Elsevier

Methods

Volume 171, 15 January 2020, Pages 68-76
Methods

Fabrication and characterization of extracellular matrix scaffolds obtained from adipose-derived stem cells

https://doi.org/10.1016/j.ymeth.2019.07.004Get rights and content

Highlights

  • Extracellular matrix (ECM) scaffolds are fabricated from cultured adipose-derived stem cells (ASCs).

  • Methods to assess the molecular composition and biological properties of the ECM are presented.

  • The microstructure and native constituents are preserved in the scaffolds.

  • The decellularized scaffolds provide a useful platform to study the role of ECM deposited by ASCs in wound healing.

Abstract

Chronic non-healing wounds are detrimental for the quality of life of the affected individuals and represent a major burden for the health care systems. Adipose-derived stem cells (ASCs) are being investigated for the development of novel treatments of chronic wounds, as they have shown several positive effects on wound healing. While these effects appear to be mediated by the release of soluble factors, it is has also become apparent that the extracellular matrix (ECM) deposited by ASCs is essential in several phases of the wound healing process. In this work, we describe an approach to produce ECM scaffolds derived from ASCs in culture. Upon growth of ASCs into an overconfluent cell layer, a detergent-based cell extraction approach is applied to remove the cellular components. The extraction is followed by an enzymatic treatment to remove the residual DNA. The resultant cell-derived scaffolds are depleted of cellular components, display low DNA remnant, and retain the native fibrillar organization of the ECM. Analysis of the molecular composition of the ECM scaffolds revealed that they are composed of collagens type I and III, and fibronectin. The decellularized scaffolds represent a substrate that supports adhesion and proliferation of primary human fibroblasts and dermal microvascular endothelial cells, indicating their potential as platforms for wound healing studies.

Introduction

Normal wound healing progresses through a series of highly regulated partially overlapping phases involving a complex interplay between the different cells residing in or migrating into the wound bed [1]. A multitude of conditions, including diabetes, trauma or vascular insufficiency, may perturb the healing process in such a manner that the wound healing halts, and the wound becomes chronic [2]. Chronic wounds are defined as wounds that have failed to heal within 3 months and, as such, they impose a major strain on patients, whose quality of life becomes invariably deteriorated. Importantly, chronic wounds represent a major burden to the health care systems, since the associated costs represent a relatively big proportion of the health budget. In the EU, for example, it is estimated that the treatment of chronic wounds drains approximately 2% of the financial resources [3], [4].

The current treatment approaches, which are largely based on dressings to maintain the humidity of the wound and to protect against further trauma, are often inefficient [1]. In the quest to develop new and more efficient treatments, the use of adipose-derived stem cells (ASCs) seems to offer a new promise. ASCs have namely been shown to exert a number of properties that are critical for would healing, such as the capability to reduce inflammation, promote angiogenesis and fibroblast proliferation and migration [5], [6]. Not surprisingly, the augmentation of healing has been demonstrated in vitro, and was initially attributed to the soluble factors secreted from ASCs [7], [8]. However, as ASCs produce both structural elements as well as factors involved in extracellular matrix (ECM) maturation [9], and ECM integrity and composition play a role in wound healing, it is plausible that the wound healing effects of ASCs are, at least in part, mediated through effects on the ECM [10]. Although there is an increasing body of evidence from preclinical and early-stage clinical trials, which strongly indicate that application of ASCs to chronic wounds accelerate the wound closure [11], little is known about the specific contribution of the ECM in this process. Interestingly, cell-free ECM scaffolds obtained from cultured bone marrow-derived mesenchymal stem cells (BM-MSCs), which share similarities to ASCs in terms of wound healing properties [12], have been shown to promote cutaneous wound healing in an animal model [13]. While the improved healing appears to be mediated by enhanced re-epithelialization and angiogenesis, the detailed molecular picture of the processes remains elusive, and it would be of interest to obtain a deeper understanding of the role played by the ECM. To that end, the establishment of a wound healing model that would allow exploration of decellularized ASC ECM would be highly desirable.

Decellularized matrices derived from cell cultures have gained increasing attention in biomedical research over the past years [14]. They have been mainly employed as in vitro models in studies aimed at investigating the role of ECM components in the control of cell fate [15], [16], [17]. In general, following a period of culture of the cells on an appropriate substrate, the cellular components are removed by physical, chemical and/or enzymatic treatments. The resulting matrix, often referred as a cell-derived ECM scaffold, consists of a complex assembly of fibrillar proteins, associated macromolecules and growth factors resembling the natural ECM microenvironment [18], [19], [20]. The major parameters that influence the properties and composition of the ECM scaffold are the cell type used, the culture conditions, and the decellularization approach [21]. Various protocols for the production of cell-derived ECM scaffolds, based on fibroblasts of BM-MSCs cultures, are available in the literature [20], [22], [23]. However, given the different origin and location of these cells, their ECM display compositional and structural differences to that produced by ASCs [24], [25]. Furthermore, none of the previously reported protocols fits accurately the requirements of our intended application in terms of preservation of the ECM structural integrity, removal of cellular remnant, and suitability for downstream wound healing relevant assays.

In this paper, we present an approach for the production and decellularization of ECM from ASCs, which constitute a platform to study the particular properties of the ECM in the absence of the confounding effects of ASCs and their continuous production of soluble factors. The focus has been to devise an approach that produces an ECM scaffold suitable for the development of in vitro wound healing models.

Section snippets

Culture of ASCs

Human ASCs cultures were initiated from adipose tissue obtained from three healthy donors undergoing elective liposuction. The cell isolation protocol has been described in detail elsewhere [26]. The protocol was approved by the regional Committee on Biomedical Research Ethics of Northern Jutland, Denmark (project no. VN 2005/54). These ASC cultures have been thoroughly characterized by our group in terms of their stem cell properties, which include multilineage differentiation capacity,

Assessment of culture conditions to prevent cell detachment

In contrast to ASCs in the control wells (Fig. 1, control) or in the PLL-coated wells (Fig. 1, PLL), supplementation of the culture medium with AA over the 10-day period supported the growth of an overconfluent cell layer (Fig. 1, AA, PLL). The significant effect of AA on promoting cell growth and ECM synthesis has been described in the literature for ASCs and various other types of adherent cells, including BM-MSCs and fibroblasts [35], [36], [37]. AA has a well-documented role as coenzyme in

General considerations and hints for troubleshooting

For the induction of cells for ECM production, we recommend the preparation of a sterile stock solution of AA (20 mM, for instance), which must be stored at 4 °C and protected from light. AA should be added to the medium immediately before media changes. It is possible to increase the induction period for more than 10 days to increase the matrix thickness. However, longer incubation times appear to increase the risk of dislodgment of the matrices.

A critical point in the fabrication process

Conclusions

The present work is describing a reliable method to obtain extracellular matrices produced by cultured ASCs. The decellularization treatment efficiently removed the cellular components while maintaining a considerable amount of well-preserved extracellular matrix components. The ECM displayed a dense network of fibrillar components (type I and III collagens), as well as a mesh of fibronectin fibrils. Cell growth experiments showed that the decellularized matrices supported growth of fibroblasts

Acknowledgments

The authors would like to acknowledge Ole Jensen and Lisa Engen for technical assistance during the laboratory experiments.

Funding

The work was supported in part by funds from the Toyota Foundation and the Obelske Family Foundation. The funding sources had no influence on neither study design, collection, analysis, interpretation of data, writing the report, nor decision to submit the paper for publication.

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