Different Effects of Sugars and Methods to Preserve Post-Thaw Functional Properties of Cryopreserved Caprine Spermatogonial Stem Cells

Spermatogonial stem cells (SSCs) are the male germline stem cells of the seminiferous tubules in the testis and are the creator cells of spermatogenesis [Kanatsu-Shinohara et al., 2008]. The culture of SSCs is required for their expansion, differentiation [Singh et al., 2021], and manipulation. However, subsequent sub-cultures over time resulted in a gradual decline in proliferation rate, differentiation into other cell types, and higher senescence and apoptosis of putative SSCs [Zhu et al., 2013]. Therefore, an effective method for SSC preservation is required for the long-term maintenance of their functional properties and utilized in advanced biotechnological and therapeutic applications in reproductive biology research such as germline gene therapy or male fertility restoration.

Cryopreservation represents the gold standard in cell storage and transportation, and by maintaining the functional properties of cells, cryopreservation is the preferred method for the long-term storage of SSCs [Yokoyama et al., 2012]. Moreover, cryopreservation has been an essential part of any cell transplantation procedure, assisting to overcome the challenges related to the place and time barriers between recipient and donor.

The post-thaw viability of SSCs depends on the cryopreservation methods as well as the species analyzed [Onofre et al., 2016]. In contrast to single cells, tissue fragments have dissimilar heat transfer properties that are required during the process of cryopreservation, and thus this makes them different for cooling rates and distribution of cryoprotective agents [Onofre et al., 2016]. Therefore, to accomplish the improved recovery rate of target cells, the sample volume, rate of cooling (slow or fast), type (extracellular or intracellular), and concentrations of cryoprotective agents (alone or in combination) should be optimized.

Several studies suggest that cryopreservation does not affect post-thaw metabolic activity Chinnadurai et al. [2014] or proliferation potential of stem cells [Lauterboeck et al., 2016; Bahsoun et al., 2019]; nonetheless, lower [Heino et al., 2012] or higher [Jung et al., 2020] proliferation rate of stem cells has also been reported. The species-dependent variations in cell viability and metabolic activities of cryopreserved SSCs are described earlier [Liu et al., 2011]. Human studies suggest no difference in the level of senescent mesenchymal stromal cells after cryopreservation [Mamidi et al., 2012]. However, information about the senescent profile of cryopreserved SSC using different sugars as cryoprotectant and the effect on different cryoprotectants while using slow freezing (SF) or fast freezing (FF) methods on maintaining post-thaw functional properties such as recovery, proliferation, and metabolic viability of caprine SSCs (cSSCs) is not available.

Therefore, the present study was planned to investigate the effect of sugars (trehalose and sucrose) as additive cryoprotectant agents (ACAs) and/or permeable cryoprotectant agents (dimethyl sulfoxide [DMSO] and ethylene glycol [EG]) on cryopreservation of cSSCs (experiment 1) and testicular tissue (experiment 2) by FF or SF methods. For this, we have evaluated and compared the recovery rate, metabolic viability, senescence, proliferation, and expression of stemness markers in the post-thaw cultured cSSCs among control and different treatment groups.

Testes of the prepubertal Barbari bucks (age ~3 months; n = 6) were obtained through castration and immediately brought to the laboratory (within 15 min) in warm physiological saline supplemented with antibiotics (streptomycin, 500 µg/mL). The extra tissue was trimmed off from the testis and washed six to seven times with physiological saline followed by a brief washing (~30 s) with 70% ethanol and finally with Dulbecco’s phosphate-buffered saline (DPBS) (Sigma-Aldrich, Catalog#D5773) solution. In a sterile environment, the tunica from the testis was removed by giving a longitudinal incision with a surgical blade, and a sample from testicular parenchymal tissue was obtained. The finely chopped tissue was transferred into a 15-mL centrifuge tube and washed six times at 110 g for 5 min with DPBS supplemented with antibiotics and finally once with Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12 media; Gibco, Catalog#12660012). The pellet was dissolved in DMEM/F12 (5-fold v/v) with antibiotics. Further, the two-step sequential digestion was performed by incubating the sample at 37.0°C in an orbital shaker with a cocktail of enzymes (trypsin [1 mg/mL; Sigma-Aldrich, Catalog#T7409], hyaluronidase [1 mg/mL; Sigma-Aldrich, Catalog#H2126], collagenase [1 mg/mL; Sigma-Aldrich, Catalog#C5138], and DNase I [5 µg/mL; Sigma-Aldrich, Catalog#DN25]) for first digestion (45 min) and second digestion (30 min). To stop enzymatic activity, DMEM/F12 containing 10% FBS (Gibco, Catalog#10082-147) was added to the supernatant. Then, the cell pellet was washed with DMEM/F12, and culture media (DMEM/F12, 15% FBS, 1% l-glutamine [Thermo Fisher Scientific, GlutaMAX supplement, Catalog#35050061], 1% non-essential amino acid [Sigma-Aldrich, Catalog#M7145], gentamicin [50 µg/mL, Sigma-Aldrich, Catalog#G1264], and a combination of antibiotic-antimycotic [10 µL/mL, Sigma-Aldrich, Catalog#A5955]) was added. The single-cell suspension was filtered using a 60-µm nylon filter (Merck Millipore, Catalog#NY6002500). The filtrate was analyzed for cell number and the viability of cells was evaluated by the trypan blue staining technique using 0.4% (w/v) trypan blue dye (Sigma-Aldrich, Catalog#93595) and a Countess™ II FL automatic cell counter (Invitrogen Inc., Bothell, WA, USA). Thereafter, the cell suspension was processed for cryopreservation and comparative evaluation of post-thaw culture characteristics in different treatment groups.

The cell suspension (0.5 mL) was distributed into the precooled cryovials, and an equal amount of culture media were added and centrifuged (2,650 g for 10 min at 25°C) to form a cell pellet. To the pellet, 0.5 mL of stock cryopreservation media (60% DMEM/F12, 20% DMSO, and 20% heat-inactivated FBS) and an equal volume of DMEM/F12 media containing different concentrations of ACPs (sugars: trehalose [140 mm; 140T or 400 mm; 400T] and sucrose [140 mm; 140S or 400 mm; 400S] or EG [5%]) were added drop-wise to a final volume of 1.0 mL in the 2.0 mL cryovials (Corning, Midland, MI; Catalog#430659). In the control group, only stock cryopreservation media were used. The concentrations of ACPs used were based on the outcome of earlier studies [Lee et al., 2014; Li et al., 2018; Bahsoun et al., 2019; Anjos et al., 2021]. The cryovials were kept in an ice bath during the entire procedure.

The cryopreservation of cSSCs was done following standard protocols for SF and FF methods. For cryopreservation by SF method, the cryovials containing cell suspension and cryoprotectants were kept in Nalgene Mr. Frosty^® Freezing Containers (Thermo Fisher Scientific, Catalog#5100-0001) overnight at -80°C to obtain 1°C/min cooling rate to -80°C. After overnight storage at -80°C, the cryovials were plunged into liquid nitrogen (-196°C). For the FF method, cell suspension with cryoprotectants in the cryovials was kept at -20°C for 45 min for initial equilibration and then plunged into liquid nitrogen. The cell suspensions were cryopreserved for 1 week before thawing and assessment of viability and culture characteristics of enriched cSSCs.

The cryovials containing cell suspension were removed from liquid nitrogen storage and placed in a water bath at 37°C. The vials were gently swirled in the water bath for 2 min. Then, the cryovials were taken out of the water bath, and to avoid possible contamination, the tubes were gently wiped with tissue paper absorbed in 70% ethanol.

For the cultivation of cells, 24-well culture plate (Greiner Bio-One; Cellstar, Catalog#662160) was used. After seeding of cells, plates were incubated in a CO₂ incubator at 37°C with maximum humidity. Further, the enrichment of putative cSSCs was done by differential plating method and kept overnight at 37°C in a CO₂ incubator as described earlier [Singh et al., 2022a]. The enriched cSSCs were further cultivated for 2 weeks, and culture media were changed every 3rd day. After culturing of cSSCs for 21 days, the cell viability, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT), senescence-associated ß-galactosidase (SA-ß-GAL senescence), proliferation assays, and expression of stemness markers were evaluated.

The cSSC proliferation was determined by 5-bromo-2-deoxyuridine (BrdU) incorporation assay. The cells in culture media containing 100 µm BrdU (Sigma-Aldrich, Catalog#B5002) were incubated overnight at 37°C in a CO₂ incubator. Thereafter, the content from the well was removed and cells were fixed with chilled ethanol (70%). Then, the fixed cells were incubated with HCl (1.5 m) for 30 min at RT to hydrolyze cellular DNA. Subsequently, following two washes with PBS and cell permeabilization by blocking buffer (2% BSA in 1× PBS) with 0.3% Triton X 100 for 60 min, staining of cells was performed by incubating them for 1 h in dark with FITC-conjugated secondary antibody (anti-BrdU-Alexa 488 antibody; Merck Millipore, Catalog#FCMAB101A4; 1:200). Further, the 4',6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich; Catalog#D8417, 300 ng/mL in blocking buffer) solution was added for nuclei staining. After three washes with PBS, proliferating cells were examined for BrdU incorporation under a fluorescence microscope (Zeiss Axiovert A1, Carl Zeiss Microscopy GmbH 07745 Jena, Germany).

For determining the cellular viability of cultured cSSCs, the cell colonies were washed once with PBS to remove cells in suspension (non-adherent cells). Then, the adherent cells were stained with crystal violet solution (Sigma-Aldrich, Catalog#V5265) for 30 min. After washing 5–6 times with distilled water, cell colonies were examined under the bright field microscope. Further, the stain was solubilized with 1% SDS solution for 10 min at RT and the optical density was measured at 590 nm by a microplate reader (Sun Rise, Tecan, Männedorf, Switzerland).

The cellular metabolic activity of cultured cSSCs of all the treatment groups was evaluated by a tetrazolium dye (MTT)-based colorimetric kit following the manufacturer’s instructions (Merck Millipore, Catalog#CT02). Briefly, after culturing of cSSCs at 37°C in a CO₂ incubator for 3 weeks, culture media were removed and MTT solution (40 µL) along with fresh media (2.0 mL) were added to each well of 24-well culture plate and incubated at 37°C for 4 h. Thereafter, isopropanol (100 µL) with 0.04 N HCl was added to the wells and mixed properly. Further, the colonies were washed with distilled water and observed under the microscope. The optical density was estimated at 570 nm using a microplate reader.

The cellular senescence of cultured cSSCs was assessed by evaluating SA-ß-GAL following the manufacturer’s protocol (Merck Millipore, Catalog#KAA002). Briefly, after 2 weeks of culture, fixing of cells was performed by adding a fixing solution (200 µL/well) and incubation for 15 min at RT. Then, the cells were stained with 1× SA-ß-GAL detection solution and kept at 37°C in a CO₂ incubator for 4 h in dark. The blue-stained colonies were counted against the total number of cells under a phase contrast microscope.

The immunocytochemical analyses of important germ cell-related markers, i.e., OCT-4 (octamer-binding transcription factor-4 (OCT-4) and protein gene product 9.5 (PGP9.5) were performed after 3-week culture of cryopreserved and thawed cSSCs. For this, following the removal of culture media, cells were washed once with 0.1% solution of polyvinyl alcohol in DPBS (DPBS-PVA) and fixed by treating cells with 4% paraformaldehyde for 30 min and permeabilized by Triton X-100 for another 30 min at RT. Then, blocking of unoccupied space was done by adding 4% BSA in DPBS and incubation at RT for 60 min. The cells were incubated overnight at 4°C with pluripotent marker-specific primary antibodies, i.e., rabbit anti-OCT-4 (Sigma-Aldrich, Catalog#AB3209; 1:100 dilution) and rabbit anti-PGP9.5 (Invitrogen, Catalog#PA1-10024; 1:400 dilution). After washing thrice with DPBS-PVA (5 min each), cells were incubated for 30 min at RT in dark with a secondary antibody, i.e., donkey anti-rabbit IgG (Alexa Fluor 488, Invitrogen, Catalog#R37118). Then, after five washings with DPBS-PVA, a gold antifade liquid mountant with DAPI (ProLong™, Invitrogen, Catalog#P36931) was added and the cellular staining was observed under a fluorescent microscope (Zeiss Axiovert A1, Carl Zeiss Microscopy GmbH 07745 Jena, Germany).

In the second experiment, the cryopreservation of testicular tissue of six prepubertal Barbari goats was performed to assess the effect of sugars (trehalose or sucrose) on the functional characteristics of cSSCs obtained from the cryopreserved testicular tissues.

The testes from the prepubertal Barbari bucks were obtained from an abattoir and transferred to the laboratory. The extra tissue was trimmed off before several washings with normal saline. Then, in a sterile condition, tunica was removed and the testis tissue pieces were obtained (as presented in experiment 1). Then, the tissue samples were cut into 3–4-mm³ pieces and transferred to 2.0 mL cryovials. Further, the cryovials were kept in the ice bath and 600 µL each of stock cryopreservation media (20% v/v DMSO, 60% v/v DMEM/F-12, and 20% heat-inactivated FBS) and media (DMEM/F-12) without cryoprotectant (control group) or with cryoprotectant (trehalose [140 mm trehalose [140T]] or sucrose [140 mm sucrose [140S]]) was added. The SF and FF methods for cryopreservation of testicular tissue were similar as stated in experiment 1. The cryopreserved tissues were kept for 1 week before thawing and isolation of cSSCs for assessment of their viability and culture characteristics.

After thawing, the testicular tissue pieces of treatment and control groups were taken out of the cryovials and chopped finely in a sterile glass petri dish with the help of a surgical blade. The finely minced tissue was washed six times with DPBS (3 mL each time) supplemented with antibiotic (gentamicin, 50 µg/mL, Sigma-Aldrich, Catalog#G1264) and then once with DMEM/F12 media before being subjected to a two-step digestion method for isolation of cSSCs, as mentioned in experiment 1. The number of live/dead cells against the total number of cells was determined by the trypan blue exclusion method before the enrichment of isolated cells by differential plating and their further cultivation.

After isolation and culture of cSSCs obtained from testicular tissues cryopreserved by either SF or FF method, assays for cellular viability, metabolic viability, senescence, and proliferation were performed as presented in experiment 1.

After 3 weeks of the culture of cSSCs onto the 96-well cell culture plates, the fixing of cell colonies was performed by adding a 4% solution of PFA in PBS for 20 min. Then, the cell permeabilization and blocking were done by adding Triton X-100 for 10 min and 4% BSA in PBS (blocking buffer) for 1 h at RT, respectively. The cells were then incubated with the primary antibodies (rabbit anti-OCT-4 or rabbit anti-PGP9.5) diluted in a blocking buffer, as presented in experiment 1. Further, after 3 washings with DPBS-polyvinyl alcohol, secondary antibody (donkey anti-rabbit IgG [Alexa Fluor 488, Invitrogen, Catalog#R37118]) and Dolichos biflorus agglutinin (DBA) labeled with red fluorescent rhodamine (1:100 dilution; Vector Laboratories, Inc., CA 94010, USA; Catalog#RL-1032-2) were added to incubate for 30 min in dark at RT. Then, after washing 5 times, an antifade mountant with DAPI (Invitrogen, Catalog#P36931) was applied prior to the cell imaging using a combination of fluorescence filters (green filter for OCT-4 and PGP9.5, red filter for DBA, and blue filter for DAPI) using a fluorescent microscope (Zeiss Axiovert A1, Carl Zeiss Microscopy GmbH 07745 Jena, Germany).

A Student’s t test (independent sample test) was conducted to identify the statistically significant difference between the two treatment groups (p < 0.05). In all experiments, the data were taken at least in triplicates for each treatment group. The results are presented as mean ± SEM.

The prepubertal Barbari cSSCs in control and different treatment groups were assessed for the total cell number and their recovery rate (post-thawed survival rate) by the trypan blue exclusion method using an automatic cell counter and the data are presented in Figure 1. While cryopreservation using the SF method, the number of live cells was 1.31-, 1.71-, 1.82-, 1.37-, and 1.57-fold higher in EG, 140T, 400T, 140S, and 400S groups compared with the control group, respectively. Overall, the SF and FF methods provide similar results when using trehalose as an ACA (Fig. 1a, b), whereas for the other ACAs, the cell number was 1.14-, 1.34-, and 1.64-fold higher when using EG, 140S, and 400S with the SF method than the FF method, respectively. Among SF groups, all the ACAs improved the live percentage in which the 400T group showed the best result, whereas in FF groups, both 140 and 400 mm trehalose had a better effect on the recovery rate. Overall, using trehalose provides a greater number of live cells compared with other cryoprotectants with either SF or FF method (Fig. 1b).

The representative images of the BrdU proliferation assay of post-thawed cultured cSSCs cryopreserved through SF or FF methods are presented in Figures 2a and 3a, respectively. The quantification results of SF and FF methods are presented in Figures 2b and 3b, respectively. The BrdU expression in all the groups suggests that post-thaw cSSCs were able to proliferate after cryopreservation and thawing. The cells cryopreserved by the SF method with the use of 400 mm trehalose provide better proliferation results compared with the control and other treatment groups.

To assess the cellular and metabolic viability of post-thawed cSSCs, crystal violet staining and MTT assay were performed, respectively. In both the freezing methods (SF and FF methods), significant (p < 0.05) differences were observed among the values of optical density of different groups (Fig. 4b). When cryopreserved through either SF or FF method, the OD of 140T group was significantly (p < 0.05) higher compared with the other treatment groups. This indicates a favorable effect of trehalose on the viability of the cSSCs in the culture system after thawing (Fig. 4a).

For the metabolic viability assay, an MTT assay was performed on cultured cSSCs. This assay was based on the principle that only the viable cells change the colorless MTT into formazan crystals of dark purple color (Fig. 4a). A significant (p < 0.05) difference in the number of viable cells under the above concentration of supplements as compared with the control was observed in both the freezing methods (Fig. 4c). For FF method, the OD of 140T group was significantly (p < 0.05) higher than the other groups. This suggests the relatively better favorable effect of trehalose on the metabolic viability of post-thaw cultured cSSCs.

The senescent phenotypes of cultured cSSCs were identified by the expression of SA-ß-GAL by the SA-ß-GAL of cultured cSSCs (Fig. 5a). In experiment 1, the percentage of senescent cells was higher in control, 5% EG, 400T, and 400S groups of both the freezing methods compared with the 140T group (Fig. 5b).

The cSSCs were characterized by immunostaining for SSC markers, and the representative images of immunofluorescence staining of cultured cSSCs for OCT-4, PGP9.5, and DBA are presented in Figures 6-9. The immunocytochemical analyses demonstrate the expression of the pluripotency-specific intracellular markers in post-thawed cultured cSSCs (OCT-4 and PGP9.5). The expression of germ cell markers was higher when trehalose was used as ACA compared with other additional cryoprotectants.

The cells obtained from post-thawed prepubertal Barbari buck testis tissue were assessed for cell number and recovery rate of cells by the trypan blue exclusion method using 0.4% trypan blue solution and an automatic cell counter. In this experiment, trehalose (140T group) favors an improved recovery rate when used in the SF method. The recovery rate was higher if using 140T (2.72-fold) or 140S (2.00-fold) compared with the control group, whereas using the FF method, both trehalose and sucrose provide an improved effect on live percentage and recovery rate (Fig. 10).

Representative images of BrdU proliferation assay of cSSC culture which were cultured from cryopreserved testis tissue are presented in Figure 11. The expression of BrdU in the cSSCs demonstrated that the proliferation was observed in all groups of SF, whereas in FF, proliferation was observed in 140T and 140S group but not in the control group.

To measure the cellular viability and proliferation of cSSCs, crystal violet staining was done. The values of optical density in 140T and 140S groups were significantly (p < 0.05) higher compared with the control group in both SF and FF methods (Fig. 12b), demonstrating the positive effect of ACAs on the proliferation of cultured cSSCs (Fig. 12a). The significantly (p < 0.05) higher optical densities of 140T and 140S groups (Fig. 12c) compared with the control group in both SF and FF methods indicate a higher number of viable cells and improved cellular and metabolic viability of cSSCs when these ACAs were used along with the stock cryopreservation media (Fig. 12a).

The senescent cSSCs were recognized by the expression of SA-ß-GAL. For this, the SA-ß-GAL staining was done (Fig. 13). In experiment 2, a higher percentage of senescent cells was observed in 140T and 140S groups compared to control in both SF and FF methods (Fig. 13b). The colony count at day 7 of culture representing single, paired, cluster, and rosette colonies in respective groups and freezing methods of experiment 2 are presented in Figure 13c.

The immunophenotypic characterization of cSSCs was done by staining cultured cells for GC markers (OCT-4, PGP9.5, and DBA), and the representative images of immunofluorescence staining are presented in Figures 14 and 15, respectively. The immunocytochemical analyses demonstrate the expression of the pluripotency-specific intracellular (OCT-4 and PGP9.5) markers in cSSC cultured cells, implying that cryopreservation does not adversely affect the expression of pluripotent stem cell characteristics of cSSC culture. The higher expression of OCT-4, PGP9.5, and DBA was observed when 140T was used as ACA compared with the 140S in SF as well as in the FF method.

The survival and maintenance of functional properties of SSC after cryopreservation are essential for their broader applications. However, comparative studies assessing optimal approaches for cSSC cryopreservation (single cell suspension vs. testicular tissue) using fast and slow protocols are not available. This study demonstrates the effect of two cryopreservation methods, viz., SF and FF methods, and five types of freezing media on cryopreservation of cSSC in single cell suspension or testicular tissue (non-digested) obtained from prepubertal Barbari bucks. Thus, the present study reports the workable method for effective cryopreservation of cSSCs, which may be used for their long-term preservation. Following cryopreservation, the thawed cells and isolated cells from frozen tissue were evaluated for cell quantification and viability assessment. We report that adding ACAs to the basal freezing medium containing DMSO (stock cryopreservation media) improves survival and maintains functional properties of cSSC during culture after thawing.

The cell survival during the process of cryopreservation and thawing may be affected by the type and concentration of cryoprotectant used. The sugars are non-penetrating cryoprotectants, and because of their positive influence on preserving cell membrane integrity, they have been used in cryopreservation protocols of various cell types like oocytes [Somfai et al., 2015] and sperms [Anjos et al., 2021].

In the present study, for cSSC cryopreservation by SF method, all cryoprotectant supplements enhanced the survival and proliferation compared with the control group. Among all the groups of the FF method, 400 mm trehalose showed the best effect on live count and recovery rate. This is in agreement with an earlier study that reported that cryopreservation media supplemented with 200 mm trehalose functions as a better cryoprotectant for mice [Lee et al., 2013] and pig [Lee et al., 2014] SSCs than DMSO alone.

The cells were subsequently cultured in vitro in DMEM/F12 media with growth factors. The process of differential adherence technique (differential plating) was done for enriching the stem cell population [Singh et al., 2022a, b]. The culture characteristics were studied and evaluated for cellular and metabolic viability assays, viz., proliferation, cellular viability, MTT assay, SA-ß-GAL senescence also immunophenotypic characterization.

The application of SSC transplantation for clinical applications such as restoration of spermatogenesis and fertility preservation exclusively depends on the survival of both cSSCs and related somatic cells after cryopreservation and thawing [Valli-Pulaski et al., 2019]. There are limited known phenotypic and molecular markers of spermatogonia in domestic animals. However, few markers are consistently expressed in spermatogonia from a domestic animal such as PGP9.5 (alternatively known as ubiquitin carboxyl-terminal hydrolase L1 [UCHL1]), DBA, and OCT-4. The immunocytochemistry of cultured cSSCs demonstrates the expression of the pluripotency-specific intracellular markers (OCT-4 and PGP9.5), implying that cryopreservation by either SF or FF or the use of ACAs does not adversely affect the expression of pluripotent stem cell characteristics of cSSCs in culture. This is in accordance with the previous report suggesting the expression of stem cell markers following the culture of post-thawed cells [Kim et al., 2015].

For cellular viability and proliferative potential of cSSCs, crystal violet staining was performed on cultured cells, and the extent of staining was measured through the estimation of optical density [Singh et al., 2022a]. Irrespective of freezing methods, in tissue freezing method groups, significantly higher optical density was observed in ACA-supplemented groups. This is in agreement with the earlier report suggesting that the SSCs can be preserved for a long time via cryopreservation and adding trehalose to a basal freezing medium containing DMSO increases stem cell survival after thawing [Lee et al., 2013]. The impermeable cryoprotectants such as trehalose and sucrose are unable to enter the cell due to their high molecular weight [Kazemzadeh et al., 2022]. However, they can alter the physical state of the extracellular compartment and make coverings around the cells that prevent cellular dehydration during the process of cryopreservation [Liu et al., 2021]. Trehalose and sucrose are the non-toxic disaccharides of glucose that maintain cellular integrity throughout the process of freezing and thawing [Rodrigues et al., 2008].

The enzymatic digestion step is an integral part of the SSC isolation protocol. This directly exposes single cells to digestive enzymes like collagenase and trypsin that may have increased sensitivity of cell membrane to various physicochemical changes during the process of cryopreservation (e.g., solution effects, osmolarity, and ice nucleation), thereby causing increased cell death [Brook et al., 2001]. Hence, cryopreservation of testicular tissue has been shown as the preferred method for better maintenance of the functional properties of SSCs [Onofre et al., 2020]. Moreover, by testicular tissue freezing, the cell-cell contacts remain preserved if more permeable cryoprotectants are used. Therefore, cryopreservation of testicular tissues was also tried after investigating the cryopreservation of cSSCs with different ACAs.

The metabolic viability assay (MTT assay) was performed on cultured cSSCs after thawing. The coloration was quantified by the optical density that is related to the viability of the cells. A significantly higher difference was observed in cryoprotectant-added groups in tissue and cell freezing methods irrespective of the type of cryopreservation. In testicular tissue freezing groups using both freezing methods, an increased percentage of senescent cells was observed in the treatment groups. In the present study, we report that trehalose is the most effective of the sugars tested for maintaining metabolic activity and proliferation of cryopreserved cSSCs and the results are in agreement with earlier studies, which demonstrated more beneficial effects of trehalose in survival, recovery rate, and proliferation capacity of post-thaw SSC [Lee et al., 2013, 2014].

We demonstrated maintenance of the metabolic activity and post-thaw proliferation of the cSSCs in the culture system with higher proliferation potential of cells when cryopreserved using trehalose compared with the other sugars or DMSO alone. An earlier study demonstrated the maintenance of tubular integrity and the proliferation potential of spermatogonia after cryopreservation of testicular tissue [Curaba et al., 2011]. The majority of studies suggest that careful cryopreservation does not affect the post-thaw proliferation potential of mesenchymal stem cells [Lauterboeck et al., 2016; Bahsoun et al., 2019]; nonetheless, lower [Heino et al., 2012] or higher [Jung et al., 2020] proliferation rate of cryopreserved cells is also demonstrated. The variation in the outcome may be mainly due to the difference in the target species [Liu et al., 2011] and the cryopreservation protocol used.

Here, we show the successful cryopreservation through the maintenance of post-thaw functional properties of cSSCs and testicular tissue using sugars (trehalose and sucrose) as an extracellular CPA. Overall, the outcome of the study demonstrates the superiority of 140T when used with the SF method for the preservation of post-thaw proliferation ability, viability, metabolic activity, and reduced senescence of post-thaw cSSCs or cSSCs obtained from cryopreserved tissue over other treatment groups. The method developed will serve for the long-term storage of cSSCs for ex vivo cultivation and trans-differentiation of goat and other mammalian SSCs.

The authors are thankful for the support extended by the director of the institute for providing the necessary facilities to carry out this study.

All the experiments were conducted following the guidelines of the “Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA),” Government of India. The Institutional Animal Ethics Committee (IAEC) duly approved the present investigation (year 2020; IAEC/CIRG/Sr Nr. 6/2019).

The authors of this study affirm that there were no financial or commercial affiliations that may be viewed as having a possible conflict of interest.

The study was supported by a grant from the Department of Biotechnology (DBT), Government of India, New Delhi (Grant No. BT/PR27544/AAQ/1/715/2018).

Conceptualization: Shiva P. Singh and Saleema A. Quadri; methodology: Saleema A. Quadri, Shiva P. Singh, Juhi Pathak, and Dilip Swain; formal analysis and investigation: Suresh D. Kharche, Juhi Pathak, Yogesh K. Soni, and Dilip Swain; writing – original draft preparation: Saleema A. Quadri, Shiva P. Singh, and Dilip Swain; writing – review and editing: Suresh D. Kharche and Atul Saxena; funding acquisition: Shiva P. Singh; and supervision: Suresh D. Kharche.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.