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Feb 2016
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Isolation and Long-term Cultivation of Mouse Alveolar Macrophages
小鼠肺泡巨噬细胞的分离与长期培养   

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Abstract

Alveolar macrophages (AM) are tissue-resident macrophages that colonize the lung around birth and can self-maintain long-term in an adult organism without contribution of monocytes. AM are located in the pulmonary alveoli and can be harvested by washing the lungs using the method of bronchoalveolar lavage (BAL). Here, we compared different conditions of BAL to obtain high yields of murine AM for in vitro culture and expansion of AM. In addition, we describe specific culture conditions, under which AM proliferate long-term in liquid culture in the presence of granulocyte-macrophage colony-stimulating factor. This method can be used to obtain large numbers of AM for in vivo transplantation or for in vitro experiments with primary mouse macrophages.

Keywords: Macrophage (巨噬细胞), Alveolar (肺泡), Lungs (肺), Self-renewal (自我更新), Bronchoalveolar lavage (支气管肺泡灌洗), Primary cell culture (原代细胞培养)

Background

AM are resident tissue macrophages of the lungs with critical importance for immune regulation and surfactant homeostasis (Kopf et al., 2015). Due to their localization in the airspace of the alveoli, AM are directly exposed to inhaled air and pathogens or other aerosolized particles. Consequently, AM play a crucial role in the initiation or suppression of inflammatory responses and are the subject of investigation in numerous studies that explore mechanisms of pulmonary diseases (Hodge et al., 2007; Sun and Metzger, 2008; Happle et al., 2014; Schneider et al., 2014a; Machiels et al., 2017; Yu et al., 2017). Interestingly, AM in the mouse originate from fetal monocytes and are able to self-maintain their numbers in vivo without contribution of bone-marrow-derived monocytes under steady state conditions (Guilliams et al., 2013; Hashimoto et al., 2013). AM are unique in that they reside outside of the body surface and are directly exposed to the external environment. They can therefore be isolated with minimal tissue disturbance using bronchoalveolar lavage (BAL). We previously demonstrated that the self-renewal property of AM harvested by BAL is maintained in culture by growing AM long-term in liquid media or serially re-plating AM in semi-solid media (Soucie et al., 2016; Imperatore et al., 2017). Here, we describe the methodological advancements in BAL and specific culturing conditions optimized for long-term culture and high AM yields.

To obtain sufficient quality and numbers of AM, we tested different cell harvesting conditions and developed a culture method that allows long-term maintenance of AM in vitro. Our BAL method was based on earlier studies using pre-warmed PBS and EDTA for detaching AM from lung alveoli (Steele et al., 2003; Zhang et al., 2008). Additionally, serum was added for cellular protection during the isolation period. Importantly, both omitting EDTA in the lavage buffer and using pre-cooled PBS for lavage resulted in lower AM yield in our hands. Our comparisons demonstrated that the variable with the largest effect on yield was the temperature of the BAL buffer (Figure 1). Whereas many earlier studies obtain BAL cell numbers below 2 x 105 per wild-type mouse (Table 1), our comparative analysis indicated that BAL cell numbers can be considerably increased by these optimizations. We generally obtained up to 5 x 105-7 x 105 live (Trypan-Blue-negative) cells per mouse using this method (Figure 1). FACS analysis revealed that more than 98% of cells were alive at the time of recording using the Zombie Violet Fixable Viability Kit, independent of whether the BAL was performed with 4 °C PBS or 37 °C PBS/EDTA/FBS. Our experience has shown that when performing dozens of BALs on the same day long waiting times on ice will result in higher cell death unless low amounts of FBS are added to the BAL buffer (typically 0.5%, although we have good experience with up to 2%).


Figure 1. Comparison of BAL conditions using pre-cooled or pre-warmed PBS with or without 2 mM EDTA. Either 4 °C PBS without EDTA, 4 °C PBS with 2 mM EDTA and 0.5% FBS, or 37 °C PBS with 2 mM EDTA and 0.5% FBS was used. Numbers show the total amount of living cells (Trypan-Blue-negative) per BAL treatment per mouse. Each symbol denotes the mean cell count of 3 technical replicates of an individual mouse; horizontal lines indicate the mean, error bars show standard error of the mean (SEM); one-way ANOVA with Tukey’s multiple comparisons test; ns, non-significant.

Table 1. Comparison of the efficiency of different BAL protocols. Total cell number in BAL fluid obtained in cited studies. Only counts from WT animals (or comparable conditions, such as control-treated WT animals) were considered. WT denotes wild-type mouse.


Increased efficiency in harvesting BAL cells is advantageous for in vivo reconstitution of multiple recipient animals such as strains devoid of endogenous AM, such as GM-CSF-receptor-deficient mice (Guilliams et al., 2013; van de Laar et al., 2016). High starting cell numbers and viability of harvested cells will accelerate establishment of long-term AM cultures and improve cellular yield. We also noticed several culture conditions outlined in the detailed protocol that affect the quality, yield, doubling time and durability of the cultures. In order to avoid cell activation or death, several parameters need to be controlled. Firstly, we used exclusively sterile supplies and applied sterile handling techniques to avoid activation of AM. Secondly, we use non-treated plastic ware (not tissue-culture treated plates or dishes) and a gentle detachment protocol. Together, these technical improvements will be helpful for starting and maintaining a long-term AM culture.

Biochemical and genetic manipulations of macrophages that require a large number of cells could so far only be done in cell lines, such as RAW 264.7 or J774A.1 cells (Ralph and Nakoinz, 1975; Raschke et al., 1978), oncogene-transformed cells, for example Myc (Baumbach et al., 1986; Baumbach et al., 1987) or SV40 transformed macrophages like IC-21 (Walker and Demus, 1975) or MH-S cells (Mbawuike and Herscowitz, 1989), macrophages differentiated from progenitors (Zhang et al., 2008; Fejer et al., 2013) or non-transformed but genetically modified macrophages, such as Maf-DKO macrophages (Aziz et al., 2009). The ability to obtain large numbers of normal unmodified AM in culture allows such experiments in primary resident macrophages. Our studies on macrophage self-renewal mechanisms serve as an example (Soucie et al., 2016; Imperatore et al., 2017).

Materials and Reagents

  1. 15-ml conical tubes (Corning, catalog number: 352196)
  2. Bottle-top vacuum filter with 0.22 μm membrane (Corning, catalog number: 431161)
  3. Plastic storage bottle (Corning, catalog number: 430281)
  4. 70-μm sterile cell strainer (BD, catalog number: 340633)
  5. 1-ml syringe (Braun, catalog number: 9161406V)
  6. 18-G cannula (Braun, catalog number: 4667123)
  7. Petri dish 94/16 mm (Greiner Bio-one, catalog number: 633181)
  8. Non-treated 6-well plate (NuncTM, catalog number: 150239)
  9. C57BL/6 mice (aged 6-10 weeks)
  10. PBS, pH 7.2 (Thermo Fisher Scientific, GibcoTM, catalog number: 20012019)
  11. EDTA stock solution (e.g., 0.5 M, pH 8.0)
  12. Hemolysis buffer (self-made or commercial, e.g., Morphisto, catalog number: 12146)
  13. Trypan Blue solution 0.4% (Sigma-Aldrich, catalog number: T8154)
  14. RPMI 1640 Medium, no glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 31870025)
  15. Fetal bovine serum (Testing of different batches is recommended)
  16. Gentamicin sulphate 50 mg/ml in aqueous solution (Lonza, catalog number: BE02-012E)
  17. Penicillin-Streptomycin (10,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122)
  18. Sodium Pyruvate (100 mM) (Thermo Fisher Scientific, GibcoTM, catalog number: 11360070)
  19. GlutaMAXTM Supplement (Thermo Fisher Scientific, GibcoTM, catalog number: 35050038)
  20. Conditioned medium from J558L cell line transfected with murine GM-CSF cDNA as a source for GM-CSF (Zal et al., 1994; Stockinger et al., 1996; Rayasam, 2015)
  21. ESGRO Complete Accutase (Merck, catalog number: SF006)
  22. EGTA stock solution (e.g., 0.5 M, pH 8.0)
  23. UltraComp eBeadsTM Compensation Beads (Thermo Fisher Scientific, Invitrogen, catalog number: 01-2222-41)
  24. Zombie Violet Fixable Viability Kit (BioLegend, catalog number: 423113)
  25. FACS antibodies (as indicated in Table 2)
  26. BAL buffer (see Recipes)
  27. Complete medium (see Recipes)
  28. AM culture medium (see Recipes)
  29. Detachment medium (see Recipes)

Equipment

  1. Pipettes
  2. Mouse dissection tools (scissors, forceps)
  3. Water bath set to 37 °C
  4. Refrigerated benchtop centrifuge for spinning conical tubes
  5. Hemocytometer (Roth, catalog number: T729.1)
  6. Incubator (37 °C, 5% CO2)
  7. Inverse microscope

Procedure

  1. Harvest alveolar macrophages by bronchoalveolar lavage (BAL)
    1. For each mouse, prepare a 15-ml conical tube filled with 3 ml complete medium (see Recipes).
    2. Warm-up BAL buffer (see Recipes) to 37 °C in a water bath. Keep warm during the whole procedure. 
    3. Euthanize the mouse by cervical dislocation without rupturing the jugular vein or the trachea to avoid exposing AM to CO2 or isoflurane, which could affect functional properties of AM.
    4. Using dissection tools, remove the skin, ribcage and muscles to expose both lungs and trachea. Avoid cutting or rupturing blood vessels.
      Note: Since methods for the surgical exposure of lungs and the trachea have been published previously in this journal, the reader is referred to those protocols for instructions (Han and Ziegler, 2013; Tibbitt and Coquet, 2016; Jhingran et al., 2016; Sun et al., 2017).
    5. Use a fine scissor to make a small incision in the upper part of the trachea just below the larynx. The part of the trachea facing downwards (away from the experimenter) should remain intact, do not cut through the whole trachea. 
    6. Use the incision to insert a slightly blunted 18-G cannula and direct the cannula 5 mm deeper into the trachea towards the lungs. Take care not to damage lung tissue. 
    7. Attach a 1 ml syringe filled with 1 ml warm BAL buffer onto the inserted cannula. 
    8. Inject 1 ml buffer while fixating the cannula position with the other hand. 
    9. Pull the plunger to collect BAL fluid in the syringe. About 800-900 μl can be recovered. Observe that the pressure should not be too high, otherwise the alveoli will burst and BAL fluid will be lost. Upon injection and collection, the lungs should visibly inflate and deflate. 
    10. Filter collected BAL fluid through a 70 μm cell strainer into the 15-ml tube with 3 ml complete medium from Step A1. 
    11. Repeat Steps A6-A10 for 9 more times each time with fresh warm BAL buffer. Pool cells into the same 15-ml tube.
    12. Collect cells by centrifugation at 300 x g, 5 min at 4 °C. Remove supernatant. The cell pellet should be white. A red/pink color indicates that blood was accidentally collected during the BAL.
    13. Add 1 ml hemolysis buffer for 2 min incubation at room temperature to lyse residual erythrocytes. Fill up tube with complete medium to stop lysis and collect cells by centrifugation as before. Remove supernatant. The color of the cell pellet should be white now.
    14. Resuspend cell pellet in 500 μl BAL buffer and take a sample for counting using a hemocytometer chamber after staining with Trypan Blue to exclude dead cells. Count only live (Trypan-Blue negative) cells. 
    15. Calculate the total cell number per BAL. Typically, 5 x 105-7 x 105 live cells per adult wild-type mouse aged 6-8 weeks can be recovered when using pre-warmed BAL buffer, containing PBS with 2 mM EDTA and 0.5% serum. 
    16. Proceed to cell staining and flow cytometry analysis or in vitro cultivation.

  2. Flow cytometric analysis of alveolar macrophages
    1. Block unspecific binding sites on cells with TruStain fcX and concomitantly stain with Zombie Violet in 200 μl cold PBS (without FBS) at 4 °C in the dark for 15 min (see Table 2).
    2. Wash cells with cold BAL buffer by centrifugation at 300 x g for 5 min at 4 °C. 
    3. Stain cells in a volume of 100 μl per 1 million cells according to Table 2 for 30 min at 4 °C in the dark using BAL buffer.

      Table 2. FACS reagents used for staining BAL AM


    4. Prepare compensation beads for each antibody conjugate.
    5. Wash cells with BAL buffer, resuspend in 200 μl BAL buffer for recording.
    6. Record cells by flow cytometry after acquiring the compensation beads. AM are double-positive for SiglecF and CD11c (Figures 2A-2C), and > 98% viable (Figure 2D).


      Figure 2. FACS analysis of BAL AM. A. Simple gating strategy for exclusion of doublets, dead cells. AM are SiglecF- and CD11c-positive. B-C. BAL cells harvested with pre-warmed BAL buffer containing EDTA are phenotypically not different from BAL cells harvested using pre-cooled PBS. Each symbol denotes 1 mouse. Typically, > 95% of BAL cells are AM. D. Viability analysis of BAL singlet cells assessed by staining with Zombie Violet Fixable Dye.

  3. Cultivation of alveolar macrophages
    1. Collect cells by centrifugation as before. Remove supernatant.
    2. Plate 3 x 105-4 x 105 cells per well of a non-treated 6-well plate in 3 ml pre-warmed AM culture medium (see Recipes).
      Note: Typically, 3 x 105-4 x 105 cells are plated in 1 well of a 6-well plate. If BAL cells of several mice are pooled, 1.1-1.2 million cells can be plated in a non-treated 94 mm Petri dish in 10 ml pre-warmed AM culture medium. 
    3. Add gentamicin to the AM culture (1:1,000).
      Note: Gentamicin is omitted after the first medium change. 
    4. Incubate at 37 °C, 5% CO2
    5. Replace culture supernatant after 6-18 h with fresh AM culture medium and discard the supernatant. 
    Note: Cells will adhere fully within a few hours after the first plating and we do not keep cells in suspension at the first medium exchange. However, for subsequent medium exchanges, the cells in the supernatant are collected as well since a typical AM culture consists of both adherent and suspended cells (see also Notes section).
    1. Change medium every 2 days until the cell culture reaches confluency.
    2. To change medium, transfer the medium and suspension cells into a 15 ml-tube. Add 2 ml warm AM culture medium to the well with adherent cells to prevent drying-out. Collect the suspension cells using centrifugation at 300 x g 5 min. Resuspend the pelleted cells in 1 ml warm AM culture medium and combine with adherent cells.
      Note: Freshly harvested primary AM will double every 7-10 days (Soucie et al., 2016). If the majority of AM appear stretched (spindle-like) and activated, increasing the amount of conditioned medium or adding recombinant GM-CSF might help; however, proliferative capacity will be limited and it might advisable to start a new culture (see Figure 3 for exemplary images of early AM culture).


      Figure 3. Representative images of AM culture within the first days after plating the cells. A. AM culture with predominantly round-shaped cells that are partly floating and partly adherent on Day 1 after plating. B. Same culture as (A) on Day 2. C. Same culture as (A) on Day 4. D. Example for an AM culture with a large fraction of elongated, dark cells on Day 4 after plating. Arrowheads indicate dividing cells, 100x magnification.

    3. To detach cells from a confluent well, collect suspension cells into a 15-ml tube. 
    4. Add 750 μl detachment medium (see Recipes) to 1 well of a 6-well plate (or 3 ml to a 94 mm Petri dish) and incubate for 10-30 min at 37 °C.
      Note: AM are very adherent and prone to rupture when using too harsh detachment procedures. Thus, the use of non-treated plastic ware and proper detachment medium is important (see Recipes). Ruptured cells in the culture medium might affect both activation status and proliferative capacity of AM. 
    5. Detachment of cells can be supported by pipetting on the plastic bottom gently to avoid cellular damage (see Notes).
    6. Pool detached cells with cells in suspension and centrifuge cells at 300 x g for 5 min. 
    7. Resuspend cell pellet in 1 ml warm AM cultured medium and take a sample for counting using a hemocytometer chamber after staining with Trypan Blue to exclude dead cells. Count only live (Trypan-Blue negative) cells.
    8. If the cell number has doubled, add 5 ml warm AM culture medium and split into 2 wells of a 6-well plate (or correspondingly to 2x 94 mm Petri dishes). In general, the cell number plated is maintained around the values indicated above in the note to Step C2.
      Note: Earlier, we could show that AM culture remains proliferative for at least 10 passages (Soucie et al., 2016). Since then, we have experience with AM cultures that remain proliferative even beyond 20 passages with no indication of a decline in proliferative capacity.

Data analysis

Harvested cells were counted manually using a hemocytometer and considering only Trypan-Blue-negative cells. Stained cells were recorded on a BD LSRFortessa with 5 lasers using BD FACSDiva software and analyzed using FlowJo v10. Microscope images were acquired on an inverse microscope (Leica DMi1) equipped with a digital camera (MC120). Gating was performed as indicated in Figure 2A. To test for statistically significant differences between the means of three groups (Figure 1), one-way ANOVA with Tukey’s multiple comparisons test was performed using GraphPad Prism 7. No data points were excluded.

Notes

  1. Proliferative AM are round-shaped and semi-adherent. Re-plating of suspension AM will result in part of the cells attaching to the new well, while the other part remains in suspension. Take care to not lose the suspended cells when changing medium as this will reduce the number of proliferative cells and slow the expansion of the culture.
  2. When detaching cells, do not pipet the cell suspension up and down extensively, this might affect the viability of the culture; if cells do not detach readily, collect detachment medium containing already detached cells and perform another round of incubation with fresh detachment medium and/or increase the incubation time. Late-passage cells require shorter incubation times (~ 5 min) than early-passage cultures (up to 30 min). 
  3. The percentage of conditioned medium should be titrated after preparation of each batch by testing the growth of AM in the presence of various amounts of conditioned medium (e.g., 1, 2, 5, 10% in complete medium). In our batches, we use typically 1%-3% conditioned medium diluted into complete medium (e.g., 100 μl in 10 ml), which corresponds roughly to 2-5 ng/ml purified recombinant mouse GM-CSF.
  4. We successfully replaced conditioned medium with 20 ng/ml recombinant GM-CSF (Peprotech) for long-term culture. Lower GM-CSF concentrations might be sufficient but have not been tested.

Recipes

  1. BAL buffer
    PBS
    2 mM EDTA (dilute 1:250 from 0.5 M EDTA stock solution)
    0.5% Fetal bovine serum (FBS)
    Sterile-filter using vacuum filtration and keep at 4 °C until use
  2. Complete medium
    RPMI 1640
    1x GlutaMAX
    1x Pyruvate
    1x Penicillin/Streptomycin
    10% FBS
    Sterile-filter using vacuum filtration and keep at 4 °C until use
  3. AM culture medium
    Supplement complete medium with 1-5% conditioned medium containing mouse GM-CSF (needs to be titrated)
    Pre-warm an aliquot in a water bath to 37 °C before use
  4. Detachment medium
    ESGRO Complete Accutase
    1 mM EGTA (dilute 1:500 from 0.5 M EGTA stock solution)
    Aliquot and freeze at -20 °C. Pre-warm an aliquot in the water bath to 37 °C before use

Acknowledgments

We thank Stephanie Vargas Aguilar and Sethuraman Subramanian for technical discussions and helpful comments on the manuscript, Philippe Pierre for J558L cells, and the flow cytometry and animal caretaking facilities of the Max-Delbrück-Centrum for assistance. This study was supported by institutional grants from Institut National de la Santé et de la Recherche Médicale, Centre National de la Recherche Scientifique, and Aix-Marseille University to the CIML and grants to M.H. Sieweke from the Agence Nationale de la Recherche (ANR-11-BSV3-0026 and ANR-17-CE15-0007-01), the Institut National du Cancer (InCA13-10/405/AB-LC-HS), Fondation pour la Recherche Médicale (DEQ. 20110421320), and the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement number 695093 MacAge). K. Molawi was supported by a Human Frontier Science Program long-term fellowship and Stiftung Charité, L. Geiersdottir by the Fondation ARC pour la Recherche sur le Cancer. M.H. Sieweke is a Berlin Institute of Health–Einstein visiting fellow at MDC and an Alexander von Humboldt Professor at TU Dresden.

Competing interests

The authors declare no competing financial interests.

Ethics

Animal husbandry and mouse work were conducted in accordance with the German Animal Welfare legislation, after the approval by the Landesamt für Gesundheit und Soziales (for work in Berlin, following the guidelines of the Institutional Animal Care and Use Committee of the Max Delbrück Centrum für Molekulare Medizin) and after the approval by the Landesdirektion Sachsen (for work in Dresden).

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简介

肺泡巨噬细胞(AM)是组织驻留的巨噬细胞,其在出生时定植于肺部并且可以在成体生物体中长期自我维持而没有单核细胞的贡献.AM位于肺泡中并且可以通过以下方式收获: 在这里,我们比较不同的BAL条件,以获得高产量的鼠AM用于体外培养和扩增AM。此外,我们描述了特定的培养条件,其中 AM在粒细胞 - 巨噬细胞集落刺激因子存在下长期在液体培养中增殖。该方法可用于体内移植或体外获得大量AM / em>用原代小鼠巨噬细胞进行实验。
【背景】AM对于对免疫调节和表面活性物质稳态至关重要的常驻组织巨噬细胞(Kopf et al。,2015)。由于它们定位于肺泡的空气空间,AM直接暴露于吸入AM在炎症反应的发生或抑制中起着至关重要的作用,并且是许多引起肺部疾病机制的研究中的探索性研究的主题(仿真) Happle et al。,2014; Schneider et al。,2014a; Machiels et al。,2017 Yu et al。,2017)。有趣的是,小鼠的AM来源于胎儿单核细胞,并且能够在没有骨髓贡献的情况下自我维持其体内体内的数量。 AM在稳态条件下是独特的(Gilliams et al。,2013; Hashimoto et al。,2013).AM的独特之处在于它们位于体外使用支气管肺泡灌洗(BAL)可以认为它们被隔离,组织干扰最小。我们以前证明了BAL收获的AM的自我更新特性是持续表达在这里,我们描述了AM-半固体培养基中的方法学(Soucie et al。,2016; Imperatore et al。,2017)。 BAL的进步和针对长期培养和高AM产量优化的特定培养条件。

为了获得足够的质量和数量的AM,我们测试了不同的细胞收获条件,并开发了一种培养方法,可以在体外长期维持AM 。我们的BAL方法是基于使用预热的早期研究PBS和EDTA用于从肺泡分离AM(Steele et al。,2003; Zhang et al。,2008)。另外,在分离过程中加入血清用于细胞保护重要的是,灌洗缓冲液中的天文台EDTA和使用预先冷却的PBS进行灌洗均导致我们手中的AM结果较低。我们的比较表明,对产量影响最大的变量是BAL缓冲液的温度。我们有许多早期的研究,研究每只野生型小鼠2×10 5以下的BAL细胞数量(表1),我们的比较分析表明BAL细胞数量在这些优化中被认为是无效的。获得高达5 x 10 5-7 x 10 5直播(Trypan-Blu使用该方法每只小鼠的e阴性细胞(图1).FACS分析显示,使用Zombie Violet Fixable Viability Kit记录时,超过98%的细胞存活,独立于BAL进行4° C PBS或37°C PBS / EDTA / FBS。我们的经验表明,当在冰上等待的同一天进行数十次BAL时,会导致更高的细胞死亡而没有少量的FBS被添加到BAL缓冲液中(通常0.5%,虽然我们有高达2%的良好经验)。
图1.使用预冷或预热的PBS(含或不含2 mM EDTA)比较BAL条件。 4°C不含EDTA的PBS,4°C PBS含2 mM EDTA和0.5%FBS使用数字或含有2mM EDTA和0.5%FBS的37℃PBS。数字显示每只小鼠每BAL处理的活细胞总量(锥虫蓝色阴性)。每个符号表示3个技术重复的平均细胞数。单因素方差分析采用Tukey多重比较检验; ns,无显着性。单个小鼠;水平线表示平均值,误差条表示平均值的标准误差(SEM);

表1.不同BAL方案效率的比较引用研究中获得的BAL液中的总细胞数。仅考虑来自WT动物的计数(作为可比较的条件,对照处理的WT动物) WT表示野生型小鼠。


提高收获BAL细胞的效率有利于体内重建多个受体动物,例如缺乏内源性AM的菌株,例如GM-CSF-受体缺陷型小鼠(Gilliams等。 Van de Laar et al。,2016)。高起始细胞数和收获细胞的活力将加速长期AM培养物的建立并提高细胞产量。我们还注意到几个为了避免细胞激活或死亡,需要控制几个参数。首先,我们专门使用。其次,我们使用未经处理的塑料制品(不是经过组织培养处理的板材或餐具)和温和的分离协议。这些技术改进将共同启动和维持长期的AM培养。重。

巨噬细胞的生化和遗传操作需要大量细胞,这些细胞只能在细胞系中进行,如RAW 264.7或J774A.1细胞(Ralph和Nakoinz,1975; Raschke et al。 ,1978),致癌基因转化细胞,例如Myc(Baumbach et al。,1986; Baumbach et al。,1987)或SV40转化的巨噬细胞如IC-21( Walker和Demus,1975)或MH-S细胞(Mbawuike和Herscowitz,1989),巨噬细胞与祖细胞分化(Zhang et al。,2008; Fejer et al。, 2013)或非转化但转基因的巨噬细胞,如Maf-DKO巨噬细胞(Aziz et al。,2009)。在培养中获得大量正常修饰模型的能力允许在初级中进行此类实验。我们对巨噬细胞自我更新机制的研究就是一个例子(Soucie et al。,2016; Imperatore et al。,,2017)。

关键字:巨噬细胞, 肺泡, 肺, 自我更新, 支气管肺泡灌洗, 原代细胞培养

材料和试剂

  1. 15毫升锥形管(康宁,目录号:352196)
  2. 带0.22μm膜的瓶顶真空过滤器(Corning,目录号:431161)
  3. 塑料储存瓶(康宁,目录号:430281)
  4. 70-μm无菌细胞过滤器(BD,目录号:340633)
  5. 1毫升注射器(Braun,目录号:9161406V)
  6. 18-G插管(Braun,目录号:4667123)
  7. 培养皿94/16 mm(Greiner Bio-one,目录号:633181)
  8. 未处理的6孔板(Nunc TM ,目录号:150239)
  9. C57BL / 6小鼠(6-10周龄)
  10. PBS,pH 7.2(Thermo Fisher Scientific,GibcoTM,目录号:20012019)
  11. EDTA储备溶液(例如,0.5 M,pH 8.0)
  12. 溶血缓冲液(自制或商业,例如,Morphisto,目录号:12146)
  13. 台盼蓝溶液0.4%(Sigma-Aldrich,目录号:T8154)
  14. RPMI 1640培养基,无谷氨酰胺(Thermo Fisher Scientific,GibcoTM,目录号:31870025)
  15. 胎牛血清(推荐不同批次的测试)
  16. 硫酸庆大霉素50 mg / ml水溶液(Lonza,目录号:BE02-012E)
  17. 青霉素 - 链霉素(10,000 U / ml)(Thermo Fisher Scientific,GibcoTM,目录号:15140122)
  18. 丙酮酸钠(100 mM)(Thermo Fisher Scientific,GibcoTM,目录号:11360070)
  19. GlutaMAX TM 补充剂(Thermo Fisher Scientific,Gibco TM ,目录号:35050038)
  20. 来自J558L细胞系的条件培养基转染小鼠GM-CSF cDNA作为GM-CSF的来源(Zal et al。,1994; Stockinger et al。,1996; Rayasam ,2015)
  21. ESGRO Complete Accutase(默克,目录号:SF006)
  22. EGTA储备溶液(例如,0.5 M,pH 8.0)
  23. UltraComp eBeadsTM补偿珠(Thermo Fisher Scientific,Invitrogen,目录号:01-2222-41)
  24. Zombie Violet Fixable Viability Kit(BioLegend,目录号:423113)
  25. FACS抗体(如表2所示)
  26. BAL缓冲液(见食谱)
  27. 完全中等(见食谱)
  28. AM培养基(见食谱)
  29. 支队介质(见食谱)

设备

  1. 移液器
  2. 小鼠解剖工具(剪刀,镊子)
  3. 水浴温度设定为37°C
  4. 用于旋转锥形管的冷冻台式离心机
  5. 血细胞计数器(罗斯,目录号:T729.1)
  6. 培养箱(37°C,5%CO 2 )
  7. 逆显微镜

程序

  1. 通过支气管肺泡灌洗(BAL)收获肺泡巨噬细胞
    1. 对于每只小鼠,准备一个装有3毫升完全培养基的15毫升锥形管(见食谱)。
    2. 在整个过程中保持温暖。预热BAL缓冲液(见食谱)在37°C水浴中。
    3. 通过颈椎脱位使小鼠安乐死而不破坏颈静脉或气管,以避免AM暴露于CO 2 或异氟烷,这可能影响AM的功能特性。
    4. 避免切割或破坏血管。
      使用解剖工具,去除皮肤,胸腔和肌肉,露出肺部和气管。 注意:由于手术暴露弓箭的方法已经在本期刊中发表过,读者可以从协议中获取指导(Han和Ziegler,2013; Tibbitt和Coquet,2016; Jhingran > et al。 ,2016; Sun et al。 ,2017)。
    5. 气管朝下的部分(远离实验者)应保持完整,不要穿过整个气管。 
    6. 注意不要损伤肺组织。注意不要损伤肺部5毫米深的气管朝向肺部。
    7. 将装有1 ml温热BAL缓冲液的1 ml注射器连接到插入的插管上。 
    8. 注射1 ml缓冲液,同时用另一只手固定套管位置。 
    9. 可以回收大约800-900μl。观察到压力不应该太高,否则肺泡会破裂,BAL液会流失。注射和采集时,肺部明显膨胀和放气。 
    10. 将收集的BAL液通过70μm细胞过滤器过滤到15ml管中,其中含有来自步骤A1的3ml完全培养基。 
    11. 每次用新鲜的温热BAL缓冲液重复步骤A6-A10 9次。将细胞合并到相同的15ml管中。
    12. 细胞沉淀应为白色。红色/粉红色指示血液在BAL期间意外收集。通过在300 x x x 5 ,4°C下离心5分钟收集细胞。
    13. 用完全培养基填充试管以停止裂解并如前所述通过离心收集细胞。除去上清液。细胞沉淀的颜色为白色。在室温下加入1ml溶血缓冲液孵育2分钟以裂解残留的红细胞。
    14. 仅计数活的(锥虫蓝色阴性)细胞。将细胞沉淀重悬于500μlBAL缓冲液中,并用台盼蓝染色后用血细胞计数器室取样以排除死细胞。
    15. 计算每个BAL的总细胞数。通常,每个成年6-8周的野生型小鼠可以回收5×10 5-7×10 5个活细胞。使用预热BAL缓冲液,含有2 mM EDTA和0.5%血清的PBS。 
    16. 进行细胞染色和流式细胞术分析或体外培养。

  2. 肺泡巨噬细胞的流式细胞术分析
    1. 用TruStain fcX阻断细胞上的非特异性结合位点,并在4℃,黑暗中用200μl冷PBS(不含FBS)中的Zombie Violet同时染色15分钟(参见表2)。
    2. 用冷BAL缓冲液洗涤细胞,在4℃下以300×g离心5分钟洗涤细胞。 
    3. 根据表2,在4℃,黑暗中使用BAL缓冲液以100μl/ 1百万细胞的体积染色细胞30分钟。

      表2.用于染色BAL AM的FACS试剂


    4. 为每种抗体缀合物制备补偿珠。
    5. 用BAL缓冲液洗涤细胞,重悬于200μlBAL缓冲液中进行记录。
    6. 在获得补偿珠后通过流式细胞术记录细胞。对于SiglecF和CD11c,AM是双阳性的(图2A-2C),并且> 98%存活(图2D)。


      图2. BAL AM的FACS分析。 A.排除双峰,死细胞的简单门控策略.AM是SiglecF-和CD11c-阳性.BC。用预热的BAL缓冲液收集BAL细胞每个符号表示1只小鼠。通常,> 95%的BAL细胞是AM.D.通过用EDTA染色评估的BAL单线态细胞的活力分析。

  3. 肺泡巨噬细胞的培养
    1. 如前所述通过离心收集细胞。
    2. 在3ml预热的AM培养基中,每孔3×10 5 -4×10 5个细胞未处理的6孔板(参见配方)。
      注意:通常,3x10 5 -4 x 10 5
    3. 将庆大霉素添加到AM培养物中(1:1,000)。
      注意:第一次更换介质后会发出庆大霉素。
    4. 在37°C孵育,5%CO 2 。 
    5. 用新鲜培养基替换培养上清液6-18小时后丢弃上清液。 
    注意:细胞将在第一次平板培养后几小时内完全摄取,我们不会在第一次培养基更换时将细胞保持悬浮状态。但是,对于中间培养基更换,细胞也会被收集,因为AM培养由粘附细胞和悬浮细胞组成(参见注释部分)。
    1. 每2天换一次培养基,直到细胞培养物达到融合。
    2. 向贴壁细胞中加入2 ml温热的AM培养基,以防止干涸细胞。使用300 离心收集悬浮细胞。 em> 5分钟。将沉淀的细胞重悬于1 ml温热的AM培养基中,并与贴壁细胞结合。
      注意:新收获的初级AM将每7-10天翻一番(Soucie 等。 ,,2016)。如果大部分AM出现拉伸(纺锤状)然而,增殖能力将是有限的,它可能适用于开始新的培养(参见图3的早期AM培养的示例图)。)并且激活,增加条件培养基的量或添加重组GM-CSF可能有帮助; em>


      图3.接种细胞后第一天AM培养的代表性图像。 A. AM培养,主要呈圆形细胞,在接种后第1天部分漂浮并部分粘附.B。相同箭头不分裂细胞,放大100倍,如第2天的培养物(A)所示.C。与第4天的(A)相同的培养物.D。

    3. 为了从融合孔中分离细胞,将悬浮细胞收集到15ml管中。 
    4. 将750μl脱离培养基(参见配方)加入6孔板(或3 ml至94 mm培养皿)的1孔中,并在37°C下孵育10-30分钟。
      培养基中破裂的细胞可能会出现野心注意:当破裂过于严苛的脱离过程时,AM非常容易和倾向。因此,使用未经处理的塑料制品和适当的分离介质是很重要的(参见食谱)。 AM的激活状态和增殖能力。 
    5. 通过轻轻地在塑料底部移液来支持细胞分离以避免细胞损伤(参见注释)。
    6. 将细胞悬浮于细胞中,将细胞以300μL离心细胞培养5分钟。 
    7. 仅计数活的(锥虫蓝色阴性)细胞。将细胞沉淀重悬于1ml温热的AM培养基中,并用台盼蓝染色后用血细胞计数器室取样以排除死细胞。
    8. 如果细胞数加倍,加入5ml温热的AM培养基并分成6孔板的2个孔(或对应于2×94mm培养皿)。通常,接种的细胞数保持在上述值附近。在步骤C2的说明中。
      注意:早期,我们可以证明AM培养至少传代10代(Soucie 等。 ,2016)。 AM培养物甚至超过20代仍然增殖,没有增殖能力下降的迹象。

数据分析

使用激光在用于tessa的BD LSR上记录染色的细胞并使用激光,对其进行分析,仅使用血细胞计数器进行分析,仅涉及台盼染色的细胞。如图2A所示进行门控。为了测试三组平均值之间的统计学显着差异(图1),使用GraphPad Prisma进行单向ANOVA和Tukey多重比较检验。 7.没有排除任何数据点。

笔记

  1. 悬浮液AM的重新接种将导致部分细胞附着在新孔中,而另一部分保持悬浮状态。注意表达培养基时不要丢失悬浮细胞这将减少增殖细胞的数量并减缓培养物的扩增。
  2. 如果细胞不易分离,收集含有细胞的培养基并进行另一轮抑制,当细胞没有脱落,细胞悬浮液过度上下时,这可能会影响培养的活力;晚期传代细胞比早期传代培养(最多30分钟)需要更短的孵育时间(约5分钟)。 
  3. 通过在各种量的条件培养基(例如,1,2,5,10%在完全培养基中)测试AM的生长,在每批制备后滴定条件培养基的百分比。在我们的批次中,我们通常使用1%至3%的条件培养基稀释成完全培养基(例如,100μl,10 ml),这大致相当于2-5 ng / ml纯化的重组小鼠-CSF。
  4. 较低的GM-CSF浓度可能已足够,但测试不够。
    我们成功地用20 ng / ml重组GM-CSF(Peprotech)替换条件培养基用于长期培养。

食谱

  1. BAL缓冲区
    PBS
    2mM EDTA(从0.5M EDTA储备溶液中稀释1:250)
    0.5%胎牛血清(FBS)
    使用真空过滤进行无菌过滤,并保持在4°C直至使用
  2. 完整媒体
    RPMI 1640
    1x GlutaMAX
    1x丙酮酸
    1x青霉素/链霉素
    10%FBS
    使用真空过滤进行无菌过滤,并保持在4°C直至使用
  3. AM培养基
    补充含有1-5%条件培养基的完全培养基,含有小鼠GM-CSF(需要滴定)
    使用前在水浴中预热至37°C
  4. 支队媒体
    ESGRO完成Accutase
    1mM EGTA(从0.5M EGTA储备溶液中稀释1:500)
    等分并在-20°C冷冻。使用前将水浴中的预热温度升至37°C

致谢

我们感谢Stephanie Vargas Aguilar和Sethuraman对手稿,Philippe Pierre的J558L细胞以及Max-Delbrück-Centrum的流式细胞术和动物护理功能的技术讨论和有益评论国家研究所德拉桑特等德拉RECHERCHE MEDICALE,中心法国国家科学研究和艾克斯 - 马赛大学的CIML和法新社国立德拉RECHERCHE(ANR-11-BSV3-0026补助MH Sieweke和ANR-17 -CE15-0007-01),国家癌症研究所(InCA13-10 / 405 / AB-LC-HS),RechercheMédicale基金会(DEQ.20110421320)和欧盟的欧洲研究理事会(ERC) K. Molawi得到了人类前沿科学计划长期奖学金和StiftungCharité,L。Geiersdottir基金会的支持,地平线2020研究和创新计划(授权协议编号695093 MacAge)。 M.H. Sieweke是柏林健康研究所 - 爱因斯坦访问MDC的人和德累斯顿工业大学的Alexander von Humboldt教授。

竞争利益

作者声明没有竞争性的经济利益。

道德

经LandesamtfürGesundheitund Soziales批准后,根据德国动物福利立法进行畜牧业和老鼠工作(在柏林工作,遵循委员会机构动物护理和使用委员会的指导方针) Medizin)并经Landesdirektion Sachsen批准(在德累斯顿工作)。

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引用:Busch, C. J., Favret, J., Geirsdóttir, L., Molawi, K. and Sieweke, M. H. (2019). Isolation and Long-term Cultivation of Mouse Alveolar Macrophages. Bio-protocol 9(14): e3302. DOI: 10.21769/BioProtoc.3302.
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