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1.
Curr Protoc ; 4(6): e1067, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38857108

RESUMEN

The blood-brain barrier (BBB) constitutes a crucial protective anatomical layer with a microenvironment that tightly controls material transit. Constructing an in vitro BBB model to replicate in vivo features requires the sequential layering of constituent cell types. Maintaining heightened integrity in the observed tight junctions during both the establishment and post-experiment phases is crucial to the success of these models. We have developed an in vitro BBB model that replicates the cellular composition and spatial orientation of in vivo BBB observed in humans. The experiment includes comprehensive procedures and steps aimed at enhancing the integration of the four-cell model. Departing from conventional in vitro BBB models, our methodology eliminates the necessity for pre-coated plates to facilitate cell adhesion, thereby improving cell visualization throughout the procedure. An in-house coating strategy and a simple yet effective approach significantly reduce costs and provides superior imaging of cells and corresponding tight junction protein expression. Also, our BBB model includes all four primary cell types that are structural parts of the human BBB. With its innovative and user-friendly features, our in-house optimized in vitro four-cell-based BBB model showcases novel methodology and provides a promising experimental platform for drug screening processes. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Coating and culture system Basic Protocol 2: Cell seeding and Transwell insert handling Basic Protocol 3: Assessment of model functionality.


Asunto(s)
Barrera Hematoencefálica , Humanos , Barrera Hematoencefálica/metabolismo , Uniones Estrechas/metabolismo , Técnicas de Cultivo de Célula/métodos , Modelos Biológicos , Encéfalo/citología , Células Cultivadas , Células Endoteliales/citología , Células Endoteliales/metabolismo
2.
Sci Rep ; 14(1): 10709, 2024 05 10.
Artículo en Inglés | MEDLINE | ID: mdl-38729980

RESUMEN

Three years after SARS-CoV-2 emerged as a global infectious threat, the virus has become endemic. The neurological complications such as depression, anxiety, and other CNS complications after COVID-19 disease are increasing. The brain, and CSF have been shown as viral reservoirs for SARS-CoV-2, yielding a potential hypothesis for CNS effects. Thus, we investigated the CNS pharmacology of orally dosed nirmatrelvir/ritonavir (NMR/RTV). Using both an in vitro and an in vivo rodent model, we investigated CNS penetration and potential pharmacodynamic activity of NMR. Through pharmacokinetic modeling, we estimated the median CSF penetration of NMR to be low at 18.11% of plasma with very low accumulation in rodent brain tissue. Based on the multiples of the 90% maximal effective concentration (EC90) for SARS-CoV-2, NMR concentrations in the CSF and brain do not achieve an exposure level similar to that of plasma. A median of only 16% of all the predicted CSF concentrations in rats were > 3xEC90 (unadjusted for protein binding). This may have implications for viral persistence and neurologic post-acute sequelae of COVID-19 if increased NMR penetration in the CNS leads to decreased CNS viral loads and decreased CNS inflammation.


Asunto(s)
Leucocitos Mononucleares , Ritonavir , SARS-CoV-2 , Animales , Ratas , Ritonavir/farmacocinética , SARS-CoV-2/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/virología , Humanos , Masculino , Encéfalo/metabolismo , Encéfalo/virología , Tratamiento Farmacológico de COVID-19 , COVID-19/virología , COVID-19/líquido cefalorraquídeo , Antivirales/farmacocinética , Antivirales/farmacología , Ratas Sprague-Dawley , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/virología
3.
J Neurosci Methods ; 392: 109867, 2023 05 15.
Artículo en Inglés | MEDLINE | ID: mdl-37116621

RESUMEN

The blood-brain barrier (BBB) is a protective cellular anatomical layer with a dynamic micro-environment, tightly regulating the transport of materials across it. To achieve in-vivo characteristics, an in-vitro BBB model requires the constituent cell types to be layered in an appropriate order. A cost-effective in-vitro BBB model is desired to facilitate central nervous system (CNS) drug penetration studies. Enhanced integrity of tight junctions observed during the in-vitro BBB establishment and post-experiment is essential in these models. We successfully developed an in-vitro BBB model mimicking the in-vivo cell composition and a distinct order of seeding primary human brain cells. Unlike other in-vitro BBB models, our work avoids the need for pre-coated plates for cell adhesion and provides better cell visualization during the procedure. We found that using bovine collagen-I coating, followed by bovine fibronectin coating and poly-L-lysine coating, yields better adhesion and layering of cells on the transwell membrane compared to earlier reported use of collagen and poly-L-lysine only. Our results indicated better cell visibility and imaging with the polyester transwell membrane as well as point to a higher and more stable Trans Endothelial Electrical Resistance values in this plate. In addition, we found that the addition of zinc induced higher claudin 5 expressions in neuronal cells. Dolutegravir, a drug used in the treatment of HIV, is known to appear in moderate concentrations in the CNS. Thus, dolutegravir was used to assess the functionality of the final model and cells. Using primary cells and an in-house coating strategy substantially reduces costs and provides superior imaging of cells and their tight junction protein expression. Our 4-cell-based BBB model is a suitable experimental model for the drug screening process.


Asunto(s)
Barrera Hematoencefálica , Polilisina , Animales , Bovinos , Humanos , Barrera Hematoencefálica/fisiología , Línea Celular , Polilisina/metabolismo , Polilisina/farmacología , Células Endoteliales , Microscopía Confocal
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