Formation of C2C12 myoblast spheroids by hanging drop method: role of initial cell density and culture time in myogenesis

Year 2025, Issue: 063, 14 - 23, 30.12.2025

Damla Çetin Altındal

https://doi.org/10.59313/jsr-a.1771330

Abstract

Spheroids offer significant advantages over two-dimensional (2D) cell cultures by providing a three-dimensional (3D) microenvironment that enhances cell-cell interactions and better mimics native architecture of the original tissue. C2C12 myoblasts, a well-established model for skeletal muscle regeneration, have been widely studied in 2D cultures, but systematic investigations into their spheroid formation and therapeutic potential for muscular injuries remain limited. In this study, hanging drop method was used for the preparation of C2C12 spheroids with different range of initial seeding densities (500–5000 cells per 30 µL drop). Compact, uniform spheroids formed at 2500 and 5000 cells per drop, with diameters around 100 µm as confirmed by optical microscopy and F-actin/DAPI staining. Immunostaining revealed that higher-density spheroids (5000 cells/drop) exhibited stronger and more localized expression of myogenic markers (MyoD and myogenin), indicating advanced differentiation compared to lower-density counterparts. The study results demonstrated that cell density and culture duration are important for the successful generation and myogenic differentiation of C2C12 spheroids. This spheroid model can be considered a potential cellular therapy platform for skeletal muscle regeneration studies.

Keywords

C2C12 myoblasts , spheroids , cellular therapy , skeletal muscle regeneration , tissue engineering

Project Number

Hacettepe University Scientific Researchers Project Units with Project Number FHD-2025-22010

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