A 1mm3 lung biopsy likely would contain plenty of fibroblasts if the aim were to culture the biopsy and isolate fibroblasts. I used to sometimes use a technique called unit gravity sedimentation to remove fibroblast populations from preps of primary keratinocytes. Basically, the single cell suspension was carefully layered over a step gradient made of differing amounts of BSA dissolved in PBS. After the cell suspension was layered on top of the first BSA cushion with the lowest % BSA, the cells sedimented by gravity alone. The fibroblast population would float on top of the first BSA step while other cell types sedimented further into the BSA step gradient according to their densities. The problem this approach might present for your application is that the fibroblast cell number in the biopsy will be small. Is there a particular reason why you prefer to not expand the fibroblast population?

Make a single cell suspension and then use either CD90 or CD271-antibody coated magnetic beads. It of course depends on how many cells you need for futher investigation. Good luck!

Hi Sdegh, you need to desinfect with antibiotics, http://www.gentaur.com/nieuwe_pagina_7.htm

In agreement with Suchitra, Immunomagnetic bead isolation would be advisable for small tissue biopsies, Miltenyi microbeads may prove useful for direct analysis if larger Dynabeads fail to detach easily. The following reference might be helpful for you: Kisselbach et al., 2009, Cytotechnology 59(1):31-44. The ideal choice of target antigen may be context dependent, e.g. in lung fibrosis certain antigens e.g. Thy-1 can be suppressed in stimulated fibroblasts. Primary lung fibroblasts constitutively express alpha(4)beta(7) integrin receptor (Kohan et al., 2010, FASEB J 24:4503-12).

Use this

http://www.cf.gu.se/digitalAssets/1298/1298666_ZEISS_LabProtocol_CellCulture.pdf

very expensive but was developed by Palm exactly for this problem. It uses lasers to cut out the cell (or area of cells) then 'jumps' it out for isolation

Thank you all.

In fact I don't want to change the expression profile of the cells. I want to prepare a cDNA library, then amplify it for microarray. I have seen some papers that they expand primary cells but it definitely changes the transcriptome profile of the cell. I asked some friends about the cells separated with MACS. The advised me first for a FACS to estimate the percent of the cells in a biopsy. Then, they were not hopeful if MACS work with low cell numbers. Just because of small cell number.

Then, we have Lase microdissection in our Lab. But, it seems impossible for this issue. I mean if we prepare slides, do IHC and then cut the cells, we will miss-leaded from our goal. Cause, when we sea a cell in IHC, this means that, the cell is cut. I don't know if you can imagine it or if I am wrong. Then if we use single cell suspensions, how we can distinguish cells? If any way to this, how many cells we can pick up one by one? I will check this but not hopefully.

Also, I have studied fibroblast specific markers. They are optimized for normal tissue. The in diseased tissue, other cells also express mentioned markers that makes protocol complicated. I should perform negative selections too. But we can find plenty of papers which have used them. is it right? no other way? (thank you for the papers).

then, what should be negative and positive QC?

Magnetic bead or laser capture, Sadegh!

I published a paper with a working protocol on how to do this. We isolated CAFs (carcinoma-associated fibroblasts) from murine lungs and also use this protocol for our human lung tissue specimens. You might wanna have a look and see if it's helpful.

@ Jonathon Roybal

Thank you. Very useful paper. You scored a bull's eye. I wonder if you had other similar papers or if you know sth from others to introduce me.

To establish a primary cultured cell from a small biopsy the following method will be worth to try. Plate the small biopsy onto the bottom surface of an empty culture dish. Collagen or gelatin coated dish will be preferable. Let the biopsy stick onto the bottom surface by keeping the dish in a humidified incubator for 20 - 30 min, then pour a small amount of medium on the biopsy to start culture.

Fibroblast cells will start to grow in a several days. You may need a weight to prevent the lung biopsy from floating.

I am not sure if you meant human fibroblasts. You could try incubate lung sample at 37°C overnight (or less) adding 0.5% collagenase A solution. After incubation period, gently shake (or spin down the solution in low gravity) and remove lung tissue and spin down the digested solution in a centrifuge at 300g for 10 minutes. Discard supernatant and suspend the pellet in Fibroblasts Cell Medium. To avoid cell proliferation do not use foetal calf serum in fibroblasts medium.

CCD19Lu can be used in immunocytochemistry for cell type confirmation.

Fibroblast count will be low and, you may get lung myofibroblasts too.

Hope this helps.

You have noted there is a technique called "laser microdissection" that allows you to isolate single cells from a biopsy: http://www.leica-microsystems.com/science-lab/laser-microdissection/an-introduction-to-laser-microdissection-precise-location-or-separation-of-single-cells-and-tissue-structures/ That will get you a cell or cells that you identify as "fibroblasts" by whatever criteria you are using. Now, whether you can get enough mRNA from those cells to do a cDNA library for microarray is a different question.

IF you decide to identify the fibroblast by antibody staining, and IF the antigen you are using is on the cell surface, then you don't have to fix the tisssue in order to stain it. The cells will be alive and unchanged. Just incubate with the primary antibody under physiological conditions in media (without serum), wash, and then incubate with your labeled secondary antibody. Then do the microdissection. The Leica site has examples where this has been done.

As a conclusion:

1- I should get a biopsy.

2- I should prepare a single cell suspension without expanding. I should use enzymatic treatment.

3- centrifugation.

4- then antibody treatment for performing a FACS to estimate humang lung fibroblast frequency in a biopsy.

5- I should use single prepared cell suspension for MACS or lasemicrodissection.

6- Prepare cDNA.

right?

You may isolate the spindle-shaped fibroblasts/myofibroblasts by laser-captured microdissection on the frozen sections of about 10 micrometers in thickness. The isolates pooled may be sufficient for expression profiling. Good luck!

Sadegh: If I am reading the Leica site correctly, your steps are:

1. Get a biopsy

2. Frozen section the biopsy (or fix and paraffin section, it doesn't seem to matter)

3. Use immunohistochemistry to identify the fibroblasts in several sections

4. Laser microdissect the fibroblasts.

5. Extract mRNA from the dissected fibroblasts.

6. Prepare cDNA.

It's all right there at the Leica site. If you have any other questions, I strongly suggest you just call Leica and ask for Technical Support.

You don't need any enzymatic treatment or FACS IF you have access to laser microdissection. Now, if you do NOT t have access to laser microdissection, then yes, you will need to do the enzymatic digestion and then a FACS to get the fibroblasts.

@sadegh

No problem. There are plenty of people of that use these techniques but they usually aren't specific for lung. In fact I adapted my protocol from a breast cancer group. Most of the cell separation people are in immunology groups. Just remember as you isolate certain fibroblasts, they are all marker dependent so the biology that you see may be specific to a certain population. For example CD90 (THY1) is not just specific to fibroblasts so you need to remove the other cells that also have this marker so that you only have fibroblasts left over. Other CAF markers are (FSP-1, FAP, PDGFR, etc).

Qin: Fibroblasts are spindle shaped in adherent cultures. In a frozen section of a biopsy,they would not necessarily be that shape. For instance, if the section is perpendicular to their long axis (even if spindle shaped), you would see them as a circle in the microscope.

Dear Paul, no problem for a pathologist to identify (myo)fibroblasts on the cryostat sections slightly stained or even not stained but with phase contrast lense. The protocol may be like those listed below.

1. take an biopsy;

2. prepare serial frozen sections of 10 micrometers in thickness;

3. stain the first sections with H&E staining and for vimentin, respectively;

4. microscopic observation for determing the locations of (myo)fibroblastis areas, the target cells appear spindle or in bundles;

5. microdissection of the selected areas from the remaining serial cryostat sections and pool the isolates from same selected areas;

6. extraction, purification and your analyses;

7. the epithelial and vascular components can also be isolated to do the parallele analyses as the internmal reference tissue types.

Good luck!

Below may helpful for you to isolate fibroblast.

http://link.springer.com/protocol/10.1385%2F1-59259-940-0%3A115?LI=true

I strongly recommend the same protocol proposed by Hiroshi Nagashima; I use this method for rapid and easy isolation of dermal fibroblasts from extremely small skin biopsies (i have tried this from different species). You can check some additional features on Lattanzi et al, Gene Ther. 2008 Oct;15(19):1330-43 (Free in PMC)

Good luck

What can you be doing on a single cell that you wouldn't want a clean population to draw from? Even for cloning, a few simple culture techniques allow you to exploit the vigorous proliferation of fibroblasts (most researchers want to get rid of them!) in two ways: either in a primary culture, or by exploiting their superior attachment times. Primary cell culture at W. Alton Jones Cell Science Center (Tissue Culture Assoc. of America, Lake Placid) included making cultures from tissue cubes ~ 0.5 mm in dimension. If you’re using needle biopsies this is perfect, but use small culture dishes and don’t overwhelm with medium. Epithelial cells may take 8 or more days to begin to grow out, but fibroblasts start to crawl away a lot faster.

If you take the dissociation route, I’d choose gentle trypsinization rather than collagenase, considering how essential collagen is to a fibroblast; then quench enzyme action with a serum-laced wash. As you plate, be ready with two sets of prepared plates (precoated with poly-L-lysine to promote attachment), because after you permit the attachment process to incubate for (say) 15-20 minutes, you will then pour off the lysate into another dish. Many fibroblasts should have already attached to the first dish: this is your seed culture, which you can check within an hour for flattened, happy cells. You can continue doing transfers from the lysate if the first plate wasn’t successful, allowing more time; every time you pour off it resets the attachment routine clock for the cells, particularly those that have low attachment efficiency.

An excellent reference describing establishing fibroblast cultures is: "Preparation and Isolation of Cells". In: Current Protocols in Cell Biology webdoc.nyumc.org/.../attachments/CPCB.ch02.Isolate%20cells.pdf . It describes the need for sharply cut edges of the explant as well as using coverslips to provide a microenvironment.

Of the two methods, with the understanding that your biopsies are small in quantity, the risk of dissociating the whole batch and not getting any live cells seems risky; explants may be slower, but are kinder to the tissue. Why not try both methods on control tissue until you get one to work well in your hands? All this assumes you already have media and conditions suitable for the type of tissue you’re trying to grow, but the procedures are relatively simple.

you might try to just culture whole tissue on the collagen coated dish with Fibroblast media, then you might get mixed cells, you could use Fac (or magnet method) using Fibroblast antibody to sort out…..

I have not done cell culture for a while, but I did with this method and very easy just with different tissues.

Thanks

We have three ways:

1- biopsy culture and primary fibroblast selection (I don't prefer as I don't want to change the expression profiling);

2) Laser microdissection;

3) single cell preparation and isolation using MACS or flow cytometry and sorting.

In the case of LCM (lasermicrodissection) there is one important concern. Imagine a bit microscopic. Then look at the prepared slide of IHC. What we will see? We will see a colored fibroblast from top part, then probably another cell beneath. Isn't it? We cant make sure that the separated part contains just one single fibroblast cell. Moreover, even if a single cell is separated, its not totally intact. Its cleaved by cryosector. I mean we may have a single fibroblast cleaved.

Now, in the third way we will find single cells but a bit stimulated. because cells are alive and responding to environment.

Who can make a good conclusion of this?

1) The hyperplastic (myo)fibroblats usally appear as bundles or sheets, that will provide a chance to isolate sufficient cells out of the slides. 2) For a skillful pathologist, It is not necessary to perform the LCM isolation using the slides which are already stained by immunohistochemistry, or even by the H&E staining. 3) Considering the size of the cells to be isolated and the fact that you'll collect the same target areas and pool them together for the profiling, splitting the cells involved will not be a big problem.

For the in vitro propagation approach, staying and growth in culture may also result in significant changes in expression profiles. It will need further caution for the data interpretation.

Wish you a good luck!

Dear Qin

May you introduce a good reference for non-staining determination of fibroblasts? It seems that I should be a fibroblast pathologist first. :)

Then you write (myo)fibroblasts and not fibroblasts? What do you mean? I just need fibroblasts.

1) The fibroblasts can be identifiable by morphology and pattern from the unstained cryostat scetions by phase contrast microscopy, or from the frozen sections slightly stained using a blue dye which will ne interfere with the following assays. You may approach to a surgical pathologist to help you.

2) For technical issues of the LCM, you may approach to Leica or ABI persons. For references, you can also find our experiences in this area in some of my articles which are available here. Fibroblasts and myofibroblasts are a continuous spectrum between the lineage differentiation from collagen-producing phenotypes, similar to fibrocytes, to contraction function-related phenotypes, simulating smooth muscle cells. You may find some reviews on it for updating.

Dear Qin;

Does it need a special biopsy preparation before freezing?

Yes, you need some medium to embed the tiny tissue before freezing, which will allow you to handle the tissue and cryostat section preparation.

In addition, if you can find an antibody speicific for the fibroblasts, flow sorting might also be an option. The question is how to find an antibody which is specific for these spindle-shaped mesenchymal cells named as fibroblasts.