Serum-free medium

Abstract

Provided is a serum-free medium that is composed of a fewer number of constituent components and is less expensive.　The serum-free medium comprises Dulbecco's modified Eagle's medium (DMEM), albumin (BSA), and insulin-transferrin-selenium-ethanolamine (ITS-X). The serum-free medium is suitable for the culturing of mouse ES cells that are naïve pluripotent stem cells, and bears comparison with the most superior medium on the market.

Description

Serum-free medium

[0001] The present invention relates to a serum-free medium used for culturing ES cells, which does not use fetal bovine serum (FBS).

[0002] Mouse embryonic stem cells (ES cells), derived from the undifferentiated cell population of preimplantation embryos (blastocysts), possess pluripotency, self-renewal, and unlimited proliferation potential, and are widely used as a model system for basic research on pluripotent stem cells and the development of cell therapy.

[0003] Fetal bovine serum (hereinafter referred to as "FBS") contains over 1,000 components necessary for maintaining stable proliferation and viability of many cultured cells, including not only mouse ES cells but also cancer cell lines. Therefore, even today, many researchers still culture mouse ES cells in FBS-containing medium.

[0004] However, FBS-containing media have many problems, such as variations between lots in the proliferation of ES cells and maintenance of undifferentiated state, and the inability to control the medium composition.

[0005] For this reason, chemically defined serum-free media have been proposed. The most widely used commercially available serum-free media include "KSR medium," which contains Knockout Serum Replacement (KSR, a Thermo Fisher Scientific product), developed as an FBS substitute, and "N2B27 medium," which contains N2 and B27 supplements (both Thermo Fisher Scientific products). However, because both KSR and B27 supplements are expensive and their ingredients are not publicly disclosed, researchers still face the challenge of being unable to control the medium composition according to their objectives, even though they are chemically defined media.

[0006] Patent Document 1 discloses a method for producing target cells, which comprises culturing source cells in a serum-free medium containing at least one culture factor selected from a growth factor and a differentiation-inducing factor, human serum albumin, and at least one additive selected from the group consisting of an Src inhibitor, a PKC activator, methylcellulose, Essential 8 medium, recombinant human serum albumin, and non-human animal serum albumin, to produce the target cells.

[0007] JP 2023-56008 A

[0008] Patent Document 1 does not use FBS, and its components are also known. However, there is a high demand for a medium that is cheaper and has a high culture effect.

[0009] The present invention has been conceived in view of the above problems, and aims to provide a serum-free medium which has a reduced number of constituent components and is less expensive.

[0010] Specifically, the serum-free medium according to the present invention is characterized by containing Dulbecco's modified Eagle's medium (DMEM), albumin (BSA), insulin-transferrin-selenium-ethanolamine (ITS-X), and cholesterol.

[0011] The serum-free medium according to the present invention makes it possible to culture ES cells using only a few ingredients: DMEM, albumin (BSA), and insulin-transferrin-selenium-ethanolamine (ITS-X), which are common mediums.

[0012] Furthermore, by adding an appropriate concentration of cholesterol to this serum-free medium, it is possible to achieve culture capabilities comparable to those of KSR medium, which is considered to have the highest proliferation potential for ES cells. Therefore, the serum-free medium of the present invention can provide an inexpensive and stable cell culture environment.

[0013] Examination of extracellular matrix proteins. Mouse ES cells showed sustained proliferation only on laminin-coated surfaces. Examination of cholesterol concentration that maximizes proliferation of mouse ES cells. Mouse ES cells showed high proliferation at concentrations of 5-20 μM. Comparison of proliferation between the new serum-free medium "DARP medium" and existing serum-free medium (DK10, N2B27). DARP medium is composed of DA-X medium + β-ME + cholesterol (10 μM).

[0014] The serum-free medium according to the present invention will be described below with reference to examples. Note that the following description exemplifies one embodiment of the present invention and one example, and the present invention is not limited to the following description. The following description can be modified within the scope of the present invention.

[0015] The serum-free medium according to the present invention contains Dulbecco's modified Eagle's medium (DMEM), which is a basal medium, albumin (BSA), and insulin-transferrin-selenium-ethanolamine (ITS-X).

[0016] DMEM only needs to have increased amounts of amino acids and vitamins compared to the original EME (Eagle's Medium), and the contents of other components may differ by about 10%. Furthermore, the serum-free medium of the present invention does not necessarily need to contain F-12 Ham. However, this does not preclude the inclusion of F-12 Ham.

[0017] Albumin can also be used from a wide variety of sources, including egg albumin, serum albumin, and milk albumin. Human serum albumin is more preferred.

[0018] Insulin-transferrin-selenium-ethanolamine may be a commercially available product, and for example, a composition of insulin 1000.0 mg / L, transferrin 550.0 mg / L, selenium 173.0 mg / L, and ethanolamine 61.0 mg / L can be suitably used.

[0019] The albumin content relative to DMEM is preferably 3 mg / mL to 8 mg / mL, more preferably 4 mg / mL to 7 mg / mL, and most preferably 4.5 mg / mL to 5.5 mg / mL.

[0020] Furthermore, the concentration of ITS-X relative to DMEM is preferably 4.0 mg / mL to 7.0 mg / mL, more preferably 4.5 mg / mL to 6.5 mg / mL, and most preferably 5.0 mg / mL to 6.0 mg / mL. The serum-free medium according to the present invention is called "DA-X medium."

[0021] Cholesterol can be added to DA-X medium. The cholesterol may be biologically derived or synthetic as long as it is purified. The cholesterol concentration in DMEM is preferably 5 μM to 20 μM, more preferably 10 μM to 20 μM, and most preferably 10 μM. DA-X medium containing cholesterol is called DARP medium.

[0022] The serum-free medium of the present invention can be suitably used primarily for culturing ES cells, but the following also mentions extracellular matrix proteins that serve as scaffolds during cell culture. Cells require a scaffold for proliferation. There are several proteins that can serve as scaffolds, and it has been discovered that the serum-free medium of the present invention uses laminin as a scaffold, allowing ES cells to proliferate favorably.

[0023] The effects of the present invention will be explained below with reference to examples.

[0024] Materials and Methods: Culture of Mouse ESCs in Conventional Chemical Medium. Mouse embryonic fibroblast (MEF) feeder cells were prepared from E14.5 mouse embryos. The E14tg2a mouse embryonic stem cell (ESC) line, derived from 129 / Ola (RIKEN BRC, AES0135), was maintained on mitomycin C (MMC) (Fujifilm, 139-18711)-inactivated MEF feeder cells in DF10LIF medium in a single well of a 4-well plate (SPL Life Sciences, SPL-30004).

[0025] The DF10LIF medium had the following composition: 10% fetal bovine serum (Gibco, 10270-106) in DMEM medium (Nacalai Tesque, 08458-45) containing recombinant mouse LIF (1 x 10 ３The solution was prepared by mixing the following ingredients: 1x MEM non-essential amino acid solution (NEAA) (Nacalai Tesque, 06344-58), 100 μM β-mercaptoethanol (β-ME, Nacalai Tesque, 21438-82), 2 mM L-glutamine (Nacalai Tesque, 16948-04), and 1x Penicillin-Streptomycin Mixed Solution (P / S) (Nacalai Tesque, 26253-84).

[0026] The medium was changed every 2 days, and colony-forming ESCs were dissociated using TrypLE (Gibco, 12604-013) and subcultured onto freshly prepared MMC-inactivated MEF feeder cells. They were further cultured at a split ratio of 1:20 every 4 to 5 days. The switch from DF10LIF medium to each serum-free medium was performed at a rate of 1.5 × 10 ５ Cells were seeded onto MMC-inactivated MEF feeder cells or extracellular matrix-coated plates.

[0027] The detailed compositions of the medium and extracellular matrix are as follows: DK10 medium: DMEM (Nacalai Tesque, 08458-45) containing 10% knockout serum replacement (KSR, Gibco, 10828028) supplemented with 1x MEM non-essential amino acid solution (NEAA), 100 μM β-mercaptoethanol, 2 mM L-glutamine, and 1x penicillin-streptomycin mixed solution (P / S). N2B27 medium: Neurobasal medium (Gibco, 21103049) and DMEM Ham's F-12 (Nacalai Tesque, 11581-15) were mixed at a 1:1 ratio, and N-2 supplement (1x, Gibco, 17502048), B-27 supplement (1x, Gibco, 17504044), and 2 mM L-glutamine solution were added.

[0028] 3 μM CHIR99021 (Cayman Chemical, 13122) and 1 μM PD0325901 (Cayman Chemical, 13034) were added to recombinant mouse LIF (1 × 10 ３ units / ml) and supplemented as 2iLIF.

[0029] [Pre-coating with extracellular matrix] Prior to seeding ESCs, a single well of a 4-well cell culture plate was pre-coated with the extracellular matrix at the desired concentration described below, incubated at 37°C for 1 hour, and then aspirated for use. ２ Fibronectin (Sigma-Aldrich, F1141) and vitronectin (FUJIFILM, 220-02041) were 1.25 μg / cm ２ , recombinant laminin 511-E8 fragment (iMatrix-511, MAX, 892011) at 0.5 μg / cm ２ The concentration of Matrigel matrix (Corning, 354234) was 2%, gelatin (Sigma, G1890) was 0.2%, and collagen type I (FUJIFILM, ASC-1-100) was 0.2%.

[0030] [Preparation of DA-X and DARP media] DA-X medium consists of DMEM medium (Nacalai Tesque, 08458-45) supplemented with 1x ITS-X (Insulin-Transferrin-Selenium-Ethanolamine, Gibco, 51500056), 5 mg / mL BSA (Bovine Serum Albumin, Sigma, A3059), 1x L-glutamine, and 1x Penicillin-Streptomycin Mixed Solution.

[0031] DARP (DA-X medium-based for rodent pluripotent stem cells) medium is DA-X medium supplemented with 100 μM (β-mercaptoethanol, β-ME) and 10 μM water-soluble cholesterol solution (a complex of methyl-β-cyclodextrin and cholesterol / EtOH solution, described below).

[0032] To optimize cholesterol concentrations for enhancing the proliferation of mouse ESCs, water-soluble cholesterol was administered in the range of 5–25 μM. Depending on the experiment using DA-X and DARP media, 3 μM CHIR99021 and 1 μM PD0325901 were administered as 2i, and recombinant mouse LIF (1 × 10 ３ All medium components, including 2iLIF, except for cholesterol, were mixed in advance, but only the aqueous cholesterol solution was added to the medium immediately before seeding the cells to prevent crystallization during storage at 4°C.

[0033] [Solubilization of Cholesterol Solution] To prepare a 50 mM cholesterol / EtOH solution, 19.3 mg of cholesterol (Sigma, C8667) was dissolved in 1 mL of 100% EtOH using an 80°C heat block. If residual cholesterol remains after dissolution, heating for several more minutes is required until complete dissolution. 100 μL of the 50 mM cholesterol / EtOH solution was mixed with 900 μL of 100% EtOH to obtain a 5 mM cholesterol / EtOH solution.

[0034] To solubilize the cholesterol / EtOH solution, 42 mg of methyl-β-cyclodextrin (Sigma-Aldrich, 332615) was added to 1 mL of 5 mM cholesterol / EtOH solution to prepare a 5 mM water-soluble cholesterol solution. This solution was stored at −30°C before use.

[0035] Results: DA-X Medium Supports the Growth of Mouse ESCs with 2iLIF and β-Mercaptoethanol. To determine whether DA-X medium can support not only adherent cancer cell lines but also mouse pluripotent stem cell lines, we examined the efficacy of DA-X medium on mouse ESCs in a naive pluripotent state on fibronectin-coated plates, the most efficient extracellular matrix for robust growth of adherent cancer cell lines. To minimize the medium composition, we first tested DA-X medium containing 2i and 2iLIF, which are free of other components, because these components have been reported to be sufficient to maintain mouse ESCs in an undifferentiated state.

[0036] Alkaline phosphatase staining revealed that DA-X ２ｉ Since the signal was weak in the culture medium, it became clear that the addition of LIF is essential for maintaining the undifferentiated state and proliferation of ESCs under DA-X medium conditions. ２ｉＬＩＦ The cell viability and proliferation could not be maintained even after one passage in the medium. Therefore, we added β-mercaptoethanol (β-ME), an antioxidant widely used in the culture of pluripotent stem cells, to the DA-X medium. ２ｉＬＩＦ When tested under culture conditions, they exhibited distinct dome-shaped colony morphology and were found to be effective in the proliferation of ESCs over multiple passages.

[0037] [Laminin supports the proliferation of mouse ESCs in DA-X medium and 2iLIF] The extracellular matrix is ​​an important component in regulating cell proliferation and differentiation not only in vivo but also in vitro in cell culture systems. ２ｉＬＩＦ／β－ＭＥ We sought to identify specific extracellular matrices that could conditionally support the proliferation and cell viability of ESCs in an undifferentiated state over successive passages in long-term culture.

[0038] We tested several extracellular matrices widely used to efficiently maintain human ESCs / iPSCs and mouse ESCs in an undifferentiated state. The results are shown in Figure 1. Referring to Figure 1, the horizontal axis represents the number of days in culture, and the vertical axis represents the number of cells. Circles (◯) in the graph represent laminin-coated plates, diamonds (◇) represent gelatin-coated plates, crosses (×) represent fibronectin-coated plates, squares (□) represent matelligel-coated plates, triangles (△) represent vitronectin-coated plates, and crosses (+) represent collagen-coated plates. We found that laminin-coated plates (circles (◯) in the graph) strongly supported ESC proliferation and cell viability, resulting in a unique monolayered, amoeba-like colony morphology and continuous passage for over 55 days, whereas none of the other extracellular matrices supported this.

[0039] [Cholesterol Promotes Mouse ESC Growth Under DA-X Medium-Based Conditions] Changes in cholesterol levels in various cell types are associated with changes in cell proliferation and cell division, highlighting the possibility that cholesterol metabolism may influence cell proliferation. However, the specific relationship between cholesterol and mouse pluripotent stem cell growth has not been elucidated in conventional media using cholesterol-rich FBS. Therefore, we investigated the concentration-dependence of cholesterol. The results are shown in Figure 2.

[0040] 2, the horizontal axis represents the number of days of culture, and the vertical axis represents the number of cells. In the graph, triangles (△) represent the results when cholesterol was 25 μM, crosses (×) represent the results when cholesterol was 20 μM, circles (◯) represent the results when cholesterol was 10 μM, squares (□) represent the results when cholesterol was 5 μM, and diamonds (◇) represent the results when cholesterol was not present.

[0041] DA-X ２ｉＬＩＦ／β－ＭＥ As a result of serial passage under these conditions, administration of water-soluble cholesterol in the range of 5 to 20 μM had a favorable effect on ESC proliferation (at 20 μM or more, numerous precipitates associated with toxicity were observed).

[0042] These results demonstrate that administration of cholesterol dose-dependently promotes the proliferation of mouse ESCs under serum-free medium conditions. ２ｉＬＩＦ／β－ＭＥ The medium was ２ｉＬＩＦ This medium is called DA-X-based medium for Rodent Pluripotent stem cells.

[0043] DARP ２ｉＬＩＦ To maximize growth potential in culture, we used DARP ２ｉＬＩＦ We focused on the role of cholesterol and NEAAs in ESC proliferation under culture conditions. NEAAs also play important roles in various cellular processes, including the proliferation of mouse ESCs. ２ｉＬＩＦ To examine the effects of cholesterol and NEAAs on ESC proliferation in the medium, growth curves for 30 consecutive passages were performed in DK10, another medium defined for mouse ESCs. ２ｉＬＩＦ Culture medium and N2B27 ２ｉＬＩＦ The results are shown in Figure 3.

[0044] Referring to Figure 3, the horizontal axis represents the number of days of culture, and the vertical axis represents the number of cells. ２ｉＬＩＦ The results are for the case where the black circle (●) indicates the DARP ２ｉＬＩＦ The black triangles (▲) indicate the results for DARP. ２ｉＬＩＦ The results are for NEAA (+), and the black diamonds (◆) indicate DARP ２ｉＬＩＦ The results are for NEAA (+) and cholesterol (-). The cross (x) indicates DARP. ２ｉＬＩＦ _The results are for cholesterol (-), and squares (□) indicate N2B27 ２ｉＬＩＦ The results are for Laminin and N2B27. ２ｉＬＩＦ This is the result of gelatin.

[0045] According to these results, cholesterol-containing DARP ２ｉＬＩＦ The growth ability in the medium is DK10 ２ｉＬＩＦ The results were comparable to those of the culture medium, and even on laminin-coated plates, the results were similar to those of N2B27. ２ｉＬＩＦOn the other hand, DARP ２ｉＬＩＦ When cholesterol was removed from the medium and NEAAs were administered, DARP ２ｉＬＩＦ No positive effect was observed on ESC proliferation under culture conditions.

[0046] DARP ２ｉＬＩＦ As a characteristic of ESCs maintained in culture, the colony morphology of ESCs was observed as a unique amoeba-like monolayer, rather than the conventional dome-like colony morphology. ２ｉＬＩＦ The medium achieved robust growth of ESCs with its unique colony morphology, comparable to or better than other chemically defined media.

[0047] The serum-free medium according to the present invention can be suitably used for culturing mouse ES cells, which are naive pluripotent stem cells.

Claims

1. Serum-free medium containing Dulbecco's modified Eagle's medium (DMEM), albumin (BSA), and insulin-transferrin-selenium-ethanolamine (ITS-X).

2. The serum-free medium according to claim 1, further comprising cholesterol.

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