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Microvesicle Isolation from Rat Brain Extract Treated Human Mesenchymal Stem Cells
从大鼠脑提取物处理的人间充质干细胞中分离微泡   

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Abstract

Microvesicle (MVs) are submicron-sized membranous vesicles that are either actively released from cells via secretory compartments or shed from cell surface membranes. MVs are generated by many cell types and serve as vehicles that transfer biological information (e.g., protein, mRNA, and miRNA) to distant cells, thereby affecting their gene expression, proliferation, differentiation, and function. Although their physiological functions are not clearly defined, recent studies have shown their therapeutic potential for tissue repair and regeneration. While MVs can be isolated readily from mesenchymal stem cells (MSCs) and other cell types from various sources, the yield of MVs under conventional culture condition in vitro is one of the limiting factors for both the in vivo functional study as well as in vitro molecular analysis. Here, we provide a protocol to increase the yield of microvesicles by preconditioning MSCs with rat brain extract.

Keywords: Mesenchymal stem cell(间充质干细胞), Microvesicle(微泡), Extracellular vesicles(细胞外囊泡), Sucrose gradient(蔗糖梯度), Diafiltration(透析过滤), Tissue regeneration(组织再生)

Background

Generation of neural stem cells or neural cells by direct reprogramming or utilization of mesenchymal stem cells for cell replacement therapy is potential options for neurodegenerative diseases (Adib et al., 2015). Recent studies have demonstrated that microvesicles derived from MSCs represent a novel and safe alternative to other cell replacement approaches to enhance tissue regeneration such as neuronal regeneration, immune modulation, angiogenesis in brain injury (Kim et al., 2013; Porro et al., 2015; Lee et al., 2016). Little is known about how external signals derived from damaged tissues can affect the quantity and composition of microvesicles. The contents and quantities of such functional secretome of MSCs can be significantly changed in response to their microenvironment (Qu et al., 2007). For example, ischemic brain extracts or hypoxia are known to induce the synthesis of a number of cytokines and growth factors that are beneficial to the tissue regeneration process (Chen et al., 2007; Shin et al., 2014). In the present study, normal and ischemic brain extracts as a form of brain injury signal were employed to increase the yields as well as to modulate the molecular composition of MVs from MSCs that can be beneficial for their clinical application. Indeed, the quantity of MVs in conditioned medium of MSCs was greatly increased by the treatment of normal brain extracts or ischemic brain extracts. The current protocol was mainly based on previously described methods (Choi et al., 2007; Kim et al., 2012) with a few modifications including reagents, recipes. The yield and composition of microvesicles can be significantly modulated by preconditioning of producing cells by physical, chemical or biological means. As an example, we utilized brain extract to stimulate MSCs to simulate signal for brain tissue damage and the final products (MVs) can be a potent specific therapy for brain tissue repair and regeneration. This protocol may provide a clue to develop better strategies to obtain higher yields of MVs with stronger therapeutic potential from various cell sources.

Materials and Reagents

  1. 4-0 surgical suture
  2. 0.2 µm syringe filters (Sartorius, catalog number: 17823-K )
  3. 0.45 µm syringe filters (Sartorius, catalog number: 16555-K )
  4. Falcon tubes 50 ml (Corning, Falcon®, catalog number: 352070 )
  5. Falcon tubes 15 ml (Corning, Falcon®, catalog number: 352099 )
  6. T75 culture flasks (SPL LIFE SCIENCES, catalog number: 70075 )
  7. Polyallomer tube 38 ml (Beckman Coulter, catalog number: 344058 , for gradient formation and fractionation)
  8. Polyallomer tube 13.2 ml (Beckman Coulter, catalog number: 344059 , for gradient formation and fractionation)
  9. 8-week-old male Sprague-Dawley rat (Koatech)
  10. Human adipose tissue-derived MSCs (from 2 healthy female donors, passage 4) provided from Yonsei cell therapy center or adipose derived stem cells purchased from Lonza (Lonza, catalog number: PT-5006 )
  11. Isoflurane (Hana Pharm, Korea)
  12. 80% N2O
  13. 20% O2
  14. Ketamine
  15. Xylazine
  16. Bradford protein assay kit I (Bio-Rad Laboratories, catalog number: 5000001 )
  17. Dulbecco’s phosphate buffered saline (DPBS) (Biowest, catalog number: L0615-500 )
  18. Trypsin/EDTA (0.05%, phenol red) (Thermo Fisher Scientific, InvitrogenTM, catalog number: 25300054 )
  19. Trypan blue solution 0.4% (in normal saline) (Thermo Fisher Scientific, GibcoTM, catalog number: 15250061 )
  20. DMEM low glucose (GE Healthcare, HycloneTM, catalog number: SH30021.01 )
  21. Penicillin/streptomycin (5,000 U/ml) (GE Healthcare, HycloneTM, catalog number: SV30010 )
  22. Fetal bovine serum (FBS) (GE Healthcare, HycloneTM, catalog number: SH30071.03 )
  23. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888 )
  24. HEPES (Thermo Fisher Scientific, GibcoTM, catalog number: 15630080 )
  25. 10 mM Tris-HCl (WHITLAB, catalog number: BTH-9274 )
  26. Sucrose (Sigma-Aldrich, catalog number: S9378 )
  27. EDTA (Bio-Rad Laboratories, catalog number: 1610729 )
  28. OptiPrepTM (Alere Technologies, Axis-Shield Density Gradient Media, catalog number: 1114542 )
  29. MSC culture media (see Recipes)
  30. Sucrose dilution buffer (see Recipes)
  31. Sucrose cushion (see Recipes)
  32. OptiPrepTM solution (see Recipes)

Equipment

  1. Surgical scissors–Straight sharp/blunt 12 cm (Fine Science Tools, catalog number: 14001-12 )
  2. Narrow pattern forceps–Curved 12 cm, 2 x 1.25 mm (Fine Science Tools, catalog number: 11003-12 )
  3. Iris scissors–Large loops, angled (Fine Science Tools, catalog number: 14107-09 )
  4. Scalpel handle with scalpel (Fine Science Tools, catalog numbers: 10011-00 , 10003-12 )
  5. Bone rongeur (JEUNG DO BIO & PLANT, catalog number: H-2041-1 )
  6. Adult rat brain matrix (Kent Scientific, catalog number: RBMS-300C )
  7. Tissue grinders (WHEATON, catalog number: 357546 )
  8. Pipettes
  9. 10 ml serological pipettes (SPL LIFE SCIENCES, catalog number: 91010 )
  10. Ice bucket
  11. Centrifuge (Eppendorf, model: 5804 )
  12. Minimate TFF capsule system with a 100 kDa membrane (Pall, catalog number: OA100C12 )
  13. Glass Pasteur pipettes (Fisher Scientific, catalog number: 13-678-20A )
  14. 37 °C, 5% CO2 cell culture incubator (Eppendorf, model: Galaxy® 170 S )
  15. Inverted microscope (Olympus, model: CKX41 )
  16. Hemocytometer
  17. Ultracentrifuge (Beckman Coulter, model: OptimaTM XPN-100 )
  18. Rotor: SW41Ti (Beckman Coulter, catalog number: 331362 )
  19. Rotor: SW32Ti (Beckman Coulter, catalog number: 369650 )
  20. Peristaltic pump (Poong Lim Tech, catalog number: PP-150 )
  21. Masterflex L/S Easy-Load II head for Precision Tubing, PPS/CRS (Cole-Parmer, catalog number: EW-77200-50 )
  22. Feed reservoir (100 ml or 500 ml)

Procedure

  1. Procedures for permanent middle cerebral artery occlusion and brain extracts preparation
    1. Prepare two 8-week-old male Sprague-Dawley rats, one for normal and one for ischemic brain injury.
      Note: One rat brain is enough for 5-10 experiments of the present experimental scale.
    2. Ischemic brain injury (MCAO surgery)
      1. Anesthetize rats with 3% isoflurane (Hana Pharm, Seoul, Korea) in a mixture of 80% N2O and 20% O2.
      2. Place the rat in a face-up supine position, incise midline of neck skin and retract neck muscles gently.
      3. Carefully isolate the left common carotid artery and external carotid artery.
      4. Ligate left common carotid artery and external carotid artery with a 4-0 surgical suture temporarily.
      5. Incise left internal carotid artery slightly and insert 4-0 surgical suture into the left internal carotid artery and advance carefully to the Circle of Willis.
      6. Loose the knot that tied the external carotid artery and tie tightly left common carotid artery.
      7. Suture the neck skin.
    3. 24 h after MCAO surgery, normal rats and ischemic brain injury rats are deeply anesthetized with an intraperitoneal injection of ketamine (40 mg/kg body weight) and xylazine (5 mg/kg body weight), and then are perfused with normal saline.
    4. After the rats are perfused, use surgical scissors to remove the head with a cut posterior from the ears. Quickly make a midline incision in the head skin, aseptically remove the skull and meninges by bone rongeur.
      Note: The methods for rat sacrifice and brain tissue extraction were performed as previously described by Spijker (2011).
    5. After placing whole brain on a brain matrix, make coronal dissection the region of the middle cerebral artery (bregma -1~+ 1 mm) on the ice from the ipsilateral hemisphere, collect the sliced brain tissue (Figure 1A).
    6. Homogenize tissue pieces using tissue grinders.
    7. Centrifuge tissue homogenates at 100,000 x g for 2 h at 4 °C, and then take supernatants.
    8. Centrifuge the supernatants at 100,000 x g for 1 h at 4 °C (Figure 1B).
    9. Filter the supernatants through a 0.2 μm filter. Protein content was measured using the Bradford assay (Figure 1C). (Typical total amount of protein is 120-220 mg.)


      Figure 1. Preparation of brain extract. A. Rat brain was placed on a brain matrix, coronary dissected the region of the middle cerebral artery (bregma -1~+ 1 mm) from the ipsilateral hemisphere and the sliced brain tissue was collected. B. Brain homogenates were ultracentifuged to remove rat brain-derived microvesicles, filtered to remove residual aggregates and cellular debris. C. The supernatant was finally filtered through 0.2 μm syringe filter and aliquots were stored at -80 °C.

  2. Cell culture procedure
    1. Rapidly thaw (less than 3 min) frozen human MSC (hMSC) vials in a 37 °C water bath.
    2. Pipet entire contents of the cryovials into a sterile 50 ml conical tube and carefully add 10 volume of prewarmed complete culture medium (DMEM containing 10% fetal bovine serum).
    3. After gentle swirling of tube determine the cell concentration by hemocytometer and adjust cell concentration at 3 x 105/ml with prewarmed complete culture medium.
    4. Inoculate 75 cm2 flasks at ~8 x 103 cells/cm2 and incubate at 37 °C in a 5% CO2 incubator.
    5. Exchange the medium with fresh prewarmed complete medium 24 h post thaw.
    6. Observe culture flasks routinely on an inverted microscope.
    7. When cells are nearly confluent (over 80% confluent), aspirate medium and floating debris from a confluent monolayer and discard.
    8. Wash cells with prewarmed PBS once and add 5 ml of 0.05% trypsin-EDTA to the culture flasks. Incubate at 37 °C until 50-70% detached (approximately 2-5 min).
    9. Add 5 ml of prewarmed complete medium and gently pipet up and down to detach adherent cells and disperse cells into single cell suspension.
    10. Transfer cell suspension into sterile 50 ml conical tubes. Wash flasks with additional 5 ml complete medium and combine into the conical tube.
    11. Centrifuge cell suspension at 300 x g for 5-10 min.
    12. Aspirate supernatant and resuspend the pellet in an appropriate volume of prewarmed complete medium.
    13. Inoculate total of 50 flasks (75 cm2) with 20 ml/flask of 1 x 105 viable cells/ml for microvesicle isolation and return to incubator.
      Note: Cell cultures should be re-fed every 3 days with fresh complete medium for optimal cell growth.
    14. When cells are nearly confluent (over 80% confluent), wash the cells extensively with PBS at ambient temperature or pre-warmed at 37 °C (5 times, 10 ml per wash).
    15. After final wash, culture medium was replaced with 20 ml serum- and antibiotics-free low-glucose DMEM medium with 300 μg of brain extract or the ischemic brain extracts (final concentration of 15 μg/ml). The culture supernatant was harvested 48 h later to isolate microvesicles (MVs) (Figure 2).


      Figure 2. MSC culture. A. Microscopic image of hMSCs; B. Scale of hMSCs culture; C. Experimental schedule. The primary cultured MSCs (2 x 106 cells/flask) were seeded in 75 cm2 culture flasks (total of 50 flasks). C. After culturing in complete media, culture medium was replaced with serum-free antibiotic-free medium for 48 h, and the culture supernatant was harvested to isolate microvesicles.
      Note: MVs prepared from untreated MSCs have low protein yields (< 0.3 mg/3 x 108 cells/48 h) while the amount of MVs from MSC-conditioned medium was greatly increased by treatment with normal or ischemic brain extracts (2.2 mg/3 x 108 cells/48 h for MVs from normal brain extract-treated MSCs and 2.5 mg/3 x 108 cells/48 h for MVs from ischemic brain extract-treated MSC).

  3. Isolation of microvesicles (MVs)
    1. Collect the culture supernatant from MSC cultures (conditioned media amount about 1,000 ml).
    2. To remove cell and cellular debris, centrifuge the harvest culture supernatant media once at 500 x g for 10 min and then the supernatant are collected.
    3. Centrifuge the supernatant at 800 x g for 15 min, then take the supernatants (about 1,000 ml).
    4. Concentrate the supernatants to 30 ml (about 33 fold concentration) by ultrafiltration using the Minimate Tangential Flow Filtration (TFF) capsule system with a 100 kDa cut-off membrane at 4 °C (Figure 3).
      1. Remove the caps from the feed and retentate ports of the Minimate TFF capsule.
        Note: Do not discard caps. They are required for storage.
      2. Screw a male luer-to-hose-barb connector (included) into each of the feed/retentate ports.
      3. Cut a piece of tubing 3.2 mm (1/8”) i.e., long enough to reach from the feed reservoir, through the pump head to the capsule.
        Note: Keep tubing lengths as short as possible to reduce system hold-up volume.
      4. Connect the tubing to the hose-barb on one of the feed ports. Install the tubing in the pump head. Put the other end of the tubing into the reservoir.
        Notes:
        1. If a pressure gauge or transducer is used, connect the tubing to the pressure device. Then connect the pressure device as close as possible to the feed port using suitable connectors.
        2. Feed and retentate ports are interchangeable. Depending on the orientation of the capsule, choose the port that is at the lowest elevation as the feed port. This allows for air to be easily expelled when liquid is pumped through the capsule. The recommended crossflow for the Minimate TFF capsule is 30-40 ml/min.
      5. Cut another piece of tubing, long enough to return from the retentate port to the sample reservoir.
      6. Attach the tubing to the retentate hose-barb and put the other end in the reservoir. (Again, if a pressure gauge or transducer is used, the tubing connects to the pressure device, which must then be connected to the retentate port.)
      7. Place the retentate screw clamp on the retentate tubing close to the retentate port (after the pressure gauge if installed). Secure in place but do not tighten to restrict the tubing.
      8. Remove one of the filtrate caps.
      9. Attach a female luer-to-hose-barb fitting to one of the filtrate/vent ports.

        Note: This concentration ratio was selected based on the capacity of the subsequent ultracentrifugation steps. Thus, the concentration ratio may vary depending on the ultracentrifuge (rotor and tubes), cell types and culture condition employed.


        Figure 3. Diafiltration of hMSC conditioned media. A. Schematic drawing of Tangential Flow Filtration (TFF) system; B. Photo image of diafiltration system using Minimate TFF capsule system with a 100 kDa cutoff membrane.


    5. To enrich the MVs, the concentrated supernatant is agglutinate using sucrose gradient centrifugation.
    6. Prepare 38 ml polyallomer tube and 0.8 and 2.7 M sucrose cushions (see Recipes 2 and 3).
    7. Carefully put 0.5 ml of 2.7 M sucrose into the bottom of a tube.
    8. Carefully add 0.8 M sucrose 1 ml just above the 2.7 M sucrose layer. Allow the sucrose solution to run down very slowly inside of tube wall.
    9. Add 33 ml sample on the sucrose cushion taking care not to break the sucrose layer (Figure 4A).
    10. Centrifuge the tube at 100,000 x g for 1 h at 4 °C [Rotor: 32Ti, Acceleration: max, Deceleration: slow (9)].
    11. After centrifugation, remove the supernatant (about 32 ml) carefully from sucrose fraction-enriched MVs layer.
      Note: MVs are about just above sucrose cushion (yellow line, Figure 4A) and should be handled with care because it is difficult to identify with the naked eyes (Figure 4B).


      Figure 4. Enrichment of microvesicles by sucrose gradient ultracentrifugation. A. A discontinuous sucrose density gradient was prepared by layering 1.5 ml of 0.8 M sucrose gradient upon 0.5 ml of 2.7 M sucrose density solution. Next, 33 ml of concentrated conditioned medium was added to the top lay carefully. B. After centrifugation, microvesicles are enriched in the interface between medium and sucrose layers.

    12. Prepare OptiPrepTM solution (see Recipe 4) and two 13.2 ml polyallomer tubes.
    13. For further purification of the MVs, mix the sucrose fraction−enriched MVs with OptiPrepTM (final concentration = 30%).
    14. Transfer about 2.5 ml of sucrose fraction−MVs into a 15 ml tube.
    15. Add 1.34 ml sucrose dilution buffer to 2.5 ml Sucrose fraction-enriched MVs then mix 5.76 ml of 50% OptiPrepTM to make 30% OptiPrepTM (total volume: 9.6 ml).
    16. Put 4.8 ml of 30% OptiPrepTM-sample in two 13.2 ml polyallomer tubes, each.
    17. Add 3 ml of 20% OptiPrepTM just above 30% OptiPrepTM layer, each.
    18. Add 2.5 ml of 5% OptiPrepTM on top of 20% OptiPrep layer, each (Figure 5A).
      Note: Caution! OptiPrepTM has a low viscosity, so be careful not to disturb or break the layer.


      Figure 5. Isolation of microvesicles by OptiPrep density gradient. A. Discontinuous OptiPrepTM gradients were prepared in 12 ml tube with 3 layers of gradients consisting of 5% (2.5 ml) of the 2 additional layers of 20% (3 ml) and 30% (4.8 ml) of stock OptiPrepTM. The stock solutions were diluted with 20 mM HEPES/150 mM NaCl buffer (pH 7.2) and sample solution (microvesicle enriched fraction from sucrose gradient) was mixed with 30% gradient solution. B. After centrifugation, sample tubes were arbitrarily divided into 10 equal portions and 1 ml aliquots of each portion were collected to new tubes. The major microvesicle fraction was clearly shown in the interface between 5% and 20% OptiPrepTM (F3 layer).

    19. Centrifuge the sample tube at 200,000 x g for 3 h at 4 °C (Rotor: SW41Ti).
    20. After centrifugation, divide sample tubes into 10 equal portions and taken 1 ml each. The MVs are at the interface between 5% and 20% OptiPrepTM showing the white band (F3 layer) (Figure 5B).
    21. Collect 1 ml MVs (F3), dilute 10-fold with PBS (9 ml) and centrifuge at 100,000 x g for 1 h at 4 °C (Rotor: SW41Ti).
    22. After centrifugation, identify a pellet at the bottom of the tube. Remove the PBS and dissolve the pellets with 100 μl of PBS.
    23. The protein concentration of each fraction is determined using refractometer and the Bradford dye assay.
      Note: For therapeutic purposes, quantification of final products should be expressed as weight/ml (or unit/ml), if possible. Since protein is one of the main components of the microvesicles, protein quantification was employed. Alternatively, the quantification of MVs can be done by flow cytometry, nanoparticle tracking analysis (NTA) or resistive pulse sensing combined with Raman microspectroscopy.

Data analysis

  1. The optimal time point for harvesting conditioned medium of hMSCs for later microvesicle isolation was determined based on the following assays:
    1. > 90% cell viability measured by flow cytometric analysis (Annexin V-PI staining).
    2. Protein concentration measured by Bicinchoninic acid (BCA) assay.
  2. Upon treating brain extract to the target hMSCs, cell morphology was changed significantly (Figure 6).


    Figure 6. Cell morphology untreated hMSCs to brain extract treated hMSCs. A. Untreated hMSCs; B. 20% normal brain extract treated hMSCs; C. 20% stroke brain extract treated hMSCs. All images were acquired at 48 h after medium replacement.

  3. To quantify the protein concentration in untreated-MVs and brain extract treated-MVs, samples were measured by Bradford method (Table 1).

    Table 1. Protein amount in MVs from 1 L of conditioned MSCs medium

Notes

The quantity of the resulting microvesicles can be significantly varied depending on MSC tissue source, MSC cell density, passage number, duration of culture and culture microenvironment (biological, chemical or physical stimulations) for conditioned medium harvesting and the concentration of brain extract.
The processed data and statistical analyses are published in Lee et al. (2016), which can be found at: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5016792/

Recipes

  1. MSC culture media
    DMEM low glucose
    100 U/ml Pen/Strep (100x stock)
    10% FBS
  2. Sucrose dilution buffer
    HEPES 20 mM/150 mM NaCl buffer (pH 7.2)
  3. Sucrose cushion
    0.8 M sucrose cushion: Dissolve 5.48 g of sucrose in 20 ml of sucrose dilution buffer (pH 7.2) filter sterile with 0.45 µm syringe filter and store at 4 °C before use
    2.7 M sucrose cushion: Dissolve 9.24 g sucrose in 10 ml sucrose dilution buffer (pH 7.2)
    Note: 2.7 M sucrose pre-dissolved in 55 °C oven, filter sterile with 0.45 µm syringe filter and store at 4 °C before use.
  4. OptiPrepTM solution
    Homogenization media (HM): 0.25 M sucrose, sucrose dilution buffer (pH 7.4)
    OptiPrepTM working solution: 0.25 M sucrose, 1 mM EDTA, 10 mM Tris-HCl (pH 7.4)
    50% OptiPrepTM: 30 ml (60% OptiPrepTM 25 ml + OptiPrepTM working solution 5 ml)
    20% OptiPrepTM: 20 ml (50% OptiPrepTM 8 ml + HM media 12 ml)
    5% OptiPrepTM: 20 ml (50% OptiPrepTM 2 ml + HM media 18 ml)

Acknowledgments

This protocol is adapted from ‘Microvesicles from brain-extract-treated mesenchymal stem cells improve neurological functions in a rat model of ischemic stroke’ (Lee et al., 2016). This work was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MSIP), No. 2012M3A9B4028639.

References

  1. Adib, S., Tiraihi, T., Darvishi, M., Taheri, T. and Kazemi, H. (2015). Cholinergic differentiation of neural stem cells generated from cell aggregates-derived from human bone marrow stromal cells. Tissue Eng Reg Med 12(1): 43-52.
  2. Chen, X., Li, Y., Wang, L., Katakowski, M., Zhang, L., Chen, J., Xu, Y., Gautam, S.C., Chopp, M. (2007). Ischemic rat brain extracts induce human marrow stromal cell growth factor production. Neuropathology 27: 255-363.
  3. Choi, D. S., Lee, J. M., Park, G. W., Lim, H. W., Bang, J. Y., Kim, Y. K., Kwon, K. H., Kwon, H. J., Kim, K. P. and Gho, Y. S. (2007). Proteomic analysis of microvesicles derived from human colorectal cancer cells. J Proteome Res 6(12): 4646-4655.
  4. Kim, H. O., Choi, S. M. and Kim, H. S. (2013). Mesenchymal stem cell-derived secretome and microvesicles as cell-free therapeutics for neurodegenerative disorders. Tissue Eng Reg Med 10(3): 93-101.
  5. Kim, H. S., Choi, D. Y., Yun, S. J., Choi, S. M., Kang, J. W., Jung, J. W., Hwang, D., Kim, K. P. and Kim, D. W. (2012). Proteomic analysis of microvesicles derived from human mesenchymal stem cells. J Proteome Res 11(2): 839-849.
  6. Lee, J. Y., Kim, E., Choi, S. M., Kim, D. W., Kim, K. P., Lee, I. and Kim, H. S. (2016). Microvesicles from brain-extract-treated mesenchymal stem cells improve neurological functions in a rat model of ischemic stroke. Sci Rep 6: 33038.
  7. Porro, C., Trotta, T. and Panaro, M. A. (2015). Microvesicles in the brain: Biomarker, messenger or mediator? J Neuroimmunol 288: 70-78.
  8. Qu, R., Li, Y., Gao, Q., Shen, L., Zhang, J., Liu, Z., Chen, X., Chopp, M. (2007). Neurotrophic and growth factor gene expression profiling of mouse bone marrow stromal cells induced by ischemic brain extracts. Neuropathology 27: 355-363.
  9. Shin, J.H., Park, Y.M., Kim, D.H., Moon, G.J., Bang, O.Y., Ohn, T., Kim, H.H. (2014). Ischemic brain extract increases SDF-1 expression in astrocytes through the CXCR2/miR-223/miR-27b pathway. Biochim Biophys Acta 1839: 826-936.
  10. Spijker, S. (2011). Dissection of rodent brain regions. Neuroprotemics 57: 13-26.

简介

微囊泡(MV)是亚微米尺寸的膜泡囊,其通过分泌室从细胞中积极释放或从细胞表面膜脱落。 MV由许多细胞类型产生并且用作将生物信息(例如,蛋白质,mRNA和miRNA)转移到远端细胞的载体,从而影响其基因表达,增殖,分化和功能。 虽然他们的生理功能没有明确定义,但最近的研究已经显示出其组织修复和再生的治疗潜力。 虽然MV可以从间充质干细胞(MSC)和来自各种来源的其他细胞类型容易地分离,但在体外常规培养条件下MV的产量是限制因素之一, 功能研究以及体外分析分析。 在这里,我们提供了一个通过大鼠脑提取物预处理MSC增加微泡产量的方案。
【背景】通过直接重编程或利用间充质干细胞进行细胞替代治疗来产生神经干细胞或神经细胞是神经变性疾病的潜在选择(Adib等人,2015)。最近的研究已经证明,来自MSC的微泡代表了增强组织再生,例如神经元再生,免疫调节,脑损伤中的血管发生的其他细胞替代方法的新颖且安全的替代方案(Kim等人,2013) ; Porro等,,2015; Lee等人,2016)。对受损组织外源信号如何影响微泡数量和组成的了解甚少。 MSCs的功能分泌物的含量和数量可以根据微环境的显着变化(Qu等人,2007)。例如,已知缺血性脑提取物或缺氧诱导合成有益于组织再生过程的许多细胞因子和生长因子(Chen等,2007; Shin et al。等等,,2014)。在本研究中,使用正常和缺血性脑提取物作为脑损伤信号的一种形式来提高产量,并且调节来自MSC的MV的分子组成,这可以有益于其临床应用。事实上,通过正常脑提取物或缺血性脑提取物的处理,MSC的条件培养基中的MV的量大大增加。目前的方案主要基于以前描述的方法(Choi等人,2007; Kim等人,2012),其中包括试剂,配方等几个修改。通过物理,化学或生物学手段预处理生产细胞可以显着调节微泡的产量和组成。例如,我们利用脑提取物刺激MSC来模拟脑组织损伤的信号,最终产物(MV)可以是脑组织修复和再生的有效特异性治疗。该协议可以提供一个线索,以制定更好的策略,以获得更高产量的具有来自各种细胞来源的更强治疗潜力的MV。

关键字:间充质干细胞, 微泡, 细胞外囊泡, 蔗糖梯度, 透析过滤, 组织再生

材料和试剂

  1. 4-0手术缝合线
  2. 0.2微米注射器过滤器(Sartorius,目录号:17823-K)
  3. 0.45μm注射器过滤器(Sartorius,目录号:16555-K)
  4. Falcon管50ml(Corning,Falcon ®,目录号:352070)
  5. Falcon管15ml(Corning,Falcon ®,目录号:352099)
  6. T75培养瓶(SPL LIFE SCIENCES,目录号:70075)
  7. 多聚腺管38ml(Beckman Coulter,目录号:344058,用于梯度形成和分级)
  8. 多聚腺管13.2ml(Beckman Coulter,目录号:344059,用于梯度形成和分级)
  9. 8周龄的雄性Sprague-Dawley大鼠(Koatech)
  10. 来自延世细胞治疗中心或从Lonza购买的脂肪干细胞(Lonza,目录号:PT-5006)的人类脂肪组织来源的MSC(来自2个健康女性供体,第4代)
  11. 异氟烷(Hana Pharm,韩国)
  12. 80%N 2 O
  13. 20%O 2
  14. 氯胺酮
  15. 赛拉宾
  16. Bradford蛋白测定试剂盒I(Bio-Rad Laboratories,目录号:5000001)
  17. Dulbecco的磷酸盐缓冲盐水(DPBS)(Biowest,目录号:L0615-500)
  18. 胰蛋白酶/ EDTA(0.05%,酚红)(Thermo Fisher Scientific,Invitrogen TM,目录号:25300054)
  19. 台盼蓝溶液0.4%(生理盐水)(Thermo Fisher Scientific,Gibco TM,目录号:15250061)
  20. DMEM低糖(GE Healthcare,Hyclone TM,目录号:SH30021.01)
  21. 青霉素/链霉素(5,000U / ml)(GE Healthcare,Hyclone TM,目录号:SV30010)
  22. 胎牛血清(FBS)(GE Healthcare,Hyclone TM,目录号:SH30071.03)
  23. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S9888)
  24. HEPES(Thermo Fisher Scientific,Gibco TM ,目录号:15630080)
  25. 10mM Tris-HCl(WHITLAB,目录号:BTH-9274)
  26. 蔗糖(Sigma-Aldrich,目录号:S9378)
  27. EDTA(Bio-Rad Laboratories,目录号:1610729)
  28. OptiPrep TM (Alere Technologies,Axis-Shield Density Gradient Media,目录号:1114542)
  29. MSC培养基(见食谱)
  30. 蔗糖稀释缓冲液(参见食谱)
  31. 蔗糖垫(见食谱)
  32. OptiPrep TM 解决方案(请参阅配方)

设备

  1. 手术剪刀 - 直尖/钝12厘米(精细科学工具,目录号:14001-12)
  2. 窄幅镊子 - 弯曲12厘米,2 x 1.25毫米(精细科学工具,目录号:11003-12)
  3. 虹膜剪刀 - 大环,角度(精细科学工具,目录号:14107-09)
  4. 手术刀手术刀(精细科学工具,目录号:10011-00,10003-12)
  5. 骨髓(JEUNG DO BIO&amp; PLANT,目录号:H-2041-1)
  6. 成年大鼠脑基质(Kent Scientific,目录号:RBMS-300C)
  7. 组织研磨机(WHEATON,目录号:357546)
  8. 移液器
  9. 10ml血清移液管(SPL LIFE SCIENCES,目录号:91010)
  10. 冰桶
  11. 离心机(Eppendorf,型号:5804)
  12. 具有100kDa膜的Minimate TFF胶囊系统(Pall,目录号:OA100C12)
  13. 玻璃巴斯德移液器(Fisher Scientific,目录号:13-678-20A)
  14. 37℃,5%CO 2细胞培养箱(Eppendorf,型号:Galaxy 170S)
  15. 倒置显微镜(Olympus,型号:CKX41)
  16. 血细胞计数器
  17. 超速离心机(Beckman Coulter,型号:Optima TM XPN-100)
  18. 转子:SW41Ti(Beckman Coulter,目录号:331362)
  19. 转子:SW32Ti(Beckman Coulter,目录号:369650)
  20. 蠕动泵(Poong Lim Tech,目录号:PP-150)
  21. Masterflex L / S易于加载II型精密管道头,PPS / CRS(Cole-Parmer,目录号:EW-77200-50)
  22. 饲料池(100ml或500ml)

程序

  1. 永久性大脑中动脉闭塞和脑提取物制剂的程序
    1. 准备两只8周龄的雄性Sprague-Dawley大鼠,一只为正常,一只为缺血性脑损伤。
      注意:一只大鼠的大脑足以进行本实验规模的5-10次实验。
    2. 缺血性脑损伤(MCAO手术)
      1. 用80%N 2 O和20%O 2的混合物麻醉具有3%异氟烷(Hana Pharm,Seoul,Korea)的大鼠。
      2. 将大鼠置于仰卧位置,切开颈部中线,轻轻收缩颈部肌肉。
      3. 仔细分离左颈总动脉和颈外动脉。
      4. 临时用4-0手术缝合线左颈总动脉和颈外动脉。
      5. 切开左颈内动脉,将4-0手术缝合线插入左颈内动脉,并小心前进到威利斯圈。
      6. 松开结扎外颈动脉的结,紧紧扣住颈总动脉。
      7. 缝合颈部皮肤。
    3. MCAO手术后24 h,正常大鼠和缺血性脑损伤大鼠腹腔注射氯胺酮(40 mg / kg体重)和赛拉嗪(5 mg / kg体重)深度麻醉,然后用生理盐水灌注。 br />
    4. 灌注大鼠后,用手术剪刀从耳朵后面切下头部。快速地在头皮肤中进行中线切口,用骨骼肌无菌除去头骨和脑膜。
      注意:如先前Spijker(2011)所述,进行大鼠牺牲和脑组织提取的方法。
    5. 将全脑置于脑基质上后,从同侧半球的冰上进行冠状动脉夹层中部大脑动脉区域(1〜+ 1 mm),收集切片脑组织(图1A)。
    6. 使用组织研磨机均匀化组织片。
    7. 离心组织匀浆在100,000 xg 2℃,4℃,然后取上清液。
    8. 在4℃下将100,000xg上清液离心1小时(图1B)。
    9. 通过0.2μm过滤器过滤上清液。使用Bradford测定法测量蛋白质含量(图1C)。 (蛋白质的典型总量为120-220毫克)

      图1.脑提取物的制备A.将大鼠脑放置在脑基质上,从同侧半球冠状动脉切开大脑中动脉区域(1〜+ 1毫米),收集切片的脑组织。 B.将大脑匀浆物超声离心以除去大鼠脑源性微泡,过滤以除去残留的聚集体和细胞碎片。 C.最后通过0.2微米注射器过滤器过滤上清液,将等分试样储存在-80℃
  2. 细胞培养程序
    1. 在37℃水浴中快速解冻(小于3分钟)冷冻人MSC(hMSC)小瓶。
    2. 将全部冷冻管内容物吸入无菌的50ml锥形管中,小心地加入10体积的预热完全培养基(含10%胎牛血清的DMEM)。
    3. 经温和旋转后,用血细胞计数器测定细胞浓度,并用预热的完全培养基调整3×10 5 / ml的细胞浓度。
    4. 在约8×10 3细胞/ cm 2的范围内接种75cm 2烧瓶,并在37℃下在5%CO 2中孵育, 2 孵化器。
    5. 在解冻后24小时用新鲜预热的完整培养基更换培养基。
    6. 在倒置显微镜下常规观察培养瓶。
    7. 当细胞几乎汇合(超过80%汇合)时,吸出培养基和来自汇合单层的浮选碎片并丢弃。
    8. 用预热的PBS洗涤细胞一次,并向培养瓶中加入5ml的0.05%的Trypin-EDTA。在37°C孵育至50-70%分离(约2-5分钟)
    9. 加入5ml预热的完整培养基,轻轻移液上下分离贴壁细胞,将细胞分散到单细胞悬液中。
    10. 将细胞悬浮液转移到无菌的50ml锥形管中。用另外的5ml完整培养基洗涤烧瓶并合并成锥形管
    11. 以300×g离心细胞悬浮液5-10分钟
    12. 吸出上清并将沉淀重新悬浮在适量体积的预热完全培养基中
    13. 将总共50个烧瓶(75cm 2)与20ml / 1ml10μg/ ml活细胞接种,用于微泡分离并返回培养箱。
      注意:细胞培养物应每3天用新鲜的完整培养基重新喂养,以获得最佳细胞生长。
    14. 当细胞几乎融合(超过80%汇合)时,在室温下用PBS大量洗涤细胞或在37℃预热(5次,每次洗涤10ml)。
    15. 最终洗涤后,用含有300μg脑提取物或缺血脑提取物(终浓度为15μg/ ml)的20ml具有血清和抗生素的低葡萄糖DMEM培养基代替培养基。 48 h后收获培养上清液,分离微泡(MV)(图2)

      图2. MSC培养。:一种。 hMSCs的显微镜图像; B.细胞培养的规模; C.实验时间表。将原代培养的MSC(2×10 6细胞/烧瓶)接种在75cm 2培养瓶(共50个烧瓶)中。 C.在完全培养基中培养后,用无血清抗生素培养基替换培养基48小时,收获培养上清液以分离微泡。
      注意:由未处理的MSC制备的MV具有低蛋白质产量(<0.3mg / 3×10 7细胞/ 48小时)而来自MSC条件培养基的MV的量通过用正常或缺血性脑提取物(2.2mg / 3×10 8) 细胞/来自正常脑提取物处理的MSC的MVs的48小时和来自缺血的MV的2.5mg / 3×10 7细胞/ 48小时脑提取物处理的MSC)。
  3. 分离微泡(MV)
    1. 收集来自MSC培养物的培养上清液(条件培养基约1,000ml)
    2. 为了去除细胞和细胞碎片,将收获培养上清培养基以500×g离心10分钟,然后收集上清液。
    3. 以800 x g离心上清液15分钟,然后取上清液(约1,000 ml)。
    4. 使用Minice Tangential Flow Filtration(TFF)胶囊系统通过超滤将上清液浓缩至30ml(约33倍浓度),在4℃下用100kDa截留膜(图3)。
      1. 从Minimate TFF胶囊的进料和滞留口中取出盖子。
        注意:不要丢弃上限。它们是存储所必需的。
      2. 将每个进纸/滞留端口中的一个公路由器 - 软管倒钩连接器(随附)拧入。
      3. 切割一块3.2毫米(1/8“)的管子,即长度足以从进料储存器通过泵头到达胶囊。
        注意:保持管道尽可能短,以减少系统滞留量。
      4. 将管道连接到其中一个进料口上的软管倒钩。将管道安装在泵头中。将管道的另一端放入水箱。
        注意:
        1. 如果使用压力表或换能器,将管道连接到压力装置。然后使用适当的连接器将压力设备尽可能靠近进料口进行连接。
        2. 进料和滞留端口是可互换的。根据胶囊的方向,选择位于最低高度的端口作为进纸口。这允许当液体泵送通过胶囊时空气容易排出。 Minimate TFF胶囊的推荐交叉流量为30-40 ml / min。
      5. 切割另一条管道,足够长的时间从滞留端口返回到样品池。
      6. 将管道连接到滞留软管 - 倒钩,并将另一端放在储存器中。 (再次,如果使用压力表或传感器,管道连接到压力装置,然后必须连接到滞留端口。)
      7. 将滞留螺丝钳放置在靠近滞留端口的滞留管上(如果安装了压力表)。安全就位,但不要拧紧以限制管路。
      8. 取出一个滤液盖。
      9. 在其中一个滤液/排气口上安装一个女性鲁尔 - 软管 - 倒钩配件。

        注意:该浓度比基于随后的超速离心步骤的容量来选择。因此,浓缩比可以根据超速离心机(转子和管),细胞类型和培养条件而变化。


        图3. hMSC条件培养基的渗滤 A.切向流过滤(TFF)系统示意图; B.使用具有100kDa截留膜的Minimate TFF胶囊系统的渗滤系统的照片图像。


    5. 为了丰富MV,使用蔗糖梯度离心浓缩上清液凝集。
    6. 准备38毫升聚集管和0.8和2.7 M蔗糖垫(见配方2和3)。
    7. 小心地将0.5ml的2.7M蔗糖放入管的底部。
    8. 小心地加入0.8 M蔗糖1毫升正好高于2.7 M蔗糖层。允许蔗糖溶液在管壁内缓慢下降。
    9. 在蔗糖垫上加入33ml样品,注意不要破坏蔗糖层(图4A)
    10. 在4℃下将管以100,000×g离心1小时[转子:32Ti,加速度:最大,减速度:慢(9)]。
    11. 离心后,从蔗糖级分富集的MV层中小心地去除上清液(约32ml)。
      注意:MVs仅在蔗糖垫上方(黄线,图4A),并且应该小心处理,因为用肉眼可以识别(图4B)。


      图4.通过蔗糖梯度超速离心富集微泡。A.通过在0.5ml 2.7M蔗糖密度溶液上分层1.5ml 0.8M蔗糖梯度来制备不连续的蔗糖密度梯度。接下来,仔细地将33ml浓缩条件培养基添加到顶部。 B.离心后,微泡在培养基和蔗糖层之间的界面富集
    12. 准备OptiPrep TM 溶液(参见配方4)和两个13.2 ml聚集管。
    13. 为了进一步纯化MV,将富含蔗糖级分的MV与OptiPrep TM(最终浓度= 30%)混合。
    14. 将约2.5ml蔗糖级分转移至15ml管中
    15. 加入1.34ml蔗糖稀释缓冲液至2.5ml富蔗糖馏分的MV,然后混合5.76ml 50%OptiPrep TM,以制备30%OptiPrep TM(总体积:9.6ml) 。
    16. 将4.8ml 30%OptiPrep - 样品放入两个13.2ml聚集管中,每个。
    17. 加入3ml 20%OptiPrep TM ,高于30%OptiPrep TM 层,每个。
    18. 在20%OptiPrep层的顶部加入2.5ml 5%OptiPrep TM ,每个(图5A)。
      注意:注意! OptiPrep TM 具有低粘度,因此小心不要打扰或打破图层。


      图5.通过OptiPrep密度梯度分离微泡 A.在12ml管中制备不连续的OptiPrep TM梯度,其中3层梯度由5%(2.5ml)的另外20%(3ml)和30%(4.8ml)储备OptiPrep TM的另外的层。将储备溶液用20mM HEPES / 150mM NaCl缓冲液(pH7.2)稀释,并将样品溶液(来自蔗糖梯度的富含微泡的级分)与30%梯度溶液混合。 B.离心后,样品管被任意分成10等份,每份的1ml等分试样收集到新管中。在5%至20%OptiPrep TM(F3层)之间的界面中清楚显示了主要的微泡级分。

    19. 在4℃下将样品管以200,000×g离心3小时(转子:SW41Ti)。
    20. 离心后,将样品管分成10等份,分别取1ml。 MV在5-10%OptiPrep TM之间的界面,显示白色带(F3层)(图5B)。
    21. 收集1ml MV(F3),用PBS稀释10倍(9ml),并在4℃下以100,000×g离心1小时(转子:SW41Ti)。
    22. 离心后,确定管底部的颗粒。取出PBS并用100μlPBS溶解细粒
    23. 使用折射计和Bradford染料测定法确定每个级分的蛋白质浓度 注意:出于治疗目的,如果可能,最终产品的定量应表示为重量/ ml(或单位/ ml)。由于蛋白质是微泡的主要成分之一,所以使用蛋白质定量。或者,MV的定量可以通过流式细胞术,纳米粒子跟踪分析(NTA)或电阻脉冲检测与拉曼显微光谱结合来完成。

数据分析

  1. 基于以下测定法确定用于收获用于后续微泡分离的hMSC条件培养基的最佳时间点:
    1. &GT;通过流式细胞术分析(Annexin V-PI染色)测量90%的细胞活力。
    2. 蛋白质浓度通过二喹啉酸(BCA)测定
  2. 将脑提取物处理至目标hMSCs后,细胞形态发生明显变化(图6)

    图6.未处理的hMSCs对脑提取物处理的hMSCs的细胞形态。未处理的hMSC; B. 20%正常脑提取物处理hMSCs; 20%脑卒中脑提取物治疗hMSCs。所有图像在中等替代后48小时获得。

  3. 为了量化未处理的MV和脑提取物处理的MV中的蛋白质浓度,通过Bradford方法测量样品(表1)。

    表1.来自1L条件性MSCs培养基的 的MV中的蛋白质量

笔记

根据MSC组织来源,MSC细胞密度,传代次数,培养持续时间和培养微环境(生物,化学或物理刺激),所得微泡的数量可以根据条件培养基收获和脑提取物的浓度而显着变化。 /> 经处理的数据和统计分析发表在Lee等人(2016),可以在以下网址找到: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5016792/

食谱

  1. MSC培养基
    DMEM低血糖
    100 U / ml Pen / Strep(100x库存)
    10%FBS
  2. 蔗糖稀释缓冲液
    HEPES 20mM / 150mM NaCl缓冲液(pH7.2)
  3. 蔗糖垫
    0.8 M蔗糖缓冲液:将5.48 g蔗糖溶于20ml蔗糖稀释缓冲液(pH 7.2)中,过滤,用0.45μm注射器过滤器灭菌,并在使用前在4°C储存。 2.7 M蔗糖缓冲液:将9.24 g蔗糖溶于10ml蔗糖稀释缓冲液(pH 7.2)中 注意:将2.7M蔗糖预溶解于55℃烘箱中,用0.45μm注射器过滤器过滤,并在使用前在4℃下储存。
  4. OptiPrep TM 解决方案
    均质培养基(HM):0.25M蔗糖,蔗糖稀释缓冲液(pH7.4)
    OptiPrep TM工作溶液:0.25M蔗糖,1mM EDTA,10mM Tris-HCl(pH7.4)
    50%OptiPrep TM:30ml(60%OptiPrep 25ml + OptiPrep TM工作溶液5ml)
    20%OptiPrep TM:20ml(50%OptiPrep TM 8ml + HM培养基12ml)
    5%OptiPrep TM:20ml(50%OptiPrep 2ml + HM培养基18ml)

致谢

该方案改编自“来自脑提取物处理的间充质干细胞的微泡改善缺血性卒中大鼠模型中的神经功能”(Lee等人,2016)。这项工作得到了Bio&amp;由韩国政府资助的国家研究基金会(NRF)医疗技术发展计划,第2012M3A9B4028639号。

参考

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引用:Lee, J., Choi, S. and Kim, H. (2017). Microvesicle Isolation from Rat Brain Extract Treated Human Mesenchymal Stem Cells. Bio-protocol 7(13): e2375. DOI: 10.21769/BioProtoc.2375.
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