An injectable tissue engineering scaffold composed of poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAm-g-CS)-containing alginate microparticles was prepared. The adhesive strength, swelling properties and in vitro biocompatibility are analyzed in this study. The characterization techniques developed here may be applicable to other thermogelling systems.
Injectable biomaterials are defined as implantable materials that can be introduced into the body as a liquid and solidify in situ. Such materials offer the clinical advantages of being implanted minimally invasively and easily forming space-filling solids in irregularly shaped defects. Injectable biomaterials have been widely investigated as scaffolds for tissue engineering. However, for the repair of certain load-bearing areas in the body, such as the intervertebral disc, scaffolds should possess adhesive properties. This will minimize the risk of dislocation during motion and ensure intimate contact with the surrounding tissue, providing adequate transmission of forces. Here, we describe the preparation and characterization of a scaffold composed of thermally sensitive poly(N-isopropylacrylamide)-graft-chondroitin sulfate (PNIPAAM-g-CS) and alginate microparticles. The PNIPAAm-g-CS copolymer forms a viscous solution in water at RT, into which alginate particles are suspended to enhance adhesion. Above the lower critical solution temperature (LCST), around 30 °C, the copolymer forms a solid gel around the microparticles. We have adapted standard biomaterials characterization procedures to take into account the reversible phase transition of PNIPAAm-g-CS. Results indicate that the incorporation of 50 or 75 mg/ml alginate particles into 5% (w/v) PNIPAAm-g-CS solutions quadruple the adhesive tensile strength of PNIPAAm-gCS alone (p<0.05). The incorporation of alginate microparticles also significantly increases swelling capacity of PNIPAAm-g-CS (p<0.05), helping to maintain a space-filling gel within tissue defects. Finally, results of the in vitro toxicology assay kit, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and Live/Dead viability assay indicate that the adhesive is capable of supporting the survival and proliferation of encapsulated Human Embryonic Kidney (HEK) 293 cells over 5 days.
Injectable biomaterials are those that can be conveniently delivered into the body as a liquid and solidify in situ. Such materials have been applied extensively in regenerative medicine, where they are used to deliver encapsulated cells to the affected site 1-4 and act as a three-dimensional temporary extracellular matrix for the cells 5. For the patient, injectable biomaterials are advantageous because the surgical procedures for implantation are minimally invasive and the solid phase can fill irregularly shaped tissue defects, eliminating the need for custom-size implants.
Injectability can be achieved through a variety of mechanisms. External factors, like pH, have been investigated as a trigger for the formation of gels that encapsulate cells and bioactive molecules6-8. However, pH may not be the most convenient trigger to use in all physiological environments. Another traditional alternative for achieving injectability is using in situ chemical polymerization or crosslinking. A group developed a water-soluble redox system composed of ammonium persulfate and N,N,N',N'-tetramethylethylenediamine and used it for reacting macromers composed of polyethylene glycol and poly(propylene) glycol 9,10. Zan et al. 11 developed injectable chitosan polyvinylalcohol networks crosslinked with glutaraldehyde. In such systems, the cytotoxicity of reactive components must be considered, especially for applications involving cell encapsulation. Also, exothermic polymerization could produce high enough temperatures to compromise surrounding tissue, which has been reported for polymeric bone cements 12,13.
Still other injectable polymer systems have been developed that exhibit a change from the liquid to solid state with temperature as the trigger. Known as thermogelling systems, these are aqueous polymer solutions that do not require chemical stimulus, monomers, or crosslinkers to achieve in situ formation 14. Rather, a phase transition usually occurring near physiological temperature induces the formation of a physically crosslinked three-dimensional network. Poloxamers such as Pluronic F127 are among the most widely studied polymers for thermogelling drug delivery 15-17 and cell encapsulation 18,19. However, it is well accepted that these gels lack stability at physiological conditions. Studies have demonstrated increased stability using chain extenders 20 or chemical crosslinkers 21,22. Nevertheless, the use of these reagents may limit the potential of the materials for cell encapsulation.
Poly(N-isopropylacrylamide) is a synthetic thermogelling polymer that has received significant attention in tissue engineering and drug delivery 14. Aqueous solutions of poly(N-isopropylacrylamide) (PNIPAAm) exhibit a lower critical solution temperature (LCST), typically occurring around 32 – 34 °C 23,24. Below the LCST, water hydrates PNIPAAm chains. Above the transition temperature, the polymer becomes hydrophobic, resulting in a dramatic phase separation 25-27 and formation of a solid gel without the use of toxic monomers or crosslinkers. However, PNIPAAm homopolymers exhibit poor elastic properties and hold little water at physiological temperature due to hydrophobicity 28. In this work, we choose to incorporate chondroitin sulfate covalently into the PNIPAAm network, which offers the potential for enzymatic degradability 29, anti-inflammatory activity 30,31, and increased water and nutrient absorption 32. PNIPAAm copolymers with CS were prepared in our laboratory by polymerizing the monomer NIPAAm in the presence of methacrylate-functionalized CS to form grafted copolymer (PNIPAAm-g-CS). Because of the low crosslinking density of the copolymer, PNIPAAm-g-CS forms a viscous solution in water at RT and an elastic gel at physiological temperature due to the LCST 29. The polymer solutions become flowable again upon cooling below the LCST due to the reversibility of the transition.
We have demonstrated that PNIPAAm-g-CS has the potential to function as a tissue engineering scaffold, due to mechanical properties that can be tailored, degradability, and cytocompatibility with human embryonic kidney (HEK) 293 cells 29. However, in certain load bearing areas, such as the intervertebral disc, tissue engineering scaffolds should have the ability to form a substantial interface with surrounding disc tissue to eliminate the risk of dislocation 33. This interface is also necessary for the adequate transmission of force across the interface between the implant and the tissue 33. In our work, we have suspended alginate microparticles in aqueous solutions of PNIPAAm-g-CS and found that gelation localizes the microparticles, which provide adhesion with surrounding tissue 34. In this paper, we outline the steps for preparation of the thermogelling, adhesive polymer. Standard techniques for biomaterials characterization, cell imaging, and assays for viability were adapted to take into account the temperature sensitivity of the polymer and the reversibility of the phase transition. The injectable polymer described in this paper has wide potential for drug delivery and tissue engineering applications outside of those described in this paper. Moreover, the characterization methods described here may be applicable to other thermogelling systems.
1. Poly(N-isopropylacrylamide)-g-chondroitin Sulfate Synthesis
2. Calcium-alginate Crosslinked Microparticle Synthesis
3. Preparation of the Adhesives
4. Bioadhesive Mechanical Tensile Tests
5. Swelling Study of PNIPAAm-g-CS with Alginate Microparticles
6. Qualitative Cell Viability Using a Live/Dead Assay
7. Quantitative Cell Viability Using an XTT Assay
A thermally responsive grafted co-polymer was successfully synthesized and characterized for its bioadhesive strength, swelling properties, and in vitro cytocompatibility. We chose to investigate alginate due to its well-established mucoadhesive properties. Alginate microparticles, with an average diameter of 59.7 ± 14.9 µm, were blended with 5% (w/v) PNIPAAm-g-CS at concentrations of 25, 50, and 75 mg/ml. These concentrations were based on one-half, equal to, and twice the equivalent dry weight of PNIPAAm-g-CS in aqueous solution. Microparticle concentrations above 75 mg/ml exhibited excessive viscosity and were not investigated. The maximum tensile adhesive strength of 5% (w/v) PNIPAAm-g-CS with varying concentrations of alginate microparticles were compared to the hydrogel alone. The addition of 50 or 75 mg/ml of alginate microparticles suspended in 5% (w/v) PNIPAAm-g-CS resulted in a four-fold increase in tensile strength (p<0.05), as shown in Figure 1. A microparticle concentration of 25 mg/ml did not show a significant increase in tensile strength compared to 5% (w/v) PNIPAAm-g-CS (p>0.05). There was no significant change in the tensile strength of 5% (w/v) PNIPAAm-g-CS when the concentration of alginate microparticles was increased from 50 to 75 mg/ml (p>0.05).
The swelling properties of the bioadhesive hydrogel were characterized by immersing various formulations in PBS at 37 °C. Alginate microparticle concentrations of 25 and 50 mg/ml were tested to identify any differences in swelling behavior. Alginate microparticles were measured using light microscopy and had an average diameter of 25.0 ± 14.4 µm. After 7 days, the initial and final wet mass of the hydrogel discs were recorded and a change in mass was calculated for multiple samples per adhesive formulation (n = 5). Figure 2 shows a drastic difference in the change in mass amongst all formulations. Adhesives containing both 25 and 50 mg/ml of alginate microparticles demonstrated positive changes in mass, while 5% (w/v) PNIPAAm-g-CS decreased in mass. All samples were found to be significantly different from one another (p<0.05) with the adhesive containing 50 mg/ml of alginate microparticles showing the greatest swelling effect.
The biocompatibility of the proposed adhesive was investigated both qualitatively and quantitatively through the use of a Live/Dead and XTT cytotoxicity assay. HEK-293 cells were encapsulated in both 5% (w/v) PNIPAAm-g-CS and 5% (w/v) PNIPAAm-g-CS containing 50 mg/ml of alginate microparticles with an average diameter of 59.7 ± 14.9 µm. A monolayer of cells and encapsulated cells in PNIPAAm-g-CS killed with 70% methanol were considered the positive and negative controls, respectively. Formulations seeded with cells were characterized after 5 days of growth in a humidified incubator at 37 °C with 5% CO2. Each test indicated good cell viability for both PNIPAAm-g-CS and PNIPAAm-g-CS with microparticles. Images from Figure 3 illustrate living cells fluorescing green in both adhesive formulations (Panels A and B) and the cell monolayer, serving as the positive control (Panel C). Killed cells (Panel D) fluoresce a red color and show cell death. Results from the XTT cytotoxicity tests confirmed the results of the Live/Dead assay. The relative cell viability of the adhesive containing 50 mg/ml of microparticles decreased 1.3 fold compared to PNIPAAm-g-CS as illustrated in Figure 4, but the decrease was not statistically significant (p>0.05). Both PNIPAAM-g-CS and PNIPAAM-g-CS with microparticles also had comparable viability to the cell monolayer (p>0.05). Encapsulated cells in hydrogel formulations and the cell monolayer were found to be significantly different compared to the negative control (p<0.05). Overall, these results suggest that the adhesive containing microparticles not only exhibits increased adhesive strength, but good biocompatibility as well.
Figure 1. The Effects on Tensile Strength After Incorporating Varying Concentrations of Alginate Microparticles into 5% (w/v) PNIPAAm-g-CS. The tensile strength of PNIPAAm-g-CS quadrupled with the addition of 50 or 75 mg/ml of alginate microparticles (p<0.5). Error bars represent a calculated 95% confidence interval (n = 5). Alginate microparticles had an average size of 59.7 ± 14.9 µm. Please click here to view a larger version of this figure.
Figure 2. The Effect of Varying Concentrations of Alginate Microparticles on the Swelling Capacity of 5% (w/v) PNIPAAm-g-CS. After 7 days immersion in 37 °C PBS, PNIPAAm-g-CS decreased in wet mass due to the hydrophobic properties of PNIPAAm above the LCST. PNIPAAm-g-CS with 25 or 50 mg/ml of alginate microparticles exhibited swelling capability. Increasing the concentration of microparticles significantly increased (p<0.05) the change in mass of the hydrogel over 7 days, attributed to water uptake. Error bars represent a calculated 95% confidence interval. Alginate microparticles had an average size of 25.0 ± 14.4 µm. Please click here to view a larger version of this figure.
Figure 3. Representative Live/Dead Fluorescence Images of Cultured HEK-293 cells Over a Period of 5 Days. Cells were encapsulated in (A) PNIPAAm-g-CS and (B) PNIPAAm-g-CS with 50 mg/ml of alginate microparticles. Cells were also grown in a (C) monolayer and (D) killed with methanol in PNIPAAm-g-CS to represent a positive and negative control, respectively. The Live/Dead viability experiment was repeated three times with three replicates per experiment. Scale bars represent 100 µm. Alginate microparticles had an average size of 59.7 ± 14.9 µm. Please click here to view a larger version of this figure.
Figure 4. Short-term Viability of HEK-293 Cells in PNIPAAm-g-CS with Alginate Microparticles. Survival of encapsulated HEK-293 cells within PNIPAAm-g-CS with and without alginate microparticles was assessed and compared using XTT after 5 days. A cell monolayer and encapsulated cells in PNIPAAm-g-CS killed with 70% methanol were used as positive and negative controls, respectively. Both PNIPAAm-g-CS and PNIPAAm-g-CS with alginate microparticles showed no significant reduction (p>0.05) in cell viability compared to the cell monolayer, yet showed a significant reduction (p<0.05) in cell viability compared to the killed cells. The addition of alginate microparticles does not significantly compromise the cell viability of PNIPAAm-g-CS (p>0.05). Error bars represent a calculated 95% confidence interval (n = 3, six replicates per experiment). Alginate microparticles had an average size of 59.7 ± 14.9 µm. Please click here to view a larger version of this figure.
There are several critical steps in synthesizing the hydrogel-microparticle composite and evaluating its adhesive strength, swelling ability, and cellular biocompatibility. Free radical polymerization of PNIPAAm-g-CS requires successful methacrylation of chondroitin sulfate, complete dissolution of monomer components, and oxygen-free reaction conditions. The ratio of NIPAAm monomer to methacrylated chondroitin sulfate in the reaction mixture was chosen because it has been demonstrated, in our previous work, to generate copolymers with mechanical properties similar to native intervertebral disc tissue 29. Increasing the amount of chondroitin sulfate in the hydrogel will reduce water loss during gelation and thus decrease the compressive stiffness of the gel. The selected degree of methacrylation has also been demonstrated to produce hydrogels with favorable handling properties for injectability through an 18 G needle when dissolved in aqueous solution 29. An increased degree of methacrylate substitution of the CS will increase the crosslink density of the polymer network, causing increased solution viscosity below the LCST and reducing gel swelling capability. Others have investigated different approaches in synthesizing PNIPAAm hydrogels such as atom transfer radical polymerization (ATRP) 35 or reverse addition-fragmentation chain transfer (RAFT) 36. Polymerization via ATRP has led to the development of narrow, polydisperse hydrogels with fined tuned molecular weight by altering the monomer to initiator ratio. Functional groups can be grafted in a comb-like manner using RAFT polymerization, thus impacting polymer chain entanglement and phase separation.
A homogenous water-in-oil mixture is necessary in creating uniform, cross-linked alginate microparticles. An emulsion technique is simple to perform and does not require the use of hazardous chemicals, however both microparticle shape and distribution are difficult to control. Microparticle size can be altered by increasing the emulsion stir speed, thus creating smaller particles and vice-versa. In this particular study, alginate microparticle size was investigated as a potential property that may impact swelling, mechanics, or cellular viability. Light scattering may also be used as an alternative to measuring the average microparticle size. Other emulsion properties such as the surfactant type and alginate concentration will impact microparticle size and aggregate formation 37. Decreasing the alginate concentration will reduce the microparticle size, however aggregates are more likely to form. Variations in the hydrophilic and hydrophobic nature of surfactants will affect the surface tension between the oil and water phases, hence changing the droplet size within the emulsion. Alginate microparticles may alternatively be produced using a microfluidic device, which allows for uniform particle shape and narrow size distribution 38. This technique involves dispersing an alginate phase within a continuous oil phase that flows through a channel and enters a calcium chloride bath. Spray draying is another technique which produces very small microparticles 39. It is also important to note that alginate may be loaded with proteins or growth factors and can be cross-linked with other divalent ions such as zinc or barium.
In this work, we characterize an adhesive system with potential applications in tissue engineering. Our tensile test specifically aims to quantify the adhesive strength of the composite when in contact with porcine cartilage. This type of substrate was selected to mimic adhesion of the hydrogel composite to the cartilage end plates of the intervertebral disc. Other tissue substrates, such as porcine skin, may be used as an alternative to quantify the strength of an adhesive 40, but the tissue type will vary depending on the intended application. Maintaining proper temperature control of the saline water bath and pre-cooling the composite before use are crucial in accurately measuring the mechanical properties. Elevated water bath temperatures above 37 °C will cause the hydrogel to rapidly shrink and stiffen, while composites that are mechanically tested below 37 °C will exhibit decreased tensile strength due to the lack of phase separation. Currently, we are developing methods to investigate the shear adhesive strength of various injectable adhesive formulations. Future work will include performing ex vivo mechanical tests with porcine IVDs to determine whether the adhesive resists extrusion through an annular defect and restores biomechanical functionality to the spinal motion segment.
Above 32 °C, PNIPAAm-g-CS shrinks and expels water from the hydrogel matrix due to the LCST behavior of PNIPAAm, resulting in a decreased mass over a 7 day period in PBS (Figure 2). Conversely, addition of alginate microparticles imparts a swelling effect and the adhesive significantly increased in mass (p<0.05) at a microparticle concentration of 25 mg/m and even more so at 50 mg/ml. In this swelling study, changes in wet mass of the hydrogel are likely attributable to water exchange with the surroundings, since PNIPAAm-g-CS has been shown to be selectively degradable in the presence of enzymes 29. Many of the protocol steps developed for the swelling study were adapted based on the thermoreversibility of PNIPAAm-g-CS. Hydrogel discs must fully transition at 37 °C before adding the pre-warmed PBS solution, otherwise the gel will disperse. After swelling for one week, the PBS solution must be quickly and carefully removed from the vials before the hydrogels revert back to a liquid-like state. It should be noted that the swelling protocol described in this paper does not fully simulate the in vivo environment of the native IVD. The swelling properties of the scaffold in vivo will depend on the osmotic pressure of the surrounding tissues 41 and degradation behavior of the gel. However, in general, the increased swelling capacity with alginate microparticle incorporation is considered favorable, as it will help the injectable hydrogel to stay space-filling at physiological temperature. Characteristics such as the swelling ratio and water content may be calculated with additional dry mass measurements. Future swelling studies will include immersing hydrogel discs against a polyethylene glycol (MW = 20,000 g/mol) bath, which will apply an osmotic pressure and mimic the in vivo environment of the IVD.
Aside from the mechanical and swelling properties, the adhesive must exhibit cellular biocompatibility in order to be considered as a suitable scaffold. Previous studies have demonstrated the survival of encapsulated cells in alginate 42,43 and PNIPAAm-based materials 44,45. Results from both the Live/Dead and XTT assays are consistent with these prior findings, suggesting appropriate cell viability over a period of 5 days (Figures 3 and 4). Our laboratory has also conducted preliminary investigations of viability over a longer time course (21 days) and observed similar behavior. In general, a surplus of PNIPAAm-g-CS and microparticles should be prepared to account for any material loss during pipette transfers. Once again, it is extremely critical to preserve scaffold architecture by maintaining proper temperature conditions at 37 °C. If the scaffold network reverts from a gel to a liquid-like state, cell attachment and viability may become compromised. With regards to the Live/Dead assay, sodium citrate plays an important role in the removal and deconstruction of alginate microparticles. Sodium citrate reverses the cross-linking action between calcium and alginate and causes a slurry to form 46. Additional washes with PBS to remove PNIPAAm-g-CS or alginate may be performed to enhance the quality of the pictures. During the XTT assay, polymer samples containing no cells are created within the well plates. These replicates serve as blank standards and are required to calculate an accurate absorbance value. Our preliminary viability studies are strictly based on using HEK-293 cells. In future studies, the differentiation of encapsulated adipose-derived mesenchymal stem cells (AD-MSC) into nucleus pulposus cells will be demonstrated. AD-MSC differentiation toward NP-like phenotype has been demonstrated by using dynamic compression 47 and hypoxia 48,49.
Overall, the proposed adhesive demonstrates promise for tissue engineering applications in load bearing areas, such as the intervertebral disc, where success of the regeneration strategy will depend on scaffold ability to resist dislocation during motion and loading. This system is very versatile in that it could potentially be adapted to release extracellular matrix proteins or growth factors specific to the desired application. Importantly, the methods described here can be adapted to any characterization study with thermogelling polymers.
The authors have nothing to disclose.
The authors would like to gratefully acknowledge the assistance of Dr. Jennifer Kadlowec in the development of the adhesive tensile testing protocol.
Research reported in this publication was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases and the National Institute of Biomedical Imaging and Bioengineering of the National Institutes of Health under Award Number 1R15 AR 063920-01. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
N-isopropylacrylamide, 99%, pure, stabilized | Acros Organics | 2210-25-5 | Refrigerate and remove stabilier with hexane |
Chondroitin sulfate A sodium salt (from bovine trachea) | Sigma-Aldrich | 39455-18-0 | Refrigerate |
Hexanes | Fisher Scientific | H302-4 | Store in a flammable cabinet |
50% (w/w) sodium hydroxide | Fisher Scientific | SS254-1 | Caustic in nature |
Methacrylic anhydride | Sigma-Aldrich | 276685 | Strong fumes; use in a fume hood |
Acetone | Fisher Scientific | A18-4 | Chill in a refrigerator prior to use |
Nitrogen Gas | Praxair | 7727-37-9 | Part Number: NI 4.8, cylinder style T, 99.998% pure nitrogen (Argon may be used as an alternative inert gas) |
Tetramethylethylenediamine, 99% extra pure | Acros Organics | 110-18-9 | |
Ammonium persulfate | Sigma Aldrich | A3678 | Hygroscopic and degrades in the presence of water |
Phosphate buffered saline tablets | Fisher Scientific | BP2944 | Keep dry |
Alginic acid, sodium salt | Acros Organics | 177775000 | Use heat to aid in dissolving |
Calcium chloride dihydrate | Fisher Scientific | C79 | |
Canola oil | Local store | Obtain from a local store | |
Tween 20 | Sigma-Aldrich | 93773 | |
70% (v/v) Isopropoanol | Fisher Scientific | A416-4 | |
Porcine ears | Haine's Pork Shop | Obtain from a local butcher | |
Sodium Chloride | Fisher Scientific | S271-3 | |
Human embryonic kidney 293 cells | ATCC | ATCC CRL-1573 | Store in liquid nitrogen for long-term use |
DMEM: 1X, high glucose, no pyruvate | Life Technologies | 11965126 | Refrigerate |
Fetal bovine serum | Life Technologies | 10082-147 | Refrigerate |
Penn Strep: 10,000 U/ml | Life Technologies | 15140-122 | Refrigerate |
Trypsin-EDTA: 0.5%, 10X | Life Technologies | 15400-054 | Refrigerate |
Methanol | VWR | AAA44571-K7 | |
Live/Dead Cell viability kit | Life Technologies | L3224 | Light sensitive, keep frozen |
XTT cell viability kit | Sigma Aldrich | TOX2-1KT | Light sensitive, keep frozen |
Clear DMEM: 1X, high glucose, no phenol | Life Technologies | 21063-029 | Refrigerate |
Dulbecco's PBS: 1X | Life Technologies | 14190136 | Refrigerate |
Sodium citrate | EMD | SX0445-1 | |
Positive displacement pipette | BrandTech Scientific, INC | 2702904 | Dispenses 100 – 500 µL and comes with attachable tips |
No 3. Stainless Steel scalpel handle | Sigma Aldrich | S2896 | |
Miltex sterile surgical blades | Fisher Scientific | 12-460-440 | Size 10 |
Power gem homogenizer | Fisher Scientific | 08-451-660 | Model # 125 |
Porcelain mortar and pestle | Sigma Aldrich | Z247464 | Holds 50 mL |
FreeZone 1 L benchtop freeze dry system | Labconco | 7740020 | Freeze samples prior to use |
Oil sealed rotary vane pump | Edwards | A65301906 | Model # RV5 |
Incubating orbital shaker | VWR | 12620-946 | Model # 980153 |
Benchtop refrigerated centrifuge | Forma Scientific, INC | Model # 5682 | |
Heated ovens | VWR | Model # 1235PC | |
2 N force gauge | Shimpo | FGV-0.5XY | Model # FGV-0.5XY |
E-force test stand | Shimpo | FGS-200PV | Model # FGS-200PV |
Tissue culture swinging bucket centrifuge | Beckman Coulter | 366830 | Model #6S-6KR |
Tissue culture microcentrifuge | Eppendorf | Model #5415C | |
Hemacytometer set | Hausser Scientific | 3720 | Requires replacement cover glass slips |
Slide warmer | Lab Scientific | XH-2022 | Model # XH-2002 |
Portable heating lamp | Underwriters Laboratories | Helps to maintain polymer temperature at 37°C | |
Inverted fluorescent microscope | Zeiss | Model Axiovert 25 CFL | |
Heated water bath | VWR | Model # 1235PC | |
Rocking platform | VWR | Series 100 | |
Multiskan FC microtiter plate reader | Thermo Scientific | Type 357 | |
Cell culture incubator | VWR | Model # 2350T | |
Purifier class II biosafety cabinet | Labconco | Delta Series |