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WO2004111209A1 - Procede de surveillance faisant appel a des sondes a micro-membrane - Google Patents

Procede de surveillance faisant appel a des sondes a micro-membrane Download PDF

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Publication number
WO2004111209A1
WO2004111209A1 PCT/GB2004/002510 GB2004002510W WO2004111209A1 WO 2004111209 A1 WO2004111209 A1 WO 2004111209A1 GB 2004002510 W GB2004002510 W GB 2004002510W WO 2004111209 A1 WO2004111209 A1 WO 2004111209A1
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WIPO (PCT)
Prior art keywords
tissue
marker
probe
cells
dialysate
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PCT/GB2004/002510
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English (en)
Inventor
Zhanfeng Cui
Jocelyn Penelope Urban
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Isis Innovation Limited
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Publication of WO2004111209A1 publication Critical patent/WO2004111209A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/70—Polysaccharides
    • C12N2533/74—Alginate

Definitions

  • the invention relates to methods of monitoring tissue formation and cell viability and function.
  • the methods may be used for tissue engineering, or to monitor tissue function in vivo.
  • Tissue engineering is an emerging field in which tissues and organs are produced in the laboratory to replace or support the function of defective or injured body parts.
  • Cells have been cultured outside the body for many years.
  • Cell cultures are grown in the laboratory either as a suspension or by adherence of cells to tissue culture plates, where they form two-dimensional monolayers.
  • these colonies of cells do not become organised into three-dimensional arrangements.
  • Cells need a complex range of external signals to develop into functional three- dimensional tissues or organs. In vivo, many of these signals originate from or relate to the extracellular matrix (ECM), which is produced by the cells themselves and also gives the tissues of the body their form and shape.
  • ECM extracellular matrix
  • tissue constructs in vitro that will ideally mature into fully functional tissues and organs.
  • Such constructs are based on the seeding of cells onto a scaffold, which form a base for the growth and development of the cells into the desired tissue.
  • many growth factors and other signals must be present in the construct for the tissue to develop in the correct manner.
  • microdialysis and ultrafiltration using micromembrane probes can be used to monitor tissue viability and function of cells and to monitor the formation of tissues.
  • the methods may be used in vitro for tissue engineering purposes or may be used to assess tissue function in vivo, for example following transplantation.
  • Microdialysis is based on the perfusion of a microdialysis probe containing a semipermeable membrane. Substances from the environment surrounding the probe diffuse though the membrane into the perfused solution. Perfusate can be analysed on-line or collected for ex situ analysis.
  • the related technique of ultrafiltration involves applying a small negative pressure to the fluid in the probe so that the molecules in the surrounding fluid are drawn rapidly into the probe.
  • a method of monitoring the formation of a tissue in a tissue construct in vitro or the viability or function of cells of a tissue in a tissue construct being grown in vitro comprises: contacting a micromembrane probe with the tissue construct; carrying out microdialysis and/or ultrafiltration to extract a marker into the dialysate; and analysing the marker in the dialysate; thereby to monitor the formation of the tissue or the viability or function of the cells.
  • the invention also provides the use of microdialysis and ultrafiltration to monitor the formation of a tissue in a tissue construct in vitro or the viability or function of cells of a tissue in a tissue construct being grown in vitro.
  • the invention also provides a method of monitoring the viability or function of cells of a tissue, which method comprises: contacting a micromembrane probe with the tissue construct; carrying out microdialysis and/or ultrafiltration to extract a marker into the dialysate; and analysing the marker in the dialysate; thereby to monitor the viability or function of the cells of the tissue.
  • Figure 1 is a schematic diagram of the flow cell used: a. flow cell cup
  • microdialysis probe (outside diameter (od) 30 mm); b. glass mesh (od 20mm); c. spacer (od 30mm; inside diameter (id) 20mm; width 4.5mm); d. microdialysis probe; e. isopore polycarbonate membrane filters (pore size 0.22 ⁇ m); f. site of fixation of the microdialysis probe by dental cement; g. chondrocytes in alginate gel; h. sleeves for inserting the micromembrane probes
  • Figure 2 is a schematic diagram of the microdialysis set-up.
  • Figure 3a represents typical dependence of lactic acid concentration in dialysate on concentration of the lactic acid in the solution in vitro.
  • Figure 3b shows the dependence of the recovery on lactic acid concentration.
  • Figure 4 shows recovery of the microdialysis probe before and after autoclaving.
  • Figure 5 is a typical curve of the monitored lactic acid level in alginate chondrocyte construct.
  • Figure 6 shows the concentration of (a) lactic acid, (b) 3-methyl-glucose and (c) 3 kDa dextran in the middle of the alginate gel with the time.
  • Figure 7 shows dependence of the recovery of one type of microdialysis probe under equilibrium conditions on MW of solutes.
  • Figure 8 shows the diffusion of lactic acid in an alginate gel formed inside of a bioreactor (data for four probes shown).
  • Figure 9 shows the distribution of lactic acid in a gel at different time points.
  • Figure 10 represents D app of lactic acid and glucose in comparison with their diffusion coefficients in aqueous solution (D free ).
  • Figure 11 a and b show the diffusion of lactic acid and glucose respectively in alginate gel, monitored by microdialysis probe positioned in the centre of the gel.
  • Figure 12 shows the level of lactic acid in dialysate monitored over time during the bioconstruct culture using two probes, one at the centre and one at the edge of the gel.
  • Figure 13 shows the probe permeability over the course of time.
  • Figure 14 shows the cell count across the construct.
  • Figure 15 shows the amount of glycosaminoglycan across the construct.
  • the invention provides methods of monitoring the formation of a tissue in a tissue construct in vitro or the viability or function of cells of a tissue in vitro for example, in a tissue construct.
  • the present invention provides a method of monitoring the viability or function of cells of a tissue, for example in vivo.
  • the methods can be used to monitor the performance and viability of transplanted tissue or to assess viability or function across the tissue, for example in response to different stimuli.
  • the tissue is transplanted tissue
  • a micromembrane probe is contacted with the tissue.
  • the micromembrane probe is inserted into the tissue.
  • tissue construct in accordance with the present invention is meant the cells or tissue growing in vitro.
  • tissue construct may also include a scaffold, if present.
  • a scaffold is meant a suitable three-dimensional cell support.
  • the scaffold can act as a temporary extracellular matrix (ECM) to direct the formation of the tissue in three dimensions.
  • ECM extracellular matrix
  • the scaffold is made of a natural or synthetic material and is preferably biodegradable.
  • a typical scaffold is an alginate gel.
  • the scaffold may be seeded with the cells to be grown and cell culture medium and/or any other necessary growth factors may be added to the tissue construct.
  • the present invention can also be used to monitor tissue function in vivo or in vitro.
  • the invention may be used to monitor the growth and function of cells in two or three dimensions.
  • the present invention detects markers which can be used to monitor the formation of a tissue or the viability or function of cells of a tissue.
  • Suitable markers include soluble markers.
  • the molecular weight (MW) of the marker is in the range of up to 100 kD (kilo Dalton), for example 0.01 to 100 kD, 0.02 to 50 kD, 0.05 to 10 kD, 0.09 to 5kD or 0.1 to 3 kD, although larger proteins could be of interest.
  • markers with a molecular weight of up to 500 kD, for instance 200 kD, 300 kD or 400 kD can be used.
  • Markers for use in the invention include metabolic products, cell nutrients, cell type markers, soluble proteins or other macromolecules synthesised by the cells and extracellular matrix turnover markers. By detecting the levels of such markers over time or in particular locations within the tissue construct, the viability of the cells can be assessed and the formation of the tissue can be analysed. Detection of markers can include detecting a portion of the marker.
  • the methods of the invention can be used to monitor the levels of metabolic products produced by cells in the tissue. This can be used to give an indication of cell viability and function and the rate of turnover of cells. For instance, lactic acid can be analysed to monitor glycolysis. Glycerol can be monitored as a marker for lipolysis. This allows, for example, the calculation of the metabolic rate of the cells in the tissue to give an indication of the viability of the cells. For instance, a constant metabolic rate indicates that the population of cells is stable and a fall in metabolic rate for instance warns that the cells are dying or losing activity. Oxygen tension and pH can also be used as markers.
  • Methods of the invention can also be used to monitor the levels of cell nutrients. In this way, uptake of nutrients by the cells can be analysed and this can be used to assess cell viability and function. Markers such as glucose may be monitored. Methods of the invention can also be used to monitor markers for a specific tissue. These types of markers are cell type specific markers. They may be markers of tissue formation or breakdown. For instance, for monitoring the development of cartilage, extracellular matrix (ECM) turnover can be analysed. The levels of procollagen can be monitored to track collagen synthesis. The level of proteoglycans can be monitored. The level of glycosaminoglycans (GAG) or collagen fragments can be monitored to track matrix breakdown. Other suitable markers may be used for different tissues.
  • Such markers can be monitored over particular periods of time to analyse the development or breakdown of a tissue with time.
  • the presence of the markers in specified spatial locations can also be monitored. This gives an indication of the development or breakdown of the tissue in a particular area and allows the assessment of three-dimensional tissue formation.
  • Differentiation of stem cells can also be monitored by the method of the invention. Markers of differentiation, such as osteocalcin for osteoblasts and albumin for hepatocytes can be monitored.
  • the markers that can be monitored in accordance with the present invention can be divided into small markers such as metabolic products and cell nutrients such as lactic acid or glucose (MW 0.1 to 1 kD), intermediate molecules such as growth factors, cytokines, hormones (MW 1 to 50 kD) and large molecules such as proteins or protein fragments (50 to 500 kD).
  • the micromembrane probes for such markers will be provided with an appropriate pore size (molecular weight cut-off, MWC) such that the probes for small nutrients will have a MWC of 10 kD, while those for larger proteins will have a MWC of 500 kD.
  • MWC molecular weight cut-off
  • the probe has a MWC of 50 kD, 20 kD or 10 kD.
  • small markers will be in the MW range of 0.01 to 50 kD, such as 0.03 to 20 kD, 0.05 to 5 kD, 0.07 to 10 kD, 0.09 to 5 kD or 0.1 to 3kD.
  • the present invention can also be used to monitor larger molecule such as macromolecules such as proteins.
  • the micromembrane probes are then selected to have a larger pore size, for example having a 300 kD cut off or a 100 kD cut off.
  • Markers may typically be in the molecular weight range of 2 to 300 kD, preferably 5 to 200 kD, preferably 10 to 100 kD, preferably 20 to 50 kD.
  • the micromembrane probe can be, for example, a commercial microdialysis probe.
  • a microdialysis probe generally consists of a thin capillary, typically of 200 microns in diameter, for example from 50 to 1000 microns in diameter, and has porous, semipermeable walls.
  • the pore size of the micromembrane is selected based on the particular marker to be monitored. For example, the pore size may be selected to have a cut off of between 10 to 20 kD, for monitoring smaller molecules such as glucose and lactic acid. For larger markers such as protein markers, a larger pore size is selected, for example having a cut off of 300 kD.
  • microdialysis is carried out by feeding a perfusion liquid into the microdialysis probe and removing the perfusion liquid as a dialysate from the probe.
  • dialysate is meant the perfusion liquid containing molecules from the environment surrounding the probe.
  • the perfusion liquid is typically fed into the microdialysis probe by means of a pump.
  • a negative pressure may be applied to the perfusion fluid when the perfusion liquid is withdrawn from the probe by the pump and if this is carried out the technique is called ultrafiltration. This has the effect of increasing the rate of diffusion of the molecules into the dialysate.
  • the perfusion liquid may be a physiological salt solution.
  • the perfusion liquid may be phosphate buffered saline (PBS) or another appropriate solution.
  • Osmolytes such as serum albumin or polyethylene glycol can be added to provide iso-osmotic conditions between probe and interstitial fluid.
  • Alternative perfusion liquids include physiological salt solutions such as Krebs-Ringers solution or tissue culture media such as Dulbecco's modified Eagles medium.
  • the dialysate can be analysed on line, for instance using a flow through cell. Alternatively, the dialysate can be removed from the microdialysis probe.
  • the dialysate is analysed to detect the presence, absence or level of a particular marker.
  • the microdialysis probe can be left in situ while the dialysate is being removed and analysed. Any suitable method can be used to assay the marker. Suitable methods include standard enzymatic procedures, enzyme-linked immunosorbent assay (ELISA), detection of radioactivity and detection using optical methods such as a fluorimeter or electrochemical methods. For instance, lactic acid concentration can be measured by a standard enzymatic procedure. Typically, a reagent containing lactate oxidase and colourless chromogen precursors is added to the sample obtained by collecting dialysate. The lactic acid is converted by oxidase to pyruvate and hydrogen peroxide, H 2 O 2 .
  • the H 2 O 2 formed catalyses oxidative condensation of the chromogen precursors to produce a coloured dye.
  • Absorbance can then be read at 540 nm and is proportional to the original lactic acid concentration. Absorbance is calibrated against a standard curve, produced from known concentrations of lactic acid in PBS or blank medium with components presented in the sample.
  • the microdialysis probe is contacted with the tissue.
  • the probe can be placed in the interstitial space within the scaffold in which the cells are embedded. This allows the sampling of markers which are produced or taken up by the cells.
  • the microdialysis probe may be placed near the boundary of the construct or in the centre of the construct. Gradients in nutrients and metabolites, such as oxygen and lactic acid, which develop can be measured. These gradients depend, for instance, on how quickly the marker diffuses into, out of or away from the cell and how rapidly it is metabolised or produced.
  • the level of a particular marker can be monitored spatially. This is particularly useful in the analysis of tissues and three dimensional constructs. By detecting particular markers at specific locations within the tissue or construct, the development or viability of the tissue or construct in three dimensions can be followed. Typically, more than one microdialysis probe is placed at different locations within the tissue or tissue construct and each probe monitors the same or different marker(s) at each location. Alternatively, the same probe can be inserted sequentially at different locations to detect the concentration of the marker at each location. Preferably, three or more micromembrane probes are used in order to assess the three dimensional distribution of particular markers. The distribution of the markers can be used to give an indication of the status, viability and function of the tissue under analysis.
  • the methods of the invention can also be used to monitor more than one marker at a time.
  • the dialysate removed from the microdialysis probe can be analysed for the presence, absence or level of a number of different markers.
  • more than one type of microdialysis probe is inserted into the tissue construct at different locations to monitor different markers.
  • the relationship between the extracellular concentration of the solute and dialysate concentration can be characterised by the probe recovery.
  • the probe recovery is expressed as a percentage of the extracellular concentration of the solute:
  • the levels of particular markers can be monitored over time. Measurements of the level of the marker can be made by collecting dialysate at regular intervals, for instance every 5, 10, 15, 20, 30 or 45 minutes. Alternatively, dialysate may be collected every 1, 2, 3, 4, 12 or 24 hours.
  • the microdialysis probe can be perfused continuously or for a set period of time. Typically, the time for a measurement to be taken is from 15 minutes to 3 hours, from 30 minutes to 2 hours or from 45 minutes to 1 hour.
  • the duration of perfusion depends on the marker being analysed. The duration of the monitoring may be from 1, 2, 3, 4 or 5 days and preferably at least 7 days, up to 10, 12, 14, 21, 28 or 35 days.
  • the methods of the invention can be used to monitor particular markers over time.
  • a microdialysis probe can be inserted into the scaffold or tissue and remain there for the entire culture period or for a selected period of time. Typically, the probe remains in the scaffold and new perfusion liquid is perfused and dialysate removed at desired intervals.
  • the present invention can be used to monitor the formation of a range of different tissues containing various cells.
  • the cells can be chondrocytes and the tissue can be cartilage.
  • the cells can be keratinocytes, stem cells, hepatocytes, osteoblasts, fibroblasts, muscle cells, nerve cells or thyroid cells.
  • the tissue can be skin, liver, bone, blood vessel, muscle, nerve or thyroid. In one embodiment, more than one micromembrane probe is used.
  • the probes can be used to monitor for diffusion of substances through the tissue, or the effect on cell function to stimuli, such as in the presence of selected agents, such as compounds under investigation to determine their physiological effect on the tissue. Comparative experiments can be carried out with healthy tissue with a view to identifying expected changes in marker levels in response to certain stimuli or expected diffusion rates. Differences between the control tissue and tissue under analysis can be used to assess cell function and viability in the tissue under test.
  • the microdialysis probe is used to monitor levels of markers for a construct of cells growing in a scaffold in a bioreactor.
  • lactic acid can be monitored in a chondrocyte cell culture growing in alginate gel in a bioreactor.
  • a bioreactor is meant any suitable vessel in which cells and tissues are grown in vitro.
  • the cell can be grown, for example, in a glass flow cell consisting of two identical flow cell cap-glass mesh assemblies.
  • the apparatus is suitably set up to provide culture medium, such as Dulbecco's modified Eagle medium (DMEM), to the cultured cells.
  • DMEM Dulbecco's modified Eagle medium
  • the spacer may have more than one means to introduce microdialysis probes, which can be fixed in place.
  • a multichannel peristaltic pump can be used to provide the microdialysis flow and culture medium perfusion. Dialysate is then collected and analysed by any of the methods described above.
  • Dulbecco's modified Eagle medium (DMEM) and foetal bovine serum was obtained from Gibco (Paisley, UK); antibiotic and antimycotic mixture, collagenase type II, diagnostic reagent for lactic acid assay and phosphate buffer solution (PBS) tablets were obtained from Sigma (Poole, UK); alginate was obtained from Fluka (UK); ascorbic acid was obtained from Wako (Japan); CaCl 2 was obtained from BDH Laboratory Supply (Poole, UK).
  • Autoclavable Microbiotech microdialysis probes, PEP microdialysis tubing and adapters were supplied by Royem Scientific (UK).
  • Peristaltic pump PVC tubing of the inside diameter (id) 0.25mm was supplied by Anachem (Luton, UK).
  • Peristaltic pump silicon tubing (id 2.06 mm) was supplied by Alkey (Basingstoke, UK); Isopore polycarbonate membrane filters of pore size 0.2 ⁇ m were obtained from Millipore (Watford, UK).
  • the peristaltic pump used was a Gilson 8 channel pump.
  • Articular chondrocytes were isolated from the metacarpal-phalangeal joint of 18-24 month steers using enzymatic digestion procedure with collagenase following the procedure described by Hopewell and Urban (Biorheology. 2003; 40(l-3):73-7). The isolated cells were washed and resuspended in DMEM supplemented with antibiotic-antimycotic solution (1% v/v). 1.2 % w/v alginate solution was prepared in 0.9% w/v sodium chloride solution and sterilised by filtration through polyethersulfone (PES) filters of pore size 0.22 ⁇ m. The cell suspension and alginate solution were mixed together to yield a final concentration 16x10 6 cells ml " .
  • a glass flow cell was developed to maintain chondrocyte cell culture in alginate gel (see figure 1). It consists of two identical flow cell cap-glass mesh assemblies (id 20mm) to provide DMEM supply to the cultured cells (a & b); a spacer (id 20mm, high 4.5 mm) to define the construct size (c); two isopore polycarbonate membranes to retain the gel-cell construct and separate alginate gel from the circulating solution (e).
  • the spacer has four sleeves (h) to introduce microdialysis probes (d). In this experiment, one probe was fixed inside of the sleeve by dental cement (f). Other sleeves were blocked by silicon glue.
  • the flow cell with fixed microdialysis probe was assembled and sterilised by autoclaving.
  • Microdialysis set-up The experimental set-up to monitor lactic acid concentration in the alginate gel containing bovine cartilage chondrocytes is shown in figure 2.
  • a multichannel (8 channel) peristaltic pump was used to provide the microdialysis flow and culture medium perfusion. The pump rate was 0.08 rpm.
  • a PVC tubing of id 0.25 mm produced pulse flow of PBS for microdialysis probe at a flow rate 0.6+0.08 ⁇ l/min while the silicon tubing of id 2.06 mm provided flow of DMEM above and below the spacer at 30.5 ⁇ 0.5 ⁇ l/min at the given pump rate. DMEM was not re-circulated.
  • the mixture of the cell suspension and alginate solution was injected inside of the spacer of the assembled flow cell by the syringe.
  • the flow cell was then perfused by 102 mM CaCl 2 for 45 min at the rate 2 ml/minto crosslink the alginate.
  • CaCl 2 was replaced by DMEM supplemented with antibiotic-antimycotic solution (1% v/v), 6% v/v foetal bovine serum and 0.5% v/v ascorbic acid.
  • the flow cell was perfused by DMEM in the course of the experiment at the rate 0.6 ⁇ 0.08 ⁇ l/min.
  • the relationship between the extracellular concentration of the solute and dialysate concentration was characterised by the probe recovery.
  • the probe recovery was expressed as a percentage of the extracellular concentration of the solute:
  • the microdialysis probe was perfused by PBS. Perfusion rate was 0.6 ⁇ 0.08 ⁇ l/min. Dialysate was collected every 20 min. Duration of the monitoring of the lactic acid was 11 days.
  • Lactic acid concentration was measured by a standard enzymatic procedure (Sigma procedure no. 735). A reagent containing lactate oxidase and colourless chromogen precursors was added to the sample. The lactic acid was converted by oxidase to pyruvate and hydrogen peroxide, H 2 O 2 . The H 2 O 2 formed catalysed oxidative condensation of the chromogen precursors to produce a coloured dye. Absorbance was read at 540 nm (Titertek Multiscan Plus plate reader, Diversified Equipment Co. Inc., Lorton, Virginia, USA), and was proportional to the original lactic acid concentration. Absorbance was calibrated against a standard curve, produced from known concentrations of lactic acid in PBS or blank medium with components presented in the sample. Results
  • Figure 3 a represents typical dependence of solute concentration in dialysate on concentration of the solute in stirred solution in vitro. Recovery of the probe was determined to be 79.7+ 4.5% at given perfusion rate (Fig 3b). Autoclaving slightly altered the probe recovery, but the difference was not statistically significant, as it is shown in figure 4.
  • Figure 5 represents a typical curve of the monitored lactic acid level in the middle of alginate-chondrocyte construct. Over the first 20 hours the concentration of the lactate changed from 0.73 ⁇ 0.05 mM up to 3.0 ⁇ 0.5 mM showing the cell metabolic activity increase following an initial adaptation. The concentration of the lactic acid in the construct remained on the constant level over the observed period of the monitoring. The average mean of the lactic acid concentration was 3.110.4 mM.
  • Alginic sodium salt was obtained from Fluka (UK); CaCl 2 was obtained from
  • 3 kDa fluorescent dextran was supplied by Molecular Probes Inc (Cambridge, UK).
  • Glass flow cell was used to form the alginate gel discs (Fig 1). It consists of two identical flow cell cap-glass meshes (id 20mm) to provide supply the solutions to the gel (a & b); a spacer (id 20mm, height 4.5 mm) to define the construct size (c); and two isopore polycarbonate membranes to retain the gel and separate alginate gel from the circulating solution (e).
  • the spacer has four sleeves (h) to introduce microdialysis probes (d) in the middle of the gel. In this experiment, one probe was fixed inside of the sleeve by dental cement (f). Other sleeves were blocked by silicon glue.
  • the flow cell was assembled and 1.2 % alginate solution was injected into the spacer.
  • the solution contained 102 niM CaCl 2 was pumped at the rate 0.5 ml/min through the both cap-glass meshes for 0.5 hr.
  • the alginate gel was formed as a disc with a diameter of 20 mm and a thickness of 5 mm.
  • the diffusion of the lactic acid as a main product of cell metabolism, 3-methyl-D- glucose as a model molecule of glucose and 3 kDa dextran as the model of bioactive molecules (growth factors, peptides etc) were tested.
  • the concentrations of the lactic acid, 3-methyl-D-glucose and fluorescent dextran in the buffer were 10 mM, 1.20E- 04 ⁇ Ci/ml and 4.38E-05 mg/ml respectively.
  • the lactate assay kit (Sigma) was used to determine the lactic acid concentration in collected dialysate. 3-methyl-D-glucose concentration was determined by radioactive counting; 3 kDa dextran concentration was determined by fluorimeter.
  • Hydration of the gel was calculated from: (wet weight - dry weight)/dry weight
  • Partition coefficients (K) were estimated as:
  • Example 3 The experiments described above were extended using multiple micromembrane probes.
  • Viability/Cytotoxicity kit was purchased by Molecular Probes Inc. (Cambridge, UK).
  • Bioreactor used Glass bioreactor was developed to form alginate gel discs and to maintain chondrocyte cell culture in alginate gel. It consists of two identical cap-glass mesh assemblies (id 20mm) to provide DMEM supply to the cultured cells; a spacer of id 20mm and different heights (4.5 mm, 15 mm and 16 mm) to define the construct size for specific experiment, having 4 or 8 ports or sleeves allowing position of microdialysis probes according to the aim of the experiment. Unused ports were blocked by silicon glue; two Isopore polycarbonate membranes to retain the gel-cell construct and separate alginate gel from the circulating solution. An appropriate number of microdialysis probes were introduced into the ports by introducer and fixed by Epoxy glue.
  • the middle of the microdialysis membrane was always situated along the geometrical axis (centre) of the cylindrical spacer.
  • the bioreactor with fixed microdialysis probes was assembled and clamped by clamps.
  • the bioreactor was sterilised by autoclaving for experiments conducted on chondrocytes-alginate gel constructs.
  • a multichannel (8 channel) peristaltic pump was used to provide the microdialysis flow and culture medium perfusion.
  • the pump produced pulse flow for microdialysis probe at a flow rate 0.6 - 3 ⁇ l/min (PVC tubing of id 0.25mm) and for bioreactor 30 - 60 ⁇ l/min (silicon tubing of id 2.06 mm). Buffer/DMEM to supply bioreactor was not re-circulated.
  • PVC tubing id 0.25 mm
  • syringe stainless steel needles G 19 were used to connect FEP microdialysis tubing to the peristaltic pump.
  • the bioreactor and reservoirs with buffer for probe and Buffer/DMEM for bioreactor were kept in the incubator under 37 0 C.
  • Articular chondrocytes were isolated from the metacarpal-phalangeal joint of 18-24 month steers using enzymatic digestion procedure with collagenase following the procedure described by Hopewell and Urban (2003).
  • the isolated cells were washed and re-suspended in DMEM supplemented with antibiotic-antimycotic solution (1% v/v).
  • 1.2 % w/v alginate solution was prepared in 0.9% w/v sodium chloride solution and sterilised by filtration through polyethersulfone (PES) filters pore size 0.22 ⁇ m.
  • PES polyethersulfone
  • the mixture was injected inside of the spacer of the assembled bioreactor by the syringe.
  • the bioreactor was then perfused by 102 mM CaCl 2 for 45 min at the rate 2 ml/min to crosslink the alginate.
  • CaCl 2 was replaced by DMEM supplemented with antibiotic-antimycotic solution (1% v/v), 6% v/v foetal bovine serum and 0.5% v/v ascorbic acid.
  • the bioreactor was perfused by DMEM in the course of the experiment at the rate 0.6 ⁇ 0.08 ⁇ l/min.
  • Alginate solution (1.2% w/v in saline) or isolated chondrocytes suspension in alginate solution was injected in assembled bioreactor through the port by syringe. Probes were introduced and solution of 102 mM CaCl 2 was pumped through the glass mash for periods of time ranged from 0.5-5 hr in dependence of spacer high to form alginate gel.
  • Microdialysis probe characteristics Probe used For measurement of a solute concentration in alginate gel or alginate-chondrocyte construct autoclavable microdialysis probes with 15 kDa cutoff polyethersulfone (PES) dialysis membrane of an effective length 4 mm, od 0.6mm and 35 mm in length flexible polyurethane shaft were used.
  • PES polyethersulfone
  • Ca e q was a concentration of lactic acid or 3-methyl-D-glucose in dialysate after equilibration of a gel with a solute and C g eq was concentration of a solute in a gel.
  • tissue engineered construct was removed from bioreactor, sectioned by blade and cell viability was assessed using Viability/Cytotoxicity kit for live and dead cells. Briefly, staining of living cells was based on detection of intracellular esterase activity. They convert non-fluorescent cell-permeating calcein AM to the intensely fluorescent calcein which is well retained within living cells producing bright green fluorescence at excitation/emission wavelengths of 495/515 run. Dead cells were stained by ethidium homodimer-1 (EthD-1), which penetrates damaged cell membranes and undergoes the enhancement of fluorescence upon binging to nucleic acids, producing a bright red fluorescence at excitation/emission wavelengths 495/635 nm.
  • EthD-1 ethidium homodimer-1
  • Papain digestion, hi order to determine total sulphated glycosaminoglycan (GAG) content sections of the construct were dissolved in citrate buffer containing 55 mM sodium citrate, 50 mM EDTA and 0.15 M NaCl.
  • Papain suspension was add in the proportion of l ⁇ l of papain suspension to lOO ⁇ l of dissolved construct and samples were digested at 67° C for 18 hr.
  • Glycosaminoglycan content was determined colorimetrically using dimethylmethylene blue (DMB). Papain digest (20-100 ⁇ l) were mixed with DMB reagent (pH 4.6) in a proportion (1:10) and absorbance at 576 nm was immediately read using Perking-Elmer spectrophotometer (Perkin Elmer UK Ltd, Beaconsfield, Buckinghamshire, UK). Concentrations of GAG were determined against chondroitine sulphate standards containing corresponding amount of alginate.
  • DMB dimethylmethylene blue
  • Collagen content was calculated from hydroxyproline concentration, measured using a modification of the Stegemann method. Digests were hydrolysed in 6 N HCL for 18 hr at 110° C, neutralized with sodium hydrogen carbonate, mixed with oxidant containing chloramines T and a colour reagent containing p-dimethylaminodenzaldehyde, incubated for 20 min at 67° C, left for 15 min at room temperature and the absorbance read at 550 run on Perking-Elmer spectrophotometer. Collagen concentration was calculated from hydroxyproline concentration, using a factor of 7.14 grams collagen per gram of hydroxyproline.
  • PhR is commonly used indicator for pH range 6.4- 8.2. Its dissociation in a solution can be described by: H 2 PhR ⁇ HPhR “ + H + HPhR " ⁇ PhR 2" + H +
  • HPhR " has a yellow colour and PhR 2" has a red colour. On pH > 8.2 molecules of PhR are converted predominantly to PhR 2" "red” form and absorbency read at 540 mm did not depend on pH.
  • HPhR " form in dialysate was converted to PhR 2" by adding 2 ⁇ l of 5M NaOH to 40-50 ⁇ l of dialysate (final pH 12.5). Absorbance of the samples was read at 540 nm on Plate reader.
  • Lactic acid concentration was measured by a standard enzymatic procedure (Sigma procedure no. 735). A reagent containing lactate oxidase and colourless chromogen precursors was added to the sample in a proportion sample:reagent 1 :10.
  • the lactic acid was converted by oxidase to pyruvate and hydrogen peroxide, H 2 O 2 .
  • the H 2 O 2 formed catalysed oxidative condensation of the chromogen precursors to produce a coloured dye.
  • Absorbance was read at 620 nm and was proportional to the original lactic acid concentration. Absorbance was calibrated against a standard curve, produced from known concentrations of lactic acid in PBS or blank medium with components presented in the sample.
  • D app l/4 • t • slope
  • t is the time allowed for solute diffusion from the surface along the gel.
  • the slope was obtained from a plot of ln(c ⁇ / ⁇ c ⁇ ) vs ⁇ 2 , where c ⁇ was the concentration of solute measured in dialysate collected a probe, which was situated at a distance ⁇ from the surface.
  • a ratio C g / C g eq was plotted versus t,, where t, was time interval a solute 20 needed to rich a concentration C g in the middle of the gel.
  • C 0 was initial uniform concentration within a region — I ⁇ x ⁇ I where probe was situated and / was a distance between probe and the surface of the gel, C was the concentration in a region x at time t; t was the time of diffusion of a solute from a surface of the gel to region i on a distance ⁇ l; Ci was a constant concentration of a solute on the surfaces of the gel; D was an apparent diffusion coefficient defined experimentally as described above.
  • HPhR has a yellow colour and PhR 2- has a red colour.
  • pH > 8.2 molecules of PhR are converted predominantly to PhR 2" "red” form and absorbance read at 540 mm exists in PhR "
  • both form are present in proportions depending on pH.
  • HPhR form was converted to PhR 2" by adding 2 ⁇ l of 5M NaOH to 40-50 ⁇ l of dialysate (final pH) . Absorbance of the samples was read at 540 nm on Plate reader
  • Figure 8 represents monitoring of the diffusion of lactic acid in an alginate gel formed inside of a bioreactor by 8 microdialysis probes (data from probes 1, 3, 5 and 7 are not shown).
  • time points ranged from 0.5-5 hr where chosen (Fig 8). Within this period of time the solute had not reached the edge of the gel and the construct is symmetrical we have considered the case of the diffusion occurred in a plane sheet.
  • a distribution of lactic acid in a gel at different time points is shown on Figure 9. The concentration of lactic acid in a gel of 14.8 mm high has reached the equilibrium within 27 hr.
  • Figure 10 represents D app of lactic acid and glucose in comparison with their diffusion coefficients in aqueous solution (Df ree ). Diffusion coefficients decreased with increasing of solute molecular weight.
  • the probe permeability was assessed during the experiment. Concentration of phenol red in dialysate collected from the centre probe during the experiment is shown on figure 13. The decline of probe permeability to solutes was 2.5% for 24 hr.
  • the construct has been cultured for 12 days and cell viability, gel hydration, collagen and glycosaminoglycan content were determined.

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Abstract

L'invention concerne un procédé destiné à surveiller la formation d'un tissu dans une structure tissulaire ou la viabilité ou la fonction de cellules dans un tissu. Ce procédé consiste à mettre une sonde à micro-membrane en contact avec la structure tissulaire, à réaliser une microdialyse et/ou une ultrafiltration en vue d'extraire un marqueur dans le dialysat, puis à analyser le marqueur dans le dialysat. Ces procédés peuvent être utilisés pour surveiller la formation, la viabilité ou la fonction de tissus in vitro, ou pour surveiller la viabilité ou la fonction in vivo.
PCT/GB2004/002510 2003-06-13 2004-06-14 Procede de surveillance faisant appel a des sondes a micro-membrane WO2004111209A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010118150A1 (fr) * 2009-04-07 2010-10-14 Carnegie Mellon University Système de micro-dialyse en temps réel
EP2515109A3 (fr) * 2007-10-29 2012-11-14 Taipei Veterans General Hospital Système et procédés pour le criblage ou l'analyse de cibles
US8865460B2 (en) 2005-08-12 2014-10-21 Clemson University Research Foundation Co-culture bioreactor system
CN107275679A (zh) * 2017-07-13 2017-10-20 深圳市恒翼能科技有限公司 新型化成针床探针模块
WO2025056633A1 (fr) 2023-09-12 2025-03-20 The Cultivated B. Gmbh Procédé de surveillance continue en temps réel de concentrations chimiques dans des bioréacteurs et appareil et sonde s'y rapportant

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001000783A2 (fr) * 1999-06-25 2001-01-04 Advanced Tissue Sciences, Inc. Echafaudages tridimensionnels surveillables et systemes de cultures cellulaires
WO2001003763A1 (fr) * 1999-07-14 2001-01-18 Cma/Microdialysis Ab Sonde de microdialyse
US20020082490A1 (en) * 2000-08-04 2002-06-27 Josef Roeper Microdialysis system

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001000783A2 (fr) * 1999-06-25 2001-01-04 Advanced Tissue Sciences, Inc. Echafaudages tridimensionnels surveillables et systemes de cultures cellulaires
WO2001003763A1 (fr) * 1999-07-14 2001-01-18 Cma/Microdialysis Ab Sonde de microdialyse
US20020082490A1 (en) * 2000-08-04 2002-06-27 Josef Roeper Microdialysis system

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PARK JONG-CHUL ET AL: "Viability evaluation of engineered tissues", YONSEI MEDICAL JOURNAL, vol. 41, no. 6, December 2000 (2000-12-01), pages 836 - 844, XP002297029, ISSN: 0513-5796 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8865460B2 (en) 2005-08-12 2014-10-21 Clemson University Research Foundation Co-culture bioreactor system
EP2515109A3 (fr) * 2007-10-29 2012-11-14 Taipei Veterans General Hospital Système et procédés pour le criblage ou l'analyse de cibles
WO2010118150A1 (fr) * 2009-04-07 2010-10-14 Carnegie Mellon University Système de micro-dialyse en temps réel
US8246541B2 (en) 2009-04-07 2012-08-21 Carnegie Mellon University Real-time microdialysis system
CN107275679A (zh) * 2017-07-13 2017-10-20 深圳市恒翼能科技有限公司 新型化成针床探针模块
CN107275679B (zh) * 2017-07-13 2023-07-04 广东恒翼能科技股份有限公司 新型化成针床探针模块
WO2025056633A1 (fr) 2023-09-12 2025-03-20 The Cultivated B. Gmbh Procédé de surveillance continue en temps réel de concentrations chimiques dans des bioréacteurs et appareil et sonde s'y rapportant

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