CN114181889A - Primary sweat gland cell conditioned medium and primary sweat gland cell culture method - Google Patents
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Abstract
The invention provides a primary sweat gland cell conditioned medium and a primary sweat gland cell culture method, wherein the primary sweat gland conditioned medium comprises: cell basal medium, serum, Glutamax, buffer solution, cell culture additive, penicillin/streptomycin, epidermal growth factor, basic fibroblast factor, non-essential amino acid, insulin, ROCK inhibitor and NF-kappa B agonist. In the invention, epidermal growth factor, basic fibroblast growth factor, NF-kB agonist and ROCK inhibitor are added in a primary sweat gland cell basic culture medium. The combination of the growth factors and the small molecules realizes the in-vitro long-term amplification culture of the human primary sweat gland cells under the two-dimensional condition, maintains the biological characteristics of the human primary sweat gland cells in passage, delays the aging process of the primary sweat gland cells, and provides a precondition for rapidly preparing a large amount of high-quality human sweat gland cells which can be transplanted, and meeting the requirement of large-scale clinical treatment.
Description
Technical Field
The invention 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 significant role in the aspects of substance metabolism, water electrolyte balance, maintenance of homeostasis of the body and the like. However, the regeneration capacity of sweat glands is limited, and the scar repair of the skin after trauma further destroys the structure and the function of the sweat glands, so that the skin can not sweat normally, the body temperature is adjusted to be unbalanced, and huge physical and psychological damage is brought to patients.
Currently, strategies such as growth factors and gene therapy 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, after large-area burn and trauma, the number of sweat gland cells remained in the wound surface is insufficient, and the regeneration potential is limited. The cascade reaction of wound repair can not be started, and the structure and the function of the damaged sweat gland can not be repaired. Therefore, autologous or allogeneic sweat gland transplantation becomes an ideal choice, and a new opportunity is brought for solving the rehabilitation of patients with large-area deep burn and trauma and recovering the perspiration function of the patients. However, the source of donor tissues is short, the acquisition process of primary sweat glands is complicated, the multiplication capacity of sweat gland cells in vitro is weak, and the long-term amplification culture and the maintenance of the biological characteristics are difficult. These drawbacks hinder further clinical popularization of the related art. Therefore, how to effectively amplify and obtain high-quality and high-purity sweat gland cells in vitro to meet the requirement of large-scale clinical treatment is one of the most critical scientific problems in the clinical application of sweat gland transplantation and one of the difficult problems that the development of sweat gland-containing tissue engineering skin must overcome.
Disclosure of Invention
The embodiment of the invention provides a primary sweat gland cell conditioned medium and a primary sweat gland cell culture method for realizing the in vitro long-term culture and amplification of human sweat gland cells, and aims to solve the problems that in the prior art, sweat gland cells which are subjected to in vitro separation culture have weak proliferation capacity, are difficult to perform long-term amplification culture and maintain the biological characteristics of the sweat gland cells.
In order to solve the above problems, the present application is implemented as follows:
in a first aspect, the embodiments of the present invention provide a conditioned medium of primary sweat gland cells, wherein the conditioned medium includes a cell basal medium, serum, Glutamax, a buffer, a cell culture additive, penicillin/streptomycin, an epidermal growth factor, a basic fibroblast growth factor, a non-essential amino acid, insulin, a ROCK inhibitor, and an NF- κ B agonist.
Optionally, in the conditioned medium of primary sweat gland cells, the NF- κ B agonist is phorbol 12-myristate 13-acetate.
Optionally, in the conditioned medium of primary sweat gland cells, phorbol 12-myristate 13-acetate is 0.01-0.05 μ g/mL
Optionally, in the primary sweat gland cell conditioned medium, the cell basal medium is DMEM/F12 medium.
Optionally, in the conditioned medium of primary sweat gland cells, the cell culture additive is B27.
Optionally, in the conditioned medium of primary sweat gland cells, the ROCK inhibitor is Y-33075.
Optionally, in the primary sweat gland cell conditioned medium, the content of the epidermal 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 μ M.
In a second aspect, the embodiments of the present invention provide a conditioned medium for realizing in vitro long-term culture and amplification of human sweat gland cells, wherein the conditioned medium comprises the following steps:
taking a cell culture dish, and adding the primary sweat gland cell conditioned medium;
adding primary sweat gland tissues into the cell culture dish, supplementing the primary sweat gland cell conditioned medium after the sweat gland tissues adhere to the wall, and changing the liquid once every 2-3 days;
and when the fusion degree of the sweat gland cells reaches 70% -80%, carrying out digestion passage to obtain the expanded sweat gland cells.
Optionally, in the method, prior to the step of adding the conditioned medium of primary sweat gland cells, the method further comprises:
adding collagen to the cell culture dish, and then placing in CO2After coating treatment in the incubator, collagen is aspirated off and washed with phosphate buffered saline.
Optionally, in the method, the process of digestion passage comprises:
removing culture medium, washing with phosphate buffered saline solution, adding cell digestive juice, and placing in CO2Digesting in an incubator, and adding the culture medium to stop digesting 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 CO2And (3) culturing in an incubator, after the sweat gland cells adhere to the wall, changing the liquid by using the primary sweat gland cell conditioned medium, and changing the liquid once every 2-3 days.
The embodiment of the invention has the following advantages:
in the embodiment of the invention, epidermal growth factor, fibroblast factor and ROCK inhibitor are added in the conditioned medium of primary sweat gland cells, 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 NF-kB agonist so as to realize the long-term amplification culture of human primary sweat gland cells in vitro and maintain the biological characteristics of the human primary sweat gland cells in passage, thereby providing a precondition for rapidly manufacturing high-quality and transplantable human sweat gland cells in large quantity and meeting the requirement of large-scale clinical treatment.
Drawings
Fig. 1 is a first generation sweat gland cell morphology map after day 3 of in vitro expansion, bar 200 um;
fig. 2 is a first generation sweat gland cell morphology map after day 5 of in vitro expansion, bar 200 um;
fig. 3 is a morphogram of second generation sweat gland cells after 15 days of in vitro expansion, bar 200 um;
fig. 4 is a morphogram of second generation sweat gland cells after day 15 of in vitro expansion, bar 50 um;
FIG. 5 is a diagram of the morphology of third generation sweat gland cells after 30 days of in vitro amplification, bar 200 um;
FIG. 6 is a diagram of the morphology of third generation sweat gland cells after 30 days of in vitro amplification, bar 50 um;
fig. 7 is a morphogram of fourth generation sweat gland cells after 33 days of in vitro expansion, bar 200 um;
fig. 8 is a morphogram of fourth generation sweat gland cells after day 40 of in vitro expansion, bar 200 um;
FIG. 9 is a comparison graph of the staining effect of beta-galactosidase in primary sweat gland cells cultured in vitro for 10 days in different culture media;
FIG. 10 is a comparison graph of the staining effect of beta-galactosidase in primary sweat gland cells cultured in vitro in the culture medium of the embodiment of the present invention for different time periods;
FIG. 11 is a graph of the results of CK5 PCR of primary sweat gland cells;
FIG. 12 is a graph of the CK18 PCR results of primary sweat gland cells.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in further detail below.
The primary sweat gland cell conditioned medium provided by the embodiment of the invention comprises a cell basal medium, serum, a glutamine additive (Glutamax), a buffer solution, a cell culture additive, penicillin/streptomycin double antibody, an epidermal growth factor, a basic fibroblast growth factor, non-essential amino acid, an insulin ROCK inhibitor and an NF-kappa B agonist.
Wherein, Epidermal Growth Factor (EGF) and basic Fibroblast Growth Factor (FGF) can activate extracellular signal-regulated protein kinase (ERK) signal channels, and EGF plays an active role in the morphogenesis and homeostasis of sweat glands; FGF is used for regulating various stages of growth and development of epithelial tissues and organs and is bound to epithelial cell surface receptors as a specific paracrine medium, so that the proliferation of epithelial cells is stimulated; and the ROCK inhibitor can activate the continuous expression of growth promoting genes IGF2 and ALDH1A1, inhibit apoptosis and promote the proliferation and survival of epithelial stem cells.
Therefore, in the cell culture medium provided by the embodiment of the invention, the epidermal growth factor, the fibroblast factor, the ROCK inhibitor and the NF- κ B agonist are combined, so that the aging process of the primary sweat gland cells can be delayed under the condition of maintaining the homeostasis of the primary sweat gland cells, the extracted primary sweat glands can quickly climb out the sweat gland cells and quickly expand, the biological characteristics of the primary sweat gland cells in the culture process can be maintained, and the problems of small number of sweat gland cells, long culture time and the like obtained by the traditional tissue mass culture method are solved.
Optionally, the serum concentration is 5-10% by volume, the Glutamax concentration is 1-2 mM, the buffer solution concentration is 5-10 mM, the cell culture additive concentration is 0.5-1%, the penicillin/streptomycin dual antibody concentration is 1-2% by volume, the nonessential amino acid concentration is 1-2% by volume, and the insulin concentration is 5-10 μ g/mL.
Alternatively, in the conditioned medium of primary sweat gland cells provided in the embodiments of the present invention, the ROCK inhibitor may specifically be Y-33075.
Optionally, in the primary sweat gland cell conditioned medium, the content of the epidermal 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 μ M. Wherein too low a concentration of the components has no effect, while too high a concentration inhibits cell growth.
Optionally, in one embodiment, the conditioned medium of primary sweat gland cells provided in the examples of the present invention further comprises an NF- κ B agonist. The NF-kappa beta passage is used as the downstream of EDA/EDAR signals and can activate gene components of Shh, cyclin D1, Fox gene family and the like which play a role in various stages of sweat gland development. Therefore, the NF-kB agonist can replace the function of the sweat gland development regulatory factor EDA and maintain the biological characteristics of the sweat gland cell in-vitro amplification culture system.
Alternatively, in one embodiment, the NF-. kappa.B agonist is phorbol 12-myristate 13-acetate (PMA) which is effective for maintaining the properties of sweat gland cells.
Optionally, in a specific embodiment, the PMA is present in an amount of 0.01 to 0.05. mu.g/mL.
Optionally, in the primary sweat gland cell conditioned medium, the cell basal medium is DMEM/F12 medium.
Optionally, in the conditioned medium of primary sweat gland cells, the cell culture additive is B27 additive, which can provide nutrition and maintain cell growth.
The embodiment of the invention 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, supplementing the primary sweat gland cell conditioned medium after the sweat gland tissue adheres to the wall, and changing the liquid once every 2-3 days;
s3, when the sweat gland cell fusion degree reaches 70% -80%, carrying out digestion passage to obtain the expanded sweat gland cells.
According to the method provided by the embodiment of the invention, because the epidermal growth factor, the basic fibroblast growth factor, the ROCK inhibitor and the NF-kB agonist are added into the primary sweat gland cell conditioned medium, the primary sweat gland cells are cultured by using the primary sweat gland cell conditioned medium, the primary sweat gland cells can be treated by the combination of the epidermal growth factor, the basic fibroblast growth factor, the ROCK inhibitor and the NF-kB agonist, the long-term amplification culture of 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 and transplantable human sweat gland cells in large scale and meeting the requirement of large-scale clinical treatment.
Optionally, in step S1, collagen is added to the cell culture dish and then placed in CO before the step of adding the primary sweat gland cell culture medium2After coating treatment in the incubator, collagen is aspirated off and washed with phosphate buffered saline.
Wherein, the collagen is used for coating the cell culture dish, so that the sweat gland tissue can be attached to the wall quickly and climb out the sweat gland cells, and the culture process is accelerated. The collagen may specifically be IV collagen, and the CO may be2The temperature of the incubator is 37 ℃ and CO2The volume fraction is 5%, and the coating treatment time is 10-15 minutes.
Alternatively, in step S2, the primary sweat gland tissue may be directly extracted by using a 1mL tip and directly dropped into a cell culture dish containing the culture medium; and then the adherent growth of the sweat gland tissue can be completed after 8 hours.
Optionally, in an embodiment, the step S3 specifically includes steps S301 to S302:
s301, removing the culture medium, washing with phosphate buffer saline solution, adding cell digestive juice, and placing in CO2Digesting in an incubator, and adding the culture medium to stop digesting when sweat gland cells float;
step S302, uniformly mixing the culture solution after digestion termination, then carrying out passage according to the ratio of 1:2, adding the culture solution into a coated cell culture dish, and then placing the culture dish in CO2And (3) culturing in an incubator, and after the sweat gland cells adhere to the wall, replacing the liquid by using the primary sweat gland cell culture medium, wherein the liquid is replaced once every 2-3 days.
In the step S301, the sweat gland cell conditioned medium is discarded, the sweat gland cell conditioned medium is washed with phosphate buffered saline solution, 0.5-1 mL of cell digestive juice solution such as accutase is added, and the mixture is placed in CO with the temperature of 37 ℃ and the volume fraction of 5%2Digesting in an incubator, observing every 5 minutes, and adding 2mL of the culture medium to stop digestion when a large amount of cells float;
in the step S302, the culture solution after the digestion is stopped is mixed evenly by gentle blowing, then passage is carried out according to the ratio of 1:2, the mixture is added into a coated cell culture dish, and the coated cell culture dish is placed in CO with the temperature of 37 ℃ and the volume fraction of 5 percent2And (3) culturing in an incubator, and after about 8 hours, allowing sweat gland cells to adhere to the wall, changing the culture medium, and then changing the culture medium once every 2-3 days to realize digestion and passage.
In the above embodiment, since all sweat gland cells cannot be digested by one-time digestion, the digested cell culture dish can be washed with phosphate buffered saline solution and then cultured by adding the primary sweat gland cell conditioned medium.
The present invention will be described in detail below with reference to examples.
Example 1
Preparation of a primary sweat gland cell culture medium:
primary sweat gland cell conditioned medium was prepared by adding 10% final 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 a 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, adding CO at 37 deg.C and 5% volume fraction2And (3) coating for 15 minutes in the incubator, taking out the cell culture dish, sucking off IV collagen, washing with PBS once, adding the culture medium prepared in the embodiment 1, and ensuring that the culture medium is just submerged in the bottom of the dish.
(2) Directly beating the primary sweat gland tissue sucked by a 1mL gun head into a cell culture dish added with the culture medium, supplementing the culture medium after the sweat gland tissue adheres to the wall (about 8 hours), changing the culture medium every 2 days, and carrying out digestion and passage when the sweat gland cell fusion degree reaches 80%.
(3) Removing sweat gland culture medium, washing with PBS, adding 1mL of accutase solution, and placing at 37 deg.C and 5% volume fraction CO2Digesting in an incubator, observing once every 5 minutes, adding 2mL of the culture medium prepared in the example 1 when a large amount of cells float, stopping digestion, slightly blowing, uniformly mixing, directly carrying out passage according to the ratio of 1:2, adding the mixture into a 6cm coated cell culture dish, placing the coated 6cm cell culture dish in a CO culture dish with the temperature of 37 ℃ and the volume fraction of 5%2Culturing in an incubator, timely replacing the culture medium after the sweat gland cells adhere to the wall, and replacing the culture medium once every 2 days.
Wherein, the form of the first generation sweat gland cell after the 3 rd day of in vitro amplification is shown in figure 1, which shows that the form of the primary sweat gland cell is complete, which indicates that the growth speed is faster;
the morphology of the first generation sweat gland cells after the 5 th day of in vitro amplification is shown in fig. 2, which indicates that the primary sweat gland cells grow rapidly;
the form of the second generation sweat gland cell after 15 days of in vitro amplification is shown in fig. 3 and 4, and the form of the sweat gland cell is still intact and does not undergo apoptosis;
the form of the third generation sweat gland cell after 30 days of in vitro amplification is shown in fig. 5 and fig. 6, and it can be seen that the form of the sweat gland cell is still intact and the cell activity is high;
the form of the fourth generation sweat gland cells after 33 days of in vitro amplification is shown in fig. 7, and it can be seen that the sweat gland cells still do not lose the form of the primary sweat gland cells;
the form of the fourth generation sweat gland cell after the 40 th day of in vitro amplification is shown in fig. 8, and it can be seen that the refractive index of the cell is still strong, and no apoptosis occurs.
Comparative example 1
Preparation of a culture medium:
serum was added to a basal medium DMEM/F12 to a final concentration of 10% and penicillin/streptomycin was added to the medium to prepare a cell culture medium.
Comparative example 2
Amplification of primary sweat gland cells:
(1) taking out 6cm cell culture dish, adding 2mL IV collagen, adding CO at 37 deg.C and 5% volume fraction2And (3) coating for 15 minutes in the incubator, taking out the cell culture dish, sucking off IV collagen, washing with PBS once, adding the culture medium prepared in the comparative example 1, and ensuring that the culture medium is submerged at the bottom of the dish.
(2) Directly beating the primary sweat gland tissue sucked by a 1mL gun head into a cell culture dish added with the culture medium, supplementing the culture medium after the sweat gland tissue adheres to the wall, then changing the culture medium every 2 days, and carrying out digestion and passage when the fusion degree of the sweat gland cells reaches 80%.
(3) Removing sweat gland culture medium, washing with PBS, adding 1mL of accutase solution, and placing at 37 deg.C and 5% volume fraction CO2Digesting in an incubator, observing every 5 minutes, adding 2mL of the culture medium prepared in the comparative example 1 when a large amount of cells float, stopping digestion, slightly blowing, uniformly mixing, directly carrying out passage according to the ratio of 1:2, adding the mixture into a 6cm cell culture dish after coating, and placing the culture dish in the culture dishCO with a volume fraction of 5% at 37 ℃2Culturing in an incubator, timely replacing the culture medium after the sweat gland cells adhere to the wall, and replacing the culture medium once every 2 days.
The experimental effect is as follows:
the staining effects of beta-galactosidase on 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 shown as E and F in FIG. 9 respectively, which indicates that the NF- κ B agonist can delay senescence of the primary sweat gland cells in vitro long-term amplification culture;
the primary sweat gland cells cultured in vitro for 10 days and 40 days in example 2 are stained with beta-galactosidase, the staining effects of which are respectively shown in D10 and D40 in FIG. 10, which shows that the NF-kappa B agonist can delay the senescence of the primary sweat gland cells in the in vitro long-term amplification culture;
the real-time quantitative PCR results of CK5 of the just-isolated primary sweat gland cells, the primary sweat gland cells after 10 days in vitro culture in example 2, and the primary sweat gland cells after 10 days in vitro culture in comparative example 2 are shown in A, B, C of fig. 11, respectively;
the real-time quantitative PCR results of CK18 of the just-isolated primary sweat gland cells, the primary sweat gland cells after in vitro culture for 10 days in example 2, and the primary sweat gland cells after in vitro culture for 40 days in example 2 are shown as D0, D10, and D40 in fig. 12, respectively;
it is demonstrated by FIGS. 11 and 12 that the combination of epidermal growth factor, basic fibroblast growth factor, NF-. kappa.B agonist and ROCK inhibitor is able to maintain the biological properties of primary sweat gland cells in long-term expansion culture in vitro.
The above results illustrate that: by utilizing the primary sweat gland cell conditioned medium provided by the invention, the extracted primary sweat glands can quickly climb out the sweat gland cells and are quickly amplified, the difficulties of small number of sweat gland cells, long culture time and the like obtained by a traditional tissue block culture method are overcome, and the biological characteristics of the primary sweat gland in the culture process can be maintained for a long time.
The primary sweat gland cell conditioned medium and the primary sweat gland cell culture method provided by the invention are described in detail, specific examples are applied in the text to explain the principle and the implementation mode of the invention, and the description of the examples is only used for helping to understand the method and the core idea of the invention; meanwhile, for a person skilled in the art, according to the idea of the present invention, there may be variations in the specific embodiments and the application scope, and in summary, the content of the present specification should not be construed as a limitation to the present invention.
Claims (10)
1. A primary sweat gland cell conditioned medium is characterized by comprising a cell basal medium, serum, Glutamax, a buffer solution, a cell culture additive, penicillin/streptomycin, an epidermal cell growth factor, a basic fibroblast growth factor, nonessential amino acids, insulin, a ROCK inhibitor and an NF-kappa B agonist.
2. The primary sweat gland cell conditioned medium according to claim 1, characterized in that the NF- κ B agonist is phorbol 12-myristate 13-acetate.
3. The primary sweat gland cell conditioned medium of claim 2, wherein phorbol 12-myristate 13-acetate is present in an amount of 0.01-0.05 μ g/mL.
4. The primary sweat gland conditioned cell culture medium according to claim 1, wherein the cell basal medium is DMEM/F12 medium.
5. The primary sweat gland cell conditioned medium of claim 1, wherein the cell culture additive is B27.
6. The primary sweat gland cell conditioned medium of claim 1, wherein said ROCK inhibitor is Y-33075.
7. The primary sweat gland cell conditioned medium of claim 6, wherein the content of the epidermal 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 μ M.
8. A primary sweat gland cell culture method is characterized by comprising the following steps:
taking a cell culture dish, and adding a primary sweat gland cell conditioned medium as defined in any one of claims 1 to 7;
adding primary sweat gland tissues into the cell culture dish, supplementing the primary sweat gland cell conditioned medium after the sweat gland tissues adhere to the wall, and changing the liquid once every 2-3 days;
and when the fusion degree of the sweat gland cells reaches 70% -80%, carrying out digestion passage to obtain the expanded sweat gland cells.
9. The method of claim 8, wherein prior to the step of adding the conditioned medium of primary sweat gland cells, the method further comprises:
adding collagen to the cell culture dish, and then placing in CO2After coating treatment in the incubator, collagen is aspirated off and washed with phosphate buffered saline.
10. The method of claim 9, wherein the process of digestive passaging comprises:
removing culture medium, washing with phosphate buffered saline solution, adding cell digestive juice, and placing in CO2Digesting in an incubator, and adding the primary sweat gland cell conditioned medium to terminate digestion when the 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 CO2And (3) culturing in an incubator, and after the sweat gland cells adhere to the wall, replacing the liquid by using the primary sweat gland cell culture medium, wherein the liquid is replaced once every 2-3 days.
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