Optimizing amniotic membrane tissue banking protocols for ophthalmic use

Amniotic membrane (AM) due to its anti-inflammatory, anti-scarring and anti-angiogenic properties is used as corneal and wound grafts. When developing AM tissue banks, cell viability, membrane morphology and genomic stability should be preserved following cryopreservation. To analyze the changes rendered to the AM during the process of cryopreservation by comparing different combinations of standard cryopreservation media; fetal bovine serum (FBS), dimethyl sulfoxide (DMSO), Dulbecco’s modified eagle’s medium (DMEM) and glycerol at ?80 °C and at ?196 °C for a period of 6 weeks and at 4 °C in 70 % alcohol for 6 weeks. Following informed consent, placentae of healthy term pregnancies delivered by elective Cesarean section were collected and AM separated into 5 × 5 cm size sections and under sterile conditions stored in 9:1 DMSO:FBS and 1:1 DMEM:Glycerol at ?196 and ?80 °C for 6 weeks. Similar sections were also stored at 4 °C in 70 % alcohol for 6 weeks. After storage periods following were assessed; AM epithelial cell viability by trypan blue vital stain, epithelial cell proliferation capacity by cell doubling time, membrane morphology by haematoxylin and eosin (H&E) stain and genomic stability by conventional G-banded karyotyping. Human amniotic epithelial cells were cultured in DMEM and 10 % FBS in humidified atmosphere of 5 % carbon dioxide at 37 °C and were characterized using RT-PCR for Octamer-binding protein 4 (Oct-4) and glucose-6-phosphate dehydrogenase (G6PD) genes. All the above parameters were also assessed in fresh AM. AM obtained from 4 term placentae. Mean cell count and mean cell doubling times in days respectively; for fresh AM 3.8 × 10⁶; 1.59, after 6 weeks in DMSO:FBS at ?196 °C 3.0 × 10⁶; 2.38 and at ?80 °C 2.1 × 10⁶; 1.60, in DMEM:Glycerol at ?196 °C 3.6 × 10⁶; 2.33 at ?80 °C 23 × 10⁶; 1.66 and at 4 °C 3.3 × 10⁶; 2.14. Histology analysis of the fresh AM showed an intact epithelial monolayer, thick basement membrane (BM) and avascular stromal matrix. Amniotic membranes stored at ?196 °C showed morphology similar to fresh AM in both preservation media and AM stored at ?80 °C showed disruption of the stromal matrix. At 4 °C the epithelial monolayer showed flattening. Fresh AM karyotype was 46XX. Analyzable spreads for karyotype were not obtained from stored AMs. Human amniotic epithelial cells were positive for both Oct-4 and G6PD genes. AM is best preserved at ?196 °C either in 1:9 DMSO:FBS or 1:1 DMEM:Glycerol. In both conditions cell viability and membrane integrity were shown to be preserved up to 6 weeks. Since analyzable chromosome spreads from cell cultures were not obtained, genomic stability could not be assessed.     

Assessment of organ culture for the conservation of human skin allografts

Objective Human skin allografts are used in the treatment of severe burns and their preservation is therefore critical for optimal clinical benefit. Current preservation methods, such as 4°C storage or cryopreservation, cannot prevent the decrease of tissue viability. The aim of this study was to assess viability and function of skin allografts in a new skin organ culture model, allowing conservation parameters as close as possible to physiological conditions: 32°C, air–liquid interface and physiological skin tension. Design Twelve skin samples, harvested from 6 living surgical donors, were conserved 35 days in two conditions: conservation at 4°C and organ culture. Viability and function of skin samples were investigated at Day 0, 7, 14, 21, 28 and 35 using cell culture methods (trypan blue exclusion, Colony Forming Efficiency and Growth Rate), histopathological and histoenzymological studies (Ki67 immunostaining). Results In the two conditions, fibroblast and keratinocyte viability was progressively affected by storage, with a significant decrease observed after 35 days. No statistical difference could be observed between the two conditions. The two methods were also comparable regarding alterations of fibroblast and keratinocyte culture parameters, which were respectively significantly reduced at Day 7 and 21, compared to fresh skin. By contrast, histopathological and histoenzymological studies revealed a better preservation of skin architecture and proliferative potential at 4°C, as compared to organ culture. Conclusion These results indicate that skin organ culture does not provide significant advantages for skin allograft preservation. However, its potential use as an experimental model to study skin physiology and wound healing should be further evaluated.     

Frozen fungi: cryogenic storage is an effective method to store Fusarium cultures for the long‐term

Microbial culture collections provide a vast amount of genotypic and phenotypic information which are invaluable resources for future advancements in research. For most microbial strains, cryopreservation in the vapour phase above liquid nitrogen provides the most stable and long‐term storage method. However, in the case of fungal microbes, not all are suited for cryogenic storage and few studies have addressed the effectiveness of storage in the vapour phase above liquid nitrogen on a diverse collection of Fusarium species. In this work, a collection of 374 Fusarium strains from the Fungal Genetics Stock Center, including 24 unique species, were duplicated and sent to the National Laboratory for Genetic Resource Preservation for storage in the vapour phase above liquid nitrogen. After 5 years of storage the entire collection was tested for viability and phenotypic stability by using plating, cellular staining assays, assessing the number of viable cells and measuring the rate of growth of each isolate. Additionally, the rate of growth for ～10% of the isolates were compared with the same isolates which had been stored at ?80°C at the Fungal Genetics Stock Center over the same timeframe to determine if cryopreservation in liquid nitrogen vapour provided a comparable method of storage. All National Laboratory for Genetic Resources Preservation isolates grew after being stored at ?165°C for 5 years. In general, the isolates that were stored at ?165°C grew at a faster rate than the isolates stored at ?80°C for the same period. Of the isolates stored at ?165°C, most had greater than 80% cell viability, however, those isolates that had less than 50% cell viability generally also had fewer conidia germinate. These isolates may be at a greater risk for storage over longer times. In conclusion, storage at ?165°C liquid nitrogen provided reliable preservation of a diverse collection of Fusarium spp. over 5 years, and culture viability data indicates that they will remain viable during additional storage for longer periods.     

Study of the viability of cultured human cells in suspensions

The success of cell therapy is directly related to the viability of cells used for transplantation. The cells used for transplantation are in some cases injected in suspension. However, the optimal conditions for the preservation of cell viability upon the preparation and storage of cell suspensions for transplantation have not been defined yet. The aim of the present work consisted in the selection of optimal conditions for the storage of suspensions of human submandibular salivary gland cells, differentiated cells of the submandibular salivary gland, and dermal fibroblasts in biocompatible solutions. Standard procedures of cell isolation and cultivation were used in the study. An automatic cell counter from BioRad was used to count the cells, and viability of the cells was assessed using staining with 4% Trypan Blue. The biocompatible solutions tested included phosphate-buffered saline, physiological saline for injections, and a 2% solution of human albumin in phosphate-buffered saline. The study showed that the human cells under investigation remained viable in suspension at both +4°С and +25°С for at least 24 hours, regardless of the carrier solution used. The highest content of viable cells of the salivary gland (more than 50%) at both temperatures examined was observed when cells were suspended in phosphate-buffered saline. However, the adhesive and proliferative properties of the salivary gland cells were better preserved at +4°С in case of 24 hours of incubation under the conditions described above. Fibroblasts maintained in physiological saline formed a homogeneous single-cell suspension that remained stable for 30 hours at +4°С; virtually no loss of cell viability was observed. The addition of 2% albumin resulted in a decrease of the viability of fibroblasts. Thus, storage and transportation in phosphate- buffered saline at +4°С can be recommended for suspensions of cells of the human submandibular salivary gland, whereas human fibroblast suspensions should be maintained at +4°С in physiological saline.     

Cryopreservation by slow cooling of rat neuronal cells

Although primary neuronal cells are routinely used for neuroscience research, with potential clinical applications such as neuronal transplantation and tissue engineering, a gold standard protocol for preservation has not been yet developed. In the present work, a slow cooling methodology without ice seeding was studied and optimized for cryopreservation of rat cerebellar granular cells. Parameters such as cooling rate, plunge temperature and cryoprotective agent concentration were assessed using a custom built device based on Pye's freezer idea. Cryopreservation outcome was evaluated by post thawing cell viability/viable cell yield and in culture viability over a period of 14 days. The best outcome was achieved when 10% of Me₂SO as cryoprotective agent, a cooling rate of 3.1 ± 0.2 °C/min and a plunge temperature of −48.2 ± 1.5 °C were applied. The granular cells cryopreserved under these conditions exhibited a cell viability of 82.7 ± 2.7% and a viable cell yield of 28.6 ± 2.2%. Moreover, cell viability in culture remained above 50%, very similar to not cryopreserved cells (control). Our results also suggest that post-thaw viability (based on membrane integrity assays) not necessarily reflects the quality of the cryopreservation procedure and proper functionality tests must be carried out in order to optimize both post thaw viability/cell yield and in culture performance.     

Preservation of Phakopsora pachyrhizi Uredospores

This study compared different temperatures and dormancy‐reversion procedures for preservation of Phakopsora pachyrhizi uredospores. The storage temperatures tested were room temperature, 5°C, ?20°C and ?80°C. Dehydrated and non‐dehydrated uredospores were used, and evaluations for germination (%) and infectivity (no. of lesions/cm²) were made with fresh harvested spores and after 15, 29, 76, 154 and 231 days of storage. The dormancy‐reversion procedures evaluated were thermal shock (40°C/5 min) followed or not by hydration (moist chamber/24 h). Uredospores stored at room temperature were viable only up to a month of storage, regardless of their hydration condition. Survival of uredospores increased with storage at lower temperatures. Dehydration of uredospores prior to storage increased their viability, mainly for uredospores stored at 5°C, ?20°C and ?80°C. At 5°C and ?20°C, dehydrated uredospores showed increases in viability of at least 47 and 127 days, respectively, compared to non‐dehydrated spores. Uredospore germination and infectivity after storage for 231 days (7.7 months), could only be observed at ?80°C, for both hydration conditions. At this storage temperature, dehydrated and non‐dehydrated uredospores exhibited 56 and 28% of germination at the end of the experiment, respectively. Storage at ?80°C also maintained uredospore infectivity, based upon levels of infection frequency, for both hydration conditions. Among the dormancy‐reversion treatments applied to spores stored at ?80°C, those involving hydration allowed recoveries of 85 to 92% of the initial germination.     

A simple method to preserve algal spores of <Emphasis Type="Italic">Ulva</Emphasis> spp. in cold storage with ampicillin

Preservation of algal spores of the green seaweed Ulva fasciata and U. pertusa was enhanced by the addition of ampicillin in f/2 medium at 4°C. The viability of preserved spores was determined by a spore germination assay at various time intervals. The germination rate of U. fasciata remained at 5% to 38% for the first five days, dropping to 1% to 6% on the 10th day of storage with various preservation treatments without ampicillin at 4°C during parameter-selecting experiments. In f/2 medium, 53% of U. fasciata spores were still viable on day 5 and 23% on day 10 at 4°C. By adding 100 μg mL⁻¹ ampicillin to f/2 medium, 90% of the spores were viable at day 40 and 61% after 100 days of storage at 4°C. Spores of U. pertusa had lower preservation rates, with viabilities of 70% at day 40 and 32% at day 100. Algal spore preservation was heavily dependent on the bacterial contamination and subsequent degradation in stock solutions. Handling editor: L. Naselli-Flores     

Yeasts preservation: alternatives for lyophilisation

The aim of the study was to compare the effect of two low-cost, low technology traditional methods for drying starter cultures with standard lyophilisation. Lyophilised yeast cultures and yeast cultures preserved in dry rice cakes and dry plant fibre strands were examined for viable cell counts during 6?months storage at 4 and 25?°C. None of the yeast cultures showed a significant loss in viable cell count during 6?months of storage at 4?°C upon lyophilisation and preservation in dry rice cakes. During storage at 25?°C in the dark, yeast cultures preserved in dry rice cakes, and lyophilised cultures of Saccharomyces cerevisiae and Issatchenkia orientalis showed no significant loss of viable cells up to 4?months of storage. Yeast cultures preserved in dry plant fibre strands had the greatest loss of viable count during the 6?months of storage at 25?°C. Preservation of yeasts cultures in dry rice cakes provided better survival during storage at 4?°C than lyophilisation. The current study demonstrated that traditional methods can be useful and effective for starter culture preservation in small-scale, low-tech applications.     

Leukocyte preservation at near-zero temperatures

Intense metabolism and fast exhaustion of the energy potential of nucleate blood cells at positive temperatures substantially reduce the duration of their storage in a viable state. However, this transfusion environment is extremely necessary for clinical purposes. The popular method of cell preservation at an ultralow temperature (?196°C) is limited in application because of the use of liquid nitrogen, special equipment, and trained engineering staff. The goal of this study was to develop an efficient and simple method of leukocyte preservation at near-zero temperatures (+2°–0°C) using a standard household refrigerator and a novel cryopreservation solution.     

Mortality of bifidobacteria in boiled yogurt

Bifidobacterial strains showed various mortalities during storage at 30°C in boiled yogurt prepared with Streptococcus sp. and Lactobacillus sp. Bifidobacterium breve 203 was most stable, maintaining its initial cell number for more than 5 days. It was also stable at 4 or 10°C in fresh yogurt. B. longum 401 rapidly lost viability in the boiled yogurt and was unstable in the fresh yogurt at any temperature. Lactobacillus sp. remained fully viable for one week or more at 4–30°C while Streptococcus sp. lost viability at 20°C or above. The difference in mortality between B. breve 203 and B. longum 401 was mainly due to their sensitivity to the acidic environment, with temperature during storage having a secondary effect. Effects of lysozyme, pepsin and bile acids on the two strains were also investigated.     

Deep-supercooling for extended preservation of adipose-derived stem cells

Cell preservation is an enabling technology for widespread distribution and applications of mammalian cells. Traditional cryopreservation via slow-freezing or vitrification provides long-term storage but requires cytotoxic cryoprotectants (CPA) and tedious CPA loading/unloading, cooling, and recovering procedures. Hypothermic storage around 0–4 °C is an alternative method but only works for a short period due to its high storage temperatures. Here, we report on the deep-supercooling (DSC) preservation of human adipose-derived stem cells at deep subzero temperatures without freezing for extended storage. Enabled by surface sealing with an immiscible oil phase, cell suspension can be preserved in a liquid state at −13 °C and −16 °C for 7 days with high cell viability, retention of stemness, attachment, and multilineage differentiation capacities. These results demonstrate that DSC is an improved short-term preservation approach to provide off-the-shelf cell sources for booming cell-based medicine and bioengineering.     

Proceedings: Preservation of bacteria by freezing at moderately low temperatures

In order to get some basic information for the development of a long-term preservation method by freezing at moderately low temperatures, the viability of 259 strains belonging to 32 genera and 135 species was measured. Cells were suspended in 10% glycerol and stored at ?53 °C for 16 months. About 93%, 88%, and 74% of aerobic bacteria gave viable cell counts higher than 10⁵/ml, 10⁶/ml, and 10⁷/ml, respectively. About 10% of gram-positives and 3% of gram-negatives gave viable cell counts lower than 10⁵/ml. There seemed to be some species—and genus—specificity with respect to viability after frozen storage and liquid paraffin-seal storage. Strains of coryneform bacteria, genera of the family Enterobacteriaceae, and the genus Pseudomonas were generally resistant. Pseudomonas putrefaciens proved to be specifically sensitive. Lactic acid bacteria were subject to sublethal injury, requiring special recovery media. Psychrophilic bacteria were very susceptible to frozen storage. All the tested strains of acetic acid bacteria survived frozen storage well both in 10% glycerol and in 10% honey at ?28 °C for 4.5 years. Honey proved to be a better adjuvant for frozen storage than glycerol. It was suggested from the results that for many kinds of bacteria, long-term preservation by freezing at moderately low temperatures might be possible when appropriate procedures are applied.     

冷风干燥制备高活菌的恶臭假单胞菌菌粉

【目的】获得高活菌恶臭假单胞菌菌粉,提高菌体干燥及保藏存活率。【方法】选用冷风干燥法制备活菌粉,并优化吸附载体与保护剂。【结果】冷风干燥制备恶臭假单胞菌菌粉干燥存活率普遍达到65%以上,显著优于喷雾干燥(24%);对载体与保护剂进行正交试验优化,确定了载体为混合的硅藻土和碱处理玉米芯粉,混合比为1:2,保护剂(质量比)为甘露醇7%、谷氨酸钠5%、甘油1%,制得菌粉活菌数为1.03×1011 CFU/g,室温保藏30 d和4 °C保藏60 d存活率分别达到40.54%和71.67%。【结论】冷风干燥温度相对较低(10?40 °C),对菌体损伤小,碱处理玉米芯粉、甘露醇和谷氨酸钠是提高菌粉保藏存活率的重要因子,此法克服了革兰氏阴性菌菌粉不易制备和不耐保藏的瓶颈。     

Preservation of cell-based immunotherapies for clinical trials

In the unique supply chain of cellular therapies, preservation is important to keep the cell product viable. Many factors in cryopreservation affect the outcome of a cell therapy: (i) formulation and introduction of a freezing medium, (ii) cooling rate, (iii) storage conditions, (iv) thawing conditions and (v) post-thaw processing. This article surveys clinical trials of cellular immunotherapy that used cryopreserved regulatory, chimeric antigen receptor or gamma delta T cells, dendritic cells or natural killer (NK) cells. Several observations are summarized from the given information. The aforementioned cell types have been similarly frozen in media containing 5–10% dimethyl sulfoxide (DMSO) with plasma, serum or human serum albumin. Two common freezing methods are an insulated freezing container such as Nalgene Mr. Frosty and a controlled-rate freezer at a cooling rate of -1°C/min. Water baths at approximately 37°C have been commonly used for thawing. Post-thaw processing of cryopreserved cells varied greatly: some studies infused the cells immediately upon thawing; some diluted the cells in a carrier solution of varying formulation before infusion; some washed cells to remove cryoprotective agents; and others re-cultured cells to recover cell viability or functionality lost due to cryopreservation. Emerging approaches to preserving cellular immunotherapies are also described. DMSO-free formulations of the freezing media have demonstrated improved preservation of cell viability in T lymphocytes and of cytotoxic function in natural killer cells. Saccharides are a common type of molecule used as an alternative cryoprotective agent to DMSO. Improving methods of preservation will be critical to growth in the clinical use of cellular immunotherapies.     

Stability of biocontrol products carrying Candida sake CPA-1 in starch derivatives as a function of water activity

The preservation and shelf-life of formulations of the biocontrol agent Candida sake CPA-1 and starch derivatives as a function of water activity (a_W) were studied in terms of the physical stability of the products and cell viability. Formulations of biocontrol products (BCPs), based on combinations of potato starch and pre-gelatinised potato starch (F1 and F2) or maltodextrines (MD) (F3) containing cell protectants, were obtained by fluidised-bed drying. The carriers and the formulated products were stored at 20°C under different a_W conditions. The water sorption and water plasticization behaviour of the different products were analysed through the water sorption isotherms and glass transition temperatures (T_g). Likewise, the viability of C. sake over time was determined as a function of the a_W. The solubility of the products was also assessed. Although formulations stored at 20°C and low a_W (≤?0.33) exhibited a better shelf-life, a significant decrease in cell survival ratio after 180 storage days was observed. Cold storage (5°C) was required to better maintain the cell viability, thus prolonging the shelf-life of BCPs. Formulations containing MD were the most effective at preserving cell viability and also exhibited the highest water solubility. All the formulations were physically stable at ambient temperature; therefore, the cell stability is the critical point at which to establish both the a_W levels and temperature during storage. Packaging the product using high water vapour barrier material and under cold storage would be necessary to ensure a high number of viable cells and an effective and competitive BCP.     

Effect of storage conditions on detection of mycoplasma in biopharmaceutical products

Mycoplasma contamination affects many different aspects of cell culturing, resulting in unreliable experimental results and potentially harmful biological products. Therefore, the specificity, sensitivity, and reliability of detecting mycoplasma contamination are important aspects of quality control in biotechnological products. In this study, Mycoplasma hyorhinis was adopted as a model strain to evaluate the effects of storage on the viability of Mycoplasma species in cell culture samples. Medium X was compared with conventional media 243 and 988 for the ability to detect M. hyorhinis. The 10¹ CFU/ml of M. hyorhinis was inoculated into medium X prepared using the same lots of components and preserved for 7 d, 1 mo, and 2 mo. M. hyorhinis grew readily and typically on agar plates prepared within 1 mo. The viable mycoplasmas in samples containing different initial titers (10¹ and 10⁶ CFU/ml) after storage at 4° C and −30° C were analyzed. During storage, viable organisms were found with little or no reduction in titers after storage for 8 wk at −30° C under aerobic and anaerobic conditions. A reduction in titers of 3 log10 occurred after 4 wk storage for high-dose cultures (10⁶ CFU/ml) at 4° C. The titers of viable organisms were diminished over 8 wk at 4° C under aerobic and anaerobic conditions.     

Critical temperature for skin necrosis in experimental cryosurgery

To investigate the minimal lethal freezing temperature required to produce skin necrosis in dogs, multiple skin sites were frozen with cryosurgical equipment. Tissue temperatures were recorded from thermocouple sites placed at diverse distances, usually 5 mm from the edge of the freezing probe. In single freezing cycles of about 3 min duration, tissue temperatures in the range of 0 to ?60 °C were produced. Punch biopsies of the skin at the thermocouple sites 3 days after freezing injury provided tissues for estimation of viability by histologic examination.The histologic findings permitted classification of the biopsy tissue into three groups, that is, viable, borderline, or necrotic. When classified as borderline, the division between the necrotic and viable tissue was evident on the histologic slide. The viable specimens were scattered through the 0 to ?35 °C range. All specimens frozen to ?10 °C or warmer were viable. In biopsies classified as borderline, the range of viability extended from ?11 ° to ?50 °C. The necrotic biopsies covered a range of ?14 ° to ?50 °C. Cell death was certain at temperatures colder than ?50 °C. The data showed cryosurgical freezing conditions produced a range of temperatures in which viability or death of tissue may occur and that the ranges of viability and necrosis overlapped to a great extent.The wide range of temperatures at which cells were viable shows the need to achieve tissue temperatures in the range of ?50 °C in the cryosurgical treatment of cancer.     

Impact of storage conditions and duration on function of native and cargo-loaded mesenchymal stromal cell extracellular vesicles

Background aimsAs evidenced by ongoing clinical trials and increased activity in the commercial sector, extracellular vesicle (EV)-based therapies have begun the transition from bench to bedside. As this progression continues, one critical aspect of EV clinical translation is understanding the effects of storage and transport conditions. Several studies have assessed the impact of storage on EV characteristics such as morphology, uptake and component content, but effects of storage duration and temperature on EV functional bioactivity and, especially, loaded cargo are rarely reported.MethodsThe authors assessed EV outcomes following storage at different temperatures (room temperature, 4°C, –20°C, –80°C) for various durations as well as after lyophilization.ResultsMesenchymal stromal cell (MSC) EVs were observed to retain key aspects of their bioactivity (pro-vascularization, anti-inflammation) for up to 4–6 weeks at –20°C and –80°C and after lyophilization. Furthermore, via in vitro assays and an in vivo wound healing model, these same storage conditions were also demonstrated to enable preservation of the functionality of loaded microRNA and long non-coding RNA cargo in MSC EVs.ConclusionsThese findings extend the current understanding of how EV therapeutic potential is impacted by storage conditions and may inform best practices for handling and storing MSC EVs for both basic research and translational purposes.     

Effect of Storage Temperature on Cultured Epidermal Cell Sheets Stored in Xenobiotic-Free Medium

Cultured epidermal cell sheets (CECS) are used in regenerative medicine in patients with burns, and have potential to treat limbal stem cell deficiency (LSCD), as demonstrated in animal models. Despite widespread use, short-term storage options for CECS are limited. Advantages of storage include: flexibility in scheduling surgery, reserve sheets for repeat operations, more opportunity for quality control, and improved transportation to allow wider distribution. Studies on storage of CECS have thus far focused on cryopreservation, whereas refrigeration is a convenient method commonly used for whole skin graft storage in burns clinics. It has been shown that preservation of viable cells using these methods is variable. This study evaluated the effect of different temperatures spanning 4°C to 37°C, on the cell viability, morphology, proliferation and metabolic status of CECS stored over a two week period in a xenobiotic–free system. Compared to non-stored control, best cell viability was obtained at 24°C (95.2±9.9%); reduced cell viability, at approximately 60%, was demonstrated at several of the temperatures (12°C, 28°C, 32°C and 37°C). Metabolic activity was significantly higher between 24°C and 37°C, where glucose, lactate, lactate/glucose ratios, and oxygen tension indicated increased activation of the glycolytic pathway under aerobic conditions. Preservation of morphology as shown by phase contrast and scanning electron micrographs was best at 12°C and 16°C. PCNA immunocytochemistry indicated that only 12°C and 20°C allowed maintenance of proliferative function at a similar level to non-stored control. In conclusion, results indicate that 12°C and 24°C merit further investigation as the prospective optimum temperature for short-term storage of cultured epidermal cell sheets.     

Viability of rape (Brassica napus L.) seeds following selection on the basis of newly-emerged radicles then subsequent drying and storage

Germinating rape seeds selected on the basis of newly-emerged radicles (1 ± 0.5 mm) were dried to an equilibrium moisture content (c. 11%) in air at 20°C and 80% relative humidity without loss of viability. Storage life of these low-moisture-content germinating (LMCG) seeds at 15°C was limited to 7 days before viability was significantly reduced. However, viability of LMCG seeds was maintained for 84 days in storage at -20°C. Longer periods in store reduced viability, but 96% of seeds still remained viable after 336 days at - 20°C. Increasing periods of storage at -20°C reduced the subsequent seed longevity at 15°C, indicating a reduction in vigour during storage. Storage under reduced pressure or in a nitrogen atmosphere had little significant effect on seed longevity. Reduction of moisture content below 11% using vacuum drying at a range of temperatures reduced seed vigour.