Primary cells from the three tissues were isolated at the Faculty of Pharmacy, University of Ljubljana. The isolation protocols followed previously published studies for isolating primary cells from bone tissue [5 (link),26 (link)], synovium [17 (link),39 (link)], and periosteum [18 (link),22 (link)]. Briefly, the tissues were cut into small pieces, washed thoroughly in phosphate-buffered saline, weighed, and incubated at 37 °C in 1 mg/ mL collagenase solution (Roche, Basel, Switzerland) for 3 h (bone tissue) or 12 h (synovium and periosteum). The resulting suspensions of tissue and cells was passed through a 70 µm cell strainer (Corning Inc., Corning, NY, USA). Aliquots of freshly isolated cells were seeded using StemMACS MSC expansion media kit XF, human (Miltenyi Biotec, Bergisch Gladbach, North Rhine-Westphalia, Germany) supplemented with 1% glutamine, 2% penicillin, and streptomycin (all Biowest). The cells were incubated at 37 °C under 5% humidified CO2. The study design and the analyses are summarized in Figure 10 .
Collagenase solution
Manufactured by Roche
Sourced in Switzerland, United States
Collagenase solution is a laboratory reagent used to break down the collagen matrix in tissues. It is a mixture of enzymes that help to disrupt the extracellular matrix, allowing for the isolation and separation of cells from solid tissues. The solution is commonly used in cell culture applications, tissue engineering, and various research procedures that require the isolation of specific cell types.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Biowest (1 mentions)
Streptomycin, by Biowest (1 mentions)
70 m cell strainer, by Corning (1 mentions)
Glutamine, by Biowest (1 mentions)
Hank s balanced salt solution, by Thermo Fisher Scientific (1 mentions)
100 μm cell strainer, by BD (1 mentions)
Trypan blue, by Harvard Bioscience (1 mentions)
Dulbecco modified eagle medium (dmem), by Euroclone (1 mentions)
Phosphate buffered saline (pbs), by GE Healthcare (1 mentions)
Culture flasks, by Greiner (1 mentions)
Staining buffer, by BioLegend (1 mentions)
Live dead fixable near ir dead cell stain kit, by Thermo Fisher Scientific (1 mentions)
Cytoflex lx system, by Beckman Coulter (1 mentions)
Cytexpert 2, by Beckman Coulter (1 mentions)
Diva 8, by BD (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
M199 media, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin solution, by Thermo Fisher Scientific (1 mentions)
Histopaque 1077, by Merck Group (1 mentions)
Egta solution, by Merck Group (1 mentions)
9 protocols using collagenase solution
1
Isolation of Primary Cells from Bone, Synovium, and Periosteum
2
Isolation and Culture of Pancreatic Islets
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Mice and rats were anesthetized with CO2 and then killed by decapitation. Their abdomens were opened to expose the pancreas. Subsequently, in situ ductal perfusion was performed. Approximately, 5 and 15 ml collagenase solution (1.2 mg/ml; Roche, Basel, Switzerland) were injected into mouse and rat pancreas, respectively, through the common bile duct. The inflated pancreas was digested by shaking in collagenase solution for 20 min at 37 °C. Then, the digested pancreas was disintegrated by pipetting through a 5-ml pipette tip and rinsed with Hanks balanced salt solution (Invitrogen, Carlsbad, CA, USA) [28 (link), 29 (link)]. The harvested islets were hand-picked and some of them were dispersed into single islet cells. Thereafter, both islets and dispersed islet cells were subjected to cultivation.
3
Isolation and Expansion of Adipose-Derived Cells
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Adipose samples (n = 4) were harvested according to the Coleman technique [13 (link)] during scheduled abdominal liposuctions in patients undergoing breast reconstruction. The study was approved by the local Ethical Committee on Human Study (PROT.N. 3676/CE), and participants provided written informed consent to take part in the study. An average of 600 mg of adipose samples was washed in phosphate-buffered saline (PBS; PAA Laboratories GmbH, Pasching, Austria), minced, and digested for 60 min in Dulbecco’s modified Eagle’s medium (DMEM; Euroclone Spa, Milan, Italy) containing 1.76 UI/mL collagenase solution (Roche Diagnostics GmbH, Mannheim, Germany) and 1% penicillin–streptomycin (P/S; 104 UI/mL and 10 mg/mL; PAA) at 37 °C with gentle agitation [14 (link)]. After enzyme inactivation by the addition of blocking media, the resulting cell suspension was centrifuged, filtered through a 100 μm cell strainer (BD Falcon, Durham, NC, USA), counted using 0.4% trypan blue (Biochrom AG, Berlin, Germany), and seeded in culture flasks (Greiner Bio-One GmbH, Frickenhausen, Germany) at a density of 100,000 cells/cm2 in Quantum 333 culture medium (PAA) as reported [11 (link)]. Once an 80–90% confluence was reached, cells were detached with 0.05% trypsin/0.02% EDTA (Euroclone), counted, and seeded at a density of 6000 cells/cm2.
4
Evaluating AM Collagenase Resistance
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Collagenase assays were performed to confirm whether the hardened AMs were resistant to degradation. GA- and DAS-crosslinked AMs and normal AMs in the form of 1 × 1-cm pieces were placed in a 0.1% collagenase solution (Roche, Mannheim, Germany) and observed at room temperature. The grid paper was attached to a dish, and the size of the remaining AMs retained after degradation was calculated.
5
Endothelial Cell and PTDC Staining
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For endothelial cell and PTDC staining, the residual scaffolds in PTs were first discarded, and then, the freshly obtained PTs were crushed in staining buffer (Biolegend) with a mortar and pestle. Single-cell suspensions were obtained with an additional digestion in collagenase solution (Roche Diagnostics) at 37 °C for 30 min.
Cell suspensions were then filtered with a 40 μm cell strainer and spun at 300 × g for 5 min at 4 °C. The supernatant was removed, and the deposit was resuspended in staining buffer. Then, the resuspended single cells were stained with an antibody cocktail for 60 min at 4 °C. After incubation, the cells were washed and resuspended in adequate staining buffer again and used for flow cytometric analysis.
Flow cytometric analyses were carried out using a CytoFlex LX system equipped with CytExpert 2.3 software (Beckman Coulter) or a Symphony A5 system equipped with Diva 8.0 software (BD Biosciences). Dead cells were excluded using a Live/Dead Fixable Near-IR Dead Cell Stain Kit (Thermo Fisher Scientific) or DAPI solution (1 μg·mL−1). We used fluorescence minus one control to define the boundaries between the positively and negatively stained cell populations. Flow cytometric data were analyzed with FlowJo V10 (Three Star) or CytExpert 2.3.
Cell suspensions were then filtered with a 40 μm cell strainer and spun at 300 × g for 5 min at 4 °C. The supernatant was removed, and the deposit was resuspended in staining buffer. Then, the resuspended single cells were stained with an antibody cocktail for 60 min at 4 °C. After incubation, the cells were washed and resuspended in adequate staining buffer again and used for flow cytometric analysis.
Flow cytometric analyses were carried out using a CytoFlex LX system equipped with CytExpert 2.3 software (Beckman Coulter) or a Symphony A5 system equipped with Diva 8.0 software (BD Biosciences). Dead cells were excluded using a Live/Dead Fixable Near-IR Dead Cell Stain Kit (Thermo Fisher Scientific) or DAPI solution (1 μg·mL−1). We used fluorescence minus one control to define the boundaries between the positively and negatively stained cell populations. Flow cytometric data were analyzed with FlowJo V10 (Three Star) or CytExpert 2.3.
6
Isolation and Characterization of Pancreatic Islets
7
Isolation of Liver Cell Populations
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Liver cells were isolated from 2-month-old mTmG mouse liver using the standard two-step collagenase perfusion method (Berry and Friend, 1969 (link); Li et al., 2010 (link)). Briefly, mice were anesthetized with isoflurane, and the liver was perfused with 30 ml prewarmed EGTA solution (0.5 mM) through the inferior vena cava for 10 min, and perfused with 30 ml prewarmed pronase solution (14 mg/mouse; Sigma-Aldrich, St Louis, MO, USA), followed by 30 ml collagenase solution (3.7 U/mouse; Roche, Basel, Switzerland) for 10 min each. The liver was then removed and transferred to a sterile Petri dish and gently minced with forceps. The liver was further digested with the pronase/collagenase solution with 1% DNase I (Sigma-Aldrich) for 25 min at 40°C. Cells were filtered by a 70-μm filter and used directly as unfractionated WLCs, or centrifugated at 50 g for 5 min to pellet HCs. The supernatant was then centrifuged at 580 g for 5 min to pellet NPCs. WLCs, HCs and NPCs were washed twice in cold Advanced DMEM/F12 (Thermo Fisher Scientific, Waltham, MA, USA) and subjected to spheroid and 2D culture.
8
Isolation of Human Cardiac Progenitor Cells
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The Institutional Review Board of Loma Linda University approved the protocol for the use of tissue that was discarded during cardiovascular surgery, without identifiable patient information, for this study with a waiver of informed consent. Human neonatal and adult Islet-1+ cardiac progenitor cell clones were previously isolated from discarded surgical cardiovascular tissue [53 (link)] and were available for use in this study. Briefly, discarded atrial tissue from human neonates and adults was digested in a collagenase solution (Roche Applied Science, Indianapolis, IN, USA), and clonal cell populations were established by limiting dilution. Human CPC clones used in the current project were cultured in growth media that included 10% fetal bovine serum (FBS) or extracellular-vesicle-depleted 10% FBS (Genesee Scientific, San Diego, CA, USA), medium 199 (Thermo Fisher Scientific, Waltham, MA, USA), 100 units/mL of penicillin–streptomycin (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), 22% endothelial cell growth medium-2 BulletKit (Lonza, Basel, Switzerland), and 1% minimum essential medium non-essential amino acids solution (Life Technologies by Thermo Fisher Scientific, Waltham, MA, USA).
9
Isolation of Chondrocytes from Human Cartilage
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Human articular cartilage as part of surgical waste obtained from three donors without osteoarthritis was provided by the University of Wisconsin Hospital and Clinics with approval from the Institutional Review Board. To isolate chondrocytes, cartilage was individually minced, washed twice with D-PBS, and incubated with pronase solution (1 mg/ml; Roche, Basel, Switzerland) for 30 min at 37°C and additionally with collagenase solution (1 mg/ml; Roche) for 12 to 18 hours at 37°C with agitation. The digestion solution was collected and centrifuged at 600g for 5 min to prepare uncultured NCs for RNA-seq analysis.
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