参数的优化用于再生医学疗法更有效地利用脂肪干细胞

抽象

脂肪组织来源的干细胞（ASCs）关联到脂肪中的自体脂肪移植是有希望用于更有效的软组织重建，并且非常需要的协议来从脂肪抽吸物分离脂肪的ASC优化。我们表明，增加脂肪细胞分化是依赖于先进的结构陶瓷的数量。在10个供体的样品中，我们发现有核细胞的下腹部更高的浓度相比，侧翼（）。在6个供体的样品，我们没有发现在通过手动或泵辅助抽吸获得的细胞产量（差异）。我们建议，在重新注入脂肪的ASC数量的增加可以提高新形成的脂肪组织的效率，并从其中收获的脂肪组织需要解剖区域被视为以优化收获的组织的ASC的数量。最后，泵辅助愿望可以在没有任何显著损害细胞的活力使用。

1。Introduction

Fat transfer has been used over the past decades as an autologous dynamic filler in plastic surgery rehabilitation in several circumstances, such as in reconstruction of damaged adipose tissue due to burn injury, for craniofacial reconstruction due to congenital defects or trauma, in cancer or other tumors as well as for aesthetic reasons. Although this approach is considered successful, the need of several surgical interventions to reach the aimed result is not unusual. Therefore, the development of alternative protocols to achieve more effective reconstruction of soft tissues is of major interest. Nowadays, a current promise is to enhance adipose tissue survivability by the combination of fat transplantation and stem cell therapy, particularly with the use of adipose tissue-derived stem cells (ASCs), since stem cells potentially ameliorate neovascularization and partially halt inflammatory response [1，2]。

脂肪组织来源的干细胞（ASCs）可以通过简单的外科手术过程的人类脂肪组织的基质血管级分（SVF）容易地分离，可以重复获得，大批量，并且在一些情况下，在局部麻醉下，和能够接受体外朝当与特定的因素[处理的成骨，成脂，神经，肌原，软骨形成和分化谱系3，4]。Multipotentiality of ASCs makes them interesting and promising candidates for mesodermal defect repair and disease management [五]。

无论使用脂肪组织作为唯一实现材料或实施的ASCs的，有几个问题尚未以增强其生存能力组织再生疗法加以解决。例如，它不能很好地理解中的ASC的百分比是否在脂肪组织新生的效率重新注入脂肪组织干涉。此外，在间质细胞的数量和类型的解剖供区的影响仍不明朗：虽然有些作者认为subabdominal区域是间充质干细胞（MSCs）是最丰富的网站[6，7]，其他人指出臀部的最佳组织提取物的MSC。被评估的另一个重要问题是收获的方法，因为有些作者认为，使用泵辅助技术，降低核细胞的活力[8]。考虑到这些因素可能由外科医生来控制，他们的理解肯定有个ASC的最终浓度的影响包含在脂肪抽吸物[9]。

鉴于上面列出的问题，这项工作的范围是，以调查是否个ASC的比例影响的效率体外脂肪生成，并取决于收获面积，最后收获的方法比较有核细胞数量在SVF。

2。材料和方法

2。1。Ethics Statement

本研究通过Biosciences的人类研究伦理委员会的机构（许可证号095/2009-FR251136）。

2.2。Lipoaspiration外科手术

Lipoaspiration was done by a 10 mL syringe coupled to a cannula with a diameter of 2,5 mm, using the Coleman method [10] or by pump-assisted liposuction regulated at −350 mmHg using a cannula with a diameter of 3 mm. For pump versus manual lipoaspiration comparison experiments, 10 donors were submitted to sub-abdominal liposuction of right side with manual method and the sub-abdominal left side with pump method. For flank versus abdomen comparison experiments, 6 donors were submitted to liposuction in these distinct anatomical sites, with traditional pump suction lipoaspiration method. In order to minimize the differences between individuals, samples taken from the different locations and obtained using the different methods tested were paired in each series of subjects.

2.3。脂肪来源干细胞（ASCs）分离和扩增

Adipose tissue from sub-abdominal and flank subcutaneous lipoaspirates were obtained by traditional pump suction from six healthy women undergoing cosmetic surgery procedures. In another instance, adipose tissue from sub-abdominal region of ten healthy women was isolated by either pump-assisted liposuction, with controlled negative pressure, or manual lipoaspiration. The adipose tissue were washed extensively with sterile phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM NA2HPO4, 2 mM KH2PO4 pH 7.4, reagents from Sigma-Aldrich) to remove contaminating debris and red blood cells. Washed adipose tissue was treated with 0.075% collagenase (type IA; Sigma-Aldrich) in PBS for 40 min at 37°C with gentle agitation) as described previously [4]。The collagenase was inactivated with 2 volumes of HBSS (Invitrogen, CA, USA) and the infranatant was centrifuged for 5 min at 3000 g. Concerning the samples used to test whether there is an influence of harvesting pressure on cell concentration, the cellular pellet for seven samples obtained by pump-assisted liposuction and seven matched samples obtained by manual lipoaspiration were resuspended in DMEM F12 (Invitrogen, CA, USA) supplemented with 15% FBS (Hyclone), 1% non essential aminoacids (Invitrogen, CA, USA), 1% penicillin-streptomicyn (Invitrogen, CA, USA) and seeded on conventional tissue culture flasks for immunophenotypical characterization.

2。4。Cell Count

Viable cells were counted using the trypan blue dye exclusion assay. A freshly prepared solution of 10uL trypan blue at 0.05% (Sigma Aldrich) in distilled water was mixed with 10uL of cellular suspension for 5 min., viable cells were counted in a Neubauer chamber using a light microscope (Nikon Eclipse TS100).

2.5。免疫分型

细胞群的免疫表型特征，通过流式细胞仪分析完成。对于取决于解剖区域细胞产量的比较中使用的样品，我们执行使用新鲜分离的基质血管级分该表征和分析的每个样本是基质血管级分的从五位女性供体的池。对于取决于吸脂技术细胞产量的比较中使用的样品，镀细胞培养物用PBS洗涤，并通过胰蛋白酶溶液（0125％胰蛋白酶，在PBS中0.02％EDTA）消化。将细胞温育1小时，在4℃下用以下抗 - 人类抗体：CD29-PECy5，CD34PerCP，CD31-PE，CD45-FITC，CD90-R-PE，CD73-PE，CD105（碧迪公司，NJ，EUA）和SH2，SH3，SH4教授阿诺德·卡普兰（Case Western Reserve大学）轻轻地捐赠。匹配的对照样品与仅PBS中温育。After a second wash with PBS, samples incubated with non-conjugated primary antibodies were incubated with anti-mouse-PE secondary antibody (Guava Technologies) for additional 15 min at 4°C. Cell suspensions were washed with PBS, fixed with 1% p-formaldehyde (Sigma-Aldrich) and 5.000 labeled cells were analyzed using a Guava EasyCyte flow cytometer running the Guava Express Plus software (Guava Technologies Hayward, CA, USA).

2。6。Preparation of Heterogeneous ASC and Mature Fibroblast Culture

A coronal suture periosteal fibroblast cell lineage and an adipose derived stem cell lineage were grown to 80–90% confluence in independent flasks. The cell lineages were treated with 0,125% trypsin, 0,02% EDTA (Invitrogen, CA, USA) and the suspended cells were mixed in different concentrations of these two cell lineages, as following: 5% ASC and 95% fibroblast; 25% ASC and 75% fibroblast; 50% ASC and 50% fibroblast; 75% ASC and 25% fibroblast and 100% ASC only. Cells were plated in a concentration of 10⁴/厘米²for each experiment. These mixed cell lineages were seeded in 12 well plates and after 24 hour, the mixed cells were induced to adipogenic differentiation.

2.7。体外成脂诱导分化

为了诱导脂肪细胞分化，将细胞在脂肪形成诱导培养基高葡萄糖DMEM补充有10个％FBS（GIBCO-Invitrogen公司，CA，USA），1％青霉素 - 链霉素（Invitrogen公司，CA，USA），1中培养 μM地塞米松（Sigma-Aldrich公司），100 μ中号吲哚美辛（Sigma-Aldrich公司），500 μM 3-isobutyl-1-methylxanthine (IBMX), and 10 μ克/ mL胰岛素（Sigma-Aldrich公司），持续14天。

2.8。油红O染色和定量

对于油红O染色，将细胞在12孔培养板中培养，并且对于每个实验;使用每种条件四个孔。14天后脂肪形成分化后，将细胞用PBS洗涤，固定于4％甲醛1小时，并洗涤，然后细胞用去离子水。后，将细胞用60％的异丙醇和细胞板在室温下干燥。The cells were stained with 0.6% (w/v) Oil Red O solution (60% isopropanol, 40% water) for 1 h at room temperature. Cells were then washed with deionized water three times to remove unbound dye and photographed. Stained Oil Red O was also eluted with 100% isopropanol (v/v) and quantified by measuring the optical absorbance at 500 nm. Oil Red O staining of undifferentiated cells grown in parallel culture served as the blank sample for this assay.

2.9。统计分析

连续变量任何明示单独或通过均值和标准差，我们使用非参数Wilcoxon符号秩检验配对资料，以评估如果人口平均等级不同。为了评估泵辅助和手动吸脂之间的ASC混合细胞群，我们用百分比和脂肪细胞分化之间的相关性的统计显着性双尾Pearson相关测试。与测试values < 0.05 were considered to be statistically significant. All the applied tests were done by the use of the GraphPad Prism 5 program.

3.结果

3。1。Influence of the Concentration of Adipose-Derived Stem Cells on the体外成脂诱导分化

为了评估是否有脂肪来源干细胞的浓度的上的效率的影响体外adipogenesis, we created five heterogeneous cell cultures composed of mixed subpopulations of ASCs and mature fibroblasts derived from cranial coronal suture, induced them to adipogenic differentiation and quantified the lipid vacuoles formation by Oil Red-O staining after 14 days (Figure1）。应用线性回归模型，如在图表明2，there is a significant correlation between the percentage of ASCs and the increase in Oil Red-O staining (）。

3。2。Influence of Donor Site on Cell Concentration

为了确定的数量和比例of mesenchymal cells varies according to the donor site of the adipose tissue, we evaluated cells from SVF from six female patients with mean age of 37 years (range, 26–51 years) submitted to liposuction at the lower abdomen and at the flank following medical recommendations. We observed a significantly higher concentration of nucleated cells in fat from the lower abdomen when compared to fat from the flank (;数据图3（a）and3（b）中）。

作为显示在图4，我们观察到阳性细胞间质（79.7-84.7％）和粘附细胞标志物（CD29; 96％）的比例较高，从腹部分离样品相比，从侧翼（24.36-28.40％和50.48％得到的试样，RESP）。此外，与从腹部的那些当从侧翼样品具有造血（53.75％）和内皮（21.56％）来源的细胞的更加突出的亚群（29.38和15.8％，RESP）。

3。3。Influence of Harvesting Methodology on Cell Concentration after 1 Passage

A group formed by 10 patients, with mean age of 49 (range, 22–73 years) and mean body mass index of 24.88 (range, 22–29.6), submitted to sub-abdominal liposuction according to medical recommendation, was used to evaluate if there is an influence of the harvesting method on the yield of nucleated cells in the SVF. Liposuction was performed applying 2 commonly used harvesting methods: manual aspiration using a syringe and pump-assisted aspiration, with a controlled pressure of −350 mmHg. After 1 passage in culture, cells aspirated manually and obtained by pump-assisted aspiration showed positive staining (>89%) for mesenchymal (SH2, 3 and 4) and adhesion (CD29) markers and negative staining (<4%) for hematopoietic (CD45) and endothelial (CD31) markers. Fold change differences in the amount of cells obtained by pump-assisted method were calculated compared to manual aspiration and are represented in Figure五（一个）。应用线性回归模型（图五（b）中），对使用这些2种方法获得的细胞产量无统计学差异显著。

4。讨论

In autologous fat transplantation with large volume transfer, fat survival rather than fibrosis is desired. Other expected characteristics, which are usually highly variable, are the clinical longevity of fat graft and the maintenance of volume of the transplanted fat. To amend these variables, the combination of stem cell therapy with fat transfer, supplementing fat grafts with adipose-derived stem cells, has been reported as a method of autologous tissue transfer termed cell-assisted lipotransfer [9，11，12] and is a promise for the rehabilitation of several patients.

使用体外model of admixtured heterogeneous cell populations we found a positive correlation between the percentage of ASCs and the increase in the体外脂肪细胞分化。因此，我们建议在重新注入脂肪组织的ASC数量的增加可以提高新形成的脂肪组织的效率。因此，这些结果将支持这一想法，与间充质干细胞的脂肪组织的富集可以影响提交到自体脂肪移植患者的康复过程[五]。

因此，建立理想的参数，以优化可行的间充质细胞，如沉降，从中分离出的ASC lipoaspiration的优选供体位点和确定是否lipoaspiration干涉的间充质细胞的数量和质量的方法肯定会有助于数量更成功的使用脂肪移植的富含间充质细胞。

一个显著较高浓度的发现（）下腹部的有核细胞时相比，与来自间充质来源的细胞的富集亚群的样品中观察一起侧翼从下腹部表明SVF从下腹部可能是间充质干细胞的比脂肪组织更好的来源从侧翼隔离。我们的发现与Padoin等人的工作相一致。[7]，相对于上腹部，大腿内侧，转子间区域，膝和后刀面时，他们发现，在从下腹部获得的SVF核细胞的显著更高的浓度。尤尔根斯等人。[6] also suggested, after CFU assays, that the abdomen is preferable to the hip/thigh region for harvesting mesenchymal stem cells.

如果我们考虑到mes的数量和类型enchymal cells, it thus seems that the adipose tissue from the lower abdomen is better than those from the flank. However, further studies are necessary in order to elucidate the functional effect on tissue regeneration as we do not know if the higher proportion of hematopoietic and endothelial cells contained in the flank might actually facilitate neovascularization, which is critical for the success of the surgery procedure.

We did not find significant differences in SVF cell yields comparing manual liposuction using a syringe or pump-assisted lipoaspiration. Indeed, Fraser et al. [13]还测试了脂肪组织抽吸的这两种方法并得出结论认为克隆发生细胞的频率没有被收获方法的影响。尽管这些数据表明在SVF有核细胞数量没有这些方法的影响，通过Mojallal等的研究。[8] concluded that liposuction at a controlled pressure of −350 mmHg gives a greater cell yield when compared to power-assisted aspiration with a negative pressure of 700 mmHg and syringe aspiration. However, these results should be considered with caution, as they performed this test using only 3 patients.

In this paper, we demonstrate that even though there is no difference on the nucleated cell yield obtained by manual aspiration using a syringe or with pump-assisted aspiration with a pressure of −350 mmHg, the anatomical region from which to harvest fat tissue needs to be considered as a means to optimize the total number of nucleated cells in the SVF and, consequently, the quantity of adipose-derived stem and progenitor cells. For the foreseeable future, implementations to the cell-assisted lipotransfer will lead to improve fat-grafting outcomes for restoration of tissues for either aesthetic or reconstructive purposes. Further, our results support the hypothesis that enrichment of adipose tissue with mesenchymal stem cells might improve the regeneration process following a cell-assisted lipotransfer.

作者的贡献

M. Aguena和R. D. Fanganiello同等贡献这项工作。

参考

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Copyright

Copyright © 2012 Meire Aguena et al. This is an open access article distributed under the知识共享署名许可，其允许在任何介质无限制地使用，分发和再现时，所提供的原始工作正确的引用。