3D brain-like tissues were placed on a flat weigh boat and subjected to contusion injury using a pneumatic cylinder with a 3 mm flat tip impactor at a velocity of 6 m/s, penetration depth 0.6 mm, and a dwell time duration of 200 ms (6). Sham tissues were handled similarly to the injured tissues but without receiving the contusion injury. Following injury or sham protocol, the tissues were incubated at 37 °C in 5% CO2 in a humidified atmosphere until the indicated time marks for imaging and/or media collection for miRNA analysis.
Growth protocol
3D human tricultures were fabricated following an earlier published protocol (6, 7). Human induced neural stem cell (hiNSCs) , human primary astrocytes (Sciencell research laboratories, cat. no. 1800), and HMC3 (human microglial) cells were allowed to reach nearly 100% confluency at the day of triculture seeding. All cell types were collected and concentrated as described in the individual culture methods. Silk scaffolds freshly coated with poly-ornithine (PLO) (Sigma, cat.no. A004C) - and laminin were placed in 96-well plates, and a vacuum manifold was used to remove excess liquid (45). Each scaffold was seeded with a 40L cell suspension containing 2:0.5:0.1 million neurons, astrocytes, and microglia, followed by 30 minutes of incubation at 37°C to allow cell adhesion to the scaffolds. To ensure efficient cell attachment, the freshly seeded scaffolds were submerged in 150L of Neuronal media (composed of NeuroBasal medium supplemented with 2% B-27, 1% Anti-Anti, 1% Glutamax, all from Invitrogen, and 1% astrocyte growth factors, Sciencell research laboratories) and incubated overnight in a tissue culture incubator (37°C, 5% CO2 in a humidified atmosphere). The following day, the cell-seeded scaffolds were transferred to new 96-well plates to remove the unattached cells, embedded with 100L of collagen type I solution (Corning or R&D systems, 3 mg/mL with a pH adjusted to 7.0-7.2 with NaOH), and incubated for 30 minutes at 37°C to allow the collagen gel to crosslink. Neuronal media (150L) was added to all scaffolds and incubated for 24h at 37°C. The following day, 3D tricultures were moved into 48-well plates with 1 ml of Neuronal media in each well. Half the media volume was changed every fourth day until the tissues were used for experiments (6-7 weeks).
Extracted molecule
total RNA
Extraction protocol
miRNA isolation and purification were performed on 1 mL of triculture media collected from sham and impact samples at 6, 24, 72 hours post-injury. Samples were immediately stored in -80 °C. Once all samples were collected, samples were thawed and miRNA purification was performed using the mirVANA miRNA isolation kit (AM1560, Ambion, Life Technologies). Briefly, miRNA homogenate additive and Acid-Phenol:Chloroform was added to the media and centrifuged at 10,000 x g for 5 minutes. The upper aqueous phase containing the RNA was then collected and transferred to a new tube. Total RNA purification followed by miRNA purification was carried out using the filter cartridges from the mirVANA miRNA isolation kit per the manufacturer’s instructions. miRNA was collected by eluting the filter cartridge with 100 μl of nuclease-free water. miRNA samples were further concentrated for Nanostring analysis using the Oligo Clean and Concentrator Kit (D4060, Zymo Research) per the manufacturer’s instructions. The eluate, containing the miRNA was collected and the concentration was measured using the Quibit miRNA assay kit (Q32880, Molecular probes, Life Technology) per manufacturer’s instructions.
Label
not provided
Label protocol
N/A
Hybridization protocol
N/A
Scan protocol
Evaluation of the expression of 798 unique miRNAs was evaluated with the nCounter MAX/FLEX Analysis System using the Nanostring nCounter® Human v3 miRNA Expression Panels (NanoString Technologies, Seattle, WA, USA). Samples were prepared and ran according to the manufacturer’s instructions to determine the mean copy of each miRNA probe of interest. Housekeeping genes for normalization included: ribosomal protein L10, beta-actin, beta-2-microglobulin, glyceraldehyde 3-phosphate dehydrogenase, and ribosomal protein L19, as well as endogenous miRNAs and positive and negative controls. Raw data was analyzed by nSolver Software version 4.0 (NanoString Technologies).
Data processing
All samples passed quality control checks and normalization was carried out using the geometric mean of the ligation factors. miRNA expression differences in sham and impact injury at the different time points were measured using t-tests, with the Benjamini-Hochberg’s False Discovery Rate (FDR) method for multiple comparison corrections. Expression data were log2 transformed for presentation.
