Xenotransplantation of cryopreserved composite organs on the rabbit

Abstract

Nowadays, It is easy to define optimal conditions (cryoprotective agent, speed and steps of freezing, speed of warming) for the cryopreservation of a homogeneous cell population or a one cell-layer tissue. Meanwhile, It is still hard to obtain cryopreservation of composite organs. Each tissue has its own requirements and its own reactivity to the cryopreservation process. The challenge consists of, on the one hand, to select the ideal combination of cryoprotective agents that can fit the needs of the different tissues, and the definition of adequate technical parameters, on the other hand. All the experimental trials have studied the survival rate of non-vascularized cryopreserved tissues. The aim of our experimental work is to demonstrate the feasibility of cryopreserving a composite organ with its nutrient vessels "artery and veins" in order after thawing to revitalize it by reestablishing the blood irrigation by microsurgical vascular anastomosis. We report our experimental results on the cryopreservation of composite organs-amputated digits-xenotransplanted in the rabbit. Digital segments were cryopreserved, then revitalized after warming using vascular microsurgical techniques. Preliminary results are encouraging and may pave the way in the future to the microvascular allotransplantation of cryopreserved composite organs.

Keywords: animals; composite tissues; cryopreservation; digits; microsurgery; organs; skin; vascular anastomoses; vessels; xenotransplantation.

Figure 1

Recovered xenografts. (A) An index severed in zone II from a hand laborer, which did not fulfill conditions allowing microsurgical revascularization. (B) A stiff finger that hindered functioning of the hand was removed during a scheduled operation.

Figure 2

After recovery of the finger, the dominant palmar collateral artery was catheterized and perfused abundantly with Custodiol^® solution until flow through from the vein was clear.

Figure 3

Double walled container “Cryokit^®” used for packaging the xenotransplant allowing a safe and constant hibernation during transport.

Figure 4

After intra-arterial administration of the cryoprotector, the finger was placed in a specially adapted, three-vent bag and immersed in the cryoprotective solution with no air bubbles.

Figure 5

The rabbit was used as an animal model. Xenograft implantation was performed in the cervical region with vascular attachment to the carotid artery and the jugular vein.

Figure 6

The dominant collateral artery was again catheterized and the finger abundantly rinsed with physiological serum until tissues recovered a normal consistency. Heparin was administered intravascularly to avoid non-reflow phenomenon.

Figure 7

Following microsurgical vascular attachment, the xenograft was positioned around the neck and secured with sutures at both extremities.

Figure 8

The rabbit was placed in a cage with its head immobilized to avoid inopportune movement. A perfusion was placed in the auricular vein to administer anticoagulants and fibrinolytics.

Figure 9

(A) End-to-end arterial attachment of the dominant palmar artery of the finger to the rabbit's carotid artery. (B) Upon release of the arterial clamps, xenograft revascularization was immediate, with appearance of a venous flow-through. However, the finger became rapidly marbled and the capillary pulse was hardly perceptible.

Figure 10

Low power histology sections showed that cutaneous, hypodermal, tendon, neural and vascular tissues retained their normal architectures.

Figure 11

At higher magnification one sees spaces at the dermal-epidermal junction that indicate the onset of a foreseeable epidermolysis.

Figure 12

At very high magnification vascular thrombosis is totally absent from both venules and arterioles.