Article Cite This: ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Transferable Matrixes Produced from Decellularized Extracellular Matrix Promote Proliferation and Osteogenic Differentiation of Mesenchymal Stem Cells and Facilitate Scale-Up Gina D. Kusuma,†,‡ Michael C. Yang,†,‡ Shaun P. Brennecke,†,§ Andrea J. O’Connor,‡ Bill Kalionis,*,†,§ and Daniel E. Heath*,‡ †
Pregnancy Research Centre, Department of Maternal-Fetal Medicine, Royal Women’s Hospital, 20 Flemington Road, Parkville, Victoria 3052, Australia ‡ School of Chemical and Biomedical Engineering, Particulate Fluids Processing Centre, The University of Melbourne, Parkville, Victoria 3052, Australia § Department of Obstetrics and Gynaecology, Royal Women’s Hospital, The University of Melbourne, Parkville, Victoria 3052, Australia ABSTRACT: Decellularized extracellular matrixes (dECM) derived from mesenchymal stem cell (MSC) cultures have recently emerged as cell culture substrates that improve the proliferation, differentiation, and maintenance of MSC phenotype during ex vivo expansion. These biomaterials have considerable potential in the fields of stem cell biology, tissue engineering, and regenerative medicine. Processing the dECMs into concentrated solutions of biomolecules that enable the useful properties of the native dECM to be transferred to a new surface via a simple adsorption step would greatly increase the usefulness and impact of this technology. The development of such solutions, hereafter referred to as transferable matrixes, is the focus of this article. In this work, we produced transferable matrixes from dECM derived from two human placental MSC cell lines (DMSC23 and CMSC29) using pepsin digestion (PECM), urea extraction (U-ECM), and mechanical homogenization in acetic acid (AA-ECM). Native dECMs improved primary DMSC proliferation as well as osteogenic and adipogenic differentiation, compared with traditional expansion procedures. Interestingly, tissue culture plastic coated with P-ECM was able to replicate the proliferative effects of native dECM, while UECM was able to replicate osteogenic differentiation. These data illustrate the feasibility of producing dECM-derived transferable matrixes that replicate key features of the native matrixes and show that different processing techniques produce transferable matrixes with varying bioactivities. Additionally, these transferable matrixes are able to coat 1.3−5.2 times the surface area covered by the native dECM, facilitating scale-up of this technology. KEYWORDS: cell secreted matrix, solubilization, decellularized extracellular matrixes, mesenchymal stem cells (MSC), stem cell expansion
1. INTRODUCTION
Most studies have focused on the decellularization of whole tissues.11,22−27 Nevertheless, cells deposit extracellular matrix during in vitro culture, and these cultured cells can be decellularized to produce a biologically complex dECM layer, which coats the tissue culture plate. Recently, dECM produced by cultured mesenchymal stem cells (MSC) has emerged as a growth surface that promotes MSC proliferation, delays the onset of cellular senescence, enhances multilineage differentiation capacity, and maintains MSC-specific phenotype, when compared with traditional cell culture surfaces including tissue culture plastic, monolayers of adsorbed collagen or fibronectin, control matrixes produced by murine or human
Decellularized extracellular matrixes (dECMs) are a rapidly emerging class of materials with applications in the fields of biomaterials, tissue engineering, and regenerative medicine.1−10 These materials are produced from tissue explants or cell cultures that are exposed to chemical/physical processes to remove cells and cellular components while leaving behind a biologically complex extracellular matrix.11−13 Recent advances in biomaterials science have produced synthetic systems (e.g., tunable hydrogels, electrospun polymers, and adhesive ligand patterning) that enable control of a wide variety of cell behaviors, including adhesion, migration, proliferation, and cell fate.14−17 However, cells cultured on dECMs exhibit various desirable behaviors that cannot currently be replicated with synthetic or fully defined, artificial substrates.18−21 © XXXX American Chemical Society
Received: October 7, 2017 Accepted: April 11, 2018 Published: April 11, 2018 A
DOI: 10.1021/acsbiomaterials.7b00747 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Article
ACS Biomaterials Science & Engineering fibroblasts, and even Matrigel.7,18,28−32 These results allude to the potential impact of MSC-derived dECM in the fields of cell culture, biomaterials, tissue engineering, and regenerative medicine. Current technology produces dECMs on two-dimensional tissue culture plates, and production of these dECMs is labor intensive. Developing technologies that facilitate the transfer of dECM-derived matrixes to a new substrate, while retaining the desired bioactivity, would significantly increase the practical application of these materials. Such technologies would aid in the scale-up of this technology by allowing the generation of larger cell culture surface that possesses the desired bioactive properties of the native dECM. In this study, we process the dECM to produce a concentrated biomolecule solution that can be used to coat new surfaces and transfer the bioactivity of the native dECM to this surface. Hereafter, we call these solutions transferable matrixes. These transferable matrixes may be able to coat biomaterials/tissue engineering scaffolds with tailored mechanics and 3D architectures, which may improve performance. We use several different solubilization/dispersion techniques to create transferable matrixes that can coat plastic surfaces and maintain important, beneficial properties of the native matrix. Additionally, we provide evidence that particular solubilization/dispersion techniques generate transferable matrixes that elicit selective cell behavior responses from MSC. Placental MSC were chosen for this work because they can be readily isolated with high yield and purity from term human placentae: a source that is readily available, abundant, obtained by noninvasive means, and is unencumbered by significant ethical and moral concerns.33−38 Due to their immuneprivileged/evasive nature, placental MSC could be used in allogeneic cell therapy.33,39 In this work, we utilized two welldefined placenta-derived MSC types: fetal MSC isolated from the chorionic villi (CMSC) and maternal MSC isolated from the decidua basalis (DMSC).40−42 Further, we previously developed stable human telomerase (hTERT)-transduced cell lines from these sources, CMSC29 and DMSC23, respectively, which maintain many important stem cell functions. CMSC29 and DMSC23 were used to generate dECMs, as previously described.43,44 We explored three different solubilization/dispersion treatments to generate transferable matrixes: extraction with urea, digestion with pepsin, and mechanical homogenization in acetic acid. These solutions/suspensions were then used to coat tissue culture plastic (TCP), and their ability to promote cell attachment, proliferation, and multilineage differentiation of DMSC was assessed.
DMSC23 and CMSC29 cell lines were created by hTERT transduction from primary DMSC and chorionic villous MSC as previously described.43 CMSC29 cells were maintained in culture using Amniomax C100 medium (Life Technologies), whereas DMSC23 cells were maintained using the Mesencult Proliferation Kit (Stem Cell Technologies, Vancouver, Canada). Adult human dermal fibroblasts (HDF) were purchased from Lonza and cultured in FGM Bullet Kit supplemented medium according to the manufacturer’s instructions. All cultured cells were maintained in a humidified incubator at 37 °C with 5% CO2. All experiments were performed with DMSC and HDF up to passage 5, while the DMSC23 and CMSC29 cell lines were used up to passage 25. 2.2. Decellularization of MSC Cultures. Decellularized ECM was prepared from DMSC23, CMSC29, and HDF cells as we described previously.29,44 Briefly, cells were cultured in their respective growth medium. All media were supplemented with 50 μM ascorbic acid (Wako, Japan) to increase ECM production, and media were changed twice a week for up to 2 weeks. After washing with PBS, cells were lysed with 0.5% Triton X-100 (Thermo Scientific) solution containing 20 mM NH4OH (Sigma-Aldrich) for 5 min. Native dECMs were washed with PBS, air-dried in the sterile biosafety cabinet, and stored at 4 °C for up to 1 month. dECMs deposited by DMSC23 and CMSC29 cells were named dECM-DMSC23 and dECM-CMSC29, respectively. HDF-coated plates were prepared as fibroblast control and named dECM-HDF. In subsequent cell culture experiments, cells were seeded directly onto the dECM-covered well plates. DNase treatment was not used, as it resulted in dECM detachment from the tissue culture plate. However, our previous research showed a high degree of DNA removal using this method.44 2.3. Production of Transferable Matrixes. To prepare the transferable matrixes, dECMs were prepared from cultured cells in their respective growth medium supplemented with 50 μM ascorbic acid for up to 2 weeks. Cells were washed with PBS and subsequently lysed with 0.5% Triton X-100 solution containing 20 mM NH4OH for 5 min. dECMs were collected by scraping and were subsequently treated with 100 unit/mL DNase I (Worthington, NJ, United States) for 1 h to remove DNA contamination. Three solubilization/ dispersion methods were used as follows. Pepsin Digestion. dECM was digested with 0.1% pepsin (w/v) in 0.01 N HCl at 37 °C until the pellet was dissolved (4 mL per ECM pellet collected from a 175 cm2 flask). The final solution was neutralized using 0.1 M NaOH to pH 7.4 (named P-ECM). Urea Extraction. dECM was extracted with 2 M urea in 150 mM NaCl solution at 4 °C for 2 days (2.5 mL per ECM pellet collected from a 175 cm2 flask). The insoluble pellet was removed by centrifugation at 2000 rpm for 5 min, and the solution was named U-ECM. Both P-ECM and U-ECM were dialyzed against PBS and concentrated via ultrafiltration using Amicon centrifugal filters (3 kDa MW limit, Millipore). Homogenization in Acetic Acid. dECM was scraped into a solution of 0.02 N acetic acid, transferred to microcentrifuge tubes with metal beads (Genework Pty Ltd.), and sonicated (Mo Bio Powerlyzer 24) 10 times with 35 s pulses at 2500 rpm to mechanically homogenize the dECM contents. The final suspension was named AA-ECM. Protein yield was determined by Coomassie protein assay (Thermo Scientific) performed on each sample according to the manufacturer’s instructions. 2.4. Coating of Tissue Culture Plastic Surfaces with Transferable Matrixes. Tissue culture plastic was coated with PECM, U-ECM, or AA-ECM (25 μg/mL or equivalent to 5 μg/cm2, unless otherwise specified) by pipetting onto the plastic surfaces of tissue culture plates and then incubating the plate for 1 h at 37 °C. PECM/U-ECM/AA-ECM-coated surfaces were rinsed with PBS prior to cell seeding to remove nonadsorbed proteins or particulates of ECM. 2.5. SDS-PAGE Analysis. dECM extracts were prepared from native dECMs and transferable dECMs. Protein concentrations of each extract were measured using the Coomassie protein assay according to the manufacturer’s protocol, with bovine serum albumin
2. MATERIALS AND METHODS 2.1. MSC Isolation and Culture. Human term placentae from uncomplicated pregnancies were collected following elective Caesarean section or normal vaginal delivery. Placenta tissue was obtained with written patient consent and the study was approved by the Human Research and Ethics Committee of the Royal Women’s Hospital, Victoria. DMSC were isolated as previously described.41,42 DMSC were cultured in α-MEM medium (Sigma-Aldrich, MO, United States) with 10% fetal bovine serum (Thermo Scientific, CA, United States), 100 U/mL penicillin, 100 mg/mL streptomycin, and 2 mM L-glutamine. Medium was changed every 3−4 days, and once the cells reached 70−80% confluence, cells were detached with TrypLE Express solution (Life Technologies, CA, United States) and passaged. MSC populations isolated from individual patients were routinely tested for osteogenic, adipogenic, and chondrogenic differentiation potential as described previously.40,42,44 B
DOI: 10.1021/acsbiomaterials.7b00747 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
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ACS Biomaterials Science & Engineering
Figure 1. (A) Schematic representation of production of cell culture surface coated with native or transferable dECMs. (B) Protein concentrations in transferable dECMs. Statistical significance was determined by unpaired t test, data are presented as mean ± SEM, *p < 0.05 and **p < 0.01. (C) SDS-PAGE of native and transferable dECMs: Lane 1, DMSC23 P-ECM; lane 2, DMSC23 U-ECM; lane 3, DMSC23 AA-ECM; lane 4, DMSC23 native dECM; lane 5, CMSC29 P-ECM; lane 6, CMSC29 U-ECM; lane 7, CMSC29 AA-ECM; lane 8, CMSC29 native dECM; lane 9, protein standards. as the standard. The protein extracts were loaded into a 1.0 mm 4− 12% gradient Bis-Tris Criterion XT Precast Gel (Bio-Rad) along with Precision Plus protein standard. SDS-PAGE was performed at 150 V for at least 100 min using Bio-Rad XT MOPS buffer. The gel was stained with BioSafe Coomassie (Bio-Rad) overnight and destained in deionized H2O for 1 h. Visualization of the bands was carried out on a GE Image Scanner III densitometer, and the image was taken using ImageQuant TL software (GE Healthcare). 2.6. MSC Spreading and Morphology. After 72 h of culture, immunofluorescence of the cytoskeleton and nuclei was assessed using TRITC-conjugated Phalloidin (Millipore, 1:500 dilution) for 1 h and DAPI (Millipore, 1:2000 dilution) for 5 min. All steps were carried out at room temperature. An Olympus IX81 microscope was used for fluorescence imaging, and the resulting multicolor images were composited using Cell R Software (Olympus). The cell spreading areas were analyzed using ImageJ software (National Institute of Health, Bethesda, MD). 2.7. MSC Viability. To determine cell viability, the enzymatic activity of the adherent cells was measured using Cell Counting Kit-8 (CCK-8, Sigma-Aldrich). Increased enzymatic activity leads to an increase in the amount of formazan dye, which is measured with a spectrophotometer. DMSC were seeded onto dECM-coated 96-well plates at a density of 1 × 103 cells/cm2. After a 6 day incubation period, the cell culture medium was aspirated and fresh medium was added along with CCK-8 reagent (10:1 ratio). Plates were then incubated for 3 h at 37 °C. After incubation, 100 μL of solution from
each well was transferred into a new 96-well plate, and the absorbance was measured at 450 nm with a SpectraMax Plus microplate reader (Molecular Devices). Background signal was determined from wells containing media alone, incubated with CCK-8 reagent. 2.8. Osteogenic Differentiation. DMSC were seeded onto dECM-coated surfaces at a density of 3 × 103 cells/cm2. Mesencult osteogenic medium (Stem Cell Technologies) was added on the following day. After a 14 day induction period, cells were washed with PBS, fixed with 4% paraformaldehyde, and incubated with the fluorescent OsteoImage staining reagent (Lonza, Basel, Switzerland) as per the manufacturer’s instructions. Fluorescence detection was performed at 492/520 nm excitation/emission wavelengths on a FluoStar Optima plate reader (BMG Labtech, Offenburg, Germany). Alternatively, DMSC were seeded onto dECM-coated wells of 8well Permanox chamber slides (Thermo Scientific). After a 14 day induction period, medium was aspirated from each well, and cells were fixed in 10% formalin. Cells were stained with Alizarin Red solution for 30 min to detect calcium deposits. Slides were visualized using Axioskop 2 (Carl Zeiss) with bright field optics, and images were captured on a Nikon DXM 1200C camera. 2.9. Adipogenic Differentiation. Adipogenic differentiation was carried out using adipogenic induction medium consisting of α-MEM complete medium with the addition of adipogenic stimulatory supplements (containing hydrocortisone, isobutylmethylxanthine, and indomethacin in 95% ethanol, R&D Systems). Briefly, 1 × 104 cells were plated in each well of an 8-well chamber slide (Ibidi) in C
DOI: 10.1021/acsbiomaterials.7b00747 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Article
ACS Biomaterials Science & Engineering adipogenic medium and incubated for 21 days at 37 °C with 5% CO2. After the induction period, medium was aspirated from the chamber slides, and cells were fixed in 4% formalin. Cells were then stained with Oil Red O solution (Sigma-Aldrich) for 5 min. Lipid droplets were visualized under an Olympus IX81 microscope with DIC optics to obtain optical contrast of the cell outline. Images were captured using an Axiocam HRC (Carl Zeiss) camera. For quantification of triglyceride accumulation, the deposited Oil Red O droplets were solubilized by treating the cells with 100% isopropanol for 10 min. The optical density of the eluted sample was measured at 520 nm with a SpectraMax Plus microplate reader (Molecular Devices). 2.10. Statistical Analysis. All experiments were conducted at least 3 times (n ≥ 3) unless otherwise stated. All quantitative data are presented as mean ± standard error of the mean. One-way ANOVA with Tukey’s multiple comparison tests was performed using GraphPad Prism software. A p-value of
