Date post: | 21-Dec-2015 |
Category: |
Documents |
View: | 218 times |
Download: | 3 times |
Development of a high-throughput bioreactor
system for biomedical applications
Mazzei Daniele Interdepartmental Research Center “E.Piaggio”
Tissue Engineering
Aim: Transplant Pharmacological Testing Artificial Organs
Basic steps• Biopsy• Cells extraction• Cell seeding• Cell Growing• Cell monolayer
High Throughput Screening (HTS)
HTS, is a method used in experimentation , such as drug testing
Is based on a brute-force approach to collect a large amount of experimental data in a short time and with few animals
HTS is achieved nowadays using multi-well equipment that contains cell cultures
?Does it work? The multi-well system presents a
principal problem, the environment discrepancy problem, that affects the relevance of tests
the environment discrepancy problem implies that the collected data are not directly usable in drug tests, because the cell monolayer grown in wells is only a brutal approximation of biological reality
This seems to be a paradox since the multi-well has been the core element of the HTS
Our IdeaTransfor
m a bioreactor
system in a HTS
machine
• Autonomous
• Safe• Usef
ull• Easy
to use
A bioreactor is a system able to maintain a cell culture in acontrolled environment, that mimics a living organism.
The Bioreactors A bioreactor is a more
realistic approximation of in-vivo Physiology.
Bioreactors have not been designed for HTS
We present a new bioreactor, called MCB, able to perform HTS experiment in a in-vivo-like simulated environment for long time
The MCB System
The MCB bioreactor system consists of the following parts:Cell culture chamberMixing chamberElectronic board and
electro-valve systemPeristaltic pump, PCHeating system
The mixing chamber The mixing chamber
allows regulation of pH and Oxygen flow
The medium is perfused with gas according to the measured pH
pH regulation is performed by inlet of two different gases : CO2 and Air
The Control System
The micro controlled board runs the μTNetOS operating system
The goal of this application is to realise an autonomous bioreactor by a real-time control of environmental variables
µTNetOS μTNetOS is a generated
operating system: the system generator takes as
input the description of an XML based protocol,
The system uses cooperative multi-tasking to run concurrent activities
μTNetOS is based on a robotics programming framework called Robotics4.NET
void TimerDoStep(Timer* t) { Heap *h = &t-> priorityQueue; while (h->pos && heap_Top(h) ->val <= GETTIME()) { HeapElement e = heap_Remove(h); int d = e.task->nextState(e.task); if (d) { e.val = GETTIME() + d; heap_Insert(h, e); } } }
Robotics4.Net and the Bioreactors
Bioreactors µTNetOS[Roblet]
PC [Brain
and bodymap]
The framework proposes a software architecture inspired to the architecture of the human nervous system.•The Brain: The core of control system•The Bodymap: A sort of black board used to send and receive messages•The Roblets: The appendix of the system, as the parts of our nervous system, read data from the environment and convert the Brain signal into an action
The High Throughput Bioeactor
Grazie a Robotics4.Net ogni bioreattore sarà visto come un sistema autonomo
Attraverso il PC sarà possbile controllare più bioreattori
Il controllo del sistema rimane però a bordo del bioreattore
Bioreactor
1
• Roblet: Bioreactor_1
Bioreactor 2
• Roblet: Bioreator_2
Bioreactor 3
• Roblet Bioreactor_3
A High Throughput bioreactor experiment, employs many bioreactors with different cell culture chambers
The network connections allows remote monitoring of experiments (through Internet)
A pathology can be simulated in one of the Bioreactor, and the others can be used as control
The user interface The Graphical User
Interface is developed in C#.Net
It is based on a multi tab structure
GUI is used to read data from the bioreactors and to setup the experimental variables
There are tools for sensor calibration
Manual control is possible
Preliminar Controll Results 48h parallel experiment with 4 bioreactorsThe data extracted from the bioreactors during
the experiment shows how the new system is able to correctly control the environmental variables
Preliminar Controll ResultsEndothelial cells (HUVECS) on a laminar flow culture chamber after 48h We can observe the
morphology of the cells, they do not show any type of cell membrane damage
We can use the cells as sensors of the environment, in the micrograph we can observe how the endothelial cells, are oriented with the medium flow and have an elliptical shape similar to their in-vivo morphology.
New Modular MCB Chamber Same dimension of Multiwell Modular connection in serie and
parallel configuration Easy to use Microfluid-dynamic modelled
New Modular MCB Chamber
New Modular MCB Chamber
1mm Inlet and Outlet
1mm inlet , 2mm outlet
Velocity reduced of 0,005 m/s !
New Modular MCB Chamber
How to control pHpH = -log10[H+]
H2O + H2O H3O+(aq) + OH-
(aq)
The culture medium is added with a BufferA buffer is a solution able to stabilized the pH of a liquid
Phosphate buffer H2CO3/HCO3 Is a water solution of Carbonic Acid
and sodium bicarbonate NaHCO3.
Key Points:•The Solubility of Oxygen in water is very Low •The Solubility of Carbon Dioxide in water is very High •Water + O2 pH increase slowly•Water + CO2 pH decrease very fast
How to control pHCO2 + H2O HCO3
- + H+ 4H+ + OH- + O2 2H2O + OH-
•High delay•Only few CO2 mole decrease pH•High risk for cell culture!•CO2 and O2 kinetich are very different
Impossible to use a simple On-Off Controll!Impossible to find a controll transfer function!
ASM Environment Simulator
Developed of an ASM Environment Simulator Simulate the fluid/gas environmentTesting the pH controll algorithm
Arti AhluwaliaGiovanni VozziFederico VozziBruna Vinci Daniele MazzeiFrancesca MontemurroCarmelo De MariaMariangela Guzzardi
Interdepartmental Research Center “E. Piaggio” Bio- Group
http://dionisio.ing.unipi.it