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Isolation and Culture of Human CD133+ Non-adherent Endothelial Forming Cells
分离和培养人 CD133+非粘连内皮形成细胞

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Abstract

Circulating endothelial progenitor cells (EPCs) have been the focus of many clinical trials due to their roles in revascularisation following ischemic events such as acute myocardial infarction as well as their contribution to vascular repair during organ transplantation. Research on EPCs has been controversial due to the lack of distinct markers expressed at the cell surface and varying methods for isolation and culture have resulted in the identification of a multitude of cell types, with differing phenotype and function, all falling under the label of “EPCs”. The most widely documented EPCs isolated for cell therapy are adherent in nature and lacking the progenitor markers such as CD133 and therefore unlikely to represent a true circulating EPC, the cells mobilised in response to a vascular injury.
We recently published the isolation and extensive characterisation of a population of non-adherent endothelial forming cells (naEFCs) (Appleby et al., 2012) (Figure 1). These cells expressed the progenitor cell markers (CD133, CD34, CD117, CD90 and CD38) together with mature endothelial cell markers (VEGFR2, CD144 and CD31). These cells also expressed low levels of CD45 but did not express the lymphoid markers (CD3, CD4, CD8) or myeloid markers (CD11b and CD14) which distinguishes them from ‘early’ EPCs, the ‘late outgrowth EPC’ [more recently known as endothelial colony forming cells (ECFCs)] as well as mature endothelial cells (ECs). Figure 2A exemplifies the surface expression profile of the naEFCs. Functional studies demonstrated that these naEFCs (i) bound Ulex europaeus lectin (Figure 2A), (ii) demonstrated acetylated-low density lipoprotein uptake, (iii) increased vascular cell adhesion molecule (VCAM-1) surface expression in response to tumor necrosis factor and (iv) in co-culture with mature ECs increased the number of tubes, tubule branching and loops in a 3-dimensional in vitro matrix. More importantly, naEFCs placed in vivo generated new lumen containing vasculature lined by CD144 expressing human ECs and have contributed to various advances in scientific knowledge (Appleby et al., 2012; Barrett et al., 2011; Moldenhauer et al., 2015; Parham et al., 2015). Here, we describe the isolation and enrichment of a non-adherent CD133+ endothelial forming population of cells from human cord blood.

Keywords: Endothelial progenitor cells(内皮祖细胞), Expansion(膨胀), Human(人类), CD133(CD133), Non-adherent(非贴壁)



Figure 1. Enrichment of human naEFCs. A. Umbilical cord blood derived CD133+ enriched cells (naEFCs) at 4 days of culture and human umbilical vein endothelial cells (ECs) were compared for cell size by light microscopy. Scale bar=200 µm B. The cells were assessed for heterogeneity of enrichment process (0-10 days) via forward scatter and side scatter profiling using flow cytometric analysis and compared to mature ECs.


Figure 2. Surface expression phenotype of human naEFCs. A. CD133+ enriched cells at 4 days of culture were assessed for progenitor and endothelial markers by flow cytometry. Histograms show a representative experiment from ≥3 biological replicates where grey dashed lines represent isotype controls and solid black lines represent cells stained with the indicated marker. B. The function of the naEFCs was assessed by flow cytometry and compared to mature ECs, detecting the ability of cells to uptake DiI labelled acetylated low density lipoprotein (Ac-LDL) and bind FITC labelled Ulex europaeus agglutinin I (UEA-1) lectin. Density plots represent stained cells of one representative experiment from ≥3 biological replicates.

Materials and Reagents

  1. For manual cell sorting
    MS Columns (Miltenyi Biotec, catalog number: 130-042-201 )
  2. MacoPharma cord blood collection bags (MacoPharma, catalog number: MSC1201DU )
  3. 50 ml tubes (Corning, Falcon®, catalog number: 352070 )
  4. 10 ml tubes (Sarstedt AG, catalog number: 62.9924.284 )
  5. 2 ml soft sterile bulb transfer pipette, sterile (Stephen Gould corporation, catalog number: 222-1S )
    Note: Currently, it is “Capitol Scientific, catalog number: 222-1S”.
  6. 20 µm filter (Sartorius Stedim Biotec, Minisart, catelog number: 16534-K )
  7. 24-well plate (Corning, Falcon®, catalog number: 353047 )
  8. Microfuge tubes
  9. 20 ml syringes
  10. 0.2 µm filter
  11. Human umbilical cord blood (40-250 ml)
  12. 20x Dulbecco’s phosphate buffered (DPBS) (Life Technologies, Gibco®, catalog number: 14200-075 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 14200-075”.
  13. Sterile water (Baxter, catalog number: UKF7114 )
  14. LymphoprepTM (Axis-Shield, catalog number: 114547 )
  15. CD133 microbeads including human FcR blocking reagent (Miltenyi Biotec, catalog number: 130-050-801 )
  16. AutoMACS Pro Washing Solution (Miltenyi Biotec, catalog number: 130-092-987 )
  17. AutoMACS Running Buffer (Miltenyi Biotec, catalog number: 130-091-221 )
    Note: If the AutoMACS Pro separator is not available, cells of interest can be isolated by manual sorting (see below). This manual method, however, is not necessarily optimal for naEFC cell sorting with lower cell viability and number observed; thus the AutoMACS method is preferred.
  18. Endothelial growth media with Bullet kit (EGM-2) (Lonza, catalog number: cc-3162 )
  19. Fetal bovine serum, characterized (FBS) (VWR International, HycloneTM, catalog number: SH30071.03 )
  20. Recombinant human Vascular endothelial growth factor (VEGF) (Sigma-Aldrich, catalog number: V7259 )
  21. Recombinant human Insulin-like growth factor-1 (IGF-I) (R&D Systems, catalog number: 291-G1-200 )
  22. Recombinant human fibroblast growth factor basic (FGFb) (R&D Systems, catalog number: 233-FB-025 )
  23. L-Ascorbic acid (Sigma-Aldrich, catalog number: A5960 )
  24. Bovine Serum Albumin (BSA) (Sigma-Aldrich, catalog number: A6003 )
  25. EDTA (Merck Millipore Corporation, catalog number: 1.08418 )
  26. Medium 199 (Sigma-Aldrich, catalog number: M4530 )
  27. Sodium bicarbonate (7.5%) (Life Technologies, Gibco, catalog number: 25080-094 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 25080-094 ”.
  28. HEPES (1 M) (Life Technologies, Gibco, catalog number: 15630-080 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 15630-080”.
  29. Pen Strep 100x (Life Technologies, Gibco, catalog number: 15140-122 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 15140-122”.
  30. MEM Non-essential amino acid solution 100x (Sigma-Aldrich, catalog number: M7145 )
  31. Sodium pyruvate 100 mM (Sigma-Aldrich, catalog number: S8636-100 )
  32. GlutaMAXTM 100x (Life Technologies, Gibco, catalog number: 35050-061 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 35050-061”.
  33. Acetic acid, glacial (Chem Supply, catalog number: AA009-2.5 L )
  34. Crystal violet (Sigma-Aldrich, catalog number: C3886-25 g )
  35. 1x DPBS (see Recipes)
  36. 0.1% BSA/DPBS (see Recipes)
  37. EGM-2 Media with bullet kit (see Recipes)
  38. White blood cell counting fluid (see Recipes)
  39. Fibronectin (Roche Diagnostics, catalog number: 10838039001 ) (see Recipes)
  40. VEGF (see Recipes)
  41. FGFb (see Recipes)
  42. Ascorbic acid (see Recipes)
  43. IGF-I (see Recipes)
  44. HUVE media + 20% FBS (see Recipes)
  45. EGM-2 Media + FBS and growth factors (see Recipes)
  46. MACS buffer (see Recipes)

Equipment

  1. Certified biological safety cabinet
  2. AutoMacs® Pro with chill 15 rack (Miltenyi Biotec, catalog number: 130-092-545 )
  3. Pipettes
  4. Pipette gun with ability to set to slow
  5. Centrifuge with lids (Eppendorf AG, model: 5810R ) with A-4-81 rotor
  6. Cell counting device (i.e. Haemocytometer)
  7. Microscope
  8. CO2 incubator
    For manual cell sorting
  9. MiniMACSTM separator (Miltenyi Biotec, catalog number: 130-042-102 )
  10. MACS MultiStand separator (Miltenyi Biotec, catalog number: 130-042-303 )

Procedure

  1. Cell Isolation
    Note:The AutoMacs® automatic cell sorting is the preferred method for isolating naEFCs however a manual method using the MS columns has also been included if an AutoMacs® is not accessible.

    AutoMacs® automatic cell sorting
    1. Collect 40-250 ml of human cord blood from the umbilical vein of placentas from healthy pregnant women, preferably from caesarean section, into cord blood collection bags.
    2. Transfer cord blood into 50 ml tubes by cutting the tube of the collection bag and draining 25 ml of blood directly into each tube (note 20 ml of this volume is due to the anticoagulant in the collection bags).
    3. Dilute blood 1:1 with sterile DPBS and mix by inverting the tubes 3-4 times.
    4. In fresh 50 ml tubes add 15 ml of lymphoprepTM (use 15 ml of lymphoprepTM regardless of blood volume in tube).
    5. Carefully layer 35 ml of the diluted blood onto the lymphoprepTM using a 25 ml pipette with the pipette gun set to slow. Do this very slowly so the layer of blood and the layer of lymphoprepTM do not mix (see Figure 3). Repeat until all the diluted blood is layered onto lymphoprepTM in tubes. Minimise the time the blood remains on the lymphoprepTM.


      Figure 3. Before and after density centrifugation to separate mononuclear cells. As seen in (A) blood is layered on top of the lymphoprepTM carefully to avoid mixing. Red blood cells can be seen starting to settle down. (B) shows the expected layers following density centrifugation. The buffy layer containing the mononuclear cells is then taken for cell sorting. It is not unusual for cord blood samples to have red blood cells contamination in the buffy layer, have a chunky appearance to the buffy layer or for the plasma layer to appear red from lysed red blood cells, this is all normal.

    6. Immediately centrifuge at 700 x g for 20 min at room temperature with both the brake and acceleration turned to zero to allow the mononuclear cells (MNCs) to separate.
    7. After centrifugation the MNCs should be visible as a buffy coat at the interphase between the lymphoprep and the plasma/DPBS layer (see Figure 3).
    8. Carefully collect the MNCs using a soft sterile bulb transfer pipette by slowly sucking up the buffy layer of cells and transferring the cells to a clean 50 ml tube (this may require multiple tubes it there is a large sample of blood with multiple lymphoprepTM tubes).
    9. Repeat transfer until no buffy coat remains in each lymphoprepTM tube.
    10. Dilute transferred buffy coat cells 1:1 with HUVE media in 50 ml tube/s.
    11. Centrifuge at 450 x g for 5 min at room temperature.
    12. Aspirate the supernatant, resuspend cells in 10 ml of HUVE media.
    13. Combine cells into one 50 ml tube, if required.
    14. Take a sample for cell counting to determine total cell number. Counting a range of 50-250 cells to calculate cell number should give an accurate enough result for this stage of the protocol, an appropriate volume and dilution to achieve this range may need to be worked out.
    15. Dilute cells 1:1 (as a minimum dilution) with the white blood cell counting fluid. Leave mix for 2 min so red blood cells can lyse for a more accurate cell count. All nucleated cells will appear blue for counting on a haemocytometer or equivalent. Count cells and determine total cell number.
    16. Wash cells twice more by resuspending in 25 ml HUVE media followed by centrifugation at 450 x g for 5 min at room temperature. When aspirating supernatant do not remove all media from the pellet before resuspension to improve cell yield.
    17. Aspirate supernatant and resuspend ≤ 1 x 108 cells in 500 μl of HUVE media by pipetting up and down slowly and transfer to a 10 ml tube (if there is more than 1 x 108 cells add 1 ml of HUVE media and split into 2 tubes and continue for each tube).
    18. Add 100 μl of human FcR blocking reagent followed by 100 μl of resuspended CD133 microbeads and mix well. Incubate for 30 min at 4 °C in the dark.
    19. Centrifuge tubes at 4 °C at 450 x g for 5 min (for manual cell sorting proceed to protocol below).
    20. Meanwhile turn on AutoMACS® Pro and attach running buffer and perform rinse cycle as per manufacturer’s instructions.
    21. Aspirate supernatant carefully and resuspend cells in 3 ml sterile MACS buffer by gently pipetting up and down.
    22. On the AutoMACS® Pro select “possel-s” (positive selection-sensitive) and set the desired number of samples as per manufacturer’s instructions.
    23. Use chilled AutoMACS® Chill 15 rack and insert sample tube with cells into position 1 and empty 10 ml tubes into position 2 (CD133 negative) and 3 (CD133 positive). Run program.
    24. Combine all CD133 positively sorted cells and perform a cell count with the white blood cell counting fluid as per step 15 to determine media volume for seeding.
    25. Centrifuge at 450 x g for 5 min at room temperature.

    For manual cell sorting
    1. Attach miniMACSTM magnetic separator to the MultiStand and insert a MS column (1 per sample) into the separator and place a collection tube under the column (this can be seen in the manufacturers product information sheet).
    2. To prepare column, add 500 μl MACS buffer to the top of the column and allow to flow through the column (it will not run dry). Discard flow through from collection tube.
    3. Aspirate supernatant (from step A19) carefully and resuspend cells in 1 ml sterile MACS buffer by gently pipetting up and down.
    4. Add the cell suspension to the top of the column 500 μl at a time. Unlabelled cells will flow through into the collection tube.
    5. Wash the column by adding 500 μl of MACS buffer to the column. Repeat 3 times.
    6. Remove the MS column from the separator and place on a new collection tube.
    7. Add 1 ml of MACS buffer to the column and using the plunger supplied with the column, immediately flush out positively labelled cells by firmly pushing the plunger down the column.
    8. Proceed to step A24.

  2. Initial cell seeding
    1. Coat a 24 well plate with 300 µl of fibronectin for each well (about 1-6 wells will be required depending on sample size) and leave well plate for at least 30 min in a 5% CO2 incubator set to 37 °C.
    2. Make up required amount of EGM-2 media with growth factors.
    3. Resuspend CD133+ cells at a density of 1 x 106 cells/ml in EGM-2 with growth factors.
    4. Aspirate the fibronectin from the wells, no additional washing is required.
    5. Add cells to the well/s (1-2 ml per well).
    6. Place well plate into a 5% CO2 incubator set to 37 °C this is considered as day 0.

  3. Cell culture (separating non-adherent and adherent cells)
    1. After 48 h (day 2) from initial seeding, cells need to be transferred to a new fibronectin coated well to separate the desired non-adherent cells from the small number of adherent cells.
    2. Coat new a 24 well plate with 300 µl of fibronectin for each well leave well plate for at least 30 min in a 5% CO2 incubator set to 37 °C.
    3. Separate non-adherent cells by collecting all non-adherent cells from all wells into a 10 ml tube.
    4. Wash all wells with 1 ml per well of DPBS by pipetting gently up and down in each well and add to the tube of non-adherent cells.
    5. Centrifuge at 450 x g for 5 min at room temperature.
    6. Aspirate supernatant carefully to avoid sucking up any of the cell pellet and resuspend cells in fresh EGM-2 with growth factors at a density of 1 x 106 cells/ml.
    7. Aspirate the fibronectin from the wells (no additional washing is required) and add cells (1-2 ml per well) to the newly fibronectin coated well/s.
    8. Place well plate into a 5% CO2 incubator set to 37 °C until day 4-5 when a homogeneous population of cells is formed (see Figure 2 for examples of the cell population at this stage).

Notes

  1. All procedures aside from blood collection and AutoMacs® Pro cell sorting should be performed under sterile conditions in a certified biological safety cabinet using aseptic technique.
  2. Relevant human ethics for human cord blood collection and research use needs to be in place prior to collection.
  3. There is extensive donor variation in both overall mononuclear cell and CD133 positive cell numbers and some samples appear a lot darker or thicker than others. For example you might have a higher cell yield from a smaller volume of blood compared to a different sample. From 40-250 ml of blood the expected CD133 positive cell yield would be from 5 x 105 to 8 x 106 cells. Ideally the largest volume of blood should be collected but this if often limited by donors and birthing techniques as cord blood collection should not compromise the health of the donor.
  4. Blood can be collected and left at room temperature overnight in the collection bag at room temperature on a rocker but cell numbers recovered may be lower.
  5. Do not do a specific red blood cell lysis step as it affects the naEFCs viability. The slight RBC and other cell contamination seemingly help cell viability and the CD133+ cells become homogeneous after 4 days in the selective media.

Recipes

  1. 1x DPBS
    Dilute 20x DPBS 1:20 in sterile water to get 1x DPBS
  2. 0.1% BSA/DPBS
    DPBS
    100 ml
    BSA
    0.1 g
    Dissolve BSA into DPBS and then sterilise through a 0.2 µm filter
  3. EGM-2 Media with bullet kit
    As per manufacturer’s instructions, add all components of the bullet kit to the EBM-2 media bottle to make the EGM-2 media
    Aliquot and store at -20 °C until use
  4. White blood cell counting fluid
    Acetic acid, glacial
    1 ml
    Water
    49 ml
    Crystal violet
    7.5 mg
    Dissolve and mixing well then sterilise with a 0.2 µm filter
    Stored at room temperature
    Dilute cells into this preparation and count with hemocytometer
  5. Fibronectin
    Reconstitute with sterile water to make 1 mg/ml and leave at 37 °C for 30-60 min to dissolve without agitation
    Store stock at -20 °C
    Dilute 1 mg/ml stock to 50 mg/ml in sterile DPBS for use
    Stored at 4 °C
  6. VEGF
    Reconstitute to 5 µg/ml in sterile 0.1% BSA/DPBS
    Aliquot and stored at -20 °C
  7. FGFb
    Reconstitute to 25 µg/ml in sterile 0.1% BSA/ DPBS
    Aliquot and stored at -80 °C
  8. Ascorbic acid
    Reconstitute to 0.5 M in sterile water
    Aliquot and stored at -20 °C
  9. IGF-I
    Reconstitute to 100 µg/ml in sterile DPBS
    Dilute to 5 µg/ml in sterile DPBS
    Aliquot and stored at -80 °C
  10. HUVE media + 20% FBS
    Medium 199
    400 ml
    Fetal bovine serum
    100 ml
    1 M HEPES
    10 ml
    7.5% sodium bicarbonate
    7.5 ml
    100x GlutaMAXTM
    5 ml
    100x MEM Non-essential amino acid solution
    5 ml
    100x Pen strep
    5 ml
    100 mM sodium pyruvate
    5 ml
  11. EGM-2 media + FBS and growth factors

    Table 1. EGM-2 Media, FBS and Growth Factors recipe

    Dilution
    To make 5 ml
    Final concentration
    EGM-2 media with Bullet kit added

    4.5 ml

    Fetal bovine serum
    (1/10)
    500 μl
    10%
    VEGF (5 µg/ml)
    (1/1,000)
    5 μl
    5 ng/ml
    IGF-1 (5 µg/ml)
    (1/5,000)
    1 μl
    1 ng/ml
    Ascorbic acid (0.5 M)
    (1/5,000)
    1 μl
    0.1mM
    bFGF (25 µg/ml)
    (1/25,000)
    10 μl
    1 ng/ml

    0.2 µm sterile filter and use immediately
  12. MACS buffer
    100 ml 1x DPBS
    0.5 g BSA (0.5%)
    0.074 g EDTA (2 mM)
    pH 7.2
    0.2 µm sterile filter
    Stored at 4 °C

Acknowledgements

We thank Dr Sarah Appleby, Dr Katie Tooley, Dr Jeffrey Barrett and Samantha Escarbe for their assistance in developing this protocol; Dr Rosalie Grivell, the staff and consenting donors at Women’s and Children’s Hospital and Burnside Memorial Hospital for collection of the umbilical cord blood. This project was funded by a Heart Foundation Fellowship to CSB (CR10A4983) as well as a project grant from the Co-operative Research Centre for Biomarker Translation (Trans Bio Ltd).

References

  1. Appleby, S. L., Cockshell, M. P., Pippal, J. B., Thompson, E. J., Barrett, J. M., Tooley, K., Sen, S., Sun, W. Y., Grose, R., Nicholson, I., Levina, V., Cooke, I., Talbo, G., Lopez, A. F. and Bonder, C. S. (2012). Characterization of a distinct population of circulating human non-adherent endothelial forming cells and their recruitment via intercellular adhesion molecule-3. PLoS One 7(11): e46996.
  2. Barrett, J. M., Parham, K. A., Pippal, J. B., Cockshell, M. P., Moretti, P. A., Brice, S. L., Pitson, S. M. and Bonder, C. S. (2011). Over-expression of sphingosine kinase-1 enhances a progenitor phenotype in human endothelial cells. Microcirculation 18(7): 583-597.
  3. Moldenhauer, L. M., Cockshell, M. P., Frost, L., Parham, K. A., Tvorogov, D., Tan, L. Y., Ebert, L. M., Tooley, K., Worthley, S., Lopez, A. F. and Bonder, C. S. (2015). Interleukin-3 greatly expands non-adherent endothelial forming cells with pro-angiogenic properties. Stem Cell Res 14(3): 380-395.
  4. Parham, K. A., Zebol, J. R., Tooley, K. L., Sun, W. Y., Moldenhauer, L. M., Cockshell, M. P., Gliddon, B. L., Moretti, P. A., Tigyi, G., Pitson, S. M. and Bonder, C. S. (2015). Sphingosine 1-phosphate is a ligand for peroxisome proliferator-activated receptor-gamma that regulates neoangiogenesis. FASEB J 29(9): 3638-3653.

简介

循环内皮祖细胞(EPCs)已经成为许多临床试验的焦点,因为它们在缺血事件例如急性心肌梗死后的血管再形成中的作用以及它们在器官移植期间对血管修复的贡献。由于缺乏在细胞表面表达的不同标记物,EPCs的研究已经引起争议,并且用于分离和培养的不同方法导致鉴定了具有不同表型和功能的多种细胞类型,所有这些都属于" EPCs"。分离用于细胞治疗的最广泛记载的EPCs本质上是粘附的,缺乏祖细胞标志物例如CD133,因此不太可能代表真正的循环EPC,所述细胞响应于血管损伤而动员。
我们最近发布了非粘附内皮形成细胞(naEFCs)群体的分离和广泛表征(Appleby等人,2012)(图1)。这些细胞与成熟内皮细胞标记(VEGFR2,CD144和CD31)一起表达祖细胞标记(CD133,CD34,CD117,CD90和CD38)。这些细胞还表达低水平的CD45,但不表达将其与"早期"EPC区分开的淋巴标志物(CD3,CD4,CD8)或骨髓标志物(CD11b和CD14),"晚期生长EPC"[最近称为内皮细胞集落形成细胞(ECFCs)]以及成熟内皮细胞(ECs)。图2A例示了naEFC的表面表达谱。功能研究证明这些naEFC(i)结合于玻璃体凝集素(图2A),(ii)显示乙酰化低密度脂蛋白摄取,(iii)增加血管细胞粘附分子(VCAM-1)响应于肿瘤坏死因子的表达和(iv)与成熟EC的共培养增加了在三维体外基质中的管,小管分支和环的数目。更重要的是,放置在体内的naEFC产生了包含由表达CD144的人EC内衬的脉管系统的新腔,并且有助于科学知识的各种进步(Appleby等人,2012; Barrett 等人,2011; Moldenhauer等人,2015; Parham等人,2015)。在这里,我们描述从人脐带血的细胞的非粘附CD133 + 内皮形成群体的分离和富集。

关键字:内皮祖细胞, 膨胀, 人类, CD133, 非贴壁



图1.人naEFC的富集。 A。通过光学显微镜比较在培养4天时的脐带血衍生的CD133 +富集的细胞(naEFC)和人脐静脉内皮细胞(EC)的细胞大小。比例尺=200μmB.通过使用流式细胞术分析的前向散射和侧向散射分析来评估细胞的富集过程的异质性(0-10天),并与成熟ECs比较。

图2.人naEFC的表面表达表型。通过流式细胞术评估在培养4天时的CD133 +富集的细胞的祖细胞和内皮标记物。直方图显示来自≥3个生物复制品的代表性实验,其中灰色虚线代表同种型对照,实黑色线代表用所示标记染色的细胞。 B.通过流式细胞术评价naEFC的功能,并与成熟EC相比,检测细胞吸收DiI标记的乙酰化低密度脂蛋白(Ac-LDL)和结合FITC标记的Ulex europaeus凝集素的能力I(UEA-1)凝集素。密度图代表来自≥3个生物学重复的一个代表性实验的染色细胞。

材料和试剂

  1. 对于手动细胞分选
    MS柱(Miltenyi Biotec,目录号:130-042-201)
  2. MacoPharma脐带血收集袋(MacoPharma,目录号:MSC1201DU)
  3. 50ml管(Corning,Falcon ,目录号:352070)
  4. 10ml管(Sarstedt AG,目录号:62.9924.284)
  5. 2ml软无菌球转移移液管,无菌(Stephen Gould corporation,目录号:222-1S)
    注意:目前,它是"Capitol Scientific,目录号:222-1S"。
  6. 20μm滤器(Sartorius Stedim Biotec,Minisart,目录号:16534-K)
  7. 24孔板(Corning,Falcon ,目录号:353047)
  8. 微透镜管
  9. 20ml注射器
  10. 0.2μm过滤器
  11. 人脐带血(40-250 ml)
  12. 20x Dulbecco's磷酸盐缓冲(DPBS)(Life Technologies,Gibco ,目录号:14200-075)
    注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:14200-075"
  13. 无菌水(Baxter,目录号:UKF7114)
  14. Lymphoprep TM (Axis-Shield,目录号:114547)
  15. CD133微珠,包括人FcR阻断试剂(Miltenyi Biotec,目录号:130-050-801)
  16. AutoMACS Pro洗涤溶液(Miltenyi Biotec,目录号:130-092-987)
  17. AutoMACS运行缓冲液(Miltenyi Biotec,目录号:130-091-221) 注意:如果AutoMACS Pro分离器不可用,可以通过手动排序分离感兴趣的细胞(见下文)。然而,这种手动方法不一定是最佳的naEFC细胞分选与较低的细胞活力和数量观察;因此优选AutoMACS方法。
  18. 使用Bullet试剂盒(EGM-2)(Lonza,目录号:cc-3162)的内皮生长培养基
  19. 胎牛血清,表征(FBS)(VWR International,Hyclone ,目录号:SH30071.03)
  20. 重组人血管内皮生长因子(VEGF)(Sigma-Aldrich,目录号:V7259)
  21. 重组人胰岛素样生长因子-1(IGF-I)(R& D Systems,目录号:291-G1-200)
  22. 重组人成纤维细胞生长因子碱性(FGFb)(R& D Systems,目录号:233-FB-025)
  23. L-抗坏血酸(Sigma-Aldrich,目录号:A5960)
  24. 牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A6003)
  25. EDTA(Merck Millipore Corporation,目录号:1.08418)
  26. 培养基199(Sigma-Aldrich,目录号:M4530)
  27. 碳酸氢钠(7.5%)(Life Technologies,Gibco,目录号:25080-094)
    注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:25080-094"
  28. HEPES(1M)(Life Technologies,Gibco,目录号:15630-080) 注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:15630-080"
  29. Pen Strep 100x(Life Technologies,Gibco,目录号:15140-122)
    注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:15140-122"
  30. MEM非必需氨基酸溶液100x(Sigma-Aldrich,目录号:M7145)
  31. 丙酮酸钠100mM(Sigma-Aldrich,目录号:S8636-100)
  32. GlutaMAX TM 100x(Life Technologies,Gibco,目录号:35050-061)
    注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:35050-061"
  33. 乙酸,冰(Chem Supply,目录号:AA009-2.5L)
  34. 结晶紫(Sigma-Aldrich,目录号:C3886-25g)
  35. 1x DPBS(请参阅配方)
  36. 0.1%BSA/DPBS(参见配方)
  37. EGM-2带子弹套件的介质(参见配方)
  38. 白细胞计数液(见配方)
  39. 纤连蛋白(Roche Diagnostics,目录号:10838039001)(参见Recipes)
  40. VEGF(参见食谱)
  41. FGFb(参见配方)
  42. 抗坏血酸(见配方)
  43. IGF-I(参见配方)
  44. HUVE媒体+ 20%FBS(见配方)
  45. EGM-2培养基+ FBS和生长因子(参见配方)
  46. MACS缓冲区(参见配方)

设备

  1. 认证的生物安全柜
  2. AutoMacs ? Pro with chill 15 rack(Miltenyi Biotec,目录号:130-092-545)
  3. 移液器
  4. 移液枪具有设置缓慢的能力
  5. 用盖子离心机(Eppendorf AG,型号:5810R),用A-4-81转子
  6. 细胞计数装置(即血细胞计数器)
  7. 显微镜
  8. CO <2>孵化器
    对于手动细胞分选
  9. MiniMACS TM分离器(Miltenyi Biotec,目录号:130-042-102)
  10. MACS MultiStand分离器(Miltenyi Biotec,目录号:130-042-303)

程序

  1. 单元隔离
    注意:AutoMacs 自动细胞分选是分离naEFCs的首选方法,但是使用MS列的手动方法包括是否无法访问AutoMacs ?


    1. 从脐静脉收集40-250毫升的人脐带血 来自健康孕妇,优选来自剖腹产的胎盘 部分,进入脐带血收集袋
    2. 转移脐带血 通过切割收集袋的管和排水到50ml管中 25毫升血液直接进入每个管(注意,这个体积20毫升到期 ?到收集袋中的抗凝剂)
    3. 用无菌DPBS稀释血液1:1,倒置管3-4次混合
    4. 在新鲜的50ml管中加入15ml的淋巴细胞粘附分子(使用15ml的淋巴细胞粘附分子TM,无论管中的血容量如何)。
    5. 使用25ml移液管小心地将35ml稀释的血液层置于lymphoprep TM上,用移液枪设定为慢速。做这个非常 慢慢地,所以血液层和淋巴细胞层 TM 层不混合 (参见图3)。重复,直到所有稀释的血液分层 lymphoprep TM 。最小化血液保留在的时间 lymphoprep TM


      图3.密度离心前后 单独的单核细胞。如(A)所示,血液层叠在上面 小心地避免混合淋巴细胞。可以看到红细胞 开始沉降。 (B)显示了密度之后的预期层 ?离心。含有单核细胞的血沉棕黄层 然后进行细胞分选。这是不寻常的脐血样本 ?有红细胞污染的血沉棕黄层,有大块 外观至血沉棕黄层或血浆层出现红色 ?裂解的红细胞,这都是正常的
    6. 立即 在700离心机在室温下用制动器20分钟 ?并且加速度变为零以允许单核细胞(MNC) 分隔。
    7. 离心后,MNCs应该是可见的 ?在淋巴细胞增多物和血浆/DPBS之间的界面处的血沉棕黄层 ?层(见图3)。
    8. 使用软件小心收集MNC 无菌球管转移吸管通过缓慢吸取血沉棕黄层 细胞并将细胞转移到干净的50ml管(这可能需要 ?多个管,它有一个大样本的血液与多个 lymphoprep TM 管)
    9. 重复转移,直到每个淋巴管 TM 管中没有血沉棕黄层。
    10. 用50ml管中的HUVE培养基稀释转移的血沉棕黄层细胞1:1
    11. 在室温下以450xg离心5分钟
    12. 吸出上清液,将细胞重悬于10ml HUVE培养基中
    13. 如果需要,将细胞合并到一个50ml管中。
    14. 取样品进行细胞计数以确定总细胞数。 计算50-250个细胞的范围以计算细胞数应该给出 ?足够准确的结果为本阶段的协议,适当 体积和稀释以达到这个范围可能需要计算
    15. 稀释细胞1:1(作为最小稀释)与白细胞 计数液。离开混合2分钟,所以红细胞可以裂解为 更准确的细胞计数。所有有核细胞将显示蓝色 在血细胞计数器或等价物上计数。计数细胞并确定 总细胞数。
    16. 洗涤细胞两次以上重悬在25 ml HUVE培养基,随后在室温下以450×g离心5分钟 温度。吸入上清液时,不要从中取出所有培养基 重悬浮之前的沉淀以提高细胞产量
    17. 吸出 上清液,并在500μlHUVE培养基中重悬<1×10 8个细胞 慢慢地上下吸取并转移到10ml管(如果有的话) 超过1×10 8个细胞加入1ml的HUVE培养基并分成2个管 并继续每个管)
    18. 加入100μl的人FcR阻断 然后加入100μl重悬的CD133微珠并混合均匀。 ?在4℃在黑暗中孵育30分钟。
    19. 在4℃下以450×g离心管离心5分钟(用于手动细胞分选,进行下面的方案)。
    20. 同时打开AutoMACS ? Pro,并附上运行缓冲液,并按照制造商的说明进行冲洗循环。
    21. 小心吸出上清液,通过轻轻地上下吹打,将细胞重悬在3ml无菌MACS缓冲液中
    22. 在AutoMACS ? Pro上选择"possel-s"(正数 选择敏感),并按照设置所需的样本数 制造商的说明
    23. 使用冷冻AutoMACS ? Chill 15机架 ?并将具有细胞的样品管插入位置1并且空的10ml管 ?到位置2(CD133阴性)和3(CD133阳性)。运行程序。
    24. 组合所有CD133阳性分选细胞并执行细胞计数 与根据步骤15的白细胞计数液进行测定 播种的媒体量。
    25. 在室温下以450xg离心5分钟。

    对于手动细胞分选
    1. 将miniMACS TM 磁性分离器连接到MultiStand并插入MS 柱(每个样品1个)装入分离器并放置收集管 下面列(这可以在制造商产品中看到 信息表)。
    2. 为了准备柱,加入500μlMACS缓冲液 列的顶部,并允许流过列(它不会 干涸)。丢弃收集管中的流体。
    3. 吸出 上清液(来自步骤A19)并将细胞重悬于1ml中 无菌MACS缓冲液,轻轻地上下吹打
    4. 添加单元格 ?悬浮液在柱顶部每次500μl。未标记的细胞 将流入收集管。
    5. 通过向柱中加入500μlMACS缓冲液来洗涤柱子。重复3次。
    6. 从分离器中取出MS柱,并置于新的收集管上。
    7. 向柱中加入1ml MACS缓冲液,并使用提供的柱塞 ?与柱,立即冲出积极标记的细胞 牢牢地将柱塞向下推入柱子
    8. 继续步骤A24。

  2. 初始细胞接种
    1. 用每孔300μl的纤连蛋白涂布24孔板(约1-6 ?根据样品大小将需要孔)并留下孔板 在设定为37℃的5%CO 2培养箱中至少30分钟
    2. 用生长因子补充所需量的EGM-2培养基
    3. 在具有生长因子的EGM-2中以1×10 6个细胞/ml的密度重悬CD133 +细胞。
    4. 从孔中吸出纤连蛋白,不需要额外的洗涤
    5. 向孔中加入细胞(每孔1-2ml)。
    6. 将孔板置于设定为37℃的5%CO 2培养箱中,这被认为是第0天。

  3. 细胞培养(分离非粘附和粘附细胞)
    1. 在从初始接种48小时(第2天)后,需要转移细胞 ?新的纤连蛋白包被孔以分离所需的非粘附物 来自少量贴壁细胞的细胞
    2. 外套新一个24孔 ?每个孔用300μl纤连蛋白离开孔板 在设定为37℃的5%CO 2培养箱中至少30分钟。
    3. 分离非粘附细胞,收集所有孔的所有非粘附细胞到10ml管中
    4. 用1ml /孔的DPBS清洗所有孔,轻轻向上轻轻吹打 ?在每个孔中,并添加到非粘附细胞的管
    5. 在室温下以450xg离心5分钟
    6. 小心吸出上清液以避免吸出任何细胞 沉淀和重悬细胞在新鲜EGM-2中与生长因子在a 密度为1×10 6个细胞/ml
    7. 从中吸出纤连蛋白 孔(不需要另外的洗涤),并加入细胞(1-2ml / 孔)至新的纤连蛋白包被的孔
    8. 放置孔板 进入设定为37℃的5%CO 2培养箱中,直到第4-5天,当均质时 形成细胞群(参见图2的细胞实例 人口在这个阶段)。

笔记

  1. 除血液收集和AutoMacs ? Pro细胞分选之外的所有程序应在无菌条件下在使用无菌技术的认证生物??安全柜中进行。
  2. 在收集之前,需要有相关的人类伦理用于人类脐带血收集和研究使用。
  3. 在整个单核细胞和CD133阳性细胞数目中存在广泛的供体变化,并且一些样品显示出比其他样品更深或更厚。例如,与不同的样品相比,您可能从较小体积的血液中获得较高的细胞产量。从40-250ml血液,预期的CD133阳性细胞产量为5×10 5至8×10 6个细胞。理想地,应该收集最大体积的血液,但是如果经常受到捐赠者和分娩技术的限制,则脐带血收集不应该损害捐赠者的健康。
  4. 可以收集血液,并在室温下在摇床上在室温下在收集袋中放置过夜,但回收的细胞数目可以更低。
  5. 不要做特定的红细胞裂解步骤,因为它影响naEFCs生存力。轻微的RBC和其他细胞污染似乎有助于细胞活力,并且在选择性培养基中4天后CD133 + 细胞变得均质。

食谱

  1. 1x DPBS
    在无菌水中稀释20x DPBS 1:20以获得1x DPBS
  2. 0.1%BSA/DPBS
    DPBS
    100 ml
    BSA
    0.1 g
    将BSA溶解于DPBS中,然后通过0.2μm过滤器灭菌
  3. EGM-2带子弹套件的介质
    根据制造商的说明,将弹头套件的所有组件添加到EBM-2培养基瓶中,制成EGM-2培养基。
    等分并存储在-20°C,直到使用
  4. 白细胞计数液
    乙酸,冰醋
    1 ml

    49 ml
    水晶紫
    7.5 mg
    溶解并混合好,然后用0.2μm过滤器灭菌 在室温下贮存
    稀释细胞进入这个准备和计数血细胞计数器
  5. 纤连蛋白
    用无菌水重构1mg/ml,并在37℃放置30-60分钟,不搅拌溶解
    将商品储存在-20°C
    用无菌DPBS稀释1 mg/ml原液至50 mg/ml,用于
    储存在4°C
  6. VEGF
    在无菌的0.1%BSA/DPBS中重构为5μg/ml 等分并储存在-20°C
  7. FGFb
    在无菌的0.1%BSA/DPBS中重构至25μg/ml 分装并储存在-80℃下
  8. 抗坏血酸
    在无菌水中重构至0.5 M 等分并储存在-20°C
  9. IGF-I
    在无菌DPBS中重构至100μg/ml 在无菌DPBS中稀释至5μg/ml
    分装并储存在-80℃下
  10. HUVE媒体+ 20%FBS
    中等199
    400 ml
    胎牛血清
    100 ml
    1 M HEPES
    10 ml
    7.5%碳酸氢钠 7.5 ml
    100x GlutaMAX TM
    5 ml
    100x MEM非必需氨基酸溶液
    5 ml
    100x Pen strep
    5 ml
    100mM丙酮酸钠 5 ml
  11. EGM-2培养基+ FBS和生长因子
    表1. EGM-2媒体,FBS和生长因子配方

    稀释
    要做到5ml
    最终浓度
    添加Bullet试剂盒的EGM-2培养基

    4.5 ml

    胎牛血清
    (1/10)
    500微升
    10%
    VEGF(5μg/ml) (1/1,000)
    5微升
    5 ng/ml
    IGF-1(5μg/ml) (1/5,000)
    1微升
    1 ng/ml,
    抗坏血酸(0.5M)
    (1/5,000)
    1微升
    0.1 mM
    bFGF(25μg/ml) (1/25,000)
    10微升
    1 ng/ml

    0.2μm无菌过滤器,立即使用
  12. MACS缓冲区
    100 ml 1x DPBS
    0.5克BSA(0.5%) 0.074g EDTA(2mM) pH 7.2
    0.2μm无菌过滤器
    储存在4°C

致谢

我们感谢Sarah Appleby博士,Katie Tooley博士,Jeffrey Barrett博士和Samantha Escarbe在制定本议定书方面提供的帮助; Rosalie Grivell博士,妇女儿童医院和伯恩赛德纪念医院的工作人员和同意捐赠者收集脐带血。这个项目是由心脏基金会奖学金CSB(CR10A4983),以及来自生物标记物翻译合作研究中心(Trans Bio Ltd)的项目资助。

参考文献

  1. Appleby,SL,Cockshell,MP,Pippal,JB,Thompson,EJ,Barrett,JM,Tooley,K.,Sen,S.,Sun,WY,Grose,R.,Nicholson,I.,Levina, ,I.,Talbo,G.,Lopez,AFand Bonder,CS(2012)。 循环人类非粘附内皮形成细胞的独特群体及其通过细胞间粘附分子的募集的表征-3。 PLoS One 7(11):e46996。
  2. Barrett,J.M.,Parham,K.A.,Pippal,J.B.,Cockshell,M.P.,Moretti,P.A.,Brice,S.L.,Pitson,S.M.and Bonder,C.S。(2011)。 鞘氨醇激酶-1的过表达增强人内皮细胞中的祖细胞表型。微循环 18(7):583-597。
  3. 这些研究结果表明,这些研究结果表明,在不同的温度条件下,不同温度条件下, 。 白细胞介素3大大扩展了非粘附内皮形成细胞,具有促血管生成特性。 Stem Cell Res 14(3):380-395。
  4. Parham,K.A.,Zebol,J.R.,Tooley,K.L.,Sun,W.Y.,Moldenhauer,L.M.,Cockshell,M.P.,Gliddon,B.L.,Moretti,P.A.,Tigyi,G.,Pitson,S.M.and Bonder, 鞘氨醇1-磷酸是过氧化物酶体增殖物激活受体-γ的配体,其调节新血管生成。 a> FASEB J 29(9):3638-3653。
  • English
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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。
引用:Cockshell, M. P. and Bonder, C. S. (2016). Isolation and Culture of Human CD133+ Non-adherent Endothelial Forming Cells. Bio-protocol 6(7): e1772. DOI: 10.21769/BioProtoc.1772.
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