TISSUE ENGINEERING: Part C Volume 18, Number 12, 2012 ª Mary Ann Liebert, Inc. DOI: 10.1089/ten.tec.2011.0541
Human Platelet Lysate Gel Provides a Novel Three Dimensional-Matrix for Enhanced Culture Expansion of Mesenchymal Stromal Cells Gudrun Walenda, M.Sc.,1,* Hatim Hemeda, Ph.D.,1,* Rebekka K. Schneider, M.D.,2 Rudolf Merkel, Ph.D.,3 Bernd Hoffmann, Ph.D.,3 and Wolfgang Wagner, M.D., Ph.D.,1
Cell culture in regenerative medicine needs to facilitate efficient expansion according to good manufacturing practice requirements. Human platelet lysate (HPL) can be used as a substitute for fetal calf serum without the risk of xenogeneic immune reactions or transmission of bovine pathogens. Heparin needs to be added as anticoagulant before addition of HPL to culture medium; otherwise, HPL-medium forms a gel within 1 h. Here, we demonstrated that such HPL-gels provide a suitable 3D-matrix for cell culture that—apart from heparin— consists of the same components as the over-layered culture medium. Mesenchymal stromal cells (MSCs) grew in several layers at the interface between HPL-gel and HPL-medium without contact with any artificial biomaterials. Notably, proliferation of MSCs was much higher on HPL-gel compared with tissue culture plastic. Further, the frequency of initial fibroblastoid colony forming units (CFU-f) increased on HPL-gel. The viscous consistency of HPL-gel enabled passaging with a convenient harvesting and reseeding procedure by pipetting cells together with their HPL-matrix—this method does not require washing steps and can easily be automated. The immunophenotype and in vitro differentiation potential toward adipogenic, osteogenic, and chondrogenic lineage were not affected by culture-isolation on HPL-gel. Taken together, HPL-gel has many advantages over conventional plastic surfaces: it facilitates enhanced CFU-f outgrowth, increased proliferation rates, higher cell densities, and nonenzymatic passaging procedures for culture expansion of MSCs.
Introduction
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he rising field of cellular therapy requires optimized cell culture methods—they have to enable fast expansion of cell preparations under good manufacturing practice-conform conditions and need to be safe, reliable, and highly standardized. These prerequisites differ from traditional cell culture techniques. Optimized culture methods are of specific interest for mesenchymal stromal cells (MSCs; alternatively termed ‘‘mesenchymal stem cells’’), which are concurrently tested in many clinical trials for a broad range of diseases. Their clinical application in either autologous or allogeneic settings necessitates large-scale in vitro expansion prior to application.1,2 Biomaterials have impact on proliferation and differentiation of cell preparations.3 MSCs are routinely cultured on conventional tissue culture plastic (TCP) with biofunctionalized polystyrene surfaces. In these dishes, plastic adherent cell growth is restricted to two dimensions and preferentially occurs at the rim of colonies due to contact inhibition.4 For
passaging, adherent cells need to be loosened from the substrate, which is usually performed by enzymatic treatment with peptidases such as trypsin. Recently, hydrogels and fibrous scaffolds based on natural or synthetic polymers were developed for expansion of primary cells.5–7 Such biomaterials provide new perspectives for efficient culture expansion with homogeneous cell growth at higher cell densities. Culture media are at least equally important for MSC preparations.8,9 Since the pioneering work on chemically defined culture media serums supplements are usually added.10 To date, fetal calf serum (FCS; alternatively termed ‘‘fetal bovine serum’’) is the most commonly used growth additive for culture media. It is isolated from clotted blood of unborn bovine fetuses and has been shown to be rich in growth factors. In light of therapeutic applications, there is a growing interest to avoid the use of FCS due to potential xenogeneic immune reactions, bovine pathogens (viruses and prions), and a high lot-to-lot variability that hampers reproducibility of results.11,12 Human platelet lysate (HPL)
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Helmholtz-Institute for Biomedical Engineering, RWTH Aachen University Medical School, Aachen, Germany. Institute of Pathology, RWTH Aachen University Medical School, Aachen, Germany. 3 Institute of Complex Systems, ICS-7: Biomechanics, Forschungszentrum Ju¨lich GmbH, Ju¨lich, Germany. *These authors equally contributed to this work. 2
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PLATELET LYSATE GEL PROVIDES A CELL CULTURE MATRIX has been shown to be an efficient nonxenogeneic replacement for FCS.13–20 It is generated from platelet units by a simple freeze-thaw procedure. The disrupted platelets contribute growth factors (such as PDGF, FGF, IGF, TGFb, PF-4, and PDEGF).14,19,21–23 Proliferation of MSCs is significantly stimulated by medium supplemented with HPL as compared with FCS.4,19,22,24 Since the first description in 2005,17 many laboratories have changed their culture conditions for HPL— especially for therapeutic cell preparations. Notably, HPLmedium has already been implemented in clinical trials.24–26 Apart from the platelet fraction HPL also comprises some human plasma. In contrast to serum, plasma still contains fibrinogen and other components of the coagulation cascade.27 Therefore, heparin needs to be added as anticoagulant before addition of HPL to culture medium—otherwise, HPL-medium gelatinizes. In this study, we demonstrate that such HPL-gel provides a very effective 3D-matrix for improved in vitro culture of MSCs. Materials and Methods Ethics statement All samples in this study were used after patient’s written consent using guidelines approved by the Ethic Committee of the University of Aachen (Permit numbers: EK128/09 for MSCs and EK163/07 for fibroblasts). Generation of HPL Platelet units were kindly provided by the Institute for Transfusion Medicine, RWTH Aachen University Medical School after their expiry date (5 days after harvesting). Platelet units were generated by apheresis using the Trima Accel Collection System (CaridianBCT) and comprise 2.0– 4.2 · 1011 platelets in 200 mL plasma supplemented with Acid-Citrate-Dextrose (1:11 v/v). Aliquots of 45 mL were twice frozen at - 80C and re-thawed at 37C and then centrifuged at 2600 g for 30 min. The supernatant was filtered through 0.2 mm GD/X PVDF filters (Whatman) and stored at - 80C until use.4,22 Experiments of this study were performed with three HPL-pools each consisting of lysates of four donors, but similar results were also observed using individual donor = derived HPLs. HPLs consisted predominately of blood type A but a systematic analysis did not reveal any impact of the blood group.28 To avoid HPL gel formation, 2 U/mL heparin (Ratiopharm) was added as an anticoagulant before addition to culture medium. Culture medium and HPL-gel Culture medium consisted of Dulbecco’s modified Eagle’s medium-Low Glucose (DMEM-LG; PAA) with 2 mM L-glutamine (Sigma Aldrich) and 100 U/mL penicillin/ streptomycin (pen/strep; Lonza) and HPL (10% if not indicated otherwise). Cells were cultured on TCP (Greiner). For gel preparation, HPL containing cell culture medium without heparin was filled in cell culture plates. Due to the calcium comprised in the medium the coagulation cascade was initiated and the HPL-medium gelatinized within 1 h at 37C. The resulting HPL-gel was used as cell culture substrate that was seeded with the same amount of mononuclear cell (MNC) and overlaid with the same HPL-medium supplemented with heparin. For comparison, gels consisting of 80% collagen G
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(3 mg/mL collagen type I/III in 12 mM HCl; Biochrome) were used as described before.29 Further, we used fibrin-gels that were made from lyophilized human fibrinogen powder (F3789 Sigma Aldrich) dissolved in Tris-buffered saline with an addition of 3.75 mM CaCl2 and 3 IU/mL thrombin powder (T1063 Sigma Aldrich) dissolved in sterile water for crosslinking.30 Cells were cultured at 37C in six-well plates or T75 tissue culture flasks (Nunc Thermo Fisher Scientific). Elasticity measurements of HPL gels For elasticity measurements, HPL-gels were formed in 5%, 10%, and 20% concentrations using plastic cylinders (14 mm in diameter and 2 mm in height) as mold on top of thin cover slides. After gelatinization as described above, mold cylinders were removed and HPL-gels were overlaid with a cover slide of known mass. Gel thickness was determined using a confocal laser scanning microscope (LSM 510 Meta, Zeiss) equipped with a long distance 40 · Achroplan (NA 0.6, PH 2) objective. Placement of additional cover slides of known weight and subsequent thickness measurement were repeated at least twice for each HPL-gel cylinder. From the measured quantities of gel thickness (h), total mass of cover slides (m), thickness reduction due to loading (Dh), and gel cross-section (A), we calculated the elastic modules of the gel according to E = (m*g)/A*(h/DL), where g denotes the acceleration due to gravitation. Isolation of human MSCs from bone marrow MSCs were isolated from MNCs by plastic adherence. In brief, bone fragments from the caput femoris of patients undergoing femoral head prosthesis were flushed with phosphate-buffered saline (PBS). Subsequently, MNCs were isolated by Ficoll density gradient centrifugation (Biocoll Separating Solution, density 1.077g/L, Biochrom AG) and washed twice with PBS. At least 5 · 105 MNC were then resuspended in culture medium either on TCP, HPL-gel, or collagen-gels in six-well plates. Cells were cultured at 37C in a humidified atmosphere with 5% CO2. The first medium exchange was performed after 48 h to remove nonadherent cells. Thereafter, half-medium changes were performed twice per week and MSCs were further passaged as described below. Isolation of human dermal fibroblasts Fibroblasts were isolated from skin samples of patients undergoing cosmetic surgery as described in our previous work.31 Initially the cells were expanded in DMEM-LG supplemented with glutamine (PAA), penicillin/streptomycin (PAA) and 10% FCS (Biochrom). Cells at passage 3 to 5 were then transferred to HPL-culture conditions as described above. Scanning electron microscopy Human MSC/HPL-gel matrix were fixed in 3% glutaraldehyde for at least 1 h at room temperature, rinsed with PBS, and dehydrated by incubating consecutively in 30%, 50%, 70%, 90%, and 100% ethanol for 10 min at room temperature. HPL-gels were then critical point dried in liquid CO2 and sputter coated with a 30 nm-gold layer using an ion sputter coater (LEICA EM SCD 500). Samples were analyzed using an environmental scanning electron microscope at the
926 electron microscope facility, RWTH Aachen University (ESEM XL 30 FEG, FEI, Philips). Histosections—hematoxilin and eosin staining For histological analysis, HPL-gels with cultured cells at the surface were fixed in 3.7% formaldehyde for 24 h. The constructs were paraffin embedded, orientated on the perpendicular plane, cut with a rotating microtome at 2 mm thickness (Leica), and stained with hematoxilin and eosin according to routine histology protocols. Proliferation assay and vitality analysis
WALENDA ET AL. plasmin (stock solution 2.76 mg/mL; Enzyme Research Laboratories) for 18 h at 37C. To compare different cell propagation methods, MSCs were seeded in parallel on TCP and HPL gel (1000 cells/ well) and adhered overnight. Cells on TCP were then passaged with trypsin, whereas MSCs on HPL-gel were harvested by pipetting with or without additional incubation with trypsin/EDTA for 5 min, or with plasmin as described above. All cells were reseeded on TCP and after 1 day the number of adherent and nonadherent cells was estimated using the MTT assay. Immunophenotypic analysis
Cell proliferation was assessed via the methyl thiazolyl tetrazolium (MTT) assay as described previously.22 Briefly, MSC of passage 3–6 were seeded in 96-well plates (3000 cells/cm2) either on TCP, collagen-gel, fibrin-gel, or HPLgels in culture medium supplemented with 1%, 5%, 10%, or 20% HPL. After 7 days, cells were washed with PBS and incubated with 1 mM MTT (Sigma Aldrich) for 3.5 h at 37C. The excess solution was discarded and formazan crystals were resolved in 4 mM HCl in isopropanol (both from Roth). The absorbance was measured at 590 nm using a Tecan Infinite 200 plate reader (Tecan Trading). Each measurement included four technical replicas. Alternatively, proliferation was estimated by carboxyfluorescein diacetate N-succinimidyl ester (CFSE) staining as described before.32 Further, nuclear staining or live dead staining was performed with 4¢,6-Diamidin-2-phenylindol (DAPI; Molecular Probes) or with 5 mg/mL fluorescein diacetate (Sigma) and 0.5 mg/mL Propidium iodide (BD Pharmingen) respectively, after 7 days of culture on TCP or HPL-gels and analyzed with a Leica DM IL HC fluorescence microscope.
For characterization of MSCs we have used a panel of surface markers. MSCs were isolated and cultured for three passages either on TCP or on HPL-gel. To remove the HPLgel prior to flowcytometric analysis we have either seeded the cells for one additional passage on TCP or we have harvested the HPL-gel by pipetting and washed the cells once in culture medium. Each cell preparation was stained in parallel with the following monoclonal antibodies: mouse anti-human CD14-APC (clone M5E2), CD29-PE (clone MAR4), CD31-PE (clone WM59), CD34-APC (clone 8G12), CD45-APC (clone HI30), CD73-PE (clone AD2), CD90-APC (clone 5E10) (all BD Bioscience), and CD105-FITC (clone MAR-226; ImmunoTools). Analysis was performed using a FACS Canto II cytometer (BD Bioscience) and the collected data were analyzed with WinMDI 2.9 software (Windows Multiple Document Interface for Flow Cytometry). For statistical comparison of different measurements we have normalized the fluorescence intensities to the corresponding autofluorescence measurements.
Fibroblastoid colony-forming unit assay
In vitro differentiation
The fibroblastoid colony-forming unit (CFU-f) assay was performed by plating MNCs in a limiting dilution series in 96-well plates (330,000; 100,000; 33,000; 10,000; 3000; and 1000 cells per well; at least 24-wells per dilution step in each experiment) in parallel on both TCP and HPL-gel. Medium was exchanged after 48 h for the first time and then twice per week. After 14 days in culture, cells were washed twice with PBS and labeled with MTT. All wells with cell growth scored positive and the CFU-f frequency was determined by Poisson statistics (L-calc; Stem Cell Technologies).33
Freshly isolated MSCs were directly seeded on either HPLgels or TCP and expanded for three passages prior to in vitro differentiation. Differentiation toward osteogenic, adipogenic, and chondrogenic lineage was then induced as previously described.22,35 This differentiation of HPL-gel-MSCs was either performed on HPL-gel or upon transfer to TCP. Chondrogenic differentiation was performed in 3D pellet culture upon centrifugation at 400 g for 7 min.31 After 3 weeks, differentiation was analyzed by Alizarin Red stain (osteogenic differentiation), with green fluorescent dye BODIPY (4,4-difluoro-1,2,5,7,8-pentamethyl-4-bora-3a,4a-diazas-indacene; Molecular Probes; adipogenic differentiation), or with Alcian blue and nuclear fast red (for sections of chondrogenic differentiated samples) according to routine histology protocols and analyzed with a Leica DM IL HC fluorescence microscope (Leica). Further, adipogenic differentiation was quantified on TCP and HPL-gel by counterstaining all cells with DAPI and direct counting of nondifferentiated in relation to differentiated cells as described before (at least 300 cells per experiment).22,33
Passaging and long-term culture of MSCs MSCs on TCP were continuously passaged after reaching a confluency level of 80% using trypsin-ethylenediaminetetraacetic acid (EDTA) solution 0.25% (Sigma Aldrich). Cells on HPL-gel were harvested together with the gel by pipetting. Thereby, the gel-MSC mix was diluted with culture medium and then plated onto fresh HPL-gel-coated well plates. To determine the MSC cell number, a fraction of the MSC/HPL-gel mixture was seeded on TCP for 24 h to facilitate attachment to the plastic surface. The plastic-adherent cells were then trypsinized and the cell number was finally determined using Neubauer counting chamber (Brand). Cell population doublings per passage and cumulative population doublings were calculated as previously described.34 Alternatively, MSC/HPL-gel was incubated with 20 mg
Statistics Results are expressed as mean – standard deviation of independent experiments. To estimate the probability of differences we have adopted the paired two-sided Student’s t-test.
PLATELET LYSATE GEL PROVIDES A CELL CULTURE MATRIX Results HPL-gel provides 3D-scaffold for MSCs Translucent gels were formed within 30 to 60 min upon addition of HPL to culture medium in the absence of anticoagulants. The resulting HPL-gels had a very soft and viscous consistency. The substrate elasticity was extremely low and increased with higher percentages of HPL (5%
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HPL: 20 Pa; 10% HPL: 28 Pa; and 20% HPL: 32 Pa; Fig. 1A). These gels were over-layered with the same HPL-medium supplemented with heparin to prevent gelatinization of the liquid phase. When MSCs were seeded on HPL-gel, they grew at the interface between HPL-gel and HPL-medium without any contact to the plastic surface. Live/dead staining hardly revealed any nonvital cells after 7 days of culture. Histological analysis demonstrated multilayered
FIG. 1. Mesenchymal stromal cells (MSCs) grow at the interface of human platelet lysate (HPL)-gel and HPL-medium. HPL containing cell culture medium without anticoagulant was gelatinized in thin bottom cell culture wells at indicated concentrations. Each HPL concentration was analyzed for elasticity on two independent samples with one to three measurements per sample. The substrate elasticity of HPL-gel was very low and correlated with HPL-concentrations (A). Mean elasticity values are indicated as thin line. Then, MSCs were seeded on top with the same culture medium supplemented with heparin. Histological sections after 4 days of culture revealed multilayered cell growth at the interface of HPL-gel and HPLmedium (B). Scanning electron microscopy (SEM) demonstrated the reticular fibrin scaffold of HPL-gel (C). MSCs on tissue culture plastic (TCP) (D) and on HPL-gel (E) showed the typical spindle-shaped MSC-morphology. Color images available online at www.liebertpub.com/tec
928 growth at this interface between gel and medium. Cell densities were much higher in HPL-gel in comparison to two-dimensional growth on TCP (Fig. 1B). Scanning electron microscopy demonstrated the fibrin scaffold of HPLgel and supported the notion that MSCs covered this matrix densely in a multilayered manner (Fig. 1C–E). MSCs may secrete enzymes that degrade fibrin and therefore, we assessed the maintenance of a thin HPL-gel upon culture expansion: within the first 4 days hardly any degradation was observed, whereas after 8 days, some HPL-gels were partly degraded and cells got in contact with the plastic surface. This was particularly observed in larger culture wells whereas hardly any degradation occurred in multititer plates.
WALENDA ET AL. HPL-gel increases proliferation and CFU-f frequency Proliferation was analyzed by seeding plastic adherent MSCs in parallel on TCP, collagen scaffolds, fibrin-gel, and HPL-gel. Culture medium was supplemented with different HPL-concentrations (1%, 5%, 10%, and 20%) and the HPLconcentrations in gel and medium were adjusted correspondingly (n = 7). After 7 days, the MTT-assay revealed that proliferation of MSCs increased at higher HPL-concentrations and this is in line with previous observations.4,22 Notably, cell expansion was significantly higher on HPL-gel than on TCP, collagen-gel (Fig. 2A), or fibrin-gel (Fig. 2B). Similar results were observed with dermal fibroblasts (Fig. 2C). Additionally, we tested proliferation in the course of
FIG. 2. Proliferation rate and fibroblastoid colony forming units (CFU-f) frequency increase on HPL-gel. MSCs were seeded in 96-well plates on TCP, collagen-gel, or HPL-gel in culture medium supplemented with 1%, 5%, 10%, or 20% HPL. After 7 days, proliferation was assessed with the methyl thiazolyl tetrazolium (MTT) assay (please note that this assay provides a relative measure that may vary between experiments). Overall, cell growth was stimulated with higher concentrations of HPL, and it was significantly increased on HPL-gel in comparison to TCP and collagen-gel (A; n = 7). Further, proliferation of MSCs was higher on HPL-gel than on fibrin-gel (B; n = 3). A similar growth supportive effect of HPL-gel was also observed for dermal fibroblasts (C; n = 4). The Proliferation rate of MSCs was also analyzed in the course of culture expansion after 2, 5, 7, and 9 days in 10% HPL (D; n = 3). To quantify initial colony formation on either TCP or HPL-gel the cells were seeded in limiting dilution in 96-well plates. After 14 days, the wells were scored for colony formation using MTT assay and the CFU-f frequency was determined by Poisson statistics (E; n = 3) *p < 0.05; **p < 0.005; ***p < 0.0005.
PLATELET LYSATE GEL PROVIDES A CELL CULTURE MATRIX culture expansion after 2, 5, 7, and 9 days. The proliferative advantage on HPL-gel became more and more apparent— especially after 9 days when MSC were already confluent on TCP (Fig. 2D). It needs to be taken into account that the MTTassay is only an indirect indicator for the cell number by measuring the metabolic activity. However, same tendency was also observed when measuring the residual fluorescence upon staining with CFSE or counting of DAPI-stained nuclei. Thus, proliferation was greatly increased on HPL-gel. Subsequently, we compared initial outgrowth of MSCs by seeding mono-nuclear cells from human bone marrow in parallel on TCP and HPL-gel to determine the number of CFU-f.36 This analysis was performed with limiting-dilution assays in multiwell-format to exclude effects by proliferation or migratory activity. The average CFU-f frequency on TCP was 88.35 per 106 MNC. Interestingly, the CFU-f frequency was 2.24-fold higher on HPL-gel compared with TCP ( p = 0.02; n = 3; Fig. 2E). These results indicate that HPL-gel enhances outgrowth of MSCs. Tissue culture expansion on HPL-gel matrix Long-term culture and large-scale expansion of cells require serial passaging steps. This is usually performed by harvesting the cells with trypsin/EDTA by the time when the cells reach a certain level of confluency. HPL-gel is too fragile for such harvesting procedures from the gel-surface. Instead, the fibrin-matrix of HPL-gel was degraded by addition of plasmin to the liquid phase overnight—thereby, the fibrin scaffold of the HPL-gel was dissolved and the cells relocated on TCP, which then facilitated conventional passaging procedures. However, the semi-fluid consistency of HPL-gel enabled a more convenient way for passaging without intermediate contact of the cells to the plastic surface: the viscous HPL-gel was pipetted together with the cells and reseeded without separation of MSCs from their matrix (Fig. 3A). This method was used over four subsequent pas-
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sages and the expansion rates were similar as compared with the conventional method on TCP with trypsin (Fig. 3B). To evaluate the cell recovery rate after passaging we determined how many cells remained associated with nondissolved HPL-gel plugs using the MTT assay: about 1/3 of the cells remained detached with the pipetting procedure, whereas the majority of cells were capable of plastic adherent outgrowth. The percentage of outgrowing cells was slightly increased by additional treatment of trypsin that might disrupt cell–cell adhesion on the HPL-gel. Notably, cell activity was lower upon treatment with plasmin, which might be due to fact that long-term treatment with this peptidase is detrimental to MSC survival (Fig. 3C).37 Comparison of MSC preparations on TCP and HPL-gel Culture conditions may affect the composition of subpopulations and they have direct impact on cellular characteristics.38 For morphological comparison of culture-isolated MSC preparations on either TCP or on HPL-gel we have transferred both for one passage to the same conventional TCP conditions—no differences in plastic adherent growth or cellular morphology were observed. Immunophenotypic analysis of MSC on HPL-gel was either performed with cells that were directly harvested from HPL-gel or upon transferring the cells to TCP for one additional passage. All MSC preparations (on TCP, HPL-gel, or HPL-gel with transfer to TCP) revealed the same CD14 + , CD31 - , CD34 - , CD45 - , CD29 + , CD73 + , CD90 + , and CD105 + immunophenotype without any significant differences (n = 4; Fig. 4A). In vitro differentiation toward adipogenic and osteogenic lineages was initially compared on TCP to exclude potential effects of the different substrates—no differences were observed between MSCs that were isolated on TCP or on HPL-gel with transfer to TCP for one passage (n > 3; Fig. 4B, C). Further, adipogenic differentiation was directly induced on HPL-gel and there was no significant difference in the percentage of
FIG. 3. Culture expansion and passaging of MSCs in HPL-gel. Scheme of culture expansion on TCP or HPL-gel: MSCs on TCP were continuously passaged after reaching a confluency level of 80% with trypsin-ethylenediaminetetraacetic acid (EDTA). In parallel, cells on HPL-gel were harvested together with the gel by pipetting and reseeding without separation of MSCs from their matrix (A). Long-term culture of MSCs was performed in parallel with both methods over four consecutive passages. Cumulative population doublings (cPD) were calculated from the first passage onward. Representative data of three independent experiments are shown (B). For comparison of different passaging methods 1000 MSCs were cultured overnight on either TCP or HPL-gel. MSCs on TCP were then passaged with trypsin, whereas the cells on HPL-gel were propagated back to TCP by pipetting, additional treatment with trypsin/EDTA, or incubation with plasmin. After one further day, adherent and nonadherent cells were measured using the MTT-assay (C).
FIG. 4. Comparison of MSCs cultured on tissue culture plastic and HPL-gel. Cells were isolated and expanded for three passages on TCP, on HPL-gel, or on HPL-gel with transfer to TCP for one additional passage (A). Representative histograms are presented for each cell preparation. All MSCs revealed the same CD14 + , CD31 - , CD34 - , CD45 - , CD29 + , CD73 + , CD90 + , and CD105 + immunophenotype without significant differences in the mean signal intensity (n = 4; normalized to the corresponding autofluorescence). MSCs isolated on either TCP or HPL-gel were then induced toward adipogenic (B) or osteogenic (C) lineage on conventional TCP for 3 weeks, whereas chondrogenic differentiation was induced in pellet culture for 3 weeks (D). Differentiation was analyzed with the green fluorescent dye BODIPY, by Alizarin Red staining, or by Alcian blue staining, respectively. Representative data of three independent experiments are shown. Scale bar: 200 mm. Color images available online at www.liebertpub.com/tec 930
PLATELET LYSATE GEL PROVIDES A CELL CULTURE MATRIX cells with lipid droplets on TCP and HPL-gel (42% vs. 37%; n = 3). Osteogenic differentiation was also induced directly on HPL-gel without apparent differences between TCP and HPL-gel (n = 3). Chondrogenic differentiation was induced in pellet culture upon harvesting the cells directly from TCP or from HPL-gel—analysis of proteoglycan deposition with Alcian blue did not reveal significant differences (Fig. 4D). These results demonstrate that the modified culture procedures on HPL-gel facilitate isolation of cells with typical MSC characteristics. Discussion HPL gel provides a new matrix for culture expansion of MSCs. The main constituent of the gel is a fibrin scaffold, which normally polymerizes into a mesh-like clot during final stages of the hemostatic coagulation cascade. Fibrinogen derives from the plasma fraction of platelet units and the coagulation cascade is inhibited by chelation of calcium ions with citrate. Upon addition to DMEM-culture medium, the excess of calcium ions stimulates polymerization into a fibrin matrix. This matrix has many advantages: (1) it is naturally occurring—especially in areas of wound formation and regeneration; (2) it is biocompatible and biodegradable;39,40 (3) it is translucent facilitating microscopic observations; and (4) there is increasing interest in fibrin scaffolds for tissue engineering applications.41–43 Further, HPL-gel may be injected in combination with embedded cells in transplantation settings that might facilitate higher recovery and survival of cells than injection of cell pellets in aqueous solutions.44 Efficient expansion and high proliferation rates are important for many cell applications. We demonstrate that proliferation was higher on HPL-gel in comparison to culture on TCP, fibrin-gel, or collagen-gel. HPL has been demonstrated to be a very effective serum supplement, which further enhances proliferation of MSCs in comparison to FCS.4,13,14,17,19,22,24 The platelet fraction comprises relevant growth factors such as platelet-derived growth factors (PDGFs), which support this tremendous growth stimulation in comparison to human plasma or serum.14,45,46 The increased proliferation on HPL-gel might be due to the following reasons: (1) in HPL-gel, nutrients and growth factors are accessible to the cells from all directions and their supply is buffered by the underlying matrix;47 (2) cell growth is not strictly limited to two dimensions with contact inhibition upon confluent growth. Instead, the cells proliferate more homogeneously and reach higher cell densities on HPL-gel; and (3) HPL-gel might facilitate better adhesive interaction with specific adhesion proteins such as integrins.48 This might also contribute to the significantly enhanced outgrowth of CFU-f on HPL-gel. Taken together, HPL-gel strongly enhances expansion of MSCs—by a better recovery of initial colonies, faster proliferation rates, and higher cell densities in multiple layers. Propagation of cells over subsequent passages usually involves enzymatic treatment, for example, with trypsin or Accutase, to harvest the cells from the cell culture surface— after washing steps the cell suspension is then seeded on new culture dishes.49 This procedure has little impact on cell survival but it stalls them in their proliferative cycle. Another alternative for enzyme-free cell harvest is the use of thermoresponsive polymers that can release cells after a tem-
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perature shift.50 If required, HPL-gel can be dissolved by plasmin, which is a physiological peptidase although it has been shown that exogenous plasmin impairs survival of MSCs over time.37 More importantly, the very soft and viscous nature of HPL-gel facilitates an alternative procedure by pipetting cells together with their matrix on new culture plates. This was only possible due to the extremely soft nature of HPL-gel—conventional collagen and fibrin-gels are more rigid. Initially, we expected that cell recovery might be hindered as cells can be trapped in HPL-gel-fragments or they might be disrupted during the pipetting procedure. Despite some cell loss in the HPL-gel fragments, the expansion rates were in a similar range as with the conventional methods using trypsin on TCP—probably due to the largely increased proliferation rate. The passaging procedure without separating cells from their matrix has several advantages as washing steps and centrifugation are not required. This is of specific interest for improved protocols for cell culture automation. Automation approaches may help to serve the increasing and costly needs in cellular therapy in the future and a simple pipetting procedure is much easier to implement on such platforms. It is not a trivial task to determine precise cell numbers on HPL-gel. For most experiments, we used the MTT-assay that does not require separation of cells from the gel matrix. However, this assay provides only an indirect estimate for cell numbers by measuring the conversion of MTT into formazan. Alternatively, we counted DAPI-stained nuclei but reliable quantification was impaired by the localization of cells in different levels on the HPL-gel. Plasmin can be used to degrade the HPL-gels but this may compromise cell viability over time and correspondingly the MTT-signal was lower after plasmin treatment. Alternatively, we diluted the HPL-gel with culture medium to transfer these cells to conventional TCP surfaces. As described above, this passaging procedure worked efficiently and the majority of MSCs could transfer to the TCP surface—potentially by migration or thinning of HPL-gel with the pipetting procedure. However, about 1/3 of the cells remained nonadherent in floating HPL-fragments. This cell loss could be slightly but not significantly reduced by additional treatment with trypsin/ EDTA potentially due to disruption of cell–cell adhesion between MSCs on HPL-gel. Another important aspect is the stability of HPL-gel, which can be partially degraded by fibrinolytic enzymes secreted by MSCs. On the other hand, our study demonstrated that it is possible to use HPL-gel even for long-term culture over 3 weeks without addition of antifibrinolytic agents, although the stability might be enhanced by aprotinin or tranexamic acid.51 Standardization of protocols is an important issue in generation of therapeutic cell products. Serum supplements such as FCS and HPL notoriously reveal batch-to-batch variation. In our previous work, we demonstrated differences in HPL generated from individual platelet units22 and the stimulatory effect significantly declined with HPL-donor age.28 It has to be noted that we also observed some variation in the gelatinization process, which might result from the plasma-fraction of HPL. Further research will enable better control of gel-formation and standardization of HPL-gels and it will provide better insight in the HPL-donor associated variation. This variation may be leveled by pooling of several HPLs,20,24 whereas, it may be favorable to use single
932 donor-derived HPL-gel—potentially even from the transplant-recipient—to reduce the risk of infections with human pathogens. Cell growth and in vitro differentiation can be influenced by surface elasticity.52 HPL-gel revealed a substrate elasticity of about 30 Pa—this is extremely low and even below the estimated stiffness of adipose tissue. It might be anticipated that adipogenic differentiation is more pronounced on HPLgel than on TCP, but this effect did not become evident in our analysis. Overall, we did not detect clear effects on morphology, immunophenotype or differentiation potential, indicating that the composition of MSCs might be similar on HPL-gel and TCP. Either way, HPL-gel facilitates isolation of cells with typical MSC characteristics. In conclusion, HPL-gel is an extremely soft matrix providing various new perspectives for cell culture. It facilitates cell growth embedded in culture medium components without any contact to artificial biomaterials such as polystyrene. Further, this medium does not include xenogeneic components such as FCS.13,19 It is even conceivable to use HPL-gel in an autologous setting to further minimize the risk of immunological effects or infections. The initial outgrowth and the proliferation rate of MSCs were much higher on HPL-gel than on TCP. The mechanical consistency allows a simple passaging procedure without the need of separating cells from their matrix or additional washing steps—this convenient propagation procedure is of specific relevance for cell culture automation. HPL-gel may facilitate faster generation of high cell numbers in cellular therapy.
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Acknowledgments The authors would like to thank Manfred Bovi for excellent technical assistance in electron microscopy, Isabelle Leisten for help with chondrogenic differentiation (both RWTH Aachen University Medical School), and Nico Hampe (Forschungszentrum Ju¨lich) for intense help during HPL elasticity measurements. This work was supported by the German Ministry of Education and Research (BMBF) within the supporting program ‘‘cellbased regenerative medicine’’ (CB-HERMES), by the state North Rhine Westphalia within the BioNRW2 program (StemCellFactory) and the Stem Cell Network NRW.
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Disclosure Statement RWTH Aachen Medical School has applied for a patent for the method described in this article. There are no products in development or marketed products to declare. No competing financial interest exists.
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References
14.
1. Sensebe, L., Krampera, M., Schrezenmeier, H., Bourin, P., and Giordano, R. Mesenchymal stem cells for clinical application. Vox Sang 98, 93, 2009. 2. Dominici, M., Le Blanc, K., Mueller, I., Slaper-Cortenbach, I., Marini, F., Krause, D., Deans, R., Keating, A., Prockop, D., and Horwitz, E. Minimal criteria for defining multipotent mesenchymal stromal cells. The international society for cellular therapy position statement. Cytotherapy 8, 315, 2006. 3. Neuss, S., Apel, C., Buttler, P., Denecke, B., Dhanasingh, A., Ding, X., Grafahrend, D., Groger, A., Hemmrich, K., Herr,
15.
16.
A., Jahnen-Dechent, W., Mastitskaya, S., Perez-Bouza, A., Rosewick, S., Salber, J., Woltje, M., and Zenke, M. Assessment of stem cell/biomaterial combinations for stem cellbased tissue engineering. Biomaterials 29, 302, 2008. Cholewa, D., Stiehl, T., Schellenberg, A., Bokermann, G., Joussen S, Koch C, Walenda T, Pallua, N., Marciniak-Czochra, A., Suschek, C.V., and Wagner W. Expansion of adipose mesenchymal stromal cells is affected by human platelet lysate and plating density. Cell Transplant 20, 1409, 2011. Toh, W.S., Lim, T.C., Kurisawa, M., and Spector, M. Modulation of mesenchymal stem cell chondrogenesis in a tunable hyaluronic acid hydrogel microenvironment. Biomaterials 33, 3835, 2012. Zhang, J., Skardal, A., and Prestwich, G.D. Engineered extracellular matrices with cleavable crosslinkers for cell expansion and easy cell recovery. Biomaterials 29, 4521, 2008. Leisten I., Kramann S., Ventura Ferreira M.S., Ziegler P., Wagner W., Neuss S., Knu¨chel R., and Schneider R. Threedimensional co-culture of hematopoietic stem and progenitor cells and mesenchymal stem cells in collagen scaffolds: an in vitro model of the hematopoetic niche. Biomaterials 33, 1736, 2012. Wagner, W., Roderburg, C., Wein, F., Diehlmann, A., Frankhauser, M., Schubert, R., Eckstein, V., and Ho, A.D. Molecular and secretory profiles of human mesenchymal stromal cells and their abilities to maintain primitive hematopoietic progenitors. Stem Cells 10, 2638, 2007. Wagner, W., Feldmann, R.E., Jr., Seckinger, A., Maurer, M.H., Wein, F., Blake, J., Krause, U., Kalenka, A., Burgers, H.F., Saffrich, R., Wuchter, P., Kuschinsky, W., and Ho, A.D. The heterogeneity of human mesenchymal stem cell preparations-Evidence from simultaneous analysis of proteomes and transcriptomes. Exp Hematol 34, 536, 2006. Eagle, H. Nutrition needs of mammalian cells in tissue culture. Science 122, 501, 1955. Heiskanen, A., Satomaa, T., Tiitinen, S., Laitinen, A., Mannelin, S., Impola, U., Mikkola, M., Olsson, C., Miller-Podraza, H., Blomqvist, M., Olonen, A., Salo, H., Lehenkari, P., Tuuri, T., Otonkoski, T., Natunen, J., Saarinen, J., and Laine, J. N-glycolylneuraminic acid xenoantigen contamination of human embryonic and mesenchymal stem cells is substantially reversible. Stem Cells 25, 197, 2007. Sundin, M., Ringden, O., Sundberg, B., Nava, S., Gotherstrom, C., and Le, B.K. No alloantibodies against mesenchymal stromal cells, but presence of anti-fetal calf serum antibodies, after transplantation in allogeneic hematopoietic stem cell recipients. Haematologica 92, 1208, 2007. Schallmoser, K., Bartmann, C., Rohde, E., Reinisch, A., Kashofer, K., Stadelmeyer, E., Drexler, C., Lanzer, G., Linkesch, W., and Strunk, D. Human platelet lysate can replace fetal bovine serum for clinical-scale expansion of functional mesenchymal stromal cells. Transfusion 47, 1436, 2007. Bieback, K., Hecker, A., Kocaomer, A., Lannert, H., Schallmoser, K., Strunk, D., and Kluter, H. Human alternatives to fetal bovine serum for the expansion of mesenchymal stromal cells from bone marrow. Stem Cells 27, 2331, 2009. Carrancio, S., Lopez-Holgado, N., Sanchez-Guijo, F.M., Villaron, E., Barbado, V., Tabera, S., ez-Campelo, M., Blanco, J., San Miguel, J.F., and Del Canizo, M.C. Optimization of mesenchymal stem cell expansion procedures by cell separation and culture conditions modification. Exp Hematol 36, 1014, 2008. Avanzini, M.A., Bernardo, M.E., Cometa, A.M., Perotti, C., Zaffaroni, N., Novara, F., Visai, L., Moretta, A., Del, F.C.,
PLATELET LYSATE GEL PROVIDES A CELL CULTURE MATRIX
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27. 28.
Villa, R., Ball, L.M., Fibbe, W.E., Maccario, R., and Locatelli, F. Generation of mesenchymal stromal cells in the presence of platelet lysate: a phenotypic and functional comparison of umbilical cord blood- and bone marrow-derived progenitors. Haematologica 94, 1649, 2009. Doucet, C., Ernou, I., Zhang, Y., Llense, J.R., Begot, L., Holy, X., and Lataillade, J.J. Platelet lysates promote mesenchymal stem cell expansion: a safety substitute for animal serum in cell-based therapy applications. J Cell Physiol 205, 228, 2005. Holzwarth, C., Vaegler, M., Gieseke, F., Pfister, S.M., Handgretinger, R., Kerst, G., and Muller, I. Low physiologic oxygen tensions reduce proliferation and differentiation of human multipotent mesenchymal stromal cells. BMC Cell Biol 11, 11, 2010. Muller, I., Kordowich, S., Holzwarth, C., Spano, C., Isensee, G., Staiber, A., Viebahn, S., Gieseke, F., Langer, H., Gawaz, M.P., Horwitz, E.M., Conte, P., Handgretinger, R., and Dominici, M. Animal serum-free culture conditions for isolation and expansion of multipotent mesenchymal stromal cells from human BM. Cytotherapy 8, 437, 2006. Lange, C., Cakiroglu, F., Spiess, A.N., Cappallo-Obermann, H., Dierlamm, J., and Zander, A.R. Accelerated and safe expansion of human mesenchymal stromal cells in animal serum-free medium for transplantation and regenerative medicine. J Cell Physiol 213, 18, 2007. Perez-Ilzarbe, M., ez-Campelo, M., Aranda, P., Tabera, S., Lopez, T., del, C.C., Merino, J., Moreno, C., Andreu, E.J., Prosper, F., and Perez-Simon, J.A. Comparison of ex vivo expansion culture conditions of mesenchymal stem cells for human cell therapy. Transfusion 49, 1901, 2009. Horn, P., Bokermann, G., Cholewa, D., Bork, S., Walenda, T., Koch, C., Drescher, W., Hutschenreuther, G., Zenke, M., Ho, A., and Wagner W. Comparison of individual platelet lysates for isolation of human mesenchymal stromal cells. Cytotherapy 12, 888, 2010. Schallmoser K., and Strunk D. Preparation of pooled human platelet lysate (pHPL) as an efficient supplement for animal serum-free human stem cell cultures. J Vis Exp 30, 1523, 2009. Schallmoser, K., Rohde, E., Reinisch, A., Bartmann, C., Thaler, D., Drexler, C., Obenauf, A.C., Lanzer, G., Linkesch, W., and Strunk, D. Rapid large-scale expansion of functional mesenchymal stem cells from unmanipulated bone marrow without animal serum. Tissue Eng Part C Methods 14, 185, 2008. Bonin, M., Stolzel, F., Goedecke, A., Richter, K., Wuschek, N., Holig, K., Platzbecker, U., Illmer, T., Schaich, M., Schetelig, J., Kiani, A., Ordemann, R., Ehninger, G., Schmitz, M., and Bornhauser, M. Treatment of refractory acute GVHD with third-party MSC expanded in platelet lysate-containing medium. Bone Marrow Transplant 43, 245, 2009. Lucchini, G., Introna, M., Dander, E., Rovelli, A., Balduzzi, A., Bonanomi, S., Salvade, A., Capelli, C., Belotti, D., Gaipa, G., Perseghin, P., Vinci, P., Lanino, E., Chiusolo, P., Orofino, M.G., Marktel, S., Golay, J., Rambaldi, A., Biondi, A., D’Amico, G., and Biagi, E. Platelet-lysate-expanded mesenchymal stromal cells as a salvage therapy for severe resistant graft-versus-host disease in a pediatric population. Biol Blood Marrow Transplant 16, 1293, 2010. Harrison, P., and Cramer, E.M. Platelet alpha-granules. Blood Rev 7, 52, 1993. Lohmann, M., Walenda, G., Hemeda, H., Joussen, S., Drescher, W., Jockenhoevel, S., Hutschenreuter, G., Zenke, M., and Wagner, W. Donor age of human platelet lysate affects
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
933
proliferation and differentiation of mesenchymal stem cells. PLoS One 2012. Schneider, R.K., Puellen, A., Kramann, R., Raupach, K., Bornemann, J., Knuechel, R., Perez-Bouza, A., and Neuss, S. The osteogenic differentiation of adult bone marrow and perinatal umbilical mesenchymal stem cells and matrix remodelling in three-dimensional collagen scaffolds. Biomaterials 31, 467, 2010. Koroleva, A., Gittard, S., Schlie, S., Deiwick, A., Jockenhoevel, S., and Chichkov, B. Fabrication of fibrin scaffolds with controlled microscale architecture by a two-photon polymerization-micromolding technique. Biofabrication 4, 015001, 2012. Koch, C., Suschek, C.V., Lin, Q., Bork, S., Goergens, M., Joussen, S., Pallua, N., Ho, A.D., Zenke, M., and Wagner, W. Specific age-associated DNA methylation changes in human dermal fibroblasts. PLoS One 6, e16679, 2011. Walenda, T., Bork, S., Horn, P., Wein, F., Saffrich, R., Diehlmann, A., Eckstein, V., Ho, A.D., and Wagner, W. CoCulture with mesenchymal stromal cells increases proliferation and maintenance of hematopoietic progenitor cells. J Cell Mol Med 14, 337, 2010. Schellenberg, A., Stiehl, T., Horn, P., Joussen, S., Pallua, N., Ho, A., and Wagner W. Population dynamics of mesenchymal stromal cells during culture expansion. Cytotherapy 14, 401, 2012. Wagner, W., Horn, P., Castoldi, M., Diehlmann, A., Bork, S., Saffrich, R., Benes, V., Blake, J., Pfister, S., Eckstein, V., and Ho, A.D. Replicative senescence of mesenchymal stem cells a continuous and organized process. PLoS One 5, e2213, 2008. Pittenger, M.F., Mackay, A.M., Beck, S.C., Jaiswal, R.K., Douglas, R., Mosca, J.D., Moorman, M.A., Simonetti, D.W., Craig, S., and Marshak, D.R. Multilineage potential of adult human mesenchymal stem cells. Science 284, 143, 1999. Friedenstein, A.J., Petrakova, K.V., Kurolesova, A.I., and Frolova, G.P. Heterotopic of bone marrow. Analysis of precursor cells for osteogenic and hematopoietic tissues. Transplantation 6, 230, 1968. Copland, I.B., Lord-Dufour, S., Cuerquis, J., Coutu, D.L., Annabi, B., Wang, E., and Galipeau, J. Improved autograft survival of mesenchymal stromal cells by plasminogen activator inhibitor 1 inhibition. Stem Cells 27, 467, 2009. Wagner, W., and Ho, A.D. Mesenchymal stem cell preparations-comparing apples and oranges. Stem Cell Rev 3, 239, 2007. Bensaid, W., Triffitt, J.T., Blanchat, C., Oudina, K., Sedel, L., and Petite, H. A biodegradable fibrin scaffold for mesenchymal stem cell transplantation. Biomaterials 24, 2497, 2003. Jockenhoevel, S., Zund, G., Hoerstrup, S.P., Chalabi, K., Sachweh, J.S., Demircan, L., Messmer, B.J., and Turina, M. Fibrin gel—advantages of a new scaffold in cardiovascular tissue engineering. Eur J Cardiothorac Surg 19, 424, 2001. Shaikh, F.M., Callanan, A., Kavanagh, E.G., Burke, P.E., Grace, P.A., and McGloughlin, T.M. Fibrin: a natural biodegradable scaffold in vascular tissue engineering. Cells Tissues Organs 188, 333, 2008. Lucarelli, E., Beretta, R., Dozza, B., Tazzari, P.L., O’Connel, S.M., Ricci, F., Pierini, M., Squarzoni, S., Pagliaro, P.P., Oprita, E.I., and Donati, D. A recently developed bifacial platelet-rich fibrin matrix. Eur Cell Mater 20, 13, 2010. Koch, S., Flanagan, T.C., Sachweh, J.S., Tanios, F., Schnoering, H., Deichmann, T., Ella, V., Kellomaki, M., Gronloh, N.,
934
44.
45.
46.
47.
48.
WALENDA ET AL. Gries, T., Tolba, R., Schmitz-Rode, T., and Jockenhoevel, S. Fibrin-polylactide-based tissue-engineered vascular graft in the arterial circulation. Biomaterials 31, 4731, 2010. Dozza, B., Di, B.C., Lucarelli, E., Giavaresi, G., Fini, M., Tazzari, P.L., Giannini, S., and Donati, D. Mesenchymal stem cells and platelet lysate in fibrin or collagen scaffold promote non-cemented hip prosthesis integration. J Orthop Res 29, 961, 2011. Coppinger, J.A., Cagney, G., Toomey, S., Kislinger, T., Belton, O., McRedmond, J.P., Cahill, D.J., Emili, A., Fitzgerald, D.J., and Maguire, P.B. Characterization of the proteins released from activated platelets leads to localization of novel platelet proteins in human atherosclerotic lesions. Blood 103, 2096, 2004. Kocaoemer, A., Kern, S., Kluter, H., and Bieback, K. Human AB serum and thrombin-activated platelet-rich plasma are suitable alternatives to fetal calf serum for the expansion of mesenchymal stem cells from adipose tissue. Stem Cells 25, 1270, 2007. Wirz, S., Dietrich, M., Flanagan, T.C., Bokermann, G., Wagner, W., Schmitz-Rode, T., and Jockenhoevel, S. Influence of platelet-derived growth factor-AB on tissue development in autologous platelet-rich plasma gels. Tissue Eng Part A 17, 1891, 2011. Gorodetsky, R., Vexler, A., Shamir, M., An, J., Levdansky, L., Shimeliovich, I., and Marx, G. New cell attachment peptide sequences from conserved epitopes in the carboxy termini of fibrinogen. Exp Cell Res 287, 116, 2003.
49. Weber, C., Pohl, S., Portner, R., Wallrapp, C., Kassem, M., Geigle, P., and Czermak, P. Expansion and Harvesting of hMSC-TERT. Open Biomed Eng J 1, 38, 2007. 50. Takahashi, H., Nakayama, M., Yamato, M., and Okano, T. Controlled chain length and graft density of thermoresponsive polymer brushes for optimizing cell sheet harvest. Biomacromolecules 11, 1991, 2010. 51. Cholewinski, E., Dietrich, M., Flanagan, T.C., Schmitz-Rode, T., and Jockenhoevel, S. Tranexamic acid—an alternative to aprotinin in fibrin-based cardiovascular tissue engineering. Tissue Eng Part A 15, 3645, 2009. 52. Engler, A.J., Sen, S., Sweeney, H.L., and Discher, D.E. Matrix elasticity directs stem cell lineage specification. Cell 126, 677, 2006.
Address correspondence to: Wolfgang Wagner, M.D., Ph.D. Helmholtz-Institute for Biomedical Engineering RWTH Aachen University Medical School Pauwelsstrasse 20 52074 Aachen Germany E-mail:
[email protected] Received: September 26, 2011 Accepted: May 22, 2012 Online Publication Date: July 16, 2012
