Microfluidic detection chip and method for automated culture and differentiation of mesenchymal stem cells

Abstract

The application belongs to the technical field of microfluidic chip application, and relates to an automatic culture and detection chip for mesenchymal stem cell multi-lineage differentiation based on microfluidics, which comprises a culture main body and multiple culture zones, is used for continuous automatic culture and differentiation under the premise of avoiding pollution, and comprises an interdigital electrode, a mechanical peristalsis device and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the application of microfluidic chip, and relates to a microfluidic detection chip and method for automatic culture and differentiation of mesenchymal stem cells. BACKGROUND

[0002] Mesenchymal stem cells (MSCs) are adult stem cells derived from mesoderm, mainly existing in dental pulp, bone marrow, fat, umbilical cord, amniotic membrane, menstrual blood and other tissues. Its clinical development has made great progress in China. As "seed cells", MSCs can provide repair capacity for damaged organs and accelerate the healing of tissues. Therefore, MSCs are of great significance for the treatment of diseases, and their clinical popularity has gradually increased and breakthroughs have been made. Mesenchymal stem cells are also applied to treat various diseases such as diabetes, stroke, Alzheimer's disease, pulmonary fibrosis and the like.

[0003] In recent years, due to the preclinical research and clinical research of mesenchymal stem cells carried out by scientific institutions and biopharmaceutical companies all over the world, the extraction, separation and identification of mesenchymal stem cells have become an important basic skill for all researchers. The identification of stem cells is the basis for carrying out stem cell research. At present, the identification of MSCs can be carried out by morphological observation (such as fibroblast-like growth, oval nucleus in the center of the cell, and spiral or radial growth when the density is high), surface marker detection (such as CD29, CD44, etc.) and three-line differentiation (osteogenic differentiation, adipogenic differentiation and chondrogenic differentiation) potential identification. Among them, the three-line differentiation potential identification is the most powerful evidence for determining whether the cell is MSCs.

[0004] Microfluidic chip, as a rapidly developing science and technology, has shown its unique advantages in the field of biology. Its cell size matching, similar physiological environment, simple operation to avoid human error and other characteristics enable it to provide more precise manipulation and direct detection of results.

[0005] The conventional tri-lineage differentiation detection adopts manual operation, consumes a lot of manpower and time in the process of cell culture, and increases the risk of contamination in the process of opening bottle and replacing liquid. The tri-lineage differentiation of stem cells mainly includes the following steps: first, the stem cells are cultured and inoculated in a suitable well plate; second, the stem cells are inoculated in a suitable well plate; third, the stem cells are inoculated in a suitable well plate; fourth, the stem cells are inoculated in a suitable well plate; fifth, the stem cells are inoculated in a suitable well plate; sixth, the stem cells are inoculated in a suitable well plate; seventh, the stem cells are inoculated in a suitable well plate; eighth, the stem cells are inoculated in a suitable well plate; ninth, the stem cells are inoculated in a suitable well plate; tenth, the stem cells are inoculated in a suitable well plate; eleventh, the stem cells are inoculated in a suitable well plate; twelfth, the stem cells are inoculated in a suitable well plate; thirteenth, the stem cells are inoculated in a suitable well plate; fourteenth, the stem cells are inoculated in a suitable well plate; fifteenth, the stem cells are inoculated in a suitable well plate; sixteenth, the stem cells are inoculated in a suitable well plate; seventeenth, the stem cells are inoculated in a suitable well plate; eighteenth, the stem cells are inoculated in a suitable well plate; nineteenth, the stem cells are inoculated in a suitable well plate; twentieth, the stem cells are inoculated in a suitable well plate; twenty-first, the stem cells are inoculated in a suitable well plate; twenty-second, the stem cells are inoculated in a suitable well plate; twenty-third, the stem cells are inoculated in a suitable well plate; twenty-fourth, the stem cells are inoculated in a suitable well plate; twenty-fifth, the stem cells are inoculated in a suitable well plate; twenty-sixth, the stem cells are inoculated in a suitable well plate; twenty-seventh, the stem cells are inoculated in a suitable well plate; twenty-eighth, the stem cells are inoculated in a suitable well plate; twenty-ninth, the stem cells are inoculated in a suitable well plate; thirtieth, the stem cells are inoculated in a suitable well plate; thirty-first, the stem cells are inoculated in a suitable well plate; thirty-second, the stem cells are inoculated in a suitable well plate; thirty-third, the stem cells are inoculated in a suitable well plate; thirty-fourth, the stem cells are inoculated in a suitable well plate; thirty-fifth, the stem cells are inoculated in a suitable well plate; thirty-sixth, the stem cells are inoculated in a suitable well plate; thirty-seventh, the stem cells are inoculated in a suitable well plate; thirty-eighth, the stem cells are inoculated in a suitable well plate; thirty-ninth, the stem cells are inoculated in a suitable well plate; and fortieth, the stem cells are inoculated in a suitable well plate. SUMMARY

[0006] The present application discloses a mesenchymal stem cell automatic culture and differentiation microfluidic detection chip and method, to solve any of the above and other potential problems of the prior art.

[0007] In order to solve the above problems, the technical scheme of the present application is: a mesenchymal stem cell automatic culture and differentiation microfluidic detection chip, characterized in that the microfluidic detection chip comprises a culture main body, an interdigital electrode and a mechanical peristalsis device.

[0008] The culture main body is provided with a plurality of culture zones, and the plurality of culture zones can complete the culture and differentiation of mesenchymal stem cells under the premise of avoiding pollution.

[0009] The interdigital electrode is used for data acquisition of the differentiation results in the culture zone.

[0010] The impedance analysis device is used for detecting the change of cell (ball) impedance before and after induction differentiation, and determining the differentiation effect.

[0011] The mechanical peristalsis device is used for flushing cells with flowing culture medium, so that the cells are subjected to shear force of the culture medium to promote differentiation and enhance the differentiation effect of the differentiation medium.

[0012] Further, the culture main body comprises a first culture zone, a second culture zone, a third culture zone, a flow guide channel and a spacing device.

[0013] The first culture zone, the second culture zone, the third culture zone and the flow guide channel are arranged on the main body, and the flow guide channel is communicated with both ends of the first culture zone, the second culture zone and the third culture zone, respectively.

[0014] several said spacing devices are arranged inside one of said first culture area, second culture area, third culture area,

[0015] a plurality of said interdigital electrodes are arranged on the lower end surface of said first culture area, second culture area and third culture area, and the interdigital electrodes are arranged in positive and negative alternation,

[0016] said first culture area, second culture area and third culture area are all communicated with one end of at least one said interdigital electrode, and the other end of the interdigital electrode is connected with impedance analysis device;

[0017] both ends of said flow channel are provided with said mechanical peristalsis device.

[0018] Further, said first culture area, second culture area and third culture area are all circular grooves, and the depths of the first culture area and the second culture area are equal, and the depth of the third culture area is greater than the depths of the first culture area and the second culture area.

[0019] Further, said spacing device is asymmetric X-shaped, and a flow channel is arranged in the middle, the narrowest part of the flow channel is 0.3-0.7mm wide, and the widest part is 2-3mm, which is smaller than the particle size of the culture, and the waste liquid can be discharged.

[0020] Further, said flow channel includes a first flow channel and a second flow channel;

[0021] wherein said first flow channel is communicated with both ends of said first culture area and second culture area, and the other end is open, for setting peristalsis device to carry out liquid inlet or liquid discharge,

[0022] said second flow channel is communicated with both ends of said third culture area.

[0023] Further, said first flow channel includes a first branch flow channel, a second branch flow channel, a third branch flow channel, a fourth branch flow channel and a fifth branch flow channel;

[0024] wherein one end of said first branch flow channel is open; the other end is communicated with one end of said second branch flow channel and third branch flow channel,

[0025] one end of said second branch flow channel and third branch flow channel is respectively communicated with one end of said first culture area and second culture area,

[0026] one end of said fourth branch flow channel and fifth branch flow channel is respectively communicated with the other end of said first culture area and second culture area, and the other end is open.

[0027] Further, the width of said first branch flow channel is 0.2-0.5mm, and the height is 0.2-0.5mm;

[0028] The width of the second branch flow guide groove and the third branch flow guide groove is 0.1-0.2mm, and the height is 0.2-0.5mm;

[0029] The width of the fourth branch flow guide groove and the fifth branch flow guide groove is 2-3mm, and the diameter is 15-25mm.

[0030] Further, the second flow guide groove comprises a sixth branch flow guide groove and a seventh branch flow guide groove;

[0031] One end of the sixth branch flow guide groove and the seventh branch flow guide groove is communicated with two ends of the third culture area respectively,

[0032] The width of the sixth branch flow guide groove is 3-5mm, and the height is 2-3mm; the width of the seventh branch flow guide groove is 2-3mm, and the height is 2-3mm.

[0033] Further, the culture main body is a composite structure of polydimethylsiloxane and glass, that is, the polydimethylsiloxane is a culture layer, and is bonded on the lower layer of glass.

[0034] Another object of the application is to provide a detection method using the above-mentioned detection chip, which specifically comprises the following steps:

[0035] S1) first inject a cell suspension with a concentration of 5×10 5 -1×10 6 cells / mL from one end of the culture main body into the culture area, close the peristaltic device, stand for 1-3h to allow the cells to adhere and grow, then start the peristaltic device to discharge all the waste liquid;

[0036] S2) continuously inject an induction culture medium from the other end of the culture main body into the culture area through the peristaltic device, close the peristaltic device after the induction culture medium fills the culture area, connect the interdigital electrode to collect the impedance values at each position, continue to culture, and in the process, discharge the waste liquid from one end of the culture main body through the peristaltic device, and after at least 15 days of culture, complete the induction culture;

[0037] S3) electrical impedance detection: after 15 days of culture, connect the interdigital electrode, measure the impedance values at each position after differentiation is completed, compare the ratio with the electrical impedance measured in S2), and obtain the detection result according to the result.

[0038] It can also be: S1) first inject a cell suspension with a concentration of 5×10 5 cells / mL from one end of the culture main body into the culture area, close the peristaltic device, stand for 1-3h to allow the cells to adhere and grow, then start the peristaltic device to discharge all the waste liquid;

[0039] S2) again through the peristaltic device from the other end of the culture medium from the continuous injection of culture zone, after the induction of culture medium filled culture zone, through the peristaltic device waste from the culture main end of the process, culture at least 15 days, complete induction culture;

[0040] S3) induction culture 15d, with 20ul / min flow rate by the fourth branch of the guide groove continuous input 1ml PBS cleaning, after by the fourth branch of the guide groove with 20ul / min flow rate input 4% neutral formaldehyde solution to fill the culture area, fixed 30min; again with 20ul / min flow rate by the fourth branch of the guide groove continuous input 1ml PBS cleaning, finally with 20ul / min flow rate by the fourth branch of the guide groove continuous input alizarin red to fill the culture area staining 5min, again with 20ul / min flow rate by the fourth branch of the guide groove continuous input 1ml PBS cleaning, using microscope observation differentiation effect.

[0041] The beneficial effects of the present application are: due to the adoption of the above technical scheme, the present application has simple structure, convenient to use, can avoid pollution on the premise of continuous automatic culture and differentiation, simplify the manual operation steps, for the quality control of mesenchymal stem cells. And combined with microfluidic chip using dynamic culture, that is, the flowing culture medium flushes the cells, so that they are subjected to the shear force of the culture medium to promote differentiation, enhance the differentiation effect of the differentiation culture medium, shorten the experimental time. At the same time, after the culture is completed, the three system differentiation result detection can be directly carried out on the interdigital electrode below the microfluidic chip. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 It is a front view structural schematic diagram of a kind of mesenchymal stem cell automatic culture and differentiation microfluidic detection chip.

[0043] Figure 2 It is a rear view structural schematic diagram of a kind of mesenchymal stem cell automatic culture and differentiation microfluidic detection chip.

[0044] Figure 3 It is the schematic diagram of osteogenic staining result 1 of the detection chip culture using the present application.

[0045] Figure 4 It is the schematic diagram of osteogenic staining result 2 of the detection chip culture using the present application.

[0046] Figure 5 It is the schematic diagram of adipogenic staining result 1 of the detection chip culture using the present application.

[0047] Figure 6 It is the schematic diagram of adipogenic staining result 2 of the detection chip culture using the present application.

[0048] Figure 7A schematic diagram of chondroblast staining results of the detection chip cultured according to the present application.

[0049] Figure 8 A schematic diagram of chondroblast staining results 2 of the detection chip cultured according to the present application.

[0050] Figure 9 A graph of osteogenic and adipogenic impedance results of the detection chip cultured according to the present application.

[0051] Figure 10 A graph of chondroblast impedance results of the detection chip cultured according to the present application.

[0052] In the figure:

[0053] 1. culture main body, 2. first culture area, 3. second culture area, 4. third culture area, 5. flow guide channel, 5-1. first branch flow guide groove, 5-2. second branch flow guide groove, 5-3 third branch flow guide groove, 5-4. fourth branch flow guide groove, 5-5. fifth branch flow guide groove, 5-6. first branch flow guide groove, 5-7. second branch flow guide groove, 6. interdigital electrode, 7. spacing device. DETAILED DESCRIPTION

[0054] The technical solutions of the present application will be further described below in combination with the drawings and specific embodiments.

[0055] As shown in 1- Figure 2 The mesenchymal stem cell automatic culture and differentiation microfluidic detection chip of the present application comprises a culture main body, an interdigital electrode and a mechanical peristalsis device;

[0056] The culture main body is provided with multiple culture areas, and the multiple culture areas can complete the culture and differentiation of mesenchymal stem cells under the premise of avoiding pollution;

[0057] The interdigital electrode is used for data acquisition of the differentiation results in the culture area;

[0058] The mechanical peristalsis device is used for flushing cells with flowing culture medium, so that the cells are subjected to shear force of the culture medium to promote differentiation and enhance the differentiation effect of the differentiation culture medium.

[0059] The upper end surface of the culture main body is provided with multiple culture areas and a flow guide channel,

[0060] Multiple interdigital electrodes are respectively arranged on the lower end surface of the culture main body, and each culture area is connected with one end of at least one interdigital electrode, and the other end of the interdigital electrode is connected with an impedance analysis device;

[0061] The mechanical peristalsis device is arranged at both ends of the culture main body and is communicated with the multiple culture main bodies through the flow guide channel.

[0062] The culture main body comprises a first culture area, a second culture area, a third culture area and a spacer device;

[0063] The first culture area, the second culture area and the third culture area are all arranged on the culture main body, and the flow guide channels are respectively communicated with two ends of the first culture area, the second culture area and the third culture area,

[0064] The spacer device is arranged in one of the first culture area, the second culture area and the third culture area,

[0065] When detection is needed, the impedance analysis device is connected with the interdigital electrode, so that the impedance value of the cells in the culture area can be collected,

[0066] The first culture area, the second culture area and the third culture area are all circular grooves, and the depths of the first culture area and the second culture area are equal, and the depth of the third culture area is greater than the depths of the first culture area and the second culture area.

[0067] The spacer device is an asymmetric X shape, and a flow channel is arranged in the middle, the narrowest part of the flow channel is 0.3-0.7mm wide, and the widest part is 2-3mm wide.

[0068] The flow guide channel comprises a first flow guide groove and a second flow guide groove;

[0069] The first flow guide groove is respectively communicated with two ends of the first culture area and the second culture area,

[0070] The second flow guide groove is communicated with two ends of the third culture area.

[0071] The first flow guide groove comprises a first branch flow guide groove, a second branch flow guide groove, a third branch flow guide groove, a fourth branch flow guide groove and a fifth branch flow guide groove;

[0072] One end of the first branch flow guide groove is an opening, and the other end is communicated with one end of the second branch flow guide groove and the third branch flow guide groove,

[0073] One end of the second branch flow guide groove and the third branch flow guide groove is respectively communicated with one end of the first culture area and the second culture area,

[0074] The other end of the fourth branch flow guide groove and the fifth branch flow guide groove is an opening.

[0075] The width of the first branch flow guide groove is 0.2-0.5mm, and the height is 0.2-0.5mm;

[0076] The width of the second branch flow guide groove and the third branch flow guide groove is 0.1-0.2mm, and the height is 0.2-0.5mm;

[0077] The fourth branch flow guide groove and the fifth branch flow guide groove have a width of 2-3mm and a diameter of 15-25mm;

[0078] The second flow guide groove comprises a sixth branch flow guide groove and a seventh branch flow guide groove;

[0079] One end of the sixth branch flow guide groove and the seventh branch flow guide groove is respectively communicated with two ends of the third culture area,

[0080] The sixth branch flow guide groove has a width of 3-5mm and a height of 2-3mm, and the seventh branch flow guide groove has a width of 2-3mm and a height of 2-3mm.

[0081] The culture main body is a composite structure of polydimethylsiloxane and glass.

[0082] A detection method using the detection chip, which specifically comprises the following steps:

[0083] S1) The concentration of 5×10 5 The cell suspension is injected into the culture area from one end of the culture main body, the peristaltic device is closed, and the cells are allowed to adhere and grow for 1-3h, and then the peristaltic device is started to discharge all the waste liquid;

[0084] S2) The induction medium is continuously injected into the culture area from the other end of the culture main body by the peristaltic device, and after the induction medium fills the culture area, the peristaltic device is closed, the interdigital electrode is connected to collect the impedance values at each position, and the culture is continued, and the waste liquid is discharged from one end of the culture main body by the peristaltic device during the culture, and after at least 15 days of culture, the induction culture is completed;

[0085] S3) Electrical impedance detection: after 15 days of culture, the interdigital electrode is connected, and the impedance values at each position after differentiation are measured, which are compared with the electrical impedance measured in S2), and the differentiation effect is obtained according to the results.

[0086] The first branch flow guide groove has a width of 5×10 5The cell suspension was injected at a rate of 1 cell / mL, flowed through the second branch of the flow guide into and filled the cell culture pool, and was allowed to stand for 2 h for cell adhesion and growth. The remaining waste liquid was discharged from the fourth branch of the flow guide. The osteogenic induction medium was continuously introduced from the fourth branch of the flow guide at a flow rate of 10 μL / min, and the culture waste liquid was discharged through the second branch of the flow guide and the first branch of the flow guide. After 15 d of induction culture, 1 mL of PBS was continuously introduced from the fourth branch of the flow guide at a flow rate of 20 μL / min, followed by continuously introducing 4% neutral formaldehyde solution from the fourth branch of the flow guide at a flow rate of 20 μL / min until the culture area was filled, and was allowed to stand for 30 min. Then, 1 mL of PBS was continuously introduced from the fourth branch of the flow guide at a flow rate of 20 μL / min, and finally, alizarin red was continuously introduced from the fourth branch of the flow guide at a flow rate of 20 μL / min until the culture area was filled, and was allowed to stand for 5 min. Then, 1 mL of PBS was continuously introduced from the fourth branch of the flow guide at a flow rate of 20 μL / min

[0087] Example 1

[0088] The chip was made as follows:

[0089] The cell culture area had a depth of 1 mm and a diameter of 15 mm. The first branch of the flow guide had a width of 0.2 mm and a height of 0.2 mm. The second branch of the flow guide and the third branch of the flow guide, which branched from the first branch of the flow guide, had a width of 0.1 mm and a height of 0.2 mm. The fourth branch of the flow guide and the fifth branch of the flow guide had a width of 0.05 mm and a height of 0.05 mm. The cell sphere culture area had a depth of 2 mm and a diameter of 15 mm. The sixth branch of the flow guide had a width of 3 mm and a height of 2 mm. The seventh branch of the flow guide had a width of 2 mm and a height of 2 mm. The unconnected asymmetric "X" shape had a width of 0.3 mm at the narrowest part and a width of 2 mm at the widest part.

[0090] The electrodes with a width of 0.05 mm were laid out at an interval of 0.1 mm under the cell culture area, and were arranged alternately in positive and negative directions, and a total of six groups were laid out, which were used for detecting the osteogenic and adipogenic effects of MSCs. The electrodes with a width of 0.05 mm were laid out at an interval of 0.1 mm from the narrowest part to the widest part of each unconnected asymmetric "X" shape in the cell sphere culture area, and were arranged alternately in positive and negative directions, which were used for detecting the chondrogenic effect of the cell spheres.

[0091] Example 2

[0092] The chip was made as follows:

[0093] The cell culture area is 1.5 mm in depth and 20 mm in diameter; the first branch flow guide is 0.25 mm in width and 0.25 mm in height; the second branch flow guide and the third branch flow guide are 0.15 mm in width and 0.25 mm in height, which are branched from the first branch flow guide; the fourth branch flow guide and the fifth branch flow guide are 0.07 mm in width and 0.07 mm in height. The first cell sphere culture area is 2.5 mm in depth and 20 mm in diameter; the sixth branch flow guide is 4 mm in width and 2.5 mm in height; the seventh branch flow guide is 2.5 mm in width and 2.5 mm in height; the narrowest part of the disconnected asymmetric "X" shape isolation device is 0.4 mm in width, and the widest part is 2.5 mm in width.

[0094] Six groups of electrodes with a width of 0.05 mm are laid out under the cell culture area at intervals of 0.15 mm, and are alternately positive and negative, for detecting the osteogenic and adipogenic effects of MSCs; electrodes with a width of 0.05 mm are laid out in each disconnected asymmetric "X" shape at intervals of 0.15 mm from the narrowest part to the widest part, and are alternately positive and negative, for detecting the chondrogenic effect of the cell spheres.

[0095] Example 3:

[0096] The chip manufacturing method is as follows:

[0097] The chip design is as follows: the cell culture area is 3 mm in depth and 25 mm in diameter; the first branch flow guide is 0.5 mm in width and 0.5 mm in height; the second branch flow guide and the third branch flow guide are 0.2 mm in width and 0.5 mm in height, which are branched from the first branch flow guide; the fourth branch flow guide and the fifth branch flow guide are 0.1 mm in width and 0.1 mm in height. The cell sphere culture area is 3 mm in depth and 25 mm in diameter; the sixth branch flow guide is 5 mm in width and 3 mm in height; the seventh branch flow guide is 3 mm in width and 3 mm in height; the narrowest part of the disconnected asymmetric "X" shape is 0.7 mm in width, and the widest part is 3 mm in width.

[0098] Twenty-five electrodes with a width of 0.05 mm are laid out under the two shallower cell culture areas at intervals of 0.2 mm, and are alternately positive and negative, for detecting the osteogenic and adipogenic effects of MSCs; electrodes with a width of 0.05 mm are laid out in each disconnected asymmetric "X" shape at intervals of 0.2 mm from the narrowest part to the widest part, and are alternately positive and negative, for detecting the chondrogenic effect of the cell spheres.

[0099] Example 4:

[0100] Cell inoculation and osteogenic differentiation induction:

[0101] From the first branch flow guide, 5×10 5Cells / mL of cell suspension were injected, flowing through the second guide channel into and filling the cell culture tank. After 2 hours of inertia to allow cell adhesion and growth, the remaining waste liquid was drained through the fourth guide channel. Osteogenic induction medium was continuously introduced through the fourth guide channel at a flow rate of 10 μL / min. Once the culture area was filled with the induction medium, each set of interdigitated electrodes was connected to an impedance detection device to measure the impedance values ​​at various locations before differentiation. During the culture process, culture waste liquid flowed through the second and first guide channels and was discharged. After 15 days of induction culture, each set of interdigitated electrodes was connected to an impedance detection device to measure the impedance values ​​at various locations after differentiation. The ratio of these values ​​to the impedance values ​​measured before differentiation was used to assess the differentiation effect. Figure 9 (As shown), then continuously 1 mL of PBS is introduced through the fourth channel at a flow rate of 20 μL / min for washing. Next, 4% neutral formaldehyde solution is introduced through the fourth channel at a flow rate of 20 μL / min until the culture area is filled, and fixed for 30 min. Then, 1 mL of PBS is continuously introduced through the fourth channel at a flow rate of 20 μL / min for washing. Finally, alizarin red is continuously introduced through the fourth channel at a flow rate of 20 μL / min until the culture area is filled for staining for 5 min. Then, 1 mL of PBS is continuously introduced through the fourth channel at a flow rate of 20 μL / min for washing. The staining results can then be observed under a microscope to determine the osteogenic differentiation status. (As shown) Figure 3 and Figure 4 (As shown)

[0102] Example 5:

[0103] Cell seeding and adipogenic differentiation induction:

[0104] From the first guide channel at 5×10 5 Cells / mL of cell suspension were injected, flowing through the third channel into and filling the cell culture tank. The cells were allowed to adhere and grow for 2 hours. Residual waste liquid was then discharged through the fifth channel. Adipogenic induction media A and B were continuously introduced through the fifth channel at a flow rate of 10 μL / min, with the media type changed every three days. After the induction medium filled the culture area, each set of interdigitated electrodes was connected to an impedance detection device to measure the impedance values ​​at various locations before differentiation. During culture, culture waste liquid was discharged through the third and first channels. After 15 days of induction culture, each set of interdigitated electrodes was connected to an impedance detection device to measure the impedance values ​​at various locations after differentiation. The ratio of these values ​​to the impedance values ​​measured before differentiation was used to assess the differentiation effect. Figure 9 (as shown), Again1 mL of PBS was continuously circulated through the fifth channel at a flow rate of 20 μL / min for washing. Then, 4% neutral formaldehyde solution was continuously circulated through the fifth channel at a flow rate of 20 μL / min until the culture area was filled, and the solution was fixed for 30 min. Next, 1 mL of PBS was continuously circulated through the fifth channel at a flow rate of 20 μL / min for washing. Finally, Oil Red O working solution was continuously circulated through the fifth channel at a flow rate of 20 μL / min until the culture area was filled for staining for 5 min. Then, 1 mL of PBS was continuously circulated through the fifth channel at a flow rate of 20 μL / min for washing. The staining results can then be observed under a microscope to determine the adipogenic differentiation status. (e.g.) Figure 5 and Figure 6 (As shown)

[0105] Example 6:

[0106] Cell seeding and chondrogenic differentiation induction:

[0107] Pre-cultured cell suspension was injected into the sixth channel, allowing it to settle in the center of an asymmetrical "X" shape as it flowed through the third culture zone. This process was repeated until all cell suspensions settled in the center of the asymmetrical "X" shape within the culture zone. The remaining waste liquid was then discharged from the seventh channel. Chondrogenic induction medium was continuously introduced into the sixth channel at a flow rate of 60 μL / min. Once the culture zone was filled with the induction medium, each set of interdigitated electrodes was connected to an impedance detection device to measure the impedance values ​​at each location before differentiation. During the culture process, culture waste liquid was discharged from the seventh channel. After 15 days of induction culture, each set of interdigitated electrodes was connected to an impedance detection device to measure the impedance values ​​at each location after differentiation. The ratio of these values ​​to the impedance values ​​measured before differentiation was used to assess the differentiation effect. Figure 10 (As shown), PBS is then introduced through the seventh channel at a flow rate of 300 μL / min, causing the MSCs spheres in the asymmetric "X" shape in the third culture zone to detach from the microfluidic chip through the sixth channel. The MSCs spheres to be tested are then fixed in 4% neutral formaldehyde for 2 hours, embedded in OCT for frozen sectioning, stained with Allis blue, and observed under a microscope to determine the degree of cartilage differentiation. (As shown) Figure 7 and Figure 8 (As shown).

[0108] The above provides a detailed description of a microfluidic detection chip and method for automated culture and differentiation of mesenchymal stem cells, as provided in the embodiments of this application. The descriptions of the embodiments above are merely for the purpose of helping to understand the method and its core ideas; furthermore, those skilled in the art will recognize that, based on the ideas of this application, there will be changes in the specific implementation methods and application scope. Therefore, the content of this specification should not be construed as a limitation of this application.

[0109] As used in the specification and claims, certain terms have particular meanings. One skilled in the art will understand that different manufacturers can refer to a component by different names. The specification and claims should not be construed as limited to components by a particular name, but should be construed by the component's function. As used in the specification and claims, "comprising" and "including" are meant to be interpreted as specifying open-ended claims that are not limited to the listed elements. "Approximately" means within an acceptable error range for the corresponding function, which will vary from one context to another. The description that follows is intended to provide a better understanding of the preferred embodiments of the present application, and is not intended to be a complete description of all possible embodiments of the present application. The description serves only to illustrate the general principles of the present application, and is not meant to limit the present application to specific embodiments.

[0110] It should also be noted that the terms "comprising," "including," and "having" or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a process, product, or composition that comprises a list of elements is not necessarily limited to only those elements but can include other elements not expressly listed or inherent to such process, product, or composition. An element proceeded by "comprises... a" does not, without more constraints, exclude the existence of additional identical elements in the process, product, or composition that contains the element.

[0111] It should be understood that the term "and / or" as used herein is merely an open joining of two independent items, and is not intended to imply that the joined items are to be combined in the same manner. In addition, the term "or" as used herein is intended to mean an inclusive "or" rather than an exclusive "or." That is, unless explicitly stated to the contrary, the term "or" means an inclusive "or" and includes the possibility of more than one of the items listed in the "or" statement.

[0112] The above specification and description of the application discloses and describes several preferred embodiments of the application. However, it is understood that the application is not limited to the precise forms described and shown in the specification and drawings. Rather, the application is intended to cover any and all modifications and variations of the application which become apparent to those skilled in the art upon reading the description and understanding the concepts disclosed herein. It is therefore desired that what is claimed be understood broadly by reference to the specification and drawings and by reference to the appended claims, rather than by reference to the limited examples described and shown in the specification and drawings.

Claims

1. A microfluidic detection chip for automated culture and differentiation of mesenchymal stem cells, characterized in that, The microfluidic detection chip includes a culture medium, interdigitated electrodes, and a mechanical peristalsis device; The culture medium is equipped with multiple culture zones, and these multiple culture zones are capable of culturing and differentiating mesenchymal stem cells without contamination. The interdigitated electrodes are used to collect data on the differentiation results within the culture region; The mechanical peristaltic device is used to flush cells with flowing culture medium, subjecting them to shear force from the medium to promote differentiation and enhance the differentiation effect of the differentiation medium. The upper surface of the culture body is provided with multiple culture zones and flow channels. Multiple interdigitated electrodes are respectively disposed on the lower surface of the culture body, and each culture zone is connected to one end of at least one interdigitated electrode. The other end of the interdigitated electrode is connected to an impedance analysis device. The mechanical peristaltic device is disposed at both ends of the culture body and is connected to the culture body through the flow channels. The culture body includes a first culture zone, a second culture zone, a third culture zone, and a spacer. The first, second, and third culture zones are all disposed on the culture body, and the flow channels are respectively connected to both ends of the first, second, and third culture zones. A plurality of spacers are disposed inside the third culture zone. The first, second, and third culture zones are all circular grooves, and the depths of the first and second culture zones are equal, while the depth of the third culture zone is greater than the depths of the first and second culture zones. The spacer is an asymmetrical X-shape and has a flow channel in the middle, with the narrowest part of the flow channel being 0.3-0.7 mm wide and the widest part being 2-3 mm.

2. The microfluidic detection chip according to claim 1, characterized in that, The flow channel includes a first flow channel and a second flow channel; wherein the first flow channel is connected to both ends of the first culture zone and the second culture zone, and the second flow channel is connected to both ends of the third culture zone.

3. The microfluidic detection chip according to claim 2, characterized in that, The first guide channel includes a first guide channel, a second guide channel, a third guide channel, a fourth guide channel, and a fifth guide channel; wherein, one end of the first guide channel is open; the other end is connected to one end of the second and third guide channels, one end of the second and third guide channels is connected to one end of the first culture zone and the second culture zone, respectively, and one end of the fourth and fifth guide channels is connected to the other end of the first and second culture zones, respectively, and the other end is open.

4. The microfluidic detection chip according to claim 3, characterized in that, The width of the first guide channel is 0.2-0.5 mm and the height is 0.2-0.5 mm; the width of the second and third guide channels is 0.1-0.2 mm and the height is 0.2-0.5 mm. The width and height of the fourth and fifth guide channels are 0.07 mm and 0.07 mm respectively. The width and height of the fourth and fifth guide channels are 0.05 mm and 0.05 mm respectively. The width and height of the fourth and fifth guide channels are 0.1 mm and 0.1 mm respectively.

5. The microfluidic detection chip according to claim 2, characterized in that, The second guide channel includes a sixth guide channel and a seventh guide channel; wherein, one end of the sixth guide channel and the seventh guide channel are respectively connected to both ends of the third culture zone, the width of the sixth guide channel is 3-5mm and the height is 2-3mm; the width of the seventh guide channel is 2-3mm and the height is 2-3mm.

6. The microfluidic detection chip according to claim 2, characterized in that, The culture medium is a composite structure of polydimethylsiloxane and glass.

7. A detection method using a microfluidic detection chip as described in any one of claims 1-6, characterized in that, The method specifically includes the following steps: S1) First, using a peristaltic device, the concentration is 5×10 5- 1×10 6 Inject the cell suspension (cells / mL) into the culture zone from one end of the culture body, turn off the peristaltic device, let it stand for 1-3 hours to allow the cells to adhere and grow, and then turn on the peristaltic device to drain all waste liquid. S2) The induction medium is continuously injected into the culture area from the other end of the culture body again through the peristaltic device. After the induction medium fills the culture area, the peristaltic device is turned off, the interdigital electrodes are connected to collect the impedance values ​​at each position, and the culture continues. During the process, the waste liquid is discharged from one end of the culture body through the peristaltic device. The induction culture is completed after at least 15 days of culture. S3) Impedance detection: After 15 days of culture, the interdigital electrodes are connected, and the impedance values ​​at each position after differentiation are measured. The ratio of these values ​​to the impedance measured in S2) is compared, and the differentiation effect is detected based on the results.

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