Exploring Alternative Perfusion Solutions Using Next-Generation Polymerized Hemoglobin-Based Oxygen Carriers in a Model of Rat Ex Vivo Lung Perfusion

Summary

Here, we describe the application of a polymerized human hemoglobin (PolyhHb)-based oxygen carrier as a perfusate and the protocol in which this perfusion solution can be tested in a model of rat ex vivo lung perfusion.

Abstract

Lung transplantation is hampered by the lack of suitable donors. Previously, donors that were thought to be marginal or inadequate were discarded. However, new and exciting technology, such as ex vivo lung perfusion (EVLP), offers lung transplant providers extended assessment for marginal donor allografts. This dynamic assessment platform has led to an increase in lung transplantation and has allowed providers to use donors that were previously discarded, thus expanding the donor pool. Current perfusion techniques use cellular or acellular perfusates, and both have distinct advantages and disadvantages. Perfusion composition is critical to maintaining a homeostatic environment, providing adequate metabolic support, decreasing inflammation and cellular death, and ultimately improving organ function. Perfusion solutions must contain sufficient protein concentration to maintain appropriate oncotic pressure. However, current perfusion solutions often lead to fluid extravasation through the pulmonary endothelium, resulting in inadvertent pulmonary edema and damage. Thus, it is necessary to develop novel perfusion solutions that prevent excessive damage while maintaining proper cellular homeostasis. Here, we describe the application of a polymerized human hemoglobin (PolyhHb)-based oxygen carrier as a perfusate and the protocol in which this perfusion solution can be tested in a model of rat EVLP. The goal of this study is to provide the lung transplant community with key information in designing and developing novel perfusion solutions, as well as the proper protocols to test them in clinically relevant translational transplant models.

Introduction

Like any field in solid organ transplantation, lung transplantation suffers from a shortage of donor organs. In order to increase the donor pool, significant research has been dedicated to investigating the potential of allografts that were once thought to be unsuitable for transplantation, i.e., extended criteria donors (ECD). These allografts can be considered ECD for a milieu of reasons, including questionable quality, poor function, infection, trauma, prolonged warm or cold ischemic times, and advanced age^1,2. In certain cases, where these lungs are suitable for immediate transplant³

Protocol

Sprague-Dawley rats (300 g body weight) were commercially obtained and housed under pathogen-free conditions at The Ohio State University Wexner Medical Center Animal Facility. All procedures were humanely performed according to the NIH and the National Research Council's Guide for the Humane Care and Use of Laboratory Animals and with the approval of The Ohio State University Institutional Animal Care and Use Committee (IACUC Protocol 2023A00000071).

1. PolyhHb synthesis and purific.......

Discussion

The development and testing of perfusion solutions is a novel endeavor that many throughout the globe are embarking on. Traditionally, standard perfusates offer the ability to suspend ischemic time and mitigate the associated injuries with ischemia, as well as reperfusion¹⁸. However, the next evolution of EVLP is to improve current perfusate technology as well as incorporate repair and reconditioning therapies^39,40,

Materials

Name	Company	Catalog Number	Comments
10 cc insulin syringe 29 G x 1/2" needle	B-D	309301
30 L Glass Batch Bioreactor	Ace Glass
30g Needle	Med Needles	BD-305106
Baytril (enrofloxacin) Antibacterial Tablets	Elanco	NA
Calcium Chloride dihydrate (CaCl2.2H2O)	Sigma Aldrich	10035-04-8	For modified Ringer's lactate
CFBA carrier frequency bridge amplifier type 672	Harvard Apparatus	731747
Connect kit D150	Cole-Parmer	VK 73-3763
Dumont #5 Forceps	Fine Science tools	11252-50
Dumont Medical #5/45 Forceps - Angled 45°	Fine Science tools	11253-25
Ecoline Star Edition 003, E100 Water Heater	Lauda	LCK 1879
Expired human leukoreduced, packed RBC units	Wexner Medical Center Canadian Blood Services Zen-Bio Inc
Fiberoxygenator D150	Hugo Sachs Elektronik	PY2 73-3762
Forceps	Fine Science tools	11027-12
Glutaraldehyde (C5H8O2 70 wt%)	Sigma Aldrich	111-30-8 (G7776)
Halsted-Mosquito Hemostat	Roboz Surgical	RS-7112
Heparin 30,000 units per 30 ml	APP Pharmaceuticals
Human Serum Albumin (HSA)	OctaPharma Plasma		Perfusate additive
IL2 Tube set for perfusate	Harvard Apparatus	733842
IPL-2 Basic Lung Perfusion System	Harvard Apparatus
Ketamine 500 mg per 5 ml	JHP Pharmaceuticals
Left Atrium cannula	Harvard Apparatus	730712
Liqui-Cel EXF Series G420 Membrane Contactor	3M	G420	gas contactor
low potassium dextran glucose solution (perfadex)	XVIVO		solution flushing the lung
Masterflex Platinum Coated Tubing(Size: 73,17,16,24)	Cole-Palmer
N-Acetyl-L-cysteine (NALC, C5H9NO3S)	Sigma Aldrich	616-91-1 (A7250)	For modified Ringer's lactate
Nalgene Vessels (10L, 20L)	Nalgene		Filtration vessels
Peristaltic Pump	Ismatec	ISM 827B
PES, 0.65 µm TFF module	Repligen	N02-E65U-07-N
PhysioSuite	Kent Scientific Corporation	PS-MSTAT-RT
polyethersulfone (PES), 0.2 µm TFF module	Repligen	N02-S20U-05-N
Polysulfone (PS), 500 kDa TFF module	Repligen	N02-P500-05-N
Potassium Chloride (KCl)	Fisher Scientific	7447-40-7	For PBS
PowerLab 8/35	ADInstruments	730045
Pulmonary Artery cannula	Harvard Apparatus	730710
Pump Head tubing (Size: 73,17,16,24)	PharMed BPT
Puralube Ophthalmic Ointment	Dechra	NA
Scissors	Fine Science tools	14090-11
SCP Servo controller for perfusion type 704	Harvard Apparatus	732806
Small Animal Ventilator model 683	Harvard Apparatus	55-000
Sodium Chloride (NaCl)	Fisher Scientific	7647-14-5 (S271-10)	For PBS and saline
Sodium cyanoborohydride (NaCNBH3)	Sigma Aldrich	25895-60-7
Sodium Dithionite (Na2S2O4)	Sigma Aldrich	7775-14-6
Sodium Hydroxide (NaOH)	Fisher Scientific	1310-73-2	For modified Ringer's lactate
Sodium Lactate (NaC3H5O3)	Sigma Aldrich	867-56-1	For modified Ringer's lactate
Sodium phosphate dibasic (Na2HPO4)	Fisher Scientific	7558-79-4	For PBS
Sodium phosphate monobasic (NaH2PO4)	Fisher Scientific	7558-80-7	For PBS
SomnoSuite Small Animal Anesthesia System	Kent Scientific Corporation	SS-MVG-Module
Sprague-Dawley rats	Envigo
TAM-A transducer amplifier module type 705/1	Harvard Apparatus	73-0065
TAM-D transducer amplifier type 705/2	Harvard Apparatus	73-1793
TCM time control module type 686	Harvard Apparatus	731750
Tracheal cannula	Harvard Apparatus	733557
Tube set for moist chamber	Harvard Apparatus	73V83157
Tubing Cassette	Cole-Parmer	IS 0649
Tweezer #5 Dumostar	Kent Scientific Corporation	INS500085-A
Tweezer #5 stainless steel, curved	Kent Scientific Corporation	IND500232
Tweezer #7 Titanium	Kent Scientific Corporation	INS600187
Tygon E-3603 Tubing 2.4 mm ID	Harvard Apparatus	721017	perfusate line entering lung
Tygon E-3603 Tubing 3.2 mm ID	Harvard Apparatus	721019	perfusate line leaving lung
Vannas-Tubingen Spring Scissors	Fine Science Tools	15008-08
VCM ventilator control module type 681	Harvard Apparatus	731741
William's E Media	Gibco, ThermoFisher Scientific	A12176-01	Perfusate additive
Xylazine 100 mg per 1 ml	Akorn