Structural integrity after cold storage of human epidermal model in hypothermosol: A correlative ultrastructural and fluorescent assay

1994 ◽  
Vol 52 ◽  
pp. 270-271
Author(s):  
Henry H. Eichelberger ◽  
John G. Baust ◽  
Robert G. Van Buskirk
Keyword(s):  
Cold Storage ◽  
Structural Integrity ◽  
Culture Media ◽  
Cell Structure ◽  
Natural State ◽  
Sodium Cacodylate ◽  
Ruthenium Tetroxide ◽  
Cacodylate Buffer ◽  
Before And After

For research in cell differentiation and in vitro toxicology it is essential to provide a natural state of cell structure as a benchmark for interpreting results. Hypothermosol (Cryomedical Sciences, Rockville, MD) has proven useful in insuring the viability of synthetic human epidermis during cold-storage and in maintaining the epidermis’ ability to continue to differentiate following warming.Human epidermal equivalent, EpiDerm (MatTek Corporation, Ashland, MA) consisting of fully differentiated stratified human epidermal cells were grown on a microporous membrane. EpiDerm samples were fixed before and after cold-storage (4°C) for 5 days in Hypothermosol or skin culture media (MatTek Corporation) and allowed to recover for 7 days at 37°C. EpiDerm samples were fixed 1 hour in 2.5% glutaraldehyde in sodium cacodylate buffer (pH 7.2). A secondary fixation with 0.2% ruthenium tetroxide (Polysciences, Inc., Warrington, PA) in sodium cacodylate was carried out for 3 hours at 4°C. Other samples were similarly fixed, but with 1% Osmium tetroxide in place of ruthenium tetroxide. Samples were dehydrated through a graded acetone series, infiltrated with Spurrs resin (Polysciences Inc.) and polymerized at 70°C.

scholarly journals A High Throughput Approach Based on Dynamic High Pressure for the Encapsulation of Active Compounds in Exosomes for Precision Medicine

2021 ◽  
Vol 22 (18) ◽  
pp. 9896
Author(s):  
Eugenia Romano ◽  
Paolo Antonio Netti ◽  
Enza Torino
Keyword(s):  
Cell Culture ◽  
High Pressure ◽  
Culture Media ◽  
Preliminary Test ◽  
Bilayer Membrane ◽  
Microfluidic System ◽  
Culture Model ◽  
Before And After ◽  
Dynamic High Pressure

In recent decades, endogenous nanocarrier-exosomes have received considerable scientific interest as drug delivery systems. The unique proteo-lipid architecture allows the crossing of various natural barriers and protects exosomes cargo from degradation in the bloodstream. However, the presence of this bilayer membrane as well as their endogenous content make loading of exogenous molecules challenging. In the present work, we will investigate how to promote the manipulation of vesicles curvature by a high-pressure microfluidic system as a ground-breaking method for exosomes encapsulation. Exosomes isolated from Uppsala 87 Malignant Glioma (U87-MG) cell culture media were characterized before and after the treatment with high-pressure homogenization. Once their structural and biological stability were validated, we applied this novel method for the encapsulation in the lipidic exosomal bilayer of the chemotherapeutic Irinotecan HCl Trihydrate-CPT 11. Finally, we performed in vitro preliminary test to validate the nanobiointeraction of exosomes, uptake mechanisms, and cytotoxic effect in cell culture model.

scholarly journals Sodium cacodylate as antimitotic agent

2014 ◽  
Vol 57 (3) ◽  
pp. 329-340
Author(s):  
Jadwiga A. Tarkowska
Keyword(s):  
Allium Cepa ◽  
Mitotic Spindle ◽  
Sodium Cacodylate ◽  
Antimitotic Agent ◽  
Ultrastructural Studies ◽  
Meristematic Cells ◽  
Cacodylate Buffer ◽  
Root Meristematic Cells ◽  
Dividing Cells

The effect of pure sodium cacodylate on dividing cells was studied. The root meristematic cells of <em>Allium cepa</em> L. (the roots were squashed in acetoorcein) and endosperm cells of <em>Haemanthus katherinae</em> Bak. (<em>in vitro</em> observations) were used. Serious disturbances in karyokinesis and cytokinesis were found that led most often to the formation of polyploid or multinucleate (<em>A. cepa</em>) cells. These results point to damage of the mitotic spindle and phragmoplast. Careful use of cacodylate buffer in ultrastructural studies of microtubules is advised.

Enzymolysis–Microwave-Assisted Hydrodistillation for Extraction of Volatile Oil from Atractylodes Chinensis and Its Hypoglycemic Activity in vitro

2021 ◽  
Author(s):  
Yitong Wang ◽  
Meixing Yan ◽  
Ruiqing Qin ◽  
Yanling Gong
Keyword(s):  
Cell Structure ◽  
Volatile Oil ◽  
Enzyme Concentration ◽  
Perennial Herb ◽  
Gas Chromatography Mass Spectrometry ◽  
Solid Ratio ◽  
Microwave Assisted ◽  
Before And After ◽  
Microwave Time

Abstract Background Atractylodes chinensis (family Asteraceae) is a perennial herb with many pharmacological effects. Objective Extraction of volatile oil from Atractylodes chinensis was optimized and its hypoglycemic activities were studied. Methods Enzymolysis–microwave-assisted hydrodistillation (EMAHD) was adopted to extract the volatile oil, and the technology was optimized using a single-factor experiment that incorporated response surface methodology (RSM). The extraction rates of volatile oil by EMAHD, microwave-assisted hydrodistillation (MAHD), and hydrodistillation (HD) methods were compared at different times. The ingredients of Atractylodes chinensis volatile oil were analyzed by gas chromatography–mass spectrometry. Scanning electron microscopy (SEM) were used to analyze the microstructural changes in Atractylodes chinensis residue before and after extraction. The inhibition of α-amylase activity was determined. Results The obtained optimal extraction conditions for EMAHD were as follows: enzyme concentration 1.6%, pH 7, enzymolysis time 20 min, enzymolysis temperature 50°C, liquid–solid ratio 30:1, microwave power 455 W, and microwave time 40 min. The levels of the main ingredients and the in vitro inhibition of α-amylase activities were higher for Atractylodes chinensis volatile oil extracted by EMAHD than by HD or MAHD. The powder residue of Atractylodes chinensis remaining after EMAHD showed a ruptured and collapsed cell structure, indicating accelerated dissolution of the volatile oil. Conclusions and Highlights EMAHD is deemed a method with many advantages for extraction of volatile oil from Atractylodes chinensis. The volatile oil of Atractylodes chinensis is a promising component for treating hyperglycemia.

scholarly journals In Vitro Enzymatic Digestibility of Glutaraldehyde-Crosslinked Chitosan Nanoparticles in Lysozyme Solution and Their Applicability in Pulmonary Drug Delivery

Molecules ◽  
2019 ◽  
Vol 24 (7) ◽  
pp. 1271 ◽  
Author(s):  
Nazrul Islam ◽  
Hui Wang ◽  
Faheem Maqbool ◽  
Vito Ferro
Keyword(s):  
Electron Microscopy ◽  
Drug Delivery ◽  
Structural Integrity ◽  
Chitosan Nanoparticles ◽  
Morphological Characterization ◽  
Pulmonary Drug Delivery ◽  
Oil Emulsion ◽  
Vis Spectroscopy ◽  
Before And After

Herein, the degradation of low molecular weight chitosan (CS), with 92% degree of deacetylation (DD), and its nanoparticles (NP) has been investigated in 0.2 mg/mL lysozyme solution at 37 °C. The CS nanoparticles were prepared using glutaraldehyde crosslinking of chitosan in a water-in-oil emulsion system. The morphological characterization of CS particles was carried out using scanning electron microscopy (SEM) and Transmission Electron Microscopy (TEM) techniques. Using attenuated total reflectance Fourier transform infrared (ATR-FTIR) and UV-VIS spectroscopy, the structural integrity of CS and its NPs in lysozyme solution were monitored. The CS powder showed characteristic FTIR bands around 1150 cm−1 associated with the glycosidic bridges (C-O-C bonds) before and after lysozyme treatment for 10 weeks, which indicated no CS degradation. The glutaraldehyde crosslinked CS NPs showed very weak bands associated with the glycosidic bonds in lysozyme solution. Interestingly, the UV-VIS spectroscopic data showed some degradation of CS NPs in lysozyme solution. The results of this study indicate that CS with a high DD and its NPs crosslinked with glutaraldehyde were not degradable in lysozyme solution and thus unsuitable for pulmonary drug delivery. Further studies are warranted to understand the complete degradation of CS and its NPs to ensure their application in pulmonary drug delivery.

Sur la variabilité de la capacité rhizogène d'explantats racinaires de Cichorium intybus (var. Witloof) cultivés in vitro : influence de la dimension des explantats initiaux et de la durée de conservation des racines au froid

1986 ◽  
Vol 64 (1) ◽  
pp. 242-246 ◽  
Author(s):  
Jacques Vasseur ◽  
René Lefebvre ◽  
Enoch Backoula
Keyword(s):  
Cold Storage ◽  
Adventitious Root ◽  
Adventitious Roots ◽  
Root Formation ◽  
Culture Media ◽  
Adventitious Root Formation ◽  
Cichorium Intybus ◽  
Root Production ◽  
Root Explants

On Cichorium intybus root explants of different size, it is possible to demonstrate the existence of a relation between the volume/surface ratio and adventitious root formation capacities. With a volume/surface ratio equal to one, the highest number of adventitious roots and percentage of explants able to produce roots have been observed. When this ratio deviates from unity, adventitious root formation declines. Cold storage of chicory roots causes breakdown of fructosans and accumulation of sucrose, glucose, and fructose. At the same time, adventitious root formation on explants cultured in vitro decreases. Inclusion of glucose in culture media increases adventitious root production whatever the duration of chicory root cold storage may have been. Results are discussed and the hypothesis of a regulation of adventitious roots by sucrose and reducing sugars is advanced.

scholarly journals Effectiveness Assessment of a Modified Preservation Solution Containing Thyrotropin or Follitropin Based on Biochemical Analysis in Perfundates and Homogenates of Isolated Porcine Kidneys after Static Cold Storage

2021 ◽  
Vol 22 (16) ◽  
pp. 8360
Author(s):  
Aneta Ostróżka-Cieślik ◽  
Barbara Dolińska ◽  
Florian Ryszka
Keyword(s):  
Protective Effect ◽  
Cold Storage ◽  
Biochemical Parameters ◽  
Biochemical Analysis ◽  
Structural Integrity ◽  
Biochemical Tests ◽  
Preservation Solution ◽  
Kidney Perfusion ◽  
Effectiveness Assessment

In this paper, we assess the nephroprotective effects of thyrotropin and follitropin during ischaemia. The studies were performed in vitro in a model of isolated porcine kidneys stored in Biolasol (FZNP, Biochefa, Sosnowiec, Poland) and modified Biolasol (TSH: 1 µg/L; FSH 1 µg/L). We used the static cold storage method. The study was carried out based on 30 kidneys. The kidneys were placed in 500 mL of preservation solution chilled to 4 °C. The samples for biochemical tests were collected during the first kidney perfusion (after 2 h of storage) and during the second perfusion (after 48 h of storage). The results of ALT, AST, and LDH activities confirm the effectiveness of Biolasol + p-TSH in maintaining the structural integrity of renal cell membranes. Significantly reduced biochemical parameters of kidney function, i.e., creatinine and protein concentrations were also observed after 48 h storage. The protective effect of Biasol + p-TSH is most pronounced after 2 h of storage, suggesting a mild course of damage thereafter. A mild deterioration of renal function was observed after 48 h. The results of our analyses did not show any protective effect of Biolasol + p-FSH on the kidneys during ischaemia.

High-pressure freezing of cells in suspension for low-voltage scanning electron microscopy (LVSEM)

1991 ◽  
Vol 49 ◽  
pp. 64-65
Author(s):  
Marek Malecki ◽  
James Pawley ◽  
Hans Ris
Keyword(s):  
Electron Microscopy ◽  
High Pressure ◽  
Low Voltage ◽  
Culture Media ◽  
Cell Structure ◽  
Freeze Substitution ◽  
Ice Crystal ◽  
High Pressure Freezing ◽  
Cell Culture Media ◽  
Voltage Scanning

The ultrastructure of cells suspended in physiological fluids or cell culture media can only be studied if the living processes are stopped while the cells remain in suspension. Attachment of living cells to carrier surfaces to facilitate further processing for electron microscopy produces a rapid reorganization of cell structure eradicating most traces of the structures present when the cells were in suspension. The structure of cells in suspension can be immobilized by either chemical fixation or, much faster, by rapid freezing (cryo-immobilization). The fixation speed is particularly important in studies of cell surface reorganization over time. High pressure freezing provides conditions where specimens up to 500μm thick can be frozen in milliseconds without ice crystal damage. This volume is sufficient for cells to remain in suspension until frozen. However, special procedures are needed to assure that the unattached cells are not lost during subsequent processing for LVSEM or HVEM using freeze-substitution or freeze drying. We recently developed such a procedure.

Oligodendrocytes in Developing Hameter Cerebral Cortex

1976 ◽  
Vol 34 ◽  
pp. 126-127
Author(s):  
MB. Tank Buschmann
Keyword(s):  
Cerebral Cortex ◽  
Optic Nerve ◽  
Frontal Cortex ◽  
Osmium Tetroxide ◽  
Sodium Cacodylate Buffer ◽  
Sodium Cacodylate ◽  
Tissue Samples ◽  
Cacodylate Buffer ◽  
Layer V ◽  
Adult Hamster

Development of oligodendrocytes in rat corpus callosum was described as a sequential change in cytoplasmic density which progressed from light to medium to dark (1). In rat optic nerve, changes in cytoplasmic density were not observed, but significant changes in morphology occurred just prior to and during myelination (2). In our study, the ultrastructural development of oligodendrocytes was studied in newborn, 5-, 10-, 15-, 20-day and adult frontal cortex of the golden hamster (Mesocricetus auratus).Young and adult hamster brains were perfused with paraformaldehyde-glutaraldehyde in sodium cacodylate buffer at pH 7.3 according to the method of Peters (3). Tissue samples of layer V of the frontal cortex were post-fixed in 2% osmium tetroxide, dehydrated in acetone and embedded in Epon-Araldite resin.

Electron microscopic study of the membrane glycoproteins in the rat kidney after cisplatin treatment

1989 ◽  
Vol 47 ◽  
pp. 912-913
Author(s):  
S.K. Aggarwal
Keyword(s):  
Alkaline Phosphatase ◽  
Rat Kidney ◽  
Light And Electron Microscopy ◽  
Kidney Tissue ◽  
Membrane Glycoproteins ◽  
Electron Microscopic ◽  
Before And After ◽  
Interstrand Cross Links ◽  
Cross Links

The proposed primary mechanism of action of the anticancer drug cisplatin (Cis-DDP) is through its interaction with DNA, mostly through DNA intrastrand cross-links or DNA interstrand cross-links. DNA repair mechanisms can circumvent this arrest thus permitting replication and transcription to proceed. Various membrane transport enzymes have also been demonstrated to be effected by cisplatin. Glycoprotein alkaline phosphatase was looked at in the proximal tubule cells before and after cisplatin both in vivo and in vitro for its inactivation or its removal from the membrane using light and electron microscopy.Outbred male Swiss Webster (Crl: (WI) BR) rats weighing 150-250g were given ip injections of cisplatin (7mg/kg). Animals were killed on day 3 and day 5. Thick slices (20-50.um) of kidney tissue from treated and untreated animals were fixed in 1% buffered glutaraldehyde and 1% formaldehyde (0.05 M cacodylate buffer, pH 7.3) for 30 min at 4°C. Alkaline phosphatase activity and carbohydrates were demonstrated according to methods described earlier.

Inhibitory effects of bioaccessible anthocyanins and procyanidins from apple, red grape, cinnamon on α-amylase, α-glucosidase and lipase

2020 ◽  
pp. 1-9
Author(s):  
Pınar Ercan ◽  
Sedef Nehir El
Keyword(s):  
Inhibitory Activity ◽  
Digestive Enzymes ◽  
Positive Control ◽  
Anthocyanin Content ◽  
Inhibitory Effects ◽  
Total Anthocyanin ◽  
Total Anthocyanin Content ◽  
Before And After ◽  
Red Grape

Abstract. The goals of this study were to determine and evaluate the bioaccessibility of total anthocyanin and procyanidin in apple (Amasya, Malus communis), red grape (Papazkarası, Vitis vinifera) and cinnamon (Cassia, Cinnamomum) using an in vitro static digestion system based on human gastrointestinal physiologically relevant conditions. Also, in vitro inhibitory effects of these foods on lipid (lipase) and carbohydrate digestive enzymes (α-amylase and α-glucosidase) were performed with before and after digested samples using acarbose and methylumbelliferyl oleate (4MUO) as the positive control. While the highest total anthocyanin content was found in red grape (164 ± 2.51 mg/100 g), the highest procyanidin content was found in cinnamon (6432 ± 177.31 mg/100 g) (p < 0.05). The anthocyanin bioaccessibilities were found as 10.2 ± 1%, 8.23 ± 0.64%, and 8.73 ± 0.70% in apple, red grape, and cinnamon, respectively. The procyanidin bioaccessibilities of apple, red grape, and cinnamon were found as 17.57 ± 0.71%, 14.08 ± 0.74% and 18.75 ± 1.49%, respectively. The analyzed apple, red grape and cinnamon showed the inhibitory activity against α-glucosidase (IC50 544 ± 21.94, 445 ± 15.67, 1592 ± 17.58 μg/mL, respectively), α-amylase (IC50 38.4 ± 7.26, 56.1 ± 3.60, 3.54 ± 0.86 μg/mL, respectively), and lipase (IC50 52.7 ± 2.05, 581 ± 54.14, 49.6 ± 2.72 μg/mL), respectively. According to our results apple, red grape and cinnamon have potential to inhibit of lipase, α-amylase and α-glucosidase digestive enzymes.

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