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Isolation and Cultivation of Primary Brain Endothelial Cells from Adult Mice
从成年小鼠分离和培养原代脑内皮细胞   

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Abstract

Brain endothelial cells are the major building block of the blood-brain barrier. To study the role of brain endothelial cells in vitro, the isolation of primary cells is of critical value. Here, we describe a protocol in which vessel fragments are isolated from adult mice. After density centrifugation and mild digestion of the fragments, outgrowing endothelial cells are selected by puromycin treatment and grown to confluence within one week.

Keywords: Primary culture(原代培养), Blood-brain barrier(血脑屏障), Tight junctions(紧密连接), CD31(CD31), Occludin(闭合蛋白), Claudin-5(紧密连接蛋白-5), ZO-1(ZO-1), VE-cadherin(VE钙粘蛋白)

Background

The blood-brain barrier protects the brain from uncontrolled entry of cells and substances. This is mainly achieved by brain endothelial cells that form a barrier composed of tight and adherens junctions to restrict paracellular transport.

This protocol was developed to overcome the limited availability of mouse brain endothelial cell lines that maintain their key characteristics, e.g., the expression of sufficient amounts of tight junction proteins such as occludin, ZO-1 or claudin-5 to induce a high transendothelial resistance.

In addition, the isolation of brain endothelial cells from genetically modified mice allows investigating of gene-specific functions in vitro.

Using this method, we previously complemented in vivo studies demonstrating the importance of NF-κB signaling in brain endothelial cells for maintaining normal blood-brain barrier function (Ridder et al., 2015).

Materials and Reagents

  1. Materials
    1. Multiwell plate (cell culture grade) (Greiner Bio One International, catalog number: 662160 )
    2. Cellulose chromatography paper (sterilize at 180 °C) (Whatman, catalog number: 3030-931 )
    3. 50 ml centrifuge tubes (cell culture grade) (Greiner Bio One International, catalog number: 210261 )
    4. 10 ml disposable pipette (Greiner Bio One International, catalog number: 607160 )
    5. Mice (C57BL/6, age 6 weeks up to 1 year from Charles River, Germany or the in-house breeding facility)
    6. Ice

  2. Reagents

    Reagents 
    Manufacturer 
    Brand 
    Catalog number
    Preparation 
    Aliquots 
    Storage 
    Stock concentration
    Working concentration
    Dilution

    Hydrochloric acid (HCl) 1 N (sterile filtered)
    Carl Roth 

    1 L
    RT 

    0.05 N 
    1 ml HCl 1 N + 19 ml H2O, sterile
    2
    Dulbecco’s PBS (DPBS)
    Biowest 

    500 ml 

    4 °C 
    1x 
    1x 
    undiluted 
    3
    70% EtOH (denatured)
    Th. Geyer 



    RT



    4
    Isoflurane
    Baxter



    RT



    Collagenase/dispase
    (sterile filtered) 
    Roche Diagnostics 

    500 mg/5 ml H2O, sterile 
    200 µl 
    -20 °C 
    100 mg/ml 
    1 mg/ml 
    100 µl collagenase/dispase in 10 ml medium
    6

    DNase I 
    Roche Diagnostics

    100 mg/10 ml H2O, sterile
    100 µl 
    -20 °C

    10 mg/ml 
    4 µg/ml 
    40 µl DNase I in 10 ml medium

    Nα-Tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK)
    Sigma-Aldrich 

    14.7 mg/10 ml H2O, sterile
    200 µl 
    -20 °C 
    1.47 mg/ml diluted to 14.7 µg/ml
    0.147 µg/ml 
    100 µl TLCK in 10 ml medium 
    Puromycin 
    Sigma-Aldrich 

    2.5 mg/10 ml H2O, sterile
    500 µl 
    -20 °C 
    0.25 mg/ml 
    8 µg/ml 
    32 µl puromycin in 1 ml medium
    Trypsin-EDTA 0.25% 
    Thermo Fisher Scientific
    GibcoTM 
    100 ml 

    -20 °C 
    1x 
    1x 
    undiluted 
    10 
    4% paraformaldehyde 
    Merck Millipore 

    4% paraformaldehyde in DPBS

    -20 °C 



    11 
    CD31 
    BD 
    BD Pharmingen
    557355 





    1:500 
    12 
    α-smooth muscle Actin (α-SMA)
    Acris Antibodies







    13 
    Iba1 
    Wako Pure Chemical Industries






    14 
    Glial Fibrillary Acidic Protein (GFAP)
    EMD Millipore 







    15
    Zona occludens-1
    (ZO-1) 
    Thermo Fisher Scientific
    Invitrogen 





    16 
    VE-Cadherin 
    Santa Cruz Biotechnology






    17
    Claudin-5 (Cldn-5) 
    Thermo Fisher Scientific
    Invitrogen 






    18
    Occludin (Ocln)
    Sigma-Aldrich







    19 
    Mouse collagen, type IV 
    Corning 

    890 mg/ml 0.05 N HCl
    100 µl
    -80 °C 
    890 mg/ml 
    50 µg/ml 
    56 µl collagen IV + 944 µl 0.05 N HCl 
    20
    Dextran MW 60,000-90,000
    Alfa Aesar 

    1 kg

    RT

    18% 
    5.4 g dextran in 30 ml DPBS
    21 
    Penicillin/streptomycin (100x)
    Biochrom 

    100 ml
    1 ml
    -20 °C 
    100x 
    1x 
    100 µl pen/strep in 10 ml medium/dextran
    22
    L-glutamine 
    Thermo Fisher Scientific
    GibcoTM 
    100 ml
    1 ml
    -20 °C 
    200 mM (100x) 
    2 mM (1x) 
    100 µl L-glutamine in 10 ml medium 
    23 
    DMEM-F12 w/o glutamine 
    Thermo Fisher Scientific
    GibcoTM
    500 ml

    4 °C 
    1x 
    1x 
    undiluted 
    24
    DMEM w/o glucose 
    Thermo Fisher Scientific
    GibcoTM 
    500 ml

    4 °C 
    1x 
    1x 
    undiluted 
    25 
    Plasma-derived bovine serum (PDS)
    First Link 

    500 ml
    10 ml 
    -20 °C 
    100% 
    20% 
    10 ml PDS in 50 ml medium
    26
    Antibiotic/antimycotic (100x) 
    Thermo Fisher Scientific
    GibcoTM 
    100 ml 
    1 ml 
    -20 °C 
    100x 
    1x 
    100 µl AA in 10 ml medium/dextran 
    27 
    Heparin-sodium 
    Ratiopharm 

    PZN 003029843
    1 ampule 

    4 °C 
    5,000 I.E./ml 
    750 I.E./50 ml 
    150 µl heparin in 50 ml medium
    28
    Endothelial Cell Growth Supplement (ECGS)
    Sigma-Aldrich 

    15 mg/5 ml DPBS 
    500 µl 
    -20 °C 
    3 mg/ml 
    30 µg/ml 
    500 µl ECGS in 50 ml medium 
    29 
    18% dextran solution (see Recipes)









    30
    Working medium (see Recipes)









    31 
    Digestion medium (see Recipes)









    32
    Full medium (see Recipes)









    Note: Mouse collagen, type IV: Defrost stock vial slowly on ice at 4 °C overnight. Vortex thoroughly. Aliquot and store at -80 °C. The collagen concentration varies from lot to lot. Therefore, the amount of HCl added has to be adjusted for every new lot.

Equipment

  1. Refrigerator (4 °C)
  2. Shaker
  3. Sterile beakers 100-150 ml (sterilize at 180 °C)
  4. Laminar flow work bench
  5. Dounce tissue grinder, 15 ml, autoclave (Sigma-Aldrich, catalog number: D9938 )
  6. Scalpel
  7. Tweezers (sterilize at 180 °C)
  8. Centrifuge (Hettich Lab Technology, model: UNIVERSAL 320 R ),
  9. Fixed-angle rotor (Hettich Lab Technology, catalog number: 1620A )
  10. Big scissors (sterilize at 180 °C)
  11. Small scissors (sterilize at 180 °C)
  12. Pipette or vacuum pump
  13. Water bath
  14. Microwave oven

Procedure

  1. Preparations (Day 1)–Coating of wells with collagen
    1. Defrost one collagen aliquot for 2 wells of a 6-well plate or an according volume for other well sizes (see Table 1) slowly (2-3 h) on ice in a refrigerator (4 °C)
      If necessary, defrosting aliquots in the refrigerator without ice is possible.

      Table 1. Volume adjustment according to well size


    2. Dilute collagen to 50 µg/ml with 0.05 N HCl.
      Note: 0.05 N HCl aliquots can be stored at -20 °C.
    3. Vortex thoroughly. At least 10 sec until small bubbles form (turn the tube to ensure that the viscous collagen stock solution does not continuously stick to the bottom).
    4. Coat wells evenly with collagen solution (volume see Table 1).
    5. Put the plate on a shaker, 1 h at room temperature, 25 rpm.
    6. Move the plate to 4 °C for storage (overnight possible).
      Note: It is also possible to coat the plates on the day of endothelial cell preparation.

  2. Isolation (Day 2)
    1. Preparations:
      1. Ice
      2. Sterilization of instruments
      3. Approx. 50 ml DPBS in a 150 ml sterile beaker on ice (for each sample).
      4. Disinfectant (70% EtOH) in a sterile beaker on ice (approx. 50 ml in 150 ml sterile beaker)
      5. Switch on laminar flow and prepare cellulose chromatography paper, tissue grinder, scalpel, tweezers and 50 ml centrifuge tubes (one for each sample).
      6. Precool centrifuge to 4 °C.
    2. Anesthetize mice according to your local animal regulations. We use an overdose of isoflurane, which leads to breathing arrest within one minute. Decapitate the mouse with a big scissor and dip the head in ethanol (on ice). Remove the brain swiftly (Figure 1A) and store it in DPBS on ice. Repeat for all brains.
      1. Cut off cerebellum and olfactory bulb.
      2. Remove meninges by rolling the brains on cellulose chromatography paper using blunt tweezers.
      3. Cut cerebrum in 2 to 4 pieces and put the pieces in 5 ml working medium (4 °C). Repeat for all brains.
      4. Transfer brains with 5 ml working medium (4 °C) into a tissue grinder (Figure 1E) and homogenize (30 strokes with pistil A, 25 strokes with pistil B, Figure 1F). Use a maximum of 10 brains in one tissue grinder.
      5. Transfer homogenate into a 50 ml centrifuge tube. Rinse tissue grinder with 5 ml working medium (4 °C) and add to the homogenate (10 ml altogether).
      6. Centrifuge homogenate at 1,350 x g, 5 min, 4 °C. Remove supernatant carefully using a pipette or vacuum pump.
      7. Resuspend the pellet in 15 ml dextran solution and vortex extensively (2 min). The result is a white, cloudy, homogenous suspension (Figure 1G).
      8. Centrifuge at 6,080 x g, 10 min, 4 °C. In the meantime, supplement digestion medium with 100 µl collagenase/dispase, 40 µl DNase I and 100 µl TLCK each per 10 ml digestion medium. Pre-warm digestion medium to 37 °C.
      9. After centrifugation, remove the fluffy myelin layer (top, black arrows in Figures 1H and 1I) and the dextran as completely as possible. Use a 10 ml disposable pipette. Remove the filter of the pipette first if necessary.
      10. Resuspend the pellet (white arrows in Figures 1H and 1I) in 10 ml digestion medium (37 °C).
      11. Digest the tissue for 1 h 15 min in a 37 °C water bath (shake from time to time for 2 to 3 sec–approx. every 15 min).
      12. Centrifuge cell suspension at 1,350 x g, 5 min, room temperature. In the meantime, get the pre-coated plate from the refrigerator, fill sterile DPBS (10 ml per sample) in a centrifuge tube and heat it to 37 °C. Optionally, supplement full medium with puromycin and pre-warm to 37 °C (see step B2q).
      13. Remove digestion medium.
      14. Resuspend pellet in 10 ml warm DPBS.
      15. Centrifuge at 1,350 x g, 5 min, room temperature. In the meantime, remove collagen from the coated wells and wash twice with DPBS. DPBS from the second wash is left in the wells until cells are ready for seeding.
      16. Remove DPBS and resuspend the pellet in full medium. 2.5 ml full medium per well for a 6-well plate. Use 4-6 brains per culture plate.
      17. Mix cell suspension carefully before seeding to ensure even distribution.
      18. Add puromycin as indicated in Table 2. (Can be added directly to the full medium, see step B2l).

        Table 2. Culture volume according to well size



        Figure 1. Typical images for the preparation of the brain, removal of meninges, homogenization and subsequent dextran gradient centrifugation. The images depict the first steps of the isolation procedure showing the brain in situ after removal of the skullcap (A), before (B. cut planes indicated by dashed line) and after removal of cerebellum, olfactory bulb (C) and the meninges (D). Then, collect the brains in a Dounce tissue grinder (E), homogenize them (F), and centrifuge the tissue homogenate. Next, resuspend the cells in the dextran solution and vortex extensively (G). Following the centrifugation, the resulting myelin layer is at the top while the vessel fragments collect around the edge of the tube bottom (H + I, black arrows: myelin layer, white arrows: pellet location). The size of the vessel fragment pellet depends on the number of brains used. In E-I, two brains were used for the preparation.

    3. Day 3
      1. Wash cells twice with DPBS.
      2. Change full medium.
      3. Add puromycin (alternatively, puromycin can be added in advance to the full medium).
    4. Day 4
      1. Change full medium.
        Note: No puromycin needed anymore.

  3. Cultivation
    1. Change medium 1-2 times per week, first time approx. 4-6 days after isolation.
    2. Split the culture 1:2 (or 1:3) if the cells are confluent. Use trypsin 5-10 min and inactivate with full medium.
    3. Plate cells and change medium the next day.

  4. Purity of the cell culture (Figures 2 and 3)
    Endothelial cells (CD31+) > 95%
    Pericytes (α-SMA+) < 5%
    No astrocytes (GFAP+), microglia (Iba1+), neurons (NeuN+)


    Figure 2. Representative immunofluorescence images of primary mouse brain endothelial cells. Cells were fixed with 4% paraformaldehyde 14 days after isolation and subsequently stained for CD31 (BD, 1:500) as an endothelial cell specific marker in combination with α-SMA (pericytes and smooth muscle cells, Acris, 1:200, upper row), Iba1 (microglia, Wako Pure Chemical Industries, 1:100, middle row) and GFAP (astrocytes, Millipore, 1:400, lower row). Scale bars represent 50 µm.


    Figure 3. Primary mouse brain endothelial cells maintain expression of tight and adherens junction proteins. Cells were fixed 6-8 days after isolation with ice-cold methanol and subsequently stained for the tight junction proteins Ocln (Sigma-Aldrich, 1:500), ZO-1 (Thermo Fisher Scientific, 1:500), Cldn-5 (Thermo Fisher Scientific, 1:500) and the adherens junction protein VE-Cadherin (Santa Cruz Biotechnology, 1:500). Scale bars represent 50 µm.

Data analysis

Primary mouse brain endothelial cells isolated by this method can be used for a variety of methods that include protein and gene expression analysis, assessment of transendothelial resistance using transwell inserts and transmigration or adhesion assays. In addition, these cells can also be grown on glass coverslips coated with collagen IV for live imaging, e.g., to monitor intracellular calcium dynamics.

Notes

  1. This protocol was developed to isolate brain endothelial cells from adult mice. We successfully isolated and cultured cells from young mice (6-8 weeks) as well as old mice (up to 1 year) in our laboratory without any modifications to the protocol. Using brains from other mouse strains has not been tested in our laboratory.
  2. Cells usually reach confluence within 6-8 days (see Figure 4). They can be maintained in culture but are eventually overgrown by pericytes after several weeks.
  3. After approximately 10 days cells do not adhere as strongly as before and are more likely to detach during staining procedures.


    Figure 4. Bright field images of primary murine brain endothelial cells 2 days (left) or 7 days (right) after isolation. Note the attached vessel fragment and its radially outgrowing endothelial cells after 2 days in culture. Scale bars represent 100 µm.

Recipes

  1. 18% dextran solution (for 2 preparations)
    5.4 g dextran dissolved in 30 ml DPBS by heating (microwave)
    300 µl penicillin/streptomycin (100x)
    300 µl L-glutamine (200 mM)
    The solution can be stored at -20 °C and defrosted before usage, but penicillin/streptomycin and L-glutamine should be added after thawing
  2. Working medium (for 2 preparations)
    20 ml DMEM-F12
    200 µl penicillin/streptomycin (100x)
    200 µl L-glutamine (200 mM)
  3. Digestion medium (for 2 preparations)
    20 ml DMEM
    200 µl penicillin/streptomycin (100x)
    200 µl collagenase/dispase–add right before digestion
    200 µl TLCK–add right before digestion
    80 µl DNase I–add right before digestion
  4. Full medium (max. storage time 3-4 weeks at 4 °C)
    40 ml DMEM-F12
    10 ml PDS
    500 µl antibiotic/antimycotic (100x)
    500 µl L-glutamine (200 mM)
    150 µl heparin (5,000 U/ml)
    500 µl ECGS

Acknowledgments

The protocol described here has been modified based on the method published by Song and Pachter (2003). We would like to thank Beate Lembrich for expert technical assistance. This work was funded by the Deutsche Forschungsgemeinschaft (SCHW416/5-2, 416/9-1).

References

  1. Ridder, D. A., Wenzel, J., Müller, K., Töllner, K., Tong, X. K., Assmann, J. C., Stroobants, S., Weber, T., Niturad, C., Fischer, L., Lembrich, B., Wolburg, H., Grand'Maison, M., Papadopoulos, P., Korpos, E., Truchetet, F., Rades, D., Sorokin, L. M., Schmidt-Supprian, M., Bedell, B. J., Pasparakis, M., Balschun, D., D’Hooge, R., Löscher, W., Hamel, E. and Schwaninger, M. (2015). Brain endothelial TAK1 and NEMO safeguard the neurovascular unit. J Exp Med 212(10): 1529-1549.
  2. Song, L. and Pachter, J. S. (2003). Culture of murine brain microvascular endothelial cells that maintain expression and cytoskeletal association of tight junction-associated proteins. In Vitro Cell Dev Biol Anim 39(7): 313-320.

简介

脑内皮细胞是血脑屏障的主要组成部分。 为了研究脑内皮细胞在体外的作用,原代细胞的分离是临床价值。 在这里,我们描述了一种方案,其中血管片段与成年小鼠分离。 在密度离心和片段的温和消化后,通过嘌呤霉素处理选择出生长的内皮细胞,并在一周内生长至汇合。
【背景】血脑屏障保护大脑免受细胞和物质的不受控制的侵入。 这主要是由脑内皮细胞形成的屏障组成的紧密和粘附连接限制细胞旁运输。
该方案被开发以克服维持其关键特征的小鼠脑内皮细胞系的有限可用性,例如表达足够量的紧密连接蛋白如闭合蛋白,ZO-1或紧密连接蛋白-5以诱导高跨内皮细胞抗性。
此外,从内含基因修饰的小鼠中分离脑内皮细胞可以在体外研究基因特异性功能。
使用这种方法,我们以前补充了体内研究,证明NF-κB信号在脑内皮细胞中维持正常血脑屏障功能的重要性(Ridder等,2015)。

关键字:原代培养, 血脑屏障, 紧密连接, CD31, 闭合蛋白, 紧密连接蛋白-5, ZO-1, VE钙粘蛋白

材料和试剂

  1. 物料
    1. 多孔板(细胞培养级)(Greiner Bio One International,目录号:662160)
    2. 纤维素层析纸(180℃灭菌)(Whatman,目录号:3030-931)
    3. 50ml离心管(细胞培养级)(Greiner Bio One International,目录号:210261)
    4. 10 ml一次性移液管(Greiner Bio One International,目录号:607160)
    5. 小鼠(C57BL/6,来自德国Charles River或内部养殖场的6周龄至1年)


  2. 试剂

    试剂

    制造商

    品牌

    目录号
    准备

    等分试样

    存储

    库存浓度
    工作集中
    稀释
    1


    盐酸(HCl)1 N(无菌过滤)
    Carl Roth



    K025.1


    1 L



    RT


    0.05 N

    1ml HCl 1N + 19ml H 2 O,无菌
    2

    Dulbecco的PBS(DPBS)
    Biowest


    L0615-500

    500 ml


    4°C

    1x

    1x

    未稀释的

    3

    70%EtOH(变性)
    钍。盖尔


    2270



    RT




    4
    异氟烷
    巴克斯特

    KDG9623


    RT



    5


    胶原酶/分散素
    (无菌过滤)

    罗氏诊断


    11097113001


    500毫克/毫升H 2 O,无菌

    200μl


    -20°C


    100毫克/毫升


    1 mg/ml


    100μl胶原酶/分散在10ml培养基中
    6

    DNase I

    罗氏诊断

    11284932001

    100毫克/毫升H 2 O,无菌
    100μl

    -20°C

    10毫克/毫升

    4μg/ml

    40μlDNase I在10ml培养基中
    7


    α-甲基-L-赖氨酸氯甲基酮盐酸盐(TLCK)
    Sigma-Aldrich



    90182


    14.7mg/10ml H 2 O,无菌
    200μl

    -20 °C

    1.47 mg/ml稀释至14.7μg/ml
    0.147μg/ml


    100μlTLCK在10ml培养基中
    8

    霉素

    Sigma-Aldrich


    P8833

    2.5mg/10ml H 2 O,无菌
    500 μl

    -20 °C

    0.25 mg/ml

    8μg/ml

    32μl嘌呤霉素在1 ml培养基中
    9


    胰蛋白酶-EDTA 0.25%


    赛默飞世尔科技
    Gibco TM


    25200-056


    100 ml



    -20 °C


    1x


    1x


    未稀释的


    10


    4%多聚甲醛


    默克·米利波尔


    1040051000


    DPBS中4%多聚甲醛

    -20 °C





    11

    CD31

    BD

    BD Pharmingen
    557355






    1:500

    12

    α-平滑肌肌动蛋白(α-SMA)
    Acris抗体


    DM001-05






    13


    Iba1


    和光纯药业


    019-19741







    14

    胶质纤维酸性蛋白(GFAP)
    EMD Millipore



    AB5541






    15


    Zona occludens-1
    (ZO-1)

    赛默飞世尔科技
    Invitrogen



    40-2200







    16

    VE-Cadherin

    Santa Cruz Biotechnology


    sc-6458






    17


    Claudin-5(Cldn-5)


    赛默飞世尔科技
    Invitrogen



    34-1600







    18
    Occludin(Ocln)
    Sigma-Aldrich


    SAB3500301





    19




    小鼠胶原蛋白,IV型



    康宁





    354233



    890 mg/ml 0.05 N HCl
    -80°C



    890 mg/ml



    50μg/ml



    56μl胶原蛋白IV + 944μl0.05 N HCl


    20

    葡聚糖MW 60,000-90,000
    阿尔法阿萨尔



    J14495

    1公斤

    RT


    18%

    5.4 g葡聚糖在30 ml DPBS中
    21

    青霉素/链霉素(100x)
    生物色素



    A2212

    100 ml

    -20°C

    100x

    1x

    100μlpen/strep在10ml培养基/葡聚糖中
    22


    L-谷氨酰胺


    赛默飞世尔科技
    Gibco TM



    25030024


    100 ml


    -20°C


    200 mM(100x)


    2 mM(1x)


    100μlL-谷氨酰胺在10ml培养基中
    23


    DMEM-F12 w/o谷氨酰胺

    赛默飞世尔科技
    Gibco TM



    21331020


    500 ml


    4°C


    1x


    1x


    未稀释的


    24


    DMEM w/o葡萄糖


    赛默飞世尔科技
    Gibco TM



    11966025


    500 ml


    4°C


    1x


    1x


    未稀释的


    25

    血浆衍生牛血清(PDS)
    第一连接


    60-00-810

    500 ml

    10毫升

    -20°C

    100%

    20%

    10 ml PDS在50ml培养基中
    26

    抗生素/抗真菌药(100x)

    赛默飞世尔科技
    Gibco TM


    15240062


    100 ml


    1 ml


    -20°C


    100x


    1x


    100μlAA在10ml培养基/葡聚糖中
    27

    肝素钠

    Ratiopharm


    PZN 003029843
    1安瓿


    4°C

    5,000 I.E./ml

    750 I.E./50 ml

    150μl肝素在50ml培养基中
    28


    内皮细胞生长补充剂(ECGS)
    Sigma-Aldrich



    E2759


    15毫克/5毫升DPBS


    500μl


    -20°C


    3 mg/ml


    30μg/ml


    500μlECGS在50ml培养基中
    29

    18%葡聚糖溶液(见配方)









    30

    工作介质(见配方)









    31

    消化培养基(参见食谱)









    32

    完整介质(见配方)









    注意:小鼠胶原蛋白,IV型:在冰上,4℃下将霜冻缓慢清除过夜。彻底旋涡等分并储存于-80°C。胶原浓度因批次而异。因此,每个新批次必须调整添加的HCl量。

设备

  1. 冰箱(4°C)
  2. 振动器
  3. 无菌烧杯100-150毫升(180度消毒)
  4. 层流工作台
  5. Dounce组织研磨机,15ml,高压釜(Sigma-Aldrich,目录号:D9938)
  6. Scalpel
  7. 镊子(180°C灭菌)
  8. 离心机(Hettich Lab Technology,型号:UNIVERSAL 320 R),
  9. 固定角转子(Hettich Lab Technology,目录号:1620A)
  10. 大剪刀(180°C灭菌)
  11. 小型剪刀(180°C灭菌)
  12. 移液器或真空泵
  13. 水浴
  14. 微波炉

程序

  1. 准备(第1天) - 用胶原蛋白加油
    1. 在冰箱(4℃)中冰上缓慢(2-3小时)除霜6孔板2孔或其他孔尺寸(见表1)的一个胶原等分试样
      如果需要,可以在冰箱中除冰等分试样,无冰。

      表1.根据井号调整体积


    2. 用0.05N HCl将胶原蛋白稀释至50μg/ml 注意:0.05 N HCl等分试样可以在-20°C储存。
    3. 彻底旋涡至少10秒钟,直到形成小气泡(转动管,以确保粘性胶原蛋白原液不会持续粘在底部)。
    4. 用胶原蛋白溶液均匀涂抹(体积见表1)
    5. 将板放在振荡器上,室温1小时,25rpm
    6. 将板移动到4°C进行存储(可能过夜)。
      注意:也可以在内皮细胞制备当天涂上平板。

  2. 隔离(第2天)
    1. 准备工作:

      1. 仪器灭菌
      2. 约。 50毫升DPBS在冰上150毫升无菌烧杯(每个样品)
      3. 在冰上的无菌烧杯中(约150ml无菌烧杯中的约50ml)消毒剂(70%EtOH)
      4. 打开层流,制备纤维素层析纸,组织研磨机,手术刀,镊子和50ml离心管(每个样品一个)。
      5. 预冷离心机至4°C
    2. 根据当地的动物规定麻醉老鼠。我们使用过量的异氟烷,这会在一分钟内导致呼吸停止。用大剪刀取下鼠标,将头浸于乙醇(冰上)。迅速取出大脑(图1A)并将其存放在DPBS上。重复所有的大脑。
      1. 切断小脑和嗅球。
      2. 使用钝镊子在纤维素层析纸上滚动脑部去除脑膜。
      3. 切割大脑2至4片,将片放在5ml工作介质(4℃)中。重复所有大脑。
      4. 将大脑用5ml工作培养基(4℃)转移到组织研磨机(图1E)中,并匀化(雌蕊A为30次,雌蕊B为25次,图1F)。在一个组织研磨机中最多使用10个大脑。
      5. 将匀浆转移到50ml离心管中。用5ml工作介质(4℃)冲洗组织研磨机,并加入匀浆(共10ml)。
      6. 离心机在1,350℃,5分钟,4℃下匀浆。用移液器或真空泵小心取出上清液。
      7. 将沉淀重悬于15ml葡聚糖溶液中并大量旋转(2分钟)。结果是白色,多云,均匀的悬浮液(图1G)。
      8. 以6,080 x g,10分钟,4℃离心。同时,每10 ml消化培养基中加入100μl胶原酶/分散酶,40μlDNase I和100μlTLCK的消化培养基。预热消化培养基至37°C
      9. 离心后,尽可能地去除蓬松髓磷脂层(图1H和1I中的顶部,黑色箭头)和葡聚糖。使用10ml一次性移液器。如有必要,先取出移液器的过滤器。
      10. 在10ml消化培养基(37℃)中将沉淀(图1H和1I中的白色箭头)重悬。
      11. 在37℃水浴中将组织消化1小时15分钟(不时摇动2至3秒 - 约15分钟)。
      12. 以1,350×g离心细胞悬浮液,5分钟,室温。同时,从冰箱中取出预涂板,在离心管中加入无菌DPBS(每个样品10ml),并加热至37℃。补充完全培养基与嘌呤霉素并预热至37℃(见步骤B2q)
      13. 去除消化介质。
      14. 将沉淀重悬于10ml温热DPBS中
      15. 以1,350×g离心,5分钟,室温。同时,从涂覆的孔中除去胶原蛋白,并用DPBS洗涤两次。 DPBS从第二次洗涤留在井中,直到细胞准备播种
      16. 去除DPBS并将沉淀物重新悬浮在完全培养基中。对于6孔板,每孔2.5ml完全培养基,每孔1-2脑(每个培养板约4只脑)。
      17. 播种前仔细混合细胞悬液,以确保均匀分布。
      18. 加入嘌呤霉素,如表2所示。(可直接加入完整培养基,见步骤B2)
        表2.根据井尺寸的文化体积



        图1.用于制备脑的典型图像,去除脑膜,均匀化和随后的葡聚糖梯度离心。图像描绘了分离程序的第一步骤,其显示脑原位在去除骷髅帽(A)之前,(B.虚线所示的切割平面)和除去小脑,嗅球(C)和脑膜(D)之后。然后,在大脑组织研磨机(E)中收集脑,使其均质(F),并离心组织匀浆。接下来,将细胞重悬在葡聚糖溶液中并大量旋转(G)。离心后,所得髓鞘层位于顶部,血管碎片聚集在管底部的边缘(H + I,黑色箭头:髓鞘层,白色箭头:颗粒位置)。血管碎片颗粒的大小取决于所用脑的数量。在E-I中,两只脑被用于准备。

    3. 第3天
      1. 用DPBS洗涤细胞两次。
      2. 更改完整媒介。
      3. 添加嘌呤霉素(或者,嘌呤霉素可以预先加入到完全培养基中)
    4. 第4天
      1. 更改完整媒介。
        注意:不需要嘌呤霉素。

  3. 养殖
    1. 每周更换1-2次,每次约1次。隔离后4-6天。
    2. 如果细胞融合,分裂培养物1:2(或1:3)。使用胰蛋白酶5-10分钟,并用完全培养基灭活。
    3. 板细胞和第二天更换培养基。

  4. 细胞培养物的纯度(图2和3)
    内皮细胞(CD31 + )> 95%
    周细胞(α-SMA + ) 5%
    无星形胶质细胞(GFAP + ),小胶质细胞(Iba1 + ),神经元(NeuN +


    图2.原代小鼠脑内皮细胞的代表性免疫荧光图像细胞在分离后14天用4%多聚甲醛固定,随后染色为CD31(BD,1:500)作为内皮细胞特异性标记与α-SMA(周细胞和平滑肌细胞,Acris,1:200,上排),Iba1(小胶质细胞,和光纯药化学工业株式会社,中号行)和GFAP(星形胶质细胞,Millipore,1:400,较低行)。比例尺表示50μm。


    图3.原代小鼠脑内皮细胞维持紧密和粘附连接蛋白的表达。细胞在用冰冷的甲醇分离后6-8天固定,随后染色紧密连接蛋白Ocln(Sigma- Aldrich,1:500),ZO-1(Thermo Fisher Scientific,1:500),Cldn-5(Thermo Fisher Scientific,1:500)和Adherens连接蛋白VE-钙粘蛋白(Santa Cruz Biotechnology,1:500)。比例尺表示50μm。

数据分析

通过该方法分离的原代小鼠脑内皮细胞可用于多种方法,包括蛋白质和基因表达分析,使用transwell插入片段进行跨内皮细胞耐药性评估,并进行迁移或粘附测定。此外,这些细胞也可以在涂覆有胶原IV的玻璃盖玻片上生长,用于活检成像,例如以监测细胞内钙动态。

笔记

  1. 该方案被开发用于从成年小鼠中分离脑内皮细胞。我们成功地从我们的实验室中分离和培养来自年轻小鼠(6-8周)和老鼠(长达1年)的细胞,而不对该方案进行任何修改。使用其他小鼠品系的大脑没有在我们的实验室进行测试。
  2. 细胞通常在6-8天内达到汇合(见图4)。他们可以维持文化,但最终在周末后长满周末。
  3. 大约10天后,细胞不像以前一样强烈地粘附,并且在染色过程中更可能分离

    图4.分离后2天(左)或7天(右)初次鼠脑内皮细胞的明视野图像。注意培养2天后连接的血管片段及其径向成长的内皮细胞。比例尺表示100μm。

食谱

  1. 18%葡聚糖溶液(2份制剂)
    通过加热(微波)将5.4g葡聚糖溶解在30ml DPBS中 300μl青霉素/链霉素(100x)
    300μlL-谷氨酰胺(200 mM)
    溶液可以储存在-20°C,使用前要除霜,但解冻后应加入青霉素/链霉素和L-谷氨酰胺。
  2. 工作介质(2个准备)
    20毫升DMEM-F12
    200μl青霉素/链霉素(100x)
    200μlL-谷氨酰胺(200 mM)
  3. 消化培养基(2个制剂)
    20ml DMEM
    200μl青霉素/链霉素(100x)
    消化之前200μl胶原酶/分散 - 添加 消化前200μlTLCK-添加 消化前80μlDNase I添加即可
  4. 完全培养基(最高储存时间4周,4°C)
    40毫升DMEM-F12
    10 ml PDS
    500μl抗生素/抗真菌剂(100x)
    500μlL-谷氨酰胺(200 mM)
    150μl肝素(5,000 U/ml)
    500μlECGS

致谢

这里描述的协议已经基于Song and Pachter(2003)发表的方法进行了修改。我们要感谢Beate Lembrich的专家技术援助。这项工作由Deutsche Forschungsgemeinschaft(SCHW416/5-2,416/9-1)资助。

参考

  1. Ridder,DA,Wenzel,J.,Müller,K.,Töllner,K.,Tong,XK,Assmann,JC,Stroobants,S.,Weber,T.,Niturad,C.,Fischer,L.,Lembrich,B ,Wolburg,H.,Grand'Maison,M.,Papadopoulos,P.,Korpos,E.,Truchetet,F.,Rades,D.,Sorokin,LM,Schmidt-Supprian,M.,Bedell,BJ,Pasparakis ,M.,Balschun,D.,D'Hooge,R.,Löscher,W.,Hamel,E。和Schwaninger,M。(2015)。  脑内皮细胞TAK1和NEMO保护神经血管单位。 J Exp Med 212(10) :1529-1549。
  2. Song,L. and Pachter,JS(2003)。  维持紧密连接相关蛋白的表达和细胞骨架关联的鼠脑微血管内皮细胞的培养物。体外细胞发育生物学动画 39(7):313-320。
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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。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Assmann, J. C., Müller, K., Wenzel, J., Walther, T., Brands, J., Thornton, P., Allan, S. M. and Schwaninger, M. (2017). Isolation and Cultivation of Primary Brain Endothelial Cells from Adult Mice. Bio-protocol 7(10): e2294. DOI: 10.21769/BioProtoc.2294.
  2. Khan, M. A., Schultz, S., Othman, A., Fleming, T., Lebron-Galan, R., Rades, D., Clemente, D., Nawroth, P. P. and Schwaninger, M. (2016). Hyperglycemia in Stroke Impairs Polarization of Monocytes/Macrophages to a Protective Noninflammatory Cell Type. J Neurosci 36(36): 9313-9325.
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