参见作者原研究论文

本实验方案简略版
May 2021
Advertisement

本文章节


 

Isolation of CD31+ Bone Marrow Endothelial Cells (BMECs) from Mice
从小鼠中分离 CD31+ 骨髓内皮细胞 (BMECs)   

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

In the bone marrow microenvironment, endothelial cells (ECs) play a pivotal role in regulating the production of both growth and inhibiting factors. They are held together by adherence molecules that interact with hematopoietic progenitor cells. The study of ECs in the hematopoietic stem cell niche is limited due to the lack of efficient protocols for isolation. In this protocol, we developed a two-step approach to extract bone marrow endothelial cells (BMECs) to unlock the challenges researchers face in understanding the function of the endothelial vascular niche in in-vitro studies.

Keywords: Bone marrow endothelial cells (BMECs) (骨髓内皮细胞(BMECs)), Endothelial cells (ECs) (内皮细胞(ECs)), Extraction (抽取), Mouse bone marrow (小鼠骨髓), Isolation (分离), Endothelial-vascular niche (内皮血管微环境)

Background

The bone marrow microenvironment (BMM) is the compilation of cells and vascular networks within the marrow that acts as a vital and dynamic support system to assist hematopoiesis. The cellular component of the bone marrow consists of hematopoietic and non-hematopoietic cells, such as endothelial cells (ECs), osteoblasts, osteoclasts, adipocytes, and fibroblasts (Mishra et al., 2011; Birbrair and Frenette, 2016). The presence of ECs within the bone marrow environment regulates the trafficking and homing of the hematopoietic progenitor and stem cells. Alteration of the bone marrow endothelial cells (BMECs) due to malignant cells causes activation of endothelial-derived signaling pathways that favor the proliferation, differentiation, migration, and survival of neoplastic cells. It also stimulates angiogenesis, disrupting homeostasis within the BMM (Coşkun and Hirschi, 2015; Isern and Mendez-Ferrer, 2011; Passweg et al., 2016). In this protocol, we describe the isolation of BMECs and characterize their morphology.

Materials and Reagents

  1. 1.5 ml Eppendorf centrifuge tube (Eppendorf, catalog number: NA)

  2. 0.5 ml Nest micro-centrifuge tube (Biosharp, Life Science, catalog number: 201214)

  3. 18-gauge needle and syringe (Lujie®, 2015315175)

  4. Sterile 15 ml and 50 ml conical tubes (Beijing Labgic Technology, catalog numbers: CT-002-15 and CT-002-50)

  5. 100 mm cell culture dish (Thermo Fisher Scientific) and 12-48 well plates (Costar®, Corning, catalog number: 430161)

  6. Micro-dissecting board and pins

  7. Sterile gloves and gauze

  8. 70 µm cell strainer (Biologix, catalog number: 15-1070)

  9. Pipette calibrators P1000 (N253062), P200 (MZ26504), and P10 (N252362)

  10. Magnetic separation column and MACS® Separator (MACS, Miltenyi Biotec)

  11. Wild-type mice (C57BL/6)

    Male and female wild-type mice aged 8-12 weeks old were obtained from Jackson laboratory. Mice were housed under the regulation of Xuzhou Medical University, Jiangsu Province, China.

  12. 70-80% ethanol

  13. Milli-Q water (Quantum® Tix, catalog number: QTUMOTIX1)

  14. Miltenyi Biotec CD 31 microbeads, MS column (miniMACSTM Separator, Miltenyi Biotec)

  15. Fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific)

  16. Bovine serum albumin (BSA) (Solarbio® Life Science)

  17. Distilled water

  18. Accutase solution (Absin, Biochemical Company/ Thermo Fisher Scientific)

  19. Hydrochloride acid (HCl)

  20. Potassium chloride (KCl)

  21. Sodium chloride (NaCl)

  22. Sodium phosphate Dibasic (Na2HPO4)

  23. Potassium phosphate monobasic (KH2PO4)

  24. 10,000 U/ml penicillin /streptomycin (Gibco, Thermo Fisher Scientific)

  25. Rat-tail collagen type-I 3 mg/ml (Thermo Fisher Scientific, catalog number: A10483-01)

  26. Human plasma fibronectin purified protein (EMD Millipore, catalog number: 3383980 or FC010)

  27. PECAM-1 (CD31), rabbit-polyclonal (Affinity Bioscience, catalog number: AF6191)

  28. VE-CADHERIN (CD144) rabbit-polyclonal (Affinity Bioscience, catalog number: AF6265)

  29. Vascular endothelial growth factor receptor-2 (VEGFR2 (FlK-1) rabbit-polyclonal (Bioworld Technology, catalog number: Q1169)

  30. Secondary antibodies: Goat Anti-rabbit (IgG) Alexa Fluor® 594 (green; Abcam, catalog number: Ab15008) and Alexa Fluor®488 (red; Abcam, catalog number: Ab15007)

  31. Endothelial cells medium (EBM-2) (Lonza, Clonetics®)

  32. 1× Dulbecco’s phosphate buffer solution (DPBS) (see Recipes)

  33. Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Thermo Fisher Scientific) (see Recipes)

  34. 0.5 M Disodium Ethylenediaminetetraacetic acid (EDTA)·2H2O (see Recipes)

  35. 0.001 M Ethylenediaminetetraacetic acid (EDTA)/DBPS (see Recipes)

  36. Sterile harvesting buffer solution (see Recipes)

  37. Complete endothelial cell medium (see Recipes)

  38. 4% Paraformaldehyde (PFA) solution (Sinopharm Chemical Reagent) (see Recipes)

  39. 1% bovine serum albumin (BSA/DPBS) (see Recipes)

  40. 0.5 M EDTA (see Recipes)

  41. Dish pre-coating reagents (see Recipes)

Equipment

  1. Surgical scissors/forceps (10-11 cm long stainless steel dissecting scissors and 10-11 cm long stainless steel dissecting straight forceps)

  2. Cell culture incubator (CO2 incubator) (Thermo Fisher Scientific, model: Heracell 150i, catalog number: 41629032)

  3. Pipettes: P10, P200, and P1000 (Thermo Fisher Scientific, catalog numbers: 464000, 4640050, 4640060)

  4. Cell counting chamber (hemocytometer)/automated cell counting machine (Mindray)

  5. Refrigerator (2-8°C) (Haier, Bio-Medical)

  6. -20°C freezer (Haier)

  7. Laminar flow hook (Thermo Fisher Scientific, SN-194564, model -1381)

  8. High-speed centrifuge (Beckman Coulter, model: Microfuge®20R, catalog number: MRZ14H028, and low-speed centrifuge AIRTECH-KDC-40 Anhui USTC Zonkia Scientific instrument co.Ltd, catalog number: 02241700049)

  9. Nikon Eclipse microscope (Nikon, model: Eclipse Ti)

  10. Sterilization machine (Hirayama, model: HICLAVETM HVE-50, catalog number: 30613065826)

  11. pH meter and automated weighing machine (Metter Toledo)

  12. Magnetic stirrer (IKA® RCT Basic, 0317090005661)

  13. Graduate cylinder

Software

  1. Flow cytometry software

  2. FlowJo software version 7.6.2

Procedure

Note: Sterilize all necessary equipment and reagents beforehand.

  1. Preparation of adherent bone marrow cells

    1. Euthanize mouse (aged 8-12 weeks, Figure 1-i) by cervical dislocation and soak the whole mouse in 70% ethanol for 2-5 min. Place the sterile microdissection board, forceps, and scissors in the laminar flow hook. Clip the mouse onto the dissecting board with the pins, which were disinfected with 70% ethanol. Peel the skin off the mouse from the top of the hind leg down to the foot. Separate the hind legs with scissors and gently cut off the foot delicately to maintain the integrity of the bones. Place the hind legs in a 100 mm diameter dish containing 4-5 ml of the sterile room temperature DBPS for 2-3 min.

    2. Detach the muscle from the bones with forceps and scissors, dissect the bones with sterile tooth forceps, and place them in a sterile 100 mm dish diameter containing 2-3 ml of the DPBS (Figure 1-ii). Clean the bones with separate sterile forceps in DPBS and transfer them into a sterile 100 mm diameter dish containing 3 ml of 0.002 M EDTA/DPBS/antibiotic solution. Cut off the distal end of the femora and tibias (Figure 1-iii).

    3. Take the sterilized 0.5 ml nest microcentrifuge tube and push an 18-gauge needle down to the bottom of the tube to create a hole. Insert the cut edge of the femora and tibias downward, with a maximum of four bones per tube, and close the lid tightly (Figure 1-iv).

    4. Place the closed 0.5 ml nest microcentrifuge tube into a 1.5 ml Eppendorf tube and seal with Parafilm disinfected with 70% ethanol. Centrifuge the 1.5 ml Eppendorf tube at 15,000 × g for 30 s at 37°C (Figure 1-v). Remove the Eppendorf tube and verify that the bones are white (Figure 1-vi); if this is not the case, repeat the centrifugation. Discard the 0.5 ml nest microcentrifuge tube, suspend the noticeable pellet at the bottom of the 1.5 ml Eppendorf tube gently with 100-200 µl of sterile room temperature DPBS/EDTA solution containing antibiotics, and transfer the cells into a new clean tube.

    5. Gently pipet the suspended bone marrow cells with 3-4 ml of sterile room temperature DPBS/EDTA solution and filter the cells with a 70 µm cell strainer into a sterile 50 ml conical tube (Figure 1-vii). Transfer the purified bone marrow cells into a 10-15 ml clean tube (Figure 1-viii) and centrifuge at 300 × g for 5 min at room temperature (37°C) (Figure 1-ix). Discard the supernatant and resuspend the visible pellet with 2-3 ml of complete DMEM (see Recipes) (Figure 1-x). Count the total number of cells with an automated cell counter or hemocytometer.

    6. Seed 50-100 million bone marrow cells into a sterile 100 mm diameter cell culture dish containing 10-12 ml of complete DMEM (see Recipes) and incubate in a 5% CO2 humidified incubator for 8-12 h or overnight (Figure 1-xi).



      Figure 1. Illustration of Procedure A: preparation of bone marrow adherent cells.


  2. Isolation of cultured adherent BMECs

    1. Remove the 100 mm cell culture dish from the cell culture incubator after 8-12 h or overnight incubation. Patiently pipette the non-adherent cells from the cell culture dish with P1000 and discard them. Wash the cultured adherent bone marrow cells with 2-3 ml of sterile room temperature DPBS twice (Figure 2-i). Place 2-3 ml of accutase solution into a 100mm cell culture dish and incubate for 15 min at room temperature.

    2. Observe the cultured adherent bone marrow cells under the light microscope. If the cells have detached from the dish, suspend them in 1-2 ml of the harvesting buffer solution. Transfer the cells into 10-15 ml tubes and add 2-3 ml of the harvesting buffer solution to resuspend the remaining cells from the cell culture dish (Figure 2-ii). Confirm that all cells have been detached by looking at the cell culture dish under the light microscope. Centrifuge the resuspended cells 300 × g for 10 min at room temperature (Figure 2-iii). Resuspended the pellet (Figure 2-iv) with 100-200 µl of harvesting buffer and determine the total number of cells by an automated cell counter or hemocytometer.

    3. Aliquot approximately 107-109 cultured adherent marrow cells in 90 µl with 10 µl of CD31 microbeads into a 1.5 ml Eppendorf tube and mix gently with a P200 pipette. Then, incubate the mix at 4°C for 15 min in the dark (Figure 2-v).

    4. After incubation, transfer the CD31 microbeads cells into 10-15 ml conical tubes and resuspend the cells with 10 ml of harvesting buffer solution (Figure 2-vi). Centrifuge at 300 × g for 10 min at room temperature to wash out the excess magnetic beads from the cells (Figure 2-vii). Resuspend the pellet with 500-1,000 µl of the harvesting buffer solution (Figure 2-viii).

    5. Sanitize the magnetic (MACSTM Separator) and miniMACSTM Separator with 70% ethanol and place it in the laminar flow hook. Attach the MS column (miniMACSTM Separator) to the MACSTM Separator and rinse the column once with 500 µl of the harvesting buffer solution. Load the resuspended marrow cells into the column to allow single cells to pass through. Rinse the MS column three times with 500 µl of harvesting buffer solution, followed by a solitary wash with 100-200 µl of the complete endothelial cell medium (see Recipes) (Figure 2-ix).

      Note: The flow-through of non-magnetic CD31 microbeads cells (CD31 negative cells) is comprised of other marrow adherent cells such as macrophages, mesenchymal cells, and endothelial progenitor cells, which can be stored for other experiments or discarded.

    6. Push the magnetic beads cells into a new sterile tube with 1,000 µl of complete endothelial cell medium (see Recipes) (Figure 2-x). Determine the total number of BMECs by pipetting the cells into the well of the hemocytometer counting chamber or automated cell counter.

      Note: For the immediate experimental use of the isolated BMECs, we advise that the positive magnetic bead cells be resuspended with 1,000 µl of the harvesting buffer solution and repeat step 5. If the isolated cells are to remain in culture for several days after isolation, move to step 7. The minimal contaminated adherent cells with isolated BMECs will die off after 4-7 days in cell culture.

    7. If the 12-48 well plates are pre-coated with rat-tail collagen type-I, rinse the plate with 0.5-1ml of sterile room temperature DPBS solution twice and once with 100-200 µl of complete endothelial cell medium (see Recipes). Seed approximately 2 × 105-3 × 105 cells per well (Figure 2-xi) and leave the cells undisturbed for 3-4 days to maintain their number. Then, change the medium without a wash and incubate the cells for an additional 7-10 days (Figure 2-xii). Alternatively, if the plates were pre-coated with human plasma fibronectin, there is no need to wash, and cells can be seeded directly into the well as described above.

      Notes:

      1. The total number of BMECs per mouse is approximately 3 × 104-5 × 104 cells.

      2. The prepared complete endothelial cell medium (50 ml, see Recipes) should be pre-warmed at room temperature.

      3. The cells should be distributed at the side of the wells to have equivalent dispensation. Agitation or shaking of the plates must be avoided to prevent aggregation of the cells in some regions of the wells.



      Figure 2. Illustration of Procedure B: isolation of cultured adherent BMECs.


  3. Treatment of BMECs

    1. Cells should be cultured in pre-coated 12-48 wells plate for seven days before being treated with pharmacological drugs or inhibitors. Before treatment of the cells, wash the cells with pre-warmed endothelial cells medium. Add 100-300 µl of the endothelial cell medium containing the inhibitor or drug and 100-300 µl of endothelial cell medium containing 2% FBS for the vehicle cells as described in our publication (Smith et al., 2021). After the duration of treatment, replace the medium with complete endothelial cell medium (see Recipes). For external treatment such as radiation exposure, replace complete endothelial cell medium (see Recipes) with the pre-warmed medium at room temperature.

      Notes:

      1. Aspiration and release treads of the media must be executed at the side of the wells to avoid aggregation of the cells, as shown in Figure 3.

      2. Perform a serial dilution of the drugs or inhibitors to ascertain the non-toxic concentration for the primary BMECs.

      3. The time scale for individual experiments needs to be established to attain optimal results.

      4. An experiment involving protein extraction from BMECs should be cultured in 12-48 wells plate without coating for excellent results. The coating of the wells with fibronectin/ rat tail collagen type I will affect the experimental outcome.



    Figure 3. Aspiration and release of the medium should be performed at the side of the wells.

    Hold the plate at an approximate angle of 20-30 degrees and hold the pipette at 120 degrees to avoid detachment of the cells.


  4. Fixation and imaging of BMECs (Figure 4)

    1. Aspirate the supernatant from the adherent BMECs.

    2. Drop 50-100 µl of 4% PFA solution (see Recipes) into the wells and incubate at room temperature for 20 min. Wash cells with DBPS and incubate with 50-100 µl of 90% methanol for 20-25 min. Fellow the link to see the entire procedure: https://rdcu.be/cni0O or https://doi.org/10.1186/s13287-021-02352-3.

      Note: Do not culture the primary BMECs in a glass slide. The cells do not adhere even to slides pre-coated with either fibronectin or rat tail collagen type I.



      Figure 4. Characterization of BMECs by immunofluorescence staining and flow cytometry.

      (A) Imaging of BMEC specific markers after seven days of cell culture. Primary antibodies: PECAM-1 (CD31) Rabbit-polyclonal, VE-CADHERIN (CD144) Rabbit-polyclonal, and vascular endothelial growth factor receptor-2 (VEGFR2/FlK-1) Rabbit-polyclonal. Secondary antibodies: Goat Anti-rabbit (IgG) Alexa Fluor® 594 (green, Ab15008) and Goat Anti-rabbit Alexa Fluor®488 (red, Ab15007). Dilution in 1% BSA/DPBS of 1:50 for primary antibodies and of 1:500 for secondary antibodies. Scale bars = 5 µm, 20× magnification. (B) Flow cytometry analysis of BMEC surface markers: CD31 (PE, anti-mouse, eBioscienceTM), CD106 (PE, Rat-anti-mouse, BD-PharmingenTM), CD144 (APC, anti-mouse, eBioscienceTM) and endothelial selective adhesion molecule (ESAM) (APC, anti-mouse, Biolegend®) antibodies. 5 × 105 cells were collected per tube, washed with DPBS, and centrifuged at 500 × g for 5 min at 37°C (this procedure was repeated twice). The cell was diluted with the antibodies or a blank control at 4°C for one hour. Data were analyzed by flow cytometry (BD LSRFortessaTM) within 24 h. The lower frequency was used to exclude dead cells/debris by forward scatter (FSC) × side scatter (SSC), followed by graphical histogram presentation. Cytometry data were analyzed by FlowJo software version 7.6.2.


Data analysis

Each independent experiment should be executed with at least three experimental repeats and the data normalized with vehicle cells or untreated cells as described in our publication (Smith et al., 2021). A bar graph and suitable statistical tests can be used to represent the data. Follow the link to see statistical analysis: https://rdcu.be/cni0O or https://doi.org/10.1186/s13287-021-02352-3.

Note: At least six (Smith et al., 2021) mice per group for each experimental setup are necessary to attain optimal results.

Recipes

  1. 1× Dulbecco’s phosphate buffer solution (DPBS) sodium (1 L)

    NaCl 8.0 g

    KCl 0.2 g

    KH2PO4 0.2 g

    Na2HPO4 1.15 g

    Dissolve in 1,000 ml of Milli-Q water

    Adjusted pH to 7.2-7.6 with HCl, sterilize, and store at 4°C

  2. Complete DMEM (50 ml)

    Dulbecco’s Modified Eagle Medium (DMEM)

    20% fetal bovine serum (FBS)

    500 µl of 10,000 U/ml penicillin/streptomycin

    Keep at 4°C

  3. 0.5 M Ethylenediaminetetraacetic acid (EDTA) solution, pH 8.0 (500 ml)

    93.05 g of Na2EDTA·2H2O

    400 ml of Milli-Q water

    Adjust pH with NaOH, sterilize and store at 4°C.

  4. 0.001 M Ethylenediamine tetra-acetic acid (EDTA)/DBPS

    50 ml of DPBS

    100 µl of 0.5 M EDTA

    100 µl of 10,000 U/ml penicillin/streptomycin

    Store at 4°C

  5. Sterile harvesting buffer solution

    0.002 M EDTA/DBPS

    2% FBS

    1% bovine serum albumin

    100 µl of 10,000 U/ml penicillin/streptomycin

    Store at 4°C for 4 weeks

  6. Complete endothelial cell medium (50 ml)

    25 ml of Fetal bovine serum (FBS)

    0.2 ml of hydrocortisone

    0.5 ml of vascular endothelial growth factor (VEGF)

    Human fibroblast growth factor (hFGF)

    Ascorbic acid

    Human epidermal growth factor (hEGF)

    Heparin

    Gentamicin/Amphotericin

    Store at 4°C for 4 weeks only

  7. 1% bovine serum albumin (BSA/DPBS)

    50 ml of sterile DPBS

    0.5 g of bovine serum albumin (BSA)

  8. 4% Paraformaldehyde (PFA) solution

    Add 4 g of graded Paraformaldehyde to 50 ml of distilled water or Milli-Q-water.

    Add 1 ml of 1 M NaOH, stir gently with a magnetic stirrer at 60°C until the PFA has dissolved.

    Add 10 ml of 10× DPBS solution.

    Allow the mixture to cool at room temperature.

    Adjust the pH to 7.4 with 1 M HCl, then raise the volume to 100 ml.

    Filter solution through 0.45 µm membrane to get rid of any residual particles.

    Aliquot into appropriate volumes and store at 4°C for one month or at -20°C for several months.

  9. 0.5 M EDTA

    Add 93.05 g of Na2(EDTA)·2H2O to 400 ml of distilled water and stir with a magnetic stirrer. Adjust pH to 8.0 with NaOH, raise the volume to 500 ml, and store the solution at 4°C.

  10. Dish pre-coating reagents

    1. The Rat-tail collagen type-I 3 mg/ml plus 0.2 M sterile HCl (50-100 µg/ml).

      Preparation of 50 µg/ml of Rat-tail collagen type-I in 0.02 M HCl:

      Prepare 0.02 M HCl = 1 ml of 0.2 M HCl in 9 ml of distilled water

      Aliquots ~17 µl of 3 mg/ml Rat-tail collagen type-I add to 983 µl of 0.02 M HCl

    2. Alternatively, human fibronectin coating solution 1 mg/ml with 2-10 µg/cm2.

      Preparation of 20 µg/ml of the human fibronectin coating solution:

      Aliquots 200 µl of human fibronectin solution and add to 3.8 ml of sterile room temperature DBPS.

      Note: Overnight pre-coating of the above coating reagents produced optimal results, and the table below shows the volume ranges of pre-coating solution recommended.

      Plates/well Recommended volume
      48 well 250-300 µl
      12 well 400-500 µl
      24 well 0.5-1 ml
      6 well 1-2 ml

Acknowledgment

The protocol was modified from Smith et al. (2021) . The authors would like to acknowledge the support and expert advice of the corresponding authors: Jianlin Qiao, Kailin Xu, and Lingyu Zeng.

Funding: This study was supported by the National Natural Science Foundation of China [Grant number 31872795, 81570096 and 81700178]; Major Basic Research Project of the Natural Science[Grant number 17KJA320008]; Jiangsu Provincial Key Research and Development Program[Grant number BE2018637]; Jiangsu Province’s Key Provincial Talents Program under Grant [number ZDRCA2016054]; Foundation of the Jiangsu Higher Education Institutions Natural Science Foundation of Jiangsu Province under Grant [number BK20170259]; China Postdoctoral Science Foundation Grant [number 2018M632380]; and Jiangsu Postdoctoral Science Foundation under Grant [number 1701064B].

Competing interest

The authors declared no competing interest in these protocols.

Ethics

The National Institute of Health guide for the care and use of laboratory animals and use committee of Xuzhou Medical University (Xuzhou, China) and Jiangsu province, China, approved the studies with acceptance number: XZMC20130226.

References

  1. Birbrair, A. and Frenette, P. S. (2016). Niche heterogeneity in the bone marrow. Ann N Y Acad Sci 1370(1): 82-96.
  2. Coşkun, S. and Hirschi, K. K. (2015). Vascular Niche in HSC Development, Maintenance, and Regulation. In: Tissue-Specific Stem Cell Niche. Springer International Publishing. pp191-219.
  3. Isern, J. and Mendez-Ferrer, S. (2011). Stem cell interactions in a bone marrow niche. Curr Osteoporos Rep 9(4): 210-218.
  4. Mishra, A., Shiozawa, Y., Pienta, K. J. and Taichman, R. S. (2011). Homing of cancer cells to the bone. Cancer Microenviron 4(3): 221-235.
  5. Passweg, J. R., Baldomero, H., Bader, P., Bonini, C., Cesaro, S., Dreger, P., Duarte, R. F., Dufour, C., Kuball, J., Farge-Bancel, D., et al. (2016). Hematopoietic stem cell transplantation in Europe 2014: more than 40 000 transplants annually. Bone Marrow Transplant 51(6): 786-792.
  6. Smith, A. O., Adzraku, S. Y., Ju, W., Qiao, J., Xu, K. and Zeng, L. (2021). A novel strategy for isolation of mice bone marrow endothelial cells (BMECs). Stem Cell Res Ther 12(1): 267.

简介

[摘要]在骨髓微环境中,内皮细胞(ECs)在调节生长因子和抑制因子的产生中起着举足轻重的作用。它们通过与造血祖细胞相互作用的粘附分子结合在一起。由于缺乏有效的隔离方案,对造血干细胞生态位中 EC 的研究受到限制。在该协议中,我们开发了一种两步法来提取骨髓内皮细胞 (BMECs),以解开研究人员在体外研究中理解内皮血管壁龛功能所面临的挑战。

[背景]骨髓微环境 (BMM) 是骨髓内细胞和血管网络的集合,作为辅助造血的重要和动态支持系统。骨髓的细胞成分由造血和非造血细胞组成,例如内皮细胞 (EC)、成骨细胞、破骨细胞、脂肪细胞和成纤维细胞(Mishra等,2011;Birbrair 和 Frenette,2016 )。骨髓环境中内皮细胞的存在调节造血祖细胞和干细胞的运输和归巢。恶性细胞引起的骨髓内皮细胞 (BMEC) 的改变导致内皮源性信号通路的激活,这些信号通路有利于肿瘤细胞的增殖、分化、迁移和存活。它还刺激血管生成,破坏BMM 内的稳态(Coşkun 和 Hirschi,2015;Isern 和 Mendez-Ferrer,2011;Passweg等,2016)。在这个协议中,我们描述了 BMECs 的隔离并描述了它们的形态。

关键字:骨髓内皮细胞(BMECs), 内皮细胞(ECs), 抽取, 小鼠骨髓, 分离, 内皮血管微环境

材料和试剂

1.     1.5 ml Eppendorf 离心管(Eppendorf目录号:NA 

2.     0.5 ml Nest 微量离心管(BiosharpLife Science目录号:201214 

3.     18 号针头和注射器Lujie ® 2015315175 )

4.     无菌 15 ml 50 ml 锥形管(Beijing Labgic Technology,目录号:CT-002-15 CT-002-50

5.     100毫米细胞培养皿(赛默飞世尔科技)和12-48孔板(Costar ® ,康宁,目录号:430161 

6.     显微解剖板和针脚

7.     无菌手套和纱布

8.     70 µm 细胞过滤器(Biologix ,目录号:15-1070

9.     移液器校准器 P1000 (N253062)P200 (MZ26504) P10 (N252362)

10.  磁分离柱和 MACS ®分离器(MACSMiltenyi Biotec

11.  野生型小鼠C57BL/6)

8-12周龄雄性和雌性野生型小鼠获自Jackson实验室。小鼠在中国江苏省徐州医科大学的规定下饲养。

12.  70-80% 乙醇

13.  Milli-Q Quantum ® Tix,目录号:QTUMOTIX1

14.  Miltenyi Biotec CD 31 微珠,MS 柱(miniMACS TM SeparatorMiltenyi Biotec

15.  胎牛血清 (FBS)ibcoThermo Fisher Scientific

16.  牛血清白蛋白 (BSA) (Solarbio ® Life Science)

17.  蒸馏水

18.  Accutase 溶液(bsinBiochemical Company/Thermo Fisher Scientific

19.  盐酸(HCl

20.  氯化钾 (KCl)

21.  氯化钠 (NaCl)

22.  磷酸氢二钠 (Na HPO )

23.  磷酸二氢钾 (KH PO )

24.  10,000 U/ml 青霉素/链霉素(ibcoThermo Fisher Scientific

25.  鼠尾胶原蛋白 I 3 mg/ml Thermo Fisher Scientific,目录号:A10483-01

26.  人血浆纤连蛋白纯化蛋白(EMD Millipore,目录号:3383980 FC010

27.  PECAM-1CD31),兔多克隆(Affinity Bioscience,目录号:AF6191

28.  VE-CADHERINCD144)兔多克隆(Affinity Bioscience,目录号:AF6265

29.  血管内皮生长因子受体-2VEGFR2FlK-1)兔多克隆(Bioworld Technology,目录号:Q1169

30.  二抗:山羊抗兔(IgGAlexa Fluor ® 594(绿色;Abcam,目录号:Ab15008)和 Alexa Fluor ® 488(红色;Abcam,目录号:Ab15007

31.  内皮细胞培养基(EBM-2 Lonza公司,Clonetics ® 

32.  1 × Dulbecco 磷酸盐缓冲液(DPBS)(见配方)

33.  Dulbecco 的改良 Eagle 培养基(DMEMibcoThermo Fisher Scientific(见食谱)

34.  0.5 M乙二胺四乙酸二钠(EDTA ·2 (见配方)

35.  0.001 M 乙二胺四乙酸(EDTA/DBPS(见配方)

36.  无菌收获缓冲液(见配方)

37.  完整的内皮细胞培养基(见食谱)

38.  4%多聚甲醛(PFA)溶液(国药化学试剂)(见配方)

39.  1% 牛血清白蛋白(BSA/DPBS)(见食谱)

40.  0.5 M EDTA(见配方)

41.  盘子预涂层试剂(见配方)

 

设备

 

1.     手术剪刀/镊子(10-11 厘米长不锈钢解剖剪刀和10-11 厘米长不锈钢解剖直钳)

2.     细胞培养箱(CO 2培养箱)(Thermo Fisher Scientific型号:Heracell 150i 目录号:41629032

3.     移液器:P10P200 P1000Thermo Fisher Scientific,目录号:46400046400504640060             

4.     细胞计数室(血细胞计数器)/自动细胞计数机(迈瑞)

5.     冰箱(2-8 °C )(海尔、生物医药)

6.     -20°C冰箱(海尔)

7.     层流钩(Thermo Fisher ScientificSN-194564,型号 -1381 

8.     高速离心机(Beckman Coulter型号:Microfuge ® 20R,目录号:MRZ14H028,低速离心机AIRTECH-KDC-40 AnhuiUSTC Zonkia Scientific Instruments co.Ltd目录号:02241700049

9.     Nikon Eclipse 显微镜(Nikon,型号:Eclipse Ti

  1. 灭菌机(Hirayama,型号:HICLAVE TM HVE-50目录号:30613065826
  2. pH计和自动称重机(Metter Toledo
  3. 磁力搅拌器(IKA ® RCT Basic0317090005661
  4. 量筒

 

软件

 

1.     流式细胞术软件

2.     FlowJo 软件版本 7.6.2

 

程序

 

注意:事先对所有必要的设备和试剂进行消毒。

A.    贴壁骨髓细胞的制备

1.     通过颈椎脱位对小鼠8-12 周龄,图 1-i)实施安乐死并将整只小鼠浸泡在 70% 乙醇中 2-5 分钟。将无菌显微切割板、镊子和剪刀放在层流钩中。用针脚将鼠标夹在解剖板上,用 70% 乙醇消毒。剥下鼠标从后腿顶部到脚的皮肤。用剪刀将后腿分开,轻轻地将脚剪掉,以保持骨骼的完整性。将后腿放入含有 4-5 毫升无菌室温 DBPS 100 毫米直径盘中 2-3 分钟。

2.     用镊子和剪刀将肌肉从骨骼中分离出来,用无菌牙钳解剖骨骼,并将它们放在一个直径为 2-3 毫升的 DPBS 的无菌 100 毫米盘中(图 1-ii)。在 DPBS 中用单独的无菌镊子清洁骨头,并将它们转移到含有 3 ml 0.002 M EDTA/DPBS/抗生素溶液的无菌 100 mm 直径盘中。切断股骨和胫骨的远端 ( 1-iii)

3.     取出已消毒的 0.5 毫升巢式微量离心管,将 18 号针头向下推至管底部以形成一个孔。将股骨和胫骨的切缘向下插入,每管最多四根骨头,并盖紧盖子(图 1-iv)。

4.     将封闭的 0.5 ml Nest 微量离心管放入 1.5 ml Eppendorf 管中,并用用 70% 乙醇消毒的封口膜密封。将 1.5 ml Eppendorf 管以 15,000 × g离心30 秒,37 (图 1-v)。取出 Eppendorf 管,确认骨头是白色的(图 1-vi);如果不是这种情况,请重复离心。丢弃 0.5 ml 巢式微量离心管,用 100-200 µl 无菌室温 DPBS/EDTA 溶液轻轻悬浮在 1.5 ml Eppendorf 管底部的明显沉淀物,并将细胞转移到新的干净管中。

5.     3-4 ml 无菌室温 DPBS/EDTA 溶液轻轻吸取悬浮的骨髓细胞,并用 70 µm 细胞过滤器将细胞过滤到无菌 50 ml 锥形管中(图 1-vii)。将纯化的骨髓细胞转移到 10-15 ml 干净的管中(图 1-viii),并在室温(37°C)下以 300 × g离心5 分钟(图 1-ix)。丢弃上清液并用 2-3 ml 完整 DMEM(参见食谱)重悬可见颗粒(图 1-x)。使用自动细胞计数器或血细胞计数器计算细胞总数。

6.     50-1 亿个骨髓细胞接种到含有 10-12 ml 完整 DMEM(参见配方)的无菌 100 毫米直径细胞培养皿中,并在 5% CO 2加湿培养箱中孵育 8-12 小时或过夜(图 1-十一)。

 

 

1. 程序 A 的图示:骨髓贴壁细胞的制备。

 

B.    培养贴壁 BMEC 的分离

1.     8-12 小时或过夜孵育后,从细胞培养箱中取出100 毫米细胞培养皿。耐心地用 P1000 从细胞培养皿中吸取非贴壁细胞并丢弃它们。用 2-3 ml 无菌室温 DPBS 清洗培养的贴壁骨髓细胞两次(图 2-i)。将 2-3 ml accutase 溶液放入 100mm 细胞培养皿中,并在室温下孵育 15 分钟。

2.     在光学显微镜下观察培养的贴壁骨髓细胞。如果细胞已经从培养皿中分离出来,将它们悬浮在 1-2 ml的收获缓冲液中。将细胞转移到 10-15 ml 管中,并添加 2-3 ml 收获缓冲液,以重新悬浮细胞培养皿中的剩余细胞(图 2-ii)。在光学显微镜下观察细胞培养皿,确认所有细胞均已分离。在室温下将重悬的细胞 300 × g离心10 分钟(图 2-iii)。ř esuspended佩尔等(图2-iv)与100-200微升收获缓冲液中,确定细胞的总数通过自动细胞计数器或血细胞计数器。

3.     将大约 10 -10 9培养的粘附骨髓细胞在 90 µl 中与 10 µl CD31 微珠分装到 1.5 ml Eppendorf 管中,并用 P200 移液器轻轻混合。然后,将混合物在 4°C 下在黑暗中孵育 15 分钟(图 2-v)。

4.     孵育后,将 CD31 微珠细胞转移到 10-15 ml 锥形管中,并用 10 ml收获缓冲液重新悬浮细胞(图 2-vi)。在室温下以300 × g离心10 分钟,以洗掉细胞中多余的磁珠(图 2-vii)。用 500-1,000 µl 收获缓冲液重悬沉淀(图 2-viii)。

5.     70% 乙醇对磁性(MACS TM分离器)和 miniMACS TM分离器进行消毒,并将其放入层流钩中。将 MS 色谱柱(miniMACS TM Separator)连接到 MACS TM Separator 并用 500 µl 收获缓冲液冲洗色谱柱一次。将重悬的骨髓细胞装入柱中,让单个细胞通过。用 500 µl 收获缓冲液冲洗 MS 色谱柱 3 次,然后用 100-200 µl 完整内皮细胞培养基单独洗涤(参见食谱)(图 2-ix)。

注意:非磁性 CD31 微珠细胞(CD31 阴性细胞)的流通由其他骨髓贴壁细胞组成,如巨噬细胞、间充质细胞和内皮祖细胞,可储存用于其他实验或丢弃

6.     磁珠细胞入装有 1,000 µl 完整内皮细胞培养基的新无菌管中(参见食谱)(图 2-x)。通过将细胞移入血球计数室或自动细胞计数器的井中来确定 BMECs 的总数。

注意:对于分离的 BMEC 的直接实验用途,我们建议将阳性磁珠细胞用 1,000 µl 收获缓冲液重新悬浮并重复步骤 5。如果分离的细胞在分离后要保持培养几天,转到第 7 步。带有分离的 BMEC 的最小污染贴壁细胞将在细胞培养 4-7 天后死亡。

7.     如果 12-48 孔板预先涂有 I 型鼠尾胶原蛋白,则 0.5-1ml 无菌室温 DPBS 溶液冲洗板两次,然后用 100-200 μl 完整内皮细胞培养基冲洗一次(参见食谱)。每孔播种大约 2 × 10 -3 × 10 5 细胞(图 2-xi),并使细胞不受干扰 3-4 天以保持其数量。然后,无需洗涤即可更换培养基,并将细胞再孵育 7-10 天(图 2-xii)。或者,如果板预先涂有人血浆纤连蛋白,则无需洗涤,细胞可以直接接种到上述孔中。

笔记:

a.     每只小鼠的 BMEC 总数约为 3 × 10 -5 × 10 4 细胞。

b.     制备的完整内皮细胞培养基(50 ml,参见食谱)应在室温下预热

c.     细胞应分布在孔的侧面以具有等效的分配。必须避免搅动或摇晃板子,以防止细胞在孔的某些区域聚集。

 

 

2. 程序 B 的说明:分离培养的贴壁 BMEC

 

C.    BMEC 的治疗

1.     在用药物或抑制剂处理之前,细胞应在预包被的 12-48 孔板中培养 7 天。在处理细胞之前,用预热的内皮细胞培养基清洗细胞。添加 100-300 µl 含有抑制剂或药物的内皮细胞培养基和 100-300 µl 含有 2% FBS 的内皮细胞培养基,如我们的出版物中所述(Smith2021)。治疗持续时间后,用完整的内皮细胞培养基更换培养基(参见食谱)。对于辐射暴露等外部治疗,在室温下用预热的培养基替换完整的内皮细胞培养基(参见食谱)。

笔记:

a.     介质的吸入和释放踏板必须在井的一侧执行,以避免细胞聚集,如图 3 所示。

b.     对药物或抑制剂进行连续稀释,以确定主要 BMEC 的无毒浓度。

c.     需要确定单个实验的时间尺度以获得最佳结果。

d.     涉及从 BMEC 中提取蛋白质的实验应在 12-48 孔板中培养,无需涂层,以获得出色的结果。用纤连蛋白/鼠尾胶原蛋白 I 型对孔进行涂层会影响实验结果。

 

 

3. 介质的吸入和释放应在孔的一侧进行。

将板保持在大约 20-30 度的角度,并将移液器保持在 120 度以避免细胞脱离。

 

D.    BMECs 的固定和成像(图 4

1.     从粘附的 BMEC 中吸出上清液。

2.     50-100 µl 4% PFA 溶液(参见配方)滴入孔中,并在室温下孵育 20 分钟。用 DBPS 洗涤细胞并用 50-100 µl 90% 甲醇孵育 20-25 分钟。访问链接以查看整个程序:https : //rdcu.be/cni0Ohttps://doi.org/10.1186/s13287-021-02352-3

注意:不要在载玻片中培养主要的 BMEC。即使在预先涂有纤连蛋白或 I 型鼠尾胶原蛋白的载玻片上,细胞也不会粘附。

 

 

4. 通过免疫荧光染色和流式细胞术表征 BMEC

(A)细胞培养 7 天后 BMEC 特定标记的成像一抗:PECAM-1 (CD31)兔多克隆抗体VE-钙粘蛋白(CD144)兔多克隆和血管内皮生长因子受体-2VEGFR2 / FLK-1)的兔多克隆二抗:山羊抗兔 (IgG) Alexa Fluor ® 594(绿色,Ab15008)和山羊抗兔 Alexa Fluor ® 488(红色,Ab15007。在 1% BSA/DPBS 中稀释 1:50 的初级抗体和 1:500 的二级抗体。比例尺 = 5 µm20 ×放大倍数。(B) BMEC 表面标志物的流式细胞术分析:CD31PEanti-mouseeBioscience TM ),CD106PERat-anti-mouseBD-Pharmingen TM ),CD144APCanti-mouseeBioscience TM )和内皮选择性粘附分子 (ESAM) (APC, anti-mouse, Biolegend ® ) 抗体。每管收集× 10 5 细胞,用 DPBS 洗涤,并在 37 °C 500 × g离心5 分钟(此过程重复两次)。细胞在°C下用抗体或空白对照稀释1 小时。在 24 小时内通过流式细胞术 (BD LSRFortessa TM )分析数据。较低的频率用于通过前向散射 (FSC) ×侧向散射 (SSC)排除死细胞/碎片,然后是图形直方图呈现。流式细胞术数据由 FlowJo 软件 7.6.2 版进行分析。               

 

数据分析

 

每个独立实验应至少进行三个实验重复,并且数据用载体细胞或未经处理的细胞标准化,如我们的出版物(史密斯等人2021)中所述。可以使用条形图和适当的统计测试来表示数据点击链接查看统计分析:https : //rdcu.be/cni0Ohttps://doi.org/10.1186/s13287-021-02352-3

注意:对于每个实验设置,每组至少需要六只(Smith 等人,2021)小鼠才能获得最佳结果。

 

食谱

 

1.     1× Dulbecco 磷酸盐缓冲溶液 (DPBS) (1 L)

氯化钠 8.0

氯化钾 0.2

KH PO 0.2

Na HPO 1.15

溶于 1,000 毫升 Milli-Q 水中

HCl pH 值调节至 7.2-7.6 ,灭菌,并在°C 储存

2.     完整的 DMEM50 毫升)

Dulbecco 改良 Eagle 培养基 (DMEM)

20% 胎牛血清 (FBS)

500 µl 10,000 /ml 青霉素/链霉素

保持在°C

3.     0.5 M 乙二胺四乙酸 (EDTA) 溶液,pH 8.0500 毫升)

93.05 Na EDTA·2H O

400 毫升 Milli-Q

NaOH 调节 pH 值,灭菌并在°C 下储存

4.     0.001 M 乙二胺四乙酸 (EDTA)/DBPS

50 毫升 DPBS

100 µl 0.5 M EDTA

100 µl 10,000 U/ml 青霉素/链霉素

储存°C

5.     无菌收获缓冲液

0.002 M EDTA/DBPS

2% 胎牛血清

1% 牛血清白蛋白

100 µl 10,000 U/ml青霉素/链霉素

°C 储存4

6.     完全内皮细胞培养基 (50 ml)

25 毫升胎牛血清 (FBS)

0.2 毫升氢化可的松

0.5 ml 血管内皮生长因子 (VEGF)

人成纤维细胞生长因子 (hFGF)

抗坏血酸

人表皮生长因子 (hEGF)

肝素

庆大霉素/两性霉素

仅在°C 储存4

7.     1% 牛血清白蛋白 (BSA/DPBS)

50 毫升无菌 DPBS

0.5 克牛血清白蛋白 (BSA)

8.     4% 多聚甲醛 (PFA) 溶液

4 g 分级多聚甲醛添加到 50 ml 蒸馏水或 Milli-Q-水中。

加入 1 ml 1 M NaOH,在 60 °C 用磁力搅拌器轻轻搅拌,直到 PFA 溶解。

加入 10 ml 10 × DPBS 溶液。

让混合物在室温下冷却。

1 M HCl pH 值调节至 7.4,然后将体积增加至 100 ml

通过 0.45 µm 膜过滤溶液以去除任何残留颗粒。

分装成适当的体积,并在°C 储存一个月或在 -20 °C储存几个月。

9.     0.5 M乙二胺四乙酸

93.05 g Na (EDTA) · 2H O 添加到 400 ml 蒸馏水中并用磁力搅拌器搅拌。用 NaOH 调节 pH 8.0,将体积增加至 500 ml,并将溶液储存在°C 

10.  碟子预涂试剂

a.     鼠尾胶原蛋白 I 3 mg/ml 0.2 M 无菌 HCl (50-100 µg/ml) 

0.02 M HCl 中制备 50 µg/ml 鼠尾胶原蛋白 I 型:

9 ml 蒸馏水中制备 0.02 M HCl = 1 ml 0.2 M HCl

~17 µl 3 mg/ml 鼠尾胶原蛋白 I 型加入 983 µl 0.02 M HCl

b.     或者,人纤连蛋白涂层溶液 1 mg/ml 2-10 µg/cm 

制备 20 µg/ml 的人纤连蛋白涂层溶液:

200 µl 人纤连蛋白溶液分装到 3.8 ml 无菌室温 DBPS 中。

注:上述包衣试剂过夜预包衣效果最佳,下表列出了推荐的预包衣液体积范围。

/

推荐音量

48

250-300 微升

12

400-500 微升

24

0.5-1毫升

6

1-2毫升

 

致谢

 

该协议是从史密斯等人修改的。(2021)。作者感谢通讯作者:乔建林、徐开林凌宇的支持和专家建议。

经费资助:本研究得到国家自然科学基金项目[批准号318727958157009681700178];自然科学重大基础研究项目[批准号17KJA320008];江苏省重点研发计划[批准号BE2018637];江苏省省级重点人才计划[编号ZDRCA2016054];江苏省高等学校自然科学基金资助项目[编号BK20170259];中国博士后科学基金资助[编号2018M632380];江苏省博士后科学基金[编号1701064B]

 

竞争利益

 

作者声明对这些协议没有竞争利益。

 

伦理

 

徐州医科大学(中国徐州)和中国江苏省卫生研究院实验动物护理和使用指南和使用委员会批准了该研究,验收号:XZMC20130226

 

参考

1.     Birbrair, A. Frenette, PS (2016)骨髓中的利基异质性。Ann NY Acad Sci 1370(1): 82-96

2.     Coşkun, S. Hirschi, KK (2015)HSC 开发、维护和调节中的血管利基。在:组织特异性干细胞壁龛。施普林格国际出版社。第 191-219 页。

3.     Isern, J. Mendez-Ferrer, S. (2011)骨髓壁龛中的干细胞相互作用。Curr Osteoporos 代表9(4)210-218              

4.     Mishra, A., Shiozawa, Y., Pienta, KJ Taichman, RS (2011)癌细胞归巢到骨骼。 癌症微环境4(3): 221-235

5.     Passweg, JR, Baldomero, H., Bader, P., Bonini, C., Cesaro, S., Dreger, P., Duarte, RF, Dufour, C., Kuball, J., Farge-Bancel, D.,(2016)2014 年欧洲造血干细胞移植:每年移植超过 40 000 次。骨髓移植51(6): 786-792 

6.     Smith, AOAdzraku, SYJu, W.Qiao, J.Xu, K. Zeng, L. (2021)一种分离小鼠骨髓内皮细胞 (BMECs) 的新策略。干细胞水疗12(1): 267

登录/注册账号可免费阅读全文
  • 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。
Copyright: © 2021 The Authors; exclusive licensee Bio-protocol LLC.
引用:Smith, A. O., Adzraku, S. Y., Ju, W., Qiao, J., Xu, K. and Zeng, L. (2021). Isolation of CD31+ Bone Marrow Endothelial Cells (BMECs) from Mice. Bio-protocol 11(22): e4227. DOI: 10.21769/BioProtoc.4227.
提问与回复

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。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的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。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的形式来说明遇到的问题。