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Mesenchymal Stem Cell (MSC) Aggregate Formation in vivo
间充质干细胞(MSC)聚合体的体内形成试验

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Abstract

Human mesenchymal stem/progenitor cells (MSCs) isolated from various adult tissues show remarkable therapeutic potential and are being employed in clinical trials for the treatment of numerous diseases (Prockop et al., 2010). While routes of cell administration vary, profound beneficial effects of MSCs in animal models have been observed following intraperitoneal injections of the cells (Roddy et al., 2011). Similar to MSC spheres formed in culture under conditions where attachment to plastic is not permitted (Bartosh et al., 2010), MSCs injected into the peritoneum of mice spontaneously aggregate into 3D sphere-like structures (Bartosh et al., 2013). During the process of sphere assembly and compaction, MSCs upregulate expression of numerous therapeutic anti-inflammatory and immune modulatory factors. Here we describe the method we previously used for the generation of human bone marrow-derived MSC aggregates/spheres in vivo (Bartosh et al., 2013). By tagging the MSCs with green fluorescent protein (GFP), the aggregates formed can be easily visualized, collected and analyzed for changes in cellular properties and interactions with host immune cells.

Keywords: MSCs(骨髓间充质干细胞), Spheroids(球状细胞), Immunomodulatory(免疫调节), Peritoneum(腹膜), Antiinflammatory(抗炎)

Materials and Reagents

  1. Human bone marrow mesenchymal stem cells expressing green fluorescent protein (GFP-MSCs) from The Center for the Preparation and Distribution of Adult Stem Cells (http://medicine.tamhsc.edu/irm/msc-distribution.html)
  2. C57BL/6J or BALB/C mice (2-3 months of age) (The Jackson Laboratory)
  3. Phosphate-buffered saline (PBS) without Ca2+ and Mg2+ (pH 7.4) (Life Technologies, catalog number: 10010 )
  4. Hank’s Balanced Salt Solution (HBSS) without Ca2+ and Mg2+ (Lonza, catalog number: 04-315Q )
  5. 0.25% trypsin with 1x EDTA (Life Technologies, catalog number: 25200 )
  6. Minimum Essential Medium alpha (Life Technologies, catalog number: 12561 )
  7. Premium select fetal bovine serum (Atlanta Biologicals, catalog number: S11550 )
  8. Penicillin-streptomycin (Life Technologies, catalog number: 15140 )
  9. 100x L-glutamine (Life Technologies, catalog number: 25030 )
  10. Complete culture medium (CCM) for MSC growth (see Recipes)

Equipment

  1. Stericup-GP 0.22 µm vacuum filtration device (EMD Millipore, catalog number: SCGPU05RE )
  2. Water bath set to 37 °C
  3. Centrifuge with swinging-bucket rotor and adaptors for 50 ml conical tubes
  4. 50 ml sterile conical tube (BD Biosciences, Falcon®, catalog number: 352070 )
  5. Humidified cell culture incubator set to 37 °C and 5% CO2
  6. Upright microscope with 4x and 10x objectives and a filter set to visualize GFP
  7. 29 gauge needle with 1 ml syringe (Terumo Europe N.V., catalog number: 05M2913 )
  8. Isoflurane anesthesia system with nose cone for mouse
  9. Sterile dissecting scissors, pins, and curved forceps with a serrated edge
  10. Rubber or styrofoam platform
  11. Dissecting microscope with optional camera and monitor (Figure 1)
  12. Illumatool Bright Lights Systems LT 9900 with epi-fluorescence attachment (Lightools Research) and GFP filter set (Figure 1)


    Figure 1. Equipment required to visualize and collect GFP-MSC aggregates/spheres from the mouse peritoneum. GFP-MSC aggregates/spheres can be visualized in the mouse peritoneum using a dissecting microscope with an epi-fluorescence attachment and GFP filter set. High quality images can be acquired with an appropriate camera mounted to the dissecting scope (a camera is not a requirement for collecting the aggregates).

Procedure

  1. Determine the number of animals that will be injected with the GFP-MSCs and the number of cells that will be required.
    Note: We have successfully injected 1-3 x 106 GFP-MSCs per animal to achieve the desired results.
  2. Expand the GFP-MSCs in CCM from low-density seeding (100-300 cells/cm2) for 6-7 days. Upon reaching 70% confluence, harvest the GFP-MSCs using trypsin/EDTA then collect the cells by centrifugation at 450 x g for 5-7 min.
    Note: Proper MSC culture method is detailed in references below. It is important to prevent MSC cultures from becoming confluent. If necessary, the MSCs can be passaged to avoid the formation of confluent cultures.
  3. Aspirate the supernatant and suspend the GFP-MSC pellet in a low volume of CCM for cell counts.
    Note: A viability dye such as trypan blue is recommended to discriminate between live and dead cells. GFP-MSCs are typically greater than 95% viable.
  4. After counting the cells, add up to 45 ml HBSS to the cells and centrifuge at 450 x g for 5-7 min.
  5. Aspirate the supernatant and suspend the cells in HBSS at a concentration of 5,000-10,000 viable cells per µl.
  6. With the mouse under isoflurane anesthesia, inject 200-300 µl of GFP-MSCs (1-3 x 106 cells) from the cell suspension into the peritoneal cavity using a 29 gauge needle.
    Notes:
    1. For optimal MSC viability, do not mix the cell suspension with the needle. Instead, mix cells using a pipet or simply by flicking the vial.
    2. Intraperitoneal injections should be performed following procedures set forth by your Institutional Animal Care and Use Committee or equivalent.
  7. Immediately following GFP-MSC injection, disperse the cells within the peritoneum by gently massaging the abdomen for 5-10 sec.
    Note: To massage the abdomen, gently press on the abdomen with 1 or 2 fingers. Start from an area in close proximity to the injection site and progress distally. Do not press forcefully to avoid causing trauma to the internal organs.
  8. Repeat procedure with additional mice.
  9. Accommodate the mice for 4-72 h on a standard 12 h light/dark cycle and with standard nutrition.
    Notes:
    1. Aggregates collected at 72 h are compact and often adhere to the tissues making them more difficult to obtain. Aggregates collected at earlier time points are less compact and more abundant.
    2. Aggregates collected from the peritoneum will vary in size and number. MSC aggregates typically range in size from several hundreds of microns to 2-3 mm in diameter although smaller aggregates composed of fewer cells can be obtained. The number of aggregates that can be collected from the peritoneum gradually decreases with time. Following injection of 1-3 x 106 GFP-MSCs, 15-30 aggregates can be obtained at 4 h, 10-15 aggregates at 24 h, and less than 10 aggregates at 72 h.
  10. After 4-72 h, anesthetize the mouse by isoflurane inhalation then euthanize the animal by cervical dislocation.
    Note: Other methods of euthanasia can be performed however it is not recommended to use a method that introduces fluid into the peritoneum.
  11. Using dissecting pins stabilize the euthanized mouse on a rubber or styrofoam platform (ventral side up).
  12. Expose the peritoneal cavity using dissecting forceps and scissors (Figure 2). Avoid disturbing the internal organs or cell aggregates.
  13. Visualize the GFP signal using a dissecting microscope with an epi-fluorescence attachment and appropriate filter set (Figure 2).
    Note: The localization of the aggregates can be variable between animals. Under some circumstances, visualization of aggregates requires careful repositioning of the internal organs.


    Figure 2. Formation of GFP-MSC aggregates in the peritoneal cavity. Images of the mouse peritoneal cavity uncovered to reveal the (A) internal organs and (B) GFP-MSC aggregates. (C) Magnified view of a GFP-MSC aggregate (arrow) in the peritoneum of a BALB/c mouse 4 h after intraperitoneal injection of 2 x 106 GFP-MSCs.

  14. Collect the aggregates using forceps and transfer to a 50 ml tube containing ice cold HBSS.
    Notes:
    1. Due to surface tension, the aggregates adhere readily to the forceps without the need to squeeze the forceps around the cells.
    2. Aggregate formation by MSCs in the peritoneal cavity is not the only fate of the cells.  MSCs will adhere to certain structures of the peritoneum. Free floating aggregates can be distinguished from adherent cells with careful examination. Moreover, the peritoneum can be washed repeatedly with 1-2 ml HBSS to distinguish the free floating aggregates that often reposition during the wash from adherent cells that will not move.
  15. After collecting the GFP-MSC aggregates of interest, make certain that most of the cells descended to the bottom of the tube.
    Note: Aggregates connected to fatty tissue will remain in suspension and can be removed by careful aspiration. Aggregates that are void of fatty tissue will rapidly fall to the bottom of the tube.  
  16. Aspirate the HBSS supernatant to remove cell aggregates containing fatty tissue while avoiding contact with the cell pellet.
  17. Wash the cells in HBSS and collect aggregates by centrifugation at 400 x g for 5 min.
  18. Aspirate the supernatant. The aggregates are now prepared for downstream applications and analysis. For analyzing changes in gene and protein expression, the cell pellet can by lysed in appropriate lysis solution. Alternatively, aggregates can be transferred to a cell culture dish (Figure 3) or to a cryomold and frozen for immunofluorescence applications. MSC aggregates can also be dissociated using trypsin/EDTA and analyzed by microscopy (Figure 4) or flow cytometry. Other applications have not been tested.


    Figure 3. Transfer of GFP-MSC aggregates in vitro. Images of a GFP-MSC aggregate 1 h after transfer from the peritnoneum of a BALB/c mouse to a cell culture dish. The aggregates form sturdy micro-tissue structures. Scale bar = 200 µm


    Figure 4. Microscopic examination of GFP-MSCs derived from peritoneal aggregates. Images of the cells obtained 12 h following dissociation of aggregates harvested from the peritoneum. GFP-MSCs appear as large flat cells that readily attach to a cell culture dish. Small round cells (GFP negative) are also obtained from peritoneal aggregates. The majority of the small round cells are host immune cells.  Scale bar = 100 µm

Notes

  1. All animal procedures were approved by the Animal Care and Use Committe of Texas A&M Health Science Center and in accordance with guidelines set forth by the National Institutes of Health.
  2. The protocol was performed using MSCs obtained from human bone marrow aspirates. The cells were transduced with a lentiviral reporter harboring GFP. Other types of cells have not been thoroughly tested in this model.
  3. Aggregates/spheres will show a range in size from several hundreds of microns to 2-3 mm in diameter with each aggregate containing thousands of cells. The size and number of aggregates can be augmented by increasing the number of cells injected into the peritoneum. We typically inject 1-3 x 106 GFP-MSCs per animal and readily collect 10-30 aggregates/spheres of variable size.
  4. Aggregates/spheres collected from the peritoneum generally consist of both the GFP-MSCs that were administered and resident peritoneal immune cells.

Recipes

  1. Complete Culture Medium (CCM)
    Minimum Essential Medium alpha, 1 L
    Premium select fetal bovine serum, 200 ml
    Penicillin-streptomycin, 12 ml
    100x L-glutamine, 12 ml
    Filter sterilize (0.22 µm)
    Stored at 4 °C for up to 1 month
    Pre-warm to 37 °C in water bath prior to use

Acknowledgments

This protocol was adapted from our previous work (Bartosh et al., 2013) and was supported by an NIH grant (P40RR17447) to Darwin J. Prockop, Institute for Regenerative Medicine, Texas A&M University Health Science Center, and a grant from the Cancer Prevention and Research Institute of Texas (RP110553-P1).

References

  1. Bartosh, T. J., Ylostalo, J. H., Bazhanov, N., Kuhlman, J. and Prockop, D. J. (2013). Dynamic compaction of human mesenchymal stem/precursor cells into spheres self-activates caspase-dependent IL1 signaling to enhance secretion of modulators of inflammation and immunity (PGE2, TSG6, and STC1). Stem Cells 31(11): 2443-2456.
  2. Bartosh, T. J., Ylostalo, J. H., Mohammadipoor, A., Bazhanov, N., Coble, K., Claypool, K., Lee, R. H., Choi, H. and Prockop, D. J. (2010). Aggregation of human mesenchymal stromal cells (MSCs) into 3D spheroids enhances their antiinflammatory properties. Proc Natl Acad Sci U S A 107(31): 13724-13729.
  3. Prockop, D. J., Kota, D. J., Bazhanov, N. and Reger, R. L. (2010). Evolving paradigms for repair of tissues by adult stem/progenitor cells (MSCs). J Cell Mol Med 14(9): 2190-2199.
  4. Roddy, G. W., Oh, J. Y., Lee, R. H., Bartosh, T. J., Ylostalo, J., Coble, K., Rosa, R. H., Jr. and Prockop, D. J. (2011). Action at a distance: systemically administered adult stem/progenitor cells (MSCs) reduce inflammatory damage to the cornea without engraftment and primarily by secretion of TNF-alpha stimulated gene/protein 6. Stem Cells 29(10): 1572-1579.

简介

从各种成人组织分离的人间充质干/祖细胞(MSC)显示出显着的治疗潜力,并且正用于治疗许多疾病的临床试验(Prockop等人,2010)。尽管细胞施用途径不同,但是在腹膜内注射细胞后已经观察到MSC在动物模型中的深远有益效果(Roddy等人,2011)。类似于在不允许附着到塑料的条件下在培养物中形成的MSC球体(Bartosh等人,2010),注射到小鼠腹膜中的MSC自发聚集成3D球状结构(Bartosh < em> et al。,2013)。在球体装配和压实的过程中,MSCs上调多种治疗性抗炎和免疫调节因子的表达。在这里我们描述了我们以前用于在体内产生人骨髓衍生的MSC聚集体/球体的方法(Bartosh等人,2013)。通过用绿色荧光蛋白(GFP)标记MSC,形成的聚集体可以容易地可视化,收集和分析细胞性质的变化和与宿主免疫细胞的相互作用。

关键字:骨髓间充质干细胞, 球状细胞, 免疫调节, 腹膜, 抗炎

材料和试剂

  1. 来自成人干细胞的制备和分布中心的表达绿色荧光蛋白(GFP-MSC)的人骨髓间充质干细胞( http://medicine.tamhsc.edu/irm/msc-distribution.html
  2. C57BL/6J或BALB/C小鼠(2-3个月龄)(The Jackson Laboratory)
  3. 没有Ca 2+和Mg 2+(pH 7.4)的磷酸盐缓冲盐水(PBS)(Life Technologies,目录号:10010)
  4. 没有Ca 2+和Mg 2+ 2+(Lonza,目录号:04-315Q)的Hank's平衡盐溶液(HBSS)
  5. 0.25%胰蛋白酶与1x EDTA(Life Technologies,目录号:25200)
  6. Minimum Essential Medium alpha(Life Technologies,目录号:12561)
  7. Premium select胎牛血清(Atlanta Biologicals,目录号:S11550)
  8. 青霉素 - 链霉素(Life Technologies,目录号:15140)
  9. 100x L-谷氨酰胺(Life Technologies,目录号:25030)
  10. 用于MSC生长的完全培养基(CCM)(参见Recipes)

设备

  1. Stericup-GP0.22μm真空过滤装置(EMD Millipore,目录号:SCGPU05RE)
  2. 水浴设置为37℃
  3. 带有摆动斗转子和适配器的离心机用于50ml锥形管
  4. 50ml无菌锥形管(BD Biosciences,Falcon ,目录号:352070)
  5. 将加湿的细胞培养箱设定为37℃和5%CO 2/h
  6. 具有4x和10x物镜的直立显微镜和用于可视化GFP的过滤器
  7. 29号针与1ml注射器(Terumo Europe N.V.,目录号:05M2913)
  8. 异氟烷麻醉系统与鼻锥鼻子
  9. 无菌解剖剪刀,针和弯曲镊子与锯齿边
  10. 橡胶或聚苯乙烯泡沫塑料平台
  11. 用可选的相机和显示器(图1)解剖显微镜
  12. Illumatool Bright Lights系统LT 9900具有荧光附件(Lightools Research)和GFP滤光片组(图1)

    图1.从小鼠腹膜可视化和收集GFP-MSC聚集体/球体所需的设备。可以使用具有落射荧光附件的解剖显微镜在小鼠腹膜中可视化GFP-MSC聚集体/球体和GFP滤光片组。可以使用安装在解剖镜体上的适当的相机获取高质量的图像(相机不是收集聚集体的必要条件)。

程序

  1. 确定将注射GFP-MSC的动物数量和所需的细胞数。
    注意:我们已成功为每只动物注射1-3 x 10 6 GFP-MSCs,
  2. 在CCM中从低密度接种(100-300个细胞/cm 2)扩增GFP-MSC 6-7天。在达到70%汇合时,使用胰蛋白酶/EDTA收获GFP-MSC,然后通过在450×g离心5-7分钟收集细胞。
    注意:适当的MSC培养方法详述于下面的参考文献中。重要的是要防止MSC培养物变得汇合。如果需要,可以传代MSC以避免形成融合培养物。
  3. 吸出上清液并悬浮GFP-MSC沉淀在小体积的CCM中进行细胞计数 注意:建议使用活性染料如台盼蓝来区分活细胞和死细胞。 GFP-MSC通常大于95%存活。
  4. 计数细胞后,向细胞中加入45ml HBSS,并在450×g离心5-7分钟。
  5. 吸出上清液,并悬浮在HBSS中的细胞,浓度为5,000-10,000个活细胞/μl
  6. 使用小鼠在异氟烷麻醉下,使用29号针将200-300μl来自细胞悬浮液的GFP-MSC(1-3×10 6个细胞)注射到腹膜腔中。
    注意:
    1. 对于最佳的MSC活力,不要将细胞悬浮液与 针。相反,使用移液管或简单地通过轻弹小瓶混合细胞。
    2. 腹膜内注射应按照程序进行 由您的机构动物护理和使用委员会或 等效。
  7. 在GFP-MSC注射后立即通过轻轻按摩腹部5-10秒使细胞分散在腹膜内。
    注意:要按摩腹部,用1或2个手指轻轻按压腹部。 从接近注射部位的区域开始并向远侧进展。 不要用力按压,以免对内脏造成创伤。
  8. 对其他小鼠重复操作。
  9. 在标准的12小时光照/黑暗周期和标准营养,容纳小鼠4-72小时 注意:
    1. 在72小时收集的聚集体是紧凑的并且经常粘附于 组织使得它们更难获得。收集的聚集 较早的时间点不太紧凑和更丰富。
    2. 从腹膜收集的聚集体在大小和数量上不同。 MSC聚集体的尺寸通常在几百微米的范围内 直径为2-3mm,尽管较小的聚集体由较少的聚集体组成 细胞。可以收集的聚合数 从腹膜逐渐减少随时间。注射后 的1-3×10 6 GFP-MSC,15-30个聚集体可在4h,10-15 在24小时聚集,在72小时小于10聚集
  10. 4-72小时后,通过异氟醚吸入麻醉小鼠,然后通过颈椎脱位使动物安乐死。
    注意:可以执行安乐死的其他方法,但不建议使用将液体引入腹膜的方法。
  11. 使用解剖销将安乐死的小鼠稳定在橡胶或聚苯乙烯泡沫平台上(腹侧朝上)。
  12. 使用解剖钳和剪刀暴露腹膜腔(图2)。避免干扰内脏或细胞聚集体
  13. 使用具有落射荧光附件和适当的滤光片组的解剖显微镜可视化GFP信号(图2)。
    注意:聚集体的定位可以在动物之间变化。在某些情况下,聚集体的可视化需要仔细重新定位内脏。


    图2.在腹膜腔中形成GFP-MSC聚集体。小鼠腹膜腔的图像被揭示以显示(A)内部器官和(B)GFP-MSC聚集体。 (C)在腹膜内注射2×10 6个GFP-MSC后4小时,在BALB/c小鼠的腹膜中GFP-MSC聚集体(箭头)的放大视图。
  14. 使用镊子收集聚集体,并转移到含有冰冷HBSS的50ml管 注意:
    1. 由于表面张力,聚集体很容易粘附在镊子上,而不需要挤压细胞周围的镊子。
    2. 腹膜腔中MSC的聚集形成不是唯一的 细胞的命运。 MSC将坚持某些结构 腹膜。自由浮动聚集体可以与粘附体区分开 细胞仔细检查。此外,可以洗涤腹膜 重复用1-2ml HBSS以区分自由浮动的聚集体 其通常在洗涤期间从不粘附的细胞重新定位 移动。
  15. 收集感兴趣的GFP-MSC聚集物后,确保大多数细胞下降到管底部。
    注意:连接到脂肪组织的聚集体将保持悬浮状态,并可以通过小心抽吸除去。没有脂肪组织的聚集体将迅速落到管的底部。  
  16. 吸出HBSS上清液以去除含有脂肪组织的细胞聚集体,同时避免与细胞沉淀物接触
  17. 在HBSS中洗涤细胞并通过在400×g离心5分钟收集聚集体。
  18. 吸出上清液。聚集体现在准备用于下游应用和分析。为了分析基因和蛋白质表达的变化,可以通过在合适的裂解溶液中裂解细胞沉淀。或者,可以将聚集体转移至细胞培养皿(图3)或冷冻模具中,并冷冻用于免疫荧光应用。 MSC聚集体也可以使用胰蛋白酶/EDTA解离,并通过显微镜(图4)或流式细胞术分析。其他应用程序尚未测试。


    图3.体外转移GFP-MSC聚集体。 GFP-MSC聚集体从BALB/c小鼠的腹膜转移到细胞培养皿后1小时的图像。聚集体形成坚固的微组织结构。比例尺=200μm


    图4.源自腹膜聚集体的GFP-MSC的显微镜检查从腹膜收集的聚集体解离后12小时获得的细胞图像。 GFP-MSC表现为容易附着于细胞培养皿的大扁平细胞。小圆形细胞(GFP阴性)也从腹膜聚集物获得。大多数小圆细胞是宿主免疫细胞。比例尺=100μm

笔记

  1. 所有动物程序由Texas A& M Health Science Center的Animal Care and Use Committe批准并且根据美国国立卫生研究院提出的指导。
  2. 使用从人骨髓抽出物获得的MSC进行方案。用携带GFP的慢病毒报道基因转导细胞。其他类型的细胞在这个模型中没有被彻底测试
  3. 聚集体/球体将显示尺寸范围,从几百微米到2-3mm的直径,每个聚集体含有数千个孔。聚集体的大小和数量可以通过增加注射到腹膜中的细胞的数量来增加。我们通常每个动物注射1-3×10 6个 GFP-MSC,并且容易收集10-30个可变大小的聚集体/球体。
  4. 从腹膜收集的聚集体/球体通常由施用的GFP-MSC和驻留的腹膜免疫细胞组成。

食谱

  1. 完全培养基(CCM)
    最小必需培养基α,1 L
    高级精选胎牛血清,200 ml
    青霉素 - 链霉素,12ml
    100x L-谷氨酰胺,12ml
    过滤灭菌(0.22μm)
    储存于4°C长达1个月
    使用前在水浴中预温至37°C

致谢

该方案改编自我们以前的工作(Bartosh等人,2013),并且由得克萨斯大学健康再生医学研究所Darwin J.Prockop的NIH授权(P40RR17447)支持 科学 中心,以及得克萨斯癌症预防和研究所(RP110553-P1)的资助。

参考文献

  1. Bartosh,T.J.,Ylostalo,J.H.,Bazhanov,N.,Kuhlman,J.and Prockop,D.J。(2013)。 将人间质干细胞/前体细胞动态压实成球体自激活半胱天冬酶依赖性IL1信号以增强分泌炎症和免疫调节剂(PGE2,TSG6和STC1)。 干细胞 31(11):2443-2456。
  2. Bartosh,T.J.,Ylostalo,J.H.,Mohammadipoor,A.,Bazhanov,N.,Coble,K.,Claypool,K.,Lee,R.H.,Choi,H.and Prockop,D.J。(2010)。 人间质基质细胞(MSC)聚集成3D球状体增强了它们的抗炎性质。 Proc Natl Acad Sci USA 107(31):13724-13729。
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引用:Bartosh, T. J. and Ylostalo, J. H. (2014). Mesenchymal Stem Cell (MSC) Aggregate Formation in vivo. Bio-protocol 4(14): e1181. DOI: 10.21769/BioProtoc.1181.
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