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)

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Bu Makaleden Alıntı Yapın
Esophageal 3D Raft Culture Model: An In Vitro Culture Technique to Generate Human Esophageal Mucosa Model. J. Vis. Exp. (Pending Publication), e20495, doi: (2023).

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