Creation of a long-lifespan ciliated epithelial tissue structure using a 3D collagen scaffold
Introduction
Ciliary activity, one of the most primitive forms of locomotion in nature, was first discovered in the mammalian respiratory tract by Purkinje and Valentin in 1834 [1]. Ciliated cells are adapted in the respiratory tract to form an epithelial lining of coordinated metachronal ciliary activity that provides the propelling force for the transport of mucus along the airways. This mechanism is central to the maintenance of patent airways and pulmonary homeostasis. Impaired mucociliary transport is an inherent pathological characteristic of patients with chronic obstructive pulmonary disease, asthma and cystic fibrosis [2].
A ciliated epithelial tissue or cell culture system that mimics in vivo metachronal ciliary activity is critical to the understanding of the physiology and biophysics of this phenomenon. However, the preservation of active functional cilia and the induction of ciliogenesis of airway epithelial cell cultures remains one of the most elusive culturing and tissue engineering problems. Cells dissociated from their natural milieu of 3D geometrical supporting structures usually do not grow into tissue-like cultures. This is particularly obvious in cells that exhibit contractility or motility functions such as cardiac myocytes and ciliated cells. Thus, to develop a tissue culture system for ciliated epithelial tissue structure (CETS), we explored a process in which we used different biomaterials as scaffolds, including collagen [3], [4], [5], [6], chitosan [7], and gelatin [8], in combination with the air–liquid interface (ALI) culturing technique that has been uniquely developed for epithelial ciliated cell culture [9], [10], [11], [12], [13].
We herein describe a tissue engineering method in which we used dissociated primary mammalian airway ciliated epithelial cells and collagen, in association with the right constituents and the ALI culturing condition, to create a CETS. This 3D CETS with cilia exhibited vigorous, coordinated beating, viable cell interactions, polarity and morphological features closely resembling those found in vivo and has never before been reported. The CETS structure can be maintained in culture for a minimum of 4 months. Such a prolonged lifespan is especially useful for pulmonary cell-based drug discovery for the investigation of agents with long-acting physiopharmacological effects on mucociliary clearance. We have validated this method by using dissociated primary canine and porcine tracheal ciliated epithelial cells.
Section snippets
Preparation of scaffold matrix using collagen, chitosan, and gelatin
Type I collagen (0.3% at pH 3, ICN Biochemicals, Aurora, OH) was mixed with Dulbecco's Modified Eagle Medium (DMEM) and phosphate-buffered saline (PBS) to reach a final concentration of 2.0 mg/ml collagen in 10% PBS (v/v). A 1% solution of penicillin-streptomycin (10,000 units/ml penicillin, 10,000 μg/ml streptomycin) was then added and the pH adjusted to 7–8 using 4.5% (w/v) NaHCO3. The resulting collagen mixture was stored at 4 °C to prevent premature fibril assembly.
A day before culture, the
The collagen scaffold material
We evaluated 4 tissue culture protocols (Table 1). In each of the protocols, culture inserts for the ALI were used to establish ciliated cell cultures. With a ciliated cell culture using only ALI (Protocol 1), a limited ciliogenesis with a sparse ciliated cell structure was observed, consistent with all previous reports [9], [11], [13]. In Protocols 2 and 3, ciliated cells were cultured on the respective chitosan gel and gelatin gel on the apical chamber of the ALI inserts. There was no
Discussion
Biodegradable synthetic polymers and natural polymers are the two types of common scaffold materials used for structural support in tissue engineering [20]. For example, synthetic poly (N-isopropylacrylamide) has been applied successfully to generate cultures of electrically communicating 3D cardiac tissues [21], [22]. However, synthetic polymers may not interact well with ciliated epithelial cells and they are laborious to produce. Our initial focus was on natural scaffold materials, such as
Conclusions
The airway epithelial tissue system reported herein closely mimics the in vivo morphological characteristics and function of ciliated epithelia. In our studies, a simple 3D tissue engineering approach to generate a CETS has been described. This collagen gel scaffold, ALI tissue culture system enables dissociated primary ciliated cells to develop and differentiate into a 3D CETS. The CETS system is robust and straightforward to maintain. It also appears to preserve the integrity of the cellular
Acknowledgements
This work was supported by grants from the US National Institutes of Health, National Heart, Lung and Blood Institute (Grants No. R43, R44 HL 67595) awarded to HM. The authors would like to thank Dr. Barlow of SDSU for the skillful assistance and support of SEM work.
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