Organotypic co-cultures allow for immortalized human gingival keratinocytes to reconstitute a gingival epithelial phenotype in vitro

Roesch‐Ely, Mariana; Steinberg, Thorsten; Bosch, F. Xavier; Müssig, Eva; Whitaker, Noel J.; Wiest, T.; Kohl, Annette; Komposch, Gerda; Tomakidi, Pascal

doi:10.1111/j.1432-0436.2006.00099.x

Cited by 38 publications

(43 citation statements)

References 57 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Similar cultivation times were used in previous studies for organotypic gingival cocultures. 17,31,32 These preliminary tests on the aforementioned corneal cocultures revealed an only marginal maturation before day 5, and epithelial disarrangements starting from day 10 onward. Consequently, three cultivation times were chosen to investigate the characteristics of balanced epithelial morphogenesis: (1) 5 days for premature epithelial differentiation, (2) 6 days for progression of differentiation, and (3) 9 days for completion of differentiation, including terminal differentiation.…”

Section: Resultsmentioning

confidence: 75%

“…17 Briefly, 4.5 · 10 5 IHCF and 1 · 10 5 IHCE (holistic mode) or only 4.5 · 10 5 IHCF (nonholistic mode) were trypsinized, counted, and resuspended in 1 mL of fetal calf serum (Biochrome, Berlin, Germany). This suspension was mixed in a beaker chilled on ice with 8 mL of collagen solution (5 mg/ mL; Gibco, Life Technologies, Grand Island, NY) and 1 mL HBSS 10 · (Gibco, Life Technologies).…”

Section: Cocultures Comprising Combinations Of Corneal Keratinocytes mentioning

confidence: 99%

“…By using this operation mode, 3D spatially separated growth of IHCK from holistic and nonholistic microenvironments was warranted. In analogy to the cocultures established on collagen gels, cells here can also only communicate through diffusible growth factors, 17 and keratinocyte RNA contaminations with microenvironmental cell RNA can be excluded. These setups were cultivated for a time period of 5, 6, and 9 days.…”

Section: Cocultures Comprising Combinations Of Corneal Keratinocytes mentioning

confidence: 99%

“…In this study, distinct cell morphologies correspond to respective layers, and in conjunction with the expression of biomarkers, for example, stem cell markers like cornea-attributed ABCG2, cytokeratins (CKs), involucrin, and filaggrin indicate certain stages of keratinocyte differentiation. [6][7][8][9][10][11] Circumvention of these limitations is achieved by human keratinocyte immortalization, [12][13][14] and generation of stratified epithelia in interactive 3D cell cocultures as described for skin, 15 oral cavity, 16,17 and cornea. [18][19][20][21][22] Following immortalization, the resulting keratinocyte cell line should express most of the biomarkers, assigning to tissue homeostatic parameters such as differentiation, to reflect an authentic in vitro model system, suitable as an animal experiment substitute.…”

mentioning

confidence: 99%

“…11 In this context, spontaneous keratinocyte immortalization, a very seldom observed phenomenon, and the use of HPV-16 E6 and E7 genes as immortalizing agents are reported to maintain the cells' normal differentiation behavior. 17,23 By contrast, employment of the SV40 T-antigen early region genes has been described, for instance, for mammary epithelial cells to be accompanied with aberrant differentiation, loss of DNA damage response, karyotypic instability and, in some cases, tumorigenicity with matched hTERT/catalytic telomerase subunit-immortalized counterparts. 24 Thus, it appears likely that optimized tissue-authentic in vitro models should consist of target cells immortalized by a smooth modus operandi, that is, a biomarker-conservative immortalizing agent.…”

mentioning

confidence: 99%

See 4 more Smart Citations

Natural Corneal Cell-Based Microenvironment as Prerequisite for Balanced 3D Corneal Epithelial Morphogenesis: A Promising Animal Experiment-Abandoning Tool in Ophthalmology

Schulz

Beck

Laird

et al. 2014

Tissue Engineering Part C: Methods

Self Cite

View full text Add to dashboard Cite

To achieve durable recognition as a promising animal experiment-abandoning tool in ophthalmology, in vitro engineered tissue equivalents of the human cornea should exhibit proper morphogenesis. Regarding this issue, we were seeking for the natural cell microenvironment fulfilling the minimum requirements to allow human corneal keratinocytes to develop a balanced epithelial morphology with regular spatial appearance of tissue homeostatic biomarkers. Hence, we established cocultures of 3D cell-based collagen scaffolds comprising immortalized corneal keratinocytes combined with a gradual cornea-derived in vivo-like cell microenvironment, together with immortalized stromal fibroblasts alone (nonholistic) or fibroblasts and immortalized endothelial cells (holistic). With matched non-holistic microenvironments revealing mostly flattened cells and putative apical cell ablation foci at day 6, and 9 in HE stains, holistic counterparts yielded proper epithelial stratification with cell flattening restricted to apical layers. Concordantly, RT 2 -PCR showed a tremendous increase in gene expression for progressive and terminal biomarkers of corneal keratinocyte differentiation, cytokeratin (CK) 12, and filaggrin (FIL), in response to nonholistic environments, while involucrin (INV) was moderately but significantly upregulated. Although visible, this increase was moderate in corneal keratinocytes with a holistic environment. On the protein level, indirect immunofluorescence revealed that only epithelia of holistic environments showed diminishment in CK19, counteracted by CK12 rising over time. This time-dependent progression in differentiation coincided with declined proliferation and tissue-regular focus of differentiation biomarkers inv and fil to suprabasal and apical cell layers. Our novel findings suggest the interplay of native tissue forming cell entities, important for balanced corneal epithelial morphogenesis. In addition, they provide evidence for a holistic cell microenvironment as a prerequisite for development of an in vitro engineered corneal epithelial tissue equivalent, exhibiting a regular appearance of tissue homeostatic biomarkers. Such equivalents will be promising tools in ophthalmology, for example, for mechanistic studies in basic research and/or testing of generics or preclinical validation of innovative cornea-tailored biomaterials, desired for regenerative strategies.

show abstract

Section: Resultsmentioning

confidence: 75%

Section: Cocultures Comprising Combinations Of Corneal Keratinocytes mentioning

confidence: 99%

Section: Cocultures Comprising Combinations Of Corneal Keratinocytes mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Natural Corneal Cell-Based Microenvironment as Prerequisite for Balanced 3D Corneal Epithelial Morphogenesis: A Promising Animal Experiment-Abandoning Tool in Ophthalmology

Schulz

Beck

Laird

et al. 2014

Tissue Engineering Part C: Methods

Self Cite

View full text Add to dashboard Cite

show abstract

Severe Deformations of Malignant Bone and Skin Cells, as well as Aged Cells, on Micropatterned Surfaces

Davidson

Haraguchi

Koujin

et al. 2013

Biomaterials Surface Science

View full text Add to dashboard Cite

Human Organs‐on‐Chips: A Review of the State‐of‐the‐Art, Current Prospects, and Future Challenges

et al. 2021

View full text Add to dashboard Cite

New emerging technologies, remarkably miniaturized 3D organ models and microfluidics, enable simulation of the real in vitro microenvironment ex vivo more closely. There are many fascinating features of innovative organ‐on‐a‐chip (OOC) technology, including the possibility of integrating semipermeable and/or stretchable membranes, creating continuous perfusion of fluids into microchannels and chambers (while maintaining laminar flow regime), embedding microdevices like microsensors, microstimulators, micro heaters, or different cell lines, along with other 3D cell culture technologies. OOC systems are designed to imitate the structure and function of human organs, ranging from breathing lungs to beating hearts. This technology is expected to be able to revolutionize cell biology studies, personalized precision medicine, drug development process, and cancer diagnosis/treatment. OOC systems can significantly reduce the cost associated with tedious drug development processes and the risk of adverse drug reactions in the body, which makes drug screening more effective. The review mainly focus on presenting an overview of the several previously developed OOC systems accompanied by subjects relevant to pharmacy‐, cancer‐, and placenta‐on‐a‐chip. The challenging issues and opportunities related to these systems are discussed, along with a future perspective for this technology.

show abstract

Organotypic co-cultures allow for immortalized human gingival keratinocytes to reconstitute a gingival epithelial phenotype in vitro

Cited by 38 publications

References 57 publications

Natural Corneal Cell-Based Microenvironment as Prerequisite for Balanced 3D Corneal Epithelial Morphogenesis: A Promising Animal Experiment-Abandoning Tool in Ophthalmology

Natural Corneal Cell-Based Microenvironment as Prerequisite for Balanced 3D Corneal Epithelial Morphogenesis: A Promising Animal Experiment-Abandoning Tool in Ophthalmology

Severe Deformations of Malignant Bone and Skin Cells, as well as Aged Cells, on Micropatterned Surfaces

Human Organs‐on‐Chips: A Review of the State‐of‐the‐Art, Current Prospects, and Future Challenges

Contact Info

Product

Resources

About