Extracellular cues play a role in many aspects of neural development in vivo36–38
as well an in vitro
The substrate chosen for in vitro
neuron studies is thus highly important because phenotypic, electrophysiological, and molecular changes have been identified in neural cells when cultured on various proteins or synthetic coatings.38–40
Indeed, cell–matrix interactions of neurons derived from human embryonic stem cells were seen to strongly affect differentiation, as neurons demonstrated increased differentiation on laminin-rich substrates, and higher expansion and neurite outgrowth in a dose-dependent manner.41
As the culture substrate has demonstrated effects on morphology, differentiation, and function of neurons, the development of a complex tissue-matched culture substrate may be beneficial for in vitro
assays and neural growth.
The native ECM is a complex combination of proteins and polysaccharides that play an important role in cellular behavior such as attachment, proliferation, and differentiation. Current cell culture methods and tissue engineering scaffolds conventionally use purified proteins and do not mimic the complexity of the brain extracellular microenvironment. Combinations of purified proteins have been shown to improve cell proliferation and differentiation, which indicates that complex coatings are beneficial, and thus there has been a shift toward more complex materials.42,43
As there are limitless potential combinations, using a naturally derived matrix may be more physiologically relevant. A variety of tissues have been decellularized and used as cell culture coatings to provide a closer mimic to the in vivo
These coatings have shown tissue-specific effects on cellular behavior, and in some instances increased maturation when compared with conventional substrates.8,10
In this study, we isolated and solubilized ECM from porcine brain using a detergent decellularization method and processed it to a liquid form using enzymatic digestion. We used SDS detergents to remove cellular content, and though SDS has been shown to denature ECM proteins,44
others have reported that SDS decellularization was milder than other techniques such as those using Triton X-100 and trypsin.45,46
Our initial trials attempting to decellularize brain with other detergents such as sodium deoxycholate and Triton X-100 did not remove as much cellular content. We found many differences in developing a decellularization protocol for brain compared with other tissues, as the brain ECM is very weak and the tissue fell apart readily, leading to difficulties in rinsing and recovering the brain matrix. Through this method we were able to isolate brain ECM, but could not maintain the original structure of the brain. However, the isolated ECM was able to be processed into a cell culture coating and an injectable scaffold. The matrix that was retained in our method was rinsed and processed, and while there were no visible nuclei present in the H&E sections, not all nuclear content was fully removed from the brain matrix material. The remaining DNA content was, however, less than that reported with other decellularization techniques.10
Overall, brain ECM is composed mostly of GAGs and proteoglycans with relatively small amounts of fibrous proteins such as collagen and fibronectin. Our results indicated that the decellularized material contains protein components that are found in the native brain ECM, and also retains sulfated GAGs. Multiple collagen isoforms and laminin as well as the proteoglycan perlecan were retained postprocessing, though there may be other components that were not identified. The retention of laminin may prove to be important, as laminin has been shown to increase neurite expansion, survival and outgrowth for neurons,40,41
as well as retinal explant attachment and axonal outgrowth.47
The decellularization process was able to retain sulfated GAGs, despite the use of SDS, and was found to have one of the highest GAG contents when compared with other reported tissues that have been decellularized.8–10,27,44
GAGs have been shown to have an effect on cell behavior, either alone or through association with other molecules.48
While our decellularization protocol likely reduces protein and GAG content relative to native tissue, we are still able to retain many of the components while removing over 95% of DNA. The presence of the proteoglycans and GAGs may be an important mediator of the cellular behavior seen in our studies. While there is minor DNA content remaining in our brain matrix, a recent study demonstrated that several commercially available decellularized ECM scaffolds, although contained measureable amounts of DNA, could still be used successfully in the clinic.49
This may indicate that there is a threshold of DNA levels to avoid a negative immune response, or that the detergents may disrupt the structure of DNA so that the immune response will not be triggered.
The brain matrix can be used as an in vitro
coating and it was shown to support the culture and maturation of neurons. iPSC neurons were studied, as these cells have the potential to be an autologous source for neuronal formation. Neurons expressed synapsin, a protein marker that identifies formation of synapses and maturation of the neurons.50
Synapsin expression increased over time in culture, demonstrating that the neurons matured on the brain matrix coating. Interestingly, extensive dendritic processes were observed on the decellularized brain matrix, supporting complex arborization, but were not seen on Matrigel. Branching complexity has been theorized to have an important effect on the electrophysiology of dendritic neurons,51
though mechanisms controlling dendritic architecture are not fully known. It should be noted that the PO coating was important for this cell type, as the iPSC-derived neurons cultured on Matrigel or brain matrix coatings alone resulted in lower attachment.
While there are no clinically used materials for brain tissue reconstruction yet,52
several naturally derived and synthetic materials have been studied as scaffolds in small animal models. None of these scaffolds offer the complexity of native brain ECM, and require major surgery for implantation. Injectable scaffolds23–25
have been developed for minimally invasive delivery, but again do not recapitulate the composition of the native microenvironment. Thus, we tested proof-of-concept for utilizing our solubilized decellularized brain ECM as an injectable tissue engineering scaffold. While the brain matrix remains liquid at room temperature, when brought to physiological pH and injected subcutaneously, the material self-assembles into a gel in vivo
. However, the brain matrix material was unable to form a gel in vitro
, despite the fact that it was able to self-assemble upon injection. SEM analysis shows that the structure of the injectable scaffold is different from native brain tissue, but is porous and fibrous which may be suitable for endogenous cell infiltration. The advantage of using an injectable hydrogel is that it will allow minimally invasive delivery of the material, injection into multiple sites, and could be tailored to fit the size of the brain lesion. As the material has been decellularized and ECM proteins are largely conserved across species, a negative immune response would not be expected. In fact, many decellularized ECM scaffolds have already been FDA approved for use in the clinic.49
Though only injected subcutaneously, this preliminary work supports the potential for the use of this material as a tissue-matched scaffold, and for minimally invasive therapy. In addition to potential use as an acellular scaffold, the brain matrix may also have the potential to be used as a cell delivery vehicle given our in vitro
results. However, long-term in vivo
studies will be critical to assess in vivo
biocompatibility, degradation time, and cell infiltration and survival. With this work, we demonstrate a method to decellularize and isolate brain ECM, the development of cell culture coatings derived from brain ECM, and in vivo
feasibility of a brain matrix scaffold for tissue engineering applications.