Effect of Cell Density on Post-Thaw Viability in Cryopreserved Artificial Tissue

2000 ◽  
Author(s):  
Masanobu Ujihira ◽  
Yumi Matsumura ◽  
Chinatsu Kuroda ◽  
Koji Okaniwa ◽  
Kiyoshi Mabuchi
Keyword(s):  
Cooling Rate ◽  
Cell Density ◽  
Latent Heat ◽  
Human Fibroblasts ◽  
Test Chamber ◽  
Freezing Process ◽  
Cell Contact ◽  
Slow Cooling ◽  
Trypan Blue Exclusion ◽  
Artificial Tissue

Abstract The effect of cell density on the post-thaw viability of cells in cryopreserved artificial tissue was studied. Human fibroblasts were three-dimensionally cultured for 2 days in a collagen sponge (ϕ20×1mm) as an extracellular matrix to imitate biological tissue (artificial tissue). Different cell densities for the artificial tissue were used, from 104 to 107 cells/cm3. Four artificial tissues were first stacked in a test chamber, then frozen at a slow or fast cooling rate (either 1 or 50°C/min) in a solution of Dulbecco’s Modified Eagle Medium, 20% Fetal Bovine Serum, and 10% dimethylsulfoxide, then kept frozen at −196°C for 2 hours, and finally thawed. The collagen matrix of the artificial tissue was dissolved using collagenase. Post-thaw viability of fibroblasts was evaluated by using a trypan blue exclusion assay. The experiments were prepared, and then the latent heat of artificial tissue (3.5×3.5×1mm) during the freezing process was measured by using a differential scanning calorimeter. Results show that with increasing cell density, the post-thaw viability decreased, whereas the latent heat was almost independent of cell density. With increasing cell density at the slow cooling rate, the degree of supercooling of the intracellular solution increased with decreasing temperature, possibly leading to intracellular freezing. Moreover, when the cell density was high, cell-to-cell contact or an obstruction to dehydration seemed to induce intracellular freezing. Therefore, the post-thaw viability seems to decrease as the number of cells exhibiting intracellular freezing increased.

Using Encapsulation to Improve the Viability of Cryopreserved Cells

1999 ◽  
Author(s):  
Yoshifumi Matsumoto ◽  
Yukihiro Morinaga ◽  
Masanobu Ujihira ◽  
Kotaro Oka ◽  
Kazuo Tanishita
Keyword(s):  
Cooling Rate ◽  
Pc12 Cells ◽  
Latent Heat ◽  
Ice Crystals ◽  
Freezing Process ◽  
Biological Cell ◽  
Scanning Calorimetry ◽  
Encapsulated Cells ◽  
Pheochromocytoma Pc12 ◽  
Suspended Cells

Abstract The purpose of this study is to clarify whether encapsulated cells have an advantage over suspended cells in cryopreservation. Rat pheochromocytoma (PC12) cells were selected for test biological cell and microencapsulated in alginate-polylysine-alginate membranes. Microencapsulated PC12 cells were frozen with differential scanning calorimetry (DSC) at a cooling rate of 0.5 to 10°C/min, their latent heat was measured among the freezing process over the temperature range 4 to −80°C. Their post-thaw viability were evaluated by dye exclusion assay and dopamine release. As a result, latent heat of encapsulated cells was lower than that of suspended cells at a cooling rate of 0.5 and l°C/min. This is because extra-capsule was frozen and intra-capsule unfrozen, as ice crystals forms in extra-capsule space. Post-thaw viability of microencapsulated PC12 cells was improved at 0.5 and l°C/min compared with that of suspended cells. Therefore, in microencapsulated PC12 cells, achievement of intra-capsule unfrozen condition during freezing leads to reducing the solution effect and improving the viability.

Comparison of Ocular Surface Cytological Appearance in Glaucoma Patient Treated with Timolol Maleat 0,5% Latanoprost 0,005% and Timolol-Latanoprost Fixed Combination Preservative Free Eye Drop

2019 ◽  
Vol 44 (2) ◽  
pp. 82
Author(s):  
Maretha Amrayni ◽  
Elsa Gustianty ◽  
Susi Heryati ◽  
Andika Prahasta ◽  
Maula Rifada ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cell Density ◽  
Goblet Cell ◽  
Ocular Surface ◽  
Fixed Combination ◽  
Cell Contact ◽  
Corneal Epithelial ◽  
Preservative Free ◽  
Goblet Cell Density ◽  
Epithelial Metaplasia

Introduction : The longterm use of topical antiglaucoma might cause ocular surface instability due to active substance or preservative used. Impression cytology examination may reveal superficial epithelial cells on conjunctiva and cornea, including goblet cells. Goblet cell density decrease is the most important parameter on evaluation of ocular surface disorder. Objective : This study was to understand ocular surface remodeling due to active substance of topical antiglaucoma with impression cytology examination among the patient prior and 3 months after therapy. Methods : This was a randomized controlled trial study with single blind masking. A total of 45 eyes from 31 patients were used as subject and distributed onto three groups treatment, which were timolol maleat 0.5%, latanoprost 0.005%, and latanoprost-timolol maleat fixed combination. All topical antiglaucoma in this study were preservative free. Result : There were differences between 3 groups in goblet cells density after 3 months therapy (p=0,030). Goblet cell density in timolol group was lower than latanoprost (p=0,041) and fixed combination (p=0,045). There was no significantly difference between 3 groups in conjunctival epithelial metaplasia degree (p=0,706) and cell to cell contact degree in corneal epithelial cells (p=0.66) after 3 months therapy. Conjunctival epithelial metaplasia degree were increased among group of timolol (p=0,008) and fixed combination (p=0,046). Conclusion : Timolol maleat 0,5% caused lower goblet cell density after 3 months therapy compare with latanoprost and fixed combination. There was no significantly difference in conjunctival epithelial metaplasia and cell to cell contact degree in corneal epithelial cells among these glaucoma treatment groups.

scholarly journals Calorimetric analysis of ice onset temperature during cryoablation: a model approach to identify early predictors of effective applications

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Elena Campagnoli ◽  
Andrea Ballatore ◽  
Valter Giaretto ◽  
Matteo Anselmino
Keyword(s):  
Cooling Rate ◽  
Latent Heat ◽  
Bovine Liver ◽  
Onset Temperature ◽  
Ice Formation ◽  
Scanning Calorimetry ◽  
Calorimetric Analysis ◽  
Early Predictors ◽  
Latent Heat Capacity ◽  
Model Approach

AbstractAim of the present study is to analyze thermal events occurring during cryoablation. Different bovine liver samples underwent freezing cycles at different cooling rate (from 0.0075 to 25 K/min). Ice onset temperature and specific latent heat capacity of the ice formation process were measured according to differential scanning calorimetry signals. A computational model of the thermal events occurring during cryoablation was compiled using Neumann’s analytical solution. Latent heat (#1 = 139.8 ± 7.4 J/g, #2 = 147.8 ± 7.9 J/g, #3 = 159.0 ± 4.1 J/g) of all liver samples was independent of the ice onset temperature, but linearly dependent on the water content. Ice onset temperature was proportional to the logarithm of the cooling rate in the range 5 ÷ 25 K/min (#3a = − 12.2 °C, #3b = − 16.2 °C, #3c = − 6.6 °C at 5K/min; #3a = − 16.5 °C, #3b = − 19.3 °C, #3c = − 11.6 °C at 25 K/min). Ice onset temperature was associated with both the way in which the heat involved into the phase transition was delivered and with the thermal gradient inside the tissue. Ice onset temperature should be evaluated in the early phase of the ablation to tailor cryoenergy delivery. In order to obtain low ice trigger temperatures and consequent low ablation temperatures a high cooling rate is necessary.

scholarly journals Effect of cell density on binding and uptake of low density lipoprotein by human fibroblasts.

1979 ◽  
Vol 83 (3) ◽  
pp. 588-594 ◽  
Author(s):  
H S Kruth ◽  
J Avigan ◽  
W Gamble ◽  
M Vaughan
Keyword(s):  
Cell Density ◽  
Low Density Lipoprotein ◽  
Human Fibroblasts ◽  
Density Lipoprotein ◽  
Low Density ◽  
Cellular Lipid ◽  
Cholesterol Accumulation ◽  
Ldl Receptors ◽  
Ldl Binding ◽  
In Cells

The effect of cell density on low density lipoprotein (LDL) binding by cultured human skin fibroblasts was investigated. Bound LDL was visualized by indirect immunofluorescence. Cellular lipid and cholesterol were monitored by fluorescence in cells stained with phosphine 3R and filipin, respectively. LDL binding and lipid accumulation were compared in cells in stationary and exponentially growing cultures, in sparsely and densely plated cultures, in wounded and non-wounded areas of stationary cultures, and in stationary cultures with and without the addition of lipoprotein-deficient serum. We conclude that LDL binding and cholesterol accumulation induced by LDL are influenced by cell density. It appears that, compared to rapidly growing cells, quiescent (noncycling) human fibroblasts exhibit fewer functional LDL receptors.

Measurement and Modeling of Latent Heat Release During Freezing of Aqueous Solutions in a Small Container

2000 ◽  
Author(s):  
Ramachandra V. Devireddy ◽  
John C. Bischof ◽  
Perry H. Leo ◽  
John S. Lowengrub
Keyword(s):  
Mass Transport ◽  
Heat Release ◽  
Latent Heat ◽  
Pure Water ◽  
Heat Of Fusion ◽  
Freezing Process ◽  
Latent Heat Release ◽  
Aqueous Systems ◽  
Heat And Mass Transport ◽  
Latent Heat Of Fusion

Abstract The latent heat of fusion, ΔHf of a cryobiological medium (a solute laden aqueous solution) is a crucial parameter in the cryopreservation process. The latent heat has often been approximated by that of pure water (∼ 335 mJ/mg). However, recent calorimetric (DSC - Pyris 1) measurements suggest that the actual magnitude of latent heat of fusion during freezing of solute laden aqueous systems is far less. Fourteen different pre-nucleated solute laden aqueous systems (NaCl-H2O, Phosphate Buffered Saline or PBS, serum free RPMI, cell culture medium, glycerol and Anti Freeze Protein solutions) were found to have significantly lower ΔHf than that of pure water (Devireddy and Bischof, 1998). In the present study additional calorimetric experiments are performed at 1, 5 and 20 °C/min in five representative cryobiological media (isotonic or 1× NaCl-H2O, 10× NaCl-H2O, 1× PBS, 5× PBS and 10× PBS) to determine the kinetics of ice crystallization. The temperature (T) and time (t) dependence of the latent heat release is measured. The experimental data shows that at a fixed temperature, the fraction of heat released at higher cooling rates (5 and 20 °C/min) is lower than at 1 °C/min for all the solutions studied. We then sought a simple model that could predict the experimentally measured behavior and examined the full set of heat and mass transport equations during the freezing process in a DSC sample pan. The model neglects the interaction between the growing ice crystals and is most appropriate during the early stages of the freezing process. An examination of the coefficients in the heat and mass transport equations shows that heat transport occurs much more rapidly than solute transport. Hence, the full model reduces to one in which the temperature profile is constant in space while the solute concentration profile obeys the full time and space dependent diffusion equation. The model reveals the important physical parameters controlling the mass transport at the freezing interface and further elucidates the experimental results, i.e. the temperature and time dependence of the latent heat release.

scholarly journals Correlation of Processing, Inner Structure, and Part Properties of Injection Moulded Thin-Wall Parts on Example of Polyamide 66

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Dietmar Drummer ◽  
Steve Meister
Keyword(s):  
Cooling Rate ◽  
Injection Moulding ◽  
Degree Of Crystallinity ◽  
Process Conditions ◽  
Thin Wall ◽  
Polyamide 66 ◽  
Slow Cooling ◽  
Cooling Conditions ◽  
Internal Structures ◽  
Wall Injection

In micro- and thin-wall injection moulding the process conditions affect the developed internal structures and thus the resulting part properties. This paper investigates exemplarily on polyamide 66 the interactions of different cooling conditions on the morphological and crystalline structures. The investigations reveal that a slow cooling rate of the melt results in a homogeneous morphology and a higher degree of crystallinity and also a favoured crystalline structure. Consequently, the dielectric behaviour and light transmitting part properties are affected.

Controlled Freezing of Nonideal Solutions With Application to Cryosurgical Processes

1991 ◽  
Vol 113 (4) ◽  
pp. 430-437 ◽  
Author(s):  
H. M. Budman ◽  
J. Dayan ◽  
A. Shitzer
Keyword(s):  
Cooling Rate ◽  
Freezing Process ◽  
Data Sets ◽  
Tracking Accuracy ◽  
Loop Control ◽  
Set Point ◽  
New Device ◽  
Loop Control System ◽  
The Stability ◽  
Controlled Freezing

Success of a cryosurgical procedure, i.e., maximal cell destruction, requires that the cooling rate be controlled during the freezing process. Standard cryosurgical devices are not usually designed to perform the required controlled process. In this study, a new cryosurgical device was developed which facilitates the achievement of a specified cooling rate during freezing by accurately controlling the probe temperature variation with time. The new device has been experimentally tested by applying it to an aqueous solution of mashed potatoes. The temperature field in the freezing medium, whose thermal properties are similar to those of biological tissue, was measured. The cryoprobe temperature was controlled according to a desired time varying profile which was assumed to maximize necrosis. The tracking accuracy and the stability of the closed loop control system were investigated. It was found that for most of the time the tracking accuracy was excellent and the error between the measured probe temperature and the desired set point is within ±0.4°C. However, noticeable deviations from the set point occurred due to the supercooling phenomenon or due to the instability of the liquid nitrogen boiling regime in the cryoprobe. The experimental results were compared to those obtained by a finite elements program and very good agreement was obtained. The deviation between the two data sets seems to be mainly due to errors in positioning of the thermocouple junctions in the medium.

scholarly journals Analysis of isothermal and cooling rate dependent immersion freezing by a unifying stochastic ice nucleation model

2015 ◽  
Vol 15 (9) ◽  
pp. 13109-13166
Author(s):  
P. A. Alpert ◽  
D. A. Knopf
Keyword(s):  
Cooling Rate ◽  
Surface Area ◽  
Ice Nucleation ◽  
Rate Dependence ◽  
Nucleation Theory ◽  
Freezing Process ◽  
Nucleation Kinetics ◽  
Rate Dependent ◽  
Immersion Freezing ◽  
The Impact

Abstract. Immersion freezing is an important ice nucleation pathway involved in the formation of cirrus and mixed-phase clouds. Laboratory immersion freezing experiments are necessary to determine the range in temperature (T) and relative humidity (RH) at which ice nucleation occurs and to quantify the associated nucleation kinetics. Typically, isothermal (applying a constant temperature) and cooling rate dependent immersion freezing experiments are conducted. In these experiments it is usually assumed that the droplets containing ice nuclei (IN) all have the same IN surface area (ISA), however the validity of this assumption or the impact it may have on analysis and interpretation of the experimental data is rarely questioned. A stochastic immersion freezing model based on first principles of statistics is presented, which accounts for variable ISA per droplet and uses physically observable parameters including the total number of droplets (Ntot) and the heterogeneous ice nucleation rate coefficient, Jhet(T). This model is applied to address if (i) a time and ISA dependent stochastic immersion freezing process can explain laboratory immersion freezing data for different experimental methods and (ii) the assumption that all droplets contain identical ISA is a valid conjecture with subsequent consequences for analysis and interpretation of immersion freezing. The simple stochastic model can reproduce the observed time and surface area dependence in immersion freezing experiments for a variety of methods such as: droplets on a cold-stage exposed to air or surrounded by an oil matrix, wind and acoustically levitated droplets, droplets in a continuous flow diffusion chamber (CFDC), the Leipzig aerosol cloud interaction simulator (LACIS), and the aerosol interaction and dynamics in the atmosphere (AIDA) cloud chamber. Observed time dependent isothermal frozen fractions exhibiting non-exponential behavior with time can be readily explained by this model considering varying ISA. An apparent cooling rate dependence ofJhet is explained by assuming identical ISA in each droplet. When accounting for ISA variability, the cooling rate dependence of ice nucleation kinetics vanishes as expected from classical nucleation theory. The model simulations allow for a quantitative experimental uncertainty analysis for parameters Ntot, T, RH, and the ISA variability. In an idealized cloud parcel model applying variability in ISAs for each droplet, the model predicts enhanced immersion freezing temperatures and greater ice crystal production compared to a case when ISAs are uniform in each droplet. The implications of our results for experimental analysis and interpretation of the immersion freezing process are discussed.

scholarly journals Hyaluronate synthetase inhibition by normal and transformed human fibroblasts during growth reduction

1987 ◽  
Vol 104 (4) ◽  
pp. 1105-1115 ◽  
Author(s):  
K Matuoka ◽  
M Namba ◽  
Y Mitsui
Keyword(s):  
Cell Proliferation ◽  
Cell Density ◽  
Human Fibroblasts ◽  
Transformed Cells ◽  
Synthetase Activity ◽  
Intact Cells ◽  
Free System ◽  
Glycosaminoglycan Synthesis ◽  
Cell Free System ◽  
A Cell

To establish the relation of glycosaminoglycan synthesis to cell proliferation, we investigated the synthesis of individual glycosaminoglycan species by intact cells and in a cell-free system, using normal and transformed human fibroblasts under differing culture conditions. Reducing serum concentration brought about a marked decline in the synthesis of hyaluronate (HA) as well as cell proliferation on both normal and transformed cells. Both HA synthesis and proliferation decreased with increasing cell densities markedly (in inverse proportion to cell density) in normal cells but gradually in transformed cells. This noticeable congruity of the changes in HA synthesis and proliferation indicates that the change in HA synthesis is related primarily to cell proliferation rather than to cell density or cellular transformation. Examination of HA synthesis in a cell-free system demonstrated that the activity of HA synthetase also fluctuated in conjunction with cell proliferation. Furthermore, growth-reduced cells (except crowded transformed cells) inhibited cell-free HA synthesis and this inhibition was induced coincidentally with a decrease in both HA synthetase activity and proliferation. These findings suggest that the change in HA synthesis is significant in the regulation of cell proliferation.

Enhancing the electrical conductivity of PP/CNT nanocomposites through crystal-induced volume exclusion effect with a slow cooling rate

2020 ◽  
Vol 183 ◽  
pp. 107663 ◽  
Author(s):  
Jun Wang ◽  
Yasamin Kazemi ◽  
Sai Wang ◽  
Mahdi Hamidinejad ◽  
Mayesha B. Mahmud ◽  
...  
Keyword(s):  
Electrical Conductivity ◽  
Cooling Rate ◽  
Slow Cooling ◽  
Exclusion Effect
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