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The enhanced availability of functional fibroblasts from precious tissue samples requires an ideal cell-culture system. Therefore, this study was designed to investigate the performance of caprine adult fibroblast cells (cadFibroblast) when cultivated in different culture media. The cadFibroblast cell lines from adult Barbari bucks were established and the effect of different media viz. DMEM/F-12 [with low glucose (5.5 mM; DL) and high glucose (30 mM; DH)], α-MEM [with low glucose (5.5 mM; ML) and with high glucose (30 mM; MH)], and fibroblast growth medium (FGM) were evaluated and compared for growth characteristics and in-vitro dynamics through cellular morphology, proliferation, population doubling time, double-immunocytochemistry, colony forming units, wound healing, transwell migration and differential expression of fibroblast-specific markers [FSP-1 and vimentin]. The results of immunocytochemistry, transwell migration/invasion, and wound healing assays showed the superiority of DH over DL and other media tested. Whereas, similar effects of glucose supplementation and expression of FSP-1 were not observed in α-MEM. Transwell migration was significantly (p

The present study aimed to identify the effects of sugar and methods [slow freezing (SF) vs fast freezing (FF)] on post-thaw in-vitro functional characteristics of cryopreserved caprine SSCs (cSSCs) and the cells obtained from cryopreserved testis tissue of pre-pubertal Barbari bucks. For this, in experiment-1, cSSCs were isolated and cryopreserved by either SF or FF method with different nonpermeable [sugars; trehalose (140 mM; 140T or 400 mM; 400T), and sucrose (140 mM; 140S or 400 mM; 400S)] or/and permeable [5% ethylene glycol (EG) and DMSO] cryoprotectants. After one-wk of cryopreservation, the cSSCs were thawed and cultured for evaluation of their characteristics. Further, in experiment-2, the effectiveness of sugars [trehalose (140 mM) or sucrose (140 mM)] for cryopreservation of testicular tissues of pre-pubertal Barbari bucks using the SF or FF method was evaluated. After one-wk of cryopreservation, the tissues were thawed and cSSCs were isolated and cultured for 3-wk. In both experiments, cSSCs were evaluated for recovery rate, proliferation, metabolic viability, senescence, and stemness markers expression. The recovery rate was 1.3, 1.3, and 1.1-folds higher in the 140T group compared with EG, 140S, and 400S groups, respectively. Similarly, the expression of stemness markers (PGP9.5 and OCT-4) was relatively higher in 140T group compared with the other groups. In experiment 2, the recovery rate of cells per unit tissue wt. was significantly (P

The present study aims to evaluate culture temperature-dependent variation in survival, growth characteristics and expression of stress, pluripotency, apoptosis, and adhesion markers in enriched caprine male germline stem cells (cmGSCs). For this, testes from pre-pubertal bucks (4–5 months; n = 4) were used to isolated cells by a two-step enzymatic digestion method. After enrichment of cmGSCs by multiple methods (differential platting, Percoll density gradient centrifugation, and MACS), viability of CD90(+) cells was assessed before co-cultured onto the Sertoli cell feeder layer at different temperatures (35.5, 37.0, 38.5, and 40.0 °C). The culture characteristics of cells were compared with MTT assay (viability); cluster-forming activity assay, SA-β1-gal assay (senescence), BrdU assay (proliferation), and transcript expression analyses by qRT-PCR. Moreover, the co-localization of pluripotency markers (UCHL-1, PLZF, and DBA) was examined by a double-immunofluorescence method. The cells grown at 37.0 °C showed faster proliferation with a significantly (p

The milieu of testicular germline stem cells (mGSCs) is characterized as low oxygen (O2) environment, whereas, there in-vitro expansion is typically performed under normoxia (20-21% O2). Here, we evaluated and compared the culture and multilineage differentiation characteristics of enriched (through differential platting and percoll density centrifugation) caprine mGSCs (cmGSCs) under hypoxic (5% O2) and normoxic (21% O2) culture conditions. For this, in addition to growth characteristics and population-doubling time (PDT); viability, proliferation, senescence, and expression of key-markers of adhesion (β-integrin and E-Cadherin) and stemness (OCT-4, THY-1 and UCHL-1) were evaluated and compared under normoxia and hypoxia. Moreover, the extent of multilineage differentiation (neurogenic, adipogenic, and chondrogenic differentiation) was assessed. The survival, viability and proliferation were significantly promoted and PDT was reduced (p < 0.05), thus yielding a higher number of viable cells with larger colonies under hypoxia. Furthermore, expression of stemness and adhesion markers was distinctly increased under lowered O2 condition. Conversely, the presence of differentiated regions and expression of differentiation specific key genes [C/EBPα (adipogenic), nestin and β-tubulin (neurogenic), and COL2A1 (chondrogenic)] were significantly (p < 0.05) reduced under hypoxic conditions. These data demonstrate that culturing cmGSCs under hypoxia augments the growth characteristics, and stemness but not the multilineage differentiation potential of cmGSCs as compared with normoxia. These data are important for the development of robust methodologies for ex-vivo expansion and lineage-committed differentiation of cmGSCs for clinical applications.

The present study aims to evaluate season- and reproductive-stage dependent variation in culture characteristics and expression of pluripotency and adhesion markers in caprine-male germline stem cells (cmGSCs). For this, testes from pre-pubertal (4–6 months) and adult (~ 2 years) bucks during non-breeding (July–August; n = 4 each) and breeding (October–November; n = 4 each) seasons were used to isolated testicular cells by two-step enzymatic digestion. After cmGSCs enrichment by multiple methods (differential platting, Percoll density gradient centrifugation, and MACS), cell viability of CD90(+) cells was assessed before co-cultured onto the Sertoli cell feeder layer up to 3(rd)-passage (P-3). The culture characteristics of cmGSCs were compared during primary culture (P-0) and P-3 with different assays [BrdU-assay (proliferation), MTT-assay (senescence), and Cluster-forming activity-assay] and transcript expression analyses by qRT-PCR. Moreover, the co-localization of UCHL-1, CD90, and DBA was examined by a double-immunofluorescence method. In adult bucks, significantly (p