Design and Production of a 37° C Heated Sample Container for Cell and Tissue Transport
Mark Finch, Miso Yang, Dr. Julia Lyubovitsky
Design Team A BIEN 175B SPRING 2010
We would like to extend our appreciation and acknowledge the following individuals.
Acknowledgements
Dr. Jerome Schultz
Dr. Julia Lyubovitsky
Dr. Hyle Park
Hong Xu
Prashanthi Vandrangi
Table of Contents
Abstract 3
Project Objectives3
Background 4
Prior Art Review6
Functional and Performance Specifications7
Block Diagram of Problem12
Evolution of the Final Design 12
Methods and Materials13
Description of Final Solution20
Method of Prototyping Discussion23
Performance Testing Protocol Discussion23
Performance Testing Results Discussion24
Financial Considerations25
Conclusions25
Future Work26
Statement of Societal Impact26
Abstract
A feasible design is required for a portable heated sample container that will be cost-efficient and effective. Heated sample containers are widely used in the study of animal cell and tissue. Heating the sample container and controlling the desired temperature of around 37 degrees C is important because the constant temperature can keep mammalian cell samples inside a BD Biosciences well plate comfortable in its environment and reduce dramatic temperature changes that can drastically affect the cells. Here, we build a 37 degree Celsius temperature controlled box powered by 12 Volts 5 amps yielding 60 Watts. It is found that using ten 110 Kohm resistive heaters coupled with a switching relay, LP339 comparator and 50K thermistor will provide adequate controlled heating to maintain 37 °C with an accuracy of half a degree inside each well. LoggerPro software was used to calculate real-time measurements of temperature inside the heated container as well as inside the well-plate simultaneously. Temperature inside the wells reach equilibrium in an average time of 42 minutes from when the device is powered on. Data shows a three degree higher difference in setpoint temperature inside the heated container than actual temperature inside the well plate. Here we present a method to maintain target temperature of 37 °C inside the wells with time plots depicting accurate representations of warm-up time for cells to reach equilibrium inside wells, recovery-time from opening lid of container, and heat distribution across each well.
Project Objectives
A 37 °C heated sample container is required for cell/tissue transportation to the characterization facilities and/or customer sites.Our goal is to design a portable 37 °C temperature controlled box fitting one standard BD Biosciences 24-well tissue culture plate with lid. The container must be completely portable so that researchers may be able to move samples station to station within a laboratory setting without the restriction of an electrical cord and power outlet. The device will have to be tested with a controller-based resistively heated container while incorporating a sold-state relay. The switching device must be capable of switching reliably at high speeds with maximum reduction of associated noise to electromechanical relays. In essence, the faster the switching device is the more efficient the system will be because the measured temperature of the container will not fluctuate as much. Thermocouples provide real-time feedback.
Background
Many cell and tissue applications require maintaining physiological body temperatures at approximately 37 degrees Celsius. Mammalian cell culturing involves keeping all cell incubation at a constant temperature of 37 degrees Celsius. At this temperature, mammalian cells tend to exhibit maximized cell growth which alludes to the fact that 37 degrees is an ideal temperature where physiological functions can be carried out. Since the mid 1900’s scientists have been culturing mammalian cells for laboratory experimentation. With recent technological innovations in cell biology and tissue engineering, there is growing need for a portable heated sample container capable of maintaining cell samples at a constant 37 degree C in laboratory applications. The well-being of suspended cells and/or tissue during transportation to the characterization facilities or customer sites rely heavily on maintaining a closed environment with constant temperature of37 degree C. Specifically, lipid bilayers change in response to environmental conditions, mainly temperature during growth and development. If environmental conditions are altered beyond normal limits, cell membranes undergo structural changes including phase separation of the membrane constituents and characteristic disturbances of function such as loss of selective permeability and transport processes. Cell membranes structures include proteins that are crucial for the transport of nutrients in and out of the cell. If these functions are disrupted, the cell will cease to function properly and exhibit necrosis. The changes in phase separation and membrane constituents are direct responses to temperature changes in the cells environment, however CO2 and pH are also dependent factors. Dissolved CO2 in the cell media affects the overall pH of the media. High concentrations of carbon dioxide dissolved in water form high concentrations of carbonic acid (CO2 + H2O H2CO3). Increasing CO2 also decreases the pH. Cell function operates in a narrow pH range thus it would need to be regulated. When mammalian cells are cultured in a standard BD Biosciences 24-well culture plate, temperature has the most immediate effect on physiological functions thus the device must be optimized for said parameter. Because cell media contains buffer solutions, pH regulation is negligible to reach our goals. Cell sensitivity requires maintaining a closed 37 °C heated container.
Certain commercial heating systems that might satisfy this goal are prohibitively expensive therefore construction of an affordable heated sample container for cells and tissue transportation is needed. Our ultimate goal is to construct a portable heated cell tissue sample container where the heated surface area of the container must maintain temperature of 37 degrees C and that of standard BD Biosciences 24-well tissue culture plate effectively for maximum cell sample survival. A BD Biosciences 24-well tissue culture plate is a common cell culturing plate used by researchers; however, other common sample dishes may be used. In sum, we wish to minimize the time mammalian cell samples are kept outside an enclosed 37 degrees C environment where the dramatic change in temperature could be hazardous to the well-being of the cells. The lids of such well plates must be able to fit within the container for suitable transportation of sample size.
Prior Art Review
Most laboratory incubation products on the market today are prohibitively expensive ranging in the thousands of dollars. They are bulky and largely overweight, making it impossible to carry by hand with only one person. Our method provides for a portable device that is capable of carrying by hand to different characterization sites or laboratories without the interference of wind, temperature changes in the air, or excess humidity that might drastically affect cell samples. Not only does the device solve the cost problem, but it also decreases the transportability limitation as well.
Functional and Performance Specifications
TECHNICAL SPECIFICATIONS
Temperature variation (time) / +/-
°C / 0,1
Temperature deviation (spatial) / +/- % / 1
Readability/ Setability / °C / N/A
Temperature range / °C / 38-64
Sensor 50KΩ RTD thermistor / Standard
Controller / PID
Indicator Display / RED LED
Safety / System
Temperature variation / +/-°C / 3
Sensor 50KΩ RTD thermistor / Standard
Adjustment / Manual (25KΩ Tunable Potentiometer)
Fan / System
Fan speed / Standard
Features / System
12 V Fan / +/- outputs
Optional External Heating Element
Input / DC V / 12
Heating up time to 37°C / min / 42
Power / System
Nominal power / W / 20
Nominal DC voltage / V / 12
Inlet Current / A / 16x 0.109
Dimensions / System
Exterior W,L,H / cm / 8.890,15.875,6.985
Interior W,L,H / cm / 325,260, 270
Volume working chamber / liter / 1
Aluminum chamber plate W, L, H / cm / 15.240,8.890
Weights / System
Net weight / kg / 0.5
All technical data subject to empty container and 22 °C ambient temperature.
Fig 3. A schematic of Heated Sample Container system depicting relay connections between each major circuit component.
The complete layout of the circuit controller shown in Figure 2 is placed at the base of the container. A 50 KΩ RTD Thermistor mounted to the controller board varies resistance with respect to temperature. With an increase in temperature, there is a decrease in resistance, allowing for lower voltages to pass through to the comparator. Once the comparator recognizes a decrease in Voltage, it sends a signal to the relay to switch Off. When the temperature inside the container decreases, the sensor resistance increases allowing for higher voltages to pass to the comparator. Once the comparator recognizes the increase in Voltage, a signal is sent to the relay to switch On. The temperature adjustment potentiometer sets the Voltage quantity of the comparator, programming the comparator to switch power to the 110 Ω resistive heaters On of Off relative to the voltage supplied. Thus, the temperature adjustment potentiometer dictates the setpoint temperature for the controller and the system will activate the heaters by supplying power until the average temperature inside the container reaches the set-point temperature.
Figure 1. Controller kit components with resistive heaters.
Fig 2. A controller circuit.
Fig 3. A complete sketch of the controller circuit.
Length (cm) / Width (cm) / Height (cm)Well plate(Lid) / 12.928 / 8.514 / 1.008
Well plate (Bottom) / 12.763 / 8.547 / 2.019
Aluminum / 15.240 / 8.890 / 3.810
Box(Neoprene) / 15.875 / 8.890 / 6.985
Table 1. Dimensions of interior materials.
Block Diagram of Project
Evolution of Final Design
Our initial design of the container consisted of a box with a rigid frame with insulation material. After biomaterial literary research and consultation with supervising professor, we were able to first fit a Styrofoam box with 1 liter volume and test with our heater and thermostat kit. We used expanded polystyrene as an insulating material first, in order to test the relative effectiveness of our incubation kit. Because of the durability and low thermal heat conductivity of the material, it served as a viable insulation material to run a preliminary test procedure of the kit.
The final design container consisted of a Coolsafe Deluxe box, mtmscientific thermostat and heater controller, and power supply. The Coolsafe design made of high polymer plastic and neoprene insulation provided enough insulation to maintain an accurate temperature range. Steel posts raised the BD Biosciences 24 well cell culture plate in the container so that the air inside the container would circulate around the well plate through convection. Since the entire controller and heating unit rests inside the Coolsafe box, the efficiency of the container is 100%, meaning all the energy put into the system is converted to heat energy or is lost somewhere else inside the system.
Methods and Materials
Our testing procedure consisted of Vernier thermocouple measuring instruments fed into a Vernier LabPro testing module. LoggerPro software was used to collect data samples in real time throughout the duration of all experiments. Thermocouples are factory calibrated and have an accuracy of 0.2 degree C. For our first experiment, we placed a thermocouples inside our heater device kit with Styrofoam insulation. Three thermocouples were situated inside the container to show relevant heat distribution within the container. We recorded temperature vs time for 20 minutes. Results showed a 9 minute warm-up time deadband with initial spike in temperature before heat cycling begins. Results can be seen below.
Fig 4. Initial heat cycling test run with expanded polystyrene container.
Four curves present on temperature plot. Three thermocouples placed inside the container measure temperature during heating cycles while a fourth probe measure mean ambient temperature outside the container in the lab. Orange colored curve represents mean ambient while Green, Red, and Blue measure temperature inside. Temperature 3, 1, and 2 are allocated closest to the heaters respectively with 3 inch spacing between them.
Fig 5. Expanded view of heat cycling from Figure 3.
When you take a closer look at the temperature curves, one can see cycles with mean close to 37 degrees however it may be slightly above that. Although the min and max peak of each cycle measures a 4 degree temperature differential, it does hold within this range therefore we have a working prototype.
The mean temperature of the air inside the expanded polystyrene container showed temperature distribution within the container. Results showed one degree overshoot in setpoint temperature. With temperature of the air inside the container exhibiting cycling effect, the next experiment is to measure temperature of solution inside the wellplate. With a mean temperature of 38 degree C, mean ambient temperature of the wells will eventually reach the same value. However, the time to reach equilibrium within the wells will vary depending on number of heaters, and position.
Figure 6: Preliminary temperature measurement of heating cycling of air inside container.
After we completed fabrication of the final design, real-time temperature measurements were taken inside the container. First, temperature of the air inside the container was recorded using the LoggerPro apparatus for 30 minutes before measurements of solution inside well plate were taken. Vernier LabPro temperature sensors with accuracy of 0.2 °C were used to record temperature measurements over time. The system showed warm-up time of 9.5 minutes (570 seconds) of air inside container before data enters heat cycling pattern. Thermostat controller was set at “High Setpoint” first where average temperature was 41.92 °C. After 18 minutes, lid of the container was opened for one minute to tune the temperature adjustment potentiometer. A decrease of 12.5 KΩ resistance of the potentiometer yielded a two degree drop in temperature. Once lid was closed, temperature of air inside the container recovered to heat cycling in less than 1 minute. The same procedure was done at 22 minutes yielding a momentary drop in temperature inside the container (down to 37 °C) however when closed, the temperature recovered in less than one minute. Low Setpoint shows heating cycling with an average temperature of 38.78 °C inside the Coolsafe Deluxe box.
Peak to peak temperature range of the heating cylces inside the container showed a maximum of two degrees. However, since the fluctuation of temperature is exhibited in the air, temperature of solution inside the well plate will remain steady with maintaining an accuracy of 0.5 °C.
Figure 7A. High Setpoint segment of heating cycling.
Figure 7B. Low Setpoint segment of heating cycling.
For our next experiment, we conducted real-time temperature measurements of solution inside the wells of the well-plate using the LoggerPro interface. For this we filled each well with 1.5 mL of water to simulate solution with which mammalian cells will rest in while inside the well-plate. Three temperature sensors were used in conjunction with the LoggerPro interface to record temperature. One sensor was used to record ambient room temperature outside the container while two sensors were to record temperature inside two different wells. Because the performance testing equipment was limited to using only three sensors, we were limited to measuring only two different wells while simultaneously measuring ambient temperature. Ideally, every well will need to be measured simultaneously to gain the most accurate heat distribution layout. Since only two sensors are used, one was placed in a well on the outermost row while the other remained in a well closest to the middle of the plate. For this experiment we will call the well on the outermost edge Well 1 and the middle well, Well 2. After recording temperature measurements for one hour in Wells 1, 2 and of the ambient, from an off position, it was found that Well 1 exhibited a higher equilibrium temperature than Well 2. After 40 minutes from switching the device On, temperature inside Well 1 reached equilibrium after 40 minutes at 35.20 °C while Well 2 reached equilibrium at 44 minutes with a temperature of 32.22 °C.
Figure 8A. Well 1 equilibrated temperature measurement.
Figure 8B. Well 2 equilibrated temperature measurement.
Final Design
Figure9: Coolsafe Deluxe system.
As mentioned previously, Coolsafe Deluxe Box exhibits adequate neoprene insulation and rigid formality to form the container for our heated device.
Figure 10: A. Side view of BD Biosciences 24-well cell culture plate on aluminum chamber plate. B. Top View.
An Aluminum plate with dimensions 15.240 cm x 8.890 cm x 0.016cm was machined to hold the well-plate. Because Aluminum is an excellent conductor, it will heat uniformly and distribute heat evenly across its surface to the well-plate. Two side pieces were fastened on the plate to prevent the well-plate from sliding off the heating platform while being carried inside the container. The metal is galvanized to prevent corrosion.
Figure 11: Completed Coolsafe Final Design with temperature control. A. Top View, lid open. B. Side View, lid closed. C. Side View, no insulation, lid closed. D. Back View.
Final design for the temperature controlled system included all components inside the Coolsafe Deluxe box. The Thermostat controller and heating element was situated at the base of the box. Four steel posts with 1 cm diameter and standing four cm tall, raised the Aluminum chamber plate platform which holds the BD Biosciences 24-Well Cell culture plate. 12V fan provides air circulation so heat distributes evenly around the well plate through convection. As shown in the image above, the device is turned On as indicated by the glowing red LED. When the LED is illuminated, DC power is supplied to the device and heaters are active and will remain active until the device is turned Off or 12V DC power is no longer being supplied to the device.
Method of Prototyping Discussion
The methods we used to prototype the final design was derived from literature searches and current products on the market. Each component to the final design of the product was legitimized through internet searches and consultation from Bioengineering professors. The thermodynamic properties of every material was examined and verified with other material databases found online or through literary search. With our two hundred dollar budget, we were limited to the thermostat controller and heating unit fabricated for this project.