CN117625526A - Mesenchymal stem cell culture composition, atomization composition and application - Google Patents
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Abstract
The invention provides a mesenchymal stem cell culture composition, an atomization composition and application. Wherein the mesenchymal stem cell culture composition is composed of a biomembrane wrapped sacculus secreted in the mesenchymal stem cell culture, and the content of the sacculus in the mesenchymal stem cell culture composition is 1.0x10 9 ‑1.0×10 10 particles/ml. Atomized composition and mesenchymal stem cell culture combinationAnd (3) an object. Both the mesenchymal stem cell culture composition and the atomized composition can be applied to preparing medicines and/or conditioning liquid for treating rhinitis.
Description
Technical Field
The invention belongs to application of mesenchymal stem cells, and particularly relates to a mesenchymal stem cell culture composition, an atomization composition and application.
Background
Allergic diseases develop civilization in the last few decades in humans, and their incidence shows a significant rising trend, becoming a major public health problem of global concern. The World Health Organization (WHO) classified allergic diseases as one of the major diseases that need to be studied and controlled in the 21 st century.
According to published data (world allergic organization World Allergy Organization, WAO) it has been shown that the incidence of global allergic diseases has reached a fairly high level, up to 20% of which means that 1 out of every 5 people in the global population are afflicted with allergic diseases. At least 30% to 40% of the population worldwide has been or is experiencing the affliction of allergic diseases, and this trend is also continuously rising. It is expected that over 40 million people worldwide will suffer from allergic diseases such as asthma, allergic rhinitis and atopic dermatitis in the next 25 years. In view of this, the World Health Organization (WHO) has emphasized that allergic diseases are the major diseases of research and prevention in the 21 st century.
Epidemiological investigation results of 33 nations allergic diseases published by the world allergic organization show that: of the 13.9 million general population in these countries, about 2.5 million people have food allergy, and asthma patients reach 3.58 million, with allergic asthma patients accounting for 2/3 of the general population of asthma. And patients suffering from allergic rhinitis worldwide are more than 4-5 billion. And the incidence of allergic diseases has increased by at least 3 times over the last 30 years. It is expected that after 20 years, 50% of the population in industrialized countries will develop allergic disease.
Allergic rhinitis (allergic rhinitis, AR) refers to a non-infectious inflammatory disease of the nasal mucosa in which IgE-mediated mediators (mainly histamine) are released after an atopic individual contacts an allergen, and various immunocompetent cells, cytokines, etc. are involved. There is no particularly effective treatment in medicine at present, and the medical recommended solution mainly consists in avoiding exposure to known allergens, reducing the outgoing, keeping the room ventilated, avoiding exposure to animals, taking drugs including antihistamines or gradually achieving immune tolerance by repeatedly exposing the patient to allergens; however, the existing scheme can not thoroughly cure allergic rhinitis, and can only partially control or improve clinical symptoms of patients, and patients still face the difficult situation of respiratory obstruction, especially the problem that the patients cannot normally ventilate when sleeping every day, and can cause choking or late night awakening.
Disclosure of Invention
It is an object of the present invention to provide a mesenchymal stem cell culture composition that can be used for the treatment of allergic diseases, in particular for the treatment of allergic rhinitis. The mesenchymal stem cell culture composition is composed of a capsule wrapped by a biological membrane secreted in the culture of the mesenchymal stem cells, and the content of the capsule in the mesenchymal stem cell culture composition is 1.0x10 9 -1.0×10 10 particles/ml。
The mesenchymal stem cell culture composition is a composition formed by biomembrane-coated sacs secreted in the culture of the mesenchymal stem cells, various substances are secreted in the culture process of the mesenchymal stem cells, the biomembrane-coated sacs refer to lipid-inlaid vesicles secreted by cells and coated by double-molecular-layer biomembranes, the diameters of the lipid-inlaid vesicles are about 30-200nm, and the lipid-inlaid vesicles comprise various substances such as proteins, lipids, polysaccharides and nucleic acids, and the like, so that the lipid-inlaid vesicles participate in the transportation and information communication among the cells. Allergic rhinitis is a nasal mucosa inflammation caused by an immune system excessive reaction, and common symptoms include nasal obstruction, runny nose, sneeze, nasal itching and the like. The composition is different from the cell treatment of mesenchymal stem cells, belongs to non-cell treatment, can be used for treating allergic diseases, and particularly can be used for relieving symptoms of allergic rhinitis and effectively relieving respiratory disorder and the like caused by the allergic rhinitis to patients through anti-inflammatory action, immunoregulation, repairing nasal mucosa injury, regulating immune balance and the like during the treatment of the allergic rhinitis: 1) Anti-inflammatory action: since allergic rhinitis is related to abnormal reactions and inflammatory reactions of the immune system, the sacculus body in the mesenchymal stem cell culture composition contains various anti-inflammatory factors, such as anti-inflammatory proteins and anti-inflammatory small molecules, and the substances can inhibit the inflammatory reactions of nasal mucosa and relieve the symptoms of the allergic rhinitis by regulating the signal paths of the inflammatory reactions; 2) Immunomodulation: since allergic rhinitis is caused by abnormal reaction of an immune system to an allergen, the sacculus in the mesenchymal stem cell culture composition can regulate the immune reaction, inhibit activation and proliferation of immune cells and reduce the immune reaction caused by the allergen; 3) Repairing nasal mucosa injury: because the inflammatory reaction of allergic rhinitis can cause the injury of nasal mucosa, the sacculus body in the mesenchymal stem cell culture composition contains various factors for promoting cell proliferation and repair, such as growth factors and extracellular matrix components, so that the repair and regeneration of the nasal mucosa can be promoted, and the function of the nasal mucosa can be improved; 4) Regulating immune balance: since occurrence of allergic rhinitis is related to imbalance of immune balance, the sacculus in the mesenchymal stem cell culture composition can regulate immune balance, promote normal functions of an immune system and reduce the occurrence of allergic rhinitis.
The treatment means is not only dependent on direct contact among cells, but also a plurality of researches find that the mesenchymal stem cells can secrete various mediums, the mediums are wrapped in extracellular vesicles, and the treatment effect is exerted through the migration of the extracellular vesicles, so that a key conclusion is laid for developing cell-free products derived from the mesenchymal stem cells.
Further, the mesenchymal stem cell culture composition is prepared by supernatant of the mesenchymal stem cell culture solution.
In the process of culturing mesenchymal stem cells, after the cells grow to a certain extent, the cells can precipitate in the precipitate formed at the bottom of the culture, and the precipitate of the mesenchymal stem cell culture can contain cell fragments and/or cell residues, wherein the cell fragments can cause immune reaction or other side effects, the cell residues such as cell membranes, organelles and the like can have adverse effects on human bodies, and the composition and quality of the precipitate are difficult to accurately control, so that the instability of the therapeutic effect can be caused. Compared with mesenchymal stem cell culture sediment or cell products, the composition has the advantages of high safety, stable treatment effect, low immunogenicity and the like, and is an ideal medicament for treating allergic rhinitis; specifically, 1) the safety is high: the supernatant can avoid the safety problems such as immune rejection, tumor formation and the like which can occur when living cells are used, and the cell residues and/or cell fragments are not or less present, so that the risks of potential immune reactions and other side effects are further reduced; 2) The treatment effect is stable: the components in the supernatant are relatively stable, so that the problems of unstable cell survival rate and the like possibly occurring when the mesenchymal stem cells are used can be avoided, the freezing and transportation and long-term storage can be performed, and the transportation and the use are convenient; 3) Low immunogenicity: the supernatant fluid does not contain HLA antigen on the surface of the mesenchymal stem cells, has low immunogenicity, and can avoid the problem of immunological rejection possibly occurring when the mesenchymal stem cells are used.
Further, the mesenchymal stem cell culture composition is prepared by a method comprising the steps of:
and culturing the mesenchymal stem cells in a serum-free culture medium, collecting supernatant, filtering and concentrating to obtain the mesenchymal stem cell culture composition.
Specifically, cell culture supernatant was obtained: taking mesenchymal stem cells, placing the mesenchymal stem cells in a serum-free culture medium containing 5% -10% of serum substitutes for subculturing, and collecting supernatant when the cells reach 80% -90% of fusion degree;
and (3) filtering and concentrating: the supernatant collected in the step of obtaining the cell culture supernatant is firstly adopted
And (3) performing rough filtration on a filter membrane with the concentration of 0.45 mu M to 0.65 mu M, performing primary purification through a filter membrane with the concentration of 100KD, circularly washing out waste liquid with the concentration of 3 to 10 times, transferring primary pure liquid, performing fine purification on the primary pure liquid by using a filter with the concentration of 0.1 mu M to 0.2 mu M, and collecting the fine pure liquid to obtain the mesenchymal stem cell culture composition.
The mesenchymal stem cell culture composition is mainly prepared by the steps of obtaining cell culture supernatant and filtering and concentrating, wherein in the step of obtaining the cell culture supernatant, the mesenchymal stem cells are cultured by adopting a serum-free culture medium containing 5% -10% of serum substitutes, and the mesenchymal stem cell culture composition has the advantages of avoiding serum related risks, improving the purity of products, reducing immunogenicity, being better in consistency and the like, and is more suitable for clinical and research applications; in particular, the serum-free culture medium can effectively avoid potential related infection risks such as viruses, bacteria and the like possibly existing in serum, and is safer; since serum contains many complex components that may affect the purity and activity of cytokines and bioactive substances in the supernatant, the use of serum-free media according to the present invention may reduce the presence of these interfering substances and thereby increase the purity of the cell culture supernatant obtained; proteins and other components in serum may trigger an immune response, resulting in increased immunogenicity of the supernatant, and the invention uses serum-free medium to reduce immunogenicity of the obtained cell culture supernatant, reducing potential immune response; the serum components may be different from batch to batch, which results in unstable supernatant quality and effect.
In the step of filtering and concentrating, the supernatant is firstly subjected to rough filtration by adopting a filter membrane of 0.45 mu M to 0.65 mu M, so that large particulate matters such as cell fragments, cell nuclei, bacteria and the like in the supernatant can be removed, and then the supernatant is subjected to primary purification by a filter membrane of 100KD, so that the large particulate matters such as proteins, polypeptides, nucleic acids and the like are removed; and (3) circularly washing out waste liquid with the volume of 3-10 times, transferring the primary pure liquid, finely purifying the primary pure liquid by using a 0.1 mu M-0.2 mu M filter, removing smaller particulate matters such as microorganisms and viruses in the primary pure liquid, and then collecting the fine pure liquid.
The mesenchymal stem cell culture composition is mainly prepared by the steps of obtaining cell culture supernatant and filtering and concentrating, and the obtained composition is sterile, nontoxic and high in purity, and can be effectively applied to preparation of medicines and/or conditioning liquid.
Further, in the step of "obtaining a cell culture supernatant", the mesenchymal stem cells include cells of a first tissue mass adherence culture and cells of a second tissue mass adherence culture, the cells of the first tissue mass adherence culture and the cells of the second tissue mass adherence culture are obtained by:
obtaining cells of a first tissue mass adherence culture: placing the tissue blocks of the mesenchymal stem cells in a culture container, placing for 4-8 hours, adding a serum-free culture medium containing 5-10% of serum substitutes for culture, obtaining cells of the first tissue block in an adherence culture when the cells around the adherence tissue reach 80-90% of fusion degree, and transferring the cultured tissue blocks into a new culture container;
obtaining cells of the second tissue mass adherence culture: placing the tissue blocks transferred to a new culture container for 4-8 hours, then adding a serum-free culture medium containing 5% -10% of serum substitutes for culture, and obtaining cells of the second tissue block adherence culture when the cells around the adherence tissues reach 80% -90% of fusion degree;
the inventor finds that the tissue blocks of the mesenchymal stem cells are placed in a culture container, and after the tissue blocks are placed for 4 to 8 hours, a serum-free culture medium containing 5 to 10 percent of serum substitutes is added for culture, instead of the tissue blocks of the mesenchymal stem cells being placed in the culture container and then the culture medium being added for culture, the mesenchymal stem cells can adapt to the culture environment more quickly, the survival rate and the growth speed are improved, and meanwhile, the adhesion capability of the mesenchymal stem cells and the reconstruction of the extracellular matrix of the mesenchymal stem cells are also maintained, and the adhesion effect and the expansion efficiency of the mesenchymal stem cells are improved.
Specifically, 1) when a tissue block of the mesenchymal stem cells is just connected into a culture container, the growth environment and the in-vivo environment of the tissue block have larger difference, a certain time is needed to adapt to the growth environment in the culture container, and in the process, the tissue block is not added with a culture medium, so that the stress response of the cells can be reduced, the cells can adapt to the culture environment more quickly, and the survival rate and the growth speed of the cells are improved; 2) The adhesion capability and the extracellular matrix of the tissue block are the key for maintaining the growth and the expansion of cells, if the tissue block is added with the culture medium when the tissue block is just connected into the culture dish, the adhesion capability and the extracellular matrix of the tissue block can be damaged, so that the cells fall off and die; 3) In the process of tissue block adherence culture, the tissue block can release extracellular matrix components which can help cells to reestablish the extracellular matrix, promote the growth and the expansion of the cells, if the tissue block is just added with a culture medium when being connected into a culture dish, the extracellular matrix components can be diluted to influence the reconstruction process of the extracellular matrix, and the culture medium is added after the tissue block is placed for 4 to 8 hours, so that the reconstruction of the extracellular matrix is facilitated, and the growth and the expansion effect of the cells are improved.
In addition, in the step of obtaining the cell culture supernatant, mesenchymal stem cells are obtained through the steps of obtaining cells of the first tissue mass adherence culture and obtaining cells of the second tissue mass adherence culture, wherein the mesenchymal stem cell tissue mass cultured in the step of obtaining the cells of the second tissue mass adherence culture is the mesenchymal stem cell tissue mass cultured in the step of obtaining the cells of the first tissue mass adherence culture, namely, the mesenchymal stem cell tissue mass cultured by the culture medium is subjected to secondary culture, and the method can fully utilize the mesenchymal stem cell tissue mass and effectively improve the quantity of the obtained mesenchymal stem cells; secondly, by secondary culture of tissue blocks of the mesenchymal stem cells, the better growth state of cells in the whole system can be effectively maintained, and the functions and the characteristics of the cells are maintained; again, this culture approach can reduce the number of passages and processing steps of the cells, thereby reducing the risk of cell damage and variation, helping to maintain the stability and consistency of the cells throughout the system.
In the step of obtaining the cells for the first tissue block adherence culture, the mesenchymal stem cell tissue block is placed in a culture container, is placed for 4-8 hours, is added with serum-free culture medium containing 5-10% of serum replacement for culture, is subjected to half liquid exchange after 2-3 days of culture, is subjected to half liquid exchange again after 6-7 days of culture, is subjected to half liquid exchange for 1 time every 2-3 hours, when the cells around the adherence tissue reach the fusion degree of 80-90%, the cells for the first tissue block adherence culture are obtained, and are subjected to pancreatin digestion passage in the proportion of 1:2-1:3, and the cultured tissue block is transferred into a new culture container;
the step of obtaining the cells for the second tissue mass adherence culture is as follows: obtaining cells of the second tissue mass adherence culture: placing the tissue blocks transferred to a new culture container for 4-8 hours, adding a serum-free culture medium containing 5% -10% of serum replacement for culture, changing the liquid once every 2-3 days, and performing pancreatin digestion and passage by adopting the ratio of 1:2-1:3 when the cells around the adherent tissues reach the fusion degree of 80% -90%, so as to obtain cells of the second tissue block adherent culture.
The composition can maintain the stability of extracellular matrix and cell-cell interaction in the process of obtaining the mesenchymal stem cells, namely in the process of culturing the mesenchymal stem cells by adopting a half-quantity liquid exchange mode, reduce the stress reaction of the mesenchymal stem cells, improve the stability and consistency of the mesenchymal stem cells and save the culture medium and the cost.
Specifically, 1) maintenance of extracellular matrix and cell-cell interactions: the invention adopts a culture mode of half liquid exchange to reduce the dilution and replacement frequency of the culture medium, is beneficial to the continuous existence of extracellular matrix and cell-cell interaction, plays an important role in regulating and controlling the growth, differentiation and function of cells, thus maintaining the stability of the interactions and improving the growth and function expression of the cells; 2) Reducing cellular stress response: the invention adopts a culture method with half liquid exchange, which can reduce the stress response of the culture medium to cells, and frequent culture medium replacement can lead to the stress response of the cells, such as apoptosis, disorder of cell cycle and the like; 3) Improving the stability and consistency of cells: the invention adopts the culture method of half liquid exchange, can reduce the change of culture conditions, improve the stability and consistency of cells, and can reduce the replacement of the culture medium due to the possible difference of trace components in the culture medium of different batches, thereby reducing the influence of the difference of the components of the culture medium on the cells and ensuring that the expression and the functions of the cells of different batches are more consistent. 4) Saving culture medium and cost: the invention adopts a half-liquid-changing culture method, so that the use amount of the culture medium can be reduced, thereby saving the culture medium and reducing the cost, the culture medium is an important component of cell culture, and the use amount of the culture medium can be saved and the economic cost can be reduced by reducing the replacement of the culture medium.
In the step of obtaining the cells for the first tissue block adherence culture, the composition of the invention is used for half liquid exchange after 2-3 days of culture, half liquid exchange again after 6-7 days of culture, and half liquid exchange for 1 time every 2-3 hours, so that fresh nutrients and growth factors can be provided, metabolic products are removed, pollution is prevented, the stability of culture environment is maintained, and the normal growth and functional expression of mesenchymal stem cells are ensured.
In particular, the mesenchymal stem cells are in a proliferation stage after 0-7 days of culture, the number of the cells is rapidly increased, and the liquid is respectively changed in half after 2-3 days and after 6-7 days, so that fresh nutrients and growth factors can be provided to meet the requirements of the cells, promote the rapid growth and proliferation of the cells, ensure that the cells obtain enough nutrients and growth factors and maintain a good growth state of the cells. And then the growth speed of the mesenchymal stem cells is gradually slowed down, the mesenchymal stem cells enter a stabilization period, and the culture medium is replaced every 2-3 hours, so that metabolites can be effectively removed, new nutrients are provided, the health state of the cells is maintained, and the stability of a culture environment is maintained.
One of the objects of the present invention is to provide a culture method of a mesenchymal stem cell culture composition, comprising the steps of:
cell culture supernatant was obtained: taking mesenchymal stem cells, placing the mesenchymal stem cells in a serum-free culture medium containing 5% -10% of serum substitutes for culture, and collecting supernatant when the cells reach 80% -90% of fusion degree;
and (3) filtering and concentrating: the supernatant collected in the step of obtaining the cell culture supernatant is firstly subjected to rough filtration by adopting a filter membrane with the concentration of 0.45 mu M to 0.65 mu M, then is primarily purified by a filter membrane with the concentration of 100KD, and is transferred to primary purified liquid after the waste liquid with the volume of 3 to 10 times is circularly washed out, and the primary purified liquid is subjected to fine purification by using a filter with the concentration of 0.1 mu M to 0.2 mu M, so that the fine purified liquid is collected.
Further, in the "obtaining cell culture supernatant" step, the mesenchymal stem cells are obtained by:
obtaining cells of a first tissue mass adherence culture: placing the tissue blocks of the mesenchymal stem cells in a culture container, placing for 4-8 hours, adding a serum-free culture medium containing 5-10% of serum substitutes for culture, obtaining cells of the first tissue block in an adherence culture when the cells around the adherence tissue reach 80-90% of fusion degree, and transferring the cultured tissue blocks into a new culture container;
obtaining cells of the second tissue mass adherence culture: placing the tissue blocks transferred to a new culture container for 4-8 hours, then adding a serum-free culture medium containing 5% -10% of serum substitutes for culture, and obtaining cells subjected to wall-attached culture of the tissue blocks for the second time when the cells around the wall-attached tissues reach 80% -90% of fusion degree;
in the step of obtaining the cells for the first tissue block adherence culture, the mesenchymal stem cell tissue block is placed in a culture container, is placed for 4-8 hours, is added with serum-free culture medium containing 5-10% of serum replacement for culture, is subjected to half liquid exchange after 2-3 days of culture, is subjected to half liquid exchange again after 6-7 days of culture, is subjected to half liquid exchange for 1 time every 2-3 hours, when the cells around the adherence tissue reach the fusion degree of 80-90%, the cells for the first tissue block adherence culture are obtained, and are subjected to pancreatin digestion passage in the proportion of 1:2-1:3, and the cultured tissue block is transferred into a new culture container;
the step of obtaining the cells for the second tissue mass adherence culture is as follows: obtaining cells of the second tissue mass adherence culture: placing the tissue blocks transferred to a new culture container for 4-8 hours, adding a serum-free culture medium containing 5% -10% of serum replacement for culture, changing the liquid once every 2-3 days, and performing pancreatin digestion and passage by adopting the ratio of 1:2-1:3 when the cells around the adherent tissues reach the fusion degree of 80% -90%, so as to obtain cells of the second tissue block adherent culture.
It is an object of the present invention to provide an aerosolized composition comprising the mesenchymal stem cell culture composition described above.
The atomized composition of the invention comprises the mesenchymal stem cell culture composition, and is an atomizing agent when the atomized composition is used by an atomizer. When the aerosol is inhaled through the atomizer, injection or other complex operation is not needed, and the aerosol is convenient and simple to use and is particularly suitable for special people, such as the elderly or children; when the atomized composition inhaled by the atomizer can directly enter the respiratory tract and contact with the epithelial cells of the respiratory tract, so that local treatment is realized, and the local treatment mode can better target respiratory diseases and effectively treat rhinitis, in particular allergic rhinitis; the atomized composition inhaled through the atomizer can be absorbed and utilized more quickly, and the tiny particles generated by the atomizer can be absorbed by respiratory tract better, enter blood circulation, and thus exert therapeutic effect rapidly.
Further, the mesenchymal stem cell culture composition in the atomized composition is a mesenchymal stem cell culture composition subjected to freeze-drying treatment.
The intermediate mesenchymal stem cell culture composition of the atomized composition is a mesenchymal stem cell culture composition subjected to freeze-drying treatment, and has the advantages of long-term storage, convenience in transportation, high stability, convenience in use and the like, and specifically, 1) long-term storage: the lyophilization process removes moisture from the mesenchymal stem cell culture composition, and converts it into a solid state, thereby prolonging its shelf life. The freeze-dried mesenchymal stem cell culture composition can be stored for a long time under the low-temperature condition, and is not easy to be polluted and deteriorated by microorganisms; 2) The transportation is convenient: the freeze-dried mesenchymal stem cell culture composition does not contain water, has small volume and light weight, is easy to transport and carry, and compared with a liquid state, the freeze-dried state can reduce the risk of liquid leakage and damage, and is convenient for remote transportation; 3) Stability is high: the freeze-dried mesenchymal stem cell culture composition has higher stability, is not easy to be influenced by factors such as temperature, illumination, oxygen and the like, and can keep the activity and the function of active ingredients in the mesenchymal stem cell culture composition, so that the mesenchymal stem cell culture composition can keep a consistent effect when required; 4) The use is convenient: the freeze-dried mesenchymal stem cell culture composition can be redissolved when needed, is convenient to use, can recover the liquid state by only adding a proper amount of solvent, and is convenient to use.
Further, the atomized composition further comprises a solvent, and the mesenchymal stem cell culture composition is dissolved in the solvent for use after freeze-drying; preferably, the solvent is water; more preferably, the water is obtained after filtration of purified water.
The solvent in the atomized composition is used for dissolving the freeze-dried mesenchymal stem cell culture composition, is convenient to use, and enables the mesenchymal stem cell culture composition to be recovered into a liquid state. The solvent is preferably water, particularly the purified water is obtained after filtration, and the filtered purified water passes through a microporous filter, so that the impurities such as microorganisms, particles, organic matters and the like in the water can be removed, the purity of the solvent is ensured, and the filtered purified water does not contain harmful substances and has no toxic and damaging effects on cells and human bodies.
The invention aims at providing application of the mesenchymal stem cell culture composition in preparation of medicines and/or conditioning fluid for treating rhinitis.
The invention aims to provide the application of the atomized composition in preparing medicines and/or conditioning liquid for treating rhinitis.
The nebulized composition of the invention is administered 24-72 hours apart, preferably 24 hours apart or 48 hours apart.
The atomized composition can act on the upper respiratory tract after atomized inhalation, condition breath, dredge blockage, break up stasis and energize immunity, can effectively relieve nasal obstruction caused by allergic rhinitis, clear residual nasal discharge of nasal cavity and bring a feeling of smooth breathing to a user; the use method is simple, the atomization device can be assembled, the atomization process can be started for about 20-30 minutes, the space is not occupied, and other complicated dangerous operations are not needed; the whole preparation process is a serum-free culture system, does not contain any animal serum components, and is quite reliable in terms of safety; can play a positive promoting role in scientific research and conditioning and relieving of allergic rhinitis.
Drawings
Fig. 1 shows the data analysis results of the present atomization conditioning.
Detailed Description
Unless otherwise indicated or defined, all terms used have the usual meaning in the art, which will be understood by those skilled in the art. Reference is made, for example, to Sambrook et al, "Molecular Cloning: A Laboratory Manual"; lewis, "Genes VIII"; and Roitt et al, "Immunology" (8 th edition), stewart seal et al, "Stem Cells Handbook"; massimiliano Gnecchi et al, "Mesenchymal Stem Cells Methods and Protocols" and the general prior art cited herein; moreover, unless otherwise indicated, all methods, steps, techniques and operations not specifically detailed may be, and have been, performed in a manner known per se, which will be appreciated by those skilled in the art. Reference is also made to, for example, standard handbooks, the above-mentioned general prior art and other references cited therein.
Stem cells are cells derived from an embryo or a living body and having the ability to self-renew, proliferate and differentiate under certain conditions, and are capable of producing progeny cells having the same phenotype and genotype as those of the stem cells themselves, and differentiating to produce specialized cells constituting tissues or organs of the living body.
Mesenchymal stem cells, also called pluripotent stromal cells, abbreviated as MSCs, are a type of pluripotent stem cells, which have the common characteristics of stem cells, have the ability to self-renew and the ability to multiply differentiate, belong to the mesodermal class of pluripotent stem cells, and are mainly present in connective tissues and organ mesenchyme, and comprise: bone marrow, placenta, umbilical cord, fat, mucous membrane, bone, muscle, lung, liver, pancreas, amniotic fluid, amniotic membrane, umbilical cord blood, etc.; the source of the mesenchymal stem cells is not particularly limited in the present invention.
The capsule wrapped by the biological film is a vesicle secreted by cells and wrapped by a bilayer biological film, and the vesicle is inlaid with lipid, has the diameter of about 30-200nm, contains various substances such as protein, lipid, polysaccharide, nucleic acid and the like, and participates in the transportation and information communication among cells.
All the operation processes in the invention are required to be completed under the condition of hundred-level cleanliness.
The invention will be further illustrated with reference to specific examples.
EXAMPLE 1 preparation of mesenchymal Stem cell culture composition in aerosolized composition
1. Source of the location of mesenchymal stem cells
Umbilical cord-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, and adipose-derived mesenchymal stem cells: the donor guardian signed the donor consent, and was checked for hepatitis C virus antibodies, HIV I/II antibodies, hepatitis B virus antigens, and treponema pallidum antibodies prior to donation. And after donation, simultaneously collecting tissue samples with qualified inspection results for detecting bacteria, viruses and mycoplasma, and ensuring that the donated tissues have no bacteria, viruses and mycoplasma pollution.
2. The preparation method comprises the following steps:
step 1: obtaining a supernatant of mesenchymal stem cell culture:
1) Under the aseptic condition, cleaning the part where the obtained mesenchymal stem cells are located with PBS for 3 times, and removing residual blood cells; cutting the part where the washed mesenchymal stem cells are located into tissue blocks with the length of about 2.0cm, and removing blood vessels; taking out the gel-like tissue, and cutting into pieces of about 1mm in size 3 Then placed in a T75 flask;
2) Obtaining cells of a first tissue mass adherence culture: after placing the tissue mass in a T75 flask for 6 hours, 12ml of complete medium (MSCBM serum-free medium (Dake 6114021) +5% -10% serum substitute (AventaCell, HPCFDCGL)) was added, and the mixture was placed in a CO2 incubator at 37℃and a partial pressure of 5% for cultivation (Day 0); tilting the culture flask for 45 degrees, slowly sucking half volume of liquid culture medium from the upper liquid level by using a pipette to store in a collecting flask to avoid contacting with one side of bottom cells, replacing a new pipette, sucking half volume of fresh complete culture medium, slowly adding along the inner wall of the culture flask to avoid blowing up bottom adherent cells, closing a bottle cap, putting back into an incubator to continue culture, and performing half volume exchange again after the 7 th Day (Day 7), and performing half volume exchange for 1 time every 3 hours; observing the growth condition of cells around the adherent tissues, when the cells reach 80% -90% of fusion degree, adopting a ratio of 1:2-1:3 to digest and passage with pancreatin (Gibco, 15090046), obtaining cells (generation P1) of the first tissue mass adherent culture, and transferring the tissue mass into a new culture bottle;
3) Obtaining cells of the second tissue mass adherence culture: transferring the tissue block to a new culture flask in the step 2) for 6 hours, adding 12ml of complete culture medium (MSCBM serum-free culture medium plus 5% -10% serum substitute), and placing in a CO2 incubator with the partial pressure of 5% at 37 ℃ for culture; half liquid change is carried out every 2-3 days, wherein adherent cells can be seen on the 2-3 th day, obvious cell clones can be formed on the 5 th day, when the cells reach 80% -90% of fusion degree, pancreatin digestion passage (generation P1) is carried out by adopting the proportion of 1:2-1:3, and cells of the second tissue mass adherent culture are obtained;
4) Subculture: obtaining cells of the first tissue block adherence culture and cells of the second tissue block adherence culture, respectively carrying out subculture to P6 generation times, when the cells of the P6 generation reach 80% -90% fusion degree, respectively obtaining culture supernatant of the cells of the P6 generation, obtaining the cell culture supernatant of the first tissue block adherence culture and the cell culture supernatant of the second tissue block adherence culture, and mixing the cell culture supernatant of the first tissue block adherence culture and the cell culture supernatant of the second tissue block adherence culture to obtain the cell culture supernatant; then the cells were digested with pancreatin and then treated to 1X 10 7 The tube was then frozen by adding serum-free cryopreservation solution (MSCBM serum-free medium+5% -20% serum substitute+8% DMSO (sigma, D2650-100 ML)).
Specifically, cells subjected to the first tissue mass adherence culture are P1 generation, 12ml of complete culture medium (MSCBM serum-free culture medium plus 5% -10% serum substitute) is added into the P1 generation cells, the cells are placed in a CO2 incubator with the temperature of 37 ℃ and the partial pressure of 5%, and when the cells reach 80% -90% fusion degree, pancreatin (Gibco, 15090046) digestion and passage are carried out by adopting the ratio of 1:2-1:3, so that P2 generation cells are obtained; adding 12ml of complete culture medium (MSCBM serum-free culture medium plus 5% -10% serum substitute) into cells of the generation P2, placing the cells in a CO2 incubator with the partial pressure of 5% at 37 ℃ for culture, and when the cells reach the fusion degree of 80% -90%, adopting the proportion of 1:2-1:3 to carry out pancreatin (Gibco, 15090046) digestion and passage to obtain the cells of the generation P3; until the cells are cultivated to the P6 generation, when the cells of the P6 generation reach 80 to 90 percent of fusion degree, firstly, harvesting culture supernatant of the cells of the P6 generation, and obtaining cell culture supernatant of the first tissue mass adherence culture;
obtaining cells subjected to second tissue mass adherence culture as P1 generation, adding 12ml of complete culture medium (MSCBM serum-free culture medium+5-10% serum substitute) into the cells of the P1 generation, placing the cells in a CO2 incubator with the temperature of 37 ℃ and the partial pressure of 5%, culturing, and when the cells reach 80-90% fusion degree, adopting the proportion of 1:2-1:3 to digest and passge with pancreatin (Gibco, 15090046) to obtain P2 generation cells; adding 12ml of complete culture medium (MSCBM serum-free culture medium plus 5% -10% serum substitute) into cells of the generation P2, placing the cells in a CO2 incubator with the partial pressure of 5% at 37 ℃ for culture, and when the cells reach the fusion degree of 80% -90%, adopting the proportion of 1:2-1:3 to carry out pancreatin (Gibco, 15090046) digestion and passage to obtain the cells of the generation P3; and (3) culturing until the cell reaches the P6 generation, and when the cell reaches 80% -90% of fusion degree after the P6 generation, firstly harvesting culture supernatant of the cell after the P6 generation, thus obtaining cell culture supernatant of the second tissue block adherence culture.
Step 2: filtering and concentrating
1) Uniformly mixing the obtained supernatant with a conformation stabilizer (LASCO, living organism, 1201228-001) according to the ratio of 4:1-6:1, performing rough filtration by using a 0.45 mu M filter, performing primary purification by using a 100KD tangential flow membrane package, and transferring the primary pure liquid into a biosafety cabinet after circulating and washing out waste liquid with the volume of 3 times;
2) The primary pure liquid is refined by using a 0.1 mu M filter, and the refined pure liquid is collected;
3) After finishing the purification, subpackaging into penicillin bottles, wherein each bottle is about 3.0 ml.
Step 3: freeze-drying
1) Sealing the penicillin bottles in a box, and freezing in a-80 refrigerator for 18-24 hours;
2) Placing the frozen penicillin bottle in a freeze-dryer cold hydrazine, sealing and vacuumizing, setting the temperature to-60 ℃, and the vacuum degree to 30pa, wherein the freeze-drying process lasts for 72 hours;
3) And after the freeze-drying is finished, taking out the penicillin bottle, placing the penicillin bottle in a biological safety cabinet, plugging, capping, and labeling NR powder to obtain the capsule for extracting the mesenchymal stem cells in the atomized composition.
EXAMPLE 2 preparation of solvent in aerosolized pharmaceutical composition
Filtering the purified water with 0.1 μm filter, packaging with 8 ml/bottle into penicillin bottle, adding plug, capping, and labeling NR solvent to obtain the solvent in the atomized composition.
Example 3 nebulized composition
The atomized composition of the present invention comprises a mesenchymal stem cell-extracted capsule prepared by the method of example 1 and a solvent obtained by the method of example 2, and comprises 1 bottle of mesenchymal stem cell-extracted capsule (NR powder) and 1 bottle of solvent (NR solvent).
1. Product inspection
In the invention, the packaged NR powder and NR solution are delivered to a third-party CNAS detection mechanism to detect appearance, sterility, mycoplasma, endotoxin, osmotic pressure, total protein and the content of the capsule wrapped by the biological film;
the detection results are shown in the following table 1, and the results meet the release requirements;
table 1: detection result
| Sequence number | Detection item | Detection standard | Detection result |
| 1 | Appearance of | Pale yellow or colorless transparent liquid | Pale yellow transparent liquid |
| 2 | Sterile | Negative of | Negative of |
| 3 | Mycoplasma species | Negative of | Negative of |
| 4 | Endotoxin (endotoxin) | ≤10EU/ml | ≤0.1EU/ml |
| 5 | Osmotic pressure | 280-330mOsm/Kg | 298mOsm/Kg |
| 6 | Total protein | 2.0-4.0g/L | 2.8g/L |
| 7 | Content of capsules | 1.0×10 9 -1.0×10 10 particles/ml | 6.8×10 9 particles/ml |
2. Conditioning effect of the atomized composition on allergic rhinitis;
1. assembly and use of the atomizing device.
(1) Placing a compression atomizer (Guangdong instrument standard 20142080360) on a firm and stable desktop or ground, confirming that a power switch is in a closed state, and inserting a power plug into a matched socket;
(2) Tearing the package (Guangdong instrument injection 20162080041) of the disposable atomization device, taking out the atomization mask and the elastic rope, tying the elastic rope on the mounting holes on two sides of the atomization mask, then tightly inserting the atomization mask into the air outlet hole of the atomization cup, and connecting the air inlet at the bottom of the atomization cup with the air outlet of the compression atomizer through a vent pipe;
(3) Taking out an NR powder and an NR solvent, lifting the sealing cover according to the arrow indication on the sealing cover, carefully pulling open the metal skin, and then stripping the whole cover body;
(4) Pulling out the rubber plug, pouring all the solution in the NR solvent into an NR powder bottle, covering the rubber plug, pinching the rubber plug and the bottle bottom, reversing the force for about 8-10 times up and down, and standing for 2 minutes;
(5) Opening a liquid inlet of the atomizing cup, pouring the uniformly mixed solution, closing the liquid inlet of the atomizing cup, wearing the atomizing device, and ensuring that the nasal cavity is correctly wrapped;
(6) Starting a power supply, starting atomization conditioning, and enabling a person to breathe normally after about 20-30 minutes in the whole single atomization conditioning process;
2. an atomization conditioning regimen;
(1) The conditioning is carried out by setting 4 conditioning groups, wherein 5 conditioning persons in each group are half of men and women, the age distribution is 20-67 years old, and the specific schemes are shown in Table 2;
table 2: atomized conditioning grouping scheme
(2) In the atomization conditioning stage, the times of adverse events such as cough, nasal obstruction, nasal discharge, itching of the canthus, dizziness, headache and the like of each person conditioning every day are counted respectively, and finally, group comparison analysis is carried out, wherein specific atomization conditioning results are shown in a figure 1, wherein the calculated P value results of a BLANK group and other three groups are smaller than 0.0001, and the results of the BLANK group and the other three groups are statistically significant differences; the calculated P value of the G24H group and the G72H group is 0.0001, and the calculated P value of the G48H group and the G72H group is 0.0008, which shows that the atomization conditioning result at intervals of 24 hours or 48 hours is superior to the atomization conditioning at intervals of 72 hours; the P value calculation result of the G24H group and the G48H group is 0.8089, no statistically significant difference exists, and the conditioning scheme of the G48H group should be adopted in consideration of the cost control factor.
Claims (10)
1. A mesenchymal stem cell culture composition, which is composed of a capsule wrapped by a biological membrane secreted in the culture of mesenchymal stem cells, wherein the content of the capsule in the mesenchymal stem cell culture composition is 1.0x10 9 -1.0×10 10 particles/ml。
2. The mesenchymal stem cell culture composition of claim 1, wherein the mesenchymal stem cell culture composition is prepared by supernatant of a mesenchymal stem cell culture solution.
3. Mesenchymal stem cell culture composition according to one of claims 1-2, characterized in that the mesenchymal stem cell culture composition is prepared by a method comprising the steps of:
cell culture supernatant was obtained: taking mesenchymal stem cells, placing the mesenchymal stem cells in a serum-free culture medium containing 5% -10% of serum substitutes for subculturing, and collecting supernatant when the cells reach 80% -90% of fusion degree;
and (3) filtering and concentrating: the supernatant collected in the "obtain cell culture supernatant" step was first subjected to a step of 0.45. Mu.M
And (3) carrying out rough filtration by a filter membrane with the concentration of 0.65 mu M, then carrying out primary purification by a filter membrane with the concentration of 100KD, circularly washing out waste liquid with the concentration of 3-10 times of the volume, transferring primary pure liquid, carrying out fine purification on the primary pure liquid by a filter with the concentration of 0.1 mu M to 0.2 mu M, and collecting the fine pure liquid to obtain the mesenchymal stem cell culture composition.
4. A mesenchymal stem cell culture composition according to claim 3, wherein in the step of "obtaining a cell culture supernatant", the mesenchymal stem cells comprise cells of a first tissue mass adherence culture and cells of a second tissue mass adherence culture, the cells of the first tissue mass adherence culture and the cells of the second tissue mass adherence culture being obtained by:
obtaining cells of a first tissue mass adherence culture: placing the tissue blocks of the mesenchymal stem cells in a culture container, placing for 4-8 hours, adding a serum-free culture medium containing 5-10% of serum substitutes for culture, obtaining cells of the first tissue block in an adherence culture when the cells around the adherence tissue reach 80-90% of fusion degree, and transferring the cultured tissue blocks into a new culture container;
obtaining cells of the second tissue mass adherence culture: placing the tissue blocks transferred to a new culture container for 4-8 hours, then adding a serum-free culture medium containing 5% -10% of serum substitutes for culture, and obtaining cells subjected to wall-attached culture of the tissue blocks for the second time when the cells around the wall-attached tissues reach 80% -90% of fusion degree.
5. The mesenchymal stem cell culture composition according to claim 4, wherein in the step of obtaining cells for the first tissue mass adherence culture, the mesenchymal stem cell tissue mass is placed in a culture container, cultured by adding a serum-free culture medium containing 5% -10% of serum replacement after 4-8 hours, half-changed after 3-4 days, half-changed after 6-7 days, half-changed after 1 time every 2-3 hours, when the cells around the adherence tissue reach a fusion degree of 80% -90%, the cells for the first tissue mass adherence culture are obtained, pancreatin digestion passage is performed by a ratio of 1:2-1:3, and the cultured tissue mass is transferred into a new culture container;
the step of obtaining the cells for the second tissue mass adherence culture is as follows: obtaining cells of the second tissue mass adherence culture: placing the tissue blocks transferred to a new culture container for 6 hours, adding a serum-free culture medium containing 5% -10% of serum substitutes for culture, changing the liquid once every 2-3 days, and performing pancreatin digestion passage by adopting the proportion of 1:2-1:3 when the cells around the adherent tissues reach the fusion degree of 80% -90%, so as to obtain cells of the second tissue block adherent culture.
6. An aerosolized composition comprising the mesenchymal stem cell culture composition of any one of claims 1-5.
7. The aerosolized composition of claim 6, wherein the mesenchymal stem cell culture composition is a freeze-dried mesenchymal stem cell culture composition.
8. The aerosolized composition of any of claims 6-7, wherein the aerosolized composition further comprises a solvent, and wherein the mesenchymal stem cell culture composition is lyophilized for dissolution in the solvent; preferably, the solvent is water; more preferably, the water is obtained after filtration of purified water.
9. Use of a mesenchymal stem cell culture composition according to any one of claims 1 to 8 for the preparation of a medicament and/or conditioning fluid for treating rhinitis.
10. Use of an aerosolized composition according to any of claims 1-8 in the manufacture of a medicament and/or conditioning fluid for the treatment of rhinitis.
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