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, Fibronectin, Vitronectin, Collagen IV, Formaldehyde, Fixation
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
Equipment
Procedure
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
Recipes
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
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