WO2024045404A1 - Surnageant de moelle osseuse et son utilisation dans la culture cellulaire - Google Patents

Surnageant de moelle osseuse et son utilisation dans la culture cellulaire 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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Chinese (zh)
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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
    • C12N5/0634—Cells from the blood or the immune system
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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
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    • 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
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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

L'invention concerne un surnageant de moelle osseuse et son utilisation dans la culture de cellules souches. Le surnageant de moelle osseuse est obtenu au moyen du traitement d'un échantillon de moelle osseuse d'un mammifère nouveau-né, est utilisé en tant qu'additif de milieu de culture pour la culture cellulaire, peut obtenir une amplification efficace, et présente des perspectives d'application clinique.
PCT/CN2022/137352 2022-09-02 2022-12-07 Surnageant de moelle osseuse et son utilisation dans la culture cellulaire WO2024045404A1 (fr)

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