WO2021227573A1

WO2021227573A1 - Xeno-free culture medium and method for expansion of mesenchymal stem cells by means of using same

Info

Publication number: WO2021227573A1
Application number: PCT/CN2021/075252
Authority: WO
Inventors: 钟树根; 邝纬阳
Original assignee: 梦芊科技知识产权有限公司
Priority date: 2020-05-14
Filing date: 2021-02-04
Publication date: 2021-11-18
Also published as: CN113667635A

Abstract

Provided is a xeno-free culture medium, which contains a basal medium and at least 50 ng/mL of a fibroblast growth factor. Also provided is a culture medium supplementation preparation, which contains at least 50 ng/mL of a fibroblast growth factor. Also provided is a method for the expansion of mesenchymal stem cells by means of using the xeno-free culture medium or the culture medium supplementation preparation.

Description

Non-heterogeneous culture medium and method for amplifying mesenchymal stem cells using the same

Technical field

The present disclosure generally relates to non-heterologous media, and more specifically to media and methods for expanding mesenchymal stem cells.

Background technique

Stem cells are a group of undifferentiated cells that can regenerate somatic cells through cell division and differentiation. In the stem cell lineage, human mesenchymal stem cells (hMSC) are adult stem cells that can be isolated from human tissues such as bone marrow, adipose tissue, and amniotic fluid.

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]. However, 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]. In addition, uncertain serum components also carry the risk of spreading infectious agents or contamination by bacteria, fungi or viruses. In view of the above shortcomings of serum-containing growth media [11,12], a new type of cell culture medium needs to be established under serum-free conditions to improve consistency, reduce operating costs and avoid infection.

In the art, there is still a need for non-exogenous culture media and methods that can cost-effectively expand hMSC without reducing its differentiability and potential.

Summary of the invention

In one aspect, 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.

In one embodiment, the fibroblast growth factor is basic fibroblast growth factor (FGF2), especially human FGF2.

In one embodiment, the heterologous-free medium of the present disclosure may further comprise human platelet lysate and/or human serum. In one embodiment, the content of the human platelet lysate and/or human serum is 0.5-5% v/v, such as 0.5-1% v/v.

In one embodiment, the basic medium may be selected from MEM medium, α-MEM medium, DMEM medium, IMDM medium, HAM F12 medium, DMEM/F12 mixed medium, PRMI1640 medium, StemSpan ^™ Medium and any combination thereof.

In one embodiment, 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.

In one embodiment, the heterologous-free medium of the present disclosure does not contain additional cell growth factors or hormones.

In another aspect, 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.

In one embodiment, the medium supplement formulation of the present disclosure may further comprise human platelet lysate and/or human serum. In one embodiment, 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.

In one embodiment, 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.

In one embodiment, the medium supplement formulation of the present disclosure does not contain additional cell growth factors or hormones.

In yet another aspect, 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.

In one embodiment, 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.

In one embodiment, after passage of at least 50 generations, the mesenchymal stem cells are substantially undifferentiated and the proliferation efficiency remains substantially unchanged.

In one embodiment, the mesenchymal stem cells are human mesenchymal stem cells.

Description of the drawings

The drawings only illustrate the present invention more clearly, but do not limit the scope of disclosure and protection of the present invention in any way.

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. (B) At the 10th ^{passage, 5,000 cells/cm 2} hMSC were plated on a 6-well plate and placed in commercial human mesenchymal XF expansion medium containing 8ng/mL FGF2 or conditioned medium containing DMEM Culture, where DMEM contains 2% (v/v) human platelet lysate, 1% Glutamax, 1% penicillin/streptomycin, and 0, 8, 20, 50, and 100 ng/mL FGF2. At designated time points, cells were harvested and viable cells were counted.

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.

Figure 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. (C) Western blot of hMSC grown on different media probed with antibodies against CD44, THY-1 and CD146. Lane 1: Commercial medium; Lane 2: Conditioned medium with 0 ng/mL FGF2; Lane 3: Conditioned medium with 100 ng/mL FGF2.

Detailed ways

For the purpose of promoting an understanding of the principles of the present invention, reference is now made to the embodiments illustrated in the drawings, and the embodiments will be described in detail. However, it will be understood that these descriptions are not intended to limit the scope of the present invention in any way.

The following provides a description of non-heterogeneous media and methods that can be used for culturing mesenchymal stem cells. These media and methods meet at least one need in the art.

The chapter headings used in this article are for organizational purposes only and should not be construed as limiting the subject matter in any way.

Unless clearly defined otherwise, the terms used herein should be understood according to their usual meanings in the art. Unless otherwise specified or instructed in the context, nouns that are not modified by a quantifier represent one or more types.

Unless otherwise stated, the term "about" as used herein refers to +/-10% of the specified value, more preferably +/-5%, such as +/-4%, +/-3%, +/-2 % Or +/-1%.

As used herein, the term "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. For example, mesenchymal stem cells can differentiate into mesenchymal cells such as bone, cartilage, muscle and fat cells, and fibrous connective tissue. In some embodiments, 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. Due to the high differentiation ability to differentiate into multiple lineages of different cell types, human mesenchymal stem cells have a high value in regenerative medicine. Therefore, in one embodiment, the mesenchymal stem cells are human mesenchymal stem cells (hMSC).

It should be noted that, in the case of no conflict, the features in the embodiments and examples in the present disclosure can be combined with each other.

The present disclosure demonstrates a well-defined, heterologous-free conditioned medium containing human basic fibroblast growth factor (FGF2) for hMSC culture. 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. In the present disclosure, 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.

In one aspect, 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. In some embodiments, the heterologous-free medium of the present disclosure can be used to expand mesenchymal stem cells.

Culture medium

As used herein, 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.

As used herein, the term "xeno-free medium" refers to a medium that does not contain components from heterologous species. For example, when used to culture human MSCs, the "heterologous-free medium" does not contain components of animal origin, such as animal serum such as fetal bovine serum. In some embodiments, the heterologous-free medium may contain additives of human origin, such as human serum or human platelet lysate. In some embodiments, the heterologous-free medium contains only human additives.

The medium of the present disclosure can be prepared by using a basal medium. As used herein, 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, α-modified MEM (α-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.

In some embodiments, 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.

In some embodiments, 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. In one embodiment, the cell culture medium used in the present disclosure may be supplemented with Glutamax.

In some embodiments, 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.

In some embodiments, 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.

In some embodiments, the cell culture medium of the present disclosure may contain inorganic ions, such as sodium, potassium, magnesium, calcium, phosphorus, and the like.

In some embodiments, 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). In one embodiment, each milliliter of culture broth may contain 100 U penicillin and 100 ug streptomycin.

Those skilled in the art will understand that in order to obtain the best results, 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

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 (bFGF, also known as FGF2) is a well-known growth factor that can replicate various types of stem cells from different sources [13-19]. In the case of feeder-free culture, adding FGF2 to the cell culture medium can effectively promote the proliferation, self-renewal and pluripotency of human embryonic stem cells [20,21]. Based on the recently developed 2293T expression system of FGF with high yield and high biological activity [15], the inventors further tried to use FGF2 to formulate a cost-effective growth medium for hMSC culture.

In one embodiment, 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. In one embodiment, the fibroblast growth factor is basic fibroblast growth factor (FGF2), especially human FGF2. In one embodiment, FGF2 can be natural or recombinant.

In some embodiments, 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. In other embodiments, 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.

In one embodiment, 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.

Human additives

In one embodiment, the culture medium of the present disclosure contains human-derived additives. For example, human platelet lysate and/or human serum can be added to the medium of the present disclosure.

Surprisingly, when the amount of FGF2 of the present disclosure is added, 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). In one embodiment, when the amount of FGF2 of the present disclosure is added, 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. As used herein, 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.

In some embodiments, 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).

In some embodiments, it is contemplated to use a mixture of human platelet lysate and human serum. In this case, 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). %, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, Use at a concentration of about 2%, about 2.5%, about 3%, about 4%, or about 5%), preferably at a concentration of about 0.5%-1.0% v/v, or more preferably at a concentration of about 0.5%-0.8% The 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. As used herein, 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)). In one embodiment, 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. In one embodiment, 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 ^™ human AB serum, GemCell Plus ^™ human AB serum, and the like.

Medium supplement

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. Alternatively, the cell culture medium components may be provided separately and mixed with a suitable basal medium before or during cell culture.

In one aspect, 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. In some embodiments, the medium supplement formulation further comprises human platelet lysate and/or human serum. In some embodiments, 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. In some embodiments, the medium supplement formulation further contains nutrients required for cell growth, such as amino acids, vitamins, carbohydrates, and/or inorganic ions. In some embodiments, the medium supplement formulation does not contain additional cell growth factors or hormones.

In one embodiment, 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.

Method of expanding mesenchymal stem cells

In one aspect, 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.

In one embodiment, 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. In one embodiment, after at least 50 generations, the mesenchymal stem cells are substantially undifferentiated and the proliferation efficiency remains substantially unchanged. As used herein, the term "proliferation efficiency remains substantially unchanged" means that the 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. For each round of proliferation, 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} .

In one embodiment, 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. In one embodiment, 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.

In one embodiment, 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.

The present disclosure is based at least in part on the surprising finding: When adding FGF2 in the amount of the present disclosure, 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 In this case, the amount of human platelet lysate and/or human serum can be as low as 0.5% (v/v). Starting with 50 ng/mL FGF2 in the media of the present disclosure, the cultured cells showed a proliferation rate comparable to that of commercial media. When 100 ng/mL FGF2 was provided to the conditioned medium, the maximum hMSC proliferation rate was shown. The results show that 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. When the 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. What is exciting is that 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.

Example

The present invention is described herein through the following examples, which are only intended to illustrate and do not limit the scope of the present invention.

Example 1

Materials and Method

Cell culture and transfection

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.

hMSC proliferation rate

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.

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.

Immunocytochemistry

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. After washing three times with PBS, 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.

Morphological analysis

The 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.

Fluorescence activated cell sorting (FACS)

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).

mRNA quantification

According to the manufacturer's instructions, total 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.

result

Optimization of FGF2 concentration in non-exogenous medium

Previous reports have shown that the addition of fibroblast growth factor 2 (FGF2) inhibits the differentiation of human mesenchymal stem cells (hMSC), thereby maintaining complete differentiation [24]. According to the manufacturer's instructions, hMSC grows best in commercial human mesenchymal XF expansion medium containing human serum, and 8ng/mL FGF2 is added to maintain the highest proliferation rate. The present disclosure aims to reduce the cost of culturing hMSC by adjusting the concentration of FGF2 in the basal medium, such as DMEM, to replace the use of a proprietary medium. Therefore, 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. Compared with the commercial XF expansion medium, when the FGF2 concentration is low, the conditioned medium of the present disclosure reduces the proliferation rate of hMSC (Figure 1A). However, starting with 50 ng/mL FGF2 in the conditioned medium, the cultured cells showed a proliferation rate comparable to that of the commercial medium. When 100 ng/mL FGF2 was supplied to the conditioned medium, it showed the largest hMSC proliferation rate, while the conditioned medium containing 150 ng/mL or more FGF2 showed no significant effect on cell proliferation (Figure 1A). The cell proliferation assay was repeated by counting live cells on different days of subculture at passage 10. Similar results showed that a FGF2 concentration of 100 ng/mL provided sufficient growth stimulation (Figure 1B). Live hMSCs cultured in conditioned media with lower FGF2 concentrations of 0, 8, and 20 ng/mL could only reach 6,000, 6,200, and 9,500 cells per square centimeter, respectively (Figure 1B). 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.

Verify hMSC with a microscope

On the basis of verifying that the conditioned medium containing high concentration of FGF2 supports the growth of hMSC, 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). In order to further characterize the morphology of hMSC in different media, 100 cells were randomly selected from each medium, and their cell area and maximum cell diameter were measured. The results are shown on the dot plot (Figure 2D). In the cells cultured in the conditioned medium containing 100ng/mL FGF2, more than 90% of the cells showed ^{an area of <8000μm 2} and a maximum diameter of <250μm (Figure 2B), which is less than the conditioned medium containing 0ng/mL FGF2 Cultured cells. Then hMSCs cultured in different media were stained with mesenchymal stem cell surface marker CD44 and epithelial marker CD146. Cells cultured in commercial XF expansion medium and conditioned medium containing 100 ng/mL FGF2 were stained positive for mesenchymal stem cell marker CD44 (Figure 3A), while staining for epithelial marker CD146 was negative (Figure 3B). At the same time, the mesenchymal stem cell marker staining of cells cultured without FGF2 was negative (Figure 3A), and the staining of non-MSC markers was positive (Figure 3B).

Verify hMSC with FACS

In order to further verify the pluripotent stem cell populations in different media, fluorescence assisted cell sorting (FACS) was used to measure the stem cell markers of hMSCs cultured in commercial XF expansion media and conditioned media. Consistent with immunocytochemical data, at least 90% of cells cultured in commercial XF expansion medium and conditioned medium containing 100 and 500 ng/mL FGF2 were counted as stem cell markers THY-1 (Figure 4A) and STRO- 1 (Figure 4B) is positive. The cells were also stained for the epithelial marker CD146, and it was found that the cells were negative for the epithelial marker CD146 (Figure 4C). In contrast, it was found that cells grown in conditioned medium without FGF2 supplements were negative for mesenchymal stem cell markers and positive for non-MSC markers (Figure 4).

Validation of hMSC with qPCR and Western Blot

In order to eliminate possible artifacts caused by antibody staining, quantitative polymerase chain reaction (qPCR) was used to further verify the pluripotent stem cell populations in different culture media. Target-specific probes with high specificity to positive stem cell markers CD44, THY-1 and STRO-1 were used (Figure 5A). The mRNA expression level of the designated markers was quantified, which further confirmed that hMSC cultured in commercial XF expansion medium and conditioned medium with 100ng/mL FGF2 showed similar expression levels for these stem cell markers. These cells did not show mRNA expression of epithelial marker CD146 (Figure 5B) and hematopoietic stem cell markers CD14 and CD19 (Figure 5B). However, 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).

In order to confirm the results of qPCR, Western blot was used to check the expression levels of stem cell markers CD44 and THY-1. Antibodies specific to stem cell markers are used. Consistent with the previous results, hMSCs cultured in commercial XF expansion medium and conditioned medium containing 100ng/mL FGF2 were positive for stem cell markers CD44 and THY-1, but negative for epithelial marker CD146. In addition, cells cultured in a medium without FGF2 were negative for mesenchymal stem cell markers and positive for non-MSC markers (Figure 5C).

discuss

Although 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.

The data obtained from cell proliferation analysis (Figure 1) confirmed the important role of FGF2 in supporting the growth of hMSC. The growth medium without FGF2 is not sufficient to maintain the growth of undifferentiated hMSC. In the hMSC method of the present disclosure, the addition of 100 ng/mL FGF2 resulted in an amazing yield of ^{0.8×10 5 hMSCs per square centimeter on the culture plate within 96 hours.}

It is worth noting that compared with cells grown in FGF2-free medium, cells grown in FGF2 conditioned medium at a concentration of 100ng/mL or higher are more elongated and spindle-shaped (Figure 2 ). Compared with the medium without FGF2 supplement, the cells cultured in the medium containing 100ng/mL FGF2 showed a smaller maximum cell diameter and a smaller cell area. This morphology is the inherent standard for hMSC to grow at a much higher density and exhibit pluripotency [27].

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 results of Western blotting and qPCR analysis further verified the undifferentiated characteristics of hMSC derived in cultures supplemented with FGF2. What's more exciting is that when hMSCs grown in FGF2 supplemented medium were passaged for 50 generations, no obvious differentiation was found, and the proliferation efficiency remained unchanged.

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. In addition, 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.

Although various embodiments of exogenous media and methods for expanding hMSC have been described in great detail herein, these embodiments are only provided as non-limiting examples of the disclosure described herein. Therefore, those skilled in the art will understand that various changes and changes can be made to the solutions described in the present disclosure without departing from the spirit of the present invention. In fact, this disclosure is not intended to be exhaustive or to limit the scope of the invention.

Further, in the description of the representative embodiments, the present disclosure has presented the method and/or process of the present invention in a specific step sequence. However, 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. Furthermore, 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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Claims

A non-heterogeneous medium, which contains a basic medium and at least 50ng/mL fibroblast growth factor.
The heterologous-free medium according to claim 1, wherein the concentration of the fibroblast growth factor is 80-150 ng/mL, for example, about 100 ng/mL.
The heterogeneous culture medium according to claim 1 or 2, wherein the fibroblast growth factor is basic fibroblast growth factor (FGF2), especially human FGF2.
The heterologous-free medium according to any one of claims 1 to 3, which further comprises human platelet lysate and/or human serum.
The heterogeneous culture medium according to claim 4, wherein 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 heterologous-free medium according to any one of claims 1 to 5, wherein the basic medium is selected from the group consisting of MEM medium, α-MEM medium, DMEM medium, IMDM medium, HAM F12 medium, DMEM /F12 mixed medium, PRMI1640 medium, StemSpan TM medium and any combination thereof.
The non-heterogeneous medium according to any one of claims 1 to 6, which further comprises nutrients required for cell growth, such as amino acids, vitamins, carbohydrates and/or inorganic ions.
The heterologous-free medium according to any one of claims 1-7, wherein the heterologous-free medium does not contain additional cell growth factors or hormones.
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, it is present at a concentration of at least 50ng/mL, for example 80-150ng/mL, such as about 100ng/mL; preferably, the fibroblast growth factor is basic fibroblast growth factor (FGF2), especially human FGF2; preferably, the medium supplement preparation further comprises human platelet lysate and/or human The content of serum, preferably the human platelet lysate and/or human serum is such that it is present at a concentration of 0.5-5% v/v, for example 0.5-1% v/v after being added to the basal medium.
The medium supplement preparation according to claim 9, further comprising nutrients required for cell growth, such as amino acids, vitamins, carbohydrates and/or inorganic ions.
The medium supplement preparation according to claim 9 or 10, which does not contain additional cell growth factors or hormones.
A method for expanding human mesenchymal stem cells, which comprises without adding animal serum, under conditions suitable for the growth of the mesenchymal stem cells (1) according to any one of claims 1-8 The mesenchymal stem cells are cultured in a heterologous-free medium, or (2) the mesenchymal stem cells are cultured in a basic medium supplemented with the medium supplement preparation of any one of claims 9-11, to Expansion of the mesenchymal stem cells.
The method according to claim 12, wherein mesenchymal stem cells of about 0.8×10 5 cells/mL are obtained within 96 hours and the mesenchymal stem cells maintain about 99% of cell viability and mesenchymal stem cell identity.
The method according to claim 12 or 13, wherein the mesenchymal stem cells are substantially undifferentiated and the proliferation efficiency remains substantially unchanged after passage for at least 50 generations.