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WO2008028241A1 - Microbioréacteur - Google Patents

Microbioréacteur Download PDF

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Publication number
WO2008028241A1
WO2008028241A1 PCT/AU2007/001317 AU2007001317W WO2008028241A1 WO 2008028241 A1 WO2008028241 A1 WO 2008028241A1 AU 2007001317 W AU2007001317 W AU 2007001317W WO 2008028241 A1 WO2008028241 A1 WO 2008028241A1
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WO
WIPO (PCT)
Prior art keywords
cell
layer
chamber
cells
transport layer
Prior art date
Application number
PCT/AU2007/001317
Other languages
English (en)
Inventor
Michael Robert Doran
Justin John Cooper-White
Original Assignee
The University Of Queensland
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from AU2006904891A external-priority patent/AU2006904891A0/en
Application filed by The University Of Queensland filed Critical The University Of Queensland
Publication of WO2008028241A1 publication Critical patent/WO2008028241A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/14—Scaffolds; Matrices
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/12—Well or multiwell plates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/46—Means for regulation, monitoring, measurement or control, e.g. flow regulation of cellular or enzymatic activity or functionality, e.g. cell viability

Definitions

  • This invention relates to a device for controlling the microenvironment around one or more cells and/or tissue and a method of assessing the effects of parameters in a microenvironment on the cells and/or tissue.
  • Tissue engineering offers the prospect of replacing missing or non functional body parts and organs with newly created tissue.
  • the strategy most often used to generate these new tissues is the seeding and culture of antilogous cells within highly engineered biocompatible bioabsorbable polymeric scaffolds.
  • the scaffolds are designed with the aims of mimicking the microenvironment created by the extracellular matrix which encases all cells in normal tissue as well as maintaining the required space for supporting new tissue growth.
  • Tissue engineering involves four clearly defined steps:
  • the engineered scaffold is resorbed (biosorbable) or degraded (biodegradable) in situ, leaving only healthy, functional generated tissue.
  • Steps (i) and (ii) are aimed at creating the appropriate microenvironment for optimum cell physiology and steps (iii) and (iv) then provide the appropriate microenvironment to support cell viability, phenotype and tissue cultivation.
  • factors such as the cell- surface and cell-cell interactions control cell attachment, migration, proliferation, differentiation and functionality of newly formed cells within the 3D spaces of a scaffold.
  • the effects of the physical characteristics of the scaffold such as pore size, pore size distribution, pore connectivity and tortuosity on cell regeneration and repair are expected to not only be cell specific and culture systems specific but also dependent on surface characteristics such as the topography, biochemical interaction and mobility of the scaffold.
  • an integrated systematic approach is needed which allows a number of these variables to be modified whilst maintaining others invariant allowing true assessment of the variables of interest.
  • US patent application publication no. 2003/0186217 discloses a method and device for growing and/or treating cells.
  • a cell culture plate is disclosed as having cells located at the base of a cell culture chamber and in one embodiment O 2 , air and nutrients to the cell culture chamber are supplied through membranes in the top and bottom of the chamber by pressure applied to those membranes.
  • This reference is not capable of controlling the microenvironment around a cell and quantifying the cellular response to the microenvironment nor would it be possible to assess scaffold design variables in such a device.
  • a device for assessing cellular response including a first transport layer and a second transport layer for carrying, supplying or removing a flow of at least one cell metabolite, a cell housing layer having at least one cell chamber for cultivating cells, the cell housing layer enabling the at least one cell metabolite to transfer from the first transport layer and/or second transport layer to at least the cell chamber, and means to measure at least one of the physical process parameters of the fluid flowing through the at least the first transport layer.
  • the cell chamber of the cell housing layer may be separated from the first transport and second transport layer by a first and second membrane respectively permeable to the at least one metabolite.
  • cell metabolites is intended to include molecules which are essential to cell homeostasis, survival and growth as well as cell type determinants. Such metabolites include oxygen/air and nutrient medium, growth factors, mitogens, cytokines and chemokines.
  • the term 'transfer is intended to cover transport mechanisms such as diffusion and/or convection.
  • the physical process parameters of the fluid are preferably at least one variable selected from the group including pressure, temperature, oxygen concentration, carbon dioxide concentration and pH. All of the above variables may be measurable in real time.
  • the above apparatus may further include a means to control the measured variable or variables preferably in real time.
  • the first transport layer includes a fluid passage contacting the membrane between the chamber of the cell housing layer and the first transport layer.
  • the second transport layer includes a second fluid passage contacting the second membrane between the cell chamber of the cell housing layer and the second transport layer.
  • the cell housing layer may further include at least one access channel communicating with the cell chamber.
  • the at least one channel may be capable of further providing a flow of cell metabolites to the cell within the cell chamber.
  • the access channel may further provide a means by which waste products from the cell can be removed from the cell chamber and cell metabolites generally not capable of passing through the permeable membranes are supplied to the cell chamber. Additionally the access channel may be used for seeding of one or more cells into the cell chamber.
  • the height of the cell chamber is sufficient to not physically interfere with the cell but sufficiently thin to allow predictable determination of the conditions around the cells in the cell chamber.
  • the interior of the chamber of the housing layer comprises a 2 dimensional support surface or a 3 dimensional scaffold for supporting cells. With this construction, any flow within cell chamber and in particular in proximity to the cells may be kept in the laminar region.
  • the surface or scaffold is located within the laminar flow regions of the cell chamber. It is anticipated that the height of the cell chamber would be in the range of 10 ⁇ m to 1000 ⁇ m (preferably less than 200 ⁇ m and most preferably less than 100 ⁇ m) in order to fulfil this requirement. In most circumstances, the cells on the surface or scaffold need to be no greater than 200 ⁇ m from the source of oxygen (membrane) to enable oxygen to diffuse to the cell through the culture at a sufficient rate to ensure survival of the cell.
  • the device is a micro fluidic device in which each of the layers of the device are produced from layers of materials commonly used in such reactor construction including without limitation silicon, glass, metal, polymer using known manufacturing techniques for producing micro fluidic devices.
  • the cell housing layer includes a plurality of cell chambers.
  • the cell chambers are preferably circular to maximise the cell chamber volume relative to its surface area, thereby enabling rapid equilibration of cell metabolite(s) within the cell chamber.
  • the cell chambers are preferably arranged in a two dimensional array, with the cell chambers aligned across the cell housing layer and parallel to the first transport layer and/or second transport layer. Once a metabolite gradient has been established over the cell housing layer, each column of cell chambers which are parallel to the first transport layer and/or second transport layer will have the same metabolite concentration.
  • a metabolite concentration gradient is established across the row of cell chambers which span the cell housing layer, thereby, enabling different cell chamber environments (eg. scaffold architecture or culture medium) to be evaluated against varying metabolite levels. Further, as each chamber cell will have a relatively small metabolite concentration gradient, the migration of cells in response to metabolite concentration changes (eg. cytokines, such as vascular endothelial growth factor) may be conveniently assessed.
  • metabolite concentration changes eg. cytokines, such as vascular endothelial growth factor
  • the device may further include a probe able to assess the metabolite concentration across the cell housing layer.
  • the probe includes a spectrometer with a fibre optic component which may extend across the cell housing layer.
  • the probe may be used to assess the metabolite concentration across each column of cell chambers, parallel to the first transport layer. It may also be maintained above the cell chambers to monitor, detect and record any disturbances in metabolite concentration over time.
  • the cell housing layer is preferably contiguous with the cell chambers.
  • the transport of the metabolite(s) across a fluid permeable solid cell housing layer extending across the cell housing layer enables a ⁇ /ery steep, yet stable metabolite concentration gradient to be established thereover.
  • a micro fluidic device for cultivating cells including:
  • a first transport layer for carrying, supplying or removing a flow of fluid containing at least one cell metabolite and a second transport layer for carrying, supplying or removing a flow of fluid containing at least one cell metabolite separated by a cell housing layer, the cell housing layer having at least one cell chamber for cultivating cells, the cell housing layer being permeable to the at least one cell metabolite required by the cell; and means to measure the physical process parameters of the fluid flowing through the transport layer wherein the dimension across the cell chamber is in the range of 10 ⁇ m to 1000 ⁇ m, preferably less than 200 ⁇ m.
  • the cell housing layer may further include at least one channel communicating with the cell chamber.
  • the at least one access channel is preferably capable of further providing a flow of cell metabolites to the cell within the cell chamber.
  • the access channel may further be used to seed one or more cells into the cell chamber.
  • This aspect of the invention is provided with a second transport layer adjacent the cell housing layer, the second transport layer and the cell chamber being separated by a membrane (cell housing layer) permeable to the at least one cell metabolite within a flow passage within the second transport layer.
  • the monitored physical process parameters of the fluid flowing in the first and second transport layer are variables selected from the group including pressure, temperature, pH, oxygen concentration and carbon dioxide concentration and other process variables useful for controlling conditions within the cell housing layer. All of the above variables are measurable in real time.
  • the above apparatus may further include a means to control the measured variables preferably in real time.
  • the flux of cell metabolites into the cell chamber side of the membrane can be determined. Furthermore, due to the physical dimensions of the cell chamber and the positioning of the cells in the cell chamber, flow of fluid in the cell chamber is substantially laminar enabling a more predictable and precise determination of flux of cell metabolites from the membrane surface to the surface of the cells.
  • process parameters within the transport passage the conditions or process variables within the cell microenvironment can be individually controlled. Alternatively process parameters such as temperature can be monitored and/or controlled by one or more heating elements positioned in the transport layer or layers, the cell housing layer or within the permeable membrane.
  • the cell chamber may include an elongated channel.
  • the elongated channel preferably includes a plurality of parallel elongated sections (aligned parallel to the cell housing layer) which travel across the cell housing layer. These elongated channel sections align to specific metabolite concentrations and thus enables the optimum metabolite concentration to be determined over a relatively short distance.
  • the width of the channels is preferably between 10 ⁇ m to 100 ⁇ m and more preferably between 20 ⁇ m and 40 ⁇ m.
  • the distance between the parallel elongated sections is preferably 10 ⁇ m to 100 ⁇ m and more preferably between 20 ⁇ m and 40 ⁇ m.
  • the elongated channel may further include a plurality of parallel elongated sections (aligned perpendicular to the cell housing layer) which travel across the cell housing layer. These elongated channel sections may enable cells to migrate towards an metabolite source, such as oxygen if the cells are capable of aerotaxis.
  • the width of the channels is preferably between 10 ⁇ m to 100 ⁇ m and more preferably between 20 ⁇ m and 40 ⁇ m.
  • the distance between the parallel elongated sections is preferably 10 ⁇ m to 100 ⁇ m and more preferably between 20 ⁇ m and 40 ⁇ m.
  • a method of controlling process variables within a cell chamber including the steps of providing a device having a permeable cell housing layer between at least one side of the cell chamber, and a flow passage of cell metabolites, providing a flow of at least one cell metabolite through the flow passage to permeate through the permeable cell housing layer into the cell chamber, monitoring the process variables within the flow of cell metabolites in the flow passage, and/or the cell housing layer and determining the process variables within the cell chamber based on the known characteristics of the permeable cell housing layer and the mass transfer gradients of the nutrients established across the cell housing layer.
  • the preferred form of the invention requires at least the two sides of the cell housing layer to comprise permeable membranes to separate the cell chambers from the flow passages of nutrients .
  • the above aspect of the invention further provides the step of locating one or more cells or a tissue culture on a surface or scaffold positioned within the one or more cell chambers.
  • the above method of controlling the conditions within the cell chamber enables the conditions at the surface of the cell to be controlled.
  • the process parameters are preferably monitored in real time and the measured variable controlled by feed back control preferably in real time
  • a method of assessing the effect of surface conditions on the growth of one or more cells including the steps of introducing or creating in a cell chamber a microenvironment having predetermined environmental conditions at, or in proximity to the surface of one or more cells and monitoring and assessing the effect of the surface condition on the growth of such cells.
  • the predetermined micro-environmental conditions being assessed may be parameters including the physical characteristics of the surface such as surface chemistry and topography.
  • the method may further provide for recreating the known environmental conditions in a cell chamber, varying at least one of the environmental conditions in a known manner to monitor and assess the effect of the varying environmental conditions on one or more cells.
  • the above method may further comprise the step of monitoring the effect of the cell on the condition being monitored.
  • a method of assessing a scaffold including the steps of introducing a scaffold into the cell chamber of a device for cultivating cells, establishing a predetermined microenvironment with the cell chamber, seeding one or more cells onto the scaffold and monitoring and assessing the effect the scaffold and microenvironment have on the behaviour of the one or more cells.
  • the features of the scaffold being assessed may be parameters including the physical characteristics of the scaffold such as the nature of the scaffold and properties of the scaffold material, the pore size, pore distribution, pore connectivity and tortuosity.
  • the method according to this aspect of the invention may further include the step of varying at least one of the microenvironment conditions in a known manner to monitor and access the effect of varying the environmental condition on the one or more cells.
  • Figures 1(a)-1(g) are plan views of the components of an embodiment of the invention with Figures 1(b) and 1(d),
  • Figure 2 is a sectional view of the embodiment of Figures 1 (a)-1 (g),
  • Figure 3 is a second embodiment of the invention
  • Figure 4 is a sectional view of a third embodiment of the invention showing the control circuit layout
  • Figure 5 is a sectional view of the embodiment of Figure 4,
  • Figure 6 is a fourth embodiment a cell housing layer for incorporation into the invention.
  • Figures 7 and 8 are further alternative embodiments of fluid transport layers for incorporation into the invention
  • Figures 9, 11-15 are plan views of alternative designs of surface and scaffold for incorporation into the cell chamber in the apparatus of the invention
  • Figure 10 is a plan view of a grid sensor pattern for use with the cell chamber in the apparatus of the invention
  • Figure 16 is a plan view of an optional mask which may be applied to the membrane separating the cell housing layer from the first and/or second transport layer/s,
  • FIGS 17, 18 and 19 illustrate possible micro heater configurations which could be used in the present invention
  • Figure 20 is a schematic diagram of a device of the present invention.
  • Figure 21 is a perspective view of the device of Figure 20
  • Figure 22 is a graph of the oxygen concentration gradient over the cell housing layer of the device of Figure 21 under the conditions described in Example 1 ,
  • Figure 23 is a graph of the Fibroblast 3T3 fold expansion under discreet oxygen concentrations as described in Example 1 .
  • Figure 24 is a schematic diagram of a microbioreactor of the present invention constructed from an integral piece of PDMS material
  • Figure 25 is a schematic diagram of the microreactor of Figure 24 further including a glass plate and polycarbonate shell,
  • Figure 26 is a graph of the oxygen concentration gradient across the cell housing layer for the three by three well array of Figure 25,
  • Figure 27 is a graph of the oxygen concentration gradient across the cell housing layer for the two by three well array of Figure 25
  • Figure 28 is a graph of the estimated oxygen profile through a microbioreactor of Figure 25 maintained in an anaerobic atmosphere
  • Figure 29 is a graph of the cell expansion data for Experiment 1 (Example 2)
  • Figure 30 is a schematic diagram of an embodiment of the invention used in Experiment 3.
  • Figure 31 is a schematic diagram of an embodiment of the invention used in Experiment 4.
  • Figure 32 is a plan view of serpentine channels with imposed oxygen gradient of a microfluidic microbioreactor of the present invention.
  • Figure 33 is a schematic diagram of the cell housing layer with a serpentine cell chamber as shown in figure 32;
  • Figure 34 is a side view of a microbioreactor similar to Figure 32;.
  • Figure 35 is a photograph showing cell expansion in a serpentine cell chamber between and oxygen gradient of 0% to 20%vol.
  • Figure 36 shows the layers of the device used in this experiment 5.
  • the microbioreactor 1 includes a cell housing layer 2 between a first transport layer 3 and preferably a second transport layer 4.
  • a membrane 7 permeable to at least one cell metabolite is provided between the cell housing layer 2 and the first transport layer 3 and when present a membrane 6 is present between the cells housing layer 2 and the second transport layer 4.
  • the membrane material is selected and produced at a thickness to allow these metabolites to permeate from the transport layers 3, 4 into the cell housing layer 2.
  • the transport layers and the membranes have similar light transmission properties in the wavelengths at which analysis is to take place.
  • the transport layer 3, 4 and membrane should be transparent.
  • a suitable membrane material for such an analytical technique is PDMS.
  • the transport layers and membranes should allow transmission of the relevant wavelengths.
  • the cell housing layer 2 further includes a cell chamber 5 within which cells introduced via a well or port within the cell chamber (which may contain either a surface or a scaffold) are able to grow.
  • the height of the cell chamber may be altered to closely mimic the in vivo microenvironment and/or enable the control and/or monitoring of the conditions.
  • the cell housing layer may also be provided with an auxiliary passage 8 for the delivery of cell metabolites to the cell and may be used for removal of waste products from the cell. This auxiliary passage is not intended to provide the majority of the cell metabolites to the cell and while laminar conditions are generally maintained through the cell chamber, turbulent conditions can be induced if required.
  • the cell chamber 5 is shown as having a functional surface 15.
  • the surface 15 may be hyaluronic acid/chitsan layer which has been added or layered on the interior of the cell chamber.
  • the functional surface provides a surface upon which the cells may be seeded and/or cultivated on thus separating them from interacting with the membrane.
  • the functional layer may also serve as a secondary membrane offering increased molecular selectivity over the membrane bordering the cell housing layer 2.
  • the functional surface layer is preferably formed of multiple oriented layers up to 20 ⁇ m thick. For example, a hyaluronic acid/acid layer of 20 ⁇ m would consist of approximately 130 layers.
  • the layered functional surface is made up of alternating polycationic substance and polyanionic substance layers.
  • the first transport layer 3 is provided with a passage 9 through which fluid is able to pass.
  • the passage is preferably open adjacent to the cell chamber and separated from the cell chamber by the permeable membrane. In this way, mass transfer gradients established between the first transport layer 3, second transport layer 4 and the cell chamber 5 enable material to transfer from the transport layer or layers to the cell chamber 5. Furthermore, the flux of material transferring across the permeable membrane can be accurately determined.
  • the passage 9 through the first transport layer 3 and corresponding passage 10 in the second transport layer 4 is provided with inlets 11 , 13 and outlets 12, 14 respectively, to enable the flow of fluid containing cell metabolites through the passages 9, 10.
  • the positioning of the inlet and outlet into the fluid passages 9 and 10 will depend on the proximity of other equipment to the apparatus.
  • a plurality of microbioreactors of the present invention may be stacked into a multi reactor column.
  • the respective inlets 11 1 , 13 1 and outlets 12 1 , 14 1 are in through the side of the transport layers as shown in Figure 3.
  • a number of microbioreactors may be placed side by side, an inlet 11 , 13" and outlet 12, 14" arrangement shown in Figure 5 could be used optional with multiple reactors 20, 21 with inlets 22 and outlets 23 on a single cell housing layer 24 (Figure 6).
  • the flux of material transported from the transport layers across the membrane interface to the growth cell layer can be reliably predicted and determined. Due to the laminar flow conditions within the cell chamber, the transfer of these materials from the membrane interface to the surface of the cell is a linear distance related relationship. Therefore the concentration of cell metabolites at the cell surface can be reliably predicted, determined and included within associated process control methodologies. Other variables such as temperature may be more accurately measured directly in order to define the environmental state within the cell.
  • the process variables in the first transport layer are monitored to establish the nature of the mass transfer gradients from the transport layers 3, 4 to the cell chamber 5. These variables may be selected from the group of variables including pressure within the fluid transport passage 9, 10, the pH, oxygen and carbon dioxide concentration of the fluid as well as the concentration of other cell metabolites entering and leaving the entrance 11 , 13 and exit 12, 14 ports of the transport layers 3, 4. Temperature may also be measured in the transport layer although as mentioned above, it is preferable to measure temperature of the cell housing layer directly. These conditions are monitored during operation of the device of the invention.
  • any fluid entering within, and leaving the auxiliary passage is monitored to enable a mass balance to be established around the entire vessel and thereby provide information on the cell consumption of cell metabolites and also the effect in which predetermined changes in the microenvironment have on the cells within the cell housing layer.
  • These monitoring systems further enable levels of material entering the system to be set or varied in a known manner over a predetermined period of time. Once these material levels have been varied in a known manner, the effect of these and other variables on the cells and the response of the cell can be monitored.
  • a more complex integrated measurement and control apparatus as shown in Figure 4 can be used.
  • process parameters such as O 2 , CO 2 , glucose, temperature etc
  • concentration within the cell chamber can be directly measured with micro fluidic optic fibre sensors using fluorescent lead technology at multiple sensor locations.
  • the sensors may be arranged as a grid of sensors ( Figure 10) providing local measurements on a range of process variables over the surface of the cell chamber or membrane.
  • the microenvironment within cell chambers can be controlled directly through a central environmental controller controlling the flow of fluid media into the transport layer. For convenience only one master controller 20 is shown controlling the flow of media through inlet 13".
  • a slave controller feeds information back to a master controller which compares the readings against set points for the variables being measured. Variations from the set points results in changes to the flow rates through the inlet. Changes in the flow rate into inlet 13 11 are then fed back to the slave controller which resets the set points until the next measurement cycle.
  • Figures 17, 18 and 19 show possible micro heater configurations which could be used in the present invention.
  • the micro heater 51 is located in the cell housing layer 50 surrounding the cell chamber.
  • a two chambered layer is shown.
  • micro heater 53 is located in the membrane layer above and/or below the cell chamber and in Figure 19, micro heaters 54, 55 are positioned in the transport layers 56 and 57 respectively.
  • a temperature sensor or sensors may be incorporated into the control apparatus.
  • the feedback from the control sensor may be used to directly control heating elements located in close proximity to or embedded in the transport layer or layers, membrane or cell chamber layer. This enables localised heating to be provided to the cell chamber.
  • the heating elements may be any available micro heater design.
  • the effect of variations in environmental conditions on the cells can be determined for different cell types. Furthermore the microenvironment around the cell can be reliably reproduced.
  • cells are introduced via a port (not shown) on to a surface or into a well positioned within the cell chamber.
  • the conditions for cultivation in the cell are set by a combination of establishing the required variables in the fluid flowing through the transport layers and the auxiliary passage of the cell housing layer as well as other surface related conditions on which the cells exist. Any one of a number of conditions may then be varied in a known manner to monitor the effect of the variable change on the condition of the cells.
  • a method of determining the effect of one or more microenvironmental conditions on the cell can be performed.
  • the cell chamber includes highly programmable pores emanating from the central or off centre where cells are introduced. Properties or conditions of the pores are known and their effect on the cell can be observed. As the other microenvironmental conditions of the cell are controlled and known, the effect of changes to the properties or conditions of the pores is directly observable.
  • the properties or conditions of pores which are variable and hence can be found to have an observable effect are the size, length, shape, tortuosity, surface biochemistry and topology and pore interconnectivity.
  • the pore sizes and characteristics may be representative of structures found in nature or those in manufactured rigid and hydrogel based scaffolds.
  • the characteristics and features of surface biochemistry include the inclusion of growth factors, growth factor complexes, peptidomimetics, oligopeptides, protein segments, proteins (glycoproteins etc), glycosamines (e.g. GAG, HA), carbohydrates or mixtures thereof in constant concentrations or concentration gradients on the surface via various surface treatment techniques such as wet chemistry, block copolymer templating, plasma templating, and plasma polymerisation. These surface treatment techniques may be applied to create surface structures with controlled functionality and topology at defined locations throughout the highly defined pores.
  • a number of variations and configurations of the cell chamber can be established within a single microbioreactor in order to observe the actual effect of selected surfaces and/or scaffold materials on the cells.
  • a radial disc Figure 9 of selected surfaces and/or scaffold materials 31 around the disc with the cells initially centrally located can be used to observe the preference of certain types of cell behaviour to different surface or scaffold characteristics.
  • the differing selected surfaces can be arranged as sectors of a radial disc or may be arranged as concentric layers and these differing selected surfaces may incorporate different growth factor complexes and/or extracellular matrix molecules with specific distributions at specific locations.
  • the cells would be inoculated into the centre of the scaffold but they will be distributed at specific locations throughout the scaffold to ascertain the most effective inoculation technique for functional tissue growth.
  • different scaffold architectures can be designed to provide the effect of different conditions of surface chemistry and pore size distribution and tortuosity ( Figures 11 , 12, 13, 15).
  • the cell chamber can be filled with the scaffold ( Figure 14) and then seeded with cells. The growth of the cell and the effect of various parameters as the cell creates pores in the hydrogel can be monitored and the suitability of such a scaffold assessed.
  • Another method of providing or designing microenvironments within the cell chamber is to use baffles, tortuous pathways or structures within the first and second transport layers.
  • An example of two possible arrangements is shown in Figures 7 and 8 although a variety of arrangements could be conceived depending on the microenvironment and concentrating gradients to be constructed within the cell chamber.
  • the concentration of cell metabolites closest to the inlet 33 would be at its greatest.
  • a greater lateral concentration gradient of cell metabolites in the cell chamber can be created.
  • the cells in the cell chamber 5 are dosed with a digestive proteinase such as trypsin through the inlet access port of the cell chamber to detach the cells from the scaffold. This allows the cells to be removed through the access passage or other port designed in the cell housing layer for that purpose.
  • a digestive proteinase such as trypsin
  • an optional masking layer 40 adjacent or on the membrane layer is provided.
  • the masking layer effectively allows for the passage of cell metabolites through a predetermined region of the membrane.
  • An example of such a masking layer 40 is shown in figure 16 where only a thin annulus 41 of membrane is available for the transfer of cell metabolites into the cell chamber.
  • the masking layer 40 may be applied directly to the membrane layer 40 or provided on a separate membrane layer having the same or different permeability properties to the first membrane layer. It would be appreciated that the masking layer could be shaped and sized so that only a point source is available for the transfer of cell metabolites into the cell chamber.
  • the microbioreactor of the present invention may be manufactured using microfabrication technology which is know to those in the art and preferably would be sterilisable, optically clear and include sensors for environmental monitoring and hence control of the process variables.
  • the microbioreactor of the present invention can be readily assembled and disassembled, for post hoc histological tissue processing. Any surface or scaffold material can be assessed in the cell chamber of the present invention as described above.
  • microbioreactors of the present invention allow for systematic investigations into the effects of surface and scaffold structure and function on cell culture and tissue growth and allow for the development of reliable wet control manufacturing techniques for programmed microbioreactors.
  • This example relates to a device (microbioreactor) of the present invention designed to provide a tool to study the response of mammalian cells to various oxygen concentrations in otherwise identical culture conditions, including maintenance of constant CO 2 concentrations.
  • the microbioreactor is:
  • the device generates and maintains an oxygen gradient over an array of discreet tissue culture micro wells.
  • An oxygen gradient is maintained in the gas phase of the device by flowing gases having differing oxygen composition through the ports at opposing ends of the device.
  • Figure 20 illustrates how this diffusion gradient is maintained within the device while Figure 21 provides a perspective drawing of the actual device.
  • the oxygen gradient achieves rapid equilibrium within the gas layer of a culture/cell chamber.
  • the actual oxygen content within the gas phase can be measured above any row of wells (cell chambers) 66 during operation without disturbing the gradient through use of the oxygen probe 68.
  • the culture medium contained within the micro wells equilibrates rapidly due to the small volume relative to surface area.
  • An alternative approach to studying the response of mammalian cells to various oxygen concentrations would be to generate a gradient by diffusion in a liquid phase. This is not feasible on a reasonable time scale, thus devices which do use a liquid phase gradient rely on convection. In a convective system measurements of the local oxygen concentration are more challenging making control of the gradient limited and often the measurement process disrupts the gradient.
  • Figures 20 and 21 show that the oxygen concentration in the gas phase can be measured at any time along the length of the chamber using a fiber optic oxygen probe 68 which slides freely in the axial direction.
  • the semi rigid aluminium casing on the probe keeps it at a constant vertical position above the wells and also keeps it from contacting the culture medium.
  • the probe 68 accesses the culture environment though a narrow stainless steel tube on the same end in which the gas providing the oxygen source is fed. This placement mitigates any concern over the quality of the gas seal at the probe access point.
  • the precise oxygen concentration over each row of wells 66 is measured and recorded. Having established the culture conditions throughout the device, the probe 68 is locked in place over a central well and data logged to detect any disturbances in the gradient over the culture period.
  • the aim of the culture well design of this embodiment is to maximize the medium surface area to volume ratio for rapid equilibrium with the gas phase and to minimize medium depth such that oxygen tension at the cell layer most closely reflects the concentration in the gas phase.
  • This aim was achieved by fabricating the base of the wells from glass and the walls from polydimethylsiloxane (PDMS) (Sylgard 184, Dow Corning Corporation, Ml, USA).
  • PDMS polydimethylsiloxane
  • the glass base provides a suitable substrate for cell adhesion, while the PDMS repels culture medium, resulting in an inverted drop geometry which maximizes the surface area to volume ratio.
  • Well dimensions are 4 mm diameter by 2 mm deep. This diameter was selected as the entire well could be observed in the 4x field of view on a light microscope.
  • the well array occupies an area 5 cm by 2 cm while the total chamber length is 6 cm.
  • the head space in the chamber above the wells is 1.5 mm, allowing for gas diffusion and space for the oxygen probe.
  • the glass plate 70 on which the wells are situated can simply be lifted in and out of the polycarbonate shell 72 in which the oxygen gradient is maintained. This provides flexibility in that multiple cultures can be initiated in separate well arrays and transferred into the gradient device for evaluation under different gradient conditions, thus facilitating the rapid analysis of many cultures.
  • Sterility of the wells and polycarbonate shell can be achieved through autoclaving as all materials are heat stable.
  • the gases pass through 0.22 ⁇ m polycarbonate membranes (Polycarbonate lsopore membrane filters, 0.22 ⁇ m, Millipore, Australia) situated under the gas manifolds 64.
  • the filter/manifold design ensures sterility while minimizing convective mass transport within the chamber.
  • the gas which provides either the oxygen source or sink preferably contains a constant concentration of 5% carbon dioxide such that a CO2/PH gradient is not formed along the length of the device.
  • the flow rate of pressurized gas mixes into the device are measured using conventional rotameters and controlled manually through needle valves. Prior to entering the device, gasses were humidified by bubbling through water in order to mitigate evaporative effects on the cultures.
  • the cells selected for this example were NIH mouse fibroblast 3T3's. Culture slides containing the well array were sterilized and seeded with 20 ⁇ l droplets containing 1000 cells. Culture medium was DMEM (GIBCO, Grand Island, NY) supplemented with 10% FBS (Serum Supreme, BioWhittaker, Walkersville, MD). Cells cultures were established in a standard 5% CO 2 incubator prior to being subjected to an oxygen gradient. Photos were taken at time zero to demonstrate that all wells contained similar numbers of cells and to determine the density of the starting cell population.
  • the doubling time of the NIH 3T3 fibroblast is roughly 22 hours and thus this time was selected as the duration for which the culture would be subjected to the oxygen gradient.
  • cell numbers were determined by counting those cells attached in 16 randomly selected 1 mm 2 regions. Cell viability was assessed through evaluation of morphological features and through the use of live/dead stain Trypan blue.
  • Cell numbers at time zero and at 22 hours were determined by the manual counting of cells attached in randomly selected regions. Fold expansion was determined by dividing the number of cells per unit area at time 22 hours by those at time 0 hours.
  • Figure 23 shows that the full expansion potential of NIH 3T3 fibroblasts is achieved at oxygen concentrations greater than 1%. While less than 1% oxygen inhibits normal rates of expansion, loss of adherent cell populations does not occur until concentrations of less than 0.5% oxygen (less than 1-fold expansion or maintenance of the population). Generally these cells, which are no longer adhering to the substrate, have compromised viability, however in this example the viability of any floating cells was not assessed.
  • Figures 24 and 25 illustrate one of the designs which we have utilised to study cell migration.
  • the device contains a three by three array 80 and a two by three 82 array of culture wells.
  • the chambers 84 flanking the well array function as either an oxygen sink or as an oxygen source.
  • the oxygen concentration in each well can be estimated through a steady state model as shown in Figure 26 for the three by three well array and in Figure 27 for the two by three well array.
  • the source gas contains 3% oxygen and the sink gas contains 0% oxygen. Both source and sink gas contain 5% CO 2 thus eliminating a CO 2 /pH gradient.
  • Figures 24 and 25 show the microbioreactor in an inverted position. In this position the culture wells are loaded. Once loaded with cells and medium the glass plate is placed over the wells and the entire device is inverted such that the cells settle and attach on the glass.
  • Cell culture medium is DMEM (GIBCO, Grand Island, NY) supplemented with 10% FBS
  • NIH 3T3 fibroblasts suspended at 10,000 cells per ml in culture medium are introduced into individual culture wells of the device shown in Figure 1. Cultures are stabilized for 24 hours in a 20% O 2 , 5% CO 2 atmosphere. A gradient is imposed across the device for a 24 hour period by a source gas containing 3% O 2 , 5% CO 2 and sink gas containing 0% O 2 , 5% CO 2 in nitrogen.
  • Cell expansion data is provided in Table 1 and graphically in Figure 29.
  • Cell culture medium is DMEM (GIBCO 1 Grand Island, NY) supplemented with 10% FBS (Serum Supreme, BioWhittaker, Walkersville, MD).
  • NIH 3T3 fibroblasts suspended at 20,000 cells per ml in culture medium were loaded into individual culture wells 86. Cultures were stabilized for 24 hours in a 20% O 2 , 5% CO 2 atmosphere. A gradient was imposed across the device for a 72 hour period using a source gas containing 5% O 2 , 5% CO 2 and sink gas containing 0% O 2 , 5% CO 2 in nitrogen. Pictures of the cells in each well were taken before and after subjecting the cultures to the gradient.
  • hypoxic environment is one that contains less than 1% oxygen.
  • a 0.5% oxygen environment appears sufficient for cell survival and 1% for proliferation (see Example 1). It is likely that the aerotaxis phenomenon is terminated in environments having oxygen levels sufficient for normal cell function (or at roughly ⁇ 1%). This proposition is supported by the observation that the cell migration front appears to be terminated at roughly 0.8% oxygen.
  • a glass Petri dish was modified by drilling a hole through the base of the dish providing access to the outside atmosphere. This hole is sealed by a channel which extends parallel to the well array functioning to provide oxygen equally to each column of cell chambers. This is shown in figure 30.
  • a model was generated for cell migration towards a point source.
  • a simple point source culture chamber was developed
  • This chamber included 5 tissue culture treated polystyrene wells, each with a 300 ⁇ m hole in the centre. On the bottom of the well array, there was a 100 ⁇ m
  • PDMS membrane which provides both gas exchange and a barrier from foreign pathogens.
  • the headspace above the wells was sealed off with the exception of an inlet and exit feed for a controlled atmosphere.
  • the headspace in the vessel was replaced with an atmosphere containing 5% CO2 in nitrogen, the single hole in the middle of each well became the only oxygen source (point source).
  • Cell culture medium is DMEM (GIBCO, Grand Island, NY) supplemented with 10% FBS (Serum Supreme, BioWhittaker, Walkersville, MD).
  • A7r5 cells were utilized (A7r5 is a smooth muscle cell line derived from rat aorta) (Kimes and Brandt 1976). Each well is equivalent to that in a 24 well plate. Cells were seeded at 20,000 cells per ml and cultures permitted to establish for 24 hours in a standard 20% O 2 , 5% CO 2 incubator. Each well contained 0.5 ml medium. For the next 48 hours the headspace in the chamber was exchanged with humidified 5% CO 2 in nitrogen at a flow rate of 2-3 ml/minute. Pictures were taken at 24 and 48 hours.
  • the modified point source design shown in Figure 31 is equivalent to the device used in Experiment 4 above. Essentially the membrane filter is replaced by a PDMS membrane in Figure 31. With this modification ( Figure 31), it is possible to reduce the thickness of the cell layer allowing greater control of the oxygen environment by rapid purging and the elimination of convection.
  • Figure 36 shows the layers of a three layer device to explore NIH 3T3 fibroblast morphology used in this experiment.
  • the figure shows the layers of the device. From left to right the layers are: (1) glass to isolate from external atmosphere, (2) fluid layer containing specifically conditioned fluid through the left and right chamber, (3) a polydimethylsiloxane (PDMS) gas permeable membrane, (4) cell culture array or cell layer, (5) a cuprophan or nutrient permeable membrane, (6) a medium layer, and (7) glass to isolate the external environment.
  • PDMS polydimethylsiloxane
  • NIH 3T3 fibroblasts were subjected to the following environments.
  • the behaviour of the fibroblast were contrasted between 20% and 5% oxygen environments.
  • the NIH 3T3 fibroblasts were cultured for 48 hours in the device of figure 36 at the oxygen levels defined above and on the above specified gel scaffolds or surfaces. Cells were loaded into cell layer at 10 5 cells/ml.
  • the design incorporates an array of micro channels formed using PDMS fused onto a glass substrate.
  • the serpentine channels double back on themselves first in an arrangement parallel to the gas membrane (cell housing layer) where the gradient is maintained and then perpendicularly.
  • the serpentine channels have an oxygen gradient superimposed above the cell chamber (layer 2), as oxygen diffuses between the first and second transport layers (layer 3 and 1).
  • the cell housing layer forms a contiguous and permeable connection between the first and second transport layers and the cell chamber to enable a stable oxygen gradient to be imposed across the cell chamber.
  • the aerobic transport layer (layer 3) and anaerobic layer (layer 1) are preferably supplied an aerobic and anaerobic gas supply respectively, which contains 5% carbon dioxide to maintain the pH level of the system.
  • FIG 32 and 33 An enlarged view of the channels parallel to the cell housing layer is shown in Figure 32 and 33.
  • the cell housing layer thickness is minimal as this facilitates rapid equilibrium.
  • soft lithography it is possible to fabricate channels 25 ⁇ m in width and 25 ⁇ m spacing between channels. Using this thickness and spacing it is possible to place 20 channels beneath a 1 mm thick membrane. Such an arrangement allows for one channel for each percent oxygen concentration from 20% to 1 %.
  • One of the other strengths of this design is that controls are maintained in both the source gas (first transport layer) and the sink gas (second transport layer).
  • the cell chamber (cell layer) is confined to the region between the first and second transport layers, in which a metabolite concentration gradient exists.
  • the cell chambers may extend into each of the transport layers. This configuration provides control environments in each of the fully defined atmospheres.
  • FIG. 35 is a diagram of cell expansion in a channel network shown in figure 32 with the aerobic conditions represented as 20% oxygen on the right of the diagram and 0% oxygen or anaerobic conditions on the left of the diagram.

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Abstract

Dispositif d'évaluation de réponse cellulaire comprenant une première couche de transport et une seconde couche de transport permettant d'acheminer, de fournir ou d'éliminer un flux d'au moins un métabolite cellulaire, une couche d'hébergement de cellule à au moins une chambre cellulaire pour la culture cellulaire, cette couche permettant au(x) métabolite(s) de passer au moins de la première couche de transport à au moins la chambre cellulaire, et un système de mesure d'au moins un des paramètres de processus physique du fluide s'écoulant à travers au moins la première couche de transport.
PCT/AU2007/001317 2006-09-06 2007-09-06 Microbioréacteur WO2008028241A1 (fr)

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WO2010013016A3 (fr) * 2008-07-31 2010-04-22 Heriot Watt University Appareil et procédé destinés au traitement ou au stockage d’échantillons
FR2972117A1 (fr) * 2011-03-04 2012-09-07 Centre Nat Rech Scient Systeme microfluidique pour controler un profil de concentration de molecules susceptibles de stimuler une cible
WO2012120102A1 (fr) * 2011-03-08 2012-09-13 Universiteit Leiden Appareils et procédés destinés à traiter des cellules et des structures associées
FR2974360A1 (fr) * 2011-04-22 2012-10-26 Centre Nat Rech Scient Systeme microfluidique pour controler une carte de concentration de molecules susceptibles de stimuler une cible
CN103080294A (zh) * 2010-09-08 2013-05-01 株式会社岛津制作所 细胞培养容器及使用该容器的细胞培养方法
CN103146576A (zh) * 2013-02-04 2013-06-12 中国科学院大学 一种可机械拉伸的微流控芯片细胞培养装置及其应用
JP2015231354A (ja) * 2014-06-10 2015-12-24 一般財団法人生産技術研究奨励会 組織片の機能を発現・維持する方法および組織片培養デバイス
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US11634677B2 (en) 2016-06-07 2023-04-25 Terumo Bct, Inc. Coating a bioreactor in a cell expansion system
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US11685883B2 (en) 2016-06-07 2023-06-27 Terumo Bct, Inc. Methods and systems for coating a cell growth surface
US11795432B2 (en) 2014-03-25 2023-10-24 Terumo Bct, Inc. Passive replacement of media
US11965175B2 (en) 2016-05-25 2024-04-23 Terumo Bct, Inc. Cell expansion
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US11773363B2 (en) 2010-10-08 2023-10-03 Terumo Bct, Inc. Configurable methods and systems of growing and harvesting cells in a hollow fiber bioreactor system
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WO2012120424A1 (fr) * 2011-03-04 2012-09-13 Centre National De La Recherche Scientifique Systeme microfluidique pour controler un profil de concentration de molecules susceptibles de stimuler une cible
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US9404914B2 (en) 2011-03-04 2016-08-02 Centre National De La Recherche Scientifique-Cnrs Microfluidic system for controlling a concentration profile of molecules capable of stimulating a target
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US11795432B2 (en) 2014-03-25 2023-10-24 Terumo Bct, Inc. Passive replacement of media
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