Vitrocol

Manufactured by Advanced BioMatrix

Sourced in United States

VitroCol is a type of collagen solution used for cell culture applications. It is derived from bovine sources and provides an extracellular matrix component to support cell growth and differentiation in vitro.

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Lab products found in correlation

15 protocols using vitrocol

Biomimetic Scaffolds for Tissue Engineering

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Scaffolds were created using human dermal collagen type I (VitroCol, Advanced Biomatrix, Carlsbad, CA) and recombinant human dermal tropoelastin (rhTE, Protein Genomics, Sedona, AZ). The proteins were dissolved into 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, Oakwood Chemical, Estill, SC). The biomimetic WHDs were prepared using a ratio of 9:1 collagen to rhTE. The solution was loaded into a 5 mL syringe and dispensed with a syringe pump at a constant rate of 3mL/hr. A voltage of 24–27kV was used to create circular scaffolds onto a circular aluminum foil target. The scaffolds were dried in a desiccator at room temperature overnight. The scaffolds were then sterilized by exposure to ultraviolet (UV, 254 nm) light for one hour on each side and prepared using a sterile 8 mm biopsy punch.

Kellar R.S., Diller R.B., Tabor A.J., Dominguez D.D., Audet R.G., Bardsley T.A., Talbert A.J., Cruz N.D., Ingraldi A.L, & Ensley B.D. (2020). Improved Wound Closure Rates and Mechanical Properties Resembling Native Skin in Murine Diabetic Wounds Treated with a Tropoelastin and Collagen Wound Healing Device. Journal of diabetes and clinical research, 2(3), 86-99.

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Biomimetic Scaffolds for Tissue Engineering

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Evaluating Cell Stiffness Changes in HASM Cells

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Dynamic changes in cell stiffness were measured in isolated HASM cells using forced motions of functionalized beads anchored to the cytoskeleton through cell surface integrin receptors, as previously described (An et al., 2016 (link)). The increase or decrease in stiffness is considered an index of single-cell smooth muscle contraction and relaxation, respectively. For these studies, serum-deprived, post-confluent cultured HASM cells were plated at 30,000 cells/cm² on plastic wells (96-well Removawell, Immulon II, Dynetech Laboratories Inc, Chantilly, VA, USA) previously coated with type I collagen (VitroCol; Advanced BioMatrix, Inc., Carlsbad, CA, USA) at 500 ng/cm², and maintained in serum-free media for 24 hours at 37 °C in humidified air containing 5% CO₂. These conditions have been optimized for seeding cultured cells on collagen matrix and for assessing their mechanical properties (An et al., 2016 (link)). For each individual cell, baseline stiffness was measured for the first 60 seconds and then measured continuously for the next 240 seconds after β-agonist addition. To evaluate desensitization, HASM cells were pretreated with or without 10 μM agonist for 0.5, 4, or 24 hours, washed, and then stiffness was measured after addition of 10 μM isoproterenol as described above.

De Pascali F., Ippolito M., Wolfe E., Komolov K.E., Hopfinger N., Lemenze D., Kim N., Armen R.S., An S.S., Scott C.P, & Benovic J.L. (2022). β-agonist profiling reveals biased signalling phenotypes for the β2-adrenergic receptor with possible implications for the treatment of asthma. British journal of pharmacology, 179(19), 4692-4708.

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Analyzing Platelet Aggregation with GPVI-CD39

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The effect of GPVI‐CD39 and control proteins on platelet aggregation was analyzed using the Multiplate (Roche) device.25 1:1 diluted hirudin‐anticoagulated blood was preincubated with antagonist for 3 minutes in the test cell without stirring to avoid platelet preactivation.26 Agonist was added, and samples were incubated for 6 minutes at 37°C with stirring. The following agonists were used: 6.5 μmol/L ADP, 12 μg/mL collagen isolated from rabbit aorta, 103 μg/mL collagen type I secreted by human fibroblasts (VitroCol; Advanced BioMatrix), or 333 μg/mL pooled human plaque homogenate. Platelet aggregation was measured in arbitrary units over the time period (arbitrary units×minutes; cumulative aggregation values).

Degen H., Borst O., Ziegler M., Mojica Munoz A., Jamasbi J., Walker B., Göbel S., Fassbender J., Adler K., Brandl R., Münch G., Lorenz R., Siess W., Gawaz M, & Ungerer M. (2017). ADPase CD39 Fused to Glycoprotein VI‐Fc Boosts Local Antithrombotic Effects at Vascular Lesions. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 6(8), e005991.

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Isolation and Culture of Nasal Epithelial Cells

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NECs were procured by curettage of the inferior turbinate using a rhinoprobe (Arlington Scientific, Inc.), pooling cells from both nostrils into a 15 mL conical filled with Media A (Table E1) and storing on ice for <24 h. Curettage was chosen based on site expertise.
Curettes were rinsed with media and the collected cells were centrifuged at 360 ×g for 5 min. Cell pellets were re-suspended in Accutase (Innovative Cell Technologies, Inc.) and centrifuged at 360 ×g for 5 min. The resulting pellet was re-suspended in Media A and placed into VitroCol (Advanced BioMatrix) coated petri dishes pre-seeded with irradiated mouse embryonic fibroblast feeder cells (Globalstem). Cells were maintained in Media A for five days, then Media B until confluent, changing media daily. Once confluent, media was removed and cells were exposed to 0.1% trypsin (Sigma-Aldrich) for 5 min to remove them from the dish. This mixture was centrifuged at 360 ×g for 5 min and the supernatant was removed. Cells were then passaged to a new dish using the protocol above or to spheroid or monolayer culture. All experiments utilized cells of passage 1 or 2.

Brewington J.J., Filbrandt E.T., LaRosa FJ I.I.I., Ostmann A.J., Strecker L.M., Szczesniak R.D, & Clancy J.P. (2017). Detection of CFTR function and modulation in primary human nasal cell spheroids. Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society, 17(1), 26-33.

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Epidermal Plug Cell Isolation and Culture

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Epidermal plugs were dissociated into a single cell solution as noted above. The cell solution was then cultured in a VitroCol (Advanced Biomatrix, Carlsbad, CA, USA) coated plate with EpiLife media (ThermoFisher Scientific, Waltham, MA), EpiLife Defined Growth Supplement EDGS, and 1% penicillin/streptomycin. Media was changed every two days and cells were passaged using 0.05% trypsin when 60–70% confluency was reached.

Riedl J.A., Riddle M., Xia L., Eide C., Boull C., Ebens C.L, & Tolar J. (2022). Interrogation of RDEB epidermal allografts after BMT reveals co-expression of collagen VII and keratin 15 with proinflammatory immune cells and fibroblasts. The Journal of investigative dermatology, 142(9), 2424-2434.

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Scaffold-based cardiac cell culture

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Low-adherent well plates were prepared
by coating them with 5 mg/mL poly(2-hydroxyethyl methacrylate) solution
(Sigma) and stored sealed at 4 °C until use. BC and BC-Ppy materials
were sterilized with UV irradiation for 15 min on each side and secured
by polytetrafluoroethylene rings at the bottom of each well.
The scaffolds were immersed in DMEM containing 4.5 g/L glucose, 50%
FBS (Gibco, Invitrogen), and incubated overnight inside a cell culture
incubator at 37 °C with 5% CO₂. Scaffolds were coated
with collagen (100 mg/mL, VitroCol, Advanced Biomatrix) prior to myoblast
seeding.
Thirty thousand cardiomyoblasts/cm² or fifteen
thousand cardiac fibroblasts/cm² were seeded on top of
each scaffold. Culture plastic wells (without scaffolds) were used
as controls. Adult human cardiac fibroblasts (p5-p6, PromoCell) were
cultured in fibroblast growth medium (PromoCell), whereas H9c2 rat
cardiac myoblasts (p2-p9, ATCC) were cultured with DMEM containing
4.5 g/L glucose, 10% FBS, 1% l-glutamine (Gibco, Thermo Fisher),
and 1% penicillin/streptomycin (Gibco, Invitrogen). Cultures were
maintained in a humidified atmosphere with 5% CO₂ at 37
°C, and culture medium was changed every 2 days.

Srinivasan S.Y., Cler M., Zapata-Arteaga O., Dörling B., Campoy-Quiles M., Martínez E., Engel E., Pérez-Amodio S, & Laromaine A. (2023). Conductive Bacterial Nanocellulose-Polypyrrole Patches Promote Cardiomyocyte Differentiation. ACS Applied Bio Materials, 6(7), 2860-2874.

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Measuring Cell Stiffness via Bead Assay

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Changes in cell stiffness were measured in isolated HASM cells using forced motions of functionalized beads anchored to the cytoskeleton through cell surface integrin receptors, as previously described (22 (link)). An increase or decrease in stiffness is considered an index of single-cell contraction and relaxation, respectively. For these studies, serum-deprived, post-confluent cultured HASM cells were plated at 30,000 cells/cm² on plastic wells (96-well Removawell, Immulon II, Dynetech Laboratories Inc, Chantilly, VA, USA) coated with type I collagen (VitroCol; Advanced BioMatrix, Inc., Carlsbad, CA, USA) at 500 ng/cm², and maintained in serum-free media for 24 h at 37 °C in humidified air containing 5% CO₂. To evaluate functional desensitization of the β₂ AR, in-real time, ISO-induced stiffness changes were monitored continuously for 30 min in the presence or absence of DFPQ. HASM cells were pretreated with or without 1 μM DFPQ for 30 min. Following 30 min preincubation, cell stiffness was measured for 60 s, and after ISO addition (10 μM, t = 60 s), stiffness was continuously measured for the next 1740 s (data were collected at every 1.3 s intervals). Cell stiffness is expressed as Pascal per nanometer (Pa/nm) and, for each cell, stiffness was normalized to its baseline stiffness prior to ISO stimulation.

Ippolito M., De Pascali F., Hopfinger N., Komolov K.E., Laurinavichyute D., Reddy P.A., Sakkal L.A., Rajkowski K.Z., Nayak A.P., Lee J., Lee J., Cao G., Donover P.S., Reichman M., An S.S., Salvino J.M., Penn R.B., Armen R.S., Scott C.P, & Benovic J.L. (2023). Identification of a β-arrestin-biased negative allosteric modulator for the β2-adrenergic receptor. Proceedings of the National Academy of Sciences of the United States of America, 120(31), e2302668120.

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Culturing 16-HBE Human Bronchial Cells

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16HBE14o (16-HBE) cells, an SV-40 transformed human bronchial epithelial cell line (Cozens et al. 1994 (link)), were a gift from the laboratory of Dr. Ilona Jaspers (UNC Chapel Hill, NC, USA). 16-HBE cells were cultured in minimal essential media (MEM) with GlutaMax (Thermo Fisher Scientific), 10% FBS and 1% penicillin-streptomycin (Sigma). All vessels used to culture 16-HBE cells were coated with a fibronectin solution (LHC Basal Medium (Life Technologies, Inc.), 0.01% BSA (Sigma), 1% Vitrocol (Advanced Bio Matrix, San Diego, CA, USA), and 1% human fibronectin (Sigma)). Cells were cultured in a humidified incubator at 37° C in 5% carbon dioxide (CO₂). For live-cell exposures, cells were plated in 35 mm Wilco Well glass-bottomed dishes with a 12 mm #1.5 glass aperture (Ted Pella Inc). For other O₃ exposures, cells were plated in 10 cm² cell culture dishes (Techno Plastic Products AG, Trasadingen, Switzerland).

Corteselli E.M., Gold A., Surratt J., Cui T., Bromberg P., Dailey L, & Samet J.M. (2020). Supplementation with Omega-3 Fatty Acids Potentiates Oxidative Stress in Human Airway Epithelial Cells Exposed to Ozone. Environmental research, 187, 109627.

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Air-Liquid Interface Culture of Bronchial Epithelial Cells

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The bronchial epithelial cell line VA10 was previously established at the laboratory [25] (link). The VA10 cell line is available upon request for all academic investigators for non-commercial purposes. Cells were cultured in LHC9 medium (Invitrogen, NY, USA) supplemented with 50 IU/ml penicillin and 50 µg/ml streptomycin (Invitrogen). For air-liquid interface cultures, cells were seeded on the upper layer of Transwell cell culture filters (Corning®Costar®) pore size 0.4 µm, 12 mm diameter, polyester membrane) (Sigma-Aldrich, St. Louis, USA) at density of 2×10⁵ cells per well. The cultures were maintained on LHC9 for 5 days, 0,5 ml in the upper chamber and 1.5 ml in the lower chamber. After 5 days medium was changed to DMEM/F-12 (Invitrogen), supplemented with 2% Ultroser G (Cergy-Saint-Christophe, France) for another 5 days. For air-liquid interface culture, the medium was aspired from the apical side and rinsed with PBS. Primary cells were isolated from patient material with enzyme digestion and cultured in chemically defined bronchial cell growth medium as previously described [29] (link) Isolated cells were maintained on collagen coated flasks (VitroCol, Advanced BioMatrix) and cultured in chemically defined bronchial epithelial cell medium (BEGM, Life Technologies/Sigma) as previously described [30] .

Arason A.J., Jonsdottir H.R., Halldorsson S., Benediktsdottir B.E., Bergthorsson J.T., Ingthorsson S., Baldursson O., Sinha S., Gudjonsson T, & Magnusson M.K. (2014). deltaNp63 Has a Role in Maintaining Epithelial Integrity in Airway Epithelium. PLoS ONE, 9(2), e88683.

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