Transplantation of Bioengineered Lung Using Decellularized Mouse Lungs and Primary Human Endothelial Cells

Summary

This paper describes how to create bioengineered mouse lungs using decellularization and recellularization methods. It also details subsequent orthotopic lung transplantation.

Abstract

Lung transplantation is a critical treatment for patients with end-stage lung diseases like idiopathic pulmonary fibrosis, but challenges such as donor shortages and posttransplant complications persist. Bioengineered lungs, integrating patient-specific cells into decellularized animal scaffolds, present a promising alternative. Despite progress in using bioengineered lungs in animal models, functionality and structure remain immature. This protocol addresses a critical barrier in organ bioengineering: the need for a cost-effective experimental platform. By using mouse models instead of larger animals like rats or swine, researchers can significantly reduce the resources required for each experiment, accelerating research progress.

The protocol outlines a detailed procedure for lung bioengineering using mouse heart-lung blocks and human primary cells, focusing on isolation strategy for the mouse heart-lung block, decellularization, bioreactor setup, perfusion-based organ culture, and orthotopic transplantation of bioengineered lungs. This mouse-scale platform not only reduces experimental costs but also provides a viable framework for optimizing cell types and numbers for recellularization, testing different cell types using histological and molecular methods, and ensuring blood flow post-transplantation. The method holds potential for broad applications, including studying cell interactions in three-dimensional culture conditions, cell-matrix interactions, and ex vivo cancer modeling, thereby advancing the field of organ bioengineering.

Introduction

Lung transplantation has been the decisive cure for patients having end-stage lung disease¹ such as idiopathic pulmonary fibrosis, where drug treatment is ineffective to stop the deterioration of respiratory function. More eligible patients add up to the waiting list every year; however, the number of organ donations from deceased donors has been trailing the increasing number of waiting patients^2,3. Even after undergoing lung transplantation, quite a few problems would degrade the function of transplanted lungs, including primary organ dysfunction, reactive allogenic syndrome, and infe....

Protocol

All experiments followed the Regulations for Animal Experiments and Related Activities at Tohoku University (15^th edition), published by Tohoku University²³. This study was approved by the Institutional Animal Care and Use Committee at Tohoku University (#2020AcA-041-01).

1. Preparation of materials for decellularization

1. Preparation of decellularization solutions (1,000 mL format in a 1 L autoclavable glass bottle)
   1. Sterile deionized (DI) water: Add 1,000 mL of distilled or DI water to 1 L autoclavable glass bottles. Autoclave for 20 min at 121 °C.
   2. Tr....

Results

Following the decellularization protocol, mouse lungs are visibly white and translucent (Figure 6A). Cellular components should be entirely removed, but the alveolar structure remains intact in the histological observation (Figure 6B,C). Recellularized mouse lungs using 3 × 10⁷ HUVECs with 2 day perfusion-based bioreactor culture show a homogeneous distribution of HUVECs (Figure 7A). HUVECs migrate i.......

Discussion

Organ bioengineering is a demanding enterprise. The costly screening process has been hindering this field's research and development cycle. By using mice as an experimental platform, space, cells, and media are significantly reduced compared to the previously used rat platform. Although measuring detailed physical parameters such as gas exchange, vascular resistance, or lung compliance has not been achieved yet, the mouse lung model allows for accelerated research timelines as it enables rapid iteration of experimen.......

Materials

Name	Company	Catalog Number	Comments
DECELLULARIZATION
27 G x 1/2 in. BD PrecisionGlide Needle	BD	305109	Or equivalent 27 G injection needle
BD Insyte IV Catheter 20 GA X 1.8 8IN	BD	381237	Or equivalent 20 G IV catheter
Blade silk suture (4-0)	Nesco	GA04SB	Or equivalent
CaCl2	Sigma-Aldrich	C5670
Catheter for rat jugular vein, PU 2Fr 10 cm	Instech	C20PU-MJV1301	Recommended for mice weighs 30 g and under.
Catheter for rat jugular vein, PU 3Fr 10 cm	Instech	C30PU-RJV1307	Recommended for mice weighs over 30 g.
DNase I	Sigma-Aldrich	DN25
MgSO4	Sigma-Aldrich	M7506
NaCl	Sigma-Aldrich	S3014
PinPort injectors	Instech	PNP3M
PinPorts, 22 G	Instech	PNP3F22-50	Fits C30PU-RJV1307
PinPorts, 25 G	Instech	PNP3F25-50	Fits C20PU-MJV1301
Sodium deoxycholate	Sigma-Aldrich	D6750
Sterile syringe, 5 mL	Generic
Triton X-100	Sigma-Aldrich	9036-19-5
CELL CULTURE
EGM-2 Endothelial Cell Growth Medium-2 BulletKit	Lonza	CC-3162
HUVEC – Human Umbilical Vein Endothelial Cells	Lonza	C2519A
PERFUSION-BASED BIOREACTOR
20 G needle	Generic
3-way stopcock	Generic
Cork borer	Generic		Boring size, 6-10 mm
EasyLoad III pump head	Cole-Parmer	243934
Glass canister	Hario	SCN-200T	Inner diameter: 80 mm
Heating magnetic stirrer	Generic
Lure fitting, PVDF, For Soft Tube	Nordson Medical	2-9965-01	Female, fits tubing with I.D. 1.5 mm (L/S 14)
Lure fitting, PVDF, For Soft Tube	Nordson Medical	2-9964-01	Male, fits tubing with I.D. 1.5 mm (L/S 14)
Lure fitting, PVDF, For Soft Tube	Nordson Medical	2-9965-03	Female, fits tubing with I.D. 3 mm (L/S 16)
Lure fitting, PVDF, For Soft Tube	Nordson Medical	2-9964-03	Male, fits tubing with I.D. 3 mm (L/S 16)
Magnetic stirring bar	Generic
Masterflex L/S Digital Precision Modular Drive with Remote I/O and Benchtop Controller	Cole-Parmer	07557-00
Masterflex L/S Precision Pump Tubing, PharMed BPT, L/S 16	Cole-Parmer	06508-16
Masterflex L/S Pricision Pump Tubing, Platinum-Cured Silicone, L/S 14	Cole-Parmer	96410-14
Millex-GP Syringe Filter Unit, 0.22 µm, polyethersulfone, 33 mm, gamma sterilized	Millipore	SLGPR33RS
Pyrex 250 mL grass bottle, GL-45 screw cap	Corning	1395-250
Silicon Septa for GL45 Open Top PBT Screw Cap	Corning	1395-455S
Silicone Light Stopper	IMG	07763-18	Upper diameter: 87 mm, Lower diameter: 75 mm
Sterile syringe, 10 mL, 50 mL	Generic
MOUSE SURGERY (Isolation of the heart-lung block | Lung transplantation)
10-0 Nylon ties	Kono Seisakusho	N/A
10-0 Silk ties	Kono Seisakusho	N/A
4-0 Silk ties	Kono Seisakusho	N/A
Arterial clamp, 45 mm curved, grooved	Natsume seisakusyo	C-17-45
BD Insyte IV Catheter 24GA	BD	381512	Or equivalent 24G i.v. catheter
Bulldog Vascular Forceps 45mm curved	Natsume seisakusyo	M2
Butorphanol tartrate	Meiji Seika Pharma	N/A
Cefazolin Sodium	Otsuka Pharmaceutical	N/A
Dumont forceps #5/45	Fine Science Tools	1251-35
Fine vannas style spring scissors	Fine Science Tools	15403-08	45° tip, 0.01 x 0.06 mm
Gemini Cautery Kit	Harvard Apparatus	RS-300
Halsted-Mosquito clamp curved tip, 125 mm	Bioresearch center	16181670
Hegar needle holder, 150 mm	B Braun/Aesculap	BM065R
Heparine solution	Mochida Seiyaku	N/A
Medetomidine	Nippon Zenyaku Kogyo	N/A
Micro forceps straight	B Braun/Aesculap	BD33R
Midazolam	Sandoz	N/A
Mouse Ventilator	Harvard Apparatus	Model 687™
Normal Saline, Clinical grade	Otsuka Pharmaceutical	N/A
Petri dish, 60 x 15 mm	BD	351007
Safelet Cath PU 20 gauge polyurethan catheter	Nipro	09-031
Sakaki stainless scissors curved 14 cm	Bioresearch center	64152034
Scalpel holder	Bioresearch center	16101040
Small animal retraction system	Fine Science Tools	18200-20
Spare blade scalpel #11	Muranaka Medical Instruments	567-001-03
Spring scissors, 15 cm	Bioresearch center	PRI13-3736
Stereomicroscope	Leica Microsystems	M525	Clinical-grade surgical microscope with a flexible arm system is preferable.
Sugita titanium aneurysm clip curved slim, No.98	Mizuho medical	17-001-98
Sugita titanium clip applier, 110 mm	Mizuho medical	17-013-53
Temperature-adjustable electric warmer	Generic
Ultrafine cotton swab	Generic
VASCULAR AND BRONCHIAL CUFF
Fine sandpaper	Generic
Venula 20 gauge Teflon angiocatheter	Top	1160
Venula 22 gauge Teflon angiocatheter	Top	1161
Venula 24 gauge Teflon angiocatheter	Top	1124