CN114591897B - Method for three-dimensional culture of mesenchymal stem cells using xeno-free medium - Google Patents

Abstract

The present invention relates to a method for three-dimensionally culturing mesenchymal stem cells using a xeno-free medium. Specifically, the present invention provides a three-dimensional culture method of mesenchymal stem cells, comprising three-dimensionally culturing the human mesenchymal stem cells in a xeno-free medium without adding animal serum to expand the mesenchymal stem cells, wherein the xeno-free medium comprises a basal medium and at least 50ng/mL of fibroblast growth factor.

Description

Method for three-dimensional culture of mesenchymal stem cells using xeno-free medium

Technical Field

The present disclosure relates generally to methods of expanding mesenchymal stem cells, and more particularly to methods of three-dimensionally and stereoscopically culturing mesenchymal stem cells using xeno-free medium.

Background

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

Hmscs have been widely used as reliable cell sources for stem cell therapy, tissue engineering, drug discovery and disease modeling due to their high accessibility, proliferative potential, inherent immunomodulatory and repair properties, and multipotency. Hmscs are readily and directly available from bone marrow, adipose tissue, amniotic Fluid (AF), umbilical cord and placenta for seed culture of cell expansion [1,10]. Although many researchers have demonstrated the therapeutic potential of hmscs, challenges remain in stable and scalable cell expansion in order to fully meet the ever-increasing clinical demands. The low consistency maintenance of stem cell differentiation capacity and potential and expensive stem cell culture have hampered the research and application of hmscs in the current medical field. Thus, it is highly desirable to find a more defined, low cost medium for the expansion of hmscs without reducing their differentiation and potential.

Traditionally, two-dimensional adherent culture conditions have been used for hMSC expansion. However, two-dimensional adherent culture is a highly artificial and off-physiological environment due to the loss or impairment of certain in vivo features and traits. Three-dimensional (3D) cell culture is believed to have more physiological properties and may better preserve cellular properties.

Although the use of animal serum can promote the growth of 3D hmscs, the composition of animal serum is not fixed and unstable and uncertainty in the composition of animal serum carries the risk of infectious disease transmission or bacterial, viral and fungal contamination. In view of the above-mentioned shortcomings of serum-containing growth media, it is desirable to establish new cell culture media under serum-free conditions to improve consistency, reduce operating costs and avoid infection.

The present disclosure addresses at least one of the above problems.

Disclosure of Invention

In one aspect, the present disclosure provides a xeno-free medium for three-dimensional culture of mesenchymal stem cells comprising a basal medium and at least 50ng/mL of a fibroblast growth factor, such as 80-150ng/mL, such as about 100ng/mL.

In one embodiment, the fibroblast growth factor is basic fibroblast growth factor (FGF 2), particularly human FGF2.

In one embodiment, the xeno-free medium of the present disclosure may further comprise human platelet lysate and/or human serum. In one embodiment, the human platelet lysate and/or human serum is present in an amount of 0.5-5% v/v, e.g. 0.5-1% v/v.

In one embodiment, the basal medium can be selected from MEM medium, α - Μ e m medium, DMEM medium, IMDM medium, HAM F12 medium, DMEM/F12 mixed medium, PRMI1640 medium, stemspan medium, and any combination thereof.

In one embodiment, the xeno-free medium of the present disclosure may further comprise nutrients required for cell growth, such as amino acids, vitamins, carbohydrates, and/or inorganic ions.

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

In another aspect, the present disclosure provides a culture medium supplement formulation for three-dimensional culture of mesenchymal stem cells comprising a fibroblast growth factor, wherein the fibroblast growth factor is present in an amount such that it is present at a concentration of at least 50ng/mL, such as 80-150ng/mL, e.g., about 100ng/mL, after addition to a basal medium.

In one embodiment, the fibroblast growth factor is basic fibroblast growth factor (FGF 2), particularly human FGF2.

In one embodiment, the culture medium supplement formulation of the present disclosure may further comprise human platelet lysate and/or human serum. In one embodiment, the human platelet lysate and/or human serum is present in an amount such that it is present at a concentration of 0.5-5% v/v, e.g. 0.5-1% v/v, after addition to the basal medium.

In one embodiment, the culture medium supplement formulation of the present disclosure may further comprise nutrients required for cell growth, such as amino acids, vitamins, carbohydrates, and/or inorganic ions.

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

In yet another aspect, the present disclosure provides a method of three-dimensional culturing of mesenchymal stem cells comprising three-dimensionally culturing the human mesenchymal stem cells in a xeno-free medium without adding animal serum to expand the mesenchymal stem cells, wherein the xeno-free medium comprises a basal medium and at least 50ng/mL of fibroblast growth factor.

In one embodiment, wherein the concentration of fibroblast growth factor is 80-150ng/mL, such as about 100ng/mL.

In one embodiment, the fibroblast growth factor is basic fibroblast growth factor (FGF 2), particularly human FGF2.

In one embodiment, the xenfree medium further comprises human platelet lysate and/or human serum.

In one embodiment, the human platelet lysate and/or human serum is present in an amount of 0.5% to 5% v/v, e.g. 0.5% to 1% v/v, based on the xeno-free medium.

In one embodiment, the basal medium is selected from MEM medium, α - Μ medium, DMEM medium, IMDM medium, HAM F12 medium, DMEM/F12 mixed medium, PRMI1640 medium, stemspan medium, and any combination thereof.

In one embodiment, the xeno-free medium does not contain additional cell growth factors or hormones.

In one embodiment, the three-dimensional culture is performed using a hanging drop method.

In one embodiment, the culturing results in three-dimensional human mesenchymal stem cell spheres.

In one embodiment, the resulting mesenchymal stem cells express CD44, THY-1 and STRO-1 and do not express CD146, CD14 and CD19.

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

Drawings

The drawings are only for the purpose of illustrating the invention more clearly and are not to be taken as limiting the scope of the invention as regards its disclosure and protection in any way.

FIG. 1 shows a schematic diagram of a plasmid construction vector (10.4 kb) expressing an H6-DnaE-bFGF insert expression cassette according to an embodiment of the present disclosure. ori = origin of replication of bacillus subtilis; ampR = ampicillin resistance gene; lacI = lacI gene; t7rnap=t7 ribonucleic acid polymerase gene; bFGF = bFGF gene; asp DnaE = Asp DnaE inteins; h6 =6xhis tag; RBS = ribosome binding site. Arrows indicate the direction of gene expression.

Fig. 2 shows bFGF western blot assay results in a bacillus subtilis host cell lysate sample according to one embodiment of the present disclosure. Lanes 0h, 2h, 4h, 6h and 8h: samples collected from cultures at 0h, 2h, 4h, 6h and 8h after induction, respectively, were represented, and 5 μl of cell lysate was loaded per lane. Lane-ve: mu.l of cell lysate from pECBS1 vector culture 8 hours after induction.

Fig. 3 shows a time course study of shake flask culture of bacillus subtilis bFGF according to one embodiment of the present disclosure. Culture samples were obtained at different time points before and after IPTG induction. Fig. 3A: western blot analysis of bFGF present in Cell Lysate (CL) sample, wherein each Each lane was loaded with 5. Mu.l of cell lysate. Fig. 3B: quantification of cellular activity and bFGF.Indicating the detected bFGF level; CFU refers to colony forming units. Viable cell counts were measured on normal agar plates and kanamycin-added plates, respectively, with +.>And->And (3) representing. The transformant growth test was repeated 3 times and standard error bars were shown.

Fig. 4 shows a time course study of bFGF protein expression in fed-batch fermentation bacillus subtilis according to one embodiment of the disclosure. Culture samples were obtained at different time points before and after IPTG induction. Fig. 4A: results of western blot analysis of bFGF present in Cell Lysate (CL) samples, 5 μl of cell lysate per lane. Fig. 4B: quantification of cell activity and bFGF,indicating the detected bFGF level; CFU means colony forming units, viable cell counts were measured on ordinary agar plates and plates supplemented with kanamycin, respectively, in +.>And->And (3) representing. The transformant growth test was repeated 3 times and standard error bars were shown.

FIG. 5 shows mass spectrometry results (molecular size) of purified bFGF samples derived from pECBS1-H6-DnaE-bFGF construct according to an embodiment of the present disclosure.

Fig. 6 shows the results of mitogenic activity experiments of bFGF proteins according to one embodiment of the present disclosure. The effect of different concentrations of purified bFGF protein samples derived from pECBS1-H6-DnaE-bFGF construct on fibroblast proliferation is shown.

FIG. 7 shows the results of the cleavage assay for construct pECBS 1-H6-DnaE-bFGF.

FIG. 8 shows WB results of bFGF protein expression using H6 and CBD affinity tags, respectively. Lanes 0h, 4h, 8h: represents samples collected from cultures at 0h, 4h, 8h after induction, respectively; lanes +ve and-ve represent positive and negative controls, respectively.

FIG. 9 shows WB results of bFGF protein expression using H6 and GST affinity tags, respectively. Lanes 0h, 4h, 8h: represents samples collected from cultures at 0h, 4h, 8h after induction, respectively; lanes +ve and-ve represent positive and negative controls, respectively.

Fig. 10 shows the results of purification using heparin-sepharose chromatography alone.

Fig. 11 shows verification of 3D hMSC with a microscopic wide field image: (a) 3D hMSC cultured in a heterogeneous 3D hMSC-free medium; (B) 3D hMSC were re-cultured in DMEM with FBS using 2D dishes.

Fig. 12 shows the verification of hMSC with fluorescence microscopy. Cells were stained with MSC marker CD44, MSC and STRO-1, and epithelial cell marker CD146, and counterstained with DAPI.

Fig. 13 shows the validation of hmscs with qPCR. mRNA from different hMSC cultures was extracted and assayed by qPCR and expressed as control gene ACTB. The expression levels of MSC markers CD44, THY-1 and STRO-1, epithelial markers CD146 and hematopoietic stem cell markers CD14 and CD19 are shown.

Fig. 14 shows the validation of hmscs with Western blot. Western blots of 2D and 3D hMSC grown on different media were probed with antibodies directed against MSC-labeled CD44, THY-1 and epithelium-labeled CD 146. Lane 1: commercial XF amplification medium (2D hMSC); lane 2: serum-supplemented DMEM medium (3D hmsc+serum); lane 3: heterologous 3D hMSC-free medium (3D hMSC + conditioned medium).

Detailed Description

For the purposes of promoting an understanding of the principles of the invention, reference will now be made to the embodiments illustrated in the drawings and specific descriptions thereof. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended.

A description of xeno-free media and methods that can be used to culture mesenchymal stem cells is provided below. These media and methods meet at least one need in the art.

The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way.

Unless explicitly defined otherwise, terms used herein should be construed according to their usual meaning in the art. Unless the context indicates otherwise or indicated, nouns without quantitative word modifications denote one or more than one.

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

As used herein, the term "mesenchymal stem cell" refers to an undifferentiated pluripotent cell isolated from human or mammalian tissue that has the ability to self-renew while retaining the ability to multipotent and differentiate into multiple cell types of mesenchymal origin (e.g., osteoblasts, chondrocytes, adipocytes, stromal cells, fibroblasts, and tendons) or non-mesodermal origin (e.g., hepatocytes, neural cells, and epithelial cells), and may be derived from a variety of tissues. For example, mesenchymal stem cells can differentiate into mesenchymal cells such as bone, cartilage, muscle and adipocytes, 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, nerve-derived mesenchymal stem cells, skin-derived mesenchymal stem cells, amniotic-derived mesenchymal stem cells, and placenta-derived mesenchymal stem cells. Techniques for isolating stem cells from various tissues are known in the relevant art. Human mesenchymal stem cells have high value in regenerative medicine due to their high capacity to differentiate into multiple lineages of different cell types. Thus, in one embodiment, the mesenchymal stem cells are human mesenchymal stem cells (hmscs).

It should be noted that, without conflict, the features of the embodiments and examples in the present disclosure may be combined with each other.

In view of the above-described shortcomings of serum-containing growth media, the inventors of the present disclosure have focused on establishing novel cell culture media under serum-free conditions, resulting in better consistency, lower operating costs and infection-free conditions. The inventors used Fibroblast Growth Factor (FGF), such as human basic fibroblast growth factor hbFGF, to develop a highly efficient heterogeneous free 3D hMSC medium that can be used for large scale culture of 3D human mesenchymal stem cell spheres. The data demonstrate an important role for hbFGF in supporting 3D hMSC growth.

The present disclosure demonstrates well-defined xeno-free conditioned medium comprising human basic fibroblast growth factor (bFGF or FGF 2) for hMSC three-dimensional culture. The results of the present disclosure demonstrate that hMSC in three-dimensional culture in FGF 2-supplemented conditioned medium successfully obtained hMSC spheres and maintained the elongated and clostridial morphology of the recultured hMSC. More importantly, the undifferentiated nature of hmscs was also verified by microscopy, qPCR and Western blotting. The growth medium of the present disclosure may be used for large-scale three-dimensional production of hmscs.

In one aspect, the present disclosure provides a xeno-free medium for three-dimensional culture of mesenchymal stem cells comprising a basal medium and at least about 50ng/mL of a fibroblast growth factor, for example about 80-150ng/mL such as about 100ng/mL. In some embodiments, the xeno-free medium of the present disclosure can be used to expand mesenchymal stem cells, such as human mesenchymal stem cells, by three-dimensional culture.

Culture medium

As used herein, the term "culture medium" refers to a medium such as a solid, liquid, or gel designed to support cell growth. The medium is structured and/or provided with conditions suitable to allow cell growth. The medium may be solid, liquid or a mixture of various phases and materials. The medium may comprise a solid or liquid growth medium. The medium also includes gel-like media such as agar, agarose, gelatin and collagen matrices. The term "medium" also refers to a material intended for cell culture, i.e. it has not 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 "xeno-free medium" is free of components of animal origin, e.g., animal serum such as fetal bovine serum. In some embodiments, the xeno-free medium may contain additives of human origin, such as human serum or human platelet lysate. In some embodiments, the xeno-free medium contains only human-derived additives.

The media of the present disclosure may 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., MSCs). The basal Medium includes, for example, an Eagle Minimum Essential (MEM) Medium, an α Modified MEM (α - Μ e) Medium, a Darber Modified Eagle's Medium (DMEM), an Iscove's Modified Dulbecco's Medium (IMDM), a HAM F12 Medium, a DMEM/F12 mixed Medium, a prim 1640 Medium, a stemspan Medium, and any combination thereof, and is not particularly limited as long as it can be used for culturing stem cells. Other basal media suitable for MSC culture are known in the art.

In some embodiments, the cell culture media of the present disclosure may further comprise nutrients required for cell growth, such as amino acids, vitamins, carbohydrates, and/or inorganic ions, as well as components such as antibiotics to prevent bacterial contamination.

In some embodiments, the cell culture media of the present disclosure comprise 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 media of the present disclosure may comprise vitamins, for example, 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, and the like.

In some embodiments, the cell culture media of the present disclosure may comprise a carbohydrate. Carbohydrates are the primary energy source for cell growth, some of which are components of synthetic proteins and nucleic acids, such as glucose, ribose, deoxyribose, sodium pyruvate, and acetic acid.

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

In some embodiments, the cell culture media of the present disclosure may comprise antibiotics, such as penicillin, streptomycin, kanamycin (e.g., at a concentration of 50 ug/ml), and/or nystatin (e.g., at a concentration of 25U/ml). In one embodiment, 100U penicillin and 100ug streptomycin may be present per milliliter of culture broth.

Those skilled in the art will appreciate that for optimal results, the basal medium needs to be suitable for the cell line of interest, with the critical nutrients being of sufficient level to sustain cell proliferation. For example, if glucose is found to be depleted of such energy sources and thus limit cell proliferation, it may be desirable to increase the level of glucose (or other energy source) in the basal medium or to add glucose (or other energy source) during the culture process. It will also be appreciated by those skilled in the art that periodic medium exchanges may also be performed, typically adding/removing fresh/spent medium to the cells, in order to ensure adequate levels of nutrients and metabolites required for MSC expansion.

Fibroblast growth factor

Fibroblast growth factor is a class of polypeptides consisting of about 150-200 amino acids that exist in two closely related forms, basic fibroblast growth factor (bFGF) and acidic fibroblast growth factor (aFGF). Basic fibroblast growth factor (bFGF, also known as FGF 2) is a well known growth factor that is capable of replicating various types of stem cells from different sources. In the case of feeder layer-free culture, FGF2 is added to the cell culture medium to effectively promote proliferation, self-renewal and pluripotency of human embryonic stem cells. Based on the recently developed expression systems for FGF that can be produced in high yield and high bioactivity, the inventors have further attempted to formulate an economical and efficient growth medium for three-dimensional hMSC culture using FGF 2.

In one embodiment, the basic fibroblast growth factor useful in the present disclosure is human bFGF. In one embodiment, the basic fibroblast growth factor useful in the present disclosure is recombinant human bFGF. Recombinant human bFGF useful in the present disclosure may be obtained using methods known in the art. In a particularly preferred embodiment, the recombinant human bFGF useful in the present disclosure is a recombinant human bFGF obtained using the recombinant methods described herein (as described in detail below) that is stable, highly active and has biological identity to wild-type human bFGF.

The present disclosure provides a method of expressing recombinant human bFGF, comprising the steps of: (a) Culturing a transformed bacillus subtilis (Bacillus subtilis) under conditions that allow expression of human bFGF, the transformed bacillus subtilis comprising an expression vector comprising a nucleic acid construct, wherein the nucleic acid construct comprises, from 5 'to 3' end, a trans-splicing intein derived from Anabaena sp, and a polynucleic acid sequence of human bFGF, wherein the short peptide affinity tag is an N-terminal extein of the trans-splicing intein and the human bFGF is a C-terminal extein of the trans-splicing intein; and (b) isolating the expressed human bFGF.

Inteins useful in the present disclosure may be trans-spliced inteins derived from anabaena. In one embodiment, the intein may be an intein derived from an anabaena DNA polymerase III unit (Asp DnaE). As used herein, the term "trans-spliced intein" refers to an intein having trans-splicing activity.

As used herein, the term "intein of anabaena DNA polymerase III unit" refers to an intein derived from anabaena DNA polymerase III unit. In one aspect, the nucleotide encoding an intein of the present disclosure may have or comprise or consist of the sequence set forth in SEQ ID No. 1 or a complement thereof, or may have or comprise or consist of a sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the nucleotide sequence set forth in SEQ ID No. 1. In one embodiment, the intein may have or comprise, or may consist of, an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence set forth in SEQ ID NO. 2.

In one embodiment, the human bFGF of the present disclosure may be encoded by the following nucleotide sequence: which comprises or consists of the nucleotide sequence shown in SEQ ID NO. 3 or a nucleotide sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the nucleotide sequence shown in SEQ ID NO. 3. In one embodiment, bFGF useful in the present disclosure may have or comprise an amino acid sequence set forth in SEQ ID No. 4, or may have or comprise an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence set forth in SEQ ID No. 4, or may consist of the amino acid sequences set forth above.

As used herein, the terms "affinity tag," "purification tag," and "protein tag" are used interchangeably to refer to a protein or polypeptide that is expressed in fusion with a protein of interest during the preparation of a recombinant protein. The affinity tag can be used for promoting the solubility and stability of the target protein, and is convenient for the target protein to be detected and purified. Without intending to be bound by theory, a short peptide affinity tag of relatively small molecular weight is beneficial for obtaining mature and biologically identical (native) foreign proteins or polypeptides.

In some embodiments, the affinity tag useful in the present disclosure may be a short peptide affinity tag, which may have a length of about 4-15, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids. In some embodiments, the short peptide affinity tag includes, but is not limited to: HIS tag, HA tag (e.g., YPYDVP), FLAG tag (e.g., dykdddk), HSV tag (e.g., QPELAPEDPED), MYC tag (e.g., ILKKATAYIL or EQKLISEEDL), V5 tag (e.g., GKPIPNPLLGLDST), xpress tag (e.g., dlddk or DLYDDDDK), thrmbin tag (e.g., LVPRGS), BAD (biotin receptor domain) (e.g., GLNDIFEAQKIEWHE), factor Xa tag (e.g., IEGR or IDGR), VSVG tag (e.g., YTDIEMNRLGK), SV40 NLS tag (e.g., PKKKRKV or PKKKRKVG), protein C tag (e.g., EDQVDPRLIDGK), S tag (e.g., KETAAAKFERQHMDS), SB1 tag (e.g., PRPSNKRLQQ), etc. In one aspect, the affinity tag may be a 5-15 xHis tag, more specifically a 6 xHis tag (H6).

In some embodiments, the short peptide affinity tag of the present disclosure is as an N-terminal extein of a trans-splicing intein, and bFGF is as a C-terminal extein of a trans-splicing intein. In one example, the H6 tag is fused to the N-terminus of the Asp DnaE intein, and bFGF is fused to the C-terminus of the Asp DnaE intein.

In one embodiment, the present disclosure provides an expression vector comprising a nucleic acid construct of the present disclosure. As used herein, the terms "vector," "expression vector," "recombinant vector," and "recombinant system" are used interchangeably to refer to a carrier through which a polynucleotide or DNA molecule can be manipulated or introduced into a host cell. The vector may be a linear or circular polynucleotide, or may be a large-sized polynucleotide or any other type of construct, such as DNA or RNA from a viral genome, virion, or any other biological construct, that allows manipulation of the DNA or its introduction into a cell.

Those skilled in the art will appreciate that there is no limitation on the type of vector that can be used, as long as the vector can be a cloning vector suitable for propagation, capable of obtaining sufficient polynucleotide or genetic construct, or an expression vector suitable for purification of the fusion protein in a different heterologous organism. In one embodiment, suitable vectors according to the present disclosure include expression vectors in prokaryotes, such as prokaryotic expression vectors, including, but not limited to: pET14, pET21, pET22, pET28, pET42, pMAL-2c, pTYB2, pGEX-4T-2, pGEX-6T-1, pQE-9, pBAD-his, pBAD-Myc, pECB series vectors, pRB series vectors, etc., such as pUC18, pUC19, bluescript and derivatives thereof, mp18, mp19, pBR322, pBR374, pMB9, coIE1, pCR1, RP4, phages and "shuttle" vectors (e.g. pSA3 and pAT 28).

In one embodiment, the present disclosure also contemplates a shuttle vector. As used herein, the term "shuttle vector" is a class of vectors that can replicate and amplify in two different host cells (e.g., e.coli and b.subtilis), thereby enabling the same expression vector to be transformed into different host cells. Shuttle vectors contemplated by the present disclosure may include, but are not limited to, pECBS1.

The vector component may generally include, but is not limited to, one or more expression control elements as follows: promoters, enhancers, operators, ribosome binding sites, transcription termination sequences and the like. Exemplary promoters useful in the present disclosure may include promoters active in prokaryotes, such as T7 promoters, phoA promoters, beta-lactamase and lactose promoter systems, alkaline phosphatase, tryptophan (trp) promoter systems, and hybrid promoters such as tac promoters. Exemplary operons useful in the present disclosure include, but are not limited to, lactose operon, arabinose operon, tryptophan operon, and the like. Lactose operon is a group of genes involved in lactose decomposition, consisting of repressors and operator sequences of the lactose system, such that a group of genes associated with lactose metabolism are synchronously regulated. As used herein, the term "ribosome binding site" (ribosome binding site, abbreviated RBS) refers to a sequence located upstream of the start codon of an mRNA that can be used to bind to the ribosome at the start of translation.

Expression vectors according to the present disclosure may also comprise polynucleotides encoding marker proteins. Suitable marker proteins for the present disclosure include proteins that are resistant to antibiotics or other toxic compounds. Examples of marker proteins with antibiotic resistance include neomycin phosphotransferase, which phosphorylates neomycin and kanamycin, or hpt, which phosphorylates hygromycin, or proteins which confer resistance to, for example, bleomycin, streptomycin, tetracycline, chloramphenicol, ampicillin, gentamycin, geneticin (G418), spectinomycin or blasticidin. In one example, the protein confers resistance to chloramphenicol. For example, the protein is a gene from E.coli designated CmR, such as Nilsen et al, J.Bacteriol,178:3188-3193, 1996.

Polynucleotides encoding polypeptides of interest may be cloned into vectors of the present disclosure using standard techniques well known to those of skill in the art. For example, polynucleotides encoding the polypeptides of interest are produced using Polymerase Chain Reaction (PCR). Methods of PCR operation are known in the art.

In some embodiments, the nucleic acid construct of the present disclosure may further comprise a first cloning site located upstream of the insert and a second cloning site located downstream of the insert, wherein the first cloning site and the second cloning site allow for insertion of the nucleic acid construct into an expression vector. The cloning site allows cloning of polynucleotides encoding heterologous polypeptides. Preferably, the cloning sites are combined together to form a multiple cloning site. As used herein, the term "multiple cloning site" refers to a nucleic acid sequence comprising a series of two or more restriction endonuclease target sequences positioned adjacent to each other. The multiple cloning site comprises a restriction endonuclease target that allows insertion of fragments having a blunt end, a cohesive 5 'end, or a cohesive 3' end. Insertion of the target polynucleotide is performed using standard molecular biology methods, e.g., as described by Sambrook et al (Sambrook et al molecular Cloning: A Laboratory Manual, cold Spring Harbour Laboratory Press, 1989) and/or Ausubel et al (Current Protocols in Molecular Biology, greene pub. Associates and Wiley-Interscience (1988).

As used herein, the term "restriction endonuclease" or "restriction endonuclease" refers to a class of enzymes that can recognize and attach a particular sequence of deoxyribonucleotides and cleave a phosphodiester bond between two deoxyribonucleotides at a particular position in each strand. The cleavage method is to cleave the bond between the sugar molecule and the phosphate, thereby generating a nick on each of the two DNA strands without damaging the nucleotide and the base. There are two types of cleavage formats, each of which produces a sticky end with protruding single-stranded DNA and a smooth end with a flat end without protrusions. Since the broken DNA fragments can be joined by DNA ligase, different restriction fragments on the chromosome or DNA can be joined together by splicing. Restriction enzymes useful in the present disclosure may include, but are not limited to: ecoRI, pstI, xbaI, bamHI, hindIII, taqI, notI, hinfI, sau3, A, povII, smaI, haeIII, aluI, salI, dra, etc.

Methods of ligating nucleic acids are apparent to those skilled in the art and are described, for example, in Sambrook et al molecular Cloning, A Laboratory Manual, cold Spring Harbour Laboratory Press,1989 and/or Ausubel et al (editors), current Protocols in Molecular Biology, greene Pub.associates and Wiley-Interscience (1988). In one example, the nucleic acid is ligated using a ligase (e.g., T4DNA ligase).

In some embodiments, the present disclosure provides a transformed host cell comprising an expression vector of the present disclosure. Since bacillus subtilis is not endotoxin-free and is considered "generally recognized as safe", bacillus subtilis is employed as a host cell in one embodiment of the present disclosure.

As used herein, the term "transformation" means introducing DNA into a prokaryotic host as an extrachromosomal element or by chromosomal integration so that the DNA may replicate. Depending on the host cell used, transformation is performed using standard techniques appropriate for such cells. Calcium treatment with calcium chloride is typically used for bacterial cells containing a strong cell wall barrier. Another method for transformation uses polyethylene glycol/DMSO. Another technique that may also be used is electroporation.

The prokaryotic host cells used to produce bFGF of the present disclosure are cultured in media known in the art and suitable for use in such host cell culture. Examples of suitable media may include Luria-Bertani (LB) media supplemented with essential nutritional supplements. In some embodiments, the medium further comprises a selection agent, selected based on the constructed expression vector, to selectively allow the growth of prokaryotic cells containing the expression vector. For example, ampicillin and/or kanamycin are added to a medium for cell growth, which expresses ampicillin and/or kanamycin resistance genes. Any necessary supplements other than carbon, nitrogen and inorganic phosphorus sources may also be included in suitable concentrations, either alone or in admixture with another supplement or medium such as a complex nitrogen source.

In some embodiments, the present disclosure provides a method of producing bFGF comprising culturing the transformed bacillus subtilis of the present disclosure under conditions that allow expression of the bFGF.

For accumulation of expressed gene products, the host cell is cultured under conditions sufficient to accumulate the gene products. Such conditions may include, for example, temperature, nutrient, and cell density conditions that allow the cells to express and accumulate proteins. Furthermore, as known to those skilled in the art, such conditions are conditions under which the cell may perform basic cellular functions, such as transcription, translation, and intracellular expression. The prokaryotic host cell is cultured at a suitable temperature. For bacillus subtilis cultivation, for example, the temperature is typically about 20 ℃ to about 39 ℃. In one embodiment, the temperature is from about 25 ℃ to about 37 ℃, such as 37 ℃. For induction, the cells are typically cultured until a defined optical density is reached, e.g., a55tl of about 80-100, at which point induction begins (e.g., by addition of an inducer, by depletion of a repressor, inhibitor, or medium component, etc.) to induce expression of the gene encoding bFGF. After product accumulation, the cells present in the culture may be mechanically lysed using any mechanical means known in the art to release the protein from the host cell. Alternatively, other lysis methods may be employed, including, but not limited to, alkaline lysis, SDS lysis, and the like. Cell lysates used to lyse cells may include, but are not limited to, tris-HCl, EDTA, naCl, glucose, lysozyme, and the like. Optionally, the lysate is incubated for a sufficient time to allow bFGF contained in the cells to be released prior to product recovery. The lysate may be subjected to further processing, such as dilution with water, addition of buffers or flocculants, PH adjustment, or changing or maintaining the temperature of the lysate/homogenate in the preparation for subsequent recovery steps.

In one embodiment, the xeno-free medium of the present disclosure comprises at least about 50ng/mL of a fibroblast growth factor, e.g., about 80-150ng/mL such as about 100ng/mL. In one embodiment, the fibroblast growth factor is basic fibroblast growth factor (FGF 2), particularly human FGF2. In one embodiment, FGF2 can be natural or recombinant.

In some embodiments, the level of FGF2 is at least about 80ng/ml, at least about 90ng/ml, at least about 95ng/ml, or at least about 100ng/ml. In some embodiments, the level of FGF2 is no more than about 500ng/ml, such as no more than about 400ng/ml, no more than about 300ng/ml, no more than about 200ng/ml, no more than about l50 ng/ml, no more than about 140ng/ml, no more than about 130ng/ml, no more than about 120ng/ml, or no more than about 110ng/ml. In other embodiments, FGF2 is provided at a level of about 80-150ng/mL, e.g., 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 xeno-free medium of the present disclosure does not contain additional cell growth factors or hormones. In one embodiment, the xeno-free medium of the present disclosure does not contain a cell growth factor or hormone other than FGF2. As used herein, the term "free of additional cell growth factors or hormones" refers to the absence of other cell growth factors or hormones other than the fibroblast growth factor such as FGF2 added to the media of the present disclosure.

Human-derived additive

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

Surprisingly, when FGF2 is added in the amounts of the present disclosure, the culture medium of the present disclosure may not contain additional cell growth factors or hormones, or if human platelet lysate and/or human serum is added, the amount of human platelet lysate and/or human serum may be as low as 0.5% (v/v). In one embodiment, when FGF2 is added in the amount of the present disclosure, the amount of human platelet lysate or human serum added to the media of the present disclosure can be as low as 0.5% (v/v), and no additional cell growth factors or hormones are required or contained. As used herein, the term "without or containing additional cell growth factors or hormones" refers to the absence or presence of other cell growth factors or hormones in addition to the fibroblast growth factors such as FGF2 and the cell growth factors in human platelet lysate and human serum added to the media of the present disclosure.

In some embodiments, the human platelet lysate or human serum is used at a concentration of 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%, 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% by volume), preferably at a concentration of about 0.5% -1.0% v/v, or more preferably at a concentration of about 0.5% -0.8% v/v (e.g., 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), for example, at a concentration of 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%, 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 at a concentration of about 0.5% -1.0% v/v, or more preferably at a concentration of about 0.5% -0.8% v/v (e.g., about 0.5% v/v).

Platelet lysate may be obtained from any suitable source. Suitable commercial sources are PLT Max from Mill Creek Life Sciences (Rochester, minnesota, USA) or platelet lysate from Millipore. Platelet lysate is derived from the same species as the cultured MSCs. As used herein, the term "platelet lysate derived from … …" is used to describe a platelet lysate that has been prepared from a blood sample, for example by separating platelets from the blood sample, followed by 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 from which it is derived may be from adipose tissue used to prepare the MSC. Typically, the blood sample is from a different individual than the individual from which the MSC or adipose tissue was obtained.

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 may be from a single donor, or may be from pooled blood or cells. Platelet lysate may be prepared from infusible whole blood or platelets, for example, about 5 to 7 days after collection. Platelet lysate can be prepared from blood using commercially available kits (e.g., the platelet lysate kit from MacoPharma (France)). In one embodiment, the platelet lysate is prepared from blood collected in the presence of an anticoagulant (e.g., heparin sodium or citrate). The blood is centrifuged under appropriate conditions, for example at 200g for about 20 minutes, after which the platelets (top layer) are collected and then subjected to freeze-thawing to lyse the cells. Typically, multiple rounds of freeze-thaw are performed, such as two, three, four or more rounds. The lysed platelets are centrifuged to allow the precipitated cell fragments to be discarded, for example, at 4000g for about 10 minutes. 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 ℃) until use.

Human serum may be obtained from any suitable source, such as commercial sources. In one embodiment, the human serum is human AB serum, e.g., human AB serum from Sigma or Gibco company; or a human AB serum series from Gemini, including GemCellTM human AB serum, gemCellPlusTM human AB serum, etc.

Culture medium supplement preparation

The media of the present disclosure may be provided as complete media, wherein the basal medium and other ingredients have been mixed together prior to cell culture. Alternatively, the cell culture medium components may be provided separately and mixed with a suitable basal medium prior to or during cell culture.

In one aspect, the present disclosure provides a culture medium supplemented formulation comprising a fibroblast growth factor, wherein the fibroblast growth factor is present in an amount sufficient to be present at a concentration of at least 50ng/mL, such as 80-150ng/mL, e.g., about 100ng/mL, after addition to a basal medium. In some embodiments, the culture medium supplement formulation further comprises human platelet lysate and/or human serum. In some embodiments, the human platelet lysate and/or human serum is present at a concentration of about 0.5-5% v/v, e.g., about 0.5-1% v/v, after addition to the basal medium. In some embodiments, the medium supplement formulation further comprises 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 culture medium or culture medium supplement formulation of the present disclosure may be packaged in or with a suitable solvent or in lyophilized form. The cell culture media and/or culture medium supplement formulations disclosed herein may optionally be packaged in a suitable container, along with instructions for use for the desired purpose.

Method for amplifying mesenchymal stem cells

In one aspect, the present disclosure provides a method of expanding mesenchymal stem cells by a three-dimensional culture method, comprising culturing the mesenchymal stem cells by a three-dimensional culture method under conditions suitable for growth of the mesenchymal stem cells without adding animal serum, (1) in a xeno-free medium described in the present disclosure, or (2) in a basal medium to which a medium supplement formulation described in the present disclosure is added, to expand the mesenchymal stem cells.

The three-dimensional culture method is a culture method for growing cells in a spatial stereoscopic manner using various methods and materials, which allows the cells to more closely approach an in vivo growth mode, form structures resembling in vivo tissues, and exert their functions.

In one embodiment, the three-dimensional culture method is a hanging drop method. As used herein, the term "hanging drop" refers to a three-dimensional culture of cells that builds up a three-dimensional environment of cells with hanging drops without the aid of additional material scaffolds, wherein the cells proliferate or expand in the hanging drops. The cells cultured by the three-dimensional hanging drop method are closer to the real environment in the body, and the functions of the cells are favorably exerted. In some embodiments, the stentless pendant drop method may be performed using a multiwell plate, such as the multiwell plate commercially available under the trade name Gravityplus (TM) from InSphero AG, schlieren, CH.

In some embodiments, the three-dimensional culture method may also employ co-culturing different carriers (e.g., microcarriers) with cells having a three-dimensional structure in vitro to enable migration and growth of cells in the three-dimensional spatial structure of the carrier, thereby forming a three-dimensional cell carrier complex.

Microcarriers may be small, generally spherical particles, providing a larger surface area for cell adhesion and proliferation. Microcarriers useful in the present disclosure may be produced from a variety of materials including, but not limited to, gelatin, dextran, polystyrene, and alginate, and in some embodiments are made non-porous (e.g.)Plastic microcarriers), macropores (e.g. +.>) Or micropores (e.g.)>)。/>

A variety of natural and synthetic polymeric materials have been used as the backbone component of commercial microcarriers for stem cell culture, including dextran (Cytodex-1, cytodex-3), gelatin (i.e., cultiSpher-S), cellulose (DE-53, cytopore-2), polystyrene (Cytodex-1, cytodex-3), plastic (FACT III), polyester (fibri-CelVR), hydroxylated methacrylate (Tosoh 10 PR), poly (lactic-co-glycolic acid) (PLGA), alginate, and chitosan. The microcarrier scaffold may also be crosslinked with other compounds, such as with cationic groups (DEAE) or other extracellular matrices (gelatin and collagen) to enhance cell adhesion.

Compared with the traditional two-dimensional culture which often leads to the damage of the multipotency and the secretion capacity of the cells, the mesenchymal stem cell sphere obtained by adopting the three-dimensional culture such as a hanging drop culture method or a carrier (such as a microcarrier) culture not only can well inherit the advantages of the mesenchymal stem cells, but also can expand the repairing effect of the mesenchymal stem cells in various tissue injuries through the enhanced paracrine capacity and proliferation differentiation capacity.

In one embodiment, the methods described herein obtain about 0.8x105 cells/mL of mesenchymal stem cells within 96 hours and the expanded mesenchymal stem cells maintain at least about 99% of cellular activity and mesenchymal stem cell identity. In one embodiment, the mesenchymal stem cells are substantially undifferentiated and the proliferation efficiency remains substantially unchanged after passage for at least 50 passages. As used herein, the term "the 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 more of the proliferation efficiency of the originally isolated mesenchymal stem cells.

The culture according to this aspect of the disclosure may be performed for a limited amount of time such that no expansion occurs, e.g. only during the cell seeding phase, or for a longer period of time to allow expansion of the mesenchymal stem cells, thereby obtaining an increased cell number. For each round of proliferation, adherent cells (when cultured with a carrier) may be harvested using trypsin/EDTA or by cell scraping, and dissociated by pipette, and the three-dimensional culture re-performed, for example, at a density of about 100 to about 10,000 cells/cm 2.

In one embodiment, culturing 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, cells are expanded by at least two population doublings, at least four population doublings, at least six population doublings, at least eight population doublings, at least ten population doublings, at least 15 population doublings, at least 20 population doublings, at least 25 population doublings, at least 30 population doublings, at least 35 population doublings, at least 40 population doublings, at least 45 population doublings.

In one embodiment, mesenchymal stem cells may be selected and validated based on the expression of a mesenchymal stem cell surface marker. Selecting or sorting may include selecting Mesenchymal Stem Cells (MSCs) from a mixed population of cells by one or more such surface markers. In one embodiment, the mesenchymal stem cell surface marker may be selected from the group consisting of CD44, THY-1, STRO-1, and any combination thereof.

The present disclosure is based, at least in part, on the surprising discovery that: when FGF2 is added in the amounts of the present disclosure, the culture medium of the present disclosure may not contain additional cell growth factors or hormones, or if human platelet lysate and/or human serum is added, the amount of human platelet lysate and/or human serum may be as low as 0.5% (v/v).

Exemplary sequences in this disclosure are shown in the following table.

Examples

The disclosure is described herein by the following examples, which are intended to be illustrative only and are not limiting on the scope of the disclosure.

Coli strain DH 5. Alpha. Was purchased from New England Biolabs (Ipswich, mass.). Obtaining a Bacillus subtilis strain as described in the previous report

WB800 (Nguyen TT, quyen TD, le HT (2013) Cloning and enhancing production of a detergent-and organic-solvent-resistant nattokinase from Bacillus subtilis VTCC-DVN-12-01by using an eight-protease-gene-defcient Bacillus subtitle WB800.Microb cell face 12:79). Synthetic DNA fragments, restriction enzymes and antibodies to bFGF were purchased from Thermo Fisher Scientific (Ipswich, MA). All other chemicals were purchased from Sigma-Aldrich (st.louis, MO) unless otherwise indicated.

Example 1: expression vector construction and host cell transformation

Construction and design of E.coli/B.subtilis expression shuttle vector

pRB374 and pBR322 were used as starting vectors for E.coli/Bacillus subtilis expression shuttle vectors, respectively (Bruckner, R. (1992). A series of shuttle vectors for Bacillus subtilis and Escherichia coll. Gene,122 (1), 187-192.). Specifically, pECBS1 was constructed by the following modification procedure: first, pRB374 (5.9 kb) was digested with SalI and BglII; after digesting both sites with the same SalI and BglII, a fragment (5.3 kb) of the T7 ribopolymerase-Lac promoter-LacI gene-LacIq promoter-bleomycin resistance gene-part of the neomycin resistance gene, generated from the shotgun polymerase chain reaction, was substituted to form a pECBSi vector. Then, the resulting pECBSi vector and pBR322 vector were digested with EcoRI and BglI, respectively, and the pECBSi digested fragment was replaced with a fragment obtained by digesting pBR322 (4.3 kb), thereby forming a pECBS1 shuttle vector.

Construction of bFGF expression vector

The construction method of the E.coli/Bacillus subtilis expression shuttle vector (pECBS 1-H6-DnaE-bFGF) is as follows: a DNA fragment encoding the EcoRI-T7 promoter (T7) -lactose operon (LacO) -Ribosome Binding Site (RBS) -6x-His tag (H6) -Asp-DnaE int-c (DnaE) -bFGF-T7 transcription terminator-XbaI sequence was synthesized by Thermo Fisher Scientific as shown in SEQ ID NO: 5. The aforementioned synthesized DNA fragment was digested with EcoRI and XbaI, and then the Bacillus subtilis/Escherichia coli shuttle vector pECBS1 was ligated by digesting with the same two restriction enzymes. The pECBS1-H6-DnaE-bFGF construct was finally obtained (see FIG. 1). The results of the cleavage assay of the obtained construct are shown in FIG. 7.

Transformation of bacillus subtilis

Individual colonies of WB800 were inoculated into 5ml of medium a (containing 1x Spizizen salt solution, 0.5% glucose, 0.005% tryptophan, 0.02% casamino acid, 0.5% yeast extract, 0.8% arginine, 0.4% histidine) and incubated overnight at 37 ℃, 200 rpm. Then 0.5ml of overnight culture was subcultured into 50ml of medium a and incubated at 37 ℃ at 200rpm until a600=1.7. 1ml of 87% glycerol was added to 10ml of culture and placed on ice for 15 minutes. 1ml of the culture was then further subcultured into 20ml of medium B (containing 1 XSpizizezen salt, 0.5% glucose, 0.0005% tryptophan, 0.01% casamino acid, 0.1% yeast extract, 2.5mM MgCl2, 0.5mM CaCl2) and incubated at 30℃for 2 hours at 150 rpm. 1ml of the culture was transferred to a microcentrifuge tube, and EGTA was added at a final concentration of 1mM and incubated for 5 minutes at room temperature. 2ug of plasmid DNA was then added to 1ml of competent WB800 and allowed to grow for 2 hours at 37℃and 200 rpm. The transformed WB800 was then collected by centrifugation at 5000rpm at room temperature and resuspended in 100ul of culture supernatant. Transformed WB800 was plated on kanamycin resistance plates and incubated overnight at 37 ℃.

Example 2: expression of bFGF

Shake flask culture

The bacillus subtilis transformants were grown in 200ml of 2x LB medium supplemented with 25 μg/ml kanamycin at 37 ℃ (250 rpm) (He, q., fu, a.y., & Li, t.j. (2015) & Expression and one-step purification of the antimicrobial peptide cathelicidin-BF using the intein system in Bacillus sublis. Journal of industrial microbiology & biotechnology,42 (4), 647-653). When the a600 value reached 1.0, IPTG was added at a final concentration of 0.2mM, followed by collection of 1ml of culture samples every 3 hours for bFGF expression analysis. The cell pellet was resuspended in 200. Mu.l of resuspension buffer (50 mM Tris-Cl,200mM EDTA,pH 8.0) and then incubated on ice for 5 minutes. The mixture was then treated with 120. Mu.l of lysozyme solution (10 mg/mL) at 37℃for 20 minutes. Then 80. Mu.l of lysis buffer (10mM EDTA,10%Triton X-100 and 50mM Tris-Cl, pH 8.0) was added. The tube containing the solution was gently inverted and then centrifuged at 14,800rpm for 5 minutes. Cell lysate samples were analyzed for bFGF protein expression by western blotting.

In order to successfully express the soluble bFGF protein, the inventors have also experimentally examined the combination of different inteins and exogenous polypeptides, and finally found that the Asp DnaE intein is beneficial. The fusion of bFGF to the C-terminus of the Asp DnaE intein was chosen because in vitro cleavage at the C-terminus of DnaE can be controlled by pH change or treatment with a reducing agent. In addition, the inventors tried several different expression tags, including GST, chitin Binding Domain (CBD) and H6 affinity tag. With the first two expression tags, the construct produced only the precursor in insoluble form (see fig. 8 and 9), while with the relatively small size of the H6 tag, the experimental results gave positive expression results for mature and biologically identical bFGF (see in particular table 1). The results of the shake flask culture experiments showed (see fig. 2): the construct pECBS1-H6-DnaE-bFGF expressed satisfactory levels of the final product bFGF under induction, whereas the precursor form was not detected from Western blots (see FIGS. 8 and 9).

Table 1: purified bFGF was analyzed by liquid chromatography-mass spectrometry.

a. After trypsin partially digested purified bFGF, N-and C-terminal sequences were identified by Mascot search engine

Fed-batch fermentation

The bacillus subtilis transformant was grown in 200ml of 2x LB medium supplemented with 25 μg/ml kanamycin at 37 ℃ (rotational speed 250 rpm) until a600=1.0. Then 50ml of the culture was transferred to a 2L flask (containing 450ml of 2 XLB medium supplemented with 25. Mu.g/ml kanamycin, culture was continued at 37℃and 250rpm rotation until the A600 value reached 1.0. The whole culture was inoculated into a 5L fermenter containing 3.5L of 2 XLB medium supplemented with 25. Mu.g/ml kanamycin, 1M NaOH was added to maintain the pH of the culture at 7.0. The pO2 value (partial pressure of oxygen) in the culture was set at 1.5vvm. Furthermore, when the pH began to rise, a 50% glucose feed solution was added to maintain the pH of the culture at 7.0. When A600=8, followed by induction culture with 0.2mM IPTG concentration, pH adjustment was maintained with 1M H2SO4, and culture samples were collected at 2-hour intervals for analysis of bFGF expression.

The results show that: the expression quantity and the cell quantity of bFGF protein of bacillus subtilis are obviously increased. Specifically, from shake flask culture (FIG. 3) to large scale culture (FIG. 4), the total bFGF protein production and the final Colony Forming Units (CFU) of the expression construct were increased 2-fold (from 64mg/L to 113 mg/L) and 6-fold, respectively. It follows that the constructs obtained in the present disclosure achieve unexpected technical effects in both shake flask culture and large-scale fermentation culture.

Example 3: purification and Structure determination of bFGF protein

Cation exchange chromatography and heparin-sepharose chromatography were used for purification of bFGF. First, the protein concentration of the eluted fraction was measured using a Nanodrop Microvolume spectrophotometer. In addition, the eluted fractions with significant readings (about 1 mg/ml) were pooled and dialyzed against 0.1 XPB. Thereafter, purified bFGF bands were obtained by electrophoresis on 10% SDS-PAGE gels stained with Coomassie Brilliant blue R-250. The bands containing bFGF protein in the SDS-PAGE gel were recovered for subsequent analysis by LC-MS.

The western blot analysis showed that: the soluble bFGF protein extracted from the lysate had the same molecular weight as the bFGF protein purchased from Thermofisher Scientific (fig. 4). Purified bFGF protein samples were subjected to LC-MS for N-and C-terminal protein sequencing and MALDI-TOF mass determination. The results show that: the final bFGF protein product obtained from expression of the H6-DnaE-bFGF construct had a biological structure of 146 amino acids (table 1) and a size of 16.4kda (fig. 5), consistent with the native human bFGF protein. The purification of the present disclosure using cation exchange chromatography and heparin-sepharose chromatography yields bFGF protein of high purity compared to purification using heparin-sepharose chromatography alone (fig. 10).

Example 4: biological Activity detection of bFGF protein

The effect of purified bFGF protein on NIH/3T3 fibroblast proliferation was examined by MTT method (also known as MTT colorimetric method). The method comprises the following specific steps: NIH/3T3 cells (density of 2X 104 cells) were seeded in 96-well plates, starved cultured in DMEM medium supplemented with 1% fetal bovine serum at 37℃for 24 hours, and then treated with bFGF at various concentrations for 3 days. 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT) was added to each well of the well plate at a final concentration of 0.5mg/mL and incubated at 37℃for 4h at 5% CO 2. All solutions were then removed from the wells of the wells and 150 μl DMSO was added to dissolve the purple crystals. The plate was continuously shaken in the dark for 10 minutes and the absorbance was read with a microplate reader at 570 nm.

The results show that: purified bFGF protein expressed in bacillus subtilis was able to induce cell proliferation of NIH/3T3 cells (fig. 6), and also human mesenchymal stem cells (data not shown). Thus, it can be seen that the purified bFGF protein obtained in the present disclosure has biological activity (mitogenic activity).

The above results show that the purified bFGF protein of the present disclosure has the same primary sequence of 146 amino acids as the wild-type protein, is in the form of mature soluble protein, and has high biological activity in the aspect of inducing NIH/3T3 cell proliferation. Furthermore, the inventors have tried fermentation culture at different scales at the same time, and have obtained unexpected technical effects in terms of both the expression amount and the cell amount of bFGF protein.

Example 5: three-dimensional culture hMSC

Preparation of culture medium

The hbFGF obtained in example 4 was added to a xeno-free DMEM medium (Life Technologies, CA, USA) at 100mg/mL to obtain a xeno-free 3D hMSC medium.

Commercial XF amplification medium was used as control 2D medium and DMEM medium supplemented with serum was used as control 3D medium.

Cell culture

Human mesenchymal stem cells extracted from bone marrow were purchased from Merck Millipore. Cells were cultured in human mesenchymal XF expansion medium in humidified incubator maintained at 37 ℃ and 5% co 2. When 80% confluence was reached, cells were plated at a density of 5,000 cells/cm 2. All plates were coated with 0.1% gelatin solution for 30 minutes at room temperature prior to plating. Cell culture medium was changed daily.

Approximately 25,0000 2D hMSC cells were harvested and diluted into non-heterologous 3D hMSC medium and control 3D medium. Cells were pipetted onto the underside of the petri dish cover to form hanging drops and cultured without interference for at least 3 days in a humidified incubator maintained at 37 ℃ and 5% co 2. After 24 hours, a 3D hMSC was formed.

Real-time PCR of stem cell marker expression

The obtained 3D hmscs were harvested and lysed, and their total RNA was then extracted with RNAzol reagent (Molecular Research Center). The yield of RNA was quantified using Qubit. 100ug of RNA was reverse transcribed with GoScript reverse transcriptase (Promega) using Oligodt primers. cDNA samples were analyzed by pre-designed real-time PCR primers/probes to detect CD44, THY-1 and CD146 expression with Quantum studio 3 and SYBR Green I premix. All samples were run in triplicate. Average gene expression was calculated in 3 independent experiments.

Immunocytochemistry

Cells were plated on sterile gelatin-coated coverslips for imaging. DPBS washed the cells three times and then fixed in methanol at-20℃for 10 minutes. Subsequently, cells were blocked in 5% fbs (fetal bovine serum) in DPBS for 1 hour at room temperature. Cells were washed three times with DPBS. The mouse monoclonal CD44 antibody (1:500, CBL 154), CD146 antibody (1:500, MAB 16985) or STRO-1 antibody (1:500, MAB 4315) was mixed with 1% FBS, then added to the cells and incubated overnight at 4 ℃. Cells on coverslips were gently washed three times in DPBS. Alexa Fluor 488 rabbit anti-mouse secondary antibody (1:1000,Life Technology) was prepared in 1% fbs. Cells were incubated with secondary antibodies overnight at 4 ℃. Cells were counterstained with DAPI for 1 hour at room temperature. Finally, the cells were washed three times with DPBS. Coverslips were mounted on slides with ProLong anti-fade mounting agent (ThermoFisher). Fluorescence signals were observed under an EVOS M5000 imaging system (thermo fisher).

Morphological analysis of 3D hMSC

As described above, in order to produce 3D hmscs, 2D MSCs were cultured in 3D hMSC medium in a hanging manner on a dish cover, during which the cells spontaneously assembled into spheres. As shown in fig. 11A, the obtained cells were spherical, and the size of the sphere increased with time. After 3 days of incubation, the average diameter of the spheres was 60um. To determine if sphere production can induce adhesive migration, the obtained 3D hmscs were re-cultured in 2D dishes in DMEM supplemented with FBS. As shown in fig. 11B, the cells can reattach to the surface of the dish, thus preserving the ability to migrate and adhere.

Microscopic validation of 3D hMSC

The 3D hMSC-derived recultured 2D hmscs cultured in 3D hMSC medium were subsequently stained with mesenchymal stem cell surface marker CD44 and STRO-1 and epithelial marker CD 146. As shown in fig. 12, both mesenchymal stem cell markers CD44 and STRO-1 stained positive, while epithelial marker CD146 stained negative.

Verification of 3D hMSC Using qPCR and Western blotting

To eliminate possible artifacts due to antibody staining, hMSC populations in each medium were further validated using quantitative polymerase chain reaction (qPCR): (1) 2D hmscs cultured in commercial XF expansion medium; (2) 3D hmscs cultured in DMEM with FBS; and (3) 3D hmscs cultured in the xeno-free 3D hMSC-free medium of the invention. Target specific probes with high specificity for positive stem cell markers CD44, THY-1 and STRO-1 were used. The mRNA expression level of the indicated markers was quantified. As shown in fig. 13, the expression level of stem cell markers in the heterologous 3D hMSC-free medium of the invention was even higher than 2D hmscs as well as 3D hmscs cultured in serum-containing 3D medium. mRNA expression of the epithelial marker CD146 and hematopoietic stem cell markers CD14 and CD19 was not shown for all cells.

To confirm the results of qPCR, the expression levels of the stem cell markers CD44 and THY-1 were examined by Western blotting. Antibodies specific for stem cell markers are used. As shown in FIG. 14, consistent with previous results, hMSC cultured in each medium was positive for CD44 and THY-1 markers, and negative for the epithelial marker CD 146.

Discussion of the invention

Although stem cells have advantages in cell therapy, the use of stem cells has so far been limited by the high production costs due to the need for expensive commercial growth media such as XF media, minimal media, and the like. While the use of serum can reduce costs, unknown variables (e.g., the presence of viruses, allergens) can be problematic when entering clinical trials. To facilitate the transition of stem cells from basic research to clinical application, it is important to develop cost-effective xeno-free media for the strong expansion of human stem cells.

The hMSC spheres successfully obtained, i.e., successfully maintained 3D hMSC morphology, in the non-heterologous 3D hMSC medium supplemented with only hbFGF of the present disclosure (fig. 11A), and the 2D hmscs re-cultured from the obtained spheres retained the ability to migrate and adhere, as well as the elongated and clostridial cell morphology (fig. 11B).

CD44, THY-1 and STRO-1 are the most commonly used hMSC markers. The multijunction glycoproteins CD44 and THY-1 expressed on the membrane surface can trigger a variety of cellular functions including differentiation, proliferation, cell adhesion and apoptosis. STRO-1 enriched hmscs promote cell differentiation into various mesenchymal lineages, such as bone marrow stromal cells, adipocytes, osteoblasts, fibroblasts, and myoblasts. Hmscs supplemented with FGF2 were positive for hMSC surface marker CD44 and negative for hematological marker CD19 under fluorescence microscopy. The results of Western blot and qPCR analysis further confirmed the undifferentiated nature of the derived hmscs in FGF 2-added cultures.

The above results demonstrate that addition of hbFGF (without additional growth factors or hormones) to basal medium is effective in promoting growth of 3D hmscs. More importantly, the undifferentiated nature of 3D hmscs was also verified by fluorescent staining of biomarkers, qPCT and Western blot (fig. 12-14). Notably, the expression level of stem cell markers in the heterologous 3D hMSC-free medium of the invention was even higher than 2D hmscs as well as 3D hmscs cultured in serum-containing 3D medium (fig. 13). Cells cultured in medium containing 100ng/mL FGF2 showed smaller maximum cell diameter and smaller cell area compared to medium without FGF2 supplement. This morphology is an intrinsic standard for hmscs to be able to grow at much higher densities and to exhibit pluripotency.

Although various embodiments of exogenous-free media and methods for three-dimensional culture of hmscs have been described in great detail herein, these embodiments are provided merely as non-limiting examples of the disclosure described herein. Accordingly, those skilled in the art will appreciate that various changes and modifications can be made to the arrangements described in the present disclosure without departing from the spirit of the invention. Indeed, the present disclosure is not intended to be exhaustive or to limit the scope of the invention.

Further, in the description of representative embodiments, the present disclosure has presented the methods and/or processes of the present invention in a particular sequence of steps. However, the method or process should not be limited to the particular sequence of steps described. Other sequences of steps are possible. Therefore, the particular order of the steps disclosed herein should not be construed as limitations on the invention. Furthermore, the disclosure directed to methods and/or processes should not be limited to performing their steps in the order described. Such order may be altered and still be within the scope of the invention.

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Claims (5)

1. A three-dimensional culture method of human mesenchymal stem cells, comprising three-dimensionally culturing the human mesenchymal stem cells in a xeno-free medium without adding animal serum to expand the human mesenchymal stem cells, wherein the xeno-free medium comprises a basal medium and at least 50ng/mL of human basic fibroblast growth factor, (i) the xeno-free medium does not contain cell growth factors or hormones other than human basic fibroblast growth factor, and (ii) the xeno-free medium does not comprise human platelet lysate and human serum, the basal medium being DMEM medium.

2. The method of claim 1, wherein the concentration of fibroblast growth factor is 80-150ng/mL.

3. The method of any one of claims 1-2, wherein the three-dimensional culture is performed using a hanging drop method.

4. The method according to any one of claims 1-2, wherein the culturing results in three-dimensional human mesenchymal stem cell spheres.

5. The method of any one of claims 1-2, wherein the obtained human mesenchymal stem cells express CD44, THY-1 and STRO-1 and do not express CD146, CD14 and CD19.