فایل ورد کامل حوزه سلول های بنیادی جنینی: یک روش کنترل شده برای تولید انبوه سلول های بنیادین جنینی موش برای تمایزیابی

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تعداد صفحات این فایل: ۱۸ صفحه

تحقیقات  اخیر استفاده قابل توجه از  آلژینات را در کشت و تمایز ESCs  های انسانی علاوه بر  واسکلوژنز (۲۰) و در تشکیل کندروسیت ها،  بافت های غضروفی ( ۲۱-۲۴)، سلول های بنیادین مزونشیمی ( ۲۵-۲۷)، هپاتوسیت ها( ۲۸-۳۰) بافت پاراتیروییدی(۳۱) و جزایر لانگرهانس (۳۲-۳۳) گزارش کرده اند. هم چنین اثبات شده است که بعد از انکپسوله سازی آلژینات، سلول ها فنوتیپ خود را حفظ کرده و تمایز سلول های بنیادین را افزایش داده و  عملکرد سلول های کشت شده بهبود پیدا کرده و از رد سلول جلوگیری می کند(۳۲). به علاوه، آلژینات یک ماتریکس جالب برای بی تحرک سازی و یا کپسوله سازی پروتین هاست.  مزیت اصلی آن ها این است که فرایند  ژلاسیون آلژینات  تنها  تحت شرایط متوسط رخ می دهد که بدون استفاده از دماهای بالا و یا   مواد شیمیایی است(۸). آلژینات برای کپسوله سازی فاکتور های رشد آنژیوژنی نظیر فاکتور رشد فیبروبلاست (bFGF) (34) و فاکتور رشد  اندوتلیال وریدی (VEGF) (35-37) برای درمان ایسکمی مورد استفاده قرار گرفته است. آلژینات دارای خصوصیات چسبندگی زیستی بوده و می تواند در حفاظت از غشاهای موکوزی دستگاه گوارشی کارامد باشد(۳۸). اخیرا مطالعات به توصیف  تولید سیستم های کشت کنترل شده با استفاده از  انکپسوله سازی  آلژینات polyL-lysine (PLL) ( 39-40) پرداخته اند.

عنوان انگلیسی:Embryonic stem cell sphere: A controlled method for production of mouse embryonic stem cell aggregates for differentiation~~en~~

INTRODUCTION Mouse embryonic stem cells (ESCs) are clonal cell lines derived from the preimplantation embryos (1). They are able to differentiate to virtually all cell and tissue types in vivo as well as in vitro (2). The basic strategy for in vivo differentiation of ESCs is the formation of multicellular aggregates called embryoid bodies (EBs). EBs can be generated by using the hanging drop method, by culturing ESC colonies detached from cultures grown without a feeder cell layer, or by the spontaneous aggregation of ESC in suspension culture. These methods have some limitations, including (i) limited production of EBs, (ii) delivery of EBs with a large variation in size and differentiation state, and (iii) the need for frequent passages. Moreover, frequent passaging conditions are associated with a heightened risk of contamination and, most importantly, may lead to uncontrolled cell-differentiation responses due to variation in the cellular microenvironment (3, 4). To facilitate the investigation and exploitation of ESC-derived cells in research, a scaling up of cell production and optimization of culture conditions are necessary (5). Microencapsulation of animal cells into hydrogels has been used with a large number of different cell types (6-8). Hydrogels are cross-linked hydrophilic polymers that contain large amounts of water without dissolution. They should allow nutrients and metabolic wastes to diffuse through them, so that the encapsulated cells remain viable and functional while also allowing the molecules produced by the encapsulated cells to diffuse out to the environment. The naturally derived polymers (macromolecules) most frequently used as hydrogel scaffolds in tissue engineering are alginate, hyaluronate, collagen, and their derivatives (9). Alginates are a family of linear natural copolymers of 1,4- linked -D-mannuronic acid (M) and -L-glucuronic acid (G) of varying compositional and sequential structures (10, 11). They have several unique properties for cell microencapsulation, including a relatively inert aqueous environment within the matrix, a mild room temperature encapsulation process free of organic solvents (12), a high gel porosity which allows for high diffusion rates of macromolecules, and the ability to control this porosity with simple coating procedures (13). Immobilization of ESCs into alginate microbeads in an undifferentiating condition is assumed to give rise to ESC spheres, which offers significant advantages over the hanging drop method and the spontaneous aggregation of ESCs. The immobilization process can be optimized to (i) provide continuous mass production of undifferentiated ESC aggregates for differentiation and (ii) deliver ESC aggregates with a narrow distribution in size and differentiation state without the need for frequent passages. Here we describe a large-scale production of ESC spheres by immobilization of ESCs into alginate microbeads in an undifferentiating condition for tissue-engineering approaches. Microencapsulated ESCs can grow as compact colonies within the beads with the ability to differentiate. Alginate microbeads might be simple bioreactors which can lead to mass production of ESC spheres without the need for frequent passages for differentiation. MATERIALS AND METHODS Culture of undifferentiated ESCs The ESC line Royan B1 (14) derived from the C57BL/6 mouse strain was used throughout the present study. ES cells were kept in an undifferentiated, pluripotent state using a mitomycin C–inactivated feeder layer (M0503; Sigma) of primary cultures of mouse embryonic fibroblasts (MEFs). They were cultivated on gelatin-coated (0.1%, G2500; Sigma) plastic flasks (Falcon) in ESC medium containing of knockout Dulbecco’ s modified Eagle’ s medium (DMEM; 10829-018; Gibco) supplemented with 15% ESCqualified fetal bovine serum (FBS; 16141-079; Gibco), 0.1 mM -mercaptoethanol (M7522; Sigma), 2 mM glutamine (15039-027; Gibco), 0.1 mM non-essential amino acids (M7145; Sigma), 0.1 mM penicillin-streptomycin (15070- 063; Gibco), and 1,000 IU/mL leukemia inhibitory factor (LIF; Esgro, ESG1107; Chemicon). Cultures were grown in 5% CO2, 95% humidity, and were routinely passaged every 2 days.ESC microencapsulation For encapsulation, ESCs were isolated from MEFs by treatment of the cells with trypsin-ethylenediaminetetraacetic acid (EDTA) solution (15305-014; GIBCO), and replated for 1.5 hours onto precoated gelatin plates (0.1% W/V) containing ESC medium. The supernatant was then collected and centrifuged (6 minutes, 1,200 rpm). The isolated cells were resuspended in 1.2% alginate solution (A7003; Sigma) at 4×۱۰۶ cells/mL. Beads were formed by dispensing the alginate/cell suspension dropwise into a 102 mM CaCl2 solution via a 22-gauge needle attached to a syringe. After 10 minutes, the newly formed beads (70 beads containing approximately 40,000 cells/bead) were washed once with a sterile 0.9% saline solution[AUTHORS: please advise: phosphate-buffered saline (PBS)] followed by 2 washes with ESC medium and 10 beads were cultured in each well of 12-well plate (TPP, 92412) in ESC medium on the MEF feeder layer. The cultures were maintained for up to 20 days at 37°C in a humidified atmosphere of 5% CO2, with daily changes of medium. On day 3 after suspension of ESCs in alginate, embryonic spherical structures (ESC spheres) were observed in each well. From day 3 up to 20 days, alginate beads were transferred daily onto new feeder layers, and the number of ESC spheres emerging from the alginate beads was counted. Further, ESC spheres were collected for differentiation and flow cytometry to clarify the status of ESC spheres.

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