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Cite This: ACS Appl. Bio Mater. 2019, 2, 2791−2801
Microparticles for Suspension Culture of Mammalian Cells Daniel Smith,† Chase Herman,† Sidharth Razdan,† Muhammad Raisul Abedin,† William Van Stoecker,‡ and Sutapa Barua*,† †
Department of Chemical and Biochemical Engineering, Missouri University of Science and Technology, Rolla, Missouri 65409, United States ‡ Phelps County Regional Medical Center, Rolla, Missouri 65401, United States
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S Supporting Information *
ABSTRACT: The focus of this work is to develop a technology for the synthesis of polymer microcarriers that demonstrate mammalian cell culture adhesion on the surface of the microcarriers. Most mammalian cells are adherent in nature that requires multilayer vessels, large volume, expensive cell culture media, high manufacturing time, and high costs of cell culture supplies for the commercial-scale manufacturing of cells. The development of an efficient, scalable technology for producing large volumes of cells is a need in bioprocess industries to improve product potency. We developed a method of synthesizing soft biocompatible US FDA approved polymer based microparticle carrier system of approximately 260 ± 27 μm in diameter that serves as an adherent platform for human umbilical vein endothelial cells (HUVEC) to grow in suspension. Our preliminary experimental results showed that using the polymeric microcarrier system cell adhesion to the surface of the microcarriers was 2−3-fold higher than conventional cell culture flasks while using 10-fold lower cell culture media in a bioreactor than a tissue-culture treated flask. The survival of HUVEC on microparticles was confirmed by live cell staining (green fluorescent calcein AM), dead cell staining (ethidium homodimer-1), nuclear DAPI staining, actin cytoskeleton staining, confocal microscopy, and flow cytometry analysis. This technology will provide high cell culture productivity while reducing the costs of growing adherent cells. KEYWORDS: cell suspension, gelatin, human umbilical vein endothelial cells (HUVEC), microcarrier, microparticle, polylactic-co-glycolic acid (PLGA) and poly L-lysine (PLL)
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bioconjugation,10 physical adsorption,11 plasma polymerization,12 or alkaline treatment.8 Both natural and synthetic materials have been explored for the production of cell microcarriers. Natural materials such as alginate,13 chitosan,14 and gelatin15 are advantageous for anchoring cells to a surface but they suffer from weak structural integrity.16 Synthetic materials are mechanically stronger, easier to fabricate, and modifiable to a specific function. Common synthetic, biodegradable materials, which are FDA approved for use in vivo,17 consist of poly(lactic-co-glycolic acid) (PLGA),10 poly(lactic acid) (PLA),18,19 polycaprolactone (PCL),20 and polyethylene glycol (PEG).21 PLGA has been widely used for cell anchorage, proliferation, and differentiation.22,23 However, bare PLGA materials typically suffer from poor cell attachment unless the surface is chemically modified. The ends of PLGA monomers are capped with carboxylic acid (−COOH) groups, which afford PLGA a unique property in that it can be chemically modified and functionalized. A variety of techniques have been shown to improve cell association with PLGA based materials including soaking in ethanol or growth medium,24 polymerization,8,19,25 conjugation chemistry,10,26 or physical
INTRODUCTION Mammalian cell culture technology is devoted to a variety of applications from the production of therapeutic proteins to in vitro drug testing to cell therapy and tissue engineering.1,2 Vascular tissue regeneration participates in endothelial cell remodeling, which demands the delivery of a large number of healthy endothelial cells for seeding at the damaged blood vessels to accelerate reendothelialization.3,4 To date, there are only a handful of techniques developed for the cultivation of endothelial cells because the cells are difficult to grow in culture.3,5 Therefore, generating functional adherent endothelial cells is a prerequisite for blood vessel construction through in vitro cell culture in bioreactors. Large scale mammalian cell culture industry uses batch, fed-batch, or perfusion techniques for culturing viable cell population in bioreactors.6,7 However, these processes suffer from the inconvenience of operation, low cell population, high cell culture medium consumption, and poor product stability. An alternative technology to the bioreactors is to grow scalable mammalian cells in suspension on the surface of polymer microparticles, often referred to as microcarriers.8,9 The advantages of using the polymer microparticle approach are high cell adhesion capacity, biocompatibility, and scalability in a biomanufacturing workflow. The surface topography of microparticles is tunable through surface functionalization by facile methods such as © 2019 American Chemical Society
Received: March 13, 2019 Accepted: May 22, 2019 Published: May 22, 2019 2791
DOI: 10.1021/acsabm.9b00215 ACS Appl. Bio Mater. 2019, 2, 2791−2801
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ACS Applied Bio Materials adsorption.27 Modifying the surface is paramount in maximizing cell attachment to PLGA, which is a critical factor in binding cells. Additionally, lathering proteins on the surface of biomaterials should improve cell health and prevent pathological phenotypic changes in the cell through better mimicking the environment sensed by the cell in vivo. We explored the possibility of an emulsion-based technique to improve the microparticle size using a home-built flowfocusing device. Using this technique, PLGA microparticles of (165 ± 40) μm were synthesized that were further conjugated with poly L-lysine (PLL) polycations and coated with a thin layer of gelatin. Since PLL is cytotoxic depending on its molecular weight, charge density, and concentration28,29 but promotes cellular adhesion and angiogenesis,30,31 it is hypothesized that incorporation of a small noncytotoxic dose of PLL below its 50% inhibitory concentration (IC50 ≈ 40 μg/ mL)29 in combination with gelatin resolves the shortcoming of PLL while improving cellular adherence on the surface of PLGA microparticles and maintaining the cell viability. We verified this hypothesis by allowing human umbilical vein endothelial cells (HUVEC) as a model of therapeutic angiogenic cells to attach and spread on the microparticle surface.
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measurements (Figure S2). The number of microparticles per gram (#/g) was determined by the following equation: number of particles 6 × 1012 = g ρπd3
(1)
Where ρ is particle density in g/cm and d is particle diameter in μm. Surface Coating of Microparticles. PLL (Sigma; MW ∼150 000−300 000 Da) and gelatin from bovine (Sigma) were physically adsorbed onto the surface of PLGA microparticles to increase cell−particle interactions. This commercially available PLL has been widely used for coating solid surfaces to study cellular attachment, spreading, growth, morphology, differentiation, and motility.32−34 Briefly, 35 mg of microparticles was weighed and added to three centrifuge tubes for PLGA−gelatin, PLGA−PLL, and PLGA−PLL−gelatin particles. All tubes were coated in Sigmacote (Sigma) to minimize product loss. All formulations were treated with gelatin, PLL, or a mixture of both at a constant 6.86 μg coating/mg of particles. To prepare, PLGA−gelatin particle formulations, 75 μL of a 0.32% w/v solution of gelatin was added to the particles. PLGA−PLL formulations were prepared by adding 240 μL of a 0.1% w/v PLL solution. PLGA−PLL−gelatin particles were coated with 45 μL of a 0.32% w/v gelatin solution and 96 μL of 0.1% w/v PLL for a total of 240 μg of protein at a mass ratio of 4:6 PLL/gelatin. The total volume used in the adsorption process was 9 mL with the remainder consisting of PBS. Samples were incubated at 37 °C and shaken (Fisherbrand Multi-Platform Shaker, Fisher Scientific) at 150 rpm for 100 min. Particles were then centrifuged at 4700g for 20 min and washed three times with PBS, and the supernatant was collected. Finally, microparticles were lyophilized, weighed, and stored at 4 °C. NMR Analysis. To effectively determine the amount of PLL and gelatin adsorbed on the surface of PLGA microparticles, nuclear magnetic resonance (NMR) analysis using 1H NMR (INOVA 400 MHz FT/NMR, Varian, Inc.) was performed. First, standard curves for both gelatin and PLL were constructed (Figure S3). Gelatin was prepared in 10, 7.5, 5, 2.5, and 1 mg/mL solutions and subsequently analyzed by NMR. PLL was prepared in 2, 1.75, 1.5, 1.25, and 1 mg/ mL and subsequently analyzed by NMR. An appropriate volume of 0.1% w/v PLL solution was lyophilized until a constant mass was achieved and subsequently dissolved in 1 mL of RO water. Initially, PLL was prepared at the same concentrations as gelatin; however, it was found that PLL solutions above ∼2 mg/mL produced sediment that was not soluble. The absolute integral value of peaks from the NMR spectra data were measured and used in the standard curves. During construction of the standard curve, it was found that samples below a concentration of 1 mg/mL did not produce peaks strong enough such that they could be distinguished from background noise. Supernatant collected during the washing of coated particles was used in coating quantification. The supernatant was frozen at −80 °C and lyophilized until a constant weight was achieved. This product was then diluted in 700 μL of deuterium oxide (D2O, Sigma, St. Louis, MO), transferred to NMR pipettes, and analyzed. Spectral data were collected, and the absolute integrals for the respective molecule were assessed over the same spectral range that was used for the standards. Using the integral value associated with the characteristic peak, the amount of gelatin and PLL was determined using the standard curve previously constructed. The percent adsorption efficiency was then determined using the following equation: 3
MATERIALS AND METHODS
Synthesis of PLGA Microparticles. To prepare PLGA microparticles, the single-emulsion solvent evaporation method was used with a simple flow-focusing apparatus to synthesize polymer microparticles. Briefly, 110 mL of a 1% w/v aqueous solution (W1) of poly(vinyl alcohol) (PVA; Sigma-Aldrich, 30−70 kDa, 87−90% hydrolyzed) was prepared as a hardening bath. While PVA is a known water-soluble polymer, the solution was first heated to >80 °C, and then PVA powder was added slowly to the solution until a homogeneous solution was formed. Thereafter, a flow-focusing apparatus was assembled using a 9 mm glass Pasteur pipet, surgical tubing, one 3 mL syringe (National Scientific Company, Germany), one 10 mL syringe (National Scientific Company, Germany), two needles (25G, BD PrecisionGlide Needle, BD Biosciences, New Jersey), and a two-syringe pump (kdScientific, KDS-200, Holliston, MA) (Figure S1). Then 200 mg of PLGA (∼19 kDa; Acros Organics) was dissolved in 3 mL of ethyl acetate (Fisher Scientific) to form the organic phase (O). The PVA solution was used as a carrier stream to surround the organic (O) phase as it was pumped through the apparatus. By using an equal drive block velocity on both syringes, the two streams were injected into the Pasteur pipet at a 3:10 flow rate differential. For 10 min of injection, the organic droplets were dispersed into the continuous aqueous phase to form an emulsion (W1/O). This droplet emulsion was further emulsified in the 1% PVA hardening bath forming a double emulsion ((W1/O)/W2). The Pasteur pipet was then removed, and the emulsion was stirred for an additional 15 min. The mixture was left undisturbed overnight (>18 h) to facilitate evaporation of residual ethyl acetate at room temperature. PVA was removed by centrifugation at 7000g, followed by five washes with reverse osmosis (RO) water. Microparticles were then lyophilized, weighed, and stored at 4 °C. Microparticle Characterization. The size and surface topography of PLGA microparticles were analyzed by scanning electron microscopy (SEM) (Hitachi S-4700) and stereo microscopy (Hirox KH-8700). Particle size and shape were also visualized using light microscopy (Zeiss Axio Observer). The microparticle surface charge was measured in water and phosphate buffer saline (PBS; Fisher Scientific) with dynamic light scattering (DLS; Malvern NanoSeries Zetasizer ZS90). DLS measurements were performed at 25 °C in disposable capillary cells (Malvern) using the backscattering detection at 90°. The zeta potential was measured for 20 successive runs. Data were analyzed using means and standard deviations of three
% adsorption efficiency Initial Coating Mass − Sample Mass = × 100 Initial Coating Mass
(2)
Cell Attachment, Proliferation, and Quantification. HUVEC were chosen as a model cell type because they had been widely used in vascular tissue engineering applications both in vitro and in vivo.35−37 HUVEC (Lonza) were maintained in endothelial growth medium (PromoCell), supplemented with 2% v/v fetal calf serum (FCS), 1 μg/mL hydrocortisone, 0.1 ng/mL human epidermal growth factor, 1 ng/mL basic fibroblast growth factor, 90 μg/mL 2792
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ACS Applied Bio Materials heparin, and 1% penicillin−streptomycin in an incubator at 37 °C and 5% CO2. The medium was changed regularly every 2−3 days, and cells were subcultured at ∼80% confluence according to manufacturer’s protocol. Cells were collected via trypsinization, centrifuged at 200g for 5 min, and resuspended in fresh media at the appropriate concentration for bioreactor addition. The bioreactors used in this study were sterile 50 mL bioreaction tubes (CELLTREAT, Pepperell, MA) with a hydrophobic membrane cap to facilitate oxygen and CO2 gas transfer into the bioreactors. Bioreactors were not tissue culture treated such that cells would not attach to the surface of the tube. Cells were seeded at a constant cell density of 35 000 cells/cm2 in bioreactor samples as well as T-25 tissue treated cell culture flasks coated in gelatin (Corning) as a control. Eight-well chambered borosilicate coverglass slides (LabTek), also coated with gelatin, were used in fluorescence microscope imaging of controls. Microparticles were sterilized by ultraviolet (UV) treatment for 15 min before commencing cell attachment. The cell− particle mixture was stirred intermittently for 110 min at 50 rpm and 10 min at rest in an incubator at 37 °C and 5% CO2. Data were collected at 0, 4, 8, and 72 h of culture. At 0, 4, 8, and 72 h after initial seeding, particles were aspirated from their bioreactors and investigated for cell viability, proliferation, and cell loading capacity. Additionally, control flasks were trypsinized, collected, and subject to the same analysis. Cell viability was determined by staining with calcein-AM (CAM) and ethidium homodimer-1 (EthD-1) for live and dead cells, respectively, and visualized by fluorescence microscopy.38,39 Cell proliferation was determined by performing flow cytometry analysis in conjunction with a live cell assay using CAM staining. Briefly, 180 μL of each bioreactor sample and control was aspirated and collected in 0.6 mL microcentrifuge tubes. CAM was added to each sample such that the final concentration was 2 μM and incubated at room temperature for 30 min. Subsequently, samples were analyzed using a flow cytometer (BD Accuri C6 Plus, BD Biosciences). Samples were analyzed at 54 μL/min using a core diameter of 30 μm, and at least 10 000 events were collected. Gates were determined for each control (Figure S4), and data were analyzed to determine cell concentration, density, and extent of attachment. Cell viability was measured using MTT assay. Further, cell−particle samples were fixed with 4% paraformaldehyde and analyzed by SEM. SEM samples were gold sputter coated for 60 s at 8 mA. Images were processed with ImageJ software (NIH) to indicate areas of cell attachment. Additionally, fixed samples were perforated with 0.1% v/v Triton X-100 in PBS and stained with 30 μL of 5 mg/mL 4′,6-diamidino-2-phenylindole (DAPI) for 5 m as well as 205 μL of 6.6 μM tetramethylrhodamine isothiocyanate (rhodamine phalloidin, Abcam) for 30 min. Samples were washed with PBS, mounted, and dried under vacuum desiccation and subsequently visualized using confocal laser microscopy (TCS SP8, Leica Camera AG). Statistical Analysis. Each experiment was carried out in independent repetitions to have at least triplicates valid measurements. The mean differences and standard deviations were verified using a two-sample t-test. P values less than 0.05 were considered statistically significant.
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165 ± 40.4 μm (Table 1). Coating particles in gelatin and PLL saw a marked increase with PLGA−gelatin, PLGA−PLL, and Table 1. Particle Characteristicsa particle type PLGA PLGA−gelatin PLGA−PLL PLGA−PLL−gelatin
average particle diameter 165 205 261 206
± ± ± ±
40.4 42.1 26.8 45.0
μm μm μm μm
no. of particles/mL 7070 6930 2900 4730
± ± ± ±
500 400 500 300
a
Size distribution of PLGA, PLGA−gelatin, PLGA−PLL, and PLGA−PLL−gelatin microspheres. The average particle diameter is the mean value of 50 or more microparticles. The number of microparticles per gram was determined using eq 1, which was based on polymer density and average microparticle diameter. The number of particles per milliliter reflects the concentration of microparticles used in bioreactors.
PLGA−PLL−gelatin particles increasing in size to 205 ± 42.1, 261 ± 26.8, 206 ± 45.0 μm, respectively. Upon microscopic imaging of the particles, it is clear that gelatin and PLL coating modified the surface of the particles (Figure 1). SEM images indicate that the bare particles are indeed spherical and slightly porous, which is most likely due to the diffusion of ethyl acetate prior to washing and sublimation of entrapped water during lyophilization. Coated particles show a distinctly different morphology as their surfaces look woven like a web with gaps in the coated areas rather than pores into the internal structure of the particle. The brightfield images of each coated particle, whether gelatin, PLL, or a combination of the two, display a central section surrounded by a lighter area. This result suggests a few things about these particles. One is that the particles are coated sufficiently with proteins, causing an expansion in particle size (Table 1). Second, the particles most likely have undergone some level of degradation during the 100 min incubation period at elevated temperature. Upon comparison of the images of the PLGA particles (column A, Figure 1) with PLGA−gelatin, PLGA−PLL, and PLGA−PLL−gelatin, the PLGA particles are black and devoid of light passing through, whereas the coated particles are substantially lighter, which indicates that more light is passing through to the aperture and particles are less dense. Additionally, coated particles are much less regular in shape when compared to the bare PLGA particles, which indicates that protein coating induces a shape change in the particles. Upon comparing the SEM images in columns B, C, and D, the bare PLGA particles are more regular in shape and lack the degree of particle debris/disintegration in the coated formulations. Most notable from column B, the act of shaking particles in an elevated temperature environment seems to induce particle deformation and dissociation. PLGA− PLL particles especially experience this sort of degradation because much of the particles have fragmented into irregularly shaped chunks. Finally, it is quite apparent that physical adsorption of proteins to microparticles increases the surface roughness of the particles (column D, Figure 1). Adsorption Efficiency. Particles fabricated from PLGA typically repel cells because of the hydrophobic nature of the polymer,22,26 unless the particle surface is modified. To improve interactions between particles and cells, PLGA microparticles were coated with PLL, gelatin, or a mixture of both, and analyzed quantitatively using NMR (Table 2). Particles coated with gelatin experienced the highest total
RESULTS
Particle Size Determination. Microparticles in this study were designed to attain a peak size in which the particles would reach a maximum surface area while not sacrificing cell binding ability. Initially, 10% w/v PVA was used as the concentration for particle formulation; however, these microparticles never surpassed 40 μm in diameter. This problem was alleviated by decreasing the PVA concentration from 10% w/v to 1% w/v, which is known to increase particle size, as well as reducing the stirring time of particles after pumping the carrier and organic streams through the flow-focusing device and allowing solvent diffusion and evaporation to occur via natural convection. This resulted in a particle size increase of bare PLGA particles to 2793
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Figure 1. Microscope images of particles. (A) Light microscopic images of PLGA microparticles with and without adsorbed coating (10×); SEM images of each particle formulation at (B) 150×, (C) 500×, and (D) 1500× magnifications; (B, C) particle morphology, indicating porous structures in bare particles and a rough surface for coated particles; (D) single-particle surface image comparing the roughness and porosity of each particle formulation. Coated particle (PLGA−gelatin, PLGA−PLL, and PLGA−PLL−gelatin) surfaces are rough and irregular, indicating a coating has formed on the particle, whereas PLGA particles are homogeneous and porous, indicating a lack of such coating. Scale bar = 100 μm
Table 2. Adsorption Efficiency of Coatinga particle
initial mass loading (μg/mg particles)
adsorption efficiency
coating density (μg/cm2 particle)
adsorbed coating (μg/mg particles)
PLGA−gelatin PLGA−PLL PLGA−PLL−gelatin
6.86 6.86 6.86
62.9 ± 4.53% 30.6 ± 1.51% 76.8 ± 4.94%
9.51 ± 0.70 5.41 ± 0.22 12.0 ± 0.79
4.19 ± 0.310 2.08 ± 0.083 5.13 ± 0.338
a
Microparticles were loaded with a uniform ratio of total coating mass to microparticle mass. PLGA−PLL−gelatin particles were prepared using a 60/40 mixture of gelatin/PLL, based on mass ratio. Adsorption efficiency and coating density on the microparticle surface were determined using a standard curve prepared by NMR analysis (Figure S3). Coating density was calculated using the surface area per mg of microparticles. The surface area was based on spherical particles. The adsorbed coating was calculated using eq 1 and the average surface area per particle.
adsorption efficiency at 62.9% ± 4.53% and 76.8% ± 4.94%, while an adsorption efficiency of 30.6% ± 4.94% was calculated for PLL coated particles. Given the adsorption efficiency, PLGA−PLL−gelatin particles, which were a 40/60 mix of PLL/gelatin, had the highest coating density and therefore the most gelatin and PLL adsorbed. Curiously, the highest adsorption efficiency did not correlate to the large particle size. It is thought that the method of preparation of the gelatin solution used in the coating procedure may potentially degrade or break the polypeptide chains in the gelatin protein structure. Small strands of polypeptide constituents of gelatin remain in solution and are available for adsorbing onto the surface of particles. This is in direct contrast to the PLL solution, which does not require heat treatment to form a solution such that the integrity of the polymeric lysine chains should be maintained. The high molecular weight PLL saturates the particle surface at a low concentration (2−5 μg/mg of particles), which provides a high density of cationic lysine for cell adherence to the particle surface.40 In the aqueous solution, the long chain of PLL moves freely, which might result in an increase in particle size and a more corrugated particle surface than the bare PLGA particles as depicted by column D in Figure 1. Despite the minor increase in size between PLGA−gelatin and PLGA−PLL−gelatin microparticles, there must exist a synergistic adsorption effect for PLL and gelatin on the surface of PLGA microparticles, which may translate to other mixtures of proteins and cationic polymers. Zeta Potential. One of the notable changes when modifying microparticle surfaces is the zeta potential measure-
ment. Zeta potential describes the electric potential between a particle surface and the suspension medium at the plane in which counterions to the particle surface do not move with the particle.41 All particles were measured for zeta potential in water and PBS. PBS was used to mimic in vivo conditions such as pH and ion concentration. Zeta potential measurements of bare PLGA particles were −26.9 mV and −16.7 mV in water and PBS, respectively (Figure 2). These values are lower in absolute value than those typically reported for PLGA,42 which is most likely due to the relatively large size of the microparticles, which decreases the surface charge density. Coated particles displayed an interesting trend of zeta potential and mainly reported values higher in absolute value than bare PLGA. Zeta potential measurements for PLGA−gelatin, PLGA−PLL, and PLGA−PLL−gelatin particles in RO water were −35.1 ± 14.3, 27.9 ± 20.4, and −26.1 ± 17.4 mV, respectively. Measurements for PLGA−gelatin, PLGA−PLL, and PLGA−PLL−gelatin particles in PBS were −25.1 ± 19.1, −23.4 ± 11.4, and −19.9 ± 12.8 mV, respectively. When placed in PBS, all particles had a decrease in zeta potential, relative to when water is the suspension medium, suggesting that the ionic content of PBS reduces the stability of microparticle solutions. Interestingly, particles coated with PLL experienced the most negative zeta potential value, which is unusual for PLL coated materials.27 It is likely that the coating solutions used in PLGA−PLL particles were sufficiently low in PLL that the amount adsorbed to the particle surface did not incur a significant impact on zeta potential measurements. The zeta potential of gelatin coated 2794
DOI: 10.1021/acsabm.9b00215 ACS Appl. Bio Mater. 2019, 2, 2791−2801
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To confirm HUVEC attachment to particles, SEM (Figure 3b) and confocal laser microscopy (Figure 4) were used to analyze 4 h samples. SEM images depict PLGA−PLL−gelatin particles with a change in surface topography and cell spreading, supporting that cells have effectively bound to the particles. Approximately 5 μm in diameter spherical cells are observed (yellow circle on the zoomed-in image) on the surface of ∼200 μm PLGA−PLL−gelatin microparticles. PLGA−PLL−gelatin particles display adequate cell attachment in fluorescence microscopy (Figure 4 and Movie 1). Samples were stained for actin (phalloidin) and nuclei (DAPI). Blue staining represents cell nuclei and red staining represents actin cytoskeleton network within the cell. Multiple cells have attached to the particle surface and have begun to spread as indicated by an expanding cytoskeleton network. Movie 1 depicting multiple HUVEC on the surface of PLGA−PLL− gelatin microparticles is presented in the Supporting Information. Further confirmation of cell spreading and attachment on PLGA−PLL and PLGA−PLL−gelatin particles is confirmed through fluorescent microscopy (Figure S5). Images represent three-dimensional (3D) renderings of z-stack compiled images. The first two rows depict the bright field and EGFP fluorescence channels, respectively, and the third row is the merged image. The bottom row resembles a heat map of the fluorescent channel depicting the elevation of cells within the 3D rendering confirming that cells have attached and spread on the surface of the particles in the z-axis, or vertical direction. Quantitative Analysis of Cells on Microparticles. Cell−particle mixtures were analyzed using flow cytometry to quantify the extent of cell attachment to particles and assess HUVEC proliferation in a bioreactor system (Figure 5). Flow cytometer data represents characterization of samples after 4 h of bioreactor culture which was chosen as these samples encompassed the best HUVEC binding capacity. Panel A of Figure 5 summarizes the dot plots generated by the flow cytometer. SSC vs FSC plots resemble the shape and size of the particles and cells giving an indication to the difference in the diameter as well as granularity of the samples. The FSC value indicates how much of the laser passes around the sample while the SSC value indicates how much of the laser beam is reflected off organelles and particulates in the cell and the matrix composing the core of the particles. Dot plots from flow cytometer are colored green for HUVEC and purple for particles, while red indicates a mixture of particles and debris (Figure 5a). PLGA, PLGA−gelatin, and PLGA−PLL particles all have a higher SSC value than cells, however, PLGA−PLL− gelatin samples are overlapped with HUVEC in the sample. This indicates that cells are strongly bound to PLGA−PLL− gelatin particles, which is further exemplified by the FITC count graph of PLGA−PLL−gelatin where the overlap is so strong that the three populations have meshed into a single peak. The higher SSC value means that the particles reflect more laser than the cells suggesting that particles have a solid core, whereas HUVEC have more empty space in their interior. Flow cytometry results for samples at 0 and 8 h of bioreactor culture can be found in the Supporting Information (Figure S6). Flow cytometry data were further analyzed to assess the proliferative capacity of HUVEC bound to microparticles and cultured in a conical bioreactor (Figure 5b). Initial seeding densities (time point 0) is the basis cell density of 35 000 cells/ cm2, which comprises the entire bioreactor sample. Samples
Figure 2. Zeta potential for each particle formulation measured by dynamic light scattering (DLS) and electrophoretic mobility. Each sample was measured in both RO H2O and PBS, represented by blue and gray bars, respectively. Error bars are a mean ± standard deviation. ∗ represents statistically significant groups with p < 0.05.
particles is negative and higher in absolute value than that measured for bare PLGA as well. This suggests that coating with proteins like gelatin and amino acid polymers like PLL increases the stability of PLGA microparticles since a higher absolute value of zeta potential prevents particle aggregation and flocculation. Apparently, these stability data may seem contradictory to the SEM and microscopic images of the particles as shown in Figure 1 that displays particle aggregation; however, one must keep this in mind that SEM imaging involves drying of the particles and coating, whereas the zeta potential measurement permits the examination of particles in the hydrated form in biological solutions without prior sample preparation or conductive coating. While the microscopic images provide the size and morphological features of particles, the zeta potential data are more applicable to the surface charge in colloidal suspensions of particles in solutions. Cell Attachment and Proliferation. Particle−cell mixtures were prepared using a cell density of 35 000 cells/ cm2. Controls consisted of T-25 flasks for flow cytometer and eight-well chamber borosilicate cover-glass slides for microscopy. Microscope images were taken at 0 and 4 h (Figure 3). Images taken at initial seeding (0 h) show viable cells fluorescing green; however, most cells in nontissue culture plate control are rounded and not clearly associated with particles, indicating attachment has not occurred. Control HUVEC are rounded indicating they have not yet attached completely to the chamber surface. At 4 h, HUVEC associate with PLGA particles but are clustered in aggregates, indicating a higher affinity for cell−cell interactions rather than particle binding. PLGA−gelatin samples display a similar trend in that there are many HUVEC around the particles, but few have adhered, noted by their round shape. HUVEC are attached strongly to PLGA−PLL and PLGA−PLL−gelatin particles at 4 h, whereas PLGA and PLGA−gelatin particles show some cell attachment mixed with cell aggregates, with many cells in the fluid surrounding the particles (Figure 3a). This suggests that while cells are present in the samples, they do not bind as well to bare PLGA and PLGA−gelatin particles. 2795
DOI: 10.1021/acsabm.9b00215 ACS Appl. Bio Mater. 2019, 2, 2791−2801
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Figure 3. (a) Fluorescent microscopic characterization of cell-laden particles. Fluorescent microscope images of cell−particle samples. Control images were taken in eight-well coverglass chambers, particle samples were aspirated from bioreactor tubes, stained with 2 μM CAM (green fluorescent) and 1 μM EthD-1 (red fluorescent) for live and dead cells, respectively, and plated on microscope slides. All images are taken at 10× magnification, scale bar represents 200 μm. (b) SEM images of HUVEC on PLGA−PLL−gelatin particles. SEM imaging confirmed the cellular attachment of HUVEC on the surface of PLGA−PLL−gelatin particles. There are approximately 10−15 cells seen per particle.
may appear to be constant, but one must keep this mind that HUVEC are primary cells that usually have longer specific growth rate than other cell types and are difficult to grow cells.43 The cell density after 72 h is >1.2 × 106 cells/cm2, which is comparable with the previously published data.2,5,43 The cell viability on PLGA−PLL−gelatin particles is found greater than 70% after 72 h (Figure 5c), indicating PLGA− PLL−gelatin do not induce cellular cytotoxicity. Control cells display a similar trend to PLGA−PLL−gelatin particles as the cell density increases at 4 h and decreases slightly at 8 and 72 h. This is attributed to T-25 flasks reaching 100% confluence and some HUVEC being lost to apoptosis after 72 h.
taken at 4, 8, and 72 h represent cells associated with particles, thus large variations exist between measurements. PLGA (cross line columns) and PLGA−gelatin (vertical line) particles realize a significant decrease in cell density at 4 h but recovered to just above the initial density for PLGA particles at 8 h. As noted in Figure 3, both PLGA and PLGA− gelatin particles do not bind HUVEC well but may have associated with them loosely; thus, this result is not surprising. PLGA−PLL (hatched) and PLGA−PLL−gelatin particles (filled) show an increase in cell density at 4 and 8 h culture time. The microparticles improve the initial cell adhesion for the first 8 h after which a significant number of dead cells are seen in free suspension for all particle types except PLGA− PLL−gelatin, which show the highest cell density. After 72 h, the increase in cell density reached about 3- and 3.5-fold higher in control T-25 flasks as well as on PLGA−PLL−gelatin particles, respectively, than the initial cell density, demonstrating the in vitro live cell proliferation and retention on the microparticles over time. Compared with the 8 h time point, the final cell density on PLGA−PLL−gelatin microparticles
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DISCUSSION Microparticles offer a unique solution by providing adequate growth surfaces for the expansion of cells during culture and hence scaling up the culture production at high cell densities for therapeutic use.1,44−46 For example, a large scale culture of endothelial cells has applications in the recovery of injured endothelium.33,47 There are only a handful of reports about 2796
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interactions.54 The plasma membrane surrounding cells is typically negatively charged, while PLL is positively charged due to the ε-ammonium group on the lysine side chain.55 The cationic nature of PLL attracts cells at the plasma membrane via electrostatic forces,56 the extent of which is pH dependent.57,58 Thus, coating the surface of biomaterials with PLL causes more cells to attach and proliferate.59 After attachment, cells typically spread on a surface and use special protein domains to anchor themselves. However, PLL only participates in nonspecific cationic adherence of cells,56 whereas other molecules bind cells through integrins.60,61 Integrins are a class of transmembrane proteins that bind cells using cellrecognition motifs.62 The motifs are composed of specific amino acid sequences like arginine−glycine−aspartate (RGD)63 in fibronectin and YIGSR in laminin.64,65 When integrins bind to these motifs the cell undergoes phenotypic changes that cause anchoring and spreading on the surface. Gelatin contains linear RGD cell adhesive motifs that recognize several integrins, primarily α5β1 and α5β3 integrins.3,4,51 Interestingly, PLGA−gelatin particles only coated with gelatin did not bind HUVEC well compared to other particle formulations (Figure 3), which may be due to a noninteractive configurational presentation of RGD sequences toward integrin receptors when used alone for PLGA surface coatings without cations.66 Moreover, PLGA−gelatin particles had a negative zeta potential (Figure 2), thus indicating that the surface of these particles carries a negative charge restricting the cells to adhere. The surface charge of gelatin depends on its isoelectric point (pI), which varies between 5− 6.7 and the solution pH.52 This means that an increase in solution pH above the pI leads to a decrease in zeta potential, which is what happened in the case of gelatin-coated particles both in water (pH ≈ 6.5) and PBS (pH ≈ 7.4). The plasma membrane of cells also carries a negative charge, and consequently should repel the surface of the PLGA−gelatin particles. Cross-linking gelatin with cationic PLL on the surface of PLGA promotes the cell binding, which indicates the important cell−substrate interactions through RGD recognition sequences as well as the content of PLL. PLGA−PLL− gelatin particles, which are coated in both PLL and gelatin, attached cells well, most likely due to the cationic charge of PLL attracting cells and the amino acid sequencing of gelatin enabling anchoring and spreading of cells once they are in proximity to the particle surface. PLGA−PLL particles, coated only with PLL, also saw distinct cell association with the particle surface. In a parallel investigation, we studied the ability for PLL to attach 75 000 MDA-MB-231 breast cancer cells per cm2 of the bioreactor over 72 h (Figure S7). These cancer cells lost viability after 3 days in culture on the PLL only coated particles. However, these particles were coated with 15 μg PLL/mg of microparticles, which was higher than the 6.86 μg/mg used in this study due to higher cancer cell density than HUVEC. Combinedly, all these results indicate that the PLGA−PLL−gelatin microcarrier system we developed enables HUVEC to be expanded in suspension up to 3.5fold using only 5 mL of the medium while maintaining healthy cytoskeleton and >70% cell viability after 72 h. The data are consistent with the previously reported HUVEC expansion using beads in tissue culture treated dishes.2,47 Microparticles can also be fabricated as spheres, disks, and rods (Figure S8).67,68 In this investigation, microrods (Figure S8a) and microdisks (Figure S8b) have the advantage of maintaining the same surface area as a sphere but having a
Figure 4. Confocal laser microscopy of cell growth on PLGA−PLL− gelatin particles. The image represents PLGA−PLL−gelatin microparticles taken at 20× magnification. Microparticles were incubated with HUVEC in a bioreactor for 4 h and stained with 4,6-diamidino2-phenylindole (DAPI) for nuclei and tetramethylrhodamine phalloidin for F-actin. Red and blue fluorescent channel overlap depicting cell nuclei attached to the surface of microparticle and cytoskeleton structure of adherent cells spreading across the microparticle surface. The dotted line represents the microparticle outline. Scale bar = 40 μm
suspended cell culture systems of functionally active human endothelial cells.3,5,48 Most of these studies use beads in regular tissue culture treated dishes, which anyway are designed for cell attachment and as a result, create background noises by attaching cells on stagnant beads in the dishes instead of growing cells on the beads in suspension in nontissue culture bioreactors. In addition, traditional microcarriers suffer from size limitation (≤100 μm in diameter), fragility, and cytotoxicity.49 In the present study, we developed a novel method of synthesizing stable, large size (≥200 μm) biocompatible microcarriers using PLGA, an FDA approved biocompatible and biodegradable polymer to grow HUVEC on the surface of the particles in suspension.50 PLGA microparticles were fabricated using a home-built flow-focusing device and coated with PLL, a cationic polymer, gelatin, a remnant of the extracellular matrix protein collagen, or a combination of both that together formed a stable particle diameter ≥200 μm (Figure 1 and Table 1). Gelatin was incorporated on the surface of PLGA to support cell adhesion and proteolytic degradation and also because it was relatively inexpensive compared to collagen and fibrinogen.4,10,51 However, because of gelatin’s anionic nature under physiological conditions at pH ≈ 7.4,52 it is postulated that the complexation with positively charged PLL below its IC50 (40 μg/mL)29 promotes the cell attachment. HUVEC were cultured with the microparticles in nontissue culture treated bioreactors for a facile and portable approach to attaching cells to a scaffold. Interestingly, a combination of both PLL and gelatin yielded the highest cell attachment to the microparticles, compared to the bare and separately coated particles (Figure 3). Cationic polymers,53 like PLL, are known to induce cell association with coated surfaces through cationic 2797
DOI: 10.1021/acsabm.9b00215 ACS Appl. Bio Mater. 2019, 2, 2791−2801
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Figure 5. Flow cytometry characterization. (a) Qualitative flow cytometry data for cells mixed with particles at 4 h. Dot plots are color labeled as purple = particles; green = cells; M2 = percentage of samples that are not cells. SSC versus FSC graphs depict granularity, or surface roughness, compared to the size of the sample. Particles consistently report a higher degree of granularity as well as a bigger size. FITC count data show the number of events that report a certain fluorescence value: cells stained with CAM report higher values, while particles report low values. However, some overlap is observed, especially in PLGA−gelatin and PLGA−PLL−gelatin which indicates cells and particles are closely associated. SSC versus FITC data aid in determining the extent at which particles and cells are associated. ∗ represents p < 0.05 with a statistically significant difference between groups. (b) Cell density increase. Data were quantified from flow cytometry dot plots to calculate the number of cells and the number of particles present in a sample. The surface area in square centimeters was determined by making a basis that particle dot plot populations represented a single particle with a diameter equal to that mentioned in Table 2. The base 10 log of each cell density was taken and then plotted. (c) Cell viability: HUVEC viability was assessed using MTT assay. No noticeable decrease in cell viability was observed at different time points. Error bars at the early stage of incubation time of 100% because the mean sample values were calculated based on the average live cell control. The standard deviations may be resulted from variations in initial cell density or sampling volumes.
the underlying curvature. It has been shown that on a nonspherical surface cells bend and stress the cytoskeletal fibers along the direction of maximum curvature, which could recapitulate the alignment pattern of the cytoskeleton found in
more intense curvature. Cells like to associate with surfaces that have intense curvature as the surface curvature aids in spreading as well as mimic the environment present in the body. Cells can change their cytoskeleton and orientation to 2798
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vivo.69 Together with our findings of microparticle surface curvature on HUVEC binding, this has opened an interesting direction to pursue further how geometric cues can be optimized for cell attachment, proliferation, and growth on microcarriers for the implication of various diseases including vascular and cancer. Additionally, other cell lines, like chondrocytes and human microvascular endothelial cells (HMVEC), should be investigated and compared quantitatively to determine the extent at which these particles can bind a range of cells. Modifying the microparticle surface with different extracellular matrix proteins, like collagen or laminin, and cationic polymers could prove beneficial. Not much is known about combining cationic polymers with extracellular matrix proteins and their effect on cell phenotype. Particles can be modified with different extracellular matrix constituents or various cationic polymers to optimize attachment of each cell type. PLGA particles can be copolymerized with other functional polymers such as polyethylene glycol (PEG)21,70 or polyethylenimine (PEI)53 to aid in cell association for delivery to tissues in vivo. Further analysis is necessary to assess the potential of these microparticles cultured with cells to regenerate tissue. In vivo or ex vivo studies should be conducted to assess the therapeutic potential of PLGA microparticles loaded with HUVEC to remediate blood vessel damage as well as induce vascularization and angiogenesis in ischemic tissues.
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Sutapa Barua: 0000-0002-2385-0222 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by funding from the Ozark Biomedical Initiative at Missouri University of Science and Technology and the University of Missouri Research Board. We would like to thank Ming Huang for assisting with the NMR.
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REFERENCES
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CONCLUSION Microparticle-based cell culture system is economical to explore because it combines the potential ease of scalability, less consumption of expensive cell culture medium than cellculture flasks, and use of high surface area-to-volume ratio. In this report, PLGA−PLL−gelatin microcarriers of approximately 200 μm in diameter were synthesized by developing a simple flow-focusing device to improve the cultivation of human endothelial cells in liquid suspension. When cultured on the PLGA−PLL−gelatin microspheres in small medium (5 mL) of bioreactors, HUVEC adhered the microcarrier surface, grew rapidly, and could be cultured on the microcarriers for 72 h without significant toxicity. The cell density was multiplied up to 3.5-fold and maintained healthy morphology as depicted from actin and cell nucleus staining. The HUVEC carrying microparticles cultured in liquid suspension are envisioned to be implanted at the damaged blood vessel site for the reconstruction of vascular structures, construction of an anticoagulable artificial blood vessel or an artificial skin with good transplantability. Further efforts are required to implement this strategy.
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Article
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsabm.9b00215. Animation of 3D arrangement from z-stack confocal scanning laser experiment (AVI) Laboratory set up of microparticles synthesis, graphical presentation of surface zeta potential data, standard curves, particle controls using flow cytometry, 3D rendering of cell attachment to cells, additional flow cytometry data, cancer cell binding to microparticles and HUVEC binding to microparticles of different shapes (PDF) 2799
DOI: 10.1021/acsabm.9b00215 ACS Appl. Bio Mater. 2019, 2, 2791−2801
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