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Porous Scaffold Seeding and Chondrogenic Differentiation of BMSC-seeded Scaffolds
BMSC结籽支架的多孔支架播种和软骨分化   

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Abstract

Bone marrow-derived mesenchymal stromal stem cells (BMSCs) are a promising cell source for treating articular cartilage defects (Bornes et al., 2014). BMSCs can be seeded within porous biomaterial scaffolds that support three-dimensional cell organization, chondrogenic differentiation and extracellular matrix deposition for the creation of engineered cartilage. This protocol describes our defined methods for isolation and expansion of human and ovine BMSCs, seeding of BMSCs within porous scaffolds and in vitro chondrogenic differentiation (Adesida et al., 2012; Bornes et al., 2015).

Keywords: Mesenchymal stem cell(间充质干细胞), Scaffold(支架), Cell seeding(细胞接种), Chondrogenesis(软骨), Cartilage(软骨)

Materials and Reagents

  1. Culture plate, 24 wells (Becton Dickinson Labware, catalog number: 353047 )
    Note: Currently, it is “Corning, Falcon®, catalog number: 353047”.
  2. Pipette tips, 1,000 μl, 200 μl and 10 μl volumes (Corning, DeckWorksTM, catalog numbers: 4124 , 4121 and 4120 )
  3. Pasteur pipettes, 230 mm length (WHEATON, catalog number: 4500448667 )
    Note: Currently, it is “WHEATON, catalog number: 357335”.
  4. Conical tube, 50 ml volume (Corning, Falcon®, catalog number: 352070 )
  5. Cell strainer, nylon with 100 μm pores (Corning, BD Biosciences, catalog number: 352360 )
  6. Tissue-culture flask, 150 cm2 surface area (T150) (Corning, Falcon®, catalog number: 355000 )
  7. Conical microtube, 1.5 ml volume (Bio Basic Canada, catalog number: TC152SN )
  8. Biopsy punch, circular with 6 mm diameter (Southern Anesthesia & Surgical, Miltex, catalog number: 3336 )
  9. Bone marrow aspirate, human or ovine, collected through needle aspiration at the iliac crest
  10. Crystal violet solution (Sigma-Aldrich, catalog number: HT90132 )
  11. Alpha minimal essential medium (αMEM), containing Earle’s salts, ribonucleosides, deoxyribonucleosides and L-glutamine (Thermo Fisher Scientific, Corning, Mediatech,catalog number: 10022CV )
  12. Fetal bovine serum (FBS), heat inactivated at 56 °C in the laboratory (Life Technologies, Gibco®, catalog number: 12483 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 12483 ”.
  13. 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) (1 M) (Life Technologies, Gibco®, catalog number: 15630 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 15630”.
  14. Sodium pyruvate, 100 mM (Life Technologies, Gibco®, catalog number: 11360 )
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 11360”.
  15. Fibroblast growth factor-two (FGF-2), human recombinant (Neuromics, catalog number: PR80001 )
  16. Dulbecco’s phosphate buffered saline (PBS), sterile filtered (Sigma-Aldrich, catalog number: D8537 )
  17. Human serum albumin (Sigma-Aldrich, catalog number: A4327 )
  18. Transforming growth factor-beta three (TGF-β3), human recombinant, HEK (Prospecbio, ProSpec, catalog number: CYT-113 )
  19. L-Ascorbic acid 2-phosphate sesquimagnesium salt hydrate (Sigma-Aldrich, catalog number: A8960 )
  20. Dexamethasone-Water Soluble (Sigma-Aldrich, catalog number: D2915 )
  21. L-proline (Sigma-Aldrich, catalog number: P5607 )
  22. Trypan blue solution, 0.4% (Sigma-Aldrich, catalog number: T8154 )
  23. Porous scaffolds, collagen I or esterified hyaluronic acid (described in detail in Bornes et al., 2015)
  24. Penicillin-streptomycin-glutamine (Life Technologies, Gibco®, catalog number: 1248310378 ) (see Recipes)
    Note: Currently, it is “Thermo Fisher Scientific, GibcoTM, catalog number: 1248310378 ”.
  25. Trypsin-ethylenediaminetetraacetic acid (EDTA) (Thermo Fisher Scientific, Corning, catalog number: 25052 ) (see Recipes)
  26. Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, catalog number: D6429 ) (see Recipes)
  27. Insulin-transferrin-selenium (ITS+) premix (Corning, BD Biosciences, catalog number: 354352 ) (see Recipes)
  28. Expansion medium (see Recipes)
  29. Serum-free medium (see Recipes)
  30. TGF-β3 working solution (see Recipes)
  31. Chondrogenic medium (see Recipes)

Equipment

  1. Biosafety cabinet (Microzone Corporation, catalog number: BK-2-6 A2 )
  2. Incubator, containing humidified air at 37 °C with 5% carbon dioxide, and 3% oxygen (Thermo Fisher Scientific, catalog number: Forma Series II Water Jacket CO2 )
  3. Centrifuge, 1,500 revolutions per min (rpm) (Beckman Coulter, AllegraTM, catalog number: X-22R )
  4. Light microscope (Microscope, Omano, catalog number: OM159T )
  5. Pipette (Drummond Scientific Company, catalog number: Pipet Aid XP )
  6. Micropipettes, volumes of 100-1,000 μl, 20-200 μl, 2-20 μl, and 0.5-10 μl (Bio-Rad Laboratories, catalog numbers: 1660508 , 1660507 , 1660506 , and 1660505 )
  7. Suction source
  8. Forceps
  9. Water bath, set to 37 °C (VWR International, catalog number: 89501 )
  10. Neubauer hemacytometer, 0.1 mm deep (Reichert Bright-Line) (Sigma-Aldrich, catalog number: Z359629 )

Procedure

Methods involving manipulation of bone marrow aspirate, cells and porous scaffolds must be performed within a biosafety cabinet. Instruments, solutions and media in contact with bone marrow aspirate, cells and porous scaffolds must be sterile. Solutions and medium should be preheated to 37 °C in a closed container submersed in a water bath.


  1. Isolation and expansion of BMSCs
    1. Obtain a sterile bone marrow aspirate. Collection of heparinized human and ovine iliac crest aspirates has been described in detail previously (Buda et al., 2010; Bornes et al., 2015). Aspirate volumes of 15-60 ml and 20-40 ml may be obtained from human and ovine donors, respectively (Wakitani et al., 2007; Buda et al., 2010; Nejadnik et al., 2010; Zscharnack et al., 2010; Bornes et al., 2015). Aspirates should be taken to the laboratory directly following collection for processing in order to avoid cell death.
    2. Filter the bone marrow aspirate using a 100 μm cell strainer to remove clots and tissue. Collect the filtrate in a sterile 50 ml conical tube.
    3. For mononucleated cell counting, micropipette 50 μl of diluted bone marrow aspirate filtrate (1:10 in PBS; dilution might have to be increased to 1:50 if cells are highly concentrated) and 50 μl of diluted crystal violet solution (1:50 in PBS) into a 1.5 ml conical microtube and mix thoroughly. Micropipette 20 μl of this mixture into a hemacytometer. Count the number of mononucleated cells within the four grid squares of the hemacytometer using a light microscope. Divide the number of cells counted by four (number of grid squares in hemacytometer), multiply by 20 (dilution of aspirate filtrate) and multiply by 10,000 (hemacytometer factor) to calculate the concentration of mononucleated cells per ml of aspirate filtrate. The total number of mononucleated cells is then determined my multiplying the concentration by the total volume (in ml) of the aspirate filtrate.
    4. Calculate the volume of bone marrow aspirate filtrate containing 15 million human mononucleated cells (Adesida et al., 2012) or 80 million ovine mononucleated cells (Bornes et al., 2015) and pipette this volume into a sterile 50 ml conical tube. Pipette 20 ml of expansion medium into the 50 ml conical tube and mix with the aspirate filtrate. Transfer the aspirate filtrate-medium mixture into a T150 flask (passage 0). If large numbers of mononucleated cells are present in the aspirate filtrate, multiple T150 flasks may be seeded.
    5. Statically incubate each T150 flask for seven days undisturbed at 37 °C. No media changes should be performed during this period.
    6. After seven days, aspirate off medium and wash adherent BMSCs with 10 ml of PBS. Pipette 20 ml of fresh expansion medium into each T150 flask.
    7. Statically incubate each T150 flask at 37 °C, and change the medium twice per week until 80% confluence is obtained based on microscopy.
    8. Once 80% confluence has been reached, aspirate off medium and wash cells with 10 ml of PBS.
    9. Pipette 6 ml of trypsin-EDTA into each T150 flask and incubate at 37 °C for 5 min. Agitate the flask to promote detachment of BMSCs from the flask surface. Microscopy may be used to confirm detachment of cells.
    10. Pipette the BMSC-trypsin-EDTA mixture into a 50-ml conical tube and add 2 ml of serum-containing medium (expansion medium or αMEM with FBS) to deactivate trypsin.
    11. Centrifuge the resulting mixture for 10 min (1,500 rpm) and aspirate off the liquid component. The BMSC collection will be present at the bottom of the 50 ml conical tube.
    12. For BMSC counting, re-suspend the BMSC collection within a known quantity of expansion medium (e.g., 10 ml). Micropipette 50 μl of BMSC collection and 50 μl of trypan blue into a 1.5-ml conical microtube and mix thoroughly. Micropipette 20 μl of this mixture into a hemacytometer. Count the number of BMSCs within the four grid squares of the hemacytometer using a light microscope. Divide the number of cells by four (number of grid squares), multiply by 2 (dilution of BMSC suspension) and multiply by 10,000 (hemacytometer factor) to calculate the concentration of cells (BMSCs per ml of suspension). The total number of BMSCs is then determined by multiplying this concentration by total volume (in ml) of the BMSC suspension. Each T150 flask can be expected to yield 1-3 million BMSCs for human donors and 3-10 million BMSCs for ovine donors.
    13. Add expansion medium to the BMSCs and re-suspend. BMSCs derived from one T150 flask during passage 0 should be re-suspended in 40 ml of expansion medium and divided into two T150 flasks for passage 1. There are a total of two T150 flasks during passage 1 per one T150 flask during passage 0.
    14. Repeat steps A7-12 for passage 1.
    15. Add expansion medium to the BMSCs and re-suspend. BMSCs derived from each T150 flask during passage 1 should be re-suspended in 40 ml of expansion medium and divided into two T150 flasks for passage 2. There are a total of four T150 flasks during passage 2 per one T150 flask during passage 0.
    16. Repeat steps A7-12 for passage 2 BMSCs (Figures 1A-1B). Subsequent passaging may be performed beyond passage 2 to increase the number of BMSCs available for use, although prolonged BMSC expansion has been shown to result in de-differentiation and loss of multipotent differentiation capacity of BMSCs (Wagner et al., 2008; Tsai et al., 2011). Therefore, the authors recommend using passage 2 BMSCs for use in chondrogenic differentiation.

  2. Seeding of porous biomaterial scaffolds with BMSCs and chondrogenic differentiation
    1. Porous scaffold composition and size should be based on the goals of the study. For in vitro assessment of chondrogenesis, porous scaffold sheets composed of collagen I sponge or esterified hyaluronic acid mesh (Bornes et al., 2015) may be cut into 6 mm-diameter cylinders using a biopsy punch. Dimensions of the scaffold of choice must be known to calculate BMSC seeding density.
    2. Calculate the number of BMSCs required to create a seeding density of 10 million BMSCs per cm3 of scaffold. Other densities may be considered, although the authors recommend a seeding density of 5-10 million BMSCs per cm3 of scaffold to be used in chondrogenic differentiation. For cylindrical collagen I scaffolds with a diameter of 6 mm and height of 3.5 mm, 989,602 BMSCs are required per scaffold for a density of 10 million BMSCs per cm3. For cylindrical esterified hyaluronic acid scaffolds with a diameter of 6 mm and height of 2 mm, 565,487 BMSCs are required per scaffold.
    3. Following counting of passage 2 BMSCs, centrifuge the resulting mixture for 10 min (1,500 rpm) and aspirate off the liquid component. The BMSC collection will be present at the bottom of the 50 ml conical tube.
    4. Re-suspend BMSCs in chondrogenic medium with a total volume dependent on the number of scaffolds to be seeded (Figure 1C). For each 6 mm diameter scaffold, BMSCs (number calculated in step B2) should be re-suspended in 20 μl of chondrogenic medium.
    5. Place scaffolds within empty wells of a 24 well culture plate using forceps (Figure 1D).
    6. Micropipette the 20 μl BMSC-medium suspension onto the central area of the flat surface of each scaffold (Figure 1E). If the suspension does not spread throughout the entirety of the surface of the scaffold, pre-soaking the scaffold with 20 μl of cell-free chondrogenic medium may be required to promote full dispersion of the BMSC-medium suspension over the scaffold. If a larger scaffold is to be used, multiple BMSC-medium suspensions may be micropipetted onto different areas of the scaffold to promote uniform seeding.
    7. Incubate BMSC-seeded scaffolds at 37 °C for 15 min.
    8. Micropipette 100 μl of chondrogenic medium onto the base of each BMSC-seeded scaffold.
    9. Incubate BMSC-seeded scaffolds at 37 °C for 30 min.
    10. Micropipette 1 ml of chondrogenic medium into each well to submerse the BMSC-seeded scaffolds.
    11. Statically incubate 24 well plates at 37 °C for 2-3 weeks. Change the chondrogenic medium twice per week. BMSCs will differentiate into cells capable of producing cartilaginous extracellular matrix (Figure 1F).


      Figure 1. Isolation, expansion, seeding, and chondrogenic differentiation of BMSCs. A. A tissue-culture flask (T150) containing isolated BMSCs and expansion medium. B. Adherent, human BMSCs on the surface of a tissue-culture flask demonstrating a characteristic spindle-shaped morphology during expansion (10x magnification). C. Re-suspension of BMSCs in chondrogenic medium within a 50 ml conical tube prior to scaffold seeding. D. Collagen I scaffold placement into the empty well of a 24 well culture plate using forceps. E. Micropipetting of a BMSC-chondrogenic medium suspension onto the central area of a collagen I scaffold. F. Extracellular cartilaginous proteoglycans stained with safranin O following three weeks of chondrogenic differentiation of human BMSCs seeded within a collagen I scaffold. Remnant collagen I scaffold is stained with fast green counterstain [staining protocol described in detail by Bornes et al. (2015); 10x magnification].

Recipes

  1. Penicillin-streptomycin-glutamine
    10,000 units/ml penicillin
    10 mg/ml streptomycin
    29.2 mg/ml L-glutamine
  2. Trypsin-ethylenediaminetetraacetic acid (EDTA)
    0.05% trypsin
    0.53 mM EDTA without sodium bicarbonate
    Calcium and magnesium
  3. Dulbecco’s modified Eagle’s medium (DMEM)
    4.5 mg/ml glucose
    110 μg/ml sodium pyruvate
    L-glutamine
  4. Insulin-transferrin-selenium (ITS+) premix
    625 μg/ml insulin
    625 μg/ml transferrin
    625 μg/ml selenium
    125 μg/ml bovine serum albumin
    535 μg/ml linoleic acid
  5. Expansion medium, 565 ml total volume with final concentrations listed
    500 ml αMEM
    50 ml FBS [8.8% volume/volume (v/v)]
    5 ml penicillin-streptomycin-glutamine
    a. 88.5 units/ml penicillin
    b. 88.5 μg/ml streptomycin
    c. 258.4 μg/ml L-glutamine
    5 ml HEPES (8.8 mM)
    5 ml sodium pyruvate (885.0 μM)
    282.5 μl FGF-2, 10 μg/ml stock solution (5 ng/ml)
  6. Serum-free medium, 182 ml total volume with final concentrations listed
    166 ml DMEM
    2 ml HEPES (11.0 mM)
    2 ml penicillin-streptomycin-glutamine
    a. 109.9 units/ml penicillin
    b. 109.9 μg/ml streptomycin
    c. 320.9 μg/ml L-glutamine
    2 ml ITS+ premix
    10 ml human serum albumin, 25 mg/ml stock solution (1.4 mg/ml)
  7. TGF-β3 working solution, 10 ml total volume with final concentrations listed
    9.4 ml DMEM
    100 μl TGF-β3, 10 μg/ml stock solution (100 ng/ml)
    500 μl human serum albumin, 25 mg/ml stock solution (1.3 mg/ml)
  8. Chondrogenic medium, 100 ml total volume with final concentrations listed
    87 ml serum-free medium
    1. DMEM
    2. 9.6 mM HEPES
    3. 95.6 units/ml penicillin
    4. 95.6 μg/ml streptomycin
    5. 279.2 μg/ml L-glutamine
    6. 1 ml ITS+ premix
    10 ml TGF-β3 working solution
    1. DMEM
    2. 10.0 ng/ml TGF-β3
    3. 125 μg/ml human serum albumin
    1 ml ascorbic acid 2-phosphate, 3.65 mg/ml stock solution (365 μg/ml)
    1 ml dexamethasone, 10 μM stock solution (100 nM)
    1 ml L-proline, 4 mg/ml stock solution (40 μg/ml)

Acknowledgments

Research related to this protocol was funded by the University Hospital Foundation at the University of Alberta Hospital, Canadian Institutes of Health Research and the Edmonton Orthopaedic Research Committee. Integra LifeSciences Corp. and Anika Therapeutics Inc. have generously provided in-kind biomaterials for our laboratory. Stipend support for Troy Bornes was provided by Alberta Innovates - Health Solutions and Canadian Institutes of Health Research.

References

  1. Adesida, A. B., Mulet-Sierra, A. and Jomha, N. M. (2012). Hypoxia mediated isolation and expansion enhances the chondrogenic capacity of bone marrow mesenchymal stromal cells. Stem Cell Res Ther 3(2): 9.
  2. Bornes, T. D., Adesida, A. B. and Jomha, N. M. (2014). Mesenchymal stem cells in the treatment of traumatic articular cartilage defects: a comprehensive review. Arthritis Res Ther 16(5): 432.
  3. Bornes, T. D., Jomha, N. M., Mulet-Sierra, A. and Adesida, A. B. (2015). Hypoxic culture of bone marrow-derived mesenchymal stromal stem cells differentially enhances in vitro chondrogenesis within cell-seeded collagen and hyaluronic acid porous scaffolds. Stem Cell Res Ther 6: 84.
  4. Buda, R., Vannini, F., Cavallo, M., Grigolo, B., Cenacchi, A. and Giannini, S. (2010). Osteochondral lesions of the knee: a new one-step repair technique with bone-marrow-derived cells. J Bone Joint Surg Am 92(Suppl 2): 2-11.
  5. Nejadnik, H., Hui, J. H., Feng Choong, E. P., Tai, B. C. and Lee, E. H. (2010). Autologous bone marrow-derived mesenchymal stem cells versus autologous chondrocyte implantation: an observational cohort study. Am J Sports Med 38(6): 1110-1116.
  6. Tsai, C. C., Chen, Y. J., Yew, T. L., Chen, L. L., Wang, J. Y., Chiu, C. H. and Hung, S. C. (2011). Hypoxia inhibits senescence and maintains mesenchymal stem cell properties through down-regulation of E2A-p21 by HIF-TWIST. Blood 117(2): 459-469.
  7. Wagner, W., Horn, P., Castoldi, M., Diehlmann, A., Bork, S., Saffrich, R., Benes, V., Blake, J., Pfister, S., Eckstein, V. and Ho, A. D. (2008). Replicative senescence of mesenchymal stem cells: a continuous and organized process. PLoS One 3(5): e2213.
  8. Wakitani, S., Nawata, M., Tensho, K., Okabe, T., Machida, H. and Ohgushi, H. (2007). Repair of articular cartilage defects in the patello-femoral joint with autologous bone marrow mesenchymal cell transplantation: three case reports involving nine defects in five knees. J Tissue Eng Regen Med 1(1): 74-79.
  9. Zscharnack, M., Hepp, P., Richter, R., Aigner, T., Schulz, R., Somerson, J., Josten, C., Bader, A. and Marquass, B. (2010). Repair of chronic osteochondral defects using predifferentiated mesenchymal stem cells in an ovine model. Am J Sports Med 38(9): 1857-1869.

简介

骨髓来源的间充质干细胞(BMSCs)是治疗关节软骨缺陷的有希望的细胞来源(Bornes等人,2014)。 BMSCs可以种植在支持三维细胞组织,软骨形成分化和细胞外基质沉积的多孔生物材料支架中,用于创建工程化软骨。 该方案描述了我们定义的用于分离和扩增人和绵羊BMSCs,在多孔支架内接种BMSCs和在体外软骨形成分化的方法(Adesida等人,2012; Bornes et al。,2015)。

关键字:间充质干细胞, 支架, 细胞接种, 软骨, 软骨

材料和试剂

  1. 培养板,24孔(Becton Dickinson Labware,目录号:353047)
    注意:目前,"Corning,Falcon
  2. 移液管吸头,1,000μl,200μl和10μl体积(Corning,DeckWorks TM ,目录号:4124,4121和4120)
  3. 巴斯德移液管,230mm长(WHEATON,目录号:4500448667)
    注意:目前为"WHEATON,目录号:357335"。
  4. 锥形管,50ml体积(Corning,Falcon ,目录号:352070)
  5. 细胞过滤器,具有100μm孔的尼龙(Corning,BD Biosciences,目录号:352360)
  6. 组织培养瓶,150cm 2表面积(T150)(Corning,Falcon ,目录号:355000)
  7. 锥形微管,1.5ml体积(Bio Basic Canada,目录号:TC152SN)
  8. 活检穿孔器,直径6mm的圆形(Southern Anaesthesia& Surgical,Miltex,目录号:3336)
  9. 骨髓抽吸物,人或羊,通过针头抽吸在髂嵴
    收集
  10. 结晶紫溶液(Sigma-Aldrich,目录号:HT90132)
  11. 含有Earle's盐,核糖核苷,脱氧核糖核苷和L-谷氨酰胺的α最小必需培养基(αMEM)(Thermo Fisher Scientific,Corning,Mediatech,目录号:10022CV)
  12. 胎牛血清(FBS),在实验室中在56℃热灭活(Life Technologies,Gibco ,目录号:12483)
    注意:目前,是"Thermo Fisher Scientific,Gibco TM ,目录号:12483" />
  13. 4-(2-羟乙基)-1-哌嗪乙磺酸(HEPES)(1M)(Life Technologies,Gibco ,目录号:15630) 注意:目前,"Thermo Fisher Scientific,Gibco TM ,目录号:15630" />
  14. 丙酮酸钠,100mM(Life Technologies,Gibco ,目录号:11360)
    注意:目前,是"Thermo Fisher Scientific,Gibco TM ,目录号:11360" />
  15. 成纤维细胞生长因子二(FGF-2),人重组体(Neuromics,目录号:PR80001)
  16. Dulbecco's磷酸盐缓冲盐水(PBS),无菌过滤(Sigma-Aldrich,目录号:D8537)
  17. 人血清白蛋白(Sigma-Aldrich,目录号:A4327)
  18. 转化生长因子β3(TGF-β3),人重组体HEK(Prospecbio,ProSpec,目录号:CYT-113)
  19. L-抗坏血酸2-磷酸倍半镁盐水合物(Sigma-Aldrich,目录号:A8960)
  20. 地塞米松 - 水溶性(Sigma-Aldrich,目录号:D2915)
  21. L-脯氨酸(Sigma-Aldrich,目录号:P5607)
  22. 台盼蓝溶液,0.4%(Sigma-Aldrich,目录号:T8154)
  23. 多孔支架,胶原I或酯化透明质酸(在Bornes等人,2015年详细描述)
  24. 青霉素 - 链霉素 - 谷氨酰胺(Life Technologies,Gibco ,目录号:1248310378)(参见配方)
    注意:目前,"赛默飞世尔科技,Gibco TM ,目录号:1248310378" />
  25. 胰蛋白酶 - 乙二胺四乙酸(EDTA)(Thermo Fisher Scientific,Corning,目录号:25052)(参见配方)
  26. Dulbecco改良的Eagle培养基(DMEM)(Sigma-Aldrich,目录号:D6429)(参见Recipes)
  27. 胰岛素 - 转铁蛋白 - 硒(ITS +)预混物(Cornig,BD Biosciences,目录号:354352)(参见配方)
  28. 膨胀介质(参见配方)
  29. 无血清培养基(见配方)
  30. TGF-β3工作溶液(参见配方)
  31. 软骨形成介质(参见配方)

设备

  1. 生物安全柜(Microzone Corporation,目录号:BK-2-6 A2)
  2. 孵育器,其含有在37℃,5%二氧化碳和3%氧气的加湿空气(Thermo Fisher Scientific,目录号:Forma Series II Water Jacket CO 2)
  3. 离心机,每分钟1500转(rpm)(Beckman Coulter,AllegraTM,目录号:X-22R)
  4. 光学显微镜(显微镜,Omano,目录号:OM159T)
  5. Pipette(Drummond Scientific Company,目录号:Pipet Aid XP)
  6. 微量移液管,体积为100-1,000μl,20-200μl,2-20μl和0.5-10μl(Bio-Rad Laboratories,目录号:1660508,1660507,1660506和1660505)
  7. 吸引源
  8. 镊子
  9. 设定为37℃的水浴(VWR International,目录号:89501)
  10. Neubauer血细胞计数器,0.1mm深(Reichert Bright-Line)(Sigma-Aldrich,目录号:Z359629)

程序

涉及操作骨髓抽出物,细胞和多孔支架的方法必须在生物安全柜内进行。与骨髓抽吸物,细胞和多孔支架接触的器械,溶液和介质必须是无菌的。溶液和介质应在浸没在水浴中的密闭容器中预热至37℃。


  1. 骨髓基质干细胞的分离和扩增
    1. 获得无菌骨髓抽吸物。肝素化人类的集合 和髂嵴抽吸物已经在前面详细描述 (Buda等人,2010; Bornes等人,2015)。吸出体积15-60 ml 和20-40ml可分别从人和绵羊供体获得 (Wakitani等人,2007; Buda等人,2010; Nejadnik等人,2010; Zscharnack等人,2010; Bornes ,,2015)。应该采取Aspirates ?到实验室直接按顺序收集处理 ?以避免细胞死亡
    2. 使用100过滤骨髓抽吸物 ?μm细胞过滤器以去除凝块和组织。收集滤液在a 无菌50ml锥形管
    3. 对于单核细胞计数, 微量移液管50μl稀释的骨髓抽吸液(1:10英寸 PBS;稀释可能必须增加到1:50,如果细胞是高度 浓缩)和50μl稀释的结晶紫溶液(1:50的PBS溶液) ?倒入1.5ml锥形微管中并充分混合。微量移液器20μl 的混合物加入血球计。计数单核细胞的数目 细胞在血细胞计数器的四个网格正方形内使用光 显微镜。将计数的细胞数除以4(网格数 在血细胞计数器中的平方),乘以20(稀释的吸出物 滤液)并乘以10,000(血细胞计数因子)以计算 ?每毫升抽吸滤液的单核细胞浓度。的 然后测定单核细胞的总数 浓度乘以吸出物滤液的总体积(以ml计)
    4. 计算骨髓抽吸液的体积含15 (Adesida等人,2012)或8000万个 绵羊单核细胞(Bornes等人,2015)并移取该体积 装入无菌的50ml锥形管中。吸取20ml的膨胀介质 进入50ml锥形管并与吸出的滤液混合。转让 ?将吸出的滤液 - 培养基混合物倒入T150烧瓶(第0代)。如果 抽吸物中存在大量单核细胞 滤液,可以接种多个T150烧瓶
    5. 静态孵育 ?每个T150烧瓶在37℃下静置7天。无媒体更改 应在此期间执行。
    6. 七天后, 吸出培养基并用10ml PBS洗涤贴壁BMSCs。吸管 将20ml新鲜的扩增培养基加入每个T150烧瓶中。
    7. 静态 ?在37℃下孵育每个T150烧瓶,并且每周更换培养基两次 ?直到获得基于显微镜的80%汇合
    8. 一旦达到80%汇合,吸出培养基并用10ml PBS洗涤细胞
    9. 吸取6毫升胰蛋白酶-EDTA到每个T150烧瓶中并在37℃下孵育 ?℃5分钟。搅动烧瓶以促进骨髓间充质干细胞脱离 ?烧瓶表面。显微镜可用于确认细胞的分离
    10. 吸取BMSC胰蛋白酶-EDTA混合物到50毫升锥形管和 ?加入2ml含血清培养基(扩增培养基或含FBS的αMEM) ?以停用胰蛋白酶。
    11. 将所得混合物离心10分钟 min(1,500rpm),并抽出液体组分。 BMSC 收集将存在于50ml锥形管的底部
    12. 对于BMSC计数,在已知的情况下重新挂起BMSC收集 扩增培养基的量(例如10ml)。微量移液器50微升BMSC 收集和50微升锥虫蓝到1.5毫升锥形微管和 充分混合。微量移液器20微升的混合物到血细胞计数器。 ?计数四个网格方格内的骨髓基质干细胞的数量 血细胞计数器使用光学显微镜。将单元格数除以 四个(网格正方形数),乘以2(稀释BMSC 悬浮)并乘以10,000(血细胞计数因子)进行计算 细胞(BMSCs/ml悬浮液)的浓度。总数 然后通过将该浓度乘以来确定BMSC的数量 BMSC悬浮液的总体积(以ml计)。每个T150烧瓶可以 预期为人类供体产生1-3百万个骨髓基质干细胞,3-10万个 用于绵羊供体的BMSC
    13. 添加扩展介质到骨髓基质干细胞和 重新暂停。在第0代期间来自一个T150烧瓶的BMSC应该是 ?重悬于40ml的膨胀介质中并分成两个T150 烧瓶中通过1.在期间共有两个T150烧瓶 在通道0期间每个T150烧瓶通道1
    14. 对通过1重复步骤A7-12。
    15. 添加扩展介质到骨髓基质干细胞并重新挂起。 BMSCs衍生 从每个T150烧瓶在第1代应重新悬浮在40毫升 ?膨胀介质并分成两个T150烧瓶用于第2代 在每个T150烧瓶的第2代通道期间总共有四个T150烧瓶 在通过0期间。
    16. 对于第2代BMSC重复步骤A7-12 (图1A-1B)。随后的传代可以超过传代2 以增加可供使用的骨髓基质干细胞的数量,虽然延长 BMSC扩增已经显示导致去分化和丧失 的BMSCs的多潜能分化能力(Wagner等人,2008; Tsai等人,2011)。因此,作者建议使用第2节 BMSCs用于软骨形成分化。

  2. 多孔生物材料支架与BMSCs和软骨形成分化的种子
    1. 多孔支架组成和尺寸应根据目标 ?研究。对于软骨形成的多孔支架的体外评估 片由胶原I海绵或酯化透明质酸网组成 (Bornes等人,2015)可以使用a切成6mm直径的圆柱体 活检穿刺。必须知道所选择的支架的尺寸 计算BMSC播种密度
    2. 计算骨髓基质干细胞的数量 需要产生一千万个BMSCs/cm 3的接种密度 脚手架。其他密度可以考虑,虽然作者 推荐5-10cm BMSCs每cm 3支架的接种密度 ?用于软骨形成分化。对于圆柱形胶原I 支架直径6毫米和高度3.5毫米,989,602骨髓基质干细胞 对于每平方厘米3百万个BMSCs的密度需要每个支架。对于 ?圆柱形酯化透明质酸支架,直径为6 mm,高度为2 mm,每个脚手架需要565,487个骨髓间充质干细胞
    3. 在计数第二代BMSCs后,离心所得混合物 10分钟(1,500rpm),并抽出液体组分。 BMSC 收集将存在于50ml锥形管的底部
    4. 在具有总体积依赖性的软骨形成培养基中重悬浮BMSCs ?对待接种的支架的数量(图1C)。每6 mm 直径支架,BMSCs(在步骤B2中计算的数字)应当是 重悬于20μl软骨形成培养基中
    5. 使用镊子将支架置于24孔培养板的空孔中(图1D)
    6. Micropipette 20微升BMSC-培养基悬浮液在中心区域 的每个支架的平坦表面(图1E)。如果暂停 ?不遍及支架的整个表面, 预浸泡支架与20微升无细胞软骨形成培养基可能 ?以促进BMSC-介质悬浮液的完全分散 在支架上。如果要使用更大的支架,则多个 BMSC-培养基悬浮液可以被吸取到不同区域上 ?支架促进均匀播种
    7. 孵育BMSC种子支架在37℃下15分钟
    8. 微量移液器100微升软骨形成培养基到每个BMSC种子支架的基础上
    9. 孵育BMSC种子支架在37℃下30分钟
    10. Micropipette 1毫升软骨形成培养基到每个孔中,以浸没BMSC种子支架。
    11. 将24孔板在37℃静置孵育2-3周。更改 软骨形成培养基每周两次。骨髓基质干细胞将分化成 细胞能够产生软骨细胞外基质 1F)。


      图1.隔离,扩张,接种和软骨形成 BMSCs的分化。 A.组织培养瓶(T150)含有 分离的BMSCs和扩增培养基。 B.粘附,人类BMSCs上 表现出特征的组织培养瓶的表面 扩张期间(10×放大率)的纺锤形形态。 C。 重悬浮的骨髓基质干细胞在软骨形成培养基在50毫升锥形 管。 D.胶原I脚手架放入 使用镊子的24孔培养板的空孔。 E. 将BMSC-软骨形成性培养基悬浮液微量移液到中心 ?面积的胶原蛋白I支架。 F.胞外软骨 三周后用safranin O染色的蛋白聚糖 软骨形成分化的人类BMSCs接种在胶原蛋白I 脚手架。残余胶原I支架用快速绿染色 复染[由Bornes等人(2015)详细描述的染色方案; 10x放大]。

食谱

  1. 青霉素 - 链霉素 - 谷氨酰胺 10,000单位/ml青霉素
    10mg/ml链霉素 29.2mg/ml L-谷氨酰胺
  2. 胰蛋白酶 - 乙二胺四乙酸(EDTA)
    0.05%胰蛋白酶 0.53mM EDTA,无碳酸氢钠
    钙和镁
  3. Dulbecco改良的Eagle培养基(DMEM) 4.5mg/ml葡萄糖 110μg/ml丙酮酸钠
    L-谷氨酰胺
  4. 胰岛素 - 转铁蛋白 - 硒(ITS +)预混物
    625μg/ml胰岛素
    625μg/ml转铁蛋白
    625μg/ml硒
    125μg/ml牛血清白蛋白
    535μg/ml亚油酸
  5. 扩增培养基,总体积为565ml,终浓度列于
    500 mlαMEM
    50ml FBS [8.8%体积/体积(v/v)]
    5ml青霉素 - 链霉素 - 谷氨酰胺 一个。 88.5单位/ml青霉素
    b。 88.5μg/ml链霉素 C。 258.4μg/ml L-谷氨酰胺 5ml HEPES(8.8mM) 5ml丙酮酸钠(885.0μM) 282.5μlFGF-2,10μg/ml储备溶液(5ng/ml)
  6. 无血清培养基,总体积182 ml,最终浓度列于
    166 ml DMEM
    2ml HEPES(11.0mM) 2ml青霉素 - 链霉素 - 谷氨酰胺 一个。 109.9单位/ml青霉素
    b。 109.9μg/ml链霉素 C。 320.9μg/ml L-谷氨酰胺 2ml ITS +预混物
    10ml人血清白蛋白,25mg/ml储备溶液(1.4mg/ml)
  7. TGF-β3工作溶液,10ml总体积,最终浓度列于
    9.4 ml DMEM
    100μlTGF-β3,10μg/ml储备溶液(100ng/ml)
    500μl人血清白蛋白,25mg/ml储备溶液(1.3mg/ml)
  8. 软骨形成培养基,总体积为100ml,终浓度列于
    87毫升无血清培养基
    1. DMEM
    2. 9.6 mM HEPES
    3. 95.6单位/ml青霉素
    4. 95.6μg/ml链霉素
    5. 279.2μg/ml L-谷氨酰胺
    6. 1ml ITS +预混物
    10ml TGF-β3工作溶液
    1. DMEM
    2. 10.0ng/ml TGF-β3
    3. 125μg/ml人血清白蛋白
    1ml抗坏血酸2-磷酸盐,3.65mg/ml储备溶液(365μg/ml)
    1ml地塞米松,10μM储备溶液(100nM) 1ml L-脯氨酸,4mg/ml储备溶液(40μg/ml)

致谢

与该方案有关的研究由阿尔伯塔大学医院,加拿大卫生研究院和埃德蒙顿骨科研究委员会的大学医院基金会资助。 Integra LifeSciences公司和Anika Therapeutics公司为我们的实验室慷ously地提供了实物生物材料。 Stubend对Troy Bornes的支持由Alberta Innovates - 健康解决方案和加拿大健康研究所提供。

参考文献

  1. Adesida,A.B.,Mulet-Sierra,A。和Jomha,N.M。(2012)。 低氧介导的分离和扩增增强了骨髓间充质基质细胞的软骨形成能力。 em> Stem Cell Res Ther 3(2):9.
  2. Bornes,T. D.,Adesida,A. B.和Jomha,N. M.(2014)。 间充质干细胞在创伤性关节软骨缺损的治疗中:综合评价。 em> Arthritis Res Ther 16(5):432.
  3. Bornes,T.D.,Jomha,N.M.,Mulet-Sierra,A。和Adesida,A.B。(2015)。 骨髓间充质间质干细胞的缺氧培养在体外差异增强[em] >细胞接种的胶原和透明质酸多孔支架内的软骨形成。 Stem Cell Res Ther 6:84.
  4. Buda,R.,Vannini,F.,Cavallo,M.,Grigolo,B.,Cenacchi,A.and Giannini,S。(2010)。 膝盖骨软骨病变:使用骨髓衍生细胞的新一步修复技术。 J Bone Joint Surg Am 92(Suppl 2):2-11。
  5. Nejadnik,H.,Hui,J.H.,Feng Choong,E.P.,Tai,B.C.and Lee,E.H。(2010)。 自体骨髓间充质干细胞与自体软骨细胞植入:观察性队列研究。 Am J Sports Med 38(6):1110-1116。
  6. Tsai,C.C.,Chen,Y.J.,Yew,T.L.,Chen,L.L.,Wang,J.Y.,Chiu,C.H。和Hung,S.C。 缺氧抑制衰老并通过HIF-TWIST下调E2A-p21维持间充质干细胞特性。 Blood 117(2):459-469。
  7. Wagner,W.,Horn,P.,Castoldi,M.,Diehlmann,A.,Bork,S.,Saffrich,R.,Benes,V.,Blake,J.,Pfister,S.,Eckstein, Ho,AD(2008)。 间充质干细胞的复制性衰老:连续而有组织的过程。一个 3(5):e2213。
  8. Wakitani,S.,Nawata,M.,Tensho,K.,Okabe,T.,Machida,H.and Ohgushi,H。(2007)。 用自体骨髓间充质细胞移植修复髌股关节中的关节软骨缺损:三例报告涉及五个膝盖中的九个缺陷。 J Tissue Eng Regen Med 1(1):74-79。
  9. Zscharnack,M.,Hepp,P.,Richter,R.,Aigner,T.,Schulz,R.,Somerson,J.,Josten,C.,Bader,A.and Marquass, 在羊模型中使用预分化间充质干细胞修复慢性软骨缺损 Am J Sports Med 38(9):1857-1869。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Bornes, T. D., Jomha, N. M., Mulet-Sierra, A. and Adesida, A. B. (2015). Porous Scaffold Seeding and Chondrogenic Differentiation of BMSC-seeded Scaffolds. Bio-protocol 5(24): e1693. DOI: 10.21769/BioProtoc.1693.
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