The Technologies in Cryopreservation - Lab Report Example

Cryopreservation of Zebrafish Ovarian Follicles Practical Session on Cryopreservation of Zebrafish Ovarian Follicles Using Controlled Slow-Cooling and Vitrification 1. Controlled Slow-Cooling: Introduction: Cryopreservation is a technology that allows humans to interfere with the biological clockwork to stop “biological time” (Ozkavukcu & Erdemli 2002: 187). It plays a significant role in tissue banking and is expected to assume higher significance in the future when more tissue-engineered products will enter the clinical arena. The most common concept that underlies tissue engineering is combining the living cells with a scaffold or matrix to form a tissue-engineered construct in order to promote the regeneration and repair of tissues, which plays a key role in the treatment of many diseases as well as in the treatment of problems like infertility . The technologies in cryopreservation have undergone tremendous advances over the last decade. In addition, cryopreservation of oocytes and embryos is also being used as an effective means of treating infertility. In this technology, the clinical application seeks to ensure the optimal survival of embryos and oocytes that are subsequently thawed and stored for transfer . The aim of this practical experimentation is to compare the slow-cooling procedures with vitrification to analyze and evaluate the most effective and safest procedure as well as to endorse suitable recommendation for the adoption of best practices. To do this, it is necessary to test and calculate the viability for control, slow-cooling and vitrification samples. Determining the number of cells in the culture is also important for standardising culture conditions and performing accurate quantitation experiments . The use of viability test with hemacytometer and typan blue staining will enable us to determine the cell number, the correctness of which is inevitable for accurate test results. Live cells appear colourless and bright under phase contrast, while the dead cells sustain blue stains and are non-refractive. To facilitate accuracy and consistency of cell counts, we have used a viability counting system. This involves counting viable, live and dead cells in one or more large corner squares and recording the cell counts. In order to obtain an accurate cell count, 40 to 70 cells will be counted during the test phase. Therefore, it may be necessary to count more than one large corner square. The controlled technique, which is the conservative method used for the purpose of cryopreservation of cells and tissues, is based on the slow-cooling approach. It needs to be appreciated that a large number of non-sensitive cells can be preserved in liquid nitrogen with little damage through slow-cooling method. For this, they are usually cooled at a predetermined cooling rate, in a programmable freezing device, where they are surrounded by ice crystals. The slow-cooling rate allows the cells to dehydrate by maintaining equilibrium with the partially frozen extra-cellular solution . Methods and Materials: The materials used for the test have included 1 tube containing zebrafish ovarian follicles, 1 tube containing 50% L-15 medium, pH 7.2, 4 tubes containing2M, 3M, 6M and 8M DMSO in 50% L-15 medium, 1 tube containing 8 M methanol in 50% L-15 medium, 1 tube containing Trypan Blue solution Cryo-straws, 0.5 and 0.25 ml Micro pipettes and pipette tips, 1 ml and 20 l, Pasteur pipettes, microscope slides and cover-slips, one 6-well culture plate and a programmable cooler (Planer KRYO 550). To obtain ovarian follicles, a gravid female zebrafish has been anaesthetised with a lethal dose of tricaine (0.6 mg/ml) for 5min and decapitated before the ovaries were removed. The ovaries were then gently and immediately placed into a Petri dish containing L15 medium at 22°C. Oocytes were separated manually using forceps and scissors. Subsequently, membrane integrity was assessed, using Trypan Blue (TB). Results: 2. Cryopreservation of zebrafish ovarian follicles: The TB test is a membrane integrity test. Cells with an intact membrane are able to exclude the dye, while cells without an intact membrane take up the colouring agent. We incubated the ovarian follicles were incubated in 0.2% Trypan Blue for 3-5 min at room temperature, and then washed in L-15 medium and the viability was determined on the basis of presence or absence of stains. Unstained follicles were considered viable, while the follicles stained blue were considered non-viable. By assessing ovarian follicle viability of the control group using TB staining, the viability of oocytes was calculated based on the following formula: Viability (%) = Number of viable oocytes X 100% Total number of oocytes The results are shown in the table below: 3. Controlled Slow-Cooling: After incubating 2 batches of ovarian follicles at 22°C in 4M Methanol solution for 30 min, the follicles were loaded into 0.5 ml plastic straws and put them into a programmable cooler (Planer KRYO 550), where the following cooling protocol was used: cooled at 5°C/min from 20°C to -12.5°C, manually seeded and held for 3 min, frozen from -12.5°C to -80°C at 10°C/min, then plunged into liquid nitrogen (LN2) at -196°C and held in LN2 for at least 10 min. The samples were thawed using a water bath at 27°C, the cryoprotectants were removed, the samples were transferred to L15 medium and their viability was assessed by TB staining. This procedure has yielded the following results: This figure is difficult to read. Total no = 32, No. Alive follicles= 11. Viability = (11*100)/32 = 34.3% which considered high viability, compared with vitrification. Discussion: As shown in table 1, levels of 33.3%, 42.9%, and 85.7% were seen for control viability, whereas the viability results were 52.6% and 25% for controlled slow-cooling samples in a stepwise procedure. On the other hand, it gave the result of 0% for the two slides when the fast procedure was used. The viability of controlled slow-cooling samples proved that a stepwise method with a slow-cooling technique is better than the fast one. Viability in the control samples was higher than the viability in the slow-cooling samples, with a significant difference. [DISCUSSION WILL BE INCUDED IN THE OTHER PAPER] 1) Vitrification: Introduction: The ability to cryogenically preserve tissues, cells and even whole organs is an very important aspect of cryopreservation technique. Until recently, the cryopreservation of organs has seemed a remote prospect to most observers, although the developments over recent years are rapidly changing the basis for preserving even the most delicate and difficult organs for long periods of time. Animal ovaries and intestines have been frozen, thawed, and shown to function after transplantation, but the preservation of organs will most likely require vitrification. In the vitrification method, any possibility for ice formation is ruled out and the organ is preserved in a glassy state below the glass transition temperature. Vitrification has been successful for many tissues such as veins, cartilage, arteries and heart valves. Moreover, preservation of whole ovaries has been attained successfully using vitrification. A significant recent milestone for vitrification in the case of vital organs has been found in its ability to routinely recover rabbit kidneys after cooling to temperature of about -45°C, as verified by life support function after transplantation. For long-term banking, this temperature is not low enough, but researches are under way for preservation below -45°C. Full development of organ generation and tissue engineering from stem cells, when combined with the ability to bank these laboratory products can have significant outcomes for progress in this field . Vitrification is the process of solidifying a liquid without allowing it to crystallize. It is understandable that cryopreservation is more efficient when the solutions used in the process are enclosed in constructs and cells are maintained in ice-free conditions during the entire procedure. Vitrification is achieved by the partial replacement of water by penetrating agents, which are easy glass formers, and by accompanying the dehydration of biological materials with non-penetrating cryoprotectants. Such a brief procedure is usually performed at room temperature in routine daily practices. Methods and Materials: The following materials were used: 1 tube containing zebrafish ovarian follicles, 1 tube containing 50% L-15 medium, pH 7.2, 4 tubes containing2M, 3M, 6M and 8M DMSO in 50% L-15 medium, 1 tube containing 8 M methanol in 50% L-15 medium, 1 tube containing Trypan Blue solution Cryo-straws, 0.5 and 0.25 ml micropipettes and pipette tips, 1 ml and 20 l, Pasteur pipettes, microscope slides and cover-slips and one 6-well culture plate. DMSO was used as a cryoprotectant at 3 different concentrations (3M, 6M and 8M prepared in 50% L-15 medium). More detail required to allow somebody to repeat what you did. The vitrification ability of DMSO solutions and L-15 medium was tested. In stage two, the equilibration of ovarian follicles incubated in 2M concentration of DMSO in 50% L-15 medium at room temperature for 10 min was performed. Results: 1- Vitrification of Solutions: After loading solutions in unsealed 0.25ml plastic straws at room temperature, with a syringe using a 20μl pipette, the loaded straws were plunged directly into liquid nitrogen. A transparent glassy appearance was seen, which indicated a vitrified solution (V) and a milky appearance indicated de-vitrification, when the solution showed ice formation (C). 3M 6M 8M Batch 1 C C V Batch 2 C C V Table 1: Vitrification of solutions 2. Vitrification of Stage III Zebrafish Ovarian Follicles (3M, 6M and 8M Straws): After equilibration, the ovarian follicles were transferred to 3M, 6M or 8M DMSO for 5 min and then loaded into the unsealed 0.25ml plastic straws with a syringe using a 20μl pipette. Subsequently, the loaded straws were plunged directly into liquid nitrogen and then transferred to a water bath at 27°C. The cryoprotectants were diluted gradually in 4 steps (2M, 1M and 0.5M DMSO in 50% L-15 medium, 2.5 min for each step) or in a single step before the final transfer of ovarian follicles to L-15 medium. Finally, the viability was assessed by TB staining and recorded the results in the table below. Total no = 28, No. Alive follicles= 2 Viability = (2*100)/28 = 7.1% which is considered very low viability, probably due to personal factors and toxicity. Discussion: In table 3, it is obvious that 8M DMSO attained the transparent glassy appearance, while there was a milky appearance in 3M and 6M DMSO, although it was clearer in 3M straws. This suggested that the 8M DMSO could be better in the laboratory. So far, vitrification seemed to be the most promising single approach. However, it is clear that the problem of eliminating or sufficiently limiting ice formation throughout samples without inducing unacceptable toxicity is a complex and multifaceted process, as shown by the results in table 4. [Explain!] It became crucial only with the development of vitrification technology, since this method required the employment of a high concentration of chemicals. Different agents have been tested and new formulations have been developed for the application of vitrification . As mentioned before in table 1, levels were 33.3%, 42.9%, and 85.7% for control viability, whereas the viability was 0%, 25% and 20% for the vitrification samples in a stepwise procedure. In contrast, it gave the result of 0% for all samples in the fast procedure of viability of vitrification. Viability in the control samples was higher than viability in the vitrification samples, with a significant difference. These variations in the results could be due to the influence of a personal factor such as any bias or a specific interest in a certain field. 2) Extended Discussion: It is about 50 years since the first reliable recovery of living cells frozen to cryogenic temperatures has been reported. Tremendous growth has been seen in the use of cryobiology in medicine, horticulture, agriculture, forestry, and the conservation of endangered or economically important species since then . Cryoprotectants were formulated with the purpose of replacing water in cells and tissues. During the cooling–cryostorage–warming cycle, the damage was minimised by encouraging the formation of an amorphous state in cells, rather than ice crystals. The discovery of the effectiveness of glycerol for red blood cell freezing brought a much-needed focus to low temperature biology, which had been largely disparate and lacked rigor until that time . Over the past 40 years, scientists have tested and developed a range of cryopreservation techniques for preserving cells and tissues. Nowadays, LN is used, which is considered as a relatively new practice . Controlled slow-cooling, vitrification, encapsulation dehydration, dormant bud preservation, or combinations of these techniques are now directly applied to materials and tissues. Cryopreservation is commonly used for the storage of suspension and callus cultures, and is now becoming more useful for organised tissues . Cryopreservation in liquid nitrogen (LN, − 196°C) is the most commonly used technique that is currently available to ensure safe and cost-efficient long-term conservation of the material. All cellular metabolic and division processes are stopped at this temperature. Thus, the material can be stored without modification or alteration for an unlimited period of time. Moreover, it can be stored in a small volume, protected from contamination, and requires very limited maintenance. Cryopreservation is considered a valuable addition to the current conventional methods employed for the maintenance of clonal tissue and materials. Also, cryopreservation has great potential to improve the quality of conservation of materials and tissues . When embryos and oocytes are to be cryopreserved, they are suspended in a solution of one of several low-molecular-weight solutes and their permeability to various low-molecular-weight compounds varies. These differences determine how cells take up these compounds, which influences how these compounds act to protect cells from damage when the cells are subjected to cryopreservation. Since these compounds have the protective elements, they are referred to as cryoprotective additives. Another principal variable is the rate at which the cells are cooled to subzero temperatures. After being stored for some time at −196°C in liquid nitrogen, the cryopreserved embryos and oocytes are warmed to liquefy the medium. The rate at which the specimens are warmed is highly significant in determining the ultimate survival of the embryos and oocytes. The effects are described as the physical variables on cell survival . Various types of organs and tissues can be cryopreserved, including cell suspensions, embryogenic cultures, pollen, zygotic embryos, seeds, shoot tips, dormant buds and somatic . Over the last 20 years, cryopreservation protocols have been established for several hundreds of species. In particular, the vitrification and controlled slow-cooling methods have been continuously improved and are thus the most frequently employed for cryopreservation . However, there are a growing number of examples of testing experiments under way in different countries for various species. Progress in the further application and development of cryopreservation techniques will be made through a better understanding involved in the induction of tolerance to cryopreservation and dehydration in frozen samples . It is expected that cryopreservation will become more frequently employed for the long-term conservation of resources, because of its high potentiality . This laboratory report is mainly based on a comparison of the principles, procedures, and results of controlling slow-cooling and the vitrification of cryopreservation. Both slow-cooling and vitrification procedures have resulted in the successful cryopreservation of Zebrafish ovarian follicles, although their vitrification offers very low success rates. The fulfilment of a transparent glassy appearance in cells is the purpose of both techniques to protect them from damage caused by ice crystals . [Explain the relevance of this. Discuss in the context of the practical aims!] The report compared two groups of zebrafish ovarian follicles that underwent a slow-cooling protocol (n=32) and vitrification (n=28). The paper evaluated the effectiveness of the two freezing methods and the influence of the viability of zebrafish ovarian follicles rate. The study showed higher viability of the slow-cooling protocol (34.4%) than the significant low viability of vitrification (7.1%). However, those results may have some errors due to a number of factors occurring during the process of conducting the tests. The zebrafish model (Fig 1) has been used more frequently than other vertebrate animal model systems . Zebrafish are easily maintained, small and breed well under laboratory conditions. Hundreds of eggs can be produced per day, which are fertilised externally. Besides, the embryos develop rapidly and most of the organ systems will be formed within the next five days. This makes the zebrafish embryos accessible for genetic manipulation by the microinjection of DNA, mRNA or morpholinos, which are antisense DNA oligonucleotides that can alter protein synthesis in the developing embryo . When the embryos are transparent, it allows microscopic imaging at the sub-cellular level, specifically when performed in combination with fluorescent labelling of specific proteins or cells. Moreover, an increasing number of transgenic, mutant zebrafish lines and several zebrafish cell lines derived from embryos and adult tissues are available, which can allow more refined biochemical characterisations . Figure 1 Vitrification is a promising technique in assisted reproductive technology, but comparative success rates are yet to be established . The application of controlling slow-cooling involving a range of cooling rates was compared with vitrification using rapid cooling in simple containers. The success of the slow-cooling technique requires freezing with low cooling rates to allow the efflux of water from cells during ice formation. The slow-cooling method came into practice many years ago as it was very well understood and was compatible with straightforward warming. However, this method is not economical, as it requires a controlled rate freezer. In addition, it is extremely difficult to preserve their integrity as well as that of the biomaterials of neo-tissues during the liquid-ice stage transition intact . It is apparent that the role of non-penetrating cryoprotectants is especially significant in the vitrification protocols, since samples require fast dehydration before immersion in liquid nitrogen. This provides a simple and effective strategy for reducing or eliminating the risk of contamination during cooling, storage and warming. There is no doubt that the application of vitrification in the cryopreservation of cells, tissues and TECs has its disadvantages and limitations too. One major problem is the toxicity of high concentrations of the penetrating cryoprotectants that are required in the vitrification method and the osmotic damage during the addition and removal of cryoprotectants. The early development in vitrification involved the use of long pre-equilibration procedures. The use of mixtures of penetrating and non-penetrating solutes resulted in improved methods over a range of cooling rates they also eliminate toxicity. The most important advantage of vitrification is that it is a simple procedure, which is safe, requires less time and is more cost effective than slow-cooling . In conclusion, the development of appropriate complementary conservation methods will still require more research to refine the methods as well as the criteria, and test their suitability for practical application for a range of gene pools and situations . References: BENELLI, C., DE CARLO, A. & ENGELMANN, F. 2013. Recent advances in the cryopreservation of shoot-derived germplasm of economically important fruit trees of Actinidia, Diospyros, Malus, Olea, Prunus, Pyrus and Vitis. Biotechnology Advances, 31, 175-185. DRIEVER, W. & RANGINI, Z. 1993. Characterisation of a cell line derived from zebrafish (Brachydaniorerio) embryos. In Vitro Cell DevBiolAnim, 29A, 749-54. ENGELMANN, F. 2004. Plant cryopreservation: Progress and prospects. In Vitro Cellular & Developmental Biology - Plant, 40, 427-433. FAHY, G. M., WOWK, B. & WU, J. 2006. Cryopreservation of complex systems: the missing link in the regenerative medicine supply chain. Rejuvenation Res, 9, 279-91. FULLER, B. 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