WO2018223830A1 - 一种人间充质干细胞外泌体冻干粉的制备方法、使用方法 - Google Patents

一种人间充质干细胞外泌体冻干粉的制备方法、使用方法 Download PDF

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WO2018223830A1
WO2018223830A1 PCT/CN2018/087402 CN2018087402W WO2018223830A1 WO 2018223830 A1 WO2018223830 A1 WO 2018223830A1 CN 2018087402 W CN2018087402 W CN 2018087402W WO 2018223830 A1 WO2018223830 A1 WO 2018223830A1
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mesenchymal stem
human mesenchymal
stem cell
exosomes
lyophilized powder
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黄兵
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广州白泽科技有限公司
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    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0021—Intradermal administration, e.g. through microneedle arrays, needleless injectors
    • A—HUMAN NECESSITIES
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    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
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    • C12N2501/20—Cytokines; Chemokines
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    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/24—Interferons [IFN]

Definitions

  • the invention relates to the field of stem cell technology, in particular to a preparation method and a use method of a human mesenchymal stem cell exosome lyophilized powder.
  • Mesenchymal stem cells are a group of mesenchymal pluripotent stem cells with multi-directional differentiation potential.
  • mesenchymal stem cells exist in almost all tissues and organs, such as bone marrow, liver, spleen, lung and kidney, gastrointestinal, Skeletal muscle, peripheral blood, fat, ligament, placenta, umbilical cord, cord blood, pulp and skin.
  • Human umbilical cord mesenchymal stem cells are a kind of stem cells derived from neonatal umbilical cord with self-renewal and multi-directional differentiation potential. These cells are rich in source, easy to collect and transport, and proliferate compared with mesenchymal stem cells from other sources.
  • mesenchymal stem cells can secrete some different sizes of vesicles, such as small vesicles (80-1000 nm) and exosomes (30-150 nm), in addition to the above-mentioned cytological features.
  • the secretion of exosomes was first discovered by Johnstone et al. in 1987 to study the maturation of reticulocytes. In the supernatant of cultured sheep reticulocytes in vitro, centrifugation of 60 nm in diameter can be collected after centrifugation at 100,000 g for 90 minutes.
  • the vesicles and vesicles contain the membrane structure released during the maturation of reticulocytes.
  • the surface is rich in lipids such as cholesterol, sphingomyelin and ceramide.
  • lipids such as cholesterol, sphingomyelin and ceramide.
  • the vesicle is a nanometer-sized membranous vesicle that is released into the extracellular environment by fusion of multivesicular bodies (MVBs) of eukaryotic cells with the cell membrane, and can be secreted by various types of cells. It contains different kinds of proteins (such as growth factors, cytokines, transcription factors and enzymes), lipids (cholesterol cholesterol, sphingomyelin, and ceramide), RNAs such as LincRNA, mRNAs and microRNAs, signaling molecules, etc.
  • MVBs multivesicular bodies
  • Biologically active substances which are more easily fused with the cell membrane of adjacent cells, selectively deliver biologically active substances to recipient cells, transmit information between different cells, regulate signal transduction between cells, and exert various biology.
  • exosomes have more potential biological functions in addition to removing excess proteins in the cells.
  • exosomes as an information complex can transport cytoplasm and play a key role in intercellular signal transduction. The role of this signal pattern is different from the classic mode.
  • the exosomes are not only a liposome, but also a nanocomposite with multiple biological activities.
  • exosomes usually secrete more when cells grow vigorously, and the principles and regulatory mechanisms of their formation are still unclear. Therefore, how to increase the quantity and quality of exosomes is a new challenge.
  • the separation of exosomes is mostly carried out by ultracentrifugation, magnetic bead immunocapture, precipitation filtration and kits.
  • Ultracentrifugation Time-consuming and labor-intensive, often takes 8-30 hours; can only process up to 6 samples at a time; requires a large amount of starting material; the yield is not high (1010/ml). A supercentrifuge is required.
  • Magnetic bead immunocapture using surface markers specific to the exosomes (such as CD63, CD9, 81 proteins), and binding to the exosomal vesicles with magnetic beads coated with anti-marker antibodies The body is adsorbed and separated.
  • the magnetic bead method has the advantages of high specificity, simple operation, and no influence on the morphological integrity of the exosomes, but the efficiency is low, and the biological activity of the exosomes is easily affected by pH and salt concentration, which is not conducive to downstream experiments and is difficult to be widely popularized.
  • Exosomes Isolation Kit The exosomes isolation kit separates exosomes from body fluids, blood, urine, and cells by centrifugation, and then purified through two columns to remove albumin and the like to obtain exosomes.
  • centrifugation The exosomes isolation kit separates exosomes from body fluids, blood, urine, and cells by centrifugation, and then purified through two columns to remove albumin and the like to obtain exosomes.
  • albumin and the like to obtain exosomes.
  • the present invention provides a method and a method for preparing a human mesenchymal stem cell exosomal lyophilized powder.
  • the concentrated culture solution after refrigeration is prepared as a lyophilized powder, and a composite liquid is added to the lyophilized powder for storage.
  • the specific operation method of step 2) selecting the third to fifth generation human mesenchymal stem cells with strong proliferative ability, and placing them in a 37 ° C incubator with an oxygen content of 21%, using DMEM/F12 containing 5% FBS.
  • the culture medium is cultured until the cell confluence is 60 to 70%, and culture is carried out for 3 to 5 days under culture conditions for promoting synthesis and secretion of exosomes, and then the supernatant is collected.
  • the culture condition for promoting exosome synthesis and secretion is: serum-free starvation medium, while adding a promoting factor to the starvation medium, and the oxygen content in the starvation medium is 1% to 5 %, pH value is 6.5-7.
  • the promoting factor is: 1-100 ng/ml gamma interferon, 1-10 mg/ml polylysine, 1-6 ng/ml MG-CSF, 2-5 ng/ml IL-6, 1- 5 ng/ml HGF, 1-10 mM ATP, 2.8-3.8 mmol (14 mg/dl) calcium ion.
  • the starvation medium is a medium having the application number CN2017100038074.
  • the concentration method in the step 3) centrifuging the product of the step 2) by using a centrifuge to remove cell debris and bacteria, filtering, and filtering the filtrate by ultrafiltration to obtain a concentrated liquid, and adding the concentrated liquid to the concentrated liquid.
  • the vehicle was chilled at -80 °C.
  • the excipient is mannitol in an amount of 5 g of mannitol per 100 ml of the concentrate.
  • the culture of human mesenchymal stem cells in step 1) uses a serum-free medium using the medium of application number CN2017100038074.
  • the invention further proposes a method for using a human mesenchymal stem cell exosomal lyophilized powder, which uses a microneedle, a needle roller or a water light needle to introduce the exosomes into the deep layer of the epidermis and the dermis layer.
  • a technical scheme for inducing a large number of mesenchymal stem cells to synthesize and secrete functional exosomes includes using physicochemical properties and biological properties that change various culture conditions, such as reducing oxygen content in cell culture, Use serum-free medium for starvation to increase the concentration of ATP and calcium ions in the medium, change the pH of the medium and add some stimulating factors such as gamma interferon, polylysine, MG-CSF (macrophage) And granulocyte colony-stimulating factor), IL-6 (interleukin-6), HGF (liver cell growth factor).
  • physicochemical properties and biological properties that change various culture conditions, such as reducing oxygen content in cell culture, Use serum-free medium for starvation to increase the concentration of ATP and calcium ions in the medium, change the pH of the medium and add some stimulating factors such as gamma interferon, polylysine, MG-CSF (macrophage) And granulocyte colony-stimulating factor),
  • the exosomes of umbilical cord mesenchymal stem cells can be recovered quickly, easily and on a large scale, and the recovery concentration can reach 10 13 /ml, which is much larger than the recovery rate and quantity of other technologies. It enables the application of stem cell paracrine secretions, avoiding the shortcomings of the currently used time-consuming and labor-saving ultracentrifugation and low-dose kit purification techniques, using ultrafiltration freeze-drying method for large-scale concentration, long-term storage and simple Transporting biological active ingredient exosomes;
  • exosomes are much smaller than the cells, only 30-150 nm in size, but the gap between the surface cells of the skin is only about 40 nm, and they are difficult to introduce and penetrate the skin.
  • the novelty of the present invention lies in the initiative to successfully use the microneedle, the needle roller and the water-light needle to perforate the skin first, and as a feasible method for opening the barrier channel of the human skin, the bioactive macromolecule such as various cytokines and sub-efficiencies can be efficiently utilized.
  • Organelles, such as exosomes are introduced into the epidermal or dermal layers of the skin, greatly traversing the skin barrier that has been plaguing people, allowing macromolecules and larger subcellular organomers to truly interact and interact with various cells in the skin. , exerting their biological and physiological functions to improve the cortex, uniformity and whitening complexion.
  • a method for preparing human mesenchymal stem cell exosomal lyophilized powder comprises the following steps:
  • the concentrated culture solution after refrigeration is prepared as a lyophilized powder, and a composite liquid is added to the lyophilized powder for storage.
  • the umbilical cord mesenchymal stem cells were cultured at 37 ° C under 5% CO 2 , and the whole amount was changed for the first time after 36 hours of culture, and then changed every three or four days.
  • the medium was the medium using the application number CN2017100038074.
  • the cell growth and morphological changes were observed under an inverted microscope. When the cells were grown to 80%-90% of the bottom of the flask, the cells were digested with 0.05% trypsin and subcultured.
  • CD90 monoclonal antibody was incubated in the dark for 60 min, the cells were washed with PBS, the unbound antibody was washed away, centrifuged, the supernatant was discarded, the isotype control monoclonal antibody was used as the negative control group, and the flow cytometry was used to detect each marker. Expression, the results are shown in Figure 1, demonstrating that these cells are indeed mesenchymal stem cells, not other cells.
  • the third to fifth generation human umbilical cord blood mesenchymal stem cells with strong proliferative ability were selected and cultured in a normal oxygen content 37 ° C incubator in DMEM/F12 medium containing 5% FBS until the cell confluence was 60. -70%, the culture conditions for promoting exosome synthesis and secretion are switched to: serum-free starvation medium using medium No.
  • 3mmol (14mg/dl) calcium ion cultured for 3 days, then collect 500mL of culture supernatant, centrifuge at 2000r/min for 30min to remove the cells, and then filter through 0.45 ⁇ m sterile filter to 50mL specification ultrafiltration centrifuge tube, 2000r After centrifugation for 30 min for 30 min, 100 ml of a concentrate containing exosomes was obtained, and 6 g of excipient mannitol was added to each 100 ml of the concentrate, and the mixture was stored at -80 ° C for 1 ml per ml.
  • the third to fifth generation human umbilical cord blood mesenchymal stem cells with strong proliferative ability were selected and cultured in a normal oxygen content 37 ° C incubator in DMEM/F12 medium containing 5% FBS until the cell confluence was 60. -70%, the culture conditions for promoting exosome synthesis and secretion are switched to: serum-free starvation medium using medium No.
  • the oxygen content in the starvation medium is 5%, pH The value was 7, and the exosome promoting factor was added: 20 ng/ml gamma interferon, 5 mg/ml polylysine, 4 ng/ml MG-CSF, 3 ng/ml IL-6, 5 ng/ml HGF, 6 mM ATP.
  • 3.5mmol (14mg/dl) calcium ion cultured for 3 days, then collected 500mL of culture supernatant, centrifuged at 2000r/min for 30min to remove the cells, and then filtered through a 0.45 ⁇ m sterile filter into a 50mL ultrafiltration centrifuge tube.
  • the main observation indicators of exosomes 1 TEM observation of exosomes. 2Western Blotting detects the expression level of marker proteins. 3 ELISA detects the content of related protein polypeptides. 4 flow cytometry to identify exosome surface related antigens.
  • Exosomal morphology Exosomes-like vesicles were isolated from different media of umbilical cord mesenchymal stem cells (UC-MSCs). The exudate vesicles from three different cell culture media were observed under transmission electron microscope.
  • UC-MSCs umbilical cord mesenchymal stem cells
  • exosome protein concentration The total protein content of exosomes was detected by ND-2000 ultra-micro UV-visible spectrophotometer using BCA protein quantification kit. Parameters: Specimen 0.1 mL + reagent 2.0 mL; reaction time 30 min; reaction temperature 37 ° C; wavelength 562 nm. The results showed that 50 ml of the culture medium of 10% FBS DMEM/F12 medium and ExoQuick-TC reagent method obtained the exosome protein concentration of (872 ⁇ 30) ug/ml. 50 ml of an exosome solution derived from a 5% FBS low serum medium-2 was calculated to be (1.57 ⁇ 0.27) mg/ml in terms of protein concentration. A serum-free culture supernatant of human umbilical cord blood mesenchymal stem cells can be isolated to obtain about 50 ml of exosomal suspension, and the protein concentration thereof is (2.66 ⁇ 0.42) mg/ml.
  • ELISA The culture medium of the mesenchymal stem cells treated under the above special conditions was collected, centrifuged at 2000 ⁇ g for 30 min, and the supernatant was collected. The absorbance of each group of samples at a specific wavelength was measured by ELISA according to the instruction, and the supernatant was calculated according to the standard curve. , IL-11, TNF, HGF, bFGF, IGF-1, GM-CSF, TGF- ⁇ , TPO, VEGF, LIF, EGF, KGF, IFN- ⁇ and IFN- ⁇ concentrations. The ELISA results (Fig.
  • control exosomes group (10% serum medium and conventional culture conditions) were compared with the secretion amount of the above cytokines in the exosome supernatant of the special culture conditions of the present invention.
  • the difference was very significant, and the P values of the 13 cytokine concentrations in the figure were less than 0.01, indicating that the difference was statistically significant.
  • Flow cytometry for detection of exosome surface markers derived from human umbilical cord blood mesenchymal stem cells 20 ul of exosome-containing concentrate was diluted with 1 ml of PBS, and the resulting suspension was incubated with the relevant monoclonal antibody, and homologous IgG was used. Negative control, detected by flow cytometry.
  • exosome surface markers derived from human umbilical cord blood mesenchymal stem cells Flow cytometry results showed that exosome expression of exosomes of human umbilical cord blood mesenchymal stem cells derived from three different media sources Surface adhesion molecules CD90, CD73, CD105 of CD9, CD63, CD81, Calnexin and Flotillin-1 and mesenchymal stem cells. The results are shown in Table 1.
  • the medium-1 in Table 1 is 10% FBS DMEM/F12 medium.
  • Base-2 is 5% FBS DMEM/F12 low serum medium
  • medium-3 is the medium of the second embodiment of the present invention.
  • exosome-specific molecular markers The vesicles extracted from different culture supernatants of umbilical cord mesenchymal cells express exosome-specific molecular markers CD63 and Flotillin-1, further demonstrating these vesicular forms.
  • the substance may be an exosome. It is inferred from the above results that the application of special culture conditions has greatly promoted the synthesis and secretion of exosomes of UC-MSCs, and the exosomes and activities in the supernatant of UC-MSCs culture medium by simple and easy ultrafiltration freeze-drying technique. Large-scale preparation of ingredients.
  • the Westenblot assay ( Figure 6) showed that exosomes from different culture supernatants expressed surface marker proteins.
  • the lyophilized powder component is: the main material is the above-mentioned exosome concentrate and the excipient mannitol, the mass ratio of the two is 95:5, and the exosome lyophilized powder is prepared as follows: The exosomal supernatant in the -80 degree refrigerator was equilibrated at -60 degrees for 1 hour, and then quickly loaded into the dry box of the lyophilizer, at which time the temperature of the lyophilizer dry box was controlled at about -48 °C ( The product freezes below the eutectic point), and then the temperature of the condensing cylinder of the lyophilizer is rapidly reached -50 to 55 ° C.
  • the vacuum of the lyophilizer dry box is vacuumed to a vacuum of 15 Pa to dry the product and control the shelf temperature.
  • time 8 lasts for a short time; finally the product is desorbed and dried, the temperature is slowly heated to -20 ° C at a constant rate within 10 hours, kept for 2 hours, and then heated to 5 ° C at a constant rate within 5 hours.
  • the degree of vacuum is less than 10 Pa, so that the water content is less than 3%, and the package is put into storage after passing the test (Fig. 7).
  • the prepared lyophilized powder has no defect appearance, the surface is flat, the volume is basically equal to the volume when frozen, the color is uniform, the solubility is good, the clarity is good, the stability is good, the pollution is not easy, and the shelf life is long.
  • UC-MSCs exosome supernatant lyophilized powder promotes proliferation of skin keratinocyte HeCat:
  • UC-MSCs were seeded in 24-well plates, and after 24 h, 1) PBS (as a control), 2) 60 ug/ml exosomes, 3) exosomes and 10 umol/LSB431542, one EGF/KGF
  • the inhibitor of the body is the exosomes + inhibition group.
  • the results showed that after 1, 3, 5, 7 and 9 days, the proliferative capacity of HeCat was significantly higher than that of the control group (P ⁇ 0.01).
  • this up-regulation of proliferation can be inhibited by an inhibitor of the EGF/KGF receptor (Fig. 8).
  • Cell proliferation was measured using Cell Counting Kit-8 (CCK-8, Dojindo, Japan). The curve was calculated and calculated based on the absorbance (A) value at 450 nm measured by a microplate reader.
  • UC-MSCs exosome supernatant lyophilized powder promotes improvement of human facial skin
  • the specific implementation steps are as follows: disinfect the skin with 75% medical alcohol, soak the microneedle in 75% medical alcohol disinfectant for about 15-30 minutes, and freeze the exosome supernatant lyophilized powder and exosomes resuscitation liquid 1: 3 After merging, apply to the skin of the face. Then, use the microneedles to roll from bottom to top in sequence, about 2 times for each part, and then wash the whole face in a rice shape. Then apply a special mask for the US plastic 40-60min, no need to clean. The skin's elasticity, pore size, moisture, wrinkles and skin tone were measured with a dermabrasion (CBS-807 Skin Analysis System) after 1, 3, 7, 15, and 30 days. The results are shown in Table 2. These indicators show significant improvement.

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Abstract

提供了一种人间充质干细胞外泌体冻干粉的制备方法，包括以下步骤：1)人间充质干细胞的培养、纯化及传代；2)诱导人间充质干细胞大量合成和分泌有功能的外泌体；3)超滤浓缩人间充质干细胞外泌体，制得浓缩培养液并进行冷藏；4)将冷藏后的所述浓缩培养液制备成冻干粉，向所述冻干粉中添加复合液进行保存。

Description

一种人间充质干细胞外泌体冻干粉的制备方法、使用方法 技术领域
本发明涉及干细胞技术领域，特别是指一种人间充质干细胞外泌体冻干粉的制备方法、使用方法。
背景技术
间充质干细胞是一群中胚层来源的具有多向分化潜能的多能干细胞，大量研究已经证明间充质干细胞存在于几乎所有的组织和器官，如骨髓、肝脏、脾脏、肺肾、胃肠、骨骼肌、外周血、脂肪、韧带、胎盘、脐带、脐血、牙髓和皮肤等组织中。人脐带间充质干细胞是一类来源于新生儿脐带，具有自我更新和多向分化潜能的干细胞，这类细胞与其他来源的间充质干细胞相比，具有来源丰富，易于采集和运输，增殖能力强，本身无异体排斥反应、不涉及伦理问题等诸多优点，近年来逐渐成为倍受人们关注的成体干细胞。目前研究显示，人脐带间充质干细胞可作为多种疾病治疗的细胞来源，修复多种原因引起的组织损伤，激活体内的休眠细胞，更新和替代衰老和死亡的细胞，但是长期移植入体内后，由于增殖分化和异体抗原的出现，或发生不良分化致瘤的缺点，限制了其在临床广泛应用。
近来研究发现间充质干细胞除了上述细胞学的特征外，还能向细胞外分泌一些不同大小的囊泡，如小囊泡(80-1000nm)和外泌体(30-150nm)等。外泌体的分泌最早是由Johnstone等在1987年研究网织红细胞的成熟过程中发现的，在体外培养羊网织红细胞的上清中经过100000g离心90分钟后可以收集到直径60纳米的囊性小泡，小泡中含有网织红细胞成熟过程中放出的胞膜结构，表面富含胆固醇、神经鞘磷脂、神经酰胺等脂类物质.Johnstone将这些纳米级的囊泡命名为外泌体(exosomes)。这种囊泡是一种由真核细胞的多泡内涵体(multivesicular bodies，MVBs)与细胞膜融合后释放到细胞外环境中的纳米量级的膜性小囊泡，可由多种类型细胞分泌，其内含有不同种类的蛋白质(如生长因子，细胞因子、转录因子和酶等)、脂质(胆固醇cholesterol，神经鞘磷脂sphingomyelin，和ceramide等)、RNAs如LincRNA，mRNAs和microRNAs、 信号分子等具有生物学活性的物质，较易与邻近细胞的细胞膜发生融合，将生物学活性物质选择性地递送至受体细胞，在不同细胞间进行信息传递，调节细胞间的信号传导，发挥多种生物学功能。研究还发现外泌体除了清除细胞内多余的蛋白质外，还具有更多潜在的生物功能，很多实验证实了外泌体作为信息复合体能够转运胞质并在胞间信号转导中起关键性的作用，这种信号模式与经典的模式不同，外泌体不仅是一个脂质体，还是一个具有多种生物活性的纳米复合体。有文献报道，移植间充质干细胞能够通过旁分泌有效生物学活性物质-外泌体，从而激活细胞的增殖和迁移，抵抗应急氧和自由基的损伤，防止细胞的凋亡，和修复组织的损伤等多种效果。可见，人脐带间充质干细胞来源的外泌体具有多种重要的生理功能，将其开发成为一种既具有间充质干细胞特点而又能克服其缺陷的新型治疗方式，是今后具有重要探讨价值的研发方向。
研究进一步发现外泌体通常在细胞生长旺盛时分泌较多，其形成的原理和调控机制尚不清楚。所以，如何提高外泌体的数量和质量乃是人们面临的新挑战。目前，外泌体的分离多采用超速离心、磁珠免疫捕获、沉淀过滤和试剂盒等方法。超速离心：耗时耗力，往往需要8-30个小时；每次最多只能处理6个样品；需要大量的起始材料；产量不高(1010/ml)。必备一台超速离心机。磁珠免疫捕获：利用外泌体表面特有的表面标记物(如CD63、CD9、81蛋白)，用包被抗标记物抗体的磁珠与外泌体囊泡孵育后结合，即可将外泌体吸附并分离出来。磁珠法具有特异性高、操作简便、不影响外泌体形态完整等优点，但是效率低，外泌体生物活性易受pH和盐浓度影响，不利于下游实验，难以广泛普及。外泌体分离试剂盒：外泌体分离试剂盒通过离心，从体液、血液、尿液、细胞中分离外泌体，再经过两次柱子纯化，去除白蛋白等从而获得外泌体。但不能进行规模化生产和相对成本较高。
所以，需要新的方法来提高外泌体分离回收的效率和规模，使之产业化用于医疗和大健康，是未来人类健康的一种全新的诊断、治疗和预后评估的新技术和新方法。再者，如何将这种全新的外泌体亚细胞器导入机体的皮肤作为抗衰老护肤美容的一种自然、安全和高效方法和技术也是人们关注的新问题。
发明内容
为了解决上述问题，本发明提出一种人间充质干细胞外泌体冻干粉的制备方法、使用方法。
本发明的技术方案是这样实现的：一种人间充质干细胞外泌体冻干粉的制备方法，包括以下步骤：
1)人间充质干细胞的培养、纯化及传代；
2)诱导人间充质干细胞大量合成和分泌有功能的外泌体；
3)超滤浓缩人间充质干细胞外泌体，制得浓缩培养液并进行冷藏；
4)将冷藏后的所述浓缩培养液制备成冻干粉，向所述冻干粉中添加复合液进行保存。
优选的，步骤2)的具体操作方法：选择增殖能力强的第3～5代人间充质干细胞，置于含氧量为21％的37℃培养箱中，用含有5％FBS的DMEM/F12培养基进行培养，直到细胞汇合度为60～70％时，改用促进外泌体合成和分泌的培养条件进行培养3～5天，后收集上清液。
优选的，所述促进外泌体合成和分泌的培养条件：无血清的饥饿培养基，同时向所述饥饿培养基中添加促进因子，且所述饥饿培养基中含氧量为1％～5％，pH值为6.5～7。
优选的，所述促进因子为：1-100ng/ml伽马干扰素，1-10mg/ml多聚赖氨酸，1-6ng/ml MG-CSF，2-5ng/ml IL-6，1-5ng/ml HGF，1-10mM ATP，2.8-3.8mmol(14mg/dl)钙离子。
优选的，所述饥饿培养基采用申请号为CN2017100038074的培养基。
优选的，步骤3)中浓缩方法：采用离心机对步骤2)的产物进行离心，去除细胞碎片以及细菌后，过滤，将滤液进行超滤离心，制得浓缩液，向所述浓缩液中添加赋形剂并置于-80℃进行冷藏。
优选的，所述赋形剂为甘露醇，添加量为每100ml浓缩液中添加甘露醇5g。
优选的，步骤1)中人间充质干细胞的培养采用无血清培养基，所述无血清培养基采用申请号为CN2017100038074的培养基。
本发明又提出一种人间充质干细胞外泌体冻干粉的使用方法，采用微针、滚针或水光针将外泌体导入表皮深层和真皮层。
本发明的有益效果：
1)诱导间充质干细胞大量合成和分泌有功能的外泌体的技术方案；它包括采用改变多种培养条件的理化性质和生物学特性来达到此目的，如降低细胞培养中氧的含量，采用无血清培养基的饥饿措施，提高培养基中ATP和钙离子的浓度，改变培养基的pH值和外加某种刺激因素如伽马干扰素、多聚赖氨酸，MG-CSF(巨噬和粒细胞集落刺激因子)、IL-6(白细胞介素-6)，HGF(肝脏细胞生长因子)。它们能调节和控制其它细胞的生长发育和增值分化，可以有效地诱导间充质干细胞最大限度的合成和分泌外泌体，有助于大规模地获取这些活性成分，便于直接地用于大健康的方方面面，如疾病的诊治和人体的美容整形；
2)采用了超滤和冻干技术联动，使脐带间充质干细胞的外泌体能快速、简便和大规模的回收，其回收浓度可达到10 13/ml，远大于其他技术的回收率和数量，使干细胞旁分泌物的应用成为可能，避开了目前常用的费时费力的超速离心和小剂量的试剂盒纯化技术的短板，采用超滤冷冻干燥法来大批量浓缩、长时间保存和简易运输生物有效成分外泌体；
3)外泌体比起细胞要小得多，只有30-150nm的大小，但是皮肤表层细胞之间的间隙只有40nm左右，它们导入和透过皮肤乃具有一定的困难。本发明的新颖性在于自主率先成功利用微针、滚针和水光针先将皮肤打孔，作为打开人体皮肤屏障通道的可行方法，能够高效地将生物活性大分子如各种细胞因子和亚细胞器如外泌体导入皮肤的表皮层或真皮层，从而极大地穿越了一直困扰人们的皮肤屏障，使大分子和更大的亚细胞器都能真正地和皮肤中的各种细胞接触和相互作用，发挥它们的生物学和生理学功能，达到改善皮质、均匀和美白肤色的功效。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其他的附图。
图1.脐带间充质干细胞表面标志物的流失细胞仪检测；
图2.不同培养条件下，脐带间充质干细胞分泌的外泌体的电镜照片；
图3.在本发明的特殊培养条件下，脐带间充质干细胞分泌的外泌体的大小分布情况；
图4.不同培养条件下，脐带间充质干细胞分泌外泌体量的比较；
图5.比较不同培养条件下，脐带间充质干细胞分泌的细胞因子量的差异；
图6.外泌体标志物的Westernbloting检测结果；
图7.脐带间充质干细胞外泌体冻干粉的外形；
图8.外泌体对皮肤角质细胞的生长的促进作用。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
一种人间充质干细胞外泌体冻干粉的制备方法，包括以下步骤：
1)人间充质干细胞的培养、纯化及传代；
2)诱导人间充质干细胞大量合成和分泌有功能的外泌体；
3)超滤浓缩人间充质干细胞外泌体，制得浓缩培养液并进行冷藏；
4)将冷藏后的所述浓缩培养液制备成冻干粉，向所述冻干粉中添加复合液进行保存。
人脐血间充质干细胞的培养、纯化及传代：
将脐带间充质干细胞培养在37℃，5％CO 2的条件下，培养36h后首次全量换液，以后每隔三四天换液。培养基为采用申请号为CN2017100038074的培养基。倒置显微镜下观察细胞生长情况及形态变化，待细胞长至培养瓶底80％-90％面积时，用0.05％胰酶进行常规细胞消化，传代培养。人脐血间充质干细胞的形态学观察及鉴定：取第4代人脐血间充质干细胞，用0.05％胰酶消化1.0-2.0min，待细胞变圆加入血清培养基中和胰酶作用，吹打管壁至细胞脱落，制成细胞悬液，以1000转/min，离心5min，使细胞沉淀，PBS冲洗细胞，室温下分别与CD34、CD38、CD45、CD166、CD44、FLK1、CD29、HLA-ABC、CD90单克隆抗体避光孵育60min，PBS清洗细胞，洗去未结合抗体，离心，弃去上清液，用同型对照单克隆抗体作为阴性对照组，流式细胞仪检测各标志物的表达，结果如图1所示，证明这些细胞确实是间充质干细胞，而不是其他细胞。
外泌体的制备、超滤浓缩，后进行保藏，本发明以两个实施例来具体介绍该技术方案。
实施例一
选择增殖能力强的第3～5代人脐血间充质干细胞，在正常含氧量的37℃培养箱中，用含有5％FBS的DMEM/F12培养基来培养，直到细胞汇合度为60-70％时，改用促进外泌体合成和分泌的培养条件：无血清的饥饿培养基，该培养基采用申请号为CN2017100038074的培养基，且饥饿培养基中含氧量为1％，pH值为6.8，添加外泌体促进因子为：5ng/ml伽马干扰素，1mg/ml多聚赖氨酸，2ng/ml MG-CSF，4ng/ml IL-6，3ng/ml HGF，3mM ATP，3mmol(14mg/dl)钙离子，培养3天，后收集培养上清液500mL，2000r/min离心30min除去细胞，再经0.45μm无菌滤膜过滤到50mL规格的超滤离心管中，2000r/min离心30min得到含外泌体的浓缩液100ml，每100ml浓缩液中添加赋形剂甘露醇6g，按每1毫升分装，于-80℃冷藏备用。
实施例二
选择增殖能力强的第3～5代人脐血间充质干细胞，在正常含氧量的37℃培养箱中，用含有5％FBS的DMEM/F12培养基来培养，直到细胞汇合度为60-70％时，改用促进外泌体合成和分泌的培养条件：无血清的饥饿培养基，该培养基 采用申请号为CN2017100038074的培养基，且饥饿培养基中含氧量为5％，pH值为7，添加外泌体促进因子为：20ng/ml伽马干扰素，5mg/ml多聚赖氨酸，4ng/ml MG-CSF，3ng/ml IL-6，5ng/ml HGF，6mM ATP，3.5mmol(14mg/dl)钙离子，培养3天，后收集培养上清液500mL，2000r/min离心30min除去细胞，再经0.45μm无菌滤膜过滤到50mL规格的超滤离心管中，2000r/min离心30min得到含外泌体的浓缩液100ml，每100ml浓缩液中添加赋形剂甘露醇6g，按每1毫升分装，于-80℃冷藏备用。
外泌体的形态特征和标志物鉴定
外泌体的主要观察指标：①透射电镜观察外泌体形态。②Western Blotting检测标志蛋白的表达水平。③ELISA检测相关蛋白多肽的含量。④流式细胞仪鉴定外泌体表面相关抗原。
外泌体的形态学观察：取上述PBS悬浮液50μL，向样品中滴入染色剂醋酸铀水溶液(1％，pH＝4.0)，混匀，用移液枪滴加1滴在复膜铜网上，自然干燥后在透射电子显微镜上进行观察，并拍摄电镜照片。外泌体形态：从脐带间充质干细胞(UC-MSCs)的不同培养基中均分离得到了外泌体样的泡状物。透射电镜下观察3种不同细胞培养液来源的外泌体泡状物，均可见大小较为均一，明显异质性圆形小囊泡，直径20-140nm，有完整膜，内有低电子密度成分。见图2：培养基1即为10％FBS DMEM/F12培养基，培养基2即为5％FBS DMEM/F12低血清培养基，培养基3即为本发明实施例二的培养基，10％FBS DMEM/F12培养基1来源泡状物平均直径最大，可见140nm，泡膜光滑清晰，泡内电子致密物多；5％FBS DMEM/F12低血清培养基2来源泡状物平均直径稍小，可见110nm，泡膜较为粗糙模糊，泡内电子致密物较少；本发明实施例二的培养基3(上述的特殊无血清培养条件)来源泡状物平均直径最小，大多数为90nm，泡膜光滑清晰，泡内电子致密物较多。利用Nanosight纳米粒度分析仪对本发明的特殊培养条件下提取到的外泌体进行粒子分布范围的分析结果如图3所示。比较在不同培养基中的外泌体浓度表明特殊培养条件能大大促进干细胞合成和分泌外泌体(图4)。这些结果证明本发明的特殊培养条件是一种可行的和有效的方法，它能提高外体的产量。
外泌体蛋白质浓度测定：采用BCA蛋白定量试剂盒，ND-2000超微量紫外可见分光光度计检测外泌体总蛋白量。参数：标本0.1mL+试剂2.0mL；反应时间30min；反应温度37℃；波长为562nm。结果显示：来源10％FBS DMEM/F12培养基的培养液50ml，ExoQuick-TC试剂法得到外泌体蛋白浓度为(872±30)ug/ml。5％FBS低血清培养基-2来源的外泌体溶液50ml，以蛋白浓度计算为(1.57±0.27)mg/ml。人脐血间充质干细胞无血清培养上清可分离获得约50ml外泌体悬液，其蛋白质浓度为(2.66±0.42)mg/ml。
ELISA：收集经上述特殊条件处理后的间充质干细胞的培养液，2000xg离心30min，收集上清，根据说明书使用ELISA法检测各组样品在特定波长处的吸光度，根据标准曲线计算上清中IL6、IL-11、TNF、HGF、bFGF、IGF-1、GM-CSF、TGF-β、TPO、VEGF、LIF、EGF、KGF、IFN-α和IFN-γ等浓度。ELISA结果(图5)揭示，对照外泌体组(10％的血清培养基和传统地培养条件)与本发明的特殊培养条件外泌体组上清中上述细胞因子的分泌量相比，后者中的非常显著升高，图中13种细胞因子浓度的P值小于0.01，说明这种差异具有统计学意义。
流式细胞术检测人脐血间充质干细胞来源的外泌体表面标志：将20ul含外泌体的浓缩液用1ml PBS稀释，所得悬液与相关单克隆抗体温育，同时用同型IgG作阴性对照，经流式细胞仪检测。人脐血间充质干细胞来源的外泌体表面标记物的表达：流式细胞仪检测结果显示，三种不同培养基来源的人脐血间充质干细胞的外泌体表达外泌体共性标志CD9，CD63，CD81，Calnexin和Flotillin-1及间充质干细胞的表面黏附分子CD90、CD73、CD105，结果见表1，表1中培养基-1即为10％FBS DMEM/F12培养基，培养基-2即为5％FBS DMEM/F12低血清培养基，培养基-3即为本发明实施例二的培养基。
表1.外泌体标志物的流式细胞仪检测结果(％)
  CD9 CD63 CD73 CD81 CD90 CD105 Caln Flot
培养基-1 59.2 96.9 29.1 79.3 18.1 9.7 69.0 79.1
培养基-2 57.5 95.2 38.2 76.2 17.6 8.8 65.1 79.4
培养基-3 68.3 97.6 38.8 86.0 19.2 9.1 78.9 82.5
外泌体特异性分子标志物的表达从脐带间充质细胞不同培养上清液中提取的泡状物都能表达外泌体特异性分子标志物CD63和Flotillin-1，进一步证明了这些泡状物可能为外泌体。由上述结果推断，本研究应用特殊培养条件，大大促进了UC-MSCs合成和分泌外泌体，并用简单易行的超滤冷冻干燥技术将UC-MSCs培养液上清中的外泌体和活性成分大规模的制备。Westenblot检测(图6)显示不同培养上清液来源的外泌体都能表达表面标志蛋白。
外泌体冻干粉的制备及保存
该冻干粉组分为：主料为上述的外泌体浓缩液和赋形剂甘露醇，二者的质量比为95∶5，所述外泌体冻干粉制备方法如下：将上述置于-80度冰箱中的外泌体上清液置于-60度平衡1小时，然后快速装入冻干机的干箱内，此时冻干机干箱的温度控制在-48℃左右(制品冻结到共晶点以下)，随后使得冻干机冷凝筒的温度迅速达-50～55℃，对冻干机干箱抽真空，使其真空度达15Pa使制品升华干燥，控制搁板温度在-55～35℃，时间8持续小时；最后制品解吸干燥，温度在10小时内匀速缓慢升温至-20℃，保温2小时，再在5小时内匀速升温至5℃。真空度低于10Pa，使含水量小于3％，检测合格后包装入库(图7)。制备出的冻干粉外观无缺损，表面平整，体积与冻结时的体积基本相等，颜色均匀一致，溶解度好，澄明度好，稳定性好，不易受到污染，保质期长。
UC-MSCs外泌体上清液冻干粉促进皮肤角质细胞HeCat的增殖：
为了研究UC-MSC细胞来源的外泌体对HeCat细胞的影响，首先检测HeCat的细胞增殖能力是否发生变化。将UC-MSCs接种在24孔板中，24h后分别1)加入PBS(作为对照)，2)60ug/ml外泌体，3)同时加入外泌体与10umol/LSB431542，一种EGF/KGF受体的抑制剂为外泌体+抑制组。结果显示，在1、3、5、7和9天之后，HeCat的增殖能力相比于对照组显著上升(P＜0.01)。并且，这种增殖上调能够被EGF/KGF受体的抑制剂所抑制(图8)。使用Cell Counting Kit-8(CCK一8，Dojindo，日本)检测细胞增殖情况。根据酶标仪测定的450nm处吸光度(A)值进行计算并绘制曲线。
UC-MSCs外泌体上清液冻干粉促进人面部皮肤的改善
具体实施步骤如下：用75％的医用酒精消毒皮肤，将微针浸泡在75％的医用酒精消毒液中约15-30min，将外泌体上清液冻干粉与外泌体复苏液1∶3融合后，涂抹在面部皮肤上。继而，用微针按顺序由下至上滚动，每个部位约2次，然后呈米字型全脸洗一遍。然后敷美塑专用面膜40-60min，无须清洗。用后1、3、7、15、30天用皮肤镜(CBS-807皮肤分析系统)检测皮肤的弹性、毛孔大小、水分、皱纹和肤色。结果见表2显示这些指标均有明显的改善。
表2. 30人次使用间充质干细胞培养液前后的面部皮肤改善情况
  用前 用后-3天 用后-7天 用后-15天 用后-30天
弹性 ++ +++ +++ ++++ ++++
水分 ++ +++ +++ ++++ ++++
皱纹 +++ +++ ++ + -
斑点 +++ +++ +++ ++ +
毛孔 ++++ +++ +++ ++ ++
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (9)

  1. 一种人间充质干细胞外泌体冻干粉的制备方法，其特征在于，包括以下步骤：
    1)人间充质干细胞的培养、纯化及传代；
    2)诱导人间充质干细胞大量合成和分泌有功能的外泌体；
    3)超滤浓缩人间充质干细胞外泌体，制得浓缩培养液并进行冷藏；
    4)将冷藏后的所述浓缩培养液制备成冻干粉，向所述冻干粉中添加复合液进行保存。
  2. 如权利要求1所述的一种人间充质干细胞外泌体冻干粉的制备方法，其特征在于，
    步骤2)的具体操作方法：选择增殖能力强的第3～5代人间充质干细胞，置于含氧量为21％的37℃培养箱中，用含有5％FBS的DMEM/F12培养基进行培养，直到细胞汇合度为60～70％时，改用促进外泌体合成和分泌的培养条件进行培养3～5天，后收集上清液。
  3. 如权利要求2所述的一种人间充质干细胞外泌体冻干粉的制备方法，其特征在于，
    所述促进外泌体合成和分泌的培养条件：无血清的饥饿培养基，同时向所述饥饿培养基中添加促进因子，且所述饥饿培养基中含氧量为1％～5％，pH值为6.5～7。
  4. 如权利要求3所述的一种人间充质干细胞外泌体冻干粉的制备方法，其特征在于，
    所述促进因子为：1-100ng/ml伽马干扰素，1-10mg/ml多聚赖氨酸，1-6ng/ml MG-CSF，2-5ng/ml IL-6，1-5ng/ml HGF，1-10mM ATP，2.8-3.8mmol(14mg/dl)钙离子。
  5. 如权利要求3所述的一种人间充质干细胞外泌体冻干粉的制备方法，其特征在于，
    所述饥饿培养基采用申请号为CN2017100038074的培养基。
  6. 如权利要求1所述的一种人间充质干细胞外泌体冻干粉的制备方法，其特征在于，
    步骤3)中浓缩方法：采用离心机对步骤2)的产物进行离心，去除细胞碎片以及细菌后，过滤，将滤液进行超滤离心，制得浓缩液，向所述浓缩液中添加赋形剂并置于-80℃进行冷藏。
  7. 如权利要求6所述的一种人间充质干细胞外泌体冻干粉的制备方法，其特征在于，
    所述赋形剂为甘露醇，添加量为每100ml浓缩液中添加甘露醇5g。
  8. 如权利要求1所述的一种人间充质干细胞外泌体冻干粉的制备方法，其特征在于，
    步骤1)中人间充质干细胞的培养采用无血清培养基，所述无血清培养基采用申请号为CN2017100038074的培养基。
  9. 如权利要求1至8中任一项所述一种人间充质干细胞外泌体冻干粉的使用方法，其特征在于：
    采用微针、滚针或水光针将外泌体导入表皮深层和真皮层。
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