WO2017091043A1 - Système de culture cellulaire par perfusion microfluidique - Google Patents

Système de culture cellulaire par perfusion microfluidique Download PDF

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Publication number
WO2017091043A1
WO2017091043A1 PCT/KR2016/013732 KR2016013732W WO2017091043A1 WO 2017091043 A1 WO2017091043 A1 WO 2017091043A1 KR 2016013732 W KR2016013732 W KR 2016013732W WO 2017091043 A1 WO2017091043 A1 WO 2017091043A1
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Prior art keywords
microfluidic
channel
cell culture
substrate
fluid
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PCT/KR2016/013732
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English (en)
Korean (ko)
Inventor
고정상
전형진
김문정
박동혁
Original Assignee
부산대학교 산학협력단
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Application filed by 부산대학교 산학협력단 filed Critical 부산대학교 산학협력단
Priority to US15/774,501 priority Critical patent/US10934512B2/en
Priority claimed from KR1020160158138A external-priority patent/KR101878569B1/ko
Publication of WO2017091043A1 publication Critical patent/WO2017091043A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/10—Perfusion

Definitions

  • the present invention relates to a system for cell culture, and more particularly, to a perfusion cell culture system using a microfluidic system to enable 3D cell culture.
  • the existing cell culture experiments can be said to be an experiment in a different environment than in vivo to grow and observe the cells while periodically changing the culture medium on a common plate (plate).
  • micro fluidic systems grow cells through the flow of culture in a very small area of microunits.
  • the microfluidic system provides a bio-like physical environment due to the very small size of the microfluidic channels included in the system, providing sufficient oxygen and at the same time helping the diffusion of nutrients.
  • Such conditions provide more accurate experimental results by providing a more suitable environment for cells than typical macro-scale experiments in cell-related experiments.
  • Many studies have been conducted on cell chips for this purpose, but until now, many experiments have been conducted on 2D systems by coating on the surface of a microfluidic channel.
  • microfluidic technology that is, microfluidic system
  • 3D cell culture technology cell migration, cell interactions, and cell-cell interactions observed in various phenomena in the human body such as angiogenesis, immune response, and cancer metastasis Interaction with extracellular matrix can be directly observed and analyzed in high resolution, real time, and can be applied to a variety of physical and chemical stimuli to determine how a particular element, environment, or combination thereof affects the cell.
  • angiogenesis angiogenesis
  • immune response immune response
  • cancer metastasis Interaction with extracellular matrix can be directly observed and analyzed in high resolution, real time, and can be applied to a variety of physical and chemical stimuli to determine how a particular element, environment, or combination thereof affects the cell.
  • Patent Document 1 Republic of Korea Patent Registration 10-1412155
  • An object of the present invention is to provide a perfusion cell culture system capable of accurate concentration distribution and evaluation, and precisely simulated microenvironment.
  • the present invention relates to a perfusion cell culture system using a microfluidic channel system.
  • the present invention utilizes a microfluidic system.
  • Microfluidic system refers to a technology that controls the flow in an extremely miniaturized device, such as nanoliter or picoliter, of liquid or gas, which has a streamlined flow characteristic, large surface area, and greater inertia than macro systems. It shows unusual characteristics such as surface tension characteristics.
  • the present invention precisely distributes the correct nutrients to the cell culture system through this microfluidic system to enable control and evaluation of the perfusion cell culture system.
  • the perfusion cell culture system of the present invention includes a substrate; A microfluidic injection channel configured to guide a fluid in the substrate along a planar direction of the substrate; Two or more microfluidic branching channels branched into the microfluidic injection channel in the substrate; Microfluidic outlet channels extending in a direction from an end of each branch channel to an upper surface of the substrate to penetrate the upper surface of the substrate to form a through opening; And well plates located on an upper surface of the substrate and connected in fluid communication with each of the outlet channels.
  • Nutrients and / or oxygen necessary for cell culture are supplied to the well plates through the microfluidic channels, and the cells are cultured in the well plates.
  • the culture of the well plate is discharged through the discharge means at the top of the well plate. This provides a perfusion system that continues to be supplied to the microchannels and continues to be discharged through the discharge means.
  • the branch channels are different in length from each other, and are configured to have different flow rates between the branch channels according to the Poiseuille law.
  • the law of fool-guilt is shown in Equation 1 below.
  • R is the resistance
  • P is the pressure drop
  • Q is the flow rate
  • d is the diameter of the channel
  • ⁇ is the viscosity coefficient
  • L is the length of the channel.
  • each branch channel is configured such that the flow rate into the branch channel increases linearly or decreases linearly with respect to the order of the flow direction of the fluid in the injection channel.
  • the difference in flow rate per branch channel is set to increase or decrease linearly, which facilitates comparative evaluation of the feed nutrients of each wellplate.
  • the substrate of the present invention includes an inlet channel for injecting microfluid into the injection channel, and includes a bubble tab between the inlet channel and the injection channel.
  • the bubble tap is, for example, a macro-sized space formed on the side of the microchannel and formed higher than the channel. If a gas (bubble) is contained in the fluid flowing through the microchannel, the bubble gas meets this space and the bubble rises and exits the space and the fluid continues to move into the microchannel.
  • the upper surface of the substrate has a plurality of cavities, through-holes formed by the outlet channels are formed in the inner surface of the cavity, and the bottom of the well plate has two or more holes, and the longest width of the cavity is the well.
  • the well plate is smaller than the shortest width of the lower surface of the plate and forms a space formed between the lower surface of the well plate and the cavity while the well plate is positioned above the cavity, and the fluid discharged from the through opening is stored in the cavity space, and the fluid of the cavity is stored in the well. And flows into the well plate through one or more openings on the bottom surface of the plate.
  • the well plate includes an opening on top of its sidewalls and is configured to discharge the well plate culture of the well plate through the opening.
  • the substrate further comprises a second microfluidic injection channel and two or more second microfluidic branching channels branched to the second microfluidic injection channel on the other side of the wellplate row that is not located on the injection channel; End portions of the two branch channels are joined to the end portions of the branch channel, and is connected in fluid communication with the microfluidic outlet channel.
  • the second branch channels are different in length from each other, and are configured to have different flow rates between the second branch channels according to the Poiseuille law.
  • each second branch channel is configured such that the flow rate into the second branch channel increases or decreases linearly with respect to the order of the flow direction of the fluid in the injection channel.
  • the substrate includes an inlet channel for injecting microfluid into the injection channel, and includes a bubble tab between the inlet channel and the injection channel.
  • the mixing channel includes a meander section, thereby improving the efficiency of mixing.
  • the perfusion cell culture system of the present invention is controllable and configured to compare and observe the nutritional component according to the exact concentration distribution, and can observe and evaluate tumor proliferation for various nutritional component and oxygen concentration inputs.
  • FIG. 1 is an exploded perspective view showing the configuration of the perfusion cell culture microfluidic system of the present invention.
  • FIG. 2 is a plan view of an upper plate of the substrate shown in FIG. 1.
  • FIG. 3 is a bottom perspective view of the upper plate of the substrate shown in FIG. 1.
  • FIG. 4 is a plan view of the cell culture kit shown in FIG. 1.
  • FIG. 5 is a bottom perspective view of the cell culture kit shown in FIG. 1.
  • FIG. 6 is a cross-sectional view showing a state in which the cell culture kit is coupled to the substrate shown in FIG.
  • Figure 7 shows the test results of whether the fluid is correctly supplied at different flow rates through the perfusion cell culture microfluidic system according to the present invention.
  • Figure 8a shows the test results of whether the fluid is supplied at the correct concentration distribution when the fluid is injected through the perfusion cell culture microfluidic system according to the present invention.
  • FIG. 8B is a graph showing the results of quantitative analysis of the fluid in each well plate according to the test of FIG. 8A.
  • FIG. 9A shows the well plates injected with different concentrations of the culture solution and the drug.
  • Figure 9b is a graph showing the result of confirming the amount of DNA extracted from the culture in each well plate shown in Figure 9a through PI staining.
  • Figure 9c is a graph showing the protein quantitative analysis results contained in cells according to the concentration of the drug supplied into each well plate.
  • FIG. 10 is a diagram illustrating a mixing channel of the present invention.
  • FIG. 11 is a view showing mixing efficiency according to the bending characteristics of the mixing channel of the present invention.
  • FIG. 12 is a photograph showing the state of injection into the system of the present invention through a syringe pump (Harvard PHD 2000) for quantitatively injecting the fluid used in Example 2.
  • FIG. 12 is a photograph showing the state of injection into the system of the present invention through a syringe pump (Harvard PHD 2000) for quantitatively injecting the fluid used in Example 2.
  • Example 13 is a photograph showing the results of fluorescence analysis of Example 2.
  • FIG. 14 is a photograph of a standard sample prepared for the calibration curve preparation of Example 2.
  • FIG. 16 (a) is a result of fluorescence analysis of Example 2, and FIG. 16 (b) is a result expressed in concentration ratio by fitting to a calibration curve.
  • FIG. 18 is a photograph showing that in the case of the system of the present invention to which the bubble trap is applied, the bubbles injected together with the injection fluid after the syringe is replaced are collected in the bubble trap.
  • 19 and 20 show the system of the present invention used in Example 2 containing a mixing channel.
  • first and second may be used to describe various components, but the components should not be limited by the terms. The terms are used only for the purpose of distinguishing one component from another.
  • the first component may be referred to as the second component, and similarly, the second component may also be referred to as the first component.
  • FIG. 1 is a view showing the configuration of the perfusion cell culture microfluidic system of the present invention
  • Figure 2 is a plan view of the top plate of the substrate shown in Figure 1
  • Figure 3 is a bottom perspective view of the top plate of the substrate shown in Figure 1
  • 4 is a plan view of the cell culture kit shown in FIG. 1
  • FIG. 5 is a bottom perspective view of the cell culture kit shown in FIG.
  • a perfusion cell culture microfluidic system includes a substrate 100, a microfluidic injection channel 210, a microfluidic branching channel 220, and a microfluidic outlet. Channels 230 and well plates 310.
  • Substrate 100 is a micro fluid injection channel 210, micro fluid branching channels 220 and micro fluid outlet channels 230 to supply a fluid, that is, nutrients required for cell culture to the well plates 310 This is the fluid supply plate that is formed.
  • the substrate 100 has a predetermined thickness to form the microfluidic injection channel 210, the microfluidic branch channels 220, and the microfluidic outlet channels 230 therein.
  • the substrate 100 may be formed of an upper plate 100a and a lower plate 100b having a rectangular plate shape having a predetermined length.
  • the microfluidic injection channel 210, the microfluidic branch channels 220, and the microfluidic outlet channels 230 may be formed on the upper plate 100a or the lower plate 100b, preferably the well plates. It may be formed on the lower surface of the upper plate (100a) to facilitate the supply of fluid to the (310).
  • the upper plate 100a and the lower plate 100b may be stacked and integrated.
  • the substrate 100 may be integral and the channels may be formed inside the substrate.
  • the microfluidic injection channel 210 is configured to guide the fluid injected into the substrate 100 along the planar direction of the substrate 100.
  • the microfluidic injection channel 210 may be engraved on the bottom surface of the upper plate 100a of the rectangular plate shape constituting the substrate 100 to guide the fluid in the long axis direction of the rectangular plate shape.
  • the microfluidic branch channels 220 supply the fluid flowing along the microfluidic injection channel 210 at a predetermined flow rate toward the microfluidic outlet channels 230.
  • the microfluidic branch channels 220 may consist of two or more channels branched to the microfluidic injection channel 210, wherein each of the microfluidic branch channels 220 is separated from the microfluidic injection channel 210. Extend toward the microfluidic outlet channels 230.
  • the microfluidic branch channels 220 may be configured to have different flow rates between the branch channels according to the Poiseuille law.
  • the law of fool-guilt is the same as Equation 1 described above.
  • the fluid flowing through the microfluidic injection channel 210 is divided into the microfluidic branch channels 220 and flows into the microfluidic branch channels 220 depending on the length of each microfluidic branch channel 220.
  • the microfluidic branching channels 220 are designed according to the Poisson's law, such that the flow rate of the microfluidic branching channels 220 increases or decreases linearly with respect to the order according to the flow direction of the fluid of the injection channel.
  • the length of the fluid branch channel 220 may be configured.
  • the microfluidic branch channels 220 may be formed in four, and the length of each of the four microfluidic branch channels 220 may be sequentially set to 69 mm, 40 mm, 21 mm, and 16 mm according to the flow direction of the fluid.
  • the flow rate of the fluid is such that a 69 mm channel provides a flow rate of 40%, a 40 mm channel flows 30%, a 21 mm channel flows 20%, and a 16 mm channel flows 10%. This may be configured in a linear decreasing form.
  • microfluidic branching channels 220 facilitates a comparative evaluation of the feed nutrients of each well plate 310.
  • microfluidic outlet channels 230 discharge fluid moving along the microfluidic branch channels 220 toward the well plates 310.
  • the microfluidic outlet channels 230 extend in the direction of the top surface of the substrate 100 at the end of each microfluidic branch channel 220 to form a through opening through the top surface of the substrate 100.
  • These microfluidic outlet channels 230 are arranged along the flow direction of the fluid.
  • the substrate 100 includes an inflow channel 110 and a cavity 120 to continuously supply the fluid to the well plates 310 from the time when the fluid is supplied into the substrate 100.
  • Inlet channel 110 is a channel for injecting fluid into the microfluidic injection channel 210.
  • the inflow channel 110 penetrates toward the microfluid injection channel 210 from the end of the fluid inlet 111 and the end of the fluid inlet 111 extending from the side of the substrate 100 toward the inside of the substrate 100.
  • the fluid inlet 111 may be positioned higher than the micro fluid injection channel 210, and may have a form of a thread formed on an inner surface thereof so that an adapter connected to a hose for supplying fluid may be coupled.
  • the cavity 120 is a space in which the fluid discharged from the microfluidic outlet channels 230 collects.
  • the cavity 120 may be equal to the number of microfluidic outlet channels 230, and each cavity 120 is formed on the top surface of the substrate 100 to be positioned above the microfluidic outlet channels 230. .
  • the longest width of the cavity 120 is smaller than the shortest width of the bottom surface of the well plate 310, so that the space between the bottom surface of the well plate 310 and the inner surface of the cavity 120 is formed while the well plate 310 is positioned above the cavity 120.
  • the through openings formed by the microfluidic outlet channels 230 are formed on the inner surface of the cavity 120. As a result, fluid discharged from the microfluidic outlet channels 230 may be stored in the cavity 120.
  • the well plates 310 store cells for culture, and supply the fluid for cell culture through the microfluidic injection channel 210, the microfluidic branch channels 220, and the microfluidic outlet channels 230. Receive. To this end, the well plates 310 are formed on the upper surface of the substrate 100 in the same number as the number of the microfluidic outlet channels 230 and are connected in fluid communication with the respective microfluidic outlet channels 230. .
  • Figure 6 is a cross-sectional view showing a state in which the cell culture kit is coupled to the substrate shown in Figure 1, this state is shown well in FIG.
  • the well plates 310 may include a bottom plate 311 constituting a bottom surface of the well plate 310 and a sidewall 312 extending vertically from the bottom plate 311.
  • the well plates 310 may have an opening 312a formed at an upper end of the sidewall 312 so that the culture in the well plates 310 may be discharged, and the bottom plate 311 may include one or more openings 311a.
  • One or more openings 311a may be in fluid communication with the cavity 120 of the substrate 100 so that fluid in the cavity 120 may enter the well plates 310 through the one or more openings 311a. have.
  • the well plates 310 may be provided as one cell culture kit 300.
  • the cell culture kit 300 may be in the form of constituting a plurality of cells (cell) for cell culture. Some of the cells that make up the cell culture kit 300 may be composed of well plates 310 connected to the microfluidic outlet channels 230, and some of the remaining cells may be formed of the well plates 310.
  • the culture receiving cell 320 may be configured to receive a culture discharged through the opening 312a at the top of the sidewall 312.
  • the culture receiving cell 320 may have a form including a bottom plate 321 and sidewalls 322 to form the same shape as the well plates 310, for example, two, each culture
  • the bottom plate 321 of the receiving cell 320 may be formed with a discharge hole (321a) for discharging the supplied culture.
  • the well plates 310 may be arranged to constitute one row among the plurality of cells of the cell culture kit 300, and the culture receiving cell 320 may be different. May be arranged in columns.
  • an opening 322a formed by cutting from the top of the sidewall 322 to the bottom plate 321 may be formed in the sidewall 322 of the culture accommodation cells 320, and the culture accommodation cells 320 may be formed.
  • the bottom between the disposed row and the row of well plates 310 may be blocked, and the opening 312a formed in the sidewall 312 of the well plates 310 faces the culture receiving cells 320.
  • Discharge holes 322a disposed on the sidewalls 322 of the culture accommodation cells 320 may be disposed to face the well plates 310.
  • the culture discharged from the well plates 310 by this structure may be introduced into the culture receiving cells 320.
  • the fluid can provide a perfusion system that continues to be supplied to the microchannels and continues to be discharged through the discharge means.
  • the substrate 100 includes a bubble tab 130.
  • Bubble tab 130 is a space for receiving the bubbles contained in the fluid injected through the inlet channel (110).
  • the bubbletab 130 may be positioned on the path of the microfluidic injection channel 210.
  • the bubble tab 130 may be disposed at the end of the microfluid injection channel 210 connected to the inlet channel 110 so as to be disposed between the inflow channel 110 and the microfluid injection channel 210 for injecting the microfluid.
  • the bubble tab 130 may be formed higher than the micro fluid injection channel 210.
  • the bubble tap 130 includes gas (bubble) in the fluid injected from the inflow channel 110 to the microfluidic injection channel 210, the bubble gas meets the bubble tap 130 and the bubble gas is As it rises into and exits the bubble tap 130, the fluid moves into the microfluidic injection channel 210.
  • gas bubble
  • the perfusion cell culture microfluidic system may include a second microfluid injection channel 240, second microfluidic branch channels 250, a second inflow channel 140, and a second bubbletab 150. ).
  • the second microfluidic injection channel 240 is configured to guide the fluid injected into the substrate 100 along the planar direction of the substrate 100, such as the microfluidic injection channel 210.
  • the second microfluidic injection channel 240 may be located on the other side of the substrate 100 in which the injection channel 210 is not located, or in the same side, the second microfluid injection channel 240 may be positioned to sandwich the rows in which the well plates are placed. .
  • the second microfluidic injection channel 240 is parallel to the microfluidic injection channel 210 and is engraved on the lower surface of the upper plate 100a of the rectangular plate shape constituting the substrate 100 in the longitudinal direction of the rectangular plate shape.
  • the fluid can be guided.
  • the second microfluidic injection channel 240 may guide the fluid in a direction opposite to the flow direction of the fluid flowing along the microfluidic injection channel 210.
  • the second microfluidic branch channels 250 divide and supply the fluid flowing along the second microfluid injection channel 240 at a predetermined flow rate toward the microfluidic outlet channels 230.
  • the second microfluidic branching channels 250 may be formed of two or more channels branched to the second microfluidic injection channel 240, wherein each of the second microfluidic branching channels 250 may be a second channel. It extends from the microfluidic injection channel 240 toward the microfluidic outlet channels 230. That is, the ends of the second microfluidic branch channels 250 are joined to the ends of the microfluidic branch channels 220 and are connected in fluid communication with the microfluidic outlet channels 230.
  • the second microfluidic branch channels 250 may be configured to be different in length from each other so that flow rates between the branch channels are different according to the Poisson's law. That is, the flow rate into the second microfluidic branch channels 250 is increased or decreased linearly with respect to the order of the flow direction of the fluid of the second microfluidic injection channel 240. Length can be configured. Since the configuration of the second microfluidic branch channels 250 is the same as that of the microfluidic branch channels 220, a detailed description thereof will be omitted.
  • the second inflow channel 140 is a channel for injecting fluid into the second microfluidic injection channel 240.
  • the second inflow channel 140 may include a second fluid injection hole extending toward the inside of the substrate 100 from the other side of the substrate 100, that is, the side opposite to the side on which the inflow channel 110 is formed. 141 and a second opening 142 penetrated from the end of the second fluid inlet 141 toward the second microfluid injection channel 240. Since the shape of the second fluid inlet 141 is the same as that of the fluid inlet 111, a detailed description thereof will be omitted.
  • the second bubble tab 150 is a space for receiving the bubbles contained in the fluid injected through the second inflow channel 140.
  • the second bubbletab 150 may be positioned on a path of the second microfluidic injection channel 240.
  • the second bubble tab 150 is connected to the second inflow channel 140 to be disposed between the second inflow channel 140 and the second microfluid injection channel 240 for injecting the microfluid.
  • 2 may be positioned at the end of the micro fluid injection channel 240, wherein the second bubble tap 150 may be formed higher than the second micro fluid injection channel 240.
  • the bubble gas When a gas (bubble) is included in the fluid injected from the second inflow channel 140 to the second microfluid injection channel 240 by the second bubble tap 150, the bubble gas is formed in the second bubble tap ( 150, the bubble gas rises into and exits the second bubble tap 150, and the fluid moves into the second microfluid injection channel 240.
  • a gas bubble
  • nutrients and oxygen required for cell culture can be provided in different precise concentration distributions into each well plate 310 for culturing cells, and at different concentrations.
  • the proliferation of each cultured cell can be observed and evaluated routinely by the flow rate of the fluid dispensed and supplied.
  • FIG. 10 illustrates the system of the present invention further comprising a mixing channel.
  • 20 illustrates a state in which FIG. 19 is coupled.
  • 10 shows an enlarged view of the branch channel.
  • the mixing channel 1010 is further included from the branch point of the branch channel end and the end of the second branch channel to the microfluidic outlet channel.
  • the mixing channel 1010 may be located at the same level as the fluid injection channels and the branching channels, the end of the mixing channel is connected to the start point of the outlet channel and the outlet channel extends in the thickness direction of the substrate so that the top surface of the substrate Penetrates.
  • the mixing channel includes a meander section.
  • the grain section means a section having a serpentine shape as illustrated in FIG. 10, and facilitates mixing of different fluids introduced into the two channels, thereby reducing the length of the mixing channel.
  • the design of the channel of 120 mm is made to confirm that the mixing is performed according to the design.
  • the CV Coefficient of variation
  • the length of the channel is long.
  • the bending shape further applies the mixing according to the flow (fluid convection) in the channel, so that the CV falls below 5% at 60 mm and is uniformly mixed. You can check it. That is, in the case of applying the mixing channel of the grain form, even if the length of 60 mm can be seen evenly mixed, the length of the channel also has the advantage of reduced.
  • FIG. 1 The system illustrated in FIG. 1 was fabricated to evaluate the system of the present invention.
  • Figure 7 shows the test results of whether the fluid is correctly supplied at different flow rates through the perfusion cell culture microfluidic system according to the present invention. The dye was used for the test of FIG.
  • FIG. 7A illustrates test results when a blue dye is injected through the inlet channel 110, and each cavity 120 is shown through the microfluidic outlet channels 230 as shown. It can be seen that the flow rate of the fluid discharged divided into the gradual decrease with respect to the order according to the flow direction of the fluid.
  • FIG. 7B illustrates a case where yellow dye is injected through the second inflow channel 140, and is divided into the respective cavity 120 through the microfluidic outlet channels 230 as shown. It can be seen that the flow rate of the discharged fluid gradually decreased with respect to the order according to the flow direction of the fluid.
  • FIG. 7 (c) shows a case where blue dye and yellow dye are injected through the inflow channel 110 and the second inflow channel 140, respectively, and the microfluidic outlet channels as shown in FIG.
  • the flow rate of the fluid discharged by dividing into each cavity 120 through 230 is linearly decreased in the order of the flow direction of the microfluid injection channel 210 and the flow direction of the second microfluid injection channel 240, respectively. You can see that.
  • Figure 8a shows the test results of whether the fluid is supplied at the correct concentration distribution when the fluid is injected through the perfusion cell culture microfluidic system according to the present invention.
  • ultrapure water DI-water
  • bromine phenol blue Bromphenol blue
  • FIG. 8B is a graph showing the results of quantitative analysis of the fluid in each well plate according to the test of FIG. 8A.
  • the perfusion cell culture microfluidic system according to the present invention can be effectively used for three-dimensional perfusion cell culture experiments. To confirm this, while continuously injecting the culture solution and the drug (hydrogen peroxide solution) to confirm the cell death results according to the concentration of the drug.
  • Figure 9a shows the state in which the culture medium and the drug is injected into the correct concentration distribution different from the well plate
  • Figure 9b is a graph showing the result of confirming the amount of DNA extracted from the culture in each well plate through PI staining
  • Figure 9c is a graph showing the protein quantitative analysis results contained in cells according to the concentration of the drug supplied into each well plate.
  • a syringe pump such as the photograph of FIG. 12 (Harvard PHD 2000 ) was used. Two 10 ml syringes were used for long infusion. The experiment was prepared by connecting the syringe and the device using a silicon tube. In addition, a pipette and a 96 well plate were prepared so that the fluid from each well of the device could be collected at a predetermined time for fluorescence analysis.
  • One syringe was injected with DI water into the first microfluid injection channel, and the other syringe was injected with a mixture of methanol and ultrapure water in which fluorescent substance rhodamine 110 was dissolved into the second microfluid injection channel.
  • the channel was filled with ultrapure water so that the concentration gradient functioned smoothly as soon as the pump was running. If the channel is not filled with ultrapure water, the inside of the bubble trap is placed in a bubble-filled environment before the experiment, making it difficult to achieve normal concentration gradient operation. Therefore, the syringe pump was operated in an environment filled with ultrapure water to check the function of the concentration gradient and bubble trap of rhodamine 110.
  • Samples collected in 96 well plates were fluorescence analyzed using a FLUOstar OPTIMA microplate reader. Considering that the excitation and emission wavelengths of rhodamine 110 are 496 and 520 nm, respectively, the excitation and emission wavelengths of the reader are set to 485 and 520 nm, and the gain value is set to 100. The luminous intensity of rhodamine 110 was measured.
  • the perfusion cell culture microfluidic system according to the present invention can observe various cell cultures and cell proliferation, and can quantitatively evaluate cell proliferation.
  • the microenvironment in the living body can be simulated to observe and evaluate tumor proliferation in the living body.

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Abstract

L'invention concerne un système de culture cellulaire par perfusion microfluidique. Le système de culture cellulaire par perfusion microfluidique comprend : un substrat ; un canal d'injection microfluidique formé à l'intérieur du substrat pour guider un fluide le long d'une direction plane du substrat ; au moins deux canaux ramifiés microfluidiques ramifiés à partir du canal d'injection microfluidique à l'intérieur du substrat ; des canaux de sortie microfluidiques s'étendant dans la direction de la surface supérieure du substrat depuis l'extrémité de chacun des canaux ramifiés et pénétrant dans la surface supérieure du substrat de façon à former des trous traversants ; et des plaques à puits disposées sur la surface supérieure du substrat et en communication fluidique avec chacun des canaux de sortie. Un tel système de culture cellulaire par perfusion microfluidique met en œuvre un système microfluidique. Le système microfluidique se rapporte à une technologie permettant de commander un écoulement d'une trace de liquide ou de gaz (de l'ordre du nanolitre ou du picolitre) dans un dispositif microminiaturisé, et présente des caractéristiques uniques telles qu'un écoulement rationnel, des caractéristiques de surface importante et une tension de surface supérieure à l'inertie, contrairement à un système macro. La présente invention peut commander et évaluer un système de culture cellulaire par perfusion en répartissant avec précision les bons nutriments dans un système de culture cellulaire par le biais du système microfluidique.
PCT/KR2016/013732 2015-11-26 2016-11-25 Système de culture cellulaire par perfusion microfluidique WO2017091043A1 (fr)

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US15/774,501 US10934512B2 (en) 2015-11-26 2016-11-25 Microfluidic perfusion cell culture system

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KR10-2015-0166102 2015-11-26
KR20150166102 2015-11-26
KR10-2016-0158138 2016-11-25
KR1020160158138A KR101878569B1 (ko) 2015-11-26 2016-11-25 관류 세포 배양 마이크로 유체 시스템

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CN113046244A (zh) * 2021-03-30 2021-06-29 上海睿钰生物科技有限公司 培养装置及基于培养装置的培养方法
WO2021223150A1 (fr) * 2020-05-07 2021-11-11 元锦生物科技股份有限公司 Récipient pour ajuster la distribution de liquide et procédé associé

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021223150A1 (fr) * 2020-05-07 2021-11-11 元锦生物科技股份有限公司 Récipient pour ajuster la distribution de liquide et procédé associé
CN113046244A (zh) * 2021-03-30 2021-06-29 上海睿钰生物科技有限公司 培养装置及基于培养装置的培养方法

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