CN114053306A - Preparation method of exosome freeze-dried powder from human induced pluripotent stem cells - Google Patents
Preparation method of exosome freeze-dried powder from human induced pluripotent stem cells Download PDFInfo
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Abstract
The invention discloses a preparation method of exosome freeze-dried powder derived from human induced pluripotent stem cells, belongs to the technical field of biotechnology stem cell processing, and solves the problem of high production cost of the existing exosome freeze-dried powder, wherein the exosome freeze-dried powder comprises the following raw materials in percentage by weight: 70-90% of human induced pluripotent stem cell exosome, 1-3% of trehalose, 1-4% of PVP40 and 5-25% of PBS buffer solution, and the preparation method comprises the following steps: the preparation method is characterized in that the raw materials are prepared into a solution, the solution is filtered and then filled into a vacuum bottle, the vacuum bottle is pre-frozen and then placed into a freeze dryer, and freeze drying is carried out under the condition of below-50 to obtain the exosome freeze-dried powder.
Description
Technical Field
The invention relates to the technical field of biotechnology stem cell processing, in particular to the technical field of preparation of exosome freeze-dried powder of human induced pluripotent stem cells.
Background
Human induced pluripotent stem cells (hipscs) are transformed into a stem cell state by introducing four transcription factors Oct4, Sox2, Klf4, and c-Myc into somatic cells. Advantages of hiPSCs include their human origin, ready availability, scalability and ability to generate almost all desired cell types, avoidance of ethical issues associated with human embryonic stem cells, and the potential to develop personalized drugs for personalized therapy using patient-specific iPSCs. The exosome freeze-dried powder is the most ideal cosmetic for repairing damaged skin at present, has magical repairing and regenerating effects on the skin, and also has the effects of tightening skin, removing wrinkles, moisturizing, enhancing resistance and moisturizing power, soothing sensitive, thin horny layer, red blood streak, changing skin, sunburning skin, and treating wound surfaces, depressed scars and anti-wrinkle treatment of aging skin formed by laser, freezing, eyebrow tattooing, eyebrow washing, acne and the like after a cosmetic operation.
At present, some exosome freeze-dried powders are prepared, for example, a chinese patent with application number 202110432805.3 discloses a "a dry cell exosome freeze-dried powder for beauty treatment and skin care and a preparation method thereof", and the formula thereof comprises: the preparation method comprises the following steps of firstly, selecting materials and mixing; step two, mixing and stirring; step three, sterile filtration; step four, filling the stock solution; step five, freeze drying; compared with the existing stem cell exosome freeze-dried powder for beautifying and protecting skin, the technical scheme has the advantages that the plant extract component with the skin beautifying effect is added into the preparation formula of the freeze-dried powder, the skin whitening and protecting effect of the freeze-dried powder is effectively improved, and meanwhile, in the freeze-drying process, the active ingredients of the freeze-dried powder are prevented from being oxidized and lost by filling protective gas into the preparation environment.
Further, as the Chinese patent with application number 202110293553.0, the invention discloses an exosome freeze-dried powder, a preparation method and a skin care product thereof, wherein the exosome freeze-dried powder is prepared from the following raw materials in percentage by weight: 40-80% of umbilical cord mesenchymal stem cell exosome; 10-30% of mannitol; 10-30% of trehalose; sodium hyaluronate, oligopeptide-1 and oligopeptide-3 in balance. The preparation method comprises preparing exosome lyophilized powder into solution, pre-freezing the solution at-80 deg.C for 12-15 hr, placing in a freeze dryer with vacuum degree of less than 80mT, and freeze-drying at-50 deg.C for 26-32 hr. The exosome freeze-dried powder can be transported at low temperature, can be stored at 2-8 ℃ for at least 12 months, and can be used by dissolving exosomes with menstruum.
In addition, the Chinese patent with the application number of 202011146426.X discloses an exosome freeze-drying protective agent, a freeze-drying powder resuscitation liquid and application thereof, wherein the freeze-drying protective agent consists of sugar and a surfactant, the resuscitation liquid comprises normal saline, hydroxyethyl starch and liposome, the hydroxyethyl starch accounts for 0.5-2.5% of the resuscitation liquid, and the liposome accounts for 1-20% of the resuscitation liquid. According to the technical scheme, the liposome which has a structure similar to that of the vesicle membrane is added into the resuscitation solution and is used for repairing the damaged vesicle, the process follows the characteristic of biological membrane fluidity, the vesicle membrane structure of the exosome is effectively repaired, and the activity of the exosome is kept.
In addition, the Chinese patent with the application number of 201910742324.5 discloses an exosome freeze-dried powder, a preparation method thereof and a preparation containing the exosome freeze-dried powder, wherein the preparation method of the exosome freeze-dried powder comprises the following steps: preparation of platelet factor-rich plasma: adopting autoblood, centrifuging to prepare an exosome; self-taking autologous fat to prepare adipose mesenchymal stem cells, and culturing the mesenchymal stem cells to prepare the adipose mesenchymal stem cells; preparing a freeze-drying solution: mixing platelet factor-rich plasma and exosome, adding trehalose, and mixing; and (3) freeze drying: and (4) freeze-drying the freeze-drying solution to obtain the freeze-dried powder. The preparation comprises exosome freeze-dried powder, hyaluronic acid and normal saline, and can effectively solve the problems of poor filling effect, short maintenance time and large immunological rejection of the existing cosmetic preparation.
The above patent documents all have the following problems: 1. the preparation process of the exosome freeze-dried powder without human induced pluripotent stem cell sources; 2. at present, the exosome freeze-dried powder preparation has more components, and the production cost is increased; 3. the preparation process of the exosome freeze-dried powder is complex, the production efficiency is low, the preparation of the existing exosome freeze-dried powder is not facilitated, and the existing production requirements cannot be met. Therefore, the optimization and improvement of the existing preparation method of the exosome freeze-dried powder of the human induced pluripotent stem cells have very important significance in the aspects of reducing the production cost and improving the production efficiency.
Disclosure of Invention
The invention aims to: in order to solve the technical problems, the invention provides a preparation method of exosome freeze-dried powder derived from human induced pluripotent stem cells.
The invention specifically adopts the following technical scheme for realizing the purpose:
the exosome freeze-dried powder derived from the human induced pluripotent stem cells comprises the following raw materials in percentage by weight: 70-90% of human induced pluripotent stem cell exosome, 1-3% of trehalose, 1-4% of PVP40 and 5-25% of PBS buffer solution.
The exosome freeze-dried powder comprises the following raw materials in percentage by weight: 85% of human induced pluripotent stem cell exosome, 2% of trehalose, 2.5% of PVP40 and 10.5% of PBS buffer.
The preparation method of the exosome freeze-dried powder comprises the following steps:
s1: preparation of human induced pluripotent stem cell exosomes:
s101: collecting 300ml urine, centrifugally collecting exfoliated epithelial cells, and performing amplification culture;
s102: transfecting the urine cells with the Sendai virus-packaged Oct4, Sox2, Klf4 and c-Myc transcription factors for logarithmic growth period;
s103: observing the morphological change and the aggregation growth condition of the cells within one week after transfection;
s104: after cell cloning is formed, selecting a single clone to be cultured in a new culture container, recording the single clone as F1 generation, then carrying out amplification culture and passage, and carrying out expression detection of stem cell markers, karyotype analysis and teratoma detection;
s105: after the detection result meets the standard, collecting the culture medium and carrying out centrifugal ultrafiltration to obtain the human induced pluripotent stem cell exosome;
s2: adding trehalose into a PBS buffer solution, and uniformly stirring to obtain a mixed solution for later use;
s3: adding the human induced pluripotent stem cell exosome and PVP40 into the mixed solution prepared in the step S2, and uniformly stirring to obtain a freeze-dried solution;
s4: filtering the freeze-dried solution prepared in the step S3 at room temperature, collecting filtrate, and filling the collected filtrate into a vacuum bottle;
s5: pre-freezing the vacuum bottle filled with the freeze-drying liquid at-80 ℃ for 12-15h, then placing the vacuum bottle in a freeze dryer, and freeze-drying the vacuum bottle at-50 to-60 ℃ and 60-70 Pa for 26-32h to obtain the exosome freeze-drying powder.
The invention has the following beneficial effects:
the invention improves the preparation process of the existing exosome freeze-dried powder, reduces the components of the freeze-dried powder preparation, effectively reduces the production cost of the exosome freeze-dried powder, can maximally retain the activity of the human induced pluripotent stem cell exosomes, improves the production efficiency of the exosome freeze-dried powder, and ensures the production quality of the exosome freeze-dried powder.
Drawings
FIG. 1 is a graph showing the identification results of the induced pluripotent stem cell markers of the present inventors;
FIG. 2 is a morphogram of urine shedding epithelial cells before induction according to the present invention;
FIG. 3 is a morphological diagram of induced pluripotent stem cells of the invention.
FIG. 4 is a schematic diagram of the particle size analysis of the exosomes derived from the induced pluripotent stem cells of the present inventors;
FIG. 5 is a graph showing the identification of exosome markers derived from induced pluripotent stem cells by the present inventors;
FIG. 6 is a transmission electron microscope image of exosomes derived from induced pluripotent stem cells of the present inventors.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are some embodiments of the present invention, but not all embodiments.
Thus, the following detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Examples
The invention provides exosome freeze-dried powder derived from human induced pluripotent stem cells, which comprises the following raw materials in percentage by weight: 70-90% of human induced pluripotent stem cell exosome, 1-3% of trehalose, 1-4% of PVP40 and 5-25% of PBS buffer solution.
The method for inducing the human induced pluripotent stem cells comprises the following steps:
1. collecting 300ml urine, centrifuging, collecting the desquamated epithelial cells, and performing amplification culture.
2. The Sendai virus-packaged Oct4, Sox2, Klf4 and c-Myc transcription factors are used to transfect the urine cells with log-growth period.
3. Cell morphology changes and aggregate growth were observed within one week after transfection.
4. After the cell clone was formed, individual clones were picked up and cultured in a new culture vessel and designated as F1 generation. Then, carrying out amplification culture and passage, and carrying out expression detection of stem cell markers, karyotype analysis and teratoma detection, wherein the detection results are shown in figures 1-3.
The preparation method of the human induced pluripotent stem cell exosome comprises the following steps:
1: collecting culture medium of human induced pluripotent stem cells, centrifuging the collected culture medium at 4 ℃ for 15min at 1000 Xg, removing cell debris, collecting supernatant, centrifuging the collected supernatant at 4 ℃ for 70min again at 10000 Xg to further remove the cell debris, collecting supernatant, and filtering to obtain concentrated solution containing exosome;
2: centrifuging the concentrated solution at 100000 Xg for 70min at 4 ℃, collecting bottom precipitate, washing the precipitate with PBS, centrifuging at 100000 Xg for 70min again, collecting the exosomes of the human induced pluripotent stem cells, and suspending the collected exosomes of the human induced pluripotent stem cells in PBS again and extracting for subsequent use;
the preparation method of the exosome freeze-dried powder comprises the following steps:
s1: adding trehalose into a PBS buffer solution, and uniformly stirring to obtain a mixed solution for later use;
s2: adding the human induced pluripotent stem cell exosome and PVP40 into the mixed solution prepared in the step S1, and uniformly stirring to obtain a freeze-dried solution;
s3: filtering the freeze-dried solution prepared in the step S2 at room temperature, collecting filtrate, and filling the collected filtrate into a vacuum bottle;
s5: pre-freezing the vacuum bottle filled with the freeze-drying liquid at-80 ℃ for 12-15h, then placing the vacuum bottle in a freeze dryer, and freeze-drying the vacuum bottle at-50 to-60 ℃ and 60-70 Pa for 26-32h to obtain the exosome freeze-drying powder.
The specific embodiment of the invention is as follows:
example 1
The embodiment provides exosome freeze-dried powder derived from human induced pluripotent stem cells, which comprises the following raw materials in percentage by weight: 85% of human induced pluripotent stem cell exosome, 2% of trehalose, 2.5% of PVP40 and 10.5% of PBS buffer.
The preparation method of the exosome freeze-dried powder comprises the following steps:
s1: weighing human induced pluripotent stem cell exosomes, trehalose, PVP40 and PBS buffer solution according to a proportion, adding the trehalose into the PBS buffer solution, and uniformly stirring to obtain a mixed solution for later use;
s2: adding the human induced pluripotent stem cell exosome and PVP40 into the mixed solution prepared in the step S1, and uniformly stirring to obtain a freeze-dried solution;
s3: filtering the freeze-dried solution prepared in the step S2 at room temperature, collecting filtrate, and filling the collected filtrate into a vacuum bottle;
s4: pre-freezing the vacuum bottle filled with the freeze-drying liquid at-80 ℃ for 12-15h, then placing the vacuum bottle in a freeze dryer, and freeze-drying the vacuum bottle at-50 to-60 ℃ and 60-70 Pa for 26-32h to obtain the exosome freeze-drying powder.
Example 2
The embodiment provides exosome freeze-dried powder derived from human induced pluripotent stem cells, which comprises the following raw materials in percentage by weight: 90% of human induced pluripotent stem cell exosomes, 2% of trehalose, 2.5% of PVP40 and 5.5% of PBS buffer.
The preparation method of the exosome freeze-dried powder comprises the following steps:
s1: weighing human induced pluripotent stem cell exosomes, trehalose, PVP40 and PBS buffer solution according to a proportion, adding the trehalose into the PBS buffer solution, and uniformly stirring to obtain a mixed solution for later use;
s2: adding the human induced pluripotent stem cell exosome and PVP40 into the mixed solution prepared in the step S1, and uniformly stirring to obtain a freeze-dried solution;
s3: filtering the freeze-dried solution prepared in the step S2 at room temperature, collecting filtrate, and filling the collected filtrate into a vacuum bottle;
s4: pre-freezing the vacuum bottle filled with the freeze-drying liquid at-80 ℃ for 12-15h, then placing the vacuum bottle in a freeze dryer, and freeze-drying the vacuum bottle at-50 to-60 ℃ and 60-70 Pa for 26-32h to obtain the exosome freeze-drying powder.
Example 3
The embodiment provides exosome freeze-dried powder derived from human induced pluripotent stem cells, which comprises the following raw materials in percentage by weight: 85% of human induced pluripotent stem cell exosome, 1% of trehalose, 1% of PVP40 and 13% of PBS buffer.
The preparation method of the exosome freeze-dried powder comprises the following steps:
s1: weighing human induced pluripotent stem cell exosomes, trehalose, PVP40 and PBS buffer solution according to a proportion, adding the trehalose into the PBS buffer solution, and uniformly stirring to obtain a mixed solution for later use;
s2: adding the human induced pluripotent stem cell exosome and PVP40 into the mixed solution prepared in the step S1, and uniformly stirring to obtain a freeze-dried solution;
s3: filtering the freeze-dried solution prepared in the step S2 at room temperature, collecting filtrate, and filling the collected filtrate into a vacuum bottle;
s4: pre-freezing the vacuum bottle filled with the freeze-drying liquid at-80 ℃ for 12-15h, then placing the vacuum bottle in a freeze dryer, and freeze-drying the vacuum bottle at-50 to-60 ℃ and 60-70 Pa for 26-32h to obtain the exosome freeze-drying powder.
The exosome freeze-dried powder prepared in the embodiment 1-3 is weighed respectively, distilled water is used for reviving the exosome freeze-dried powder, the purity and the activity of the revived exosome freeze-dried powder are detected by high performance liquid chromatography, and the detection results are shown in table 1.
TABLE 1 results of purity and Activity measurements of exosome lyophilized powder after resuscitation in examples 1-3
| Item | National standard | Example 1 | Example 2 | Example 3 |
| Specific Activity of EGF/mg | Not less than 50 ten thousand IU | 80.3 ten thousand IU | 79.8 ten thousand IU | 79.1 ten thousand IU |
| Purity of | >95％ | 97.40％ | 97.18％ | 96.40％ |
And (4) conclusion: by detecting the purity and activity of the resuscitated exosome freeze-dried powder in the embodiments 1-3, the detection results both meet the national calibration standard.
Claims (3)
1. The exosome freeze-dried powder derived from the human induced pluripotent stem cells is characterized by comprising the following raw materials in percentage by weight: 70-90% of human induced pluripotent stem cell exosome, 1-3% of trehalose, 1-4% of PVP40 and 5-25% of PBS buffer solution.
2. The exosome lyophilized powder derived from human induced pluripotent stem cells according to claim 1, comprising the following raw materials in percentage by weight: 85% of human induced pluripotent stem cell exosome, 2% of trehalose, 2.5% of PVP40 and 10.5% of PBS buffer.
3. The exosome lyophilized powder derived from human induced pluripotent stem cells according to claim 1, which is prepared by the following steps:
s1: preparation of human induced pluripotent stem cell exosomes:
s101: collecting 300ml urine, centrifugally collecting exfoliated epithelial cells, and performing amplification culture;
s102: transfecting the urine cells with the Sendai virus-packaged Oct4, Sox2, Klf4 and c-Myc transcription factors for logarithmic growth period;
s103: observing the morphological change and the aggregation growth condition of the cells within one week after transfection;
s104: after cell cloning is formed, selecting a single clone to be cultured in a new culture container, recording the single clone as F1 generation, then carrying out amplification culture and passage, and carrying out expression detection of stem cell markers, karyotype analysis and teratoma detection;
s105: after the detection result meets the standard, collecting the culture medium and carrying out centrifugal ultrafiltration to obtain the human induced pluripotent stem cell exosome;
s2: adding trehalose into a PBS buffer solution, and uniformly stirring to obtain a mixed solution for later use;
s3: adding the human induced pluripotent stem cell exosome and PVP40 into the mixed solution prepared in the step S2, and uniformly stirring to obtain a freeze-dried solution;
s4: filtering the freeze-dried solution prepared in the step S3 at room temperature, collecting filtrate, and filling the collected filtrate into a vacuum bottle;
s5: pre-freezing the vacuum bottle filled with the freeze-drying liquid at-80 ℃ for 12-15h, then placing the vacuum bottle in a freeze dryer, and freeze-drying the vacuum bottle at-50 to-60 ℃ and 60-70 Pa for 26-32h to obtain the exosome freeze-drying powder.
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