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Cellular biocompatibility of cyanophycin substratum prepared with recombinant Escherichia coli
Biochemical Engineering Journal 105 (2016) 97–106
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Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Regular article
Cellular biocompatibility of cyanophycin substratum prepared with recombinant Escherichia coli Wen-Chi Tseng a,∗ , Tsuei-Yun Fang b,∗∗ , Sheng-Yang Chen a a b
Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei, Taiwan Department of Food Science, National Taiwan Ocean University, 2 Pei-Ning Rd., Keelung 202, Taiwan
a r t i c l e
i n f o
Article history: Received 5 June 2015 Received in revised form 28 August 2015 Accepted 16 September 2015 Available online 24 September 2015 Keywords: Tissue cell culture Biomedical Protein Biomimetics Macrophage activation
a b s t r a c t Cyanophycin from recombinant Escherichia coli is composed of aspartic acid as a backbone with arginine and lysine as the side chains. Cyanophycin exists in insoluble and soluble forms based on its solubility in aqueous solution. This study aims to assess the physical properties and cellular biocompatibility of cyanophycin prepared with recombinant E. coli. The decomposition temperature of cyanophycin was around 230 ◦ C for both forms of cyanophycin, as measured by thermogravimetric analysis. Soluble cyanophycin showed no toxicity to Chinese Hamster Ovary (CHO) cells at a concentration of 5 mg/mL as revealed by the thiazolyl blue tetrazolium bromide method. When the insoluble cyanophycin formed thin films, the films exhibited a structure of stacking lamellae. CHO cells grown on the films had a higher relative cell density, or 107–142% that of those grown on tissue culture polystyrene (TCPS), 48 h after seeding. After the removal of serum-containing medium, the CHO cells maintained cell morphology for up to 72 h in Dulbecco’s modified Eagle medium without serum, and the relative cell density was 150–170% that of the cells grown on TCPS 48 h after serum removal, indicating that the cyanophycin substratum could provide sustained cell growth. When RAW 246.7 cells were grown on the films of insoluble cyanophycin for 96 h, nitric oxide concentration released from the macrophages was below 2 mM/mg protein, suggesting that a minimal immune response was elicited. The results showed that cyanophycin might have the potential to serve as a biocompatible, degradable material in biomedical applications, such as tissue engineering and drug delivery. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Cyanophycin (multi-l-arginyl-poly-l-apspartic acid) is a nonribosomal polypeptide that exists as granules in inclusion bodies mostly inside cyanobacteria [1] and serves as an intracellular energy nutrient when the organisms are grown under nitrogen limitations [2–4]. Cyanophycin has a comb-shaped molecular structure. The backbone consists of aspartic acid of which carboxylic group is linked to the amine group of arginine [5,6]. The synthesis of cyanophycin is directed by a single enzyme, cyanophycin synthetase [7–9]. Recently, cyanophycin has been produced by recombinant DNA technology in other organisms such as Escherichia coli [8,10–12], Saccharomyces cerevisiae [13],
∗ Corresponding author at: Department of Chemical Engineering, National Taiwan University of Science and Technology, No. 43, Sec. 4, Keelung Rd., Taipei 106, Taiwan. Fax: +886 2 2462 2586. ∗∗ Corresponding author. Fax: +886 2 2462 2192. E-mail addresses: [email protected] (W.-C. Tseng), [email protected] (T.-Y. Fang). http://dx.doi.org/10.1016/j.bej.2015.09.012 1369-703X/© 2015 Elsevier B.V. All rights reserved.
and Pichia pastoris [14]. Corynebacterium glutamicum, Ralstonia, Eutropha, Pseudomonas putida [15], and plants [16,17]. Cyanophycin has been shown to be resistant to several commercially available endoproteases and exopeptidases [9]. However, cyanophycin could be degraded into aspartic acid-arginine dipeptides and free arginine extracellularly by cyanophycinase from cyanobacteria. Several previous studies reported that cyanophycinase from other strains also has the capability of digesting cyanophycin extracellularlly [18–21]. The cyanophycin in nature contains an equal molar ratio of aspartic acid and arginine, and has a molecular weight distribution from 25 to 100 kDa [22,23]. The recombinant cyanophycin, with a less dispersed distribution of molecular weight from 14 to 45 kd, can incorporate lysine into the side chains during its synthesis [13,24]. According to the solubility at physiological pH (pH 7), cyanophycin can be classified as soluble and insoluble forms. The soluble form from recombinant E. coli has a high lysine/arginine molar ratio and a lower molecular weight distribution than the insoluble form [25]. The primary amine of lysine residues can also allow reactions under moderate conditions, such as at room temperature.
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Several previous studies have demonstrated the applications of cyanophycin [26–28]. The dipeptides produced from the partial digestion of cyanophycin could provide some nutritional benefits [21,27]. However, the use of cyanophycin as a biomaterial has received relatively little attention. In this study, we examine the cellular biocompatibility of both soluble and insoluble forms of cyanophycin. Cyanophycin was prepared with the culture of recombinant E. coli harboring the gene coding for cyanophycin synthetase. The powder of purified cyanophycin was subjected to thermogravimetric analysis. Cellular cytotoxicity of cyanophycin was examined by the MTT method for the CHO cells grown in the medium containing soluble cyanophycin, and on the films of insoluble cyanophycin, respectively. The release of nitric oxide from RAW 246.7 cells grown on the films of insoluble cyanophycin was used to assess the immune response. 2. Materials and methods 2.1. Materials Both soluble and insoluble forms of cyanophycin were prepared with the culture of recombinant E. coli harboring cyanophycin synthetase gene on pET21b as previously described [25]. Fetal bovine serum, Penicillin–Streptomycin–Amphotericin B antibiotic solution, and Dulbecco’s modified Eagle medium (DMEM) were obtained from Thermo Fisher Scientific (Waltham, MA). Multi-well microplates were from Corning (Tewksbury, MA). Other chemicals were purchased from Sigma–Aldrich (St. Louis, MO) and used as received. Water was deionized by a Milli-Q water purification system (Bedford, MA). 2.2. SDS polyacrylamide gel electrophoresis The purified cyanophycin was mixed with an equal volume of the loading buffer. The mixture was heated in boiling water bath for 5 min, and 20 L of the mixture was applied to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). 2.3. Thermogravimetric analysis Approximately 3–5 mg of cyanophycin powder was placed in an aluminum pan in a thermogravimetric analyzer (PerkinElmer Diamond TG/DTA) operating under 20 mL/min nitrogen flow. After equilibrium at 25 ◦ C for 30 min, the sample was heated to 750 ◦ C at 10 ◦ C/min. The decomposition temperatures were then analyzed. 2.4. Gel permeation chromatography The molecular weight distribution of soluble cyanophycin was determined by a gel permeation chromatography (GPC) equipped with Ultrahydrogel columns and a refractive index detector (Waters; Milford, MA). An aliquot of 200 L aqueous solution containing 0.5 mg/mL of purified soluble cyanophycin was injected into the GPC system with pullulan as calibration standards.
2.6. Preparation of insoluble cyanophycin film Different concentrations of insoluble cyanophycin were dissolved in 0.1 N HCl. An aliquot of 200 L was then added into each well of 12-well tissue culture plates to obtain four different films, with cyanophycin concentrations of 0.25, 0.5, 1, and 2 mg/cm2 . After the water was evaporated to dryness at room temperature for 24 h, 1 mL of 0.15 M sodium bicarbonate in 50% ethanol was added into each well to neutralize HCl. Then cross-linking was performed in 1 mL of 70% ethanol containing 0.5% glutaraldehyde for 1 h. After the removal of glutaraldehyde solution, 1 mL of 70% ethanol was added for disinfection for 4 h followed by washing with 1.5 mL phosphate buffered saline (PBS) twice to remove the residual ethanol. The film was further equilibrated with DMEM overnight prior to the application of cells. 2.7. Scanning electron microscopy observation The films of insoluble cyanophycin were peeled off by forceps, immersed in liquid nitrogen, and snapped to smaller pieces. The snapped film was sputtered with platinum, and both the top surface and the cross section were examined under a scanning electron microscope (JSM-6390, JOEL). 2.8. Measurement of cellular viability Cellular viability was monitored using thiazolyl blue tetrazolium bromide (MTT) [29]. CHO cells were harvested from the T-75 flasks and seeded at a density of 10,000 cells/cm2 . For the cells on the films of insoluble cyanophycin, the medium was replaced with fresh complete medium every 48 h, and was further cultured until the specified time period. For soluble cyanophycin, the medium was replaced with the complete medium containing various concentrations of soluble cyanophycin at 0.001, 0.01, 0.1, 1, and 5 mg/mL 24 h post seeding. For the measurement of cellular viability, the cells were washed twice with 1 mL phosphate buffered saline (PBS; pH 7.2). An aliquot of 0.5 mL PBS solution containing 0.5 mg of MTT was added to the cultured cells. After 4 h incubation at 37 ◦ C, 1 mL dimethyl sulfoxide was added to solubilize the colored formazan product, and the absorbance was measured at 540 nm by a spectrophotometer (Jasco, Tokyo, Japan). A correlation of the cell number and the absorbance from MTT assay was established by applying various amounts of cells onto the multi-well plates. A series of two wells containing the same amount of seeding cells were employed in pairs to establish the quantitative correlation. After 24 h culture, one of the wells was subject to MTT assay, and the cells on the other well were trypsinized and counted with a hemocytometer under a microscope. 2.9. Protein determination The cells were washed twice with PBS, and then incubated with 0.5 mL of lysis buffer (0.2 M Tris–HCl, 1% Triton X-100, 5 mM EDTA, and protease inhibitors, pH 7.8) for 5 min followed by centrifugation at 10,000 × g, 4 ◦ C for 10 min to prepare the cell lysate. The protein content of the lysate supernatant was quantitated using the Bradford’s method [30].
2.5. Cell culture 2.10. NO release measurement CHO-K1 cells (a Chinese Hamster Ovary cell line, ATCC-CCL-61) and RAW 246.7 cells (a mouse leukaemic monocyte macrophage cell line, ATCC-TIB-71) were maintained at 37 ◦ C, 5% CO2 , and 100% humidity in complete media containing DMEM supplemented with 10% fetal bovine serum, 2 mM glutamine, and 1% Penicillin–Streptomycin–Amphotericin B antibiotic solution.
The amounts of NO released from RAW 246.7 cells into medium were measured by 2,3 diaminonaphthalene (DAN) as previously described [31]. Briefly, 200 L of the culture medium of RAW 246.7 cells was collected and brought to 1 mL with water. The mixture was mixed with 100 L of 50 g/mL DAN in 0.62 N HCl. The reaction
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Fig. 1. Distribution of molecular weight of cyanophycin. The purified cyanophycin was subjected to the SDS-PAGE analysis as shown in panel A (M: protein markers; insol: insoluble cyanophycin; sol: soluble cyanophycin). The molecular weights of protein standards () were plotted along with the Rf positions of both soluble (䊐) and insoluble cyanophycin () to estimate their molecular weights (panel B). Panel C shows the elution profile of the soluble cyanophycin (dashed line) analyzed by GPC with pullulan as the standards (solid line) which molecular weights are 708000, 107000, 21100, and 6100.
was allowed to take place in the dark for 10 min, and then terminated by adding 200 L of 2.8 N NaOH. The fluorescence intensity was measured at an excitation wavelength 520 nm and emission wavelength 605 nm with a 5 nm slit by a fluorimeter (Eclipse; Varian, Palo Alto, CA). The standard curve was constructed by using different concentrations of sodium nitrite. 3. Results and discussion 3.1. Molecular weight distributions of cyanophycin The molecular weight distributions of purified cyanophycin were examined by SDS–PAGE and GPC. The Rf of each band on the SDS–PAGE gel of Fig. 1A was plotted with its corresponding molecular weight of protein markers in a logarithmic scale in Fig. 1B. The molecular weights of soluble and insoluble cyanophycin that were calculated with the linear regression curve of standard protein markers ranged from 25 to 45 kDa and from 8 to 30 kDa,
respectively. Because the insoluble cyanophycin became soluble in a condition as acid as 0.1 N HCl which prevented its GPC analysis due to the limitations of applicable pH ranges of the columns, only the molecular weight of soluble cyanophycin was examined by GPC. The weight-average molecular weight, Mw, is 18250, and the number-average molecular weight, Mn, is 10280 as calculated from the elution profiles of Fig. 1C. The soluble cyanophycin exhibits a polydispersity index of 1.78. Both SDS–PAGE and GPC measures gave rather consistent molecular weight distributions of cyanophycin. 3.2. Thermo-decomposition temperatures of cyanophycin In an attempt to understand the thermal stability of cyanophycin, thermogravimetric analysis (TGA) was carried out to measure the decomposition temperature of cyanophycin. The TGA thermogram showed that both soluble and insoluble cyanophycin had similar decomposition temperatures, around 230 ◦ C (Fig. 2).
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Fig. 2. Thermogram of cyanophycin. Both insoluble (—) and soluble (- - -) cyanophycin prepared with recombinant E. coli were analyzed by a thermogravimetric analyzer under a nitrogen stream.
The soluble cyanophycin has a lower molecular weight than the insoluble cyanophycin, and the lysine molar content of soluble cyanophycin is higher than that of the insoluble form [25]. The small differences in the decomposition temperatures indicated that both molecular weight distributions and lysine contents only minimally affected the thermal decomposition of cyanophycin. Both forms of cyanophycin also showed an approximate 10% weight loss during the onset of the heating process. The weight loss was presumably due to the evaporation of water, suggesting a moisture content of 10% water associated with the lyophilized cyanophycin. On the other hand, the slow slopes of the TGA thermogram also suggested that the cyanophycin might exhibit amorphous forms, not crystalline. Unlike other proteins, the recombinant cyanophycin might be in a denatured state because the purification process involved an acidic condition of 0.1 N HCl. Compared with other proteins which require a defined conformation to achieve biological activity, the high decomposition temperature might allow further modifications of cyanophycin to proceed at higher temperatures without the loss of biological activity. 3.3. Cellular toxicity of soluble cyanophycin CHO cells were cultured in the presence of various concentrations of soluble cyanophycin to examine its potential cytotoxicity. Because of the poor solubility of soluble cyanophycin in DMEM, the concentration was limited to below 0.8% (w/v). As depicted in Fig. 3, the soluble cyanophycin exhibited almost no toxicity to CHO cells at a concentration as high as 5 mg/mL, when the relative cell density on the tissue culture polystyrene (TCPS) was taken as 100% at each sampling time. Interestingly, the relative cell density was slightly increased when the cells were cultured for 96 h at a concentration of soluble cyanophycin above 1 mg/mL, suggesting that high concentrations of soluble cyanophycin might promote the cell growth. 3.4. Cell growth on the films of insoluble cyanophycin The minimal cytotoxicity of soluble cyanophycin prompted the examination of cell growth on cyanophycin substratum. Various concentrations of insoluble cyanophycin were employed to prepare thin films for cell growth. Before being cross-linked by glutaraldehyde, the dried cyanophycin films were brittle, fragile, and tended to become detached from the dishes when solvents were added. For the insoluble form, which was dissolved in 0.1 N HCl, the residual acidity on the dried films might have caused a local redissolution when an aqueous solvent was added. Therefore, bicarbonate buffer was used in an attempt to neutralize the acidity before the
Fig. 3. Cellular toxicity of soluble cyanophycin. CHO cells were grown in the medium containing additional soluble cyanophycin at various concentrations. The cell density was obtained from the correlation of cell number and the MTT assay at different time periods (48 h: white; 72 h: \\\; 96 h:///) after seeding, and then is expressed by taking the cell density of cells on TCPS at each sampling time as 100%. The corresponding cell densities on TCPS for 100% were approximate 36500, 78600, and 167500 cells/cm2 for 48 h, 72 h, and 96 h, respectively. An asterisk indicates the significant difference between the viability in the presence of soluble cyanophycin and that on TCPS (p < 0.05) (mean ± S.D.; n = three independent experiments).
application of glutaraldehyde solution. For 20 min of cross-linking, cracked films were still observed after glutaraldehyde solution was replaced with aqueous solution, presumably due to insufficient cross- linking. Therefore, the period of cross-linking was prolonged to one hour. The dried films remained intact, and appeared to be transparent at low concentrations of cyanophycin and slightly yellowish to brownish around the edges for those with high concentrations of cyanophycin after washing with ethanol and PBS. The films were peeled from the TCPS, immersed in liquid nitrogen, and snapped to small pieces for the observation of the cross sections. SEM observation revealed the lack of porous structures inside the films as shown in Fig. 4. Most films exhibited nearly smooth surfaces (Fig. 4A–C) although some roughness was found on the surface of the thick film containing 2 mg/cm2 cyanophycin (Fig. 4D). When the cross section was examined, no pores were observed, and the film thickness was found to increase almost proportional to the contents of cyanophycin except for the lowest content of 0.25 mg/cm2 as shown in Fig. 4E–H. The results indicate that most cyanophycin remained intact after crosslinking, but insufficient crosslinking might occur when the cyanophycin content was too low to form a rigid structure which could avoid being removed during the subsequent washing processes. Interestingly the cross section showed a structure of stacking thin lamellae which run almost in parallel irrespective of the cyanophycin contents (Fig.4E–H). The stacking lamellae were not observed on the top surface of cyanophycin films which shows a contrastingly smooth surface. Formation of the stacking structure remains unclear, but might presumably relates with the side chains of cyanophycin, mainly the interactions among the amine group of lysine and the guanidino group of arginine. CHO cells were seeded onto the films. The cell proliferation revealed that the films of insoluble cyanophycin had almost no toxic effect on the cells (Fig. 5A). Furthermore, when compared with the cells grown on TCPS (which relative cell density was taken as 100% at each sampling time), the cells on the cyanophycin films exhibited higher relative cell densities as analyzed by the MTT method at the time period of 48 h after seeding (Fig. 5A). Specifically, the relative cell densities on the films containing high cyanophycin contents, such as 1 and 2 mg/cm2 were noted as high as 135% that of TCPS at a short time period of 48 h. After a longer period of culture, the differences in the relative cell density on TCPS and those on cyanophycin
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Fig. 4. Film structure of insoluble cyanophycin by SEM observation. The films of cyanophycin after cross-linking were observed under SEM. The top surfaces were shown in panels A–D and the corresponding cross sections were shown in panels E–H for the films prepared with 0.25, 0.5, 1, and 2 mg/cm2 insoluble cyanophycin, respectively.
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Fig. 5. Cellular viability on the films of insoluble cyanophycin. In panel A, CHO cells were seeded onto the films prepared with different amounts of insoluble cyanophycin. The cell density was obtained from the correlation of cell number and the MTT assay at different time periods (48 h: white; 72 h: \\\; 96 h:///) after seeding, and then is expressed by taking the cell density of cells on TCPS at each sampling time as 100%. The corresponding cell densities on TCPS for 100% were approximate 36500, 78600, and 167500 cells/cm2 for 48 h, 72 h, and 96 h, respectively. An asterisk indicates the significant difference between the viability on the films of insoluble cyanophycin and that on TCPS (p < 0.05). (mean ± S.D.; n = three independent experiments) The cell morphology 96 h post seeding was photographed under a microscope at a magnification of 10 × 20. Panels B1–B5 show the CHO cells on TCPS and on the films of insoluble cyanophycin at 0.25, 0.5, 1, and 2 mg/cm2 , respectively.
films almost disappeared. The cell morphologies were indistinguishable for the cells on the TCPS and on the cyanophycin films (Fig. 5B), suggesting that the cells on cyanophycin films presumably maintained a similarly healthy state as those on TCPS. After CHO cells were seeded and cultured in complete medium for 24 h, the culture medium was replaced with DMEM without serum. The relative cell density on TCPS after an additional 48 h culture in the absence of serum was set as 100%. Higher relative cell densities were observed for those cells grown on the films containing insoluble cyanophycin. As the culture continued, the cells grown on TCPS gradually became apoptotic as the relative cell density dropped to 60%, and then to below 20% after an
additional 96 h culture (Fig. 6A). The cell number diminished and the cell shapes became rounded (Fig. 6B1). On the other hand, the cells on the cyanophycin films maintained a healthier state as the relative cell density stayed almost unchanged after an additional culture in the absence of serum for 72 h, and dropped slightly after 96 h (Fig. 6A). For the cells grown on the insoluble cyanophycin films, the changes in confluence over time were less than those on TCPS, and most of the cell shapes remained unchanged (Fig. 6B2–5), especially on the films containing higher concentrations of cyanophycin, suggesting that those films provided a sustained environment for cell growth. The basis of the ability of cyanophycin to support sustained cell growth remains
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Fig. 6. Sustained cell growths on the films of insoluble cyanophycin after the removal of serum medium. In panel A, CHO cells were seeded onto TCPS (white) and the films prepared with different amounts of insoluble cyanophycin (0.25 mg/cm2 : \\\; 0.5 mg/cm2 : ////; 1 mg/cm2 : ×××; 2 mg/cm2 : black). After 24 h culture, the medium containing serum was replaced by DMEM without serum. The cell density was obtained from the correlation of cell number and the MTT assay at different time periods after serum removal and is expressed by taking the cell density on TCPS 48 h after serum removal as 100%, approximate 33100 cells/cm2 (mean ± S.D.; n = three independent experiments). The cell morphology 72 h after serum removal was photographed under a microscope at a magnification of 10 × 20. Panels B1–B5 show the CHO cells on TCPS and on the films of insoluble cyanophycin of 0.25, 0.5, 1, and 2 mg/cm2 , respectively.
unclear, but might be attributable to the unique comb structure of arginine and lysine side chains along the aspartic backbone; the lysine and arginine could provide a charged environment to which the cells might more readily anchor. Most of the adherent cells require anchorage to a suitable substratum for proliferation. In addition to TCPS, some polypeptides, such as collagen, gelatin, fibronectin, have been employed to coat the surface in order to provide such an environment. A previous report showed that HeLa cell line had approximate proliferation rates on collagen films and on TCPS, and human skin fibroblasts proliferated at a slightly higher rate on collagen films than on TCPS [32]. Another report indicated approximate cell densities of 3T3 fibroblast grown on TCPS and on gelatin films after culturing for
1 and 3 days [33]. The components of extracellular matrix such as collagen can assist the modulation of cell behavior. Cyanophycin exhibits an equivalent capability to provide a suitable environment for cell proliferation when being compared with those two commonly used natural polypeptides, collagen and gelatin. Additionally, cyanophycin can be chemically conjugated onto other polymers through its lysine side chains, and might be expected to become another alternative used in preparing substratum for in vitro cell expansion. The films of insoluble cyanophycin can furthermore sustain CHO cell growth for a prolonged period of time after the replacement of DMEM containing no serum. Such a characteristic might provide a platform for those studies which intend to investigate the
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Fig. 7. Levels of nitric oxide released from macrophages grown on the films of insoluble cyanophycin. Macrophages (RAW 246.7 cells) were grown on TCPS (white) and the films prepared with different amounts of insoluble cyanophycin (0.25 mg/cm2 : \\\; 0.5 mg/cm2 : ////; 1 mg/cm2 : +++; 2 mg/cm2 : ×××). The cells grown on TCPS alone and in the presence of LPS (black) were used as controls. The protein contents of the cell lysate (panel A) and the levels of nitric oxide (panel B) released from the macrophages were determined at different time periods (mean ± S.D.; n = three independent experiments). The cell morphology 96 h post seeding was photographed under a microscope at a magnification of 10 × 20. Panels C1–C6 show the cells on TCPS, and on the films of insoluble cyanophycin of 0.25, 0.5, 1, and 2 mg/cm2 , and in the presence of 0.1 mg/mL LPS, respectively.
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interactions of reagents with cells and require the exclusion of the interferences of serum. Cells can be grown on the films for a period of time and then the regular medium can be replaced with DMEM containing the specific reagents.
Appendix A. Supplementary data
3.5. Nitric oxide (NO) release by macrophages grown on the films of insoluble cyanophycin
References
In order to examine whether cyanophycin stimulates the release of nitric oxide from macrophages, RAW 246.7 cells were cultured on the films containing different amounts of insoluble cyanophycin. Both the protein content and NO release were monitored at different time intervals. The protein content was used as an indicator for cell proliferation. After a 48 h culture, macrophages proliferated at higher rates on the cyanophycin films, specifically for those containing higher cyanophycin concentrations (Fig. 7A). However, the advantages of cyanophycin for the growth of macrophages became marginal after 72 h and 96 h in culture (Fig. 7A). This observation was in agreement with the results obtained in the cultures of CHO cells grown on cyanophycin films (Fig. 6A). The NO release from the macrophages grown on TCPS alone and from those grown on the cyanophycin films stayed at comparable levels for up to 72 h in culture (Fig. 7B). After a 96 h culture, NO release slightly increased to around 2 mM/mg protein for the macrophages grown on the cyanophycin films. On the other hand, the presence of lipopolysaccharide (LPS) in the medium slightly limited the growth of macrophages (Fig. 7A), and the NO release continued to increase with time to 11 mM/mg protein after a 96 h culture (Fig. 7B). The morphology of macrophage tends to become dendritic upon activation. The cells exhibited a dendritic shape in the presence of LPS (Fig. 7C6), whereas most of the cells remained inactivated in the absence of LPS (Fig. 7C1) and on the cyanophycin films (Fig. 7C2–5), even after a 96 h culture. Although the cyanophycin was prepared with recombinant E. coli, the amounts of NO release suggested that the residual lipopolysaccharides of E. coli became minimally associated with the purified cyanophycin. LPS has been reported to be labile in a strongly acidic environment. Most of the LPS might be destroyed or removed during the purification process in the presence of 0.1 N HCl. Consequently, the morphology of very few RAW 246.7 cells possessed a dendritic shape after 96 h culture on the films of insoluble cyanophycin.
4. Conclusion Cyanophycin, a degradable biopolymer, was prepared in recombinant E. coli, and was subsequently used for the preparation of cyanophycin films. The films of insoluble cyanophycin elicited minimal NO release from macrophages grown on the films, suggesting that minimal macrophage activation might be evoked. In addition, cyanophycin exerted no cellular toxicity in these cultures, and furthermore the cyanophycin films provided a culture condition for sustained and high relative cell viability in comparison with those cells grown on the TCPS. This study showed that cyanophycin might have the potential of serving as a biocompatible, degradable material for future use in biomedical applications.
Acknowledgement This research was supported by grant 103-2221-E-011-136 from the Ministry of Science and Technology at Taiwan.
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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bej.2015.09.012.
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Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Regular article
Cellular biocompatibility of cyanophycin substratum prepared with recombinant Escherichia coli Wen-Chi Tseng a,∗ , Tsuei-Yun Fang b,∗∗ , Sheng-Yang Chen a a b
Department of Chemical Engineering, National Taiwan University of Science and Technology, Taipei, Taiwan Department of Food Science, National Taiwan Ocean University, 2 Pei-Ning Rd., Keelung 202, Taiwan
a r t i c l e
i n f o
Article history: Received 5 June 2015 Received in revised form 28 August 2015 Accepted 16 September 2015 Available online 24 September 2015 Keywords: Tissue cell culture Biomedical Protein Biomimetics Macrophage activation
a b s t r a c t Cyanophycin from recombinant Escherichia coli is composed of aspartic acid as a backbone with arginine and lysine as the side chains. Cyanophycin exists in insoluble and soluble forms based on its solubility in aqueous solution. This study aims to assess the physical properties and cellular biocompatibility of cyanophycin prepared with recombinant E. coli. The decomposition temperature of cyanophycin was around 230 ◦ C for both forms of cyanophycin, as measured by thermogravimetric analysis. Soluble cyanophycin showed no toxicity to Chinese Hamster Ovary (CHO) cells at a concentration of 5 mg/mL as revealed by the thiazolyl blue tetrazolium bromide method. When the insoluble cyanophycin formed thin films, the films exhibited a structure of stacking lamellae. CHO cells grown on the films had a higher relative cell density, or 107–142% that of those grown on tissue culture polystyrene (TCPS), 48 h after seeding. After the removal of serum-containing medium, the CHO cells maintained cell morphology for up to 72 h in Dulbecco’s modified Eagle medium without serum, and the relative cell density was 150–170% that of the cells grown on TCPS 48 h after serum removal, indicating that the cyanophycin substratum could provide sustained cell growth. When RAW 246.7 cells were grown on the films of insoluble cyanophycin for 96 h, nitric oxide concentration released from the macrophages was below 2 mM/mg protein, suggesting that a minimal immune response was elicited. The results showed that cyanophycin might have the potential to serve as a biocompatible, degradable material in biomedical applications, such as tissue engineering and drug delivery. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Cyanophycin (multi-l-arginyl-poly-l-apspartic acid) is a nonribosomal polypeptide that exists as granules in inclusion bodies mostly inside cyanobacteria [1] and serves as an intracellular energy nutrient when the organisms are grown under nitrogen limitations [2–4]. Cyanophycin has a comb-shaped molecular structure. The backbone consists of aspartic acid of which carboxylic group is linked to the amine group of arginine [5,6]. The synthesis of cyanophycin is directed by a single enzyme, cyanophycin synthetase [7–9]. Recently, cyanophycin has been produced by recombinant DNA technology in other organisms such as Escherichia coli [8,10–12], Saccharomyces cerevisiae [13],
∗ Corresponding author at: Department of Chemical Engineering, National Taiwan University of Science and Technology, No. 43, Sec. 4, Keelung Rd., Taipei 106, Taiwan. Fax: +886 2 2462 2586. ∗∗ Corresponding author. Fax: +886 2 2462 2192. E-mail addresses: [email protected] (W.-C. Tseng), [email protected] (T.-Y. Fang). http://dx.doi.org/10.1016/j.bej.2015.09.012 1369-703X/© 2015 Elsevier B.V. All rights reserved.
and Pichia pastoris [14]. Corynebacterium glutamicum, Ralstonia, Eutropha, Pseudomonas putida [15], and plants [16,17]. Cyanophycin has been shown to be resistant to several commercially available endoproteases and exopeptidases [9]. However, cyanophycin could be degraded into aspartic acid-arginine dipeptides and free arginine extracellularly by cyanophycinase from cyanobacteria. Several previous studies reported that cyanophycinase from other strains also has the capability of digesting cyanophycin extracellularlly [18–21]. The cyanophycin in nature contains an equal molar ratio of aspartic acid and arginine, and has a molecular weight distribution from 25 to 100 kDa [22,23]. The recombinant cyanophycin, with a less dispersed distribution of molecular weight from 14 to 45 kd, can incorporate lysine into the side chains during its synthesis [13,24]. According to the solubility at physiological pH (pH 7), cyanophycin can be classified as soluble and insoluble forms. The soluble form from recombinant E. coli has a high lysine/arginine molar ratio and a lower molecular weight distribution than the insoluble form [25]. The primary amine of lysine residues can also allow reactions under moderate conditions, such as at room temperature.
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Several previous studies have demonstrated the applications of cyanophycin [26–28]. The dipeptides produced from the partial digestion of cyanophycin could provide some nutritional benefits [21,27]. However, the use of cyanophycin as a biomaterial has received relatively little attention. In this study, we examine the cellular biocompatibility of both soluble and insoluble forms of cyanophycin. Cyanophycin was prepared with the culture of recombinant E. coli harboring the gene coding for cyanophycin synthetase. The powder of purified cyanophycin was subjected to thermogravimetric analysis. Cellular cytotoxicity of cyanophycin was examined by the MTT method for the CHO cells grown in the medium containing soluble cyanophycin, and on the films of insoluble cyanophycin, respectively. The release of nitric oxide from RAW 246.7 cells grown on the films of insoluble cyanophycin was used to assess the immune response. 2. Materials and methods 2.1. Materials Both soluble and insoluble forms of cyanophycin were prepared with the culture of recombinant E. coli harboring cyanophycin synthetase gene on pET21b as previously described [25]. Fetal bovine serum, Penicillin–Streptomycin–Amphotericin B antibiotic solution, and Dulbecco’s modified Eagle medium (DMEM) were obtained from Thermo Fisher Scientific (Waltham, MA). Multi-well microplates were from Corning (Tewksbury, MA). Other chemicals were purchased from Sigma–Aldrich (St. Louis, MO) and used as received. Water was deionized by a Milli-Q water purification system (Bedford, MA). 2.2. SDS polyacrylamide gel electrophoresis The purified cyanophycin was mixed with an equal volume of the loading buffer. The mixture was heated in boiling water bath for 5 min, and 20 L of the mixture was applied to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). 2.3. Thermogravimetric analysis Approximately 3–5 mg of cyanophycin powder was placed in an aluminum pan in a thermogravimetric analyzer (PerkinElmer Diamond TG/DTA) operating under 20 mL/min nitrogen flow. After equilibrium at 25 ◦ C for 30 min, the sample was heated to 750 ◦ C at 10 ◦ C/min. The decomposition temperatures were then analyzed. 2.4. Gel permeation chromatography The molecular weight distribution of soluble cyanophycin was determined by a gel permeation chromatography (GPC) equipped with Ultrahydrogel columns and a refractive index detector (Waters; Milford, MA). An aliquot of 200 L aqueous solution containing 0.5 mg/mL of purified soluble cyanophycin was injected into the GPC system with pullulan as calibration standards.
2.6. Preparation of insoluble cyanophycin film Different concentrations of insoluble cyanophycin were dissolved in 0.1 N HCl. An aliquot of 200 L was then added into each well of 12-well tissue culture plates to obtain four different films, with cyanophycin concentrations of 0.25, 0.5, 1, and 2 mg/cm2 . After the water was evaporated to dryness at room temperature for 24 h, 1 mL of 0.15 M sodium bicarbonate in 50% ethanol was added into each well to neutralize HCl. Then cross-linking was performed in 1 mL of 70% ethanol containing 0.5% glutaraldehyde for 1 h. After the removal of glutaraldehyde solution, 1 mL of 70% ethanol was added for disinfection for 4 h followed by washing with 1.5 mL phosphate buffered saline (PBS) twice to remove the residual ethanol. The film was further equilibrated with DMEM overnight prior to the application of cells. 2.7. Scanning electron microscopy observation The films of insoluble cyanophycin were peeled off by forceps, immersed in liquid nitrogen, and snapped to smaller pieces. The snapped film was sputtered with platinum, and both the top surface and the cross section were examined under a scanning electron microscope (JSM-6390, JOEL). 2.8. Measurement of cellular viability Cellular viability was monitored using thiazolyl blue tetrazolium bromide (MTT) [29]. CHO cells were harvested from the T-75 flasks and seeded at a density of 10,000 cells/cm2 . For the cells on the films of insoluble cyanophycin, the medium was replaced with fresh complete medium every 48 h, and was further cultured until the specified time period. For soluble cyanophycin, the medium was replaced with the complete medium containing various concentrations of soluble cyanophycin at 0.001, 0.01, 0.1, 1, and 5 mg/mL 24 h post seeding. For the measurement of cellular viability, the cells were washed twice with 1 mL phosphate buffered saline (PBS; pH 7.2). An aliquot of 0.5 mL PBS solution containing 0.5 mg of MTT was added to the cultured cells. After 4 h incubation at 37 ◦ C, 1 mL dimethyl sulfoxide was added to solubilize the colored formazan product, and the absorbance was measured at 540 nm by a spectrophotometer (Jasco, Tokyo, Japan). A correlation of the cell number and the absorbance from MTT assay was established by applying various amounts of cells onto the multi-well plates. A series of two wells containing the same amount of seeding cells were employed in pairs to establish the quantitative correlation. After 24 h culture, one of the wells was subject to MTT assay, and the cells on the other well were trypsinized and counted with a hemocytometer under a microscope. 2.9. Protein determination The cells were washed twice with PBS, and then incubated with 0.5 mL of lysis buffer (0.2 M Tris–HCl, 1% Triton X-100, 5 mM EDTA, and protease inhibitors, pH 7.8) for 5 min followed by centrifugation at 10,000 × g, 4 ◦ C for 10 min to prepare the cell lysate. The protein content of the lysate supernatant was quantitated using the Bradford’s method [30].
2.5. Cell culture 2.10. NO release measurement CHO-K1 cells (a Chinese Hamster Ovary cell line, ATCC-CCL-61) and RAW 246.7 cells (a mouse leukaemic monocyte macrophage cell line, ATCC-TIB-71) were maintained at 37 ◦ C, 5% CO2 , and 100% humidity in complete media containing DMEM supplemented with 10% fetal bovine serum, 2 mM glutamine, and 1% Penicillin–Streptomycin–Amphotericin B antibiotic solution.
The amounts of NO released from RAW 246.7 cells into medium were measured by 2,3 diaminonaphthalene (DAN) as previously described [31]. Briefly, 200 L of the culture medium of RAW 246.7 cells was collected and brought to 1 mL with water. The mixture was mixed with 100 L of 50 g/mL DAN in 0.62 N HCl. The reaction
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Fig. 1. Distribution of molecular weight of cyanophycin. The purified cyanophycin was subjected to the SDS-PAGE analysis as shown in panel A (M: protein markers; insol: insoluble cyanophycin; sol: soluble cyanophycin). The molecular weights of protein standards () were plotted along with the Rf positions of both soluble (䊐) and insoluble cyanophycin () to estimate their molecular weights (panel B). Panel C shows the elution profile of the soluble cyanophycin (dashed line) analyzed by GPC with pullulan as the standards (solid line) which molecular weights are 708000, 107000, 21100, and 6100.
was allowed to take place in the dark for 10 min, and then terminated by adding 200 L of 2.8 N NaOH. The fluorescence intensity was measured at an excitation wavelength 520 nm and emission wavelength 605 nm with a 5 nm slit by a fluorimeter (Eclipse; Varian, Palo Alto, CA). The standard curve was constructed by using different concentrations of sodium nitrite. 3. Results and discussion 3.1. Molecular weight distributions of cyanophycin The molecular weight distributions of purified cyanophycin were examined by SDS–PAGE and GPC. The Rf of each band on the SDS–PAGE gel of Fig. 1A was plotted with its corresponding molecular weight of protein markers in a logarithmic scale in Fig. 1B. The molecular weights of soluble and insoluble cyanophycin that were calculated with the linear regression curve of standard protein markers ranged from 25 to 45 kDa and from 8 to 30 kDa,
respectively. Because the insoluble cyanophycin became soluble in a condition as acid as 0.1 N HCl which prevented its GPC analysis due to the limitations of applicable pH ranges of the columns, only the molecular weight of soluble cyanophycin was examined by GPC. The weight-average molecular weight, Mw, is 18250, and the number-average molecular weight, Mn, is 10280 as calculated from the elution profiles of Fig. 1C. The soluble cyanophycin exhibits a polydispersity index of 1.78. Both SDS–PAGE and GPC measures gave rather consistent molecular weight distributions of cyanophycin. 3.2. Thermo-decomposition temperatures of cyanophycin In an attempt to understand the thermal stability of cyanophycin, thermogravimetric analysis (TGA) was carried out to measure the decomposition temperature of cyanophycin. The TGA thermogram showed that both soluble and insoluble cyanophycin had similar decomposition temperatures, around 230 ◦ C (Fig. 2).
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Fig. 2. Thermogram of cyanophycin. Both insoluble (—) and soluble (- - -) cyanophycin prepared with recombinant E. coli were analyzed by a thermogravimetric analyzer under a nitrogen stream.
The soluble cyanophycin has a lower molecular weight than the insoluble cyanophycin, and the lysine molar content of soluble cyanophycin is higher than that of the insoluble form [25]. The small differences in the decomposition temperatures indicated that both molecular weight distributions and lysine contents only minimally affected the thermal decomposition of cyanophycin. Both forms of cyanophycin also showed an approximate 10% weight loss during the onset of the heating process. The weight loss was presumably due to the evaporation of water, suggesting a moisture content of 10% water associated with the lyophilized cyanophycin. On the other hand, the slow slopes of the TGA thermogram also suggested that the cyanophycin might exhibit amorphous forms, not crystalline. Unlike other proteins, the recombinant cyanophycin might be in a denatured state because the purification process involved an acidic condition of 0.1 N HCl. Compared with other proteins which require a defined conformation to achieve biological activity, the high decomposition temperature might allow further modifications of cyanophycin to proceed at higher temperatures without the loss of biological activity. 3.3. Cellular toxicity of soluble cyanophycin CHO cells were cultured in the presence of various concentrations of soluble cyanophycin to examine its potential cytotoxicity. Because of the poor solubility of soluble cyanophycin in DMEM, the concentration was limited to below 0.8% (w/v). As depicted in Fig. 3, the soluble cyanophycin exhibited almost no toxicity to CHO cells at a concentration as high as 5 mg/mL, when the relative cell density on the tissue culture polystyrene (TCPS) was taken as 100% at each sampling time. Interestingly, the relative cell density was slightly increased when the cells were cultured for 96 h at a concentration of soluble cyanophycin above 1 mg/mL, suggesting that high concentrations of soluble cyanophycin might promote the cell growth. 3.4. Cell growth on the films of insoluble cyanophycin The minimal cytotoxicity of soluble cyanophycin prompted the examination of cell growth on cyanophycin substratum. Various concentrations of insoluble cyanophycin were employed to prepare thin films for cell growth. Before being cross-linked by glutaraldehyde, the dried cyanophycin films were brittle, fragile, and tended to become detached from the dishes when solvents were added. For the insoluble form, which was dissolved in 0.1 N HCl, the residual acidity on the dried films might have caused a local redissolution when an aqueous solvent was added. Therefore, bicarbonate buffer was used in an attempt to neutralize the acidity before the
Fig. 3. Cellular toxicity of soluble cyanophycin. CHO cells were grown in the medium containing additional soluble cyanophycin at various concentrations. The cell density was obtained from the correlation of cell number and the MTT assay at different time periods (48 h: white; 72 h: \\\; 96 h:///) after seeding, and then is expressed by taking the cell density of cells on TCPS at each sampling time as 100%. The corresponding cell densities on TCPS for 100% were approximate 36500, 78600, and 167500 cells/cm2 for 48 h, 72 h, and 96 h, respectively. An asterisk indicates the significant difference between the viability in the presence of soluble cyanophycin and that on TCPS (p < 0.05) (mean ± S.D.; n = three independent experiments).
application of glutaraldehyde solution. For 20 min of cross-linking, cracked films were still observed after glutaraldehyde solution was replaced with aqueous solution, presumably due to insufficient cross- linking. Therefore, the period of cross-linking was prolonged to one hour. The dried films remained intact, and appeared to be transparent at low concentrations of cyanophycin and slightly yellowish to brownish around the edges for those with high concentrations of cyanophycin after washing with ethanol and PBS. The films were peeled from the TCPS, immersed in liquid nitrogen, and snapped to small pieces for the observation of the cross sections. SEM observation revealed the lack of porous structures inside the films as shown in Fig. 4. Most films exhibited nearly smooth surfaces (Fig. 4A–C) although some roughness was found on the surface of the thick film containing 2 mg/cm2 cyanophycin (Fig. 4D). When the cross section was examined, no pores were observed, and the film thickness was found to increase almost proportional to the contents of cyanophycin except for the lowest content of 0.25 mg/cm2 as shown in Fig. 4E–H. The results indicate that most cyanophycin remained intact after crosslinking, but insufficient crosslinking might occur when the cyanophycin content was too low to form a rigid structure which could avoid being removed during the subsequent washing processes. Interestingly the cross section showed a structure of stacking thin lamellae which run almost in parallel irrespective of the cyanophycin contents (Fig.4E–H). The stacking lamellae were not observed on the top surface of cyanophycin films which shows a contrastingly smooth surface. Formation of the stacking structure remains unclear, but might presumably relates with the side chains of cyanophycin, mainly the interactions among the amine group of lysine and the guanidino group of arginine. CHO cells were seeded onto the films. The cell proliferation revealed that the films of insoluble cyanophycin had almost no toxic effect on the cells (Fig. 5A). Furthermore, when compared with the cells grown on TCPS (which relative cell density was taken as 100% at each sampling time), the cells on the cyanophycin films exhibited higher relative cell densities as analyzed by the MTT method at the time period of 48 h after seeding (Fig. 5A). Specifically, the relative cell densities on the films containing high cyanophycin contents, such as 1 and 2 mg/cm2 were noted as high as 135% that of TCPS at a short time period of 48 h. After a longer period of culture, the differences in the relative cell density on TCPS and those on cyanophycin
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Fig. 4. Film structure of insoluble cyanophycin by SEM observation. The films of cyanophycin after cross-linking were observed under SEM. The top surfaces were shown in panels A–D and the corresponding cross sections were shown in panels E–H for the films prepared with 0.25, 0.5, 1, and 2 mg/cm2 insoluble cyanophycin, respectively.
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Fig. 5. Cellular viability on the films of insoluble cyanophycin. In panel A, CHO cells were seeded onto the films prepared with different amounts of insoluble cyanophycin. The cell density was obtained from the correlation of cell number and the MTT assay at different time periods (48 h: white; 72 h: \\\; 96 h:///) after seeding, and then is expressed by taking the cell density of cells on TCPS at each sampling time as 100%. The corresponding cell densities on TCPS for 100% were approximate 36500, 78600, and 167500 cells/cm2 for 48 h, 72 h, and 96 h, respectively. An asterisk indicates the significant difference between the viability on the films of insoluble cyanophycin and that on TCPS (p < 0.05). (mean ± S.D.; n = three independent experiments) The cell morphology 96 h post seeding was photographed under a microscope at a magnification of 10 × 20. Panels B1–B5 show the CHO cells on TCPS and on the films of insoluble cyanophycin at 0.25, 0.5, 1, and 2 mg/cm2 , respectively.
films almost disappeared. The cell morphologies were indistinguishable for the cells on the TCPS and on the cyanophycin films (Fig. 5B), suggesting that the cells on cyanophycin films presumably maintained a similarly healthy state as those on TCPS. After CHO cells were seeded and cultured in complete medium for 24 h, the culture medium was replaced with DMEM without serum. The relative cell density on TCPS after an additional 48 h culture in the absence of serum was set as 100%. Higher relative cell densities were observed for those cells grown on the films containing insoluble cyanophycin. As the culture continued, the cells grown on TCPS gradually became apoptotic as the relative cell density dropped to 60%, and then to below 20% after an
additional 96 h culture (Fig. 6A). The cell number diminished and the cell shapes became rounded (Fig. 6B1). On the other hand, the cells on the cyanophycin films maintained a healthier state as the relative cell density stayed almost unchanged after an additional culture in the absence of serum for 72 h, and dropped slightly after 96 h (Fig. 6A). For the cells grown on the insoluble cyanophycin films, the changes in confluence over time were less than those on TCPS, and most of the cell shapes remained unchanged (Fig. 6B2–5), especially on the films containing higher concentrations of cyanophycin, suggesting that those films provided a sustained environment for cell growth. The basis of the ability of cyanophycin to support sustained cell growth remains
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Fig. 6. Sustained cell growths on the films of insoluble cyanophycin after the removal of serum medium. In panel A, CHO cells were seeded onto TCPS (white) and the films prepared with different amounts of insoluble cyanophycin (0.25 mg/cm2 : \\\; 0.5 mg/cm2 : ////; 1 mg/cm2 : ×××; 2 mg/cm2 : black). After 24 h culture, the medium containing serum was replaced by DMEM without serum. The cell density was obtained from the correlation of cell number and the MTT assay at different time periods after serum removal and is expressed by taking the cell density on TCPS 48 h after serum removal as 100%, approximate 33100 cells/cm2 (mean ± S.D.; n = three independent experiments). The cell morphology 72 h after serum removal was photographed under a microscope at a magnification of 10 × 20. Panels B1–B5 show the CHO cells on TCPS and on the films of insoluble cyanophycin of 0.25, 0.5, 1, and 2 mg/cm2 , respectively.
unclear, but might be attributable to the unique comb structure of arginine and lysine side chains along the aspartic backbone; the lysine and arginine could provide a charged environment to which the cells might more readily anchor. Most of the adherent cells require anchorage to a suitable substratum for proliferation. In addition to TCPS, some polypeptides, such as collagen, gelatin, fibronectin, have been employed to coat the surface in order to provide such an environment. A previous report showed that HeLa cell line had approximate proliferation rates on collagen films and on TCPS, and human skin fibroblasts proliferated at a slightly higher rate on collagen films than on TCPS [32]. Another report indicated approximate cell densities of 3T3 fibroblast grown on TCPS and on gelatin films after culturing for
1 and 3 days [33]. The components of extracellular matrix such as collagen can assist the modulation of cell behavior. Cyanophycin exhibits an equivalent capability to provide a suitable environment for cell proliferation when being compared with those two commonly used natural polypeptides, collagen and gelatin. Additionally, cyanophycin can be chemically conjugated onto other polymers through its lysine side chains, and might be expected to become another alternative used in preparing substratum for in vitro cell expansion. The films of insoluble cyanophycin can furthermore sustain CHO cell growth for a prolonged period of time after the replacement of DMEM containing no serum. Such a characteristic might provide a platform for those studies which intend to investigate the
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Fig. 7. Levels of nitric oxide released from macrophages grown on the films of insoluble cyanophycin. Macrophages (RAW 246.7 cells) were grown on TCPS (white) and the films prepared with different amounts of insoluble cyanophycin (0.25 mg/cm2 : \\\; 0.5 mg/cm2 : ////; 1 mg/cm2 : +++; 2 mg/cm2 : ×××). The cells grown on TCPS alone and in the presence of LPS (black) were used as controls. The protein contents of the cell lysate (panel A) and the levels of nitric oxide (panel B) released from the macrophages were determined at different time periods (mean ± S.D.; n = three independent experiments). The cell morphology 96 h post seeding was photographed under a microscope at a magnification of 10 × 20. Panels C1–C6 show the cells on TCPS, and on the films of insoluble cyanophycin of 0.25, 0.5, 1, and 2 mg/cm2 , and in the presence of 0.1 mg/mL LPS, respectively.
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interactions of reagents with cells and require the exclusion of the interferences of serum. Cells can be grown on the films for a period of time and then the regular medium can be replaced with DMEM containing the specific reagents.
Appendix A. Supplementary data
3.5. Nitric oxide (NO) release by macrophages grown on the films of insoluble cyanophycin
References
In order to examine whether cyanophycin stimulates the release of nitric oxide from macrophages, RAW 246.7 cells were cultured on the films containing different amounts of insoluble cyanophycin. Both the protein content and NO release were monitored at different time intervals. The protein content was used as an indicator for cell proliferation. After a 48 h culture, macrophages proliferated at higher rates on the cyanophycin films, specifically for those containing higher cyanophycin concentrations (Fig. 7A). However, the advantages of cyanophycin for the growth of macrophages became marginal after 72 h and 96 h in culture (Fig. 7A). This observation was in agreement with the results obtained in the cultures of CHO cells grown on cyanophycin films (Fig. 6A). The NO release from the macrophages grown on TCPS alone and from those grown on the cyanophycin films stayed at comparable levels for up to 72 h in culture (Fig. 7B). After a 96 h culture, NO release slightly increased to around 2 mM/mg protein for the macrophages grown on the cyanophycin films. On the other hand, the presence of lipopolysaccharide (LPS) in the medium slightly limited the growth of macrophages (Fig. 7A), and the NO release continued to increase with time to 11 mM/mg protein after a 96 h culture (Fig. 7B). The morphology of macrophage tends to become dendritic upon activation. The cells exhibited a dendritic shape in the presence of LPS (Fig. 7C6), whereas most of the cells remained inactivated in the absence of LPS (Fig. 7C1) and on the cyanophycin films (Fig. 7C2–5), even after a 96 h culture. Although the cyanophycin was prepared with recombinant E. coli, the amounts of NO release suggested that the residual lipopolysaccharides of E. coli became minimally associated with the purified cyanophycin. LPS has been reported to be labile in a strongly acidic environment. Most of the LPS might be destroyed or removed during the purification process in the presence of 0.1 N HCl. Consequently, the morphology of very few RAW 246.7 cells possessed a dendritic shape after 96 h culture on the films of insoluble cyanophycin.
4. Conclusion Cyanophycin, a degradable biopolymer, was prepared in recombinant E. coli, and was subsequently used for the preparation of cyanophycin films. The films of insoluble cyanophycin elicited minimal NO release from macrophages grown on the films, suggesting that minimal macrophage activation might be evoked. In addition, cyanophycin exerted no cellular toxicity in these cultures, and furthermore the cyanophycin films provided a culture condition for sustained and high relative cell viability in comparison with those cells grown on the TCPS. This study showed that cyanophycin might have the potential of serving as a biocompatible, degradable material for future use in biomedical applications.
Acknowledgement This research was supported by grant 103-2221-E-011-136 from the Ministry of Science and Technology at Taiwan.
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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bej.2015.09.012.
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