CN114181889B - Primary sweat gland cell conditioned medium and primary sweat gland cell culture method - Google Patents
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Abstract
The application provides a primary sweat gland cell conditioned medium and a primary sweat gland cell culture method, wherein the primary sweat gland conditioned medium comprises the following components: cell basal medium, serum, glutamax, buffer, cell culture additives, penicillin/streptomycin, epidermal growth factor, basic fibroblast factor, nonessential amino acids, insulin, ROCK inhibitors, and NF- κb agonists. The application adds the epidermal cell growth factor, the basic fibroblast growth factor, the NF- κB agonist and the ROCK inhibitor into the primary sweat gland cell basic culture medium. Through the combination of the growth factors and the small molecules, the in-vitro long-term expansion culture of the primary sweat gland cells of the human body under the two-dimensional condition is realized, the biological characteristics of the primary sweat gland cells are maintained in passage, the aging process of the primary sweat gland cells is delayed, and preconditions are provided for rapidly preparing the sweat gland cells of the high-quality implantable human body in a large quantity and meeting the requirements of large-scale clinical treatment.
Description
Technical Field
The application relates to the technical field of sweat gland cell culture, in particular to a primary sweat gland cell conditioned medium and a primary sweat gland cell culture method for realizing in-vitro long-term culture and amplification of human sweat gland cells.
Background
Sweat glands are one of the most important accessory organs of the skin, and play a role in the aspects of metabolism of substances, balance of water and electrolyte, maintenance of homeostasis in the body, and the like. However, sweat glands have limited regeneration capacity, and scar repair of the skin after wounds further damages the structure and function of sweat glands, so that the skin cannot sweat normally, and the body temperature is regulated and unbalanced, thereby bringing huge physiological and psychological injuries to patients.
At present, strategies such as growth factors, gene therapy and the like are mainly applied to activate the regeneration potential of in-situ tissue cells of an organism so as to realize the functional repair of the wounded skin. However, the number of sweat gland cells remained in the wound surface after large-area burn and wound is insufficient, and the regeneration potential is limited. The cascade reaction of wound repair cannot be started, and the structure and the function of damaged sweat glands are repaired. Therefore, autogenous or allogenic sweat gland transplantation becomes an ideal choice, and brings a new opportunity for solving the rehabilitation of patients with large-area deep burn wounds and recovering the sweat discharging function. Nevertheless, the source of donor tissue is short, the primary sweat gland acquisition process is complicated, the in vitro proliferation capacity of sweat gland cells is weak, and long-term expansion culture and maintenance of biological characteristics are difficult. These drawbacks prevent further clinical popularization of the related art. Therefore, how to effectively amplify and obtain sweat gland cells with high quality and high purity in vitro so as to meet the requirement of large-scale clinical treatment is one of the most critical scientific problems in clinical application of sweat gland transplantation, and is one of the difficult problems that research and development of sweat gland-containing tissue engineering skin is necessary to overcome.
Disclosure of Invention
The embodiment of the application provides a primary sweat gland cell conditioned medium and a primary sweat gland cell culture method for realizing in-vitro long-term culture and amplification of human sweat gland cells, which are used for solving the problems that sweat gland cells isolated and cultured in vitro in the prior art are weak in proliferation capacity, difficult to be cultured in a long-term amplification way and maintain biological characteristics.
In order to solve the above problems, the present application is achieved as follows:
in a first aspect, embodiments of the present application provide a primary sweat gland cell conditioned medium comprising a cell basal medium, serum, glutamax, buffer, cell culture additive, penicillin/streptomycin, epidermal growth factor, basic fibroblast growth factor, nonessential amino acids, insulin, ROCK inhibitor, and NF- κb agonist.
Optionally, in the primary sweat gland cell conditioned medium, the NF- κB agonist is phorbol 12-tetradecanoate 13-acetate.
Optionally, the phorbol 12-tetradecanoate 13-acetate in the primary sweat gland cell conditioned medium is 0.01-0.05 mug/mL
Optionally, in the primary sweat gland cell conditioned medium, the cell basal medium is a DMEM/F12 medium.
Optionally, in the primary sweat gland cell conditioned medium, the cell culture additive is B27.
Optionally, in the primary sweat gland cell conditioned medium, the ROCK inhibitor is Y-33075.
Optionally, in the primary sweat gland cell conditioned medium, the content of the epidermal cell growth factor is 20-50 ng/mL, the content of the basic fibroblast growth factor is 10-20 ng/mL, and the content of the Y-33075 is 0.5-1 mu M.
In a second aspect, an embodiment of the present application provides a conditioned medium for achieving in vitro long-term culture and expansion of human sweat gland cells, comprising the steps of:
taking a cell culture dish, and adding the primary sweat gland cell conditioned medium;
adding primary sweat gland tissue into the cell culture dish, and supplementing the primary sweat gland cell conditioned medium after the sweat gland tissue adheres to the wall, and changing liquid once every 2-3 days;
and when the fusion degree of sweat gland cells reaches 70-80%, performing digestion and passage to obtain amplified sweat gland cells.
Optionally, in the method, before the step of adding the primary sweat gland cell conditioned medium, the method further comprises:
adding collagen into the cell culture dish, and placing in CO 2 After the coating treatment in the incubator, the collagen was sucked off and washed with phosphate buffer saline.
Optionally, in the method, the digestion and passage process comprises:
discarding the culture medium, washing with phosphate buffer solution, adding cell digestive juice, and placing in CO 2 Digestion is carried out in an incubator, and the culture medium is added to stop digestion when sweat gland cells float;
mixing the culture solution after digestion termination, then carrying out passage according to the ratio of 1:2, adding the culture solution into a cell culture dish after coating treatment, and then placing the cell culture dish in CO 2 Culturing in an incubator, and changing the liquid by using the primary sweat gland cell conditioned medium after sweat gland cells adhere to the wall, wherein the liquid is changed once every 2-3 days.
The embodiment of the application has the following advantages:
in the embodiment of the application, the epidermal growth factor, the fibroblast cell factor and the ROCK inhibitor are added in the primary sweat gland cell conditioned medium, and the primary sweat gland cells are treated by the combination of the epidermal growth factor, the basic fibroblast growth factor, the ROCK inhibitor and the NF- κB agonist, so that the human primary sweat gland cells can be amplified and cultured in vitro for a long time, and the biological characteristics of the human primary sweat gland cells are maintained in passage, thereby providing preconditions for rapidly manufacturing high-quality transplantable human sweat gland cells in a large scale and meeting the requirements of large-scale clinical treatment.
Drawings
Fig. 1 is a morphology of first generation sweat gland cells after in vitro expansion on day 3, bar=200 um;
fig. 2 is a morphology of first generation sweat gland cells after in vitro expansion on day 5, bar=200 um;
fig. 3 is a morphology of second generation sweat gland cells after in vitro expansion on day 15, bar=200 um;
fig. 4 is a morphology of second generation sweat gland cells after in vitro expansion on day 15, bar=50 um;
fig. 5 is a third generation sweat gland cell morphology after in vitro expansion on day 30, bar=200 um;
fig. 6 is a third generation sweat gland cell morphology after in vitro expansion on day 30, bar=50um;
fig. 7 is a fourth generation sweat gland cell morphology after in vitro expansion at day 33, bar=200 um;
fig. 8 is a fourth generation sweat gland cell morphology after in vitro expansion at day 40, bar=200 um;
FIG. 9 is a graph showing the comparison of the effect of beta-galactosidase staining of primary sweat gland cells cultured for 10 days outside different culture substrates;
FIG. 10 is a graph showing the comparison of the effect of beta-galactosidase on primary sweat gland cells cultured in vitro for different times in the culture medium of the example of the present application;
FIG. 11 is a graph showing the result of CK5 PCR of primary sweat gland cells;
FIG. 12 is a graph showing the result of CK18 PCR on primary sweat gland cells.
Detailed Description
In order that the above-recited objects, features and advantages of the present application will become more readily apparent, a more particular description of the application will be rendered by reference to the appended drawings and appended detailed description.
The primary sweat gland cell conditioned medium provided by the embodiment of the application comprises a cell basal medium, serum, a glutamine additive (Glutamax), a buffer solution, a cell culture additive, penicillin/streptomycin double antibody, an epidermal cell growth factor, an alkaline fibroblast growth factor, nonessential amino acid, an insulin ROCK inhibitor and an NF- κB agonist.
Wherein, both Epidermal Growth Factor (EGF) and basic Fibroblast Growth Factor (FGF) can activate extracellular signal regulated protein kinase (ERK) signal pathway, EGF plays a positive role in the morphogenesis and homeostasis of sweat glands; FGF regulates the growth and development stages of epithelial tissue organ, and binds to epithelial cell surface receptor as specific paracrine medium to stimulate epithelial cell proliferation; the ROCK inhibitor can activate the continuous expression of growth promoting genes IGF2 and ALDH1A1 and inhibit apoptosis, so that proliferation and survival of epithelial stem cells are promoted.
Therefore, in the cell culture medium provided by the embodiment of the application, the epidermal growth factor, the fibroblast factor, the ROCK inhibitor and the NF- κB agonist are combined, so that the aging process of the primary sweat gland cells can be delayed under the condition of maintaining the self-steady state of the primary sweat gland cells, the extracted primary sweat glands quickly climb out of the sweat gland cells and expand, the biological characteristics of the primary sweat gland culture process can be maintained, and the problems of small quantity of sweat gland cells, long culture time and the like obtained by the traditional tissue block culture method are solved.
Alternatively, the serum concentration is 5-10% by volume, the Glutamax concentration is 1-2 mM, the buffer concentration is 5-10 mM, the cell culture additive concentration is 0.5-1%, the penicillin/streptomycin diabody concentration is 1-2% by volume, the non-essential amino acid concentration is 1-2% by volume, and the insulin concentration is 5-10 μg/mL.
Optionally, in the primary sweat gland cell conditioned medium provided by the embodiment of the application, the ROCK inhibitor may specifically be Y-33075.
Optionally, in the primary sweat gland cell conditioned medium, the content of the epidermal cell growth factor is 20-50 ng/mL, the content of the basic fibroblast growth factor is 10-20 ng/mL, and the content of Y-33075 is 0.5-1 mu M. Wherein, too low a concentration of each component has no effect, while too high a concentration inhibits cell growth.
Optionally, in one embodiment, the primary sweat gland cell conditioned medium provided by the examples of the application further comprises an NF- κB agonist. The NF-. Kappa.pathway, downstream of EDA/EDAR signals, activates the genome components of Shh, cyclin D1, fox gene family, etc., that function at various stages of sweat gland development. Therefore, the NF- κB agonist can replace EDA to maintain the biological characteristic in sweat gland cell in-vitro amplification culture system.
Alternatively, in one embodiment, the NF- κB agonist is phorbol 12-tetradecanoate 13-acetate (PMA) and is effective to maintain sweat gland cell characteristics.
Alternatively, in one embodiment, the PMA is present in an amount of 0.01 to 0.05 μg/mL.
Alternatively, in the primary sweat gland cell conditioned medium described above, the cell basal medium is DMEM/F12 medium.
Optionally, in the primary sweat gland cell conditioned medium, the cell culture additive is a B27 additive, so that nutrition can be provided and cell growth can be maintained.
The embodiment of the application also provides a primary sweat gland cell culture method, which comprises the following steps:
s1, taking a cell culture dish, and adding the primary sweat gland cell conditioned medium;
s2, adding the primary sweat gland tissue into the cell culture dish, and supplementing the primary sweat gland cell conditioned medium after the sweat gland tissue adheres to the wall, and changing liquid once every 2-3 days;
s3, when the fusion degree of sweat gland cells reaches 70% -80%, digestion and passage are carried out, and amplified sweat gland cells are obtained.
According to the method provided by the embodiment of the application, the primary sweat gland cells are cultured by adding the epidermal cell growth factor, the basic fibroblast growth factor, the ROCK inhibitor and the NF-kappa B agonist into the primary sweat gland cell conditioned medium, and can be treated by the combination of the epidermal cell growth factor, the basic fibroblast growth factor, the ROCK inhibitor and the NF-kappa B agonist, so that the long-term expansion culture of the human primary sweat gland cells in vitro is realized, the biological characteristics of the human primary sweat gland cells are maintained in passage, and the precondition is provided for rapidly manufacturing high-quality implantable human sweat gland cells in a large scale and meeting the requirements of large-scale clinical treatment.
Optionally, in step S1 above, collagen is added to the cell culture dish before the step of adding the primary sweat gland cell culture medium, and then placed in CO 2 After the coating treatment in the incubator, the collagen was sucked off and washed with phosphate buffer saline.
The collagen is used for coating the cell culture dish, so that sweat gland tissues can be quickly attached to the cell culture dish and climb out of sweat gland cells, and the culture process is quickened. The collagen may be IV collagen, and the CO 2 The temperature of the incubator is 37 ℃ and CO 2 The volume fraction is 5%, and the coating treatment time is 10-15 minutes.
Alternatively, in the step S2, the primary sweat gland tissue may be sucked by a 1mL gun head and directly beaten into the cell culture dish to which the culture medium is added; then the adherent growth of sweat gland tissues can be completed after 8 hours.
Optionally, in one embodiment, the step S3 specifically includes steps S301 to S302:
step S301, discarding the culture medium, washing with phosphate buffer solution, adding cell digestive juice, and placing in CO 2 Digestion is carried out in an incubator, and the culture medium is added to stop digestion when sweat gland cells float;
step S302, uniformly mixing the culture solution after digestion termination, then carrying out passage according to a ratio of 1:2, adding the culture solution into a coated cell culture dish, and then placing the culture solution in CO 2 Culturing in an incubator, and changing the liquid by using the primary sweat gland cell culture medium after sweat gland cells adhere to the wall, wherein the liquid is changed once every 2-3 days.
In the step S301, the sweat gland cell conditioned medium is firstly discarded, then the sweat gland cell conditioned medium is washed once by phosphate buffer salt solution, then 0.5-1 mL of cell digestive juice solution such as accutase and the like is added, and then the mixture is placed in CO with the volume fraction of 5% at 37 DEG C 2 Digestion is carried out in an incubator, and the digestion is stopped by adding 2mL of the culture medium when the cells float in a large amount after observing every 5 minutes;
in the step S302, the culture solution after digestion is evenly mixed by a light blowing method, then passage is carried out according to the ratio of 1:2, the mixture is added into a coated cell culture dish, and then the mixture is placed in CO with the volume fraction of 5% at the temperature of 37 DEG C 2 Culturing in an incubator, and after about 8 hours, changing the liquid by using the culture medium, and changing the liquid once every 2-3 days to realize digestion passage.
In the above embodiment, since all sweat gland cells cannot be digested at a time, the cell culture dish after digestion may be washed once with phosphate buffer solution, and then the above primary sweat gland cell conditioned medium may be added to continue the culture.
The present application will be described in detail with reference to examples.
Example 1
Preparation of primary sweat gland cell culture medium:
a primary sweat gland cell conditioned medium was prepared by adding final concentration of 10% serum, 1mM Glutamax, 5mM HEPES, 01% B27, 1% penicillin/streptomycin, 50ng/mL EGF, 20ng/mL FGF, 1% NEAA, 10. Mu.g/mL insulin, 1. Mu.M Y-33075, 0.05. Mu.g/mL PMA to the cell basal medium DMEM/F12.
Example 2
Long-term expansion of primary sweat gland cells:
(1) Taking out 6cm cell culture dish, adding 2mL IV collagen, placing at 37deg.C, and CO with volume fraction of 5% 2 Coating in an incubator for 15 minutes, taking out the cell culture dish, sucking out the IV collagen, washing the cell culture dish once with PBS, and then adding the culture medium prepared in the embodiment 1 to ensure that the culture medium does not pass through the bottom of the dish.
(2) The primary sweat gland tissue sucked by a 1mL gun head is directly beaten into a cell culture dish added with the culture medium, after the sweat gland tissue adheres to the wall (about 8 hours), the culture medium is added, then the liquid is changed once every 2 days, and when the sweat gland cell fusion degree is observed to reach 80%, digestion and passage can be carried out.
(3) Removing sweat gland culture medium, washing with PBS once, adding 1mL of accutase solution, standing at 37deg.C with volume fraction of 5% CO 2 Digestion is carried out in an incubator, the cells are observed once every 5 minutes, when floating in a large quantity, 2mL of the culture medium prepared in the example 1 is added to stop digestion, gently blown and evenly mixed, then passage is directly carried out according to the ratio of 1:2, the cells are added into a 6cm cell culture dish after being coated, and the cells are placed in CO with the volume fraction of 5 percent at 37 DEG C 2 Culturing in an incubator, and after sweat gland cells adhere to the wall, timely changing the liquid with the culture medium, and then changing the liquid once every 2 days.
The form of the first generation sweat gland cells after in vitro expansion on the 3 rd day is shown in the figure 1, and the original sweat gland cells are complete in form, which shows that the growth speed is higher;
the morphology of the first generation sweat gland cells after the 5 th day of in vitro expansion is shown in figure 2, which shows that the first generation sweat gland cells grow rapidly;
the form of the second generation sweat gland cells after in vitro expansion on the 15 th day is shown in fig. 3 and 4, and the form of the sweat gland cells is still complete and apoptosis is not generated;
the form of the third generation sweat gland cells after in vitro expansion on the 30 th day is shown in fig. 5 and 6, and the sweat gland cells are still complete in form and high in cell activity;
the morphology of the fourth generation sweat gland cells after the 33 th day of in vitro expansion is shown in fig. 7, and the sweat gland cells still do not lose the morphology of the first generation sweat gland cells;
the morphology of fourth generation sweat gland cells after in vitro amplification on day 40 is shown in fig. 8, and it can be seen that the refractive index of the cells is still very strong, and apoptosis phenomenon does not occur.
Comparative example 1
Configuration of the culture medium:
a cell culture medium was prepared by adding 10% serum and 1% penicillin/streptomycin to the minimal medium DMEM/F12.
Comparative example 2
Expansion of primary sweat gland cells:
(1) Taking out 6cm cell culture dish, adding 2mL IV collagen, placing at 37deg.C, and CO with volume fraction of 5% 2 Coating in an incubator for 15 minutes, taking out the cell culture dish, sucking out the IV collagen, washing the cell culture dish once with PBS, and then adding the culture medium prepared in the comparative example 1 to ensure that the culture medium does not pass through the bottom of the dish.
(2) The primary sweat gland tissue sucked by a 1mL gun head is directly beaten into a cell culture dish added with the culture medium, after the sweat gland tissue adheres to the wall, the culture medium is supplemented, then the liquid is changed once every 2 days, and when the sweat gland cell fusion degree is observed to reach 80%, digestion and passage can be carried out.
(3) Removing sweat gland culture medium, washing with PBS once, adding 1mL of accutase solution, standing at 37deg.C with volume fraction of 5% CO 2 Digestion is carried out in an incubator, the digestion is carried out once every 5 minutes, 2mL of the culture medium prepared in the comparative example 1 is added to stop digestion when the cells float in a large amount, after gentle blowing and uniform mixing, passage is directly carried out according to the ratio of 1:2, the cells are added into a 6cm cell culture dish after being coated, and the cells are placed in CO with the volume fraction of 5 percent at 37 DEG C 2 Culturing in incubator, changing liquid with the culture medium after sweat gland cells adhere to wall, and then every 2 daysThe liquid is changed once.
Experimental effect:
the dyeing effects of the beta-galactosidase of the primary sweat gland cells cultured in vitro for 10 days in example 2 and the primary sweat gland cells cultured in vitro for 10 days in comparative example 2 are respectively shown as E and F in figure 9, which shows that the NF- κB agonist can delay the aging of the primary sweat gland cells in the in vitro long-term expansion culture;
the primary sweat gland cells cultured in vitro for 10 days and 40 days in example 2 are subjected to beta-galactosidase staining, and the staining effects are shown as D10 and D40 in FIG. 10 respectively, which shows that the NF- κB agonist can delay the aging of the primary sweat gland cells in the in vitro long-term expansion culture;
the CK5 real-time quantitative PCR results of the primary sweat gland cells just separated, the primary sweat gland cells after 10 days of in vitro culture in example 2, and the primary sweat gland cells after 10 days of in vitro culture in comparative example 2 are shown in fig. 11, A, B, C, respectively;
the CK18 real-time quantitative PCR results of the primary sweat gland cells just separated, the primary sweat gland cells after 10 days of in vitro culture in example 2 and the primary sweat gland cells after 40 days of in vitro culture in example 2 are shown as D0, D10 and D40 in FIG. 12 respectively;
the combination of epidermal growth factor, basic fibroblast growth factor, NF- κB agonist and ROCK inhibitor can maintain the biological characteristic of primary sweat gland cell in vitro long-term expansion culture as shown in FIGS. 11 and 12.
The above results illustrate: the primary sweat gland cell conditioned medium provided by the application can enable the extracted primary sweat glands to quickly climb out of sweat gland cells and quickly expand, overcomes the difficulties of small number of sweat gland cells, long culture time and the like obtained by the traditional tissue block culture method, and can maintain the biological characteristics of the primary sweat glands in the culture process for a long time.
The primary sweat gland cell condition culture medium and the primary sweat gland cell culture method provided by the application are described in detail, and specific examples are applied to illustrate the principle and the implementation mode of the application, and the description of the examples is only used for helping to understand the method and the core idea of the application; meanwhile, as those skilled in the art will have variations in the specific embodiments and application scope in accordance with the ideas of the present application, the present description should not be construed as limiting the present application in view of the above.
Claims (4)
1. A primary sweat gland cell conditioned medium is prepared by adding 10% serum, 1mM Glutamax, 5mM buffer, 1% B27, 1% penicillin/streptomycin, 50ng/mL epidermal cell growth factor, 20ng/mL basic fibroblast growth factor, 1% nonessential amino acid, 10 μg/mL insulin, 1 μg/mL Y-33075, 0.05 μg/mL phorbol 12-tetradecanoate 13-acetate to a cell basal medium DMEM/F12.
2. A method for culturing primary sweat gland cells, comprising the steps of:
taking a cell culture dish, and adding the primary sweat gland cell conditioned medium according to claim 1;
adding primary sweat gland tissue into the cell culture dish, and supplementing the primary sweat gland cell conditioned medium after the sweat gland tissue adheres to the wall, and changing liquid once every 2-3 days;
and when the fusion degree of sweat gland cells reaches 70-80%, performing digestion and passage to obtain amplified sweat gland cells.
3. The method of claim 2, wherein prior to the step of adding the primary sweat gland cell conditioned medium, the method further comprises:
adding collagen into the cell culture dish, and placing in CO 2 After the coating treatment in the incubator, the collagen was sucked off and washed with phosphate buffer saline.
4. A method according to claim 3, wherein the digestion passaging process comprises:
discarding the culture medium, washing with phosphate buffer solution, adding cell digestive juice, and placing in CO 2 Digestion is carried out in an incubator, and when the cells float, the primary sweat gland cell conditioned medium is added to terminate the digestion;
mixing the culture solution after digestion termination, then carrying out passage according to the ratio of 1:2, adding the culture solution into a cell culture dish after coating treatment, and then placing the cell culture dish in CO 2 Culturing in an incubator, and changing the liquid by using the primary sweat gland cell culture medium after sweat gland cells adhere to the wall, wherein the liquid is changed once every 2-3 days.
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