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Primary epithelial cell models for cystic fibrosis research.

Methods in molecular biology (Clifton, N.J.) (2011-05-07)
Scott H Randell, M Leslie Fulcher, Wanda O'Neal, John C Olsen
ABSTRACT

When primary human airway epithelial (hAE) cells are grown in vitro on porous supports at an air-liquid interface (ALI), they recapitulate in vivo morphology and key physiologic processes. These cultures are useful for studying respiratory tract biology and diseases and for testing new cystic fibrosis (CF) therapies. This chapter gives protocols enabling creation of well-differentiated primary CF and non-CF airway epithelial cell cultures with non-proprietary reagents. We also discuss the production of retroviral and lentiviral vectors, the derivation of hAE cell lines, reporter gene assays, and the evolving science of gene overexpression and knockdown in ALI hAE cultures.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
holo-Transferrin human, powder, BioReagent, suitable for cell culture, ≥97%
Sigma-Aldrich
Hydrocortisone-Water Soluble, BioReagent, suitable for cell culture
Sigma-Aldrich
Trypsin inhibitor from Glycine max (soybean), lyophilized powder
Sigma-Aldrich
Protease from Streptomyces griseus, Type XIV, ≥3.5 units/mg solid, powder
Sigma-Aldrich
Collagenase from Clostridium histolyticum, suitable for release of physiologically active rat epididymal adipocytes, Type II, 0.5-5.0 FALGPA units/mg solid, ≥125 CDU/mg solid
Sigma-Aldrich
Bovine Serum Albumin, lyophilized powder, essentially globulin free, ≥99% (agarose gel electrophoresis)
Sigma-Aldrich
3,3′,5-Triiodo-L-thyronine sodium salt, powder, BioReagent, suitable for cell culture
Sigma-Aldrich
(−)-Epinephrine
Sigma-Aldrich
Sodium metasilicate nonahydrate, BioReagent, suitable for plant cell culture, ≥98%
Sigma-Aldrich
Adenosine 5′-triphosphate disodium salt hydrate, Grade II, ≥97% (HPLC), crystalline, from microbial
Sigma-Aldrich
Deoxyribonuclease I from bovine pancreas, lyophilized powder, Protein ≥85 %, ≥400 Kunitz units/mg protein
Sigma-Aldrich
Collagen from human placenta, Bornstein and Traub Type IV, powder