CN115463698A - Microfluidic chip for detecting stem cell deformation performance and preparation method thereof - Google Patents
Microfluidic chip for detecting stem cell deformation performance and preparation method thereof Download PDFInfo
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- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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- 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
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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
- B01L3/502707—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 the manufacture of the container or its components
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- 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
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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
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/46—Means for regulation, monitoring, measurement or control, e.g. flow regulation of cellular or enzymatic activity or functionality, e.g. cell viability
Abstract
The invention discloses a micro-fluidic chip for detecting stem cell deformation performance, which adopts array columns to form a limited micro-flow channel to simulate the capillary vessel environment in a human body, and the preparation method comprises the following steps: step 1: preparing stem cell suspension to be detected; step 2: preparing a micro-fluidic chip; and step 3: building a visualized stem cell flow deformation experiment system; and 4, step 4: finally, a stem cell flow deformation experiment is carried out. The invention realizes the purpose of observing the deformation of the stem cells when the stem cells are extruded to pass through a narrow channel in vitro by combining the microfluidic chip technology, can simulate the extrusion deformation condition of the stem cells at a slit between endothelial cells in vitro, and has low experience requirement on operators, high experimental stability and good repeatability.
Description
Technical Field
The invention relates to the technical field of microfluidic chip preparation, in particular to a microfluidic chip for detecting stem cell deformation performance and a preparation method thereof.
Background
A microfluidic chip (Microfluidics), also called a Lab-on-a-chip (Lab-a-chip), is a micro-technology platform which flexibly combines and integrates a plurality of technical units relating to the fields of biology, chemistry, medicine and the like, such as sample preparation, reaction, separation, detection and the like, on a large scale and completes control analysis.
The micro-fluidic chip can simulate normal physiological and pathological conditions under experimental conditions, so that a new technical platform is provided for cell performance analysis from single cell and multi-cell levels. Therefore, with the continuous development of microfluidic technology, microfluidic chips are also gradually applied to the fields of cell manipulation, cell analysis and the like. The micro-channel with micro-flow control chip and micron size is suitable for single cell introduction, control and performance analysis.
The stem cells have multiple functions of self-renewal, tissue repair, multidirectional differentiation, inflammation inhibition, immunoregulation and the like, and have great potential in clinical application. Hundreds of clinical trials have evaluated the effectiveness of mesenchymal stem cells or multi-component stromal cells for the treatment of various inflammatory and cardiovascular diseases, myocardial infarction, brain injury, spinal cord injury, and the like. Preclinical animal studies have shown that mesenchymal stem cells are injected into the blood circulation preferentially into inflamed or ischemic tissues, i.e., "homing", which is critical to the therapeutic effect. Stem cell homing requires transendothelial migration, also called "extravasation," a behavior that requires extrusion through the gap formed by extracellular matrix fibers and endothelial cells. In this process, the stem cells will undergo large deformation.
Currently, the main methods for detecting the deformation performance of stem cells include atomic force microscopy based on force spectroscopy and micropipette sucking based on micropipette sucking technology. Atomic force microscopy is a common device for measuring intercellular forces, mainly relies on detecting a "force-distance curve" to obtain an interaction force, and measures the degree of cell deformation by contacting stem cells with a matrix and measuring the degree of bending of a cantilever during the action. The micropipette technology uses a microtube with the inner diameter smaller than that of a cell to be detected, and the cell is sucked into the microtube by utilizing negative pressure, so that the deformability of the stem cell is measured. However, both of the above techniques require mechanical contact with stem cells when measuring adhesion, and have potential interference factors and changes the functional state of the stem cell surface; furthermore, the detection throughput of these techniques is relatively low, and it is difficult to quickly obtain detection data of a sample when facing a large amount of samples. Therefore, the technical means for detecting the deformability of stem cells still needs to be improved and perfected continuously.
Disclosure of Invention
1. Technical problem to be solved
The invention aims to solve the problems that the detection flux is relatively low and the detection data of a sample is difficult to obtain quickly when a large number of samples face in the prior art, and provides a micro-fluidic chip for detecting the deformation performance of stem cells and a preparation method thereof.
2. Technical scheme
In order to achieve the purpose, the invention adopts the following technical scheme:
a stem cell deformation performance detection microfluidic chip comprises a PDMS microfluidic chip, wherein the PDMS microfluidic chip comprises an array column microfluidic channel.
The invention also provides a preparation method of the microfluidic chip for detecting the stem cell deformation performance, which comprises the following steps:
step 1: preparing a suspension of mesenchymal stem cells to be detected, placing the collected human mesenchymal stem cells in a centrifuge to obtain packed stem cells, and discarding supernatant; then, washing the stem cells by using 0.01M Phosphate (PBS) buffer solution, centrifuging for 5 minutes by using centrifugation 140 Xg again, and sucking out supernatant after washing; diluting with isotonic phosphate buffer solution to prepare stem cell suspension with the concentration of 5w/ml for later use;
step 2: preparing a micro-fluidic chip, namely preparing the PDMS micro-fluidic chip by adopting Polydimethylsiloxane (PDMS) as a material and adopting a photosensitive dry film soft lithography method;
and step 3: constructing a visualized mesenchymal stem cell flow deformation experiment system, and constructing a microfluidic visualized detection system capable of observing deformation characteristics of stem cells in a microfluidic channel in real time by adopting an inverted microscope microscopic imaging technology and a high-speed and high-resolution imaging technology of an sCMOS camera;
and 4, step 4: finally, a stem cell deformation experiment is carried out.
Preferably, in the step 1, 2ml of cell suspension is centrifuged in each centrifuge tube; and storing according to the storage operation specification of the cell suspension sample.
Preferably, the packed stem cells are obtained by centrifugation at 140 Xg relative centrifugal force for 5 minutes at 4 ℃ using density gradient centrifugation in step 1.
Preferably, the preparation of the microfluidic chip in the step 2 mainly comprises the following steps:
s2.1: laminating a photosensitive dry film layer with the thickness of 100 mu m on a substrate, and manufacturing a pattern on a transparent film into a photomask through a printer;
s2.2: stripping the cured PDMS substrate, and punching holes at two ends of the microfluidic channel by using a flat-head puncher to form a fluid inlet and a fluid outlet;
s2.3; and performing irreversible bonding with a glass slide after surface treatment by using oxygen plasma to prepare the PDMS microfluidic chip, wherein the PDMS on the upper layer of the microfluidic channel contains 1 parallel straight channel, and a column is arranged in the middle of the channel.
Preferably, the length x width x height of the parallel straight channels is 3mm x 1.326mm x 0.015mm, and a row of rectangular column columns are arranged in the middle of the channels to form a series of slits, and the length x width x height of the slits is 5-30 μm x 5-10 μm x 10 μm.
Preferably, the inverted microscope in the step 3 is a japanese olympus IX73, and the objective lens is selected to be a 40-fold oil lens, and the observation is performed by using a 10-fold eyepiece.
Preferably, the sCMOS camera in step 3 is Orca-flash2.8, hamamatsu, japan.
Preferably, the stem cell flow deformation experiment in the step 4 comprises the following steps:
s4.1: injecting 200 mul of stem cell suspension into a liquid storage tank by using a pipette;
s4.2: connecting a microfluid PDMS chip, a sample liquid storage tank and a pressure controller by using a microfluid conduit, and installing an experiment system;
s4.3: turning on a pressure pump, loading at a low speed for 1 minute, discharging bubbles in the system, and keeping the temperature of the container at 25 ℃;
s4.4: the pressure of the gas is regulated through a pressure regulating valve, and the gas pressure drop gradient dP/dL is further controlled;
s4.5: controlling the flow rate Qflow of the fluid by adjusting the gas pressure drop gradient;
s4.6: observing the process of stem cells passing through a narrow channel in real time through a microfluidic visual detection system;
s4.7: according to the speed of the stem cells passing through the middle slit of the channel and the elongation EI, the deformability of the stem cells is characterized:
wherein D is A The major axis dimension of the stem cell, D T Is the short axis dimension of the stem cell.
3. Advantageous effects
Compared with the prior art, the invention has the advantages that:
in the invention, the purpose of observing the deformation of the stem cells generated when the stem cells are extruded to pass through the narrow channel in vitro is realized by combining a micro-fluidic chip technology, and the micro-fluidic chip is adopted to simulate the size of a slit between an in vivo capillary vessel and an endothelial cell of the capillary vessel, so that the deformation of the stem cells generated when the stem cells pass through the narrow channel can be observed in vitro, and the extrusion condition of the stem cells at the slit between the endothelial cells is predicted. Meanwhile, the technology has low requirements on experience of operators, and is high in experimental stability and good in repeatability.
Drawings
FIG. 1 is a technical route chart for detecting stem cell deformation performance based on a microfluidic chip technology;
fig. 2 is an experimental quick chart for observing the deformation process of the mesenchymal stem cells through the narrow channel in real time by the microfluidic visual detection system.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.
Example 1:
a stem cell deformation performance detection microfluidic chip comprises a PDMS microfluidic chip, wherein the PDMS microfluidic chip comprises an array column microfluidic channel.
In the invention, referring to fig. 1, a method for preparing a microfluidic chip for detecting stem cell deformability comprises the following steps:
step 1: preparing a to-be-detected mesenchymal stem cell suspension, placing collected human mesenchymal stem cells in a centrifuge, centrifuging for 5 minutes under the temperature condition of 4 ℃ and the relative centrifugal force of 140 Xg by using a density gradient centrifugation method to obtain packed stem cells, and centrifuging 2ml of cell suspension in each centrifuge tube; storing according to the storage operation specification of the cell suspension sample, and discarding the supernatant; then, washing the stem cells by using 0.01M Phosphate (PBS) buffer solution, centrifuging for 5 minutes by using centrifugation 140 Xg again, and sucking out supernatant after washing; diluting with isotonic phosphate buffer solution to prepare stem cell suspension with the concentration of 5w/ml for later use;
step 2: preparing a micro-fluidic chip, namely preparing the PDMS micro-fluidic chip by adopting Polydimethylsiloxane (PDMS) as a material and adopting a photosensitive dry film soft lithography method;
and step 3: constructing a visualized mesenchymal stem cell flow deformation experiment system, adopting an inverted microscope microscopic imaging technology and an sCMOS camera high-speed and high-resolution imaging technology, wherein the inverted microscope is a Japanese Olympus IX73, an objective lens is a 40-time oil lens, and the objective lens is observed by using a 10-time eyepiece, the inverted microscope is the Japanese Olympus IX73, the objective lens is a 40-time oil lens, and the objective lens is observed by using a 10-time eyepiece, and constructing a microfluidic visual detection system capable of observing the deformation characteristics of stem cells in a microfluidic channel in real time;
and 4, step 4: finally, a stem cell deformation experiment is carried out.
In the invention, the purpose of observing the deformation of the stem cells generated when the stem cells are extruded to pass through the narrow channel in vitro is realized by combining a micro-fluidic chip technology, and the micro-fluidic chip is adopted to simulate the size of a slit between an in vivo capillary vessel and an endothelial cell of the capillary vessel, so that the deformation of the stem cells generated when the stem cells pass through the narrow channel can be observed in vitro, and the extrusion condition of the stem cells at the slit between the endothelial cells is predicted. Meanwhile, the technology has low requirements on experience of operators, and is high in experimental stability and good in repeatability.
Example 2:
the implementation contents of the above embodiments can be referred to the above description, and the embodiments herein are not repeated in detail; in the embodiment of the present application, the difference from the above embodiment is:
referring to fig. 2, the preparation of the microfluidic chip mainly comprises the following steps:
s2.1: laminating a photosensitive dry film layer with the thickness of 100 mu m on a substrate, and manufacturing a pattern on a transparent film into a photomask through a printer;
s2.2: stripping the cured PDMS substrate, and punching holes at two ends of the microfluidic channel by using a flat-head puncher to form a fluid inlet and a fluid outlet;
s2.3; the PDMS microfluidic chip is prepared by performing irreversible bonding on a glass slide after surface treatment by oxygen plasma, wherein the PDMS on the upper layer of the microfluidic channel contains 1 parallel straight channel, a column is arranged in the middle of the channel, the length, the width and the height of the parallel straight channel are 3mm, 1.326mm and 0.015mm, a row of cuboid column is arranged in the middle of the channel to form a series of slits, and the length, the width and the height of the slits are 5-30 mu m, 5-10 mu m and 10 mu m.
Example 3:
the implementation contents of the above embodiments can be referred to the above description, and the embodiments herein are not repeated in detail; in the embodiment of the present application, the difference from the above embodiment is:
the stem cell flow deformation experiment comprises the following steps:
s4.1: injecting 200 mul of stem cell suspension into a liquid storage tank by using a pipette;
s4.2: connecting a microfluid PDMS chip, a sample liquid storage tank and a pressure controller by using a microfluid conduit, and installing an experiment system;
s4.3: turning on a pressure pump, loading at a low speed for 1 minute, discharging bubbles in the system, and keeping the temperature of the container at 25 ℃;
s4.4: the pressure of the gas is regulated through a pressure regulating valve, and the gas pressure drop gradient dP/dL is further controlled;
s4.5: controlling the flow rate Qflow of the fluid by adjusting the gas pressure drop gradient;
s4.6: observing the process of stem cells passing through a narrow channel in real time through a microfluidic visual detection system;
s4.7: according to the speed of the stem cells passing through the middle slit of the channel and the elongation EI, the deformability of the stem cells is characterized:
wherein D is A The major axis dimension of the stem cell, D T Is the short axis dimension of the stem cell.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (9)
1. A stem cell deformation performance detection microfluidic chip comprises a PDMS microfluidic chip and is characterized in that the PDMS microfluidic chip comprises an array column microfluidic channel.
2. The method for preparing the microfluidic chip for detecting the deformability of the stem cells according to claim 1, including the steps of:
step 1: preparing a suspension of mesenchymal stem cells to be detected, placing the collected human mesenchymal stem cells in a centrifuge to obtain packed stem cells, and discarding supernatant; then, washing the stem cells by using 0.01M Phosphate (PBS) buffer solution, centrifuging for 5 minutes by using centrifugation 140 Xg again, and sucking out supernatant after washing; diluting with isotonic phosphate buffer solution to prepare stem cell suspension with the concentration of 5w/ml for later use;
step 2: preparing a micro-fluidic chip, namely preparing the PDMS micro-fluidic chip by adopting Polydimethylsiloxane (PDMS) as a material and adopting a photosensitive dry film soft lithography method;
and step 3: constructing a visualized mesenchymal stem cell flow deformation experiment system, and constructing a microfluidic visualized detection system capable of observing the deformation characteristics of stem cells in a microfluidic channel in real time by adopting an inverted microscope microscopic imaging technology and an sCMOS camera high-speed and high-resolution imaging technology;
and 4, step 4: finally, stem cell deformation experiments are carried out.
3. The method for preparing a microfluidic chip for detecting stem cell deformability as claimed in claim 2, wherein in step 1, each centrifuge tube is centrifuged with 2ml of cell suspension; and storing according to the storage operation specification of the cell suspension sample.
4. The method for preparing a microfluidic chip for detecting stem cell deformability as claimed in claim 2, wherein the packed stem cells are obtained by centrifugation at 140 × g relative centrifugal force for 5 minutes at 4 ℃ using density gradient centrifugation in step 1.
5. The method for preparing the microfluidic chip for detecting the stem cell deformability as claimed in claim 2, wherein the step 2 of preparing the microfluidic chip mainly comprises the steps of:
s2.1: laminating a photosensitive dry film layer with the thickness of 100 mu m on a substrate, and manufacturing a pattern on a transparent film into a photomask through a printer;
s2.2: stripping the cured PDMS substrate, and punching holes at two ends of the microfluidic channel by using a flat-head puncher to form a fluid inlet and a fluid outlet;
s2.3; and performing irreversible bonding with a glass slide after surface treatment by using oxygen plasma to prepare the PDMS microfluidic chip, wherein the PDMS on the upper layer of the microfluidic channel contains 1 parallel straight channel, and a column is arranged in the middle of the channel.
6. The method for preparing a microfluidic chip for detecting stem cell deformability as claimed in claim 5, wherein the length x width x height of the parallel straight channel is 3mm x 1.326mm x 0.015mm, and a row of rectangular columns are arranged in the middle of the channel to form a series of slits, and the length x width x height of the slits is 5-30 μm x 5-10 μm x 10 μm.
7. The method for preparing a microfluidic chip for detecting the deformability of stem cells according to claim 2, wherein the inverted microscope in step 3 is olympus IX73, and the objective lens is selected to be a 40-fold oil lens, and the stem cells are observed through a 10-fold eyepiece.
8. The method for preparing a microfluidic chip for detecting the deformability of stem cells as claimed in claim 2, wherein the sCMOS camera in step 3 is Orca-flash2.8 of Bingson.
9. The method for preparing the microfluidic chip for detecting the stem cell deformability as claimed in claim 2, wherein the stem cell flow deformation experiment in the step 4 includes the following steps:
s4.1: injecting 200 mul of stem cell suspension into a liquid storage tank by using a pipette;
s4.2: connecting a microfluid PDMS chip with a sample liquid storage tank and a pressure controller by using a microfluid conduit, and installing an experiment system;
s4.3: turning on a pressure pump, loading at a low speed for 1 minute, discharging bubbles in the system, and keeping the temperature of the container at 25 ℃;
s4.4: the pressure of the gas is regulated through a pressure regulating valve, and the gas pressure drop gradient dP/dL is further controlled;
s4.5: controlling the flow rate Qflow of the fluid by adjusting the gas pressure drop gradient;
s4.6: observing the process of stem cells passing through a narrow channel in real time through a microfluidic visual detection system;
s4.7: stem cell deformability was characterized by the speed of the stem cells through the slit in the middle of the channel and the degree of elongation EI:
wherein D is A The major axis dimension of the stem cell, D T The minor axis dimension of the stem cell.
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