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Hydrodynamics considerations in cells systems from ocean flow to perfusion cultivation process
KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Industrial Biotechnology.
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Microorganisms and animal cells are grown surrounded by fluid, which is providing them with nutrients and removing their waste products. In nature and industry processes, cells/microbes can be subject to aggressive environments, such as turbulent flow or shear flow. Hydrodynamics force generated in these flows can affect the distribution of cells/microbes and even lead to cell damage. Understanding the mechanism and exploring the effect of hydrodynamic force in these environments could make the prediction of cells’ hydrodynamic response more systematic. In pharmaceutical industry, perfusion process is recognized as an attractive option for biologics production due to its high productivity. However, there are still some challenges and limitations for further process improvement due to lack of information of cell response to hydrodynamic force and nutrients. In both cases, hydrodynamics plays an important role and similar tool can be used to achieve a deeper understanding of these processes. This thesis is mainly aiming to elucidate the influence of hydrodynamic forces on microorganisms or cells in nature and during bioprocesses. In particular, shear stress in a natural environment and in a bioreactor operated in perfusion mode is studied.

This work mainly investigates hydrodynamics in nature and bioprocess including three flow cases. The first study investigates the effect of turbulence on marine life by performing direct numerical simulations (DNS) of motile micro-organisms in isotropic homogeneous turbulence. The clustering level of micro-organisms with one preferential swimming direction (e.g. gyrotaxis) is examined. The second study uses Computation Fluid Dynamics (CFD) to simulate the fluid flow inside a Wave bioreactor bag. The phenomenon of mixing, oxygen transfer rate and shear stress in nine different operating conditions of rocking speeds and angles are discussed. In the third study, the cellular response to shear force including growth and metabolism in a cell retention device such as hollow fiber filters during a perfusion process is analyzed. Theoretical calculations and experiment validation is performed to compare two filtration modes, tangential flow filtration (TFF) or alternating tangential flow filtration (ATF). Further optimizations regarding mixing and feeding are performed in a screening scale of in a perfusion system of stirred tank bioreactor with cell separation device.

The main findings can be summarized as that spherical gyrotaxis swimmers show significant clustering, whereas prolate swimmers remain more uniformly distributed due to their large sensitivity to the local shear. These results could explain how pure hydrodynamic effects can alter the ecology of micro-organisms for instance by varying shape and their preferential orientation (paper I). The simulations of Wave bioreactors show that the mixing and shear stress increase with the rocking angle but that increasing rocking speeds are not systematically associated with increasing mixing and shear stress. A resonance phenomenon is responsible for the fact that the lowest studied rocking speed generates the highest fluid velocity, mixing and shear stress (paper II). Theoretical velocity profile-based calculations suggested a lower shear stress for ATF by a factor 0.637 compared to TFF. This is experimentally validated by cultures of HEK (human embryonic kidney) 293 cells subjected to shear stress by a perfusion system that affects growth and metabolism using these cell separation devices (paper III). Thanks to optimization of mixing and oxygen transfer in a screening system for perfusion process, very high cell densities above 100 x 106 cells/mL of mammalian cells were achieved (paper IV).

Place, publisher, year, edition, pages
Stockholm, Sweden: KTH Royal Institute of Technology, 2019. , p. 57
Series
TRITA-CBH-FOU ; 35
Keywords [en]
Microorganism, cells, hydrodynamics, turbulence, numerical simulation, CFD, Resonance, Wave bioreactor, ATF, TFF, perfusion, design feeding strategy
National Category
Industrial Biotechnology
Research subject
Biotechnology
Identifiers
URN: urn:nbn:se:kth:diva-252023ISBN: 978-91-7873-233-3 (print)OAI: oai:DiVA.org:kth-252023DiVA, id: diva2:1317585
Public defence
2019-06-14, FA32, Albanova, Department of Mechanics, Royal Institute of Technology, Stockholm, 13:00 (English)
Opponent
Supervisors
Funder
AstraZenecaKnut and Alice Wallenberg Foundation
Note

QC 2019-05-23

Available from: 2019-05-23 Created: 2019-05-23 Last updated: 2019-05-23Bibliographically approved
List of papers
1. Accumulation of motile elongated micro-organisms in turbulence
Open this publication in new window or tab >>Accumulation of motile elongated micro-organisms in turbulence
2014 (English)In: Journal of Fluid Mechanics, ISSN 0022-1120, E-ISSN 1469-7645, Vol. 739, p. 22-36Article in journal (Refereed) Published
Abstract [en]

We study the effect of turbulence on marine life by performing numerical simulations of motile micro-organisms, modelled as prolate spheroids, in isotropic homogeneous turbulence. We show that the clustering and patchiness observed in laminar flows, linear shear and vortex flows, are significantly reduced in a three-dimensional turbulent flow mainly because of the complex topology; elongated micro-organisms show some level of clustering in the case of swimmers without any preferential alignment whereas spherical swimmers remain uniformly distributed. Micro-organisms with one preferential swimming direction (e.g. gyrotaxis) still show significant clustering if spherical in shape, whereas prolate swimmers remain more uniformly distributed. Due to their large sensitivity to the local shear, these elongated swimmers react more slowly to the action of vorticity and gravity and therefore do not have time to accumulate in a turbulent flow. These results show how purely hydrodynamic effects can alter the ecology of micro-organisms that can vary their shape and their preferential orientation.

Keywords
biological fluid dynamics, micro-organism dynamics
National Category
Engineering and Technology
Identifiers
urn:nbn:se:kth:diva-143737 (URN)10.1017/jfm.2013.608 (DOI)000331624200002 ()2-s2.0-84910642794 (Scopus ID)
Funder
Swedish Research CouncilSwedish e‐Science Research Center
Note

QC 20140327

Available from: 2014-03-27 Created: 2014-03-27 Last updated: 2019-05-23Bibliographically approved
2. Study of hydrodynamics in wave bioreactors by computational fluid dynamics reveals a resonance phenomenon
Open this publication in new window or tab >>Study of hydrodynamics in wave bioreactors by computational fluid dynamics reveals a resonance phenomenon
2019 (English)In: Chemical Engineering Science, ISSN 0009-2509, E-ISSN 1873-4405, Vol. 193, p. 53-65Article in journal (Refereed) Published
Abstract [en]

Culture of mammalian or human cells in Wave bioreactor is widely used for cell expansion or for biologics manufacturing. Wave bioreactor cultivation of sensitive cells such as stem cells, immune cells or anchorage-dependent cells, is recognized as an attractive option for culture in suspension or adherently on microcarriers. A systematic optimization of the mixing, oxygen transfer rate and shear stress, most favorable for the cells requires a deep understanding of the hydrodynamics inside the Wave bioreactor bag, i.e. cellbag. Numerical simulation by Computation Fluid Dynamics (CFD), is considered as an inexpensive and efficient tool for predicting the fluid behavior in many fields. In the present study, we perform numerical simulations by Ansys-FLUENT to characterize the flow conditions in a 10 L cellbag. The numerical simulations are carried out to investigate the fluid structures for nine different operating conditions of rocking speed and angle. The influence of these operating parameters on the mixing and the shear stress induced by the liquid motion are studied. We find that the mixing and shear stress increase with the cellbag angle from 4° to 7° but that increasing rocking speeds are not systematically associated with increasing mixing and shear stress. It is concluded that a resonance phenomenon is responsible for the fact that the lowest studied rocking speed, 15 rpm, generates the highest fluid velocity, mixing and shear stress compared to the higher speeds of 22 and 30 rpm.

Place, publisher, year, edition, pages
Elsevier, 2019
Keywords
Computation Fluid Dynamics (CFD), Hydrodynamic, Resonance, Volume of fluid (VOF), Wave bioreactor, Bioconversion, Bioreactors, Cytology, Hydrodynamics, Mammals, Mixing, Numerical models, Shear flow, Shear stress, Stem cells, Anchorage-dependent cells, Computation fluid dynamics, Different operating conditions, Operating parameters, Oxygen transfer rate, Systematic optimization, Volume of fluids, Wave bioreactors, Computational fluid dynamics
National Category
Mechanical Engineering
Identifiers
urn:nbn:se:kth:diva-236330 (URN)10.1016/j.ces.2018.08.017 (DOI)000447171800006 ()2-s2.0-85052913142 (Scopus ID)
Funder
VINNOVA, 2016-05181
Note

QC 20181120

Available from: 2018-11-20 Created: 2018-11-20 Last updated: 2019-05-23Bibliographically approved
3. Hydrodynamic shear stress in hollow filter for perfusion culture of human cells
Open this publication in new window or tab >>Hydrodynamic shear stress in hollow filter for perfusion culture of human cells
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

High cell density perfusion process is an economical way to produce biopharmaceuticals at high yield. To achieve high density of healthy cells, the cell culture conditions should be free from mechanically detriment. Human embryonic kidney (HEK) K293 cells, interesting for the production of therapeutic glycoproteins, are known as shear sensitive. In order to obtain the optimal hydrodynamics conditions with reduced mechanical damage, we investigated the fact of the shear stress compatible with HEK293 cells. We reviewed hollow filter based tangential flow filtration strategies, tangential flow filtration (TFF) and alternating tangential flow filtration (ATF). We studied shear stress introduced by these two flow filtration methods. By theoretical study, we obtained that lower shear stress introduced by alternating tangential flow filtration result in lower average shear stress comparing to tangential flow filtration with same flow rate.  In our experimental runs, we achieved different shear stress levels by applying different flow rates. 5-Days batch cultivations were performed to examine the influence of shear stress on cell growing and metabolic behaviour. We identified that the shear stress potentially reduce the growth rate and productivity of HEK293 cells and found the cell metabolism associated with shear stress levels. By documenting these cell responses to shear stress, we confirmed our theoretical results and could further optimize the hydrodynamic conditions for perfusion process of HEK 293 cells. 

Keywords
shear stress, perfusion, HEK293 cells, tangential flow filtration
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-252020 (URN)
Note

QC 20190523

Available from: 2019-05-23 Created: 2019-05-23 Last updated: 2019-05-27Bibliographically approved
4. Small-scale bioreactor supports high density HEK293 cell perfusion culture for the production of recombinant Erythropoietin
Open this publication in new window or tab >>Small-scale bioreactor supports high density HEK293 cell perfusion culture for the production of recombinant Erythropoietin
Show others...
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Process intensification in mammalian cell culture-based recombinant protein production has been achieved by high cell density perfusion exceeding 108 cells/mL in the recent years. As the majority of therapeutic proteins are produced in Chinese Hamster Ovary (CHO) cells, intensified perfusion processes have been mainly developed for this type of host cell line. However, the use of CHO cells can result in non-human posttranslational modifications of the protein of interest, which may be disadvantageous compared with human cell lines.

In this study, we developed a high cell density perfusion process of Human Embryonic Kidney (HEK293) cells producing recombinant human Erythropoietin (rhEPO). Firstly, a small-scale perfusion system from commercial bench-top screening bioreactors was developed for <250 mL working volume. Then, after the first trial runs with CHO cells, the system was modified for HEK293 cells (more sensitive than CHO cells) to achieve a higher oxygen transfer under mild aeration and agitation conditions. Steady states for medium (20 x 106 cells/mL) and high cell densities (80 x 106 cells/mL), normal process temperature (37 °C) and mild hypothermia (33 °C) as well as different cell specific perfusion rates (CSPR) from 10 to 60 pL/cell/day were applied to study the performance of the culture. The volumetric productivity was maximized for the high cell density steady state but decreased when an extremely low CSPR of 10 pL/cell/day was applied. The shift from high to low CSPR strongly reduced the nutrient uptake rates. The results from our study show that human cell lines, such as HEK293 can be used for intensified perfusion processes. 

Keywords
Chinese Hamster Ovary (CHO) cells, Erythropoietin, High cell density culture, Human Embryonic Kidney 239 (HEK293) cells, Perfusion process, Small-scale bioreactor
National Category
Industrial Biotechnology
Identifiers
urn:nbn:se:kth:diva-252022 (URN)
Note

QC 20190523

Available from: 2019-05-23 Created: 2019-05-23 Last updated: 2019-05-23Bibliographically approved

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