- Saeed heidari keshel added an answer:3Why do cryopreserved stem cells take a long time to restore their morphology and function?
Considering three cryopreserved MSCs with a passage number of four and timeline with two in 2015 and one with 2014. After a week of thawing and maintainence, I could see very few cells changing from spherical to spindle. Is it normal and is there any reason behind this?
P.S. the cryopreservation was done by someone else.
MSCs from bone marrow and cord blood are cryopreservable by the methods used to cryopreserve their haematopoietic analogues, and slow freezing protocols utilising DMSO as cryoprotectant are in use by many groups . Recent studies by Liu et al. have confirmed that 10% DMSO and slow cooling/rapid warming does not affect the viability or differentiation potential of adipose-derived MSCs. Adult stem cells derived from human dental pulp also showed high post-thaw recovery and trilineage differentiation potential after slow cooling in 1–1.5 mol/1 DMSO (∼7.5–10%). Furthermore, DMSO was shown to be superior to both ethylene glycol and propylene glycol under these conditions .
DMSO is known to have an effect on the epigenetic profile of, and induce differentiation in, murine stem cells . This, together with its the known adverse reactions when employed as a cryoprotectant for haematopoietic cell therapy, plus its cellular toxicity, have led to attempts to cryopreserve MSCs with other cryoprotectants; either on their own or in combination with DMSO at lowered concentrations. Many of the alternative cryoprotectant formulations have also attempted to remove animal serum from the cryoprotectant solution; both to reduce cost and to improve clinical utility through reducing the likelihood of zoonotic infections in the absence of terminal sterilisation procedures. This has led to substitution with human serum or human serum albumin. However, this too is costly and introduces at least the potential for transmission of human pathogens.
The use of polyvinylpyrrolidone (PVP), an extracellular cryoprotectant, has been investigated as an alternative to cryopreservation with DMSO and foetal calf serum (FCS). Cellular recovery and differentiation capacity were studied after equilibration in a number of different cryopreservation media followed by ‘dump’ freezing to −80 °C and storage in liquid nitrogen. Recovery of cells cryopreserved in 10% PVP with human serum was comparable with (though slightly lower than) cells cryopreserved in DMSO with animal serum. A similar study utilising methylcellulose either alone or in conjunction with reduced levels of DMSO indicated that human serum could replace FCS in standard DMSO mixtures without affecting the recovery of cells and that 1% methylcellulose produced comparable results with DMSO concentrations as low as 2% when an annexin V apoptosis assay was used to analyses cells 24 h post-thaw. Cells also maintained their adipogenic and osteogenic potential . Lui et al. have also adopted a reduced DMSO approach in a recent study to produce a well-defined and xeno-free cryopreservation media for cell therapy with bone marrow-derived MSCs. They replaced FCS with combinations of polyethylene glycol (PEG) and trehalose, with DMSO concentration ranging from 2.5 to 7.5%. The recovery, metabolic activity, proliferative capacity and differentiation potential were measured against the standard slow cooling protocol of DMSO and 10% FCS. Results replacing FCS with PEG in reduced concentrations of DMSO were comparable with those of the control, but this was achieved only in the presence of 2% bovine serum albumin. Moreover, cryopreservation in 10% DMSO with 90% FCS was more effective in their hands than any other combination. Trehalose was ineffective, in contrast to studies on haematopoietic cells derived from cord blood, bone marrow or mobilised peripheral blood where its effectiveness in combination with reduced DMSO concentration has been demonstrated . Nevertheless, these laboratory studies indicate that there is a potential for the development of xeno-free cryoprotectant solutions utilising lowered concentrations of DMSO and slow cooling.
- Paul Cizdziel added an answer:8Best way to reprogram fibroblats and PBMC?
I currently use Sendai virus to reprogram my fibroblasts and PBMC to iPSCs. As I have translational aspirations, I need to switch to other approaches. Any thoughts about episomal plasmids? or other non integrative methods? What do you do in your labs?
About the only way to do PBMC is with virus. The cells do not transfect with enough efficiency to use any other technique. However, it is possible to isolate endothelial progenitor cells (EPC) from the blood and expand a culture of them. They resemble fibroblasts and easily reprogram using RNA. The Stemgent srRNA Reprogramming System is optimized for that and yields cells that typically have 10-20X greater genomic stability (less copy number variations) than by any other technique. Furthermore, being RNA, the vector clears from the cytoplasm from all cells in 3-5 passages. You can also keep back-up frozen vials of the original EPC for future use, if it becomes required. With regard to fibroblasts, the Stemgent mRNA Reprogramming kit is a bit more cumbersome to use because it requires many transfections, but it is very effective and well published. Recently the NY stem cell foundation published an article creating a high-through-put reprogramming robotic system, that employs this technology. RNA is the way to go. DISCLAIMER - I work for ReproCell Japan, the company that owns Stemgent. That aside, prove it to yourself, you will be satisfied!Following
- Viktor Y Butnev added an answer:2How long to incubate primary antibody, abcam alpha-SMA, at room temperature for western blot?
I'm performing a western blot testing for alpha-SMA. I purchased the antibody from abcam (ab5694), and was wondering if it could be incubated at room temperature; and if so, how long it should be incubated?
My understanding is that certain antibodies can degrade at room temperature. Therefore, I wanted to see if someone had already figured out the optimal time for primary antibody incubation.
It depends on the affinity and concentration of each particular antibody. In our lab we use even low-affinity purified monoclonal antibody at the concentration 1 ug/ml for 30 min in western blot ( 1 ug antigen loaded) and it works just fine. You don't have to worry about any possible degradation of your Ab at room t°. But you need to test your Ab in experiments at different times of incubation to draw any conclusion. Usually, commercial companies test any antibodies for stability (including room t°) before they get a clearance for sale.Following
- Alys Jones added an answer:5I am inducing iPS cells into cortial neurons and was wondering if anyone know why after neural induction cells form rosettes?
I am inducing iPS cells into cortical neurons and was wondering if anyone know why after neural induction cells form rosettes? Why do they polarise in this way? Are cells that don't form rosettes not successfuly induced?
Ok thanks James. We have been able to form the rosettes but have had some issues of other cell types in the culture (possibly neuroepithelial cells). We are optimising our dispase step at the moment and this is helping with increasing our final neuron efficiency.Following
- Bhawna Chaubey added an answer:13What is the minimum methanol concentration that can be used as control for tissue culture?I am testing my crude plant extract. It is dissolved in methanol so I am taking methanol as a control. I am using 100ul for 2 ml media.
thanks Mr. Gustavo, but lower concentration of methanol will lead the drug stay insoluble. what protocol should one follow to see interaction between any protein and any compound that insoluble in water. What are other suitable solvents for dissolving protein other than pbs that dont affect its strucutre. How to deal with solvent selection for such system?Following
- Amira Othman added an answer:5Does any have experience with Ki67 stainng in stem cell ?
I am working with cardiac proginator cells and I need to do immunoflourscent staining with Ki67, I need to know the best way how to fix the cells ? does it need antigen retrieval step .
I appreciate if any one provide me with a protocol.
Thank you in advance
Ok thank you so much , I will follow this protocol.Following
- Gary Lee Gilmore added an answer:5Hello everybody,does anyone have an explanation for the loss of surface expression(by FACS) after thawing?
Primary human stem cells expressing c-kit (also known as CD117) were obtained by cell sorting (99% purity ,using CD117 antibody),seeded,expanded and frozen(10%DMSO,90% FCS) at confluence.
Surprisingly,after thawing these cells don't expresse c-kit(DC117) evaluated by FACS analysis.
Does anyone have an explanation for that ?
I work with human UCB stem cells [CD34+/CD133+] that are also CD117+ [c-Kit]. I often use frozen mononuclear cells [post-Ficoll] and have never had any problem seeing CD17 on the surface of the CD34+ CD133+ cells. That being said, you are dealing with an adherent cell population. c-Kit is a receptor tyrosine kinase, like c-fms [CD115] and I know from studying mouse c-fms that freezing of an adherent c-fms+ cell line [RAW 264.7] results in loss of c-fms. Since RAW 264.7 is a transformed line, it eventually recovers c-fms expression after culture for a while. I do not know if your cell population will behave in a similar fashion or notFollowing
- Christopher Lange added an answer:9How can I dissociate very adherent differentiated stem cells?
hiPSCs are plated on Matrigel coated 6 Well Plates and differentiated using a Dual SMAD Inhibition protocol to induce neurectoderm. After approximately 14 days, the cells are passaged onto chamber slides for immunofluorescence analysis.
While this protocol has worked for other members of the lab using cell lines, I am having trouble dissociating the differentiated cells from the 6 Well Plate. I have tried Accutase, 0.5mM EDTA, and a Trypsin(0.05%)/EDTA solution. The cells simply do not lift off the plate, and I am forced to use a cell scraper to remove them. While the cells lift off with scrapping, they form whispy white threads/clumps which can not be plated. The cells that are not contained in those clumps have a very low survival rate.
I am hoping to find suggestions for improved dissociation of these cells and any ideas as to why this is happening.
Note: These cells passage fine with either 0.5mM EDTA or Accutase before they are exposed to the differentiation protocol.
The collagenase and TrypLe (separately) should be prewar med to 37 deg before use. It is also better than standard teypsin because most teypsins contain a xhymotrypsin contaminant that digests the trypsin while it is at 37 deg C. The genetically engineered active moiety of trypsin, in TrypLe is pure and does not self digest, so your stock remains at the same strength o er long periods of time.Following
- Dong-Jiunn Jeffery Truong Zhang added an answer:2Did anyone try the lentivirus for CRISPR and nCas9 of abm?
I want to knockout a gen and I would like to know how to select the clones without the protein. There is a antibiotic sistem in the plasmid but that does not provide that the mutation take place in every cell if I am using a pool of them. Thank you!!
If your GOI is expressed in your target cell line already (because some people first modify one easy to transfect/harvest cell type and afterwards transdifferentiate it to another), you can create a "Donor" sequence like this:
5'-HA_SA_P2A_Marker_2xpA_3'-HA when targeting intronic sequence with already at least one coding exon before.
5'-HA_SA_Marker_2xpA_3'-HA when targeting 5'-UTR (Don't forget start codon for Marker)
5'-HA_P2A_Marker_2xpA_3'-HA when targeting exonic coding sequences
HA: Homology Arms
SA: Splice Acceptor
P2A: Porcine Teschoivirus 2A peptide
Marker: Can also be fluorecent proteins (especially when you have FACS sorting systems in your facility) and not only selection markers. I prefer using fluorescent proteins since selection often leads to multiple non-specific integrations especially when you use too much antibiotics.
2xpA: 2x strong pA signal to really terminate the transcript at this point (e.g. SV40 late pA + bGH pA), because you might have very few times a read-through
Pay attention that all is in-frame which is important when using SA and P2A sequences. Also be aware of isoforms and alternative TSS.
I prefer to method targeting an intron inlcuding loxP sites flanking the "SA_P2A_Marker_2xpA" region because then you can show elegantly that you can rescue the phenotype or whatever by cutting this "stop-cassette" out with Cre and showing the reviewers that your actually phenotype is because of your knoch-in...Following
- Corey Fee added an answer:2How can I create a mouse model of a Dopamine Transporter 3'-UTR variant (VNTR) that doesn't exist in mice?
I'm trying to model the human DAT1 Variable Number of Tandem Repeats (VNTR) 9- and 10-repeat alleles in mice (this is purely theoretical and for a project - will not be carried out in practice). This variant is localized in the human 3'-UTR, but does not exist in mice (the gene does). Different alleles of this variant have been associated with different levels of DAT1 expression, but results are not conclusive. I have already proposed in vitro work to examine transcriptional activity (Luc assay) and mRNA stability (pulse-chase), but I would like to create an in vivo model.
Can I simply replace the mouse 3'-UTR with the human code? What in vivo considerations do I need to include? Is there another way to mimic this variant e.g. site-directed mutagenesis? siRNA?
Thank you. I ended up coming across a paper that had expressed the human variant in a mouse ES line. http://onlinelibrary.wiley.com/doi/10.1046/j.1471-4159.2001.00647.x/fullFollowing
- Rezvan mobasseri added an answer:4How much GAGs (glycosaminoglycans) is expected of MSCs in their ECM?
Does anyone know how much GAGs I can collect if I decellularize MSCs in their stemness state and can I stain this GAGs with Alcian Blue?
Thank you all.Following
- Diptiman Choudhury added an answer:4Can anyone please suggest me a method for synthesizing nanoporous polysaccharide nanomaterial?
For my work I need a porous carbohydrate nanomaterial. Can anyone please suggest me a method for the same?
Thanks a lot Prof. Renard,
I will definitely try your suggestion. Lets just hope it will work for me.Following
- Aditi Nadkarni added an answer:9ProtocolHi , do anyone have protocol for chondrocytes differentiation from MSC?
This may help: http://www.currentprotocols.com/WileyCDA/CPUnit/refId-sc01h03.htmlFollowing
- Apurva Kulkarni added an answer:14Which marker can I use in conjunction with Nestin for staining Neural Stem/Progenitor Cells (NSPCs)?I need to double stain for NSPCs as a confirmatory test that the cells that I am culturing are NSPCs. Please suggest a another protein that I could stain for. I have read about Sox-1/2. Would these be appropriate?
I used nestin, SOX2 and Pax 6. These have worked really well for my characterization. I think you should make these three as starting points. Although, I believe the age at which you take these NSPCs would also afftect the expression of these markers. Also, like many others have stated in this discussion, NSPCs would also be oct 4-. So I would also include that as a negative marker.Following
- Aslam Khan added an answer:42Nitric Oxide Assay?I would like to quantify NO production from cell lines. I found a couple kits on the market and I am not sure what one to use. Does anyone have any experience using these kits. Can I use colored media?
Would you please be more specific? As you Nitric Oxide pathway is dependent on arginine and citruline pathway. If your compounds are iNOS2 substrates then you need use rINOS2 enzyme to test effect on your compounds.Following
- Saleh Alkarim added an answer:4Neural differentiation of induced pluripotent stem cells?
I'm searching for quality control test about neural differentiation of induced pluripotent stem cells?
# reverse transcription-polymerase chain reaction (RT-PCR) analysis
# electrophysiological testing
# microRNA (miRNA)Following
- Sawsan Mohammed Elhalawani added an answer:4Expansion of rat bone MSCs, what are the best conditions?
I'm trying to expand rat BM MSCs, using DMEM, low glucose, 10 % FBS (Hyclone) + glutamax, but I'm facing some detachment of cells specially after passage #6. Any advice for the best conditions?
Hi Dr/ Masi
this is very very clear :-), i will try this protocol in my next isolation...
thanks a lot for your quick replyFollowing
- Nadjia Bekkari added an answer:1How long can human macrophages survive under hypoxic condition in culture?
Hi, everyone. How long can human macrophages (differentiated from human blood monocytes) survive under 1 or 3% O2 hypoxic condition in culture? Other conditions are the same as those for standard macrophage culture.
Hello, it may resiste to 2 or 3hours. under hypoxic condition cells have involved some other signaling pathways mainly HSP pathway, you must to test this experiment according cinétic (15 minutes, 30 minutes, 1h, 2h, 3h) to confirm result and search what pathway can human macrophage use in this condition, best wishs.Following
- Dushyant Tomer asked a question:OpenWhat should I do to make CVD grown MoS2 flatter and cleaner?
I am growing MoS2 by using CVD method ( MoO3+ S as precursors). Getting good triangles but these is something on top of those triangles so I am looking for suggestions to find a way to get rid of these features.
Thanks in advance.
- Wulligundam Praveen added an answer:4What must be the initial seeding number of mESCs for a kill curve expreriment ?
I am trying to figure out the puromycin concentration for selection of puro-resistant mESCs. For the kill curve, I am confused about the initial cell number and the confluence % required to start the treatment.Syed A Ali,
Thank you for the article.Following
- Wulligundam Praveen asked a question:OpenIs there any alternate for OP9 cells in hematopoieitc differentiation of ESCs ? How good is the OP9-conditioned medium in this regard?
I currently use OP9 cells for hematopoietic differentiation of mESCs, which is taking a lot of time. Multiple times, the OP9-mESC co-cultures go bad and show an altered morphology and spatial arrangement, leading to floating of the differentiating cells, which puts my time and efforts in vain. Currently I cannot afford for defined medium (with external addition of cytokines). Hence, I would like to know any alternatives in this regard.Following
- Joe Graymer added an answer:9Which marker is best for staining of breast cancer stem cells by immunohistochemistry?
I have been collecting breast tumor tissues from patients and am going to study the expression profiling of stem cell regulatory genes. Also, i would like to do assessment of breast cancer stem cells in tissue section by immunohistochemistry.
but am in little bit of confusion regarding markers. Which marker is best for assessment ? ( ALDH, CD133, CD24, CD44)
There was a 2012 Astra-Zeneca slide presentation in a series of: 'Early Breast Cancer', first unit was about: 'Overview and assessment of Early Breast cancer', perhaps you can contact directly them, I can't send you the slides, because I'm not the author nor the copyright owner. Good day!Following
- Dmitriy Sheyn added an answer:1What is the replacement curve for BMP4 for adipogenic differentiation of C3HT101/2 cells?
I am trying to induce adipogenic differentiation of c3ht101/2 cells by using IBMX, Dex and ınsulin. The protocol requires BMP4 addition to the media. I could not find the replacement curve for BMP4. Should I change the media twice for a six day incubation period.
Standard adipogenesis protocol doesn't require BMP4, look for the ITS+/Dexa/Insulin protocol. C3H are easy to differentiate.
- Dmitriy Sheyn added an answer:3Any advice on the morphological changes of human IPS cells culture on feeder layer?
I used primary human feeder cells, MSCs, to maintain human IPS cells.
I wonder why the morphology of human IPS cells on MSC is flatter and thinner than on MEF feeder.
I used same density of both feeder layers.
is that associate with "naive" and "primed" state?
I know that primed ESC colony is flat and also late passages pluripotent stem cells are flat.
please tell me your opinion about that phenomenon.
There feeder free media these days... I would expect them to differentiate on MSCs. try the feeder free system and check if your cells look better. Sometime they change without particular reason, but here the feeder layer seems to be the factor.
- Hamed Ghazavi added an answer:4Which stem cells are best to do cell culture studies on wound healing?
I need to know whether co-culture is the best way or only one cell culture on the prepared scaffolds is a way to do for wound healing?Hi,
Adipose drived mesenchymal stem cells due to their valuable advantages can be a suitable candidate .Following
- Eric Lyle Weiss added an answer:10Re-use Miltenyi columns?Does anyone have a protocol for reusing Miltenyi columns? I heard that it is doable and popular but just could not find a detailed protocol for that. thanks.
How about microMACS columns used for protein purification or sub cellular fractionation? Is there a special protocol for cleaning these up?Following
- Hemant Agrawal added an answer:19My mesenchymal stem cells(h-MSCs, P4) express CD90 and CD73, but only 30% of the MSCs express CD105.The result is weird. What type of cells express CD90 and CD73 without CD105? Can anybody share experience?
wharton's jelley is digested with 1mg/ml collagenase supplemented with 500ug(total amount) hyaluronidase for 10 hours
medium is LG-DMEM+10%FBS+2mM Gln+5ng bFGF
Which clone of Cd105 (company) you are using? Is your protocol has a fixation step before or surface staining? As everyone said CD105 is expressed in all h-MSC.Following
- Amita Ajit added an answer:6What do you think is the best economical cocktail/niche for differentiating MSCs to Endothelial Cells?
I am working on therapeutic angiogenesis and lately i have been differentiating MSCs to ECs. I have come across numerous strategies and i would like to know your valuable opinion on the most economical & effective strategy based on personal observations.
Thank you Mr. Todd.Following
- Swati Chitrangi added an answer:8What do you think about these human MSC I've cultured?
Hi! This is my first time to culture MSC(from MDS patients' BM), I notice there are three kinds of MSC: 1. stretched and big,irregular in shape,with sandy granule near nucleus, most frequently seen 2. thin and long,spindle-like. 3. flat and faint in color,with spider like reticulate structure in cytoplasm. These can form extraordinarily orginaized structure can they are confluent, some expand as colonies, with adipocytes in the center. What do you think of these cell,are they typical MSC in morphology? Do the different morphology signify anything? Thanks!
Cells in all four figures are looking like mesenchymal stem cell, you can confirm by immunophenotyping CD105, CD 73 and CD 90 positive. Figure 1 is looking like 100% confluent viable cells. Cells in figure 2 and 3 are looking like growth arrested cells.
About Stem Cell Biology
All about stem cell biology! Stem Cell Protocols and Methods.