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Formaldehyde Fixation of Extracellular Matrix Protein Layers for Enhanced Primary Cell Growth
甲醛法固定细胞外基质蛋白层以增强原代细胞生长   

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Abstract

Coating tissue culture vessels with the components of the extracellular matrix such as fibronectin and collagens provides a more natural environment for primary cells in vitro and stimulates their proliferation. However, the effects of such protein layers are usually rather modest, which might be explained by the loss immobilized proteins due to their weak non-covalent association with the tissue culture plastic. Here we describe a simple protocol for a controlled fixation of fibronectin, vitronectin and collagen IV layers by formaldehyde, which substantially enhances the stimulation of primary cell proliferation by these extracellular proteins.

Keywords: Mesenchymal stem cells(间充质干细胞), Fibronectin(纤维连接蛋白), Vitronectin(玻连蛋白), Collagen IV(胶原蛋白IV), Formaldehyde(甲醛), Fixation(固定)

Background

The components of the extracellular matrix (ECM) such as fibronectin, laminin, vitronectin and collagens are often used for coating tissue culture vessels since they provide a more natural environment for primary cells in vitro and stimulate their proliferation (Sawada et al., 1987; Rajaraman et al., 2013). However, the observed stimulation of cell proliferation by these protein layers is usually fairly modest. This might be explained by their weak non-covalent association with the tissue culture plastic resulting in delamination and loss of immobilized protein molecules. Recently, it has been shown that the retention of ECM produced by the cells might be significantly increased by covalent immobilization of fibronectin to the plastic surface (Prewitz et al., 2013). However, ECM production is cumbersome and may be difficult to standardize. We have demonstrated that simple formaldehyde fixation under controlled conditions of layers formed by the selected individual ECM proteins can substantially enhance positive effects of these proteins on cell proliferation (Andreeva et al., 2016). Here we describe a detailed protocol of culture plastic coating and formaldehyde fixation for three individual components of ECM, namely fibronectin, vitronectin and collagen IV. Although we did not test the effects of controlled fixation with other protein constituents of the ECM, the positive results obtained with our protocol for three proteins of vastly differing molecular and biological properties provide sufficient reasons to assume the general applicability of the described procedure for enhancing proliferation stimulatory properties of a wider range of ECM proteins.

Materials and Reagents

  1. 3.5 cm cell culture dish (Greiner Bio One International, CELLSTAR®, catalog number: 627160 )
  2. Sterile pipette filter tips 200 and 1,000 μl (Greiner Bio One International, catalog numbers: 739288 and 740288 , respectively)
  3. Parafilm M (Sigma-Aldrich, catalog number: P7668 )
  4. 15 ml centrifuge tube (Greiner Bio One International, CELLSTAR®, catalog number: 188261 )
  5. 50 ml centrifuge tube (Greiner Bio One International, CELLSTAR®, catalog number: 227261 )
  6. 1.8 ml round bottom cryogenic tubes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 375418 )
  7. 0.22 μm syringe filter (GVS, catalog number: 1213641 )
  8. 10 and 20 ml syringes (SFM Hospital Products, catalog numbers: 534235 and 534236 , respectively)
  9. 2 and 10 ml Serological pipets (Greiner Bio One International, CELLSTAR®, catalog numbers: 710180 and 607107 , respectively)
  10. Phosphate-buffered saline (PBS), pH 7.4 tablets (Thermo Fisher Scientific, GibcoTM, catalog number: 18912014 )
  11. Paraformaldehyde (Sigma-Aldrich, catalog number: 76240 )
    Note: This product has been discontinued.
  12. Sodium hydroxide (NaOH), 10 M (Sigma-Aldrich, catalog number: 72068 )
  13. Fibronectin human (Imtek, catalog number: H Fne-C )
  14. Vitronectin human (Imtek, catalog number: H Vne-C )
  15. Collagen IV bovine (Imtek, catalog number: B C44-C )
  16. Acetic acid (Sigma-Aldrich, catalog number: 695092 )
  17. Ethanol 96% (Sigma-Aldrich, catalog number: 24105 )
    Note: This product has been discontinued.
  18. Sterile distilled water
  19. 1x phosphate-buffered saline (PBS) pH 7.4 (see Recipes)
  20. Formaldehyde solution (see Recipes)
  21. Human fibronectin solution (prepare freshly) (see Recipes)
  22. Human vitronectin solution (prepare freshly) (see Recipes)
  23. Bovine collagen IV solution (prepare freshly) (see Recipes)

Equipment

  1. Pipette controller (Corning, catalog number: 4091 )
  2. Automatic single-channel pipettes, 20-200 and 100-1,000 μl (Gilson-compatible)
  3. Magnetic hot plate stirrer (Sigma-Aldrich, catalog number: Z168580 )
  4. Magnetic stirring bar, 7 x 2 mm (Fisher Scientific, FisherbrandTM, catalog number: 14-513-63 )
  5. Laminar flow tissue culture hood
    Note: This item can be ordered from any qualified company.
  6. Refrigerator with interior power outlets
    Note: This item can be ordered from any qualified company.
  7. CO2 incubator (Sanyo, catalog number: MCO-18AIC )
    Note: This product has been discontinued. Possible substitute: Panasonic Healthcare, model: MCO-18AC.
  8. Centrifuge 5810 R (Eppendorf, model: 5810 R , catalog number: 5811000320)
  9. Autoclave
    Note: This item can be ordered from any qualified company.

Procedure

  1. Coating Petri dishes with fibronectin followed by formaldehyde fixation
    Note: All procedures below should be performed under a laminar flow hood.
    1. Add 1 ml of the 50 µg/ml fibronectin solution (see Recipes) to each of the 3.5-cm Petri dishes. Make sure the entire dish surface is completely covered by gently swirling the solution or redistributing it over the surface using pipette tip.
      Note: The fibronectin layer is only about 1-mm deep for the indicated volume, thus it is recommended to perform this operation with Petri dishes placed on a strictly horizontal surface to ensure even coverage of all areas of the dish.
    2. Incubate dishes with the lid on for 1.5 h at 37 °C in a humidified CO2 incubator.
    3. Aspirate carefully and save the fibronectin solution.
      Note: The saved fibronectin solution can be re-used at least once. Store it at 4 °C and use within one week.
    4. Add 1 ml of 0.4% formaldehyde solution (see Recipes). Incubate dishes with the lid on for 20 min under a sterile laminar flow hood.
    5. Aspirate carefully the formaldehyde solution, wash twice with 2 ml of sterile PBS and then 4 times with 4 ml of sterile PBS.
      Note: Formaldehyde solution should be disposed of as a hazardous waste.
    6. Dry dishes for 1 h under the laminar flow hood.
      Note: Fibronectin-covered dishes can be sealed with Parafilm and stored at 4 °C for up to 1 week before use.

  2. Coating Petri dishes with vitronectin followed by formaldehyde fixation
    Note: All procedures below should be performed under a laminar flow hood.
    1. Add 0.5 ml of the 10 µg/ml vitronectin solution (see Recipes) to each of the 3.5-cm Petri dishes. Make sure the entire dish surface is completely covered by gently swirling the solution or redistributing it over the surface using pipette tip.
      Note: The vitronectin layer is only about 0.5-mm deep for the indicated volume, thus it is recommended to perform this operation with Petri dishes placed on a strictly horizontal surface to ensure an even coverage of all areas of the dish.
    2. Incubate dishes with the lid on for 1.5 h at 37 °C in a humidified CO2 incubator.
    3. Aspirate carefully and save the vitronectin solution.
      Note: The saved vitronectin solution can be re-used at least once. Store it at 4 °C and use within one week.
    4. Add 1 ml of 0.4% formaldehyde solution (see Recipes). Incubate dishes with the lid on for 20 min under a sterile laminar flow hood.
    5. Aspirate carefully the formaldehyde solution, wash twice with 2 ml of sterile PBS and then 4 times with 4 ml of sterile PBS.
      Note: Formaldehyde solution should be disposed of as a hazardous waste.
    6. Dry dishes for 1 h under the laminar flow hood.
      Note: Vitronectin-covered dishes can be sealed with Parafilm and stored at 4 °C for up to 1 week before use.

  3. Сoating Petri dishes with collagen IV followed by formaldehyde fixation
    Note: Coating Petri dishes overnight is performed in the refrigerator, the rest of procedures should be performed under a laminar flow hood while keeping collagen solution on ice and minimizing exposure to room temperature conditions.
    1. Add 0.5 ml of the 50 µg/ml collagen IV solution (see Recipes) to each of 3.5-cm Petri dishes. Make sure the entire dish surface is completely covered by gently swirling the solution or redistributing it over the surface using pipette tip. Immediately transfer the dishes to the refrigerator.
    2. Incubate dishes with the lid on overnight at 4 °C.
      Note: The collagen layer is only about 0.5-mm deep for the indicated volume, thus it is important to perform this operation with Petri dishes placed on a strictly horizontal surface to ensure even coverage of all areas of the dish.
    3. Transfer dishes to the laminar flow hood, carefully aspirate and save collagen solution.
      Note: The saved collagen solution can be re-used at least once. Store it at 4 °C and use within one week.
    4. Add 1 ml of 0.4% formaldehyde solution (see Recipes). Incubate for 20 min under sterile laminar flow hood.
    5. Aspirate carefully the formaldehyde solution, wash twice with 2 ml of sterile PBS and then 4 times with 4 ml of sterile PBS.
      Note: Formaldehyde solution should be disposed of as a hazardous waste.
    6. Dry dishes for 1 h under the laminar flow hood.
      Note: Fibronectin-covered dishes can be sealed with Parafilm and stored at 4 °C for up to 1 week before use.

Data analysis

The assessment of the success of ECM protein coating and fixation includes analysis of MSC or other primary cell proliferation rate. Proliferation rates are determined by seeding 15 to 30 thousand cells per 3.5-cm dish followed by culturing for 1 or 2 days. A minimal testing setup includes: a) control untreated dishes; b) dishes, coated with ECM protein but unfixed; c) dishes coated with ECM and fixed with formaldehyde. Each variant is performed in triplicate, cells after culturing are collected by trypsinization followed by manual or automatic cell counting. It is recommended to perform counting of each cell sample two or three times using independent cell aliquots to increase the accuracy of analysis. It is important to assess the cell proliferation while the cells are within the exponential growth phase, that is, with cell confluence not exceeding 50%. The proliferation rate can be represented as an expansion factor (ratio of harvested to seeded cells) or population doublings (log2 of expansion factor). The results are analyzed by determining mean, standard deviations and performing Student’s t-tests. The typical result of such analysis is shown in Figure 1.


Figure 1. Controlled fibronectin fixation significantly enhances expansion of mesenchymal stem cells (MSCs). Mouse MSC growth under hypoxia conditions (5% O2) on control uncoated dishes (TCP) and dishes coated with fibronectin at a concentration of 50 µg/ml. Fibronectin-coated dishes were left unfixed (FN/0%) or fixed with 0.2% formaldehyde (FN/0.2%) or 0.4% formaldehyde (FN/0.4%). Y axis depicts the number of population doublings underwent by the culture within 24 h. Data are presented as mean ± SD.

Notes

  1. Imtek is a Russian company producing, among others, extracellular matrix proteins for cell culture. Their website: http://new.imtek.ru/eng. We have as yet no experience with the extracellular matrix proteins from other companies.
  2. The formaldehyde concentrations producing best results in our setup for 20-min fixation time are 0.3%-0.4%. Higher formaldehyde concentrations result in diminished effects on cell proliferation (Andreeva et al., 2016). When working with other extracellular matrix proteins not tested by us, it is highly advisable to perform test fixations with formaldehyde concentrations ranging from 0.2% to 0.6% to find the optimal fixation conditions.
  3. The measured temperature during formaldehyde fixation (i.e., under laminar flow hood) in our setup is 24 °C to 26 °C. Since the level of fixation depends on the temperature, it is recommended to determine the temperature under the laminar hood to make sure the deviation from the above temperature does not exceed 2 °C. If the temperature is out of this range, it is recommended to perform test fixations for somewhat different periods of time to determine the optimal fixation time for a given experimental setup.
  4. According to our results (Andreeva et al., 2016), coating Petri dishes with fibronectin at the 50 µg/ml concentration produced better results as compared to the 10 µg/ml concentration, whereas increasing fibronectin concentration to 250 µg/ml produced only a small additional effect. Although we did not test this yet, it is fairly possible that extending time for coating dishes over the 1.5-h period used in our protocol may produce a sizeable increase in the effects of fibronectin layers at 10 µg/ml concentration.
  5. Coating with vitronectin, due to a high cost of this protein, was performed at lower concentration (10 µg/ml) as compared to fibronectin and collagen IV (50 µg/ml), and produced smaller effects than those observed for the latter two proteins (Andreeva et al., 2016). Based on analogy with fibronectin results, it is likely that increasing concentration of vitronectin during plastic coating will produce larger effects.
  6. Since the treatment of plastic surfaces for different types vessels and by various manufacturers may differ, it is fairly possible that the effects of fixed extracellular matrix protein layers on cell proliferation may vary. In our hands, fibronectin coating followed by fixation was more efficient for 3.5-cm Petri dishes than for 6-well plates.
  7. Formaldehyde during storage may polymerize at lower temperatures and decompose at higher temperatures, thus decreasing the effective formaldehyde concentration. It is therefore recommended to use the prepared formaldehyde solution within one week and, preferably, prepare formaldehyde solution freshly for each fixation experiment.
  8. It is important that collagen IV is processed at low temperature to prevent its slow denaturation which occurs even at room temperature.
  9. Among the cell types tested by us, the most pronounced effects were observed for mesenchymal stem cells, both murine and human ones, smaller effects were obtained for fibroblasts, and no positive effects were observed for established cell lines (Andreeva et al., 2016).

Recipes

  1. 1x phosphate-buffered saline (PBS) pH 7.4
    2 tablets PBS (10 g)
    Add distilled water to a final volume of 1 L
    Sterilize by autoclaving
    Store at room temperature
  2. Formaldehyde solution
    Add 4.0 g paraformaldehyde to a beaker with magnetic stirring bar
    Add distilled water to a final volume 100 ml
    Boil until complete dissolution on a hot plate magnetic stirrer, cool down
    Add 10 N NaON dropwise to pH 7.4
    Store at 5 °C, use within 1 week
    Dilute prior to the experiment to the desired concentration (usually, 0.4%) with sterile 1x PBS
  3. Human fibronectin solution (prepare freshly)
    1 ml fibronectin sterile solution 1 mg/ml
    19 ml sterile 1x PBS
    Store at 4 °C, use within one week
  4. Human vitronectin solution (prepare freshly)
    0.25 mg sterile vitronectin dry powder
    25 ml sterile 1x PBS
    Store at 4 °C, use within one week
  5. Bovine collagen IV solution (prepare freshly)
    Notes:
    1. All manipulations should be performed at temperatures 0 °C to 5 °C using pre-chilled solutions.
    2. Sterilize the magnet before use in 70% ethanol for 1 h, then under UV irradiation for 1 h.
    1. Add 1.0 ml of 20 mM acetic acid (filter-sterilized) to 0.5 mg sterile collagen IV dry powder in a 1.8-ml round-bottom cryovial
    2. Add a small-size magnetic stirring bar and stir the collagen solution overnight on a magnetic stirrer at 4 °C
    3. Dilute the prepared collagen IV solution 10-fold with cold sterile 20 mM acetic acid to a final concentration of 50 µg/ml
    4. Store the final solution at temperature 4 °C, use within one week

Acknowledgments

The protocol described herein was adapted from Andreeva et al. (2016). This work was supported by the grant (No. 14-04-01855) from the Russian Foundation for Basic Research and by the Program of fundamental research for state academies for 2013-2020 years (Task 0103-2014-0006 Subprogram #58 Molecular genetics, mechanisms of realization of genetic information, bioengineering).

References

  1. Andreeva, N. V., Leonova, O. G., Popenko, V. I. and Belyavsky, A. V. (2016). Controlled formaldehyde fixation of fibronectin layers for expansion of mesenchymal stem cells. Anal Biochem 514: 38-41.
  2. Prewitz, M. C., Seib, F. P., von Bonin, M., Friedrichs, J., Stissel, A., Niehage, C., Muller, K., Anastassiadis, K., Waskow, C., Hoflack, B., Bornhauser, M. and Werner, C. (2013). Tightly anchored tissue-mimetic matrices as instructive stem cell microenvironments. Nat Methods 10(8): 788-794.
  3. Rajaraman, G., White, J., Tan, K. S., Ulrich, D., Rosamilia, A., Werkmeister, J. and Gargett, C. E. (2013). Optimization and scale-up culture of human endometrial multipotent mesenchymal stromal cells: potential for clinical application. Tissue Eng Part C Methods 19(1): 80-92.
  4. Sawada, N., Tomomura, A., Sattler, C. A., Sattler, G. L., Kleinman, H. K. and Pitot, H. C. (1987). Effects of extracellular matrix components on the growth and differentiation of cultured rat hepatocytes. In Vitro Cell Dev Biol 23(4): 267-273.

简介

用诸如纤连蛋白和胶原的细胞外基质的组分涂覆组织培养容器为体外原代细胞提供更自然的环境并刺激它们的增殖。 然而,这种蛋白质层的作用通常相当适中,这可能由于与组织培养塑料的非共价结合弱而被固定蛋白质损失所解释。 在这里我们描述一个简单的协议,通过甲醛控制纤维连接蛋白,玻连蛋白和胶原IV层的固定,这大大增强了这些细胞外蛋白对原代细胞增殖的刺激。
【背景】细胞外基质(ECM)如纤连蛋白,层粘连蛋白,玻连蛋白和胶原蛋白的组分通常用于包被组织培养容器,因为它们为体外原代细胞提供更自然的环境并刺激它们的增殖( Sawada等人,1987; Rajaraman等人,2013)。然而,观察到的这些蛋白质层对细胞增殖的刺激通常是相当适度的。这可能是由于它们与组织培养塑料的弱非共价结合而导致的,导致分离和失去固定的蛋白质分子。最近,已经表明,通过纤连蛋白共价固定到塑料表面,可以显着增加由细胞产生的ECM的保留(Prewitz等人,2013)。然而,ECM生产是麻烦的,可能难以标准化。我们已经证明,在所选择的各个ECM蛋白形成的层的控制条件下的简单甲醛固定可以显着增强这些蛋白质对细胞增殖的积极作用(Andreeva等人,2016)。在这里我们描述了ECM的三个单独组分的培养塑料涂层和甲醛固定的详细方案,即纤连蛋白,玻连蛋白和胶原IV。虽然我们没有测试与ECM的其他蛋白质成分的控制固定的效果,但是通过我们的方案获得的三种具有极大不同分子和生物学特性的蛋白质的阳性结果提供了足够的理由来假定所述的增强增殖过程的一般适用性更广泛的ECM蛋白的刺激性质。

关键字:间充质干细胞, 纤维连接蛋白, 玻连蛋白, 胶原蛋白IV, 甲醛, 固定

材料和试剂

  1. 3.5厘米细胞培养皿(Greiner Bio One International,CELLSTAR ®,目录号:627160)
  2. 无菌吸管过滤嘴200和1,000μl(Greiner Bio One International,目录号分别为739288和740288)
  3. Parafilm M(Sigma-Aldrich,目录号:P7668)
  4. 15ml离心管(Greiner Bio One International,CELLSTAR ®,目录号:188261)
  5. 50ml离心管(Greiner Bio One International,CELLSTAR ®,目录号:227261)
  6. 1.8毫升圆底低温管(Thermo Fisher Scientific,Thermo Scientific TM,目录号:375418)
  7. 0.22μm注射器过滤器(GVS,目录号:1213641)
  8. 10和20毫升注射器(分别为SFM医院产品,目录号:534235和534236)
  9. 2和10ml血清移液管(Greiner Bio One International,CELLSTAR ®,目录号:710180和607107)
  10. 磷酸缓冲盐水(PBS),pH 7.4片(Thermo Fisher Scientific,Gibco TM,目录号:18912014)
  11. 多聚甲醛(Sigma-Aldrich,目录号:76240)
    注意:本产品已停产。
  12. 氢氧化钠(NaOH),10M(Sigma-Aldrich,目录号:72068)
  13. 纤连蛋白人类(Imtek,目录号:H Fne-C)
  14. Vitronectin人类(Imtek,目录号:H Vne-C)
  15. 胶原蛋白IV牛(Imtek,目录号:B C44-C)
  16. 乙酸(Sigma-Aldrich,目录号:695092)
  17. 乙醇96%(Sigma-Aldrich,目录号:24105)
    注意:本产品已停产。
  18. 无菌蒸馏水
  19. 1x磷酸盐缓冲盐水(PBS)pH 7.4(参见食谱)
  20. 甲醛溶液(见配方)
  21. 人纤连蛋白溶液(新鲜制备)(参见食谱)
  22. 人玻连蛋白溶液(新鲜制备)(参见食谱)
  23. 牛胶原IV溶液(新鲜制备)(参见食谱)

设备

  1. 移液器控制器(Corning,目录号:4091)
  2. 自动单通道移液器,20-200和100-1,000μl(Gilson兼容)
  3. 磁性热板搅拌器(Sigma-Aldrich,目录号:Z168580)
  4. 7×2mm的电磁搅拌棒(Fisher Scientific,Fisherbrand TM,目录号:14-513-63)
  5. 层流组织培养罩
    注意:该项目可以从任何合格的公司订购。
  6. 带内置电源插座的冰箱
    注意:该项目可以从任何合格的公司订购。
  7. CO 3培养箱(Sanyo,目录号:MCO-18AIC)
    注意:本产品已停产。可能的替代品:Panasonic Healthcare,型号:MCO-18AC。
  8. 离心机5810 R(Eppendorf,型号:5810 R,目录号:5811000320)
  9. 高压灭菌器
    注意:该项目可以从任何合格的公司订购。

程序

  1. 涂布纤维连接素培养皿,然后甲醛固定 注意:以下所有步骤均应在层流罩下进行。
    1. 加入1 ml的50μg/ ml纤连蛋白溶液(参见食谱)到每个3.5厘米培养皿中。确保整个菜表面完全覆盖,轻轻地旋转溶液或使用移液器吸头将其重新分布在表面上。
      注意:纤连蛋白层对于指定体积只有约1毫米深,因此建议用放置在严格水平表面上的培养皿进行此操作,以确保均匀覆盖该盘的所有区域。 >
    2. 在加湿的CO 2培养箱中,将盖子与37℃孵育1.5小时。
    3. 小心吸出并保存纤连蛋白溶液。
      注意:保存的纤连蛋白溶液可以重复使用至少一次。将其储存在4°C,并在一周内使用。
    4. 加入1 ml的0.4%甲醛溶液(参见食谱)。在无菌层流罩下用盖子孵育20分钟
    5. 小心吸出甲醛溶液,用2 ml无菌PBS洗涤两次,然后用4 ml无菌PBS洗涤4次。
      注意:甲醛溶液应作为危险废物处理。
    6. 在层流罩下干燥1小时。
      注意:纤连蛋白覆盖的菜肴可以用Parafilm密封,并在使用前在4℃下储存长达1周。

  2. 涂覆维生素后培养皿,然后甲醛固定 注意:以下所有步骤均应在层流罩下进行。
    1. 向每个3.5厘米培养皿中加入0.5毫升10微克/毫升的玻连蛋白溶液(参见食谱)。确保整个菜表面完全覆盖,轻轻地旋转溶液或使用移液器吸头将其重新分布在表面上。
      注意:针对所指定的体积,玻连蛋白层的深度仅为0.5毫米深,因此建议使用放置在严格水平表面的培养皿进行此操作,以确保均匀覆盖该盘的所有区域。 / >
    2. 在加湿的CO 2培养箱中,将盖子与37℃孵育1.5小时。
    3. 仔细吸出并保存玻连蛋白溶液。
      注意:保存的玻连蛋白溶液可以至少重新使用一次。将其储存在4°C,并在一周内使用。
    4. 加入1 ml的0.4%甲醛溶液(参见食谱)。在无菌层流罩下用盖子孵育20分钟
    5. 小心吸出甲醛溶液,用2 ml无菌PBS洗涤两次,然后用4 ml无菌PBS洗涤4次。
      注意:甲醛溶液应作为危险废物处理。
    6. 在层流罩下干燥1小时。
      注意:使用玻璃纤维素覆盖的菜肴可以用Parafilm密封,并在使用前在4℃下储存长达一周。

  3. 用胶原蛋白IV培养培养皿,然后用甲醛固定 注意:在冰箱中进行涂层培养皿过夜,其余步骤应在层流罩下进行,同时将胶原蛋白溶液保持在冰上,并尽量减少暴露于室温条件。
    1. 向每个3.5厘米培养皿中加入0.5毫升50微克/毫升胶原蛋白IV溶液(见食谱)。确保整个菜表面完全覆盖,轻轻地旋转溶液或使用移液器吸头将其重新分布在表面上。立即将菜肴转移到冰箱。
    2. 在4℃下将盖子与盖子一起孵育。
      注意:指定体积的胶原蛋白层的深度仅为0.5毫米深,因此使用放置在严格水平表面上的培养皿进行此操作非常重要,以确保均匀覆盖该盘的所有区域。 >
    3. 将盘子转移到层流罩上,小心吸出并保存胶原蛋白溶液 注意:保存的胶原蛋白溶液可以至少重新使用一次。将其储存在4°C,并在一周内使用。
    4. 加入1 ml的0.4%甲醛溶液(参见食谱)。在无菌层流罩下孵育20分钟。
    5. 小心吸出甲醛溶液,用2 ml无菌PBS洗涤两次,然后用4 ml无菌PBS洗涤4次。
      注意:甲醛溶液应作为危险废物处理。
    6. 在层流罩下干燥1小时。
      注意:纤维连接蛋白覆盖的菜肴可以用Parafilm密封,并在使用前在4℃下储存长达一周。

数据分析

ECM蛋白涂层和固定成功的评估包括MSC或其他原代细胞增殖率的分析。通过每3.5cm培养皿接种15-30万个细胞,然后培养1或2天来确定增殖速率。最小的测试设置包括:a)控制未经处理的菜肴; b)涂有ECM蛋白但不固定的餐具; c)涂有ECM并用甲醛固定的盘子。每个变体一式三份进行,培养后的细胞通过胰蛋白酶消化收集,随后手动或自动细胞计数。建议使用独立细胞等分试样对每个细胞样品进行两次或三次的计数,以提高分析的准确性。当细胞处于指数生长期,即细胞汇合不超过50%时,评估细胞增殖是重要的。增殖率可以表示为扩增因子(收获到种子细胞的比例)或群体倍增(扩增因子的log 2)。通过确定平均值,标准偏差和执行Student's 测试来分析结果。这种分析的典型结果如图1所示

图1.受控的纤连蛋白固定显着增强间充质干细胞(MSC)的扩增。 在对照未涂覆的培养皿(TCP)和50μg/ ml浓度的纤连蛋白包被的培养皿上,在缺氧条件(5%O 2 2)下的小鼠MSC生长。将纤连蛋白包被的培养皿未固定(FN / 0%)或用0.2%甲醛(FN / 0.2%)或0.4%甲醛(FN / 0.4%)固定。 Y轴描绘在24小时内由文化进行的群体倍增数。数据以平均值±SD表示。

笔记

  1. Imtek是一家俄罗斯公司,其中生产用于细胞培养的细胞外基质蛋白。他们的网站: http://new.imtek.ru/eng 。我们还没有其他公司的细胞外基质蛋白的经验。
  2. 我们设置20分钟固色时间的甲醛浓度最高,为0.3%-0.4%。较高的甲醛浓度导致对细胞增殖的影响减弱(Andreeva等人,2016)。当与未经我们测试的其他细胞外基质蛋白一起使用时,建议使用甲醛浓度范围为0.2%至0.6%的测试固定剂,以找到最佳的固定条件。
  3. 我们设置的甲醛固定(即层流罩下)的测量温度为24°C至26°C。由于固定水平取决于温度,因此建议确定层流罩下的温度,以确保与上述温度的偏差不超过2°C。如果温度超出此范围,建议在稍微不同的时间段内执行测试固定,以确定给定实验装置的最佳固定时间。
  4. 根据我们的结果(Andreeva等人,2016),与50μg/ ml浓度的纤维连接蛋白涂布培养皿与10μg/ ml浓度相比产生更好的结果,而增加纤连蛋白浓度250μg/ ml只产生一个小的附加效应。虽然我们还没有测试,但是在我们的方案中使用的1.5小时时间内延长涂布时间是非常有可能产生10μg/ ml浓度的纤连蛋白层效应的显着增加。
  5. 与纤连蛋白和胶原IV(50μg/ ml)相比,以较低的浓度(10μg/ ml)进行玻连蛋白的涂覆,由于该蛋白质的高成本,并且产生比后两种蛋白质观察到的效果更小的效果(Andreeva等人,,2016)。基于与纤连蛋白结果的类比,塑料涂层中玻连蛋白浓度的增加可能会产生更大的影响。
  6. 由于用于不同类型血管和各种制造商的塑料表面的处理可能不同,固定的细胞外基质蛋白质层对细胞增殖的影响是可能变化的。在我们手中,纤连蛋白涂层,然后固定,对于3.5厘米培养皿比6孔板更有效。
  7. 储存期间甲醛可在较低温度下聚合,并在较高温度下分解,从而降低有效甲醛浓度。因此,建议在一周内使用所制备的甲醛溶液,最好每次固定实验新鲜制备甲醛溶液。
  8. 重要的是,在低温下处理胶原蛋白IV以防止其甚至在室温下发生的慢变性。
  9. 在我们测试的细胞类型中,观察到间充质干细胞(鼠类和人类)中最显着的作用,对于成纤维细胞获得较小的效果,并且对已建立的细胞系没有观察到阳性效应(Andreeva等人,2016)

食谱

  1. 1x磷酸盐缓冲盐水(PBS)pH 7.4 2片PBS(10克)
    加入蒸馏水至最终体积为1升 高压消毒灭菌
    在室温下存放
  2. 甲醛溶液
    用磁力搅拌棒将4.0g多聚甲醛加入到烧杯中 加入蒸馏水至最终体积100 ml 煮沸直到完全溶解在热板磁力搅拌器上,冷却下来
    将10 N NaON滴加至pH 7.4 储存于5°C,1周内使用
    在无菌1x PBS下将实验前稀释至所需浓度(通常为0.4%)
  3. 人纤连蛋白溶液(新鲜准备)
    1 ml纤连蛋白无菌溶液1 mg / ml
    19 ml无菌1x PBS
    储存于4°C,一周内使用
  4. 人玻连蛋白溶液(新鲜准备)
    0.25毫克无菌玻连蛋白干粉
    25 ml无菌1x PBS
    储存于4°C,一周内使用
  5. 牛胶原IV溶液(新鲜准备)
    注意:
    1. 所有操作应在0℃至5℃的温度下使用预冷的溶液进行。
    2. 在70%乙醇中使用磁铁1小时之前,然后在紫外线照射下1小时,将磁铁灭火。
    1. 将1.0ml 20mM乙酸(过滤灭菌)加入到1.8ml圆底冰箱中的0.5mg无菌胶原IV干粉中,
    2. 加入一个小尺寸的磁力搅拌棒,并在4°C的磁力搅拌器下将胶原溶液搅拌过夜
    3. 用冷无菌20mM乙酸稀释制备的胶原IV溶液10倍至终浓度为50μg/ ml
    4. 将最终溶液储存在4°C的温度下,在一周内使用

致谢

本文描述的方案改编自Andreeva等人。(2016)。这项工作得到俄罗斯基础研究基金会的资助(第14-04-01855号)和2013-2020年度国家学院基础研究计划的支持(任务0103-2014-0006子程序#58分子遗传学遗传信息的实现机制,生物工程)。

参考

  1. Andreeva,NV,Leonova,OG,Popenko,VI和Belyavsky,AV(2016)。  用于扩展间充质干细胞的纤连蛋白层的受控甲醛固定。 Anal Bioch 514:38-41。
  2. Prewitz,MC,Seib,FP,von Bonin,M.,Friedrichs,J.,Stissel,A.,Niehage,C.,Muller,K.,Anastassiadis,K.,Waskow,C.,Hoflack,B.,Bornhauser ,M.和Werner,C。(2013)。紧密锚定的组织模拟基质作为指导性的干细胞微环境。 Nat方法 10(8):788-794。
  3. Rajaraman,G.,White,J.,Tan,KS,Ulrich,D.,Rosamilia,A.,Werkmeister,J.and Gargett,CE(2013)。< a class =“ke-insertfile”href = http://www.ncbi.nlm.nih.gov/pubmed/22738377“target =”_ blank“>人子宫内膜多能间充质基质细胞的优化和放大培养:临床应用的潜力。组织工程C部分方法 19(1):80-92。
  4. Sawada,N.,Tomomura,A.,Sattler,CA,Sattler,GL,Kleinman,HK和Pitot,HC(1987)。< a class =“ke-insertfile”href =“http://www.ncbi .nlm.nih.gov / pubmed / 2883170“target =”_ blank“>细胞外基质组分对培养的大鼠肝细胞的生长和分化的影响。体外细胞发育生物 23( 4):267-273。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Andreeva, N. V. and Belyavsky, A. V. (2017). Formaldehyde Fixation of Extracellular Matrix Protein Layers for Enhanced Primary Cell Growth. Bio-protocol 7(13): e2374. DOI: 10.21769/BioProtoc.2374.
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