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

Damla Çetin Altındal

doi:10.59313/jsr-a.1771330

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

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

Project Number

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

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Details

Primary Language

English

Subjects

Animal Cell Culture and Tissue Engineering

Journal Section

Research Article

Authors

Damla Çetin Altındal ^*
0000-0001-9437-0931
Türkiye

Publication Date

December 30, 2025

Submission Date

August 24, 2025

Acceptance Date

October 6, 2025

Published in Issue

Year 2025 Number: 063

DOI

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

IZ

https://izlik.org/JA54WS25SN

Cite

RIS / Bibtex

APA

Çetin Altındal, D. (2025). Formation of C2C12 myoblast spheroids by hanging drop method: role of initial cell density and culture time in myogenesis. Journal of Scientific Reports-A, 063, 14-23. https://doi.org/10.59313/jsr-a.1771330

AMA

1.Çetin Altındal D. Formation of C2C12 myoblast spheroids by hanging drop method: role of initial cell density and culture time in myogenesis. JSR-A. 2025;(063):14-23. doi:10.59313/jsr-a.1771330

Chicago

Çetin Altındal, Damla. 2025. “Formation of C2C12 Myoblast Spheroids by Hanging Drop Method: Role of Initial Cell Density and Culture Time in Myogenesis”. Journal of Scientific Reports-A, nos. 063: 14-23. https://doi.org/10.59313/jsr-a.1771330.

EndNote

Çetin Altındal D (December 1, 2025) Formation of C2C12 myoblast spheroids by hanging drop method: role of initial cell density and culture time in myogenesis. Journal of Scientific Reports-A 063 14–23.

IEEE

[1]D. Çetin Altındal, “Formation of C2C12 myoblast spheroids by hanging drop method: role of initial cell density and culture time in myogenesis”, JSR-A, no. 063, pp. 14–23, Dec. 2025, doi: 10.59313/jsr-a.1771330.

ISNAD

Çetin Altındal, Damla. “Formation of C2C12 Myoblast Spheroids by Hanging Drop Method: Role of Initial Cell Density and Culture Time in Myogenesis”. Journal of Scientific Reports-A. 063 (December 1, 2025): 14-23. https://doi.org/10.59313/jsr-a.1771330.

JAMA

1.Çetin Altındal D. Formation of C2C12 myoblast spheroids by hanging drop method: role of initial cell density and culture time in myogenesis. JSR-A. 2025;:14–23.

MLA

Çetin Altındal, Damla. “Formation of C2C12 Myoblast Spheroids by Hanging Drop Method: Role of Initial Cell Density and Culture Time in Myogenesis”. Journal of Scientific Reports-A, no. 063, Dec. 2025, pp. 14-23, doi:10.59313/jsr-a.1771330.

Vancouver

1.Damla Çetin Altındal. Formation of C2C12 myoblast spheroids by hanging drop method: role of initial cell density and culture time in myogenesis. JSR-A. 2025 Dec. 1;(063):14-23. doi:10.59313/jsr-a.1771330