CN112210531B - Stem cell induction culture solution and preparation method and application thereof - Google Patents
Stem cell induction culture solution and preparation method and application thereof Download PDFInfo
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- CN112210531B CN112210531B CN202010957658.7A CN202010957658A CN112210531B CN 112210531 B CN112210531 B CN 112210531B CN 202010957658 A CN202010957658 A CN 202010957658A CN 112210531 B CN112210531 B CN 112210531B
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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- C12N2509/10—Mechanical dissociation
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Abstract
The invention belongs to the field of cell experiments, and particularly discloses a stem cell induction culture solution, and a preparation method and application thereof, wherein the stem cell culture solution comprises an inducer, an induction culture medium and a PH buffer solution, the content of the inducer is 0.4-1.4 mu g/ml, the content of the induction culture medium is 0.6-2.8 mu g/ml, the content of the PH buffer solution is 0.03-0.5 mu g/ml, the inducer is sulfonated beta-cyclodextrin or oleic acid, and the induction culture medium contains any two or more of fetal calf serum, amino acid and penicillin. The induction culture solution has high induction speed and high induction rate, can be used for inducing fat cells with different sizes, has good compatibility, and is beneficial to ensuring the activity of the cells and simultaneously keeping the difference of cell functions.
Description
Technical Field
The invention relates to the field of cell experiments, in particular to a stem cell induction culture solution and a preparation method and application thereof.
Background
Cell culture techniques refer to the growth of cells under in vitro conditions, during which the cells no longer form tissues (animals). The cell culture technology, as a technical method for culturing cells, has been extensively and extensively applied to observing the morphological structure and the life activities of living cells and various subject researches such as cytology, genetics, immunology, experimental medicine, oncology and the like in the field of biology, and has important significance for clinical and basic researches.
The emergence of stem cell regenerative medicine research provides a new treatment approach for repairing organ damage, and the basic method is to culture and expand autologous normal tissue cells in vitro. However, the traditional culture method has low induced differentiation rate and long time.
Disclosure of Invention
The invention aims to provide a stem cell induction culture solution, a preparation method and application thereof, and aims to solve the problems in the background art.
In order to achieve the purpose, the invention provides the following technical scheme: a stem cell induction culture solution comprises an inducer, an induction culture medium and a PH buffer solution, wherein the content of the inducer is 0.4-1.4 mu g/ml, the content of the induction culture medium is 0.6-2.8 mu g/ml, and the content of the PH buffer solution is 0.03-0.5 mu g/ml. .
Preferably, the inducer is sulfonated beta-cyclodextrin or oleic acid, and the induction culture medium contains any two or more of fetal calf serum, amino acid and penicillin.
Preferably, the pH buffer is a sterile phosphate buffer and has a pH of 7.2 to 7.5.
Preferably, the stem cell culture solution is prepared by isolating and culturing tissue cells including tissue cells of mammals such as human.
The preparation method and application of the stem cell induction culture solution comprise the following steps:
s1: firstly, removing blood vessels, fascia and other useless tissues from adipose tissues, preparing the adipose tissues into paste, and adding collagenase for digestion treatment;
s2: then adding a culture medium for neutralization treatment, and performing centrifugal treatment by using a centrifugal device;
s3: removing supernatant, adding cell culture solution, and culturing cells;
s4: digestion passage is carried out when the cells are fused to 80-90%, and when the second generation cells are grown to a fused state, the cells are cultured in a growth medium added with an inducer, an induction medium and a pH buffer solution.
Preferably, the centrifugal device comprises a box body, a first centrifugal shell, a second centrifugal shell and centrifugal structures, wherein the first centrifugal shell and the second centrifugal shell are arranged in the box body, the centrifugal structures are arranged at the bottom sides of the first centrifugal shell and the second centrifugal shell, the first centrifugal shell and the second centrifugal shell are consistent in structure and are symmetrically distributed, and a driving assembly for driving the first centrifugal shell and the second centrifugal shell to operate is arranged at the inner side of the box body; the driving assembly comprises a first motor and two rotating rods, one ends of the two rotating rods are fixedly connected with the tops of the first centrifugal shell and the second centrifugal shell, and the other ends of the two rotating rods are rotatably connected with the top of the box body through rotating shafts; two all the cover is equipped with driving pulley on the dwang, and links through the belt between two driving pulley, and first motor is installed in the dwang at box outside and its output transmission connection second centrifugal housing top.
Preferably, the bottom parts of the first centrifugal shell and the second centrifugal shell are provided with openings, and the middle parts of the inner sides of the first centrifugal shell and the second centrifugal shell are fixedly provided with fixed seats; the centrifugal structure comprises a screw rod, a screw sleeve and a handle, wherein the screw rod is rotatably connected among the side walls of the first centrifugal shell and the second centrifugal shell and the side end of the fixed seat; the centrifugal pipe fitting is arranged on the mounting support, the end of the mounting support is movably connected with a connecting rod through a rotating seat, and the free end of the connecting rod is movably connected with the end of a threaded sleeve.
Preferably, the centrifugal pipe fitting comprises a lower test tube support and an upper test tube support which are arranged on the mounting support, the upper test tube support is fixed on the upper part of the mounting support, a pipe clamp is arranged at the inner side end of the upper test tube support and used for fixing the centrifugal test tube, and racks are distributed on the outer side of the pipe clamp; the test tube upper bracket facial make-up is equipped with the second motor, and second motor output is connected with the gear, and gear and rack toothing.
Preferably, hold in the palm under the test tube and be fixed in the installing support lower part, hold in the palm under the test tube and have a cavity that is used for bearing centrifugal test tube.
Compared with the prior art, the invention has the beneficial effects that:
1. the induction culture solution has high induction speed and high induction rate, can be used for inducing fat cells with different sizes, has good compatibility, is favorable for ensuring the activity of the cells and simultaneously keeping the difference of cell functions, and has stronger differentiation capability.
2. The centrifugal device adopted by the invention can enable the movement of the centrifugal test tubes on the first centrifugal shell and the second centrifugal shell to be more complicated, can more thoroughly mix and stir the test tubes to be centrifuged, and improves the centrifugation effect.
Drawings
FIG. 1 is a schematic view showing the structure of a centrifugal apparatus in example 2 of the present invention;
fig. 2 is a schematic structural view of a first centrifugal case in embodiment 2 of the present invention;
fig. 3 is a schematic structural view of a test tube mounting tray in embodiment 2 of the present invention.
In the figure: 1. a box body; 2. a first centrifugal housing; 3. a second centrifuge housing; 4. a first motor; 5. rotating the rod; 6. a drive pulley; 7. a fixed seat; 8. a screw; 9. a threaded sleeve; 10. mounting a bracket; 11. a connecting rod; 12. a test tube is put on a support; 13. the test tube is lifted; 14. a pipe clamp; 15. a rack; 16. a second motor; 17. a gear.
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.
In the description of the present invention, it should be noted that the terms "vertical", "upper", "lower", "horizontal", and the like indicate orientations or positional relationships based on those shown in the drawings, and are only for convenience of describing the present invention and simplifying the description, but do not indicate or imply that the referred device or element must have a specific orientation, be constructed in a specific orientation, and be operated, and thus, should not be construed as limiting the present invention.
In the description of the present invention, it should also be noted that, unless otherwise explicitly specified or limited, the terms "disposed," "mounted," "connected," and "connected" are to be construed broadly and may, for example, be fixedly connected, detachably connected, or integrally connected; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.
Example 1: the invention provides a technical scheme that: a stem cell induction culture solution comprises an inducer, an induction culture medium and a PH buffer solution, wherein the content of the inducer is 0.4-1.4 mu g/ml, the content of the induction culture medium is 0.6-2.8 mu g/ml, and the content of the PH buffer solution is 0.03-0.5 mu g/ml. .
In this embodiment, the inducer is sulfonated beta-cyclodextrin or oleic acid, and the induction medium contains any two or more of fetal calf serum, amino acids, and penicillin.
In this example, the pH buffer is a sterile phosphate buffer and has a pH of 7.2 to 7.5.
In the present example, the stem cell culture solution was prepared by isolated culture of tissue cells including human and mammalian tissue cells.
The preparation method and application of the stem cell induction culture solution comprise the following steps:
s1: firstly, removing blood vessels, fascia and other useless tissues from adipose tissues, preparing the adipose tissues into paste, and adding collagenase for digestion treatment;
s2: then adding a culture medium for neutralization treatment, and performing centrifugal treatment by using a centrifugal device;
s3: removing supernatant, adding cell culture solution, and culturing cells;
s4: digestion passage is carried out when the cells are fused to 80-90%, and when the second generation cells are grown to a fused state, the cells are cultured in a growth medium added with an inducer, an induction medium and a pH buffer solution.
Example 2: referring to fig. 1-3, in the present embodiment, the centrifugal device in S2 includes a box body 1, a first centrifugal shell 2 and a second centrifugal shell 3 disposed in the box body 1, and centrifugal structures mounted at bottom sides of the first centrifugal shell 2 and the second centrifugal shell 3, the first centrifugal shell 2 and the second centrifugal shell 3 have the same structure and are symmetrically distributed, and a driving assembly for driving the first centrifugal shell 2 and the second centrifugal shell 3 to operate is disposed at an inner side of the box body 1; the driving component comprises a first motor 4 and two rotating rods 5, one ends of the two rotating rods 5 are fixedly connected with the tops of the first centrifugal shell 2 and the second centrifugal shell 3, and the other ends of the two rotating rods are rotatably connected with the top of the box body 1 through rotating shafts; two all the cover is equipped with driving pulley 6 on dwang 5, and links through the belt between two driving pulley 6, and first motor 4 is installed in the dwang 5 at the 3 tops of box 1 outside and its output transmission connection second centrifugal housing.
In this embodiment, first motor 4 can drive two dwang 5 operations when starting, makes the first centrifugal shell 2 of 5 tip of two dwang, the operation of second centrifugal shell 3, makes the motion of centrifugation test tube more become complicated on first centrifugal shell 2, the second centrifugal shell 3, can be more thorough treat that the centrifugation test tube mixes and stir.
In this embodiment, the bottom of each of the first centrifugal shell 2 and the second centrifugal shell 3 is provided with an opening, and the middle part of the inner side of each of the first centrifugal shell and the second centrifugal shell is fixedly provided with a fixed seat 7; the centrifugal structure comprises a screw 8, a threaded sleeve 9 and a handle, wherein the screw 8 is rotatably connected between the side walls of the first centrifugal shell 2 and the second centrifugal shell 3 and the side end of the fixed seat 7, the threaded sleeve is sleeved on the screw 8, and the handle is arranged on the outer sides of the first centrifugal shell 2 and the second centrifugal shell 3 and is used for driving the screw 8 to rotate; the end part of the bottom side of the fixed seat 7 is hinged with a mounting bracket 10, the mounting bracket 10 is provided with a centrifugal pipe fitting, the end part of the mounting bracket 10 is movably connected with a connecting rod 11 through a rotating seat, and the free end of the connecting rod 11 is movably connected with the end part of the threaded sleeve 9.
In this embodiment, make the screw rod 8 operation through the handle that sets up, make the screw rod 8 drive swivel nut 9 and remove along its length direction, swivel nut 9 drives connecting rod 11, makes connecting rod 11 drive installing support 10, and the inclination that drives centrifugal pipe spare on it through installing support 10 changes, even centrifugal pipe spare's inclination is adjustable for centrifugal effect is better.
In this embodiment, the centrifugal tube includes a lower test tube holder 12 and an upper test tube holder 13 mounted on the mounting bracket 10, the upper test tube holder 13 is fixed on the upper portion of the mounting bracket 10, a tube clamp 14 is disposed at the inner end of the upper test tube holder 13, the tube clamp 14 is used for fixing the centrifugal test tube, and racks 15 are distributed outside the tube clamp 14; the test tube is held in the palm 13 facial make-up and is equipped with second motor 16, and second motor 16 output is connected with gear 17, and gear 17 and rack 15 meshing.
In this embodiment, the tube bottom 12 is fixed to the lower part of the mounting bracket 10, and the tube bottom 12 has a cavity for holding the centrifugal tube.
In this embodiment, second motor 16 drives gear 17 and moves to make gear 17 mesh in rack 15, drive pipe clamp 14 rotatory, utilize pipe clamp 14 to drive centrifugal test tube relative operation, make the centrifugal effect of centrifugal test tube better, the cooperation makes the motion of first centrifugal casing 2, second centrifugal casing 3, and what the centrifugal test tube of treating that can be more thorough mixes and stirs, has improved centrifugal effect.
In this embodiment, whole centrifugal device is connected with power and total control button, and it realizes controlling it through total control button, because the equipment that control button matches is equipment commonly used, belongs to current mature technology, no longer gives unnecessary details its electric connection relation and specific circuit structure here.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (1)
1. A method for inducing and culturing stem cells by using a culture solution is characterized by comprising the following steps:
s1: firstly, removing blood vessels, fascia and other useless tissues from adipose tissues, preparing the adipose tissues into paste, and adding collagenase for digestion treatment;
s2: then adding a culture medium for neutralization treatment, and performing centrifugal treatment by using a centrifugal device;
s3: removing supernatant, adding cell culture solution, and culturing cells;
s4: carrying out digestion passage when the cells are fused to 80-90%, and culturing in a growth culture medium added with an induction culture solution when the second generation cells grow to a fused state;
the induction culture solution comprises an inducer, an induction culture medium and a pH buffer solution, wherein the content of the inducer is 0.4-1.4 mu g/ml, the content of the induction culture medium is 0.6-2.8 mu g/ml, and the content of the pH buffer solution is 0.03-0.5 mu g/ml;
the S2 middle centrifugal device comprises a box body (1), a first centrifugal shell (2) and a second centrifugal shell (3) which are arranged in the box body (1), and centrifugal structures which are arranged at the bottom sides of the first centrifugal shell (2) and the second centrifugal shell (3), wherein the first centrifugal shell (2) and the second centrifugal shell (3) have the same structure and are symmetrically distributed, and a driving component for driving the first centrifugal shell (2) and the second centrifugal shell (3) to operate is arranged at the inner side of the box body (1); the driving assembly comprises a first motor (4) and two rotating rods (5), one ends of the two rotating rods (5) are fixedly connected with the tops of the first centrifugal shell (2) and the second centrifugal shell (3), and the other ends of the two rotating rods are rotatably connected with the top of the box body (1) through rotating shafts; the two rotating rods (5) are sleeved with driving belt wheels (6), the two driving belt wheels (6) are linked through a belt, the first machine (4) is arranged on the outer side of the box body (1), and the output end of the first machine is in transmission connection with the rotating rod (5) at the top of the second centrifugal shell (3);
openings are formed in the bottoms of the first centrifugal shell (2) and the second centrifugal shell (3), and a fixed seat (7) is fixedly arranged in the middle of the inner side of each centrifugal shell; the centrifugal structure comprises a screw rod (8) rotatably connected between the side walls of the first centrifugal shell (2) and the second centrifugal shell (3) and the side end of the fixed seat (7), a threaded sleeve (9) sleeved on the screw rod (8), and a handle which is arranged on the outer sides of the first centrifugal shell (2) and the second centrifugal shell (3) and used for driving the screw rod (8) to operate; the end part of the bottom side of the fixed seat (7) is hinged with a mounting bracket (10), the mounting bracket (10) is provided with a centrifugal pipe fitting, the end part of the mounting bracket (10) is movably connected with a connecting rod (11) through a rotating seat, and the free end of the connecting rod (11) is movably connected with the end part of a threaded sleeve (9);
the centrifugal pipe fitting comprises a test tube lower support (12) and a test tube upper support (13) which are arranged on a mounting support (10), the test tube upper support (13) is fixed on the upper portion of the mounting support (10), a pipe clamp (14) is arranged at the inner side end of the test tube upper support (13), the pipe clamp (14) is used for fixing the centrifugal test tube, and racks (15) are distributed on the outer side of the pipe clamp (14); a second motor (16) is arranged on the test tube upper support (13), the output end of the second motor (16) is connected with a gear (17), and the gear (17) is meshed with the rack (15);
the test tube lower support (12) is fixed at the lower part of the mounting bracket (10), and the test tube lower support (12) is provided with a cavity for supporting a centrifugal test tube;
the inducer is oleic acid, and the induction culture medium contains any two or more of fetal calf serum, amino acid and penicillin;
the pH buffer solution is sterile phosphate buffer solution, and the pH value of the pH buffer solution is 7.2-7.5.
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| CN202010957658.7A CN112210531B (en) | 2020-09-10 | 2020-09-10 | Stem cell induction culture solution and preparation method and application thereof |
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| CN202010957658.7A CN112210531B (en) | 2020-09-10 | 2020-09-10 | Stem cell induction culture solution and preparation method and application thereof |
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Citations (3)
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|---|---|---|---|---|
| CN105779387A (en) * | 2016-05-10 | 2016-07-20 | 张君 | Method of induced differentiation of rat bone marrow mesenchymal stem cells into adipocytes |
| CN108384750A (en) * | 2018-02-08 | 2018-08-10 | 深圳市益鑫智能科技有限公司 | A kind of stem cell medium and preparation method thereof |
| CN110665651A (en) * | 2019-10-14 | 2020-01-10 | 山东中医药大学附属医院 | Automatic draw type cell experiment centrifuge |
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2020
- 2020-09-10 CN CN202010957658.7A patent/CN112210531B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105779387A (en) * | 2016-05-10 | 2016-07-20 | 张君 | Method of induced differentiation of rat bone marrow mesenchymal stem cells into adipocytes |
| CN108384750A (en) * | 2018-02-08 | 2018-08-10 | 深圳市益鑫智能科技有限公司 | A kind of stem cell medium and preparation method thereof |
| CN110665651A (en) * | 2019-10-14 | 2020-01-10 | 山东中医药大学附属医院 | Automatic draw type cell experiment centrifuge |
Non-Patent Citations (2)
| Title |
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| Multifunctional aliphatic polyester nanofibers for tissue engineering;Jiannan Zhan et al.;《Biomatter》;20121231;第202-212页 * |
| 骨髓间充质干细胞诱导内皮细胞与自体骨髓间充质干细胞共培养后的成骨特性;孙源等;《中国临床康复》;20060910;第68-71页 * |
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Denomination of invention: A stem cell induction culture medium and its preparation method and application Effective date of registration: 20230425 Granted publication date: 20221118 Pledgee: Industrial and Commercial Bank of China Limited Weihai Branch Pledgor: Weihai Jiansheng Biotechnology Co.,Ltd. Registration number: Y2023980039089 |
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