WO2021227573A1 - Milieu de culture exempt de xeno et procédé de multiplication de cellules souches mésenchymateuses au moyen de celui-ci - Google Patents

Milieu de culture exempt de xeno et procédé de multiplication de cellules souches mésenchymateuses au moyen de celui-ci Download PDF

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WO2021227573A1
WO2021227573A1 PCT/CN2021/075252 CN2021075252W WO2021227573A1 WO 2021227573 A1 WO2021227573 A1 WO 2021227573A1 CN 2021075252 W CN2021075252 W CN 2021075252W WO 2021227573 A1 WO2021227573 A1 WO 2021227573A1
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mesenchymal stem
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stem cells
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钟树根
邝纬阳
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梦芊科技知识产权有限公司
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    • C—CHEMISTRY; METALLURGY
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)

Definitions

  • the present disclosure generally relates to non-heterologous media, and more specifically to media and methods for expanding mesenchymal stem cells.
  • Stem cells are a group of undifferentiated cells that can regenerate somatic cells through cell division and differentiation.
  • human mesenchymal stem cells hMSC
  • hMSC human mesenchymal stem cells
  • hMSC Due to its high accessibility, proliferation potential, inherent immune regulation and repair properties, and multi-differentiation ability [1-4], hMSC has been widely used in stem cell therapy, tissue engineering [5,6], and drug discovery [3,4] ,7] and a reliable cell source for disease modeling [8,9]. It is easy to obtain hMSCs conveniently and directly from bone marrow, adipose tissue, amniotic fluid (AF), umbilical cord and placenta for seed culture for cell expansion [1,10]. Although many researchers have confirmed the therapeutic potential of hMSC, in order to fully meet the growing clinical needs, the challenge still lies in stable and scalable cell expansion. The low consistency maintenance of stem cell differentiation ability and potential and expensive stem cell culture hinder the research and application of hMSC in the current medical field. Therefore, it is urgent to find a more defined, low-cost medium for the expansion of hMSC without reducing its differentiation and potential.
  • the choice of growth medium is a critical step for amplification efficiency.
  • Animal serum is widely used as a source of growth factors in growth media [11,12].
  • the composition of animal serum is easy to change and unstable, and it is necessary to test its suitability before use, which will greatly increase operating costs [11,12].
  • uncertain serum components also carry the risk of spreading infectious agents or contamination by bacteria, fungi or viruses.
  • a new type of cell culture medium needs to be established under serum-free conditions to improve consistency, reduce operating costs and avoid infection.
  • the present disclosure provides a heterologous-free medium comprising a basal medium and at least 50 ng/mL fibroblast growth factor, such as 80-150 ng/mL, such as about 100 ng/mL.
  • the fibroblast growth factor is basic fibroblast growth factor (FGF2), especially human FGF2.
  • FGF2 basic fibroblast growth factor
  • the heterologous-free medium of the present disclosure may further comprise human platelet lysate and/or human serum.
  • the content of the human platelet lysate and/or human serum is 0.5-5% v/v, such as 0.5-1% v/v.
  • the basic medium may be selected from MEM medium, ⁇ -MEM medium, DMEM medium, IMDM medium, HAM F12 medium, DMEM/F12 mixed medium, PRMI1640 medium, StemSpan TM Medium and any combination thereof.
  • the heterologous-free medium of the present disclosure may further include nutrients required for cell growth, such as amino acids, vitamins, carbohydrates, and/or inorganic ions.
  • the heterologous-free medium of the present disclosure does not contain additional cell growth factors or hormones.
  • the present disclosure provides a medium supplement preparation comprising fibroblast growth factor, wherein the content of the fibroblast growth factor is such that after being added to the basal medium at a concentration of at least 50ng/mL Exist, for example 80-150 ng/mL, such as about 100 ng/mL.
  • the fibroblast growth factor is basic fibroblast growth factor (FGF2), especially human FGF2.
  • FGF2 basic fibroblast growth factor
  • the medium supplement formulation of the present disclosure may further comprise human platelet lysate and/or human serum.
  • the content of the human platelet lysate and/or human serum is such that it is present at a concentration of 0.5-5% v/v, such as 0.5-1% v/v after being added to the basal medium.
  • the medium supplement formulation of the present disclosure may further include nutrients required for cell growth, such as amino acids, vitamins, carbohydrates, and/or inorganic ions.
  • the medium supplement formulation of the present disclosure does not contain additional cell growth factors or hormones.
  • the present disclosure provides a method for expanding mesenchymal stem cells, which includes, without adding animal serum, under conditions suitable for the growth of the mesenchymal stem cells (1) according to the present invention.
  • the mesenchymal stem cells are cultured in the heterologous-free medium described in the disclosure, or (2) the mesenchymal stem cells are cultured in a basal medium supplemented with the medium supplement preparation described in the present disclosure to expand The mesenchymal stem cells.
  • the method of the present disclosure can obtain mesenchymal stem cells of about 0.8 ⁇ 10 5 cells/mL within 96 hours and the mesenchymal stem cells maintain about 99% of cell viability and mesenchymal stem cell identity.
  • the mesenchymal stem cells after passage of at least 50 generations, are substantially undifferentiated and the proliferation efficiency remains substantially unchanged.
  • the mesenchymal stem cells are human mesenchymal stem cells.
  • Figure 1 shows the proliferation of hMSC in different media.
  • A Culture hMSC in commercial human mesenchymal XF expansion medium containing 8ng/mL FGF2 or conditioned medium containing DMEM, where DMEM is supplemented with 2% (v/v) human platelet lysate, 1% Glutamax, 1% penicillin/streptomycin and 0, 8, 20, 50, 100, 150, 200, and 500 ng/mL FGF2 for 20 generations.
  • the proliferation rate is measured by MTT assay.
  • Figure 2 shows the verification of hMSC by microscope. Shows the growth in (A) commercial human mesenchymal XF expansion medium containing 8ng/mL FGF2, (B) conditioned medium with 0ng/mL FGF2, and (C) conditioned medium with 100ng/mL FGF2 Wide-field image of hMSC. (D) The dot plot shows the morphological analysis of hMSC grown in different media.
  • Figures 3A-3B show the verification of hMSC by microscope. Fluorescence images of hMSCs cultured in commercial human mesenchymal XF expansion medium containing 8 ng/mL FGF2, and conditioned medium with 0 and 100 ng/mL FGF2 are shown. The cells were stained with (A) MSC labeled CD44 and (B) epithelial labeled CD146 and counterstained with DAPI.
  • Figure 4 shows the verification of hMSC with FACS. Shown are cultured in commercial human mesenchymal XF expansion medium containing 8ng/mL FGF2, conditioned medium with 0 and 100ng/mL FGF2 and labeled with MSC (A)THY-1 and (B)STRO-1 and (C) Histogram of hMSC stained with epithelial marker CD146.
  • FIG. 5 shows the verification of hMSC with qPCR and Western blot.
  • MRNA was extracted from hMSCs cultured in commercial human mesenchymal XF amplification medium containing 8ng/mL FGF2, conditioned medium with 0 and 100ng/mL FGF2, and measured by qPCR.
  • the expression levels of (A) MSC markers CD44, THY-1 and STRO-1 and (B) epithelial marker CD146 and hematopoietic stem cell markers CD14 and CD19 are shown.
  • Lane 1 Commercial medium
  • Lane 2 Conditioned medium with 0 ng/mL FGF2
  • Lane 3 Conditioned medium with 100 ng/mL FGF2.
  • the term "about” as used herein refers to +/-10% of the specified value, more preferably +/-5%, such as +/-4%, +/-3%, +/-2 % Or +/-1%.
  • mesenchymal stem cells refers to undifferentiated pluripotent cells isolated from human or mammalian tissues, which have the ability to self-renew while maintaining pluripotency and differentiation into mesenchymal origin (for example, into Osteocytes, chondrocytes, adipocytes, stromal cells, fibroblasts, and tendons) or a variety of cell types of non-mesodermal origin (such as hepatocytes, nerve cells, and epithelial cells), and can be derived from various tissues.
  • mesenchymal stem cells can differentiate into mesenchymal cells such as bone, cartilage, muscle and fat cells, and fibrous connective tissue.
  • the mesenchymal stem cells may be umbilical cord-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, muscle-derived mesenchymal stem cells Mesenchymal stem cells, nerve-derived mesenchymal stem cells, skin-derived mesenchymal stem cells, amniotic membrane-derived mesenchymal stem cells, and placental-derived mesenchymal stem cells. Techniques for isolating stem cells from various tissues are known in the related art.
  • the mesenchymal stem cells are human mesenchymal stem cells (hMSC).
  • the present disclosure demonstrates a well-defined, heterologous-free conditioned medium containing human basic fibroblast growth factor (FGF2) for hMSC culture.
  • FGF2 human basic fibroblast growth factor
  • the results of the present disclosure indicate that hMSC cultured in a conditioned medium supplemented with a specific amount of FGF2 has enhanced proliferation activity and successfully maintained its elongated and spindle-shaped morphology. More importantly, the undifferentiated nature of hMSC has also been verified by FACS, microscopy, qPCR and Western blotting.
  • the fine-tuned growth medium of the present disclosure can be used for large-scale production of hMSC.
  • the inventors successfully used a fine-tuned growth medium containing a specific amount of FGF2 for hMSC expansion. The results showed that 0.8 ⁇ 10 5 /mL hMSC cells could be reached within 96 hours, and it was confirmed that 99% of cell viability and hMSC identity can be maintained.
  • the present disclosure provides a heterologous-free medium comprising a basal medium and at least about 50 ng/mL fibroblast growth factor, for example, about 80-150 ng/mL, such as about 100 ng/mL.
  • the heterologous-free medium of the present disclosure can be used to expand mesenchymal stem cells.
  • the term "medium” refers to a medium designed to support cell growth, such as a solid, liquid, or gel.
  • the medium constitutes and/or provides conditions suitable for allowing cell growth.
  • the culture medium can be solid, liquid or a mixture of various phases and materials.
  • the medium may include solid or liquid growth medium.
  • the culture medium also includes gel-like culture media such as agar, agarose, gelatin and collagen matrix.
  • the term “medium” also refers to material intended for cell culture, ie it has not yet been in contact with cells.
  • the term "xeno-free medium” refers to a medium that does not contain components from heterologous species.
  • the heterologous-free medium does not contain components of animal origin, such as animal serum such as fetal bovine serum.
  • the heterologous-free medium may contain additives of human origin, such as human serum or human platelet lysate.
  • the heterologous-free medium contains only human additives.
  • the medium of the present disclosure can be prepared by using a basal medium.
  • the term "basal medium” refers to a non-supplemented medium suitable for exposure to cells (e.g., MSC).
  • the basic medium includes, for example, Eagle's Minimum Essential (MEM) medium, ⁇ -modified MEM ( ⁇ -MEM) medium, Dulbecco's Modified Eagle's Medium (DMEM), and Ithev's modified medium Dulbecco's Medium (Iscove's Modified Dulbecco's Medium, IMDM), HAM F12 medium, DMEM/F12 mixed medium, PRMI1640 medium, StemSpan TM medium, and any combination thereof, and are not particularly limited, as long as it can be used to cultivate stem cells .
  • Other basic media suitable for MSC culture are known in the art.
  • the cell culture medium of the present disclosure may further contain nutrients required for cell growth, such as amino acids, vitamins, carbohydrates and/or inorganic ions, and antibiotics to prevent bacterial contamination.
  • nutrients required for cell growth such as amino acids, vitamins, carbohydrates and/or inorganic ions, and antibiotics to prevent bacterial contamination.
  • the cell culture medium of the present disclosure contains one or more or all essential amino acids, and may also contain one or more non-essential amino acids.
  • Amino acids include essential amino acids such as Thr, Met, Val, Leu, Ile, Phe, Trp, Lys, and His; and non-essential amino acids such as Gly, Ala, Ser, Cys, Gln, Asn, Asp, Tyr, Arg, and Pro.
  • the cell culture medium used in the present disclosure may be supplemented with Glutamax.
  • the cell culture medium of the present disclosure may contain vitamins, such as fat-soluble vitamins such as A, D, E, K; and/or water-soluble vitamins such as B1, B2, B6, B12, pantothenic acid, folic acid, biotin , C, Nicotinamide, etc.
  • vitamins such as fat-soluble vitamins such as A, D, E, K
  • water-soluble vitamins such as B1, B2, B6, B12, pantothenic acid, folic acid, biotin , C, Nicotinamide, etc.
  • the cell culture medium of the present disclosure may include carbohydrates.
  • Carbohydrates are the main energy source for cell growth, some of which are the components of protein and nucleic acid synthesis, such as glucose, ribose, deoxyribose, sodium pyruvate and acetic acid.
  • the cell culture medium of the present disclosure may contain inorganic ions, such as sodium, potassium, magnesium, calcium, phosphorus, and the like.
  • the cell culture medium of the present disclosure may include antibiotics, such as penicillin, streptomycin, kanamycin (for example, at a concentration of 50 ug/ml), and/or nystatin (for example, at a concentration of 25 U/ml).
  • antibiotics such as penicillin, streptomycin, kanamycin (for example, at a concentration of 50 ug/ml), and/or nystatin (for example, at a concentration of 25 U/ml).
  • each milliliter of culture broth may contain 100 U penicillin and 100 ug streptomycin.
  • the basal medium needs to be suitable for the target cell line, where the key nutrients have sufficient levels to maintain cell proliferation. For example, if it is found that the energy source of glucose is depleted and thus restricts cell proliferation, it may be necessary to increase the level of glucose (or other energy source) in the basal medium or add glucose (or other energy source) during the culture process. .
  • Fibroblast growth factor is a kind of polypeptide composed of about 150-200 amino acids, and exists in two closely related forms, namely basic fibroblast growth factor (bFGF) and acidic fibroblast growth factor (aFGF).
  • Basic fibroblast growth factor also known as FGF2
  • FGF2 is a well-known growth factor that can replicate various types of stem cells from different sources [13-19].
  • FGF2 basic fibroblast growth factor
  • FGF2 also known as FGF2
  • FGF2 acidic fibroblast growth factor
  • FGF2 acidic fibroblast growth factor
  • the heterologous-free medium of the present disclosure contains at least about 50 ng/mL fibroblast growth factor, for example, about 80-150 ng/mL, such as about 100 ng/mL.
  • the fibroblast growth factor is basic fibroblast growth factor (FGF2), especially human FGF2.
  • FGF2 can be natural or recombinant.
  • the level of FGF2 is at least about 80 ng/ml, at least about 90 ng/ml, at least about 95 ng/ml, or at least about 100 ng/ml. In some embodiments, the level of FGF2 does not exceed about 500 ng/ml, for example, does not exceed about 400 ng/ml, does not exceed about 300 ng/ml, does not exceed about 200 ng/ml, does not exceed about 150 ng/ml, does not exceed about 140 ng/ml. ml, not more than about 130ng/ml, not more than about 120ng/ml, or not more than about 110ng/ml.
  • the level of FGF2 is about 80-150ng/mL, for example about 90-140ng/mL, about 95-130ng/mL, about 100-120ng/mL, about 100-110ng/mL, about 100-105ng /mL or about 100ng/mL.
  • the heterologous-free medium of the present disclosure does not contain additional cell growth factors or hormones. In one embodiment, the heterologous-free medium of the present disclosure does not contain cell growth factors or hormones other than FGF2. As used herein, the term "does not contain additional cell growth factors or hormones" means that it does not contain other cell growth factors or hormones other than fibroblast growth factors such as FGF2 added to the culture medium of the present disclosure.
  • the culture medium of the present disclosure contains human-derived additives.
  • human platelet lysate and/or human serum can be added to the medium of the present disclosure.
  • the medium of the present disclosure may not contain additional cell growth factors or hormones, or if human platelet lysate and/or human serum are added, human platelets are lysed
  • the amount of food and/or human serum can be as low as 0.5% (v/v).
  • the amount of human platelet lysate or human serum added to the medium of the present disclosure can be as low as 0.5% (v/v), and does not require or contain Additional cell growth factors or hormones.
  • the term "does not need or does not contain additional cell growth factors or hormones” means that it does not need or does not contain fibroblast growth factors such as FGF2 and human platelet lysates other than those added to the culture medium of the present disclosure. Cell growth factors or hormones other than cell growth factors in serum.
  • the human platelet lysate or human serum is at about 0.5%-5% v/v (e.g., about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1% by volume). %, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2%, about 2.5%, about 3%, about 4%, Or about 5%), preferably about 0.5%-1.0% v/v, or more preferably about 0.5%-0.8% v/v (for example, about 0.5% v/v).
  • the concentration of the mixture of human platelet lysate and human serum may be as low as 0.5% (v/v), such as about 0.5%-5% v/v (for example, about 0.6% by volume, about 0.7% by volume).
  • concentration of v/v (for example, about 0.5% v/v) is used.
  • the platelet lysate can be obtained from any suitable source.
  • a suitable commercial source is PLT Max from Mill Creek Life Sciences (Rochester, Minnesota, USA) or platelet lysate from Millipore.
  • the platelet lysate is derived from the same species as the cultured MSC.
  • the term "derived from " platelet lysate is used to describe platelet lysate that has been prepared from a blood sample, for example by separating platelets from the blood sample and then lysing the separated platelets.
  • the blood sample from which the platelet lysate is derived may or may not be from the same individual as the MSC, or when the MSC is prepared from adipose tissue, the blood sample derived may be from the adipose tissue used to prepare the MSC. Generally, the blood sample is from an individual different from the individual from which the MSC or adipose tissue was obtained.
  • the platelet lysate can be prepared from fresh whole blood or from stored whole blood using methods or kits known to those skilled in the art.
  • the platelet lysate can be from a single donor, or it can be from pooled blood or cells.
  • the platelet lysate can be prepared by transfusing whole blood or platelets, for example, about 5 to 7 days after collection.
  • Platelet lysates can be prepared from blood using commercially available kits (for example the platelet lysate kit from MacoPharma (France)).
  • a platelet lysate is prepared from blood collected in the presence of an anticoagulant, such as sodium heparin or citrate.
  • the blood is centrifuged under appropriate conditions, for example, at 200 g for about 20 minutes, after which the platelets (top layer) are collected, and then the platelets are frozen-thawed to lyse the cells. Generally, multiple rounds of freezing-thawing are performed, for example, two rounds, three rounds, four rounds or more.
  • the lysed platelets are centrifuged to allow the pelleted cell fragments to be discarded, for example, at 4000 g for about 10 minutes.
  • the platelet lysate can be sterilized, for example, by filtration through a suitable matrix (e.g., 0.22 micron filter), and stored under appropriate conditions (e.g., -80°C) until use.
  • Human serum can be obtained from any suitable source, such as a commercial source.
  • the human serum is human AB serum, such as human AB serum from Sigma or Gibco; or a human AB serum series from Gemini, including GemCell TM human AB serum, GemCell Plus TM human AB serum, and the like.
  • the medium of the present disclosure may be provided as a complete medium, in which the basic medium and other ingredients have been mixed together before cell culture.
  • the cell culture medium components may be provided separately and mixed with a suitable basal medium before or during cell culture.
  • the present disclosure provides a medium supplement preparation comprising a fibroblast growth factor, wherein the content of the fibroblast growth factor is sufficient to be present at a concentration of at least 50 ng/mL after being added to the basal medium, for example 80-150ng/mL, such as about 100ng/mL.
  • the medium supplement formulation further comprises human platelet lysate and/or human serum.
  • the content of human platelet lysate and/or human serum is such that it is present at a concentration of about 0.5-5% v/v, such as about 0.5-1% v/v after being added to the basal medium.
  • the medium supplement formulation further contains nutrients required for cell growth, such as amino acids, vitamins, carbohydrates, and/or inorganic ions.
  • the medium supplement formulation does not contain additional cell growth factors or hormones.
  • the medium or medium supplement formulation of the present disclosure can be packaged in or with a suitable solvent or packaged in a lyophilized form.
  • the cell culture medium and/or medium supplement formulations disclosed herein may optionally be packaged in a suitable container together with instructions for use for the desired purpose.
  • the present disclosure provides a method for amplifying mesenchymal stem cells, which includes, without adding animal serum, under conditions suitable for the growth of the mesenchymal stem cells (1) described in the present disclosure
  • the mesenchymal stem cells are cultured in a heterogeneous culture medium, or (2) the mesenchymal stem cells are cultured in a basal medium supplemented with the medium supplement described in the present disclosure to expand the mesenchymal stem cells.
  • the method described in the present disclosure obtains mesenchymal stem cells of about 0.8 ⁇ 10 5 cells/mL within 96 hours, and the expanded mesenchymal stem cells maintain about 99% of cell viability and mesenchymal stem cells identity.
  • the mesenchymal stem cells are substantially undifferentiated and the proliferation efficiency remains substantially unchanged.
  • proliferation efficiency is at least about 95%, at least about 96%, at least about 97%, at least about 98%, At least about 99% or higher.
  • Culturing according to this aspect of the present disclosure can be performed for a limited amount of time so that expansion does not occur, for example, only during the cell seeding phase, or for a longer period of time to allow expansion of mesenchymal stem cells, thereby obtaining increased Number of cells.
  • the adherent cells can be harvested using trypsin/EDTA or by a cell scraper, and the cells are dissociated by a pipette, and re-plated , for example, at a density of about 100 to about 10,000 cells/cm 2 .
  • the cultivation is performed for at least one day, at least two days, at least three days, at least four days, at least five days, at least six days, at least one week, at least two weeks, at least three weeks, at least four weeks, or At least five weeks.
  • the cells are expanded by at least two population doubling numbers, at least four population doubling numbers, at least six population doubling numbers, at least eight population doubling numbers, at least ten population doubling numbers, at least 15 population doubling numbers , At least 20 population doubling numbers, at least 25 population doubling numbers, at least 30 population doubling numbers, at least 35 population doubling numbers, at least 40 population doubling numbers, and at least 45 population doubling numbers.
  • mesenchymal stem cells can be selected and confirmed based on the expression of mesenchymal stem cell surface markers. Selection or sorting can include selecting mesenchymal stem cells (MSC) from a mixed cell population by one or more such surface markers. In one embodiment, the mesenchymal stem cell surface marker may be selected from CD44, THY-1, STRO-1, and any combination thereof.
  • MSC mesenchymal stem cells
  • the present disclosure is based at least in part on the surprising finding:
  • the medium of the present disclosure may not contain additional cell growth factors or hormones, or if adding human platelet lysate and/or human serum
  • the amount of human platelet lysate and/or human serum can be as low as 0.5% (v/v).
  • the cultured cells showed a proliferation rate comparable to that of commercial media.
  • 100 ng/mL FGF2 was provided to the conditioned medium, the maximum hMSC proliferation rate was shown.
  • hMSC grown in commercial media and conditioned media containing 50ng/mL FGF2 can reach 0.7 ⁇ 10 5 viable cells per square centimeter in approximately 96 hours.
  • concentration of FGF2 added to the medium of the present disclosure is 100ng/mL, it only takes 96 hours to reach 0.8 ⁇ 10 5 live hMSCs per square centimeter, and the cell survival rate is greater than 99%, thereby further improving the proliferation rate and Confirmed that 99% of cell viability and hMSC identity can be maintained.
  • the hMSC cultured with the medium of the present disclosure is still basically undifferentiated and the proliferation efficiency remains basically unchanged when it is passaged for at least 50 times. It can be seen that the non-exogenous medium of the present disclosure can replace or even be better than commercial XF expansion medium.
  • Human mesenchymal stem cells extracted from bone marrow were purchased from Merck Millipore. Place the cells in human mesenchymal XF expansion medium or supplemented with 0.5% v/v human platelet lysate (Millipore), 1% w/v Glutamax (Life Technologies, California, USA), 1% w/v penicillin -Streptomycin (Life Technologies, California, USA) and DMEM medium (Life Technologies, CA, USA) with different concentrations of FGF2 were cultured in a humidified incubator maintained at 37°C and 5% CO 2. When 80% confluence was reached, the cells were plated at a density of 5,000 cells/cm 2. Before plating, all culture plates were coated with 0.1% gelatin solution for 30 minutes at room temperature. The cell culture medium is changed daily.
  • the hMSCs were plated on 96-well plates and cultured in human mesenchymal XF expansion medium or non-exogenous conditioned medium.
  • the cell viability was determined by adding MTT to a final concentration of 1 mg/mL [22], and incubated at 37°C for 6 hours. Then the medium was replaced with DMSO, and the absorbance was measured at 540nm in a microplate reader.
  • hMSC To measure the proliferation of hMSC, cells were cultured in different media for 2, 3, and 4 days before treatment with trypsin. Then, trypan blue stained the trypan-digested cells, and a Thermo Fisher Countess II automatic cell counter was used to count the living cells.
  • the cells were plated on gelatin-coated coverslips for imaging.
  • the cells were washed twice with ice-cold PBS, and then fixed in 4% w/v paraformaldehyde for 30 minutes at room temperature. Subsequently, the cells were blocked in 4% w/v blocking donkey serum and combined with mouse monoclonal CD44 antibody (1:500, CBL154), THY-1 antibody (1:500, CBL415), STRO-1 antibody (1:500, CBL415), and STRO-1 antibody. : 500, MAB4315), CD146 antibody (1:500, MAB16985), CD14 antibody (1:500, MAB1219) or CD19 antibody (1:500, MAB1794) incubate overnight at 4°C.
  • the cells were stained with Alexa Fluor 647-conjugated goat anti-mouse antibody (1:1000, Invitrogen), and counter-stained with DAPI for 1 hour at room temperature. Then the cells were washed three times with PBS, and then fixed on a glass slide with ProLong Antifade Mountant (Thermo Fisher) for observation.
  • phase contrast image was taken with a Nikon Eclipse Ti inverted microscope. As previously described, the captured images were analyzed using ImageJ software [23]. In short, the cell area and Feret diameter were measured using the plug-in "Measure and Lavel". The hMSC was plated and allowed to grow for 3 days, and then the measurement was performed. 100 cells from each medium were measured, and the experiment was repeated three times independently. Then plot the data on a dot plot of area versus maximum cell diameter.
  • the cells in a 100 mm petri dish were trypsinized and washed three times with ice-cold PBS. The cells were then fixed with ice-cold ethanol at 4°C for 30 minutes. After fixation, the cells were incubated in 1% w/v BSA in PBS, and then blocked in 4% w/v blocking donkey serum for 30 minutes. The cells were then stained with the same primary antibody in the immunocytochemistry section. After washing, the cells were stained with Alexa Fluor 488-conjugated goat anti-mouse antibody (Invitrogen) at room temperature for 30 minutes. Then the cells were washed and resuspended in PBS, and analyzed on BD FACSAria III (BD Bioscience).
  • Alexa Fluor 488-conjugated goat anti-mouse antibody Invitrogen
  • RNA was extracted from the hMSC monolayer using RNAzol reagent (Molecular Research Center). The yield of RNA was quantified with Nanodrop (Thermo Fisher). According to the manufacturer's instructions, 100 ug RNA was reverse transcribed with GoScript reverse transcriptase (Promega) using oligonucleotide (dT) 15 primers. According to the manufacturer's instructions, the LightCycler 480 SYBR Green I premix was used, and the LightCycler 480 qPCR instrument (Roche) was used to quantify the mRNA level of the target gene. Gene expression was normalized to 18S rRNA and calculated using 2'ct. All samples were run in triplicate. The average gene expression was calculated in 3 independent experiments.
  • FGF2 fibroblast growth factor 2
  • hMSC human mesenchymal stem cells
  • the inventors used DMEM to formulate a non-heterogeneous formula of conditioned medium containing 0.5% human platelet lysate, 1% Glutamax, 1% penicillin/streptomycin and 0, 8, 20, 50 , 100, 150, 200 and 500ng/mL FGF2 for 50 passages.
  • Cell viability was measured by MTT assay.
  • the conditioned medium of the present disclosure reduces the proliferation rate of hMSC ( Figure 1A).
  • the cultured cells showed a proliferation rate comparable to that of the commercial medium.
  • the hMSC grown in commercial medium and conditioned medium containing 50ng/mL FGF2 takes about 96 hours to reach 0.7 ⁇ 10 5 viable cells per square centimeter.
  • the results show that when the final concentration is 100ng/mL FGF2, it only takes 96 hours to reach 0.8 ⁇ 10 5 live hMSCs per square centimeter, and the cell survival rate is greater than 99%, which further improves the proliferation rate. It can be seen that the non-exogenous medium of the present disclosure is even better than the commercial XF expansion medium.
  • the stem cell markers were further verified.
  • the hMSCs were maintained in commercial XF expansion medium and conditioned medium containing 0 or 100 ng/mL FGF2 supplements until passage 10 and cultured for 4 days. Observe the cells under a phase contrast microscope. Cells cultured in commercial XF expansion medium and conditioned medium containing 100 ng/mL FGF2 both showed spindle and pointed morphology ( Figures 2A and 2C). The cells cultured in the conditioned medium without FGF2 showed a flatter epithelial morphology ( Figure 2B).
  • FACS fluorescence assisted cell sorting
  • the cells cultured in the medium without FGF2 did not show detectable mRNA levels after using the mesenchymal stem cell marker (Figure 5A); in contrast, they showed positive mRNA levels for the epithelial marker CD146 ( Figure 5B). None of the cells expressed hematopoietic stem cell markers CD14 and CD19 ( Figure 5B).
  • stem cells have advantages in cell therapy [3,4,8,10], so far, the application of stem cells has been limited by high production due to the need for expensive commercial growth media such as XF media, minimal media, etc. Cost [25,26]. Although the use of serum can reduce costs, unknown variables (such as the presence of viruses and allergens) may become problems when entering clinical trials [11,12]. In order to promote the transition of stem cells from basic research to clinical applications, it is important to develop cost-effective, heterologous-free media for the strong expansion of human stem cells.
  • CD44, THY-1 and STRO-1 are the most commonly used hMSC markers [10,27-29].
  • the multi-structure glycoproteins CD44 and THY-1 expressed on the membrane surface can trigger various cell functions, including differentiation, proliferation, cell adhesion and apoptosis.
  • STRO-1-rich hMSC promotes cell differentiation into a variety of mesenchymal mass lines, such as bone marrow stromal cells, adipocytes, osteoblasts, fibroblasts and myoblasts [28]. Under a fluorescence microscope, hMSCs cultured with FGF2 were positive for the hMSC surface marker CD44 and negative for the hematological marker CD19.
  • the combination of cell viability, flow cytometry, morphology, and immunocytochemical characteristics indicate that the addition of FGF2 is essential for the growth of undifferentiated hMSCs.
  • using the fine-tuned medium supplemented with FGF2 of at least 50 ng/mL, preferably at least 100 ng/mL of the present disclosure can not only effectively promote the expansion of hMSC, but also maintain the pluripotency of its differentiation into multiple lineages. This development can help achieve low-cost and scalable production of hMSC cultures for commercial and therapeutic applications.
  • the present disclosure has presented the method and/or process of the present invention in a specific step sequence.
  • the method or process should not be limited to the specific sequence of steps described.
  • Other sequence of steps are possible. Therefore, the specific sequence of steps disclosed herein should not be construed as limiting the present invention.
  • the disclosure of methods and/or processes should not be limited to performing their steps in the described order. Such order may vary and still be within the scope of the present invention.

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Abstract

L'invention concerne un milieu de culture exempt de xeno, qui contient un milieu de base et au moins 50 ng/mL d'un facteur de croissance des fibroblastes. L'invention concerne également une préparation de supplémentation en milieu de culture, qui contient au moins 50 ng/mL d'un facteur de croissance des fibroblastes. L'invention concerne en outre un procédé de multiplication de cellules souches mésenchymateuses au moyen du milieu de culture exempt de xeno ou de la préparation de supplémentation de milieu de culture.
PCT/CN2021/075252 2020-05-14 2021-02-04 Milieu de culture exempt de xeno et procédé de multiplication de cellules souches mésenchymateuses au moyen de celui-ci WO2021227573A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008129563A2 (fr) * 2007-04-23 2008-10-30 Stempeutics Research Private Limited, Cellules souches mésenchymales humaines et leur préparation
CN101384703A (zh) * 2005-10-07 2009-03-11 塞拉提斯股份公司 用于获得无异源物质的hBS细胞系的方法
CN104894064A (zh) * 2015-07-08 2015-09-09 河南中科干细胞基因工程有限公司 一种用于培养间充质干细胞的培养基
CN105200008A (zh) * 2015-10-23 2015-12-30 新乡医学院 干细胞培养基
CN107475185A (zh) * 2016-06-07 2017-12-15 杨旅军 一种用于培养人间充质干细胞的无动物源成分培养基
CN110564680A (zh) * 2019-08-27 2019-12-13 西安艾尔菲生物科技有限公司 人脐带间充质干细胞无血清培养基和制备方法及人脐带间充质干细胞无血清培养液获取方法

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009145761A1 (fr) * 2008-05-27 2009-12-03 Mayo Foundation For Medical Education And Research Procédés et matériaux destinés à utiliser des cellules dans le traitement du tissu cardiaque
WO2011101834A1 (fr) * 2010-02-22 2011-08-25 Advanced Neuro-Science Allies Private Limited Procédé d'obtention de cellules souches mésenchymateuses, milieux utilisés, procédés et composition associés
CN105154395B (zh) * 2015-09-28 2018-09-28 丛秀丽 一种增强MSCs免疫调节功能的临床级别细胞制备方法
CN106701671A (zh) * 2016-11-14 2017-05-24 天津市康婷生物工程有限公司 一种利于间充质干细胞应用于骨再生的培养体系
LT3573630T (lt) * 2017-01-27 2021-05-25 Xintela Ab Kaulų ir kremzlių pažeidimų arba ligų prevencija ir gydymas
CN106591230A (zh) * 2017-02-09 2017-04-26 上海爱萨尔生物科技有限公司 人脐带间充质干细胞培养液及其培养方法
CN107056896B (zh) * 2017-06-05 2020-05-26 沃昕生物科技(深圳)有限公司 多肽及含有多肽的培养基
CN107519126B (zh) * 2017-07-13 2021-05-28 沃昕生物科技(深圳)有限公司 一种脐带干细胞除皱剂及其制备方法
CN109897815B (zh) * 2019-02-20 2021-04-16 北京京蒙高科干细胞技术有限公司 一种无需包被的脂肪内皮祖细胞的高效分离和培养方法
CN111139221B (zh) * 2020-01-09 2022-05-10 赛瑞诺(北京)生物科技有限公司 一种羊膜间充质干细胞的培养及冻存方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101384703A (zh) * 2005-10-07 2009-03-11 塞拉提斯股份公司 用于获得无异源物质的hBS细胞系的方法
WO2008129563A2 (fr) * 2007-04-23 2008-10-30 Stempeutics Research Private Limited, Cellules souches mésenchymales humaines et leur préparation
CN104894064A (zh) * 2015-07-08 2015-09-09 河南中科干细胞基因工程有限公司 一种用于培养间充质干细胞的培养基
CN105200008A (zh) * 2015-10-23 2015-12-30 新乡医学院 干细胞培养基
CN107475185A (zh) * 2016-06-07 2017-12-15 杨旅军 一种用于培养人间充质干细胞的无动物源成分培养基
CN110564680A (zh) * 2019-08-27 2019-12-13 西安艾尔菲生物科技有限公司 人脐带间充质干细胞无血清培养基和制备方法及人脐带间充质干细胞无血清培养液获取方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
RAJENDRAN ARUN KUMAR, ARISAKA YOSHINORI, ISEKI SACHIKO, YUI NOBUHIKO: "Sulfonated Polyrotaxane Surfaces with Basic Fibroblast Growth Factor Alter the Osteogenic Potential of Human Mesenchymal Stem Cells in Short-Term Culture", ACS BIOMATERIALS SCIENCE & ENGINEERING, AMERICAN CHEMICAL SOCIETY, US, vol. 5, no. 11, 11 November 2019 (2019-11-11), US , pages 5652 - 5659, XP055869712, ISSN: 2373-9878, DOI: 10.1021/acsbiomaterials.8b01343 *
WU DI: "In Vitro Expansion of Human Mesenchymal Stem Cells in Animal Serum-Free Media", CHINESE JOURNAL OF TISSUE ENGINEERING RESEARCH, vol. 18, no. 28, 2 July 2014 (2014-07-02), pages 4583 - 4587, XP055852100 *

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