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Feb 2019

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Mesenchymal Stromal Cells Derived from Bone Marrow and Adipose Tissue: Isolation, Culture, Characterization and Differentiation
骨髓和脂肪间充质干细胞:分离、培养、鉴定和分化   

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Abstract

Since their discovery, mesenchymal stromal cells (MSCs) have received a lot of attention, mainly due to their self-renewal potential and multilineage differentiation capacity. For these reasons, MSCs are a useful tool in cell biology and regenerative medicine. In this article, we describe protocols to isolate MSCs from bone marrow (BM-MSCs) and adipose tissues (AT-MSCs), and methods to culture, characterize, and differentiate MSCs into osteoblasts, adipocytes, and chondrocytes. After the harvesting of cells from bone marrow by flushing the femoral diaphysis and enzymatic digestion of abdominal and inguinal adipose tissues, MSCs are selected by their adherence to the plastic tissue culture dish. Within 7 days, MSCs reach 70% confluence and are ready to be used in subsequent experiments. The protocols described here are easy to perform, cost-efficient, require minimal time, and yield a cell population rich in MSCs.

Keywords: Adipose tissue (脂肪组织), Adipocyte (脂肪细胞), Bone (骨), Bone marrow (骨髓), Cell culture (细胞培养), Chondrocyte (软骨细胞), Mesenchymal Stromal cell (间充质干细胞), Osteoblast (成骨细胞)

Background

The concept of stem cells dates back to the 19th century, but their existence was confirmed in the 1960s and 1970s following experiments by Friedenstein and collaborators, which showed the presence of stem cells in the bone marrow (Friedenstein, 1970; Bianco et al., 2008). Afterward, Caplan (1991) named them as mesenchymal stem cells (here, called mesenchymal stromal cells–MSCs) and proposed their use in regenerative medicine. In the bone marrow, the percentage of MSCc is estimated to be 0.001 to 0.01% of the total mononuclear cells. Because of their scarcity, alternative sources have been described, although bone marrow remains as the main source of MSCs (Nancarrow-Lei et al., 2017). Adipose tissue is a very promising source because it contains a large number of MSCs that are relatively easy to harvest with minimal discomfort and risk for donors (Zuk et al., 2001). The protocols used to harvest and culture MSCs from either bone marrow (BM-MSCs) or adipose tissue (AT-MSCs), may vary among different species or even among different strains of the same species. The most commonly used methods for obtaining MCSs involve using flow cytometry (Schrepfer et al., 2007), multipotent adult progenitor cell media (Harting et al., 2008), the ficoll-paque gradient centrifugation method (Pierini et al., 2012), and immunomagnetic beads (Wadajkar et al., 2014). Here, we describe cost-efficient protocols that are relatively easy and fast to perform and can be used to obtain cell populations rich in MSCs from bone marrow and adipose tissues. These protocols can be used to study several cellular and molecular aspects of MSCs, such as their proliferation, differentiation, and signaling pathways (Abuna et al., 2016; Fideles et al., 2019), the biological effects of growth factors and drugs on MSCs (Oliveira et al., 2012; Zhang et al., 2017), the interactions between MSCs and natural or synthetic biomaterials (Hu et al., 2018; Lopes et al., 2019), and the application of MSCs in regenerative medicine strategies (Almeida et al., 2019; Freitas et al., 2019).

Materials and Reagents

  1. Sterile surgical drape
  2. Aluminum foil
  3. Coat (ProtDesc, catalog number: 80404440020), storage temperature: RT
  4. Mask (ProtDesc, catalog number: 80404440006), storage temperature: RT
  5. Cap (ProtDesc, catalog number: 80404440004), storage temperature: RT
  6. Gloves (Maxitec, Kevenol, catalog number: 80748910002), storage temperature: RT
  7. 20-ml syringe (BD Plastipak, catalog number: 990687), storage temperature: RT
  8. 21G needle (BD PrecisionGlide, catalog number: 300054), storage temperature: RT
  9. Glass tissue culture dish (Pyrex, catalog number: HX0004-00376), storage temperature: RT
  10. Corning® 75 cm2, U-Shaped canted neck cell culture flask with vent cap (Corning, catalog number: 430641U), storage temperature: 15/30 °C
  11. 24-well cell culture plates (Corning, catalog number: 3524), storage temperature: 15/30 °C
  12. 12-well plates (Corning, catalog number: 3512), storage temperature: 15/30 °C
  13. 6-well culture plates (Corning, catalog number: 3335), storage temperature: 15/30 °C
  14. 50-ml conical tube (Sarstedt, catalog number: 62.547.254), storage temperature: 15/30 °C
  15. Microtube 1.5-ml (Eppendorf, catalog number: Z606340), storage temperature: RT
  16. Micropipette tips (Eppendorf, catalog numbers: 0030000811/0030000854/0030000870/0030000919), storage temperature: RT
  17. Ultra-low attachment, 96-well (Costar, catalog number: CLS7007), storage temperature: 15/30 °C
  18. Alpha minimum essential medium (α-MEM) (Thermo Fisher Scientific, catalog number: 12000-022), storage temperature: 2/8 °C
  19. Dulbecco’s modified Eagle’s medium (D-MEM) (Thermo Fisher Scientific, catalog number: 12100-046), storage temperature: 2/8 °C
  20. Dulbecco’s phosphate-buffered saline (PBS) (Thermo Fisher Scientific, catalog number: 21600-010), storage temperature: 15/30 °C
  21. Sodium bicarbonate (Sigma-Aldrich, Sigma, catalog number: S5761-1KG), storage temperature: 15/30 °C
  22. Gentamycin reagent solution (Thermo Fisher Scientific, catalog number: 15710-064), storage temperature: -20/-5 °C
  23. Penicillin-Streptomycin (Thermo Fisher Scientific, catalog number: 15140-122), storage temperature: 15/30 °C
  24. Dexamethasone (Sigma-Aldrich, catalog number: D8893), storage temperature: 2/8 °C
  25. FBS qualified fetal calf serum (Thermo Fisher Scientific, catalog number: 12657-029), storage temperature: -10 °C
  26. Amphotericin B 250 μg/ml (Thermo Fisher Scientific, catalog number: 15290-018, storage temperature: -20/-5 °C)
  27. 0.25% Trypsin (1x) (Thermo Fisher Scientific, catalog number: 15050-057), storage temperature: -20/-5 °C
  28. Collagenase type II lyophilized (Thermo Fisher Scientific, catalog number: 17101-015), storage temperature: 2/8 °C
  29. 2.5% Chlorhexidine (Bioflora Manipullarium), storage temperature: RT
  30. β-Glycerophosphate disodium salt pentahydrate 98.0% (NT) (Sigma-Aldrich, catalog number: 50020-100G), storage temperature: 2/8 °C
  31. L-Ascorbic acid (Sigma-Aldrich, catalog number: 33034-100G), storage temperature: 15/30 °C
  32. Ethanol 96% (Merck, catalog number: 100971), storage temperature: 5/30 °C
  33. Formaldehyde solution 37% (Merck, catalog number: 104002), storage temperature: 15/25 °C
  34. Isopropanol (Merck Millipore, catalog number:1096341000), storage temperature: 5/30 °C
  35. Alizarin red S (Sigma-Aldrich, catalog number: A5533-25G), storage temperature: 15/30 °C
  36. Acetic acid (Merck, catalog number: 199061), storage temperature: 15/25 °C
  37. 3-Isobutyl-1-methylxanthine (Sigma-Aldrich, catalog number: I7018-1000MG), storage temperature: -20 °C
  38. Methanol (Merck, catalog number: 1.06009), storage temperature: 5/30 °C
  39. Insulin human (Sigma-Aldrich, catalog number: I2643-50MG), storage temperature: -20 °C
  40. Hydrochloric acid fuming 37% (Merck, catalog number: 1.00317), storage temperature: 5/30 °C
  41. Indomethacin (Sigma-Aldrich, catalog number: I7378-5G, storage temperature: 15/30 °C)
  42. Oil red O (Sigma-Aldrich, catalog number: O0625-25G, storage temperature: 15/30 °C)
  43. Trichome stain (Masson) Kit (Sigma-Aldrich, catalog number: HT15-1KT, storage temperature: RT)
  44. Sodium pyruvate (Sigma-Aldrich, catalog number: S8636, storage temperature: 2/8 °C)
  45. Human albumin (Institute Grifols, catalog number: A4AFC03441, storage temperature: 2/25 °C)
  46. Transforming growth factor-β3 (Peprotech Inc., catalog number: 100-36E, storage temperature: -20 °C)
  47. 4% Paraformaldehyde (Electron Microscopy Sciences, catalog number: 157-4-100), storage temperature: 2/8 °C
  48. Xylene (LabSynth, catalog number: X1001.01.BJ), storage temperature: 16/26 °C
  49. Paraffin (EasyPath, catalog number: EP-21-20068A), storage temperature: 15/30 °C
  50. Eosin (Sigma-Aldrich, catalog number: HT110132), storage temperature: RT
  51. Monoclonal anti-rat antibody: anti-CD29 (BD Biosciences, catalog number: 562154, storage temperature: 4 °C)
  52. Monoclonal anti-rat antibody: anti-CD31 (BD Biosciences, catalog number: 555027, storage temperature: 4 °C)
  53. Monoclonal anti-rat antibody: anti-CD34 (Invitrogen, catalog number: 11-0341-81, storage temperature: 4 °C)
  54. Monoclonal anti-rat antibody: anti-CD45 (BD Biosciences, catalog number: 554878, storage temperature: 4 °C)
  55. Monoclonal anti-rat antibody: anti-CD90 (BD Biosciences, catalog number: 554898, storage temperature: 4 °C)
  56. Monoclonal anti-rat antibody: anti-CD106 (BD Biosciences, catalog number: 559229, storage temperature: 4 °C)
  57. Transport medium (see Recipes)
  58. Collagenase solution (see Recipes)
  59. Trypsin solution (see Recipes)
  60. Ascorbic acid and β-Glycerophosphate solution (see Recipes)
  61. Growth medium (10% MEM) (see Recipes)
  62. Osteogenic differentiation medium (see Recipes)
  63. Chondrogenic differentiation medium (see Recipes)
  64. Adipocyte differentiation medium (see Recipes)
  65. Dexamethasone stock solution (200 μM) (see Recipes)
  66. Ascorbic acid stock solution (20 mM) (see Recipes)
  67. TGF-β3 (see Recipes)
  68. Oil red O staining (see Recipes)

Equipment

  1. Scissors (Quinelato, catalog number: QT.109.14)
  2. Forceps (Quinelato, catalog number: QC.301.14)
  3. Erv-Mount® (EasyPath, catalog number: EP-51-05041), storage temperature: 20 °C
  4. Micropipette (Eppendorf, catalog numbers: 4921000028/4921000044/4921000079/4921000109/4921000117/4921000150)
  5. Analytical balance M214A (BEL, catalog number: BL0003)
  6. RT basic series magnetic stirrers (Thermo Fisher Scientific, catalog number: 88880009)
  7. Bench meter for pH (Hanna, catalog number: HI5522-01)
  8. Stericup quick release vacuum driven disposable filtration system (Merck, catalog number: S2GPU05RE)
  9. Vacuum pump and compressor (Prismatec, catalog number: 132)
  10. Airstream class II biohazard safety cabinet (Esco Micro Pte.Ltd., model: AC2-4E8)
  11. Microprocessor water bath (Quimis, catalog number: Q215M)
  12. CO2 incubator (Panasonic, Panasonic/Sanyo, model: MCO-19AIC)
  13. Eppendorf® Centrifuge 5702 (Sigma-Aldrich, catalog number: Z606936)
  14. Axiovert 25 inverted microscope for advanced routine (Carl Zeiss)
  15. Compact digital microplate shaker (Thermo Fisher Scientific, catalog number: 88880023)
  16. Epoch 2 microplate spectrophotometer (BioTek, catalog number: BTEPOCH2)
  17. Centrifuge 5418 R (Eppendorf, catalog number: 5401000013)
  18. Gas exhaust chapel (Lutech, catalog number: LCE-15)
  19. Vertical freezer, 231 liters (Consul, catalog number: CVU26EB)
  20. Refrigerator frost free, 342 liters (Consul, catalog number: CRB39AB)
  21. Ultra-low freezer (Panasonic, catalog number: MDF-U500VXC-PA)
  22. FACSCantoTM II (BD Biosciences, catalog number: 338962)
  23. Paraffin dispenser (Oma, catalog number: IO-88)
  24. Microtome (Micron, GMI, catalog number: 8243-30-0001)

Software

  1. Gen 5 TS 2.06 (BioTek Instruments Inc./BioTek, https://www.biotek.com/products/software-robotics-software/gen5-microplate-reader-and-imager-software/)
  2. BD FACSDivaTM Software v8.0.3 (https://www.bdbiosciences.com/en-us/instruments/research-instruments/research-software/flow-cytometry-acquisition/facsdiva-software)
  3. StepOne Software v2.3 (Thermo Fisher Scientific/Applied Biosystems, https://www.thermofisher.com/br/en/home/technical-resources/software-downloads/StepOne-and-StepOnePlus-Real-Time-PCR-System.html)

Procedure

  1. Surgical procedure
    1. Euthanize the rat using isoflurane according to the local regulations.
    2. Disinfect the rat by completely bathing with 1% iodized ethanol (Figure 1A) and wipe the abdomen and lower limbs with 2.5% chlorhexidine.
    3. Transfer the rat in a sterile surgical drape.
    4. Wear a sterile coat, mask, cap, and gloves.
    5. To prevent contamination, use sterile scissors and forceps, to make a small bilateral incision in the skin of the femorotibial joint region (Figure 1B).
    6. Use this incision as an access point to perform a bilateral divulsion toward the abdominal and inguinal region.
    7. Make a horizontal cut to join the two previously made incisions (Figure 1B).
    8. Using a #15 scalpel blade attached to cable #3, cut the patellar tendons, and the lateral and medial collateral ligaments bilaterally to expose the joint capsules.
    9. Perform joint capsule divulsion bilaterally.
    10. Remove the muscle tissue to expose the anterior part of the femur (Figure 1C).
    11. Cut the remaining ligaments and disarticulate the femoral hip joint.
    12. Remove the femur, and quickly clean off the majority of muscle and connective tissues attached to the bone.
    13. Transfer the femur to a 50-ml conical tube containing 15 ml of transport medium.
    14. Retract the skin from the abdominal and inguinal regions (Figure 1D).
    15. Carefully remove all adipose tissue without puncturing the abdominal wall and transfer it to a 50-ml conical tube containing 15 ml of transport medium.
    16. Take the conical tubes containing the fat tissue and femurs to the laminar flow hood.


      Figure 1. Surgical procedures for harvesting the femur and adipose tissue of a Wistar rat weighing 150-200 g. A. Disinfection of the animal with 1% iodized ethanol after euthanasia. B. Schematic representation of the incisions. C. Muscle removal and femur exposure. D. Skin retraction and exposure of the abdominal and inguinal adipose tissues.

  2. BM-MSC isolation and culture procedures
    1. Transfer the femurs from the conical tubes to a glass tissue culture dish filled with 70% ethanol.
    2. Within 1 min, remove the remaining connective tissue with sterile scissors, forceps and a #15 scalpel blade.
    3. Transfer the femurs to a new glass tissue culture dish filled with 2.5% chlorhexidine.
    4. Within 1 min, clean the remaining connective tissue with sterile scissors, forceps and a #15 scalpel blade (Figure 2A).
    5. Transfer the femurs to new conical tubes containing 15 ml of transport medium and incubate for 15 min at RT.
    6. Again, transfer the femurs to a new conical vial containing 15 ml transport medium and incubate for 15 min at RT.
    7. Lastly, transfer the femurs to a new conical vial containing 15 ml transport medium and incubate for 15 min at RT.
    8. Transfer the contents of this conical tube to a glass tissue culture dish.
    9. Fill a 20-ml syringe with growth medium and attach a 21G needle.
    10. Hold the femur with tweezers and cut the epiphyses using sterile scissors (Figure 2B).
    11. Insert the needle of the syringe filled with growth medium into the diaphysis and flush all bone marrow into a new 50-ml conical tube (Figure 2C).
    12. Centrifuge this tube for 5 min at 600 x g at RT.
    13. Discard the supernatant and resuspend the pellet in new growth medium (2 ml per femur).
    14. Transfer 2 ml of this suspension in a 75 cm2 cell culture flask filled with 10 ml of growth medium.
    15. Incubate this flask in an incubator at 37 °C in a humidified atmosphere containing 5% CO2 and 95% air.
    16. After 24 h, gently rinse the flask three times with 1x PBS and replace with fresh growth medium.
    17. Change the culture medium every 2 days until the cells grow to 70% confluence.
    Note: After 7 days of culture in growth medium, approximately 5 x 106 MSCs were generated from each femur of each animal.


    Figure 2. The harvesting of bone marrow from femur. A. Cleaned femur. B. Marrow cavity exposure after epiphyseal sectioning. C. Bone marrow flushing with growth medium using a needle and a syringe.

  3. AT-MSC isolation and culture procedures
    1. Transfer the adipose tissue from the 50-ml conical tube to a glass tissue culture dish filled with 1x PBS to rinse the tissue.
    2. Transfer the adipose tissue to a new glass tissue culture dish (Figure 3A).
    3. Use sterile scissors to mince the adipose tissue into small pieces, around 1-2 mm3 (Figure 3B).
    4. Transfer the minced pieces to a 50-ml conical tube containing 20 ml of collagenase solution (Figure 3C).
    5. Place the tube in a water bath for 40 min at 37 °C, with shaking.
    6. Add 20 ml of growth medium to the 50-ml conical tube containing the adipose tissue and collagenase solution.
    7. Centrifuge the conical tube containing the adipose tissue, collagenase solution, and growth medium for 5 min at 600 x g.
    8. Discard the supernatant and resuspend the pellet in new growth medium (5 ml per adipose tissue removed from 1 animal).
    9. Transfer 5 ml of this suspension into a 75 cm2 cell culture flask filled with 10 ml of growth medium.
    10. Incubate this flask in an incubator at 37 °C in a humidified atmosphere containing 5% CO2 and 95% air.
    11. After 24 h, gently wash the flask three times with 1x PBS and replace with fresh growth medium.
    12. Replace the culture medium every 2 days until cells are 70% confluent.
    Notes:
    1. After 7 days of culture in growth medium, approximately 5 x 106 MSCs were generated from the adipose tissue of each animal.
    2. Typically, within 7 days, MSCs reach 70% of confluence and are ready to be used in subsequent experiments (Figure 4).


    Figure 3. The enzymatic digestion of abdominal and inguinal adipose tissue. A. Harvested adipose tissue. B. Mincing of adipose tissue into small pieces using sterile scissors and tweezers. C. Transfer adipose tissue pieces to collagenase type II solution for enzymatic digestion and cell isolation.


    Figure 4. Phase-contrast micrographs showing the morphology of BM-MSCs and AT-MSCs that were cultured in growth medium and on polystyrene dishes for up to 7 days. After 24 h, both BM-MSCs and AT-MSCs have attached to the polystyrene dish, and their morphology is round/oval. As the cells were cultured, they proliferated and became elongated, polygonal, and spindle-shaped. Scale bar = 100 µm.

  4. Characterization of BM-MSCs and AT-MSCs
    1. Wash the flask three times with 1x PBS.
    2. Add 5 ml of trypsin solution into the flask and incubate for 5 min at 37 °C.
    3. Add 2.5 ml of fresh growth medium into the flask, transfer the cell suspension into a 50-ml conical tube, and centrifuge for 5 min at 600 x g.
    4. Discard the supernatant.
    5. Wash the cell pellet once with 1x PBS.
    6. Centrifuge the cell suspension for 5 min at 600 x g.
    7. Discard the supernatant.
    8. Add 5 ml of 1x PBS to the cell pellet and mix the cell suspension.
    9. Count the cells in a hemocytometer (Neubauer Chamber).
    10. Adjust the concentration of the cell suspension to obtain a density of 2 x 105 cells/ml with 1x PBS.
    11. Add 1 ml of cell suspension to each flow cytometer tube (one tube for each specific antibody, one tube for isotype control, and one tube with cells that will not be labeled with antibody).
    12. Centrifuge the flow cytometer tubes for 5 min at 600 x g.
    13. Discard the supernatant.
    14. Add 100 µl of 1x PBS to the cell pellet and mix by flicking/tapping the tube.
    15. Incubate each tube for 30 min at RT in the dark with 2 µl of the following monoclonal anti-rat antibodies: anti-CD29, -CD31, -CD34, -CD45, and -CD106, directly conjugated with a fluorophore (antibody final dilution: 1:50). For monoclonal anti-rat antibody -CD90 directly conjugated with a fluorophore: dilute the antibody 1:5 in 1x PBS and then add 2 µl to the cell suspension (antibody final dilution: 1:250).
    16. Add 2 µl of isotype control to the corresponding tube.
    17. Wash the cells with 2 ml of 1x PBS.
    18. Centrifuge for 5 min at 600 x g.
    19. Discard the supernatant.
    20. Add 0.5 ml of formaldehyde solution (4%) diluted to 1% in 1x PBS.
    21. Analyze the cells by flow cytometry (Figures 5 and 6).


      Figure 5. Flow cytometry analysis of BM-MSCs cultured in growth medium on a polystyrene culture dish for 7 days. Histograms show the expression of the surface markers CD29, CD90, CD106, CD31, CD34, and CD44 after incubation with the respective antibodies. Cells were also incubated with the isotypes FITC-A and PE-A, which were used as negative controls. A high percentage of BM-MSCs expressed CD29, CD90, and CD106 (98.7%, 98.7%, and 28.7%, respectively) and a low percentage expressed CD31, CD34, and CD44 (7.8%, 0.4%, and 0.3%, respectively).


      Figure 6. Flow cytometry analysis of AT-MSCs cultured in growth medium on a polystyrene dish for 7 days. Histograms show the expression of the surface markers CD29, CD90, CD106, CD31, CD34, and CD44 after incubation with the respective antibodies. Cells were also incubated with the isotypes FITC-A and PE-A, which were used as negative controls. A high percentage of AT-MSC expressed CD29, CD90, and CD106 (99.9%, 99.3%, and 41.8%, respectively) and a low percentage expressed CD31, CD34, and CD44 (7.6%, 5.3%, and 0.2%, respectively).

  5. Osteoblast differentiation
    1. When BM-MSCs or AT-MSCs reach 70% confluence, remove the growth medium.
    2. Wash the flask three times with 1x PBS.
    3. Add 5 ml of trypsin solution into the flask and incubate for 5 min at 37 °C.
    4. Add 2.5 ml of fresh growth medium into the flask, transfer the cell suspension to a 50-ml conical tube, and centrifuge for 5 min at 600 x g.
    5. Discard the supernatant and resuspend the cell pellet in new growth medium.
    6. Count the cells and plate them at a cell density of 2 x 104 cells/well in 24-well culture plates in 1 ml of osteogenic medium or 1 x 105 cells/well in 6-well culture plates in 2 ml of osteogenic medium.
    7. Incubate the plates in an incubator at 37 °C in a humidified atmosphere containing 5% CO2 and 95% air during the time-course of the experiment.
    8. Replace the culture medium every 2 days.
    9. The extracellular matrix mineralization can be observed after 21 days in culture.

  6. Alizarin red staining
    To confirm osteoblast differentiation, one of the methods we used is the detection of mineralized extracellular matrix by alizarin red staining.
    1. Remove the culture medium from each well and gently wash the cells 3 times with 1x PBS.
    2. Add 10% formalin and incubate at 4 °C for 24 h (24-well plates–500 µl; 12-well plates–1 ml; 6-well plates–2.4 ml).
    3. Remove the 10% formalin and dehydrate the cells using increasing concentrations of ethanol (30%, 50%, 70%, and 96%) for 1 h each (24-well plates–500 µl; 12-well plates–1 ml; 6-well plates–2.4 ml).
    4. Remove the 96% ethanol and incubate at RT until the wells are dry.
    5. Cover the well with alizarin red staining and incubate at RT for 10 min.
    6. Wash once with deionized water and incubate at RT until the wells are dry.
    7. Take macroscopic (Figure 7) and microscopic photos of the wells.
    Note: Typically, BM-MSCs are more committed to osteoblast differentiation compared to AT-MSCs, as we previously observed (Abuna et al., 2016).


    Figure 7. Mineralized extracellular matrix detected by alizarin red staining in BM-MSC and AT-MSC cultures after 21 days of culture in osteogenic medium on polystyrene dishes


  7. Chondroblast differentiation
    1. When BM-MSC and AT-MSC cultures reach 70% confluence, remove the growth medium.
    2. Wash the flask three times with 1x PBS.
    3. Add 5 ml of trypsin solution into the flask and incubate for 5 min at 37 °C.
    4. Add 2.5 ml of fresh growth medium into the flask.
    5. Transfer this cell suspension to a 50-ml conical tube and centrifuge for 5 min at 600 x g.
    6. Discard the supernatant.
    7. Resuspend the cells in chondroblast differentiation medium at a density of 1.25 x 106 cells/ml.
    8. Using a pipette, dispense 200 µl aliquots of the cell suspension (2.5 x 105 cells) into each well of polypropylene 96-well plates.
    9. Centrifuge the plates at 500 x g for 5 min.
    10. Add 200 μl of 1x PBS into the empty wells to minimize the evaporation of the culture medium.
    11. Incubate the plates in an incubator at 37 °C in a humidified atmosphere containing 5% CO2 and 95% air during the time-course of the experiment.
    12. After 24 h of incubation, cell aggregates were visible, and the chondrogenic phenotype was observed after 30 days in culture.
    13. Replace the culture medium every 2 days by carefully aspirating the expired medium using a sterile 200 μl pipette and adding 200 μl of fresh chondrogenic medium to each well.

  8. Trichrome staining
    To confirm chondroblast differentiation, one of the methods we used is the detection of collagen fibers with trichrome staining.
    1. Using a micropipette, remove the chondroblast differentiation medium.
    2. Add 200 μl of 1x PBS into each well.
    3. Using a micropipette, remove the PBS.
    4. Add 200 μl of 4% paraformaldehyde for 5 min at RT.
    5. Remove the paraformaldehyde.
    6. Wash each well with 1x PBS, 2 times for 3 min each.
    7. Stain the cell aggregates with eosin for 5 min at RT.
    8. Remove the eosin and wash with 1x PBS, 2 times for 3 min each.
    9. Using a 1,000 µl micropipette, harvest the aggregates and transfer them to 1.5-ml microtubes.
    10. Dehydrate the cells aggregates in 300 µl of a graded ethanol series (70%, 80%, 90%, 95%, and 100%, 5 min each) by placing them into 1.5-ml microtubes.
    11. Remove the 100% ethanol and perform three clarification steps in 300 µl xylene for 3 min each.
    12. Paraffin-embed the aggregates into a mold in a hot surface for 5 min then transfer each mold to a cold surface.
    13. Cut adjacent 5 μm sections using a microtome.
    14. Deparaffinize the sections overnight in an incubator at 60 °C.
    15. Deparaffinize the sections in three steps of xylene for 5 min each, and rehydrate the sections by incubation in an ethanol series (100%, 95%, 90%, 80%, and 70%, 3 min each).
    16. Wash the sections with deionized water for 5 min.
    17. Stain with acid fuchsin (HT15-1) for 5 min at RT.
    18. Wash with water for 5 min.
    19. Stain with a working solution of phosphomolybdic acid (HT15-3) and phosphotungstic acid (HT15-2) for 5 min at RT.
    20. Stain the sections with an aniline blue solution for 5 min at RT.
    21. Remove the excess aniline blue, and add a 1% acetic acid solution for 3 min.
    22. Rinse with sections with tap water for 3 min.
    23. Dehydrate sections in a graded ethanol series (70%, 80%, 90%, 95%, and 100%, 1 min each), followed by three clarification steps in xylene for 1 min each.
    24. Mount the slides using Erv-Mount®.
    25. Take microscopic photos of the histological sections (Figure 8).
    Note: Cytoplasm is stained in red, and collagen fibers are stained in blue.


    Figure 8. Collagen fibers (blue) and cytoplasm (red) detected by trichrome staining in BM-MSCs and AT-MSCs cultured in chondrogenic medium on ultra-low cluster 96-well plates for 30 days. Scale bar = 100 µm.

  9. Adipocyte differentiation
    1. When BM-MSC and AT-MSC cultures reach 70% confluence, remove the growth medium.
    2. Wash the flask three times with 1x PBS.
    3. Add 5 ml of trypsin solution into the flask and incubate for 5 min at 37 °C.
    4. Add 2.5 ml of fresh growth medium into the flask.
    5. Transfer this cell suspension to a 50-ml conical tube and centrifuge for 5 min at 600 x g.
    6. Discard the supernatant and resuspend the cell pellet in fresh growth medium.
    7. Count the cells and plate them at a cell density of 2 x 104 cells/well in 24-well culture in 1 ml of adipogenic medium plates or 1 x 105 cells/well in 6-well culture plates in 2 ml of adipogenic medium.
    8. Incubate the plates in an incubator at 37 °C in a humidified atmosphere containing 5% CO2 and 95% air during the time-course of the experiment.
    9. Replace the culture medium every 2 days. The intracytoplasmic lipid droplets were observed after 10 days in culture.

  10. Oil red O staining
    To confirm adipocyte differentiation, one of the methods we used is the detection of intracytoplasmic lipid droplets with oil red O staining.
    1. Remove the culture medium from each well.
    2. Add 10% formalin and incubate for 5 min at RT (24-well plates–500 µl, 12-well plates–1 ml, 6-well plates–2.4 ml).
    3. Discard the 10% formalin and add the same volume of fresh 10% formalin. Incubate for at least 1 h.
      Note: Cells can be kept in formalin for a couple of days before staining. Wrap parafilm around the plate to prevent the cells from drying out and cover the plate with aluminum foil.
    4. Remove the 10% formalin using a small transfer pipette.
    5. Wash the wells with 60% isopropanol (24-well plates–500 µl, 12-well plates–1 ml, 6-well plates–2.4 ml).
    6. Let the wells dry completely.
    7. Add the oil red O staining working solution for 10 min (do not touch walls of the wells).
    8. Remove the oil red O staining, and immediately add deionized water (repeat this step 4 times).
    9. Remove all deionized water and incubate at RT to dry.
    10. Image the wells using a phase-contrast microscope (Figure 9).
    Note: Typically, AT-MSCs are more committed to adipogenic differentiation compared to BM-MSCs, as we previously observed (Abuna et al., 2016).


    Figure 9. Intracytoplasmic lipid droplets detected by oil red O staining in BM-MSCs and AT-MSCs cultured in adipogenic medium on a polystyrene dish for 10 days. Scale bar = 100 µm.

Recipes

  1. Transport medium
    Note: Prepared fresh just prior to use and kept at 37 °C.
    57 ml of alpha minimum essential medium (α-MEM)
    3 ml of gentamycin (50 µg/ml)
    720 µl of amphotericin B (0.3 µg/ml)
  2. Collagenase solution
    Note: Prepared fresh just prior to use and placed at RT.
    15 mg of type II collagenase (0.075%)
    20 ml of 1x PBS
    Filter this solution in the laminar flow hood into a 50-ml conical tube using a 20-ml syringe and 0.2 µm filter
  3. Trypsin solution
    Note: Prepared fresh just prior to use and placed at RT.
    19 ml of trypsin (0.25%)
    500 µl of type II collagenase (1.3 mg/ml)
    1 ml of EDTA (1 mM)
  4. Ascorbic acid and β-Glycerophosphate solution
    Note: Previously prepared, kept at 4 °C for up to 7 days.
    5 mg of ascorbic acid (5 µg/ml)
    2.16 g of β-glycerophosphate (7 mM)
    10 ml of deionized water
    Filter this solution in the laminar flow hood into a 50-ml conical tube using a 20-ml syringe and 0.2 µm filter
  5. Growth medium (10% MEM)
    Note: Previously prepared, kept at 4 °C for up to 30 days.
    360 ml of α-MEM
    40 ml of fetal calf serum
    2 ml of gentamycin (50 µg/ml)
    500 µl of amphotericin B (0.3 µg/ml)
  6. Osteogenic differentiation medium
    Note: Previously prepared, kept at 4 °C for up to 30 days.
    400 ml of growth medium
    4 ml of dexamethasone (10-7 M)
    1% ascorbic acid and β-glycerophosphate solution
    Note: The ascorbic acid and β-glycerophosphate solution are added immediately before using the medium.
  7. Chondrogenic differentiation medium
    Note: Previously prepared, kept at 4 °C for up to 30 days.
    100 ml of D-MEM
    100 µl of sodium pyruvate (100 mM)
    100 µl of dexamethasone (1 mM)
    250 µl of ascorbic acid (20 mM)
    1 ml of human albumin (0.02%)
    20 µl/ml of transforming growth factor β3 (TGF-β3, 1 µg/ml).
    Note: The TGF-β3 is added immediately before using the medium.
  8. Adipocyte differentiation medium
    Note: Previously prepared, kept at 4 °C for up to 30 days.
    180 ml of D-MEM
    20 ml of fetal calf serum (10%)
    2 ml of gentamycin (50 µg/ml)
    250 µl of amphotericin B (0.3 µg/ml)
    2 ml of dexamethasone (10-6 M)
    2 ml of 3-isobutyl-1-methylxanthine (0.5 mM)
    260 µl of indomethacin (0.1 M)
    150 µl of insulin (10 mg/ml)
  9. Dexamethasone stock solution (200 µM)
    Note: Previously prepared, kept at -20 °C.
    Dexamethasone is dissolved at 200 µM in absolute ethanol and deionized water
  10. Ascorbic acid stock solution (20 mM)
    Note: Previously prepared, kept at 4 °C for up to 7 days.
    A stock solution of 20 mM ascorbic acid is prepared in 1x PBS
  11. TGF-β3
    Note: Prepared fresh just prior to use and placed at RT.
    A 1 μg/ml stock solution of TGF-β3 is prepared in 1x PBS and 0.5% human albumin.
  12. Oil red O staining
    Oil red O stock solution
    Note: Previously prepared, kept at RT up to 6 months.
    Oil red O (700 mg) is added to 200 ml of isopropanol, stirred overnight, and then passed through a 0.2 µm filter.
    Oil red O work solution
    Mix 6 parts of oil red O stock solution with 4 parts deionized water and incubate at RT for 20 min.

Acknowledgments

This study was funded by the State of Sao Paulo Research Foundation (FAPESP, Brazil, #2017/12622-7), National Council for Scientific and Technological Development (CNPq, Brazil, # 305523/2013-9 and 404318/2016-9), and Coordination of Improvement of Higher Education Personnel (CAPES, Brazil). The English language review was carried out by ENAGO (www.enago.com) funded by FAPESP (#2018/17356-6).This protocol was adapted from these works (Maniatopoulos et al., 1988; Huang et al., 2002; Penick et al.,2005).

Competing interests

The authors declare no conflict of interest.

Ethics

All procedures performed were conducted in accordance with the ethical standards of the international, national, and/or institutional animal care guidelines. The Committee of Ethics in Animal Research of the School of Dentistry of Ribeirão Preto, University of São Paulo (#2018.1.30.58.8) reviewed and approved all animal procedures we have done here.

References

  1. Abuna, R. P., De Oliveira, F. S., Santos Tde, S., Guerra, T. R., Rosa, A. L. and Beloti, M. M. (2016). Participation of TNF-α in inhibitory effects of adipocytes on osteoblast differentiation. J Cell Physiol 231(1): 204-214.
  2. Almeida, A. L. G., Freitas, G. P., Lopes, H. B., Gimenes, R., Siessere, S., Sousa, L. G., Beloti, M. M. and Rosa, A. L. (2019). Effect of stem cells combined with a polymer/ceramic membrane on osteoporotic bone repair. Braz Oral Res 33: e079.
  3. Bianco, P., Robey, P. G. and Simmons, P. J. (2008). Mesenchymal stem cells: revisiting history, concepts, and assays. Cell Stem Cell 2(4): 313-319.
  4. Caplan, A. I. (1991). Mesenchymal stem cells. J Orthop Res 9(5): 641-650.
  5. Fideles, S. O. M., Ortiz, A. C., Assis, A. F., Duarte, M. J., Oliveira, F. S., Passos, G. A., Beloti, M. M. and Rosa, A. L. (2019). Effect of cell source and osteoblast differentiation on gene expression profiles of mesenchymal stem cells derived from bone marrow or adipose tissue. J Cell Biochem 120(7): 11842-11852.
  6. Freitas, G. P., Lopes, H. B., Souza, A. T. P., Oliveira, P., Almeida, A. L. G., Souza, L. E. B., Coelho, P. G., Beloti, M. M. and Rosa, A. L. (2019). Cell therapy: effect of locally injected mesenchymal stromal cells derived from bone marrow or adipose tissue on bone regeneration of rat calvarial defects. Sci Rep 9(1): 13476.
  7. Friedenstein, A. J., Chailakhjan, R. K. and Lalykina, K. S. (1970). The development of fibroblast colonies in monolayer cultures of guinea-pig bone marrow and spleen cells. Cell Tissue Kinet 3(4): 393-403.
  8. Harting, M., Jimenez, F., Pati, S., Baumgartner, J. and Cox, C., Jr. (2008). Immunophenotype characterization of rat mesenchymal stromal cells. Cytotherapy 10(3): 243-253.
  9. Hu, Q., Liu, M., Chen, G., Xu, Z. and Lv, Y. (2018). Demineralized bone scaffolds with tunable matrix stiffness for efficient bone integration. ACS Appl Mater Interfaces 10(33): 27669-27680.
  10. Huang, J. I., Beanes, S. R., Zhu, M., Lorenz, H. P., Hedrick, M. H., Benhaim, P. (2002). Rat extramedullary adipose tissue as a source of osteochondrogenic progenitor cells. Plast Reconstr Surg 109(3):1033-1041.
  11. Lopes, H. B., Freitas, G. P., Fantacini, D. M. C., Picanço-Castro, V., Covas, D. T., Rosa, A. L. and Beloti, M. M. (2019). Titanium with nanotopography induces osteoblast differentiation through regulation of integrin αV. J Cell Biochem 120(10): 16723-16732.
  12. Maniatopoulos, C., Sodek, J., Melcher, A. H. (1988). Bone formation in vitro by stromal cells obtained from bone marrow of young adult rats. Cell Tissue Res 254(2): 317-330.
  13. Nancarrow-Lei, R., Mafi, P., Mafi, R. and Khan, W. (2017). A systemic review of adult mesenchymal stem cell sources and their multilineage differentiation potential relevant to musculoskeletal tissue repair and regeneration. Curr Stem Cell Res Ther 12(8): 601-610.
  14. Oliveira, F. S., Bellesini, L. S., Defino, H. L., da Silva Herrero, C. F., Beloti, M. M. and Rosa, A. L. (2012). Hedgehog signaling and osteoblast gene expression are regulated by purmorphamine in human mesenchymal stem cells. J Cell Biochem 113(1): 204-208.
  15. Penick, K. J., Solchaga, L. A. and Welter, J. F. (2005). High-throughput aggregate culture system to assess the chondrogenic potential of mesenchymal stem cells. Biotechniques 39(5): 687-691.
  16. Pierini, M., Dozza, B., Lucarelli, E., Tazzari, P. L., Ricci, F., Remondini, D., di Bella, C., Giannini, S. and Donati, D. (2012). Efficient isolation and enrichment of mesenchymal stem cells from bone marrow. Cytotherapy 14(6): 686-693.
  17. Schrepfer, S., Deuse, T., Lange, C., Katzenberg, R., Reichenspurner, H., Robbins, R. C. and Pelletier, M. P. (2007). Simplified protocol to isolate, purify, and culture expand mesenchymal stem cells. Stem Cells Dev 16(1): 105-107.
  18. Wadajkar, A. S., Santimano, S., Tang, L. and Nguyen, K. T. (2014). Magnetic-based multi-layer microparticles for endothelial progenitor cell isolation, enrichment, and detachment. Biomaterials 35(2): 654-663.
  19. Zhang, Y. D., Zhao, S. C., Zhu, Z. S., Wang, Y. F., Liu, J. X., Zhang, Z. C. and Xue, F. (2017). Cx43- and smad-mediated TGF-beta/BMP signaling pathway promotes cartilage differentiation of bone marrow mesenchymal stem cells and inhibits osteoblast differentiation. Cell Physiol Biochem 42(4): 1277-1293.
  20. Zuk, P. A., Zhu, M., Mizuno, H., Huang, J., Futrell, J. W., Katz, A. J., Benhaim, P., Lorenz, H. P. and Hedrick, M. H. (2001). Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 7(2): 211-228.

简介

[摘要 ] 自从发现以来,间充质基质细胞(MSC)受到了广泛的关注,主要是由于它们具有自我更新的潜能和多系分化能力。由于这些原因,MSC是细胞生物学和再生医学中的有用工具。在本文中,我们描述了从骨髓(BM-MSC)和脂肪组织(AT-MSC)分离MSC的协议,以及将MSC培养,鉴定和区分为成骨细胞,脂肪细胞和软骨细胞的方法。通过冲洗股骨干骨并通过酶消化腹部和腹股沟脂肪组织从骨髓中收获细胞后,通过粘附在塑料组织培养皿上选择MSC。在7天内,MSC融合度达到70%,并准备用于后续实验。这里描述的协议易于执行,具有成本效益,需要最少的时间并产生富含MSC的细胞群。

背景 ] 干细胞可以追溯到19的概念个世纪,但它们的存在被证实在20世纪60年代和70年代以下通过实验Friedenstein 和合作者,这表明干细胞在骨髓中(存在Friedenstein ,1970; Bianco的等人,2008)。此后,Caplan(1991)将它们命名为间充质干细胞(此处称为间充质基质细胞-MSCs),并提出将其用于再生医学。在骨髓中,MSCc 的百分比估计为单核细胞总数的0.001%至0.01%。由于它们的稀缺性,尽管骨髓仍然是MSC的主要来源,但已描述了其他来源(Nancarrow- Lei 等人,2017)。脂肪组织是非常有前途的来源,因为它包含大量相对容易收获的MSC,给其带来的不适和风险极小(Zuk 等,2001)。用于从骨髓(BM-MSC)或脂肪组织(AT-MSC)收获和培养MSC的协议在不同物种之间,甚至在同一物种的不同菌株之间也可能有所不同。用于获得的MCS的最常用的方法包括使用流式细胞术(Schrepfer 等人,2007),多能成体祖细胞培养基(哈丁等人。,2008),在菲可帕克密度梯度离心法(Pierini 等人。,2012)和免疫磁珠(Wadajkar 等人,2014)。在这里,我们描述了具有成本效益的协议,该协议相对容易且快速地执行,可用于从骨髓和脂肪组织获得富含MSC的细胞群体。这些协议可用于研究MSC的细胞和分子方面的几个方面,例如其增殖,分化和信号传导途径(Abuna 等人,2016; Fidel e s 等人,2019),生长因子的生物学效应和MSCs上的药物(Oliveira 等人,2012; Zhang 等人,2017),MSCs与天然或合成生物材料之间的相互作用(Hu 等人,2018; Lopes 等人,2019)以及MSCs在MSCs中的应用再生医学策略(Almeida 等,2019; Freitas 等,2019)。

关键字:脂肪组织, 脂肪细胞, 骨, 骨髓, 细胞培养, 软骨细胞, 间充质干细胞, 成骨细胞

材料和试剂


 


无菌手术单
铝箔
外套(ProtDesc ,目录号:80404440020),储存温度:RT
面膜(ProtDesc ,目录号:80404440006),储存温度:RT
瓶盖(ProtDesc ,目录号:80404440004),储存温度:RT
手套(Maxitec ,Kevenol ,目录号:80748910002),存储温度:RT
20 ml注射器(BD Plastipak,目录号:990687),存储温度:RT
21G针(BD PrecisionGlide ,目录号:300054),存储温度:RT
玻璃组织培养皿(派热克斯(Pyrex),目录号:HX0004-00376),储存温度:RT
康宁® 75厘米2 ,具有排气孔盖U型倾斜颈细胞培养瓶(Corning,目录号:430641U),贮存温度:15/30 ℃下
24孔细胞培养板(Corning,目录号:3524),储存温度:15/30 °C
12孔板(Corning,目录号:3512),存储温度:15/30°C
6孔培养板(Corning,目录号:3335),存储温度:15/30 °C
50 ml锥形管(Sarstedt ,目录号:62.547.254),储存温度:15/30°C
1.5 ml 微量管(Eppendorf,目录号:Z606340),储存温度:RT
微量移液器吸头(Eppendorf,目录号s :0030000811/0030000854/0030000870/0030000919 ),储存温度:RT
超低附件,96孔(Costar ,目录号:CLS7007),储存温度:15/30 °C
Alpha最低基本培养基(α-MEM)(Thermo Fisher Scientific,目录号:12000-022),存储温度:2/8 °C
Dulbecco改良的Eagle培养基(D-MEM)(Thermo Fisher Scientific,目录号:12100-046),存储温度:2/8 °C
Dulbecco的磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,目录号:21600-010),储存温度:15/30 °C
碳酸氢钠(Sigma-Aldrich,Sigma,目录号:S5761-1KG),储存温度:15/30 °C
庆大霉素试剂溶液(Thermo Fisher Scientific,目录号:15710-064),存储温度:-20 / -5 °C
青霉素-链霉素(Thermo Fisher Scientific,目录号:15140-122),储存温度:15/30 °C
地塞米松(Sigma-Aldrich,目录号:D8893),储存温度:2/8 °C
FBS合格的胎牛血清(赛默飞世尔科技,产品目录号:12657-029),贮存温度:- 10 ℃下
两性霉素B 250 μ 克/毫升(赛默飞世尔科技,产品目录号:15290-018,储存温度:- 20 / - 5 ℃下)
0.25%胰蛋白酶(1×)(赛默飞世尔科技,产品目录号:15050-057),贮存温度:- 20 / - 5 ℃下
II型胶原酶冻干(Thermo Fisher Scientific,目录号:17101-015),储存温度:2/8 °C
2.5%洗必泰(Bioflora M anipullarium ),储存温度:室温
β- 甘油磷酸二钠五水合物98.0%(NT)(Sigma-Aldrich,目录号:50020-100G),储存温度:2/8 °C
L-抗坏血酸(Sigma-Aldrich,目录号:33034-100G),储存温度:15/30 °C
乙醇96%(默克(Merck),货号:100971),存储温度:5/30 °C
甲醛溶液37%(默克(Merck),货号:104002),存储温度:15/25 °C
异丙醇(默克密理博(Merck Millipore),目录号:1096341000),存储温度:5/30°C
茜素红S(Sigma-Aldrich,目录号:A5533-25G),储存温度:15/30 °C
醋酸(默克(Merck),目录号:199061),储存温度:15/25 °C
3-异丁基-1-甲基黄嘌呤(Sigma-Aldrich公司,目录号:I7018-1000MG),贮存温度:- 20 ℃下
甲醇(Merck,目录号:1.06009),存储温度:5/30 °C
人胰岛素(Sigma-Aldrich公司,目录号:I2643-50MG),贮存温度:- 20 ℃下
盐酸发烟37%(默克,目录号:1.00317),存储温度:5/30 °C
消炎痛(Sigma-Aldrich,目录号:I7378-5G,储存温度:15/30 °C )
油红色O(Sigma-Aldrich,目录号:O0625-25G,存储温度:15/30 °C )
Trichome染色剂(Masson)试剂盒(Sigma-Aldrich,目录号:HT15-1KT,存储温度:RT )
丙酮酸钠(Sigma-Aldrich,目录号:S8636,储存温度:2/8 °C )
人白蛋白(Institute Grifols,目录号:A4AFC03441,储存温度:2/25 °C )
转化生长因子β 3(派普泰克公司制公司,目录号:100-36E,存储温度:- 20 ℃下)
4%多聚甲醛(Electron Microscopy Sciences,目录号:157-4-100),储存温度:2/8 °C
二甲苯(LabSynth ,目录号:X1001.01.BJ),储存温度:16/26 °C
石蜡(EasyPath ,目录号:EP-21-20068A),储存温度:15/30 °C
曙红(Sigma-Aldrich,目录号:HT110132),储存温度:RT
单克隆抗大鼠抗体:抗CD29(BD Biosciences,目录号:562154,存储温度:4 °C )
单克隆抗大鼠抗体:抗CD31(BD Biosciences,目录号:555027,存储温度:4 °C )
单克隆抗大鼠抗体:抗CD34(Invitrogen,目录号:11-0341-81,储存温度:4 °C )
单克隆抗大鼠抗体:抗CD45(BD Biosciences,目录号:554878,存储温度:4 °C )
单克隆抗大鼠抗体:抗CD90(BD Biosciences,目录号:554898,存储温度:4 °C )
单克隆抗大鼠抗体:抗CD106(BD Biosciences,目录号:559229,储存温度:4 °C )
传输介质(请参阅食谱)
胶原酶溶液(请参阅食谱)
胰蛋白酶溶液(请参阅食谱)
抗坏血酸和β-甘油磷酸酯溶液(参见食谱)
生长培养基(10%MEM)(请参阅食谱)
成骨分化培养基(请参见食谱)
软骨分化培养基(请参阅食谱)
脂肪细胞分化培养基(请参阅食谱)
地塞米松原液(200 μ中号)(见配方)
抗坏血酸储备溶液(20 mM)(请参阅食谱)
TGF-β3 (请参阅食谱)
油红色O染色(请参阅配方)
 


设备


 


剪刀(Quinelato ,目录号:QT.109.14)
镊子(Quinelato ,目录号:QC.301.14)
ERV -mount ® (的EasyPath ,目录号:EP-51-05041),存储温度:20 ℃的
微量移液器(Eppendorf,目录号s :4921000028/4921000044/4921000079/4921000109/4921000117/4921000150)
分析天平M214A(BEL,货号:BL0003)
RT basic系列磁力搅拌器(Thermo Fisher Scientific,目录号:88880009)
pH台式测量仪(汉娜,目录号:HI5522-01)
Stericup 快速释放真空驱动的一次性过滤系统(Merck,目录号:S2GPU05RE)
真空泵和压缩机(Prismatec ,目录号:132)
气流II级生物危害安全柜(Esco Micro Pte.Ltd 。,型号:AC2-4E8)
微处理器水浴箱(Quimis ,目录号:Q215M)
CO 2 培养箱(Panasonic,Panasonic / Sanyo,型号:MCO-19AIC)
的Eppendorf ® 离心机5702(Sigma-Aldrich公司,目录号:Z606936)
Axiovert 25倒置显微镜用于高级常规操作(卡尔·蔡司)
紧凑型数字微孔板振荡器(Thermo Fisher Scientific,目录号:88880023)
Epoch 2微孔板分光光度计(BioTek ,目录号:BTEPOCH2)
5418 R离心机(埃彭多夫(Eppendorf),目录号:5401000013)
排气教堂(Lutech ,目录号:LCE-15)
立式冷冻机,231升(领事,目录号:CVU26EB)
无霜冰箱,342升(领事,目录号:CRB39AB)
超低温冰箱(松下,目录号:MDF-U500VXC-PA)
FACSCantoTM II(BD Biosciences,目录号:338962)
石蜡分配器(奥马,货号:IO-88)
切片机(Micron,GMI,目录号:8243-30-0001)
 


软件


 


第5代TS 2.06(BioTek Instruments Inc./ BioTek ,https: //www.biotek.com/products/software-robotics-software/gen5-microplate-reader-and-imager-software/ )
BD FACSDiva TM 软件v8.0.3(https://www.bdbiosciences.com/zh-cn/instruments/research-instruments/research-software/flow-cytometry-acquisition/facsdiva-software)
StepOne 软件v2.3(Thermo Fisher Scientific / Applied Biosystems,https://www.thermofisher.com/br/en/home/technical-resources/software-downloads/StepOne-and-StepOnePlus-Real-Time-PCR-System .html)
 


程序


 


手术程序
根据当地法规,使用异氟烷对大鼠实施安乐死。
用1%的碘化乙醇完全沐浴消毒大鼠(图1A),并用2.5%的洗必泰擦拭腹部和下肢。
将大鼠转移到无菌手术单中。
戴无菌外套,口罩,帽子和手套。
为防止污染,用无菌剪刀和镊子,以做一个小切口,双边的腿胫关节区域(图1B)的皮肤。
使用该切口作为进入点,向腹部和腹股沟区域进行双侧渗出。
进行水平切割以连接先前制作的两个切口(图1B)。
使用#15手术刀刀片连接到电缆#3,双侧切断tell骨肌腱,外侧和内侧副韧带,露出关节囊。
进行双侧关节囊泄漏。
去除肌肉组织以暴露股骨的前部(图1C)。
切下剩余的韧带,使股骨髋关节脱节。
去除股骨,并迅速清除附着在骨骼上的大部分肌肉和结缔组织。
将股骨转移到装有15 ml运输介质的50 ml锥形管中。
从腹部和腹股沟区域收回皮肤(图1D)。
小心地去除所有脂肪组织而不刺破腹壁,并将其转移到装有15 ml传输介质的50 ml锥形管中。
将包含脂肪组织和股骨的锥形管移至层流罩。
 


D:\ Reformatting \ 2020-1-6 \ 1902730--1292 Adalberto Rosa 753853 \ Figs jpg \图1.jpg


图1.收获股骨和一个只Wistar大鼠体重150的脂肪组织的外科手术- 200克。A.安乐死后用1%碘化乙醇对动物进行消毒。B.切口的示意图。C.肌肉去除和股骨暴露。D.皮肤回缩以及腹部和腹股沟脂肪组织的暴露。


 


BM-MSC分离和培养程序
将股骨从锥形管转移到装有70%乙醇的玻璃组织培养皿中。
在1分钟内,用无菌剪刀,镊子和15号手术刀刀片除去剩余的结缔组织。
将股骨转移至装有2.5%洗必泰的新玻璃培养皿中。
1分钟内,用无菌剪刀,镊子和15号手术刀刀片清洁剩余的结缔组织(图2A)。
将股骨转移到新的锥形管中,该锥形管中含有15 ml的运输培养基,并在室温下孵育15分钟。
再次,将股骨转移至含有15 ml转运培养基的新锥形瓶中,并在室温下孵育15分钟。
最后,将股骨转移至含有15 ml转运培养基的新锥形瓶中,并在室温下孵育15分钟。
将该锥形管的内容物转移到玻璃组织培养皿中。
用生长培养基填充20毫升注射器,并连接21G针头。
用镊子夹住股骨,并用无菌剪刀剪掉骨epi(图2B)。
将充满生长培养基的注射器针头插入骨干,并将所有骨髓冲洗到新的50 ml锥形管中(图2C)。
在室温下以600 xg离心5分钟。
弃去上清液,将沉淀重悬于新的生长培养基中(每股股骨2 ml)。
将2 ml这种悬浮液转移到装有10 ml生长培养基的75 cm 2 细胞培养瓶中。
在37°C的恒温箱中,在含有5%CO 2 和95%空气的潮湿气氛中,将烧瓶保温。
24小时后,用1x PBS轻轻冲洗烧瓶三遍,并用新鲜的生长培养基替换。
每两天更换一次培养基,直到细胞生长到70%融合为止。
注意:在生长培养基中培养7天后,从每只动物的每个股骨中产生大约5 x 10 6个MSC。


 


D:\ Reformatting \ 2020-1-6 \ 1902730--1292 Adalberto Rosa 753853 \ Figs jpg \图2.jpg


图2.从股骨中提取骨髓。答:清洁股骨。B.骨epi切片后骨髓腔暴露。C.使用针头和注射器用生长培养基冲洗骨髓。


 


AT-MSC分离和培养程序
将脂肪组织从50 ml锥形管转移到装有1x PBS的玻璃组织培养皿中,以冲洗组织。
将脂肪组织转移到新的玻璃组织培养皿中(图3A)。
使用无菌剪刀剁碎的脂肪组织切成小块,大约1 - 2毫米3 (图3B)。
将切碎的碎块转移到装有20 ml胶原酶溶液的50 ml锥形管中(图3C)。
将试管置于37°C水浴中振摇40分钟。
向含有脂肪组织和胶原酶溶液的50 ml锥形管中加入20 ml生长培养基。
将含有脂肪组织,胶原酶溶液和生长培养基的锥形管以600 xg离心5分钟。
弃去上清液,然后将沉淀重悬于新的生长培养基中(每只脂肪组织从1只动物中取出5毫升)。
将5毫升这种悬浮液转移到装有10毫升生长培养基的75 cm 2 细胞培养瓶中。
在37°C的恒温箱中,在含有5%CO 2 和95%空气的潮湿气氛中,将烧瓶保温。
24小时后,用1 x PBS 轻轻洗涤烧瓶3次,并用新鲜的生长培养基替换。
每两天更换一次培养基,直到细胞融合至70%。
注意小号:


在生长培养基中培养7天后,从每只动物的脂肪组织中产生了大约5 x 10 6个MSC。
通常,在7天内,MSC达到融合的70%,并准备用于后续实验(图4)。
 


D:\ Reformatting \ 2020-1-6 \ 1902730--1292 Adalberto Rosa 753853 \ Figs jpg \图3.jpg


图3.腹部和腹股沟脂肪组织的酶消化。A.收获的脂肪组织。B.用无菌剪刀和镊子将脂肪组织切成小块。C.将脂肪组织碎片转移至II型胶原酶溶液中,以进行酶消化和细胞分离。


 


D:\ Reformatting \ 2020-1-6 \ 1902730--1292 Adalberto Rosa 753853 \ Figs jpg \图4.jpg


图4.相差显微照片,显示了在生长培养基中和在聚苯乙烯培养皿上培养长达7天的BM-MSC和AT-MSC的形态。24小时后,BM-MSC和AT-MSC均已附着在聚苯乙烯培养皿上,其形态为圆形/椭圆形。培养细胞时,它们增殖并变得细长,多边形和纺锤形。比例尺= 100 µm。


 


BM-MSC和AT-MSC的表征
用1x PBS洗涤烧瓶3次。
将5 ml胰蛋白酶溶液加入烧瓶中,并于37孵育5分钟 ° Ç 。
向烧瓶中加入2.5 ml新鲜生长培养基,将细胞悬浮液转移到50 ml锥形管中,并以600 xg离心5分钟。
丢弃上清液。
用1x PBS洗涤细胞沉淀一次。
将细胞悬液以600 x g 离心5分钟。
丢弃上清液。
将5 ml 1x PBS加入细胞沉淀并混合细胞悬液。
在血细胞计数器(Neubauer Chamber)中计数细胞。
调整细胞悬液的浓度,用1x PBS 获得2 x 10 5个细胞/ ml 的密度。     
向每个流式细胞仪管中加入1 ml细胞悬液(每种特异性抗体一个管,一个同种型对照管,一个不带有抗体标记细胞的管)。
将流式细胞仪试管以600 x g 离心5分钟。
丢弃上清液。
向细胞沉淀中加入100 µl 1x PBS,并轻拂/轻敲试管进行混合。
将每支试管与2 µl以下单克隆抗大鼠抗体在黑暗中于室温孵育30分钟:抗CD29,-CD31,-CD34,-CD45和-CD106,直接与荧光团偶联(抗体最终稀释度: 1:50)。对于直接与荧光团偶联的单克隆抗大鼠抗体-CD90 :在1x PBS中按1:5稀释抗体,然后将2 µl加入细胞悬液中(抗体最终稀释度:1:250)。
将2 µl同型对照加入相应的试管中。
用2毫升1x PBS洗涤细胞。
以600 xg 离心5分钟。
丢弃上清液。
加入在1x PBS中稀释至1%的0.5 ml甲醛溶液(4%)。
通过流式细胞仪分析细胞(图5和6)。
 


 


D:\ Reformatting \ 2020-1-6 \ 1902730--1292 Adalberto Rosa 753853 \ Figs jpg \图5.jpg


图5.在聚苯乙烯培养皿上的生长培养基中培养7天的BM-MSC的流式细胞仪分析。直方图显示与相应抗体孵育后表面标记CD29,CD90,CD106,CD31,CD34和CD44的表达。还将细胞与同型FITC-A和PE-A孵育,用作阴性对照。高百分比的BM-MSC表达CD29,CD90和CD106(分别为98.7%,98.7%和28.7%),低百分比表达CD31,CD34和CD44(分别为7.8%,0.4%和0.3%) )。


 


D:\ Reformatting \ 2020-1-6 \ 1902730--1292 Adalberto Rosa 753853 \ Figs jpg \图6.jpg


图6.在聚苯乙烯培养皿上的生长培养基中培养7天的AT-MSC的流式细胞仪分析。直方图显示与相应抗体孵育后表面标记CD29,CD90,CD106,CD31,CD34和CD44的表达。还将细胞与同型FITC-A和PE-A孵育,用作阴性对照。高百分比的AT-MSC表达CD29,CD90和CD106(分别为99.9%,99.3%和41.8%),低百分比表达CD31,CD34和CD44(分别为7.6%,5.3%和0.2%) )。


 


成骨细胞分化
当BM-MSC或AT-MSC达到70%融合时,请去除生长培养基。
用1x PBS洗涤烧瓶3次。
将5 ml胰蛋白酶溶液加入烧瓶中,并在37 °C 下孵育5分钟。
将2.5 ml新鲜的生长培养基添加到烧瓶中,将细胞悬浮液转移到50 ml锥形管中,并以600 x g 离心5分钟。
丢弃上清液并将细胞沉淀重悬在新的生长培养基中。
计数细胞,并将其以2 x 10 4个细胞/ 孔的细胞密度接种在1 ml成骨培养基中的24孔培养板中或1 x 10 5个细胞/孔的2 ml成骨培养基中的6孔培养板中。
在实验过程中,将板在培养箱中于37°C,含有5%CO 2 和95%空气的潮湿气氛中孵育。
每2天更换一次培养基。
Ť 他的细胞外基质矿化能够在培养21天观察。
 


茜素红染色
为了确认成骨细胞的分化,我们使用的方法之一是通过茜素红染色检测矿化的细胞外基质。


从每个孔中移出培养基,并用1x PBS轻轻洗涤细胞3次。
加入10%福尔马林,并在4 °C 下孵育24小时(24孔板– 500 µl; 12孔板–1 ml; 6孔板– 2.4 ml)。
除去10%福尔马林,并使用浓度递增的乙醇(30 %,50 %,70 %和96 %)将细胞脱水,每次1小时(24孔板– 500 µl; 12 孔板– 1 ml; 10孔板– 500 ml)。 6孔板– 2.4毫升)。
除去96%的乙醇,在室温下孵育直至孔干燥。
用茜素红染色覆盖孔,在室温下孵育10分钟。
用去离子水洗涤一次,并在室温下孵育直至孔干燥。
拍摄孔的宏观(图7)和微观照片。
注意:通常,与我们之前观察到的相比,与AT-MSC相比,BM-MSC更致力于成骨细胞分化(Abuna 等,2016)。


 


D:\ Reformatting \ 2020-1-6 \ 1902730--1292 Adalberto Rosa 753853 \ Figs jpg \图7.jpg


图7. 在聚苯乙烯培养皿中在成骨性培养基中培养21天后,在BM-MSC和AT-MSC培养物中通过茜素红染色检测到的矿化细胞外基质


 


软骨母细胞分化
当BM-MSC和AT-MSC培养物达到70%融合时,请去除生长培养基。
用1x PBS洗涤烧瓶3次。
将5 ml胰蛋白酶溶液加入烧瓶中,并在37°C 下孵育5分钟。
向烧瓶中加入2.5 ml新鲜生长培养基。
将此细胞悬液转移至50 ml锥形管中,并以600 xg 离心5分钟。
丢弃上清液。
将细胞重悬于软骨母细胞分化培养基中,密度为1.25 x 10 6 细胞/ ml。
用移液器将200 µl细胞悬液等分试样(2.5 x 10 5个细胞)分配到聚丙烯96孔板的每个孔中。
将板以500 xg离心5分钟。
加入200 微升1×PBS中的入空孔,以最小化培养基的蒸发。
在实验过程中,将板在培养箱中于37°C,含有5%CO 2 和95%空气的潮湿气氛中孵育。
温育24小时后,可见细胞聚集体,并且在培养30天后观察到软骨形成表型。
每两天更换一次培养基,方法是使用200μl 无菌移液管小心吸出过期的培养基,并向每个孔中添加200μl 新鲜的软骨形成培养基。
 


三色染色
为了确认软骨母细胞的分化,我们使用的方法之一是用三色染色法检测胶原纤维。


使用微量移液器,去除软骨细胞分化培养基。
加入200 微升1×PBS中的到每个孔中。
使用微量移液器除去PBS。
在室温下添加200μl4 %多聚甲醛5分钟。
除去多聚甲醛。
用1x PBS清洗每个孔,每次2次,每次3分钟。
在室温下用曙红对细胞聚集体染色5分钟。
除去曙红,用1x PBS洗涤2次,每次3分钟。
使用1 ,000微升微量,收获所述聚集体,并将它们传送到1.5毫升的微管。
将细胞聚集体放入1.5 ml微量管中,以300 µl梯度乙醇系列(分别为70 %,80 %,90 %,95 %和100 %,每5分钟)脱水。
除去100 %乙醇,并在300 µl二甲苯中进行三个澄清步骤,每个步骤3分钟。
将石蜡在热表面上将聚集体嵌入模具中5分钟,然后将每个模具转移到冷表面上。
使用切片机切割相邻的5μm 切片。
在60°C的培养箱中将石蜡切片脱蜡过夜。
分三步在二甲苯中对石蜡进行脱蜡,每次5分钟,然后通过在乙醇系列(100 %,95 %,90 %,80 %和70 %,每次3分钟)中孵育使切片重新水化。
用去离子水清洗切片5分钟。
在室温下用酸性品红(HT15-1)染色5分钟。
用水洗涤5分钟。
在室温下用磷钼酸(HT15-3)和磷钨酸(HT15-2)的工作溶液染色5分钟。
在室温下用苯胺蓝溶液将切片染色5分钟。
除去过量的苯胺蓝,并加入1%的乙酸溶液3分钟。
用自来水冲洗切片3分钟。
以梯度乙醇系列(分别为70 %,80 %,90 %,95 %和100 %,每次1分钟)脱水切片,然后在二甲苯中进行三个澄清步骤,每次1分钟。
安装使用幻灯片ERV -mount ® 。
拍摄组织切片的显微照片(图8)。
注意:细胞质染成红色,胶原纤维染成蓝色。


 


D:\ Reformatting \ 2020-1-6 \ 1902730--1292 Adalberto Rosa 753853 \ Figs jpg \图9.jpg


图8.在超低簇96孔板的软骨形成培养基中培养30天的BM-MSC和AT-MSC中,通过三色染色检测的胶原纤维(蓝色)和细胞质(红色)。比例尺= 100 µm。


 


脂肪细胞分化
当BM-MSC和AT-MSC培养物达到70%融合时,请去除生长培养基。
用1 x PBS 洗涤烧瓶三遍。
将5 ml胰蛋白酶溶液加入烧瓶中,并在37°C 下孵育5分钟。
向烧瓶中加入2.5 ml新鲜生长培养基。
将此细胞悬液转移至50 ml锥形管中,并以600 xg 离心5分钟。
丢弃上清液并将细胞沉淀重悬在新鲜的生长培养基中。
计数细胞,并将其以2 x 10 4个细胞/ 孔的细胞密度接种于1 ml 脂肪形成培养基板中的24孔培养皿中,或以1 x 10 5个细胞/孔置于2 ml 脂肪形成培养基中的6孔培养皿中接种。。
在实验过程中,将板在培养箱中于37°C,含有5%CO 2 和95%空气的潮湿气氛中孵育。
每2天更换一次培养基。牛逼,在培养了十天时间观察他胞浆内脂滴。
 


油红O染色
为了确认脂肪细胞的分化,我们使用的方法之一是用油红O染色检测胞浆内脂质滴。


从每个孔中移出培养基。
加入10%福尔马林,在室温下孵育5分钟(24孔板– 500 µl,12孔板– 1 ml,6孔板– 2.4 ml)。
丢弃10%福尔马林,并加入相同体积的新鲜10%福尔马林。孵育至少1小时。
注意:在染色前,细胞可以在福尔马林中保存几天。将平膜包裹在板上,以防止细胞干燥,并用铝箔覆盖板。


使用小型移液管移出10%福尔马林。
用60%异丙醇(24孔板– 500 µl,12孔板– 1 ml,6孔板– 2.4 ml)洗涤孔。
让孔完全干燥。
加入油红色的O染色工作溶液10分钟(请勿触摸孔壁)。
去除油红色的O染色,并立即添加去离子水(重复此步骤4次)。
除去所有去离子水,在室温下孵育干燥。
使用相差显微镜对孔成像(图9)。
注意:通常,与我们之前观察到的相比,与BM-MSC相比,AT-MSC更致力于成脂分化(Abuna 等,2016)。


 


D:\ Reformatting \ 2020-1-6 \ 1902730--1292 Adalberto Rosa 753853 \ Figs jpg \图8.jpg


图9. 在聚苯乙烯培养皿中的成脂培养基中培养10天的BM-MSC和AT-MSC 中,通过油红O染色检测到的胞质内脂质滴。比例尺= 100 µm。


 


菜谱


 


运输介质
注意:刚使用前准备新鲜,并保持在37 °C下。


57毫升的阿尔法最低必需培养基(α-MEM)


3毫升庆大霉素(50微克/毫升)


720 µl两性霉素B(0.3 µg / ml)


胶原酶溶液
注意:刚使用前准备新鲜,并置于RT 。


15 mg II型胶原酶(0.075%)


20毫升1x PBS


使用20 ml注射器和0.2 µm过滤器在层流罩中将该溶液过滤到50 ml锥形管中。


胰蛋白酶溶液
注意:刚使用前准备新鲜,并置于RT 。


19毫升胰蛋白酶(0.25%)


500 µl II型胶原酶(1.3 mg / ml)


1毫升EDTA(1毫米)


抗坏血酸和β-甘油磷酸酯溶液
注意:事先准备好,在4 °C下保存最多7天。


5 mg抗坏血酸(5 µg / ml)


2.16克β-甘油磷酸酯(7毫米)


10毫升去离子水


使用20 ml注射器和0.2 µm过滤器在层流罩中将该溶液过滤到50 ml锥形管中。


生长培养基(10%MEM)
注意:事先准备好,在4 °C下保存30天。


360毫升的α-MEM


40毫升胎牛血清


2毫升庆大霉素(50微克/毫升)


500 µl两性霉素B(0.3 µg / ml)


成骨分化培养基
注意:事先准备好,在4°C下保存长达30天。


400 ml生长培养基


4毫升地塞米松(10 - 7 M)


1%抗坏血酸和β-甘油磷酸酯溶液


注意:在使用培养基之前,应立即添加抗坏血酸和β-甘油磷酸溶液。


软骨分化培养基
注意:事先准备好,在4 °C下保存30天。


100毫升D-MEM


100 µl丙酮酸钠(100 mM)


100 µl地塞米松(1 mM)


250 µl抗坏血酸(20 mM)


1 ml人白蛋白(0.02%)


20 µl / ml转化生长因子β3(TGF-β3,1 µg / ml)。


注意:在使用培养基之前,应立即添加TGF-β3。


脂肪细胞分化培养基
注意:事先准备好,在4 °C下保存30天。


180毫升D-MEM


20毫升胎牛血清(10%)


2毫升庆大霉素(50微克/毫升)


250 µl两性霉素B(0.3 µg / ml)


2毫升地塞米松(10 - 6 M)


2毫升3 - 异丁基-1-甲基黄嘌呤(0.5毫摩尔)


260毫升吲哚美辛(0.1 M)


150微升胰岛素(10毫克/毫升)


地塞米松原液(200 µM)
注意:预先制备,保持在- 20 ℃。


地塞米松以200 µM的浓度溶于绝对的乙醇和去离子水中


抗坏血酸原液(20 mM)
注意:事先准备好,在4 °C下保存最多7天。


在1x PBS中制备20 mM抗坏血酸的储备溶液


转化生长因子β3
注意:刚使用前准备新鲜,并置于RT 。


A 1 微克/ ml储备TGF-β3的溶液在1×PBS和0.5%人白蛋白制备。


油红O染色
油红O原液


注意:事先准备好,在室温下保存长达6个月。


将油红色O(700毫克)添加到200毫升异丙醇中,搅拌过夜,然后通过0.2微米过滤器。


油红O工作液


将6份油红色O储备溶液与4份去离子水混合,并在室温下孵育20分钟。    


 


致谢


 


这项研究由圣保罗州研究基金会(FAPESP,巴西,#2017 / 12622-7),国家科学技术发展委员会(CNPq ,巴西,#305523 / 2013-9和404318 / 2016-9)资助,以及提高高等教育人员的协调能力(CAPES,巴西)。由FAPESP(#2018 / 17356-6)资助的ENAGO (www.enago.com)进行了英语审查,该协议是根据这些工作改编的(Maniatopoulos 等,1988; Huang 等,2002; Penick 等,2005)。


 


 


利益争夺


 


作者宣称没有利益冲突。


 


伦理


 


所执行的所有程序均根据国际,国家和/或机构动物保健指南的道德标准执行。圣保罗大学RibeirãoPreto 牙科学院动物研究伦理委员会(#2018.1.30.58.8)审查并批准了我们在此所做的所有动物程序。


 


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引用:Freitas, G. P., Souza, A. T. P., Lopes, H. B., Trevisan, R. L. B., Oliveira, F. S., Fernandes, R. R., Ferreira, F. U., Ros, F. A., Beloti, M. M. and Rosa, A. L. (2020). Mesenchymal Stromal Cells Derived from Bone Marrow and Adipose Tissue: Isolation, Culture, Characterization and Differentiation. Bio-protocol 10(4): e3534. DOI: 10.21769/BioProtoc.3534.
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Yuko Kaku
Nippi Research Institute of Biomatrix
I'd like to confirm concentration of indomethacin and insulin in 'Adipocyte differentiation medium'.
Is it final concentration of these components given in parentheses in 'Recipes' No. 8 ?
2020/7/8 0:50:59 回复
Gileade Freitas
Oral & Maxillofacial Surgery, School of Dentistry of Ribeirao Preto - University of Sao Paulo, 2019-

Dear colleagues,

This is the correct recipe:

Adipocyte differentiation medium
180 ml of D-MEM
20 ml of fetal calf serum (final concentration = 10%)
2 ml of gentamycin (final concentration = 50 μg / ml)
250 μl of amphotericin B (final concentration = 0.3 μg / ml)
2 ml of dexamethasone (final concentration = 10E-6 M )
2 ml of 3-isobutyl-1-methylxanthine (final concentration = 0.5 mM)
260 μl of indomethacin (final concentration = 60 µM)
150 μl of insulin (final concentration = 10 µg / ml)

We apologize for the mistake and the inconvenience.
Best regards.

2020/7/10 17:25:25 回复


Yuko Kaku
Nippi Research Institute of Biomatrix

Dear Dr. Freitas,

Thank you for taking the time to reply.
As it's first time for me to isolate and differentiate MSC, your article was very helpful.

Best Regards.

2020/7/14 17:46:27 回复