Measurement of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in cultured rat mesencephalic neurons by high-performance liquid chromatography with electrochemical detection

An HPLC method with electrochemical detection for the simultaneous measurement of serotonin (5-hydroxytryptamine) and 5-hydroxyindoleacetic acid in primary mesencephalic cell culture is described. The serotonin and 5-hydroxyindoleacetic acid cell content was measured on different days of growth in vitro; after twelve days in culture the amounts of serotonin and 5-hydroxyindoleacetic acid detected were 916.0 ± 70.2 and 215.8 ± 15.5 pg per well, respectively. The heterogeneity of neurons in our cultures and their capacity to take up serotonin were assessed by measuring the amounts of exogenous serotonin taken up in the presence of different monoamine uptake inhibitors. This method, sensitive and reliable, can represent a valid alternative to the use of labelled compounds.     

Semi-continuous biohydrogen production as an approach to generate electricity

In this work, a semi-continuous biological system was established to produce hydrogen and generate electricity by coupling the bioreactor to a fuel cell. Heat and acid pretreatments (at 35 and 55 °C) of a seed sludge used as inoculum were performed in order to increase hydrogen producers. Different initial glucose concentrations (IGC) were tested for heat pretreated inoculum at 35 °C to determine the optimum concentration of glucose that supported the highest hydrogen production. Results showed that the heat pretreated inoculums (35 °C) reached the highest hydrogen molar yield of 2.85 mol H₂/mol glucose (0.014 L/h), which corresponds to the acetic acid pathway. At the optimum IGC (10 g/L, 35 °C) the hydrogen molar yield was 3.6 mol H₂/mol glucose (0.023 L/h). The coupled bioreactor-fuel cell system yielded an output voltage of 1.06 V, power of 0.1 W (25 °C) and a current of 68 mA. The overall results suggest that high hydrogen molar yields can be obtained through the acetic acid pathway and that is feasible to generate electricity using hydrogen from the semi- continuous bioreactor.     

Upper lethal temperature limits of the common furniture beetle Anobium punctatum (Coleoptera: Anobiidae)

The susceptibility of the egg, larval and adult stages of Anobium punctatum De Geer (Coleoptera: Anobiidae) to heat (46–54°C, 25–30% RH) was investigated. The larval stage was found to be most tolerant to heat. Very short exposure (5 min) of the larvae to temperatures of 52°C and above led to 100% mortality of the larvae. Treatment for 1.5 and 1 h at 47° and 48°C, respectively, led to 100% mortality. At 46°C, longer treatment time (2.5 h) was necessary. Exposure to 45°C for 3.5 h did not lead to immediate death of the larvae. The actual mortality is assumed to be greater than the results actually reported, as most of the larvae had not resumed normal activity at the end of the two-week observation period. The egg and adult stages were more sensitive to heat than the larvae; shorter exposure times were sufficient to obtain 100% mortality.     

Temperature compensation in the mammalian cell cycle

Random and synchronous V79 cells were shifted from 37.5 °C to temperatures between 29 ° and 41 °C. Intermitotic time determinations of random cultures showed an increase in generation time and a broadening in the distribution of generation times in cells whose cycle spanned the temperature shift, but only a slight increase in generation time after one generation at temperatures between 34 °–40 °C. At 33.5 °C and below there was a stepwise increase in generation time. When cells grown at non-standard temperatures were allowed to habituate for 48 h at the altered temperature prior to analysis, the increase in median intermitotic time was slightly less in comparison to analyses done after only one generation following the temperature step. The Q₁₀ for cell division of cells growing at temperatures from 34 ° to 40 °C was between 1.15 and 1.26, suggesting that the mammalian cell cycle is temperature compensated over a limited (6–7 °C) temperature span. Mammalian cells in culture appear to have the same capacity for temperature compensation in their cell cycle as do unicellular eukaryotes. The fact that cycle time at lower temperatures increases in a discrete manner is taken as evidence for a quantal clock.     

Effects of prostaglandin E1 and isoproterenol on cyclic AMP content of human fibroblasts modified by time and cell density in subculture

In confluent cultures of “young” (< 30 generations) human fibroblasts, maximally effective concentrations of prostaglandin E₁ (5.6 μM) and isoproterenol (2 μM) increased cyclic AMP content several hundred-fold and approximately 30-fold, respectively. On the first day after initiation of cultures at either low (approx. 3 · 10⁵ cells) or high (approx. s · 10⁶ cells) cell density the magnitude of the isoproterenol effect was similar to that in confluent cultures. It increased during the next few days, reaching a maximum around day 2–3, and then declined. On any day during the period of subculture, the magnitude of the isoproterenol effect was inversely related to cell density. Alterations in response to prostaglandin E₁ as a function of time in subculture or cell density were less dramatic. The effects of prostaglandin E₁ were, however, smaller at some point during the first few days of subculture than after day 7, and when effects of prostaglandin E₁ were minimal, those of isoproterenol were maximal and approached those of prostaglandin E₁. On any day of subculture, cells in cultures of higher density tended to accumulate more cyclic AMP in response to prostaglandin E₁ than did those in low density cultures. The effects of prostaglandin E₁ and isoproterenol on cyclic AMP content were qualitatively similar in “young” and in “old” (> 60 generations in culture) human fibroblasts although the changes associated with duration of subculture and cell density tended to be less marked with “old” cells. In the “young” fibroblasts responsiveness to isoproterenol and prostaglandin E₁ appears to correlate with cell morphology and with the fractional rate of growth in subcultures. It is suggested that the capacities of the fibroblasts to respond to these two agents may be altered independently during growth of human fibroblasts.     

Adaptation to different growth temperatures modifies some mammalian cell survival responses

Chinese hamster (HA-1) cells that have been grown at 37 °C since explant several years ago can adapt themselves to grow at temperatures ranging from 32 to 41 °C. This growth adaptation is accompanied by major phenotypic changes in, for exampie, the cellular responses to 43 and 45 °C heat challenges and to ethanol challenges (0–10% in concentration). Cells grown at 39.5 °C are seen to acquire substantial heat resistance when compared with cells grown at 37 °C; resistance is even more pronounced if the growth temperature is at 41 °C. On the other hand, cells grown at 32 °C become more sensitive to heat than controls. Our results also indicate an increased resistance to ethanol of the 41 °C grown cells. By contrast the cells' X-ray survival response is affected only minimally. The changes seen are phenotypic; upon being returned to 37 °C, HA-1 cells within 34 h regain their ‘normal’ heat responses.     

Testosterone modifies response to chronic heat exposure in rats

Eight weeks of heat exposure (34±0.5°C) in sham-orchiectomized rats leads to an increase of body temperature, slowing of body growth rate, and decrease of serum corticosterone level, as compared with animals maintained at 21±2°C. Orchiectomy decreases body temperature, slows growth rate, and increases plasma corticosterone concentration both in control and heat exposed animals. Testosterone administration reverts these parameters to initial values. We conclude that testosterone plays a role in the regulation of heat balance in male rats.     

Evaluation of prostaglandin-receptors in human and rat liver: Interspecies differences at the prostaglandin receptor-level

The properties of PGE1-, PGE2- and iloprost (stable PGI2-analogue)-binding sites on normal human and rat liver surface cell membranes were investigated. The specific binding of [3H]PGE1 to human (rat) liver surface cell membranes could be displaced most effectively by unlabeled PGE1 (IC-50: 2.5±1.7, (6.1±2.1) μM) and the specific binding of [3H]PGE2 by unlabeled PGE2 (IC-50: 1.9±0.9 (2.0±0.8) μM. The Scatchard analysis on [3H]PGE1- as well as on [3H]ilioprost-binding was curvilinear whereas it was clearly linear on [3H]PGE2-binding in both the species. The high-affinity [3H]PGE1-sites showed a Bmax of 36.3±5.2 (21.3±4.3) fmol/mg protein and a Kd of 2.1±1.8 (1.9±0.7) nM, the low-affinity [3H]PGE1-sites a Bmax of 93.4±18.2 (86.1±13.2) fmol/mg protein and a Kd of 10.5±2.9 (15.3±3.2) nM. The high-affinity [3H]iloprost-sites exhibited a Bmax of 71.4±13.9 (35.9±8.2) fmol/mg protein and a Kd of 4.1±1.2 (1.7±1.8) nM, the low-affinity [3H]iloprost-sites a Bmax of 217.3±42.1 (142.9±17.8) fmol/mg protein and a Kd of 16.3±4.9 (9.2±7.2) nM. The [3H]PGE2-sites showed a Bmax of 135.4±51.9 (38.8±7.4) fmol/mg protein and a Kd of 16.2±3.2 (2.5±1.2) nM.It is assumed that prostaglandins of the E-series are promising substances in the regulation of human and rat liver function since liver cells are stable to bind reasonable amounts of these substances in a high affinity manner. However, interspecies differences in the affinity of the prostaglandins to their receptor-sites make it strange to assume that the same biological findings claimed several times for the rat liver are relevant for human too.     

Tubulin synthesis and heat shock-induced cell synchrony in Tetrahymena

This paper offers the suggestion that heat shock inhibition of tubulin synthesis accounts for the molecular mechanism by which periodic heat shocks induce cell synchrony in Tetrahymena. Each heat shock (34 °C) represses tubulin synthesis and blocks the division cycle at the point when the oral structure, rich in microtubules, would normally begin to assemble. Recovery (at 28 °C) from each heat shock is characterized by parallel derepression of tubulin synthesis and of oral development. Changes in protein synthesis patterns are complex when the temperature is shifted up and down between 28 and 34 °C and further experimental support is required in support of the hypothesis here forwarded.     

Selective isolation of rat aortic wall layers and their cell types in culture—Application to converting enzyme activity measurement

The rat aorta, whose three wall layers can be separated by microdissection offers the rare possibility of comparing physiological characteristics of in vivo tissular cell components and corresponding cells after culture.We developed a technique allowing the dissociation of the three tunicae (intima, media and adventitia) of the rat aorta and the culture of their main cell types i.e: endothelial cells (EC) from intima, smooth muscle cells (SMC) from media and fibroblasts (Fib) from adventitia. Comparison between selected tunicae in vivo and their corresponding cells in vitro was performed via arterial angiotensin converting enzyme (ACE) activity measurements in Wistar rats.In vivo microsomial ACE activity for each tunica was as follows: 368.9 ± 34.3 (endothelium), 10.5 ± 1.9 (media) and 10.2 ± 4.9 (adventitia) pmol/mg protein/min. Corresponding cell primary culture values were 1.2 ± 0.1 (EC), 0.06 ± 0.02 (SMC) and 0.24 ± 0.01 (Fib) pmol/mg protein/min. Incubation of serum-deprived cells with Dexamethasone (10⁻⁷M) over 48 hr induced a statistically significant shift of total ACE activity from controls to stimulated cells of 2.9 ± 0.3 to 9.7 ± 1.0 in EC, 0.8 ± 0.1 to 32.1 ± 4.9 in SMC and 1.03 ± 0.65 to 57.2 ± 2.1 pmol/ mg prot/min in fibroblasts.In the rat aorta, ACE was present not only in the intimal endothelial cell lining, but also in the media and the adventitia. ACE activity levels in primary cultured vascular cells were about 100-fold less than those found in the ex vivo tissues. Nevertheless, ACE expression seems to be more constitutive in endothelial cells and more inducible in smooth muscle cells and fibroblasts. This methodological approach should be of interest in studying environmental or genetic regulation of protein expression in the three layers/three cell types of the vascular wall.     

X-ray diffraction data for (1→3)-α-d-glucan triacetate

The crystal structures of (1→3)-α-d-glucan triacetates were studied by X-ray diffraction measurements on fibre diagrams. The oriented films annealed in water at high temperature were of higher crystallinity and occurred as two crystalline polymorphs (GTA I and GTA II) depending on the samples and also the annealing temperature. All reflections in GTA I were indexed with a pseudo-orthorhombic unit cell with a = 1·753, b = 3·018 and c(fibre axis) = 1·205 nm. From the fibre repeat data coupled with the density data and the presence of only the (003) reflection on the meridian, an extended three-fold helical structure was proposed. Although some reflections in GTA II split from the layer lines, the basic unit cell was a monoclinic system with a = 1·685, b = 3·878, c (fibre axis) = 1·210 nm and γ = 112·2°. A similar three-fold structure to GTA I was proposed from the almost identical fibre repeat and the conformational analysis on (1→3)-α-d-glucan. It was concluded that, on acetylation, the d-glucan structure changed from the fully extended two-fold helix to the extended three-fold accompanied by some extent of chain shrinking.     

Effects of Epidermal Growth Factor and Phorbol Ester on Thyroid Epithelial Integrity

The effects of epidermal growth factor (EGF) and phorbol ester (tetra-O-decanoylphorbol-16-acetate; TPA) on thyroid epithelial integrity were studied in filter-cultured monolayers of porcine thyrocytes, which before experiments were growth-arrested and had a high transepithelial resistance (R_TE > 6 · 10³ Ω · cm²) and polarized, thyroid-specific functions. Both EGF and TPA stimulated dose-dependently the cellular incorporation of [³H]thymidine, which maximally (at 10 ng/ml EGF for 48 h) corresponded to a 65% increase of the DNA content. The EGF-treated cells proliferated mainly within the original monolayer, which became folded due to the increased cell number; clusters of epithelial cells also assembled between the monolayer and the filter. Although the transepithelial potential difference was reduced, from 15-30 mV in controls to 2-10 mV, the epithelial barrier function was maintained (R_TE 1-3 · 10³ Ω · cm²; impermeability to [³H]inulin). EGF did not change the ultrastructural polarity of the plasma membrane or the distinct distribution of ZO-1 and cadherin immunoreactivities to junctions, but cytoplasmic cadherin present in controls disappeared after EGF. In cultures acutely depleted of extracellular Ca²⁺ EGF pretreatment for 48 h antagonized the preventive effect of thyrotropin on paracellular leakage and loss of cell-cell adhesion. TPA (0.1 μM) induced a temporary barrier dysfunction (maximal after 24 h) accompanied by pronounced alterations of cell shape and actin-based cytoskeleton, dissociation of junctional cadherin, and shedding of cells into the apical medium. In long-term (2-5 days) TPA-treated cultures the epithelial morphotype and barrier function recovered. The combined stimulation with EGF and TPA caused a persistent derangement of the cell layer including attenuation of ZO-1 at cell-cell contacts, paracellular leakage of [³H]inulin, and cell detachment. We conclude that EGF is able to release porcine thyroid epithelial cells from contact inhibition of growth along with intact cell polarity and tight junctions. Yet, when acting together with phorbol ester EGF provokes a lasting morphological transformation. Impaired positive control of Ca²⁺-dependent cell-cell adhesion in EGF-treated cultures suggests a latent defect with possible transforming potential in the cadherin-based regulation of the junctional complex.     

Influence of process temperature on recombinant enzyme activity in Escherichia coli fed-batch cultures

The influence of proteolysis over recombinant protein quality has been studied using rhamnulose 1-phosphate aldolase (RhuA) production as case example. Progressive induction by means of continuous isopropyl-β-d-thiogalactopyranoside (IPTG) dosage in Escherichia coli fed-batch cultures led to high specific levels of recombinant protein. However, the specific activity profile did not correlate to the specific protein content when the process was run at 37 °C and there was a decrease of the enzyme activity along the induction phase. Specific activity loss depending on the presence of an energy source was observed at short term, but protein degradation due to the action of energy-independent metalloproteases occurred after a longer time period. The effects of lowering the temperature were analysed on both mechanisms, and a reduction of the specific activity loss was observed when the process temperature was decreased to 28 °C. Lower plasmid copy number and specific production rates probably alleviated the metabolic load on host cell during recombinant protein overexpression, and a high increase of the enzyme activity was achieved in high cell density fed-batch cultures under these conditions.     

Simultaneous exposure of Xenopus A6 kidney epithelial cells to concurrent mild sodium arsenite and heat stress results in enhanced hsp30 and hsp70 gene expression and the acquisition of thermotolerance

In this study, we examined the effect of concurrent low concentrations of sodium arsenite and mild heat shock temperatures on hsp30 and hsp70 gene expression in Xenopus A6 kidney epithelial cells. RNA blot hybridization and immunoblot analysis revealed that exposure of A6 cells to 1–10 µM sodium arsenite at a mild heat shock temperature of 30 °C enhanced hsp30 and hsp70 gene expression to a much greater extent than found with either stress individually. In cells treated simultaneously with 10 µM sodium arsenite and different heat shock temperatures, enhanced accumulation of HSP30 and HSP70 protein was first detected at 26 °C with larger responses at 28 and 30 °C. HSF1 activity was involved in combined stress-induced hsp gene expression since the HSF1 activation inhibitor, KNK437, inhibited HSP30 and HSP70 accumulation. Immunocytochemical analysis revealed that HSP30 was present in a granular pattern primarily in the cytoplasm in cells treated simultaneously with both stresses. Finally, prior exposure of A6 cells to concurrent sodium arsenite (10 µM) and heat shock (30 °C) treatment conferred thermotolerance since it protected them against a subsequent thermal challenge (37 °C). Acquired thermotolerance was not observed with cells treated with the two mild stresses individually.     

Prostaglandin F2α, oxytocin and progesterone secretion by bovine luteal cells at several stages of luteal development: Effects of oxytocin, luteinizing hormone, prostaglandin F2α and estradiol-17β

Bovine luteal cells from Days 4, 8, 14 and 18 of the estrous cycle were incubated for 2 h (1 × 10⁵ cells/ml) in serum-free media with one or a combination of treatments [control (no hormone), prostaglandin F₂α (PGF), oxytocin (OT), estradiol-17β (E) or luteinizing hormone (LH)]. Luteal cell conditioned media were then assayed by RIA for progesterone (P), PGF, and OT. Basal secretion of PGF on Days 4, 8, 14 and 18 was 173.8 ± 66.2, 111.1 ± 37.8, 57.7 ± 15.4 and 124.3 ± 29.9 pg/ml, respectively. Basal release of OT and P was greater on Day 4 (P<0.01) than on Day 8, 14 and 18 (rmOT: 17.5 ± 2.6 versus 5.6 ± 0.7, 6.0 ± 1.4 and 3.1 ± 0.4 pg/ml; P: 138.9 ± 19.5 versus 23.2 ± 7.5, 35.4 ± 6.5 and 43.6 ± 8.1 ng/ml, respectively). Oxytocin increased (P<0.01) PGF release by luteal cells compared with control cultures irrespective of day of estrous cycle. Estradiol-17β stimulated (P<0.05) PGF secretion on Days 8, 14 and LH increased (P<0.01) PGF production only on Day 14. Prostaglandin F₂α, E and LH had no effect on OT release by luteal cells from any day. Luteinizing hormone alone or in combination with PGF, OT or E increased (P<0.01) P secretion by cells from Days 8, 14 and 18. However on Day 8, a combination of PGF + OT and PGF + E decreased (P<0.05) LH-stimulated P secretion. These data demonstrate that OT stimulates PGF secretion by bovine luteal cells in vitro. In addition, LH and E also stimulate PGF release but effects may vary with stage of estrous cycle.     

Cytogenetic analysis of ovulated mouse oocytes following hyperthermic stress during meiotic maturation

Effects of hyperthermia on maturing oocytes of a random-bred stock of mice were investigated to determine if those effects might in part be responsible for the decreased reproductive efficiency observed in animals during periods of high ambient temperatures. Oocytes were collected from virgin mice following synchronization of ovulation with Pregnant Mare Serum Gonadotropin (PMSG) and Human Chorionic Gonadotropin (HCG). Stressed animals were exposed to hyperthermic conditions (35 ± 1 °C, 65 ± 3% relative humidity (RH)) immediately following the injection of HCG until the time of oocyte recovery. Prior to heat exposure all animals were maintained at control conditions of 21 ± 2 °C and 65 ± 5% RH. Meiotic maturation was disrupted in a significant proportion (>25%) of oocytes from stressed animals. Apparent disruption of the spindle mechanism resulted in the cessation of the meiotic process at metaphase I in 12.28% of the oocytes from heat-stressed mice with 4.87% oocytes exhibiting subnucalei. Other nuclear forms presumed to be non-viable occurred in an additional 8.58% of the oocytes. Two oocytes exhibited retained polar body chromatin and several oocytes at metaphase II exhibited atypical configuration. The remaining oocytes were in normal metaphase II configuration.     

Heat-sensitive development of the phagocytotic organelle in a Tetrahymena mutant

A laboratory-induced mutant with heat-sensitive development of the phagocytotic organelle has been isolated in Tetrahymena pyriformis, syngen 1; the mutant cells form food vacuoles at 30 °C, but not after incubation at 37 °C. Mutant cells transferred to 37 °C undergo a maximum of 3–5 doublings, but a sizeable fraction remains viable for several days. Results of temperature shift-up experiments reveal that an oral apparatus (OA) constructed at 30 °C remains functional at 37 °C, while one constructed at 37 °C is non-functional with regard to phagocytosis. Preliminary cytological observations reveal severe structural abnormalities of the OA. Thus the mutant appears to be primarily affected in the morphogenesis of the OA. The phenotypic effect of the mutation is reversible by a temperature shiftdown. Changes in phenotype caused by temperature shifts in either direction can occur even in stationary or starved cultures. Cell division is not required for the resumption of phagocytosis after a temperature shiftdown. Null-formers obtained at the first doubling after a temperature shift-up can divide at least once more, indicating that a functional OA is not required for cell division at any stage of the cell cycle. Mutants defective in phagocytosis may prove useful in gaining deeper understanding of this mechanism and its relationship to other cellular processes.     

Single crystals of dextran: High temperature polymorph

Lamellar single crystals of a high temperature polymorph of synthetic dextran were prepared at temperatures ranging from 120 to 200°C in a mixture of water and polyethylene glycol. Individual crystals with lath-like shapes gave well resolved electron diffraction diagrams from which the reciprocal unit cell parameters a^*, b^* and γ^* could be measured. The direct cell parameters were then determined from a series of electron diffraction diagrams obtained by sequential tilting of the crystal about the b^* axis. This gave a = 0·922 ± 0·001 nm, b = 0·922 ± 0·001 nm, c (chain axis) = 0·78 ± 0·01 nm, α = γ = 90° and β = 91·3° ± 0·5°. The crystal symmetry was P2₁ with b as the unique monoclinic axis. These data coupled with the observed density of the crystals, indicated that the unit cell contained two antiparallel dextran chains of two residues each. When the crystals were grown at temperatures between 90 and 120°C, a percentage of crystals containing both low and high temperature polymorphs were obtained. These mixed crystals had most likely grown in syntaxy.     

Laser-assisted selection and passaging of human pluripotent stem cell colonies

The derivation of somatic cell products from human embryonic stem cells (hESCs) requires a highly standardized production process with sufficient throughput. To date, the most common technique for hESC passaging is the manual dissection of colonies, which is a gentle, but laborious and time-consuming process and is consequently inappropriate for standardized maintenance of hESC. Here, we present a laser-based technique for the contact-free dissection and isolation of living hESCs (laser microdissection and pressure catapulting, LMPC). Following LMPC treatment, 80.6 ± 8.7% of the cells remained viable as compared to 88.6 ± 1.7% of manually dissected hESCs. Furthermore, there was no significant difference in the expression of pluripotency-associated markers when compared to the control. Flow cytometry revealed that 83.8 ± 4.1% of hESCs isolated by LMPC expressed the surface marker Tra-1-60 (control: 83.9 ± 3.6%). In vitro differentiation potential of LMPC treated hESCs as determined by embryoid body formation and multi-germlayer formation was not impaired. Moreover, we could not detect any overt karyotype alterations as a result of the LMPC process. Our data demonstrate the feasibility of standardized laser-based passaging of hESC cultures. This technology should facilitate both colony selection and maintenance culture of pluripotent stem cells.     

Continued expression of plant-made vaccines following long-term cryopreservation of antigen-expressing tobacco cell cultures

Production of vaccines in plant cells provides an alternative system that has several advantages when compared to current vaccine production methods. Establishment of stable seed stocks for a continuous supply of a vaccine is a critical part of production systems. Therefore, a vitrification method for cryopreservation was applied to non-transgenic and three different antigen-expressing transgenic Nicotiana tabacum (NT-1) lines. Preculture of the suspension cultures 1 d prior to vitrification was sufficient for cell survival through the cryopreservation process. Inclusion of 0.3 M mannitol in the preculture medium was necessary for maintenance of cell viability. Cultures were also treated with and without heat shock prior to vitrification, and it was found that heat shock was unnecessary for growth recovery post cryopreservation. All cultures survived storage in liquid nitrogen at intervals ranging from 1 h to 1 yr. Antigen expression was measured by enzyme-linked immunosorbent assay for cultures that grew post cryopreservation and those that had never been cryopreserved. Expression levels in cultures derived from cryopreserved material were comparable to cultures that had not been cryopreserved. Transmission electron microscopy showed that the integrity of the cell structure was maintained post cryopreservation.