Serum-free culture medium

Abstract

Provided herein are serum-free cell culture media for culturing cells in vitro, and methods of using the serum-free cell culture media.

Description

Field of the Invention

[0001] The present invention relates to culture media and solutions for cell growth of cultured cells. Background of the Invention

[0003] Cell-based in vitro assays are widely used to characterize the biological activity of molecules or to quantify the efficacy of clinical and marketed drugs, as required by regulatory agencies [1]. To evaluate drug efficacy, the assay should reflect or mimic the known or expected mechanism of action of the product as closely as possible [1]. For this reason, cell-based bioassays are performed using cell lines that respond to specific ligands or infectious agents, thereby generating a signal that can be easily measured [1].

[0004] Fetal bovine serum (FBS) is the gold standard for cell supplements and is commonly used to support cell growth in vitro. However, it is one of the most critical reagents in cell culture for different reasons [2].

[0005] First, the use of FBS involves significant ethical issues, as its production causes suffering to animals. Second, FBS is one of the largest sources of cell culture contamination. Finally, FBS composition has not been fully characterized, which may lead to batch-to-batch variability and may affect analytical precision. For all these reasons, pharmaceutical companies are working to remove FBS from different processes, as eliminating FBS appears to be a safe, cost-effective, and ethical solution [2]. Summary of the invention

[0006] The present invention provides a serum-free cell culture medium for in vitro cell culture processes and procedures. The serum-free culture medium for culturing cells comprises a) Dulbecco's Modified Eagle's Medium (DMEM), b) a nutrient mixture, c) an antioxidant and d) a supplementary glutamine source. Preferably, the nutrient mixture is an F-12 Ham nutrient mixture. In addition, it is preferred that the antioxidant is 2-mercaptoethanol. The supplementary glutamine source can be selected from glutamine or GlutaMAX.

[0007] BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 Descriptions of four SFM adaptation scenarios are shown.

[0009] Figure 2 Shown are cell viability measured throughout the HEK-293T cell adaptation process. 2A) All protocols with SFM_A and 2B) All protocols with SFM_B.

[0010] Figure 3The viability of HEK 293-T cells under different adaptation protocols using SFM_A is shown. The red line represents the 75% viability threshold.

[0011] Figure 4 Shows the use Images of HEK-293T cells in SFM Type A were acquired to determine the confluence of cells for each regimen at passage 11. Magnification: 10X.

[0012] Figure 5 . Cell growth rates under different serum-free conditions seeded at the same concentration: red: HEK-293T, yellow: regimen 1, green: regimen 2, blue: regimen 3. DETAILED DESCRIPTION OF THE INVENTION

[0014] In recent years, media supplements have been shown to play an important role in cell adaptation and proliferation. In fact, the receptors involved in cell survival, growth, and differentiation are different in cells and release different factors into their environment. In order to develop a homemade SFM for HEK-293T cells, preliminary studies on the components of the media were conducted. The first component of our homemade SFM is the basal medium, which contains a mixture of 50% DMEM and 50% F-12. The combination of the two media ensures a high amino acid content required for cell survival and growth. The basal medium is then supplemented with an insulin-transferrin-selenium (ITS) solution. Insulin is known to be essential for cell growth and metabolism in cell culture. Transferrin is an iron transporter that helps reduce the toxic levels of oxygen free radicals and peroxides produced during cell culture. Selenium protects cells from oxidative stress and reduces the production of free radicals [3].

[0015] Another important component in SFM is glutamine, which is an essential precursor for protein and ribonucleotide synthesis. However, the glutamine used in SFM may have negative effects due to its instability in solution. In fact, serum proteins avoid the breakdown and metabolism of glutamine, which can lead to the production and accumulation of ammonia that is toxic to cells. An alternative reagent to glutamine is Glutamax, which is more stable and resistant. In order to verify the optimal components, two different SFMs were developed, of which SFM_A contained Glutamax and SFM_B contained glutamine. Finally, b-mercaptoethanol was added to the formula to protect cells from oxidative stress in culture. These two culture media will be used as essential culture media to test the adaptability of HEK-293T in serum-free culture.

[0016] However, additional components can be used to better support cell survival, proliferation, and adhesion. The literature suggests the use of specific hormones and growth factors, such as epidermal growth factor (EGF) and glucocorticoids (hydrocortisone and dexamethasone), lipids, and vitamins [3]. All of these components promote different cell functions, such as proliferation, differentiation, and cell migration. In addition, some matrix components, such as fibronectin or vitronectin, can be added to maintain cell adhesion.

[0017] Although the serum-free medium shown herein was developed using HEK 293 cells, the medium can be used with a variety of cell lines, particularly those that are routinely grown in similar growth media as HEK 293 cells. Example

[0018] Cell lines

[0019] HEK-293T (Creative Bioarray, NY, USA) is a human embryonic kidney cell line that was cultured in flasks and 6-well plates. Cells used as controls were maintained in culture according to the supplier's instructions. Different adaptation protocols were used for the cells. (Chemometec, Denmark) to assess cell number and viability.

[0020] Serum-containing medium (SCM)

[0021] HEK-293T cells were cultured in the medium recommended by the supplier: Dulbecco's modified Eagle's medium (DMEM) (Merck Life Science, MO, USA) supplemented with 10% FBS (Thermo Fisher, MA, USA) and 1.5% penicillin-streptomycin (Thermo Fisher, MA, USA). Cells were maintained in a humidified incubator at 37.0°C and 5.0±1.0% CO2.

[0022] Serum-free medium (SFM)

[0023] Two serum-free media were used during the adaptation process: serum-free medium type A (SFM_A) and serum-free medium type B (SFM_B). Both media contained: Dulbecco's modified Eagle's medium / Nutrient mixture F-12 Ham (DMEM / F-12) (Merck Life Science, MO, USA), supplemented with the antioxidants 2-mercaptoethanol (Thermo Fisher, MA, USA) and insulin-transferrin-selenium (ITS-G) (Thermo Fisher, MA, USA). The difference between the two SFMs is that SFM_B contains glutamine, while SFM_A contains GlutaMAX TM Supplements (Thermo Fisher, MA, USA).

[0024] Adaptation of HEK-293T cells to serum-free medium

[0025] HEK-293T cell culture was adapted to serum-free conditions using four different protocols generally described in the literature [3]: Figure 1 ):

[0026] 1. Sequential adaptation > Cells are grown in a mixture of SCM and SFM. The percentage of SCM is gradually reduced, while the percentage of SFM is gradually increased until serum is completely removed.

[0027] 2. Serum content reduction> Cells are cultured in SFM supplemented with a certain percentage of FBS, and the percentage of FBS is reduced with each passage until the final concentration is 0%;

[0028] 3. Inside adaptation > cells are directly seeded in SFM;

[0029] 4. Adaptation with a conditioned medium> involves the use of conditioned medium where the previous passage is mixed with serum-free medium for cell growth.

[0030] Cells were monitored microscopically every 2-3 days and the medium was changed twice a week. Cells were passaged to the next generation when confluence >70% and viability >80%.

[0031] Cell proliferation

[0032] use (Sartorius) Real-time analysis of cell proliferation. It is a real-time quantitative live cell imaging and analysis platform that enables visualization and quantification of cell behavior over time by automatically collecting and analyzing images 24 / 7 in a standard laboratory incubator.

[0033] At each split, 5x103HEK-293T cells were seeded into each well of a 6-well plate using different serum-free adaptation protocols in SFM_A. Images of each well were taken every hour for a total of 4 days. For analysis, a confluence mask was created to calculate the occupied area (% confluence) of the cell images over time and generate cell growth curves.

[0034] Cell viability of HEK 293-T cells during adaptation to serum-free medium

[0035] A vial of HEK-293T cells was thawed and cell viability was determined. Since no loss of cell viability was observed, the cells were split into four regimens using two different SFMs. Cell viability was measured at each split throughout the process, as shown in Table 1. Figure 2 shown.

[0036] With SFM_A, cells were successfully adapted to 100% serum-free, maintaining high viability in 3 of the 4 protocols. With SFM_B, cells were only adapted to serum-free in protocol 2, where >75% cell viability was observed. These results suggest that SFM_A better supports HEK-293T cell adaptation in serum-free conditions. This medium will be used in the following experiments.

[0037] To identify the most promising method for the adaptation of HEK-293T cells in SFM_A, the viability obtained using the four protocols was analyzed in detail and Figure 3 The results are reported in .

[0038] Protocol Nos. 1 (3A) and 2 (3B) showed cell viability >75% during serum-free adaptation, which was comparable to that observed in cells cultured in SCM used as a control (CTRL). Figure 2 ).

[0039] Protocol No. 3 (3C) showed an initial decrease in cell viability, but recovered from passage 11. Finally, protocol 4 (3D) was able to maintain high viability only for a few passages. For this reason, this protocol was abandoned.

[0040] Based on the results obtained previously, new adaptations were made using protocols 1, 2, and 3. To monitor cell morphology and confluence, use (Sartorius) real-time analysis of cells.

[0041] like Figure 4As shown, in protocols 1 and 2, HEK-293T cell morphology remained similar to control cells. However, the cell confluence after 93 hours was reduced in protocol 2 compared to CTRL and protocol 1. On the other hand, in protocol 3, the cells were smaller in morphology and began to remain suspended rather than adherent. This adaptation protocol affected cell growth. In fact, the cell confluence reached 28% after 93 hours.

[0042] Growth curve analysis revealed differences in cell proliferation between the different regimens, confirming these observations. Figure 5 As shown, HEK-293T cells in protocol 1 (yellow line) grew similarly to those in CTRL (red line). Cells cultured using protocols 2 (green line) and 3 (blue line) showed a significant slowing of cell growth rate.

[0043] Therefore, the HEK-293T cell line was adapted to grow in serum-free medium. All experimental conditions were tested, and medium SFM_A showed the best performance in terms of cell viability. For the different protocols, 3 out of 4 protocols showed cell viability >75%, but only protocol 1 presented a cell morphology and growth rate curve similar to that of the control cells.

[0044] The possibility of having cell lines adapted to grow in a serum-free environment can become an important resource for bioassay development. In fact, serum-free cell lines can be engineered to express specific drug targets, with the ultimate goal of developing completely serum-free bioassays, improving their reproducibility and reducing their variability.

[0045] References

[0046] [1]International Pharmaceutical Quality.USP Chapter 1032.Design and development of Biological Assay.US Pharmacopeial Convention.

[0047] [2]JVDVALK, "Fetal bovine serum—a cell culture dilemma," SCIENCE, vol.375, pp.143-144, 2022.

[0048] [3]B.D.e.a.Van der Valk J,"Optimization of chemically defined cellculture media–Replacing fetal bovine serumin mammalian in vitro methods,"Toxicology in Vitro,pp.1053-1063,2010.

[0049] [4]E.J.H.K.J.v.L.J.W.H.V.J.v.Mandl,"Fibroblast growth factor-2inserum-free medium is a potent mitogen and reduces dedifferentiation of humanear chondrocytes in monolayer culture,"Matrix Biology,vol.24,p.231–241,2004.

[0050] [5]T.M.B.R.e.a.Lomba A,"Serum-Free Suspension Adaptation of HEK-293TCells:Basis for Large-Scale Biopharmaceutical Production,"Brazilian Archivesof Biology and Technology,2021.

Claims

1. A serum-free culture medium comprising: a) Dulbecco's Modified Eagle's Medium (DMEM), b) a nutrient mixture, c) an antioxidant, and d) a supplemental glutamine source.

2. The serum-free medium according to claim 1, wherein the nutrient mixture is nutrient mixture F-12 Ham.

3. The serum-free medium according to any one of the preceding claims, wherein the antioxidant is 2-mercaptoethanol.

4. The serum-free medium according to any one of the preceding claims, wherein the supplemental glutamine source is selected from glutamine or GlutaMAX.

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