Viability of human umbilical cord–derived mesenchymal stem cells in G-rich and M-rich alginates

Materials

Pharmaceutical-grade sodium alginates (FMC biopolymers, Philadelphia, PA; Protanal LF200S and Protanal LF120M); sodium chloride and calcium chloride dihydrate (Fisher scientific, Pittsburgh, PA, USA); 2,5-diphenyl tetrazolium bromide (MTT) and antibiotic and antimicotic solution as well as phosphate buffered saline (PBS; Sigma, St. Louis, MO); low-glucose Dulbecco’s modified Eagle’s medium (DMEM) and trypsin (Invitrogen, Carlsbad, CA); fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA, USA); and hUMSCs (Sciencell, Carlsbad, CA).

Bead preparation

Two percent w/v solutions of G-rich and M-rich alginate polymers were prepared in physiological saline solution and passed through a 25-µm syringe filter and then extruded dropwise through a 25-gauge needle into a calcium chloride (CaCl₂) solution under magnetic stirring. The beads were left in the gelling medium for 15 min and then washed twice in sterile double distilled water, followed by a rinse with sterile PBS.

Cell culture

The hUMSCs were cultured in monolayers in T75 flasks with low-glucose DMEM supplemented with 10% fetal bovine serum and 1% antibiotic and antimicotic solution. The cells were fed with a fresh DMEM every 2–3 days. Cultures were propagated at 37°C under humidified conditions using 5% CO₂. The cells grown up to 70%–80% confluency were treated with trypsin to dislodge them from the flask. Suspended cells were plated either in 12- or 24-well plates, according to the experimental design, where series of experiments were conducted to investigate the effects of alginates and calcium chloride on the hUMSC viability.

Cell viability in the presence of alginate beads

A hUMSC suspension (density of 30 × 10⁴) in a growth medium, containing DMEM, 10% fetal bovine serum, and 1% antibiotic and antimicotic solutions, was placed in a flat-bottom, 12-well plate. After the cells reached 30% confluency, freshly prepared alginate beads (~30, 60, and 120 mg) were added and incubated at 37°C in a 5% CO₂ incubator for 48 hours. The cell viability was evaluated by a standard colorimetric MTT assay. The same procedure was used to prepare alginate beads cross-linked at different calcium chloride solutions (50, 100, 150, and 200 mM) and used to evaluate their effect on cell viability.

Cell viability in alginate solutions

Monolayer cells in a 24-well plate with 30%–40% confluency were exposed to ~30 mg w/v of LF200S and LF120M alginate solutions. Cell viability was estimated by MTT assay (described below) after 48 hours of incubation period.

Cell viability in calcium chloride solution

The hUMSCs plated in a 24-well plate with a density of 3 × 10⁴ were exposed to 30 mg w/v of CaCl₂ (10, 50, 100, and 200 mM) solutions. Cell viability was estimated after 48 hours of incubation period.

MTT assay

Viable hUMSCs were counted using MTT colorimetric assay after being cultured in the presence of 2 wt% alginate beads for 48 hours. Following Mosmann et al., ²¹ 100 µL of 2 mg/mL of MTT solution in a PBS was added to the wells after the designated incubation times and was further incubated for 4 hours at 37°C in a CO₂ incubator. The medium containing the free dye was removed, the cell layer was briefly rinsed with PBS, and the formazan crystals were dissolved in 200 µL of dimethyl sulfoxide (DMSO), where its absorbance was measured at 570 nm. The values for each of the treatments represent the average from duplicate wells.

Protein content

The protein content of the alginates was determined utilizing bicinchoninic acid (BCA) method. To a total of 100 µL protein sample and 2 mL of BCA reagent was added and incubated for 30 min at 37°C.

Total phenols content

The total phenol content in all alginate samples was measured using Folin–Ciocalteu (FC) method. ²² Serially diluted quercetin (standard phenolic compound) was mixed with 400 µL of FC reagent followed by 800 µL of 20% sodium carbonate. The mixture was allowed to stand for 15 min and total phenol was determined by a spectrophotometer at 765 nm. The standard curve was prepared using 0, 2.5, 5, 10, 20, and 40 µg solutions of quercetin in ethanol:water mixture (50:50 v/v). Total phenol content was expressed in terms of quercetin equivalent.

Statistical Analysis

A Student’s t-test (two-tailed, paired) was used to evaluate the data, and a p < 0.05 was considered to be statistically significant.

Cell viability in the presence of alginate beads cross-linked with calcium

Cells grown in monolayer were exposed to three different quantities of alginate beads (Figure 1), and their viability was assessed (mean ± SD, n = 3) against control (cells in the absence of beads). As shown in Figure 2, the cells were comparatively viable in the presence of LF200S, especially at low and medium doses of 30 and 60 mg, respectively, while the cells were 23% less viable at the maximum gel dose of 120 mg. However, at all gel doses, the viability of the hUMSCs was significantly decreased up to 34% in the presence of LF120M alginate gels at the maximum gel dose. Regardless of the gel type, the cells showed less viability at the maximum gel dose, even the rate of survival was less in the presence of LF120M gel beads. Between the two grades of alginate, the hUMSCs were generally shown to be less viable in the presence of LF120M. This may be accounted for in terms of the high M content of this polymer or high G content of the LF200S, which affects the gel strength after complexation.^23,26 Wang et al. ²⁷ observed that higher proportions of guluronic acid provided better colonization and differentiation of bone marrow cells in rats.

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Figure 1.

Alginate gel beads.

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Figure 2.

hUMSC percentage of viability in the presence of various doses of alginates (mean ± SD, n = 4).

Since the biocompatibility of alginates depends on various factors such as alginate concentration, molecular weight,^28–31 solution viscosities,^32,33 guluronic acid content^29,34, and purity levels,^35–37 our studies revealed that the viability of hUMSCs depends on the alginate composition as well as its optimum concentration and dose. To elucidate the effect on cell viability of the G content of the polymer, another series of experiments were conducted in which the concentration of the gelling agent ranged between 50 and 200 mM.

Cell viability in the presence of alginate beads cross-linked with different calcium concentrations

At the lower CaCl₂ concentrations (50 and 100 mM), the hUMSC viability was comparable to that of the control in the presence of LF200S gel beads (Figure 3). However, cells were found to be 15% less viable at the higher CaCl₂ concentration range of 150 to 200 mM. However, the viability at all CaCl₂ concentrations was significantly less for the cells in the presence of LF120M. The minimum viability of ~75% was observed at the lowest concentration and ~15% less viability was observed in the range of 100–200 mM. Interestingly, cell viability was ~15% less for both alginate gels over a concentration range of 150–200 mM. The minimum viability of 74% for the cells in the presence of LF120M gel beads can be accounted for in terms of the weak physicomechanical properties of the gel due to the lower cross-link density. However, there is an effective CaCl₂ concentration range (50–100 mM), where comparable hUMSC viability was observed with LF200S gel beads.

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Figure 3.

hUMSC % viability in the presence of alginate gel beads (LF200S and LF120M) cross-linked with different calcium chloride concentrations and calcium chloride (CaCl₂) alone (mean ± SD, n = 4); bars from left to right: control, 50, 100, 150, and 200 mM.

When gel beads are in contact with the cells, two components of the gel can potentially be extracted into the medium over time; these are CaCl₂ residues and the water soluble (non-cross-linked fraction or sol fraction) alginate polymer, which might affect the cell viability at high concentrations. To monitor the hUMSC viability in the presence of non-cross-linked alginate polymers, experiments were conducted in the absence of calcium and in pure CaCl₂ solutions at different concentrations.

Cell viability in alginate solutions

Experiments were conducted at 30-mg gel dose using two grades of alginates. The cell viability was dramatically less in the presence of LF120M (33%) compared to LF200S (18%) (Table 1). Since the only obvious difference between the two grades is their G/M ratio building blocks, the viability is apparently related to the G or M contents of the alginate polymer.

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Table 1.

hUMSC viability in the presence of non-cross-linked 2% alginate solutions.

Alginate	Cell viability (%)	SD	p-value
LF200S	82.07	0.09	p < 0.05
LF120M	67.71	0.04	p < 0.05

hUMSC: human mesenchymal stem cells derived from umbilical cord.

Cells viability in calcium chloride solutions

The hUMSC viability was comparable to that of control over the CaCl₂ concentration ranging from 50 to 200 mM and significant at the minimum concentration of 50 mM (Figure 2). Physiologically, calcium ions seem to regulate a wide range of intracellular functions, including cell attachment, motility, and signal transductions ³⁸ as well as that of a second messenger. The intracellular free calcium concentration is ~100 nM in the cytoplasm and ~100 µM in the endoplasmic reticulum, while extracellular calcium concentration is ~1.2 mM. ³⁹ However, an excess of calcium provokes cell toxicity, leading to apoptosis followed by cell death.^38–40 In this study, cells treated with calcium chloride over the range of 50 to 200 mM did not show any significant decrease in cell viability. The hUMSC viability was decreased at high calcium concentration of 150 to 200 mM (Table 2), but the lower cell viability was apparently not due to higher calcium residues based on the viability data at high calcium concentration.

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Table 2.

G/M ratio, protein, and polyphenol contents of the two alginates.

Alginate	G/M (%)	Protein content (mg/g)	Polyphenolcontent (mg/g)
LF200S	65–75/25–35	3.585	0.190
LF120M	30–35/65–70	3.058	0.249

G: guluronic acid; M: mannuronic acid.

Alginate protein and polyphenol content

The major contaminants of sodium alginate, due to its natural origin, are proteins, polyphenols, and endotoxins. These contaminants cause inflammatory reactions in vivo. Since there was no purity data for the commercial alginates used in this study, the impurity level was measured to see if they may affect hUMSC viability. Although both alginates’ grades differed considerably in G and M content, the amounts of protein and polyphenol were similar (Table 2).