Technical Issues

Saxe and coworkers7 explored methodologic aspects of cryopreserving human parathyroid tissue. They quantitated the rate of freezing of parathyroid tissue under a variety of conditions, including placing tissue directly into vapor-phase liquid nitrogen, directly into a -70°C freezer, into a programmable freezer, and into an ethanol bath placed in a -70°C freezer. They demonstrated that when using the inexpensive ethanol bath method, the freezing rate was influenced by the volume of ethanol but not by the number of vials being frozen or by the mass of tissue within each cryopreservation vial (100 mg tissue versus 200 mg tissue per vial). By measuring the in vitro viability of cell suspensions derived from patients' samples maintained as long as 50 months, they concluded that there was no difference in cell viability between fresh tissue and tissue frozen by any technique that used a -l°C/min freezing rate. Wagner and colleagues8 demonstrated increased necrosis of parathyroid tissue frozen at -2°C/min compared with that of tissue frozen at -l°C/min. Saxe and coworkers7 also found no difference in viability among samples from the same patient assessed at varying times after cryopreservation and no difference in viability between cryopreserved adenoma and hyperplastic parathyroid tissue.

Our Current Cryopreservation Technique

Tissue is harvested in the operating room as promptly as possible and placed immediately in chilled, sterile RPMI-1640 culture medium for transport to the laboratory. If medium is not available, saline is a better alternative than water. Under a laminar flow hood, using sterile technique, tissue is bathed in chilled RPMI-1640 in a Petri dish on ice, and adherent fat and capsule are stripped from the parathyroid parenchyma. The tissue is divided (freehand) into cubes, approximately 1 to 2 mm per side. Two solutions are prepared on ice in separate test tubes: 20% (by volume) solutions of autologous serum and of DMSO in RPMI-1640. These can be conveniently prepared by placing 4 mL of RPMI-1640 in each of two test tubes on ice and adding 1 mL of autologous serum to the first and 1 mL of DMSO to the second. If large amounts (several grams) of tissue are to be cryopreserved, 2 mL of serum or DMSO added to 8 mL of RPMI-1640 is suggested.

Before introducing tissue to the vial, 0.6 mL of the 20% serum solution is added to each 2.0-mL cryopreservation vial (Corning, Corning, NY) on ice. Approximately 20 pieces of tissue are added to each cryopreservation vial while the vials are kept on ice. After tissue has been placed in all vials, 0.6 mL of the 20% DMSO solution is added to each vial in the same order so that chilled DMSO is added to tissue that has been chilled by the 20% serum RPMI-1640. Vials are mixed by gentle agitation and then left on ice for 5 minutes. If fewer than 18 vials are to be stored, we now use a Mr. Frosty Cryo 1°C Freezing Container (Nalge, Rochester, NY) by adding 250 mL of chilled isopropyl alcohol (kept overnight in a 4°C refrigerator) to the reservoir and placing the device in a -70°C freezer overnight. At a convenient time the next day, we transfer the vials for long-term storage in a -135°C freezer. If an electric -135° freezer is not available, storage in vapor-phase nitrogen is satisfactory. If more than 18 vials are to be stored, we place the vials in a test tube rack that is placed in a metal pan with internal dimensions of approximately 29 x 18 cm, to which is added 1000 mL of chilled (4°C refrigerator overnight) ethanol. The pan volume-ethanol ratio does influence freezing rate. The pan is covered with aluminum foil and placed in a -70°C freezer overnight.

Our Technique for Thawing the Tissue

A tube of 20% patient's serum (by volume) in RPMI-1640 is prepared as previously discussed and placed on ice.

Cryopreservation vials are removed from the long-term storage freezer and placed in a 37°C water bath. The vials are gently agitated until the ice has nearly completely melted and only a core of frozen RPMI-1640 remains. At that point, the vial is opened and 0.5 mL of freshly prepared serum-RPMI-1640 solution is added. The vial is returned to the water bath and agitated until the ice crystal is completely melted. Then, 0.5 mL of the liquid vial contents is aspirated with a sterile pipette and replaced with 0.5 mL of fresh serum-RPMI-1640. The vial is now placed on ice and the cycle of aspirating vial contents and replacing with fresh serum-RPMI-1640 is repeated three times. The intention is to dilute the DMSO while the tissue remains chilled. After the vial contents have been exchanged four times, the tissue is removed, placed in a fresh container with fresh RPMI-1640, and kept on ice until used.

If the tissue is to be used as an autotransplant, a piece is submitted for bacteriologic culture and for frozen section to confirm its identity as parathyroid. We have given patients perioperative antibiotics prophylactically. Using blunt dissection, several pockets are created in the selected muscle and then observed for hemostasis. In an absolutely dry pocket, 5 to 10 pieces of gland tissue are inserted, and the pocket is closed with nonabsorbable material that can serve as a marker for relocating the pocket if necessary. We believe that a hematoma in the pocket may interfere with revascularization of the tissue fragments.

Typically, 20 pieces of tissue are transplanted. Almost certainly, some cases of graft failure are due to transplantation of insufficient viable tissue. At present, however, there is no rapid method for assessing the function of cryopreserved tissue on a "per unit mass" basis and no practical method, therefore, for determining the correct number of pieces to be placed in the muscle pockets.

Variations of Cryopreservation

Variations on this technique of cryopreservation have been introduced periodically. Basile and colleagues9 reported successfully cryopreserving rat parathyroid tissue and, in a single case, human parathyroid tissue using Waymouth's solution in place of RPMI-1640 and placing cryopreservation vials with tissue into a -80°C freezer (without chilled ethanol) for 16 hours before long-term storage in liquid nitrogen.

Kapur and associates10 placed rat parathyroid tissue in 15% DMSO-Hanks basic salt solution in a -20°C freezer for 24 hours and were able to demonstrate function in 7 of 10 animals that underwent autotransplantation with cryopreserved tissue.

The rates of viability of cryopreserved parathyroid tissue in vitro and in vivo are displayed in Table 60-1.

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