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

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 technology involves different phases to form the microspheres. The continuous or collecting phase comprises an aqueous poly(vinyl alcohol) (PVA, 600 mL, 1%, w/v).

2. Droplet microfluidic device assembly and fabrication of PLGA-HOPMs

NOTE: The consumable items required for this assembly are listed in the Table of Materials.

1. Customize the droplet generator assembly.
   1. Construct a co-flow microfluidic generator using a glass capillary (OD, 1 mm), PVC tubes (ID, 1 mm), and a 26 G dispensing needle.
   2. Connect the glass capillary to the end of the PVC tube and then pierce with a needle at the connection of the above structure.
   3. Place the other end of the PVC tube in the continuous phase during the droplet generation. Using UV glue, seal the gaps at the joint after curing.
      NOTE: The needle needs to be bent curved about 45° for convenient piercing at the PVC tube, and the needlepoint stretched into the capillary tube to form the coaxial structure.
2. Prepare the droplet microfluidic platform.
   1. Use two syringe pumps, as shown in Figure 1. Use a 50 mL syringe to load the continuous phase and a 5 mL syringe for emulsion formation.
   2. Set the flow rates of the continuous and dispersion phases at 2 mL/min and 0.08 mL/min, respectively.
   3. Place a 500 mL beaker in an ice bath and fill it with a pre-cooled PVA aqueous solution (step 1.1).
      NOTE: The end of the glass capillary must be immersed in the collecting phase. It is suggested to stir (1 h, 60 rpm) the supernatant of the collecting phase with the glass rod after the emulsion-droplets formation in the platform. Emulsion droplets are produced at the tip of the needlepoint. In general, the gauge of the needle influences the size of PMs, in which the lesser gauge corresponds to the smaller size of PMs. Moreover, the pore diameter is mainly correlated to the porogen concentration, capable of increasing with appropriate gelatin concentration¹.
3. Perform the emulsification and droplet generation.
   1. Prepare the emulsion following the steps below.
      1. Prepare the emulsion by immediately decanting the aqueous gelatin solution into the DCM solution of PLGA (step 1.2) and emulsifying using the ultrasonic device (see Table of Materials).
      2. Adjust the ultrasonic power to 400 W, and set the total processing time as 90 s (interval 2 s, ultrasonic treatment 1 s), accompanied by constantly changing the probe's position manually.
      3. Stabilize the resultant emulsion for syringe suction and droplet generation in approximately 20 min.
         NOTE: Place the probe of the ultrasonic cell breaker in the oil-water interface. It is suggested not to let the ultrasonic probe contact the container.
   2. Perform the droplet generation.
      1. Load the prepared emulsion (step 2.3.1) rapidly in the 5 mL syringe of the microfluidic platform after ultrasonic treatment.
      2. Introduce the unstable W/O emulsion (which is in the course of phase-separation) into the microfluidic device as the discontinuous phase. At the same time, use the aqueous solution of PVA as the continuous phase.
      3. Collect the microspheres containing the emulsion at the bottom of the collecting beaker (PVA, 1%, w/v) after injecting proper portions of the emulsion into the microfluidic device.
      4. Place the sample at 4 °C overnight for further stabilization.
   3. Rinse and lyophilize the prepared microspheres (PLGA-HOPMs) following the steps below.
      ​NOTE: The generated PLGA-HOPMs contain arbitrary pores on the interior and surface.
      1. Store the microspheres at 4 °C before normalizing to RT and stir by a glass rod (1 h, 60 rpm) to eliminate the residual DCM.
      2. Filtrate the collecting phase in the 500 mL beaker carefully, and wash the collected microspheres thrice with deionized water to wash off the residual PVA on the surface of PMs.
      3. Place the PLGA microspheres in the 37 °C water bath for 30 min to dissolve the gelatin wrapped in the PLGA backbone.
      4. Rinse the resultant PLGA-HOPMs twice with the deionized water to remove the residual gelatin and pre-cool for lyophilization at -80 °C for 24 h.
      5. Store the dry PLGA-HOPMs at -20 °C for further experiments.

3. Scanning electron microscope (SEM) imaging

1. Deposit PLGA-HOPMs on the sample stage of SEM (see Table of Materials) at RT and put the sample stage into the sputter cell of the magnetron sputter. Switch on the transformer and magnetron one by one. Introduce the sample to the SEM after being sputter-coated with gold for 1 min.
2. Obtain the SEM images by setting the acceleration voltage at 5 kV.
3. Additionally, quantify the particle size of PLGA-HOPMs using 100 PMs at the variable field of SEM imaging. Measure the representative result to quantify the pore size distribution.
   1. Open the graphics with Image J and use the "straight line" to match the scale bar of the SEM image, which makes the software identify pixel to length.
   2. Click on analyze > set scale, set the "known distance" to the scale bar of the SEM image, and click on global > OK.
   3. Click on analyze > Tools > ROI manager... to measure the pore or particle size of PLGA PMs and click on add it. Measure the number of samples based on the requirement.
   4. Click on Measure to obtain the quantitative data of the graphics for further calculation.

4. Cell culture and fluorescence imaging

NOTE: Rat bone marrow mesenchymal stem cells (BMSCs) were used for the present work. The cells were obtained from a commercial source (see Table of Materials).

1. Culture the cells in Dulbecco's modified Eagle medium supplemented with L-glutamine (DMEM/F-12), 10% (v/v) fetal bovine serum, and 1% penicillin/streptomycin. Incubate the cells at 37 °C and 5% CO₂.
2. Passage the rat BMSCs in a 1:2 ratio after reaching 90% of confluence.
3. Perform cell adhesion following the steps below.
   1. Incubate the PLGA-HOPMs with ethyl alcohol (5 mL, 70%, v/v) and centrifuge at 500 x g for 10 min at RT to sterilize.
   2. Wash the sterilized PLGA-HOPMs twice with phosphate buffer solution.
   3. Incubate the HOPMs (five in number) with the cell suspension (5 mL, 1 x 10⁵ cells/mL) in a sterile centrifuge tube (50 mL) under dynamic culture for BMSCs adhesion to PLGA-HOPMs.
   4. Perform the dynamic culture in a thermostatic aseptic shaker (37 °C, 90 rpm) by placing a sealed 50 mL centrifuge tube in the shaker (see Table of Materials).
      NOTE: The dynamic culture intends to enhance the adherence rate of BMSCs on the scaffolds, rather than directly incubating the PMs and cells onto the plastic plate. The cells and PLGA PMs are added into a 50 mL sterile centrifuge tube, incubating the tube at a thermostatic aseptic shaker (37 °C, 90 rpm) and changing the media every 2 days.
4. Perform the live and dead dual-stain assay.
   1. Rinse the cell-laden PMs thrice for eluting BMSCs without attachment to PLGA-HOPMs.
   2. Place the cell-laden PMs on the 35 mm glass-bottom plate. Add 1 mL of staining buffer (see Table of Materials), including 1 µL of calcein-AM and propidium iodide (PI), respectively, to the PBS-prewashed sample.
   3. Assess the samples by confocal laser scanning microscopy (see Table of Materials). Switch on the excitation light of 488 nm and 552 nm at the corresponding detector. For z-overlay analysis, set the z-wide to capture the different layers of the sample by z-axis movement of the microscope.
      NOTE: It is worth noting that the other stains can be appropriately employed for analysis.

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

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