CN114525245B - CMM culture medium and application thereof - Google Patents
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- CN114525245B CN114525245B CN202210138639.0A CN202210138639A CN114525245B CN 114525245 B CN114525245 B CN 114525245B CN 202210138639 A CN202210138639 A CN 202210138639A CN 114525245 B CN114525245 B CN 114525245B
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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
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0668—Mesenchymal stem cells from other natural sources
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- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
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Abstract
The invention discloses a CMM culture medium and application thereof, wherein the CMM culture medium comprises a DMEM/F12 culture medium, dog platelet-rich plasma accounting for 1-10% of the volume percentage of the DMEM/F12 culture medium, 10-20 mug/L selenium element, 55-75 mg/LL-ascorbic acid, 200-500 mug/L hydrocortisone, 15-30 mg/L heparin, 450-500 mg/L NaHCO 3 And 250 to 400mg/LTransferrin. The invention also discloses application of the CMM culture medium in isolated culture of the canine umbilical cord blood mesenchymal stem cells. Compared with the umbilical cord mesenchymal stem cells obtained by the general culture method, the canine umbilical cord mesenchymal stem cells obtained by the isolated culture method have stronger stem cell activity and value-increasing capability. The canine umbilical cord mesenchymal stem cells obtained by the invention have obvious improvement effect on canine arthritis.
Description
Technical Field
The invention relates to a complete medium for mesenchymal stem cells and application thereof, in particular to a complete medium for canine umbilical cord mesenchymal stem cells and application thereof, belonging to the fields of animal medicine and biology.
Background
In veterinary experiments and in clinic, the use of stem cells in regenerative medicine and tissue engineering is rapidly emerging. On the one hand, preclinical efficacy and safety testing of new therapies of human stem cells must be assessed using two animal species, namely first rodent species (preferably rats) and second non-rodent large animals more similar to humans. Mesenchymal Stem Cells (MSCs) are generally considered to be superior to rodents in the study of therapeutic applications in human disease. In contrast to rodents, dogs can evaluate the therapeutic effect in more detail in terms of anatomy, pathogenesis, athletic performance, and larger joints, as compared to rodents and humans. On the other hand, dogs themselves are often afflicted with disease and require intervention with new therapies such as stem cells. To date, many existing treatment regimens for canine orthopedic and neurological diseases often fail to produce the desired clinical outcome or patient recovery from normal function. Therefore, MSCs hold great promise in this particular field. Indeed, a large number of MSCs have been used in veterinary and human cell therapy. In human medicine, 1-5 million cells/kg are injected intravenously or directly into tissue. In order to obtain cell numbers of this order, culture expansion is required. The mesenchymal stem cells obtained by the general culture method widely used at present often have the conditions of serious replicative senescence and stem loss, which hampers the effect of stem cell treatment research. Therefore, the development of a canine mesenchymal stem cell culture method capable of mass expansion and maintaining stem cell stem property is of great importance to the research of stem cell regeneration medicine and veterinary clinical practice.
Disclosure of Invention
The invention aims to: the first object of the invention is to provide a complete medium CMM medium for canine umbilical cord mesenchymal stem cells; the second object of the invention is to provide the application of the CMM culture medium in isolated culture of canine umbilical cord mesenchymal stem cells; the third object of the invention is to provide a method for separating and culturing the umbilical cord mesenchymal stem cells of dogs; the fourth object of the invention is to provide an application of the canine umbilical cord mesenchymal stem cells obtained by separating and culturing the CMM culture medium in preparing a medicament for treating canine arthritis.
The technical scheme is as follows: the complete culture medium of the canine umbilical cord mesenchymal stem cells is a CMM culture medium, and the CMM culture medium comprises a DMEM/F12 culture medium and canine platelet-rich plasma accounting for 1-10% of the volume of the DMEM/F12 culture medium.
Further comprises 10-20 mug/L selenium element, 55-75 mg/L L-ascorbic acid, 200-500 mug/L hydrocortisone, 15-30 mg/L heparin, 450-500 mg/L NaHCO 3 And 250-400 mg/L Transferrin (Transferrin).
Further, the extraction of the canine platelet rich plasma comprises the following steps: the anticoagulated whole blood of dogs is separated into three layers after centrifugation, wherein the upper layer is serum, the middle layer is a blood plasma layer rich in platelets, and the lower layer is a red blood cell layer; and (5) extracting the middle plasma layer under the aseptic condition to obtain the canine platelet-rich plasma.
Further, the centrifugation speed is 800-2000rpm, and the centrifugation time is 15-20min.
The invention also comprises the application of the complete culture medium of the canine umbilical cord mesenchymal stem cells in the isolated culture of the canine umbilical cord mesenchymal stem cells.
The invention also discloses a method for separating and culturing the canine umbilical cord mesenchymal stem cells, which comprises the following steps:
(1) Cleaning the dog umbilical cord with a cleaning solution, and cutting the dog umbilical cord into small sections to obtain dog umbilical cord sections;
(2) Removing blood vessels in the dog umbilical cord segments, and shearing to obtain dog umbilical cord fragments;
(3) Digesting the dog umbilical cord fragments with the digestive juice, sieving and discarding tissue blocks to obtain a cell solution;
(4) Centrifugally separating the cell solution, and re-suspending the cell solution by using a CMM (China mobile multimedia broadcasting) culture medium to obtain a cell tissue treated by the CMM culture medium;
(5) And (3) inoculating the cell tissues into a culture dish coated by Magrigel, and culturing by using the CMM culture medium to obtain the canine umbilical cord mesenchymal stem cells.
Further, in step (1), the washing solution is a PBS solution of double antibody, which is penicillin and streptomycin.
Further, in the step (1), the sheared segments are 3-5cm.
Further, in the step (3), the digestive juice is 0.2% collagenase I.
Further, in the step (3), the digestion time is 30-50min.
Further, in step (3), the sieving is a 70um cell sieve.
Further, in the step (4), the centrifugal separation speed is 1500-2000rpm, and the centrifugal separation time is 15-30min
Further, in the step (5), the cell seeding amount is 5×10 4 -5×10 5 Per mL, the inoculum size refers to 5X 10 per mL of CMM medium 4 -5×10 5 Individual cells.
The invention also comprises the application of the umbilical cord blood mesenchymal stem cells obtained by culturing the CMM culture medium and the separation culture method in preparing the medicine for treating the canine arthritis.
The beneficial effects are that: compared with the prior art, the invention has the following remarkable advantages: according to the invention, the canine platelet rich plasma is firstly applied to a culture medium to obtain the CMM culture medium. Compared with umbilical cord mesenchymal stem cells obtained by a common culture method, umbilical cord mesenchymal stem cells obtained by the isolated culture method provided by the CMM culture medium have stronger stem cell activity and value-increasing capability. The mesenchymal stem cells obtained by the culture of the CMM culture medium can better improve claudication and pain caused by arthritis and can better improve the function of repairing joints.
Drawings
FIG. 1 is a graph showing OP and CO cell doubling time under the culture system of example 6 and comparative example 5;
FIG. 2 is a diagram showing the expression states of the stem cell stem genes OCT4, NANOG and SOX2 at the 20 th generation of OP and CO;
FIG. 3 is a graph of the effect of OP on canine arthritis.
Detailed Description
The technical scheme of the invention is further described below with reference to the accompanying drawings.
EXAMPLE 1 preparation of canine platelet rich plasma
The canine platelet rich plasma was obtained by the following method: taking 5-50 ml of whole blood through a canine vein, adding heparin for anticoagulation, and centrifugally separating at 1500rpm for 15min, wherein the centrifugate is divided into three layers, namely, serum is taken as an upper layer, blood plasma layer rich in platelets is taken as a middle layer, and red blood cell layer is taken as a lower layer; and (5) extracting the middle plasma layer under the aseptic condition to obtain the canine platelet-rich plasma.
Example 2CMM Medium formulation
The specific concentration of each component of the culture medium is regulated to prepare a CMM-0 complete culture medium which comprises DMEM/F12 culture medium, dog platelet-rich plasma described in the example 1 accounting for 5 percent of the volume of the DMEM/F12 culture medium, 15 mug/L selenium element, 70 mg/LL-ascorbic acid, 200 mug/L hydrocortisone, 20mg/L heparin and 500mg/LNaHCO 3 And 400mg/L transferin.
Example 3CMM Medium formulation
The specific concentration of each component of the culture medium is regulated to prepare a CMM-1 complete culture medium which comprises DMEM/F12 culture medium, dog platelet-rich plasma accounting for 1 percent of the volume of the DMEM/F12 culture medium, 10 mug/L selenium element, 55mg/L L-ascorbic acid, 200 mug/L hydrocortisone, 15mg/L heparin and 450mg/L NaHCO 3 And 250mg/LTransferrin.
Example 4CMM Medium formulation
The specific concentration of each component of the culture medium is regulated to prepare a CMM-2 complete culture medium which comprises a DMEM/F12 culture medium, dog platelet-rich plasma accounting for 10 percent of the volume percentage of the DMEM/F12 culture medium, 20 mug/L selenium element, 75mg/L L-ascorbic acid, 500 mug/L hydrocortisone, 30mg/L heparin, 500mg/L NaHCO3 and 400mg/L Transferrin.
Example 5Matrigel coated cell culture dishes
The Matrigel (Matrigel) is dissolved in ultrapure water at 4 ℃ until the volume concentration is 0.1%, the bottom of the culture dish is uniformly covered, the culture dish is moved to 37 ℃ and kept stand for 30min, and the redundant coating liquid is sucked out for standby.
Example 6 isolated culture of canine umbilical cord mesenchymal Stem cells in CMM Environment
The isolated culture steps of the canine umbilical cord mesenchymal stem cells are as follows:
(1) After obtaining the dog umbilical cord tissue, rinsing the dog umbilical cord tissue with PBS solution containing double antibody (penicillin 200U/mL, streptomycin 0.2 mg/mL), and cutting the dog umbilical cord tissue into 3-5cm small segments;
(2) After separating the blood vessel, rinsing the dog umbilical cord tissue, and shearing the dog umbilical cord tissue to 2mm by using an ophthalmic scissors 3 Left and right;
(3) Digesting with 0.2% collagenase I at 37deg.C for 50min, sieving with 70 μm cell sieve, and discarding tissue block;
(4) Centrifuging at 1500rpm for 15min, discarding supernatant, and culturing with CMM-0 complete medium, CMM-1 complete medium, and CMM-2 complete medium obtained in examples 2-4 at 5×10, respectively 5 Density of individual cells/mL was resuspended;
(5) Inoculated onto Matrigel-coated 10cm prepared in example 5 2 The culture dish is changed every three days, and the cells climb out; and (3) until the cells are fused, carrying out passage, wherein the passage method is similar to the digestion passage method of general cells, and culturing the cells in a Matrigel coated culture plate respectively by using a corresponding complete culture medium of CMM-0, a complete culture medium of CMM-1 and a complete culture medium of CMM-2 after passage, thereby obtaining the canine umbilical cord mesenchymal stem cells respectively called OP, OP1 and OP2.
Comparative example 1 preparation of common Medium (COM-B)
Common media were prepared, including DMEM/F12 medium and 10% by volume Fetal Bovine Serum (FBS).
Comparative example 2 preparation of modified Medium
COM-0 complete medium comprising DMEM/F12 medium and 5% fbs by volume of DMEM/F12 medium, and further comprising 15 μg/L selenium element, 70 mg/LL-ascorbic acid, 200 μg/L hydrocortisone, 20mg/L heparin, 500mg/LNaHCO3 and 400mg/L Transferrin.
Comparative example 3 preparation of improved Medium
COM-1 complete medium comprising DMEM/F12 medium and 1% FBS in the volume of DMEM/F12 medium, 10 μg/L selenium element, 55mg/L L-ascorbic acid, 200 μg/L hydrocortisone, 15mg/L heparin, 450mg/L NaHCO 3 And 250mg/LTransferrin.
Comparative example 4 preparation of modified Medium
COM-2 complete medium comprising DMEM/F12 medium and 10% FBS,20 μg/L selenium element, 75mg/L L-ascorbic acid, 500 μg/L hydrocortisone, 30mg/L heparin, 500mg/L NaHCO3 and 400 mg/LTransferin by volume percentage of DMEM/F12 medium.
Comparative example 5 general culture method and isolated culture method of canine umbilical cord mesenchymal stem cells under improved environment canine umbilical cord mesenchymal stem cells were obtained in the general culture method as a control for comparative analysis:
(1) After obtaining the canine umbilical cord tissue, rinsing the canine umbilical cord tissue with PBS containing double antibody (penicillin 200U/mL, streptomycin 0.2 mg/mL), and cutting the canine umbilical cord tissue into 3-5cm small segments;
(2) After separating the blood vessel, rinsing the dog umbilical cord tissue, and shearing the dog umbilical cord tissue to 2mm by using an ophthalmic scissors 3 Left and right;
(3) Digesting with 0.2% collagenase I at 37deg.C for 50min, sieving with 70 μm cell sieve, and discarding tissue block;
(4) Centrifuging at 1500rpm for 15min, discarding supernatant, and concentrating at 5×10 5 Density of individual cells/mL COM-B, COM-0, COM-1, COM-2 prepared in comparative examples 1-4 were resuspended and inoculated, inoculated into Matrigel-coated dishes prepared in example 5, and the solution was changed every three days until the cells crawled out. After passage to cell fusion, the passage was carried out in the same manner as in the digestion passage method of ordinary cells, and corresponding COM-B, COM-0, COM-1 and COM-2 media were used in Matrigel-coated dishes prepared in example 5 after passage, whereby canine umbilical cord mesenchymal stem cells were obtained, which were designated as CO, CO1, CO2 and CO3, respectively.
By recording the cell number, the proliferation speed of any cell line in the CMM culture environment is higher than that of the cell line in the COM culture environment. Wherein, the proliferation speed of the CO cell strain is highest in four cells of CO, CO1, CO2 and CO3, and the proliferation speed of the OP cell strain is highest in three cells of OP, OP1 and OP2. We therefore consider the comparison of CO and OP as examples only.
The cell doubling time in the culture systems of example 6 and comparative example 5 is shown in FIG. 1, and FIG. 1 is a graph of OP and CO cell doubling time in the culture systems of example 6 and comparative example 5. As can be seen from FIG. 1, in the culture environment provided by the present invention, OP cell proliferation reaches 35 generations, only a slight extension of doubling time occurs, whereas in the method of comparative example 5, doubling time reaches 67 hours at 25 th CO generation, and signs of aging appear; indicating that CMM medium maintained better cell activity and state of proliferation for a long period of time.
EXAMPLE 7 real-time quantitative PCR detection of mesenchymal Stem cell Stem Gene expression level
Still, the total RNA kit was used to extract total RNA from both OP and CO cells of comparative example 5 of example 6 by comparing the best OP and CO cells under CMM culture conditions with the best CO cells under ordinary COM culture conditions, and then the expression of stem cell stem genes such as SOX2, OCT4 and NANOG, and bone differentiation genes ALP and RUNX2 were examined and compared, and β2 MICROGLOBULIN (MICROGLOBULIN) was used as a reference. The quantitative PCR reaction was performed using a one-step RT-qPCR kit (LM-0051, shanghai-Haimai bioengineering Co., ltd.) and the reaction system was established as required in the specification. The reaction conditions are as follows: firstly, after the treatment is completed for 5 minutes at 94 ℃, the reaction cycle is started. The circulation conditions are as follows: 94℃for 30 seconds, 55℃for 30 seconds, 68℃for 2 minutes, 30 cycles. And finally, treating at 68 ℃ for 10 minutes. The primers used were respectively:
OCT4：
Forward:GAGTGAGAGGCAACCTGGAG
Reverse:GTGAAGTGAGGGCTCCCATA
NANOG：
Forward:GAATAACCCGAATTGGAGCAG
Reverse:AGCGATTCCTCTTCACAGTTG
SOX2：
Forward：AGTCTCCAAGCGACGAAAAA
Reverse：GCAAGAAGCCTCTCCTTGAA
ALP：
Forward：TCAACAGACCCTGAAATACGC
Reverse：TCTTGGAGAGGGCCACGTAAG
RUNX2：
Forward：GTCTCCTTCCAGAATGCTTCC
Reverse：GGAACTGAGGATGAGGAGAC
B2 MICROGLOBULIN：
Forward：TCTACATTGGGCACTGTGTCAC
Reverse：AAGAGTTCAGGTCTGACCAAG
the expression states of stem cell stem gene OCT4, NANOG, SOX2 at the 20 th generation of OP of example 6 and CO of comparative example 5 are shown in fig. 2. FIG. 2 is a diagram showing the expression states of stem cell stem gene OCT4, NANOG, SOX2 at the 20 th generation of OP and CO, wherein the expression levels of the above genes are 1.4 times, 1.3 times and 1.8 times, respectively, of the expression level in OP as shown in FIG. 2; whereas the expression of the osteodifferentiation genes ALP and RUNX2 in OP is 0.9 and 0.7 times that of CO. The results show that the OP has stronger stem cell characteristics, and the lower CO stem level tends to differentiate into bone, thereby proving that the OP maintains better cell activity and proliferation state for a long time.
Example 8OP effect on canine arthritis
Dogs meeting osteoarthritis were divided into 3 groups of 10 animals each. The first group is a control group, and 1 milliliter of physiological saline is injected; the second group is OP group, and the affected part of OP mesenchymal stem cells is injected 1 time a week with 1 ml containing 10 7 Physiological saline of OP cells for 3 times as 1 treatment course; the third group is CO group, and CO mesenchymal stem cells are injected into affected part 1 ml/week with 10 7 Physiological saline of each cell, 3 times is 1 treatment course. After the end of one treatment session, day 90 was scored on the following functional scale, see in particular table 1.
TABLE 1
| Lameness of claudication | Pain and pain | Joint function |
| 1 branch-normal | 1 part-painless pain | 1 branch-normal |
| 2 minutes-intermittently | 2 points-slight pain | 2 points-slightly stiff |
| 3 minutes-continuous | 3 minutes-obvious pain | 3 minutes-stiffness |
| 4 minutes-cannot bear weight to walk | 4 minutes-obvious pain | 4 minutes-very stiff, reluctant to walk |
| 5 minutes-unable to walk independently | 5 minutes-obvious pain | 5 minutes-unable to walk independently |
The specific experimental results are shown in fig. 3, and fig. 3 is a graph of OP effect on canine arthritis. As shown in fig. 3, compared with CO mesenchymal stem cells, application of OP mesenchymal stem cells can better improve lameness and pain caused by arthritis, and can better improve the function of repairing joints.
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Claims (2)
1. A method for separating and culturing umbilical cord mesenchymal stem cells of dogs is characterized by utilizing a CMM culture medium, wherein the CMM culture medium consists of a DMEM/F12 culture medium, dog platelet-rich plasma accounting for 1-10% of the volume percentage of the DMEM/F12 culture medium, 10-20 mug/L selenium element, 55-75 mg/L L-ascorbic acid, 200-500 mug/L hydrocortisone, 15-30 mg/L heparin, 450-500 mg/L NaHCO3 and 250-400 mg/L transferrin; the separation culture method comprises the following steps:
(1) Rinsing the dog umbilical cord with PBS solution containing double antibody, and cutting into 3-5cm segments to obtain dog umbilical cord segments, wherein the double antibody in the PBS solution is 200U/mL penicillin and 0.2mg/mL streptomycin;
(2) Removing blood vessel in dog umbilical cord segment, and cutting to 2mm 3 Obtaining dog umbilical cord fragments from left to right;
(3) Digesting for 50min at 37 ℃ by using 0.2% collagenase I, sieving with a 70 mu m cell sieve, and discarding tissue blocks to obtain a cell solution;
(4) Centrifuging the cell solution at 1500rpm for 15min, discarding the supernatant, and concentrating at 5×10 5 Resuspension of individual cells/mL in CMM medium to obtain CMM medium treated cell tissue;
(5) And (3) inoculating the cell tissues into a culture dish coated by Magrigel, changing the liquid every three days, and culturing with a CMM culture medium after passage until the cells climb out until the cells are fused, and carrying out passage.
2. The method for isolated culture of canine umbilical cord mesenchymal stem cells according to claim 1, wherein the extraction of canine platelet-rich plasma comprises the steps of: and (3) separating the anticoagulated whole blood of the dogs into three layers, and extracting a middle plasma layer under the aseptic condition to obtain the platelet-rich plasma of the dogs.
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