Cryoprotective Medium Research Articles

The development of the cell cryopreservation protocol with controlled rate thawing

Thawing in the water bath is often considered as a standard procedure. The thermal history of samples thawed in this way is poorly controlled, but cryopreservation and banking of cell-based products require standardization, automation and safety of all the technological stages including thawing. The programmable freezers allow implementation of the controlled cooling as well as the controlled thawing. As the cell damage occurs during the phase transformation that takes place in the cryoprotectant medium in the process of freezing-thawing, the choice of warming rates within the temperature intervals of transformations is very important. The goal of the study was to investigate the influence of warming rates within the intervals of the phase transformations in the DMSO-based cryoprotectant medium on the cell recovery and to develop a cryopreservation protocol with controlled cooling and warming rates. The temperature intervals of phase transformations such as melting of the eutectic mixture of the cryoprotectant solution (MEMCS), melting of the eutectic salt solution (MESS), melting of the main ice mass (MMIM), recrystallization before MEMCS, recrystallization before MESS and recrystallization before MMIM were determined by thermo-mechanical analysis. The biological experiments were performed on the rat testicular interstitial cells (TIC). The highest levels of the cell recovery and metabolic activity after cryopreservation were obtained using the protocol with the high (20°C/min) warming rate in the temperature intervals of crystallization of the eutectics as well as recrystallizations and the low (1°C/min) warming rate in the temperature intervals of melting of the eutectics as well as MMIM. The total cell recovery was 65.3±2.1%, the recovery of the 3-beta-HSD-positive (Leydig) cells was 82.9±1.8%, the MTT staining was 32.5±0.9% versus 42.1±1.7%; 57.4±2.1% and 24.0±1.1% respectively, when compared to the thawing in the water bath.

Melatonin Effect on Immature Mouse Testicular Tissues, Vitrified-Thawed With Different Cryoprotectant Media

Background: Freezing of the immature testis tissue and then its transplantation can be considered a major step in fertility preservation for young boys with cancer. Objectives: The objective of this study was to investigate the antioxidant effects of melatonin on histological structure of seminiferous tubules that were vitrified-thawed with different cryoprotectant media. Materials and Methods: Male mice were randomly assigned to one of 10 experimental groups. Testes of 6-day-old BALB/c male mice pups were extracted and vitrified-thawed with different vitrification media with melatonin and without melatonin. Then, their testes were stained with haematoxylin-eosin. Results: The result showed that supplementation of glycerol based media with melatonin has reduced injury to histological structure of seminiferous tubules in the 6-day-old male BALB/c mice pups testes (P = 0.004). Conclusions: It seem that melatonin due to its antioxidant properties, if added to glycerol based media, reduces the harmful effects of freezing-thawing process, and protects seminiferous tubules histological structure from oxidative injury.

Optimization of the nanocellulose based cryoprotective medium to enhance the viability of freeze dried Lactobacillus plantarum using response surface methodology

Nanocellulose was produced from cotton lint and it was used as an agent of cryoprotective medium. The microstructures and surface morphologies were examined by field emission scanning electron and atomic force microscopes. Particles had a width of less than 50 nm and the length of hundreds of nanometers to several micrometers. According to the results of wide-angle X-ray diffraction, crystalline index was 74.07% and the average thickness of nanocellulose was 3.18 nm. Response surface coefficients and P values showed that the viability of Lactobacillus plantarum after freeze drying was highly dependent on nanocellulose concentration (P < 0.01). The highest viability of L. plantarum was obtained at the concentration of skim milk 13.75%, the concentration of trehalose 20.5% and the concentration of nanocellulose 13.75%. Under the optimized conditions, the predicted survival rate and observed experimental values for L. Plantarum, were 1.93 ± 0.1 × 109 CFU/ml and 1.85 ± 0.1 × 109 CFU/ml, respectively.

C-1013: Cryopreservation of spermic milt in the model species Ambystoma tigrinum (Tiger salamander) for application in endangered salamanders

In the face of the amphibian extinction crisis, captive assurance colonies have been established to preserve genetic diversity. Sadly, many captive assurance colonies fail due to reproductive cues which are not entirely understood. In addition, the majority of amphibian conservation and genome banking research has focused on anurans (frogs/toads), despite the fact that caudates (salamanders/newts) face a larger threat of extinction. Therefore, it is critical that assisted reproductive technologies, such as gamete cryopreservation and artificial fertilization, be developed as conservation tools for application in genetic management of threatened caudates. It was hypothesized that a modified anuran sperm freezing protocol could be successfully used in cryopreservation of Ambystoma tigrinum (tiger salamander) sperm as a caudate model. Spermiation in male tiger salamanders was hormonally induced via injection with hCG, LHRH, or a combination of these two hormones. Average spermic milt concentrations were 2.75 × 107(±7.2 × 106)/mL. Average total motility upon collection of fresh milt was 53.6% (±17.6%). After collection and initial analysis, fresh milt samples were diluted 1:1 in chilled cryoprotectant treatment solutions. They were then frozen in 0.25 mL French straws using a stepwise slow-rate freezing process. Upon thawing, milt suspensions were diluted 1:10 with a 10% Holtfreter’s solution in order to activate the sperm for post thaw analysis. Frozen-thawed sperm in the 5% Me2SO treatment group had an average total motility of 19.1% (±15.8%) and sperm in the 5% Me2SO with 0.5% BSA treatment group had an average total motility of 21.75% (±14.8%). Early trials showed that caudate sperm are not susceptible to toxicity from several cryoprotective media. Moreover, initial fertilization trials indicate that frozen-thawed caudate sperm are capable of producing early cleavage stage embryos. Optimizing cryopreservation protocols for salamander sperm is an important step in building a global genetic resource bank for endangered caudates.

Analysis of the Effect of Cryoprotectant Medium Composition to Viability of Autologous Hematopoietic Cells Collected by Leukapheresis

ObjectivesCryopreservation of hematopoietic stem cells intended for autologous transplantation is a crucial element of the banking process. Although cryopreservation techniques are well known, improvement is needed. This study was designed to optimize cryopreservation to improve the quantitative and qualitative parameters of hematopoietic stem cells in the material intended for transplantation. We used available opportunities to provide the best quantitative and qualitative parameters of hematopoietic stem cell transplants processed in a closed system. Material and methodsTwo hundred forty-eight products of hematopoietic stem cells collected by leukapheresis from patients with lymphoproliferative disorders create the basis of this report. The material was frozen in a controlled-rate freezer and stored in containers in the vapor phase of LN2 (−160°C). The composition of a cryoprotectant medium was modified. For freezing, 192 probes were used with a cryoprotective medium containing 20% dimethyl sulfoxide (DMSO) and enriched RPMI 1640. For 56 samples, we used 20% DMSO in autologous plasma harvested during leukapheresis. Products of hematopoietic stem cells and cryoprotectant medium were combined in a 1:1 ratio. The final number of nuclear cells did not exceed 2 × 108/mL. Analysis was performed after thawing the probes. Viability of nuclear cells has been assessed using the microscopic technique after incubation in Trypan blue and the CD34+ cells by flow cytometry using the 7-aminoactynomycin D. A statistical analysis has been conducted using the Statistica program (StatSoft, Cracow, Poland). Results and conclusionsThe results show that the application of autologous plasma is linked with higher viability of nuclear cells and CD34+ cells. Moreover, statistical analysis of the nuclear cells and CD34+ cells viability differs significantly between groups frozen using RPMI 1640 and autologous plasma (P < .05). To assess the viability of CD34+, cells frozen using RPMI 1640 results showed a large span of at 16.4% to 99.1% living cells.

Permeability of isolated rat hepatocyte plasma membranes for molecules of dimethyl sulfoxide

We have studied permeability of isolated rat hepatocyte membranes for molecules of dimethyl sulfoxide (DMSO) at different hypertonicity of a cryoprotective medium. The permeability coefficient of hepatocyte membranes κ1 for DMSO molecules was shown to be the differential function of osmotic pressure between a cell and an extracellular medium. Ten-fold augmentation of DMSO concentration in the cryoprotective medium causes the decrease of permeability coefficients κ1 probably associated with the increased viscosity in membrane-adjacent liquid layers as well as partial limitations appeared as a result of change in cell membrane shape after hepatocyte dehydration. We have found out that in aqueous solutions of NaCl (2246 mOsm/l) and DMSO (2250 mOsm/l) the filtration coefficient L(p) in the presence of a penetrating cryoprotectant (L(pDMSO) = (4.45 ± 0.04) x 10(-14) m3/Ns) is 3 orders lower compared to the case with electrolyte (L(pNaCl) = (2.25 ± 0.25) x 10(-11) m3/Ns). This phenomenon is stipulated by the cross impact of flows of a cryoprotectant and water at the stage of cell dehydration. Pronounced lipophilicity of DMSO, geometric parameters of its molecule as well as the presence of large aqueous pores in rat hepatocyte membranes allow of suggesting the availability of two ways of penetrating this cryoprotectant into the cells by non-specific diffusion through membrane lipid areas and hydrophilic channels.

Investigation of membrane permeability of carp spermatozoa for water molecules

The fundamentals of a photometry method for determination of membrane permeability of some fish spermatozoa for water molecules are presented. Osmotic tolerance of carp spermatozoa membranes was studied using EPR-spectroscopy and photometric analysis methods. It was shown that carp spermatozoa look like the ideal osmometers in their reaction on media of different osmolarity. The value of membrane permeability of carp spermatozoa for water molecules was determined. Data obtained can be used in cryobiology for creating cryoprotective media and regimes of fish sperm cryopreservation.

Addition of Tempol in semen cryopreservation medium improves the post-thaw sperm function

Despite extensive research carried out for optimization and commercialization of sperm cryopreservation media, percentage of motility and viability remain low following cryopreservation. These adverse effects have been partially ascribed to reactive oxygen species (ROS) production during cryopreservation. Therefore, we proposed that addition of a cell permeable antioxidant like Tempol, with superoxide dismutase (SOD) mimetic action, may overcome these effects in an optimized commercially available cryo-protective medium. Therefore, semen samples were cryopreserved in the presence or absence of Tempol. A concentration of 5 μM Tempol was defined as optimal since it significantly improved motility and viability post thawing and reduced DNA fragmented sperm. In addition, percentage of ROS positive sperm was reduced. These effects of Tempol can be attributed to cell permeability characteristic and ability to reduce superoxide production both at intra- and extra-cellular levels. Tempol may hold the potential for clinical applications.

LOW DENSITY LIPOPROTEINS AS CRYOPROTECTANTS OF RABBIT SEMEN CRYOPRESERVATION

The present study was carried out at the International livestock management training center, Sakha, Kafrelsheikh, belonging to Animal Production Research Institute, Agricultural Research Center, Ministry of Agriculture, Egypt; during the period from March to May 2013. Aim of this study was to evaluate sperm characteristics and fertility of rabbit buck semen cryopreserved in tris-extender containing different levels of low density lipoproteins (LDL) as replacement of egg yolk. Semen was collected twice weekly form twenty APRI rabbit bucks using artificial vagina. Only ejaculates with ≥ 70% mass motility were used for dilution and processing. Four extenders were used: Tris 20% egg yolk extender as a control, and substitution of whole egg yolk with 8, 10 and 12% LDL. Semen was diluted and packaged into 0.25 ml straws, cooled, held at 5◦C for 4 h , and then frozen in liquid nitrogen and stored at -196◦C. Percentages of progressive motility, livability, abnormality and intact acrosome were determined at post-dilution, -equilibration, -thawing. Results indicated that LDL extenders were more effective in preservation of rabbit sperm characteristics than whole egg yolk extender. Percentages of progressive motility, livability, abnormality and intact acrosome were much higher (P<0.05) in the 10% LDL extender, being 49.5, 61.6, 10.4 and 71.3%versus 30.5, 38.2, 13.3 and 58.6% in the control extender in post-thawed semen, respectively. Fertility rates were higher in extender containing 10% LDL compared with the control (75 vs. 60%). In conclusion, an extender containing 10% LDL extracted from hen egg yolk could be used as a cryoprotective media because it improved the freezability and fertility of rabbit spermatozoa.

Функциональная активность адренокортикоцитов крыс после инкубации в криозащитных средах на основе ДМСО, содержащих эмбриональную телячью сыворотку

Investigation of functional state of cells at various stages of cryopreservation is important for the development and improvement of freezing protocols of endocrine cells. This study assessed the effect of incubation in media with 5, 7, 10, and 15% dimethyl sulfoxide (DMSO), as well as the media with DMSO and 25% fetal bovine serum (FBS) on some indices of adrenal cortex cell suspension: viability (MTT-test), content of cells with active 3b-hydroxysteroid dehydrogenase, functionally active mitochondria (JC‑1 test), as well as the level of basal and stimulated aldosterone secretion. It was shown that presence of FBS in a cryoprotective medium reduced the toxic effect of DMSO on adrenocorticocytes during incubation, furthermore in some cases incubation in media with DMSO and FBS led to the increase in the number of cells with functionally active mitochondria and cells with active steroidogenesis if compared with the suspensions with cells not exposed to cryoprotectant solution. Probl Cryobiol Cryomed 2014; 24(1):47–56.

Towards an advanced therapy medicinal product based on mesenchymal stromal cells isolated from the umbilical cord tissue: quality and safety data

IntroductionStandardization of mesenchymal stromal cells (MSCs) manufacturing is urgently needed to enable translational activities and ultimately facilitate comparison of clinical trial results. In this work we describe the adaptation of a proprietary method for isolation of a specific umbilical cord tissue-derived population of MSCs, herein designated by its registered trademark as UCX®, towards the production of an advanced therapy medicinal product (ATMP).MethodsThe adaptation focused on different stages of production, from cell isolation steps to cell culturing and cryopreservation. The origin and quality of materials and reagents were considered and steps for avoiding microbiological and endotoxin contamination of the final cell product were implemented. Cell isolation efficiency, MSCs surface markers and genetic profiles, originating from the use of different medium supplements, were compared. The ATMP-compliant UCX® product was also cryopreserved avoiding the use of dimethyl sulfoxide, an added benefit for the use of these cells as an ATMP. Cells were analyzed for expansion capacity and longevity. The final cell product was further characterized by flow cytometry, differentiation potential, and tested for contaminants at various passages. Finally, genetic stability and immune properties were also analyzed.ResultsThe isolation efficiency of UCX® was not affected by the introduction of clinical grade enzymes. Furthermore, isolation efficiencies and phenotype analyses revealed advantages in the use of human serum in cell culture as opposed to human platelet lysate. Initial decontamination of the tissue followed by the use of mycoplasma- and endotoxin-free materials and reagents in cell isolation and subsequent culture, enabled the removal of antibiotics during cell expansion. UCX®-ATMP maintained a significant expansion potential of 2.5 population doublings per week up to passage 15 (P15). They were also efficiently cryopreserved in a DMSO-free cryoprotectant medium with approximately 100% recovery and 98% viability post-thaw. Additionally, UCX®-ATMP were genetically stable upon expansion (up to P15) and maintained their immunomodulatory properties.ConclusionsWe have successfully adapted a method to consistently isolate, expand and cryopreserve a well-characterized population of human umbilical cord tissue-derived MSCs (UCX®), in order to obtain a cell product that is compliant with cell therapy. Here, we present quality and safety data that support the use of the UCX® as an ATMP, according to existing international guidelines.

An outcome analysis and long-term viability of cryopreserved cultured epidermal allografts: assessment of the conservation of transplantable human skin allografts

To assess the viability of cultured epithelium and preserved by freezing for periods varying from one month to one year. Samples of cultured epithelium were incubated in cryoprotectant medium (Group A), packed in aluminum envelopes and packed in polystyrene boxes. The boxes were subjected to a temperature of-70 ºC. After freezing for a period of time ranging from one to 12 months, cultured epithelial samples were assessed for their viability by vital staining (Trypan blue) and metabolic analysis based on glucose consumption and lactate production. Samples of not frozen cultured epithelium (Group B) were also tested for viability and the results obtained were used as comparison parameter for the variation of viability. Statistical analysis between the group A and B indicate that the mean age of the donors (p=0.51) and the culture time (p=1.18) showed no statistical difference. In 30 days we obtained 37% of the original viability of cultured epithelium, 25% at six months and one year, less than 15%. This trend was confirmed statistically with a reduction of approximately 1.8% of the original viability epithelium cultured every 30 days of storage. In the analysis by lactate production, similar results were observed. In the analysis by the glucose consumption results were not significant. The viability indices show statistically significant difference between the group A and B (p<0.0001). Although cryopreserved cultured epithelium showed significant reduction of viability, all samples remained viable. It was also found that the viability of cryopreserved cultured epithelial decreased as a function of storage time.

032 Cryobanking of testicular tissue in men of reproductive age

Cryopreservation of ejaculated or testicular spermatozoa is recommended for fertility preservation in adolescence and adult men. However cryobanking of the properly preserved human testicular tissue in men of reproductive age is an essential source for further clinical application and additional examination for diagnostic purposes. During the last three years 107 infertile patients were referred for diagnostic testicular biopsy to the Andrology Laboratory, Center of Andrology and Sexual Medicine at Karolinska University Hospital, Stockholm, Sweden. All patients had azoospermia with a testis volume of 1–15 mL. An open biopsy was performed in all cases. The primary section of the biopsy was disaggregated for testicular spermatozoa retrieval for cryopreservation and subsequent use for intracytoplasmic sperm injection (ICSI). Two fresh pieces were fixed for histological evaluation and early cancer detection. As a part of our routine clinical protocol, we typically assess spermatogenesis in semi-thin plastic embedded sections and cryopreserve a further piece of each testis for an additional diagnostic examination. The following three protocols – conventional cooling in two different devices and vitrification, were applied and adjusted for a proper cryopreservation of the testicular tissue as a multicellular system: 1. Small 1–2 × 2 × 2 mm pieces were washed in basic solution, equilibrated in cryoprotectant medium with 0.7 M Me 2 SO for 30 min at +4 °C and cryopreserved by slow programmed cooling protocol, established for cryopreservation of prepubertal testicular tissue in 1.8 mL Nunc cryovials according Keros et al., Hum Reprod., 2007. 2. To preserve tissue for possible clinical application and avoid leaking of LN2 when storing in a liquid faze nitrogen, cryovials were replaced by CBS™ High Security tissue 2 mL straw (CryoBioSystem) and cryopreserved in a closed system using the same protocol. This resulted in a visible structural damage of the tissue. To achieve an appropriate survival of all types of testicular cells, the protocol was optimised, 1.1 M Me 2 SO was used as a cryoprotectant (CPA) and the temperature of initiated seeding was changed to −5 °C. 3. It is well established that cryopreservation of the tissue in pieces as a multicellular system is more complicated and conventional cryopreservation results in harmful ice formation in the cells and in the surrounding extracellular matrix. As an alternative to conventional cryopreservation we applied the vitrification protocol developed for human ovarian tissue (Keros et al., 2009, Hum Reprod ). For this combination of Me 2 SO, Propanediol, Ethylene glycol, PVP was applied. Ice crystal formation in human testicular tissue at ultra rapid cooling and warming rates was analysed by freeze substitution technique and transmission electron microscopy. On the basis of our experience, we summarize that human testicular tissue can be successfully cryopreserved by programmed slow freezing using cryovials or tissue straws as well as by vitrification. All types of testicular cells, including cells of different stages of spermatogenesis, motile spermatozoa, Leydig cells and Sertoli cells can survive cryopreservation and long-term storage in liquid nitrogen when an optimized cryopreservation protocol is used. Source of funding: The National council for human tissues and cells (Nationella Vävnadsrådet) at The Swedish Association of Local Authorities and Regions (SKL). Conflict of interest: None declared. Victoria.Keros@ki.se

Tumeurs malignes de localisation ORL chez l’enfant : une expression clinique initiale banale, un diagnostic pas si exceptionnel

Post-thaw motility rate, curvilinear velocity (VCL), and fertilizing ability of carp spermatozoa can be improved by short-term treatment with moderately hypotonic media prior to freezing. Before cryopreservation, carp sperm samples were treated with NaCl solutions of differing osmolalities, ranging from 100 to 300 mOsm kg−1 for 10 s, after which final osmolality was adjusted to 300 mOsm kg−1. The resulting sperm suspension was diluted 1:1 with cryoprotective medium and frozen using conventional techniques. Control samples were treated in the same way, without the pre-dilution step. Post-thaw motility rate in samples pretreated with 200 mOsm kg1 NaCl was significantly higher (44 ± 10%) than in controls (21 ± 15%) and samples pretreated with 100 mOsm kg−1 (25 ± 15%) and 300 mOsm kg−1 (25 ± 12%) NaCl. Significantly higher mean VCL were observed in samples pretreated with 100, 150, and 200 mOsm kg−1 (119 ± 24, 118 ± 22, and 115 ± 32 μm s−1, respectively) compared to controls (92 ± 27 μm s−1). Fertilization rate of frozen-thawed sperm treated with 200 mOsm kg−1 solution of 2 M NaCl was significantly higher (25 ± 18%) than that of sperm treated with 300 mOsm kg−1 NaCl solutions (12 ± 7%) and the control (9 ± 6%).

Efficient cryopreservation protocol enables accessibility of a broad range of ammonia-oxidizing bacteria for the scientific community

Long-term storage of the fastidious ammonia-oxidizing bacteria has proven difficult, which limits their public availability and results in a loss of cultured biodiversity. To enable their accessibility to the scientific community, an effective protocol for cryopreservation of ammonia-oxidizing cultures at −80 °C and in liquid nitrogen was developed. Long-term storage could be achieved using 5% DMSO as cryoprotectant, preferably in a cryoprotective preservation medium containing tenfold-diluted trypticase soy broth and 1% trehalose. As such, successful activity and growth recovery was observed for a diverse set of ammonia-oxidizing cultures.

Hypotonic treatment prior to freezing improves cryoresistance of common carp (Cyprinus carpio L.) spermatozoa

Post-thaw motility rate, curvilinear velocity (VCL), and fertilizing ability of carp spermatozoa can be improved by short-term treatment with moderately hypotonic media prior to freezing. Before cryopreservation, carp sperm samples were treated with NaCl solutions of differing osmolalities, ranging from 100 to 300mOsmkg−1 for 10s, after which final osmolality was adjusted to 300mOsmkg−1. The resulting sperm suspension was diluted 1:1 with cryoprotective medium and frozen using conventional techniques. Control samples were treated in the same way, without the pre-dilution step. Post-thaw motility rate in samples pretreated with 200mOsmkg1 NaCl was significantly higher (44±10%) than in controls (21±15%) and samples pretreated with 100mOsmkg−1 (25±15%) and 300mOsmkg−1 (25±12%) NaCl. Significantly higher mean VCL were observed in samples pretreated with 100, 150, and 200mOsmkg−1 (119±24, 118±22, and 115±32μms−1, respectively) compared to controls (92±27μms−1). Fertilization rate of frozen-thawed sperm treated with 200mOsmkg−1 solution of 2M NaCl was significantly higher (25±18%) than that of sperm treated with 300mOsmkg−1 NaCl solutions (12±7%) and the control (9±6%).

Comparative Fertility of Freshly Collected vs Frozen–Thawed Semen with Laparoscopic Oviductal Artificial Insemination in Domestic Cats

Artificial insemination (AI) is potentially invaluable as an adjunct to natural breeding for the conservation management of non-domestic felid populations. The efficacy of AI, however, must be substantially improved for applied use, especially when using frozen semen. Our recent advances in using laparoscopic oviductal AI (LO-AI) with low sperm numbers and freezing of cat semen in a soy lecithin-based cryoprotectant medium suggest that combining these two approaches might improve pregnancy outcomes with frozen-thawed spermatozoa. In this study, our objectives were to (i) assess the effect of two gonadotropin dosages (100 vs 150 IU eCG) on ovarian response in domestic cats and (ii) compare the relative fertility of frozen-thawed and fresh semen in vivo following LO-AI. All 16 females ovulated after gonadotropin treatment and were inseminated with fresh semen from one male and frozen-thawed semen from a second male. There were no differences between gonadotropin dosages in CL number, pregnancy percentage or litter size. Half (8/16) of the females conceived, with seven females giving birth to a total of 36 offspring. Paternity analysis showed that more kittens resulted from LO-AI with fresh (28/36, 78%) than frozen-thawed (8/36, 22%) semen, possibly due to impaired motility and longevity of thawed sperm. These results demonstrated that viable offspring can be produced by AI using semen frozen in a soy lecithin-based medium. Insemination with greater numbers of frozen-thawed spermatozoa, combined with further refinement of cat sperm cryopreservation methods, may be necessary to optimize pregnancy success with LO-AI in domestic and nondomestic cats.

Using Unfixed, Frozen Tissues to Study Natural Mucin Distribution

Mucins are complex and heavily glycosylated O-linked glycoproteins, which contain more than 70% carbohydrate by weight1-3. Secreted mucins, produced by goblet cells and the gastric mucosa, provide the scaffold for a micrometers-thick mucus layer that lines the epithelia of the gut and respiratory tract3,4. In addition to mucins, mucus layers also contain antimicrobial peptides, cytokines, and immunoglobulins5-9. The mucus layer is an important part of host innate immunity, and forms the first line of defense against invading microorganisms8,10-12. As such, the mucus is subject to numerous interactions with microbes, both pathogens and symbionts, and secreted mucins form an important interface for these interactions. The study of such biological interactions usually involves histological methods for tissue collection and staining. The two most commonly used histological methods for tissue collection and preservation in the clinic and in research laboratories are: formalin fixation followed by paraffin embedding, and tissue freezing, followed by embedding in cryo-protectant media.Paraffin-embedded tissue samples produce sections with optimal qualities for histological visualization including clarity and well-defined morphology. However, during the paraffin embedding process a number of epitopes become altered and in order to study these epitopes, tissue sections have to be further processed with one of many epitope retrieval methods13. Secreted mucins and lipids are extracted from the tissue during the paraffin-embedding clearing step, which requires prolong incubation with organic solvents (xylene or Citrisolv). Therefore this approach is sub-optimal for studies focusing on the nature and distribution of mucins and mucus in vivo.In contrast, freezing tissues in Optimal Cutting Temperature (OCT) embedding medium avoids dehydration and clearing of the sample, and maintains the sample hydration. This allows for better preservation of the hydrated mucus layer, and thus permits the study of the numerous roles of mucins in epithelial biology. As this method requires minimal processing of the tissue, the tissue is preserved in a more natural state. Therefore frozen tissues sections do not require any additional processing prior to staining and can be readily analyzed using immunohistochemistry methods.We demonstrate the preservation of micrometers-thick secreted mucus layer in frozen colon samples. This layer is drastically reduced when the same tissues are embedded in paraffin. We also demonstrate immunofluorescence staining of glycan epitopes presented on mucins using plant lectins. The advantage of this approach is that it does not require the use of special fixatives and allows utilizing frozen tissues that may already be preserved in the laboratory.

Using Unfixed, Frozen Tissues to Study Natural Mucin Distribution

Mucins are complex and heavily glycosylated O-linked glycoproteins, which contain more than 70% carbohydrate by weight1-3. Secreted mucins, produced by goblet cells and the gastric mucosa, provide the scaffold for a micrometers-thick mucus layer that lines the epithelia of the gut and respiratory tract3,4. In addition to mucins, mucus layers also contain antimicrobial peptides, cytokines, and immunoglobulins5-9. The mucus layer is an important part of host innate immunity, and forms the first line of defense against invading microorganisms8,10-12. As such, the mucus is subject to numerous interactions with microbes, both pathogens and symbionts, and secreted mucins form an important interface for these interactions. The study of such biological interactions usually involves histological methods for tissue collection and staining. The two most commonly used histological methods for tissue collection and preservation in the clinic and in research laboratories are: formalin fixation followed by paraffin embedding, and tissue freezing, followed by embedding in cryo-protectant media. Paraffin-embedded tissue samples produce sections with optimal qualities for histological visualization including clarity and well-defined morphology. However, during the paraffin embedding process a number of epitopes become altered and in order to study these epitopes, tissue sections have to be further processed with one of many epitope retrieval methods13. Secreted mucins and lipids are extracted from the tissue during the paraffin-embedding clearing step, which requires prolong incubation with organic solvents (xylene or Citrisolv). Therefore this approach is sub-optimal for studies focusing on the nature and distribution of mucins and mucus in vivo. In contrast, freezing tissues in Optimal Cutting Temperature (OCT) embedding medium avoids dehydration and clearing of the sample, and maintains the sample hydration. This allows for better preservation of the hydrated mucus layer, and thus permits the study of the numerous roles of mucins in epithelial biology. As this method requires minimal processing of the tissue, the tissue is preserved in a more natural state. Therefore frozen tissues sections do not require any additional processing prior to staining and can be readily analyzed using immunohistochemistry methods. We demonstrate the preservation of micrometers-thick secreted mucus layer in frozen colon samples. This layer is drastically reduced when the same tissues are embedded in paraffin. We also demonstrate immunofluorescence staining of glycan epitopes presented on mucins using plant lectins. The advantage of this approach is that it does not require the use of special fixatives and allows utilizing frozen tissues that may already be preserved in the laboratory.

Cryoprotective effects of low-density lipoproteins, trehalose and soybean lecithin on murine spermatogonial stem cells

Spermatogonial stem cells (SSCs) have the ability to self-renew and offer a pathway for genetic engineering of the male germ line. Cryopreservation of SSCs has potential value for the treatment of male infertility, spermatogonial transplantation, and so on. In order to investigate the cryopreservation effects of different cryoprotectants on murine SSCs, 0.2 M of low-density lipoproteins (LDL), trehalose and soybean lecithin were added to the cryoprotective medium, respectively, and the murine SSCs were frozen at -80°C or -196°C. The results indicated that the optimal recovery rates of murine SSCs in the cryoprotective medium supplemented with LDL, trehalose and soybean lecithin were 92.53, 76.35 and 75.48% at -80°C, respectively. Compared with freezing at -196°C, the optimum temperature for improvement of recovery rates of frozen murine SSCs, cryopreservation in three different cryoprotectants at -80°C, were 17.11, 6.68 and 10.44% respectively. The recovery rates of murine SSCs in the cryoprotective medium supplemented with 0.2 M LDL were significantly higher than that of other cryoprotectants (P < 0.05). Moreover, the recovery rates were demonstrated to be greater at -80°C compared with at -196°C (P < 0.05). In conclusion, 0.2 M of LDL could significantly protect murine SSCs at -80°C. In the freezing-thawing process, LDL is responsible for the cryopreservation of murine SSCs because it can form a protective film at the surface of membranes. However, more research is needed to evaluate and understand the precise role of LDL during the freezing-thawing of SSCs.