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Isolation and Establishment of Mesenchymal Stem Cells from Wharton’s Jelly of Human Umbilical Cord
从人脐带华通氏胶中分离及建立间充质干细胞   

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Stem Cells International
Feb 2017

Abstract

Mesenchymal stem cells (MSCs) are currently considered as ‘medicinal signaling cells’ and a promising resource in regard to cell-based regenerative therapy. Umbilical cord is a human term perinatal tissue which is easily attainable, and a promising source of stem cells with no associated ethical concerns. MSCs have been isolated from different regions of the umbilical cord and Wharton’s jelly (WJ) is the gelatinous matrix that surrounds and provides protection to the umbilical cord blood vessels. Being more primitive, MSCs from human umbilical cord exhibit greater proliferative capacity and immunosuppressive ability as compared to adult stem cells which gives them a therapeutic advantage. To meet the requirements for cell therapy, it is important to generate MSCs at a clinical scale by following steps which are not time consuming or labor intensive. Here we present a simple, efficient protocol for isolation of MSCs from WJ of human umbilical cord by explant culture method which is reproducible and also, cost effective.

Keywords: Umbilical cord (脐带), Wharton’s jelly (华通氏胶), Mesenchymal stem cells (间充质干细胞), Isolation (分离), Explant (外植体), Cell culture (细胞培养)

Background

Mesenchymal stem cells (MSCs) have a remarkable clinical potential to treat a wide range of debilitating diseases, mainly due to their unique immunomodulatory role and regenerative capacity (Caplan and Sorrell, 2015). They exist in many tissues (Hass et al., 2011) and have been observed to be perivascular in vivo (Caplan and Correa, 2011). The niche of the source or the source itself, could lead to important functional differences between the various MSC types (Kwon et al., 2016). Though bone marrow is the most well studied and best characterized source of MSCs, there are certain limitations associated with it (Liu et al., 2016). An appealing and convenient alternative choice of MSC source is the fetus-derived umbilical cord, which is discarded after birth and provides an easily accessible and non-controversial source of stem cells for therapy (El Omar et al., 2014). Umbilical cord MSC-based trials are still at early phase, though no cell rejection, tumor formation or long-term adverse effects have been reported from them (Zhang et al., 2017). Moreover, they have been successfully used in experimental animal disease models. For convincingly establishing safety and therapeutic efficacy of umbilical cord-MSCs, followed by their use in clinical applications, a vast number of MSCs need to be generated for transplantations (Bartmann et al., 2007).

MSCs have been isolated from different compartments of the umbilical cord and Wharton’s jelly (WJ), is the connective tissue surrounding the umbilical cord vessels (Troyer and Weiss, 2008). Being a primitive stromal cell population, WJ-MSCs offer the advantage of faster proliferation rate and reduced immunogenicity as compared to adult tissue derived MSCs (Liu et al., 2016). Hence, successful isolation of robustly proliferating healthy MSCs from WJ of human umbilical cord, which retain all the basic MSC properties, assumes importance. Here we describe step-by-step an explant method of isolation procedure followed by establishment of MSC culture from WJ of human umbilical cord. This method of isolation eliminates the use of any enzymatic treatment making it more economical, and getting rid of batch-to-batch variations and endotoxin contaminations likely to be associated with enzymatic preparations. The isolated WJ-MSCs expressed MSC-characteristic surface antigens and were positive for the expression of CD73, CD90 and CD105 and negative for CD34.

Materials and Reagents

  1. 15 and 50 ml centrifuge tubes (Corning, catalog numbers: 430791 and 430829 respectively)
  2. 1.5 ml microcentrifuge tube (Corning, Axygen®, catalog number: MCT-150-C )
  3. 90 mm Petri dish sterile (Tarsons, catalog number: 460090 )
  4. 35 mm cell culture dish (Corning, Falcon®, catalog number: 353001 )
  5. 1 ml blunt-end pipette tips
  6. Corning® 1.2 ml External Threaded Polypropylene Cryogenic Vial, Self-Standing with Conical Bottom (Corning, catalog number: 430658 )
  7. FACS tubes (Corning, Falcon®, catalog number: 352054 )
  8. Millex-GP Syringe Filter Unit, 0.22 µm, polyether sulfone, 33 mm, gamma sterilized (Merck, catalog number: SLGP033RS )
  9. 20 ml and 5 ml syringes*
  10. Sterile Scalpel blade No. 20 (HiMedia Laboratories, catalog number: LA771 )
  11. 0.5-10 µl microtips (Corning, Axygen®, catalog number: T-300 )
  12. 20-200 µl pipette tips (Corning, Axygen®, catalog number: T-200-C )
  13. 100-1000 µl pipette tips (Corning, Axygen®, catalog number: T-1000-C )
  14. Biohazards disposable waste bags*
  15. Plastic beaker*
  16. 0.9% w/v saline*
  17. Antibiotic-Antimycotic (100x) (Thermo Fisher Scientific, GibcoTM, catalog number: 15240096 )
  18. Isopropanol*
  19. Autoclaved distilled water
  20. Absolute ethanol (Merck, catalog number: 1.00983.0511 )
  21. Sodium hypochlorite solution (Merck, catalog number: 1.93607.1021 )
  22. Dulbecco’s phosphate-buffered saline (DPBS), No calcium, no magnesium (Thermo Fisher Scientific, GibcoTM, catalog number: 14190144 )
  23. TrypLETM Express Enzyme (1x), no phenol red (Thermo Fisher Scientific, GibcoTM, catalog number: 12604013 )
  24. Trypan blue solution 0.4% (Sigma-Aldrich, catalog number: T8154 )
  25. Flow cytometry antibodies (Table 1)

    Table 1. Flow cytometry antibodies
    S. No.
    Antigen (CD marker)
    Antibody
    Manufacturer
    Dilution
    1.
    CD73
    Anti-Human CD73 PE
    BD, BD PharmingenTM,
    catalog number: 550257  
    1.5 µl in 50 µl of cell suspension
    2.
    CD90
    Anti-Human CD90 PE 
    BD, BD PharmingenTM,
    catalog number: 555596
    1.5 µl in 50 µl of cell suspension
    3.
    CD105
    Anti-Human CD105-PE
    R&D Systems,
    catalog number: FAB10971P  
    1 µl in 50 µl of cell suspension
    4.
    CD34
    Anti-Human CD34 
    BD, BD PharmingenTM,
    catalog number: 550761  
    1 µl in 50 µl of cell suspension
    S. No.

    Isotype antibody 
    Manufacturer
    Dilution
    1.

    IgG1, kappa 
    BD, BD PharmingenTM,
    catalog number: 550617
    1µl of 1:16 dilution in 50 µl of cell suspension (for CD73 and CD34) 1.5 µl in 50 µl cell suspension for CD90
    2.

    IgG1
    R&D Systems,
    catalog number: IC002P  
    1µl of 1:16 dilution in 50 µl cell suspension

  26. KnockOutTM D-MEM high glucose no glutamine (Thermo Fisher Scientific, GibcoTM, catalog number: 10829018 )
    Note: DMEM-F12 or DMEM, high glucose could also be used in place of KnockOutTM D-MEM (Nekanti et al., 2010).
  27. L-Glutamine (200 mM) (Thermo Fisher Scientific, GibcoTM, catalog number: 25030149 )
  28. Fetal bovine serum (FBS) mesenchymal stem cell qualified (Thermo Fisher Scientific, GibcoTM, catalog number: 12662029 )
    Note: The source, grade and lot number of FBS play a critical role in MSC culture establishment.
  29. Penicillin-Streptomycin (10,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140148 )
  30. Phosphate buffered saline tablets (Sigma-Aldrich, catalog number: P4417 )
  31. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog numbers: D2650 )
  32. Sheath fluid ( BD Biosciences, BD FACS FlowTM, catalog number: 342003 )
  33. MSC isolation media (see Recipes)
  34. MSC growth media (see Recipes)
  35. 1x sterile phosphate buffer saline, pH 7.4 (see Recipes)
  36. MSC freezing mixture (see Recipes)

*Note: This item can be ordered from any standard company.

Equipment

  1. Laminar flow hood* (EuroClone, model: S@feflowTM 0.9 , catalog number: LD80000)
  2. Surgical tools including scalpel holder, forceps (large and small), pointed scissors
  3. Incubator (BINDER, model: C 150 CO2 Incubator, catalog number: 9040-0078 )
  4. Centrifuge 5810 R (Eppendorf, model: 5810 R , catalog number: 5811000320)
  5. Haemocytometer depth 0.1 mm* (Rohem Instruments Private limited, http://www.sciencecorner.co.in/Rohem.php)
  6. Mr. FrostyTM Freezing Container (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 5100-0001 )
  7. -20 °C freezer* (Celfrost, model: BSF 150 )
  8. -86 °C Upright Ultra-Low Temperature Freezer (Revco value, Thermo Fisher Scientific, Thermo ScientificTM, model: ULT-1386-3 )
  9. Liquid nitrogen tank (Thermo Fisher Scientific, Thermo ScientificTM, model: Model 8031 )
  10. Flow cytometer (BD Biosciences, BD FACSCaliburTM)
  11. Inverted microscope* (Nikon Instruments, model: Eclipse TS100 )
  12. Refrigerator* (SAMSUNG, model: RT31 , 2007)
  13. 2-20 μl single channel variable pipette* (Eppendorf, model: Research® plus , catalog number: 3120000038)
  14. 20-200 μl single channel variable pipette* (Eppendorf, model: Research® plus , catalog number: 3120000054)
  15. 100-1,000 μl single channel variable pipette* (Eppendorf, model: Research® plus , catalog number: 3120000062)
  16. Autoclave*(Tuttnauer, model: 3850 ML )

*Note: This item can be ordered from any standard company.

Software

  1. BD Cell Quest pro

Procedure

  1. Isolation of WJ-MSCs
    To isolate mesenchymal stem cells from Wharton’s jelly of human umbilical cord, use the explants culture method.
    1. Collect fresh human umbilical cord after full-term birth (normal or cesarean) with informed consent (Figure 1) and transport it to the lab in an empty sterile 50 ml centrifuge tube on ice.


      Figure 1. Fresh umbilical cord after collection

    2. Rinse off the blood and blood clots using normal saline. Cut the cord into 2-3 cm pieces (Figure 2) and incubate the pieces in normal saline containing 1x antibiotic-antimycotic (100 U/ml of penicillin, 100 μg/ml of streptomycin and 250 ng/ml of amphotericin B) at 4 °C for about 2 h.
      Note: This incubation step in saline containing antibiotic-antimycotic is carried out mainly to disinfect the umbilical cord tissue.


      Figure 2. The umbilical cord cut into 2-3 cm long pieces

    3. After 2 h, inside the laminar flow tissue culture hood, rinse the cord pieces three times with sterile PBS (Video 1).

      Video 1. Rinsing umbilical cord pieces with 70% ethanol and PBS

    4. Next disinfect the cord pieces briefly by rinsing with 70% ethanol for 30 sec. Wash thoroughly three more times with PBS to remove any traces of ethanol (Video 1).
    5. Transfer one cord piece into an empty 10 cm cell culture dish, while the tube containing the other pieces can be stored on ice. Using forceps try to straighten the twists, if any, in the cord piece and using a scalpel fixed to a scalpel holder make a longitudinal slit along the length (Video 2).

      Video 2. Removal of artery from umbilical cord piece

    6. There are two arteries and one vein inside the cord. Cut open flaps of tissue to expose an artery and then trace the artery using scissors and forceps and remove it. The underlying perivascular Wharton’s jelly tissue can now be neatly excised (Video 2).
    7. Chop the excised tissue into smaller 3-5 mm pieces with the help of the scalpel and place in an empty 35 mm tissue culture dish using a fine forceps (Video 3).

      Video 3. Plating small explants of WJ portion of human umbilical cord

    8. Allow the cord pieces to attach for about 5 min with air drying. Next add 2 ml of warm fresh MSC isolation medium carefully, drop-wise and gently, taking care not to dislodge the tissue pieces (Video 3).
    9. Place about 10-15 explants in each 35 mm tissue culture dish and use about four to five 35 mm tissue culture dishes per umbilical cord sample.
    10. Place the tissue culture dishes containing the cord explants in a CO2 incubator maintaining 37 °C and 5% CO2.
      Note: Discard the remaining tissue pieces in a biohazard waste bag. All the liquid waste should be collected in a beaker containing sodium hypochlorite solution and discarded appropriately.
    11. After 48 h, give the first medium change with 2 ml of warm fresh MSC isolation medium. Following this, media changes are given with 2 ml of fresh MSC isolation medium every 72 h.
    12. After about 7-10 days, once enough cells have come out from the explants (Figure 3), remove the cord pieces using 1 ml blunt-end tips and add 2 ml of warm fresh MSC isolation medium.


      Figure 3. Phase contrast image of MSCs emerging from an umbilical cord tissue piece explant

  2. Establishment of MSC culture
    1. After 24-48 h of cord piece removal and depending on confluency, carefully aspirate medium and wash cells twice with DPBS.
      Note: As the cells are growing out in patches from the explants at this stage, within each patch cells should be ~50-80% confluent.
    2. Add 250 µl of TrypLE to each 35 mm tissue culture dish and let the cells detach for 4-6 min at 37 °C.
      Note: Trypsin can be used as a substitute for TrypLE. TrypLE is an animal origin-free recombinant enzyme which is gentle on stem cells, thus preserving well cell’s surface proteins.
    3. Add 1 ml of warm fresh MSC growth medium to the cells and transfer them to a 15 ml centrifuge tube and centrifuge at room temperature for 2 min at 500 x g.
    4. Aspirate medium, add 500 µl of warm fresh MSC growth medium to the cell pellet and suspend the pellet. Mix a small aliquot of cell suspension with an equal volume of trypan blue, and count the live (non-blue) cells using a hemocytometer.
    5. As a next step, seed WJ-MSCs at a density of 5,000 cells/cm2 in 2 ml of warm fresh MSC growth medium in a 35 mm tissue culture dish or any appropriate cell culture vessel.
    6. Culture them for 72 h or till cells are 70-80% confluent. No medium change required in between.
    7. Detach cells with TrypLE, count and seed for the next passage, repeating Steps B2-B6.
    Note: TrypLE, MSC growth medium and DPBS need to be pre-warmed to 37 °C before start of trypsinization or detachment of cells.

  1. Freezing and reviving of WJ-MSCs
    1. Once the WJ-MSCs are 70-80% confluent, detach with 250 µl TrypLE as per Step B2.
    2. Add 1 ml of warm fresh MSC growth medium to the cells and transfer them to a 15 ml centrifuge tube. Take an aliquot for counting and centrifuge the remaining cells at 500 x g for 2 min at room temperature.
    3. Aspirate supernatant and suspend the cells directly in freezing medium at ~2 x 106 cells/ml.
    4. Aliquot 150-200 µl cell suspension into cryogenic vials and transfer the cryogenic vials to a Mr. FrostyTM freezing container containing isopropanol.
    5. Place the Mr. FrostyTM freezing container at -80 °C freezer for ~24 h before transferring the cryogenic vials to a liquid nitrogen tank for long-term storage.
    6. For revival, remove a cryogenic vial with frozen WJ-MSCs from the liquid nitrogen tank and immediately transfer it to a beaker containing water at 37 °C.
    7. Once the cell suspension has thawed, inside the laminar flow hood add 1 ml of warm fresh MSC growth medium to the cell suspension in the cryogenic vial and transfer the contents to a 15 ml centrifuge tube containing 3-4 ml of additional warm fresh MSC growth medium.
    8. Centrifuge the cells at room temperature at 500 x g for 2-3 min to wash off the DMSO.
    9. Aspirate the supernatant and resuspend the cell pellet in 0.5 ml of fresh warm MSC growth medium. Mix well by pipetting, count the live cells and plate up to 2-3 x 105 cells on a 35 mm tissue culture plate in 2 ml of warm fresh MSC growth medium.
      Note: During thawing and revival of MSCs, cells are plated at a higher density in order to maximize recovery.
    10. Transfer the dish to a CO2 incubator and replace medium with 2 ml of warm fresh MSC growth medium the next day.
    11. After 48 h of reviving and plating, detach cells with TrypLE and seed for the next passage.

  2. Immunophenotyping by flow cytometry
    1. Take WJ-MSCs at passage 4-5. Detach them with TrypLE and count the cell number as per Steps B2-B4.
    2. Wash the harvested cells 1-2 times with ice cold PBS by centrifuging at 600 x g for 2 min.
    3. Discard the supernatant and suspend cells in PBS at 2 x 106 cells/ml.
    4. Distribute 1 x 105 cells or 50 µl of cell suspension in each of the pre-labeled 5 ml FACS tubes.
    5. Add the PE or FITC labeled antibodies as per Table 1 and incubate on ice for 45-60 min. Mouse isotype antibodies can serve as controls (Figure 4).


      Figure 4. Immunophenotyping of WJ-MSCs as demonstrated by flow cytometry analysis. Open histograms represent background signal while shaded histograms indicate positive reactivity with the indicated antibodies. WJ-MSCs are positive for CD73 (A), CD90 (B) and CD 105 (C), and negative for CD34 (D).

Data analysis

WJ-MSCs, isolated from human umbilical cord, are analysed for surface marker expression profile at passage 4-6. At least 10,000 events are acquired on BD FACSCalibur flow cytometer and results are analyzed using BD Cell Quest Pro software. Cultured MSCs are known to be strongly positive for the expression of CD73, CD90 and CD105 and negative for the expression of CD34, CD45, HLADR etc. All analyses are standardized against control cells incubated with PE-conjugated mouse IgG isotype antibody. Representative data from one WJ-MSC culture at passage 5 is presented in this protocol. However, similar results have been obtained from multiple independent WJ-MSC cultures (Himal et al., 2017).

Notes

  1. For MSC isolations, cords are usually collected from healthy donors.
  2. After collection, umbilical cords should be transported to the laboratory and processed as soon as possible, preferably within one hour.
  3. Depending on cord sample, WJ-MSCs might take 6-14 days to come out from the cord pieces.
  4. Pre-warm medium to 37 °C and add only the warm medium to the cells.
  5. All plasticwares like microcentrifuge tubes and pipet tips, and surgical instruments were sterilized by autoclaving before use in cell culture.
  6. Cell concentration can be calculated using a hemocytometer as, the total number of live (non-blue) cells in the four corner squares/4 x dilution factor x 10,000 = No. cells/ml.

Recipes

  1. MSC isolation medium
    DMEM KO
    2 mM glutamine
    1x antibiotic-antimycotic
    10% MSC FBS
    Sterilize by filtration through a 0.22 μm syringe filter
    Store at 4 °C, use within 2 weeks
  2. MSC growth medium
    DMEM KO
    2 mM glutamine
    1x penicillin-streptomycin
    10% FBS
    Sterilize by filtration through a 0.22 μm syringe filter
    Store at 4 °C, use within 2 weeks
  3. 1x sterile phosphate buffered saline (PBS), pH 7.4
    Dissolve two tablets of PBS in 400 ml of distilled water
    Sterilize by autoclaving
    Store at 4 °C. Use within 1 month
  4. MSC freezing mixture
    90% FBS
    10% DMSO
    Sterilize by filtration through a 0.22 μm syringe filter
    Store at 4 °C. Use within 2 weeks

Acknowledgments

This work has been financially supported by IISER-Kolkata and SERB, DST, India. We thank CSIR, India for the fellowship of Mr. Umesh Goyal. We thank Mr. Pritam Saha and Mr. Tamal Ghosh for their assistance with cell culture facility maintenance and flow cytometry data analysis, respectively. And we are thankful to Dr. Jayanta Chatterjee, Aastha, Kalyani, for generously providing us with human umbilical cord samples. This protocol has been adapted from our previous protocol (Venugopal et al., 2011). The authors declare no potential conflicts of interest.

References

  1. Bartmann, C., Rohde, E., Schallmoser, K., Purstner, P., Lanzer, G., Linkesch, W. and Strunk, D. (2007). Two steps to functional mesenchymal stromal cells for clinical application. Transfusion 47(8): 1426-1435.
  2. Caplan, A. I. and Correa, D. (2011). The MSC: an injury drugstore. Cell Stem Cell 9(1): 11-15.
  3. Caplan, A. I. and Sorrell, J. M. (2015). The MSC curtain that stops the immune system. Immunol Lett 168(2): 136-139.
  4. El Omar, R., Beroud, J., Stoltz, J. F., Menu, P., Velot, E. and Decot, V. (2014). Umbilical cord mesenchymal stem cells: the new gold standard for mesenchymal stem cell-based therapies? Tissue Eng Part B Rev 20(5): 523-544.
  5. Hass, R., Kasper, C., Bohm, S. and Jacobs, R. (2011). Different populations and sources of human mesenchymal stem cells (MSC): A comparison of adult and neonatal tissue-derived MSC. Cell Commun Signal 9: 12.
  6. Himal I., Goyal, U. and Ta, M. (2017). Evaluating wharton’s jelly-derived mesenchymal stem cell’s survival, migration, and expression of wound repair markers under conditions of ischemia-like stress. Stem Cells Int 2017: 5259849.
  7. Kwon, A., Kim, Y., Kim, M., Kim, J., Choi, H., Jekarl, D. W., Lee, S., Kim, J. M., Shin, J. C. and Park, I. Y. (2016). Tissue-specific differentiation potency of mesenchymal stromal cells from perinatal tissues. Sci Rep 6: 23544.
  8. Liu, C. B., Huang, H., Sun, P., Ma, S. Z., Liu, A. H., Xue, J., Fu, J. H., Liang, Y. Q., Liu, B., Wu, D. Y., Lu, S. H. and Zhang, X. Z. (2016). Human umbilical cord-derived mesenchymal stromal cells improve left ventricular function, perfusion, and remodeling in a porcine model of chronic myocardial ischemia. Stem Cells Transl Med 5(8): 1004-1013.
  9. Nekanti, U., Rao, V. B., Bahirvani, A. G., Jan, M., Totey, S. and Ta, M. (2010). Long-term expansion and pluripotent marker array analysis of Wharton's jelly-derived mesenchymal stem cells. Stem Cells Dev 19(1): 117-130.
  10. Troyer, D. L. and Weiss, M. L. (2008). Wharton’s jelly-derived cells are a primitive stromal cell population. Stem Cells 26(3): 591-599.
  11. Venugopal, P., Balasubramanian, S., Majumdar, A. S. and Ta, M. (2011). Isolation, characterization, and gene expression analysis of Wharton's jelly-derived mesenchymal stem cells under xeno-free culture conditions. Stem Cells Cloning 4: 39-50.
  12. Zhang, Y. C., Liu, W., Fu, B. S., Wang, G. Y., Li, H. B., Yi, H. M., Jiang, N., Wang, G., Zhang, J., Yi, S. H., Li, H., Zhang, Q., Yang, Y. and Chen, G. H. (2017). Therapeutic potentials of umbilical cord-derived mesenchymal stromal cells for ischemic-type biliary lesions following liver transplantation. Cytotherapy 19(2): 194-199.

简介

间充质干细胞(mesenchymal stem cells,MSCs)目前被认为是“医药信号传导细胞”,在基于细胞的再生治疗方面是一种很有前景的资源。脐带是人类的围产期组织,很容易实现,是一种有前途的干细胞来源,没有相关的伦理问题。 MSC已经从脐带的不同区域分离出来,而Wharton's果冻(WJ)是包围并提供对脐带血管的保护的凝胶状基质。更原始的是,与成人干细胞相比,来自人脐带的MSC表现出更大的增殖能力和免疫抑制能力,这使其具有治疗优势。为了满足细胞疗法的要求,通过遵循不耗时或劳动强度的步骤来产生临床规模的MSC是重要的。在此我们提出了一种简单,高效的方法,通过外植体培养方法从人脐带WJ中分离出MSC,这种方法具有重现性和成本效益。

【背景】间充质干细胞(MSC)具有显着的临床潜力来治疗各种衰弱性疾病,主要是由于其独特的免疫调节作用和再生能力(Caplan and Sorrell,2015)。它们存在于许多组织中(Hass et al。,2011),并被观察到是血管周围的体内(Caplan和Correa,2011)。来源或来源本身的小生境可能导致各种MSC类型之间的重要功能差异(Kwon等人,2016年)。虽然骨髓是研究得最充分和最好的MSCs来源,但也有一定的局限性(Liu et al。,2016)。 MSC源的吸引人和方便的替代选择是胎儿衍生的脐带,其在出生后被丢弃,并为治疗提供了容易获得的且无争议的干细胞来源(El Omar等人 ,2014年)。脐带MSC为基础的试验仍处于早期阶段,虽然没有细胞排斥反应,肿瘤形成或长期不良反应的报道(Zhang等人,2017)。而且,它们已经成功用于实验动物疾病模型。为了令人信服地建立脐带MSC的安全性和治疗效果,然后将其用于临床应用,需要为移植产生大量的MSC(Bartmann等人,2007)。

MSC从脐带的不同隔室中分离出来,而Wharton's果冻(WJ)是围绕脐带血管的结缔组织(Troyer和Weiss,2008)。作为原始基质细胞群体,与成人组织来源的MSC相比,WJ-MSC提供了更快的增殖速率和降低的免疫原性的优势(Liu等人,2016)。因此,从保持所有MSC基本性质的人脐带WJ成功分离出健壮增殖的健康MSC是重要的。这里我们一步一步地描述分离过程的外植体方法,然后从人脐带WJ建立MSC培养物。这种分离方法消除了任何酶处理的使用,使其更经济,并消除了可能与酶制剂相关的批次间变化和内毒素污染。分离的WJ-MSC表达MSC特征性表面抗原,CD73,CD90和CD105表达阳性,CD34阴性。

关键字:脐带, 华通氏胶, 间充质干细胞, 分离, 外植体, 细胞培养

材料和试剂

  1. 15和50毫升离心管(康宁公司,产品目录号分别为430791和430829)
  2. 1.5ml微量离心管(Corning,Axygen,目录号:MCT-150-C)
  3. 90毫米无菌培养皿(Tarsons,目录号:460090)
  4. 35mm细胞培养皿(Corning,Falcon ,产品目录号:353001)。
  5. 1毫升钝头移液器尖端
  6. 康宁 1.2 ml外螺纹聚丙烯低温瓶,锥形底部自立式(Corning,目录号:430658)
  7. FACS管(Corning,Falcon ,产品目录号:352054)
  8. Millex-GP注射式过滤器,0.22微米,聚醚砜,33毫米,γ消毒(Merck,目录号:SLGP033RS)
  9. 20毫升和5毫升注射器*
  10. 无菌手术刀第20号(HiMedia实验室,目录号:LA771)
  11. 0.5-10μl微尖端(Corning,Axygen ,目录号:T-300)
  12. 20-200μl移液枪头(Corning,Axygen ®,目录号:T-200-C)
  13. 100-1000微升移液枪头(Corning,Axygen ,产品目录号:T-1000-C)
  14. 生物危害物一次性废物袋*
  15. 塑料烧杯*
  16. 0.9%w / v盐水*
  17. 抗生素 - 抗真菌药(100x)(Thermo Fisher Scientific,Gibco TM,目录号:15240096)
  18. 异丙醇*
  19. 蒸压蒸馏水
  20. 无水乙醇(Merck,目录号:1.00983.0511)
  21. 次氯酸钠溶液(Merck,目录号:1.93607.1021)
  22. Dulbecco's磷酸盐缓冲盐水(DPBS),不含钙,不含镁(Thermo Fisher Scientific,Gibco TM,产品目录号:14190144)
  23. TrypLETM Express酶(1x),不含酚红(Thermo Fisher Scientific,Gibco,产品目录号:12604013)
  24. 台盼蓝溶液0.4%(Sigma-Aldrich,目录号:T8154)
  25. 流式细胞术抗体(表1)

    表1.流式细胞术抗体
    S. No.
    Antigen (CD marker)
    Antibody
    Manufacturer
    Dilution
    1.
    CD73
    Anti-Human CD73 PE
    BD, BD PharmingenTM,
    catalog number: 550257 
    1.5 µl in 50 µl of cell suspension
    2.
    CD90
    Anti-Human CD90 PE 
    BD, BD PharmingenTM,
    catalog number: 555596
    1.5 µl in 50 µl of cell suspension
    3.
    CD105
    Anti-Human CD105-PE
    R&D Systems,
    catalog number: FAB10971P 
    1 µl in 50 µl of cell suspension
    4.
    CD34
    Anti-Human CD34 
    BD, BD PharmingenTM,
    catalog number: 550761 
    1 µl in 50 µl of cell suspension
    S. No.

    Isotype antibody 
    Manufacturer
    Dilution
    1.

    IgG1, kappa 
    BD, BD PharmingenTM,
    catalog number: 550617
    1µl of 1:16 dilution in 50 µl of cell suspension (for CD73 and CD34) 1.5 µl in 50 µl cell suspension for CD90
    2.

    IgG1
    R&D Systems,
    catalog number: IC002P 
    1µl of 1:16 dilution in 50 µl cell suspension

  26. KnockOut TM D-MEM高葡萄糖无谷氨酰胺(Thermo Fisher Scientific,Gibco TM,产品目录号:10829018)
    注意:DMEM-F12或DMEM,高葡萄糖也可用于代替KnockOut TM D-MEM(Nekanti等人。,2010)。
  27. L-谷氨酰胺(200mM)(Thermo Fisher Scientific,Gibco TM,目录号:25030149)
  28. 合格的胎牛血清(FBS)间充质干细胞(Thermo Fisher Scientific,Gibco TM,目录号:12662029)
    注:FBS的来源,等级和批号在MSC文化建设中起着至关重要的作用。
  29. 青霉素 - 链霉素(10,000U / ml)(Thermo Fisher Scientific,Gibco TM,目录号:15140148)
  30. 磷酸盐缓冲盐水片(Sigma-Aldrich,目录号:P4417)
  31. 二甲基亚砜(DMSO)(Sigma-Aldrich,目录号:D2650)
  32. 鞘液(BD Biosciences,BD FACS Flow TM,目录号:342003)
  33. MSC隔离介质(见食谱)
  34. MSC生长介质(见食谱)
  35. 1x无菌磷酸盐缓冲盐水,pH值7.4(见食谱)
  36. MSC冷冻混合物(见食谱)

*注意:此项目可以从任何标准公司订购

设备

  1. 层流罩*(EuroClone,型号:S @ feflow 0.9,目录号:LD80000)
  2. 手术工具,包括手术刀支架,镊子(大小),尖头剪刀
  3. 培养箱(BINDER,型号:C 150 CO 2培养箱,目录号:9040-0078)
  4. 离心机5810 R(Eppendorf,型号:5810 R,目录号:5811000320)
  5. 血细胞计数器深度0.1mm *(Rohem Instruments Private limited, http://www.sciencecorner.co.in/ Rohem.php
  6. Frosty TM冷冻容器(Thermo Fisher Scientific,Thermo Scientific TM,目录号:5100-0001)
  7. -20°C冰箱*(Celfrost,型号:BSF 150)
  8. -86°C立式超低温冷冻机(Revco值,Thermo Fisher Scientific,Thermo Scientific TM,型号:ULT-1386-3)
  9. 液氮罐(Thermo Fisher Scientific,Thermo Scientific TM,型号:Model 8031)
  10. 流式细胞仪(BD Biosciences,BD FACSCalibur TM)
  11. 倒置显微镜*(尼康仪器,型号:Eclipse TS100)
  12. 冰箱*(SAMSUNG,型号:RT31,2007)
  13. 2-20μl单通道可变移液器*(Eppendorf,型号:Research ®plus,产品目录号:3120000038)
  14. 20-200μl单通道可变移液器*(Eppendorf,型号:Research ®plus,产品目录号:3120000054)
    注意:胰蛋白酶可以用作TrypLE的替代品。 TrypLE是一种无动物源的重组酶,对干细胞温和,从而保护细胞的表面蛋白。
  15. 向细胞中加入1ml温热的新鲜MSC生长培养基,并将它们转移到15ml离心管中,并在室温下以500gxg离心2分钟。
  16. 吸出培养基,加入500微升温暖新鲜的MSC生长培养基的细胞沉淀,悬浮颗粒。将一小份细胞悬液与等体积的台盼蓝混合,并用血细胞计数器计数活的(非蓝色)细胞。
  17. 作为下一步,在35mm组织培养皿或任何合适的细胞培养皿中的2ml温热的新鲜MSC生长培养基中密度为5,000个细胞/ cm 2的种子WJ-MSC。 />
  18. 培养他们72小时或直到细胞70-80%汇合。
    中间不需要更换。

  19. 使用TrypLE分离细胞,为下一个细胞计数和种子,重复步骤B2-B6。
注意:在胰蛋白酶消化或细胞分离开始之前,TrypLE,MSC生长培养基和DPBS需要预热至37°C。

  1. WJ-MSC的冻结和恢复
    1. 一旦WJ-MSC为70-80%汇合,按照步骤B2用250μlTrypLE分离。
    2. 向细胞中加入1ml新鲜的MSC生长培养基并将其转移至15ml离心管中。取等分部分进行计数,并在室温下将剩余的细胞在500×g下离心2分钟。
    3. 吸出上清液并以约2×10 6细胞/ ml直接悬浮于冷冻培养基中。
    4. 将150-200μl细胞悬液分装到低温瓶中,并将低温瓶转移到含有异丙醇的Mr. Frosty TM冷冻容器中。
    5. 将Frosty TM冷冻容器放置在-80°C冷冻箱中约24小时,然后将低温瓶转移到液氮罐中进行长期储存。
    6. 为了复苏,从液氮罐中取出冷冻WJ-MSCs的冷冻小瓶,立即转移到含有37℃水的烧杯中。
    7. 一旦细胞悬浮液融化,在层流罩内加入1ml温热的新鲜MSC生长培养基到低温小瓶中的细胞悬液中,并将内容物转移到15ml离心管中,该离心管含有3-4ml另外的温热的新鲜MSC生长中等。
    8. 将细胞在室温下500×g离心2-3分钟以洗去DMSO。
    9. 吸出上清液,并在0.5毫升新鲜温暖的MSC生长培养基重悬细胞沉淀。计数活细胞,并在2毫升温热的新鲜MSC生长培养基中的35毫米组织培养板上平板2-3×10 5个细胞。
      注意:在MSCs解冻和复苏过程中,细胞以更高的密度接种,以最大限度地恢复。
    10. 将培养皿转移到CO 2培养箱中,第二天用2ml新鲜的MSC生长培养基代替培养基。
    11. 复苏和铺板48小时后,用TrypLE和种子分离细胞用于下一次传代。

  2. 流式细胞仪免疫分型
    1. 在第4-5代采取WJ-MSC。用TrypLE分离它们并按照步骤B2-B4计算单元格编号。
    2. 使用冰冷PBS将收获的细胞洗涤1-2次,在600×g离心2分钟。
    3. 弃去上清液并以2×10 6细胞/ ml悬浮于PBS中的细胞。
    4. 在每个预先标记的5ml FACS管中分配1×10 5个细胞或50μl细胞悬液。
    5. 按照表1添加PE或FITC标记的抗体,并在冰上孵育45-60分钟。小鼠同种型抗体可以作为对照(图4)。


      图4.流式细胞术分析显示的WJ-MSC的免疫分型。打开的直方图表示背景信号,而阴影直方图表示与所示抗体的阳性反应性。 WJ-MSC对CD73(A),CD90(B)和CD105(C)呈阳性,对CD34(D)呈阴性。

数据分析

分析从人脐带分离的WJ-MSC,在第4-6代分析表面标志物表达谱。在BD FACSCalibur流式细胞仪上至少获得10,000个事件,并使用BD Cell Quest Pro软件分析结果。已知培养的MSC对CD73,CD90和CD105的表达强烈表达,对于CD34,CD45,HLADR等的表达是不利的。所有分析均针对与PE缀合的小鼠IgG同种型抗体温育的对照细胞进行标准化。本议定书提供了第5代WJ-MSC文化的代表性数据。然而,类似的结果已经从多个独立的WJ-MSC培养物中获得(Himal等人,2017)。

笔记

  1. 对于MSC隔离,绳索通常从健康的捐助者收集。
  2. 收集后,应将脐带运送到实验室并尽快处理,最好在一小时内完成。
  3. 根据绳索样品,WJ-MSC可能需要6-14天从绳索中出来。
  4. 预热到37°C的温度,并只给细胞添加温暖的媒介。
  5. 所有的塑料制品如微量离心管和吸管尖端以及手术器械在使用之前均经过高压灭菌处理,然后用于细胞培养。
  6. 可以使用血细胞计数器计算细胞浓度,因为四个角区域中的活(非蓝色)细胞总数/ 4×稀释倍数x 10,000 = No.细胞/ ml。

食谱

  1. MSC隔离介质
    DMEM KO
    2 mM谷氨酰胺
    1x抗生素 - 抗真菌剂
    10%MSC FBS
    通过0.22微米注射器过滤器过滤消毒
    在4°C储存,2周内使用
  2. MSC生长培养基
    DMEM KO
    2 mM谷氨酰胺
    1x青霉素 - 链霉素
    10%FBS
    通过0.22微米注射器过滤器过滤消毒
    在4°C储存,2周内使用
  3. 1x无菌磷酸盐缓冲盐水(PBS),pH 7.4
    将两片PBS溶解在400毫升蒸馏水中
    通过高压灭菌来消毒
    在4°C储存。在1个月内使用
  4. MSC冷冻混合物
    90%FBS
    10%DMSO
    通过0.22微米注射器过滤器过滤消毒
    在4°C储存。 2周内使用

致谢

这项工作得到了IISER-Kolkata和印度DST SERB的资助。我们感谢CSIR,印度Umesh Goyal先生的奖学金。我们感谢Pritam Saha先生和Tamal Ghosh先生分别协助细胞培养设施维护和流式细胞术数据分析。我们感谢Kalyani的Aastha的Jayanta Chatterjee博士慷慨地为我们提供人类脐带样品。该协议已根据我们以前的协议(Venugopal et al。,2011)进行了改编。作者声明不存在潜在的利益冲突。

参考

  1. Bartmann,C.,Rohde,E.,Schallmoser,K.,Purstner,P.,Lanzer,G.,Linkesch,W。和Strunk,D。(2007)。 功能性间充质基质细胞临床应用的两个步骤 输血 47(8):1426-1435。
  2. Caplan,A.I。和Correa,D。(2011)。 MSC:一家受伤的药店 干细胞 9(1):11-15。
  3. Caplan,A.I。和Sorrell,J.M。(2015)。 停止免疫系统的MSC窗帘。 Immunol Lett > 168(2):136-139。
  4. El Omar,R.,Beroud,J.,Stoltz,J. F.,Menu,P.,Velot,E.和Decot,V.(2014)。 脐带间充质干细胞:基于间充质干细胞治疗的新金标准? Tissue Eng Part B Rev 20(5):523-544。
  5. Hass,R.,Kasper,C.,Bohm,S.和Jacobs,R.(2011)。 人类间充质干细胞(MSC)的不同人群和来源:成人和新生儿组织 - 派生的MSC。通讯信号 9:12.
  6. Himal I.,Goyal,U.和Ta,M.(2017)。 评估沃顿公司果冻衍生的间充质干细胞的存活,迁移和伤口修复标志物的表达缺血性应激的条件。 干细胞内容 2017:5259849.
  7. Kwon,A.,Kim,Y.,Kim,M.,Kim,J.,Choi,H.,Jekarl,D.W.,Lee,S.,Kim,J.M.,Shin,J.C。和Park,I.Y。(2016) 来自围生期组织的间充质基质细胞的组织特异性分化潜能 Rep 6:23544.
  8. 本研究的主要内容如下:1,研究方向:动物实验,动物实验,动物实验,动物实验,动物实验,动物实验,动物实验, XZ(2016)。 人类脐带间充质基质细胞可改善猪模型的左心室功能,灌注和重塑慢性心肌缺血的干细胞研究 5(8):1004-1013。
  9. Nekanti,U.,Rao,V.B.,Bahirvani,A.G.,Jan,M.,Totey,S.and Ta,M.(2010)。 沃顿商学院果冻衍生的间充质干细胞的长期扩增和多能标记物阵列分析 干细胞开发 19(1):117-130。
  10. Troyer,D.L。和Weiss,M.L。(2008)。沃顿商学院的果冻来源的细胞是一种原始的基质细胞群体。干细胞 26(3):591-599。
  11. Venugopal,P.,Balasubramanian,S.,Majumdar,A.S。和Ta,M。(2011)。 无异种培养下沃顿果冻衍生的间充质干细胞的分离,鉴定和基因表达分析条件。干细胞克隆 4:39-50。
  12. 本刊中的类似文章文章评论(请注意:本站实行文责自负,请不要发表与学术无关的内容!评论内容不代表本站观点。) ,Q.,Yang,Y.和Chen,GH(2017)。 脐带间充质基质细胞治疗肝移植后缺血型胆道病变的治疗潜力< / a> Cytotherapy 19(2):194-199。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Goyal, U., Jaiswal, C. and TA, M. (2018). Isolation and Establishment of Mesenchymal Stem Cells from Wharton’s Jelly of Human Umbilical Cord. Bio-protocol 8(4): e2735. DOI: 10.21769/BioProtoc.2735.
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Mervat Daood
mansoura research center for cord stem cell(MARC-CSC)
Am trial this protocol,but i used the complete media in step of isolation (DMEM+penicillin-streptomycin+L glutamine +FBS) but not FBS MSC qualified ,is this true .I don't now why this protocol failed with me . I need to know what is false in my work.Please help me thanks very much.
4/15/2018 9:22:34 AM Reply
Mervat Daood
mansoura research center for cord stem cell(MARC-CSC)
what is difference between isolation MSC media & MSC growth media ,when iam work ,I use same media(complete media prepared as DMEM+penicillin strepto1%+FBS10%+1%glutamine) , I cant know whats difference.thanks
4/6/2018 5:32:35 PM Reply
Malancha TA
Department of Biological Sciences, IISER, India, India,

The isolation medium contains antibiotic-antimycotic (as fresh human tissue is being plated in this step). The MSC growth medium, which is used to culture the MSCs which have come out of WJ explants, contains pen-step. This is the only difference between these two media compositions.

4/11/2018 2:47:15 AM


Mervat Daood
mansoura research center for cord stem cell(MARC-CSC)
Am working by your protocol 4 sample by 3 petri dish and one 12 well plate but he didn't work ,please inform me some specific data to work by this protocol .thanks
4/6/2018 4:43:36 PM Reply