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Culture of Megakaryocytes from Human Peripheral Blood Mononuclear Cells
人外周血单核细胞中巨核细胞的培养   

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Abstract

Megakaryocytes are the precursor cells of platelets and are bona fide resident cells in the bone marrow but extremely low in numbers (~1% of total nucleated cells). Upon terminal differentiation, megakaryocytes increase their size, become polyploid and develop a demarcation membrane system. Mature megakaryocytes form proplatelets, which are cytoplasmic extensions that protrude through the endothelial cell layer of venous sinusoids within the bone marrow, entering into the blood circulation and, subsequently, releasing platelets. Despite limited in numbers, megakaryocytes have been successfully isolated from bone marrow (Tolhurst et al., 2012), adult peripheral blood (Mazur et al., 1990; Thornton et al., 1999), cord blood (Sun et al., 2004) and also from embryonic stem cells (Pick et al., 2013; Eto et al., 2002). These procedures rely on immunostaining using antibodies against megakaryocyte surface markers (i.e. CD41 or CD42b) to isolate an enriched population of megakaryocytes. Here, we describe a culture method wherein megakaryocytes can be grown and differentiated in vitro from human peripheral blood mononuclear cells (PBMCs) directly without the need of initial isolation of CD34+ cells. This method is based on a previously published culture method of human erythroid progenitor cells from PBMCs (Borg et al., 2010; Leberbauer et al., 2005). Although the purity of megakaryocytes is not 100% in this culture method, an enriched fraction of megakaryocytes can be further isolated using BSA gradient or cell-sorting techniques. In addition, our method offers the possibility to freeze the cultures after minimal expansion of yet undifferentiated megakaryocytes, which will yield equal megakaryocyte cultures after thawing when compared to fresh uninterrupted cultures. As this has been proven difficult with CD34+ sorted pluripotent cells, it allows managing samples and to perform downstream analysis when human material is not always available.

Materials and Reagents

Note: *These materials/reagents can be replaced by similar alternatives.

  1. NUNC tubes-50 ml (Thermo Fisher Scientific, catalog number: 339651 )*
  2. Whole blood (aseptic) in anticoagulant (~ 50 ml volume)
  3. Phosphate buffer saline (PBS) (pH 7.4)
  4. Trisodium Citrate Dihydrate (TNC) buffer (38 gram/L demi water, pH 7.0) (Sigma-Aldrich, catalog number: S1804 )
  5. Percoll® (GE Healthcare, catalog number: 17-0891-09 )
  6. StemSpanTM SFEM (500 ml) (STEMCELL technologies, catalog number: 09650 )
  7. Lipids Cholesterol Rich from adult bovine serum (Sigma-Aldrich, catalog number: L4646 )*
  8. Penicillin-Streptomycin (Sigma-Aldrich, catalog number: P4333 )*
  9. Recombinant human stem cell factor (SCF) (Life technologies, catalog number: PHC2115 )*
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: PHC2115”.
  10. Recombinant human thrombopoietin (TPO) (Life technologies, catalog number: PHC9514 )*
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: PHC9514”.
  11. Recombinant human Erythropoietin (EPO) (R&D Systems, catalog number: 287-TC-500 )*
  12. (Optional) Recombinant Human Flt3-Ligand (FLT3-L) (Peprotech, catalog number: 30019 )*
  13. Culture media (see Recipes)
    1. Basis medium (see Recipes)
    2. Phase I medium (expansion) (see Recipes)
    3. Phase II medium (commitment) (see Recipes)
    4. Phase III medium (differentiation) (see Recipes)

Equipment

Note: *Indicated equipment can be replaced by similar alternatives.

  1. Centrifuge-Rotanta 460 (Hettich, catalog number: 5650 )*
  2. CASY Cell counter (Roche, model: CASY TTC )*

Procedure

  1. Start with approximately 50 ml of whole blood, recently collected using a closed system (sterile) from a healthy donor or a patient by a trained phlebotomist under consent. It is advisable to start the culture on the day of collection, but 1-2 days after extraction is also possible (although with donor variation). We normally use citrate phosphate dextrose, but EDTA and heparin are also compatible with this method.
  2. Centrifuge blood at 300 x g (1,000 rpm) for 15 min (using brake 3) at room temperature.
  3. Discard the Platelet Rich Plasma (PRP). Collect the buffy ring and also a little bit of red cell layer by pipetting, and mix 1:1 with PBS/TNC buffer in 50 ml tubes (Figure 1).
  4. Pipette carefully the diluted buffy ring in a proportion of 25 ml on top of 15 ml of Percoll® (1.076 g/ml) in 50 ml Nunc tubes. Centrifuge for 20 min at 1,220 x g or 2,400 rpm (accelerator 3, brake 3, room temperature).
  5. Discard upper layer (plasma) and take the buffy ring (where the peripheral blood mononuclear cells -PBMCs- are) that lays above the Percoll layer, avoiding the red cell lower layer (Figure 1).
  6. Wash PBMCs by adding 1:1 volume of PBS and centrifuge at 475 x g or 1, 500 rpm for 5 min, resuspend in the same volume of PBS.


    Figure 1. Isolation of peripheral blood mononuclear cells (PBMCs). Blood is centrifuged and the buffy ring is diluted 1:1 with PBS/TNC, and added to a proportional volume of Percoll as described. After centrifugation, distinct layers are separated based on density, including plasma (yellow), buffy layer containing PBMCs (purple), Percoll (beige), and RBCs and granulocytes (red). Buffy layer was carefully isolated for further culture of megakaryocytes.

  7. Count the cells using a cell counter. Resuspend to a density of 1-2 x 106 cells/ml in Phase I culture medium (preferably, grow cells initially in a standard 12-well culture plate).
  8. Observe cells every day. A healthy culture is characterized by round, healthy 7-10 μm cells, which grow single in suspension or in clumps (see Figure 2B). Medium should be refreshed every 2-3 days (for this, cells are collected, centrifuged at 475 x g or 1,500 rpm for 5 min at room temperature and resuspended in fresh medium). Dead cells and cell debri are washed out or diluted once the culture is refreshed with new medium, which helps to the welfare of the culture.
  9. Wash/refresh the culture at day 3-4 if necessary, and re-culture in fresh medium, or dilute in ½ volume of Phase I medium, depending on cell numbers, which should not exceed 4 x 106 cells/ml.
  10. From day 6-8, cells can be cultured in Phase II culture medium. In general, the duration of the culture in Phase II should not be longer than 4 days.
  11. Observe cells every day. Wash the culture after 3 days if necessary, and re-culture in fresh medium, or dilute in ½ volume of Phase II medium, depending on cell numbers.
  12. From day 9-12, cells can be cultured in Phase III culture medium. The start of Phase III depends on the culture development during Phase II, i.e. welfare of the culture and the majority of cells reaching a cell diameter > 13 μm (see Figures 2B and 3B).
  13. There will be appearance of bigger cells in the culture at day 13-14 or earlier and there should be gradually increasing numbers of these cells. Observe the culture every day, because depending on donor, some megakaryocytes will undergo terminal differentiation and start forming proplatelets, which implies losing cells due to cell death as well. Morphology and growth is subject to donor variability. It is estimated that from a starting culture with 50 x 106 cells, there will be around 50 x 106 megakaryocytes in average, with donor variation. This means that the culture first expands 2-4 times, and then, only 25-50% of the cells mature into megakaryocytes. See Figure 2 for an overview of the dynamics of the culture in terms of morphology and surface marker expression as measured by flow cytometer.



    Figure 2. PBMC-derived megakaryocyte culture. A. Flow cytometry analysis. A cocktail of antibodies against megakaryocyte surface markers is used to distinguish differentiation stages in human megakaryocyte cultures. The expression of these markers through maturation is depicted below. B. Cell morphology. Cells were observed under the bright field microscope and pictures were taken at day 8, day 12 and day 16 of culture.

    Important notes:
    1. Although we present this culture method with the advantage to culture and differentiate megakaryocytes from PBMCs without the need of CD34+ cell selection, it goes without saying that it can be also applied to sorted CD34+ cells (derived from cord blood, bone marrow or peripheral blood).
    2. It is possible to freeze the cultures during the expansion phase: If needed, cells can be frozen between day 3 and day 6. Cells can be frozen in 10% DMSO in fetal bovine or calf serum (FBS or FCS) and placed at -80 °C in a freezing container overnight to allow gradual temperature drop before transferring them to liquid nitrogen for long-term storage. Upon thawing, culture cells for a couple of days in Phase I and then continue as normal towards Phase II and III. The culture will resume very quickly at this stage at which it was frozen and the growth will be very similar to the original culture in terms of cell numbers, morphology and expression of MK-specific surface markers as analyzed by flow cytometry (Figure 3).

Optional complementary techniques

Materials and Reagents

  1. Bovine serum albumin-100G (BSA) (Sigma-Aldrich, catalog number: A9647)*
  2. Conjugated antibodies for flow cytometry analysis:
    1. CD41a PerCP cy5.5 (CD41) (BD Biosciences, catalog number: 333148)*
    2. CD31 Pacific Blue (CD31) (Biolegend, catalog number: 303114)*
    3. cKIT PE-Cy7 (KIT) (Biolegend, catalog number: 313212)*
    4. CD42b APC (CD42b) (BD Biosciences, catalog number: 551061)*
    5. CD71 FITC (CD71) (BD Biosciences, catalog number: 555536)*
    6. CD61 FITC (CD61) (Sanquin, catalog number: M1592)*
    7. CD49b PE (CD49b) (BD Biosciences, catalog number: 555669)*

Equipment

  1. EasySepTM Magnet (STEMCELL Technologies)
  2. Flow Cytometer (LSRII + HTS) (BD Biosciences)*
  3. Fluorescent Microscope (Life Technologies, model: EVOS FL imaging system)*
    Note: Currently, it is “Thermo Fisher Scientific, model: EVOS FL imaging system”.
    Note: *These reagents/equipment can be replaced by similar alternatives.

Procedure



Figure 3. PBMC-derived megakaryocyte culture variations. A. Fresh vs. Phase I-frozen PBMC-derived megakaryocyte cultures: Flow cytometry analysis. A cocktail of antibodies against megakaryocyte surface markers is used to distinguish differentiation stages in human megakaryocyte cultures. The profiles from both conditions (Fresh vs. Phase I-Frozen) look similar and suggest that megakaryocytes can be successfully grown with this culture method, which allows freezing of cultures during the first days of culture Phase I. B. Cell morphology of cultures derived fromCD34+ sorted cells instead of PBMCs. Cells were observed under the bright field microscope and pictures were taken. The morphology of megakaryocytes from cultured CD34+ sorted cells is similar to that from cultured total PBMCs.


  1. BSA gradient
    1. If you need to collect a purer fraction of megakaryocytes (since the cultures are always heterogeneous), you can do a BSA gradient to collect the bigger cells by sedimentation and proceed with the enriched fraction for further analysis.
    2. A BSA gradient (0%, 1.5% and 3%) can be used to further isolate relatively bigger size megakaryocytes as depicted in Figure 4. If the amount of cells is above 10 million, perform the BSA gradient on a 50 ml tube with 5 ml volume for each layer. If the amount of cells is lower than10 million, perform the BSA gradient on a 15 ml tube with 3 ml volume for each layer.


      Figure 4. Enrichment of cultured megakaryocytes using a BSA gradient. Cells from the Phase III medium were allowed to sediment through a BSA gradient for 15-30 min. Megakaryocytes will pellet at the bottom and can be collected in PBS.

    3. Allow cells to sediment for 15-30 min at 0 x g (no centrifugation) so that megakaryocytes will pellet at the bottom of the tube.
      Collect megakaryocytes with PBS, wash once again at low speed (100 x g or 700 rpm) and proceed further with the downstream analysis.
    4. Alternatively, megakaryocytes can be retrieved by magnetic separation (EasySepTM Magnet) based on positive selection using an antibody (e.g. CD41-FITC) or by FACS sorting depending on the downstream experimental procedure.

  2. Flow cytometry based megakaryocyte differentiation
    Basic flow cytometry analysis can be performed using a cocktail of antibodies that allow the analysis of the differentiation stages of megakaryocytes in culture. Figures 2-3 show examples of the gating strategies, and the sequence of expression of surface markers during megakaryocyte differentiation.

Recipes

  1. Culture media
    1. Basis medium
      StemSpan (500 ml)
      Cholesterol rich lipid mix (Add 2 ml in 500 ml StemSpan medium)
      1% Pen/Strep
    2. Phase I medium (expansion)
      Basis medium supplemented with:
      SCF 100 ng/ml
      TPO 30 ng/ml
      EPO 0.5 U/ml
      FLT3-L 50 ng/ml (optional, although it favors expansion)
    3. Phase II medium (commitment)
      Basis medium supplemented with:
      SCF 50 ng/ml
      TPO 50 ng/ml
      EPO 0.5 U/ml
      FLT3-L 25 ng/ml (optional, although it favors expansion)
    4. Phase III medium (differentiation)
      Basis medium supplemented with:
      TPO 100 ng/ml

Acknowledgments

This protocol was adapted from previous culture method (Borg et al., 2010; Leberbauer et al., 2005) on human erythroid progenitor cells and was developed by V.S., P.P., and L.G. Blood samples were obtained under informed consent, after approval by our institute medical ethics committee in accordance with the 1964 Declaration of Helsinki. This work was partly supported by an I+D Excelencia 2014 grant (SAF2014-55231-P; Ministerio de Economía y Competitividad -Spain- y Fondos Feder, L.G. and P.P.) and a Ramón y Cajal Fellowship (RYC-2013-12587; Ministerio de Economía y Competitividad -Spain-, L.G.).

References

  1. Borg, J., Papadopoulos, P., Georgitsi, M., Gutierrez, L., Grech, G., Fanis, P., Phylactides, M., Verkerk, A. J., van der Spek, P. J., Scerri, C. A., Cassar, W., Galdies, R., van Ijcken, W., Ozgur, Z., Gillemans, N., Hou, J., Bugeja, M., Grosveld, F. G., von Lindern, M., Felice, A. E., Patrinos, G. P. and Philipsen, S. (2010). Haploinsufficiency for the erythroid transcription factor KLF1 causes hereditary persistence of fetal hemoglobin. Nat Genet 42(9): 801-805.
  2. Eto, K., Murphy, R., Kerrigan, S. W., Bertoni, A., Stuhlmann, H., Nakano, T., Leavitt, A. D. and Shattil, S. J. (2002). Megakaryocytes derived from embryonic stem cells implicate CalDAG-GEFI in integrin signaling. Proc Natl Acad Sci U S A 99(20): 12819-12824.
  3. Leberbauer, C., Boulme, F., Unfried, G., Huber, J., Beug, H. and Mullner, E. W. (2005). Different steroids co-regulate long-term expansion versus terminal differentiation in primary human erythroid progenitors. Blood 105(1): 85-94.
  4. Mazur, E. M., Basilico, D., Newton, J. L., Cohen, J. L., Charland, C., Sohl, P. A. and Narendran, A. (1990). Isolation of large numbers of enriched human megakaryocytes from liquid cultures of normal peripheral blood progenitor cells. Blood 76(9): 1771-1782.
  5. Pick, M., Azzola, L., Osborne, E., Stanley, E. G. and Elefanty, A. G. (2013). Generation of megakaryocytic progenitors from human embryonic stem cells in a feeder- and serum-free medium. PLoS One 8(2): e55530.
  6. Sun, L., Tan, P., Yap, C., Hwang, W., Koh, L. P., Lim, C. K. and Aw, S. E. (2004). In vitro biological characteristics of human cord blood-derived megakaryocytes. Ann Acad Med Singapore 33(5): 570-575.
  7. Thornton, M. A. and Poncz, M. (1999). In vitro expansion of megakaryocytes from peripheral blood hematopoietic progenitors. Methods Mol Med 31: 337-345.
  8. Tolhurst, G., Carter, R. N., Miller, N. and Mahaut-Smith, M. P. (2012). Purification of native bone marrow megakaryocytes for studies of gene expression. Methods Mol Biol 788: 259-273.

简介

巨核细胞是血小板的前体细胞,并且是骨髓中的真正的驻留细胞,但数量非常低(约1%的总有核细胞)。在终末分化时,巨核细胞增加其大小,变成多倍体并形成分界膜系统。成熟巨核细胞形成前血小板,其是突出穿过骨髓内的静脉窦内皮的内皮细胞层的细胞质延伸,进入血液循环并随后释放血小板。尽管数量有限,但已经成功地从骨髓中分离出巨核细胞(Tolhurst等人,2012),成人外周血(Mazur等人,1990; Thornton (2004),以及来自胚胎干细胞(Pick等人,2013);脐带血(Sun等人,2004) Eto等人,2002)。这些方法依赖于使用针对巨核细胞表面标记(即CD41或CD42b)的抗体来分离富集的巨核细胞群的免疫染色。在这里,我们描述了一种培养方法,其中巨核细胞可以在体外从人外周血单核细胞(PBMC)直接生长和分化,而不需要初始分离CD34 + sup/+细胞。该方法基于先前公开的来自PBMC的人类红细胞祖细胞的培养方法(Borg等人,2010; Leberbauer等人,2005)。尽管在该培养方法中巨核细胞的纯度不是100%,但是可以使用BSA梯度或细胞分选技术进一步分离巨核细胞的富集级分。此外,我们的方法提供了在尚未分化的巨核细胞的最小扩增后冷冻培养物的可能性,与新鲜的不间断培养物相比,其在解冻后将产生相等的巨核细胞培养物。由于这已被证明难以与CD34 + 分选的多能细胞,它允许管理样品和执行下游分析,当人类材料不总是可用。

材料和试剂

注意:*这些材料/试剂可以替换为类似的替代品。

  1. NUNC管-50ml(Thermo Fisher Scientific,目录号:339651)*
  2. 全血(无菌)抗凝血剂(?50ml体积)
  3. 磷酸盐缓冲盐水(PBS)(pH 7.4)
  4. 柠檬酸二钠二水合物(TNC)缓冲液(38克/升demi水,pH7.0)(Sigma-Aldrich,目录号:S1804)
  5. Percoll (GE Healthcare,目录号:17-0891-09)
  6. StemSpan TM SFEM(500ml)(STEMCELL technologies,目录号:09650)
  7. 脂质来自成年牛血清(Sigma-Aldrich,目录号:L4646)的胆固醇*
  8. 青霉素 - 链霉素(Sigma-Aldrich,目录号:P4333)*
  9. 重组人干细胞因子(SCF)(Life technologies,目录号:PHC2115)*
    注意:目前,是"Thermo Fisher Scientific,Gibco TM ,目录号:PHC2115" />
  10. 重组人血小板生成素(TPO)(Life technologies,目录号:PHC9514)*
    注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:PHC9514" />
  11. 重组人红细胞生成素(EPO)(R& D Systems,目录号:287-TC-500)*
  12. (可选)重组人Flt3-配体(FLT3-L)(Peprotech,目录号:30019)*
  13. 培养基(见配方)
    1. 基本介质(参见配方)
    2. 阶段I介质(膨胀)(参见配方)
    3. 阶段II培养基(承诺)(参见配方)
    4. III期培养基(分化)(见配方)

设备

注意:*指示的设备可以用类似的替代品替换。

  1. Centrifuge-Rotanta 460(Hettich,目录号:5650)*
  2. CASY细胞计数器(Roche,型号:CASY TTC)*

程序

  1. 从大约50ml全血开始,最近使用封闭系统(无菌)从健康供体或患者由训练的抽血者在同意下收集。建议在收集当天开始培养,但提取后1-2天也是可能的(尽管有供体变异)。我们通常使用柠檬酸盐磷酸盐葡萄糖,但EDTA和肝素也与此方法兼容
  2. 在室温下以300×g(1000rpm)离心血液15分钟(使用制动器3)。
  3. 丢弃富血小板血浆(PRP)。通过移液收集白细胞环和一点红细胞层,并在PBS管中混合1:1 PBS/TNC缓冲液(图1)。
  4. 小心地在50ml的Nunc管中以25ml的比例在15ml Percoll(1.076g/ml)的顶部上小心地移动稀释的血红素环。在1,220×g /或2,400rpm(加速器3,制动器3,室温)下离心20分钟。
  5. 丢弃上层(等离子体),并采取位于Percoll层上方的血沉棕黄层(外周血单核细胞-PBMCs),避开红细胞下层(图1)。
  6. 通过加入1:1体积的PBS洗涤PBMC,并以475×g或1,500rpm离心5分钟,重悬于相同体积的PBS中。


    图1.外周血单核细胞(PBMC)的分离。离心血液,用PBS/TNC以1:1稀释血沉棕黄层,如上所述加入到比例体积的Percoll中。离心后,基于密度分离不同的层,包括血浆(黄色),含有PBMC(紫色),Percoll(米色)和RBC和粒细胞(红色)的血沉棕黄层。仔细分离白血球层以进一步培养巨核细胞
  7. 使用单元计数器计数单元格。在I期培养基中重悬至1-2×10 6个细胞/ml的密度(优选地,最初在标准12孔培养板中生长细胞)。
  8. 每天观察细胞。健康的培养物的特征在于圆形,健康的7-10μm细胞,其在悬浮液或块中单独生长(参见图2B)。培养基应每2-3天更新(为此,收集细胞,在475×g下离心或在室温下以1,500rpm离心5分钟,并重新悬浮在新鲜培养基中)。一旦用新培养基更新培养物,将死细胞和细胞碎片洗出或稀释,这有助于培养物的福利。
  9. 如有必要,在第3-4天清洗/更新培养物,并在新鲜培养基中重新培养,或稀释在1/2体积的I期培养基中,这取决于细胞数量,其不应超过4×10 6/>细胞/ml
  10. 从第6-8天,可以在阶段II培养基中培养细胞。一般来说,第二阶段培养的持续时间不应超过4天
  11. 每天观察细胞。如果必要,在3天后洗涤培养物,并在新鲜培养基中重新培养,或者稀释在1/2体积的阶段II培养基中,取决于细胞数量。
  12. 从第9-12天,可以在III期培养基中培养细胞。阶段III的开始取决于阶段II期间的培养物发育,即培养物的福利,并且大多数细胞达到细胞直径> 13μm(见图2B和3B)。
  13. 在第13-14天或更早的时候,培养物中将出现更大的细胞,并且应该逐渐增加这些细胞的数量。每天观察培养物,因为取决于供体,一些巨核细胞将经历终末分化并开始形成前血小板,这意味着由于细胞死亡也丢失细胞。形态和生长受供体变异性的影响。据估计,从具有50×10 6个细胞的起始培养物中,平均有约50×10 6个巨核细胞,具有供体变异。这意味着培养首先扩增2-4倍,然后,仅25-50%的细胞成熟为巨核细胞。参见图2,关于通过流式细胞仪测量的形态学和表面标志物表达的培养物的动力学概述。



    图2.PBMC衍生的巨核细胞培养物 A.流式细胞术分析。使用针对巨核细胞表面标志物的抗体混合物来区分人巨核细胞培养物中的分化阶段。这些标记通过成熟的表达描述如下。 B.细胞形态学。在明视野显微镜下观察细胞,并在培养的第8天,第12天和第16天拍摄照片
    重要注意事项:
    1. 虽然我们提出这种文化方法有优势文化 并且从PBMC分化巨核细胞而不需要CD34 + 细胞选择,不言而喻,它也可以应用于 分选的CD34 + 细胞(源自脐带血,骨髓或外周 血)。
    2. 在这期间可以冷冻培养物 扩增期:如果需要,可以在第3天和第6天之间冷冻细胞。 ?细胞可以在胎牛或牛血清(FBS或10%DMSO)中的10%DMSO中冷冻 FCS)中并在-80℃下在冷冻容器中放置过夜以允许 在将它们转移到液氮中之前逐渐降低温度 ?长期储存。解冻后,培养细胞几天 阶段I,然后继续正常朝向阶段II和III。的 文化将在这个冻结的阶段很快恢复 并且增长将与原始文化非常相似 细胞数目,形态和表达的MK特定表面标记 如通过流式细胞术分析(图3)。

可选补充技术

材料和试剂

  1. 牛血清白蛋白-100G(BSA)(Sigma-Aldrich,目录号:A9647)*
  2. 用于流式细胞术分析的偶联抗体:
    1. CD41a PerCP cy5.5(CD41)(BD Biosciences,目录号:333148)*
    2. CD31太平洋蓝(CD31)(Biolegend,目录号:303114)*
    3. cKIT PE-Cy7(KIT)(Biolegend,目录号:313212)*
    4. CD42b APC(CD42b)(BD Biosciences,目录号:551061)*
    5. CD71 FITC(CD71)(BD Biosciences,目录号:555536)*
    6. CD61FITC(CD61)(Sanquin,目录号:M1592)*
    7. CD49b PE(CD49b)(BD Biosciences,目录号:555669)*

设备

  1. EasySep TM 磁铁(STEMCELL Technologies)
  2. 流式细胞仪(LSRII + HTS)(BD Biosciences)*
  3. 荧光显微镜(Life Technologies,型号:EVOS FL成像系统)*
    注意:目前,它是"Thermo Fisher Scientific,型号:EVOS FL成像系统"。 注意:*这些试剂/设备可以用类似的替代品替换。

程序



图3.来自PBMC的巨核细胞培养变化 A.新鲜与相I-冷冻的PBMC衍生的巨核细胞培养物:流式细胞术分析。使用针对巨核细胞表面标志物的抗体混合物来区分人巨核细胞培养物中的分化阶段。来自两种条件(新鲜与第一阶段冷冻)的谱图看起来相似,并表明巨核细胞可以用这种培养方法成功地生长,这允许在培养的第一天期间冷冻培养物。IB阶段来自CD34的培养物的细胞形态 + 排序的细胞而不是PBMC。在明场显微镜下观察细胞并拍摄照片。来自培养的CD34 +分选的细胞的巨核细胞的形态类似于来自培养的总PBMC的巨核细胞的形态。


  1. BSA梯度
    1. 如果你需要收集一个更纯的部分 巨核细胞(因为文化总是异质的),你可以做 ?BSA梯度以通过沉降收集较大的细胞并进行 ?与富集的馏分用于进一步分析
    2. BSA梯度 (0%,1.5%和3%)可以用于进一步分离相对较大的尺寸 巨核细胞,如图4所示。如果细胞的量高于 1000万,在50ml管中用5ml体积进行BSA梯度 。如果细胞数量低于10万,执行 BSA梯度在15ml管上,每层3ml体积

      图4.使用BSA梯度富集培养的巨核细胞。使来自III期培养基的细胞通过BSA沉淀 梯度15-30分钟。巨核细胞将在底部沉淀并且可以 ?在PBS中收集
    3. 让细胞沉淀15-30分钟 在0 emg×g(未离心),使得巨核细胞在沉淀 底部。
      用PBS收集巨核细胞,再次洗涤 ?在低速(100×g /或700rpm)下进一步进行 下游分析
    4. 或者,巨核细胞可以是 通过磁分离(EasySep TM Magnet)检索 ?使用抗体(例如CD41-FITC)或通过FACS的阳性选择 根据下游实验程序进行分选。

  2. 基于流式细胞术的巨核细胞分化
    基本流式细胞术分析可以使用的混合物进行 允许分析的分化阶段的抗体 巨核细胞。图2-3显示了门控的示例 策略,以及表面标记物的表达序列 巨核细胞分化。

食谱

  1. 文化媒体
    1. 基本介质
      StemSpan(500ml)
      富含胆固醇的脂质混合物(在500ml StemSpan培养基中加入2ml) 1%Pen/Strep
    2. 阶段I介质(膨胀)
      基础培养基补充:
      SCF 100ng/ml
      TPO 30 ng/ml
      EPO 0.5U/ml
      FLT3-L 50 ng/ml(可选,尽管它有利于扩展)
    3. 第二阶段媒介(承诺)
      基础培养基补充:
      SCF 50 ng/ml
      TPO 50 ng/ml
      EPO 0.5U/ml
      FLT3-L 25 ng/ml(可选,尽管它有利于扩展)
    4. III期培养基(分化)
      基础培养基补充:
      TPO 100ng/ml

致谢

该方案改造自人类红细胞祖细胞上的先前培养方法(Borg等人,2010; Leberbauer等人,2005),并由VS,PP,和LG根据知情同意,经我们的研究所医学伦理委员会根据1964年赫尔辛基宣言批准后获得血样。

参考文献

  1. Borg,J.,Papadopoulos,P.,Georgitsi,M.,Gutierrez,L.,Grech,G.,Fanis,P.,Phylactides,M.,Verkerk,AJ,van der Spek,PJ,Scerri,CA,Cassar ,W.,Galdies,R.,van Ijcken,W.,Ozgur,Z.,Gillemans,N.,Hou,J.,Bugeja,M.,Grosveld,FG,von Lindern,M.,Felice,AE,P??atrinos ,GP和Philipsen,S。(2010)。 红细胞转录因子KLF1的半衰期导致胎儿血红蛋白的遗传性持续性。 Nat Genet 42(9):801-805。
  2. Eto,K.,Murphy,R.,Kerrigan,S.W.,Bertoni,A.,Stuhlmann,H.,Nakano,T.,Leavitt,A.D.and Shattil,S.J。(2002)。 源自胚胎干细胞的巨核细胞在整合素信号传导中牵涉CalDAG-GEFI。 Proc Natl Acad Sci USA 99(20):12819-12824。
  3. Leberbauer,C.,Boulme,F.,Unfried,G.,Huber,J.,Beug,H。和Mullner,E.W。(2005)。 不同类固醇共同调节原代人类红细胞祖细胞的长期扩增与终末分化。 Blood 105(1):85-94。
  4. Mazur,E.M.,Basilico,D.,Newton,J.L.,Cohen,J.L.,Charland,C.,Sohl,P.A.and Narendran,A。(1990)。 从正常外周血祖细胞的液体培养物中分离大量富集的人巨核细胞。 Blood 76(9):1771-1782。
  5. Pick,M.,Azzola,L.,Osborne,E.,Stanley,E.G.and Elefanty,A.G。(2013)。 在无饲养层和无血清培养基中从人胚胎干细胞产生巨核细胞祖细胞。 a> PLoS One 8(2):e55530。
  6. Sun,L.,Tan,P.,Yap,C.,Hwang,W.,Koh,L.P.,Lim,C.K.and Aw,S.E。(2004)。 体外人脐带血衍生巨核细胞的生物学特征。 a> 33(5):570-575。
  7. Thornton,M.A。和Poncz,M。(1999)。 体外扩增来自外周血造血祖细胞的巨核细胞。 Methods Mol Med 31:337-345
  8. Tolhurst,G.,Carter,R.N.,Miller,N.和Mahaut-Smith,M.P。(2012)。 纯化天然骨髓巨核细胞用于基因表达研究 方法Mol Biol 788:259-273。
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引用:Salunkhe, V., Papadopoulos, P. and Gutiérrez, L. (2015). Culture of Megakaryocytes from Human Peripheral Blood Mononuclear Cells. Bio-protocol 5(21): e1639. DOI: 10.21769/BioProtoc.1639.
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