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Transwell polyester inserts

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Transwell polyester inserts are a cell culture accessory used to create a barrier between two chambers. They are made of polyester material and are designed to be inserted into a well or culture plate, allowing for the exchange of soluble factors between the chambers while maintaining a physical separation between cells.

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Lab products found in correlation

Laminin, by Merck Group (2 mentions) Pronase, by Merck Group (2 mentions) L glutamine, by Thermo Fisher Scientific (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions) Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions) Penicillin streptomycin, by Merck Group (1 mentions) Dulbecco s modified eagle medium, by Lonza (1 mentions) Rat tail collagen solution, by Thermo Fisher Scientific (1 mentions) Rpmi 2650, by American Type Culture Collection (1 mentions) F7524, by Merck Group (1 mentions) 75 cm2 culture flask, by Corning (1 mentions) Bronchial epithelial growth medium (begm), by Lonza (1 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions) Singlequot kit, by Lonza (1 mentions) Millicell ers voltohmmeter, by Merck Group (1 mentions) Human cytokine 30 plex panel, by Thermo Fisher Scientific (1 mentions) Nuserum 4, by BD (1 mentions) Sodium pyruvate, by Harvard Bioscience (1 mentions) Fetal bovine serum (fbs), by Harvard Bioscience (1 mentions)

Close spelling variants of this product

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15 protocols using transwell polyester inserts

1

Caco-2 Cell Culture Protocol

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The Caco-2 cell line (HTB-37), derived from a human colorectal adenocarcinoma, was obtained from The American Type Culture Collection (ATCC, Manassas, VA, USA). They were grown (at passage number 29-45) in Dulbecco's Modified Eagle Medium (Lonza) supplemented with 10% heat-inactivated fetal bovine serum (Gibco, Waltham, MA USA), 1% penicillin–streptomycin (Sigma), 1% non-essential amino acids (Gibco), further referred to as DMEM+.
The cells were seeded at density of 40 000 cells per cm2 in 12 well Transwell polyester inserts (0.4 μm pore size, 1.12 cm2 surface area, Corning Amsterdam, The Netherlands) cultured in DMEM+. During culture period, medium was changed for every other day.
In the microfluidic chip cells were seeded at a density of 75 000 cell per cm2, the cells were allowed to attach to the membrane without flow for 72 h and then were perfused with low sodium bicarbonate (10 mM) DMEM+ (Sigma) for optimizing pH buffering capacity, through the AP and BL side for 7 days.
Kulthong K., Duivenvoorde L., Mizera B.Z., Rijkers D., Dam G.T., Oegema G., Puzyn T., Bouwmeester H, & van der Zande M. (2018). Implementation of a dynamic intestinal gut-on-a-chip barrier model for transport studies of lipophilic dioxin congeners. RSC Advances, 8(57), 32440-32453.
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2

Isolation and Culture of Mouse Choroid Plexus Epithelial Cells

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Primary mouse CPE cells were isolated from P2‐P7 pups as previously described (Balusu et al, 2016 (link)). Choroid plexus tissue was isolated from the lateral and fourth ventricles, pooled, and digested with pronase (Sigma‐Aldrich) for 7 min. For monolayer cultures, cells were plated on 24‐well plate or Transwell polyester inserts (pore size, 0.4 μm; surface area, 33.6 mm2; Corning) coated with laminin (Sigma‐Aldrich). Cells were grown in DMEM/F12 medium supplemented with 10% FBS, 2 mM L‐Glutamine (Gibco), 1% penicillin/streptomycin at 37°C and 5% CO2 for 1–2 weeks until their trans‐epithelial electrical resistance (TEER) values reached a plateau.
Xie J., Bruggeman A., De Nolf C., Vandendriessche C., Van Imschoot G., Van Wonterghem E., Vereecke L, & Vandenbroucke R.E. (2023). Gut microbiota regulates blood‐cerebrospinal fluid barrier function and Aβ pathology. The EMBO Journal, 42(17), e111515.
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3

Primary Mouse Choroid Plexus Epithelial Cell Isolation

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Primary mouse CP epithelial cells were isolated from P2-P4 WT pups as previously described [2 (link)]. CP tissue was isolated from the lateral and fourth ventricles, pooled and digested with pronase (53,702, Sigma-Aldrich) for 7 min. For monolayer cultures, cells were plated in 24-well Transwell polyester inserts (pore size, 0.4 μm; surface area, 33.6 mm2; Corning) coated with laminin (L2020, Sigma-Aldrich). Cells were grown in DMEM/F12 supplemented with 10% FBS, 2 mM L-Glutamine (Gibco), 1% penicillin/streptomycin at 37 °C and 5% CO2 for 1 to 2 weeks until their TEER values reached a plateau.
Xie J., Gorlé N., Vandendriessche C., Van Imschoot G., Van Wonterghem E., Van Cauwenberghe C., Parthoens E., Van Hamme E., Lippens S., Van Hoecke L, & Vandenbroucke R.E. (2021). Low-grade peripheral inflammation affects brain pathology in the AppNL-G-Fmouse model of Alzheimer’s disease. Acta Neuropathologica Communications, 9, 163.
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4

Osteoblast-like Cell Viability Assay

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Human osteoblast-like osteosarcoma cells MG63 were obtained from in-house stocks at the Eastman Dental Institute, University College London. Two different assays were applied; Alamar Blue and Live/Dead assay with confocal laser scanning microscopy. Cells were prepared and then seeded calculating a density of 3000 cells per 𝑐𝑚 : in 1 ml of medium.
Then, three discs from each composition were placed in a conventional 24 well tissue culture test plate (Transwell® polyester inserts, Corning, USA) with one disc per well. Seeding of cells on the discs was carried out calculating 5300 cells in each well.
Aldaadaa A., Al Qaysi M., Georgiou G., Ma Leeson R, & Knowles J.C. (2018). Physical properties and biocompatibility effects of doping SiO2 and TiO2 into phosphate-based glass for bone tissue engineering. Journal of biomaterials applications, 33(2).
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5

Mesenchymal Stem Cell Characterization

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Human mesenchymal stem cells were obtained from in-house stocks at the Eastman Dental Institute, University College London. Two different assays were used which are alkaline phosphatase and cell counting Kit 8 (CCK). Cells were prepared and then seeded calculating a density of 3000 cells per 𝑐𝑚 : of 1 ml medium. Then, three discs from each composition were placed in a conventional 24 well tissue culture test plate (Transwell® polyester inserts, Corning, USA) as in one disc per well. Seeding of cells on the discs was carried out calculating about 5000 cells in each well.
Aldaadaa A., Al Qaysi M., Georgiou G., Ma Leeson R, & Knowles J.C. (2018). Physical properties and biocompatibility effects of doping SiO2 and TiO2 into phosphate-based glass for bone tissue engineering. Journal of biomaterials applications, 33(2).
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6

Establishment of ALI Model for Respiratory Epithelial Cells

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Calu-3 and RPMI 2650 cells (ATCC, Manassas, VA, USA) were grown in Minimum Essential Medium and DMEM/F-12 for each cell line, respectively. Media contained 10% v/v fetal bovine serum, 1% v/v nonessential amino acid solution and 2 mM L-glutamine, according to the manufacturer's protocol. Cells were incubated in a humidified atmosphere of 95% air and 5% CO2 at 37°C. The medium was replaced three times a week and cells were passaged at a ratio of 1/3. To stablish an air–liquid interface (ALI) model, both Calu-3 and RPMI 2650 cells were seeded at a density of 1.65×105 cells per insert on Transwell polyester inserts (Corning Costar, Lowell, MA, USA). For the RPMI 2650 cells, transwells were previously coated with 200 μL 1 μg·mL−1 rat tail collagen solution (Life Technologies, Sydney, Australia) in PBS. After 24 h of seeding cells, the ALI model was established by aspirating the apical medium and the cells were maintained with 0.5 mL culture medium in the basolateral chamber. The ALI conditions stimulated differentiation of the epithelial cells and these were ready for the experiments after 11 days [7 (link), 8 (link)].
Grau-Bartual S., Al-Jumaily A.M., Young P.M., Traini D, & Ghadiri M. (2020). Effect of continuous positive airway pressure treatment on permeability, inflammation and mucus production of human epithelial cells. ERJ Open Research, 6(2), 00327-2019.
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7

Calu-3 Epithelial Barrier Formation

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The Calu-3 human adenocarcinoma epithelial cell line (ATCC HTB-55, LGC Standard, France) was used for the experiment at cell passage 24–40. All products used for cell culture were provided by Thermo Fisher Scientific unless stated otherwise. Calu-3 cells were cultured in Eagle’s Minimum Essential Medium (MEM) supplemented with 10% v/v fetal bovine serum (FBS) (F7524, Sigma-Aldrich), 1% non-essential amino acids (NEAA) 100×, 1% sodium pyruvate, 1% Glutamax, 1% penicillin streptomycin 100×, and 1% HEPES buffer 100×. Cells were culture at a density of 40,000 cells/cm2 in 25 or 75 cm2 culture flasks (Corning) at 37 °C in a humidified 5% CO2 atmosphere and passaged weekly before confluence. To form the epithelial barrier, Calu-3 cells were seeded on Transwell polyester inserts with a 3 µm pore diameter (Corning Costar) at a density of 500,000 cells/insert (with 500 µL of cell suspension in the apical compartment and 1.5 mL of medium in the basolateral compartment). The culture medium was changed every 2–3 days in both compartments until TEER > 700 Ω cm2. The medium was removed from the apical compartment to create an air–liquid interface. The medium in the basolateral compartment was replaced by 0, 2, 4 or 8% FBS one day after ALI. Calu-3 cells cultured on Transwell membrane were maintained for 21 days after ALI.
Sanchez-Guzman D., Boland S., Brookes O., Mc Cord C., Lai Kuen R., Sirri V., Baeza Squiban A, & Devineau S. (2021). Long-term evolution of the epithelial cell secretome in preclinical 3D models of the human bronchial epithelium. Scientific Reports, 11, 6621.
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8

Calu-3 Cell Transport Study Protocol

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Calu-3 cell line (HTB-55) was purchased from the American Type Cell Culture Collection (ATCC, Rockville, USA). The cells (passage 35-40) were grown in 75 cm 2 asks in Ham's F-12 with the following growth supplements: epidermal growth factor 0.5 ng ml À1 , bovine pituitary extract 50 mg ml À1 , hydrocortisone 0.5 mg ml À1 , epinephrine 0.5 mg ml À1 , transferrin 10 mg ml À1 , insulin 5 mg ml À1 , retinoic acid 0.1 ng ml À1 , triiodothyronine 6.5 ng ml À1 (Sigma, MO, USA). Cells were maintained in a humidied 95% air 5% CO 2 atmosphere of at 37 C and were propagated according to ATCC recommendations.
Cells were seeded onto 24 well Transwell polyester inserts (0.33 cm 2 polyester, 0.4 mm pore size) (Corning Costar, MA, USA) at a density of 5 Â 10 5 cells per cm 2 with 100 mL and 500 mL of the above-mentioned fat-free medium in the apical and basolateral compartment, respectively. The culture was fed every alternate day with fresh medium. The monolayers were allowed to form over 12-14 days as determined by Mamlouk. 18 Aer this period, the cells were used for transport study.
Haghi M ., Traini D ., Wood LG ., Oliver B ., Young PM , & Chrzanowski W . (2015). A 'soft spot' for drug transport: modulation of cell stiffness using fatty acids and its impact on drug transport in lung model. Journal of materials chemistry. B, 3(13).
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9

Differentiation of Bronchial Epithelial Cells

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Bronchial epithelial cells at passage 2 or 3 were seeded onto Transwell ® polyester inserts (0.33 cm 2 , 0.4 µm pore size) (Corning Costar, Lowell, MA, USA) (density: 3×10 5 cells per insert). The growth medium in the apical chamber was removed when the cells were confluent and the bronchial epithelial growth medium (BEGM) (Lonza, Melbourne, Australia) in the basal chamber was replaced by bronchial epithelial differentiation medium (BEDM) (Lonza, Melbourne, Australia). hBE cells were allowed to differentiate under air-interface over 21-28 days. Differentiation was confirmed through mucus secretion and cilia formation and beating (see supplementary materials).
Haghi M., Hittinger M., Zeng Q., Oliver B., Traini D., Young P.M., Huwer H., Schneider-Daum N, & Lehr C.M. (2015). Mono- and Cocultures of Bronchial and Alveolar Epithelial Cells Respond Differently to Proinflammatory Stimuli and Their Modulation by Salbutamol and Budesonide. Molecular pharmaceutics, 12(8).
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10

In Vitro BBB and Co-culture Models

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ECV304 and C6 cell lines were incubated with M199 containing 10% FBS and 1% penicillin-streptomycin while bEnd3 cells were grown in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. Cell culture was performed in a humidified atmosphere of 5% CO2 in air at 37°C and the confluent cells were passaged by the trypsin (0.25%)-EDTA (0.02%) solution at a split ratio of 1:5~1:10.
For the generation of mono-culture models, ECV304 or bEnd3 cells were seeded on the upper surface of the membrane in Polyester Transwell inserts (0.4 μM pore size, 6.5 mm diameter, 24 well, Costar, Kennebunk, ME, USA) at a density of 5 × 104 and 8 × 104/cm2, respectively. For the generation of co-culture models, C6 cells were seeded on the lower surface of the membrane at a density of 5 × 104/cm2 and incubated for 2 h to allow cell attachment before the seeding of ECV304 or bEnd3 cells. The culture medium (0.25 ml in the apical compartment and 1 ml in the basolateral compartment to avoid hydrostatic pressure) was replenished every day.
Yang S., Mei S., Jin H., Zhu B., Tian Y., Huo J., Cui X., Guo A, & Zhao Z. (2017). Identification of two immortalized cell lines, ECV304 and bEnd3, for in vitro permeability studies of blood-brain barrier. PLoS ONE, 12(10), e0187017.
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