One of the conclusions from chapter 3.6 stated that performing certain decellularisation steps at 37°C could be beneficial and therefore methods to warm the decellularisation solutions and bioreactor were explored. The early attempts at decellularisations were initially carried out in a 37°C, 5% CO2, humidified cell culture incubators to warm the decellularisation solutions and the internal and external chambers to 37°C (Figure 4.16 A). Any leaks from the bioreactor system required the cleaning and decontamination of the entire incubator, rendering it unuseable for several days. Peristaltic pumps are also not best suited to the heated and humidified environment and can fail as a result. Additionally, if the pump started
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to overheat then this had a subsequent effect of preventing the incubator from maintaining its set temperature. An ideal solution would be to have access to a non- humidified incubator/oven capable of maintaining 37oC to prevent corrosion of the internal components of the pump. Also to prevent the pump from overheating it would be preferable to position the pump outside of the 37°C incubator whilst fitting the media reservoir and bioreactor inside the incubator and running the tubing through an access port. However exclusive access to a non-humidified 37°C was not feasible and the tubing and access requirements to house the pump outside of the current incubator made doing so prohibitive. The alternative solution to achieve running the process at 37°C was to set up a water bath (Grant Instruments, Shepreth, UK) adjacent to the peristaltic pump for the solution reservoir to be heated to 37°C before the solutions were pumped through the bioreactor (Figure 4.16 B). The bioreactor, and the ancillary parts of the bioreactor, were also set up in a sink drainage area so any leaks could be easily contained and cleaned up (Figure 4.16 B). Because the decellularisation process was to be performed on the laboratory bench, it became essential to monitor the temperature of the internal chamber. Online temperature monitoring using temperature probes, (TC-02 Biorep, Miami, USA or PIT-6 Physitemp Instruments Inc, Clifton, USA) with tuohy borst adapters (Biorep, Miamo, USA (80409 Qosina, Edgewood, USA)) to allow connection to the bioreactor, were set up to monitor the temperature of the solutions pre-entry to and post-exit from the internal chamber to determine any temperature difference across the chamber (Figure 4.16 C and D). The temperature was displayed using either the Mon-a-therm 6510 (Mallinckrodt Medical, St. Louis, USA) (Figure 4.16 B), which was only capable of measuring the current temperature at the two sites, or the ORCA Bioreactor system (Harvard Apparatus, Holliston), which could measure and digitally record the temperatures.
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Figure 4.16: Early experiments were performed in a 37oC CO2 to maintain the experiment at 37oC (A). This potentially caused instrument failure of the incubator. Therefore the experiment was moved to a laboratory bench where a water bath was used to the heat the decellularisation solution (B). Temperature probes from Harvard Apparatus (C) and Biorep (D) were used to monitor the solution temperature in the internal chamber.
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As expected, the addition of a water bath to warm the decellularisation solutions allowed the bioreactor system to be positioned on a laboratory bench (Figure 4.16 B) and nullified the dependency on a 37°C incubator (Figure 4.16 A). Additionally, by setting the system up on a laboratory bench, it should reduce the incidence of pump or incubator failure. Integration of temperature probes positioned immediately upstream and immediately downstream of the internal bioreactor chamber allowed for monitoring of the solution temperature across the chamber. However, relying solely on a water bath to provide a heated environment did have its limitations. Most predominately there was a temperature drop of 9.31°C of the solution temperature from the solution reservoir in the water bath to the temperature probe positioned upstream of the internal chamber, due to the exposure of the tubing to a room temperature environment. In addition there was a temperature loss of 0.84°C detected between the upstream and downstream temperature probes. To compensate for this, the water bath was set to 42.9°C, however due to the extent of temperature loss and the desire to not overheat or potentially denature the enzymes in the decellularisation solutions, this resulted in an average internal chamber temperature of 33.35°C over the course of decellularisation process instead of the required temperature of 37°C (Figure 4.17). Employment of a pump that was suited to use in a 37°C incubator and performing the decellularisation in a 37°C incubator could have resolved this issue. Alternatively, sourcing a heat jacket with an integrated solution heating or cooling device with an online feedback mechanism to achieve the desired temperature of 37°C in the luminal and abluminal chambers, as commonly utilised in stirred tank bioreactor systems, would provide an ideal resolution to this issue. After use in several PTD experiments, the Biorep temperature probe (Figure 4.16 D) was deemed unsuitable for the pressurised transmural decellularisation process as the application of pressure caused the passage of the decellularisation solution inside the temperature probe casing, which would then leak out at T-type connector on the temperature probe. As this was a safety concern, this temperature probe was replaced by a solid metal temperature probe (Figure 4.16 C) which had no such issues.
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Figure 4.17: Readings from the ORCA controller from the temperature probes positioned pre and post the internal chamber (IC), or tracheal lumen, demonstrated the average pre-IC temperature was 33.59 ± 0.66°C and the average post-IC temperature was 33.10 ± 1.31°C with an average difference of 0.84°C between the two temperatures probes.