There are good protocols published especially those coming from the Univ North Carolina for growing hBECs as human bronchial epithelial cells are generally referred. Clump disruption relies on enzymatic digestion with Pronase and DNase, or alternatively mechanical disruption. After these simple steps, mucus will float and epithelial cells can then be grown on collagen- coated plates for later transferance to Transwell inserts for Air-Liquid Interface and attendant cell differentiation and polarization. - I don't understand your point about cell depletion since mainly Basal p63+; Cytokeratin 5/14+ cells are the ones that grow and are able to reconstitute the epithelium in vitro.

Thanks for your answer, Oscar. We're doing direct characterisation of the isolated epithelial cells, so we won't be growing them up as a primary culture. I know most labs use selective culturing techniques, but I won't be able to use these. I've considered using DTT to remove mucus and subsequently use trypsin, dispase and DNase digestion to de-clump the cells. Magnetic particle-conjugated antibody isolation against MUC1, cytokeratin or other surface antigens could then be used to obtain the hBECs. I'm curious if anybody has tried this before on this tissue and if they have any tips or alternative methods.

Hi Sebastian, I'm sorry I misunderstood the question. - I'm not aware of any protocol attempting to do what you want, that would be interesting to see. - From my perspective, I think it's better to take the whole epithelium since viability of these cells is already low (20-30% by Trypan blue) before processing. Moreover, cells are tightly associated by adherens junctions of all kinds so significant proteolytic damage can occur to cell surface receptors during enzymatic dissociation hampering immunomagnetic isolation. In fact, prolonged or high concentration enzyme can make the cultures negative so it's a reflection of poor cell viability. -  In the normal epithelium greater than 70% of cells are ciliated with the remainder being Basal, Clara and Goblet cells. Asthmatic or COPD individuals might have reciprocally fewer ciliated cells with more of the mucous type, thus getting rid of any particular cell type would remove any disease-relevant gene expression differences or protein content. In that regard I think it's best to keep the whole epithelium and measure what you want and correct by a normalizer or housekeeping gene like OAZ1.  - If you want to get rid of red cells you can try a hypotonic red cell lysis but it might further decrease viability. - Another idea that I once wanted to try was to pass cells through a Ficoll cushion so that dense-r cells like erythrocytes would be pelleted while epithelial cells and mucous cells would not pass the cushion. Mucous can be easily removed by simple centrifugation: it floats. However, in the clinical trials in which I worked for, none used density gradients. Or as you correctly suggest, use DTT but I really don't know how it might affect hBECs. Sputolysin containing DTT is used routinely in making mucous fluid in sputum processing protocols and it doesn't significantly affect leukocytes that are the majority of 'sputocytes'.  Good luck.

Hi Oscar, thanks for your reply. Unfortunately I can only work with the brushings as we're talking about human samples. I indeed would like to capture all cell types of the epithelium and was hoping to identify a marker that would enable me to do that. I believe MUC1 could be one such marker. Alternatively, negative selection for leukocytes and erythrocytes could be an option. I like the Ficoll cushion idea, would this also filter out the leukocytes or would they not be dense enough?

Hi Sebastian, when I recommended taking the whole epithelium I meant the whole epithelial material you get in a brushing, in fact  my experience is almost exclusively with human cells from asthma patients and healthy controls. -  Usually you might get greater than 1 million cells with 4-5 passes with a rather large brush, long strokes give greater yields. -  I haven't attempted to get rid of leukocytes but possibly negative selection with common leukocyte antigens like CD45, CD19 and CD3 might do the trick. - I'm not familiar with MUC1 as a capture antigen. Have you stained for it?  I think it might be a good idea to immunostain paraffin sections using a panel of potential membrane markers that you want to try in your isolation just to make sure that cells abundantly express those markers. E-Cadherin forms tight junctions and is known to be abundant in ciliated cells, however, I don't know if they survive the enzymatic dissociation, or are not differentially expressed in the cells you want to remove from your isolates. - Please let us know how it goes.

have you tried something as basic as decanting into 5 ml normal saline and then centrifuging ?  the mucus will separate from the cells and the cells concentrate at the bottom allowing for easier isollation.

@Oscar

We are indeed looking to deplete using anti leukocyte/lymphocyte antigens. Have you any experience with erythrocyte contamination of the bronchial brushing? MUC1 should be attached to the matrix of all three epithelial cell types, but might indeed dissociate during de-clumping.

@Dave

We're considering this as an option and may be all that is necessary. I'm talking to my collaborator about this.

Hi Sebastian, RBC contamination in bronchial epithelial bushings is very common in bronchoscopies from asthmatic individuals, the more severe the asthma, the greater chance of bleeding during the brushing maneuver. - I have tried hypotonic cell lysis but it lowers epithelial cell viability. - In a message that was lost I replied that if you wanted to get rid of leukocytes with immunomagnetic beads you could at the same time get rid of RBCs by targeting Blood Group or Rh antigens, and if you wanted a more general target, the abundant transmembrane erythrocyte protein glycophorin would be a good one. However, I realized that in my lab we had experience with a Ficoll-type cushion from STEM CELL TECHNOLOGIES that took advantage of RBCs to get rid of specific types of leukocytes (you choose which ones) by forming rossettes with RBCs. Thus you get rid of leukocytes and RBC in the pellet and above the cushion you may get the less dense epithelial cells. It's important that neither RBCs or leukocytes express the negatively selected antigen. The product line is called Rossette-Sep and you choose the one that suits your needs. Please let us know how it goes. Good luck!.

Many thanks for your help, Oscar. I'm actually talking to STEM CELL TECHNOLOGIES and we're going to try to combine two of their kits: CD45 and Glycophorin negative selection kits. This should hopefully get rid of all lympho/leuko/erythrocytes. I'll let you know how it goes.

Sebastian, this is great!. Removal of non-epithelial cells would allow you to have a pure source of cells for proteomic and other 'omic' approaches. 

Oscar, could I ask, I'm going to optimise a brief dispase treatment of the bronchial brushings in order to declump the cells. I can't decide to perform this in BEGM or Hank's Balanced Salt Solution. Do you have any tips?

Thanks

Hi Sebastian, if the dispase requires Calcium ions then BEGM is the way to go, but if calcium ions should be absent then HBSS is preferable. I only have experience with Pronase and BEGM is a good media for this. Good luck.

Dear Dr Sebastian Vencken

I am trying to isolate bronchial epithelial cells from a sputum sample. Any advice from your experience working with this type of cells ?

De Dr. Sebastian Vencken,

I would like to ask if you find a way to get rid of mucus in your sample? I have the same problem. Do you have any suggestion?

Best wishes,

Kang