WO2022018743A1 - Système et procédé d'imagerie et de croissance simultanées de cellules vivantes - Google Patents

Système et procédé d'imagerie et de croissance simultanées de cellules vivantes Download PDF

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WO2022018743A1
WO2022018743A1 PCT/IN2021/050589 IN2021050589W WO2022018743A1 WO 2022018743 A1 WO2022018743 A1 WO 2022018743A1 IN 2021050589 W IN2021050589 W IN 2021050589W WO 2022018743 A1 WO2022018743 A1 WO 2022018743A1
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cells
live cells
microfluidic chip
imaging
culture
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Ikram Khan S.I
Anil Prabhakar
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INDIAN INSTITUTE OF TECHNOLOGY MADRAS (IIT Madras)
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    • 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
    • B01L3/502761—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 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules
    • 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
    • B01L3/502715—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 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y80/00—Products made by additive manufacturing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/12—Well or multiwell plates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
    • 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
    • C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06—Fluid handling related problems
    • B01L2200/0689—Sealing
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/12—Specific details about manufacturing devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0829—Multi-well plates; Microtitration plates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/18—Means for temperature control
    • B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
    • B01L2300/1822—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks using Peltier elements
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0633—Valves, specific forms thereof with moving parts
    • B01L2400/0666—Solenoid valves

Definitions

  • the present invention is related to microfluidics. More particularly, it is related to a system and method for providing live cell growing support and simultaneously imaging the growing cells in a microfluidic chip, without any external disturbances or contamination.
  • the stem cell-based research has revolutionized the treatment for diseases like spinal cord injury, diabetes, rheumatoid arthritis, cerebral palsy, Alzheimer's, Parkinson's, and targeting cancer treatment.
  • Dr. H.V. Wilson demonstrated the potential of dissociated silicious sponges cells to self-organize and to regenerate to a complete organism.
  • the in-vitro growing of pluripotent stem cells gives the possibility to grow miniaturized versions of organs called organoids (live cells).
  • pluripotent stem cells have the potential to self-organize itself virtually to form any kind of organoids like a brain, kidney, retinal and heart. This initiated significant innovations and led to a Nobel prize- winning discovery on reprogramming mature cells to induced pluripotent stem cell (IPSC).
  • ISC induced pluripotent stem cell
  • Organoid based modeling has given the possibility to examine the development of diseases closely. The organoids can be grown in a controlled incubator environment and requires careful monitoring for better understanding the nature of the diseases to be modeled. Such kind of modelling helps in the disease diagnosis and is useful for pharmaceutical screening of medicines.
  • organoids are grown on the Petri-dish, which is not very efficient and requires a large volume of culture media.
  • bio-reactors like rotary cell culture systems (RCCS), spin infinity, and spin-omega had shown significant long-time organoid growth.
  • RCCS rotary cell culture systems
  • all these bio reactors have a major inherent drawback in handling the organoids.
  • These bio-reactors support only for culturing of organoids and there are no provisions for examining/imaging as it grows, it requires physical transferring of them to the separate imaging chamber. During the transfer process, the organoids can get damaged or contaminated, which can alter the final results and leads to inaccurate estimation.
  • United States Patent and Patent Application numbers 20160312165, 2019374944 and 8951781 deal with a system and method for imaging the cells growing in the microfluidic chamber through a transparent material.
  • the primary object of the invention is to provide a novel, compact and simple system for providing a live cell growth culture platform and simultaneously imaging the non-perturbed live cells growing in the microfluidic chip by using an optically transparent glass disk window.
  • Another objective of the present invention is to provide a cost-effective 3D printed or moulded microfluidic chip for imaging each single cell growing in the chip without physical transferring of cells to prevent perturbation and contamination.
  • Another object of the present invention is to provide a portable, stand-alone system comprising cell culture incubation, imaging/sensing support and nutrient delivery all in one platform to provide highly efficient live cell growth.
  • the present invention also provides a microfluidic system for cell culture with lower amounts of nutrient fluid utilization and to support a long-time live cell growth.
  • Yet another object of the present invention is a way to mimic drug/chemical screening/selecting and to identify the viral interactions with the live cells in real time, rapid and in a controlled environment.
  • the present invention provides a novel microfluidic bioreactor system for growing live cells in the culture platform and simultaneously imaging the live cells that are growing in the chamber.
  • the system comprises a 3D printed or molded microfluidic chip with plurality of wells, wherein each well comprises live cells which are covered by a transparent glass window for imaging the live cells, a metallic/ air/liquid coolant base with thermally conductive interface for thermal regulation of microfluidic chip and an oven cap to isolate the microfluidic chip from ambient temperature fluctuations.
  • the preheated fluid or preheated air is used for the thermal regulation of the culture well by circulating it between adjacent wells to provide convective heating.
  • a method for growing live cells and simultaneously imaging the live cells comprising the steps of (i) fabricating a microfluidic chip to support live cell culture and imaging, (ii) embedding and positioning the live cells in the center of the extracellular gel scaffold present in the culture well, (iii) maintaining the culture media supply and other parameters of the culture well to support growth of the live cells and (iv) imaging the live cells in the microfluidic chip using the imaging window for drug/chemical screening and for identifying the viral interactions with the cells.
  • the microfluidic chip is fabricated by additive manufacturing (3D printing) with scope for mass production using moulding technology.
  • the system comprises of a microfluidic chip placed in a micro- incubator environment combined with the nutrient delivery, imaging support to enhance the efficiency of the system by maintaining long term culture without any contamination.
  • FIG. la shows the 3D printed microfluidic chip that supports both live cells culture and imaging
  • FIG. lb shows the 3D printed microfluidic chip with live cells placement in the well
  • FIG. 2 illustrates the overall system for simultaneous live cell imaging and culturing
  • FIG. 3 shows the (a) exploded view of microfluidics bioreactor system, (b) incubation mode of the system and (c) imaging mode of the system;
  • FIG. 4a shows the 3D model of oven temperature controller
  • FIG 4b shows the circuit diagram of temperature controller of the system
  • FIG. 5a shows the thermal simulation of the oven of heat distribution on microfluidic chip
  • FIG. 5b represents the graph showing the thermal gradient maintained within 0.2 °C
  • FIG. 6a represents the graph showing real time temperature data from the oven
  • FIG. 6b represents the histogram showing the temperature tolerance over a set temperature
  • FIG. 7 shows the image of the microfluidics bio reactor system (a) Organoids deployment in microfluidic chip (b) Bio-reactor integration with control system and (c) Compact standalone bio reactor.
  • the present invention provides a novel, highly efficient, compact, portable microfluidic bioreactor system and method for growing live cells (organoids) in a microfluidic chamber and simultaneously imaging the growing cells without any external disturbance.
  • the system is very useful in the drug selection for a particular disease and it helps in viewing the real-time interaction of pathogens (e.g. Coronavirus) with the live cell growing in the wells.
  • pathogens e.g. Coronavirus
  • FIG. 1 illustrates the overall system that is provided with a microfluidic chip 1 with plurality of culture wells 2 growing the live cells 3, where each well in the microfluidic chip consisting of (i) a microfluidic channel 4 to deliver nutrients and/or fluids to the wells, (ii) a transparent glass window 5 for imaging the live cells which is hermetically sealed with bio compatible surgical adhesives or sealed with rubber gasket 6, (iii) a solenoid valve 7 drive to select a particular culture well for culture media delivery and (iv) a plurality of tubes 8 or docking station with gaskets are used for the delivering drugs or chemicals and for sensing or controlling the parameters inside the chip inlet/outlet port 9 using sensors based control.
  • a microfluidic chip consisting of (i) a microfluidic channel 4 to deliver nutrients and/or fluids to the wells, (ii) a transparent glass window 5 for imaging the live cells which is hermetically sealed with bio compatible surgical adhesives or sealed with rubber gasket 6, (ii
  • the system further comprises a metallic/ air/liquid coolant base 10 with thermal connective interface for holding the microfluidic chip and for regulating the temperature of the microfluidic chip.
  • Each microfluidic culture well contains an extracellular scaffold 11 or biocompatible material for holding the live cells 3 inside the well against the current of culture medium 12.
  • a small indent 27 are added on the inner surface of the culture wells as shown in Fig. 2 (b).
  • the live cell 3 can be an organoid that mimics the real human cells, a part of tissue, stem cell, a cell, an embryo or cancer cell.
  • the temperature of the microfluidic chip 1 is controlled by a pre-heated metal 10 (preferably black anodized aluminium). In another embodiment, temperature of the microfluidic chip 1 is controlled by a pre-heated fluid or air that flows between adjacent culture wells to provide convective heating.
  • a pre-heated metal 10 preferably black anodized aluminium
  • temperature of the microfluidic chip 1 is controlled by a pre-heated fluid or air that flows between adjacent culture wells to provide convective heating.
  • the live cell 3 growing in the system can be imaged by any kind of imaging techniques, but not limited only to microscopy.
  • the microfluidic chip 1 is fabricated using additive manufacturing technology (3D Printing) or moulding technology to make the microfluidic chips cost effective and suitable for mass production.
  • method for growing live cells 3 and simultaneously imaging the live cells comprising the steps of fabricating a microfluidic chip to support live cell culture and imaging, embedding and positioning.
  • FIG. 2(a) shows a novel design of a microfluidic chip 1 cross section view which supports both the imaging and live cell culture on the same chip. This is achieved by using a thin transparent glass disk 5 on the wells, which acts both as the water seal for culture medium 12 and as an optical window for live cell (e.g.: organoid) imaging.
  • the cell e.g. an organoid
  • extracellular scaffold gel 11 e.g. Matrigel
  • the culture medium 12 around the organoid contains all the required growth nutrients. As the organoids grow in size and health, the nutrients in the culture media get depleted. This can be observed by the change in the color of the culture media.
  • a small electric pump 21 is provided to infuse the new nutrient rich media. Used culture media is collected from each well in an autoclaved container 20.
  • FIG. 2b shows the placement or positioning of organoids inside the microfluidic chip by a two- level process, where the first layer 29 is created as bed inside the culture well using extracellular scaffold gel 11 (e.g. Matrigel) and is left to be solidified at room temperature. Then, the cell is positioned at the geometrical center of the well and then it is placed over the solid scaffold gel bed. A second layer 30 of extracellular scaffold gel is then used to cover the placed cell, e.g. organoid.
  • This two-level process gives the user the option to position the cell at particular height to adopt the microscope objective 25 working distance.
  • the top of the well is sealed with a glass disk using bio-compatible surgical adhesives or sealed with a rubber gasket 6.
  • a small indent 27 is added on the inner surface of the wells to restrict the detachment of the scaffold from the well for a long time cell culture.
  • each of the wells is provided with a plurality of ports, which helps in continuous temperature 24 logging of each well and selective drug delivery 39 to the individual respective well.
  • a standard cannula is inserted inside the drug delivery port 39 and sealed using biocompatible surgical adhesive. This cannula provides a hermetic sealing support and acts as a one-way valve for the drug delivery.
  • the unique design of the system demonstrates the ability to simultaneously grow and image cells on-chip (e.g.: Organoids), with excellent isolation from environmental perturbation and thus supports the examination closely through the transparent optical window 5 for imaging 26.
  • microfluidic chip After loading the chip with cells, the chip is docked to a micro-incubator environment for the growth of cells.
  • This complete incubator and the supporting devices were designed to fit in the compact form factor for easy transferability between different instruments.
  • the prototype of microfluidic chip has four wells with inlet for culture media and outlets for each well.
  • One of the advantages of this system is its scalability of the design.
  • the wells count can be increased to n numbers and they can be arranged in a matrix fashion with independent access for fluid flow in groups or individually.
  • each well is independently triggered using connection array 23 of drivers 14 of the solenoid valves 7.
  • the cells require a temperature controlled environment (e.g.: for human cells 37 °C) for growth, a benchtop micro- incubator environment was created with an oven manufactured preferably using aluminum blocks 10, where the microfluidic chip is placed in close contact with holding mechanical like direct screwing, docking with spring lock or vacuum succession based lock or magnetic.
  • a resistive heater 33 or semiconductor peltier thermoelectric device is connected to oven plate and a thermistor 34 is used for feedback to regulate the temperature of the oven by direct conduction or through circulating gas/coolant liquid.
  • the culture media or the gas mixture itself can be used for regulating the temperature in micro-incubator environment.
  • the culture media 12 entering the microfluidic chip will be at an ambient temperature of around 25 °C, which is lower than that of the temperature required for the cells growth. Such sudden temperature change can cause a thermal shock for the organoid.
  • a pre-heater was implemented on- chip, a coil of microfluidic channels 13 that has culture media which gets regulated to optimum temperature value to avoid thermal shock to cells, while feeding the media into the cells.
  • the culture media is defused with a gas mixture of 5 % CC , 21 % C , and balanced N2 19 through gas permeable membrane or through agitation based gas mixing in a bottle with filter 22.
  • a DC motor based small peristaltic pump 21 is used for feeding the culture media to wells.
  • the fluid flow is precisely controlled using a pulse width modulation (PWM) 36 signal from the microcontroller.
  • PWM pulse width modulation
  • a dedicated temperature controller 16 is programmed with a PID algorithm and can be controlled through computer PC 18 via main microcontroller
  • FIG. 3(a) shows the overall mechanical assembly of the microfluidic bioreactor system. It has three main components: an oven 10, a microfluidic chip 1 and an acrylic sheet 32 for integration to a microscope.
  • the oven has two parts, the first is a oven base heater 10 which is in contact with the microfluidic chip wells and its temperature is regulated to be around optimum cell temperature (e.g.: 37 °C for human cells).
  • the second one is an oven cap 31 as shown in Fig. 3(b) which is used to isolate the wells from ambient temperature fluctuations.
  • the oven cap 31 is opened while imaging the organoids as shown in FIG. 3(c) and is kept closed otherwise.
  • the heat transfer through the objective lens 25 can cause local temperature drop at the well under imaging and hence the temperature of the lens should be regulated.
  • NTC negative resistance coefficient
  • a dedicated temperature controller 16 designed by PID control algorithm was programmed on a microcontroller 16 and a H-bridge driver 15 is used for controlling the current through the heater. This controller measures the temperature of the oven and compares it with the target temperature (e.g.: 37 °C for human cells), thus computes the error signal e(t). Based on the error, response signal u(t) is calculated with proportional (P), integral (I) and derivative (D) terms as given below, The magnitude of u(t) is mapped to the duty cycle of the PWM signal pin, as shown in FIG. 4(b).
  • P proportional
  • I integral
  • D derivative
  • the duty cycle will be adjusted such that the error reaches zero inside thermoelectric process 35.
  • the P block is a gain factor to amplify the error
  • I block is for calculating cumulative error overtime to eliminate the residual error
  • the D block gives the rate of error change (more rapid the error change, greater damping effect).
  • the Kp, Ki, and Kd constants are optimized in the program 38 to make precise temperature control with fast settling time. On scaling to number of wells per chip (e.g.: 12 well, 48 well 96 wells 386 wells) for uniform temperature distribution preheated air/coolant liquid to be circulated around the wells.
  • FIG. 5(a) shows the steady state simulation results of microfluidics chip with human organoids model
  • FIG. 5(b) shows their corresponding thermal gradient along white dotted lines in Fig. 5(a). It is observed that the temperature is maintained around 37 °C, with the maximum temperature difference across the four wells only being 0.2 °C.
  • FIG. 6(a) shows the experimental measured temperature inside the four wells.
  • a PID temperature controller is connected to the oven heater 33 and to the feedback thermistor 34. It can be noticed that after around 12 minutes, the oven reaches a steady state.
  • the box-and- whisker plot computed for the data at steady state temperature is shown as an inset in FIG. 6(a). This steady state temperature data is acquired for around 12 hours, and from the box plot, it can be observed that the median is around 37.6 °C and 50 % of the data is within 0.2 °C around the mean. The maxima and minima shown by the whiskers are within 0.4 °C.
  • the steady state data temperature tolerance is computed as shown in FIG. 6(b). It can be noted that the oven is regulating the temperature within ⁇ 0.5 % tolerance.
  • the flow of culture medium 12 through the microfluidic chip is characterized.
  • the culture medium containing 191 pL inside each well is replaced within 6 seconds by using a calibrated flow rate of around 33.75 pL/s.
  • Such precise calibration is achieved by using PWM based control with change of PWM duty cycle translating to the speed of the peristaltic pump.
  • the Reynolds number for this microfluidic channel of 2 mm x 0.5 mm and 33 pL/s flow rate is around 263, indicating laminar flow inside the channel.
  • FIG. 7(a) shows the microfluidic bioreactor system with all the organoids positioned inside the well provided with culture medium and the thermistor probe 28 This process was carried out inside the clean room under a laminar hood after cleaning the hood with 70 % ethanol. Initially, extracellular scaffold gel of around 45 pL is filled inside each well and let to solidify for 20 minutes at room temperature. Then, the organoids are placed in the center of each well. Then, another layer of extracellular scaffold gel is applied on top of the organoids and let to solidify at room temperature. This sandwiching process is done to give an extracellular scaffold for organoids to grow in 3D spheroid and it holds organoids inside the well against the current of culture medium.
  • this two layer process helps in positioning the cells at particular height by volume of material bed to match the objective lens 25 working distance.
  • a transparent glass disk 5 was kept on the top of the well and was sealed with bio-compatible surgical adhesives or sealed with rubber gaskets 6.
  • ports are provided for each well through which the thermistor 28 or the cannula can be inserted and sealed using bio compatible surgical adhesives.
  • the culture medium 12 is pumped through each well. Now the chip is ready to be combined with the pre-heated oven for regulating the temperature at around 37 °C for human organoids, as shown in FIG. 7(b). Before filling the media inside the well, the chip should not be placed inside the oven. If so, this would cause the water content in extracellular scaffold gel to evaporate and condense on the glass surface, causing air to get trapped, affecting the quality of imaging.
  • FIG. 7(c) shows the complete system developed for cell culture with a micro-incubator environment and its control electronics 41
  • a styrofoam box 40 is used to isolate from the ambient temperature and to have a clean environment while moving between imaging and IPSC facility rooms. All components are sterilized and provide an isolated environment for an efficient organoid's growth.
  • the advantages of the present invention including, but not limited to the following, are: a novel, simple, portable, standalone microfluidic bioreactor system useful for simultaneously imaging the organoids and also provides long time organoid growth with drug delivery support. This has led to a path for a standard model for screening drugs for various diseases. The drug delivery systems showed the possibility to study the organoids in real-time.
  • the system's superior isolation and ability to closely examine through the optical window can find its applications in many researches or pharmalabs where organoids can be modeled to study interactions between human host organoids to pathogens like the coronavirus.
  • the microfluidic chip can also be scaled for multiple wells. Also, additional features like electrophysiology can be integrated in this system to study the organoid model. Further, the microfluidic valves and pumps can be made on-chip to reduce the amount of culture medium utilization.
  • the system gives the possibility to examine the organoids closely and the fully isolated design of simultaneous live cell imaging and culture platform enables a way to mimic interactions between cells (e.g.: human organoids) host with pathogens in a controlled environment and also has potential to accelerate the development of vaccinations.
  • cells e.g.: human organoids

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Abstract

La présente invention concerne un nouveau système de bioréacteur microfluidique simple, autonome, compact, rentable et très efficace, ainsi qu'un procédé permettant de cultiver des cellules vivantes (3) dans la plate-forme de culture et de former simultanément des images des cellules vivantes qui se développent dans le puits. Le système, tel qu'illustré à la figure 3, comprend une puce microfluidique (1) avec une pluralité de puits (2), une base métallique / fluide préchauffé ou air / liquide de refroidissement (10) avec une interface thermoconductrice pour la régulation thermique de la puce microfluidique et un couvercle de four pour isoler la puce microfluidique des fluctuations de la température ambiante. Le système combine l'apport de nutriments, le dispositif d'imagerie et la chambre d'incubation en une seule plateforme permettant la croissance à long terme de cellules vivantes et évitant le transfert de cellules vivantes pour l'imagerie, ce qui réduit la contamination et les dommages aux cellules. La présente invention est utile pour le dépistage des médicaments et pour identifier l'interaction des particules virales avec des cellules vivantes dans un environnement régulé.
PCT/IN2021/050589 2020-07-22 2021-06-17 Système et procédé d'imagerie et de croissance simultanées de cellules vivantes WO2022018743A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2023211926A3 (fr) * 2022-04-25 2023-12-07 The Regents Of The University Of Colorado A Body Corporate Système et procédés de mesure de la viabilité cellulaire dans un débit élevé par géométrie continue

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US20050221281A1 (en) * 2003-01-08 2005-10-06 Ho Winston Z Self-contained microfluidic biochip and apparatus
WO2013151616A1 (fr) * 2012-04-01 2013-10-10 Emd Millipore Corporation Procédés et dispositifs d'analyse de culture de cellules et de migration de gradient
WO2016172454A1 (fr) * 2015-04-22 2016-10-27 Berkeley Lights, Inc. Structure cellulaire microfluidique
RU2612904C1 (ru) * 2016-04-15 2017-03-13 Евгений Александрович Тоневицкий Способ и микрофлюидный чип для культивирования клеток или клеточной модели
EP3321681A1 (fr) * 2016-11-09 2018-05-16 University of Macau Système de criblage microfluidique

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050221281A1 (en) * 2003-01-08 2005-10-06 Ho Winston Z Self-contained microfluidic biochip and apparatus
WO2013151616A1 (fr) * 2012-04-01 2013-10-10 Emd Millipore Corporation Procédés et dispositifs d'analyse de culture de cellules et de migration de gradient
WO2016172454A1 (fr) * 2015-04-22 2016-10-27 Berkeley Lights, Inc. Structure cellulaire microfluidique
RU2612904C1 (ru) * 2016-04-15 2017-03-13 Евгений Александрович Тоневицкий Способ и микрофлюидный чип для культивирования клеток или клеточной модели
EP3321681A1 (fr) * 2016-11-09 2018-05-16 University of Macau Système de criblage microfluidique

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023211926A3 (fr) * 2022-04-25 2023-12-07 The Regents Of The University Of Colorado A Body Corporate Système et procédés de mesure de la viabilité cellulaire dans un débit élevé par géométrie continue

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