Stem cell culture system purification method based on double-helix inertial micro-fluidic chip

Abstract

The invention relates to a stem cell culture system purification method based on a double-helix inertial micro-fluidic chip. The double-helix inertial micro-fluidic chip comprises a base and five sequentially connected micro-pipelines fixed on the base, the five sequentially connected micro-pipelines are respectively an inlet micro-pipeline, a first helix micro-pipeline, an S-shaped micro-pipeline, a second helix micro-pipeline and an outlet micro-pipeline, and the first helix micro-pipeline and the second helix micro-pipeline are distributed in a staggered manner. The S-shaped micro-pipeline is positioned in the center of the double-spiral channel formed by the first spiral micro-pipeline and the second spiral micro-pipeline; the front end of the outlet micro-pipeline is divided into a first stage and a second stage, the width of the second stage is larger than that of the first stage, and the tail end of the outlet micro-pipeline is divided into a collection outlet micro-pipeline and a waste outlet micro-pipeline. According to the method, the stem cells are sorted and purified from a stem cell-feeder layer cell co-culture system in a high-flux and label-free manner.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of cell culture, in particular to a stem cell culture system purification method based on a double helix inertia microfluidic chip. BACKGROUND

[0002] Pulmonary structural damage caused by respiratory diseases poses a significant threat to human health. Lung repair and regeneration rely on a variety of lung epithelial stem progenitor cells, so lung stem progenitor cell transplantation has broad clinical application prospects. p63-positive lung precursor cells (p63 + lung progenitor cell, p63 + LPC) have an important function in repairing bronchial and alveolar tissue, and are the only type of lung stem progenitor cell that has been proven to be isolated from patients with respiratory diseases and cultured on a clinical scale, making them the most clinically promising lung stem progenitor cells.

[0003] Methods for producing medical p63 + LPC rely on growth-arrested mouse fibroblasts (3T3-J2) as feeders (GAF) to maintain their proliferation and differentiation potential. Therefore, this method requires the separation of p63 + LPC and GAF at each cell passage, and the elimination of residual GAF through terminal short-term feeder-free passage. In clinical trials, this separation is achieved by differential enzymatic digestion (DED), while laboratory research uses fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS). However, all of the above methods have inherent limitations.

[0004] Differential enzymatic digestion (DED) separates p63 + LPC and GAF based on the difference in adhesion to the culture dish surface during digestion enzyme-induced detachment. Although this method is relatively low-cost and scalable to clinical-scale production, its stability is affected by the variability of cell detachment time in different batches of experiments, and it is more dependent on the experience and skills of the experimenter, posing a challenge to standardized operating procedures. In contrast, fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) achieve precise separation through specific surface antigens, with higher stability, but have issues such as poor sorting efficiency, limited scalability, and high operating costs. Therefore, developing a technology that integrates the advantages of both methods and overcomes their shortcomings is of great value to advancing the clinical-scale production of p63 + LPC. SUMMARY

[0005] The application aims to provide a stem cell culture system purification method based on a double spiral inertia microfluidic chip, so as to realize high-throughput and label-free sorting and purification of stem cells from a stem cell-feeder cell co-culture system.

[0006] To achieve the above-mentioned purpose, the application adopts the following technical solutions:

[0007] In a first aspect, the application provides a double spiral inertia microfluidic chip, which comprises a base and five sequentially connected micro-pipes fixed on the base, the five sequentially connected micro-pipes are respectively an inlet micro-pipe, a first spiral micro-pipe, an S-shaped micro-pipe, a second spiral micro-pipe and an outlet micro-pipe, the first spiral micro-pipe and the second spiral micro-pipe are staggered and distributed to form a double spiral channel, and the S-shaped micro-pipe is located at the center of the double spiral channel formed by the first spiral micro-pipe and the second spiral micro-pipe; the front end of the outlet micro-pipe is divided into a first stage and a second stage, the width of the second stage is greater than that of the first stage, the end of the outlet micro-pipe is divided into a collection outlet micro-pipe and a waste outlet micro-pipe, and the width of the waste outlet micro-pipe is not less than that of the collection outlet micro-pipe.

[0008] Further, the channel cross section of the collection outlet micro-pipe is a rectangle with a width of 0.52 mm to 0.69 mm and a height of 0.18 mm to 0.26 mm, and the channel cross section of the waste outlet micro-pipe is a rectangle with a width of 0.69 mm to 0.87 mm and a height of 0.18 mm to 0.26 mm.

[0009] Further, the channel cross section of the collection outlet micro-pipe is a rectangle with a width of 0.52 mm to 0.69 mm and a height of 0.18 mm to 0.26 mm, and the channel cross section of the waste outlet micro-pipe is a rectangle with a width of 0.69 mm to 0.87 mm and a height of 0.18 mm to 0.26 mm.

[0010] Further, the S-shaped micro-pipe is formed by reversely connecting two semicircular pipes with the same diameter.

[0011] Further, the diameter of the two semicircular pipes is 4.6 mm.

[0012] Further, the channel cross section of the second spiral micro-pipe is a right-angled trapezoid, and the height of the side close to the center is less than the height of the side far from the center.

[0013] Further, the base length of the right-angled trapezoid is 0.8 mm, the height of the side close to the center is 0.12 mm to 0.18 mm, and the height of the side far from the center is 0.18 mm to 0.26 mm.

[0014] Further, the bottom side length of the right trapezoid is 0.8 mm, the height of the side close to the center is 0.14 mm, and the height of the side far from the center is 0.21 mm.

[0015] Further, the channel cross section of the first spiral micro-pipe is a rectangle with a width of 0.8 mm and a height of 0.08 mm-0.125 mm.

[0016] Further, the flow rate of the mixed cell suspension injected into the inlet micro-pipe of the inertial microfluidic chip is controlled at 3 mL / min-4 mL / min by using a micro-injection pump and a syringe.

[0017] Further, the flow rate of the mixed cell suspension injected into the inlet micro-pipe is controlled at 3.5 mL / min.

[0018] In a second aspect, the application provides a stem cell culture system purification method based on a double-spiral inertial microfluidic chip, comprising the following steps:

[0019] Step S11, install the microfluidic device, connect the syringe to the inlet micro-pipe of the inertial microfluidic chip using a Tygon hose, and install the syringe on a micro-injection pump; connect the collection outlet micro-pipe and the waste outlet micro-pipe of the inertial microfluidic chip to a collection tube and a waste liquid tube, respectively, using Tygon hoses;

[0020] Step S12, slowly pump 1% Pluronic F-68 solution into the chip, and introduce the solution into the waste liquid tube for 1 minute of pretreatment to prevent cell adhesion; ensure that there are no air bubbles in the pipe and no air in the outlet hose;

[0021] Step S13, pump 4 mL of PBS buffer to flush the chip pipe, completely remove the residual Pluronic F-68 solution in the pipe, and introduce the PBS buffer into the waste liquid tube;

[0022] Step S21, resuspend the mixed cells containing p63 positive lung precursor cells (p63 + lung progenitor cell, p63 + LPC) and growth-arrested feeder cells (GAF) in p63 + LPC culture medium containing 0.1% Pluronic F-68, and the cell concentration is 1×10 6 - 5×10 6 cells / mL;

[0023] Step S22, another sterile syringe is taken, the sterile syringe is connected with a blunt needle and a proper length of Tygon hose, the plunger is pulled out, the cell suspension is poured into the syringe, the plunger is put back, the syringe is placed upright, and air is discharged;

[0024] Step S23, the syringe containing the cell suspension is fixed on the microsyringe pump, the syringe containing the cell suspension is connected with the inlet microchannel of the inertial microfluidic chip using the Tygon hose, the model of the syringe is input into the microsyringe pump, and the injection speed of the microsyringe pump is set to 3 mL / min ~ 4 mL / min;

[0025] Step S24, after starting injection, the Tygon hoses of the two outlets are both introduced into the waste liquid pipe first, and after 5 seconds, the cells are accelerated and form stable focusing in the chip, the Tygon hose of the collection outlet is introduced into the collection pipe to start collecting p63 positive lung precursor cells (p63 + LPC).

[0026] Further, in step S23, the injection speed of the microsyringe pump is set to 3.5 mL / min.

[0027] Advantages of the present application:

[0028] First, the present application is suitable for purifying stem cell-feeder cell co-culture system, which realizes performance balance between the existing differential enzyme digestion method (DED) and fluorescence activated cell sorting method (FACS) purification method, combines high throughput, superior scalability, cost effectiveness, and good purity and cell recovery rate, and is a more suitable solution for clinical scale stem cell production.

[0029] Second, using the present method can not only effectively exclude feeder cells in the stem cell culture system, but also exclude senescent stem cells with impaired proliferation and differentiation function, which is beneficial to guarantee the quality attributes of stem cell products and has advantages that existing purification methods cannot achieve.

[0030] Third, the present application designs a unique double helix inertial microfluidic chip composed of five sequentially connected microchannels, which can realize high-throughput, label-free sorting and purification of stem cells from stem cell-feeder cell co-culture system.

[0031] Fourth, the geometric size and sorting flow rate of the double helix inertial microfluidic chip of the present application are designed and optimized based on the size of p63 + LPC and growth-inhibited 3T3-J2 cells, which improves the performance balance of existing methods in sorting throughput, purity, cell recovery rate, etc. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1p63 is the p63 in the embodiment of the present application + LPC and GAF cell size difference comparison chart.

[0033] Figure 2 is the structural schematic diagram of the double helix inertial microfluidic chip in the embodiment of the present application.

[0034] Figure 3 is the structural schematic diagram of the inlet micro-pipe of the double helix inertial microfluidic chip in the embodiment of the present application.

[0035] Figure 4 is the structural schematic diagram of the first spiral micro-pipe of the double helix inertial microfluidic chip in the embodiment of the present application.

[0036] Figure 5 is the structural schematic diagram of the S-shaped micro-pipe of the double helix inertial microfluidic chip in the embodiment of the present application.

[0037] Figure 6 is the structural schematic diagram of the second spiral micro-pipe of the double helix inertial microfluidic chip in the embodiment of the present application.

[0038] Figure 7 is the structural schematic diagram of the outlet micro-pipe of the double helix inertial microfluidic chip in the embodiment of the present application.

[0039] Figure 8 is the separation principle diagram of the double helix inertial microfluidic chip in the embodiment of the present application.

[0040] Figure 9 is the cell separation effect diagram under different sorting flow rates in the embodiment of the present application.

[0041] Figure 10 is the performance comparison result diagram between the purification method of the present application and the existing differential enzyme digestion method and fluorescence activated cell sorting purification method.

[0042] Figure 11 is the p63 recovered from the collection outlet micro-pipe and the waste outlet micro-pipe in the embodiment of the present application + Comparison chart of β-galactosidase staining of cell senescence related to LPC.

[0043] Figure 12 is the p63 recovered from the collection outlet micro-pipe and the waste outlet micro-pipe in the embodiment of the present application + Comparison chart of real-time fluorescent quantitative PCR detection of senescence genes of LPC.

[0044] Figure 13 is the p63 recovered from the collection outlet micro-pipe and the waste outlet micro-pipe in the embodiment of the present application + Comparison chart of colony forming ability of LPC.

[0045] Figure 14 p63 recovered from the collection outlet microchannel and the waste outlet microchannel in the embodiments of the present application + LPC organoid formation ability comparison results map.

[0046] Figure 15 p63 recovered from the collection outlet microchannel and the waste outlet microchannel in the embodiments of the present application + LPC differentiation ability comparison results map.

[0047] Explanation of reference signs: 1-inlet microchannel, 2-first spiral microchannel, 3-S-shaped microchannel, 4-second spiral microchannel, 5-outlet microchannel, 51-first stage, 52-second stage, 53-collection outlet microchannel, 54-waste outlet microchannel, 6-base. DETAILED DESCRIPTION

[0048] In order to enable a clearer understanding of the above-mentioned purposes, features and advantages of the present application, the present application will be further described in detail below in combination with the drawings and specific embodiments. In the following description, a large number of specific details are set forth in order to facilitate a full understanding of the present application, however, the present application can also be implemented in other ways different from those described herein, therefore, the present application is not limited to the specific embodiments disclosed below.

[0049] p63 based on experimental measurement + Cell size difference of LPC and GAF as shown in Figure 1 , wherein, Figure 1 (A) in is p63 + Cell size distribution map of LPC and GAF. Figure 1 (B) in is p63 + Cell average size map of LPC and GAF of different batches of culture.

[0050] The present application designs a double spiral inertial microfluidic chip according to the principle of Dean vortex micro-particle separation, as shown in Figure 2As shown, the inertial microfluidic chip comprises a base and five sequentially connected microchannels, which are an inlet microchannel 1, a first spiral microchannel 2, an S-shaped microchannel 3, a second spiral microchannel 4, and an outlet microchannel 5, respectively. All the microchannels are fixed on the base. The first spiral microchannel and the second spiral microchannel are staggered with each other to form a double spiral channel. The S-shaped microchannel 3 is located at the center of the double spiral channel formed by the first spiral microchannel 2 and the second spiral microchannel 4, and is used to change the rotation direction of the fluid in the double spiral channel. The front end of the outlet microchannel 5 is divided into a first stage 51 and a second stage 52, and the end of the outlet microchannel 5 is divided into a collection outlet microchannel 53 and a waste outlet microchannel 54. The width of the second stage 52 is greater than that of the first stage 51, and an inclined surface is arranged at the joint between the second stage 52 and the first stage 51 for smooth transition. The width of the waste outlet microchannel 54 is greater than or equal to that of the collection outlet microchannel 53. The five microchannels are made of polydimethylsiloxane (PDMS) material, and the base is made of glass material.

[0051] The geometric shape parameters of the microchannels of the inertial microfluidic chip are as follows:

[0052] The structural diagram of the inlet microchannel 1 is as shown in Figure 3 , wherein, Figure 3 (A) in Figure 3 is a structural diagram of the top surface of the inlet microchannel 1,

[0053] The structural diagram of the first spiral microchannel 2 is as shown in Figure 4 , wherein, Figure 4 (A) in Figure 4 is a structural diagram of the top surface of the first spiral microchannel 2,

[0054] The structural diagram of the S-shaped microchannel 3 is as shown in Figure 5 , wherein, Figure 5 (A) in Figure 5(B) is a structural diagram of the channel cross section of the S-shaped micro-pipe 3; in this embodiment, the S-shaped micro-pipe 3 is formed by reversely connecting two semicircular pipes with a diameter of 4.6 mm, and the channel cross section of the S-shaped micro-pipe 3 is a rectangle with a size of 0.8 mm × 0.1 mm.

[0055] A structural diagram of the second spiral micro-pipe 4 is shown in Figure 6 wherein, Figure 6 (A) is a structural diagram of the top view of the second spiral micro-pipe 4, Figure 6 (B) is a structural diagram of the channel cross section of the second spiral micro-pipe 4; in this embodiment, the second spiral micro-pipe 4 has 3 spiral turns, the radius of the outermost spiral turn is 14 mm, and the interval between the spiral turns is 3 mm; the channel cross section of the second spiral micro-pipe 4 is a right trapezoid, the length of the base of the right trapezoid is 0.8 mm, the height of the inner side (the side close to the center) is 0.14 mm, and the height of the outer side (the side far from the center) is 0.21 mm.

[0056] A structural diagram of the outlet micro-pipe 5 is shown in Figure 7 wherein, Figure 7 (A) is a structural diagram of the top view of the outlet micro-pipe 5, Figure 7 (B) is a structural diagram of the channel cross section of the outlet micro-pipe 5; in this embodiment, in the outlet micro-pipe 5, the first stage 51 (at position a) has a length of 5 mm and a channel cross section of a rectangle with a size of 0.8 mm × 0.21 mm; the second stage 52 (at position b) has a length of 5 mm and a channel cross section of a rectangle with a size of 1.6 mm × 0.21 mm; the third stage is bifurcated into a collection outlet micro-pipe 53 (at position c) and a waste outlet micro-pipe 54 (at position d), both having a length of 10 mm, wherein the channel cross section of the collection outlet micro-pipe 53 is a rectangle with a size of 0.52 mm × 0.21 mm; the channel cross section of the waste outlet micro-pipe 54 is a rectangle with a size of 0.87 mm × 0.21 mm, and the included angle between the collection outlet micro-pipe 53 and the waste outlet micro-pipe 54 is 60°. The channel height of the outlet micro-pipe 5 is consistent with the height of the side far from the center of the channel of the second spiral micro-pipe 4.

[0057] wherein, the cross sections at the connection between the S-shaped micro-pipe 3 and the second spiral micro-pipe 4 and the connection between the second spiral micro-pipe 4 and the outlet micro-pipe 5 are rectangular-trapezoidal and trapezoidal-rectangular sudden changes, respectively.

[0058] The double-spiral inertial microfluidic chip realizes p63 +LPC and GAF are separated. During the process of cells passing through the spiral microchannel with fluid, the cells move laterally on the cross section of the channel under the combined action of fluid lift force and Dean vortex, and stay at the balance position where the resultant force is zero. The fluid lift force and Dean vortex force experienced by the cells are related to the channel geometry, fluid velocity, and the diameter of the cells suspended in the fluid. Therefore, at a certain flow rate, different sizes of cells will be at different positions on the cross section of the channel when passing through the spiral microchannel with a certain geometry, and separation occurs.

[0059] The specific separation principle of the double-spiral inertial microfluidic chip of the present application is shown in Figure 8 First, the mixed p63 + LPC and GAF cells enter the inlet microchannel 1 in a disordered manner, and after flowing into the first spiral microchannel 2, they rotate counterclockwise along the spiral direction of the channel. The first spiral microchannel 2 is responsible for separating the mixed p63 + LPC and GAF cells into the inner side of the microchannel; at the end of the first spiral microchannel 2, all cells converge to the inner side of the channel (near the center of the circle); then, the mixed cells gathered on the inner side of the channel pass through the S-shaped microchannel 3 into the second spiral microchannel 4, and rotate in the reverse direction of the spiral direction of the channel (from counterclockwise rotation to clockwise rotation), at which time the mixed cells transition from gathering on the inner side of the channel (near the center of the circle) to gathering on the outer side of the channel (far from the center of the circle); through the spiral vortex effect of the second spiral microchannel 4, the p63 + LPC and GAF cells in the second spiral microchannel 4 separate based on cell size, causing the larger GAF cells to move to the inner side of the channel, and the smaller p63 + LPC undergoes lateral separation. Finally, through the expansion of the width of the outlet microchannel 5 from the first stage 51 to the second stage 52, the separation distance of the two types of cells is increased, and the two types of cells flow out from the two outlet branches, respectively, wherein the p63 + LPC flows out from the collection outlet microchannel 53 arranged on the side far from the center of the circle, and the GAF flows out from the waste outlet microchannel 54 arranged on the side near the center of the circle.

[0060] Using the double-spiral inertial microfluidic chip of the present application to purify p63 + The sorting flow rate of LPC and the cross-sectional position of the two bifurcated outlets of the outlet microchannel 5 are obtained by experimental optimization, and the experimental results are shown in Figure 9 The focusing positions of LPC and GAF before passing through the chip outlet at different flow rates are tracked by using fluorescently labeled p63 + from different donors. +Multiple experiments were conducted using LPC and GAF ​​cultured from different batches, and it was found that the two separated at a flow rate of 3 mL / min ~ 4 mL / min. (See [link to relevant documentation]). Figure 9 As shown in (A), the optimal position of the 5-branch microchannel at the chip outlet is determined based on the location of the fluorescently labeled cells, as shown in Figure (A). Figure 7 As shown. Press Figure 7 The parameters shown indicate the bifurcation positions of the outlet microchannel 5 of the double-helix inertial microfluidic chip. Fluorescently labeled p63 is then introduced at a flow rate of 3.5 mL / min. + LPC and GAF ​​were pumped into a double-helix inertial microfluidic chip, and the focusing positions of the two cell types were observed under a fluorescence microscope. The focusing positions of the two cell types are as follows: Figure 9 As shown in (B) of the document, p63 + LPC mainly enters the collection outlet microchannel 53, while GAF mainly enters the waste outlet microchannel 54; p63 is collected from both outlets. + The ratio of LPC to GAF is as follows: Figure 9 As shown in (C), the collection outlet microchannel 53 and the discarded outlet microchannel 54 are located on page 63. + The proportions of LPC were 85.2 ± 3.6% and 14.8% ± 3.6%, respectively; the proportions of GAF were 17.4 ± 2.5% and 82.6% ± 2.5%, respectively.

[0061] The stem cell culture system purification method (MDDS) based on a double-helix inertial microfluidic chip of the present invention includes the following steps:

[0062] Step S1, Prepare the chip:

[0063] Step S11: Install the inlet microchannel 1 of the inertial microfluidic chip using Tygon tubing to connect a syringe and install the syringe on a microinjection pump (e.g., Reward R462); connect the collection outlet microchannel 53 and the waste outlet microchannel 54 of the inertial microfluidic chip to the collection tube and waste liquid tube respectively using Tygon tubing.

[0064] Step S12: Slowly pump 1% Pluronic F-68 solution (a nonionic surfactant) into the chip and direct the solution into the waste tubing for 1 minute of pretreatment to prevent cell adhesion. Ensure there are no air bubbles in the tubing and no air in the outlet hose.

[0065] Step S13: Pump in 4 mL of PBS buffer (phosphate buffer) to flush the chip tubing, thoroughly removing any residual Pluronic F-68 solution from the tubing, and then transfer the PBS buffer into the waste tube.

[0066] Step S2, Cell sorting:

[0067] Step S21, remove the p63 + LPC and GAF mixed cells are resuspended in p63 + LPC medium, and the cell concentration is 1 x 10 6 - 5 x 10 6 cells / mL.

[0068] Step S22, take another sterile syringe, connect the blunt needle and the appropriate length of Tygon hose to the sterile syringe, pull out the plunger, pour the cell suspension into the syringe, put back the plunger, and place the syringe upright to remove the air.

[0069] Step S23, fix the syringe containing the cell suspension on the microsyringe pump, use the Tygon hose to connect the syringe containing the cell suspension to the inlet microchannel of the inert microfluidic chip, input the model of the syringe into the control program of the microsyringe pump, and set the injection speed of the microsyringe pump to 3.5 mL / min.

[0070] Step S24, after starting injection, first introduce the Tygon hoses of the two outlets into the waste liquid pipe, and after 5 seconds (after the cells are accelerated and form a stable focus in the chip), introduce the Tygon hose of the collection outlet into the collection pipe to start collecting p63 + LPC.

[0071] In this embodiment, the performance of the stem cell-feeder layer cell co-culture system purified by the method of the present application (MDDS) and the existing differential enzyme digestion method (DED) and fluorescence activated cell sorting purification method (FACS) are compared. The experimental results are shown in Table 1 and Figure 10 When using the differential enzyme digestion method (DED) for purification, first remove the stem cell culture medium in the culture bottle, wash the cells with PBS buffer for 2 times; then add 0.05% trypsin-EDTA, act at 37°C for 3 minutes to digest GAF, collect the digested cells into the waste pipe, and terminate the digestion with serum-containing medium; then add 0.25% trypsin-EDTA, act at 37°C for 5 minutes to digest p63 + LPC, terminate the digestion with serum-containing medium, collect the digested cells into the collection pipe; finally, count the cells in the waste pipe and the collection pipe, and use FACS to detect the p63 + LPC purity and recovery rate based on the positive rate of CD326. When using the fluorescence activated cell sorting purification method (FACS) for purification, first use 0.25% trypsin-EDTA to act at 37°C for 5 minutes, and then use 0.05% trypsin-EDTA to act at 37°C for 3 minutes to digest GAF, collect the digested cells into the waste pipe, and terminate the digestion with serum-containing medium; then use 0.25% trypsin-EDTA to act at 37°C for 5 minutes to digest p63 +The mixed cells of LPC and GAF were co-digested, then the mixed cells were stained with phycoerythrin-conjugated anti-human CD326 antibody, and then the stained cells were resuspended in PBS buffer at a density of 1 x 10 6 / mL, and CD326 positive and negative cells were separated by a Beckman CytoFLEX SRT full-automatic flow cytometer at a setting of 2000 events / second, the positive cells were collected into a collection tube, and the negative cells were collected into a waste tube; finally, the cells in the waste tube and the collection tube were counted, and the CD326 positive rate was detected again by FACS based on p63 + LPC purity and recovery rate. Table 1:

[0072] Figure 10 The stem cell purity and recovery rate data of the three purification methods summarized in Table 1 were obtained by using different donor-derived p63 + LPC for multiple tests, wherein the stem cell purity obtained by the MDDS method was 79.7 ± 5.7%, and the recovery rate was 83.1 ± 2.7%; the stem cell purity obtained by the FACS method was 95.9 ± 3.1%, and the recovery rate was 57.4 ± 3.2%; the stem cell purity obtained by the DED method was 59.6 ± 17.4%, and the recovery rate was 83.2 ± 17.2%. From these data, it can be seen that the FACS method has the relatively most stable purification efficiency (exhibiting the smallest standard deviation), ensuring high purity, but has the lowest recovery rate; the DED method can obtain a higher recovery rate, but is relatively lowest in terms of stability of purity and recovery rate; the MDDS method maintains a relatively high stability while ensuring a relatively high purity and recovery rate. Table 1 also summarizes other characteristics of the three purification methods. The FACS method uses the difference in p63 + LPC and GAF surface antigens in the sorting principle, and the sorting throughput is on the order of 10 5 cells / minute, which is the lowest among the three methods; at the same time, the FACS method needs to stain and label the mixed cells, which additionally increases the cost of antibody reagents; the low sorting efficiency and high cost also limit its scalability for large-scale cell sorting. The DED method uses the difference in p63 + LPC and GAF adhesion in the culture vessel in the sorting principle, and the sorting throughput depends on the volume of the culture vessel. For example, using a T-175 culture flask commonly used in the laboratory, the sorting throughput can reach 10 5- 10 6 cells / minute; the DED method does not need cell labeling, has relatively low cost, and has strong scalability, and has been used for clinical p63 + LPC preparation. The MDDS method uses the difference in p63 +The difference in fluid inertial force received by the LPC and GAF due to the difference in cell size, according to the method described in the application, the sorting flux can reach 10 6- 10 7 cells / min order; the MDDS method also does not require cell labeling, the cost is relatively low, and a larger number of cells can be purified by multiple chips working at the same time, and the scalability is strong; the MDDS in purifying p63 + LPC also has the function of excluding senescent p63 + The unique functional properties of LPC.

[0073] Figures 11-15 The p63 + The multiple functional properties of LPC.

[0074] Figure 11 is a comparison result map of cell senescence-related β-galactosidase staining. The p63 + LPC is sorted by the collection outlet micro-pipe 53 and the waste outlet micro-pipe 54 at a density of 8x10 4 cells / well in a 6-well plate, and after 12 hours, both are stained using a cell senescence-related β-galactosidase staining kit (MCE, HY-K1089). The results show that the p63 + LPC has an increased degree of senescence-related β-galactosidase staining.

[0075] Figure 12 is a comparison result map of real-time fluorescent quantitative PCR detection of senescence-related gene expression levels. The total RNA of the p63 + LPC is extracted using the TransZol Up RNA extraction kit. The total RNA is reverse transcribed into cDNA using the Hifair® AdvanceFast one-step RT-gDNA digestion SuperMix kit. Subsequently, the relative expression levels of senescence-related genes p16, p21 and p53 are detected by quantitative real-time PCR using the HieffUNICONTM Universal Blue qPCR SYBR Green Master Mix on the Quant Studio Dx PCR system; the GAPDH gene is used as an internal reference, and the 2 −ΔΔCt The results show that the p63 + LPC has a significantly higher expression level of senescence-related genes p53, p21 and p16 than the p63+ LPC, where p53 increased 1.27 ± 0.12-fold, p21 increased 1.38 ± 0.15-fold, and pl6 increased 1.56 ± 0.35-fold.

[0076] Figure 13 is a comparative plot of stem cell colony formation rate. p63 + LPC were seeded at a density of 600 cells per dish in 60 mm dishes previously coated with collagen type I. After 10 days of culture, the number of colonies formed was calculated by staining with 0.1% crystal violet solution. The results show that p63 + LPC had a colony formation rate of 23.9 ± 9.4%, while p63 + LPC had a colony formation rate of 17.3 ± 6.8%, the former being significantly higher than the latter.

[0077] Figure 14 is a comparative plot of lung organoid formation rate. p63 + LPC were seeded at a density of 8 x 105 4 cells were resuspended in 200 μL reduced growth factor Matrigel®, seeded in 24-well plates, and incubated in a 37°C, 7.5% CO2 incubator for 20 minutes, after which p63 + LPC medium was added. After 21 days of culture, images of the p63 + LPC formed organoids were taken under phase contrast microscopy, and the number of organoids in each image was counted. The results show that p63 + LPC had a number of organoids of 31.4 ± 6.0% per field, while p63 + LPC had a number of organoids of 4.9 ± 1.5% per field, the former being significantly higher than the latter.

[0078] Figure 15 is a comparative plot of the ability of p63 + LPC to differentiate into multiple bronchial epithelial cell types under air-liquid interface conditions. p63 + LPC were seeded at a density of 2 x 105 5 cells were seeded in the Transwell® 24-well plate chambers, and p63 +LPC medium (medium in both top and basal chambers) was incubated at 37°C in a 7.5% CO2 incubator for 48 hours. The medium in the top chamber was aspirated to expose the cells to air, and the medium in the basal chamber was replaced with PneumaCult-ALI differentiation medium. After 21 days of differentiation, the Transwell® membrane carrying the differentiated bronchial epithelium was excised and prepared for standard histological fixation, tissue processing and paraffin embedding to prepare 5-micron tissue sections. Immunofluorescence staining was performed using anti-human CC10, anti-human a-Tub antibody and DAPI stain for p63 + Club cells and ciliated cells formed by differentiation of LPC were immunofluorescently stained for nuclei. Photographs were taken using a fluorescence microscope, and the number of various differentiated cells was calculated. The results showed that the number of p63 + Club cells formed by differentiation of LPC were 21.4 ± 4.5% per field, and the number of ciliated cells was 30.0 ± 11.2% per field; whereas p63 + Club cells formed by differentiation of LPC were 9.1 ± 4.7% per field, and the number of ciliated cells was 5.5 ± 4.7% per field. p63 + Club cells and ciliated cells formed by differentiation of LPC were significantly higher than p63 + LPC.

[0079] Figures 11-15 The data shown above support the characteristics of the present application of actively excluding senescent stem cells with impaired clonogenicity and differentiation function, and suggest that the effective stem cell recovery rate is higher.

[0080] The present application can be used to purify p63-positive lung precursor cells (p63 + LPC) cultured using feeder layer cells, and has the potential to be applied to purification of other stem cell co-culture systems, such as sorting and purification of human induced pluripotent stem cells (iPSC) cultured using murine embryonic fibroblasts (MEF) as feeder layer cells.

[0081] The above only describes preferred embodiments of the present application and is not intended to limit the present application. Those skilled in the art can make various modifications and changes to the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A double-helix inertial microfluidic chip, characterized in that, The device includes a base and five sequentially connected microchannels fixed to the base. These five microchannels are an inlet microchannel, a first spiral microchannel, an S-shaped microchannel, a second spiral microchannel, and an outlet microchannel. The first and second spiral microchannels are staggered to form a double spiral channel. The S-shaped microchannel is located at the center of the double spiral channel formed by the first and second spiral microchannels. The front end of the outlet microchannel is divided into a first stage and a second stage, with the width of the second stage being greater than the width of the first stage. The end of the outlet microchannel is divided into a collection outlet microchannel and a waste outlet microchannel; the width of the waste outlet microchannel is not less than the width of the collection outlet microchannel.

2. The double-helix inertial microfluidic chip according to claim 1, characterized in that, The cross-section of the collection outlet microchannel is a rectangle with a width of 0.52 mm to 0.69 mm and a height of 0.18 mm to 0.26 mm; the cross-section of the waste outlet microchannel is a rectangle with a width of 0.69 mm to 0.87 mm and a height of 0.18 mm to 0.26 mm.

3. The double-helix inertial microfluidic chip according to claim 2, characterized in that, The cross-section of the collection outlet microchannel is a rectangle of 0.52 mm × 0.21 mm; the cross-section of the waste outlet microchannel is a rectangle of 0.87 mm × 0.21 mm, and the included angle between the collection outlet microchannel and the waste outlet microchannel is 60°.

4. The double-helix inertial microfluidic chip according to claim 3, characterized in that, The S-shaped microchannel is formed by connecting two semi-circular channels of the same diameter in opposite directions.

5. The double-helix inertial microfluidic chip according to claim 4, characterized in that, The diameter of the two semi-circular pipes is 4.6 mm.

6. The double-helix inertial microfluidic chip according to claim 5, characterized in that, The cross-section of the second spiral microchannel is a right trapezoid, and the height of the side of the right trapezoid near the center is less than the height of the side away from the center.

7. The double-helix inertial microfluidic chip according to claim 6, characterized in that, The right trapezoid has a base length of 0.8 mm, a height of 0.12 mm to 0.18 mm on the side closer to the center, and a height of 0.18 mm to 0.26 mm on the side farther from the center.

8. The double-helix inertial microfluidic chip according to claim 7, characterized in that, The cross-section of the first spiral microchannel is a rectangle with a width of 0.8 mm and a height of 0.08 mm to 0.125 mm.

9. A method for purifying a stem cell culture system based on the double-helix inertial microfluidic chip according to any one of claims 1 to 8, characterized in that, Includes the following steps: Step S11: Install the microfluidic device. Connect the inlet microchannel of the inertial microfluidic chip to the syringe using a Tygon tubing, and install the syringe on the microinjection pump. Connect the collection outlet microchannel and the waste outlet microchannel of the inertial microfluidic chip to the collection tube and waste liquid tube respectively using a Tygon tubing. Step S12: Slowly pump 1% Pluronic F-68 solution into the chip and introduce the solution into the waste liquid tube for 1 minute of pretreatment to prevent cell adhesion; ensure that there are no air bubbles in the tube and no air in the outlet hose; Step S13: Pump in 4 mL of PBS buffer to rinse the chip tubing, thoroughly removing any residual Pluronic F-68 solution from the tubing, and then transfer the PBS buffer into the waste tube. Step S21: Resuspend the mixed cells containing p63-positive lung progenitor cells and growth-inhibiting feeder cells in p63 containing 0.1% Pluronic F-68. + In LPC medium, the cell concentration is 1×10⁻⁶. 6 - 5×10 6 cells / mL; Step S22: Take another sterile syringe, connect the sterile syringe to a blunt needle and a Tygon tubing of appropriate length, pull out the plunger, pour the cell suspension into the syringe, put the plunger back in, turn the syringe upright, and expel the air. Step S23: Fix the syringe containing cell suspension onto the microinjection pump, connect the syringe containing cell suspension to the inlet microchannel of the inertial microfluidic chip using Tygon tubing, input the syringe model into the microinjection pump, and set the injection speed of the microinjection pump to 3 mL / min ~ 4 mL / min. Step S24: After the injection begins, first guide both Tygon tubing outlets into the waste liquid tube. After 5 seconds, the cells accelerate and form a stable focus in the chip. Then, guide the Tygon tubing from the collection outlet into the collection tube to begin collecting p63-positive lung progenitor cells.

10. The purification method for the stem cell culture system according to claim 9, characterized in that, In step S23, the injection rate of the microinjection pump is set to 3.5 mL / min.

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