WO2015021513A1 - Device for storing a tissue sample - Google Patents

Device for storing a tissue sample Download PDF

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Publication number
WO2015021513A1
WO2015021513A1 PCT/AU2014/050188 AU2014050188W WO2015021513A1 WO 2015021513 A1 WO2015021513 A1 WO 2015021513A1 AU 2014050188 W AU2014050188 W AU 2014050188W WO 2015021513 A1 WO2015021513 A1 WO 2015021513A1
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WO
WIPO (PCT)
Prior art keywords
storage device
fluid
chamber
storage
storage chamber
Prior art date
Application number
PCT/AU2014/050188
Other languages
French (fr)
Inventor
Yossi BUSKILA
Paul Breen
James Wright
Original Assignee
University Of Western Sydney
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from AU2013903100A external-priority patent/AU2013903100A0/en
Application filed by University Of Western Sydney filed Critical University Of Western Sydney
Publication of WO2015021513A1 publication Critical patent/WO2015021513A1/en

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M45/00—Means for pre-treatment of biological substances
    • C12M45/22—Means for packing or storing viable microorganisms
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0236—Mechanical aspects
    • A01N1/0242—Apparatuses, i.e. devices used in the process of preservation of living parts, such as pumps, refrigeration devices or any other devices featuring moving parts and/or temperature controlling components
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0236—Mechanical aspects
    • A01N1/0263—Non-refrigerated containers specially adapted for transporting or storing living parts whilst preserving, e.g. cool boxes, blood bags or "straws" for cryopreservation

Definitions

  • the present disclosure relates to a device to provide optimal conditions for the survival of a tissue sample to extend the lifespan of the tissue for experimental purposes.
  • Tissue samples and slices are used for various investigations including
  • a storage device for a sample of animal tissue including a storage chamber configured to receive a tissue sample and fluid therein; a control system to control environmental conditions in at least the storage chamber, said environmental conditions including at least one of temperature, oxygenation and pH; and an antimicrobial system.
  • a method of storing a tissue sample from an animal comprising: immersing said tissue sample in a fluid; controlling environmental conditions of said fluid including controlling at least one of pH. oxygenation and temperature of said fluid; and treating the fluid to reduce the number of or remove microorganisms therein.
  • a storage device for a sample of animal tissue including a storage chamber configured to receive a tissue sample and fluid therein; a control system to control the environmental conditions in at least the storage chamber, said environmental conditions including temperature, oxygenation and pH.
  • the storage device is typically configured to automatically monitor and control environmental conditions it provides for the tissue sample in accordance with pre -determined environmental parameters. The environmental conditions may vary depending upon the type of tissue sample stored therein.
  • the storage device typically includes a main housing.
  • the main housing may include a storage chamber to receive and store the tissue sample.
  • the storage chamber may also be configured to receive a fluid and may further comprise a removable structure to house the tissue sample.
  • the removable stucture may include a mesh tissue holding platform to receive the tissue sample.
  • the removable structure of the storage chamber may be configured such that when a sample of tissue is housed thereon or therein, the removable structure allows complete immersion of the tissue sample into the fluid received within the storage chamber.
  • the storage chamber has a base and sidewalls extending therefrom to an open top end.
  • the storage chamber typically has an upper portion adjacent the top end and a lower portion adjacent to the base.
  • the removable structure may be sized such that the tissue holding platform is positionable within the lower portion of the storage chamber. This embodiment may allow the use of less fluid to fully immerse the tissue sample.
  • the main housing may comprise a lid on an upper surface to allow placement of the tissue sample in the storage chamber, and removal therefrom.
  • the lid may form a tight seal with the remainder of the main housing.
  • the lid may not tightly seal the main housing such as to avoid a build up of pressure in the housing during use.
  • the entire storage device is surrounded by an insulating member.
  • the insulating member may provide thermal insulation while also preventing UV
  • the insulating member may insulate only the main housing particularly in an embodiment wherein the antimicrobial system is spaced from the main housing.
  • the insulating member may insulate various parts o the device including but not limited to the antimicrobial system.
  • T e insulating member may be made from any suitable material to thermally insulate and in some instance prevent UV transmission.
  • the insulating member may comprise a foam material. Examples include but are not limited to polystyrene foam and polyurethanc foams. In another embodiment the insulating member may be made from fibreglass.
  • the fluid may be tissue specific, that is, it may provide an optimal environment for storing a particular (issue type.
  • the tissue type may comprise connective, neural, muscle or epithelial tissue.
  • tissue samples include but are not limited to Mood, bone, smooth, skeletal or cardiac muscle, neural tissue from cither the central nervous system or the peripheral nervous system, squamous epithelium, cuboidal epithelium, columnar epithelium, glandular epithelium or ciliated epithelium.
  • the tissue may be sampled from a number of organs of a subject including but not limited to the brain, heart, lung, liver, kidney, skin or eye.
  • the sample chamber may receive an artificial cerebrospinal fluid (aCSF).
  • aCSF cerebrospinal fluid
  • the tissue may comprise a brain tissue slice.
  • the aCSF typically provides a particular ionic environment suitable tor neural tissue, including any one or a plurality of ions selected from sodium, potassium, calcium, magnesium, phosphorus and chloride.
  • the aCSF may be prepared by dissolving a number of compounds in water, said compounds including any one or a combination of sodium chloride, potassium chloride, disodium phosphate, calcium chloride, sodium carbonate.
  • the aCSF may further include any one of monosodium phosphate or sodium bicarbonate.
  • the aCSF includes glucose
  • the control system may comprise one or more inlets from a gaseous source to the interior of the device.
  • the one or more inlets may be configured to deliver gas to the storage chamber.
  • the inlet may deliver oxygen to the storage chamber.
  • a mixture of oxygen and carbon dioxide may be delivered to the storage container.
  • the ratio of oxygen to carbon dioxide may vary and includes 95% oxygen to 5% carbon dioxide
  • the device may further include an inlet control mechanism to control the flow of gas from the gaseous source to the storage chamber.
  • the control may comprise a sensor to sense a particular environmental condition in the storage chamber.
  • the sensor may be coupled to a controller.
  • the control mechanism may comprise an output from the controller to cause a variation in the flow of gas depending upon input signals from th sensor.
  • die concentration of a particular gas in the storage chamber may affect the pH of the fluid held therein.
  • the inlet control mechanism may, therefore, include a pH sensor coupled to the controller. Variations of the pH may be sensed by the sensor and then processed by the controller. In accordance with pre-calculated values, the controller may cause the rate of flow of die gas into the storage chamber to vary relative to input pH values sensed by the pH sensor. The controller may control the flow by control of a valve in the inlet. For example, the valve in the inlet may be caused to move between a closed and an open configuration to control the flow of gas into the fluid depending upon the pH of the fluid as sensed by the pH sensor.
  • the pH of the fluid is controlled to between pH 7 and pH 8. Still further, the pH may be controlled to be between pH 7 and pH 7.5; or between pH 7.1 and pH 7.4; or between pH 7.2 and pH 7.4; or between pH 7.2 and pH 7.3.
  • the device may further include a temperature control mechanism to maintain the tissue sample at a desired temperature.
  • the temperature control mechanism may comprise a heating element.
  • the device may include a cooling member.
  • the heating element or cooling member may be positioned within the storage container or, alternatively, adjacent thereto.
  • the temperature control mechanism may further include a thermometer to measure the temperature of the fluid and a feedback mechanism comprising a controller configured to receive an input from the thermometer and send an output signal to the heating clement or cooling member. Variation of the temperature of the fluid will cause a variation in the output of heat from the heating element to either increase heat output or decrease heat output. Similarly, if a cooling member is present, the output signal may cause a cooling of the fluid in the storage chamber. Further, the controller may control the on/off function of the heating dement or cooling member.
  • a safety mechanism may be in-built in the temperature control mechanism to cause the heating element to switch off if a temperature of the fluid reaches a pre ⁇ determined temperature.
  • the device includes one or more heating and cooling plate members.
  • the device may comprise a Peltier device.
  • the device typically includes at least a first plate attached to a heat sink and a fan member and at least a second plate attached to the storage chamber. Said plates may be attached to the beat sink and fan and the storage chamber respectively by a thermo-conductive compound. Applying a current to the plates causes one of said plates to heat up and the other plate to cool. The direction of the current applied determines which plate heat and which cools.
  • the temperature of the storage chamber may be varied ami is controllable to achieve a desired temperature.
  • the plates of the Peltier device may attach to a third plate, said third plate in connection with the storage chamber. This embodiment enables removal of the storage chamber without affecting the temperature control plates.
  • the temperature may be prc-selected by a user depending upon the type of tissue to be stored.
  • the temperature may range between 4 C and 40' C. Furthermore, the temperature may vary over a time frame.
  • the device may further include a pump.
  • the pump may be configured to pump fluid within the storage chamber to achieve a flow of fluid therein. Further, the pump may be configured to pump said fluid from the storage chamber to another chamber of the device.
  • the antimicrobial control system may include an ultraviolet ⁇ UV) tight source.
  • the UV light source may be housed within an antimicrobial chamber.
  • the antimicrobial chamber is typically spaced from the storage chamber.
  • the two chambers may be fluidly connected to each other such that a fluid may be transferred therebetween.
  • the pump may pump the fluid from the storage chamber to the antimicrobial chamber for exposure to UV light. Such exposure may kill microoraganims present in the fluid.
  • the pump may continually pump the fluid between the antimicrobial chamber and the storage chamber. Alternatively, the pump may intermittently pump the fluid between the two chambers.
  • the pump may have a variable output to pump the fluid at a variable flow rate.
  • the flow of fluid within the storage chamber provides a physical "wash" of the tissue sample which further aids in removing bacteria from the surface of the tissue.
  • the antimicrobial system may also include a microbial sensor which senses the presence of or the concentration of certain gases in the fluid as an indicator of microbial presence/activity. An increase in concentration of a gas in this embodiment is indicative of an increase in microbial numbers.
  • the sensor may comprise a nitric oxide (NO> sensor.
  • the antimicrobial system comprises a UV chamber which has a main housing and an inner housing.
  • the UV chamber houses a UV bulb.
  • the UV bulb may be substantially encased in the inner housing.
  • the inner housing is typically spaced from the main housing such as to define a fluid path.
  • the fluid pad may extend from an inlet in the main housing to an outlet in the main housing.
  • the fluid path typically substantially surrounds the UV bulb.
  • the inner housing may be made from any suitably resilient material and in one embodiment, the inner housing is made from quartz. Other UV and in particular UVC transparent materials are also envisaged.
  • the UV bulb may be connected to a power source typically through a sealed lid of the UV chamber.
  • the UV bulb may be powered to provide constant UV irradiation.
  • the application of UV light may be intermittent.
  • the application of U V light may be randomised.
  • the application may be more ordered and follow a predetermined pattern.
  • the UV light may be applied in a pulsed fashion.
  • a non- constant application of UV light may prevent excessive heating of the fluid in the fluid pathway in addition to preventing bacterial UV resistance.
  • the UV bulb is controlled by a programmable timer to turn on at varying times. Intervals of "on" time may be selected from between 2 and 30 minutes and preferably between 15 and 26 minutes.
  • the frequency of illumination may vary and may include intervals in the range of every 15 minutes through to every 30 minutes.
  • the OV light may have a wavelength of between about 280nm and about lOOnm.
  • the UV light may be UVC light
  • the main housing of the If V chamber may also comprise a U V blocking layer. It is preferred that the application of U V light is substantially contained within the UV chamber to prevent irradiation of any tissue in the device because neuronal tissue may be damaged by UV irradiation.
  • the UV blocking layer may comprise an outer layer or film.
  • the main housing wall may be made from a suitable material to block the transmission of UV wavelengths.
  • the UV blocking layer may comprise an aluminium foil cover which extends around the main housing.
  • the device may further include a titter to catch debris.
  • debris includes dead microorganisms or parts thereof.
  • At least any one of the components of the device including the storage chamber, any part of the control system or the antimicrobial system may be removable from the device to facilitate washing or other required actions such as repair of components.
  • the device is configured such that all the components (herein are removable together as a unit.
  • the storage device may be powered from a mains supply. Further, however, the device may include a re-chargeable power back up to allow for movement between locations such as different laboratories without disturbing the environment of the tissue sample.
  • the storage device may further comprise a user interface.
  • the user interface may comprise a display screen to provide real time data in relation to the environmental conditions in the device.
  • the data may include one or a combination of temperature. pH, fluid flow rate, microbial presence and activity and power status of the device.
  • the device may also include a manual over-ride system to allow a user to over-ride the automatic control of the
  • the device may be controlled remotely such as via an internet connection.
  • the control may be wireless.
  • Data from the device may be obtainable and one or more parameters of d e device may be controllable via a smart phone or other smart device.
  • a particular protocol may be initiated remotely by a user. Further, a particular protocol of the device may be modified and implemented remotely depending upon monitored conditions in the device. Such remote monitoring and control of the device enables a user to modify the conditions in real time and continuously to optimise the conditions therein.
  • the device may also include an alarm to represent an event.
  • the alarm may be in a number of forms and includes visual and auditory signals.
  • the alarm may be deliveied to a user via a smart device or phone.
  • the alarm may represent the end of a cycle; the malfunction of any components: and any other desired condition in the device to allow a user to determine the condition of the tissue sample.
  • Figure 1 is a schematic depiction of an embodiment of the storage device of the present disclosure
  • Figure 2 is a flow diagram to depict the various elements of the device of Figure 1 ;
  • Figure 3a and 3b are schematic representations of another embodiment of the device.
  • Figure 4 is a graph showing the results of cell viability for cells stored in a control environment compared to cells stored in the device of the disclosure:
  • Figures 5a and 5b arc graphs showing bacterial cell count over time for slices stored in the device and in a control device.
  • the storage device of the present invention is generally depicted as 1 in the attached drawings.
  • the device 1 is used to store a tissue sample and in one embodiment described herein, the tissue sample comprises a brain slice.
  • the usable lifespan of a brain slice is 6-8 hours.
  • the device of the present disclosure is configured to extend the lifespan of a brain slice sample beyond the current 8 hour window which therefore provides opportunities to study long term effects of various metabolic conditions or pharmaceuticals on brain tissue.
  • the present device provides an environment for maintaining a healthy brain slice over a long period by providing the control of at least one of temperature, pH and oxygenation. By increasing the lifespan by optimising the environmental conditions, microbial levels in die fluid in which the tissue sample is immersed may increase.
  • An initial Lag Phase is a period of slow growth during which the bacteria are adapting to the conditions in the fresh medium.
  • a Log Phase during which bacterial growth is exponential, doubling every replication cycle.
  • the Stationary Phase occurs when the supply of nutrients becomes a limiting factor and the rate of multiplication equals the rate of death.
  • the Logarithmic Decline Phase occurs when bacteria die taster than they are replicated.
  • recordings from brain slices are constrained to the Lag phase, in which the amount of bacteria is low and not affecting ceil viability.
  • the device therefore includes an antimicrobial system to address the problem of increasing microbial numbers and resultant cell death during storage of the tissue sample.
  • the storage device 1 includes a main housing 10 and a storage chamber 11 which is configured to receive a tissue sample (not shown).
  • the storage chamber 11 also receives a fluid 12 to immerse the tissue sample.
  • the device 10 also has a control system which controls a number of different functions and environmental parameters in the device 1. This is also shown in the flow diagram in Figure 2. Temperature
  • the device 1 includes a heating and/or cooling element 13, which is controlled by a microcontroller 34.
  • Microcontroller 34 receives input signals from a thermometer IS in the storage chamber 11.
  • the heating and/or cooling element heats or cools fluid 12 until a desired temperature is achieved.
  • microcontroller 34 may effect switching off the heating and/or cooling element 1 or may reduce the output of the heating and or cooling element to an output that maintains the desired temperature of the fluid.
  • heating and or cooling element 13 it is envisaged that this encompasses a Peltier device as described herein.
  • the control stem of device 1 also controls the del i very of a selected gas from gaseous source 16.
  • the gas is introduced through inlet 17 and microcontroller 34 controls the flow via valve 8.
  • the gas in device 1 is a mixture of oxygen and carbon dioxide to perfuse the tissue in storage chamber 11. Control of the gas may be determined in relation to other parameters such as pH as discussed below. pH
  • pH is an important environmental factor in preserving a tissue sample and it may vary over time. Further, variations in pH occur as a result of gas introduced into fluid 12, particularly the introduction of a mixture of oxygen and carbon dioxide.
  • Microcontroller 34 controls the pH by receiving pH input and effecting an output to maintain a constant pH.
  • the output in tins embodiment comprises controlling the flow of gas via valve 8.
  • Storage chamber 11 also includes a removable tray 18 which is introduced into and removed from the storage chamber 11 via lid 20 in main housing 10.
  • the removable uay 18 is configured to hold a tissue sample therein.
  • Handle 19 enables a user to place the tray 8 and sample into the storage chamber 1 1 until the sample is fully immersed in fluid 12.
  • the removable tray 18 in the depicted embodiment is made from a mesh material to allow fluid to immerse the sample as well as to allow fluid to drain through it when removed from the storage chamber.
  • fluid 12 is an artificial cerebrospinal fluid and includes (in mM) 124 NaCl. 3.5 KCI, 2 MGS04, 1.25 Na HP0 .2 CaCfe, 26 NaHC(1 ⁇ 4 and 10 glucose
  • the device also includes an antimicrobial system 22.
  • the antimicrobial system treats the fluid 12 to remove or kill microorganisms therein. Microorganisms are considered to include but are not limited to bacteria, viruses, fungi, yeasts and protozoa.
  • the antimicrobial system includes a conduit 23 extending from the storage chamber I to a UV filter chamber 24. Storage chamber 11 and U V filter chamber 24 are fluidly connected via conduit 23 which further includes a pump 21 to pump the fluid from the storage chamber 11 to the UV filter chamber 24.
  • UV filter chamber 24 me fluid is irradiated with UV light from a UV light source 25 to kill microorganisms that are present in the fluid. From here, the fluid is pumped back into the storage chamber 11. As can be seen, the fluid is pumped back via return conduit 26 and re-introduced back into the storage chamber 11. Conduit 26 may further include a filter to trap dead microorganisms and other debris therein and avoid re- introduction into the storage chamber 11.
  • the conduit 26 connects with a perforated head member 27 such that the fluid is returned to essentially "shower” the sample in the storage chamber. This has the benefit of further washing microorganisms from the surface of the sample.
  • the height of the perforated head member 27 may be adjustable relative to the storage chamber.
  • Figure 2 shows a flow diagram of the main components of device I which is typically powered by an external power source 31 although a re-chargeable battery 32 is also provided for back up and transport of the device.
  • Device 1 has a power control 33 to power the various components and
  • microcontroller 34 The microcontroller controls output to pump 21, UV source 25 and valve 8.
  • Microcontroller 34 receives input signals from thermometer 15 and a pH meter 35 and effects change in temperature and pH accordingly via heating element 1 and gas in flow via valve 8 respectively.
  • Microcontroller 34 furtljcr receives input signals from and effects output to lid 20 for opening and closing of lid 20.
  • FIG. 3a depicts antimicrobial system 101 and Figure 3b depicts main bousing 102.
  • Antimicrobial system 101 comprises a main housing 103 and an inner housing 104.
  • a UVC bulb 105 is housed within inner housing 103.
  • Main bousing 103 is covered with a U V blocking layer (not shown).
  • the system 101 also includes a lid 106 which is sealed to die main housing 103.
  • UVC bulb 105 is connected to a power source via cable 107 which passes through ltd 106.
  • the material of the inner bousing is thermally insulating and includes a quartz material to prevent over-heating of the fluid 108 in the main housing 103. Fluid 108 is passed into the main housing 103 through inlet 109 and removed from the main housing through outlet 110.
  • the main housing 102 of Figure 3b includes a mesh layer 120 to suppor the sample of tissue to be stored.
  • This embodiment also includes a series of probe mounting units 1 1 which ate configured to receive probes such as temperature or pH probes.
  • the main housing also includes a fluid inlet 122 and fluid outlet 123. Carbogen inlet 124 is also depicted.
  • the entire main housing 102 in this embodiment is insulated by a polystyrene foam casing 130 which provides thermal insulation.
  • Wistcr rats were deeply anesthetized by inhalation of isoflurane (5%), decapitated, and their brains were quickly removed and placed into ice-cold physiological solution (artificial CSF) containing (in niM): 125 NaCJ, 2.5 KCI, 1 MgC12. 1.25 NaH2P04. 2 CaCI2, 25 NaHC03, 25 dextrose and saturared with carbogen (95% 02 -5% C02 mixture; pH 7.4). Parasagittal brain slices (300 ⁇ thick) were cut with a vibrating microtome (Camden Instruments, UK) and transferred to a holding chamber containing carbogenated aCS for 30 min at 35 "C.
  • slices weie either allowed to cool to room temperature (-22 °C) in the same recovery chamber (Control chamber) or transferred into a custom-made incubation system mat closely monitored and controlled pH levels, carbogen flow and temperature as well as irradiating bacteria through a separate UV chamber as herein defined as device 1 or 100.
  • Slices were kept either the control chamber or in the chamber of device 1 or 100 for at least 30 nun before any measurement.
  • the fluid for both the control and as used in device 1,100 was the same aCSF.
  • Acute slices were incubated for 15 min with the selective dead cell fluorescent marker propidium iodide (PI, 1 jig/ml). For total cell counts, slices were co-incubated with the nuclear marker DAPI (lug/ml). Following incubation, slices were washed with fresh aCSF for 10 min. Images were acquired using a Zeiss LS -510 Meta confocal microscope (Carl Zeiss, Obcrkochen, Germany) using a 40x-oil immersion objective in the inverted configuration. Z plane optical sections (4 urn) were taken at -20 to -70 ⁇ depth from the surface of the cerebral cortex to produce an image stack.
  • PI selective dead cell fluorescent marker propidium iodide
  • the DAPI signal was obtained using Argon laser excitation at 488 nm; PI was excited with 543-nm HeNe laser. Images were visualized using ZEN software and processed. Slice viability was assessed as the ratio between dead/total cells in the visual field.
  • a recording chamber was mounted on an Olympus BX-51 microscope equipped with I.R/D1C optics. During recordings the vSlices were kept at room temperature, ⁇ 22 0 C. and constantly perfused (2-3 ml/min with oxygenated solution. Whole cell recordings were perforated from the soma of layer 5 pyramidal neurons in the somatosensory cortex with patch pipettes (5-7 Mil) containing ⁇ in mMi 130 K-Metbansulfate, 10 HEPES.0.05 EOT A. 7 CI, 0.5 Na2GTP, 2 Na2ATP, 2 MgATP.
  • a 20 second subthreshold sinusoidal current with a linear increase in frequency from 0.1 - 20 Hz was applied through the recording electrode.
  • the impedance amplitude profile (ZAP) was generated by transforming the input current (I) and the voltage response (V) into the frequency domain using a fast Fourier transform (KPT), and then dividing the voltage transformation FFT(V) by the current stimulus transformation FFT(l).
  • the stimulus file was generated by Python based software, imported into pClamp, and applied as described above.
  • the resonance frequency (fR) was determined as the peak of the ZAP profile.
  • mEPSC's spontaneous miniature excitatory postsynaptic currents in layer 5 pyramidal neurons was recorded.
  • mEPS s were recorded in the whole-cell voltage-clamp configuration at a holding potential of -70 mV to avoid NMDA conductance and in the presence of 1 ⁇ tetrodotoxin (TTX) and 50 ⁇ picroroxin, to block GABAergic receptors.
  • TTX 1 ⁇ tetrodotoxin
  • mEPSCs were analyzed off-line using pClamp 10 software. The frequency and amplitude of events was calculated over 5 min periods.
  • aCSF and acute brain slice samples were collected from either the device as herein disclosed or the control chamber at set time points (1, 6. 12, 1 , 20, 24, 30 & 36 hrs) after slicing.
  • J niL of solution was aspirated by sterile syringe, diluted and cultured on agar plates for 24 hrs at 37 C C.
  • For brain slices. 1 mL of solution together with the brain slice was aspirated from each group at set time point by sterile syringe, smashed into a thick suspension and centriftiged ⁇ 000 rpm for S min). Supernatant was collected, diluted and cultured on agar plates for 24 hrs at 37 °C. Following incubation, colony-forming units (CFU) were counted and inspected qualitatively for common colonies. Sequentially, colonies were smeared (in triplicate) with a sterile toothpick onto a matrix-assisted laser
  • Slice viability was assessed as the ratio of live cells (PI negative) out of the total visible cells (DAP1 positive + PI positive) in die field of view. To avoid discrepancy between different areas in the slice, measurements were confined to the cerebral cortex. 54 slices which had been stored in the device 1 or J 00 were imaged as were 54 slices from the control chamber. Acute brain slices were imaged at time points of 1, 6, 12. 16, 20, 24. 28, 2 and 36 hours post slicing. As shown in the graph in Figure 4 slice deterioration was reduced significantly in those stored in device 1 or 100 when compared to slices incubated in the control chamber.
  • a significant difference of the live/total cell ratio was detected (11.3%; p ⁇ 0.007; student t test), reaching a maximal difference of 28% after 24 hours (45 ⁇ 3% vs I7 ⁇ 3%, pcO.OOOI. two-tailed student t test).
  • some of the dead cells morphology was changed, probably due to membrane deterioration.
  • Electrophysiological properties of layer 5 pyramidal neurons are Electrophysiological properties of layer 5 pyramidal neurons.
  • the resonance frequency is dependent on the interplay between two currents, a slowly activating and non-inactivating K+ current and a fast persistent Na+ current.
  • the resonance frequency was measured by injecting chirp stimulation of 60pA (peak to peak). Sample traces of spontaneous synaptic activity recorded from a neuron after 31hrs in the device are shown in Figure 6c.
  • Figures 6d and 6e show bar graphs depicting the spontaneous mEPSC's frequency and amplitude across different incubation groups.
  • Miniature postsynaptic currents mPSCs
  • mEPSC's characteristics were assessed as a means of assessing the network activity impacting individual neurons in the slice. These characteristics did not show any significant changes between different time groups (two tailed student t test), implying that the majority of synaptic inputs onto layer V neurons were intact and functional even 36 hrs after the slicing procedure.

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Abstract

A storage device for a sample of animal tissue and specifically brain tissue. The device has a storage chamber which houses a tissue sample and a control system to control environmental conditions including temperature, oxygenation and/or pH in the chamber. The device also includes an antimicrobial system.

Description

"Device for storing a tissue sample"
Cross-Reference to Related Applications
[0001] The present application claims priority from Australian Provisional Patent
Application No 2013903100 filed on 16 August 2013. Australian Provisional Patent Application No 2014900375 filed on 7 February 2014. Australian Provisional Patent Application No 2014900649 filed on 27 February 2014, the contents of which are incorporated herein by reference.
Technical Field
[0002] The present disclosure relates to a device to provide optimal conditions for the survival of a tissue sample to extend the lifespan of the tissue for experimental purposes.
Background
[0003] Tissue samples and slices are used for various investigations including
pharmacological, neurophysiological and pathological studies. To optimise such studies, it is desirable to keep the tissue cells alive for as long as possible.
[0004] In brain research, neuroscientists use in vitro brain slice preparations as a means to preserve the native architecture of the brain and therefore serve as a tool to study brain networks that would otherwise be difficult to manipulate at the synaptic and cellular levels.
[0005] A major problem with tissue samples and slices, including brain slices, is their short lifespan which limits the time available to study the properties of the tissue or the effects of various elements on that tissue. There is a need therefore, to provide a device and method to optimise conditions for the survival of tissue cells in vitro.
[0006] Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each claim of this application. Summary
[0007] In one aspect, there is provided, a storage device for a sample of animal tissue, including a storage chamber configured to receive a tissue sample and fluid therein; a control system to control environmental conditions in at least the storage chamber, said environmental conditions including at least one of temperature, oxygenation and pH; and an antimicrobial system.
[0008] In a further aspect, there is provided, a method of storing a tissue sample from an animal comprising: immersing said tissue sample in a fluid; controlling environmental conditions of said fluid including controlling at least one of pH. oxygenation and temperature of said fluid; and treating the fluid to reduce the number of or remove microorganisms therein.
[0009] In a still further aspect, there is provided a storage device for a sample of animal tissue, including a storage chamber configured to receive a tissue sample and fluid therein; a control system to control the environmental conditions in at least the storage chamber, said environmental conditions including temperature, oxygenation and pH.
[00 0] The storage device is typically configured to automatically monitor and control environmental conditions it provides for the tissue sample in accordance with pre -determined environmental parameters. The environmental conditions may vary depending upon the type of tissue sample stored therein. [0012] The storage device typically includes a main housing. The main housing may include a storage chamber to receive and store the tissue sample. The storage chamber may also be configured to receive a fluid and may further comprise a removable structure to house the tissue sample.
[0012] The removable stucture may include a mesh tissue holding platform to receive the tissue sample. The removable structure of the storage chamber may be configured such that when a sample of tissue is housed thereon or therein, the removable structure allows complete immersion of the tissue sample into the fluid received within the storage chamber. In this regard, it is envisaged that the storage chamber has a base and sidewalls extending therefrom to an open top end. The storage chamber typically has an upper portion adjacent the top end and a lower portion adjacent to the base.
[0013] The removable structure may be sized such that the tissue holding platform is positionable within the lower portion of the storage chamber. This embodiment may allow the use of less fluid to fully immerse the tissue sample.
[0014] The main housing may comprise a lid on an upper surface to allow placement of the tissue sample in the storage chamber, and removal therefrom. The lid may form a tight seal with the remainder of the main housing. Alternatively, the lid may not tightly seal the main housing such as to avoid a build up of pressure in the housing during use.
[0015] In an embodiment, the entire storage device is surrounded by an insulating member. The insulating member may provide thermal insulation while also preventing UV
transmission from the antimicrobial system. Alternatively, the insulating member may insulate only the main housing particularly in an embodiment wherein the antimicrobial system is spaced from the main housing. The insulating member may insulate various parts o the device including but not limited to the antimicrobial system.
[0016] T e insulating member may be made from any suitable material to thermally insulate and in some instance prevent UV transmission. The insulating member may comprise a foam material. Examples include but are not limited to polystyrene foam and polyurethanc foams. In another embodiment the insulating member may be made from fibreglass. [0017] The fluid may be tissue specific, that is, it may provide an optimal environment for storing a particular (issue type. The tissue type may comprise connective, neural, muscle or epithelial tissue. Examples of tissue samples include but are not limited to Mood, bone, smooth, skeletal or cardiac muscle, neural tissue from cither the central nervous system or the peripheral nervous system, squamous epithelium, cuboidal epithelium, columnar epithelium, glandular epithelium or ciliated epithelium. The tissue may be sampled from a number of organs of a subject including but not limited to the brain, heart, lung, liver, kidney, skin or eye.
[0018] In one embodiment wherein the tissue is neural tissue, the sample chamber may receive an artificial cerebrospinal fluid (aCSF). In this embodiment the tissue may comprise a brain tissue slice.
[001 ] The aCSF typically provides a particular ionic environment suitable tor neural tissue, including any one or a plurality of ions selected from sodium, potassium, calcium, magnesium, phosphorus and chloride.
[0020] The aCSF may be prepared by dissolving a number of compounds in water, said compounds including any one or a combination of sodium chloride, potassium chloride, disodium phosphate, calcium chloride, sodium carbonate. The aCSF may further include any one of monosodium phosphate or sodium bicarbonate.
[0021] In another embodiment, the aCSF includes glucose.
[0022] The control system may comprise one or more inlets from a gaseous source to the interior of the device. The one or more inlets may be configured to deliver gas to the storage chamber. In this embodiment, the inlet may deliver oxygen to the storage chamber. In a further embodiment, a mixture of oxygen and carbon dioxide may be delivered to the storage container. The ratio of oxygen to carbon dioxide may vary and includes 95% oxygen to 5% carbon dioxide
[0023] The device may further include an inlet control mechanism to control the flow of gas from the gaseous source to the storage chamber. The control may comprise a sensor to sense a particular environmental condition in the storage chamber. The sensor may be coupled to a controller. As pan of a feedback mechanism, the control mechanism may comprise an output from the controller to cause a variation in the flow of gas depending upon input signals from th sensor.
[0024] In one embodiment, die concentration of a particular gas in the storage chamber may affect the pH of the fluid held therein.
[0025] For example, when one of the gases delivered is carbon dioxide, the pH of a fluid held in the storage chamber may vary with the concentration of carbon dioxide. In this embodiment:, the inlet control mechanism may, therefore, include a pH sensor coupled to the controller. Variations of the pH may be sensed by the sensor and then processed by the controller. In accordance with pre-calculated values, the controller may cause the rate of flow of die gas into the storage chamber to vary relative to input pH values sensed by the pH sensor. The controller may control the flow by control of a valve in the inlet. For example, the valve in the inlet may be caused to move between a closed and an open configuration to control the flow of gas into the fluid depending upon the pH of the fluid as sensed by the pH sensor.
[0026] Preferably the pH of the fluid is controlled to between pH 7 and pH 8. Still further, the pH may be controlled to be between pH 7 and pH 7.5; or between pH 7.1 and pH 7.4; or between pH 7.2 and pH 7.4; or between pH 7.2 and pH 7.3.
[0027] The device may further include a temperature control mechanism to maintain the tissue sample at a desired temperature. The temperature control mechanism may comprise a heating element. Further, the device may include a cooling member.
[0028] The heating element or cooling member may be positioned within the storage container or, alternatively, adjacent thereto. The temperature control mechanism may further include a thermometer to measure the temperature of the fluid and a feedback mechanism comprising a controller configured to receive an input from the thermometer and send an output signal to the heating clement or cooling member. Variation of the temperature of the fluid will cause a variation in the output of heat from the heating element to either increase heat output or decrease heat output. Similarly, if a cooling member is present, the output signal may cause a cooling of the fluid in the storage chamber. Further, the controller may control the on/off function of the heating dement or cooling member. In this regard, a safety mechanism may be in-built in the temperature control mechanism to cause the heating element to switch off if a temperature of the fluid reaches a pre~determined temperature.
[0029] In one embodiment, the device includes one or more heating and cooling plate members. The device may comprise a Peltier device. The device typically includes at least a first plate attached to a heat sink and a fan member and at least a second plate attached to the storage chamber. Said plates may be attached to the beat sink and fan and the storage chamber respectively by a thermo-conductive compound. Applying a current to the plates causes one of said plates to heat up and the other plate to cool. The direction of the current applied determines which plate heat and which cools. In this embodiment, the temperature of the storage chamber may be varied ami is controllable to achieve a desired temperature.
[0030] Rather than the plates attach to the storage camber directly, it is also envisaged that the plates of the Peltier device may attach to a third plate, said third plate in connection with the storage chamber. This embodiment enables removal of the storage chamber without affecting the temperature control plates.
[0031] The temperature may be prc-selected by a user depending upon the type of tissue to be stored. The temperature may range between 4 C and 40' C. Furthermore, the temperature may vary over a time frame.
[0032] The device may further include a pump. The pump may be configured to pump fluid within the storage chamber to achieve a flow of fluid therein. Further, the pump may be configured to pump said fluid from the storage chamber to another chamber of the device.
[0033] The antimicrobial control system may include an ultraviolet <UV) tight source. The UV light source may be housed within an antimicrobial chamber. The antimicrobial chamber is typically spaced from the storage chamber. However, in one embodiment, the two chambers may be fluidly connected to each other such that a fluid may be transferred therebetween.
[0034] The pump may pump the fluid from the storage chamber to the antimicrobial chamber for exposure to UV light. Such exposure may kill microoraganims present in the fluid. The pump may continually pump the fluid between the antimicrobial chamber and the storage chamber. Alternatively, the pump may intermittently pump the fluid between the two chambers.
[0035] The pump may have a variable output to pump the fluid at a variable flow rate. The flow of fluid within the storage chamber provides a physical "wash" of the tissue sample which further aids in removing bacteria from the surface of the tissue.
[0036] The antimicrobial system may also include a microbial sensor which senses the presence of or the concentration of certain gases in the fluid as an indicator of microbial presence/activity. An increase in concentration of a gas in this embodiment is indicative of an increase in microbial numbers. In one embodiment, the sensor may comprise a nitric oxide (NO> sensor.
[0037] In an embodiment, the antimicrobial system comprises a UV chamber which has a main housing and an inner housing. The UV chamber houses a UV bulb. The UV bulb may be substantially encased in the inner housing. The inner housing is typically spaced from the main housing such as to define a fluid path. The fluid pad) may extend from an inlet in the main housing to an outlet in the main housing. The fluid path typically substantially surrounds the UV bulb. In this embodiment, when fluid is passed from the inlet to the outlet, it is substantially illuminated with UV light from the UV bulb. The inner housing may be made from any suitably resilient material and in one embodiment, the inner housing is made from quartz. Other UV and in particular UVC transparent materials are also envisaged.
[0038] The UV bulb may be connected to a power source typically through a sealed lid of the UV chamber. The UV bulb may be powered to provide constant UV irradiation.
Alternatively, the application of UV light may be intermittent. The application of U V light may be randomised. Alternatively, the application may be more ordered and follow a predetermined pattern. The UV light may be applied in a pulsed fashion. In this regard, a non- constant application of UV light may prevent excessive heating of the fluid in the fluid pathway in addition to preventing bacterial UV resistance.
[0039] In one embodiment, the UV bulb is controlled by a programmable timer to turn on at varying times. Intervals of "on" time may be selected from between 2 and 30 minutes and preferably between 15 and 26 minutes. The frequency of illumination may vary and may include intervals in the range of every 15 minutes through to every 30 minutes.
[0040 The OV light may have a wavelength of between about 280nm and about lOOnm. The UV light may be UVC light
[0041] The main housing of the If V chamber may also comprise a U V blocking layer. It is preferred that the application of U V light is substantially contained within the UV chamber to prevent irradiation of any tissue in the device because neuronal tissue may be damaged by UV irradiation.
[0042] The UV blocking layer may comprise an outer layer or film. Alternatively, the main housing wall may be made from a suitable material to block the transmission of UV wavelengths. In one embodiment, the UV blocking layer may comprise an aluminium foil cover which extends around the main housing.
[0043] The device may further include a titter to catch debris. An example of debris includes dead microorganisms or parts thereof.
[0044] At least any one of the components of the device including the storage chamber, any part of the control system or the antimicrobial system may be removable from the device to facilitate washing or other required actions such as repair of components. Preferably, the device is configured such that all the components (herein are removable together as a unit.
[0045] The storage device may be powered from a mains supply. Further, however, the device may include a re-chargeable power back up to allow for movement between locations such as different laboratories without disturbing the environment of the tissue sample.
[0046] The storage device may further comprise a user interface. The user interface may comprise a display screen to provide real time data in relation to the environmental conditions in the device. The data may include one or a combination of temperature. pH, fluid flow rate, microbial presence and activity and power status of the device. The device may also include a manual over-ride system to allow a user to over-ride the automatic control of the
environmental conditions in die device. [0047] The device may be controlled remotely such as via an internet connection. The control may be wireless. Data from the device may be obtainable and one or more parameters of d e device may be controllable via a smart phone or other smart device. A particular protocol may be initiated remotely by a user. Further, a particular protocol of the device may be modified and implemented remotely depending upon monitored conditions in the device. Such remote monitoring and control of the device enables a user to modify the conditions in real time and continuously to optimise the conditions therein.
[0048] The device may also include an alarm to represent an event. The alarm may be in a number of forms and includes visual and auditory signals. In one embodiment, the alarm may be deliveied to a user via a smart device or phone. The alarm may represent the end of a cycle; the malfunction of any components: and any other desired condition in the device to allow a user to determine the condition of the tissue sample.
Brief Description of Drawings
[0049] Figure 1 is a schematic depiction of an embodiment of the storage device of the present disclosure;
[0050] Figure 2 is a flow diagram to depict the various elements of the device of Figure 1 ;
[0051] Figure 3a and 3b are schematic representations of another embodiment of the device;
[0052] Figure 4 is a graph showing the results of cell viability for cells stored in a control environment compared to cells stored in the device of the disclosure: and
[0053] Figures 5a and 5b arc graphs showing bacterial cell count over time for slices stored in the device and in a control device.
Description of Embodiments
[0054] The storage device of the present invention is generally depicted as 1 in the attached drawings. [0055] The device 1 is used to store a tissue sample and in one embodiment described herein, the tissue sample comprises a brain slice. Using existing technology, the usable lifespan of a brain slice is 6-8 hours. The device of the present disclosure is configured to extend the lifespan of a brain slice sample beyond the current 8 hour window which therefore provides opportunities to study long term effects of various metabolic conditions or pharmaceuticals on brain tissue.
[0056] The present device provides an environment for maintaining a healthy brain slice over a long period by providing the control of at least one of temperature, pH and oxygenation. By increasing the lifespan by optimising the environmental conditions, microbial levels in die fluid in which the tissue sample is immersed may increase.
[0057] The presence of bacteria in the fluid can ultimately lead to death of the cells of the sample. In this regard, bacteria display a characteristic four-phase pattern of growth in liquid media. An initial Lag Phase is a period of slow growth during which the bacteria are adapting to the conditions in the fresh medium. This is followed by a Log Phase during which bacterial growth is exponential, doubling every replication cycle. The Stationary Phase occurs when the supply of nutrients becomes a limiting factor and the rate of multiplication equals the rate of death. Finally, the Logarithmic Decline Phase occurs when bacteria die taster than they are replicated. Typically, using current techniques, recordings from brain slices are constrained to the Lag phase, in which the amount of bacteria is low and not affecting ceil viability. However, by extending the viability of the tissue sample using the device of the present disclosure, the Lag phase of microbial growth will be extended. In the embodiment depicted, the device, therefore includes an antimicrobial system to address the problem of increasing microbial numbers and resultant cell death during storage of the tissue sample.
[0058] The storage device 1 includes a main housing 10 and a storage chamber 11 which is configured to receive a tissue sample (not shown). The storage chamber 11 also receives a fluid 12 to immerse the tissue sample.
(00S9] The device 10 also has a control system which controls a number of different functions and environmental parameters in the device 1. This is also shown in the flow diagram in Figure 2. Temperature
[0060] To provide optimal conditions in the storage chamber i 1. the temperature is kept within a pre-determined range depending upon the type of tissue being stored. The device 1 includes a heating and/or cooling element 13, which is controlled by a microcontroller 34. Microcontroller 34 receives input signals from a thermometer IS in the storage chamber 11. The heating and/or cooling element heats or cools fluid 12 until a desired temperature is achieved. At this point microcontroller 34 may effect switching off the heating and/or cooling element 1 or may reduce the output of the heating and or cooling element to an output that maintains the desired temperature of the fluid. By "heating and or cooling element 13" it is envisaged that this encompasses a Peltier device as described herein.
Deliver}' of gas to the tissue sample
[0061] The control stem of device 1 also controls the del i very of a selected gas from gaseous source 16. The gas is introduced through inlet 17 and microcontroller 34 controls the flow via valve 8. The gas in device 1 is a mixture of oxygen and carbon dioxide to perfuse the tissue in storage chamber 11. Control of the gas may be determined in relation to other parameters such as pH as discussed below. pH
[0062] pH is an important environmental factor in preserving a tissue sample and it may vary over time. Further, variations in pH occur as a result of gas introduced into fluid 12, particularly the introduction of a mixture of oxygen and carbon dioxide. Microcontroller 34 controls the pH by receiving pH input and effecting an output to maintain a constant pH. The output in tins embodiment comprises controlling the flow of gas via valve 8.
[0063] Storage chamber 11 also includes a removable tray 18 which is introduced into and removed from the storage chamber 11 via lid 20 in main housing 10. The removable uay 18 is configured to hold a tissue sample therein. Handle 19 enables a user to place the tray 8 and sample into the storage chamber 1 1 until the sample is fully immersed in fluid 12. [0064] The removable tray 18 in the depicted embodiment is made from a mesh material to allow fluid to immerse the sample as well as to allow fluid to drain through it when removed from the storage chamber.
[0065] In device 1. fluid 12 is an artificial cerebrospinal fluid and includes (in mM) 124 NaCl. 3.5 KCI, 2 MGS04, 1.25 Na HP0 .2 CaCfe, 26 NaHC(¼ and 10 glucose
[0066] The device also includes an antimicrobial system 22. The antimicrobial system treats the fluid 12 to remove or kill microorganisms therein. Microorganisms are considered to include but are not limited to bacteria, viruses, fungi, yeasts and protozoa. The antimicrobial system includes a conduit 23 extending from the storage chamber I to a UV filter chamber 24. Storage chamber 11 and U V filter chamber 24 are fluidly connected via conduit 23 which further includes a pump 21 to pump the fluid from the storage chamber 11 to the UV filter chamber 24.
[0067] Once in the UV filter chamber 24, me fluid is irradiated with UV light from a UV light source 25 to kill microorganisms that are present in the fluid. From here, the fluid is pumped back into the storage chamber 11. As can be seen, the fluid is pumped back via return conduit 26 and re-introduced back into the storage chamber 11. Conduit 26 may further include a filter to trap dead microorganisms and other debris therein and avoid re- introduction into the storage chamber 11.
[0068] The conduit 26 connects with a perforated head member 27 such that the fluid is returned to essentially "shower" the sample in the storage chamber. This has the benefit of further washing microorganisms from the surface of the sample.
[0069] The height of the perforated head member 27 may be adjustable relative to the storage chamber.
[0070] Figure 2 shows a flow diagram of the main components of device I which is typically powered by an external power source 31 although a re-chargeable battery 32 is also provided for back up and transport of the device.
[0071] Device 1 has a power control 33 to power the various components and
microcontroller 34. The microcontroller controls output to pump 21, UV source 25 and valve 8. Microcontroller 34 receives input signals from thermometer 15 and a pH meter 35 and effects change in temperature and pH accordingly via heating element 1 and gas in flow via valve 8 respectively.
[0072] Microcontroller 34 furtljcr receives input signals from and effects output to lid 20 for opening and closing of lid 20.
[0073] A further embodiment of a device 100 is shown in Figures 3a and 3b. Figure 3a depicts antimicrobial system 101 and Figure 3b depicts main bousing 102.
[0074] Antimicrobial system 101 comprises a main housing 103 and an inner housing 104. A UVC bulb 105 is housed within inner housing 103. Main bousing 103 is covered with a U V blocking layer (not shown). The system 101 also includes a lid 106 which is sealed to die main housing 103. UVC bulb 105 is connected to a power source via cable 107 which passes through ltd 106.
[0075] The material of the inner bousing is thermally insulating and includes a quartz material to prevent over-heating of the fluid 108 in the main housing 103. Fluid 108 is passed into the main housing 103 through inlet 109 and removed from the main housing through outlet 110.
[0076] The main housing 102 of Figure 3b includes a mesh layer 120 to suppor the sample of tissue to be stored. This embodiment also includes a series of probe mounting units 1 1 which ate configured to receive probes such as temperature or pH probes. The main housing also includes a fluid inlet 122 and fluid outlet 123. Carbogen inlet 124 is also depicted.
[0077] The entire main housing 102 in this embodiment is insulated by a polystyrene foam casing 130 which provides thermal insulation.
Experiment 1
Slice preparation and recording
[0078] Wistcr rats were deeply anesthetized by inhalation of isoflurane (5%), decapitated, and their brains were quickly removed and placed into ice-cold physiological solution (artificial CSF) containing (in niM): 125 NaCJ, 2.5 KCI, 1 MgC12. 1.25 NaH2P04. 2 CaCI2, 25 NaHC03, 25 dextrose and saturared with carbogen (95% 02 -5% C02 mixture; pH 7.4). Parasagittal brain slices (300 μηι thick) were cut with a vibrating microtome (Camden Instruments, UK) and transferred to a holding chamber containing carbogenated aCS for 30 min at 35 "C. Sequentially, slices weie either allowed to cool to room temperature (-22 °C) in the same recovery chamber (Control chamber) or transferred into a custom-made incubation system mat closely monitored and controlled pH levels, carbogen flow and temperature as well as irradiating bacteria through a separate UV chamber as herein defined as device 1 or 100. Slices were kept either the control chamber or in the chamber of device 1 or 100 for at least 30 nun before any measurement. The fluid for both the control and as used in device 1,100 was the same aCSF.
Evaluating slice viability
[0079] Acute slices were incubated for 15 min with the selective dead cell fluorescent marker propidium iodide (PI, 1 jig/ml). For total cell counts, slices were co-incubated with the nuclear marker DAPI (lug/ml). Following incubation, slices were washed with fresh aCSF for 10 min. Images were acquired using a Zeiss LS -510 Meta confocal microscope (Carl Zeiss, Obcrkochen, Germany) using a 40x-oil immersion objective in the inverted configuration. Z plane optical sections (4 urn) were taken at -20 to -70 μηι depth from the surface of the cerebral cortex to produce an image stack. The DAPI signal was obtained using Argon laser excitation at 488 nm; PI was excited with 543-nm HeNe laser. Images were visualized using ZEN software and processed. Slice viability was assessed as the ratio between dead/total cells in the visual field.
[0080] Following incubation, slices were incubated for an additional 10 minutes with the fluorescent cell death marker Propidium Iodide ( I Mg ml) and then mounted on Olympus BX- 1 microscope equipped with both IR DIC and fluorescence optics.
Electrophysiological recording and stimulation
[0081] A recording chamber was mounted on an Olympus BX-51 microscope equipped with I.R/D1C optics. During recordings the vSlices were kept at room temperature, ~220C. and constantly perfused (2-3 ml/min with oxygenated solution. Whole cell recordings were perforated from the soma of layer 5 pyramidal neurons in the somatosensory cortex with patch pipettes (5-7 Mil) containing {in mMi 130 K-Metbansulfate, 10 HEPES.0.05 EOT A. 7 CI, 0.5 Na2GTP, 2 Na2ATP, 2 MgATP. 7 phosphocreatine.0.1 Alexa Fluor-488 (Molecular Probes) and titrated with OH to pH 7.2 (-285 mOsm). Stimulation protocols were designed using the pClamp 10 software suit (Molecular devices, Sunnyvale, CA) and stimulation currents were injected through the recording electrodes. Voltages were recorded in current clamp mode using a multiclamp 700B dual patch-clamp amplifier (Axon instruments, Foster city, C A). digitally sampled at 30-50 kHz, filtered at 10 kHz, and analysed off-line using pClamp software. The access resistance was corrected on-line and recordings were included in the analysis if the access resistance was <30ΜΩ. Cells were considered stable and suitable for analysis if the access resistance, input resistance and resting membrane potential did not change by more than 20% from their initial value during recording. At the termination of each experiment, the location and morphology of neurons were examined by fluorescence microscopy and digitally recorded (ROLERA-XR. Q-Imaging).
Determining the resonance frequency
[0082] In order to reveal the resonance frequency of the cells, a 20 second subthreshold sinusoidal current with a linear increase in frequency from 0.1 - 20 Hz (chirp stimulation) was applied through the recording electrode. The impedance amplitude profile (ZAP) was generated by transforming the input current (I) and the voltage response (V) into the frequency domain using a fast Fourier transform (KPT), and then dividing the voltage transformation FFT(V) by the current stimulus transformation FFT(l). The stimulus file was generated by Python based software, imported into pClamp, and applied as described above. The resonance frequency (fR) was determined as the peak of the ZAP profile.
Detecting spontaneous activity
[0083] To evaluate the functional activity of the slice, the spontaneous miniature excitatory postsynaptic currents (mEPSC's) in layer 5 pyramidal neurons was recorded. mEPS s were recorded in the whole-cell voltage-clamp configuration at a holding potential of -70 mV to avoid NMDA conductance and in the presence of 1 μΜ tetrodotoxin (TTX) and 50 μΜ picroroxin, to block GABAergic receptors. mEPSCs were analyzed off-line using pClamp 10 software. The frequency and amplitude of events was calculated over 5 min periods. Bacteria Culturing and Detection
[0084] aCSF and acute brain slice samples were collected from either the device as herein disclosed or the control chamber at set time points (1, 6. 12, 1 , 20, 24, 30 & 36 hrs) after slicing. For aCSF samples, J niL of solution was aspirated by sterile syringe, diluted and cultured on agar plates for 24 hrs at 37 CC. For brain slices. 1 mL of solution together with the brain slice was aspirated from each group at set time point by sterile syringe, smashed into a thick suspension and centriftiged Π000 rpm for S min). Supernatant was collected, diluted and cultured on agar plates for 24 hrs at 37 °C. Following incubation, colony-forming units (CFU) were counted and inspected qualitatively for common colonies. Sequentially, colonies were smeared (in triplicate) with a sterile toothpick onto a matrix-assisted laser
desorprion/iontzation <M ALDI) stainless steel target plate and allowed to dry at room temperature in a fume hood. 1L of 70% formic acid was overlaid onto the dried sample and allowed to dry. Automated spectrum acquisition was performed using a Daltonik MALDI Biotyper (Bruker. Germany), and analysed with Flex Analysis MALD! Biotyper software (Brukcr, Germany).
Results
Brain Slice Viability
[0085] Slice viability was assessed as the ratio of live cells (PI negative) out of the total visible cells (DAP1 positive + PI positive) in die field of view. To avoid discrepancy between different areas in the slice, measurements were confined to the cerebral cortex. 54 slices which had been stored in the device 1 or J 00 were imaged as were 54 slices from the control chamber. Acute brain slices were imaged at time points of 1, 6, 12. 16, 20, 24. 28, 2 and 36 hours post slicing. As shown in the graph in Figure 4 slice deterioration was reduced significantly in those stored in device 1 or 100 when compared to slices incubated in the control chamber. After one hour, the average cell viability ratio in the device 1 or 100 was 73 ± 4%; <n= ) and no changes were observed between control and device slices (Figure 2). However, during later incubation periods (>6 hrs). a significant difference of the live/total cell ratio was detected (11.3%; p < 0.007; student t test), reaching a maximal difference of 28% after 24 hours (45±3% vs I7±3%, pcO.OOOI. two-tailed student t test). Moreover, after 24 hours under control conditions, some of the dead cells morphology was changed, probably due to membrane deterioration.
Bacterial Growth
[0086] Two types of gram-negative bacteria were identified: Pseodomona* species and Stenotrophomonas maltophilia. To assess the impact of UVC light and low temperature on bacterial growth, two experimental regimes were compared. In the device of the present disclosure, slices were kept at 15 -16°C and the aCSF was constantly filtered through a UVC chamber, while in control conditions, slices were kept at room temperature (-22 CC) and without UVC filtration. As seen in figure 5b, under control conditions, the exponential bacterial growth (Log phase) in both slices and solution started between 2-16 hrs, reaching a maximal growth after 36 hrs. Although bacterial growth in die present device was comparable to control conditions until 6 hrs. from 12 hrs onwards the difference was significant (p<0.0l ; two tailed student t test), hence extending the growth Lag Phase. Figure 5c shows the significant changes in bacterial growth which started after 12 hours.
Electrophysiological properties of layer 5 pyramidal neurons.
[0087] To evaluate individual cell viability following different incubation time in the device, both passive (Vm; Rin; Tau) and active (action potential properties) membrane properties of layer V pyramidal neurons were measured. AH cells had the morphology of pyramidal neurons and responded to a depolarizing current stimulus with tonic, adapting patterns of action potentials, which categorize them as regular splicing (RS) neurons. Figure 6a shows I- V traces wherein increasing step currents of 500ms were injected into the soma through a recording electrode to reveal the input resistance and tiring properties.
[0088] Only neurons with resting membrane potentials greater than -60 mV and action potentials that overshot 0 mV were included in the analysis. The recorded neurons were divided to four groups according to the time they spent in the device of the present disclosure (Table I ). To evaluate significant alterations between groups, die first group (incubation time between 1-3 hrs) was compared to the other groups (two-tailed student t test). In general, all passive and active membrane properties remained constant between different incubation time groups (Table 1). [0089] The membrane resonance frequency was also compared, which is a property determined by the interplay between neuronal active (voltage gated currents) and passive (capacitance; leak currents) properties and describes the ability of neurons to respond selectively to inputs at preferred frequencies. In cortical neurons, the resonance frequency is dependent on the interplay between two currents, a slowly activating and non-inactivating K+ current and a fast persistent Na+ current. In Figure 7b the resonance frequency was measured by injecting chirp stimulation of 60pA (peak to peak). Sample traces of spontaneous synaptic activity recorded from a neuron after 31hrs in the device are shown in Figure 6c.
[0090] The average resonance frequency ranged between 1 and 3 Hz (average 1.7±0.1 Hz; n=38h and did not differ between groups (two-tailed student t test).
[0091] Figures 6d and 6e show bar graphs depicting the spontaneous mEPSC's frequency and amplitude across different incubation groups. Miniature postsynaptic currents (mPSCs) are generated as a consequence of spontaneous vesicular release, hence reflecting the functional synapses onto layer V pyramidal neurons. mEPSC's characteristics were assessed as a means of assessing the network activity impacting individual neurons in the slice. These characteristics did not show any significant changes between different time groups (two tailed student t test), implying that the majority of synaptic inputs onto layer V neurons were intact and functional even 36 hrs after the slicing procedure.
[0092] It wilt be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the above-described embodiments, without departing from the broad general scope of the present disclosure. The present embodiments are. therefore, to be considered in all respects as illustrative and not restrictive.
[0093] Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Claims

CLAIMS:
1. A storage device tor a sample of animal tissue, including:
a storage chamber configured to receive a tissue sample and fluid therein;
a control system to control environmental conditions in at least the storage chamber, said environmental conditions including at least one of temperature, oxygenation and pH; and an antimicrobial system.
2. The storage device of claim 1 comprising a main housing, said main housing including a storage chamber to receive and store the tissue sample.
3. The storage device of claim 2 wherein the storage chamber includes a mesh platform structure to receive the tissue sample.
4. The storage device of any one of the preceding claims wherein the fluid is tissue specific.
5. The storage device of any one of the preceding claims wherein the fluid is artificial cercbro-spinal fluid.
6. The storage system of claim 5 wherein the tissue is neural tissue.
7. The storage device of claim 5 wherein the artificial cerebro-spinal fluid includes any one or a plurality of ions selected from sodium, potassium, calcium, magnesium, phosphorus ami chloride.
8. The storage device of claim 7 wherein the artificial cerebrospinal fluid further includes glucose.
9. The storage device of any one of the preceding claims comprising one or more inlets in fluid communication with a gaseous source and the interior of the device to deliver gas from the gaseous source to the storage chamber.
10. The storage device of claim 9 wherein the inlet delivers oxygen to the storage chamber.
11. The storage device of claim 10 wherein the inlet delivers a mixture of oxygen and carbon dioxide to dtc storage chamber.
12. The storage device of any one of claims 9 to 11 further comprising an inlet control mechanism to control the flow of gas. from the gaseous source to the storage chamber.
13. The storage device of claim 12 wherein said inlet control mechanism includes a valve positioned in said inlet which moves between an open position wherein gas flows through the inlet and a closed position to prevent the flow of gas through the inlet and into the device.
14. The storage device of claim 13 further comprising a sensor to sense the conditions in the storage chamber wherein the valve in the inlet is caused to move between said open and closed positions dependent upon the sensed conditions.
15. The storage device of claim 14 wherein said sensor is a pH sensor.
16. The storage device of any one of the preceding claims further comprising a temperature control system to maintain the tissue sample at a desired temperature.
17. The storage device of claim 16 wherein the temperature control mechanism includes a heating element having an output to heat the fluid in the storage chamber.
18. The storage device of claim 1 wherein the temperature control mechanism includes a cooling element having an output to cool the fluid in the storage chamber.
19. The storage device of claim 16 wherein the temperature control system includes a Peltier device.
20. The storage device of any one of the preceding claims further including a pump configured to pump fluid within the storage chamber to achieve a flow of fluid therein.
2 J . The storage device of any one of claims 1 to 19 wherein the device further includes a pump configured to pump said fluid from the storage chamber to a second chamber of the device.
22. The storage device of claim 21 wherein said second chamber comprises the antimicrobial control system.
23. The storage device of any one of the preceding claims wherein the antimicrobial control system includes an ultraviolet (UV) light source.
24. The storage device of any one of the preceding claims wherein the antimicrobial system comprises a UV chamber having a main housing and an inner housing.
25. The storage device of claim 24 wherein the antimicrobial system includes a UV bulb in the inner housing.
26. The storage device of claim 24 of claim 25 wherein the inner housing is spaced from the main housing to define a fluid path therebetween, said fluid path extending from an inlet in the main housing to an outlet in the main housing.
27. The storage device of claim 25 wherein the application of UV light is intermittent.
28. The storage device of any one of claims 24 to 28 wherein the main housing of the UV chamber comprises a UV blocking layer.
29. The storage device of any one of the preceding claims wherein the antimicrobial system includes a microbial sensor to sense the presence of or the concentration of certain gases in the fluid as an indicator of microbial presence/activity.
30. The storage device of claim 29 wherein the gas comprises nitric oxide and the sensor is a nitric oxide sensor.
31. The storage device of any one of the preceding claims wherein the antimicrobial system further includes a filter to catch microbes or microbial debris.
32. The storage device of any one of the preceding claims wherein at least any one or all of the components of the device including the storage chamber, any part of the control system ami the antimicrobial system arc removable from the device.
33. The storage device of any one of the preceding claims further including a user interface comprising a display screen to provide real time data of the environmental conditions in the device.
34. The storage device of claim 33 wherein the data includes one or a combination of temperature, pH, fluid flow rate, microbial presence and activity and power status of the device.
35. The .storage device of any one of (he preceding claims wherein one or more Junctions of the device arc controllable remotely.
36. The storage device of any one of the preceding claims further including a thermal insulating member.
37. A method of storing a tissue sample from an animal comprising:
immersing said tissue sample in a fluid;
controlling environmental conditions of said fluid including controlling at least one of pH. oxygenation and temperature of said fluid: and
treating the fluid to reduce the number of or remove microorganisms therein.
38. A storage device for a sample of animal tissue, including
a storage chamber configured to receive a tissue sample and fluid therein;
a control system to control the environmental conditions in at least the storage chamber, said environmental conditions including temperature, oxygenation and pH.
PCT/AU2014/050188 2013-08-16 2014-08-15 Device for storing a tissue sample WO2015021513A1 (en)

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105717192A (en) * 2016-04-11 2016-06-29 爱德森（厦门）电子有限公司 Sensor for online and high-temperature eddy current monitoring
WO2018127675A1 (en) * 2017-01-09 2018-07-12 Universite De Franche Comte Device and process for preserving and transporting at least one human or animal tissue with a view to transplantion or ex-vivo experimentation
CN109819978A (en) * 2019-03-29 2019-05-31 杭州谈笑电子商务有限公司 One kind being used for immunoregulatory stem cell safety storage apparatus
CN110568873A (en) * 2019-08-26 2019-12-13 广东顺德工业设计研究院(广东顺德创新设计研究院) Online monitoring system for isolated organ perfusion pH
WO2020252148A1 (en) * 2019-06-11 2020-12-17 Paragonix Technologies, Inc. Organ transport container with antiviral therapy
CN114252324A (en) * 2022-03-02 2022-03-29 中国人民解放军军事科学院军事医学研究院 Device is hatched to porous brain piece that facilitates use
US11632951B2 (en) 2020-01-31 2023-04-25 Paragonix Technologies, Inc. Apparatus for tissue transport and preservation
US11659834B2 (en) 2017-06-07 2023-05-30 Paragonix Technologies, Inc. Apparatus for tissue transport and preservation
USD1031028S1 (en) 2022-09-08 2024-06-11 Paragonix Technologies, Inc. Tissue suspension adaptor

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999015011A1 (en) * 1997-09-23 1999-04-01 Hassanein Waleed H Compositions, methods and devices for maintaining an organ
US6280604B1 (en) * 2000-03-10 2001-08-28 Duke University Electrode materials, systems and methods for the electrochemical detection of nitric oxide
US6500809B1 (en) * 1999-11-12 2002-12-31 Neuron Therapeutics, Inc. Hyperoncotic artificial cerebrospinal fluid and method of treating neural tissue edema therewith
US20030136716A1 (en) * 2002-01-18 2003-07-24 Aquaresearch Canada Ltd./ Aquarecherche Canada Ltee Growing and conditioning system for bioaugmentation products used for the treatment of waste water and water quality improvement
US20050255442A1 (en) * 2004-05-14 2005-11-17 Organ Recovery Systems Apparatus and method for perfusion and determining the viability of an organ
US20100062519A1 (en) * 2006-12-18 2010-03-11 Franklin Jr Thomas D Portable organ and tissue preservation apparatus, kit and methods
US8354221B2 (en) * 2007-07-12 2013-01-15 Warsaw Orthopedic, Inc. Methods and compositions for the preservation of cells and tissues

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999015011A1 (en) * 1997-09-23 1999-04-01 Hassanein Waleed H Compositions, methods and devices for maintaining an organ
US6500809B1 (en) * 1999-11-12 2002-12-31 Neuron Therapeutics, Inc. Hyperoncotic artificial cerebrospinal fluid and method of treating neural tissue edema therewith
US6280604B1 (en) * 2000-03-10 2001-08-28 Duke University Electrode materials, systems and methods for the electrochemical detection of nitric oxide
US20030136716A1 (en) * 2002-01-18 2003-07-24 Aquaresearch Canada Ltd./ Aquarecherche Canada Ltee Growing and conditioning system for bioaugmentation products used for the treatment of waste water and water quality improvement
US20050255442A1 (en) * 2004-05-14 2005-11-17 Organ Recovery Systems Apparatus and method for perfusion and determining the viability of an organ
US20100062519A1 (en) * 2006-12-18 2010-03-11 Franklin Jr Thomas D Portable organ and tissue preservation apparatus, kit and methods
US8354221B2 (en) * 2007-07-12 2013-01-15 Warsaw Orthopedic, Inc. Methods and compositions for the preservation of cells and tissues

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105717192A (en) * 2016-04-11 2016-06-29 爱德森（厦门）电子有限公司 Sensor for online and high-temperature eddy current monitoring
CN110402081B (en) * 2017-01-09 2022-05-17 弗朗什-孔泰大学 Device and method for storing and transporting at least one human or animal tissue for transplantation or ex vivo experiments
WO2018127675A1 (en) * 2017-01-09 2018-07-12 Universite De Franche Comte Device and process for preserving and transporting at least one human or animal tissue with a view to transplantion or ex-vivo experimentation
FR3061633A1 (en) * 2017-01-09 2018-07-13 Universite De Franche Comte DEVICE AND METHOD FOR PRESERVING AND TRANSPORTING AT LEAST ONE HUMAN OR ANIMAL TISSUE FOR EX VIVO TRANSPLANT OR EXPERIENCE
CN110402081A (en) * 2017-01-09 2019-11-01 弗朗什-孔泰大学 The device and method of transplanting or isolated experiment are carried out for storing and transporting at least one human or animal tissues
US11800865B2 (en) 2017-01-09 2023-10-31 Universite De Franche Comte Device and process for preserving and transporting at least one human or animal tissue with a view to transplantion or ex-vivo experimentation
AU2018205755B2 (en) * 2017-01-09 2022-12-08 Tissueaegis Device and process for preserving and transporting at least one human or animal tissue with a view to transplantion or ex-vivo experimentation
US11659834B2 (en) 2017-06-07 2023-05-30 Paragonix Technologies, Inc. Apparatus for tissue transport and preservation
CN109819978A (en) * 2019-03-29 2019-05-31 杭州谈笑电子商务有限公司 One kind being used for immunoregulatory stem cell safety storage apparatus
WO2020252148A1 (en) * 2019-06-11 2020-12-17 Paragonix Technologies, Inc. Organ transport container with antiviral therapy
CN110568873A (en) * 2019-08-26 2019-12-13 广东顺德工业设计研究院(广东顺德创新设计研究院) Online monitoring system for isolated organ perfusion pH
US11632951B2 (en) 2020-01-31 2023-04-25 Paragonix Technologies, Inc. Apparatus for tissue transport and preservation
CN114252324A (en) * 2022-03-02 2022-03-29 中国人民解放军军事科学院军事医学研究院 Device is hatched to porous brain piece that facilitates use
USD1031028S1 (en) 2022-09-08 2024-06-11 Paragonix Technologies, Inc. Tissue suspension adaptor

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