प्राथमिक मानव ब्रोन्कियल उपकला Explants से विकसित कोशिकाओं

Summary

यहाँ हम मानव ब्रोन्कियल airway ऊतक के एक हवा तरल इंटरफेस पर विभेदित विकास सहित explants से प्राथमिक मानव ब्रोन्कियल उपकला कोशिकाओं से बढ़ के लिए एक विस्तृत विधि का वर्णन. इस विधि मानव फेफड़े के स्वास्थ्य और रोग में airway उपकला की भूमिका की जांच के लिए एक प्राथमिक कोशिकाओं के प्रचुर स्रोत प्रदान करता है.

Abstract

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the function and structure of human epithelial cells. Here we describe in detail the method of growing bronchial epithelial cells from bronchial airway tissue that is harvested by the surgeon at the times of lung surgery (e.g. lung cancer or lung volume reduction surgery). With ethics approval and informed consent, the surgeon takes what is needed for pathology and provides us with a bronchial portion that is remote from the diseased areas. The tissue is then used as a source of explants that can be used for growing primary bronchial epithelial cells in culture. Bronchial segments about 0.5-1cm long and ≤1cm in diameter are rinsed with cold EBSS and excess parenchymal tissue is removed. Segments are cut open and minced into 2-3mm³ pieces of tissue. The pieces are used as a source of primary cells. After coating 100mm culture plates for 1-2 hr with a combination of collagen (30 μg/ml), fibronectin (10 μg/ml), and BSA (10 μg/ml), the plates are scratched in 4-5 areas and tissue pieces are placed in the scratched areas, then culture medium (DMEM/Ham F-12 with additives) suitable for epithelial cell growth is added and plates are placed in an incubator at 37°C in 5% CO₂ humidified air. The culture medium is changed every 3-4 days. The epithelial cells grow from the pieces forming about 1.5 cm diameter rings in 3-4 weeks. Explants can be re-used up to 6 times by moving them into new pre-coated plates. Cells are lifted using trypsin/EDTA, pooled, counted, and re-plated in T75 Cell Bind flasks to increase their numbers. T75 flasks seeded with 2-3 million cells grow to 80% confluence in 4 weeks. Expanded primary human epithelial cells can be cultured and allowed to differentiate on air-liquid interface. Methods described here provide an abundant source of human bronchial epithelial cells from freshly isolated tissues and allow for studying these cells as models of disease and for pharmacology and toxicology screening.

Protocol

मानव ब्रोन्कियल खंड प्राथमिक ब्रोन्कियल उपकला कोशिकाओं का एक प्रचुर स्रोत प्रदान करते हैं. इस अनुच्छेद में हम विकास और हौसले से अलग मानव ब्रोन्कियल क्षेत्रों से मानव ब्रोन्कियल उपकला कोशिकाओं (HbE) के …

Discussion

इस अध्ययन में हम संस्कृति और प्राथमिक मानव ब्रोन्कियल उपकला कोशिकाओं के विस्तार के लिए विस्तृत तरीके प्रस्तुत किया. हम दिखा दिया कैसे explants और ब्रोन्कियल ऊतक, मीडिया जो उपकला सेल के विकास को बढ़ावा देने…

Materials

Material Name	Tipo	Company	Catalogue Number	Comment
Albumin from bovine serum, powder		Sigma	A4919	Use for coating solution
COLLAGEN TYPE I 0.1% Solution Sterile-filtered		Sigma	C8919	Use for coating solution
Fibronectin from human plasma		Sigma	F2006	Use for coating solution
Earle’s Balanced Salt Solution (EBSS, sterile)		Sigma	28888	Use for rinsing tissue and for coating solution
DMEM/Ham F-12		Sigma	D8437
FBS		Sigma	F1015	Inactivate, then aliquot and freeze (-20°C); use fresh when needed
Antibiotic-Antimycotic Stabilized		Sigma	A5955	Aliquot into 2 ml vials and freeze (-20°C); use fresh when needed
Albumin solution from bovine serum		Sigma	A8412	BSA
Pituitary Extract Bovine		Sigma	P1476	BPE
Epidermal growth factor		Sigma	E9644	EGF
(±)-Epinephrine		Sigma	E1635
Insulin		Sigma	I6634
Tranferrin Human		Sigma	T8158
Triiodo-L-thyronine		Sigma	T6397
Hydorcortisone		Sigma	H0888
Retinoic Acid		Sigma	R2625
Trypsin		Sigma	T9935
EDTA		Sigma	E6758	EDTA
Phosphate buffered saline		Sigma	P5368	PBS
70% ethanol				Use for disinfecting and cleaning
24 well Transwells		Corning	3470
Cell Culture Flasks (T75) CLLBND from Corning		Fisher Scientific	05-539-104	These flasks have a Cell Bind coating which promotes cell attachment and growth
Monoclonal Anti-Cytokeratin, pan-FITC antibody		Sigma	F3418	Permeabilization: 0.2% TRITON X-100/PBS; Use 1:250 dilution
Antibody: E-Cadherin (H108)		Santa Cruz	sc-7870	Do not permeabilize; Use 1:250 dilution and A21207 (Invitrogen) as secondary antibody
Alexa Fluor 594 donkey anti-rabbit IgG		Invitrogen	A21207	Use 1:400 dilution
Antibody: Monoclonal mouse Anti- α-Smooth Muscle Actin-Cy3		Sigma- Aldrich	C6198	Permeabilization: 0.2% TRITON X-100/PBS; Use 1:100 dilution
Antibody: Vimentin (RV202)		Santa Cruz	Sc-32322	Permeabilization: 0.2% TRITON X-100/PBS; Use 1:100 dilution and T2402 (Sigma- Aldrich) as secondary antibody
Rabbit anti-mouse TRITC		Sigma- Aldrich	T2402	Use 1:400 dilution
Antibody: CD31 or PECAM-1 (M-20)		Santa-Cruz	Sc-1506	Do not permeabilize; Use 1:200 dilution and Donkey anti-goat IgG-FITC as secondary antibody
Donkey anti-goat IgG-FITC		Santa-Cruz		Use 1:100 dilution
Vectashield Mounting medium with DAPI		Vector Laboratories	H-1200	Refrigerate in the dark; stains nuclei and retains fluorescence during prolonged storage
Vectashield Mounting medium		Vector Laboratories	H-1000	Refrigerate in the dark; retains fluorescence during prolonged storage
Hoechst Stain solution		Sigma	H6024	Stains nuclei; use 1:10 dilution and Vectashield H-1000

Other requirements: incubator, biological safety cabinet (BSC), centrifuge, 100mm culture plates, sterile tubes (15 ml, 50 ml, and 2 ml), sterile pipette tips, scalpel handle and blades, small sharp scissors, lab coats and gloves. These can be obtained from your preferred suppliers.