Yonsei Med J.  2006 Apr;47(2):179-183. 10.3349/ymj.2006.47.2.179.

A Comparative Study of Magnetic-Activated Cell Sorting, Cytotoxicity and Preplating for the Purification of Human Myoblasts

• Park YG
• Moon JH
• Kim J

• KMID: 1110739
• DOI: http://doi.org/10.3349/ymj.2006.47.2.179

Abstract

Although cultured myoblast transplantation has been extensively studied as a gene complementation approach to muscular dystrophy treatment, clinical success has still been limited. The inability to adequately isolate and purify myoblasts presents a major limitation to the production of sufficient myoblasts for engrafting purposes. This study attempted to purify myoblasts from primary culture by magnetic-activated cell sorting (MACS), complement-mediated cytotoxicity, and a preplating technique. As a result of positive myoblasts selection by MACS, the average percentage of myoblasts in mixed culture was increased from 30.0% to 41.7%. We observed both myoblast lysis and fibroblast lysis after complement-mediated cytotoxicity. Enrichment of myoblasts in mixed culture was found to increase to 83.1% by using the preplating technique. In addition, higher purification (92.8%) was achieved by following the preplating technique with MACS. Thus, preplating in combination with magnetic-activated cell sorting allows for a rapid and effective isolation of myoblasts from human muscle tissue.

Keyword

Muscular dystrophy; myoblast; magnetic-activated cell sorting; complement-mediated cytotoxicity; preplating

MeSH Terms

Time Factors
Myoblasts/*cytology
Muscle, Skeletal/*cytology
Models, Statistical
Magnetics
Immunomagnetic Separation/methods
Immunohistochemistry
Humans
Genetic Complementation Test
Fibroblasts/cytology
Complement System Proteins
Cells, Cultured
Cell Separation/*methods
Cell Differentiation

Figure

• 

  Fig. 1 Percentage increase of myoblasts prepared by MACS, complement-mediated cytotoxicity, preplating and combined preplating and MACS. Myoblast percentages were determined by immunochemical staining for desmin. All but complement-mediated cytotoxicity showed an increase in the myoblast fraction after treatment. Asterisks indicate statistical significance (p < 0.05).

• 

  Fig. 2 Characterization of the percentage of myoblasts obtained by preplating (pp1 to pp5). The myoblast populations displayed different desmin immunoreactivities ranging from the primary culture (21.3%) to the fifth preplate (pp5; 83.1%). The first preplate (pp1) contained only 29.9% desmin-positive cells, whereas the sequential preplates contained incrementally higher levels of desmin-positive cells.

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