CN118185867A - Culture method and culture medium of fibroblast from behind-the-ear skin - Google Patents
Culture method and culture medium of fibroblast from behind-the-ear skin Download PDFInfo
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- CN118185867A CN118185867A CN202410593724.5A CN202410593724A CN118185867A CN 118185867 A CN118185867 A CN 118185867A CN 202410593724 A CN202410593724 A CN 202410593724A CN 118185867 A CN118185867 A CN 118185867A
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Abstract
The invention belongs to the technical field of cell culture, and particularly relates to a culture method and a culture medium of a fibroblast from skin behind ear. The invention discloses a fibroblast culture medium from behind-the-ear skin, which comprises the following components: the culture medium comprises a basic culture medium and an exogenous additive composition, wherein the basic culture medium is a DMEM (DMEM) culture medium or an F12 culture medium or a mixed culture medium consisting of the DMEM culture medium and the F12 culture medium according to a volume ratio of 1-2:1-2, and the exogenous additive composition is a combination of multiple components. The invention can realize rapid proliferation culture of the fiber cells, shortens the culture time and improves the cell activity through reasonable compatibility of the obtained culture medium and the corresponding culture method.
Description
Technical Field
The invention belongs to the technical field of cell culture, and particularly relates to a culture method and a culture medium of a fibroblast derived from skin behind an ear.
Background
The fibroblast of the skin is widely distributed in the dermis tissue, and can secrete cell growth factors, collagen, stress proteins and other extracellular proteins in an autocrine or paracrine mode, and nutrient substances secreted by the cells can act on the skin in a synergistic way, so that regeneration of hyaluronic acid, collagen and elastin in the skin tissue is stimulated, damaged cells are repaired, and wound healing is promoted. Fibroblasts play an important role in human life, such as guiding skin formation in embryonic period, participating in maintenance of skin homeostasis after organ maturation, promoting repair of damaged site structure and function, etc. Therefore, the in vitro cultured autologous skin fibroblast has wide application in the fields of tissue engineering, cosmetology, medical treatment and the like.
At present, the culture medium used in the method for culturing the fibroblasts in vitro is mainly prepared from a basal medium and fetal bovine serum. The use of fetal bovine serum can bring about the risk of animal-derived pathogens, affecting the safety of clinical application; meanwhile, the culture period is long, and the primary activity of cells is easy to be poor.
Disclosure of Invention
The invention aims to overcome the problems in the prior art and provide a culture method and a culture medium of fibroblast from skin behind ear. The fibroblast culture method and the culture medium can realize rapid proliferation culture of the fibroblasts and shorten the culture time.
The aim and the technical problems of the invention are realized by adopting the following technical proposal.
In one aspect, the invention provides a post-aural skin-derived fibroblast culture medium comprising the following composition: a basal medium and an exogenous additive composition, the basal medium comprising one of:
(i) DMEM medium;
(ii) F12 medium;
(iii) A mixed culture medium consisting of a DMEM culture medium and an F12 culture medium according to a volume ratio of 1-2:1-2;
the exogenous additive composition comprises 10-50 ng/mL of glutamine, 1-10 mu M of dexamethasone, 10-50 ng/mL of carvacrol, 1-20 mu g/mL of fibroblast growth factor, 1-20 mu g/mL of epidermal growth factor, 1-5 mu M of isoprenaline, 1-5 ng/mL of ferulic acid, 100-200 mu g/mL of transferrin, 1-10 mu g/mL of transforming growth factor beta and 1-5 ng/mL of hexapeptide.
In another aspect, the present invention provides a method for preparing a fibroblast cell culture medium derived from skin behind the ear, the method comprising:
Providing one of the following basal media:
(i) DMEM medium;
(ii) F12 medium;
(iii) A mixed culture medium consisting of a DMEM culture medium and an F12 culture medium according to a volume ratio of 1-2:1-2;
Adding the exogenous additive composition into the basic culture medium, mixing, and sterilizing at 121-122 deg.C for 20-25min.
In still another aspect, the present invention provides a method for culturing a fibroblast from behind the ear, using the aforementioned fibroblast culture medium from behind the ear skin, the method comprising the steps of:
S1: tissue collection: cutting skin tissue after taking ear under aseptic condition, then placing in digestive enzyme solution for digestion, centrifuging after digestion, washing and centrifuging the obtained cells, repeating for 3 times to obtain cell suspension;
S2: primary culture: the primary culture medium is used for resuspension of cells, the cell concentration is 2-4 multiplied by 10 5/mL, and the cells are cultured in an incubator containing 5% CO 2 and at 37 ℃ for 5-10 days, wherein the culture medium is changed every 2-3 days;
S3: expansion culture of fibroblasts: discarding the culture medium after the primary culture in the step S2 is finished, adding 1-2 mL of dispersing enzyme, standing for 30min at 37 ℃, centrifuging, discarding the supernatant, digesting for 5min by using 1-2 mL of trypsin-EDTA, then resuspending cells by using a fibroblast culture medium to obtain 10-20 mu L of cell suspension with the concentration of 1-4X 10 5/mu L, adding 25-40 mu L of proliferation matrigel, mixing, adding 1-2 mL of fibroblast culture medium again after the proliferation matrigel is solidified, and finally culturing in an incubator containing 5% CO 2 and 37 ℃ for 10-15 days, wherein the culture medium is replaced every 2-3 days.
In a preferred embodiment of the present invention, the digestive enzyme liquid composition in step S1 is: 92-95 mu L of PBS buffer solution, 1.5-2 mu L of 20 mg/mL type II collagenase, 2-3 mu L of 15 mg/mL cell lyase and 1.5-3 mu L of 50mM sodium chloride;
the digestive enzyme solution is added according to the amount of adding 100 mu L of the digestive enzyme solution to 1mg of skin tissue behind the ear;
the digestion conditions are: 37℃for 30min.
In a preferred embodiment of the present invention, the composition of the primary medium in step S2 comprises: a basal medium and an exogenous additive composition, the basal medium comprising one of:
(i) DMEM medium;
(ii) F12 medium;
(iii) A mixed culture medium consisting of a DMEM culture medium and an F12 culture medium according to a volume ratio of 1-2:1-2;
The exogenous additive composition comprises 10-20 mu g/mL of glutathione, 1-10 mu M of hydrocortisone, 1-10 ng/mL of basic fibroblast growth factor, 1-10 ng/mL of recombinant human epidermal growth factor, 1-5 ng/mL of isorhamnetin, 1-10 ng/mL of phytic acid, 5-20 ng/mL of insulin-like growth factor-II, 50-150 ng/mL of adenosine phosphate, 20-50 ng/mL of cortisol, 10-20 ng/mL of transforming growth factor beta, 1-5 ng/mL of interleukin IL-1, 2-15 ng/mL of ferrous sulfate and 1-5 mu g/mL of arginine.
In a preferred embodiment of the present invention, the preparation method of the proliferation matrigel in step S3 includes:
Dissolving gelatin in water to obtain a gelatin solution with the mass content of 40-60%, then adding diammonium hydrogen phosphate, heating at 150-200 ℃ for reaction for 1-3 hours, and cooling to room temperature after the reaction is finished to obtain modified gelatin;
Uniformly mixing the obtained modified gelatin with N, N-dimethylformamide and vinyl sulfonate monomer according to the mass ratio of 1:50-100:1-10, reacting for 2-6 hours at 40-60 ℃ in the presence of an initiator, keeping the temperature unchanged, adding a silane coupling agent to react for 1-5 hours, cooling to room temperature after the reaction is finished, standing for 2-6 hours, performing suction filtration, and washing the obtained jelly with deionized water to obtain the proliferation matrigel.
In a preferred embodiment of the invention, the gelatin and the diammonium phosphate are added according to the mass ratio of 1:0.02-0.04.
In a preferred embodiment of the present invention, the initiator is selected from any one of sodium persulfate, potassium persulfate, and sodium persulfate; the addition amount of the initiator is 0.03-0.05 times of the mass of the modified gelatin.
In a preferred embodiment of the present invention, the vinyl sulfonate monomer is selected from at least one of sodium 2-acrylamido-2-methylpropane sulfonate, sodium allylsulfonate, sodium styrenesulfonate, and sodium hydroxysulfonate.
In a preferred embodiment of the present invention, the silane coupling agent is selected from any one of vinyltriethoxysilane, vinyltrimethoxysilane, vinyltris (β -methoxyethoxy) silane, and the amount of the silane coupling agent added is 0.1 to 0.3 times the mass of the modified gelatin.
By means of the technical scheme, the invention has at least the following advantages:
1. The invention provides two culture mediums, one is a primary culture medium and the other is a proliferation culture medium, and the culture medium provided by the invention is free from adding exogenous serum, so that the risk of animal pathogens is effectively avoided, the cost of the culture medium is reduced, and the large-scale culture of cells is facilitated.
2. The culture medium of the invention adds the combined cell growth factors, hormones, phenols, polypeptides, amino acids, antioxidants and the like on the original basic culture medium, and various components are reasonably compatible, which is favorable for maintaining and stimulating proliferation and activity of the fibroblast.
3. The invention also provides a proliferation matrigel used in the proliferation culture process of the fibroblast, the proliferation matrigel of the invention takes gelatin as a raw material, the structure of gelatin molecules is changed through the modification effect of ammonium dihydrogen phosphate at high temperature to obtain modified gelatin, then the modified gelatin is subjected to free radical polymerization reaction under the action of functional monomers, and the silane coupling agent can help the polymer molecules to reform a new three-dimensional space structure, so that the obtained product can keep the activity of the cell in the proliferation process of the fibroblast, reduce the influence of external environment, maintain a better culture environment and improve the survival rate of the cell.
The foregoing description is only an overview of the present invention, and is intended to provide a more thorough understanding of the present invention, and is to be accorded the full scope of the present invention.
Drawings
FIG. 1 shows the proliferation of cells of examples and comparative examples of the present invention;
FIG. 2 shows cell viability of examples of the present invention and comparative examples.
Detailed Description
In order to make the technical means, the creation features, the achievement of the purposes and the effects of the present invention easy to understand, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The DMEM medium used in the following examples is a high sugar version, cat No.: 11965118; f12 medium, cat No.: 31765035 both media were purchased from Gibco corporation. The dispase is dispase II, product number: 17105041; type II collagenase, cat No.: 17101015; cell lyase, cat No.: 88702; trypsin-EDTA is trypsin-EDTA (0.5%), cat# 15400054; the above products were all purchased from moeicose. Gelatin was purchased from Shenzhen Lefu Biotechnology Co. Other materials, reagents, etc., unless otherwise specified, are commercially available.
The cell proliferation and cell viability assays in the examples below were as follows, unless otherwise indicated: taking 1mL of sample, adding 10 mu L of AO dye liquor and 20 mu LPI dye liquor, uniformly mixing, incubating at 4 ℃ for 10min in a dark place, and then placing into a multifunctional cell counter for detection.
Example 1
Preparing a primary culture medium: glutathione 15 mug/mL, hydrocortisone 5 mug/mL, basic fibroblast growth factor 5ng/mL, recombinant human epidermal growth factor 5ng/mL, isorhamnetin 3ng/mL, phytic acid 5ng/mL, insulin-like growth factor-II 15ng/mL, adenosine phosphate 100 ng/mL, cortisol 35 ng/mL, transforming growth factor-beta 15ng/mL, interleukin IL-1 3ng/mL, ferrous sulfate 8 ng/mL and arginine 3 mug/mL are added to DMEM medium, and the mixture is sterilized at 121 ℃ for 25min after uniform mixing.
Preparation of fibroblast culture medium: the DMEM culture medium and F12 culture medium are mixed according to the volume ratio of 1:1, and then added with glutamine 30 ng/mL, dexamethasone 5 mu M, carvacrol 30 ng/mL, fibroblast growth factor 10 mu g/mL, epidermal growth factor 10 mu g/mL, isoprenaline 3 mu M, ferulic acid 3 ng/mL, transferrin 150 mu g/mL, transforming growth factor-beta 5 mu g/mL and hexapeptide-2 ng/mL, and sterilized at 121 ℃ for 25min.
Preparing proliferation matrigel: dissolving gelatin in water to obtain a gelatin solution with the mass content of 50%, adding diammonium hydrogen phosphate, heating at 180 ℃ for reaction for 2 hours, and cooling to room temperature after the reaction is finished to obtain modified gelatin; in the process, gelatin and diammonium phosphate are added according to the mass ratio of 1:0.03. Uniformly mixing the obtained modified gelatin with N, N-dimethylformamide and 2-acrylamide-2-methylpropanesulfonic acid sodium in a mass ratio of 1:75:5, reacting for 4 hours at 50 ℃ in the presence of sodium persulfate (the addition amount is 0.04 times of the mass of the modified gelatin), keeping the temperature unchanged, adding vinyltriethoxysilane (the addition amount is 0.2 times of the mass of the modified gelatin) for reacting for 3 hours, cooling to room temperature after the reaction is finished, standing for 4 hours, and then performing suction filtration on the obtained jelly, washing the obtained jelly with deionized water to obtain the proliferation matrigel.
Preparing digestive enzyme liquid: 95. mu.L of PBS buffer, 1.5. Mu.L of 20 mg/mL type II collagenase, 2. Mu.L of 15 mg/mL cytolytic enzyme, and 1.5. Mu.L of 50 mM sodium chloride. The digestive juice was added in an amount of 100. Mu.L of the digestive juice per 1mg of the skin tissue after ear.
A method of culturing post-aural derived fibroblasts, comprising the steps of:
S1: tissue collection: cutting skin tissue after taking ear under aseptic condition, then placing in digestive enzyme solution, digesting for 30min at 37deg.C, centrifuging after digestion, washing and centrifuging the obtained cells, repeating for 3 times to obtain cell suspension;
S2: primary culture: the cells were resuspended using primary medium to a cell concentration of 3X 10 5 cells/mL and cultured in an incubator containing 5% CO 2 at 37℃for 5 days, with medium being changed every 2 days;
S3: expansion culture of fibroblasts: discarding the culture medium after the primary culture in the step S2 is finished, adding 1mL of dispersing enzyme, standing for 30min at 37 ℃, centrifuging, discarding the supernatant, digesting for 5min by using 1mL of trypsin-EDTA (0.5%), then resuspending cells by using a fibroblast culture medium to obtain 15 mu L of cell suspension with the concentration of 4X 10 5/mu L, adding 30 mu L of proliferation matrigel, mixing, adding 1mL of fibroblast culture medium again after the proliferation matrigel is solidified, and culturing in an incubator with 5% CO 2 and 37 ℃ for 10 days, wherein the culture medium is replaced every 2 days during the culture period.
In the culture process, counting the cell growth every two days, as shown in figure 1; and cell viability was measured on day 10, see FIG. 2.
Example 2
Preparing a primary culture medium: adding 20 mug/mL of glutathione, 1 mug/mL of hydrocortisone, 10ng/mL of basic fibroblast growth factor, 1 ng/mL of recombinant human epidermal growth factor, 5ng/mL of isorhamnetin, 1 ng/mL of phytic acid, 20 ng/mL of insulin-like growth factor-II, 50 ng/mL of adenosine phosphate, 50 ng/mL of cortisol, 10ng/mL of transforming growth factor-beta, 1 ng/mL of interleukin IL-1, 2 ng/mL of ferrous sulfate and 5 mug/mL of arginine into a DMEM culture medium, uniformly mixing, and sterilizing at 121 ℃ for 25min.
Preparation of fibroblast culture medium: the DMEM culture medium and F12 culture medium are mixed according to the volume ratio of 1:2, and then added with glutamine 50 ng/mL, dexamethasone 1 mu M, carvacrol 50 ng/mL, fibroblast growth factor 1 mu g/mL, epidermal growth factor 20 mu g/mL, isoprenaline 1 mu M, ferulic acid 5 ng/mL, transferrin 100 mu g/mL, transforming growth factor-beta 10 mu g/mL and hexapeptide-2 ng/mL, and sterilized at 121 ℃ for 25min.
Preparing proliferation matrigel: dissolving gelatin in water to obtain a gelatin solution with the mass content of 40%, adding diammonium hydrogen phosphate, heating at 200 ℃ for reaction for 1h, and cooling to room temperature after the reaction is finished to obtain modified gelatin; in the process, gelatin and diammonium phosphate are added according to the mass ratio of 1:0.04. Mixing the obtained modified gelatin with N, N-dimethylformamide and sodium styrenesulfonate according to a mass ratio of 1: mixing the materials in a ratio of 50:1 uniformly, reacting for 2 hours at 60 ℃ in the presence of sodium persulfate (the addition amount is 0.03 times of the mass of the modified gelatin), keeping the temperature unchanged, adding vinyltriethoxysilane (the addition amount is 0.1 times of the mass of the modified gelatin) for reacting for 5 hours, cooling to room temperature after the reaction is finished, standing for 6 hours, performing suction filtration, and washing the obtained jelly with deionized water to obtain the proliferation matrigel.
Preparing digestive enzyme liquid: 95. mu.L of PBS buffer, 1.5. Mu.L of 20 mg/mL type II collagenase, 2. Mu.L of 15 mg/mL cytolytic enzyme, and 1.5. Mu.L of 50 mM sodium chloride. The digestive juice was added in an amount of 100. Mu.L of the digestive juice per 1mg of the skin tissue after ear.
A method of culturing post-aural derived fibroblasts, comprising the steps of:
S1: tissue collection: cutting skin tissue after taking ear under aseptic condition, then placing in digestive enzyme solution, digesting for 30min at 37deg.C, centrifuging after digestion, washing and centrifuging the obtained cells, repeating for 3 times to obtain cell suspension;
S2: primary culture: the cells were resuspended using primary medium to a cell concentration of 3X 10 5 cells/mL and cultured in an incubator containing 5% CO 2 at 37℃for 5 days, with medium being changed every 2 days;
S3: expansion culture of fibroblasts: discarding the culture medium after the primary culture in the step S2 is finished, adding 1mL of dispersing enzyme, standing for 30min at 37 ℃, centrifuging, discarding the supernatant, digesting for 5min by using 1mL of trypsin-EDTA (0.5%), then resuspending cells by using a fibroblast culture medium to obtain 15 mu L of cell suspension with the concentration of 4X 10 5/mu L, adding 30 mu L of proliferation matrigel, mixing, adding 1mL of fibroblast culture medium again after the proliferation matrigel is solidified, and culturing in an incubator with 5% CO 2 and 37 ℃ for 10 days, wherein the culture medium is replaced every 2 days during the culture period.
In the culture process, counting the cell growth every two days, as shown in figure 1; and cell viability was measured on day 10, see FIG. 2.
Example 3
Preparing a primary culture medium: glutathione 10 mug/mL, hydrocortisone 10 mug/mL, basic fibroblast growth factor 1ng/mL, recombinant human epidermal growth factor 10 ng/mL, isorhamnetin 1ng/mL, phytic acid 10 ng/mL, insulin-like growth factor-II 5 ng/mL, adenosine phosphate 150 ng/mL, cortisol 20ng/mL, transforming growth factor-beta 20ng/mL, interleukin IL-1 ng/mL, ferrous sulfate 15 ng/mL and arginine 1 mug/mL are added to DMEM medium, and the mixture is sterilized at 121 ℃ for 25min after uniform mixing.
Preparation of fibroblast culture medium: the DMEM culture medium and F12 culture medium are mixed according to the volume ratio of 2:1, and then 10 ng/mL of glutamine, 10 mu M of dexamethasone, 10 ng/mL of carvacrol, 20 mu g/mL of fibroblast growth factor, 1 mu g/mL of epidermal growth factor, 5 mu M of isoprenaline, 1 ng/mL of ferulic acid, 200 mu g/mL of transferrin, 1 mu g/mL of transforming growth factor and 2 ng/mL of hexapeptide are added to be uniformly mixed, and then sterilized at 121 ℃ for 25min.
Preparing proliferation matrigel: dissolving gelatin in water to obtain gelatin solution with mass content of 60%, adding diammonium hydrogen phosphate, heating at 150deg.C for reaction for 3 hr, and cooling to room temperature after reaction to obtain modified gelatin; in the process, gelatin and diammonium phosphate are added according to the mass ratio of 1:0.02. Uniformly mixing the obtained modified gelatin with N, N-dimethylformamide and sodium allylsulfonate according to the mass ratio of 1:100:10, reacting for 6 hours at 40 ℃ in the presence of sodium persulfate (the addition amount is 0.05 times of the mass of the modified gelatin), keeping the temperature unchanged, adding vinyltriethoxysilane (the addition amount is 0.3 times of the mass of the modified gelatin) for reacting for 1 hour, cooling to room temperature after the reaction is finished, standing for 2 hours, and then carrying out suction filtration, and washing the obtained colloid by adopting deionized water to obtain the proliferation matrigel.
Preparing digestive enzyme liquid: 95. mu.L of PBS buffer, 1.5. Mu.L of 20 mg/mL type II collagenase, 2. Mu.L of 15 mg/mL cytolytic enzyme, and 1.5. Mu.L of 50 mM sodium chloride. The digestive juice was added in an amount of 100. Mu.L of the digestive juice per 1mg of the skin tissue after ear.
A method of culturing post-aural derived fibroblasts, comprising the steps of:
S1: tissue collection: cutting skin tissue after taking ear under aseptic condition, then placing in digestive enzyme solution, digesting for 30min at 37deg.C, centrifuging after digestion, washing and centrifuging the obtained cells, repeating for 3 times to obtain cell suspension;
S2: primary culture: the cells were resuspended using primary medium to a cell concentration of 3X 10 5 cells/mL and cultured in an incubator containing 5% CO 2 at 37℃for 5 days, with medium being changed every 2 days;
S3: expansion culture of fibroblasts: discarding the culture medium after the primary culture in the step S2 is finished, adding 1mL of dispersing enzyme, standing for 30min at 37 ℃, centrifuging, discarding the supernatant, digesting for 5min by using 1mL of trypsin-EDTA (0.5%), then resuspending cells by using a fibroblast culture medium to obtain 15 mu L of cell suspension with the concentration of 4X 10 5/mu L, adding 30 mu L of proliferation matrigel, mixing, adding 1mL of fibroblast culture medium again after the proliferation matrigel is solidified, and culturing in an incubator with 5% CO 2 and 37 ℃ for 10 days, wherein the culture medium is replaced every 2 days during the culture period.
In the culture process, counting the cell growth every two days, as shown in figure 1; and cell viability was measured on day 10, see FIG. 2.
Comparative example 1
The only difference between this comparative example and example 1 is the composition of the fibroblast medium components, specifically:
Preparation of fibroblast culture medium: the DMEM culture medium and the F12 culture medium are mixed according to the volume ratio of 1:1, and then the mixed culture medium is added with 2% fetal bovine serum with the volume fraction and uniformly mixed, and then the sterilization treatment is carried out for 25min at 121 ℃.
All other things were consistent with example 1.
In the culture process, counting the cell growth every two days, as shown in figure 1; and cell viability was measured on day 10, see FIG. 2.
Comparative example 2
The only difference between this comparative example and example 1 is the composition of the primary medium, specifically:
preparing a primary culture medium: adding 2% fetal bovine serum into DMEM culture medium, mixing, and sterilizing at 121deg.C for 25min.
All other things were consistent with example 1.
In the culture process, counting the cell growth every two days, as shown in figure 1; and cell viability was measured on day 10, see FIG. 2.
Comparative example 3
This comparative example differs from example 1 in the composition of the primary medium and the fibroblast medium components, specifically:
preparing a primary culture medium: adding 2% fetal bovine serum into DMEM culture medium, mixing, and sterilizing at 121deg.C for 25min.
Preparation of fibroblast culture medium: the DMEM culture medium and the F12 culture medium are mixed according to the volume ratio of 1:1, and then the mixed culture medium is added with 2% fetal bovine serum with the volume fraction and uniformly mixed, and then the sterilization treatment is carried out for 25min at 121 ℃.
All other things were consistent with example 1.
In the culture process, counting the cell growth every two days, as shown in figure 1; and cell viability was measured on day 10, see FIG. 2.
Comparative example 4
The only difference between this comparative example and example 1 is that proliferation matrigel was not added during the expansion culture of fibroblasts.
All other things were consistent with example 1.
In the culture process, counting the cell growth every two days, as shown in figure 1; and cell viability was measured on day 10, see FIG. 2.
Comparative example 5
The only difference between this comparative example and example 1 is that the proliferation matrigel used in the proliferation culture process of fibroblasts was prepared as follows: dissolving gelatin in water to obtain a gelatin solution with the mass content of 50%, adding diammonium phosphate, heating at 180 ℃ for reaction for 2 hours, cooling to room temperature after the reaction is finished, standing for 4 hours, performing suction filtration, and washing the obtained gelatin with deionized water to obtain modified gelatin serving as proliferation matrigel; in the process, gelatin and diammonium phosphate are added according to the mass ratio of 1:0.03.
All other things remain the same as in example 1
In the culture process, counting the cell growth every two days, as shown in figure 1; and cell viability was measured on day 10, see FIG. 2.
Comparative example 6
The only difference between this comparative example and example 1 is that the proliferation matrigel used in the proliferation culture process of fibroblasts was prepared as follows: dissolving gelatin in water to obtain a gelatin solution with the mass content of 50%, uniformly mixing the gelatin solution with N, N-dimethylformamide and 2-acrylamide-2-methylpropanesulfonic acid sodium according to the mass ratio of 1:75:5, reacting for 4 hours at 50 ℃ in the presence of sodium persulfate (the addition amount is 0.04 times of the mass of the modified gelatin), keeping the temperature unchanged, adding vinyltriethoxysilane (the addition amount is 0.2 times of the mass of the modified gelatin) for reacting for 3 hours, cooling to room temperature after the reaction is finished, standing for 4 hours, performing suction filtration, and washing the obtained gelatin with deionized water to obtain the proliferation matrigel.
All other things were consistent with example 1.
In the culture process, counting the growth of cells at regular intervals of two days, as shown in figure 1; and cell viability was measured on day 10, see FIG. 2.
Comparative example 7
The only difference between this comparative example and example 1 is that isorhamnetin was not added to the primary medium, all other things being consistent with example 1.
In the culture process, counting the growth of cells at regular intervals of two days, as shown in figure 1; and cell viability was measured on day 10, see FIG. 2.
Comparative example 8
The only difference between this comparative example and example 1 is that no carvacrol was added to the fibroblast medium, all other things being consistent with example 1.
In the culture process, counting the growth of cells at regular intervals of two days, as shown in figure 1; and cell viability was measured on day 10, see FIG. 2.
Comparative example 9
The only difference between this comparative example and example 1 is that no ferulic acid was added to the fibroblast medium, all other things being consistent with example 1.
In the culture process, counting the growth of cells at regular intervals of two days, as shown in figure 1; and cell viability was measured on day 10, see FIG. 2.
Comparative example 10
The only difference between this comparative example and example 1 is that no hexapeptide was added to the fibroblast medium, all other things being consistent with example 1.
In the culture process, counting the growth of cells at regular intervals of two days, as shown in figure 1; and cell viability was measured on day 10, see FIG. 2.
As shown in fig. 1 and 2, compared with comparative examples 1 to 3, the cell proliferation and viability of the fibroblasts of examples 1 to 3 of the present invention are not greatly different, which means that the culture medium of the present invention can have an equivalent culture effect to that of a fetal bovine serum culture medium. Compared with comparative examples 4-6, the fibroblast cells of examples 1-3 of the invention have better cell proliferation and greater activity in the process of culturing, which shows that the proliferation matrigel has different obvious effects on the proliferation condition and activity of the cells, and the proliferation matrigel can obviously improve the proliferation quantity and activity of the cells by adding the proliferation matrigel. Compared with comparative examples 7-10, the fibroblast cells of examples 1-3 of the present invention have better cell proliferation and greater viability during the culture process, which means that the primary culture medium and the fibroblast cell culture medium of the present invention are the optimal combination obtained after optimization, and lack of any one of them will affect cell growth and activity.
While the invention has been described with respect to preferred embodiments, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention, and that any such changes and modifications as described in the above embodiments are intended to be within the scope of the invention.
Claims (10)
1. A post-aural skin-derived fibroblast culture medium, wherein the composition of the culture medium comprises: a basal medium and an exogenous additive composition, the basal medium comprising one of:
(i) DMEM medium;
(ii) F12 medium;
(iii) A mixed culture medium consisting of a DMEM culture medium and an F12 culture medium according to a volume ratio of 1-2:1-2;
the exogenous additive composition comprises 10-50 ng/mL of glutamine, 1-10 mu M of dexamethasone, 10-50 ng/mL of carvacrol, 1-20 mu g/mL of fibroblast growth factor, 1-20 mu g/mL of epidermal growth factor, 1-5 mu M of isoprenaline, 1-5 ng/mL of ferulic acid, 100-200 mu g/mL of transferrin, 1-10 mu g/mL of transforming growth factor beta and 1-5 ng/mL of hexapeptide.
2. The method of preparing a skin-friendly fibroblast culture medium according to claim 1, wherein the method comprises:
Providing one of the following basal media:
(i) DMEM medium
(Ii) F12 medium;
(iii) A mixed culture medium consisting of a DMEM culture medium and an F12 culture medium according to a volume ratio of 1-2:1-2;
Adding the exogenous additive composition into the basic culture medium, mixing, and sterilizing at 121-122 deg.C for 20-25min.
3. A method for culturing post-aural derived fibroblasts using the post-aural skin derived fibroblast medium of claim 1, said method comprising the steps of:
S1: tissue collection: cutting skin tissue after taking ear under aseptic condition, then placing in digestive enzyme solution for digestion, centrifuging after digestion, washing and centrifuging the obtained cells, repeating for 3 times to obtain cell suspension;
S2: primary culture: the primary culture medium is used for resuspension of cells, the cell concentration is 2-4 multiplied by 10 5/mL, and the cells are cultured in an incubator containing 5% CO 2 and at 37 ℃ for 5-10 days, wherein the culture medium is changed every 2-3 days;
S3: expansion culture of fibroblasts: discarding the culture medium after the primary culture in the step S2 is finished, adding 1-2 mL of dispersing enzyme, standing for 30min at 37 ℃, centrifuging, discarding the supernatant, digesting for 5min by using 1-2 mL of trypsin-EDTA, then resuspending cells by using a fibroblast culture medium to obtain 10-20 mu L of cell suspension with the concentration of 1-4X 10 5/mu L, adding 25-40 mu L of proliferation matrigel, mixing, adding 1-2 mL of fibroblast culture medium again after the proliferation matrigel is solidified, and finally culturing in an incubator containing 5% CO 2 and 37 ℃ for 10-15 days, wherein the culture medium is replaced every 2-3 days.
4. The method of culturing post-aural source of fibroblasts of claim 3, wherein said digestive enzyme liquid composition in step S1 is: 92-95 mu L of PBS buffer solution, 1.5-2 mu L of 20 mg/mL type II collagenase, 2-3 mu L of 15 mg/mL cell lyase and 1.5-3 mu L of 50mM sodium chloride;
the digestive enzyme solution is added according to the amount of adding 100 mu L of the digestive enzyme solution to 1mg of skin tissue behind the ear;
the digestion conditions are: 37℃for 30min.
5. A method of culturing post-aural source of fibroblasts as described in claim 3 wherein said primary medium in step S2 comprises: a basal medium and an exogenous additive composition, the basal medium comprising one of:
(i) DMEM medium;
(ii) F12 medium;
(iii) A mixed culture medium consisting of a DMEM culture medium and an F12 culture medium according to a volume ratio of 1-2:1-2;
The exogenous additive composition comprises 10-20 mu g/mL of glutathione, 1-10 mu M of hydrocortisone, 1-10 ng/mL of alkaline fibroblast growth factor, 1-10 ng/mL of recombinant human epidermal growth factor, 1-5 ng/mL of isorhamnetin, 1-10 ng/mL of phytic acid, 5-20 ng/mL of insulin-like growth factor-II, 50-150 ng/mL of adenosine phosphate, 20-50 ng/mL of cortisol, 10-20 ng/mL of transforming growth factor beta, 1-5 ng/mL of interleukin IL-1, 2-15 ng/mL of ferrous sulfate and 1-5 mu g/mL of arginine.
6. The method of culturing post-aural source of fibroblasts of claim 3, wherein said method of preparing proliferation matrigel in step S3 comprises:
Dissolving gelatin in water to obtain a gelatin solution with the mass content of 40-60%, then adding diammonium hydrogen phosphate, heating at 150-200 ℃ for reaction for 1-3 hours, and cooling to room temperature after the reaction is finished to obtain modified gelatin;
Uniformly mixing the obtained modified gelatin with N, N-dimethylformamide and vinyl sulfonate monomer according to the mass ratio of 1:50-100:1-10, reacting for 2-6 hours at 40-60 ℃ in the presence of an initiator, keeping the temperature unchanged, adding a silane coupling agent to react for 1-5 hours, cooling to room temperature after the reaction is finished, standing for 2-6 hours, performing suction filtration, and washing the obtained jelly with deionized water to obtain the proliferation matrigel.
7. The method for culturing post-aural source of fibroblasts according to claim 6, wherein said gelatin and diammonium phosphate are added in a mass ratio of 1:0.02-0.04.
8. The method of culturing post-aural source of fibroblasts of claim 6, wherein said initiator is selected from any one of sodium persulfate, potassium persulfate, and sodium persulfate; the addition amount of the initiator is 0.03-0.05 times of the mass of the modified gelatin.
9. The method of culturing post-aural source of fibroblasts of claim 6, wherein said vinylsulfonate monomer is selected from at least one of sodium 2-acrylamido-2-methylpropanesulfonate, sodium allylsulfonate, sodium styrenesulfonate, and sodium hydroxysulfonate.
10. The method for culturing post-aural source of fibroblasts according to claim 6, wherein said silane coupling agent is selected from any one of vinyltriethoxysilane, vinyltrimethoxysilane and vinyltris (β -methoxyethoxy) silane, and the addition amount of said silane coupling agent is 0.1 to 0.3 times the mass of the modified gelatin.
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