Methodologies

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Mechano-Bioreactors

The design of the bioreactor was motivated by the idea to mimic the mechanical situation during bone healing in an established sheep osteotomy model by an in vitro system. The system allows the application of various compression and load patterns to 3D constructs mimicking the fracture hematoma or soft callus. This happens in a closed environment under dynamic cultivation (medium circulation) and control of cell culture parameters like oxygen and pH. As a special feature, the mechanical properties of cell seeded constructs can be measured in situ and monitored online during cultivation. This allows analyzing the impact of cellular behavior on the macroscopic mechanical properties. By implementing the gas exchange into the medium reservoir and by utilizing gas mixing devices, specific environments (e.g. hypoxia) can be realized. Additionally the individual bioreactors units can be run in any temperature controlled environment and outside of CO2 incubators.

Bioreactor System 1 (design: A. Petersen, running since 2011):

  • 8 independent units
  • Positioning accuracy 1µm
  • Stimulation mode: monoaxial compression
  • Stimulation forces up to 5N
  • Load sensitivity 1.5mN
  • Max sample size: 5mm height, 13mm diameter
  • optical port for online confocal microscopy

Bioreactor System 2 (design: A. Petersen, running since 2016):

  • 6 independent units
  • Positioning accuracy 10nm
  • Stimulation mode: monoaxial compression
  • Stimulation forces up to 100N
  • Load sensitivity 1.5mN
  • Max sample size: 5mm height, 13mm diameter

3D culture

The use of porous 3D biomaterials for studying cell behaviour features several challenges for the researcher. A key element is the application and optimization of widely used biochemical and cell biological techniques and protocols that have been established for analysis purposes in 2D.

We have developed and optimized several procedures that allow us to use the following methods in a porous 3D cell carrier:

  • Confocal microscopy (second harmonic generation, immunofluorescent microscopy, timelapse microscopy)
  • Gene expression analysis (RNA isolation, q-RT-PCR)
  • Protein analysis (SDS-PAGE/Western blotting, ELISA)
  • Proliferation analysis (Alamar Blue, CyQuant, KI67, BrdU)
  • Mechanical loading (see Bioreactor)

Live-cell imaging in 3D

Timelapse microscopy offers the opportunity to investigate dynamic cellular processes. In our setup a Leica TCS SP5 confocal / multiphoton laser scanning microscope is equipped with an incubator chamber providing a cell culture environment that allows long-term imaging over many days. We developed and optimized culture chambers, sample holders and cell visualization protocols for live-cell imaging in 3D to gain insights in cell migration, actin remodeling and matrix formation/remodeling in a physiologically relevant environment.