作者感谢墨西哥社会保障研究所（IMSS）和墨西哥儿童医院，Federico Gomez（HIMFG）和IMSS研究协调的Bioterio工作人员为开展该项目提供的支持。我们感谢国家科学技术委员会提供的AOC（815290）奖学金和安东尼奥·杜阿尔特·雷耶斯在视听材料方面的技术支持。

所有实验程序均按照墨西哥动物护理指南进行，该指南基于国际实验动物护理评估和认证协会（墨西哥官方规范NOM-062-200-1999，墨西哥）的建议。该协议由墨西哥社会研究所健康研究伦理委员会审查、批准和注册 （R-2021-785-092）。
1.通过手术切除从大鼠身上去除脂肪组织
<ol><li>用 20 mL 无菌 1x 磷酸盐缓冲盐水-抗生素抗真菌剂 （PBS-AA） 溶液（1 x PBS、庆大霉素 [20 μg/mL] 和两性霉素 [0.5 μg/mL]）制备两个锥形管，并保持在冰上。</li>
	<li>选择两只健康状况良好的成年雄性Sprague Dawley大鼠 （R. norvegicus）。 确保大鼠在3-4个月大之间，体重为350-450克。</li>
	<li>用20mg / kg盐酸甲苯噻嗪进行镇静，5分钟后，腹膜内给予剂量为120mg / kg盐酸氯胺酮的麻醉剂。用眼药膏润滑双眼以防止干燥。然后，通过缺乏踏板反射确认麻醉深度。</li>
	<li>用碘溶液剃须和消毒腹部和腹股沟区域。使用手术单以保持无菌。然后，从胸骨到睾丸区域做一个正中纵向切口。</li>
	<li>用无菌镊子去除附睾和肾脏周围的脂肪组织，并放入1x PBS-AA溶液中。将样品放在冰上。</li>
	<li>如果需要，根据机构指南缝合切口，并用40mg / kg戊巴比妥钠的心内剂量对动物实施安乐死。一旦确认死亡，按照机构程序处理尸体。 注意：死亡引发影响间充质细胞免疫表型、分化能力和旁分泌作用的信号传导机制。因此，组织收集是在安乐死之前进行的。</li>
</ol>2. 从脂肪组织中分离间充质细胞
<ol><li>在生物安全柜内用无菌镊子取出脂肪组织，并将其放在直径为100毫米的培养皿中的无菌滤纸上以吸收多余的PBS。</li>
	<li>将脂肪组织转移到另一个培养皿中，并使用10mL移液管用10mL的1x PBS-AA溶液进行三次洗涤，每次洗涤5分钟。</li>
	<li>使用剪刀和手术刀将组织机械分解成约1cm2 的碎片。</li>
	<li>在 20 mL 未补充的 Dulbecco 改良鹰培养基 （DMEM） 中制备 IV 型胶原酶 （0.075%）。通过0.22μm注射器过滤器过滤并保持在37°C。</li>
	<li>将步骤2.4中制备的胶原酶溶液（20mL）加入带有碎裂组织和磁棒的水晶烧杯中。密封容器并在37°C下缓慢和连续搅拌孵育。 注意：酶消化大约需要90分钟（保持缓慢搅拌以保持细胞膜的完整性）。</li>
	<li>通过不锈钢，40目，0.38mm孔径过滤器在100mm培养皿上过滤匀浆，并将细胞悬液收集在无菌的50mL锥形管中。</li>
	<li>加入 20 mL 温热的补充 DMEM（10% 胎牛血清 [FBS]、2 mM 谷氨酰胺、2 mM 丙酮酸钠、100 x 非必需氨基酸溶液和 100x 抗生素抗真菌溶液 [AA]）。小心地悬浮基质血管部分（SVE）。</li>
	<li>以 290 x g 离心细胞悬液 10 分钟，用 25 mL 移液管弃去上清液，并用 40 mL 未补充的 DMEM 培养基轻轻洗涤细胞。</li>
	<li>重复此步骤。在步骤之间使用新的无菌锥形管，以拖动最少量的细胞碎屑和脂肪。</li>
	<li>用 5 mL 移液管将沉淀悬浮在 5 mL 补充的 DMEM 中，并转移到 T-25 cm2 瓶中。在37°C和5%CO2下孵育24小时。</li>
	<li>第二天，用 5 mL 加热的 1x PBS-AA 进行三次洗涤，用未补充的 DMEM 进行两次洗涤，以去除细胞碎片和非贴壁细胞。用 5 mL 移液管加入 5 mL 新鲜、温热、补充的 DMEM，每 3-4 天更换一次培养基。</li>
</ol>3. ASC原代培养物的维持和扩展
<ol><li>一旦细胞达到90%-100%汇合（8±2天），用5mL未补充的DMEM洗涤两次。加入1mL加热的0.025%胰蛋白酶-2mM乙二胺四乙酸（EDTA），并在37°C孵育5-7分钟。</li>
	<li>当细胞单层分离时，加入 4 mL 补充的 DMEM 并轻轻分解细胞悬液。</li>
	<li>将细胞悬液转移到 15 mL 锥形管中，加入 5 mL 加热、补充的 DMEM，轻轻悬浮以匀浆。</li>
	<li>以290 x g 离心5分钟。弃去上清液，将沉淀轻轻混合在 1 mL 补充的 DMEM 中。用台盼蓝染色后，在Neubauer室中计数细胞。</li>
	<li>最后，在T-75烧瓶中以1：4的分流比接种2.5 x 103 细胞/cm 2 。此步骤可确保菌落形成和高增殖率。</li>
</ol>4. ASC原代培养物的形态学表征
<ol><li>在倒置显微镜下观察ASC的形态。 注意：在安装进行组织学和ASC鉴定之前，请用1%明胶溶液处理盖玻片。用紫外线照射15分钟。<ol><li>从70%乙醇溶液中取出盖玻片，并使其干燥5分钟。</li>
		<li>将盖玻片浸入无菌的 1% 明胶溶液中，沥干水分，并在室温 （RT） 下干燥。</li>
		<li>将每个盖玻片放入6孔板的每个孔中，并用紫外线照射板15分钟。</li>
		<li>在孔中心播种含有 25 x 103 个 细胞的液滴，等待 1 分钟，然后加入 1 mL 补充的 DMEM 培养基。在37°C和5%CO2下孵育96小时。</li>
		<li>取出培养基，并使用移液管用1x PBS-AA进行三次洗涤。</li>
		<li>向每个孔中加入 1 mL 3.5% 中性福尔马林，并在室温下孵育 1 小时。 小心地取出福尔马林并向每个孔中加入 1 mL 冷的 70% 乙醇。</li>
		<li>用封口膜密封板直到使用，以防止变干。 注意：ASC的细胞形态也通过不同传代期间的苏木精 - 伊红染色进行评估。</li>
		<li>从每个孔中取出70%乙醇，并用蒸馏水水合细胞5分钟。同时，过滤染色溶液。</li>
		<li>除去水，用哈里斯苏木精溶液缓慢搅拌染色细胞15分钟。</li>
		<li>取出染色溶液，用1x PBS进行两次洗涤。</li>
		<li>用伊红溶液缓慢搅拌1分钟对细胞进行染色。然后，取出溶液并用1x PBS进行两次洗涤。</li>
		<li>小心地从每个孔中取出盖玻片，并将载玻片安装在一滴PBS-甘油安装溶液（1：1 v / v）上。</li>
		<li>在盖玻片周围放置一行指甲油，并在光学显微镜下观察组织学载玻片。</li>
	</ol></li>
</ol>5. 免疫荧光对ASC的表达标志物
<ol><li>制备含有1x PBS-0.05%吐温20的洗涤缓冲液。在室温下进行洗涤并缓慢摇动。用 2 mL 缓冲液/孔洗涤板 3 次（每次洗涤孵育 3 分钟）。</li>
	<li>放入 1 mL 封闭试剂 （1：10） 并缓慢搅拌孵育 20 分钟。</li>
	<li>向每个孔中加入 200 μL（1：50 稀释度）以下一抗：CD9（单克隆小鼠）、CD34（多克隆兔）和抗 CD63（多克隆山羊）。在4°C孵育过夜。</li>
	<li>再次洗涤板三次，并与以下二抗的 200 μL（1：50 稀释液）孵育：抗小鼠 IgG FITC偶联山羊、驴抗山羊 IgG DyeLight 550 和山羊抗兔 IgG Cy3。</li>
	<li>用 1x PBS-0.05% 吐温 20 洗涤 3 次，并用 100 μL DRAQ-7（稀释：1 μL 在 1 mL 双蒸水中）复染细胞核 20 分钟。</li>
	<li>再清洗三次，然后按照步骤4.1.12-4.1.13安装载玻片。将样品避光保存并冷冻直至使用。</li>
	<li>使用本设备推荐的正确设置和软件在落射荧光共聚焦显微镜下可视化样品。</li>
</ol>6. 流式细胞术对ASC的表达标志物
<ol><li>从胰蛋白酶消化或解冻的细胞（子传代 3 或 4）中调整单细胞悬液，浓度为 1 x 106 细胞/mL 的 1x PBS-5% FBS。将 100 μL 与 100，000 个细胞分装在 1.5 mL 离心管中。使用一个不带标记的等分试样进行自发荧光对照。</li>
	<li>根据制造商的说明，添加所有适量的抗CD105 AF-594和抗CD31 AF-680。在4°C下在黑暗中孵育20分钟。</li>
	<li>用 1 mL 的 1x PBS-5% FBS 洗涤多余的污渍，以 250 x g 离心 5 分钟，并悬浮在 300 μL 1% 福尔马林中。</li>
	<li>在流式细胞仪上进行样品采集。细胞的门控策略基于FSC-A与SSC-A门控，单细胞使用SSC-H与SSC-A进行门控，然后FSC-A与FSC-H。</li>
	<li>将 Y610-mCherry-A 检测器用于 CD105 AF-594，将 R660-APC-A 检测器用于 CD31 AF-680。</li>
</ol>7. ASC与脂肪谱系的分化
<ol><li>在6孔板中，每cm 2接种1 x 104个细胞（P -4），并在37°C，5%CO2下孵育，直到60-80%汇合（~72-96小时）。诱导分化，直接应用于培养基：4 μM 胰岛素、1 μM 地塞米松 21-乙酸酯 （Dxa） 和 0.5 mM 3-异丁基-1-甲基黄嘌呤 （MIX）。每2-3天更换一次分化培养基。</li>
	<li>在2mL无菌超纯水中制备800μM胰岛素的储备溶液（加入20μL的1N HCl以确保完全溶解）并储存在-20°C。 加入 10 μL 胰岛素原液（先前以 1：100 稀释），并以每孔 2 mL 的最终体积施用 10 μL。</li>
	<li>在 5 mL 无菌超纯水中制备 1 mM Dxa 的储备溶液（请注意，不会发生完全溶解）。储存在4°C。 加入 40 μL/孔的 1：4 稀释度的 Dxa 储备液。</li>
	<li>在 2.5 mL 的 0.1 N NaOH 中制备 100 mM MIX 储备液，涡旋，然后加入 40 μL 1 N NaOH 以完全溶解。储存在-70°C。 加入 10 μL/孔的 MIX 储备液。</li>
	<li>储存前通过0.22μm无菌注射器过滤器过滤储备溶液。</li>
	<li>在第12天，用1x PBS洗涤细胞三次，并在室温下与500μL的4%福尔马林孵育1小时。 用蒸馏水洗涤每个孔两次，然后用60%异丙醇洗涤5分钟，然后晾干。</li>
	<li>加入在60%异丙醇（500μL/孔）中稀释的0.5%油红O，并在室温下孵育20分钟。 用1mL蒸馏水洗涤三次，并在倒置显微镜下观察。 注意：有关所需的试剂、设备和材料，请参阅 材料表。</li>
</ol>

脂肪组织来源的干细胞分离和表征

<ol>
	<li>Djian, P., Roncari, A. K., Hollenberg, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+\anatomic+site+and+age+on+the+replication+and+differentiation+of+rat+adipocyte+precursors+in+culture.">Influence of \anatomic site and age on the replication and differentiation of rat adipocyte precursors in culture.</a> The Journal of Clinical Investigation. 72 (4), 1200-1208 (1983).</li><li>Greenwood, M. R., Hirsch, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+development+of+adipocyte+cellularity+in+the+normal+rat.">Postnatal development of adipocyte cellularity in the normal rat.</a> Journal of Lipid Research. 15 (5), 474-483 (1974).</li><li>Gonz&#225;lez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Engineering of the cartilage tissue: application of mesenchymal stem cells derived from adipose tissue and bone marrow for use in cartilage tissue regeneration. , (2014).</li><li>Oedayrajsingh-Varma, M. 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=Adipose+tissue-derived+mesenchymal+stem+cell+yield+and+growth+characteristics+are+affected+by+the+tissue-harvesting+procedure.">Adipose tissue-derived mesenchymal stem cell yield and growth characteristics are affected by the tissue-harvesting procedure.</a> Cytotherapy. 8 (2), 166-177 (2006).</li><li>Sherman, L. S., Conde-Green, A., Naaldijk, Y., Lee, E. S., Rameshwar, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+enzyme-free+method+for+isolation+and+expansion+of+human+adipose-derived+mesenchymal+stem+cells.">An enzyme-free method for isolation and expansion of human adipose-derived mesenchymal stem cells.</a> Journal of Visualized Experiments. (154), e59419 (2019).</li><li>Cowan, C. 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=Adipose-derived+adult+stromal+cells+heal+critical-size+mouse+calvarial+defects.">Adipose-derived adult stromal cells heal critical-size mouse calvarial defects.</a> Nature Biotechnology. 22 (5), 560-567 (2004).</li><li>Alstrup, T., Eijken, M., Bohn, A. B., Moller, B., Damsgaard, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+adipose+tissue-derived+stem+cells:+enzymatic+digestion+in+combination+with+mechanical+distortion+to+increase+adipose+tissue-derived+stem+cell+yield+from+human+aspirated+fat.">Isolation of adipose tissue-derived stem cells: enzymatic digestion in combination with mechanical distortion to increase adipose tissue-derived stem cell yield from human aspirated fat.</a> Current Protocols in Stem Cell Biology. 48 (1), 68 (2019).</li><li>Gittel, 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=Isolation+of+equine+multipotent+mesenchymal+stromal+cells+by+enzymatic+tissue+digestion+or+explant+technique:+comparison+of+cellular+properties.">Isolation of equine multipotent mesenchymal stromal cells by enzymatic tissue digestion or explant technique: comparison of cellular properties.</a> BMC Veterinary Research. 9, 221 (2013).</li><li>Aliborzi, G., Vahdati, A., Mehrabani, D., Hosseini, S. E., Tamadon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+characterization+and+growth+kinetic+comparison+of+bone+marrow+and+adipose+tissue+mesenchymal+stem+cells+of+guinea+pig.">Isolation, characterization and growth kinetic comparison of bone marrow and adipose tissue mesenchymal stem cells of guinea pig.</a> International Journal of Stem Cells. 9 (1), 115-123 (2016).</li><li>Jurgens, W. 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=Effect+of+tissue-harvesting+site+on+yield+of+stem+cells+derived+from+adipose+tissue:+implications+for+cell-based+therapies.">Effect of tissue-harvesting site on yield of stem cells derived from adipose tissue: implications for cell-based therapies.</a> Cell and Tissue Research. 332 (3), 415-426 (2008).</li><li>Secunda, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+expansion+and+characterisation+of+mesenchymal+stem+cells+from+human+bone+marrow,+adipose+tissue,+umbilical+cord+blood+and+matrix:+a+comparative+study.">Isolation, expansion and characterisation of mesenchymal stem cells from human bone marrow, adipose tissue, umbilical cord blood and matrix: a comparative study.</a> Cytotechnology. 67 (5), 793-807 (2015).</li><li>Tholpady, S. S., Katz, A. J., Ogle, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+from+rat+visceral+fat+exhibit+multipotential+differentiation+in+vitro.">Mesenchymal stem cells from rat visceral fat exhibit multipotential differentiation in vitro.</a> The Anatomical Record. Part A, Discoveries in Molecular, Cellular, and Evolutionary Biology. 272 (1), 398-402 (2003).</li><li>Yoshimura, 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=Cell-assisted+lipotransfer+for+cosmetic+breast+augmentation:+supportive+use+of+adipose-derived+stem/stromal+cells.">Cell-assisted lipotransfer for cosmetic breast augmentation: supportive use of adipose-derived stem/stromal cells.</a> Aesthetic Plastic Surgery. 32 (1), 48-57 (2008).</li><li>Si, Z., 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=Adipose-derived+stem+cells:+Sources,+potency,+and+implications+for+regenerative+therapies.">Adipose-derived stem cells: Sources, potency, and implications for regenerative therapies.</a> Biomedicine &amp; Pharmacotherapy. 114, 108765 (2019).</li><li>Hammoud, S. H., AlZaim, I., Al-Dhaheri, Y., Eid, A. H., El-Yazbi, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perirenal+adipose+tissue+inflammation:+Novel+insights+linking+metabolic+dysfunction+to+renal+diseases.">Perirenal adipose tissue inflammation: Novel insights linking metabolic dysfunction to renal diseases.</a> Frontiers in Endocrinology. 12, 707126 (2021).</li><li>Liu, B. X., Sun, W., Kong, X. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perirenal+fat:+A+unique+fat+pad+and+potential+target+for+cardiovascular+disease.">Perirenal fat: A unique fat pad and potential target for cardiovascular disease.</a> Angiology. 70 (7), 584-593 (2019).</li><li>Lee, J., Han, D. J., Kim, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+differentiation+of+human+adipose+tissue-derived+stem+cells+into+cells+with+pancreatic+phenotype+by+regenerating+pancreas+extract.">In vitro differentiation of human adipose tissue-derived stem cells into cells with pancreatic phenotype by regenerating pancreas extract.</a> Biochemical and Biophysical Research Communications. 375 (4), 547-551 (2008).</li><li>Chandra, V., 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=Islet-like+cell+aggregates+generated+from+human+adipose+tissue+derived+stem+cells+ameliorate+experimental+diabetes+in+mice.">Islet-like cell aggregates generated from human adipose tissue derived stem cells ameliorate experimental diabetes in mice.</a> PLoS One. 6 (6), e20615 (2011).</li><li>Zuk, P. 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=Multilineage+cells+from+human+adipose+tissue:+implications+for+cell-based+therapies.">Multilineage cells from human adipose tissue: implications for cell-based therapies.</a> Tissue Engineering. 7 (2), 211-228 (2001).</li><li>Huang, S. J., Yang, W. S., Lin, Y. W., Wang, H. C., Chen, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increase+of+insulin+sensitivity+and+reversal+of+age-dependent+glucose+intolerance+with+inhibition+of+ASIC3.">Increase of insulin sensitivity and reversal of age-dependent glucose intolerance with inhibition of ASIC3.</a> Biochemical and Biophysical Research Communications. 371 (4), 729-734 (2008).</li><li>Jang, H. J., Cho, K. S., Park, H. Y., Roh, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+tissue-derived+stem+cells+for+cell+therapy+of+airway+allergic+diseases+in+mouse.">Adipose tissue-derived stem cells for cell therapy of airway allergic diseases in mouse.</a> Acta Histochemica. 113 (5), 501-507 (2011).</li><li>Haasters, 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=Morphological+and+immunocytochemical+characteristics+indicate+the+yield+of+early+progenitors+and+represent+a+quality+control+for+human+mesenchymal+stem+cell+culturing.">Morphological and immunocytochemical characteristics indicate the yield of early progenitors and represent a quality control for human mesenchymal stem cell culturing.</a> Journal of Anatomy. 214 (5), 759-767 (2009).</li><li>Zhang, 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=Identification+and+characterization+of+pig+adipose-derived+progenitor+cells.">Identification and characterization of pig adipose-derived progenitor cells.</a> Canadian Journal of Veterinary Research. 80 (4), 309-317 (2016).</li><li>Varma, M. 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=Phenotypical+and+functional+characterization+of+freshly+isolated+adipose+tissue-derived+stem+cells.">Phenotypical and functional characterization of freshly isolated adipose tissue-derived stem cells.</a> Stem Cells and Development. 16 (1), 91-104 (2007).</li><li>Palumbo, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+of+isolation,+characterization+and+expansion+of+human+adipose-derived+stem+cells+(ASCs):+An+overview.">Methods of isolation, characterization and expansion of human adipose-derived stem cells (ASCs): An overview.</a> International Journal of Molecular Sciences. 19 (7), 1897 (2018).</li><li>Yu, B., Zhang, X., Li, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+derived+from+mesenchymal+stem+cells.">Exosomes derived from mesenchymal stem cells.</a> International Journal of Molecular Sciences. 15 (3), 4142-4157 (2014).</li><li>Maumus, 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=Native+human+adipose+stromal+cells:+localization,+morphology,+and+phenotype.">Native human adipose stromal cells: localization, morphology, and phenotype.</a> International Journal of Obesity. 35 (9), 1141-1153 (2011).</li><li>Helmy, M. A., Mohamed, A. F., Rasheed, H. M., Fayad, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+primary+isolation+and+culture+of+adipose-derived+stem+cells+and+their+phenotypic+profile.">A protocol for primary isolation and culture of adipose-derived stem cells and their phenotypic profile.</a> Alexandria Journal of Medicine. 56 (1), 42-50 (2020).</li><li>He, Q., Ye, Z., Zhou, Y., Tan, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+of+mesenchymal+stem+cells+from+rat+bone+marrow+and+adipose+tissue.">Comparative study of mesenchymal stem cells from rat bone marrow and adipose tissue.</a> Turkish Journal of Biology. 42 (6), 477-489 (2018).</li></ol>

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自发现间充质干细胞以来的过去四十年中，几组研究人员已经描述了从不同组织和物种获得间充质干细胞的程序。使用大鼠作为动物模型的优点之一是它们易于维护和快速发育，以及易于从脂肪组织获得MSC。已经描述了用于获得ASC的不同组织来源，例如内脏，肾周，附睾和皮下脂肪12，13，14，15，16。...

间充质干细胞（MSCs）因其自我更新，增殖，迁移和分化为不同细胞谱系的高能力而强烈影响再生医学1，2。目前，大量的研究集中在它们治疗和诊断各种疾病的潜力上。
间充质细胞有不同的来源：骨髓、骨骼肌、羊水、毛囊、胎盘和脂肪组织等。它们是从不同的物种获得的，包括人类、小鼠、大鼠、狗和马3.骨髓来源的间充质干细胞（BMSC）多年来一直被用作再生医学中干细胞的主要来源，并作为胚胎干细胞使用的替代品4。然而，脂肪来源的MSCs或脂肪来源的干细胞（ASCs）是一种重要的替代品，由于其易于收集和分离，以及每克脂肪组织获得的细胞产量5，6，具有很大的优势。据报道，ASC的收获率普遍高于BMSCs7。最初提出ASC的修复/再生能力是由于它们分化成其他细胞谱系的能力8。然而，近年来的研究加强了ASC释放的旁分泌因子在其修复潜力中的主要作用9，10。
脂肪组织（AT）除了作为能量储备外，还与内分泌，神经和心血管系统相互作用。它还参与出生后的生长发育、维持组织稳态、组织修复和再生。AT 由脂肪细胞、血管平滑肌细胞、内皮细胞、成纤维细胞、单核细胞、巨噬细胞、淋巴细胞、前脂肪细胞和 ASC 组成。后者由于其低免疫原性而在再生医学中具有重要作用11，12。ASC可以通过酶消化和机械加工或通过脂肪组织外植体获得。ASC的原代培养物易于维持、生长和扩增。通过使用免疫荧光和流式细胞术等方法评估特定膜标志物的表达，ASC 的表型表征对于验证细胞的身份至关重要13.国际脂肪治疗与科学联合会（IFATS）和国际细胞治疗学会（ISCT）已经定义ASC表达CD73，CD90和CD105，而缺乏CD11b，CD14，CD19，CD45和HLA-DR14的表达。因此，这些阳性和阴性标志物被认为对ASC的表征是可靠的。
该项目的重点是描述从大鼠AT中提取的成年间充质细胞的分离和鉴定程序，因为与胚胎干细胞不同，这种细胞来源不存在伦理挑战。这巩固了该程序作为可行选择的地位，因为与骨髓来源的干细胞相比，该方法易于访问和微创方法。 
来自这种组织来源的间充质细胞因其免疫调节能力和低免疫排斥反应而在再生医学中起着重要作用。因此，本研究是未来研究其分泌组及其在不同疾病（包括糖尿病等代谢性疾病）中作为再生疗法应用的基础部分。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amphotericin B</td>
			<td>HyClone</td>
			<td>SV30078.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical balance</td>
			<td>Sartorius</td>
			<td>AX224</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody anti- CD9 (C-4)</td>
			<td>Santa Cruz</td>
			<td>Sc-13118</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody anti-CD34 (C-18)</td>
			<td>Santa Cruz</td>
			<td>Sc-7045</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody anti-C63</td>
			<td>Santa Cruz</td>
			<td>Sc-5275</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody anti-Endoglin/CD105 (P3D1) Alexa Fluor 594 </td>
			<td>Santa Cruz</td>
			<td>Sc-18838A594</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody anti-CD31/PECM-1 Alexa Fluor 680</td>
			<td>Santa Cruz</td>
			<td>Sc-18916AF680</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody Goat anti-rabitt IgG (H+L) Cy3 </td>
			<td>Novus</td>
			<td>NB 120-6939</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody Donkey anti-goat IgG (H+L) DyLight 550 </td>
			<td>Invitrogen</td>
			<td>SA5-10087</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody anti-mouse IgG FITC conjugated goat F (ab&#180;)</td>
			<td>RD Systems.</td>
			<td>No. F103B</td>
			<td></td>
		</tr>
		<tr>
			<td>Bottle Top Filter Sterile</td>
			<td>CORNING</td>
			<td>10718003</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell and Tissue Culture Flasks</td>
			<td>BIOFIL</td>
			<td>170718-312B</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Counter Bright-Line Hemacytometer with cell counting chamber slides</td>
			<td>SIGMA Aldrich </td>
			<td>Z359629</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell wells: 6 well with Lid</td>
			<td>CORNING</td>
			<td>25810</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge conical tubes</td>
			<td>HeTTICH</td>
			<td>ROTANA460R</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge eppendorf tubes</td>
			<td>Fischer Scientific</td>
			<td>M0018242_44797</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen IV </td>
			<td>Worthington</td>
			<td>LS004186</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryovial</td>
			<td>SPL Life Science</td>
			<td>43112</td>
			<td></td>
		</tr>
		<tr>
			<td>Culture tubes</td>
			<td>Greiner Bio-One </td>
			<td>191180</td>
			<td></td>
		</tr>
		<tr>
			<td>CytExpert 2.0</td>
			<td>Beckman Coulter</td>
			<td>Free version</td>
			<td></td>
		</tr>
		<tr>
			<td>CytoFlex LX cytometer</td>
			<td>Beckman Coulter</td>
			<td>FLOW-2463VID03.17</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>GIBCO</td>
			<td>31600-034</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>SIGMA Aldrich</td>
			<td> 67-68-5</td>
			<td></td>
		</tr>
		<tr>
			<td>DraQ7 Dye</td>
			<td>Thermo Sc. </td>
			<td>D15106</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>SIGMA Aldrich</td>
			<td>60-00-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin yellowish</td>
			<td>Hycel</td>
			<td>300</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 96%</td>
			<td>Baker</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon tubes 15 mL</td>
			<td>Greiner Bio-One </td>
			<td>188271</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon tubes 50 mL</td>
			<td>Greiner Bio-One </td>
			<td>227261</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum </td>
			<td>CORNING</td>
			<td>35-010-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>SIGMA Aldrich</td>
			<td>128111163</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>GIBCO</td>
			<td>15750045</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerin-High Purity</td>
			<td>Herschi Trading </td>
			<td>56-81-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>AMRESCO </td>
			<td>0701-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Heracell 240i CO2 Incubator </td>
			<td>Thermo Sc.</td>
			<td>50116047</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamin Pet (Ketamine clorhidrate)</td>
			<td>Aranda</td>
			<td>SV057430</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine </td>
			<td>GIBCO/ Thermo Sc. </td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>LSM software Zen 2009 V5.5</td>
			<td></td>
			<td>Free version</td>
			<td></td>
		</tr>
		<tr>
			<td>Biological Safety Cabinet Class II</td>
			<td>NuAire</td>
			<td>12082100801</td>
			<td></td>
		</tr>
		<tr>
			<td>Epifluorescent microscope </td>
			<td>Zeiss Axiovert 100M</td>
			<td>21.0028.001</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope </td>
			<td>Olympus CK40</td>
			<td>CK40-G100 </td>
			<td></td>
		</tr>
		<tr>
			<td>Non-essential amino acids 100X</td>
			<td>GIBCO</td>
			<td>11140050</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro tubes 2 mL </td>
			<td>Sarstedt </td>
			<td>72695400</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro tubes 1,5 mL</td>
			<td>Sarstedt </td>
			<td>72706400</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipettes 0.2-2 &#956;L</td>
			<td>Finnpipette</td>
			<td>E97743</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipettes 2-20 &#956;L</td>
			<td>Finnpipette</td>
			<td>F54167</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipettes 20-200 &#956;L</td>
			<td>Finnpipette</td>
			<td>G32419</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipettes 100-1000 &#956;L</td>
			<td>Finnpipette</td>
			<td>FJ39895</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrogen tank liquid</td>
			<td>Taylor-Wharton</td>
			<td>681-021-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>SIGMA Aldrich</td>
			<td>SLBC3029V</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin / Streptomycin </td>
			<td>GIBCO/ Thermo Sc. </td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish Cell culture</td>
			<td>CORNING Inc</td>
			<td>480167</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet Tips</td>
			<td>Axygen Scientific</td>
			<td>301-03-201</td>
			<td></td>
		</tr>
		<tr>
			<td>Pisabental (pentobarbital sodium)</td>
			<td>PISA Agropecuaria</td>
			<td>Q-7833-215</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride </td>
			<td>J.T.Baker</td>
			<td>7447-40-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate Dibasic</td>
			<td>J.T Baker</td>
			<td>2139900</td>
			<td></td>
		</tr>
		<tr>
			<td>S1 Pipette Fillers</td>
			<td>Thermo Sc</td>
			<td>9531</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette 5 mL</td>
			<td>PYREX</td>
			<td>L010005</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette 10 mL</td>
			<td>PYREX</td>
			<td>L010010</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>J.T Baker</td>
			<td>144-55-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride </td>
			<td>J.T.Baker</td>
			<td>15368426</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate Dibasic Anhydrous</td>
			<td>J.T Baker</td>
			<td>7558-79-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>GIBCO BRL</td>
			<td>11840-048</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe Filter Sterile</td>
			<td>CORNING</td>
			<td>431222</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>PerkinElmer Lambda 25</td>
			<td>L6020060</td>
			<td></td>
		</tr>
		<tr>
			<td>Titer plate shaker</td>
			<td>LAB-LINE</td>
			<td>1250</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipets</td>
			<td>Samco/Thermo Sc</td>
			<td>728NL</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue stain</td>
			<td>GIBCO</td>
			<td>1198566</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin From Porcine Pancreas</td>
			<td>SIGMA Aldrich</td>
			<td>102H0234</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>SIGMA Aldrich</td>
			<td>9005-64-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Universal Blocking Reagent 10x</td>
			<td>BioGenex</td>
			<td>HK085-GP</td>
			<td></td>
		</tr>
		<tr>
			<td>Xilapet 2% (xylazine hydrochloride)</td>
			<td>Pet&#39;s Pharma</td>
			<td>Q-7972-025</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation and identification of mesenchymal stem cells derived from adipose tissue of sprague dawley rats

近几十年来，成人间充质细胞彻底改变了分子和细胞生物学。它们可以分化成不同的特化细胞类型，此外还有强大的自我更新、迁移和增殖能力。脂肪组织是间充质细胞侵入性最小、最容易获得的来源之一。据报道，与其他来源相比，它具有更高的产量，以及优越的免疫调节特性。最近，已经发表了从不同组织来源和动物物种获得成年间充质细胞的不同程序。在评估了一些作者的标准后，我们标准化了一种适用于不同目的且易于复制的方法。来自肾周和附睾脂肪组织的基质血管分数（SVF）池使我们能够开发具有最佳形态和功能的原代培养物。观察到细胞粘附在塑料表面上24小时，并表现出成纤维细胞样形态，具有延长和形成集落的趋势。流式细胞术（FC）和免疫荧光（IF）技术用于评估膜标志物CD105，CD9，CD63，CD31和CD34的表达。脂肪来源的干细胞（ASCs）分化成成脂肪谱系的能力也使用多种因素（4μM胰岛素，0.5mM 3-甲基异丁基黄嘌呤和1μM地塞米松）进行评估。48小时后，观察到成纤维细胞样形态逐渐丧失，并且在12天时，确认存在对油红色染色阳性的脂滴。总之，提出了一种程序来获得最佳和功能性ASC培养物以应用于再生医学。

Mesenchymal stem cells (MSCs) have strongly impacted regenerative medicine due to their high capacity for self-renewal, proliferation, migration, and differentiation into different cell lineages1,2. Currently, a great deal of research is focusing on their potential for the treatment and diagnosis of various diseases.
There are different sources of mesenchymal cells: bone marrow, skeletal muscle, amniotic fluid, hair follicles, placenta, and adipose tissue, among others. They are obtained from different species, including humans, mice, rats, dogs, and horses3. Bone marrow-derived MSCs (BMSCs) have been used for many years as a major source of stem cells in regenerative medicine and as an alternative to the use of embryonic stem cells4. However, adipose-derived MSCs, or adipose-derived stem cells (ASCs), are an important alternative with great advantages due to their ease of collection and isolation, as well as the yield of cells obtained per gram of adipose tissue5,6. It has been reported that the harvest rate of ASCs is generally higher than that of BMSCs7. It was initially proposed that the reparative/regenerative capacity of ASCs was due to their ability to differentiate into other cell lineages8. However, research in recent years has reinforced the primary role of paracrine factors released by ASCs in their reparative potential9,10.
Adipose tissue (AT), in addition to being an energy reserve, interacts with the endocrine, nervous, and cardiovascular systems. It is also involved in postnatal growth and development, the maintenance of tissue homeostasis, tissue repair, and regeneration. The AT is composed of adipocytes, vascular smooth muscle cells, endothelial cells, fibroblasts, monocytes, macrophages, lymphocytes, preadipocytes, and ASCs. The latter possess an important role in regenerative medicine due to their low immunogenicity11,12. ASCs can be obtained by enzymatic digestion and mechanical processing or by adipose tissue explants. Primary cultures of ASCs are easy to maintain, grow, and expand. Phenotypic characterization of ASCs is essential to verify the identity of the cells by assessing the expression of specific membrane markers using methods such as immunofluorescence and flow cytometry13. The International Federation for Adipose Therapeutics and Science (IFATS) and the International Society for Cellular Therapy (ISCT) have defined that ASCs express CD73, CD90, and CD105, while lacking the expression of CD11b, CD14, CD19, CD45, and HLA-DR14. These markers, both positive and negative, are therefore considered reliable for the characterization of ASCs.
This project was focused on describing a procedure for the isolation and identification of adult mesenchymal cells extracted from rats' AT, as this source of cells does not present ethical challenges, unlike embryonic stem cells. This solidifies the procedure as a viable option because of the ease of access and minimally invasive method compared to bone marrow-derived stem cells. 
Mesenchymal cells from this tissue source have an important role in regenerative medicine because of their immunomodulatory capabilities and low immune rejection. Therefore, the present study is a fundamental part of future research into their secretome and their application as regenerative therapy in different diseases, including metabolic diseases such as diabetes.

作者聚焦：分离和鉴定源自 Sprague Dawley 大鼠脂肪组织的间充质干细胞  

斯普拉格道利大鼠脂肪组织间充质干细胞的分离和鉴定

Mesenchymal stem cells have a really broad role in cellular communication due to their paracrine function. This protocol obtains adipose-derived mesenchymal stem cells with morphological, immunophenotypical, and adequate functionality for use in studying therapies for pancreatic regeneration and diabetes. There has been increased use of cell therapies in the treatment of diabetes.These therapies include allogenic transplants of pancreatic cells, autologous stem cell-derived products of insulin-producing cells, and 3D-printed pancreatic tissue using biological inks. Most research primarily studied the secretome components and the role in the regulation of different cellular processes. New techniques like microarrays and next-generation sequencing can investigate the function of miRNAs.A challenge in this area is to achieve greater stimulation of mesenchymal cells, reaching precise molecular composition in the secretome, and directing it to the treatment of a specific conditions. We will be evaluating how an miRNAs contribute to pancreatic tissue degeneration exposed to oxidative stress. The study will involve usage of stem cells from adipose tissue.

脂肪组织取自3-4个月大的成年Sprague Dawley大鼠，体重为401±41g（几何平均值±SD）。附睾和肾周脂肪组织的平均值为3.8g对应于15次实验提取的分析。培养24小时后，细胞群仍然粘附在塑料表面上并表现出异质形态。第一次传代在8±2天实现，在总共8个实验中±0.6 x 106 个细胞的产量为1.4。直接明场观察（图1）和苏木精-伊红染色（图2）有助于用光学?...

Watch this Scientific Journal Video about Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats at JoVE.com

该协议描述了从Sprague Dawley大鼠中分离和鉴定脂肪组织来源的间充质干细胞（MSCs）的方法。

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in ...

间充质干细胞由于其旁分泌功能而在细胞通讯中具有非常广泛的作用。该方案获得具有形态学，免疫表型和足够功能的脂肪来源的间充质干细胞，用于研究胰腺再生和糖尿病的治疗方法。细胞疗法在糖尿病治疗中的使用有所增加。这些疗法包括胰腺细胞的同种异体移植、产生胰岛素的细胞的自体干细胞衍生产物以及使用生物墨水的 3D 打印胰腺组织。大多数研究主要研究分泌组成分及其在不同细胞过程调节中的作用。微阵列和下一代测序等新技术可以研究miRNA的功能。该领域的一个挑战是实现对间充质细胞的更大刺激，在分泌组中达到精确的分子组成，并将其引导到特定疾病的治疗。我们将评估miRNA如何导致暴露于氧化应激的胰腺组织变性。该研究将涉及使用来自脂肪组织的干细胞。

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Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats

1.8K 次观看.  Universidad Autónoma Metropolitana. 间充质干细胞由于其旁分泌功能而在细胞通讯中具有非常广泛的作用。该方案获得具有形态学，免疫表型和足够功能的脂肪来源的间充质干细胞，用于研究胰腺再生和糖尿病的治疗方法。细胞疗法在糖尿病治疗中的使用有所增加。这些疗法包括胰腺细胞的同种异体移植、产生胰岛素的细胞的自体干细胞衍生产物以及使用生物墨水的 3D 打印胰腺组织。大多数研究主要研究...