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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 ( 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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References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53]
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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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[54] [55] [56] [57] [58] [59]
A.I. Hoch, J.K. Leach, Stem Cells Transl. Med. 3 (5) (2014) 643–652. D.C. Ding, W.C. Shyu, S.Z. Lin, Cell Transplant. 20 (1) (2011) 5–14. A. Derfoul et al., Stem Cells 24 (6) (2006) 1487–1495. T. Fink et al., Stem Cells 22 (7) (2004) 1346–1355. M. Neubauer et al., Tissue Eng. 11 (11–12) (2005) 1840–1851. M.S. Friedman, M.W. Long, K.D. Hankenson, J. Cell. Biochem. 98 (3) (2006) 538– 554. J.M. Gimble et al., J. Cell. Biochem. 58 (3) (1995) 393–402. J.H. Lee, P.A. Kosinski, D.M. Kemp, Exp. Cell Res. 307 (1) (2005) 174–182. E. Schipani, H.M. Kronenberg, Adult mesenchymal stem cells, in: StemBook, Cambridge (MA), 2008. I. McNiece, Semin. Cell Dev. Biol. 18 (6) (2007) 839–845. H.A. Awad et al., Biomaterials 25 (16) (2004) 3211–3222. N. Beyer Nardi, L. da Silva Meirelles, Handb. Exp. Pharmacol. 174 (2006) 249– 282. A.B. Parson, Cell 132 (4) (2008) 511–513. B. Short et al., Arch. Med. Res. 34 (6) (2003) 565–571. B. Parekkadan, J.M. Milwid, Annu. Rev. Biomed. Eng. 12 (2010) 87–117. S.K. Tae et al., Biomed. Mater. 1 (2) (2006) 63–71. L. Jackson et al., J. Postgrad. Med. 53 (2) (2007) 121–127. R. Zweigerdt, Adv. Biochem. Eng. Biotechnol. 114 (2009) 201–235. A.W. Lund et al., J. Biomed. Mater. Res. B Appl. Biomater. 87 (1) (2008) 213– 221. S.Q. Liu et al., Biomaterials 31 (28) (2010) 7298–7307. H. Zhao et al., Biomed. Mater. 3 (1) (2008) 015001. D. Seliktar, Science 336 (6085) (2012) 1124–1128. G. Zhang et al., Tissue Eng. 12 (1) (2006) 9–19. L. Almany, D. Seliktar, Biomaterials 26 (15) (2005) 2467–2477. J.B. Leach et al., J. Biomed. Mater. Res. A 70 (1) (2004) 74–82. J.B. Leach, C.E. Schmidt, Biomaterials 26 (2) (2005) 125–135. T. Cordonnier et al., J. Mater. Sci. – Mater. Med. 21 (3) (2010) 981–987. I. Pountos et al., Injury 38 (Suppl 4) (2007) S23–S33. D. Dikovsky, H. Bianco-Peled, D. Seliktar, Biomaterials 27 (8) (2006) 1496– 1506. M. Gonen-Wadmany, R. Goldshmid, D. Seliktar, Biomaterials 32 (26) (2011) 6025–6033. A.J. Engler et al., Methods Cell Biol. 83 (2007) 521–545. A.J. Engler et al., Cell 126 (4) (2006) 677–689. M.W. Tibbitt, K.S. Anseth, Biotechnol. Bioeng. 103 (4) (2009) 655–663. B.D. Markway et al., Cell Transplant. 19 (1) (2010) 29–42. I. Frisman et al., Langmuir 27 (11) (2011) 6977–6986. M. Plotkin et al., Biomaterials 35 (5) (2014) 1429–1438. Y. Shachaf, M. Gonen-Wadmany, D. Seliktar, Biomaterials 31 (10) (2010) 2836–2847. J. Kopecek, Biomaterials 28 (34) (2007) 5185–5192. L. Klouda, A.G. Mikos, Eur. J. Pharm. Biopharm. 68 (1) (2008) 34–45. S. Halstenberg et al., Biomacromolecules 3 (4) (2002) 710–723. P. Pozarowski, Z. Darzynkiewicz, Methods Mol. Biol. 281 (2004) 301–311. C.J. Hewitt et al., Biotechnol. Lett. 33 (11) (2011) 2325–2335. L. Boo et al., J. Mater. Sci. – Mater. Med. 22 (5) (2011) 1343–1356. A.K. Chen et al., Curr. Protoc. Stem Cell Biol. (2010). Chapter 1: p. Unit 1C 11. S.K. Oh et al., Stem Cell Res. 2 (3) (2009) 219–230. S. Sart et al., J. Tissue Eng. Regen. Med. 7 (7) (2013) 537–551. D. Schop et al., J. Tissue Eng. Regen. Med. 2 (2–3) (2008) 126–135. H.S. Yang et al., Cell Transplant. 19 (9) (2010) 1123–1132. F. Cellesi, N. Tirelli, J.A. Hubbell, Biomaterials 25 (21) (2004) 5115–5124. F. Cellesi et al., Biotechnol. Bioeng. 88 (6) (2004) 740–749. K.W. Chun et al., Biotechnol. Prog. 20 (6) (2004) 1797–1801. G. Altankov, I. Brodvarova, I. Rashkov, J. Biomater. Sci. Polym. Ed. 2 (2) (1991) 81–89. M. Kiremitci, E. Piskin, Biomater. Artif. Cells Artif. Organs 18 (5) (1990) 599– 603. B. Roder et al., J. Biomater. Sci. Polym. Ed. 5 (1–2) (1993) 79–88. N.P. Smit et al., J. Invest. Dermatol. 92 (1) (1989) 18–21. D.S. Jones, Int. J. Pharm. 179 (2) (1999) 167–178. T.K. Meyvis et al., Int. J. Pharm. 244 (1–2) (2002) 163–168. D. Kesselman et al., Acta Biomater. 9 (8) (2013) 7630–7639. I. Mironi-Harpaz et al., Acta Biomater. 8 (5) (2012) 1838–1848.
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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 ( 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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A
B
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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References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53]
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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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[54] [55] [56] [57] [58] [59]
A.I. Hoch, J.K. Leach, Stem Cells Transl. Med. 3 (5) (2014) 643–652. D.C. Ding, W.C. Shyu, S.Z. Lin, Cell Transplant. 20 (1) (2011) 5–14. A. Derfoul et al., Stem Cells 24 (6) (2006) 1487–1495. T. Fink et al., Stem Cells 22 (7) (2004) 1346–1355. M. Neubauer et al., Tissue Eng. 11 (11–12) (2005) 1840–1851. M.S. Friedman, M.W. Long, K.D. Hankenson, J. Cell. Biochem. 98 (3) (2006) 538– 554. J.M. Gimble et al., J. Cell. Biochem. 58 (3) (1995) 393–402. J.H. Lee, P.A. Kosinski, D.M. Kemp, Exp. Cell Res. 307 (1) (2005) 174–182. E. Schipani, H.M. Kronenberg, Adult mesenchymal stem cells, in: StemBook, Cambridge (MA), 2008. I. McNiece, Semin. Cell Dev. Biol. 18 (6) (2007) 839–845. H.A. Awad et al., Biomaterials 25 (16) (2004) 3211–3222. N. Beyer Nardi, L. da Silva Meirelles, Handb. Exp. Pharmacol. 174 (2006) 249– 282. A.B. Parson, Cell 132 (4) (2008) 511–513. B. Short et al., Arch. Med. Res. 34 (6) (2003) 565–571. B. Parekkadan, J.M. Milwid, Annu. Rev. Biomed. Eng. 12 (2010) 87–117. S.K. Tae et al., Biomed. Mater. 1 (2) (2006) 63–71. L. Jackson et al., J. Postgrad. Med. 53 (2) (2007) 121–127. R. Zweigerdt, Adv. Biochem. Eng. Biotechnol. 114 (2009) 201–235. A.W. Lund et al., J. Biomed. Mater. Res. B Appl. Biomater. 87 (1) (2008) 213– 221. S.Q. Liu et al., Biomaterials 31 (28) (2010) 7298–7307. H. Zhao et al., Biomed. Mater. 3 (1) (2008) 015001. D. Seliktar, Science 336 (6085) (2012) 1124–1128. G. Zhang et al., Tissue Eng. 12 (1) (2006) 9–19. L. Almany, D. Seliktar, Biomaterials 26 (15) (2005) 2467–2477. J.B. Leach et al., J. Biomed. Mater. Res. A 70 (1) (2004) 74–82. J.B. Leach, C.E. Schmidt, Biomaterials 26 (2) (2005) 125–135. T. Cordonnier et al., J. Mater. Sci. – Mater. Med. 21 (3) (2010) 981–987. I. Pountos et al., Injury 38 (Suppl 4) (2007) S23–S33. D. Dikovsky, H. Bianco-Peled, D. Seliktar, Biomaterials 27 (8) (2006) 1496– 1506. M. Gonen-Wadmany, R. Goldshmid, D. Seliktar, Biomaterials 32 (26) (2011) 6025–6033. A.J. Engler et al., Methods Cell Biol. 83 (2007) 521–545. A.J. Engler et al., Cell 126 (4) (2006) 677–689. M.W. Tibbitt, K.S. Anseth, Biotechnol. Bioeng. 103 (4) (2009) 655–663. B.D. Markway et al., Cell Transplant. 19 (1) (2010) 29–42. I. Frisman et al., Langmuir 27 (11) (2011) 6977–6986. M. Plotkin et al., Biomaterials 35 (5) (2014) 1429–1438. Y. Shachaf, M. Gonen-Wadmany, D. Seliktar, Biomaterials 31 (10) (2010) 2836–2847. J. Kopecek, Biomaterials 28 (34) (2007) 5185–5192. L. Klouda, A.G. Mikos, Eur. J. Pharm. Biopharm. 68 (1) (2008) 34–45. S. Halstenberg et al., Biomacromolecules 3 (4) (2002) 710–723. P. Pozarowski, Z. Darzynkiewicz, Methods Mol. Biol. 281 (2004) 301–311. C.J. Hewitt et al., Biotechnol. Lett. 33 (11) (2011) 2325–2335. L. Boo et al., J. Mater. Sci. – Mater. Med. 22 (5) (2011) 1343–1356. A.K. Chen et al., Curr. Protoc. Stem Cell Biol. (2010). Chapter 1: p. Unit 1C 11. S.K. Oh et al., Stem Cell Res. 2 (3) (2009) 219–230. S. Sart et al., J. Tissue Eng. Regen. Med. 7 (7) (2013) 537–551. D. Schop et al., J. Tissue Eng. Regen. Med. 2 (2–3) (2008) 126–135. H.S. Yang et al., Cell Transplant. 19 (9) (2010) 1123–1132. F. Cellesi, N. Tirelli, J.A. Hubbell, Biomaterials 25 (21) (2004) 5115–5124. F. Cellesi et al., Biotechnol. Bioeng. 88 (6) (2004) 740–749. K.W. Chun et al., Biotechnol. Prog. 20 (6) (2004) 1797–1801. G. Altankov, I. Brodvarova, I. Rashkov, J. Biomater. Sci. Polym. Ed. 2 (2) (1991) 81–89. M. Kiremitci, E. Piskin, Biomater. Artif. Cells Artif. Organs 18 (5) (1990) 599– 603. B. Roder et al., J. Biomater. Sci. Polym. Ed. 5 (1–2) (1993) 79–88. N.P. Smit et al., J. Invest. Dermatol. 92 (1) (1989) 18–21. D.S. Jones, Int. J. Pharm. 179 (2) (1999) 167–178. T.K. Meyvis et al., Int. J. Pharm. 244 (1–2) (2002) 163–168. D. Kesselman et al., Acta Biomater. 9 (8) (2013) 7630–7639. I. Mironi-Harpaz et al., Acta Biomater. 8 (5) (2012) 1838–1848.
Please cite this article in press as: R. Goldshmid et al., Methods (2015), http://dx.doi.org/10.1016/j.ymeth.2015.04.027
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