Validation of double freezing protocol for Beetal buck (Capra hircus) spermatozoa.

The study aimed to validate the double versus single freezing protocol for Beetal buck (Capra hircus) spermatozoa in tris-citric acid (TCA) based extender both in terms of quality and fertilization potential. Computer-assisted sperm motion and kinematic (CASA) variables, i.e. total (%), and progressive motilities (TM and PM, %) and rapid velocity (RV, %), average path (VAP, µm/s), straight line (VSL, µm/s) and curved line velocities (VCL, µm/s), straightness, (VSL/VAP, %) and linearity, (VSL/VCL, %) as well as supra-vital plasma membrane integrity (SV-PMI, %), mitochondrial membrane potential (MMP, %), viable/intact acrosome (V-IACR, %) and DNA integrity (DNA-I, %) had significantly greater values (p < .05) during single freeze-thawing as compared with the double freeze-thawing at 0, 30, 90, 150 and 210 days, respectively. All CASA and other assays alone did not show significant differences (p > .05) between both freeze-thaw cycles at all treatment durations, respectively. No statistical significance (p > .05) was observed for the in vivo fertility between single (n = 84/141 = 59.72%) and double freeze-thawing (n = 72/136 = 52.9%) cycles, respectively. In conclusion, sperm motion, kinematics, plasma membrane, acrosome, mitochondria and DNA integrities and in vivo fertility are acceptable after the double freezing protocol despite being lower than after one freeze cycle in Beetal buck.

Evaluation of a new freezing protocol containing 20% dimethyl sulphoxide concentration to cryopreserve human ovarian tissue.

RESEARCH QUESTION: Could a modification in the ovarian tissue freezing protocol improve follicle survival after cryopreservation and xenotransplantation? DESIGN: Ovarian tissue was used from 13 adult patients, frozen either with our original protocol, or a modified version involving a higher concentration of dimethyl sulphoxide (DMSO), larger volume of cryopreservation solution and lower seeding temperature. After thawing, the ovarian fragments were xenotransplanted to six mice with severe combined immunodeficiency (SCID) for 3 weeks. RESULTS: The proportion of primordial follicles decreased, and the proportion of growing follicles increased significantly (all P < 0.01) after cryopreservation and xenografting compared with fresh controls for both protocols. Follicle density, development, ultrastructure and function were similar between treatments. CONCLUSIONS: This study showed that, although the higher DMSO concentration did not improve survival of preantral follicles, it did not seem to induce any major toxicity in the follicle population either.

Cryopreservation of Dental Stem Cells.

Nowadays, regenerative and reparative medicine has grown in popularity. Dental stem cells are easily accessible source of adult stem cells. They can be harvested by a tooth extraction or spontaneous deciduous tooth exfoliation. They have to be isolated, expanded and stored until time they would be needed for individual stem cell therapy. Cryopreservation is both a short-term and long-term storage of tissues or cells at sub-zero temperatures. There are several methods of cryopreservation requiring different technologies. The objective of this review is to compare them and highlight their advantages and disadvantages.

Cryopreserving Rabbit Semen: Impact of Varying Sperm Concentrations on Quality and the Standardization of Protocol.

This study aimed to investigate the impact of sperm concentrations on the in vitro quality of cryopreserved rabbit semen. The semen pools (n = 8, from 80 donors) were split into five aliquots with final sperm concentrations of 15, 25, 35, 55, and 75 × 106 per straw. The sperm motility parameters (CASA system) and membrane integrity (flow cytometric analysis) were both evaluated at various stages of the cryopreservation process: fresh semen dilution, cooling, equilibration, and immediately after and 30 min post-thawing. The results indicated the significant influence of the sperm concentration on the total motility (TM) and progressive motility (PM), with a consistent decline in all sperm variables over the time points. Notably, the semen with a final concentration of 15 × 106 exhibited a higher TM and PM after cooling and equilibration. The post-thawing quality (TM, PM) was higher (p < 0.05) in the mid-range sperm concentrations of 25 × 106 (49.9% and 19.7%) and 35 × 106 (46.2% and 19.7%) compared to the other concentrations. This study demonstrated that the sperm concentration per straw played a significant role in specific phases of the cryopreservation process. These findings contribute valuable insights for refining and standardizing the cryopreservation protocol for rabbit semen, emphasizing the importance of the sperm concentration.

Improving Cell Recovery: Freezing and Thawing Optimization of Induced Pluripotent Stem Cells.

Achieving good cell recovery after cryopreservation is an essential process when working with induced pluripotent stem cells (iPSC). Optimized freezing and thawing methods are required for good cell attachment and survival. In this review, we concentrate on these two aspects, freezing and thawing, but also discuss further factors influencing cell recovery such as cell storage and transport. Whenever a problem occurs during the thawing process of iPSC, it is initially not clear what it is caused by, because there are many factors involved that can contribute to insufficient cell recovery. Thawing problems can usually be solved more quickly when a certain order of steps to be taken is followed. Under optimized conditions, iPSC should be ready for further experiments approximately 4-7 days after thawing and seeding. However, if the freezing and thawing protocols are not optimized, this time can increase up to 2-3 weeks, complicating any further experiments. Here, we suggest optimization steps and troubleshooting options for the freezing, thawing, and seeding of iPSC on feeder-free, Matrigel™-coated, cell culture plates whenever iPSC cannot be recovered in sufficient quality. This review applies to two-dimensional (2D) monolayer cell culture and to iPSC, passaged, frozen, and thawed as cell aggregates (clumps). Furthermore, we discuss usually less well-described factors such as the cell growth phase before freezing and the prevention of osmotic shock during thawing.

Effect of Using Two Cryopreservation Methods on Viability and Fertility of Frozen Stallion Sperm.

Studies involving different methods and techniques of cryopreservation and its interactions with the conception rates in artificial insemination (AI) programs are reported in the literature. This study evaluated the sperm kinetics, plasma membrane integrity, and fertility rates of mares inseminated with cryopreserved stallion semen subjected to different freezing methods. For this, four ejaculates from five stallions were collected and frozen in conventional (Styrofoam box) or automated system in Mini-Digitcool ZH 400. Seminal samples were evaluated after thawing for sperm motion parameters by CASA and plasma membrane integrity by epifluorescence microscopy. For the fertility trial, a cross-over model was performed using 100 cycles of 50 mares, which were inseminated by one the two freezing methods. No differences were observed for sperm motion parameters and plasma membrane integrity between groups (P > .05). The pregnancy rate using the conventional method was 56% (28/50) and did not differ (P = .5406) from the pregnancy rate (64%, 32/50) obtained using the automatized method. The use of semen from fertile stallions may not illustrate small differences in the two freezing methods evaluated. Conventional and automated freezing systems did not differ in the quality and viability of fertile stallion semen and conception rates, indicating that the two methodologies can be safely used in AI programs.

Improved GMP compliant approach to manipulate lipoaspirates, to cryopreserve stromal vascular fraction, and to expand adipose stem cells in xeno-free media.

BACKGROUND: The stromal vascular fraction (SVF) derived from adipose tissue contains adipose-derived stromal/stem cells (ASC) and can be used for regenerative applications. Thus, a validated protocol for SVF isolation, freezing, and thawing is required to manage product administration. To comply with Good Manufacturing Practice (GMP), fetal bovine serum (FBS), used to expand ASC in vitro, could be replaced by growth factors from platelet concentrates. METHODS: Throughout each protocol, GMP-compliant reagents and devices were used. SVF cells were isolated from lipoaspirates by a standardized enzymatic protocol. Cells were cryopreserved in solutions containing different albumin or serum and dimethylsulfoxide (DMSO) concentrations. Before and after cryopreservation, we analyzed: cell viability (by Trypan blue); immunophenotype (by flow cytometry); colony-forming unit-fibroblast (CFU-F) formation; and differentiation potential. ASC, seeded at different densities, were expanded in presence of 10% FBS or 5% supernatant rich in growth factors (SRGF) from platelets. The differentiation potential and cell transformation grade were tested in expanded ASC. RESULTS: We demonstrated that SVF can be obtained with a consistent yield (about 185 × 103 cells/ml lipoaspirate) and viability (about 82%). Lipoaspirate manipulation after overnight storage at +4 °C reduced cell viability (-11.6%). The relative abundance of ASC (CD34+CD45-CD31-) and endothelial precursors (CD34+CD45-CD31+) in the SVF product was about 59% and 42%, respectively. A period of 2 months cryostorage in autologous serum with added DMSO minimally affected post-thaw SVF cell viability as well as clonogenic and differentiation potentials. Viability was negatively affected when SVF was frozen at a cell concentration below 1.3 × 106 cells/ml. Cell viability was not significantly affected after a freezing period of 1 year. Independent of seeding density, ASC cultured in 5% SRGF exhibited higher growth rates when compared with 10% FBS. ASC expanded in both media showed unaltered identity (by flow cytometry) and were exempt from genetic lesions. Both 5% SRGF- and 10% FBS-expanded ASC efficiently differentiated to adipocytes, osteocytes, and chondrocytes. CONCLUSIONS: This paper reports a GMP-compliant approach for freezing SVF cells isolated from adipose tissue by a standardized protocol. Moreover, an ASC expansion method in controlled culture conditions and without involvement of animal-derived additives was reported.