Validation of in vitro assays in three-dimensional human dermal constructs

Idrees, Ayesha; Chiono, Valeria; Ciardelli, Gianluca; Sh, Shah; Viebahn, Richard; Zhang, Xiang; Salber, Jochen

doi:10.1177/0391398818775519

Cited by 25 publications

(24 citation statements)

References 37 publications

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“…The quantity or concentration of the marker to be measured may be beyond the linear range for an assay reagent detection chemistry designed for monolayers of cells. Although there are examples of using several commercially available offthe-shelf assays being used on large dermal constructs (Idrees et al 2018), detailed description of the validation for nonintended use is often lacking. For example, some dermal equivalent models that are assembled on filter inserts and used for skin irritation or cosmetics testing may approach~100-μm thick and 1 cm diameter.…”

Section: Sample Massmentioning

confidence: 99%

Factors to consider when interrogating 3D culture models with plate readers or automated microscopes

Trask

2021

In Vitro Cell.Dev.Biol.-Animal

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Along with the increased use of more physiologically relevant three-dimensional cell culture models comes the responsibility of researchers to validate new assay methods that measure events in structures that are physically larger and more complex compared to monolayers of cells. It should not be assumed that assays designed using monolayers of cells will work for cells cultured as larger three-dimensional masses. The size and barriers for penetration of molecules through the layers of cells result in a different microenvironment for the cells in the outer layer compared to the center of three-dimensional structures. Diffusion rates for nutrients and oxygen may limit metabolic activity which is often measured as a marker for cell viability. For assays that lyse cells, the penetration of reagents to achieve uniform cell lysis must be considered. For live cell fluorescent imaging assays, the diffusion of fluorescent probes and penetration of photons of light for probe excitation and fluorescent emission must be considered. This review will provide an overview of factors to consider when implementing assays to interrogate three dimensional cell culture models.

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Section: Sample Massmentioning

confidence: 99%

Factors to consider when interrogating 3D culture models with plate readers or automated microscopes

Trask

2021

In Vitro Cell.Dev.Biol.-Animal

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“…1A) were visualized by applying F-actin/nuclei staining. This was performed using Phalloidin/DAPI stain from Promokine following a standard protocol [47]. The fluorescence microscopy and Z-stack imaging was performed using Olympus XM10 with cellSense Standard software.…”

Section: Morphological Analysismentioning

confidence: 99%

“…HDC was previously developed based on Col. I from rat tail tendons, mimicking skin ECM thanks to its irregular fibrils with in vivo-like structure [47]. The development of HSE with dermal-epidermal compartments has been improved by using commercially available primary cells and ready-to-use media provided according to Good Laboratory Practice (GLP) and Good Manufacturing Practice (GMP) criteria to form a well-differentiated epidermis accessible to everyone.…”

Section: Epidermal Differentiation Of Hsementioning

confidence: 99%

“…The expression of differentiation-specific keratins causes keratin filament network reorganization resulting in denser bundle formation [74]. In more detail, cells from inner layers of the epidermis have small keratins (46)(47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58), while cells from the outer layers contain large keratins (63-67 kDa) [75]. These changes in keratin composition and synthesis occur during the course of terminal differentiation.…”

Section: Protein Distribution In Hsementioning

confidence: 99%

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Fundamentalin vitro3D human skin equivalent tool development for assessing biological safety and biocompatibility – towards alternative for animal experiments

Idrees

Schmitz

Zoso

et al. 2021

4open

Self Cite

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Nowadays, human skin constructs (HSCs) are required for biomaterials, pharmaceuticals and cosmetics in vitro testing and for the development of complex skin wound therapeutics. In vitro three-dimensional (3D) dermal-epidermal based interfollicular, full-thickness, human skin equivalent (HSE) was here developed, recapitulating skin morphogenesis, epidermal differentiation, ultra-structure, tissue architecture, and barrier function properties of human skin. Different 3D cell culture conditions were tested to optimize HSE maturation, using various commercially available serum/animal component-free and/or fully defined media, and air-liquid interface (ALI) culture. Optimized culture conditions allowed the production of HSE by culturing normal human dermal fibroblasts (NHDFs) for 5–7 days in CELLnTEC-Prime Fibroblast (CnT-PR-F) medium and then culturing normal human epidermal keratinocytes (NHEKs) for 3 days in CELLnTEC-Prime Epithelial culture (CnT-PR) medium on them. Co-culture was then submerged overnight in CELLnTEC-Prime-3D barrier (CnT-PR-3D) medium to stimulate cell-cell contact formation and finally placed at ALI for 15–20 days using CnT-PR-3D medium. Histological analysis revealed uniform distribution of NHDFs in the dermal layer and their typical elongated morphology with filopodia. Epidermal compartment showed a multi-layered structure, consisting of stratum basale, spinosum, granulosum, and corneum. NHDFs and keratinocytes of basal layer were positive for the proliferation marker Kiel 67 (Ki-67) demonstrating their active state of proliferation. The presence of typical epidermal tissue proteins (keratins, laminins, filaggrin, loricin, involucrin, and β-tubulin) at their correct anatomical position was verified by immunohistochemistry (IHC). Moreover, transmission electron microscopy (TEM) analyses revealed basement membrane with lamina lucida, lamina densa, hemidesmosomes and anchoring fibers. The epidermal layers showed abundant intracellular keratin filaments, desmosomes, and tight junction between keratinocytes. Scanning electron microscopy (SEM) analyses showed the interwoven network of collagen fibers with embedded NHDFs and adjacent stratified epidermis up to the stratum corneum similar to native human skin. HSE physiological static contact angle confirmed the barrier function. The developed HSE represents a fundamental in vitro tool to assess biocompatibility of biomaterials, pharmacotoxicity, safety and effectiveness of cosmetics, as well as to investigate skin biology, skin disease pathogenesis, wound healing, and skin infection.

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“…Cell viability assays were performed in 2D using Realtime-Glo™ MT Cell Viability RT-Glo (Promega UK, G9711) and in 3D using Celltiter-Glo 3D (Promega UK, G9682). Both methods were performed according to the manufacturer's instructions [94,95]. Readings were taken using a FLUOstar Omega microplate reader at 0-, 24-, 48-and 72 h time points.…”

Section: Drug Release Pro Lementioning

confidence: 99%

Nanoparticle encapsulation of HDACi with HA targeting in endometrial cancer models

Edwards

Yao

Pisano

et al. 2020

Preprint

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Bespoke nanoparticle systems can enhance drug delivery, preventing systemic exposure by modifying release kinetics and increasing tumour penetration. Flexible nano vector systems have led to selective tumour targeting through surface modifications and the addition of target moieties over expressed in the cancer microenvironment. As such nanomedicines are enabling drug re-purposing and renewed applications in solid tumour cancers. Here we present the nanoscale encapsulation of Vorinostat within a F127 Pluronic polymer particle modified to incorporate a Hyaluronic Acid moiety, targeting increased CD44 expression in endometrial cancer cells. Encapsulation within a stable micellar structure stabilized SAHA activity, demonstrating increased cytotoxic efficacy in 2-D and 3-D cultures. In addition, nano delivery enhanced spheroid penetration, suppression in cell growth, P21 associated cell cycle arrest and overcome EMT associated phenotype formation observed in the free drug treated type II Hec50 cells. Together, this study clearly demonstrates that HDAC nanoparticle encapsulation and HA targeting may offer a solution to overcoming the toxicity and side effects issues observed in clinical trials. Given the demonstrated involvement of epigenetic processes in oncology, this study is an important demonstration of this class of anti-cancerous agents for the treatment of endometrial cancers.

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Validation of in vitro assays in three-dimensional human dermal constructs

Cited by 25 publications

References 37 publications

Factors to consider when interrogating 3D culture models with plate readers or automated microscopes

Factors to consider when interrogating 3D culture models with plate readers or automated microscopes

Fundamentalin vitro3D human skin equivalent tool development for assessing biological safety and biocompatibility – towards alternative for animal experiments

Nanoparticle encapsulation of HDACi with HA targeting in endometrial cancer models

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