Esophageal 3D Raft Culture Model: An In Vitro Culture Technique to Generate Human Esophageal Mucosa Model
Published: April 30, 2023
Abstract
Source: Green, N. H. et al. Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model. J. Vis. Exp. (2015)
This video describes an in vitro culture technique to generate human organotypic esophageal constructs by co-culturing primary human esophageal fibroblast and squamous epithelial cells within a decellularized porcine scaffold. These 3D culture models mimic the function and physiology of their in vivo tissue counterparts.
Protocol
1. Production of the Human Esophageal Mucosa Model Perform all work in a laminar flow hood using aseptic technique to maintain a sterile environment. Sterilize all tools by soaking in 70% ethanol for 5 min, before rinsing in sterile PBS. The day before it is required, rehydrate the acellular porcine esophageal scaffold. One piece of scaffold is required for each construct to be prepared. To rehydrate the tissue, place sufficient pieces of porcine scaffold into a pot cont…
Açıklamalar
The authors have nothing to disclose.
Materials
| Trypsin | BD Biosciences | 215240 | Prepare 0.1% w/v solution in PBS and filter sterilize. Warm in 37 °C water bath before use. |
| DMEM | Labtech | LM-D1112 | Warm in 37 °C water bath before use |
| Foetal Calf Serum | Labtech | FB-1090 | |
| Epidermal Growth Factor | R+D Systems | 236-EG-200 | Prepare 200 µg/ml stock solution in 10 mM acetic acid 1% FCS |
| L-Glutamine | Sigma-Aldrich | G7513 | |
| Penicillin-Streptomycin | Sigma-Aldrich | P0781 | |
| Amphotericin B | Gibco | 15290-026 | Brand name Fungizone |
| PBS | Oxoid | BR0014 | Dissolve 1 tablet in 100 ml water and autoclave to sterilize |
| Collagenase A | Roche | 10103578001 | |
| Povidone-iodine solution | Ecolab | 10830E | Brand name Videne |
| Ethanol | Sigma-Aldrich | E7023 | |
| Progesterone | Sigma-Aldrich | P8783 | Prepare stock solution in DMEM and filter sterilize before use |
| Ethanolamine | Sigma-Aldrich | E9508 | Prepare stock solution in DMEM and filter sterilize before use |
| Hydrocortisone | Sigma-Aldrich | H0888 | Prepare stock solution in DMEM and filter sterilize before use use |
| O-phosphorylethanolamine | Sigma-Aldrich | P0503 | Prepare stock solution in DMEM and filter sterilize before use |
| ITS (insulin, transferrin, selenium) | Lonza | 17-838Z | Used for composite media preparation |
| Trypsin-EDTA | Sigma-Aldrich | T3924 | Warm in 37 °C water bath before use |
| EDTA 0.02% solution | Sigma-Aldrich | E8008 | Warm in 37 °C water bath before use |
| T75 culture flask | VWR | 734-2313 | |
| 50 ml centrifuge tube | Fisher | 11819650 | |
| 15 ml universal tube | SLS | SLS7504 | |
| 180 ml pot | VWR | 216-2603 | |
| Petri dish | SLS | 150350 | |
| 6 well plate | VWR | 734-2323 | |
| stainless steel rings | Manufactured in house – medical grade stainless steel. internal diameter 10 mm. external diameter 20 mm | ||
| steel mesh grids | Manufactured in house – sheets have 0.3 cm diameter holes. bent to produce grid 2 cm (w) x 2 cm (d) x 0.5 cm (h) |
Etiketler
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Esophageal 3D Raft Culture Model: An In Vitro Culture Technique to Generate Human Esophageal Mucosa Model. J. Vis. Exp. (Pending Publication), e20495, doi: (2023).