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 biomaterials4,5,6. 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 PMs10,11. 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 spheres13,14. On the one hand, PMs were produced using gas-forming agents, such as NH4HCO3, which were restrained due to insufficient interconnectivity15,16,17. 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.
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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 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.
- Customize the droplet generator assembly.
- Construct a co-flow microfluidic generator using a glass capillary (OD, 1 mm), PVC tubes (ID, 1 mm), and a 26 G dispensing needle.
- Connect the glass capillary to the end of the PVC tube and then pierce with a needle at the connection of the above structure.
- 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.
- Prepare the droplet microfluidic platform.
- 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.
- Set the flow rates of the continuous and dispersion phases at 2 mL/min and 0.08 mL/min, respectively.
- 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.
- Perform the emulsification and droplet generation.
- Prepare the emulsion following the steps below.
- 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).
- 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.
- 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.
- Perform the droplet generation.
- Load the prepared emulsion (step 2.3.1) rapidly in the 5 mL syringe of the microfluidic platform after ultrasonic treatment.
- 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.
- 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.
- Place the sample at 4 °C overnight for further stabilization.
- 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.- 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.
- 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.
- Place the PLGA microspheres in the 37 °C water bath for 30 min to dissolve the gelatin wrapped in the PLGA backbone.
- 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.
- Store the dry PLGA-HOPMs at -20 °C for further experiments.
- Prepare the emulsion following the steps below.
3. Scanning electron microscope (SEM) imaging
- 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.
- Obtain the SEM images by setting the acceleration voltage at 5 kV.
- 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.
- 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.
- Click on analyze > set scale, set the "known distance" to the scale bar of the SEM image, and click on global > OK.
- 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.
- 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).
- 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.
- Passage the rat BMSCs in a 1:2 ratio after reaching 90% of confluence.
- Perform cell adhesion following the steps below.
- 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.
- Wash the sterilized PLGA-HOPMs twice with phosphate buffer solution.
- 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.
- 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.
- Perform the live and dead dual-stain assay.
- Rinse the cell-laden PMs thrice for eluting BMSCs without attachment to PLGA-HOPMs.
- 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.
- 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.
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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, Supplementary Figure 1A,B). Subsequently, the morphological attributes of PLGA-HOPMs were observed by SEM imaging. It was observed that the PLGA-HOPMs exhibited large sizes (350-500 µm) with arbitrary-sized pores (10-100 µm) and interconnected windows, which could facilitate high efficiency in oxygen/metabolic waste transport (Figure 1B). These morphological results were in agreement with the reported results1.
As a proof-of-concept, the BMSCs were employed to tentatively culture within the PLGA-HOPMs. It was believed that PMs' interior structure could allow the cells to adhere to and ensure the proliferation during dynamic culture8. In addition, the rationale for selecting BMSCs is that these cells are characterized by multipolar differentiation and repair damaged tissues, and are also commonly employed to study the biological behavior of various bioactive materials23. The BMSCs were used to construct in vitro microtissues with the microspheres and visualized cell distribution and survival state by z-analysis of CLSM (Figure 1C). It was observed that a large number of cells adhered to scaffolds in 1 d, and the green fluorescence was maintained, representing extensive adhesion and migration capabilities of cells in PLGA-HOPMs. The cytoplasm was indirectly imaged with calcein-AM staining since the acetoxymethyl ester (AM) group provides improved hydrophobicity and facilitates the penetration through the living cell membrane. Although calcein-AM has no fluorescence, it can be hydrolyzed by the endogenous esterase intracellularly to generate the polar molecule calcein with a strong negative charge and emit strong green fluorescence24. Therefore, the cytoplasm is indirectly visualized with calcein-AM staining (Figure 1C, Supplementary Figure 1C).
Figure 1: Fabrication and cytocompatibility of PLGA-HOPMs. (A) Schematic illustrating of the fabrication of the PLGA-HOPMs. (B) The pore diameter of PMs based on SEM imaging (10-100 µm). Scale bar = 100 µm. (C) Live (calcein-AM, green) and dead (propidium iodide, PI, red) staining of BMSCs cocultured with PLGA-HOPMs for 24 h. Scale bar = 250 µm. Please click here to view a larger version of this figure.
Supplementary Figure 1: Illustrations of the real microfluidic platform, emulsion, and interlayer imaging of cell-laden PLGA-HOPMs. (A) Scheme of real droplet microfluidics platform. (B) W/O precursor emulsified by ultrasound treatment. (C) The interlayer of live and dead staining of BMSCs cocultured with PLGA-HOPMs for 24 h. Scale bar = 250 µm. Please click here to download this File.
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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 state of porogen is affected by environmental temperature, the stability of the emulsion might be undesirable. Together, the convenient assembly of the droplet microfluidic platform can substantially facilitate the fabrication of cell-laden microgels for tissue engineering and drug screening applications. PLGA is frequently applied as a delivery vehicle in fabricating microspheres since it is a Food and Drug Administration (FDA)-approved drug delivery commodity25. In addition, the aliphatic polyester offers harmless degradation attributes via hydrolysis either in vitro or in vivo, which is dependent on the proportion of lactide acid and glycolic acid during synthesis procedures26.
To fabricate PLGA-HOPMs, the aqueous and organic phases were homogenized to form a W/O emulsion using the ultrasonic probe. Then, the W/O emulsion was introduced into the droplet microfluidic device and evolved into water-in-oil-in-water (W/O/W) droplets at the needle tip (Figure 1A, Supplementary Figure 1A, B). The obtained W/O/W droplets could be solidified by extracting and evaporating DCM in the collecting phase. The pre-cooled collection phase in an ice bath could allow the gelatin to remain in the soft-gel state, directly retaining the well-distributed emulsion during solvent-evaporation, resulting in the PLGA-HOPMs after further lyophilization (Figure 1B).
In addition, the emulsion-templating method contains volatile organic solvents, which could be removed by volatilization. Also, the residue of DCM in the PLGA-HOPMs could be nearly entirely removed, calculated from gas chromatography measurement2. Moreover, the microfluidic technology would not affect the cyto/biocompatibility of PLGA. In fact, the PLGA-HOPMs cultured with rat BMSCs showed that the number of live cells was extensive, with only a few dead cells and the stretching morphology. Moreover, cytoplasm could be imaged by the live fluorescent dye, indicating stretching on the HOPMs.
Together, PLGA-HOPMs withexcellent morphological attributes can meet the requirements concerning cellular adhesion and spreading abilities throughout the HOPMs for tissue regeneration and various biomedical applications.
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Disclosures
The authors have nothing to disclose.
Acknowledgments
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
| 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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