欢迎您, 登录 | 注册

首页 | English

X
加载中

To assess the capacity of multipotent stromal cells (MSC) to induce the generation of Tregs, transwell co-cultures were performed as well as cultures with MSC-conditioned medium (CM). In short, peripheral blood mononuclear cells (PBMC) were co-cultured with allogeneic MSC or CM for one week followed by one week of culture in the absence of MSC.

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

PBMC-MSC Co-cultures for Induction of Treg Generation
PBMC-MSC共培养诱导Treg细胞的生成

干细胞 > 成体干细胞 > 造血干细胞
作者: Sara M. Melief
Sara M. MeliefAffiliation: Medical Center, Leiden University, Leiden, The Netherlands
Bio-protocol author page: a1952
C. L. M. Schrama
C. L. M. SchramaAffiliation: Medical Center, Leiden University, Leiden, The Netherlands
Bio-protocol author page: a1953
 and Helene Roelofs
Helene RoelofsAffiliation: Medical Center, Leiden University, Leiden, The Netherlands
For correspondence: h.roelofs@lumc.nl
Bio-protocol author page: a1954
Vol 5, Iss 2, 1/20/2015, 3282 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.1383

[Abstract] To assess the capacity of multipotent stromal cells (MSC) to induce the generation of Tregs, transwell co-cultures were performed as well as cultures with MSC-conditioned medium (CM). In short, peripheral blood mononuclear cells (PBMC) were co-cultured with allogeneic MSC or CM for one week followed by one week of culture in the absence of MSC.

[Abstract]

Materials and Reagents

  1. Peripheral blood mononuclear cells (PBMC) [isolated from a healthy donor using Ficoll-Paque (own pharmacy) density gradient (1.077 g/cm3)]
  2. Multipotent stromal cells (MSC) from healthy donors
  3. Roswell Park Memorial Institute (RPMI) 1640 medium (Life Technologies, catalog number: 31870-082 )
  4. Penicillin/streptomycin (5,000 U/ml) (Life Technologies, catalog number: 15070-063 )
  5. L-glutamin (200 mM) (Life Technologies, catalog number: 25030-024 )
  6. Fetal calf serum (FCS) (Greiner Bio-One GmbH)
  7. Phosphate buffered saline (PBS)
  8. Trypsin/EDTA (1x 0.05% Trypsin-EDTA, phenol red) (Life Technologies, catalog number: 25300-096 )

Equipment

  1. T75 culture flasks
  2. 12-well transwell plates (pore size 0.4 μM) (Sigma-Aldrich, catalog number: CLS3460 )
  3. 10 K Centriprep Centrifugal filters (Millipore, catalog number: 4304 )
  4. 12/24/48-well plates (Sigma-Aldrich, Corning Costar cell culture plates)
  5. 37 °C, 5% CO2 cell culture incubator
  6. Microscope
  7. Centrifuge
  8. 10 K Centriprep centrifugal filters
  9. Hemocytometer (counting chamber) or Sysmex F-820

Procedure

  1. Harvesting and counting of MSC
    1. Wash the cells once with PBS to remove culture medium.
    2. Incubate for 5 min with trypsin/EDTA.
    3. Check under the microscope whether MSC are detached.
    4. Harvest cells in culture medium diluted 5x in PBS.
    5. Spin down at 350 x g and resuspend cells in 1-3 ml of culture medium.
    6. Count cells using a hemocytometer.

  2. PBMC-MSC co-culture in transwell system
    Culture medium: RPMI medium + 10% FCS + P/S (100 U/ml) + L-glutamin (100 U/ml). L-glutamin is added freshly at day 0.
    1. At day 0 100,000 MSC are plated in the lower well of a 12-wells transwell plate in 1.5 ml culture medium.
    2. 400,000 PBMC are added in the transwell insert in 500 µl of culture medium.
    3. Co-cultures are cultured for one week at 37 °C in a 5% CO2 cell culture incubator.
    4. At day 7, PBMC are collected from the inserts.
    5. Spin down PBMC at 350 x g for 10 min.
    6. Add 120 µl of PBS and count PBMC with sysmex or hemocytometer.
    7. Calculate total number of PBMC per condition and replate the PBMC in fresh culture medium and fresh plates:
      1. 350,000-700,000 PBMC: 24-wells in 1 ml medium
      2. < 350,000 PBMC: 48-wells in 750 µl medium
      Note: Plating too low numbers of cells will result in bad Treg generation.
    8. Co-cultures are cultured for one week at 37 °C in a 5% CO2 cell culture incubator.
    9. At day 14, collect PBMC.
    10. Spin down PBMC 350 x g for 10 min.
    11. Add 120 µl of PBS and count PBMC with sysmex or hemocytometer.
    12. Further analyze PBMC with flowcytometry.

  3. MSC-PBMC co-culture with MSC conditioned medium (CM)
    1. Let MSC grow to near-confluence in a T75 flask.
    2. Change the medium with culture medium and culture for 7 days without changing the medium.
    3. Collect the culture medium after 7 days (= day 0), this the MSC conditioned medium (CM).
    4. Harvest and count the MSC.
    5. Spin down the CM 350 x g for 10 min to get rid of cell debris.
    6. Concentrate the cell free CM using 10 K Centriprep centrifugal filters (according to manufacturers’ instructions).
      1. Wash the Centriprep centrifugal filters with 10 ml PBS; spin down 3,000 x g 10 min.
      2. Add MSC-CM (14 ml) and spin down for 30 min at 3,000 x g.
      3. Collect concentrated CM and calculate the volume of CM that is equivalent to 100,000 MSC.
        1. Example: In step C5 1.0 x 106 MSC are counted and 25 ml CM is collected in step C4.
        2. After concentration the CM has a volume of 5 ml (= 5 times concentrated).
        3. 5 ml of concentrated CM is equivalent to 1.0 x 106 MSC, so 500 µl of concentrated CM is equivalent to 100,000 MSC.
    7. Plate 400,000 PBMC in CM that is equivalent to 100,000 MSC and add 1 ml of fresh culture medium in a 12-wells plate.
    8. Co-cultures are cultured for one week at 37 °C in a 5% CO2 cell culture incubator.
    9. At day 7, PBMC are collected.
    10. From this point onwards, continue as described in the co-culture in a transwell system starting at step C4.

References

  1. Melief, S. M., Schrama, E., Brugman, M. H., Tiemessen, M. M., Hoogduijn, M. J., Fibbe, W. E. and Roelofs, H. (2013). Multipotent stromal cells induce human regulatory T cells through a novel pathway involving skewing of monocytes toward anti-inflammatory macrophages. Stem Cells 31(9): 1980-1991.

材料和试剂

  1. 外周血单核细胞(PBMC)[使用Ficoll-Paque(自己的药学)密度梯度(1.077g/cm 3 )从健康供体分离]
  2. 来自健康捐献者的多功能基质细胞(MSC)
  3. Roswell Park Memorial Institute(RPMI)1640培养基(Life Technologies,目录号:31870-082)
  4. 青霉素/链霉素(5,000U/ml)(Life Technologies,目录号:15070-063)
  5. L-谷氨酰胺(200mM)(Life Technologies,目录号:25030-024)
  6. 胎牛血清(FCS)(Greiner Bio-One GmbH)
  7. 磷酸盐缓冲盐水(PBS)
  8. 胰蛋白酶/EDTA(1×0.05%胰蛋白酶-EDTA,酚红)(Life Technologies,目录号:25300-096)

设备

  1. T75培养瓶
  2. 12孔transwell板(孔径0.4μM)(Sigma-Aldrich,目录号:CLS3460)
  3. 10K Centriprep离心过滤器(Millipore,目录号:4304)
  4. 12/24/48孔板(Sigma-Aldrich,Corning Costar细胞培养板)
  5. 37℃,5%CO 2细胞培养箱中培养
  6. 显微镜
  7. 离心机
  8. 10 K Centriprep离心过滤器
  9. 血细胞计数器(计数室)或Sysmex F-820

程序

  1. MSC的收获和计数
    1. 用PBS洗涤细胞一次以去除培养基。
    2. 用胰蛋白酶/EDTA孵育5分钟
    3. 在显微镜下检查MSC是否脱落
    4. 将培养基中的收获细胞在PBS中稀释5倍
    5. 在350×g下旋转,并将细胞重悬在1-3ml培养基中
    6. 使用血细胞计数器计数细胞。

  2. 在transwell系统中的PBMC-MSC共培养
    培养基:RPMI培养基+ 10%FCS + P/S(100U/ml)+ L-谷氨酰胺(100U/ml)。 在第0天新鲜加入L-谷氨酰胺。
    1. 在第0天,将100,000个MSC接种在12孔transwell板的下孔中的1.5ml培养基中。
    2. 将400,000个PBMC加入到500μl培养基中的transwell插入物中。
    3. 将共培养物在37℃下在5%CO 2细胞培养箱中培养一周。
    4. 在第7天,从插入物收集PBMC
    5. 在350×g下旋转PBMC 10分钟。
    6. 加入120μlPBS,并用sysmex或血球计数器计数PBMC
    7. 计算每种条件的PBMC总数,并在新鲜培养基和新鲜平板中重复PBMC:
      1. 350,000-700,000 PBMC:在1ml培养基中的24孔
      2. < 350,000 PBMC:在750μl培养基中的48孔
      注意:电镀数量太少会导致Treg产生不良。
    8. 将共培养物在37℃下在5%CO 2细胞培养箱中培养一周。
    9. 在第14天,收集PBMC。
    10. 离心PBMC 350 x g 10分钟。
    11. 加入120μlPBS,并用sysmex或血球计数器计数PBMC
    12. 进一步分析与流式细胞术的PBMC。

  3. MSC-PBMC与MSC条件培养基(CM)共培养
    1. 让MSC在T75烧瓶中生长至接近汇合。
    2. 用培养基更换培养基并培养7天,而不更换培养基
    3. 7天后(=第0天)收集培养基,这是MSC条件培养基(CM)
    4. 收获并计数MSC。
    5. 旋转CM 350 x g 10分钟,以摆脱细胞碎片。
    6. 使用10 K Centriprep离心过滤器(根据制造商的说明书)浓缩无细胞的CM。
      1. 用10ml PBS洗涤Centriprep离心过滤器; 减少3,000 x g 10分钟。
      2. 加入MSC-CM(14ml)并以3000×g离心30分钟。
      3. 收集集中的CM并计算相当于100,000 MSC的CM的体积
        1. 实施例:在步骤C5中,计数1.0×10 6 6 MSC,并在步骤C4中收集25ml CM。
        2. 浓缩后,CM的体积为5ml(= 5倍浓缩)
        3. 5ml浓缩的CM相当于1.0×10 6个MSC,因此500μl浓缩的CM相当于100,000MSC。
    7. 在CM中平板400,000个PBMC,相当于100,000个MSC,并在12孔板中加入1ml新鲜培养基。
    8. 将共培养物在37℃下在5%CO 2细胞培养箱中培养一周。
    9. 在第7天,收集PBMC。
    10. 从该点向前,如在共培养中所述继续,在从步骤C4开始的transwell系统中。

参考文献

  1. Melief,S.M.,Schrama,E.,Brugman,M.H.,Tiemessen,M.M.,Hoogduijn,M.J.,Fibbe,W.E.and Roelofs,H。(2013)。 多功能基质细胞通过涉及单核细胞向抗炎巨噬细胞倾斜的新途径诱导人类调节性T细胞 。 Stem Cells 31(9):1980-1991。
English
中文翻译

免责声明

为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。

X


How to cite this protocol: Melief, S. M., Schrama, C. L. and Roelofs, H. (2015). PBMC-MSC Co-cultures for Induction of Treg Generation. Bio-protocol 5(2): e1383. DOI: 10.21769/BioProtoc.1383; Full Text



可重复性反馈:

  • 添加图片
  • 添加视频

我们的目标是让重复别人的实验变得更轻松,如果您已经使用过本实验方案,欢迎您做出评价。我们鼓励上传实验图片或视频与小伙伴们(同行)分享您的实验心得和经验。(评论前请登录)

问题&解答:

  • 添加图片
  • 添加视频

(提问前,请先登陆)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。


登陆 | 注册
引用格式
分享
Twitter Twitter
LinkedIn LinkedIn
Google+ Google+
Facebook Facebook
Sara M. Melief的其他实验方案(1)
C. L. M. Schrama的其他实验方案(1)
Helene Roelofs的其他实验方案(1)