3-D Tissue ModelingNov 13, 2014 at 12:00 PM ET
The emergence of 3-D tissue modeling raises new possibilities for the study of complex physiological processes in vitro. Advances in cell isolation, media development, substrates, and growth surfaces are leading to protocols that provide more functionality than traditional 2-D cell culture. These models may provide a more predictive analysis and result in a more streamlined process of drug discovery and development. In this webinar, we will discuss recent developments in 3-D modeling using ATCC primary and hTERT immortalized cells in areas such as angiogenesis, wound healing, and respiratory studies.
John Pulliam, PhD
Field Application Scientist, ATCC
Can the human telomerase (hTERT)-, cyclin-dependent kinase 4 (CDK4)-immortalized human neonatal foreskin keratinocytes (Ker-CT, ATCC CRL-4048) be differentiated into epidermal structures in 2-D culture?
Yes. Ker-CT cells can differentiate in 2-D culture by increasing the Ca2+ concentration in the medium to 1.2 mM. We have observed by immunocytochemistry and western blot that elevated Ca2+ increased several markers of differentiation (KRT1, KRT10, and gamma-catenin).
For air-liquid interface airway cell plating, do we need to coat the membrane?
Yes. A collagen I solution is applied at 0.03 mg/mL in a thin layer to coat the inserts and incubated at 37°C for 4-18 hours prior to seeding primary HBECs or small airway cells.
Have you evaluated 3-D culture models of primary human brain microvascular endothelial cells with primary human brain cortical astrocytes?
We have not worked with these cells types yet.
Have you observed a significant difference between primary small airway and bronchial-tracheal cells?
Yes. You would expect more cilia formed in the primary small airway. Both cell types have similar cilia forming potential, but the small airway cells are smaller in size and are able to occupy more cells per cm2. There may be variability in the potential of the cells based on the donor source of the cells.
Have you tried co-culturing immune cells, such as alveolar macrophages, with lung epithelial cells in a 3-D model to study lung infectious disease?
We have not co-cultured immune cells with lung epithelial cells.
How do Normal Human Small Airway Epithelial Cells and Normal Human Bronchial/Tracheal Epithelial Cells behave under relevant O2 tensions?
We have not cultured the cells under airway O2 tensions.
How many population doublings did the human bronchial epithelial cells (HBECs) undergo via your method? What was the transepithelial resistance achieved?
ATCC primary cells, including HBECs, are tested for the capacity to undergo at least 15 population doublings. For the ALI method, population doublings are not tested; rather, the cells are plated on Corning Transwell membrane inserts at 75% confluence. The cells are then grown to 100% confluence before we begin differentiating them, usually lasting for 2-5 days. Transepithelial resistance was not assayed.
How was the GFP-expressing hTERT-immortalized human aortic endothelial cell line (TeloHAEC-GFP, ATCC CRL-4054) created?
The TeloHAEC-GFP cell line was created by stably expressing GFP under the EF1α promoter in telomerase-immortalized TeloHAEC (ATCC CRL-4052) cells. An advantage of the TeloHAEC-GFP cell line is that the GFP signal is stable and bright, even after the cells have undergone differentiation, and is therefore suitable for live imaging and fluorescent microscopy.
In addition to the 2-D co-culture, do you have a 3-D model for angiogenesis?
The co-culture model (endothelial cells co-cultured with fibroblasts or hTERT-MSCs) of angiogenesis is a 3-D model; the tubular structures formed under this condition have a real lumen, unlike the structures that form when the endothelial cells are cultured on top of cell matrix.
Is your airway air-liquid interface protocol available online?
Yes. There is an application note on the ATCC website that provides a detailed protocol of how primary human bronchial/tracheal epithelial cells were differentiated.
What is the earliest day you observed the cilia formation in the airway model?
The earliest beating cilia are observed 18-20 days post air-lift for primary Human Small Airway epithelial cells (ATCC PCS-301-010) and 20-22 days post air-lift for primary Human Bronchial/Tracheal epithelial cells (ATCC PCS-300-010).