Bovine myoblast cell production in a microcarriers-based system

Abstract

For several tissue engineering applications, in particular food products, scaling up culture of mammalian cells is a necessary task. The prevailing method for large scale cell culture is the stirred tank bioreactor where anchor dependent cells are grown on microcarriers suspended in medium. We use a spinner flask system with cells grown on microcarriers to optimize the growth of bovine myoblasts. Freshly isolated primary cells were seeded on microcarriers (Synthemax^®, CellBIND^® and Cytodex^® 1 MCs). In this study, we provide proof of principle that bovine myoblasts can be cultured on microcarriers. No major differences were observed between the three tested microcarriers, except that sparsely populated beads were more common with CellBIND^® and Synthemax^® II beads suggesting a slower initiation of exponential growth than on Cytodex^®. We also provide direct evidence that bovine myoblasts display bead-to-bead transfer. A remarkable pick up of growth was observed by adding new MCs. Bovine myoblasts seem to behave like human mesenchymal stem cells. Thus, our results provide valuable data to further develop and scale-up the production of bovine myoblasts as a prerequisite for efficient and cost-effective development of cultured meat. Applicability to other anchorage dependent cells can extend the importance of these results to cell culture for medical tissue engineering or cell therapy.

Keywords: Bioreactor; Cell culture; Microcarriers; Myoblast.

Fig. 1

Myoblasts seeded on Cytodex^® 1, Synthemax^® II and CellBIND^® microcarriers using growth medium; seeded at a density of 1 × 10⁶ cells/ml. a The growth curve of cells for the three microcarriers. DNA (μg/ml) was measured and normalised to the value at day 1 (n = 3 for each day). b Photomicrographs of the cell-laden microcarriers at day 1 and day 6. The cells were stained with Hoechst and appear as fluorescent dots (white in B&W). c 2D proliferation of myoblasts. d Attachment of cells to the microcarriers at 24 h expressed as percentage of the total amount of cells added (n = 3 for each type of microcarrier). e The amount of cells per bead after 24 h (n = 3)

Fig. 2

Myoblasts seeded on Cytodex^® 1 microcarriers with different seeding densities. a Photomicrographs of Cytodex^® 1 microcarriers with different seeding densities, at day 1 and day 6. b The growth curve of myoblasts with different seeding densities (n = 3/density/time point). DNA (μg/ml) was measured and normalised to the value at day 1. On day 7, cell numbers were significantly higher for densities of ≥10⁶ cells/condition. c Percentage attached cells to the beads after 24 h. Asterisks indicate p < 0.05

Fig. 3

Bead-to-bead transfer of Myoblasts seeded on Synthemax^® II microcarriers. Synthemax^® II microcarriers were chosen for these experiments because they can be labeled with rhodamine. a The growth curve of the cells expressed as DNA concentration (μg/ml) where empty beads were added at day 3 or day 7. b Bead-to-bead transfer of myoblasts onto rhodamine (red) labeled Synthemax^® II beads. c Photomicrographs of the cells on beads at day 3, 7 and 8. Asterisk indicate significant difference for the growth when beads were added at day 7 compared to no extra beads added. (Color figure online)

Fig. 4

Myoblasts seeded on Cytodex^® 1 microcarriers, with new beads added on day 3 or day 7. a MyoD and Myogenin RNA expression by RT-QPCR. b The cells are trypsinized and then seeded on a plate in differentiation medium for 4 days to check myotube formation. Note the elongated structures that represent merged myoblasts. Scale bar = 1000 µm. “prol. Ctrl” is a 2D culture condition optimized for cell proliferation and “diff Ctrl” is optimized for myocyte differentiation. The arrows point to the myotubes