A placenta-umbilical cord mesenchymal stem cell culture composition and preparation method thereof

Abstract

The present invention relates to the field of cell culture technology, and specifically provides a placenta-umbilical cord mesenchymal stem cell culture composition and a preparation method thereof, wherein the placenta-umbilical cord mesenchymal stem cell culture composition is composed of a Chinese medicine extract having a mass fraction of 25-33% and a DMEM culture medium that makes up the surplus. The specific proportions of deer placenta, astragalus, salvia miltiorrhiza, snow lotus, antler glue, epimedium, saffron and dalbergia odorifera of the present invention cooperate with each other, according to the unique extraction and preparation process of the present invention, and then mixed with DMEM culture medium according to the proportion of the present invention, can simultaneously enhance the proliferation ability of placenta and umbilical cord mesenchymal stem cells and resist differentiation.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, and in particular to a placenta-umbilical cord mesenchymal stem cell culture composition and a preparation method thereof. Background Art

[0002] Mesenchymal stem cells (MSCs) are a type of adult stem cells that are widely found in various tissues throughout the body. They can be cultured and expanded in large quantities in vitro and differentiate into cells from the inner, middle and outer germ layers under specific conditions. They have potential application value in hematopoietic cell transplantation, organ transplantation, bone and cartilage tissue repair, myocardial infarction and liver damage. Compared with mesenchymal stem cells derived from bone marrow, the advantages of mesenchymal stem cells derived from placenta and umbilical cord are: the collection process is relatively simple and non-invasive, the placental barrier reduces the contamination of the umbilical cord with viruses and bacteria, and the primitiveness of the umbilical cord blood immune system reduces the chance of rejection of the recipient after transplantation.

[0003] However, umbilical cord mesenchymal stem cells and placental mesenchymal stem cells are similar in morphology, both are long spindle-shaped, and there is no apparent difference between them. Both have very strong proliferation capabilities and can be expanded for more than 30 generations. They are easily mixed when collected, difficult to distinguish after collection, and require complex separation methods. Because both are perinatal tissues with similar capabilities, there is generally no need to separate them when used, which requires an environment where the two are mixed and cultured.

[0004] The existing technology does not focus on the problem of mixed culture of placenta-umbilical cord mesenchymal stem cells. Therefore, it is of great practical significance to provide a culture composition of placenta-umbilical cord mesenchymal stem cells with strong proliferation ability and resistance to differentiation and a preparation method thereof. Summary of the invention

[0005] In order to solve the above problems, on the one hand, the present invention provides a method for preparing a placenta-umbilical cord mesenchymal stem cell culture composition, wherein the method for preparing the placenta-umbilical cord mesenchymal stem cell culture composition is:

[0006] Take 23-29% by weight of deer fetus, 5-8% by weight of astragalus, 4-7% by weight of salvia miltiorrhiza, 13-15% by weight of snow lotus, 11-15% by weight of antler glue, 6-9% by weight of epimedium, 4-7% by weight of saffron, 2-4% by weight of dalbergia odorifera and make up the balance of pure water, add 80% ethanol with a mass of 10 times the mass of the above 8 medicinal materials, soak at 50°C for 24h, then add into a flash extractor with a speed of 6800r / min, extract for 30s and filter to obtain a coarse filtration extract; distill the coarse filtration extract under reduced pressure at 72°C and a vacuum degree of 800mbar until no liquid drips continuously, collect the distillation residue, add activated carbon for injection at 0.005g / ml, sterilize at 115°C for 30min, filter through a titanium rod filter when cooled to 30°C, and obtain a traditional Chinese medicine extract;

[0007] The traditional Chinese medicine extract having a mass fraction of 25-33% and the DMEM culture medium making up the balance are mixed to obtain the placenta-umbilical cord mesenchymal stem cell culture composition.

[0008] Furthermore, the mass fraction of the deer fetus is 26%.

[0009] Furthermore, the mass fraction of the astragalus is 7%.

[0010] Furthermore, the mass fraction of the Salvia miltiorrhiza is 6%.

[0011] Furthermore, the mass fraction of the snow lotus is 14%.

[0012] Furthermore, the mass fraction of the antler glue is 13%.

[0013] Furthermore, the mass fraction of the epimedium is 8%.

[0014] Furthermore, the mass fraction of the saffron is 6%.

[0015] Furthermore, the mass fraction of the dalbergia wood is 3%.

[0016] On the other hand, the present invention provides a placenta-umbilical cord mesenchymal stem cell culture composition prepared by the preparation method of the placenta-umbilical cord mesenchymal stem cell culture composition as described above, wherein the placenta-umbilical cord mesenchymal stem cell culture composition consists of 25-33% by weight of the traditional Chinese medicine extract and DMEM culture medium to make up the balance.

[0017] The present invention can bring the following beneficial effects:

[0018] Zhang Aiguo et al. mentioned the promoting effect of various Chinese medicines on stem cell proliferation in "Effects of Four Chinese Medicine Decoctions and Snow Lotus Alcohol Extract on the Proliferation Activity of Bone Marrow Mesenchymal Stem Cells" and "Screening Study on the Effects of Five Chinese Medicines (or Prescriptions)-Containing Serum on the Proliferation Activity of Bone Marrow Mesenchymal Stem Cells in Vitro", and the addition of Chinese medicines may also promote the differentiation of stem cells. The present invention further studies the culture composition and its preparation method that simultaneously enhance the proliferation ability of placenta and umbilical cord mesenchymal stem cells and resist differentiation in the environment of mixed culture of placenta-umbilical cord mesenchymal stem cells.

[0019] Combined with the experimental data of the present invention, the specific proportions of deer placenta, astragalus, salvia miltiorrhiza, snow lotus, antler glue, epimedium, saffron and dalbergia odorifera in the present invention work together, according to the unique extraction and preparation process of the present invention, and then mixed with DMEM culture medium in the proportions of the present invention, can simultaneously enhance the proliferation ability of placental and umbilical cord mesenchymal stem cells and resist differentiation. DETAILED DESCRIPTION

[0020] In order to more clearly explain the overall concept of the present invention, the overall scheme of the present invention is described in detail in the form of embodiments below; in the following description, a large number of specific details are given to provide a more thorough understanding of the present invention; however, it is obvious to those skilled in the art that the present invention can be implemented without one or more of these details; in other examples, in order to avoid confusion with the present invention, some technical features known in the art are not described.

[0021] In the present invention, placenta and umbilical cord mesenchymal stem cells were purchased from Hefei Wanwu Biotechnology Co., Ltd., with the product numbers Delf-10765 and Delf-10770, and the viable cell density was 5×10 5 / ml; DMEM culture medium was purchased from Shenzhen Haozhi Biotechnology Co., Ltd., model XB01, high sugar type; deer fetus was purchased from Changchun Guangyu Deer Products Co., Ltd., which is a powder with a water content of 5%; deer antler glue was purchased from Yongkang Shencao Lotus Seed E-commerce Company; astragalus, salvia miltiorrhiza, snow lotus, epimedium, saffron and agarwood were purchased from Bozhou Mingzhou Pharmaceutical Co., Ltd.; the above-mentioned Chinese medicines all comply with the standards of the "Good Manufacturing Practice for Chinese Medicinal Materials" of the State Food and Drug Administration and the "Chinese Pharmacopoeia".

[0022] Unless otherwise specified, all raw material components in the following examples can be purchased through commercial channels, the experimental instruments used are conventional laboratory experimental instruments, and the performance testing methods are the testing methods known in the art.

[0023] The preferred implementation is as follows:

[0024] Embodiment 1:

[0025] The Chinese medicine extract is prepared by the following method:

[0026] Take 26% by weight of deer fetus, 7% by weight of astragalus, 6% by weight of salvia miltiorrhiza, 14% by weight of snow lotus, 13% by weight of antler glue, 8% by weight of epimedium, 6% by weight of saffron, 3% by weight of dalbergia odorifera and make up the balance of pure water, add 80% ethanol with a mass 10 times that of the above 8 kinds of medicinal materials, soak at 50°C for 24h, then add into a flash extractor with a speed of 6800r / min, extract for 30s and filter to obtain a coarse filtrate extract; distill the coarse filtrate extract under reduced pressure at 72°C and a vacuum degree of 800mbar until no liquid drips continuously, collect the distillation residue, add activated carbon for injection at 0.005g / ml, sterilize at 115°C for 30min, filter through a titanium rod filter when cooled to 30°C, and obtain the Chinese medicine extract.

[0027] Prepare the placenta-umbilical cord mesenchymal stem cell culture composition as follows:

[0028] The placenta-umbilical cord mesenchymal stem cell culture composition is composed of 30% by weight of a traditional Chinese medicine extract and the rest of a DMEM culture medium, which are mixed evenly.

[0029] Embodiment 2-19:

[0030] The only difference between Example 2 and Example 1 is that the placenta-umbilical cord mesenchymal stem cell culture composition is composed of 25% by weight of the Chinese herbal medicine extract and the rest of the DMEM culture medium;

[0031] The only difference between Example 3 and Example 1 is that the placenta-umbilical cord mesenchymal stem cell culture composition is composed of 33% by weight of the Chinese herbal medicine extract and the rest of the DMEM culture medium;

[0032] The only difference between Example 4 and Example 1 is that the mass fraction of deer fetus is 23%;

[0033] The only difference between Example 5 and Example 1 is that the mass fraction of deer fetus is 29%;

[0034] The only difference between Example 6 and Example 1 is that the mass fraction of Astragalus is 5%;

[0035] The only difference between Example 7 and Example 1 is that the mass fraction of Astragalus is 8%;

[0036] The only difference between Example 8 and Example 1 is that the mass fraction of Salvia miltiorrhiza is 4%;

[0037] The only difference between Example 9 and Example 1 is that the mass fraction of Salvia miltiorrhiza is 7%;

[0038] The only difference between Example 10 and Example 1 is that the mass fraction of snow lotus is 13%;

[0039] The difference between Example 11 and Example 1 is that the mass fraction of snow lotus is 15%;

[0040] The only difference between Example 12 and Example 1 is that the mass fraction of antler glue is 11%;

[0041] The only difference between Example 13 and Example 1 is that the mass fraction of antler glue is 15%;

[0042] The only difference between Example 14 and Example 1 is that the mass fraction of Epimedium is 6%;

[0043] The only difference between Example 15 and Example 1 is that the mass fraction of Epimedium is 9%;

[0044] The only difference between Example 16 and Example 1 is that the mass fraction of saffron is 4%;

[0045] The only difference between Example 17 and Example 1 is that the mass fraction of saffron is 7%;

[0046] The only difference between Example 18 and Example 1 is that the mass fraction of Dalbergia odorifera is 2%;

[0047] The only difference between Example 19 and Example 1 is that the mass fraction of dalbergia woodii is 4%.

[0048] Comparative Examples 1-32:

[0049] The difference between Comparative Example 1 and Example 1 is that the placenta-umbilical cord mesenchymal stem cell culture composition is composed of 20% by weight of the Chinese herbal medicine extract and the rest of the DMEM culture medium;

[0050] The difference between Comparative Example 2 and Example 1 is that the placenta-umbilical cord mesenchymal stem cell culture composition is composed of 40% by weight of the Chinese herbal medicine extract and the rest of the DMEM culture medium;

[0051] The difference between Comparative Example 3 and Example 1 is that the mass fraction of deer fetus is 17%;

[0052] The difference between Comparative Example 4 and Example 1 is that the mass fraction of deer fetus is 35%;

[0053] The difference between Comparative Example 5 and Example 1 is that the mass fraction of Astragalus is 2%;

[0054] The difference between Comparative Example 6 and Example 1 is that the mass fraction of Astragalus is 13%;

[0055] The only difference between Comparative Example 7 and Example 1 is that the mass fraction of Salvia miltiorrhiza is 2%;

[0056] The only difference between Comparative Example 8 and Example 1 is that the mass fraction of Salvia miltiorrhiza is 10%;

[0057] The only difference between Comparative Example 9 and Example 1 is that the mass fraction of snow lotus is 8%;

[0058] The only difference between Comparative Example 10 and Example 1 is that the mass fraction of snow lotus is 20%;

[0059] The difference between Comparative Example 11 and Example 1 is that the mass fraction of antler glue is 6%;

[0060] The only difference between Comparative Example 12 and Example 1 is that the mass fraction of antler glue is 20%;

[0061] The difference between Comparative Example 13 and Example 1 is that the mass fraction of Epimedium is 3%;

[0062] The only difference between Comparative Example 14 and Example 1 is that the mass fraction of Epimedium is 12%;

[0063] The only difference between Comparative Example 15 and Example 1 is that the mass fraction of saffron is 2%;

[0064] The only difference between Comparative Example 16 and Example 1 is that the mass fraction of saffron is 11%;

[0065] The only difference between Comparative Example 17 and Example 1 is that the mass fraction of Dalbergia odorifera is 1%;

[0066] The only difference between Comparative Example 18 and Example 1 is that the mass fraction of Dalbergia odorifera is 8%;

[0067] The only difference between Comparative Example 19 and Example 1 is that the speed of the flash extractor is 2000 r / min;

[0068] The only difference between Comparative Example 20 and Example 1 is that the speed of the flash extractor is 9000 r / min;

[0069] The difference between Comparative Example 21 and Example 1 is that the reduced pressure distillation is carried out at 60° C. and a vacuum degree of 800 mbar;

[0070] The only difference between Comparative Example 22 and Example 1 is that the reduced pressure distillation is carried out at 85° C. and a vacuum degree of 800 mbar;

[0071] The difference between Comparative Example 23 and Example 1 is that the Chinese herbal medicine extract is replaced with an equal mass of DMEM medium;

[0072] The difference between Comparative Example 24 and Example 1 is that astragalus, salvia miltiorrhiza, snow lotus, antler glue, epimedium, saffron and dalbergia odorifera are replaced with deer fetus of equal mass;

[0073] The difference between Comparative Example 25 and Example 1 is that deer fetus, salvia miltiorrhiza, snow lotus, antler glue, epimedium, saffron and dalbergia odorifera are replaced with astragalus of equal mass;

[0074] The difference between Comparative Example 26 and Example 1 is that deer fetus, astragalus, snow lotus, antler glue, epimedium, saffron and dalbergia odorifera are replaced with salvia miltiorrhiza of equal quality;

[0075] The difference between Comparative Example 27 and Example 1 is that deer fetus, astragalus, salvia miltiorrhiza, antler glue, epimedium, saffron and dalbergia odorifera are replaced with snow lotus of equal mass;

[0076] The difference between Comparative Example 28 and Example 1 is that deer fetus, astragalus, salvia miltiorrhiza, snow lotus, epimedium, saffron and dalbergia odorifera are replaced with deer antler glue of equal mass;

[0077] The difference between Comparative Example 29 and Example 1 is that the deer fetus, astragalus, salvia miltiorrhiza, snow lotus, antler glue, saffron and dalbergia odorifera are replaced with epimedium of equal mass;

[0078] The difference between Comparative Example 30 and Example 1 is that deer fetus, astragalus, salvia miltiorrhiza, snow lotus, antler glue, epimedium and dalbergia odorifera are replaced with saffron of equal quality;

[0079] The difference between Comparative Example 31 and Example 1 is that deer fetus, astragalus, salvia miltiorrhiza, snow lotus, antler glue, epimedium and saffron are replaced with aloeswood of equal quality;

[0080] In Comparative Example 32, ScienCell brand MSCM mesenchymal stem cell culture medium (fetal bovine serum type) was used to replace the placenta-umbilical cord mesenchymal stem cell culture composition.

[0081] Placenta and umbilical cord mesenchymal stem cells purchased from outside were mixed at a ratio of 1:1, and the cells were resuspended in 2 ml of DMEM medium. 6 Each cell was inoculated into a corresponding T175 culture flask, and 40 ml of the placenta-umbilical cord mesenchymal stem cell culture composition of each example was added, and placed in a CO2 incubator at 37°C, 5% CO2, and 95% humidity for 72 hours. When the cell confluence reached 80%, the cells were cultured in a large flask, and microscopic sampling was performed to observe whether obvious differentiation occurred. After trypsin digestion, the cells were resuspended, and the number of live cells was estimated using the hemocytometer method after trypan blue staining. The number of live cells was rounded to one significant digit after the decimal point, and the unit was 10 7 indivual.

[0082] Table 1: The number of viable cells and the results of differentiation observation in each example:

[0083]

[0084] It can be seen from the data in Table 1 that, compared with other examples, the embodiments of the present invention, especially Example 1 of the present invention, have strong proliferation ability and resistance to differentiation of placenta and umbilical cord mesenchymal stem cells; Comparative Example 23 proves the positive effect of the Chinese medicine extract of the embodiment of the present invention on cell culture, and Comparative Example 32 proves the advantage of the embodiment of the present invention over the existing culture scheme; the present invention is aimed at the environment of mixed culture of placenta-umbilical cord mesenchymal stem cells, which can simultaneously enhance the proliferation ability and resistance to differentiation of placenta and umbilical cord mesenchymal stem cells; the medicinal materials of the present invention have a synergistic effect of the formula, which cannot be effective by simple replacement.

[0085] The above description is only an embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modification, equivalent substitution, improvement, etc. made within the spirit and principle of the present invention should be included in the scope of the claims of the present invention.

Claims

1. A method for preparing a placenta-umbilical cord mesenchymal stem cell culture composition, characterized in that: The preparation method of the placenta-umbilical cord mesenchymal stem cell culture composition is: Take 26% by weight of deer fetus, 7% by weight of astragalus, 6% by weight of salvia miltiorrhiza, 14% by weight of snow lotus, 13% by weight of antler glue, 8% by weight of epimedium, 6% by weight of saffron, 3% by weight of dalbergia odorifera and make up the balance of pure water, add 80% ethanol with a mass 10 times that of the above 8 kinds of medicinal materials, soak at 50°C for 24h, then add into a flash extractor with a speed of 6800r / min, extract for 30s and filter to obtain a coarse filtration extract; distill the coarse filtration extract under reduced pressure at 72°C and a vacuum degree of 800mbar until no liquid drips continuously, collect the distillation residue, add activated carbon for injection at 0.005g / ml, sterilize at 115°C for 30min, filter through a titanium rod filter when cooled to 30°C, and obtain a traditional Chinese medicine extract; The traditional Chinese medicine extract having a mass fraction of 25-33% and the DMEM culture medium making up the balance are mixed to obtain the placenta-umbilical cord mesenchymal stem cell culture composition.

2. A placenta-umbilical cord mesenchymal stem cell culture composition prepared by the method for preparing a placenta-umbilical cord mesenchymal stem cell culture composition according to claim 1, characterized in that: The placenta-umbilical cord mesenchymal stem cell culture composition is composed of 25-33% by mass of the traditional Chinese medicine extract and the rest of the DMEM culture medium.

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