Scaffold-Free Technologies

Scaffold-free technologies are based on cell aggregation and do not need to add extra extracellular matrix proteins. Cells will produce endogenous extracellular matrix (ECM) proteins and continue to aggregate to form large aggregates. The formed aggregates are also called spheroids, and in particular multicellular spheroids (MCS), recapitulate physiological characteristics of tissues and tumors concerning cell-cell contact. The size and composition of the spheroids depend on the number of starting cells, incubation time and cell proliferation rate. Scaffold-free technologies rely on the self-aggregation of cells in specialized culture plates, such as hanging drop microplates, low adhesion plates with ultra-low attachment coating that promotes the spheroid formation and micropatterned plates that allow for microfluidic cell culture.

Figure 1．A model of the spheroid formation process (Shen, et al. 2021).

Scaffold-free technologies are especially suitable for the generation of multicellular spheres with specific size, cell number and composition, as well as the study of sphere assembly, tumor invasion, the interaction of two different cell types, and cell or molecular events in the process of stem cell embryoid angiogenesis induced by tumorspheres. CD BioSciences has a professional and experienced service support team in the field of 3D cell culture, which can provide you with high-quality scaffold-free culture service. If you have any needs, please feel free to contact us.

Comparison of Spheroid-Forming Techniques

Method	Advantages	Disadvantages
Nonadhesive surface	Inexpensive Simple to perform Easy to scale up	Variation in size/cell number/shape
Spinner flasks	Simple to perform Massive production Long-term culture Dynamic control of culture conditions Co-culture of different cell types	Require specialized equipment Variation in size/cell number High shear force
Hanging drop	Inexpensive Simple to perform Well-controlled spheroid size Fast spheroid formation Co-culture of different cell types Easy to trace spheroid assembly	Labor intensive Massive production difficult
Micromolding	Well-controlled spheroid size Designed aggregate geometry Co-culture of different cell types	Require specialized facilities
Pellet culture	Simple to perform Rapid aggregation of large number of cells	Shear force Massive production difficult
Monoclonal growth	Little work involved Some are useful morphogenesis models	Only occurs in certain cell types Long incubation periods Require extra procedures to harvest the MCS
External force enhancement	Rapid cell aggregation	Potentially undefined effects to cells Require specialized equipment and culture conditions

Application of Hanging Drop Culture

Toxicity testing in hepatocytes: Enhance the growth of hepatocytes, extend the specific functions of hepatocytes, and contribute to the current understanding of the biology and toxicity of hepatocytes similar to in vivo conditions.

Drug discovery: Screening cytotoxic and pharmaceutical compounds in vitro, the production of biologically active molecules in "bioreactors" and the construction of extracorporeal liver assist device.

Transplantation or implantation: Exploring alternative uses of human livers found to be unsuitable for transplantation following organ retrieval and more new possibilities in regenerative medicine for the use of donor human livers currently unsuitable for transplantation.

Engineering cardiac spheroids: The cardiac spheroids obtained through hanging drop culture mimicked important in vivo features of the human heart biochemically and pharmacologically offering a 3D cell culture model to study toxic effects in human heart tissue.

1. Lin, Ruei‐Zhen, and Hwan‐You Chang. "Recent advances in three‐dimensional multicellular spheroid culture for biomedical research." Biotechnology Journal: Healthcare Nutrition Technology 3.9‐10 (2008): 1172-1184.
2. Shri, Meena, et al. "Hanging drop, a best three-dimensional (3D) culture method for primary buffalo and sheep hepatocytes." Scientific reports 7.1 (2017): 1-14.
3. Shen, Honglin, et al. "Recent Advances in Three-Dimensional Multicellular Spheroid Culture and Future Development." Micromachines 12.1 (2021): 96.

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Please note: Our services can only be used for research purposes. Do not use in diagnostic or therapeutic procedures!