Fabricating Highly Open Porous Microspheres (HOPMs) via Microfluidic Technology

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 porous microspheres (PMs) have been developed using various biomaterials^4,5,6. Additionally, in dynamic culture, the scaffolds suffer from excessive shear stress⁷, and the unstable state of the culture medium might break the fidelity of PMs. Alternatively, poly(lactic-co-glycolic acid) (PLGA) could be used to process PMs with good mechanical strength for dynamic culture¹. For example, we demonstrated co-injection of mouse myoblast (C2C12)-laden PLGA highly open PMs (HOPMs) and human umbilical vein endothelial cell (HUVEC)-laden poly(ethylene glycol) hollow microrods to heal volumetric muscle loss, achieving remarkable improvement of in situ skeletal muscle regeneration⁸.

Notably, PMs are characterized by large surface areas and high porosities, which is of specific interest for cell adhesion and growth towards minimally invasive cell delivery⁹. In view of these aspects, various biocompatible materials have been employed to fabricate the PMs^10,11. These designable PMs cocultured with cells offer excellent adhesion, considerable mechanical strength, and highly interconnected windows, which could improve cell proliferation for repairing damaged tissues¹². In this regard, various technologies have also been developed to fabricate porous spheres^13,14. On the one hand, PMs were produced using gas-forming agents, such as NH₄HCO₃, which were restrained due to insufficient interconnectivity^15,16,17. On the other hand, PMs were directly sheared after emulsification, which led to polydisperse PMs¹⁸. In the end, the droplet microfluidic technology based on the emulsion-templating approach is perhaps an efficient method for constructing PMs, as it often results in uniform-sized particles¹⁹. Notably, the morphological attributes of the microspheres often depend on the quality of the generated emulsion droplets (i.e., water-in-oil, W/O, or oil-in-water, O/W), which may significantly affect the attributes of the biomaterials²⁰. It is worth noting that the predesigned microfluidic platform can be applied to generate the microfibers or microspheres. In an instance, Yu et al. demonstrated the production of cell-laden microfibrous structures based on capillary-based microfluidic platforms, which could be used to assemble cellular networks for mimicking natural tissues²¹. In another instance, Ye et al. fabricated photonic crystal microcapsules by the template replication of silica colloidal crystal beads through microfluidic technologies, which could overcome many limitations of current techniques that require complex labeling and specific apparatus²².

Indeed, the rationale behind the utilization of this technique is due to various advantages, such as being facile in nature, requiring no sophisticated equipment, and its convenience in synthesizing uniform-sized PMs for cell delivery and regenerative medicine applications. In this context, with predesigned components of emulsion-templating, PMs with high porosities and interconnectivity can be conveniently obtained from a microfluidic device assembled from poly(vinyl chloride) (PVC) tubing, a glass capillary, and a needle. A W/O emulsion-precursor is prepared by homogenizing an aqueous solution of gelatin and an organic solution of PLGA. By selectively injecting the applicable portion of the emulsion into the microfluidic platform, the PMs with uniform particle sizes and interconnected pores throughout the surface to the interior are fabricated. The present protocol aims to fabricate the PLGA-HOPMs by emulsion-templating in the microfluidic platform. It is believed that this protocol allows reproducible production of PLGA-HOPMs and will potentially be applicable in their related fields of tissue engineering and drug screening.

Protocol

1. Preparation of solutions 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. 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…

Materials

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