体外细胞模型。事实上, 从长骨的骨髓腔中冲出的塑料黏附细胞的培养, 在许多研究中保持着分化成成骨、破骨、软骨或脂肪的能力2,3,4,5. 然而, 骨髓是一种独特的组织, 由许多不同的细胞组成, 包括但不限于骨髓基质干细胞、干细胞、内皮细胞和免疫干细胞。因此, 分离和培养技术可以产生不同均匀性的细胞群。使用这种技术来测试细胞的分化潜能可能是有挑战性的。例如, 当比较不同基因型的小鼠的细胞时, 从混合细胞的数量开始限制对数据的解释。相反地, 获得骨髓基质干细胞和干细胞的同源种群在技术上是困难的, 可能不代表前体模型。
在我们的实验室里, 我们主要关注骨髓基质干细胞的利用, 因为它们有可能被分化成成骨细胞、破骨和脂肪。在这里, 我们提出了骨髓基质干细胞和干细胞分离和培养技术, 用于评估 osteoblastogenesis 或成的体外, 以及干细胞的培养, 以分化为破骨细胞。一种方法是利用混合的骨髓细胞 (bmc), 其中含有骨髓基质干细胞和干细胞直接适合成, osteoblastogenesis 和破 (称为总 bmc)。这种方法更接近于在骨髓微环境的细胞中发现的异质性的活体。另一种方法是将黏附在换药细胞中, 试图培养 "更纯净" 的骨髓基质干细胞和干细胞 (称为黏附骨髓基质干细胞) 的种群。后来的方法允许细胞培养实验开始与更准确的数量的骨髓基质干细胞或干细胞, 并降低潜在的复杂的间接影响的其他细胞的人口留在文化中。这两种方法以前都已发布, 用于解决不同的研究问题6,7,8,9。

<table><tbody><tr><td>200&amp;mu;L pipet tips&amp;nbsp;</td><td>Rainin</td><td>17014401</td><td></td></tr><tr><td>1.5 mL centrifuge tubes</td><td>USA Scientific</td><td>1615-5500</td><td></td></tr><tr><td>15 mL conical tube&amp;nbsp;</td><td>VWR</td><td>89039-668</td><td></td></tr><tr><td>50 mL conical tube</td><td>VWR</td><td>89039-660</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>21-040-CM</td><td></td></tr><tr><td>Ethanol</td><td>Fisher Science</td><td>04-355-451</td><td></td></tr><tr><td>Dissection tools</td><td></td><td></td><td></td></tr><tr><td>70 &amp;mu;m filters</td><td>BD Falcon</td><td>352350</td><td></td></tr><tr><td>0.25% trypsin</td><td>Gibco</td><td>25200</td><td></td></tr><tr><td>Kimwipe</td><td>VWR</td><td>82003-820</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Electron Microscopy Science</td><td>15710</td><td></td></tr><tr><td>Alkaline Phosphatase kit</td><td>Sigma-Aldrich</td><td>86R</td><td></td></tr><tr><td>Silver Nitrate</td><td>Sigma-Aldrich</td><td>S6506</td><td></td></tr><tr><td>Sodium Thiosulfate</td><td>Sigma-Aldrich</td><td>S7026</td><td></td></tr><tr><td>Oil Red O</td><td>Sigma-Aldrich</td><td>O0625</td><td></td></tr><tr><td>Isopropanol</td><td>Sigma-Aldrich</td><td>190764</td><td></td></tr><tr><td>10% Neutral Buffered Formalin</td><td>Sigma-Aldrich</td><td>F5554-4L</td><td></td></tr><tr><td>Whatman filter Grade 1</td><td>Sigma-Aldrich</td><td>Z274852</td><td></td></tr><tr><td>Tartrate-Resistant Acid Phosphatase kit</td><td>Sigma-Aldrich</td><td>387A</td><td></td></tr><tr><td>Glutaraldehyde</td><td>Electron</td><td>16220</td><td></td></tr><tr><td>MEM&amp;alpha;</td><td>Gibco</td><td>12571</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>VWR</td><td>97068-085</td><td></td></tr><tr><td>Penicillin/Streptomycin</td><td>Invitrogen</td><td>15140-122</td><td></td></tr><tr><td>&amp;beta;-glycerol phosphate</td><td>Sigma-Aldrich</td><td>G9891</td><td></td></tr><tr><td>Ascorbic acid</td><td>Sigma-Aldrich</td><td>A4544</td><td></td></tr><tr><td>DMEM High Glucose&amp;nbsp;</td><td>Sigma-Aldrich</td><td>D5796</td><td></td></tr><tr><td>Rosiglitazone</td><td>Cayman Chemical</td><td>717410</td><td></td></tr><tr><td>Insulin</td><td>Sigma-Aldrich</td><td>I6634</td><td></td></tr><tr><td>IBMX</td><td>Sigma-Aldrich</td><td>I5879</td><td></td></tr><tr><td>Dexamethasone</td><td>Sigma-Aldrich</td><td>D4902</td><td></td></tr><tr><td>RANKL</td><td>Peprotech</td><td>310-01</td><td></td></tr><tr><td>mCSF</td><td>Peprotech</td><td>315-02</td><td></td></tr><tr><td>Axio Observer inverter microscope</td><td>Zeiss</td><td></td><td></td></tr></tbody></table>

isolation, culture, and differentiation of bone marrow stromal cells and osteoclast progenitors from mice

骨髓基质细胞是一种细胞群, 通常用于骨髓间充质干细胞的表达,体外.他们居住在骨髓腔内的造血干细胞 (干细胞), 它可以产生红细胞, 免疫祖细胞和破骨。因此, 从骨髓中提取细胞的数量会导致不同细胞群的混杂, 从而在实验设计和混淆数据解释方面提出挑战。为了获得更多或较不均匀的骨髓基质干细胞和干细胞体, 实验室开发了几种分离和培养技术。在这里, 我们提出两种方法分离骨髓基质干细胞和干细胞从小鼠长骨: 一种方法, 产生混合的人口骨髓基质干细胞和干细胞和一种方法, 试图分离的两个细胞的人口为基础的坚持。这两种方法都提供适合于成骨和脂分化实验的细胞以及功能性的化验。

细胞培养概述 这两种培养技术允许对由骨髓基质细胞和干细胞 (总 bmc,图 1A) 或由干细胞 (黏附的骨髓基质细胞,图 1B) 分裂的骨髓基质干细胞的混合种群的 bmc 进行鉴别。48小时在细胞从骨髓被隔绝了并且被镀 (图 1C) 之后, 他们迅速地附有在塑料文化板材的底部并且倾向于形成单层。一旦细胞?...

Watch this Scientific Journal Video about Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice at JoVE.com

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

本文介绍了从小鼠长骨分离分化骨髓基质细胞和造血干细胞的方法。两种不同的协议, 产生不同的细胞群体, 适合扩展和分化成成骨细胞, 脂肪和破骨。

小鼠骨髓基质细胞与骨破骨祖的分离、培养及分化

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

isolation-culture-differentiation-bone-marrow-stromal-cells

Research

JoVE Journal

Developmental Biology

30.8K 次观看.  Maine Medical Center Research Institute. 本文介绍了从小鼠长骨分离分化骨髓基质细胞和造血干细胞的方法。两种不同的协议, 产生不同的细胞群体, 适合扩展和分化成成骨细胞, 脂肪和破骨。

视频: Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Stimulation of Notch Signaling in Mouse Osteoclast Precursors

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro investigation of its varying and sometimes contradictory roles a challenge. As a component of direct cell-cell communication with both receptors and ligands bound to the plasma membrane, Notch signaling cannot be activated in vitro by simple addition of ligands to culture media, as is possible with many other signaling pathways. Instead, Notch ligands must be presented to cells in an immobilized state. 
Variations in methods of Notch signaling activation can lead to different outcomes in cultured cells. In osteoclast precursors, in particular, differences in methods of Notch stimulation and osteoclast precursor culture and differentiation have led to disagreement over whether Notch signaling is a positive or negative regulator of osteoclast differentiation. While closer comparisons of osteoclast differentiation under different Notch stimulation conditions in vitro and genetic models have largely resolved the controversy regarding Notch signaling and osteoclasts, standardized methods of continuous and temporary stimulation of Notch signaling in cultured cells could prevent such discrepancies in the future.
This protocol describes two methods for stimulating Notch signaling specifically in cultured mouse osteoclast precursors, though these methods should be applicable to any adherent cell type with minor adjustments. The first method produces continuous stimulation of Notch signaling and involves immobilizing Notch ligand to a tissue culture surface prior to the seeding of cells. The second, which uses Notch ligand bound to agarose beads allows for temporary stimulation of Notch signaling in cells that are already adhered to a culture surface. This protocol also includes methods for detecting Notch activation in osteoclast precursors as well as representative transcriptional markers of Notch signaling activation.

Notch signaling is a form of cellular communication that relies upon direct contact between cells. To properly induce Notch signaling in vitro, Notch ligands must be presented to cells in an immobilized state. This protocol describes methods for in vitro stimulation of Notch signaling in mouse osteoclast precursors.

在鼠标破骨细胞前体Notch信号的刺激

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro ...

科研

发育生物学

Identification and Isolation of Oligopotent and Lineage-committed Myeloid Progenitors from Mouse Bone Marrow

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for hematological and immunological analyses. For example, studies of the cellular and molecular properties of myeloid progenitor populations can reveal mechanisms underlying leukemic transformation, or demonstrate how the immune system responds to pathogen exposure. Previously described flow cytometry strategies for myeloid progenitor identification have enabled significant advances in many fields, but the fractions they identify are very heterogeneous. The most commonly used gating strategies define bone marrow fractions that are enriched for the desired populations, but also contain large numbers of &#34;contaminating&#34; progenitors. Our recent studies have resolved much of this heterogeneity, and the protocol we present here permits the isolation of 6 subpopulations of oligopotent and lineage-committed myeloid progenitors from 2 previously described bone marrow fractions. The protocol describes 3 stages: 1) isolation of bone marrow cells, 2) enrichment for hematopoietic progenitors by magnetic-activated cell sorting (lineage depletion by MACS), and 3) identification of myeloid progenitor subsets by flow cytometry (including fluorescence-activated cell sorting, FACS, if desired). This approach permits progenitor quantification and isolation for a variety of in vitro and in vivo applications, and has already yielded novel insight into pathways and mechanisms of neutrophil, monocyte, and DC differentiation.

We demonstrate how to identify and isolate 6 subsets of myeloid progenitors from murine bone marrow using a combination of magnetic and fluorescence sorting (MACS and FACS). This protocol can be used for in vitro culture assays (methylcellulose or liquid cultures), in vivo adoptive transfer experiments, and RNA/protein analyses.

小鼠骨髓 Oligopotent 和世系髓样祖细胞的鉴定与分离

Myeloid progenitors that yield neutrophils, monocytes and dendritic cells (DCs) can be identified in and isolated from the bone marrow of mice for ...

免疫与感染

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

表型分析和分离小鼠造血干细胞和传承的承诺祖

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

生物学

Economical and Efficient Protocol for Isolating and Culturing Bone Marrow-derived Dendritic Cells from Mice

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method for isolating DCs primarily involves inducing bone marrow (BM) differentiation into DCs by injecting large doses (&#62;10 ng/mL) of granulocyte-macrophage colony-stimulating factor/interleukin-4 (GM-CSF/IL-4), making the procedure complex and expensive. In this protocol, using all BM cells cultured in 10 ng/mL GM-CSF/IL-4 medium, after 3-4 half-culture exchanges, up to 2.7 x 107 CD11c+ cells&#160;(DCs) per mouse (two femurs) were harvested with a purity of 80%-95%. After 10 days in culture, the expression of CD11c, CD80, and MHC II increased, whereas the number of cells decreased. The number of cells peaked after 7 days of culture. Moreover, this method only took 10 min to harvest all bone marrow cells, and a high number of DCs were obtained after 1 week of culture.

Here, we present an economical and efficient method to isolate and generate high-purity bone marrow-derived dendritic cells from mice after 7 days of culture with 10 ng/mL GM-CSF/IL-4.

从小鼠中分离和培养骨髓衍生树突状细胞的经济有效的方案

The demand for dendritic cells (DCs) is gradually increasing as immunology research advances. However, DCs are rare in all tissues. The traditional method ...

Isolation and Culture of Bone Marrow-Derived Macrophages from Mice

Macrophages have important effector functions in homeostasis and inflammation. These cells are present in every tissue in the body and have the important ability to change their profile according to the stimuli present in the microenvironment. Cytokines can profoundly affect macrophage physiology, especially IFN-&#947; and interleukin 4, generating M1 and M2 types respectively. Because of the versatility of these cells, the production of a population of bone marrow-derived macrophages can be a basic step in many experimental models of cell biology. The aim of this protocol is to help researchers in the isolation and culture of macrophages derived from bone marrow progenitors. Bone marrow progenitors from pathogen-free C57BL/6 mice are transformed into macrophages upon exposure to macrophage colony-stimulating factor (M-CSF) that, in this protocol, is obtained from the supernatant of the murine fibroblast lineage L-929. After incubation, mature macrophages are available for use from the 7th to the 10th day. A single animal can be the source of approximately 2 x 107 macrophages. Therefore, it is an ideal protocol for obtaining large amounts of primary macrophages using basic methods of cell culture.

The present protocol describes the isolation and culture of bone marrow-derived macrophages from mice.

从小鼠中分离和培养骨髓来源的巨噬细胞

Macrophages have important effector functions in homeostasis and inflammation. These cells are present in every tissue in the body and have the important ...

优化从胸腺和骨髓中分离基质细胞

Stromal Cell Isolation From Hematopoietic Organs

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens through which to study previously unresolved heterogeneity at steady state, as well as changes in cell type representation induced by extrinsic stresses or during aging. Here, we present step-wise protocols for the isolation of high-quality stromal cell populations from murine and human thymus, as well as murine bone and bone marrow. Cells isolated through these protocols are suitable for generating high-quality single-cell multiomics datasets. The impacts of sample digestion, hematopoietic lineage depletion, FACS analysis/sorting, and how these factors influence compatibility with single-cell sequencing are discussed here. With examples of FACS profiles indicating successful and inefficient dissociation and downstream stromal cell yields in post-sequencing analysis, recognizable pointers for users are provided. Considering the specific requirements of stromal cells is crucial for acquiring high-quality and reproducible results that can advance knowledge in the field.

作者聚焦：使用基质细胞分离进行单细胞测序推进造血研究

Here we present protocols that enable isolation of stromal cells from murine bone, bone marrow, thymus and human thymic tissue compatible with single-cell multiomics.

从造血器官中分离基质细胞

Single-cell sequencing has enabled the mapping of heterogeneous cell populations in the stroma of hematopoietic organs. These methodologies provide a lens ...

Osteoclast Derivation from Mouse Bone Marrow

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease.
As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation.
An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases. 
Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts.

Osteoclasts are the principal bone-resorbing cell in the body. An ability to isolate osteoclasts in large numbers has resulted in significant advances in the understanding of osteoclast biology. In this protocol, we describe a method for isolation, cultivating and quantifying osteoclast activity in vitro.

小鼠骨髓破骨细胞的推导

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the ...

Isolation, Purification, and Differentiation of Osteoclast Precursors from Rat Bone Marrow

Osteoclasts are large, multinucleated, and bone-resorbing cells of the monocyte-macrophage lineage that are formed by the fusion of monocytes or macrophage precursors. Excessive bone resorption is one the most significant cellular mechanisms leading to osteolytic diseases, including osteoporosis, periodontitis, and periprosthetic osteolysis. The main physiological function of osteoclasts is to absorb both the hydroxyapatite mineral component and the organic matrix of bone, generating the characteristic resorption appearance on the surface of bones. There are relatively few osteoclasts compared to other cells in the body, especially in adult bones. Recent studies have focused on how to obtain more mature osteoclasts in less time, which has always been a problem. Several improvements in the isolation and culture techniques have developed in laboratories in order to obtain more mature osteoclasts. Here, we introduce a method that isolates bone marrow in less time and with less effort compared to the traditional procedure, using a special and simple device. With the use of density gradient centrifugation, we obtain large amounts of fully differentiated osteoclasts from rat bone marrow, which are identified by classical methods.

Osteoclasts are tissue-specific macrophage polykaryons derived from the monocyte-macrophage lineage of hematopoietic stem cells. This protocol describes how to isolate bone marrow cells so that large quantities of osteoclasts are obtained while reducing the risk of accidents found in traditional methods.

小鼠骨髓基质细胞与骨破骨祖的分离、培养及分化

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