CN108753681B - Nasal epithelial stem cell culture method and nasal epithelial stem cell proliferation culture medium - Google Patents
Nasal epithelial stem cell culture method and nasal epithelial stem cell proliferation culture medium Download PDFInfo
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Abstract
The invention discloses a nasal epithelial stem cell culture method, which comprises the following steps: 1) plating trophoblast cells in a culture dish; 2) isolating primary nasal epithelial stem cells from nasal mucosal tissue; 3) spreading the primary nasal epithelial stem cell on a trophoblast cell, replacing a culture medium with a nasal epithelial stem cell proliferation culture medium, and removing the rest tissue cells; 4) when the nasal epithelial stem cells are proliferated to 80% density, the nasal epithelial stem cells are digested by pancreatin and continuously amplified by passage; 5) the nasal epithelial stem cells are transferred to a gas-liquid phase culture system and a Pneumaacult-ALI differentiation culture medium for differentiation culture. The invention also discloses a nasal epithelial stem cell proliferation culture medium, which comprises the following components: DMEM/F12, fetal bovine serum, penicillin, streptomycin, insulin, epidermal growth factor, hydrocortisone, 3', 5-triiodo-L-thyronine sodium, and ROCK inhibitor. The invention provides a key implementation basis for the transformation application of the clinical transplantation of the nasal epithelial stem cells.
Description
Technical Field
The invention belongs to the technical field of adult stem cells, and particularly relates to a nasal epithelial stem cell culture method and a nasal epithelial stem cell proliferation culture medium.
Background
Adult stem cells refer to undifferentiated cells present in a tissue, which are capable of self-renewal and under certain conditions, are programmed to differentiate to form new functional cells, thereby maintaining a dynamic balance of growth and apoptosis in the tissue or organ. The embryonic stem cells have totipotency, can establish lines and passages, can be immortalized and have strong proliferation capacity, but have obvious defects, such as immunological rejection, non-localized differentiation, possible canceration and the like. In contrast, adult stem cells are derived from individuals, do not have the problem of histocompatibility, avoid graft rejection, have tissue-specific proliferation and directional differentiation potential, and are the most suitable clinical applications for tissue regeneration after local organ injury (especially irreversible injury).
At present, epithelial stem cells of the skin are used for repairing and regenerating epidermis, and animal experiments prove that the lung stem cells can be applied to lung epithelial regeneration after lung injury. Adult stem cells must be obtained from specific tissues, such as epidermal, pulmonary and intestinal epithelial stem cells, and must be separated from skin, pulmonary and intestinal tissues, respectively, so that adult stem cells also belong to a class of primary cells. At present, there are commercial primary cell culture media aiming at different tissue sources, which can specifically culture primary cells of the tissue sources to enable the cells to proliferate and differentiate into functional cells in vitro, but only satisfy relevant basic and clinical researches aiming at the primary cells, and are not applied to clinical stem cell therapy (such as cell transplantation).
The nasal mucosal epithelium is the first line of defense in the human respiratory tract, maintaining important mucosal defense functions and homeostasis through ciliary beat of ciliated cells and mucus secretion of goblet cells. In response to the damage caused by external stimuli (such as viruses, bacteria and environmental stimuli), the nasal epithelial stem cells have self-repairing capacity and achieve the re-epithelialization of the nasal mucosa through the migration, proliferation and directional differentiation of the epithelial stem cells. However, damage caused by pathological conditions (such as acute and chronic inflammation of the nasal mucosa, infection of viruses or bacteria, stimulation of environmentally harmful substances, trauma and trauma after nasal sinus surgery, and mucosal damage after radiotherapy of nasopharyngeal carcinoma), damage or loss of repair and regeneration capacity of the nasal mucosa epithelium, difficulty in re-epithelialization or prolonged re-epithelialization time can cause that inflammation of the mucosa is difficult to relieve, and the life quality of a patient is greatly influenced. Therefore, how to make the nasal mucosal epithelium re-epithelialize rapidly after injury and restore function is the key to solve the inflammation injury lesion of the mucosa.
While epithelial-directed therapies may be considered in the direction of targeting inflammatory factors or optimizing existing anti-inflammatory treatment regimens, these approaches may be appropriate for specific chronic nasal mucosal inflammations, and new solutions need to be investigated for cases of irreversible epithelial injury or reduced epithelial regeneration capacity (e.g., acute infection or massive epithelial stem cell death due to environmental pollutants, post-operative mucosal trauma or trauma, and radiation-induced injury).
The nasal epithelial stem cell is used as an adult stem cell, and has good clinical application prospect due to the in-vitro proliferation and differentiation capacity. Nasal primary epithelial stem cells need to be isolated from nasal mucosal tissue and cultured in vitro, and commercial culture media (e.g., BEGM from LONZA, Pneuma Cult from Stemcell) are available for the culture of nasal epithelial primary cells; the applicant's topic group has also developed an optimized culture system in the early stage, which has a great improvement on maintaining the dryness of the nasal epithelial stem cells. The above culture methods and systems can only satisfy the conventional studies related to the function and characteristics of epithelial stem cells.
The existing culture systems and methods have several limitations: 1) commercial media can only sustain expanded cultures of undifferentiated nasal epithelial stem cells for 2-3 passages, and generally after passage 2, the cells have difficulty differentiating into ciliated or goblet epithelial cells. In a strict sense, such a medium cannot be called a "stem cell medium". 2) Although the culture system developed earlier by the applicant can maintain the growth culture of undifferentiated epithelial stem cells for 3 to 5 generations, it is difficult to ensure the differentiation ability of the cells after the growth of the cells exceeds 3 generations. Although a certain stem cell proliferative property can be maintained, the functional property (mucociliary clearance function) of the epithelial stem cell is difficult to maintain due to the decreased differentiation ability. 3) In expected clinical applications, in some cases (e.g., large mucosal lesions), a large number of stem cells are required, and thus, the existing culture medium cannot provide a sufficient number of stem cells under the condition of limited number of passages (i.e., expansion). 4) Whether in commercial culture or in culture systems developed in individual laboratories, the rate of proliferation of nasal epithelial stem cells is relatively slow, and obtaining a sufficient number of epithelial stem cells with stem cell characteristics in a short period of time (e.g., within 4 weeks) can be a problem.
In summary, the existing nasal epithelial stem cell culture system and method cannot maintain the stem cell function and characteristics, i.e. proliferation and differentiation, for a long time, and it is difficult to meet the needs of clinical stem cell transplantation application, which is also a common technical bottleneck of clinical application of adult stem cells. Therefore, there is an urgent need to develop new culture systems and methods for nasal epithelial stem cells, particularly to develop stem cells that can be expanded in vitro for many generations and still maintain normal differentiation capacity, and obtain a sufficient number of stem cells in a short period of time to meet the needs of clinical application, and to lay a key and solid foundation for clinical application.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a nasal epithelial stem cell culture method, which can more effectively maintain the proliferation capacity and the differentiation capacity of the nasal epithelial stem cells.
Another object of the present invention is to provide a nasal epithelial stem cell proliferating culture medium which is simple in formulation and can maintain the proliferating ability of the epithelial stem cells more effectively than the culture medium currently used commercially.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a nasal epithelial stem cell culture method, which comprises the following steps:
1) laying trophoblast cells in a culture dish;
2) isolating primary nasal epithelial stem cells from nasal mucosal tissue;
3) spreading the primary nasal epithelial stem cell on a trophoblast cell, replacing a culture medium with a nasal epithelial stem cell proliferation culture medium, and removing the rest tissue cells;
4) when the nasal epithelial stem cells are proliferated to 80% density, the nasal epithelial stem cells are digested by pancreatin and continuously amplified by passage;
5) the nasal epithelial stem cells are transferred to a gas-liquid phase culture system and a Pneumaacult-ALI differentiation culture medium for differentiation culture.
Further, in the step 1), the trophoblast cells are mitomycin-treated 3T3 cells and can be attached to the wall smoothly within 12-24 hours.
Preferably, in step 1), the trophoblast cells are cultured specifically as follows: culturing 3T3 cells by adopting a DMEM culture medium, 10% fetal calf serum and 1x penicillin-streptomycin complex liquid; proliferating 3T3 cells to 90% density, and treating with mitomycin at concentration of 0.5-20 ug/mL; the growth-inhibited trophoblast cells 3T3 cells were digested at a density of 0.1-1X104cells/cm2Previously plated in cell culture dishes.
Further, in step 3), the components of the nasal epithelial stem cell proliferation medium and the final concentrations thereof are as follows: DMEM/F12, fetal bovine serum, penicillin: 100x, streptomycin: 100x, insulin: 1-10mg/mL, epidermal growth factor: 1-20 μ g/mL, hydrocortisone: 50-1000 μ g/mL, 3', 5-triiodo-L-sodium thyronine: 1-20x10-4M, ROCK inhibitor: 0.5-10 mM.
Further, in the step 3), after the trophoblast cells are attached to the wall, the primary nasal epithelial stem cells are subjected to the treatment of the attaching process according to the density of 1-5x103/cm2Spreading on the nourishing layerReplacing the culture medium with nasal epithelial stem cell culture medium, and removing the remaining tissue cells by liquid replacement; the culture conditions were 37 ℃ and 5% CO2The liquid is changed every 2-3 days.
Further, the step 3) comprises adding cholera toxin into the first generation nasal epithelial stem cell proliferation culture medium, and purifying the nasal epithelial stem cells.
Preferably, the cholera toxin is used in a concentration of 0.005-0.05 μ g/mL.
Further, in step 5), the nasal epithelial stem cells are treated according to the cell density of 1-10x104/cm2Transferring to TRANSWELL chamber, proliferating for 3-5 days by using nasal epithelial stem cell proliferation culture medium, removing culture medium in the chamber, replacing the chamber with Pneumauglt-ALI differentiation culture medium, so that the cell surface is not covered with culture medium, and contacting the cells with culture medium through the permeable membrane of TRANSWELL chamber to form a gas-liquid phase culture system.
The invention provides a nasal epithelial stem cell proliferation culture medium, which comprises the following components in final concentration: DMEM/F12, fetal bovine serum, penicillin: 100x, streptomycin: 100x, insulin: 1-10mg/mL, epidermal growth factor: 1-20 μ g/mL, hydrocortisone: 50-1000 μ g/mL, 3', 5-triiodo-L-sodium thyronine: 1-20x10-4M, ROCK inhibitor: 0.5-10 mM.
Further, the components of the nasal epithelial stem cell proliferation culture medium further comprise cholera toxin, wherein the cholera toxin is used at a concentration of 0.005-0.05 μ g/mL and is used for inhibiting the fibroblasts from the tissue; in the epithelial stem cells expanded after the second generation, cholera toxin may not be added.
Compared with the prior art, the invention has the following beneficial effects:
(1) by using the novel nasal epithelial stem cell culture method and system, the proliferation capacity of the nasal epithelial stem cells can be more effectively maintained and can reach 8 generations;
(2) by using the novel nasal epithelial stem cell culture method and system, the differentiation capability of the nasal epithelial stem cells can be more effectively maintained, namely the stem cells can be differentiated into normal ciliated cells and goblet cells within 6 generations;
(3) by using the novel nasal epithelial stem cell culture method and system, a sufficient number of epithelial stem cells can be expanded in a short period (within 4 weeks);
(4) by using the novel nasal epithelial stem cell culture method and system, the key technical bottleneck of the current application of the nasal epithelial stem cell culture before clinic can be effectively broken through, and a key implementation basis is provided for the transformation application of the clinical transplantation of the nasal epithelial stem cells.
Drawings
FIG. 1 is a schematic diagram of the contents of the nasal epithelial stem cell culture method and system of the present invention;
FIG. 2 is a cell morphology map of cell clonogenic ratios in example 1;
FIG. 3 is a cell morphology map of cell clonogenic ratios in example 2;
FIG. 4 is a cell morphology map of cell clonogenic ratios in example 3.
Detailed Description
The invention will now be further described with reference to the accompanying drawings and specific embodiments.
The invention is developed aiming at the problem that the existing nasal epithelial stem cell culture method can not meet the requirements of clinical transformation application, and the technical content is to establish a novel nasal epithelial stem cell culture method and a novel nasal epithelial stem cell culture system, which mainly comprise 2 aspects:
1) preparing components of an epithelial stem cell proliferation medium;
2) establishing an epithelial stem cell-trophoblast cell culture system.
As shown in figure 1, trophoblast cells are firstly cultured and mitomycin is treated to inhibit growth, nasal epithelial stem cells are seeded on the trophoblast cells, cloning growth is carried out on the nasal epithelial stem cells under the condition of a proliferation culture medium, and after proliferation culture is completed, the cells are transferred to a gas-liquid phase culture system for differentiation. The technical scheme of the invention is as follows:
1) epithelial stem cell proliferation medium components.
The main components of the nasal epithelial stem cell culture medium comprise: DMEM/F12, fetal bovine serum, penicillin (100x)Streptomycin (100x), insulin (1-10mg/mL), epidermal growth factor (1-20 mug/mL), hydrocortisone (50-1000 mug/mL), 3', 5-triiodo-L-sodium thyronine (1-20x 10)-4M), ROCK inhibitor (0.5-10 mM).
2) The concentration of the media components used.
DMEM/F12 was a liquid and purchased from THERMO;
fetal bovine serum, 5-15% in use, purchased from THERMO;
the penicillin-streptomycin complex solution with the use concentration of 1x is purchased from THERMO company;
insulin (dry powder) used at a concentration of 0.2-10 μ g/mL, purchased from SIGMA;
epidermal growth factor (dry powder) with a concentration of 1-20ng/mL, purchased from SIGMA;
hydrocortisone (dry powder) was used at a concentration of 1-20 μ g/mL and purchased from SIGMA;
3,3', 5-Triiodo-L-thyronine sodium (dry powder) at a concentration of 1-10x10-7M, purchased from SIGMA corporation;
ROCK inhibitors (dry powder) were used at concentrations of 0.5-10uM and were purchased from STEMCELL.
3) Function and action of the media components:
DMEM/F12: maintaining essential required nutrients for the cells;
fetal bovine serum: hormones used for maintaining cell growth, providing binding protein, recognizing vitamins, lipids, metals and the like, helping cell adherence and playing a role of pH buffer solution;
penicillin-streptomycin complex liquid: the function of preventing and inhibiting the growth and the reproduction of bacteria in a culture medium is realized in the cell culture process;
insulin (dry powder): promoting the glucose and amino acid uptake of cells, and facilitating the mitosis of cells;
epidermal growth factor (dry powder): promoting the proliferation of adherent cells;
hydrocortisone (dry powder): the cell adhesion and proliferation are promoted;
3,3', 5-triiodo-L-thyronine sodium (dry powder): is helpful for promoting cell proliferation;
ROCK inhibitor (dry powder): contribute to the inhibition of cell differentiation;
the proliferation culture medium of the nasal epithelial stem cells is added with 3,3', 5-triiodo-L-thyronine sodium and ROCK inhibitor, so that the proliferation capacity and the differentiation capacity of the nasal epithelial stem cells can be more effectively maintained, the nasal epithelial stem cells can proliferate for 8 generations, can differentiate into normal cilia cells and goblet cells within 6 generations, and can proliferate sufficient numbers of epithelial stem cells within 4 weeks.
4) Trophoblast cell culture conditions and treatments.
The trophoblast cells were selected from 3T3 cells (a type of murine fibroblast) and purchased from ATCC.
The culture conditions were DMEM medium (purchased from THERMO) and 10% fetal bovine serum and 1 Xpenicillin-streptomycin complex.
After 3T3 cells are full, mitomycin treatment is needed to inhibit the growth of 3T3 cells, and the treatment concentration of mitomycin is 0.5-20 ug/mL. After the growth-inhibited trophoblast cells are digested, they are expressed at a density (0.1-1X 10)4cells/cm2) Previously plated in cell culture dishes.
5) Primary epithelial stem cell culture (proliferation phase).
After trophoblast cell adherence, nasal epithelial stem cells were at the appropriate density (1-5x 10)3/cm2) Spread on top of trophoblast cells and replaced with nasal epithelial stem cell proliferation medium. The cells separated from the tissue are expanded within 5-7 days, and after passage, the cells are expanded within 5 days for each generation, namely 5 generations of cells can be expanded within about 4 weeks; the culture conditions were 37 ℃ and 5% CO2The liquid is changed every 2-3 days.
6) Primary epithelial stem cell culture (differentiation stage).
Epithelial stem cells at a certain cell density (1-10X 10)4/cm2) Transferred to TRANSWELL chamber (purchased from CORNING) and first proliferated (using nasal epithelial stem cells for proliferation) for 3-5 daysCulture medium), the culture medium in the chamber was removed, and the chamber was replaced with Pneumaacult-ALI differentiation medium (purchased from STEMCELL); thus, the cell surface was not covered with the medium, and the cells were contacted with the medium through the permeable membrane of the TRANSWELL cell to form a gas-liquid phase culture system.
The epithelial stem cells will differentiate into ciliated cells and goblet cells in this system, with a time of about 20 days. Through the culture of differentiation, whether the obtained nasal epithelial stem cells have normal directional differentiation potential can be verified.
On the basis of the technical scheme, cholera toxin (with the use concentration of 0.005-0.05 mu g/mL) can be added into the first generation nasal epithelial stem cell culture medium (namely, the nasal epithelial stem cell culture medium is just separated from the tissue) to inhibit the fibrocytes from the tissue to achieve the purified nasal epithelial stem cells; in the epithelial stem cells expanded after the second generation, cholera toxin may not be added.
The in vitro culture of the nasal epithelial stem cells is divided into two stages of proliferation and differentiation, wherein the proliferation stage uses the nasal epithelial stem cell proliferation culture medium and a trophoblast cell culture system, and the differentiation stage uses a currently common gas-liquid phase culture system and a Pneumaacult-ALI differentiation culture medium of STEMCELL company.
The nasal epithelial stem cell proliferation culture medium and the trophoblast cell system are evaluated for maintaining the dryness and normal differentiation characteristics of the nasal epithelial stem cells through the analysis of cell and molecular layers; and the effect of the present invention on the culture of nasal epithelial stem cells was compared with that of a commercial nasal epithelial stem cell proliferation medium (BEGM of Lonza corporation).
The used cell material is derived from nasal mucosa tissue samples of different individual patients, the patients mainly diagnose chronic nasosinusitis, nasal polyp or inferior turbinate hypertrophy and have definite surgical indications, the tissue acquisition way is inflammatory nasal mucosa tissue excised by the patients in the surgical process, and partial tissue is tissue obtained by preoperative biopsy.
The cell culture steps are as follows:
1) 3T3 cells treated by mitomycin are paved in a culture dish to be used as trophoblast cells, and the cells can be attached to the wall smoothly within 12-24 hours;
2) isolating primary cells from nasal mucosal tissue;
3) the primary cells are laid on the trophoblast cells, only the epithelial stem cells can survive under the condition of a nasal epithelial stem cell proliferation culture medium, and the rest tissue cells can be removed by changing liquid;
4) the nasal epithelial stem cells are proliferated to 80% density, and are digested by pancreatin and continuously amplified by passage;
5) at the same time of passage, a part of the cells was separated and transferred to a gas-liquid culture system to evaluate the differentiation state.
The indexes and methods for evaluating the proliferation and differentiation of the nasal epithelial stem cells have the following points:
1) description of cell morphology.
The cells are cobblestone-shaped, have uniform cell contour, uniform shape and close arrangement and are the characteristics of normal stem cells, and the cell proliferation capacity is strong; the cells are in a fried egg shape, irregular cell outlines, loose arrangement, different forms, even the death of part of the cells is in a poor state, which indicates that the proliferation capacity of the stem cells is reduced and the stem cells are in a differentiated or aged state.
2) And (5) cell proliferation.
The proliferation of the cells can be shown by calculating the growth density of the cells on different days.
3) Cell clonogenic rate.
The growth of the nasal epithelial stem cells is clonal proliferation, namely scattered clones are formed in the early stage of cell proliferation; the proliferative capacity of stem cells was assessed by calculating the percentage of colonies present per unit time to the number of cells plated.
4) Expression level of stem cell factor (p 63).
The p63 is a characteristic factor of the nasal epithelial stem cells, and the number of the stem cells can be evaluated by calculating the ratio of the number of p63 positive cells and the total number of the cells in different generations through immunofluorescence staining.
The present invention is further explained by the following specific embodiments and the accompanying drawings, but the technical solution of the present invention is not limited to the specific embodiments.
The following examples each evaluate the difference in effect by comparing the nasal epithelial stem cell culturing method and the nasal epithelial stem cell proliferating culture medium of the present invention with commercial culture media used in the market.
Example 1
The nasal epithelial stem cells are derived from a tissue sample of sinusitis, and the patient is diagnosed with chronic sinusitis.
1) Treating trophoblast cells
The trophoblast cell is 3T3 cell cultured in DMEM culture medium, 10% fetal calf serum and 1x penicillin-streptomycin compound liquid.
② 3T3 cells grow to 100% density, mitomycin treatment is carried out to inhibit the growth of 3T3 cells, the treatment concentration of mitomycin is 0.5-20 ug/mL.
③ digesting the trophoblast cells treated with mitomycin according to a certain density (0.1-1x 10)4cells/cm2) Previously plated in cell culture dishes.
And fourthly, 3T3 cells treated by mitomycin are spread in a culture dish, and can be attached to the wall smoothly within 12 to 24 hours.
2) Proliferation culture of nasal epithelial stem cells (as the culture medium of the present invention and BEGM culture medium, respectively)
After the feeder layer cells are attached to the wall, the epithelial stem cells are laid on the feeder layer cells at an appropriate density, and replaced with an epithelial stem cell proliferation medium.
② primary epithelial stem cells according to proper density (1x 10)3/cm2) Spread on already adherent trophoblast cells;
③ only the epithelial stem cells can survive under the condition of the nasal epithelial stem cell proliferation culture medium, and the rest histiocytes can be removed by changing the liquid; the culture conditions were 37 ℃ and 5% CO2The liquid is changed every 2-3 days.
Fourthly, the growth density of the nasal epithelial stem cells is about 90 to 100 percent within 5 to 7 days.
Fifthly, the nasal epithelial stem cells are digested by pancreatin and continuously amplified after passage.
Sixthly, the culture condition after passage is still 37 ℃ and 5 percent CO2After the expansion of each generation is finished within 5-7 days, the cells of 4-5 generations can be expanded within about 4 weeks;
3) differentiation culture of nasal epithelial stem cells
During passage, one part of cells is separated and transferred to a gas-liquid phase culture system to evaluate the differentiation characteristics of the cells.
② the nasal epithelial stem cells according to a certain cell density (1-10x 10)4/cm2) Transferred to TRANSWELL chamber, and first propagated through 3-5 days (using the nasal epithelial stem cell proliferation medium and BEGM medium of the present invention, respectively).
Thirdly, removing the culture medium in the small chamber, and replacing the small chamber with a PneumaCult-ALI differentiation culture medium; thus, the cell surface was not covered with the medium, and the cells were contacted with the medium through the permeable membrane of the TRANSWELL cell to form a gas-liquid phase culture system.
Fourthly, the nasal epithelial stem cells are completely differentiated within about 21 to 28 days.
The cultivation effect evaluation results are specifically as follows:
the cell culture method and system of the present invention are shown in FIG. 1. The culture results are shown in FIG. 2, the nasal epithelial stem cells are cobblestone-shaped, uniform in cell contour, uniform in morphology and densely arranged in the 1 st to 8 th generations. As shown by BEGM culture medium, the nasal epithelial stem cells can maintain normal stem cell morphology in the 1 st to 2 nd generations, but from the 3 rd generation, the epithelial stem cells gradually appear egg-fried cells, have irregular outlines, are loosely arranged and have nonuniform morphology; from passage 5, the cells died and were unable to passage.
Cell growth Density As shown in Table 1, the cell culture method of the present invention showed no significant difference in growth density per generation (within 8 generations) per day, whereas BEGM medium maintained similar growth density in the 1-2 generations of cells, but showed a significant drop in growth density in the 4-5 generations.
TABLE 1 growth Density
In the aspect of evaluating the cell cloning efficiency (as shown in FIG. 2) and the ratio of the number of p63 positive cells, the cell culture method of the present invention showed no significant difference between the number of clones appearing in each generation (within 8 generations) and the number of p63 positive cells, whereas BEGM medium maintained similar numbers of clones and p63 positive cells in the 1 st-2 th generation cells, but the numbers of clones were significantly reduced in the 4 th-5 th generations. The details are shown in table 2 below:
TABLE 2 cell clonogenic ratio and number of p 63-positive cells
Differentiation phenotype of cells, the cell culture method of the present invention can present ciliated cells and high transmembrane resistance in each generation (within 6 passages), while the BEGM medium can present ciliated cells in 1-2 passages, but substantially no ciliated cells are present from 3 passages, and the transmembrane resistance is low. The details are shown in table 3 below:
TABLE 3 differentiation phenotype assessment of cells
Example 2
Nasal epithelial stem cells were derived from nasal polyp tissue samples and patients were diagnosed with nasal polyps.
1) Treating trophoblast cells
The trophoblast cell is 3T3 cell cultured in DMEM culture medium, 10% fetal calf serum and 1x penicillin-streptomycin compound liquid.
② 3T3 cells grow to 100% density, mitomycin treatment is carried out to inhibit the growth of 3T3 cells, the treatment concentration of mitomycin is 0.5-20 ug/mL.
③ digesting the trophoblast cells treated with mitomycin according to a certain density (0.1-1x 10)4cells/cm2) Previously plated in cell culture dishes.
And fourthly, 3T3 cells treated by mitomycin are spread in a culture dish, and can be attached to the wall smoothly within 12 to 24 hours.
2) Proliferation culture of nasal epithelial stem cells (as the culture medium of the present invention and BEGM culture medium, respectively)
After the feeder layer cells are attached to the wall, the epithelial stem cells are laid on the feeder layer cells at an appropriate density, and replaced with an epithelial stem cell proliferation medium.
② primary epithelial stem cells according to proper density (1x 10)3/cm2) Spread on already adherent trophoblast cells;
③ only the epithelial stem cells can survive under the condition of the nasal epithelial stem cell proliferation culture medium, and the rest histiocytes can be removed by changing the liquid; the culture conditions were 37 ℃ and 5% CO2The liquid is changed every 2-3 days.
Fourthly, the growth density of the nasal epithelial stem cells is about 90 to 100 percent within 5 to 7 days.
Fifthly, the nasal epithelial stem cells are digested by pancreatin and continuously amplified after passage.
Sixthly, the culture condition after passage is still 37 ℃ and 5 percent CO2After the expansion of each generation is finished within 5-7 days, the cells of 4-5 generations can be expanded within about 4 weeks;
3) differentiation culture of nasal epithelial stem cells
During passage, one part of cells is separated and transferred to a gas-liquid phase culture system to evaluate the differentiation characteristics of the cells.
② the nasal epithelial stem cells according to a certain cell density (1-10x 10)4/cm2) Transferred to TRANSWELL cells, and proliferated for 3-5 days (using the hair conditioner separately)Clear nasal epithelial stem cell proliferation medium and becm medium).
Thirdly, removing the culture medium in the small chamber, and replacing the small chamber with a PneumaCult-ALI differentiation culture medium; thus, the cell surface was not covered with the medium, and the cells were contacted with the medium through the permeable membrane of the TRANSWELL cell to form a gas-liquid phase culture system.
Fourthly, the nasal epithelial stem cells are completely differentiated within about 21 to 28 days.
The cultivation effect evaluation results are specifically as follows:
the cell culture method and system of the present invention are shown in FIG. 1. The culture results are shown in FIG. 3, the nasal epithelial stem cells are cobblestone-shaped, uniform in cell contour, uniform in morphology and densely arranged in the 1 st to 8 th generations. As shown by BEGM culture medium, the nasal epithelial stem cells can maintain normal stem cell morphology in the 1 st to 2 nd generations, but from the 3 rd generation, the epithelial stem cells gradually appear egg-fried cells, have irregular outlines, are loosely arranged and have nonuniform morphology; from passage 4, the cells died and were unable to passage.
Cell growth Density As shown in Table 4, the cell culture method of the present invention showed no significant difference in growth density per generation (within 8 generations) per day, whereas BEGM medium maintained similar growth density in the 1-2 generations of cells, but showed a significant drop in growth density in the 4-5 generations.
TABLE 4 growth Density
In the evaluation of cell clonogenic efficiency (as shown in FIG. 3) and the number of p63 positive cells, the cell culture method of the present invention showed no significant difference in the number of clones per generation (within 8 generations) from the number of p63 positive cells, whereas BEGM medium maintained similar numbers of clones and p63 positive cells within 1-2 generations of cells, but the number of clones decreased significantly at 4-5 generations. The details are shown in table 5 below:
TABLE 5 cell clonogenic Rate and number of p 63-positive cells
Differentiation phenotype of cells, the cell culture method of the present invention can present ciliated cells and high transmembrane resistance in each generation (within 6 passages), while the BEGM medium can present ciliated cells in 1-2 passages, but substantially no ciliated cells are present from 3 passages, and the transmembrane resistance is low. Specifically, the following table 6 shows:
TABLE 6 evaluation of differentiation phenotype of cells
Example 3
Nasal epithelial stem cells were derived from the inferior turbinate tissue sample and the patient was diagnosed with inferior turbinate hypertrophy.
1) Treating trophoblast cells
The trophoblast cell is 3T3 cell cultured in DMEM culture medium, 10% fetal calf serum and 1x penicillin-streptomycin compound liquid.
② 3T3 cells grow to 100% density, mitomycin treatment is carried out to inhibit the growth of 3T3 cells, the treatment concentration of mitomycin is 0.5-20 ug/mL.
③ digesting the trophoblast cells treated with mitomycin according to a certain density (0.1-1x 10)4cells/cm2) Previously plated in cell culture dishes.
And fourthly, 3T3 cells treated by mitomycin are spread in a culture dish, and can be attached to the wall smoothly within 12 to 24 hours.
2) Proliferation culture of nasal epithelial stem cells (as the culture medium of the present invention and BEGM culture medium, respectively)
After the feeder layer cells are attached to the wall, the epithelial stem cells are laid on the feeder layer cells at an appropriate density, and replaced with an epithelial stem cell proliferation medium.
② primary epithelial stem cells according to proper density (1x 10)3/cm2) Spread on already adherent trophoblast cells;
③ only the epithelial stem cells can survive under the condition of the nasal epithelial stem cell proliferation culture medium, and the rest histiocytes can be removed by changing the liquid; the culture conditions were 37 ℃ and 5% CO2The liquid is changed every 2-3 days.
Fourthly, the growth density of the nasal epithelial stem cells is about 90 to 100 percent within 5 to 7 days.
Fifthly, the nasal epithelial stem cells are digested by pancreatin and continuously amplified after passage.
Sixthly, the culture condition after passage is still 37 ℃ and 5 percent CO2After the expansion of each generation is finished within 5-7 days, the cells of 4-5 generations can be expanded within about 4 weeks;
3) differentiation culture of nasal epithelial stem cells
During passage, one part of cells is separated and transferred to a gas-liquid phase culture system to evaluate the differentiation characteristics of the cells.
② the nasal epithelial stem cells according to a certain cell density (1-10x 10)4/cm2) Transferred to TRANSWELL chamber, and first propagated through 3-5 days (using the nasal epithelial stem cell proliferation medium and BEGM medium of the present invention, respectively).
Thirdly, removing the culture medium in the small chamber, and replacing the small chamber with a PneumaCult-ALI differentiation culture medium; thus, the cell surface was not covered with the medium, and the cells were contacted with the medium through the permeable membrane of the TRANSWELL cell to form a gas-liquid phase culture system.
Fourthly, the nasal epithelial stem cells are completely differentiated within about 21 to 28 days.
The cultivation effect evaluation results are specifically as follows:
the cell culture method and system of the present invention are shown in FIG. 1. The culture results are shown in FIG. 4, the nasal epithelial stem cells are cobblestone-shaped, uniform in cell contour, uniform in morphology and densely arranged in the 1 st to 8 th passages. As shown by BEGM culture medium, the nasal epithelial stem cells can maintain normal stem cell morphology in the 1 st to 2 nd generations, but from the 3 rd generation, the epithelial stem cells gradually appear egg-fried cells, have irregular outlines, are loosely arranged and have nonuniform morphology; from passage 5, the cells died and were unable to passage.
Cell growth Density As shown in Table 7, the cell culture method of the present invention showed no significant difference in growth density per day in each generation (within 8 generations), whereas BEGM medium maintained similar growth density in the 1-3 generations of cells, but showed a significant drop in growth density in the 4-5 generations.
TABLE 7 growth Density
In the evaluation of cell clonogenic efficiency (as shown in FIG. 4) and the number of p63 positive cells, the cell culture method of the present invention showed no significant difference in the number of clones per generation (within 8 generations) from the number of p63 positive cells, whereas BEGM medium maintained similar numbers of clones and p63 positive cells within 1-2 generations of cells, but the number of clones decreased significantly at 4-5 generations. The details are shown in table 8 below:
TABLE 8 cell clonogenic ratios and number of p63 positive cells (example 3)
Differentiation phenotype of cells, the cell culture method of the present invention can present ciliated cells and high transmembrane resistance in each generation (within 6 passages), while the BEGM medium can present ciliated cells in 1-2 passages, but substantially no ciliated cells are present from 3 passages, and the transmembrane resistance is low. The details are shown in table 9 below:
TABLE 9 differentiation phenotype assessment of cells
The present invention is not limited to the above-described embodiments, and various changes and modifications of the present invention are intended to be included within the scope of the claims and the equivalent technology of the present invention if they do not depart from the spirit and scope of the present invention.
Claims (9)
1. A method for culturing nasal epithelial stem cells is characterized by comprising the following steps:
1) laying trophoblast cells in a culture dish;
2) isolating primary nasal epithelial stem cells from nasal mucosal tissue;
3) spreading the primary nasal epithelial stem cell on a trophoblast cell, replacing a culture medium with a nasal epithelial stem cell proliferation culture medium, and removing the rest tissue cells;
4) when the nasal epithelial stem cells are proliferated to 80% density, the nasal epithelial stem cells are digested by pancreatin and continuously amplified by passage;
5) transferring the nasal epithelial stem cells to a gas-liquid phase culture system and a Pneumaacult-ALI differential culture medium for differential culture;
the nasal epithelial stem cell proliferation culture medium in the step 3) comprises the following components in percentage by weight: DMEM/F12, fetal bovine serum, penicillin: 100x, streptomycin: 100x, insulin: 1-10mg/mL, epidermal growth factor: 1-20 μ g/mL, hydrocortisone: 50-1000 μ g/mL, 3', 5-triiodo-L-sodium thyronine: 1-20x10-4M, ROCK inhibitor: 0.5-10 mM.
2. The method of culturing nasal epithelial stem cells according to claim 1, wherein: in the step 1), the trophoblast cells are mitomycin-treated 3T3 cells and can be attached to the wall smoothly within 12-24 hours.
3. The method of culturing nasal epithelial stem cells according to claim 2, wherein: in the step 1), the trophoblast cells are cultured specifically as follows: culturing 3T3 cells by adopting a DMEM culture medium, 10% fetal calf serum and 1x penicillin-streptomycin complex liquid; proliferating 3T3 cells to 90% density, and treating with mitomycin at concentration of 0.5-20 ug/mL; the growth-inhibited trophoblast cells 3T3 cells were digested at a density of 0.1-1X104cells/cm2Previously plated in cell culture dishes.
4. The method of culturing nasal epithelial stem cells according to claim 1, wherein:
in step 3), after the trophoblast cells are attached to the wall, the primary nasal epithelial stem cells are subjected to density 1-5x103/cm2Plating on the trophoblast cells, replacing the culture medium with nasal epithelial stem cell culture medium, and removing the rest tissue cells by liquid replacement; the culture conditions were 37 ℃ and 5% CO2The liquid is changed every 2-3 days.
5. The method of culturing nasal epithelial stem cells according to claim 1, wherein:
in the step 3), cholera toxin is added into the first generation nasal epithelial stem cell proliferation culture medium, and the nasal epithelial stem cells are purified.
6. The method of claim 5, wherein the culture of the nasal epithelial stem cells comprises: the cholera toxin is used at a concentration of 0.005-0.05. mu.g/mL.
7. The method of culturing nasal epithelial stem cells according to claim 1, wherein:
in the step 5), the nasal epithelial stem cells are subjected to cell density of 1-10x104/cm2Transferring to TRANSWELL chamber, proliferating for 3-5 days with nasal epithelial stem cell proliferation culture medium, removing culture medium in the chamber, and replacing with Pneumaacult-ALI differentiation culture medium to form a gas-liquid phase culture mediumAnd (5) a nutrient system.
8. A nasal epithelial stem cell proliferation culture medium is characterized by comprising the following components in final concentration: DMEM/F12, fetal bovine serum, penicillin: 100x, streptomycin: 100x, insulin: 1-10mg/mL, epidermal growth factor: 1-20 μ g/mL, hydrocortisone: 50-1000 μ g/mL, 3', 5-triiodo-L-sodium thyronine: 1-20x10-4M, ROCK inhibitor: 0.5-10 mM.
9. The nasal epithelial stem cell proliferation medium according to claim 8, characterized in that: the components of the nasal epithelial stem cell proliferation culture medium further comprise cholera toxin, and the usage concentration of the cholera toxin is 0.005-0.05 mu g/mL.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013066802A2 (en) * | 2011-10-27 | 2013-05-10 | Agency For Science, Technology And Research (A*Star) | Compositions and methods for lung regeneration |
| KR20150042535A (en) * | 2013-10-11 | 2015-04-21 | 고려대학교 산학협력단 | Mesenchymal Stem Cells Drived from Nasal polyp and Method for Isolating the Same |
| CN105505860A (en) * | 2016-01-13 | 2016-04-20 | 河南科技大学第一附属医院 | Method for isolated culture of esophagus epithelial stem cells |
| CN106609258A (en) * | 2015-10-23 | 2017-05-03 | 江苏齐氏生物科技有限公司 | Rat nasal mucosa epithelial cell isolation and culture method |
| CN107267442A (en) * | 2008-11-20 | 2017-10-20 | 詹森生物科技公司 | Multipotential stem cell culture on microcarrier |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110143415A1 (en) * | 2009-12-14 | 2011-06-16 | Sergei Paylian | Reprogramming normal and cancerous human cell lines into human induced poluripotent stem cells by co-electroporation with living xenopus laevis frog oocytes |
| CN102505005B (en) * | 2011-12-24 | 2013-04-24 | 南方医科大学 | Culture medium for keeping primary airway epithelial cells in physiological state in vivo |
| CN107129967A (en) * | 2017-06-18 | 2017-09-05 | 广东博溪生物科技有限公司 | A kind of serum free medium system for primary human tonsillar cell |
-
2018
- 2018-04-28 CN CN201810400123.2A patent/CN108753681B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107267442A (en) * | 2008-11-20 | 2017-10-20 | 詹森生物科技公司 | Multipotential stem cell culture on microcarrier |
| WO2013066802A2 (en) * | 2011-10-27 | 2013-05-10 | Agency For Science, Technology And Research (A*Star) | Compositions and methods for lung regeneration |
| KR20150042535A (en) * | 2013-10-11 | 2015-04-21 | 고려대학교 산학협력단 | Mesenchymal Stem Cells Drived from Nasal polyp and Method for Isolating the Same |
| CN106609258A (en) * | 2015-10-23 | 2017-05-03 | 江苏齐氏生物科技有限公司 | Rat nasal mucosa epithelial cell isolation and culture method |
| CN105505860A (en) * | 2016-01-13 | 2016-04-20 | 河南科技大学第一附属医院 | Method for isolated culture of esophagus epithelial stem cells |
Non-Patent Citations (9)
| Title |
|---|
| Basal cells of the human adult airway surface epithelium retain transit-amplifying cell properties;Rodolphe Hajj;《Stem Cells》;20070131;第25卷(第1期);第139-148页 * |
| Reduced growth and proliferation dynamics of nasal epithelial stem/progenitor cells in nasal polyps in vitro;Xue Min Yu等;《Scientific Reports》;20140409(第4期);第1-8页 * |
| The use of nasal epithelial stem/progenitor cells to produce functioning ciliated cellsin vitro;Xuening Zhao等;《American Journal of Rhinology & Allergy》;20121031;第26卷(第5期);摘要，第345页左栏第1段-第346页右栏第2段，第347-第349页"结果"、"讨论" * |
| Xuening Zhao等.The use of nasal epithelial stem/progenitor cells to produce functioning ciliated cellsin vitro.《American Journal of Rhinology & Allergy》.2012,第26卷(第5期), * |
| 人细支气管上皮细胞的ROCK激酶抑制剂培养体系扩增及气液相模型的建立;贾元元等;《中国组织工程研究》;20170702;第19卷(第28期);摘要，第4583页左栏第1段-第4584页右栏第2段 * |
| 人鼻上皮干细胞体外模型的构建及Ki67在人鼻上皮中表达及分布的研究;赵莉;《中国博士学位论文全文数据库 医药卫生科技辑》;20170815(第08期);摘要，第11页"前言"-第22页"结论" * |
| 人鼻黏膜上皮细胞2种原代体外培养方案的比较研究;王振霖等;《临床耳鼻咽喉头颈外科杂志》;20080930;第22卷(第17期);第776-779页 * |
| 气液界面培养的鼻黏膜上皮细胞的纤毛分化;郭永清等;《临床耳鼻咽喉科杂志》;20040229;第18卷(第2期);第88-90页 * |
| 赵莉.人鼻上皮干细胞体外模型的构建及Ki67在人鼻上皮中表达及分布的研究.《中国博士学位论文全文数据库 医药卫生科技辑》.2017,(第08期), * |
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