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Scaffold-Free v/s Scaffold-Based 3D Culture : Can AXTEX-4D™ Potentially Bridge the Gap?
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Types Of 3D Culture

3D Culture can be classified into two parts
i.) Scaffold-free (non-scaffold based) 3D cell culture which gathers all the methods where no exogenous artificial platforms are used for promoting cell growth, and
ii.) Scaffold-based 3D culture in which 3D cell cultures are obtained by promoting the cells growth on artificial 3D structures (artificial ECM).

This blog examines both of these 3D cultures, as well as their functions, advantages, and disadvantages. We will also discuss our innovative three-dimensional cell culture platform, AXTEX-4Dᵀᴹ.

Scaffold-free 3D Culture

Scaffold‐free or non-scaffold‐based 3D cell cultures gather all the methods where no exogenous artificial platforms are used for promoting cell growth. These methods
•Promote the spheroids formation by avoiding the cells adhesion to the surfaces and favoring the cell–cell interactions and cells self‐aggregation.
•Promote the formation of 3D microtissues as cellular aggregates known as spheroids or multicellular tumor spheroids, in which cells produce their own ECM i.e., no artificial ECM is provided.
Scaffold-free techniques are cell aggregate-based, such as hanging drops, ultra-low attachment plates, microfluidics, liquid overlay and magnetic biolevitation, and rely on physical forces to bring the cells together, and cell-cell adhesions to form the aggregate.


• No external biomaterials are required.
• ECM is produced by the cells.
• High number of cells–ECM interactions are established.
• High number of cells–cell interactions are established.
• Gradients of gases, nutrients, and pH are present.
• Spheroids can be formed without specific equipment and tools.
• The majority of the techniques are inexpensive, compatible with HTS.
• Mathematical models can be applied.


• Optimizations may be required for the formation of uniform spheroids.
• Spheroids may be destroyed during their analysis and manipulation.
• Only few protocols and assays are standardized.

Scaffold-based 3D Culture

In this 3D cell cultures are obtained by promoting the cells growth on artificial 3D structures (artificial ECM). The artificial 3D structures can be produced with bulk materials including i) ceramics, ii) glasses, iii) polymers, and iv) metals. Polymers are of 2 types:

1) Natural polymers such as silk, alginate, gelatin, matrigel, collagen, hyaluronic acid etc.
• Excellent biocompatibility
• Poor mechanical strength

2)Artificial polymers such as polystyrene (transparent hence suitable for imaging), polycaprolactone (biodegradable), Polytetrafluoroethylene (desirable mechanical and thermochemical stability but poor cell adhesiveness)
• Main challenges for in vivo biocompatibility is the toxic moieties and chemicals used in their polymerization


Physicochemical (pH, porosity, hydrophobicity, surface area) and biological properties (biocompatibility) are customizable.


• ECM is artificial.
• Gradient of gases, nutrients, and pH is not always reproduced.
• Specific equipment and tools may be required, such as 3D bioprinter.
• Some biomaterials can interfere with the therapeutics response.
• Low reproducibility.
• Techniques are laborious, expensive.
• Difficult to scale up, not compatible with HTS.
• Difficult to isolate and recover the cells from the structure for further assays.
• Investigated mainly for tissue engineering purposes, where they can be used as delivery vehicles for cells and drugs; should meet the needs of regeneration and repair.

Irrespective of excellent biocompatibility, the poor mechanical performance of the biological scaffolds is still a major limitation. Similarly, in case of synthetic scaffolds, one of the main challenges for in vivo biocompatibility is the toxic moieties and chemicals used in their polymerization. Hence, developing a robust 3D model for cell culture is still a challenge. In contrast, a disadvantage of the wide range of spheroid based tumor models is the lack of standard protocols, difficulties in measurement and microscopic analysis.


To this end, the cells were grown on our novel AXTEX-4D™ platform. Patterns like intercellular bridges were observed in cultured cells which mimic the real tissue morphology and helped in creating an ECM and thus emulated TME. Therefore, we called the system a “tissueoids cell culture system. The platform that consists of a chamber containing a non-woven fabric base matrix for receiving and supporting the growth of tissueoids. Nonwoven fabric is mechanically bonded together by entangling fibres resemble some of ECM characteristics and is therefore promising scaffolds. It should be noted that fibrous materials are attractive for biomedical applications owing to their structural superiorities, which include large surface-area-to-volume ratio, high porosity, and pore interconnectivity in a controlled manner.

The tissueoids cell culture system has several advantages over other 3D cell culture approaches, such as Matrigel coating and spheroid cultures, for the following reasons:
1) Cells grow as a tissueoids on non-woven fabric in a non-influential manner
2) The fabric of the device is not chemically treated, which provides an additional advantage of being non-toxic and biocompatible to the cells
3) Sterilization of the fabric can be achieved in a very simple and effective manner
4) Platform is compatible with standard biological assays such as flow cytometry, western blot, ELISA, sequencing, proteomics, confocal microscopy with proper z stack, labeling of cells etc.
5) high-throughput
6) Convenient for various cell culture formats (6-96 well) and cell numbers (25 cells to 10 5cells) with optimized porosity.

3D Tissueoids are formed on AXTEX-4Dᵀᴹ platform by using hanging drop methods. Thus, this platform fills the gap that exists in scaffold free and scaffold-based system.

By Reeshu Gupta
Lead-Content Generation

14 July 2021

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