Tissue-specific extracellular matrix coatings for the promotion of cell proliferation and maintenance of cell phenotype
Introduction
Cell-extracellular matrix (ECM) interactions play a fundamental role in cell growth, organ development, tissue regeneration, and wound healing as well as in malignant growth processes. In vivo, cells attach to proteins and carbohydrate moieties present in the extracellular matrix (ECM). Examples of native forms of ECM include interstitial matrix and basal lamina. These structures provide support and anchorage for cells, segregate tissues from one another and regulate intercellular communication. However, once cells are isolated from tissue and removed from the native matrix, differentiated cells rapidly lose important characteristics when cultured without an adequate supportive microenvironment such as a substrate coating or a feeder layer. Cultured cells are influenced by soluble factors (e.g. growth factors and cytokines), physical factors (e.g. stress and strain) [1], [2] and by the insoluble matrix microenvironment [3]. However, the overwhelming majority of current research on this subject has focused on the use of soluble factors to influence cell growth [4], [5] although numerous groups have shown the importance of providing tissue-specific forms of ECM as a substratum for culturing cells in order to maintain phenotypic and functional characteristics [6], [7], [8], [9], [10], [11]. For example, it is particularly difficult to maintain functional hepatocytes in culture, but when these cells are cultured with hormones, growth factors, serum free medium and ECM, the hepato-cellular physiology (including albumin synthesis and urea metabolism) can be maintained [11]. However, ECM cues are multi-factorial and complex, and mimicking them in a culture system is exceedingly difficult. When functional cells, such as liver cells, were cultured in vitro, the influences of hormones, growth factors, serum free medium and ECM, regulated the hepato-cellular physiology [11].
Specific components from the ECM are commonly used as a culture substratum and are commercially available. Individual matrix components (e.g. collagens, fibronectins, laminins) have been used in cell culture for many years and have been shown to have profound effects on cells, both with respect to attachment and survival as well as for the maintenance of various functions [12], [13]. Certain tissue extracts enriched in matrix (e.g. Matrigel, extracts from amnions) have well-documented, dramatic effects on cells in culture [14]. However, these extracts are not tissue-specific. Moreover, non-human tissue extracts enriched in matrix, such as Matrigel, cannot be considered for clinical purposes.
Tissue-specific ECM coatings for tissue culture dishes and scaffolds for supporting cell growth have attracted attention in recent years [7]. Subtle differences in ECM composition from one type of tissue to another can affect cellular interactions in a lineage-specific manner. Due to the unique capability of each tissue's ECM to provide an optimal substrate for specific cell types to attach and grow in vivo, cell culture systems seeking to maintain normal cell function should make use of similar strategies. This type of culture system would provide desirable cell–substrate interactions and would also sustain cell growth while maintaining phenotype and function. It has already been shown that tissue-specific ECM can improve the reliability and efficiency of cell culture and stem/progenitor cell differentiation [7]. Once inexpensive alternatives to conventional coatings are developed, use of this methodology may prove to be more economical for use in production of bio-pharmaceuticals and cell therapy.
The intricate and highly ordered nature of the ECM makes it difficult to reproduce using synthetic or purified components. In an attempt to reconstitute the mature cell niche in vitro, we prepared tissue-specific ECMs from skin, muscle and liver, and used these tissue-specific ECMs to coat culture dishes in which cells from each tissue could be grown. The goals of this study were to demonstrate that specific ECM derived from target tissues can produce an optimal substrate for in vitro culture and to develop optimal ECM-based culture systems for skin, skeletal muscle and liver cells for regenerative therapies and drug development.
Section snippets
Skin, skeletal muscle and liver tissue harvest
Institutional Review Board and Institutional Animal Care and Use Committee approvals for this project were obtained for human and animal surgery, including the collection of skeletal muscle, skin and liver tissue samples. Fresh and frozen organs and tissues were used to obtain the ECMs and cells for culture. Skeletal muscle tissue was harvested from the quadriceps and hamstring muscle of adult Fischer 344 × Brown Norway F1 rats. Liver tissues were harvested from the same rats. Skin tissues were
Histological evaluation of decellularized matrices
The overall scheme of the experimental design of our study is shown in Fig. 1. In order to evaluate the efficiency of decellularization, histological staining of tissue sections was carried out. In fresh-frozen control tissues, intense cellular remnants, specifically nuclear material, were obvious in H&E (Fig. 2a,c,e) and DAPI (Fig. 2g,i,k) stained sections. There was minimal inter-fascicular and intra-fascicular space present in the H&E-stained sections of the fresh-frozen muscle and liver
Discussion
Although remarkable advances have been made in cell culture techniques that have allowed for the proliferation and maintenance of specific cell phenotypes and functions, the current conservative culture system does not offer the ability to enhance cell expansion while retaining cellular functionality for multiple culture passages or in long-term cultures [18], [19]. The development of new culture conditions, including modification of culture surface coatings, would be beneficial in culturing
Conclusions
Synthetic and natural biomaterials have commonly been used as substrates for culturing primary mammalian cells. While isolate differentiated cell types grow well in culture, their phenotypic and functional characteristics may change during long-term culture without an inadequate supportive microenvironment or feeder layer. This study demonstrated that tissue-specific matrix components cause significant differences in adhesion efficiencies, growth rates, and morphology and phenotypes of skin,
Acknowledgements
The study has been supported by NIH STTR 1R41EB005900-01A1. The authors would like to thank Dr. Lola M. Reid for her valuable comments and Dr. Jennifer Olson for editorial assistance with this manuscript.
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