Separation of human endothelial cells from fibroblasts by centrifugation in Percoll gradients

Sbarbati, R

doi:10.1007/bf01116944

Cited by 7 publications

(2 citation statements)

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“…The discontinuous Percoll gradient is based on buoyant densities for epithelial cells (1.065–1.07 g/mL) and fibroblasts (1.04–1.055 g/mL) from previous publications. 1 , 2 , 3 The Percoll gradient is prepared during the 40 min incubation for tissue dissociation. Prepare the stock isotonic Percoll (SIP) and duplicate Percoll dilutions according to Table 1 in the materials and equipment section.…”

Section: Step-by-step Methods Detailsmentioning

confidence: 99%

Protocol to establish an oviduct epithelial cell line derived from Gallus gallus using Percoll for in vitro validation of recombinant proteins

Loubser¹,

Nikitina²

2023

STAR Protocols

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Section: Step-by-step Methods Detailsmentioning

confidence: 99%

Protocol to establish an oviduct epithelial cell line derived from Gallus gallus using Percoll for in vitro validation of recombinant proteins

Loubser¹,

Nikitina²

2023

STAR Protocols

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“…Our technique for long-term culture of retinal microvascular endothelial cells was developed after modify-ing several reported methods (Bowman et al, 1982;Sbarbati, 1985;Carson and Haudenschild, 1986;Capetandes and Gerritsen, 1990). Briefly, cell suspensions were gently loaded on Percoll gradients and centrifuged at 2,000 rpm for 10 minutes using a swinging bucket rotor.…”

Section: Materials and Methods Isolation And Culture Of Retinal Micromentioning

confidence: 99%

Effect of hypoxia on the proliferation of retinal microvessel endothelial cells in culture

1997

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Background To determine if hypoxia stimulates the proliferation of retinal microvessel endothelial cells in culture. Methods Bovine retinal microvessel endothelial cells were cultured in normoxic (95% air, 5% CO2) and hypoxic (2% O2, 5% CO2, 93% N2) conditions. Endothelial cells were identified by acetylated LDL and Factor VIII‐related antigen immunocytochemical staining. Cells from passages three to eight were used in these experiments. Proliferation assays included cell counts by hemocytometer and autoradiographic analysis of incorporated 3H‐thymidine (3H‐TdR). Results At day 4, cell counts of endothelial cells in hypoxia showed a 133% increase over those grown in normoxic conditions (N = 25, P < 0.01). Cell counts per day for 5 days were 121‐181% greater in hypoxia. Autoradiography of endothelial cells exposed to 3H‐TdR and counted every 12 hours for 60 hours exhibited labeling indices 112‐118% higher in hypoxic conditions (P < 0.0001). Endothelial cells cultured under hypoxic conditions were smaller and spindle‐shaped, whereas those grown under normoxic conditions were larger and more polygonal. Conclusions Hypoxia increases DNA synthesis and stimulates proliferation of retinal microvessel endothelial cells in vitro and induces alterations in morphology. These results may be relevant to microvessel angiogenesis, which occurs in vivo under ischemic conditions. Anat. Rec. 248:366‐373, 1997. © 1997 Wiley‐Liss, Inc.

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Characterization of a primary cellular airway model for inhalative drug delivery in comparison with the established permanent cell lines CaLu3 and RPMI 2650

Martin,

Rittersberger,

Treitler

et al. 2024

In vitro models

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Purpose For optimization of respiratory drug delivery, the selection of suitable in vitro cell models plays an important role in predicting the efficacy and safety of (bio)pharmaceutics and pharmaceutical formulations. Therefore, an in-depth comparison of different primary and permanent in vitro cellular airway models was performed with a focus on selecting a suitable model for inhalative antibodies. Methods Primary cells isolated from the porcine trachea were compared with the established human cell lines CaLu3 and RPMI 2650. The in vitro models were characterized for different epithelial markers by real-time quantitative polymerase chain reaction, which provides insight into the cellular composition of each model. For a few selected markers, the results from RT-qPCR were confirmed via immunofluorescence. Barrier integrity was assessed by transepithelial electrical resistance measurements and FITC-dextran permeability. Results Primary cell models retain key features of the respiratory epithelium, e.g., the formation of a tight epithelial barrier, mucin production, and the presence of club/basal cells. Furthermore, the expression of Fc receptors in the primary cell models closely resembles that in respiratory mucosal tissue, an essential parameter to consider when developing therapeutic antibodies for inhalation. Conclusion The study underlines the importance of selecting wisely appropriate in vitro models. Despite the greater effort and variability in cultivating primary airway cells, they are far superior to permanent cells and a suitable model for drug development.

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Separation of human endothelial cells from fibroblasts by centrifugation in Percoll gradients

Abstract: Human endothelial cells from the umbilical vein and skin fibroblasts can be separated by means of centrifugation in a density gradient of Percoll. Cells show a good recovery in culture. Viability is not impaired.

Cited by 7 publications

References 2 publications

Protocol to establish an oviduct epithelial cell line derived from Gallus gallus using Percoll for in vitro validation of recombinant proteins

Protocol to establish an oviduct epithelial cell line derived from Gallus gallus using Percoll for in vitro validation of recombinant proteins

Effect of hypoxia on the proliferation of retinal microvessel endothelial cells in culture

Characterization of a primary cellular airway model for inhalative drug delivery in comparison with the established permanent cell lines CaLu3 and RPMI 2650

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