As the gap between donors and patients in need of an organ transplant continues to widen, research in regenerative medicine seeks to provide alternative strategies for treatment. One of the most promising techniques for tissue and organ regeneration is decellularization, in which the extracellular matrix (ECM) is isolated from its native cells and genetic material in order to produce a natural scaffold. The ECM, which ideally retains its inherent structural, biochemical, and biomechanical cues, can then be recellularized to produce a functional tissue or organ. While decellularization can be accomplished using chemical and enzymatic, physical, or combinative methods, each strategy has both benefits and drawbacks. The focus of this review is to compare the advantages and disadvantages of these methods in terms of their ability to retain desired ECM characteristics for particular tissues and organs. Additionally, a few applications of constructs engineered using decellularized cell sheets, tissues, and whole organs are discussed.
Tissue and organ failure is currently one of the biggest health issues our society faces. Arising from disease or trauma, complete treatment typically requires the reparation or replacement of the affected organ. Conventional practices utilize organs from either live or deceased donors for the procedure of transplantation. However, the deficit between donors and patients requiring a new organ has grown substantially in recent years, so much that there are more than 100,000 patients on waiting lists for organs in the United States alone [
Tissue engineering aims at replacing or regenerating human tissues or organs in order to restore or establish normal function [
Because many challenges are associated with preparing synthetic scaffolds that recapitulate the complexity of the cell microenvironment, there has been increasing interest in utilizing naturally derived extracellular matrix (ECM) itself. This biologic scaffold is obtained through the process of decellularization. The ultimate goal of decellularization is to rid the ECM of native cells and genetic materials such as DNA while maintaining its structural, biochemical, and biomechanical cues. The decellularized ECM can then be repopulated with a patient’s own cells to produce a personalized tissue. Decellularized ECM has been successfully used to recreate various types of tissues and organs including blood vessel [
Figure
Process of engineering tissues using decellularized ECM.
Decellularization has been performed through chemical, physical, or combinative methods. An evaluation of these strategies will first focus on the removal of cells and genetic material followed by the maintenance of structural proteins. In addition, mechanical properties and ultrastructure of the tissue or organ before and after treatment will be compared. Emphasis will be placed upon the method’s ability to retain the characteristics necessary for successful recreation of the tissue or organ. Table
Summary of decellularization agents and techniques.
Category | Agents and techniques | Mechanism/description | Significant effects | References |
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Chemical and enzymatic | (1) |
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Sodium dodecyl sulfate (SDS) | (i) Ionic | (i) Cytotoxic: requires extensive wash process | [ | |
(ii) Alters microstructure (i.e., collagen fibers) | [ | |||
Triton X-100 | (i) Nonionic | (i) Less damaging to structure of tissue than ionic surfactants | [ | |
(ii) Commonly used with ammonium hydroxide | ||||
Sodium deoxycholate (SD) | (i) Ionic | (i) Causes agglutination of DNA when used without DNase | [ | |
(ii) Commonly used with DNase | (ii) Remnant DNA fragments | [ | ||
CHAPS | (i) Zwitterionic | (i) Maintains structural ECM proteins | [ | |
(ii) Remnant cytoplasmic proteins | [ | |||
(iii) Maintains ultrastructure | [ | |||
(2) |
|
|||
Peracetic acid | (i) Highly corrosive | (i) Insufficient cell removal | [ | |
(ii) Oftentimes used for sterilization | (ii) Increases stiffness of ECM | [ | ||
Ethylenediaminetetraacetic acid (EDTA) | (i) Commonly used with trypsin | (i) Decreases salt- and acid-soluble ECM proteins | [ | |
Reversible alkaline swelling | (i) Induces negative charge on collagen to cause swelling | (i) Alters mechanical properties | [ | |
(ii) Used with tridecyl alcohol ethoxylate | ||||
(3) |
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|||
Trypsin | (i) Breaks cell-matrix adhesions | |||
(ii) Commonly used with EDTA | ||||
Deoxyribonuclease (DNase) | (i) Breaks down DNA fragments | |||
(ii) Commonly used with SD | ||||
Ribonuclease (RNase) | (i) Breaks down RNA fragments | |||
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||||
Mechanical | High hydrostatic pressure (HHP) | (i) Pressures greater than 600 MPa applied to lyse cells | (i) Remnant DNA fragments | [ |
(ii) Denatures ECM proteins | [ | |||
Supercritical carbon dioxide | (i) Applies CO2 at pressures above 7.40 MPa and temperatures above 31.1°C | (i) Requires entrainer to remove polar phospholipid membrane | [ | |
(ii) Maintains ECM proteins & mechanical properties | [ | |||
Freeze-thaw | (i) Alternate between freezing temperatures (−80°C) and biological temperatures (~37°C) | (i) Maintains ECM proteins & mechanical properties | [ | |
(ii) Remnant DNA | [ |
Several types of chemicals have been used in decellularization, including surfactants, acids, and bases. Surfactants, the most common decellularizing agents, typically work by lysing cells through disarranging the phospholipid cell membrane [
The widely used ionic surfactant, sodium dodecyl sulfate (SDS), has been successful in a number of applications in decellularization because of its ability to efficiently remove cells and genetic material. For example, it was the major agent used in the whole rat heart perfusion decellularization [
Although SDS can successfully remove unwanted native constituents of the tissue, it can be damaging to the structural and signaling proteins. For instance, the collagen in SDS-treated heart valves became compacted [
The nonionic surfactant Triton X-100 is oftentimes utilized to remove the remnant SDS. This practice has been especially prevalent in the perfusion decellularization of whole organs [
Sodium deoxycholate (SD) is another ionic surfactant that works by solubilizing the cell membrane. Unlike SDS, SD produced scaffolds that were highly biocompatible, as cells seeded on the SD-decellularized matrices exhibited higher metabolic activity compared to those decellularized via SDS [
The zwitterionic, nondenaturing detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), has been applied in both immersion and perfusion decellularization procedures. Human- and porcine-derived lung tissues [
Acid- and base-containing protocols utilize agents like peracetic acid and techniques like reversible alkaline swelling. Peracetic acid is a highly corrosive and strong oxidizer oftentimes used for sterilization. Thinner tissues such as the small intestine submucosa (SIS) [
Like the use of DNase I to prevent the agglutination of DNA in SD treatments, other types of enzymes have been used in decellularization protocols to supplement the chemicals’ properties. For example, Triton X-100 and SD were used in combination with DNase in the decellularization of both normal and emphysematous human lungs [
Trypsin is an enzyme commonly used with EDTA which works by breaking the cell-matrix adhesions. Used in the treatment of porcine pulmonary valves, complete cell and genetic material removal was observed after 24 hours [
While chemical and enzymatic approaches are the most widely applied decellularization methods, there are concerns regarding the possible toxicity of the chemicals and destruction of ECM proteins. Other methods that can physically or mechanically decellularize the ECMs are therefore being developed. These methods include temperature and pressure treatments that work to eliminate cells through a combination of lysing the cells and destroying cell-matrix adhesive proteins. In particular, physical treatments include the use of freeze-thaw, high hydrostatic pressure, or supercritical carbon dioxide (CO2) to fully remove a tissue’s constituent cells and genetic materials. While each specific method is unique, every mechanical decellularization protocol involves a wash process to remove any cellular debris that may remain. In many cases, the washing step is critical in determining the efficacy of the procedure.
Freezing and thawing tissues lyses, and subsequently eliminates, the cells to produce a decellularized matrix. Freeze-thaw procedures oftentimes involve alternating between freezing temperatures around −80°C and biological temperatures around 37°C. Specific protocols can be altered by increasing the temperature difference or changing the number of freeze-thaw cycles performed. Two particular freeze-thaw studies involved the decellularization of fibroblast cell sheets carried out with three cycles [
High hydrostatic pressure (HHP) has become an increasingly prevalent method that applies pressures greater than 600 MPa to destroy cell membranes. In one study, porcine corneas were decellularized using HHP at 980 MPa for 10 minutes at 10 or 30°C [
Difference in the temperature can lead to alterations in protein content and structure. At 10°C, both collagen and GAG content were better maintained in the cornea decellularization than at 30°C [
With the unique transport properties, that is, a liquid-like density and a gas-like diffusivity, supercritical fluids have been used in extraction applications in industry [
Because each aforementioned technique has its advantages and disadvantages, several techniques have been combined to complement one another with the goal of retaining desired characteristics in the engineered tissue. For example, mechanical methods are typically less damaging to the tissue’s structure; however, they fail to meet the requirements for immunogenicity. On the other hand, surfactants at low concentrations or enzymes used alone may not completely remove all cellular debris. To this regard, combining the two treatments in a multistep process can yield a decellularized ECM appropriate for its particular application. While the combined method may demand more chemicals and a longer processing time than single-treatment protocols, each constituent parameter needs to be optimized to suit a particular tissue. These tissue-specific protocols can thus be more practical and effective.
Thicker tissues, in particular those composed of multiple layers, oftentimes require multistep decellularization procedures involving chemical, enzymatic, and mechanical treatments. Adipose, or fat tissue, is one type of thick tissue and is desired in reconstructive surgeries. While its characteristics are simple compared to more complex tissues such as lung or heart, it is necessary to maintain adipose tissue’s mechanical properties and biochemical factors in order to regulate adipogenesis. In one particular method, a multistep process was carried out on porcine adipose tissue [
Multistep chemical, enzymatic, and mechanical combinative methods have been proven to be detrimental to different properties of some tissues. In a protocol involving the treatment of porcine cartilage disks, two different hypotonic buffers were used, the first supplemented with 100 mM KCl, 5 mM MgCl2, and 100 mM dithiothreitol (DTT) and the second supplemented with 0.5% SDS, in order to solubilize the cell membranes [
Reducing the scaffold’s immunogenicity is one of the most critical requirements of decellularization. This particular aspect has been crucial in preventing the use of decellularized ECM as scaffolds in clinical applications. Ideally, xenogeneic scaffolds may be used, as they are highly abundant and thus have the potential to be manufactured. If their immunogenicity is not sufficiently reduced, they may be rejected in vivo, leading to functional failure and the need for immediate replacement or removal. The two components capable of inducing an immunogenic response include remnant genetic materials such as DNA and RNA and antigens. In terms of eliminating genetic materials, Crapo and colleagues have suggested that the decellularized ECM should contain less than 50 ng dsDNA per mg ECM and DNA fragment lengths less than 200 bp [
Native antigens must also be reduced in the scaffolds to prevent immunorejection. Hyperacute rejection of scaffolds, occurring shortly after implantation and caused by circulating antibodies within the host, and acute immune rejection, occurring days to weeks after implantation, are of particular concern [
In regenerating a tissue or organ via decellularization, maintaining the mechanical characteristics of the native tissue is of vital importance in ensuring proper functionality. Primary properties of interest include elastic modulus, viscous modulus, tensile strength, and yield strength; however, the most crucial properties ultimately depend on the nature of the tissue or organ’s desired function. In particular, stiffness is desired in the design of a trachea to provide an unobstructed airway. Furthermore, the anisotropic or isotropic characteristics of the tissue are also necessary to modulate, as they can oftentimes dictate the orientation of reseeded cells: such is the case with cardiomyocytes in myocardium regeneration [
Both chemical and mechanical methods have been shown to damage the structural proteins of the ECM. In particular, SDS has been shown to alter the ECM’s microstructure by causing collagen to become compacted [
Recellularization of the decellularized ECM must also be optimized in order to produce a functional tissue or organ. The cell type used to repopulate the matrix and method of recellularization is largely dependent on the complexity of the cell sheet, tissue, or organ of interest. Cell sheets for skin grafts or blood vessels may only require a single cell type, whereas whole organs such as the heart or liver necessitate the seeding of multiple cell types. Furthermore, recellularization of cell sheets can be accomplished by simply applying the cell suspension onto the monolayer surface, and three-dimensional constructs can be created through alternating between the cell suspension and additional cell sheets as in the “sandwich model” for cartilage construction [
Decellularization has been performed on a variety of cell sheets, tissues, or organs, depending on the necessary replacement or regenerative treatment. Table
Summary of ECM type treated using main decellularization strategies.
Agents and techniques | Cell sheet/tissue/organ |
---|---|
SDS | Fibroblast cell sheet [ |
Triton X-100 | Bovine pericardium [ |
SD/DNase | Porcine blood vessel [ |
CHAPS | Rat lung [ |
Peracetic acid | Porcine SIS [ |
EDTA/trypsin | Porcine pulmonary valve [ |
Freeze-thaw | Fibroblast cell sheet [ |
High hydrostatic pressure | Porcine cornea [ |
Cell sheets can be prepared and subsequently decellularized to be used in a number of applications. They typically consist of a single cell type that can be used alone to facilitate regeneration of minor injuries or combined with many sheets and structured to form complex constructs. For example, to generate an ECM to promote periodontium regeneration, periodontal ligament cell sheets were prepared and then decellularized to conserve the native growth factor-, collagen-, and fibronectin-laden matrix [
Another promising application involving the creation of blood vessels from fibroblast cell sheets emphasizes the ability of simple decellularized matrices to form more complex structures. In one study, fibroblast monolayer cell sheets were prepared and rolled into vessels before being decellularized and seeded with endothelial cells [
Decellularization methods have been used on different types of tissues either to examine the treatment on a less complex tissue or to create a scaffold for simpler applications. For example, adipose ECM has been utilized in testing breast cancer treatments by providing simulated in vivo microenvironments [
In another example of adipose tissue regeneration, adipose tissue was successfully decellularized, recellularized, and implanted in vivo. Human adipose tissue was obtained from reconstructive surgeries and decellularized using a protocol that first required three freeze-thaw cycles, a two-day agitation period, and two four-hour saline solution washes of different concentrations repeated over two days. The sample was then treated with 0.25% trypsin/EDTA for two hours, washed, and submersed in isopropyl alcohol overnight. Finally, the sample underwent treatment in 1% Triton X-100 for three days and was washed in ultrapure water for two days and PBS for one day [
The complexity of organ function requires the most extensive decellularization and reconstruction techniques, in particular necessitating the maintenance of the organ’s ultrastructure and vasculature. A variety of organs have been engineered in this way and have subsequently been tested in vivo on the small animal level. In one example, the vasculature of decellularized porcine bladder matrices was resurfaced using epithelial progenitor cells. These scaffolds supported reseeded bladder smooth muscle and urothelial cells, and the resultant bladders were successfully implanted for one to three hours in pigs [
One particular study observed an engineered pancreas in mice that had been achieved through perfusion decellularization [
Clinical applications of decellularized ECM are becoming more prevalent; however, they have been limited to less complex tissues primarily functioning in structural or reconstructive roles. Several FDA-approved products on the market aimed at tissue regeneration and replacement are derived from xenogeneic or allogeneic decellularized ECM, including LifeCell’s AlloDerm® Regenerative Tissue Matrix (human dermal graft), DSM’s Meso BioMatrix® Surgical Mesh (porcine mesothelium), and CryoLife’s SynerGraft® (human pulmonary heart valve). The former two scaffolds serve to facilitate cell proliferation, whereas the lattermost product serves as a replacement for the existing structure. Furthermore, clinical trials have been carried out for more complex structures. For example, the first tissue-engineered trachea to be implanted was generated using decellularization [
While great advancements are being made in regenerative medicine through decellularization, it is essential that protocols be further developed to optimize the process. Current decellularization methods are beneficial in some regards but oftentimes lack in others. Ideal methods will produce cell- and genetic material-free ECM that retains important structural, biochemical, and biomechanical properties crucial to its inherent function. Several methods may be optimized in such a way as to achieve this balance, specifically in eliminating the components individualized to the tissue which could cause an immunogenic response, while maintaining those that will support and regulate the reconstruction of a new tissue. In particular, developing methods that consist of combined strategies with chemicals, enzymes, and mechanical techniques may improve decellularization efficiency and limit the negative effects caused by simpler methods. This improvement may be achieved through dynamic processes in which parameters are adjusted over time based upon the component in need of removal or maintenance. Thus, the tissue may be examined and assessed at predetermined time points throughout treatment. For example, supercritical CO2 may be utilized for cell lysis; then the ECM may undergo brief exposure to lower concentrations of surfactants to eliminate remnant cellular materials. Further enzymatic or wash agents can then be applied. The goal is ultimately to administer the minimum amount of harsh chemicals or mechanical treatments so as to prevent unnecessary damage to the ECM’s microstructure and ultrastructure.
By optimizing the decellularization process, tissue and organ scaffolds derived from humans and animals may be utilized, thus minimizing the donor-patient specificity required to ensure compatible transplants. However, truly bridging the gap between donors and patients in need of transplants through bioscaffold tissue engineering will require the development of scaled-up methods, since current methods are usually limited to small tissues or organs. In addition, recellularization methods will need to be improved in order to distribute the proper cell types uniformly through the tissue and enable sufficient nutrient and oxygen delivery for optimal cell viability. Finally, with the continuous innovation in decellularization strategies and advancements in uses for human induced pluripotent stem cells, off-the-shelf tissues and organs may be made available, thereby aiding in, and potentially eliminating, the ever-increasing need for transplants.
The authors declare that there are no conflicts of interest regarding the publication of this paper.
The authors would like to acknowledge funding support from the National Institute of Health (R15GM122953) and Anna Gilpin’s NASA Space Grant Scholarship.