CN218687190U - Liquid gel spray preparation and treatment system for stem cell exosomes and secretion factors - Google Patents

Abstract

The invention relates to a stem cell exosome and secretion factor liquid gel spray preparation and treatment system, which comprises a liquid gel spray preparation system and a treatment system; liquid gel spraying preparation system include aseptic preparation room, operation platform, aseptic preparation room be provided with the stem cell culture fluid container that connects gradually, from purifying thick treater, first total flow filtration membrane pond, second total flow filtration membrane pond, negative pressure pump, the concentrated subassembly of exosome concentrate, growth factor receiving flask, treatment system including the treatment platform, the treatment platform include the cabinet body, spectrum therapeutic instrument, exosome spraying appearance, growth factor spraying appearance. The device combines the preparation and the use of the stem cell exosome and the secretion factor liquid gel, so that the use of the stem cell exosome and the secretion factor liquid gel is more efficient.

Description

Liquid gel spray preparation and treatment system for stem cell exosomes and secretion factors

Technical Field

The invention relates to the application fields of bioengineering technology and plastic beauty and skin anti-aging, in particular to a stem cell exosome and secretion factor liquid gel spray preparation and treatment system.

Background

However, no matter which type of stem cells, the cell diameter is mostly above 15 μm, together with the preconditions for the application of stem cells, not only seed cells but also microenvironment and growth scaffolds for cell growth are required for survival and growth, proliferation and differentiation at the target site of action, particularly, human stem cells have relative limitations in the maintenance of bioactivity, quantification of substances and logistics for clinical treatment. Making it possible to use it in skin tissues, in particular in aged and wounded skin tissues.

The human stem cell exosome is derived from human stem cells, is a subcellular component of a vesicular structure in which one outer membrane in an outer vesicle is a double-layer phospholipid membrane, and contains various bioactive substances such as a main proteome, a genome, lipids, nucleic acids and the like of a stem cell matrix. The exosome has the appearance of irregular oval or small cup shape, small volume, average particle size of only about 30-150 nm, average particle size of about 100nm and density of 1.09-1.18g/ml. During in vitro culture and passage, the stem cells secrete exosomes (exosomes may also be present in human body fluids). The exosomes contain specific proteins, lipids and nucleic acids of the mother cells, can be used as signal molecules to conduct signal transduction, substance transfer and interconnection, interaction and mutual influence among cells, and transfer biological information of the mother cells to other cells, so that the functions of other cells are changed, and important physiological and biological functions of the mother cells are played; moreover, the interaction between cells and adjacent and distant cell organs through chemical substances by means of and by utilizing an exosome system enables exosomes to be integrated into cells to transmit chemical information and simultaneously participate in the integration processes of cell physiology and biology, including intercellular connection, cell proliferation, differentiation, regeneration and renewed signaling, angiogenesis, anti-apoptosis, antigen presentation, immune regulation and the like. In particular, exosomes exist as a form of cell-free system, are intercellular junctions that transport proteins, lipids and nucleic acids to target cells and play important roles in a variety of biological processes. The possible mechanism of action of stem cell exosomes is primarily the action and influence on recipient cells via two metabolic pathways: one is the interaction and influence of ligand-receptor patterns between exosomes and receptor cells, which can exert their biological effects by modulating the activation or inhibition of target cell signaling pathways; the other is that exosomes enter cells through membrane fusion or endocytosis with target cells or receptor cells, transduce and release their bioinformatics or active ingredients into host cells or receptor cytoplasm, and influence host cells or receptor cells through regulating specific gene expression and signal paths, finally resulting in the change of functions or phenotypes of the cells. In addition to the above features and functions, exosomes themselves belong to a double-layer phospholipid structure, and the vesicle membrane of the exosome is an ideal carrier as a natural liposome, and can be specifically absorbed by target cells and enhance the specific targeting effect of the cells by virtue of the homing capability of the exosome.

The exosome has the advantages that the cytotherapy does not have, particularly in the aspects of fibroblast proliferation and division, collagen synthesis and decomposition, wound scar reduction and inhibition, matrix metalloproteinase activation and inhibition, neovascularization and the like related to tissue regeneration repair, skin anti-aging, facial rejuvenation and the like, compared with stem cells, the exosome has the advantages of more stable property, smaller volume, more single type, longer half-life period in systemic circulation, lower immunogenicity or no immunogenicity, easy coating of target functional components and therapeutic substances for targeted precise treatment, easy penetration of blood brain barriers and skin barriers, easier preparation, storage and transportation, participation in cell differentiation, neovascularization, promotion of blood flow recovery, neovascularization and the like; meanwhile, exosome does not contain living cells, has no possibility of malignant change, has no tumorigenicity even in large-dose and high-frequency application and the like, and is a new choice of cell-free treatment with similar curative effect to human stem cells in regenerative medicine, transformation medicine, anti-aging and biological treatment. In recent years, exosomes derived from different types of mesenchymal stem cells are reported to promote migration, proliferation and angiogenesis of endothelial cells by using microRNAs (microRNAs) carried by the exosomes, such as miR31, miR125a and the like, as stimulation signals. Meanwhile, miR-21, miR-23a, miR-125b, miR-145 and the like carried by the stem cell exosomes can also inhibit a TGF-beta 2/SMAD2 signal pathway, so that fibroblast proliferation is inhibited, and the scar formation of injured skin tissues is prevented.

In addition, it has been reported that the stem cell exosome can effectively repair sensitive skin tissue, regenerate damaged skin tissue, resist skin aging and beautify skin when applied to the skin surface, and has the effects of anti-inflammation, soothing, repairing, skin barrier reconstruction and the like. The stem cell exosome can partially penetrate the stratum corneum barrier of the skin to enter the dermis, and the stem cell exosome can promote the generation, expression and secretion of collagen (type I collagen, type II collagen, type III collagen and type V collagen) and elastin of human skin tissues through stimulating cells of the dermis layer structure, and obviously reduce the expression levels of galactosidase (SA-BETA-GAL) and metalloproteinase (MMP-1/3) related to aging, so that the skin aging condition, the elasticity, the thickness and the skin quality are effectively improved. It has also been found that subcutaneous injection of exosomes stimulates ceramide synthesis in skin tissues. Ceramide is an important component for increasing the hydration degree of the stratum corneum of the skin, maintaining the skin barrier, resisting aging, whitening and the like. Therefore, the stem cell exosome has great significance for skin anti-aging, facial rejuvenation and the like, and also has wide application potential and prospect.

The temperature-sensitive gel is a semisolid gel preparation which is applied in a solution state and naturally undergoes phase transition by taking the temperature-sensitive gel as a response of a high molecular material or organic micromolecule to the external environment temperature, namely, the temperature-sensitive gel is converted from a liquid gel into non-chemical crosslinking. Temperature sensitive gels can be formed by weaker interaction forces (such as intermolecular hydrogen bonding, pi-pi interactions, van der waals forces, and hydrophobic interactions, among others). When the temperature is changed, the interaction force is directly influenced, so that the gel state is changed. It is characterized in that it is in the form of a gel liquid below the Lowest Critical Solution Temperature (LCST) and in the form of a semisolid or solid gel above the Lowest Critical Solution Temperature (LCST). When the ratio of the hydrophilic group to the hydrophobic group is appropriate, gel transition can occur in an aqueous solution. As the temperature increases, the water's ability to dissolve the polymer decreases and the interaction between the polymers becomes dominant, thereby forming a gel. At present, the wide application of the temperature-sensitive gel for the medicine and the cosmetics becomes a development direction which is concerned, valued and favored in various subject fields. Because the temperature-sensitive gel can be directly used as a good carrier of the functional substances, the active ingredients and the natural medicines to protect the biological activity of the functional substances, the active ingredients and the natural medicines and promote the penetration and absorption of the functional substances, the active ingredients and the natural medicines, and the temperature-sensitive gel can also increase the solubility, the stability, the targeting property, the plasticity and the slow release/controlled release performance of the functional substances, the active ingredients and the natural medicines; meanwhile, the composition can be combined with other dosage forms to play the functions of advantage complementation, synergy and combined synergism. Compared with the traditional preparation forms such as aqueous solution, microemulsion, submicroemulsion, cream, carbomer gel and the like, the temperature-sensitive gel preparation for skin has more characteristics and advantages. In addition, compared with the traditional microemulsion which is added with thickening agents such as carbomer and the like, the temperature-sensitive gel is more rapid to release, permeate and absorb, is more uniform and more complete, is more beneficial to uniform coating on the surface of the skin, has no toxic or side effect and irritation on the skin, and can accelerate the transdermal permeation and transdermal absorption of functional substances, active ingredients and natural medicines. Research reports that exosomes (exosomes) secreted by adipose-derived mesenchymal stem cells, various cytokines, growth factors, trophic factors, small-molecular bioactive substances and temperature-sensitive gel (temperature-sensitive hydrogel taking chitosan, sodium alginate, beta-sodium glycerophosphate and hyaluronic acid as main components) are jointly applied to promote repair, regeneration and renewal of epidermis and dermal tissues in damaged skin tissues and inhibit excessive inflammatory reaction and scar formation.

Infrared is an invisible, harmless radiation and electromagnetic wave, has a certain warming effect, and has a wavelength range of about 0.75 to 1000 μm. According to its biological characteristics, the infrared ray can be near infrared ray, its wavelength is 0.75-1.4 μm: middle infrared ray with wavelength of 1.5-3.9 micron; far infrared rays having a wavelength of 4 to 1000 μm. The wavelength range of infrared radiation commonly used in medicine is generally between 4 and 400 μm. The far infrared ray can penetrate through the skin to the tissue depth of about 3-5 mm, and has no harm to human body and no toxic side effect. It has been reported that, when applied to the skin surface, the far infrared spectrum can convert absorbed energy of the far infrared spectrum into heat energy, which causes skin epidermis temperature to be slightly raised, capillary vessels in the skin tissue to expand, blood flow speed is increased, local blood circulation is improved, microcirculation is obviously improved, metabolism of skin tissue substances is enhanced, and skin nutrition supply state is improved. Moreover, research reports that the far infrared spectrum can effectively promote the proliferation and regeneration of fibroblasts and fibroblasts in the dermis layer of the skin tissue, and promote the synthesis, expression and secretion of skin anti-aging substances such as collagen, elastin, acid mucopolysaccharide and the like in the cells, so that the repair and regeneration functions of the skin tissue and the anti-aging effect of the skin are enhanced.

Although a great deal of experimental research data support the application of stem cell exosomes in the fields of skin tissue engineering, skin injury repair, skin aging resistance, facial rejuvenation and the like, the prior art still has certain limitations in the separation and purification of stem cell exosomes, so that it is very difficult to achieve effective therapeutic dose and purity of the stem cell exosomes, and particularly, the highest yield and yield of the stem cell exosomes in one milliliter of stem cell culture solution are usually very difficult to exceed 1 microgram of the stem cell exosomes by using the current international ultracentrifugation method or polyethylene glycol kit method for preparing the stem cell exosomes. In addition, the purity of the stem cell exosomes extracted and separated from the stem cell culture solution is poor, the high heterogeneity contained in the stem cell exosomes is relatively high, and the purity of the stem cell exosomes is low due to the fact that the stem cell exosomes are obtained through microvesicles, nuclear exosomes, protein aggregates, autophagy corpuscles, apoptotic corpuscles and the like which are mixed with certain non-exosomes, and in addition, certain pollutants or specific subgroups can be entrained while the original composition of the stem cell exosomes is kept prepared, so that adverse reactions and side effects of the stem cell exosomes can be possibly caused. Meanwhile, in the process of local application of the stem cell exosome, the high action concentration and local retention state of the stem cell exosome are difficult to maintain by using a general dosage form, and the stem cell exosome needs to be applied for many times, so that certain difficulty is brought to application, and the effect is limited.

In addition, although the centrifugal supernatant of stem cells after in vitro culture contains abundant stem cell secretion factors, it is necessary to standardize extraction and separation procedures in application, and to ensure that various components contained therein do not contain components harmful to the human body, such as microorganisms having potential safety problems. In addition, whether the prepared stem cell exosome and secretion factor still have good biological functions after being stored for a certain time is also an important precondition and condition for popularization and application. Particularly, because the skin tissue damage repair and skin aging mechanism are complicated and have multiple action mechanisms and participation factors, when the aqueous solution of the pure stem cell exosome is applied to the skin tissue, the long-term curative effect and the short-term curative effect are limited, and the biological effect and the positive effect function of the pure stem cell exosome are difficult to be exerted to the maximum extent, therefore, the long-term curative effect and the short-term curative effect can be more obvious only by compounding and matching related synergistic preparations and synergistic carriers.

Meanwhile, whether the stem cell exosome or the stem cell secretion factor is applied to the skin surface, two important conditions must be met: firstly, the transdermal penetration and the transdermal absorption of the medicine are promoted to the maximum extent, and secondly, the longer retention time of the medicine on the local part is ensured to the maximum extent; and thirdly, the biological activity, the stability and the compliance of the medicines are kept to the maximum extent. However, the application of stem cell exosomes and stem cell secreted factors to the skin surface, such as by painting or spraying in an aqueous liquid medium and by conventional methods, is difficult to achieve and achieve in promoting their transdermal penetration and absorption, ensuring their longer residence time in the affected area, and maintaining their biological activity and stability and compliance.

Disclosure of Invention

The present invention is directed to improvements and innovations directed to the disadvantages, drawbacks, and problems of the background art, and provides a system for preparing and treating a liquid gel spray of exosomes and secreted factors of stem cells, which is highly effective for application to the surface of the skin.

The technical scheme of the invention is to construct a stem cell exosome and secretion factor liquid gel spray preparation and treatment system, which comprises a liquid gel spray preparation system and a treatment system;

the liquid gel spray preparation system comprises an aseptic preparation chamber and operation platforms arranged at two ends of the aseptic preparation chamber, wherein the aseptic preparation chamber comprises a shell, a stem cell culture solution container, a self-purification rough processor, a first full-flow filtration membrane pool, a second full-flow filtration membrane pool, a negative pressure pump, an exosome concentrated solution concentration component and a growth factor collecting bottle are sequentially arranged in the shell, the exosome concentrated solution concentration component comprises an exosome concentrated solution collecting bottle, a circulating pump, a three-way switching valve and a circulating membrane pool which are sequentially connected end to end, the circulating membrane pool comprises a membrane pool inlet, a first membrane pool outlet and a second membrane pool outlet, the membrane pool inlet is communicated with the first membrane pool outlet, a filter membrane is arranged between the second membrane pool outlet and the membrane pool inlet and between the second membrane pool outlet and the first membrane pool outlet, the membrane pool inlet is connected with the three-way switching valve, the first membrane pool outlet is connected with the exosome concentrated solution collecting bottle, and the second membrane pool outlet is connected with the growth factor collecting bottle;

the filtration precision of the self-purification rough filtration processor is 10-15 microns, the filtration precision of the first full-flow filtration membrane pool is 1-1.5 microns, the filtration precision of the second full-flow filtration membrane pool is 0.3-0.4 microns, and the filtration precision of the filtration membrane in the circulating membrane pool is 0.01-0.02 microns;

the treatment system include the treatment platform, the treatment platform include the cabinet body, spectrum therapeutic instrument, exosome spraying instrument, growth factor spraying instrument, the spectrum therapeutic instrument pass through the bracing piece setting in the top of the cabinet body, exosome spraying instrument and growth factor spraying instrument activity respectively set up the both sides at the cabinet body, exosome spraying instrument pass through pipe connection tee bend diverter valve, growth factor spraying instrument pass through pipe connection growth factor receiving flask, growth factor spraying instrument and growth factor receiving flask between still be provided with the trace liquid feeding pump.

In one embodiment, the back of the shell is provided with a door, and a hand sleeve is arranged on the shell or the door, the hand sleeve is positioned in the shell, and the opening is connected with the shell or the door in a sealing mode.

In one embodiment, the top of the shell is provided with a lighting lamp and an ultraviolet germicidal lamp.

In one embodiment, the pipelines between the growth factor spraying instrument and the micro liquid adding pump and between the exosome spraying instrument and the three-way switching valve are respectively provided with a bacteria filter, and the filtering precision of the bacteria filter is 0.22-0.45 micrometer.

In one of them embodiment, self-purification colating treater include the colating membrane of barrel, agitator, tube-shape, the lateral wall upper portion of barrel is provided with the liquid outlet, the agitator including take the motor and the scraper blade of (mixing) shaft, the scraper blade connect the (mixing) shaft and with the inner wall contact of coarse filtration membrane, the top and the barrel top of coarse filtration membrane are connected, lateral wall and bottom are provided with the filtration pore, the top of barrel is provided with the inside waste liquid escape orifice of intercommunication coarse filtration membrane, and the waste liquid escape orifice is provided with the solenoid valve, the bottom of coarse filtration membrane be provided with the inlet that pierces through the barrel bottom, the liquid outlet connect first full-flow filtration membrane pond, the dry cell culture liquid container is connected to the inlet.

In one embodiment, the exosome atomizer and the growth factor atomizer are of the same structure and respectively comprise a support and a material bottle, the support is of an L-shaped structure and comprises a cross arm and a vertical support body, the ends of the cross arm and the vertical support body are connected, an operating platform is arranged on the vertical support body, a rechargeable battery module is arranged at the bottom of the vertical support body, an atomizing and atomizing device is arranged in the cross arm, one end of the atomizing and atomizing device is connected with a nozzle located at the end of the cross arm, the other end of the atomizing and atomizing device extends out of the cross arm to be connected with the material bottle, and a horn mouth is further arranged at the nozzle.

In one embodiment, the material bottle comprises a bottle body and a stirring base, the stirring base is arranged at the lower part of the bottle body, a material inlet, a material outlet and an air inlet are formed in the bottle body, an inlet pipe connected with an atomizing and spraying device is arranged in the material outlet, a bacteria filtering film is arranged at the air inlet, a magnetic stirring rod matched with the stirring base is arranged in the bottle body, and the stirring base comprises an electrically-driven rotary magnetic pole and can drive the magnetic stirring rod in the bottle body to rotate.

In one embodiment, two sides of the cabinet body are respectively provided with a mounting box, a hook is arranged above the mounting box, and the vertical distance between the hook and the mounting box is as follows: when the exosome spray instrument or the growth factor spray instrument is arranged in the placing box, the hook is supported below the cross arm of the bracket.

In one embodiment, the frequency spectrum therapeutic apparatus is connected with the cabinet body through a support pipe which can be telescopically bent.

Advantages and advantageous effects of the invention

The invention relates to a stem cell exosome and secretion factor liquid gel spray preparation and application system, which has innovative new and beneficial values mainly expressed in the following points:

firstly, the invention adopts an automatic integrated preparation and application system, not only can synchronously obtain the stem cell exosomes and the secretion factors in the supernatant fluid of the in vitro subculture stem cell culture, but also can prepare the stem cell exosomes and the secretion factors into liquid gel spray for direct application, and the liquid gel spray preparation and application system for forming the stem cell exosomes and the secretion factors has obvious scientificity, advancement, practicability and innovation.

Secondly, the invention can synchronously obtain the stem cell exosome and the secretion factor in the culture supernatant of the in vitro subculture stem cells, and the stem cell exosome and the secretion factor are used as byproducts or derivatives prepared from the stem cells and are discarded after the stem cell culture and separation are finished in the past, thereby causing the waste of precious biological resources and missing a resource with wide treatment value. The invention not only recycles a large amount of waste stem cell culture solution in the original laboratory; moreover, a large amount of valuable biological resources are saved; meanwhile, the water quality pollution and the environmental damage caused by discharging a large amount of or discarding stem cell culture solution are reduced; in addition, the invention can fully exert the advantages of biological action, make the best use of things, avoid the waste of resources and save the application cost.

Thirdly, the invention adopts cascade membranes with different apertures to carry out microfiltration, ultrafiltration, nanofiltration and cross-flow ultrafiltration technology, not only can effectively extract and separate the stem cell exosomes and stem cell secretory factors in the supernatant of the in vitro subculture stem cell culture, but also can remove cell components (hybrid cells and dead cells), cell fragments, microvesicles, apoptotic bodies, autophagosomes, protein aggregates and the like of non-stem cell exosomes and secretory factors, so that the purity of the exosomes and the secretory factors is further improved. Furthermore, the stem cell exosomes can be concentrated to the quantity or density required for treatment, thereby improving the treatment effect and effect in actual application.

Fourthly, the invention adopts a modern variable-frequency micro-power pump to guide operation according to the requirement of a preparation and application system, thereby not only accelerating and improving the extraction, separation and preparation speeds, but also greatly improving the product output efficiency and the working efficiency; meanwhile, the noise is reduced, the power is saved, the energy consumption is reduced, and the like.

Fifthly, the invention can be widely used for clinical treatment, such as skin tissue burn or wound, bedsore, skin tissue ulcer caused by diabetes, diabetic foot and other skin wound repair; moreover, the skin care product can be widely used for medical cosmetology, skin anti-aging, facial rejuvenation and the like, such as skin repair and skin barrier reconstruction for removing spots and dead skin and the like of facial laser, dermabrasion, chemical exfoliation and the like, and also has the functions of improving facial microcirculation, nourishing skin, resisting wrinkles, moisturizing skin, resisting inflammation and the like. Therefore, the method has good social benefit and medical value.

Sixth, stem cell technology was once the first of the ten leading technologies in the world. The invention synchronously and efficiently extracts and separates stem cell exosomes and secretion factors from stem cell in-vitro subculture solution, and simultaneously completes the preparation of the stem cell exosomes and secretion factor liquid spray and an application system carrying automatic spraying and far infrared spectrum film forming and promoting percutaneous permeation and percutaneous absorption, which is not reported at present at home and abroad.

Seventh, many growth factors, cytokines and trophic factors such as Keratinocyte Growth Factor (KGF), epidermal Growth Factor (EGF), basic fibroblast growth factor (bFGF), vascular Endothelial Growth Factor (VEGF) and the like can promote the repair of aged skin (including skin photoaging), the reconstruction of skin barrier structures, the repair of tissue cells, the healing of skin wounds and the like, but the effect of using a single growth factor is very limited. The paracrine function of the stem cells can not only secrete a plurality of growth factors, cell factors, trophic factors and the like with high biological activity, but also directly participate in the repair, regeneration and renewal of aging or damaged skin tissues and cells and the neovascularization of skin wounds, thereby promoting the improvement of the structure and function of aging skin and the repair and healing of damaged wounds. The supernatant of the stem cell in vitro culture contains various abundant paracrine products, including stem cell exosomes and paracrine factors (such as cell factors, growth factors and trophic factors), and has practical and useful application value. They are different from stem cell therapy and application of single exogenous growth factor, and have good synergistic and combined synergistic effects.

Eighth, the invention can synchronously prepare the stem cell exosome and the temperature-sensitive gel spray of the secretion factor, not only saves the preparation time, but also improves the working efficiency, has simple, rapid and stable preparation process, and is beneficial to the process amplification and industrialization of the preparation technology; moreover, the liquid temperature-sensitive gel spray at low temperature is easy to store, transport and use; the film-forming gel state under the body surface temperature rising state is beneficial to long-time detention and controllable release of stem cell exosomes and secretory factors, reduces the use frequency, improves the compliance and comfort of a user, and has higher conversion value in multiple subject fields including skin injury repair, plastic beauty, skin radiation injury, skin photoaging, facial rejuvenation and the like. In addition, the liquid gel spray based on the stem cell exosomes and the stem cell secretion factors belongs to an application mode without cells, has more advantages, not only has low immunogenicity, fast wound healing, no scar and the like, but also can improve the directness, targeting property and high efficiency of the functions of the stem cell exosomes and the stem cell secretion factors when being locally applied to skin tissues, enhances the stability and biological activity of the exosomes under the assistance of the gel after the skin forms a film, is favorable for the stem cell exosomes and the stem cell secretion factors to exert the biological function and the curative effect function thereof to the maximum extent, and has wide application development prospect in the future.

Drawings

Fig. 1 is a schematic view of the overall structure of the present invention.

FIG. 2 is a schematic view of a liquid gel spray preparation system in an example.

Fig. 3 is a schematic view of the back of the housing in the liquid gel spray preparation system of the example.

FIG. 4 is a schematic diagram of a self-cleaning rough filter treatment unit in the liquid gel spray preparation system according to the embodiment.

Fig. 5 is a schematic structural diagram of the treatment system in the embodiment.

Fig. 6 is a schematic perspective view of an exosome nebulizer and a growth factor nebulizer in the example.

FIG. 7 is a schematic diagram of the structure of the exosome nebulizer and the growth factor nebulizer in the example.

Detailed Description

To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

It will be understood that when an element is referred to as being "disposed" on another element, it can be directly disposed or attached to the other element or intervening elements may also be present. The terms "center," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," and the like are used in an orientation or positional relationship indicated in the drawings for ease of description and simplicity of description, but do not indicate or imply that the referenced device or element must have a particular orientation, be constructed in a particular orientation, and be operated.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

Example 1

As shown in figures 1-7, a stem cell exosome and secreted factor liquid gel spray preparation and treatment system comprises a liquid gel spray preparation system and a treatment system.

The liquid gel spray preparation system comprises an aseptic preparation chamber 1 and operation platforms 2 arranged at two ends of the aseptic preparation chamber 1.

Specifically, as shown in fig. 2, the sterile preparation chamber 1 includes a housing 11, a stem cell culture solution container 12, a self-purification crude processor 13, a first full-flow filtration membrane pool 14, a second full-flow filtration membrane pool 15, a negative pressure pump 16, an exosome concentrated solution concentrating assembly 17, and a growth factor collecting bottle 18 are sequentially arranged in the housing 11, the exosome concentrated solution concentrating assembly 17 includes an exosome concentrated solution collecting bottle 171, a circulation pump 172, a three-way switching valve 173, and a circulation membrane pool 174 which are sequentially connected end to end, the circulation membrane pool includes a membrane pool inlet 174a, a first membrane pool outlet 174b, a second membrane pool outlet 174c, the membrane pool inlet 174a is communicated with the first membrane pool outlet 174b, a filtration membrane 174d is arranged between the second membrane pool outlet 174c and the membrane pool inlet 174a, and the first membrane pool outlet 174b, the membrane pool inlet 174a is connected with the three-way switching valve 173, the first membrane pool outlet 174b is connected with the exosome concentrated solution collecting bottle 171, and the second membrane pool outlet 174c is connected with the growth factor collecting bottle 18.

The filtration precision of the self-purification rough filtration processor 13 is 10-15 microns, the filtration precision of the first full-flow filtration membrane pool 14 is 1-1.5 microns, the filtration precision of the second full-flow filtration membrane pool 15 is 0.3-0.4 microns, and the filtration precision of the filter membrane 174d in the circulating membrane pool 174 is 0.01-0.02 microns.

Specifically, as shown in fig. 5-7, the treatment system includes a treatment table 3, the treatment table 3 includes a cabinet 31, a spectrum treatment apparatus 32, an exosome nebulizer 33, and a growth factor nebulizer 34, the spectrum treatment apparatus 32 is disposed above the cabinet 31 through a support rod 35, the exosome nebulizer 33 and the growth factor nebulizer 34 are respectively movably disposed at two sides of the cabinet 31, the exosome nebulizer 33 is connected to a three-way switching valve 173 through a pipeline, the growth factor nebulizer 34 is connected to a growth factor collection bottle 18 through a pipeline (as shown in fig. 1), and a micro liquid feeding pump 4 is further disposed between the growth factor nebulizer 34 and the growth factor collection bottle 18.

As shown in fig. 3, a door 111 and a hand sleeve 112 are disposed on the back of the housing 11 or on the door 111, the hand sleeve 112 is located in the housing 11, the opening is hermetically connected to the housing 11 or the door 111, an external operator can operate the article of the housing by inserting his hand into the hand sleeve, and the hand does not directly contact with the article in the housing, so that the contamination in the housing is avoided. The top of the inside of the housing 11 is provided with a lighting lamp 113 and an ultraviolet germicidal lamp 114, as shown in fig. 2.

The pipelines between the growth factor spraying instrument 34 and the micro liquid adding pump 4 and between the exosome spraying instrument 33 and the three-way switching valve 173 are provided with bacteria filters 5, and the filtering precision of the bacteria filters 5 is 0.22-0.45 micron.

Specific structure as shown in fig. 4 of self-purification coarse filtration treater 13, rough filtration membrane 133 including barrel 131, agitator, tube-shape, the lateral wall upper portion of barrel 131 is provided with liquid outlet 134, the agitator including take (mixing) shaft 135 motor 136 and scraper blade 137, scraper blade 137 connect (mixing) shaft 135 and with rough filtration membrane 133's inner wall contact, rough filtration membrane 133's top and barrel 131 top are connected, and lateral wall and bottom are provided with filtration pore 138, the top of barrel 131 is provided with the inside waste liquid escape orifice 139 of intercommunication rough filtration membrane 133, and waste liquid escape orifice 139 is provided with solenoid valve 1391, and rough filtration membrane 133's bottom is provided with the inlet 1331 that pierces through barrel 131 bottom, liquid outlet 134 connect first full-flow filtration membrane pond 14, inlet 1331 is connected dry cell culture liquid container 12 (shown in fig. 2).

As shown in fig. 6 and 7, the exosome nebulizer 33 and the growth factor nebulizer 34 have the same structure, and both include a support 331 and a material bottle 332, the support 331 has an L-shaped structure, and includes a cross arm 333 and a vertical support 334 connected at their ends, the vertical support 334 is provided with an operation platform 335, the bottom is provided with a rechargeable battery module 336, an atomizing and nebulizing device 337 is provided in the cross arm 333, one end of the atomizing and nebulizing device 337 is connected to a nozzle 338 at the end of the cross arm 333, the other end of the atomizing and nebulizing device 337 extends out of the cross arm 333 and is connected to the material bottle 339, and the nozzle is further provided with a bell-mouth 3331.

The material bottle 339 comprises a bottle body 50 and a stirring base 51, the stirring base 51 is arranged at the lower part of the bottle body 50, the bottle body 50 is provided with a material inlet 52, a material outlet 53 and an air inlet 54, the material outlet 53 is internally provided with an inlet pipe 55 connected with an atomizing and spraying device 337, the air inlet 54 is provided with a bacteria filtering membrane 56, and the bottle body 50 is internally provided with a magnetic stirring rod matched with the stirring base 51. The stirring base 51 comprises an electrically driven rotary magnetic pole, which can drive the magnetic stirring rod in the bottle 50 to rotate.

The both sides of the cabinet body 31 respectively set up a settling box 60, the top of mounting box 60 sets up a couple 61, couple 61 and settling box 60 the distance from top to bottom do: when the exosome nebulizer 33 or the growth factor nebulizer 34 is set in the housing box 60, the hook 61 is supported under the cross arm 333 of the holder 331.

The support tube 35 is a tube structure capable of being bent and extended.

The working principle of the device is as follows: the stem cell culture solution is separated in the liquid gel spray preparation system to obtain a growth factor solution (in a growth factor collecting bottle) and an exosome concentrated solution (in an exosome concentrated solution collecting bottle), the growth factor solution and the exosome concentrated solution are led into a growth factor spray instrument and an exosome spray instrument in the treatment system, a proper amount of temperature-sensitive gel is filled in material bottles of the growth factor spray instrument and the exosome spray instrument in advance, the growth factor solution, the exosome concentrated solution and the temperature-sensitive gel solution are stirred and mixed and then sprayed to an affected part in a mist form, and the mixture becomes a gel under the illumination of a spectrum treatment instrument to cover the affected part to achieve the treatment effect.

The preparation process of the growth factor solution and the exosome concentrated solution comprises the following steps: and (3) filling the stem cell culture solution into a stem cell culture solution container, and allowing the stem cell culture solution to sequentially pass through a self-purification rough filtration processor, a first full-flow filtration membrane pool and a second full-flow filtration membrane pool under the drive of a negative pressure pump and enter an exosome concentrated solution collecting bottle. Wherein the filtering process of the self-purification rough filtering processor is as follows: the material gets into inside the coarse filtration membrane through the inlet, filters out through the filtration pore of coarse filtration membrane, overflows through the oil liquid outlet and gets into first full flow membrane pond again, and the motor drives the scraper blade rotation during the coarse filtration, scrapes the material that will adhere to at the coarse filtration membrane inner wall down, avoids blockking up the membrane pore, discharges from the waste liquid escape orifice through filterable material by solenoid valve control. The material in the exosome concentrated solution collecting bottle passes through the circulation membrane pond, and littleer growth factor filters from the export outflow of second membrane pond entering growth factor collecting bottle through the filter membrane in circulation membrane pond, and the material that does not pass through the filter membrane flows back to exosome concentrated solution collecting bottle from first membrane pond export, through the circulation of certain time after, growth factor gets into the growth factor collecting bottle, and the exosome concentration in the exosome concentrated solution collecting bottle also obtains improving.

The operation platform of the present invention is typically configured as two computers or tablet computers, one of which is responsible for controlling the process flow and recording the system data, and the other is used for recording and storing the treatment data of the subject or patient, including some epidemiological data, treatment records, etc.

The invention innovatively improves the preparation technology of the application type stem cell exosomes and stem cell secretion factors, not only improves the preparation efficiency, yield and purity of the stem cell exosomes, but also improves the number, density and purity of the exosomes in the unit volume of the extracted and obtained stem cell exosomes, which is one of the aims and targets to be achieved by the invention. In the invention, multiple cascade ultrafiltration is adopted in a relatively closed sterile environment to remove possible impurity components of non-stem cell exosomes such as live cells, dead cells, cell fragments, multiple vesicles, microbubbles, nuclear exosomes, protein aggregates, autophagy bodies, apoptosis bodies and the like in a stem cell culture solution to the maximum extent, so that the purity of the stem cell exosomes is maximized; meanwhile, the stem cell exosomes are effectively intercepted and circularly concentrated by adopting a nanofiltration membrane (the aperture is 10-20 nm), so that not only is the microbial infection or cross contamination of the application-type stem cell exosomes reduced, but also the number, density and purity of the stem cell exosomes in unit volume can be improved to the maximum extent; and simultaneously, when the stem cell exosome is prepared, various biological active ingredients such as growth factors, cytokines and polypeptide factors secreted by the stem cell in a stem cell culture solution are synchronously extracted and separated, so that the coordination and the combined synergy of the stem cell exosome at the action site are increased.

The invention is innovative and improved in the application technology of the application type stem cell exosome and the stem cell secretory factor. Not only the therapeutic dose of the stem cell exosome and the stem cell secreted factor is required to meet and meet the application requirements and standards, but also the stem cell exosome and the stem cell secreted factor applied to the local tissues of the skin have better transdermal penetration and transdermal absorption effects and are kept for a longer residence time at the action part. The temperature-sensitive gel is organically combined with a stem cell exosome and a stem cell factor respectively, the stem cell exosome and the stem cell factor temperature-sensitive gel are automatically prepared, and under the synchronous action of a spraying system and a far infrared spectrum system, a stem cell exosome gel membrane and a stem cell factor gel membrane with gel membrane protection are formed at the local part and the action part of the applied skin, the stability and the slow release/controlled release action of the stem cell exosome and the stem cell secretion factor are maintained, and the transdermal permeation of the stem cell exosome and the stem cell secretion factor is promoted or accelerated under the synergistic action of infrared spectrum/frequency spectrum.

The invention adopts uniform and same conditions to prepare the stem cell exosomes, the secretion factors and the liquid gel spray thereof, which are usually colorless transparent liquid at the temperature of 30 ℃, have moderate viscosity and are easy to spray and coat. However, the gel spray of the stem cell exosome and the secretion factor is changed into solid gel after the gel spray is subjected to gel behavior at the temperature of more than 34 ℃. The stem cell exosome and the secretion factor are sprayed by gel to form a film shape, and then the film shape is solidified to present an irregular pore channel structure, the pore diameter of the pore channel structure is about 50, and the pore channel structure has certain pore wall thickness, so that better conditions are provided for compounding and loading the stem cell exosome and the stem cell secretion factor. Moreover, the whole preparation process is operated in a relatively sealed environment with a disinfection function, and a bacteria filtering device is additionally arranged, so that the consistency, the stability and the safety of the preparation process are ensured.

In addition, the liquid spray prepared by the invention is bottled or canned in a sealable manner, so that the liquid spray is very favorable for low-temperature storage and transportation, and is also helpful and significant for ensuring the biological activity of stem cell exosomes and secretory factors. Moreover, the coagulation film-forming mechanism of the stem cell exosome and secretion factor temperature-sensitive gel liquid spray on the skin surface is mainly characterized in that protons are transferred from chitosan to glycerophosphoric acid through the heat induction effect of a far infrared spectrum in the invention, so that the repulsion force between amino groups with positive charges is reduced, and the strong interaction between chitosan chains is formed, so that the chitosan chains can be converted into a gel state with a porous three-dimensional structure from a liquid state. The preparation has good histocompatibility, can effectively improve the adhesiveness and film-forming property of bioactive substances such as exosomes, secretory factors and the like separated and purified from a human mesenchymal stem cell conditioned medium, prolongs the retention time of the bioactive substances on the surface of the skin and the release time and action time of the exosomes and cell growth factors, and thus plays better roles in repairing, regenerating and updating the skin tissue. Exosomes, cytokines, growth factors and trophic factors secreted by stem cells have a wide range of skin biological effects, and can inhibit inflammatory processes, keep skin tissues compact, promote re-epithelialization processes, promote the reconstruction of skin tissues rich in new blood vessels, reduce skin fibrosis, inhibit scar tissue formation, and the like. The chitosan, trehalose water and hyaluronic acid in the temperature-sensitive gel also have the effects of resisting wrinkles, preserving moisture, moistening and protecting skin.

On the basis of the techniques and structures disclosed in the present invention, those skilled in the art can determine the relevant operation and electrical control parts in the prior art by themselves without inventive efforts.

The embodiments of the present invention are described only for the preferred embodiments of the present invention, and not for the limitation of the concept and scope of the present invention, and various modifications and improvements made to the technical solution of the present invention by those skilled in the art without departing from the design concept of the present invention shall fall into the protection scope of the present invention, and the technical content of the present invention which is claimed is fully set forth in the claims.

Claims (9)

1. A stem cell exosome and secretion factor liquid gel spray preparation and treatment system is characterized in that: comprises a liquid gel spray preparation system and a treatment system;

the liquid gel spray preparation system comprises an aseptic preparation chamber and operation platforms arranged at two ends of the aseptic preparation chamber, wherein the aseptic preparation chamber comprises a shell, a stem cell culture solution container, a self-purification rough processor, a first full-flow filtration membrane pool, a second full-flow filtration membrane pool, a negative pressure pump, an exosome concentrated solution concentration component and a growth factor collecting bottle are sequentially arranged in the shell, the exosome concentrated solution concentration component comprises an exosome concentrated solution collecting bottle, a circulating pump, a three-way switching valve and a circulating membrane pool which are sequentially connected end to end, the circulating membrane pool comprises a membrane pool inlet, a first membrane pool outlet and a second membrane pool outlet, the membrane pool inlet is communicated with the first membrane pool outlet, a filter membrane is arranged between the second membrane pool outlet and the membrane pool inlet and between the second membrane pool outlet and the first membrane pool outlet, the membrane pool inlet is connected with the three-way switching valve, the first membrane pool outlet is connected with the exosome concentrated solution collecting bottle, and the second membrane pool outlet is connected with the growth factor collecting bottle;

the filtration precision of the self-purification rough filtration processor is 10-15 microns, the filtration precision of the first full-flow filtration membrane pool is 1-1.5 microns, the filtration precision of the second full-flow filtration membrane pool is 0.3-0.4 microns, and the filtration precision of the filtration membrane in the circulating membrane pool is 0.01-0.02 microns;

the treatment system include the treatment platform, the treatment platform include the cabinet body, spectrum therapeutic instrument, exosome spraying instrument, growth factor spraying instrument, the spectrum therapeutic instrument pass through the bracing piece setting in the top of the cabinet body, exosome spraying instrument and growth factor spraying instrument activity respectively set up the both sides at the cabinet body, exosome spraying instrument pass through pipe connection tee bend diverter valve, growth factor spraying instrument pass through pipe connection growth factor receiving flask, growth factor spraying instrument and growth factor receiving flask between still be provided with the trace liquid feeding pump.

2. The stem cell exosome and secreted factor liquid gel spray preparation and therapy system according to claim 1, characterized in that: the back of the shell is provided with a door and hand sleeves arranged on the shell or the door, the hand sleeves are positioned in the shell, and the opening is connected with the shell or the door in a sealing mode.

3. The stem cell exosome and secreted factor liquid gel spray preparation and therapy system according to claim 1, characterized in that: the top in the shell is provided with a lighting lamp and an ultraviolet germicidal lamp.

4. The stem cell exosome and secreted factor liquid gel spray preparation and therapy system according to claim 1, characterized in that: and the pipelines between the growth factor spraying instrument and the micro liquid adding pump and between the exosome spraying instrument and the three-way switching valve are respectively provided with a bacteria filter, and the filtering precision of the bacteria filter is 0.22-0.45 micrometer.

5. The stem cell exosome and secreted factor liquid gel spray preparation and therapy system according to claim 1, characterized in that: self-purification colating treater include the coarse filtration membrane of barrel, agitator, tube-shape, the lateral wall upper portion of barrel is provided with the liquid outlet, the agitator including take the motor and the scraper blade of (mixing) shaft, the scraper blade connect the (mixing) shaft and with the inner wall contact of coarse filtration membrane, the top and the barrel top of coarse filtration membrane are connected, lateral wall and bottom are provided with the filtration pore, the top of barrel is provided with the inside waste liquid escape orifice of intercommunication coarse filtration membrane, and the waste liquid escape orifice is provided with the solenoid valve, and the bottom of coarse filtration membrane is provided with the inlet that pierces through the barrel bottom, the liquid outlet connect first full current filtration membrane pond, dry cell culture liquid container is connected to the inlet.

6. The stem cell exosome and secreted factor liquid gel spray preparation and therapy system according to claim 1, characterized in that: the exosome spraying instrument and the growth factor spraying instrument are of the same structure and respectively comprise a support and a material bottle, the support is of an L-shaped structure and comprises a cross arm and a vertical support body, the end portions of the cross arm are connected, an operating platform is arranged on the vertical support body, a rechargeable battery module is arranged at the bottom of the vertical support body, an atomizing and spraying device is arranged in the cross arm, one end of the atomizing and spraying device is connected with a nozzle located at the end portion of the cross arm, the other end of the atomizing and spraying device extends out of the cross arm to be connected with the material bottle, and a horn mouth is further arranged at the position of the nozzle.

7. The stem cell exosome and secreted factor liquid gel spray preparation and therapy system according to claim 6, characterized in that: the material bottle include bottle and stirring base, stirring base set up the lower part at the bottle, the bottle on be provided with material import, material export and air intlet, material export in be provided with the import pipe of connecting atomizing and atomizer, air intlet department be provided with the fungus membrane of straining, the bottle in be provided with stirring base matched with magnetism stirring rod, stirring base include electrically driven rotatory magnetic pole, can drive the magnetism stirring rod rotation in the bottle.

8. The stem cell exosome and secreted factor liquid gel spray preparation and therapy system according to claim 6, characterized in that: the both sides of the cabinet body respectively set up a settling box, the top of mounting box sets up a couple, couple and settling box the upper and lower distance do: when the exosome spray instrument or the growth factor spray instrument is arranged in the placing box, the hook is supported below the cross arm of the bracket.

9. The stem cell exosome and secreted factor liquid gel spray preparation and therapy system according to claim 1, characterized in that: the frequency spectrum therapeutic apparatus is connected with the cabinet body through a supporting tube which can be bent in a telescopic way.

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