Are 3D Scaffolds Better Than 2D Membranes?

Bio-Spun™ Scaffold Provides Novel, Animal-Free, Extracellular Matrix Environment


Today’s cell culture plates and vessels come in a variety of styles and sizes. The majority of cell culture plates used to grow human cells are made from borosilicate glass or clear plastic, typically polystyrene or polycarbonate. Using these Petri dishes, culture plates, and Transwell® devices, cells can spread out and grow on the flat 2D surface of the vessels. Producing novel, animal free, scaffold materials that more faithfully replicate natural 3D in vivo extracellular matrix compared to the 2D microporous membranes utilized in currently available Transwell® inserts products can be accomplished using BioSurfaces’s proprietary electrospinning technology.


In Vitro cell culture devices as described above have become indispensable tools in biological research, and have played a key role in uncovering numerous fundamental cellular and biological processes. However, in recent years, there has been a growing appreciation that 2D surfaces do not adequately support or reproduce the in vivo structure and functions that cells possess in their native 3D environment in the body. For example, 2D plastic and glass surfaces are excessively stiff compared to the typical in vivo environment, and do not promote the normal 3D interactions that cells maintain in the body (Figure 1). Cells cultured in such an environment often display abnormal behaviors, and are not amenable to conducting studies that require the cells to maintain their normal in vivo architecture and organ function.


The desire to reproduce the 3D form and function that cells have in the body has led to efforts to develop culture methods and devices that are able to recreate an in vivo-like 3D of environment. These include devices that utilize hanging drops, low adhesion plate surfaces and/or hydrogel matrices for creating 3D cell spheroids. An attractive option for reproducing an in vivo-like 3D environment is the creation of 3D scaffolds using the electrospinning techniques. Electrospinning is a method of electrostatically producing polymer nanofibers by applying a high voltage to a liquid polymer solution. Using collection surfaces of various shapes and sizes as molds, this technology can produce standalone materials and devices, or can be used to coat nanofibers onto existing materials and devices. Electrospun scaffolds can also be added to the wells of traditional tissue culture devices, or in place of 2D microporous membranes in Transwell® devices (Click Here for Bio-Spun Membranes on Plates).


Advantages of Electrospinning Scaffold Technology for Production of Complex 3D In Vitro Tissue Models


Electrospinning technology can produce novel, animal free, scaffold materials that more faithfully replicate natural 3D in vivo extracellular matrix compared to the 2D surfaces of cell culture plates or microporous membranes utilized in currently available Transwell® inserts products. Transwell® inserts products constructed with electrospun scaffolds in place of traditional film-based membranes will overcome limitations of traditional membranes as described above. These new products will allow development of advanced 3D organotypic tissue models that are more reproducible and stable, and that more faithfully reproduce in vivo structure, function and behavior.

Important advantages of electrospun scaffolds compared to film-based scaffolds currently utilized for tissue culture inserts include the following:

·The diameter of electrospun scaffold fibers is similar to natural extracellular matrix. Therefore, cells do not perceive the materials as foreign, and are less prone to mount inflammatory and/or fibrotic responses.

·Electrospun scaffolds are more similar to in vivo extracellular matrix in terms of stiffness and compliance properties that are important regulators of cell signaling and behavior.

·Electrospun scaffolds can be tuned to produce in vivo-like porosity to allow more natural nanoparticle/drug penetration and cell migration.

·Electrospun scaffolds can be made from a wide variety of materials that can be non-degradable or biodegradable.

·Electrospun scaffolds can be produced in a wide variety of thickness, and can be utilized as 3D scaffolds to produce stable stromal tissue components without the use of animal collagen.


Use of Electrospun Scaffolds to Produce Full-Thickness Human Models Such as Skin


Transwell® inserts containing Bio-Spun™ PET scaffolds were utilized to produce full-thickness skin models and were found to overcome the limitation mentioned above. Full-thickness skin models were prepared utilizing electrospun PET scaffolds with different thicknesses, ranging from 150 to 400 m. The scaffolds were seeded with normal human dermal fibroblasts (NHDF). The scaffolds were then cultured for 2 weeks to allow the NHDF to proliferate and secrete extracellular matrix, which self-assembled into a robust stromal matrix. Normal human epidermal keratinocytes (NHEK) cells were next seeded onto the stromal component, which was subsequently raised to the air-liquid interface to produce an organotypic skin model with stratified differentiated epidermis, a robust fibroblast populated dermal component and a well-developed basement membrane, which is often lacking in full-thickness in vitro skin models (Figure 2). This co-culture tissue model did not suffer from issues of contraction, and the stromal component was formed entirely from in-situ produced human extracellular matrix components without the need for exogenous animal-derived extracellular matrix.


Conclusion

These results demonstrate the remarkable potential of our Bio-Spun™ scaffold technology for use in production of 3D tissue models possessing advanced structural and functional capabilities. These unique scaffold materials and culture devices will find utility for production of a wide variety of additional 3D in vitro tissue models such as airway, intestine, ocular, renal, hepatic and others.