A device for freezing and resuscitating umbilical cord mesenchymal stem cells

Abstract

The present invention relates to the field of biotechnology, and in particular to a method for differentiating frozen umbilical cord mesenchymal stem cells into neural stem cells, comprising the following steps: 1) obtaining and preparing umbilical cord mesenchymal stem cells; 2) freezing the umbilical cord mesenchymal stem cells; 3) resuscitating and sampling the umbilical cord mesenchymal stem cells: after resuscitating the umbilical cord mesenchymal stem cells using a freezing and resuscitation box, taking out a non-destructive sampling cryopreservation tube, and detecting cell activity; 4) culturing the umbilical cord mesenchymal stem cells using an MSC culture medium until the culture dish is covered; 5) taking F3-F5 generations of umbilical cord mesenchymal stem cells in a logarithmic growth phase, digesting them, centrifuging them, and washing them to obtain mesenchymal stem cells; 3) inducing the mesenchymal stem cells obtained in step 2) into differentiated stem cells using an induction culture medium; 4) culturing the differentiated stem cells in a first differentiation culture medium at 37° C. and 5% CO2 for 3-7 days, and then transferring them to a second differentiation culture medium at 37° C. and 5% CO2 for further culturing for 3-7 days to finally obtain neural stem cells.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a device for freezing and resuscitating umbilical cord mesenchymal stem cells. Background Art

[0002] Neurodegenerative diseases are a type of progressive central nervous system disease in clinical practice. They are mainly caused by the degeneration of specific systemic nerve cell subpopulations. Common disease types include Parkinson's disease, hereditary chorea, Alzheimer's disease, etc. Their common feature is the loss of nerve cell function or malfunction. Stem cells have great potential in the treatment of these diseases.

[0003] Under specific induction conditions in vivo or in vitro, mesenchymal stem cells can differentiate into various tissue cells such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, myocardium, endothelium, etc. They still have multidirectional differentiation potential after continuous subculture and cryopreservation. Therefore, mesenchymal stem cells can be used as ideal seed cells and differentiate into neural cells under certain induction conditions.

[0004] Umbilical cord mesenchymal stem cells (MSCs) have high differentiation potential and can differentiate in multiple directions. They have broad clinical application prospects in tissue engineering such as bone, cartilage, muscle, tendon, ligament, nerve, liver, endothelium and myocardium. There are reports that MSCs were isolated from human umbilical cords, and the cell content and proliferation ability are better than bone marrow MSCs, the immunogenicity is lower than that of bone marrow MSCs, and it has the advantages of easy collection and no ethical controversy. Therefore, it has attracted more and more attention from researchers.

[0005] After the stem cells in the umbilical cord blood are separated, they need to be stored in cryopreservation tubes through gradient cooling. However, in the traditional gradient cooling method, the cryopreservation tubes need to be placed in different refrigerants in turn for gradient cooling. In the process of being taken out and transferred from the refrigerant, the cryopreservation tubes will enter the external environment. In the extremely high temperature difference environment, the opening and closing of the door will bring heat and moisture into the sample storage environment, which will have a great impact on the ultra-low temperature environment, greatly shortening the life cycle of the sample and making it impossible to preserve it for a long time, affecting the cryopreservation effect of the stem cells and causing cell activity after subsequent cell recovery. Summary of the invention

[0006] The purpose of the present invention is to solve the problems in the background technology and provide a device for freezing and resuscitating umbilical cord mesenchymal stem cells.

[0007] The above technical objectives of the present invention are achieved through the following technical solutions:

[0008] A method for differentiating cryopreserved umbilical cord mesenchymal stem cells into neural stem cells comprises the following steps:

[0009] 1) Obtaining and preparing umbilical cord mesenchymal stem cells;

[0010] 2) Cryopreservation of umbilical cord mesenchymal stem cells: After the obtained umbilical cord mesenchymal stem cells are placed in a non-destructive sampling cryopreservation tube (200), they are placed in a cryopreservation recovery box for cryopreservation. The cryopreservation sequence is: cryopreservation in ice water for 1-3 hours, dry ice for 12-24 hours, and finally transferred to liquid nitrogen for cryopreservation;

[0011] 3) Umbilical cord mesenchymal stem cell resuscitation and sampling detection: After resuscitation of umbilical cord mesenchymal stem cells using a cryopreservation resuscitation box, take out the non-destructive sampling cryopreservation tube and detect the cell activity;

[0012] 4) Take the revived umbilical cord mesenchymal stem cells with cell activity greater than 80% for culture, and culture the umbilical cord mesenchymal stem cells with MSC culture medium until they fill the culture dish;

[0013] 5) Obtaining F3-F5 generation umbilical cord mesenchymal stem cells in the logarithmic growth phase, digesting them, centrifuging them, and washing them to obtain mesenchymal stem cells;

[0014] 6) inducing the mesenchymal stem cells obtained in step 2) into differentiated stem cells using an induction medium;

[0015] 7) The differentiated stem cells are cultured in a first differentiation medium at 37°C, 5% CO2 for 3-7 days, and then transferred to a second differentiation medium at 37°C, 5% CO2 for a further 3-7 days to ultimately obtain neural stem cells.

[0016] Preferably, the preparation steps of umbilical cord mesenchymal stem cells in step 1) are as follows: ① remove blood vessels and epidermis from human umbilical cord tissue, wash it with 0.9% saline, cut it into 5mm×5mm tissue blocks, inoculate it in a 100mm culture dish, and culture it at 37°C and 5% CO2; ② wait until the cell confluence reaches 80%-90%, pass it, digest it with 0.05% trypsin, and adjust the cell number to 1×10 6 ml -1 The cells were inoculated into 100 mm culture dishes at 37°C and 5% CO2, and cultured every 3 days.

[0017] Preferably, in step 2), 0.125% trypsin / 0.05% disodium ethylenediaminetetraacetic acid (EDTA) is used for digestion, centrifuged at 1200 rpm / min for 5 min, the supernatant is removed, and the solution is rinsed once with 0.9% saline.

[0018] Preferably, the MSC culture medium preparation method is: ① DMEM culture medium and F12 culture medium are prepared into DMEM / F12 basic culture medium in a ratio of 1:1; ② 10% fetal bovine serum, 1% penicillin and 1% streptomycin are added to the DMEM / F12 basic culture medium.

[0019] Preferably, the preparation method of the induction medium is as follows: ① DMEM medium and F12 medium are prepared into DMEM / F12 basic medium in a ratio of 1:1; ② 20 ng / mL of epidermal growth factor, 20 ng / mL of basic fibroblast growth factor, 1×B27, and 1×N2 are added to the DMEM / F12 basic medium.

[0020] Preferably, the preparation method of the first differentiation medium is: ① DMEM medium and F12 medium are prepared in a ratio of 1:1 to form DMEM / F12 basal medium; ② basic fibroblast growth factor 20 ng / mL, nerve growth factor 10 ng / ml and brain-derived neurotrophic factor 10 ng / ml are added to the DMEM / F12 basal medium.

[0021] Preferably, the preparation method of the second differentiation medium is as follows: ① DMEM medium and F12 medium are prepared into DMEM / F12 basal medium in a ratio of 1:1; ② retinoic acid 10 μg / ml, 2-mercaptoethanol 95 μg / ml, folic acid 70 μg / ml, gentamicin 50 μg / ml, and fluoroquinolone 70 μg / ml are added to the DMEM / F12 basal medium.

[0022] Preferably, the culture density of the stem cells is 1.8×10 4 cells / cm 2 .

[0023] A device for freezing and resuscitating umbilical cord mesenchymal stem cells comprises a freezing and resuscitation box and a non-destructive sampling cryopreservation tube, wherein the freezing and resuscitation box comprises a partition box body and a rotating mounting frame, wherein the partition box body is a hollow box body, comprising a plurality of temperature control zones and a resuscitation zone, wherein the partition box body is provided with an opening at the top of the resuscitation zone, and a box cover is provided at the opening; the rotating mounting frame comprises a central column and a rotating frame, wherein the central column is arranged at the center of the partition box body; transfer chambers are provided between the temperature control zones and between the resuscitation zone and the temperature control zone, and an electric heating wire is provided on the side wall of the resuscitation zone; the rotating frame comprises a rotating shaft, a rotating hanger and a freezing box, wherein the rotating shaft is arranged in the central column, the freezing box is connected to the rotating shaft through the rotating hanger, and the non-destructive sampling cryopreservation tube is installed in the freezing box; a boss is provided on the side of the central column, and the transfer chamber is provided on the outside of the boss; there are three temperature control zones in total, namely, an ice water zone, a dry ice zone and a liquid nitrogen zone.

[0024] Preferably, the transfer chamber includes a first sealed door plate and a second sealed door plate, a discharge port is arranged at the bottom of the transfer chamber, the first sealed door plate and the second sealed door plate are hinged at the inner side wall of the separation box body, and the first sealed door plate and the second sealed door plate are driven by a door plate rotation motor arranged outside the separation box body.

[0025] Preferably, the temperature control zone is connected to a temperature control mechanism, which includes a cooling medium bin, an input pipeline and a recovery pipeline, the input pipeline outlet is arranged at the top of the temperature control zone, and the recovery pipeline inlet is arranged at the bottom of the temperature control zone.

[0026] Preferably, the non-destructive sampling cryopreservation tube includes a cryopreservation tube body and a sampling tube body arranged on the outside of the bottom, a sealing cap is arranged on the top of the cryopreservation tube body, and a one-way valve body is arranged on the bottom of the cryopreservation tube body; a sealing cover is arranged on the top of the sampling tube body, a tube hole is arranged in the middle of the sealing cover, the cryopreservation tube body is inserted into the sampling tube body through the tube hole, a sampling outlet is arranged at the bottom of the sampling tube body, and a blocking cover is arranged outside the sampling outlet; the one-way valve body includes a straight pipe section, a trapezoidal pipe section and a leak plate arranged in sequence from top to bottom, a spherical valve core is arranged in the trapezoidal pipe section, and the diameter of the straight pipe section is smaller than the diameter of the spherical valve core.

[0027] Preferably, a rotating valve is provided between the outer side of the bottom of the freezing tube body and the sampling tube body, and the rotating valve comprises a slider, a connecting rod and a baffle plate, and the slider is connected to the baffle plate via the connecting rod.

[0028] Preferably, an annular groove is provided on the outer wall at the bottom of the freezing tube body, a sliding groove is provided on the inner wall of the sampling tube body, and both ends of the sliding block are respectively arranged in the annular groove and the sliding groove.

[0029] Preferably, the baffle plate corresponds to the opening on the leakage plate.

[0030] Preferably, the sealing cover is provided with an air inlet, and a sealing plug is provided at the air inlet.

[0031] In summary, the beneficial effects of the present invention are:

[0032] 1. The method of differentiating cryopreserved umbilical cord mesenchymal stem cells into neural stem cells described in the present invention uses a special cryopreservation recovery box during the cryopreservation of stem cells, which can effectively ensure the activity of cells after recovery and improve the subsequent differentiation effect.

[0033] 2. The method of differentiating cryopreserved umbilical cord mesenchymal stem cells into neural stem cells described in the present invention uses non-destructive sampling cryopreservation tubes to ensure that the test sample is consistent with the actual cell fluid composition in the cryopreservation tube, thereby ensuring the accuracy of the test. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Schematic diagram of the cryopreservation and resuscitation box of the present invention

[0035] Figure 2 It is a structural schematic diagram of the temperature control zone of the present invention;

[0036] Figure 3 It is a structural schematic diagram of the non-destructive sampling cryopreservation tube of the present invention;

[0037] Figure 4 It is a structural schematic diagram of the rotary valve of the present invention. DETAILED DESCRIPTION

[0038] The following specific embodiments are merely explanations of the present invention and are not limitations of the present invention. After reading this specification, those skilled in the art may make modifications to the embodiments without any creative contribution as needed. However, such modifications are protected by the patent law as long as they are within the scope of the claims of the present invention.

[0039] The present invention will be described in detail below with reference to the accompanying drawings by way of embodiments. Example

[0040] A method for differentiating cryopreserved umbilical cord mesenchymal stem cells into neural stem cells comprises the following steps:

[0041] 1) Umbilical cord mesenchymal stem cells were cultured in MSC culture medium until they filled the culture dish. The preparation steps of umbilical cord mesenchymal stem cells were as follows: ① Remove the blood vessels and epidermis of human umbilical cord tissue, wash it with 0.9% saline, cut it into 5mm×5mm tissue blocks, inoculate it in a 100mm culture dish, and culture it at 37℃, 5% CO2; ② When the cell confluence reached 80%-90%, subculture it, digest it with 0.05% trypsin, and adjust the cell number to 1×10 6 ml -1 The concentration was inoculated in a 100 mm culture dish and cultured at 37°C with 5% CO2, and passaged every 3 days. The MSC culture medium preparation method was as follows: ① DMEM culture medium and F12 culture medium were prepared in a 1:1 ratio to form DMEM / F12 basic culture medium; ② 10% fetal bovine serum, 1% penicillin and 1% streptomycin were added to the DMEM / F12 basic culture medium;

[0042] 2) Cryopreservation of umbilical cord mesenchymal stem cells: After the obtained umbilical cord mesenchymal stem cells are placed in a non-destructive sampling cryopreservation tube 200, they are placed in a cryopreservation recovery box 100 for cryopreservation. The cryopreservation sequence is: cryopreservation in ice water for 1-3 hours, dry ice for 12-24 hours, and finally transferred to liquid nitrogen for cryopreservation;

[0043] 3) Umbilical cord mesenchymal stem cell resuscitation and sampling detection: After the umbilical cord mesenchymal stem cells are resuscitated using the cryopreservation resuscitation box 100, the non-destructive sampling cryopreservation tube 200 is taken out to detect the cell activity;

[0044] 4) Take the revived umbilical cord mesenchymal stem cells with cell activity greater than 80% for culture, take the umbilical cord mesenchymal stem cells of the F3-F5 generation in the logarithmic growth phase, use 0.125% trypsin / 0.05% disodium ethylenediaminetetraacetic acid (EDTA) for digestion, centrifuge at 1200rpm / min for 5min, remove the supernatant, and rinse once with 0.9% saline to obtain mesenchymal stem cells;

[0045] 5) Use the induction medium to induce the mesenchymal stem cells obtained in step 2) into differentiated stem cells at a culture density of 1.8×10 4 cells / cm 2 ; The preparation method of the induction medium is as follows: ① DMEM medium and F12 medium are prepared into DMEM / F12 basic medium in a ratio of 1:1; ② 20 ng / mL of epidermal growth factor, 20 ng / mL of basic fibroblast growth factor, 1×B27, and 1×N2 are added to the DMEM / F12 basic medium.

[0046] 6) The differentiated stem cells were cultured in a first differentiation medium at 37°C and 5% CO2 for 3-7 days. The preparation method of the first differentiation medium is as follows: ① DMEM medium and F12 medium were prepared in a ratio of 1:1 to form DMEM / F12 basal medium; ② 20 ng / mL of basic fibroblast growth factor, 10 ng / ml of nerve growth factor and 10 ng / ml of brain-derived neurotrophic factor were added to the DMEM / F12 basal medium;

[0047] 7) Transfer to the second differentiation medium at 95% humidity, 37°C, 5% CO2 and continue culturing for 3-7 days. The preparation method of the second differentiation medium is as follows: ① DMEM medium and F12 medium are prepared in a 1:1 ratio to form DMEM / F12 basal medium; ② Retinoic acid 10μg / ml, 2-mercaptoethanol 95μg / ml, folic acid 70μg / ml, gentamicin 50μg / ml, and fluoroquinolone 70μg / ml are added to the DMEM / F12 basal medium to finally obtain neural stem cells.

[0048] In the above steps, according to Figure 1 to Figure 4 As shown, the cryopreservation resuscitation box 100 includes a partition box body 1 and a rotating mounting frame. The partition box body 1 is a hollow box body, including a plurality of temperature control zones 11 and a resuscitation zone 12. The partition box body 1 is provided with an opening at the top of the resuscitation zone 12, and a box cover is provided at the opening; the rotating mounting frame includes a central column 21 and a rotating frame 22, and the central column 21 is provided at the center of the partition box body 1;

[0049] Transfer chambers 3 are provided between each temperature control zone 11 and between the resuscitation zone 12 and the temperature control zone 11, and electric heating wires are provided on the side walls of the resuscitation zone 12; the rotating frame 22 includes a rotating shaft 221, a rotating hanger 222 and a freezing box 223, the rotating shaft 221 is provided in the central column 21, the freezing box 223 is connected to the rotating shaft 221 through the rotating hanger 222, the non-destructive sampling cryogenic tube 200 is installed in the freezing box 223, and a sealing strip 2221 is provided in the middle of the rotating hanger 222; a boss 211 is provided on the side of the central column 21, and the transfer chamber 3 is provided on the outside of the boss 211; there are three temperature control zones 11, namely, the ice water zone 11A, the dry ice zone 11B and the liquid nitrogen zone 11C.

[0050] The transfer chamber 3 includes a first sealed door plate 31 and a second sealed door plate 32. A discharge port 33 is arranged at the bottom of the transfer chamber 3. The first sealed door plate 31 and the second sealed door plate 32 are hinged at the inner wall of the partition box body 1. The first sealed door plate 31 and the second sealed door plate 32 are driven by a door plate rotating motor 34 arranged outside the partition box body 1.

[0051] The temperature control zone 11 is connected to a temperature control mechanism 4, which includes a cooling medium bin 41, an input pipeline 42 and a recovery pipeline 43. The outlet of the input pipeline 42 is arranged at the top of the temperature control zone 11, and the inlet of the recovery pipeline 43 is arranged at the bottom of the temperature control zone 11. A separation device 431 is arranged in the recovery pipeline 43.

[0052] according to Figure 3 , Figure 4 As shown, the non-destructive sampling cryopreservation tube 200 includes a cryopreservation tube body 51 and a sampling tube body 52 arranged on the outer side of the bottom, a sealing cap 511 is arranged on the top of the cryopreservation tube body 51, and a one-way valve body 53 is arranged on the bottom of the cryopreservation tube body 51; a sealing cover 521 is arranged on the top of the sampling tube body 52, a tube hole is arranged in the middle of the sealing cover 521, the cryopreservation tube body 51 is inserted into the sampling tube body 52 through the tube hole, a sampling outlet 522 is arranged at the bottom of the sampling tube body 52, and a blocking cover 523 is arranged outside the sampling outlet 522; the one-way valve body 53 includes a straight tube section 531, a trapezoidal tube section 532 and a leak plate 533 arranged in sequence from top to bottom, a spherical valve core 534 is arranged in the trapezoidal tube section 532, and the diameter of the straight tube section 531 is smaller than the diameter of the spherical valve core 534. An air inlet 5111 is provided at the cover 511 , and a sealing plug 5112 is provided at the air inlet 5111 . The cryopreservation tube body 51 is a transparent tube body, and a scale bar is provided on its side wall.

[0053] A rotating valve 54 is also provided between the outer side of the bottom of the freezing tube body 51 and the sampling tube body 52. ​​The rotating valve 54 includes a slider 541, a connecting rod 542 and a baffle plate 543. The slider 541 is connected to the baffle plate 543 through the connecting rod 542. An annular groove 512 is provided on the outer wall of the bottom of the freezing tube body 51, and a slide groove 524 is provided on the inner wall of the sampling tube body 52. ​​Both ends of the slider 541 are respectively arranged in the annular groove 512 and the slide groove 524. The baffle plate 543 corresponds to the opening on the leakage plate 533.

[0054] When freezing the device:

[0055] 1. Drive the rotating shaft 221 to rotate the cryopreservation box 223 into the resuscitation area 12, open the box cover on the top of the resuscitation area 12, put the cryopreservation tube in, and then close the box cover.

[0056] 2. Open the first sealed door plate 31 of the transfer chamber 3 between the temperature control zone 11 and the ice water zone 11A, connect the transfer chamber 3 with the resuscitation zone 12, drive the rotating shaft 221 to rotate the cryopreservation box 223 into the transfer chamber 3, and close the first sealed door plate 31. After starting the temperature control mechanism 4 to fill the ice water zone 11A with ice water, open the second sealed door plate 32 of the transfer chamber 3, connect the transfer chamber 3 with the ice water zone 11A, and then drive the rotating shaft 221 to rotate the cryopreservation box 223 into the ice water zone 11A for the first cooling. At the same time, close the second sealed door plate 32, and discharge the residual ice water in the transfer chamber 3 through the discharge port 33.

[0057] 3. After the first cooling is completed, the first sealed door panel 31 of the transfer chamber 3 between the ice water area 11A and the dry ice area 11B is opened, and after the cryopreservation box 223 is rotated into the transfer chamber 3, the first sealed door panel 31 is closed, and the dry ice area 11B is filled with dry ice through the temperature control mechanism 4. After the ice water in the transfer chamber 3 is discharged through the discharge port 33, the second sealed door panel 32 is opened to transfer the cryopreservation box 223 to the dry ice area 11B for the second cooling, and at the same time, the second sealed door panel 32 is closed, and the residual dry ice in the transfer chamber 3 is discharged through the discharge port 33.

[0058] 4. Use the same method to transfer the cryopreservation box 223 to the liquid nitrogen area 11C for the third cooling and cryopreservation.

[0059] When the device warms up and recovers:

[0060] 1. Move the cryopreservation box 223 to the resuscitation zone and heat the resuscitation zone by means of an electric heating wire until the cryopreservation tube returns to normal temperature.

[0061] When sampling with cryotubes:

[0062] 1. Rotate the sampling tube body 52, and at the same time drive the slider 541 to rotate the baffle plate 543 to a certain angle, so that the holes on the baffle plate 543 and the leakage plate 233 are aligned. 2. Open the air inlet 5111, pull the sampling tube body 52 downward, and under the action of pressure, the cell fluid in the cryopreservation tube body 51 enters the sampling tube body 52 from the cryopreservation tube body 51 through the one-way valve body 53. 3. Rotate the sampling tube body 52 again, so that the holes on the baffle plate 543 and the leakage plate 533 are staggered, that is, the leakage plate 533 is covered by the baffle plate 543. 4. Remove the baffle 523 to open the sampling outlet 522, press the cryopreservation tube body 51, and under the action of pressure, the cell fluid flows out of the sampling tube body 52. A small amount of cell fluid that flows back from between the orifice plate 543 and the leakage orifice plate 533 pushes up the spherical valve core 534 and is blocked in the one-way valve body 53 , and cannot flow back into the cryopreservation tube body 51 .

Claims

1. A device for cryopreservation and resuscitation of umbilical cord mesenchymal stem cells, characterized in that: include: A cryopreservation and resuscitation box (100) and a non-destructive sampling cryopreservation tube (200), wherein the cryopreservation and resuscitation box (100) comprises a partition box body (1) and a rotating mounting frame, wherein the partition box body (1) is a hollow box body, comprising a plurality of temperature control zones (11) and a resuscitation zone (12), wherein the partition box body (1) is provided with an opening at the top of the resuscitation zone (12), and a box cover is provided at the opening; the rotating mounting frame comprises a central column (21) and a rotating frame (22), wherein the central column (21) is provided at the center of the partition box body (1); and the temperature control zones (11) are provided between two of each other. A transfer chamber (3) is provided between the resuscitation zone (12) and the temperature control zone (11); an electric heating wire is provided on the side wall of the resuscitation zone (12); the rotating frame (22) comprises a rotating shaft (221), a rotating hanger (222) and a freezing box (223); the rotating shaft (221) is provided in the central column (21); the freezing box (223) is connected to the rotating shaft (221) via the rotating hanger (222); a non-destructive sampling cryogenic tube (200) is installed in the freezing box (223); a boss is provided on the side of the central column (21); (211), the transfer chamber (3) is arranged outside the boss (211); the temperature control zone (11) has three zones, namely, an ice water zone (11A), a dry ice zone (11B) and a liquid nitrogen zone (11C); the non-destructive sampling cryopreservation tube (200) comprises a cryopreservation tube body (51) and a sampling tube body (52) arranged outside the bottom, the cryopreservation tube body (51) is provided with a sealing cap (511) at the top, and a one-way valve body (53) is provided at the bottom of the cryopreservation tube body (51); the sampling tube body (52) is provided with a sealing cover (521) at the top, and the sealing cover ( A tube hole is provided in the middle of the freezing tube body (51), the freezing tube body (51) is inserted into the sampling tube body (52) through the tube hole, a sampling outlet (522) is provided at the bottom of the sampling tube body (52), and a blocking cover (523) is provided outside the sampling outlet (522); the one-way valve body (53) comprises a straight tube section (531), a trapezoidal tube section (532) and a leak plate (533) arranged in sequence from top to bottom, a spherical valve core (534) is arranged in the trapezoidal tube section (532), and the diameter of the straight tube section (531) is smaller than the diameter of the spherical valve core (534).

Images