**1. Introduction**

In many adult animal tissues, the main component in terms of volume is not the cells, but the cell-secreted three-dimensional (3D) structure known as the extracellular matrix (ECM). Structural and specialized proteins and proteoglycans are some of the ECM's macromolecular components, and they interact in this network with multiple key roles and functions. During homeostasis, the ECM provides structural integrity and mechanical support for tissues and organ architecture. In parallel, the ECM is the reservoir and the place for the active exchange of ions, nutrients, waters, metabolites, and signals [1]. In this way, the ECM serves as the environment in which tissue-resident cells attach, communicate, and interact, thereby regulating cell dynamics and behavior, and contributing to the maintenance of tissue-specific cell phenotypes and functions (Figure 1). Notably, the ECM provides a niche for tissue-resident stem cells and drives their fate decisions, a property particularly relevant during homeostasis and tissue repair–regeneration processes.

**Figure 1.** Structure, components, and functions of extracellular matrix (ECM) (MMP, matrix metalloprotease; GF, growth factor).

The properties of the ECM have been thoroughly used in tissue engineering and regenerative medicine research, aiming to restore the function of damaged or dysfunctional tissues [2]. In this context, applications of ECM-derived components are multiple, from in vitro stem cell basic research to clinical settings. For example, ECM-derived components have been used as surface coatings for cell adhesion purposes; gel matrices for establishing organoid cultures; 3D environments for cell seeding and growth factor delivery approaches; and as biocompatible and biomimetic implantable allograft or xenograft scaffolds with in vivo tissue regeneration properties. Often, strategies are based on specific ECM components, such as collagens, fibrin, hyaluronic acid, or even cell-culture-derived ECM. Using these materials, multiple fabrication techniques have been implemented to generate successful ECM-derived biomimetic structures such as hydrogels, or even more sophisticated engineering approaches such as micropatterned surfaces, electrospinning, 3D printing, and bioprinting-designed scaffolds, among others [3].

Interestingly, tissue decellularization is becoming a common technique to obtain decellularized extracellular matrix (dECM) [4] and it is a research field gaining momentum in recent years (Figure 2). The rationale for decellularization is related to the adverse response that cell waste may induce when tissue-derived material is used for implantation procedures, including immune reaction and inflammation, leading to implant rejection. Therefore, dECM is usually obtained by chemical, enzymatic, and/or physical decellularization methods, developed to eliminate the cells and their waste, mainly DNA [5]. These procedures yield decellularized materials formed by the multiple ECM components, which are maintained similar to the original tissue in composition, even in architecture, if required. dECM-related advantages are often associated with better performance and applicability as implants for tissue repair, and also with better mechanical/biochemical properties for the intended use. Initially, research in this field was focused precisely on developing proper decellularization methods and techniques, while more recently, the field is moving to implement approaches related to bioengineering and which tackle applied research aims.

**Figure 2.** Research items per year with the words "decellularization" or "decellularized" in the title (Source: https://scholar.google.es "allintitle: decellularized OR decellularization").

Notably, regenerative medicine research can now take advantage of approaches based on the use of the targeted tissue-specific dECM [6,7]. The ECM is different from one tissue to another, and therefore, decellularization methods and techniques to obtain dECM have been extensively studied and tissue-specifically improved, aiming to preserve the ECM molecules and structures relevant for the intended use. In this sense, research has been mainly based on empirical testing of tissue-specific decellularization methods intended to achieve specific aims or applications.

In this review, we provide an overview of the methods used to obtain the dECM, and we summarize the most common strategies adopted to decellularize specific tissues, aiming to provide a helpful guide for future research development.

#### **2. Organ Decellularization and Tissue Decellularization Approaches for Biomedical Applications**

Whole tissue or intact tissue pieces or sections are a common starting point for decellularization, especially when the final purpose is whole organ decellularization for bioengineering purposes. In these cases, the resulting dECM is a tissue scaffold generally created to keep its structure as intact as possible. Note that dECM tissue scaffold decellularization processes tend to be long, due to the need to be sure about all the reagents reaching the target cells in order to achieve complete decellularization [5]. The bigger the tissue pieces, the longer it takes to make sure that they are completely decellularized. Moreover, the longer the period of chemicals and enzymatic reactions, the higher the chance of damaging the ECM components.

In tissue pieces, it is easy to assess by histology the decellularization and integrity of the remaining ECM. However, even if there are no histologically visible nuclei in the tissue, it is still important to quantify the DNA content by molecular techniques, with the safe limit of DNA content in a decellularized tissue established as below 50 pg DNA per milligram of dry tissue. Regarding macromolecules, they need to be assessed both in quantity and quality by histology, spectrophotometry, and other techniques, while the other ECM components as growth factors may need to be assessed and quantified as well [8]. In some cases, tissue-specific tests are also required to characterize those properties key to the intended biomedical application. For example, a mechanical stress test is required to assess the mechanical/elastic properties after the decellularization of tendons, muscles, and cartilage tissues [9].

Keeping the ECM as intact as possible can be a problem when dECM tissue scaffolds are meant to be used for cell colonization purposes [10]. The ECM is grown by and around the cells, providing physical support, and therefore keeping intact the intricate ECM net, which may impede proper cell seeding of decellularized material. Moreover, potential implantation may be physically limited by the dECM structure and form. Therefore, the decellularization strategy is often designed to degrade some of the ECM components, aiming to maximize the further cell seeding strategy [11].

Tissue decellularization is achieved using as starting material tissues treated with mechanical or chemical methods for tissue grinding, pulverization, or homogenization before decellularization. This approach is gaining relevance, especially in strategies aiming to use the dECM in postprocessing fabrication approaches (hydrogels, 3D printing, electrospinning, and similar). The outcome of decellularization in these cases is dECM powder, an intermediate product mainly used to artificially generate further ECM coatings or 3D structures [12]. Note that tissue powder processing yields dECM powder with multiple components, but does not keep the tissue architecture and a ffects the structure of the ECM macromolecules. This is because the aim of approaches using this processing method is not to keep the structural proteins untouched, but rather to use the properties of the relevant ECM components to improve biocompatibility, adhesion, di fferentiation, and/or other properties or purposes.

The applications of decellularized materials and matrices in regenerative medicine context are multiple, including clinically used implantable materials, and continue to expand. For example, whole decellularized pieces are mainly used as sca ffolds for transplantation purposes; dECM processed to form sheets and/or patches is useful in soft tissue and cardiac repair; Powder of demineralized bone matrix can be resuspended and be used to fill and heal bone defects; dECM-derived hydrogels are useful as injectable materials with regenerative properties; Hydrogels can be processed too, to generate inks and bioinks useful in 3D printing and electrospun-based strategies; dECM-derived sca ffolds can be used as cell carriers for in vitro modeling or in vivo regenerative purposes [2,13–19].

In any case, the decellularization procedure selected and the further characterization needs depend on the final aim and approach (Figure 3). Whole organ, tissue pieces, or powdered ECM are the most common starting materials and multiple possible decellularization methods can be applied, all of them with advantages and disadvantages to be taken into account in light of the specific aim and context [18].

**Figure 3.** Schematic of organ decellularization and tissue decellularization approaches.

#### **3. ECM Decellularization and Sterilization Methods**

Most protocols describe combinatorial and sequential use of di fferent physical, chemical, and enzymatic techniques in order to achieve tissue-specific decellularization. In general, chemical and enzymatic techniques are mainly responsible for successful decellularization in most protocols. Physical techniques are generally used to complement chemical and enzymatic techniques and therefore increase the decellularization e ffects (Table 1). Physical techniques can produce damage in the matrix, while chemical techniques can produce reactions that change the chemical composition of the ECM [20–22]. For this reason, setting up the decellularization protocol is of paramount importance in each specific approach.

#### *3.1. Chemical and Enzymatic Methods for Decellularization*

Detergents are chemical agents used to solubilize cell membranes and to dissociate their inner structure. Among them, Triton X-100 is the most commonly used detergent in decellularization processes. It targets the lipid–lipid and lipid–protein interactions, but it leaves the protein–protein interaction intact [9,23]. It is a very useful agen<sup>t</sup> in those tissues where the key matrix components are primarily proteins. It is an effective detergent to eliminate cells from many tissues, but it is generally avoided in tissues with glycosaminoglycans (GAGs) as a key component in their matrix.

Side by side with the Triton X-100, sodium dodecyl sulfate (SDS) is the other most commonly used detergent in decellularization procedures. SDS solubilizes both the external and nuclear membranes, but also tends to denature proteins and may alter the native structure of the matrix [24,25]. For these reasons, short time SDS treatment is the most common, aiming to minimize the possible damage to proteins and the overall matrix structure [26]. Nevertheless, it is very efficient in removing nuclear and cytoplasmic waste.

Other detergents are useful for specific tissues and applications in a decellularization context. CHAPS reagen<sup>t</sup> has properties of both ionic and nonionic detergents, and therefore, it targets lipid–lipid and lipid–protein interactions, while also solubilizes membranes [27]. CHAPS is not good as a permeating agent, and is therefore mainly used to decellularize thin tissues, for which it is more effective. Triton X-200 is another detergent, less used than its X-100 counterpart because it is more prone to alter the ECM's structure, but it is highly effective for tissues such as neural tissue [28].

Enzymes are also used in most decellularization protocols, mainly to eliminate cell waste and other undesirable components of the ECM. However enzymatic treatments can often lead to additional problems related to enzyme removal [20], a problem usually tackled with further steps using nonenzymatic agents or detergent treatment. Among such enzymes, trypsin is the most commonly used in decellularization procedures [29]. Trypsin targets the C-side bonds in arginine and lysine amino acids and is mostly used combined with EDTA, a chemical agen<sup>t</sup> able to break cell–matrix interactions. Of note, the prolonged exposure to trypsin–EDTA treatment can significantly alter the structure of the matrix, destroy laminin, and remove GAGs, resulting in severe mechanical weakness of the tissue [30]. Pepsin is another useful enzyme in decellularization processes. It is a highly aggressive protease commonly found in the stomach and, like trypsin, it targets the bounds between peptides. Thus, it may cause damage to the ECM if used under long exposition times [31].

Hypotonic and hypertonic solutions [32] use osmotic properties to make the cells explode. The osmotic shock kills the cells, but it does not remove the cell waste that it releases to the matrix, which should be taken into account in the design of a decellularization procedure. Moreover, the treatment of the DNA waste is of paramount relevance in all decellularization processes, due to the tendency of the nuclear material to remain stuck to ECM proteins. In this sense, endonucleases and exonucleases are other kinds of enzymes that are of grea<sup>t</sup> use to eliminate the waste of nuclear components [8,30].

Finally, chemical acid–base [33] and TBP [32] treatments are rarely used, because they are very aggressive toward the proteins of the matrix. Specifically, acid–base solutions damage collagen and TBP disrupts protein–protein bonds.

#### *3.2. Physical Methods for Decellularization*

As previously mentioned, physical techniques are not enough to decellularize the tissue, but they can help in combination with chemical and enzymatic processes. For example, when big tissue pieces or whole organs are the target of decellularization, perfusion is recommended in order to better reach all tissue areas [21,34].



The most commonly used physical technique is snap freezing, or freeze–thawing, as the first step of a decellularization process. By freezing tissue, intracellular ice crystals are formed, thereby disrupting cellular membranes and causing cell lysis. Thus, freezing is a common and effective method for cell lysis and it eases further uniform decellularization. Protocols using this approach have to

carefully control the rate of temperature change to control the size of the formed ice crystals, therefore preventing excessive damage to the ECM [35–38].

Cells can be lysed by applying direct pressure to tissue, but this method is only e ffective for tissues or organs that are not characterized by a densely organized ECM (e.g., liver and lung). Mechanical force has also been used to delaminate layers of tissue from organs that are characterized by natural planes of dissection, such as the small intestine and the urinary bladder. These methods are e ffective and cause minimal disruption to the three-dimensional architecture of the ECM within these tissues [39].

Mechanical agitation and sonication are useful in combination with a chemical treatment to assist in cell lysis and the removal of cellular debris [40,41]. Mechanical agitation can be applied by using a magnetic stir plate, an orbital shaker, or a low-profile roller. There are no studies to determine the optimal magnitude or frequency of sonication for the disruption of cells. However, the standard ultrasonic cleaner appears to be as e ffective at removing cellular material as the movement of an orbital shaker. In all of these procedures, the optimal speed, the volume of reagent, and the length of mechanical agitation are dependent on the composition, volume, and density of the tissue.

Vacuum-assisted decellularization (VAD) cannot decellularize, but it is highly e ffective in enabling chemical agents to reach the whole tissue [42]. Hydrostatic pressure, on the other hand, is an e ffective decellularization method, but it is usually combined with enzymes such as DNases to achieve complete decellularization [43].
