Confluence Rate Research Articles

A Microfluidic Cell Co-Culture Chip for the Monitoring of Interactions between Macrophages and Fibroblasts

Macrophages and fibroblasts are two types of important cells in wound healing. The development of novel platforms for studying the interrelationship between these two cells is crucial for the exploration of wound-healing mechanisms and drug development. In this study, a microfluidic chip composed of two layers was designed for the co-culturing of these two cells. An air valve was employed to isolate fibroblasts to simulate the wound-healing microenvironment. The confluence rate of fibroblasts in the co-culture system with different macrophages was explored to reflect the role of different macrophages in wound healing. It was demonstrated that M2-type macrophages could promote the activation and migration of fibroblasts and it can be inferred that they could promote the wound-healing process. The proposed microfluidic co-culture system was designed for non-contact cell-cell interactions, which has potential significance for the study of cell-cell interactions in biological processes such as wound healing, tumor microenvironment, and embryonic development.

Passaging capability of human corneal endothelial cells derived from old donors with and without accelerating cell attachment

In a recent report, we showed that it is possible to establish the culture of Human Corneal Endothelial Cells (HCEnCs) from older donor corneas (usually over 65 year olds) when left to attach in the presence of a viscoelastic solution, potentially increasing the donor pool for culturing HCEnCs. Therefore, we set out to evaluate the outcome of using a viscoelastic solution (Viscoat) to accelerate the attachment of passaged cultured human corneal endothelial cells (HCEnCs). The cells from 28 donor tissues were isolated using peel-and-digest method and evenly seeded into two wells of an 8-well chamber slide. The cells were left to attach after topical application of Viscoat. At confluence, one well was subjected to end-stage characterization, whereas the other well was passaged into another two wells. The cells at P1 were attached with and without the use of Viscoat. The growth rate was monitored; and at confluence, morphometric analysis, corneal endothelial specific (CD166-Tag1A3 & PRDX6-Tag2A12), mitochondrial and respiration assessment (Tom-20 and Seahorse); function-associated (Na+/K+ATPase & ZO-1); proliferative (Ki-67) marker analysis, and viability (Hoechst, Ethidium Homodimer and Calcein AM-HEC) studies were performed. Cells at P0 (with Viscoat) showed 100% confluence at day 9. Cells at P1 with and without Viscoat showed significant difference of confluence 67.0% v 18.8% respectively (p < 0.05). Confluence rate, cell density, hexagonality, Ki-67 positivity and mitochondrial intensity was significantly higher (p < 0.05), whereas cell-area and polymorphism was significantly lower (p < 0.05) in the cells attached with Viscoat compared with the cells attached without Viscoat. There was no significant difference in oxygen consumption rate between the groups. In conclusion, we observed that acceleration in the attachment of passaged HCEnCs with the assistance of Viscoat, could be beneficial for the propagation of HCEnCs isolated from older donors, to increase their propensity to proliferate, without loss of the expression of vital proteins and heterogeneity in cellular morphology.

Increasing Donor Endothelial Cell Pool by Culturing Cells from Discarded Pieces of Human Donor Corneas for Regenerative Treatments.

Purpose To investigate if the peripheral corneal endothelium that is usually discarded after a corneal transplant could be used for endothelial cell culture. Methods Donor corneas (n = 19) with a mean age of 72 years, male : female ratio of 15 : 4, and death-to-preservation time of 10 hours were assessed for endothelial cell density (ECD) and number of dead cells before isolation. Alizarin red staining (n = 3) was performed to check the morphology of cells in the center and periphery. Descemet's membrane-endothelial complex was peeled from the center (8.25 mm) and the periphery (2.75 mm) and plated in two different wells of an 8-well chamber slide with media refreshed every alternate day. The confluence rate was monitored by microscopy. Live/dead analysis was performed (n = 3) at confluence. Tag-2A12 as a monoclonal antibody against peroxiredoxin-6 (Prdx-6) (n = 4), ZO-1 (zonula occludens-1) as a tight junction protein (n = 4), and Ki-67 as a proliferative cell marker (n = 4) were used to characterize the cells at confluence. Results At confluence, 8.25% average increase in the number of cells was observed from the central zone compared with 16.5% from the peripheral zone. Proliferation rate, hexagonality, Ki-67 positivity, and the cell area did not significantly differ between the groups (p > 0.05). All the proteins corresponding to the biomarkers tested were expressed in both the groups. Conclusions Although there are significantly fewer amounts of peripheral cells available after graft preparation for keratoplasty, these cells can still be used for endothelial cell culture due to their proliferative capability. The peripheral cells that are discarded after graft preparation can thus be utilized to increase the donor endothelial cell pool for regenerative treatments.

Culturing Discarded Peripheral Human Corneal Endothelial Cells From the Tissues Deemed for Preloaded DMEK Transplants.

To investigate if the peripheral corneal endothelium that is discarded after the preparation of preloaded Descemet membrane endothelial keratoplasty (DMEK) grafts for transplantation could be successfully used for corneal endothelial cell culture. Complete Descemet membrane-endothelial complex (11.00 mm) was peeled from research-grade tissues (n = 15). The periphery (2.75 mm) of clinical-grade tissues (n = 15) deemed for preloaded DMEK transplants was gently peeled and preserved for 48 hours in tissue culture media, followed by centrifugation at 1000 rpm for 5 minutes. After enzymatic digestion, the cells from each group were plated in 2 different wells of an 8-well chamber slide. Media were refreshed and the confluence rate was monitored every alternate day. Live/dead staining and the expression of ZO-1, Tag1A3, Tag2A12, and Ki-67 markers were used to assess the viability, morphology, tight-junctions, cell area, and number of proliferative cells. The Wilcoxon and Student's t test were applied, where P < 0.05 was deemed statistically significant. Average endothelial cell density at confluence was 2,352 cells/mm2 from complete endothelium and 2,510 cells/mm from peripheral endothelium (P = 0.0351). The confluence rate (%), hexagonality (%), polymorphism (%), cell area (μm), and Ki-67 positivity (%) did not differ between both groups (P > 0.05). All the antibodies were expressed in both groups at confluence. The discarded peripheral endothelial cells obtained after preparing a preloaded DMEK graft for clinical application has a huge reservoir of healthy endothelial cells having proliferative potential. Using these discarded tissue pieces from donor tissues will significantly increase the primary source of healthy donor endothelial cells for regenerative treatments, which are otherwise difficult to obtain.

Effects of corneal preservation conditions on human corneal endothelial cell culture

The purpose of this study was to investigate the growth capacity of human corneal endothelial cells (HCEnCs) isolated from old donor corneas preserved in 4 different storage conditions. The following conditions were evaluated, A) cold storage (CS) (Optisol GS) for 7 days at 4 °C [n = 6]; B) organ culture (OC) (Cornea Max) for 7 days at 31 °C [n = 6]; C) OC for 28 days at 31 °C [n = 6] and; D) CS for 7 days at 4 °C followed by OC for 28 days at 31 °C [n = 6]. Following preservation, the Descemet membrane-endothelium complex was peeled and digested using Collagenase-Type1 and was subsequently trypsinized before being plated into 2 wells (from each cornea) of an 8-well chamber slide. Media was refreshed every alternate day. The confluence rate (%) was assessed, and overall viability was determined using Hoechst, Ethidium Homodimer and CalceinAM staining. HCEnC-associated markers ZO-1, Na+/K+-ATPase, CD166 (Tag1A3), PRDX-6 (Tag2A12) and proliferative marker Ki-67 were used to analyse the cultures established from each condition. Donor tissues preserved in hypothermia (condition A) resulted in 9.3% ± 4.0% trypan-blue positive cells (TBPCs) hence lower number of HCEnCs was plated. <1% TBPCs were observed in conditions B, C and D. Indicatively, confluence in conditions A, B, C and D was 14.0%, 24.8%, 23.4% and 25.4% respectively (p = 0.9836) at day 1. By day 9, HCEnCs established from all conditions became confluent except cells from condition A (94.2% confluence). All HCEnCs in the 4 conditions were viable and expressed HCEnC-associated markers. In conclusion, OC system has advantages over hypothermic media for the preservation of older donor corneas rejected for corneal transplant and deemed suitable for corneal endothelial cell expansion, with lower TBPCs before peeling and longer period of tissue preservation over hypothermic storage system.

Effect of a microwave warming of cell culture media on cell viability and confluence rate

Here we present a method for rapidly and stably warming up a small volume of cell culture media that can maintain cell viability and confluence rate. This method uses microwave radiation for warming without any direct contact with water, preventing the potential issue of contamination induced by the use of a water bath. To demonstrate the proof of concept validation, we used a conventional microwave oven for warming cell culture media. In our experiments, it took only 10 s to warm a 50 mL-media tube (mostly proper volume for the use of microfluidic cell culture experiments) up to 37 °C. Multiple tubes can also be used to increase the volume of cell culture media by placing them in a plastic support within the oven at the same time in a scalable manner. The results show that there was no jump discontinuity to a higher temperature than 37 °C within 10 s. Both apoptosis and necrosis were monitored and examined to confirm whether the new method can affect cell viability and metabolism. The proposed method is fast, easy and user-friendly in conventional cell culture process, even scalable for the use of large media volume, and free of biological contamination due to water contact occurred by use of conventional water baths. We furthermore believe that this approach can be potentially helpful for advancing on-chip cell culture process that may require a small volume of cell culture media often used in microfluidic devices.

Drug-induced cholestasis detection in cryopreserved rat hepatocytes in sandwich culture.

In vitro identification of compounds that cause cholestasis in vivo still remains a problem in pharmaceutical R&D. Currently existing in vitro systems show poor predictivity towards the clinical situation. Recently, our research group developed a model, based on sandwich-cultured (rat) hepatocytes (SC(R)H), to detect compounds causing cholestasis via altered bile acid (BA) homeostasis (Chatterjee et al., 2014). In the present study, we assessed whether this model performs equally well with freshly-isolated and cryopreserved hepatocytes. We exposed sandwich cultures from rat hepatocytes before and after cryopreservation to the cholestatic compounds, cyclosporin A (CsA) and troglitazone (Tro), in the presence and in the absence of a BA mixture. At the end of the incubations, the capability of the hepatocytes to produce urea was measured to determine changes in the drug-induced cholestasis index (DICI). The mean (± SEM) urea production was significantly higher in sandwich cultures from freshly-isolated hepatocytes (27.88 (± 0.96) nmol/cm(2)), compared to cultures from cryopreserved hepatocytes (22.86 (± 1.91) nmol urea/cm(2)). However, after normalization for confluence rate (based on light microscopic image analysis), it appeared that the urea production was similar for all the batches of SCRH. The mean (± SEM) DICI values for CsA 10 μM and Tro 75 μM were 0.89 (± 0.03) and 0.93 (± 0.03), respectively. Higher concentrations, CsA (≥ 15 μM) and Tro (≥ 100 μM), elicited a significant decrease in urea production when incubated in the presence of a BA mixture compared to the compound alone. This was the case for all the batches of SCRH, irrespective of cryopreservation history. In conclusion, no significant differences were seen when the previously described in vitro cholestasis model was applied in SCRH before or after cryopreservation. This study demonstrates the robustness of the model, which implies that it can be used with SCRH obtained from both freshly-isolated and cryopreserved hepatocytes.

Correlation between morphological and biological characteristics of mesenchymal stem cells and hepatocytes derived from rat mesenchymal stem cells in vitro

Hepatocyte dysfunction with the possibility of eventual organ failure is created from most liver diseases. Images of cell morphology can be obtained nondestructively using a conventional inverted microscope. Therefore, this study attempted to investigate several morphological parameters of mesenchymal stem cells (MSCs) and MSCs-derived hepatocytes, in vitro, and their correlation with biological activities. Our results show that MSCs are clonogenic and self-renewal and these cells showed apoptotic property. There were significant difference in confluence rate and doubling time rate among serial passages (P<0.01). When MSCs were cultured with fibroblast growth factor – 4 (FGF-4) and hepatocyte growth factor (HGF), approximately 80-90% of cells became small, round and epithelioid on day 21 that was increasingly similar to hepatocytes in appearance. Compared with the control, levels of alpha-fetoprotein (AFP), albumin (ALB) and urea increased significantly from day 12, 16 and 20, respectively and were higher on day 24 (P < 0.01). The AFP, ALB and urea production level of the large polygonal cells was markedly higher than that of cells with other morphologies. In conclusion, morphological parameters such as polygonal index, cell adhesion area, morphologic changes, proliferation and double nucleoli rate might be use as an indication of differentiation of bone marrow-MSCs into hepatocytes and their functions. Key words : Hepatocyte, stem cell, morphological parameter, polygonal index.

Correlation between morphological and biological characteristics of mesenchymal stem cells and hepatocytes derived from rat mesenchymal stem cells in vitro

Hepatocyte dysfunction with the possibility of eventual organ failure is created from most liver diseases. Images of cell morphology can be obtained nondestructively using a conventional inverted microscope. Therefore, this study attempted to investigate several morphological parameters of mesenchymal stem cells (MSCs) and MSCs-derived hepatocytes, in vitro, and their correlation with biological activities. Our results show that MSCs are clonogenic and self-renewal and these cells showed apoptotic property. There were significant difference in confluence rate and doubling time rate among serial passages (P<0.01). When MSCs were cultured with fibroblast growth factor – 4 (FGF-4) and hepatocyte growth factor (HGF), approximately 80-90% of cells became small, round and epithelioid on day 21 that was increasingly similar to hepatocytes in appearance. Compared with the control, levels of alpha-fetoprotein (AFP), albumin (ALB) and urea increased significantly from day 12, 16 and 20, respectively and were higher on day 24 (P < 0.01). The AFP, ALB and urea production level of the large polygonal cells was markedly higher than that of cells with other morphologies. In conclusion, morphological parameters such as polygonal index, cell adhesion area, morphologic changes, proliferation and double nucleoli rate might be use as an indication of differentiation of bone marrow-MSCs into hepatocytes and their functions. Key words : Hepatocyte, stem cell, morphological parameter, polygonal index.

Effects of the confluence rate on the FTIR spectrum of PC-3 prostate cancer cells in culture

Recently, the possibility of using IR spectroscopy to fingerprint the mode of action of potent antitumor drugs on cancer cells at sub-lethal concentrations has been demonstrated by comparing spectra recorded from untreated or drug-treated cells. The present study investigates the potential interference of the cell culture confluence rate on cell FTIR signature. Significant spectral differences were observed on cells harvested at different confluence rates. Yet, these differences were weak when compared to those induced by sub-lethal ouabain concentrations, used as a model of cardenolide drug. Furthermore, principal component analysis reveals that the impact of the confluence rate, above 50% coverage, on the FTIR spectra is essentially unique and orthogonal to the one induced by the drug model.

Evidence of enhanced turbulent dissipation in the frontogenetic Kuroshio Front thermocline

Enhanced turbulent dissipation, O(10−8–10−7) Wkg−1 in the thermocline on the cyclonic side of the Kuroshio Front was observed during a period of frontogenesis, using a microstructure profiler, XBT and ADCP along 143°E across the Kuroshio Front in August 2008. The eddy diffusivity corresponding to the mixing below the central jet is estimated to be O(10−4–10−3) m2 s−1. The strong turbulent mixing we observed in the Kuroshio is in sharp contrast to previous field measurements which found that small scale diapycnal mixing in western boundary currents remains at levels typically measured in the open ocean thermocline. The turbulence in the Kuroshio is attributed to frontogenesis arising out of an estimated confluence rate of O(10−5) s−1 based on satellite altimeters, suggesting the ubiquity of a forward energy cascade from mesoscale to microscale turbulence near ocean fronts as indicated by recent theoretical studies.

Análise do cultivo de fibroblastos de pterígios primários e recidivados e da cápsula de Tenon normal

To evaluate fibroblast proliferation activity of the Tenon capsule from primary and recurrent pterygia. A randomized prospective study was performed with 43 surgical samples from 30 primary and 13 recurrent pterygia, and samples of normal Tenon capsule from the same pterygium patients. Proliferation, migration, and confluence rate, and patient age, lesion location and type (fleshy or involutive; primary or recurrent) were evaluated. Data were submitted to statistical analysis. Of the 30 cultivated primary pterygia, 21 migrated and proliferated, and 18 (60%) went to confluence. Migration, proliferation and confluence started much later in fibroblasts from normal Tenon than from pterygia lesion. The recurrent pterygia showed migration, proliferation, and confluence starting much earlier than the primary ones. There was no statistically significant difference in the start of migration, proliferation and confluence between fleshy and involuted pterygia, or between pterygias of Grades I-II and III-IV. Pterygium fibroblasts are more viable than normal Tenon in cell culture. Migration, proliferation, and confluence differ in primary and recurrent pterygia. Fleshy and involutive pterygia are similar in culture; there is also no difference between pterygia according to lesion size in cell culture.

Effect of surgical technique on in vitro posterior capsule opacification

Purpose To compare the effect of different cataract extraction surgical techniques on residual lens epithelial cell (LEC) density and cell regrowth rates using an in vitro model of posterior capsule opacification (PCO). Setting Comparative Ophthalmology Research Laboratories, North Carolina State University, Raleigh, North Carolina, USA. Methods Lens capsule explants were prepared from freshly enucleated canine globes after extracapsular cataract extraction (ECCE), phacoemulsification, or phacoemulsification followed by capsule vacuuming. Initial cell density on the capsule and cell proliferation were determined by phase contrast microscopy. The effects of the surgical technique on time to confluent growth of the cells across the posterior lens capsule were determined. Results Residual cell density on the remaining anterior capsule immediately after lens removal was 31.6% ± 19.3%, 16.1% ± 8.9%, and 7.7% ± 5.7% in the ECCE, phacoemulsification, and phacoemulsification/capsule-vacuuming groups, respectively. Time to confluence (range 5.0 to 6.3 days) was not significantly different among the 3 groups when the lens capsules were cultured in serum-supplemented media. The confluence rate was significantly longer (by approximately 5 to 7 days) in the phacoemulsification/capsule-vacuuming group than in the other 2 groups when the capsules were cultured in serum-free media. Conclusions Phacoemulsification with and without anterior and equatorial capsular vacuuming led to less initial LEC density in the capsular bag than ECCE. However, because cell proliferation rates among the 3 groups were only marginally affected, near 100% removal of LEC at the time of cataract extraction may be necessary to prevent PCO.

A stochastic model to simulate the growth of anchorage dependent cells on flat surfaces

A stochastic model is proposed to simulate the growth of anchorage dependent cells on a flat surface. The model, based on representing the cell shapes on the surface as external irregular polygons with the nuclei distributed as a set of Poisson points (producing a modified Voronoi tessellation of 2 space) and incorporating a distribution function to describe cell division of the perimeter cells of the colony, provides data not only on population dynamics but also on the patterns produced by clusters of cells in the colony. These patterns produced by the model are qualitatively similar to observations reported for some cell cultures. The periods of induction, rapid growth, and decreasing growth asymptoting to zero as confluence is reached are predicted by the model. Quantitative comparison with published experimental data for this is good. The specific growth rate computed for the period of rapid growth predicted by the model is dependent on the distribution function describing the cell division time. As the standard deviation of this increases, the specific growth rate decreases as with a consequent increase in time to achieve confluence. The removal of cells from the colony by shear forces or death is considered in the model. As the probability for removal increases, the cell density at confluence and specific growth rate decrease. The clusters of cells, patterns, in the colony are very sensitive to cell removal. By analyzing these patterns in experiments, an estimate of cell removal can be made. The areas covered by cells on a substrate are fractal patterns. The fractal dimension is always greater than 1 and is a function of the removal probability.