The Culture Force Monitor (CFM) is an apparatus whereby real-time force contraction of 3D cellular collagen constructs can be accurately measured. The original CFM is now well documented in the literature (Eastwood, et al., 1996) however this system used 5ml larger gels requiring greater numbers of cells.
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A new CFM system was set up using smaller gels in order to reduce the number of cells required and increase the cost effectiveness of the system.
2.7.1 Rig construction
A rig which could support all the required components of the CFM was constructed using a PTFE base and glass fibre poles (G.W. Cowler), cut to specification. The rig allowed for the positioning of the force transducer and opposing fixed point in a single plane, to accurately measure the force generated as a consequence of contraction of the gel. During experiments, the alignment of the force transducer and the opposing fixed point was achieved through the measurement of distances along the rig using digital calipers (Silverline, UK). This accurate measurement allowed for the controlled set up of the system between experiments, to minimise variation in the alignment and forces transduced.
2.7.2 The Force Transducer
The central measuring beam (Fig. 2-4) of the CFM was manufactured from a 0.15 mm thick Copper-Beryllium sheet (Goodfellow Metals Ltd, Cambridge, UK) cut into strips of 100 x 10 mm. Transducer class strain gauges (Wellyn Strain Measurement, Basingstoke, Hants, UK) were attached to the Copper-Beryllium beam in a full bridge electrical network at 20 mm from one end, to give a
maximum lever arm for maximum sensitivity. A hook was soft soldered onto the opposite end of the measuring beam to facilitate connection to the culture through the custom made stainless steel wire A-frame. The force transducer was attached through a wired mechanism to the Model P3 Strain Indicator and Recorder (Vishay, Basingstoke, UK) connected to a computer via USB. This unit acted as a bridge amplifier, static strain indicator, and digital data logger. Micro- strain (µƐ) and time data was collected at a rate of 1 Hz (one reading per second) for all measurements.
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2.7.2.1 Calibration of the Force Transducers
Before each experiment the force transducers need to be calibrated, in order to check the integrity of the force transducer and also to provide an equation to convert the micro-strain (µƐ) reading to micro-Newton’s (µN). Prior to calibration the force transducers were left in the incubator for a period of 24 hrs to
thermally adjust to the normal operational temperature, humidity and CO2
content.
The force transducers were calibrated against known masses which ranged between 0.5 g and 0.03 g (Table 2-1). Using the output software the correct channel was ‘zeroed’. Each mass was attached to the force transducer via the attachment hook and then data was recorded for a period of 10 mins. The collected data was averaged and this value was recorded as the output for the particular mass. This process was repeated for each of the masses on the force transducers. Each of the average readings in micro-strain were plotted against theoretical value of force that each of the known masses would produce, in accordance with acceleration due to gravity (Table 2-1).
Table 2-1. CFM Force Calibration
g kg Expected Force (N) Expected Force (μN) μƐ 0.5 0.0005 0.004905 4905 244.2433 0.3 0.0003 0.002943 2943 139.222 0.2 0.0002 0.001962 1962 92.79642 0.05 0.00005 0.000491 491 25.96563 0.03 0.00003 0.000294 294 24.09446
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Figure 2-4. Typical CFM Calibration Curve. This curve allows for the conversion
of recorded micro-strain (µƐ) to µN. It also allows for the assessment of the integrity of the strain gauge electrical bridge network on the transducer.
The r2 value represents the extent to which the two variables are correlated. If a value any less than 0.95 is observed, the force transducer should be returned to the manufacturer for testing, as the accuracy and/or sensitivity of the force transducer is no longer effective. If the r2 value in ≥ 0.95, the equation obtained from the trend line can be used for experimentation when converting the raw data in micro-strain to micro-Newton’s.
Equation 2-3 Conversion of data from micro-strain(µƐ)tomicro-Newton’s(µN)
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Figure 2-5. The Culture Force Monitor (CFM). A) Schematic of the CFM
illustrating the constructed rig to hold the force transducer (left) and fixed point (right). The dashed arrowed line between the force transducer and fixed point illustrates the requirement for alignment B) Image of the actual CFM set-up with a collagen construct tethered ready for experimentation. The alignment of the A- frames in all planes is of paramount importance to allow for the transfer of longitudinal force to be accurately detected by the force transducer.
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2.7.3 Tethering the Construct to the CFM
Stainless steel wire ‘A-frames’ attached to the flotation bars facilitated a
connection between the construct and the CFM. The force measurement system for the CFM is extremely sensitive, therefore when tethering the construct to both the fixed point and the measuring beam, it is important that the mechanics are correct. If not, the force output may not represent the contraction from the construct. The two A-frames must be aligned in both a superior view along with a lateral view when looking into the incubator.
2.7.4 CFM Data Analysis
The raw data collected at 1 Hz was processed in Microsoft Excel in order to obtain a mean reading for each minute of experimentation. This data was then converted to micro-Newton’s and plotted on a graph to represent the force-time contraction profile of the constructs (Fig. 3-1). Data was also analysed for initial rate of force development for each minute up to 60 min of contraction (µN/min). Data was also analysed for peak force and relative peak force according to cell seeding density. For compairsons in generated force between seeding densities or other conditions detailed in expiermental chapters, inferential statistical analysis was performed. Data was analysed for normal distrubtion and compliance to test assunmptions prior to presentation of data.
2.8 The Tensioning Culture Force Monitor (t-CFM)