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Jan 2018
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Generation of Human Mesenchymal Stem Cell 3D Spheroids Using Low-binding Plates
利用低结合培养板培养产生人间充质干细胞3D球状体   

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Abstract

The 3D culture of human mesenchymal stem cells (hMSCs) represents a more physiological environment than classical 2D culture and has been used to enhance the MSC secretome or extend cell survival after transplantation. Here we describe a simple and affordable method to generate 3D spheroids of hMSCs by seeding them at high density in a low-binding 96-well plate.

Spheroids of hMSCs cultured in low-binding 96-well plates can be used to study the basic biology of the cells and to generate conditioned media or spheroids to be used in transplantation therapeutic approaches. These MSCs or their secretome can be used as a regenerative therapy and for tissue repair across multiple disease areas, including neurodegeneration.

In comparison to other methods (hanging drop, use of gels or biomaterials, magnetic levitation, etc.), the method described here is simple and affordable with no need to use specialized equipment, expensive materials or complex reagents.

Keywords: Low-binding plate (低结合培养板), Spheroid (球状体), 3D culture (3D 培养), Human mesenchymal stem cells (人间充质干细胞), High density (高密度 )

Background

Mesenchymal stem cells (MSCs) are an attractive candidate for the development of novel regenerative therapies for diseases such as stroke or amyotrophic lateral sclerosis (Chen et al., 2001; Bang et al., 2005; Boido et al., 2014). Their versatility makes the optimization and standardization of techniques essential to ensure MSC therapies can provide as much benefit as possible. One possible way to maximize the therapeutic potential (e.g., enhanced secretion of anti-inflammatory mediators) of MSCs is to culture them in 3D (Bartosh et al., 2010). Cells do not normally grow in monolayers in physiological conditions, therefore culturing them in 3D provides a more realistic environment, and increases secretion of certain factors such as vascular endothelial growth factor (VEGF) or granulocyte-colony stimulating factor (GCSF), amongst others (Caplan and Correa, 2011; Redondo-Castro et al., 2018). Some of these factors exert beneficial actions leading to an enhanced repair response (Torres-Espín et al., 2013; Kalladka and Muir, 2014) and by modulating the inflammatory component (Bernardo and Fibbe, 2013; Mathew et al., 2017).

Several methods have been developed to generate spheroids including magnetic levitation (Haisler et al., 2013); nanoparticles (Daquinag et al., 2013), hanging drop techniques (Bartosh et al., 2010; Murphy et al., 2014), suspension methods (Carpenedo et al., 2007) and hydrogels (Laschke et al., 2013; Tseng et al., 2017). Some of these methods, despite being effective, are time consuming or expensive as they require complex reagents or equipment (Cha et al., 2017). For this reason, we have been culturing spheroids using a very simple method (Redondo-Castro et al., 2018) that only requires a low-binding 96-well plate combined with a high-density suspension of cells.

With this method, we are able to obtain mature spheroids in a few days, with a very high rate of efficiency and reproducibility. Moreover, phenotypic characterization of spheroids shows that this method could be really useful for researchers developing cell therapies (either cell suspensions for transplants or generating cell-derived products such as conditioned media), as well as in other research fields.

Materials and Reagents

  1. Cell culture plasticware
    1. T75 and/or T25 flasks (Corning, catalog numbers: 430641U for T75 and 3056 for T25)
    2. Plates low cell binding, 96 wells, round bottom (Thermo Fisher Scientific, NuncTM, catalog number: 145399 )
    3. Centrifuge tubes (15 ml; 50 ml, Corning, catalog numbers: 430790 ; 430828 )
    4. Cryovials (STARLAB, catalog number: E3110-6122 )
    5. Plastic stripettes (5 ml; 10 ml; 25 ml, Corning, Costar®, catalog numbers: 4487 ; 4488 ; 4489 )
    6. Pipette tips (TipOne, STARLAB, catalog numbers: S1111-3700 ; S1111-1706 ; S1111-6701 )
    7. Non-adherent microfuge tubes (Eppendorf, catalog number: 0030108116 )

  2. Reagents
    1. DMEM low glucose (Sigma-Aldrich, catalog number: D6046 )
    2. Fetal bovine serum (FBS, Thermo Fisher Scientific, GibcoTM, catalog number: 10500064 )
    3. Gelatin, Analar (BDH, catalog number: 440454B )
    4. L-Glutamine, 200 mM (Sigma-Aldrich, catalog number: G7513 )
    5. MesenPRO RSTM Medium (Thermo Fisher Scientific, GibcoTM, catalog number: 12746012 )
    6. PBS, without calcium and magnesium (Sigma-Aldrich, catalog number: D8537 )
    7. Penicillin-streptomycin (P/S), 10,000 units penicillin and 10 mg streptomycin per ml (Sigma-Aldrich, catalog number: P0781 )
    8. Trypsin/EDTA 10x (Sigma-Aldrich, catalog number: T4174 )
    9. Triton X-100 (Sigma-Aldrich, catalog number: T8787 )
    10. Trypan blue solution (Sigma-Aldrich, catalog number: T8154 , 0.4% [w/v] solution)
    11. Paraformaldehyde (Sigma-Aldrich, catalog number: P6148 )
    12. Methanol (Fisher Scientific, CAS: 67-56-1)
    13. Fish skin gelatin (Sigma-Aldrich, catalog number: G7041 )
    14. Antibodies:
      Mouse anti-fibrillin (used at 1/200, Merck, catalog number: MAB1919 )
      Rabbit anti-fibronectin (used at 1/200, Sigma-Aldrich, catalog number: F3648 )
      Donkey anti-mouse 680 nm (used at 1/400, LI-COR, catalog number: 926-68072 )
      Donkey anti-rabbit 488 nM (used at 1/500, Thermo Fisher Scientific, InvitrogenTM, catalog number: R37118 )
    15. DAPI ( used at 1/100,000,Thermo Fisher Scientific, InvitrogenTM, catalog number: D1306 )
    16. Gelatin solution (see Recipes)
    17. MesenPRO RSTM Medium (see Recipes)
    18. DMEM low glucose (see Recipes)
    19. Trypsin (see Recipes)

Equipment

  1. Water bath, 37 °C (Grant JB Nova)
  2. CO2 Incubator (Eppendorf, New BrunswickTM, model: Galaxy® 170 S )
  3. Glass hemocytometer (Brand)
  4. Laminar flow hood (ENVAIR, model: Envair Eco Safe Basic Plus )
  5. Inverted microscope (Olympus, model: CKX31 )
  6. Moticam 2300 camera coupled to Motic Images Plus 2.0 ML software (Motic, model: Moticam 2300 )
  7. Cell culture centrifuge (Sigma Laborzentrifugen, model: 3-16KL )
  8. Aspirator (dry vacuum pump/compressor, Welch Vacuum - Gardner Denver, model: 2511 )
  9. Autoclave (Prestige Medical, model: Classic 2100 Extended, catalog number: 210004UK )

Software

  1. Motic Images Plus 2.0 ML software, Motic®

Procedure

  1. Preparing the cells
    1. Culture the cells in flasks (cells may come from a frozen vial or from a previously established culture. Cells identity can be confirmed by using specific markers, detailed in Dominici et al., 2006 or Redondo-Castro et al., 2017). Recommended initial densities are around 2,500 cells/cm2, but numbers can be adjusted (depending on the donors and their proliferation properties). Ensure cells are evenly distributed and incubate at 37 °C in MesenPRO RSTM Medium (or any other suitable culture media for MSCs).
    2. When cells are being cultured (from a previous passage sub-culture or from the frozen vial) and reach 70-80% confluency, they are ready to be sub-cultured or used to generate spheroids. One passage normally takes one week, but this time can change depending on the donor; proliferation of cells from some donors can be as slow as three weeks between passages. Cells should be used up to passage 6, and the medium should be changed every 3-6 days (slower growing cells require less changes of media).
    3. Warm gelatin solution in a water bath (~37 °C), and add enough gelatin solution to cover the entire surface of the flask/well. Ideally, leave them in an incubator overnight (37 °C). Aspirate the gelatin and rinse wash with warm or room temperature PBS. Flasks and plates coated with gelatin can be kept in the incubator until cells are ready (ideally this step needs to be done on the same day, but can be done the day before if needed. Flasks can be kept in the incubator ON, at 37 °C).
    4. Once cells are ready, aspirate media and wash cells twice with warm PBS. Then add the solution containing trypsin, diluted 1/10 in PBS, from the 10x stock [final concentration of trypsin is 0.5% (w/v)]. Use the minimum volume necessary to cover the flask surface (normally 1-2 ml for a T25 or 5 ml for a T75, volumes may vary). It should be warm, as trypsin works optimally at 37°C. Return flasks to the incubator for 5 min, checking them every 2 min for detachment of cells. 
    5. Once detached, transfer the solution containing cells and trypsin to a falcon tube. Add the double of the volume of DMEM low glucose (containing FBS, check recipe section. MesenPRO RSTM medium can be used at this step, but DMEM is normally cheaper) to the flask, to wash out the remaining cells in the flask (if you added 5 ml of trypsin, add then 10 ml of media to stop the reaction). Transfer the whole volume to the tube and centrifuge at 770 x g for 5 min. 
    6. Discard the supernatant and resuspend the cells in 1-2 ml of media. Count the viable cells (using trypan blue) in a glass hemocytometer and divide the cells into new flasks or seed them to generate spheroids if they are in the right passage (P5-P6). 

  2. Spheroid formation
    1. Resuspend the pellet in MesenPRO RSTM medium (14,000-60,000 cells per well [Figure 1C] do not need further modifications of this protocol–suspension volumes or incubation days), and add 50 µl of cell suspension to each well of the low cell binding plate. Variations of density can be used, according to specific requirements. Mixing and resuspending the cell suspension during the plating can help obtaining more reproducible spheroid sizes across the plate.
    2. Spheroids will begin to appear in less than 24 h (Figures 1A and 1D). Just few hours after seeding, most of the wells should contain small spheroids that will progressively converge into one large spheroid. After 5 days in culture, spheroids are visible by eye and are stable enough for experimental investigation (Figure 2). Cells from different donors may grow faster or slower (Figures 1A and 1B), therefore culture times may need to be adjusted. If longer periods of culture are needed, or big numbers of cells are used, media can be changed by slowly removing the medium without disrupting the spheroid. Small preparations (14,000-25,000 cells) do not need media change during those 5-6 days.


      Figure 1. Formation of spheroids. A. Area of spheroids from 2 different human donors (in white dots for the cells purchased from Lonza and black dots for the cells purchased from 3H Biomedical), at days 1, 4 and 6 after cell seeding. Notice the difference in size between donors, the progressive reduction on size and the exclusion criteria for all structures smaller than 0.15 mm2 (indicated with a dotted line). B. Average area of spheroids from the same donors at the same time points; donor from 3H Biomedical in black, donor from Lonza in grey. C. Representative images of spheroids formed with 14,000 and 60,000 cells, after 5 days. Scale bar = 500 µm. D. Time course of the formation of a spheroid (20,000 cells) up to 6 days in vitro. Notice the reduction on size. Scale bar = 500 µm.

  3. Treating and collecting spheroids
    1. Treatments can be added directly to the media, without need of moving the spheroids. Make sure to adjust volumes and concentrations. Be sure that your incubator has water to avoid evaporation which could lead to changes in the final volume of media surrounding the spheroid and give rise to inaccuracies in the results.
    2. Supernatants can be easily collected with a plastic pipette tip (200 µl or 1 ml size will be suitable), just taking care to avoid the spheroid. Collect spheroids individually or together with the supernatants and transfer them to a tube using a plastic pipette tip (Figure 2).


      Figure 2. Working with spheroids. Spheroids can be seen in the wells from above (A) and below (B) the plate (5 days spheroids in the picture, 20,000 cells). Spheroids can be easily aspirated with a pipette tip (C) and transferred into centrifuge tubes (D). Spheroids are indicated with arrows. As a reference, the wells from (A) and (B) are from a 96-well plate.

  4. Immunostaining of spheroids
    1. Transfer spheroids to non-adherent microfuge tubes containing 4% paraformaldehyde in PBS containing 1% Triton X-100 at 4 °C for 4 h or overnight with gentle rocking (any tube rotator can be used here, use ~500 µl to ensure proper fixation of the spheroids).
    2. Wash spheroids in 0.1% Triton X-100 in PBS, at least a couple of times (~15 min/wash; all washes require ~500 µl to ensure efficient washing; no centrifugation is needed, wait 1 min before aspirating the liquid so the spheroids will sediment into the bottom of the tube).
    3. Dehydrate by exposure to an increasing series of methanol (10%, 20%, 50%, 75% and 95%) in PBS solution for 15-20 min each at 4 °C(~500 µl of each concentration should be enough). 
    4. Incubate spheroids in 100% methanol at 4° C for 4 h or overnight with gentle rocking. 
    5. Rehydrate spheroids (involve the same decreasing methanol series). 
    6. Block unspecific staining by incubating spheroids in 3% fish skin gelatin (FSG) in 0.1% Triton X-100/PBS solution at 4 °C for 10 min-1 h to overnight with gentle rocking. 
    7. Incubate with primary antibody diluted in 0.1% Triton X-100/PBS + 3% FSG, overnight at 4°C with gentle rocking. Optimal concentration of antibodies should be tested by users. We present an example using fibronectin and fibrillin-1, but other proteins can be detected by this method.
    8. Wash spheroids three times in 0.1% Triton X-100 each for 30 min at room temperature. 
    9. Incubate spheroids with secondary antibody in 0.1% Triton X-100/PBS for 3-24 h at 4°C with gentle rocking. DAPI or other staining steps can be added at this point.
    10. Wash three times using 0.1% Triton X-100/PBS. Spheroids are ready to be imaged (e.g., by fluorescence or confocal microscopy, see examples in Figure 3). We imaged the spheroid by using dipping confocal lenses whilst the spheroid was immersed in PBS solution, but different approaches can be used and every researcher needs to adapt this step to their own microscopes and resources.


      Figure 3. Example of immunofluorescent staining of spheroids. Confocal images of an immunostained spheroid (A, scale bar = 100 µm), and details at higher magnification (scale bars = 50 µm) of fibronectin (B) and fibrillin-1 staining (C), as well as the merge image (D).

Data analysis

  1. Measuring spheroids: spheroid growth can be monitored by taking photos and measuring their maximum sectional area using a camera attached to the inverted microscope. Bright field images are sufficient to achieve reliable measurements. All structures smaller than 0.15 mm2 are not considered spheroids and excluded from further analysis (Figures 1A and 1B). Further details on this process can be found in Redondo-Castro et al., 2018.
  2. Biological measurements: supernatants can be directly used in different assays, such as ELISAs, or Western blots. Spheroids can be lysed to use them in molecular biology assays (ELISAs, Western blots, PCR, etc.) or processed for histological or immunocytochemistry protocols (Figure 4).
  3. Conditioned media: supernatants can be used as conditioned medium for cell and in vivo treatments.


    Figure 4. Spheroid formation and applications. Scheme depicting the main stages of spheroid formation and some of the main applications.

Notes

  1. Cells from different donors may behave differently (Figure 1). Some donors require longer incubation times between passages or different incubation times before spheroids form. Adjust the times to your donor characteristics.
  2. As the cells are contained in a small volume of media, it is important that evaporation is not an issue, as that can affect the concentrations of treatments and therefore cell behavior. Ensure your incubator tray always contains water. It is a good idea to use all the outer wells as an extra reservoir of liquid containing PBS and 1-2% of P/S.
  3. Immunofluorescence protocols may require some further optimization by the user, as some epitopes may be more accessible than others (especially the ones on the surface of the spheroid). So we recommend optimization of the concentrations and timings of incubations to ensure proper penetration and staining of the right targets.

Recipes

  1. Gelatin solution
    1. Dissolve gelatin (0.1%) in distilled water and autoclave the solution
    2. Store the solution at 4 °C and use within two weeks
  2. MesenPRO RSTM Medium
    1. Defrost the MesenPro supplement (it is sold with the media) and add it to a 500 ml bottle
    2. Add 5 ml of P/S
    3. Store the medium in 50 ml aliquots and keep at 4 °C
    4. Add 5 ml of fresh glutamine, just before feeding the cells (glutamine in media can last up to 14 days)
  3. DMEM low glucose
    1. Add 50 ml of FBS and 5 ml of P/S to a 500 ml bottle of media
    2. Store the media at 4 °C if not use immediately
    3. Add 5 ml of fresh glutamine, just before adding it to the cells (glutamine in media can last up to 14 days)
  4. Trypsin
    Dilute trypsin/EDTA10x to 1x in PBS, just before use

Acknowledgments

The work was supported with funds from the Stroke Association (grant TSA 2017/03, UK), the Engineering and Physical Sciences Research Council (EPSRC, UK), the Medical Research Council Centre for Doctoral Training (MRC-CDT) in Regenerative Medicine studentship grant EP/ L014904/1, and the Manchester Regenerative Medicine Network (MaRMN).
Author contributions statements: All authors contributed to the writing, editing and testing of this protocol.

Competing interests

The authors declare that there are no conflicts of interest or competing interests.

References

  1. Bang, O. Y., Lee, J. S., Lee, P. H. and Lee, G. (2005). Autologous mesenchymal stem cell transplantation in stroke patients. Ann Neurol 57(6): 874-882.
  2. Bartosh, T. J., Ylostalo, J. H., Mohammadipoor, A., Bazhanov, N., Coble, K., Claypool, K., Lee, R. H., Choi, H. and Prockop, D. J. (2010). Aggregation of human mesenchymal stromal cells (MSCs) into 3D spheroids enhances their antiinflammatory properties. Proc Natl Acad Sci U S A 107(31): 13724-13729.
  3. Bernardo, M. E. and Fibbe, W. E. (2013). Mesenchymal stromal cells: sensors and switchers of inflammation. Cell Stem Cell 13(4): 392-402.
  4. Boido, M., Piras, A., Valsecchi, V., Spigolon, G., Mareschi, K., Ferrero, I., Vizzini, A., Temi, S., Mazzini, L., Fagioli, F. and Vercelli, A. (2014). Human mesenchymal stromal cell transplantation modulates neuroinflammatory milieu in a mouse model of amyotrophic lateral sclerosis. Cytotherapy 16(8): 1059-1072.
  5. Caplan, A. I. and Correa, D. (2011). The MSC: an injury drugstore. Cell Stem Cell 9(1): 11-15.
  6. Carpenedo, R. L., Sargent, C. Y. and McDevitt, T. C. (2007). Rotary suspension culture enhances the efficiency, yield, and homogeneity of embryoid body differentiation. Stem Cells 25(9): 2224-2234.
  7. Cha, J. M., Park, H., Shin, E. K., Sung, J. H., Kim, O., Jung, W., Bang, O. Y. and Kim, J. (2017). A novel cylindrical microwell featuring inverted-pyramidal opening for efficient cell spheroid formation without cell loss. Biofabrication 9(3): 035006.
  8. Chen, J., Li, Y., Wang, L., Zhang, Z., Lu, D., Lu, M. and Chopp, M. (2001). Therapeutic benefit of intravenous administration of bone marrow stromal cells after cerebral ischemia in rats. Stroke 32(4): 1005-1011.
  9. Daquinag, A. C., Souza, G. R. and Kolonin, M. G. (2013). Adipose tissue engineering in three-dimensional levitation tissue culture system based on magnetic nanoparticles. Tissue Eng Part C Methods 19(5): 336-344.
  10. Dominici, M., Le Blanc, K., Mueller, I., Slaper-Cortenbach, I., Marini, F., Krause, D., Deans, R., Keating, A., Prockop, D. and Horwitz, E. (2006). Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 8(4): 315-317. 
  11. Haisler, W. L., Timm, D. M., Gage, J. A., Tseng, H., Killian, T. C. and Souza, G. R. (2013). Three-dimensional cell culturing by magnetic levitation. Nat Protoc 8(10): 1940-1949.
  12. Kalladka, D. and Muir, K. W. (2014). Brain repair: cell therapy in stroke. Stem Cells Cloning 7: 31-44.
  13. Laschke, M. W., Schank, T. E., Scheuer, C., Kleer, S., Schuler, S., Metzger, W., Eglin, D., Alini, M. and Menger, M. D. (2013). Three-dimensional spheroids of adipose-derived mesenchymal stem cells are potent initiators of blood vessel formation in porous polyurethane scaffolds. Acta Biomater 9(6): 6876-6884.
  14. Mathew, B., Poston, J. N., Dreixler, J. C., Torres, L., Lopez, J., Zelkha, R., Balyasnikova, I., Lesniak, M. S. and Roth, S. (2017). Bone-marrow mesenchymal stem-cell administration significantly improves outcome after retinal ischemia in rats. Graefes Arch Clin Exp Ophthalmol 255(8): 1581-1592.
  15. Murphy, K. C., Fang, S. Y. and Leach, J. K. (2014). Human mesenchymal stem cell spheroids in fibrin hydrogels exhibit improved cell survival and potential for bone healing. Cell Tissue Res 357(1): 91-99.
  16. Redondo-Castro, E., Cunningham, C. J., Miller, J., Brown, H., Allan, S. M. and Pinteaux, E. (2018). Changes in the secretome of tri-dimensional spheroid-cultured human mesenchymal stem cells in vitro by interleukin-1 priming. Stem Cell Res Ther 9(1): 11.
  17. Redondo-Castro, E., Cunningham, C., Miller, J., Martuscelli, L., Aoulad-Ali, S., Rothwell, N. J., Kielty, C. M., Allan, S. M. and Pinteaux, E. (2017). Interleukin-1 primes human mesenchymal stem cells towards an anti-inflammatory and pro-trophic phenotype in vitro. Stem Cell Res Ther 8(1): 79. 
  18. Torres-Espin, A., Hernandez, J. and Navarro, X. (2013). Gene expression changes in the injured spinal cord following transplantation of mesenchymal stem cells or olfactory ensheathing cells. PLoS One 8(10): e76141.
  19. Tseng, T. C., Wong, C. W., Hsieh, F. Y. and Hsu, S. H. (2017). Biomaterial substrate-mediated multicellular spheroid formation and their applications in tissue engineering. Biotechnol J 12(12).

简介

人间充质干细胞(hMSC)的3D培养代表比经典2D培养更生理的环境,并且已经用于增强MSC分泌组或移植后延长细胞存活。 在这里,我们描述了一种简单且经济实惠的方法,通过在低密度96孔板中高密度接种hMSC来生成三维球状体。

在低结合96孔板中培养的hMSC的球状体可用于研究细胞的基本生物学并产生用于移植治疗方法的条件培养基或球状体。 这些MSC或其分泌蛋白组可用作再生疗法和用于多个疾病区域的组织修复,包括神经变性。

与其他方法(悬滴,使用凝胶或生物材料,磁悬浮,等)相比,此处描述的方法简单且经济实惠,无需使用专用设备,昂贵材料或复杂试剂。

【背景】 间充质干细胞(MSCs)是开发针对中风或肌萎缩侧索硬化等疾病的新型再生疗法的有吸引力的候选者(Chen et al。,2001; Bang et al。,2005; Boido et al。,2014)。它们的多功能性使得技术的优化和标准化对于确保MSC疗法可以提供尽可能多的益处是必不可少的。使MSC的治疗潜力(例如,增强的抗炎介质分泌)最大化的一种可能方式是以3D方式培养它们(Bartosh 等人,,2010)。细胞通常不会在生理条件下以单层生长,因此在3D中培养它们可提供更真实的环境,并增加某些因子的分泌,如血管内皮生长因子(VEGF)或粒细胞集落刺激因子(GCSF)等( Caplan和Correa,2011; Redondo-Castro et al。,2018)。其中一些因素发挥了有益的作用,导致修复反应增强(Torres-Espín et al。,2013; Kalladka和Muir,2014)和调节炎症成分(Bernardo和Fibbe,2013; Mathew) et al。,2017)。

已经开发了几种方法来产生包括磁悬浮的球状体(Haisler et al。,2013);纳米粒子(Daquinag et al。,2013),悬滴技术(Bartosh et al。,2010; Murphy et al。,2014),悬浮法(Carpenedo et al。,2007)和水凝胶(Laschke et al。,2013; Tseng et al。,2017)。尽管这些方法有效,但其中一些方法耗时或昂贵,因为它们需要复杂的试剂或设备(Cha et al。,2017)。出于这个原因,我们一直在使用一种非常简单的方法(Redondo-Castro et al。,2018)培养球状体,只需要一个低结合的96孔板结合高密度悬浮液细胞。

使用这种方法,我们能够在几天内获得成熟的球状体,具有非常高的效率和再现性。此外,球状体的表型特征表明,该方法对于开发细胞疗法(用于移植的细胞悬浮液或产生细胞衍生产物,例如条件培养基)以及其他研究领域的研究人员非常有用。

关键字:低结合培养板, 球状体, 3D 培养, 人间充质干细胞, 高密度

材料和试剂

  1. 细胞培养塑料器皿
    1. T75和/或T25烧瓶(康宁,目录号:T75为430641U,T25为3056)
    2. 板低细胞结合,96孔,圆底(Thermo Fisher Scientific,Nunc TM ,目录号:145399)
    3. 离心管(15毫升; 50毫升,Corning,目录号:430790; 430828)
    4. Cryovials(STARLAB,目录号:E3110-6122)
    5. 塑料条纹(5毫升; 10毫升; 25毫升,Corning,Costar ®,目录号:4487; 4488; 4489)
    6. 移液器吸头(TipOne,STARLAB,目录号:S1111-3700; S1111-1706; S1111-6701)
    7. 非粘附微量离心管(Eppendorf,目录号:0030108116)

  2. 试剂
    1. DMEM低葡萄糖(Sigma-Aldrich,目录号:D6046)
    2. 胎牛血清(FBS,Thermo Fisher Scientific,Gibco TM ,目录号:10500064)
    3. 明胶,Analar(BDH,目录号:440454B)
    4. L-谷氨酰胺,200 mM(Sigma-Aldrich,目录号:G7513)
    5. MesenPRO RS TM 培养基(Thermo Fisher Scientific,Gibco TM ,目录号:12746012)
    6. PBS,不含钙和镁(Sigma-Aldrich,目录号:D8537)
    7. 青霉素 - 链霉素(P / S),10,000单位青霉素和10毫克链霉素/ ml(Sigma-Aldrich,目录号:P0781)
    8. 胰蛋白酶/ EDTA 10x(Sigma-Aldrich,目录号:T4174)
    9. Triton X-100(Sigma-Aldrich,目录号:T8787)
    10. 台盼蓝溶液(Sigma-Aldrich,目录号:T8154,0.4%[w / v]溶液)
    11. 多聚甲醛(Sigma-Aldrich,目录号:P6148)
    12. 甲醇(Fisher Scientific,CAS:67-56-1)
    13. 鱼皮明胶(Sigma-Aldrich,目录号:G7041)
    14. 抗体:
      小鼠抗原纤维蛋白(用于1/200,默克,目录号:MAB1919)
      兔抗纤连蛋白(用于1/200,Sigma-Aldrich,目录号:F3648)
      驴抗小鼠680 nm(用于1/400,LI-COR,目录号:926-68072)
      驴抗兔488 nM(1/500使用,Thermo Fisher Scientific,Invitrogen TM ,目录号:R37118)
    15. DAPI(1 / 100,000,Thermo Fisher Scientific,Invitrogen TM ,目录号:D1306)
    16. 明胶溶液(见食谱)
    17. MesenPRO RS TM 培养基(参见食谱)
    18. DMEM低葡萄糖(见食谱)
    19. 胰蛋白酶(见食谱)

设备

  1. 水浴,37°C(Grant JB Nova)
  2. CO 2 培养箱(Eppendorf,New Brunswick TM ,型号:Galaxy ® 170 S)
  3. 玻璃血细胞计数器(品牌)
  4. 层流罩(ENVAIR,型号:Envair Eco Safe Basic Plus)
  5. 倒置显微镜(奥林巴斯,型号:CKX31)
  6. Moticam 2300相机耦合到Motic Images Plus 2.0 ML软件(Motic,型号:Moticam 2300)
  7. 细胞培养离心机(Sigma Laborzentrifugen,型号:3-16KL)
  8. 吸气器(干式真空泵/压缩机,Welch Vacuum - Gardner Denver,型号:2511)
  9. 高压灭菌器(Prestige Medical,型号:Classic 2100 Extended,目录号:210004UK)

软件

  1. Motic Images Plus 2.0 ML软件,Motic ®

程序

  1. 准备细胞
    1. 在烧瓶中培养细胞(细胞可来自冷冻小瓶或来自先前建立的培养物。细胞特性可通过使用特定标记物确认,详见Dominici 等人,,2006或Redondo-Castro et al。,2017)。推荐的初始密度约为2,500个细胞/ cm 2, 2 ,但数量可以调整(取决于供体及其增殖特性)。确保细胞均匀分布并在37℃下在MesenPRO RS TM 培养基(或任何其他合适的MSC培养基)中孵育。
    2. 当培养细胞(来自先前的传代亚培养物或来自冷冻的小瓶)并达到70-80%汇合时,它们准备进行传代培养或用于产生球状体。一段通常需要一个星期,但这一次可能会根据捐赠者而改变;来自一些供体的细胞增殖可以与传代之间的三周一样慢。细胞应该使用至第6代,培养基应每3-6天更换一次(生长缓慢的细胞需要较少的培养基更换)。
    3. 在水浴(~37℃)中加热明胶溶液,并加入足够的明胶溶液以覆盖烧瓶/孔的整个表面。理想情况下,将它们放在培养箱中过夜(37°C)。吸出明胶并用温热或室温PBS冲洗。涂有明胶的烧瓶和平板可以保存在培养箱中,直到细胞准备好(理想情况下,这一步骤需要在同一天完成,但如果需要,可以在前一天完成。烧瓶可以保存在培养箱中,37℃ C)。
    4. 细胞准备好后,吸出培养基并用温PBS洗涤细胞两次。然后加入含有胰蛋白酶的溶液,在PBS中稀释1/10,从10x原液中[胰蛋白酶的最终浓度为0.5%(w / v)]。使用覆盖烧瓶表面所需的最小体积(对于T25通常为1-2 ml或对于T75为5 ml,体积可能不同)。它应该是温暖的,因为胰蛋白酶在37°C时效果最佳。将烧瓶放回培养箱中5分钟,每隔2分钟检查一次,以便细胞分离。 
    5. 分离后,将含有细胞和胰蛋白酶的溶液转移到falcon管中。加入体积为DMEM的低倍葡萄糖(含有FBS,检查配方部分.MesenPRO RS TM 培养基可用于此步骤,但DMEM通常更便宜)到烧瓶中,洗掉烧瓶中剩余的细胞(如果加入5ml胰蛋白酶,则加入10ml培养基以终止反应)。将整个体积转移至管中,并以770 x g 离心5分钟。 
    6. 弃去上清液,将细胞重悬于1-2ml培养基中。在玻璃血细胞计数器中计数活细胞(使用台盼蓝)并将细胞分成新的培养瓶或播种,如果它们位于正确的通道中则生成球状体(P5-P6)。 

  2. 球体形成
    1. 将沉淀重悬于MesenPRO RS TM 培养基中(每孔14,000-60,000个细胞[图1C]不需要进一步修改该方案 - 悬浮液体积或孵育天数),并加入50μl细胞悬液至低细胞结合板的每个孔。根据具体要求,可以使用密度的变化。在电镀期间混合和重悬细胞悬浮液可以帮助在整个板上获得更可重复的球体尺寸。
    2. 球体将在不到24小时内开始出现(图1A和1D)。播种后几个小时,大多数井应该包含小球状体,这些小球状体将逐渐收敛成一个大的球状体。在培养5天后,通过眼睛可见球状体并且足够稳定以进行实验研究(图2)。来自不同供体的细胞可以更快或更慢地生长(图1A和1B),因此可能需要调整培养时间。如果需要更长时间的培养,或者使用大量细胞,可以通过缓慢移除培养基而不破坏球体来改变培养基。在5-6天内,小剂量(14,000-25,000个细胞)不需要更换培养基。


      图1.球状体的形成。 :一种。在细胞接种后第1,4和6天,来自2个不同人供体的球状体的面积(对于从Lonza购买的细胞为白点,对于购自3H Biomedical的细胞为黑点)。注意供体之间的尺寸差异,尺寸的逐渐减小和小于0.15mm 2 的所有结构的排除标准(用虚线表示)。 B.同一时间点来自同一捐助者的球状体的平均面积;来自黑色的3H Biomedical捐赠者,灰色的Lonza捐赠者。 C.5天后用14,000和60,000个细胞形成的球状体的代表性图像。比例尺=500μm。 D.体外形成球状体(20,000个细胞)长达6天的时间过程。请注意尺寸减小。比例尺=500μm。

  3. 治疗和收集球体
    1. 处理可以直接添加到介质中,而无需移动球状体。确保调整体积和浓度。确保您的培养箱中有水以避免蒸发,这可能会导致球体周围介质的最终体积发生变化,从而导致结果不准确。
    2. 可以使用塑料移液器吸头(200μl或1 ml尺寸适合)轻松收集上清液,只需注意避免球状体。单独或与上清液一起收集球状体,并使用塑料移液管尖端将其转移到管中(图2)。


      图2.使用球状体。从板上方(A)和下方(B)的孔中可以看到球状体(图中5天的球状体,20,000个细胞)。可以用移液管尖端(C)容易地吸出球状体并将其转移到离心管(D)中。球体用箭头表示。作为参考,(A)和(B)的孔来自96孔板。

  4. 球状体的免疫染色
    1. 将球体转移至含有4%多聚甲醛的含有1%Triton X-100的PBS中的非粘附微量离心管,4°C或过夜,轻轻摇动(此处可使用任何管旋转器,使用~500μl以确保正确固定的球体)。
    2. 用PBS中的0.1%Triton X-100洗涤球体,至少两次(约15分钟/洗涤;所有洗涤需要~500μl以确保有效洗涤;不需要离心,等待1分钟然后吸入液体,这样球状体将沉积到管的底部)。
    3. 通过在4℃下暴露于增加系列的甲醇(10%,20%,50%,75%和95%)在PBS溶液中脱水15-20分钟(每种浓度~500μl应该足够)。  
    4. 将球状体在4%的100%甲醇中孵育4小时或轻轻摇动过夜。 
    5. 再水化球状体(涉及相同的甲醇系列减少)。 
    6. 通过将3%鱼皮明胶(FSG)中的球状体在0.1%Triton X-100 / PBS溶液中于4℃温育10分钟-1小时至温和摇动过夜来阻断非特异性染色。 
    7. 与在0.1%Triton X-100 / PBS + 3%FSG中稀释的一抗孵育,在4℃下轻轻摇动过夜。用户应测试抗体的最佳浓度。我们提供了使用纤连蛋白和原纤蛋白-1的实例,但是通过该方法可以检测其他蛋白质。
    8. 在0.1%Triton X-100中洗涤球体三次,每次在室温下洗涤30分钟。 
    9. 将球状体与0.1%Triton X-100 / PBS中的二抗孵育3-24小时,在4°C轻轻摇动。此时可以添加DAPI或其他染色步骤。
    10. 使用0.1%Triton X-100 / PBS洗涤三次。球状体已准备好成像(例如,通过荧光或共聚焦显微镜,参见图3中的实例)。我们通过使用浸渍共焦透镜对球体进行成像,同时将球体浸没在PBS溶液中,但可以使用不同的方法,并且每个研究人员都需要使这一步适应他们自己的显微镜和资源。


      图3.球状体的免疫荧光染色实例免疫染色球体的共聚焦图像(A,比例尺=100μm),以及纤维连接蛋白(B)的更高放大倍数(比例尺=50μm)的细节和原纤维蛋白-1染色(C),以及合并图像(D)。

数据分析

  1. 测量球体:可以通过拍摄照片并使用连接到倒置显微镜的相机测量其最大截面积来监测球体生长。明场图像足以实现可靠的测量。所有小于0.15 mm 2 的结构都不被视为球状体,不包括在进一步分析中(图1A和1B)。关于该过程的更多细节可以在(Redondo-Castro 等人,,2018)中找到。
  2. 生物测量:上清液可直接用于不同的测定,如ELISA或Western印迹。可以裂解球状体以在分子生物学测定(ELISAs,Western印迹,PCR,等)中使用它们或者用于组织学或免疫细胞化学方案(图4)。
  3. 条件培养基:上清液可用作细胞和体内处理的条件培养基。


    图4.球体形成和应用。描绘球体形成的主要阶段和一些主要应用的方案。

笔记

  1. 来自不同捐赠者的细胞可能表现不同(图1)。在形成球状体之前,一些供体需要更长的传代时间或不同的孵育时间。根据您的捐赠者特征调整时间。
  2. 由于细胞包含在少量培养基中,重要的是蒸发不是问题,因为这会影响处理的浓度并因此影响细胞行为。确保您的培养箱托盘始终盛水。使用所有外部孔作为含有PBS和1-2%P / S的额外液体储库是个好主意。
  3. 免疫荧光方案可能需要使用者进一步优化,因为一些表位可能比其他表位更易接近(尤其是球状体表面上的表位)。因此,我们建议优化孵育的浓度和时间,以确保正确的目标正确渗透和染色。

食谱

  1. 明胶溶液
    1. 将明胶(0.1%)溶解在蒸馏水中并对溶液进行高压灭菌
    2. 将溶液储存在4°C并在两周内使用
  2. MesenPRO RS TM Medium
    1. 解冻MesenPro补充剂(与介质一起出售)并将其加入500毫升瓶中
    2. 加入5毫升P / S.
    3. 将培养基储存在50ml等分试样中并保持在4℃
    4. 在喂食细胞之前加入5毫升新鲜谷氨酰胺(培养基中的谷氨酰胺可持续长达14天)
  3. DMEM低葡萄糖
    1. 将50ml FBS和5ml P / S加入500ml瓶中
    2. 如果不立即使用,请将介质保存在4°C
    3. 添加5毫升新鲜谷氨酰胺,然后将其添加到细胞中(培养基中的谷氨酰胺可持续长达14天)
  4. 胰蛋白酶
    在使用前将PBS中的胰蛋白酶/ EDTA10x稀释至1x

致谢

这项工作得到了卒中协会(授予TSA 2017/03,英国),工程和物理科学研究委员会(EPSRC,英国),医学研究委员会博士培训中心(MRC)的资助。 -CDT)在再生医学学生资助计划EP / L014904 / 1和曼彻斯特再生医学网络(MaRMN)。
作者贡献声明:所有作者都参与了本协议的编写,编辑和测试。
利益冲突声明:作者声明没有利益冲突或竞争利益。

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引用:Redondo-Castro, E., Cunningham, C. J., Miller, J., Cain, S. A., Allan, S. M. and Pinteaux, E. (2018). Generation of Human Mesenchymal Stem Cell 3D Spheroids Using Low-binding Plates. Bio-protocol 8(16): e2968. DOI: 10.21769/BioProtoc.2968.
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