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Relative Stiffness Measurements of Cell-embedded Hydrogels by Shear Rheology in vitro
在体外试验中采用剪切流变学测量载有细胞水凝胶的相对硬度   

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Abstract

Hydrogel systems composed of purified extracellular matrix (ECM) components (such as collagen, fibrin, Matrigel, and methylcellulose) are a mainstay of cell and molecular biology research. They are used extensively in many applications including tissue regeneration platforms, studying organ development, and pathological disease models such as cancer. Both the biochemical and biomechanical properties influence cellular and tissue compatibility, and these properties are altered in pathological disease progression (Cox and Erler, 2011; Bonnans et al., 2014). The use of cell-embedded hydrogels in disease models such as cancer, allow the interrogation of cell-induced changes in the biomechanics of the microenvironment (Madsen et al., 2015). Here we report a simple method to measure these cell-induced changes in vitro using a controlled strain rotational rheometer.

Keywords: Shear rheology(剪切流变学), Matrix stiffness(基质硬度), Cancer-associated fibroblasts(癌症相关成纤维细胞), Hydrogels(水凝胶)

Background

Fibrosis and solid tumours are both accompanied by pathological remodelling of their native tissue (Cox and Erler, 2011; Bonnans et al., 2014). In both pathological conditions, the local tissue environment experiences physico-chemical as well as biological changes, resulting in increased tissue stiffness (elastic modulus) (Humphrey et al., 2014). The strengthened tissue/matrix regulates mechano-signaling that leads to altered cell behaviour, cell morphology, differentiation state, proliferation, migration and stemness. In preclinical animal models of cancer, these changes can drive malignant progression and metastatic spread (Bonnans et al., 2014). Not surprisingly, targeting matrix stiffening has received substantial attention in recent years, and several clinical trials have been initiated (Kai et al., 2016).

The elasticity and mechanical properties of a matrix component can readily be examined using atomic force microscopy (AFM), which is a technique that provides nanometre resolution and concurrent measurement of the applied force with picoNewton resolution (Kasas and Dietler, 2008). However, AFM is not applicable to understand the elastic properties of larger 3D matrices. The mechanical properties of bulk 3D matrices can more accurately be examined using shear rheology (Picout and Ross-Murphy, 2003). Rheology is the study of how materials deform when forces are applied to them. Thus applying shear stress to a 3D matrix can determine the elastic modulus (stiffness) of a bulk 3D matrix. In this protocol we describe a method to measure cell-induced changes on matrix stiffness of hydrogels embedded with cancer-associated fibroblasts by shear rheology.

Materials and Reagents

  1. NuncTM cell-culture treated multidishes, 24-well (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 142475 )
  2. 100 μl sterile pipet tip
  3. 1,000 μl sterile pipet tip
  4. 1.5 ml sterile microcentrifuge tubes
  5. 8 mm disposable biopsy punch (KAI, catalog number: BP-80F )
  6. Syringe filter, minisart, 0.20 µm (VWR, catalog number: 514-7011 )
  7. Cells: immortalized human cancer-associated fibroblasts (CAFs) (Gaggioli et al., 2007)
  8. Collagen type I, high concentration, rat tail (Corning, catalog number: 354249 )
  9. Matrigel® basement membrane matrix, *LDEV-Free (Corning, catalog number: 354234 )
  10. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10270106 )
  11. Sterile PBS, pH 7.2 (Thermo Fisher Scientific, catalog number: 20012068 )
  12. Trypsin-EDTA (0.25%), phenol red (Thermo Fisher Scientific, GibcoTM, catalog number: 25200056 )
  13. DMEM (Thermo Fisher Scientific, catalog number: 41966-052 )
  14. Insulin-transferrin-selenium (Thermo Fisher Scientific, GibcoTM, catalog number: 41400045 )
  15. Penicillin-streptomycin (Thermo Fisher Scientific, GibcoTM, catalog number: 15140-122 )
  16. Y-27632 (Sigma-Aldrich, catalog number: Y0503 )
  17. MEM α, nucleosides (Thermo Fisher Scientific, GibcoTM, catalog number: 11900-073 )
  18. Sodium bicarbonate, NaHCO3 (Sigma-Aldrich, catalog number: S5761 )
  19. 1 M HEPES buffer (Thermo Fisher Scientific, GibcoTM, catalog number: 15630080 )
  20. 5x collagen buffer (see Recipes)
  21. Growth medium (see Recipes)
  22. 1 ml collagen type I/Matrigel hydrogel (+/- cancer-associated fibroblasts) (see Recipes)

Equipment

  1. Timer
  2. Centrifuge
  3. Pipette  
  4. Cell incubator at 37 °C, 5% CO2
  5. Discovery Series Hybrid rheometer (TA Instruments, model: DHR-2 )
  6. 8 mm geometry, Figure 1a (TA Instruments)
  7. 8 stepped mm Peltier plate, Figure 1a (TA Instruments)
  8. Stainless Steel Spatula, One End Flat, One End Bent, 6 in. in length (UNITED SCIENTIFIC SUPPLIES, model: SSFB06 )
  9. Hemocytometer

Procedure

  1. Fabrication of collagen type I/Matrigel hydrogels embedded with cancer-associated fibroblasts
    1. Keep all reagents on ice (collagen type I, Matrigel, growth medium, 5x collagen buffer and FBS).
    2. Aspirate the growth medium from the cells and wash the cells once briefly with PBS.
    3. Aspirate the PBS, and add trypsin-EDTA (0.25%) enough to just cover the cells.
    4. Once the cells have detached, resuspend them in normal growth medium and count the cells.
    5. Prepare the gels on ice. For a 1 ml volume of gel add in the following order:
      1. 100 µl HN-CAFs (500,000 cells) – cell number will need to be optimized according to the cell line (see Notes). Use 100 µl growth medium for hydrogels without embedded cells.
      2. 120 µl growth medium (cell line dependent, but typically DMEM, 10% FBS, insulin-transferrin-selenium, penicillin-streptomycin).
      3. 100 µl FBS (100%).
      4. 80 µl 5x collagen buffer (or according to the collagen amount) – vortex before aspirating.
      5. 200 µl Matrigel (or according to the desired concentration, see Notes).
      6. 400 µl collagen type I (or according to the desired concentration, see Notes).    
    6. Mix the collagen type I/Matrigel hydrogels very well by pipetting up and down without introducing air-bubbles. In case bubbles occur, centrifuge the hydrogel solution a few seconds at 200 x g.
    7. Transfer 1 ml collagen type I/Matrigel hydrogel to one well in a NuncTM Cell-Culture Treated Multidishes, 24-well plate. Avoid bubbles. In case bubbles occur aspirate the bubbles using a 100 µl pipette tip.
    8. Place the lid on the 24-well plate and transfer the plate to an incubator without adding growth medium.
    9. Let the gel polymerize for 1 h at 37 °C, 5% CO2.
    10. Add 1 ml growth medium and transfer the plate back to the incubator (washing step).
    11. Let the gel wash for 1 h at 37 °C, 5% CO2.
    12. Aspirate the growth medium without touching the gel.
    13. Add 1 ml fresh growth medium and transfer the plate to the incubator at 37 °C, 5% CO2.
    14. Leave the cells to remodel their surrounding gel in the incubator at 37 °C, 5% CO2 for 24-72 h (or until a measurement is desired). The media does not need to be replaced during the period when using the CAFs, however, it will depend on the specific cell type used.

  2. Measuring relative stiffness of cell-remodelled gels
    Rheological characterization was performed on all hydrogel samples using a TA Instruments DHR-2 controlled strain rotational rheometer using an 8 mm sand-blasted parallel plate geometry. Table 1 below outlines the testing parameters which we have determined to be optimal for the hydrogel setup described above using a Discovery Series Hybrid rheometer (TA Instruments).

    Table 1. Rheometer settings


    1. Start and calibrate the rheometer according to manufacturer instructions.
    2. Attach the stepped lower geometry to the peltier plate (Figure 1a).
    3. Attach the 8 mm diameter upper geometry (Figure 1a).
    4. Set the peltier temperature to required temperature (Table 1).
    5. Zero the axial force.
    6. Take the hydrogel out of the 24-well plate without damaging the gel using the stainless steel spatula (Figures 1b and 1c).
    7. Using an 8 mm disposable biopsy punch trim the gel to the correct size (Figures 1d and 1e).
    8. Carefully load the hydrogel into the stepped lower geometry (Figure 1f).
    9. Set a logarithmic oscillation strain sweep as per Table 1.
    10. Set a fixed angular frequency as per Table 1.
    11. Move the 8 mm upper geometry down until it just contacts the top surface of the gel (Figure 1g).
    12. Decrease the gap by small 50 microns increments to increase the axial force applied to the hydrogel (Figure 1h).
    13. Continue until a stable axial force of 0.03 N is reached as detailed in Table 1.
    14. Begin measurement.

  3. Analysis of the relative stiffness
    1. Ensure a linear viscoelastic (storage modulus [G’]) response within the strain range evaluated (Figures 1i and 1j).
    2. Extract the storage modulus (G’) at 1% strain when comparing multiple gel measurements (Figures 1i and 1j).
    3. The elastic moduli (E) can be determined from the storage modulus (G’) using:
      E = 2 x G’ (1 + υ)
      Where,
      υ = Poisson’s ratio of 0.5 for hydrogels.


      Figure 1. Rheology set-up. a. Lower geometry, peltier plate and upper geometry (8 mm in diameter). b. CAF-embedded hydrogels in a 24-well plate covered with growth medium. c. CAF-embedded hydrogel taken out of the 24-well plate using a spatula. d. Using an 8 mm disposable biopsy punch to excise an 8 mm diameter CAF-embedded hydrogel biopsy for profiling. e. Image illustrating the CAF-embedded hydrogel after excision. f. Excised hydrogel biopsy placed on the lower diameter geometry (8 mm in diameter). g. Lowering of the upper geometry (8 mm in diameter). h. Upper geometry in contact with the hydrogel. Applying axial force to the hydrogel until reaching a stable force of 0.03 N. i. Representative example of CAF-induced remodelling of collagen type I/Matrigel hydrogels. The storage modulus (G’) of collagen type I/Matrigel hydrogels is measured after 72 h of CAF remodelling. CAFs are still alive within the hydrogels upon measurement (see Notes). The storage modulus is measured over a decade of oscillation strain from 0.2% to 2%. The bar chart represents the storage modulus at 1% strain, and illustrates that increasing the number of CAFs induces stiffening of the hydrogel. j. Representative example of how CAF-induced remodelling of collagen type I/Matrigel hydrogels, and in turn stiffening can be blocked by perturbing the ROCK1/2 kinases (Rho-associated kinases1 and 2). The storage modulus (G’) of collagen type I/Matrigel hydrogels is measured after 72 h of CAF remodelling has taken place under the inhibition of ROCK1/2 kinases using 10 µM Y-27632. CAFs are still alive within the hydrogels upon measurement. The graph represents the storage modulus at 1% strain, and illustrates that ROCK1/2-inhibition prevents CAFs from remodelling and stiffening their environment.

Data analysis

  1. To ensure reliable data make sure to perform three technical repeats in each experiment. Extract the storage modulus (G’) at 1% strain for each technical repeat when comparing multiple gel measurements (Figures 1i and 1j). Make sure to conduct the experiment three biological times using the appropriate controls.
  2. Ensure a linear viscoelastic (storage modulus [G’]) response within the strain range evaluated (Figures 1i and 1j). If this is not the case disregard the measurement. If this is a recurrent issue, one should lower the strain range (to less than 1%) and extract the storage modulus (G’) at i.e., 0.1-0.5% strain.

Notes

  1. It is very important to ensure the collagen type I and Matrigel solutions remain ice-cold. Thaw aliquots of Matrigel on ice (or at 4 °C overnight).
  2. Always vortex the 5x collagen buffer solution just before use. This ensures good resuspension of precipitated NaHCO3.
  3. Optimization of cell numbers depends on the set-up of the experiment. One has to decide how quick the remodelling will take. The more cells that are incorporated into the gels, the quicker the remodelling will occur. We normally suggest 48-72 h of remodelling for highly active cells such as CAFs (Madsen et al., 2015).
  4. The use of different concentrations of collagen type I and Matrigel will affect the initial properties of the hydrogel. The higher the concentration, the higher the stiffness of the gels. As a consequence, a greater number of cells, or a longer period of remodelling may be needed to effectively detect small changes in the biomechanical properties of the hydrogel during the experimental time frame.
  5. When using different volumes of collagen type I and Matrigel, the final volume is adjusted with growth medium.
  6. The viability of hydrogels embedded CAFs can be determined in various ways (Ruedinger et al., 2015): 1) Cells can be dissociated from the hydrogels using i.e., collagenase/dispase treatment for 1 h at 37 °C, followed by cell counting using either hemocytometer, automated cell counters or flow cytometry. 2) Total DNA/RNA levels can be determined upon extraction. 3) Various metabolic assays e.g., MTT, CellTiter-Blue and ATP assays (Ruedinger et al., 2015).
  7. The gels were only minimally frequency dependent within the range of testing and showed a linear viscoelastic response within the strain range evaluated (see Figure 1).
  8. When applying an axial force to the gels prior to starting the measurements, ensure this is consistent across measurements. A value of 0.03 N for gels described above is sufficient.
  9. Always make sure to compare measurements from paired experiments of gels made at the same time.

Recipes

  1. 5x collagen buffer (5x refers to the collagen volume used in the hydrogel)
    2.5 g MEM α, nucleosides
    5 ml 1 M HEPES buffer (pH 7.5)
    1 g NaHCO3
    Water up to 50 ml
    Dissolve well and filter sterilize
    Note: Store master stocks at -20 °C. Store smaller working solutions at 4 °C.
  2. Growth medium
    DMEM
    Fetal bovine serum (FBS) (10%)
    Penicillin-streptomycin (100 U/ml)
    Insulin-transferrin-selenium (1x)
  3. 1 ml collagen type I/Matrigel hydrogel with cells
    220 µl growth medium (+/- cells)
    80 µl 5x collagen buffer (or according to the collagen amount) – vortex before aspirating
    100 µl FBS (100%)
    200 µl Matrigel (store aliquots at -80 °C)
    400 µl collagen type I (store at 4 °C)

Acknowledgments

This protocol was adapted from previous published papers (Madsen et al., 2015; Cox et al., 2013; Baker et al., 2013). TRC is supported by an NHMRC New Investigator grant, Australia. CDM is supported by the Ragnar Söderberg Foundation, BioCARE, Cancerfonden, and Åke Wiberg foundation, all Sweden. We also thank Professor Janine Erler at the Biotech Research & Innovation Centre, University of Copenhagen for providing access to the rheometer.

References

  1. Baker, A. M., Bird, D., Lang, G., Cox, T. R. and Erler, J. T. (2013). Lysyl oxidase enzymatic function increases stiffness to drive colorectal cancer progression through FAK. Oncogene 32(14): 1863-1868.
  2. Bonnans, C., Chou, J. and Werb, Z. (2014). Remodelling the extracellular matrix in development and disease. Nat Rev Mol Cell Biol 15(12): 786-801.
  3. Cox, T. R. and Erler, J. T. (2011). Remodeling and homeostasis of the extracellular matrix: implications for fibrotic diseases and cancer. Dis Model Mech 4(2): 165-178.
  4. Cox, T. R., Bird, D., Baker, A. M., Barker, H. E., Ho, M. W., Lang, G. and Erler, J. T. (2013). LOX-mediated collagen crosslinking is responsible for fibrosis-enhanced metastasis. Cancer Res 73(6): 1721-1732.
  5. Gaggioli, C., Hooper, S., Hidalgo-Carcedo, C., Grosse, R., Marshall, J. F., Harrington, K. and Sahai, E. (2007). Fibroblast-led collective invasion of carcinoma cells with differing roles for RhoGTPases in leading and following cells. Nat Cell Biol 9(12): 1392-1400.
  6. Humphrey, J. D., Dufresne, E. R. and Schwartz, M. A. (2014). Mechanotransduction and extracellular matrix homeostasis. Nat Rev Mol Cell Biol 15(12): 802-812.
  7. Kai, F., Laklai, H. and Weaver, V. M. (2016). Force matters: Biomechanical regulation of cell invasion and migration in disease. Trends Cell Biol 26(7): 486-497.
  8. Kasas, S. and Dietler, G. (2008). Probing nanomechanical properties from biomolecules to living cells. Pflugers Arch 456(1): 13-27.
  9. Madsen, C. D., Pedersen, J. T., Venning, F. A., Singh, L. B., Moeendarbary, E., Charras, G., Cox, T. R., Sahai, E. and Erler, J. T. (2015). Hypoxia and loss of PHD2 inactivate stromal fibroblasts to decrease tumour stiffness and metastasis. EMBO Rep 16(10): 1394-1408.
  10. Picout, D. R. and Ross-Murphy, S. B. (2003). Rheology of biopolymer solutions and gels. ScientificWorldJournal 3: 105-121.
  11. Ruedinger, F., Lavrentieva, A., Blume, C., Pepelanova, I. and Scheper, T. (2015). Hydrogels for 3D mammalian cell culture: a starting guide for laboratory practice. Appl Microbiol Biotechnol 99(2): 623-636.

简介

由纯化的细胞外基质(ECM)组分(如胶原,纤维蛋白,Matrigel和甲基纤维素)组成的水凝胶系统是细胞和分子生物学研究的支柱。它们广泛用于许多应用,包括组织再生平台,研究器官发育和病理疾病模型如癌症。生物化学和生物力学性质都影响细胞和组织相容性,并且这些性质在病理疾病进展中发生改变(Cox和Erler,2011; Bonnans等人,2014)。在诸如癌症的疾病模型中使用细胞嵌入的水凝胶允许询问细胞诱导的微环境生物力学变化(Madsen等人,2015)。在这里,我们报告一种使用受控应变旋转流变仪测量这些细胞诱导的体外变化的简单方法。

背景 纤维化和实体瘤伴随着其天然组织的病理重塑(Cox和Erler,2011; Bonnans等人,2014)。在两种病理状况下,局部组织环境经历物理化学和生物学变化,导致组织刚度(弹性模量)增加(Humphrey等人,2014)。增强的组织/基质调节导致细胞行为改变,细胞形态,分化状态,增殖,迁移和干性的机械信号。在癌症的临床前动物模型中,这些变化可以驱动恶性进展和转移性扩散(Bonnans等人,2014)。不足为奇的是,靶基质硬化近年来受到了极大的关注,几项临床试验已经开始(Kai等人,2016)。
 基质组分的弹性和机械性能可以使用原子力显微镜(AFM)进行检查,原子力显微镜(AFM)是一种提供纳米分辨率并以picoNewton分辨率同时测量施加力的技术(Kasas和Dietler,2008)。然而,AFM不适用于理解较大的3D矩阵的弹性特性。使用剪切流变学可以更精确地检查体积3D矩阵的机械性能(Picout和Ross-Murphy,2003)。流变学是研究当施加力时材料如何变形。因此,将剪切应力施加到3D矩阵可以确定体积3D矩阵的弹性模量(刚度)。在该方案中,我们描述了一种通过剪切流变学测量细胞诱导的与癌相关成纤维细胞嵌入的水凝胶的基质刚度变化的方法。

关键字:剪切流变学, 基质硬度, 癌症相关成纤维细胞, 水凝胶

材料和试剂

  1. Nunc TM 细胞培养处理的多片剂,24孔(Thermo Fisher Scientific,Thermo Scientific TM,目录号:142475)
  2. 100μl无菌吸头 -
  3. 1,000μl无菌移液管尖端
  4. 1.5 ml无菌微量离心管
  5. 8毫米一次性活检穿孔器(KAI,目录号:BP-80F)
  6. 注射器过滤器,minisart,0.20μm(VWR,目录号:514-7011)
  7. 细胞:永生化的人类癌相关成纤维细胞(CAFs)(Gaggioli等人,2007)
  8. 胶原I型,高浓度,大鼠尾巴(Corning,目录号:354249)
  9. Matrigel ®基底膜基质,* LDEV-Free(Corning,目录号:354234)
  10. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:10270106)
  11. 无菌PBS,pH 7.2(Thermo Fisher Scientific,目录号:20012068)
  12. 胰蛋白酶-EDTA(0.25%),苯酚红(Thermo Fisher Scientific,Gibco TM,目录号:25200056)
  13. DMEM(Thermo Fisher Scientific,目录号:41966-052)
  14. 胰岛素转铁蛋白硒(Thermo Fisher Scientific,Gibco TM,目录号:41400045)
  15. 青霉素 - 链霉素(Thermo Fisher Scientific,Gibco TM,目录号:15140-122)
  16. Y-27632(Sigma-Aldrich,目录号:Y0503)
  17. MEMα,核苷(Thermo Fisher Scientific,Gibco TM,目录号:11900-073)
  18. 碳酸氢钠,NaHCO 3(Sigma-Aldrich,目录号:S5761)
  19. 1 M HEPES缓冲液(Thermo Fisher Scientific,Gibco TM,目录号:15630080)
  20. 5x胶原蛋白缓冲液(参见食谱)
  21. 生长培养基(见食谱)
  22. 1ml胶原I型/Matrigel水凝胶(+/-癌症相关成纤维细胞)(参见食谱)

设备

  1. 计时器
  2. 离心机
  3. 移液器
  4. 细胞培养箱在37℃,5%CO 2
  5. 发现系列混合流变仪(TA仪器,型号:DHR-2)
  6. 8 mm几何,图1a(TA Instruments)
  7. 8步mm帕尔帖板,图1a(TA Instruments)
  8. 不锈钢螺旋桨,一端平头,一端弯头,长6英寸(UNITED SCIENTIFIC SUPPLIES,型号:SSFB06)
  9. 血细胞计数器

程序

  1. 含有癌相关成纤维细胞的胶原I型/Matrigel水凝胶的制备
    1. 将所有试剂置于冰上(胶原I型,Matrigel,生长培养基,5x胶原缓冲液和FBS)。
    2. 从细胞吸出生长培养基,并用PBS短暂洗涤细胞。
    3. 吸出PBS,并加入足以覆盖细胞的胰蛋白酶-EDTA(0.25%)。
    4. 一旦细胞分离,将其重悬于正常生长培养基中并计数细胞
    5. 准备凝胶在冰上。对于1ml体积的凝胶按以下顺序添加:
      1. 100μlHN-CAF(500,000个细胞) - 细胞数量需要根据细胞系进行优化(见注释)。使用100μl生长培养基,无需嵌入细胞的水凝胶。
      2. 120μl生长培养基(依赖细胞系,但通常为DMEM,10%FBS,胰岛素转铁蛋白硒,青霉素 - 链霉素)。
      3. 100μlFBS(100%)。
      4. 80μl5x胶原蛋白缓冲液(或根据胶原量计算) - 吸出前旋涡。
      5. 200μl基质胶(或根据所需浓度,见附注)。
      6. 400μlI型胶原蛋白(或根据所需浓度,见附注)。   
    6. 通过上下移动,不引入气泡,非常好地混合胶原I型/Matrigel水凝胶。在发生气泡的情况下,水凝胶溶液在200 x g下离心几秒钟。
    7. 将1ml I型胶原I/Matrigel水凝胶转移到Nunc TM细胞培养处理的Multidishes,24孔板中的一个孔中。避免气泡。如果发生气泡,使用100微升移液管吸头吸出气泡。
    8. 将盖子放在24孔板上,将板转移到培养箱中,不加入生长培养基。
    9. 让凝胶在37℃,5%CO 2下聚合1小时。
    10. 加入1 ml生长培养基,并将板转移回培养箱(洗涤步骤)
    11. 让凝胶在37℃,5%CO 2 洗涤1小时
    12. 吸收生长培养基而不接触凝胶。
    13. 加入1毫升新鲜生长培养基,并将板转移到培养箱中,37℃,5%CO 2。
    14. 使细胞在37℃,5%CO 2的培养箱中重塑其周围的凝胶24-72小时(或直到需要测量)。在使用CAF期间不需要更换介质,但这取决于使用的特定单元格类型。

  2. 测量细胞改造凝胶的相对刚度
    使用TA Instruments DHR-2控制应变旋转流变仪对所有水凝胶样品进行流变学表征,其使用8mm喷砂平行板几何形状。下表1概述了使用Discovery Series混合流变仪(TA Instruments)确定的上述水凝胶装置的最佳测试参数。

    表1.流变仪设置


    1. 根据制造商的说明启动和校准流变仪。
    2. 将阶梯式下几何形状连接到珀耳帖板(图1a)。
    3. 连接8 mm直径的上几何体(图1a)。
    4. 将珀尔帖温度设置为所需温度(表1)
    5. 零轴向力。
    6. 将水凝胶从24孔板中取出,不要使用不锈钢刮刀损坏凝胶(图1b和1c)。
    7. 使用8毫米一次性活检穿孔将凝胶修剪成正确的尺寸(图1d和1e)。
    8. 小心地将水凝胶装入阶梯式下部几何体(图1f)
    9. 根据表1设置对数振荡应变扫描。
    10. 根据表1设置固定角度频率。
    11. 向上移动8毫米的上几何,直到它接触凝胶的顶部表面(图1g)
    12. 将间隙减小50微米,增加施加于水凝胶的轴向力(图1h)
    13. 继续,直到达到稳定的0.03 N的轴向力,详见表1.
    14. 开始测量。

  3. 相对刚度分析
    1. 确保在评估的应变范围内的线性粘弹性(储能模量[G'])响应(图1i和1j)。
    2. 当比较多个凝胶测量时,提取1%应变下的储能模量(G')(图1i和1j)。
    3. 弹性模量(E)可以使用:
      从储能模量(G')确定 E = 2×G'(1 +υ)
      哪里,
      υ=水凝胶的泊松比为0.5

      图1.流变学设置 a。较低的几何形状,peltier板和上部几何(直径8 mm)。 b。 CAF嵌入的水凝胶在一个覆盖有生长培养基的24孔板中。 C。使用刮刀将CAF嵌入的水凝胶从24孔板中取出。 d。使用8毫米一次性活检穿刺切割直径为8毫米的CAF嵌入式水凝胶活检用于分析。 e。图片说明切除后CAF-嵌入的水凝胶。 F。切下的水凝胶活检置于直径较小的几何(直径8毫米)上。 G。降低上几何形状(直径8 mm)。 H。与水凝胶接触的上几何形状。向水凝胶施加轴向力直到达到0.03N的稳定力。 CAF诱导的I型/Matrigel水凝胶重塑的代表性实例。在CAF重塑72小时后测量胶原型I/Matrigel水凝胶的储能模量(G')。 CAF在测量后仍然存在于水凝胶中(见注释)。储能模量在十年的振荡应变中测量为0.2%至2%。条形图表示在1%应变下的储能模量,并且示出增加CAF的数量引起水凝胶的硬化。 j。如何通过扰乱ROCK1/2激酶(Rho相关激酶1和2)阻止CAF诱导的I型胶原纤维素水凝胶重塑以及反过来的硬化的代表性实例。在使用10μMY-27632的ROCK1/2激酶的抑制下进行72小时CAF重塑后,测量胶原I/Matrigel水凝胶的储能模量(G')。测量后,水凝胶中的CAF仍然存在。该图表示在1%应变下的储能模量,并且示出了ROCK1/2-抑制防止CAF重塑和加强其环境。

数据分析

  1. 为了确保可靠的数据,请确保在每个实验中执行三个技术重复。当比较多个凝胶测量时,每个技术重复提取1%应变下的储能模量(G')(图1i和1j)。确保使用适当的对照进行实验三个生物学时间。
  2. 确保在评估的应变范围内的线性粘弹性(储能模量[G'])响应(图1i和1j)。如果不是这种情况,不考虑测量。如果这是一个复发问题,应该降低应变范围(小于1%),并提取 的储能模量(G'),0.1-0.5%的应变。

笔记

  1. 确保胶原I型和Matrigel溶液保持冰冷非常重要。将基底胶在冰上解冻等分(或4℃过夜)。
  2. 始终在使用前旋转5x胶原蛋白缓冲溶液。这确保了沉淀的NaHCO 3的良好再悬浮。
  3. 细胞数的优化取决于实验的设置。人们必须决定重塑将采取多快的速度。结合到凝胶中的细胞越多,重塑就越快。我们通常建议48-72 h重建高活性细胞如CAF(Madsen等人,2015)。
  4. 使用不同浓度的I型和Matrigel胶原将影响水凝胶的初始性质。浓度越高,凝胶的刚度越高。因此,可能需要更多的细胞或更长的重塑周期才能有效地检测实验时间范围内水凝胶的生物力学性质的微小变化。
  5. 当使用不同体积的I型和Matrigel胶原时,用生长培养基调整最终体积。
  6. 水凝胶嵌入的CAF的活力可以以各种方式确定(Ruedinger等人,2015):1)可以使用胶原酶/在37℃下分散处理1小时,然后使用血细胞计数器,自动细胞计数器或流式细胞术进行细胞计数。 2)总DNA/RNA水平可以在提取后测定。 3)各种代谢测定,例如MTT,CellTiter-Blue和ATP测定(Ruedinger等人,2015)。
  7. 在测试范围内,凝胶的最小频率依赖性,并在所评估的应变范围内显示出线性粘弹性响应(见图1)。
  8. 在开始测量之前向凝胶施加轴向力时,请确保测量结果一致。上述凝胶的0.03N值就足够了
  9. 始终确保比较同时进行凝胶配对实验的测量。

食谱

  1. 5x胶原蛋白缓冲液(5x是指水凝胶中使用的胶原体积)
    2.5克MEMα,核苷
    5 ml 1M HEPES缓冲液(pH 7.5)
    1g NaHCO 3
    水可达50 ml
    溶解好并过滤灭菌
    注意:将主库存存储在-20°C。在4°C储存更小的工作解决方案。
  2. 生长培养基
    DMEM
    胎牛血清(FBS)(10%)
    青霉素 - 链霉素(100U/ml)
    胰岛素 - 转铁蛋白硒(1x)
  3. 1ml具有细胞的I型胶原I型/Matrigel水凝胶 220微升生长培养基(+/-细胞)
    80μl5x胶原蛋白缓冲液(或根据胶原量) - 吸出之前的涡流 100μlFBS(100%)
    200μlMatrigel(在-80℃下储存等分试样)
    400μlI型胶原蛋白(4°C储存)

致谢

该协议改编自以前发表的论文(Madsen等人,2015; Cox等人,2013; Baker等人,2013年)。 TRC由澳大利亚NHMRC新研究者补助金支持。 CDM由瑞典所有的RagnarSöderberg基金会,BioCARE,Cancerfonden和ÅkeWiberg基金会提供支持。我们还感谢Janine Erler教授在生物技术研究与生物技术研究哥德堡大学创新中心提供流变仪。

参考文献

  1. Baker,AM,Bird,D.,Lang,G.,Cox,TR and Erler,JT(2013)。  重塑细胞外基质在发育和疾病中的应用。 Nat Rev Mol Cell Biol 15(12):786-801。
  2. Cox,TR和Erler,JT(2011)。  重塑和细胞外基质的体内平衡:对纤维化疾病和癌症的影响。 Dis Model Mech 4(2):165-178。
  3. Cox,TR,Bird,D.,Baker,AM,Barker,HE,Ho,MW,Lang,G.and Erler,JT(2013)。< a class ="ke-insertfile"href ="http: /www.ncbi.nlm.nih.gov/pubmed/23345161"target ="_ blank"> LOX介导的胶原交联负责纤维化增强的转移。癌症研究73(6) ):1721-1732。
  4. Gaggioli,C.,Hooper,S.,Hidalgo-Carcedo,C.,Grosse,R.,Marshall,JF,Harrington,K。和Sahai,E。(2007)。以成纤维细胞为主导的癌细胞的集落侵袭在前导细胞和后续细胞中的RhoGTPase具有不同的作用。 Nat Cell Biol 9(12):1392-1400。
  5. Humphrey,JD,Dufresne,ER和Schwartz,MA(2014)。  机械转导和细胞外基质体内平衡。 Nat Rev Mol Cell Biol 15(12):802-812。
  6. Kai,F.,Laklai,H.和Weaver,VM(2016)。  强制事项:疾病中细胞侵袭和迁移的生物力学调节。趋势细胞周期 26(7):486-497。
  7. Kasas,S。和Dietler,G.(2008)。从生物分子到活细胞探测纳米力学性质。 Pflugers Arch 456(1):13-27。
  8. Madsen,CD,Pedersen,JT,Venning,FA,Singh,LB,Moeendarbary,E.,Charras,G.,Cox,TR,Sahai,E。和Erler,JT(2015)。< a class = -insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/26323721"target ="_ blank">缺氧和缺失PHD2使基质成纤维细胞失活以降低肿瘤的僵硬度和转移。 em> EMBO Rep 16(10):1394-1408。
  9. Picout,DR和Ross-Murphy,SB(2003)。  生物聚合物溶液和凝胶的流变学。"科学世界日记" 3:105-121。
  10. Ruedinger,F.,Lavrentieva,A.,Blume,C.,Pepelanova,I. and Scheper,T。(2015)。  3D哺乳动物细胞培养的水凝胶:实验室实践的起始指南。 Appl Microbiol Biotechnol 99(2):623 -636。
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引用:Cox, T. R. and Madsen, C. D. (2017). Relative Stiffness Measurements of Cell-embedded Hydrogels by Shear Rheology in vitro. Bio-protocol 7(1): e2101. DOI: 10.21769/BioProtoc.2101.
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