Scalable Microgravity Simulator Used for Long-Term Musculoskeletal Cells and Tissue Engineering.

Alessandra Cazzaniga,Sara Castiglioni,Jeanette A Maier,Adrian Koller,Monica Zocchi,Marcel Egli,Simon Wuest,Carsten Haack,Fabian Ille,Christina Giger-Lange

doi:10.3390/ijms21238908

Abstract

We introduce a new benchtop microgravity simulator (MGS) that is scalable and easy to use. Its working principle is similar to that of random positioning machines (RPM), commonly used in research laboratories and regarded as one of the gold standards for simulating microgravity. The improvement of the MGS concerns mainly the algorithms controlling the movements of the samples and the design that, for the first time, guarantees equal treatment of all the culture flasks undergoing simulated microgravity. Qualification and validation tests of the new device were conducted with human bone marrow stem cells (bMSC) and mouse skeletal muscle myoblasts (C2C12). bMSC were cultured for 4 days on the MGS and the RPM in parallel. In the presence of osteogenic medium, an overexpression of osteogenic markers was detected in the samples from both devices. Similarly, C2C12 cells were maintained for 4 days on the MGS and the rotating wall vessel (RWV) device, another widely used microgravity simulator. Significant downregulation of myogenesis markers was observed in gravitationally unloaded cells. Therefore, similar results can be obtained regardless of the used simulated microgravity devices, namely MGS, RPM, or RWV. The newly developed MGS device thus offers easy and reliable long-term cell culture possibilities under simulated microgravity conditions. Currently, upgrades are in progress to allow real-time monitoring of the culture media and liquids exchange while running. This is of particular interest for long-term cultivation, needed for tissue engineering applications. Tissue grown under real or simulated microgravity has specific features, such as growth in three-dimensions (3D). Growth in weightlessness conditions fosters mechanical, structural, and chemical interactions between cells and the extracellular matrix in any direction.

Highlights

Used static two-dimensional (2D) cell culture techniques have contributed substantially to many breakthroughs in cell biology during the last decades
Simulated microgravity has been proposed as a challenging opportunity to grow cells as well as to generate organoids, spheroids, or tissues with and without scaffolds, which can be used for drug testing, tissue engineering, and regenerative medicine [2,3,4]
The rotating wall vessel (RWV), a suspension culture system that permits the cultivation of cells under low physiological fluid shear conditions, has been widely utilized [5,6]

Summary

Introduction

Used static two-dimensional (2D) cell culture techniques have contributed substantially to many breakthroughs in cell biology during the last decades. 3D dynamic bioreactors overcome diffusional limitations of static culture, improving the quality of different types of engineered tissues and optimizing cell proliferation and differentiation, e.g., for regenerative medicine. Some of these 3D bioreactors have been used to simulate microgravity. It is noteworthy that experiments performed in the RVW and RPM yield similar results and corroborate data reported in real microgravity [10,12] Both the RWV and the RPM have proven to be elegant tools for tissue engineering [13]. Our new MGS can be used for cell biological experiments under simulated microgravity conditions, which can be specified as a mechanically unloaded environment

Description of the MGS

C2C12 Myoblasts

Discussion

Real-Time PCR

Statistical Analysis