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Jul 2020
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A Novel Method to Make Polyacrylamide Gels with Mechanical Properties Resembling those of Biological Tissues
一种制备具有类似生物组织力学性能的聚丙烯酰胺凝胶的新方法     

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Abstract

Studies characterizing how cells respond to the mechanical properties of their environment have been enabled by the use of soft elastomers and hydrogels as substrates for cell culture. A limitation of most such substrates is that, although their elastic properties can be accurately controlled, their viscous properties cannot, and cells respond to both elasticity and viscosity in the extracellular material to which they bind. Some approaches to endow soft substrates with viscosity as well as elasticity are based on coupling static and dynamic crosslinks in series within polymer networks or forming gels with a combination of sparse chemical crosslinks and steric entanglements. These materials form viscoelastic fluids that have revealed significant effects of viscous dissipation on cell function; however, they do not completely capture the mechanical features of soft solid tissues. In this report, we describe a method to make viscoelastic solids that more closely mimic some soft tissues using a combination of crosslinked networks and entrapped linear polymers. Both the elastic and viscous moduli of these substrates can be altered separately, and methods to attach cells to either the elastic or the viscous part of the network are described.


Graphic abstract:



Polyacrylamide gels with independently controlled elasticity and viscosity.


Keywords: Viscoelasticity (粘弹性), Polyacrylamide (聚丙烯酰胺), Viscosity sensing (粘度传感), Mechanosensing (机械传感), Extracellular matrix (细胞外基质)

Background

Biological tissues are viscoelastic materials that combine features of elastic solids and viscous fluids. Different tissues contain different amounts of the components that contribute to both viscosity (e.g., hyaluronic acid) and elasticity (e.g., collagen fibers), and their structure and proportion change during pathological processes. The widespread use of elastomers or hydrogels as substrates for cell culture (Pelham and Wang, 1997) has revealed the strong impact of substrate mechanics on cell structure and function and focused attention on the limited view of cell biology derived from studies of cells on traditional glass or plastic substrates (Janmey et al., 2020). Until recently, studies of cellular response to substrate or matrix stiffness only examined how cells respond to elastic modulus alterations, with the viscous contribution being either neglected or uncontrolled. Theories of cell mechanosensing have also generally focused on the elastic resistance of the substrate. These limitations were largely due to the lack of suitable materials in which viscosity can be systematically varied within a viscoelastic substrate. There now exist three general approaches for introducing viscous dissipation in a viscoelastic substrate. In one, a polymerizing and crosslinking system, like a polyacrylamide gel, is designed to produce a network near the threshold of the sol-gel transition, such that there are significant elastic properties but also significant viscosity (Cameron et al., 2011). A second method forms crosslinked polymer networks with two classes of crosslinkers: one is covalent and static and the other is non-covalent and dynamic. These first two classes of material are viscoelastic fluids, or viscoplastic material, meaning that if subjected to constant stress, they can deform without limits, as the crosslinks or entanglements rearrange to minimize energy in the deformed state (Chaudhuri et al., 2016). The third class of material, which is the topic of this report, is viscoelastic solids. These materials dissipate substantial energy when they are deformed and slowly continue to deform in response to constant stress; however, eventually, they reach a steady state of deformation (strain) that depends on the magnitude of stress but not on the duration that it is applied. A detailed description of the differences between viscoelastic liquids and solids and a summary of how these three classes of material are constructed are provided in Chaudhuri et al. (2020).


Synthetic polyacrylamide (PAAm) hydrogels are widely used as a model system to study the effect of tissue elasticity on its behavior in health and disease (Beningo et al., 2002). PAAm can be easily tuned to the range of stiffness that reflects the physiological environment of cells, i.e., from hundreds of Pascals up to tens of kilo Pascals. While the role of tissue elasticity in cell biology has been powerfully illustrated, little is known about the viscous aspect of tissue mechanics and how it determines cell structure and fate.


Upon polymerization, polyacrylamide hydrogels create a nearly purely elastic network, with a viscosity several orders of magnitude smaller than the elasticity (Basu et al., 2011). While many reports focus on controlling the elastic properties of hydrogels, our aim was to develop a strategy to introduce and control the viscosity in a viscoelastic network. Our protocol describes the preparation of high molecular weight linear polyacrylamide chains, which can be subsequently sterically entrapped in the crosslinked polyacrylamide network to serve as a viscous component of the gel that dissipates energy. This strategy allows the independent tuning of both the elastic and viscous properties of the hydrogel using the same chemical components as for the preparation of purely elastic polyacrylamide hydrogels. Moreover, it is possible to attach adhesive ligands using classical crosslinkers that can be bound to only the viscous part of the hydrogels (linear polyacrylamide chains), the elastic part of the hydrogels (polyacrylamide network), or both the elastic and viscous elements (as presented in Figure 1).




Figure 1. Illustration of the three methods to make viscoelastic gels presenting adhesion proteins. (1) Linear PAAm, where activated linear PAAm is used for the gel mix preparation; (2) the elastic network of crosslinked PAAm, where unactivated linear PAAm is mixed with an appropriate acrylamide/bis-acrylamide solution and NHS-acrylate monomers; or (3) both types of PAAm, where an unactivated gel mix is prepared and sulfo-SANPAH is used post-polymerization.

Materials and Reagents

  1. 18-mm diameter glass coverslips (Menzel-Glaser, VWR, catalog number: MENZCB00190RA020)

  2. 6-well plates (Corning, Fischer Scientific, catalog number: 07-200-83)

  3. Pipette tips

  4. 50-ml glass bottle

  5. 1.5-ml and 0.5-ml conical tubes (Eppendorf, catalog numbers: 00030 120 086; 00030 121 023)

  6. Aluminum foil

  7. 0.1 M NaOH (Sigma-Aldrich, catalog number: 211465, store at room temperature)

  8. (3-Aminopropyl)trimethoxysilane (3-APTMS) (Sigma, catalog number: 281778, store in a hood at room temperature)

  9. Glutaraldehyde (Sigma, catalog number: 340855; store at 4°C)

  10. SurfaSil Siliconized Fluid (Thermofisher Scientific, catalog number: TS 42800; store at room temperature)

  11. Acetone (Sigma, catalog number: 179124; store at room temperature)

  12. Methanol (Sigma, catalog number: 322415; store at room temperature)

  13. 40% acrylamide solution (Bio-Rad, catalog number: 1610141; store in a hood at 4°C)

  14. 2% bis-acrylamide solution (Bio-Rad, catalog number: 1610142; store in a hood at 4°C)

  15. TEMED (Bio-Rad, catalog number: 1610801; store in a hood at room temperature)

  16. Ammonium persulfate (APS) powder (Bio-Rad, catalog number: 1610700; store desiccated at room temperature)

  17. Acrylic acid N-hydroxysuccinimide ester (NHS-acrylate) (Sigma, catalog number: A8060; store at -20°C)

  18. Sulfo-SANPAH (sulfosuccinimidyl 6-(4′-azido-2′-nitrophenylamino)hexanoate) (Sigma, catalog number: 803332-50MG; store at -20°C)

  19. Dimethyl sulfoxide (DMSO) (Sigma, catalog number: 472301; store at room temperature)

  20. HEPES (50 mM pH 8.2) (Sigma, catalog number: 83264; store at room temperature)

  21. Phosphate-buffered saline (PBS) (Sigma, catalog number: 806552; store at room temperature)

  22. MiliQ water

  23. Protein of interest, e.g., collagen I (Corning, catalog number: 354236) or fibronectin (MP Biomedicals, catalog number: MP215112601)

Equipment

  1. 250-ml glass beaker

  2. Set of micropipettes (from P1 to P1000)

  3. Coverslip mini-rack

  4. Shear rheometer (e.g., Malvern Instruments, Kinexus stress-controlled rheometer)

  5. Tweezers

  6. Chemical hood

  7. 4°C refrigerator

  8. -20°C freezer

  9. Incubator set at 37°C

  10. UV light source at 320-365 nm

  11. Centrifuge at 10,000 × g

  12. Desiccator

Procedure

  1. Glass coverslip functionalization

    1. Adhesive coverslips

      1. Pour 100 ml 0.1 M NaOH into a 250-ml glass beaker and preclean each coverslip by immersing in NaOH; allow them to air-dry.

      2. Under the chemical hood, pour 100 ml 3-APTMS solution into a 250-ml glass beaker and immerse the coverslips for 3 min.

      3. Wash the coverslips 3-5 times with 100 ml MilliQ water until no foam is observed in the water.

        CRITICAL STEP: Residual 3-APTMS will react with glutaraldehyde in the next step and produce an orange precipitate. If this happens, discard the coverslips and start from step a).

      4. Prepare 200 ml 0.5% glutaraldehyde in MilliQ water in a 250-ml glass beaker. Place the coverslips in a mini-rack and immerse in glutaraldehyde solution for 1 h.

      5. Air-dry the coverslips and store under vacuum at room temperature.

    2. Non-adhesive coverslips

      1. Under a chemical hood, prepare 100 ml 5% SurfaSil Siliconized Fluid in acetone.

      2. Immerse the coverslips in SurfaSil solution for 10 s and agitate to ensure a uniform coat.

      3. Rinse the coverslips with clean acetone.

      4. Rinse the coverslips with methanol.

      5. Air-dry the coverslips and store at room temperature.


  2. Preparation of the linear polyacrylamide solution

    1. Prepare a 10% (w/v) solution of APS in H2O before use. For long-term storage, aliquot the solution into 500-µl Eppendorf tubes and store in a -20°C freezer. Avoid freeze-thaw cycles. Solutions stored at room temperature are not stable. For the most effective polymerization, we recommend preparing a fresh 10% (w/v) solution of APS immediately prior to each gel preparation.

    2. In a 50-ml glass bottle, mix acrylamide, H2O, TEMED, and 10% APS according to the recipe in Table 1.

      CAUTION: The linear polyacrylamide solution can be made as inert or activated with the capability of binding adhesive proteins. If you aim to prepare activated linear polyacrylamide, substitute 1 ml H2O with 1 ml 4% NHS-acrylate in DMSO, according to Table 1.

      CRITICAL STEP: It is important to use very low amounts of polymerization initiator (APS) to ensure the formation of very long linear polyacrylamide chains with radii of gyration much larger than the mesh size of the crosslinked network of polyacrylamide. This prevents the linear chains from diffusing out of the polyacrylamide network during the time of the experiment.


      Table 1. Composition of linear polyacrylamide solution in ml for a total volume of 10 ml

      40% acrylamide solution
      H2O

      TEMED

      10% APS
      4% NHS in DMSO
      Inert 1.25 8.72 0.005 0.025 -
      Activated 1.25 7.72 0.005 0.025 1


    3. Polymerize for 1 h at 37°C.

    4. Cover the bottle with aluminum foil to protect it from light. Store at 4°C. If linear polyacrylamide is properly polymerized, it can be stored for months.

      CAUTION: The linear polyacrylamide solution is highly viscous; therefore, it is difficult to pipette it accurately. We suggest cutting the pipette tip before transferring and transferring the solution slowly.


  3. Preparation of viscoelastic polyacrylamide gels

    1. In an Eppendorf tube, mix acrylamide, bis-acrylamide, linear polyacrylamide, H2O, and 4% NHS-acrylate in DMSO (if applicable) according to the recipe presented in Table 2.

      CRITICAL STEP: Mix the solution very gently by pipetting it up and down. It is important not to introduce air bubbles into the mixture. If you fail, you might need to degas the solution in a vacuum desiccator. The presence of air bubbles in the mixture will disrupt polymerization and change the mechanical properties of the resulting gels.


      Table 2. Composition of viscoelastic polyacrylamide gels in µl for a total volume of 500 µl


    2. Add TEMED and 10% APS according to Table 2, gently mix and pipet 100 µl gel mix onto the adhesive coverslip, and place the non-adhesive coverslip on top of the droplet.

    3. After 15 min, add MilliQ water around the gel to avoid drying on the sides.

    4. After an additional 15 min, remove the non-adhesive coverslip from the top of the gel, place the gel in a 6-well plate, and immerse in water.

      CAUTION: The mechanical properties of the gels depend strongly on the quality of the reagents used for the gel formulation, accuracy in pipetting, and mixing of the mixture. Most of the failures in gel polymerization come from mistakes in APS and TEMED pipetting and mixing or inappropriate storage of these solutions. Briefly, TEMED should be kept in the dark, and APS, which is not stable in water, should be freshly prepared for the most effective polymerization. We strongly recommend directly measuring the viscoelasticity of the gels using a rheometer after every separate preparation. In our experience, mixing and pipetting is not always identical, and G’ tends to decrease as reagents get old, apparently due to oxidation. Thus, discrepancies in the measured G’ and G’’ values can be as large as 50%.


  4. Attachment of the adhesion proteins

    Viscoelastic polyacrylamide gels consist of an acrylamide and bis-acrylamide network polymerized in the presence of linear polyacrylamide chains that are incorporated into the network, which determine its viscous properties. Our method allows for the covalent binding of proteins to the polyacrylamide network, linear polyacrylamide, or both.

    1. Attachment of adhesion proteins to only the linear polyacrylamide

      1. To prepare activated linear polyacrylamide that can be crosslinked to the protein of interest, follow section “B. Preparation of the linear polyacrylamide solution” and the recipe from Table 1 for activated linear polyacrylamide. The resulting linear polyacrylamide chains will covalently bind proteins upon incubation with the protein of interest diluted to 0.1 mg/ml protein in 50 mM HEPES pH 8.2 for 2 h at room temperature or overnight at 4°C (both incubations result in uniform coating). For protein incubation, prepare a minimum of 80 µl 0.1 mg/ml protein solution per 18-mm diameter gel. The larger the gel, the more protein solution is needed.

        CAUTION: Incubation with the proteins is performed following polymerization of viscoelastic gels using activated linear polyacrylamide chains immediately after their incorporation within the acrylamide/bis-acrylamide network. Linear polyacrylamide chains of the viscoelastic gel cannot be selectively activated after polymerization of the crosslinked network.

      2. After incubation with the protein, rinse the gels 3 times with 100 µl PBS and store in PBS at 4°C until cell seeding, but not longer than a couple of days.

    2. Attachment of the proteins to only the crosslinked polyacrylamide network

      1. 4% NHS in DMSO method

        1. Prepare fresh 4% NHS solution in DMSO.

        2. Add 50 µl 4% NHS in DMSO per 500 µl gel mix volume according to Table 2.

          CAUTION: 4% NHS solution in DMSO is added to the gel mix prior to polymerization. The crosslinked polyacrylamide network of the viscoelastic gel cannot be selectively activated post-polymerization.

        3. When APS and TEMED are mixed in, cast the gels between adhesive and non-adhesive coverslips, as previously described (Section C: Preparation of viscoelastic polyacrylamide gels).

        4. Once the gel is rinsed and soaked in 50 mM HEPES pH 8.2, illuminate with UV light under the hood for 15 min. From this step forward, gels that will be used for cell seeding should be maintained under aseptic conditions.

        5. Immerse the gels in the protein of interest diluted to 0.1 mg/ml protein in 50 mM HEPES pH 8.2 and incubate for 2 h at room temperature or overnight at 4°C (both incubations result in uniform coating).

        6. Rinse 3 times with PBS and store at 4°C until cell seeding, but not longer than a couple of days.

      2. NHS-acrylate in toluene method

        1. Under the chemical hood, prepare 0.5 ml 2% NHS-acrylate in toluene solution.

        2. Prepare the gel mix without APS and TEMED following the recipe from Table 2, with one modification, i.e., replace 50 µl 4% NHS in DMSO with 50 µl MilliQ water.

        3. Add 100 µl 2% NHS-acrylate in toluene solution per 500 µl gel mix and agitate until the solution becomes uniformly turbid.

        4. Leave for 5 min at room temperature and then centrifuge at 10,000 × g for 5 min to separate the toluene from the gel mix.

        5. Remove the toluene (upper) layer using a 200-µl pipette and transfer the gel mix to a new tube.

        6. Add APS and TEMED to initiate polymerization and cast the gels between adhesive and non-adhesive coverslips, as previously described (Section C: Preparation of viscoelastic polyacrylamide gels).

        7. Once the gel is rinsed and soaked in 50 mM HEPES pH 8.2, illuminate with UV light under the hood for 15 min. From this step forward, gels that will be used for cell seeding should be maintained under aseptic conditions.

        8. Immerse the gels in the protein of interest diluted to 0.1 mg/ml protein in 50 mM HEPES pH 8.2 and incubate for 2 h at room temperature or overnight at 4°C (both incubations result in uniform coating). For protein incubation, prepare a minimum of 80 µl 0.1 mg/ml protein solution per 18-mm diameter gel. The larger the gel, the more protein solution is needed.

        9. Rinse with 100 µl PBS 3 times and store in PBS at 4°C until cell seeding, but not longer than a couple of days.

    3. Attachment of proteins to both the linear polyacrylamide and crosslinked polyacrylamide networks

      1. Soak the gels in 50 mM HEPES pH 8.2 for 15 min.

      2. Prepare 5 mM sulfo-SANPAH in 25% DMSO and 75% 50 mM HEPES pH 8.2 (first dissolve sulfo-SANPAH in DMSO, then add HEPES in MilliQ water). A minimum of 80 µl 5 mM sulfo-SANPAH solution per 18-mm diameter gel is needed.

        CAUTION: We strongly recommend preparing fresh sulfo-SANPAH solution prior to each gel preparation and not storing it longer than 1 day.

      3. Cover the surface of each gel with 80 µl sulfo-SANPAH solution (for an 18-mm diameter gel) and illuminate with UV light (320-365 nm) for 10-15 min under the hood. From this step forward, gels that will be used for cell seeding should be maintained under aseptic conditions. Properly activated sulfo-SANPAH should change color from bright orange to a darker burnt orange or brown.

        CAUTION: Do not overexpose the gels to UV light and do not allow the gels to dry during irradiation.

      4. Rinse the gels 3-5 times with 100 µl 50 mM HEPES pH 8.2 until all the sulfo-SANPAH has been removed.

      5. Immerse the gels in the protein of interest diluted to 0.1 mg/ml protein in 50 mM HEPES pH 8.2 and incubate for 2 h at room temperature or overnight at 4°C (both incubations result in uniform coating). For protein incubation, prepare a minimum of 80 µl 0.1 mg/ml solution per 18-mm diameter gel. The larger the gel, the more protein solution is needed.

      6. Rinse 3 times with 100 µl PBS and store in PBS at 4°C until cell seeding.

        CAUTION: We recommend using the gels within a couple of days of preparation and not placing them in long-term storage. On long time scales (day to weeks), linear polyacrylamide chains can diffuse out of the gel surface and alter their mechanical properties.


  5. Cell seeding

    1. Thirty minutes before seeding the cells, soak the gels in media and keep at 37°C.

    2. Trypsinize the cells and prepare a cell suspension at the concentration of interest (to study cell-substrate interactions, single cells are typically needed).

    3. Remove the media used for soaking the gels and replace with the cell suspension. Keep the gels in the incubator at 37°C. Cells will start to spread upon contact with the gel surface.

Data analysis

The primary data that characterize the gels are their shear storage or loss modulus as functions of time and shear strain, as introduced in Charrier et al. (2018). The magnitude of the viscous dissipation will be highly time dependent and also depends on the length of the linear polyacrylamide chains. The polymer length is highly dependent on the amount and activity of the APS initiator and is impossible to control precisely. Determination of the hydrodynamic radius from dynamic light scattering of very dilute solutions will provide a measure of this length, as described in Charrier et al. (2018).

    For cells cultured on viscoelastic substrates, the most commonly measured quantities are cell area, shape, size of focal adhesions, motility, traction force, and proliferation. Examples of how these features change with substrates and vary among different cell types are provided elsewhere (Gong et al., 2018; Charrier et al., 2020 and 2021; Mandal et al., 2020). Additionally, in Figure 2, we show the morphology and spreading area of the glioma cell line LBC3 grown for 24 h on 1 kPa and 5 kPa elastic and viscoelastic substrates. As can be seen, glioma cells are sensitive to substrate viscosity in an elastic modulus-dependent manner. For example, on G’ = 1 kPa purely elastic gels, glioma cells were smaller than those on 1 kPa viscoelastic gels; on higher stiffness (G’ = 5 kPa), the introduction of viscosity to the substrate did not cause a statistically significant increase in cell spreading area.



Figure 2. The effect of substrate viscosity on cell morphology and spreading area. LBC3 glioma cells grown for 24 h on 1 kPa elastic (A), viscoelastic (B), 5 kPa elastic (D), and viscoelastic (E) hydrogels, with 0.1 mg/ml fibronectin presented only on the crosslinked network of PAAm. Panels C and F show quantitation of the LBC3 cell spreading area for 1 kPa and 5 kPa elastic and viscoelastic gels.

Acknowledgments

This work was funded by the National Institutes of Health (R01EB017753) and the NSF Center for Engineering Mechanobiology (CMMI-154857). KP was supported by the National Science Center, Poland, under Grant No. UMO-2017/26/D/ST4/00997. This protocol was adapted from Charrier et al. (2020).

Competing interests

None of the authors have any competing interests to declare.

Ethics

No human or animal subjects were used in this study.

References

  1. Basu, A., Wen, Q., Mao, X. M., Lubensky, T. C., Janmey, P. A. and Yodh, A. G. (2011). Nonaffine displacements in flexible polymer networks. Macromolecules 44(6) 1671-9.
  2. Beningo, K. A., Lo, C. M. and Wang, Y. L. (2002). Flexible polyacrylamide substrata for the analysis of mechanical interactions at cell-substratum adhesions. Methods Cell Biol 69: 325-339.
  3. Cameron, A. R., Frith, J. E. and Cooper-White, J. J. (2011). The influence of substrate creep on mesenchymal stem cell behaviour and phenotype. Biomaterials 32(26): 5979-5993.
  4. Charrier, E. E., Pogoda, K., Li, R., Park, C. Y., Fredberg, J. J. and Janmey, P. A. (2020). A novel method to make viscoelastic polyacrylamide gels for cell culture and traction force microscopy. APL Bioeng 4(3): 036104.
  5. Charrier, E. E., Pogoda, K., Li, R., Wells, R. G. and Janmey, P. A. (2021). Elasticity-dependent response of malignant cells to viscous dissipation. Biomech Model Mechanobiol. 20(1) 145-54.
  6. Charrier, E. E., Pogoda, K., Wells, R. G. and Janmey, P. A. (2018). Control of cell morphology and differentiation by substrates with independently tunable elasticity and viscous dissipation. Nat Commun 9(1): 449.
  7. Chaudhuri, O., Cooper-White, J., Janmey, P. A., Mooney, D. J. and Shenoy, V. B. (2020). Effects of extracellular matrix viscoelasticity on cellular behaviour. Nature 584(7822): 535-546.
  8. Chaudhuri, O., Gu, L., Klumpers, D., Darnell, M., Bencherif, S. A., Weaver, J. C., Huebsch, N., Lee, H. P., Lippens, E., Duda, G. N. and Mooney, D. J. (2016). Hydrogels with tunable stress relaxation regulate stem cell fate and activity. Nat Mater 15(3): 326-334.
  9. Gong, Z., Szczesny, S. E., Caliari, S. R., Charrier, E. E., Chaudhuri, O., Cao, X., Lin, Y., Mauck, R. L., Janmey, P. A., Burdick, J. A. and Shenoy, V. B. (2018). Matching material and cellular timescales maximizes cell spreading on viscoelastic substrates. Proc Natl Acad Sci U S A 115(12): E2686-E2695.
  10. Janmey, P. A., Fletcher, D. A. and Reinhart-King, C. A. (2020). Stiffness sensing by cells. Physiol Rev 100(2) 695-724.
  11. Mandal, K., Gong, Z., Rylander, A., Shenoy, V. B. and Janmey, P. A. (2020). Opposite responses of normal hepatocytes and hepatocellular carcinoma cells to substrate viscoelasticity. Biomater Sci 8(5): 1316-1328.
  12. Pelham, R. J., Jr. and Wang, Y. (1997). Cell locomotion and focal adhesions are regulated by substrate flexibility. Proc Natl Acad Sci U S A 94(25): 13661-13665.

简介

[摘要]研究characteriz荷兰国际集团如何细胞所处环境的力学性能响应已经通过使用软弹性体和水凝胶作为细胞培养基质的功能。大多数此类基质的局限性在于,虽然它们的弹性特性可以精确控制,但它们的粘性特性却不能,并且细胞对它们所结合的细胞外材料的弹性和粘性都有反应。一些赋予软基材粘度和弹性的方法是基于在聚合物网络内串联耦合静态和动态交联或形成具有稀疏化学交联和空间缠结组合的凝胶。这些材料形成粘弹性流体,揭示了 粘性耗散对细胞功能的显着影响;然而,它们并不能完全捕捉软实体组织的机械特征。在本报告中,我们描述了一种使用交联网络和夹带的线性聚合物的组合来制造更接近地模拟一些软组织的粘弹性固体的方法。这些基材的弹性和粘性模量都可以单独改变,并且描述了将细胞连接到网络的弹性或粘性部分的方法。

图文摘要:

P olyacrylamide凝胶具有独立控制的弹性和粘性。

[背景]生物组织是结合了弹性固体和粘性流体特征的粘弹性材料。不同组织中含有不同量有助于兼顾粘度(组分例如,透明质酸)和弹性(例如,胶原纤维),以及它们的结构和比例变化过程中的病理过程ES 。普遍使用的弹性体或水凝胶的作为细胞培养基材(佩勒姆和Wang,1997)公顷小号揭示基板力学对细胞结构的强烈冲击次函数和着眼于从对细胞的研究中获得的细胞生物学的有限视图传统的玻璃或塑料基板(Janmey等人,2020 年)。直到最近,细胞应答的研究,以基体或母体的刚度检测的细胞只如何响应弹性模量的变化,与粘性贡献被要么忽视或未被控制。细胞机械传感理论也普遍关注基材的弹性阻力。这些限制主要是由于缺乏合适的材料,其中粘度可以在粘弹性基材内系统地改变。现在存在三种用于在粘弹性基材中引入粘性耗散的一般方法。在一个中,聚合和交联体系,如聚丙烯酰胺凝胶,被设计成产生近溶胶-凝胶转变的阈值的网络,S UCH有显著弹性性能也显著粘度(卡梅伦等人。,2011). 第二种方法与两类交联剂形成交联聚合物网络:一种是共价和静态的,另一种是非共价和动态的。这些第一两类材料是粘弹性的流体,或粘塑性材料,这意味着如果对象编恒定应力,它们可以变形而不限制小号,作为交联或缠结重新排列,以在变形状态最小化能量(乔赫里等人。,2016 ) 。第三类材料,即本报告的主题,是粘弹性固体。这些材料在变形时会耗散大量能量,并在恒定应力下缓慢地继续变形;然而,最终它们会达到稳定的变形(应变)状态,这取决于应力的大小而不是施加的持续时间。的粘弹性液体和固体,以及如何这三个类材料的构造的概要之间的区别的详细描述中所提供乔赫里等。( 2020) 。

合成聚丙烯酰胺 (PAAm) 水凝胶被广泛用作模型系统来研究组织弹性对其在健康和疾病中的行为的影响(Beningo等,2002)。PAAm可以很容易地调整到反映细胞生理环境的刚度范围,即从数百帕斯卡到数十千帕斯卡。虽然组织弹性在细胞生物学中的作用得到了有力的说明,但对组织力学的粘性方面以及它如何决定细胞结构和命运知之甚少。

在聚合时,聚丙烯酰胺水凝胶创建几乎纯弹性网络,具有幅度小于的粘度几个数量级的弹性(巴苏等人。,2011) 。虽然许多报告侧重于控制水凝胶的弹性特性,但我们的目标是开发一种策略来引入和控制粘弹性网络中的粘度。我们的方案描述的高分子量线性聚丙烯酰胺链,其可以随后的制备空间中截留的交联聚丙烯酰胺网络到作为凝胶消散能量的粘性成分。该策略允许在使用相同的化学成分,作为纯弹性的聚丙烯酰胺水凝胶的制备中的水凝胶的两个弹性和粘性特性的独立调谐。此外,也可以使用能够结合经典交联剂附着粘合剂的配体,以仅在所述水凝胶(线性聚丙烯酰胺链),水凝胶(聚丙烯酰胺网络)的弹性部分的粘性部分,或两者的弹性和粘性元件(如图呈现URE 1)。

图 1.制作具有粘附蛋白的粘弹性凝胶的三种方法的图示。(1)大号inear聚丙烯酰胺,其中活化线性聚丙烯酰胺被用于凝胶制备混合物; (2)交联的聚丙烯酰胺,其中未活化线性聚丙烯酰胺与混合的弹性网络的适当的丙烯酰胺/双丙烯酰胺溶液和NHS-丙烯酸酯单体; 或(3)这两种类型的聚丙烯酰胺,其中未激活的凝胶混合物制备和sulfo-SANPAH使用后-聚合。

关键字:粘弹性, 聚丙烯酰胺, 粘度传感, 机械传感, 细胞外基质

材料和试剂
 
1. 18 -毫米直径的玻璃盖玻片(Menzel的-格拉泽,VWR,目录号:MENZCB00190RA020)      
2. 6-孔板(Corning,费小号系统求解,目录号:07-200-83)      
3.移液器吸头      
4. 50 -毫升玻璃瓶      
5. 1.5 - ml和0.5 -毫升锥形管仪(Eppendorf,目录号小号:00030 120 086; 00030 121 023)      
6.铝箔      
7. 0.1 M NaOH (Sigma-Aldrich ,目录号:211465,室温储存)      
8. (3-氨基丙基)三甲氧基硅烷(3-APTMS)(Sigma ,目录号:281778,在室温下储存在通风橱中)      
9.戊二醛(Sigma,目录号:340855 ;在4°C下储存)      
10. SurfaSil 硅化液(Thermofisher Scientific,目录号:TS 42800 ;在室温下储存)   
11.丙酮(Sigma,目录号:179124 ;在室温下储存)   
12.甲醇(Sigma,目录号:322415 ;在室温下储存)   
13. 40% 丙烯酰胺溶液(Bio-Rad,目录号:1610141 ;在 4°C 下储存在通风橱中)   
14. 2% 双丙烯酰胺溶液(Bio-Rad,目录号:1610142 ;在 4°C 下储存在通风橱中)   
15. TEMED(Bio-Rad,目录号:1610801 ;在室温下储存在通风橱中)   
16.过硫酸铵(APS)粉末(Bio-Rad,目录号:1610700 ;在室温下干燥储存)   
17.丙烯酸N-羟基琥珀酰亚胺酯(NHS-丙烯酸酯)(Sigma,目录号:A8060 ;储存在-20°C)   
18. Sulfo-SANPAH(磺基琥珀酰亚胺6-(4'-叠氮基-2'-硝基苯基氨基)己酸酯)(Sigma,目录号:803332-50MG ;储存在-20 °C )   
19.二甲基亚砜(DMSO)(Sigma,目录号:472301 ;在室温下储存)   
20.    HEPES(50 mM pH 8.2)(Sigma,目录号:83264 ;在室温下储存)
21.磷酸盐-缓冲盐水(PBS)(Sigma,目录号:806552 ;在室温下储存)   
22. MiliQ 水   
23.蛋白质的兴趣,例如,胶原蛋白I(康宁,目录号:354236)或纤连蛋白(MP Biomedicals公司,目录号:MP215112601)   
 
设备
 
250 -毫升玻璃烧杯中
微量移液器组(从 P1 到 P1000)
盖玻片迷你架
剪切流变仪(例如,Malvern Instruments、Kinexus 应力控制流变仪)
镊子
化学罩
4℃冰箱
-20 °C冰箱
培养箱设置在 37 °C
320 - 365 nm紫外光源
以 10 , 000 × g离心
干燥器
 
程序
 
玻璃盖玻片功能化
胶粘剂盖玻片小号
倾100毫升0.1M NaOH中放入250 -毫升玻璃烧杯中,通过在NaOH中浸渍预清洁每个盖玻片; 让他们到风干。
下的化学通风橱,倒入100mL三APTMS溶液放入250 -毫升玻璃烧杯中,浸入盖玻片3分钟。
用 100 毫升M illiQ 水清洗盖玻片3-5 次,直到在水中观察不到泡沫。
关键步骤:残留的 3-APTMS 将在下一步与戊二醛反应并产生橙色沉淀。如果发生这种情况,请丢弃盖玻片并从步骤 a) 开始。
制得200毫升0.5%的戊二醛在中号illiQ水在250 -毫升玻璃烧杯中。将盖玻片放在迷你架中,并在戊二醛溶液中浸泡 1 小时。
风干的在室温下在真空盖玻片和存储。
非粘合盖玻片小号
下化学通风橱,预PARE 100毫升5%SurfaSil硅化流体在丙酮中。
将盖玻片浸入 SurfaSil 溶液中10 秒并搅拌以确保涂层均匀。
用干净的丙酮冲洗盖玻片。
冲洗的用甲醇盖玻片。
风干盖玻片并在室温下储存。
 
线性聚丙烯酰胺溶液的制备
制备一个10%(W / V)在APS H的溶液2 ö之前使用。对于长-长期储存,分装所述溶液到500 -微升Eppendorf管中并存储在一个-20℃的冰箱中。一个v Ø ID冻融循环。室温下储存的溶液不稳定。为了达到最有效的聚合,我们建议prepar荷兰国际集团一个新鲜的10%(W / V)的APS溶液立即之前到每个凝胶制剂。
在一个50 -毫升玻璃瓶,混合丙烯酰胺,H 2 O,TEMED ,和10%根据表1的配方的AP。
注意:线性聚丙烯酰胺溶液可以制成惰性或活化的,具有结合粘附蛋白的能力。如果您打算制备活化的线性聚丙烯酰胺,请根据表 1 用 DMSO 中的 1 ml 4% NHS-丙烯酸酯代替 1 ml H 2 O。
关键步骤:使用非常少量的聚合引发剂 (APS) 以确保形成非常长的线性聚丙烯酰胺链,其回转半径远大于聚丙烯酰胺交联网络的网眼尺寸,这一点很重要。这防止了线性链从弥漫性荷兰国际集团的聚丙烯酰胺网络的出期间实验的时间。
 
表1.组成以ml线性聚丙烯酰胺溶液为一总体积为10毫升
 
在37 °C 下聚合 1 小时。
用铝箔盖住瓶子以避光。储存在 4 °C 。如果线性聚丙烯酰胺聚合得当,它可以储存数月。
注意: 线性聚丙烯酰胺溶液粘度高; 因此,很难对其进行准确的移液。我们建议切吸量管末端转移之前,和转让环溶液缓慢。
 
粘弹性聚丙烯酰胺凝胶的制备
在 Eppendorf管中,根据表2 中提供的配方在 DMSO(如果适用)中混合丙烯酰胺、双丙烯酰胺、线性聚丙烯酰胺、H 2 O和4% NHS-丙烯酸酯。
关键的一步:混合解决方案非常轻轻地通过吸管婷它向上和向下。重要的是不要将气泡引入混合物中。如果失败,您可能需要在真空干燥器中对溶液进行脱气。混合物中气泡的存在会破坏聚合并改变所得凝胶的机械性能。
表2组合物中的粘弹性微升聚丙烯酰胺凝胶的用于一个总体积500的微升
 
根据表 2 添加 TEMED 和 10% APS,轻轻混合并吸取 100 µl凝胶混合物到粘性盖玻片上,并将非粘性盖玻片放在液滴顶部。
15分钟后,添加中号illiQ水凝胶周围,以避免在侧面上干燥。
后的15分钟,从凝胶的顶部除去非粘性盖玻片,放置于6孔板中的凝胶,在水和浸没。
注意:所述的机械性能的凝胶上用于凝胶制剂,精度移液试剂的质量强烈依赖,并将该混合物的混合。大多数凝胶聚合失败来自 APS 和 TEMED 移液和混合中的错误或这些溶液的不当储存。简而言之,TEMED 应保持在黑暗中,而在水中不稳定的 APS 应新鲜制备以进行最有效的聚合。我们强烈建议在每次单独制备后直接使用流变仪测量凝胶的粘弹性。根据我们的经验,混合和移液并不总是相同的,而且 G' 往往会随着试剂变旧而降低,这显然是由于氧化。因此,差异在所测量的G”和G‘’值可以大到50%。
 
粘附蛋白的附着
粘弹性聚丙烯酰胺凝胶包括一个在被掺入到所述网络线性聚丙烯酰胺链的存在下聚合丙烯酰胺和双丙烯酰胺的网络,这决定了其粘稠的性质。我们的方法允许的共价蛋白的聚丙烯酰胺网络,线性聚丙烯酰胺的结合,或两者。
粘附蛋白的附着到仅线性聚丙烯酰胺
Ť ø制备能够交联到蛋白质激活线性聚丙烯酰胺兴趣,后续部分“B. 线性聚丙烯酰胺溶液的制备”和表 1 中活性线性聚丙烯酰胺的配方。所得线性聚丙烯酰胺链在与稀释至 0.1 mg/ml 蛋白质的目标蛋白质一起在室温下在 50 mM HEPE S pH 8.2 中孵育2 小时或在 4°C 下过夜后将与蛋白质共价结合(两种孵育均产生均匀涂层) . 对于蛋白质温育,准备至少微升每80 0.1mg / ml的蛋白质溶液的18 -毫米直径的凝胶。该LARG尔胶,就需要越多的蛋白质溶液。
小心:孵化与蛋白质进行以下polymeriz的通货膨胀使用活性线性聚丙烯酰胺凝胶的粘弹性的内它们掺入后立即链丙烯酰胺/双丙烯酰胺网络。粘弹性凝胶的线性聚丙烯酰胺链在交联网络聚合后不能被选择性激活。
与蛋白质孵育后,用 100 µl PBS 冲洗凝胶 3 次,并在 4°C 下储存在 PBS 中直至细胞接种,但不得超过几天。
仅将蛋白质连接到交联的聚丙烯酰胺网络
4% NHS 的 DMSO 方法
在 DMSO 中制备新鲜的 4% NHS 溶液。
根据表 2,每 500 µl 凝胶混合物体积添加 50 µl 4% NHS 的 DMSO。
注意:在聚合前将 DMSO 中的 4% NHS 溶液添加到凝胶混合物中。粘弹性凝胶的交联的聚丙烯酰胺网络不能被选择性地激活后-聚合。
当 APS 和 TEMED 混合时,在粘性和非粘性盖玻片之间浇注凝胶,如前所述(C 部分:粘弹性聚丙烯酰胺凝胶的制备)。
一旦凝胶被冲洗并浸泡在 50 mM HEPES pH 8.2 中,在引擎盖下用紫外线照射15 分钟。从这一步开始,用于细胞接种的凝胶应保持在无菌条件下。
将凝胶浸入在 50 mM HEPES pH 8.2 中稀释至 0.1 mg/ml 蛋白质的目标蛋白质中,并在室温下孵育 2 小时或在 4°C 下孵育过夜(两种孵育均产生均匀涂层)。
用 PBS 冲洗 3 次并在 4°C 下储存直至细胞接种,但不得超过几天。
NHS-丙烯酸酯甲苯法
在化学罩下,准备在甲苯溶液中的 0.5 毫升 2% NHS-丙烯酸酯。
制备无APS和TEMED的凝胶混合物从表2的配方下面,用一个修改中,即,代替50微升4%NHS在DMSO与50μl中号illiQ水。
每 500 µl 凝胶混合物加入 100 µl 2% NHS-丙烯酸酯的甲苯溶液并搅拌直至溶液变得均匀混浊。
勒AVE在室温下5分钟,然后离心以10 ,000 ×g下5分钟,以甲苯从凝胶混合物中分离。
使用200除去甲苯(上部)层-微升移液管和凝胶混合物转移到一个新试管中。
添加 APS 和 TEMED 以启动聚合并在粘性和非粘性盖玻片之间浇注凝胶,如前所述(C 部分:粘弹性聚丙烯酰胺凝胶的制备)。
一旦凝胶漂洗和在50mM HEPES pH 8.2的浸泡,照亮与UV光引擎盖下15分钟。从这一步开始,用于细胞接种的凝胶应保持在无菌条件下。
将凝胶浸入在 50 mM HEPES pH 8.2 中稀释至 0.1 mg/ml 蛋白质的目标蛋白质中,并在室温下孵育2 小时或在 4°C 下孵育过夜(两种孵育均产生均匀涂层)。对于蛋白质温育,准备至少微升每80 0.1mg / ml的蛋白质溶液的18 -毫米直径的凝胶。在LAR GER凝胶,就需要越多的蛋白质溶液。
用 100 µl PBS 冲洗3 次,并在 4 °C 下储存在 PBS 中直至细胞接种,但不超过几天。
蛋白质与线性聚丙烯酰胺和交联聚丙烯酰胺网络的连接 
将凝胶浸泡在 50 mM HEPES pH 8.2 中 15 分钟。
制备5mM的磺基SAN PAH在25%DMSO和75%的50mM HEPES pH 8.2的(第一溶解磺基SANPAH在DMSO,然后在添加HEPES中号illiQ水)。每80微升的5mM磺基SANPAH溶液最少18 -毫米直径的需要凝胶。
注意:我们强烈建议每个凝胶制备之前准备新鲜磺SANPAH的解决方案,而不是STOR荷兰国际集团就超过1天。
覆盖80μl的磺基- SANPAH溶液(对于18各自凝胶的表面-毫米直径凝胶),并用UV光照射(320 - 365纳米)10 -引擎盖下15分钟。从这一步开始,用于细胞接种的凝胶应保持在无菌条件下。正确激活磺SANPAH应该从明亮的橙色改变颜色,以一个较深的桔红色或棕色。
注意:不要将凝胶过度暴露在紫外线下,也不要让凝胶在照射过程中变干。 
用 100 µl 50 mM HEPES pH 8.2冲洗凝胶3-5 次,直到去除所有的 sulfo-SANPAH 。
将凝胶浸入在 50 mM HEPES pH 8.2 中稀释至 0.1 mg/ml 蛋白质的目标蛋白质中,并在室温下孵育 2 小时或在 4°C 下孵育过夜(两种孵育均产生均匀涂层)。对于蛋白质温育,准备至少微升每80 0.1mg / ml的溶液18 -毫米直径的凝胶。在LAR GER凝胶,就需要越多的蛋白质溶液。
用 100 µl PBS 冲洗 3 次,并在 4°C 下储存在 PBS 中直至细胞接种。
注意:我们建议在制备后的几天内使用凝胶,而不是将它们长期存放。在很长一段时间内(几天到几周),线性聚丙烯酰胺链可以从凝胶表面扩散出来并改变它们的机械性能。
 
细胞接种
在接种细胞前 30 分钟,将凝胶浸泡在培养基中并保持在37°C。
胰蛋白酶化的细胞,并准备一个在所关注的浓度的细胞悬浮液(至研究细胞-底物相互作用,被单个细胞通常需要)。
去除用于浸泡凝胶的介质并替换为细胞悬液。将凝胶保存在 37°C 的培养箱中。细胞在与凝胶表面接触后开始扩散。
 
数据分析
 
主数据即characteriz Ë凝胶是其剪切储能或损耗模量作为时间和剪切应变的功能,如在引入Charrier等。( 2018) 。粘性耗散的大小将高度依赖于时间,并且还取决于线性聚丙烯酰胺链的长度s 。钍ë聚合物长度是高度依赖于APS的量和活性引发剂和是即时通讯可以精确地控制。d从非常稀溶液的动态光散射的流体动力学半径的etermination将提供这一长度的量度,如在Charrier等人。( 2018) 。
  对于在粘弹性基质上培养的细胞,最常测量的量是细胞面积、形状、粘着斑的大小、运动性、牵引力和增殖。这些参考文献中提供了这些特征如何随基质变化以及如何在不同细胞类型之间变化的示例(Gong等人,2018 年;Charrier等人,2020 年和2021 年;Mandal等人,2020 年)。此外,我Ñ图URE 2 ,我们表明生长24胶质瘤细胞系LBC3的形态和铺展面积1千帕和5千个帕的弹性和粘弹性基材小时。可以看出,胶质瘤细胞以依赖弹性模量的方式对底物粘度敏感,例如。,在G” = 1千帕纯弹性凝胶,神经胶质瘤细胞是小于那些在1千个帕的粘弹性凝胶; 而在较高的刚度(G” = 5千帕),引入粘度到衬底没有引起细胞铺展面积统计学显著增加。
 
图URE 2 。底物粘度对细胞形态和铺展面积的影响。LB C3神经胶质瘤细胞生长24 H于1千帕的弹性(A) ,粘弹性(B) ,5千帕的弹性(d) ,和粘弹性(E)的水凝胶,用0.1mg / ml的纤连蛋白仅聚丙烯酰胺的交联网络上呈现。图C和F显示孔定量吨为1千帕和5千个帕的弹性和粘弹性凝胶的LBC3细胞扩散区域的通货膨胀。
 
致谢
 
这项工作由美国国立卫生研究院 (R01EB017753) 和 NSF 工程机械生物学中心 (CMMI-154857) 资助。KP 得到了波兰国家科学中心的支持,资助号为 UMO-2017/26/D/ST4/00997。该协议是从Charrier等人那里采用的。(2020 年)。
 
利益争夺
 
没有一个作者有任何竞争利益要声明。
 
伦理
 
本研究中未使用人类或动物受试者。
 
参考
Basu, A., Wen, Q., Mao, XM, Lubensky, TC, Janmey, PA 和 Yodh, AG (2011)。柔性聚合物网络中的非仿射位移。大分子44(6) 1671-9。
Beningo, KA, Lo, CM 和 Wang, YL (2002)。用于分析细胞-基质粘附的机械相互作用的柔性聚丙烯酰胺基质。方法细胞生物学69:325-339。
Cameron, AR, Frith, JE 和 Cooper-White, JJ (2011)。底物蠕变对间充质干细胞行为和表型的影响。生物材料32(26):5979-5993。
Charrier, EE, Pogoda, K., Li, R., Park, CY, Fredberg, JJ 和 Janmey, PA (2020)。一种制备用于细胞培养和牵引力显微镜的粘弹性聚丙烯酰胺凝胶的新方法。APL 生物工程4(3):036104。
Charrier, EE, Pogoda, K., Li, R., Wells, RG 和 Janmey, PA (2021)。恶性细胞对粘性耗散的弹性依赖反应。Biomech Model Mechanobiol 。20(1)145-54。
Charrier, EE, Pogoda, K., Wells, RG 和 Janmey, PA (2018)。通过具有独立可调弹性和粘性耗散的基质控制细胞形态和分化。国家通讯社9(1): 449。
Chaudhuri, O., Cooper-White, J., Janmey, PA, Mooney, DJ 和 Shenoy, VB (2020)。细胞外基质粘弹性对细胞行为的影响。自然584(7822):535-546。
Chaudhuri, O., Gu, L., Klumpers, D., Darnell, M., Bencherif, SA, Weaver, JC, Huebsch, N., Lee, HP, Lippens, E., Duda, GN 和 Mooney, DJ ( 2016)。具有可调应力松弛功能的水凝胶可调节干细胞的命运和活性。国家材料15(3): 326-334。
Gong, Z., Szczesny, SE, Caliari, SR, Charrier, EE, Chaudhuri, O., Cao, X., Lin, Y., Mauck, RL, Janmey, PA, Burdick, JA 和 Shenoy, VB (2018) . 匹配材料和细胞时间尺度可最大限度地提高细胞在粘弹性基材上的扩散。Proc Natl Acad Sci USA 115(12):E2686-E2695。
Janmey, PA, Fletcher, DA 和 Reinhart-King, CA (2020)。细胞的刚度传感。生理学修订版100(2) 695-724。
Mandal, K.、Gong, Z.、Rylander, A.、Shenoy, VB 和 Janmey, PA(2020 年)。正常肝细胞和肝细胞癌细胞对底物粘弹性的相反反应。生物材料科学8(5): 1316-1328。
Pelham, RJ, Jr. 和 Wang, Y. (1997)。细胞运动和粘着斑受基质柔性调节。Proc Natl Acad Sci USA 94(25): 13661-13665。
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引用:Pogoda, K., Charrier, E. E. and Janmey, P. A. (2021). A Novel Method to Make Polyacrylamide Gels with Mechanical Properties Resembling those of Biological Tissues. Bio-protocol 11(16): e4131. DOI: 10.21769/BioProtoc.4131.
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