Investigation of subfertility in the female Nsmf knockout mouse.

OBJECTIVE: To study if a pituitary or ovarian defect contributes to subfertility of the female Nsmf knockout (KO) mouse, an animal model of the hypogonadotropic hypogonadism gene NSMF. DESIGN: Analysis of hypothalamic, pituitary and ovarian gene expression at baseline, serum gonadotropin levels before and after gonadotropin-releasing hormone (GnRH) stimulation, ovarian response and implantation after superovulation, gonadotropin effects after ovariectomy, and ovarian NSMF protein expression. SETTING: University research laboratory. PATIENTS: None; mice were used. INTERVENTIONS: Gonadotropin-releasing hormone stimulation, superovulation, and ovariectomy in separate experiments. MAIN OUTCOME MEASURES: Gene expression in the hypothalamus, pituitary, and ovary; ovarian response and implantation after superovulation; serum gonadotropins after GnRH stimulation and ovariectomy; Western blot to measure ovarian NSMF expression. RESULTS: We found increased hypothalamic Kiss1, Gnrh1, and Jak2 mRNA expression in female Nsmf KO vs. wild type (WT) mice. However, pituitary gonadotropin, and GnRH receptor gene expression was not affected, and serum gonadotropin levels were normal. Gonadotropins increased after ovariectomy for both groups. Baseline Kiss1, Fshr, Prkaca, Prkar1a, and Gdf9 ovarian mRNA expression was increased and Cyp19a1 expression was decreased in Nsmf KO mice, while superovulated Nsmf KO mice had reduced ovarian Kiss1r, Prkar1a, and Fshr mRNA expression, 50% less oocytes, and normal implantation. Western blot demonstrated NSMF protein expression in the ovary of WT mice. CONCLUSIONS: Altered hypothalamic and ovarian gene expression was demonstrated in female Nsmf KO mice. It is possible that increased hypothalamic Gnrh1 and Kiss1 mRNA expression could compensate for reduced NSMF enabling a normal pituitary gonadotropin response. Impaired superovulation response, altered ovarian gene expression, and decreased number of oocytes indicate ovarian dysfunction, but a uterine factor cannot be excluded. These findings provide an anatomic basis for future mechanistic studies of subfertility in female Nsmf KO mice.

Permeation of individual cryoprotectants and their different combinations into mouse liver tissue.

Development of successful tissue cryopreservation methods requires specific knowledge regarding tissue permeation of individual cryoprotective agents (CPAs) and their combinations. The present study assessed the permeation of dimethyl sulfoxide, ethylene glycol, and propylene glycol into liver tissue, and addressed whether the diffusion coefficient of individual CPAs changes when combining CPAs. To do this, mouse liver slices were exposed at room temperature to 3.5 mol/L concentrations of CPAs individually or in combination for 15, 30, 45, and 60 min. Subsequently, tissue CPA concentrations were determined using a gas chromatography/mass spectrometry (GC/MS) method. Our results show that (1) the GC/MS method allows measurement of multiple CPA concentrations in a single small tissue sample, (2) dimethyl sulfoxide has a higher diffusion coefficient than ethylene glycol and propylene glycol, and (3) the CPA diffusivity appears to decrease in mixtures with multiple CPAs. These findings may help the development of effective tissue cryopreservation methods.

Hidden Diversity in a Narrow Valley: Description of New Endemic Palearctic Rock Lizard Darevskia (Squamata: Lacertidae) Species from Northeastern Turkey.

Darevskia is a particularly species-rich radiation of Palearctic rock lizards from the Caucasus region. Thanks to intense systematic and taxonomic research, the knowledge of species-level diversity within this genus has increased over the last quarter century. Here, we described a new species, Darevskia salihae sp. nov. from northeastern Turkey. The new taxon is differentiated from other nearby taxon by the low number of dorsal scales in the middle of the body, the shorter body length, and the absence of blue dots both on the lateral region above the forelimbs and on the margin of the ventral plates. In addition to their morphological differences, the new taxon is phylogenetically different from close groups. It is located in a separate subclade from the rudis-valentini-portschinskii subclade. This distinction is supported by both a high bootstrap value (100) and a high posterior probability value (1.00). These two subclades are separated from each other by a genetic distance of almost 4%. This separation is supported not only genetically and morphologically, but also geographically. Since the habitat of the new taxon is limited to a high mountain and a narrow valley, it does not provide an opportunity for a different Darevskia species to shelter because it creates geographical isolation. However, Darevskia parvula that live closest to the habitat of the new taxon live only at the habitat boundaries and do not enter areas where the new taxon is found. Therefore, it might be possible that while it was separated from the rudis-valentini-portschinskii group during the evolutionary transformation, it remained as a refuge and relict in a narrow area as a result of the collapse of the valleys and the partial uplift of the Kaçkar Mountains.

Multiple cryoprotectant toxicity model for vitrification solution optimization.

Vitrification is a promising cryopreservation technique for complex specimens such as tissues and organs. However, it is challenging to identify mixtures of cryoprotectants (CPAs) that prevent ice formation without exerting excessive toxicity. In this work, we developed a multi-CPA toxicity model that predicts the toxicity kinetics of mixtures containing five of the most common CPAs used in the field (glycerol, dimethyl sulfoxide (DMSO), propylene glycol, ethylene glycol, and formamide). The model accounts for specific toxicity, non-specific toxicity, and interactions between CPAs. The proposed model shows reasonable agreement with training data for single and binary CPA solutions, as well as ternary CPA solution validation data. Sloppy model analysis was used to examine the model parameters that were most important for predictions, providing clues about mechanisms of toxicity. This analysis revealed that the model terms for non-specific toxicity were particularly important, especially the non-specific toxicity of propylene glycol, as well as model terms for specific toxicity of formamide and interactions between formamide and glycerol. To demonstrate the potential for model-based design of vitrification methods, we paired the multi-CPA toxicity model with a published vitrification/devitrification model to identify vitrifiable CPA mixtures that are predicted to have minimal toxicity. The resulting optimized vitrification solution composition was a mixture of 7.4 molal glycerol, 1.4 molal DMSO, and 2.4 molal formamide. This demonstrates the potential for mathematical optimization of vitrification solution composition and sets the stage for future studies to optimize the complete vitrification process, including CPA mixture composition and CPA addition and removal methods.

Evaluation the association of facet tropism in multi-sports athletes with cervical disc hernia.

BACKGROUND: Facet tropism (FT) can be defined as the angular difference between the orientation of the right and left facet joints in axial or sagittal planes. Most studies discuss about the relationship with lumbar disc hernia and facet joint angle. However, little is known about the association of facet tropism with disc herniation in the cervical spine in multisports athletes. In this study, We aimed to investigate the relationship between cervical facet tropism and disc hernia in athletes of different branches between the ages of 20-40 from the cervical MR images of the cases. METHODS: This is a retrospective study performed on athletes who applied our hospital between January 2014-2019 with neck pain and have MR imaging of the cervical spine. Cervical MR images of the patients were evaluated by an experienced radiologist from the hospital system database and archives. 79 cases (52 men and 27 women) were included in the study. RESULTS: No statistically significant difference was found between the facet joint angles of both groups at all levels (p˃0.05). Only left C6-7 disc angles of CDH group were measured as 92.99° ± 10.770 (620-1130) and 88.58° ± 7.65° (67°-110°) for the normal group and this difference was found statistically significant (p = 0.007). CONCLUSION: In this study, we did not predict that cervical facet tropism may be a factor associated with cervical disc hernia in young athletes with CDH.

Effectiveness of Ultrasound-Guided Corticosteroid Injections, Prolotherapy, and Exercise Therapy on Partial-Thickness Supraspinatus Tears.

CONTEXT: To investigate the effects of steroid injection (STE), prolotherapy (PRO), and exercise therapy in the treatment of partial tears of the supraspinatus. DESIGN: A retrospective cohort study. METHODS: A total of 64 patients with clinically and radiologically diagnosed partial-thickness supraspinatus tear who received either a cortisone injection (STE), dextrose PRO, or physical therapy combined with home-based exercise therapy were included. Main outcome measures were patients' visual analog scale scores, Western Ontario Rotator Cuff (WORC) Index scores, and the Shoulder Pain and Disability Index scores at the baseline, 3 weeks, and 3 months. RESULTS: The effect of group, time, and group-time interaction on visual analog scale, WORC, and Shoulder Pain and Disability Index scores was statistically significant (P < .001). Visual analog scale and Shoulder Pain and Disability Index scores were the lowest in the STE group at week 3, and the lowest in the PRO group at month 3 (P < .001). WORC scores of the STE group were the highest at week 3 (P < .001). At month 3, WORC scores of STE and PRO groups were similar (P = .089), but significantly higher than exercise therapy. CONCLUSIONS: Corticosteroids provide a fast pain-relieving effect and improvement in function in partial-thickness rotator cuff tears, but these effects diminish over time, whereas PRO provides a long-lasting effect.

General tissue mass transfer model for cryopreservation applications.

Successful cryopreservation of complex specimens, such as tissues and organs, would greatly benefit both the medical and scientific research fields. Vitrification is one of the most promising techniques for complex specimen cryopreservation, but toxicity remains a major challenge because of the high concentration of cryoprotectants (CPAs) needed to vitrify. Our group has approached this problem using mathematical optimization to design less toxic CPA equilibration methods for cells. To extend this approach to tissues, an appropriate mass transfer model is required. Fick's law is commonly used, but this simple modeling framework does not account for the complexity of mass transfer in tissues, such as the effects of fixed charges, tissue size changes, and the interplay between cell membrane transport and transport through the extracellular fluid. Here, we propose a general model for mass transfer in tissues that accounts for all of these phenomena. To create this model, we augmented a previously published acellular model of mass transfer in articular cartilage to account for the effects of cells. We show that the model can accurately predict changes in CPA concentration and tissue size for both articular cartilage and pancreatic islets, tissue types with vastly different properties.

Photobiomodulation has rejuvenating effects on aged bone marrow mesenchymal stem cells.

The plasticity and proliferative capacity of stem cells decrease with aging, compromising their tissue regenerative potential and therapeutic applications. This decline is directly linked to mitochondrial dysfunction. Here, we present an effective strategy to reverse aging of mouse bone marrow mesenchymal stem cells (BM-MSCs) by restoring their mitochondrial functionality using photobiomodulation (PBM) therapy. Following the characterization of young and aged MSCs, our results show that a near-infrared PBM treatment delivering 3 J/cm2 is the most effective modality for improving mitochondrial functionality and aging markers. Furthermore, our results unveil that young and aged MSCs respond differently to the same modality of PBM: whereas the beneficial effect of a single PBM treatment dissipates within 7 h in aged stem cells, it is lasting in young ones. Nevertheless, by applying three consecutive treatments at 24-h intervals, we were able to obtain a lasting rejuvenating effect on aged MSCs. Our findings are of particular significance for improving autologous stem cell transplantation in older individuals who need such therapies most.

Chemically Defined and Xeno-Free Cryopreservation of Human-Induced Pluripotent Stem Cells.

Human-induced pluripotent stem cells (hiPSCs) can be derived from a variety of biopsy samples and have an unlimited capacity for self-renewal and differentiation into almost any cell type in the body. Therefore, hiPSCs offer unprecedented opportunities for patient-specific cell therapies, modeling of human diseases, biomarker discovery, and drug testing. However, clinical applications of hiPSCs require xeno-free and, ideally, chemically defined methods for their generation, expansion, and cryopreservation. In this chapter, we present a chemically defined and xeno-free slow freezing method for hiPSCs along with a chemically undefined protocol. Both approaches yield reasonable post-thaw viability and cell growth.

Chemically Defined, Clinical-Grade Cryopreservation of Human Adipose Stem Cells.

Adipose-derived stem cells (ASCs) reside in the stromal compartment of adipose tissue and can be easily harvested in large quantities through a clinically safe liposuction procedure. ASCs do not induce immunogenic reactions and rather exert immunosuppressive effects. Therefore, they can be used for both autologous and allogeneic transplantations. They hold great promise for cell-based therapies and tissue engineering. A prerequisite to the realization of this promise is the development of successful cryopreservation methods for ASCs. In this chapter, we describe a xeno-free- and chemically defined cryopreservation protocol, which can be used for various clinical applications of ASCs.

Preservation of Mammalian Sperm by Freeze-Drying.

Long-term preservation of mammalian sperm at suprazero temperatures is desired to save storage and space costs, as well as to facilitate transport of preserved samples. This can be accomplished by the freeze-drying of sperm samples. Although freeze-drying results in immotile and membrane-compromised sperm, intracytoplasmic sperm injection (ICSI) can be used to introduce such an immotile sperm into an oocyte and thus start the fertilization process. So far, it has been shown that improved freeze-drying protocols preserve chromosomal integrity and oocyte-activating factor(s) in rodent and mammalian species at 4 °C for several years and at ambient temperature for up to 1 year depending on species, which permits shipping freeze-dried samples at ambient temperature. This chapter concisely reviews freeze-drying of mammalian sperm first and then presents a simple freeze-drying protocol.

Rapid quantification of multi-cryoprotectant toxicity using an automated liquid handling method.

Cryopreservation in a vitrified state has vast potential for long-term storage of tissues and organs that may be damaged by ice formation. However, the toxicity imparted by the high concentration of cryoprotectants (CPAs) required to vitrify these specimens remains a hurdle. To address this challenge, we previously developed a mathematical approach to design less toxic CPA equilibration methods based on the minimization of a toxicity cost function. This approach was used to design improved methods for equilibration of bovine pulmonary artery endothelial cells (BPAEC) with glycerol. To fully capitalize on the toxicity cost function approach, it is critical to describe the toxicity kinetics of additional CPAs, including multi-CPA mixtures that are commonly used for vitrification. In this work, we used automated liquid handling to characterize the toxicity kinetics of five of the most common CPAs (glycerol, dimethyl sulfoxide (DMSO), propylene glycol, ethylene glycol, and formamide), along with their binary and ternary mixtures for BPAEC. In doing so, we developed experimental methods that can be used to determine toxicity kinetics more quickly and accurately. Our results highlight some common CPA toxicity trends, including the relatively low toxicity of ethylene glycol and a general increase in toxicity as the CPA concentration increases. Our results also suggest potential new approaches to reduce toxicity, including a surprising toxicity neutralization effect of glycerol on formamide. In the future, this dataset will serve as the basis to expand our CPA toxicity model, enabling application of the toxicity cost function approach to vitrification solutions containing multiple CPAs.

The effect of plasma melatonin levels in the treatment of lifelong premature ejaculation with selective serotonin reuptake inhibitors.

The aim of our study was to compare melatonin levels of patients with lifelong premature ejaculation (LPE) (n:60) with healthy controls (n:30) and to investigate the changes of melatonin levels in the treatment with dapoxetine and sertraline. Age, body mass index, duration of marriage, weekly intercourse number, International Index of Erectile Function scores, Intravaginal Ejaculation Latency Time (IELT) and melatonin levels were recorded. LPE patients were divided into two treatment groups. The first group was included 30 patients, who received 60 mg dapoxetine for six weeks, twice a week, an hour before intercourse. The second group received 50 mg of sertraline daily, for six weeks. IELT and melatonin measures were repeated after the treatment. IELT (dapoxetine group: 41.22 ± 21.3 s, sertraline group: 48 ± 23.11 s, control group: 195.54 ± 84.14 s; p < .001) and melatonin levels (dapoxetine group: 5.75 ± 2.04 pg/mL, sertraline group: 5.49 ± 2.88 pg/mL, control group: 13.4 ± 12.09 pg/mL; p < .001) of both LPE groups were significantly lower than control group. Following the six-week sertraline (before: 48 ± 23.11 s, after: 101.01 ± 59.55 s; p < .001) and dapoxetine (before: 41.22 ± 21.3 s, after: 97.39 ± 44.1 s; p < .001) treatments, IELT increased. The melatonin levels increased in the sertraline group (before: 5.49 ± 2.88 pg/mL, after: 10.6 ± 7.37 pg/mL; p < .001). Our results indicate that melatonin levels of LPE patients are lower than levels of healthy volunteers. Furthermore, we found a significant increase in melatonin levels following sertraline treatment.

Can serum procalcitonin levels be useful in predicting spontaneous ureteral stone passage?

BACKGROUND: Medical expulsive therapy (MET) is recommended for ureteral stones when there is no indication for interventional treatment. Spontaneous passage (SP) may not always be perceived in patients undergoing MET. We aimed to demonstrate the effects of inflammatory factors on spontaneous ureteral stone passage in patients undergoing MET. METHODS: Our study was conducted between August and November, 2016, in healthy volunteers and patients with a single distal ureteral stone between 5 and 10 mm in diameter and no indications for interventional therapy. Blood and urine samples from all patients and healthy volunteers were tested. The patients were followed up every 2 weeks for 1 month unless emergency situations appeared. Patients with stone-free status at follow-up were concluded to have achieved complete stone passage [SP(+)], and failure [SP(-)] was concluded if the patient had not passed the stone by the end of the study. Blood samples of the patients and the control group were analyzed, recording WBC (white blood cell), CRP (c-reactive protein), SED (sedimentation), MPV (mean platelet volume), NLR (neutrophil-to-lymphocyte ratio), and serum procalcitonin levels. Abnormalities in urine samples were recorded. All patients received diclofenac sodium 75 mg/day, tamsulosin 0.4 mg/day, and at least 3 l/day fluid intake. Patients were followed for a month with kidney, ureter, bladder (KUB) plain films, ultrasonography (USG), and unenhanced abdominal CT scans while undergoing MET. Comparative statistical analyses were performed between the SP(+) and SP(-) groups. RESULTS: The procalcitonin levels of the SP(-) group were significantly higher (207 ± 145.1 pg/ml) than in the SP(+) group (132.7 ± 28.1 pg/ml) (p = 0.000). The leucocyturia rate of the SP(-) group was significantly higher than in the SP(+) group (p = 0.004). Based on the ROC curve analysis, 160 pg/ml (86.7% sensitivity, 70.8% specificity, p < 0.001; AUC: 0.788 95% CI (0.658-0.917) was identified as the optimal cut-off value for procalcitonin. In logistic regression analysis, a significant efficacy of procalcitonin and leucocyturia was observed in the univariate analysis on spontaneous passage. In the multivariate analysis, significant independent activity was observed with procalcitonin. (p < 0.05). CONCLUSION: Our findings suggest that high procalcitonin levels and the presence of leucocyturia have a strong negative effect on SP of ureteral stones between 5 and 10 mm in diameter. This relationship can be explained by stone impaction, possibly caused by increased mucosal inflammation.

Probing lasting cryoinjuries to oocyte-embryo transcriptome.

Clinical applications of oocytes cryopreservation include preservation of future fertility of young cancer patients, substitution of embryo freezing to avoid associated legal and ethical issues, and delaying childbearing years. While the outcome of oocyte cryopreservation has recently been improved, currently used vitrification method still suffer from increased biosafety risk and handling issues while slow freezing techniques yield overall low success. Understanding better the mechanism of cryopreservation-induced injuries may lead to development of more reliable and safe methods for oocyte cryopreservation. Using the mouse model, a microarray study was conducted on oocyte cryopreservation to identify cryoinjuries to transcriptionally active genome. To this end, metaphase II (MII) oocytes were subjected to standard slow freezing, and then analyzed at the four-cell stage after embryonic genome activation. Non-frozen four-cell embryos served as controls. Differentially expressed genes were identified and validated using RT-PCR. Embryos produced from the cryopreserved oocytes displayed 200 upregulated and 105 downregulated genes, associated with the regulation of mitochondrial function, protein ubiquitination and maintenance, cellular response to stress and oxidative states, fatty acid and lipid regulation/metabolism, and cell cycle maintenance. These findings reveal previously unrecognized effects of standard slow oocyte freezing on embryonic gene expression, which can be used to guide improvement of oocyte cryopreservation methods.

Freeze-Dried Extracellular Vesicles From Adipose-Derived Stem Cells Prevent Hypoxia-Induced Muscle Cell Injury.

Cellular therapies have tremendous potential for the successful treatment of major extremity wounds in the combat setting, however, the challenges associated with transplanting stem cells in the prolonged field care (PFC) environment are a critical barrier to progress in treating such injuries. These challenges include not only production and storage but also transport and handling issues. Our goal is to develop a new strategy utilizing extracellular vesicles (EVs) secreted by stem cells that can resolve many of these issues and prevent ischemic tissue injury. While EVs can be preserved by freezing or lyophilization, both processes result in decrease in their bioactivity. Here, we describe optimized procedures for EVs production, isolation, and lyophilization from primary human adipose-derived stem cells (hADSCs). We compared two isolation approaches that were ultrafiltration (UF) using a tangential fluid filtration (TFF) system and differential ultracentrifugation (UC). We also optimized EVs lyophilization in conjunction with trehalose and polyvinylpyrrolidone 40 (PVP40) as lyoprotectants. Bioactivity of EVs was assessed based on reversal of hypoxia-induced muscle cell injury. To this end, primary human myoblasts were subjected to hypoxic conditions for 6 h, and then treated with hADSC-derived EVs at a concentration of 50 µg/mL. Subsequently, muscle cell viability and toxicity were evaluated using MTS and LDH assays, respectively. Overall, nanoparticle tracking data indicated that UF/TFF yields threefold more particles than UC. Lyophilization of EVs resulted in a significantly reduced number of particles, which could be attenuated by adding lyoprotections to the freeze-drying solution. Furthermore, EVs isolated by UF/TFF and freeze-dried in the presence of trehalose significantly increased viability (P < 0.0193). Taken together, our findings suggest that the isolation and preservation methods presented in this study may enhance therapeutic applications of EVs.

Comparison of ultrasound-guided platelet-rich plasma, prolotherapy, and corticosteroid injections in rotator cuff lesions.

BACKGROUND: Injections are a good alternative to conventional treatment-resistant cases with rotator cuff (RC) lesions before operation. Currently, different injection methods are used in RC lesions. OBJECTIVE: To evaluate the efficacy of different injection methods (platelet-rich plasma [PRP], corticosteroid [COR] and prolotherapy [PRO]) in RC tendon lesions. METHODS: One hundred and twenty-nine patients were divided into 4 groups as PRP, COR, PRO and the lidocaine group. Subacromial injection was applied to all groups. They were evaluated using the Visual Analogue Scale (VAS), American Shoulder and Elbow Surgeons Standardized Shoulder Assessment Form (ASES), and Western Ontario Rotator Cuff Index (WORC) at 3, 12 and 24 weeks post-injection. RESULTS: In the COR group in the 3rd week, VAS and WORC scores were significantly lower than the other groups (p< 0.01 and p< 0.05 respectively). In the PRP group in the 24th week, VAS and WORC scores were found to be significantly lower than the COR group (p< 0.01 and p< 0.05 respectively). In the COR group in the 3rd week the ASES score was found to be significantly higher than the PRP and PRO group (p< 0.01). CONCLUSION: In patients with RC lesions, corticosteroid injection provides short-term relief for pain, function, and quality of life, while PRP injection works for long-term wellbeing. For all types of applied injections, improvement in pain, function and quality of life were observed.

Phylogeography of the red-bellied lizard, Darevskia parvula in Turkey.

The groups of red-bellied lizards had a small distribution area in the Pontic zone. The several studies performed on these lizard groups are based on taxonomy and systematics. Although there were several taxonomic or systematic researches on some species of this group, the phylogeographical pattern and species disturbing boundaries of this group is still not clear. In the present study, we aimed to resolve the taxonomic and phylogenetic relationships of the red-bellied lizards in Turkey, based on two combined mitochondrial gene fragments and one protein-coding nuclear gene (rag1). Also, we evaluated ecological niches differentiations among red-bellied lizard groups. The mitochondrial DNA genes were found to be highly polymorphic in this group. One hundred and one variable nucleotide sites were detected on the combined gene sequences. According to phylogenetic trees based on the maximum likelihood (ML) and Bayesian inference (BI), the red-bellied lizards group have three species groups; Darevskia parvula, D. adjarica and unnamed Darevskia sp. (candidate species for Darevskia genus). This situation was supported by high bootstrap and posterior probability values in the trees of mitochondrial DNA gene fragments. However, no genetic variation was detected according to nuclear DNA (rag1) sequence. Because the species groups have no overlaps in terms of their ecological niches, ecological niche modelling (ENM) results revealed differences among the groups of D. parvula, D. adjarica, and unnamed Darevskia sp. Besides, we detected no geographical overlaps among three species groups, since there were geographical isolation zones among the species groups of red-bellied lizard.

Effect of water content on the glass transition temperature of mixtures of sugars, polymers, and penetrating cryoprotectants in physiological buffer.

Long-term storage of viable mammalian cells is important for applications ranging from in vitro fertilization to cell therapy. Cryopreservation is currently the most common approach, but storage in liquid nitrogen is relatively costly and the requirement for low temperatures during shipping is inconvenient. Desiccation is an alternative strategy with the potential to enable viable cell preservation at more convenient storage temperatures without the need for liquid nitrogen. To achieve stability during storage in the dried state it is necessary to remove enough water that the remaining matrix forms a non-crystalline glassy solid. Thus, the glass transition temperature is a key parameter for design of cell desiccation procedures. In this study, we have investigated the effects of moisture content on the glass transition temperature (Tg) of mixtures of sugars (trehalose or raffinose), polymers (polyvinylpyrrolidone or Ficoll), penetrating cryoprotectants (ethylene glycol, propylene glycol, or dimethyl sulfoxide), and phosphate buffered saline (PBS) solutes. Aqueous solutions were dried to different moisture contents by equilibration with saturated salt solutions, or by baking at 95°C. The glass transition temperatures of the dehydrated samples were then measured by differential scanning calorimetry. As expected, Tg increased with decreasing moisture content. For example, in a desiccation medium containing 0.1 M trehalose in PBS, Tg ranged from about 360 K for a completely dry sample to about 220 K at a water mass fraction of 0.4. Addition of polymers to the solutions increased Tg, while addition of penetrating cryoprotectants decreased Tg. Our results provide insight into the relationship between relative humidity, moisture content and glass transition temperature for cell desiccation solutions containing sugars, polymers and penetrating cryoprotectants.

A toxicity cost function approach to optimal CPA equilibration in tissues.

There is growing need for cryopreserved tissue samples that can be used in transplantation and regenerative medicine. While a number of specific tissue types have been successfully cryopreserved, this success is not general, and there is not a uniform approach to cryopreservation of arbitrary tissues. Additionally, while there are a number of long-established approaches towards optimizing cryoprotocols in single cell suspensions, and even plated cell monolayers, computational approaches in tissue cryopreservation have classically been limited to explanatory models. Here we develop a numerical approach to adapt cell-based CPA equilibration damage models for use in a classical tissue mass transport model. To implement this with real-world parameters, we measured CPA diffusivity in three human-sourced tissue types, skin, fibroid and myometrium, yielding propylene glycol diffusivities of 0.6 × 10-6 cm2/s, 1.2 × 10-6 cm2/s and 1.3 × 10-6 cm2/s, respectively. Based on these results, we numerically predict and compare optimal multistep equilibration protocols that minimize the cell-based cumulative toxicity cost function and the damage due to excessive osmotic gradients at the tissue boundary. Our numerical results show that there are fundamental differences between protocols designed to minimize total CPA exposure time in tissues and protocols designed to minimize accumulated CPA toxicity, and that "one size fits all" stepwise approaches are predicted to be more toxic and take considerably longer than needed.