DE202018103067U1 - Serum-free cell culture medium suitable for propagation and recovery of mesenchymal stem cells - Google Patents

Abstract

A cell culture medium suitable for culturing mesenchymal stem cells, comprising: a serum-free basal medium; a serum-free supplement, the B-27 ™ supplement; a basic fibroblast growth factor (bFGF); and an epidermal growth factor (EGF).

Description

Field of the invention

The present invention relates to a cell culture medium which is suitable for the propagation and recovery of mesenchymal stem cells.

State of the art

Mesenchymal stem cells (MSCs) are non-hematopoietic multipotent stromal cells that can differentiate in vitro or in vivo into various cell types, including osteoblasts, chondrocytes, myocytes and adipocytes. MSCs have the advantages of being well-available, easy to isolate, pain-free, and highly legal and ethical. Therefore, MSCs are currently being tested for their potential use in gene therapy and regenerative medicine for the treatment of a variety of human and animal diseases and defects. However, there are some concerns about MSC-based treatment that need to be overcome. For example, MSCs are traditionally recovered in serum-containing culture media, such culture media typically having an addition of 5 to 20% fetal calf serum (FCS). This raises particular concerns about the use of MSCs for therapeutic applications, as serum-containing culture media always run the risk of introducing contaminants, pathogens, toxins and other undesirable substances.

Various efforts have been made to replace serum-containing media for culturing MSCs by serum-free media. For example, a defined serum-free medium has been proposed for human MSCs primary cultures to which the recombinant human platelet growth factor BB, basic fibroblast growth factor (bFGF) and transforming growth factor (TGF) -β1 have been added ( Chase LG, et al., Stem Cell Res Ther 2010; 11: 8 ). The publication WO 2011/111787 A1 discloses a cell culture medium for MSC culture containing growth factors such as FGF, PDGF, TGF-8, HGF and EGF in combination with a phospholipid and a fatty acid.

However, in terms of manufacturing cost and ability to maintain cell viability and activity, these conventional culture media can not meet user requirements. Thus, there remains a need to provide a serum-free cell culture medium that is suitable in the application to maintain the desired regenerative and differentiating properties of the MSCs while minimizing potential safety risks.

Object of the invention

The invention is thus based on the object to provide a novel serum-free cell culture medium, in particular a serum-free cell culture medium which is suitable for the propagation and recovery of mesenchymal stem cells.

Technical solution

• ein serumfreies Basalmedium;
• ein serumfreies Ergänzungsmittel, welches B-27™ - Supplement ist;
• den basischen Fibroblasten-Wachstumsfaktor (bFGF); und
• den epidermalen Wachstumsfaktor (EGF).

The inventor has extensively studied the problems of the prior art and has developed a novel cell culture medium suitable for propagation and recovery of mesenchymal stem cells, which serum-free cell culture medium suitable for culturing mesenchymal stem cells substantially includes the following components:

• a serum-free basal medium;
• a serum-free supplement which is B-27 ™ supplement;
• the basic fibroblast growth factor (bFGF); and
• the epidermal growth factor (EGF).

In a preferred embodiment, the serum-free basal medium is selected from a group consisting of Basal Medium Eagle (BME), Minimum Essential Medium (MEM), Alpha Minimum Essential Medium (α-MEM), Dulbecco's Modified Eagle's Medium (DMEM), Nutrient Mixture F -10 (HAM's F-10) and the nutrient mixture F-12 (HAM's F-12). Particularly preferred are embodiments in which the basal medium is selected from a group comprising DMEM and α-MEM.

In a preferred embodiment, the serum-free supplement, which is B-27 ™ supplement, is present in an amount of 1 to 3% (v / v), based on the total volume of the cell culture medium.

In a preferred embodiment, bFGF is present in an amount of 10 to 50 ng / ml, based on the total volume of the cell culture medium.

In a preferred embodiment, EGF is present in an amount of 10 to 50 ng / ml, based on the total volume of the cell culture medium.

In a preferred embodiment, the cell culture medium further comprises fetuin in an amount of 10 to 50 ng / ml, based on the total volume of the cell culture medium.

In a preferred embodiment, the cell culture medium further comprises 50 to 150 units / ml of penicillin and 50 to 150 units / ml of streptomycin, based on the total volume of the cell culture medium.

Detailed Description of the Preferred Embodiments of the Invention

As explained above, herein is disclosed a composition useful as a cell culture medium for cultivating mesenchymal stem cells. The term "mesenchymal stem cells" or abbreviated "MSCs" as used herein refers to multipotent stem cells derived from adult stromal tissue and includes, but is not limited to, bone marrow, adipose tissue, muscle tissue, dental pulp, umbilical cord blood, amniotic fluid, skeletal muscle, synovium, and wharton Sulze and has the property of comprehensive self-renewal and the ability to differentiate into cells of the mesenchymal line. In particular, the cell culture medium of the invention is suitable for culturing MSCs derived from dental pulp tissues. The MSCs cultured in the cell medium according to the invention can be derived from humans, rats, mice, sheep, cattle, pigs, dogs, cats, horses or non-human primates such as monkeys, gorillas and chimpanzees.

The MSCs cultured in the cell medium according to the invention can be obtained from various sources by methods known to those skilled in the art. For example, to obtain bone marrow-derived MSCs, bone marrow may be first harvested using a needle from the iliac crest of a human or non-human animal subject, with appropriate anesthesia, followed by density gradient centrifugation and selection of adherent cells. Alternatively, if recovery of MSCs from dental pulp is desired, tissue samples from the gums of a human or non-human subject can be harvested using a biopsy device and then subjected to collagenase digestion. The complete process of obtaining MSCs further includes isolating MSCs from cell culture based on differences in surface antigen labeling, such as magnetic cell sorting (MACS), fluorescence activated cell sorting (FACS), and flow cytometry sorting (FCS).

The cell culture medium disclosed herein is suitable for culturing MSCs by standard procedures that employ aseptic processing and handling. The cell culture medium comprises a serum-free basal medium, a serum-free supplement, namely B-27 ™ supplement, basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF).

As used herein, the term "basal medium" refers to any type of liquid culture medium containing inorganic salts, amino acids, and vitamins, which are commonly required to promote the growth of mammalian cells. Examples of suitable basal media include, but are not limited to, Basal Medium Eagle (BME), Minimum Essential Medium (MEM), Alpha Minimum Essential Medium (α-MEM), Dulbecco's Modified Eagles Medium (DMEM), Nutrient Mixture F-10 (HAM's F-10), Nutrient Mixture F-12 (HAM's F-12) or a combination thereof. In a preferred embodiment, the basal medium is selected from a group comprising DMEM and α-MEM.

In the cell culture medium disclosed herein, the serum-free basal medium is supplemented with the serum-free supplement B-27 ™ supplement to obtain a serum replacement. B-27 ™ supplement is commercially available from Thermo Fisher Scientific (Hemel Hempstead, UK) and is usually sold in the form of a 50X stock fluid. B-27 ™ supplement was originally developed to promote the growth and viability of hippocampal and other CNS neurons, and is added to the cell culture medium of the present invention to promote the cultivation of MSCs. The amount of B-27 ™ supplement present in the cell culture medium disclosed herein is sufficient to promote the growth and viability of the MSCs cultured therein. In a preferred embodiment, B-27 ™ supplement is present in an amount of 1 to 3% (v / v) based on the total volume of the cell culture medium.

Basic fibroblast growth factor (bFGF), also known as FGF2 and FGF-β, belongs to the signaling proteins and has broad mitogenic and cellular functions and is involved in a variety of biological processes, including embryonic development, cell growth, morphogenesis and the tissue repair. The term "bFGF" as used herein includes native bFGF polypeptides as well as recombinant variants and fragments thereof that have similar biological activity but contain inadvertently or deliberately induced alterations such as deletions, additions, extensions or substitutions of amino acid residues. The amount of bFGF present in the cell culture medium disclosed herein is sufficient to promote the growth and viability of the MSCs cultured therein. In a preferred embodiment, bFGF is in in an amount of 10 to 50 ng / ml based on the total volume of the cell culture medium.

The term "epidermal growth factor" or "EGF" as used herein includes native EGF polypeptides as well as recombinant variants and fragments thereof which have similar biological activity but contain inadvertently or deliberately induced alterations such as deletions, additions, extensions or substitutions of amino acid residues. The amount of EGF present in the cell culture medium disclosed herein is sufficient to promote the growth and viability of the MSCs cultured therein. In a preferred embodiment, EGF is present in an amount of 10 to 50 ng / ml based on the total volume of the cell culture medium.

In a preferred embodiment, the cell culture medium disclosed herein may further comprise fetuin. The term "fetuin" as used herein includes human α ₂ -HS glycoprotein (AHSG) and all mammalian fetuin protein equivalents as well as recombinant variants thereof and fragments thereof that have similar biological activity but contain accidentally or deliberately induced alterations such as deletions. Additions, extensions or substitutions of amino acid residues. In a preferred embodiment, the fetuin is human or bovine. Fetuin has been identified in serum and has been found to contribute to the attachment and spread of cells in a culture medium. In a preferred embodiment, fetuin is present in an amount of 0.5 to 2 mg / ml based on the total volume of the cell culture medium.

The cell culture medium may further antibiotics, such as. As penicillin, streptomycin and / or tetracycline, fungicides and / or antioxidants may be added. In a preferred embodiment, the cell culture medium may comprise 50 to 150 units / ml of penicillin and 50 to 150 units / ml of streptomycin based on the total volume of the cell culture medium.

The cell culture medium disclosed herein has a defined chemical composition that reduces the problems associated with the raw materials and enables reproducible application to MSC culture. The serum-free composition of the cell culture medium has the additional advantage of eliminating the risk of disease transmission. Another advantage of using the cell culture medium disclosed herein to grow MSCs is that, contrary to the results obtained using conventional cell culture media, cell growth and activity of the MSCs can be maintained after multiple culture runs. As shown in the examples below, the cell culture medium disclosed herein is particularly suitable for the propagation of MSCs isolated from adipose tissue and tooth pulp tissue.

The following examples are presented only for the purpose of illustrating the use of the cell culture medium of the present invention.

example 1

20 ml of adipose tissue are washed three times with phosphate buffered saline and treated with type II collagenase (50 to 100 U / ml) for one hour. The tissue is then centrifuged at 200 rpm for 4 minutes to obtain a cell mass, which is then washed three times with phosphate buffered saline. The cells are transferred to a T-75 flask with the cell medium of the invention consisting of 7 ml of α-MEM (Invitrogen, Carlsbad, CA) with 1% (v / v) B-27 ™ supplement (Thermo Fisher Scientific, Hemel Hempstead, UK), 10 ng / ml bFGF, 10 ng / ml EGF, 0.5 mg / ml fetuin, 100 units / ml penicillin and 100 units / ml streptomycin. The cell culture medium is renewed every three days until the number of MSCs has reached more than 1 × 10 ⁶ .

Example 2

The upper tooth of a 3-week-old male Wistar rat is completely pulled. Thereafter, the tissue is extracted using a syringe needle and transferred to a 25 cm ² flask (Corning, Inc. NY, USA). The tooth pulp is washed three times with phosphate buffered saline and treated with collagenase type II (50 to 100 U / ml) for one hour. The tissue is then centrifuged at 200 rpm for 4 minutes to obtain a cell mass, which is then washed three times with phosphate buffered saline. The cells are transferred to a T-75 flask containing the cell medium of the invention, which consists of 10 ml of DMEM / F12 (Invitrogen, Carlsbad, CA) with 3% (v / v) B-27 ™ supplement (Thermo Fisher Scientific, Hemel Hempstead, UK), 50 ng / ml bFGF, 50 ng / ml EGF, 2 mg / ml fetuin, 150 units / ml penicillin and 150 units / ml streptomycin. The cell culture medium is renewed every three days until the number of MSCs has reached more than 1 × 10 ⁶ .

Example 3

4 x 10 ⁵ fatty tissue-derived mesenchymal stem cells are thawed at 37 ° C and washed twice with phosphate buffered saline. The cells are transferred to a T-150 flask with the cell medium according to the invention, which consists of 20 ml of α-MEM (Invitrogen, Carlsbad, CA) with 2% (v / v) B-27 ™ supplement (Thermo Fisher Scientific, Hemel Hempstead, UK), 20 ng / ml bFGF, 20 ng / ml EGF, 1 mg / ml fetuin, 100 units / ml penicillin and 100 units / ml streptomycin. The cell culture medium is refreshed every three days until the number of AD-MSCs has reached more than 3 × 10 ⁶ .

In summary, the present invention thus relates to a serum-free cell culture medium, which is particularly suitable for the propagation and recovery of mesenchymal stem cells. The cell culture medium comprises a serum-free basal medium, a serum-free supplement, namely B-27 ™ supplement, basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). Optionally, fetuin and antibiotics may additionally be present in the cell culture medium according to the invention. The cell culture medium is advantageous in that it is able to maintain the desired regenerative and differentiating properties of MSCs in cell culture while minimizing the potential safety risks associated with serum.

Although the invention has been described with reference to the above preferred embodiments, it should be understood that the described preferred embodiments are illustrated by way of example only for purposes of illustration and are not intended to limit the scope of the invention, with various modifications and changes those skilled in the art can be made without departing from the spirit and scope of the invention.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• WO 2011/111787 A1 [0003]

Cited non-patent literature

• Chase LG, et al., Stem Cell Res Ther 2010; 11: 8 [0003]

Claims (8)

Cell culture medium suitable for culturing mesenchymal stem cells comprising: a serum-free basal medium; a serum-free supplement that is B-27 ™ supplement; a basic fibroblast growth factor (bFGF); and an epidermal growth factor (EGF). Cell culture medium after Claim 1 in which the serum-free basal medium is selected from a group consisting of Basal Medium Eagle (BME), Minimum Essential Medium (MEM), Alpha Minimum Essential Medium (α-MEM), Dulbecco's Modified Eagles Medium (DMEM), Nutrient Mixture F-10 (HAM's F-10) and nutrient mixture F-12 (HAM's F-12). The cell culture medium of any one of the preceding claims, wherein the basal medium is selected from the group consisting of DMEM and α-MEM. The cell culture medium of any one of the preceding claims, wherein the B-27 ™ supplement is present in an amount of 1 to 3% (v / v) based on the total volume of the cell culture medium. A cell culture medium according to any one of the preceding claims, wherein bFGF is present in an amount of 10 to 50 ng / ml based on the total volume of the cell culture medium. A cell culture medium according to any one of the preceding claims, wherein EGF is present in an amount of 10 to 50 ng / ml based on the total volume of the cell culture medium. Cell culture medium according to any one of the preceding claims, further comprising fetuin in an amount of 10 to 50 ng / ml based on the total volume of the cell culture medium. The cell culture medium of any one of the preceding claims, further comprising 50 to 150 units / ml of penicillin and 50 to 150 units / ml of streptomycin, based on the total volume of the cell culture medium.