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

doi:10.21203/rs.3.rs-5130290/v1

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

https://doi.org/10.21203/rs.3.rs-5130290/v1

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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)

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.

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 cm², placed in 50mL conical tubes up to the 15mL mark for each tube, and stored at -80 ^oC 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 neurons⁴⁹. The cells were grown in 25 mm² 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% CO₂. 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 maturation⁴³. 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% O₂ in a hypoxia chamber with a ProOx110 oxygen-sensor controller (Biospherix, Parish, NY, USA), evacuated with N₂ inside a regular water-jacketed 5% CO₂ 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.

3.1 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. 2A). In terms of cell number per brain region, we observed that the cortex had an average of 2.18 x 10⁹ cells (37.59 ± 0.53%); the cerebellum, 1.82 x 10⁹ cells (39.59 ± 0.70%); and the remaining areas, 0.89 x 10⁹ cells (22.84 ± 0.60%) (Fig. 2B).

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. 3A-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. 3D-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. 3G-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. 4A). 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. 4B). 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. 4C).

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. 5A-F), as seen in the H&E nuclear quantification histogram (Fig. 5G). 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. 5H-M), as seen in the DAPI nuclei quantification histogram (Fig. 5N). 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. 6A-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. 6G-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. 6M). 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. 6M). Finally, we also quantified the presence of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) (Fig. 6N-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. 6N). 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. 6O). 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. 7A-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. 7A-C). When quantified, viability remained close to 100% in PC12 cell cultures exposed to decellularized ECM from specific brain regions (Fig. 7D). 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. 7E).

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. 8A-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. 8A). 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. 8B-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. 8D-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. 8G).

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.

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. 10A). 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. 10B-D), which may better represent the natural organization of the cells in brain.

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.

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.

Data Availability

The data that support our findings are available on request from the corresponding author.

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

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Abstract

Figures

1. INTRODUCTION

2. MATERIALS & METHODS

2.1 Neuroanatomical dissection of porcine brain tissue

2.2 Total cell quantification of the dissected brain regions

2.3 Processing and characterization of region-specific brain decellularized ECM

2.3.1 Brain Tissue Decellularization Protocol

2.3.2 Characterization of the Decellularization Efficiency: DNA Content

2.3.3 Characterization of the Decellularization Efficiency: Western Blot

2.3.4 Structural Characterization: Histological Analysis

2.3.5 Morphological Characterization: Scanning Electron Microscopy

2.3.6 Neurotrophin Levels in Decellularized Cerebral Tissue

2.4 In vitro cell response to region-specific brain decellularized ECM

2.4.1 PC12 Cell Culture

2.4.2 Cytotoxicity of Region-Specific Brain Decellularized ECM

2.4.3 Differentiation of PC12 Cells after Treatment with Region-Specific Brain Decellularized ECM

2.4.4 Cell Viability and Recovery after the Oxygen-Glucose Deprivation (OGD) Model

2.5 Statistical Analysis

3. RESULTS

3.1 Quantitative assessment of mass, cellular composition of porcine brain subregions

3.2 Macroscopic evaluation of the cerebral decellularization process

3.3 Decellularization efficiency in cerebral extracellular matrix

3.4 Brain decellularized ECM maintains tissue ultrastructure after processing

3.5 Soluble brain decellularized ECM can support in vitro cell viability.

3.6 Soluble brain decellularized ECM can support unidirectional cell differentiation

3.7 Recovery of PC12 cells in the presence of brain decellularized ECM after oxygen-glucose deprivation (OGD)

4. DISCUSSION

CONCLUSIONS

Declarations

DISCLOSURES

FUNDING

Author Contribution

Acknowledgement

Data Availability

References

Additional Declarations

Supplementary Files

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