Region‐specific brain decellularized scaffolds can recover cell viability in an oxygen-glucose deprivation model

Region‐specific brain decellularized scaffolds can recover cell viability in an oxygen-glucose deprivation model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Region‐specific brain decellularized scaffolds can recover cell viability in an oxygen-glucose deprivation model Diego Reginensi, Didio Ortiz, Solangel Castillo, Andrea Burillo, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5130290/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 07 Apr, 2025 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract Brain decellularized extracellular matrix (ECM) can be an attractive scaffold capable of mimicking the native ecosystem of the central nervous system tissue. In this study, we studied the in vitro response of neural lineage cells exposed to region-specific brain decellularized ECM scaffolds from three distinct neuroanatomical sections: cortex, cerebellum and remaining areas. First, the evaluation of each brain subregion was performed with the isotropic fractionator method to understand the cellular composition of the different cerebral areas. Second, each of the cerebral subregions was subjected to the decellularization process and their respective characterization using molecular, histological, and ultrastructural techniques. Third, the presence of neurotrophic factors in the decellularized brain scaffold was analyzed. Finally, we studied the region-specific brain decellularized ECM as a mimetic platform for the maturation of PC12 cells and for the recovery of cell viability in an oxygen-glucose deprivation model. Our results show that region-specific brain decellularized ECM can serve as a biomimetic scaffold capable of promoting the growth of neural lineage cells and, in addition, it possesses a combination of structural and biochemical signals ( e.g. , neurotrophic factors) that are capable of inducing cell phenotypic changes that can promote cell recovery and viability in a stroke/ischemia model in vitro . Biological sciences/Neuroscience/Regeneration and repair in the nervous system Physical sciences/Materials science/Biomaterials/Bioinspired materials Tissue engineering decellularized scaffold brain extracellular matrix oxygen-glucose deprivation (OGD) Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. INTRODUCTION The extracellular matrix (ECM) serves as a scaffold that gives structural support to the tissue and serves as a biochemical reservoir that helps maintain the tissue specific cell phenotype 1 . The ECM can be decellularized and still retain the biochemical and biomechanical properties of native tissue; therefore, it has been used as a platform for tissue engineering 2 , 3 . The concept of decellularized ECM can be simply described as a tissue that has been treated to remove cells and their cellular components while maintaining as much ECM components as possible. It is these cellular components (e.g., DNA, cytoplasmic and membrane-bound proteins) that could hinder cell development and differentiation around these decellularized ECM scaffolds in the case of regular cell culture, 3D bioprinting and lab-on-a-chip applications 4 , 5 or cause an adverse response in the immune system of a host organism that will receive it at an injured site 6 , 7 No decellularization protocol is perfect. Some cellular remnants most certainly will be left behind (ideally below the acceptable threshold of 50 ng DNA per mg of dry tissue 8 , 9 and/or the remaining DNA fragments must be less than 200 base pairs 8 ), and some (potentially important) ECM proteins and soluble factors will be lost (Fig. 1 ). Biologic scaffolds composed of decellularized ECM have become a novel platform to promote the innate regenerative capacities of most tissues by providing structural support and site-specific ligands that can promote cell attachment, differentiation and local signaling 10 , 11 . Various protocols for the preparation of decellularized ECM have been developed in vitro and tailored for different tissues of interest, including skin, bladder, liver, adipose and neural tissue 8 , 12 – 15 . Thus, the use of decellularized ECM as an in-situ implant in damaged tissues offers a versatile and flexible scaffolding platform that minimizes tissue mismatch and immune complications that, in many cases, can lead to rejection of the transplanted tissue/organ 16 , 17 . One of the most promising approaches to generate artificial tissues for grafting is by reseeding decellularized scaffolds with specified cells depending on the biomedical problem being addressed 18 – 20 . Cell therapy, or the use of autologous or allogeneic cells as a therapeutic treatment, can offer a promising solution to highly complex medical conditions, such as neurodegenerative diseases 21 , 22 . The effects associated with cell therapy in neural regeneration can be divided into the following main categories: immune modulation, support of cell survival, axonal growth, cell differentiation and cellular replacement 23 . However, the use of cells by themselves can leave them vulnerable to unwanted phenotypic changes based on the harsh environment of the injury site, as well as prone to quick removal by the host’s immune cells 24 . The administration of cell therapy in combination with decellularized ECM scaffolds can promote a microenvironment that enhances cell survival and maintains a more robust phenotypic profile, such as in the case of glial and neuronal cells and the promotion of axonal regeneration 25 , 26 . Another example is the co-administration of stem cells and scaffold matrices as therapy for brain trauma and spinal cord injury, which has been shown to be beneficial 27 – 29 . Several reviews of different types of materials used as scaffolds to support stem cell survival and proliferation in different models of nerve injury have recently been published 30 , 31 . However, most of these materials are based on synthetic derivatives that do not resemble the structural complexity of the tissue microenvironment of the central nervous system (CNS) 32 . Therefore, the use of decellularized ECM derived from CNS tissue is presented as a biomimetic substrate for in vitro models of neurological disorders, with the potential to become a treatment for injuries in the brain such as stroke 32 – 34 . Most biomaterials scaffolds such as collagen-based matrix, gelatin, matrigel and fibrin enhance cell survival, providing a temporary mimic of the extracellular matrix in therapies for traumatic brain injury and stroke 8 , 35 . Nonetheless, this single-molecule approach does not recapitulate the synergistic effects that can be found in the complex native ECM of the brain, with a unique combination of proteins and proteoglycans contained in its specific regions (e.g., cortex, cerebellum or remaining areas). Brain-derived decellularized ECM has been applied as an effective therapeutic method for spinal cord injury, traumatic brain injury, and stroke in vitro and rat models 36 – 38 . However, the capacity of regeneration of the decellularized ECM from the different brain subregions has never been tested separately to evaluate the regulation of stem neuronal cell survival and differentiation. Brain cells, to function properly, require a constant supply of oxygen and glucose 39 , 40 . The lack of these components or exposure to levels below normal physiological conditions experienced during brain ischemia ( i.e. , stroke), produces deleterious effects on the cell’s metabolism 40 , 41 . Depending on the severity of the ischemic state ( i.e. , mild, moderate or severe) and its duration, cells suffer injuries that can lead to chronic, degenerative cell death through necrosis and apoptosis 42 in a very localized manner. This makes brain ischemia a good candidate to develop regenerative therapies based on decellularized ECM that, in the worst-case scenario, could stop the chronic cell death; or, in the best-case scenario, could promote tissue regeneration and neuronal maturation in the site of the injury. Our long-term goal is to develop new regenerative medicine therapies for the treatment of neurological disease ( e.g. , brain ischemia/ reperfusion and stroke) on the basis of a comprehensive methodology focused on two interrelated pillars: ( i ) development of novel biomaterials to modulate the cellular response using different anatomical sections of the brain extracellular matrix ( i.e. , cortex, cerebellum and remaining areas) and ( ii ) to define the effects in vitro of decellularized scaffold on culture of neural lineage cells. In this study we present and characterize an optimized protocol to produce cerebral decellularized extracellular matrix from porcine source and confirm its capacity to enhance neuronal maturation of PC12 cells. Finally, we compare the role of the region-specific brain decellularized ECM in functional cellular recovery after a simulated ischemic stroke in vitro using the oxygen/glucose deprivation model. 2. MATERIALS & METHODS The methodology was divided into 4 experimental protocols: (i) neuroanatomical dissection of porcine brain tissue; (ii) total cell quantification of each of the dissected brain regions; (iii) processing and characterization of region-specific brain decellularized ECM and (iv) in vitro cell response to the brain decellularized ECM in regular cell culture and in simulated stroke conditions using the oxygen/glucose deprivation model. All methods were performed in accordance with relevant guidelines and regulations. The detailed description of the methods is as follows: 2.1 Neuroanatomical dissection of porcine brain tissue All animal tissue handling was performed with approval from the Institutional Animal Care and Use Committee (IACUC) of INDICASAT-AIP (for porcine tissue) and the School of Medicine of the University of Miami (for rodent tissue), strictly complying with the ARRIVE guidelines. Porcine brains were collected fresh at the Macelo S. A. abattoir, in Panama City, Panama, as previously described 43 . Briefly, the animals were euthanized through electric shock stunning and cardiac arrest using electrodes set at 0.5 A and 220 V. Brain tissue is a byproduct of the operations of the abattoir, so the material used does not pose a risk to any additional animals. The porcine brains were collected fresh, less than 5 min after euthanasia, as hemispheres cut along the mid-sagittal plane and brought immediately to the lab in ice to be processed in a biosafety cabinet where the meninges were removed and the brains were dissected in three sections: cortex ( i.e. , cortical gray and white matter and hippocampus), cerebellar structure and remaining areas ( i.e. , the sum of diencephalon, basal ganglia, mesencephalon, pons and medulla) under aseptic conditions. Each section was cut into pieces smaller than 1x1 cm 2 , placed in 50mL conical tubes up to the 15mL mark for each tube, and stored at -80 o C for at least 24 h before decellularization. 2.2 Total cell quantification of the dissected brain regions The total number of brain cells was estimated using the isotropic fractionator method, as previously described 44 . Immediately after arriving at the laboratory, the brain halves that were separated for isotropic fractionator were weighed and dissected into the three brain sections: cortex, cerebellum and remaining areas. Next, each section was fixed in 4% paraformaldehyde in 0.1M phosphate buffer (PB, pH 7.4) overnight, cryoprotected in 30% sucrose in 0.1 M PB at 4°C and stored in an antifreeze solution at -20°C until processing 45 , if necessary. Subsequently, tissue samples were homogenized with a Tenbroeck homogenizer in a saline detergent solution to dissolve the cytoplasmic membrane but not the nuclear membrane to produce a homogeneous solution of free nuclei (isotropic solution). The total number of nuclei in suspension, which correspond to the number of cells in the original tissue, was determined by staining with the DNA marker DAPI (4-6-diamidino-2-phenylindole dihydrochloride) and counting with a hemocytometer (Neubauer chamber) under a fluorescence microscope 46 , 47 . 2.3 Processing and characterization of region-specific brain decellularized ECM 2.3.1 Brain Tissue Decellularization Protocol An optimized brain decellularization method has been developed based on our previous work 43 comparing two commonly used protocols: a 1-day enzymatic-based protocol 48 and a 4-day detergent-based method 35 . Initially, the dissected tissue was submerged in 0.1% sodium dodecyl sulfate (SDS, Sigma- Aldrich Corp., St. Louis, MO, USA) in medical grade injectable water (PISA Farmaceutica, Guadalajara, Mexico) supplemented with 1% streptomycin/penicillin (Gibco™ catalog # 15140122, Thermo Fisher Scientific, Waltham, MA, USA) and washed at 4ºC with gentle orbital agitation (50 rpm) for 6 h before replenishing the solution and continue washing with the same conditions for an additional 14 h ( i.e. , overnight). Subsequently, the following series of timed washes were performed: 0.02% trypsin/0.05% EDTA (Invitrogen Corp., Carlsbad, CA, USA) for 75 min; 3.0% Triton X-100 (Sigma- Aldrich Corp) for 75 min; 1.0 M sucrose (Thermo Fisher Scientific) for 30 min and 0.1% SDS for 16 h. All these washes were performed with an agitation of 80 rpm at room temperature, except for the step with trypsin, which was performed at 37°C. The final washes involved once with 0.1% peracetic acid (Rochester Midland Corp., Rochester, NY, USA) in 4.0% ethanol for 120 min, which was used as a sterilizing agent; twice with PBS (Thermo Fisher Scientific) for 20 min and twice with sterile injectable water for 20 min; again, with all these steps at RT and 80 rpm. The decellularized tissue obtained was frozen overnight at -80 ºC, lyophilized in aseptic conditions and stored dry at -80 ºC until use. 2.3.2 Characterization of the Decellularization Efficiency: DNA Content See supplementary material for details. 2.3.3 Characterization of the Decellularization Efficiency: Western Blot See supplementary material for details. 2.3.4 Structural Characterization: Histological Analysis See supplementary material for details. 2.3.5 Morphological Characterization: Scanning Electron Microscopy See supplementary material for details. 2.3.6 Neurotrophin Levels in Decellularized Cerebral Tissue Nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), two neurotrophic proteins involved in neuronal maturation, were quantified using DuoSet® enzyme-linked immuno-sandwich assay (ELISA) kits (catalog # DY256-05 and DY248, respectively) (R&D Systems, Minneapolis, MN, USA), following the manufacturer’s instructions 43 . For BDNF and NGF ELISAs, 15 mg of lyophilized native and decellularized tissue were used per mL RIPA lysis buffer plus protease inhibitors, (catalog # sc-24948, Santa Cruz Biotechnology) 43 . Samples were homogenized on ice using a Polytron PT10-35 ultrasonic disruptor (Kinematica, Eschbach, Germany) and centrifuged at 12,000 g and 4°C for 15 minutes. Supernatants were individually collected, placed in new microcentrifuge tubes, and stored at -80°C until used 43 , 48 . 2.4 In vitro cell response to region-specific brain decellularized ECM 2.4.1 PC12 Cell Culture PC12 cells are clonal cells originating from a transplantable rat pheochromocytoma, a neoplastic rat cell line arising from neural crest tissue. An important feature of PC12 cells is that they respond to neurotrophic factors with a dramatic change in phenotype and acquire several properties characteristic of sympathetic neurons 49 . The cells were grown in 25 mm 2 tissue culture flasks with complete RPMI medium containing 5% fetal bovine serum (FBS) (catalog # 30-2020, ATCC, Manassas, VA, USA), 10% heat-inactivated horse serum (HS catalog # 30-2040, ATCC), and 1% penicillin-streptomycin (pen-strep, Thermo Fisher Scientific). Cultures were maintained according to standard protocols at 37 ºC in a 95% humidified incubator with 5% CO 2 . PC12 cells were treated with 50 ng/mL NGF (Cat # N-100, Alomone Labs, Jerusalem, Israel) or 50 ng/mL BDNF (Cat # B-250, Alomone Labs), mixed with differentiation media ( i.e. , RPMI supplemented with 1% HS, 1% pen-strep) every two days for a week. Control cells without neurotrophic factors were also grown under the same conditions 50 , 51 . 2.4.2 Cytotoxicity of Region-Specific Brain Decellularized ECM See supplementary material for details. 2.4.3 Differentiation of PC12 Cells after Treatment with Region-Specific Brain Decellularized ECM The PC12 cell line was used as a unidirectional model of differentiation to evaluate the potential of region-specific brain decellularized ECM to promote neuronal maturation 43 . Experimental groups included a negative control (PBS), positive controls (NGF and BDNF), and brain decellularized ECM groups ( i.e. , cortex, cerebellum, and remaining areas). All treatments were delivered as soluble factors to the differentiation media and were used throughout cell culture for 7 days, with feedings every 48 h. To visualize PC12 cell morphology at harvest, we stained them with fluorochrome-conjugated Phalloidin, a mushroom toxin that has a high affinity to polymerized F-actin. For this, the coverslip-adhered cells were immersion-fixed with 4% paraformaldehyde, rinsed with sterile water, and treated with 0.1% Triton X-100. The cells were then incubated in Alexa-488 Phalloidin (catalog # 59-6559, Thermo Fisher Scientific) overnight at 4ºC. The coverslips were rinsed with water, mounted on glass slides, and cover-slipped with Fluoromount-G with DAPI (catalog # 00-4959, Thermo Fisher Scientific). Cells were visualized in an Olympus BX-60 fluorescent microscope and analyzed with ImageJ to quantify the percent of cell maturation as determined by neurite-like processes extending from their cytoplasm 43 , 52 . 2.4.4 Cell Viability and Recovery after the Oxygen-Glucose Deprivation (OGD) Model PC12 cells were cultured in 96-well plates until they reached 80% of monolayer confluence. Next, cells were treated with 100 ng/ml NGF added to the growth media, and they were allowed to differentiate for 6 days. Subsequently, the cells were washed twice with DMEM/F12, supplied with hypoxic, serum- and glucose-free DMEM/F12 medium, and exposed to hypoxic conditions for 6 hours, which involved incubation at 37°C with 0.3% O 2 in a hypoxia chamber with a ProOx110 oxygen-sensor controller (Biospherix, Parish, NY, USA), evacuated with N 2 inside a regular water-jacketed 5% CO 2 incubator 53 – 55 . After 6 hours, cell cultures were removed from the hypoxia chamber. For cell recovery studies, the hypoxic conditioned media in the wells were maintained and supplemented with an additional 100 µL recovery media consisting of DMEM/F12 treated with 0.1 mg lyophilized decellularized ECM of each brain region ( i.e. , cortex, cerebellum and remaining areas), to stimulate the cells. A negative control group was treated with 100 µL additional hypoxic serum- and glucose-free DMEM/F12 medium alone, and two different positive control groups were treated with additional 100 µL DMEM/F12 medium stimulated with either 100 ng/ml NGF or BDNF. Serum- and glucose-free media conditioned during the 6 h-hypoxia was not removed from the cultures to allow the secreted molecules to remain in the medium. The rate of recovery was monitored at 8, 12, 24, and 48 hours. Cell viability after hypoxic conditions and recovery treatment was measured using a mitochondrial reduction assay based on the reduction of tetrazolium salts, using the second-generation tetrazolium salt XTT (sodium 2,3,-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium) inner salt) and an optimized intermediate electron carrier. The test was performed using the manufacturer’s instructions (ATCC) 56 . After 6 h in the OGD model (0 h) and at each indicated recovery monitoring time (8, 12, 24 and 48 h), the cells were removed from the incubator, treated with the activated-XTT solution and incubated for 4 hours at 37°C. Before reading the absorbance, plates were put on an orbital shaker for 2 minutes, and the absorbance was measured at 450 nm using a Multiskan FC Microplate Photometer (catalog # 51119000, Thermo Fisher Scientific) to assess the amount of formazan product on all the wells. The results were expressed as the percentage of viability ( i.e. , absorbance measured) and compared between controls under normal culture conditions, as well as those exposed to neurotrophic factors ( i.e. , NGF, BDNF) and the region-specific brain decellularized ECM from the cortex, cerebellum and remaining areas. The cellular viability of PC12 cell cultures in the presence of region-specific brain decellularized ECM before the OGD model was also evaluated as a baseline for comparison. 2.5 Statistical Analysis Data from experiments examining nuclear counting are presented as a total count for ten individual fields of view per specimen. All other experiments were independently repeated three times to ensure the validity of the observations, and the results from one of the experiments were presented. Significant differences between groups were determined using one-way ANOVA and Tukey’s modified t-test for experiments with equal sample size or a Tukey-Kramer test for experiments with different sample sizes (i.e., isotropic fractionator), with p < 0.05 considered to indicate a statistically significant difference. 3. RESULTS 3.1 Quantitative assessment of mass, cellular composition of porcine brain subregions The isotropic fractionator method was used to obtain the number of cells in each of the brain’s three dissected regions of interest: cortex, cerebellum, and remaining areas. Comparing the mass of each region to the total porcine brain, we found that the cerebral cortex corresponds to 52.30 ± 0.83 g (65.18 ± 1.19%); the cerebellum, to 10.12 ± 0.58 g (12.68 ± 0.47%); and the remaining areas, to 17.33 ± 0.87 g (22.14 ± 0.89%) (Fig. 2 A). In terms of cell number per brain region, we observed that the cortex had an average of 2.18 x 10 9 cells (37.59 ± 0.53%); the cerebellum, 1.82 x 10 9 cells (39.59 ± 0.70%); and the remaining areas, 0.89 x 10 9 cells (22.84 ± 0.60%) (Fig. 2 B). 3.2 Macroscopic evaluation of the cerebral decellularization process Following the dissection of the brain, each section was decellularized separately. Initially, the native tissue exhibited pink-red coloration with clear visual cues from each section. The cortex showed the characteristic pattern between the gray and white matter, while the cerebellum had the typical branching pattern (Fig. 3 A-C). After the decellularization process, these tissue morphological cues and coloration were lost. The resulting brain decellularized ECM was white/transparent in color, accompanied by a reduction in size/volume mainly due to loss of the cellular components and some of the structural molecules removed by the mechanical and chemical forces during the washes (Fig. 3 D-F). The brain decellularized ECM samples were immediately frozen at -80°C and lyophilized after the decellularization for the subsequent molecular, microscopy, and cellular analysis (Fig. 3 G-I). 3.3 Decellularization efficiency in cerebral extracellular matrix The effectiveness of our decellularization protocol in removing cellular components was confirmed with different assays. DNA quantitation using the PicoGreen double-strand DNA assay after the decellularization process revealed a considerable decrease in DNA content. The native brain tissue exhibited DNA concentrations of 1,029 ± 165 ng / mg-dry-tissue in the cerebral cortex, 1,912 ± 273 ng / mg-dry-tissue in the cerebellum and 801 ± 95 ng / mg-dry-tissue in the remaining areas; while the brain decellularized ECM presented values of 52 ± 5 ng / mg-dry-tissue; 71 ± 7 ng / mg-dry-tissue and 56 ± 4 ng / mg-dry-tissue, respectively for each brain section (Fig. 4 A). For all subregions studied, electrophoresis on agarose gels confirmed that DNA chains in native specimens maintained high number of base pairs, while DNA fragments from decellularized specimens did not exceed 200 bp in length (Fig. 4 B). In addition, we searched for certain nuclear and cytosolic proteins by Western Blot, such as the cytoskeletal protein tubulin and the neuronal nuclear protein NeuN, which showed an important reduction after the decellularization process that serves as an additional confirmation of the removal of neurons from brain sections (Fig. 4 C). The efficiency of the decellularization protocol was also evaluated histologically, with hematoxylin and eosin (H&E) staining, as well as by immunofluorescence with the DAPI marker. A total of 10 visual fields with H&E staining were used to calculate the average number of cell nuclei on native and the corresponding decellularized brain subregion. We observed a decrease in cell nuclei in the cortex from 248 to 16; in cerebellum, from 569 to 11; and in remaining areas, from 175 to 16 nuclei (Fig. 5 A-F), as seen in the H&E nuclear quantification histogram (Fig. 5 G). In addition, quantification of fluorescent DAPI-labeled nuclei in brain tissues was performed. The results revealed a decrease in nuclei in the cortex, from 133 to 14; in the cerebellum, from 309 to 17; and in the remaining areas, from 146 to 19 (Fig. 5 H-M), as seen in the DAPI nuclei quantification histogram (Fig. 5 N). The results obtained from the molecular analysis of DNA and nuclear/cytosolic protein content indicate that the decellularization process was effective at removing the cellular components of the tissue. 3.4 Brain decellularized ECM maintains tissue ultrastructure after processing After confirming the efficiency of our decellularization protocol to remove cellular components, we then studied the structural morphology of the brain decellularized ECM and its capacity to conserve growth factors. The structural and biochemical integrity of the scaffolds is important to promote cell attachment, proliferation, and differentiation into the neuronal lineage. Electron micrographs of the different brain sub-regions did not show any evident differences between the native tissues and the corresponding decellularized cerebral tissue (Fig. 6 A-F). In addition, histological evaluation through Coomasie Blue staining, used for non-specific labeling of total proteins, resulted in a strong and continuous blue color in the native tissue that was comparable to that of the decellularized ECM (Fig. 6 G-L). All histological sections were also stained with hematoxylin to highlight the efficiency of cell removal after the decellularization process. We also quantified the loss of protein after decellularization using the bicinchoninic acid biochemical assay (Fig. 6 M). The protein levels of the native brain tissue registered at 1,342 µg/mL in the cortex, 1,826 µg/mL in the cerebellum and 1,814 µg/mL in the remaining areas, which decreased considerably after decellularization to 871 µg/mL, 535 µg/mL and 858 µg/mL, respectively. These results translate to a greater retention of proteins in the cortex and the remaining areas, with 65.0% and 47.3% respectively, while the cerebellum only retained 29.3% of the initial protein content (Fig. 6 M). Finally, we also quantified the presence of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) (Fig. 6 N-O). All native brain sections presented measurable levels of NGF, including the cortex with 5.1 ng/mg-dry-tissue, the cerebellum with 7.4 ng/mg-dry-tissue and the remaining areas with 5.7 ng/mg-dry-tissue (Fig. 6 N). However, NGF could not be detected in the decellularized ECM samples. In the case of BDNF, it was also present in all native brain sections and at higher levels than NGF, especially in the remaining areas, with the cortex showing 24 ng/mg-dry-tissue, the cerebellum 81 ng/mg-dry-tissue and the remaining areas 138 ng/mg-dry-tissue (Fig. 6 O). Brain decellularized ECM did maintain measurable levels of BDNF, with 63 ng/mg-dry-tissue in cortex, 20 ng/mg-dry-tissue in cerebellum and 62 ng/mg-dry-tissue in remaining areas. Interestingly, we observed that BDNF levels seemed to increase after decellularization in the case of the cortex, reaching as much as 2.7-times the original BDNF level. 3.5 Soluble brain decellularized ECM can support in vitro cell viability. The effect of brain decellularized ECM from cortex, cerebellum and remaining areas on PC12 cell viability was assessed through live/dead staining (Calcein AM / Propidium Iodide) (Fig. 7 A-D). The decellularized ECM did not have any evident effects on PC12 viability under regular conditions, with most cells exhibiting the characteristic green fluorescence of the “live” stain, and almost no cells showing the red “dead” stain (Fig. 7 A-C). When quantified, viability remained close to 100% in PC12 cell cultures exposed to decellularized ECM from specific brain regions (Fig. 7 D). The mitochondrial activity was also measured with the XTT assay to analyze cellular viability in PC12 cells treated with decellularized ECM from the three different regions in a period of 24 hours. The results did not show considerable differences between cells stimulated with vehicle solution (control), and the experimental treatments with decellularized ECM from cortex, cerebellum and remaining areas, respectively. These results suggest that the brain decellularized ECM used as a soluble factor can maintain cellular viability in PC12 cell cultures ( Fig. 7 E ). 3.6 Soluble brain decellularized ECM can support unidirectional cell differentiation The differentiation potential of brain decellularized ECM on PC12 cells was also assessed (Fig. 8 A-G). PC12 cells mature and generate neurite-like extensions when they are stimulated, typically through exposure to neurotrophic factors, thus providing a simple unidirectional differentiation model. Indeed, when grown on poly-L-lysine (PLL)-coated coverslips and treated with vehicle (PBS), the cells had a rounded morphology and grew in clusters (Fig. 8 A). When PC12 cells were treated with neurotrophic factors known to stimulate neuronal maturation ( i.e. , positive controls NGF and BDNF), the cells generated long neurite-like processes that extended several micrometers and connected with extensions coming from neighboring cells (Fig. 8 B-C). The cells were also exposed to brain decellularized ECM from cortex, cerebellum and remaining areas, which were all able to promote morphological changes and neurite-like extensions compared to vehicle ( i.e. , negative control) (Fig. 8 D-F). However, when quantified by image analysis, neurite extensions promoted by brain decellularized ECM were reduced compared to cells treated with positive controls ( i.e. , NGF, BDNF) (Fig. 8 G). 3.7 Recovery of PC12 cells in the presence of brain decellularized ECM after oxygen-glucose deprivation (OGD) Control groups in an in vitro model in homeostasis (Normoxia) were compared to the experimental groups in an in vitro model for stroke (OGD) exposed to hypoxic conditions. After 6 h of culture, the experimental treatments were provided to the cells and the responses were monitored at 0, 8, 12, 24 and 48 h (Fig. 9 ; Supplementary Fig. 1 ). PC12 cell viability significantly decreased from 99.4% (Normoxia) to 55.1% after oxygen and glucose deprivation for 6 h (OGD – 0 h), and this value was used as a baseline for the change calculations (↑) of the evaluated treatments. Treatment with PBS (Vehicle) did not promote cell recovery even after 48 h (56.1% viability, ↑1%, after 8 h; 58.5% viability, ↑3.4%, after 12 h; 53.2% viability, ↑-1.9%, after 24 h; 56.8% viability, ↑1.7%, after 48 h). Treatment with the positive control NGF registered a significant increase in viability from 12 h after treatment (86.4% viability, ↑31.3%), reaching complete recovery at 24 and 48 h (96.5% viability, ↑41.4%, after 24 h; 103.5% viability, ↑48.4%, after 48 h). In the case of treatment with the other positive control, BDNF, cell recovery was also noticeable after 12 h (78.2% viability, ↑23.1%), although final cell recovery after 48 h (88.8% viability, ↑33.7%) was more moderate than for NGF. Finally, the experimental treatment with brain decellularized ECM had a similar impact in the recovery of cell viability as that of positive controls, and specifically that of BDNF, with a slight increase in cell viability after 8 h (72.0% viability, ↑16.9%, for cortex; 71.2% viability, ↑16.1%, for cerebellum; and 68.9% viability, ↑13.8%, for remaining areas) that continued to improve at 12 h with higher recovery rates than BDNF but lower than NGF (85.0% viability, ↑29.9%, for cortex; 80.7% viability, ↑25.6%, for cerebellum; and 80.3% viability, ↑25.2%, for remaining areas). The viability improvements continued until 48 h for the three region-specific decellularized ECM treatments (88.1% viability, ↑33.0%, for cortex; 85.5% viability, ↑30.4%, for cerebellum; and 84.1% viability, ↑29.0%, for remaining areas). Treatment with cortex ECM exhibited slightly, the most controlled and robust cell recovery of the three decellularized treatments, but still significantly lower than the normoxia group, similar to the BDNF positive control treatment. 4. DISCUSSION Decellularized ECM has been studied as a potential regenerative therapy for several decades 3 , 57 – 59 . However, few studies have characterized the effects of brain decellularized ECM scaffold properties on neural lineage development, stem cell differentiation 35 , 43 , 48 or its therapeutic potential in neurological diseases such as stroke 60 , 61 . In other words, the application of brain decellularized ECM for the regeneration of CNS tissue still holds several frontiers that need to be explored. In this study, we established an optimized protocol to produce brain decellularized ECM from three specific regions and evaluated their effect on neuronal maturation and recovery capacity in an in vitro model of brain ischemia. The original total number of cells in a tissue that is targeted for decellularization is seldom reported. In addition, even less studies have considered the number of cells in different regions of a porcine brain 62 . This initial information may be useful to determine the efficiency and quality of the decellularization process. In our case, using the isotropic fractionator technique, we were able to quantify the total number of cells per specific brain region ( i.e. , cortex, cerebellum, remaining areas) 63 . Our data indicated that the cortex represented more than 65% of the mass of the brain, while the cerebellum accounted for just over 10%. Interestingly, the cerebellum contained close to 40% of all brain cells, despite representing the smallest section of the brain. Our results were in good agreement with previously published studies that have used the isotropic fractionator to characterize the brain cellular composition for several species, including the porcine ( Sus scrofa domesticus ) brain 62 . Neuroanatomical studies have established that the cortex may possess 45% of the cell population, while the cerebellum accounts for up to 40% of the cellular presence with only 1/5 of the mass of the cerebral cortex. This relationship also seems to apply to the human brain and other species in the evolutionary ladder that show a similar pattern between the cortex and the cerebellum 62 , 64 . Our isotropic fractionator results correlate well with the values of DNA content measured in each brain section in a per-mg-dry-tissue basis, as well as in the quantification of histological sections after labeling with eosin/hematoxylin. Our decellularization protocol successfully eliminated most of the DNA content present in all three highly-cellularized studied brain regions, although none was under the threshold value of 50 ng/mg-dry-tissue, established as the gold standard for other tissues 65 . Previous studies with porcine brain decellularized ECM have reached the threshold value 43 , 48 , so our protocol could be tailored to be more aggressive or extended for a longer period to reach the before mentioned threshold. However, the risk is to further compromise the tissue’s integrity and bioactivity. The cell density of the tissue of interest is an important consideration for decellularization purposes, since removing the cellular components from the decellularized ECM is important to minimize possible cytotoxicity or immunological rejection for the culture/host. Also, native tissue with a higher cell number/mg-tissue ratio may be prone to more considerable loss of tissue ultrastructure over the course of the decellularization process regardless of the aggressiveness of the protocol. We observed such behavior in the cerebellar tissue, the native tissue with the highest cell density compared to the cortex and the remaining areas. The cerebellum decellularized ECM presented the greatest protein and structural loss after decellularization. Thus, relative cell density of the native tissue may serve as an indirect predictor of biological performance, considering that one of the most important aspects of decellularization is the conservation of the tissue's structural and bioactive proteins at the end of the process 43 . Brain decellularized ECM scaffolds generated with our decellularization protocol maintained a high degree of morphological and ultrastructural integrity, as assessed by SEM and Coomassie Blue staining, despite losing significant amounts of proteins in general and specific neurotrophic factors evaluated by ELISA. NGF and BDNF serve as growth factors that promote the development and regeneration of CNS and support crucial neuronal processes including synaptic activity, neuronal growth, neuronal regeneration and plasticity 66 . In the present study, we measured NGF and BDNF to determine if they were retained after the decellularization process. As expected, NGF, which is a well-known soluble neurotrophin reported to be involved in the constitutive pathway, the activity-dependent pathway, or in both 67 , was mostly eliminated from all decellularized scaffolds in comparison to native tissues. In contrast, certain levels of BDNF were retained in all brain decellularized ECM scaffolds, and the decellularized cortex and remaining areas presented even higher levels of BDNF per mg of dry tissue than its native counterpart, which has been reported previously as a relative effect of the loss of cell-associated proteins and other less tightly-bound ECM components 43 . For example, in cortical extracts BDNF has been found enriched in a vesicular fraction isolated from lysed synaptosomes 68 , which could explain its stronger interaction with the ECM and its resilience to the detergents and other chemicals used during decellularization. As we only found higher BDNF retention in the cortex and remaining areas ECM, this may suggest a differential storage of BDNF depending on associated brain regions. Also, the brain decellularized ECM scaffolds promoted high cell viability in PC12 cells and supported appropriate attachment of cortical primary cell cultures. In addition, the fact that brain decellularized ECM could elicit a morphological change indicative of PC12 stimulation and neuronal maturation confirms the presence of supportive proteins and neurotrophins, such as BDNF, that are known to modulate the behavior and differentiation in neural cell lineages 69 . However, there may be other extracellular components that could be triggering the development and maturation of the PC12 cells that are worth exploring. If matched correctly, the biochemical composition and structural properties of brain decellularized ECM scaffolds may be able to induce differentiation into site-appropriate functional cells that can replace lost CNS tissue in cases of brain injury or neurodegenerative pathology 70 . The effects of region-specific brain decellularized ECM have not been tested in in vitro or in vivo as a recovery treatment in cerebral ischemia or hypoxia models. We established an OGD model with PC12 cells to analyze the cell recovery potential of soluble brain decellularized ECM and other control treatments, after the hypoxic insult (i.e., oxygen-glucose deprivation). Previous studies determined that an OGD model using 6 h of hypoxia falls in the category of cytotoxicity/apoptotic response, with more than 25% loss of PC12 cell viability measured by MTT assay 71 that could still be recovered 71 , 72 , making it an ideal ischemia cell recovery model. All three region-specific brain decellularized ECM treatments had a considerable cell recovery effect, with the cerebellum decellularized ECM having the best response at 91.5% cell viability after 48 h, an increase of 36.4% compared to the vehicle group at the same timepoint. This response was similar to the positive control BDNF, which is a neural growth factor that can block caspase-3, a protein that is a major player in apoptosis 73 , and has been detected in early stages of brain ischemia associated to neuroprotection in neuronal networks 74 – 76 . After our decellularization process, all region-specific brain decellularized ECM still contained measurable levels of BDNF, which could explain the similar responses. However, the levels of other neurotrophins ( e.g. , NT-3, NT-4, CTNF) in the decellularized ECM were not measured and their role in the cell recovery results is still unknown. Taken together, our results show that region-specific brain decellularized ECM can retain structural and biochemical cues that can promote neuronal maturation under normal in vitro conditions, and robust cell recovery after oxygen-glucose deprivation using the PC12 cell line. Indeed, in vitro models using cell lines can be very convenient and biologically useful; however, often they have characteristics that are different from primary cells, and the changes needed to immortalize cell lines can sometimes allow them to withstand harsher treatments that may not be viable for primary cells or in vivo applications 77 . We have performed preliminary experiments to study the effects of region-specific brain decellularized brain ECM as a substrate on mixed primary cortical cultures, which are much more delicate to culture (Fig. 10 ). All experiments with animal to isolate primary cells were carried out under an approved protocol by the Institutional Animal Care and Use Committee (IACUC) of the School of Medicine of the University of Miami, strictly complying with the ARRIVE guidelines. Our results showed that primary cells were able to adhere, grow and express classic neural biomarkers, such as the neuronal nuclear antigen (NeuN + ) and glial fibrillary acidic protein (GFAP + ), over the course of 7 days. Primary cells on the control substrate (PLL) exhibited a homogeneous coverage of the coverslip surface, with the presence of neurons and glial cell well distributed throughout the surface (Fig. 10 A). Attachment to the decellularized ECM-coated coverslips was also well-distributed; however, NeuN + expression seemed to be concentrated in certain clusters of cells throughout the surface (Fig. 10 B-D), which may better represent the natural organization of the cells in brain. CONCLUSIONS The present study reported a decellularization method that was successfully applied to three different brain sections: cortex, cerebellum and remaining areas. Brain tissue can be decellularized while preserving various components of the ECM, including neurotrophic factors such as BDNF. Brain decellularized ECM offered many advantages to enhance neuronal cell culture in 2D ( i.e. , as a soluble treatment) and 3D models ( i.e. , as a scaffold/substrate) due to its complex biomolecular composition and retention of neurotrophic factors and diverse biochemical cues, under normal conditions to promote neuronal maturation or under hypoxic conditions to promote cell recovery. These results suggest that brain decellularized ECM may be an alternative in the future for various clinical applications in diseases associated with lesions of the CNS. Declarations DISCLOSURES The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. FUNDING The results presented in this study were supported with funds from NIH/NINDS Fogarty International Center (grant number 1R21NS098896-02), which supported training on the OGD model; SENACYT, Panama, (grants number PFID-INF-2020-43, Contract DDCCT-No-101-2021, FID17-078, IDDS22-09, PFID-INF-2020-22, APY-NI-2018-17, and APY-NI-2019B-02) and SNI-SENACYT (grant numbers SNI-12-2020, SNI-051-2023 and Res-84-2022). Funding agencies were not involved in the study design. Author Contribution DR and RAG wrote the main manuscript text and did most of the project administration. DR, KRD, MAPP and RAG were responsible for most of the funding acquisition and designed the methodology. DR, DO, SC, AB, BD, NK, JX, AH, RAG were involved in data acquisition and figure preparation. All authors reviewed the manuscript. Acknowledgement We thank the Macelo S. A. abattoir for their support supplying the porcine brains. 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Supplementary Files SciRepdecellECMOGDSupplrev1.docx Cite Share Download PDF Status: Published Journal Publication published 07 Apr, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 04 Dec, 2024 Reviews received at journal 30 Nov, 2024 Reviewers agreed at journal 26 Nov, 2024 Reviews received at journal 03 Nov, 2024 Reviewers agreed at journal 21 Oct, 2024 Reviewers agreed at journal 19 Oct, 2024 Reviewers invited by journal 17 Oct, 2024 Editor assigned by journal 17 Oct, 2024 Editor invited by journal 17 Oct, 2024 Submission checks completed at journal 17 Oct, 2024 First submitted to journal 21 Sep, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5130290","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":367922542,"identity":"fa643af3-90ec-4cde-8d79-50d318bd382b","order_by":0,"name":"Diego Reginensi","email":"","orcid":"","institution":"Universidad de Panamá","correspondingAuthor":false,"prefix":"","firstName":"Diego","middleName":"","lastName":"Reginensi","suffix":""},{"id":367922543,"identity":"f5d106e2-4b8c-4161-915d-ac28219715d1","order_by":1,"name":"Didio Ortiz","email":"","orcid":"","institution":"University of Navarra","correspondingAuthor":false,"prefix":"","firstName":"Didio","middleName":"","lastName":"Ortiz","suffix":""},{"id":367922544,"identity":"b1d83a27-00a0-4f8f-9d90-fd31f77f1721","order_by":2,"name":"Solangel Castillo","email":"","orcid":"","institution":"Universidad de Panamá","correspondingAuthor":false,"prefix":"","firstName":"Solangel","middleName":"","lastName":"Castillo","suffix":""},{"id":367922545,"identity":"ef402f2f-b446-4ab5-ab84-bbde241a798f","order_by":3,"name":"Andrea Burillo","email":"","orcid":"","institution":"Instituto de Investigaciones Científicas y Servicios de Alta Tecnología","correspondingAuthor":false,"prefix":"","firstName":"Andrea","middleName":"","lastName":"Burillo","suffix":""},{"id":367922546,"identity":"a57db760-7027-4a79-834f-44698d75c91f","order_by":4,"name":"Bernardino Denis","email":"","orcid":"","institution":"Instituto de Investigaciones Científicas y Servicios de Alta Tecnología","correspondingAuthor":false,"prefix":"","firstName":"Bernardino","middleName":"","lastName":"Denis","suffix":""},{"id":367922547,"identity":"0036a6e6-06ba-49b4-a476-0b8156aa3221","order_by":5,"name":"Nathalie Khoury","email":"","orcid":"","institution":"University of Miami Miller School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Nathalie","middleName":"","lastName":"Khoury","suffix":""},{"id":367922548,"identity":"4c9b3162-de85-4322-aae1-17dce8eacf51","order_by":6,"name":"Jing Xu","email":"","orcid":"","institution":"University of Miami Miller School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Xu","suffix":""},{"id":367922549,"identity":"8d4062e4-fd09-4c18-9eae-5b2c194c380f","order_by":7,"name":"Anthony A. Hurtado Escobar","email":"","orcid":"","institution":"Instituto de Investigaciones Científicas y Servicios de Alta Tecnología","correspondingAuthor":false,"prefix":"","firstName":"Anthony","middleName":"A. Hurtado","lastName":"Escobar","suffix":""},{"id":367922550,"identity":"d71a9007-2b5a-4a6d-8bf4-c2611e3bc669","order_by":8,"name":"Kunjan R. Dave","email":"","orcid":"","institution":"University of Miami Miller School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Kunjan","middleName":"R.","lastName":"Dave","suffix":""},{"id":367922553,"identity":"79b08ea5-1e45-446e-adea-2d5d25bd0958","order_by":9,"name":"Miguel A. Perez-Pinzon","email":"","orcid":"","institution":"University of Miami Miller School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Miguel","middleName":"A.","lastName":"Perez-Pinzon","suffix":""},{"id":367922557,"identity":"09939a76-2100-42b3-bba7-e9b0f175ded1","order_by":10,"name":"Rolando A. Gittens","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4ElEQVRIiWNgGAWjYFACxgYwxc9MgpbGhgNASrKZFD1gLQYHiNVi3r+4/fGHijv2xsf5D3/4wGAnzyDd/ACvFpkbD4EOO/MscdthZjbJGQzJhg0yxwzwapGQONjYcLDtcIIZUAszDwNzAoNEAjFa/h22N25mZv78h6EeqCX9A34t/I1ALQ2HGTcwMzNIMzAcBmrJIWQLY+OMM8cOJ844zGwm2WNw3LBN5kwBAVuOP/hQUXPYnr//4OMPPyqq5fml2zfg1QL0LDIP6CQ2CfwagAnlAIYhhLSMglEwCkbBSAMARPRGv56pbnQAAAAASUVORK5CYII=","orcid":"","institution":"Instituto de Investigaciones Científicas y Servicios de Alta Tecnología","correspondingAuthor":true,"prefix":"","firstName":"Rolando","middleName":"A.","lastName":"Gittens","suffix":""}],"badges":[],"createdAt":"2024-09-21 22:53:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5130290/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5130290/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41598-025-95656-w","type":"published","date":"2025-04-07T16:04:50+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":67131914,"identity":"a9403b5a-0578-49f3-b84b-126fbb359ce0","added_by":"auto","created_at":"2024-10-21 12:57:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":67791,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic illustration of the decellularization process.\u003c/strong\u003e After decellularization, most cells and soluble factors are lost, while the ECM, matrix-bound factors and some cellular remnants are maintained.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5130290/v1/d40232ff67e5b741fcbc3e68.png"},{"id":67132346,"identity":"22ceab20-ff7c-4723-9f3d-215cd7e26e93","added_by":"auto","created_at":"2024-10-21 13:05:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":74459,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eQuantification of brain regions by mass, and by cell number using the isotropic fractionator method.\u003c/strong\u003e The porcine brain was dissected in three sections: cortex, cerebellum, and remaining areas. (A) The mass and (B) the number of cells of each brain section of the adult porcine brain were quantified and compared in percent format. Significant differences were determined between all groups shown by one-way ANOVA with Tukey-Kramer post hoc analysis (p\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5130290/v1/83f57fac377e0024133de379.png"},{"id":67132350,"identity":"fbfd46a6-e770-4423-862b-15933d42b52e","added_by":"auto","created_at":"2024-10-21 13:05:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1335382,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMacroscopic evaluation of the cerebral decellularization process. \u003c/strong\u003e(A-C)\u003cstrong\u003e \u003c/strong\u003ePhotographic record of the native brain regions (Native Tissue) that were dissected: cortex, cerebellum, and remaining brain areas. Macroscopically, a pinkish-reddish color is observed in brain tissue. (D-F) Photographs of the decellularized brain sections right after processing (Decellularized Tissue). The resulting decellularized ECM shows a loss of coloration at the macroscopic level. (G-I) Photographs of the freeze-dried decellularized brain scaffolds, state in which they were used for the characterization assays (Lyophilized Tissue). Scale bar: 10 mm.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5130290/v1/62d3d438cd0519c5c4ff1a19.png"},{"id":67131904,"identity":"5ae88adf-e0d2-489f-a00c-a6c33275416b","added_by":"auto","created_at":"2024-10-21 12:57:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":393653,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMolecular analysis of the decellularization process efficiency.\u003c/strong\u003e (A) DNA concentration graph of the cortex, cerebellum and remaining areas before (■) and after (□) decellularization. (B) Gel electrophoresis showed that residual DNA fragments did not exceed 200 bp in cortex, cerebellum or remaining areas. Arrows (left side) denote 2500 bp, 1000 bp, 500 bp, 200 bp and 100 bp. (C) Western blot analysis of tubulin and NeuN levels of native and decellularized brain tissue. Significant differences were determined between all groups shown by one-way ANOVA with Tukey post hoc analysis (p\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5130290/v1/401984d825aabe42048c3f46.png"},{"id":67132347,"identity":"493a20dc-90e9-4a99-9f63-02a790c957f5","added_by":"auto","created_at":"2024-10-21 13:05:56","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":6912403,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistological evaluation of cell removal after decellularization. \u003c/strong\u003eAfter H\u0026amp;E staining, cell nuclei were visible in native (A) cortex, (B) cerebellum and (C) remaining areas tissues but were not as evident in (D-F) the corresponding decellularized tissues. (G) The quantification of the average H\u0026amp;E nuclei counted confirmed the decrease in cell content after decellularization. DAPI showed similar results in native (H) cortex, (I) cerebellum and (J) remaining areas compared to (K-M) decellularized ECM from the corresponding regions, and (N) the quantification of the average DAPI\u003csup\u003e+\u003c/sup\u003e nuclei also confirmed this observation. Significant differences were determined between all groups shown by one-way ANOVA with Tukey post hoc analysis (p\u0026lt;0.05). Scale bar is 20 μm.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5130290/v1/4da377f01fd8ce0902899c13.png"},{"id":67133425,"identity":"0ee05446-e939-4c03-ab45-bc2a27eb6282","added_by":"auto","created_at":"2024-10-21 13:13:56","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":896893,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStructural and biochemical integrity of decellularized ECM\u003c/strong\u003e. (A-F) Electron micrographs show that the morphology of the tissue was not altered considerably after the decellularization process. (G-L) Protein ultrastructure evaluated with Coomassie Blue to stain for non-specific proteins, exhibited a well-preserved structure after the decellularization process. Tissue from porcine brains before (■ Native) and after decellularization (□ Decellularized) were analyzed for (M) general protein content with the bicinchoninic acid assay, which confirmed the loss of proteins during processing; as well as for (N) NGF and (O) BDNF protein levels, which were quantified by enzyme-linked immunosorbent assay (ELISA). Significant differences were determined between all groups shown by one-way ANOVA with Tukey post hoc analysis (p\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5130290/v1/a79686a1029ba6186c531ab2.png"},{"id":67131906,"identity":"974afad3-e73f-4a49-9449-725994e37d13","added_by":"auto","created_at":"2024-10-21 12:57:56","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":261990,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCell viability of PC12 cells treated with brain decellularized ECM as a soluble factor.\u003c/strong\u003e Cell viability in the presence of brain decellularized ECM obtained from (A) cortex, (B) cerebellum and (C) remaining areas was assessed through live/dead staining (Calcein AM / Propidium Iodide), showing mainly green fluorescence (\u003cem\u003ei.e.\u003c/em\u003e, “live” cells) and no evident red staining (\u003cem\u003ei.e.\u003c/em\u003e, “dead” cells). (D) Quantification of cellular viability based on the live/dead staining showed that PC12 cells remained close to 100% viable when treated with the negative control or in presence of decellularized ECM. (E) The XTT assay was preformed to analyze mitochondrial activity using decellularized ECM as a soluble factor, and there were no significant changes in cell viability.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-5130290/v1/19ba547e80c3100f7b2256a3.png"},{"id":67132349,"identity":"05aae2fb-9f2a-4377-946a-cbdd11aab85d","added_by":"auto","created_at":"2024-10-21 13:05:56","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":524038,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEvaluation of PC12 cell differentiation response to decellularized ECM from cortex, cerebellum and remaining areas.\u003c/strong\u003e (A) DMEM-F12, was used as vehicle (negative control), while (B) NGF and (C) BDNF were used as positive controls. (D-F) The experimental groups included brain decellularized ECM from cortex, cerebellum, and remaining areas added to the culture media in soluble form (100 μg/mL). (G) Quantification of the maturation levels of PC12 cells when exposed to the different treatments, with n \u0026gt; 250 cells for each condition. The scale bar for all images is equal to 100 μm. *refers to a statistically significant p-value below 0.05 versus vehicle (control); # refers to a statistically significant p-value below 0.05 versus NGF; $ refers to a statistically significant p-value below 0.05 versus BDNF.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-5130290/v1/37afe1776e11e3dd6abcb6ae.png"},{"id":67131909,"identity":"3c4dc6c1-d165-4ce0-b7de-7f71787140dd","added_by":"auto","created_at":"2024-10-21 12:57:56","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":207726,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eXTT metabolic activity for NGF-differentiated PC12 cells after oxygen-glucose deprivation insult. A)\u003c/strong\u003e Timeline of experiment comparing control cells in an \u003cem\u003ein vitro\u003c/em\u003e model in homeostasis (Normoxia) to the experimental groups in an in vitro model for stroke (OGD) exposed to hypoxic conditions. After 6 h of culture under normal or hypoxic conditions, the experimental treatments were provided to the cells and their responses were monitored at 0, 8, 12, 24 and 48 hours. \u003cstrong\u003eB).\u003c/strong\u003e Percentage of cell viability before (Control) and after 6 h of hypoxic conditions (OGD, 0h), as well as with the recovery treatments (\u003cem\u003ei.e.\u003c/em\u003e, Vehicle, NGF, BDNF, and decellularized ECM from Cortex, Cerebellum and Remaining Areas) monitored at 0, 8, 12, 24 and 48 hours. Results were expressed as Mean \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;SD (n=9). *refers to a statistically significant p-value below 0.05 versus control (normoxia) (0h); # refers to a statistically significant p-value below 0.05 versus OGD (0h); $ refers to a statistically significant p-value below 0.05 versus vehicle (8h); @ refers to a statistically significant p-value below 0.05 versus vehicle (12h); \u0026amp; refers to a statistically significant p-value below 0.05 versus vehicle (24h); % refers to a statistically significant p-value below 0.05 versus vehicle (48h); ? refers to a statistically significant p-value below 0.05 versus NGF (8h); ! refers to a statistically significant p-value below 0.05 versus NGF (24h); Ç refers to a statistically significant p-value below 0.05 versus NGF (48h); + refers to a statistically significant p-value below 0.05 versus BDNF (48h); = refers to a statistically significant p-value below 0.05 versus cortex ECM (8h); // refers to a statistically significant p-value below 0.05 versus cortex ECM (24h); \u0026gt; refers to a statistically significant p-value below 0.05 versus cortex ECM (48h); \u0026lt; refers to a statistically significant p-value below 0.05 versus cerebellum ECM (8h); º refers to a statistically significant p-value below 0.05 versus cerebellum ECM (12h); ^ refers to a statistically significant p-value below 0.05 versus cerebellum ECM (24h) and () refers to a statistically significant p-value below 0.05 versus cerebellum ECM (48h).\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-5130290/v1/e4ae652050907c1a768a4486.png"},{"id":67131912,"identity":"70165bf7-eacf-46e7-b550-283d722c6736","added_by":"auto","created_at":"2024-10-21 12:57:56","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":683922,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCortical primary cell culture on brain decellularized ECM. \u003c/strong\u003eCortical primary cell culture on (A) conventional PLL (control) or on (B-D) brain decellularized ECM-coated coverslips were able to attach, grow and express both neuronal (NeuN, red) and glial (GFAP, green) classical markers, assessed by fluorescence microscopy. Scale bar at 100 μm.\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-5130290/v1/a246b10bcd47990396abdb39.png"},{"id":80558140,"identity":"95290454-5772-4a87-96fe-0e54dc0fdc4b","added_by":"auto","created_at":"2025-04-14 16:07:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":15305126,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5130290/v1/03cb16fc-239a-4210-8f5e-88f0fad68b26.pdf"},{"id":67133424,"identity":"68b99625-374a-45ba-b53e-5b897e8b2f95","added_by":"auto","created_at":"2024-10-21 13:13:56","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":130923,"visible":true,"origin":"","legend":"","description":"","filename":"SciRepdecellECMOGDSupplrev1.docx","url":"https://assets-eu.researchsquare.com/files/rs-5130290/v1/c0d876008e8091e649329fff.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Region‐specific brain decellularized scaffolds can recover cell viability in an oxygen-glucose deprivation model","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eThe extracellular matrix (ECM) serves as a scaffold that gives structural support to the tissue and serves as a biochemical reservoir that helps maintain the tissue specific cell phenotype \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. The ECM can be decellularized and still retain the biochemical and biomechanical properties of native tissue; therefore, it has been used as a platform for tissue engineering \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. The concept of decellularized ECM can be simply described as a tissue that has been treated to remove cells and their cellular components while maintaining as much ECM components as possible. It is these cellular components (e.g., DNA, cytoplasmic and membrane-bound proteins) that could hinder cell development and differentiation around these decellularized ECM scaffolds in the case of regular cell culture, 3D bioprinting and lab-on-a-chip applications \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e or cause an adverse response in the immune system of a host organism that will receive it at an injured site \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e No decellularization protocol is perfect. Some cellular remnants most certainly will be left behind (ideally below the acceptable threshold of 50 ng DNA per mg of dry tissue \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e and/or the remaining DNA fragments must be less than 200 base pairs \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e), and some (potentially important) ECM proteins and soluble factors will be lost (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBiologic scaffolds composed of decellularized ECM have become a novel platform to promote the innate regenerative capacities of most tissues by providing structural support and site-specific ligands that can promote cell attachment, differentiation and local signaling \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Various protocols for the preparation of decellularized ECM have been developed \u003cem\u003ein vitro\u003c/em\u003e and tailored for different tissues of interest, including skin, bladder, liver, adipose and neural tissue \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Thus, the use of decellularized ECM as an \u003cem\u003ein-situ\u003c/em\u003e implant in damaged tissues offers a versatile and flexible scaffolding platform that minimizes tissue mismatch and immune complications that, in many cases, can lead to rejection of the transplanted tissue/organ \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. One of the most promising approaches to generate artificial tissues for grafting is by reseeding decellularized scaffolds with specified cells depending on the biomedical problem being addressed \u003csup\u003e\u003cspan additionalcitationids=\"CR19\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Cell therapy, or the use of autologous or allogeneic cells as a therapeutic treatment, can offer a promising solution to highly complex medical conditions, such as neurodegenerative diseases \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe effects associated with cell therapy in neural regeneration can be divided into the following main categories: immune modulation, support of cell survival, axonal growth, cell differentiation and cellular replacement \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. However, the use of cells by themselves can leave them vulnerable to unwanted phenotypic changes based on the harsh environment of the injury site, as well as prone to quick removal by the host\u0026rsquo;s immune cells \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. The administration of cell therapy in combination with decellularized ECM scaffolds can promote a microenvironment that enhances cell survival and maintains a more robust phenotypic profile, such as in the case of glial and neuronal cells and the promotion of axonal regeneration \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Another example is the co-administration of stem cells and scaffold matrices as therapy for brain trauma and spinal cord injury, which has been shown to be beneficial \u003csup\u003e\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eSeveral reviews of different types of materials used as scaffolds to support stem cell survival and proliferation in different models of nerve injury have recently been published \u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. However, most of these materials are based on synthetic derivatives that do not resemble the structural complexity of the tissue microenvironment of the central nervous system (CNS) \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Therefore, the use of decellularized ECM derived from CNS tissue is presented as a biomimetic substrate for \u003cem\u003ein vitro\u003c/em\u003e models of neurological disorders, with the potential to become a treatment for injuries in the brain such as stroke \u003csup\u003e\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMost biomaterials scaffolds such as collagen-based matrix, gelatin, matrigel and fibrin enhance cell survival, providing a temporary mimic of the extracellular matrix in therapies for traumatic brain injury and stroke \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Nonetheless, this single-molecule approach does not recapitulate the synergistic effects that can be found in the complex native ECM of the brain, with a unique combination of proteins and proteoglycans contained in its specific regions (e.g., cortex, cerebellum or remaining areas). Brain-derived decellularized ECM has been applied as an effective therapeutic method for spinal cord injury, traumatic brain injury, and stroke \u003cem\u003ein vitro\u003c/em\u003e and rat models \u003csup\u003e\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. However, the capacity of regeneration of the decellularized ECM from the different brain subregions has never been tested separately to evaluate the regulation of stem neuronal cell survival and differentiation.\u003c/p\u003e \u003cp\u003eBrain cells, to function properly, require a constant supply of oxygen and glucose \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. The lack of these components or exposure to levels below normal physiological conditions experienced during brain ischemia (\u003cem\u003ei.e.\u003c/em\u003e, stroke), produces deleterious effects on the cell\u0026rsquo;s metabolism \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e. Depending on the severity of the ischemic state (\u003cem\u003ei.e.\u003c/em\u003e, mild, moderate or severe) and its duration, cells suffer injuries that can lead to chronic, degenerative cell death through necrosis and apoptosis \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e in a very localized manner. This makes brain ischemia a good candidate to develop regenerative therapies based on decellularized ECM that, in the worst-case scenario, could stop the chronic cell death; or, in the best-case scenario, could promote tissue regeneration and neuronal maturation in the site of the injury.\u003c/p\u003e \u003cp\u003eOur long-term goal is to develop new regenerative medicine therapies for the treatment of neurological disease (\u003cem\u003ee.g.\u003c/em\u003e, brain ischemia/ reperfusion and stroke) on the basis of a comprehensive methodology focused on two interrelated pillars: (\u003cem\u003ei\u003c/em\u003e) development of novel biomaterials to modulate the cellular response using different anatomical sections of the brain extracellular matrix (\u003cem\u003ei.e.\u003c/em\u003e, cortex, cerebellum and remaining areas) and (\u003cem\u003eii\u003c/em\u003e) to define the effects \u003cem\u003ein vitro\u003c/em\u003e of decellularized scaffold on culture of neural lineage cells. In this study we present and characterize an optimized protocol to produce cerebral decellularized extracellular matrix from porcine source and confirm its capacity to enhance neuronal maturation of PC12 cells. Finally, we compare the role of the region-specific brain decellularized ECM in functional cellular recovery after a simulated ischemic stroke \u003cem\u003ein vitro\u003c/em\u003e using the oxygen/glucose deprivation model.\u003c/p\u003e"},{"header":"2. MATERIALS \u0026 METHODS","content":"\u003cp\u003eThe methodology was divided into 4 experimental protocols: (i) neuroanatomical dissection of porcine brain tissue; (ii) total cell quantification of each of the dissected brain regions; (iii) processing and characterization of region-specific brain decellularized ECM and (iv) \u003cem\u003ein vitro\u003c/em\u003e cell response to the brain decellularized ECM in regular cell culture and in simulated stroke conditions using the oxygen/glucose deprivation model. All methods were performed in accordance with relevant guidelines and regulations. The detailed description of the methods is as follows:\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Neuroanatomical dissection of porcine brain tissue\u003c/h2\u003e \u003cp\u003e All animal tissue handling was performed with approval from the Institutional Animal Care and Use Committee (IACUC) of INDICASAT-AIP (for porcine tissue) and the School of Medicine of the University of Miami (for rodent tissue), strictly complying with the ARRIVE guidelines. Porcine brains were collected fresh at the Macelo S. A. abattoir, in Panama City, Panama, as previously described \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Briefly, the animals were euthanized through electric shock stunning and cardiac arrest using electrodes set at 0.5 A and 220 V. Brain tissue is a byproduct of the operations of the abattoir, so the material used does not pose a risk to any additional animals. The porcine brains were collected fresh, less than 5 min after euthanasia, as hemispheres cut along the mid-sagittal plane and brought immediately to the lab in ice to be processed in a biosafety cabinet where the meninges were removed and the brains were dissected in three sections: cortex (\u003cem\u003ei.e.\u003c/em\u003e, cortical gray and white matter and hippocampus), cerebellar structure and remaining areas (\u003cem\u003ei.e.\u003c/em\u003e, the sum of diencephalon, basal ganglia, mesencephalon, pons and medulla) under aseptic conditions. Each section was cut into pieces smaller than 1x1 cm\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e, placed in 50mL conical tubes up to the 15mL mark for each tube, and stored at -80 \u003csup\u003eo\u003c/sup\u003eC for at least 24 h before decellularization.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Total cell quantification of the dissected brain regions\u003c/h2\u003e \u003cp\u003eThe total number of brain cells was estimated using the isotropic fractionator method, as previously described \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Immediately after arriving at the laboratory, the brain halves that were separated for isotropic fractionator were weighed and dissected into the three brain sections: cortex, cerebellum and remaining areas. Next, each section was fixed in 4% paraformaldehyde in 0.1M phosphate buffer (PB, pH 7.4) overnight, cryoprotected in 30% sucrose in 0.1 M PB at 4\u0026deg;C and stored in an antifreeze solution at -20\u0026deg;C until processing \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e, if necessary. Subsequently, tissue samples were homogenized with a Tenbroeck homogenizer in a saline detergent solution to dissolve the cytoplasmic membrane but not the nuclear membrane to produce a homogeneous solution of free nuclei (isotropic solution). The total number of nuclei in suspension, which correspond to the number of cells in the original tissue, was determined by staining with the DNA marker DAPI (4-6-diamidino-2-phenylindole dihydrochloride) and counting with a hemocytometer (Neubauer chamber) under a fluorescence microscope \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Processing and characterization of region-specific brain decellularized ECM\u003c/h2\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1 \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eBrain Tissue Decellularization Protocol\u003c/span\u003e\u003c/h2\u003e \u003cp\u003eAn optimized brain decellularization method has been developed based on our previous work \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e comparing two commonly used protocols: a 1-day enzymatic-based protocol \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e and a 4-day detergent-based method \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Initially, the dissected tissue was submerged in 0.1% sodium dodecyl sulfate (SDS, Sigma- Aldrich Corp., St. Louis, MO, USA) in medical grade injectable water (PISA Farmaceutica, Guadalajara, Mexico) supplemented with 1% streptomycin/penicillin (Gibco\u0026trade; catalog # 15140122, Thermo Fisher Scientific, Waltham, MA, USA) and washed at 4\u0026ordm;C with gentle orbital agitation (50 rpm) for 6 h before replenishing the solution and continue washing with the same conditions for an additional 14 h (\u003cem\u003ei.e.\u003c/em\u003e, overnight). Subsequently, the following series of timed washes were performed: 0.02% trypsin/0.05% EDTA (Invitrogen Corp., Carlsbad, CA, USA) for 75 min; 3.0% Triton X-100 (Sigma- Aldrich Corp) for 75 min; 1.0 M sucrose (Thermo Fisher Scientific) for 30 min and 0.1% SDS for 16 h. All these washes were performed with an agitation of 80 rpm at room temperature, except for the step with trypsin, which was performed at 37\u0026deg;C. The final washes involved once with 0.1% peracetic acid (Rochester Midland Corp., Rochester, NY, USA) in 4.0% ethanol for 120 min, which was used as a sterilizing agent; twice with PBS (Thermo Fisher Scientific) for 20 min and twice with sterile injectable water for 20 min; again, with all these steps at RT and 80 rpm. The decellularized tissue obtained was frozen overnight at -80 \u0026ordm;C, lyophilized in aseptic conditions and stored dry at -80 \u0026ordm;C until use.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2 \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eCharacterization of the Decellularization Efficiency: DNA Content\u003c/span\u003e\u003c/h2\u003e \u003cp\u003eSee supplementary material for details.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.3.3 \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eCharacterization of the Decellularization Efficiency: Western Blot\u003c/span\u003e\u003c/h2\u003e \u003cp\u003eSee supplementary material for details.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.3.4 \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eStructural Characterization: Histological Analysis\u003c/span\u003e\u003c/h2\u003e \u003cp\u003eSee supplementary material for details.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.3.5 \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eMorphological Characterization: Scanning Electron Microscopy\u003c/span\u003e\u003c/h2\u003e \u003cp\u003eSee supplementary material for details.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.3.6 \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eNeurotrophin Levels in Decellularized Cerebral Tissue\u003c/span\u003e\u003c/h2\u003e \u003cp\u003eNerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), two neurotrophic proteins involved in neuronal maturation, were quantified using DuoSet\u0026reg; enzyme-linked immuno-sandwich assay (ELISA) kits (catalog # DY256-05 and DY248, respectively) (R\u0026amp;D Systems, Minneapolis, MN, USA), following the manufacturer\u0026rsquo;s instructions \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. For BDNF and NGF ELISAs, 15 mg of lyophilized native and decellularized tissue were used per mL RIPA lysis buffer plus protease inhibitors, (catalog # sc-24948, Santa Cruz Biotechnology) \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Samples were homogenized on ice using a Polytron PT10-35 ultrasonic disruptor (Kinematica, Eschbach, Germany) and centrifuged at 12,000 g and 4\u0026deg;C for 15 minutes. Supernatants were individually collected, placed in new microcentrifuge tubes, and stored at -80\u0026deg;C until used \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.4\u003c/b\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003ecell response to region-specific brain decellularized ECM\u003c/b\u003e\u003c/h2\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1 \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003ePC12 Cell Culture\u003c/span\u003e\u003c/h2\u003e \u003cp\u003ePC12 cells are clonal cells originating from a transplantable rat pheochromocytoma, a neoplastic rat cell line arising from neural crest tissue. An important feature of PC12 cells is that they respond to neurotrophic factors with a dramatic change in phenotype and acquire several properties characteristic of sympathetic neurons\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. The cells were grown in 25 mm\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e tissue culture flasks with complete RPMI medium containing 5% fetal bovine serum (FBS) (catalog # 30-2020, ATCC, Manassas, VA, USA), 10% heat-inactivated horse serum (HS catalog # 30-2040, ATCC), and 1% penicillin-streptomycin (pen-strep, Thermo Fisher Scientific). Cultures were maintained according to standard protocols at 37 \u0026ordm;C in a 95% humidified incubator with 5% CO\u003csub\u003e2\u003c/sub\u003e. PC12 cells were treated with 50 ng/mL NGF (Cat # N-100, Alomone Labs, Jerusalem, Israel) or 50 ng/mL BDNF (Cat # B-250, Alomone Labs), mixed with differentiation media (\u003cem\u003ei.e.\u003c/em\u003e, RPMI supplemented with 1% HS, 1% pen-strep) every two days for a week. Control cells without neurotrophic factors were also grown under the same conditions \u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2 \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eCytotoxicity of Region-Specific Brain Decellularized ECM\u003c/span\u003e\u003c/h2\u003e \u003cp\u003eSee supplementary material for details.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.4.3 \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eDifferentiation of PC12 Cells after Treatment with Region-Specific Brain Decellularized ECM\u003c/span\u003e\u003c/h2\u003e \u003cp\u003eThe PC12 cell line was used as a unidirectional model of differentiation to evaluate the potential of region-specific brain decellularized ECM to promote neuronal maturation\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Experimental groups included a negative control (PBS), positive controls (NGF and BDNF), and brain decellularized ECM groups (\u003cem\u003ei.e.\u003c/em\u003e, cortex, cerebellum, and remaining areas). All treatments were delivered as soluble factors to the differentiation media and were used throughout cell culture for 7 days, with feedings every 48 h. To visualize PC12 cell morphology at harvest, we stained them with fluorochrome-conjugated Phalloidin, a mushroom toxin that has a high affinity to polymerized F-actin. For this, the coverslip-adhered cells were immersion-fixed with 4% paraformaldehyde, rinsed with sterile water, and treated with 0.1% Triton X-100. The cells were then incubated in Alexa-488 Phalloidin (catalog # 59-6559, Thermo Fisher Scientific) overnight at 4\u0026ordm;C. The coverslips were rinsed with water, mounted on glass slides, and cover-slipped with Fluoromount-G with DAPI (catalog # 00-4959, Thermo Fisher Scientific). Cells were visualized in an Olympus BX-60 fluorescent microscope and analyzed with ImageJ to quantify the percent of cell maturation as determined by neurite-like processes extending from their cytoplasm \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e2.4.4 \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eCell Viability and Recovery after the Oxygen-Glucose Deprivation (OGD) Model\u003c/span\u003e\u003c/h2\u003e \u003cp\u003ePC12 cells were cultured in 96-well plates until they reached 80% of monolayer confluence. Next, cells were treated with 100 ng/ml NGF added to the growth media, and they were allowed to differentiate for 6 days. Subsequently, the cells were washed twice with DMEM/F12, supplied with hypoxic, serum- and glucose-free DMEM/F12 medium, and exposed to hypoxic conditions for 6 hours, which involved incubation at 37\u0026deg;C with 0.3% O\u003csub\u003e2\u003c/sub\u003e in a hypoxia chamber with a ProOx110 oxygen-sensor controller (Biospherix, Parish, NY, USA), evacuated with N\u003csub\u003e2\u003c/sub\u003e inside a regular water-jacketed 5% CO\u003csub\u003e2\u003c/sub\u003e incubator \u003csup\u003e\u003cspan additionalcitationids=\"CR54\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. After 6 hours, cell cultures were removed from the hypoxia chamber. For cell recovery studies, the hypoxic conditioned media in the wells were maintained and supplemented with an additional 100 \u0026micro;L recovery media consisting of DMEM/F12 treated with 0.1 mg lyophilized decellularized ECM of each brain region (\u003cem\u003ei.e.\u003c/em\u003e, cortex, cerebellum and remaining areas), to stimulate the cells. A negative control group was treated with 100 \u0026micro;L additional hypoxic serum- and glucose-free DMEM/F12 medium alone, and two different positive control groups were treated with additional 100 \u0026micro;L DMEM/F12 medium stimulated with either 100 ng/ml NGF or BDNF. Serum- and glucose-free media conditioned during the 6 h-hypoxia was not removed from the cultures to allow the secreted molecules to remain in the medium.\u003c/p\u003e \u003cp\u003eThe rate of recovery was monitored at 8, 12, 24, and 48 hours. Cell viability after hypoxic conditions and recovery treatment was measured using a mitochondrial reduction assay based on the reduction of tetrazolium salts, using the second-generation tetrazolium salt XTT (sodium 2,3,-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium) inner salt) and an optimized intermediate electron carrier. The test was performed using the manufacturer\u0026rsquo;s instructions (ATCC) \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. After 6 h in the OGD model (0 h) and at each indicated recovery monitoring time (8, 12, 24 and 48 h), the cells were removed from the incubator, treated with the activated-XTT solution and incubated for 4 hours at 37\u0026deg;C. Before reading the absorbance, plates were put on an orbital shaker for 2 minutes, and the absorbance was measured at 450 nm using a Multiskan FC Microplate Photometer (catalog # 51119000, Thermo Fisher Scientific) to assess the amount of formazan product on all the wells. The results were expressed as the percentage of viability (\u003cem\u003ei.e.\u003c/em\u003e, absorbance measured) and compared between controls under normal culture conditions, as well as those exposed to neurotrophic factors (\u003cem\u003ei.e.\u003c/em\u003e, NGF, BDNF) and the region-specific brain decellularized ECM from the cortex, cerebellum and remaining areas. The cellular viability of PC12 cell cultures in the presence of region-specific brain decellularized ECM before the OGD model was also evaluated as a baseline for comparison.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Statistical Analysis\u003c/h2\u003e \u003cp\u003eData from experiments examining nuclear counting are presented as a total count for ten individual fields of view per specimen. All other experiments were independently repeated three times to ensure the validity of the observations, and the results from one of the experiments were presented. Significant differences between groups were determined using one-way ANOVA and Tukey\u0026rsquo;s modified t-test for experiments with equal sample size or a Tukey-Kramer test for experiments with different sample sizes (i.e., isotropic fractionator), with p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered to indicate a statistically significant difference.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.1 \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eQuantitative assessment of mass, cellular composition of porcine brain subregions\u003c/span\u003e\u003c/h2\u003e \u003cp\u003eThe isotropic fractionator method was used to obtain the number of cells in each of the brain\u0026rsquo;s three dissected regions of interest: cortex, cerebellum, and remaining areas. Comparing the mass of each region to the total porcine brain, we found that the cerebral cortex corresponds to 52.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.83 g (65.18\u0026thinsp;\u0026plusmn;\u0026thinsp;1.19%); the cerebellum, to 10.12\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58 g (12.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.47%); and the remaining areas, to 17.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87 g (22.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.89%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). In terms of cell number per brain region, we observed that the cortex had an average of 2.18 x 10\u003csup\u003e9\u003c/sup\u003e cells (37.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53%); the cerebellum, 1.82 x 10\u003csup\u003e9\u003c/sup\u003e cells (39.59\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70%); and the remaining areas, 0.89 x 10\u003csup\u003e9\u003c/sup\u003e cells (22.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.60%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.2 \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eMacroscopic evaluation of the cerebral decellularization process\u003c/span\u003e\u003c/h2\u003e \u003cp\u003eFollowing the dissection of the brain, each section was decellularized separately. Initially, the native tissue exhibited pink-red coloration with clear visual cues from each section. The cortex showed the characteristic pattern between the gray and white matter, while the cerebellum had the typical branching pattern (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-C). After the decellularization process, these tissue morphological cues and coloration were lost. The resulting brain decellularized ECM was white/transparent in color, accompanied by a reduction in size/volume mainly due to loss of the cellular components and some of the structural molecules removed by the mechanical and chemical forces during the washes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-F). The brain decellularized ECM samples were immediately frozen at -80\u0026deg;C and lyophilized after the decellularization for the subsequent molecular, microscopy, and cellular analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG-I).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Decellularization efficiency in cerebral extracellular matrix\u003c/h2\u003e \u003cp\u003eThe effectiveness of our decellularization protocol in removing cellular components was confirmed with different assays. DNA quantitation using the PicoGreen double-strand DNA assay after the decellularization process revealed a considerable decrease in DNA content. The native brain tissue exhibited DNA concentrations of 1,029\u0026thinsp;\u0026plusmn;\u0026thinsp;165 ng / mg-dry-tissue in the cerebral cortex, 1,912\u0026thinsp;\u0026plusmn;\u0026thinsp;273 ng / mg-dry-tissue in the cerebellum and 801\u0026thinsp;\u0026plusmn;\u0026thinsp;95 ng / mg-dry-tissue in the remaining areas; while the brain decellularized ECM presented values of 52\u0026thinsp;\u0026plusmn;\u0026thinsp;5 ng / mg-dry-tissue; 71\u0026thinsp;\u0026plusmn;\u0026thinsp;7 ng / mg-dry-tissue and 56\u0026thinsp;\u0026plusmn;\u0026thinsp;4 ng / mg-dry-tissue, respectively for each brain section (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). For all subregions studied, electrophoresis on agarose gels confirmed that DNA chains in native specimens maintained high number of base pairs, while DNA fragments from decellularized specimens did not exceed 200 bp in length (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). In addition, we searched for certain nuclear and cytosolic proteins by Western Blot, such as the cytoskeletal protein tubulin and the neuronal nuclear protein NeuN, which showed an important reduction after the decellularization process that serves as an additional confirmation of the removal of neurons from brain sections (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe efficiency of the decellularization protocol was also evaluated histologically, with hematoxylin and eosin (H\u0026amp;E) staining, as well as by immunofluorescence with the DAPI marker. A total of 10 visual fields with H\u0026amp;E staining were used to calculate the average number of cell nuclei on native and the corresponding decellularized brain subregion. We observed a decrease in cell nuclei in the cortex from 248 to 16; in cerebellum, from 569 to 11; and in remaining areas, from 175 to 16 nuclei (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-F), as seen in the H\u0026amp;E nuclear quantification histogram (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). In addition, quantification of fluorescent DAPI-labeled nuclei in brain tissues was performed. The results revealed a decrease in nuclei in the cortex, from 133 to 14; in the cerebellum, from 309 to 17; and in the remaining areas, from 146 to 19 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH-M), as seen in the DAPI nuclei quantification histogram (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eN). The results obtained from the molecular analysis of DNA and nuclear/cytosolic protein content indicate that the decellularization process was effective at removing the cellular components of the tissue.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.4 \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eBrain decellularized ECM maintains tissue ultrastructure after processing\u003c/span\u003e\u003c/h2\u003e \u003cp\u003eAfter confirming the efficiency of our decellularization protocol to remove cellular components, we then studied the structural morphology of the brain decellularized ECM and its capacity to conserve growth factors. The structural and biochemical integrity of the scaffolds is important to promote cell attachment, proliferation, and differentiation into the neuronal lineage. Electron micrographs of the different brain sub-regions did not show any evident differences between the native tissues and the corresponding decellularized cerebral tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-F). In addition, histological evaluation through Coomasie Blue staining, used for non-specific labeling of total proteins, resulted in a strong and continuous blue color in the native tissue that was comparable to that of the decellularized ECM (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG-L). All histological sections were also stained with hematoxylin to highlight the efficiency of cell removal after the decellularization process.\u003c/p\u003e \u003cp\u003eWe also quantified the loss of protein after decellularization using the bicinchoninic acid biochemical assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eM). The protein levels of the native brain tissue registered at 1,342 \u0026micro;g/mL in the cortex, 1,826 \u0026micro;g/mL in the cerebellum and 1,814 \u0026micro;g/mL in the remaining areas, which decreased considerably after decellularization to 871 \u0026micro;g/mL, 535 \u0026micro;g/mL and 858 \u0026micro;g/mL, respectively. These results translate to a greater retention of proteins in the cortex and the remaining areas, with 65.0% and 47.3% respectively, while the cerebellum only retained 29.3% of the initial protein content (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eM). Finally, we also quantified the presence of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eN-O). All native brain sections presented measurable levels of NGF, including the cortex with 5.1 ng/mg-dry-tissue, the cerebellum with 7.4 ng/mg-dry-tissue and the remaining areas with 5.7 ng/mg-dry-tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eN). However, NGF could not be detected in the decellularized ECM samples. In the case of BDNF, it was also present in all native brain sections and at higher levels than NGF, especially in the remaining areas, with the cortex showing 24 ng/mg-dry-tissue, the cerebellum 81 ng/mg-dry-tissue and the remaining areas 138 ng/mg-dry-tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eO). Brain decellularized ECM did maintain measurable levels of BDNF, with 63 ng/mg-dry-tissue in cortex, 20 ng/mg-dry-tissue in cerebellum and 62 ng/mg-dry-tissue in remaining areas. Interestingly, we observed that BDNF levels seemed to increase after decellularization in the case of the cortex, reaching as much as 2.7-times the original BDNF level.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Soluble brain decellularized ECM can support \u003cem\u003ein vitro\u003c/em\u003e cell viability.\u003c/h2\u003e \u003cp\u003eThe effect of brain decellularized ECM from cortex, cerebellum and remaining areas on PC12 cell viability was assessed through live/dead staining (Calcein AM / Propidium Iodide) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-D). The decellularized ECM did not have any evident effects on PC12 viability under regular conditions, with most cells exhibiting the characteristic green fluorescence of the \u0026ldquo;live\u0026rdquo; stain, and almost no cells showing the red \u0026ldquo;dead\u0026rdquo; stain (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-C). When quantified, viability remained close to 100% in PC12 cell cultures exposed to decellularized ECM from specific brain regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). The mitochondrial activity was also measured with the XTT assay to analyze cellular viability in PC12 cells treated with decellularized ECM from the three different regions in a period of 24 hours. The results did not show considerable differences between cells stimulated with vehicle solution (control), and the experimental treatments with decellularized ECM from cortex, cerebellum and remaining areas, respectively. These results suggest that the brain decellularized ECM used as a soluble factor can maintain cellular viability in PC12 cell cultures \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE\u003cb\u003e).\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.6 \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eSoluble brain decellularized ECM can support unidirectional cell differentiation\u003c/span\u003e\u003c/h2\u003e \u003cp\u003eThe differentiation potential of brain decellularized ECM on PC12 cells was also assessed (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-G). PC12 cells mature and generate neurite-like extensions when they are stimulated, typically through exposure to neurotrophic factors, thus providing a simple unidirectional differentiation model. Indeed, when grown on poly-L-lysine (PLL)-coated coverslips and treated with vehicle (PBS), the cells had a rounded morphology and grew in clusters (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). When PC12 cells were treated with neurotrophic factors known to stimulate neuronal maturation (\u003cem\u003ei.e.\u003c/em\u003e, positive controls NGF and BDNF), the cells generated long neurite-like processes that extended several micrometers and connected with extensions coming from neighboring cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB-C). The cells were also exposed to brain decellularized ECM from cortex, cerebellum and remaining areas, which were all able to promote morphological changes and neurite-like extensions compared to vehicle (\u003cem\u003ei.e.\u003c/em\u003e, negative control) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD-F). However, when quantified by image analysis, neurite extensions promoted by brain decellularized ECM were reduced compared to cells treated with positive controls (\u003cem\u003ei.e.\u003c/em\u003e, NGF, BDNF) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.7 \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eRecovery of PC12 cells in the presence of brain decellularized ECM after oxygen-glucose deprivation (OGD)\u003c/span\u003e\u003c/h2\u003e \u003cp\u003eControl groups in an \u003cem\u003ein vitro\u003c/em\u003e model in homeostasis (Normoxia) were compared to the experimental groups in an \u003cem\u003ein vitro\u003c/em\u003e model for stroke (OGD) exposed to hypoxic conditions. After 6 h of culture, the experimental treatments were provided to the cells and the responses were monitored at 0, 8, 12, 24 and 48 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e; \u003cb\u003eSupplementary Fig.\u0026nbsp;1\u003c/b\u003e).\u003c/p\u003e \u003cp\u003ePC12 cell viability significantly decreased from 99.4% (Normoxia) to 55.1% after oxygen and glucose deprivation for 6 h (OGD \u0026ndash; 0 h), and this value was used as a baseline for the change calculations (\u0026uarr;) of the evaluated treatments. Treatment with PBS (Vehicle) did not promote cell recovery even after 48 h (56.1% viability, \u0026uarr;1%, after 8 h; 58.5% viability, \u0026uarr;3.4%, after 12 h; 53.2% viability, \u0026uarr;-1.9%, after 24 h; 56.8% viability, \u0026uarr;1.7%, after 48 h). Treatment with the positive control NGF registered a significant increase in viability from 12 h after treatment (86.4% viability, \u0026uarr;31.3%), reaching complete recovery at 24 and 48 h (96.5% viability, \u0026uarr;41.4%, after 24 h; 103.5% viability, \u0026uarr;48.4%, after 48 h). In the case of treatment with the other positive control, BDNF, cell recovery was also noticeable after 12 h (78.2% viability, \u0026uarr;23.1%), although final cell recovery after 48 h (88.8% viability, \u0026uarr;33.7%) was more moderate than for NGF.\u003c/p\u003e \u003cp\u003eFinally, the experimental treatment with brain decellularized ECM had a similar impact in the recovery of cell viability as that of positive controls, and specifically that of BDNF, with a slight increase in cell viability after 8 h (72.0% viability, \u0026uarr;16.9%, for cortex; 71.2% viability, \u0026uarr;16.1%, for cerebellum; and 68.9% viability, \u0026uarr;13.8%, for remaining areas) that continued to improve at 12 h with higher recovery rates than BDNF but lower than NGF (85.0% viability, \u0026uarr;29.9%, for cortex; 80.7% viability, \u0026uarr;25.6%, for cerebellum; and 80.3% viability, \u0026uarr;25.2%, for remaining areas). The viability improvements continued until 48 h for the three region-specific decellularized ECM treatments (88.1% viability, \u0026uarr;33.0%, for cortex; 85.5% viability, \u0026uarr;30.4%, for cerebellum; and 84.1% viability, \u0026uarr;29.0%, for remaining areas). Treatment with cortex ECM exhibited slightly, the most controlled and robust cell recovery of the three decellularized treatments, but still significantly lower than the normoxia group, similar to the BDNF positive control treatment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eDecellularized ECM has been studied as a potential regenerative therapy for several decades \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan additionalcitationids=\"CR58\" citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e–\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. However, few studies have characterized the effects of brain decellularized ECM scaffold properties on neural lineage development, stem cell differentiation \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e or its therapeutic potential in neurological diseases such as stroke \u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e,\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. In other words, the application of brain decellularized ECM for the regeneration of CNS tissue still holds several frontiers that need to be explored. In this study, we established an optimized protocol to produce brain decellularized ECM from three specific regions and evaluated their effect on neuronal maturation and recovery capacity in an \u003cem\u003ein vitro\u003c/em\u003e model of brain ischemia.\u003c/p\u003e \u003cp\u003eThe original total number of cells in a tissue that is targeted for decellularization is seldom reported. In addition, even less studies have considered the number of cells in different regions of a porcine brain \u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. This initial information may be useful to determine the efficiency and quality of the decellularization process. In our case, using the isotropic fractionator technique, we were able to quantify the total number of cells per specific brain region (\u003cem\u003ei.e.\u003c/em\u003e, cortex, cerebellum, remaining areas) \u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e. Our data indicated that the cortex represented more than 65% of the mass of the brain, while the cerebellum accounted for just over 10%. Interestingly, the cerebellum contained close to 40% of all brain cells, despite representing the smallest section of the brain. Our results were in good agreement with previously published studies that have used the isotropic fractionator to characterize the brain cellular composition for several species, including the porcine (\u003cem\u003eSus scrofa domesticus\u003c/em\u003e) brain \u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. Neuroanatomical studies have established that the cortex may possess 45% of the cell population, while the cerebellum accounts for up to 40% of the cellular presence with only 1/5 of the mass of the cerebral cortex. This relationship also seems to apply to the human brain and other species in the evolutionary ladder that show a similar pattern between the cortex and the cerebellum \u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e,\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOur isotropic fractionator results correlate well with the values of DNA content measured in each brain section in a per-mg-dry-tissue basis, as well as in the quantification of histological sections after labeling with eosin/hematoxylin. Our decellularization protocol successfully eliminated most of the DNA content present in all three highly-cellularized studied brain regions, although none was under the threshold value of 50 ng/mg-dry-tissue, established as the gold standard for other tissues \u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. Previous studies with porcine brain decellularized ECM have reached the threshold value \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e,\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e, so our protocol could be tailored to be more aggressive or extended for a longer period to reach the before mentioned threshold. However, the risk is to further compromise the tissue’s integrity and bioactivity.\u003c/p\u003e \u003cp\u003eThe cell density of the tissue of interest is an important consideration for decellularization purposes, since removing the cellular components from the decellularized ECM is important to minimize possible cytotoxicity or immunological rejection for the culture/host. Also, native tissue with a higher cell number/mg-tissue ratio may be prone to more considerable loss of tissue ultrastructure over the course of the decellularization process regardless of the aggressiveness of the protocol. We observed such behavior in the cerebellar tissue, the native tissue with the highest cell density compared to the cortex and the remaining areas. The cerebellum decellularized ECM presented the greatest protein and structural loss after decellularization. Thus, relative cell density of the native tissue may serve as an indirect predictor of biological performance, considering that one of the most important aspects of decellularization is the conservation of the tissue's structural and bioactive proteins at the end of the process \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBrain decellularized ECM scaffolds generated with our decellularization protocol maintained a high degree of morphological and ultrastructural integrity, as assessed by SEM and Coomassie Blue staining, despite losing significant amounts of proteins in general and specific neurotrophic factors evaluated by ELISA. NGF and BDNF serve as growth factors that promote the development and regeneration of CNS and support crucial neuronal processes including synaptic activity, neuronal growth, neuronal regeneration and plasticity \u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e. In the present study, we measured NGF and BDNF to determine if they were retained after the decellularization process. As expected, NGF, which is a well-known soluble neurotrophin reported to be involved in the constitutive pathway, the activity-dependent pathway, or in both \u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e, was mostly eliminated from all decellularized scaffolds in comparison to native tissues. In contrast, certain levels of BDNF were retained in all brain decellularized ECM scaffolds, and the decellularized cortex and remaining areas presented even higher levels of BDNF per mg of dry tissue than its native counterpart, which has been reported previously as a relative effect of the loss of cell-associated proteins and other less tightly-bound ECM components \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. For example, in cortical extracts BDNF has been found enriched in a vesicular fraction isolated from lysed synaptosomes \u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e, which could explain its stronger interaction with the ECM and its resilience to the detergents and other chemicals used during decellularization. As we only found higher BDNF retention in the cortex and remaining areas ECM, this may suggest a differential storage of BDNF depending on associated brain regions.\u003c/p\u003e \u003cp\u003eAlso, the brain decellularized ECM scaffolds promoted high cell viability in PC12 cells and supported appropriate attachment of cortical primary cell cultures. In addition, the fact that brain decellularized ECM could elicit a morphological change indicative of PC12 stimulation and neuronal maturation confirms the presence of supportive proteins and neurotrophins, such as BDNF, that are known to modulate the behavior and differentiation in neural cell lineages \u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e. However, there may be other extracellular components that could be triggering the development and maturation of the PC12 cells that are worth exploring. If matched correctly, the biochemical composition and structural properties of brain decellularized ECM scaffolds may be able to induce differentiation into site-appropriate functional cells that can replace lost CNS tissue in cases of brain injury or neurodegenerative pathology \u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe effects of region-specific brain decellularized ECM have not been tested in \u003cem\u003ein vitro\u003c/em\u003e or \u003cem\u003ein vivo\u003c/em\u003e as a recovery treatment in cerebral ischemia or hypoxia models. We established an OGD model with PC12 cells to analyze the cell recovery potential of soluble brain decellularized ECM and other control treatments, after the hypoxic insult (i.e., oxygen-glucose deprivation). Previous studies determined that an OGD model using 6 h of hypoxia falls in the category of cytotoxicity/apoptotic response, with more than 25% loss of PC12 cell viability measured by MTT assay \u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e that could still be recovered \u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e,\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e, making it an ideal ischemia cell recovery model. All three region-specific brain decellularized ECM treatments had a considerable cell recovery effect, with the cerebellum decellularized ECM having the best response at 91.5% cell viability after 48 h, an increase of 36.4% compared to the vehicle group at the same timepoint. This response was similar to the positive control BDNF, which is a neural growth factor that can block caspase-3, a protein that is a major player in apoptosis \u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e, and has been detected in early stages of brain ischemia associated to neuroprotection in neuronal networks \u003csup\u003e\u003cspan additionalcitationids=\"CR75\" citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e–\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e. After our decellularization process, all region-specific brain decellularized ECM still contained measurable levels of BDNF, which could explain the similar responses. However, the levels of other neurotrophins (\u003cem\u003ee.g.\u003c/em\u003e, NT-3, NT-4, CTNF) in the decellularized ECM were not measured and their role in the cell recovery results is still unknown.\u003c/p\u003e \u003cp\u003eTaken together, our results show that region-specific brain decellularized ECM can retain structural and biochemical cues that can promote neuronal maturation under normal \u003cem\u003ein vitro\u003c/em\u003e conditions, and robust cell recovery after oxygen-glucose deprivation using the PC12 cell line. Indeed, \u003cem\u003ein vitro\u003c/em\u003e models using cell lines can be very convenient and biologically useful; however, often they have characteristics that are different from primary cells, and the changes needed to immortalize cell lines can sometimes allow them to withstand harsher treatments that may not be viable for primary cells or \u003cem\u003ein vivo\u003c/em\u003e applications \u003csup\u003e\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/sup\u003e. We have performed preliminary experiments to study the effects of region-specific brain decellularized brain ECM as a substrate on mixed primary cortical cultures, which are much more delicate to culture (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e). All experiments with animal to isolate primary cells were carried out under an approved protocol by the Institutional Animal Care and Use Committee (IACUC) of the School of Medicine of the University of Miami, strictly complying with the ARRIVE guidelines. Our results showed that primary cells were able to adhere, grow and express classic neural biomarkers, such as the neuronal nuclear antigen (NeuN\u003csup\u003e+\u003c/sup\u003e) and glial fibrillary acidic protein (GFAP\u003csup\u003e+\u003c/sup\u003e), over the course of 7 days. Primary cells on the control substrate (PLL) exhibited a homogeneous coverage of the coverslip surface, with the presence of neurons and glial cell well distributed throughout the surface (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eA). Attachment to the decellularized ECM-coated coverslips was also well-distributed; however, NeuN\u003csup\u003e+\u003c/sup\u003e expression seemed to be concentrated in certain clusters of cells throughout the surface (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eB-D), which may better represent the natural organization of the cells in brain.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e "},{"header":"CONCLUSIONS","content":"\u003cp\u003eThe present study reported a decellularization method that was successfully applied to three different brain sections: cortex, cerebellum and remaining areas. Brain tissue can be decellularized while preserving various components of the ECM, including neurotrophic factors such as BDNF. Brain decellularized ECM offered many advantages to enhance neuronal cell culture in 2D (\u003cem\u003ei.e.\u003c/em\u003e, as a soluble treatment) and 3D models (\u003cem\u003ei.e.\u003c/em\u003e, as a scaffold/substrate) due to its complex biomolecular composition and retention of neurotrophic factors and diverse biochemical cues, under normal conditions to promote neuronal maturation or under hypoxic conditions to promote cell recovery. These results suggest that brain decellularized ECM may be an alternative in the future for various clinical applications in diseases associated with lesions of the CNS.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eDISCLOSURES\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003ch2\u003eFUNDING\u003c/h2\u003e\n\u003cp\u003eThe results presented in this study were supported with funds from NIH/NINDS Fogarty International Center (grant number 1R21NS098896-02), which supported training on the OGD model; SENACYT, Panama, (grants number PFID-INF-2020-43, Contract DDCCT-No-101-2021, FID17-078, IDDS22-09, PFID-INF-2020-22, APY-NI-2018-17, and APY-NI-2019B-02) and SNI-SENACYT (grant numbers SNI-12-2020, SNI-051-2023 and Res-84-2022). Funding agencies were not involved in the study design.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eDR and RAG wrote the main manuscript text and did most of the project administration. DR, KRD, MAPP and RAG were responsible for most of the funding acquisition and designed the methodology. DR, DO, SC, AB, BD, NK, JX, AH, RAG were involved in data acquisition and figure preparation. All authors reviewed the manuscript.\u003c/p\u003e\n\u003ch2\u003eAcknowledgement\u003c/h2\u003e\n\u003cp\u003eWe thank the Macelo S. A. abattoir for their support supplying the porcine brains.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eThe data that support our findings are available on request from the corresponding author.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBonnans, C., Chou, J. \u0026amp; Werb, Z. Remodelling the extracellular matrix in development and disease. \u003cem\u003eNat. 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Group II Metabotropic Glutamate Receptors Reduce Apoptosis and Regulate BDNF and GDNF Levels in Hypoxic-Ischemic Injury in Neonatal Rats. \u003cem\u003eInt. J. Mol. Sci.\u003c/em\u003e \u003cb\u003e23\u003c/b\u003e (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGiordano, G. \u0026amp; Costa, L. G. in \u003cem\u003eIn Vitro Neurotoxicology: Methods and Protocols\u003c/em\u003e (eds Lucio G. Costa, Gennaro Giordano, \u0026amp; Marina Guizzetti) 13\u0026ndash;27Humana Press, (2011).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Tissue engineering, decellularized scaffold, brain extracellular matrix, oxygen-glucose deprivation (OGD)","lastPublishedDoi":"10.21203/rs.3.rs-5130290/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5130290/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBrain decellularized extracellular matrix (ECM) can be an attractive scaffold capable of mimicking the native ecosystem of the central nervous system tissue. In this study, we studied the \u003cem\u003ein vitro\u003c/em\u003e response of neural lineage cells exposed to region-specific brain decellularized ECM scaffolds from three distinct neuroanatomical sections: cortex, cerebellum and remaining areas. First, the evaluation of each brain subregion was performed with the isotropic fractionator method to understand the cellular composition of the different cerebral areas. Second, each of the cerebral subregions was subjected to the decellularization process and their respective characterization using molecular, histological, and ultrastructural techniques. Third, the presence of neurotrophic factors in the decellularized brain scaffold was analyzed. Finally, we studied the region-specific brain decellularized ECM as a mimetic platform for the maturation of PC12 cells and for the recovery of cell viability in an oxygen-glucose deprivation model. Our results show that region-specific brain decellularized ECM can serve as a biomimetic scaffold capable of promoting the growth of neural lineage cells and, in addition, it possesses a combination of structural and biochemical signals (\u003cem\u003ee.g.\u003c/em\u003e, neurotrophic factors) that are capable of inducing cell phenotypic changes that can promote cell recovery and viability in a stroke/ischemia model \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Region‐specific brain decellularized scaffolds can recover cell viability in an oxygen-glucose deprivation model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-21 12:57:51","doi":"10.21203/rs.3.rs-5130290/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-12-04T05:53:28+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-12-01T00:01:32+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"238952620132021888681230814745067491463","date":"2024-11-26T13:07:34+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-03T11:37:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"234347690398333820357552429956571256409","date":"2024-10-21T15:56:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"107776507105101944334545154922239890243","date":"2024-10-19T09:03:46+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-10-17T15:35:14+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-17T15:06:22+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-10-17T15:00:08+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-10-17T14:57:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-09-21T22:48:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c2411e8e-1ce0-47b6-8403-5c9292f6b779","owner":[],"postedDate":"October 21st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":39152714,"name":"Biological sciences/Neuroscience/Regeneration and repair in the nervous system"},{"id":39152715,"name":"Physical sciences/Materials science/Biomaterials/Bioinspired materials"}],"tags":[],"updatedAt":"2025-04-14T16:06:06+00:00","versionOfRecord":{"articleIdentity":"rs-5130290","link":"https://doi.org/10.1038/s41598-025-95656-w","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-04-07 16:04:50","publishedOnDateReadable":"April 7th, 2025"},"versionCreatedAt":"2024-10-21 12:57:51","video":"","vorDoi":"10.1038/s41598-025-95656-w","vorDoiUrl":"https://doi.org/10.1038/s41598-025-95656-w","workflowStages":[]},"version":"v1","identity":"rs-5130290","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5130290","identity":"rs-5130290","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}