Minimizing the variability in the particle fraction within granular scaffolds facilitates reproducible experimentation. This work describes methods for generating granular scaffolds with controlled particle fractions for in vitro tissue engineering applications.
Microgels are the building blocks of microporous annealed particle (MAP) scaffolds, which serve as a platform for both in vitro cell culture and in vivo tissue repair. In these granular scaffolds, the innate porosity generated by the void space between microgels enables cell infiltration and migration. Controlling the void fraction and particle fraction is critical for MAP scaffold design, as porosity is a bioactive cue for cells. Spherical microgels can be generated on a microfluidic device for controlled size and shape and subsequently freeze-dried using methods that prevent fracturing of the polymer network. Upon rehydration, the lyophilized microgels lead to controlled particle fractions in MAP scaffolds. The implementation of these methods for microgel lyophilization has led to reproducible studies showing the effect of particle fraction on macromolecule diffusion and cell spreading. The following protocol will cover the fabrication, lyophilization, and rehydration of microgels for controlling particle fraction in MAP scaffolds, as well as annealing the microgels through bio-orthogonal crosslinking for 3D cell culture in vitro.
Microporous annealed particle (MAP) scaffolds are a subclass of granular materials in which the microgel (µgel) building blocks are interlinked to form a bulk, porous scaffold. With the unique microarchitecture of these granular scaffolds, the innate porosity generated by the void space between interlinked spherical microgel supports accelerated cell infiltration and migration1. The microgel building blocks of MAP scaffolds can be fabricated from both synthetic and natural polymers with chemical modifications2. The methods described here specifically highlight the use of microgels comprised of a hyaluronic acid (HA) backbone modified with functional norbornene (NB) handles. The NB functional handle on the HA polymer supports click chemistry reactions for forming microgels and linking them together to generate MAP scaffolds3,4. Numerous schemes have been employed for linking the microgels together (i.e., annealing), such as enzymatic1, light-based5,6, and additive-free click chemistry3,7 reactions. Additive-free click chemistry is described in this work, using the tetrazine-norbornene inverse electron demand Diels-Alder conjugation for interlinking the HA-NB microgels.
To fabricate MAP scaffolds, users first generate the microgel building blocks using reverse emulsions either in batch systems or within microfluidic devices, as well as with electrohydrodynamic spraying, lithography, or mechanical fragmentation2. The production of spherical HA-NB microgels has been well described and previously reported using both batch emulsion2 and microfluidic droplet generation techniques8,9,10,11. In this work, spherical HA-NB microgels were generated on a flow-focusing microfluidic platform for controlled size and shape, as previously described8,9,10. After purification, the microgels exist in an aqueous suspension and must be concentrated to induce a jammed state. When jammed, microgels exhibit shear-thinning properties, which allow them to function as injectable, space-filling materials1. One method of inducing a jammed state is to dry the microgels via lyophilization, or freeze-drying, then subsequently rehydrate the dried product in a controlled volume12. Alternatively, excess buffer can be removed from the microgel slurry via centrifugation over a strainer or with manual removal of the buffer from the microgel pellet either by aspiration or using an absorbent material. However, using centrifugation to dry the microgels can generate a highly variable range of particle fractions and void fractions when making granular scaffolds12. Techniques for lyophilizing microgels have been described using 70% IPA for polyethylene glycol (PEG) microgels13, fluorinated oils for gelatin methacryloyl (GelMa) microgels14, and 70% ethanol for HA microgels12. This protocol highlights methods for freeze-drying spherical HA microgels using 70% ethanol, a standard laboratory reagent, to retain the original microgel properties during the drying process. The freeze-dried HA microgels can be weighed and rehydrated at user-defined weight percentages to control the final particle fractions in MAP scaffolds12.
The final step in MAP scaffold formation relies on annealing the microgels to create a bulk, porous scaffold1. By utilizing native extracellular matrix components and employing bio-orthogonal annealing schemes, MAP scaffolds serve as a biocompatible platform for both in vitro cell culture and in vivo tissue repair3. Through these approaches, MAP scaffolds can be fabricated from HA-NB building blocks with user-defined particle fractions for their employment in tissue engineering applications12. The following protocol describes the microfluidic production of HA-NB microgels followed by lyophilization and rehydration for controlling particle fraction in MAP scaffolds. Lastly, steps for annealing the microgels are described using bio-orthogonal chemistry for in vitro 3D cell culture experiments.
1. Microfluidic device fabrication
2. Microfluidic production of hyaluronic acid (HA) microgels with norbornene (NB) functional handles
Figure 1: Microfluidic production of hyaluronic acid (HA) microgels with norbornene (NB) functional handles. (A) Approximately 31% of HA repeat units were successfully modified with NB, as determined by proton NMR analysis performed in deuterium oxide. 1H NMR shifts of pendant norbornenes at δ6.33 and δ6.02 (vinyl protons, endo), and δ6.26 and δ6.23 ppm (vinyl protons, exo) were compared to the HA methyl group δ2.05 ppm to determine functionalization. Reprinted from Anderson et al.12 with permission from Elsevier. (B) Schematic of the flow-focusing microfluidic device used to generate HA-NB µgels. (C) Maximum intensity projections from confocal microscopy were used to visualize fluorescently labeled µgels (scale bar = 500 µm). (D) Frequency distributions of microgel diameter from independent runs on the microfluidic setup demonstrate control over microgel size ~50 µm or ~100 µm depending on the device used. (E) Microgel diameter is reported as the mean and standard deviation for each independent run. Reprinted from Wilson et al.9 with permission from Wiley. Please click here to view a larger version of this figure.
3. Purifying and drying microgels
Figure 2: Drying HA-NB microgels. (A) Maximum intensity projection of µgels in aqueous solution (scale bar = 100 µm). (B) Purified µgels can be incubated 1:1 by volume in the lyophilization medium of choice and lyophilized. (C) Maximum intensity projection of dried lyo-µgels (scale bar = 100 µm). (D) Microgels are resuspended after lyophilization. EtOH (70%) is recommended for retaining the original properties of the µgels throughout the lyophilization process; however, other media such as isopropyl alcohol (IPA), water, and acetonitrile (MeCN) can be used interchangeably to facilitate cryogel formation (scale bar = 100 or 50 µm as noted). (E) Measurement of HA-NB microgel diameter before (gray) and after lyophilization (green) in 70% EtOH shown as frequency distributions for three microgel populations. Reprinted from Anderson et al.12 with permission from Elsevier. Please click here to view a larger version of this figure.
4. MAP scaffold fabrication
Figure 3: Synthesis of tetrazine linker for the fabrication of microporous annealed particle (MAP) scaffolds. (A) Schematic of HA-NB µgels being interlinked with a tetrazine linker to form MAP scaffolds. (B) Reaction scheme for HA-Tet synthesis. (C) The HA-Tet reaction was setup and allowed to react overnight followed by (D) precipitation of HA-Tet in ethanol. (E) Once purified and dried, the HA-Tet was rehydrated and lyophilized to yield (F) a dried, light pink product. (G) Proton NMR analysis shows successful modification of 11% of HA repeat units. Reprinted from Anderson et al.12 with permission from Elsevier. Please click here to view a larger version of this figure.
Figure 4: Rehydration of lyophilized microgels for MAP scaffold fabrication. (A) Maximum intensity projection of dried lyo-µgels (scale bar = 100 µm). (B) After freeze-drying, rehydration of lyo-µgels is shown to take approximately 20 min (scale bar = 100 µm). (C) Lyo-µgels can be rehydrated at varying wt% MAP to produce jammed µgels (scale bar = 100 µm). (D) Increasing the wt% MAP when rehydrating lyo-µgels alters the particle fraction in MAP scaffolds, as shown by single Z-slices of MAP scaffolds and volume projections (scale bar = 100 µm). (E) Using these user-defined wt% MAP scaffolds, unique particle fractions can be achieved (NL = non-lyophilized µgels). A one-way ANOVA with Tukey HSD was performed on the samples (n = 3), with significance reported at p < 0.05 (*), p < 0.01 (**), p < 0.005 (***), and p < 0.001 (****). Reprinted from Anderson et al.12 with permission from Elsevier. Please click here to view a larger version of this figure.
5. 3D cell culture in map scaffolds
Figure 5: Cell culture in MAP scaffolds. (A) The mold for creating cell culture wells can be 3D printed and cast with PDMS. The entire mold is 95 mm in diameter, the large wells are 6 mm in diameter, and the small inner wells are 4 mm in diameter. (B) Once cast with PDMS, the cell culture devices are plasma bonded to coverslips for improved microscopy capabilities. (C) The cross section of a cell culture well depicts the reservoir for cell media (~50 µL) and a smaller reservoir for seeding MAP scaffold with cells (~10 µL). (D) The process of seeding cells in MAP scaffolds first relies on the rehydration of lyo-µgels at the user's desired wt%, followed by mixing with cells and the crosslinker for interlinking the µgels. (E) Cells can be encapsulated in MAP scaffolds (green) with varied wt% MAP. Representative images are from day 5 of D1 cell culture in MAP scaffolds (scale bar = 100 µm). (F) Single Z-slices show differences in cell growth in scaffolds comprising different wt% MAP (scale bar = 50 µm). Reprinted from Anderson et al.12 with permission from Elsevier. Please click here to view a larger version of this figure.
The aim of this protocol is to demonstrate the preparation of microporous annealed particle (MAP) scaffolds with a bio-orthogonal crosslinking scheme as well as controlled particle fractions for 3D cell culture. First, HA was modified with norbornene pendant groups to be used in both microgel formation and interlinking to form MAP scaffolds. Using these methods, approximately 31% of HA repeat units were successfully modified with a norbornene functional handle (Figure 1A). Microfluidic devices with a flow-focusing region (Figure 1B) were shown to produce HA-NB µgels of either ~50 µm or ~100 µm in diameter (Figure 1C,D). The µgels used throughout the rest of this work had a mean diameter of 92 µm (Q1 = 79 µm, Q3 = 103 µm) (Figure 1E).
To control the particle fraction, µgels were dried via lyophilization (Figure 2A–C) to produce a product that could be weighed by the user and rehydrated to achieve swollen µgels. The medium for lyophilizing the µgels was optimized to prevent cryogel formation (i.e., internal defects) by using 70% ethanol, but it has also been demonstrated that cryogels were achieved using other media for lyophilization if cryogels are desired by the user (Figure 2D). A comprehensive study of different lyophilization media for microgel cryogel formation can be found in the work by Anderson et al.12. Using confocal microscopy, HA-NB microgel diameter was quantified both before and after lyophilization with 70% ethanol (Figure 2E), which indicated no significant change in microgel size with this drying process.
To facilitate bio-orthogonal interlinking of HA-NB µgels, a linear HA-Tet crosslinker was synthesized (Figure 3). Proton NMR spectroscopy showed successful modification of 11% of HA repeat units with a tetrazine pendant group using the steps detailed in this work (Figure 3G), and the reaction yield was 95%. Using the HA-Tet linker, the dried lyo-microgel product (Figure 4A) was rehydrated with specified volumes to achieve different wt% (w/v) formulations of µgels (Figure 4B,C) in MAP scaffolds ranging from 10 µL (4 mm diameter) to 50 µL (8 mm diameter) in size for cell culture experiments and material characterization, respectively. These user-defined wt% of µgels in MAP scaffolds corresponded to unique particle fractions in the scaffolds (Figure 4D,E).
This protocol also detailed the process for seeding cells in MAP scaffolds for 3D culture (Figure 5D) using bio-orthogonal annealing schemes. The lyo-µgels were rehydrated at the desired wt% in the cell media, and then mixed with the cell pellet and HA-Tet for interlinking the µgels. This mixture was then plated in cell culture devices (Figure 5A-C) to yield cells encapsulated in the void space between µgels in MAP scaffolds. Cells in 3D culture in MAP scaffolds were fixed on day 5, stained, and imaged on a confocal microscope as shown in Figure 5E. An example of the cells in the void space of the MAP scaffolds for different wt% formulations is shown in Figure 5F.
Supplemental Coding File 1: CAD file used to 3D print the mold for cell culture devices. Please click here to download this File.
Microfluidic production of HA-NB microgels has been shown to generate microgels with a narrower range of size distribution than emulsion batch production3,9. The microgels described in this protocol were formulated using an MMP-cleavable crosslinker (Ac-GCRDGPQGIWGQDRCG-NH2) to support material degradation. However, HA-NB microgels can also be crosslinked using an alternative di-thiol linker such as dithiothreitol (DTT), which is non-degradable. Similarly, other photo-initiators, such as Irgacure 2959 2-hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone, can be used in the HA-NB precursor instead of LAP to facilitate thiol-ene crosslinking10. These microgels were composed of 3.5 wt% HA based on a previous work that demonstrated the viscosity of a 3.5 wt% precursor solution was amenable for pinching into droplets on a microfluidic platform6. Others have described HA-NB microgel fabrication on flow-focusing microfluidic devices using lower wt% formulations (3 wt% HA-NB) as well8,10,11. While this method is beneficial for producing microgel populations with low polydispersity, it is more time- and labor-intensive than batch emulsion techniques that can be used for HA-NB microgel production as well3.
The microgel lyophilization process relies on freezing crosslinked polymer networks. Defects can occur during freezing when crystal structures in the lyophilization medium form and act as porogens, and the resulting cryogels exhibit enhanced porosity and different mechanical properties compared to the original material19,20. To date, there have been methods described for freeze-drying microgels that prevent cryogel formation for polyethylene glycol (PEG)13, GelMa14, and HA formulations12 using different mediums for lyophilization. For this protocol, the user could interchange 70% ethanol with another lyophilization medium of their choosing if cryogels are desired. While cryogels can be advantageous for some applications, 70% ethanol was highlighted in this protocol as the lyophilization medium because it is a common lab reagent, it incorporates the benefit of sterilization in the process of MAP fabrication, and it allows the HA microgels to retain their original size, shape, and stiffness while also minimizing internal defects once rehydrated12. In addition to these assessments of microgel characteristics, environmental scanning electron microscopy (SEM) could be implemented as a potential tool to assess microgel morphology before and after lyophilization.
The methods described in this work for drying HA-NB microgels were introduced to circumvent the variability in particle fraction and produce consistent MAP scaffolds with user-defined wt% formulations. The study of controlled particle fraction at different wt% rehydration was limited to the study of spherical microgels. Other microgel shapes, such as rods8,21 or irregular shapes10,22, have not been investigated to assess the relationship between wt% MAP and particle fraction. Particle fraction has been assessed in MAP scaffolds using consistent proportions of rehydrated lyo-microgels (84%) and HA-Tet crosslinker (16%) based on a previous study2; however, these fractions could be altered at the user's discretion as long as the HA-Tet volume is sufficient for dissolving HA-Tet at the desired crosslinking ratio. The norbornene-tetrazine click chemistry reaction has been effective in annealing microgels to form MAP scaffolds using bi-functional23, multi-arm3, as well as the linear12 tetrazine linker described in these methods. If desired, the molar ratio of Tet:HA-NB can be varied to achieve different stiffnesses of the bulk MAP scaffolds12. These techniques for rehydrated lyo-microgels have yet to be assessed for other annealing chemistries besides tetrazine-norbornene.
3D cell culture in MAP scaffolds has been described previously with numerous cell types, such as endothelial cells8, fibroblasts1,3,4,24, neural progenitor cells9, and mesenchymal stem cells25,26. Residual ethanol was observed via proton NMR after HA-NB microgels were lyophilized in 70% ethanol; however, it was also confirmed that the trace amounts of ethanol did not impact cell viability12. Culture in MAP scaffolds comprising lyo-microgels has only been demonstrated thus far with mouse mesenchymal stem cells12; however, this protocol for seeding cells in MAP scaffolds with lyo-microgels could be interchanged with other cell types and their corresponding cell media. For D1 cell culture, F-actin intensity and total cell volume has been shown to decrease as the wt% MAP increases12. It has also been shown that increasing the wt% MAP corresponds to an increase in bulk scaffold stiffness (not local microgel stiffness) as the void space between microgels becomes smaller, leading to less cell proliferation in high wt% MAP scaffolds12.
This work described methods for producing HA-NB microgels on a microfluidic platform followed by freeze-drying the microgels to either retain the original microgel properties or produce cryogels. This protocol outlined the synthesis of a tetrazine linker used to interlink the lyo-microgels once they have been rehydrated at user-defined weight percentages to create MAP scaffolds with controlled particle fractions. Lastly, this work detailed the steps for culturing cells within these MAP scaffolds with user-defined microarchitectures. Creating granular scaffolds with consistent particle fractions improves the reproducibility of experimental results for MAP users, extending beyond cell responses to mass transport and mechanical properties as well12. MAP scaffolds generated using these techniques can be used moving forward for in vivo applications. The use of a lyophilized product for granular scaffold fabrication is beneficial for improving the material shelf-life and will also be advantageous for future translation of MAP scaffolds to a clinical setting.
The authors have nothing to disclose.
The authors would like to thank the National Institutes of Health, the National Institutes of Neurological Disorders and Stroke (1R01NS112940, 1R01NS079691, R01NS094599), and the National Institute of Allergy and Infectious Disease (1R01AI152568). This work was performed in part at the Duke University Shared Materials Instrumentation Facility (SMIF), a member of the North Carolina Research Triangle Nanotechnology Network (RTNN), which is supported by the National Science Foundation (award number ECCS-2025064) as part of the National Nanotechnology Coordinated Infrastructure (NNCI). The authors would like to thank the lab's former post-doc Dr. Lucas Schirmer as well as Ethan Nicklow for their assistance in generating the 3D printed device for cell culture experiments.
1 mL Luer-Lok syringe sterile, single use, polycarbonate | BD | 309628 | |
5 mL Luer-Lok syringe sterile, single use, polycarbonate | BD | 309646 | |
Alexa Fluor 488 C5 maleimide | Invitrogen | A10254 | For synthesis of fluorescently-labeled tetrazine |
Alexa Fluor 647 Phalloidin | Invitrogen | A22287 | For staining cell culture samples |
Aluminum foil | VWR | 89107-726 | |
Biopsy punch with plunger, 1.0 mm | Integra Miltex | 69031-01 | |
Biopsy punch, 4 mm | Integra Miltex | 33-34 | |
Blunt needle, 23 G 0.5", Non-Sterile, Capped | SAI Infusion Technologies | B23-50 | |
Bottle-top vacuum filter, 0.22 μm | Corning | CLS430521 | |
Calcium chloride | VWR | 1B1110 | For microgel washing buffer |
Capillary-piston assemblies for positive-displacement pipettes, 1000 μL max. volume | Rainin | 17008609 | |
Capillary-piston assemblies for positive-displacement pipettes, 25 μL max. volume | Rainin | 17008605 | |
Capillary-piston assemblies for positive-displacement pipettes, 250 μL max. volume | Rainin | 17008608 | |
Countess Cell Counting Chamber Slides | Invitrogen | C10228 | |
Countess II FL Automated Cell Counter | Invitrogen | AMQAF1000 | |
Centrifuge tube, 15 mL | CELLTREAT | 667015B | |
Centrifuge tube, 50 mL | CELLTREAT | 229421 | |
Chloroform, ACS grade, Glass Bottle | Stellar Scientific | CP-C7304 | For synthesis of fluorescently-labeled tetrazine |
Corona plasma gun, BD-10A High Frequency Generator | ETP | 11011 | |
CryoTube Vials, Polypropylene, Internal Thread with Screw Cap | Nunc | 368632 | |
D1 mouse mesenchymal cells | ATCC | CRL-12424 | Example cell line for culture in MAP gels |
DAPI | Sigma-Aldrich | D9542 | For staining cell culture samples |
Deuterium oxide, 99.9 atom% D | Sigma-Aldrich | 151882 | For NMR spectroscopy |
Dialysis tubing, regenerated cellulose membrane, 12-14 kDa molecular weight cut-off | Spectra/Por | 132703 | For purifying HA-NB and HA-Tet |
Diethyl ether | VWR | BDH1121-4LPC | For synthesis of fluorescently-labeled tetrazine |
Dimethylformamide | Sigma-Aldrich | 277056 | For synthesis of fluorescently-labeled tetrazine |
4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) | TCI-Chemicals | D2919 | For modifying HA |
Dithiothreitol (DTT) | Thermo Scientific | R0861 | Non-degradable dithiol linker (substitute for MMP-cleavable peptide) |
Dulbecco's Modified Eagle's Medium (DMEM), high glucose, w/ 4500 mg/L glucose, L-glutamine, sodium pyruvate, and sodium bicarbonate, liquid, sterile-filtered, suitable for cell culture | Sigma-Aldrich | D6429-500ML | For D1 cell culture |
EMS Paraformaldehyde, Granular | VWR | 100504-162 | For making 4% PFA |
Ethanol absolute (200 proof) | KOPTEC | 89234-850 | |
Fetal bovine serum (FBS) | ATCC | 30-2020 | For D1 cell culture |
Heating Plate | Kopf Instruments | HP-4M | |
Hemacytometer with coverglass | Daigger Scientific | EF16034F | |
2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) | Sigma-Aldrich | H3375 | |
Sodium hyaluronate, 79 kDa average molecular weight, produced in bacteria Streptococcus zooepidemicus, pharmaceutical grade, microbial contamination <100 CFU/g, bacterial endotoxins <0.050 IU/mg | Contipro | N/A | 79 kDa average molecular weight was used for HA-Tet synthesis, but these methods could be adapted for other molecular weights. |
IMARIS Essentials software package | Oxford Instruments | N/A | Microscopy image analysis software |
Infusion pump, dual syringe | Chemyx | N/A | |
Kimwipe | Kimberly-Clark | 34120 | |
Laboratory stand with support lab clamp | Geyer | 212100 | |
Liquid nitrogen | Airgas | NI 180LT22 | |
Lithium Phenyl(2,4,6-trimethylbenzoyl)phosphinate | TCI-Chemicals | L0290 | |
Lyophilizer | Labconco | N/A | Labconco FreeZone 6 plus has been discontinued, but other lab grade console freeze dryers could be used for this protocol. |
Methyltetrazine-PEG4-maleimide | Kerafast | FCC210 | For synthesis of fluorescently-labeled tetrazine |
2-(4-Morpholino)ethane Sulfonic Acid (MES) | Fisher Scientific | BP300-100 | For modifying HA |
Micro cover glass, 24 x 60 mm No. 1 | VWR | 48393-106 | |
Microfluidic device SU8 master wafer | FlowJem | Custom design made either in-house in clean room or outsourced | |
Mineral oil, heavy | Sigma-Aldrich | 330760 | |
MMP-cleavable dithiol crosslinker peptide (Ac-GCRDGPQGIWGQDRCG-NH2) | GenScript | N/A | |
5-Norbornene-2-methylamine | TCI-Chemicals | 95-10-3 | For HA-NB synthesis |
Packing tape | Scotch | 3M 1426 | |
Parafilm | Bemis | PM996 | |
PEG(thiol)2 | JenKem Technology USA | A4001-1 | For synthesis of fluorescently-labeled tetrazine |
Penicillin-Streptomycin, 10,000 units/mL | Thermo Fisher Scientific | 15140122 | For D1 cell culture |
Petri dish, polystyrene, disposable, Dia. x H=150 x 15 mm | Corning | 351058 | |
Pluronic F-127 | Sigma-Aldrich | P2443 | For washing HMPs |
Phosphate buffered saline (PBS) 1x | Gibco | 10010023 | |
RainX water repellent glass treatment | Grainger | 465D20 | Synthetic hydrophobic treatment solution for microfluidic device treatment |
RGD peptide (Ac-RGDSPGERCG-NH2) | GenScript | N/A | |
Rubber bands | Staples | 112417 | |
Sodium chloride | Chem-Impex | 30070 | For dialysis |
Span 80 for synthesis | Sigma-Aldrich | 1338-43-8 | |
Sylgard 184 Silicone Elastomer | Electron Microscopy Science | 4019862 | polydimethylsiloxane (PDMS) elastomer for making microfluidic devices and tissue culture devices |
Syringe filter, Whatman Uniflo, 0.2 μm PES, 13 mm diameter | Cytvia | 09-928-066 | |
Tetraview LCD digital microscope | Celestron | 44347 | |
Tetrazine-amine HCl salt | Chem-Impex | 35098 | For HA-Tet synthesis |
Triethylamine | Sigma-Aldrich | 471283 | For synthesis of fluorescently-labeled tetrazine |
Tris(2-carboxyethyl)phosphine (TCEP) | Millipore Sigma | 51805-45-9 | |
Triton X-100 | VWR | 97063-864 | |
Trypan blue solution, 0.4% | Thermo Fisher Scientific | 15250061 | |
Trypsin EDTA (0.25%), Phenol red | Fisher Scientific | 25-200-056 | For lifting adherent cells to seed in MAP gels |
Tygon ND-100-80 Non-DEHP Medical Tubing, Needle Gauge=23, Wall Thickness=0.020 in, Internal diameter = 0.020, Outer diameter = 0.060 in | Thomas Scientific | 1204G82 | |
UV curing system controller, LX500 LED | OmniCure | 010-00369R | |
UV curing head, LED spot UV | OmniCure | N/A | |
UV light meter, Traceable | VWR | 61161-386 | |
Vacuum dessicator | Bel-Art | 08-594-15C | |
X-Acto Z Series Precision Utility Knife | Elmer's | XZ3601W |