Dear Maxim,

I have to agree with all of the above comments. Rat and murine ASCs are more difficult to maintain in culture than human ASCs for instance, but if you see a decrease in earlier passages, I would suspect something's wrong with your culture conditions (contamination?; mycoplasma?).

I wouldn't go above 20% FBS, you should add glutamine to the medium, platelet lysate does increase cell proliferation but you can get you medium gellified  and you cannot use it for differentiation assays.

FACS is the best tool you have to assure pure sub-populations within ASCs, but if you want to work with the full population of ASCs I wouldn't worry to much about doing it (also the expression of the referred markers varies and most, if not all, are not specific of stem cells or MSCs..only by looking at the combined expression you can withdraw some conclusions).

Good luck with your culture!

Dear Maxim,

The isolation of ASCs of high purity from adherent/cultured adipose-derived cells can only be achieved after FACS sorting for CD44+CD73+CD90+CD105+CD31-CD45- population of cells. Fresh isolation of ASCs from adipose tissue can be carried out by sorting for CD34+ cells as the fresh cells express a high level of CD34 protein in addition to their expression of MSC surface markers. In regard to a crude isolation of preadipocytes, according to the method you have described, the cells will proliferate or can differentiate into mature adipocytes though they may contain a small percentage of  other contaminating cell types including immune cells, endothelial cells, and fibroblasts that may not differentiate but grow along with preadipocytes. Unlike mouse bone marrow MSCs that retain their pluripotency for a long period, the ASCs irrespective of their purity tend to lose their potential to proliferate and differentiate when allowed to continuously grow under in vitro conditions for a long time. I would only use the cultured ASCs up to 3 or 4 passages beyond which the cells display a flattened morphology and exhibit reduced potential to proliferate and differentiate.

Dear Maxim, 

I would also sort the cells by FACS or by manipulating the centrifugation speed since your culture could be contaminate by endothelial cells, pre adipocites and fibroblasts that have similar morphology. I will suggest to use platelet lysate instead of fbs since, in my own experience, enance very much the cell proliferation.

 Dear Maxim,

 I have only isolated and cultured human and rat ASC. In both cases I would say the culture is rather simple and after 2-3 passage you can get a population expressing high levels of CD44. CD73 and CD90 (typical mesenchymal markers) and negative for CD31 and CD45. If you perform the isolation of the fat pads properly, contamination with other cell types should not be a concern. 

 In any case, I have read reports before of mouse ASC being particularly difficult to culture. Sometimes adding 1 ng/ml of bFGF helps. Also, testing of your serum batches can be important as you already point out.

 Regarding your pictures, the cells do not look very happy but I have seen worse, what strikes me is the difference before and after washing away the non-adherent cells.

 Lastly, how many times has this happened? 

 Best,

Manu

Thank you all for your valuable answers!

@ Muruganandan Shanmugam: Thank you for confirming the presence of other cells in the culture (immune cells, endothelial cells, fibroblasts). I have indeed read about the FACS, but in some articles they don't mention this step, so I assume they just used all cells from the culture (incl. the other cell types) and considered them ASC.

@ Manuel Mazo: Regarding the first picture, I wrote an error. The picture is taken after washing, but also after the 2 minutes incubation with trypsin. Hence you don't see any attached cells. Sorry for the confusion! This is my first murine SVF/ASC culture, by following this protocol, so I can't compare with pictures from other samples.

Dear Maxim Geerons,

To obtain the 100% purity you need start with single adipose derived mesenchymal stem cells (ADMSC) from sorting FACS; otherwise you could use the BIDs and isolate the cells with immunophenotyping chacarteristics of ADMSC and then made cultivation. If you can not do that, you will have some fibroblasts and endothelial cells; you could minimize those cells variety withou FACS and BIDs, if you isolate those cells from explant and do not use the enzymatic dissociation method.

The ADMSC cultivation not need the FGF, those cells produces their growing factors!

Good Luck!

Hi Maxim, I have no experience with mouse ASC but with human ones. I used to grow them in DMEM/F12/FCS (45/45/10) with Glutamax and with antibiotics and 10ng/mL FGF2.

For the first plating, I used to replace the medium in the first 2 hours in order to remove non adherent cells (SVF is a very heterogenous cell suspension) and add fresh medium. I plated the cells at 4,000 cells/cm² for the susbsequent subcultures but at 40,000 cells/cm² for the first one (SVF plating). 

Do you have any results regarding CFU assays to check the clonogenicity of your cell suspension? I mean at each passage? This is a very informative test.

Keep in mind that even if you have a "pure-like population of cells" regarding the classical markers, don't forget that if you look at Stro-1 or CD271 or others, you will only have a small population stained.

In my opinion and when I look your pics, you cell are not in a good shape. Don't let them grow to much because they will inhibit each others by cell-cell contact. 

Nevertheless, in your culture condition, you will not be able to grow your cells after the 4-5th passage. If you want to put them forward, use xeno-free media with ECM coating in your dishes.

Regards

Dear Maxim,

I have to agree with all of the above comments. Rat and murine ASCs are more difficult to maintain in culture than human ASCs for instance, but if you see a decrease in earlier passages, I would suspect something's wrong with your culture conditions (contamination?; mycoplasma?).

I wouldn't go above 20% FBS, you should add glutamine to the medium, platelet lysate does increase cell proliferation but you can get you medium gellified  and you cannot use it for differentiation assays.

FACS is the best tool you have to assure pure sub-populations within ASCs, but if you want to work with the full population of ASCs I wouldn't worry to much about doing it (also the expression of the referred markers varies and most, if not all, are not specific of stem cells or MSCs..only by looking at the combined expression you can withdraw some conclusions).

Good luck with your culture!

Dear Maxim,

I think your cells are not pure, and part has differentiated or undergone senescence. You can look at the morphology, they are not all fibroblastic, and some have become big and fat.

I do not have experience with mouse, but with human AT MSC. In my lab, we just plated as you did, but I used alpha MEM and Platelet lysate + heparin to prevent clotting, instead of FBS, and + Glutamax + AB and fungison. At early passage, they are mixture of cells, but after 4-5 passages, they become homogenous. 

Using platelet lysate, the cells look very nice, and small, and not easily undergo senescence. We have compared to FBS, FBS caused the cells to undergo senescence faster, sometimes after P2-3

Dear Maximum,

I have some tips that I hope will help you.

1)VERY important, when you collect the inguinal fat pads be careful in removing all the lymphonodes which represent a source for cell contamination. You may need a dissection microscope...

2)After the first centrifugation step you have separation of adipocytes on the top. Mix troughly for 10 seconds by hand and centrifuge again. This help complete separation of adipocytes.

3)The choose of the appropriate FBS is critical. Try with a FBS batch test and you will notice a lot of difference in cell growth. To evaluate cell growth, plate  in 12well at a density of 2500cells/cm2 and every 2 days for 10 total days count a well. Also calculate the Population Doubling Level(PDL) at each passage  and plot the cumulative PDL in function of time in cultutre  Also try DMEM high glucose.

4)Which strain and at wich age are you collecting the tissue? These make big differences.

5)To characterize the cells I use CD90, 44, 29, 45, 31, 34, 45 and 106 at passage 5. There are not exclusive MSC markers. However you can check CD31 (ebdothelial), Cd45,cd11b, cd34 (hematopoietic). Cd106 is expressed by hBM-MSC but not by hADSCs. I found that mADSCs do express CD106.  I would not go for CD105, unless you are interested in the two different populations (105+ or 105-)

6) Do not go to low with plating density. Around 5000/6000cm2 you should split the cells every 4 days. Do not let the cells get confluent (max 80%). 

7) Finally ADSC is a mixed population, so you cannot aim to obtain a PURE cell population. You can reduce heterogeneity by washing away non-adherent cells before 24h after plating the SVF.

For the protocol I found very usefull this paper:

http://www.bio-protocol.org/e1642

Hope I have been of some help. If you want to ask something else please do not esitate. Good luck!

BW

Yuri

Hi Maxim,

I've lot of experience with murine and human ASCs. Check out the link(research paper) below for more instructions/information. Good luck with your research. In Fig.1, cells look senescent/dying. Other figures look just fine.

http://stemcellres.biomedcentral.com/articles/10.1186/s13287-016-0343-y

Let me know if you have more questions.

BP

Dear Maxim

I don't have experience with murine ASCs, but I have worked with rat ASCs since 2006. As the other answers have indicated, your cells definitely don't look happy and there seems to be senescent cells in your culture. I also don't understand why you lose all the cells only by washing, as we have experienced the senescent cells to be super-adherent and we struggle to lift them even by trypsinization. I can give you the following tips:

1) We add 20% FBS to the DMEM for 24 hours after isolation. After that we use DMEM plus 10% FBS, no other additives. 

2) In our experience, these cells are sensitive to low cell density. We plate the cell pellet from 1 inguinal fat pad (from rat) into 2 100mm dishes. This takes approx 6 days to reach 80-90% confluence. Then we trypsinize and subculture at 1:4 dilution. Anything higher than that does not work very well and often induces early cell death or senescence. 

3) We have also found the cells to be sensitive to trypsinization. We use Lonza trypsin (170000 units / L). We only use 1 ml trypsin in a 100ml dish, for approx 1 minute before neutralizing.

4) We only use the cells at passage 2. We have found that by then, the adherent cells form a single population on flow cytometry (Sadie-Van Gijsen et al., Mol Cell Endocrinol 2012). You can see from passage 0 to passage 2 how the other cells like haemopoietic cells and mature adipocytes that came along with the isolation don't survive the successive rounds of trypsinizing. We have tested a few cell surface markers, and the other answers here also suggest other cell surface markers. I would think that for publication, you would need to at least test a few markers for characterization using flow cytometry, even if you don't use any of them for FACS sorting. Our cells remain proliferative until passage 5, but the drug responses and differentiation potential become unpredictable from passage 3 onwards.

Good luck, I hope this helps!

Regards

Hanél Sadie-Van Gijsen

Stellenbosch University

South Africa

Hi Maxim,

I agree with the comments above. I thing that one of your problems is that you allow them to overgrow. Never allow them to get more then 80% I know it can be difficult at times. What do you use to passage them? Trypsin? What percentage? How long do you do it for? In my personal experience if one uses 0.25% Trypsin the viability and future expansion strongly diminishes in these cells.

Second is how long do you keep them in collagenase and what do you mix your collagenase with, if you have it in powder version, when you dilute it do you syringe filter it? Also how long do you incubate

In my experience, as also mentioned by some other researchers above, the quality of FBS is essential. I always get MSC grade FBS from Thermo Fisher and it worked great for me, while whenever I used other types of FBS.

Let me know if you have any questions!

Maciej

Hi Maxim

Of course there will be a mix of cells in the isolated SVF. These will gradually get weeded out as you passage them in the appropriate media.

To answer your query on cell viability - Your photos look fine except the last one in which the cells look a little stressed. However, it is hard to estimate cell viability in the absence of a viability dye like trypan blue

Also you would not be able to confirm the identity of the cells or contamination with fibroblasts etc. in the absence of a flow cytometric evaluation. But before you do that, I have a couple of questions and suggestions,

QUERIES

1. Are you using MSC qualified FBS

2. At what confluence do you normally harvest your cells

3. How do you trypsinize the cells, are they getting over-trypsinized

4. Have you done mycoplasma testing of your cells

SUGGESTIONS,

1. Try using MSC qualified FBS

2. Add L-glutamine or Glutamax to the cells if you are not already doing so

3. Try initial seeding of the SVF at 1-2 x 10^5 cells/cm2. Change media every second day. Once you get a confluent monolayer (try harvesting at 70-80% confluence - if you go beyond generally cells undergo senescence), trypsinize and seed at 1000-5000 c/cm2

4. DMEM media containing 10% FBS is fine but is you want to maintain cells for a longer time you could try adding FGF-2 to the media

5. You could try the above and then change media to DMEM-F12 or aMEM if you want a better cell yield

Best of luck!

Preeti

Thank you for the remarks you all made.

I repeated my experiment 3 times. I used new stromal medium containing

• DMEM (Gibco, Life Technologies) including 4.5g/L D-glucose, L-glutamine and 110 mg/L sodium pyruvate
• 10% FBS (Cell Culture Bioscience, Nichirei Bioscience, Tokyo)
• 1% penicillin-streptomycin.

I used different mouse ages and had the best results with fat from young mice (5.5 weeks old).

As pointed out by Manuel Mazo and Yuri Ciervo, I removed carefully the inguinal lymph nodes before harvesting the adipose tissue with a new set of sterile instruments. After the collagenase digestion, I also centrifugated the sample twice with manually shaking in between as suggested by Yuri Ciervo, which gave me a very good yield of SVF-cells.

I plated the SVF and the ASC at different densities and compared the increase in cell number and the viability. I got the best results when I initially plated 1.0 x 10^6 SVF-cells in the 100-mm cell culture dish (confluency after 1 week) followed by plating 1.5 x 10^5 ASC in the 100-mm dish at P1, P2, P3. After an initial decrease at P0, the cells expanded very well with 70% confluency and doubling every 2-3 days. These different plating densities are similar to what Jonathan Rodriguez has indeed suggested here with his human ASC.

24 hours after plating the SVF I indeed removed non-adherent cells from my culture.

I did flow cytometry analysis after the third passage and it was positive for MSC markers CD73, CD90.2 and CD105. Other markers I tested were CD31, CD45, CD11b and CD34. These were are all negative (90% after staining with trypan blue.

While I work with cells that are less differentiated than MSCs, your comments and those above suggest that your cultures may be contaminated with other less differentiated cell types, that were unexpected and unrecognized.

- I would suggest running flow cytometric analysis with CD56, CD13, CD90, CD10, and CD66e (markers for the lineage-uncommitted stem cells that I work with).

- With flow cytometry I would suggest using propridium iodide to determine cell viability, rather than Trypan blue. Trypan blue is based on the idea that a viable cell will pump out the dye, while a dead cell will not. However, there are cells that lack the pumping mechanism and therefore the Trypan blue accumulates within the cell giving a false negative. This can be tested on Trypan blue viable cells by pre-treating cells with agents that inhibit the pump. Cells are still viable, but they accumulate Trypan blue, giving a false negative. Alternatively, one could use CD95, which is the apoptotic cell marker for a dead or dying cell.

- Some caveats are that FBS contains bioactive factors (various concentrations of inductive factors, proliferative factors, inhibitory factors, depending on particular lot of serum) that can alter the characteristics of the cells.  Therefore, I would suggest using heat inactivated serum to destroy these activities. My serum of choice can be obtained from Atlas Biologicals, Fort Collins, CO.

- Be very careful as to the use of bioactive factors such as FGF and TGF-b. They have multiple activities, dependent on the differentiative state of the cell. In cells that have already committed to a particular tissue type, i.e., progenitor cell ("x-blast"), such as a chondroblast, osteoblast, myoblast, etc, these two reagents stimulate proliferation (about 2-3 times above control values). However, when applied to cells that are lineage-uncommitted, these two factors induce fibrogenesis, which can contaminate your cultures, especially if you have unexpected/unrecognized lineage-uncommitted stem cells present.

- If you are interested in proliferating your cells without the potential for inducing differentiation in contaminating unexpected/unrecognized lineage-uncommitted stem cells in your cultures I would suggest using PDGF-BB (R&D Labs). It only has a single activity - induces cellular proliferation, about 10-12 times above control values in lineage-committed cells as well as lineage-uncommitted cells.

Protocols and explanations can be found in the following articles:

Young HE, Duplaa C, Romero-Ramos M, Chesselet M-F, Vourc’h P, Yost MJ, Ericson K, Terracio L, Asahara T, Masuda H, Tamura-Ninomiya S, Detmer K, Bray RA, Steele TA, Hixson D, El-Kalay M, Tobin BW, Russ RD, Horst MN, Floyd JA, Henson NL, Hawkins KC, Groom J, Parikh A, Blake L, Bland LJ, Thompson AJ, Kirincich A, Moreau C, Hudson J, Bowyer III FP, Lin TJ, Black Jr AC. Adult reserve stem cells and their potential for tissue engineering. Cell Biochem Biophys, 40(1):1-80, 2004.

Young HE, Black AC. Pluripotent Stem Cells, Endogenous versus Reprogrammed, a Review. MOJ Orthop Rheumatol 1(4): 00019, 2014. 

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Henry E Young, thank you for your these insights. Your recommendations are very useful for my upcoming research.