Cartilage storage at 4 °C with regular culture medium replacement benefits chondrocyte viability of osteochondral grafts in vitro

Maintenance of articular cartilage allografts in culture media is a common method of tissue storage; however, the technical parameters of graft storage remain controversial. In this study, we examined the optimal temperature and culture medium exchange rate for the storage of osteochondral allografts in vitro. Cylindrical osteochondral grafts (n = 120), harvested from the talar joint surface of ten Boer goats, were randomly classified into four groups and stored under the following conditions: Group A1 was maintained at 4 °C in culture medium that was refreshed every 2 days; Group A2 was maintained at 4 °C in the same culture medium, without refreshing; Group B1, was maintained at 37 °C in culture medium that was refreshed every 2 days; Group B2, was maintained at 37 °C in the same culture medium, without refreshing. Chondrocyte viability in the grafts was determined by ethidium bromide/fluorescein diacetate staining on days 7, 21, and 35. Proteoglycan content was measured by Safranin-O staining. Group A1 exhibited the highest chondrocyte survival rates of 90.88 %, 88.31 % and 78.69 % on days 7, 21, and 35, respectively. Safranin O staining revealed no significant differences between groups on days 21 and 35. These results suggest that storage of osteochondral grafts at 4 °C with regular culture medium replacement should be highly suitable for clinical application.     

Evaluation of storage conditions for refrigerated rabbit skin

An evaluation of various refrigerated (4 °C) storage solutions and conditions was conducted using rabbit skin. Two in vitro methods to assay skin viability are presented: one which directly measures basal cell viability and one which assesses the skin's ability to grow in culture following storage. The superiority of storage in nutrient medium supplemented with fetal bovine serum over conventional storage in saline is clearly demonstrated. Storage in nutrient medium with 10% fetal calf serum resulted in basal cell viabilities which were over 30% higher than viabilities of skin stored by conventional methods in saline. Skin stored in saline failed to grow in culture, while 100% of the cultures of skin stored in medium plus fetal calf serum grew. Although addition of fetal calf serum to the saline improved the basal cell viability, growth in culture occurred only when the skin was stored in a capped tube. Skin stored in medium without serum gave viability results which were not significantly different from the unstored control, but growth rates in culture did differ significantly from the control values. Our study shows that the viability of rabbit skin and its ability to grow in vitro are depressed when the tissue is maintained at 4 °C in saline or in petri dishes, and optimal when refrigerated in nutrient medium supplemented with FBS in a sealed tube.     

Viability of cryopreserved human skin allografts: effects of transport media and cryoprotectant

Human skin allografts can be preserved by different methods. In our clinical practice, human skin allografts are harvested on multi-organ and tissue donors, transferred at +4°C in Ringer Lactate, cryopreserved with 15% Glycerol and held in the vapor phase of a liquid nitrogen freezer until delivery to the burn center. The aim of this experimental study was to evaluate the impact of transport medium and cryoprotectant on the viability of human skin allografts. For this purpose, we compared skin samples harvested from 19 multi-organ and tissue donors with two different transport media and two different cryoprotectants. Viability was assessed by the MTT assay after harvesting at laboratory reception, during storage (at +4°C) at day 2 and day 7, and after cryopreservation and thawing. Histopathological analysis was performed for each MTT assay. Results indicate that, when stored at +4°C, skin retains more viability with RPMI, whereas Glycerol and DMSO are equivalent cryoprotectants regardless of the transport medium. In conclusion, our protocol could be improved by the utilization of RPMI as transport medium.     

Viability of commercial wine yeasts during freezer storage in glycerol-based media

Glycerol-based medium (BM) with and without the addition of 1 g/L ascorbic acid (Asc) and/or 100 mg/L (±)-catechin (Cat) was tested for the storage of three commercial wine yeasts at −20 °C. The medium supplemented with Asc was also used to store 706 strains to verify the maintenance of the liquid state. A decline in survival throughout the storage period was observed. The media containing Asc maintained viability better than the other three. The BM caused a loss of viability of 7 orders for one strain and of 6 orders for the other two. All three strains exhibited a loss of viability of 4 orders when stored in BM+Asc. Two strains decreased viability by 5 orders while one strain by 4 orders, when stored in BM+Cat. Two strains decreased viability by 6 orders while one strain by 5 orders, when stored in BM+Asc+Cat. Regarding the physical state of the medium tested on 706 yeast strains, three cases were observed: completely liquid (56.5 %), liquid with only the upper part frozen (40.4 %) without involving the yeast biomass settled at the bottom, and completely frozen (3.12 %). It is practicable to prepare a BM that remains liquid at −20 °C enhancing yeast viability when Asc is added as cryoprotectant.     

A non-activating diluent to prolong in vitro viability of Apis mellifera spermatozoa: Effects on cryopreservation and on egg fertilization

A non-activating semen diluent does not cause motility or acrosomal reaction or capacitate the sperm cell. The effects of such a diluent on the viability of honey bee spermatozoa stored in ambient conditions were assessed 60 days pre-cryopreservation and 24 h post-cryopreservation. Seven variations of a Tris-based non-activating diluents (FEM1 – FEM7) were compared to samples treated with conventional activating diluent and untreated semen. Semen viability (membrane integrity) was assessed after short- and long-term storage at 14.0 ± 0.2 °C. The non-activating medium FEM7 contained more viable spermatozoa than the activating medium, 24 h after cryopreservation (67.6 ± 10.9% and ~4%, respectively). After 60 days, 22.0 ± 7.8% of spermatozoa was viable in non-activating medium versus 0.0 and 60.8 ± 12.3%, in conventional media and untreated controls, respectively. Hence FEM7 was used to cryopreserve bee semen and subsequently inseminate honey bee queens. The quality of brood produced by the queens was assessed 30–60 days after insemination. The percentage of worker-bee offspring (produced from successfully fertilized eggs) was ~75% for both the non-activating medium and the conventional extender medium. Our results indicate that a non-activating medium possesses significant advantage over the conventional activating medium if the semen requires storage after treatments such as cryopreservation. The percentage of female offspring (from fertilized eggs) produced by queens inseminated with semen diluted in either the activating or non-activating medium did not differ from one another.     

The effect of storage on the biomechanical behavior of articular cartilage--a large strain study.

The transplantation of stored shell osteochondral allografts is a potentially useful alternative to total joint replacements for the treatment of joint ailments. The maintenance of normal cartilage properties of the osteochondral allografts during storage is important for the allograft to function properly and survive in the host joint. Since articular cartilage is normally under large physiological stresses, this study was conducted to investigate the biomechanical behavior under large strain conditions of cartilage tissue stored for various time periods (i.e., 3, 7, 28, and 60 days) in tissue culture media. A biphasic large strain theory developed for soft hydrated connective tissues was used to describe and determine the biomechanical properties of the stored cartilage. It was found that articular cartilage stored for up to 60 days maintained the ability to sustain large compressive strains of up to 40 percent or more, like normal articular cartilage. Moreover, the equilibrium stress-strain behavior and compressive modulus of the stored articular cartilage were unchanged after up to 60 days of storage.     

Factors influencing myeloid stem cell (CFU-C) survival after cryopreservation of human marrow and chronic granulocytic leukemia cells

Human marrow stem cells obtained from 20 patients (9 with nonhematological malignancy, 11 with acute leukemia in remission) and peripheral blood stem cells from 27 patients with chronic granulocytic leukemia were cryopreserved in 10% dimethyl sulfoxide (Me₂SO). It was found that the optimal cooling rate for the human myeloid stem cells (CFU-C) ranged from 1 to 3 °C per minute. The myeloid stem cells (CFU-C) maintained their viability for up to one year of storage in liquid nitrogen, after an initial 20% reduction due to the freezing procedure. Myeldoid stem cells survived better when thawed and diluted at room temperature (RT) than at 4 °C. However, the viability of thawed stem cells decreased when stored at RT for more than 1 hr. The viability of stem cells cryopreserved in bags and ampoules was similar. No differences were noted in the surivial of normal human marrow stem cells and cells from patients with chronic granulocytic leukemia when cryopreserved under similar conditions.     

Refrigerated storage of European common frog Rana temporaria oocytes

Reproduction technologies (RTs) for the storage and use of amphibian gametes have rapidly developed since the recognition of the amphibian conservation crisis in the late 20th Century. Of these RTs, the refrigerated storage of oocytes and sperm can help to achieve reliable pair-matching when unexpected deaths could lead to critical gaps in studbook programs, and also to enable gamete transport between facilities or when sampled from field populations. Viable sperm can be reliably stored in vitro in testes, as suspensions in refrigerators for weeks and in situ in refrigerated carcasses for days. However, oocytes have only been reliably stored in vitro and then only for a few hours. We stored mature oocytes of the European common frog Rana temporaria refrigerated at 4 °C: in situ in the oviduct of carcasses for 1–5 days, in vivo in the oviduct of live frogs for 30 days, and in vitro in plastic boxes for 1–5 days. Oocyte viability was measured as the percentage of fertilisation relative to controls and as the percentage hatch of fertilised oocytes. Rana temporaria oocytes in situ or in vitro retained some viability to hatch for up to 5 days. In contrast, when stored in vivo, oocytes showed little loss of viability to hatch after 10 days and moderate viability up to 30 days.     

Viability and virulence of blastospores of Metarhizium anisopliae (Metch.) Sorokin after storage in various liquids at different temperatures

Blastospores of three strains of Metarhizium anisopliae were stored in 18 liquids at 4°C, 20°C and 35°C for 18 weeks, 12 weeks or 9 days respectively. Viability was quantified by determination of their germination. In bioassays the virulence of stored blastospores was studied using adults and third instars of Locusta migratoria migratorioides (R. & F.) and compared to those of freshly produced blastospores and conidia. Generally, there was great variability in the viability of blastospores, depending on the fungal strain and the liquids used. Blastospores survived best at 4°C in 10% hydroxyethyl starch; for example, germination of M. anisopliae strain 97 still amounted to more than 80% after storage for 18 weeks. Other suitable liquids were deionized water, 25% Ringer's solution and 1% sodium alginate. The viability of blastospores stored at 20°C was considerably shorter than at 4°C. During storage for 12 weeks at 20°C the best protective liquids for M. anisopliae strain 97 were 25% Ringer's solution (43% germination), deionized water (23%) and 10% hydroxyethyl starch (23%). At 35°C, 45% of M. anisopliae strain 97 blastospores still germinated after storage for 7 days in 25% glycerol. The bioassays revealed that the virulence of blastospores after storage was comparable to that of fresh ones and even better than that of fresh conidia. In general, the LT₅₀ was about 4–6 days at an alternating day/night temperature of 28/20°C.     

Effect of storage time and pretreatment on seed germination of the threatened coniferous species Fokienia hodginsii

We report the effects of storage time and pretreatment on seed germination of Fokienia hodginsii. Lower mean germination was observed in seeds stored for 2 years (6.41 ± 1.23 seeds/replicate) compared with those stored for 1 year (8.52 ± 1.06 seeds/replicate). Seeds collected from a southern location had statistically higher mean germination (9.67 ± 1.28 seeds/replicate) than those collected from a northern location (7.99 ± 1.36 seeds/replicate). Higher mean T₅₀ was observed in seeds stored for 2 years (37.02 ± 4.43 days) compared with those stored for 1 year (30.69 ± 5.06 days). Mean germination of untreated fresh seeds was 9.97 ± 1.34 seeds/replicate and that of treated fresh seeds in 60°C water was 12.95 ± 1.24 seeds/replicate. Fresh seeds treated with 50°C and 70°C water had a significantly lower mean germination compared with untreated seeds and seeds treated in 60°C water. Mean T₅₀ was lowest in seeds treated with 60°C water.     

Effect of long-term cold storage of the predatory mite Neoseiulus californicus at high relative humidity on post-storage biological traits

The present study investigated whether long-term cold storage at high relative humidity (RH) affected the quality of the predatory mite Neoseiulus californicus (McGregor) (Acari: Phytoseiidae) in terms of its survival and reproduction. For this purpose, we examined biological traits at the end of storage and during the post-storage period. Mated females three?days after adult emergence were stored individually in 1.5-ml vials for 15, 30, 45, 60, or 75?days at 5.0?±?0.3°C and RH of 99?±?0.1% under continuous darkness. At the end of the storage period, 94–100% of females had survived when the storage period was ≤30?days, but percent survival decreased with longer storage. After storage, female survival and oviposition rates were equivalent to un-stored females at 24?±?1°C, RH of 93?±?2%, and a photoperiod of LD 16:8?h. The quality of progeny (hatchability, survival to adulthood, and sex ratio) of stored females was not affected by storage periods as long as 60?days. These results indicate that storage using the tested method can preserve N. californicus for at least 30?days without any degradation.     

Development of a cryopreservation protocol for long-term storage of black tiger shrimp (Penaeus monodon) spermatophores

The objectives of this study were to determine the effect of cryoprotectants on sperm viability and develop a freezing protocol for long-term storage of P. monodon spermatophores. Spermatophores suspended for 30 min in calcium-free saline (Ca-F saline) containing the cryoprotectants dimethyl sulfoxide (DMSO), ethylene glycol (EG), 1,2-propylene glycol (PG), formamide, and methanol at concentrations of 5, 10, 15, or 20% were studied using a modified eosin-nigrosin staining technique. The smallest reductions in apparent sperm viability occurred with DMSO; therefore, a freezing protocol was developed using Ca-F saline containing 5% DMSO. Spermatophores were cryopreserved using three protocols; cooling to a final temperature of -30, -80 or -80 degrees C and immediately stored in liquid nitrogen (cooling rates of -2, -4, -6, -8, -10, -12, -14 or -16 degrees C/min). Frozen spermatophores were thawed (2 min) at 30, 60, 70, or 90 degrees C. Successful cryopreservation of spermatophores in liquid nitrogen was achieved by a one-step cooling rate of -2 degrees C/min between 25 and -80 degrees C before storing in liquid nitrogen. Optimal thawing was in a 30 degrees C water bath for 2 min; this yielded live sperm after storage in liquid nitrogen for 210 days. Average sperm viability for fresh (97.8+/-2.9%) and cryopreserved spermatophores held for less than 60 days (87.3+/-4.1%) did not differ (P>0.05); however, that for spermatophores stored in liquid nitrogen between 90 and 210 days were lower (P<0.05) and varied from 27.3+/-3.4 to 53.3+/-4.3%. Thawed spermatophores previously held in liquid nitrogen for less than 62 days fertilized eggs (fertilization and hatching rates of 71.6-72.2% and 63.6-64.1%, respectively) at rates comparable to fresh spermatophores (70.8-78.2% and 66.3-67.8%, respectively). In conclusion, sperm within cryopreserved spermatophores stored in liquid nitrogen retained their viability for up to 210 days.     

Culture substrate dependence of mouse fibroblasts survival at 4° C

Summary When L-929 mouse fibroblasts grown in Eagle’s medium (MEM) supplemented with 10% fetal bovine serum (FBS) were stored in a monodisperse suspension at 4° C, the viability decreased rapidly from the beginning of storage. The viability in this study was determined by counting electronically the number of cells with the capacity to attach to glass substrate and with the membrane boundary resistant to a proteolytic digestion. When, however, the dissociated cells were preincubated briefly at 37° C, and subsequently stored at 4° C as they were attaching on a glass substrate, the rapid loss of viability could be reduced effectively. A biphasic survival profile consisting of an initial phase of slowly decreasing viability and the subsequent phase of rapidly decreasing viability were then observed. The rapid viability loss occurred not only when the cell suspension was prepared by mechanical dislodging but also after trypsinization or dispase treatment. Such viability loss was also observed when the dissociated cells were not stored at 4° C directly but preincubated in a monodisperse suspension at 37° C in a siliconized plate and then stored at 4° C. The above results show that the rapid loss of viability is associated closely with the fact that the cells were not attached to the substrate but in suspension. This work was supported in part by grants-in-aids from the Institute of Physical and Chemical Research and from the Mitsubishi Foundation.     

Germination and Storage of Pollen of Phytolacca dodecandra L. (endod)

The effect of sucrose, H₂BO₃, KNO₃, Ca(NO₂)₂.4H₂O and MgSO₄.7H₂O on pollen germination of Phytolacca dodecandra L. (endod)in a liquid medium was investigated. Sucrose and H₃BO₃ werecritical to pollen germination. A concentration of 10% sucroseand 161.8 µm H₂BO₃ gave over 70% germination. The germinationof pollen was not enhanced by Ca(NO₃)₂.4H₂O, KNO₃ and MgSO₄.7H₂O.Endod pollen was dehydrated over CaCl₂ and stored in gelatincapsules in cryogenic vials at –175 °C, 1±1°C and 24±2 °C. The pollen moisture content atcollection was approx. 7.8% (f. wt basis) and dehydration overCaCl₂ reduced it to about 1.4%. Pollen stored at 1±1°C and –175 °C maintained viability for over 6months. Pollen stored at room temperature lost viability within4 weeks of storage. Pollination with cryopreserved pollen resultedin normal fruit set. Phytolacca dodecandra, endod, pollen germination, pollen storage     

Influence of storage time and nutrient medium on recovery of fibroblast-like cells from refrigerated collared peccary (<Emphasis Type="Italic">Pecari tajacu</Emphasis> Linnaeus, 1758) skin

Animal cloning is a promising technology for biodiversity conservation, and its success depends on the recovery of nucleus donor cells. Specifically for collared peccaries, found sometimes in regions that are difficult to access, the storage at 4–6°C of skin tissues would be an alternative for the conservation of genetic material. Therefore, we aimed to evaluate different storage periods and the presence of a nutrient medium at 4–6°C on the recovery of somatic cells from the skin of collared peccaries. To analyze cell recovery rates, ear explants were distributed in non-refrigerated samples and samples refrigerated for 10, 30, and 50 d in the absence or presence of nutrient medium. All explants were analyzed by histologically and cultured. Only the fragments stored for 50 d without medium showed an increase in the total thickness of skin. Moreover, increased storage period, regardless of the presence of medium, increased the halo number and reduced the metabolic activity. After culture, only the fragments stored without medium for 50 d did not yield any somatic cells. Cells recovered from explants stored for 10 d showed similar characteristics to these recovered from non-refrigerated explants, regardless of the presence of medium, including the day at which explants achieved attachment and the total time to reach subconfluence. In conclusion, viable cells can be recovered from somatic tissues of collared peccaries stored for up to 50 d in the presence of medium, and tissues refrigerated for up to 10 d in the presence of medium yielded more viable cells.     

Improving germination and vigour of aged and stored onion seeds by matriconditioning

Germination and vigour of accelerated aged (AA) and naturally stored onion seeds were examined. Accelerated ageing was conducted at 40 °C and 100 % relative humidity (RH). Non aged seeds were stored for 34 months at 3 or 15 °C and 40, 60 or 90 % RH. To restore seed viability, stored and aged seeds were matriconditioned with Micro-Cel E. A distinct loss of germination was observed after 5 days of accelerated ageing. Naturally stored seeds maintained high viability for 34 months, when stored at 3 °C and 40, 60 and 90 % RH or at 15 °C and 40 %. An increase of RH to 60 and 90 % at 15 °C caused loss of germination and vigour. Matriconditioning improved germination and increased endogenic ethylene release and in vivo ACC oxidase activity of both aged and stored seeds.     

Synthetic seed technology for short term conservation of medicinal orchid Dendrobium densiflorum Lindl. Ex Wall and assessment of genetic fidelity of regenerants

Artificial seeds were obtained through encapsulation of protocorm-like bodies (PLBs) of Dendrobium densiflorum in calcium alginate beads. This paper demonstrates the alginate-encapsulation and conversion (complete plantlet regeneration) from PLBs, the effect of storage conditions (at different temperature; 4, 8, 16 °C, RT and duration; 15, 30, 45, 60, 75, 90 days) on viability of encapsulated plant materials as well as the assessment of genetic fidelity of the regenerants. Individual PLBs were encapsulated in calcium alginate beads for mass propagation, short-term storage and germplasm sharing. The superior gel matrix for encapsulation was obtained using 3 % sodium alginate and 100 mM calcium chloride (CaCl₂·2H₂O). The highest percentage of conversion (100 %) of encapsulated PLBs (capsules) was obtained on MS2 medium (MS medium + 2 mg/l BAP). Capsules were successfully stored till 60 days at 8 °C with conversion frequency of 95.5 %. Plantlets regenerated from encapsulated beads were acclimatized successfully with 95 % survival rate. A total of 40 primers were screened, out of which 10 primers successfully generated 39 scorable bands, ranging from 0.2 to 1.3 kb amplicons. The uniform RAPD banding profile among the plantlets derived from encapsulated PLBs following 60 days of storage confirmed genetic fidelity.     

Effect of medium and temperature of storage on viability of lactic acid bacteria immobilized in κ-carrageenan-locust bean gum gel beads

Summary The influence of various storage solutions and temperature (4°C and 25°C) on viability ofStreptococcus salivarius subsp.thermophilus andLactobacillusdelbrueckii subsp.bulgaricus entrapped in κ-carrageenan-locust bean gum mixed gel beads was studied. The immobilized strains could be stored at 4°C in all storage solutions studied for at least 14 and 11 days respectively before counts decreased to 10⁵c.f.u./mL, which was considered to be the practical limit for their use as inoculum in a fermentation process. The most effective storage solutions for preserving cell viability at 4°C were NaCl, glycerol and sorbitol solutions forS. thermophilus, and PO₄ buffer and sorbitol solutions forL. bulgaricus. At 25°C,S. thermophilus could be stored for over 14 days in all solutions except glycerol, andL. bulgaricus for 4 days in 10% sorbitol.     

Serum- and albumin-free cryopreservation of endothelial monolayers with a new solution

Cryopreservation is the only long-term storage option for the storage of vessels and vascular constructs. However, endothelial barrier function is almost completely lost after cryopreservation in most established cryopreservation solutions. We here aimed to improve endothelial function after cryopreservation using the 2D-model of porcine aortic endothelial cell monolayers.?The monolayers were cryopreserved in cell culture medium or cold storage solutions based on the 4°C vascular preservation solution TiProtec^®, all supplemented with 10% DMSO, using different temperature gradients. After short-term storage at ?80°C, monolayers were rapidly thawed and re-cultured in cell culture medium.?Thawing after cryopreservation in cell culture medium caused both immediate and delayed cell death, resulting in 11 ± 5% living cells after 24 h of re-culture. After cryopreservation in TiProtec and chloride-poor modifications thereof, the proportion of adherent viable cells was markedly increased compared to cryopreservation in cell culture medium (TiProtec: 38 ± 11%, modified TiProtec solutions ≥ 50%). Using these solutions, cells cryopreserved in a sub-confluent state were able to proliferate during re-culture. Mitochondrial fragmentation was observed in all solutions, but was partially reversible after cryopreservation in TiProtec and almost completely reversible in modified solutions within 3 h of re-culture. The superior protection of TiProtec and its modifications was apparent at all temperature gradients; however, best results were achieved with a cooling rate of ?1°C/min.?In conclusion, the use of TiProtec or modifications thereof as base solution for cryopreservation greatly improved cryopreservation results for endothelial monolayers in terms of survival and of monolayer and mitochondrial integrity.     

In vitro response of encapsulated and non-encapsulated somatic embryos of Kinnow mandarin (Citrus nobilis Lour × C. deliciosa Tenora)

The aim of present investigation was to study the effect of storage conditions on percentage germination of encapsulated and non-encapsulated somatic embryos of Kinnow mandarin (Citrus nobilis Lour × C. deliciosa Tenora). Different batches of encapsulated and non-encapsulated embryos were preserved at room temperature, 4°C, in liquid nitrogen as such and by embedding in liquid paraffin. In the encapsulated somatic embryos stored at room temperature in sealed Petri plates, percentage of germination was 24.99%, but 5.55% in non-encapsulated embryos after 3 days of storage. Encapsulated embryos stored in vials containing liquid paraffin at room temperature were germinated at 18.05% after 60 days of storage, while it was 13.88% in non-encapsulated embryos after 45 days of storage. Encapsulated somatic embryos stored at 4°C in sealed Petri plates remained viable for up to 75 days with 6.94% germination, whereas non-encapsulated embryos remained viable for up to 45 days with 24.99% germination. Encapsulated embryos stored at 4°C in vials filled with paraffin germinated at 11.11% after 120 days of storage, but 5.55% in non-encapsulated embryos after 90 days of storage. Encapsulated and non-encapsulated embryos stored in liquid nitrogen showed 58.33 and 51.38% survival, respectively, after 7 months of storage. The plantlets developed from these embryos were transplanted after acclimatization and are growing normal.