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Comparison of the enzymatic efficiency of Liberase TM and tumor dissociation enzyme: effect on the viability of cells digested from fresh and cryopreserved human ovarian cortex

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The aim of this study was to examine the effectiveness of Tumor Dissociation Enzyme (TDE) on the viability of follicles after digestion of fresh and cryopreserved ovarian cortex fragments (OCFs).


Fresh and thawed OCF from 14 patients (29 ± 6 years), sized 20 to 210 mm3 were randomly distributed into four treatment groups and digested with 16% TDE or 0.05 mg/ml Liberase TM: Group 1, frozen OCF digested with TDE; Group 2, frozen OCF digested with LiberaseTM; Group 3, fresh OCF digested with TDE; and Group 4, fresh OCF digested with Liberase TM. Evaluation of follicle viability was performed under light microscope after staining with Neutral red. For visualization of viable and dead cells under a confocal laser scanning microscope, the follicles were stained with Calcein AM and ethidium homodimer-1.


The results showed that the number of retrieved follicles was significantly higher (990 vs 487; P < 0.01) in the TDE-treatment group compared to the Liberase TM-group. The presence of intense neutral red stained follicles was significantly higher in Group 1 and Group 3 compared to Group 2 and Group 4 (70.3% ± +/− 6.22 vs 53,1% ± 2.03 and 94.2% ± 6.6 vs 79.1% ± 2.1; P < 0.01). The percentage of Calcein AM stained follicles of class V1 was significantly higher in Group 1 and Group 3 compared to Group 2 and Group 4 (95.97% ± 7.8 vs 87.87% ± 2.4; 97.1% ± 6.8 vs 91.3% ± 2.3; P < 0.01).


The enzymatic digestion of ovarian cortex with TDE provides recovery of a higher number of healthy preantral follicles in contrast to earlier described Liberase TM procedure.


Over the last decades, research in medicine has made a great progress in cancer therapy. Long-time survival of cancer patients has increased significantly [1, 2] and after convalescence many patients can lead normal lives. However, highly aggressive and gonadotoxic chemotherapy and radiotherapy have strong side effects such as premature ovarian failure and infertility [3, 4]. At present, the only possibility to maintain fertility as well as hormonal cyclicity and endocrinological function is the cryopreservation and autologous re-transplantation of ovarian cortical fragments taken before chemotherapy or radiotherapy. This method has resulted in large number of live births worldwide [5, 6].

However, this procedure runs the risk of re-transplanting of malignant cells, which makes it unsuitable for patients suffering from neoplastic diseases whose malignant cells are likely to metastasize into the ovary. Cancers that are considered to have a high risk for ovarian metastases are leukaemia, lymphoma, carcinoma coli and cervical as well as uterine cancers [7,8,9,10]. In the same time, the re-transplantation of isolated primordial follicles could be an alternative to safe fertility restoration in women with premature ovarian failure [11,12,13]. It is possible because the basal lamina surrounding the follicles separates them from the surrounding ovarian stroma, blood vessels and nerves, which prevents the invasion of metastatic cells [14]. Therefore, primordial follicles can be isolated and used for the generation of a transplantable artificial human ovary [15, 16]. Construction of an artificial ovary could be the optimal way of preserving fertility as well as protecting female cancer patients, against the re-transplantation of malignant cells, independent from type of neoplastic diseases [9].

This possibility was shown by Laronda et al. [17]. It was demonstrated the real possibility for successful re-transplantation of follicles seeded with scaffolds to SCID-mice with subsequent pups born through natural mating. Successful isolation of preantral follicles from cryopreserved tissue is especially important in global practice: a large amount of ovarian tissues have been already conserved from patients with different types of cancers. According to data, preantral follicles can be successfully cryopreserved before or after their isolation without impairing their ability to survive and grow in vitro [18, 19].

The important step of the artificial ovary creation from preantral follicles is the isolation of these follicles from fresh or cryopreserved (frozen and thawed) ovarian tissues.

In fact, ovarian stromal cells play an important role in the process of development of preantral follicles. According to Young and McNeilly [20], the thecal cells around the secondary preantral follicles appear to be recruited from the surrounding stromal tissue layer by factors that are secreted by activated primary follicles. The growth and development of secondary follicles is affected by steroid hormones that are produced by interactions between granulosa and thecal cells [21, 22]. Ovarian medulla is composed by dense fibrotic stroma consisted from spindle-shaped cells (fibroblasts) and follicles of different stages of maturity [23]. Development of follicles, their ovulation and luteinisation are finely controlled by physiologic hormonal milieu. Therefore, stromal cells of adult ovary actively contribute to the synthesis and phase remodelling both extracellular matrix and blood vessels [23]. Ovarian stromal and endothelial cells are essential to ensure graft survival and quality [24].

In fact, two arteries (arteria ovarica and the branch of the arteria uterina) penetrate the ovarian hilus and form network of blood vessels [23]. They are an opening port for malignant cells [24] which could preferentially generate special kind of stroma cells for aberrant proliferation and invasion [24]. In this case the including of stromal cells by creating of an artificial ovary plays important role only for healthy patients, because of the risk of transfer of malignant cells included in ovarian stroma [7, 8]. The fibroblasts and some other cell types (for example, pre-vascular cells and mesenchymal stem cells) potentially include cancer-associated fibroblasts [25].

At present, for isolation of preantral follicles from human and animal ovarian cortex, a number of mechanical, enzymatic and a combination of both methods have been described [26,27,28,29,30,31,32,33,34,35,36,37,38,39,40].

The human ovarian tissue has a relatively high density and the current mechanical isolation of follicles demonstrates unsatisfactory performance. However, the combination of mechanical isolation technique with enzymatic digestion significantly improved the viability of isolated follicles [36, 41, 42].

Our own experience supports the effectiveness of the use of a commercial enzyme -cocktail “Tumor Dissociation Enzyme Reagent” (TDE, Innovative Diagnostic Systems Dr. Christian Sartori, Hamburg, Germany) for enzymatic digestion of gynaecological solid tumors with good viability and developmental rate of cancer -and non -cancer cells (non-published data).

This especially designed commercial enzyme - cocktail was developed for gentle enzymatic digestion of solid tumors [43,44,45] and allows to isolate tumor cells without appearance of apoptosis or necrosis. Despite the highly secured composition of TDE due to patent [46], it was discerned that the contents of this drug are various types of highly purified collagenase (personal communication with Dr. Christian Sartori Labor, Hamburg, Germany) that allowed the digested tissue suspensions to be not sticky and not viscous, and easy to handle and thus, can obtain a good number of viable cells.

The aim of our experiments was to study the Tumor Dissociation Enzyme effects on the integrity and viability of cell -complexes (follicles) after digesting of these cells from cryopreserved ovarian cortex.


Except where otherwise stated, all chemicals were obtained from Sigma (Sigma-Aldrich Chemie, GmbH, Schnelldorf, Germany).

Tissue collection, dissection, and distribution into groups

This study was approved by the Ethics Boards of Cologne University (applications 99,184 and 13–147).

Written informed consents were obtained from all study participants aged 18 and over.

Tissues were obtained from 14 patients aged between 22 and 39 (29.1± 5.9) years. According to our approved protocol, 10% of ovarian tissues (ovarian tissue biopsies, OTBs) collected from patients were used for ‘patient-oriented’ research. This refers to research done in order to assess the viability of the tissues for re-transplantation.

The patients with following diseases were indicated: breast cancer (3 patients), Hodgkin lymphoma (2 patients), non-Hodgkin lymphoma (2 patients), acute lymphoblastic leukemia (3 patient), uterine cancer (1 patient), nasopharyngeal cancer (1 patient) and soft tissue sarcoma (2 patient). For our research, the OTBs from three patients were used after surgery without cryopreservation, and OTBs for patient-oriented research from eight patients were cryopreserved long before these experiments.

The medium used for OTBs transport and dissection (the basal medium) was composed by Leibovitz L-15 with 5% Dextran Serum Substitute (Irvine Scientific, Santa Ana, USA). After collection, fresh ovarian tissue fragments were transported at 32 °C to 34 °C to the laboratory within 20 min of surgery.

Using sterile surgical tweezers and no. 22 scalpels, the medullary part of the fragment was removed to achieve ~ 1 mm thickness of the cortical part. A small piece of fragment (~ 1 mm3) was fixed in Bouin solution for histological evaluation and served as a fresh control. Depending on the size of ovarian fragment intended for this research, the size of OCFs for patient-oriented research was ranged from 20 to 210 mm3 (Table 1). Each OCF from each patient was cut into two equal parts and cooled to 5 °C for 24 h. After this, the cooled OCF was enzymatically digested or frozen (one OCF per cryo-vial) with subsequent storage in liquid nitrogen until thawing and use. Before each enzymatic treatment, the OCF was weighted using analytical balance.

Table 1 Total number of follicles isolated from frozen/thawed ovarian tissues after enzymatic digestion with different kinds of enzymes in 14 experiments

Histological examination of ovarian tissue cortex

For histological investigation, the OCFs were fixed in Bouin’ solution, imbedded in paraffin wax, serially sectioned at 5 mm, stained with haematoxylin/eosin, and analyzed under a light inverted stereomicroscope Nikon SMZ1270 (Nikon, Düsseldorf, Germany) under 400× magnification. The following types of preantral follicles were evaluated: (1) primordial follicles composed of an oocyte surrounded by a layer of flattened follicular cells, (2) primary and secondary follicles that are similar to primordial follicles, but in which the oocyte is surrounded by one to two layers of cuboidal granulosa cells. Morphology of the follicles was evaluated on the basis of parameters previously described [47]. The number of viable and damaged follicles was counted. To avoid overcounting of the same follicles, only the section with a visible oocyte nucleus was counted. Normality of follicles was evaluated based on the parameters previously described by Paynter et al. [47]. Three types of follicles were distinguished: Type I follicle is spherical with randomly distributed granulosa cells around the oocytes. The cytoplasm is homogenous with slightly granulated nucleus, in the center of which condensed chromatin is detected in the form of dense spherical structure. Type II follicle is spherical; however, granulosa cells do not cover the oocytes regularly. The oocytes can be flat, and condensed chromatin is not detected in the cytoplasm. Type III follicle has partly or fully disrupted cytoplasm and pyknotic nucleus. The cytoplasm of granulosa cells has damages similar to oocytes. Follicles of Type I and Type II were denoted as normal, and those of Type III were denoted as degenerated.

Tissue cryopreservation (freezing and thawing)

This procedure was performed as published previously [48,49,50,51,52,53,54,55,56,57,58]. In our protocol, we used DMSO and ethylene glycol as cryoprotective cocktail to support a multi-cellular structure of ovarian tissues to protect all types of cells [59]. On the day of freezing, OCFs were placed for 30 min at room temperature in a 20-ml freezing medium composed of basal medium supplemented with 6% dimethyl sulfoxide, 6% ethylene glycol and 0.15-M sucrose. Then pieces were put into a standard 5-ml cryo-vials (Thermo Fisher Scientific, Rochester, NY, USA) previously filled by 4.5 ml freezing medium and frozen in an IceCube 14S freezer (Sy-Lab, Neupurkersdorf, Austria). The slow cooling profile started at − 6 °C, and the samples were then cooled from − 6 °C to − 34 °C at a rate of 0.3 °C/min. At − 34 °C, the cryo-vials were finally plunged into liquid nitrogen and stored until thawing. The freezing protocol for cryopreservation of this ovarian tissue included an auto-seeding step at − 6 °C.

To thaw the samples, cryo-vials were removed from liquid nitrogen and held for 30 s at room temperature; they were then immersed in a 100 °C (boiling) water bath for 60 s. The exposure time in the boiling water was visually controlled by the presence of ice in the medium; as soon as the ice was 2 to 1 mm apex, the cryo-vial was removed from the boiling water, at which point the final temperature of the medium was between 4 °C and 10 °C. Within 5–10 s after thawing, the pieces from the cryo-vials were transferred to a 10 ml thawing solution (basal medium containing 0.5-M sucrose) in a 100 ml specimen container (Sarstedt, Nümbrecht, Germany). For stepwise dilution of cryoprotectants, the container was placed on a shaker and continuously agitated with 200 osc/min for 15 min at room temperature.

Rehydration of the tissue by stepwise rehydration followed. This was also performed using the same, previously published [5, 48,49,50,51,52,53,54,55,56,57,58] ‘dropping’ methodology: slow addition of basal medium to the solution of sucrose with ovarian pieces. For ‘dropping’, we used 50-ml of basal medium in a 50-ml tube (Greiner Bio-One GmbH, Frickenhausen, Germany). The final sucrose concentration was 0.083-M, resulting in almost isotonic conditions [5, 48,49,50,51,52,53,54,55,56,57,58]. The last step involved three washes in a basal medium for 10 min immediately prior to preparation.

Preparation of fresh and thawed ovarian tissue and isolation of follicles

We compared the influence of the enzyme Liberase TM Research Grade (Thermolysin Medium Concentration, Roche Diagnostics GmbH, Roche Applied Science, Mannheim, Germany) with the Tumor Dissociation Enzyme (TDE) from the DCS ATP-Chemo-sensitivity Assay Kit (DCS Innovative Diagnostik-Systeme, Dr. Christian Sartori Labor, Hamburg, Germany) on the survival of different stages of isolated follicles after enzymatic digestion of fresh and cryopreserved human ovarian cortex.

The TDE (commercially purchased from the Firma Dr. Christian Sartori Labor, Hamburg, Germany) is a commercial enzyme -cocktail for enzymatic digestion of any solid tumors [43,44,45].

All enzyme solutions were prepared on Leibovitz L-15 medium of appropriate concentrations in combination with 50 μg/ml neutral red dye, a vitality dye that shows a deep red colour in acid cell structures for the express- visualization of viable follicles (Fig. 1a, b).

Fig. 1

Example of follicles treated with neutral red dye and live/dead fluorochrome assay. a neutral red positive follicle; b B neutral red negative follicle. Neutral red dye is a viability stain which shows a deep red colour in acid cell structures like lysosomes. c V1, fully viable isolated follicle (granulosa cells as well as oocyte show bright green fluorescence) positive to Calcein AM, negative to ethidium homodimer-1; d V2, minimally damaged follicle with two cells positive (bright red colour) to ethidium homodimer-1; e negative control: dead follicle showing bright red fluorescence (positive to ethidium homodimer-1, negative to Calcein AM); f group of follicles, mostly V1, arrow indicates a follicle that was mechanically disrupted during the manipulation process. Bar = 50 μm

Twenty-two frozen/thawed and six fresh OCFs obtained from 11 patients were divided into four treatment groups: Group 1: 11 frozen OCFs for enzymatic digestion with TDE; Group 2: 11 frozen OCFs for enzymatic digestion with Liberase TM: and two fresh control groups: Group 3: 3 fresh OCFs for enzymatic digestion with TDE; Group 4: 3 fresh OCFs for enzymatic digestion with Liberase TM (Fig. 2).

Fig. 2

Experimental design

The treatment procedure was performed as previously described [60] with some modification. OCFs were mechanically cut into ~ 0.5 × 1 to 1 × 1 mm3 pieces onto a sterile 100 mm Falcon Petri dish (Falcon, Corning Incorporated-Life Science, Corning, USA) pre-cooled on ice to + 4 °C and put onto a cold plate to stabilize the temperature. The cutting of OCFs was performed with quick simultaneous movements of two scalpels no. 22. The ovarian tissue pieces were then suspended in basal medium containing 16% TDE (Group 1 and Group 3) or in basal medium containing 0.05 mg/ml Liberase TM (Group 2 and Group 4). The optimal concentration for digestion with TDE was titrated in previous experiments (unpublished data). Incubation with enzymes was performed in CO2 -incubator at 37 °C on KS 260 basic shaker (IKA, Staufen, Germany) with 100 rotations/min for 75 min. Enzymatic digestion was inhibited by the addition of an equal volume of 4 °C cold Leibovitz L-15 medium containing 20% fetal calf serum (FCS). The cell suspension was immediately put onto ice, gently aspirated up and down with a 1-ml pipette, filtered through 100-μm cell strainers (Falcon, Corning Incorporated-Life Science, NY, USA) and examined by two experienced co-workers under a light inverted stereomicroscope Nikon SMZ1270 (Nikon, Düsseldorf, Germany) in the presence of follicles stained and non-stained with neutral red follicles.

Collection and morphological evaluation of follicles

Isolated follicles were collected with 135 μm V-denuded capillaries (Vitromed GmbH, Jena, Germany) and washed four times in pre-cooled to 4 °C basal medium to discard stromal cells. The number of stained and unstained follicles was calculated for both groups. To identify the isolated primordial follicles, the classification described by Gougeon and Fortune [61, 62] was used: primordial follicle (< 60 μm), oocytes surrounded by a single layer of flattened pre-granulosa cells; primary follicle (> 60 μm - ≤75 μm), oocytes with single layer of cuboidal granulosa cells; secondary follicle (> 75 μm - < 200 μm).

Immediately after isolation of follicles, they were observed under Nikon microscope SMZ25. The follicle diameter was measured by Program Zen (Nikon, Düsseldorf, Germany).

Confocal laser scanning microscopy

The neutral red staining method provides a fairly rough estimation of viability. However, dead granulose cells are difficult to identify [39] and we have chosen a live/dead fluorochrome assay for exact analysis of viability using confocal laser scanning microscopy. To check the viability of cellular structures of isolated ovarian follicles (oocytes surrounded by flat or cubical follicular cells) collected follicles were stained with Calcein AM for visualization of viable cells and ethidium homodimer-1 for visualization of dead cells as previously described by Cortvrindt and Smitz [63]. The follicles were exposed to 2 μM of Calcein AM and 5 μM of ethidium homodimer-1 in Dulbecco’s phosphate buffer saline (DPBS) for 15 min at 37 °C in the dark. During this time, living cells for intracellular esterase activity converted the non -fluorescent cell-permeable Calcein AM in fluorescent Calcein producing an intense bright uniform green fluorescence (ex/em, 495 nm/515 nm). In contrast to Calcein AM, ethidium homodimer-1 shows a red fluorescence when bound to the DNA of dead cells (ex/em, 528 nm/617 nm) (Fig. 1c-f). After exposure to these dyes, the follicles were washed in PBS and visualized using a confocal laser scanning microscope Olympus Fluoview FV 1000 (Olympus, Hamburg, Germany) with a multi-line argon laser (458, 488, 515 nm) to record the fluorescent images. Follicle viability was done as described by Paulini [64], as follows. Viable follicles of class V1: oocytes and all granulose cells are viable; minimally damaged follicles of class V2: the presence of less than 10% dead follicular cells; moderately damaged follicles of class V3: 10–50% dead follicular cells; and follicles of class V4: both the oocytes and all follicular cells are dead.

Confocal images were processed and analyzed using the program Fiji, an open-source platform for scientific image processing, which is an advanced version of the commonly used Image J.

Statistical analysis

The enzymatic digestion of ovarian cortex with two types of enzymes was repeated at least three times on different days. Results were expressed as mean ± standard deviation (SD). Statistical evaluation of flow cytometrical results was performed with the GraphPad Prism 5 software package (GraphPad, La Jolla, USA), applying D’Agostino’s K2 test to assess Gaussian distribution. To evaluate the effect of two different enzymatic treatments on the cellular viability of ovarian cortex (stromal cells and follicles) immediately after digestion, a T-test for equal variances was performed. P-values less than 0.05 were considered statistically significant.

The correlation test (EXCEL – 2010) was performed for multi-variables (recovered follicle number, patient’s age, size and volume of biopsies) to investigate the relationship between these parameters. The value of correlation coefficient (r) was always between + 1 and − 1 inclusive, where 1 is total positive linear correlation, 0 is no linear correlation, and − 1 is total negative linear correlation. A commonly used scale for the interpretation of correlation coefficient is the following: no correlation: r from 0 to 0.1 or from 0 to − 0.1; weak correlation exists: r from 0.1 to 0.3 or from − 0.1 to − 0.3; moderate correlation exists: r from 0.3 to 0.5 or from − 0.3 to − 0.5; and strong correlation exists: r from 0.5 to 1 or from − 0.5 to − 1.


Retrieval rate of isolated follicles

In experiments total of 1477 follicles were isolated from fresh and frozen OCFs (169 follicles were isolated from 6 fresh and 1308 follicles were isolated from 22 cryopreserved OCFs). From fresh biopsies, 122 follicles were recovered using TDE and 46 follicles were recovered using Liberase TM. From frozen biopsies, 868 follicles were recovered using TDE and 440 follicles using Liberase TM (P < 0.05) (Table 1). It was established that the retrieval rate of follicles from the biopsies of patients 22–39 years old has a strong negative (r = − 0.6) correlation with age. However, the relationship between the patient age and number of follicles per 1 mm3 had no correlation (r = 0.06). The correlation between the volume of biopsies and number of retrieved follicles is a moderate positive (r = 0.3). The same moderate correlation (r = 0.4) was found between the volume of biopsies and number of follicles per 1 mm3. The correlation between the weight of biopsies and number of retrieved follicles was very weak (r = 0.1). However, between the weight of biopsies and number of follicles per 1 mm3, medium correlation (r = 0.) was found. Between the age of patient and volume of biopsies or weight of biopsies, the same very weak correlation (r = 0.1 and r = 0.2, respectively) was observed. However, the relationship between the weight of biopsies and its volume has a strong positive correlation (r = 1) (Table 1).

Quantity and morphology of isolated follicles

As shown in Fig. 3a the most of follicles in Group 1 are fully isolated (P < 0.01).

Fig. 3

Typical view of follicle suspension after enzymatic digestion. a follicles isolated from frozen ovarian cortex with TDE-enzyme cocktail; b follicles isolated from frozen ovarian cortex with Liberase TM. The black arrows show the clustered and partially isolated from incompletely digested stroma follicles. Bar = 50 μm

Compared to digestion with TDE, the digestion with Liberase TM (Fig. 3b) has resulted in incomplete tissue digestion (black arrows). Extruded oocytes were found in both treatment groups, but it was extremely rare (< 3%). Note that apart from the good three-dimensional structure, the general morphology of isolated follicles was well maintained independently from the type of enzymatic treatment. After evaluation of isolated follicles under an inverted microscope, it was noted their normal spherical form with mostly one layer of granulosa cells around the oocyte. The data for the quantity of retrieved follicles of different maturity inside each treatment are shown (Fig. 4a). It was also demonstrated that the number of retrieved follicles independently from their maturity was significantly (P < 0.01) higher in the TDE -treatment group in comparison with Liberase TM groups (Group 1[n = 122] vs Group 2 [n = 46], Group 3 [n = 868] vs Group 4 [n = 440]. However, these differences in all treatment groups were no significant (P > 0.1) in the distribution of follicles according to their maturity (Fig. 4b).

Fig. 4

Distribution of follicles according to their maturity in each treatment group. a Percent of retrieved follicles of different maturity inside of each treatment group. b Number of retrieved follicles of different maturity inside of each treatment group. Bars (mean ± SD) with different superscripts in respective treatment group represent significant differences (P < 0.05)

Viability of follicles

The viability assessment of follicles was performed using two techniques: (1) express technique for visualization of live follicles with the application of neutral red dye and subsequent evaluation of follicles under a light inverted stereomicroscope and (2) by fluorescence-staining technique with Calcein AM and ethidium homodimer-1 for visualization of viable and dead cells under a confocal laser microscope.

Express technique for visualization of follicle vitality

Immediately after enzymatic treatment and simultaneous staining with Neutral red dye, the suspension of ovarian stromal cells and follicles were examined under an inverted microscope. Follicles were collected and neutral red -uptake was investigated based on the optical presence of their red staining.

The data on Fig. 5a show that the presence of intense red -stained follicles in the suspension from fresh ovarian tissues digested with TDE was significantly higher than in the suspension of tissues digested with Liberase TM (94.2 ± 6.6% in Group 1 vs 79.1 ± 2.1% in Group 2, respectively).

Fig. 5

Influence of the type of enzymatic treatment of ovarian cortex on the vitality of isolated follicles of different maturity tested applying of Neutral Red dye. a Vitality of follicles in different treatment groups independent of their maturity stage; b Comparison of follicle vitality in Group 1 (fresh ovarian tissues digested with TDE) compared to Group 2 (fresh ovarian tissues digested with Liberase TM) depending on their maturity stage, c Comparison of follicle vitality in Group 3 (frozen ovarian tissues digested with TDE) compared to Group 4 (frozen ovarian tissues digested with Liberase TM) depending on their maturity stage. Bars (mean ± SD) with different superscripts in respective treatment group represent significant differences (P < 0.05)

The percent of light red- stained follicles was significantly higher (P < 0.05) in the suspension of tissues digested with Liberase TM than after digestion with TDE (Group 2 vs Group 1: 16.6 ± 0.6% vs 4.3 ± 0.4%, respectively), while the presence of non -stained follicles was not significantly different in these groups (Group 2 vs Group 1: 4.2 ± 0.3% vs 1.4 ± 0.3%, respectively). The presence of intense red -stained follicles in the suspension of cryopreserved ovarian tissues digested with TDE was significantly higher (P < 0.05) in frozen ovarian tissues (70.3 ± 6.2% in Group 3 vs 53.1 ± 2.0% in Group 4, respectively) than in the suspension of tissues digested with Liberase TM.

The amount of light red -stained follicles was not significantly different (P > 0.1) between Group 3 and Group 4 (17.7 ± 1.5% vs 20.2 ± 1.0%, respectively), while the presence of non-stained follicles was significantly higher (P < 0.05) (12.0 ± 1.3% in Group 3 vs 26.8 ± 0.9% in Group 4, respectively) in the suspension of tissues digested with Liberase TM. The data on Fig. 5b (digested fresh tissue) and Fig. 5c (digested frozen tissue) characterize the vitality of follicles according to their maturity. No significant difference (P > 0.1) according to the vitality of different maturity stages of follicles between treatment groups was found.

Vitality visualisation with confocal laser scanning microscopy

All isolated follicles after assessment of vitality using the Neutral red dye were stained with fluorescent dye to identify the presence of living and dead cells in each follicle. The data on Fig. 6a show that the percent of recovered class V1 follicles in the suspension from fresh ovarian tissues are not significantly different between Group 1 and Group 2 (97.1 ± 6.8% vs 91.3 ± 2.1%, respectively) (P > 0.1). However, a significantly higher rate (96.0 ± 7.8% vs 87.9 ± 2.4%, respectively) (P < 0.05) of recovered follicles of class V1 in Group 3 (cortical tissue suspension digested with TDE) compared to Group 4 was found. The amount of class V2 and V3 follicles in all treatment groups was not significantly different (P > 0.1). Class V4 follicles were absent in all treatment groups.

Fig. 6

Influence of the type of enzymatic treatment of ovarian cortex on the vitality of isolated follicles of different maturity tested using of Calcein AM for visualization of viable cells and ethidium homodimer-1 for visualization of dead cells. a Viability of follicles in different treatment groups. b Comparison of follicle vitality in Group 1 (fresh ovarian tissues digested with TDE) compared to Group 2 (fresh ovarian tissues digested with Liberase TM) depending on their maturity stage, c Comparison of follicle vitality in Group 3 (frozen ovarian tissues digested with TDE) compared to Group 4 (frozen ovarian tissues digested with Liberase TM) depending on their maturity stage. Bars (mean ± SD) with different superscripts in respective treatment group represent significant differences (P < 0.05)

The data on Fig. 6b (digested fresh tissue) and Fig. 6c (digested frozen tissue) characterize the vitality of follicles according to their maturity stage.

No significant difference (P > 0.1) between class V1 primordial follicles was found in ovarian tissues of Group 1 (fresh, TDE-digested ovarian tissues) (58.5 ± 4.2%), Group 2 (fresh, Liberase TM-digested ovarian tissues) (55.8 ± 1.3%), Group 3 (frozen, TDE-digested ovarian tissues) (53.9 ± 4.2%), and Group 4 (frozen, Liberase TM-digested ovarian tissues) (52.1 ± 1.3%).

The presence of class V2 and V3 follicles was not significantly different between all treatment groups, independent of the stage of preantral follicles, (P > 0.1). Class V4 preantral follicles in all treatment groups were absent.

Histological evaluation of fresh and frozen ovarian tissue

Histological evaluation of non-treated pieces of ovarian cortex (fresh control) shows that the most of exanimated follicles (96.8 ± 2.5%) were morphologically normal.

Histologic analysis of haematoxylin-eosin stained ovarian cortical tissues showed morphologically normal preantral follicles. The follicles were surrounded by non-disrupted intact basement membrane. The oocytes were slightly stained, without signs of degeneration or retraction. The percentage of morphologically normal follicles was not significantly different (P > 0.1) between the fresh and frozen ovarian tissues samples (96.8 ± 2.5% vs 97.1 ± 5.1%, respectively).


The latest work in the field of follicle isolation from human and animal ovarian cortical tissue indicates their excellent survival after thawing and the ability to further development both in culture and after xenotransplantation [18, 19, 64, 65]. An important task is isolation of the follicles from ovarian cortex to recover a maximal number of high quality viable follicles.

In this work, we applied our original routine cryopreservation protocol for human ovarian tissue, which presupposes the long-time tissue pre-cooling step before freezing. The reason is our previous establishment that the 24 h cooling to 5 °C before cryopreservation is beneficial for the cryopreservation of human ovarian tissues, especially of follicles [5, 48,49,50,51,52,53,54,55,56,57,58].

Enzymatic digestion is commonly used for tissue dissociation and cell harvesting and offers the advantages of unattended quick sample preparation, potential automation, and is low cost with the possibility to obtain more cells of interest [66,67,68]. The feasibility of enzymatic digestion is a good alternative tissue sample preparation method to the technique of mechanical tissue dissection.

It is well known that the human ovarian cortex possesses a highly dense and fibrous structure. Therefore, using of special enzymatic digestion technique is necessary for effective isolation of the follicles from the surrounding tissue.

The widely used enzyme collagenase is known to degrade connective tissues to allow tissue dissolution and to get the single-celled suspensions. However, enzymatic digestion with the use of collagenase also has serious disadvantages. These disadvantages are well described [69] on the example of hepatocytes.

Thus, it is known that most isolation protocols result in damage of cell junctions, cell membranes, to cell surface receptors and antigens, and cytosolic contents [70, 71].

By enzymatic follicle isolation, the basal membrane disruption occurs often [72]. Collagenase digestion also induces oxidative stress observed 4-8 h after isolation of hepatocytes, leading to a loss of cytochrome enzyme activity [73].

This could be why many isolated preantral follicles degenerate within the first 24 h of in vitro culture and only a few of them could reach the early antral stage [34, 35, 38, 41].

In attempts to standardize the protocol of enzymatic digestion and to improve the quality of isolated follicles, the various types of collagenase (Ia, II, IX, XI) alone [36, 37, 39, 74,75,76] or in combinations with DNA-se [35, 38, 41, 76,77,78] were used. However, it was reported about increased amount of lipid droplets in granulosa cells of isolated follicles (collagenase IX and deoxyribonuclease IV, [35]) and about high amounts of premature oocyte extrusions from the enzymatically isolated follicles (collagenase Type II) [37] that shows that the enzyme collagenase has a batch-to-batch variation in effectiveness [79].

According to the latest data [36, 40, 42, 80], the viability of enzymatically isolated follicles depends on the level of purity, the type of collagenase and on combination with other enzymes, which could reduce its toxicity. Such products are Liberase Research Grade Purified Enzyme Blends and are mixtures of highly purified collagenase and neutral protease enzymes, formulated for efficient, gentle, and reproducible dissociation of tissues from a wide variety of sources (Roche Diagnostics GmbH, Mannheim, Germany).

The works of Dolmans et al. [36], Vanacker [40] and Kristensen [39] have shown the beneficial role of different types of Liberase for follicle isolation during enzymatic digestion. In the work of Lierman [60] both Liberase TM combined with collagenase IV and Liberase DH were shown to be better for isolating high-quality primordial follicles, compared with mono-enzyme collagenase IV. The Liberase TM (Thermolysin Medium, 0.04 mg/ml, Roche Diagnostics GmbH, Mannheim, Germany), we used in our experiments belongs to the group of Liberase Research Grade Purified Enzyme Blends with reduced endotoxin levels and is a mixture of highly purified Collagenase I and Collagenase II with a medium concentration of Thermolysin (a non-clostridial neutral extremely stable Zn-metalloendopeptidase ( formulated for efficient, gentle, and reproducible dissociation of tissues from a wide variety of sources (; Worthington Enzyme Manual, V Worthington Biochemical Corporation). Their activity is directed to break the peptide bonds in collagen (Collagenase), fibronectin, collagen IV, and to a lower extent collagen I, however, it does not cleave collagen V or laminin of neutral protease (V Worthington Biochemical Corporation).

Compared to Liberase TM, TDE is a commercial enzyme -cocktail [46] for enzymatic digestion of any solid tumors [43,44,45]. Due to highly secured patenting [46], we could not obtain the full composition of this enzyme cocktail and we can only assume, according to the quality of the digested material (not sticky, not viscous, and easy to handle), that this cocktail, together with highly purified types of collagenase, may also include enzymes such as protease, dispase or DNA-se.

The comparison of two digestion protocols according to the quality of digestion of stromal tissues showed that TDE allows complete digestion of the stromal tissue with good preservation of follicle integrity. The digested tissue suspension is easy to handle, not sticky and able to obtain a good number of viable follicles. In contrast, the tissue digested with Liberase TM remained poorly dissolved, was sticky and stretchy, and many follicles were still tightly embedded in the tissue and was very difficult to isolate, and this support the finding described [39]. The comparison of the two treatment groups also showed that the number of fully isolated follicles in the TDE groups was significantly higher than in the Liberase TM groups both for fresh and frozen tissues, considering that the size of digested ovarian cortex was similar in both groups.

For evaluation of the quality of recovered follicles from cryopreserved ovarian cortex, we used two techniques: (1) vital staining with neutral red dye for visualization of live follicles and (2) fluorescence -staining of follicles with Calcein AM and ethidium homodimer-1 for visualization of viable and dead cells under a confocal laser microscope.

We have decided to apply the neutral red dye in our experiments, because this vital dye has no deleterious effects on enzymatic activity within cellular organelles [81] and proves to be nontoxic with no long-term negative effects on the follicles [39, 82]. Neutral red (toluylene red, Basic Red 5, or C.I. 50,040) is a eurhodin dye used for many staining methods in histology. Neutral red is used also as a vital stain because the live cells incorporate neutral red into their lysosomes [83]. As cells begin to die, their ability to incorporate neutral red diminishes [84, 85]. This vital dye allows also easy visualization of the follicles in cell suspension. This technique was first applied by Kristensen [39] for follicle visualization and their vitality assessment, and it allows accelerating the process of follicle collection. Our results show that the presence of intense red stained follicles in the ovarian cortical tissue suspension digested with TDE for both fresh and frozen ovarian tissues was significantly higher than in the suspension of tissues digested with Liberase TM.

Visualization of viable and dead cells under a confocal laser scanning microscopy with Calcein AM and ethidium homodimer-1 showed that digestion ovarian tissue with TDE for both fresh and frozen samples provides a significantly higher integrity rate of recovered follicles (class V1) compared to the Liberase TM -groups. The effectiveness of the TDE technique is similar to the data for the Liberase follicles isolated from fresh cortical tissues [36] and with the data for follicles isolated from frozen tissues [18, 86,87,88].

According to the latest research data, the best survival of the cells of an oocyte-granulosa-cells complex immediately after enzymatic isolation of preantral follicles from fresh ovarian tissue with subsequent one-week in vitro culture provides the Liberase DH enzymatic cocktail [40]. Our data presented here with use of TDE commercial enzyme-cocktail for isolation of preantral follicles are not principally different from the results achieved with use of Liberase DH for digestion of fresh ovarian tissue (95% TDE vs 95% Liberase DH, P > 0.5) [40]. The use of highly purified commercial enzyme-cocktails allows eliminating the possibility of obtaining undesirable results through the fault of the drug manufacturer [35, 37, 41, 79]. In fact, there are not principal differences between these two enzymatic cocktails: they content seemingly different types of collagenase.

In the future work we suppose to perform a comparative evaluation of effectiveness of these two commercial enzymatic cocktails (TDE and Liberase DH) with detailed investigation of all apoptotic degenerations (early apoptotic, late apoptotic and necrotic cells) which could take place in enzymatically isolated follicles using specific surface markers for follicular cells by applying flow cytometry and confocal microscopy.


New method for isolation of preantral follicles with tumor dissociation enzyme (TDE) that can be used for the construction of artificial ovary, provides a higher number of healthy preantral follicles from cryopreserved ovarian cortex in contrast with the earlier described Liberase Thermolysin Medium method.



Deoxyribonucleic acid

Liberase TM:

Liberase Thermolysin Medium


Ovarian tissue cortical fragment


24 ovarian tissue biopsies


Phosphate buffer saline Dulbecco


Standard deviation


Tumor dissociation enzyme


  1. 1.

    Abir R, Nitke S, Ben-Haroush A, Fisch B. In vitro maturation of human primordial ovarian follicles: clinical significance, progress in mammals, and methods for growth evaluation. Histol Histopathol. 2006;21:887–98.

  2. 2.

    Feigin E, Freud E, Fisch B, Orvieto R, Kravarusic D, Avrahami G. Fertility preservation in female adolescents with malignancies. In: Cancer in Female Adolescents. Hauppauge: USA Science Publishers Inc; 2008. p. 38–101.

  3. 3.

    Donnez J, Jadoul P, Squifflet J, van Langendonckt A, Donnez O, Van Eyck AS, et al. Ovarian tissue cryopreservation and transplantation in cancer patients. Best Pract Res Clin Obstet Gynaecol. 2010;24:87–100.

  4. 4.

    Wallace WH, Thomson AB, Saran F, Kelsey TW. Predicting age of ovarian failure after radiation to a field that includes the ovaries. Int J Radiat Oncol Biol Phys. 2005;62:738–44.

  5. 5.

    Isachenko V, Todorov P, Isachenko E, Rahimi G, Tchorbanov A, Mihaylova N, et al. Long-time cooling before cryopreservation decreased translocation of phosphatidylserine (Ptd-L-Ser) in human ovarian tissue. PLoS One. 2015;10:e0129108.

  6. 6.

    Donnez J, Dolmans MM. Fertility preservation in women. N Engl J Med. 2017;377:1657–65.

  7. 7.

    Meirow D, Hardan I, Dor J, Fridman E, Elizur S, Ra'anani H, et al. Searching for evidence of disease and malignant cell contamination in ovarian tissue store from hematologic cancer patients. Hum Reprod Oxf Engl. 2008;23:1007–13.

  8. 8.

    Dolmans MM, Marinescu C, Saussoy P, Van Langendonckt A, Amorim C, Donnez J. Reimplantation of cryopreserved ovarian tissue from patients with acute lymphoblastic leukemia is potentially unsafe. Blood. 2010;116:2908–14.

  9. 9.

    Dolmans MM, Jadoul P, Gilliaux S, Amorim CA, Luyckx V, Squifflet J, et al. A review of 15 years of ovarian tissue bank activities. J Assist Reprod Genet. 2013;30:305–14.

  10. 10.

    Rosendahl M, Andersen MT, Ralfkiaer E, Kjeldsen L, Andersen MK, Andersen CY. Evidence of residual disease in cryopreserved ovarian cortex from female patients with leukemia. Fertil Steril. 2010;94:2186–90.

  11. 11.

    Amorim CA, Van Langendonckt A, David A, Dolmans MM, Donnez J. Survival of human pre-antral follicles after cryopreservation of ovarian tissue, follicular isolation and in vitro culture in a calcium alginate matrix. Hum Reprod. 2009;24:92–9.

  12. 12.

    Soares M, Saussoy P, Maskens M, Reul H, Amorim CA, Donnez J, et al. Eliminating malignant cells from cryopreserved ovarian tissue is possible in leukaemia patients. Br J Haematol. 2017;178:231–9.

  13. 13.

    Amorim CA, Shikanov A. The artificial ovary: current status and future perspectives. Future Oncol. 2016;12:2323–32.

  14. 14.

    Rodgers RJ, Irving-Rodgers HF, Russell DL. Extracellular matrix of the developing ovarian follicle. Reproduction. 2003;126:415–24.

  15. 15.

    Luyckx V, Dolmans MM, Vanacker J, Scalercio SR, Donnez J, Amorim CA. First step in developing a 3D biodegradable fibrin scaffold for an artificial ovary. J Ovar Res. 2013;6:83–92.

  16. 16.

    Camboni A, Van Langendonckt A, Donnez J, Vanacker J, Dolmans MM, Amorim CA. Alginate beads as a tool to handle, cryopreserve and culture isolated human primordial/ primary follicles. Cryobiology. 2013;67:64–9.

  17. 17.

    Laronda MM, Rutz AL, Xiao S, Whelan KA, Duncan FE, Roth EW, et al. A bioprosthetic ovary created using 3D printed microporous scaffolds restores ovarian function in sterilized mice. Nat Commun. 2017;8:15261.

  18. 18.

    Vanacker J, Luyckx V, Amorim C, Dolmans MM, Van Langendonckt A, Donnez J, et al. Should we isolate human preantral follicles before or after cryopreservation of ovarian tissue? Fertil Steril. 2013;99:1363–8.

  19. 19.

    Lunardi FO, de Aguiar FL, Duarte AB, Araújo VR, de Lima LF, Ribeiro de Sá NA, et al. Ovine secondary follicles vitrified out the ovarian tissue grow and develop in vitro better than those vitrified into the ovarian fragments. Theriogenology. 2016;85:1203–10.

  20. 20.

    Young JM, McNeilly AS. Theca: the forgotten cell of the ovarian follicle. Reproduction. 2010;140:489–504.

  21. 21.

    Kent J, Ryle M. Histochemical studies on three gonadotrophin-responsive enzymes in the infantile mouse ovary. J Reprod Fertil. 1975;42:519–36.

  22. 22.

    Blanco LZ Jr, Kuhn E, Morrison JC, Bahadirli-Talbott A, Smith-Sehdev A, Kurman RJ. Steroid hormone synthesis by the ovarian stroma surrounding epithelial ovarian tumors: a potential mechanism in ovarian tumorigenesis. Mod Pathol. 2017;30:563–76.

  23. 23.

    Female reproductive system. In. Sorenson RL, Brelje TC, editors. Atlas of human histology. A guide to microscopic structure of cells, tissues and organs. 2014. Minneapolis: University of Minnesota Bookstore; p. 305–28.

  24. 24.

    Furuya M. Ovarian cancer stroma: pathophysiology and the roles in cancer development. Cancers. 2012;4:701–24.

  25. 25.

    Xouri G, Christian S. Origin and function of tumor stroma fibroblasts. Semin Cell Dev Biol. 2010;21:40–6.

  26. 26.

    Figueiredo JR, Hulshof SC, Van den Hurk R, Ectors FJ, Fontes RS, Nusgens B, et al. Development of a combined new mechanical and enzymatic method for the isolation of intact preantral follicles from fetal, calf and adult bovine ovaries. Theriogenology. 1993;40:789–99.

  27. 27.

    Carambula SF, Goncalves PB, Costa LF, Figueiredo JR, Wheeler MB, Neves JP, et al. Effect of fetal age and method of recovery on isolation of preantral follicles from bovine ovaries. Theriogenology. 1999;52:563–71.

  28. 28.

    Amorim CA, Lucci CM, Rodrigues AP, Carvalho FC, Figueiredo JR, Rondina D, et al. Quantitative and qualitative analysis of the effectiveness of a mechanical method for the isolation of preantral follicles from ovine ovaries. Theriogenology. 2000;53:1251–62.

  29. 29.

    Yin H, Kristensen SG, Jiang H, Rasmussen A, Andersen CY. Survival and growth of isolated pre-antral follicles from human ovarian medulla tissue during long-term 3D culture. Hum Reprod. 2016;31:1531–9.

  30. 30.

    Demeestere I, Delbaere A, Gervy C, Van Den Bergh M, Devreker F, Englert Y. Effect of preantral follicle isolation technique on in-vitro follicular growth, oocyte maturation and embryo development in mice. Hum Reprod. 2002;17:2152–9.

  31. 31.

    Santos SS, Biondi FC, Cordeiro MS, Miranda MS, Dantas JK, Figueiredo JR, et al. Isolation, follicular density, and culture of preantral follicles of buffalo fetuses of different ages. Anim Reprod Sci. 2006;95:1–15.

  32. 32.

    Nagano M, Atabay EP, Atabay EC, Hishinuma M, Katagiri S, Takahashi Y. Effects of isolation method and pre-treatment with ethylene glycol or raffinose before vitrification on in vitro viability of mouse preantral follicles. Biomed Res. 2007;28:153–60.

  33. 33.

    Aerts JM, Martinez-Madrid B, Flothmann K, De Clercq JB, Van Aelst S, Bols PE. Quantification and viability assessment of isolated bovine primordial and primary ovarian follicles retrieved through a standardized biopsy pick-up procedure. Reprod Domest Anim. 2008;43:360–6.

  34. 34.

    Abir R, Franks S, Mobberley MA, Moore PA, Margara RA, Winston RM. Mechanical isolation and in vitro growth of preantral and small antral human follicles. Fertil Steril. 1997;68:682–8.

  35. 35.

    Abir R, Fisch B, Nitke S, Okon E, Raz A, Ben Rafael Z. Morphological study of fully and partially isolated early human follicles. Fertil Steril. 2001;75:141–6.

  36. 36.

    Dolmans MM, Michaux N, Camboni A, Martinez-Madrid B, Van Langendonckt A, Nottola SA, et al. Evaluation of Liberase, a purified enzyme blend, for the isolation of human primordial and primary ovarian follicles. Hum Reprod. 2006;21:413–20.

  37. 37.

    Hovatta O, Wright C, Krausz T, Hardy K, Winston RM. Human primordial, primary and secondary ovarian follicles in long-term culture: effect of partial isolation. Hum Reprod. 1999;14:2519–24.

  38. 38.

    Roy SK, Treacy BJ. Isolation and long-term culture of human preantral follicles. Fertil Steril. 1993;59:783–90.

  39. 39.

    Kristensen SG, Rasmussen A, Byskov AG, Andersen CY. Isolation of pre-antral follicles from human ovarian medulla tissue. Hum Reprod. 2011;26:157–66.

  40. 40.

    Vanacker J, Camboni A, Dath C, Van Langendonckt A, Dolmans MM, Donnez J, et al. Enzymatic isolation of human primordial and primary ovarian follicles with Liberase DH: protocol for application in a clinical setting. Fertil Steril. 2011;96:379–83.

  41. 41.

    Abir R, Roizman P, Fisch B, Nitke S, Okon E, Orvieto R, et al. Pilot study of isolated early human follicles cultured in collagen gels for 24 hours. Hum Reprod. 1999;14:1299–301.

  42. 42.

    Rice S, Ojha K, Mason H. Human ovarian biopsies as a viable source of pre-antral follicles. Hum Reprod. 2008;23:600–5.

  43. 43.

    Andreotti PK, Cree IA, Kurbacher CM, Hartmann DM, Linder D, Harel G, et al. Chemosensitivity testing of human tumors using a microplate adenosine triphosphate luminescence assay: clinical correlation for cisplatin resistance of ovarian carcinoma. Cancer Res. 1995;55:52w76–5282.

  44. 44.

    Kurbacher CM, Cree IA, Bruckner HW, Brenne U, Kurbacher JA, Müller K, et al. Use of an ex vivo ATP luminescence assay to direct chemotherapy for recurrent ovarian cancer. Anticancer Drug Des. 1998;9:51–7.

  45. 45.

    Nicolantonio FD. Multidrug resistance in solid tumours. A thesis submitted to the University of London for the degree of Doctor of Philosophy in the Faculty of Medicine Department of Pathology Institute of Ophthalmology, University of London, 2004. p. 372.

  46. 46.

    Bruckner HW. Methods and systems for guiding selection of chemotherapeutic agents. Patent US20060058966A1, 2006.

  47. 47.

    Paynter SJ, Cooper A, Fuller BJ, Shaw RW. Cryopreservation of bovine ovarian tissue: structural normality of follicles after thawing and culture in vitro. Cryobiology. 1999;38:301–9.

  48. 48.

    Isachenko V, Isachenko E, Reinsberg J, Montag M, van der Ven K, Dorn C, et al. Cryopreservation of human ovarian tissue: comparison of rapid and conventional freezing. Cryobiology. 2007;55:261–8.

  49. 49.

    Isachenko V, Isachenko E, Mallmann P, Rahimi G. Long-time cooling of human ovarian tissue before cryopreservation as obvious procedure: stimulation of follicular development and neo-vascularisation. Clin Lab. 2012;58:1293–300.

  50. 50.

    Isachenko V, Isachenko E, Mallmann P, Rahimi G. Increasing follicular and stromal cell proliferation in cryopreserved human ovarian tissue after long-term precooling prior to freezing: in vitro versus chorioallantoic membrane (CAM) xenotransplantation. Cell Transplant. 2013;22:2053–61.

  51. 51.

    Isachenko V, Isachenko E, Reinsberg J, Montag M, Braun F, van der Ven H. Cryopreservation of human ovarian tissue: effect of spontaneous and initiated ice formation. Reprod BioMed Online. 2008;16:336–45.

  52. 52.

    Isachenko V, Isachenko E, Reinsberg J, Montag M, Weiss J, Braun F, van der Ven H. Simplified technique of human ovarian tissue freezing: quick cooling from −36-degree C. CryoLetters. 2008;29:261–8.

  53. 53.

    Isachenko V, Lapidus I, Isachenko E, Krivokharchenko A, Kreienberg R, Woriedh M, et al. Human ovarian tissue vitrification versus conventional freezing: morphological, endocrinological, and molecular biological evaluation. Reproduction. 2009;138:319–27.

  54. 54.

    Isachenko V, Isachenko E, Weiss JM, Todorov P, Kreienberg R. Cryobanking of human ovarian tissue for anti-cancer treatment: comparison of vitrification and conventional freezing. CryoLetters. 2009;30:449–54.

  55. 55.

    Isachenko V, Mallmann P, Petrunkina AM, Rahimi G, Nawroth F, Hancke K, Felberbaum R, Genze F, Damjanoski I, Isachenko E. Comparison of in vitro- and chorioallantoic membrane (CAM)-culture systems for cryopreserved medulla-contained human ovarian tissue. PLoS One. 2012;7:e32549.

  56. 56.

    Isachenko V, Dittrich R, Keck G, Isachenko E, Rahimi G, van der Ven H, et al. Cryopreservation of ovarian tissue: detailed description of methods for transport, freezing and thawing. Geburtsh Frauenheilk. 2012;72:927–32.

  57. 57.

    Isachenko V, Isachenko E, Keck G, Dittrich R, Montag M, van der Ven H, et al. First live birth in Germany after re-transplantation of cryopreserved ovarian tissue: original device for initiation of ice formation. Clin Lab. 2012;58:933–8.

  58. 58.

    Isachenko V, Todorov P, Isachenko E, Rahimi G, Hanstein B, Salama M, et al. Cryopreservation and xenografting of human ovarian fragments: medulla decreases the phosphatidylserine translocation rate. Reprod Biol Endocrin. 2016;14:79.

  59. 59.

    Gook DA, Edgar DH. Ovarian tissue cryopreservation. In: Donnez J, Kim S, editors. Principles and practice of fertility preservation. New York: Cambridge University Press; 2011. p. 342–56.

  60. 60.

    Lierman S, Tilleman K, Cornelissen M, De Vos WH, Weyers S, T’Sjoen G, et al. Follicles of various maturation stages react differently to enzymatic isolation: a comparison of different isolation protocols. Reprod BioMed Online. 2015;30:181–90.

  61. 61.

    Gougeon A. Regulation of ovarian follicular development in primates: facts and hypotheses. Endocr Rev. 1996;17:121–55.

  62. 62.

    Fortune JE. The early stages of follicular development: activation of primordial follicles and growth of preantral follicles. Anim Reprod Sci. 2003;78:135–63. 43

  63. 63.

    Cortvrindt RG, Smitz JE. Fluorescent probes allow rapid and precise recording of follicle density and staging in human ovarian cortical biopsy samples. Fertil Steril. 2001;75:588–93.

  64. 64.

    Paulini F, Vilela JM, Chiti MC, Donnez J, Jadoul P, Dolmans MM, et al. Survival and growth of human preantral follicles after cryopreservation of ovarian tissue, follicle isolation and short-term xenografting. Reprod BioMed Online. 2016;33:425–32.

  65. 65.

    Sampaio da Silva AM, Bruno JB, de Lima LF, Ribeiro de Sá NA, Lunardi FO, Ferreira AC, et al. Connexin 37 and 43 gene and protein expression and developmental competence of isolated ovine secondary follicles cultured in vitro after vitrification of ovarian tissue. Theriogenology. 2016;85:1457–67.

  66. 66.

    Yu C, Penn LD, Hollembaek J, Li W, Cohen LH. Enzymatic tissue digestion as an alternative sample preparation approach for quantitative analysis using liquid chromatography-tandem mass spectrometry. Anal Chem. 2004;76:1761–7.

  67. 67.

    Telfer EE, Zelinski MB. Ovarian follicle culture: advances and challenges for human and non-human Primates. Fertil Steril. 2013;99:1523–33.

  68. 68.

    Hornick JE, Duncan FE, Shea LD, Woodruff TK. Multiple follicle culture supports primary follicle growth through paracrine-acting signals. Reproduction. 2013;145:19–32.

  69. 69.

    Soldatow VY, LeCluyse EL, Griffith LG, Rusyn I. In vitro models for liver toxicity testing. Toxicol Res (Camb). 2013;2:23–39.

  70. 70.

    Jauregui HO, McMillan PN, Hevey K, Naik S. A quantitative analysis of lectin binding to adult rat hepatocyte cell surfaces. In vitro Cell Dev Biol. 1988;24:401–12.

  71. 71.

    Lerche-Langrand C, Toutain HJ. Precision-cut liver slices: characteristics and use for in vitro pharmaco-toxicology. Toxicology. 2000;153:221–53.

  72. 72.

    Demeestere I, Delbaere A, Gervy C, Englert Y. Effect of pre-antral follicle isolation technique on follicular growth, oocyte maturation and fertilization in vitro in the mouse. Hum Reprod. 2000;15(Abst. book 1):89–90.

  73. 73.

    Tirmenstein MA, Nicholls-Grzemski FA, Schmittgen TD, Zakrajsek BA, Fariss MW. Characterization of nitric oxide production following isolation of rat hepatocytes. Toxicol Sci. 2000;53:56–62.

  74. 74.

    Merdassi G, Mazoyer C, Guerin JF, Saad A, Salle B, Lornage J. Examination of viability and quality of ovarian tissue after cryopreservation using simple laboratory methods in ewe. Reprod Biol Endocrinol. 2011;9:78.

  75. 75.

    Courbiere B, Odagescu V, Baudot A, Massardier J, Mazoyer C, Salle B, et al. Cryopreservation of the ovary by vitrification as an alternative to slow-cooling protocols. Fertil Steril. 2006;86(4 Suppl):1243–51.

  76. 76.

    Milenkovic M, Wallin A, Ghahremani M, Brännström M. Whole sheep ovary cryopreservation: evaluation of a slow freezing protocol with dimethylsulphoxide. J Assist Reprod Genet. 2011;28:7–14.

  77. 77.

    Abir R, Garor R, Felz C, Nitke S, Krissi H, Fisch B. Growth hormone and its receptor in human ovaries from fetuses and adults. Fertil Steril. 2008;90(Suppl):1333–9.

  78. 78.

    Oktay K, Nugent D, Newton H, Salha O, Chatterjee P, Gosden RG. Isolation and characterization of primordial follicles from fresh and cryopreserved human ovarian tissue. Fertil Steril. 1997;67:481–6.

  79. 79.

    McShane P, Sutton R, Gray DW, Morris PJ. Protease activity in pancreatic islet isolation by enzymatic digestion. Diabetes. 1989;38(Suppl 1):126–8.

  80. 80.

    Hornick JE, Duncan FE, Shea LD, Woodruff TK. Isolated primate primordial follicles require a rigid physical environment to survive and grow in vitro. Hum Reprod. 2012;27:1801–10.

  81. 81.

    Nemes Z, Dietz R, Luth JB, Gomba S, Hackenthal E, Gross F. The pharmacological relevance of vital staining with neutral red. Experientia. 1979;35:1475–6.

  82. 82.

    Chambers EL, Gosden RG, YapC PHM. In situ identification of follicles in ovarian cortex as a tool for quantifying follicle, viability and developmental potential in strategies to preserve female fertility. Hum Reprod. 2010;25:2559–68.

  83. 83.

    Winckler J. Vital staining of lysosomes and other cell organelles of the rat with neutral red. Prog Histochem Cytochem. 1974;6:1–89.

  84. 84.

    Park DH, Zeikus JG. Utilization of electrically reduced neutral red by Actinobacillus succinogenes: physiological function of neutral red in membrane-driven fumarate reduction and energy conservation. J Bacteriol. 1999;181:2403–10.

  85. 85.

    Repetto G, del Peso A, Zurita JL. Neutral red uptake assay for the estimation of cellviability/cytotoxicity. Nat Protoc. 2008;3:1125–31.

  86. 86.

    Soares M, Sahrari K, Amorim CA, Saussoy P, Donnez J, Dolmans MM. Evaluation of a human ovarian follicle isolation technique to obtain disease-free follicle suspensions before safely grafting to cancer patients. Fertil Steril. 2015;104:672–80.

  87. 87.

    Amorim CA. Artificial ovary. In: Donnez J, Kim S, editors. Principles and practice of fertility preservation. New York: Cambridge University Press; 2011. p. 448–58.

  88. 88.

    Amorim CA, David A, Dolmans MM, Camboni A, Donnez J, van Langendonckt A. Impact of freezing and thawing of human ovarian tissue on follicular growth after long term xenotransplantation. J Assist Reprod Genet. 2011;28:1157–65.

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Conceived and designed the experiments: EI, VS, GR, VI, PM; performed the experiments: VS, EI, GR; analyzed the data: GR, GR, BH, BM; contributed materials/analysis tools: GR, VS, PM, wrote the manuscript: EI, VI, VS; manuscript drafting and critical discussion: EI, VI, VS, GR; finally approved of the version to be published: EI, VI. All authors read and approved the final manuscript.

Correspondence to Evgenia Isachenko.

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Schmidt, V.M., Isachenko, V., Rappl, G. et al. Comparison of the enzymatic efficiency of Liberase TM and tumor dissociation enzyme: effect on the viability of cells digested from fresh and cryopreserved human ovarian cortex. Reprod Biol Endocrinol 16, 57 (2018).

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  • Artificial ovary
  • Follicle
  • Ovarian tissue
  • Enzymatic isolation
  • Cryopreservation
  • Liberase TM
  • Tumor dissociation enzyme