Methanol fixed fibroblasts serve as feeder cells to maintain stem cells in the pluripotent state in vitro

Area of Science:

• Stem Cell Biology
• Cell Culture Technology

Background:

• Traditional methods for preparing mouse embryonic fibroblast (MEF) feeder cells for pluripotent stem cell (PSC) culture are time-consuming and raise ethical concerns.
• Existing feeder cell preparation methods often involve harsh treatments like mitomycin C or gamma irradiation.

Purpose of the Study:

• To develop a novel, efficient, time-saving, and cost-effective method for preparing feeder cells for PSC culture.
• To evaluate the efficacy of methanol-fixed MEF (MT-MEF) feeder cells in maintaining pluripotency and supporting long-term expansion of PSCs.
• To explore the applicability of MT-MEF for culturing PSCs from different species and for downstream applications like genetic manipulation.

Main Methods:

• Mouse embryonic fibroblasts (MEFs) were fixed with methanol for 5 minutes and air-dried for 5 minutes.
• The prepared methanol-fixed MEF (MT-MEF) cells were used directly for PSC culture or stored at room temperature.
• Pluripotent stem cells (PSCs) were cultured on MT-MEF, and their pluripotency and expansion capacity were assessed over multiple passages.
• The reusability of culture dishes and the application of MT-MEF in antibiotic resistance screening assays were investigated.
• Immortalized cell lines like NIH3T3 were also tested as methanol-fixed feeder cells.

Main Results:

• Methanol fixation provides an efficient, time-saving, and low-cost method for preparing feeder cells.
• PSCs cultured on MT-MEF maintained naïve pluripotency and were continuously expanded for over 40 passages.
• MT-MEF effectively supported the culture of human and pig induced PSCs (iPSCs).
• Culture dishes with MT-MEF were reusable up to 4 times and suitable for establishing stable transfected PSC lines.
• Methanol-fixed feeder cells offer a viable alternative to mitomycin C or gamma radiation-treated MEFs for maintaining PSCs from various species.

Conclusions:

• Methanol fixation is a superior method for preparing feeder cells, offering significant advantages in efficiency, time, and cost.
• This novel approach ensures stable long-term culture and maintenance of pluripotency in PSCs.
• The MT-MEF method is versatile, applicable to PSCs from different species and various downstream applications, presenting a significant advancement in stem cell culture technology.