CN113969260A - Mixed frozen crystal powder combining platelet exosomes and stem cell exosomes and preparation method thereof - Google Patents
Mixed frozen crystal powder combining platelet exosomes and stem cell exosomes and preparation method thereof Download PDFInfo
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- CN113969260A CN113969260A CN202110755875.2A CN202110755875A CN113969260A CN 113969260 A CN113969260 A CN 113969260A CN 202110755875 A CN202110755875 A CN 202110755875A CN 113969260 A CN113969260 A CN 113969260A
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Abstract
The invention provides a mixed frozen crystal powder combining platelet exosome and stem cell exosome and a preparation method thereof, and the preparation method comprises the following steps: centrifuging a platelet plasma sample to obtain a first platelet exosome; centrifuging a whole blood sample to obtain a platelet sample, and then centrifuging the platelet sample to obtain a second platelet exosome; centrifuging a stem cell culture medium sample to obtain a first stem cell exosome; performing ultrasonic oscillation and centrifugation on a stem cell sample to obtain a second stem cell exosome; and respectively freeze-drying the exosomes to obtain mixed cryogel powder of the platelet exosomes and the stem cell exosomes.
Description
Technical Field
The invention relates to the technical field of frozen crystal powder, in particular to mixed frozen crystal powder combining platelet exosomes and stem cell exosomes and a preparation method thereof.
Background
Exosomes (exosomes) are vesicles which are released by fusion of a multivesicular body (MVB) containing a plurality of vesicles formed by inward recession of cells in a budding manner after the cells engulf heterologous substances, and are extracellular vesicles with a diameter of about 30-100 nm. Exosomes can be released from different types of cells such as stem cells, tumor cells, macrophages, dendritic cells, mast cells, human amniotic epithelial cells, endothelial progenitor cells and the like, and are widely distributed in various body fluids such as saliva, ascites, pericardial effusion, urine, amniotic fluid, breast milk, cerebrospinal fluid, blood and the like. Exosomes also carry multiple molecular components that participate in physiological and pathological processes such as cellular communication, cell migration, angiogenesis, and tumor cell growth. The exosome structure contains various substances such as information nucleic acid structure (mRNA), microribonucleic acid (microRNA), Deoxyribo Nucleic Acid (DNA) fragment, protein, lipid and the like, wherein the microRNA is a main regulating substance for intercellular communication, can regulate the gene expression of receptor cells through self degradation and re-expression, can be used as a biomarker for early diagnosis and prognosis of cancers in lung cancer, colorectal cancer, prostate cancer and the like, and researches show that the exosome plays an important role in the processes of tumor cell occurrence and development and nerve cell signal transduction.
Platelet Rich Plasma (PRP) contains a large amount of bioactive platelets, and under certain conditions, these platelets can release various cytokines stored therein through an activation process into the surrounding environment to promote cell proliferation, differentiation and tissue reconstruction and repair, thus being a novel and convenient technology for treatment and medical and aesthetic purposes.
The autologous platelet plasma technology can be applied to orthopedic treatment, and also can be widely applied to the treatment of chronic wounds such as diabetic foot, bedsore and the like in the aspect of wound treatment, meanwhile, the autologous platelet plasma technology is also applied to the treatment of corneal ulcer or xerophthalmia in ophthalmology, and the autologous platelet plasma technology can be applied to the filling and repairing of tooth extraction tooth root wounds in dentistry. In addition, in the field of medical cosmetology, this technique can be applied to eliminate wrinkles and improve baldness.
Mesenchymal Stem Cells (MSCs) in stem cells are cells derived from mesoderm and have the ability to self-renew and differentiate in multiple directions. And the MSC can be separated from various tissues, and the common MSC is generally extracted from tissues such as bone marrow, fat, umbilical cord, cord blood and the like, thereby promoting the application of the MSC. Paracrine action of MSCs is observed in a number of animal models and related studies demonstrate that MSCs exert their therapeutic effect by secreting factors, membrane vesicles known as exosomes, to reduce cell damage and enhance repair. In studies of clinical tissue regeneration applications, it was suggested that the described cell growth factors promote hair growth. In addition, the cell growth factor has a certain degree of help for local tissue ischemia caused by diabetics and repair of wounds and scars in plastic surgery. MSC as a source of exosomes have two significant features, immunomodulatory properties and low production cost. The exosome from the MSC can regulate innate immunity and adaptive immune response, reduce B cell activation, proliferation and secretion by inhibiting T lymphocyte functions, influence macrophage differentiation and dendritic cell maturation, and inhibit cytotoxic activity of natural killer cells to play the role of immune regulation, thereby prolonging the service life of the MSC exosome-derived drug delivery carrier and improving the bioavailability of the drug. Moreover, there is increasing evidence that MSCs produce large amounts of exosomes compared to other cells.
Therefore, the high-purity exosome is beneficial to analysis and function research of contents, and the efficiency of obtaining the secretion factor by the high-purity exosome is high. However, in the prior art, the exosome purity obtained from the mixed cryogel powder made of exosomes extracted from platelets is low. In view of the above, the present invention provides a customized mixed cryo-crystal powder combining platelet exosomes and stem cell exosomes and a preparation method thereof.
Disclosure of Invention
The invention provides a mixed frozen crystal powder combining platelet exosomes and stem cell exosomes and a preparation method thereof. According to an embodiment of the present invention, the mixed cryo-powder comprises a powder of platelet exosomes and a powder of stem cell exosomes.
Wherein the platelet exosome is obtained from blood of a user who wants to use the mixed cryogel powder.
Wherein the stem cell exosome is obtained from cells such as skeletal stem cells, umbilical cord stem cells or adipose-derived stem cells; wherein the stem cell exosomes are derived from stem cells comprising mammalian bone, mammalian umbilical cord, mammalian fat, or mammalian blood; wherein the stem cell exosomes are stem cells obtained from human bones, human umbilical cords, human fat or human blood.
The platelet exosome powder and the stem cell exosome powder are obtained by respectively extracting the platelet exosome and the stem cell exosome through an ultracentrifugation method, and then the platelet exosome and the stem cell exosome are subjected to quick freezing at the temperature of-80 ℃ and freeze drying at the temperature of-55 ℃ for 12-16 hours and then mixed to form the mixed frozen crystal powder.
The invention also provides a preparation method of the mixed cryogel powder for combining the platelet exosomes and the stem cell exosomes. According to another embodiment of the present invention, a method for preparing a mixed cryogel powder comprises the steps of centrifuging a platelet plasma sample in a sterile environment to obtain a first platelet exosome; centrifuging a whole blood sample in a sterile environment to obtain a platelet sample, and then centrifuging the platelet sample in the sterile environment to obtain a second platelet exosome; centrifuging a stem cell culture medium sample in a sterile environment to obtain a first stem cell exosome; obtaining a second stem cell exosome from a stem cell sample by ultrasonic oscillation and centrifugation; respectively freeze-drying the first platelet exosome, the second platelet exosome, the first stem cell exosome and the second stem cell exosome to form powder of the first platelet exosome, powder of the second platelet exosome, powder of the first stem cell exosome and powder of the second stem cell exosome; and mixing one of the powder of the first platelet exosome and the powder of the second platelet exosome with one of the powder of the first stem cell exosome and the powder of the second stem cell exosome to form a mixed cryo-crystal powder.
Wherein the step of centrifuging the platelet plasma sample in a sterile environment to obtain the first platelet exosomes further comprises the substeps of: centrifuging the platelet plasma sample at an acceleration of 300xg for 15 minutes to obtain a first platelet plasma sample; centrifuging the first platelet plasma sample at an acceleration of 2000xg for 15 minutes to obtain a second platelet plasma sample; filtering the second platelet plasma sample with a filter having a pore size of 0.22 μm; centrifuging the filtered second platelet plasma sample at an acceleration of 4000xg for 15 minutes to obtain a third platelet plasma sample; and separating the third platelet plasma sample with a Tangential Flow Filtration System (TFF) to obtain first platelet exosomes.
Wherein the step of centrifuging the whole blood sample in a sterile environment to obtain a platelet sample, and then centrifuging the platelet sample in a sterile environment to obtain a second platelet exosome further comprises the substeps of: centrifuging the whole blood sample at room temperature for 30 minutes at an acceleration of 2600g to obtain a whole blood sample pellet and a supernatant; adding the supernatant to 2U/mL heparin and centrifuging twice at an acceleration of 500Xg for 10 minutes to produce a first supernatant; centrifuging the first supernatant twice for 15 minutes at an acceleration of 2000xg to produce a second supernatant; centrifuging the second supernatant twice for 30 minutes at an acceleration of 10000xg to produce a third supernatant; centrifuging the third supernatant at 30000rpm for 1 hour at a temperature of 4 ℃ to produce a fourth supernatant; filtering the fourth supernatant with a filter having a pore size of 0.22 μm; centrifuging the filtered fourth supernatant at an acceleration of 4000xg for 90 minutes to produce a fifth supernatant; and, separating the fifth supernatant with a Tangential Flow Filtration System (TFF) to obtain second platelet exosomes.
Wherein the step of centrifuging the stem cell culture medium sample in a sterile environment to obtain the first stem cell exosomes further comprises the substeps of: after culturing the stem cell culture medium sample for 48 hours, filtering the stem cell culture medium sample with a filter having a pore size of 0.22 μm to obtain a first stem cell culture medium sample; centrifuging the first stem cell culture medium sample at an acceleration of 200xg to obtain a second stem cell culture medium sample; centrifuging the second stem cell culture medium sample at an acceleration of 2000xg to obtain a third stem cell culture medium sample; and, isolating the third stem cell culture medium sample with a Tangential Flow Filtration System (TFF) to obtain the first stem cell exosomes.
Wherein, the step of obtaining the second stem cell exosome from the stem cell sample by ultrasonic oscillation and centrifugation further comprises the following substeps: culturing the stem cell sample for 48 hours, performing ultrasonic oscillation on the stem cell sample for 10 minutes at a preset frequency, stopping the ultrasonic oscillation for two minutes, and performing ultrasonic oscillation on the stem cell sample for 10 minutes at an amplitude of 25% of the maximum amplitude set by the ultrasonic oscillation again to obtain a first stem cell sample; placing the first stem cell sample at room temperature, standing, incubating for 15 minutes, performing MNase digestion, and centrifuging at an acceleration of 200Xg to obtain a second stem cell sample; centrifuging the second stem cell sample at an acceleration of 2000xg to obtain a third stem cell culture medium sample; and, isolating the third stem cell culture medium sample with a Tangential Flow Filtration System (TFF) to obtain a second stem cell exosome.
Wherein the step of freeze-drying the first platelet exosome, the second platelet exosome, the first stem cell exosome and the second stem cell exosome respectively comprises the following substeps: rapidly freezing a first platelet exosome, a second platelet exosome, a first stem cell exosome and a second stem cell exosome to-80 ℃ respectively; and drying the quick-frozen platelet exosomes and stem cell exosomes at the temperature of-55 ℃ to obtain first platelet exosome powder, second platelet exosome powder, first stem cell exosome powder and second stem cell exosome powder.
The invention also provides a preparation method of the mixed cryogel powder for combining the platelet exosomes and the stem cell exosomes. According to another embodiment of the present invention, a method for preparing a mixed frozen powder comprises the following steps: centrifuging a platelet plasma sample in a sterile environment to obtain a first platelet exosome; centrifuging the whole blood sample in a sterile environment to obtain a platelet sample, and then centrifuging the platelet sample in a sterile environment to obtain a second platelet exosome; centrifuging a sample of the stem cell culture medium in a sterile environment to obtain a first stem cell exosome; obtaining a second stem cell exosome from the stem cell sample by ultrasonic oscillation and centrifugation; mixing one of the first platelet exosome and the second platelet exosome with one of the first stem cell exosome and the second stem cell exosome to form a mixed substance; and, subjecting the mixed substance to a freezing and drying process to form the mixed cryo-crystalline powder.
The invention at least achieves the following beneficial effects:
compared with the prior art, the mixed frozen crystal powder rich in platelet exosomes and combined with stem cell exosomes and the preparation method thereof utilize the frozen crystal powder of autologous platelet exosomes to be mixed with the frozen crystal powder of interstitial stem cell exosomes so as to customize the required mixed frozen crystal powder, and have the characteristics of simple subsequent storage, simple use by only matching with solvent injection or direct wiping when in use, and the like so as to simplify the use steps. Furthermore, the exosome source of the mixed frozen crystal powder is autologous, and the rejection phenomenon caused by allogenic injection cannot be generated. Meanwhile, the culture solution used for cell culture is human platelet plasma instead of animal fetal calf serum, so that the occurrence of protein variation of the fetal calf serum can be reduced.
The advantages and spirit of the present invention can be further understood by the following detailed description of the invention and the accompanying drawings.
Drawings
FIG. 1 is a flow chart of steps of a method of making a mixed cryo-powder according to an embodiment.
Fig. 2 is a flowchart illustrating a further step of obtaining first platelet exosomes of the method of fig. 1.
Fig. 3 is a flowchart illustrating a further step of obtaining second platelet exosomes of the method of manufacturing the mixed cryo-powder of fig. 1.
FIG. 4 is a flowchart illustrating a further step of obtaining first stem cell exosomes of the method of FIG. 1.
FIG. 5 is a flowchart illustrating a further step of obtaining second stem cell exosomes of the method of FIG. 1.
FIG. 6 is a flowchart illustrating a further step of a method of making a blended frozen powder according to an embodiment.
FIG. 7 is a flow chart of the steps of a method of making a blended frozen powder according to another embodiment.
FIG. 8 is a flowchart illustrating further steps in a method of making a blended frozen powder according to one embodiment.
Description of the drawings:
S1-S6, S11-S15, S21-S27, S31-S34, S41-S44, S51-S52, S61-S62, S5 '-S6': and (5) flow steps.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and examples.
Referring to fig. 1, fig. 1 is a flow chart illustrating steps of a method for manufacturing a mixed frozen crystal powder according to an embodiment of the invention. As shown in FIG. 1, in the present embodiment, the method for manufacturing the mixed cryo-powder includes the following steps: centrifuging a platelet plasma sample in a sterile environment to obtain a first platelet exosome; step S2: centrifuging the whole blood sample in a sterile environment to obtain a platelet sample, and then centrifuging the platelet sample in a sterile environment to obtain a second platelet exosome; step S3: centrifuging a sample of the stem cell culture medium in a sterile environment to obtain a first stem cell exosome; step S4: obtaining a second stem cell exosome from the stem cell sample by ultrasonic oscillation and centrifugation; step S5: freeze-drying the first platelet exosome, the second platelet exosome, the first stem cell exosome and the second stem cell exosome to form powder of the first platelet exosome, powder of the second platelet exosome, powder of the first stem cell exosome and powder of the second stem cell exosome; and step S6, mixing one of the powder of the first platelet exosome and the powder of the second platelet exosome with one of the powder of the first stem cell exosome and the powder of the second stem cell exosome to form the mixed cryo-crystal powder.
The following further describes each step of the method for producing the mixed frozen crystal powder according to the present embodiment in detail.
Referring to fig. 2, fig. 2 is a flowchart illustrating a further step of step S1 of fig. 1. As shown in fig. 2, in the present embodiment, the step S1 of the method for making the mixed frozen crystal powder of fig. 1 further includes the following steps: step S11: centrifuging the platelet plasma sample at an acceleration of 300xg for 15 minutes to obtain a first platelet plasma sample; step S12: centrifuging the first platelet plasma sample at an acceleration of 2000xg for 15 minutes to obtain a second platelet plasma sample; step S13: filtering the second platelet plasma sample with a filter having a pore size of 0.22 μm; step S14: centrifuging the filtered second platelet plasma sample at an acceleration of 4000xg for 70 minutes to obtain a third platelet plasma sample; step S15: the third platelet plasma sample was separated with a Tangential Flow Filtration System (TFF) to obtain the first platelet exosomes.
In practice, the platelet plasma sample of step S11 can be obtained by self-centrifugation of a whole blood sample in a sterile environment, and the detailed steps are as follows: drawing a whole blood sample from a user's body to which the mixed cryo-crystal powder is to be applied; then, centrifuging the whole blood sample for 5-15 minutes in a sterile environment at a rotating speed of 1,000-1,500 revolutions per minute; and finally, taking out supernatant of the centrifuged platelet plasma-rich whole blood sample in a sterile environment to obtain the platelet plasma sample.
In one embodiment, the platelet plasma sample of step 15 is separated in the TFF system by separating a third platelet plasma sample (1-250 ml) with a Hollow Fiber membrane (Hollow Fiber) and a multi-tube dialysis concentrating membrane (Midikors FiliterModule) to obtain a first platelet exosome (Mw:500KD), and finally concentrating the first platelet exosome to 2-10 ml.
In practice, the whole blood sample may contain an anticoagulant to prevent coagulation during centrifugation or other processing. For example, a whole blood sample containing an anticoagulant can be centrifuged at 1,200 rpm for 10 minutes to separate plasma and blood cells, and the supernatant fluid rich in platelet plasma can be removed in a sterile environment for subsequent processing to obtain a platelet plasma sample. Therefore, the platelet plasma sample in step S11 is obtained from the autologous blood of the user who wants to use the mixed cryo-crystal powder, and the obtained first platelet exosomes do not cause the problem of blood infection such as hepatitis and aids due to poor source, and do not generate exclusivity, thereby further ensuring the absorption and safety of the human body.
Referring to fig. 3, fig. 3 is a flowchart illustrating a further step of step S2 of fig. 1. As shown in fig. 3, in the present embodiment, the step S2 of the method for making the mixed frozen crystal powder of fig. 1 further includes the following steps: step S21: centrifuging the whole blood sample at room temperature for 30 minutes at an acceleration of 2600g to obtain a whole blood sample pellet and a supernatant; step S22: adding the supernatant to 2U/mL heparin and centrifuging twice at an acceleration of 500Xg for 10 minutes to produce a first supernatant; step S23: centrifuging the first supernatant twice for 15 minutes at an acceleration of 2000xg to produce a second supernatant; step S24: centrifuging the second supernatant twice for 30 minutes at an acceleration of 10000xg to produce a third supernatant; step S25: centrifuging the third supernatant at 30000rpm for 1 hour at a temperature of 4 ℃ to produce a fourth supernatant; step S26: filtering the fourth supernatant with a filter having a pore size of 0.22 μm, and centrifuging the filtered fourth supernatant at an acceleration of 4000xg for 90 minutes to produce a fifth supernatant; and step S27: the fifth supernatant was separated with a Tangential Flow Filtration System (TFF) to obtain second platelet exosomes.
In practice, the whole blood sample in step S21 is obtained from autologous blood of the user, and an anti-coagulation agent may be added to prevent coagulation during centrifugation or subsequent processing. Because the whole blood sample is obtained from the autologous blood of the user, the obtained second platelet exosomes do not cause the problem of blood infection such as hepatitis, AIDS and the like due to unclean sources, and the exclusivity is not generated, thereby further ensuring the absorption and safety of the human body.
In one embodiment, step 27 is performed by separating the fifth supernatant (1-250 ml) with Hollow Fiber membrane (Hollow Fiber) and multi-tube dialysis concentrating membrane (Midikors FiliterModule) to obtain the second platelet exosome (Mw:500KD) and finally concentrating the second platelet exosome to 2-10ml in TFF system separation. Referring to fig. 4, fig. 4 is a flowchart illustrating a further step of step S3 of fig. 1. As shown in fig. 4, in the present embodiment, the step S3 of the method for making the mixed frozen crystal powder of fig. 1 further includes the following steps: step S31: after culturing the stem cell culture medium sample for 48 hours, filtering the stem cell culture medium sample with a filter having a pore size of 0.22 μm to obtain a first stem cell culture medium sample; step S32: centrifuging the first stem cell culture medium sample at an acceleration of 200xg to obtain a second stem cell culture medium sample; step S33: centrifuging the second stem cell culture medium sample at an acceleration of 2000xg to obtain a third stem cell culture medium sample; step S34: the third stem cell culture medium sample is separated with a tangential flow filtration system to obtain the first stem cell exosomes.
In practice, the human mesenchymal stem cells may be cultured in a growth medium in an aseptic environment, and the culture medium is collected after the cells of the human mesenchymal stem cells in the growth medium are cultured for 48 hours, and the culture medium is the stem cell culture medium sample of step S31. In addition, the mesenchymal stem cells are obtained from human bone, umbilical cord, fat or fibroblast, and further obtained from bone, umbilical cord, fat or fibroblast of a user who wants to use the mixed cryo-powder, so as to improve the adaptability of the first stem cell exosome.
In one embodiment, step 34 is to separate a third sample (1-250 ml) of stem cell culture medium from a Hollow Fiber membrane (Hollow Fiber) and a multi-tube dialysis concentrating membrane (Midikors FiliterModule) to obtain a first stem cell exosome (Mw:500KD) during the TFF system separation, and finally concentrating the first stem cell exosome to 2-10 ml.
In practice, the growth solution may comprise DMEM medium, 5% human platelet plasma and 1mM of optional amino acids. The DMEM medium used contains 4.0mM L-glutamic acid (L-Glutamine) and 4500mg/L Glucose (Glucose), and does not contain Sodium Pyruvate (Sodium Pyruvate), Phenol Red (Phenol Red) and antibiotics, so that immune rejection phenomenon and easiness can be avoided. The addition of 5% human platelet plasma and 1mM of an optional amino acid accelerates cell growth, and the 5% human platelet plasma used is not limited to those obtained from users who want to use the mixed cryo-powder, and is typically commercially available.
Referring to fig. 5, fig. 5 is a flowchart illustrating a further step of step S4 of fig. 1. As shown in fig. 5, in the present embodiment, the step S4 of the method for making the mixed frozen crystal powder of fig. 1 further includes the following steps: step S41: culturing the stem cell sample for 48 hours, performing ultrasonic oscillation on the stem cell sample for 10 minutes at a preset frequency, stopping the ultrasonic oscillation for two minutes, and performing ultrasonic oscillation again at an amplitude of 25% of the maximum amplitude set by the ultrasonic oscillation for 10 minutes to obtain a first stem cell sample; step S42: placing the first stem cell sample at room temperature, standing, incubating for 15 minutes, performing MNase digestion, and centrifuging at an acceleration of 200Xg to obtain a second stem cell sample; step S43: centrifuging the second stem cell sample at an acceleration of 2000xg to obtain a third stem cell sample; step S44: a third sample of stem cell culture medium was separated using a Tangential Flow Filtration System (TFF) to obtain a second stem cell exosome.
In practice, the stem cell sample of step S41 is cultured in a growth medium in a sterile environment to obtain human stem cells, and after culturing the human stem cells in the growth medium for 48 hours, the cell sample is collected and placed in 1ml of saline solution. In addition, step S42 may utilize Micrococcal Nuclease (MNase) to perform cell lysis on the cells in the first stem cell sample to obtain the substance released in the cells, which is MNase digestion.
In one embodiment, step S44 is to separate a third stem cell culture medium sample (1-250 ml) with a Hollow Fiber membrane (Hollow Fiber) and a multi-tube dialysis concentrating membrane (Midikors FiliterModule) to obtain a second stem cell exosome (Mw:500KD) during the TFF system separation, and finally, to concentrate the second stem cell exosome to 2-10 ml.
Referring to fig. 6, fig. 6 is a flowchart illustrating a further step of step S4 of fig. 1. As shown in fig. 6, in the present embodiment, the step S5 of the method for making the mixed frozen crystal powder of fig. 1 further includes the following steps: step S51: rapidly freezing a first platelet exosome, a second platelet exosome, a first stem cell exosome and a second stem cell exosome to-80 ℃ respectively; and step S52: drying the quick-frozen platelet exosomes and stem cell exosomes at the temperature of-55 ℃ to obtain powder of the first platelet exosomes, powder of the second platelet exosomes, powder of the first stem cell exosomes and powder of the second stem cell exosomes.
In practice, step S52 may be performed by drying and rapidly freezing the first platelet exosome, the second platelet exosome, the first stem cell exosome and the second stem cell exosome at-55 ℃ for 12-16 hours to obtain powder of the first platelet exosome, powder of the second platelet exosome, powder of the first stem cell exosome and powder of the second stem cell exosome.
As mentioned above, the platelet exosome and stem cell exosome of the present invention can be cultured from the platelet, mesenchymal stem cell or fibroblast of the user himself who wants to use the mixed cryo-powder, and since most of the raw materials are from the user himself, the problem of body rejection is not easy to occur. In addition, in practice, the mixed frozen crystal powder of the present invention can be dissolved in a solvent for injection or mixed in a slurry for wiping.
Referring to fig. 7, fig. 7 is a flow chart showing steps of a method for manufacturing a mixed frozen crystal powder according to another embodiment of the invention. As shown in fig. 7, the difference between the present embodiment and the previous embodiment is that the method for manufacturing the mixed frozen crystal powder of the present embodiment further includes the following steps: step S5', mixing one of the first platelet exosome and the second platelet exosome with one of the first stem cell exosome and the second stem cell exosome to form a mixed substance; and, step S6', freeze-drying and flash-freezing the mixture to form a mixed frozen crystal powder. Please note that, other steps of the method of this embodiment are substantially the same as those of the previous embodiment, and thus are not described herein again.
In addition, referring to fig. 8, fig. 8 is a flowchart illustrating a further step of step S6' of fig. 7. As shown in fig. 8, in the present embodiment, the step S6' of the method for making the mixed frozen crystal powder of fig. 7 further includes the following steps: step S61: respectively rapidly freezing the mixture to-80 deg.C; and a step S62 of drying the mixed material at-55 ℃ to form the mixed frozen crystal powder.
In practice, step S62 is to dry and rapidly freeze the first platelet exosome, the second platelet exosome, the first stem cell exosome and the second stem cell exosome at-55 ℃ for 12-16 hours.
In this embodiment, because the mixed material is prepared and then frozen and dehydrated, the required process steps can be reduced, thereby reducing the possibility of contamination during the process.
In summary, the mixed cryogel powder combining platelet and plasma exosomes and the preparation method thereof of the invention utilize autologous blood of a user as a material to improve the human body absorption benefit and safety of the mixed cryogel powder, and utilize a freeze-drying technology to overcome the deterioration problem caused by heat sensitivity and time efficiency. Meanwhile, the mixed frozen crystal powder can be further used after being mixed with a required solvent or slurry according to different customized mixing proportions of subsequent injection use or wiping absorption modes.
Compared with the prior art, the mixed frozen crystal powder combining the platelet plasma exosomes and the stem cell exosomes and the preparation method thereof utilize the frozen crystal powder of the autologous platelet plasma to be mixed with the frozen crystal powder of the stem cell exosomes so as to customize the required mixed frozen crystal powder, have a method for improving the purity of the exosomes, and the prepared frozen crystal powder has the characteristics of simple subsequent storage, simple use steps by only matching with solvent injection or direct wiping when in use and the like. Furthermore, the sample for preparing the stem cell exosome frozen crystal powder is autologous, and the rejection phenomenon caused by allogenic injection cannot be generated. Meanwhile, the culture solution used for cell culture is human platelet plasma instead of animal fetal calf serum, so that the occurrence of protein variation of the fetal calf serum can be reduced.
The above detailed description of the preferred embodiments is intended to more clearly illustrate the features and spirit of the present invention, and is not intended to limit the scope of the present invention by the preferred embodiments disclosed above. On the contrary, it is intended to cover various modifications and equivalent arrangements included within the scope of the claims.
Claims (11)
1. A mixed cryo-crystal powder, comprising:
a platelet exosome; and
a stem cell exosome.
2. The mixed cryogel powder of claim 1, wherein the platelet exosomes are obtained from blood of a user.
3. The mixed cryogel powder of claim 1, wherein the stem cell exosomes are derived from stem cells comprising mammalian bone, mammalian umbilical cord, mammalian fat, or mammalian blood.
4. The blended frozen crystal powder of claim 1, wherein the blended frozen crystal powder is obtained by a method comprising:
extracting a platelet plasma sample or a whole blood sample by an ultracentrifugation method to obtain the platelet exosome; and a process for the preparation of a coating,
extracting a stem cell sample or a stem cell culture medium sample by an ultracentrifugation method to obtain the stem cell exosome; followed by
(1) Mixing the platelet exosome and the stem cell exosome, and then quickly freezing at-80 ℃ and freeze-drying at-55 ℃, or (2) quickly freezing the platelet exosome and the stem cell exosome at-80 ℃ and freeze-drying at-55 ℃ and then mixing to obtain the mixed cryo-crystal powder.
5. The preparation method of the mixed frozen crystal powder is characterized by comprising the following steps:
centrifuging a platelet plasma sample to obtain a first platelet exosome;
centrifuging a whole blood sample to obtain a platelet sample, and then centrifuging the platelet sample to obtain a second platelet exosome;
centrifuging a stem cell culture medium sample to obtain a first stem cell exosome;
performing ultrasonic oscillation and centrifugation on a stem cell sample to obtain a second stem cell exosome;
respectively freeze-drying the first platelet exosome, the second platelet exosome, the first stem cell exosome and the second stem cell exosome to form a powder of the first platelet exosome, a powder of the second platelet exosome, a powder of the first stem cell exosome and a powder of the second stem cell exosome; and
mixing one of the powder of the first platelet exosomes and the powder of the second platelet exosomes with one of the powder of the first stem cell exosomes and the powder of the second stem cell exosomes to form the mixed cryo-crystallized powder.
6. The method for preparing a platelet-rich plasma as claimed in claim 5, wherein the step of centrifuging a platelet plasma sample to obtain a first platelet exosome further comprises the substeps of:
centrifuging the platelet plasma sample at an acceleration of 300Xg for 15 minutes to obtain a first platelet plasma sample;
centrifuging the first platelet plasma sample at an acceleration of 2000xg for 15 minutes to obtain a second platelet plasma sample;
filtering the second platelet plasma sample with a filter having a pore size of 0.22 μm;
centrifuging the filtered second platelet plasma sample at an acceleration of 4000Xg for 15 minutes to obtain a third platelet plasma sample; and
the third platelet plasma sample was separated with a Tangential Flow Filtration System (TFF) to obtain the first platelet exosomes.
7. The method of claim 5, wherein the step of centrifuging a whole blood sample to obtain a platelet sample and then centrifuging the platelet sample to obtain a second platelet exosome further comprises the sub-steps of:
centrifuging the whole blood sample at room temperature for 30 minutes at an acceleration of 2600Xg to obtain a whole blood sample precipitate and a supernatant;
adding 2U/mL heparin to the supernatant and centrifuging at an acceleration of 500Xg for 10 minutes twice to produce a first supernatant;
centrifuging the first supernatant twice for 15 minutes at an acceleration of 2000Xg to produce a second supernatant;
centrifuging the second supernatant twice for 30 minutes at an acceleration of 10000xg to produce a third supernatant;
centrifuging the third supernatant at 30000rpm for 1 hour at 4 ℃ to produce a fourth supernatant;
filtering the fourth supernatant with a filter having a pore size of 0.22 μm; and
centrifuging the filtered fourth supernatant at an acceleration of 4000Xg for 90 minutes to produce a fifth supernatant;
separating the fifth supernatant with a tangential flow filtration system to obtain the second platelet exosomes.
8. The method of claim 5, wherein the step of centrifuging a sample of stem cell culture medium to obtain a first stem cell exosome further comprises the substeps of:
culturing the stem cell culture medium sample for 48 hours, and filtering the stem cell culture medium sample by using a filter with the pore size of 0.22 mu m to obtain a first stem cell culture medium sample;
centrifuging the first stem cell culture medium sample at an acceleration of 200xg to obtain a second stem cell culture medium sample;
centrifuging the second stem cell culture medium sample at an acceleration of 2000xg to obtain a third stem cell culture medium sample; and
separating the third stem cell culture medium sample with a tangential flow filtration system to obtain the first stem cell exosomes.
9. The method of claim 5, wherein the step of ultrasonically oscillating and centrifuging a stem cell sample to obtain a second stem cell exosome further comprises the following sub-steps:
culturing the stem cell sample for 48 hours, performing ultrasonic oscillation on the stem cell sample for 10 minutes, stopping the ultrasonic oscillation for two minutes, and performing ultrasonic oscillation again at an amplitude of 25% for 10 minutes to obtain a first stem cell sample;
placing the first stem cell sample at room temperature, standing, incubating for 15 minutes, performing MNase digestion, and centrifuging at an acceleration of 200Xg to obtain a second stem cell sample; centrifuging the second stem cell sample at an acceleration of 2000xg to obtain a third stem cell sample; and
separating the third stem cell culture medium sample with a tangential flow filtration system to obtain the second stem cell exosomes.
10. The method for preparing according to claim 5, wherein the step of freeze-drying the first platelet exosome, the second platelet exosome, the first stem cell exosome and the second stem cell exosome respectively comprises the sub-steps of:
rapidly freezing the first platelet exosomes, the second platelet exosomes, the first stem cell exosomes and the second stem cell exosomes to-80 ℃ respectively; and
drying the first platelet exosome, the second platelet exosome, the first stem cell exosome and the second stem cell exosome after the rapid freezing at a temperature of-55 ℃ to obtain powder of the first platelet exosome, powder of the second platelet exosome, powder of the first stem cell exosome and powder of the second stem cell exosome.
11. The preparation method of the mixed frozen crystal powder is characterized by comprising the following steps:
centrifuging a platelet plasma sample to obtain a first platelet exosome;
centrifuging a whole blood sample to obtain a platelet sample, and then centrifuging the platelet sample to obtain a second platelet exosome;
centrifuging a stem cell culture medium sample to obtain a first stem cell exosome;
performing ultrasonic oscillation and centrifugation on a stem cell sample to obtain a second stem cell exosome;
mixing one of the first platelet exosome and the second platelet exosome with one of the first stem cell exosome and the second stem cell exosome to form a mixed substance; and
freezing and drying the mixed substance to form the mixed frozen crystal powder.
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