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3D PETG Cell Culture Scaffolds

24-Well, 3D PETG Cell Culture Scaffolds by Copner Biotech

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In July 2021, Copner Biotech was presented with the Global Health and Pharma International Life Sciences Award for Innovation, for their work in 3D cell culture technologies. Most notably, their method of fabrication of 3D PETG scaffolds, which has been shown to optimise cell capture and attachment whilst using low cell numbers for seeding (patent-pending). The company has now entered several high value research projects in the field of bioprinting, with support from Welsh Government.

2D v 3D Cell Culture

The benefits of 3D cell culture over conventional 2D culture are widely accepted in the scientific community. Traditional 2D cell culture methods subject cells to an un-physiological architectural state. When cultured in 2D, mammalian cells assume a bipolar state with a basal and apical side. In order to tackle this unnatural morphology, cytoskeletal re-modelling takes place and the subsequent ultrastructure of the cell is greatly altered (Ravi et al., 2015). Cells cultured in 2D also have unlimited access to oxygen, nutrients and metabolites –which is not the case in their respective tissues in vivo. These limitations ultimately mean those laboratories undertaking assays on cells in this unnatural environment yield less reliable results in vitro.

3D cell culture creates a 3D architecture for cell growth, offering a closer knit and physiologically relevant environment. Growth of cells on 3D scaffolds promotes accurate cell-to-cell and cell-to-extracellular environment interactions with variable access to oxygen, nutrients and metabolites. Mammalian cells that sit within a more representative environment, reflective of their host tissue in vivo, exhibit more relevant gene expression, splicing topology and biomarkers in vitro, which in turn offers far greater reliability in terms of drug and toxicity modelling in the lab (Lv et al., 2016).

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Scaffold Design

Recent studies demonstrate the importance of discrete oxygen gradients in cell movement across the scaffold interface (Ardakani et al, 2014). Cells grown on woodpile structures typically have a heterogenous distribution, with clusters common. By introducing a discrete oxygen gradient across the interface of our 3D PETG scaffold, we encourage cell proliferation from the centre to the periphery. The result is a much more balanced system of cells on the scaffold, with confluency patterns like that of in vivo tissue.

Copner Biotech have developed a bespoke 3D modelling software and next generation 3D printing technique in order to create these complex architectures. Our additive manufacturing process demonstrates high batch-tobatch consistency, minimising variables in your 3D cell models.

Scaffold microstructure validation work -

(Left) Top view of PETG scaffold using a standard light microscope. (Right) Cross section of a PETG scaffold captured using scanning electron microscopy (SEM).

Cellular Adherence

Copner Biotech’s 3D PETG scaffolds have been shown to efficiently mediate mammalian cell attachment, over multiple cell lines, all using low cell seeding concentrations.

Scanning Electron Microscopy (SEM) images of L929 cells grown on a PETG scaffold, at day 7 (50K cells seeded). Cells have demonstrated a successful migration from the centre to the periphery of the scaffold.

Our PETG material has been modified to include discrete regions where cells become temporarily trapped. These regions then give rise to the first successfully attached cells, and ultimately lead to the formation of high-density cell populations.
From here, cells begin to migrate toward the periphery, due to discrete oxygen and nutrient gradients across the scaffold interface. These gradients form due to the controlled, heterogenous pore size/distribution across the scaffold.

Cellular Viability

Our PETG material has been altered in order to optimise surface roughness and subsequently improve cell attachment and proliferation. This final inert, noncytotoxic material has been confirmed safe for use in cell culture, showing no negative effects on cell growth or function, making your transition from 2D to 3D cell culture easier.
Scaffolds also show rigidity and do not degrade, making them very user-friendly for long term culture of cells.

Cytotoxicity (A) LDH release (n=3) noted by optical density at 490 nm (B) Relative fluorescence intensity of alamarBlue assay (n=3). L929 cell line at a density of 1 x 104 cells were cultured with control media, day 1 conditioned media or day 7 conditioned media for 24h at 37°C.

Figure above shows:
(A) LDH release (n=3) to each treatment condition as noted by optical density at 490 nm
(B) Relative fluorescence intensity of almarBlue assay (n=3) to each treatment condition. Statistical significance determined by Friedmans test; * p < 0.05.

Cellular Proliferation

L929 fibroblast cells seeded on PETG scaffolds significantly proliferate, whilst exhibiting important biomarkers. Our PETG material mediates cell division events, whilst minimising cell loss, resulting in significant expansion of cell cultures over a short space of time. This is particularly beneficial to those looking to experiment on cells in an exponential growth phase.(Left) Relative fluorescence intensity of alamarBlue assay (n=3) at each time point. Statistical 315 significance determined by paired t-test; * p < 0.05, ** p < 0.01 (Right) Elongated focal adhesion points, indicating a healthy fibroblast morphology in 3D culture.

Spheroid Formation

When using high cell seeding concentrations, Copner Biotech's scaffold technology allows users to establish reliable spheroids, with high batch-to-batch consistency. Spheroids are a type of three-dimensional cell modeling that better simulate a live cell's environmental conditions compared to a two-dimensional cell model, specifically with the reactions between cells and the reactions between cells and the matrix.

Dermal Spheroids established on uncoated 3D PETG ScaffoldDermal Spheroid harvested from uncoated 3D PETG Scaffold

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