compromised cartilage [3, 4]. More recent studies suggest a plau-

sible alternative approach of partial decellularization in which a

hybrid graft is generated via removal of only the epithelium while

maintaining the cartilage alive [5–7]. We have recently reported a

novel bioreactor-based partial decellularization approach to gener-

ate hybrid tracheal grafts [8]. Using a short detergent-based treat-

ment with sodium dodecyl sulfate (SDS), we remove the

epithelium and maintain the structural integrity of the donor grafts

by keeping the cartilage alive. The partial decellularization process

is completed in an established double-chamber perfusion bioreac-

tor system in which the inner tracheal lumen is treated with SDS

while the outer chamber maintains the cartilage bathed in culture

media [8–10]. Herein, we describe our tracheal bioreactor system

and our de-epithelialization process in step-by-step detail.

2

Materials

Place the bioreactor main components and the bioreactor extra

components in autoclavable bags and autoclave. The autoclavable

bags are to be opened and used inside the biosafety cabinet (BSC).

Other materials and tools are to be used under sterile conditions in

the biosafety cabinet as well but cannot be autoclaved (such as

electronic components, pulleys, and other materials listed in Sub-

heading 2.3). Those materials are to be disinfected with a 10%

bleach solution followed by a wash in 70% ethanol solution before

placement in the BSC.

2.1

Bioreactor Main

Components (Fig. 1)

1. Stainless-steel body, lid, and silicone gasket.

2. Two chemical-resistant Viton^® fluoroelastomer brown O-rings

(3/32 fractional width #116).

3. Long and short rotating anchors.

4. Shaft holder, shaft, and corresponding rod.

5. Two stainless-steel rods with washer welded at one end.

2.2

Bioreactor Extra

Components

1. Two tight-seal moisture-resistant plastic tube fitting reducers

(1/8⁰⁰  1/16⁰⁰ tube ID).

2. One plastic barbed swivel adapter tube fitting (1/8⁰⁰ tube

ID  1/8 NPT male).

3. Three plastic barbed tube fitting adapters (1/8⁰⁰ tube ID  1/

8 NPT male).

4. Seven plastic barbed tube fittings, polypropylene tee connector

for 1/8⁰⁰ tube ID.

5. Four plastic barbed tube fittings, polypropylene 90^ elbow for

1/8⁰⁰ tube ID.

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Alba E. Marin-Araujo et al.