Preparation of Decellularized Kidney Scaffolds in Rats

Summary

This protocol introduces a method to develop a scaffold using decellularized rat kidneys. The protocol includes decellularization and recellularization processes to confirm bioavailability. Decellularization is performed using Triton X-100 and sodium dodecyl sulfate.

Abstract

Tissue engineering is a cutting-edge discipline in biomedicine. Cell culture techniques can be applied for regeneration of functional tissues and organs to replace diseased or damaged organs. Scaffolds are needed to facilitate the generation of three-dimensional organs or tissues using differentiated stem cells in vivo. In this report, we describe a novel method for developing vascularized scaffolds using decellularized rat kidneys. Eight-week-old Sprague-Dawley rats were used in this study, and heparin was injected into the heart to facilitate flow into the renal vessels, allowing heparin to perfuse into the renal vessels. The abdominal cavity was opened, and the left kidney was collected. The collected kidneys were perfused for 9 h using detergents, such as Triton X-100 and sodium dodecyl sulfate, to decellularize the tissue. Decellularized kidney scaffolds were then gently washed with 1% penicillin/streptomycin and heparin to remove cellular debris and chemical residues. Transplantation of stem cells with the decellularized vascular scaffolds is expected to facilitate the generation of new organs. Thus, the vascularized scaffolds may provide a foundation for tissue engineering of organ grafts in the future.

Introduction

Cell culture techniques are applied for regeneration of functional tissues and organs to replace diseased or damaged organs. Allogenic organ transplantation is currently the most common treatment for irreversible organ damage; however, this approach requires the use of immunosuppression to prevent rejection of the transplanted organ. Moreover, despite advances in transplant immunology, 20% of transplant recipients may experience acute rejection within 5 years, and within 10 years after transplantation, 40% of recipients may lose their transplanted graft or die¹.

Advances in tissue engineering technologies have yielde....

Protocol

This study was approved by the administration of Pusan National University of Medicine and was conducted in accordance with ethical guidelines for the use and care of animals. (certificate no. 2017-119). Prior to any animal studies, institutional approval should be obtained.
NOTE: All surgical and anesthetic instruments/equipment and reagents recommended for successful surgical presentation and imaging of abdominal organs are detailed in Table 1.

1. Preparation procedures for harvesting of rat kidneys

1. In preparation for surgery, place 8-week-old Sprague-Dawley rats (weighing 200–250 g) on a warming p....

Discussion

Various protocols have been used for decellularization of organs and other tissues. The optimal decellularization protocol should preserve the three-dimensional architecture of the extracellular matrix (ECM). In general, such protocols consist of lysing the cell membrane by physical processing or ionic solutions, dissociating the cytoplasm and nucleus from the ECM by enzymatic processing or detergents, and then removing cellular debris from the tissue³. Physical processes include scraping, solutio.......

Materials

Name	Company	Catalog Number	Comments
1 cc syringe (inject probes and vehicle solutions	Becton Dickinson	305217
10-0 ethilon for vessel anastomosis	Ethicon	9032G
25 gauge inch guide needle(for vascular catheters)	Becton Dickinson	305145
3-0 PDS incision closure rat	Ethicon	Z316H
3-0 Prolene incision closure rat	Ethicon	8832H
3-0 silk spool vascular access/ligation in rat	Braintree Scientific	SUT-S 110
4-0 PDS incision closure mouse	Ethicon	Z773D
4-0 Prolene incision closure mouse	Ethicon	8831H
5-0 silk spool vascular access/ligation in mouse	Braintree Scientific	SUT-S 106
Fine Scissors to cut fascia/connective tissue	Fine Science Tools	14058-09
Halsey needle holder	Fine Science Tools	12001-13
Kelly Hemostat for rats: muscle clamp to minimize bleeding when cut	Fine Science Tools	13018-14
Polyethelyne 50 tubing, catheter tubing 100 ft	Braintree Scientific	.023" × .038”
Schwartz microserrefine vascular clamps	Fine Science Tools	18052-01 (straight)
		18052-03 (curved)
Surgical Scissors to cut skin	Fine Science Tools	14002-12
Vannas-Tubingen Spring scissors for arteriotomy	Fine Science Tools	15003-08