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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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....

Protocol

1. Preparation of solutions

  1. Prepare the PVA stock solution in advance by heating the PVA solution in a 80 °C water bath and subsequently placing it in the refrigerator at 4 °C. Cool to room temperature (RT) for experimental usage.
  2. Prepare the emulsion-precursor by adding the aqueous gelatin solution (1 mL, 7.5%, w/v) to the organic phase of PLGA (2 mL, 2%, w/v in dichloromethane, DCM) (see Table of Materials).
    ​NOTE: Generally, microfluidic te.......

Representative Results

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, .......

Discussion

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.......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
Centrifuge tubeSolarbio, Beijing, China5 mL & 50 mL (sterility)
Confocal laser scanning microscopyLeica, Wetzlar, GermanyTCS SP8
DichloromethaneSinopharm Chemical Reagent Co., Ltd, Shanghai, China20161110Research Grade
Dispensing needleKindly, Shanghai, China26 G, ID: 250 μm, OD: 460 μm
DMEM/F-12Gibco; Life Technologies Corporation, Calsbad, USA15400054DMEM/F-12 50/50, 1x (Dulbecco's
Mod. Of Eagle's Medium/Ham's F12
50/50 Mix) with L-glutamine
Ethyl alcoholSinopharm Chemical Reagent Co., Ltd, Shanghai, China20210918Research Grade
Ethyl-enediaminetetraacetic acid (EDTA)-trypsinBiological Industries, Kibbutz Beit-Haemek, IsraTrypsin (0.25%), EDTA (0.02%)
Fetal bovine serum (FBS)Biological Industries, Kibbutz Beit-Haemek, IsraResearch Grade
Freeze drierBilon, Shanghai, ChinaFD-1B-50
GelatinSigma-Aldrich Co. Ltd, St. Louis, USAlot# SZBF2870VFrom porcine skin, Type A
Glass bottom plateBiosharp, Hefei, ChinaBS-15-GJM, 35 mm
Glass capillaryHuaou, Jiangsu, China0.9-1.1 × 120 mm
Incubator shakerZhicheng, Shanghai, ChinaZWYR-200D
Live dead kit cell imaging kitSolarbio, Beijing, China60421211112Green fluorescence in live cells (ex/em 488 nm/515 nm). Red fluorescence in dead cells (ex/em 570 nm/602 nm)
Low-speed centrifugeXiangyi, Hunan, ChinaTD5A
Magnetron sputterRiye electric Co. Ltd, Suzhou, ChinaMSP-2S
Microflow injection pumpHarvard Apparatus, Holliston, USAHarvard Pump 11 Plus
Penicillin-streptomycinBiological Industries, Kibbutz Beit-Haemek, Isra2135250Research Grade
Phosphate buffered saline (PBS)Servicebio Technology Co.,Ltd. Wuhan, ChinaGP21090181556PBS 1x, culture grade, no Calcium, no Magnesium
Poly(lactic-co-glycolic acid)Sigma-Aldrich Co. Ltd, St. Louis, USAlot# MKCF965166–107 kDa, lactide:glycolide 75:25
Poly(vinyl alcohol)Sigma-Aldrich Co. Ltd, St. Louis, USAlot# MKCK426613-13 kDa, 98% Hydrolyzed
PVC tubeShenchen, Shanghai, ChinaInner diameter, ID: 1 mm
Rat bone marrow mesenchyml stem cellsProcell, Wuhan, China
Scanning electron microscopePhenom pure, Eindhoven, NetherlandsSet acceleration voltage at 5 kV
Syrine for medical purposeKindly, Shanghai, China5 mL & 50 mL (with the needle)
Temperature water bathMingxiang, Shenzhen, China36 W
TransformerRiye electric Co. Ltd, Suzhou, ChinaSZ-2KVA
Ultrasonic cell breakerJY 92-IID, Scientz, Ningbo, ChinaJY 92-IID
UV curing glueZhuolide, Foshan, ChinaD-3100

References

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Highly Open Porous Microspheres HOPMsMicrofluidic TechnologyPLGACell Retention3D Tumor ModelTissue RegenerationEmulsionDroplet GenerationPolyvinyl AlcoholContinuous PhaseDispersion Phase

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