The impact of media supplement on the viability, proliferation, and differentiation potential of bone marrow-derived mesenchymal stem cells

Nibras Hatim Khamees

doi:10.4103/mj.mj_49_22

ORIGINAL ARTICLE

Year : 2022 | Volume : 21 | Issue : 2 | Page : 145-150

The impact of media supplement on the viability, proliferation, and differentiation potential of bone marrow-derived mesenchymal stem cells

Nibras Hatim Khamees
Department of Human Anatomy, College of Medicine, Mustansiriyah University, Baghdad, Iraq; School of Cellular and Molecular Medicine, Faculty of Biomedical Sciences, University of Bristol, Bristol, UK

Date of Submission	25-Sep-2022
Date of Decision	01-Oct-2022
Date of Acceptance	04-Oct-2022
Date of Web Publication	2-Jan-2023

Correspondence Address:
Dr. Nibras Hatim Khamees
Department of Human Anatomy, College of Medicine, Mustansiriyah University, P. O. Box 14132, Baghdad

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/mj.mj_49_22

Abstract

Background: Bone marrow-derived mesenchymal stem cells (bmMSCs) are one of the most promising therapeutic methods in modern medicine. These cells are grown in tissue culture media, commonly supplemented with fetal bovine serum (FBS), but such supplement carries a number of drawbacks, such as immunological reaction and composition variation. Another supplement known as insulin-transferrin-selenium could act as serum replacement and help enhance the experimental results and therapeutic value of MSCs. Objective: The objective was to investigate the effect of tissue culture supplement in regard to bone marrow MSCs viability, proliferation, and differentiation potential. Materials and Methods: Human bmMSCs were grown in tissue culture plates at seeding density of 5 × 10³ cells/cm². The cells were divided into two groups, 10% FBS supplement or insulin-transferrin-sodium (ITS) supplement. The viability of the cells was assessed with live/dead cells kit (ethidium homodimer-1 and calcein). Cell proliferation was assessed with MTS assay, while multilineage differentiation potential was assessed with human MSC functional identification kit. Results: Our results showed that the viability of MSCs was comparable between FBS and ITS supplement groups at 24 h and 48 h intervals. Both groups showed similar proliferation capacity after 48 h and 72 h of incubation with no significant statistical difference. The cells from both groups were able to differentiate into osteoblasts, adipocytes, and chondrocytes. Conclusion: Insulin-transferrin-selenium supplement could be used as an alternative to FBS in laboratory experiments and clinical applications (such as cell therapy) to avoid the drawbacks of FBS and enhance the outcome of these applications.

Keywords: Differentiation, fetal bovine serum, insulin-transferrin-selenium, proliferation, stem cells

How to cite this article:
Khamees NH. The impact of media supplement on the viability, proliferation, and differentiation potential of bone marrow-derived mesenchymal stem cells. Mustansiriya Med J 2022;21:145-50

How to cite this URL:
Khamees NH. The impact of media supplement on the viability, proliferation, and differentiation potential of bone marrow-derived mesenchymal stem cells. Mustansiriya Med J [serial online] 2022 [cited 2023 Jun 4];21:145-50. Available from: https://www.mmjonweb.org/text.asp?2022/21/2/145/366637

Introduction

One of the most promising new therapeutic methods in modern medicine is the use of stem cells. This approach requires a number of factors to be considered before it can be implemented. Some of these include the type of cells that will be used, its therapeutic potential, and its biological safety.^[1],[2] One of the most common types of stem cells that will be used in this process is adult stem cells, mainly mesenchymal stem cells (MSCs).

One of the most important factors that will be considered when it comes to the use of MSCs is the quality of the cell. In addition to being ethical, the process of obtaining multiple types of stem cells, such as those derived from bone marrow, is also less challenging than that of induced pluripotent stem cells and embryonic stem cells. These cells have a low teratogenic index and a high potential for proliferative activity.^{[2],[3],[4],[5]} MSCs are also known to be ideal candidates for the treatment of various conditions such as osteoarthritis, osteomyelitis, neurodegenerative disorders, and burn. Due to their unique properties, such as their ability to differentiate into different types of cells, these cells can be used to replace damaged tissues.^[6],[7] This makes MSCs handy tools in regenerative medicine.

MSCs are a heterogeneous population of cells that resemble fibroblasts in shape. They can differentiate into various types of cells such as cartilage, fat, and bone. The first known human multipotent stem cells were isolated from a bone marrow aspirate. Currently, bone marrow-derived MScs (bmMSCs) are the most commonly studied type of stem cells.^[8],[9]

In vitro and in vivo studies have shown that multilineage stem cells can differentiate into various types of cells, in contrast to the initial belief that these cells could only differentiate into mesodermal lineage cells.^[10],[11] Recent studies have shown that these cells can differentiate into other types of cells, such as neurons and hepatocytes.^[12]

The process of multilineage stem cells being able to differentiate into different types of cells involves the use of various factors. These include growth factors, cytokines, and extracellular matrix (ECM) molecules.^[13]

The interactions between the ECM and stem cells are known to be bidirectional. The availability of certain binding sites and the microenvironment's effect on the behavior of bmMSCs are some of the factors that can influence this interaction. It is believed that the large dissimilarity in the tissue microenvironment can affect the differentiation and proliferation of stem cells.^[14]

The role of the ECM in the development of bmMSCs is known to be its structural framework that regulates cell development and maintains the niche.^[15] It can help guide the development of the bmMSCs by providing a base for their various activities, such as their homing and adhesion. On the other hand, it can also affect the remodeling of the matrix by enzymatic degradation.^[16]

In in vitro experiments, the culture media serve as the microenvironment in which MSCs proliferate and differentiate. Thus, the composition of this media could have an impact on cells differentiation potential and function.^[17] Most culture media will be supplemented with fetal bovine serum (FBS) at the time of cell culture as it provides an important source of proteins, growth factors, and different nutrients. On the other hand, FBS could have a negative impact in some in vitro experiments due to variations in its purity and components, and it could elicit an immunological reaction if these cultured MSCs are used in cell therapy.^[18]

Currently, there are a number of commercially available serum-free media with not well-recognized components. A possible alternative is to use ordinary media with insulin-transferrin-sodium (ITS) supplement (insulin-transferrin and selenium), which has been investigated in previous studies for their role in the proliferation of different cell types as a source of growth factors and ECM proteins.^[19],[20]

Our aim was to compare ITS versus FBS as media supplement in regard to bmMSCs viability, proliferation, and differentiation potential.

Materials and Methods

Primary culture of bone marrow-derived MSCs

These experiments were performed on bone marrow aspirates, which were obtained from patients who had undergone hip replacement surgery at Southmead Hospital in Bristol, UK. The samples were collected under the ethical guidelines of North Bristol NHS Trust. To avoid sample clotting, the aspirates were placed in sterile conditions in heparinized tubes.

One milliliter of aspirates was allotted in T175 flasks, which were filled with 25 ml of low-glucose DMEM (Sigma, Dorset, UK) supplemented with 10% (v/v) FBS (Sigma, Dorset, UK), 1% penicillin/streptomycin (Sigma, Dorset, UK), 1% (v/v) GlutaMAX (GIBCO, Paisley, UK), and 10 ng/ml FGF-2 (PeproTech, London, UK).

The flasks were placed at a temperature of 37°C and a humidity of 95% for 4 days. The first medium change was carried out to remove nonadherent and suspended blood cells. The medium was changed every 2–3 days to maintain the confluency of the adherent bmMSCs.

The bmMSCs were then passed through the expansion stage, which involved the use of 0.05% trypsin/ethylenediaminetetraacetic acid for 3 min and a centrifuge for 5 min. They were then reseeded into the culture media and subjected to a density of about 5000 cells/cm². Throughout the 4 days of the experiments, the culture medium was changed regularly, and the incubation environment was maintained at optimal conditions. Before the cells were used in the experiments, the population purity and phenotype of the bmMSCs were analyzed by flow cytometry.

Cell viability assay

48-well plates were seeded with bmMSCs and labeled to indicate assay time points. Individual wells were assigned as FBS supplement or ITS supplement groups. Cells were incubated at 37°C in 5% CO₂ for 24 and 48 h with either 10% FBS-supplemented media or ITS-supplemented media. At the end of the specified incubation period, wells were washed once with phosphate-buffered saline (PBS). 200 μl of the detection dye, consisting of 30 μl of ethidium homodimer-1 and 7.5 μl calcein AM in 15 ml of PBS, was added to each well and incubated for 30 min at room temperature in the dark. The viability of the cells was checked according to the manufacturer's instructions using inverted fluorescent microscope with the attached digital camera to obtain cell images. Live cells stain green, while dead cells stain red.

Cell proliferation assay

Cell proliferation assay was assessed with CellTiter 96^® AQ_ueous One Solution Cell Proliferation Assay (MTS) (Promega, USA). Human bmMSCs were seeded in 96-well plates at seeding density of 5000 cells/cm² in either 10% FBS-supplemented media or ITS-supplemented media at 37°C in 5% CO_2. After 48 h from cell culture, MTS solution was added to the wells of the test and control groups. Then, after 3 h of incubation, the optical density (as an estimation of cell proliferation) was measured by ELISA at 450-nm absorbance and plotted in Excel.

Mesenchymal stem cell differentiation

bmMSCs differentiation was carried out according to the protocol provided with human MSC functional identification kit (R&D Systems, Inc.). The cells were divided into two groups, control group (with 10% FBS as supplement) and test group (with ITS as supplement).

Induction of osteogenesis

bmMSCs were seeded in 96-well plates at a density of 4.2 × 10³ cells/cm² in osteogenic medium that consisted of α minimum essential medium supplemented with 10% FBS (or ITS), 1% penicillin/streptomycin, 1% (v/v) GlutaMAX, and 50 μl/ml osteogenic supplement. The medium was changed every 3–4 days for 3 weeks. The cells phenotype was checked by immunocytochemistry using anti-osteocalcin antibody stain with the appropriate controls.

Induction of adipogenesis

bmMSCs were seeded in 96-well plates at a density of 2.1 × 10⁴ cells/cm² in adipogenic medium that consisted of α minimum essential medium supplemented with 10% FBS (or ITS), 1% penicillin/streptomycin, 1% (v/v) GlutaMAX, and 10 μl/ml adipogenic supplement. The medium was changed every 3–4 days for 3 weeks. The cells phenotype was checked by immunocytochemistry using anti-FAB4 antibody staining with the appropriate controls.

Induction of chondrogenesis

bmMSCs were seeded in 96-well plates at a density of 2.1 × 10⁴ cells/cm² in monolayer culture with chondrogenic medium. The medium consisted of high-glucose DMEM supplemented with 1% penicillin/streptomycin, 1% (v/v) GlutaMAX, 1% 100 mM sodium pyruvate, 1% ITS supplement, and 1 μl/ml from each of transforming growth factor beta, ascorbic acid, and dexamethasone. The medium was changed every 3–4 days for 3 weeks. The cells phenotype was checked by immunocytochemistry using anti-aggrecan antibody staining with the appropriate controls.

Statistical analysis

All experiments were performed in triplicate using cells from three different patients. Data were evaluated using GraphPad Prism software version 9 (GraphPad Software, La Jolla, CA, USA). Data are expressed as mean ± standard error of the mean unless otherwise specified. Paired t-test was used to determine the statistical significance. P ≤ 0.05 were considered statistically significant.

Results

Cell viability assay

After 24 h of incubation, the viability of bmMSCs was similar in both FBS-supplemented media and ITS-supplemented media, with most cells stained with calcein (green) and few cells stained with ethidium homodimer-1 (red), as shown in [Figure 1]a and [Figure 1]b.

Figure 1: Viability of bmMSCs after 24 and 48 h incubation. (a) 24 h incubation in FBS-supplemented media, (b) 24 h incubation in ITS-supplemented media, (c) 48 h incubation in FBS-supplemented media, (d) 48 h incubation in ITS-supplemented media. Viable cells stain green and dead cells stain red. Calcein and ethidium homodimer-1 stain. ×40. FBS: Fetal bovine serum, ITS: Insulin-transferrin-sodium, bmMSCs: Bone marrow-derived mesenchymal stem cells

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After 48 h of incubation, the number of dead cells increased slightly in both conditions (FBS-supplemented media and ITS-supplemented media) compared to the cells at 24 h time interval. Although, the percentage of viable cells was comparable between both culture conditions [Figure 1]c and [Figure 1]d.

Cell proliferation assay

In both control and test groups, bmMSCs proliferate at comparable rates and exhibit fibroblast-like cell morphology, which is typical of MSCs grown in vitro [Figure 2]a and [Figure 2]b. The cells were spindle shaped and grown in colonies.

The results of the MTS assay showed that bmMSCs grown in FBS-supplemented media proliferate faster than that grown in ITS-supplemented media; however, the difference in proliferation capacity did not reach statistical significance, as shown in [Figure 3]a and [Figure 3]b.

Figure 2: Proliferation and morphology of unstained bmMSCs showing the confluent layer of fibroblast-like cells. (a) Unstained bmMSCs in 10% FBS-supplemented media. (b) Unstained bmMSCs in ITS-supplemented media. ×100. FBS: Fetal bovine serum, bmMSCs: Bone marrow-derived mesenchymal stem cells

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Figure 3: MTS assay showing the proliferation of bmMSCs in 10% FBS versus ITS. bmMSCs were incubated in either 10% FBS- or ITS-supplemented media, and cell proliferation was estimated after 24 h (a) and 48 h (b) using MTS assay at 450-nm absorbance with ELISA. Ns: Nonsignificant, FBS: Fetal bovine serum, ITS: Insulin-transferrin-sodium, bmMSCs: Bone marrow-derived mesenchymal stem cells

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Cell differentiation potential

After 21 days of induction of bmMSCs differentiation in the appropriate differentiation induction media, results showed that bmMSCs successfully differentiated into osteoblasts, adipocytes, and chondrocytes in both culture conditions, namely 10% FBS-supplemented media and ITS-supplemented media. There was no difference in differentiation in relation to cell type. Osteogenic differentiation was confirmed by positive staining of differentiated cells with anti-osteocalcin [Figure 4]a and [Figure 4]b. Adipogenic differentiation was confirmed by positive staining with anti-FAB4 and the presence of lipid vacuoles [Figure 4]c and [Figure 4]d, while chondrogenic differentiation was confirmed by positive staining with anti-aggrecan [Figure 4]e and [Figure 4]f.

Figure 4: Multilineage differentiation potential of bmMSCs in FBS- versus ITS-supplemented media using immunocytochemistry staining. (a and b) Differentiated osteoblasts showing calcium crystals with cells nuclei. Red areas indicate osteoblasts detected mouse anti-osteocalcin. (c and d) differentiated adipocytes showing lipid vacuoles with cells nuclei. Red areas indicate adipocytes detected by goat antifatty acid-binding protein Ab. (e and f) differentiated chondrocytes with cells nuclei. Red areas indicate chon

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Discussion

Studies on the therapeutic potential of bmMSCs have been conducted in the context of various human disorders.^[21],[22] Due to the emergence of antibiotic resistance and the increasing number of superbug infections, modern medicine is in need of new strategies to treat these diseases. In vitro and in vivo studies have been conducted on the various aspects of this issue. One of the most promising new therapeutic methods that can be used in the treatment of various human diseases is the use of human stem cells. These cells, known as bmMSCs, are a type of adult stem cell that can be used in the treatment of various conditions. They are very small and can potentially migrate to sites of inflammation and infection.^[21],[23]

Although the use of a standard culture medium supplement known as a 10% FBS is commonly used in the generation of human MSCs for basic research, it is not ideal due to various safety concerns. These include the presence of xenogeneic proteins, high variability in the batches, the risk of contamination, and the possible presence of growth inhibitors.^{[24],[25],[26]}

In vitro culture, media containing ITS is commonly used as a supplement for the generation of stem cells in various species of mammals.^[27] Insulin is a polypeptide that is known to play a role in the differentiation of cells and promote the uptake of glucose and amino acids. It can also stimulate mitosis through its effects on the mitogenic process.^[28] Transferrin is an iron-transporting protein that needs to be bound to iron in order for iron to nourish the cells in culture.^[29] Selenium is also an essential element that can help protect cells from free radical production and lipid peroxidation, so it is used as culture media supplement.^[28]

One of the important factors in cell culture is the viability of cells during the culture period and during expansion. We found that the use of ITS as culture media supplement did not interfere with cell viability and was comparable to that obtained with 10% FBS-supplemented culture media. This is the result of ITS components that influence cell growth, as explained earlier. The observed increase in dead cells at 48 h interval in both culture condition is due to the increase in the number of cells in the cells monolayer, which result in competition for the available nutrients with subsequent death of some cells without effecting the overall cells viability.^[30]

We cultured bmMSCs in ITS-supplemented media and measured the cells proliferation capacity. bmMSCs exhibited normal MSCs shape and proliferated rapidly with normal doubling time and proliferation rate similar to that of FBS-supplemented media as shown by MTS assay results. This indicates that ITS supplement does not impact bmMSCs proliferation and could be used in various applications, in which cell culture is required for research purposes or for clinical applications such as cell therapy. These findings are constant with other studies.^[31]

Finally, the multilineage differentiation potential is one of the important aspects of bmMSCs therapeutic potential as it incorporates bmMSCs as a versatile therapeutic tool that can help in tissue regeneration, and cell therapy in different disease conditions. Our results showed that bmMSCs exhibit similar differentiation potential in both 10% FBS-supplemented media and ITS-supplemented media, and the cells were able to differentiate into osteoblasts, adipocytes, and chondrocytes. This implies that the use of serum-free media in these situations does have a negative impact on bmMSCs, which will minimize the drawbacks associated with the use of serum-supplemented media, as explained earlier. As a result, a more positive result could be obtained in in vitro experiments and therapeutic situations that require the absence of FBS.

Conclusion

The use of ITS supplement in serum-free culture media does not interfere with bmMSCs viability, proliferation, and multilineage differentiation potential. As a result, such a media could be used as an alternative to FBS in laboratory experiments and clinical applications (such as cell therapy) to avoid the drawbacks of FBS and enhance the outcome of these applications.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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