Fig. 34.1
Intraoperative defatting of the parathyroid tissue
Fig. 34.2
Fragmentation of the parathyroid tissue
Fig. 34.3
Transportation to the laboratory in a tube with Dulbecco’s modified Eagle medium
In most institutions, the final preservation medium used is the Roswell Park Memorial Institute solution (RPMI) 1640, at concentrations ranging between 60 and 80 %, with the addition of 2 mM of glutamine and 5 μg/ml of penicillin streptomycin or 50 μg/ml of gentamicin [9]. Between 40 and 60 particles of 1–2 mm3 explants are distributed in various vials containing that medium [3]. It is estimated that each vial contains about 15 particles of tissue, and the equivalent of two normal parathyroid glands, weighing 15–30 mg each, should be implanted [10]. Other authors recommend the use of Dulbecco’s modified Eagle medium as final preservation medium [7, 11]. Dimethyl sulfoxide (DMSO) at a concentration of 10 % is added to the medium as a cytoplasmic stabilizer, as well as 10–30 % of autologous serum.
Regarding the cooling process, almost all authors agree that vials should be placed in a programmable freezer, that decreases the temperature from −1 to −80 °C. Once they have cooled, the vials are transferred to a liquid nitrogen freezer, where they reach a final temperature of −170 to −196 °C [2, 12].
As to the manner of performing cryopreservation and its timing, there is no universal protocol. An experience carried out by Santa Ritta Barreira et al. did not show changes in the ultrastructure of parathyroid samples stored for periods of up to 12 h in Dulbecco’s medium (DMEM) before cryopreservation [8]. However, it is recommended to run the process as soon as possible after obtaining the tissue sample in the operating room. Several authors have found bacterial contamination in up to 23 % of samples; this reinforces the argument in favor of a swift processing [13].
As to the thawing and preparation process, either for a structure and functionality “in vitro” experiments or for autoimplantation in a patient, the cryovials, according to the number of fragments needed, are quickly thawed in a water bath at 37 °C. Then they are washed three times with Hank’s balanced salt solution (HBSS, Sigma®) at 37 °C, to remove the DMSO, before processing or implanting it under sterile conditions [14].
Assessment of Cell Viability and Parathyroid Function After Cryopreservation
The possibility of cryopreserving animal parathyroid cells preserving their morphology was demonstrated by Blumenthal and Walsh in 1950 [15]. Since that first experience, many authors have explored the possibility of stimulating “in vitro” and autotransplanting parathyroid tissue that had been cryopreserved for various time periods. But it was Brennan who for the first time showed that “in vitro” PTH suppression in the presence of high calcium concentrations in cryopreserved human parathyroid cells could be a good indicator of cell vitality and functionality before autotransplantation [15].
Therefore, two aspects should be considered. One is the preservation of cell function and structure after various periods of cryopreservation. Another is finding whether there is a maximum period of cryopreservation after which histological and functional characteristics begin to deteriorate.
Herrera, Grant, and Van Heerden published their experience in 1992, and showed that parathyroid cell morphology and functional activity were maintained, regardless of the duration of cryopreservation, up to a period of 24 months [9].
In 1997, Mc Henry, Stenger, and Calandro cryopreserved bovine tissue for periods of 1–52 weeks, comparing the number of viable cells per mg of cryopreserved tissue and the release of cytosolic calcium and parathormone in baths with extracellular calcium concentrations ranging from 0.5 to 3 mM. They compared a group of cryopreserved fragments of 1–2 mm3 with another group of cells dispersed after treatment with collagenase. They concluded that cryopreservation, regardless of its duration, decreased the number of viable cells, and the functional response was better for tissue preserved as fragments than as dispersed cells [4].
In 2005, Cohen et al. observed that no parathyroid samples autotransplanted in humans were functional after a cryopreservation period longer than 22 months. Marlon Guerrero et al. attempted to confirm such findings “in vitro” in 2008; among 501 samples cryopreserved between 1991 and 2006, they selected 106 at random (21 %), which were heated, centrifuged, and suspended in a medium containing 90 % of bovine serum and 10 % of DMSO, and finally stained with trypan blue to assess cell viability. Results obtained validated Cohen’s observations, since samples cryopreserved for less than 24 months exhibited a 71 % viability, compared to 1 % seen in samples preserved for longer periods [9]. The strength of this study is that it examined a period of 15 years of cryopreservation; that is also its weakness, since preservation techniques have probably changed throughout that time.
Also, Alvarez-Hernandez et al., in 2008 published an investigation with samples, both fresh and cryopreserved, cultured during 60 h, obtained from 18 patients with secondary hyperparathyroidism. Cell viability was similar in fresh and cryopreserved samples after culture. However, cryopreserved samples exhibited worse secretory response of intact PTH (10 %) than fresh samples (60 %) when exposed to a medium with 0.6 mM of calcium compared to one with 1.2 mM. As to the inhibitory response of PTH when exposed to a medium with calcitriol, it was similar in both groups. Hence, the authors propose to use this test to select cryopreserved samples during the process of “in vivo” implantation [7].
In a very successful series of metachronous autotransplantation of tissue cryopreserved for an average time of 23 months (see comments on these results later), among 15 samples studied before transplantation only one exhibited a necrosis index of 70 %, whereas 14 exhibited a 100 % cell viability [3].
In our personal experience (unpublished to date), of the 216 samples stored in 2013 in a bank of cryopreserved parathyroid tissue, 15 that were no longer necessary for autotransplantation were examined. The 15 samples had been cryopreserved for 3–11 years, and several fragments of each one were analyzed. All samples were assessed for cell viability using H&E and Tunnel techniques before culture; subsequently they were cultured in a medium with 0.6 mM of calcium to determine secretion of intact PTH, and in another medium with 1.2 mM of calcium and 108 of calcitriol to study calcitriol receptors. A control group as well as a final sample were available to assess post culture viability. Overall results showed that cryopreservation did not alter cell viability, independently of the duration of preservation, and post culture viability was close to 80 %. Most samples were functionally able to secrete intact PTH and exhibited calcitriol receptors.
Later we will further discuss other authors’ opinions related to the cost-effectiveness of maintaining an institutional bank of cryopreserved parathyroid tissue.
Evolution of Parathyroid Autotransplantation
Autotransplantation of fresh or cryopreserved parathyroid tissue is a common resource in modern surgery of thyroid and parathyroid disorders. It was described for the first time in human beings by Lahey [1].
In the case of thyroid surgery, whenever the surgeon has questions regarding vitality of the preserved parathyroid gland, immediate autotransplantation to the muscle cervical tissue is the rule. This approach applies particularly in cases in which dissection of the central compartment is indicated, especially regarding the inferior parathyroid glands, where vascularization is difficult to preserve.
For the treatment of primary hyperparathyroidism due to diffuse hyperplasia, it is usually recommended to preserve half of a well vascularized gland, preferably one of the upper glands (as we have already mentioned, cryopreservation is generally indicated for a potential metachronous autotransplantation).
In chronic renal patients, secondary hyperparathyroidism presents additional problems, and in recent years several lines of work have been proposed: subtotal parathyroidectomy (with less metabolic impact but higher chances of recurrence), total parathyroidectomy without autotransplantation (which offers better chances of cure and relatively low recurrence rates), and total parathyroidectomy with autotransplantation, occasionally with additional thymectomy (here, recurrence rates are 80 % because of the autoimplanted graft). Obviously, the three variants must be accompanied by cryopreservation in case of an eventual deferred transplant. In 2007, a group of German universities started a prospective, randomized, and multicenter trial (TOPAR PILOT trial ISRCTN86202793), comparing the last two options mentioned. The trial has not recruited patients since 2013, and no results have been reported so far [16].
One European survey published in 2013 to assess the conduct of endocrine surgeons in cases of secondary HPT showed very heterogeneous results. Of the 86 surgeons who responded to the survey, 60 % performed more than 50 procedures for HPT per year, but only 37 % operated on more than 16 cases of secondary HPT annually. Although all the surgical procedures described were listed among the answers, differences among the various responders were marked. Among 72.7 % of the surgeons surveyed, immediate autotransplantation was the rule, but only 27.4 % of them performed cryopreservation [17].
The first reference to the use of cryopreserved parathyroid tissue dates from 1977 and was published by Wells, Gunnels, and Gutman, who used it to autotransplant a patient with secondary hyperparathyroidism 6 weeks after parathyroidectomy. Wells himself performed autotransplantation in 6 hypoparathyroid patients following surgery for primary hyperplasia, 2–6 months after the original surgery; functional results were adequate in 5 patients [18].
In 2006 the group of Menezes Montenegro and Ferraz published successful results in two renal patients with postoperative hypoparathyroidism, using tissue that had been cryopreserved for 21 and 30 months respectively. The two patients, followed for 3 and 6 years post autoimplantation, had normal serum calcium values and PTH levels of 37 and 95 μg/ml [18].
Agarwal et al. reported in 2013 the Cleveland Clinic experience with more than 2000 operations for hyperparathyroidism. They cryopreserved samples in 30 % of their population (multiglandular disease and parathyroid reoperations) and in 9 patients (1.5 % of cryopreserved samples and 1 % of the population) they performed deferred autotransplantation 3–22 months after the original operation. All patients normalized their calcemia and PTH levels and most experienced symptomatic improvement [10].
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