Platelet lysate and application of preparation method thereof in skin aging resistance

Abstract

The invention belongs to the field of medicine health. Specifically, the invention provides preparation and application of a lysate of platelets differentiated in vitro. Specifically, the invention provides application of the lysate in effectively promoting migration of cells to a skin injury, reducing inflammation, limiting matrix degradation and promoting collagen synthesis to support wound healing.

Description

Technical Field

[0001] The present invention belongs to the field of medicine and health, and particularly relates to the application of a platelet lysate and a preparation method thereof in skin anti-aging. Background Art

[0002] Skin injury repair is an extremely complex biological process, involving the orderly interaction of various different types of cells, extracellular matrix, and cytokines in time and space, and finally completing skin barrier repair and tissue homeostasis maintenance. Skin injury repair, also known as tissue repair, aims to restore the continuity of cells.

[0003] Platelets are important participants in many pathophysiological processes. The research on platelet-rich plasma (PRP) in improving skin texture, delaying and improving cell aging has been relatively rich, and it has been gradually applied to the treatment of acne marks, refractory wound healing, helping fat cells survive after autologous fat transplantation, wound repair after skin laser treatment, smoothing skin fine lines and other curative effects.

[0004] Currently, platelets mainly come from the donation of donors. Due to the short storage time of donor-derived platelets and the insufficient number of donors, platelets have become a scarce resource. At the same time, there is a risk of bacterial contamination and deterioration during the process of platelet processing, preparation, and infusion.

[0005] Therefore, for promoting cell migration to the skin injury site and thus promoting skin injury repair, there is an urgent need to provide platelet substitutes that are easy to prepare and preserve, safe and effective, and with controllable cost and stability. Summary of the Invention

[0006] The present invention provides a platelet lysate prepared by in vitro differentiation and its application, and based on this lysate, provides a method for promoting cell migration to the skin injury site and / or promoting skin injury repair.

[0007] In the first aspect of the present invention, there is provided the use of a platelet lysate prepared by in vitro differentiation for preparing a composition for the following uses:

[0008] (Z1) Promoting cell migration to the skin injury site; and / or

[0009] (Z2) Promoting skin injury repair.

[0010] In another preferred example, the cells include: adipose stem cells, keratinocytes, fibroblasts, endothelial cells, skin stem cells, or a combination thereof.

[0011] In another preferred example, the keratinocytes include: melanocytes, Langerhans cells, Merkel cells, or a combination thereof.

[0012] In another preferred example, the cells include spinous cells.

[0013] In another preferred example, the cells are not senile dermal fibroblasts.

[0014] In another preferred example, the cells are dermal fibroblasts.

[0015] In another preferred example, the platelet lysate is a lysate of platelets differentiated in vitro from human pluripotent stem cells ESC / iPSC cells and / or hematopoietic stem cells.

[0016] In another preferred example, the hematopoietic stem cells include hematopoietic stem cells, multipotent progenitor cells, common myeloid progenitor cells, megakaryocyte-erythroid progenitor cells, or a combination thereof.

[0017] In another preferred example, the hematopoietic stem cells are CD34+ cells.

[0018] In another preferred example, the lysate of the platelets differentiated in vitro has one or more of the following characteristics:

[0019] (i) The level of bFGF is significantly increased;

[0020] (ii) The level of VEGF is significantly increased.

[0021] In another preferred example, "the level of bFGF is significantly increased" means that the bFGF level X1 of the platelet lysate compared to the bFGF level X0 of the human platelet lysate, X1 / X0 ≥ 1.2, more preferably, X1 / X0 ≥ 1.5, most preferably, X1 / X0 ≥ 2.

[0022] In another preferred example, "the level of VEGF is significantly increased" means that the VEGF level Y1 of the platelet lysate compared to the VEGF level Y0 of the human platelet lysate, Y1 / Y0 ≥ 1.2, more preferably, Y1 / Y0 ≥ 1.5, most preferably, Y1 / Y0 ≥ 2.

[0023] In another preferred example, the promotion of skin injury repair includes promoting skin wound healing and / or promoting skin function repair.

[0024] In another preferred example, the promotion of skin injury repair includes:

[0025] (Z2-1) Promoting skin wound healing and / or promoting skin function repair;

[0026] (Z2-2) Promoting cell migration to the skin injury site, promoting cell proliferation, promoting the synthesis of matrix for filling the wound, or a combination thereof; and / or

[0027] (Z2-3) significantly down-regulates the levels of inflammatory factors, significantly down-regulates the levels of matrix degradation, significantly increases the levels of collagen, or a combination thereof.

[0028] In another preferred embodiment, the inflammatory factors include: IL-6, IL-8, IL1α-1, or a combination thereof.

[0029] In another preferred embodiment, the significant down-regulation of the levels of inflammatory factors means that the level of inflammatory factors M1 in the subject after administering the composition is compared with the level of inflammatory factors M0 in the subject before administering the composition, and M1 / M0 ≤ 0.5. More preferably, M1 / M0 ≤ 0.3, and most preferably, M1 / M0 ≤ 0.2.

[0030] In another preferred embodiment, the significant down-regulation of the levels of matrix degradation means that the MMP-1 level N1 in the subject after administering the composition is compared with the MMP-1 level N0 in the subject before administering the composition, and N1 / N0 ≤ 0.8. More preferably, N1 / N0 ≤ 0.6, and most preferably, N1 / N0 ≤ 0.5.

[0031] In another preferred embodiment, the significant increase in the levels of collagen means that the type I collagen level H1 in the subject after administering the composition is compared with the type I collagen H0 in the subject before administering the composition, and H1 / H0 ≥ 1.2. More preferably, H1 / H0 ≥ 1.5, and most preferably, H1 / H0 ≥ 2.

[0032] In another preferred embodiment, the "significant" means that there is a statistically significant difference.

[0033] In another preferred embodiment, the statistically significant difference means that when the significance level α is 0.05, P ≤ 0.05.

[0034] In another preferred embodiment, the in vitro differentiated platelets refer to platelets obtained by in vitro differentiation of a platelet in vitro differentiation culture system, and the culture system includes StageI medium, StageII medium, StageIII medium, and / or StageIV medium.

[0035] Among them, the StageI medium includes a basal medium, BMP4, VEGF, activin A, CHIR-99021.

[0036] The StageII medium includes StemSpan TM -ACF Erythroid expansion medium, BMP4, VGEF, bFGF.

[0037] The StageIII medium comprises a basal medium, TPO, SCF, Flt3, IL3, IL6, PFHM-II, PVA;

[0038] The StageIV medium comprises a basal medium, TPO, SCF, PFHM-II.

[0039] In another preferred example, the StageI medium comprises STEMdiff TM APEL TM 2 medium, 30 ng / mL of BMP4, 50 ng / mL of VEGF, 25 ng / mL of activin A, 1.5 uM of CHIR-99021, 1% PS.

[0040] In another preferred example, the StageII medium comprises StemSpan TM -ACF Erythroid expansion medium, 30 ng / mL of BMP4, 50 ng / mL of VEGF, 50 ng / mL of bFGF, 1% PS.

[0041] In another preferred example, the StageIII medium comprises STEMdiff TM APEL TM 2 medium, 50 ng / mL of TPO, 50 ng / mL of SCF, 25 ng / mL of Flt3, 10 ng / mL of IL3, 10 ng / mL of IL6, 5% of PFHM-II, 100 ug / mL of PVA, 1% PS.

[0042] In another preferred example, the StageIV medium comprises STEMdiff TM APEL TM 2 medium, 50 ng / mL of TPO, 50 ng / mL of SCF, 5% of PFHM-II, 1% PS.

[0043] In another preferred example, the differentiation comprises the following steps:

[0044] (S1-1) Culturing and expanding ESC / iPSC cells with a stem cell medium;

[0045] (S1-2) Performing mesoderm differentiation culture on the cells obtained in step (S1-1) with the StageI medium;

[0046] (S1-3) Performing hematoendothelial and hematopoietic stem and progenitor cell differentiation culture on the cells obtained in step (S1-2) with the StageII medium;

[0047] (S1-4) Culture the cells obtained in step (S1-3) with the StageIII medium to obtain megakaryocytes; (S1-5) Culture the cells obtained in step (S1-4) with the StageIV medium to obtain platelets.

[0048] In a second aspect of the present invention, there is provided a culture system for obtaining platelet lysates that promote cell migration to skin injury sites, and the culture system includes StageI medium, StageII medium, StageIII medium, and / or StageIV medium.

[0049] Among them, the StageI medium includes a basal medium, BMP4, VEGF, activin A, CHIR-99021;

[0050] The StageII medium includes StemSpan TM -ACF Erythroid expansion medium, BMP4, VGEF, bFGF;

[0051] The StageIII medium includes a basal medium, TPO, SCF, Flt3, IL3, IL6, PFHM-II, PVA;

[0052] The StageIV medium includes a basal medium, TPO, SCF, PFHM-II.

[0053] In another preferred example, the basal medium includes E8 medium, complete medium, StemSpanTM SFEM hematopoietic cell medium, StemPro-34, STEMdiff TM APEL TM 2 medium, and / or BPEL.

[0054] In another preferred example, the basal medium is STEMdiff TM APEL TM 2 medium.

[0055] In another preferred example, the StageI medium includes STEMdiff TM APEL TM 2 medium, 20-40 ng / mL of BMP4, 40-60 ng / mL of VEGF, 20-30 ng / mL of activin A, 1-3 μM of CHIR-99021.

[0056] In another preferred example, the StageII medium includes StemSpan TM-ACF Erythroid expansion medium with 20 - 40 ng / mL of BMP4, 40 - 60 ng / mL of VEGF, and 40 - 60 ng / mL of bFGF.

[0057] In another preferred embodiment, the StageIII medium comprises STEMdiff TM APEL TM 2 medium, 40 - 60 ng / mL of TPO, 40 - 60 ng / mL of SCF, 20 - 30 ng / mL of Flt3, 5 - 15 ng / mL of IL3, 5 - 15 ng / mL of IL6, 1% - 10% of PFHM-II, 50 - 200 μg / mL of PVA.

[0058] In another preferred embodiment, the StageIV medium comprises STEMdiff TM APEL TM 2 medium, 40 - 60 ng / mL of TPO, 40 - 60 ng / mL of SCF, 1% - 10% of PFHM-II.

[0059] In another preferred embodiment, the medium comprises 0.5% - 3% of PS.

[0060] In another preferred embodiment, the StageI medium comprises STEMdiff TM APEL TM 2 medium, 30 ng / mL of BMP4, 50 ng / mL of VEGF, 25 ng / mL of activin A, 1.5 μM of CHIR-99021, 1% PS.

[0061] In another preferred embodiment, the StageII medium comprises StemSpan TM -ACF Erythroid expansion medium with 30 ng / mL of BMP4, 50 ng / mL of VEGF, 50 ng / mL of bFGF, 1% PS.

[0062] In another preferred embodiment, the StageIII medium comprises STEMdiff TM APEL TM 2 medium, 50 ng / mL of TPO, 50 ng / mL of SCF, 25 ng / mL of Flt3, 10 ng / mL of IL3, 10 ng / mL of IL6, 5% of PFHM-II, 100 μg / mL of PVA, 1% PS.

[0063] In another preferred embodiment, the StageIV medium comprises STEMdiff TM APEL TM2 medium, 50 ng / mL of TPO, 50 ng / mL of SCF, 5% of PFHM-II, 1% PS.

[0064] In another preferred example, the culture system comprises M1 medium, and the medium contains StemSpan TM SFEMII medium, IL-3, IL-6, SCF, TPO and FLt.

[0065] In another preferred example, the culture system comprises M2 medium, and the medium contains IMDM medium, N2B27, NEAA, glutamic acid, ITS, ascorbic acid, IL-6, SCF, TPO.

[0066] In another preferred example, the M1 medium contains StemSpan TM SFEMII medium, 5 - 50 ng / ml of IL-3, 5 - 50 ng / ml of IL-6, 5 - 50 ng / ml of SCF, 5 - 50 ng / ml of TPO and 5 - 50 ng / ml of FLt.

[0067] In another preferred example, the M2 medium contains IMDM medium, N2B27, NEAA, glutamic acid, ITS, ascorbic acid, 5 - 50 ng / ml of IL-6, 5 - 50 ng / ml of SCF, 5 - 50 ng / ml of TPO.

[0068] In another preferred example, the culture system comprises a stem cell medium.

[0069] In another preferred example, the stem cell medium is mTeSR1 medium.

[0070] In the third aspect of the present invention, a method for preparing a platelet lysate for promoting cell migration to a skin injury site is provided, comprising the following steps:

[0071] (S1) By means of the culture system described in the second aspect of the present invention, in vitro differentiated platelets are provided;

[0072] (S2) The platelets are lysed to obtain the platelet lysate.

[0073] In another preferred example, the lysis refers to lysing the provided platelets using a homogenizer to obtain a lysate.

[0074] In another preferred example, the lysis is carried out by repeatedly freezing and thawing the provided platelets to obtain a lysate.

[0075] In another preferred example, the lysis further comprises ultrafiltration of the lysate.

[0076] In another preferred example, the lysis further includes centrifuging and filtering the lysate.

[0077] In another preferred example, the centrifugation speed is 5000 - 8000g.

[0078] In another preferred example, the centrifugation time is 20 - 40 min.

[0079] In another preferred example, the filtration is performed using a 0.22 μm filter, and the filtrate is saved.

[0080] In another preferred example, step (S1) includes the following sub - steps:

[0081] (S1 - 1) Culturing and expanding ESC / iPSC cells with a stem cell medium;

[0082] (S1 - 2) Performing mesoderm differentiation culture on the cells obtained in step (S1 - 1) with the StageI medium in the reaction system described in the second aspect of the present invention;

[0083] (S1 - 3) Performing hemogenic endothelium and hematopoietic stem and progenitor cell differentiation culture on the cells obtained in step (S1 - 2) with the StageII medium in the reaction system described in the second aspect of the present invention;

[0084] (S1 - 4) Continuing to culture the cells obtained in step (S1 - 3) with the StageIII medium in the reaction system described in the second aspect of the present invention to obtain megakaryocytes;

[0085] (S1 - 5) Continuing to culture the cells obtained in step (S1 - 4) with the StageIV medium in the reaction system described in the second aspect of the present invention to obtain platelets.

[0086] In another preferred example, the stem cell medium includes: mTesR1 medium containing Y27632 or mTesR1 medium without Y27632.

[0087] In another preferred example, step (S1 - 1) includes: culturing ESC / iPSC cells with mTesR1 medium containing Y27632 for 12 - 24 h; then continuing to culture with mTesR1 medium without Y27632 until the confluence reaches 70% - 80%.

[0088] In another preferred example, the culture time in step (S1 - 2) is 2 days.

[0089] In another preferred example, the culture time in step (S1 - 3) is 5 days.

[0090] In another preferred example, the culture time in step (S1 - 4) is 7 days.

[0091] In another preferred embodiment, the culturing time in step (S1-5) is 0-6 days, preferably 3-6 days, and most preferably 4-6 days.

[0092] In another preferred embodiment, step (S1) includes the following sub-steps:

[0093] (S1-A) Culturing hematopoietic stem cells in M1 medium in the reaction system described in the second aspect of the present invention to obtain megakaryocyte progenitor cells;

[0094] (S1-B) Continuing to culture the megakaryocyte progenitor cells obtained in step (S1-A) in M2 medium in the reaction system described in the second aspect of the present invention to obtain platelets.

[0095] In another preferred embodiment, the hematopoietic stem cells are CD34+ cells.

[0096] In another preferred embodiment, the culturing time in step (S1-A) is 7 days.

[0097] In another preferred embodiment, the culturing time in step (S1-B) is 10-12 days.

[0098] In the fourth aspect of the present invention, there is provided a platelet lysate for promoting the migration of cells to skin injury sites in vitro differentiation, and the platelet lysate has the following characteristics:

[0099] (i) The level of bFGF is significantly increased; and / or

[0100] (ii) The level of VEGF is significantly increased.

[0101] In another preferred embodiment, the "significantly increased bFGF level" means that the bFGF level X1 of the platelet lysate is compared with the bFGF level X0 of the human platelet lysate, and X1 / X0 ≥ 1.2, more preferably, X1 / X0 ≥ 1.5, and most preferably, X1 / X0 ≥ 2;

[0102] The "significantly increased VEGF level" means that the VEGF level Y1 of the platelet lysate is compared with the VEGF level Y0 of the human platelet lysate, and Y1 / Y0 ≥ 1.2, more preferably, Y1 / Y0 ≥ 1.5, and most preferably, Y1 / Y0 ≥ 2.

[0103] In another preferred embodiment, the platelet lysate is prepared by the method described in the third aspect of the present invention.

[0104] In the fifth aspect of the present invention, there is provided a composition for promoting the migration of cells to skin injury sites, and the composition contains the platelet lysate described in the fourth aspect of the present invention.

[0105] In another preferred example, the content of the platelet lysate in the composition or preparation is 1%-20%, preferably 2%-16%, and most preferably 4%-12%.

[0106] In another preferred example, the content of the platelet lysate in the composition or preparation is 10%.

[0107] In another preferred example, the composition is a pharmaceutical composition.

[0108] In another preferred example, the form of the composition is a liquid, solid, spray, or semi-solid.

[0109] In another preferred example, the composition further comprises: a skin-acceptable water-insoluble matrix.

[0110] In another preferred example, the skin-acceptable water-insoluble matrix is a band-aid.

[0111] In the sixth aspect of the present invention, a kit is provided, and the kit comprises:

[0112] (a) The platelet lysate described in the fourth aspect of the present invention and / or the composition described in the fifth aspect of the present invention;

[0113] (b) Optionally, a disinfection reagent.

[0114] In another preferred example, the kit further contains an instruction manual, and the instruction manual guides the dosage and frequency of use of the kit for promoting skin injury repair and / or promoting cell migration to the skin injury site.

[0115] In the seventh aspect of the present invention, a method for promoting skin injury repair is provided, and the method comprises administering to a subject the platelet lysate described in the fourth aspect of the present invention or the composition described in the fifth aspect of the present invention.

[0116] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. Description of the Drawings

[0117] Figure 1 It shows that the hPL of the present invention improves the viability of HDF cells.

[0118] Wherein, Figure 1A shows the cell viability of HDFs cultured with 10% FBS or 10% hPL for 72 hours evaluated by the CCK-8 assay. Statistical significance was calculated using an unpaired t-test (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001); Figure 1 B shows representative images of EdU-labeled cells (yellow) and DAPI-stained cell nuclei (blue) in HDFs treated with 10% FBS or 10% hPL for 72 hours, and the percentage of EdU-positive cells was quantified. Scale bar = 20 μm. Statistical significance was calculated using an unpaired t-test (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

[0119] Figure 2 It shows that the hPL of the present invention improves the cell migration ability of HDFs. Among them, Figure 2 A shows representative images of HDF migration in a transwell assay after treatment with 10% FBS or 10% hPL for 24 hours. Scale bar = 20 μm; DAPI-stained cell nuclei (blue); Figure 2 B shows the quantitative results of the number of migrated cells. Statistical significance was calculated using an unpaired t-test (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

[0120] Figure 3 It shows that the hPL of the present invention promotes the migration of HDF cells towards the wound. Figure 3 A shows representative images of scratch assays in HDFs treated with 10% FBS (labeled as the Blank group in the figure) or 10% hPL for 24 hours, showing the wound area. Scale bar = 200 μm; Figure 3 B quantifies the cell-free area remaining after 24 hours. Statistical significance was calculated using an unpaired t-test (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

[0121] Figure 4 It shows that the hPL of the present invention promotes wound recovery at the molecular level. The mRNA levels of interleukin-6 (IL6), interleukin-8 (IL8), interleukin-1A (IL1A), matrix metalloproteinase-1 (MMP1), and type I collagen (COL1A1) in HDFs treated with 10% FBS or 10% hPL for 72 hours. The GAPDH mRNA level was used as a standard. Statistical significance was calculated using an unpaired t-test (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

[0122] Figure 5 Shows the comparison of the growth factor concentrations of hPL and human platelets of the present invention. Detailed implementation manners

[0123] Through extensive and in-depth research, the inventors of the present invention unexpectedly discovered for the first time that a lysate of in vitro differentiated platelets can promote cells. By optimizing the preparation conditions of the lysate, the platelet lysate of the present invention obtained can efficiently promote cell migration to skin injury sites, reduce inflammation, limit matrix degradation, and promote collagen synthesis to support wound healing. Therefore, the present invention provides the preparation of the platelet lysate and its application in repairing skin injuries. The present invention was completed on this basis.

[0124] The platelet lysate of the present invention is derived from the in vitro differentiation of pluripotent stem cells and / or hematopoietic stem / progenitor cells, has a stable source, is simple to prepare, has small differences between batches, controllable costs, and has a significant and efficient effect in treating skin aging.

[0125] Terms

[0126] To make the present disclosure easier to understand, certain terms are first defined. As used in this application, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below. Other definitions are set forth throughout the application.

[0127] As used herein, the term "and / or" relates to and encompasses any and all possible combinations of one or more of the related listed items.

[0128] 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 cells ESC / iPSC cells or hematopoietic stem cells HSC provided by the present invention.

[0129] As used herein, the terms "human platelets" and "human-derived platelets" are used interchangeably and both refer to platelets obtained from fresh human blood.

[0130] Platelets

[0131] Platelets are a type of small circulating anucleate cells, differentiated from mature megakaryocytes, and stop bleeding by aggregating and forming plugs in vascular injuries. The lifespan of a single platelet in vivo is about 7 - 10 days, so the human body produces a large number of fresh platelets every day to maintain the normal platelet count. As an inherent component in the living body, platelets can avoid being cleared by the immune system and are closely related to physiological processes such as vascular endothelial injury repair, immune response, atherosclerotic plaque formation, neurodegeneration, and tumor growth and metastasis, and have broad application prospects.

[0132] Platelet transfusion is a life-saving method used for preventing bleeding or stopping continuous bleeding in patients with thrombocytopenia or functional platelet disorders; meanwhile, it can be used to prevent bleeding in patients with thrombocytopenia caused by chemotherapy for malignant tumors or hematopoietic stem cell transplantation.

[0133] The platelet lysate of the present invention

[0134] The platelet lysate of the present invention refers to the lysate of platelets differentiated in vitro according to the present invention. Specifically, the platelet lysate of the present invention includes lysates of ESC / iPSC-derived and / or HSC-derived platelets. In a specific embodiment, hematopoietic stem cells are intermediate products of the 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 ESC / iPSC-derived platelets. In a specific embodiment, the lysates of platelets differentiated from ESC / iPSC in vitro and the lysates of platelets directly differentiated from HSC in vitro are comparable. In another preferred example, the comparability means comparable performance in improving skin aging.

[0135] In another preferred example, the preparation of the platelets comprises the following steps:

[0136] (O1) Culturing ESC / iPSC cells in mTesR1 medium containing Y27632 for 12 - 24 h; then continuing to culture in mTesR1 medium without Y27632 until the confluence reaches 70% - 80%.

[0137] (O2) Continuing to culture the cells obtained in step (O1) in StageI medium (STEMdiff TM APEL TM 2 medium, 30 ng / mL of BMP4, 50 ng / mL of VEGF, 25 ng / mL of activin A, 1.5 μM of CHIR-99021, 1% PS) for 2 days to obtain mesoderm-stage cells;

[0138] (O3) Continuing to culture the cells obtained in step (O2) in StageII medium (StemSpan TM -ACF Erythroid expansion medium, 30 ng / mL of BMP4, 50 ng / mL of VEGF, 50 ng / mL of bFGF, 1% PS) for 5 days to obtain hemogenic endothelium and hematopoietic stem and progenitor cell-stage cells;

[0139] (O4) The cells obtained in step (O3), in StageIII medium (STEMdiff TM APEL TMCultured in 2 medium, 50 ng / mL of TPO, 50 ng / mL of SCF, 25 ng / mL of Flt3, 10 ng / mL of IL3, 10 ng / mL of IL6, 5% of PFHM-II, 100 μg / mL of PVA, 1% PS for 7 days to obtain megakaryocytes;

[0140] (O5) The cells obtained in step (O4) were cultured in StageIV medium (STEMdiff TM APEL TM 2 medium, 50 ng / mL of TPO, 50 ng / mL of SCF, 5% of PFHM-II, 1% PS) for another 4 - 6 days to obtain platelets;

[0141] (O6) Lysed the platelets to obtain the platelet lysate.

[0142] In another preferred example, the lysis refers to using a homogenizer to lyse the provided platelets to obtain a lysate.

[0143] In another preferred example, the lysis was performed by repeatedly freezing and thawing the provided platelets to obtain a lysate.

[0144] In another preferred example, the lysis further includes ultrafiltration of the lysate.

[0145] In another preferred example, the lysis further includes centrifugation and filtration of the lysate.

[0146] In another preferred example, the centrifugation speed is 5000 - 8000 g.

[0147] In another preferred example, the centrifugation time is 20 - 40 min.

[0148] In another preferred example, the filtration is performed using a 0.22 μm filter and the filtrate is saved.

[0149] Skin injury repair

[0150] Skin injuries can be classified into two categories according to whether there is a wound communicating with the outside world. When the skin or mucosa of the injured part is ruptured, the wound communicates with the outside world, and there is tissue fluid exudation or blood flowing out of the wound, it is called an open injury, such as abrasions, stabbings, etc.; when the skin or mucosa of the injured part is intact, there is no wound communicating with the outside world, and the bleeding after injury accumulates in the tissue, it is called a closed injury, such as sprains of joint ligaments, muscle strains, etc.

[0151] Skin injury can be described as an interruption of cell continuity. Once it occurs, it will lead to the following results: loss of all or part of organ function, initiation of sympathetic nerve stress response, bleeding or bleeding and blood coagulation, bacterial contamination of the wound and / or cell death.

[0152] Skin injury repair occurs mainly in two ways, namely regeneration and fibrosis.

[0153] Regeneration refers to the replacement of damaged tissues with the same type of cells to complete the repair, which is characterized by the epidermal layer. Fibrosis, on the other hand, involves the repair of fibrous connective tissue, especially the formation of scar tissue, which is characterized by the wound healing through fibrosis and scar formation.

[0154] The two repair methods often coexist. Regeneration is the repair by the same type of cells around the injury. If the original tissue structure and function are completely restored, it is called complete regeneration. Tissue fibrosis or scar repair is common in tissues with relatively weak or lacking regenerative ability. When there is a defect, it cannot be repaired by the original tissue regeneration, but is filled by granulation tissue to form a scar.

[0155] Skin injury is different from the efficacy in the field of skin care products such as improving skin aging. The repair of skin injury has higher requirements for the cell migration ability. Specifically, skin injury repair requires an environment or condition that is conducive to the migration of cells to the wound site, promotes the efficient proliferation ability and vitality of the migrated cells, and has a low level of inflammatory factors in the cells, thus reducing the possibility of inflammation.

[0156] HDF cells

[0157] HDF cells (human dermal fibroblasts) are an important cell type present in the human dermis and have a variety of important physiological and pathological functions. They are mainly distributed in the human dermis and can be obtained through various methods, such as resection of surgical tissues, biopsies, liver aspiration, or reprogramming from peripheral blood mononuclear cells. HDF cells can be obtained in large quantities through culture and passage.

[0158] Dermal fibroblasts are responsible for producing the extracellular matrix that forms the skin connective tissue and play a key role in the wound healing process.

[0159] Specifically, dermal fibroblasts are crucial in wound healing and promote healing through proliferation and migration, synthesis of extracellular matrix, angiogenesis, and immune regulation.

[0160] Cell migration, cell proliferation, and cell invasion are different. Cell proliferation is the proliferative behavior of cells through division. Cell migration refers to the process by which cells move from one location to another, often verified by cell scratch. In normal physiological processes, cell migration is necessary, such as in tissue repair and development. In injury repair, cell migration usually means that the cells at the scratch edge will gradually enter the blank area to heal the "scratch". Cell invasion often refers to the ability of cells to enter the surrounding normal tissue, often verified by Transwell invasion assay.

[0161] The main advantages of the present invention include:

[0162] (a) The present invention provides an application of a lysate of in vitro differentiated platelets in skin injury repair. The lysate can effectively promote the migration of HDF cells to the wound site, significantly improve the viability of HDF cells, and promote the proliferation of HDF cells.

[0163] (b) At the molecular level, the in vitro differentiated platelet lysate provided by the present invention optimizes the efficiency of each differentiation stage from iPSC or CD34+ cells to megakaryocytes and then to the maturation of functional platelets, can effectively reduce the inflammatory factors produced by HDF, limit matrix degradation, and significantly improve the collagen synthesis ability of HDF.

[0164] (c) The lysate of the present invention does not contain plasma components, does not cause immune reactions caused by plasma, and also has the characteristics of high batch stability and can be stably stored. Therefore, it has high safety and stable effects when applied to skin injury repair.

[0165] (d) The lysate of the present invention can be obtained by in vitro differentiation and lysis of cells such as iPSC or CD34+. It has rich sources and is convenient for large-scale production, which can effectively reduce costs and improve economic benefits when used for skin injury repair.

[0166] (e) The present invention provides a method for repairing skin damage based on the platelet lysate of the present invention. This method is not limited by the extraction of autologous platelets, has simple and convenient steps, and is easy to apply.

[0167] The following further elaborates the present invention in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions noted in the following embodiments are usually in accordance with conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or in accordance with the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts.

[0168] Example 1: Preparation of platelet lysate (hPL).

[0169] The platelet lysate of the present invention includes the lysate of platelets produced by in vitro differentiation of human pluripotent stem cells ESC / iPSC cells and the lysate of platelets produced by differentiation of hematopoietic stem cells (HSC). The preparation methods of the platelet lysate of the present invention and human platelet lysate are shown as follows respectively.

[0170] For the in vitro differentiation of human pluripotent stem cells ESC / iPSC cells into platelets:

[0171] Culture ESC / iPSC cells in a culture dish coated with Matrigel (a soluble preparation of Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells), Vitronectin, or Laminin521 without feeder cells, and culture and expand them with stem cell medium (mTeSR1).

[0172] After single-cell passage of 1.0x10 5 ESC / iPSC cells per well for 24 hours, use StageI medium (STEMdiff TM APEL TM 2 medium + BMP4 (30 ng / mL) + VEGF (50 ng / mL) + Activin A (25 ng / mL) + CHIR-99021 (1.5 uM) + 1% PS) to culture for 2 days for mesoderm differentiation without changing the medium in the middle.

[0173] Culture the obtained mesoderm-stage cells with StageII medium (StemSpan TM -ACF Erythroid expansion medium + BMP4 (30 ng / mL) + VEGF (50 ng / mL) + bFGF (50 ng / mL) + 1% PS) for 5 days for the differentiation culture of hematopoietic endothelium and hematopoietic stem and progenitor cells, and change the medium every day.

[0174] On the 7th - 12th day, cells will be suspended in the supernatant. When changing the medium, centrifuge at 400 g for 3 minutes to discard the supernatant, and resuspend the precipitate with 2 ml of Stage III medium (STEMdiff TM APEL TM 2 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) and then add it to the original well plate.

[0175] On the 12th day, the suspended cells in the supernatant and the adherent cells in the lower layer were treated separately. The suspended cells in the supernatant were centrifuged at 400 g for 3 min and collected into an ultra-low attachment 6-well plate, and still cultured with 2 mL of Stage III medium until the 14th day without changing the medium in the middle; the adherent cells in the lower layer were digested with 1 mL of collagenase IV in the culture at 37 °C for 30 min, and the bottom of the well plate was gently blown to dissociate the cells. The suspension was collected into a 15 mL centrifuge tube, 1 mL of DMEM (high glucose, pyruvate) was added and mixed evenly, centrifuged at 400 g for 3 min, the supernatant was discarded, 1 mL of Trypsin-EDTA was added to the centrifuge tube, placed in an incubator at 37 °C for 3 min, and then 2 mL of Trypsin-EDTA digestion termination solution (1 mL of DMEM high glucose + 1 mL of fetal bovine serum) was added. After mixing, it was centrifuged at 400 g for 3 min, the supernatant was discarded, and then 2 mL of Stage III medium was added to resuspend the cells, and then inoculated into a 6-well plate pre-coated with Matrigel at a ratio of 1:1. 10 μM Y27632 was required on the first day, and when changing the medium on the 13th day, the supernatant was discarded by centrifugation at 400 g for 3 min, and the precipitate was resuspended with 2 mL of Stage III medium and then added to the original well plate.

[0176] On the 14th day, all suspended cells (automatically suspended and suspended cells after digestion) were collected by centrifugation at 400 g for 3 min, and cell counting was performed. Take 1×10 5 cells to measure CD34 / CD41. The total amount of suspended cells differentiated from iPS in a single well of a 6-well plate was 4×10^6 - 8×10^6, the cell viability was more than 80%, and CD34 + / CD41 + was 50% - 80%. The obtained cells were differentiated and cultured with Stage IV medium (STEMdiff TM APEL TM 2 medium + TPO (50 ng / mL) + SCF (50 ng / mL) + PFHM-II (5%) + 1% PS) until the 18th day, and a large number of platelets could be obtained.

[0177] For the differentiation of platelets from hematopoietic stem cells (HSC): CD34 + cells were cultured with differentiation medium I (StemSpan TM SFEMII medium + IL-3 + IL-6 + SCF + TPO + FLt) for 7 days, and semi-medium change was performed with differentiation medium I every 3 days to obtain megakaryocyte progenitor cells (MKP); the obtained MKP cells were cultured in differentiation medium II (IMDM + N2B27 + NEAA + glutamic acid + ITS + ascorbic acid + IL-6 + SCF + TPO) for 5 days; then continuously cultured at 39 °C and 5% CO2 for 5 - 7 days, and a large number of platelets could be obtained.

[0178] Use a homogenizer to lyse the differentiated platelets. After centrifuging the lysate at 6000g for 30 min in a high-speed refrigerated centrifuge, filter it through a 0.22 μm filter head, and aliquot and store it in a -80 °C refrigerator for later use.

[0179] In human platelet lysate, the platelets are derived from the blood of healthy donors.

[0180] Specifically, collect an appropriate amount of healthy donor blood using a sodium citrate blood collection tube. Obtain platelet-rich plasma (PRP) through a two-step centrifugation method (① centrifuge at 200g for 10 min at room temperature, and retain the upper plasma layer; ② centrifuge the plasma layer at 200g for 10 min at room temperature, discard about 3 / 4 of the supernatant, and resuspend the precipitate in the remaining liquid). Approximately 700 μL of PRP can be obtained from 5 mL of whole blood. The prepared PRP should ensure that the platelet concentration is greater than 10 9 cells / mL.

[0181] Freeze the PRP at -80 °C for 24 h and then thaw it at room temperature. Repeat this process 3 times to lyse the platelets. The lysed plasma is ultrafiltered to remove macromolecular proteins, obtaining a high-concentration human platelet lysate. Filter the human hPL through a 0.22 μm sterile filter, aliquot and store it. Store it short-term at 4 °C and long-term at -20 / -80 °C.

[0182] Example 2: Application of the hPL of the present invention in skin injury.

[0183] Dermal fibroblasts are cells located in the dermis of the skin. They play a crucial role in wound healing by proliferating, migrating to the injured site, and filling the wound with newly synthesized matrix. To explore the potential of the hPL of the present invention in wound healing, this example detected its effect on human dermal fibroblasts (HDF).

[0184] The proliferation of HDF is an important process in wound healing repair. The effect of hPL on the cell viability of HDF was evaluated by the CCK-8 assay, and the effect of hPL on the proliferation potential of HDF was evaluated by the Edu staining method.

[0185] The results are as Figure 1 shown. The results show that hPL significantly increased the cell viability in HDF.

[0186] As Figure 1 shown in A, compared with the cells cultured in 10% FBS, the cell viability of HDF cells cultured in 10% hPL increased by approximately 42%, reaching 142.10 ± 5.99%, indicating that hPL has the potential to promote the proliferation of HDF cells. To further examine this effect, EdU staining analysis was performed on the cells, and the results are as Figure 1As shown in Figure B. The percentage of EdU-positive cells in the 10% FBS group was 20.69 ± 5.49%, while it was significantly increased in the hPL treatment group, with 37.73 ± 5.28% of the cells incorporating EdU, confirming that hPL treatment significantly enhanced the proliferation of HDF cells.

[0187] The migration of HDF towards the wound is also an important part of the healing and repair process. In this example, a transwell assay was used to detect the effect of hPL on the migration ability of HDF.

[0188] The results are shown in Figure 2 Figure A and Figure 2 Figure B. The lower chamber of the transwell model contained 10% FBS or 10% hPL medium. The results showed that the migration of HDF towards the hPL chamber was significantly increased compared to the FBS chamber, indicating that hPL enhanced the migration ability of HDF.

[0189] To comprehensively evaluate the repair effect of hPL on skin injury, in this example, a scratch model was used to establish an in vitro skin healing model to evaluate the repair effect of hPL on skin injury.

[0190] The results are shown in Figure 3 Figure A and Figure 3 Figure B. After 24 hours of treatment, the cell-free area in the hPL treatment group was significantly smaller than that in the 10% FBS control group. The quantitative results showed that the remaining wound area in the hPL group was approximately 28%, much lower than that in the control group. This indicates that the hPL of the present invention effectively stimulates the proliferation and migration of HDF cells at the wound site, thus accelerating wound healing.

[0191] Finally, real-time fluorescence quantitative reverse transcription polymerase chain reaction was used to detect the changes in the ability of the hPL of the present invention to produce inflammatory factors and collagen synthesis in HDF.

[0192] The results are shown in Figure 4 Figure. The results showed that in the HDF cells treated with the hPL of the present invention, the levels of IL-6, IL-8, IL1α-1, and MMP1 were all significantly downregulated, while the level of type I collagen COL1A1 was significantly upregulated, indicating that hPL can reduce inflammation, limit matrix degradation, and promote collagen synthesis to support wound healing.

[0193] Example 3: Growth factor content of the hPL of the present invention and hPL derived from peripheral blood

[0194] This example explored the differences in growth factors between the hPL of the present invention and hPL derived from peripheral blood.

[0195] The results are shown in Figure 5As shown, the results indicate that in the hPL induced by the present invention, the content of bFGF has increased by 2 times compared to hPL derived from peripheral blood, and the content of VEGF has increased by 2 times.

[0196] Since bFGF and VEGF are key growth factors closely related to wound healing, the increase in their content significantly enhances the role of the hPL of the present invention in epithelial regeneration, angiogenesis, and granulation tissue formation, demonstrating higher therapeutic potential.

[0197] All documents mentioned in the present invention are incorporated herein by reference as if each document was individually incorporated by reference. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims

1. Use of a lysate of in vitro differentiated platelets, characterized in that, For preparing a composition for the following uses: (Z1) Promoting cell migration to skin injury sites; and / or (Z2) Promoting skin injury repair.

2. The use according to claim 1, characterized in that, The promotion of skin injury repair includes: (Z2-1) Promoting skin wound healing and / or promoting skin function repair; (Z2-2) Promoting cell migration to skin injury sites, promoting cell proliferation, promoting the synthesis of matrix for filling wounds, or a combination thereof; and / or (Z2-3) Significantly downregulating the levels of inflammatory factors, significantly downregulating the levels of matrix degradation, significantly increasing the levels of collagen, or a combination thereof.

3. The use according to claim 1, characterized in that, The lysate of the in vitro differentiated platelets has the following characteristics: (i) The level of bFGF is significantly increased; and / or (ii) The level of VEGF is significantly increased.

4. The use according to claim 1, characterized in that, The in vitro differentiated platelets refer to platelets obtained by in vitro differentiation of a platelet in vitro differentiation culture system, and the culture system includes StageI medium, StageII medium, StageIII medium, and / or StageIV medium. Among them, the StageI medium includes a basal medium, BMP4, VEGF, activin A, CHIR-99021; The Stage II medium includes StemSpan TM -ACF Erythroid expansion medium, BMP4, VGEF, bFGF; The StageIII medium includes a basal medium, TPO, SCF, Flt3, IL3, IL6, PFHM-II, PVA; The StageIV medium includes a basal medium, TPO, SCF, PFHM-II.

5. The use according to claim 4, characterized in that, The differentiation includes the following steps: (S1-1) Culturing and expanding ESC / iPSC cells with a stem cell medium; (S1-2) Performing mesoderm differentiation culture on the cells obtained in step (S1-1) with the StageI medium; (S1-3) Performing hemogenic endothelium and hematopoietic stem and progenitor cell differentiation culture on the cells obtained in step (S1-2) with the StageII medium; (S1-4) Continuing to culture the cells obtained in step (S1-3) with the StageIII medium to obtain megakaryocytes; (S1-5) Continuing to culture the cells obtained in step (S1-4) with the StageIV medium to obtain platelets.

6. A culture system for obtaining platelet lysate that promotes cell migration to skin injury sites, characterized in that, The culture system includes StageI medium, StageII medium, StageIII medium, and / or StageIV medium. Among them, the StageI medium includes a basal medium, BMP4, VEGF, activin A, CHIR-99021; The Stage II medium includes StemSpan TM -ACF Erythroid expansion medium, BMP4, VGEF, bFGF; The StageIII medium includes a basal medium, TPO, SCF, Flt3, IL3, IL6, PFHM-II, PVA; The StageIV medium includes a basal medium, TPO, SCF, PFHM-II.

7. A method for preparing platelet lysate that promotes cell migration to skin injury sites, characterized in that, It includes the following steps: (S1) Providing the culture system according to claim 6 to provide in vitro differentiated platelets; (S2) Lysing the platelets to obtain the platelet lysate as described above.

8. An in vitro differentiated platelet lysate for promoting cell migration to skin injuries, characterized in that, The platelet lysate has the following characteristics: (i) The bFGF level X1 of the platelet lysate, compared with the bFGF level X0 of the human platelet lysate, satisfies X1 / X0 ≥ 1.2, more preferably X1 / X0 ≥ 1.5, and most preferably X1 / X0 ≥ 2; and / or (ii) The VEGF level Y1 of the platelet lysate, compared with the VEGF level Y0 of the human platelet lysate, satisfies Y1 / Y0 ≥ 1.2, more preferably Y1 / Y0 ≥ 1.5, and most preferably Y1 / Y0 ≥ 2.

9. A composition for promoting cell migration to a skin injury, characterized in that, The composition comprises the platelet lysate as claimed in claim 8.

10. A kit, characterized in that, The kit as described above comprises: (a) The platelet lysate as claimed in claim 8 and / or the composition as claimed in claim 9; (b) Optionally, a disinfection reagent.

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