Thread: Primary culture & Scratch wound assays.

Hi all,

This is a long shot - Do any of you have experience with establishing primary cultures of adult murine skin fibroblasts, or with scratch wound assays? I am currently researching these protocols and will be performing them in the near future.

Just wondering if you have any tips or advice?

Best wishes,

Tridimity

I have experience in making primary cultures, I at least know the protocol

I also have experience working with the SC-1 cell line which is an adult mouse fibroblast cell line

Ace C15ka, would you mind if I ask you some questions as they arise when I first attempt the primary culture? I have created a unified protocol from 10 published protocols, sounds straight forward enough - except for peeling the epidermis from the dermis following incubation in trypsin at 4*C overnight.

By any chance, do you know how to separate the dermis from the epidermis?

Best wishes,

Tridimity

Sure Tridimity, I am happy to help, we also have a lab protocol that we've used hundreds of times now which probably takes less than an hour to complete and it can be used when establishing ANY type of primary culture (with adaptation of the enzymes specifically to the tissue)...

Before you start however, there are some things that might set you back. (I've talked to a few of my lab colleagues)

1) What tissues are you using to establish the cell culture? Is it embryonic or adult, and if your using adult mouse then you may have a big problem with the hair... the skin segment that you will be using has to be VERY CLEAN (via dissection), no fat, no necrotic tissue and NO hair should be present

If you've done that, you can start with the process of establishing the primary culture

You do not really have to separate the dermis from the epidermis, fibroblasts are the most resilient cells and are usually the cells that survive when you establish your primary culture, if you wanted another cell type to survive (like keratinocytes) you'd have to use a selective medium. (This is actually a draw back when you're trying to create a primary culture of a specific organ - damn fibroblasts are everywhere!!)

If you really want to seperate the dermis and epidermis, I suggest you also look into using dispase instead of trypsin... - This step however has to be very optimized, the trypsin concentration has to be very low and you incubation time should be very short....

If you want you can send me the protocol you've created so I can compare it to ours, and provide some background on the research, I also suggest you practice preparing primary cultures using chick embryos so that you can get used to and be comfortable with the sterile working technique and all the equipment

Remember, the advice I am providing here was not found in any research article or lab manual, its based on experience so I'm just telling you what works for us...

Thanks C15ka,

I will be establishing the primary culture from skin excised from the backs of adult mice. Might be more difficult than using embryos as the fibroblasts may proliferate at a lower rate, however one of the factors under consideration is age of the mice, so there's really no getting around this. I could consider using embryos to test the effect of genotype alone, independent of age. As for the hair, I plan to shave the backs of the mice and use hair removal cream and then disinfect with ethanol. Hopefully this should be sufficient. I'm confident that I can make a clean dissection of the skin, making sure to remove any residual fat or muscle tissue etc. - will just have to be mega-careful.

As far as separating the dermis from the epidermis is concerned, the study is looking at dermal atrophy specifically, so I would have to do this. And yeah, I don't want keratinocyte contamination... will need to use media that selects against keratinocytes.

The protocol that I plan to use is as follows:

1. Sacrifice the mouse by overdose of Euthatol (0.3ml) or by neck dislocation.
2. Remove hair on the back of the mouse using a shaver and hair removal cream.
3. Disinfect skin with ethanol
4. Excise back skin (3x3cm)making sure to separate skin from internal membrane
5. Scrape dissected skin free of fat and muscle
6. Flatten the skin and spread dermis side down in a sterile cell culture dish
7. Incubate overnight with 0.25% trypsin at 4*C
8. Discard the epidermis
9. Mince the dermis
10. Dissociate into a cellular suspension with collagenase (1mg/ml) in DMEM at 37*C for 1 hour with constant agitation
11. Pass the cell suspension through a cell strainer
12. Add an equal volume of DMEM (10% FBS plus antimicrobial)
13. Centrifuge cells for 5 minutes at 300 x g at 4*C to remove the trypsin and collagenase
14. Seed fibroblasts into cell culture dishes
15. Change medium one day after plating, and then every other day
16. Culture in media that is inhibitory to keratinocyte growth
17. Maintain in DMEM plus 10% FBS and 4mM L-glutamine (antimicrobials: optional) at subconfluent densities at 37*C in a humidified atmosphere containing 5% CO2
18. After 2-3 passages, gross morphology of cells will consist entirely of spindle-shaped fibroblasts by light microscopy
19. Split cells 1/3 and use between passages 3 and 6

Ref.

1. Baxter RM, Crowell TP, McCrann ME, Frew EM & Gardner H (2005) Analysis of the tight skin (Tsk1/þ) mouse as a model for testing antifibrotic agents. Laboratory Investigation, 85:1199-1209
2. Collins CA, Jensen KB, MacRae EJ, Mansfield W & Watt FM (2012) Polyclonal origin and hair induction ability of dermal papillae of neonatal and adult mouse back skin. Developmental Biology, http://dx.doi.org/10.1016/j.ydbio.2012.03.016
3. Echtermeyer F, Streit M, Wilcox-Adelman S, Saoncella S, Denhez F, Detmar M & Goetinck PF (2000) Delayed wound repair and impaired angiogenesis in mice lacking syndecan-4. The Journal of Clinical Investigation, 107(2):R9-R14
4. Gallucci RM, Lee EG & Tomasek JJ (2006) IL-6 Modulates Alpha-Smooth Muscle Actin Expression in Dermal Fibroblasts from IL-6-Deficient Mice. Journal of Investigative Dermatology, 126: 561-568
5. Marut WK, Kavian N, Servettaz A, Nicco C, Ba LA, Doering M, Chereau C, Jacob C, Weill B & Batteux F (2012) The Organotelluride Catalyst (PHTE)2NQ Prevents
HOCl-Induced Systemic Sclerosis in Mouse. Journal of Investigative Dermatology, 132:1125-1132
6. Prowse KR & Gredier CW (1995) Developmental and tissue-specific regulation of mouse telomerase and telomere length. Proc. Natl. Acad. Sci. USA, 92:4818-4822
7. Rogers KM, Black DH & Eberle R (2007) Primary mouse dermal fibroblast cell cultures as an in vitro
model system for the differential pathogenicity of cross-species herpesvirus papio 2 infections. Archives of Virology, 152:543-552
8. Trejo-Skalli AV, Velasco PT, Murthy SNP, Lorand L & Goldman RD (1995) Association of a transglutaminase-related antigen with intermediate filaments. Proc. Natl. Acad. Sci. USA, 92:8940-8944
9. Yang Z, Kyriakides TR & Bornstein P (2000) Matricellular Proteins as Modulators of Cell–Matrix Interactions: Adhesive Defect in Thrombospondin 2-null Fibroblasts is a Consequence of Increased Levels of Matrix Metalloproteinase-2. Molecular Biology of the Cell, 11:3353-3364
10. Yoneda M, Yamagata M, Suzuki S & Kimata K (1988) Hyaluronic acid modulates proliferation of mouse dermal fibroblasts in culture. Journal of Cell Science, 90: 265-273

So I plan to incubate the fibroblasts in 0.25% trypsin at 4*C overnight - do you think that this will be too harsh? Will it reduce viability too much?

Might take a look at dispase.

We don't have any chicks here... only rats, mice and zebra fish

Thanks for your help,

Tridimity

If your planning to incubate overnight then use dispase, trypsin is harsh on the cells...

Also you don't need specific medium, you can use DMEM, keratinocytes won't grow in it, and after a few passages you should have a clean fibroblast culture. Keratinocytes need special medium to grow, not for inhibition.

And the fibroblasts from the dermis and epidermis are exactly alike... for the sake of relevance I understand why you need to seperate the two layers, but if its a fibroblast culture your looking for, in the end, fibroblasts are fibroblasts...and fibroblasts are exactly the same through out the body... but the overnight incubation with dispase should be fine in seperating the two layers, can make sure about the exact concentration for you, after incubation you should be able peel it off.

The protocol looks great, obviously have a few differences but the basic principles are the same so you shouldn't have a problem. Just one recommendation - antimicrobials are NEVER optional and always a must... contamination can be a real setback especially after all that hard work and "nurturing"

Have you ever done any other cell culture work?

Sorry to interject, I just love the wording here:

Sacrifice the mouse by overdose of Euthatol (0.3ml) or by neck dislocation.

I know...

Thanks C15ka,

I will consider dispase and use antimicrobials. Yeah, so far I have cultured and transiently transfected WT and Bax/Bak DKO MEFs, also cultured and differentiated C2C12s

And Kalster, what's up with my wording? Too direct?

Best wishes,

Tri

Originally Posted by tridimity

And Kalster, what's up with my wording? Too direct?

No, just a funny choice of words. Is it standard jargon?

so, instead of a sacrificial lamb, it's a sacrificial mouse ?

Yep, it's standard wording

Well, you were right, C15ka. It seems that the trypsin step is too harsh on the cells. I followed the protocol above, and today the dishes were clean enough to eat off of, excepting a few rounded-up apoptotic cells. One of my colleagues has supplied a different protocol that involves only a 1 hour incubation in trypsin, so I am going to try that tomorrow.

C15ka - please could you provide me with the protocol that your Lab uses?

I am getting cells but not as many as would be required for efficient experimentation

Best wishes,

Tri

For some reason, sometimes when I perform a scratch assay, only cells on one side of the scratch will migrate into the open space. It is as if the other side is stuck behind an invisible wall. the "stuck" side maintains the perfect scratch line.

Does anyone have any idea why this might be?

Thanks

I have heard of the entire cell monolayer being lifted by the scratch when plated at roo hogh a confluency...never heard of asymmetrical migration thoygh. Bizarre, there will be a logical explanation though. Just don't know what it is. Are the tips you are using just regular tips?

Yes, just regular 1000uL pipet tips. One possibility I have considered is that the cells get stuck behind dead cell debri from the scratch, but that is just a guess. And It doesn't occur for the entire scratch, just sections along the length of the scratch. But of course there is no way to know until after the migration period.
Thanks.
Ice

Do you wash with PBS after thw scratch? That should help to remove any detached/dead cells or cellular debrisBest wishes,Tridimity

Gots fibroblasts

For anyone who needs to establish primary cultures of murine dermal fibroblasts in future, this protocol works a treat:

1. Reconstitute collagenase type I (Worthington Biochemical Corporation) powder in Hanks' Balanced Salt Solution 1x (Calcium chloride/Magnesium chloride -)
2. Aliquot out into 10ml aliquots and store at -20 (helps to avoid repeated freeze-thaw cycles, which can kill enzyme activity)
3. Prepare a Universal tube containing 10ml blank (i.e. serum-free) DMEM-F12 and penicillin/streptomycin (pen strep; 1:100 stock)
4. On morning of primary culture, prepare cell culture Hood - spray Hood down, spray eveything (stripettes, pipette man, 50ml Falcons, cell culture flasks and Petri dishes) that goes into Hood - obviously
5. Decant 70% ethanol into 50ml Falcons and use to sterilise scissors, forceps etc.
6. Place cell culture media (both blank and total (i.e. serum-supplemented)) DMEM-F12 with L-glut and pen strep into waterbath at 37*C - at least 30 minutes prior to primary culture
7. Also place a 10ml aliquot of collagenase type 1 into a water-filled beaker in waterbath, to thaw
8. Sacrifice the mouse
9. Shave its back and spray with ethanol (be conservative not liberal with ethanol - tends to kill cells)
10. Excise a relatively large area of skin (~3x3cm^2 - doesn't matter too much the exact area, so long as you are consistent between mice so as to get an approximately equal cell yield between samples)
11. Note: do not use hair removal cream (what am I going to do with that Veet now? )
12. Scrape skin free of any residual fat or muscle
13. Place excised skin into prepared Universal tube (step 3)
14. In cell culture, pour skin (and media in which skin was collected) into a sterile Petri dish and wash in blank DMEM-F12 with pen strep
15. Trim off any residual fat tissue and chop thoroughly using a large pair of scissors
16. Add skin to Universal containing 10ml collagenase type I at 2mg/ml (step 7)
17. Note: do not use a trypsin step
18. Incubate at 37*C with constant agitation for 2 hours
19. After 2 hours, centrifuge for 5 minutes at 1,000 revolutions per minute
20. Pipette off collagenase
21. Tap the pellet
22. Re-suspend pellet in 10ml total media
23. Note: do not use a gauze after centrifugation)
24. Pass through a cell-strainer into a 50ml Falcon
25. Centrifuge again for 5 minutes at 1,000 revolutions per minute
26. Pipette off media and re-suspend in 5ml total media
27. Seed into 25cm^2 orange Cell Bind flask

Best wishes,

Tridimity

Originally Posted by Wall of Ice

For some reason, sometimes when I perform a scratch assay, only cells on one side of the scratch will migrate into the open space. It is as if the other side is stuck behind an invisible wall. the "stuck" side maintains the perfect scratch line.

Does anyone have any idea why this might be?

Thanks

I have found that pushing too hard with the pipet tip while making the scratch may cause single-side or no-side migration.