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The present protocol describes the fabrication of poly(lactic-co-glycolic acid)-based highly open porous microspheres (HOPMs) via the single-emulsion formulation based facile microfluidic technology. These microspheres have potential applications in tissue engineering and drug screening.
Compared to bulk scaffolds and direct injection of cells alone, the injectable modular units have garnered enormous interest in repairing malfunctioned tissues due to convenience in the packaging of cells, improved cell retention, and minimal invasiveness. Moreover, the porous conformation of these microscale carriers could enhance the medium exchange and improve the level of nutrients and oxygen supplies. The present study illustrates the convenient fabrication of poly(lactic-co-glycolic acid)-based highly open porous microspheres (PLGA-HOPMs) by the facile microfluidic technology for cell delivery applications. The resultant monodispersed PLGA-HOPMs possessed particle sizes of ~400 µm and open pores of ~50 µm with interconnecting windows. Briefly, the emulsified oil droplets (PLGA solution in dichloromethane, DCM), wrapped with the 7.5% (w/v) gelatin aqueous phase, were introduced into the 1% (w/v) continuous flowing poly(vinyl alcohol) (PVA) aqueous solution through the coaxial nozzle in the customized microfluidic setup. Subsequently, the microspheres were subjected to solvent extraction and lyophilization procedures, resulting in the production of HOPMs. Notably, various formulations (concentrations of PLGA and porogen) and processing parameters (emulsifying power, needle gauge, and flow rate of dispersed phase) play crucial roles in the qualities and characteristics of the resulting PLGA HOPMs. Moreover, these architectures might potentially encapsulate various other biochemical cues, such as growth factors, for extended drug discovery and tissue regeneration applications.
Cell-laden microspheres offer favorable advantages, such as enhanced cell retention capacity in situ, efficient delivery of cells, and subsequent ability of cell proliferation in vivo1. To date, numerous investigations have been put forward for developing a successful scaffolding structure to support a conducive environment for cells for tissue regeneration or drug screening applications2. However, the hypoxia environment is oftentimes inevitable in the interiors due to insufficient supplies of nutrients/oxygen and metabolic waste accumulation3. To overcome these problems, highly....
1. Preparation of solutions
Based on previous work that optimized the major parameters1, PLGA was dissolved in the evaporable DCM solvent. The primary W/O emulsion was prepared by homogenizing with gelatin under ultrasonic probe treatment. The customized co-flow fluidic structure was simplistically assembled, in which a syringe was employed to introduce the flows constantly. Furthermore, sufficient rinsing procedures were carried out to eliminate PVA and gelatin of PLGA microspheres (Figure 1A, .......
This article describes an efficient strategy to fabricate PLGA-based architectures, namely the PLGA-HOPMs. It is to be noted that several critical steps must be taken carefully, including avoiding solvent volatilization of PLGA and gentle adjustment of the ultrasonic power to the target position during the preparation of the emulsion. In addition, the liquid outlet of the 20 mL syringe can be adjusted to a certain extent to solve the phase separation of emulsified precursors. However, a limitation is that, since the stat.......
SCL, YW, RKK, and AZC acknowledge financial support from the National Natural Science Foundation of China (NSFC, 32071323, 81971734, and U1605225) and Program for Innovative Research Team in Science and Technology in Fujian Province University. YSZ was neither supported by any of these programs nor received payment of any type; instead, support from the Brigham Research Institute is acknowledged.
....Name | Company | Catalog Number | Comments |
Centrifuge tube | Solarbio, Beijing, China | 5 mL & 50 mL (sterility) | |
Confocal laser scanning microscopy | Leica, Wetzlar, Germany | TCS SP8 | |
Dichloromethane | Sinopharm Chemical Reagent Co., Ltd, Shanghai, China | 20161110 | Research Grade |
Dispensing needle | Kindly, Shanghai, China | 26 G, ID: 250 μm, OD: 460 μm | |
DMEM/F-12 | Gibco; Life Technologies Corporation, Calsbad, USA | 15400054 | DMEM/F-12 50/50, 1x (Dulbecco's Mod. Of Eagle's Medium/Ham's F12 50/50 Mix) with L-glutamine |
Ethyl alcohol | Sinopharm Chemical Reagent Co., Ltd, Shanghai, China | 20210918 | Research Grade |
Ethyl-enediaminetetraacetic acid (EDTA)-trypsin | Biological Industries, Kibbutz Beit-Haemek, Isra | Trypsin (0.25%), EDTA (0.02%) | |
Fetal bovine serum (FBS) | Biological Industries, Kibbutz Beit-Haemek, Isra | Research Grade | |
Freeze drier | Bilon, Shanghai, China | FD-1B-50 | |
Gelatin | Sigma-Aldrich Co. Ltd, St. Louis, USA | lot# SZBF2870V | From porcine skin, Type A |
Glass bottom plate | Biosharp, Hefei, China | BS-15-GJM, 35 mm | |
Glass capillary | Huaou, Jiangsu, China | 0.9-1.1 × 120 mm | |
Incubator shaker | Zhicheng, Shanghai, China | ZWYR-200D | |
Live dead kit cell imaging kit | Solarbio, Beijing, China | 60421211112 | Green fluorescence in live cells (ex/em 488 nm/515 nm). Red fluorescence in dead cells (ex/em 570 nm/602 nm) |
Low-speed centrifuge | Xiangyi, Hunan, China | TD5A | |
Magnetron sputter | Riye electric Co. Ltd, Suzhou, China | MSP-2S | |
Microflow injection pump | Harvard Apparatus, Holliston, USA | Harvard Pump 11 Plus | |
Penicillin-streptomycin | Biological Industries, Kibbutz Beit-Haemek, Isra | 2135250 | Research Grade |
Phosphate buffered saline (PBS) | Servicebio Technology Co.,Ltd. Wuhan, China | GP21090181556 | PBS 1x, culture grade, no Calcium, no Magnesium |
Poly(lactic-co-glycolic acid) | Sigma-Aldrich Co. Ltd, St. Louis, USA | lot# MKCF9651 | 66–107 kDa, lactide:glycolide 75:25 |
Poly(vinyl alcohol) | Sigma-Aldrich Co. Ltd, St. Louis, USA | lot# MKCK4266 | 13-13 kDa, 98% Hydrolyzed |
PVC tube | Shenchen, Shanghai, China | Inner diameter, ID: 1 mm | |
Rat bone marrow mesenchyml stem cells | Procell, Wuhan, China | ||
Scanning electron microscope | Phenom pure, Eindhoven, Netherlands | Set acceleration voltage at 5 kV | |
Syrine for medical purpose | Kindly, Shanghai, China | 5 mL & 50 mL (with the needle) | |
Temperature water bath | Mingxiang, Shenzhen, China | 36 W | |
Transformer | Riye electric Co. Ltd, Suzhou, China | SZ-2KVA | |
Ultrasonic cell breaker | JY 92-IID, Scientz, Ningbo, China | JY 92-IID | |
UV curing glue | Zhuolide, Foshan, China | D-3100 |
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