Side Effects of Culture Media Antibiotics on Cell Differentiation

Introduction

The protocols for cell differentiation frequently require long-lasting cultures, which increase the risk of microbial contamination. Antibiotics were introduced into culture media to reduce the frequency of contamination. An antibiotic mix commonly used in cell culture contains 100 U/mL penicillin, a β-lactam that inhibits bacterial cell wall synthesis; 100 μg/mL streptomycin, an aminoglycoside that inhibits bacterial protein synthesis; and sometimes, 0.25 μg/mL amphotericin B, a macrolide polyene that causes disintegration of the fungal lipid membranes.^1,2 The aminoglycoside gentamicin is also used in cell culture protocols.^3,4 Thus, gentamicin 5 μg/mL is recommended to prepare the complete adipogenic differentiation medium for human adipose tissue-derived stem cells (hASCs) (STEMPRO^® human adipose-derived stem cells; Invitrogen).

Despite their effectiveness to avoid bacterial and fungal growth, antibiotics can have a number of significant disadvantages. One of them is a potential influence on cultured cell biochemistry. We accomplished a very extensive literature review and, surprisingly, only found a very small number of reports on this topic, and none of them in human cells. Thus, in rat primary hepatocyte cultures, penicillin 360 μM (∼210 U/mL)–streptomycin 175 μM (250 μg/mL) inhibited protein synthesis. ⁵ In primary adipoblasts from young obese Zucker rats, the penicillin 1 mg/mL (∼1785 U/mL)–streptomycin 33 μg/mL mix modified the heparin-releasable lipoprotein lipase activity. ⁶ In mouse B16/F10 melanoma cells, penicillin 100 U/mL–streptomycin 100 μg/mL causes a moderate stimulation in dopa oxidase and tyrosine hydroxylase activities, but a slight inactivation in the dopachrome tautomerase activity. ⁷ The absence of studies on this issue maybe is the cause for the unawareness about the effect of antibiotics used in cell culture on many different aspects of cell biochemistry.

Since the influence of these antibiotics on cell differentiation, at concentrations found in cell culture, has not been usually analyzed, we have studied their effect on adipocyte differentiation of hASCs.

Materials and Methods

Results

The hASCs cultured in the adipogenic differentiation medium slowed down proliferation and showed a morphological change with an important rise in cell volume, mainly due to a lipid accumulation, as demonstrated by the significant increase in Nile Red staining (p<0.0001), a vital stain for the detection of intracellular lipid droplets, ¹¹ and cell triglycerides (p<0.0001) (Fig. 1A, B). Moreover, the levels of secreted leptin, another adipocyte marker, ¹² were significantly higher after the differentiation protocol (p=0.0004) (Fig. 1C). All these results support the adipocyte differentiation of hASCs.

OPEN IN VIEWER

FIG. 1.

Adipocyte markers. Dot, white, gray, and black bars code for undifferentiated, differentiated, penicillin–streptomycin–amphotericin-treated differentiated, and gentamicin-treated differentiated human adipose tissue-derived stem cells (hASCs), respectively. The mean value for differentiated hASCs has been represented as 100%. ^#* denote significant differences versus differentiated hASCs. (A) Intracellular lipid droplets. (B) Triglyceride amount. (C) Secreted leptin levels.

At concentrations used in cell culture, the penicillin 100 U/mL–streptomycin 100 μg/mL–amphotericin 0.25 μg/mL mix significantly increased Nile Red staining (136%, p=0.0274) and leptin secretion (390%, p=0.0027), although it did not affect triglyceride levels (Fig. 1). During the adipocyte differentiation, gentamicin 5 μg/mL significantly increased leptin production (244%, p=0.0441), but it had no effect on triglyceride levels (Fig. 1). This gentamicin concentration is within the range of concentrations (0.9–11.4 μg/mL) found in the blood of some patients, for example, in neonates with hypoxic ischemic encephalopathy treated for presumptive infection, and therefore, it could have physiologic consequences on medicated individuals. ¹³

Although considered selective for the A-site of the prokaryotic ribosome, most aminoglycosides also bind to the A-site of the eukaryotic cytosolic ribosome, but with lower affinities. Nevertheless, their binding to the A-site of the eukaryotic mitochondrial ribosome, whose sequence is very close to the bacterial one, might inhibit mitochondrial protein synthesis and negatively affect mammal cells. ¹⁴ However, the CIV-specific activity and quantity, an enzyme with mitochondrial DNA (mtDNA)-encoded subunits and, therefore, potentially more susceptible to aminoglycosides, were not affected in hASCs differentiated in a culture with the aminoglycoside streptomycin or gentamicin (Fig. 2). Maybe, there was a relative decrease in the CIV activity and quantity but compensated, in some way, by a general mitochondrial biogenesis. However, these antibiotics did not affect the CS-specific activity, a nuclear DNA-encoded enzyme that is a marker of mitochondrial biogenesis (Fig. 2).

OPEN IN VIEWER

FIG. 2.

Mitochondrial enzymes. White, gray, and black bars code for differentiated, penicillin–streptomycin–amphotericin-treated differentiated, and gentamicin-treated differentiated hASCs, respectively. The mean value for differentiated hASCs has been represented as 100%. (A) Complex IV (CIV)-specific activity. (B) CIV quantity. (C) Citrate synthase (CS)-specific activity.

Experimental evidence in animals has indicated that reactive oxygen species (ROS) may be one of the factors responsible for the development of aminoglycoside toxicity.^15,16 Therefore, we checked the ROS amount in hASCs differentiated in the presence/absence of antibiotics. The antibiotics mix, or gentamicin, provokes a significant increase in ROS production (∼140%, p≤0.0238) (Fig. 3A). Many evidences from the literature claim that ROS are responsible for rise in different variables related to adipogenic differentiation.^17–24 Therefore, we differentiated hASCs in the presence of these antibiotics with or without NAC. This antioxidant significantly decreased Nile Red staining (>70%, p<0.0001) and secreted leptin concentrations (>60%, p<0.0001) (Fig. 3B, C).

OPEN IN VIEWER

FIG. 3.

Reactive oxygen species effects on adipocyte differentiation. White, gray, stripped gray, black, and stripped black bars code for differentiated, penicillin–streptomycin–amphotericin-treated differentiated, penicillin–streptomycin–amphotericin-N-acetylcysteine (NAC)-treated differentiated, gentamicin-treated differentiated, and gentamicin-NAC-treated differentiated hASCs, respectively. The mean value for differentiated hASCs has been represented as 100%. *denotes significant differences versus differentiated hASCs. ^#denotes significant differences between untreated and NAC-treated hASCs differentiated in the presence of antibiotics. (A) Hydrogen peroxide production. (B) Intracellular lipid droplets. (C) Secreted leptin levels.

Discussion

At very high concentrations, penicillin G [≥730 μg/mL or ≥1300 U/mL ²⁵ ] had an inhibitory effect on mouse embryonic stem cell (mESC) differentiation into cardiomyocytes, as determined by contracting cardiomyocytes and quantitative expression of the myosin heavy chain (MHC) gene. This penicillin concentration also inhibited neuronal, osteoblast, and chondrocyte differentiation as determined by quantitative expression of the neurofilament 160, osteocalcin, and aggrecan genes, respectively. ²⁶ However, these concentrations are not relevant for normal cell cultures.

At concentrations usually found in cell culture, the combination of penicillin 200 U/mL–streptomycin 50 μg/mL, streptomycin 50 μg/mL, or gentamicin 50 μg/mL interfered with the synthesis of sulfatide, a component of the myelin membrane and indicator of cell differentiation, in brain cell cultures of newborn mice. ²⁷ The accumulation of MHC, often used as a marker of myogenic differentiation of chick embryo myogenic cells, was significantly reduced in cultures with streptomycin 100 μg/mL. ²⁸ Penicillin 100 U/mL–streptomycin 100 μg/mL or gentamicin 100 μg/mL decreased the growth rate of mESC and, after their differentiation to type II pneumocytes, the mRNA levels for surfactant protein C. ²⁹

Our results confirm that antibiotics commonly used in cell culture, at concentrations usually employed, can affect cell differentiation. Moreover, they extend previous results obtained in other animal cells to encompass human cells.

Compounds from the aminoglycoside family are the antibiotics shared in all these reports. It has been previously shown that particular mtDNA mutations can increase aminoglycoside toxicity, but the absence of a CIV defect rules out an effect on mitochondrial protein synthesis as the cause for the altered cell differentiation. However, in the absence of these pathologic mtDNA mutations, high concentrations or after long periods of exposure, aminoglycosides are also usually toxic. It has been proposed that ROS can act to promote both the initiation of adipocyte lineage commitment of stem cells and the terminal differentiation of preadipocytes to mature adipose cells.^17–24 Our results on the adipocyte differentiation in NAC presence support that the increase in ROS levels provoked by aminoglycosides is responsible for the rise in other variables related with adipogenic differentiation, such as Nile Red staining and leptin secretion. The origin of aminoglycoside side effects on adipocyte differentiation and general cell differentiation probably results from a combination of different factors. Aminoglycosides are positively charged molecules that can interact with other negatively charged molecules, such as nucleic acids or phospholipids, or inhibit phospholipases.^30–32 Moreover, it has been shown that streptomycin ≥80 μg/mL increases read through of stop codons and gentamicin 5 μg/mL induces read through of a premature termination codon found in an expressed human pseudogene.^33,34

Therefore, aminoglycosides can modify cell physiology in unknown ways.^30–32 To avoid undesired phenotypic effects, in general, cultured cells should be grown without antibiotics. The use of aseptic techniques makes antibiotics unnecessary.