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A method for preparation of hydrogel microcapsules for stem cell bioprocessing and stem cell therapy
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Contents lists available at ScienceDirect
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A method for preparation of hydrogel microcapsules for stem cell bioprocessing and stem cell therapy
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Revital Goldshmid a,b, Iris Mironi-Harpaz a, Yonatan Shachaf a, Dror Seliktar a,⇑
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a b
The Faculty of Biomedical Engineering, Technion – Israel Institute of Technology, Haifa 32000, Israel The Interdisciplinary Program for Biotechnology, Technion – Israel Institute of Technology, Haifa 32000, Israel
a r t i c l e
i n f o
Article history: Received 11 December 2014 Received in revised form 20 April 2015 Accepted 21 April 2015 Available online xxxx Keywords: Biomaterial Suspension bioreactor 3D culture Crosslinking Pluronic-fibrinogen PEG
a b s t r a c t A method for the preparation of suspension culture microcapsules used in the bioprocessing of human mesenchymal stem cells (hMSCs) is reported. The microcapsules are prepared from a semi-synthetic hydrogel comprising PluronicÒF127 conjugated to denatured fibrinogen. The Pluronic-fibrinogen adducts display a lower critical solubility temperature (LCST) at 30 °C, thus enabling mild, cell-compatible physical crosslinking of the microcapsules in a warm gelation bath. Cell-laden microgels were prepared from a solution of Pluronic-fibrinogen hydrogel precursor and hMSCs; these were cultivated for up to 15 days in laboratory-scale suspension bioreactors and harvested by reducing the temperature of the microcapsules to disassemble the physical polymer network. The viability, proliferation and cell recovery yields of the hMSCs were shown to be better than photo-chemically crosslinked microcapsules made from a similar material. The cell culture yields, which exceeded 300% after 15 days in suspension culture, were comparable to other microcarrier systems used for the mass production of hMSCs. The simplicity of this methodology, both in terms of the cell inoculation and mild recovery conditions, represent distinct advantages for stem cell bioprocessing with suspension culture bioreactors. Ó 2015 Published by Elsevier Inc.
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1. Introduction
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An emerging approach in regenerative medicine involves the replacement of damaged cells with healthy ones that have the potential to restore function of damaged tissues [1]. In this context, stem cells are anticipated to become the ideal cell source for this approach [2]. They have shown efficacy in a variety of experimental models of tissue regeneration, including bone, cartilage, fat and muscle [3–9]. Hematopoietic stem and progenitor cells (HSC) are already routinely used in the clinic [10]; and pluripotent human mesenchymal stem cell (hMSCs) therapies are expected to provide far more treatment options [9,11,12]. This potential of cultured stem cells has already begun to materialize into clinical products poised to reach the market in just a few short years [13–16]. Given the high priority for commercialization of cell therapy in general and stem cell therapy in particular, one of the toughest tasks facing this field is how to generate the large numbers of cells required for the eventual treatment of large patient populations. Most research-stage projects working with stem cells can generate only a limited number of cells that support proof-of-concept
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⇑ Corresponding author at: Technion – Israel Institute of Technology, Faculty of Biomedical Engineering, Technion City, Haifa 32000, Israel. E-mail address: [email protected] (D. Seliktar).
clinical trials [17]. With technologies based on autologous cells, it is not clear how theses cells can be efficiently extracted, expanded, differentiated in vitro, and then delivered back into the patient. Regardless of the advanced stages of some stem cell technologies, a commercial medical delivery infrastructure for stem cell therapies is still very much in its infancy. The realization of stem cell therapies will therefore require robust, efficient and reproducible bioprocessing methodologies. In this context, suspension culture bioreactors are highly favoured in process scale-up because established culture conditions in lab-scale can often be transferred to much higher volumes with relative ease [18]. A key factor used to control stem cell growth in vitro is the matrix provided for cell attachment. With the exception of HSC, which are generally expanded without a cell attachment matrix, most other stem cell types have been isolated under conditions dependent on surface adherence. Amongst these are hMSCs, which are adherent cells that require culture surface enlargement to ensure efficient and reasonable mass production. Hence, the expansion of anchorage-dependent cells on two-dimensional (2D) substrates is a central challenge in bioreactor design. Moreover, the considerable cost with respect to consumables, labour and time as well as the inherent variability in manual processes of 2D culture not only make this option unattractive, but also render it commercially unviable. In this regard, automation
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and the use of an efficient bioprocessing paradigm are imperative for the creation of successful clinical products. A variety of three-dimensional (3D) cell carriers for the suspension culture of hMSCs have been developed during the past few years [19,20]. When used for 3D cell cultivation, these platforms can also provide a physical support for cell attachment, proliferation and differentiation [21]. Both synthetic and biological polymers have been studied in order to find the ideal scaffold material, which integrates structural properties of synthetic polymers with bio-functionality of natural extracellular matrix (ECM) constituents [22–26]. For example, ECM proteins or polysaccharides such as alginate, hyaluronic acid, collagen, and fibrin have been used as culture scaffolds of hMSCs [24–27]. Biodegradable protein hydrogels that directly encapsulate cells within a 3D microenvironment (i.e. microcapsules) have many advantages [19,20,28], including easy inoculation, precise control of the cell microenvironment, and straightforward cell recovery. Additionally, microencapsulation of cells in these biomaterials can mimic the body’s extracellular environment better, and capture the ability of cells to proliferate in this more natural milieu [29,30]. Moreover, such scaffolds can provide physical protection and better control of biodegradation [29,30], two very important features that are beneficial in the often-harsh hydrodynamic environment of suspension bioreactors. When culturing MSCs in microcapsules, another factor that should be considered from the scaffold material is the influence of the material modulus on the cultured cells [31,32]. Previous studies demonstrated that MSCs are highly responsive to matrix modulus, and can often show unnatural behavior when they are removed from their tissue microenvironment [33]. Culturing MSCs in 3D embryoid bodies, for example, enhances chondrogenesis [34], suggesting that MSCs may be guided to this and other differentiation pathways during their suspension culture using simple modifications to the microcapsule properties. Finding a way to guide stem cell fate determination using the stiffness of culture substrates is a focus of much research, and will likely become essential aspect of future suspension culture paradigms for stem cell bioprocessing. Premised on these concepts, our group has recently developed microcapsules made from a bioactive semi-synthetic hydrogel. The biomaterial is made by conjugating the PluronicÒF127 to fibrinogen [35–37]. The PluronicÒF127 is a stimuli-responsive synthetic block copolymer that exhibits lower critical solution temperature (LCST) behavior [38,39]. When PluronicÒF127 is conjugated to fibrinogen, the semi-synthetic precursor retains bioactivity from the fibrinogen and LCST properties from the PluronicÒF127 [35,37]. In the current study, we exploit this material for developing bioprocessing methodologies for hMSCs, through their microencapsulation in hydrogel microcapsules and subsequent cultivation in suspension bioreactors. The fast LCST gelation, combined with photo-initiated chemical crosslinking, and multi-functional protein-like bioactive domains provides the features that help to facilitate the cell inoculation, control the cultivation microenvironment, and expedite the recovery of the cells from the microcarriers at the end of the culture period. This combined approach aims to provide a routine, efficient, and scalable solution for hMSC bioprocessing in suspension cell culture systems.
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2. Materials and methods
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2.1. Synthesis of PluronicÒF127-DA and PluronicÒF127-Fibrinogen adducts
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Temperature-responsive bioactive hydrogels were made from fibrinogen conjugated to PluronicÒF127 (F127), a poly(ethylene
oxide) (PEO)-poly(propylene oxide) (PPO)-PEO tri-block co-polymer that exhibits lower critical solubility temperature (LCST) properties. The F127 was end-functionalized with acryl groups to form F127-diacrylate (F127-DA), and then reacted with denatured bovine fibrinogen (Bovagen, Melbourne, Australia) via a Michaeltype addition reaction to form the Pluronic-fibrinogen biosynthetic copolymer [37]. A 7 mg/ml fibrinogen concentration was used for making Pluronic-fibrinogen hydrogels. Poly (ethylene glycol)-diacrylate (PEG-DA) was prepared from 10 kDa PEG-OH (Merck) as described elsewhere [40].
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2.2. Shear modulus measurements
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Mechanical properties of the Pluronic-fibrinogen were characterized with a strain-rate controlled shear rheometer (AR-G2, TA Instruments, Delaware, USA) equipped with a Peltier plate temperature controlled base, an overhead UV curing assembly and a transparent geometry. Time-sweep oscillatory tests were performed in 50 mm parallel-plate quartz geometry using 600 lL of Pluronicfibrinogen hydrogel precursor solution containing 0.1% w/v IrgacureÒ2959 photoinitiator (Ciba, Basel, Switzerland). In order to find the linear viscoelastic region of the time-sweep tests, oscillatory strain (0.1–10%) and frequency sweeps (0.1–10 Hz) were conducted. Rheology experiments were performed at 2% strain and 1 Hz.
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2.3. Cell culture and microencapsulation procedure
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Human mesenchymal stem cells (hMSCs, Lonza, Basel, Switzerland) were purchased and expanded in MSC growth medium (MSCGM, Lonza) containing 1% Pen-Strep (Kibbutz Beit Haemek, Israel) for 4 passages (12–14 days of expansion for each passage, corresponding to 4 doublings from P4 to P8). Cells were harvested and removed from 2D Flasks (Nunc, New York, USA) using trypsin EDTA solution B (Biological Industries). Centrifugation of the cells was performed at 1000 RPM for 5 min at room temp to obtain the hMSC pellet for proliferation experiments. In order to prepare the microcarriers, pelleted hMSCs were mixed with Pluronic-fibrinogen hydrogel precursor containing 0.1% IrgacureÒ2959 photoinitiator (Ciba, Basel, Switzerland). Two types of hydrogel microcapsules were prepared from the cell/polymer solution: a chemically crosslinked version and a physically crosslinked version. For physically crosslinked microcapsules, 400 lL cell/polymer mixture was dripped through a 30 gauge syringe needle into a warm (37 °C) gelation bath containing continually stirred culture medium (Supplementary video 1), forming beads with a diameter ranging from 500 to 1000 lm. The typical microcapsule volume was 0.25–0.5 lL (the polydispersity of the bead size and volume was not characterized). For the chemically crosslinked microcapsules, the cell/polymer solution was supplemented with PEG-DA (at a concentration of 0.2–0.5% w/v) and similarly dripped onto a super-hydrophobic surface at RT, followed by UV-light-activated photopolymerization (365 nm, 4–5 mW/cm2) for 1.5 min. The procedure created droplets of 0.25–0.5 lL and diameter of 500–1000 lm. The hMSC microcapsules (physically or chemically crosslinked) were cultured in laboratory-scale stirred-flask bioreactors (500 ml) for up to 15 days using expansion MSC growth medium (Lonza). The microcapsules were stirred at 1 RPM to ensure proper transport in the reactor volume. The cells were harvested from the hydrogel microcapsules on days 1, 3, 7 and 15, either by cooling down to 4 °C for physically crosslinked microcapsules (Supplementary video 2) or by using collagenase incubation for the chemically crosslinked microcarriers, as detailed below.
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Fig. 1. A schematic illustrating the methodological approach for using hydrogel microcapsules in the suspension culture of hMSCs. The main steps include cell inoculation, suspension culture, and cell recovery.
Fig. 2. A schematic illustration of the cell inoculation steps used for preparing (A) physically (UV) and (B) chemically crosslinked (+UV) Pluronic-fibrinogen microcapsules.
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2.4. hMSC viability
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Harvested hMSCs were pelleted by centrifugation, resuspended in 100 lL of phosphate buffered saline (PBS) and processed by flow cytometry for viability measurements. The cells were kept on ice until they were stained and analyzed. Propidium iodide (PI) staining solution was added to each sample prior to analysis for 10 min of incubation at 37 °C. The samples were measured in an LSR-II flow cytometer (Becton Dickinson, New Jersey, USA), and data was analyzed using FCS-express software (version 4.7). A control of unstained cells was used in order to adjust the flow cytometer settings. In situ cell viability was confirmed by calcein/ ethidium live/dead assay. Briefly, hMSCs in microcapsules were incubated in PBS solution containing 4 mM calcein acetoxymethyl ester and 2 mM ethidium homodimer-1 (Sigma–Aldrich, Buchs,
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Switzerland) for 40 min on an orbital shaker at 37 °C. After staining, the cells were washed twice with PBS (2 10 min) and microscopically imaged on an inverted fluorescence microscope (Nikon Eclipse TS100, Nikon, Japan) using a digital camera (Digital Sight, Nikon, Japan) and Nikon Nis-Elements F3.00 software (Nikon, Japan).
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2.5. % yield calculation
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The hMSCs were harvested from the physically crosslinked microcapsules by cooling them to 4 °C, incubating in 0.5 mg/ml collagenase solution (Sigma) for 60 min at 4 °C, followed by centrifugation for 5 min at 1000 RPM. For chemically crosslinked microcapsules, the microgels were incubated in 0.5 mg/ml collagenase solution for 120 min at 37 °C, followed by 5 min
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Fig. 3. Shear rheometry data showing different crosslinking schemes for Pluronicfibrinogen hydrogel precursors. Time sweep experiments of the shear storage modulus (G0 ) were initiated at 25 °C for 5 min, followed by physical crosslinking at 37 °C (blue triangles) or UV photopolymerization-based chemical crosslinking at 25 °C, with and without additional PEG-DA added to the Pluronic-fibrinogen precursor (red squares and orange circles, respectively).
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centrifugation at 1000 RPM. The % yield of the cell recovery was calculated according to the following equitation:
% Yield ¼
Harvested hmSCs=ml gel Seeded hMSCs=ml gel
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where the numerator represents the harvested live cell density at the conclusion of the experiment and the denominator represented the initial live cell seeding density.
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2.6. Proliferation by cell cycle analysis
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DNA staining and cell cycle analysis were carried out using PI and flow cytometry according to a previously described protocol [30,41]. The harvested cells were collected by centrifugation, washed in PBS and fixed in 70% ethanol at 4 °C for 20 min. The fixed cells were collected by centrifugation and stained with PI staining solution (Sigma) according to manufacturer’s instructions. The PI fluorescence staining was measured in a flow cytometer and scatter plots showing the populations of cells in the various cellcycle stages were obtained for sub G1 (apoptotic cells), G1/G0 phase, S phase and G2/M phase (proliferating cells phases). The different cell populations were plotted in single-parameter histograms of fluorescence PI levels (DNA content), and used to determine the number of cells in the apoptotic population (<2N DNA), in G0/G1 (2N DNA), in S (2–4N DNA) and in G2/M (4N DNA). Proliferation was quantified as the percent of cells in the S and G2/M phases.
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2.7. hMSCs proliferation kinetics (BrdU assay)
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Proliferation was quantified by bromodeoxyuridine (BrdU) incorporation into hMSCs residing within the microcarriers in suspension culture. The BrdU is an analog of the DNA precursor thymidine, which incorporates into newly synthesized DNA of dividing cells. The BrdU was supplemented into the microcarrier culture medium 17 h before detection as per the Flow Kit Staining Protocol (BD pharmingen, New Jersey, USA). Cells were then harvested from microcarriers (as described earlier) at days 1, 3, 7 and 15. The cells were then fixed and stained with FITC antiBrdU antibodies according to instructions provided by BD pharmingen. In addition, cells were stained with 7-aminoactinomycin D (7AAD), a dye that binds to all DNA and is used in conjunction with the BrdU staining to quantify the total number of cells. The levels of cell-associated BrdU and 7AAD were then measured by flow cytometry. The percent of BrdU-positive cells was reported
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as the number of proliferating cells within the total cell population of each treatment.
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2.8. Morphology analysis
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Cultures of hMSCs in microcarriers were rinsed in PBS, permeabilized with 0.1% Triton X-100 in PBS for 10 min, and stained with TRITC-labeled phalloidin (TRITC-phalloidin, 1 lg/ml, Sigma) for 1 h at room temperature followed by Sytox-green addition for 30 min. Microcarriers were then washed three times with PBS (at RT) and placed at 4 °C for an overnight wash in PBS. Stained cells were imaged using a Zeiss LSM 700 confocal microscope (Carl Zeiss, Oberkochen, Germany). Scans using a 20 objective were performed at a z-step size of 2.3 lm with a typical depth of 100–200 lm. All scanning areas were randomly selected and images sampled at a resolution of 1024 1024 pixels.
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2.9. Statistical analysis
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Statistical analysis was performed using MicrosoftÒ Excel software; FACS data analysis was performed using FCS express 4 software. Significant differences between data sets were found using the Student’s t-test (two way); the minimal criterion for significance was set at p < 0.05.
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3. Results
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The Pluronic-fibrinogen biopolymers were used as a three-dimensional (3D) microencapsulation system enabling straightforward cell inoculation, suspension culture in bioreactors and efficient cell recovery (Fig. 1). The microcarriers were made with two hydrogel variants including a physically crosslinked version produced by elevating the temperature of the cell/polymer droplets to 37 °C in a gelation bath (Fig. 2a); and a chemically crosslinked hydrogel produced by UV photopolymerization of the cell/ polymer droplets on superhydrophobic surfaces (Fig. 2b). The two ways of crosslinking (physical vs. chemical) resulted in significantly different bulk mechanical properties of the hydrogels, as measured by strain-rate controlled shear rheology. Fig. 3 shows the characteristic bulk mechanical properties of the two different crosslinking routes. The chemically crosslinked matrix (+UV) reached a plateau shear storage modulus (G0 ) of 68 ± 17 Pa, whereas the physically crosslinked matrix (UV) reached a plateau G0 of 152 ± 60 Pa. In order to focus on the effects of chemical vs. physical crosslinking of this system (+UV and UV, respectively) we normalized the mechanical properties of the +UV hydrogels by adding additional PEG-DA crosslinker to the chemically crosslinked system. The PEG-DA added to the Pluronic-fibrinogen increased the G0 of the crosslinked hydrogel to 175 ± 30 Pa, thus obtaining similar bulk mechanical properties for the physical and chemical hydrogels (n P 3 from at least 2 different batches of Pluronic-fibrinogen materials). We first investigated the viability of hMSCs in the microcapsules using both a qualitative live/dead assay and a quantitative PI staining assay with FACS analysis. Optimization of seeded cell density was explored by viability analysis of different cell concentrations (1, 3, 6 and 12 106 cells/ml). The qualitative live/dead assay revealed a highly viable cell population in the microgels, irrespective of concentration used (Fig. 4). The highest quantitative viability results were achieved at a 3 106 cells/ml concentration (data not shown); thus this concentration was chosen for further experiments. The cells in the physically crosslinked material showed a somewhat higher viability then those in the chemically crosslinked material, particularly during the first week in culture, with a statistical difference on day 1 (Fig. 5). Apparently, the
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Fig. 4. Microencapsulation of cells in Pluronic-fibrinogen hydrogel microcapsules. Phase contrast micrographs (top) are shown with live/dead staining (middle) using calcein (green) and ethidium (red). The superimposition of the phase contrast and live/dead images clearly shows the cells residing and spreading within the microgels after 3 days in culture. Different cell concentrations and microcapsule sizes are shown from left to right, to exemplify the versatility of the system. Scale bar = 50 lm.
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photochemistry had an initial adverse effect on the cells; however, the cells did recover after one week in suspension culture. Between days 11 and 15, there was a slight decrease in viability of the cells in both systems, most likely stemming from mass transport limitations associated with increasing cell density (due to high proliferation within the microcarriers). We further used laser scanning confocal microscopy (LSCM) in order to visualize the hMSC morphogenesis within the hydrogel microcarriers. hMSCs were stained with f-actin dye (TRITC-labeled phalloidin, red) and nuclear dye (Sytox-green) in order to characterize their typical morphology in the microcapsules (Fig. 6). On the first day the cells were homogenously dispersed in the hydrogel microcapsules, showing a predominantly rounded morphology. On day five, the cells appeared much more spindled with distinct lamellipodia. By day eight, the cellular lamellipodia became much more branched and continued to display these branching patterns throughout day 20. There were no distinguishable differences between the morphology patterns of
hMSCs in the physically and chemically crosslinked microcapsules (Fig. 6). Cell cycle analysis was carried out for hMSCs in microcapsules using both a PI assay (Fig. 7) and a 7AAD/BrdU-FITC assay (Fig. 8). The DNA staining using PI clearly indicated a substantial portion of the hMSCs in the proliferative phases of the cell cycle (S + G2/M) in both physically and chemically crosslinked microcapsules. In the physically crosslinked system, 25–27% of the cells are in the proliferation phases, while for the chemically crosslinked microcapsules, only 20–23% are in the proliferation phases. For both systems, the proliferating cell populations showed a statistical difference on day 1 (n P 6, p < 0.05), and were unaffected by the 2-week duration of the experiment. The kinetics of cell proliferation was measured by incorporating FITC-labeled BrdU for 17 h (the cell generation time was found to be 28 h). This DNA pre-labeling with BrdU (at set time intervals) provided a direct measure of the average rate of proliferation in the microcapsules at each
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Fig. 5. Viability of hMSCs in the physically (UV) and chemically (+UV) crosslinked Pluronic-fibrinogen microcapsules. Live/dead staining with calcein (green) and ethidium (red) show a relatively high number of viable cells within the microgels, when cultured for up to 21 days in suspension bioreactors (A). Quantitative viability with DNA staining and cell cycle analysis using propidium iodide (PI) confirms the qualitative data (B). Statistically significant difference between treatments is observed at day 1 (p < 0.05, n P 6, two different experiments).
Fig. 6. Morphogenesis of hMSCs in the physically (UV) and chemically (+UV) crosslinked Pluronic-fibrinogen microcapsules. The inoculated hMSCs are initially rounded in the microcapsules; they become elongated and highly spindled over the course of three weeks in culture, irrespective of the crosslinking method.
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time-point of the experiment. The BrdU results indicated a very slow rate of proliferation after the first day in suspension culture, irrespective of the crosslinking method (Fig. 7). Both systems showed an increased proliferation rate at day 3, which was maintained at a constant rate at the later time-points. However, the cells in both systems showed a statistical difference in their
proliferation rate from day 3 onward: in the physically crosslinked system the cells exhibited a proliferation rate of 75%, whereas cells in the chemically crosslinked microcapsules displayed a proliferation rate of 45% (p < 0.05, n P 6, at least two different experiments). These data suggested that both variants of the Pluronicfibrinogen microcapsules were able to maintain proliferative
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Fig. 7. Proliferation of hMSCs in the physically (UV) and chemically (+UV) crosslinked Pluronic-fibrinogen microcapsules as determined by cell cycle analysis. Proliferation of the hMSCs in the suspension culture bioreactors was quantified as the percent of cells in the S and G2/M phases (A); Representative cell cycles of hMSCs in the microcapsules are shown for each time point and each respective treatment (B). A statistically significant difference between treatments was observed at day 1 (p < 0.05, n P 6, two different experiments).
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hMSCs in 3D suspension culture for at least a couple of weeks. Furthermore, the differences between the proliferation at day 1 and the latter time-points suggested that the hMSCs in both systems need some time to recover from the inoculation procedure. The recovery of the cells from the microcarriers after 15 days in suspension culture was determined by calculating the percent yield, which was the number of retrieved cells divided by number of inoculated cells in each microcapsule variant (Fig. 9). The cells in both the physically and chemically crosslinked systems show similar percent yields on day 1 and day 3 (80–90% and 115–125%, respectively) and exhibit increasing percent yields over the duration of the experiment. On day 15, the hMSCs cultured in the physically crosslinked microcapsules showed an average yield of 300%, whereas the cells cultured in the chemically crosslinked microcapsules showed an average yield of 190%. Due to the high variability in the results, there were no significant differences found between the two systems (n P 6, p > 0.05). These results indicate an ability of the Pluronic-fibrinogen microcapsules to facilitate the straightforward and efficient recovery of hMSCs after two weeks of 3D suspension culture in laboratory-scale bioreactors.
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4. Discussion
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The need for large quantities of therapeutically viable stem cells will increase as more clinical uses for these cells are realized in pre-clinical and clinic studies. The mass production of stem cells in bioreactors, already a major focus of development in academia and industry, is expected to be the gold standard for the bioprocessing of stem cell therapy products. However, because most
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mesenchymal cells are dependent on adhesion (i.e. anchorage dependent), they cannot be cultivated in traditional suspension bioreactors without the use of a microcarrier [42]. As such, there has been a steady increase in the development of microcarriers for hMSC cultivation and expansion [43–48]. Suspension culture microcarriers, which provide an alternative to the static expansion of stem cells in the 2D culture flasks [43], are made from a variety of synthetic and natural biomaterials and come in different shapes and size [42,44–46]. Used in combination with suspension bioreactors, these cell culture systems produce a unique environment that enhances the proliferation of the cells without producing alterations in the phenotype and differentiation potential of the cells [28]. Microcarriers come in two forms: hydrogels microcapsules that embed the cells [49,50], and solid polymer constructs that support cell adhesion on the surface and within pores [46]. Microcarriers made from synthetic polymers such as polystyrene are designed for culture on the surface using various surface modifications techniques that make the material more adherent to cells [51]. Natural microcarriers made from polysaccharides or proteins can support cell adhesion both on the surface as well as within the pores of the matrix, but may require additional surface modifications to enhance cell adhesion [52–55]. The inoculation of cells in these materials is straightforward; however, the removal of the densely packed cells from the surfaces and pores can be challenging and detrimental to the overall yields of the suspension culture system. Additionally, the surface cultivation of cells in a harsh hydrodynamic environment of the bioreactor can be detrimental to cell phenotype and survival. Hydrogel microcapsules are designed for microencapsulation of the cells during inoculation, thereby providing protection from hydrodynamics of the bioreactor and better
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Fig. 8. Proliferation of hMSCs in the physically (UV) and chemically (+UV) crosslinked Pluronic-fibrinogen microcapsules as determined by BrdU uptake. Pre-staining with BrdU for 17 h prior to the measurement provides a measure of proliferating cell populations at different time points in the suspension culture bioreactor. The summary of the % BrdU stained hMSCs showed a marked increase in the physically cross-linked (UV) treatment (A). Representative flow cytometry profiles of BrdU-FITC stained hMSCs in the microcarriers are shown for each time point and each respective treatment (B). Statistically significant differences between treatments were observed at day 3, 8 and 15 (p < 0.05, n P 6, two different experiments).
Fig. 9. The percent yield of the recovery phase from the physically (UV) and chemically (+UV) crosslinked Pluronic-fibrinogen microcarriers is shown. The % yield is calculated by dividing the harvested live cell density at the conclusion of the experiment by the inoculation cell seeding density. No statistically significant differences between treatments were observed at any time point (p > 0.05, n P 6, two different experiments).
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control of the cell microenvironment. Cell inoculation in hydrogel microcapsules can be more difficult based on the mechanism of crosslinking; however, cell recovery can be easily managed by the dissolution of the polymer network. We explored a method of preparing hydrogel microcapsules premised on thermoresponsive semi-synthetic polymers [37]. The thermoresponsive nature of the Pluronic-fibrinogen matrix enables straightforward cell inoculation in a temperature-
controlled gelation bath, and efficient cell recovery and harvesting (Supplementary video 2). The method of inoculating cells by temperature induced phase transition was compared with more conventional methods for crosslinking the hydrogel microcapsules by photochemistry. Oscillatory shear rheology was used to characterize the bulk mechanical properties of all the materials prior to cell inoculation (Fig. 3) [56,57]. The bulk mechanical properties of the physically or chemically cross-linked hydrogels were normalized with the addition of PEG-DA cross-linker to the chemical gels (+UV). It is important to note that the micromechanical properties around the cells within the microcapsules may be different than the bulk mechanical properties measured by rheology. The cell culture results indicated that the mild physical crosslinking of the polymer resulted in more favorable inoculation conditions, which had consequences on the downstream suspension culture outcomes. In particular, the advantage of the physically crosslinked system was underscored by the increased viability and proliferation of the hMSCs in these microcapsules, when compared to their chemically crosslinked counterparts. Given that the morphogenesis of the cells within the two hydrogel systems were similar – and the largest differences in viability were observed in the first few days after inoculation – we speculate that these difference were attributed to the photochemistry reaction. Consequently, several recent studies have demonstrated that mild photochemistry can be used to inoculate cells in hydrogels with excellent viability results [58,59]. Nevertheless, in a side-by-side comparison, the physical crosslinking was superior to the
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photochemistry approach both in terms of viability and methodological simplicity. Another critical component of the microcapsule suspension culture paradigm is the cell recovery phase of the bioprocessing. Whilst most conventional microcarriers require harsh enzymatic treatment to recover the cells during harvesting [48], the Pluronic-fibrinogen materials were incubated at 4 °C for 60 min to facilitate the rapid enzymatic dissolution of the polymer network (Supplementary video 2). Cell harvesting from these microcapsules was thus facilitated by short collagenase incubation in reduced temperatures followed by mild centrifugation. In contrast, the chemically crosslinked microcapsules required longer enzymatic dissolution in collagenase at 37 °C, increasing the likelihood of cell damage and phenotype reversion during this process. In a side-by-side comparison of the cell recovery from both systems, the physically crosslinked microcapsules yielded better cell harvests, although this could be attributed to the higher proliferation rates observed with these cultures. Further investigations are thus required to ascertain the distinct advantages of the simpler recovery protocols from the physically crosslinked hydrogels, particularly in terms of cell viability and cell phenotype.
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5. Conclusions
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Bioactive LCST polymers made form Pluronic-fibrinogen adducts were adapted for making physically crosslinked microcapsules to be used in suspension culture of hMSCs. The straightforward methodology to inoculate hMSCs in the microcapsules using a temperature-controlled gelation bath and mild physical crosslinking enabled suspension cultivation of the cells for at least two weeks in bioreactors, and efficient recovery during cell harvesting. This method of cell inoculation was demonstrated to be better to conventional biopolymer encapsulation of cells by photochemistry. Thus, the simplicity of the inoculation and mild recovery methodology represents an improvement over other microencapsulation systems, including porous polymeric substrates and chemically crosslinked hydrogels.
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Acknowledgments
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This work was supported by the Singapore National Research Foundation (NRF)-Technion-NUS Grant for Regenerative Medicine Initiative in Cardiac Restoration Therapy, the Lorry I. Lokey Interdisciplinary Center for Life Sciences and Engineering, Russell Berrie Nanotechnology Institute, as well as by EC-IP FP7 grants Angioscaff and Biodesign.
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Appendix A. Supplementary data
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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.ymeth.2015.04. 027.
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Methods journal homepage: www.elsevier.com/locate/ymeth 5 6
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A method for preparation of hydrogel microcapsules for stem cell bioprocessing and stem cell therapy
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Revital Goldshmid a,b, Iris Mironi-Harpaz a, Yonatan Shachaf a, Dror Seliktar a,⇑
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a b
The Faculty of Biomedical Engineering, Technion – Israel Institute of Technology, Haifa 32000, Israel The Interdisciplinary Program for Biotechnology, Technion – Israel Institute of Technology, Haifa 32000, Israel
a r t i c l e
i n f o
Article history: Received 11 December 2014 Received in revised form 20 April 2015 Accepted 21 April 2015 Available online xxxx Keywords: Biomaterial Suspension bioreactor 3D culture Crosslinking Pluronic-fibrinogen PEG
a b s t r a c t A method for the preparation of suspension culture microcapsules used in the bioprocessing of human mesenchymal stem cells (hMSCs) is reported. The microcapsules are prepared from a semi-synthetic hydrogel comprising PluronicÒF127 conjugated to denatured fibrinogen. The Pluronic-fibrinogen adducts display a lower critical solubility temperature (LCST) at 30 °C, thus enabling mild, cell-compatible physical crosslinking of the microcapsules in a warm gelation bath. Cell-laden microgels were prepared from a solution of Pluronic-fibrinogen hydrogel precursor and hMSCs; these were cultivated for up to 15 days in laboratory-scale suspension bioreactors and harvested by reducing the temperature of the microcapsules to disassemble the physical polymer network. The viability, proliferation and cell recovery yields of the hMSCs were shown to be better than photo-chemically crosslinked microcapsules made from a similar material. The cell culture yields, which exceeded 300% after 15 days in suspension culture, were comparable to other microcarrier systems used for the mass production of hMSCs. The simplicity of this methodology, both in terms of the cell inoculation and mild recovery conditions, represent distinct advantages for stem cell bioprocessing with suspension culture bioreactors. Ó 2015 Published by Elsevier Inc.
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1. Introduction
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An emerging approach in regenerative medicine involves the replacement of damaged cells with healthy ones that have the potential to restore function of damaged tissues [1]. In this context, stem cells are anticipated to become the ideal cell source for this approach [2]. They have shown efficacy in a variety of experimental models of tissue regeneration, including bone, cartilage, fat and muscle [3–9]. Hematopoietic stem and progenitor cells (HSC) are already routinely used in the clinic [10]; and pluripotent human mesenchymal stem cell (hMSCs) therapies are expected to provide far more treatment options [9,11,12]. This potential of cultured stem cells has already begun to materialize into clinical products poised to reach the market in just a few short years [13–16]. Given the high priority for commercialization of cell therapy in general and stem cell therapy in particular, one of the toughest tasks facing this field is how to generate the large numbers of cells required for the eventual treatment of large patient populations. Most research-stage projects working with stem cells can generate only a limited number of cells that support proof-of-concept
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⇑ Corresponding author at: Technion – Israel Institute of Technology, Faculty of Biomedical Engineering, Technion City, Haifa 32000, Israel. E-mail address: [email protected] (D. Seliktar).
clinical trials [17]. With technologies based on autologous cells, it is not clear how theses cells can be efficiently extracted, expanded, differentiated in vitro, and then delivered back into the patient. Regardless of the advanced stages of some stem cell technologies, a commercial medical delivery infrastructure for stem cell therapies is still very much in its infancy. The realization of stem cell therapies will therefore require robust, efficient and reproducible bioprocessing methodologies. In this context, suspension culture bioreactors are highly favoured in process scale-up because established culture conditions in lab-scale can often be transferred to much higher volumes with relative ease [18]. A key factor used to control stem cell growth in vitro is the matrix provided for cell attachment. With the exception of HSC, which are generally expanded without a cell attachment matrix, most other stem cell types have been isolated under conditions dependent on surface adherence. Amongst these are hMSCs, which are adherent cells that require culture surface enlargement to ensure efficient and reasonable mass production. Hence, the expansion of anchorage-dependent cells on two-dimensional (2D) substrates is a central challenge in bioreactor design. Moreover, the considerable cost with respect to consumables, labour and time as well as the inherent variability in manual processes of 2D culture not only make this option unattractive, but also render it commercially unviable. In this regard, automation
http://dx.doi.org/10.1016/j.ymeth.2015.04.027 1046-2023/Ó 2015 Published by Elsevier Inc.
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and the use of an efficient bioprocessing paradigm are imperative for the creation of successful clinical products. A variety of three-dimensional (3D) cell carriers for the suspension culture of hMSCs have been developed during the past few years [19,20]. When used for 3D cell cultivation, these platforms can also provide a physical support for cell attachment, proliferation and differentiation [21]. Both synthetic and biological polymers have been studied in order to find the ideal scaffold material, which integrates structural properties of synthetic polymers with bio-functionality of natural extracellular matrix (ECM) constituents [22–26]. For example, ECM proteins or polysaccharides such as alginate, hyaluronic acid, collagen, and fibrin have been used as culture scaffolds of hMSCs [24–27]. Biodegradable protein hydrogels that directly encapsulate cells within a 3D microenvironment (i.e. microcapsules) have many advantages [19,20,28], including easy inoculation, precise control of the cell microenvironment, and straightforward cell recovery. Additionally, microencapsulation of cells in these biomaterials can mimic the body’s extracellular environment better, and capture the ability of cells to proliferate in this more natural milieu [29,30]. Moreover, such scaffolds can provide physical protection and better control of biodegradation [29,30], two very important features that are beneficial in the often-harsh hydrodynamic environment of suspension bioreactors. When culturing MSCs in microcapsules, another factor that should be considered from the scaffold material is the influence of the material modulus on the cultured cells [31,32]. Previous studies demonstrated that MSCs are highly responsive to matrix modulus, and can often show unnatural behavior when they are removed from their tissue microenvironment [33]. Culturing MSCs in 3D embryoid bodies, for example, enhances chondrogenesis [34], suggesting that MSCs may be guided to this and other differentiation pathways during their suspension culture using simple modifications to the microcapsule properties. Finding a way to guide stem cell fate determination using the stiffness of culture substrates is a focus of much research, and will likely become essential aspect of future suspension culture paradigms for stem cell bioprocessing. Premised on these concepts, our group has recently developed microcapsules made from a bioactive semi-synthetic hydrogel. The biomaterial is made by conjugating the PluronicÒF127 to fibrinogen [35–37]. The PluronicÒF127 is a stimuli-responsive synthetic block copolymer that exhibits lower critical solution temperature (LCST) behavior [38,39]. When PluronicÒF127 is conjugated to fibrinogen, the semi-synthetic precursor retains bioactivity from the fibrinogen and LCST properties from the PluronicÒF127 [35,37]. In the current study, we exploit this material for developing bioprocessing methodologies for hMSCs, through their microencapsulation in hydrogel microcapsules and subsequent cultivation in suspension bioreactors. The fast LCST gelation, combined with photo-initiated chemical crosslinking, and multi-functional protein-like bioactive domains provides the features that help to facilitate the cell inoculation, control the cultivation microenvironment, and expedite the recovery of the cells from the microcarriers at the end of the culture period. This combined approach aims to provide a routine, efficient, and scalable solution for hMSC bioprocessing in suspension cell culture systems.
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2. Materials and methods
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2.1. Synthesis of PluronicÒF127-DA and PluronicÒF127-Fibrinogen adducts
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Temperature-responsive bioactive hydrogels were made from fibrinogen conjugated to PluronicÒF127 (F127), a poly(ethylene
oxide) (PEO)-poly(propylene oxide) (PPO)-PEO tri-block co-polymer that exhibits lower critical solubility temperature (LCST) properties. The F127 was end-functionalized with acryl groups to form F127-diacrylate (F127-DA), and then reacted with denatured bovine fibrinogen (Bovagen, Melbourne, Australia) via a Michaeltype addition reaction to form the Pluronic-fibrinogen biosynthetic copolymer [37]. A 7 mg/ml fibrinogen concentration was used for making Pluronic-fibrinogen hydrogels. Poly (ethylene glycol)-diacrylate (PEG-DA) was prepared from 10 kDa PEG-OH (Merck) as described elsewhere [40].
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2.2. Shear modulus measurements
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Mechanical properties of the Pluronic-fibrinogen were characterized with a strain-rate controlled shear rheometer (AR-G2, TA Instruments, Delaware, USA) equipped with a Peltier plate temperature controlled base, an overhead UV curing assembly and a transparent geometry. Time-sweep oscillatory tests were performed in 50 mm parallel-plate quartz geometry using 600 lL of Pluronicfibrinogen hydrogel precursor solution containing 0.1% w/v IrgacureÒ2959 photoinitiator (Ciba, Basel, Switzerland). In order to find the linear viscoelastic region of the time-sweep tests, oscillatory strain (0.1–10%) and frequency sweeps (0.1–10 Hz) were conducted. Rheology experiments were performed at 2% strain and 1 Hz.
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2.3. Cell culture and microencapsulation procedure
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Human mesenchymal stem cells (hMSCs, Lonza, Basel, Switzerland) were purchased and expanded in MSC growth medium (MSCGM, Lonza) containing 1% Pen-Strep (Kibbutz Beit Haemek, Israel) for 4 passages (12–14 days of expansion for each passage, corresponding to 4 doublings from P4 to P8). Cells were harvested and removed from 2D Flasks (Nunc, New York, USA) using trypsin EDTA solution B (Biological Industries). Centrifugation of the cells was performed at 1000 RPM for 5 min at room temp to obtain the hMSC pellet for proliferation experiments. In order to prepare the microcarriers, pelleted hMSCs were mixed with Pluronic-fibrinogen hydrogel precursor containing 0.1% IrgacureÒ2959 photoinitiator (Ciba, Basel, Switzerland). Two types of hydrogel microcapsules were prepared from the cell/polymer solution: a chemically crosslinked version and a physically crosslinked version. For physically crosslinked microcapsules, 400 lL cell/polymer mixture was dripped through a 30 gauge syringe needle into a warm (37 °C) gelation bath containing continually stirred culture medium (Supplementary video 1), forming beads with a diameter ranging from 500 to 1000 lm. The typical microcapsule volume was 0.25–0.5 lL (the polydispersity of the bead size and volume was not characterized). For the chemically crosslinked microcapsules, the cell/polymer solution was supplemented with PEG-DA (at a concentration of 0.2–0.5% w/v) and similarly dripped onto a super-hydrophobic surface at RT, followed by UV-light-activated photopolymerization (365 nm, 4–5 mW/cm2) for 1.5 min. The procedure created droplets of 0.25–0.5 lL and diameter of 500–1000 lm. The hMSC microcapsules (physically or chemically crosslinked) were cultured in laboratory-scale stirred-flask bioreactors (500 ml) for up to 15 days using expansion MSC growth medium (Lonza). The microcapsules were stirred at 1 RPM to ensure proper transport in the reactor volume. The cells were harvested from the hydrogel microcapsules on days 1, 3, 7 and 15, either by cooling down to 4 °C for physically crosslinked microcapsules (Supplementary video 2) or by using collagenase incubation for the chemically crosslinked microcarriers, as detailed below.
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Fig. 1. A schematic illustrating the methodological approach for using hydrogel microcapsules in the suspension culture of hMSCs. The main steps include cell inoculation, suspension culture, and cell recovery.
Fig. 2. A schematic illustration of the cell inoculation steps used for preparing (A) physically (UV) and (B) chemically crosslinked (+UV) Pluronic-fibrinogen microcapsules.
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2.4. hMSC viability
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Harvested hMSCs were pelleted by centrifugation, resuspended in 100 lL of phosphate buffered saline (PBS) and processed by flow cytometry for viability measurements. The cells were kept on ice until they were stained and analyzed. Propidium iodide (PI) staining solution was added to each sample prior to analysis for 10 min of incubation at 37 °C. The samples were measured in an LSR-II flow cytometer (Becton Dickinson, New Jersey, USA), and data was analyzed using FCS-express software (version 4.7). A control of unstained cells was used in order to adjust the flow cytometer settings. In situ cell viability was confirmed by calcein/ ethidium live/dead assay. Briefly, hMSCs in microcapsules were incubated in PBS solution containing 4 mM calcein acetoxymethyl ester and 2 mM ethidium homodimer-1 (Sigma–Aldrich, Buchs,
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Switzerland) for 40 min on an orbital shaker at 37 °C. After staining, the cells were washed twice with PBS (2 10 min) and microscopically imaged on an inverted fluorescence microscope (Nikon Eclipse TS100, Nikon, Japan) using a digital camera (Digital Sight, Nikon, Japan) and Nikon Nis-Elements F3.00 software (Nikon, Japan).
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2.5. % yield calculation
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The hMSCs were harvested from the physically crosslinked microcapsules by cooling them to 4 °C, incubating in 0.5 mg/ml collagenase solution (Sigma) for 60 min at 4 °C, followed by centrifugation for 5 min at 1000 RPM. For chemically crosslinked microcapsules, the microgels were incubated in 0.5 mg/ml collagenase solution for 120 min at 37 °C, followed by 5 min
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Fig. 3. Shear rheometry data showing different crosslinking schemes for Pluronicfibrinogen hydrogel precursors. Time sweep experiments of the shear storage modulus (G0 ) were initiated at 25 °C for 5 min, followed by physical crosslinking at 37 °C (blue triangles) or UV photopolymerization-based chemical crosslinking at 25 °C, with and without additional PEG-DA added to the Pluronic-fibrinogen precursor (red squares and orange circles, respectively).
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centrifugation at 1000 RPM. The % yield of the cell recovery was calculated according to the following equitation:
% Yield ¼
Harvested hmSCs=ml gel Seeded hMSCs=ml gel
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where the numerator represents the harvested live cell density at the conclusion of the experiment and the denominator represented the initial live cell seeding density.
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2.6. Proliferation by cell cycle analysis
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DNA staining and cell cycle analysis were carried out using PI and flow cytometry according to a previously described protocol [30,41]. The harvested cells were collected by centrifugation, washed in PBS and fixed in 70% ethanol at 4 °C for 20 min. The fixed cells were collected by centrifugation and stained with PI staining solution (Sigma) according to manufacturer’s instructions. The PI fluorescence staining was measured in a flow cytometer and scatter plots showing the populations of cells in the various cellcycle stages were obtained for sub G1 (apoptotic cells), G1/G0 phase, S phase and G2/M phase (proliferating cells phases). The different cell populations were plotted in single-parameter histograms of fluorescence PI levels (DNA content), and used to determine the number of cells in the apoptotic population (<2N DNA), in G0/G1 (2N DNA), in S (2–4N DNA) and in G2/M (4N DNA). Proliferation was quantified as the percent of cells in the S and G2/M phases.
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2.7. hMSCs proliferation kinetics (BrdU assay)
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Proliferation was quantified by bromodeoxyuridine (BrdU) incorporation into hMSCs residing within the microcarriers in suspension culture. The BrdU is an analog of the DNA precursor thymidine, which incorporates into newly synthesized DNA of dividing cells. The BrdU was supplemented into the microcarrier culture medium 17 h before detection as per the Flow Kit Staining Protocol (BD pharmingen, New Jersey, USA). Cells were then harvested from microcarriers (as described earlier) at days 1, 3, 7 and 15. The cells were then fixed and stained with FITC antiBrdU antibodies according to instructions provided by BD pharmingen. In addition, cells were stained with 7-aminoactinomycin D (7AAD), a dye that binds to all DNA and is used in conjunction with the BrdU staining to quantify the total number of cells. The levels of cell-associated BrdU and 7AAD were then measured by flow cytometry. The percent of BrdU-positive cells was reported
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as the number of proliferating cells within the total cell population of each treatment.
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2.8. Morphology analysis
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Cultures of hMSCs in microcarriers were rinsed in PBS, permeabilized with 0.1% Triton X-100 in PBS for 10 min, and stained with TRITC-labeled phalloidin (TRITC-phalloidin, 1 lg/ml, Sigma) for 1 h at room temperature followed by Sytox-green addition for 30 min. Microcarriers were then washed three times with PBS (at RT) and placed at 4 °C for an overnight wash in PBS. Stained cells were imaged using a Zeiss LSM 700 confocal microscope (Carl Zeiss, Oberkochen, Germany). Scans using a 20 objective were performed at a z-step size of 2.3 lm with a typical depth of 100–200 lm. All scanning areas were randomly selected and images sampled at a resolution of 1024 1024 pixels.
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2.9. Statistical analysis
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Statistical analysis was performed using MicrosoftÒ Excel software; FACS data analysis was performed using FCS express 4 software. Significant differences between data sets were found using the Student’s t-test (two way); the minimal criterion for significance was set at p < 0.05.
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The Pluronic-fibrinogen biopolymers were used as a three-dimensional (3D) microencapsulation system enabling straightforward cell inoculation, suspension culture in bioreactors and efficient cell recovery (Fig. 1). The microcarriers were made with two hydrogel variants including a physically crosslinked version produced by elevating the temperature of the cell/polymer droplets to 37 °C in a gelation bath (Fig. 2a); and a chemically crosslinked hydrogel produced by UV photopolymerization of the cell/ polymer droplets on superhydrophobic surfaces (Fig. 2b). The two ways of crosslinking (physical vs. chemical) resulted in significantly different bulk mechanical properties of the hydrogels, as measured by strain-rate controlled shear rheology. Fig. 3 shows the characteristic bulk mechanical properties of the two different crosslinking routes. The chemically crosslinked matrix (+UV) reached a plateau shear storage modulus (G0 ) of 68 ± 17 Pa, whereas the physically crosslinked matrix (UV) reached a plateau G0 of 152 ± 60 Pa. In order to focus on the effects of chemical vs. physical crosslinking of this system (+UV and UV, respectively) we normalized the mechanical properties of the +UV hydrogels by adding additional PEG-DA crosslinker to the chemically crosslinked system. The PEG-DA added to the Pluronic-fibrinogen increased the G0 of the crosslinked hydrogel to 175 ± 30 Pa, thus obtaining similar bulk mechanical properties for the physical and chemical hydrogels (n P 3 from at least 2 different batches of Pluronic-fibrinogen materials). We first investigated the viability of hMSCs in the microcapsules using both a qualitative live/dead assay and a quantitative PI staining assay with FACS analysis. Optimization of seeded cell density was explored by viability analysis of different cell concentrations (1, 3, 6 and 12 106 cells/ml). The qualitative live/dead assay revealed a highly viable cell population in the microgels, irrespective of concentration used (Fig. 4). The highest quantitative viability results were achieved at a 3 106 cells/ml concentration (data not shown); thus this concentration was chosen for further experiments. The cells in the physically crosslinked material showed a somewhat higher viability then those in the chemically crosslinked material, particularly during the first week in culture, with a statistical difference on day 1 (Fig. 5). Apparently, the
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Fig. 4. Microencapsulation of cells in Pluronic-fibrinogen hydrogel microcapsules. Phase contrast micrographs (top) are shown with live/dead staining (middle) using calcein (green) and ethidium (red). The superimposition of the phase contrast and live/dead images clearly shows the cells residing and spreading within the microgels after 3 days in culture. Different cell concentrations and microcapsule sizes are shown from left to right, to exemplify the versatility of the system. Scale bar = 50 lm.
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photochemistry had an initial adverse effect on the cells; however, the cells did recover after one week in suspension culture. Between days 11 and 15, there was a slight decrease in viability of the cells in both systems, most likely stemming from mass transport limitations associated with increasing cell density (due to high proliferation within the microcarriers). We further used laser scanning confocal microscopy (LSCM) in order to visualize the hMSC morphogenesis within the hydrogel microcarriers. hMSCs were stained with f-actin dye (TRITC-labeled phalloidin, red) and nuclear dye (Sytox-green) in order to characterize their typical morphology in the microcapsules (Fig. 6). On the first day the cells were homogenously dispersed in the hydrogel microcapsules, showing a predominantly rounded morphology. On day five, the cells appeared much more spindled with distinct lamellipodia. By day eight, the cellular lamellipodia became much more branched and continued to display these branching patterns throughout day 20. There were no distinguishable differences between the morphology patterns of
hMSCs in the physically and chemically crosslinked microcapsules (Fig. 6). Cell cycle analysis was carried out for hMSCs in microcapsules using both a PI assay (Fig. 7) and a 7AAD/BrdU-FITC assay (Fig. 8). The DNA staining using PI clearly indicated a substantial portion of the hMSCs in the proliferative phases of the cell cycle (S + G2/M) in both physically and chemically crosslinked microcapsules. In the physically crosslinked system, 25–27% of the cells are in the proliferation phases, while for the chemically crosslinked microcapsules, only 20–23% are in the proliferation phases. For both systems, the proliferating cell populations showed a statistical difference on day 1 (n P 6, p < 0.05), and were unaffected by the 2-week duration of the experiment. The kinetics of cell proliferation was measured by incorporating FITC-labeled BrdU for 17 h (the cell generation time was found to be 28 h). This DNA pre-labeling with BrdU (at set time intervals) provided a direct measure of the average rate of proliferation in the microcapsules at each
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Fig. 5. Viability of hMSCs in the physically (UV) and chemically (+UV) crosslinked Pluronic-fibrinogen microcapsules. Live/dead staining with calcein (green) and ethidium (red) show a relatively high number of viable cells within the microgels, when cultured for up to 21 days in suspension bioreactors (A). Quantitative viability with DNA staining and cell cycle analysis using propidium iodide (PI) confirms the qualitative data (B). Statistically significant difference between treatments is observed at day 1 (p < 0.05, n P 6, two different experiments).
Fig. 6. Morphogenesis of hMSCs in the physically (UV) and chemically (+UV) crosslinked Pluronic-fibrinogen microcapsules. The inoculated hMSCs are initially rounded in the microcapsules; they become elongated and highly spindled over the course of three weeks in culture, irrespective of the crosslinking method.
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time-point of the experiment. The BrdU results indicated a very slow rate of proliferation after the first day in suspension culture, irrespective of the crosslinking method (Fig. 7). Both systems showed an increased proliferation rate at day 3, which was maintained at a constant rate at the later time-points. However, the cells in both systems showed a statistical difference in their
proliferation rate from day 3 onward: in the physically crosslinked system the cells exhibited a proliferation rate of 75%, whereas cells in the chemically crosslinked microcapsules displayed a proliferation rate of 45% (p < 0.05, n P 6, at least two different experiments). These data suggested that both variants of the Pluronicfibrinogen microcapsules were able to maintain proliferative
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Fig. 7. Proliferation of hMSCs in the physically (UV) and chemically (+UV) crosslinked Pluronic-fibrinogen microcapsules as determined by cell cycle analysis. Proliferation of the hMSCs in the suspension culture bioreactors was quantified as the percent of cells in the S and G2/M phases (A); Representative cell cycles of hMSCs in the microcapsules are shown for each time point and each respective treatment (B). A statistically significant difference between treatments was observed at day 1 (p < 0.05, n P 6, two different experiments).
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hMSCs in 3D suspension culture for at least a couple of weeks. Furthermore, the differences between the proliferation at day 1 and the latter time-points suggested that the hMSCs in both systems need some time to recover from the inoculation procedure. The recovery of the cells from the microcarriers after 15 days in suspension culture was determined by calculating the percent yield, which was the number of retrieved cells divided by number of inoculated cells in each microcapsule variant (Fig. 9). The cells in both the physically and chemically crosslinked systems show similar percent yields on day 1 and day 3 (80–90% and 115–125%, respectively) and exhibit increasing percent yields over the duration of the experiment. On day 15, the hMSCs cultured in the physically crosslinked microcapsules showed an average yield of 300%, whereas the cells cultured in the chemically crosslinked microcapsules showed an average yield of 190%. Due to the high variability in the results, there were no significant differences found between the two systems (n P 6, p > 0.05). These results indicate an ability of the Pluronic-fibrinogen microcapsules to facilitate the straightforward and efficient recovery of hMSCs after two weeks of 3D suspension culture in laboratory-scale bioreactors.
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The need for large quantities of therapeutically viable stem cells will increase as more clinical uses for these cells are realized in pre-clinical and clinic studies. The mass production of stem cells in bioreactors, already a major focus of development in academia and industry, is expected to be the gold standard for the bioprocessing of stem cell therapy products. However, because most
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mesenchymal cells are dependent on adhesion (i.e. anchorage dependent), they cannot be cultivated in traditional suspension bioreactors without the use of a microcarrier [42]. As such, there has been a steady increase in the development of microcarriers for hMSC cultivation and expansion [43–48]. Suspension culture microcarriers, which provide an alternative to the static expansion of stem cells in the 2D culture flasks [43], are made from a variety of synthetic and natural biomaterials and come in different shapes and size [42,44–46]. Used in combination with suspension bioreactors, these cell culture systems produce a unique environment that enhances the proliferation of the cells without producing alterations in the phenotype and differentiation potential of the cells [28]. Microcarriers come in two forms: hydrogels microcapsules that embed the cells [49,50], and solid polymer constructs that support cell adhesion on the surface and within pores [46]. Microcarriers made from synthetic polymers such as polystyrene are designed for culture on the surface using various surface modifications techniques that make the material more adherent to cells [51]. Natural microcarriers made from polysaccharides or proteins can support cell adhesion both on the surface as well as within the pores of the matrix, but may require additional surface modifications to enhance cell adhesion [52–55]. The inoculation of cells in these materials is straightforward; however, the removal of the densely packed cells from the surfaces and pores can be challenging and detrimental to the overall yields of the suspension culture system. Additionally, the surface cultivation of cells in a harsh hydrodynamic environment of the bioreactor can be detrimental to cell phenotype and survival. Hydrogel microcapsules are designed for microencapsulation of the cells during inoculation, thereby providing protection from hydrodynamics of the bioreactor and better
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Fig. 8. Proliferation of hMSCs in the physically (UV) and chemically (+UV) crosslinked Pluronic-fibrinogen microcapsules as determined by BrdU uptake. Pre-staining with BrdU for 17 h prior to the measurement provides a measure of proliferating cell populations at different time points in the suspension culture bioreactor. The summary of the % BrdU stained hMSCs showed a marked increase in the physically cross-linked (UV) treatment (A). Representative flow cytometry profiles of BrdU-FITC stained hMSCs in the microcarriers are shown for each time point and each respective treatment (B). Statistically significant differences between treatments were observed at day 3, 8 and 15 (p < 0.05, n P 6, two different experiments).
Fig. 9. The percent yield of the recovery phase from the physically (UV) and chemically (+UV) crosslinked Pluronic-fibrinogen microcarriers is shown. The % yield is calculated by dividing the harvested live cell density at the conclusion of the experiment by the inoculation cell seeding density. No statistically significant differences between treatments were observed at any time point (p > 0.05, n P 6, two different experiments).
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control of the cell microenvironment. Cell inoculation in hydrogel microcapsules can be more difficult based on the mechanism of crosslinking; however, cell recovery can be easily managed by the dissolution of the polymer network. We explored a method of preparing hydrogel microcapsules premised on thermoresponsive semi-synthetic polymers [37]. The thermoresponsive nature of the Pluronic-fibrinogen matrix enables straightforward cell inoculation in a temperature-
controlled gelation bath, and efficient cell recovery and harvesting (Supplementary video 2). The method of inoculating cells by temperature induced phase transition was compared with more conventional methods for crosslinking the hydrogel microcapsules by photochemistry. Oscillatory shear rheology was used to characterize the bulk mechanical properties of all the materials prior to cell inoculation (Fig. 3) [56,57]. The bulk mechanical properties of the physically or chemically cross-linked hydrogels were normalized with the addition of PEG-DA cross-linker to the chemical gels (+UV). It is important to note that the micromechanical properties around the cells within the microcapsules may be different than the bulk mechanical properties measured by rheology. The cell culture results indicated that the mild physical crosslinking of the polymer resulted in more favorable inoculation conditions, which had consequences on the downstream suspension culture outcomes. In particular, the advantage of the physically crosslinked system was underscored by the increased viability and proliferation of the hMSCs in these microcapsules, when compared to their chemically crosslinked counterparts. Given that the morphogenesis of the cells within the two hydrogel systems were similar – and the largest differences in viability were observed in the first few days after inoculation – we speculate that these difference were attributed to the photochemistry reaction. Consequently, several recent studies have demonstrated that mild photochemistry can be used to inoculate cells in hydrogels with excellent viability results [58,59]. Nevertheless, in a side-by-side comparison, the physical crosslinking was superior to the
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photochemistry approach both in terms of viability and methodological simplicity. Another critical component of the microcapsule suspension culture paradigm is the cell recovery phase of the bioprocessing. Whilst most conventional microcarriers require harsh enzymatic treatment to recover the cells during harvesting [48], the Pluronic-fibrinogen materials were incubated at 4 °C for 60 min to facilitate the rapid enzymatic dissolution of the polymer network (Supplementary video 2). Cell harvesting from these microcapsules was thus facilitated by short collagenase incubation in reduced temperatures followed by mild centrifugation. In contrast, the chemically crosslinked microcapsules required longer enzymatic dissolution in collagenase at 37 °C, increasing the likelihood of cell damage and phenotype reversion during this process. In a side-by-side comparison of the cell recovery from both systems, the physically crosslinked microcapsules yielded better cell harvests, although this could be attributed to the higher proliferation rates observed with these cultures. Further investigations are thus required to ascertain the distinct advantages of the simpler recovery protocols from the physically crosslinked hydrogels, particularly in terms of cell viability and cell phenotype.
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Bioactive LCST polymers made form Pluronic-fibrinogen adducts were adapted for making physically crosslinked microcapsules to be used in suspension culture of hMSCs. The straightforward methodology to inoculate hMSCs in the microcapsules using a temperature-controlled gelation bath and mild physical crosslinking enabled suspension cultivation of the cells for at least two weeks in bioreactors, and efficient recovery during cell harvesting. This method of cell inoculation was demonstrated to be better to conventional biopolymer encapsulation of cells by photochemistry. Thus, the simplicity of the inoculation and mild recovery methodology represents an improvement over other microencapsulation systems, including porous polymeric substrates and chemically crosslinked hydrogels.
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Acknowledgments
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This work was supported by the Singapore National Research Foundation (NRF)-Technion-NUS Grant for Regenerative Medicine Initiative in Cardiac Restoration Therapy, the Lorry I. Lokey Interdisciplinary Center for Life Sciences and Engineering, Russell Berrie Nanotechnology Institute, as well as by EC-IP FP7 grants Angioscaff and Biodesign.
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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.ymeth.2015.04. 027.
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