甲醛法固定细胞外基质蛋白层以增强原代细胞生长

Natalia V.  Andreeva; Alexander V.  Belyavsky

doi:10.21769/BioProtoc.2374

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Peer-reviewed

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

Natalia V. Andreeva

Alexander V. Belyavsky email

发布: 2017年07月05日第7卷第13期 DOI: 10.21769/BioProtoc.2374 浏览次数: 8619

评审: Yanjie LiHui ZhuAnonymous reviewer(s)

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Cover of Analytical Biochemistry, featuring study using the protocol.

Dec 2016

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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)

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

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

Equipment

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

Procedure

English

中文翻译

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如何引用

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.