In Vitro Cell.Dev.Biol.—Animal (2015) 51:433–440 DOI 10.1007/s11626-014-9853-z
REPORT
Construction of an in vitro primary lung co-culture platform derived from New Zealand white rabbits Joshua D. Powell & Becky M. Hess & Janine R. Hutchison & Timothy M. Straub
Received: 12 August 2014 / Accepted: 23 November 2014 / Published online: 10 December 2014 / Editor: Tetsuji Okamoto # The Society for In Vitro Biology 2014
Introduction Co-culturing of primary lung epithelial cells with monocytes provides an avenue to study lung-associated responses to carcinogens and pathogens in vitro. Historically, a major obstacle in the development of tissue culture-based lung model development was epithelial proliferation without culturing unwanted fibroblasts (Wise and Lechner 2002). Advances in cell culture media formulations and the advent of the transwell dish now enable convenient culturing of lung epithelial cells at air liquid interface (ALI). Due to the high numbers of bronchial epithelial cells that can be isolated from lung postmortem for human (Lechner and LaVeck 1985) (Gruenert et al. 1995), horse (Shibeshi et al. 2008), or pig (Hauser et al. 2013), a sufficient number of cells can be isolated at low passage for experimental use. Unfortunately in smaller mammalian species, lung epithelial-based experiments can be challenging because cells from the trachea and small bronchiole tubes can be difficult to culture to substantial quantities prior to the onset of cell senescence. In light of this challenge posed from smallersized animals, it is crucial that optimal growth conditions are deduced to ensure enough cells at low passage are obtainable for cryopreservation and experimental use. Primary lung epithelial cell protocols have been established for mice (Lam et al. 2011); however, the culturing of rabbit lung epithelial at ALI has not been reported. Numerous studies encompassing a wide-range of research fields utilize the Electronic supplementary material The online version of this article (doi:10.1007/s11626-014-9853-z) contains supplementary material, which is available to authorized users. J. D. Powell (*) : B. M. Hess : J. R. Hutchison : T. M. Straub Chemical and Biological Signature Sciences Group, Pacific Northwest National Laboratory, 902 Battelle Boulevard, MSIN: P7-50, P.O. Box 999, Richland, WA 99352, USA e-mail: [email protected]
rabbit lung model; these include but are not limited to anthrax spore inhalation (Phipps et al. 2004), tuberculosis (Subbian et al. 2012), lung metastasis (Huang et al. 2013), computational respiratory modeling (Corley et al. 2012), or for partially fulfilling animal efficacy requirements for FDA approval (Aebersold 2012). Considering the widespread use of the rabbit lung model, particularly New Zealand white rabbits (Oryctolagus cuniculus), we report results on the successful isolation and differentiation of O. cuniculus lung epithelial cells at ALI, referred to hereafter as normal rabbit bronchial epithelial cells (NRaBE). NRaBE culture conditions were optimized enabling ten serial passages representing >5×107 usable cells from a single rabbit trachea-lung. Even with limited rabbit-specific antibodies commercially available, we were able to confirm successful co-culturing for each rabbit’s respective peripheral blood mononuclear monocytes (PBMCs) deposited on top of NRaBE cells that resulted in differentiation into CD11c+ cells.
Materials and Methods Animal use. The housing and humane euthanasia of 2.5–5 kg New Zealand white rabbits (Harlan Laboratories, Indianapolis, IN) was approved by the Pacific Northwest National Laboratory Institutional Animal Care and Use Committee (IACUC: 2014–04) under Animal Welfare Assurance number A3353-01. After anesthesia rabbits were exsanguinated and blood (~30 ml) collected in sodium heparin tubes for further processing. Excised lungs, representing both the trachea and lower bronchial regions were washed twice with 1×PBS buffer and processed as described below. A total of five rabbits were used with each rabbit’s respective cells harvested, cultured, and stored in liquid nitrogen individually for experimental use.
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Isolation, NRaBE. Extraction, protease treatment, and culturing of Rabbit NRaBE cells were similar to that reported for swine lung (Hauser et al. 2013) with the exception that protease treatment with pronase (Roche Applied Science, Penzberg, Germany) was for 16 h instead of 72 h. Briefly, the lungs were excised with the trachea and lung being separated from the remaining tissue. The trachea, and after careful extraction of major visible bronchiole regions, was incubated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1.5 mg/ml pronase with end-over-end incubation in 50 ml conical tubes for 16 h at 4°C. Next, the inner surface of the trachea and entire bronchiole tubes were gently scraped with a sterile scalpel in DMEM. Cells were strained through a 100 μM cell strainer (Falcon, VWR International, Radnor, PA) in DMEM supplemented with 10% fetal bovine serum (FBS), Primocin antibiotic (InvivoGen, San Diego, CA), and Fungizone antimycotic (Life Technologies, Carlsbad, CA) at manufacturer recommendations. Of note, serum was specifically used to neutralize pronase enzymatic activity. Next, cells were pelleted at 300×g for 5 min before cell culturing. Culturing NRaBE in undifferentiated state. Resuspension of cell pellets and culturing of NRaBE was performed according to the manufacturers suggestions for human NHBE cells (Lonza, Basel, Switzerland) in BronchiaLife B/T media with the specified cocktail of serum-free growth supplements (Lifeline Cell Technologies, Carlsbad, CA; LL-0023), Primocin antibiotic, and Fungizone antimycotic. Before passage 1, an epithelial enrichment step was performed with 2-h incubation in non-collagen-treated tissue culture T-150 flasks at 37°C, 5% CO2 to preferentially adhere unwanted fibroblasts while unbound epithelial cells were collected for further culturing. Of note, unwanted fibroblasts selected through adherence to non-collagen-treated tissue culture flasks for 2 h were performed every passage after trypsinizing cells and never represented more than 1% of the total epithelial cell population. At passage 3, a subset of NRaBEs were placed in cryostorage medium [50% BronchiaLife B/T, 40% fetal bovine serum (FBS), 10% dimethyl sulfoxide (DMSO)] and stored in liquid nitrogen for long-term use. Of note, and also shown in Fig. 1D, before differentiation, NRaBEs were supplemented with 1.0 nM retinoic acid (Sigma-Aldrich, St. Louis, MO) from passages 2–4 before differentiation at passage 4 in all subsequent reported results. Differentiation of NRaBEs. For ALI experiments, NRaBEs at passage 4 were cultured to 100% confluency from initial seeding at 1×105 cells (12-well) or 5×104 cells (24-well) on polycarbonate transwell inserts (Corning Inc., Corning, NY). BronchiaLife B/T media used during growth in the undifferentiated state had 1 nM RA, but higher 50 nM RA concentration after ALI. Cells were brought to ALI at 100% confluency at day 5 or 6 by leaving the apical surfaces without media to
induce differentiation and basal media changed every 72 h thereafter. Epithelial resistance was measured with a MillicellERS VOLT-OHM meter (EMD Millipore, Billerica, MA) during growth, and differentiation in 12-well transwells and apical surfaces were washed with PBS after measurements to remove excess mucus. NRaBE cells for results reported in Fig. 2 were cultured in specified RA and collagen conditions at time of seeding until day 18 end-point. In all cases, cells were 100% confluent at days 5–6 with additional culturing until day 8 before ALI. Unless otherwise stated in the results section, all transwell and tissue culture flasks were coated with 20-μg/cm2 rat tail collagen (Sigma-Aldrich) in 0.02 M acetic acid for 1 h, aspirated, and washed once in PBS before cell seeding. All cell culture conditions reported were incubated at 37°C, 5% CO2. NRaBE proliferation studies. As depicted in Fig. 1D, E, after undifferentiated epithelial cells reached 75–85% confluency in T-25 flasks at passage 1, a series of experimental conditions related to RA concentration and percent media replacement was assessed to deduce and optimize NRaBE culture conditions. 12-well plates were initially seeded with 5×104 cells (passage 2). When cells reached 90–95% confluency, they were trypsinized and then seeded again at 5×104 cells on 12-well plates (passage 3); this process was repeated a third time (passage 4). The cumulative days from initial passage 2 seeding to 95% confluency at passage 4 with standard deviation was determined with n=5 wells for each RA and media replacement experimental condition. Additionally, passages 5–7 experiments using 80 versus 100% media replacement with or without 1.0 nm RA was also assessed (Fig. 1E). In all cases, the media were replaced every 48 h through the duration of proliferation studies to maintain cell health in specified culture conditions. Isolation, culturing, and differentiation of PBMCs. Rabbit PBMCs were isolated by Ficoll-Paque gradient according to the manufacturer’s instructions (GE Healthcare, Buckinghamshire, UK). A 2-h adherence to tissue culture-treated flasks for monocyte enrichment was performed before downstream use. PBMCs were cultured in RPMI-1640 media (Lonza) supplemented with 10% FBS. Dendritic cells differentiation from PBMCs were cultured in LGM-3 media +2 mM L-glutatmine (Lonza) supplemented with 50 ng/mL granulocytemacrophage colony-stimulating factor (GM-CSF) (R&D Systems, Minneapolis, MN) and 50 ng/mL interleukin 4 (IL-4) (R&D Systems) for 7 d with media replacement at day 3. Monocyte to macrophage differentiation was performed through the use of phorbol myristate acetate (PMA) 20 ng/ mL (Sigma-Aldrich) or macrophage colony- stimulating factor (M-CSF) 20 ng/mL (R&D Systems) in Macrophage-SFM media (Life Technologies) for 6 d with media replaced at day 3. No exogenous cytokines were added on day 7 before use.
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Figure 1. Isolation and proliferation of NRaBE cells in vitro. A Rabbit trachea and lungs were extracted in one piece and were relatively devoid of surrounding tissue. B After protease digestion, epithelial cells were grown in culture; by passage 4, NRaBEs were largely devoid of unwanted fibroblasts and showed characteristic epithelial morphology. C Beginning at passage 10, NRaBE proliferation decreased significantly and by passage 12 cells appeared either highly polarized or showed visible signs of duress. D Increasing concentrations of retinoic acid (RA) representing no RA (−), 0.1, 1, and 100 nM in media with removal of either 100, 80, or 50% of media after 48 h and replacement with fresh media revealed optimal proliferation for 80% media replacement with 0.1
and 1.0 nM RA at passages 2–4. Shown are cumulative days after seeding from passage 2 to until 100% confluency was reached at passage 4. Standard deviation was calculated based on n=5 wells for each experimental condition. E Cumulative day totals representing passages 5–7 revealed no significant difference for 20% media carry-over and 1.0 nM retinoic acid suggesting earlier passages are more impacted by changes in these respective conditions, n=5 wells. F NRaBE cells cultured in a higher 100 nM RA concentration were substantially polarized by passage 4 and reached cell senescence at passage 7; therefore, they never reach confluency as shown in Fig. 2E experiments. Scale bars=100 μm.
For co-culture studies, 5×104 PBMCs were deposited on top of 4×105 confluent NRaBEs (5% of total) at day 8 postculture which represented initial day 0 for ALI differentiation. Co-cultures were then allowed to incubate for an additional 5 d before analysis. All media used in PBMC differentiation was supplemented with 0.5% Penicillin-Streptomycin antibiotic (Life Technologies).
according to the manufacturer’s suggested protocols. All other fluorescent staining entailed fixation in 4% PFA for 30 min. Immunofluorescence blocking and primary and secondary steps were performed in PBS buffer supplemented with 3% bovine serum albumin (BSA) using standard immunofluorescence incubation and wash times. With the exception of cellsurface lectin staining and CellMask Orange Plasma Membrane stain (Life Technologies), cells were first permeablized in PBS, 0.5% Triton-X for 20 min. Primary antibody concentrations are specified with all secondary antibody staining
Cell staining. Cell viability was performed with Invitrogen Live/Dead® Viability/Cytoxicity kit (Life Technologies)
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POWELL ET AL.
Figure 2. Characterization of NRaBE cultured in transwells with and without collagen and brought to ALI (day 8 line) in the presence or absence of either A 50 nM retinoic acid or B without RA. Peak TEER for the mean of three independent wells for the specified condition, asterisk (*) denotes significance p value
REPORT
Construction of an in vitro primary lung co-culture platform derived from New Zealand white rabbits Joshua D. Powell & Becky M. Hess & Janine R. Hutchison & Timothy M. Straub
Received: 12 August 2014 / Accepted: 23 November 2014 / Published online: 10 December 2014 / Editor: Tetsuji Okamoto # The Society for In Vitro Biology 2014
Introduction Co-culturing of primary lung epithelial cells with monocytes provides an avenue to study lung-associated responses to carcinogens and pathogens in vitro. Historically, a major obstacle in the development of tissue culture-based lung model development was epithelial proliferation without culturing unwanted fibroblasts (Wise and Lechner 2002). Advances in cell culture media formulations and the advent of the transwell dish now enable convenient culturing of lung epithelial cells at air liquid interface (ALI). Due to the high numbers of bronchial epithelial cells that can be isolated from lung postmortem for human (Lechner and LaVeck 1985) (Gruenert et al. 1995), horse (Shibeshi et al. 2008), or pig (Hauser et al. 2013), a sufficient number of cells can be isolated at low passage for experimental use. Unfortunately in smaller mammalian species, lung epithelial-based experiments can be challenging because cells from the trachea and small bronchiole tubes can be difficult to culture to substantial quantities prior to the onset of cell senescence. In light of this challenge posed from smallersized animals, it is crucial that optimal growth conditions are deduced to ensure enough cells at low passage are obtainable for cryopreservation and experimental use. Primary lung epithelial cell protocols have been established for mice (Lam et al. 2011); however, the culturing of rabbit lung epithelial at ALI has not been reported. Numerous studies encompassing a wide-range of research fields utilize the Electronic supplementary material The online version of this article (doi:10.1007/s11626-014-9853-z) contains supplementary material, which is available to authorized users. J. D. Powell (*) : B. M. Hess : J. R. Hutchison : T. M. Straub Chemical and Biological Signature Sciences Group, Pacific Northwest National Laboratory, 902 Battelle Boulevard, MSIN: P7-50, P.O. Box 999, Richland, WA 99352, USA e-mail: [email protected]
rabbit lung model; these include but are not limited to anthrax spore inhalation (Phipps et al. 2004), tuberculosis (Subbian et al. 2012), lung metastasis (Huang et al. 2013), computational respiratory modeling (Corley et al. 2012), or for partially fulfilling animal efficacy requirements for FDA approval (Aebersold 2012). Considering the widespread use of the rabbit lung model, particularly New Zealand white rabbits (Oryctolagus cuniculus), we report results on the successful isolation and differentiation of O. cuniculus lung epithelial cells at ALI, referred to hereafter as normal rabbit bronchial epithelial cells (NRaBE). NRaBE culture conditions were optimized enabling ten serial passages representing >5×107 usable cells from a single rabbit trachea-lung. Even with limited rabbit-specific antibodies commercially available, we were able to confirm successful co-culturing for each rabbit’s respective peripheral blood mononuclear monocytes (PBMCs) deposited on top of NRaBE cells that resulted in differentiation into CD11c+ cells.
Materials and Methods Animal use. The housing and humane euthanasia of 2.5–5 kg New Zealand white rabbits (Harlan Laboratories, Indianapolis, IN) was approved by the Pacific Northwest National Laboratory Institutional Animal Care and Use Committee (IACUC: 2014–04) under Animal Welfare Assurance number A3353-01. After anesthesia rabbits were exsanguinated and blood (~30 ml) collected in sodium heparin tubes for further processing. Excised lungs, representing both the trachea and lower bronchial regions were washed twice with 1×PBS buffer and processed as described below. A total of five rabbits were used with each rabbit’s respective cells harvested, cultured, and stored in liquid nitrogen individually for experimental use.
434
POWELL ET AL.
Isolation, NRaBE. Extraction, protease treatment, and culturing of Rabbit NRaBE cells were similar to that reported for swine lung (Hauser et al. 2013) with the exception that protease treatment with pronase (Roche Applied Science, Penzberg, Germany) was for 16 h instead of 72 h. Briefly, the lungs were excised with the trachea and lung being separated from the remaining tissue. The trachea, and after careful extraction of major visible bronchiole regions, was incubated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1.5 mg/ml pronase with end-over-end incubation in 50 ml conical tubes for 16 h at 4°C. Next, the inner surface of the trachea and entire bronchiole tubes were gently scraped with a sterile scalpel in DMEM. Cells were strained through a 100 μM cell strainer (Falcon, VWR International, Radnor, PA) in DMEM supplemented with 10% fetal bovine serum (FBS), Primocin antibiotic (InvivoGen, San Diego, CA), and Fungizone antimycotic (Life Technologies, Carlsbad, CA) at manufacturer recommendations. Of note, serum was specifically used to neutralize pronase enzymatic activity. Next, cells were pelleted at 300×g for 5 min before cell culturing. Culturing NRaBE in undifferentiated state. Resuspension of cell pellets and culturing of NRaBE was performed according to the manufacturers suggestions for human NHBE cells (Lonza, Basel, Switzerland) in BronchiaLife B/T media with the specified cocktail of serum-free growth supplements (Lifeline Cell Technologies, Carlsbad, CA; LL-0023), Primocin antibiotic, and Fungizone antimycotic. Before passage 1, an epithelial enrichment step was performed with 2-h incubation in non-collagen-treated tissue culture T-150 flasks at 37°C, 5% CO2 to preferentially adhere unwanted fibroblasts while unbound epithelial cells were collected for further culturing. Of note, unwanted fibroblasts selected through adherence to non-collagen-treated tissue culture flasks for 2 h were performed every passage after trypsinizing cells and never represented more than 1% of the total epithelial cell population. At passage 3, a subset of NRaBEs were placed in cryostorage medium [50% BronchiaLife B/T, 40% fetal bovine serum (FBS), 10% dimethyl sulfoxide (DMSO)] and stored in liquid nitrogen for long-term use. Of note, and also shown in Fig. 1D, before differentiation, NRaBEs were supplemented with 1.0 nM retinoic acid (Sigma-Aldrich, St. Louis, MO) from passages 2–4 before differentiation at passage 4 in all subsequent reported results. Differentiation of NRaBEs. For ALI experiments, NRaBEs at passage 4 were cultured to 100% confluency from initial seeding at 1×105 cells (12-well) or 5×104 cells (24-well) on polycarbonate transwell inserts (Corning Inc., Corning, NY). BronchiaLife B/T media used during growth in the undifferentiated state had 1 nM RA, but higher 50 nM RA concentration after ALI. Cells were brought to ALI at 100% confluency at day 5 or 6 by leaving the apical surfaces without media to
induce differentiation and basal media changed every 72 h thereafter. Epithelial resistance was measured with a MillicellERS VOLT-OHM meter (EMD Millipore, Billerica, MA) during growth, and differentiation in 12-well transwells and apical surfaces were washed with PBS after measurements to remove excess mucus. NRaBE cells for results reported in Fig. 2 were cultured in specified RA and collagen conditions at time of seeding until day 18 end-point. In all cases, cells were 100% confluent at days 5–6 with additional culturing until day 8 before ALI. Unless otherwise stated in the results section, all transwell and tissue culture flasks were coated with 20-μg/cm2 rat tail collagen (Sigma-Aldrich) in 0.02 M acetic acid for 1 h, aspirated, and washed once in PBS before cell seeding. All cell culture conditions reported were incubated at 37°C, 5% CO2. NRaBE proliferation studies. As depicted in Fig. 1D, E, after undifferentiated epithelial cells reached 75–85% confluency in T-25 flasks at passage 1, a series of experimental conditions related to RA concentration and percent media replacement was assessed to deduce and optimize NRaBE culture conditions. 12-well plates were initially seeded with 5×104 cells (passage 2). When cells reached 90–95% confluency, they were trypsinized and then seeded again at 5×104 cells on 12-well plates (passage 3); this process was repeated a third time (passage 4). The cumulative days from initial passage 2 seeding to 95% confluency at passage 4 with standard deviation was determined with n=5 wells for each RA and media replacement experimental condition. Additionally, passages 5–7 experiments using 80 versus 100% media replacement with or without 1.0 nm RA was also assessed (Fig. 1E). In all cases, the media were replaced every 48 h through the duration of proliferation studies to maintain cell health in specified culture conditions. Isolation, culturing, and differentiation of PBMCs. Rabbit PBMCs were isolated by Ficoll-Paque gradient according to the manufacturer’s instructions (GE Healthcare, Buckinghamshire, UK). A 2-h adherence to tissue culture-treated flasks for monocyte enrichment was performed before downstream use. PBMCs were cultured in RPMI-1640 media (Lonza) supplemented with 10% FBS. Dendritic cells differentiation from PBMCs were cultured in LGM-3 media +2 mM L-glutatmine (Lonza) supplemented with 50 ng/mL granulocytemacrophage colony-stimulating factor (GM-CSF) (R&D Systems, Minneapolis, MN) and 50 ng/mL interleukin 4 (IL-4) (R&D Systems) for 7 d with media replacement at day 3. Monocyte to macrophage differentiation was performed through the use of phorbol myristate acetate (PMA) 20 ng/ mL (Sigma-Aldrich) or macrophage colony- stimulating factor (M-CSF) 20 ng/mL (R&D Systems) in Macrophage-SFM media (Life Technologies) for 6 d with media replaced at day 3. No exogenous cytokines were added on day 7 before use.
RABBIT LUNG IN VITRO MODEL
435
Figure 1. Isolation and proliferation of NRaBE cells in vitro. A Rabbit trachea and lungs were extracted in one piece and were relatively devoid of surrounding tissue. B After protease digestion, epithelial cells were grown in culture; by passage 4, NRaBEs were largely devoid of unwanted fibroblasts and showed characteristic epithelial morphology. C Beginning at passage 10, NRaBE proliferation decreased significantly and by passage 12 cells appeared either highly polarized or showed visible signs of duress. D Increasing concentrations of retinoic acid (RA) representing no RA (−), 0.1, 1, and 100 nM in media with removal of either 100, 80, or 50% of media after 48 h and replacement with fresh media revealed optimal proliferation for 80% media replacement with 0.1
and 1.0 nM RA at passages 2–4. Shown are cumulative days after seeding from passage 2 to until 100% confluency was reached at passage 4. Standard deviation was calculated based on n=5 wells for each experimental condition. E Cumulative day totals representing passages 5–7 revealed no significant difference for 20% media carry-over and 1.0 nM retinoic acid suggesting earlier passages are more impacted by changes in these respective conditions, n=5 wells. F NRaBE cells cultured in a higher 100 nM RA concentration were substantially polarized by passage 4 and reached cell senescence at passage 7; therefore, they never reach confluency as shown in Fig. 2E experiments. Scale bars=100 μm.
For co-culture studies, 5×104 PBMCs were deposited on top of 4×105 confluent NRaBEs (5% of total) at day 8 postculture which represented initial day 0 for ALI differentiation. Co-cultures were then allowed to incubate for an additional 5 d before analysis. All media used in PBMC differentiation was supplemented with 0.5% Penicillin-Streptomycin antibiotic (Life Technologies).
according to the manufacturer’s suggested protocols. All other fluorescent staining entailed fixation in 4% PFA for 30 min. Immunofluorescence blocking and primary and secondary steps were performed in PBS buffer supplemented with 3% bovine serum albumin (BSA) using standard immunofluorescence incubation and wash times. With the exception of cellsurface lectin staining and CellMask Orange Plasma Membrane stain (Life Technologies), cells were first permeablized in PBS, 0.5% Triton-X for 20 min. Primary antibody concentrations are specified with all secondary antibody staining
Cell staining. Cell viability was performed with Invitrogen Live/Dead® Viability/Cytoxicity kit (Life Technologies)
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POWELL ET AL.
Figure 2. Characterization of NRaBE cultured in transwells with and without collagen and brought to ALI (day 8 line) in the presence or absence of either A 50 nM retinoic acid or B without RA. Peak TEER for the mean of three independent wells for the specified condition, asterisk (*) denotes significance p value
