Here we describe the isolation, amplification, and differentiation of primary human nasal epithelial (HNE) cells at the air-liquid interface and a biobanking protocol allowing to successfully freeze and then thaw amplified HNE. The protocol analyzes electrophysiological properties of differentiated HNE cells and CFTR-related chloride secretion correction upon different modulator treatments.
Human nasal epithelial (HNE) cells are easy to collect by simple, non-invasive nasal brushing. Patient-derived primary HNE cells can be amplified and differentiated into a pseudo-stratified epithelium in air-liquid interface conditions to quantify cyclic AMP-mediated Chloride (Cl–) transport as an index of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) function. If critical steps such as quality of nasal brushing and cell density upon cryopreservation are performed efficiently, HNE cells can be successfully biobanked. Moreover, short-circuit current studies demonstrate that freeze-thawing does not significantly modify HNE cells' electrophysiological properties and response to CFTR modulators. In the culture conditions used in this study, when less than 2 x 106 cells are frozen per cryovial, the failure rate is very high. We recommend freezing at least 3 x 106 cells per cryovial. We show that dual therapies combining a CFTR corrector with a CFTR potentiator have a comparable correction efficacy for CFTR activity in F508del-homozygous HNE cells. Triple therapy VX-445 + VX-661 + VX-770 significantly increased correction of CFTR activity compared to dual therapy VX-809 + VX-770. The measure of CFTR activity in HNE cells is a promising pre-clinical biomarker useful to guide CFTR modulator therapy.
Cystic Fibrosis (CF) is an autosomal recessive disorder resulting from mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene leading to the absence or dysfunction of the CFTR protein, an anion channel located at the apical surface of epithelia1,2. Recent advances in CFTR therapy have improved the prognosis of the disease, and the last approved drugs combining CFTR correctors and CFTR potentiators led to major improvements in lung function and quality of life for CF patients carrying the most frequent mutation p.Phe508del mutation (F508del)3,4. Despite this promising therapeutic progress, around 10% of CF patients are ineligible as they carry mutations that are unrescuable by these CFTR modulators. For these patients, there is a need to test other drugs or drug combinations to find the most efficient combination for specific mutations, highlighting the importance of personalized therapies.
Human nasal epithelial (HNE) cells are easy to collect by simple, non-invasive nasal brushing and allow quantification of cyclic AMP-mediated Chloride (Cl−) transport as an index of CFTR function. HNE cells yield an accurate model of human airway, but their lifespan is limited in culture. Thanks to the optimization of culture techniques, patient-derived primary HNE cells can be conditionally reprogrammed with Rho-associated kinase inhibitor (ROCKi), amplified, and differentiated into a pseudo-stratified epithelium in air-liquid interface (ALI) conditions on microporous filters5,6. Numerous culture protocols for HNE culture exist (commercially available, serum-free, "homemade", co-culture with feeder-cells, etc.), and choice of media and culture conditions have been described to impact growth, cell population differentiation and epithelial function7,8. The protocol here presents a simplified, feeder-free, ROCKi amplification method that allows to successfully obtain a large number of HNE cells that are then differentiated at ALI for CFTR function assays.
We have demonstrated that, in differentiated HNE cells, a 48 h treatment with CFTR modulators is sufficient to induce electrophysiological correction of CFTR dependent Cl– current and that the correction observed in vitro may be correlated with the patient's clinical improvement9. HNE cells, therefore, represent an appropriate model not only for fundamental CF research but for pre-clinical studies with patient-specific CFTR modulator testing. In this context of personalized therapy, the goal of the protocol was to validate that cryopreserved HNE cells from CF patients, grown in our conditions, were an appropriate model for CFTR correction studies, and similar results could be expected when comparing CFTR dependent Cl– transport from fresh and frozen-thawed cells. The study also assessed different CFTR modulators' efficacy when using dual and triple therapies.
All experiments were performed following the guidelines and regulations described by the Declaration of Helsinki and the Huriet-Serusclat law on human research ethics.
1. Preparation of flasks and different media
2. Nasal brushing
NOTE: Ensure that nasal brushing is performed while the participant does not have an acute infection. If CF patients present a pulmonary infection, refer to the patient's antibiogram and add additional antibiotics to the amplification medium. Human nasal epithelial cells were collected by nasal brushing from F508del/F508del patients as previously described9.
3. Isolation of HNE
NOTE: HNE were isolated from the cytology brush, washed with Mg2+– and Ca2+-free DPBS, dissociated with 0.25% Trypsin, and seeded onto a 25 cm2 plastic flask as previously described10.
4. Amplification and passaging of human nasal epithelial cells
5. Cryopreservation of primary nasal epithelial cells
NOTE: Grow HNE cells until passage 1 before biobanking to obtain enough cells to facilitate cell growth when thawed. Biobanking is, however, possible at initial passage 0 or passage 2. The following cryopreservation steps are adapted to all passages.
6. Thawing frozen amplified HNE cells
7. Differentiation of human nasal epithelial cells at the air-liquid interface
NOTE: HNE were differentiated at the air-liquid interface as previously described10.
8. CFTR modulators
9. Ussing chamber studies
10. Immunocytochemistry
11. Statistical analysis
Fresh HNE cells cultured at the air-liquid interface display typical features of the polarized and differentiated respiratory epithelium as assessed by immunostaining (Figure 1). HNE cells re-differentiate into a heterogeneous layer of epithelial cells (positive keratin 8 immunostaining) that mimic the in vivo situation of a pseudo-stratified respiratory epithelium composed of ciliated (positive alpha-tubulin staining) and non-ciliated mucus-producing goblet cells (positive Muc5Ac immunostaining). The overall epithelium presents tight junctions (assessed by ZO-1, zona-occludens-1 staining). Strong apical CFTR staining is visible in wild-type (WT) HNE cells, and decreased CFTR staining both at the apical surface and in the cytoplasm is observed in F508del/F508del HNE.
We considered positive outcomes as samples not only successfully expanding but also successfully differentiating into a pseudo-stratified epithelium after 3 weeks at air-liquid interface and presenting a TEER higher than 200 Ω·cm2, allowing short-circuit current measurements. As only a few dedicated labs perform CFTR-related Cl– secretion assays by short-circuit current measurements, samples often require transport from hospitals to dedicated facilities.
In the 78 fresh HNE cell samples, success rates were compared in nasal brushings seeded immediately and in brushings seeded after 1-7 days of transport in flushing medium at room temperature. The majority of samples (55%) were seeded 1 day after brushing, and 82% of samples were seeded within 48 h. The mean percentage of successful cultures in all 78 samples was 82% and reached 87% in samples seeded between day 0 and day 5 (Table 2).
The success and failure rates for identical samples differentiated in both fresh and freeze-thawed conditions were compared. 83% of samples that differentiated successfully in fresh conditions also were successful in cryopreserved samples. Likewise, in samples that failed to differentiate in fresh conditions, 71% were also unsuccessful in freeze-thaw conditions (Table 2). All these data suggest that the quality of nasal brushing is the key factor to a successful culture.
We also wanted to determine the optimal number of cells per cryovial. Samples frozen at passage 0, from a single 25 cm2 generally only allow to reach 1-1.5 x 106 cells per flask. Samples frozen at passage 1 and amplified to three 75 cm2 flasks generally allowed to reach 10 x 106 cells, thus allowing to freeze several vials containing at least 3 x 106 cells. In the culture conditions described in this protocol, 50% of samples frozen between 2 x 106 and 3 x 106 cells per cryovial failed to grow when thawed, reaching 80% when below 2 x 106 cells per cryovial (Table 2).
CFTR-related Cl– secretion reflects CFTR function and can be measured by short circuit current (Isc) experiments in Ussing chambers. The Isc variation in response to Forskolin (ΔIscForskolin) and CFTR inhibitor inh172 (ΔIscCFTRinh172) are the main endpoints for CFTR corrector efficacy assessment.
Short-circuit-current experiments were performed on either fresh HNE, expanded in amplification medium and seeded onto filters at passage 2 and grown at air-liquid interface for 3 weeks in air-liquid medium, or from HNE that had been previously frozen at passage 1 in enriched freezing medium, thawed, expanded, and seeded on porous filters at passage 3. Cryopreservation times ranged from 3 months to 16 months, with a mean of 9.8 months. In the culture and cryopreservation conditions detailed in this study, no significant changes in growth or morphology were observed between different passages or fresh or frozen-thawed F508del/F508del HNE cultures.
For both mean ΔIscForskolin (Figure 2A) and mean ΔIscCFTRinh172 (Figure 2B) in DMSO treated HNE cells, no significant difference was observed between fresh or frozen-thawed HNE. To assess whether functional correction of CFTR was influenced by cryopreservation, mean Isc changes were measured in VX-809 + VX-770 treated cells compared to DMSO treated HNE cells in fresh (HNE cells derived from n =78 patients) or frozen-thawed HNE (HNE cells derived from n = 9 patients) (Figure 2). Both frozen-thawed and fresh cells displayed significant correction upon treatment, with a significant increase in ΔIscForskolin (p < 0.05) (Figure 2A), and a significant decrease in the ΔIscCFTRinh172 (p < 0.05) (Figure 2B). The mean fold increase for ΔIscForskolin (ΔIscForskolin VX-809 + VX-770/ΔIscForskolin DMSO) reached 2.9 ± 1.6 in frozen-thawed HNE cells and was not significantly different from the fold increase in fresh HNE cells (2.5 ± 2.0). Similarly, the fold decrease in ΔIscCFTRinh172 (ΔIscCFTRinh172 VX-809 + VX-770/ΔIscCFTRinh172 DMSO) was not significantly affected by cryopreservation: 1.49 ± 0.9 in frozen-thawed HNE compared to 2.5 ± 3.7 in fresh HNE.
The functional correction activity of several modulator therapies was further investigated to assess whether this HNE cell model could discriminate between different treatments. The results here combine HNE cells from both fresh and frozen-thawed conditions as the response to treatment was not significantly altered by cryopreservation (Figure 2).
We first assessed the corrector response of two approved CFTR modulator therapies on Cl– transport after a 48 h treatment with either VX-809 (3 µM) + VX-770 (100 nM) or VX-661 (3 µM) + VX-770 (100 nM) in HNE cells from 10 F508del/F508del patients (Figure 3). As compared to DMSO treated HNE cells, ΔIscForskolin (Figure 3A) and ΔIscCFTRinh172 (Figure 3B) were significantly improved by both VX-809 + VX-770 and VX-661 + VX-770. Both treatments functionally corrected CFTR-related Cl– secretion to a similar level, with a significant mean fold increase in ΔIscForskolin of 1.75 ± 1.39 for VX-809 + VX-770 (p < 0.001) and 0.97 ± 0.93 for VX-661 + VX-770 (p < 0.01). Mean ΔIscCFTRinh172 fold decrease was 1.79 ± 2.04 for VX-809 + VX-770 (p < 0.001) and was not significantly different from the mean ΔIscCFTRinh172 fold decrease of 1.16 ± 1.44 for VX-661 + VX-770 (p < 0.001 vs. DMSO).
Three different CFTR modulator therapies in HNE from six F508del/F508del patients (Figure 4) were further compared. The results here combine HNE cells from both fresh and frozen-thawed conditions. Both Vertex and Abbvie CFTR modulators, when used as a combination of a CFTR corrector and a CFTR potentiator, corrected ΔIscForskolin (Figure 4A) and ΔIscCFTRinh172 (Figure 4B) to a similar extent. The partial correction observed in Figure 3 by VX-809 + VX-770 was confirmed in these HNE cells with a mean ΔIscForskolin fold increase of 2.87 ± 1.67 (p < 0.05). ABBV-2222 (1 µM) + ABBV-974 (10 µM) treatment induced a similar correction representing 91% of VX-809 + VX-770 correction for ΔIscForskolin (mean ΔIscForskolin fold increase of 2.6 ± 1.4, p < 0.05 vs. DMSO) and 85% of VX-809 + VX-770 for ΔIscCFTRinh172 (mean ΔIscCFTRinh172 fold decrease of 4.2 ± 5.7, p < 0.05 vs. DMSO).
Interestingly, when HNE were treated with the triple combination VX-445 (3 µM) + VX-661 (3 µM) + VX-770 (100 nM), correction efficacy significantly improved, reaching 312% of VX-809 + VX-770 ΔIscForskolin correction (mean ΔIscForskolin fold increase of 9.0 ± 4.1, p < 0.05 vs. VX-809 + VX-770) and 372% of ΔIscCFTRinh172 (mean ΔIscCFTRinh172 fold decrease of 15.5 ± 10.5, p < 0.05 vs. VX-809 + VX-770).
Figure 1: Differentiation markers in HNE cultures. Representative confocal microscopy images of immune-fluorescent staining of CFTR (green), Zona-Occludens-1 (ZO-1, red), alpha-tubulin (green), Muc5AC (red) and cytokeratin 8 (K8, green) in wild-type (WT) (first panel) and F508del (panels 2 -4) HNE cells grown at air-liquid interface for 3 weeks. Nuclei were stained blue with DAPI. Projections of confocal microscopy images, top view (upper panels) and side view (lower panels) of three-dimensional (3D) Z stacks reconstitutions. Scale bar (20 µm) applies to both upper and lower panels. Please click here to view a larger version of this figure.
Figure 2: Effect of freezing-thawing on CFTR-related Cl– secretion in HNE cells. Summary of short-circuit current (Isc) recordings (mean ± SD) in HNE cultures derived from F508del/F508del patients and grown at air-liquid interface for 3 weeks. Cells were treated for 48 h with vehicle alone (DMSO) or VX-809 (3 µM) + VX-770 (100 nM) in fresh (light grey, patient-derived HNE cells from 78 patients) or frozen-thawed (dark grey, patient-derived HNE cells from 9 patients). Data represent the mean (± SD) response to acutely added (A) Forskolin (10 µM)/3-isobutyl-1-methylxanthine (IBMX, 100 µM), ΔIscForskolin or (B) CFTR inhibitor inh172 (5 µM) ΔIscCFTRinh172. A minimum of two filters were analyzed per patient and per condition. Asterisks indicate the statistical difference between correctors and control (DMSO-treated) * p < 0.05 (unpaired t-test). Please click here to view a larger version of this figure.
Figure 3: Correction of CFTR function by dual therapies. Summary of short-circuit current (Isc) recordings (mean ± SD) in HNE cultures derived from 10 F508del/F508del patients and grown at air-liquid interface for 3 weeks (combined results from both fresh and frozen-thawed cultures). Cells were treated for 48 h with vehicle alone (DMSO); VX-809 (3 µM) + VX-770 (100 nM) or VX-661 (3µM) + VX-770 (100 nM). Data represent the mean (± SD) response to acutely added (A) Forskolin (10 µM)/IBMX (100 µM), ΔIscForskolin or (B) CFTR inhibitor inh172 (5 µM), ΔIscCFTRinh172. A minimum of two filters were analyzed per patient and per condition. Asterisks indicate a statistical difference between correctors and control (DMSO-treated) ** p < 0.01, *** p < 0.001, **** p < 0.0001 (Wilcoxon matched pairs signed rank test). Please click here to view a larger version of this figure.
Figure 4: Efficiency of triple-therapy vs. dual therapies on CFTR correction. Summary of short-circuit current (Isc) recordings (mean ± SD) in HNE cultures derived from 6 F508del/F508del patients and grown at air-liquid interface for 3 weeks (combined results from both fresh and frozen-thawed cells). Cells were treated for 48 h with vehicle alone (DMSO); VX-809 (3 µM) + VX-770 (100 nM); ABBV-2222 (1 µM) + ABBV-974 (10 µM); or VX-661 (3 µM) + VX-445 (3 µM) + VX-770 (100 nM). Data represent the mean (± SD) response to acutely added (A) Forskolin (10 µM)/IBMX (100 µM), ΔIscForskolin or (B) CFTR inhibitor inh172 (5 µM), ΔIscCFTRinh172. A minimum of two filters were analyzed per patient and per condition. Asterisks indicate a statistical difference between treatments. * p < 0.05 (Wilcoxon matched pairs signed rank test). Please click here to view a larger version of this figure.
Media Component | Final concentration |
AMPLIFICATION MEDIUM | |
Advanced DMEM/F-12 Nutrient mixture | 86% |
Fetal Bovine Serum | 10% |
Penicillin | 100 U/mL |
Streptomycin | 100 µg/mL |
Tazocillin | 10 µg/mL |
Amphotericin B | 2.5 µg/mL |
Colimycin | 16 µg/mL |
Ciprofloxacin | 3 µg/mL |
Hydrocortisone | 0.2 µM |
Insulin | 5 µg/mL |
Epinephrine | 0.5 µg/mL |
EGF | 10 ng/mL |
Y-27632 (Rho-associated kinase inhibitor) | 10 µM |
AIR-LIQUID MEDIUM | |
Advanced DMEM/F-12 Nutrient mixture | 94% |
UltroserG | 2% |
Penicillin | 100 U/mL |
Streptomycin | 100 µg/mL |
Tazocillin | 10 µg/mL |
Amphotericin B | 2.5 µg/mL |
Colimycin | 16 µg/mL |
ENRICHED FREEZING MEDIUM | |
F12 Nutrient Mixture | 77% |
HEPES | 3% |
Fetal Bovine Serum | 10% |
DMSO | 10% |
Table 1: Amplification, air-liquid, and enriched freezing medium composition. List of reagents and corresponding final concentrations.
Length of conservation of nasal brushing before seeding (n = 78) | |
Days before seeding | Success rates (%) |
0 | 83 |
1 | 84 |
2 | 67 |
3 | 100 |
4 | 89 |
5 | 100 |
7 | 0 |
Outcomes from the same initial nasal brushing grown in both fresh and frozen-thawed conditions (n = 13) | |
Outcome in fresh conditions | Success rates upon freeze-thawing (%) |
success | 83 |
failure | 29 |
Impact of cell number on success rates (n = 36) | |
Number of cells per cryovial | Success rates upon freeze-thawing (%) |
<2 x 106 | 20 |
2–3 x 106 | 50 |
3–4 x 106 | 67 |
>4 x 106 | 79 |
Table 2: Parameters influencing success rates in fresh and frozen-thawed HNE cell cultures. Success rates were defined as nasal brushings that successfully expanded and differentiated into a pseudo-stratified epithelium after 3 weeks at the air-liquid interface and presented a TEER higher than 200 Ω·cm2. Before seeding, samples were shipped at room temperature in flushing medium with antibiotics. Results represent data from 78 fresh samples and 36 frozen-thawed samples. 13 nasal brushings were analyzed in both fresh and frozen-thawed conditions.
The use of patient-derived nasal epithelial cells as surrogates for human bronchial epithelial (HBE) cells to measure CFTR activity in the context of personalized medicine has been proposed as HNE reproduce cells' properties in culture9,11. The strong advantage of HNE over HBE cell cultures is that they are easily and non-invasively sampled. Short-circuit current measurements in HNE cell cultures enable assessment of CFTR-dependent Cl– transport across the epithelium, which was shown to significantly correlate to CFTR in vivo activity assessed by nasal potential difference in patients with various genotypes9. The rescue of CFTR activity in HNE cells upon VX-809 treatment also significantly correlated to the improvement of clinical outcome FEV1 in lumacaftor-ivacaftor treated F508del homozygous patients12.
Importantly, this study shows that freshly harvested HNE cells easily tolerate transport and standard shipping conditions, (i.e., they survive 72 h at room temperature) which allows the testing of patient's cells originating from various geographical locations. The critical factors for successful culture are mainly the quality of nasal brushing and the number of living cells at sampling. A number of protocols have been published on growing and differentiating HNE cell cultures; some with no conditional reprogramming during the expansion phase13,14,15,16,17,18,19,20, some with conditional reprogramming in feeder9,11,12,21,22,23,24,25,26 or feeder-free5,8,27,28,29 conditions, and several comparing different methods7,8. They all allow obtaining a relatively large number of ALI cultures with correct differentiation and polarization as well as good TEER values. Similar to this study, many used cryopreserved HNE cells frozen at passage 113,28. Interestingly, several authors report that ROCKi can improve survival after cryopreservation when added to the post-thaw culture medium and increase the number of cells that remain attached5,29, suggesting that the amplification medium used in this study is particularly appropriate for cryopreservation.
Here, the study shows that HNE cells can be successfully biobanked, the critical factor again being the quality of the nasal brushing. A good correlation between the outcome of fresh samples and frozen-thawed ones is shown, suggesting that in order to optimize the quality of HNE cells that are going to be biobanked for further studies, a preliminary study could be performed on fresh samples if they fail to achieve correct ALI differentiation and acceptable TEER value; a second nasal brushing would be preferable rather than freezing cells that have a high probability of poor outcome. A second critical factor for cryopreservation is cell number; in the culture conditions used in this study, when less than 2 x 106 cells are frozen per cryovial, the failure rate is very high. Freezing at least 3 x 106 cells per cryovial is recommended.
The results in this study suggest that freeze-thawing does not significantly modify HNE cells' electrophysiological properties or response to CFTR modulators. The range of measures in HNE cell cultures from the same F508del-homozygous patient obtained without freezing or after thawing was not different upon VX-809 + VX-770 treatment. Similar results were previously reported for HBE cells where Forskolin-stimulated Isc were stable after several thaw/culture cycles of non-treated F508del cells30. This provides evidence that HNE cells can be frozen and stored in a biobank and studied later.
An increasing number of studies show that HNE cell cultures can be used to test the efficacy of different therapies in the context of personalized medicine. The analysis showed that dual therapies combining a corrector with a potentiator all have comparable correction efficacy for CFTR-dependent Cl– secretion in F508del-homozygous HNE cells. However, an important interpatient variability was noticed, confirming published results9,12,20,25. By contrast, VX-445 + VX-661 + VX-770 tripled correction of CFTR activity compared to VX-809 + VX-770. A similar level of correction of F508del-CFTR in HNE cells upon triple combination was also observed by Laselva et al.28. As reported by Veit et al.26, correction of Cl– channel function upon VX-445 + VX-661 + VX-770 treatment was even higher, reaching 62% of mean WT-CFTR activity (range of 19-36 µA/cm2 in corrected F508del-HNE and 14-50 µA/cm2 in WT-HNE)26.
HNE cell cultures also served to test the efficacy of CFTR modulators for other class II mutations. An important improvement of CFTR activity upon VX-445 + VX-661 + VX-770 was observed in HNE ALI cultures harboring G85E/G85E, V520F/1717-1G>A, Y569/Y569D, M1101K/M1101K, and N1303K/N1303K genotypes26. Similarly, Laselva et al.28 showed a significant correction of CFTR with M1101K and N1303K mutations but no significant correction in G85E HNE cells. Galapagos/AbbVie correctors were evaluated for these lumacaftor-ivacaftor-resistant mutants31. AbbVie corrector AC2-1 and especially bi-corrector AC1+AC2-1 effectively improved Cl– secretion in M1101K and G85E HNE cells, whereas N1303K derived cells were corrected by a combination of AC1+AC2-2.
Measures of CFTR activity in HNE cells are promising pre-clinical biomarkers predictive for the patient's response to tested treatment. Evidence that freezing-thawing cycles do not alter the correction level provides evidence that these cells can be biobanked and used as a powerful tool in the context of personalized medicine programs. A unique nasal brushing could therefore allow for sufficient material to compare drug efficacies, and patient's HNE cells could be thawed as new drugs become available with no need to resample the patients; this is particularly interesting in infants.
The authors have nothing to disclose.
We warmly thank all patients and their families for participation in the study. This work was supported by grants from French Association Vaincre la Mucoviscidose; French Association ABCF 2 and Vertex Pharmaceuticals Innovation Awards.
ABBV-2222 | Selleckchem | S8535 | |
ABBV-974 | Selleckchem | S8698 | |
Advanced DMEM/F-12 | Life Technologies | 12634010 | |
Alexa 488 goat secondary antibody | Invitrogen | A11001 | |
Alexa 594 goat secondary antibody | Invitrogen | A11012 | |
Amphotericin B | Life Technologies | 15290026 | |
Anti-alpha-tubulin antibody | Abcam | ab80779 | |
Anti-CFTR monoclonal antibody (24-1) | R&D Systems | MAB25031 | |
Anti-cytokeratin 8 antibody | Progen | 61038 | |
Anti-Muc5AC antibody | Santa Cruz Biotech | sc-20118 | |
Anti-ZO-1 antibody | Santa Cruz Biotech | sc-10804 | |
Ciprofloxacin | provided by Necker Hospital Pharmacy | ||
Colimycin | Sanofi | provided by Necker Hospital Pharmacy | |
Collagen type IV | Sigma-Aldrich Merck | C-7521 | |
cytology brush | Laboratory GYNEAS | 02.104 | |
DMSO | Sigma-Aldrich Merck | D2650 | |
EGF | Life Technologies | PHG0311 | |
Epinephrin | Sigma-Aldrich Merck | E4375 | |
F12-Nutrient Mixture | Life Technologies | 11765054 | |
FBS | Life Technologies | 10270106 | |
Ferticult | Fertipro NV | FLUSH020 | |
Flasks 25 | Thermo Scientific | 156.367 | |
Flasks 75 | Thermo Scientific | 156.499 | |
Glacial acetic acid | VWR | 20104.298 | |
HEPES | Sigma-Aldrich Merck | H3375 | |
Hydrocortisone | Sigma-Aldrich Merck | SLCJ0893 | |
Insulin | Sigma-Aldrich Merck | I0516 | |
Mg2+ and Ca2+-free DPBS | Life Technologies | 14190094 | |
Penicillin/Streptomycin | Life Technologies | 15140130 | |
Tazocillin | Mylan | provided by Necker Hospital Pharmacy | |
Transwell Filters | Sigma-Aldrich Merck | CLS3470-48EA | |
Triton-X100 | Sigma-Aldrich Merck | T8787 | |
Trypsin 0,25% | Life Technologies | 25200056 | |
Vectashield mounting medium with DAPI | Vector Laboratories | H-1200 | |
VX-445 | Selleckchem | S8851 | |
VX-661 | Selleckchem | S7059 | |
VX-770 | Selleckchem | S1144 | |
VX-809 | Selleckchem | S1565 | |
Xylocaine naphazoline 5% | Aspen France | provided by Necker Hospital Pharmacy | |
Y-27632 | Selleckchem | S1049 |