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Stem Cell Culture - Science method

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Do anyone can share the ES cell culture protocol with proper dilution explaining like 1:6, 1:12 etc?
And if I split the passage from 6 well plate to 10cm or 15cm plate what would be the dilution?
Thanks in advance.
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Thank you very much
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I always use F-12 for stem cell culture , can I use DMEM high glucose instead ?
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Hello Shaimaa Mohamed Good Morning
Though high-glucose DMEM is the conventional basal medium for stem cells, it could encourage differentiation. You can use DMEM: F12 (DMEM/F-12 is a 1:1 mixture of DMEM and Ham's F-12) which can support greater growth rates. DMEM and F12 are often mixed to combine the higher concentrations of components in DMEM with the wider range of Ham’s F12 ingredients. For instance, the addition of Ham’s F12 provides components such as biotin, putrescine, lipoic acid, proline, copper, and zinc that are not present in DMEM.
DMEM/F-12 contains no proteins, lipids, or growth factors. Therefore, DMEM/F-12 may require supplementation, commonly with 10% FBS.
You can refer to the research article attached for more details.
Good Luck.
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Hello everyone,
In my upcoming project, I will use poly-L-lysine coated culture flasks due to the culture conditions of neural stem cells.
One thing that comes to my mind is if I need to perform a viability test such as MTT or SRB on these cells, do the 96-well plates have to be poly-L-lysine coated as well? Buying manufactured coated plates increases the cost a lot. I am guessing my deviations may increase if I coat them myself. Also, does poly-L-lysine interfere with these kinds of tests? I do not have much experience with this subject. I would be very happy if you share your information not only for the viability tests but also for any kind of plate-based tests for the neural stem cells.
Thank you very much.
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For your first question, I think yes you should coat the 96-well plates with poly-L-lysine as well because in MTT assay, for example, you will seed your cells overnight so that they can be attached to the flask and this poly-L-lysine will help the neural stem cells to attach better to the flask and will increase their adhesions.
For your second question, as far as I know, coating the plates with poly-L-lysine will not interfere with cell viability tests, there are some articles that coated plates with poly-L-lysine and did the MTT or other viability tests.
Also, in order to reduce the risk of your deviation when coating the plates with poly-L-lysine, I recommend following the protocol provided by Sigma :
1. Add 10 ml sterile tissue culture grade water to 1 mg of poly-L-lysine(CAT:7280),
NOTE: (this step is not necessary for poly-L-lysine Solutions(like cat: P4707))
2. Coat surface with 1ml/25 cm2
3. Rock gently
4. After 5 minutes, remove solution and rinse with sterile culture grade water
5. Allow drying at least 2 hours before using.
Hope this can be helpful for your task.
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Hello,
In our ASC (adipose derived stem cells) culture we observed different morphology than usual. What could it be ? Rounded shape cells instead of typical fibroblast-like cells?
No medium/supplements/ culture condition changed.
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I would like to ask about the culture period of these cells as they seem to be at the beginning f culture and it is normal to see round cells at this stage of culture
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Hallo! Does someone have experience culturing cells derived from infectious samples?
I would like to isolate the bone marrow mesenchymal stem cells from patients diagnosed with bone infection and culture them. The only problem is that the tissue would be infectious with bacteria inside. Some of the bacteria might be also multiresistant. If I culture them in the incubator, there might generate bacteria membrane, and even contaminate other cell wells. Is there any good idea how I can eliminate these bacteria and culture these stem cells?
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Dear Ren,
In addition to outstanding recommendations from Henry, I would recommend changing media frequently, like everyday if possible until you see very clear and clean growth of cells with no bacteria. It will help to dilute the bacteria and wash them away from cells. If encountered contamination and it's a precious sample, try to treat with 2x concentration of antibiotics and anitmycotic. Cells can recover from bacterial induced changes in long term if you get rid of contaminants by doing so.
Hope it helps!
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We are experiencing a significant amount of cell death upon differentiation of h9 hESCs to definitive endoderm cells. We grow h9 cells in feeder free conditions and induce differentiation to endoderm using RPMI 1640 supplemented with 2mM glutamine, 1X NEAA, 100ng/ml Activin A, and .1% B27. After 1 day of induction we add .1X ITS, after 2 days induction we add 1x ITS. We are trying to perform an initial RT-qPCR batch assay on day 3 cells, however by that time we have only a few cells left with endodermal morphology - most of our initial stem cell culture has lifted off the plate and died. Does anyone have any successful strategies for increasing cell count at day 3, while maintaining a relatively high degree of differentiation? Would greatly appreciate any insight - Thanks!
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Dear McCabe,
We used ipsc cells for our differentiation, but we do not differentiation into endoderm. Sometimes, first few days, you can add ROCK inhibitor (10uM). Maybe decrease the dead cells.
BW
Xuezhi
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So, I will be culturing H1 over geltrex, which will be subjected to electroporation, and subsequently FACS-single cell sorting.
I am currently deciding which of these two medium shall I work with..., any suggestions will be very much appreciated! Thanks!!!!!!
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We've tested both media for our gene editing experiments on iPSCs. Stemflex (in combination with CloneR (Stemcell)) worked best in our hands. Furthermore, we noticed that iPSCs cultured in Stemflex recover more easily after cryopreservation, or (as mentioned above), after single cell seeding. Good look with your experiments!
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Hello,
I am doing fibroblast, stem cell culture for sequencing. (From primary culture to the end)
Since FBS is expensive, there are some protocols that mix FBS and horse serum (1:1)
I have done mixing them, and culture results were not that different. They grew well like in normal FBS containing medium.
But I have to do WGS with the cells I grew, so I am suspicious about the result.
Even if the growth rate and cell morphologies are the same, do you think the DNA sequencing results are different when the FBS is mixed with other serum?
Thank you.
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No problem with DNA sequencing. If you are doing RNAseq or gene expression analysis, then yes it may.
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I am growing organoids in monolayer but honestly couldn't manage to make it confluent. the protocol i followed made use of collagen. they first look "like confluent" with about 70-80% cell, but after washing or changing media, they just turn from 40 to 10% remaining cells.
i thought there could be a problem with collagen? what is the best collagen concentration and how much collagen should be used to coat per well? Thank you.
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If we seed human pluripotent stem cells (hPSCs) in absolute Matrigel and allow them to form embryoid bodies over the next ~5 days then how much time it takes for them to spontaneously differentiate when we remove the stem cell culture media and maintain them in spontaneous differentiation media? Also, I'd like to know different media for spontaneous differentiation.
Please let me know if you have any information regarding this or if you've come across any articles or know anybody in this field who can help me.
Thanks in advance!
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Well, they'll BEGIN to visibly differentiate within just a few days but the process will continue for weeks. Sometimes you'll see cardiac beating within a couple weeks and others it might be well over a month. Of course it also will depend on the media used.
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I've been working with some bone marrow derived MSCs I got from Texas A&M and I've wondered what other's experience has been with bone marrow derived MSCs (or MSCs from adipose tissue, hUCB etc. doesn't matter). In my experience they tend to be slow growers (plated at ~2000 cells/cm^2 it'll take ~10 days to reach 80% confluency) and senesce quickly (I get like 7-10 passages out of them). I change the media weekly and don't use a pH indicator. I've been using stemlife MSC medium for undifferentiated expansion. This media is interesting to me because I assume its probably pretty basic but it contains what they call "life-factors" which is proprietary of course.
Has anyone on here created their own media or found a particular method of culturing to be the most robust and productive when working with bone marrow derived MSC's?
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We have tried with both alpha mem and glutamax as basal media plus 10% FBS, 1ng of FGF2 and antibiotics.
Change twice weekly or once in three days
Our loading density vary between 5000-10,000 cells/cm2..
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March 18, 2020: COVID-19: Why can't we ramp up production of gamma globulin from recovered individuals, to treat those in imminent danger of Covid-19 morbidity and mortality? Same question for production from Stem Cell labs, using those from recovered patients, in the manner that we used to us, for example, ALF to treat infections in immune compromised patients.
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In today's lead article in JAMA It worked very well.
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How can we determine either to use of Rock inhibitor or Blebbistatin for cell culture experiment? Since both of them prevent the cell apoptosis is there any preference for one over another? If yes why?
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The addition of ROCK inhibitor or Blebbistatin can promote cell survival in the initial stage and support high clone formation efficiency. The addition of transferrin and selenium can also improve the efficiency of clone formation. If this type of ROCK inhibitor is included in the culture medium of conventionally cultured cells, it may be necessary to continuously add the inhibitor to the daily culture
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Does anyone know that how many cells lines and primary culture can be grown in L-15 medium? I need the complete list of cell lines and tissue types.
Our CO2 incubator in cell culture facility is not working and we need to grow our cells in L-15 medium which does not require CO2 for buffering. Another medium is CO2 independent medium by Thermofisher but the information about this medium is quite scarce.
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You can also just get your usual media (DMEM, F12, MEM, etc) as a powder without bicarbonate, supplement with 25-30 mM HEPES and adjust pH to 7.5-7.6 (will decrease ~0.2 units at 37°C). In principle, most media (esp. F and MCDB series) are more "physiological" than L15 in their overall composition and will work fine or maybe even better than L15 this way.
"Normal" mammalian cells typically need CO2 (HCO3-) as a nutrient and thus need the 2-5% CO2 atmosphere to proliferate. But quite a few transformed/cancer cell lines can also grow at ambient air CO2 (some may have changes in morphology). The ones known or developed to grow in L15 will certainly do, some others will too, many won't.
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We are trying to run a blot for LC3 [MAP1LC3B], however unlike the two 14 & 16 KD band we are getting only one band (picture attached). This blot has 5 treatments: Lane1: Control, Lane2-5: Treatment in increasing order Cell used for this expt: HEK293 Gel % = 10% in 1mm spacer glass Development: Manually in Dark Room on X-ray film Primary Antibody dilution= 1: 1000 (as recommended), overnight treatment at 4C Secondary Antibody dilution= 1;10,000 (recommended= 1;10,000 to 20,000) 1 hr at RT Chemi-luminescent: ECL plus 1:1 in 1 ml H2O Please kindly assist why we are getting only one band in all the samples. Also why all the samples seems to be attached with each an other and why they appear zig-zag is structure rather than straight
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Amit Kumar Shrivastava Use 12% gel and run your gel at 70V. Do not run the gel its entire length. Stop running when the dye front is around 1.5 cm above from the bottom of the gel. The LC3 bands will not become fuzzy in this way.
Use 0.2 µ PVDF membrane for transfer instead of 0.45µ Nitrocellulose membrane.
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I have tried to weigh out lipolyzed collagenase to be used for stem cell culture, but it seems to either fall off- or blow off the sides of the spatula inside the fume hood where we measure out our chemicals. Does anyone have any techniques to reduce losing so much of the samples? I am using a very small and thin spatula, so that I am able to reach down inside the container and place 10 mg of collagenase into a 15 mL tube.
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Hi Eden Klepper . Fluffy protein powder in small quantities can be very difficult to measure accurately. My solution was to make a stock solution, make single-use aliquots and freeze at -80oC. Collagenase is fine at -80oC for a long time.
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I was freezing Wharton Jelly stem cell in Mr.Frosty and forgot them in -20 degree for 48hours (due to weekend). I transferred them to -80 degree in Monday morning. Does anyone has experienced this?
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What a pity.
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Hello, I have stem cells in culture in a plastic flask and I want to observe it in a Scanning Electron Microscope (SEM), i want to know if there is a special preparation to the culture whitout using glass slides or scaffolds?
thanks
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As Ajay mentioned, you have to cross linked them with glutaraldehyde. I normally do it with 2% (w/v) in pbs.
remove the medium, add the glutaraldehyde on the cells and leave them like that for 2 hours.
Remove the glutaraldehyde and wash gently with pbs. Then add alcohol 100% and leave it for one 1hr.
Then remove the alcohol and add HMDS (hexamethyldisilazane) to dry the samples overnight and they are ready and completely dried.
now, before SEM, you have to Souter coat them with 5nm of platinium or gold for conductivity. And now, you can finally observe them under SEM
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We are doing fruit stem cell culture and need to keep some of our cultures going for up to 1 months. For one week, we have struggled with yeast contamination in our cultures. However, we wear gloves when handling cultures and media. Yet we continue yeast in our cultures. Is it possible to clear the yeast contamination?
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Thanks the answers. Really appreciated it.
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I went through a basic protocol provided by Brewer et al., 1994
1. The media was stored according to instruction (-4 to -20 for B27, -20 for trypsin and 2-8C for neurobasal and 15C for Glutamax)
2. The hippocampus of mature rat (6W) was extracted under the sterile condition
3. The extracted hippocampus was stored in HANK and Pen/Step for 2 min
4. The hippocampus was homogenized for 15min using trypsin-EDTA 0.05 (using micropipette with blue and yellow tips). i had extrac care about buble formation
5. The trypsin was inactivated and centrifuged, the pellet was re-suspended in 500ul hank+pen
6. The cells was counted (2 * 10E6) and inoculated into the culture media (5ml neurobasal+ 2% b27+glutamax 1%) in a T25 flask (adherent + filter cap)
7. The cells were incubated at 37C and 5% Co2
* No antibiotic in culture medium / even addition of antibiotic dosen't make change. Furthermore, i didnt have contamination
* No serum in the medium
* No bFGF/ even addition of bFGF dosen't make change
Still i have some growth that i don't know what they are. I think they are not typical neural cells. In my though, the problem is that the cells are die within few days.
what went wrong?
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According to your pic, cell shape is circle and it looks like they did not attach to bottom and died. In my opinion, cell number is too low and T25 flask is too big. Use the 8-10 cover slips and 100pi petri dish (which is treated by after alcohol and baking) before spreading neuronal cell. You can easily deal neuron in coverslip with 12 well plate to perform other experiments.
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I want to know how to prepare the poly-HEMA for cancer stem cell culture?
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1. 48mg poly-HEMA in 40ml 95% EtOH;
2. Shaking 200rpm at 65℃ for at least 6-8 hours until all poly-HEMA powders are dissolved;.
3.
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I am using the TeSR-E8 medium for the maintenance and growth of iPSCs. I made the aliquotes of complete medium without adding Pen/Strp and Normocine, and stored at -20C. On need, I thaw one of them and store the remaining volume at 4C where it remains free of contamination for one week. But it gets contaminated after that even though I use and store the vial very carefully. Can I please get any suggestions regarding the use and store of stem cell medium? Is it ok for iPSCs to use medium containing Pen/Str and Normocine?
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Hello
We routinely culture iPSC in medium without any antibiotics but If you need to use them, it is recommended to use lower concentrations ( 1% pen/strep )
Good Luck
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I wish to induce stress conditions to Adipose derived mesenchymal stem cells and for that i have these three available chemicals. Your expertise are needed.
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Hi Numan
We used H2O2 with success
Effects of H2O2 exposure on human sperm motility parameters, reactive oxygen species levels and nitric oxide levels
SS Du Plessis, DA McAllister, A Luu, J Savia, A Agarwal, F Lampiao
Andrologia 42 (3), 206-210
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Instead of using PBS or sterile water, I use DMEM to prepare bFGF. Is it would be stable if it is prepared in DMEM/culture media? Does anyone have any ideas or information?
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Hello Nur.
Yes, you could use if you add 1mg BSA/ml DMEM as a carrier protein and a preventing bFGF from losing activity by attaching to the container walls. Thus, make 5ul aliquot tubes (1,000~2,000X stock solution) and store at -80C until use. Do not re-store at 4 or -20 or -80C.
However, some of DMEM components are degrading as time goes. Therefore, it is best make 5ul bFGF in PBS containing 1mg BSA/ml (PBS + BSA). It will goes forever at -80C without deterioration.
Make PBS + BSA, millipore (0.22 um) and dilute bFGF with the millipored PBS + BSA to make 1,000~2,000X stock solution to be added to the cell culture.
Best wishes.
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Update: Cells are growing fine in our lab now!
I'm the only person in my lab starting on iPSc culture (we received a vial from another lab and all I have is some the medium they use for culture) and I'd like some tips and help. I have human iPS cells, and I culture in mTeSR1 on matrigel coated plates (so feeder free), replacing media daily. I try to remove what I think is differentiation by marking on the plate while looking through a microcscope. One cell line shows nice colonies like I saw on suppliers webpages (Like Stemcell or Invitrogen), the other I'm not so sure... 
I've attached some pictures, can someone tell me if this is what it's supposed to look like? And can give someone tips on how to passage? I've passaged one with gently scraping, like i saw mentioned in literature and tried picking the colonies with a p1000 tip (but didn't know if I actually picked up a colony or not, so I tried scraping). Maybe i just need a bit more patience :) 
While awesome to start things up, it's frustrating having no one to ask if things are supposed to be this way :)
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"When you plate the cells in low densities use ROCK inhibitor to prevent cell differentiation and/or death. With higher densities you should be fine without any. Cell passaging can be done using TrypLE, Accutase or dPBS + EDTA, I would let them grow till around 70%, more and they start to differentiate."
Sorry, but this answer makes no sense. Density is not the problem that is resolved by ROCK inhibitor. Clump size is the determinant. You can have a very dense concentration of single cells and they all will die, while larger clumps will survive.
The same is true for susceptibility to differentiation. Percentage of confluency is not the problem. The size and age of the individual colonies is what determines the likelihood of differentiation.
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Which concentration of Matrigel you use for iPSC culture? I was using 500 ul in 15ml and then, I have added 2 ml in 6 cm dish. However, my colonies did not attach.
Now, I am using 1.5ml of matrigel in 12 ml and so far, nothing.
:)
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Using a dilution ratio is meaningless unless you know the starting concentration of the matrigel you have on hand. I am not aware of any vendor that supplies it at a constant concentration that would allow you to use a constant dilution ratio.
All that matters is the protein concentration of the specific lot of matrigel that you have. From that you can calculate the proper dilution. In general, I use one mg of matrigel per 100 mm plate, which is the same surface area as one 6 or 24 well, or three 60 mm plates.
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I am adding (EGTA 2mM-10mM) to my mouse neural stem cell culture (media is made up of DMEM +10%FBS + 5% Horse Serum + 1% Sodium Pyruvate). I see the media changing color to orange, and the cells are rounding up and dying. However, when I PH the media has not changed in PH. What else could be causing this effect?
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Hiiiiiiiiiiii
Most cell attachment is calcium mediated , Caffein addition may be of interest as it opens up Calcium stores
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Currently I am working on mechanism of astrocyte development. For this I am making neural stem cell cultures (mouse).I am using CRISPR/Cas9 strategy to knock out genes. My plasmid is puromycin resistant. So I am planing to do Puromycin titration for my experiment. But I don't have a standard protocol for puromycin treatment. can anyone help me.
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Hi, John.
I've began recently to work with hiPS derived neurons.
My first experiments show that differentiated neurons
are dying during 3 to 4 days at Puromycin concentration
2 to 3 µg/ml. I'd suggest that neuronal precursors should be
even more sensitive. Any way, I guess quite simple titration experiments
in the range 1-5 µg/ml can completely solve your problem.
Best
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Real-Time is done and Mesenchymal stem cells had expression of cTNT, CTNN1 and other Cardiomyocyte cells markers
is it possible or cTNT is an specific Cardiomyocyte marker and something wrong is in this result ?
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As mentioned by others something in your culture conditions is causing your MSCs to express cardiomyocyte markers. You can try changing the culture conditions and then assay for the markers
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I am trying to transfect canine Ad-MSCs and NSCs with Bmi-1 plasmid by using lipofectamine 3000, but every time i do the transfected cells start dying after 48hrs, i am using following method for 6-well plate
· Per well
1. 125µL opti MEM + 5µL lipofectamine
2. 125µL opti MEM + 10µg P3000 + 5µg DNA
3. Mix both 1 & 2 and incubation for 20 min then put in cell culture drop by drop
4. after 6 hr serum & antibiotic free culture media is changed to full culture media
the first picture is of cell culture after 24-hrs and 2nd is after 48-hr
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Sir i am using 6 well plate with 4ml of culture media in each well ... surface are of each well 9 cm2
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RWPE-1 and WPE-stem are two prostate cell lines which need K-SFM(Gibco 17005-042) as their culture medium,which is in the protocol provided by ATCC.Problem is that in China mainland,because the medium has BPE(bovine pituitary extract) as a component,it is prohibited to import from overseas.My colleagues told me KM(sciencell 2101) or defined K-SFM(Gibco 10744-019) could be used as equivalence.But I asked technical support of Gibco to assure it,they gave me a negative answer.Could anyone tell me is there substitute medium which also could be used to culture these two cells? Thanks!
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Hi, Have u tried to grow them in other media like RPMI??
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Could you recommend is the best kit for isolation of CD34+ cells from bone marrow samples, using beads magnetic?
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What are the best culturing media, supplements, and protocols for expansion of HSCs?
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Dear Farshid,
I just briefly wanted to email you concerning your HSC culture question. In the attached review, you find a rather comprehensive overview over a) different supplements for HSC culture b) culture periods and c) subsequent assays that have been used to assess successful expansion, which you can hopefully tailor to your research question.
A company that supposedly has superb media for HSC culture is Stemcell Diagnostics from Canada. My fav choice (that always worked like a charm) is StemMACS from Miltenyi Biotec.
Unfortunately, they both don't come cheap!
All the best & good luck with your experiment,
Michael
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Hey all,
I'm an undergraduate researcher at UCLA and I was assigned to start a stem cell culture and differentiate those cells into cardiomyocytes. Currently using the GiWi method (CHIR and IWR1). I find that a large number of my xc-HUF-1 cells begin to die after Mesoderm induction by CHIR occurs after D0 (2 days after single cell seeding). In contrast, the H9 cells that I'm also differentiating seem to have minimal cell death after CHIR addition. I'm currently in the process of modifying the protocol using varying amounts of CHIR concentration and hopefully I'll be able to obtain beating cardiomyocytes. For the time being, I was wondering if any of you all have any advice / suggestions regarding my differentiation protocol. I was able to reach Day 15 with my last Xc-HUF-1 Differentiation protocol but was unable to get beating cardiomyocytes. Ran qPCR on the cells and saw that cTNT was expressed.
Thanks,
Jimmy Zhou
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1.You can check mycoplasma in your xc-HUF1 ipsc cell line.
2. Did your positive control (H9) work (get beating cardiomyocytes) ?
3. Single cell seed, what is the cell density? try more cell seed or add CHIR Day 3 or 4.
4. Single cell: with accurate and Y27632?
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I am trying to generate iPSCs by retransfecting my target cells (HEK293T) grown on feeder cells. I grew Mit-C treated feeder cells (70% confluent) on Gelatin-coated plate followed by seeding HEK293T. I am transfecting the plate with my reprogramming factor containing plasmid after every 2 days. The growth medium is KO-DMEM + 20% SR + NEAA + L-glutamine + b-marceptoethanol. This medium is supplemented with small molecules like sodium butyrate, Ascorbic acid, LIF and rhFGF. I am changing medium after every 24 hours. Moreover, my plasmid is Dox-inducible so I am treating with DOX after every 12 hours. After induction, I can see the GFP signal from my transfected cells but no appearance of iPSCS. Instead of iPSCs, the cells are dying. I am using the amounts of all the above mentioned components according to the literature.
Is there anything wrong with my medium or strategy? Is it a good strategy to re-transfect my target cell line in a mixture of feeder + target? Are my cells sensitive to the applied cocktail of chemicals? Please share your expert opinions. It would be very helping if you share some optimized protocol. Thank you.
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Hello Nasir.
The idea of iPSCs from a fairly stable cell line cells is not a solid. The cell has polyploidy feature of over 60 chromosomes and would not be easy to change their differemtiated state although it derived from 'embryonic' adrenal glandular cells. The original study using HEK293T cell line (check your cells are HEK293?) may also be different from your cell line due to the many sublines. And it did not demonstrated complete iPSCs from HEK293T cells with no further subsequent studies.
It is just like using human MCF7 or HeLa cancer cell lines that have highly polyploidy feature, except for adenovirus-transformed.
However, death of your cells may arise from the acidity caused by ascorbic acid and toxicties caused by sodium butyrate and frequent transfections. I'd rather use blood-derived cells for the iPSC line generation with many successful and well-established standard protocols.
Regards.
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I've been attempting to expand an iPSC line but unfortunately, compared to other lines I've worked with, it grows very slowly. I nearly lost the line as it started to detach due to (I think) the matrigel giving up after 12 days. I managed to save it and get it to a nice stage with good colonies but to avoid detachment again I split the cells 1:1 onto fresh matrigel. However it is still very slow and I keep feeling I'm going back to square one.
I use StemMACs iPS Brew media and matrigel as per the recommendation of the provider of the line.
I'm relatively inexperienced with iPSC work so would appreciate any advice or suggestions!
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That colony looks beautiful, a perfectly normal PSC morphology. That's so strange they grow so slow. Coating underneath Matrigel with vitronectin probably wouldn't help so I'd just seed the cells directly on vitronectin, either rhVTN-N from Thermo or VTN-XF from StemCell Tech. Incubating it at RT for an hour is good.
What's your concentration of Matrigel you're using for coating? You passage them as clumps and not as single cells, is that correct?
I have worked with an ESC line that was derived on VTN and I could never make it to grow on Matrigel, the clumps would never attach and the cells would just die, unlike all other PSC lines I used that could seamlessly transition between the two matrices.
I wouldn't want to accuse Miltenyi of not doing a good job with the StemMACS medium but its formulation is not published and I don't think it's as widely used as other media formulations like mTESR-1 (richer) or E8 (low-protein and xeno-free). Next time you're ready to passage them, I think I'd go for the 12-well plate and split the cells into 2 wells, one with your Matrigel and StemMACS medium and the other one with VTN and E8. The cells should be able to make the transition into the new condition relatively seamlessly, with no need for adaptation.
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Differential gene/protein expression profiles on same cell line always shows varied results, why does this happen?
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In both our H1 and H9 cells (just bought/thawed) we have noticed that sometimes the colonies will die/lift after 2-3 days of passaging or thawing. Usually the whole well is affected, sometimes only the outer edges, other times only the center. We are not sure what is causing this.
They are both mycoplasma free.
We're pretty sure it's not our media, we make our own E8 but tested with Stemflex and mTeSR and we still see this.
Our matrix is laminin-E8 (0.1 ug/cm2), but we see it (but to a less exptreme) in geltrex (1:100) as well. Is it matrix related maybe?
Any help/input/advice woud be greatly appreciated!
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Dear Shahab,
I think it is matrix related problem. TQ
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I am trying to grow Embryonic stem cells and and I have read in the literature that rho inhibitor are used to preserve "stemness" and viability. These were the most commonly cited chemicals, but I have no experience with these pathways. 
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Rock inhibitor does not preserve "stemness." It preserves survival during the passaging of colonies and allows for the survival of single cells that normally would commence apoptosis due to cell adhesion signalling being disturbed.
I have not noted any changes in colony morphology because I never use Y-27632 for longer than 12-18 hours. There is no benefit to using it any longer than that. In fact, a one hour exposure is sufficient.
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Hi has anyone had any issues using this kit? My negative control of the kit gives a higher value than the values suggested for positive control. Both controls from the kit itself. Tested for the recommended wavelengths of 490 and 650 nm. Also of course, all my derived mESCs show a high positive value. Suggestions/ Comments, please do share. 
Thanks!
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DEAR POOJA
GREETINGS
Here is the link to trouble shooting .Hopefull it should have remedy for your issue.please explote it.good luck.
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Just aCSF media will work or need some additives?
Regards
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Hello Maqsood.
You may use CSF if they are from fetal sources. However, spin it at 12,000rpm in eppendorf aliquots to precipitate any possible cell debris or particles. Take upper clear fluid into a lager tube together. Add 1 mg glucose, PenP+ StrepM mix/ml CSF to maintain ES cells. Mix well, make aliquots and store 4C until use. If you obtained from adult, you may need some other GFs for induced neural differentiation.
Best wishes.
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Hi
I used two cell lines hESC cutured in E8 medium feeder-free condition.When I convert into naive state use protocal by 3iL or NHSM media,I find the colonies shows continuous apoptosis and cannot passage even though forming a little dense colonies as picture.First I think the high concentration of every inhibitor harms to cells in consideration of individual cell line,but when I reduce the dose,it does not work.
Does anyone have similar experience or can give me advices?
Many thanks.
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Hi,
Have you figure out the way to convert the cells to naive state without using MEF feeders? I am now trying to use the 5iLAF in MEF-conditioned medium. Some domed colonies appeared, but very small along with massive of cell death.
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Im currently culturing dental pulp stem cells but to get the media is kind of difficult. a colleague offers me alpha MEM instead of DMEM. can I change the media or the cells will die. I also have F-12 but i have heard that cells culture can fail. thanks for help or advise.
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I search your question in google, so I think this article can help you to reach the answer:
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In terms of maintenance and differentiation, the potential of hESCs and mESCs differ. What are the differences between these two cell types and what are the ways to culture these two different cells? Please suggest a detailed protocol. Kindly cite any article if it is possible. Thank for your kind co-operation.
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I have isolated tumor-initiating (cancer stem) cells from a solid tumor and have hypothesized that our molecule of interest mediates the differentiation of these cancer stem cells into neo-vascular tissue.  I plan to incubate them in a hypoxic environment and measure the production of vascular markers and tube formation as described in the literature as markers for vasculogenesis with and without siRNA knockdown or inhibitor treatment
However, I can not find a clear idea about what media or factors I should add.  There are many protocols for maintenance of these cells once they grow, but which media should I be growing them in?  To force differentiation I see many researchers use VEGF, but A) I'm planning to measure VEGF production as an output for formation of these cells and B)I don't want to force them to become vascular cells, I want to see if they do it on their own.
If anyone has any direction or advice I would really appreciate it!
EDIT: I'm not starting with bone marrow or mesenchymal ES cells, these are de-differentiated astrocytes.  
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I would like to talk about the experience with human breast cancer MCF7 cells, however, I have no experience with a solid tumour.
Firstly, your culture system to be used will be hypoxia condition. This may be enough to induce differentiation pre-tumour cells without any additional peptides or chemicals among the mixed cell population of your sample. That is mimicking the in vivo niche condition forming spontaneous cell-cell and cell-ECM interactions. Some cells in your culture condition would make their own way from the mesodermal origin of cells hidden the mixed cells.The mesodermal cells could differentiate into endothelial ball of cells filled with fluid. However, number of those cells will be very small among the cell population. By increasing density, and so the hypoxia state, you could see floating cells of ball. or enclosed shape of  cells with a space on the dish bottom under low density. Obviously you are going to use biomarkers for the detection to be verified.
Second, I have seen this types of fluid-filled ball of cells with MCF7 cells which may contain stem-like phenotypes in the cell line. This happened in the medium we routinely used. You could use a similar medium people used for their culture of the origin of the solid tumour. that you are interested in. By the way, MCF7 is so-called epithelial cells, which are also heterogenous cells. They tend to show subtle EMT or MET under certain condition (niche gradually formed during proliferation and differentaition). Your mixed cells would show considerable selection process during culture, becoming much less heterogenous cells during culture condition and duration of culture.
It may be possible to demonstrate what's in your mind although it would be formidable task. So, my advice are to eliminate all the odds you may come across and perhaps to select cells from the tissues after chracterization for proper main experiments. Characterize the probable cell lineages within the dispersed cell suspension from the solid. then isolate by MACS or FACS for rather uniform cell population. It sounds like looking for a needle in a haystack.
You will see the light if you use less heterogenous cells. You can tell exactly what is possible or not with the characterized cells. The cells in neuroblastoma and glioma cell line are very uniform. Just compare your suspended cells prepared from the solid before you do anything. Good luck to you!
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I want to culture Balb/c mouse bone marrow derived mesenchymal stem cells in Low glucose DMEM media but i am unable to culture them. Although when cultured in IMDM media these cells efficiently grow . Why I am not able to grow these cells in Low glucose DMEM media ?
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Currently in the market for a protocol on how to culture hematopoietic stem cells isolated from mice OR if anyone has any suggestions on an immortal cell line representing immature myeloid cells. I am trying to study how cytokines change chemokine receptor expression in these cells.
Thanks!
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Hi all,
I am working with neural stem cells from SVZ in mice. Usually I get these cells from 4-7 days neonatal mice. this time I got cells from 11 days neonatal and result of my cell culture is completely different. All the primary plates show just clumps of cell.
my media is DMEM(F12)+B27+ gentamycine+ glutamin. 
Dose anyone know how I can treat these clumps and gets neurospheres?
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Hi Azadeh,
we use DMEM/F12+N2+P/S + EGF/FGF2 for neurospheres of adult SVZ and SGZ. SVZ usually does not make big problems in these conditions. Maybe they are growing more slowly than the early postnatal ones (we usually don't touch the cultures for 7-10 days before checking for spheres), and probably there is more tissue debris (myelin, dead cells, ...) in the initial cultures. Else might there be a problem with the tissue dissociation, which is probably different for the adult tissue. We use the Milteny adult brain dissociation kit, which works quite well.
I hope this helps.
Cheers,
Michael
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H!
We are using NS-A proliferation medium (Human) for neurosphere stem cell culture. Cell culture medium is becoming pink and little cloudy after every 5days during cell-proliferation. I am using sterilized condition all the time.
I was wondering if anyone can tell what could be the possible reason for this?
Thanks in advance ,
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Agree to all! Sound a lot like bacterial contamination. 
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Hi everybody,
We are struggling with a huge problem since a long time in our cell culture. Although we culture the our iPS cell (E8 and its supplement with Geltrex Coating) according to the same protocol we always do, cells do not attach or come off day after thawing/passaging. We do not have any contamination, we have tried different incubators, different lot number of the cell media/coating/plates, different vial of cells (partially differentiated or pure iPSCs)  and different cell culture rooms. Unfortunately we have got the same results (de-attaching or non attaching cells) each time.
Has anyone experienced that kind of problem before? If so could you give me some advice?
Thank you very much in advence
Tugce
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Make sure to add some ROCK inhibitor on the first day of passages or thaws, and give  the geltrex plenty of time to coat before seeding the cells!  As mentioned, matrigel also works, as well as vitronectin. Also make sure you're seeding them not too sparsely-- stem cells like having neighbors.  Good luck! 
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iPS cells were grown according to manufacturer's instructions:
E8 - vitronectin @ 400-500ng/cm2
mTeSR1 - matrigel (not specific concentration)
iPS cells in E8 apparently differentiate at a higher rate at similar conditions.
Room T-Culture media are changed every day.
Cells are split at 1:6
mTeSR1 seems to propagate and favor the growth of pluripotent cells while reducing the population of differentiated cells.
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I have been maintaining several cell lines of hiPSC in both E8 (E8 Flex as well as E8, and with daily media change as per Thermofisher for early passages) and mTeSR1 GMP (this is new GMP certified mTeSR1 by Stemcell).  The reason for this is that I wanted to test culture conditions for efficiency of differentiation into cardiomyocytes.
What I noticed was that a transition from "ideal" colony morphology (in E8 Flex or E8) to an alarmingly different morphology (in mTeSR) was cell line dependent, even from a single donor.
Colonies in E8 maintained it's ideal appearance whereas those in mTeSR eventually did not. However, when testing for pluripotency markers. both morphologies expressed NANOG, SSEA4, OCT3/4, and SOX2.
An important observation was that successful differentiation into iPSC derived CM could not be determined by morphology alone.  One cell line we had yielded many many more iPSC-CM when cultured in mTeSR (even with 'bad' morphology), whereas the opposite was found with another cell line—only E8 cultured cells with ideal morphology yielded a large amount of iPSC-CM.
I have attached images of the same cell line in E8 Flex and mTeSR, same passage number.
I spoke with our StemCell Technologies rep and showed them these images and discussed the issues we were having and I have not yet been provided with a response that addresses these differences.
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I am starting my post-doc using mESCs in a lab where they mostly culture human cells. They have a lot of human LIF from thermo fisher (PHC9481). I've read that mouse LIF receptor is activated by human LIF (but not viceversa), so I guess hLIF should be OK to grow mESCs in chemically defined media with 2i. Does anybody know for sure if this is OK? I am thinking on using 10 ng of hLIF / ml of medium. 
Thanks!!!
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we use human LIF for our ESC without any problem. do not worry
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I am using Mouse (ICR) Inactivated Embryonic Fibroblasts (Thermofisher, Cat No:A24903) to culture ES-E14TG2a Cell Line ( Sigma, Cat No: 08021401-1VL). It is suggested that approximately 1.5*10^6 MEF cells should be plated to culture mESC cells in T25 flask. If I increase density of MEF cells to 4*10^6 per T25 flask during culturing of mESC, Do I encounter any problem such as differentiation of mESC, low MEF depletion efficiency during MEF Depletion etc.?
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If using feeders, you should stick to the recommanded density. Feeders are there for LIF and other secreted factors, for providing a suitable matrix (that means not too many and not much). They should not exhaust the nutriments in detriment of the ESC.
Why would you change the feder concentration?
PS: for ES-E14TG2a, you can remove completely the feeders and growth only on gelatin coated plates. but then you need to have 2i in your medium.
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Hello every body, I want to perform a fibroblast-like colony unit formation or CFU assay or clonogenic assay for mesenchymal stem cells.
Personally I am working with equine adipose-derived stem cells, I would like to know the optimal numbers to perform the assay in passage 3 because I am afraid of seeding too much and I cannot count them or too few and I do not see any colony. I thought about a range of serial dilutions (800,400,200,100,50 and maybe 25).
Another question I have, some people perform this assay with the stromal vascular fraction, which is better? Which number of cells I must use in this second case?
Thank you,
Sergio.
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Being in "close proximity" would defeat the purpose of clonogenicity. Try doing a limited dilution technique to identify the optimal number of cells for whatever plate size you are attempting to use. We used this method on 100mm culture dishes and we were successful. Hope this helps!
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i want to culture neural stem cells from E14 mouse cortex. which neural stem cell culture gives the best result? what growth medium components are to be used? 
for serum free media supplements? what do you recommend to use? 
would using only GS21 be enough? or should i use b27 and N2 for better results? and also which vendor do you recommend to buy the supplements from? 
thanks in advance
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Hello,
We use Esc medium (with KSR), Neurobasal medium, N2 and B27 supp. to make dopaminergic neurons, but must be used in different times of differentiation.
I recommend you follow a article that alredy did this culture and diffrentiation (oligodendrocytes) from mouse cells.
You will spend less time following a protocol already established.
Regards,
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I'm dealing with human induced pluripotent stem cells culture using Matrigel or vitronectin XF coated plates. I passage the cells as small clusters and used ROCK inhibitor with mTeSR1 medium for the first two days of each passage. It has been fine for the past but recently, cells would detach and die. At first I through it was because they were too crowded and do the passage before it was too dense. It seemed to work. Then last time during passaging, I broke cell clusters into mostly single cells and supplemented with ROCKi all the way during culture. They grown normally for the first 5 days, then detach before reaching optimal confluency. I checked literatures but find nothing about long term ROCKi usage would cause detachment. Please help me find the reason.
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Hello,
Yes, rock is used only in passage or defrost (first 24 hours). After 24 hours you must changing meium take off it. Normally diluated in DMSO that can differentiate your iPSC too.
I agree, maybe the problem is with your sample of the medium or matrix.
We work with iPCS in clamps and single cells, both works good.
In our lab we use E8, mTeSr1, L7 médium, gtx, matrigel, vitronectin, L7 matrix...
When we have problem first we check reagents (see with others researchers if has same problem. If yes, use new samples that you are using same) and incubator (water, CO2, O2, temperature...)
To dissossiate cells we use:
- Vensene, Accutase , Tryple, Relesr = passage in single cells
- Versene, Relesr, Gentle = passage in clumps
- Dispase, Collagenase IV = to make Embryoid Bodies
Regards
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My mouse embryonic stem cell line are not working. They are not able to attach to the surface of coated dishes ( I have tried both Geltrex and 0.5% gelatin) remain in suspension. I am using ESGRO LIF. Please help 
Media composition =  KO-DMEM+15% FBS+1xGluatmax+1xNEAA+BME+PS 
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You should start by checking your FBS.  Always use ES-qualified FBS.  We have had batched of regular FBS that do not promote adhesion of ES cells.  Different lots of ES-qualified FBS still give variability, but at least they are screened to confirm adhesion.
You should also check how the ESCs were cultured before they were frozen.  If they were on MEF feeder cells, you will want to thaw them on feeders.  If they were adapted to feeder-free, confirm the matrix. There will be less stress to maintain the same conditions they were adapted to before frozen.
Confirm your final concentration of b-mercapto.  I have had techs use the wrong bottle at the wrong dilution and wonder why the cells are not attaching.
ES cell culture is less forgiving than other cell cultures.  If you are still having problems, find a PI or mentor for more hands-on training and practice.
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Hi,
I have successfully isolated stem cells from fresh urine but I am trying to figure out if the urine sample can be refrigerated for a period of time equivalent to shipping on ice and still get cells out. I am setting up an experiment to try and isolate cells from urine that is refrigerated for 24, 48 and 72 hours but I was wondering if people have done this. I haven't found many papers online. Any suggestions are welcome.
Thanks!
Elisa
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Hi Elisa, you could consider freezing them at-80oC in the presence of DMSO.
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Is the only way to make essential 8 medium without FGF2, TGFbeta1 and insulin (I want to use insulin at 5ug/mL, not 19.4mg/L as in the original essential 8 medium protocol in the Chen et al 2011 publication) to make the medium from scratch? The commercially available essential 6 media (LifeTech) still contains insulin at 19.4mg/L if I'm not mistaken?
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There's TeSR-E5 from STEMCELL technologies that does not contain FGF2, TGFbeta 1 and insulin:
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Hello,
I am culturing stem cells for 96 hours and there are currently several Problems I am experiencing:
1- the humidity (even under the CO2/humidity/temperature control of cell culture incubator) does nto seem to be enough. Lecault et al. made an iso-osmothic bath, however, this is very time consuming and needs the whole design of the chip to be redone. Does anyone have other methods/tricks to Keep the environment humid enough for the cells in the chip? It seems the cell line cells surviving such environmental Change much better.
2- what are some surface covering techniques that have worked for you to reduce the pdms toxicity as well as reduce adsorbtion of small hydrophobic molecules into pdms? (the PDMS i have is 1:20 for control & 1:5 for flow layer)
thank you in advance for sharing any of your advices
i am new to microfluidics, so protocols, advices, tips and tricks on two-layer soft lithography are highly appreciated.¨
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Hi Anar,
How thick is your PDMS membrane separating your cells from the atmosphere? From what I've read in the literature, you want to keep it under 200 um for optimal gas exchange.
Also, is this a static or flow system? If static, you may need continuous perfusion of culture media in order to keep the cells viable by supplying fresh nutrients and removing waste products.
Best,
Allan
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I am establishing a mo-DC protocol, and there is too many option in regards of maturation. I would like some help deciding. Since later in downstream applications I would be combining IFNg, I would like to include this cytokine in the protocol. However, I've seen that in order to truly create maDC i should activate the other canonical pathways. That is why I though of using TNFa and LPS. Before jumping and trying it out, I was wondering if anyone has experience with this type of cocktail or IFNg+TNFa.
Thanks
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Hi
Try LPS + IFNg + Poly I:C for max TLR stimulation. I would not try TNFa if you want a good TH1 output from your DC. TNFa used to be used along with IL-6 and PGE2, but now you don't see them used as they can create a DC2 profile to Tregs. 
Bruce 
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Does anyone culture op9 without FBS? When I removed FBS or added bad FBS, OP9 was abnormal and could not support the hematopoietic differentiation. Why KSR can not replace FBS?
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Has anyone tried to culture iPSC on Geltrex with mTeSR ??
The data sheet suggest to use Matrigel or vitronectin for mTeSR but I would like to know if anyone has tried to use this medium with Geltrex too?
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Hi,
Geltrex can be used as an alternative to Matrigel, both are the extracellular matrix (ECM) from EHS tumor.
Best regards
Justyna
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Good evening, friends.
I did MSC differentiation from human iPSC. but difficult to maintain and subculture. 
I followed the papers, 
I just followed some published methods.
"PLOS ONE, January 2013 | Volume 8 | Issue 1 | e54524" 
after 12 days, i subcultured on 0.1% gelatin coated dished and maintained in MSC media (Low glucose DMEM supplemented 10% FBS + rock inhibitor).
After 3 passaging, cells were not good.
After MSC differentiation, cell size was bigger than iPSC and numbers were decreased. there was no cell proliferation. I think cells are senescence. So it is hard to subculture and hard to maintain.
how to subculture iPSC-MSC? is there any media supplement or extra matrix?
Thanks in advance......
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Hi,
I think that you should use differentiation medium with mitogens: basic fibroblast growth factor (bFGF) and/or epidermal growth factor (EGF).
Good luck!
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I want to culture spheroids and isolate stem cells from immortalized human proximal tubule cells. I have been able to get single suspension of human proximal tubule and seed them into ultra low attachment flasks but I want to pick one sphere and grow them on a 6-well plate to see if they can produce a clonal population. I am just beginning to do this, I do not have a lot of idea on these type of isolation techniques to obtain a stem cell population, so I would appreciate any comments/ suggestion. Thank you
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Dear Mai
Great, I wish you a lot of success! If you need assistance, please let me know.
Regards,
Patrick
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i'm currently looking for what would be the best vector/plasmid for silencing genes in rat stem cells culture. I found some paper where they use lipofectamine, plus reagent, vector pCU and pCH. I also found that genscript make vectors. I would like to know f anyone does silencing with lipofecanie method, and also what vector would be perfect for silencing.
Thanks!!! and sorry for bad english haha
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I am seeking a method to generate microglia from human mesenchymal stem cells (preferably bone marrow derived MSCs).
Does anyone have a method to suggest?
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Dear Lauren
It is very interesting question "Generation of microglia from human mesenchymal stem cells?"
But I have some question.
1- Why microglia from MSCs not from HSC (hematopoetic stem cells).
Hinze A, Stolzing A. Microglia differentiation using a culture system for the expansion of mice non-adherent bone marrow stem cells. Journal of inflammation. 2012 Apr 02;9(1):12. PubMed PMID: 22471998. Pubmed Central PMCID: 3495406.
Muffat J, Li Y, Yuan B, Mitalipova M, Omer A, Corcoran S, et al. Efficient derivation of microglia-like cells from human pluripotent stem cells. Nature medicine. 2016 Nov;22(11):1358-67. PubMed PMID: 27668937. Pubmed Central PMCID: 5101156.
Hinze A, Stolzing A. Differentiation of mouse bone marrow derived stem cells toward microglia-like cells. BMC cell biology. 2011 Aug 19;12:35. PubMed PMID: 21854582. Pubmed Central PMCID: 3175184.
The references may help you .
2- Microglia belongs to hematopietic lineage
HSCs------myeloid progenitor cells- -----yold sac microglia cell progenitor-------microglia. 
I SUGGEST YOU TO DIFFERENTIATE MSCS TO MYELOID PROGENITOR CELLS THEN IT IS VERY EASY TO DIFFERENTIATE MYELLID PROGENITOR TO MICROGLIA CELLS
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I recently tested the EdU alternative AmdU in MEF cells and tried to detect it using DBCO-Sulfo-Cy3 all from the company Jena Bioscience. However, the DBCO-Sulfo-Cy3 gave super strong unspecific staining everywhere in the cell. There are a few publications that used AmdU but different dyes for detection.
Can anybody provide helpful hints or a protocol to improve the staining?
Thank you very much,
Patrick
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Hey Ali,
thank you for commenting! Still hoping to get some help or suggestions on that.
Best,
Patrick
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Hello! I would like to ask about the quantification of sGAG after the chondrogenesis of mesenchymal stem cells.
My principal doubt is how to extract the sGAG to performs an assay with Dimethylmethylene Blue Assay (DMMB) because I have found protocols for that and they talk about the complex sGAG-DMMB but they do not mention how to obtain the sGAG in solution... Maybe with the application of the reagent the sGAG are realising to the medium as sGAG-DMMB complex directly after following the protocol?
Another issue, if I do Alcian blue/nuclear fast red stain. I cannot use them for the abovementioned assay right? I should have two pellets (one for the histology and one for the quantification)
Thanks a lot,
Sergio.
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Alternatively, you could use an Alcian blue based assay. But be forewarned they stopped making Alcian blue back in 1979 due to its highly carcinogenic activities during manufacture (large number of employees making stain were getting cancers). So any Alcain blue purchased after 1979 is not anywhere near to being 100% stain, it is mostly fillers. Alcian blue assay protocols can be found in:
Young HE, Dalley BK, Markwald RR. Glycoconjugates in normal wound tissue matrices during the initiation phase of limb regeneration in adult Ambystoma. Anatomical Record, 223:231-241, 1989.
Young HE, Young VE, Caplan AI. Comparison of fixatives for maximal retention of  glycoconjugates for autoradiography, including use of sodium sulfate to release unincorporated radiolabeled [35S]sulfate. Journal of Histochemistry and Cytochemistry, 37:223-228, 1989.
Young HE, Carrino DA, Caplan AI. Histochemical analysis of newly synthesized and resident sulfated glycosaminoglycans during musculogenesis in the embryonic chick leg. Journal of Morphology, 201:85-103, 1989.
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Hi, so I have hESC colonies growing on MEF feeder layer. I'm wondering approximately how many cells a colony of about 1mm in diameter would contain?
Thanks in advance.
Wei Xing 
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I think you have to count. It depend of dencity, day, passage.... We are working with placental MSCs  - monolayer is 24000-50000 cells\cm2
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Is my first time working with stem cells and I don't what kind of stem cells are better for this. 
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It would be helpful if you specify which kind of neurones do you want to obtain. There are plenty of protocols published and some commercial kits that can be useful (I would recommend you to better find a protocol than a commercial kit since they are quite expensive). 
And also what kind of stem cells: if you want to start with pluripotent stem cells (embryonic or iPS) or if you're starting directly with neural stem cells, if they are human or from any other source, etc. Then I guess people might give you more specific answer.
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I'm trying to do a live/dead cell assay (grown on 96 well plates) and I have problems with my cells adhering to the plate. I'm trying to establish a method that will allow addition of live/dead detection reagents, and then immediate fixation without washing out the live/dead reagents.
I need to fix the cells because this is for high throughput screening and I can't image quickly enough for accurate comparison.
Any thoughts?
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you can use 'CellTracker™ and CellTrace™ Reactive Probes' from Fisher. Live cells will be fluorescent and this compound is stable after fixation...
good luck!
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i m searching for papers that have an injection dose of stem cells that used to treat colon cancer ..
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i think using mesenchymal cells may enhance tumour progress if they act as immune supressants
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I'm working on immobilize graphene oxide onto polydimethylsiloxane (PDMS) for cell line/ stem cell culture purpose. I have graphene oxide solution (not sure what the solvent is, but solvent is not highly volatile) and PDMS membrane/sheet. If I directly spin-coating or dip-coating graphene oxide solution on PDMS and dry it by heating, does it work to immobilize graphene oxide onto it? If it does work and durable for cell culture, I wonder the interaction force between the graphene oxide flakes and PDMS substrate. Or should I modify PDMS surface before graphene oxide coating? Any references?