CN111979193A - Rat bone marrow-derived mast cell culture method - Google Patents
Rat bone marrow-derived mast cell culture method Download PDFInfo
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0642—Granulocytes, e.g. basopils, eosinophils, neutrophils, mast cells
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/22—Colony stimulating factors (G-CSF, GM-CSF)
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2303—Interleukin-3 (IL-3)
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
Abstract
The invention discloses a method for culturing rat bone marrow-derived mast cells, which comprises the following steps: killing SD young rats born for 3-5 weeks by neck-removing method, soaking in 75% alcohol for 3min for sterilization, transferring into an ultra-clean workbench, cutting skin of hind limb, removing muscle of leg to expose femur, peeling off external muscle of femur, and cutting femur; cutting off femoral heads at two ends of a femur, sucking a DMEM/F12 basic culture medium by using an injector, blowing out femoral bone marrow, and washing the bone marrow in the blowing process; using a Pasteur pipette to blow and beat the bone marrow for 30 times to prepare single cell suspension, centrifuging through a centrifugal machine, collecting cells, and discarding supernatant; then adding erythrocyte lysate for lysis for 3min at 4 ℃. According to the invention, the mast cells are cultured and centrifugally separated for many times by the mast cell induction culture medium and the mast cell maturation promoting culture medium, so that the purity of the cultured mast cells is higher, and the culture period is greatly shortened.
Description
Technical Field
The invention relates to the technical field of cell culture, in particular to a method for culturing rat bone marrow-derived mast cells.
Background
Mast cells are widely distributed around microvessels under skin and visceral mucosa, secrete various cytokines, participate in immune regulation, express MHC molecules, B7 molecules, have APC function, express a large amount of IgE Fc receptors, release allergic mediators, have weak phagocytosis function, and like basophils of blood, histocytes with strong basophils, in the past, researches on the mast cells mostly focus on the effect of the mast cells in hypersensitivity diseases, but since the fact that mast cell defect mice are difficult to induce skin transplantation tolerance for the first time in 2006, the transplantation tolerance of MC normal mice reconstructed by infusing bone marrow-derived MC is induced, the key effect of the mast cells in organ transplantation tolerance is proved, but the mast cells cultured by the existing rat bone marrow-derived mast cell culture method have low purity and long culture period, and the experimental progress is influenced, so the prior art needs to be improved, to solve the above problems.
Disclosure of Invention
The invention aims to provide a method for culturing rat bone marrow-derived mast cells, which aims to solve the problems that the mast cells cultured by the existing method for culturing rat bone marrow-derived mast cells in the background art have low purity, long culture period and influence on the experimental progress.
In order to achieve the purpose, the invention provides the following technical scheme: a method of culturing rat bone marrow-derived mast cells, the method comprising the steps of:
s1: killing SD young rats born for 3-5 weeks by neck-removing method, soaking in 75% alcohol for 3min for sterilization, transferring into an ultra-clean workbench, cutting skin of hind limb, removing muscle of leg to expose femur, peeling off external muscle of femur, and cutting femur;
s2: cutting off femoral heads at two ends of a femur, sucking a DMEM/F12 basic culture medium by using an injector, blowing out femoral bone marrow, and washing the bone marrow in the blowing process;
s3: using a Pasteur pipette to blow and beat the bone marrow for 30 times to prepare single cell suspension, centrifuging through a centrifugal machine, collecting cells, and discarding supernatant;
s4: adding erythrocyte lysate for lysis for 3min at 4 ℃, centrifuging by a centrifuge, collecting cells, discarding supernatant, absorbing mast cell induction culture medium to resuspend cell sap, and cleaning to remove residual erythrocyte lysate;
s5: sucking the mast cell induction culture medium through a syringe to resuspend the cell fluid, inoculating the cell fluid into a T25 bottle for culture, changing the fluid once every 3 days in the culture process, and collecting cells rounded and shed in the culture medium when the fluid is changed to 8 th time, namely immature mast cells;
S6: transferring a culture medium containing immature mast cells into a centrifuge tube, centrifuging by a centrifuge, collecting cells after centrifugation is finished, then sucking the mast cells by using a syringe to promote mature culture medium to resuspend cell fluid, inoculating the cell fluid into a T25 bottle for culture, changing the fluid once every 3 days during the culture process, and culturing the changed fluid for 5 times to obtain the differentiated mature mast cells.
Preferably, the specific operation method of the necking method in S1 is set as follows: the neck of the rat is clamped by forceps with the left hand, the tail of the rat is grasped by the right hand, and the cervical vertebrae of the rat are pulled off by the two hands simultaneously.
Preferably, the rotation speed of the centrifuges in the S3 and the S4 is set to be 1000rmp, and the centrifugation time is set to be 5 min.
Preferably, the mast cell induction medium in S4 and S5 is formulated as follows: taking a certain amount of DMEM/F12 basic culture medium from a clean bottle, adding FBS, 50U/ml penicillin, 100U/ml mitomycin, 0.07% beta-mercaptoethanol, 20ng/ml CSF and 30ng/ml IL-3 into the bottle, filtering the mixture by a filter, transferring the mixture into a new bottle, and storing the new bottle in a refrigerator at 4 ℃ for later use.
Preferably, the preparation method of the culture medium for promoting maturation of mast cells in S6 is set as follows: a certain amount of 1640 basic culture medium is taken from an ultra-clean workbench, and is added with FBS, 1ml double antibody, 0.07% beta-mercaptoethanol, 30ng/ml CSF and 50ng/ml IL-3 in a sterile and clean bottle, and the mixture is filtered by a filter and then transferred to a new bottle and is stored in a refrigerator at 4 ℃ for later use.
Preferably, the rotation speed of the centrifuge in the S6 is set to 1200rmp, and the centrifugation time is set to 5 min.
Compared with the prior art, the invention has the beneficial effects that.
(1) According to the method, the mast cells are cultured in the mast cell induction culture medium and the mast cell maturation promoting culture medium in sequence, a large amount of high-purity mast cells can be obtained quickly and economically, the structural characteristics of the surface and the inner membrane of the cultured mast cells are observed through a light mirror and an electron microscope, the mast cells are uniform in size, the internal structure of the mast cells conforms to the characteristics of the mast cells, and the method is subjected to two steps of induction and maturation promoting, so that the mature time of the cultured mast cells is short, the service life of the cultured mast cells is long, and the maturation purity of the cultured mast cells is high.
(2) In the culture process, the cells in the rat bone marrow can be completely separated by adding the erythrocyte lysate in a certain proportion and performing multiple centrifugal separation operations, so that the resource utilization rate is high, the separation purity is higher, and the purity of the cultured mast cells is also improved.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all 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.
The invention provides the following examples:
example 1
The method for culturing rat bone marrow-derived mast cells comprises the following steps:
s1: killing SD young rats born for 3-5 weeks by neck-removing method, soaking in 75% alcohol for 3min for sterilization, transferring into an ultra-clean workbench, cutting skin of hind limb, removing muscle of leg to expose femur, peeling off external muscle of femur, and cutting femur;
s2: cutting off femoral heads at two ends of a femur, sucking a DMEM/F12 basic culture medium by using a 5ml syringe, blowing out femoral bone marrow, and washing the bone marrow in the blowing process;
s3: using a Pasteur pipette to blow and beat the bone marrow for 30 times to prepare single cell suspension, centrifuging through a centrifugal machine, collecting cells, and discarding supernatant;
s4: adding 3ml of erythrocyte lysate for lysis for 3min at 4 ℃, centrifuging by a centrifuge, collecting cells, removing supernatant, sucking 2ml of mast cell induction culture medium for resuspension of cell lysate, and cleaning to remove residual erythrocyte lysate;
s5: sucking 5ml of mast cell induction culture medium through a syringe to resuspend cell sap, inoculating the cell sap into a T25 bottle for culture, changing the cell sap once every 3 days in the culture process, and collecting cells rounded and shed in the culture medium when the cell sap is changed to 8 th time, namely immature mast cells;
S6: transferring a culture medium containing immature mast cells into a 15ml centrifuge tube, centrifuging by a centrifuge, collecting cells after centrifugation is finished, then sucking 7ml of mast cells by using a syringe to promote mature culture medium to resuspend cell sap, inoculating the cell sap into a T25 bottle for culture, changing the cell sap once every 3 days during culture, and obtaining the differentiated mature mast cells after 5 times of culture.
Further, the specific operation method of the neck removal method in S1 is set as follows: the neck of the rat is clamped by forceps with the left hand, the tail of the rat is grasped by the right hand, and the cervical vertebrae of the rat are pulled off by the two hands simultaneously.
Further, the rotation speed of the centrifuge in each of S3 and S4 was set to 1000rmp, and the centrifugation time was set to 5 min.
Further, the formulation method of mast cell induction medium in S4 and S5 was set up as follows: taking 84ml DMEM/F12 basic culture medium in a clean bottle, adding 15ml FBS, 50U/ml penicillin, 100U/ml mitomycin, 0.07% beta-mercaptoethanol, 20ng/ml CSF, 30ng/ml IL-3, filtering by a 0.22um filter, transferring into a new bottle, storing at 4 ℃ in a refrigerator for later use, wherein the specific liquid volume value can be scaled proportionally to prepare the culture medium with the required volume.
Further, the formulation of the mast cell maturation-promoting medium in S6 is set as follows: taking 84ml 1640 basic culture medium in an ultra-clean workbench, adding 15ml FBS, 1ml double antibody, 0.07% beta-mercaptoethanol, 30ng/ml CSF, 50ng/ml IL-3 and 0.22um filter, filtering, transferring to a new bottle, storing in a refrigerator at 4 ℃ for later use, wherein the specific liquid volume value can be scaled proportionally, and preparing the culture medium with the required volume.
Further, the rotation speed of the centrifuge in S6 was set to 1200rmp, and the centrifugation time was set to 5 min.
Example 2
The method for culturing rat bone marrow-derived mast cells comprises the following steps:
s1: killing SD young rats born for 3-5 weeks by neck-removing method, soaking in 75% alcohol for 3min for sterilization, transferring into an ultra-clean workbench, cutting skin of hind limb, removing muscle of leg to expose femur, peeling off external muscle of femur, and cutting femur;
s2: cutting off femoral heads at two ends of a femur, sucking a DMEM/F12 basic culture medium by using a 10ml syringe, blowing out femoral bone marrow, and washing the bone marrow in the blowing process;
s3: using a Pasteur pipette to blow and beat the bone marrow for 30 times to prepare single cell suspension, centrifuging through a centrifugal machine, collecting cells, and discarding supernatant;
S4: adding 6ml of erythrocyte lysate for lysis at 4 ℃ for 3min, centrifuging by a centrifuge, collecting cells, removing supernatant, sucking 4ml of mast cell induction culture medium for resuspension of cell lysate, and cleaning to remove residual erythrocyte lysate;
s5: sucking 10ml of mast cell induction culture medium through a syringe to resuspend cell sap, inoculating the cell sap into a T25 bottle for culture, changing the cell sap once every 3 days in the culture process, and collecting cells which are rounded and shed in the culture medium when the cell sap is changed to 8 th time, namely immature mast cells;
s6: transferring a culture medium containing immature mast cells into a 30ml centrifuge tube, centrifuging by a centrifuge, collecting cells after centrifugation is finished, sucking 14ml of mast cells by using a syringe to promote mature culture medium to resuspend cell sap, inoculating the cell sap into a T25 bottle for culture, changing the cell sap once every 3 days during culture, and culturing the cell sap for 5 times to obtain the differentiated mature mast cells.
Further, the specific operation method of the neck removal method in S1 is set as follows: the neck of the rat is clamped by forceps with the left hand, the tail of the rat is grasped by the right hand, and the cervical vertebrae of the rat are pulled off by the two hands simultaneously.
Further, the rotation speed of the centrifuge in each of S3 and S4 was set to 1000rmp, and the centrifugation time was set to 5 min.
Further, the formulation method of mast cell induction medium in S4 and S5 was set up as follows: 84ml of DMEM/F12 basic culture medium is taken from a clean bottle in a clean bench, 15ml of FBS, 50U/ml of penicillin, 100U/ml of mitomycin, 0.07 percent of beta-mercaptoethanol, 20ng/ml of CSF, 30ng/ml of IL-3 and 0.22um of filter are added, and the mixture is transferred into a new bottle after being filtered, and is stored in a refrigerator at 4 ℃ for standby.
Further, the formulation of the mast cell maturation-promoting medium in S6 is set as follows: 84ml 1640 basic culture medium is taken from an ultra-clean workbench, and is added with 15ml FBS, 1ml double antibody, 0.07% beta-mercaptoethanol, 30ng/ml CSF, 50ng/ml IL-3 and 0.22um filter, and then is transferred into a new bottle after being filtered, and is stored in a refrigerator at 4 ℃ for standby.
Further, the rotation speed of the centrifuge in S6 was set to 1200rmp, and the centrifugation time was set to 5 min.
Example 3
The method for culturing rat bone marrow-derived mast cells comprises the following steps:
s1: killing SD young rats born for 3-5 weeks by neck-removing method, soaking in 75% alcohol for 3min for sterilization, transferring into an ultra-clean workbench, cutting skin of hind limb, removing muscle of leg to expose femur, peeling off external muscle of femur, and cutting femur;
S2: cutting off femoral heads at two ends of a femur, sucking a DMEM/F12 basic culture medium by using a 15ml syringe, blowing out femoral bone marrow, and washing the bone marrow in the blowing process;
s3: using a Pasteur pipette to blow and beat the bone marrow for 30 times to prepare single cell suspension, centrifuging through a centrifugal machine, collecting cells, and discarding supernatant;
s4: adding 9ml of erythrocyte lysate for lysis at 4 ℃ for 3min, centrifuging by a centrifuge, collecting cells, removing supernatant, sucking 6ml of mast cell induction culture medium for resuspension of cell lysate, and cleaning to remove residual erythrocyte lysate;
s5: sucking 15ml of mast cell induction culture medium through a syringe to resuspend cell sap, inoculating the cell sap into a T25 bottle for culture, changing the cell sap once every 3 days in the culture process, and collecting cells which are rounded and shed in the culture medium when the cell sap is changed to 8 th time, namely immature mast cells;
s6: transferring a culture medium containing immature mast cells into a 45ml centrifuge tube, centrifuging by a centrifuge, collecting cells after centrifugation is finished, sucking 21ml of mast cells by using a syringe to promote mature culture medium to resuspend cell sap, inoculating the cell sap into a T25 bottle for culture, changing the cell sap once every 3 days during culture, and culturing the cell sap for 5 times to obtain the differentiated mature mast cells.
Further, the specific operation method of the neck removal method in S1 is set as follows: the neck of the rat is clamped by forceps with the left hand, the tail of the rat is grasped by the right hand, and the cervical vertebrae of the rat are pulled off by the two hands simultaneously.
Further, the rotation speed of the centrifuge in each of S3 and S4 was set to 1000rmp, and the centrifugation time was set to 5 min.
Further, the formulation method of mast cell induction medium in S4 and S5 was set up as follows: 84ml of DMEM/F12 basic culture medium is taken from a clean bottle in a clean bench, 15ml of FBS, 50U/ml of penicillin, 100U/ml of mitomycin, 0.07 percent of beta-mercaptoethanol, 20ng/ml of CSF, 30ng/ml of IL-3 and 0.22um of filter are added, and the mixture is transferred into a new bottle after being filtered, and is stored in a refrigerator at 4 ℃ for standby.
Further, the formulation of the mast cell maturation-promoting medium in S6 is set as follows: 84ml 1640 basic culture medium is taken from an ultra-clean workbench, and is added with 15ml FBS, 1ml double antibody, 0.07% beta-mercaptoethanol, 30ng/ml CSF, 50ng/ml IL-3 and 0.22um filter, and then is transferred into a new bottle after being filtered, and is stored in a refrigerator at 4 ℃ for standby.
Further, the rotation speed of the centrifuge in S6 was set to 1200rmp, and the centrifugation time was set to 5 min.
The working principle is as follows: the rat bone marrow cells are centrifugally separated, then are sequentially cultured by a mast cell induction culture medium and a mast cell maturation promoting culture medium, and the liquid is continuously changed in the culture process, can quickly and economically obtain a large amount of high-purity mast cells, the structural characteristics of the surface and the inner membrane of the cultured mast cells are observed through a light mirror and an electron microscope, the mast cells are observed to be uniform in size, the internal structure of the mast cells conforms to the characteristics of the mast cells, the method has two steps of induction and ripening, so that the mature time of the cultured mast cells is short, and the service life is longer, the purity of mature cells is higher, by adding a certain proportion of erythrocyte lysate and multiple centrifugal separation operations, the cells in the rat bone marrow can be completely separated, the resource utilization rate is high, the separation purity is higher through multiple times of centrifugation, and the purity of the cultured mast cells is also improved.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
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 (6)
1. The method for culturing rat bone marrow-derived mast cells is characterized by comprising the following steps: the method comprises the following steps:
s1: killing SD young rats born for 3-5 weeks by neck-removing method, soaking in 75% alcohol for 3min for sterilization, transferring into an ultra-clean workbench, cutting skin of hind limb, removing muscle of leg to expose femur, peeling off external muscle of femur, and cutting femur;
S2: cutting off femoral heads at two ends of a femur, sucking a DMEM/F12 basic culture medium by using an injector, blowing out femoral bone marrow, and washing the bone marrow in the blowing process;
s3: using a Pasteur pipette to blow and beat the bone marrow for 30 times to prepare single cell suspension, centrifuging through a centrifugal machine, collecting cells, and discarding supernatant;
s4: adding erythrocyte lysate for lysis for 3min at 4 ℃, centrifuging by a centrifuge, collecting cells, discarding supernatant, absorbing mast cell induction culture medium to resuspend cell sap, and cleaning to remove residual erythrocyte lysate;
s5: sucking the mast cell induction culture medium through a syringe to resuspend the cell fluid, inoculating the cell fluid into a T25 bottle for culture, changing the fluid once every 3 days in the culture process, and collecting cells rounded and shed in the culture medium when the fluid is changed to 8 th time, namely immature mast cells;
s6: transferring a culture medium containing immature mast cells into a centrifuge tube, centrifuging by a centrifuge, collecting cells after centrifugation is finished, then sucking the mast cells by using a syringe to promote mature culture medium to resuspend cell fluid, inoculating the cell fluid into a T25 bottle for culture, changing the fluid once every 3 days during the culture process, and culturing the changed fluid for 5 times to obtain the differentiated mature mast cells.
2. The method for culturing rat bone marrow-derived mast cells according to claim 1, wherein: the specific operation method of the neck removing method in the S1 is as follows: the neck of the rat is clamped by forceps with the left hand, the tail of the rat is grasped by the right hand, and the cervical vertebrae of the rat are pulled off by the two hands simultaneously.
3. The method for culturing rat bone marrow-derived mast cells according to claim 1, wherein: the rotating speed of the centrifuges in the S3 and the S4 is set to be 1000rmp, and the centrifugation time is set to be 5 min.
4. The method for culturing rat bone marrow-derived mast cells according to claim 1, wherein: the preparation method of the mast cell induction culture medium in the S4 and the S5 is set as follows: taking a certain amount of DMEM/F12 basic culture medium from a clean bottle, adding FBS, 50U/ml penicillin, 100U/ml mitomycin, 0.07% beta-mercaptoethanol, 20ng/ml CSF and 30ng/ml IL-3 into the bottle, filtering the mixture by a filter, transferring the mixture into a new bottle, and storing the new bottle in a refrigerator at 4 ℃ for later use.
5. The method for culturing rat bone marrow-derived mast cells according to claim 1, wherein: the preparation method of the culture medium for promoting maturation of mast cells in S6 is as follows: a certain amount of 1640 basic culture medium is taken from an ultra-clean workbench, and is added with FBS, 1ml double antibody, 0.07% beta-mercaptoethanol, 30ng/ml CSF and 50ng/ml IL-3 in a sterile and clean bottle, and the mixture is filtered by a filter and then transferred to a new bottle and is stored in a refrigerator at 4 ℃ for later use.
6. The method for culturing rat bone marrow-derived mast cells according to claim 1, wherein: the rotating speed of the centrifuge in the S6 is set to be 1200rmp, and the centrifugation time is set to be 5 min.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2004215581A (en) * | 2003-01-15 | 2004-08-05 | Univ Hiroshima | New mast cell strain and method using the same strain |
| CN1575341A (en) * | 2001-10-22 | 2005-02-02 | 阿文蒂斯药物股份有限公司 | Method for preparing heparin from mast cell cultures |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1575341A (en) * | 2001-10-22 | 2005-02-02 | 阿文蒂斯药物股份有限公司 | Method for preparing heparin from mast cell cultures |
| JP2004215581A (en) * | 2003-01-15 | 2004-08-05 | Univ Hiroshima | New mast cell strain and method using the same strain |
Non-Patent Citations (3)
| Title |
|---|
| STEFFENK. MEURER 等: "\"Isolation of Mature (Peritoneum-Derived) Mast Cells and Immature (Bone Marrow-Derived) Mast Cell Precursors from Mice\"" * |
| TIANYU YU 等: "\"The development of methods for primary mast cells in vitro and ex vivo: An historical review\"" * |
| 邵亦心 等: ""小鼠腹腔及骨髓源性肥大细胞的培养与鉴定"" * |
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