Team:TecCEM/Hardware
Hardware
BoB is a novel gadget with the potential of replacing classic cell culture well plates as scientists’ choice for executing tests of their pharmaceutical products or model diseases on 2D/3D cell cultures. Its automated sterile system facilitates the doing and tracking of tests, which result on a standardized and more precise process that will permit us to improve the quality and predict the time of cell proliferation and morfological/physiological changes in situ. The material of which it is made of will provide the opportunity for it to be an ecofriendly solution as it can be depyrogenated and sterilized for it to be used several times. Finally, we propose our hardware's improvements to perform a complete metabolite analysis such as glucose, lactate, ATP, LDH or O2 by adding specific sensors. Also we plan to build a fully functional incubator which would allow us to take cell cultures anywhere and connect several BOBs to form a linear 2D/3D cell culture automated essay. Up next, we will present our hardware which is divided in two main parts; a small glass flask named BOB and the automation system.
BOB
As mentioned previously, BOB is a small tempered glass flask designed to test molecule’s effect on cell cultures. Due to the properties of tempered glass, it can be depyrogenated which will allow us to reuse this flask more than once. This present diverse advantages in which they are included to be eco friendly, to be cheaper and to be easier to wash.
In the front side there is a small hole which will allow us to introduce the new cell culture medium whenever the old one is ready to be changed. Likewise, there is a elbow at the bottom which will help us to get rid of the old medium. Connected to this elbow we have a 3-way stopcock which will open using a servo-motor. We will talk more about it in the Automation System section.
To help BoB fulfill its function, we use small 5mm septas (white pieces in Figure 1) which allow us to introduce the new cell culture medium inside it via a syringe and also prevent the medium from flowing outside BoB.
Inside BoB we can find a PDMS membrane, in which the cells will rest, that is suspended ⅓ the flasks height over the bottom. The reason of the ⅓ is because we need the culture to have certain air space for correct cells’ oxygen diffusion. It was reported that optimal level of the medium is 0.2 cm, that is the equivalent to a volume of 0.2 mL/cm2 [1]. The membranes recipe is shown in Figure 2.
To keep the PDMS membrane suspended, we use orthodontic archwire to supports the membrane from below. This archwire is then attached to BoB’s lid with the help of two holes and 2 septas as seen in Figure 1.
The third hole on Bob’s lid is used to introduce the cells, using a syringe, inside the flask; a septa is also used in this hole so that the entire system is closed.
To ensure the correct system’s behavior we created a combined base for Bob and the servo-motor that will open the 3-way stopcock. BoB’s part of the base has an inclination of 7.3 degrees to allow the medium to flow outside the flask, it also permits us to put a color sensor below the flask to measure the medium’s color. This part of the base also grabs the 3-way stopcock to prevent the servo from twisting it.
Automation System
For the correct use of the system, the input and output of the culture medium is required; these inputs and outputs are automatized to save time, reagents and preserve the sterile conditions of the environment thanks to the low interaction with humans. Using standardized times, volumes and parameters we implemented the automation of the system, resulting in a device that operates specifically for these objectives.
To make the automation system we used the following material:
- Arduino Nano
- Three Way Stopcock
- Three Piece Syringe
- Acrylic
- MG995 Servo Motor
- Stepper Motor
- Arduino Nano
- Endless Screw
- Axis
- Linear Bearings
- Cople
- 7805 5V Regulator
- NodeMCU WIFI Module
- Rubber Septas
The Arduino Nano is the main device, providing the system with enough processing power to take the decisions. This board is the one in charge of processing the color sensor input, controlling the stepper motor and the servo motor, and dispatching the data from the sensor to the Nodemcu board. A friendly diagram of the electronics involved is shown below.
The Nodemcu take input from the arduino and sends the data to a Pyhton’s Flask server hosted in Heroku. This data can then be visualized in an web application built in React JS-Redux. This application is further explained in the software section.
The pumping system is composed by 3 main elements. The first one is the bipolar stepper motor which provides enough torque, precision and is used to inject the medium. The second part is the mechanism itself, shown in the below figure, which is made out of the acrylic base, the syringe and the endless screw. The last element is the servo motor, utilized to control the output of the medium.
The sensing of the system is made with the help of a TCS3200 color sensor. This sensor returns an RGB code which will then be transformed into a single value using the next function.
The medium change sequence was programmed for it to be emitted when ever the sensor recognizes an orangish tone from the medium. When this happens, the arduino executes this sequence and the change occurs.
The sequence follows the next order:
- Open the control valve.
- Introduce cleaning medium (2ml).
- Close the outlet valve.
- Introduce 6.5ml of medium to fill the flask again.
In conclusion, all hardware built during this time works perfectly allowing us to fully automate the change and sensing of the medium while providing this data to a server. The advantages of this is enormous if applied correctly, in the below Improvement section we talk more about this topic and the future of this hardware.
Arduino's code
Github BoB AutomationVideo
Improvement
As part of the improvements, we will build a fully functional glass incubator. To replicate the same behavior of an already existing one, we will control temperature and CO2 flux and support an air filtration system. We will also integrate a microscope to watch the cell culture.
To control the temperature, we will design a close loop control system, using a NTC10KB3950K sensor to measure this variable as close to the culture as possible. In the same way, we will design a close loop control for the CO2 flux using a ASINPPOKAL25818 sensor to measure this variable.
The purpose of this incubator is to build a single interaction cell culture system that can be place anywhere in the lab. An image can be seen below showing a mock of the incubators size .
