Platelet lysate and its preparation method and application thereof in skin anti-aging

Abstract

The present application belongs to the field of medicine and health. Specifically, the present application provides a lysate of in-vitro differentiated platelets, which can promote proliferation of senescent cells, promote cell migration, improve the phenotype of senescent cells, reduce the proportion of senescent cells, and improve cell senescence at the molecular level. Based on this, the present application provides preparation and application of the platelet lysate.

Description

Technical Field

[0001] This invention belongs to the field of medicine and health, specifically relating to the application of a platelet lysate and its preparation method in skin anti-aging. Background Technology

[0002] The skin is the largest organ in the human body. As people age, the skin, like other organs, gradually shows signs of aging. Skin aging is reflected at the cellular level as cellular aging. The root cause of aging is an insufficient number of new cells to completely replace aging or dead cells, resulting in a decrease in the total number of cells in the body, reduced cell vitality, and a series of aging symptoms, even age-related diseases.

[0003] Platelets are important participants in many pathophysiological processes. Research on platelet-rich plasma (PRP) in improving skin texture and delaying and improving cellular aging is relatively extensive, and it is increasingly being applied to treat acne scars, heal refractory wounds, aid fat cell survival after autologous fat transplantation, repair wounds after laser skin treatment, and smooth fine lines.

[0004] Currently, platelets mainly come from donor donations. Due to the short shelf life of platelets from donors and the insufficient number of recipients, platelets have become a scarce resource. At the same time, there are risks of bacterial contamination and deterioration during the processing, preparation and transfusion of platelets.

[0005] Therefore, there is an urgent need to provide substances and methods that are easy to prepare and store, safe and effective, and have controllable cost and stability in order to delay and improve skin aging. Summary of the Invention

[0006] This invention provides a platelet lysate prepared by in vitro differentiation and its application, and provides an anti-aging method based on the lysate.

[0007] In a first aspect of the invention, a platelet lysate for improving skin aging is provided, said platelet lysate being a lysate of platelets differentiated in vitro from human pluripotent stem cells (ESC / iPSC) and / or hematopoietic stem cells.

[0008] In another preferred embodiment, the hematopoietic stem cells include hematopoietic stem cells, pluripotent progenitor cells, common myeloid progenitor cells, megakaryocyte-erythroid progenitor cells, or combinations thereof.

[0009] In another preferred embodiment, the hematopoietic stem cells are CD34. + cell.

[0010] In another preferred embodiment, the platelet lysate shows a significant increase in the levels of one or more growth factors, including FGF, VEGF, and PDGF-BB.

[0011] In another preferred embodiment, the growth factors are VEGF and PDGF-BB.

[0012] In another preferred embodiment, "significant increase" means that the growth factor level Y1 of the platelet lysate is (Y1-Y0) / Y1≥25% compared with the growth factor level Y0 of the human platelet lysate, more preferably (Y1-Y0) / Y1≥35%, and most preferably (Y1-Y0) / Y1≥45%.

[0013] In another preferred embodiment, the improvement of skin aging includes repairing damaged skin or tissue.

[0014] In another preferred embodiment, the improvement of skin aging includes:

[0015] (Z1) Prevent skin aging, delay skin aging, stop the process of skin aging, repair aging skin, or a combination thereof;

[0016] (Z2) Promotes cell proliferation, promotes cell migration, reduces the proportion of senescent cells, promotes collagen regeneration, or a combination thereof; and / or

[0017] (Z3) Improves the molecular level of molecules related to cellular senescence.

[0018] In another preferred embodiment, the cellular senescence-related molecules include: P21, LMNB1, IL6, IL8, MMP3, COL1A2, MMP1, or combinations thereof.

[0019] In another preferred embodiment, the improvement in the molecular level of cellular senescence-related molecules includes:

[0020] (Z3-1) significantly reduced the molecular levels of P21, LMNB1, IL6, IL8, MMP3, COL1A2, or combinations thereof; and / or

[0021] (Z3-2) significantly increased the molecular level of MMP1.

[0022] In another preferred embodiment, "improvement" means that after administration of the composition or formulation, the molecular level of cellular aging-related molecules in the subject changes towards that of a younger subject; and there is a statistically significant difference compared with the molecular level of cellular aging-related molecules in the subject before administration of the composition or formulation.

[0023] In another preferred embodiment, the young subject is a subject younger than the subject and / or a subject with a skin condition younger than the subject.

[0024] In another preferred embodiment, the subject is the person to whom the composition or formulation is administered.

[0025] In another preferred embodiment, the subject is ≥40 years old, preferably ≥60 years old, and most preferably ≥80 years old.

[0026] In another preferred embodiment, the subject is ≥15 years old.

[0027] In another preferred embodiment, "significant" means that there is a statistically significant difference.

[0028] In another preferred embodiment, the statistically significant difference means that when the significance level α is 0.05, P ≤ 0.05.

[0029] In a second aspect of the invention, a culture system for obtaining platelet lysates that improve skin aging is provided, the culture system comprising Stage I medium, Stage II medium, Stage III medium, and / or Stage IV medium.

[0030] The Stage I culture medium includes basal medium, BMP4, VEGF, activator A, and CHIR-99021;

[0031] The Stage II culture medium includes StemSpan. TM -ACF Erythroid amplification medium, BMP4, VGEF, bFGF;

[0032] The Stage III culture medium includes basal medium, TPO, SCF, Flt3, IL3, IL6, PFHM-II, and PVA;

[0033] The Stage IV medium includes basal medium, TPO, SCF, and PFHM-II.

[0034] In another preferred embodiment, the basal culture medium includes E8 medium, complete medium, and StemSpan. TM SFEM hematopoietic cell culture medium, StemPro-34, STEMdiff TM APEL TM 2. Culture medium and / or BPEL.

[0035] In another preferred embodiment, the basal culture medium is STEMdiff. TM APEL TM 2. Culture medium.

[0036] In another preferred embodiment, the Stage I culture medium comprises STEMdiff TM APEL TM2. Culture medium, 20-40 ng / mL BMP4, 40-60 ng / mL VEGF, 20-30 ng / mL activator A, 1-3 μM CHIR-99021.

[0037] In another preferred embodiment, the Stage II culture medium comprises StemSpan. TM -ACF Erythroid amplification medium contains 20-40 ng / mL BMP4, 40-60 ng / mL VEGF, and 40-60 ng / mL bFGF.

[0038] In another preferred embodiment, the Stage III culture medium comprises STEMdiff TM APEL TM 2. Culture medium, 40-60 ng / mL TPO, 40-60 ng / mL SCF, 20-30 ng / mL Flt3, 5-15 ng / mL IL3, 5-15 ng / mL IL6, 1%-10% PFHM-II, 50-200 ug / mL PVA.

[0039] In another preferred embodiment, the StageIV medium comprises STEMdiff TM APEL TM 2. Culture medium, 40-60 ng / mL TPO, 40-60 ng / mL SCF, 1%-10% PFHM-II.

[0040] In another preferred embodiment, the culture medium comprises 0.5%-3% PS.

[0041] In another preferred embodiment, the Stage I culture medium comprises STEMdiff TM APEL TM 2. Culture medium, 30 ng / mL BMP4, 50 ng / mL VEGF, 25 ng / mL activator A, 1.5 μM CHIR-99021, 1% PS.

[0042] In another preferred embodiment, the Stage II culture medium comprises StemSpan. TM -ACF Erythroid amplification medium: 30 ng / mL BMP4, 50 ng / mL VEGF, 50 ng / mL bFGF, 1% PS.

[0043] In another preferred embodiment, the Stage III culture medium comprises STEMdiff TM APEL TM2. Culture medium, 50 ng / mL TPO, 50 ng / mL SCF, 25 ng / mL Flt3, 10 ng / mL IL3, 10 ng / mL IL6, 5% PFHM-II, 100 ug / mL PVA, 1% PS.

[0044] In another preferred embodiment, the StageIV medium comprises STEMdiff TM APEL TM 2. Culture medium, 50 ng / mL TPO, 50 ng / mL SCF, 5% PFHM-II, 1% PS.

[0045] In another preferred embodiment, the culture system comprises M1 medium containing StemSpan. TM SFEMII medium, IL-3, IL-6, SCF, TPO and FLT.

[0046] In another preferred embodiment, the culture system includes M2 medium, which contains IMDM medium, N2B27, NEAA, glutamate, ITS, ascorbic acid, IL-6, SCF, and TPO.

[0047] In another preferred embodiment, the M1 culture medium contains StemSpan. TM SFEMII medium, 5-50 ng / ml IL-3, 5-50 ng / ml IL-6, 5-50 ng / ml SCF, 5-50 ng / ml TPO and 5-50 ng / ml FLT.

[0048] In another preferred embodiment, the M2 medium comprises IMDM medium, N2B27, NEAA, glutamic acid, ITS, ascorbic acid, 5-50 ng / ml IL-6, 5-50 ng / ml SCF, and 5-50 ng / ml TPO.

[0049] In another preferred embodiment, the culture system includes a stem cell culture medium.

[0050] In another preferred embodiment, the stem cell culture medium is mTeSR1 medium.

[0051] In another preferred embodiment, the improvement of skin aging includes: preventing skin aging, delaying skin aging, halting the process of skin aging, repairing aging skin, or a combination thereof.

[0052] In a third aspect of the invention, a method for preparing platelet lysates that improve skin aging is provided, comprising the following steps:

[0053] (S1) Platelets differentiated in vitro are provided by the culture system described in the second aspect of the present invention;

[0054] (S2) The platelets provided are lysed to obtain the platelet lysate.

[0055] In another preferred embodiment, the lysis refers to lysing the provided platelets using a homogenizer to obtain lysate.

[0056] In another preferred embodiment, the lysis is achieved by repeatedly freezing and thawing the provided platelets to obtain lysates.

[0057] In another preferred embodiment, the pyrolysis further includes ultrafiltration of the pyrolyte.

[0058] In another preferred embodiment, the pyrolysis further includes centrifuging and filtering the pyrolytes.

[0059] In another preferred embodiment, the centrifugal speed is 5000-8000g.

[0060] In another preferred embodiment, the centrifugation time is 20-40 min.

[0061] In another preferred embodiment, the filtration is performed using a 0.22 μm filter, and the filtrate is preserved.

[0062] In another preferred embodiment, step (S1) includes the following sub-steps:

[0063] (S1-1) ESC / iPSC cells were cultured and expanded using stem cell culture medium;

[0064] (S1-2) The cells obtained in step (S1-1) are cultured for mesodermal differentiation using the Stage I medium described in the second aspect of the present invention;

[0065] (S1-3) The cells obtained in step (S1-2) are cultured for differentiation into hematopoietic endothelial cells and hematopoietic stem and progenitor cells using the Stage II culture medium described in the second aspect of the present invention;

[0066] (S1-4) Continue culturing the cells obtained in step (S1-3) in the Stage III medium described in the second aspect of the present invention to obtain megakaryocytes;

[0067] (S1-5) Continue culturing the cells obtained in step (S1-4) in Stage IV medium as described in the second aspect of the present invention to obtain platelets.

[0068] In another preferred embodiment, the stem cell culture medium comprises: mTesR1 culture medium containing Y27632 or mTesR1 culture medium not containing Y27632.

[0069] In another preferred embodiment, step (S1-1) includes: culturing ESC / iPSC cells in mTesR1 medium containing Y27632 for 12-24 hours; and then continuing to culture them in mTesR1 medium without Y27632 until the confluence is 70%-80%.

[0070] In another preferred embodiment, the incubation time for step (S1-2) is 2 days.

[0071] In another preferred embodiment, the incubation time for step (S1-3) is 5 days.

[0072] In another preferred embodiment, the incubation time for step (S1-4) is 7 days.

[0073] In another preferred embodiment, the incubation time for step (S1-5) is 0-6 days, preferably 3-6 days, and most preferably 4-6 days.

[0074] In another preferred embodiment, step (S1) includes the following sub-steps:

[0075] (S1-A) Hematopoietic stem cells are cultured in the M1 culture medium described in the second aspect of the present invention to obtain megakaryocyte progenitor cells;

[0076] (S1-B) Continue culturing the megakaryocyte progenitor cells obtained in step (S1-A) using the M2 culture medium described in the second aspect of the present invention to obtain platelets.

[0077] In another preferred embodiment, the hematopoietic stem cells are CD34. + cell.

[0078] In another preferred embodiment, the incubation time for step (S1-A) is 7 days.

[0079] In another preferred embodiment, the incubation time for step (S1-B) is 10-12 days.

[0080] In a fourth aspect of the invention, a platelet lysate prepared by the method of the third aspect of the invention is provided, wherein the platelet lysate has significantly increased levels of one or more growth factors, including FGF, VEGF, and PDGF-BB.

[0081] In another preferred embodiment, the growth factors are VEGF and PDGF-BB.

[0082] In another preferred embodiment, "significant increase" means that the growth factor level Y1 of the platelet lysate is (Y1-Y0) / Y1≥25% compared with the growth factor level Y0 of the human platelet lysate, more preferably (Y1-Y0) / Y1≥35%, and most preferably (Y1-Y0) / Y1≥45%.

[0083] In a fifth aspect of the invention, the use of the platelet lysate described in the first or fourth aspect of the invention is provided for the preparation of a composition or formulation for improving skin aging.

[0084] In another preferred embodiment, the composition or formulation is used to repair damaged skin or tissue.

[0085] In another preferred embodiment, the improvement of skin aging includes:

[0086] (Z1) Prevent skin aging, delay skin aging, stop the process of skin aging, repair aging skin, or a combination thereof;

[0087] (Z2) Promotes cell proliferation, promotes cell migration, reduces the proportion of senescent cells, promotes collagen regeneration, or a combination thereof; and / or

[0088] (Z3) Improves the molecular level of molecules related to cellular senescence.

[0089] In another preferred embodiment, the cellular senescence-related molecules include: P21, LMNB1, IL6, IL8, MMP3, COL1A2, MMP1, or combinations thereof.

[0090] In another preferred embodiment, the improvement in the molecular level of cellular senescence-related molecules includes:

[0091] (Z3-1) significantly reduced the molecular levels of P21, LMNB1, IL6, IL8, MMP3, COL1A2, or combinations thereof; and / or

[0092] (Z3-2) significantly increased the molecular level of MMP1.

[0093] In another preferred embodiment, "improvement" means that after administration of the composition or formulation, the molecular level of cellular aging-related molecules in the subject changes towards that of a younger subject; and there is a statistically significant difference compared with the molecular level of cellular aging-related molecules in the subject before administration of the composition or formulation.

[0094] In another preferred embodiment, the young subject is a subject younger than the subject and / or a subject with a skin condition younger than the subject.

[0095] In another preferred embodiment, the subject is the person to whom the composition or formulation is administered.

[0096] In another preferred embodiment, the subject is ≥40 years old, preferably ≥60 years old, and most preferably ≥80 years old.

[0097] In another preferred embodiment, the subject is ≥15 years old.

[0098] In another preferred embodiment, "significant" means that there is a statistically significant difference.

[0099] In another preferred embodiment, the statistically significant difference means that when the significance level α is 0.05, P ≤ 0.05.

[0100] In a sixth aspect of the invention, a composition or formulation for improving skin aging is provided, said composition or formulation comprising the platelet lysate described in the fourth aspect of the invention.

[0101] In another preferred embodiment, the content of the platelet lysate in the composition or formulation is 1%-20%, more preferably 2%-16%, and most preferably 4%-12%.

[0102] In another preferred embodiment, the platelet lysate is present in a concentration of 10% in the composition or formulation.

[0103] In another preferred embodiment, the composition further includes a skin-acceptable water-insoluble matrix.

[0104] In another preferred embodiment, the composition and formulation are skin care products.

[0105] In another preferred embodiment, the dosage form of the skin care product includes (but is not limited to): face cream, face mask, water, lotion, powder and paste.

[0106] In a seventh aspect of the invention, a method for improving skin aging is provided, the method comprising administering to a subject the platelet lysate described in the first or fourth aspect of the invention or the composition or formulation described in the sixth aspect of the invention.

[0107] In another preferred embodiment, the method is a non-diagnostic, non-treatment method.

[0108] In another preferred embodiment, the method is a cosmetic procedure.

[0109] In another preferred embodiment, the dose of the platelet lysate administered is 1%-20%, more preferably 2%-16%, and most preferably 4%-12%.

[0110] In another preferred embodiment, the dose of the platelet lysate administered is 10%.

[0111] In another preferred embodiment, the application period is ≥3 days, preferably ≥4 days, and most preferably ≥5 days.

[0112] In another preferred embodiment, the method of application includes: applying, smearing, injecting, or a combination thereof.

[0113] In another preferred embodiment, the method of application is by smearing or applying.

[0114] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0115] Figure 1 The growth factor levels of platelet lysates and human platelet lysates of the present invention are shown.

[0116] Figure 2 The cell viability of AG05247 cells cultured in the control group (10% fetal bovine serum), negative control group (treated with serum-containing medium and then replaced with serum-free medium after cell adhesion), hPL 5% group and hPL 10% group after (A) 72 hours, (B) 96 hours and (C) 120 hours is shown. Figure 2 D shows Figure 2 A, Figure 2 B Figure 2 C summarizes the line graph obtained from the data.

[0117] Figure 3 The results of SA-β-gal and EdU staining in AG05247 cells from (A) the control group (10% fetal bovine serum), the hPL 5% group, and the hPL 10% group are shown, along with (B) SA-β-gal staining. + EdU - Quantitative statistical results, (C)SA-β-gal - EdU + Quantitative statistical results and (D)EdU + The quantitative statistical results.

[0118] Figure 4 The results of fluorescence staining of migrating cells in (A) the control group (10% fetal bovine serum), the hPL 5% group, and the hPL 10% group are shown, along with their (B) quantitative statistics.

[0119] Figure 5 The relative expression levels of cellular senescence-related molecules were shown in the control group (10% fetal bovine serum), the hPL 5% group, and the hPL 10% group.

[0120] Figure 6 The study showed differences in growth factor concentrations in platelet lysates at 14, 17, and 20 days of differentiation.

[0121] Figure 7 The amplification capacity of cells treated with platelet lysates at 14, 17, and 20 days of differentiation was demonstrated. Detailed Implementation

[0122] Through extensive and in-depth research, the inventors unexpectedly discovered for the first time a lysate of in vitro differentiated platelets. By optimizing the preparation conditions of this lysate, the platelet lysate obtained according to this invention can efficiently promote cell proliferation and migration, reduce the proportion of senescent cells, promote collagen regeneration, and simultaneously delay skin aging at the molecular level. Therefore, this invention provides the preparation of this platelet lysate and its application in anti-aging. This invention was completed based on this.

[0123] The platelet lysate of the present invention is derived from the in vitro differentiation of pluripotent stem cells and / or hematopoietic stem / progenitor cells. It has a stable source, is simple to prepare, has small batch-to-batch differences, and is cost-controllable. It has a significant and efficient effect on treating skin aging.

[0124] the term

[0125] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0126] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0127] As used herein, the terms "platelets of the present invention", "differentiated platelets", and "differentiated platelets" are used interchangeably and all refer to platelets obtained by in vitro differentiation of human pluripotent stem cell ESC / iPSC cells or hematopoietic stem cell HSC cells provided by the present invention.

[0128] As used in this article, the terms "human platelets" and "human-derived platelets" are used interchangeably, both referring to platelets obtained from fresh human blood.

[0129] As used in this article, "treatment of skin aging," "anti-skin aging," "delaying skin aging," and "improving skin aging" can be used interchangeably. They refer to preventing the occurrence of aging, delaying the aging process, stopping the aging process, repairing aging cells, promoting cell iteration, improving skin condition, preventing wrinkles, stopping the formation of wrinkles, slowing down the rate of wrinkle formation, reducing wrinkles, shrinking or minimizing pores, protecting the skin, improving skin barrier function, reducing skin irritation, promoting skin cell proliferation and regeneration, improving antioxidant capacity, increasing collagen synthesis, slowing down or stopping collagen loss, and improving skin elasticity, thus adjusting the skin to a firm and elastic, baby-like state.

[0130] As used herein, “prevention” refers to all actions that inhibit or delay the occurrence of wrinkles, and the term “improvement” as used in this invention refers to all actions that improve or change the state of skin aging or wrinkles in a favorable direction, or make the skin more elastic, or delay the rate of loss of skin elasticity due to aging.

[0131] As used in this article, "wrinkles" refers to fine lines caused by skin aging, which can be caused by genetic factors, a decrease in collagen and elastin in the dermis, and external environmental factors.

[0132] As used herein, the term "elasticity" refers to the elasticity exhibited by elastic fibers composed of elastin, a protein found in the dermis of the skin. These elastic fibers, like rubber, have a very low modulus of elasticity, making them easily deformable even with small forces, and readily returning to their original shape when the force is removed. These elastic fibers possess a morphology in which microfibrils are embedded within an amorphous matrix called elastin, a protein composed of very unique amino acids derived from lysine, called desmosine and isodesmosine, found only in elastic fibers. These desmosine and isodesmosine form cross-links in long peptide chains, a structure that gives elastin its rubber-like properties.

[0133] In this invention, "differentiation for 14 days" means that the culture time for step (S1-5) is 0 days.

[0134] In this invention, "differentiation for 17 days" means that the culture time for step (S1-5) is 3 days.

[0135] In this invention, "differentiation for 20 days" means that the culture time in step (S1-5) is 6 days.

[0136] In this invention, the culture time for step (S1-5) corresponding to "differentiation culture to day 18" is 4 days.

[0137] platelets

[0138] Platelets are small, circulating anucleate cells that differentiate from mature megakaryocytes. They stop bleeding by aggregating and forming emboli in cases of vascular injury. The lifespan of a single platelet in the body is approximately 7-10 days; therefore, the body produces a large number of fresh platelets daily to maintain a normal platelet count. As an inherent component of the body, platelets can evade clearance by the immune system and are inextricably linked to physiological processes such as vascular endothelial injury repair, immune responses, atherosclerosis formation, neurodegeneration, and tumor growth and metastasis, thus possessing broad application prospects.

[0139] Platelet transfusion is a life-saving method used to prevent or stop bleeding in patients with thrombocytopenia or functional platelet dysfunction; it can also be used to prevent bleeding in patients with thrombocytopenia caused by chemotherapy for malignant tumors or hematopoietic stem cell transplantation.

[0140] The platelet lysate of the present invention

[0141] The platelet lysate of this invention refers to the lysate of in vitro differentiated platelets. Specifically, the platelet lysate of this invention includes lysates of platelets derived from ESCs / iPSCs and / or HSCs. In a specific embodiment, hematopoietic stem cells are intermediate products of in vitro differentiation of human pluripotent stem cells into platelets. In a specific embodiment, the lysate is a lysate of HSC-derived platelets. In a specific embodiment, the lysate is a lysate of ESCs / iPSC-derived platelets. In a specific embodiment, the lysate of in vitro differentiated platelets from ESCs / iPSCs is equivalent to the lysate of platelets directly differentiated from HSCs in vitro. In another preferred embodiment, "equivalent" refers to equivalent performance in improving skin aging.

[0142] In another preferred embodiment, the preparation of the platelets includes the following steps:

[0143] (O1) Culture ESC / iPSC cells in mTesR1 medium containing Y27632 for 12-24 hours; then continue culturing in mTesR1 medium without Y27632 until the confluence is 70%-80%.

[0144] (O2) The cells obtained in step (O1) are cultured in Stage I medium (STEM diff) TM APEL TM 2. Continue culturing in medium (30 ng / mL BMP4, 50 ng / mL VEGF, 25 ng / mL activin A, 1.5 μM CHIR-99021, 1% PS) for 2 days to obtain mesodermal stage cells;

[0145] (O3) The cells obtained in step (O2) are cultured in Stage II medium (StemSpan). TM -ACF Erythroid amplification medium (30 ng / mL BMP4, 50 ng / mL VEGF, 50 ng / mL bFGF, 1% PS) was used to culture the cells for 5 days to obtain hematopoietic endothelial cells and hematopoietic stem and progenitor cells.

[0146] (O4) The cells obtained in step (O3) are cultured in Stage III medium (STEM diff) TM APEL TMMegakaryotic cells were obtained by culturing them in a medium containing 50 ng / mL TPO, 50 ng / mL SCF, 25 ng / mL Flt3, 10 ng / mL IL3, 10 ng / mL IL6, 5% PFHM-II, 100 ug / mL PVA, and 1% PS for 7 days.

[0147] (O5) The cells obtained in step (O4) are cultured in Stage IV medium (STEMdiff). TM APEL TM Platelets were obtained by culturing in a medium containing 50 ng / mL TPO, 50 ng / mL SCF, 5% PFHM-II, and 1% PS for 4-6 days.

[0148] (O6) The platelets are lysed to obtain the platelet lysate.

[0149] In another preferred embodiment, the lysis refers to lysing the provided platelets using a homogenizer to obtain lysate.

[0150] In another preferred embodiment, the lysis is achieved by repeatedly freezing and thawing the provided platelets to obtain lysates.

[0151] In another preferred embodiment, the pyrolysis further includes ultrafiltration of the pyrolyte.

[0152] In another preferred embodiment, the pyrolysis further includes centrifuging and filtering the pyrolytes.

[0153] In another preferred embodiment, the centrifugal speed is 5000-8000g.

[0154] In another preferred embodiment, the centrifugation time is 20-40 min.

[0155] In another preferred embodiment, the filtration is performed using a 0.22 μm filter, and the filtrate is preserved.

[0156] The main advantages of this invention include:

[0157] (a) The present invention provides a lysate of in vitro differentiated platelets, which can effectively delay skin aging, does not contain plasma components, does not cause plasma-induced immune responses, and also has the characteristics of high batch stability.

[0158] (b) This invention provides a method for delaying skin aging based on the platelet lysate of this invention. This method can resist the aging process, promote the proliferation of senescent cells (2.5 times that of the 10% fetal bovine serum group), reduce the proportion of senescent cells (27% of the 10% fetal bovine serum group), significantly improve the levels of various cellular aging-related molecules, and promote collagen regeneration.

[0159] (c) The pyrolytes of the present invention are highly safe, have no restrictions on sources, are cost-controllable, and can be mass-produced.

[0160] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0161] Example 1: Preparation of platelet lysate (hPL)

[0162] The platelet lysates of the present invention include lysates of platelets generated from in vitro differentiation of human pluripotent stem cells (ESC / iPSC) and lysates of platelets generated from differentiation of hematopoietic stem cells (HSC). The preparation methods of the platelet lysates and human platelet lysates of the present invention are respectively shown below.

[0163] For the in vitro differentiation of human pluripotent stem cells (ESC / iPSC) to produce platelets:

[0164] ESC / iPSC cells were cultured in culture dishes coated with either a feeder-free matrix gel (a soluble preparation of Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells) or vitronectin or laminin521, and expanded using stem cell culture medium (mTeSR1).

[0165] 1.0x10 5 After single-cell passage of ESC / iPSC cells per well for 24 h, the cells were cultured in Stage I medium (STEMdiff). TM APEL TMCultured in medium + BMP4 (30 ng / mL) + VEGF (50 ng / mL) + Activin A (25 ng / mL) + CHIR-99021 (1.5 uM) + 1% PS for 2 days to allow mesodermal differentiation; no medium change is required during this period.

[0166] The obtained mesodermal cells were cultured in Stage II medium (StemSpan). TM -ACF Erythroid amplification medium + BMP4 (30ng / mL) + VEGF (50ng / mL) + bFGF (50ng / mL) + 1% PS) cultured for 5 days to perform differentiation culture of hematopoietic endothelial cells and hematopoietic stem and progenitor cells, with the medium changed daily;

[0167] Cells will be suspended in the supernatant on days 7-12. When changing the medium, centrifuge at 400g / 3min and discard the supernatant. Use 2ml of Stage III medium (STEMdiff). TM APEL TM 2. After resuspending the precipitate in the culture medium (TPO (50 ng / mL) + SCF (50 ng / mL) + Flt3 (25 ng / mL) + IL3 (10 ng / mL) + IL6 (10 ng / mL) + PFHM-II (5%) + PVA (100 ug / mL) + 1% PS), add it to the original well plate.

[0168] On day 12, the suspended cells in the supernatant and the adherent cells in the lower layer were separated. The suspended cells in the supernatant were collected by centrifugation at 400g / 3min and transferred to 6-well ultra-low adsorption plates, cultured in 2mL Stage III medium until day 14 without medium change. The adherent cells in the lower layer were digested with 1mL collagenase IV at 37℃ for 30min. Cells were gently dissociated by pipetting from the bottom of the wells, and the suspension was collected in a 15mL centrifuge tube. 1mL DMEM (high glucose, pyruvate) was added, mixed, and centrifuged at 400g for 3min. The supernatant was discarded, and 1mL Trypsin-EDTA was added. The tube was incubated at 37℃ for 3min, then 2mL Trypsin-EDTA digestion stop solution (1mL DMEM high glucose + 1mL fetal bovine serum) was added, mixed, and centrifuged at 400g for 3min. The supernatant was discarded, and 2mL Stage III medium was added to resuspend the cells. The cells were then seeded 1:1 into 6-well plates pre-coated with Matrigel, with a concentration of 10µM on day 1. Y27632, when changing the medium on day 13, centrifuge at 400g / 3min, discard the supernatant, resuspend the precipitate in 2ml Stage III medium, and add it to the original well plate.

[0169] On day 14, all suspended cells (both auto-resuspended and digested) were collected by centrifugation at 400g / 3min for cell counting, and 1*102 cells were collected. 5 Cell CD34 / CD41 ratio was measured. The total number of iPS cells differentiated in a single well of a 6-well plate was 400-800,000, with a cell viability of over 80%, and CD34 / CD41 ratio was [data missing]. + / CD41 + The percentage was 50%-80%. The resulting cells were cultured in Stage IV medium (STEMdiff). TM APEL TM A large number of platelets can be obtained by differentiating and culturing the medium (TPO (50 ng / mL) + SCF (50 ng / mL) + PFHM-II (5%) + 1% PS) to day 18.

[0170] For platelet production from hematopoietic stem cells (HSCs): CD34 + Cell differentiation medium I (StemSpan) TM Megakaryotic progenitor cells (MKPs) can be obtained by culturing them in SFEMII medium (IL-3, IL-6, SCF, TPO, and FLT) for 7 days, with a half-medium change every 3 days using differentiation medium I. The obtained MKPs are then cultured in differentiation medium II (IMDM, N2B27, NEAA, glutamate, ITS, ascorbic acid, IL-6, SCF, and TPO) for 5 days. After that, they are cultured at 39°C and 5% CO2 for 5-7 days to obtain a large number of platelets.

[0171] The differentiated platelets were lysed using a homogenizer. The lysate was centrifuged at 6000g for 30min in a high-speed refrigerated centrifuge, filtered through a 0.22μm filter, and aliquoted and stored at -80℃ for later use.

[0172] In human platelet lysate, the platelets are derived from the blood of a healthy donor.

[0173] Specifically, a suitable amount of healthy donor blood is collected using sodium citrate blood collection tubes. The blood is then centrifuged in two steps (① centrifuged at 200g for 10 minutes at room temperature, retaining the supernatant plasma layer; ② the plasma layer is centrifuged at 200g for 10 minutes at room temperature, approximately 3 / 4 of the supernatant is discarded, and the precipitate is resuspended in the remaining liquid) to obtain platelet-rich plasma (PRP). Approximately 700 μL of PRP can be obtained from 5 mL of whole blood. The prepared PRP should ensure a platelet concentration greater than 10. 9 per mL.

[0174] PRP was frozen at -80°C for 24 hours and then thawed at room temperature. This process was repeated three times to lyse platelets. The lysed plasma was ultrafiltered to remove large protein molecules, yielding a high concentration of human platelet lysate. The human hPL was filtered through a 0.22 μm sterile filter, aliquoted, and stored. It was stored for short-term storage at 4°C and for long-term storage at -20 / -80°C.

[0175] Example 2: Growth factor levels of platelet lysates and human platelet lysates of the present invention

[0176] The difference in growth factor concentrations between the platelet lysate of this invention and the human platelet lysate was detected using ELISA. The results are shown in Table 1 and... Figure 1 As shown.

[0177] The results showed that after lysis of differentiated platelets and human platelets, the concentrations of FGF and VEGF, two growth factors, in the differentiated platelet lysate were approximately twice that in the human platelet lysate; the PDGF-BB level increased by about 27%; and the concentrations of TGF-β1 and EGF, two growth factors, in the platelet lysate of this invention were basically the same as those in the human platelet lysate.

[0178] Table 1. Growth factor levels of platelet lysates and human platelet lysates of the present invention.

[0179]

[0180] Example 3: Anti-aging effect of hPL on skin fibroblasts in the elderly

[0181] This embodiment investigates the effects of hPL on the phenotypic and molecular-level responses of cellular senescence when aging fibroblasts are treated in vitro.

[0182] Skin fibroblasts from elderly individuals (AG05247) were purchased from Coriell, with samples taken from the arm skin of an 87-year-old woman. Normal culture was performed using DMEM medium supplemented with 10% fetal bovine serum and antibiotics, and passaged every 4-7 days depending on the inoculation density.

[0183] AG05247 cells were seeded at a density of 1500 cells / well in TC-treated 96-well plates and divided into a control group (10% fetal bovine serum), a negative control group (treated with serum-containing medium until cell attachment, then replaced with serum-free medium), a 5% hPL group, and a 10% hPL group. After 4 days of incubation at 37°C in a CO2 incubator, the medium was removed. CCK8 assay reagent was added, and after incubation at 37°C in a CO2 incubator for 3 hours, absorbance at 450 nm was measured using a microplate reader. Cell viability (%) = (OD value of experimental groups - OD value of zeroing well) / (OD value of control wells - OD value of zeroing well) * 100%.

[0184] The results are as follows Figure 2 A- Figure 2 As shown in Figure D, on day 3 of culture, the cell viability of both the 5% hPL group and the 10% hPL group was significantly higher than that of the 10% FBS group, indicating that 5% hPL significantly promoted cell proliferation after 3 days of culture. With continued culture, the cell viability of both the 5% hPL group and the 10% hPL group continued to increase, with the cell growth rate of the 10% hPL group being significantly higher than that of the 5% hPL group, and significantly higher than that of the control group. When cultured with 10% hPL for five days, the cell growth effect was 2.5 times that of the control group, indicating that hPL treatment significantly promoted the proliferation of AG05247.

[0185] AG05247 cells were seeded at a density of 15,000 cells / well in 24-well plates coated with cell spreaders and divided into a control group (10% fetal bovine serum), a 5% hPL group, and a 10% hPL group. After culturing at 37°C in a CO2 incubator for 4 days, the supernatant was removed, and the cells were washed with PBS, fixed with formaldehyde / glutaraldehyde fixative, washed with PBS, and stained overnight at 37°C with SA-β-gal. EdU and DAPI staining were then performed. After staining, the cells were mounted, observed under a fluorescence microscope, and counted. At least four fields of view were required for each sample (at least 300 cells per sample).

[0186] SA-β-gal and EdU staining results are as follows: Figure 3 As shown in Figure A. Figure 3 B indicates that in the 10% FBS control group, senescent cells accounted for 30% of the total cells. When treated with 5% hPL, the proportion of senescent cells decreased to about 15%. As the concentration of hPL increased to 10%, the proportion of senescent cells decreased to about 8%. Figure 3 C indicates that in the 10% FBS control group, non-senescent normal proliferating cells accounted for 20% of the total cells. When treated with 5% hPL, the proportion of non-senescent normal proliferating cells increased to about 35%. As the concentration of hPL increased to 10%, the proportion of non-senescent normal proliferating cells increased to about 45%. Figure 3 D indicates that in the 10% FBS control group, all proliferating cells accounted for 30% of the total cells. When treated with 5% hPL, the proportion of all proliferating cells slightly increased to about 38%. As the concentration of hPL increased to 10%, the proportion of non-proliferating cells rose to about 50%.

[0187] Therefore, compared with the control group, hPL treatment for 96 h significantly improved the senescence phenotype of AG05247 cells and promoted cell proliferation.

[0188] AG05247 cells were seeded at a density of 30,000 cells / well in Transwell chambers and divided into a control group (10% fetal bovine serum), a 5% hPL group, and a 10% hPL group. The culture medium in the chambers was DMEM + 10% fetal bovine serum + penicillin-drug antibiotics, with the test medium at the bottom. After incubation at 37°C for 24 hours, the chambers were removed, washed, fixed, permeabilized, stained with DAPI, and examined under a fluorescence microscope. At least four fields of view were required for each sample under a 10x microscope.

[0189] Cell migration detection results as follows Figure 4 As shown, Figure 4 B is Figure 4 Quantitative analysis of the staining results shown in Figure A. The results indicate that, compared with the control group, treatment with 5% hPL for 24 hours significantly promoted the migration ability of AG05247 cells.

[0190] Assessment of molecular-level responses to cellular senescence:

[0191] AG05247 cells were seeded at a density of 15,000 / 12-well plates and divided into a control group (10% fetal bovine serum), a 5% hPL group, and a 10% hPL group. After culturing in a 37°C CO2 incubator for 4 days, cells were lysed with Trizol, and RNA was extracted using chloroform extraction. The corresponding cDNA was then obtained by reverse transcription using a reverse transcription kit. The expression levels of P16, P21, LMNB1, IL-6, IL-8, IL-1A, MMP3, and COL1A1 were detected using qPCR, with GAPDH as an internal control. Expression levels were statistically analyzed using a 2-1 -ΔΔCT Law.

[0192] Detection of molecular levels of cellular senescence, such as Figure 5 As shown, compared to the control group, treatment with 5% hPL for 96 hours significantly regulated the levels of two cell senescence-related molecules, and treatment with 10% hPL for 96 hours significantly regulated the levels of six cell senescence-related molecules. Therefore, the hPL of this invention can improve cell senescence at the molecular level and promote collagen regeneration to a certain extent.

[0193] Example 4: Effects of platelet lysates at different differentiation times on cell senescence

[0194] The concentrations of growth factors after platelet lysis on days 14, 17, and 20 of differentiated platelets were further investigated according to the method described in Example 3.

[0195] The results are as follows Figure 6As shown in Table 2, with three replicates per group, the results indicated that when platelets lysed 17 days after differentiation, the concentrations of various growth factors in the resulting platelet lysates were more than 50% higher than those from platelets differentiated for 14 days. However, compared to platelets differentiated for 17 days, the molecular levels of EGF and FGF in the lysates from platelets differentiated for 20 days were comparable; the molecular level of TGF-β1 decreased by approximately 16%; and the molecular levels of BDNF, VEGF, PDGF-AB, PDGF-BB, and IGF all increased by more than 20%.

[0196] Table 2. Growth factor levels in platelet lysates at different differentiation times.

[0197]

[0198] The ability of platelet lysates from differentiated platelets on days 14, 17, and 20 to promote the proliferation of GM00038 cells was further investigated.

[0199] The results are as follows Figure 7 As shown, the results indicate that 10% of platelet lysates differentiated for 14 and 17 days and 10% FBS had similar effects, with no significant promotion of GM00038 cell proliferation. However, platelet lysates differentiated for more than 20 days significantly promoted GM00038 cell proliferation, with cell viability approximately 130% of the control group (10% FBS). This suggests that platelet lysates differentiated for more than 20 days also have a positive effect on adolescent skin, further confirming the good effect of the platelet lysates of this invention in improving skin.

[0200] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A platelet lysate for improving skin aging, characterized in that, The platelet lysate is a lysate of platelets differentiated in vitro from human pluripotent stem cells (ESC / iPSC) and / or hematopoietic stem cells.

2. A culture system for obtaining platelet lysates that improve skin aging, characterized in that, The culture system includes Stage I medium, Stage II medium, Stage III medium, and / or Stage IV medium. The Stage I culture medium includes basal medium, BMP4, VEGF, activator A, and CHIR-99021; The Stage II culture medium includes StemSpan. TM -ACF Erythroid amplification medium, BMP4, VGEF, bFGF; The Stage III culture medium includes basal medium, TPO, SCF, Flt3, IL3, IL6, PFHM-II, and PVA; The Stage IV medium includes basal medium, TPO, SCF, and PFHM-II.

3. A method for preparing platelet lysate to improve skin aging, characterized in that, Includes the following steps: (S1) Platelets differentiated in vitro are provided by the culture system of claim 2; (S2) The platelets are lysed to obtain the platelet lysate.

4. The method as described in claim 3, characterized in that, Step (S1) includes the following sub-steps: (S1-1) ESC / iPSC cells were cultured and expanded using stem cell culture medium; (S1-2) The cells obtained in step (S1-1) are cultured for mesodermal differentiation using the Stage I medium as described in claim 2; (S1-3) The cells obtained in step (S1-2) are cultured for differentiation into hematopoietic endothelial cells and hematopoietic stem and progenitor cells using the Stage II medium as described in claim 2; (S1-4) Continue culturing the cells obtained in step (S1-3) in the Stage III medium as described in claim 2 to obtain megakaryocytes; (S1-5) Continue culturing the cells obtained in step (S1-4) in the Stage IV medium as described in claim 2 to obtain platelets.

5. The platelet lysate prepared by the method of claim 3, characterized in that, The platelet lysate showed a significant increase in the levels of one or more growth factors, including FGF, VEGF, and PDGF-BB.

6. The use of the platelet lysate according to claim 1 or claim 5, characterized in that, Used to prepare a composition or formulation for improving skin aging.

7. The use as described in claim 6, characterized in that, The improvement in skin aging includes: (Z1) Prevent skin aging, delay skin aging, stop the process of skin aging, repair aging skin, or a combination thereof; (Z2) Promotes cell proliferation, promotes cell migration, reduces the proportion of senescent cells, promotes collagen regeneration, or a combination thereof; and / or (Z3) Improves the molecular level of molecules related to cellular senescence.

8. The use as described in claim 7, characterized in that, The cellular senescence-related molecules include: P21, LMNB1, IL6, IL8, MMP3, COL1A2, MMP1, or combinations thereof.

9. A composition or formulation for improving skin aging, characterized in that, The composition or formulation comprises the platelet lysate of claim 5.

10. A method for improving skin aging, characterized in that, The method includes administering to a subject the platelet lysate of claim 1 or 5, or the composition or formulation of claim 9.

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