CN113913374A - Serum-free culture method for umbilical cord mesenchymal stem cells - Google Patents
Serum-free culture method for umbilical cord mesenchymal stem cells Download PDFInfo
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- CN113913374A CN113913374A CN202111090278.9A CN202111090278A CN113913374A CN 113913374 A CN113913374 A CN 113913374A CN 202111090278 A CN202111090278 A CN 202111090278A CN 113913374 A CN113913374 A CN 113913374A
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Abstract
The invention discloses a serum-free culture method of umbilical cord mesenchymal stem cells, which comprises the following steps: s1: culturing the third-generation mesenchymal stem cells, and screening; s2: continuing culturing the selected third generation cells; s3: adding inducer to convert into islet cells during culture; the culture apparatus comprises an incubator, a storage assembly, a control assembly and a protection plate, wherein the storage assembly is arranged in the incubator and comprises a placing frame, the control assembly comprises a control box, a control bin is arranged in the front of the control box, a control opening is formed in one side of the top of the control bin, and a control mechanism is arranged in the control opening. The invention has the beneficial effects that: the invention provides a method for extracting mesenchymal stem cells from umbilical cords and a preparation method thereof, which have the advantages of easily obtained material source, simple, convenient and efficient operation, can effectively reduce the culture cost of MSCs and obtain a large amount of MSC cells, and provides a foundation for clinical application of the MSCs.
Description
Technical Field
The invention relates to a culture method of umbilical cord mesenchymal stem cells, in particular to a serum-free culture method of umbilical cord mesenchymal stem cells, belonging to the technical field of stem cell culture.
Background
Mesenchymal Stem Cells (MSCs) are a class of adult stem cells with multipotentiality, which can self-replicate, self-renew, stimulate tissue growth and repair, and have low pollution source, convenient availability, stable biological performance and more primitive immunogenicity. Under the appropriate in vitro experimental conditions, the kit is easy to amplify and store. The mesenchymal stem cells can proliferate immune cells in vivo by changing cytokines secreted by T lymphocytes, B lymphocytes, natural killer cells and dendritic cells, so that the mesenchymal stem cells have very wide application prospect in the field of cell therapy.
With the continuous progress of science and technology, the search for novel and more potential mesenchymal stem cells has become a research hotspot at present, the mesenchymal stem cells can promote tissue modification and improve immunity, and have obvious curative effects when being clinically used for autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis and the like. How to obtain sufficient quantity of mesenchymal stem cells with more potential is an important guarantee of the application of the MSCs in clinic, so as to further broaden the understanding of people on the biological properties of the umbilical cord mesenchymal stem cells and provide support for the clinical application of the mesenchymal stem cells.
And present cell culture device adopts the incubator to go on mostly, can conveniently carry out the cultivation of cell like this, present some for experiment incubators, the shape is similar with small-size refrigerator, very light, but this kind of incubator is also similar to the use of refrigerator to the sample form of cultivateing the sample, open the closing door and carry out direct taking, some protect slightly also only add one deck closing plate more, when carrying out partial sample and getting like this, still directly open the guard plate, all samples are all exposed, can influence subsequent cultivation result like this.
Disclosure of Invention
The present invention is directed to solving the above problems and providing a method for serum-free culture of umbilical cord mesenchymal stem cells.
The invention realizes the aim through the following technical scheme, and the serum-free culture method of the umbilical cord mesenchymal stem cells comprises the following steps:
s1: culturing the third-generation mesenchymal stem cells by using a serum-free culture medium, and screening the third-generation cells;
s2: placing the selected third generation cells into a culture medium containing the insulin protein for continuous culture;
s3: adding inducer in the process of culturing, inducing the mesenchymal stem cells to be transformed into islet cells, and screening and eliminating the untransformed cells.
Preferably, the serum-free medium comprises the following components in percentage by mass: 1-20ng/mL of fibroblast growth factor, 10-45ng/mL of epidermal growth factor, 1-8mg/mL of human insulin, 5-15ng/mL of fibronectin, 5-10mg/mL of vitamin, 5-10mg/mL of transforming growth factor, 10-100mg/mL of human serum albumin, 50-200 mu g/mL of fibronectin and 80-100ug/mL of penicillin;
the screening of the third generation cells comprises the following processes;
the first process is as follows: firstly, taking out a culture medium with third generation cells from a culture device;
and a second process: extracting a base solution in the culture medium, and then pumping the tertiary cells into an active detection reagent;
the third process: the process is carried out under a microscope, the third generation cells with stronger activity are screened out, and then the selected cells are put into a new serum-free culture medium again for proliferation culture.
Preferably, the inducing agent comprises the following components in percentage by mass: 5-25mg/mL of nicotinamide, 15-45mg/mL of insulin, 20-60mg/mL of nerve growth factor, 5-10mg/mL of activin and 5-10mg/mL of transferrin, 5-50ng/mL of insulin-like growth factor and 1-10mg/mL of recombinant human insulin.
Preferably, the culture apparatus used in S1 includes:
the front surface of the incubator is provided with a sealing door;
the storage assembly is arranged in the incubator and comprises a placing frame, a plurality of placing bins are arranged on the front side of the placing frame, supporting pads are arranged on the inner walls of the bottom ends of the placing bins, and a baffle is arranged on the back side of the placing frame;
the control assembly is arranged on the back face of the baffle and comprises a control box, the front face of the control box is provided with a control bin, the inner wall of the bottom end of the control bin is provided with two moving ports, one of the moving ports is internally connected with a first moving plate in a sliding and inserting manner, the other moving port is internally connected with a second moving plate in a sliding and inserting manner, one side of the top of the control bin is positioned on the front face and provided with a control port, and a control mechanism is arranged in the control port;
the guard plate, it is a plurality of the guard plate all sets up in the front of rack, and articulates respectively at a plurality of openly bin mouths of placing the storehouse.
Preferably, the front of incubator is provided with the display screen, the front of closing the door is provided with the handle, the front of incubator has seted up cultivates the storehouse, the front of incubator, and the one side that is located cultivates the storehouse has seted up the control hole.
Preferably, the front of rack, and correspond a plurality of storehouses of placing and seted up a plurality of interlude holes, it is a plurality of the locking hole has all been seted up to the inner wall of interlude hole both sides, and adjacent two the locking hole link up each other, and is a plurality of the inner wall of placing the storehouse both sides has all been seted up the air vent, and the air vent of adjacent both sides link up each other, the slip mouth has been seted up to one side of placing the frame top.
Preferably, a plurality of plugging plates are fixedly connected to the front faces of the first moving plate and the second moving plate, a linkage rod is fixedly connected between the bottoms of the first moving plate and the second moving plate, and a plurality of teeth are arranged on one side of the second moving plate.
Preferably, control mechanism includes the control lever, control lever and control hole rotate to alternate and connect, the lever arm of control lever is equipped with first control fluted disc and second control fluted disc respectively, first control fluted disc and a plurality of tooth meshing.
Preferably, the outer wall meshing of second control fluted disc bottom has the rack board, rack board and slip mouth sliding interpenetration are connected, the top and the bottom of rack board all are provided with a plurality of connecting strips, and are a plurality of the equal fixedly connected with locking lever in one side of connecting strip, it is a plurality of the locking lever is connected with a plurality of locking hole sliding interpenetration respectively.
Preferably, the bottom of the back of the protection plate is fixedly connected with a penetration column, the penetration column is connected with one penetration hole in a sliding penetration mode, a fixing hole is formed in one side of the penetration column, the fixing hole is connected with one locking rod in a sliding penetration mode, and the bottom of the front of the protection plate is fixedly connected with a pull block.
The invention has the beneficial effects that:
the invention provides a method for extracting mesenchymal stem cells from umbilical cords and a preparation method thereof, which have the advantages of easily obtained material source, simple, convenient and efficient operation, capability of effectively reducing the culture cost of MSCs and obtaining a large amount of MSC cells, and provides a basis for clinical application of the MSCs.
Secondly, the culture device controls the storage component in a matching way with the control component, so that the opening and closing states of the storage component and the control component are relatively independent when the storage component is used for taking and placing samples, and the closing and opening of each storage component are flexibly adjusted through the rotation adjustment of the control component, thereby ensuring that other samples which are being cultured can not be influenced when the samples are taken.
Thirdly, the culture device provided by the invention respectively carries out meshed transmission by utilizing the two control fluted discs through rotating the control rod, and then is matched with linkage between the two moving plates and the rack plate, so that when a sample needs to be taken out, the two moving plates drive the plugging plates to separate and independently place bins, and meanwhile, the rack plate drives the locking rod to unlock, thus opposite sampling operation can be realized.
Drawings
FIG. 1 is a schematic view showing the overall construction of a culture apparatus of the present invention;
FIG. 2 is a schematic view of the connection of the incubator and the storage assembly of FIG. 1 according to the present invention;
FIG. 3 is a schematic view of the connection structure of the rack and the control assembly of FIG. 2 according to the present invention;
fig. 4 is a schematic structural view of the rack of the present invention;
FIG. 5 is an enlarged view of the structure at A in FIG. 4 according to the present invention;
FIG. 6 is a schematic structural diagram of a control assembly according to the present invention;
FIG. 7 is an enlarged view of the structure of FIG. 6 at B in accordance with the present invention;
FIG. 8 is a schematic structural view of a control mechanism of the present invention;
fig. 9 is a schematic structural view of a guard plate according to the present invention.
In the figure: 1. an incubator; 101. a closing door; 2. placing a rack; 201. a baffle plate; 202. a support pad; 203. inserting holes; 204. a vent hole; 3. a control box; 301. a first moving plate; 302. a second moving plate; 303. a plugging plate; 304. a linkage rod; 305. teeth; 4. a control lever; 401. a first control fluted disc; 402. a second control fluted disc; 5. a rack plate; 501. a connecting strip; 502. a locking lever; 6. a protection plate; 601. the post is inserted.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Referring to fig. 1-9, a serum-free culture method for umbilical cord mesenchymal stem cells comprises the following steps:
s1: culturing the third-generation mesenchymal stem cells by using a serum-free culture medium, and screening the third-generation cells;
the serum-free medium comprises the following components in percentage by mass: 1-20ng/mL of fibroblast growth factor, 10-45ng/mL of epidermal growth factor, 1-8mg/mL of human insulin, 5-15ng/mL of fibronectin, 5-10mg/mL of vitamin, 5-10mg/mL of transforming growth factor, 10-100mg/mL of human serum albumin, 50-200 mu g/mL of fibronectin and 80-100ug/mL of penicillin;
epidermal Growth Factor (EGF) is a heat-resistant single-chain low-molecular polypeptide consisting of 53 amino acid residues. After EGF is specifically identified and combined with EGF receptor on target cells, a series of biochemical reactions occur, and finally, the DNA synthesis and mitosis of the target cells can be promoted. EGF has no glycosyl part, is very stable, heat-resistant and acid-resistant;
transforming Growth Factor (TGF) refers to two classes of polypeptide growth factors, transforming growth factor-alpha and transforming growth factor-beta. Transforming growth factor-alpha is produced by macrophage, brain cell and epidermal cell, and can induce cell development.
The screening of the third generation cells comprises the following processes;
the first process is as follows: firstly, taking out a culture medium with third generation cells from a culture device;
and a second process: extracting a base solution in the culture medium, and then pumping the tertiary cells into an active detection reagent;
the third process: the process is carried out under a microscope, the third generation cells with stronger activity are screened out, and then the selected cells are put into a new serum-free culture medium again for proliferation culture.
S2: placing the selected third generation cells into a culture medium containing the insulin protein for continuous culture;
s3: adding an inducer in the culture process to induce the mesenchymal stem cells to be converted into islet cells, and screening and eliminating the untransformed cells;
the inducer comprises the following components in percentage by mass: 5-25mg/mL of nicotinamide, 15-45mg/mL of insulin, 20-60mg/mL of nerve growth factor, 5-10mg/mL of activin and 5-10mg/mL of transferrin, 5-50ng/mL of insulin-like growth factor and 1-10mg/mL of recombinant human insulin.
The culture apparatus used in S1 includes:
the incubator 1 is provided with a sealing door 101 on the front side of the incubator 1;
the incubator 1 is a carbon dioxide incubator in the prior art, and can provide environmental conditions required for cell survival, and the sealing door 101 can be well sealed.
As a technical optimization scheme of the invention, the front surface of the incubator 1 is provided with a display screen, the front surface of the closed door 101 is provided with a handle, the front surface of the incubator 1 is provided with an incubation bin, and the front surface of the incubator 1 and one side of the incubation bin are provided with control holes;
the display screen can only display the environmental condition parameters in the incubator 1, and meanwhile, the corresponding condition parameters can be set and modified through touch screen keys.
The storage assembly is arranged in the incubator 1 and comprises a placing rack 2, a plurality of placing bins are arranged on the front side of the placing rack 2, supporting pads 202 are arranged on the inner walls of the bottom ends of the placing bins, and a baffle 201 is arranged on the back side of the placing rack 2;
the back of rack 2 is for being located the open design to the lateral wall in individual placing the storehouse, and rack 2 is hollow structure simultaneously, conveniently provides control assembly's activity space, and baffle 201 carries out half protection shutoff to rack 2's the back.
As a technical optimization scheme of the invention, a plurality of through holes 203 are formed in the front of the placement frame 2 corresponding to the plurality of placement bins, locking holes are formed in the inner walls of the two sides of each through hole 203, two adjacent locking holes are communicated with each other, vent holes 204 are formed in the inner walls of the two sides of each placement bin, the vent holes 204 of the two adjacent sides are communicated with each other, and a sliding port is formed in one side of the top of the placement frame 2;
a plurality of interlude holes 203 correspond to set up in a plurality of positive centers of placing storehouse bottom outer wall, and air vent 204 sets up in the inner wall department of placing storehouse top both sides, ensures to be in between each places the storehouse bottom and seals independent state, and air vent 204 is used for circulating the cultivation gas that incubator 1 provided, and the middle part of slip mouth and rack 2 link up each other.
The control assembly is arranged on the back of the baffle 201 and comprises a control box 3, a control bin is arranged on the front of the control box 3, two moving openings are formed in the inner wall of the bottom end of the control bin, a first moving plate 301 is connected in a sliding and inserting mode in one moving opening, a second moving plate 302 is connected in a sliding and inserting mode in the other moving opening, a control opening is formed in the front of one side of the top of the control bin, and a control mechanism is arranged in the control opening;
the opening width and length of the two moving openings are the same as those of the first moving plate 301 and the second moving plate 302, and the contact surfaces are smooth, so that the first moving plate 301 and the second moving plate 302 can well move in the vertical direction.
As a technical optimization scheme of the invention, the front surfaces of the first moving plate 301 and the second moving plate 302 are fixedly connected with a plurality of blocking plates 303, a linkage rod 304 is fixedly connected between the bottoms of the first moving plate 301 and the second moving plate 302, and one side of the second moving plate 302 is provided with a plurality of teeth 305;
a plurality of teeth 305 are arranged on one side of the second moving plate 302 in a vertical central symmetry manner, and are arranged near the back side.
As a technical optimization scheme of the invention, the control mechanism comprises a control rod 4, the control rod 4 is in rotary insertion connection with the control hole, a lever arm of the control rod 4 is respectively sleeved with a first control fluted disc 401 and a second control fluted disc 402, and the first control fluted disc 401 is meshed with the plurality of teeth 305;
the first control fluted disc 401 rotates in the control port, and one end of the control rod 4 is rotatably inserted and connected with the inner wall on the back of the control port.
A rack plate 5 is meshed with the outer wall of the bottom end of the second control fluted disc 402, the rack plate 5 is in sliding penetrating connection with a sliding opening, a plurality of connecting strips 501 are arranged at the top end and the bottom end of the rack plate 5, locking rods 502 are fixedly connected to one sides of the connecting strips 501, and the locking rods 502 are respectively in sliding penetrating connection with the locking holes;
the end of the locking rod 502 far from the connecting bar 501 is hemispherical, so that it can be more conveniently inserted into the locking hole and the fixing hole in a sliding manner.
The protection plates 6 are arranged on the front surface of the placing rack 2, and the protection plates 6 are respectively hinged to bin openings on the front surfaces of the placing bins;
as a technical optimization scheme of the invention, the bottom of the back surface of the protection plate 6 is fixedly connected with a penetration column 601, the penetration column 601 is slidably and penetratingly connected with one of the penetration holes 203, one side of the penetration column 601 is provided with a fixing hole, the fixing hole is slidably and penetratingly connected with one of the locking rods 502, and the bottom of the front surface of the protection plate 6 is fixedly connected with a pull block.
When the culture device is used, the culture medium is required to be stored in the process of carrying out cell culture by referring to FIGS. 1 to 9;
the first step of operation is implemented, the closing door 101 is opened first, then the control rod 4 is rotated clockwise, the control rod 4 drives the first control fluted disc 401 and the second control fluted disc 402 to rotate synchronously, and the following processes can occur;
the first process is as follows: the first control fluted disc 401 rotates clockwise, and the second moving plate 302 moves upwards through meshing transmission with the teeth 305, the second moving plate 302 drives the first moving plate 301 to move upwards synchronously through the linkage rod 304, meanwhile, the first moving plate 301 and the second moving plate 302 drive the plugging plate 303 with the front connected to the front to move upwards, the plugging plate 303 moves upwards to block between two sides of adjacent placing bins and plugs the adjacent vent holes 204, and at the moment, the placing bins are in a relatively independent state;
and a second process: the second control fluted disc 402 rotates clockwise, and makes the rack plate 5 move horizontally and transversely towards the direction close to the placing rack 2 through meshing transmission, at this moment, the connecting bar 501 moves along with the rack plate 5, and simultaneously drives the locking rod 502 to move synchronously, at this moment, the locking rod 502 can slide out from the fixing hole and zoom into the locking hole, and the locking of the inserting column 601 is released.
And implementing the second step of operation, pinching the pull block to turn the protection plate 6 upwards, and sliding the penetrating column 601 out of the penetrating hole 203 along with the protection plate 6 in the process of turning.
Implementing the third step of operation, will need to deposit the culture medium of culture and place in opening the storehouse of placing of protection plate 6, simultaneously for stable, can inlay the culture medium in holding up 202, after placing and accomplishing, overturn protection plate 6 downwards, will wear to insert post 601 and insert in the interlude hole 203.
Implementing the fourth step of operation, rotating the control rod 4 counterclockwise, the control rod 4 driving the first control fluted disc 401 and the second control fluted disc 402 to rotate counterclockwise, at this time, the first moving plate 301 and the second moving plate 302 driving the blocking plate 303 to move downward under the meshing transmission, the blocking of the vent hole 204 by the blocking plate 303 is removed, and the storage bins are communicated with each other by gas;
the rack plate 5 drives the connecting bar 501 to move towards the direction far away from the placing rack 2, meanwhile, the locking rod 502 moves from the locking hole along with the connecting bar 501 and penetrates into the fixing hole in a sliding mode, the locking rod 502 is matched with the fixing hole to lock the penetrating and inserting column 601 in the penetrating and inserting hole 203, and therefore the protection plate 6 is sealed.
And (5) implementing the fifth step of operation, closing the closing door 101, starting the incubator 1, performing cell culture, and if the culture medium in the corresponding placing bin needs to be taken out, opening the protection plate 6 at the corresponding position only after the first step and the second step of operation.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (10)
1. A serum-free culture method of umbilical cord mesenchymal stem cells is characterized by comprising the following steps: the method comprises the following steps:
s1: applying a serum-free culture medium, putting the culture medium into a culture device to culture the third-generation mesenchymal stem cells, and screening the third-generation mesenchymal stem cells;
s2: placing the selected third generation cells into a culture medium containing the insulin protein for continuous culture;
s3: adding inducer in the process of culturing, inducing the mesenchymal stem cells to be transformed into islet cells, and screening and eliminating the untransformed cells.
2. The method for culturing umbilical cord mesenchymal stem cells in serum-free mode according to claim 1, wherein the method comprises the following steps: the serum-free culture medium comprises the following components in percentage by mass: 1-20ng/mL of fibroblast growth factor, 10-45ng/mL of epidermal growth factor, 1-8mg/mL of human insulin, 5-15ng/mL of fibronectin, 5-10mg/mL of vitamin, 5-10mg/mL of transforming growth factor, 10-100mg/mL of human serum albumin, 50-200 mu g/mL of fibronectin and 80-100ug/mL of penicillin;
the screening of the third generation cells comprises the following processes;
the first process is as follows: firstly, taking out a culture medium with third generation cells from a culture device;
and a second process: extracting a base solution in the culture medium, and then pumping the tertiary cells into an active detection reagent;
the third process: the process is carried out under a microscope, the third generation cells with stronger activity are screened out, and then the selected cells are put into a new serum-free culture medium again for proliferation culture.
3. The method for culturing umbilical cord mesenchymal stem cells in serum-free mode according to claim 1, wherein the method comprises the following steps: the inducer comprises the following components in percentage by mass: 5-25mg/mL of nicotinamide, 15-45mg/mL of insulin, 20-60mg/mL of nerve growth factor, 5-10mg/mL of activin and 5-10mg/mL of transferrin, 5-50ng/mL of insulin-like growth factor and 1-10mg/mL of recombinant human insulin.
4. A serum-free culture method of umbilical cord mesenchymal stem cells, wherein the culture device used in S1 comprises:
the incubator (1), the front of the incubator (1) is provided with a closing door (101);
the storage assembly is arranged in the incubator (1) and comprises a placing rack (2), a plurality of placing bins are formed in the front face of the placing rack (2), supporting pads (202) are arranged on the inner walls of the bottom ends of the placing bins, and a baffle (201) is arranged on the back face of the placing rack (2);
the control assembly is arranged on the back of the baffle (201) and comprises a control box (3), a control bin is arranged on the front of the control box (3), two moving ports are formed in the inner wall of the bottom end of the control bin, one of the moving ports is internally and slidably connected with a first moving plate (301) in an inserting manner, the other moving port is internally and slidably connected with a second moving plate (302) in an inserting manner, one side of the top of the control bin is positioned on the front and provided with the control port, and a control mechanism is arranged in the control port;
the protection plate (6) is multiple, the protection plate (6) is arranged on the front surface of the placing frame (2) and is respectively hinged to the front bin openings of the placing bins.
5. The serum-free culture method of umbilical cord mesenchymal stem cells according to claim 4, wherein the culture method comprises the following steps: the front of incubator (1) is provided with the display screen, the front of closing door (101) is provided with the handle, the front of incubator (1) has been seted up and has been cultivateed the storehouse, the front of incubator (1), and the one side that is located cultivateing the storehouse has seted up the control hole.
6. The serum-free culture method of umbilical cord mesenchymal stem cells according to claim 4, wherein the culture method comprises the following steps: the front of rack (2), and correspond a plurality of storehouses of placing and seted up a plurality of interlude hole (203), it is a plurality of the locking hole has all been seted up to the inner wall of interlude hole (203) both sides, and adjacent two the locking hole link up each other, and is a plurality of place the inner wall of storehouse both sides and all seted up air vent (204), and air vent (204) of adjacent both sides link up each other, the slip mouth has been seted up to one side at rack (2) top.
7. The serum-free culture method of umbilical cord mesenchymal stem cells according to claim 4, wherein the culture method comprises the following steps: the front surfaces of the first moving plate (301) and the second moving plate (302) are fixedly connected with a plurality of blocking plates (303), a linkage rod (304) is fixedly connected between the bottoms of the first moving plate (301) and the second moving plate (302), and a plurality of teeth (305) are arranged on one side of the second moving plate (302).
8. The serum-free culture method of umbilical cord mesenchymal stem cells according to claim 4, wherein the culture method comprises the following steps: control mechanism includes control lever (4), control lever (4) and control hole rotate to alternate and connect, the lever arm of control lever (4) is equipped with first control fluted disc (401) and second control fluted disc (402) respectively, first control fluted disc (401) and a plurality of tooth (305) meshing.
9. The method for culturing umbilical cord mesenchymal stem cells in serum-free mode according to claim 8, wherein the culture method comprises the following steps: the outer wall meshing of second control fluted disc (402) bottom has rack plate (5), rack plate (5) and slip mouth slip interlude are connected, the top and the bottom of rack plate (5) all are provided with a plurality of connecting strip (501), and are a plurality of the equal fixedly connected with in one side of connecting strip (501) locking lever (502), it is a plurality of locking lever (502) are respectively with a plurality of locking hole slip interlude connection.
10. The serum-free culture method of umbilical cord mesenchymal stem cells according to claim 4, wherein the culture method comprises the following steps: the bottom fixedly connected with of guard plate (6) back alternates post (601), alternate post (601) and one of them interlude hole (203) slip interlude and connect, the fixed orifices has been seted up to one side of interlude post (601), the fixed orifices and one of them locking lever (502) slip interlude and connect, the positive bottom fixedly connected with of guard plate (6) draws the piece.
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