Ask the Expert – Meet the Cells – Tips and tools for working with different cell lines

By on December 7, 2015
Lego Lab

Do you have work with stem cells, CHO cells, or vero cells cells? Have you been waiting for some tips and tricks for improve your relationship with your cell line? If so, don’t miss this multi-cell interview where you can learn important tips for working with these cells. Whether its troubleshooting problems in your culture, understanding nutritional needs, cell vulnerabilities, or other issues, then visit this week’s review of some of our best cell related Ask the Expert questions from this year to learn more!

This Ask the Expert Session is Sponsored by ThermoFisher Scientific and features “interviews” with popular cell lines, which covers some of our best cell related Ask the Expert questions. ThermoFisher Scientific has a fun interactive way to learn more about cell lines in their “Life of Cells” series, which features the cells as characters with important information, fun interactive contests, and clever videos. Meet their new character, Neuro, a neural stem cell. This week we will feature “interviews” to share key tips and tricks for each cell line.

Cell Lines

Meet Vero

BioVero 1

I was established in the 1962 and my lineage is African Green Monkey Kidney. My main job is to grow viruses. My biggest accomplishment so far is that I have been used in the commercial manufacture of the seasonal flu vaccine. I am hoping to replace eggs altogether! I would love to use my skills to help produce vaccines for all the world’s most dangerous viruses.

Question:

We are working on the adaptation of adherent Vero cells to suspension culture under serum-free conditions. Especially the step towards stirred systems showed increased formation of cell clumps, decreased cell growth and, thus, cell stagnation or cell death. Do you believe that an adaptation of Vero cells into suspension is feasible? Which strategy would you target: media design/ optimization or cell adaptation/ luck of getting a clone which finally prevails?

The Answer:

Generating Vero suspension cells, will be a challenge and there are several aspects which will be very important to consider:

  • Media should include fluids which reduce shear forces, if the cells are being grown in a shaking format
  • It may be necessary to screen many cells, to find a clone which might have the ability to grow in a suspension format
  • It may be necessary to continually passage cells, until the suspension adapated cells finally prevail
  • May want to try formulations of media designed for suspension cells

Here is a link with some additional information that may be helpful – http://www.lifetechnologies.com/us/en/home/references/protocols/cell-culture/serum-protocol/adaptation-of-cell-cultures-to-a-serum-free-medium.html

Question:

Which are all the antibiotics can be used in combination in large scale Vero cell culture and at what concentration?

The Answer:

This question can be answered many ways depending on the goals. Maybe you would like to be proactive and you want to use antibiotics to prevent an infection or maybe you want to cure a current infection. Of course my recommendation is not to use antibiotics unless absolutely necessary. I prefer having good frozen stocks of cells that I can thaw if needed. Having said that though, I suspect the best general antimycotic is Fungizone (Amphptericin B). At normal working concentrations (0.5-2.5 ug/ml) it works to prevent fungal contaminations. Interestingly in Vero cells, at low concentrations (250 ng/ml) amphotericin B has been show to increase viral uptake (http:/www.ncbi.nih.gov/pmc/articles/PMC3194987/). I have provided a listing below of other antibiotics and antimycotics and their working concentrations. I will mention that the range of antibiotics and antimycotics used does vary depending of the media type used. Media containing serum can contain more antibiotic/antimycotic since the high protein, mostly albumin binds much of the reagent. If you are using a low protein medium or a serum-free medium you will need to use much less for equivalent results, perhaps 10% to 50% depending on specifics of your culture method.

NAME , CONCENTRATION, SPECTRUM, STABILITY AT 37C IN MEDIA
Anti-PPLO Agent , 10-100 ug/ul, Mycoplasma and gram positive bacteria, 3 days
Fungizone, 0.25-2.5 ug/ul, Fungi and yeast, 3 days
Gentamicin Sulfate, 5-50 ug/ml, Gram positive and gram negative bacteria and mycoplasma, 3 days
Kanamcin Sulfate, 100 ug/ml, Gram positive and gram negative bacteria and mycoplasma, 5 days
Neomycin Sulfate, 50 ug/ml, Gram positive and gram negative bacteria, 5 days
Nystatin, 100 U/ml, Fungi and yeast, 3 days
Penicillin G, 50-100 U/ml, Gram positive bacteria, 3 days
Polymixin B Sulfate, 100 U/ml, Gram negative bacteria, 5 days
Streptomycin Sulfate, 50-100 ug/ml, Gram positive and gram negative bacteria, 3 days

Question:

I have a vero cell line that I would like to transition to serum free media, can you give me some advice on where I should start.

The Answer:

This is a good question and those of you what are using VERO cell lines are lucky. GIbco’s VP-SFM is a media designed for growing VERO cells in suspension. VP–SFM is a serum-free media that requires little or no adaptation for VERO cells. The media will also grow other types of cells such as BHK-21 cells and COS-7, but some like COS-7 will require adaptation. We can talk about adaptation later on, but to answer your question directly, you can start with low passage cells that are highly viable. Gently pellet your cells grown in the old media. I would not centrifuge faster than about 1000 rpm. Leave a small amount of media above the cel pellet and use it to gently loosen the pellet. After pellet resuspension you can add the fresh SFM containing L-Glutamine or GLutaMAX. and grow your cells as you normally would in suspension. It is often helpful in the beginning to add a higher starting cell density than normal.

 

Meet CHO S.

BioRobust 1

I was established in the 1960’s and my lineage is chinese hamster ovary. My main job is expressing recombinant proteins in large amounts. My best attributes are that I love expressing and I’m a pretty easy going, robust cell. My biggest accomplishments are that they call us the “workhorse” of biomanufacturing and we are the most commonly used cell line in the manufacture of biopharmaceuticals.

Question:

Hi, I am wondering if the CHO genomic sequence known?

The Answer:

This is a good question and the answer is yes! The paper by Xun Xu et al., Nature Biotechnology, Vol 29, No 8, 201, pg 735. reports that a CHO-K1 ancestral cel line was sequenced. There were 24,383 predicted genes. What is very interesting about the paper is that the authors focused on investigating genes involved in glycosylation. Homologs of most human glycosylation-associated genes are present in the CHO-K1 genome. The authors also point out correctly that this is important since CHO cells are used to make human like therapeutic antibodies and glycosylation patterns are critical to function and turnover rates. Ultimately this research may lead to making CHO cells even better for bio-production than it already is.

Question:

I am looking for a good resource for using CRISPR in CHO genome editing. We are just beginning to look at this technology for improving cell characteristics. Any advice?

The Answer:

Here is a website with background information on using CRISPR for genome editing.
http://www.lifetechnologies.com/us/en/home/life-science/genome-editing/geneart-crispr/crispr-cas9-based-genome-editing.html

CRISPR-Cas systems have become powerful tools for genome editing and the system as a good choice for your needs. This website also has a short video that will probably be helpful. The CRISPR Nuclease vector is the system for mammalian cells if you don’t have any promoter constraints.

Question:

What is the difference between all these different types of CHO cells? I read about CHO-K1 and CHO-DG44 all the time.

The Answer:

There are many types of CHO (Chinese Hamster Ovary) cells in use today. It is a bit of a mystery when CHO cells originated but it is thought the line was derived in the 1960′s. Dr. Theodore Puck received a female Chinese Hamster from a laboratory at the Boston Cancer Research Foundation and used the animal to derive the original cell lines. One line was known as CHO-K1, was obtained from those first CHO lines. CHO-K1 is an adherent line and was traditionally grown of F12 Medium supplemented with 10% FBS. The CHO lines were thought to be important because they had few chromosomes for a mammalian cell and were used for radiation cytogenetics and they were known for their ease of culturing. CHO-K1 cells also do not express EGFR.

CHO-DG44 cells were derived from the original CHO-K1 cells by several rounds of mutagenesis that deleted both copies of the dhfr genes. Dihydrofolate reductase (dhfr) is required for the de novo synthesis of purines, thymidylic acid, and certain amino acids and is required for growth and proliferation of CHO cells. As a result CHO-DG44 cells require glycine, hypoxanthine and thymidine (GHT) for growth. This requirement allows for selection of recombinant clones by transfection of recombinant DNA plasmids containing a gene of interest and the replacement dhfr gene if cells grow on GHT minus medium then they have taken up the recombinant plasmid DNA and hopefully the gene of interest as well.

Question:

What are the advantages of using a transient CHO system over a 293 system?

The Answer:

In the past, 293 cells were the overwhelming choice of researchers performing transient protein expression because 293 cells could generate significantly higher titers than CHO cells in a transient setting. For example, Expi293 is capable of generating titers greater than 50-fold higher than the FreeStyle CHO™ transient expression system. With the introduction of the ExpiCHO transient expression system, this is no longer the case, and indeed in many/most instances ExpiCHO is significantly higher expressing than Expi293. Secondly, since CHO cells are used to manufacture >70% of current licensed therapeutic proteins today, the ability to start drug development work in CHO cells and stay in CHO cells from discovery through bioproduction reduces risk, as transient CHO-derived proteins are more similar in terms of critical quality attributes to proteins made in stable CHO cells. Thirdly, the ExpiCHO expression system offers unparalleled flexibility in terms of protocol options. Three different protocols are provided in the ExpiCHO manual, Standard, High Titer and Max Titer protocols, which allow researchers to choose the option that best fits their needs in terms of equipment, time, number of steps and amount of protein desired. Additionally, because of the robustness of the ExpiCHO system, there are many ways that the system can be further modified to meet the particular needs of a given lab.

Question:

We are having some difficulties with expression of one of our proteins in 293. We are considering trying CHO instead and possibly changing to a CHO platform for consistency with our manufacturing cell line. Is there any reason that you would recommend keeping both 293 and CHO as transient expression systems or would adopting a CHO platform be fine?

The Answer:

We have seen multiple instances where a protein that does not express in Expi293, or expresses at a very low level, is expressed well in the ExpiCHO system. At this time, there is not a consensus understanding as to which proteins express better in 293 cells vs. CHO cells, however, our experiences and some data shared from independent labs seems to suggest that if you were to run just one system to produce all of your proteins, that ExpiCHO would likely be the system to pick. Additionally, as you mention, the relevance of the protein generated in ExpiCHO to your production cell line is certainly also something that should be considered, as this allows for a more seamless workflow from discovery to clinic. Lastly, whether to keep a 293 system and a CHO system is based on the specific needs of your lab: for instance, if you are making protein reagents where human post-translational modifications are desired, then having a 293 system makes sense. If you are expressing only candidate therapeutics, then ExpiCHO would certainly be your first choice. Many/most labs that have the ability to run both systems are doing so, providing them with two options to best generate their proteins of interest in the most relevant cell line and providing the greatest likelihood that they will get the protein they need from either the Expi293 or the ExpiCHO system.

Question:

We need post-translational modifications of our transiently expressed proteins that are similar to those of our stably expressed proteins to be used for clinical trials. How can a transient CHO system help?

The Answer:

In addition to the high titers and the ability of the ExpiCHO system to express proteins that don’t express well, or at all, in 293 cells, the key reason to use CHO cells for transient protein expression is to reduce the risk of critical quality attributes of your protein changing when transitioning from early discovery into pre-clinical and finally clinical trials. One of the main goals during the development of the ExpiCHO transient expression system was to ensure a more seamless transition from early drug discovery through clinical production allowing for researchers to start their work in CHO and stay in CHO throughout the entire drug development process. To this end, it is well documented that post-translational modifications in 293 cells and CHO cells are distinct from one another, with glycosylation patterns and sialic acid content being two instances of differences between the cells types. The ExpiCHO expression system demonstrated glycosylation patterns for a human IgG that were highly comparable to those of the same antibody expressed stably in CHO-S cells; in contrast, the glycan patterns for the same antibody expressed in 293 cells were strikingly different, with significant differences observed in the percentages of G0F, G1F and G2F glycoforms compared to CHO-derived material. The maintenance of protein critical quality attributes is critical in ensuring similar bioactivity and pharmacokinetic profiles of therapeutic proteins from pre-clinical to clinical trials.

Question:

We are looking to improve our cell culture productivity in CHO cells. We want to use imaging to inform our process choices and improve our cell health, viability and of course titer. What methods would you recommend?

The Answer:

There are a number of choices for evaluating the health of your cells using fluorescent imaging assays, from simple to complex. Understanding how healthy your cells are, and determining optimal timing for passage can improve productivity. A number of assays can help provide useful information.

  • To evaluate viability, the use of a cell-impermeant DNA binding dye may be used, such as the Image-iT DEAD Green Viability stain where dead cells will fluoresce green. A dead cell stain can be combined with a marker of cell vitality that measures esterase activity, Calcein AM, as seen with the LIVE/DEAD Cell Imaging Kit where active cells fluoresce green and dead cells fluoresce red. Probably the most simple method uses ReadyProbes Dead Cell stains; these are ready-to-use reagents for fluorescent imaging that have been simplified for use (add 2 drops from the dropper bottle per mL of sample and image).
  • Cell proliferation analyses can be helpful for assessing cell growth. A very useful method uses incorporation of a thymidine analog, EdU, for direct measurement of cells in S-phase using click chemistry. The Click-iT Plus EdU assays are fully compatible with fluorescent proteins.
  • You may want to know if your cells are dying via apoptosis, the CellEvent Caspase 3/7 Green reagent is a no-wash assay compatible with labeling in media, the green fluorescence intensity increases with caspase activity.

Question:

We are having trouble determining the optimal cell density for our transient expression in CHO. Any suggestions?

The Answer:

The optimal cell density is dependent upon a number of factors, but in a typical system comprising cells, media and transfection reagent, most often 1×10^6 cells/mL is the cell density chosen for a number of reasons: 1) Typical transfection reagents do not work well at very high cell densities—this is both a consequence of the transfection reagent itself as well as how well the culture media can maintain the high level of cell health at higher densities needed for high efficiency transfection, 2) at high transfection densities, cells will rapidly overgrow the cultures post transfection and unless your culture media can handle this, the culture will crash and productivity will be poor. The ExpiCHO transient expression system is designed to deal with all of these problems and has been developed to transfect CHO cells at 6×10^6 cells/mL, or even higher, densities significantly higher than the standard 1×10^6 cells/mL transfections that are typical. ExpiCHO utilizes this high cell density to increase the number of cells expressing your protein and to ensure maximal yields.

Meet Neuro

BioNeuro

I was established in 1873 and my lineage is neural stem cell. I really like networking and making connections. I often find myself leaning on my good friends for support, Schwann and Gilal.

Question:

Do you have recommendation for getting neural stem cells to differentiate into astrocytes, not neurons?

The Answer:

Neural stem cells can be differentiated to either neurons or astrocytes following the protocol described at this weblink:

http://www.lifetechnologies.com/us/en/home/references/protocols/neurobiology/neurobiology-protocols/differentiating-neural-stem-cells-into-neurons-and-glial-cells.html

Question:

What cell culture surface do you use for neural cell differentiation?

The Answer:

For neural, generally it is poly L Lysine coating followed by laminin coating surfaces.

Question:

I have been tasked with developing a protocol for identifying and isolating different cell types from a mixed population of neural cells using flow cytometry. I have been working on using cell surface signatures, but it has been challenging. Could you recommend any materials or methods for review?

The Answer:

The Gibco® Neurobiology Protocol handbook provides a comprehensive list of methods and protocols to support neural cell research. The handbook includes protocols with topics including neural cell culture and differentiation, cell analysis, molecular characterization, and transfection. (http://www.lifetechnologies.com/us/en/home/global/forms/gibco-neurobiology-handbook.html)

In addition to marker expression, reagents such as fluorescent probes can be used to study neural health, anatomy and function. See an overview of products used for neural cell health at http://www.lifetechnologies.com/us/en/home/life-science/cell-analysis/neuroscience.html

Question:

Rapid protocol for differentiation of iPSCs into dopaminergic neurons?

The Answer:

Rapid in this case may not be as important as efficient. The developmental biology of TH+ neurons is not understood well enough to ‘speed up’ the development of different types of neurons including dopaminergic or cortical motor neurons. A recent study found that Dibutyryl cyclic-AMP (dbcAMP) was shown to induce up to 85% in vitro differentiation of neural stem cells in to neurons, but still required the same differentiation timeline as that of in vivo human brain development of these neurons. When looking for an efficient method to differentiate, you may consider starting with enriched dopaminergic precursor cells. It has been shown that the better the quantity of enriched precursors (for example FoxA2+/Lmx1a+) the better the efficiency of the final phenotype for dopaminergic neurons ( for example, TH+/Nurr1+?FoxA2+) which in turn creates a starting population which is enriched for desired lineages.

Meet Stem Cell

BioStem-guy

I was established in 1978 and I have many jobs. In research, I can be used to model different disease states and test the effects of drugs for toxicity and efficacy. I can also be used for cell therapy by being transplanted into patients. My biggest asset is that with the proper programming, I can become anything I want to be. My biggest weakness is that I can be sensitive, a bit difficult to culture and I am pretty specific about my needs.

Question:

How many times can I passage my pluripotent stem cells before the health suffers? Are there any products that can help extend the number of passages or time in culture?

The Answer:

Pluripotent stem cells, unlike primary cells, have an infinite lifespan. However, with increasing passage number there are a number of reports indicating chromosomal instability and differentiation bias that can result due to the stress associated with passaging methods and culture conditions. Therefore, we generally recommend that karyotype analysis of cultures be assessed every 10 passages and we ensure that a working bank of earlier passage PSCs be maintained as a safety stock. The most predominant large karyotypic abnormalities that have been shown to accumulate include gain of chromosomes 12 and 17 and chromosome X. There is a very nice review that was published early last year reviewing genome maintenance of pluripotent stem cells (J. Cell Biol. 204(2): 153-163).

One of the mechanisms outlined in this review which is responsible for some of the genomic instability is the build-up of reactive oxygen species (ROS) during stressful processes, which include passaging conditions, as well as recovery from cryopreservation. ROS can result in double stranded breaks which are particularly deleterious as during the repair process point mutations, as well as non-homologous end joining can result. Thermo Fisher Scientific recently launched a supplement which can be used in post-thaw recovery as well as for support of cells during passaging conditions-RevitaCell™ Supplement (Cat #A26445-01). NOTE: It should only be added to the culture medium for the first 24 hours post-thaw or post-split followed by feeding with unsupplemented medium for the remainder of time in culture. RevitaCell™ Supplement contains a specific ROCK inhibitor coupled with compounds having antioxidant and free radical scavenger properties. In general, addition of antioxidants to the culture medium has been shown to improve genome stability by reducing the ROS within the PSC during a range of processes (Stem Cell Reports 2:44-51, Scientific Reports 4:3779).

Question:

I am having trouble retaining differentiation potential of my pluripotent stem cells after thawing, any recommendations?

The Answer:

Generally it depends the efficiency of your differentiation method. But if you are seeing the problem due to freezing and thawing, try ThermoFisher’s RevitaCell™ Supplement (100X), on the day the cells are thawed. Following 24hrs in culture, there is no need to use this supplement. Detailed instructions are available. Also some limitations apply, as some specific cells may not respond. http://www.lifetechnologies.com/order/catalog/product/A2644501?ICID=search-product

Question:

We are trying to transition our iPSCs to a serum free media, how do you recommend weaning and transitioning?

The Answer:

I am assuming you are starting with some fibroblast growth medium like DMEM supplemented with ESC-qualified FBS. If you are using GIBCO Essential 8 medium as your SFM you can transition directly. Essential 8 is designed for feeder free growth. Here is a reference https://www.lifetechnologies.com/us/en/home/references/protocols/cell-culture/stem-cell-protocols/ipsc-protocols/generation-human-induced-pluripotent-stem-cells-fibroblasts.html

If not, There are two ways to do this. Try a sequential adaptation. Assume you are using old media A and want to transition to new media B. Start in media A and know something about the kinetics of growth. Transition to 80%A:20%B grow several passages then move to 60%A:40%B then 40%A:60%B and so on until your cells are in 100new medium B. The other method involves removing 50% of the old media and supplementing with 50% the new media. If you are removing serum you will need to coat the dishes with some attachment matrix like geltrex or vitronectin if you want attachment.

Question:

I am looking for a system that can detect differentiation using imaging techniques that are not invasive to the culture. Do you have any recommendations?

The Answer:

There are two possible approaches you could use for a system to detect differentiation using non-invasive imaging techniques. The first is simple phase contrast microscopy. Using this approach you would look for the spike appearance of differentiating cells. Using imaging tools you can mask the undifferentiated cells and mark the differentiated cells. You may want to look in to an imaging tool hosted by the NIH which would also allow you to quantify this method. The second method I would recommend is utilizing a tool offered by Life Technologies – the Alkaline Phosphatase Live Stain. This is a non-invasive technology by which undifferentiated cells can be stained in 20 minutes or less by fluorescence. Cells that are not picked by this stain are differentiated cells. The Alkaline Phosphatase Live Stain is a quick and easy tool to use for detecting differentiated cells.

Click for more information about the Alkaline Phosphatase Live Stain – http://www.lifetechnologies.com/order/catalog/product/A14353?CID=search-product

Click to see a selection of tools for live and fixed cell staining of pluripotent stem cells – http://www.lifetechnologies.com/us/en/home/life-science/stem-cell-research/induced-pluripotent-stem-cells/pluripotent-stem-cell-detection/pluripotent-stem-cell-antibodies.html?icid=cvc-stemcelldetection-c2t1

Question:

Many colonies of different shapes and sizes seem to express some surface markers of self renewal, how do I identify the best colonies to select and expand?

The Answer:

The use of a negative marker in conjunction with a positive marker is the most effective method of identifying fully reprogrammed colonies prior to expansion and selection. Somatic tissues like CD34+ blood cells, PBMCs, and human Fibroblasts express the surface marker CD44 at high levels. Expression of this marker has been demonstrated to be significantly down-regulated in fully reprogrammed cells. The combination of CD44 antibody staining with either SSEA4, Tra-1-60 or Tra-1-81 will more clearly distinguish fully reprogrammed colonies from partially reprogrammed colonies by the absence of CD44 and presence of self-renewal marker expression.

For a complete list of PSC markers, please visit lifetechnologies.com/stemcellantibodies

Question:

I am reprogramming hematopoietic stem cells to iPSCs. Ideally I would like to have a few different methods available for confirming pluripotency that could be either used in conjunction for more in-depth analysis or independently for a quick screen. Any thoughts?

The Answer:

Several methods are commonly used for PSC characterization {Marti et al (2013)

Characterization of Pluripotent Stem Cells, 8, 223-253}. For initial screening of emerging iPSC colonies, Alkaline Phosphatase Live Stain

(http://www.lifetechnologies.com/order/catalog/product/A14353) can be used. This is a method of identifying pluripotent stem cells without impacting cell survival or characteristics {Singh et al (2012) Stem Cell Rev. 8, 1021-1029}.

Alternately, immunostaining can be carried out on live cells using antibodies against pluripotent specific surface antibodies such as SSEA4, TRA-1-60 and TRA-1-80. (http://www.lifetechnologies.com/us/en/home/life-science/stem-cell-research/induced-pluripotent-stem-cells/pluripotent-stem-cell-detection/pluripotent-stem-cell-antibodies.html?icid=cvc-stemcelldetection-c2t1)

The final confirmation is to check for trilineage differentiation potential either using in vitro embryoid body formation or in vivo teratoma assays. Recently molecular assays have been developed as a comprehensive high-throughput method for characterization of pluripotent stem cells based on gene expression signatures. TaqMan® hPSC Scorecard™ Assay is a qRT-PCR based method that quantitatively measures the expression level of 93 genes comprised of a combination of self-renewal and lineage specific markers, against validated reference standards. (http://www.lifetechnologies.com/us/en/home/life-science/stem-cell-research/taqman-hpsc-scorecard-panel.html) This method can be used to analyze undifferentiated and differentiating PSCs to not only confirm biomarker expression in an undifferentiated state but also show expression of markers specific for the three germ layers during differentiation, thus confirming functional pluripotency.

Question:

How many passages can I expect to get out of my cells in long-term culture and on what days do you recommend splitting them? Also do you have any suggestions on how to increase the rate of expansion of cells?

The Answer:

Human pluripotent stem cells (both embryonic and induced pluripotent stem cells) can be successfully cultured and maintained for a very long time. Although they can be cultured for very long periods of time, most researchers and laboratories have guidelines as to how many passages they would use cells before they thaw a new vial (most guidelines suggest that scientists will use cells unto 50-75 passages before they will retire those cells and thaw a new vial). It is important to perform routine karyotype analysis to ensure that the cell line has not acquired an abnormal karyotype after long-term passaging.

There are no specific ways to increase the rate of expansion of cells. Most human pluripotent stem cell lines are routinely passaged on day 4 or day 5 and it is vital to keep the cells on a routine in order to avoid stress (passaging them too early or letting them overgrow will cause stress). Scientists have developed techniques to scale up the passaging by changing the split ratios and this is the optimal way to increase the yield. A good example is the use of EDTA as a dissociation agent when cells are cultured in a defined medium such as Essential 8 medium with a defined substrate such as Vitronectin. In this case, since the use of EDTA generates smaller colonies (compared to other dissociation agents such as collagenase and dispase) the passaging ratio can be adjusted to 1:8 1:10 or even 1:12.

We have attached the relevant protocols and a publication for culturing cells in Essential 8 medium on Vitronectin using EDTA.

Please find some links below which you may also find helpful:

Culturing PSCs in Essential 8™ Medium – http://tools.lifetechnologies.com/content/sfs/manuals/feeder_free_PSCs_in_essential8_medium.pdf

Frequently Asked Questions – Essential 8™ Medium and Vitronectin – http://tools.lifetechnologies.com/content/sfs/manuals/FAQ_Essen8_Medium_vitronectin_man.pdf

Scalable expansion of human induced pluripotent stem cells in the defined xeno-free E8 medium under adherent and suspension culture conditions

Authors: Wang Y, Chou BK, Dowey S, He C, Gerecht S, Cheng L, Journal: Stem Cell Res (2013) 11:1103-1116 –

http://www.ncbi.nlm.nih.gov/pubmed?cmd=Retrieve&dopt=AbstractPlus&list_uids=23973800

Question:

We are having difficulty maintaining genome integrity in our ipsc in culture. We have seen several mutational changes when the cells are in culture for longer periods. Any suggestions on how to troubleshoot.

The Answer:

It is difficult to answer this question without having details of your culturing conditions. Following are the most common reasons for mutational changes and abnormal karyotypes in human pluripotent stem cell cultures-

  • Overgrowth of cells- if cells are routinely overgrown and not passaged when they are in log phase (actively growing), the stress can cause mutations
  • Use of a harsh dissociation agent- if a harsh dissociation agent such as trypsin is used for routine passaging and maintenance of human pluripotent stem cells, the cells get dissociated as single cells and will not be able to survive in cultures. IF they do survive, they will adapt under stress and have mutations
  • In general any culture conditions that will induce inadvertent stress or selective pressure will result in mutations after long-term passaging
  • Also, passaging the cells too soon (every two or three days) can induce stress
  • I would suggest to check culture conditions, dissociation agents, dissociation methods, passaging frequency as possible stressors

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One Comment

  1. Ira Presser

    9 December, 2015 at 6:35 AM

    I would like to use LMH cells for the production of a virus. When I grew the cells using Weymouth medium in a CO2 incubator there was no problem. The problem started when I tried roller bottles. The cells require CO2 which could not be supplied. I adapted the cells to grow in M199 using half the amount of sodium bicarbonate and was successful. However the cells had a change in morphology- more fibroblast like and became reflexive towards the virus. Additionally, our supplier said that they can only supply Weymouth as a 1x due to solubility problems. Do you have any suggestions?

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