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A mechanical non-enzymatic method for isolation of mouse embryonic fibroblasts

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Abstract

Mouse embryonic fibroblasts (MEFs) accessibility coupled with their simple generation make them as a typical embryonic cell model and feeder layer for in vitro expansion of pluripotent stem cells (PSCs). In this study, a mechanical isolation technique was adopted to isolate MEFs and the efficiency of this technique was compared with enzymatic digestion method. The suspended MEFs were prepared either by mechanical method or 0.25% trypsin enzymatic digestion. The effect of tissue processing on cell apoptosis/necrosis, morphology, viable cell yield, population doubling time, surface marker expression, and the capacity to support PSCs were determined. The mechanical method yielded a significantly higher number of viable cells. However, it showed similar morphology and proliferation characteristics as compared to enzymatic digestion. The mechanical method induced slight apoptosis in MEFs; however, it did not exert the necrotic effect of trypsinization. Treatment of tissue slurry with trypsin solution caused cell lysis and subsequently cell clump formation. Mechanically isolated cells exhibited a higher expression of the MEF surface antigens Sca1, CD106, and CD105. The PSCs on mechanically isolated MEFs displayed a higher expression of pluripotency genes, and formed more compact colonies with a stronger tendency to crowding compared with those cultured on cells isolated by enzymatic digestion. The mechanical method based on tissue inter-syringe processing is relatively a rapid and simple method for MEF isolation. Compared to the enzymatic digestion, the cells obtained from this method show higher expression of embryonic fibroblasts markers and a more functional capacity in supporting PSCs culture.

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Abbreviations

FACS:

Fluorescence-activated cell sorting

iPSCs:

Induced pluripotent stem cells

MEFs:

Mouse embryonic fibroblasts

MSCs:

Mesenchymal stem cells

PBS-CM:

PBS without Ca2+ and Mg2+

PDT:

Population doubling time

PSCs:

Pluripotent stem cells

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Acknowledgements

Portion of this study was presented in the form of a poster at the 3rd International Congress on Stem Cell and Regenerative Medicine held in Tehran November 2018. This work was part of the first author’s Ph.D. dissertation with MD and MT supervision.

Funding

This study was supported by Stem Cell Research Center (95/2–5/6), the Student Research Committee at Tabriz University of Medical Sciences (59924), and the National Council for Development of Stem Cell Sciences and Technologies (394).

Author information

Authors and Affiliations

  1. Stem Cell Research Center, Tabriz University of Medical Sciences, Tabriz, 5166615573, Iran

    Vahid Hosseini, Reza Rahbarghazi & Masoud Darabi

  2. Department of Biochemistry and Clinical Laboratories, Faculty of Medicine, Tabriz University of Medical Sciences, Tabriz, 5166615573, Iran

    Vahid Hosseini, Ashkan Kalantary-Charvadeh, Saeed Nazari Soltan Ahmad & Masoud Darabi

  3. Student Research Committee, Tabriz University of Medical Sciences, Tabriz, 5166615573, Iran

    Vahid Hosseini

  4. Institute for Integrated Cell-Material Sciences (iCeMS), Institute for Advanced Study, Kyoto University, Kyoto, 606-8501, Japan

    Kouichi Hasegawa

  5. Department of Applied Cell Sciences, Faculty of Advanced Medical Sciences, Tabriz University of Medical Sciences, Tabriz, 516615731, Iran

    Reza Rahbarghazi

  6. Endocrine Research Center, Tabriz University of Medical Sciences, Tabriz, 5166615573, Iran

    Amir Mahdizadeh

  7. Department of Genetics, Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, 1665659911, Iran

    Mehdi Totonchi

Authors
  1. Vahid Hosseini
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  2. Ashkan Kalantary-Charvadeh
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  5. Reza Rahbarghazi
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  6. Amir Mahdizadeh
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  7. Masoud Darabi
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Contributions

MD conceived and designed the experiments. VH and RR performed the animal surgical procedures. VH performed cell isolation, cell culture and molecular analyses, and wrote the manuscript draft. AM, AKC and SNSA performed extractions and blotting. MD, MT, RR, AKC and KH reviewed the manuscript. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Masoud Darabi or Mehdi Totonchi.

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The authors declare that they have no competing interest

Ethical approval

All procedures performed in studies involving animals were in accordance with the ethical standards of the institution at which the experiments were conducted and ethical approval was obtained from Tabriz University of Medical Sciences ethics committee (IR.TBZMED.REC.1395.680).

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Hosseini, V., Kalantary-Charvadeh, A., Hasegawa, K. et al. A mechanical non-enzymatic method for isolation of mouse embryonic fibroblasts. Mol Biol Rep 47, 8881–8890 (2020). https://doi.org/10.1007/s11033-020-05940-3

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