TITLE: Decellularized adipose tissue solid foams for hDPSC differentiation to osteogenic and adipogenic cells in 3D culture
AUTHORS: Gaskon Ibarretxe

[DESCRIPTION]
Human Dental Pulp Stem Cells (hDPSCs) constitute one very interesting stem cell source for tissue engineering and regeneration, owing to their high multi-lineage differentiation ability, the absence of ethical issues as they are considered a biological waste from dental clinics, their high immune-tolerance upon in vivo transplantation, and their ample possibilities of implementation in personalized medicine and autologous tissue engineering therapies. Natural extracellular matrix (ECM)-based materials constitute very promising scaffolds for in vitro cell culture, enhancing the attachment and differentiation of cells as well as mimicking the in vivo environment more accurately. Among others, adipose tissue represents a potentially abundant source of human and animal origin ECM which can be easily obtained in large quantities by liposuction surgery. The combination of hDPSCs with decellularized adipose ECM-derived materials offers great possibilities for the development of novel 3D culture platforms for the tailored (re)generation of diverse tissues like bone and adipose tissue. In this chapter, we describe different 3D culture methodologies of hDPSCs on biologic scaffold materials composed of decellularized porcine and human adipose tissue. These 3D culture systems offer a much preferable alternative to traditional tissue culture treated cell culture plates in 2D and/or single protein composed biomaterials because, by better preserving the bioinductive signals that participate in the interaction between the cells and the extracellular matrix in native tissues, they reproduce a much closer situation to in vivo conditions.

[GUIDELINES]
1. Introduction
Human dental pieces are often extracted in dental clinics because of orthodontic or diverse oral health reasons. Taking into account that millions of human teeth worldwide are immediately disposed of after their extraction every year, this constitutes a great waste of a source of high quality stem cells. Since their discovery in the year 2000 [1], hDPSCs have attracted considerable attention due to their high capacity to differentiate to many mature cell lineages, including not only the classical mesenchymal cell-related lineages (i.e. bone, cartilage, adipose) but also non-mesenchymal ones [2–4]. Particularly, these cells have shown a high capacity to generate adult neural cells (i.e. glia and neuron-like cells), which opens the door to their application on therapy of central and peripheral nerve system lesions [5, 6]. HDPSCs can be cultured both under serum-containing and serum-devoid conditions. Conventional culture methodology involves the addition of Fetal Bovine Serum (FBS), which favors the acquisition of a plastic-adherent mesenchymal cell phenotype of hDPSC cultures, and ultimately their osteo/odontoblastic differentiation [7, 8]. On the contrary, when hDPSCs are cultured in FBS-free media on low-binding plastic flasks, they form floating cell aggregates, called dentospheres, which preserve much better the neural crest progenitor characteristics of these cells [5].
The design of new 3D culture systems that better mimic the interactions that occur between cells and the extracellular matrix (ECM) in live tissues is a long-standing demand of the tissue engineering and pharmaceutical industry. Many pharmaceutical compounds yielding a promising biological activity in conventional in vitro 2D cultures fail to reproduce those results when they are applied in vivo [9]. A more cost-efficient alternative would be to first test those compounds under 3D culture conditions, containing bioscaffolds that more accurately mimic the natural composition and 3D structure of tissues. Specifically, the biologic scaffold materials composed of ECM have been successfully applied in both preclinical and clinical studies [10]. Adipose tissue represents an abundant source of human origin ECM which can be obtained by liposuction [11]. Interestingly, the adipose tissue ECM composition includes a high abundance of components of the Basement Membrane (BM) [12]. Our research group had previously described the processing of human and porcine adipose tissues to obtain decellularized adipose tissue (DAT) scaffolds which may be implemented to 3D culture systems [13].
The assessment of biological activity of a pharmacological reagent under 3D culture conditions could provide very useful information before switching to in vivo studies and clinical trials. Life science research and industry are increasingly aware of the potential of the 3D culture system for the better monitoring of cell behavior [14]. Furthermore, if both the cells and the scaffold were derived from tissues of the same patient, it would be possible to make a completely autologous 3D culture, paving the way for strictly personalized medicine applications.
Modern decellularization technologies allow the isolation of ECM components from a great variety of tissues [15]. DATs are very promising biocompatible scaffold for 3D cell culture, owing to their high concentration of bioactive molecules and adhesion glycoproteins, which favor the adhesion and survival of the seeded cells [13, 16–18]. This material can be formulated in a variety of textures and incorporated to different cell culture plates and devices. In this work, we will describe the methodology of hDPSC 3D culture using DAT bioscaffolds of both porcine (pDAT) and human (hDAT) origin, which are incorporated to conventional cell culture plates. We use this system to generate in vitroreconstructions of bone-like and adipose-like tissues.

[STEPS]
1. Decellularized adipose tissue solid foams for hDPSC differentiation to osteogenic and adipogenic cells in 3D culture