Serum-free medium

Abstract

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

Description

[Technical Field]

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

[0002] In vitro cell-based 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 authorities. [1] To assess drug efficacy, assays must reflect or mimic, as closely as possible, the product's known or intended mechanism of action. [1] For this reason, cell-based bioassays are performed using cell lines that produce easily measurable signals in response to specific ligands or infectious agents. [1]

[0003] Fetal bovine serum (FBS) is a representative cell supplement commonly used to support cell growth in vitro. However, for different reasons, it is one of the most important reagents for cell culture [2].

[0004] First, the use of FBS poses significant ethical issues, as its production causes animal suffering. Second, FBS is one of the higher sources of cell culture contamination. Finally, the composition of FBS has not been fully characterized, which can lead to batch-to-batch variability and affect analytical accuracy. For all these reasons, pharmaceutical companies are working to remove FBS from different processes, as this is considered a safe, cost-effective, and ethical solution [2]. Summary of the Invention

[0005] The present invention provides a serum-free cell culture medium for use in in vitro cell culture processes and procedures. The serum-free medium for culturing cells comprises a) Dulbecco's Modified Eagle's Medium (DMEM), b) a nutrient mix, c) antioxidants, and d) a glutamine supplement. Preferably, the nutrient mix is ​​F-12 Ham's Nutrient Mix. Furthermore, the antioxidant is preferably 2-mercaptoethanol. The glutamine supplement may be selected from glutamine or GlutaMAX. [Brief explanation of the drawings]

[0006] [Figure 1] Figure 1 shows a description of the four adaptation protocols to SFM. [Figure 2] Figure 2 shows cell viability measured during the entire adaptation process of HEK-293T cells: 2A) All protocols using SFM_A, and 2B) All protocols using SFM_B. [Figure 3] Figure 3 shows the viability of HEK293-T cells following different adaptation protocols using SFM_A. The red line represents the 75% viability threshold. [Figure 4] Figure 4 shows images of HEK-293T cells in SFM Type A obtained using IncuCyte® to identify cell confluence for each protocol at passage 11. Magnification: 10x. [Figure 5] Figure 5. Cell proliferation rates under different serum-free conditions seeded at the same density: Red: HEK-293T, Yellow: Protocol 1, Green: Protocol 2, Blue: Protocol 3. DETAILED DESCRIPTION OF THE INVENTION

[0007] In recent years, media supplements have come to play an important role in cell adaptation and proliferation. Indeed, receptors involved in cell survival, proliferation, and differentiation vary among cells, releasing different factors into their environment. To develop a homemade SFM specifically for HEK-293T cells, we initially explored culture media components. The first component of the homemade SFM was a basal medium containing a mixture of 50% DMEM and 50% F-12. Combining the two media ensures a high amino acid content necessary for cell survival and proliferation. The basal medium was then supplemented with an insulin-transferrin-selenium (ITS) solution. Insulin is known to be essential in cell culture for cell growth and metabolism. Transferrin is an iron transporter and helps reduce toxic levels of oxygen radicals and peroxides generated during cell culture. Selenium protects cells from oxidative stress and reduces free radical production [3].

[0008] Another important component of SFM is glutamine, an essential precursor for protein and ribonucleotide synthesis. However, the glutamine used in SFM can have adverse effects due to its instability in solution. Indeed, serum proteins avoid the degradation and metabolism of glutamine, which leads to the production and accumulation of ammonia, which is toxic to cells. An alternative to glutamine is the more stable and tolerable GlutaMAX. To verify the best ingredients, two different SFMs were developed: SFM_A containing GlutaMAX and SFM_B containing glutamine. Finally, β-mercaptoethanol was added to the formulation to protect cells from oxidative stress during culture. These two media were used as essential media to test the adaptation of HEK-293T cells to serum-free culture.

[0009] However, additional components can be used to better support cell survival, proliferation, and adhesion. The literature suggests the use of certain 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 cellular functions, such as proliferation, differentiation, and cell migration. In addition to these, several matrix components, such as fibronectin and vitronectin, can be added to maintain cell adhesion.

[0010] As demonstrated herein, HEK293 cells were used to develop a serum-free medium that can be used with a variety of cell lines, particularly cell lines that grow in similar growth media under conventional conditions, such as HEK293 cells. [Example]

[0011] cell line Human embryonic kidney cell line HEK-293T (Creative Bioarray, NY, USA) was cultured in flasks and 6-well plates. Cells used as controls were maintained according to the supplier's instructions. Cells were cultured using various adaptation protocols. Cell number and viability were assessed using a NucleoCounter® (Chemometec, Denmark).

[0012] Serum-containing medium (SCM) HEK-293T cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Merck Life Science, MO, USA) supplemented with 10% FBS (Thermo Fisher, MA, USA) and 1.5% penicillin-streptomycin (Thermo Fisher, MA, USA), as recommended by the supplier. Cells were maintained in a 37.0°C incubator with a humidified atmosphere of 5.0 ± 1.0% CO2.

[0013] Serum-free medium (SFM) Two serum-free media, serum-free medium type A (SFM_A) and serum-free medium type B (SFM_B), were used during the adaptation process. Both media contained Dulbecco's Modified Eagle's Medium / F-12 Ham Nutrient Mixture (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 two SFMs differ in that SFM_B is supplemented with glutamine or SFM_A with GlutaMAX™ Supplement (Thermo Fisher, MA, USA).

[0014] Adaptation of HEK-293T cells to serum-free medium HEK-293T cell cultures were adapted to serum-free conditions using four different protocols (Figure 1), which are commonly described in the literature [3]: 1. Sequential Adaptation: Grow cells using a mixture of SCM and SFM. Gradually decrease the proportion of SCM while increasing the proportion of SFM until serum is completely removed; 2. Cultivate cells in SFM supplemented with a decreasing percentage of FBS at each passage until the serum content is reduced >0%; 3. Internal adaptation > Seeding cells directly into SFM; 4. Adaptation using conditioned medium>Involves the use of serum-free medium, where cells are grown for the previous passage mixed with serum-free medium.

[0015] Cells are monitored microscopically every 2-3 days, with medium changes performed twice weekly. Cells are passaged when they are >70% confluent and >80% viable.

[0016] Cell proliferation Cell proliferation is analyzed in real time using the Incucyte® (Sartorius), a real-time quantitative live-cell imaging and analysis platform that allows visualization and quantification of cell behavior over time by continuously and automatically collecting and analyzing images within a standard laboratory incubator.

[0017] In each aliquot, 5 × 10 cells were plated in each well of a 6-well plate using different serum-free adaptation protocols in SFM_A. 3 HEK-293T cells were plated in wells. Images of each well were taken every hour for a total of four days. For analysis, confluence masks were created to calculate the area occupied by the cells over time (% confluence) and generate a cell growth curve.

[0018] Cell viability during HEK293-T adaptation in serum-free medium One vial of HEK-293T cells was thawed and cell viability was measured. Since no decrease in cell viability was observed, the cells were split into four protocols using two different SFMs. Cell viability was measured in each split throughout the process and is shown in Figure 2.

[0019] In SFM_A, cells were successfully adapted to 100% serum-free conditions and maintained high viability in three of the four protocols. For SFM_B, cells were only adapted to serum-free conditions using protocol 2, with cell viability exceeding 75%. These results suggest that SFM_A better supports the adaptation of HEK-293T cells to serum-free conditions. This medium will be used in the following experiments.

[0020] To identify the most promising process for the adaptation of HEK-293T cells in SFM_A, we analyzed in detail the viability obtained with the four protocols and report the results in Figure 3.

[0021] Protocols 1 (3A) and 2 (3B) showed cell viability of over 75% during serum-free adaptation, comparable to that observed in cells cultured in SCM (CTRL), used as a control (Figure 2).

[0022] Protocol number 3 (3C) showed an initial decline in cell viability, which recovered from passage 11. Finally, protocol 4 (3D) was only able to maintain high viability for a few passages. For this reason, this protocol is discarded.

[0023] Based on the previously obtained results, new adaptations are made using protocols 1, 2, and 3. To monitor cell morphology and confluency, cells are analyzed in real time using Incucyte® (Sartorius).

[0024] As shown in Figure 4, the morphology of HEK-293T cells in Protocols 1 and 2 remains similar to that of the control. However, in Protocol 2, cell confluence after 93 hours is reduced compared to Control and Protocol 1. On the other hand, in Protocol 3, cells have a smaller morphology and begin to remain in a floating state without adhering. This adaptation protocol affects cell proliferation. Indeed, cell confluency reaches 28% after 93 hours.

[0025] These observations were confirmed by growth curve analysis, which showed differences in cell proliferation between protocols. As shown in Figure 5, HEK-293T cells in protocol 1 (yellow line) proliferated similarly compared to CTRL (red line). Cells cultured in protocols 2 (green line) and 3 (blue line) showed significantly reduced cell proliferation rates.

[0026] Thus, the HEK-293T cell line was adapted to grow in serum-free medium. Of all experimental conditions tested, SFM_A medium showed the best performance in terms of cell viability. Regarding the different protocols, three out of four showed cell viability above 75%, but only Protocol 1 showed cell morphology and growth rate curves similar to those of the control.

[0027] The feasibility of adapting cell lines to grow in a serum-free environment can be an important resource for bioassay development. Indeed, serum-free cell lines can be engineered to express specific drug targets, with the ultimate goal being to develop completely serum-free bioassays that increase reproducibility and reduce variability.

[0028] References [1] International Pharmaceutical Quality. USP Chapter 1032. Design and development of Biological Assay. US Pharmacopeial Convention. [2] JVD VALK, "Fetal bovine serum-a cell culture dilemma," SCIENCE, vol. 375, pp. 143-144, 2022. [3] BD ea Van der Valk J, "Optimization of chemically defined cell culture media - Replacing fetal bovine serum in mammalian in vitro methods," Toxicology in Vitro, pp. 1053 - 1063, 2010. [4] E. J. H. K. J. v. L. J. W. H. V. J. v. Mandl, "Fibroblast growth factor-2 in serum-free medium is a potent mitogen and reduces dedifferentiation of human ear chondrocytes in monolayer culture," Matrix Biology, vol. 24, p. 231-241, 2004. [5] T. M. B. R. e. a. Lomba A, "Serum-Free Suspension Adaptation of HEK-293T Cells: Basis for Large-Scale Biopharmaceutical Production," Brazilian Archives of Biology and Technology, 2021.

Claims

1. Serum-free medium containing a) Dulbecco's Modified Eagle's Medium (DMEM), b) a nutrient mix, c) antioxidants, and d) a glutamine supplement.

2. 2. The serum-free medium of claim 1, wherein the nutrient mix is ​​F-12 Ham nutrient mixture.

3. 3. The serum-free medium according to claim 1, wherein the antioxidant is 2-mercaptoethanol.

4. The serum-free medium according to any one of claims 1 to 3, wherein the glutamine supplement source is selected from glutamine or GlutaMAX.

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