CN112899222A - Efficient cell serum-free culture medium system and application of cell secretion thereof - Google Patents
Efficient cell serum-free culture medium system and application of cell secretion thereof Download PDFInfo
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Abstract
The invention discloses an efficient cell serum-free culture medium system and application of a cell secretion thereof. When the cell culture medium of some embodiments of the invention is used for culturing the human umbilical cord derived stem cells, the time for the P0 cells to climb out until the cells reach the passage density is only 10 days, and the cultured P4-generation umbilical cord mesenchymal stem cells are regularly arranged in a long fusiform manner and are better in state. The cultured P4 umbilical cord mesenchymal stem cells still maintain good differentiation capability and can be well differentiated into osteoblasts and adipoblasts. And the flow surface marker detection proves that the cells have the specific surface marker of the stem cells and do not have the specific surface marker of the cord blood stem cells. The cell culture medium of some examples of the invention has better growth rate and lower aging degree of cells when being used for culturing fibroblasts, and the effect is better than that of the existing commercial cell culture medium.
Description
Technical Field
The invention relates to the field of biology, in particular to an efficient cell serum-free culture medium system and application of a cell secretion thereof.
Background
The stem cell is one kind of cell with self-replicating capacity and multidirectional differentiation potential, and may be differentiated into multifunctional cell and tissue under certain condition to have the biological functions of regulating immunity, regenerating, repairing, etc. Mesenchymal Stem Cells (MSCs) are adult stem cells derived from mesoderm, widely present in the connective tissue and organ interstitium throughout the body, can be differentiated into bone, cartilage, fat, muscle, nerve cells and the like under specific induction conditions in vivo or in vitro, have functions of promoting tissue repair, immunoregulation and supporting hematopoiesis, and have low immunogenicity, so MSCs are tissue engineering seed cells with the most application prospects in regenerative medicine and immunoregulation treatment.
Mesenchymal stem cells are widely available relative to other stem cells, and are currently found in bone marrow, fat, muscle, heart, umbilical cord blood, umbilical cord, placenta and other tissues. Umbilical cord stem cells, umbilical cord blood stem cells, fat cells, epidermal cells, fibroblasts and the like can secrete various cytokines, exosomes, active proteins and the like, and the cell culture products have rich application in the aspects of aging resistance, beauty treatment, cell repair and the like. Culturing cells is a prerequisite for obtaining sufficient quantities of stem cells and cell secretions, and cell culture media is the key to cell culture.
In order to obtain a better culture effect, it is often necessary to add natural components such as serum to a basal medium. However, the addition of serum or the like may introduce exogenous components such as viruses, and the quality of cells between batches is difficult to control, and the cost is high, thereby limiting the use of the cells. The addition of serum replacement to cell basic culture medium to culture cell is becoming more and more popular. However, the culture medium is often lack of certain unknown specific nutrients, and the culture effect is poor.
At present, the variety of cell culture media is wide, the number of cell culture modes is also large, and the use of the culture media is different according to the variety of the culture media, the culture modes and the cell types. Serum-free culture medium and culture medium containing 10-15% of serum are the most widely applied culture medium systems at present, and the serum-free culture medium has the advantages of no animal-derived components, capability of reducing pollution of viruses, bacteria and the like caused by heterologous substances, higher safety, higher price and lower application range, and is generally only directed at certain cells; the serum-containing culture medium has the advantages of moderate price, wide application, suitability for most cell lines, low safety compared with serum-free medium, complex serum components, and increased quality control difficulty of mycoplasma, chlamydia, viruses, specific proteins and the like.
F12, DMEM (low sugar), DMEM (high sugar) are basic culture media commonly used for cell culture, can meet the basic requirements of cell growth, but cannot completely meet the requirements of cell proliferation, and cells are easy to degenerate or age during subculture.
There are also a large number of serum substitutes available on the market for addition in the basal medium instead of serum. Serum substitutes produced by different companies have characteristics and different culture effects aiming at different cells. Representative Serum substitutes include Ultroser G manufactured by Pall corporation and KnockOut Serum Replacement (KnockOut SR) manufactured by GIBCO corporation. However, in practical applications, there are still disadvantages such as slow cell proliferation. The cost of its use is also relatively high.
The existing commercial culture medium has various types, but according to the use instruction provided by a manufacturer, the culture medium of the same manufacturer with other types can be matched to obtain a better culture effect.
Disclosure of Invention
The invention aims to overcome at least one defect in the prior art and provide a high-efficiency cell serum-free medium system and application of cell secretion thereof.
The technical scheme adopted by the invention is as follows:
in a first aspect of the present invention, there is provided:
a cell complete medium consisting of a basal medium, a serum replacement and a supplement, wherein:
the dosage of the basic culture medium is 50-80 parts by volume, and the basic culture medium is selected from at least one of MEM, F12, low-sugar DMEM and high-sugar DMEM;
the dosage of the serum substitute is 5-50 parts by volume and is selected from KnockOutTM DMEM、Ultra CULTURETMSerum-free medium、EliteGroTM 、EliteGroTM-at least one of Adv, ultraser G;
the supplement is used in an amount of 0-5 parts by volume and is at least one selected from PEN STREP GLUTAMINE, Penicillin-Streptomyces, Sodium Pyruvate and L-GlutaMax. The supplement functions to provide certain non-essential amino acids and a source of energy other than glucose or an antibiotic that inhibits the microorganism.
In order to ensure the uniformity of product quality, commercial culture medium, serum substitute and supplement with more stable quality can be purchased. In the same year, the product can be configured by self under the condition of ensuring the product quality.
In some examples, the serum replacement is used in an amount of 15 to 35 parts by volume.
In some examples, the supplement is used in an amount of 0.5 to 4 parts by volume.
In some examples, the composition of the cell complete medium is: 65 parts by volume of basic culture medium, 33 parts by volume of serum substitute and 2 parts by volume of supplement, wherein:
the basal medium is DMEM basic (1X) (high sugar);
the serum substitute is KnockOutTM DMEM(1X)；
The supplement is PEN STREP GLUTAMINE.
In some examples, the composition of the cell complete medium is: 75 parts by volume of basic culture medium, 24 parts by volume of serum substitute and 1 part by volume of supplement, wherein:
the basic culture medium is DMEM/F12;
the Serum substitute is Ultra CURTURE Serum-free medium;
the supplement is a GlutaMax ™ polypeptide.
In a second aspect of the present invention, there is provided:
a cell culture method comprising seeding cells in a cell completion medium as described in the first aspect of the invention and replacing the medium in time.
In some examples, the cells include, but are not limited to, stem cells, fibroblasts, tissue cells beside cancer, epithelial cells, adipocytes, cancer cells, neural cells, thyroid cells, chondrocytes, keratinocytes, bone marrow cells, cardiomyocytes of different origins.
In some examples, the cells are fibroblasts, and the composition of the medium is: 75 parts by volume of basic culture medium, 24 parts by volume of serum substitute and 1 part by volume of supplement, wherein:
the basic culture medium is DMEM/F12;
the Serum substitute is Ultra CURTURE Serum-free medium;
the supplement is Penicilin-Streptomycin.
In some examples, the fibroblast is a human fibroblast.
In some examples, the cell is a mesenchymal stem cell, and the composition of the medium is: 65 parts by volume of basic culture medium, 33 parts by volume of serum substitute and 2 parts by volume of supplement, wherein:
the basal medium is DMEM basic (1X) (high sugar);
the serum substitute is KnockOutTM DMEM(1X)；
The supplement is PEN STREP GLUTAMINE.
In some examples, the mesenchymal stem cell is a human umbilical cord-derived mesenchymal stem cell.
In a third aspect of the present invention, there is provided:
a method of producing a cellular secretion comprising:
culturing the cells according to the cell culture method of the second aspect of the present invention;
collecting the supernatant of the culture solution, separating and purifying to obtain the target cell secretion.
In some examples, the cellular secretion includes, but is not limited to, exosomes, growth factors, cytokines, collagen extracts, functional proteins such as fibronectin, elastin, and the like. The cytokine includes Vascular Endothelial Growth Factor (VEGF), insulin-like growth factor (IGF), Hepatocyte Growth Factor (HGF), Epidermal Growth Factor (EGF), Fibroblast Growth Factor (FGF), Transforming Growth Factor (TGF), Endothelin (ET), stromal cell elongation factor (SDF-1), etc. The bioactive proteins include, for example, collagen, hyaluronic acid, glycoproteins, proteoglycans, laminins, thrombospondins, Glycosaminoglycans (GACs), heparin sulfate, insulin-like growth factor binding proteins (IGFBPs), superoxide dismutase 2(SOD2), interleukin-1 (IL-1), tissue metalloproteinase inhibitor (TIMP), and the like.
In some examples, the cells include, but are not limited to, stem cells, fibroblasts, tissue cells beside cancer, epithelial cells, adipocytes, cancer cells, neural cells, thyroid cells, chondrocytes, keratinocytes, bone marrow cells, cardiomyocytes of different origins.
In a fourth aspect of the present invention, there is provided:
a method for preparing a collagen extract, comprising:
s1) culturing fibroblasts according to the cell culture method of the second aspect of the present invention;
s2), collecting cell supernatant, centrifuging for the first time, and collecting supernatant;
s3) transferring the supernatant obtained by the primary centrifugation into a 3KD ultrafiltration tube, centrifuging to obtain a concentrated supernatant, adding a protease inhibitor and pepsin into the concentrated supernatant, adjusting the pH with acetic acid, and centrifuging after the enzymolysis is finished to obtain an enzymolysis supernatant;
s4) salting out the enzymolysis supernatant, centrifuging after full salting out, taking the precipitate, cleaning and desalting, and drying to obtain the collagen extract.
In a fifth aspect of the present invention, there is provided:
a skin care product comprising a matrix to which the collagen extract of the fourth aspect of the present invention is added.
The invention has the beneficial effects that:
the cell culture medium of some examples of the invention, when used for culturing human umbilical cord derived stem cells, required only 10 days for the P0 cells to climb out until the cells reach the passage density, while the LONZA culture medium containing 2% serum replacement consumed 12 days. Meanwhile, the cultured P4-generation umbilical cord mesenchymal stem cells are regularly arranged in a long fusiform manner, and the state is better; the conventional culture system has cell aging phenomenon, and the cells are in irregular wiredrawing shape. The cultured P4 umbilical cord mesenchymal stem cells still maintain good differentiation capability and can be well differentiated into osteoblasts and adipoblasts. And the flow surface marker detection proves that the cells have the specific surface marker of the stem cells and do not have the specific surface marker of the cord blood stem cells.
The cell culture medium of some examples of the invention has better growth rate and better effect than the existing commercial cell culture medium when used for culturing fibroblasts.
The cell culture medium of some embodiments of the invention, when used in fibroblast cell culture, results in a lower degree of senescence of the cells.
The cell culture medium of some examples of the invention can be used for culturing fibroblasts, and fibroblasts with higher purity can be obtained.
When the cell culture medium provided by some examples of the invention is used for fibroblast culture, the secretion of type I collagen can be better promoted, and the concentration of the type I collagen can reach 79.29ng/ml at most.
The cell culture medium of some embodiments of the invention has stable product quality and relatively low use cost.
The cell culture method of some embodiments of the invention can obtain fibroblasts with better quality and is also beneficial to obtaining better cell secretion products.
Drawings
FIG. 1 is a cell morphology (4X 10 times) of P0 fibroblasts after culturing in different culture systems;
FIG. 2 is a cell morphology (10X 10 times) of P4 fibroblasts after culturing in different culture systems;
FIG. 3 is a diagram showing the result of the identification of the type I collagen secretion function of each algebraic fibroblast;
FIG. 4 is a cell cycle profile of fifth and tenth generation fibroblasts;
FIG. 5 shows the results of enzyme-labeled detection of CCK-8;
FIG. 6 is a morphological diagram (4X 10 fold) of cells in different culture systems;
FIG. 7 is a photograph (4X 10 times) of a staining of beta-galactosidase cells in different culture systems;
FIG. 8 is a graph (10X 10 fold) showing the immunohistochemistry results of fibroblasts in example 2;
FIG. 9 is a graph (10X 10 fold) showing the immunohistochemistry results of fibroblasts of comparative example 2;
FIG. 10 is a graph of type I collagen secretion by fibroblasts of example 2;
FIG. 11 is a graph of type I collagen secretion by fibroblasts of comparative example 2;
FIG. 12 is a photograph of red region improvement effect;
fig. 13 is a photograph of wrinkle improvement effect;
fig. 14 is a cell morphology (4 × 10 times) of the P0 generation mesenchymal stem cells after being cultured in different culture systems;
fig. 15 is a cell morphology (10 × 10 times) of the P4 generation mesenchymal stem cells after being cultured in different culture systems;
fig. 16 is the results of osteogenesis (a) and adipogenesis (B) experiments of mesenchymal stem cells;
fig. 17 is a surface marker flow analysis of mesenchymal stem cells.
Detailed Description
The technical scheme of the invention is further explained by combining examples and experiments.
Example 1: the composition of the medium was as follows
65 parts by volume of basic culture medium, 33 parts by volume of serum substitute and 2 parts by volume of supplement, wherein:
the basal medium was DMEM basic (1X) (high sugar) from GIBICO, cat # C11995500 BT;
the serum substitute is KnockOut from GIBICOTMDMEM (1X), cat # 10829018;
the supplement is PEN STREP GLUTAMINE from Invitrogen, cat # DXT-10378016.
The market price for 500mL of complete medium formulated in this example 1 is about 479 yuan.
Example 2: the composition of the medium was as follows
75 parts by volume of basic culture medium, 24 parts by volume of serum substitute and 1 part by volume of supplement, wherein:
the basic culture medium is Advanced DMEM/F12 from Thermo Fisher Scientific, Cat. 12634010;
the serum substitute is UltraCurture from LONZATMSerum-free medium, cat # 12-725F;
the supplement is the GlutaMAX ™ polypeptide from GIBICO, Cat 35050061;
the market price for the 500mL complete medium formulated in this example 2 is about 643 Yuan.
Example 3: the composition of the medium was as follows
80 parts by volume of basic culture medium, 18 parts by volume of serum substitute and 2 parts by volume of supplement, wherein:
the basal medium was DMEM basic (1X) (low sugar) from GIBICO, cat # C11885500 BT;
the serum substitute is KnockOut from GIBICOTMDMEM (1X), cat # 10829018;
the supplement is PEN STREP GLUTAMINE from Invitrogen, cat # DXT-10378016.
The market price for the 500mL complete medium formulated in this example 3 is about 454 yuan.
Example 4: the composition of the medium was as follows
65 parts by volume of basal medium, 34.5 parts by volume of serum substitute and 0.5 part by volume of supplement, wherein:
the basic culture medium is Advanced DMEM/F12 from Thermo Fisher Scientific, Cat. 12634010;
the serum substitute is UltraCurture from LONZATMSerum-free medium, cat # 12-725F;
the supplement was Penicilin-Streptomyces from GIBICO, cat # 15140122.
This example 4 prepared 500mL of complete medium with a market price of about 612 Yuan.
Comparative example 1: the composition of the medium was as follows
98 parts by volume of basic culture medium and 2 parts by volume of serum substitute, wherein:
the basic culture medium is UltraCurture of LONZATMSerum-free medium, cat # 12-725F;
the Serum replacement was Ultroser G Serum Substitee from PALL, Cat 15950-017.
The market price for 500mL of complete medium formulated in this comparative example 1 is about 1346 yuan.
Comparative example 2: the composition of the medium was as follows
90 parts by volume of basic culture medium and 10 parts by volume of serum, wherein:
the basic culture medium is Advanced DMEM/F12 from Thermo Fisher Scientific, Cat. 12634010;
the Serum is Fetal bone Serum, qualified, Australia, catalog number 10099-141 from GIBICO;
this comparative example 2 prepared 500mL of complete medium with a market price of about 2031 Yuan.
Comparative example 3: the composition of the medium was as follows
98 parts by volume of basic culture medium and 2 parts by volume of supplement, wherein:
the basic culture Medium is FBM fiber basic Medium of LONZA company, the product number is CC-3131;
this comparative example 3 prepared 500mL of complete medium with a market price of about 1513 yuan.
The technical scheme of the invention is further explained by combining experiments.
Culturing fibroblasts:
pieces of human skin-derived tissue, cultured fibroblasts from primary P0 to P4 passages. The cell culture media of example 2 and comparative example 2 were used, respectively, and the culture was carried out according to a conventional method.
Cell morphology comparison: the results of the comparison of the culture are shown in FIGS. 1 and 2. In FIG. 1, A is a photograph showing the morphology of cells cultured for 10 days in example 2 of the P0 generation, and B is a photograph showing the morphology of cells cultured for 15 days in comparative example 2 of the P0 generation. In FIG. 2, A is a photograph showing the morphology of cells cultured for 3 days in example 2 of the P4 generation, and B is a photograph showing the morphology of cells cultured for 3 days in comparative example 2 of the P4 generation. As can be seen from fig. 1 and 2, the P0 cells crawl out to the time it takes for the cells to reach the passaging density, the example 2 formulation system takes 10 days, while the comparative example 2 takes 15 days, the inventive technique saves 5 days of time and reduces the time cost; meanwhile, as can be seen from the pictures, the fibroblasts cultured by the formulation technology of the embodiment 2 have better states (regular long fusiform arrangement, and the P4 conventional culture system has cell aging phenomenon, namely irregular-shaped dragline-shaped cells) and higher growth density.
Cell secretion comparison: the main functions of fibroblasts are: synthesizing and secreting collagen fibers, elastic fibers, matrix macromolecules, etc. The main component of the collagen fiber is collagen. The collagen in the skin is mainly type I collagen, which accounts for more than 70% of the total collagen content, so the type I collagen is selected as a main index for the function identification of fibroblasts. Fibroblasts of P5-P10 were all rich in type I collagen secretion, and the amount of type I collagen secretion was significantly decreased from the tenth generation (FIG. 3).
Effect of subculture on fibroblast traits: staining fibroblasts by PI solution, detecting the DNA content of cells by a flow cytometer, analyzing the cell cycle by using cylChred software, and judging the influence of subculture on the cell cycle of the fibroblasts. The experimental result shows that: from P5 to P10, the genomes of fibroblasts cultured in vitro were stable all the time, the cell cycle was normally distributed, and there was no significant difference in the cell cycle distribution of P5 and P10 (fig. 4).
Cell growth rate and cell senescence assay for different media systems
The conventional cell culture media for example 2 and 3 comparative examples were the culture media of comparative example 1, comparative example 2, and comparative example 3, respectively.
Method for detecting growth rate of fibroblast
By 1 × 104Perwell, 100. mu.L complete medium/well, 9 wells per group, were inoculated into 96-well plates (total 3 plates).
Bi yun tian-CCK-8 kit [ cat No.: c0038, and measuring the absorbance by a TECAN Infinite E Plex multifunctional microplate reader.
Pictures of growth of each component fiber cell under 4X 10 times Lycra inverted microscope for 24h, 48h and 72 h.
Cell senescence was determined using beta-galactosidase staining. Senescent cells appear dark blue. If the picture is black and white, the more dark color, the more senescent cells.
The results of the experiments are shown in fig. 5, 6 and 7, from which it can be seen that the cell growth rate: example 2 > comparative example 1 > comparative example 3. The growth rate of the fibroblast is fastest and the state is better under the culture medium formula system.
The aging degree: comparative example 3 > comparative example 1 > comparative example 2= example 2. Under the culture medium formula system, the fibroblast senescence degree is equivalent to that of an FBS system, and the senescence degree is lower than that of other systems.
Fibroblast immunohistochemical experiment
The specific marker protein of the fibroblast is vimentin, and does not express keratin and desmin. The project group carries out immunocytochemical staining on the fibroblasts of the 2 culture systems by the protein, and the darker the picture color is, the higher the positive rate of the corresponding protein is represented. The results show that: the positive rate of vimentin, keratin and desmin of example 2 was about 99.9%, 0% and 0% (fig. 8); the positive rate of vimentin, keratin and desmin of comparative example 2 was about 99.9%, 0% and 23.79%. The experimental results show that the fibroblasts cultured in example 2 have higher purity (fig. 9).
Fibroblast secretion function identification experiment
We compared the cultured fibroblasts of example 2 and comparative example 2 in order to study the type I collagen secretion ability. The experimental results show that the 2 culture methods reach the peak value of the type I collagen concentration 96 hours after cell inoculation, but the type I collagen concentration of the example 2 can reach 79.29ng/ml at most, which is obviously higher than that of the comparative example 2.
Extraction and purification of collagen from cell secretion
The collagen has good physical and chemical properties and biological characteristics, and is widely applied in the fields of medicine, food industry, cosmetics and the like. Therefore, the application of cell secretion to extraction and purification of active substances is exemplified by collagen.
Fibroblasts were cultured according to example 2, and when the cells were confluent in the cell flask, the cells were digested, the cell supernatant was collected, centrifuged at 1000g for 15min, and stored at 4 ℃ for further use. The supernatant was centrifuged for 90min at 4000rpm using a 15ml 3KD ultrafiltration tube to obtain a concentrated supernatant. To 9ml of the concentrated supernatant, 1ml of a protease inhibitor, 0.33ml of an acetic acid solution (0.5 mol/L) and 10. mu.L of a pepsin solution (10 g/L) were added, and after mixing, the mixture was gently stirred at 4 ℃ for 5 hours. Centrifuging at 16000r/min at 4 deg.C for 30min, slowly adding 410mg sodium chloride powder into the supernatant, stirring while mixing, dissolving, and salting out at 4 deg.C overnight. Centrifuging at 16000r/min at 4 deg.C for 30min, separating precipitate, and freeze drying for 2 hr to obtain collagen extract in culture solution.
1mg of collagen product is accurately weighed and dissolved by adding 1ml of physiological saline. 50 μ L of the mixture was taken, mixed with 48 μ L of 2xSDS loading buffer and 2 μ L of beta-mercaptoethanol, denatured at 100 ℃ for 5min, slightly cooled and centrifuged instantaneously. 80ml/L separation gel and 45ml/L accumulation gel are prepared. And adding 10-15 mu L of sample liquid into each hole, switching on a power supply, and carrying out electrophoresis for 4h under a constant current of 10 mA. After SDS-PAGE, the qualified collagen extract shows 3 purified protein bands composed of alpha peptide chains which are completely identical with the standard protein.
Direct smearing application of secretion stock solution
After 26 volunteers clean the face in the morning and at night each day, 1.5ml of fibroblast secretion stock solution is directly smeared on the face, and the face is revisited and image-recorded after 1 month of use (the volunteers cannot use other skin care products except the cell secretion stock solution in the use test process). The facial condition of the volunteers before and after the use of the secretion stock solution was recorded by a facial image analyzer (visia-cr), and comparative analysis of the following indices was performed. The experimental result shows that the cell secretion stock solution has certain effects of relieving, repairing, resisting senility, removing wrinkles and the like.
The results of the return visits of the volunteers were counted (the grade degree of the extensibility and the skin-contacting ability was classified as "excellent, better, general, worse, extremely poor". The grade degree of the absorbability was "easily absorbed, general, difficult to absorb, extremely difficult to absorb")
Test index definitions
The red region improving effect is shown in fig. 12, and the wrinkle improving effect is shown in fig. 13. From fig. 12, it can be seen that the color of the test part area becomes obviously lighter, the red area ratio is reduced, the skin sensitivity is obviously reduced after the subject uses the secretion stock solution, and the secretion stock solution has certain relieving and repairing efficacy. From fig. 13, it can be seen that the fine lines of the eyes of the subject are reduced after the subject uses the secretion stock solution, and the depth of the fine lines is reduced, which shows that the secretion stock solution has certain anti-aging and wrinkle-removing effects.
The collagen extract can provide necessary nutrients for a skin layer containing collagen, so that the activity of the collagen in the skin is enhanced, the moisture of the horny layer is kept, the integrity of a fiber structure is kept, the living environment of skin cells is improved, the metabolism of skin tissues is promoted, and the circulation is enhanced, so that the effects of moistening the skin, delaying senescence, removing wrinkles and nourishing hair are achieved; the affinity with skin tissues is good, and the effect of repairing the tissues is achieved; the skin-moistening cream contains a large amount of hydrophilic groups, so that the skin-moistening cream has good moisturizing effect and can achieve the purpose of keeping the skin moist; can inhibit melanin generation, and whiten skin.
Culture of human umbilical cord-derived stem cells
Human umbilical cord from healthy volunteers, cultured mesenchymal stem cells from primary P0 to P4 passages. The culture mediums of example 1 and comparative example 1 were used for the respective culture.
Cell morphology is shown in FIGS. 14 and 15. In FIG. 14, A is a photograph showing the morphology of cells cultured for 10 days in example 1 of the P0 generation, and B is a photograph showing the morphology of cells cultured for 12 days in comparative example 1 of the P0 generation; in FIG. 15, A is a photograph showing the morphology of cells cultured for 3 days in example 1 of the P4 generation, and B is a photograph showing the morphology of cells cultured for 3 days in comparative example 1 of the P4 generation. As can be seen from fig. 14 and 15, the time taken for the P0 cells to climb out to reach the passaging density is 10 days for example 1, and 12 days for comparative example 1, which saves 2 days and reduces the time cost by the present technology; meanwhile, as can be seen from the pictures, the umbilical cord mesenchymal stem cells cultured by the formulation technology of example 1 have better states (regular long fusiform arrangement, and the P4 conventional culture system has cell aging phenomenon, namely irregular-shaped wiredrawing cells), and have higher growth density (the cells cultured in comparative example 1 have local non-growth areas).
Osteogenic experiments and adipogenic experiments were performed using the P4-generation mesenchymal stem cells cultured in example 1 according to a conventional method. The results are shown in FIG. 16. In fig. 16, A, B are photographs (10 × 10 times) of the results of the osteogenesis experiment and the adipogenesis experiment, respectively. Flow analysis of surface markers of P4 generation mesenchymal stem cells as shown in fig. 17, surface markers: positive CD29, CD44, CD 105; negative CD 45. Osteogenic and adipogenic experiments prove that the cells have dry property, and flow surface marker detection proves that the cells have the specific surface marker of the stem cells and do not have the specific surface marker of the umbilical cord blood stem cells. The combination shows that the umbilical cord-derived cells cultured by using the culture medium formula system of example 1 are indeed umbilical cord mesenchymal stem cells.
The foregoing is a more detailed description of the invention and is not to be taken in a limiting sense. It will be apparent to those skilled in the art that simple deductions or substitutions without departing from the spirit of the invention are within the scope of the invention.
Claims (10)
1. A cell complete medium consisting of a basal medium, a serum replacement and a supplement, wherein:
the dosage of the basic culture medium is 50-80 parts by volume, and the basic culture medium is selected from at least one of MEM, F12, low-sugar DMEM and high-sugar DMEM;
the dosage of the serum substitute is 5-50 parts by volume and is selected from KnockOutTM DMEM、Ultra CULTURETM Serum-free medium、EliteGroTM 、EliteGroTM-at least one of Adv, ultraser G;
the supplement is used in an amount of 0-5 parts by volume and is at least one selected from PEN STREP GLUTAMINE, Penicillin-Streptomyces, Sodium Pyruvate and L-GlutaMax.
2. The cell complete medium according to claim 1, characterized in that: the composition is as follows:
65 parts by volume of basic culture medium, 33 parts by volume of serum substitute and 2 parts by volume of supplement, wherein:
the basal medium is DMEM basic (1X) (high sugar);
the serum substitute is KnockOutTM DMEM(1X)；
The supplement is PEN STREP GLUTAMINE;
or 75 parts by volume of basic culture medium, 24 parts by volume of serum substitute and 1 part by volume of supplement, wherein:
the basic culture medium is DMEM/F12;
the Serum substitute is Ultra CURTURE Serum-free medium;
the supplement is a GlutaMAX ™ polypeptide.
3. A cell culture method, comprising inoculating cells into a complete cell culture medium and timely replacing the medium, characterized in that: the cell complete medium is as defined in claim 1 or 2.
4. The cell culture method according to claim 3, characterized in that: the cells are stem cells, fibroblasts, cancer adjacent tissue cells, epithelial cells, fat cells, cancer cells, nerve cells, thyroid cells, chondrocytes, keratinocytes, bone marrow cells and cardiac muscle cells of different sources.
5. The cell culture method according to claim 3, characterized in that: the cells are fibroblasts, and the culture medium comprises the following components: 75 parts by volume of basic culture medium, 24 parts by volume of serum substitute and 1 part by volume of supplement, wherein:
the basic culture medium is DMEM/F12;
the Serum substitute is Ultra CURTURE Serum-free medium;
the supplement is Penicilin-Streptomycin.
6. The cell culture method according to claim 3, characterized in that: the cell is a mesenchymal stem cell, and the composition of the culture medium is as follows: 65 parts by volume of basic culture medium, 33 parts by volume of serum substitute and 2 parts by volume of supplement, wherein:
the basal medium is DMEM basic (1X) (high sugar);
the serum substitute is KnockOutTM DMEM(1X)；
The supplement is PEN STREP GLUTAMINE.
7. A method of producing a cellular secretion comprising:
culturing the cells according to the cell culture method of any one of claims 3 to 6;
collecting the supernatant of the culture solution, separating and purifying to obtain the target cell secretion.
8. The method of claim 7, wherein: the cell secretion is selected from exosome, growth factor, cytokine, collagen extract, functional protein.
9. A method for preparing a collagen extract, comprising:
s1) culturing the fibroblast according to the cell culture method of any one of claims 3 to 6;
s2), collecting cell supernatant, centrifuging for the first time, and collecting supernatant;
s3) transferring the supernatant obtained by the primary centrifugation into a 3KD ultrafiltration tube, centrifuging to obtain a concentrated supernatant, adding a protease inhibitor and pepsin into the concentrated supernatant, adjusting the pH with acetic acid, and centrifuging after the enzymolysis is finished to obtain an enzymolysis supernatant;
s4) salting out the enzymolysis supernatant, centrifuging after full salting out, taking the precipitate, cleaning and desalting, and drying to obtain the collagen extract.
10. A skin care product comprises a substrate, and is characterized in that: wherein the collagen extract of claim 9 is added.
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