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WO2024045404A1 - 一种骨髓上清液及其在细胞培养中的应用 - Google Patents

一种骨髓上清液及其在细胞培养中的应用 Download PDF

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WO2024045404A1
WO2024045404A1 PCT/CN2022/137352 CN2022137352W WO2024045404A1 WO 2024045404 A1 WO2024045404 A1 WO 2024045404A1 CN 2022137352 W CN2022137352 W CN 2022137352W WO 2024045404 A1 WO2024045404 A1 WO 2024045404A1
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cells
bone marrow
nbms
cell
culture
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PCT/CN2022/137352
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English (en)
French (fr)
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孟安明
张峻峰
马腾蛟
胡加鑫
柯岚
曹春伟
王海龙
张欢
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广州国家实验室
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
    • C—CHEMISTRY; METALLURGY
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
    • C—CHEMISTRY; METALLURGY
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
    • C—CHEMISTRY; METALLURGY
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0668—Mesenchymal stem cells from other natural sources
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/70—Undefined extracts
    • C12N2500/80—Undefined extracts from animals
    • C12N2500/84—Undefined extracts from animals from mammals

Definitions

  • the invention relates to a bone marrow supernatant and its application in cell culture.
  • MSCs Mesenchymal stem cells
  • MSCs Mesenchymal stem cells
  • MSCs are a type of stem cells derived from mesoderm with self-renewal and multi-directional differentiation potential.
  • MSCs can be isolated from various tissues of multiple species, such as bone marrow and adipose tissue. , peripheral blood, etc.
  • MSCs have multi-directional differentiation potential and can differentiate into osteoblasts, adipocytes, chondrocytes, etc.
  • MSCs are widely used in the field of tissue engineering and clinical experimental treatment of certain diseases. Current clinical applications mainly use fetal bovine serum as a culture additive to expand MSCs in vitro.
  • MSCs cultured in fetal bovine serum are prone to produce significant humoral immune responses after repeated injections, leading to rapid clearance of MSCs in the body.
  • FBS fetal bovine serum
  • some recipients of MSCs expanded in vitro in FBS culture are prone to immune reactions in clinical trials, such as urticaria. Therefore, some studies have tried to use other substances instead of FBS to culture MSCs, such as using human serum and plasma instead of FBS to culture MSCs, but they have not achieved good results.
  • the proliferation rate of MSCs is significantly reduced when cultured to the fourth passage, and differentiation preference appears. Therefore, it is necessary to further find suitable mesenchymal stem cell culture additives, which can not only promote the proliferation of MSCs, but also maintain their tissue repair function and be beneficial to their application in specific diseases.
  • Newborn mammalian bone marrow as a hematopoietic organ, contains different types of cells, such as bone marrow stromal cells (ie, bone marrow mesenchymal stem cells), endothelial cells, and various immune cells. These cells can produce a variety of cytokines, extracellular matrix, and a variety of small molecules, which provide a suitable microenvironment for the survival, stemness maintenance, and differentiation of a variety of stem cells. Therefore, it can be expected that bone marrow supernatant has potential as a high-quality source of additives for stem cell culture.
  • patent WO2021040735A invented a method for amplifying intermediates in a cell culture medium containing bone marrow supernatant (BMS) based on research on adult horse bone marrow supernatant.
  • BMS bone marrow supernatant
  • MSCs Mesenchymal stem cells
  • This method collects bone marrow supernatant fluid from female horses with an average age of 11.5 years (ranging from 2 to 17 years old) and finds that it can replace serum for culturing MSCs in vitro.
  • the bone marrow supernatant used as a stem cell culture additive in this patent is mainly used for homogeneous or autologous cell culture, and the materials are limited, making it difficult to apply on a large scale.
  • Patent CN103881971A invented a method for culturing and amplifying mesenchymal stem cells by adding 2-5% volume percentage of human autologous bone marrow plasma to the basic cell culture medium. However, this method is difficult to obtain materials and increases the patient's own costs in clinical application. The pain of bone marrow extraction.
  • the bone marrow supernatant used as a stem cell culture additive in this patent is mainly used for homogeneous or autologous cell culture, and the materials are limited, making it difficult to apply on a large scale.
  • the present invention provides a preparation method of bone marrow supernatant and its application in cell culture.
  • the bone marrow supernatant of the present invention can replace fetal bovine serum to effectively expand different types of cells (such as stem cells, primary cells, and immortalized cells, etc.) in vitro, especially when used to expand stem cells. It can maintain corresponding stem cell characteristics, and the effect is better than that of adult individual bone marrow supernatant prepared by existing methods.
  • the present invention uses bone marrow supernatant derived from newborn animals to culture cells, which solves the difficulty in obtaining autologous materials, makes the sources easier to obtain, and is suitable for large-scale production applications.
  • the culture products involved in the present invention (such as mesenchymal stem cells, hematopoietic stem cells and their cell products) have been widely used in the treatment of various clinical diseases such as tissue repair and stem cell transplantation. Therefore, the newborn mammalian bone marrow supernatant prepared by the present invention It has good application prospects and market value.
  • the present invention provides a bone marrow supernatant obtained by processing a bone marrow sample of a non-human neonatal mammal.
  • the processing includes the step of dissolving and releasing the bone marrow content of the bone marrow sample in a buffer.
  • the mammal is selected from bovine, ovine, porcine or equine. In some embodiments, the mammal is selected from cattle, such as cattle, dairy cows, Angus cattle, etc.
  • the mammal is selected from the group consisting of non-human neonatal mammals from 1 day to 1 month old. In some embodiments, the mammal is selected from the group consisting of 1 day to 20 days old, more preferably selected from the group consisting of 1 day to 10 days old, further preferably selected from the group consisting of 1 day to 7 days old non-human newborn animals.
  • the bone marrow sample is selected from leg bone marrow, hip bone marrow, or rib bone marrow.
  • the buffer is selected from PBS phosphate buffer solution, Hank's balanced salt solution and other physiological balanced salt solutions, such as carbonate buffer, borate buffer, citrate buffer, tartaric acid Salt buffer or Tris-HCl buffer.
  • the buffer also includes an anticoagulant.
  • the anticoagulant is selected from sodium heparin. In some embodiments, the anticoagulant is used in an amount of 10-300 IU/mL.
  • the buffer also includes a protease inhibitor.
  • the protease inhibitor is selected from one or more of EDTA, leupeptin, or aprotinin. In some embodiments, the protease inhibitor is used in an amount of 0.2-2 v/v%.
  • the dosage ratio of the bone marrow sample to the buffer is 100 g: (100-800) mL.
  • the dissolution and release are performed in an ice bath.
  • the centrifugation process includes fractionated centrifugation to remove cells and cell debris.
  • the centrifugal treatment includes first centrifuging at 800-3000 rpm and 4-6°C for 5-10 minutes, and then centrifuging the resulting supernatant at 6000-12000 rpm and 4-6°C for 25-40 minutes. minutes to remove cells and cell debris.
  • the processing includes: mixing the bone marrow sample with a buffer so that the bone marrow contents are dissolved and released in the buffer; performing solid-liquid separation on the dissolved and released mixture, and collecting the supernatant.
  • the treatment further includes: performing sterilization treatment and endotoxin removal treatment on the collected supernatant.
  • the sterilization treatment is selected from one or more of ultraviolet irradiation, radiation sterilization, activated carbon adsorption, or membrane filtration.
  • the cattle whose bone marrow is collected in the present invention are newborn cattle that are 1-7 days old.
  • the breeds of cattle include cattle, dairy cows, Angus cattle, etc., and the gender is not limited.
  • bone marrow samples are stored at temperatures of approximately -20°C to -80°C.
  • the methods are performed in a sterile environment.
  • the method for preparing the bone marrow supernatant of the present invention selects newborn mammals, extracts the supernatant with a variety of active ingredients from the bone marrow, and retains the macromolecular proteins, extracellular secretory vesicles and other naturally existing in the bone marrow. It contains non-protein active ingredients and has removed other impurities, bacteria and mycoplasma. It can meet the needs of cell culture and is suitable for large-scale preparation.
  • the mammalian bone marrow samples used in the present invention are all bone marrow from commercial sources (commercially available sources), and the bone marrow from the above sources is processed to obtain bone marrow supernatant.
  • the present invention verified that the bone marrow supernatant (Newborn bovine bone marrow supernatant, NBMS) of newborn bovines aged 1-7 days and the bone marrow supernatant (Adult bovine bone marrow supernatant, Adult bovine bone marrow supernatant, 1-3 years old, ABMS)
  • NBMS Newborn bovine bone marrow supernatant
  • ABMS Advanced bovine bone marrow supernatant
  • the present invention provides a culture medium comprising bone marrow supernatant obtained by the method described in the first aspect.
  • the culture medium contains no serum, or contains 0.1 wt% to 20 wt% serum.
  • the serum includes serum of mammalian origin.
  • the serum of mammalian origin includes bovine, ovine, porcine, equine or human serum.
  • the present invention provides a method for culturing cells, which includes the step of culturing cells in a culture medium added with the bone marrow treatment solution described in the first aspect, or in the culture medium described in the second aspect.
  • the cells are selected from stem cells, primary cells, or immortalized cells.
  • the cells include autologous cells or non-autologous cells.
  • the stem cells are selected from the group consisting of mesenchymal stem cells, hematopoietic stem cells, embryonic stem cells, neural stem cells, skin stem cells, adipose stem cells, retinal stem cells, liver stem cells, or pancreatic stem cells.
  • the stem cells are selected from mesenchymal stem cells or hematopoietic stem cells.
  • the mesenchymal stem cells are selected from one or more of bone marrow mesenchymal stem cells, umbilical cord mesenchymal stem cells, umbilical cord blood mesenchymal stem cells or adipose mesenchymal stem cells.
  • the hematopoietic stem cells are selected from one or more of bone marrow hematopoietic stem cells, cord blood hematopoietic stem cells, and peripheral blood hematopoietic stem cells.
  • the primary cells are selected from one or more of mammalian lung epithelial cells, intestinal epithelial cells, osteoblasts, and vascular endothelial cells.
  • the immortalized cells are selected from one or more of human embryonic kidney epithelial cells HEK293, human fibroblasts, and mouse fibroblasts.
  • the cells are selected from mammalian cells, including but not limited to: human, mouse, rat, canine, cat, rabbit, pig, monkey, horse, cow, pig, or sheep.
  • the bone marrow supernatant prepared in the present invention is used as a medium additive for cultured cells, and its expansion time limit can be changed, and it is better to undergo expansion for no more than 30 days.
  • the present invention uses the above bone marrow supernatant as an additive for cell culture in one or more cell culture media selected from stem cells, primary cells or immortalized cells.
  • cell culture media selected from stem cells, primary cells or immortalized cells.
  • the growth morphology, proliferation potential, expression of stemness molecular markers, cell activity and multi-directional differentiation ability of stem cells during in vitro expansion were unexpectedly found.
  • bone marrow supernatant can better maintain the stemness of mesenchymal stem cells MSCs and enhance their osteogenic/adipogenic differentiation ability; it can reduce the occurrence of cell senescence of stem cells during the culture process and maintain stem cells.
  • Characteristics Reduce the expression of stem cell immunogenicity-related genes and increase the survival time after stem cell transplantation; Cultured mesenchymal stem cells can increase the secretion of specific cytokines and have stronger immunoregulatory effects.
  • the present invention provides the use of the bone marrow supernatant of the first aspect, the culture medium of the second aspect, or the culture method of the third aspect in cell culture or expansion.
  • the present invention provides the use of cells or cell products obtained by the culture method described in the third aspect in the preparation of drugs, tumor models, health products or nursing products, or in the screening of tumor suppressor targets and drugs. .
  • the cells or cell products thereof are used for preparations for the treatment of cardiovascular diseases, liver cirrhosis, neurological diseases, partial meniscectomy injury repair of knee joints, autoimmune diseases, immunomodulation, anti-inflammation, skin tissue Drugs engineered to treat or repair tissue damage.
  • the cells or cell products thereof are used to prepare medicaments for the treatment of hematological malignancies, severe aplastic anemia, abnormal immune diseases, metabolic diseases, or very severe myeloid acute radiation sickness.
  • the present invention provides a cell or cell group, which is obtained by adding the bone marrow supernatant of the first aspect to a culture medium or a culture medium of the second aspect, or using the third aspect. Obtained by the cell culture methods described in the three aspects.
  • the present invention provides a preparation, which includes the bone marrow supernatant described in the first aspect and a pharmaceutically acceptable carrier.
  • the dosage form of the preparation is selected from one or more of aerosol, solution, spray, ointment, gel, and skin patch.
  • "pharmaceutically acceptable carrier” includes any and all solvents or dispersion media, including but not limited to water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and their suitable mixtures, as well as vegetable oils, coatings, isotonic and absorption delaying agents, liposomes, commercial cleansers, etc.
  • solvents or dispersion media including but not limited to water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and their suitable mixtures, as well as vegetable oils, coatings, isotonic and absorption delaying agents, liposomes, commercial cleansers, etc.
  • the present invention provides use of the preparation described in the seventh aspect for preparing regenerative medicine-related preparations.
  • the regenerative medicine-related preparation is selected from one or more of pluripotent cell proliferation-related preparations, wound healing-promoting preparations, cell transplantation or disease-related preparations.
  • the agents or formulations of the invention can be administered in a variety of ways, depending on whether local or systemic treatment is desired and depending on the area to be treated. Administration may be topical; intratracheal, intranasal, epidermal, transdermal, oral, or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular, intraarticular, intracranial, intrathecal, intrabursal, intratendinous, intralesional, perilesional, intratendinous sheath, or intraventricular administration.
  • Drugs or preparations for topical administration may include aerosols, solutions, skin patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powdery or oily bases, thickening agents, and the like may be necessary or desirable.
  • Drugs or preparations for oral administration include powders or granules, suspensions or solutions in aqueous or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be required.
  • Drugs or formulations for parenteral, intrathecal, or intraventricular administration may include sterile aqueous solutions, which may also contain buffers, diluents, and other suitable additives, such as, but not limited to, penetration enhancers, carrier compounds, and other pharmaceutical agents. acceptable carrier or excipient.
  • the pharmaceutical preparations of the present invention may conveniently be presented in unit dosage form, which may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredient with a pharmaceutical carrier or excipient. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers, or both, and then, if necessary, shaping the product.
  • the medicaments or preparations of the invention may also contain other auxiliary ingredients normally present in pharmaceutical compositions.
  • additional, compatible pharmaceutically active substances may be included, such as antipruritic, astringent, local anesthetic or anti-inflammatory agents, or additional substances such as dyes, dyes, etc. may be included for use in physically formulating various dosage forms of the composition.
  • the present invention has the following beneficial effects:
  • the beneficial effect of the bone marrow supernatant preparation method of the present invention is that the bone marrow supernatant preparation method of the present invention selects newborn mammals and can extract them from their bone marrow.
  • the supernatant with a variety of active ingredients retains the macromolecular proteins and exosome vesicles that naturally exist in the bone marrow to the greatest extent, as well as other non-protein active ingredients, and removes other impurities, bacteria and mycoplasma, thus Meet cell culture needs.
  • the bone marrow supernatant of the present invention is added to the stem cell in vitro culture system to replace the existing commercial fetal bovine serum, maintaining the cell growth morphology, proliferation potential, stemness molecular markers, cell activity and Multi-directional differentiation ability.
  • the bone marrow supernatant of the present invention can rejuvenate stem cells, reduce DNA damage in cell culture, reduce stem cell immunogenicity, and effectively expand a group of hematopoietic stem cells with hematopoietic potential. Cell groups.
  • the bone marrow supernatant additive of the present invention can increase the growth rate of stem cells, reduce cell volume, reduce the aging ratio of stem cells, and better maintain the expression of stemness molecular markers.
  • the present invention uses bone marrow from domestic animals, such as cattle, sheep, pigs or horses as commercial sources of bone marrow.
  • the bone marrow from the above sources is processed to obtain bone marrow supernatant.
  • the bone marrow supernatant is used to culture stem cells to solve the problem of autologous material collection. difficulties, the source is easier to obtain, and it is suitable for large-scale production applications.
  • the culture products involved in the present invention such as mesenchymal stem cells and hematopoietic stem cells or their cell products, have been widely used in the treatment of various clinical diseases such as tissue repair and stem cell transplantation. Therefore, the bone marrow supernatant prepared by the present invention It has good application prospects and market value.
  • the culture method of the present invention can not only culture autologous cells, but also culture non-autologous cells.
  • Figure 1 shows a flow chart for the preparation of newborn bovine bone marrow supernatant (NBMS) according to some embodiments of the present application.
  • NBMS newborn bovine bone marrow supernatant
  • Figure 2 shows an electron microscope diagram of exosomes contained in NBMS and FBS according to Example 2 of the present application.
  • Figure 3 shows the composition analysis results of proteins contained in NBMS and ABMS (1Y) according to Example 3 of the present application.
  • Figure 4 shows the results of specific protein functional enrichment analysis of NBMS according to Example 3 of the present application.
  • Figure 5 shows the results of quantitative analysis of cytokines contained in NBMS and FBS according to Example 4 of the present application.
  • Figures 6A to 6C show the effect of NBMS on the cell morphology of MSCs according to Example 5 of the present application, wherein Figure 6A, Figure 6B, and Figure 6C respectively show the cell morphology of UC-MSC, BM-MSC and AD-MSC treated by NBMS. picture.
  • Figure 7 shows the effect of NBMS on the in vitro expansion of MSCs according to Example 6 of the present application, wherein A, B and C in Figure 7 respectively show NBMS as a culture additive to culture UC-MSC, BM-MSC and AD-MSC. Statistical results of cell expansion fold.
  • Figure 8 shows the effect of NBMS on the senescence of MSCs according to Example 7 of the present application, wherein A in Figure 8 shows the cell senescence detection staining results of UC-MSC cultured with NBMS as a culture additive, and B in Figure 8 shows NBMS as a culture additive Statistical results of detection of cell senescence ratio of UC-MSC cultured with additives.
  • Figure 9A- Figure 9B shows the effect of NBMS on DNA damage of MSCs according to Example 8 of the present application, wherein Figure 9A and Figure 9B respectively show the flow cytometry detection of BM-MSC and AD-MSC cells ⁇ H2AX after treatment with different additives. -Fluorescence intensity of FITC.
  • Figures 10A-10B show the effect of NBMS on MSCs surface marker molecules according to Example 9 of the present application.
  • Figures 10A and 10B respectively show the flow cytometry detection of UC-MSC and BM-MSC surface marker protein expression ratios. result.
  • Figure 11 shows the effect of NBMS on the three-way differentiation ability of MSC according to Example 10 of the present application, wherein A, B and C in Figure 11 respectively show that NBMS is used as a culture additive to culture UC-MSC, BM-MSC and AD-MSC. Three-way differentiation ability test.
  • Figure 12 shows the effect of NBMS according to Example 11 of the present application on the secretion of cytokines by MSCs.
  • Figures 13A-13C show the effect of NBMS on the immunosuppressive effect of MSCs according to Example 12 of the present application, wherein Figure 13A shows a schematic diagram of the experimental flow chart of Example 12, and Figure 13B shows that NBMS-MSCs inhibit PHA-induced PBMC expansion in vitro Increasing results, Figure 13C shows the results of NBMS-MSCs inhibiting the secretion of pro-inflammatory factors by PBMC.
  • Figure 14 shows the effect of NBMS according to Example 13 of the present application on the expression of immunogenic protein HLA-DR in MSCs.
  • Figures 15A to 15D show the flow cytometry according to Example 14 of the present application to detect the effect of NMBS on the homing ability of MSCs to various organs.
  • Figures 15A, 15B, 15C and 15D respectively show the effects of NMBS on mice after transplantation. Percentage of green fluorescent protein-positive cells in peripheral blood, bone marrow, lung, and liver.
  • Figure 16A- Figure 16B shows the effect of NBMS on the in vitro expansion of hematopoietic stem cells HSC according to Example 15 of the present application, wherein Figure 16A shows the cell morphology of HSC cultured with NBMS additives observed under a microscope, and Figure 16B shows the cell morphology of HSC cultured with NBMS additives The expansion fold of HSC compared to the starting cells.
  • Figure 17A- Figure 17B shows the effect of NBMS according to Example 16 of the present application on the expression of long-term hematopoietic capacity marker molecules of hematopoietic stem cells HSC, wherein Figure 17A shows the effect of NBMS on the proportion of Lin-CD34-CD93+ cell population, Figure 17B shows The effect of NBMS on the proportion of Lin-CD34-CD166+ cell population was investigated.
  • a and an include plural referents.
  • reference to “a cell” includes a plurality of such cells and equivalents known to those skilled in the art, and the like.
  • neonanatal mammal refers to a mammal that is 1 day to 1 month old.
  • the neonatal mammal is selected from the group consisting of 1 to 20 days old, 1 day to 10 days old, or 1 day to 7 days old.
  • the neonatal mammals herein are selected from non-human mammals, such as cattle, horses, sheep, etc.
  • bone marrow contents refers to materials within the bone marrow cavity such as fat, air, bone marrow tissue, etc.
  • stem cell expansion refers to the process of isolating and purifying autologous, allogeneic or xenogeneic stem cells and culturing them under sterile conditions to increase their number.
  • stem cell refers to a type of cells with unlimited or immortal self-renewal capacity and the ability to produce at least one type of highly differentiated progeny cells.
  • stem cell group is a group (collective) composed of many stem cells. The function of the stem cell group is to control and maintain cell regeneration.
  • hematopoietic stem cells refers to mesodermal vascular stem cells, which are found in adult bone marrow, peripheral blood and umbilical cord blood.
  • hematopoietic stem cells refer to immature cells and are the origin of all hematopoietic cells and immune cells. They can not only differentiate into red blood cells, white blood cells, and platelets, but can also differentiate across systems into cells in various tissues and organs. They have the ability to self- Renewal, multi-directional differentiation and homing (i.e. directional migration to hematopoietic tissues and organs) potential.
  • Stem cells are primitive cells with the potential for self-replication and multi-directional differentiation. They are the cells of origin of the body and the ancestral cells that form various tissues and organs of the human body.
  • MSC meenchymal stem cells
  • primary culture cell refers to cells cultured immediately after being removed from the body.
  • primary cells refer to the first passage of culture and cells within 10 passages.
  • exemplary primary cells include immune cells (eg, hematopoietic cells), mammalian lung epithelial cells, intestinal epithelial cells, osteoblasts, or vascular endothelial cells, among others.
  • immortalized cell used in this article refers to cells that have acquired the ability to continue to grow and proliferate and can be passaged for a long time. Often accompanied by karyotypic changes. Cell immortalization can be formed spontaneously or by transfection of foreign genes, and is often used in cell biology research on tumors. As used herein, immortalized cells refer to cells capable of growing in culture for more than 35 generations. Exemplary immortalized cells include human embryonic kidney epithelial cells HEK293, human fibroblasts, mouse fibroblasts, and the like.
  • regenerative medicine used in this article refers to the use of theoretical methods of biology and engineering to create lost or functionally damaged tissues and organs so that they have the structure and function of normal tissues and organs.
  • exosomes refers to small membrane vesicles (30-150 nm) containing complex RNA and proteins. Exosomes are rich in cholesterol and sphingomyelin. It is mainly derived from multivesicular bodies formed by the invagination of intracellular lysosomal particles, and is released into the extracellular matrix after the fusion of the multivesicle outer membrane and the cell membrane. A variety of cells can secrete exosomes under normal and pathological conditions. The function of exosomes depends on the cell type from which they are derived, and they can participate in various aspects such as the body's immune response, antigen presentation, cell migration, cell differentiation, and tumor invasion.
  • Example 1 Preparation method of neonatal animal bone marrow supernatant NBMS:
  • This embodiment relates to a method for preparing neonatal animal bone marrow supernatant (NBMS), which includes the following steps:
  • buffer solution Use sterile physiological buffer solution, such as PBS phosphate buffer solution, Hank's balanced salt solution, etc. Add 200IU/mL heparin sodium to the above buffer solution for anticoagulation, and add protease inhibitors at a volume ratio of 1/100 to inhibit protein degradation.
  • sterile physiological buffer solution such as PBS phosphate buffer solution, Hank's balanced salt solution, etc.
  • the supernatant obtained above is bone marrow supernatant (NBMS), which can be used for subsequent cell culture and other applications.
  • NBMS bone marrow supernatant
  • Example 2 Identification of exosomes contained in the above-mentioned neonatal animal bone marrow supernatant NBMS
  • the above-mentioned neonatal animal bone marrow supernatant NBMS and FBS were compared to extract exosomes using ultracentrifugation method.
  • the specific implementation steps are: add 5ml NBMS or FBS into a centrifuge tube, centrifuge at 2000g for 15 minutes, and collect the supernatant. Then centrifuge the sample in a high-speed refrigerated centrifuge at 1000g for 30 minutes, and collect the supernatant. Transfer the sample to a special ultracentrifuge tube for an ultracentrifuge, centrifuge at 120,000g for 90 minutes at low temperature, remove the supernatant, and resuspend the pellet in PBS.
  • Example 3 Analysis of protein components contained in the above-mentioned neonatal animal bone marrow supernatant NBMS
  • Example 1 The newborn bovine bone marrow in Example 1 was replaced with one-year-old adult bovine bone marrow, and the one-year-old adult bovine bone marrow supernatant ABMS (1Y) was extracted using the method in Example 1.
  • the above-mentioned newborn bovine bone marrow supernatant NBMS and one-year-old adult bovine bone marrow supernatant ABMS (1Y) were analyzed for protein components by quantitative mass spectrometry. A total of 497 shared proteins were identified in three sets of replicate samples of newborn calf bone marrow supernatant NBMS, and a total of 411 shared proteins were identified in three sets of replicate samples of adult calf bone marrow (one age) ABMS (1Y) samples ( Figure 3).
  • NBMS Protein functional enrichment analysis was performed on it, and the differential protein distribution is shown in Figure 4.
  • the above-mentioned newborn bovine bone marrow supernatant NBMS contains 114 specific proteins, of which The protein content is significantly different from that of adult calf bone marrow (one age) ABMS (1Y).
  • the results of gene function enrichment analysis showed that the 114 NBMS-specific proteins are mainly related to biological processes such as integrin signal activation and cell adhesion. Therefore, NBMS is more suitable as a cell culture medium additive to promote cell adhesion and cell expansion.
  • Example 4 Analysis of cytokines contained in the above-mentioned neonatal animal bone marrow supernatant NBMS
  • the above-mentioned newborn bovine bone marrow supernatant NBMS and fetal bovine serum FBS were measured for cytokines by enzyme-linked immunosorbent quantitative method.
  • a variety of common cytokines in fetal bovine serum FBS can be detected in the above-mentioned newborn bovine bone marrow supernatant NBMS, including IL6, SDF-1, MIF, RANTES, bFGF, VEGF, PDGF-BB, MCP -1, G-CSF, HGF, etc.
  • the content of some cytokines (such as RANTES, etc.) is higher than that of fetal bovine serum.
  • Cytokines play an important role in regulating cell expansion and maintaining stem cell characteristics. This experimental result shows that the newborn bovine bone marrow supernatant NBMS prepared by the above method contains a large number of active protein components.
  • Example 5 Effect of the above-mentioned neonatal animal bone marrow supernatant NBMS on MSCs cell morphology
  • This embodiment is designed to use the additive containing the bone marrow supernatant in one or more cell culture media selected from mesenchymal stem cells or hematopoietic stem cells. Since P4MSCs cultured to the fourth generation are usually used for translational medicine applications, this example uses mesenchymal stem cells expanded to the fourth generation in vitro to detect the culture effect. In this embodiment, the cell morphology and size are obtained through microscopic observation to reflect the cell activity status.
  • the experimental group's "NBMS” medium composition is DMEM basic medium (Gibco), 1/100 Pen/Strep double antibody (Gibco) , the volume ratio is 10% of the above newborn bovine bone marrow supernatant NBMS;
  • the control group 1 "PBS” medium composition is DMEM basic medium (Gibco), 1/100 Pen/Strep double antibody (Gibco), the volume ratio is 10% PBS buffer liquid;
  • control group 2 "FBS” medium composition is DMEM basic medium (Gibco), 1/100 Pen/Strep double antibody (Gibco), volume ratio 10% commercial fetal bovine serum FBS (Gibco);
  • control group 3 " ABMS (1-Year old)” medium composition is DMEM basic medium (Gibco), 1/100 Pen/Strep double antibody (Gibco), volume ratio 10% adult calf bone marrow supern
  • the cell morphology and cell number graphs cultured in each culture medium were observed through a microscope (the scale bar on the graph is 500 ⁇ m).
  • the cell morphology of the NBMS group was more uniform and spindle-shaped, while the cells of the ABMS group (including the one-age ABMS (1Y) group and the three-age ABMS (3Y) group) grew slowly and the cell volume increased.
  • the experimental group's "NBMS" medium composition is DMEM basic medium (Gibco), 1/100 Pen/Strep double antibody (Gibco) , the volume ratio is 10% of the above newborn bovine bone marrow supernatant NBMS;
  • the control group 1 "PBS” medium composition is DMEM basic medium (Gibco), 1/100 Pen/Strep double antibody (Gibco), the volume ratio is 10% PBS buffer liquid;
  • the medium composition of control group 2 "FBS” is DMEM basic medium (Gibco), 1/100 Pen/Strep double antibody (Gibco), and the volume ratio is 10% commercial fetal bovine serum FBS (Gibco).
  • the experimental group's "NBMS" medium composition is DMEM basic medium (Gibco), 1/100 Pen/Strep double antibody (Gibco) , the volume ratio is 10% of the above newborn bovine bone marrow supernatant NBMS;
  • the control group 1 "PBS” medium composition is DMEM basic medium (Gibco), 1/100 Pen/Strep double antibody (Gibco), the volume ratio is 10% PBS buffer liquid;
  • the medium composition of control group 2 "FBS” is DMEM basic medium (Gibco), 1/100 Pen/Strep double antibody (Gibco), and the volume ratio is 10% commercial fetal bovine serum FBS (Gibco).
  • NBMS has a significant expansion effect on MSC from three different sources after seven days of culture, maintaining the properties of MSC adherent growth. and cell morphology.
  • the cell morphology of the NBMS group was more uniform and spindle-shaped.
  • both the PBS and ABMS groups increased the cell volume and the expansion effect was not ideal.
  • Example 6 Effect of the above-mentioned neonatal animal bone marrow supernatant NBMS on the in vitro expansion of MSCs
  • This embodiment is designed to use the additive containing the bone marrow supernatant in one or more cell culture media selected from mesenchymal stem cells or hematopoietic stem cells.
  • Human umbilical cord-derived mesenchymal stem cells (UC-MSCs), human bone marrow-derived mesenchymal stem cells (BM-MSCs) and human adipose-derived mesenchymal stem cells (AD-MSCs) that were not treated and cultured for seven days with different treatments were used with countstar.
  • the cell analyzer counts and records the total number of cells. Compare the total number of cells after seven days of culture to the total number of cells on Day 0 to calculate the cell expansion fold under each culture condition and draw a graph.
  • the experimental group's "NBMS” medium composition is DMEM basic medium (Gibco), 1/100 Pen/Strep double antibody (Gibco) , the volume ratio is 10% of the above newborn bovine bone marrow supernatant NBMS;
  • the control group 1 "PBS” medium composition is DMEM basic medium (Gibco), 1/100 Pen/Strep double antibody (Gibco), the volume ratio is 10% PBS buffer liquid;
  • control group 2 "FBS” medium composition is DMEM basic medium (Gibco), 1/100 Pen/Strep double antibody (Gibco), volume ratio 10% commercial fetal bovine serum FBS (Gibco);
  • control group 3 " "ABMS (1Y)” medium composition is DMEM basic medium (Gibco), 1/100 Pen/Strep double antibody (Gibco), volume ratio 10% adult calf bone marrow supernatant (
  • the initial number of cells in each well is 1X10 ⁇ 5.
  • the medium is replaced with the above-mentioned conditioned medium, and the medium is replaced every other day.
  • the untreated and different treated UC-MSCs were cultured for seven days using a countstar cell analyzer to count and record the total number of cells. The total number of cells after seven days of culture was compared with the total number of cells in the initial state of culture (i.e. day 0) to calculate the cell expansion fold under each culture condition and draw a graph.
  • PBS culture medium was used as a negative control
  • commercial FBS was used as a positive control.
  • the experimental group's "NBMS" medium composition is DMEM basic medium (Gibco), 1/100 Pen/Strep double antibody (Gibco) , the volume ratio is 10% of the above newborn bovine bone marrow supernatant NBMS;
  • the control group 1 "PBS” medium composition is DMEM basic medium (Gibco), 1/100 Pen/Strep double antibody (Gibco), the volume ratio is 10% PBS buffer liquid;
  • control group 2 "ABMS” medium composition is DMEM basic medium (Gibco), 1/100 Pen/Strep double antibody (Gibco), volume ratio 10% adult calf bone marrow supernatant (one age) ABMS (1Y );
  • the medium composition of control group 3 "FBS” is DMEM basic medium (Gibco), 1/100 Pen/Strep double antibody (Gibco), and the volume ratio is 10% commercial fetal
  • the initial number of cells in each well is 1X10 ⁇ 5.
  • the medium is replaced with the above-mentioned conditioned medium, and the medium is replaced every other day.
  • the total number of cells in the untreated and differently treated BM-MSCs cultured for seven days was measured using a countstar cell analyzer, counted and recorded. The total number of cells after seven days of culture was compared with the total number of cells in the initial state of culture (i.e. day 0) to calculate the cell expansion fold under each culture condition and draw a graph.
  • PBS culture medium was used as a negative control, and commercial FBS was used as a positive control.
  • the experimental group's "NBMS” medium composition is DMEM basic medium (Gibco), 1/100 Pen/Strep double antibody (Gibco) , the volume ratio is 10% of the above newborn bovine bone marrow supernatant NBMS;
  • the control group 1 "PBS” medium composition is DMEM basic medium (Gibco), 1/100 Pen/Strep double antibody (Gibco), the volume ratio is 10% PBS buffer liquid;
  • control group 2 "ABMS” medium composition is DMEM basic medium (Gibco), 1/100 Pen/Strep double antibody (Gibco), volume ratio 10% adult calf bone marrow supernatant (one age) ABMS (1Y );
  • the medium composition of control group 3 "FBS” is DMEM basic medium (Gibco), 1/100 Pen/Strep double antibody (Gibco), and the volume ratio is 10% commercial
  • the initial number of cells in each well is 1X10 ⁇ 5.
  • the medium is replaced with the above-mentioned conditioned medium, and the medium is replaced every other day.
  • the untreated and different treated UC-MSCs were cultured for seven days using a countstar cell analyzer to count and record the total number of cells. The total number of cells after seven days of culture was compared with the total number of cells in the initial state of culture (i.e. day 0) to calculate the cell expansion fold under each culture condition and draw a graph.
  • PBS culture medium was used as a negative control
  • commercial FBS was used as a positive control.
  • Example 7 Effect of the above-mentioned neonatal animal bone marrow supernatant NBMS on the senescence of MSCs
  • Mesenchymal stromal cells are prone to senescence after continuous passage in vitro, so optimizing stem cell culture conditions and culture medium components is a necessary condition for obtaining high-quality cells.
  • Senescent cells usually become larger in size and express ⁇ -galactosidase, which has high enzymatic activity at pH 6.0.
  • the experimental group's "NBMS” medium composition is DMEM basic medium (Gibco), 1/100 Pen/Strep double antibody (Gibco) , the volume ratio is 10% of the above newborn bovine bone marrow supernatant NBMS;
  • the control group 1 "PBS” medium composition is DMEM basic medium (Gibco), 1/100 Pen/Strep double antibody (Gibco), the volume ratio is 10% PBS buffer liquid;
  • control group 2 "ABMS” medium composition is DMEM basic medium (Gibco), 1/100 Pen/Strep double antibody (Gibco), volume ratio 10% adult calf bone marrow supernatant (one age) ABMS (1Y );
  • the medium composition of control group 3 "FBS” is DMEM basic medium (Gibco), 1/100 Pen/Strep double antibody (Gibco), and the volume ratio is 10% commercial fetal
  • the proportion of positive cells in the PBS group was 39%, the proportion of positive cells in the ABMS group was approximately 25%, the proportion of positive cells in the FBS group was approximately 5%, and the proportion of positive cells in the NBMS group was approximately 2%.
  • the results showed that the above-mentioned NBMS significantly improved the rejuvenation of MSC cells, while ABMS increased the proportion of MSC cell senescence.
  • Example 8 Effect of the above-mentioned neonatal animal bone marrow supernatant NBMS on DNA damage of MSCs
  • ⁇ H2AX is closely related to DNA double-strand breaks and can be used as a marker for double-strand repair.
  • This example reflects the impact of the above-mentioned neonatal animal bone marrow supernatant NBMS on DNA damage during the culture of MSCs by detecting ⁇ H2AX.
  • BM-MSCs human umbilical cord-derived mesenchymal stem cells
  • AD-MSCs human adipose-derived mesenchymal stem cells
  • the steps include:
  • the experimental group's "NBMS" medium composition is DMEM basic medium (Gibco ), 1/100 Pen/Strep double antibody (Gibco), volume ratio 10% of the above newborn bovine bone marrow supernatant NBMS;
  • control group 3 "FBS” medium composition is DMEM basic medium (Gibco), 1/100 Pen/ Strep double antibody (Gibco), volume ratio 10% commercial fetal bovine serum FBS (Gibco). Inoculate into a six-well plate, and the initial number of cells in each well is 1X10 ⁇ 5. After the cells adhere to the wall on the first day, the medium is replaced with the above-mentioned conditioned medium, and the medium is replaced every other day.
  • Example 9 Effect of the above-mentioned neonatal animal bone marrow supernatant NBMS on MSCs surface marker molecules
  • MSC cells cultured for seven days were subjected to immunophenotyping analysis using flow cytometry to detect the expression of classic mesenchymal cell surface markers CD34, CD45, CD73, CD90 and CD105, and their positive rates were measured respectively.
  • the left side (-) of the dotted line represents the negative expression rate of the molecule, and the right side (+) represents the positive expression rate.
  • UC-MSCs human umbilical cord-derived mesenchymal stem cells
  • BM-MSCs human bone marrow-derived mesenchymal stem cells
  • the steps include:
  • the experimental group's "NBMS” medium composition is DMEM basic medium (Gibco ), 1/100 Pen/Strep double antibody (Gibco), volume ratio 10% of the above newborn bovine bone marrow supernatant NBMS;
  • experimental group 2 "ABMS” medium composition is DMEM basic medium (Gibco), 1/100 Pen/ Strep double antibody (Gibco), volume ratio 10% adult calf bone marrow supernatant (one age) ABMS (1Y);
  • control group 1 "FBS” medium composition is DMEM basic medium (Gibco), 1/100 Pen/ Strep double antibody (Gibco), volume ratio 10% commercial fetal bovine serum FBS (Gibco).
  • FIG. 10A and Figure 10B show the expression of molecular markers after FBS (commercial), ABMS and NBMS treated UC-MSC and BM-MSC respectively. Experimental results showed that the NBMS-treated group maintained the stemness marker expression ratio of all MSC cells, including negative CD34 and CD45 and positive CD73, CD90, and CD105.
  • Example 10 Effect of the above-mentioned neonatal animal bone marrow supernatant NBMS on the three-lineage differentiation ability of MSC
  • the multi-lineage differentiation potential of MSC usually refers to osteogenic, adipogenic and chondrogenic differentiation, which is called the tri-lineage differentiation of MSC.
  • UC-MSC cells, BM-MSC cells, and AD-MSC cells cultured with NBMS supplement for seven days were induced to differentiate into osteogenic, adipogenic, and chondrogenic cells to detect NBMS in the bone marrow supernatant of newborn animals.
  • NBMS medium composition is: DMEM basic medium (Gibco), 1/100 Pen/Strep double antibody (Gibco), volume ratio 10% of the above-mentioned newborn bovine bone marrow supernatant NBMS; on the first day, wait for the cells to adhere to the wall Afterwards, the medium was replaced with the above conditioned medium respectively, and the medium was replaced every other day. After seven days of treatment, the three types of differentiated MSC cells were tested for osteogenic, adipogenic and chondrogenic differentiation induction and induction efficiency respectively.
  • the osteogenic differentiation of MSCs was identified by detecting alizarin-stained bone nodules with a mesenchymal stem cell osteogenic differentiation kit.
  • Long-term osteogenic induction will cause calcium ions to precipitate in the form of calcium salts, forming "bone nodules".
  • Bone nodules can be stained with alizarin red (alizarin red reacts with calcium to produce a deep red compound), and the calcium nodules deposited on the outside can be dyed deep red, and the bone formation can be expressed by the area and depth of the stain.
  • the strength of differentiation The steps are as follows: 1. Prepare the MSCs that need to be induced to differentiate.
  • MSC complete medium When the cell fusion reaches about 85%, digest the cells with digestive juice and resuspend them in MSC complete medium.
  • the components of MSC complete medium are: DMEM basic medium (Gibco) +1/100 Pen/Strep double antibody (Gibco), volume ratio 10% fetal bovine serum. 2. Seed the cells into a gelatin-treated six-well plate at a density of 4 ⁇ 10 4 cells/cm 2 , add 2 ml of MSC complete culture medium to each well, and culture in a 37°C, 5% CO 2 incubator. 3.
  • MSC osteogenic induction differentiation complete medium contains: DMEM basic medium (Gibco) + 1/100 Pen/Strep double antibody (Gibco), 10% fetal bovine serum by volume, ascorbic acid, b-glycerophosphate sodium, dexamethasone . 4.
  • the adipogenic differentiation ability is detected by detecting fat droplets in the cytoplasm of cells after adipogenesis induction using an adipogenic differentiation kit. Fat droplets appear significantly red under the microscope after being stained with Oil Red O, while undifferentiated cells have no obvious color.
  • the above induction solution A contains: DMEM basic medium (Gibco) + 1/100 Pen/Strep double antibody (Gibco), 10% fetal bovine serum by volume, insulin, 3-isobutyl-1-methylxanthine, rosine Glitazone, dexamethasone. 4. After 72 hours of induction culture, discard the original culture supernatant, add 2ml of induction solution B/well, and culture in a 37°C, 5% CO 2 incubator.
  • the above-mentioned induction solution B contains: DMEM basal medium (Gibco), 10% fetal bovine serum by volume, and insulin. 5. After 24 hours of induction culture, discard the original culture supernatant, add 2ml/well of induction solution A, and culture in a 37°C, 5% CO 2 incubator. 6. Repeat steps 4 and 5 about 3-5 times. When obvious lipid droplets appear in the cells, change to induction medium B and continue culturing. 7. Replace fresh induction medium B every 2 days and continue culturing until the lipid droplets are large enough. 8.
  • the chondrogenic differentiation ability of MSCs is mainly analyzed by detecting toluidine blue and mainly detecting the acidic mucopolysaccharide in chondroblasts and the formed cartilage lacunae structure.
  • the above chondrogenic induction premix contains: DMEM basal medium (Gibco) + 1/100 Pen/Strep double antibody (Gibco), ascorbic acid, sodium pyruvate, dexamethasone, insulin-transferrin-selenium mixture. 2.
  • Example 11 Effect of the above-mentioned neonatal animal bone marrow supernatant NBMS on the secretion of cytokines by MSCs
  • Cultured mesenchymal stem cells can increase the secretion of specific cytokines, giving them stronger anti-inflammatory effects and tissue repair capabilities.
  • MSCs regulate immune cells and participate in tissue repair by parasecreting a variety of cytokines and growth factors.
  • the cytokines secreted by the BM-MSCs cultured with the above-mentioned newborn bovine bone marrow supernatant NBMS and fetal bovine serum FBS were measured using an enzyme-linked immunosorbent assay.
  • the experimental group "NBMS-MSCs" medium composition is DMEM basic medium (Gibco), 1/100 Pen/Strep double antibody (Gibco), the volume ratio is 10% as above Newborn bovine bone marrow supernatant NBMS;
  • the medium composition of the control group "FBS-MSCs” is DMEM basic medium (Gibco), 1/100 Pen/Strep double antibody (Gibco), and the volume ratio is 10% commercial fetal bovine serum FBS ( Gibco). Inoculate into a six-well plate, and the initial number of cells in each well is 1X10 ⁇ 5.
  • the medium is replaced with the above-mentioned conditioned medium, and the medium is replaced every other day.
  • the cytokines secreted by BM-MSCs and UC-MSCs cultured under different treatments for seven days were measured.
  • the above-mentioned neonatal animal bone marrow supernatant NBMS was used as an additive to culture mesenchymal stem cells (NBMS-MSCs).
  • the cell supernatant contained SDF-1alpha, GM-CSF, Basic FGF, PDGF-BB, G -
  • cytokines such as CSF and SCF were basically equivalent to those of the mesenchymal stem cells (FBS-MSCs) group using fetal bovine serum FBS as an additive. Studies have shown that these cytokines play an important role in the process of MSCs participating in the repair of lung tissue damage.
  • Example 12 Effect of the above-mentioned neonatal animal bone marrow supernatant NBMS on the immunosuppressive effect of MSCs
  • MSCs Mesenchymal stem cells have broad-spectrum immunomodulatory effects and can affect adaptive immunity and innate immunity.
  • a large number of in vitro studies have shown that T cell proliferation stimulated with polyclonal mitogens, allogeneic cells or specific antigens can be inhibited by MSCs.
  • MSCs have also been reported to affect the cytokine secretion profiles of different T cell subsets. When MSC are added to T cell culture activated in vitro, the expression of pro-inflammatory cytokines such as INF- ⁇ and TNF-a can be reduced.
  • PBMC-MSCs co-culture model was used for verification in this example.
  • Peripheral blood mononuclear cells (PBMC) from healthy adults were collected, and phytohemagglutinin PHA was used to induce the proliferation of T cells and NK cells in PBMC.
  • PBMC peripheral blood mononuclear cells
  • BM-MSCs cultured with newborn bovine bone marrow supernatant NBMS supplement, or BM-MSCs cultured with fetal bovine serum FBS supplement were added.
  • PBMC proliferation fold was measured, and PBMC were collected and quantified.
  • PCR detected the expression levels of the main pro-inflammatory factors INF- ⁇ , TNF-a, IL1, and IL6 mRNA (see Figure 13A).
  • the steps of adding NBMS additives or FBS additives to culture BM-MSCs include:
  • the experimental group's "NBMS" medium composition is DMEM basic medium (Gibco), 1/100 Pen/Strep double antibody (Gibco), volume ratio 10% The above-mentioned newborn bovine bone marrow supernatant NBMS;
  • the control group "FBS” medium composition is DMEM basic medium (Gibco), 1/100 Pen/Strep double antibody (Gibco), volume ratio 10% commercial fetal bovine serum FBS (Gibco ). Inoculate into a six-well plate, and the initial number of cells in each well is 1X10 ⁇ 5.
  • FIG. 13C quantitative PCR results show that MSCs cultured with the addition of newborn bovine bone marrow supernatant NBMS supplement can significantly inhibit the expression of pro-inflammatory factors INF- ⁇ (IFNG) and TNF-a in PBMC, and the inhibitory effect is better than that of fetal bovine serum FBS-MSCs.
  • INF- ⁇ INF- ⁇
  • TNF-a fetal bovine serum FBS-MSCs
  • Example 13 Effect of the above-mentioned neonatal animal bone marrow supernatant NBMS on the immunogenicity of MSCs
  • HLA-DR cellular immunogenicity-related genes
  • BM-MSC and AD-MSC treated with NBMS and FBS for seven days were used to detect the expression of HLA-DR-BV421 using flow cytometry, and the results were recorded and statistically analyzed.
  • the steps include:
  • the experimental group's "NBMS culture” medium composition is DMEM basic medium ( Gibco), 1/100 Pen/Strep double antibody (Gibco), volume ratio 10% of the above newborn bovine bone marrow supernatant NBMS;
  • the control group "serum culture” medium composition is DMEM basic medium (Gibco), 1/100 Pen /Strep double antibody (Gibco), volume ratio 10% commercial fetal bovine serum FBS (Gibco).
  • Example 14 Effect of the above-mentioned neonatal animal bone marrow supernatant NBMS on the homing ability of MSCs to various organs
  • Mesenchymal stem cells have "homing" properties. After transplantation through intravenous injection and other methods, they can migrate to specific injury sites and survive and grow within the injury site. This biological property of mesenchymal stem cells is an important factor in their ability to repair body damage. This example verifies the effect of the NBMS additive in newborn bovine bone marrow supernatant on the ability of MSCs to reside and survive in various organs in the body after transplantation.
  • the bone marrow-derived mesenchymal stem cells BM-MSCs used in this example were introduced to express green fluorescent protein through lentivirus infection.
  • the cells in the experimental group were MSCs cultured for 7 days with NBMS supplement in newborn bovine bone marrow supernatant (NBMS-MSCs), and the cells in the control group were MSCs (FBS-MSCs) cultured with fetal bovine serum FBS for 7 days.
  • the medium composition of the experimental group "NBMS-MSCs” is DMEM basic medium (Gibco), 1/100 Pen/Strep double antibody (Gibco), and the volume ratio is 10% of the above-mentioned newborn bovine bone marrow supernatant NBMS;
  • the control group "FBS -MSCs” medium composition is DMEM basic medium (Gibco), 1/100 Pen/Strep double antibody (Gibco), volume ratio 10% commercial fetal bovine serum FBS (Gibco). Inoculate into a six-well plate, and the initial number of cells in each well is 1X10 ⁇ 5.
  • the medium is replaced with the above-mentioned conditioned medium, and the medium is replaced every other day.
  • Cells from the experimental group and cells from the control group were transplanted into 8-week-old NSG mice with severe immunodeficiency by tail vein injection at a quantity of 5x10e6. The mice were sacrificed at 24 hours, 7 days, and 14 days after transplantation, and peripheral blood mononuclear cells, bone marrow, lungs, and liver were collected for flow cytometry to detect green fluorescent protein-positive cells to calculate the number of transplants resident in different organs. source of MSCs.
  • Figure 15A, Figure 15B, Figure 15C and Figure 15D show the positivity of green fluorescent protein in the peripheral blood, bone marrow, lung and liver of mice after transplantation. Percentage of cells.
  • NBMS-MSCs were significantly enriched in the liver on the 7th day after transplantation and in the bone marrow on the 14th day after transplantation.
  • the above experimental results show that cultured mesenchymal stem cells with NBMS additives can promote their homing in the liver and bone marrow.
  • NBMS supplement is more suitable as a culture medium supplement for therapeutic cells than fetal bovine serum.
  • Example 15 Effect of the above-mentioned neonatal animal bone marrow supernatant NBMS on the in vitro expansion of hematopoietic stem cells HSC
  • the effect of using the above-mentioned neonatal animal bone marrow supernatant NBMS on the in vitro expansion of human hematopoietic stem cells HSC was verified.
  • Use Stemspan as the basal culture medium for hematopoietic stem cells (HSC), and add three growth factors necessary to maintain stem cell proliferation (10ng/ml SCF, 100ng/ml TPO, 100ng/ml FILT3).
  • the 10% NBMS additive was added to the basal medium and three cytokines as the experimental group to treat human cord blood-derived CD34+ cells for seven days, and 10% volume of PBS was added to the control group.
  • Example 16 Effect of the above-mentioned neonatal animal bone marrow supernatant NBMS on the expression of long-term hematopoietic capacity marker molecules of hematopoietic stem cells HSC
  • the Lin-CD34-CD93+ cell population in human hematopoietic stem cells represents a group of resting HSCs with self-renewal potential and hematopoietic ability.
  • Use Stemspan as the basal culture medium for hematopoietic stem cells (HSC), and add three growth factors necessary to maintain stem cell proliferation (10ng/ml SCF, 100ng/ml TPO, 100ng/ml FILT3).
  • the 10% NBMS additive was added to the basal medium and three cytokines as an experimental group to treat human cord blood-derived CD34+ cells for seven days, and 10% volume of PBS was added to the control group.
  • CD34+ cells derived from human cord blood supplemented with NBMS were continued to be cultured in vitro until the 11th day.
  • the expression of Lineage-FITC, CD34-PECY7, and CD93-BV750 on the HSC surface was detected by flow cytometry, and the proportion of Lin-CD34-CD93+ cell population was calculated. Compare.
  • the results in Figure 17A show that compared with the PBS control group, NBMS can increase the proportion of Lin-CD34-CD93+ cell population.
  • the Lin-CD34+CD166+ cell population among hematopoietic stem cells represents a group of resting HSCs with self-renewal potential and hematopoietic ability.
  • Use Stemspan as the basal culture medium for hematopoietic stem cells (HSC), and add three growth factors necessary to maintain stem cell proliferation (10ng/ml SCF, 100ng/ml TPO, 100ng/ml FILT3).
  • the 10% NBMS additive was added to the basal medium and three cytokines as the experimental group to treat human cord blood-derived CD34+ cells for seven days, and 10% volume of PBS was added to the control group.

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Abstract

提供了一种骨髓上清液及其在干细胞培养中的应用。该骨髓上清液通过对新生哺乳动物的骨髓样品进行处理获得，作为培养基添加物用于细胞培养，可以实现有效扩增，具有临床应用前景。

Description

一种骨髓上清液及其在细胞培养中的应用 技术领域
本发明涉及一种骨髓上清液及其在细胞培养中的应用。
背景技术
间质干细胞(Mesenchymal stem/stromal cells，MSCs)是来源于中胚层的一类具有自我更新和多向分化潜能的干细胞，可以从多个物种的多种组织中分离出MSCs，如骨髓、脂肪组织、外周血等。MSCs具有多向分化潜能，可分化为成骨细胞、脂肪细胞、软骨细胞等，MSCs被广泛应用于组织工程领域以及某些疾病的临床试验性治疗当中。目前临床应用主要使用胎牛血清作为培养添加物对MSCs进行体外扩增。但是，已有研究显示胎牛血清(FBS)培养的MSCs后反复注射容易产生明显的体液免疫反应，导致MSCs在体内被快速清除。此外，FBS培养体外扩增的MSCs在临床实验中部分受体容易发生免疫反应，例如出现荨麻疹。因此，一些研究尝试用其他物质代替FBS来培养MSCs，例如使用人血清、血浆代替FBS培养MSCs，但没有获得很好的效果，MSCs在培养至第四代时增殖速度明显降低，出现分化偏好。因此，进一步寻找适合的间充质干细胞培养添加物很有必要，其不仅能够促进MSCs的增殖、还要维持他们的组织修复功能以及有益于其在特定疾病中的应用。
新生哺乳动物骨髓作为造血器官，内部含有不同类型的细胞，如骨髓基质细胞(即骨髓间充质干细胞)，内皮细胞，以及各种免疫细胞。这些细胞可产生多种细胞因子、细胞外基质、以及多种小分子等，构成为多种干细胞存活、干性维持和分化提供适合的微环境。因此，可以预期骨髓上清液有望作为干细胞培养添加物的优质来源。目前已公开的骨髓上清液作为细胞培养添加物的专利中，专利WO2021040735A基于对成年马骨髓上清液的研究发明了一种在包含骨髓上清液(BMS)的细胞培养基中扩增间充质干细胞(MSCs)以产生无异种蛋白的间充质干细胞的方法。该方法从平均年龄11.5岁(2-17岁不等)的雌性马收集骨髓上清液体，发现其可替代血清用于体外培养MSCs。考虑到马、牛、山羊、绵羊、人成年个体、尤其是不同年龄段个体的骨髓液在活性成份组成上的存在巨大差异，可能导致其对干细胞的作用大不相同。另外，该专利中使用骨髓上清液作为干细胞培养添加物均主要用作同种或自体细胞培养，取材有限，难以大规模应用。专利CN103881971A发明了一种在基础细胞培养基中添加了2-5％体积百分比的人体自体骨髓血浆培养和扩增间充质干细胞的方法，但是此方法取材困难，在临床应用方面增加了患者自身的抽髓痛苦。另外，该专利中使用骨髓上清液作为干细胞培养添加物均主要用作同种或自体细胞培养，取材有限，难以大规模应用。
发明内容
基于现有技术中存在的问题，本发明提供了一种骨髓上清液的制备方法以及其在细胞培养中的应用。通过使用本发明的骨髓上清液作为细胞培养添加物，替代胎牛血清在体外有效扩增不同种类细胞(例如干细胞，原代细胞，以及永生化细胞等)，特别地用于扩增干细胞时能够维持相应的干细胞特性，效果优于现有方法所制备的成年个体骨髓上清液。本发明应用新生动物来源骨髓上清液培养细胞，解决自体取材中的困难，来源更易获取，适合大规模生产应用。本发明所涉及的培养产物(例如间充质干细胞与造血干细胞及其细胞产物)已广泛应用于组织修复、干细胞移植等各种临床疾病的治疗，因此本发明制备的新生哺乳动物骨髓上清液具有较好的应用前景和市场价值。
在第一方面，本发明提供了一种骨髓上清液，其通过对非人类新生哺乳动物的骨髓样品进行处理获得。
在一些实施方式中，所述处理包括使所述骨髓样品的骨髓内容物在缓冲液中溶解、释放的步骤。
在一些实施方式中，所述哺乳动物选自牛、羊、猪或马。在一些实施方式中，所述哺乳动物选自牛，例如为黄牛、奶牛或安格斯牛等。
在一些实施方式中，所述哺乳动物选自出生1天至1个月的非人类新生哺乳动物。在一些实施方式中，所述哺乳动物选自出生1天至20天，更优选选自出生1天至10天，进一步优选选自出生1天至7天的非人类新生动物。
在一些实施方式中，所述骨髓样品选自腿骨骨髓、胯骨骨髓或者肋骨骨髓。
在一些实施方式中，所述缓冲液选自PBS磷酸盐缓冲溶液、Hank's平衡盐溶液以及其他生理平衡盐溶液，例如碳酸盐缓冲液、硼酸盐缓冲液、枸橼酸盐缓冲液、酒石酸盐缓冲液或三羟甲基甲烷-盐酸缓冲液。
在一些实施方式中，所述缓冲液中还包括抗凝血剂。在一些实施方式中，所述抗凝血剂选自肝素钠。在一些实施方式中，所述抗凝血剂的用量为10-300IU/mL。
在一些实施方式中，所述缓冲液中还包括蛋白酶抑制剂。在一些实施方式中，所述蛋白酶抑制剂选自EDTA、亮抑蛋白酶肽或Aprotinin抑肽酶中的一种或多种。在一些实施方式中，所述蛋白酶抑制剂的用量为0.2-2v/v％。
在一些实施方式中，所述骨髓样品与所述缓冲液的用量比为100g：(100-800)mL。
在一些实施方式中，所述溶解、释放在冰浴中进行。
在一些实施方式中，所述离心处理包括分级离心，去除细胞以及细胞碎片。
在一些实施方式中，所述离心处理包括先在800-3000rpm，4-6℃条件下离心5-10分钟，再将所得上清液在6000-12000rpm，4-6℃条件下离心25-40分钟，去除细胞以及细胞碎片。
在一些实施方式中，所述处理包括：将所述骨髓样品与缓冲液混合，使骨髓内容物在缓冲液中溶解、释放；将溶解释放后的混合物进行固液分离，收集上清液。
在一些实施方式中，所述处理还包括：对所收集的上清液进行除菌处理和内毒素去除处理。在一些实施方式中，所述除菌处理选自紫外线照射、辐射除菌、活性炭吸附或滤膜过滤中的一种或多种。
在一些实施方式中，本发明所收集骨髓的牛为出生1-7天的新生牛，牛的品种包括黄牛、奶牛、安格斯牛等等，性别不限。一旦收集，骨髓样品将放置于约-20℃至-80℃温度下储存。
在一些实施方式中，所述方法在无菌环境中进行。
本发明所述骨髓上清液的制备方法选取新生哺乳动物，在其骨髓中提取具有多种活性成分的上清液，保留了骨髓中自然状态下存在的大分子蛋白、胞外分泌囊泡以及其他非蛋白活性成分，并去除了其他杂质、细菌以及支原体，能够达到细胞培养需求，且适合大规模制备。本发明中采用的哺乳动物的骨髓样品均为商品化来源(市售来源)的骨髓，处理上述来源的骨髓得到骨髓上清液。
在一些实施方式中，本发明验证了出生1-7天的新生牛的骨髓上清液(Newborn bovine bone marrow supernatant，NBMS)与1-3岁成年牛骨髓上清液(Adult bovine bone marrow supernatant，ABMS)蛋白质成分存在明显差异。新生牛骨髓上清液NBMS在对于干细胞培养中的作用明显优于成年牛骨髓上清液ABMS。且随着取样对象年龄的增大，其作用逐渐削减(1岁龄ABMS作用效果优于3岁龄ABMS)。因此本发明所采用的方法获取的NBMS，具有明显优于ABMS的效果，具有更为优越的临床应用价值和前景。
在第二方面，本发明提供了一种培养基，其包括通过第一方面所述的方法获得的骨髓上清液。
在一些实施方式中，所述培养基不含有血清，或者含有0.1wt％-20wt％的血清。
优选地，所述血清包括哺乳动物来源的血清。
优选地，所述哺乳动物来源的血清包括牛、羊、猪、马或人血清。
在第三方面，本发明提供了一种细胞的培养方法，其包括在添加第一方面所述的骨髓处理液的培养基中、或在第二方面所述的培养基中培养细胞的步骤。
在一些实施方式中，所述细胞选自干细胞，原代细胞，或永生化细胞。
在一些实施方式中，所述细胞包括自体细胞或非自体细胞。
在一些实施方式中，所述干细胞选自充质干细胞、造血干细胞、胚胎干细胞、神经干细胞、皮肤干细胞、脂肪干细胞、视网膜干细胞、肝干细胞或胰腺干细胞。
在一些实施方式中，所述干细胞选自充质干细胞或造血干细胞。
在一些实施方式中，所述间充质干细胞选自骨髓间充质干细胞、脐带间充质干细胞、脐带血间充质干细胞或脂肪间充质干细胞中的一种或多种。
在一些实施方式中，所述造血干细胞选自骨髓造血干细胞，脐血造血干细胞，外周血造血干细胞中的一种或多种。
在一些实施方式中，所述原代细胞选自哺乳动物肺上皮细胞、肠上皮细胞、成骨细胞、血管内皮细胞的一种或多种。
在一些实施方式中，所述永生化细胞选自人胚肾上皮细胞HEK293、人成纤维细胞、小鼠成纤维细胞中的一种或多种。
在一些实施方式中，所述细胞选自哺乳动物细胞，所述哺乳动物包括但不限于：人、小鼠、大鼠、犬、猫、兔子、猪、猴、马、牛、猪或羊。
在一些实施方式中，本发明制备的骨髓上清液作为培养细胞的培养基添加物，其扩增时限可以变化，以经历不超过30天的时间的扩增为佳。
本发明使用上述骨髓上清液作为细胞培养的添加物，用于选自干细胞、原代细胞或永生化细胞的一种或多种细胞培养基中。特别地，用于干细胞培养的细胞培养基中时，对干细胞在体外扩增过程中生长形态、增殖潜能、干性分子标记物表达、细胞活性以及多向分化能力进行检测，出乎意料地发现，骨髓上清液在扩增干细胞同时，能够促更好的维持充质干细胞MSCs的干性，增强其成骨/成脂分化能力；能够降低干细胞在培养过程中的细胞衰老的发生，维持干细胞特性；减少干细胞免疫原性相关基因表达，增加干细胞移植后存活时间；培养的间充质干细胞能增加特定细胞因子分泌，具有更强的免疫调节作用。
在第四方面，本发明提供了第一方面所述的骨髓上清液、第二方面所述的培养基或第三方面所述的培养方法在细胞培养或扩增中的应用。
在第五方面，本发明提供了通过第三方面所述的培养方法获得的细胞或其细胞产物在制备药物、肿瘤模型、保健品或护理用品或在筛选抑瘤靶点、筛选药物中的应用。
在一些实施方式中，所述细胞或其细胞产物用于制备治疗心血管疾病、肝硬化、神经系统疾病、膝关节半月板部分切除损伤修复、自身免疫性疾病、免疫调节、抗炎、皮肤组织工程治疗或组织损伤修复的药物。
在一些实施方式中，所述细胞或其细胞产物用于制备治疗恶性血液病、重型再生障碍性贫血、异常免疫病、代谢病或极重度骨髓型急性放射病的药物。
在第六方面，本发明提供了一种细胞或细胞群，其通过在添加了第一方面所述的骨髓上清液的培养基中或在第二方面所述的培养基中，或利用第三方面所述的细胞培养方法培养获得。
在第七方面，本发明提供了一种制剂，其包括第一方面所述的骨髓上清液和药学上可接受的载体。
在一些实施方式中，所述制剂的剂型选自气溶胶、溶液、喷雾剂、软膏、凝胶、皮肤贴片中的一种或多种。
在一些实施方式中，“药学上可接受的载体”包括任何和所有溶剂或分散介质，包括但不限于水、乙醇、多元醇(例如甘油、丙二醇和液体聚乙二醇等)以及它们的合适混合物，以及植物油、涂层、等渗和吸收延迟剂、脂质体、市售清洁剂等。
在第八方面，本发明提供了第七方面所述的制剂在用于制备再生医学相关制剂中的应用。
在一些实施方式中，所述再生医学相关制剂选自多潜能细胞增殖相关制剂、促进伤口愈合相关制剂、细胞移植或疾病相关制剂中的一种或多种。
在一些实施方式中，本发明的药物或制剂可以以多种方式给药，这取决于是否需要局部或全身治疗以及取决于待治疗的区域。给药可能是局部的；气管内、鼻内、表皮、透皮、口服或肠胃外。肠胃外给药包括静脉内、动脉内、皮下、腹膜内、肌肉内、关节内、颅内、鞘内、滑囊内、腱内、病灶内、病灶周围、腱内鞘或心室内。用于局部给药的药物或制剂可以包括气溶胶、溶液、皮肤贴剂、软膏、洗剂、乳膏、凝胶、滴剂、栓剂、喷雾剂、液体和粉末。常规药物载体、水性、粉末或油性基质、增稠剂等可能是必要的或合乎需要的。
用于口服给药的药物或制剂包括粉剂或颗粒剂、水或非水介质中的混悬剂或溶液、胶囊、袋剂或片剂。可能需要增稠剂、调味剂、稀释剂、乳化剂、分散助剂或粘合剂。
用于肠胃外、鞘内或心室内给药的药物或制剂可以包括无菌水溶液，其也可以含有缓冲剂、稀释剂和其他合适的添加剂，例如但不限于渗透促进剂、载体化合物和其他药学上可接受的载体或赋形剂。
本发明的药物制剂可以方便地以单位剂型存在的，其可以根据制药工业中众所周知的常规技术制备。这样的技术包括使活性成分与药物载体或赋形剂结合的步骤。通常，制剂通过将活性成分与液体载体或细碎的固体载体或两者均匀且紧密地结合，然后，如果需要，使产品成型来制备。
本发明的药物或制剂还可以包含通常存在于药物组合物中的其他辅助成分。因此，例如，可以包含额外的、相容的药学活性物质，例如止痒剂、收敛剂、局部麻醉剂或抗炎剂，或者可以包含用于物理配制组合物的各种剂型的额外物质如染料、调味剂、防腐剂、抗氧化剂、遮光剂、增稠剂和稳定剂。
相比于现有技术，本发明具有以下几方面的有益效果：
(1)与现有骨髓物提取方法相比，本发明的骨髓上清液的制备方法的有益效果在于：本发明所述骨髓上清液的制备方法选取新生哺乳动物，能在其骨髓中提取具有多种活性成分的上清液，最大程度保留了其骨髓中自然状态下存在的大分子蛋白和外泌体囊泡，以及其他非蛋白活性成分，并去除了其他杂质、细菌以及支原体，从而达到细胞培养需求。
(2)将本发明的骨髓上清液添加至干细胞体外培养体系中用以代替现有的商业化胎牛血清，维持了干细胞的细胞生长形态、增殖潜能、干性分子标记物、细胞活性以及多向分化能力。相较于目前市面商业化胎牛血清，本发明的骨髓上清液可使得干细胞年轻化、减少细胞培养中DNA损伤、降低干细胞免疫原性，并且有效扩增了造血干细胞中具有造血潜能的一群细胞群。本发明的骨髓上清液添加物可增加干细胞增长速度、减小细胞体积、降低干细胞衰老比例、更好的维持干性分子标记物表达。
(3)本发明应用家养动物的骨髓，如牛、羊、猪或马为商品化来源的骨髓，处理上述来源的骨髓得到骨髓上清液，用所述骨髓上清液培养干细胞，解决自体取材中的困难，来源更易获取，适合大规模生产应用。
(4)本发明所涉及培养产物，例如间充质干细胞与造血干细胞或其细胞产物，已广泛应用于组织修复、干细胞移植等各种临床疾病的治疗，因此，本发明制备的骨髓上清液具有较好的应用前景和市场价值。
(5)本发明的培养方法不仅可以培养自体细胞，还可以培养非自体细胞。
附图说明
图1显示了根据本申请的一些实施方式的新生牛骨髓上清液(NBMS)制备流程图。
图2显示了根据本申请实施例2的NBMS与FBS中所含外泌体的电镜示意图。
图3显示了根据本申请实施例3的NBMS与ABMS(1Y)中所含蛋白质的成分分析结果。
图4显示了根据本申请实施例3的NBMS的特异蛋白功能富集分析结果。
图5显示了根据本申请实施例4的NBMS和FBS中所含细胞因子定量分析结果。
图6A-图6C显示了根据本申请实施例5的NBMS对MSCs细胞形态的影响，其中图6A、图6B、图6C分别显示了NBMS处理UC-MSC、BM-MSC和AD-MSC的细胞形态图。
图7显示了根据本申请实施例6的NBMS对MSCs体外扩增的影响，其中图7中A、B和C分别显示了NBMS作为培养添加物培养UC-MSC、BM-MSC和AD-MSC的细胞扩增倍数统计结果。
图8显示了根据本申请实施例7的NBMS对MSCs衰老的影响，其中图8中A显示了NBMS作为培养添加物培养UC-MSC的细胞衰老检测染色结果，图8中B显示了NBMS作为培养添加物培养UC-MSC的细胞衰老比例检测统计结果。
图9A-图9B显示了根据本申请实施例8的NBMS对MSCs DNA损伤的影响，其中图9A和图9B分别显示了流式细胞仪检测不同添加物处理后BM-MSC和AD-MSC细胞γH2AX-FITC的荧光强度。
图10A-图10B显示了根据本申请实施例9的NBMS对MSCs表面标记分子的影响，其中图10A和图10B分别显示了流式细胞仪检测UC-MSC和BM-MSC表面标记蛋白表达比例的结果。
图11显示了根据本申请实施例10的NBMS对MSC三向分化能力的影响，其中图11中A、B和C分别显示了NBMS作为培养添加物培养UC-MSC、BM-MSC和AD-MSC的三向分化能力检测。
图12显示了根据本申请实施例11的NBMS对MSC分泌细胞因子的影响。
图13A-图13C显示了根据本申请实施例12的NBMS对MSC免疫抑制作用的影响，其中 图13A显示了实施例12的实验流程示意图，图13B显示了NBMS-MSCs抑制PHA诱导的PBMC体外扩增的结果，图13C显示了NBMS-MSCs抑制PBMC分泌促炎因子的结果。
图14显示了根据本申请实施例13的NBMS对MSCs免疫原性蛋白HLA-DR表达的影响。
图15A-图15D显示了根据本申请实施例14的流式细胞术检测NMBS对MSCs各器官归巢能力的影响，其中图15A、图15B、图15C和图15D分别显示了在移植后小鼠的外周血、骨髓、肺和肝脏中绿色荧光蛋白阳性细胞的百分比。
图16A-图16B显示了根据本申请实施例15的NBMS对造血干细胞HSC体外扩增的影响，其中图16A显示了显微镜观察的NBMS添加物培养HSC的细胞形态，图16B显示了NBMS添加物培养的HSC与起始细胞相比的扩增倍数。
图17A-图17B显示了根据本申请实施例16的NBMS对造血干细胞HSC长期造血能力标记分子表达的影响，其中图17A显示了NBMS对Lin-CD34-CD93+细胞群占比的影响，图17B显示了NBMS对Lin-CD34-CD166+细胞群占比的影响。
具体实施方式
为使本发明的目的、技术方案及优点更加清楚明白，以下结合实施例，对本发明进行进一步的详细说明。此处所描述的具体实施例仅用于解释本发明，并不用于构成对本发明的任何限制。
术语解释
除非另有定义，本文所用的所有技术和科学术语与通常是由普通技术人员对本发明所属的技术领域中的理解具有相同的含义。
本文所用术语“一种”和“一个”包括复数指代。例如，提及“一个细胞”包括多个这样的细胞及本领域技术人员可知晓的等同物等等。
本文所用术语“新生哺乳动物”是指出生1天至1个月的哺乳动物。在一些实施方式中，新生哺乳动物选自出生1天至20天，出生1天至10天，或出生1天至7天的新生动物。本文中的新生哺乳动物选自非人哺乳动物，例如牛、马、羊等。
本文所用术语“骨髓内容物”是指骨髓腔内的物质例如脂肪、空气、骨髓组织等。
本文所用术语“干细胞扩增”是指分离纯化自体、同种异体或异种干细胞在无菌条件下进行培养，使其数量得到增加的过程。
本文所用术语“干细胞”指的是一类具有无限的或者永生的自我更新能力的细胞、能够产生至少一种类型的、高度分化的子代细胞。“干细胞群”由许多个干细胞组成的部分(集体)，干细胞群的功能即为控制和维持细胞的再生。
本文所用术语“造血干细胞(Stem cell，SC)”来源用中胚层的血管干细胞，存在于成人的 骨髓，外周血及脐带血中。通俗地讲，造血干细胞是指尚未发育成熟的细胞，是所有造血细胞和免疫细胞的起源，它不仅可以分化为红细胞、白细胞和血小板，还可跨系统分化为各种组织器官的细胞，具有自我更新、多向分化和归巢(即定向迁移至造血组织器官)潜能。干细胞是具有自我复制和多向分化潜能的原始细胞，是机体的起源细胞，是形成人体各种组织器官的祖宗细胞。
本文所用术语“间充质干细胞(mesenchymal stem cells,MSC)”是干细胞家族的重要成员，属于成体干细胞。来源于发育早期的中胚层和外胚层。通常在骨髓中发现，也能从其他组织中分离得到，包括脐带血、外周血、输卵管以及胎儿肝脏和肺。MSC具有多向分化潜能、造血支持和促进干细胞植入、免疫调控和自我复制等特点。间充质干细胞在体内或体外特定的诱导条件下，可分化为脂肪、骨、软骨、肌肉、肌腱、韧带、神经、肝、心肌、内皮等多种组织细胞，连续传代培养和冷冻保存后仍具有多向分化潜能。
本文所用术语“原代细胞(primary culture cell)”是指从机体取出后立即培养的细胞。如本文中所用，原代细胞是指培养的第1代细胞与传10代以内的细胞。示例性原代细胞包括免疫细胞(例如造血细胞)、哺乳动物肺上皮细胞、肠上皮细胞、成骨细胞或血管内皮细胞等。
本文所用术语“永生化细胞(immortalized cell)”指获得持续生长增殖能力，可长期传代的细胞。多伴有核型改变。细胞永生化可自发形成，也可通过转染外源基因实现，常用于肿瘤的细胞生物学研究。如本文中所用，永生化细胞是指能够在培养物中生长超过35代的细胞。示例性永生化细胞包括人胚肾上皮细胞HEK293，人成纤维细胞，小鼠成纤维细胞等。
本文所用术语“再生医学”是指利用生物学及工程学的理论方法创造丢失或功能损害的组织和器官，使其具备正常组织和器官的结构和功能。
本文所用术语“外泌体”是指包含了复杂RNA和蛋白质的小膜泡(30-150nm)。外泌体富含胆固醇和鞘磷脂。其主要来源于细胞内溶酶体微粒内陷形成的多囊泡体，经多囊泡体外膜与细胞膜融合后释放到胞外基质中。多种细胞在正常及病理状态下均可分泌外泌体。外泌体的功能取决于其所来源的细胞类型，其可参与到机体免疫应答、抗原提呈、细胞迁移、细胞分化、肿瘤侵袭等方方面面。
实施例
实施例1：新生动物骨髓上清液NBMS的制备方法：
本实施例涉及一种新生动物骨髓上清液(Newborn bovine bone marrow supernatant，NBMS)的制备方法，其包括以下步骤：
1.缓冲液的配制：使用无菌的生理缓冲溶液配制，例如PBS磷酸盐缓冲溶液、Hank's平衡盐溶液等。在上述缓冲溶液中加入200IU/mL肝素钠抗凝血，以及体积比1/100加入蛋白酶抑制剂，抑制蛋白降解。
2.新生牛骨髓的获取。购买市售的出生一周内(1-7天)新生牛四肢的腿骨，取出骨髓腔 内骨髓，将骨髓置于无菌容器中。
3.在取出的骨髓中加入配制好的缓冲液，使骨髓液内容物在缓冲液溶解释放。本实施例中，100g骨髓加入500ml预冷缓冲液。充分搅拌均匀后，使骨髓内容物在缓冲液释放溶解。
4.将缓冲液和骨髓混合物分装转入无菌离心管中，使用离心机离心去除细胞组织沉淀。在2000转/分，4℃条件下离心5分钟，收取上清，将所得上清进一步在10000转/分，4℃条件下离心30分钟。将所得上清液转入新的无菌容器中。
5.在无菌条件下，对上述所得混合物溶液。依次用0.45μm滤膜、0.22μm滤膜过滤1次，再将得到的上清液使用0.01μm滤膜过滤3次，进行过滤除菌。
6.将上述所得上清液使用内毒素去除试剂盒去除内毒素，随后使用内毒素检测试剂盒检测上述所得上清液内毒素含量，其中内毒素含量<10EU/ml为合格。将提取物分装置于-20℃至-80℃保存。
7.上述所得上清液为骨髓上清液(NBMS)，可用于后续细胞培养等其他应用。
例如，新生动物骨髓上清液NBMS制备流程如图1所示。
实施例2：上述新生动物骨髓上清液NBMS中所含外泌体鉴定
上述新生动物骨髓上清液NBMS与FBS对照用超速离心方法提取外泌体，具体实施步骤为：将5ml NBMS或FBS加入离心管中，2000g离心15min，收集上清。再将样本于高速冷冻离心机中1000g离心30min，收集上清。将样本转移到超速离心机专用超离管中，低温120000g离心90min，去除上清，用PBS重悬沉淀。将所获取NBMS或FBS来源外泌体用透射电镜观察，鉴定外泌体。外泌体鉴定结果如图2所示，上述新生动物骨髓上清液NBMS与胎牛血清FBS中均含有结构相似的外泌体。
实施例3：上述新生动物骨髓上清液NBMS中所含蛋白质成分分析
将实施例1中的新生牛骨髓替换为一年龄成年牛骨髓，用实施例1的方法提取一年龄成年牛骨髓上清液ABMS(1Y)。上述新生牛骨髓上清液NBMS与一年龄成年牛骨髓上清液ABMS(1Y)通过定量质谱进行蛋白质成分分析。新生牛骨髓上清液NBMS的3组重复样本中共鉴定出497个共有蛋白，成年小牛骨髓(一年龄)ABMS(1Y)样本的3组重复样本中共鉴定出411个共有蛋白(图3)。对其进行蛋白质功能富集分析，差异蛋白分布如图4所示，相较于成年小牛骨髓(一年龄)ABMS(1Y)，上述新生牛骨髓上清液NBMS中含有特异蛋白114个，其所含蛋白质成分与成年小牛骨髓(一年龄)ABMS(1Y)存在明显差异。基因功能富集分析结果显示，该114个NBMS特异蛋白主要用于整合素信号激活、细胞黏附等生物学过程相关。因此，NBMS作为细胞培养基添加物更适合促进细胞黏附、促进细胞扩增。
实施例4：上述新生动物骨髓上清液NBMS中所含细胞因子分析
对上述新生牛骨髓上清液NBMS与胎牛血清FBS通过酶联免疫吸附定量法进行细胞因子测定。如图5所示，上述新生牛骨髓上清液NBMS中能检测到胎牛血清FBS中多种常见的细 胞因子，包括IL6，SDF-1，MIF，RANTES，bFGF，VEGF，PDGF-BB，MCP-1，G-CSF，HGF等。并且在相同总蛋白浓度条件下，部分细胞因子含量(如RANTES等)要高于胎牛血清。细胞因子在调控细胞扩增，维持干细胞特性中具有重要作用，该实验结果说明上述方法制备的新生牛骨髓上清液NBMS中包含了大量活性蛋白成分。
实施例5：上述新生动物骨髓上清液NBMS对MSCs细胞形态的影响
本实施例设计使用含有所述骨髓上清液的添加物，用于选自间充质干细胞或造血干细胞的一种或多种细胞培养基中。由于培养至第四代P4MSCs通常用于转化医学应用，本实施例使用体外扩增至第四代的间充质干细胞进行培养效果的检测。在本实施例中，通过显微镜观察获得其细胞形态大小，反映其细胞活性状态。
其中包括用于人脐带来源间充质干细胞(UC-MSCs)培养，其步骤包括：实验组“NBMS”培养基成分为DMEM基础培养基(Gibco)，1/100 Pen/Strep双抗(Gibco)，体积比10％上述新生牛骨髓上清液NBMS；对照组1“PBS”培养基成分为DMEM基础培养基(Gibco)，1/100 Pen/Strep双抗(Gibco)，体积比10％PBS缓冲液；对照组2“FBS”培养基成分为DMEM基础培养基(Gibco)，1/100 Pen/Strep双抗(Gibco)，体积比10％商业化胎牛血清FBS(Gibco)；对照组3“ABMS(1-Year old)”培养基成分为DMEM基础培养基(Gibco)，1/100 Pen/Strep双抗(Gibco)，体积比10％成年小牛骨髓上清液(一年龄)ABMS(1Y)；对照组4“ABMS(3-Year old)”培养基成分为DMEM基础培养基(Gibco)，1/100 Pen/Strep双抗(Gibco)，体积比10％成年小牛骨髓上清液(3年龄)ABMS(3Y)。接种于六孔板中，每孔起始细胞数目为1X10^5。第1天待细胞贴壁后，分别更换培养基为上述条件培养基，每隔一天更换一次培养基。待处理七天后，使用蔡司Axio Observer 7倒置显微镜拍照并记录培养之后的细胞形态和数目。PBS培养基为阴性对照，商业化FBS为阳性对照。两个不同年龄段ABMS和NBMS为实验组。如图6A所示，培养UC-MSC七天之后，通过显微镜观察各培养基培养的细胞形态和细胞数目图(图上标尺为500μm)。NBMS组与FBS组细胞形态相比，更均匀的呈梭形形态，而ABMS组(包括一年龄ABMS(1Y)组和三年龄ABMS(3Y)组)细胞增长缓慢，且细胞体积增大。
其中包括用于人骨髓来源间充质干细胞(BM-MSCs)培养，其步骤包括：实验组“NBMS”培养基成分为DMEM基础培养基(Gibco)，1/100 Pen/Strep双抗(Gibco)，体积比10％上述新生牛骨髓上清液NBMS；对照组1“PBS”培养基成分为DMEM基础培养基(Gibco)，1/100 Pen/Strep双抗(Gibco)，体积比10％PBS缓冲液；对照组2“FBS”培养基成分为DMEM基础培养基(Gibco)，1/100 Pen/Strep双抗(Gibco)，体积比10％商业化胎牛血清FBS(Gibco)。接种于六孔板中，每孔起始细胞数目为1X10^5。第1天待细胞贴壁后，分别更换培养基为上述条件培养基，每隔一天更换一次培养基。待处理七天后，使用蔡司Axio Observer 7倒置显微镜拍照并记录培养之后的细胞形态和数目。如图6B所示，NBMS培养基培养BM-MSC七天之后，通过显微镜观察获得其细胞形态和细胞数目图(图上标尺为500μm)。图上标尺为500μm。 NBMS组与FBS组细胞形态相比，更均匀的呈梭形形态，且细胞数相较于PBS组有明显扩增。
其中包括用于人脂肪来源间充质干细胞(AD-MSCs)培养，其步骤包括：实验组“NBMS”培养基成分为DMEM基础培养基(Gibco)，1/100 Pen/Strep双抗(Gibco)，体积比10％上述新生牛骨髓上清液NBMS；对照组1“PBS”培养基成分为DMEM基础培养基(Gibco)，1/100 Pen/Strep双抗(Gibco)，体积比10％PBS缓冲液；对照组2“FBS”培养基成分为DMEM基础培养基(Gibco)，1/100 Pen/Strep双抗(Gibco)，体积比10％商业化胎牛血清FBS(Gibco)。接种于六孔板中，每孔起始细胞数目为1X10^5。第1天待细胞贴壁后，分别更换培养基为上述条件培养基，每隔一天更换一次培养基。待处理七天后，使用蔡司Axio Observer 7倒置显微镜拍照并记录培养之后的细胞形态和数目。如图6C所示，NBMS培养基培养AD-MSC七天之后，通过显微镜观察获得其细胞形态和细胞数目图(图上标尺为500μm)。NBMS组与FBS组细胞形态相比，更均匀的呈梭形形态，细胞数相较于PBS组有明显扩增。
通过显微镜观察获得其细胞形态和细胞数目，从图6A、图6B、图6C可知，NBMS对于三种不同来源的MSC培养七天之后都起到了明显的扩增作用，维持了MSC贴壁生长的属性和细胞形态。NBMS组与FBS组细胞形态相比，更均匀的呈梭形形态，而PBS、ABMS组均使得细胞体积增大，扩增效果不理想。
实施例6：上述新生动物骨髓上清液NBMS对MSCs体外扩增的影响
本实施例设计使用含有所述骨髓上清液的添加物，用于选自间充质干细胞或造血干细胞的一种或多种细胞培养基中。将未处理和不同处理培养七天之后的人脐带来源间充质干细胞(UC-MSCs)、人骨髓来源间充质干细胞(BM-MSCs)和人脂肪来源间充质干细胞(AD-MSCs)用countstar细胞分析仪进行细胞总数，统计并记录。将培养七天之后的细胞总数对比Day0天的细胞总数计算出各个培养条件下的细胞扩增倍数并作图。
其中包括用于人脐带来源间充质干细胞(UC-MSCs)培养，其步骤包括：实验组“NBMS”培养基成分为DMEM基础培养基(Gibco)，1/100 Pen/Strep双抗(Gibco)，体积比10％上述新生牛骨髓上清液NBMS；对照组1“PBS”培养基成分为DMEM基础培养基(Gibco)，1/100 Pen/Strep双抗(Gibco)，体积比10％PBS缓冲液；对照组2“FBS”培养基成分为DMEM基础培养基(Gibco)，1/100 Pen/Strep双抗(Gibco)，体积比10％商业化胎牛血清FBS(Gibco)；对照组3“ABMS(1Y)”培养基成分为DMEM基础培养基(Gibco)，1/100 Pen/Strep双抗(Gibco)，体积比10％成年小牛骨髓上清液(一年龄)ABMS(1Y)；对照组4“ABMS(3Y)”培养基成分为DMEM基础培养基(Gibco)，1/100 Pen/Strep双抗(Gibco)，体积比10％成年小牛骨髓上清液(3年龄)ABMS(3Y)。接种于六孔板中，每孔起始细胞数目为1X10^5。第1天待细胞贴壁后，分别更换培养基为上述条件培养基，每隔一天更换一次培养基。待处理七天后，将未处理和不同处理培养七天之后的UC-MSC使用countstar细胞分析仪进行细胞总数，统计并记录。将培养七天之后的细胞总数对比培养起始状态(即第0天)的细胞总数计算出各个培养条件下的细 胞扩增倍数并作图。PBS培养基为阴性对照，商业化FBS为阳性对照。两个不同年龄段ABMS和NBMS为实验组。如图7中A所示为NBMS处理之后UC-MSC的扩增倍数统计图，**代表p-value小于0.01，***代表p-value小于0.001。结果显示，相较于PBS组，FBS组细胞培养7天扩增约15倍，NBMS组细胞扩增约12倍，一年龄ABMS组细胞扩增约2.5倍，三年龄ABMS组细胞扩增约1倍。
其中包括用于人骨髓来源间充质干细胞(BM-MSCs)培养，其步骤包括：实验组“NBMS”培养基成分为DMEM基础培养基(Gibco)，1/100 Pen/Strep双抗(Gibco)，体积比10％上述新生牛骨髓上清液NBMS；对照组1“PBS”培养基成分为DMEM基础培养基(Gibco)，1/100 Pen/Strep双抗(Gibco)，体积比10％PBS缓冲液；对照组2“ABMS”培养基成分为DMEM基础培养基(Gibco)，1/100 Pen/Strep双抗(Gibco)，体积比10％成年小牛骨髓上清液(一年龄)ABMS(1Y)；对照组3“FBS”培养基成分为DMEM基础培养基(Gibco)，1/100 Pen/Strep双抗(Gibco)，体积比10％商业化胎牛血清FBS(Gibco)。接种于六孔板中，每孔起始细胞数目为1X10^5。第1天待细胞贴壁后，分别更换培养基为上述条件培养基，每隔一天更换一次培养基。待处理七天后，将未处理和不同处理培养七天之后的BM-MSC使用countstar细胞分析仪进行细胞总数，统计并记录。将培养七天之后的细胞总数对比培养起始状态(即第0天)的细胞总数计算出各个培养条件下的细胞扩增倍数并作图。PBS培养基为阴性对照，商业化FBS为阳性对照。两个不同年龄段ABMS和NBMS为实验组。如图7中B所示为NBMS处理之后BM-MSC的扩增倍数统计图。结果显示，相较于PBS组，FBS组细胞培养7天约扩增15倍，NBMS组细胞扩增约10倍，一年龄ABMS组细胞扩增约2.5倍。
其中包括用于人脂肪来源间充质干细胞(AD-MSCs)培养，其步骤包括：实验组“NBMS”培养基成分为DMEM基础培养基(Gibco)，1/100 Pen/Strep双抗(Gibco)，体积比10％上述新生牛骨髓上清液NBMS；对照组1“PBS”培养基成分为DMEM基础培养基(Gibco)，1/100 Pen/Strep双抗(Gibco)，体积比10％PBS缓冲液；对照组2“ABMS”培养基成分为DMEM基础培养基(Gibco)，1/100 Pen/Strep双抗(Gibco)，体积比10％成年小牛骨髓上清液(一年龄)ABMS(1Y)；对照组3“FBS”培养基成分为DMEM基础培养基(Gibco)，1/100 Pen/Strep双抗(Gibco)，体积比10％商业化胎牛血清FBS(Gibco)。接种于六孔板中，每孔起始细胞数目为1X10^5。第1天待细胞贴壁后，分别更换培养基为上述条件培养基，每隔一天更换一次培养基。待处理七天后，将未处理和不同处理培养七天之后的UC-MSC使用countstar细胞分析仪进行细胞总数，统计并记录。将培养七天之后的细胞总数对比培养起始状态(即第0天)的细胞总数计算出各个培养条件下的细胞扩增倍数并作图。PBS培养基为阴性对照，商业化FBS为阳性对照。两个不同年龄段ABMS和NBMS为实验组。如图7中C所示为NBMS处理之后UC-MSC的扩增倍数统计图。结果显示，相较于PBS组，FBS组细胞培养7天约扩增13倍，NBMS组细胞扩增约8倍，一年龄ABMS组细胞扩增约2倍。
由不同来源间充质干细胞MSCs扩增结果可知，新生牛骨髓上清液NBMS处理组对细胞扩增的作用显著大于PBS组和ABMS组。扩增效果：NBMS>1年龄ABMS>三年龄ABMS>PBS。
实施例7：上述新生动物骨髓上清液NBMS对MSCs衰老的影响
间充质基质细胞在体外连续传代后容易发生衰老，因此优化干细胞培养条件和培养基成分是获得高质量细胞的必要条件。衰老细胞通常体积变大，并表达在pH6.0时有高酶活性的β-半乳糖苷酶。我们对于用NBMS处理后的UC-MSC以X-Gal为底物进行染色，在衰老相关的β-半乳糖苷酶(SA-β-Gal)催化下会生成深蓝色产物，此时衰老细胞会呈现出深蓝色，并使用显微镜对细胞拍照记录。
其中包括用于人脐带来源间充质干细胞(UC-MSCs)培养，其步骤包括：实验组“NBMS”培养基成分为DMEM基础培养基(Gibco)，1/100 Pen/Strep双抗(Gibco)，体积比10％上述新生牛骨髓上清液NBMS；对照组1“PBS”培养基成分为DMEM基础培养基(Gibco)，1/100 Pen/Strep双抗(Gibco)，体积比10％PBS缓冲液；对照组2“ABMS”培养基成分为DMEM基础培养基(Gibco)，1/100 Pen/Strep双抗(Gibco)，体积比10％成年小牛骨髓上清液(一年龄)ABMS(1Y)；对照组3“FBS”培养基成分为DMEM基础培养基(Gibco)，1/100 Pen/Strep双抗(Gibco)，体积比10％商业化胎牛血清FBS(Gibco)。接种于六孔板中，每孔起始细胞数目为1X10^5。第1天待细胞贴壁后，分别更换培养基为上述条件培养基，每隔一天更换一次培养基。待处理七天后，将未处理和不同处理培养七天之后的UC-MSC进行衰老相关β-半乳糖苷酶活性检测，统计并记录。图8中A所示为PBS、FBS、ABMS和NBMS处理UC-MSC一周之后，细胞衰老状态显示图。图8中B所示为各个组别细胞衰老率统计图。PBS组细胞阳性比例为39％，ABMS组阳性细胞比例约为25％，FBS组阳性细胞比例约为5％，NBMS组阳性细胞比例约为2％。结果显示上述NBMS显著提高MSC细胞年轻化，而ABMS会使得MSC细胞衰老比例增高。具体干细胞年轻化比例:NBMS>FBS>AMBS>PBS。
实施例8：上述新生动物骨髓上清液NBMS对MSCs DNA损伤的影响
γH2AX与DNA双链断裂紧密相关，可以作为双链修复的标志物。本实施例通过检测γH2AX反映上述新生动物骨髓上清液NBMS对MSCs培养过程中DNA损伤的影响。其中包括用于人脐带来源间充质干细胞(BM-MSCs)和人脂肪来源间充质干细胞(AD-MSCs)培养，其步骤包括：实验组“NBMS”培养基成分为DMEM基础培养基(Gibco)，1/100 Pen/Strep双抗(Gibco)，体积比10％上述新生牛骨髓上清液NBMS；对照组3“FBS”培养基成分为DMEM基础培养基(Gibco)，1/100 Pen/Strep双抗(Gibco)，体积比10％商业化胎牛血清FBS(Gibco)。接种于六孔板中，每孔起始细胞数目为1X10^5。第1天待细胞贴壁后，分别更换培养基为上述条件培养基，每隔一天更换一次培养基。将NBMS和FBS处理七天之后的BM-MSC和AD-MSC用流式细胞仪检测细胞γH2AX-FITC的荧光强度，记录结果并统计。图9A和图9B结果显示，NBMS相较于商业化FBS，可减少体外培养过程中干细胞的DNA双链损伤(γ H2AX-FITC阳性率的细胞占比：BM-MSC中0.23％减少到0.14％(图9A)；AD-MSC中17.2％减少到10.1％(图9B))。
实施例9：上述新生动物骨髓上清液NBMS对MSCs表面标记分子的影响
将培养七天之后的MSC细胞利用流式细胞术进行免疫表型分析，检测经典间充质细胞表面标志物CD34、CD45、CD73、CD90和CD105的表达情况，分别检测其阳性率。虚线左边(-)代表分子阴性表达率，右边(+)代表阳性表达率。其中包括用于人脐带来源间充质干细胞(UC-MSCs)和人骨髓来源间充质干细胞(BM-MSCs)培养，其步骤包括：实验组“NBMS”培养基成分为DMEM基础培养基(Gibco)，1/100 Pen/Strep双抗(Gibco)，体积比10％上述新生牛骨髓上清液NBMS；实验组2“ABMS”培养基成分为DMEM基础培养基(Gibco)，1/100 Pen/Strep双抗(Gibco)，体积比10％成年小牛骨髓上清液(一年龄)ABMS(1Y)；对照组1“FBS”培养基成分为DMEM基础培养基(Gibco)，1/100 Pen/Strep双抗(Gibco)，体积比10％商业化胎牛血清FBS(Gibco)。接种于六孔板中，每孔起始细胞数目为1X10^5。第1天待细胞贴壁后，分别更换培养基为上述条件培养基，每隔一天更换一次培养基。待处理七天后，对不同处理培养七天之后的BM-MSCs和UC-MSCs进行免疫表型分析。如图10A、图10B所示为FBS(商业)、ABMS和NBMS分别处理UC-MSC和BM-MSC之后的分子标记物表达。实验结果显示，NBMS处理组维持了所有MSC细胞的干性标志物表达比例，包括CD34、CD45阴性以及CD73、CD90、CD105阳性。超过90％的这些细胞经典间充质细胞表面标志物(CD73、CD90和CD105)呈阳性，而血细胞标志物(CD34和CD45)呈阴性。ABMS处理组分别有87.8％(UC-MSC)和48.8％(BM-MSC)的细胞表达CD34，并且虽然100％细胞表达CD90，但峰值已发生偏移。说明ABMS相较于NBMS，MSC细胞已部分丢失干细胞特性，NBMS组与FBS组相比，CD34、CD45以及CD73、CD90、CD105标志物表达细胞比例相似，仍保持较好的干细胞特性。
实施例10：上述新生动物骨髓上清液NBMS对MSC三系分化能力的影响
目前，MSC的多系分化潜能通常指成骨、成脂和成软骨分化，称为MSC的三系分化。在本实施例中分别将NBMS添加物培养七天之后的UC-MSC细胞，BM-MSC细胞，以及AD-MSC细胞进行成骨、成脂和成软骨分化诱导，以检测新生动物骨髓上清液NBMS作为培养添加物培养后细胞的分化能力。其步骤包括：NBMS培养基成分为：DMEM基础培养基(Gibco)，1/100 Pen/Strep双抗(Gibco)，体积比10％上述新生牛骨髓上清液NBMS；第1天待细胞贴壁后，分别更换培养基为上述条件培养基，每隔一天更换一次培养基。待处理七天后，对三种诱导分化的MSC细胞分别进行进行成骨、成脂和成软骨分化诱导和诱导效率检测。
在本实施例中，MSC的成骨分化通过间充质干细胞成骨分化试剂盒检测茜红素染色骨结节来鉴定。长时间的成骨诱导会使钙离子以钙盐方式沉淀下来，形成“骨结节”。骨结节可通过茜素红染色(茜素红和钙发生显色反应，产生一种深红色化合物)，把外面沉积的钙结节染成 深红色，通过着色物面积和深浅来表示成骨分化的强弱。步骤如下：1.准备所需诱导分化的MSC，当细胞融合达85％左右时，用消化液消化细胞，并用MSC完全培养基重悬，MSC完全培养基成分为：DMEM基础培养基(Gibco)+1/100 Pen/Strep双抗(Gibco)，体积比10％胎牛血清。2.按照4×10 4细胞/cm 2的密度将细胞接种于已明胶处理的六孔板中，每孔MSC完全培养基2ml，37℃、5％CO 2培养箱中培养。3.当细胞融合达60％时，弃去培养上清，每孔添加MSC成骨诱导分化完全培养基2ml/孔，37℃、5％CO 2培养箱中培养。每3天更换新鲜的MSC成骨诱导分化完全培养基，持续培养2-4周。上述MSC成骨诱导分化完全培养基包含：DMEM基础培养基(Gibco)+1/100 Pen/Strep双抗(Gibco)，体积比10％胎牛血清，抗坏血酸，b-甘油磷酸钠，地塞米松。4.诱导培养结束时，弃去培养上清，DPBS洗细胞两次，4％中性固定溶液2ml/孔室温固定细胞20分钟。5.弃去固定液，DPBS洗2次，加入茜素红染液1ml/孔染色15分钟。6.弃去茜素红染液，DPBS洗3次。7.显微镜下观察并拍照。由图11中A、B、C结果可见NBMS作为培养添加物培养的UC-MSC细胞，BM-MSC细胞，以及AD-MSC细胞能诱导出大量钙结节，说明细胞具有良好的成骨分化能力。
本实施例中通过成脂分化试剂盒检测成脂诱导后细胞的胞浆中的脂肪滴来检测成脂分化能力。脂肪滴经油红O染色后在显微镜下呈现显著的红色，未分化细胞则无明显颜色。操作步骤：1.准备所需诱导分化的MSC，当细胞融合达85％左右时，用消化液消化细胞，并用MSC完全培养基重悬。2.按照3×10 4细胞/cm 2的密度将细胞接种于已明胶处理的六孔板中，每孔MSC完全培养基2ml，37℃、5％CO 2培养箱中培养。3.当细胞融合达85％时，弃去原培养上清，添加诱导液A 2ml/孔，37℃、5％CO 2培养箱中培养。上述诱导液A包含：DMEM基础培养基(Gibco)+1/100 Pen/Strep双抗(Gibco)、体积比10％胎牛血清、胰岛素、3-异丁基-1-甲基黄嘌呤、罗格列酮、地塞米松。4.诱导培养72h后弃去原培养上清，添加诱导液B 2ml/孔，37℃、5％CO 2培养箱中培养。上述诱导液B包含：DMEM基础培养基(Gibco)、体积比10％胎牛血清、胰岛素。5.诱导培养24h后弃去原培养上清，添加诱导液A 2ml/孔，37℃、5％CO 2培养箱中培养。6.重复步骤4、5约3-5次，待细胞内出现明显脂滴时更换为诱导液B继续培养。7.每2天更换新鲜诱导液B，继续培养至脂滴足够大时培养结束。8.诱导培养结束时，弃去培养上清，DPBS洗细胞两次，4％中性固定溶液2ml/孔室温固定细胞20分钟。9.弃去固定液，DPBS洗2次，加入油红O染液1ml/孔染色15分钟。10.弃去油红O染液，DPBS洗3次。11.显微镜下观察并拍照。由图11A、图11B、图11C结果可见NBMS作为培养添加物培养的UC-MSC细胞，BM-MSC细胞，以及AD-MSC细胞能诱导出大量脂肪滴，说明细胞具有良好的成脂分化能力。
在本实施例中主要通过检测甲苯胺蓝则主要检测成软骨细胞内酸性粘多糖，以及形成的软骨陷窝结构来分析MSC成软骨分化能力。操作步骤：1.准备所需诱导分化的MSC，当细胞融合达85％左右时，用消化液消化细胞，细胞沉淀用成软骨诱导预混液重悬。上述成软骨诱导 预混液包含：DMEM基础培养基(Gibco)+1/100 Pen/Strep双抗(Gibco)、抗坏血酸、丙酮酸钠、地塞米松、胰岛素-转铁蛋白-硒混合物。2.150g离心5min，沉淀以预混液重悬，细胞密度约1×10 6细胞/ml，再次离心，弃上清。3.沉淀以成软骨诱导完全培养基重新悬浮，调整细胞密度为5×10 5细胞/ml。上述成软骨诱导完全培养基由成软骨诱导预混液加入转化生长因子-b配制。4.分别取500μl细胞悬液接种于15ml离心管中，150g离心5min。5.旋松离心管盖，将离心管轻轻置于37℃、5％CO 2培养箱中静置培养。6.诱导培养24h后，轻轻拨动离心管底，使细胞沉淀团块悬浮，重新放回培养箱继续培养。7.每2天更换新鲜上述成软骨诱导完全培养基。换液时轻轻吸取旧培养基，每管加新鲜配制的上述成软骨诱导完全培养基500μl。8.诱导培养结束时，弃去培养上清，DPBS洗细胞两次，4％中性固定溶液2ml/管室温固定细胞1小时。9.弃去固定液，DPBS洗2次，脱水、石蜡包埋后切片。10.阿利新蓝染色：将切片进行脱腊和复水，阿利新蓝染液染色30min，流水冲洗5min，脱水、透明、中性树胶封片，显微镜下观察、拍照。由图11A、图11B、图11C结果可见NBMS作为培养添加物培养的UC-MSC细胞，BM-MSC细胞，以及AD-MSC细胞经诱导能产生被阿利新蓝染色为蓝色的内酸性粘多糖，说明细胞具有良好的成软骨分化能力。
实施例11：上述新生动物骨髓上清液NBMS对MSC分泌细胞因子的影响
培养的间充质干细胞能增加特定细胞因子分泌，使其具有更强的抗炎作用和组织修复能力。MSCs通过旁分泌多种细胞因子和生长因子来调节免疫细胞和参与到组织修复。在本实施例中，对上述新生牛骨髓上清液NBMS与胎牛血清FBS培养所得BM-MSCs所分泌的细胞因子通过酶联免疫吸附定量法进行测定。其中包括用于间充质干细胞培养，其步骤包括：实验组“NBMS-MSCs”培养基成分为DMEM基础培养基(Gibco)，1/100 Pen/Strep双抗(Gibco)，体积比10％上述新生牛骨髓上清液NBMS；对照组“FBS-MSCs”培养基成分为DMEM基础培养基(Gibco)，1/100 Pen/Strep双抗(Gibco)，体积比10％商业化胎牛血清FBS(Gibco)。接种于六孔板中，每孔起始细胞数目为1X10^5。第1天待细胞贴壁后，分别更换培养基为上述条件培养基，每隔一天更换一次培养基。待处理七天后，对不同处理培养七天之后的BM-MSCs和UC-MSCs分泌的细胞因子进行测定。如图12所示，使用上述新生动物骨髓上清液NBMS作为添加物培养间充质干细胞(NBMS-MSCs)，细胞上清液中SDF-1alpha，GM-CSF，Basic FGF，PDGF-BB，G-CSF，SCF等细胞因子的表达水平，与使用胎牛血清FBS作为添加物培养间充质干细胞(FBS-MSCs)组基本相当。已有研究表明，这些细胞因子在MSCs参与肺组织损伤修复过程中发挥重要作用。
实施例12：上述新生动物骨髓上清液NBMS对MSC免疫抑制作用的影响
间充质干细胞具有广谱的免疫调节作用，可以影响适应性免疫和天然免疫。大量的体外研究显示：用多克隆丝裂原、同种异体细胞或特异性抗原刺激的T细胞增殖可以被MSC抑制。此外，MSCs也已被报道可以影响不同T细胞亚群的细胞因子分泌谱。当MSC被加入到经体 外活化的T细胞培养中时，可以降低促炎症细胞因子如INF-γ、TNF-a的表达。为检测上述新生牛骨髓上清液NBMS作为添加物培养所得的MSC是否具有免疫调节作用，本实施例中使用体外PBMC-MSCs共培养模型进行验证。取健康成人外周血单个核细胞PBMC，使用植物血凝素PHA诱导PBMC中T细胞及NK细胞增殖。同时加入新生牛骨髓上清液NBMS添加物培养所得的BM-MSCs，或者胎牛血清FBS添加物培养所得的BM-MSCs，共培养72小时后，对PBMC增殖倍数进行测定，同时收集PBMC利用定量PCR检测主要促炎因子INF-γ、TNF-a、IL1、IL6mRNA表达水平(参见图13A)。其中加入NBMS添加物或者FBS添加物培养BM-MSCs的步骤包括：实验组“NBMS”培养基成分为DMEM基础培养基(Gibco)，1/100 Pen/Strep双抗(Gibco)，体积比10％上述新生牛骨髓上清液NBMS；对照组“FBS”培养基成分为DMEM基础培养基(Gibco)，1/100 Pen/Strep双抗(Gibco)，体积比10％商业化胎牛血清FBS(Gibco)。接种于六孔板中，每孔起始细胞数目为1X10^5。第1天待细胞贴壁后，分别更换培养基为上述条件培养基，每隔一天更换一次培养基。待处理七天后，对不同处理培养七天之后的BM-MSCs和UC-MSCs与体外PBMC-MSCs进行共培养。图13B结果显示，同时加入新生牛骨髓上清液NBMS添加物培养所得的MSCs能够显著抑制PHA诱导的PBMC增殖，抑制效率与FBS-MSCs组相似(NBMS组的抑制效率为41％，FBS组的抑制效率为39％)。图13C定量PCR结果显示，同时加入新生牛骨髓上清液NBMS添加物培养所得的MSCs能够显著抑制PBMC中促炎因子INF-γ(IFNG)、TNF-a的表达，抑制效果优于胎牛血清FBS-MSCs。本实施例证明新生牛骨髓上清液NBMS添加物能够增强MSCs的免疫调节(抑制)作用。
实施例13：上述新生动物骨髓上清液NBMS对MSCs免疫原性的影响
原代MSCs具有较低细胞免疫原性相关基因如HLA-DR的表达，能够避免在移植后被受体体内的免疫细胞识别作为异源细胞快速清除。但是已有报道表明，在使用胎牛血清体外长时期的培养能够诱导HLA-DR基因的表达，减少再宿主体内驻留时间，不利于细胞移植的临床应用。将NBMS和FBS处理七天之后的BM-MSC和AD-MSC用流式细胞仪检测HLA-DR-BV421的表达，记录结果并统计。其中包括用于人脂肪来源间充质干细胞(AD-MSCs)和人骨髓来源间充质干细胞(BM-MSCs)培养，其步骤包括：实验组“NBMS培养”培养基成分为DMEM基础培养基(Gibco)，1/100 Pen/Strep双抗(Gibco)，体积比10％上述新生牛骨髓上清液NBMS；对照组“血清培养”培养基成分为DMEM基础培养基(Gibco)，1/100 Pen/Strep双抗(Gibco)，体积比10％商业化胎牛血清FBS(Gibco)。接种于六孔板中，每孔起始细胞数目为1X10^5。第1天待细胞贴壁后，分别更换培养基为上述条件培养基，每隔一天更换一次培养基。待处理七天后，对不同处理培养七天之后的BM-MSCs和UC-MSCs进行检测HLA-DR-BV421的表达。图14结果显示：NBMS相较于商业化FBS，可降低间充质干细胞的免疫原性(HLA-DR-BV421阳性率的细胞占比：AD-MSC中8.62％减少到0.17％；BM-MSC中6.16％减少到0.076％)。
实施例14：上述新生动物骨髓上清液NBMS对MSCs各器官归巢能力的影响
间充质干细胞具有“归巢”属性，通过静脉注射等途径移植后，可以迁移至特定的损伤部位，并在损伤部位内存活生长。间充质干细胞的这种生物学特性，是其修复机体损伤的重要能力。本实施例验证新生牛骨髓上清液NBMS添加物对MSCs移植后在体内各个器官驻留和存活生长能力的影响。本实施例所使用骨髓来源间充质干细胞BM-MSCs经慢病毒感染引入表达绿色荧光蛋白。实验组细胞为新生牛骨髓上清液NBMS添加物培养7天所得的MSCs(NBMS-MSCs)，对照组细胞为胎牛血清FBS培养7天所得的MSCs(FBS-MSCs)。其中，实验组“NBMS-MSCs”培养基成分为DMEM基础培养基(Gibco)，1/100 Pen/Strep双抗(Gibco)，体积比10％上述新生牛骨髓上清液NBMS；对照组“FBS-MSCs”培养基成分为DMEM基础培养基(Gibco)，1/100 Pen/Strep双抗(Gibco)，体积比10％商业化胎牛血清FBS(Gibco)。接种于六孔板中，每孔起始细胞数目为1X10^5。第1天待细胞贴壁后，分别更换培养基为上述条件培养基，每隔一天更换一次培养基。将实验组细胞与对照组细胞按5x10e6数量由尾静脉注射移植入8周龄重度免疫缺陷NSG小鼠。分别在移植后第24小时，7天，14天处死小鼠，收集外周血单个核细胞，骨髓，肺，肝脏进行流式细胞检测绿色荧光蛋白阳性细胞，以此计算在不同器官驻留的移植来源的MSCs细胞。结果如图15A、图15B、图15C和图15D所示，其中图15A、图15B、图15C和图15D分别显示了在移植后小鼠的外周血、骨髓、肺和肝脏中绿色荧光蛋白阳性细胞的百分比。结果显示，与FBS-MSCs相比，NBMS-MSCs移植后第7天在肝脏中有明显富集，移植后第14天在骨髓中有明显富集。以上实验结果显示，NBMS添加物培养间充质干细胞可促进其在肝脏和骨髓中的归巢。鉴于MSC的归巢能力是其发挥组织损伤修复的重要特性，说明NBMS添加物相比于胎牛血清更适合作为治疗性细胞的培养基添加物。
实施例15：上述新生动物骨髓上清液NBMS对造血干细胞HSC体外扩增的影响
本实施例中验证使用上述新生动物骨髓上清液NBMS对人造血干细胞HSC体外扩增的影响。用Stemspan作为造血干细胞(HSC)的基础培养基，并添加三种维持干细胞增殖必需的生长因子(10ng/ml SCF，100ng/ml TPO，100ng/ml FILT3)。在此基础培养4天之后，在基础培养基和三种细胞因子基础上添加所述10％NBMS添加物作为实验组处理人脐血来源CD34+细胞七天，对照组中加入10％体积PBS。如图16A所示为NBMS添加物培养HSC七天之后，通过显微镜观察获得其细胞形态和细胞数目图。图16B扩增7天后与起始细胞相比扩增倍数。结果显示，在现有培养基配方的基础上加入NBMS并不明显改变HSC细胞体外扩增倍数。
实施例16：上述新生动物骨髓上清液NBMS对造血干细胞HSC长期造血能力标记分子表达的影响
人造血干细胞中Lin-CD34-CD93+细胞群代表着一群静息状态的具有自我更新潜能和造血能力的HSC。用Stemspan作为造血干细胞(HSC)的基础培养基，并添加三种维持干细胞增殖必需的生长因子(10ng/ml SCF，100ng/ml TPO，100ng/ml FILT3)。在此基础培养4天之 后，在基础培养基和三种细胞因子基础上添加所述10％NBMS添加物作为实验组处理人脐血来源CD34+细胞七天，对照组中加入10％体积PBS。将添加NBMS的人脐血来源的CD34+细胞体外继续培养至第11天后，流式细胞术检测HSC表面Lineage-FITC、CD34-PECY7、CD93-BV750的表达，计算Lin-CD34-CD93+细胞群的占比。图17A结果显示：与添加PBS对照组相比，NBMS可增加Lin-CD34-CD93+细胞群的占比。
造血干细胞中Lin-CD34+CD166+细胞群代表着一群静息状态的具有自我更新潜能和造血能力的HSC。用Stemspan作为造血干细胞(HSC)的基础培养基，并添加三种维持干细胞增殖必需的生长因子(10ng/ml SCF，100ng/ml TPO，100ng/ml FILT3)。在此基础培养4天之后，在基础培养基和三种细胞因子基础上添加所述10％NBMS添加物作为实验组处理人脐血来源CD34+细胞七天，对照组中加入10％体积PBS。将添加NBMS的HSC体外继续培养至第11天后，单细胞测序技术检测HSC CD34+、CD166(ALCAM)+细胞的表达，计算CD34-CD116+细胞群的占比。图17B结果显示：与添加PBS对照组相比，NBMS可增加Lin-CD34-CD166+细胞群的占比。以上结果提示NBMS作为培养添加物，在不明显降低细胞体外扩增倍数的前提下，可增加具有造血潜能的造血干细胞分子标记物的表达。
本发明的技术方案不限于上述具体实施例的限制，凡是根据本发明的技术方案做出的技术变形，均落入本发明的保护范围之内。

Claims (14)

  1. 一种骨髓上清液，其通过对新生哺乳动物的骨髓样品进行处理获得，所述新生哺乳动物选自出生1天至1个月的非人类哺乳动物。
  2. 根据权利要求1所述的骨髓上清液，其特征在于，所述处理包括使所述骨髓样品的骨髓内容物在缓冲液中溶解、释放的步骤。
  3. 根据权利要求1所述的骨髓上清液，其特征在于，所述哺乳动物选自牛、羊、猪或马。
  4. 根据权利要求2所述的骨髓上清液，其特征在于，所述缓冲液选自PBS磷酸盐缓冲溶液、Hank's平衡盐溶液、碳酸盐缓冲液、硼酸盐缓冲液、枸橼酸盐缓冲液、酒石酸盐缓冲液或三羟甲基甲烷-盐酸缓冲液中的一种或多种。
  5. 一种培养基，其包括基础培养基和权利要求1-4中任一项所述的骨髓上清液。
  6. 根据权利要求5所述的培养基，其特征在于，所述培养基不含有血清，或含有0.1wt％-20wt％的血清。
  7. 一种细胞的培养方法，其包括在添加权利要求1-4中任一项所述的骨髓上清液的培养基中、或在权利要求5或6所述的培养基中培养细胞的步骤。
  8. 根据权利要求7所述的培养方法，其特征在于，所述细胞选自干细胞、原代细胞或永生化细胞；
    所述干细胞选自间充质干细胞、造血干细胞、胚胎干细胞、神经干细胞、皮肤干细胞、脂肪干细胞、视网膜干细胞、肝干细胞或胰腺干细胞；
    所述原代细胞选自哺乳动物肺上皮细胞、肠上皮细胞、成骨细胞、血管内皮细胞的一种或多种；
    所述永生化细胞选自人胚肾上皮细胞HEK293、人成纤维细胞、小鼠成纤维细胞中的一种或多种；
    所述细胞选自哺乳动物细胞，所述哺乳动物选自人、小鼠、大鼠、犬、猫、兔子、猪、猴、马、牛或羊。
  9. 权利要求1-4中任一项所述的骨髓上清液、权利要求5或6所述的培养基或权利要求7或8所述的培养方法在细胞培养或扩增中的应用。
  10. 权利要求7或8所述的培养方法获得的细胞或其细胞产物在制备药物、肿瘤模型、保健品或护理用品或在筛选抑瘤靶点、筛选药物中的应用。
  11. 根据权利要求10所述的应用，其特征在于，所述细胞或其细胞产物用于制备治疗心血管疾病、肝硬化、神经系统疾病、膝关节半月板部分切除损伤修复、自身免疫性疾病、免疫调节、抗炎、皮肤组织工程治疗或组织损伤修复的药物；和/或用于制备治疗恶性血液病、重型再生障碍性贫血、异常免疫病、代谢病或极重度骨髓型急性放射病的药物。
  12. 一种细胞或细胞群，其通过在添加了权利要求1-4任一项所述的骨髓上清液的培养基中或在权利要求5或6所述的培养基中，或利用权利要求7或8所述的细胞培养方法培养获得。
  13. 一种制剂，其包括权利要求1-4中任一项所述的骨髓上清液和药学上可接受的载体。
  14. 权利要求13所述的制剂在用于制备再生医学相关制剂中的应用。
PCT/CN2022/137352 2022-09-02 2022-12-07 一种骨髓上清液及其在细胞培养中的应用 WO2024045404A1 (zh)

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