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 Table of Contents  
Year : 2022  |  Volume : 6  |  Issue : 1  |  Page : 14-18

A comparative study of biological sample preparation methods conventional cryopreservation of human tissue

1 Department of Medicinal& Pharmaceutical Chemistry, University of Tripoli, P.O. Box 13645, Tripoli-, Libya
2 Department of Pharmacology, University of Tripoli, P.O. Box 13645, Tripoli-, Libya
3 Department of Medical Laboratories, Faculty of Medical Science and Technologies, University of Tripoli, Tripoli-, Libya

Date of Submission24-Jun-2022
Date of Acceptance02-Jul-2022
Date of Web Publication11-Aug-2022

Correspondence Address:
Dr. Anisa Ali Elhamili
Department of Medicinal and Pharmaceutical Chemistry, University of Tripoli, P. O. Box: 13394, Tripoli
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ljms.ljms_28_22

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Background/Aims: Data that compare the effectiveness of methodologies for cryopreservation of human tissue are very limited. Thus, two different biological sample preparation methods, the conventional cryopreservation of human ovarian tissue using either spontaneous or initiated (so-called “seeded”) ice formation, was carefully investigated and compared. Materials and Methods: Biopsies of ovarian tissue were obtained from women with indications for chemotherapy or radiotherapy, and small pieces of experimental tissue (0.5 mm × 1 mm × 1–3 mm) were randomly distributed into three different groups: group 1 immediately after biopsy, experimental pieces after cryopreservation (thawing) with spontaneous ice formation (Group 2) and cryopreservation with initiated ice formation (Group 3). The pieces of tissue were cultured in vitro for 16 days, after which follicle viability and hormonal activity were evaluated. The level of statistical significance was set at P < 0.05. Results: The obtained results indicated that culture supernatants for Groups 1, 2, and 3 showed estradiol 17-ß concentrations of 476, 465, and 459 pg/mL, respectively. Whereas pProgesterone concentrations were 9.68, 5.77, and 5.61 ng/mL, respectively. In addition, the mean primordial follicle density per mm3 for Group 1 was 12.1 ± 3.9, 3.1 ± 1.4 for Group 2 and 6.0 ± 2.3 for Group 3. Moreover, it was recognized that 91%, 16%, and 87% follicles for Groups 1, 2, and 3, respectively, were normal (P2-1, 3 < 0.05; P1-3 > 0.1). Conclusions: The obtained results revealed that for the best results using cryopreservation of human ovarian tissue, the protocol of conventional cryopreservation must include a step of initiated ice formation. Moreover, an advanced analytical detection technique of high sensitivity, mass spectrometry, can further be used in the future for an accurate determination of hormonal levels and other related compounds and for screening of possible biomarkers using the best-obtained sample preparation method.

Keywords: Biological sample preparation, cryopreservation, efficiency, human tissue

How to cite this article:
Elhamili AA, Saad SE, Eladressi FS. A comparative study of biological sample preparation methods conventional cryopreservation of human tissue. Libyan J Med Sci 2022;6:14-8

How to cite this URL:
Elhamili AA, Saad SE, Eladressi FS. A comparative study of biological sample preparation methods conventional cryopreservation of human tissue. Libyan J Med Sci [serial online] 2022 [cited 2022 Sep 26];6:14-8. Available from: https://www.ljmsonline.com/text.asp?2022/6/1/14/353689

  Introduction Top

In recent years, almost a total of 678 women were diagnosed with cancer in the United States of America alone.[1] During oncological treatment, there is a high chance of young women to restore their reproductive function via cryopreservation of ovarian tissue before their medical treatment.[2] Normal follicular development and ovulation of fertilizable oocytes after gonadotropins stimulation have been obtained in a woman who was grafted with a biopsy of ovarian tissue that had been previously frozen.[3] Childbirth after cryopreservation of the ovaries is now a reality; by grafting cortical ovarian tissue to an appropriate site after cryopreservation, it is possible to sustain ovulation and pregnancy. Live birth after the grafting of frozen/thawed ovarian tissue has also been achieved both in animals and in human beings.[4],[5],[6] The use of cryopreserved ovarian tissue is not limited to achieving a pregnancy alone; ovarian tissue can also be used for therapeutic purposes. It has been reported that patients with premature ovarian failure after cancer treatment can recover ovarian function after reimplantation of an ovarian cortex biopsy and thus avoid menopausal symptoms.[3],[7] The procedure of sample preparation for ovarian tissue using cryopreservation includes the formation of ice under controlled conditions. In general, this ice formation is artificially initiated in a super-cooled cryopreservation medium (so-called “seeding”) at a certain temperature (usually-6°C–9°C). A previously published data indicated the effectiveness and capability of cryopreservation protocols for isolated follicles and ovarian tissue with spontaneous ice formation, without automatic or manual initiation of the process.[8] Demirci et al. carried out a study in which cryopreservation with spontaneous ice formation was compared to a protocol where initiation of ice formation was semi-automatic; better results were found following semi-automatic seeding. Such data that compare the effectiveness of these two methodologies of ice formation (spontaneous and initiated) for cryopreservation of human tissue are very limited.[8]

This investigation was directed to compare the effectiveness of two different sample preparation techniques for biological objects, conventional cryopreservation of human ovarian tissue using either spontaneous or initiated ice formation. The obtained samples will further be analyzed using advanced analytical detection techniques like, for instance, mass spectrometry for an accurate determination of hormonal levels and other related compounds and also for screening of possible biomarkers.

  Materials and Methods Top

Tissue collection, dissection, and distribution to groups

Informed consent was given by seven patients aged between 21 and 32 (28.1 ± 2.4) years. Ovarian tissue fragments (1–3) were obtained from seven patients before oncology treatment during diagnostic or operative laparoscopies via biopsies of the ovarian cortex. These were transported to the laboratory (Tripoli Medical center and Alzawia Street Hospital) within 22–25 h in special, isolated transport boxes (DeltaT GmbH, Giessen, Germany). These boxes contain a special medium for transportation of ovarian tissue (Brama I, Cryo BioSystem, L'Aigle Cedex, France), which can maintain a stable temperature of between 5°C and 8°C for 36 h. Small pieces of the ovarian cortex (0.7–1 mm) were randomly distributed into three groups: control Group 1 consisted of fresh tissue immediately after receipt of the transport box, Group 2 represented experimental pieces for thawing after cryopreservation with spontaneous ice formation, and Group 3 represent cryopreservation with initiated ice formation. All three groups of tissue were cultured in vitro for 16 days. Six ovarian pieces (OPs) from each patient were used for the experiments, with two pieces allocated to each of the three groups. Fresh OPs from each patient were observed for histology, and the follicles were photographed according to our laboratory's routine protocol. Each OP was cryopreserved in a single cryovial.

Cryopreservation with spontaneous ice formation

The cryopreservation protocol was carried out according to Gosden et al.[4] Standard 1.8 cryovials (Nunc, Roskilde, Denmark) were filled with 1.8 mL of cryopreservation medium containing L-15 Medium (Leibovitz) with L-glutamine + 1.5 M dimethyl sulfoxide (DMSO) +10% serum substitute supplement (SSS, Irvine Sci., St. Ana, CA, USA) and were cooled in ice water (0°C). The OPs were then transferred to the cryovials and held in ice water for 30 min. Cryovials were subsequently placed in an IceCube 14S freezer (SyLab, Neupurkersdorf, Austria), with the freezing chamber previously stabilized at 2°C for 20–30 min. The cryopreservation program was as the following: starting with the temperature of 2°C; this temperature was used because the temperature of the cryovials increased from 0°C to 2°C after their manipulation and transfer from ice-water to the auto-seeding block. Samples were then cooled from 2°C to 6°C at a rate of −2°C/min and then held at −6°C for 10 min. This was followed by cooling the samples from −6°C to 40°C at a rate of −0.3°C/min; and cooling to −140°C at a rate of −10°C/min, followed by plunging of cryovials into liquid nitrogen.

Test of auto-seeding equipment

An IceCube 14S freezer was used for conventional cryopreservation of OPs because this freezer incorporates a feature that initiates ice formation (seeding) automatically. The auto-seeding block was specially developed for this study and was a novel piece of equipment [Figure 1]. The location of this block in the freezer chamber guarantees full contact of the upper part of the cryovial wall with the tube that conducts liquid nitrogen so that ice formation is initiated at a certain temperature. The reliability of the auto-seeding block was tested before starting the cryopreservation experiments. Cryoprotectant solution was cooled from room temperature 22°C to 40°C, with seeding at −6°C. At this temperature, the liquid nitrogen begins to enter and cool the conducting tubes, and these tubes cool the cryovials containing the cryopreservation medium. Twenty-eight cryovials with 1.8 mL cryopreservation medium were placed in the auto-seeding block. The freezer has two identical control electrodes: one electrode to monitor the chamber temperature and a second one to monitor the probe temperature. The probe electrode was placed in the vials to monitor the temperature of the cryopreservation medium. This electrode detected cryovial temperature in four different places (positions 1, 2, 3, and 4), as shown in [Figure 1]. Eight experiments were carried out with cryopreservation in two replicates: four experiments using 28 cryovials in two replicates (with four different positions 1, 2, 3, and 4 of a probe electrode). The cryovial temperatures in positions 1, 2, 3, and 4 were measured as + 5, 0, −2, and −5°C, respectively, using a Testo 950 electrical thermometer (Testo AG, Lenzkirch, Germany). The formation and growth of crystals were dependent on the position of the cryovial in the seeding block and were monitored visually through special openings in the cylinders containing the cryovials. Using these openings, it was also possible to initiate seeding manually. The following parameters to measure the reliability of auto-seeding were carefully considered:
Figure 1: Auto-seeding block of the freezer, arrows with 1, 2, 3, and 4 show different positions, at which the temperature of cryovials was measured. Black arrow: shows the direction of liquid nitrogen flow into and from the cooling elements

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  1. Start of seeding relative to the position of the cryovial in the seeding block
  2. Rate of decreasing or increasing the temperature after the beginning of crystallization relative to the position of the cryovial in the seeding block.

Cryopreservation with auto-seeding

This protocol differs from the protocol with spontaneous ice formation only by the initiation of Ice formation using an auto-seeding block, and the cryopreservation program was as described above.

Thawing and removal of cryoprotectants

The two groups of OPs were thawed using an identical procedure: The vials were held for 30 s at room temperature followed by immersion in a 100°C (boiling) water bath for 60 s, and the contents of the tubes were expelled into solutions for the removal of cryoprotectants. The exposure time in boiling water was visually controlled by monitoring the presence of ice in the medium; as soon as the ice was only ~ 2 mm from the apex, the tube was removed from the boiling water. The final temperature of the medium after warming ranged between 4°C and 10°C. After thawing, OPs were transferred within 5–7 s to a 100 mL specimen container (Sarstedt, Nuemrecht, Germany) containing 10 mL of solution for removal of cryoprotectants (0.75 M sucrose +10% SSS +L-15 medium). The cryoprotectant dilution steps were prepared using the same principle as that used for saturation by ethylene glycol. The container was placed on a shaker and continuously agitated at 200 osc/min for 15 min at room temperature. The tissue was rehydrated for 30 min at room temperature using the same stepwise dilutions starting in 50 mL of holding solution (L-15 medium +10% SSS) in a 50 ml tube (Greiner Bio-One GmbH, Frickenhausen, Germany). Drops of holding medium were slowly added to the solution of sucrose with OPs. The final sucrose concentration was 0.125 M. Finally, the OPs were each washed three times in Dulbecco's phosphate-buffered saline supplemented with 10% SSS. After warming and washing, the OP was transferred into the culture system.

Culture, hormonal assays, and histological examination

Pieces of Group 1, 2, and 3 were placed into 200 mL dishes with 30 mL of AIM-V® medium (Gibco, Grand Island, NY, USA) for suspension culture (Cellstar™, Greiner Bio-One GmbH, Frickenhausen, Germany). These were cultured in vitro for 16 days at 37°C in 5% CO2 with 75 osc/min agitation using a rotation shaker. Replicate cultures were studied by culturing two OP in each dish, each piece in the dish being from the same patient, having had the same treatment. The viability of the tissue was evaluated by assaying in vitro production of hormones during culture and the development of follicles after culture. The spent medium after culture of cryopreserved and fresh OPs was collected every second day (40 μL per collection from two cultured OP from each patient) and stored at– 80°C for 1 to 2 months for subsequent hormone assays. Before measurement of hormones, frozen samples were thawed, and culture media of respective in vitro culture days from each of the seven patients were pooled together. The level of estradiol 17-ß (E2, analytical sensitivity <10 pg/mL) and progesterone (P4, analytical sensitivity < 0.1 ng/mL) was measured in 280 μL medium (40 μL medium from severn patients) using Architect chemiluminescence microparticle immunoassay (Abbott Diagnostic, Wiesbaden-Delkenheim, Germany).

For histological investigation, tissue pieces were fixed in Bouin solution for 22–24 h at room temperature, embedded in paraffin wax, serially sectioned at 5m, stained with hematoxylin/eosin and analyzed under a microscope Zeiss (×400). The number of viable and damaged follicles was counted. To avoid overcounting the same follicles, only sections in which a complete oocyte nucleus was fully visible were evaluated for follicle counts. The follicles were evaluated by considering the parameters previously described by Dittrich et al.[9]

Two types of follicles were evaluated: primordial follicles surrounded by a single layer of flat cumulus cells and primary follicles, which are similar to primordial follicles but surrounded by 1–2 layers of spheroidal granulosa cells. The quality of the follicles was graded from 1 to 3. A follicle of Grade 1 is spherical, with granulosa cells randomly distributed around the oocyte. A follicle of Grade 2 has the same features, but granulosa cells do not cover the oocyte regularly. A follicle of Grade 3 has partially or fully disrupted granulose. The follicles of Grades 1 and 2 were classified as normal, whereas those of Grade 3 were classified as degenerate.

  Results Top

The obtained results using cryopreservation with spontaneous ice formation indicated that when the temperature of the cryopreservation medium reached − 17°C ± 1.5°C, a sharp increase in temperature to −5°C ± 1.5°C was observed. After this increase, the cooling rate was stabilized to the normal −0.3°C/min for 30 min [as shown in [Figure 2]]. During this 30 min, the rate of temperature decrease was changed from −1°C/min (for 10 min) to the given decreasing rate of −0.3°C/min. In the test of auto-seeding equipment, the growth of crystals was observed at −7.7°C ± 0.2°C in all four positions of the cryovials in the two replicates (224 cryovials were used). It was established that the process of crystallization of the cryopreservation medium begins and proceeds independently of the cryo-vial position in the seeding block [Figure 1]. The difference in temperature in the different cryovials was additionally monitored in positions 1, 2, 3, and 4 at + 5°C, 0°C,–2°C, and–5°C, respectively, using thermometer Testo 950; the measured temperature difference was not more than 0.2°C. A decrease in the chamber temperature was noted after the beginning of auto-seeding due to the cooling of the liquid nitrogen conductor. This decrease in temperature had no effect on the temperature of the cryopreservation solution [Figure 3]. During cooling, the cover of the freezing chamber was also deliberately opened for 7 s, in order to mimic additional manipulations in the chamber, as in the case of manual seeding. This operation produced no increase or decrease in the temperature of the cryopreservation medium [Figure 3].
Figure 2: The freezer display, (1) program of cooling, (2) auto-seeding temperature, (3) temperature of chamber, (4) temperature of freezing medium, (5) decrease in chamber temperature after starting of auto-seeding, (6) decrease in chamber temperature

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Figure 3: Spontaneous ice-formation. (1) Temperature of freezing medium, (2) beginning of spontaneous ice formation, unbroken line shows zone of programmed (−0.3°C) speed of cooling, broken line shows zone of elevated (−1 to − 0.5°C/min) speed of cooling

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The obtained level of hormones in the native AIM-V® medium was determined as E2 <10 pg/mL and P4 < 0.1 ng/mL. After the culture of Groups 1, 2, and 3 the supernatants showed estradiol 17-ß concentrations of 476, 465, and 459 pg/mL, respectively and progesterone concentrations of 9.68, 5.77, and 5.61 ng/mL, respectively (P1-2,3 < 0.05; P2-3 > 0.1). The obtained hormonal level is shown in [Figure 4]. In addition, according to the histological examination, the only follicles classified as viable were primordial. All preantral and antral follicles after in vitro culture were degenerate, and these follicles were not counted. It was also noted that the procedure of initiated ice formation had a positive effect on follicle viability. The mean follicle density and number classified as normal (Grades 1 and 2), as well as morphological appearance, are shown in [Table 1] and [Figure 5].
Figure 4: The estimated concentration of 17-ß estradiol and progesterone hormonal level

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Figure 5: Micrographs of ovarian tissue from the same patient. (1 and 2) Histology of fresh OP, (3) light micrograph after cryopreservation with spontaneous ice formation, (4) light micrograph after freezing with initiated ice formation, (5) histology of OP after cryopreservation with spontaneous ice formation, (6) histology of OP after cryopreservation with initiated ice formation. OP: Ovarian piece

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Table 1: Comparison of cryopreservation of ovarian tissue with spontaneous and initiated ice-formation, effect on follicle density and normality

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  Discussion Top

Based on an original protocol of cryopreservation by Demirci et al.,[8], two cryoprotectants, DMSO and propylene glycol, were tested at different concentrations of 1, 1.5, and 2 M using 2 M DMSO. A previous finding indicated that the cryopreservation protocol is more effective with initiated ice formation (manual seeding) than with semi-automatic seeding. The obtained results presented here support this approach. The cryopreservation protocol used in this study was performed in three steps: (1) step-wise dilutions for removal of permeable cryoprotectants; (2) saturation with DMSO at 0°C, and (3) rapid warming of tissues at 100°C. The step-wise addition and removal of cryoprotectants were used because human cells are sensitive to osmotic changes that accompany saturation with permeable cryoprotectants and their removal.[9],[10],[11],[12],[13],[14] The effects of the different treatments on the parameters assessed were evaluated using (ANOVA), and the level of statistical significance was set at P < 0.05.

  Conclusions Top

Two different sample preparation protocols for human tissue samples were evaluated and compared. The obtained results indicated that the optimal recovery of viable follicles and hormonal levels after cryopreservation and thawing of human ovarian tissue was obtained by using a protocol of conventional cryopreservation that includes a step of initiated ice formation.


The authors would like to acknowledge the University of Tripoli, Al Zawia Street Hospital and Tripoli Medical Centre.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

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Gosden RG, Baird DT, Wade JC, Webb R. Restoration of fertility to oophorectomized sheep by ovarian autografts stored at -196 degrees C. Hum Reprod 1994;9:597-603.  Back to cited text no. 4
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  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]

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