Effects of medium and hypothermic temperatures on preservation of isolated porcine testis 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%.

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Development of novel strategies for the isolation of piglet testis cells with a high proportion of gonocytes

Reproduction Fertility and Development ◽

10.1071/rd09316 ◽

2010 ◽

Vol 22 (7) ◽

pp. 1057 ◽

Cited By ~ 18

Author(s):

Yanfei Yang ◽

Mehran Yarahmadi ◽

Ali Honaramooz

Keyword(s):

In Vitro Study ◽

Enzymatic Digestion ◽

Cell Isolation ◽

Live Cells ◽

Germline Stem Cell ◽

Isolated Cells ◽

Cell Potential ◽

Mechanical Methods ◽

Neonatal Testis

Gonocytes have germline stem cell potential and are present in the neonatal testis, comprising 5–10% of freshly isolated testis cells. Maximising the number and proportion of gonocytes among freshly isolated testis cells will greatly facilitate their subsequent purification and in vitro study and manipulation. Seven experiments were conducted to evaluate the effects of multiple factors on the efficiency of testis cell isolation from neonatal pigs. We found that the use of a lysis buffer led to elimination of erythrocytes without adversely affecting testis cell isolation. Approximately ninefold as many live cells could be harvested by enzymatic digestion of testis tissues compared with mechanical methods. Digestion with collagenase–hyaluronidase–DNase followed by trypsin resulted in the highest recovery of live cells. However, the proportion of gonocytes (∼7%) did not differ between the mechanical and enzymatic methods of testis cell isolation. Pretreatment of the tissue with cold enzymes increased the recovery of live testis cells. New strategies of combining a gentle enzymatic digestion with two rounds of vortexing resulted in the isolation of testis cells with very high gonocyte proportion. The efficiency of these novel methods could be further optimised to collect testis cells with a gonocyte proportion of approximately 40%. This novel three-step testis cell isolation strategy can be completed within 1 h and can harvest approximately 17 × 106 live gonocytes per g testis tissue. Therefore, in addition to elucidating the effects of several factors on testis cell isolation, we developed a novel strategy for the isolation of testis cells that yielded approximately 40% gonocytes in the freshly isolated cells (i.e. four- to eight-fold higher than the proportions obtained using current strategies). This strategy has instant applications in the purification of gonocytes.

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tdLanYFP, a yellow, bright, photostable and pH insensitive fluorescent protein for live cell imaging and FRET-based sensing strategies

10.1101/2021.04.27.441613 ◽

2021 ◽

Author(s):

Y. Bousmah ◽

H. Valenta ◽

G. Bertolin ◽

U. Singh ◽

V. Nicolas ◽

...

Keyword(s):

Single Molecule ◽

Live Cell Imaging ◽

Cell Imaging ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Yellow Fluorescent Protein ◽

Resonance Energy ◽

Live Cell ◽

Live Cells

AbstractYellow fluorescent proteins (YFP) are widely used as optical reporters in Förster Resonance Energy Transfer (FRET) based biosensors. Although great improvements have been done, the sensitivity of the biosensors is still limited by the low photostability and the poor fluorescence performances of YFPs at acidic pHs. In fact, today, there is no yellow variant derived from the EYFP with a pK1/2 below ∼5.5. Here, we characterize a new yellow fluorescent protein, tdLanYFP, derived from the tetrameric protein from the cephalochordate B. lanceolatum, LanYFP. With a quantum yield of 0.92 and an extinction coefficient of 133 000 mol−1.L.cm−1, it is, to our knowledge, the brightest dimeric fluorescent protein available, and brighter than most of the monomeric YFPs. Contrasting with EYFP and its derivatives, tdLanYFP has a very high photostability in vitro and preserves this property in live cells. As a consequence, tdLanYFP allows the imaging of cellular structures with sub-diffraction resolution with STED nanoscopy. We also demonstrate that the combination of high brightness and strong photostability is compatible with the use of spectro-microscopies in single molecule regimes. Its very low pK1/2 of 3.9 makes tdLanYFP an excellent tag even at acidic pHs. Finally, we show that tdLanYFP can be a FRET partner either as donor or acceptor in different biosensing modalities. Altogether, these assets make tdLanYFPa very attractive yellow fluorescent protein for long-term or single-molecule live-cell imaging that is also suitable for FRET experiment including at acidic pH.

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Microprocessor-controlled vs. "dump-freezing" platelet and lymphocyte cryopreservation: A quantitative and qualitative comparative study

Vojnosanitetski pregled ◽

10.2298/vsp0603261b ◽

2006 ◽

Vol 63 (3) ◽

pp. 261-268 ◽

Cited By ~ 1

Author(s):

Bela Balint ◽

Dusan Vucetic ◽

Biljana Draskovic ◽

Danilo Vojvodic ◽

Goran Brajuskovic ◽

...

Keyword(s):

Functional Recovery ◽

Trypan Blue ◽

Proliferative Response ◽

Antigen Expression ◽

Intergroup Difference ◽

Cell Recovery ◽

Trypan Blue Exclusion ◽

Rate System ◽

Platelet Recovery ◽

Cell Percentage

Background/Aim. Thermodynamical and cryobiological parameters responsible for cell damages during cryopreservation (cryoinjuries) have not yet been completely explained. Thus, freezing procedures should be revised, exactly optimized to obtain an enhanced structural and functional recovery of frozen- thawed cells. The aim of this study was to compare microprocessor- controlled (controlled-rate) with the compensation of the released fusion heat and ?dump-freezing? (uncontrolled- rate) of the platelet and lymphocyte cryopreservation efficacy. Methods. Platelet quantitative recovery (post-thaw vs. unfrozen cell count), viability (using hypotonic shock response - HSR), morphological score (PMS), ultrastructural (electron microscopy) properties and expression of different surface antigens were investigated. In lymphocyte setting, cell recovery and viability (using trypan blue exclusion test) as well as functionality (by plant mitogens) were determined. Controlled- rate freezing and uncontrolled-rate cryopreservation were combined with 6% (platelets) and 10% (lymphocytes) dimethyl sulfoxide (DMSO). Results. Platelet recovery and functionality were superior in the controlled-rate system. The majority of surface antigen expression was reduced in both freezing groups vs. unfrozen cells, but GP140/CD62p was significantly higher in controlled-rate vs. uncontrolled-rate setting. Controlled- rate freezing resulted with better lymphocyte recovery and viability (trypan blue-negative cell percentage). In mitogen-induced lymphocyte proliferative response no significant intergroup difference (controlled-rate vs. uncontrolled-rate) were found. Conclusion. The data obtained in this study showned the dependence of cell response on the cryopreservation type. Controlled-rate freezing provided a superior platelet quantitative and functional recovery. Lymphocyte recovery and viability were better in the controlled-rate group, although only a minor intergroup difference for cell proliferative response was obtained.

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Ligand-displacement-based two-photon fluorogenic probe for visualizing mercapto biomolecules in live cells, Drosophila brains and zebrafish

The Analyst ◽

10.1039/c8an00453f ◽

2018 ◽

Vol 143 (14) ◽

pp. 3433-3441 ◽

Cited By ~ 3

Author(s):

Yanfei Zhao ◽

Yun Ni ◽

Liulin Wang ◽

Chenchen Xu ◽

Chenqi Xin ◽

...

Keyword(s):

Rapid Detection ◽

Live Cell ◽

Live Cells ◽

Two Photon ◽

Ligand Displacement ◽

Cell Tissue ◽

Fluorogenic Probe ◽

Displacement Based

We report the Fe(iii)-based complex TPFeS which acts as a novel ligand-displacement-based TP fluorogenic probe for the rapid detection of mercapto biomolecules both in vitro and in live cell/tissue/in vivo imaging.

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In vitro photoradiation therapy of the rat 9L gliosarcoma

Journal of Neurosurgery ◽

10.3171/jns.1986.64.5.0775 ◽

1986 ◽

Vol 64 (5) ◽

pp. 775-779 ◽

Cited By ~ 10

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.

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Purification of erythroblastic nests

Blood ◽

10.1182/blood.v57.5.922.bloodjournal575922 ◽

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.

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Interfacing Live Cells with Surfaces: A Concurrent Control Technique for Quantifying Surface Ligand Activity

10.1101/2021.04.27.441513 ◽

2021 ◽

Author(s):

Michael C. Robitaille ◽

Joseph A. Christodoulides ◽

Patrick Calhoun ◽

Jeff M. Byers ◽

Marc P. Raphael

Keyword(s):

Cellular Adhesion ◽

Live Cell ◽

Control Technique ◽

Live Cells ◽

Chip Surface ◽

Concurrent Control ◽

Activity Measurements ◽

Surface Ligand ◽

Ligand Activity

AbstractSurface ligand activity is a key design parameter for successfully interfacing surfaces with cells - whether in the context of in vitro investigations for understanding cellular signaling pathways or more applied applications in drug delivery and medical implants. Unlike other crucial surface parameters, such as stiffness and roughness, surface ligand activity currently lacks a standardized measurement approach that can be readily paired with live cell investigations. To fill this void, we have developed a concurrent control technique for characterizing in vitro ligand surface activity. Pairs of gold-coated glass chips were biofunctionalized with RGD ligand in a parallel workflow: one chip for in vitro applications and the other for surface plasmon resonance (SPR) based RGD activity characterization. Recombinant αVβ3 integrins were injected over the SPR chip surface as mimics of the cellular membrane bound receptors and the resulting binding kinetics parameterized to quantify ligand activity. These activity measurements were correlated with cell morphological features, measured by interfacing MDA-MB-231 cells with the in vitro chip surfaces on the live cell microscope. We show that the SPR concurrent control approach has multiple advantages based on the facts that SPR is a standardized technique and has the sensitivity to measure ligand activity across the most relevant range of extracellular surface densities. Furthermore, by pairing both SPR and in vitro approaches, a comparison of the results can provide biological insights into the nature of cellular adhesion and dynamics.

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Confocal Microscopy in Conjunction With Haemocytometry to Evaluate the Imperative Physical Characteristics of Multicellular Tumor Spheroids

10.21203/rs.3.rs-1107684/v1 ◽

2021 ◽

Author(s):

Aziz UR RAHMAN

Keyword(s):

Confocal Microscopy ◽

Drug Uptake ◽

Live Cells ◽

Multicellular Tumor Spheroids ◽

Tumor Spheroids ◽

Oriented Growth ◽

Trypan Blue Exclusion ◽

Trypan Blue Exclusion Method

Abstract Background: Tumor tissues resist penetration of therapeutic molecules. Multicellular tumor spheroids (MCTSs) were used as an in vitro tumor model. The aim of this study was to determine the growth of MCTSs with the age of spheroids, which could be applied and compared with in vivo drug uptake and penetration. Method: Spheroids were generated by liquid overlay techniques, and their diameter was measured by confocal microscopy for up to two weeks. The trypan blue exclusion method was used to count dead and live cells separately via a hemocytometer. Results: The pentaphysical characteristics of spheroids, including diameter, cell number, volume per cell, viability status, and estimated shell of viable and core of dead cells, were determined. The growth of spheroids was linear over the first week but declined in the 2nd week, which may be due to an overconcentration of dead cells and degraded products inside the spheroids, hence lowering the ratio of live cells in spheroids. Compaction of spheroids occurs from day 3 to day 7, with the mature spheroids having a low amount of extracellular space compared to intracellular volume. Conclusion: Age-oriented growth of MCTSs provides a rationale to predict less rapid penetration as spheroids get older and could be correlated with in vivo tumors to predict pharmaceutical and therapeutic intervention.

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Hypothermic Storage of Periportal and Perivenous Rat Hepatocytes

Cell Transplantation ◽

10.1177/096368979800700402 ◽

1998 ◽

Vol 7 (4) ◽

pp. 345-355 ◽

Cited By ~ 4

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.

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Purification of erythroblastic nests

Blood ◽

10.1182/blood.v57.5.922.922 ◽

1981 ◽

Vol 57 (5) ◽

pp. 922-927 ◽

Cited By ~ 8

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.

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