Standardized Isolation and Cytokine Priming of Murine Bone Marrow-Derived Mesenchymal Stromal Cells for Enhanced Immunomodulation

Bone marrow-derived mesenchymal stromal cells (BMSCs) have widespread applications in both preclinical and clinical studies. Their low immunogenicity, ready accessibility, and multilineage differentiation capacity make BMSCs promising candidates for use in clinical settings as cellular pharmaceuticals^1,2,3. Their immunosuppressive and regenerative properties have been extensively exploited in clinical studies to treat inflammatory disorders such as graft-versus-host disease (GVHD)⁴, sepsis⁵, and inflammatory bowel disease (IBD)⁶. The bone marrow stromal niche comprises a heterogeneous stromal cell population. Nevertheless, all MSCs conform to the criteria defined by the International Society for Cellular Therapy (ISCT), which include: plastic adherence, surface receptor expression -- CD73, CD90, and CD105 in humans, and CD29, CD44, and CD90 in mice -- without the expression of hematopoietic or endothelial markers - CD11b, CD14, CD19, CD34, CD45, CD79a, HLA-DR, and the capacity for adipogenic, chondrogenic, or osteogenic differentiation⁷.

BMSCs remain functional through the release of paracrine factors collectively known as the secretome, which includes cytokines, chemokines, growth factors, extracellular vesicles, and various other soluble and contact-dependent molecules^8,9. The MSCs' secretome modulates the niche by interacting with elements of the innate immune system in a bidirectional manner¹⁰. Factors released during MSCs-macrophage interactions can shift macrophages toward either a pro-inflammatory or anti-inflammatory phenotype^11,12. Conversely, the functional state of MSCs is also regulated by local immune cells -- particularly macrophages -- through feedback mechanisms¹¹. This MSC-macrophage crosstalk is indispensable for the immunosuppressive functionality of MSCs. Adoptively transferred MSCs only reduce tissue inflammation when macrophages are present, as observed in colitis mouse models. In contrast, MSCs' interaction with B or T lymphocytes in the absence of macrophages is insufficient to exert their therapeutic effects¹³.

The first phase III clinical trial using allogeneic BMSCs was completed by Osiris Therapeutics in the United States for the treatment of steroid-refractory GVHD (NCT00366145)¹. Since then, numerous preclinical and clinical studies have been conducted, although the success of clinical trials has not met expectations based on preclinical outcomes. To improve the success rate of MSC-based clinical trials, optimization of drug deployment strategies is essential. Understanding the optimal delivery route, therapeutic cell dose, and physiological state of the cells at the point of care is critical. For instance, in an in vivo colitis mouse model, studies have shown that cryopreserved MSCs or intravenous delivery are suboptimal methods of administration¹⁴.

Different approaches, like cytokine pre-licensing, genetic modification, or hypoxic preconditioning, have been widely studied to improve MSCs' in vivo persistence and therapeutic utility¹⁵. In the murine hind-limb ischemia model, hypoxia preconditioning (2% O₂) improved the proliferation and migration capacity of MSCs through increased expression of heat shock protein, 78-kD glucose-regulated protein (GRP78)¹⁶. IL-1β (Interleukin-1 beta) pre-licensing augments MSCs' ability to employ neutrophils, monocytes, lymphocytes, and eosinophils¹⁷. After in vivo delivery, MSCs are exposed to activated T and NK cells derived IFNγ, which enhances indoleamine 2,3-dioxygenase (IDO) production by MSCs¹⁸. IDO is an essential component in MSCs' secretome, which reduces the T cell proliferation rate. Interestingly, MSCs' immunosuppressive capacity is reduced when they are cocultured with IFNγ-/- mice-derived T lymphocytes¹⁹. Studies have identified that IFN-γ prelicensing reduces the rate of T cell-mediated apoptosis in cryopreserved MSCs^13,20. IFNγ priming improves the therapeutic outcome of BMSCs¹³ . All of this evidence collectively indicates the indispensable role of IFNγ in the production of more potent immunosuppressive MSCs.

To determine the most effective MSCs' delivery modality, animal models have been widely used for their ethical accessibility. In order to reduce variability in preclinical outcomes, it is crucial to establish an optimized method for the MSCs' manufacturing process that ensures high yield, cell viability, and consistency over time²¹. MSCs' passage is an important variable that can generate different therapeutic outcomes. While in vitro culturing, passaging can regulate the characterization and potency of MSCs, and after a certain number of passages, the cells become senescent²². Hence, in any clinical or preclinical study, it is crucial to be consistent with the usage of the MSC passage number. Similarly, consistency with a specific protocol for MSCs isolation is also a key factor in achieving reproducibility in the outcome of MSCs usage. Among many approaches, antibody-based cell sorting²³, negative selection method²⁴, enzymatic digestion²⁵ are popular methods of MSCs isolation. However, the applicability of these methods depends on the source, as improper usage reduces yield, potency, and trilineage differentiation capacity.

Here, we present a standardized protocol for the isolation and characterization of BMSCs, emphasizing the preferred passage number, cryopreservation technique, and BMSC-derived condition media collection. We also provide a protocol for IFN-γ-mediated MSC priming and finally demonstrate a comparative study of how peritoneal macrophages differentially respond to normal or IFN-γ pre-licensed BMSCs.

The present study was performed with the approval and following the guidelines of the animal ethical committee of the ICMR-National Institute for Research in Reproductive and Child Health (IAEC Project No.09/23) and the University of Wisconsin-Madison Institutional Animal Care and Use Committee. Female C57Bl/6J mice aged 6-8 weeks, bred and housed in specific pathogen-free (SPF) conditions, were used for the present study.

NOTE: We followed the biosafety protocols determined by the Institutional Biosafety Committee (IBSC) while working on the in vivo or in vitro studies. In brief, for animal study, we maintained gentle handling to reduce stress and risk of injury. We used appropriate PPE, such as safety glasses, gloves, a laboratory coat, a mask, and a disposable bouffant cap. For biohazard chemical reagents like DMSO, we used a biosafety cabinet to handle the chemicals. We used the specific waste container to discard biohazardous reagents or sharp objects.

1. Isolation of murine bone marrow mesenchymal stromal cells

NOTE: After hind limb collection, the rest of the steps should be performed in a tissue culture laminar flow hood, and sterile aseptic techniques²⁶.

1. Euthanize the mouse by cervical dislocation, followed by thorough rinsing of the mouse with 70% ethanol. Using ophthalmic forceps, gently tweak the abdominal skin between the two hip joints of the mouse and carefully cut the skin with scissors.
2. Separate the skin by gently pulling toward the foot. Cut down at the hip joints and ankle to free both the hind limbs.
3. Quickly transfer the hind limbs in ice-cold DMEM supplemented with 1x penicillin/streptomycin and 2% FBS to avoid tissue damage until the further dissection process.
4. Take each hind limb in a Petri dish containing the ice-cold media. Carefully peel off the muscle and connective tissue from the femur and tibia by gently scraping the diaphysis of the bone. Transfer the cleaned bone into a new Petri dish containing the ice-cold media.
5. Detach the femur and tibia, followed by making a cut at the ends of the tibia and femur just below the end of the marrow cavity. Protect the marrow cavity maximally during the femur and tibia dissection procedure.
6. Take a 1 mL sterile syringe with a 21 G needle and draw the media from the Petri plate. With sterile forceps, hold the excised femur and insert the needle gently into the marrow cavity. Flush the bone marrow cavity to get the bone marrow. Repeat this step until the bone is white. Flush the tibia in the same way.
7. Gently pipette in and out to disperse the cell clumps in the Petri dish. Using a 70 mm filter mesh, filter the cell suspension to remove any bone particles. Collect the filtrate and centrifuge it at 230 x g for 5 min at room temperature.
8. Discard the supernatant and resuspend the pellet with complete media (DMEM, 10% FBS, 5% L-Glutamine, penicillin, and Streptomycin). Measure the yield and viability of cells by Trypan blue exclusion and counting on a hemocytometer.
9. Seed the cells in a 75 cm² tissue culture-treated flask with a cell density of 5000 cells/cm² in complete DMEM media¹³. Remove non-adherent hematopoietic cells after 48 h of culture by changing the media. Replace the media with fresh media every alternate 3-4 days.
   NOTE: Use a cell culture flask instead of a dish to avoid contamination. Do not take out the flask from the incubator frequently to check the cells, since it disturbs cell growth.
10. To detach the adherent cells, wash the cells 1x with PBS, add 0.25% trypsin-EDTA, keep the cells in trypsin for 5 min. This helps to detach the cells from the well.
11. Add complete media, resuspend, and centrifuge at 230 x g for 5 min at room temperature. Discard the supernatant. Add complete DMEM media to the pellet, resuspend, and reseed the cells at a density of 5,000 cells/cm².
12. Passage the cells weekly by changing the medium every 3-4 days. After the 3rd passage, assay the MSCs culture by flow cytometric analysis as described in step 6. Use passage 3-5 cells for any experiment.
    NOTE: To cryopreserve the cells for future experimental use, centrifuge the cells at the desired volume, resuspend the cells in freezing media, store 1 x 10⁶ cells in 1 mL of freezing media in each cryovial at -80 °C overnight, followed by transferring the cells into nitrogen for long-term usage.
    Composition of freezing media: 70% DMEM, 20% FBS, 10% DMSO.

2. Preparation of conditioned medium from cultured MSCs

1. Seed the cells at a density of 5000 cells/ cm² in 6 well plate. Culture MSCs until 80% confluent.
2. Collect the conditioned media from the cell culture plate and centrifuge at 230 x g for 5 min at room temperature, filter, and use for treatment.

3. Preparation of IFNγ primed conditioned medium from cultured MSCs

1. Seed the cells in 6 well plate at a density of 5000 cells/cm². Treat the cells with 25 ng/mL IFNγ for 48 h. Perform a dose dependent study to get the optimal effect of IFNγ on MSCs. For this experiment, select a range of IFNγ concentrations, 5, 10, 25, 50, 100 ng/mL, to pre-license MSCs. The 5 and 10 ng/mL concentrations were significantly less effective in macrophage polarization, and 25 ng/mL was identified as the optimal concentration (Supplementary Figure 1).
2. After decanting the IFN-γ-containing media, wash the well 2x with PBS to ensure complete removal of IFN-γ. Any residual IFN-γ may interfere with the study outcome.
3. Incubate the cells for another 24 h in normal DMEM media at 37 °C. After 24 h, collect the conditioned media following step 2.

4. Isolation and culture of peritoneal cavity-derived macrophages

1. Inject 5 mL of ice-cold PBS (with 3% FBS) into the peritoneal cavity of mice, followed by gentle massage of the abdomen.
2. Recover the PBS solution using a sterile syringe. Collect in a 10 mL tube. Centrifuge the solution at 300 x g for 5 min.
3. Remove the supernatant. Dissolve the pellet in complete RPMI media (RPMI, 10% FBS, 1x Penicillin and Streptomycin). Plate the cells at 5000 cells/cm².
4. Remove non-adherent cells after 24 h and culture till confluence. Use cells for further experimental procedure as required.

5. Enzyme-linked immunosorbent assay

1. For detecting IL-10 derived from BMSCs-CM treated macrophages, follow the steps below.
   1. Treat the macrophages with BMSCs-CM for 24 h and collect the supernatant on the following day.
   2. Measure the amount of IL-10 using the IL-10 ELISA kit following the manufacturer's instructions.
2. For detecting CCL2 derived from BMSCs or IFNγ-treated BMSCs, use the CCL2 ELISA kit following the manufacturer's instructions.

6. Flow cytometry analysis for MSCs and macrophage phenotyping

1. Seed the cells in 6 well plate and make it confluent. Wash 1x with PBS (1 mL) followed by trypsinization. Add trypsin at 0.25% and incubate for 5 min in trypsin to completely lift the cells off the plate.
2. Add media to deactivate the trypsin activity. Collect the cells in a 10 mL polystyrene tube. Centrifuge at 300 x g for 5 min at 4 °C for macrophages and 230 x g for 5 min at 4 °C for BMSCs.
3. Remove the supernatant and resuspend the cells in 2 mL of stain buffer (ice-cold PBS with 2% FBS and 0.05% azide). Spin at 230 x g for 7 min. Decant and disrupt the pellet. Add 1 mL of Ghost red 780 viability dye and 50 mL of FACS buffer, resuspend it, and place it in the flow tube. Stain in the dark for 30 min.
4. After 30 min, wash the cells with 1 mL of stain buffer. Decant and disrupt the pellet. Add desired volume of antibody or isotype control. Incubate for another 30 min in the dark. Wash the cells with stain buffer, decant, and resuspend in 400 mL of stain buffer. Sample tube is ready for acquisition.
5. To prepare live-dead staining control, after trypsinization and centrifugation, dissolve the pellet in 2 mL of stain buffer. Take out 1 mL in another microcentrifuge tube and to this add 10 mL of DMSO to kill the cells. Vortex and incubate for 10 min. Now mix this with the other 1 mL of the cell-containing solution in a flow tube.
6. Add 1 mL of staining buffer and spin at 230 x g for 7 min. Decant and disrupt the pellet. Add 1 mL of Ghost red 780 viability dye and 50 mL of FACS buffer, resuspend it, and place it in the flow tube. Stain in the dark for 30 min. After 30 min, wash the cells with 1 mL of stain buffer. Decant and disrupt the pellet. Resuspend it in 400 mL of stain buffer. The live-dead stained sample is ready for acquisition.
   NOTE: For mouse macrophage staining, an additional staining step should be added before the standard staining protocol. To avoid inaccuracy in flow data, blocking of FC receptor is a critical step, which prevents non-specific antibody binding to Fc receptors on cells. To block the Fc receptor, add 1 mL of Fc blocking reagent (purified anti-mouse CD16/CD32 antibody) to the cell suspension and incubate for 15 min at room temperature. Perform the remaining flow antibody staining steps without washing off the Fc blocking reagent.
7. To analyze the flow data, at first, single cells should be selected based on the forward scattered and side scatter parameters. This step is crucial to remove debris and cell aggregates. Then the single cells will be gated based on live-dead staining. Perform further analysis based on the live cell population (Supplementary Figure 2).

The overall experimental schematic is described in Figure 1. Mouse Bone marrow was collected from both femur and tibia, seeded, passaged, and flow cytometry characterized (Figure 1A). BMSCs condition media (BMSCs-CM) with or without IFNγ pre-licensing were collected, and a few vials of BMSCs-CM were stored at -80 °C for future experimental needs (Figure 1B).

Bone marrow-derived cells were counted by hemocytometer (Figure 2A) and seeded at a density of 5000 cells/cm2 in complete DMEM media. After 48 h, the first medium was changed, and then the medium was changed every 3/4 days, until full confluency. We passaged the cells when they reached 80% confluency (Figure 2B). In passage 4, we characterized cells on the basis of MSC-specific marker expression. Cells were CD44, CD29, and CD90 positive, and negative for CD45, CD11b, and CD34 (Figure 2C). BMSCs can differentiate into adipogenic, osteogenic, and chondrogenic lineages14,27,28,29. This maintains the International Society for Cellular Therapy identity guidelines.

To understand the immunosuppressive effect of MSCs-CM, we treated peritoneal cavity-derived macrophages (PMφ) and treated them with BMSCs-CM (Figure 3A). We performed flow cytometry analysis of BMSCs-CM-treated macrophages (Figure 3B,C). Peritoneal cavity resident culture-adapted macrophages were stained by M1 and M2-specific surface markers. We prevented nonspecific antibody binding by incubating macrophages with purified anti-mouse CD32/CD16 (Mouse Fc Block before adding staining antibodies), before the actual staining of cellular markers. Expression of the M1-specific markers, CD86 and iNOS, and M2-specific markers, CD206 and CD163, was studied. Cells were first gated on the basis of the hematopoietic marker CD45.2 (for C57BL6). IL-10 is the signature cytokine of the M2 phenotype. IL-10 released by MSCs-CM-treated macrophages was measured by an ELISA study (Figure 3D). Since LPS/IFNg induces M1 polarization30, this treatment served as a negative control.

CCL2 is one of the signature cytokines identified in MSCs' secretome, which triggers macrophage polarization13. CCL2 released by MSCs was detected by ELISA (Figure 3E). To confirm the role of CCL2 in macrophage polarization, we pretreated macrophages with a CCR2 inhibitor followed by MSC-CM treatment (Figure 3F), and macrophage polarization was again detected by IL-10 ELISA study (Figure 3F).

Our results clearly demonstrate that the MSCs' secretome can induce anti-inflammatory macrophage polarization, as evidenced by increased IL-10 production in gut-derived macrophages following treatment with conditioned media (Figure 3).

Figure 1: Study flowchart. (A) C57BL/6 mice were euthanized. The marrow was flushed out from both the femurs and tibia, centrifuged, and cultured for a week with media changing every 3 days. MSCs were passaged and at passage 4 characterized by flow cytometry and adipogenesis study. (B) Passage 4 MSCs were seeded in 6-well plates, in one set with IFNγ added at a concentration of 25 ng/mL, and in another set, kept untreated. After 48 h, replace with normal fresh complete DMEM media. After 24 h, collect the media from the plate, centrifuge, and filter. Store the condition media in -80 °C for future use. Please click here to view a larger version of this figure.

Figure 2: Phenotypic characterization. (A) Bone marrow-derived cells were counted by a hemocytometer before seeding. (B) Cells were allowed to grow until 80% confluency before each passage. (C) Passage 4 cells were characterized by flow cytometry analysis with mouse MSC-specific markers. The scale bar represents 100 mM. Figure 2C has been modified from14. Please click here to view a larger version of this figure.

Figure 3: BM-MSC secretome analysis. (A) Schematic of experiments. (B) Representative plot forflow cytometric analysis of MSC-CM-treated PMφ. Peritoneal-derived cells were gated on the basis of the hematopoietic markers CD45.2-positive population. The selected population was further stained by M1-specific markers CD86 or iNOS, M2-specific markers CD206 or CD163, along with CD11b double staining in each case. (C) Percentage of M1 or M2 population of cells was quantified. A cell population treated with LPS/IFNγ, served as a negative control. (D) ELISA was performed to quantify IL-10 released by MSC-CM-treatedPMφ. ELISA was performed to quantify (E) CCL2 released by MSC-CM, (F) IL-10 released by PMφ. As indicated in the figure, one set of experiments was performed in the presence of a CCR2 antagonist. Error bars represent the mean ± SEM. Data representative of 3 independent experiments with 3 MSC samples per group. Student's t-test was used to perform statistical analysis of all of the experiments. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. Figure 3B-F has been modified from13. Please click here to view a larger version of this figure.

Supplementary Figure 1: BMSCs were pre-licensed with 0, 5, 10, 25, 50, 100 ng/mL IFNg. Macrophages were treated with IFNγ pre-licensed MSCs-CM. ELISA was performed to quantify IL-10 released by MSC-CM-treated PMφ. Error bars represent the mean ± SEM. Data representative of 3 independent experiments with 3 MSC samples per group. Please click here to download this File.

Supplementary Figure 2: (A-C) Single cells were selected based on the forward scattered and side scatter parameters. (D) Single cells were gated based on live-dead staining. Please click here to download this File.

The isolation of mouse bone marrow-derived mesenchymal stromal cells is a critical technique for understanding the intrinsic immunosuppressive and regenerative properties of MSCs in in vivo animal models. Although the method appears straightforward and easy to follow, several steps must be executed with precision to prevent bacterial contamination and ensure optimal cellular yield. Each step of the procedure should be carried out aseptically in an ultra-clean environment.

To avoid low yield of cells, select mice in the age group of 6-8 weeks. When cutting the ends of the femur and tibia, the marrow cavity must be protected as much as possible, and the flushing process should be firm yet gentle to ensure complete marrow extraction. The isolated cells should be incubated under an appropriate CO₂ concentration and in humidified conditions. During media changes, it is essential to gently wash the cells with phosphate-buffered saline (PBS) before replacing the media in order to eliminate contaminating hematopoietic cells. To avoid hematopoietic cell contamination, at the time of re-passaging, trypsinization should not exceed 5 min. In the early passage, media change should not be too rapid, since frequent media change may cause bacterial contamination.

At each passage, it is necessary to cryopreserve the cells in multiple batches to maintain consistency and availability. For IFN-γ priming, a dose-dependent experiment should be conducted to determine the most effective pre-licensing conditions for the MSC-conditioned media. In our experiments, we tested a range of IFNγ concentrations, i.e., 5, 10, 25, 50, 100 ng/mL, to pre-license MSCs. Then, 25 ng/mL was identified as the optimal concentration.

To preserve data integrity, cells from a specific passage number should consistently be used for all experimental assays. For flow cytometric characterization of MSCs, cells from passage 3 or higher should be used to ensure reliable detection of surface receptor expression. Live/dead cell discrimination during flow cytometry staining is essential, as it reduces the likelihood of false-negative results.

The results represent a key mechanism through which exogenously delivered BMSCs attenuate gut inflammation during colitis. IFN-γ priming enhances the effect of MSC-conditioned media on macrophage polarization. CCL2 released from BMSCs is a critical chemokine that acts on macrophages to induce M2 polarization. Our previous findings demonstrated that CCL2 and CXCL12 chemokines cooperatively act on macrophages to promote their polarization. This event is essential for propagating anti-inflammatory signaling through subsequent modulation of T cell and B cell phenotypes¹³.

In our study, we specifically followed a simple protocol for BMSCs isolation, based on the plastic adherent property of MSCs. Flow cytometry sorting or immunomagnetic sorting can be utilized to isolate BMSCs based on MSC-specific surface markers. However, in both of these methods, there is a chance of losing plenty of cells during the sample preparation. Since mouse bone marrow contains a very low number of MSCs (estimated to be about 0.001% to 0.08% of bone marrow cells), we preferred the standard plastic adherent property-based isolation method. However, this method has a few drawbacks. The major risk is hematopoietic cell contamination, which may reduce the therapeutic capacity of MSCs. Hence, identification of a specific passage number is crucial, where a significant population of cells expresses MSC-specific surface receptors.