WO2008005998A2 - Incubateur de diagnostic intégré à l'échelle du centimètre pour cultures biologiques - Google Patents

Incubateur de diagnostic intégré à l'échelle du centimètre pour cultures biologiques Download PDF

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Publication number
WO2008005998A2
WO2008005998A2 PCT/US2007/072777 US2007072777W WO2008005998A2 WO 2008005998 A2 WO2008005998 A2 WO 2008005998A2 US 2007072777 W US2007072777 W US 2007072777W WO 2008005998 A2 WO2008005998 A2 WO 2008005998A2
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WIPO (PCT)
Prior art keywords
perfusion
enclosure
sample chamber
perfused
incubator
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PCT/US2007/072777
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English (en)
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WO2008005998A3 (fr
Inventor
Jelena Vukasinovic
Ari Glezer
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Georgia Tech Research Corporation
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Priority claimed from US11/483,126 external-priority patent/US7855070B2/en
Application filed by Georgia Tech Research Corporation filed Critical Georgia Tech Research Corporation
Priority to US12/529,881 priority Critical patent/US20100151571A1/en
Publication of WO2008005998A2 publication Critical patent/WO2008005998A2/fr
Publication of WO2008005998A3 publication Critical patent/WO2008005998A3/fr

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    • 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
    • C12M23/22—Transparent or translucent parts
    • 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
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/10—Perfusion
    • 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
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
    • 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
    • C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L1/00—Enclosures; Chambers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip

Definitions

  • the present invention relates generally to portable, diagnostics, pocket-size incubators with integrated perfusion, and more particularly, to a biocompatible, disposable, centimeter-scale, incubator workstation 10 for in situ monitoring, handling and scientific studies of biological cultures in the field and within major laboratories.
  • centimeter-scale diagnostics incubator has small overall dimensions that would facilitate the control of the cellular microenvironment and provide the shortest possible response time to the physical changes and chemical stimuli. It would also be desirable to have a centimeter-scale, integrated diagnostics incubator that is portable and may be used in the field. Ultimately, it would also be desirable to have a centimeter- scale, integrated diagnostics incubator of a modular design to address different demands.
  • the tissue rests on a mesh at the interface between the open channel flow of perfusate, underneath the mesh holding the tissue, and, oxygenated and humidified atmosphere above the mesh.
  • Representative chambers are described by Li CL. et al. in 1957 J Physiol-London 139:178-190; Reynaud J.C. et al. in 1995 J Neurosci Meth. 58:203-208; Krimer L. S et al. in 1997 J Neurosci Meth 75:55-58.
  • submerged chambers provide more constant environment with less perturbations in fluid flow, purging of bubbles, and, draining, and, permit rapid exchanges of the bathing medium.
  • better perfusion fixation in preventing both the starvation and anoxia during the process of observation, can be achieved in submerged chambers.
  • a major drawback in submerging the tissue below the liquid surface is actually keeping it submerged, because it will float unless it is properly attached to the perfusing substrate or restrained otherwise.
  • localized injection of drugs is virtually impossible to administer with the current designs.
  • Fig. 1 is a top view of an exemplary centimeter-scale, diagnostics incubator with integrated perfusion
  • FIGs. 1 a and 1 b illustrats various views of another exemplary incubator
  • FIG. 2 is a top view of an exemplary microfluidic perfusion chamber that may be used in the diagnostics incubator and that comprises a culture chamber with fluidic ports and attached semi-permeable membrane that encapsulates the structure;
  • FIGs. 3 and 4 illustrate three-dimensional views of the exemplary perfusion chamber
  • FIG. 5 is a cross-sectional view of the exemplary perfused culture chamber with integrated microfluidics
  • Fig. 6 is a top view showing details of a cell culture life support chamber centrally integrated within the perfusion platform;
  • Fig. 7 illustrates an exemplary microscope stage insert plate that can be used to attach the centimeter scale incubator with an integrated perfusion chamber onto a microscope stage;
  • Fig. 8 illustrates an exemplary in-line auto-venting aerator and bubble trap that is used at high-exchange rates when apical gas exchange is not sufficient;
  • Figs. 9a-l show particle image velocimetry measurements of the induced flow within the culture chamber at different elevations parallel to the perfusing substrate;
  • Fig. 10 shows graphs that illustrate thermocouple temperature measurements following an initial 10-minute long controller auto-tuning
  • Fig. 1 1 shows graphs that illustrate temperature measurements while the incubator is in operation
  • Fig. 12 illustrates a disposable thermopolymer master formed by solid object printing for use in elastomehc molding of perfusion chambers
  • Fig 13 illustrates an exemplary in-line venting aerator bubble trap that may be employed with the incubator.
  • a biocompatible, centimeter-scale, diagnostics incubator 10 having an integrated microfluidic perfusion chamber 30 which allows simultaneous culturing and experiments on a bench top or a microscope stand, with full control of the environmental conditions, fluidic, optical, and, electrical accessibility.
  • Fig. 1 shows an exemplary centimeter-scale, integrated diagnostics incubator 10 comprising an exemplary integrated perfusion chamber 30.
  • an exemplary reduced-to-practice centimeter- scale, integrated diagnostics incubator 10, or mini-incubator 10 comprises a biocompatible, polystyrene, heated enclosure 11 with exemplary outside dimensions of 5 x 2.5 x 1 .8 cm (L x W x D).
  • the enclosure 1 1 has a removable top cover that facilitates the integration and packaging. Owing to the clarity of the polystyrene material, all surfaces of the enclosure 1 1 are optically transparent and enable visual inspection.
  • the incubator 10 houses a biocompatible polydimethylsiloxane (PDMS), for example, perfusion chamber 30 with a centrally placed cell culture life support perfusion chamber 20, a semi-permeable membrane aerator 21 that encapsulates the chamber 20, a miniature, thin flexible heater 16 that maintains a desired temperature within the incubator 10 or heated enclosure 1 1 , thermocouple sensors connected to a temperature controller 18, and; pertinent gas 12, 13 and fluidic interfaces 24, 25.
  • Incubator 10 may also have an integrated in-line venting bubble trap 26 (see also Fig.
  • the optically clear incubator enclosure 1 1 and a semi-permeable membrane aerator 21 permit optical examination or observation of a culture growing within the culture chamber 20.
  • Imaging accessible, semi-permeable membrane 21 (aerator 21 or oxygenator 21 ), located on the top of the perfusion chamber 30, ensures sufficient gas delivery and provides sterile, moist microenvironment of appropriate acidity.
  • membrane aerators 21 and 27 both sides (top and bottom) of the cultured tissue are exposed to gases necessary for cellular metabolism.
  • a forced convection flow of gases such as 5% CC> 2 -air mixture for example, enters through gas inlet 12 and outlet port 13.
  • the gas inlet 12 and outlet 13 ports are coupled to gas lines 14 that lead to a separate miniature box (not shown) that houses a fan 19 and contains prescribed gas mixture via shut-off valve connection to a gas tank.
  • a fan 19 can be a pair of miniature blowers with the overall dimensions of 4cm x 4cm x 4cm used in electronics cooling (Thermaltake A1899, for example). Blowers force the appropriate gas mixture through the gas lines 14 and optically clear enclosure 1 1 and allow external control of the fan speed to provide an optimal gas rate at a minimal noise level.
  • fluidic interfaces for the nutrient medium There are two fluidic interfaces for the nutrient medium, for example. They are associated with continuous infusion of the nutrients into the perfusion chamber 30 and withdrawal of used media and metabolic waste products at the same rate. All fluidic components (perfusion chamber 30, aerators, fittings and tubes) are steam autoclavable (re-usable) with stiff microbore tubes fabricated in an inert (Teflon FEP) material that minimizes the problems associated with bio- fouling (non-specific protein adhesion along the inner walls). Fluidic inlet and outlet ports 24, 25 are coupled to the infusion and withdrawal side of the syringe pump moving block respectively.
  • An exemplary syringe pump 19a with opposing syringes on a single drive may be a KdScientific pump model KDS210, for example.
  • the pump 19a mode of operation pertinent to the particular cell feeding schedule is externally controlled and can be a continuous infusion and withdrawal; programmable, time-periodic or aperiodic infusion and withdrawal; or, push/pull; with or without the re-use of spent perfusate.
  • the other syringe withdraws the perfusate at the same rate so that the pressure within the cell culture chamber 20 remains at or near the atmospheric level. This particular mode protects the cultures from prohibitively high pressures otherwise associated with the return flow from the chamber.
  • syringe pump 19a can be replaced with miniature piezoelectric pumps that can be battery operated (e.g., Bartels Mikrotechnik GmbH micropump measures only 14x14x3.5mm and provides flow rates of 50 ⁇ l /min to 3ml/min using 3V DC battery at 3OmA) and fluid delivery rates manipulated by varying the amplitude or the frequency, or both, of the power supplied using an inexpensive custom built function generator based on the operational amplifier circuits, for example.
  • battery operated e.g., Bartels Mikrotechnik GmbH micropump measures only 14x14x3.5mm and provides flow rates of 50 ⁇ l /min to 3ml/min using 3V DC battery at 3OmA
  • fluid delivery rates manipulated by varying the amplitude or the frequency, or both, of the power supplied using an inexpensive custom built function generator based on the operational amplifier circuits, for example.
  • microvascular perfusion chambers 30 can be easily configured to meet specific demands while exceeding the available exchange rates of commercial devices by a few orders of magnitude. Fluidic functionality of the perfusion chamber 30 enables the culturing of thicker tissue slices and engineered 3-D culture models that better represent the in-vivo condition via forced convection intercellular mass transport that is not feasible in commercially available interface or submerged-type perfusion chambers.
  • a simple fabrication methodology based on elastomeric polymer molding allows the integration of microfluidic and MEMS components (not shown) into the perfusion chamber 30, such as micropumps, valves, mixers, micro injectors, cellular manipulators or multielectrode arrays, for example. It also facilitates the interfacing of perfusion chambers 30 and incubators 10 and enables the integration of a number of plug-in functionalities into the mini-incubator 10 tailored to specific use in a short turn-around time.
  • the disclosed fluidic design also enables the introduction of externally controlled temporally varying 41 , or, spatially varying, temporally invariant concentration gradients 42 of injected agents into the culture chamber 20, with fluidic infrastructure and control located outside of the incubator enclosure 1 1 . Owing to the flexibility of the perfusion system, the nature of cell feedings can be radically changed.
  • a programmable time-periodic injection of nutrients can be employed, followed by times where perfusion chamber 30 acts as a static control; the media could also be cycled in and out of the chamber 30 in some periodic or aperiodic fashion to break the temporal invahance of spatial gradients; or, the ratio of fresh to used media can be varied to increase the amount of cytokines (neurotrophins, for example) if the need arises.
  • the fluidic system may be easily adapted to address and individually control multiple culture chambers 20 or perfusion chambers 30.
  • Drugs can be discretely applied to specific regions using separately addressable fluidic inputs, interfaced outside the incubator 10 and introduced into the perfusion chamber 30 through the inlet port 33 or a plurality of inlet ports 33 (not shown), as could other substances.
  • Extra- and intercellular ion concentration can be accurately controlled and easily altered.
  • Collected perfusate can be mixed with the fresh medium and re-used, particularly at high exchange rates and cell densities when neurotrophins may be depleting.
  • the centimeter-scale incubator 10 with its integrated perfusion chamber 30 permits simultaneous studies of cell culture parameters using optical diagnostics.
  • a particular microscope stage insert 49 may be used.
  • the incubator 10 or perfusion chamber 30 connects directly to the microscope stage insert plate by way of 4 screws and nuts that pass through four through-holes located on the body 31 of the perfusion chamber 30 and centimeter-scale incubator 10.
  • Figs. 1 a and 1 b illustrate various views of another exemplary incubator.
  • FIG. 1 a shown is a bottom portion 1 1 a of the incubator enclosure 1 1 (FIG. 1 ) that includes holes 56 that facilitate mounting of the perfusion chamber 30 (FIG. 2). Also, holes 58 accommodate thermocouples, a media inlet tube, a media outlet tube, or other structures. Also, the bottom portion 1 1 a includes tapped holes 60 to receive gas fittings to circulate gasses through the enclosure 1 1 .
  • the top cover 50 of the incubator can be modified to include a circular through-hole 52.
  • the through hole 52 can be covered with a gas, water and oil impermeable membrane 54.
  • Optically clear, impermeable membrane is sealed around the perimeter of the through-hole formed in the top cover of the incubator enclosure using an O-ring.
  • Impermeable membrane is stretched loosely over the through-hole during incubation.
  • one side of the impermeable membrane (not facing the culture) is covered with oil or water depending on the type of the objective used. Immersed, high numerical aperture objective is then placed in contact with the impermeable membrane.
  • the membrane aerator 21 US Patent No. 6,521 ,451 (12.7 ⁇ m thick Dupont Teflon® FEP fluorocarbon film) is fairly impermeable to water (0.004 % water absorption, using ASTM D570 test method) and water vapor (permeability of 7g/m 2 /day, ASTM E-96) and quite permeable to oxygen, nitrogen and carbon dioxide (respective permeabilities of 298, 125 and 647.5 cc-mm/m 2 -24hr-atm using ASTM D-1434 test method). Stated values are for 25 ⁇ m thick film.
  • the impermeable membrane such as 12.7 ⁇ m thick Saran WrapTM 3 Plastic Film (Dow Plastics) has a high transmission in the visible range (86% based on ASTM D1746) and is quite impermeable to gases and water vapor.
  • Respective permeabilities of oxygen (ASTM D3985), nitrogen (ASTM D1434) and carbon dioxide (ASTM D1434) are 0.472, 0.0709 and 2.13 cc-mm/m 2 -24hr-atm. Water vapor permeability is limited to 8.7g/m 2 /day, Permatran W).
  • this relatively impermeable thin film that replaces a part of the incubator enclosure 1 1 , prevents gas exchange between the ambient and the incubated (heated) environment.
  • SARAN WRAPTM 3 Plastic Film is a clear biaxially oriented monolayer barrier film, designed for tight wrap and heat- sealing applications with negligible air transmission of 0.14 cc-mm/m 2 -24hr-atm.
  • the impermeable membrane remains well above the membrane aerator 21 and the efficiency of gas exchange remains unaltered compared to polystyrene top cover.
  • direct contact between impermeable membrane and membrane aerator 21 may reduce sample exposure to gases depending on the relative surface area of the objective lens and perfusion chamber.
  • the membrane aerator 21 top surface is not fully covered and much of the gas exchange now takes places laterally via microchannels 37 formed in the body of perfusion chamber 30.
  • the incubator enclosure 11 be opaque to prevent ambient light to reach the specimen and create background noise.
  • clear polystyrene incubator enclosure 1 1 can be replaced with an opaque enclosure during life-time fluorescence imaging for example.
  • a heating element 16 such as a miniature (2cm x 2.5cm) resistive foil heater 16 (Minco HK5291 , for example) is disposed within the enclosure 1 1 and has the lead wires 17 extending outside of the enclosure 1 1 to a temperature controller 18 coupled to a DC switching relay and a DC power supply that can be replaced with a battery for portability.
  • Typical power requirements for the heating element 16 integrated within the incubator 10 are at the order of just 1W.
  • the resistive heating element 16 can be replaced with a thermoelectric pump such as Ferrotec Corporation miniature Peltier cooler (part #9502/065/012 M measuring 12 x 1 1 x 3 mm with cooling/heating capability of about 6W at 1 .2 Amps and the maximum temperature difference of 7O 0 C) to maintain the temperature below the ambient.
  • Thermoelectric heat pumps can be used to either cool or heat the sample depending on the preservation/incubation requirements by simply changing the polarity of the voltage source. Slightly larger thermoelectric modules obtain temperature difference of 100 0 C or more with heating/cooling capacity of few tens of Watts.
  • Thermoelectric pumps can be battery operated for portability.
  • the utility of the incubator 10 is much broader and extends well beyond the realm of tissue engineering.
  • fluidic functionality may not be necessary and perfusion chamber 30 can be replaced with a PDMS specimen holder similar to a cylindrical culture well, and, encapsulated by a membrane aerator 21 .
  • a PDMS specimen holder similar to a cylindrical culture well, and, encapsulated by a membrane aerator 21 .
  • various chemical, biochemical or even biological specimen can be preserved at specific atmosphere of gases at controlled temperature, but, without perfusion functionality.
  • the use of thermoelectric pumps for example, extends the use of the miniature incubator 10 to preservation and observation of viral or bacterial samples collected in the field prior to their inspection in full-scale laboratories.
  • gas interfaces 12 and 13 can be connected to miniature quick-disconnect shut-off valve couplings (such as those from Kent Systems) attached to sterile filters, to prevent the infection from spreading out during common handling, or, gas bottle replacement in the field when the specimen requires long-term preservation. This also facilitates the interfacing of gas lines 14 with the miniature incubator 10 with a low-risk of contamination.
  • the perfusion chamber 30 can be replaced with a miniature pipette whose tip protrudes through the incubator enclosure, with acquired sample delivered inside the incubator 10 and sealed from the ambient using sterile shut-off couplings.
  • cylindrical well that replaces the perfusion chamber 30 can be coated with chemoattractants or chemorepellants and easily turned into a haptotaxis chamber to investigate the directional motility or outgrowth of cells cultured in monolayers.
  • chemoattractants or chemorepellants When cellular perfusion is not necessary, cylindrical well that replaces the perfusion chamber 30, can be coated with chemoattractants or chemorepellants and easily turned into a haptotaxis chamber to investigate the directional motility or outgrowth of cells cultured in monolayers. These gradients are established by altering the concentration of cytophilic adhesion sites on a polymer substrate. For example, in case of axonal outgrowth, either up or down a gradient of a substrate-bound chemoattractant.
  • the perfusion chamber 30 may be replaced with a miniature chamber for steady-state gradient generation (see US Patent No. 6,705,357, for example). With minor modifications this utility reveals at what concentration drugs become effective, and, facilitates the maintenance of samples, repeatability of experiments, and, enables optical analyses of the cultures or other specimen over extended periods of time.
  • the temporal gradient generating device 41 or steady-state gradient generating apparatus 42 can be placed within the incubator 10 to replace the perfusion chamber 30. These devices can be easily turned into chemotaxis chambers to investigate the movement of cells, bacteria, and other single-cell or multicellular organisms towards certain intermittently or continuously injected chemicals into their microenvironment. Specific concentration of chemicals in question can be easily determined in apparatus 41 or 42 by optically inspecting the specimen.
  • perfusion chamber 30 can be modified and used for packaging of different perfusion substrates such as miniature tower arrays having fluidic and electrical functionality, to feed and record from the cultures.
  • the internally placed heating element 16 can be replaced with optically clear heaters based on Indium Tin Oxide (ITO) technology.
  • ITO heaters such as Honeywell 78000 Series or Dontech ThermaKlearTM Transparent Heaters, can be bonded to all interior surfaces of the incubator enclosure 1 1 . By doing so the active volume of the incubator enclosure 1 1 can be reduced by a half or more. Volume reduction further facilitates the temperature control and the maintenance of isothermal condition within the incubator 10. Owing to their clarity, ITO heaters attached to the walls of the incubator enclosure 1 1 , do not compromise optical diagnostics on a microscope stand or visual inspection of the samples.
  • the material of the incubator enclosure 1 1 needs to be replaced with that of polycarbonate or some higher heat-resistant plastic. This could potentially render the entire cm-scale incubator apparatus 10 fully autoclavable.
  • the incubator enclosure 1 1 may have double walls.
  • a number of perfusion chambers 30 with their respective membrane aerators 21 can be placed within inner enclosure.
  • Inner enclosure is connected to a bottle having prescribed concentration of gases.
  • ITO heaters can be attached to all interior surfaces of the outer enclosure or a plurality of thermofoil heaters can be placed in the clearance between the inner and the outer enclosure.
  • a miniature blower can be attached to one side of the outer enclosure to recirculate air flowing between the inner and the outer enclosure, thus enabling forced convection over the heaters.
  • an external box that houses the mini-blowers is not necessary.
  • Each sample may be perfused separately using miniature pumps to prevent the infection.
  • additional sensors and controllers including but not limited to glucose consumption, pH or dissolved O 2 sensing and control 18a can be easily integrated into the perfusion chamber 30 and the enclosure 1 1 depending on particular demand.
  • the modular design allows integration of all or some of the above mentioned elements and enables the packaging of a larger number of perfusion chambers 30 into a common, environmentally regulated enclosure 1 1. Higher number of independently controlled perfusion chambers 30, within an enclosure 1 1 enables high throughput processing of a number of cultures onboard a single, disposable platform.
  • FIG. 2 shows top view of the exemplary microfluidic perfusion chamber 30 and a cell culture life support chamber 20.
  • Figs. 3 and 4 illustrate three-dimensional views of the perfusion chamber 30.
  • Fig. 5 is a cross-sectional view of the perfusion chamber 30 and a cell culture chamber 20.
  • Fig. 6 illustrates a top view showing details of the culture chamber 20 centrally formed in the perfusion chamber 30.
  • the exemplary perfusion chamber 30 comprises a body 31 , which may be made of polydimethylsiloxane (PDMS), for example, and a semi-permeable membrane holder 22 that encapsulates the perfusion chamber 30.
  • a central opening is formed in the body 31 that defines the inlet port 33 (Fig. 5) with the exit port 34 (Fig. 5) embedded into the bottom of the return flow (consisting of the used media and catabolites) collecting pool 41.
  • the return flow collecting pool 41 is formed in the body 31 between cylindrical enclosures 35, 42.
  • the fluidic inlet and outlet ports 33, 34 are coupled to a syringe pump 19 via inlet and outlet tubing 24, 25 (shown in Fig. 1 ).
  • An optically clear membrane 21 such as a hydrophobic, fluorinated-polyethylene-propylene (FEP) membrane 21 , see for example, Potter S. M. et al. in 2001 J Neurosci Meth 1 10:17-24, is attached to the top of the body 31 via miniature Teflon holder 22 with O-rings to seal the chamber 30 (see Fig. 2).
  • the transparent membrane 21 contacts the top surfaces of the perfusion chamber 30 and culture chamber 20 while O-rings seal the device exterior wall 42 of the body 31 , and thus the entire perfusion chamber 30.
  • the FEP membrane 21 is impermeable to the media but permeable to gases necessary for cellular metabolism.
  • the transparent membrane 21 secured with a Teflon holder 22 permits optical examination or observation of the tissue within the culture chamber 20. Such observation is not possible using conventional, large-scale incubators. Typically, cultuhng takes place in the incubator and tissue has to be taken from the refrigerator size incubator to be observed and analyzed under a microscope.
  • the body 31 as shown in Figs. 3 and 4, of the perfusion chamber 30 is configured to have a centrally located cylindrical, cell culture life support chamber 20, an outer inclined, collecting pool 41 , and, fluidic inlet and outlet ports 33, 34 shown in Fig. 5.
  • the culture chamber 20 as schematically shown in Fig.
  • the 6 is an actively controlled incubation bath bounded by the perfusing substrate 38 that supports the tissue, located at the bottom of the culture chamber 20, cylindrical sidewall 35 that confine the culture to the area immediately above the substrate 38, and the optically clear semi-permeable membrane 21 attached to the top of the perfusion chamber 30 and held by the Teflon holder 22.
  • a smooth, continuous, circulation of the media and analytes is achieved by using a simultaneous push-pull pumping system in which the output exactly balances the input, that is, the culture feedings are in mass equilibrium with the amount of retrieved perfusate.
  • the level of bathing medium within the culture chamber 20 is controlled by the suction side of a syringe pump 19a.
  • Used media and metabolic waste products are withdrawn from the cultured chamber 20 through an array of microchannels 37 that are incorporated into the walls of the cylindrical PDMS enclosure 35 bounding the culture chamber 20.
  • Retrieved perfusate is collected within the inclined pool 41 , outside the culture chamber 20, before it is withdrawn through the outlet port 34.
  • the bottom surface of the exit flow collecting pool 41 is slanted to allow the used perfusate accumulating in the pool 41 to migrate toward the outlet port 34 formed in an exterior wall 42 of the body 31 .
  • a 6 ° inclination, for example, of the bottom surface of the pool 41 helps in steering the used media and catabolites towards the outlet port 34.
  • the cylindrical sidewall 35 that surrounds the culture chamber 20 measures 3.5 mm in diameter and raises 700 ⁇ m above the perfusion substrate 38 so that the exemplary, effective volume for 3-D culturing amounts to about 7 ⁇ l.
  • a tissue slice or a tissue-equivalent constructs is adhered to the interior of the culture chamber, i.e. see Cullen D. K at al. in 2007 J Neural Eng 4: 159-172.
  • Exemplary 150 ⁇ m wide microchannels 37 are 375 ⁇ m deep and start at the elevation of 375 ⁇ m above the perfusion substrate 38. These microchannels 38 allow used cell culture media to leave the culture chamber 20.
  • the start of the microchannels 38 at a mid-height of the cylindrical enclosure 35 enables the tissue to always be at least partly submerged. By filling the entire culture chamber 20 first, without starting the suction, one can ensure that the culture is fully submersed prior to the start of a closed-loop circulation.
  • the radial exit flow through the microchannel array 37 peripherally located within the culturing chamber 20 is facilitated by the presence of a selectively permeable membrane 21 placed over the top of the perfusion chamber 30.
  • the incubator 10 disclosed herein provides an efficient control of both convective and diffusive mass transport relying on interstitial nutrient convection induced by an array of microjets issuing from micronozzles formed in the perfusing substrate 38 that upholds the tissue and peripheral extraction of the perfusate, by an array of microchannels 37, for example, located within the cylindrical enclosure 35 that laterally confines the tissue. Actively growing high density cultures are continually perfused with the fresh medium. Volumetric flow rates for neuronal-only and neuronal/ astrocytic co-cultures are about 3 exchanges per hour or more depending on the type of the culture incubated and cellular density. The cell longevity is the dominant factor determining the flow rate of the supplied media. Collected perfusate can be mixed with the fresh medium and reused, particularly at high exchange rates and cell densities when neurotrophins may be depleting.
  • the perfusing substrate 38 may be made of gold, for example, and bonded to or otherwise sealed to the lip formed at the bottom of the culture chamber 20.
  • An exemplary gold perfusion substrate 38 (such as PELCO® center-marked grids, 300 mesh, 1 GG300, for example) measures 3 mm in diameter and contains 54 ⁇ m square openings (micronozzles) with center-to- center spacing of 85 ⁇ m. This yields 40% open area for fluid flow.
  • Different perfusion substrates 38 of different topology and surface chemistry for example, may be used to promote cellular adhesion to the bottom of the culture chamber 20 and enable perfusion of the tissue cultured inside.
  • Micronozzles can have sub-cellular dimensions, i.e., 5 ⁇ m for example, which is well below the size of the neural cell body (about 10 ⁇ m).
  • Various kinds of commercially available or custom made substrates 38 having different porosity and hydraulic diameters can be bonded to the bottom of the culture chamber 20 using a thin layer of PDMS pre-polymer/catalyst mixture. This allows manipulation of the cellular microenvironment by altering the flow conditions and cellular adhesion by way of substrate-bound cytophilic coatings.
  • a variety of biocompatible materials, other than gold, may be used as perfusion substrates 38 and to support the cells cultured within the culture chamber 20.
  • Thin porous films made of nylon, polycarbonate or polytetrafluoroethylene (PTFE) constitute one of many possible approaches. In some cases selective coating of these substrates 38 promotes preferential cell growth if desired. Cell attachment may be enhanced using a variety of biocompatible coatings, depending on the exact nature of the study and the type of cells cultured within the culture chamber 20. Dissociated cells can be grown in synthetic, biodegradable extracellular matrices (ECMs) placed above the perfusing substrates since most of the ECM materials adhere well, see for example, O'Connor S. M., et al. in 2001 Neurosci Lett 304:189-193; or, Woerly S., et al. in 1996 Neurosci Lett 205:197-201 .
  • ECMs extracellular matrices
  • Synthetic ECMs in the form of 3-D scaffolds can be used to culture high-density 3-D dissociated cultures over the perfusion substrates and their ability to regulate tissue formation can be easily observed.
  • 3-D cultures composed of cells of interest, matrix secreted by these cells, and biodegradable, synthetic ECM can be used to study the role of intercellular signaling in tissue development and may be used in clinical applications as replacement to lost or injured tissue.
  • the perfusion chamber 30 is aerated by a hydrophobic, fluorinated- polyethylene-propylene (FEP) membrane 21 (see US Patent No. 6,521 ,451 , for example) that is selectively permeable to gases such as oxygen, nitrogen, hydrogen and carbon dioxide and relatively impermeable to microbes, water and water vapor (less than 0.01 % moisture absorption).
  • FEP membrane 21 multiple functionalities of the FEP membrane 21 eliminate the need to equilibrate the media in an external bath, the most frequently used type of aerator (oxygenator for acute slices) used in conventional perfusion chambers, incorporate bubble traps, or to include the gas lids to direct the moist and saturated air over the culture like in interface-type (Haas) chambers.
  • an aerator 21 enables a fine control of the cellular climate without complicated, bulky components, thus allowing the fabrication of inexpensive compact devices.
  • Other benefits of an aerator comprising an FEP membrane 21 and a membrane holder 22 include the ability to culture in a non-humidified incubator since the media does not evaporate from the culture chamber 20 thereby reducing the risks associated with the contamination in a humid environment, and, preventing the increase in osmotic strength that can be detrimental to the cultures.
  • Optically transparent FEP membranes are also impermeable to microbes resulting in a reduction of contamination occurrences making them ideal for microscopic imaging in a sterile environment.
  • Different membrane materials and/or thicknesses may also be used as efficient, optically clear, aerators/bubble traps.
  • the FEP membrane 21 used in the culture chamber 20 enables sterile venting of small volumes, i.e., it constitutes an efficient auto-venting bubble trap on its own.
  • an in-line bubbler/outgassing device can be inserted into the perfusion circuit.
  • the male luer slip side of the barbed connector (Qosina® 1761 1 polypropylene) inserted into the inlet of the perfusion chamber 30, connects to a female luer end of an L luer adapter (Qosina® 17610 polycarbonate).
  • Horizontal leg (male slip) of this L adapter connects to a female side of a luer tee 26 having two male slips (Qosina® 88216 polycarbonate).
  • Horizontal male slip of the luer tee 26 is connected to the infusing syringe via microbore tubing and luer tight female fittings (Upchurch Scientific' P-835).
  • the vertical leg of the medical grade luer tee 26 is used to form an in-line vented aerator/bubbler/outgassing device.
  • an FEP membrane 27 is stretched over the vertical leg of the tee 26 having a male slip luer end, and, sealed with a finger-snap vented female luer end-cap 28 (Qosina® 71681 polypropylene).
  • This device prevents the bubbles from reaching the culture chamber 20, and ensures the media equilibrium with the gas environment inside the cm- scale incubator 10.
  • a larger surface area for the in-line gas exchange can be obtained alternatively.
  • an off- the-shelf filter holder such as 13 mm Swinnex filter holder, Millipore, Billerica, MA
  • the membrane aerator 27 helps the venting of perfusing lines thus eliminating the need for a bubble trap. This also enables a smooth circulation of the bathing medium using a syringe- rather than a peristaltic pump, as peristaltic pumps generally induce more fluctuations in nutrient delivery.
  • the presence of the FEP membrane 21 on the top of the culture ensures that the plethora of gas is provided from the top as is from the bottom via perfusing medium.
  • the PDMS material used in fabricating the chamber 30 is found to be highly permeable to oxygen and carbon dioxide so that the cellular exposure to the gases is, in fact, enhanced from all exposed sides of the tissue.
  • the perfusion chamber 30 exposes both sides of the tissue to relevant gases while keeping the tissue submerged.
  • All components of the PDMS perfusion chamber 30 and those of relevant fluidics hardware are reusable and steam autoclavable, although the low cost of fabrication allows it to be a disposable device.
  • the inlet and outlet fluidic connections are removable, i.e. they can be easily pushed in or disconnected from the perfusion chamber 30, which facilitates the rinsing of tubes and the perfusion chamber 30 prior to steam autoclaving them.
  • the inlet and outlet ports 33, 34 are designed to accept standard 3/32" and 1/16" barbed, nylon adapters with 10-32 male threaded, receiving ports, respectively. These adapters can be either straight-through, for vertical tube connection, or elbow- like, to connect the tubes at a side.
  • the adapter type depends on the spatial constraints during imaging on either upright or inverted microscopes with episcopic illumination source, such as epifluorescence.
  • An insert plate can be custom made to match a particular microscope stage, see exemplary insert plate in Fig. 7, thus allowing perfusion experiments, injection of drugs, stimuli, cytokines and other reagents to be introduced during imaging.
  • a receiving tefzel, 10-32 female-to-luer adapters connect to polypropylene LuerTight fittings (Upchurch Scientific). These quick-disconnect fittings conform to medical luer taper configurations with simple connection to luer lock syringes.
  • Exemplary fittings accept 1/16" OD semi-rigid fluropolymer tubing such as the inert, Teflon FEP, transparent tubes with 100 to 500 ⁇ m microbores. If soft tubing is to be used, then the fittings are less bulky with barbed chamber-to-tube connectors, and, luer-to-barb adapters to connect tubes to syringes. Barbed fittings require larger bore tubes, thus increasing the amount of dead volume in the system, and when the amounts of used reagents become important, the use of harder tubes is definitely warranted. While the interfacing of the perfusion chamber 30 with the cm-scale integrated, diagnostics incubator 10 is fairly straightforward, hard tubing facilitates the placement of the incubator 10 onto a microscope stage with no interruptions to the flow whatsoever.
  • the perfusion chamber 30 and incubator 10 exposes the cells cultured in a culture chamber 20 to a continuous, direct flow of media through an array of micronozzles formed by the orifice plate 38 and creates 3-D, convectively biased circulation towards the perimeter of the culture chamber 20 where perfusate leaves through an array of microchannels 37.
  • the flow under consideration is studied experimentally using microscopic particle image velocimetry, ⁇ -PIV (see for example, Wereley ST. et al., Micron resolution particle image velocimetry, in Diagnostic techniques in microfluidics, Editor K.
  • PIV is a well accepted, non- intrusive measurement technique where the fluid velocity is measured by recording the displacement of small tracer particles added to the fluid under the assumption that the particle density is identical or commensurate to that of the surrounding fluid, and its size small enough to follow the flow faithfully without influencing the flow itself (low Stokes number).
  • Microscopic PIV is a modification of a standard PIV technique to enable spatially resolved measurements of instantaneous velocity distributions in sub-millimeter scale flow domains and allow detection of micron-scale spatial structures within the flow.
  • velocity resolution is within 5% of the microjet ejection velocity.
  • Velocity distributions in the x-y planes, parallel to the perufsing substrate 38, and normal to the axes of microjets emanating from about 570 square micronozzles formed at the perfusing substrate 38, are shown in Figs. 9a-l with the elevation, z, measured from perfusing substrate.
  • the field of view measures 450 x 457 ⁇ m and covers the central part of the pefusing substrate 38, Fig. 9a, with an array of about 6 x 6 nozzles in focus.
  • the nominal volume flow rate through the perfusion chamber is 5 ⁇ l/min with micro-jet ejection velocity of 30 ⁇ m/s and a nozzle based Reynolds number of 0.002.
  • a small culturing volume (about 7 ⁇ l) allows rapid exchange of perfusate (about 40 exchanges per hour for the present experiments) facilitates control, and reduces the amount of spent media.
  • flow is characterized by semi-confined, submerged, laminar impinging microjets issuing from an array of about 570 micronozzles (formed at the perfusing substrate 38) at moderate target-to-nozzle spacing.
  • microjets Upon their discharge from the nozzles, normal to the perfusing substrate 38, microjets interact with the stagnant medium within the culture chamber 20 as demonstrated in the sequence of images in Figs. 9b-d normal to the jet axe. This shear driven interaction enables them to spread out.
  • Measurements confirm that microjets issuing closer to the center of the perfusing substrate 38 penetrate deeper into the culture chamber 20 specifically targeting the most starved part of the tissue, while jet interactions and peripheral withdrawal of perfusate aid in the establishment of a complex 3-D flow within the culture chamber 20.
  • the perfusion chamber 30 with the centrally forming culture chamber 20 is placed inside a cm-scale incubator 10 comprising a polystyrene enclosure 1 1 using nylon inserts such that biocompatible fluidic couplings pass through the bottom of the enclosure 11 .
  • the heated enclosure 1 1 which may have exemplary outer dimensions of 50 x 25 x 18 mm, contains a prescribed concentration of gases and maintains the temperature of the media and gases at about 37°C.
  • the mini-incubator 10 contains a thermofoil heater 16 that heats the air inside the incubator 10. Gases necessary for cellular metabolism enter the incubator 10 through its inlet port 12 (forced by a mini-blower, for example, outside the incubator 10) and leave through the outlet port 13.
  • Externally located and controlled mini blowers 19 force the appropriate gas mixture (depending on the cultured tissue) over the heater 16 located on one side of the enclosure 1 1.
  • gases blow directly onto the surface of the heater 16, and are predominantly heated by forced convection inside the mini- incubator 10.
  • Heated gas warms the perfusion chamber 30 located downstream from the heater 16.
  • the surface of perfusion chamber 30 and the culture chamber 20 reaches the thermal equilibrium with surrounding gas within the heated enclosure 1 1 so that the nutrient medium entering the incubator 10 through fluidic coupling inserted into media inlet 33 (Figs. 4 and 5), warms up to the prescribed temperature, during the slow media advance towards the cell culture chamber 20.
  • the perfusion chamber 30 remains fairly isothermal so that the fresh nutrient medium injected into the culture chamber 20 through the perfusion substrate 38 reaches the desired temperature 37 ⁇ 0.2 0 C within its fluidic inlet port 33 well before entering the cultured volume, with the heated length increasing linearly with the flow rate.
  • the required heated length for the nutrient medium to reach desired temperature within the perfusion chamber 30 is sub-millimeter scale, i.e., 1 mm fluidic path through the inlet port 33, for example, is enough to warm the injected medium to a prescribed temperature.
  • the temperature of the injected media just underneath the perfusion substrate 38 may be measured using a thermocouple that is connected to the temperature controller 18 to maintain the temperature in the range of 36.8-37.2°C.
  • the PID controller 18 may be coupled to a DC switching solid-state relay to regulate the heat flux dissipated by the heater 16. Owing to its 7 ⁇ l of volume, culture chamber 20 and the entire perfusion chamber 30 facilitate temperature control due to relaxed working conditions and low time constants with negligible delays before parameters reach desired values. Small characteristic dimensions of the centimeter-scale incubator 10 result in negligible losses to the ambient. For example, for the heated enclosure 1 1 to be maintained at 37 0 C heater 16 needs to dissipate about 1W (based on losses to the ambient at 2O 0 C). Likewise, a fraction of a milliWatt is needed to warm the media to 37 0 C at the volume flow rate of 7 ⁇ l/min (about 1 exchange/minute) with sub-millimeter scale heated length through the perfusion chamber 30.
  • Temperature measurements inside the mini-incubator 10 may be taken at four locations, for example, using Fluke Hydra data acquisition unit with better than 0.1 0 C temperature resolution.
  • the temperature of the fresh nutrient medium is measured just before its injection into the culture chamber 20 (a thermocouple is placed immediately adjacent to the bottom side of the perfusion substrate 38 and routed through the media inlet port 33 via inlet tubing 24 and a small tee, and sealed).
  • the temperature of the air inside the return flow collecting pool 41 may be measured by a thermocouple routed through the media outlet port 34.
  • the temperature of air above the culture chamber 20 i.e., above the FEP membrane 21
  • Fig. 10 shows graph 63 that illustrate thermocouple temperature measurements following an initial 10-minute long controller auto-tuning.
  • the graph 63 sets forth measurements for ambient air 63a, air above the FEP membrane 63b, air inside the collecting pool 63c, and water just underneath the grid 63d.
  • Fig. 1 1 shows graph 67 that illustrates temperature measurements of ambient air 67a, air above the FEP membrane 67b, air inside the collecting pool 67c, and water just underneath the grid 67d while the incubator is in operation.
  • the PID controller 18 For the PID controller 18 to maintain the temperature between a low and a high set point (37 ⁇ 0.2 0 C), an auto-tune is performed during which an optimal set of constants (proportional, integral and differential) is found based on the thermal response of the system. After auto-tuning is finished, a low set point temperature is reached after about 45 minutes, as shown in Figure 10. In operation, the controller 18 utilizes this set of parameters to maintain the temperature within a prescribed range in response to changes of ambient air temperature. Measurements taken during a 3-hour period, for example, demonstrate that the controller maintains the temperature of the media just before injection into the culture chamber 20 within the prescribed limits.
  • a more uniform temperature distribution throughout the volume of the mini-incubator 10 may be easily obtained by insulating the exterior walls with a layer of foam-like tape, for example. This is also advantageous in preventing the visible light from entering the culture chamber 20 and disrupting the cells unless optical measurements are under way.
  • perfusion chambers 30 may be integrated within a single enclosure 1 1 , for example, each with its own independent temperature controllers, or using the same controller, and gas sources.
  • Several syringes can be used on each side of the syringe pump 19a moving block such that each perfusion chamber has an independent set of syringes for the simultaneous infusion of the culture medium and the withdrawal of catabolic waste products. This permits diagnostic testing of multiple samples in parallel without the possibility of cross contamination as perfusion chambers 30 are in fact microbe impermeable on the gas side.
  • the volume flow rate of injected media can be different between adjacent perfusion chambers 30 by use of different pairs of syringes for particular chambers. For example, one culture chamber 20 can have exchange rate as much as 4 times higher, or more, than the other by choosing a syringe pair whose plungers have the surface area 4 times the surface area of plungers in another pair of syringes.
  • the scale-down from standard (refrigerator size) incubators to a cm- scale, portable, integrated, diagnostics incubator 10 obtains a remarkable physical enhancements such as the efficiency in heating or cooling the enclosures as they are miniaturized. This facilitates temperature control as heated or cooled centimeter-scale incubator obtains faster response times to temperature changes due to negligible inertia of an inherently miniature device, and, reduces costs associated with power consumption.
  • Biocompatible and autoclavable, microfluidic perfusion chambers 30 may be rapidly prototyped using a process methodology that combines solid object printing and polymer replica molding (commonly referred to as the soft lithography, see for example Xia Y. and Whiteside G. M. in 1998 Angewandte Chemie 37:550-575). Custom or commercially available perfusion/ cell attachment substrates are then sealed to the bottom of the cultured domain with a PDMS prepolymer, and cured.
  • a disposable master 70 for the elastomeric molding are produced by solid-object printing (SOP) of an organic thermoplastic (wax-like) polymer that solidifies upon extrusion based on a defined 3-D CAD mold, for example.
  • SOP solid-object printing
  • This convenient inkjet printing tool eliminates the need for expensive cleanroom facilities, and, unlike inherently planar 2-D photolithographic techniques, obtains truly 3-D objects whose feature resolution is 86 ⁇ m.
  • production is virtually hands free as printer fabricates the masters, layer by layer, directly onto a platform without the use of masks. In a few hours over fifty wax molds may be fabricated in a single print.
  • the resolution of the commercially available Thermojet printer (3D Systems, Valencia, CA) used herein is 300 x 400 x 600 dpi, i.e. 85, 64 and 24 ⁇ m in x, y, and, z respectively.
  • the smallest extruded features of the perfusion chamber mold is a positive relief for the elastomeric molding of 150 ⁇ m wide and 350 ⁇ m deep microchannels that are reproduced faithfully owing to the excellent shape conforming properties of the PDMS prepolymer.
  • a 10:1 mixture of the PDMS pre-polymer base and cross-linking agent (SYLGARD 184 Silicone Elastomer Kit, Dow Corning, Midland, Ml) is poured into the wax template, cured at room temperature for 48 hours, and, peeled off.
  • the use of mold release agents was deemed unnecessary owing to favorable surface chemistries of materials in contact.
  • the top of their sidewalls are fabricated at a 45° angle. This ensures that the upper most layer of the polymeric PDMS material is entirely peeled-off, thus allowing good grip in stripping subsequent layers.
  • thermoplastic masters can be replaced by reusable, polymeric, master molds.
  • Reusable templates can be fabricated using a "sandwich method", i.e., by encapsulating the prepolymer within an extruded box, and, by inserting the cast PDMS perfusion chamber 30 into the box face down so that its base acts as a box Nd.
  • This enclosure box may be extruded using a 3-D printer such that the model interior dimensions correspond to the outer dimensions of the PDMS perfusion chamber 30.
  • the excess prepolymer leaves the mold through fluidic openings located on the base of the perfusion chamber 30 (box lid).
  • the procedure is straightforward and it takes only two sacrificial thermoplastic molds to fabricate a single reusable PDMS master.
  • Polydimethylsiloxane is a rubbery, biologically compatible, microfabrication compatible, silicone elastomer that is low-cost, and generally non-toxic, non-flammable, thermally stable, chemically inert, optically clear, permeable to gases including oxygen and carbon dioxide and almost impermeable to water, see for example, Ng J. M. K. et al. in 2002 Electrophoresis 23:3461 -3473. It conforms to submicron features with no discernible pattern degradation or surface distortion upon common biological handling and represents the material of choice for versatile biological and medical applications (see for example, McDonald J. C. et al. in 2002 Ace Chem Res 35:491 -499; or Whitesides G.
  • PDMS properties offer substantial advantages over fabrication methods in hard materials. Molding of 3-D structures in PDMS is inexpensive, easy, and a versatile method of making complex geometries.
  • the sealing of fluidic devices, coupling of components, packaging and interfacing of integrated systems is fairly straightforward to employ in PDMS owing to its elasticity, watertight sealing properties and low electrical conductivity.
  • the modular design of the perfusion chamber 30 enables the use of commercially available or custom made substrates, thus introducing specific topography, flow characteristics and surface chemistry intended for particular set of experiments.
  • PDMS polymethyl methacrylate
  • PMMA polymethyl methacrylate
  • the ease of PDMS topographical and chemical patterning makes it an ideal candidate for perfusion substrate whose shape, texture and surface chemistry can be adjusted to promote cellular adhesion.
  • silane an amino-terminated silane
  • a similar technique can be employed to attach amino-terminated polyethylene glycol (PEG) to make the surface stable against surface rearrangement and hydrophilic for days in air, e.g., Donzel C, et al. in 2001 Adv Mater 13:1 164-1 168.
  • PEG polyethylene glycol
  • Bio-fouling a non-specific protein adsorption to surfaces such as the interiors of microbore tubes or microchannels, for example, represents a common problem in microfluidics as it eventually leads to device clogging. Grafting a poly(ethylene glycol)di- (thethoxy)silane onto an oxidized PDMS surface, the surface of PDMS becomes permanently hydrophilic with reduced bio-fouling properties, see for example Papra A. et al. in 2001 Langmuir 17: 4090-4095).
  • the disclosed instrumented, portable, centimeter-scale incubator 10 represents a striking departure from the standard culture environment posing virtually no limits for field applications with the convenience of being compact, and, flexibility in design and operation that allows its integration into a more robust totally integrated systems.
  • advantages of the miniaturized incubator 10 include (1 ) reduced size of the functional device, (2) flexibility in design (3) minimized response times in pharmacological and biochemical analyses owing to microliter volume culture chamber 20 and negligible time- delays in reaching steady state operation, (4) low expenditures associated with the amount of used reagents, (5) reduced production of toxic waster products, (6) decreased requirements for power, (7) reliable and reproducible operation owing to relaxed operating conditions associated with superior control of physical and chemical parameters in small scale devices, (8) increased speed of analyses, (9) the ability to operate in parallel a number of perfusion chambers 30 onboard a single centimeter-scale incubator 10, (1 1 ) low-cost devices resulting from fabrication of all components onboard a single, disposable platform 30, (12) optical, electrical and fluidic accessibility, (13) facile fabrication, packaging, integration and interfacing, and, (14) portability. Disclosed perfusion chamber 30 enables forced convection intercellular nutrient delivery thus extending the longevity of otherwise nutrient and gas depleted 3-D cultures.
  • a reduced-to-practice mini-incubator 10 enables simultaneous optical, fluidic and electrical interfacing, and data acquisition, in a controlled environment prescribed by the temperature, concentration, and flow rate of gases, nutrients or other relevant substances.
  • the temperature of the media inside the incubator 10, just before injection into the culture chamber 20, is controlled at 37 ⁇ 0.2 0 C.
  • Associated infrastructure can support a number of mini-incubators 10 with their separate perfusion chambers 30. In-situ diagnostic testing may be performed without disruption of cultured tissue within separate mini-incubators 10, thereby reducing the chances of contamination.
  • the centimeter-scale incubator 10 with its microliter volume perfusion platform 30 offers greater control over the cellular microenvironment. Using relatively small number of cells at a high plating density, cellular response to rapid changes in bathing medium composition, application of drugs, or the changes in the extracellular ionic concentration governing the cellular excitability can be accurately controlled and easily altered. While relevant scientific studies have been hindered by diffusion limited supply of the nutritive substances and gases necessary for cellular metabolism, the perfusion chamber 30 exposes the cells to continuous flow of media with rapid exchange rates to ensure plethora of nutrients and prevent the metabolic culture decay. Thus, in the centimeter-scale incubator 10, 3-D cultures can be easily maintained in vitro over extended periods of time with sufficient concentration of nutrients and gases at an optimal value of acidity and temperature so that the humoral states are not altered during the course of an experiment.
  • biocompatible perfusion chamber 30 ensures forced convection closed-loop continuous infusion of nutrients and withdrawal of waste with enhanced exposure to gases necessary for cell metabolism to extend the culture longevity beyond that achieved in large-scale incubators or small incubation baths.
  • Disposable or steam autoclavable perfusion chamber 30 can be easily re-configured to meet a particular demand with a new device in hand in a matter of hours without compromising its simplicity.
  • mini-incubator 10 facilitates plug- in functionality of various building blocks that can be easily integrated (e.g., mixing chambers, nano-volume multi-port controllable injectors, microelectrode arrays, sensors, etc.) and allows operation either in full-scale laboratories or in the field, providing a portable, fully self-contained biological workstation.
  • perfusion chamber 30 may enable temporal and spatial control of injected agents in addition to continuous delivery of the cell culture media
  • Customized sample holders can be fitted as necessary with fluidic and electrical connectivity to facilitate in situ sample manipulation and diagnostics with negligible risk of contamination.
  • the modular infrastructure can support a number of mini-incubators 10 with their separate perfusion chambers 30 that can be easily plugged in and out of a centralized system, providing maximum flexibility in experimental design and minimal capital expenditure. In-situ diagnostic testing may be performed within separate incubators 10, eliminating disruption of other cultured tissue and thereby dramatically reducing the chances of contamination.
  • Various workstation designs may be constructed and inexpensively customized to allow for concurrent optical, fluidic and electrical interfacing to permit full experimental control, including data acquisition and individual sample preservation. [0090]
  • the incubator workstation 10 has optical, environmental and fluidic accessibility and enables simultaneous culturing and analyses on a microscope stand.
  • Integrated, actively controlled, perfusion chamber 30 utilizes forced convection for intercellular nutrient supply and gas exchange thereby allowing the studies of thicker tissue slices and dissociated, high-density, three-dimensional (3-D) cultures over extended periods of time.
  • Miniature cell culture life support chamber 20 (about 7 ⁇ l) offers relaxed operating conditions with superior environmental control and reduced expenditures associated with harvesting, culture preparation, the amount of used reagents and power consumption.
  • a number of perfusion chambers 30 can be integrated onto a common platform for high-throughput culture screening with reduced risk of contamination.
  • Superior culture aeration is achieved twofold: (i) through an optically clear, microbe and water impermeable membrane 21 placed over the top of the culture chamber and via (ii) separate in-line venting bubble trap 26.
  • a thin membrane 21 that encapsulates the perfusion chamber 30 enables efficient gas exchange and keeps the tissue moist in a sterile environment. It further reduces the risks of contamination by allowing the culturing in non-humidified incubators.
  • the in-line aerator 26 ensures the equilibrium of the nutrient medium with the gas environment, via semipermeable membrane 27, before being injected into the culture chamber 20. This device 26 also serves as an auto-venting bubble trap that prevents the bubbles from ever reaching the perfusion chamber 30.
  • in-line aerator 26 eliminates the need for a separate enclosure to equilibrate the media and a bubble trap to purge the bubbles, thus greatly simplifying the design and enabling development of cost-efficient compact devices.
  • Spatially 42 or temporally varying concentrations of injected substances 41 into the culture chamber 20 can be dynamically controlled. Additional functionalities including but not limited to glucose consumption, pH or dissolved O 2 sensing and control 18a can be easily integrated into the device 30.
  • Shape conforming and contact sealing properties of the polydimethylsiloxane polymer used in the fabrication of a perfusion chamber 30 enable straightforward fabrication of complex three- dimensional structures, sealing of devices, coupling of various microfluidic and electrical components, packaging and integration into functional systems that can be easily interfaced with the macro world, see for example Beebe D.J. et al. in 2000 Proc Natl Acad Sci USA 97: 13488-13493; Chabinyc M. L. et al. in 2001 Anal Chem 73: 4491 -4498; or Zanzotto A. et al. in 2004 Biotechnol Bioeng 87:243-254.
  • the modular design with versatile plug-in-functionalities can be tailored to specific use and omitted in particular application.
  • These functionalities include but are not limited to: microfluidics, e.g. micropumps, valves, mixers and microinjectors; introduction of spatially or temporally varying concentration gradients of injected substances into the culture chamber 20; the integration of microelectrode arrays for physiological studies; biosensors for detection and control of small molecules (oxygen, pH, glucose) and large molecules (immunosensors); the capabilities of cooling below the ambient temperature to preserve the tissue for example; or the packaging of a larger number of culture chambers onto a common platform.
  • Controllable features include but are not limited to: the adjustable shape and texture of the cell culture life support chambers, specific surface chemistry or protein adsorption, and, tunable topography of extracellular matrices and chemically patterned microfabhcated scaffolds that can only be realized in a microfabrication compatible, low-cost, mass produced, dynamic perfusion platforms.
  • the disposable or autoclavable culture chamber 20 measures about 7 ⁇ l in volume and offers superior control of the cellular microenvironment, see for example Vukasinovic J. et al. in 2006 Proceedings of the 2006 ASME Summer Bioengineering Conference. Owing to the facile fabrication methodology, straightforward integration and packaging, perfusion chamber 30 and the miniature incubator 10 can be easily modified to meet different requirements.
  • the ultimate challenge of diagnostic cell assays is to incorporate biosensors and control loops to monitor temperature, humidity, pH, oxygen or ionic concentration in a perfused system with adjustable, spatially selective, steady state or temporally varying, velocity or concentration gradients, all within a common platform that contains a large number of cell arrays ready for fundamental studies, optical diagnostics, and, intra- or extracellular stimulation and recording.
  • biosensors and control loops to monitor temperature, humidity, pH, oxygen or ionic concentration in a perfused system with adjustable, spatially selective, steady state or temporally varying, velocity or concentration gradients, all within a common platform that contains a large number of cell arrays ready for fundamental studies, optical diagnostics, and, intra- or extracellular stimulation and recording.
  • the perfusion chamber 30 not only obtained high viability of perfused cortical slices, but also the perfusion of nutrient medium in larger scale chambers of a similar design proved to be a productive tissue engineering setting enabling the growth of high-density, physiologically more credible, 3-D tissue-equivalent constructs.
  • Perfused neuronal-astrocytic co-cultures grown in an extracellular matrix at cell densities close to that of the brain tissue (10,000 cells/mm 3 ) exhibited over 90% viability throughout their thickness (500-750 ⁇ m) while non-perfused, sister-cultures evidenced widespread death as described by Cullen D. K. et al. in 2006 at the 28TM Annual International Conference IEEE Engineering Medicine and Biology Society, and by Cullen D. K.
  • biocompatible perfusion chamber 30 integrated within a centimeter-scale diagnostic incubator 10 lies in its straightforward adaptability to various application challenges by incorporating versatile microfluidics and MEMS functionalities with superior control of pertinent cellular microenvironment than that achieved in standard, multi-well screening plates, Petri dishes, or Haas- or Zbicz-top incubation baths.
  • Other enabling qualities include the ability to integrate multielectrode arrays or biosensors to monitor physiological functions down to cellular level, and, generate complex, steady state e.g. Dertinger S.K.W. et al.
  • Perfusion can be continuous, cyclic in a periodic or an aperiodic fashion following a specific control curve, push/pull, and could involve the re-use of the spent media for subsequent feedings in cases where high-exchange rates may lead to depletion of trophic factors secreted by the cells to support their own growth.
  • the dynamic perfusion system 30 facilitates the control over the composition of the bathing medium, its temperature, pH and the degree of oxygenation.
  • non-invasive sensing methods may be employed in measuring the amount of dissolved oxygen and pH using either the commercially available sensor foils, or lifetime fluorescence of oxygen and pH- sensitive dyes.
  • the centimeter-scale integrated diagnostics incubator 10 is thus portable. Relevant hardware (pump, fan, controllers) may be battery operated and the appropriate gas mixture supplied in a small bottle. The same incubator 10 can be used to preserve biological specimen at a temperature that is below the ambient temperature by replacing the resistive heater with a miniature thermoelectric heat pump.

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Abstract

La présente invention concerne des incubateurs de diagnostic intégrés, portables, jetables, à l'échelle du centimètre, utilisables pour les cultures biologiques. Un incubateur à titre d'exemple comprend une enceinte optiquement accessible ayant une pluralité de ports pour fluides. Un élément chauffant installé à l'intérieur de l'enceinte est raccordé à une commande de chauffage externe. Une chambre de perfusion de micro-fluides pouvant passer à l'autoclave est installée à l'intérieur de l'enceinte qui comprend une chambre d'entretien de la vie de culture de cellules, un port d'entrée placé dans la chambre de perfusion, une chambre de prélèvement en communication avec la chambre de culture, un port de sortie raccordé à la réserve de prélèvements et un substrat de perfusion. Une membrane optiquement transparente et perméable aux gaz peut être fixée sur le haut de la chambre de perfusion. Les incubateurs sont accessibles optiquement et ils sont équipés d'une commande de l'écoulement des fluides et d'une régulation de la température, ils sont portables et modulaires et leur construction est bon marché. Les incubateurs permettent de pratiquer des essais de médicaments et des cultures de tissus biologiques sur place.
PCT/US2007/072777 2006-07-07 2007-07-03 Incubateur de diagnostic intégré à l'échelle du centimètre pour cultures biologiques WO2008005998A2 (fr)

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US85122206P 2006-10-12 2006-10-12
US60/851,222 2006-10-12

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CN110108622B (zh) * 2019-06-17 2024-02-02 辽宁中医药大学 一种离体微血管内皮屏障功能检测方法与设备
CN110108622A (zh) * 2019-06-17 2019-08-09 辽宁中医药大学 一种离体微血管内皮屏障功能检测方法与设备
CN111205980A (zh) * 2020-01-20 2020-05-29 河海大学常州校区 一种单细胞原位实时显微观测的细胞培养装置及其操作方法
CN111205980B (zh) * 2020-01-20 2024-01-26 河海大学常州校区 一种单细胞原位实时显微观测的细胞培养装置及其操作方法

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