Here we describe a method to generate three-dimensional spheroid cultures of human nasal epithelial cells. Spheroids are then stimulated to drive Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)-dependent ion and fluid secretion, quantifying the change in the spheroid luminal size as a proxy for CFTR function.
While the introduction of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) modulator drugs has revolutionized care in Cystic Fibrosis (CF), the genotype-directed therapy model currently in use has several limitations. First, rare or understudied mutation groups are excluded from definitive clinical trials. Moreover, as additional modulator drugs enter the market, it will become difficult to optimize the modulator choices for an individual subject. Both of these issues are addressed with the use of patient-derived, individualized preclinical model systems of CFTR function and modulation. Human nasal epithelial cells (HNEs) are an easily accessible source of respiratory tissue for such a model. Herein, we describe the generation of a three-dimensional spheroid model of CFTR function and modulation using primary HNEs. HNEs are isolated from subjects in a minimally invasive fashion, expanded in conditional reprogramming conditions, and seeded into the spheroid culture. Within 2 weeks of seeding, spheroid cultures generate HNE spheroids that can be stimulated with 3′,5′-cyclic adenosine monophosphate (cAMP)-generating agonists to activate CFTR function. Spheroid swelling is then quantified as a proxy of CFTR activity. HNE spheroids capitalize on the minimally invasive, yet respiratory origin of nasal cells to generate an accessible, personalized model relevant to an epithelium reflecting disease morbidity and mortality. Compared to the air-liquid interface HNE cultures, spheroids are relatively quick to mature, which reduces the overall contamination rate. In its current form, the model is limited by low throughput, though this is offset by the relative ease of tissue acquisition. HNE spheroids can be used to reliably quantify and characterize CFTR activity at the individual level. An ongoing study to tie this quantification to in vivo drug response will determine if HNE spheroids are a true preclinical predictor of patient response to CFTR modulation.
Cystic Fibrosis (CF) is a fatal, autosomal recessive disease affecting over 70,000 people worldwide1. This life-shortening genetic disease is caused by mutations in the Cystic Fibrosis Transmembrane conductance Regulator protein (CFTR)2. CFTR is a member of the adenosine triphosphate-binding cassette family, and functions as an ion channel allowing movement of chloride and bicarbonate across the apical membranes of multiple polarized epithelia including the gastrointestinal tract, sweat gland, and respiratory tree, among others3,4. As such, dysfunctional CFTR leads to multisystem epithelial dysfunction, with most mortality stemming from the respiratory disease1. In the CF lung, loss of CFTR-driven airway surface liquid (ASL) regulation and mucus release leads to thickened mucus, airway obstruction, chronic infection, and progressive airway remodeling and loss of lung function1,5.
Despite the identification of CFTR dysfunction as the cause of disease, therapies in CF traditionally focused on mitigation of symptoms (e.g., pancreatic enzyme replacement therapy, airway clearance therapies)1. This approach was recently revolutionized by the advent of novel therapeutics, termed "CFTR modulators," that directly target dysfunctional CFTR. This approach has shifted the clinical landscape from symptom management to disease-modifying care but carries several limitations6,7,8,9,10. Modulator activity is specific to the protein defect accompanying each CFTR mutation and limited to select genetic populations11. This limitation is driven by the heterogeneous nature of protein defects, but also by the impracticality of clinical trials in rare mutation groups. In addition, among subjects with well-studied genotypes (e.g., F508del/F508del CFTR, the most common CFTR mutation), there is wide variability in disease burden and modulator response6,7,8,9,11.
To overcome both of these issues, investigators have proposed the use of personalized models for preclinical testing12. This concept utilizes patient-specific tissue to generate an individualized ex vivo model system for compound testing, predicting in vivo subject response to therapies in a personalized fashion. Once validated, such a model could be used by clinicians to drive precision therapy regardless of the patient's underlying CFTR genotype.
Human bronchial epithelial (HBE) cells obtained from explant tissue at the time of lung transplant established the possibility of such a model for CF13,14. HBEs grown at an air-liquid interface (ALI) allow for functional CFTR quantification directly through electrophysiologic testing or indirectly through measures of ASL homeostasis13,15. This model has been critical to understanding CFTR biology and was a key driver in the development of CFTR modulators16. Unfortunately, HBE models are not tenable as a personalized model due to the invasive nature of acquisition (lung transplant or bronchial brushing) and lack of samples for those with rare mutations or mild disease. Conversely, intestinal tissue, obtained from rectal or duodenal biopsy specimens, can be used for intestinal current measurement (ICM) or a swelling-based organoid assay to study individualized CFTR function17,18,19. Organoid assays, in particular, are very sensitive models of CFTR activity20,21,22. Both models are limited by the tissue source (intestinal tissue, while most disease pathology is respiratory) and the semi-invasive method of acquisition. Alternatively, several investigators have studied human nasal epithelial (HNE) cells to model CFTR restoration23,24,25. HNEs can be safely harvested by brush or curettage in subjects of any age and, when cultured in ALI, recapitulate many characteristics of HBEs25,26,27,28. HNE monolayer cultures have traditionally been limited by squamous transformation and a long time to maturity29. Moreover, reported short-circuit current measurements in HNEs are smaller than those in HBEs, suggesting a smaller window to detect therapeutic efficacy25.
Given the need for a personalized, non-invasive, respiratory tissue culture model of CFTR function, we sought to merge the sensitivity of a swelling-based, organoid assay with the non-invasive and respiratory nature of HNEs. Here, we describe our method of generating 3-dimensional "spheroid" cultures of HNEs for individualized CFTR study in a swelling-based assay30. HNE spheroids polarize reliably with the epithelial apex towards the sphere center, or lumen. This model recapitulates numerous characteristics of a lower respiratory epithelium and matures more quickly than ALI cultures. As a functional assay, HNE spheroids reliably quantify a range of CFTR function, as well as modulation in well-studied mutation groups (e.g., F508del CFTR). This swelling-based assay capitalizes on the ion/salt transport properties of CFTR, indirectly measuring fluid influx into the spheroid as water follows apical salt efflux. In this fashion, stimulated spheroids with fully functional CFTR swell robustly, while those with dysfunctional CFTR swell less or shrink. This is quantified through image analysis of pre- and 1 h post-stimulation spheroids, measuring luminal area and determining the percent change. This measure can then be compared across experimental groups to screen for drug bioactivity in a patient-specific fashion.
HNE samples were procured from subjects recruited through the Cincinnati Children's Hospital Medical Center CF Research Center. All methods described here have been approved by the Institutional Review Board (IRB) of Cincinnati Children's Hospital Medical Center. Written consent was obtained from all subjects prior to testing.
1. Prepare Expansion Media and Antibiotic Media
2. Prepare Differentiation Media
3. Coat Culture Dishes and Plate Feeder Fibroblasts
NOTE: Perform all steps under clean conditions in the biosafety cabinet.
4. Obtain and Process HNE Sample
5. Process and Expand HNE Cells in Dishes
NOTE: Perform all steps under clean conditions in the biosafety cabinet.
6. Passage HNE Cells
7. Seeding Cells for HNE Spheroid Cultures
NOTE: Carry out all procedures in this step, other than centrifugation, in a clean biosafety cabinet.
8. Differentiate and Mature HNE Spheroids
9. Pretreat, Stimulate, and Image HNE Spheroids for Analysis
10. Analyze HNE Spheroid Images
HNEs should attach to the culture dish and form small islands of cells within 72 h of seeding; examples of good and poor island formation at one week are shown in Figure 1A and 1B, respectively. These islands should expand to cover the dish over the course of 15-30 days. Small or suboptimal samples may take longer, and often will not yield useful spheroids. Contamination with infectious agents is evidenced by deep yellow/cloudy media, failure of the cells to attach to the culture dish, and/or direct visualization of fungi/bacteria. Any contaminated cultures should be immediately discarded to avoid cross-contamination.
Within the first 3-4 days of culture in the basement membrane matrix, small cystic structures should begin to form in the matrix. These will mature over approximately 10 days into intact spheroids demonstrating a thin wall and a luminal surface. If plated at the described density, successful cultures will contain 50-100 spheroids per matrix drop. The lumens may be relatively clear (Figure 1C) or filled with cellular debris and mucus (Figure 1D); the former is more common in spheroids with wild-type CFTR (wtCFTR), and the latter in CF spheroids. Masking to delineate the luminal area of the spheroids in Figure 1C and 1D is demonstrated in Figure 1E and 1F, respectively. Examples of poorly formed/unsuccessful spheroid cultures are provided in Figure 1G and 1H.
Representative functional data for wild type and F508del CFTR homozygous HNE spheroids is shown in Figure 2A and 2B, respectively; this data is representative of >10 unique HNE samples in wild type and F508del CFTR homozygous subjects30. In short, spheroids with functional CFTR swell, while those with dysfunctional CFTR swell significantly less, or may shrink. Specifically, spheroids from subjects with wtCFTR should swell over an hour when stimulated, and should swell less or shrink if stimulated in the presence of the CFTR inhibitor Inh172. Conversely, spheroids from a subject homozygous for F508del CFTR should either shrink or swell very slightly, with increased swelling (or less shrinking) when pharmacologically corrected with the CFTR modulators VX809 and VX770. Previous analyses of spheroid reliability both within and between subjects of the same genotype demonstrate functional segregation of CFTR genotypes and modest variability in repeated measures30.
Media Component | Stock Solution | Amount | Storage |
Expansion Media | |||
DMEM / F-12 Nutrient Mixture "Base Media" | Use as is | 2x 500 mL containers | Store at 4 ºC up to manufacturer expiration date |
Fetal Bovine Serum | Use as is | 50 mL | Store at -20 ºC up to manufacturer expiration date |
Cholera Toxin | 10 mg in 1 mL of sterile water | 1 µL | Store stock at -20 ºC up to six months |
Epidermal Growth Factor | 500 µg in 1 mL of sterile water | 20 µL | Store stock at -20 ºC up to six months |
Hydrocortisone | 0.4 mg in 400 µL of sterile water | Entire 400 µL aliquot | Make fresh with each batch. Store powder at room temperature up to manufacturer expiration date |
Adenine | 24 mg in 1 mL of sterile water | Entire 1 mL aliquot | Make fresh with each batch. Store powder at room temperature up to manufacturer expiration date |
Y-27632 | 3.2 mg in 1 mL of sterile water | Entire 1 mL aliquot | Make fresh with each batch. Store powder at -20 ºC up to manufacturer expiration date |
Antibiotic Media | |||
Amphotericin B | Use as is | 1.2 mL | Store at 4 ºC up to manufacturer expiration date |
Ceftazidime | 15 mg in 1 mL of sterile water | Entire 1 mL aliquot | Make fresh with each batch. Store powder at -20 ºC up to manufacturer expiration date |
Tobramycin | 15 mg in 1 mL of sterile water | Entire 1 mL aliquot | Make fresh with each batch. Store powder at -20 ºC up to manufacturer expiration date |
Vancomycin | 15 mg in 1 mL of sterile water | Entire 1 mL aliquot | Make fresh with each batch. Store powder at -20 ºC up to manufacturer expiration date |
Table 1: Components of Expansion and Antibiotic Media.
Media Component | Stock Solution | Amount | Storage |
DMEM / F-12 Nutrient Mixture "Base Media" | Use as is | 2x 500 mL containers | Store at 4 ºC up to manufacturer expiration date |
Ultroser-G | 20 mL of sterile water in a single, 20 mL bottle of lyophilized Ultroser-G | Entire 20 mL aliquot | Make fresh with each batch. Store powder at 4 ºC up to manufacturer expiration date |
Fetal Clone II | Use as is | 20 mL | Store at -20 ºC up to manufacturer expiration date |
Pen Strep | Use as is | 10 mL | Store at -20 ºC up to manufacturer expiration date |
Bovine Brain Extract | Use as is | 2.48 mL | Store at -20 ºC up to manufacturer expiration date |
Transferrin | Use as is | 250 µL | Store at -20 ºC up to manufacturer expiration date |
Insulin | Use as is | 250 µL | Store at -20 ºC up to manufacturer expiration date |
Ethanolamine | Use as is | 15 µL | Store at room temperature up to manufacturer expiration date |
Epinephrine | 2.75 mg in 1 ml of sterile water | Entire 1 mL aliquot | Make fresh with each batch. Store powder at 4 ºC up to manufacturer expiration date |
Triiodothyronine | 8.4 mg in 50 µL of DMSO | Entire 50 µL aliquot | Make fresh with each batch. Store powder at -20 ºC up to manufacturer expiration date |
Hydrocortisone | 7.24 mg in 1 mL of ethanol | 1 µL | Store stock at -20 ºC up to six months |
Phsophoryletheanolamine | 35.25 mg in 1 mL of sterile water | 1 µL | Store stock at -20 ºC up to six months |
Retinoic Acid | 3 mg in 1 mL of DMSO | 1 µL | Store stock at -20 ºC up to six months |
Table 2: Components of Differentiation Medium.
Figure 1: HNE Expansion and Structural Characteristics of HNE Spheroids. Brightfield images of HNE expansion cultures taken seven days after plating demonstrate successful (white arrow, panel A) and unsuccessful (panel B) HNE colony formation on a feeder fibroblast background. Successful wtCFTR and F508del homozygous spheroids are shown in panels C and D, respectively. Masking to delineate the luminal area of spheroids from C/D is provided in panels E and F, respectively. Unsuccessful spheroid cultures are characterized by small, disorganized cellular debris (panel G) and/or disorganized clumps of cells (panel H). Scale bar = 100 µm. Please click here to view a larger version of this figure.
Figure 2: Functional Characteristics of HNE Spheroids. Representative functional responses of wtCFTR spheroids from a single donor, when stimulated with forskolin/IBMX with/without the presence of the CFTR inhibitor Inh172 are shown in panel (A) Each point represents the response in a single spheroid. Representative functional responses of F508del homozygous spheroids from a single donor, when stimulated with forskolin/IBMX with/without the presence of VX809 and VX770 are shown in panel (B). Error bars = SEM. **p <0.01; ***p <0.001. Please click here to view a larger version of this figure.
This protocol describes the generation of patient-derived nasal cell spheroid cultures able to produce an individualized, specific model of CFTR function. There are several key steps in the process that should be closely attended to avoid difficulty. First is a good sample acquisition from the patient’s nose. A good sample should have >50,000 cells, limited mucus/debris, and be ready for processing within 4 h (though success is also easily achievable with overnight shipping on ice). Practice acquiring samples with curette or brush by study staff is necessary, which is facilitated by focusing early sample acquisition into the hands of one or two providers to maximize their comfort. Second, good clean/sterile technique in all tissue culture steps is essential. The primary mode of failure, in our experience, for all HNE cultures is bacterial or fungal contamination, which can complicate both the primary sample and other growing cultures in the incubator. This risk only increases with cultures of subjects with chronic infection (e.g., CF), therefore success hinges largely on the ability to keep cultures clean and separate. Our practice is to keep one incubator separate only for infection-prone cultures within the first 5-7 days, protecting older, successful cultures from inadvertent contamination. Third, it is important to closely watch cells during the expansion phase and avoid overconfluence. If the cells are allowed to reach full confluence on the plate, there is a risk that the culture will either become senescent or cells will begin to die and detach. Finally, when plating cells as spheroids, it is important to thoroughly disperse the cells through the matrix and to plate an evenly distributed sample. Either over- or under-population of the matrix drops will lead to culture failure, and large clumps of cells will not produce successful spheroids.
While implementing this protocol, investigators may encounter a few common problems. Contamination, as mentioned above, is best avoided by procuring a good initial sample without mucus and clean culture techniques. If cultures are successful through expansion, but no differentiated spheres are formed, several issues may have occurred. If the matrix appears mostly empty, it is most likely that the cells were seeded at too low of a density; increase the seeding density of subsequent attempts by approximately 20%. Conversely, if the matrix appears to be “dirty” with copious cell debris, it is likely that the cells were seeded at too high of a density, and subsequent attempts should use a seeding density reduced by approximately 20%. A common complication of post-seeding feeding, and maintenance is the detachment of the matrix from the culture vessel. This is caused by overly aggressive media exchange and can be avoided by cautiously and manually changing media with a 1-mL pipette, not wall suction. If encountered, spheres can still be stimulated and imaged, though this may be difficult if the matrix “floats” in the well during imaging, necessitating cautious mapping of spheroids within the well.
Investigators may pursue several modifications to the protocol, depending on the needs of their lab. For higher-resolution imaging of spheroids, we have previously substituted the 4-well plates with optical glass options, including 35-mm glass-bottom dishes or chamber slides. This allows for high-resolution, live imaging of spheroids, but may reduce throughput. Alternatively, to increase throughput, smaller aliquots of cells in the matrix can be seeded into other vessels (e.g., 24-well culture plates). In our experience, spheroids grow best with the matrix in a “droplet” form as opposed to forming a sheet on the bottom of the well; as such, we have had the best success with droplets of at least 25 µL. Investigators may wish to alter the density of matrix by diluting with media. This reduces the total amount of matrix necessary, improving the cost of the assay. This may also, however, alter the spheroid composition. In our previous experience, lower concentrations of the basement membrane matrix lead to altered spheroid structure, with either partial or complete formation of spheroids in a “cell-apex-out” morphology. As such, spheroids generated through any protocol alterations in matrix concentration should be cautiously evaluated for morphology before functional testing is attempted. Finally, different stimulating or inhibitory drugs, or different concentrations of these drugs, could be employed. Forskolin, IBMX, and Inh172 were chosen for our studies at these concentrations based on previous experience in ALI cultures31. Using other drugs (e.g., isoproterenol for stimulation, GlyH101 for inhibition) may better apply to an investigator’s study. Similarly, using different concentrations of forskolin, IBMX, or Inh172 may alter the dynamic range of the assay, however, we have not systematically tested these options.
Given the number of existing ex vivo patient-derived CFTR assays, the described model is notable for several key reasons. First, it capitalizes on nasal cells as an easily obtained source of respiratory tissue. HNE procurement can be performed safely with minimal training in almost any setting (clinic, OR, research visit) in all age groups25. This facilitates robust sample procurement, and repeat sampling if necessary due to growth difficulty or contamination. Compared to intestinal organoids, HNE spheroids are less well characterized and appear to have a smaller dynamic range in the present form, however, use of respiratory instead of GI tissue may be a key benefit, as several CF disease drivers (e.g. mucociliary clearance, epithelial sodium channel expression) are not equivalent in the gut. As opposed to planar, ALI cultures of HNE cells, spheroids are grown faster and may be more representative of in vivo conditions, measuring a physiologic process (fluid homeostasis) that may be more relevant than electrophysiology alone32. By improving the time from sample procurement to testing, contamination potential is reduced, therefore increasing the likelihood of culture success. Finally, the 3-dimensional nature of the model may be amenable to the novel and/or complementary lines of research in airway development or morphology that are not feasible in ALI cultures (e.g., luminal mucus tracking, rapid studies of differentiation, etc.).
There are several key limitations to HNE spheroids as an assay of CFTR function. First, this assay remains relatively low-throughput. Using the current methods, image acquisition takes over an hour for each culture condition, and analysis takes an additional 20-30 min per condition. This is compounded by the degree of overlap between certain conditions (such as demonstrated in Figure 2A), which necessitates a larger number of measurements (this overlap in itself may also represent a limitation of the sensitivity of this assay). As such, a single experiment with 4-6 conditions requires almost two days for completion. Throughput can be improved by employing methods of automated image capture and analysis; such adaptations are currently in progress. Secondly, this model requires a large amount of matrix, which can become expensive. As mentioned above, dilution of the matrix may help overcome this barrier, but must be taken with caution, as alterations in the growth matrix will likely result in alterations in spheroid morphology. Third, this model relies on spheroid measurements in an XY plane only, and disregards swelling in the Z plane, which may introduce bias. While previous reproducibility analyses have been reassuring, this shortcoming could be overcome by use of automated imaging with Z-plane scanning to calculate volume30. Finally, and most importantly, extensive work to tie spheroid responses to the individual subject’s in vivo drug response is yet to be completed. In absence of this correlation, the predictive value of the model – while promising – is unclear.
Generation and analysis of HNE spheroids allow for ex vivo analysis of individual CFTR activity and modulation, which may ultimately be useful as a pre-clinical model of drug response. Moreover, given the extensive variability in CF disease severity, such an individualized model can provide insights into a subject’s unique airway microenvironment. In this way, such a model may be useful for studies of broader CFTR biology and aid in understanding disease heterogeneity. Future use of this model, as well as other similar models of individualized CFTR function, holds promise to better understand CFTR biology in the lab, and to drive personalized and precision medicine in the clinic.
The authors have nothing to disclose.
This work was supported by Cystic Fibrosis Foundation Therapeutics, grant number CLANCY14XX0, and through Cystic Fibrosis Foundation, grant number CLANCY15R0. The authors wish to thank Kristina Ray for her assistance in patient recruitment and regulatory oversight. The authors also wish to thank the HNE working group, supported by the Cystic Fibrosis Foundation, who assisted in generation of HNE culture capabilities: Preston Bratcher, Calvin Cotton, Martina Gentzsch, Elizabeth Joseloff, Michael Myerburg, Dave Nichols, Scott Randell, Steve Rowe, G. Marty Solomon, and Katherine Tuggle.
1.5 mL Eppendorf Tube | USA Scientific | 4036-3204 | |
150 mL Filter Flask | Midsci | TP99150 | To filter Media |
15 mL Conical Tube | Midsci | TP91015 | |
1 L Filter Flask | Midsci | TP99950 | To filter Media |
35 mm Glass-Bottom Dish | MatTek Corporation | P35G-0-20-C | Optional |
3-Isobutyl-1-Methylxanthine (IBMX) | Fisher Scientific | AC228420010 | Prepare a 100 mM stock solution of 22.0 mg in 1 mL of DMSO |
50 mL Conical Tube | Midsci | TP91050 | |
Accutase | Innovative Cell Technologies, Inc. | AT-104 | Cell detachment solution |
Adenine | Sigma-Aldrich | A2786-25G | See Table 1 |
Amphotericin B | Sigma-Aldrich | A9528-100MG | See Table 1 |
Bovine Brain Extract (9mg/mL) | Lonza | CC-4098 | See Table 2 |
Ceftazidime hydrate | Sigma-Aldrich | C3809-1G | See Table 1 |
Cell Scrapers 20 cm | Midsci | TP99010 | |
CFTR Inh172 | Tocris Bioscience | 3430 | Prepare a 10 mM stock solution of 4.0 mg in 1 mL of DMSO |
Cholera Toxin B (From Vibrio cholerae) | Sigma-Aldrich | C8052-.5MG | See Table 1 |
CYB-1 | Medical Packaging Corporation | CYB-1 | Cytology brush |
Dimethyl sulfoxide (DMSO) | Sigma-Aldrich | D5879-500ML | |
Dulbecco's Modified Eagle Media (DMEM)/F12 Hepes | Life Technologies | 11330-057 | Base Medium; See Tables 1 and 2 |
Epidermal Growth Factor (Recombinant Human Protein, Animal-Origin Free) | Thermo Fisher Scientific | PHG6045 | See Table 1 |
Epinephrine | Sigma-Aldrich | E4250-1G | See Table 2 |
Ethanol | Fisher Scientific | 2701 | |
Ethanolamine | Sigma-Aldrich | E0135-500ML | 16.6 mM solution; See Table 2 |
Ethylenediaminetetraacetic Acid (EDTA) | TCI America | E0084 | |
Fetal Bovine Serum (high performance FBS) | Invitrogen | 10082147 | See Table 1 |
Forskolin | Sigma-Aldrich | F6886-50 | Prepare a 10 mM stock solution of 4.1 mg in 1 mL of DMSO |
Growth Factor-Reduced Matrigel | Corning, Inc. | 356231 | Corning Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix, Phenol Red-Free, LDEV-Free, 10 mL. |
Hemacytometer | Hausser Scientific | 1483 | |
Human Collagen Solution, Type I (VitroCol; 3 mg/mL) | Advanced BioMatrix | 5007-A | Collagen solution |
HyClone (aka FetalClone II) | GE Healthcare | SH30066.03HI | See Table 2 |
Hydrocortisone | StemCell Technologies | 07904 | See Tables 1 and 2 |
Insulin, human recombinant, zinc solution | Life Technologies | 12585014 | 4 mg/mL solution; see Table 2 |
IVF 4-Well Dish, Non-treated | NUNC (via Fisher Scientific) | 12566350 | 4-well plate for spheroids; similar well size to a 24-well plate |
MEF-CF1-IRR | Globalstem | GSC-6001G | Irradiated murine embryonic fibroblasts |
Metamorph 7.7 | Molecular Devices | Analysis Software; https://www.moleculardevices.com/systems/metamorph-research-imaging/metamorph-microscopy-automation-and-image-analysis-software for a quote | |
Olympus IX51 Inverted Microscope | Olympus Corporation | Discontinued | Imaging Microscope. Replacment: Olympus IX53, https://www.olympus-lifescience.com/pt/microscopes/inverted/ix53/ for a quote |
Pen Strep | Life Technologies | 15140122 | See Table 2 |
Phsophoryletheanolamine | Sigma-Aldrich | P0503-5G | See Table 2 |
Retinoic Acid | Sigma-Aldrich | R2625-50MG | See Table 2 |
Rhinoprobe | Arlington Scientific, Inc. | 96-0905 | Nasal curette |
Slidebook 5.5 | 3i, Intelligent Imaging Innovations | Discontinued | Imaging Software. Replacement: Slidebook 6, https://www.intelligent-imaging.com/slidebook for a quote |
Sterile Phosphate Buffered Saline (PBS) | Thermo Fisher Scientific | 20012050 | |
Sterile Water | Sigma-Aldrich | W3500-6X500ML | |
Tissue Culture Dish 100 | Techno Plastic Products | 93100 | Tissue culture dish for expansion |
Tobramycin | Sigma-Aldrich | T4014-100MG | See Table 1 |
Transferrin (Human Transferrin 0.5 mL) | Lonza | CC-4205 | See Table 2 |
Triiodothryonine | Sigma-Aldrich | T6397-1G | 3,3′,5-Triiodo-L-thyronine sodium salt [T3]; See Table 2 |
Trypsin from Porcine Pancreas | Sigma-Aldrich | T4799-10G | |
Ultroser-G | Crescent Chemical (via Fisher Scientific) | NC0393024 | 20 mL lypophilized powder; See Table 2 |
Vancomycin hydrochloride from Streptomyces orientalis | Sigma-Aldrich | V2002-5G | See Table 1 |
VX770 | Selleck Chemicals | S1144 | Prepare a 1 mM stock solution of 0.4 mg in 1 mL of DMSO |
VX809 | Selleck Chemicals | S1565 | Purchase or prepare a 10 mM stock solution of 4.5 mg in 1 mL of DMSO |
Y-27632 Dihydrochloride ROCK inhibitor | Enzo LifeSciences | ALX-270-333-M025 | See Table 1 |