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Development of T Cells through Co-culture Lymphoid Progenitor Cells with OP9-DL1 Stromal Cells in vitro
体外与OP9-DL1间质细胞共培养的方法研究T细胞发育   

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Abstract

Scientists commonly study the development of lymphocytes in two ways, adoptive transfer of hematopoietic stem cells or progenitor cells into recipient mice and fetal thymic organ culture (FTOC). Both strategies, especially the first one, are still widely used. However there are some limitations of these two methods such as being time consuming, resulting in limited cell yield, and challenges in the technology. During the last decade, OP9 stromal cells co-culture system has been modified to support lymphocyte development in vitro. This alternative way offers researchers a simple, efficient approach to support lymphoid progenitors to develop in vitro. A more important advantage of this system is that a lot of factors involved in lymphocyte development, such as cytokines and Notch signaling pathway, can be manipulated in order to delineate the mechanisms more clearly. This protocol is based on the experience of supporting T cell development by OP9-DL1 stromal cells. As a matter of fact, this system can be used to facilitate other lymphocytes development in vitro, such as B cells and NK cells, depending on the type of stromal cells and different combinations of cytokines.
Stromal cell line, OP9, was derived from the bone marrow of op/op mice that are deficient for macrophage-colony stimulating factor (M-CSF). In order to investigate the role of Notch signaling on the differentiation of T cells in vitro, intensive studies have been recently done through the co-culture system including lymphoid progenitor cells and modified OP9 stromal cells that express Notch ligands such as Delta-like 1, Delta-like 4, or control vector. These derived OP9 stromal cells lines are termed as OP9-DL1, OP9-DL4, and OP9-V or OP9-vector, respectively.

Materials and Reagents

  1. Cell line: OP9-DL1 stromal cells (OP9-DL1 stromal cells originally made by Dr. J. C. Zuniga-Pflucker. Individual researchers can request this cell line directly from him or from others who got the cell line from him)
  2. OP9-DL1 Recombinant mouse IL-7 (R&D Systems, catalog number: 407-ML-005 )
  3. Recombinant mouse Flt-3 ligand (R&D Systems, catalog number: 427-FL-005 )
  4. Lymphoid progenitor cells
  5. Alpha medium/ Minimum essential medium (MEM) (Life Technologies, Gibco™, catalog number: 41061-037 )
  6. Fetal bovine serum (FBS)
  7. Penicillin-streptomycin-glutamine (Cellgro, catalog number: 30-009-CI )
  8. 2-Mercaptoethanol (Thermo Fisher Scientific, catalog number: ICN19024290 )
  9. Phosphate buffered saline (PBS)
  10. 0.5 M EDTA (Life Technologies, Ambion®, catalog number: AM9260G )
  11. Culture medium (see Recipes)

Equipment

  1. Centrifuges
  2. 10 cm tissue culture dish
  3. 96-well plate , 48-well plate, or 24-well plate
  4. Cell culture Incubator
  5. 15 ml centrifuge tube
  6. Incubator
  7. 42 or 80 µm filter

Procedure

  1. Thaw one tube of OP9-DL1 stromal cells onto a 10 cm dish with 10-12 ml culture medium.
  2. Passage the stromal cells when the confluence reaches about 70-80%, discard the stromal cells if over confluent. Usually one dish of 70% confluent OP9-DL1 stromal cells can be split into 3-4 dishes.
  3. Passage 2-3 times before preparation the single layer of stromal cells for co-culture.
  4. Prepare single layer of OP9-DL1 stromal cells 24 h before the coculture starts (it can be done as early as 48 h before the co-culture or as late as the night before the co-culture experiment).
    Note: The purpose is to let OP9-DL1 stromal cell reach 100% confluent before adding the lymphoid progenitor cells. The co-culture system can be performed with 96-well plate, 48-well plate, and 24-well plate. Usually 4 x 104 OP9-DL1 cells are plated onto one well of 24-well plate the day before the co-culture. One well of 24-well plate can hold 1 ml of medium.
  5. Prepare lymphoid progenitor cells by depletion and sorting.
  6. Prepare mixture of lymphoid progenitor cells and medium containing 1 ng/ml IL-7, and 5 ng/ml Flt-3L. Suck out the old medium from 24-well plate with OP9-DL1 cells and add 1 ml mixture into each well of 24-well plate.
  7. Change half volume medium after 3-4 days. Carefully take out half volume old medium from the top of the well, and slowly add equal volume of fresh medium containing the same cytokines mentioned in step 6 from the top of the side wall of each well. Try not to disturb the contact between the progenitor cells laid on the bottom of each well with the OP9-DL1 stromal cells. Cell number expansion can be observed at different time points depending on the property of the lymphoid progenitor.
  8. Harvest the progenitor cells from the co-culture system on day 7, and then transfer appropriate number of cells to new single-layer OP9-DL1 stromal cells that prepared one day before the harvest.
  9. In order to collect all cells from each well, strongly pipet all medium up and down several times at different locations, transfer the mixture into 15 ml centrifuge tube. Add 0.5 ml 5 mM EDTA (pH 8.0 in sterile PBS) into each well and incubate for 5 min in the incubator, and then pipet up and down several times and combine EDTA with the mixture in 15 ml centrifuge tube. Because OP9-DL1 stromal cells are also detached during the collection, the cell mixture and EDTA have to be filtered with 42 or 80 µm filter (after co-culture for a couple of days, the T lymphocytic progenitor cells touch with OP9-DL1 stromal cells relatively tight).
  10. Strongly pipets the medium up and down several times at different locations of each well, and then transfer everything from each well to a 15 ml centrifuge tube. In order to harvest all the rest cells, add 0.5 ml sterile 5 mM EDTA (pH 8.0 in PBS) to each well and put the whole 24-well plate in the incubator for 5 min. And then pipet up and down EDTA and combines it with the medium in the 15 ml centrifuge tube. Due to the strong pipetting, almost all the stromals cells are detached from the bottom of the 24-well plate and mixed with T lymphocyte progenitors. In order to remove the contamination of OP9-DL1 stromal cells, filters the medium and ETDA with 42 µM or 80 µM filter because OP9-DL1 stromal cells are much bigger than T lineage lymphocyte).
  11. The duration of the co-culture depends on two factors, the property of the lymphoid progenitor cells and the end stage of the co-culture. How to determine whether you have the T cells you want to differentiate and you can now stop the culture (usually, T cells development reaches the end point once CD4 and CD8 double positive cells emerge from the co-culture system. The more immature the progenitor cells are, the longer it takes to reach the end point of the co-culture. However it really depends on the special purpose of each study to determine the time point to stop the co-culture. For example, it could take one week or even shorter time window to observe the transition from one stage to another, like DN1 cells to DN2 cells).

Recipes

  1. Culture medium
    Alpha medium/Minimum essential medium (MEM)
    15% FBS
    1% Penicillin-Streptomycin-Glutamine
    15 μM 2-Mercaptoethanol

Acknowledgments

This protocol was adapted from Wang et al., (2009) and Cho and Zuniga-Pflucker (2003). This work was supported by the NIH.

References

  1. Cho, S. K. and Zuniga-Pflucker, J. C. (2003). Development of lymphoid lineages from embryonic stem cells in vitro. Methods Enzymol 365: 158-169.
  2. Wang, H. C., Perry, S. S. and Sun, X. H. (2009). Id1 attenuates Notch signaling and impairs T-cell commitment by elevating Deltex1 expression. Mol Cell Biol 29(17): 4640-4652.

简介

科学家通常以两种方式研究淋巴细胞的发育,将造血干细胞或祖细胞过继转移到受体小鼠和胎儿胸腺器官培养物(FTOC)中。这两种策略,尤其是第一种策略,仍然被广泛使用。然而,这两种方法存在一些限制,例如耗时,导致有限的细胞产量和技术中的挑战。在过去十年中,OP9基质细胞共培养系统已被修改以支持淋巴细胞在体外发育。这种替代方式为研究人员提供了一种简单,有效的方法来支持淋巴祖细胞在体外发育。该系统的更重要的优点是,涉及淋巴细胞发育的许多因子,例如细胞因子和Notch信号传导途径,可以被操作以便更清楚地描绘机制。该协议是基于支持OP9-DL1基质细胞的T细胞发育的经验。事实上,根据基质细胞的类型和细胞因子的不同组合,该系统可用于促进体外其他淋巴细胞的发育,例如B细胞和NK细胞。基质细胞系OP9源自于巨噬细胞集落刺激因子(M-CSF)缺陷的 op/op/op 小鼠的骨髓。为了研究Notch信号对体外T细胞分化的作用,最近通过共培养系统进行了深入研究,所述共培养系统包括淋巴祖细胞和表达Notch配体的修饰的OP9基质细胞例如Delta样1,Delta样4或对照载体。这些衍生的OP9基质细胞系分别被称为OP9-DL1,OP9-DL4和OP9-V或OP9-载体。

材料和试剂

  1. 细胞系:OP9-DL1基质细胞(OP9-DL1基质细胞,最初由J.C.Zuniga-Pflucker博士制造。个体研究者可以直接从他或其他获得细胞系的人那里请求这种细胞系)
  2. OP9-DL1重组小鼠IL-7(R& D Systems,目录号:407-ML-005)
  3. 重组小鼠Flt-3配体(R& D Systems,目录号:427-FL-005)
  4. 淋巴祖细胞
  5. α培养基/最小必需培养基(MEM)(Life Technologies,Gibco TM,目录号:41061-037)
  6. 胎牛血清(FBS)
  7. 青霉素 - 链霉素 - 谷氨酰胺(Cellgro,目录号:30-009-CI)
  8. 2-巯基乙醇(Thermo Fisher Scientific,目录号:ICN19024290)
  9. 磷酸盐缓冲盐水(PBS)
  10. 0.5M EDTA(Life Technologies,Ambion ,目录号:AM9260G)
  11. 培养基(见配方)

设备

  1. 离心机
  2. 10厘米组织培养皿
  3. 96孔板,48孔板或24孔板
  4. 细胞培养箱
  5. 15ml离心管
  6. 孵化器
  7. 42或80μm过滤器

程序

  1. 解冻一管OP9-DL1基质细胞到10厘米的培养皿与10-12毫升培养基
  2. 当融合达到约70-80%时通过基质细胞,如果融合超过则丢弃基质细胞。 通常一个70%汇合OP9-DL1基质细胞的菜可以分成3-4个皿
  3. 通道2-3次,然后制备单层基质细胞用于共培养
  4. 在共培养开始前24小时制备单层OP9-DL1基质细胞(可以在共培养前48小时或共培养实验前晚上晚些时候进行)。
    注意:目的是使OP9-DL1基质细胞在加入淋巴祖细胞之前达到100%汇合。共培养系统可以用96孔板,48孔板和24孔板进行。通常在共培养前一天将4×10 4个OP9-DL1细胞接种在24孔板的一个孔上。 24孔板的一个孔可容纳1ml培养基。
  5. 通过消耗和分选准备淋巴祖细胞。
  6. 制备淋巴祖细胞和含有1ng/ml IL-7和5ng/ml   Flt-3L。用OP9-DL1细胞从24孔板中取出旧培养基,并向24孔板的每个孔中加入1ml混合物。
  7. 3-4天后更换半体积培养基。从孔的顶部小心地取出一半体积的旧培养基,并从每孔的侧壁顶部缓慢加入等体积的含有步骤6中提及的相同细胞因子的新鲜培养基。尽量不要打扰放置在每个孔底部的祖细胞与OP9-DL1基质细胞之间的接触。可以在不同的时间点观察细胞数量的扩增,这取决于淋巴祖细胞的性质
  8. 在第7天从共培养系统收获祖细胞,然后将适当数量的细胞转移到在收获前一天制备的新的单层OP9-DL1基质细胞。
  9. 为了从每个孔收集所有细胞,强烈吸取所有培养基在不同位置上下数次,将混合物转移到15ml离心管中。向每个孔中加入0.5ml 5mM EDTA(pH 8.0,在无菌PBS中),并在培养箱中孵育5分钟,然后上下移动几次,并将EDTA与混合物在15ml离心管中混合。因为OP9-DL1基质细胞在收集期间也分离,细胞混合物和EDTA必须用42或80μm过滤器过滤(在共培养几天后,T淋巴细胞祖细胞与OP9-DL1基质接触细胞相对紧密)。
  10. 在每个孔的不同位置强烈吸取培养基上下数次,然后将每个孔中的一切转移到15ml离心管中。为了收获所有的休眠细胞,向每个孔中加入0.5ml无菌的5mM EDTA(pH8.0的PBS)到每个孔中,并将整个24孔板在孵育器中放置5分钟。然后用移液管上下移动EDTA,并将其与15ml离心管中的培养基混合。由于强吸取,几乎所有的基质细胞从24孔板的底部分离并与T淋巴细胞祖细胞混合。为了消除OP9-DL1基质细胞的污染,用42μM或80μM的过滤器过滤培养基和ETDA,因为OP9-DL1基质细胞比T谱系淋巴细胞大得多)。
  11. 共培养的持续时间取决于两个因素,淋巴祖细胞的性质和共培养的结束阶段。如何确定是否有你想要分化的T细胞,你现在可以停止培养(通常,一旦CD4和CD8双阳性细胞从共培养系统出现,T细胞发育到达终点)。祖细胞越不成熟细胞,到达共培养的终点所需的时间越长,然而,它确实取决于每个研究的特殊目的,以确定停止共培养的时间点,例如,它可能需要一个星期或甚至更短的时间窗口观察从一个阶段到另一个阶段的过渡,如DN1细胞到DN2细胞)

食谱

  1. 培养基
    Alpha介质/最小必需培养基(MEM)
    15%FBS
    1%青霉素 - 链霉素 - 谷氨酰胺 15μM2-巯基乙醇

致谢

该方案改编自Wang等人(2009)和Cho和Zuniga-Pflucker(2003)。 这项工作得到了NIH的支持。

参考文献

  1. Cho,S.K.and Zuniga-Pflucker,J.C。(2003)。 从胚胎干细胞在体外发展淋巴谱系。 a> Methods Enzymol 365:158-169
  2. Wang,H.C.,Perry,S.S.and Sun,X.H。(2009)。 Id1会削弱Notch信号,并通过提升Deltex1表达来削弱T细胞的承诺。 Mol Cell Biol 29(17):4640-4652。
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Wang, H. (2012). Development of T Cells through Co-culture Lymphoid Progenitor Cells with OP9-DL1 Stromal Cells in vitro. Bio-protocol 2(10): e189. DOI: 10.21769/BioProtoc.189.
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Zongyou Guo
CUMC
We are looking to differentiate iPSCs into T cells.

Can I some OP9-DL1 cells from you?

Thanks.
12/18/2013 12:37:06 PM Reply
Hello,

I would like to know if I must irradiate OP9 cells if I want to treat ALL-T cells for 7 days ?

Thanks

Claire Drullion ( claire.drullion@hotmail.fr)
3/19/2012 10:53:54 PM Reply
Hongcheng Wang
Immunobiology and Cancer Research Program, Oklahoma Medical Research Foundation, USA

I do not have any experience with ALL T cells and therefore can't answer your question. Wish you the best luck with your experiments

3/27/2012 1:18:57 PM