CN-121406440-B - Oxygen supply nutrition delivery micro-fluidic brain organoid chip and preparation method thereof

CN121406440BCN 121406440 BCN121406440 BCN 121406440BCN-121406440-B

Abstract

The invention discloses an oxygen supply nutrition delivery micro-fluidic brain organoid chip and a preparation method thereof, belonging to the technical field of biomedical engineering and micro-fluidic organoid chips, comprising the steps of inoculating microglial cells into an organoid culture cavity after oxidation stress treatment, and inoculating neurons subjected to ischemia and hypoxia treatment into a peripheral nerve culture cavity; the method comprises the steps of establishing concentration distribution of inflammatory factors or damage related factors through a nutrition mixing matrix and a distribution branch to simulate the differential microenvironment of the adjacent and distal ends of a focus, carrying out time sequence evaluation on the interaction process of microglial activation and neuron damage by combining morphological observation and molecular detection, establishing concentration distribution of inflammatory factors or damage related factors between two cavities through the nutrition mixing matrix, and obtaining morphology and molecular readout according to a time sequence of 24 to 72 hours so as to reproduce the interaction process of microglial activation and neuron damage.

Inventors

Wu Moxin
YIN XIAOPING
CHEN ZHIYING
LIU XIAOQUN
WU RONG
JIANG MIN

Assignees

九江学院附属医院

Dates

Publication Date: 20260508
Application Date: 20251023

Claims (5)

1. The utility model provides an oxygen suppliment nutrition delivery micro-fluidic brain organoid chip, includes chip base member, organoid culture chamber and with culture chamber upper and lower relative gaseous phase oxygen suppliment microchannel layer and the liquid phase nutrition perfusion microchannel layer that sets up, its characterized in that: a permeable membrane is arranged between the gas-phase oxygen supply micro-channel layer and the liquid-phase nutrition perfusion micro-channel layer, the permeable membrane is subjected to graphical treatment to form at least two oxygen permeation subareas, the ratio of oxygen permeation coefficients between the subareas is 2-8, and the alignment precision is not more than 20 microns; partition oxygen supply branches are correspondingly arranged above each membrane partition one to one in the gas-phase oxygen supply microchannel layer, each partition oxygen supply branch is communicated with the distribution header pipe and provided with a flow limiting structure and an independently adjustable pressure port, and gas path isolation is realized between adjacent partition oxygen supply branches through partition rib bodies; The organoid culture cavity is of a removable cassette structure, a guiding and sealing interface is arranged between the cassette and a chip substrate, the assembly tolerance is not more than 20 microns, a replaceable three-dimensional porous bracket and a circumferential buffer ring groove are arranged in the culture cavity, the aperture of the bracket is 20-80 microns, the porosity is 40-70%, the width of the ring groove is 100-300 microns, and the ring groove is used for limiting the average shear stress in the culture cavity to not more than 1 Pa in the rated perfusion rate range; The liquid-phase nutrient perfusion micro-channel layer comprises a nutrient mixing matrix and a metabolic clearance matrix, wherein the nutrient mixing matrix is provided with at least three inlets and is communicated with the culture cavity through at least four distribution branches, the metabolic clearance matrix is communicated with the culture cavity and is provided with at least one clearance outlet, and an on-chip calibration port, a bypass sampling channel and an optical detection window are arranged on the matrix and used for weight self-calibration; The bottom or the side wall of the culture cavity is integrated with a dissolved oxygen sensing array and a metabolite sensing array, and the pixel density of the array is not lower than 25 points per square millimeter and is provided with a reference channel for carrying out on-line measurement and calibration on dissolved oxygen and metabolic indexes; the permeable membrane is a gas permeable elastomer or a nano-pore polymer, the oxygen transmission rate is not lower than 300Barrer, the thickness is 20-100 micrometers, the boundary transition width of the membrane partition is not more than 50 micrometers, and the partition number is 3-6; The partition oxygen supply branch comprises flow limiting micropores or equivalent liquid resistance pieces with equivalent diameters of 10-50 micrometers, the equivalent liquid resistance deviation of each branch is not more than 10%, and the height between adjacent partitions is not less than the height of the channel and is not less than the height of the channel A partition rib having a thickness of 20 to 80 μm such that a cross-zone cross-flow ratio measured under a pressure difference of 1kPa is not more than 3%; The projection coincidence ratio of the partition oxygen supply branch and the corresponding film partition is not less than 95%; A selective unidirectional micro-channel array is arranged between the organoid culture cavity and the peripheral nerve culture cavity, and the micro-channel sequentially comprises an inlet contraction section, a neck limiting section, a gradual change expansion section and an outlet guide section along the flow direction, and the following geometric and surface energy constraints are satisfied: The inlet aspect ratio is greater than 1; The equivalent width of the neck is 2 to 4 micrometers; the relative change rate of the width of the transition section along the flow direction is smaller than 0.1; The outlet guide angle is 10 to 20 degrees; the inner surface of the channel is provided with a surface self-energy gradient which is gradually decreased along the channel direction so as to realize the unidirectional passage of axons and inhibit the reverse entry of cell bodies; The nutrition mixing matrix, the metabolism clearing matrix and the on-chip calibration structure form a self-calibration unit together, and the self-calibration unit comprises bypass sampling micro-channels with equal length errors of not more than 10% with all outlet branches, an optical detection window with equivalent optical path length of 0.2-1.0mm and a sampling port with the center distance of 2-10mm from a tracing injection port; and a locating pin and a thrust surface are arranged between the organoid culture cavity cassette and the chip main body to realize the resetting and locating, the fit clearance is 5-20 mu m, And calibrating the tracing sequence of the self-calibration unit to ensure that the maximum value of the matrix weight estimation error obtained based on least square solution is not more than 3%.
2. A method for preparing an oxygen supplying nutrition delivering micro-fluidic brain organoid chip according to claim 1, comprising the steps of: s1, preparing a structural master model of a gas-phase oxygen supply micro-channel layer, a liquid-phase nutrition perfusion micro-channel layer and a selective unidirectional micro-channel array, and obtaining a corresponding structural substrate through photoetching and copy molding; S2, carrying out partition graphic processing on the permeable membrane to form oxygen permeation partitions, enabling the ratio of oxygen permeation coefficients of different partitions to be 2-8, and completing alignment with a microchannel layout, wherein the positioning deviation is not more than 20 microns; S3, sequentially laminating the gas phase channel layer, the partition film and the liquid phase channel layer and bonding at 60-90 ℃ to form a three-layer stacked structure; S4, assembling an organoid culture cavity cassette, a three-dimensional porous bracket and a circumferential buffer ring groove, wherein the aperture of the bracket is 20-80 microns, the porosity is 40-70%, and the width of the ring groove is 100-300 microns so as to ensure that the average shear stress in the culture cavity is not higher than 1 Pa under the rated perfusion rate; S5, integrating an oxygen dissolving sensing array and a metabolite sensing array at the bottom or the side wall of the culture cavity and forming an optical detection window and a bypass sampling channel, wherein the pixel density of the array is not lower than 25 points per square millimeter and the sampling period is not more than 10 seconds; s6, constructing a nutrition mixing matrix and a metabolism removal matrix, setting a tracing injection port and a calibration port, injecting trace liquid according to a preset input sequence, collecting outlet concentration data, and solving a weight matrix by adopting a least square method and combining sparse regularization to ensure that the maximum value of a weight estimation error is not more than 3%; And S7, carrying out factory quality control endpoint test, wherein the factory quality control endpoint test comprises that an oxygen uniformity index UO is smaller than 0.1, a gradient linearity determining coefficient is not smaller than 0.99, the time for the gradient to reach a steady state is smaller than 2 minutes, the average shear stress in a culture cavity is not higher than 1 Pa under the specified perfusion rate, and generating batch records.
3. The method for preparing an oxygen supplying nutrition delivery microfluidic brain organoid chip according to claim 2, wherein in the membrane partition patterning process of step S2, plasma surface modification is first performed, followed by sol-gel local pore sealing to form oxygen permeation partitions; The plasma surface modification adopts a mixed gas of oxygen and argon as working gas, the volume fraction of the oxygen is 50-100%, the total flow is 50-150 standard cubic centimeters per minute, the cavity pressure is 50-200 millitorr, the radio frequency is 13.56 megahertz, the radio frequency power is 50-150 watts, the treatment time is 30-180 seconds, and the annealing is carried out for 10-30 minutes at 60-90 ℃ after the treatment; Then, depositing a silica sol precursor solution on a target partition in a spin coating mode, wherein the solid content is 0.5-3.0%, the spin coating speed is 500-2000 revolutions per minute, the spin coating time is 10-30 seconds, the curing temperature is 80-120 ℃, and the curing time is 5-20 minutes; The alignment deviation between the plasma surface modification and the subsequent sol-gel local hole sealing and the film partition layout is not more than 20 microns, the obtained partition boundary transition width is not more than 50 microns, and the ratio of the oxygen permeability coefficient of the high permeability area to the low permeability area reaches 2 to 8.
4. An oxygen-supplying nutrient-delivering microfluidic brain organoid according to any one of claims 2 or 3 The preparation method of the chip is characterized in that when the weight self-calibration is carried out on the nutrition mixing matrix and the metabolism removing matrix in the step S6, the following steps are sequentially carried out: Setting a tracing input sequence and an outlet sampling position under the condition of constant temperature of 23 ℃ and establishing isochronous sampling, wherein the sampling period is not more than 10 seconds; sequentially injecting tracer solution with mass fraction of 0.01% to 0.10% into each inlet under the pressure difference of 1kPa, and respectively maintaining the 60 second stable period; Collecting the concentration time sequence of each outlet and averaging in a steady-state interval to form observation data; Solving a weight matrix by least square and combining sparse regularization, setting a regularization coefficient to be 0.001-0.01, and applying column sum 1 and non-negative constraint to a solving result; calculating a weight estimation error, writing the weight estimation error into the on-chip storage if the maximum value is not more than 3%, and returning to the step 2 to increase the measurement time if the maximum value is more than 3%; And executing a recheck test and calculating the deviation of the target concentration of the outlet, ending the calibration if the maximum deviation is not more than 5%, and repeating the steps after 8 to 24 hours if the maximum deviation is more than 5%.
5. The method for preparing the oxygen supply nutrition delivery micro-fluidic brain organoid chip according to claim 4, wherein the reset verification process is implemented after the replacement and reset of the cassette, and the method comprises the following steps in sequence: Under the condition of 23 ℃, the pressure difference of 10kPa is established by taking deionized water as a working medium and maintained for 300 seconds, and a sealing leakage test is carried out, wherein the pressure drop rate is not more than 1% per minute and the equivalent leakage rate is not more than 2 microlitres per minute; Measuring the volume flow of each distribution branch under the pressure difference of 1kPa, and comparing the volume flow with a factory archive value, wherein the maximum relative deviation is not more than 5%; applying the same set partial pressure to each regional oxygen supply branch, collecting dissolved oxygen distribution at 23 ℃, and calculating the relative deviation of a dissolved oxygen uniformity index UO and a target field, wherein the UO is less than or equal to 0.10 and the relative deviation is not more than 10%; and when the judgment of the steps is met, marking that the reset is qualified, writing a batch record, and if any one item is not met, returning to the first step after reassembling, and repeating the steps.

Description

Oxygen supply nutrition delivery micro-fluidic brain organoid chip and preparation method thereof Technical Field The invention relates to the technical field of biomedical engineering and microfluidic organ chips, in particular to an oxygen supply nutrition delivery microfluidic brain organoid chip and a preparation method thereof. Background In recent years, the brain organoids are rapidly developed by combining a microfluidic technology, wherein a gas permeable membrane is used for transmembrane oxygen supply, tree-shaped or network-type multi-inlet compounding is used for space-time delivery of nutrients and factors, and liquid phase opposite pumping is used for metabolic elimination; to enhance observability, on-chip optical detection windows, bypass sampling ports and dissolved oxygen/metabolic sensing arrays are also introduced gradually; the general trend goes from static batch to continuously programmable and attempts are made to handle oxygen tension, nutrient supply and metabolic load simultaneously on the same platform; however, the existing device mostly realizes basic maintenance by combining whole-area or rough-area oxygen supply with single liquid phase perfusion, and many schemes still realize oxygen supply, supply and removal, on-line detection and dispersion, so that a systematic solution which can be repeatedly, traceably and conveniently realize mass production quality control is difficult to form in one chip. The prior art mainly has three bottlenecks: Firstly, the gas phase side and the permeable membrane are not subjected to fine patterning and one-to-one alignment design, and the lack of a flow limiting element and a partition rib body can inhibit cross-region crosstalk, so that the boundary of an oxygen field in a culture cavity is fuzzy, the gradient is easy to distort, and uniform and programmable oxygen tension distribution is difficult to obtain; secondly, multi-inlet nutrition mixing and downstream clearing often assume equal resistance or ideal shunt, a calibration port, a bypass sampling channel and a detection window on a chip do not form closed loop self-calibration, the outlet weight drifts along with assembly and temperature drift, and output linearity and error are difficult to quantitatively control; thirdly, the culture cavity still has difficulty in realizing low shear and long-term stability while ensuring perfusion, lacks removable cassettes and standardized reset verification processes, and lacks uniform caliber for endpoints such as dissolved oxygen uniformity, gradient reaching steady-state time, flow deviation, leakage limit value and the like. Disclosure of Invention In order to achieve the above purpose, the present invention provides the following technical solutions: An oxygen supply nutrition delivery microfluidic brain organoid chip comprises a chip substrate, an organoid culture cavity, and a gas phase oxygen supply microchannel layer and a liquid phase nutrition perfusion microchannel layer which are arranged up and down relative to the culture cavity; A permeable membrane is arranged between the gas-phase oxygen supply micro-channel layer and the liquid-phase nutrition perfusion micro-channel layer, the permeable membrane is subjected to graphical treatment to form at least two oxygen permeation subareas, the ratio of oxygen permeation coefficients between the subareas is 2-8, and the alignment precision is not more than 20 microns; A partition oxygen supply branch is correspondingly arranged above each membrane partition one to one in the gas phase oxygen supply micro-channel layer, each partition oxygen supply branch is communicated with the distribution header pipe and is provided with a flow limiting structure and an independently adjustable pressure port, gas path isolation is realized between adjacent partition oxygen supply branches through a partition rib body, and the cross-region crossflow ratio is not more than 5% under the condition of 1kPa pressure difference; The organoid culture cavity is of a removable cassette structure, a guiding and sealing interface is arranged between the cassette and a chip substrate, the assembly tolerance is not more than 20 microns, a replaceable three-dimensional porous bracket and a circumferential buffer ring groove are arranged in the culture cavity, the aperture of the bracket is 20-80 microns, the porosity is 40-70%, the width of the ring groove is 100-300 microns, and the ring groove is used for limiting the average shear stress in the culture cavity to not more than 1 Pa in the rated perfusion rate range; The liquid-phase nutrient perfusion micro-channel layer comprises a nutrient mixing matrix and a metabolic clearance matrix, wherein the nutrient mixing matrix is provided with no less than three inlets and is communicated with the culture cavity through no less than four distribution branches, the metabolic clearance matrix is communicated with the culture cavity and is provided with no less than one