Post-Thaw Viability and Functionality of Cryopreserved Rat Fetal Brain Cells Cocultured with Sertoli Cells

1997 ◽  
Vol 6 (2) ◽  
pp. 185-189 ◽  
Author(s):  
D.F. Cameron ◽  
A.I. Othberg ◽  
C.V. Borlongan ◽  
S. Rashed ◽  
A. Anton ◽  
...  
Keyword(s):  
Sertoli Cells ◽  
Trypan Blue ◽  
Fetal Brain ◽  
Trophic Factors ◽  
Cell Type ◽  
Isolated Cells ◽  
Trypan Blue Exclusion ◽  
Ventral Mesencephalic ◽  
Privileged Site

Testis-derived Sertoli cells have been used to create an immune “privileged” site outside of the testis to facilitate cell transplantation protocols for diabetes and neurodegenerative diseases. In addition to secreting immunoprotective factors, Sertoli cells also secrete growth and trophic factors that appear to enhance the posttransplantation viability of isolated cells and, likewise, the postthaw viability of isolated, cryopreserved cells (26). It would be beneficial if Sertoli cells could be cryopreserved with the transplantable cell type without deleterious effects on the cells. This report describes a protocol for the cocryopreservation of rat Sertoli cells with rat ventral mesencephalic neurons, neurons from the lateral and medial ganglionic eminences and the hNT neuron cell line, and reports on the effects of Sertoli cells on the the postthaw viability of these neurons. Results of trypan blue exclusion analysis indicated that the presence of Sertoli cells did not deleteriously effect cryopreserved neurons and may improve their postthaw recoverability and viability in general. Specifically, results of the tyrosine hydroxylase immunostaining showed that Sertoli cells significantly enhance the postthaw viability of ventral mesencephalic dopaminergic cells in vitro.

Hypothermic Storage of Periportal and Perivenous Rat Hepatocytes

1998 ◽  
Vol 7 (4) ◽  
pp. 345-355 ◽  
Author(s):  
Edgardo Elvio Guibert ◽  
María Gabriela Mediavilla ◽  
María Eugenia Mamprin ◽  
Joaquín Valentín Rodríguez
Keyword(s):  
Cold Storage ◽  
Trypan Blue ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Liver Cells ◽  
Enzyme Release ◽  
Isolated Cells ◽  
Trypan Blue Exclusion ◽  
Hypothermic Storage

High yields of intact parenchymal cells are produced by the two-step Digitonin-collagenase perfusion of whole liver, and it has gained wide acceptance for biochemical and cellular analyses of zonal hepatocytes. The development reached by this methodology is in contrast to the time-limited use of the isolated cells unless those other methods, such as primary cultures, are employed. An alternative option to have cells ready to be used for several days, is the cold storage in University of Wisconsin solution as a preservation solution. This procedure is easy, not too expensive, and does not require specialized equipment. We study the competence of this system to maintain liver cells: mixed or total cells and cell-enriched fractions. We affirm viability of hepatocytes during hypothermic storage (UW-96h-4°C) by Trypan Blue exclusion, the capacity to retain cytoplasmic enzymes, metabolic competence to maintain total Glutathione content, and immunocytochemistry (gene detection). After 96 h of cold storage, mixed cells and cell-enriched fractions, were submitted to normothermic incubation (120 min, 37°C) and we check Trypan Blue exclusion, cytoplasmic enzyme release, and the capacity of cell populations to synthesize urea. The results show that it is possible to use, after several days of storage, mixed liver cells and cell-enriched fractions in metabolic and gene expression studies. This procedure allows us to reduce the number of experimental animals needed, to save experimental time and costs, and to facilitate further studies in vitro about the basis and consequences of metabolic heterogeneity of the liver cell plate.

Effects of medium and hypothermic temperatures on preservation of isolated porcine testis cells

2010 ◽  
Vol 22 (3) ◽  
pp. 523 ◽  
Author(s):  
Yanfei Yang ◽  
Ali Honaramooz
Keyword(s):  
Trypan Blue ◽  
Cell Isolation ◽  
Live Cell ◽  
Live Cells ◽  
Cell Recovery ◽  
Trypan Blue Exclusion ◽  
Culture Studies ◽  
Porcine Testis ◽  
Peritubular Cells

The effects of medium and hypothermic temperatures on testis cells were investigated to develop a strategy for their short-term preservation. Testes from 1-week-old piglets were enzymatically dissociated for cell isolation. In Experiment 1, testis cells were stored at either room (RT) or refrigeration (RG) temperature for 6 days in one of 13 different media. Live cell recovery was assayed daily using trypan blue exclusion. In Experiment 2, three media at RG were selected for immunocytochemical and in vitro culture studies. Live cell recovery was also assayed daily for 6 days using both trypan blue exclusion and a fluorochrome assay kit. For all media tested, significantly or numerically more live cells were maintained at RG than RT. On preservation Day 3 at RG (cell isolation day as Day 0), 20% FBS-Leibovitz resulted in the highest live cell recovery (89.5 ± 1.7%) and DPBS in the lowest (60.3 ± 1.9%). On Day 6 at RG, 20% FBS- Leibovitz also resulted in the best preservation efficiency with 80.9 ± 1.8% of Day 0 live cells recovered. There was no difference in live cell recovery detected by the two viability assays. After preservation, the proportion of gonocytes did not change, whereas that of Sertoli and peritubular cells increased and decreased, respectively. After 6 days of hypothermic preservation, testis cells showed similar culture potential to fresh cells. These results show that testis cells can be preserved for 6 days under hypothermic conditions with a live cell recovery of more than 80% and after-storage viability of 88%.

Trophic Effect of Porcine Sertoli Cells on Rat and Human Ventral Mesencephalic Cells and Hnt Neurons in Vitro

1998 ◽  
Vol 7 (2) ◽  
pp. 157-164 ◽  
Author(s):  
Agneta I. Othberg ◽  
Alison E. Willing ◽  
Don F. Cameron ◽  
Alex Anton ◽  
Samuel Saporta ◽  
...  
Keyword(s):  
Sertoli Cells ◽  
Islet Cells ◽  
Treated Group ◽  
Term Survival ◽  
Teratocarcinoma Cell ◽  
Trophic Effect ◽  
Soma Size ◽  
Ventral Mesencephalic

The poor survival of embryonic dopaminergic (DA) neurons transplanted into patients with Parkinson's disease (PD) has encouraged researchers to search for new methods to affect the short- as well as long-term survival of these neurons after transplantation. In several previous rodent studies Sertoli cells increased survival of islet cells and chromaffin cells when cotransplanted in vivo. The aims of this study were to investigate whether porcine Sertoli cells had a positive effect on the survival and maturation of rat and human DA neurons, and whether the Sertoli cells had an effect on differentiation of neurons derived from a human teratocarcinoma cell line (hNT neurons). A significant increase of tyrosine hydroxylase (TH)-positive neurons of both rat and human ventral mesencephalic tissue was found when cocultured with Sertoli cells. Furthermore, there was a significantly increased soma size and neurite outgrowth of neurons in the coculture treated group. The Sertoli cell and hNT coculture also revealed an increased number of TH-positive cells. These results demonstrate that the wide variety of proteins and factors secreted by porcine Sertoli cells benefit the survival and maturation of embryonic DA neurons and suggest that cotransplantation of Sertoli cells and embryonic DA neurons may be useful for a cell transplantation therapy in PD.

In vitro photoradiation therapy of the rat 9L gliosarcoma

1986 ◽  
Vol 64 (5) ◽  
pp. 775-779 ◽  
Author(s):  
Douglas Hamilton ◽  
John D. S. McKean ◽  
John Tulip ◽  
Donald Boisvert ◽  
Judy Cummins
Keyword(s):  
Trypan Blue ◽  
Glioma Cells ◽  
Laser Source ◽  
Trypan Blue Exclusion ◽  
Content Type ◽  
9L Gliosarcoma ◽  
Trypan Blue Exclusion Test ◽  
9L Glioma ◽  
Fine Print

✓ The authors have investigated various factors involved in the photoradiation treatment of 9L glioma cells. The cells were grown in tissue culture and exposed to light from a laser source that allowed accurate quantitation of the light energy. Cell death was determined following treatment using the trypan blue exclusion test. It was shown that the treatment is very wavelength-dependent following the absorption spectrum of hematoporphyrin derivative (HPD). The absorption peaks in the lower part of the spectrum are more efficient than those of higher wavelengths. Photoradiation therapy is more effective the higher the concentration of HPD. Intensity of light is a very important factor in calculating the total dose of light necessary for this treatment.

scholarly journals Purification of erythroblastic nests

Blood ◽  
1981 ◽  
Vol 57 (5) ◽  
pp. 922-927
Author(s):  
AJ Macario ◽  
C Dugan ◽  
IL Perez-Lloret ◽  
E Conway de Macario
Keyword(s):  
Spleen Cell ◽  
Trypan Blue ◽  
Single Cells ◽  
Cell Suspensions ◽  
Differential Centrifugation ◽  
Minimum Essential Medium ◽  
Trypan Blue Exclusion ◽  
Large Numbers ◽  
Very High

We describe a method for preparing purified erythroblastic nests in large numbers (approximately 10(6)/run) in three steps: (1) induction of splenic erythropoiesis in mice, (2) preparative differential centrifugation for the removal of erythrocytes and single cells from spleen cell suspensions, and (3) sedimentation in an isokinetic gradient of Ficoll 400 in Joklik's modification of minimum essential medium. Viability of isolated EN is very high, as demonstrated by the trypan blue exclusion and in vitro erythrocyte formation methods.

scholarly journals Purification of erythroblastic nests

Blood ◽  
1981 ◽  
Vol 57 (5) ◽  
pp. 922-927 ◽  
Author(s):  
AJ Macario ◽  
C Dugan ◽  
IL Perez-Lloret ◽  
E Conway de Macario
Keyword(s):  
Spleen Cell ◽  
Trypan Blue ◽  
Single Cells ◽  
Cell Suspensions ◽  
Differential Centrifugation ◽  
Minimum Essential Medium ◽  
Trypan Blue Exclusion ◽  
Large Numbers ◽  
Very High

Abstract We describe a method for preparing purified erythroblastic nests in large numbers (approximately 10(6)/run) in three steps: (1) induction of splenic erythropoiesis in mice, (2) preparative differential centrifugation for the removal of erythrocytes and single cells from spleen cell suspensions, and (3) sedimentation in an isokinetic gradient of Ficoll 400 in Joklik's modification of minimum essential medium. Viability of isolated EN is very high, as demonstrated by the trypan blue exclusion and in vitro erythrocyte formation methods.

Dissociation of insect Malpighian tubules into single, viable cells

1984 ◽  
Vol 72 (1) ◽  
pp. 101-109
Author(s):  
W.M. Satmary ◽  
T.J. Bradley
Keyword(s):  
Single Cells ◽  
Stellate Cells ◽  
Malpighian Tubules ◽  
Cell Type ◽  
Isolated Cells ◽  
Trypan Blue Exclusion ◽  
Distinct Cell Type ◽  
Viable Cells ◽  
Distinct Cell ◽  
Aedes Taeniorhynchus

The Malpighian tubules of insects are generally composed of more than one cell type. In the hemipteran Rhodnius prolixus, the tubules are divided into two regions, termed the upper and lower tubules, each of which is composed of a distinct cell type. In the dipteran Aedes taeniorhynchus, primary and stellate cells are interspersed throughout the length of the tubules. We report here techniques for the dissociation of the Malpighian tubules of both of these species into single cells. Tubules are removed from the insect and placed for 1 h in insect Ringer containing elastase (Sigma, type III) at 4 mg/ml. This treatment fully removes the basal lamella. Mild agitation by hand produces a suspension of single cells, which remain viable as determined by Trypan Blue exclusion. Isolated cells have been maintained in cell culture for one week. Using light and scanning electron microscopy, upper and lower tubule cells of Rhodnius and primary and stellate cells of Aedes can be distinguished on the basis of size, shape, microvillar length, and the presence or absence of intracellular crystals.

scholarly journals Trypan Blue Exclusion Assay Verifies in Vitro Cytotoxicity of New Cis-Platinum (II) Complex in Human Cells

2019 ◽  
Vol 16 (3) ◽  
pp. 0555
Author(s):  
Hussein Et al.
Keyword(s):  
Cell Viability ◽  
Trypan Blue ◽  
Membrane Integrity ◽  
Platinum Complex ◽  
In Vitro Cytotoxicity ◽  
Toxic Effects ◽  
Polymorphonuclear Cells ◽  
Trypan Blue Exclusion ◽  
High Concentrations

          Various assays are used to determine the toxic effects of drugs at cellular levels in vitro.  One of these methods is the dye exclusion assay, which measures membrane integrity in the presence of Trypan blue. Trypan blue the dye which was used in this study to investigate cytotoxic effect of a new Cis –dichloroplatinum (II) complex [(Qu)2PtCl2] on the viability of polymorphonuclear cells (PMNs). Three concentrations of platinum complex were prepared (70, 35and 17.5 µg/ ml) and the results revealed that the percentage of cell viability decreased as the platinum complex concentration increased in comparison with control. The platinum complex exhibited low cytotoxic effects towards healthy cells at the concentrations of 17.5 µg/ ml and 35 µg/ ml, in which the percentage of cell viability was (77.01 ± 6.3) and (72.3± 0.50)respectively, with no significant differences as compared with the control(90.66 ±0.577). The viability was significantly decreased (67.59 ± 3.16) when the cells were treated with the concentration of 70 µg/ ml in comparison with control. These results indicated that the percentage of living cells decreased when treated with high concentrations of [(Qu)2PtCl2], which causes cells death, while low concentrations of the compound show low toxicity. This data indicates that this compound, at these concentrations may be suitable for use as a cancer treatment because it has low toxic effects on the healthy cells.        

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