Preservation of Adrenoceptor and Endothelin Receptor Mediated Vasoconstriction and of Endothelium-Dependent Relaxation after Cold Storage of Explanted Blood Vessels for ex vivo Analyses.

INTRODUCTION: Adrenoceptor and endothelin (ET) receptor-mediated vasoconstriction as well as endothelium-dependent vasodilation of human saphenous veins were compared before and after 20 h of cold storage. METHODS: Contractile responses to potassium chloride (KCl), norepinephrine (NE), and ET-1 as well as vasodilator responses to acetylcholine (ACh) were evaluated. RESULTS: Storage in HEPES-supplemented Dulbecco's modified Eagle's medium (HDMEM) diminished KCl induced contractile forces to 71% (p = 0.002) and NE induced contractions to 80% (p = 0.037), in contrast to HEPES-supplemented Krebs-Henseleit solution (HKH) and TiProtec solution. KCl-normalized NE contractions were not affected by storage. NE EC50 values were slightly lower (7.1E-8 vs. 7.5E-8, p = 0.019) after storage in HKH, with no changes after storage in the other solutions. Endothelium-dependent responses to ACh were not affected by storage. ET-1 induced contractions were attenuated after storage in HDMEM (77%, p = 0.002), HKH (75%, p = 0.020), and TiProtec (73%, p = 0.010) with no changes in normalized constrictions. ET-1 EC50 values were not affected by storage. CONCLUSION: Loss of contractility after storage in HDMEM may reflect the lower content of dextrose. There was no specific attenuation of adrenoceptor, ET-receptor, or ACh receptor mediated signal transduction after storage in any of the media. HKH or TiProtec are equally suitable cold storage solutions for ex vivo measurements.

The comparative analyses of decalcification procedures and methyl benzoate pre-treatment on tissue preservation and antigenicity in human acetabular labra.

The histological processing of musculoskeletal tissue might be challenging. The alteration of tissue composition e.g. by calcification of soft tissue in the elderly, after trauma or surgical interventions makes the histological processing of fixed tissue difficult. Additional steps of decalcification are then needed that probably affect the staining quality. In the present work, the effects of different decalcification agents and the intermedium methyl benzoate on histological staining methods and immunohistochemistry have been compared. Acetabular labra were fixed with 4% paraformaldehyde, left untreated or decalcified using 30% ethylenediaminetetraacetic acid (EDTA; Chelaplex®) or 6% trichloroacetic acid (TCA) for 1-4 days to investigate the effects of decalcification duration. Moreover, samples were pretreated with methyl benzoate or conventionally paraffin embedded independent of decalcification procedure and duration. The specimens were evaluated using hemalaun-eosin, Azur II- methylene blue staining or immunohistochemistry against ankyrin B to visualize nerve fibers. Decalcification with Chelaplex® or TCA reduced cutting artifacts without affecting the tissue morphology and proteoglycan staining but decreased antigenicity in immunohistochemistry. Interestingly, methyl benzoate further reduced cutting artifacts without altering tissue morphology and elevated antigenicity for Chelaplex® decalcified tissue samples in immunohistochemistry. The decalcification with Chelaplex® or 6% TCA preserves tissue morphology and proteoglycan staining similar to non- decalcified tissue but facilitates section processing. In immunohistochemistry both decalcification agents decreased antigenicity. Chelaplex® decalcified, methyl benzoate treated samples yielded an improved antigenicity.

Melatonin prevents deterioration in quality by preserving epigenetic modifications of porcine oocytes after prolonged culture.

Prolonged culture of metaphase II oocytes is an in vitro aging process that compromises oocyte quality. We tested whether melatonin preserves epigenetic modifications in oocytes after prolonged culture. The porcine oocytes were maturated in vitro for 44 h, and then metaphase II oocytes were continuously cultured in medium supplemented with or without melatonin for 24 h. We found that the parthenogenetic blastocyst formation rate of prolonged-culture oocytes was lower than in fresh oocytes. We further observed that methylation at H3K4me2 and H3K27me2 of oocytes enhanced after prolonged culture. However, 5mc fluorescence intensity was lower in prolonged-culture oocytes than in fresh oocytes. Moreover, the promoter of the imprinted gene NNAT exhibited a higher level of DNA methylation in prolonged-culture oocytes than in fresh oocytes, which was associated with a reduced expression level and glucose uptake capability. Conversely, melatonin improved blastocyst formation rate and preserved histone and DNA methylation modifications, as well as NNAT function in the oocytes after prolonged culture. Notably, DNA methyltransferase inhibitor 5-aza significantly attenuated the protective role of melatonin on genomic DNA methylation. In summary, our results revealed that epigenetic modifications are disrupted in oocytes after prolonged culture, but the changes are reversed by melatonin.

Effect of sodium alginate on mouse ovary vitrification.

The survival rate of vitrified-thawed ovarian tissues after autotransplantation still needs to be improved. Therefore finding an ideal cryoprectant to reduce the damage to ovaries that caused by vitrification will pave the way for application of ovary cryopreservation on clinics. Experiments were conducted to investigate the effect of sodium alginate in cryoprotectant solution on mouse ovaries during the vitrification process. The ovaries obtained from 6-weeks old CD1 were assigned into six groups from A to F. Group A without treatment was used as the normal control. Group B cryopreserved with the basic cryoprotectant solution containing 15% each Me2SO and EG was used as the experimental control. Groups C, D, E, and F cryopreserved with the basic cryoprotectant solution supplemented with 0.05%, 0.10%, 0.15%, and 0.20% of sodium alginate, respectively, were assigned for the experimental groups. The in vitro analyses showed that the developmental capability of the oocytes isolated from vitrified-thawed ovaries significantly increased with increasing concentration of sodium alginate in the cryoprotectant solution (groups: A = 70 ±â€¯2; B = 43 ±â€¯2; C = 48 ±â€¯3; D = 53 ±â€¯3; E = 60 ±â€¯3; B < C < D < A, P < 0.05), and reached its highest level in group E with 0.15% of sodium alginate (P < 0.05). The lowest developmental capability of all groups was group F (41 ±â€¯1%)(P < 0.05) with 0.20% of sodium alginate. The similar results were obtained by the autotransplantation in vivo. These finding demonstrated that sodium alginate can significantly reduce the damage to ovaries by vitrification.

Effect of melatonin supplementation in the long-term preservation of the sheep ovaries at different temperatures and subsequent in vitro embryo production.

Investigations in the past decades have shown that oocytes developmental competence following in vitro fertilization is greatly influenced by an interval between isolation of the ovaries immediately after death/slaughter and oocytes recovery from the visible follicles. In order to determine the optimal conditions for long-term preservation of ovaries, an experiment was conducted with adding different doses of melatonin (0 (C), 500 (M1), 600 (M2), 700 (M3) and 800 (M4) µM) as an antioxidant to sheep ovaries preservation medium (PBS) maintained at 4 and 20 °C for 24 h. The effects on in vitro embryo production (IVEP) parameters including maturation, fertilization, cleavage, and blastocyst rates and the total number of blastomere were evaluated after the ovaries preservation. Melatonin reduced the decline in fertilization rate as an indicator of success in vitro maturation (P ≤ 0.05). Furthermore, ovarian storage time had significant negative effect (P ≤ 0.05) on IVEP parameters. Supplementation with melatonin increased the total cell number of blastocysts as an indicator of embryo quality (i.e. mean blastomeric cells in 4°C groups: 86.00 ± 3.00, 98.50 ± 3.5, 111.5 ± 1.5, 125.5 ± 2.00 and 126.50 ± 5.5 for C, M1, M2, M3 and M4. respectively). Overall, the results showed that the use of melatonin antioxidant in the ovaries storage medium had beneficial effects on sheep oocytes development and embryos quality.

Hypothermic storage of isolated spermatogonia and oogonia from rainbow trout (Oncorhynchus mykiss).

A growing number of fish species are endangered due to human activities. A short- or long-time preservation of gametes could conserve genetic resources of threatened fish species. The aim of this study was to evaluate a hypothermic condition for short-term preservation of spermatogonia and oogonia cells isolated from immature transgenic rainbow trout, Oncorhynchus mykiss, and to determine the maximum time point for further transplantation. Viability rate of germ cells was investigated after isolation and during storage at 4 °C up to 24 h. Dulbecco's modification of Eagle's medium supplemented with Hepes fetal bovine serum and l-glutamine was used as hypothermic storage media. The results showed that while viability decreased following 24 h storage, the remaining viable cells did not vary morphologically as well as GFP intensity retained similar to those observed in freshly isolated cells. The hypothermal storage study indicated that culture medium is suitable for preserving germ cells in the short periods of time. Simplicity, easily available culture media and low cost provide new insight into hypothermic conditions for preserving and transporting of germ cells for next applied and basic studies.

Spray Dried, Pasteurised Bovine Colostrum Protects Against Gut Dysfunction and Inflammation in Preterm Pigs.

OBJECTIVE: Feeding bovine colostrum (BC) improves gut maturation and function and protects against necrotizing enterocolitis, relative to formula in newborn preterm pigs. Before BC can be used for preterm infants, it is important to test if the milk processing, required to reduce bacterial load and increase shelf life, may affect bioactivity and efficacy of a BC product. METHODS: We investigated if spray dried, pasteurised BC had protective effects on gut function in preterm pigs, relative to formula. After a 2-day total parenteral nutrition period, preterm pigs were fed formula for a few hours (to induce a proinflammatory state) followed by 2 days of formula (FORM, n = 14), BC (colostrum [COLOS], n = 14), spray-dried BC (POW, n = 8), or pasteurised, spray-dried BC (POWPAS, n = 9). RESULTS: Spray drying and pasteurisation of BC decreased the concentration of transforming growth factor-ß1, -ß2 and increased protein aggregation. All of the 3 BC groups had reduced necrotizing enterocolitis severity, small intestinal levels of IL-1ß, -8, and colonic lactic acid levels, and increased intestinal villus height, hexose absorption, and digestive enzyme activities, relative to the FORM group (all P < 0.05). All of the 3 BC diets stimulated epithelial cell migration in a wound-healing model with IEC-6 cells. CONCLUSIONS: Spray drying and pasteurisation affect BC proteins, but do not reduce the trophic and anti-inflammatory effects of BC on the immature intestine. It remains to be studied if BC products will benefit preterm infants just after birth when human milk is often not available.

Evaluation of Aloevera Gel as a Storage Medium in Maintaining the Viability of Periodontal Ligament Cells - An in Vitro Study.

OBJECTIVE: To investigate the effectiveness of aloevera gel as a new storage medium in maintaining the viability of periodontal ligament cells. STUDY DESIGN: Premolars extracted for orthodontic reason were obtained. Confluent monolayers of fibroblasts were grown by cell culture method from the PDL cells isolated from the extracted teeth. One ml of this cell suspension was transferred to wells of culture plates, incubated for 24 hrs, followed by exposure to the three experimental media, Hank's balanced salt solution (HBSS), aloevera gel, and packaged drinking water. These plates were then assessed for viable cells using trypan blue dye exclusion test with haemocytometer after 15, 30, 60, 90 and 120 mins. The results obtained were statistically analysed using one-way analysis of variance (ANOVA). RESULTS: At 15 min, HBSS presented maximum mean percentage of viable PDL cells (89%), followed by aloevera at 81% and packaged drinking water at 10%. Aloevera demonstrated 71%, 59%, 57% viable cells at 30, 60, 90 mins respectively. At 120 min, HBSS presented 57% viable cells followed by aloevera gel (45%) and packaged drinking water (3%). No statistical significant difference was observed between HBSS and aloevera gel. CONCLUSIONS: Within the parameters of this study, both aloevera gel and HBSS were effective in maintaining the viability of PDL cells. Hence, aloevera gel could be used as a storage media for avulsed tooth in situations where availability of HBSS is in question.

Effect of different drying methods on chemical composition and bioactivity of finger citron polysaccharides.

Traditionally, people like to take dried finger citron fruits (FC) as adjuvant herbal medicines to treat a diversity of chronic diseases like asthma, hypertension and respiratory tract infections. Many healing properties are attributed to FC polysaccharides (FCPs), one of the main active ingredients of FC. Three drying methods, freeze drying (FDM), hot air drying (HDM) and vacuum drying methods (VDM) were comparatively studied on the physicochemical and antioxidant properties of FCPs. The results showed these FCPs were similar in UV and FT-IR spectrum. However, they showed significant differences (p<0.05) in yields of crude polysaccharides and contents of protein and ash. Compared with VDM and HDM, FDM resulted in the properties of FCPs with lower molecular weight distribution, higher reducing power and scavenging abilities on DPPH, OH, and O2(-). Available data obtained in vitro models suggested that FDM was an appropriate and effective treatment for obtaining crude polysaccharides from FC fruits. Hence, drying methods used for preparation of FCPs can affect physicochemical and associated functional properties.

[Use of heparinized autologous blood for intraoperative rinsing and storage of vascular grafts].

UNLABELLED: In cerebral revascularization surgery in Japan, the preferred solution for rinsing and intraoperative storage of saphenous vein or radial artery grafts is a heparinized saline solution with albumin. On the other hand, most cardiac surgeons routinely use solutions of heparinized autologous blood during surgery. Here we used the latter type of solution for cerebral revascularization surgery and evaluated its efficacy. PATIENTS AND METHODS: Since December 2011, we have used heparinized autologous blood for saphenous vein grafts during cerebral revascularization surgery. For this, 20mL of the whole blood was obtained from an arterial line;this blood was then mixed with 20mL of a heparinized saline solution containing 500IU of heparin and 40mg of papaverine hydrochloride. The saphenous vein was harvested using standard procedures and immersed in the autologous blood solution just before implantation. RESULTS: Between December 2011 and March 2013, six revascularizations using saphenous vein grafts were performed using this solution. None of the anastomoses presented complications related to revascularization procedures, and all grafts were clearly present postoperatively. DISCUSSION: There is still no evidence that the storage in autologous blood is superior to the use of a saline solution with albumin. However, the national health insurance does not cover the use of albumin products, which carries an additional cost. Furthermore, the autologous blood medium is a red-colored solution that indicates the presence of unfavorable graft leaks when the wall of the graft turns red. CONCLUSION: We recommend the use of heparinized autologous blood for intraoperative rinsing and storage grafts.

Non-frozen preservation protocols for mature mouse oocytes dramatically extend their developmental competence by reducing oxidative stress.

The objective of this study was to test whether aging induces oxidative stress (OS) during oocyte preservation at different temperatures and whether the oocyte competence can be extended by antioxidant supplementation. The increase in activation susceptibility was efficiently prevented when oocytes were preserved at 37°C for 9 h in HCZB medium with 10.27 mM pyruvate and 10 µM α-tocopherol, at 25°C for 30 h with 20.27 mM pyruvate, and at 15°C for 96 h and at 5°C for 48 h with 10.27 mM pyruvate. Satisfactory blastocyst development was achieved after oocyte preservation at 37°C for 9 h, at 25°C for 30 h, at 15°C for 48 h and at 5°C for 24 h using the above protocols but with cysteamine/cystine supplementation. Transfer of blastocysts obtained from the above protocols showed no difference in pregnancy outcome between newly ovulated and preserved oocytes. Because oocytes preserved at 15°C for 48 h were fertilized after a 6-h recovery culture, aging of ovulated mouse oocytes has been successfully prevented for 54 h. Assays for ROS and glutathione indicated that in vitro preservation caused marked OS in oocytes. In conclusion, marked OS was observed following in vitro preservation of mature oocytes at different temperatures. Whereas any protocol that reduced OS could inhibit activation susceptibility, only those protocols that decreased OS while increasing glutathione synthesis could sustain oocyte competence.

Controls on gut phosphatisation: the trilobites from the Weeks Formation Lagerstätte (Cambrian; Utah).

Despite being internal organs, digestive structures are frequently preserved in Cambrian Lagerstätten. However, the reasons for their fossilisation and their biological implications remain to be thoroughly explored. This is particularly true with arthropods--typically the most diverse fossilised organisms in Cambrian ecosystems--where digestive structures represent an as-yet underexploited alternative to appendage morphology for inferences on their biology. Here we describe the phosphatised digestive structures of three trilobite species from the Cambrian Weeks Formation Lagerstätte (Utah). Their exquisite, three-dimensional preservation reveals unique details on trilobite internal anatomy, such as the position of the mouth and the absence of a differentiated crop. In addition, the presence of paired pygidial organs of an unknown function is reported for the first time. This exceptional material enables exploration of the relationships between gut phosphatisation and the biology of organisms. Indeed, soft-tissue preservation is unusual in these fossils as it is restricted to the digestive structures, which indicates that the gut played a central role in its own phosphatisation. We hypothesize that the gut provided a microenvironment where special conditions could develop and harboured a source of phosphorus. The fact that gut phosphatization has almost exclusively been observed in arthropods could be explained by their uncommon ability to store ions (including phosphorous) in their digestive tissues. However, in some specimens from the Weeks Formation, the phosphatisation extends to the entire digestive system, suggesting that trilobites might have had some biological particularities not observed in modern arthropods. We speculate that one of them might have been an increased capacity for ion storage in the gut tissues, related to the moulting of their heavily-mineralised carapace.

Red blood cell engineering in stroma and serum/plasma-free conditions and long term storage.

In vitro generation of artificial red blood cells (RBCs) is very important to overcome insufficient and unsafe blood supply. Despite recent progresses in RBCs engineering from several stem cell sources, none of them could succeed in generation of functional RBCs in the absence of serum/plasma and feeder cells. Without the elimination of serum and plasma, human RBC engineering in a large scale is impossible, especially for the future bioreactor system. Using an appropriate combination of cost-effective and safe reagents, the present study demonstrated the terminal maturation of hematopoietic stem cells into enucleated RBCs, which were functional comparable to donated human RBCs. Surprisingly, the viability of erythroid cells was higher in our serum- and feeder-free culture condition than in the previous serum-added condition. This was possible by supplementation with vitamin C in media and hypothermic conditions. Also, our report firstly presents the storability of artificial RBCs, which possibility is essential for clinical application. In summary, our report demonstrates engineering of human applicable RBCs with a dramatically enhanced viability and shelf-life in both serum- and stroma-free conditions. This innovative culture technology could contribute to the realization of the large-scale pharming of human RBCs using bioreactor systems.

Host serum is not indispensable in collagen performance in viable meniscal transplantation at 4-week incubation.

PURPOSE: Viable meniscal transplantation has been criticized as an expensive and logistically demanding technique. The purpose was to compare the standard culture medium with another culture medium that is more widely available and easier to work with and to assess the collagen net ultrastructure architecture and the capacity of the preserved cells to produce proteins. METHODS: Ten fresh lateral menisci were harvested. Each meniscus was divided into three parts; control group, fetal-bovinum-serum group and Insulin-Transferrin-Selenium group during 4 weeks. Cell metabolism was assessed with the gene expression of type I collagen, type II collagen and aggrecan. Collagen ultrastructure was assessed with transmission electron microscopy. The Collagen Meniscal Architecture scoring system was used to evaluate the degree of meniscal disarray. RESULTS: Type I collagen was expressed more in the fetal-bovinum-serum group than in the ITS group (P = 0.036). No differences were found between cultured samples and control groups. Type II collagen showed decreased expression in both cultured groups compared with the control group. No differences were observed in the gene expression of aggrecan in either group. No differences were observed when the Collagen Meniscal Architecture scoring system was applied. CONCLUSIONS: Insulin-Transferrin-Selenium-supplemented medium is at least as effective as the fetal-bovinum-serum-supplemented medium to preserve the net architecture of the meniscal tissue. Gene expression of the studied proteins was similar in the Insulin-Transferrin-Selenium group to that observed in the control group at 4 weeks. Insulin-Transferrin-Selenium might be a better alternative and might be used instead of fetal-bovinum-serum or an autologous host serum in order to preserve meniscal tissue, which precludes the necessity of obtaining host serum previously. Thus, viable meniscal transplantation would logistically be less complicated to perform.

Cryogenic and low temperature preservation of human placental villous explants - a new way to explore drugs in pregnancy disorders.

BACKGROUND: A major handicap in cell culture studies using human tissues is the insufficient availability of fresh material on site. A method was developed for cryogenic storage and low temperature preservation of human placental villous explants, facilitating multi-site distribution for functional studies. METHODS: Explants from term placentas were incubated with cryoprotectant agents (dimethyl-sulfoxide (DMSO), ethylene glycol, propanediol or Aedesta), frozen in liquid nitrogen, thawed and then cultured in-vitro. Viability was assessed by comparing frozen and thawed explants with non-frozen controls for morphological changes, lactate dehydrogenase (LDH) release, placenta protein 13 (PP13) secretion, and PCNA Western blotting. Functional studies determined the effect of oxygen and magnesium on explant viability. RESULTS: Cryoprotection by 3 M DMSO best maintained explants' viability, morphological integrity and PP13 release after freezing and thawing from liquid nitrogen. The effect of oxygen and magnesium was used to test the functional viability of cultured explants, after freezing in liquid nitrogen and transfer to dry ice for 1-5 days on site or for shipment to a remote lab. The tested parameters were similar between controls and cryogenically treated explants in the remote lab and the lab of origin, demonstrating the possibility of cryostoring explants for functional studies. CONCLUSION: Cryogenically stored placental villous explants shipped frozen can serve as a useful tool for comparative functional studies of placental villous tissues. The results of this pilot study also open the way for multi-site studies associated with drug tailoring for pregnancy disorders.

Safety of moxifloxacin and voriconazole in corneal storage media on porcine corneal endothelial cells.

PURPOSE: The aim of this study was to evaluate the safety of moxifloxacin and voriconazole as a supplement in corneal storage media for porcine corneal endothelial cells. METHODS: Twenty-eight eyes were divided into 4 groups. In the control group (the C group), corneal buttons were stored for 3 days in Optisol-GS. In the treatment group, the corneal buttons were preserved for 3 days in Optisol-GS mixed with 250 microg/mL moxifloxacin (M group), 100 microg/mL voriconazole (V group), and 250 microg/mL moxifloxacin plus 100 microg/mL voriconazole (MV group). We evaluated the samples via specular microscopy before and after 3 days of preservation. The mean changes of endothelial cell counts were compared among the 4 groups. Scanning electron microscopy was conducted after 3 days of preservation. RESULTS: Before the preservation, the endothelial cell counts did not differ among the 4 groups (P > 0.05). After 3 days of preservation, the endothelial cell count in the MV group was the lowest among the 4 groups (P < 0.05). After 3 days of preservation, the rate of corneal endothelial cell loss in the M and V groups did not differ significantly from the control group (P > 0.05). The rate of endothelial cell loss in the MV group was significantly higher than that of the control group (P < 0.05). Scanning electron microscopy revealed a normal mosaic pattern for the C, V, and F groups, but hexagonality was not preserved in the MV group. CONCLUSION: Preservation in Optisol-GS mixed with moxifloxacin (250 microg/mL) plus voriconazole (100 microg/mL) induced significant toxicity on the endothelial cells in porcine corneas, when compared with the control group.

Antioxidant treatment during preservation of bovine ovaries increased the development potential of embryos.

The overnight preservation of bovine ovaries would be highly useful in the subsequent harvest of viable oocytes for reproductive study. The present study aimed to optimize conditions for overnight preservation of bovine ovaries by examining the effects of temperature, solution and supplementation. In Experiment 1, the rate of development to the blastocyst stage of oocytes derived from ovaries preserved at 15°C was higher than that at either 5 or 25°C (p < 0.05). In Experiment 2, the rate of development to the blastocyst stage of oocytes derived from ovaries preserved in University of Wisconsin solution was higher than when PBS or saline was used (p < 0.05). In Experiment 3, oocytes preserved in saline supplemented with 0.3 mM glutathione (GSH) exhibited an increase in the rate of blastocyst formation compared with oocytes supplemented with 0 or 3 mM GSH (p < 0.05). In Experiment 4, supplementation with 10 µM epigallocatechin gallate during ovary preservation increased the rate of blastocyst formation (p < 0.05). The blastocysts derived from ovaries stored in saline supplemented with GSH at 15°C for 24 h were shown to develop into normal offsprings following transfer to recipient heifers. Our studies indicate that bovine IVM/IVF embryos derived from ovaries preserved in saline supplemented with an antioxidant at 15°C for 24 h can successfully develop to the blastocyst stage and result in offspring.

Inhibition of oxidative stress and apoptosis enables extended maintenance of integrity and function of isolated hepatocytes in suspension.

AIMS: Isolated hepatocytes in suspension may offer an alternative culture system for bioartificial liver devices. However, maintenance of isolated hepatocyte suspensions in conventional media leads to rapid loss of cell integrity. The aim of this study was to develop a modified medium to better maintain hepatocyte integrity. METHODS: Isolated rat hepatocytes were prepared by collagenase digestion. Hepatocytes were purified in a Percoll gradient, suspended in bicarbonate buffered isotonic saline supplemented with d-alpha-tocopherol succinate and glucose and medium changed at 24 h (modified medium). The properties of cells treated this way were compared with those prepared by collagenase digestion and suspension in bicarbonate buffered isotonic saline (basic medium). Both media were maintained at 30 degrees C for 48 h on an orbital shaker. Markers for oxidative stress, apoptosis and metabolic function were measured enzymatically. Cell morphology was assessed by electron microscopy. RESULTS: When compared to collagenase-isolated hepatocytes maintained in basic medium, hepatocytes purified by Percoll (Amersham Biosciences, Castle Hill, Australia) and maintained in modified medium demonstrated significantly increased glutathione (GSH) and GSH : glutathione disulphide (GSSH) ratios, decreased lipid peroxidation product formation, decreased caspase-3 protease activity, reduced uptake of trypan blue and loss of lactate dehydrogenase (LDH) and increase preservation of cellular adenosine triphosphate concentration ([ATP]), urea synthesis, ammonia removal and glycogen content. Cell morphology was substantially preserved following 48 h of maintenance in the modified medium. CONCLUSIONS: The use of Percoll and modified medium reduces cell injury and apoptosis and greatly improves maintenance of cell function and morphology. The modifications reported here and the use of isolated hepatocyte suspensions in bioartificial liver devices are worthy of further investigation.

Viability of human fibroblasts in coconut water as a storage medium.

AIM: To evaluate the effectiveness of a new storage medium for avulsed teeth, coconut water, in maintaining the viability of human fibroblasts. METHODOLOGY: Cell viability after different time periods was evaluated in the following storage media: coconut water, coconut water with sodium bicarbonate, milk, saline and still mineral water. Human fibroblasts were seeded in Eagle's minimal essential medium (EMEM) supplemented with 7.5% foetal calf serum. After trypsinisation, 100 microL of culture medium containing approximately 10(4) cells mL(-1) were collected and pipetted into the wells of 96-well plates, which were incubated overnight in 5% CO(2) and 95% air mixture at 37 degrees C. EMEM was then replaced by the storage media and the plates were incubated at 37 degrees C for 1, 2 and 4 h. Cell viability was determined using the neutral red assay. The proportions of viable cells after exposure to the storage media were analysed statistically by anova and the least significant difference (LSD) test (alpha = 5%). RESULTS: Milk had the greatest capacity to maintain cell viability (P < 0.05), followed by coconut water with sodium bicarbonate and saline. Coconut water was significantly worse at maintaining cell viability compared to milk, coconut water with sodium bicarbonate and saline. The smallest number of viable cells was observed for mineral water (P < 0.05). CONCLUSION: Coconut water was worse than milk in maintaining human fibroblast cell viability.