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Isolation, expansion and characterization of bone marrow-derived mesenchymal stromal cells in serum-free conditions

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Abstract

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) heralded a new beginning for regenerative medicine and generated tremendous interest as the most promising source for therapeutic application. Most cell therapies require stringent regulatory compliance and prefer the use of serum-free media (SFM) or xeno-free media (XFM) for the MSC production process, starting from the isolation onwards. Here, we report on serum-free isolation and expansion of MSCs and compare them with cells grown in conventional fetal bovine serum (FBS)-containing media as a control. The isolation, proliferation and morphology analysis demonstrated significant differences between MSCs cultured in various SFM/XFM in addition to their difference with FBS controls. BD Mosaic™ Mesenchymal Stem Cell Serum-Free media (BD-SFM) and Mesencult-XF (MSX) supported the isolation, sequential passaging, tri-lineage differentiation potential and acceptable surface marker expression profile of BM-MSCs. Further, MSCs cultured in SFM showed higher immune suppression and hypo-immunogenicity properties, making them an ideal candidate for allogeneic cell therapy. Although cells cultured in control media have a significantly higher proliferation rate, BM-MSCs cultured in BD-SFM or MSX media are the preferred choice to meet regulatory requirements as they do not contain bovine serum. While BM-MSCs cultured in BD-SFM and MSX media adhered to all MSC characteristics, in the case of few parameters, the performance of cells cultured in BD-SFM was superior to that of MSX media. Pre-clinical safety and efficiency studies are required before qualifying SFM or XFM media-derived MSCs for therapeutic applications.

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Acknowledgments

This work was fully funded by Stempeutics Research Pvt. Ltd, Manipal, India. The authors wish to thank Mr. Biju Uthup (BD Biosciences) for providing BD-SFM and Mr. Mithun Chandrashekar for helping in the experimental set-up.

Disclosures

The authors declare no competing financial interests.

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Authors and Affiliations

  1. Stempeutics Research Pvt. Ltd, Shirdi Sai Baba Cancer Hospital, Manipal, India

    Sanjay Gottipamula, K. M. Ashwin, Manjunatha S. Muttigi & Raviraja N. Seetharam

  2. Stempeutics Research Pvt. Ltd, Akshay Tech Park, No: 72 & 73, 2nd floor, EPIP Zone, Phase-I Area, Whitefield, Bangalore, India

    Suresh Kannan & Udaykumar Kolkundkar

Authors
  1. Sanjay Gottipamula
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  2. K. M. Ashwin
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  3. Manjunatha S. Muttigi
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  4. Suresh Kannan
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  5. Udaykumar Kolkundkar
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  6. Raviraja N. Seetharam
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Corresponding author

Correspondence to Raviraja N. Seetharam.

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Fig. S1

Cell size distribution analysis of BM-MSC of KO-FBS, BD-SFM and MSX. Viable cell distribution profile by flow cytometry and a cellular image based analyzer. a Average cell sizes analyzed by forward light scattering (FSC height), represented by the peak of each distribution, were compared, b Mean fluorescence intensity. c–e Forward and side scatter plot for BM-MSCs grown in KO-FBS, BD-SFM and MSX, respectively. f–h The size of BM-MSCs cultured in KO-FBS, BD-SFM and MSX respectively analyzed through Vi cell XR cell analyzer. (JPEG 170 kb)

Fig. S2

Phenotypic marker expression of BM-MSC of KO-FBS, BD-SFM and MSX. The marker profile, commonly referred to as characteristic of human BM-MSCs, was measured by flow cytometry. A total of 10,000 events were acquired using Guava Easycyte (Guava Technologies). Non-specific binding by PE and FITC-conjugated isotope matched control and results were analyzed using Guava software. (JPEG 102 kb)

Fig. S3

a, b Bar diagram of relative surface marker expression of negative markers and positive markers. SD ± SEM (n = 3). (JPEG 63 kb)

Fig. S4

MTT assay of BM-MSC cultured in KO-FBS, BD-SFM and MSX; Significant differences exist between KO-FBS with BD-SFM (n = 3; *p < 0.05) and KO-FBS with MSX (n = 3; *p < 0.05) at 25,000 cells per well. (JPEG 28 kb)

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Gottipamula, S., Ashwin, K.M., Muttigi, M.S. et al. Isolation, expansion and characterization of bone marrow-derived mesenchymal stromal cells in serum-free conditions. Cell Tissue Res 356, 123–135 (2014). https://doi.org/10.1007/s00441-013-1783-7

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  • DOI: https://doi.org/10.1007/s00441-013-1783-7

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