Staff-Student Exchange between MMIHS and Bournemouth University (UK)

We would like to dedicate this article to Dr. Anneyce Knight who died during the initial drafting of this article, Dr. Knight very sadly passed away from breast cancer in late 2021. The paper reports on a staff-student exchange grant between Nepal and the United Kingdom (UK), or to be more precise, between Manmohan Memorial Institute of Health Sciences (MMIHS) and Bournemouth University (BU). The grant was awarded by the British Council, National Agency for European Commission Erasmus+ Key Action 107 programme as part of their 2020 call for Learning Mobility of Individuals - International Credit Mobility. We were awarded the funds under the section ‘Higher education student and staff mobility between Programme and Partner Countries’

Automated cell cultivation on a well-plate

This thesis proposes a novel original system for cell cultivation of adherent cells. The system addresses the standardization demands posed by the future trends to move to difficult-to-culture cells, offers repeatable high-content screening testing, and ensures the compatibility of data for future data mining for systems biology purposes. The proposed system is based on a standardized platform the well plate, which allows for validation (for instance using fluorescence) with current systems and reduces market resistance towards novel platforms. The proposed system is analyzed firstly in the functional level and then in the component level. The key component is a functional lid that addresses each of the wells with individual stream of medium inflow and outflow. This enables individual environmental control in each of the wells. Due to predictable disturbances and experiment-specific demands, the system cannot ensure the environmental conditions in an open-loop. Therefore, a set of sensors has to be incorporated. There have been research efforts for sensor implementation directly into the wells. However, such a solution does not satisfy the standard well-plate requirement and more importantly, obstructs the observation area for microscopy/fluorescence studies. The author proposes a soft-sensing approach, where the sensors are placed into the microchannel network and thus, the well-plate remains fully untouched by the system. The main functions of the proposed system are heating, oxygenation and perfusion. Typical heating approaches include the hot air used by incubators or heating plates used during microscopy. Also here, the proposed system is inspired by nature, using the growth medium as the heat transport medium, which is a parallel to the blood circulation system. The oxygenation utilizes the superb permeability of poly(dimethylsiloxane), which is suggested as a suitable material for the functional lid. Two proposals of perfusion system approaches are suggested. The assessment of the proposed system brings up several challenging areas, which can be regarded as minor risks to the successful realization of the proposed cell cultivation system. They are solvable through optimization and re-design. However, there are also some major risks, which could lead to failure of the whole idea. These major risks include the suitability of the material, the heating demand versus the shear stress on cells and the temperature homogeneity within a well. The thesis focuses on these major risks and assesses them using literature surveys and wet-lab experiments. A test bench based on a standardized 12-well plate equipped with 9 temperature sensors in a single well was developed for testing purposes. It has been shown that in a 12-well plate system, the working volume has to be at least 1,5 ml per well in order to ensure homogeneity of the temperature distribution on the bottom of the well and to even out possible sudden temperature changes. From the hardware point of view, a dripping input (as opposed to an immersed input) has more predictable behavior and improves the temperature distribution and therefore, is the preferred choice for the proposed system. The shear stress at the cell level (10 μm from the bottom of the well) has been estimated using a finite-element method. As expected, the maximum shear stress occurs at the vicinity of the inlet. The shear stress decreases with increasing input diameter and with decreasing input flow-rate. Positioning of the input and output closer to the center of the well increases the shear stress. Therefore, input and output should be positioned at the edges of the well and for flow-rates above 0,4 ml/min, the input diameter should be larger than 400 μm, resulting in an average linear velocity of 0,05 m/s. A temperature soft-sensor has been developed based on a linear model. The linear model is well suited for the temperature measurements and can perform well even with one measurement down stream as long as there are no major disturbances. The suitability to other measurements, such as pH or O2, can not be directly suggested. The methodology used for the development of the temperature soft-sensor can, however, be used for the development of other soft-sensors. Poly(dimethylsiloxane) (PDMS) as the material of choice is assessed in terms of its processability as well as physical and chemical properties from the cell cultivation point of view. The critical chemical properties are further studied in wet-lab experiments. An experiment with fluorescent dye proves that native PDMS exhibits non-specific binding. The PDMS exhibited a negative weight change after immersion in the culturing medium. However, the high-performance liquid chromatography-size exclusion chromatography (HPLC-SEC) study has shown no foreign or missing components in the growth medium at given wavelengths. Given that the medium will be continuously supplied, the empty surface sites will be shortly saturated and further adsorption can take place only with compounds of stronger affinity to the surface. Despite these findings, the negative properties of PDMS are outweighed by its advantageous properties for cell cultivation. These include biocompatibility, non-toxicity, optical transparency, non-fluorescence, easy sterilization, and gas permeability. As a summary, the results of the performed analyses and experiments ensure the feasibility of the proposed system

Automated cell cultivation on a well-plate

This thesis proposes a novel original system for cell cultivation of adherent cells. The system addresses the standardization demands posed by the future trends to move to difficult-to-culture cells, offers repeatable high-content screening testing, and ensures the compatibility of data for future data mining for systems biology purposes. The proposed system is based on a standardized platform the well plate, which allows for validation (for instance using fluorescence) with current systems and reduces market resistance towards novel platforms. The proposed system is analyzed firstly in the functional level and then in the component level. The key component is a functional lid that addresses each of the wells with individual stream of medium inflow and outflow. This enables individual environmental control in each of the wells. Due to predictable disturbances and experiment-specific demands, the system cannot ensure the environmental conditions in an open-loop. Therefore, a set of sensors has to be incorporated. There have been research efforts for sensor implementation directly into the wells. However, such a solution does not satisfy the standard well-plate requirement and more importantly, obstructs the observation area for microscopy/fluorescence studies. The author proposes a soft-sensing approach, where the sensors are placed into the microchannel network and thus, the well-plate remains fully untouched by the system. The main functions of the proposed system are heating, oxygenation and perfusion. Typical heating approaches include the hot air used by incubators or heating plates used during microscopy. Also here, the proposed system is inspired by nature, using the growth medium as the heat transport medium, which is a parallel to the blood circulation system. The oxygenation utilizes the superb permeability of poly(dimethylsiloxane), which is suggested as a suitable material for the functional lid. Two proposals of perfusion system approaches are suggested. The assessment of the proposed system brings up several challenging areas, which can be regarded as minor risks to the successful realization of the proposed cell cultivation system. They are solvable through optimization and re-design. However, there are also some major risks, which could lead to failure of the whole idea. These major risks include the suitability of the material, the heating demand versus the shear stress on cells and the temperature homogeneity within a well. The thesis focuses on these major risks and assesses them using literature surveys and wet-lab experiments. A test bench based on a standardized 12-well plate equipped with 9 temperature sensors in a single well was developed for testing purposes. It has been shown that in a 12-well plate system, the working volume has to be at least 1,5 ml per well in order to ensure homogeneity of the temperature distribution on the bottom of the well and to even out possible sudden temperature changes. From the hardware point of view, a dripping input (as opposed to an immersed input) has more predictable behavior and improves the temperature distribution and therefore, is the preferred choice for the proposed system. The shear stress at the cell level (10 μm from the bottom of the well) has been estimated using a finite-element method. As expected, the maximum shear stress occurs at the vicinity of the inlet. The shear stress decreases with increasing input diameter and with decreasing input flow-rate. Positioning of the input and output closer to the center of the well increases the shear stress. Therefore, input and output should be positioned at the edges of the well and for flow-rates above 0,4 ml/min, the input diameter should be larger than 400 μm, resulting in an average linear velocity of 0,05 m/s. A temperature soft-sensor has been developed based on a linear model. The linear model is well suited for the temperature measurements and can perform well even with one measurement down stream as long as there are no major disturbances. The suitability to other measurements, such as pH or O2, can not be directly suggested. The methodology used for the development of the temperature soft-sensor can, however, be used for the development of other soft-sensors. Poly(dimethylsiloxane) (PDMS) as the material of choice is assessed in terms of its processability as well as physical and chemical properties from the cell cultivation point of view. The critical chemical properties are further studied in wet-lab experiments. An experiment with fluorescent dye proves that native PDMS exhibits non-specific binding. The PDMS exhibited a negative weight change after immersion in the culturing medium. However, the high-performance liquid chromatography-size exclusion chromatography (HPLC-SEC) study has shown no foreign or missing components in the growth medium at given wavelengths. Given that the medium will be continuously supplied, the empty surface sites will be shortly saturated and further adsorption can take place only with compounds of stronger affinity to the surface. Despite these findings, the negative properties of PDMS are outweighed by its advantageous properties for cell cultivation. These include biocompatibility, non-toxicity, optical transparency, non-fluorescence, easy sterilization, and gas permeability. As a summary, the results of the performed analyses and experiments ensure the feasibility of the proposed system

PDMS and it's suitability for analytical microfluidic devices

Poly(dimethylsiloxane) also known as PDMS is used in a wide range of biomedical applications. These range from implants through catheters to soft contact lenses. Therefore, it is understandable that PDMS has been extensively tested for these purposes. In past years, the microfluidics has moved from predominantly silicon and glass structures towards polymers due to their ease of manufacturing and moderate cost. PDMS has gained a lot of attention in various analytical applications. However, the testing of its suitability for such applications has not been as thorough as in the biomedical applications, perhaps relying on the experiments from that field. Microfluidic PDMS structures are more and more popular in various analytical devices. Such devices consume less reagents and can work with lower sample volumes. On the other hand, the surface-to-sample-volume ratio becomes larger. That increases the influence of material properties on the actual measurement. Some of the challenges include adsorption, diffusion, surface rougness, permeability and elasticity of PDMS, which are discussed in this paper.Peer reviewe

Capillary pressure microinjection of living adherent cells: challenges in automation

This paper is divided into two parts. The first part describes the current status and the general challenges of developing automatic microrobotics systems for microinjection of adherent mammalian cells. The discussion covers applications and the review and challenges of the components of a capillary pressure microinjection system: a micromanipulator, a microinjector, a microcapillary, a vision system and an environment control system. The second part of the paper describes the research performed on the automatic capillary pressure microinjection at the Tampere University of Technology. The advanced microinjection system includes two micromanipulators, a microinjector, a vision system and a control system. The control system comprises of motion control schemes for the micromanipulators to accurately position a microcapillary, to precisely penetrate a cell membrane and to deliver information on the injection to the operator. A novel injection guidance system being part of the control system comprises of an impedance measurement device and a user-interface which provide information on the detection of the capillarymembrane contact, capillary clogging and capillary breakage. Results show a remarkable increase of the injection success from 40 % to 65 % when the injection guidance system is used.Peer reviewe

Bioreporter pseudomonas fluorescens HK44 immobilized in a silica matrix

The bioluminescent bioreporter Pseudomonas fluorescens HK44, the whole cell bacterial biosensor that responds to naphthalene and its metabolites via the production of visible light, was immobilized into a silica matrix by the sol-gel technique. The bioluminescence intensities were measured in the maximum of the bioluminescence band at X = 500 nm. The immobilized cells (>105 cells per g silica matrix) produced light after induction by salicylate (cone. > 10 g/l), naphthalene and aminobenzoic acid. The bioluminescence intensities induced by 2,3-dihydroxynaphthalene 3-hydroxybenzoic acid and 4-hydroxybenzoic acid were comparable to a negative control. The cells in the silica layers on glass slides produced light in response to the presence of an inductor at least 8 months after immobilization, and >50 induction cycles. The results showed that these test slides could be used as assays for the multiple determination of water pollution