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

Abstract

BackgroundThe 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).MethodsFresh 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.ResultsThe 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).ConclusionThe 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.

Bioengineering of Human Artificial Ovary From Cryopreserved Tissue: Optimization of the Follicles Enzymatic Isolation.

“…Weight and volume of biopsies in both group was no signi cant (p > 0.05). This was similar to a recent study by Schmidt et al It showed that the number of follicles isolated by TDE digestion is also signi cantly higher than using Liberase TM [19]. By comparison, Lierman et al presented that no statistical differences were found between Liberase TM and Liberase DH, concerning the number and type of isolated follicles [26].…”

“…It is thus easy to distinguish between viable, damaged or dead cells [30]. Our study presented the percentage of bright red follicle in Group 1 digested from cryopreserved ovarian tissue was found to be signi cantly higher (p < 0.01) than Group 2 (70.9 ± 4.7% vs 63.9 ± 4.2%, respectively), similar to the observations of intense red -stained follicles in the suspension of cryopreserved ovarian tissues made by Schmidt et al (70.3 ± 6.2% in TDE vs 53.1 ± 2.0% in Liberase TM, respectively) [19].…”

“…Our prior study documented using "Tumor Dissociation Enzyme Reagent" (TDE, Innovative Diagnostic Systems Dr. Christian Sartori, Hamburg, Germany) to digested the cryopreserved ovarian tissue could yielded better viability follicles in contrast to Liberase TM [19]. Tumor Dissociation Enzyme (TDE) is a commercial enzyme-cocktail from the DCS ATP-Chemo-sensitivity Assay Kit (DCS Innovative Diagnostik-Systeme, Hamburg, Germany) for gentle enzymatic digestion [20,21].…”

“…Tumor Dissociation Enzyme (TDE) is a commercial enzyme-cocktail from the DCS ATP-Chemo-sensitivity Assay Kit (DCS Innovative Diagnostik-Systeme, Hamburg, Germany) for gentle enzymatic digestion [20,21]. Prior studies in our group reported that TDE contains the mixture of highly puri ed collagenase and other enzymes that enable the digested suspensions to be not sticky, easy to isolate, and yield a higher number of healthy cells [19,22]. However, we do not know what effect this enzyme will have on follicular growth and development after in vitro culture.…”

Identification of a Technique Optimized for the Isolation of Spermatogonial Stem Cells from Mouse Testes

“…This may be due to the enzymes used for isolation of SSCs from testicular tissues. Generally, a variety of digestive enzymes such as trypsin, pronase, dispase, and collagenase are used, which can induce minor or major damage to cellular junctions, membranes, surface receptors, antigens, and cytosolic contents (Miersch et al 2018;Schmidt et al 2018). Therefore, Theoretically, double MACS, which is conducted sequentially using antibodies detecting different cell surface proteins, is expected to result in higher purification efficiency compared with single MACS.…”