L-929 Cell Culture Guide

L-929 cells are among the earliest successfully established and widely used immortalized cell lines. They hold significant research value in various fields such as cell biology, virology, immunology, and toxicology. In this edition of the Cell Culture Academy, we provide a detailed overview of L-929 cells, including basic information, key considerations during cultivation, and their application in macrophage research, helping you quickly master cell culture techniques and successfully conduct experiments.

1. Cell Background

The full name of L-929 cells is NCTC clone 929. They were isolated in 1948 by WR. Earle and colleagues from the subcutaneous connective tissue of a 100-day-old male C3H/An mouse [1]. In its name, "L" indicates that the cells originated from loose connective tissue, and "929" is the cell line designation given at the time of establishment.

L-929 cells have a long history of use, and several derivative cell lines have been created over time. Commonly used derivatives include L-M[TK-] cells, A9 cells, L Wnt-3A cells, and L-WRN cells.

2. Basic Information

Aliases: NCTC-929L; L cell; Strain L-929; L929 (NCTC); Clone 929; Earle's L cells, etc.

Growth Characteristics: Fibroblast-like, adherent growth

Doubling Time: 28-36 hours

Cultivation Conditions:

•  Medium: MEM (with NEAA) + 10% FBS + 1% P/S

•  Atmosphere: Air, 95%; CO2, 5%

•  Temperature: 37°C

Medium Change Frequency: 2-3 times per week

Passage Ratio: 1:3 to 1:4

3. Cultivation Considerations

•  Serum Use: L-929 cells are typically cultured with 10% fetal bovine serum (FBS), but 10% horse serum can also meet their growth requirements. Under both serum conditions, L-929 cells do not show significant differences in morphology or growth rate.

•  Adaptability to Different Media: L-929 cells are highly adaptable and can grow in various basal media, such as commonly used DMEM, MEM, and RPMI-1640. However, when switching basal media, a period of adaptation and acclimatization is necessary to ensure proper cell growth.

•  Sensitivity to Serum Quality: L-929 cells are very sensitive to serum quality. When the serum is of poor quality, cell morphology and growth characteristics may change, such as becoming round (see Figure 1) or showing slower proliferation, or even stalling.

Figure 1. Normal growth morphology of L-929 cells (left) and changes in morphology when serum quality is poor (right).

4. Applications of L-929 Cells in Macrophage Research

L-929 cells are widely used not only in basic cell biology but also in immunology and inflammation research. L-929 cells secrete macrophage colony-stimulating factor (M-CSF), a key factor in inducing the differentiation of bone marrow monocytes into mature macrophages. In addition, L-929 cells secrete various cytokines, including MIF, CCL2, and CCL7, which work synergistically to promote macrophage differentiation and maturation [2]. Macrophages cultured with L-929 cell-conditioned medium generally exhibit superior biological characteristics compared to directly isolated mature macrophages [3], making L-929 cells an important tool in immunology and inflammation studies.

Preparation of L-929 Cell-Conditioned Medium

•  Seed L-929 cells into T75 flasks and culture for 7-10 days in complete L-929 medium.

•  Collect the culture supernatant, and remove cell debris by high-speed centrifugation or filtration.

•  Aliquot the supernatant and store at -20°C or -80°C.

•  When used, add the supernatant to complete medium containing 10% serum at a 20-30% ratio.

The extensive use of L-929 cells in cell biology and immunology research has made them an indispensable tool for researchers. Maintaining appropriate culture conditions and ensuring the stability of serum quality are critical for supporting healthy cell growth. Furthermore, L-929 cell-conditioned medium plays a vital role in macrophage research, providing valuable support for immunology experiments.

References

[1] Katherine K. Sanford, Wilton R. Earle, Gwendolyn D. Likely. The Growth in Vitro of Single Isolated Tissue Cells. Journal of the National Cancer Institute, 1948 Dec; 9 (3):229-246.

[2] Heap RE, Marín-Rubio JL, Peltier J, et al. Proteomics characterisation of the L929 cell supernatant and its role in BMDM differentiation [J]. Life Science Alliance, 2021 Apr 14; 4(6):e202000957.

[3] Wang W, Qin Y, Wang YR, et al. Characteristic comparison of mouse primary macrophages cultured in L929 cell conditioned medium [J]. Chinese Journal of Biotechnology, 2020, 36(7): 1431-1439.