Three-dimensional gel cultures were carried out according to the protocol published previously37 (link). Briefly, collagen gels were prepared by mixing 0.5 mL of fibroblast cell suspension (1 × 106 cells) in FBS, 2.3 mL type I collagen (Cell matrix type IA; Nitta Gelatin, Tokyo, Japan), 670 μL of 5 × DMEM, and 330 μL reconstitution buffer. The mixture (3 mL) was cast into each well of a 6-well plate. The solution was allowed to polymerize at 37 °C for 30 min. After overnight incubation, each gel was detached and cultured in serum-free DMEM. After 72 h, the surface area of the gels was quantified.
Cellmatrix type 1 a
Manufactured by Nitta Gelatin
Sourced in Japan
Cellmatrix Type I-A is a laboratory-grade collagen-based matrix derived from bovine sources. It is designed to provide a natural, three-dimensional scaffold for cell culture applications. The product is composed of purified type I collagen fibrils that mimic the extracellular matrix environment, supporting the growth and differentiation of various cell types.
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Lab products found in correlation
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (6 mentions)
Reconstitution buffer, by Nitta Gelatin (5 mentions)
Cell culture insert, by BD (5 mentions)
Matrigel, by Corning (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Collagenase, by Merck Group (2 mentions)
Osteo assay surface plate, by Corning (2 mentions)
Fv1000, by Olympus (2 mentions)
Ascorbic acid, by Merck Group (2 mentions)
Lipofectamine rnaimax transfection reagent, by Thermo Fisher Scientific (2 mentions)
Na alginate, by Fujifilm (1 mentions)
Cellsens, by Olympus (1 mentions)
Icr mice, by Samtako (1 mentions)
Prostaglandin e2, by Merck Group (1 mentions)
Centro lb 960, by Berthold Technologies (1 mentions)
Passive lysis buffer (plb), by Promega (1 mentions)
Tumor necrosis factor α tnf α, by Thermo Fisher Scientific (1 mentions)
Hepg2, by Japanese Collection of Research Bioresources Cell Bank (1 mentions)
Hek293t, by RIKEN BioResource Center (1 mentions)
Collagenase type 2, by Worthington (1 mentions)
73 protocols using cellmatrix type 1 a
1
3D Collagen Gel Culture Protocol
2
Fabrication of Core-Shell 3T3 Fibers
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The cell fibers were formed by using the double-coaxial laminar-flow microfluidic device assembled from pulled glass capillary tubes, rectangular glass tubes and connectors as previously reported4 (link). To form the core-shell 3T3 fibers, three types of solutions were prepared: (1) 3T3 cells-containing pre-gel solution of a mixture of two types of collagen, AteloCell IAC-50 (Koken, Japan) and Cellmatrix Type I-A (Nitta Gelatin, Japan) as ECM for the core; (2) pre-gel solution of 1.5% Na-alginate (Fujifilm Wako Pure Chemical, Japan) for the shell; (3) 100 mM CaCl2, 3% glucose solution for the sheath stream. Each 3T3 fiber was formed to contain 4 × 107 cells per fiber on the calculation with the certain length of both ends lacking cells (only alginate shell) to completely seal the cells. For the control fibers, only ECM without the cells was used to make the core (ECM-only fiber). The width of the shell and core parts of 3T3 fibers in randomly selected 18 fields from four different cell fiber formation trials were measured by using the imaging software cellSens (Olympus Life Science).
3
Isolation and Characterization of Murine Osteoclasts
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Primary calvarial osteoblasts were isolated from calvariae of newborn ICR mice (Samtako Bio Inc., Seoul, Korea) by using a sequential enzymatic digestion method described previously.21 (link) To obtain mature osteoclasts, bone marrow cells (1.5 × 107 cells) and calvarial osteoblasts (1.5 × 106 cells) were cocultured with 1α,25-dihydroxyvitamin D3 (10 nM, Sigma-Aldrich) and prostaglandin E2 (100 nM; Sigma-Aldrich) for 6 days in a 10-cm culture dish coated with collagen gel (Cellmatrix type I-A; Nitta Gelatin Inc., Osaka, Japan). Mature osteoclasts were detached with 0.2% collagenase (Sigma-Aldrich), placed on an Osteo Assay Surface plate (Corning Inc., Corning, NY, USA), and allowed to settle for 2 hours, and then cultured with vehicle or WEMC for another 16 hours. Cells were stained for TRAP to identify osteoclasts. After removing cells with sodium hypochlorite, resorption pits were photographed and analyzed by using ImageJ software (National Institutes of Health, ML, USA).
4
Murine Osteoblast Cell Culture Model
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Murine osteoblast cell line MC3T3-E1 clone-4 cells and primary osteoblasts were cultured in αMEM for 10 days. Cells were harvested, washed with phosphate-buffered saline (PBS), and plated at 1.0 × 105 cells/cm2 in αMEM for another 10 days with fresh medium replacement every 2 days (2D culture). Alternatively, cells were maintained in a 3D culture system in 12-well plates. The system comprised three layers: the bottom collagen gel layer, the middle collagen gel layer, and the top culture medium layer (Fig. 1A ). Collagen gels were made of 0.12% Cellmatrix Type I-A (Nitta Gelatin, Osaka, Japan) dissolved in αMEM. The bottom gel layer (0.5 mL) was cell-free. The middle gel layer (1.0 mL) contained cells (1.0 × 105 cells/mL). The top layer was αMEM (1.0 mL). Cells were cultured in 3D conditions for 10 days with fresh medium replacement every 2 days (3D culture). To recover cells from 3D cultures, gels were minced and digested with collagenase type I. Recovered cells were neutralised, washed with PBS, and plated at 1.0 × 105 cells/cm2 in αMEM for another 10 days with fresh medium replacement every 2 days (Re-2D culture). Cells were maintained in 5% CO2 at 37 °C.
5
Transduction and Luciferase Assay Protocol
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The cell lines were seeded at a density of 1 × 104 cells in each well of 96-well plates coated with collagen type I (Cellmatrix Type I-A; Nitta Gelatin, Osaka, Japan) on the day before transduction. The primary hepatocytes were seeded at a density of 5 × 104 cells in each well of 96-well plates coated with collagen type I. A vial of vector stock was thawed and diluted with DMEM containing 5% FBS immediately before the transduction experiment and was directly added to each well. The cells were lysed with 50 μL 1× Passive Lysis Buffer (Promega, Madison, WI) 48 h after the transduction and immediately stored in a deep freezer. According to a previously described study by Baatartsogt et al.,20 (link) the luciferase activity was measured using a luminometer (Centro LB 960; BERTHOLD Technologies, Bad Wildbad, Germany).
6
Collagen Gel Invasion Assay for Sarcoma and Fibroblasts
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Cell invasion was investigated using the collagen gel invasion assay. The collagen gel was prepared by Cellmatrix Type I‐A (Nitta Gelatin). Sarcoma and fibroblast cells (2 × 105 cells for each cell line) were suspended in 100 µL collagen mixture (3.5 mg/mL). A 50‐µL drop of collagen gel containing the cells was polymerized in a 24‐well microplate and incubated in 0.5 mL medium containing DMSO or 25 µmol/L AR‐A014418 for 2 days. Mean percentage of cells that migrated out of the collagen gel in five microscopic fields was calculated with SD.
7
Comparative Hepatocyte and Bile Duct Cell Iron Toxicity
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To compare the vulnerability of hepatocytes and bile duct cells to iron toxicity, we isolated and cultured these cells from intact mice or those treated with a lethal dose (600 mg/kg, p.o.) of sodium ferrous citrate 3 h prior to isolation. After the livers were enzymatically digested by the two-step collagenase perfusion technique, hepatocytes were collected and plated on collagen-coated dishes. The undigested fragments of Glisson’s sheath containing bile duct cells were minced and embedded within a collagen gel matrix (Cellmatrix type I-A; Nitta Gelatin, Osaka, Japan). Cells were cultured in Williams’ E medium supplemented with 10 mM nicotinamide, 10 % fetal bovine serum, 10 ng/mL epidermal growth factor, 10−7 M insulin, and 10−7 M dexamethasone. For bile duct cell cultures, 10 ng/mL tumor necrosis factor-α (TNF-α) (PeproTech Inc., Rocky Hill, NJ) was added to the medium.
8
Porcine Tendon Collagen Gel Protocol
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An acid-soluble type 1 collagen solution (3 mg·mL−1, pH 3) derived from porcine tendon (Cellmatrix Type I-A; Nitta Gelatin, Osaka, Japan) yielded transparent gels with consistently high gel strength after reconstitution for use in this study.
9
Collagen Gel Diffusion System for Biomineralization
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A gel diffusion system was prepared based on a previously reported model. 29 In this experiment, a collagen gel [Cellmatrix type I-A (Nitta Gelatin Inc., Osaka, Japan) mixed with a neutralizing buffer: 0.05 N NaOH/2.2% NaHCO 3 /200 mM HEPES] was prepared and poured into the opened bottom of a 50 mL tube, sealed with paraffin tape (Parafilm, Bemis NA, Neenah, WI, USA), and kept in an incubator at 37 1C for 30 min to facilitate gelation. The calcium [100 mM (CH 3 COO) 2 CaÁH 2 O] and phosphate (60 mM NH 4 H 2 PO 4 ) were then poured into the opposite sides of the collagen gel and left for 30 min. The gel was then fixed in 4% PFA, and the minerals formed in the center of the gel were observed using SEM after osmium coating. To analyze the effect of different pHs on the shape of the precipitated minerals, calcium and phosphate solutions were titrated with HCl or NaOH to obtain solutions with pH 6.5, pH 7.5 or pH 8.5 before pouring into the gel diffusion system.
10
Preparation of Porcine Collagen Gels
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An acid‐soluble type I collagen solution derived from porcine tendons was prepared to a concentration of 3.0 mg/mL at a pH of 3.0 (Cellmatrix Type I‐A; Nitta Gelatin, Osaka, Japan) for use in this study. This solution was quickly reconstituted into collagen gels with consistently high strength and transparency required for use.
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