CN211689096U - Kit for separating, amplifying and culturing umbilical cord mesenchymal stem cells - Google Patents
Kit for separating, amplifying and culturing umbilical cord mesenchymal stem cells Download PDFInfo
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- CN211689096U CN211689096U CN201922256670.0U CN201922256670U CN211689096U CN 211689096 U CN211689096 U CN 211689096U CN 201922256670 U CN201922256670 U CN 201922256670U CN 211689096 U CN211689096 U CN 211689096U
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- 210000003954 umbilical cord Anatomy 0.000 title claims abstract description 65
- 210000002901 mesenchymal stem cell Anatomy 0.000 title claims abstract description 40
- 238000012258 culturing Methods 0.000 title abstract description 4
- 238000003860 storage Methods 0.000 claims abstract description 60
- 210000004027 cell Anatomy 0.000 claims abstract description 52
- 238000004113 cell culture Methods 0.000 claims abstract description 20
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 20
- 230000001079 digestive effect Effects 0.000 claims abstract description 12
- 238000004140 cleaning Methods 0.000 claims abstract description 10
- 239000012530 fluid Substances 0.000 claims abstract description 9
- 230000001954 sterilising effect Effects 0.000 claims abstract description 9
- 238000007710 freezing Methods 0.000 claims abstract description 8
- 230000008014 freezing Effects 0.000 claims abstract description 8
- 239000012930 cell culture fluid Substances 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 46
- 238000005406 washing Methods 0.000 claims description 14
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims description 7
- 239000004033 plastic Substances 0.000 claims description 6
- 239000003761 preservation solution Substances 0.000 claims description 5
- 238000005138 cryopreservation Methods 0.000 claims description 4
- 238000010521 absorption reaction Methods 0.000 claims description 3
- 230000035939 shock Effects 0.000 claims description 3
- 125000006850 spacer group Chemical group 0.000 claims description 3
- 238000013016 damping Methods 0.000 claims description 2
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 2
- 239000007788 liquid Substances 0.000 description 10
- 230000003321 amplification Effects 0.000 description 5
- 238000003199 nucleic acid amplification method Methods 0.000 description 5
- 238000000926 separation method Methods 0.000 description 4
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 235000015110 jellies Nutrition 0.000 description 3
- 239000008274 jelly Substances 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 229930182555 Penicillin Natural products 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
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- 238000001914 filtration Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 229940049954 penicillin Drugs 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 229960005322 streptomycin Drugs 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 108090000145 Bacillolysin Proteins 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 102000029816 Collagenase Human genes 0.000 description 1
- 108060005980 Collagenase Proteins 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 102000035092 Neutral proteases Human genes 0.000 description 1
- 108091005507 Neutral proteases Proteins 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 210000002718 aborted fetus Anatomy 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
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- 210000004700 fetal blood Anatomy 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
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- 239000002504 physiological saline solution Substances 0.000 description 1
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- 210000001644 umbilical artery Anatomy 0.000 description 1
- 210000003606 umbilical vein Anatomy 0.000 description 1
Images
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- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The utility model discloses a reagent box for separating, amplifying and culturing umbilical cord mesenchymal stem cells in the related technical field of biological medical equipment, which comprises a box body, wherein a disinfection and sterilization device and at least 9 mutually independent functional areas are arranged inside the box body; an umbilical cord collecting and preserving fluid storage container, an umbilical cord cleaning solution storage container, an umbilical cord mesenchymal stem cell culture fluid storage container, a cell cleaning solution storage container, a cell digestive fluid storage container, a cell freezing solution storage container, a complete culture solution storage container, a six-hole cell culture plate and instrument consumables are sequentially stored in the 9 functional areas; the covers of the storage containers are different in color and are respectively provided with corresponding labels; the utility model has simple structure, each functional area is independent, which is convenient for storing different functional containers; the color of the covers of the containers with different functions is different and is respectively provided with corresponding labels, so that the containers are clear at a glance for users, the operation is convenient, and the mistakes are avoided.
Description
Technical Field
The utility model relates to a relevant technical field of biological medicine equipment, in particular to a kit that is used for separating and expands to cultivate umbilical cord mesenchymal stem cell.
Background
Umbilical cord Mesenchymal Stem Cells (MSCs) refer to a multifunctional Stem cell existing in umbilical cord tissues of newborns, can be differentiated into a plurality of tissue Cells, and has a wide clinical application prospect.
In the prior art, MSCs with good proliferation capacity and low immunogenicity can be isolated from placenta, umbilical cord blood in perinatal period, fat from liposuction and aborted fetuses. In addition, the umbilical cord belongs to medical waste, has the characteristics of easy acquisition, stable and controllable source and no ethical dispute problem, and has good research value and application prospect. However, the requirements of the acquisition technology of the MSCs are strict, and the matched instrument is precise and has high cost, so that the method is not suitable for application and popularization.
Therefore, a kit capable of well realizing umbilical cord mesenchymal stem cell separation and amplification culture needs to be developed to help the application and popularization of MSCs.
SUMMERY OF THE UTILITY MODEL
Need research and development a section that exists to prior art can realize umbilical cord mesenchymal stem cell separation and amplification culture kit betterly, help MSCs's application promotion's technical problem, the utility model provides a kit for umbilical cord mesenchymal stem cell is cultivateed in separation and amplification.
In order to achieve the above purpose, the technical scheme of the utility model is that:
a kit for separating and amplifying umbilical cord mesenchymal stem cells comprises a box body, wherein a disinfection and sterilization device and at least 9 mutually independent functional areas are arranged in the box body; an umbilical cord collecting and preserving fluid storage container, an umbilical cord cleaning solution storage container, an umbilical cord mesenchymal stem cell culture fluid storage container, a cell cleaning solution storage container, a cell digestive fluid storage container, a cell freezing solution storage container, a complete culture solution storage container, a six-hole cell culture plate and instrument consumables are sequentially stored in the 9 functional areas; the umbilical cord collection and preservation solution storage container, the umbilical cord washing solution storage container, the umbilical cord mesenchymal stem cell culture solution storage container, the cell washing solution storage container, the cell digestive juice storage container, the cell cryopreservation solution storage container and the cover of the complete culture solution storage container are different in color and are respectively provided with corresponding labels.
Preferably, the disinfection and sterilization device is an ultraviolet disinfection lamp.
Further, the instrument consumables comprise 1 pair of disposable sterile scissors, 2 pairs of disposable sterile plastic forceps, 4 disposable sterile 250mL plastic cups and 1 disposable sterile 100um cell filter screen.
Furthermore, at least one part between the box body and each functional area and between each adjacent functional areas is provided with a damping structure.
Preferably, the shock-absorbing structure is a sterile rubber spacer.
Furthermore, a coded lock is arranged at the position of the switch on the box body.
The utility model has the advantages of as follows:
1. the utility model has simple structure, each functional area is independent, which is convenient for storing different functional containers;
2. the color of the covers of the containers with different functions is different and is respectively provided with corresponding labels, so that the containers are clear at a glance, the operation is convenient, and the error is avoided
3. Simple structure, low cost and convenient popularization.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without creative efforts.
Fig. 1 is a schematic structural diagram of the present invention.
In the figure, 1-box body; 2-a disinfection and sterilization device; 3-functional region.
Detailed Description
The following describes the present invention with reference to the accompanying drawings. It should be noted that the description of the embodiments is provided to help understanding of the present invention, but the present invention is not limited thereto. In addition, the technical features related to the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
As shown in figure 1, a kit for separating and amplifying umbilical cord mesenchymal stem cells comprises a box body 1, wherein a disinfection and sterilization device 2 and at least 9 mutually independent functional areas 3 are arranged in the box body 1; an umbilical cord collecting and preserving fluid storage container, an umbilical cord cleaning solution storage container, an umbilical cord mesenchymal stem cell culture fluid storage container, a cell cleaning solution storage container, a cell digestive fluid storage container, a cell freezing solution storage container, a complete culture solution storage container, a six-hole cell culture plate and instrument consumables are sequentially stored in the 9 functional areas 3; the umbilical cord collecting and storing liquid storage container, the umbilical cord washing liquid storage container, the umbilical cord mesenchymal stem cell culture liquid storage container, the cell washing liquid storage container, the cell digestive juice storage container, the cell freezing storage liquid storage container and the complete culture liquid storage container are different in color and are respectively provided with corresponding labels, namely, the color of none of the covers of the storage containers is the same as the color of the covers of other storage containers.
Naturally, umbilical cord collection and preservation solution storage containers, umbilical cord washing solution storage containers, umbilical cord mesenchymal stem cell culture solution storage containers, cell washing solution storage containers, cell digestive juice storage containers, cell freezing solution storage containers and complete culture solution storage containers are respectively used for storing umbilical cord collection and preservation solution, umbilical cord washing solution, umbilical cord mesenchymal stem cell culture solution, cell washing solution, cell digestive juice, cell freezing solution and complete culture solution; specifically, the method comprises the following steps:
umbilical cord collection and preservation solution: is physiological saline containing 200U/mL penicillin and 200. mu.g/mL streptomycin which the filtration sterilization is carried out at 0.22 mu.m;
umbilical cord washing liquid: is PBS buffer containing 100U/mL penicillin and 0.01g/100mL streptomycin and sterilized by 0.22 mu m filtration;
umbilical cord mesenchymal stem cell culture solution: is 450mL of low-sugar DMEM cell culture solution, 50mL of fetal calf serum, 10mL of mixed solution of 250ng/mLbFGF and 250ng/mLEGF, is stored at-20 ℃ when not used, is thawed overnight at the temperature of 4 ℃ before use, and is mixed uniformly;
the cell washing solution is sterile normal saline;
cell digestive juice: collagenase a at a concentration of 1mg/mL and neutral protease at a concentration of 1mg/mL were added according to a 1: 2 volume ratio of mixed solution;
cell cryopreservation solution: the cell culture solution, FBS and DMSO are mixed according to the volume ratio of 1:3: 1.
The kit is stored at-20 ℃ at ordinary times, and is unfrozen in a refrigerator at 4 ℃ before use, and the specific use process is as follows:
1. collecting umbilical cord, namely obtaining 10cm umbilical cord tissue of a newborn produced by caesarean section, storing the umbilical cord tissue in an umbilical cord collecting and storing solution, and sending the umbilical cord tissue to a laboratory within 24 h;
2. taking out the umbilical cord from the superclean workbench, and fully washing the umbilical cord by using umbilical cord washing liquid to remove blood stains;
3. cutting umbilical cord into small segments of about 1-3 cm, mechanically separating, removing Wharton's jelly, removing umbilical artery and umbilical vein, and cutting into pieces of about 1-3 mm3The tissue mass of (a);
4. transferring the sheared Wharton's jelly into a six-hole cell culture plate, uniformly paving the sheared Wharton's jelly, and adding a small amount of umbilical cord mesenchymal stem cell culture solution until the tissue block just submerges into the six-hole cell culture plate after the tissue block is firmly attached to the six-hole cell culture plate;
5. continuously judging whether the culture medium is polluted or not by observing the color of the culture medium for two or three days, then sucking the tissue of the dissociated cells by observing through a microscope about 7 days, and then changing the liquid by half every other day;
6. when cell colonies appear in the wells with the free cells and the density is higher, passage is prepared;
remaining cells which are not dissociated or a small amount of dissociated cells can be continuously cultured until cell colonies appear in the holes of the dissociated cells and are subcultured when the density is higher;
7. cell passage:
7.1, sucking out the umbilical cord mesenchymal stem cell culture solution, cleaning the six-hole cell culture plate by using a cell cleaning solution, adding a cell digestive fluid, and placing at 37 ℃ and 5% CO2Digesting for 2 hours in the incubator, taking out every 20 minutes to detect the digestion condition, and stopping digestion by using complete culture solution when most cells are observed to become round under a microscope;
7.2, gently blowing and beating the six-hole cell culture plate by using a dropper, transferring the liquid to a centrifuge tube, centrifuging at the rotating speed of 2000rpm for 5 minutes, and removing the supernatant;
7.3, adding umbilical cord mesenchymal stem cell culture solution to resuspend cells, changing the solution every other day, and carrying out passage again when 80-90% of the cells are fused;
8. cryopreservation of cells
8.1, digesting the cells by using cell digestive juice, transferring the cells to a centrifuge tube, centrifuging at 2000rpm for 5min, and removing supernatant;
8.2, adding complete culture solution with proper volume to re-suspend the cells, then subpackaging the cell suspension and the frozen stock solution into frozen tubes for freezing after the ratio of the cell suspension to the frozen stock solution is 1: 1.
The utility model provides a can realize umbilical cord mesenchymal stem cells separation and amplification culture kit betterly, help MSCs's application and popularization. The above is the basic model of the embodiment of the present invention, and in order to improve the technical effect, the following improvements can be made to the basic model of the embodiment, and the following improvements can be combined together without contradiction.
Wherein, in the reagent kit for separating and amplifying umbilical cord mesenchymal stem cells, the disinfection and sterilization device 2 is an ultraviolet disinfection lamp which is arranged on the inner side of the box cover of the box body 1.
In the kit for separating and amplifying umbilical cord mesenchymal stem cells, the instrument consumables comprise 1 pair of disposable sterile scissors, 2 pairs of disposable sterile plastic forceps, 4 disposable sterile 250mL plastic cups and 1 disposable sterile 100um cell filter screen.
In the kit for separating and culturing the umbilical cord mesenchymal stem cells in an amplification way, at least one part of the shock absorption structures of sterile rubber spacers or other structures are arranged between the box body 1 and each functional area 3 and between each adjacent functional area 3, and during specific implementation, the shock absorption structures are arranged between two adjacent parts of the box body 1 and each functional area 3 and between each functional area 3.
In the kit for separating and amplifying umbilical cord mesenchymal stem cells, the position of the switch on the box body 1 is provided with the coded lock, so that the pollution caused by the random opening of the box body 1 by other people can be avoided.
The embodiments of the present invention have been described in detail with reference to the accompanying drawings, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the spirit and scope of the invention.
Claims (6)
1. A kit for separating and amplifying umbilical cord mesenchymal stem cells, which is characterized in that: comprises a box body, wherein a disinfection and sterilization device and at least 9 mutually independent functional areas are arranged in the box body; an umbilical cord collecting and preserving fluid storage container, an umbilical cord cleaning solution storage container, an umbilical cord mesenchymal stem cell culture fluid storage container, a cell cleaning solution storage container, a cell digestive fluid storage container, a cell freezing solution storage container, a complete culture solution storage container, a six-hole cell culture plate and instrument consumables are sequentially stored in the 9 functional areas; the umbilical cord collection and preservation solution storage container, the umbilical cord washing solution storage container, the umbilical cord mesenchymal stem cell culture solution storage container, the cell washing solution storage container, the cell digestive juice storage container, the cell cryopreservation solution storage container and the cover of the complete culture solution storage container are different in color and are respectively provided with corresponding labels.
2. The kit for isolating and expanding umbilical cord mesenchymal stem cells for culture according to claim 1, wherein: the disinfection and sterilization device is an ultraviolet disinfection lamp.
3. The kit for isolating and expanding umbilical cord mesenchymal stem cells for culture according to claim 1, wherein: the instrument consumables include 1 disposable aseptic scissors, 2 disposable aseptic plastic tweezers, 4 disposable aseptic 250mL plastic cups and 1 disposable aseptic 100um cell filter screen.
4. The kit for isolating and expanding umbilical cord mesenchymal stem cells for culture according to claim 1, wherein: at least one part of the box body is provided with a damping structure between each functional area and between each adjacent functional area.
5. The kit for isolating and expanding umbilical cord mesenchymal stem cells for culture according to claim 4, wherein: the shock absorption structure is a sterile rubber spacer.
6. The kit for isolating and expanding umbilical cord mesenchymal stem cells for culture according to claim 1, wherein: and a coded lock is arranged at the position of the switch on the box body.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201922256670.0U CN211689096U (en) | 2019-12-16 | 2019-12-16 | Kit for separating, amplifying and culturing umbilical cord mesenchymal stem cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201922256670.0U CN211689096U (en) | 2019-12-16 | 2019-12-16 | Kit for separating, amplifying and culturing umbilical cord mesenchymal stem cells |
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| Publication Number | Publication Date |
|---|---|
| CN211689096U true CN211689096U (en) | 2020-10-16 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201922256670.0U Expired - Fee Related CN211689096U (en) | 2019-12-16 | 2019-12-16 | Kit for separating, amplifying and culturing umbilical cord mesenchymal stem cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN211689096U (en) |
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2019
- 2019-12-16 CN CN201922256670.0U patent/CN211689096U/en not_active Expired - Fee Related
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Granted publication date: 20201016 |