Decellularized and sterilized HFL patches (N = 5) of 1 cm² were placed in sterile CellCrown™ inserts (12-well plate inserts, Sigma-Aldrich, Z742383-6EA, Saint Louis, MO, USA) and incubated in classical PM overnight in order to rehydrate and preheat them. The day after, or day zero (D0), 5 × 105 PMSCs were seeded on each patch, and the PM was added after 2 h of dry incubation at 37 °C and 5% CO2 to enhance cell adhesion. On day one (D1), the inserts with seeded patches were moved into a new blank 12-well plate to avoid the impact of cells attached to the well walls in future analyses. The PM was replaced every two days just after examining the cell growth (D1, D3, D5, and D7). The cell viability was assessed at the end of the 7th day. The final analyses after seven days also included H&E, MT staining, and mIF-IHC for Col-1/Hoechst, as described above. The whole procedure was performed in duplicate in order to perform a SEM analysis on the whole patches and to assess the cellular spread. All the experimentations included a positive control (cells seeded in wells without a scaffold) and a negative control (scaffold in wells without seeding cells).
Cellcrown inserts
Manufactured by Merck Group
Sourced in United States, Germany, Spain
CellCrown™ inserts are a type of laboratory equipment used for cell culture applications. They provide a platform for growing and maintaining cells in a controlled environment. The core function of CellCrown™ inserts is to support cell attachment and proliferation, enabling researchers to study cellular behavior and responses.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
N n dimethylformamide (dmf), by Thermo Fisher Scientific (1 mentions)
Dichloromethane, by Thermo Fisher Scientific (1 mentions)
Thymol, by Merck Group (1 mentions)
Erythromycin, by Merck Group (1 mentions)
Calcofluor white stain, by Merck Group (1 mentions)
S limonene, by Merck Group (1 mentions)
Naproxen sodium salt, by Merck Group (1 mentions)
12 well plate, by Thermo Fisher Scientific (1 mentions)
Ascorbic acid, by Thermo Fisher Scientific (1 mentions)
Nsc34, by Cedarlane (1 mentions)
N n dimethylformamide (dmf), by Merck Group (1 mentions)
Phosphoric acid, by Merck Group (1 mentions)
Tween 80, by Merck Group (1 mentions)
Ammonium hydroxide solution, by Merck Group (1 mentions)
7 protocols using cellcrown inserts
1
PMSC Seeding on Decellularized HFL Patches
2
CorMatrix ECM for Cardiac Cell Culture
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Pieces of CorMatrix® ECM® (CorMatrix Cardiovascular, Sunnyvale, CA) — a decellularized xenograft material clinically approved for use in cardiac surgery — of ≈1.5-cm diameter were cut and placed in wells of a 24-well plate. To fix the bioscaffold pieces to the bottom of the wells, CellCrown inserts were used (Sigma-Aldrich). Prior to the seeding, the bioscaffolds were incubated for 2 days with EGM-2 media. CPs (50 000) at P5 were seeded into each CorMatrix-containing well and maintained for 7 days on the bioscaffolds. The bioscaffolds were fixed in 4% Paraformaldehyde for 20 hours at 4°C and imbedded in paraffin or OCT-frozen. Eight-micrometer sections were examined for the presence of CPs using anti-vimentin, NG2, and PDGFR-β antibodies (see above), which were incubated for 20 hours at 4°C after permeabilization and blocking. Secondary antibody was incubated on the sections for 1 hour at room temperature. The nuclei were counterstained with DAPI (Sigma-Aldrich). The slides were mounted using Fluoromount-G® mounting media (Sigma-Aldrich) and immunofluorescence pictures were taken after 24 hours both at ×20 and ×40.
3
Antimicrobial Evaluation of Modified Polymers
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PCL (Mn = 80,000 Da), (S)-(-)-limonene (food grade, ≥95%), naproxen sodium salt (98-102%), phosphate buffered saline (PBS), thymol (THY, >98.5%), Erythromycin, Bovine Serum Albumin (BSA), Calcofluor White stain and CellCrown™ inserts (24-well plate inserts) were purchased from Sigma-Aldrich (Germany). Dichloromethane (DCM, >99%), N,N-dimethylformamide (DMF, >99%), Phalloidin 546 and DAPI were obtained from Fisher Scientific (USA). Tryptone soy broth (TSB) and tryptone soy agar (TSA) were purchased from Laboratorios Conda-Pronadisa S.A. (Spain). S. aureus ATCC 25923 strain was obtained from Ielab (Spain), while GFP-expressing antibiotic sensitive S. aureus (MSSA) and the two clinical strains Newman-(MSSA) and USA300-(MRSA) were kindly donated by Dr. Cristina Prat, Institut d'Investigació en Ciències de la Salut Germans Trias i Pujol (IGTP, Spain). Paraformaldehyde (PFA) 4% in PBS was acquired from Alfa Aesar (Germany).
Saponin from Quillaja Bark pure and SDS for molecular biology were purchased from AppliChem (Germany). Fetal Bovine Serum (FBS) was obtained from Gibco (UK) while penicillin-streptomycin-amphotericin B (PSA) was purchased from Biowest (France) and Dimethyl sulfoxide (DMSO) from Merck Millipore (Germany). MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was obtained from Invitrogen (USA).
Saponin from Quillaja Bark pure and SDS for molecular biology were purchased from AppliChem (Germany). Fetal Bovine Serum (FBS) was obtained from Gibco (UK) while penicillin-streptomycin-amphotericin B (PSA) was purchased from Biowest (France) and Dimethyl sulfoxide (DMSO) from Merck Millipore (Germany). MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was obtained from Invitrogen (USA).
4
MSC Seeding on 3D Scaffolds
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Eighteen CellCrown inserts (Sigma Aldrich) were prepared for each primary MSC donor for each scaffold type (PDOa, PDOr, PLGAa, and PLGAr) and placed in six‐well plates. Scaffolds were conditioned in 2 mL basal medium for 1 hour prior to seeding. About 1 mL MSCs at 2 × 105 cells/mL in basal media were then incubated on each scaffold for 1 hour before addition of 1 mL of basal growth media. Seeded scaffolds were incubated for a further 12 hours to allow cell attachment.
5
AF Cells Cultured on PCL/PLLA Scaffolds
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Due to a lack of healthy, primary human AF cells being available, animal-derived (Bovine) AF cells were obtained from the cell bank at the Division of Cell Matrix Biology and Regenerative Medicine, The University of Manchester, UK). AF cells were cultured to passage 3 at 37 °C and 5% CO2 in 75 cm2 sterile flasks with Dulbecco’s Modified Eagle’s Medium (DMEM) containing 4.5 g/L glucose, 5% sodium pyruvate 10% FBS, 1% antibiotic, and 50 g/mL ascorbic acid (Gibco). PCL/PLLA (50:50) blend scaffolds were individually held within 12-well CellCrown inserts (Sigma-Aldrich) and placed into 12-well plates (ThermoFisher). Samples were disinfected in 70% v/v ethanol in distilled water and pre-wetted for 12 h in culture media. This media was removed and 200 μL of AF cell suspension (1 × 105 cells/sample) was evenly distributed over the surface of each scaffold. Samples were left undisturbed in the incubator (Jencons-PLS, Bedfordshire, United Kingdom) for 30 min to allow initial cell attachment before 2 mL medium was added to each well. Cell-seeded scaffolds were cultured for one week with media changes every second day.
6
In Vitro Cell Adhesion on SilkBridge
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In vitro cell tests were performed using RT4-D6P2T, a schwannoma cell line (ATCC catalog number CRL-2768) and Mouse Motor Neuron NSC-34 cell Line (Cedarlane catalog number CLU140).
SilkBridge conduits were cut longitudinally and immersed in phosphate buffer saline (PBS). To allow cell adhesion, the longitudinally open conduits were pressed for a few minutes and then fixed to the culture plate-well through an insert (Sigma Aldrich, CellCrown inserts, catalogue number Z74230). For control conditions, cell lines were plated on not coated glass coverslips.
SilkBridge conduits were cut longitudinally and immersed in phosphate buffer saline (PBS). To allow cell adhesion, the longitudinally open conduits were pressed for a few minutes and then fixed to the culture plate-well through an insert (Sigma Aldrich, CellCrown inserts, catalogue number Z74230). For control conditions, cell lines were plated on not coated glass coverslips.
7
Osteoblast Culture and Characterization
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PCL and PVAc with an average molecular weight of 80000 Da and 140000 Da respectively, dicloromethane (DCM), N,N-dimethylformamide (DMF), calcium carbonate (CaCO 3 ), phosphoric acid (H 3 PO 4 ), ammonium hydroxide solution (NH4OH), TWEEN® 80 and CellCrown™ inserts (24 well plate inserts) were purchased from Sigma-Aldrich (Spain).
Clonetics™ OGM™ osteoblast growth medium (OGM), trypsin-EDTA and Clonetics™ normal human osteoblasts (NHOst) were purchased from Lonza (Belgium). Dulbecco's phosphatebuffered saline (DPBS) were purchased from Biowest (France).
Clonetics™ OGM™ osteoblast growth medium (OGM), trypsin-EDTA and Clonetics™ normal human osteoblasts (NHOst) were purchased from Lonza (Belgium). Dulbecco's phosphatebuffered saline (DPBS) were purchased from Biowest (France).
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